Você está na página 1de 115

The International Spinal Research Trust

Annual Research Review


2008

The International Spinal Research Trust


Annual Research Review
2008

Research Division
Bramley Business Centre,
Station Road, Bramley, Guildford, Surrey GU5 0AZ, UK
Telephone: +44 (0)1483 898786
Facsimile: +44 (0)1483 898763
E-mail: research@spinal-research.org
Website: http://www.spinal-research.org

Along with donations from private individuals, Spinal Research is pleased to acknowledge the generous support of
Charitable Trusts, Charitable Foundations and other organisations, including those listed below. Their generosity and
the significant part they are playing in helping us to beat paralysis is greatly appreciated.
Annett Trust
Bank of New York Mellon
Betfair
Bernard Piggott Trust
BHSF Medical Charity & Welfare Trust
Centreline Air Charter Ltd
Charles & Elsie Sykes Trust
Childwick Trust
Colin Javens Spinal Injury Trust
Corporate Action Trust
Dr. Scholl Foundation
Drandjeaw Charitable Trust
Elizabeth & Prince Zaiger Trust
Ernest Kleinwort Charitable Trust
Eveson Charitable Trust
Ford Motor Company Ltd
Freemasons Grand Charity
G C Gibson Charitable Trust
G J W Turner Trust
Gerald Palmer Eling Trust
GlaxoSmithKline
Grimmitt Trust

Hasluck Charitable Trust


Heineken International
Henry Lumley Charitable Trust
Henry Smith Charity
Inman Charity
Investec Bank (UK) Ltd
L & R Gilley Charitable Trust
Mace & Jones
Miss Joyce Kathleen Stirrup Charity Trust
Nene Valley Branch Spinal Research
Newton Investment Management Ltd
Ninth Duke of Newcastles 1986 Charitable Settlement
PF Charitable Trust
Robert Luff Foundation Limited
Sir Joseph Hotung Charitable Settlement
Steel Charitable Trust
Swire Charitable Trust
Thaxted Branch Spinal Research
Trust PA
West Midlands Branch Spinal Research
William A. Cadbury Charitable Trust
Yorkshire Branch Spinal Research

Contents
Welcome from the Chairman of the Scientific Committee

Funding for Research

Research Network Meeting


Agenda
Meeting Report
List of Delegates

7
9
12
26

Strategy Grants
Recent Awards

28
29

Nathalie Rose Barr PhD Studentships


Recent Awards

30
32

Research Reports
Nathalie Rose Barr PhD Studentships

33

The use of a YFP-expressing mouse in studies of spinal cord injury: mechanisms of chondroitinase-mediated repair
Lucy Carter, Liz Bradbury and Stephen B. McMahon

34

Overcoming the molecular inhibitors that impede axonal regeneration following spinal cord injury
Philip Duffy, Isabella Gavazzi, William Cafferty and John Wood

40

Do omega-3 fatty acids modify inflammatory changes following a spinal cord compression injury?
Jodie C.E. Hall, J.V. Priestley, V.H. Perry and A. Michael-Titus

49

AAV8shRNA-RhoA and AAV8nt-3 transfection of dorsal root ganglion neurons (DRGN) in vivo mediates
neuron survival and disinhibited regeneration of dorsal column (DC) axons
Steven J. Jacques, Ann Logan, Martin Berry and Zubair Ahmed

53

Effects of injury and chondroitinase ABC treatment on neuro-glial morphology and interactions involved in
CNS functioning, particularly of the visual pathway
Maria Cristina Ovejero-Boglione and Arthur Butt

61

Promoting long-distance axonal regeneration and functional reconnection using combined treatments for
spinal cord injury
Thomas Sardella, John S. Riddell and Susan C. Barnett

66

Project Grants

70

Promoting axon regeneration in the injured spinal cord by RNAi-mediated knockdown of receptors for neurite
growth inhibitors
B. Blits, E. Ehlert and Joost Verhaagen

71

Role of microglia in spinal cord injury pain


Fabien Marchand, Elizabeth Bradbury and Stephen McMahon

79

Standing and Stepping with Intraspinal Microstimulation after Spinal Cord Injury
Vivian K. Mushahwar

81

Can Human Lamina Propria Olfactory Ensheathing Cells Expand, Migrate and Stimulate Rat SCI Repair
as well as Mouse OECs?
Jane Roskams

84

Development of functional magnetic resonance imaging for assessing human spinal cord injuries
P.W. Stroman, R. Smith, R. Pokrupa and K. Smith

87

Improving cardiovascular function after spinal cord injury


P. Waite, P. Carrive and A. Mackay-Sim

93

Comprehensive evaluation of the physiological and functional adaptations induced by locomotor training in
incomplete spinal cord injured subjects
Bernie Conway, Ken Hunt, David Allan, Sujay Galen and Celia Caton

98

ISRT Grantholders

104

ISRT Scientific Committee Members

108

International Campaign for Cures of spinal cord injury Paralysis (ICCP)

110

ICCP Spinal Cord Injury Clinical Trials Guidelines

112

ICCP Participating Organisations

113
3

Welcome from the Chairman of the Scientific Committee


It is a great pleasure for me to be able to introduce the 2008
Annual Research Review, which contains reports of all
projects currently funded by the Trust, and of Trust activities
such as the annual Network meeting. The Trust currently
supports research into spinal cord injury under three main
funding streams, and as a guide to the detailed reports that
are contained in the Review, Ill briefly describe work in
each stream:

applications, and were able to fund 4 (Dr Elizabeth Bradbury,


Kings College London, Investigation into the conduction
properties of surviving axons following chronic spinal
cord contusion and whether therapeutic intervention can
restore normal function, Professor Ann Logan, University
of Birmingham, Comparative evaluation of surgical and
pharmacological methods for removal of a mature scar in a
chronic spinal cord injury model and subsequent regeneration
of stimulated sensory neurons through the treated wound,
Professor Mark Tuszynski, University of California, Axonal
regeneration in the chronically injured spinal cord and
Dr Ronaldo Ichiyama, University of Leeds, Locomotor
training in chronic adult spinal cord injured rats: plasticity of
interneurons and motoneurons).

Natalie Rose Barr (NRB) PhD studentships. These


awards are important for the training they provide as well as
for the research projects undertaken. In 2008 the Trust
made two new awards (Professor Gennadij Raivich from
University College London entitled; Non-integrating
lentiviral expression of GMCSF to promote spinal cord
regeneration and Dr Claudia Wheeler-Kingshott from
Institute of Neurology, UCL entitled; Spinal cord diffusion
imaging: challenging characterisation and prognostic value),
and two existing students (Philip Duffy, Maria OvejeroBoglione) completed projects and submitted, or were
awarded, PhD degrees.

The Clinical Initiative. This important collaborative


programme is focussed on developing, and evaluating, new
ways of measuring functional recovery in spinal injury
patients. Phase 1 of the programme started in 2000 and we
are currently over half way through its second phase, which
involves four groups (in London, Glasgow, Zurich,
Edmonton) and is due to finish in June 2009.

Strategy Awards. These are normally three year project


grants which are awarded in response to an annual grant
call, which is focussed each year on a different theme,
informed by the Trusts research strategy. This year we invited
applications that focussed on understanding the changes that
take place in the chronic stages of a spinal cord injury and on
experimental strategies designed to promote recovery of
function from such chronic injuries. This is an underresearched and very important topic, which is aimed at
developing therapies for existing spinal injury patients. We
received 41 outline applications, from which we invited 9 full

Thanks to the hard work of Trust staff, and the generous


donations of the Trusts many supporters, the Trust had
28 active projects during the year, with a combined budget
of 5,845,236. Great challenges remain in our efforts to
repair the damaged spinal cord, but this Report is eloquent
testimony to the important progress that is being made.
Thank you to all who have contributed to it.
Professor J.V. Priestley
Chairman of the Scientific Committee

Funding for Research


For a project to receive funding, the application must
pass through a thorough peer-review procedure. For this the
ISRT is privileged to be able to rely upon the advice of a
distinguished Scientific Committee as well as external
reviewers. Their comments, recommendations and advice
on each application are made available to the Trustees of
ISRT, who make the final decision on whether and to what
extent a project will receive funding.

The International Spinal Research Trust had its inception


at the Guildhall in the City of London in 1981, with the
sole purpose to raise funds for research into a cure for
paralysis caused by spinal cord injury. In the intervening
years the Trust has committed over 18 million pounds to
a wide range of relevant research projects, with significant
advances made through these. Considering the challenge
ahead of translating laboratory results to clinical treatments,
our funds are limited. Nevertheless the Trust has been able
to support essential background work that has, in turn,
enabled researchers to secure major grants from government
and other funding bodies.

The scientific referees take into account several factors


when making their recommendations. The feasibility of the
project, together with the ability of the applicant and their
team to undertake it, is assessed to ensure that all the work
funded is scientifically respectable and likely to reach a
tangible conclusion. In addition, the referees attach most
importance to the relevance of each project, both in relation
to the advertised topic and to advancing the field in the best
manner possible. Researchers may be expected, for example,
to transfer their experimental studies to the adult
mammalian spinal cord.

In accordance with the Trusts Research Strategy, which


was first published in 1996, developed as a major review of
the field in 2000 and recently updated (published in 2007),
applications for funding are normally accepted following an
advertised call for proposals on a specific area of interest.
The frequency of these rounds depends on the availability
of funds: regrettably the Trust is not able to fund as much
research as we would like and, at times, many excellent
applications have had to be turned away. Advertisements
calling for proposals appear in the scientific press (e.g.
Nature and the BMJ), on our website and are sent via e-mail
to hundreds of researchers on our database. A few shortlisted
applicants are then invited to submit full proposals. Further
information about our research programmes and the various
projects that are supported by ISRT is available on our
website at http://www.spinal-research.org.

Financial support typically covers the costs of salaries for


young postdoctoral scientists based in laboratories that
already have some expertise in spinal cord injury research,
plus the necessary laboratory materials and consumables to
carry out the planned research. Assistance for items of
equipment, technical support and collaborative travel
essential to the project can be considered, but need strong
justification. Institutional overheads and administrative
costs are not covered in any award. ISRT aims to be flexible
and unsolicited proposals can be considered at any time.
However, any application that falls outside our current
phase of the Strategy would need to make an exceedingly
strong case, not only in research terms but also in terms of
its relevance to Spinal Researchs ultimate goal. Applications
are considered on a competitive basis whenever possible.

The Trust has a number of funding streams that run


concurrently and are closely linked. These support projects
ranging from PhD studentships to our multicentre Clinical
Initiative programme, which is described briefly on the
next page.

For further information on the ISRT Research Strategy, see:


Adams, M. et al., (2007) International Spinal Research Trust research. III: A discussion document. Spinal Cord 45, 214.
5

Project Grants

Nathalie Rose Barr PhD Studentships

These form the mainstay of our basic science programme


and are normally awarded following an internationally
advertised competitive call for proposals based on themes
identified in our Research Strategy. Project grants are
generally for the support of postdoctoral researchers to
undertake the approved research plan over a period of up
to three years, plus necessary consumables, travel or
technical assistance. Support will also be considered for
equipment if essential to the project.

Initiated in 1998 and named in honour of a late benefactress,


these awards are aimed at encouraging the development of
talented, highly-motivated young scientists in the field of
spinal cord repair, in both clinical and basic science research
environments. Calls for project proposals are advertised as
detailed previously, with a straightforward, one-step, peer
review process. The successful project supervisors then recruit
suitable candidates. The PhD degree must be awarded from
a UK university and a high priority is given to collaborative
proposals between more than one laboratory or institution.
Support includes University fees, a stipend in line with that
offered by the Wellcome Trust, plus funds for consumables,
travel costs and IT equipment. The studentships are tenable
for either 3 or 4 years for projects in basic science, and 3 years
for those that involve clinical studies. The Trust has so far
pledged support to 29 projects under this scheme. Typically,
the PhD student is recruited to a team that is already
established in the field of spinal cord injury research, where
they should receive an excellent quality of training and
support. As well as obligations within their own institution
all students are encouraged to attend and present their data
at research conferences and to attend our annual Research
Network Meeting.

Clinical Initiative
This programme aims to set in place some of the clinical
infrastructure necessary before trials of potential therapies
can be reliably conducted. A major collaborative study in
the UK began in 2000, linking clinical researchers and
spinal injury units, with the aim of developing a set of
accurate methods to detect and monitor small changes in
function in patients with spinal cord injury. The second
stage of the Clinical Initiative, with projects that designed to
test and refine the methodologies, began at the end of 2004.
This second stage involves researchers and spinal injury
units in the UK, Canada and Switzerland.

As a member of the Association of Medical Research


Charities, the Trust follows their guidelines regarding best
practice, including peer review, monitoring, the use of
animals in medical research, and patient agreements.
Further information is available at www.amrc.org.uk. Once
a full application is approved for funding, the Trust
negotiates a legal contract with the Principal Investigators
Institution that details both parties responsibilities
regarding, for example, finance, reporting procedures and
intellectual property rights.

Guidelines for Applicants


Our guidelines for completing initial proposals or letters of
intent are straightforward. There is no standard application
form because we prefer to leave the style up to individual
applicants. However, we require a concise outline of the
proposed project on no more than two pages. This should
include:

a clear definition of the specific problem


a reasoned argument for how this problem will be
tackled
some background, with evidence from previous
published work and/or suitable preliminary data to
support the hypothesis
a brief plan of the proposed experiments
the predicted outcome(s)
potential pitfalls and how they will be overcome
how this work will move the field of spinal cord repair
forward
any potential direct clinical benefits
an outline of the proposed budget

Progress reports are of major importance to the Trust


because they are essential for both monitoring the project
and as part of our responsibility towards those who provide
the finances. For this reason fund holders are required to
submit an annual report of the work undertaken that
indicates the goals they expect to achieve in the forthcoming
year. Final reports on the work are also needed. In addition,
many of the major donors and charitable trusts who support
our work require specific progress updates.

If successful, the applicants are invited to complete a


full application form, where further details of these
considerations are required.
6

Network Meetings

added infusions of growth factors and blockers of inhibitory


molecules. Because of the necessity for direct intervention at
the lesion site it is essential that the first treatments are
delivered to a region of the cord where any collateral damage
from the surgery is unlikely to have significant adverse
effects on the patient. This makes it unlikely that the first
treatments should be delivered to patients with cervical cord
lesions, even though they are the group that would benefit
most from even minor regeneration. Spinal Research
considers that the most favourable group of patients for a
safe trial of the first treatments are those with functionally
complete lesions in the lower part of the thoracic cord.

After the first gatherings in 1999, The Trusts annual


meetings have developed into a popular and important
event where we invite those involved with all of our current
grants to meet, discuss their research and learn more about
each others projects in a confidential environment. In
particular we are keen that all the postdoctoral researchers
and PhD students that we support attend, as well as
grantholders and project supervisors. For younger scientists
it is sometimes the first chance to present their work to their
international peer-group. In addition to the traditional oral
and poster presentations, open discussion sessions on
current themes and controversial areas of research can lead
to new avenues of investigation.

Therefore we began funding studies to develop


techniques for detecting functional, physiological and
structural changes over two or three spinal segments
following spinal cord repairs, and for high resolution
imaging of the progress of lesions and the behaviour of
implanted cells. Such assessment techniques with the
necessary resolution are not presently available in routine
clinical practice and it is our aim to have these in place in
advance of trials of interventions.

The 2009 Network Meeting will be held in Glasgow on


the 45th September.

The Clinical Initiative

The first stage of this initiative began in 2000 with a


major collaborative project in the UK and the progress made
and techniques developed have been peer reviewed and
published.

Regardless of the present success in animal models of spinal


cord injuries, where axon regeneration has been induced for
up to 3 cm, there are considerable hurdles to be overcome
before any therapeutic strategy can be considered for testing
in human patients. Not the least of these is that
experimental treatments cannot be used safely until the
progress and effect of the treatments can be accurately
assessed. At present the techniques needed for this are not
sufficiently well developed. Therefore Spinal Research has
developed a unique initiative to develop techniques for the
clinical assessment of spinal injury treatments.

The second stage of the Clinical Initiative started in


2005. In this stage, researchers in spinal injury units in the
UK, Canada and Switzerland are testing and refining the
procedures developed in Stage 1. These studies involve
monitoring and assessing the effects of non-invasive
strategies such as weight-supported treadmill training,
repetitive transcranial magnetic stimulation (rTMS) and
functional electrical stimulation (FES) on patients with
spinal cord injuries.

Future therapy of spinal cord injury might involve


implantation of cells into the lesion site, to which could be

Research Network Meeting, September 2008, London

10th Research Network Meeting, 56 September 2008


PROGRAMME FRIDAY SEPT 5TH
Session I: Chronic spinal cord injury Chair: Mark Tuszynski
Vivian Mushahwar
Use of intraspinal microstimulation for restoring function after spinal cord injury: sites of
action and long-term muscular adaptations
Paul Reier
Respiratory function and chronic cervical spinal cord injury (SCI)
James Guest
Chronic spinal cord injury; overview and challenges
Session II: The influence of the innate immune system Chair: Ann Logan
Serge Rivest
Bone marrow stem cells to the rescue of brain diseases
Salvatore Cuzzocrea
Inflammation in spinal cord injury
Regino Perez-Polo
Anti-inflammation treatment of spinal cord
Session III: NRB students Chair: Peter Ellaway
Phil Duffy
Targeting ROCKII inhibition following spinal cord injury
Milan Makwana
Investigating the effects of constitutively active Ras and dominant negative MEK1 on
regeneration and functional recovery following cervical spinal cord lesion
Nick King
Use of repetitive transcranial magnetic stimulation in incomplete spinal cord injury
Session IV: Cell based therapies Chair: Geoffrey Raisman
Jane Roskams
OECs use different mechanisms to stimulate dendritic branching and axonal elongation
of regenerating corticospinal tract neurons
Wolfram Tetzlaff
Schwann cells from Skin-derived precursors (SKP-SC) for spinal cord repair
John Riddell
Olfactory ensheathing cell transplants: axonal regeneration, neuroprotection or sprouting?
Scientific Committee Meeting / Wine and Poster Session
PROGRAMME SATURDAY SEPT 6TH
Session V: Biobridges/nanoengineering/RNAi Chair: Phil Wait
Molly Shoichet
Biomaterials for spinal cord injury repair
Joost Verhaagen
A multi-step high/medium-throughput screening approach to discover novel molecular
mechanisms involved in neural regeneration
Michel Kliot
Microscale surgery on single axons: splicing instead of regenerating axons
Session VI: Auxiliary assessments of SCI & function Chair: Thomas Carlstedt
Praveen Anan
Contact Heat Evoked Potentials (CHEPS) for assessment of small fibre and spinothalamic
dysfunction in patients with neuropathy and spinal cord injury
Patrick Stroman
Development of functional magnetic resonance imaging for assessing human spinal cord
injuries
Claudia Wheeler-Kingshott
Quantification of metabolites and connectivity in spinal cord to assess disability in acute
multiple sclerosis
Session VII: Clinical issues and clinical trials Chair: James Fawcett
David Allan
A perspective from a clinician, researcher and trustee
Peter Ellaway
The Clinical Initiative an update
Treatments to aid recovery from spinal cord injury: testing improved clinical, physiological
and functional assessments
Hooshang Saberi
Preliminary results of Schwann cell transplantation for chronic spinal cord injuries
Discussion Forum: Priorities in translational research Chair: Steve McMahon
David Allan, National Spinal Injuries Unit, Glasgow, UK
James Fawcett, Cambridge University Brain Repair Centre, UK
Mark Tuszynski, University of California, USA
Summary by Naomi Kleitman, NINDS, USA / Closing Remarks: John Priestley
Reception at Churchill Museum

POSTER PRESENTATIONS
Convergence of anti-integrin and cell-based therapies promotes recovery from spinal cord injury
A. Brown, L.C. Weaver
The effects of omega-3 fatty acids on early inflammatory events after spinal cord injury in the rat
J.C.E. Hall, J.V. Priestley, V.H. Perry, A. Michael-Titus
Effects of chondroitinase treatment on axonal and glial functions
M.C. Ovejero-Boglione
Modification of N-glycosylation sites to allow secretion of bacterial chondroitinase-ABC from mammalian cells: a treatment
for spinal cord injury
P.M Warren, E.M. Muir, J. Fawcett, R. Keynes
Chondroitinase ABC promotes repair of ascending and descending pathways following spinal cord injury in the yellow
fluorescent protein (YFP-H) mouse
L.M. Carter, M.L. Starkey, M. Davies, S.B. McMahon, E.J. Bradbury
Combined effects of chondroitinase and rehabilitation on recovery after SCI
G. Gracia-Alias
The effect of Chondroitinase ABC and task specific rehabilitation on chronic spinal cord injuries
D. Wang, G. Garca-Alas, J. Fawcett
Comprehensive evaluation of spinal cord function accompanying Lokomat rehabilitation in patients with incomplete
spinal cord injury
S. Galen, C. Catton, K.J. Hunt, D.B. Allan, B.A. Conway
Hebbian rehabilitation for incomplete spinal cord injury: Using temporally linked exercises to forge functional connections
between spinal tracts
N.Y. Harel, X. Tang, K.-H. Song, S.M. Strittmatter
Specific locomotor versus unspecific weight training and their effects on gait function and corticospinal conductivity after
chronic incomplete spinal cord injury
R. Labruyre, V. Dietz, H.J.A. van Hedel
The clinical relevance and longitudinal course of semi-quantitative sensory tests in subjects with a spinal cord injury
H.J.A. van Hedel, P. Dokladal, R. Labruyre, M. Stssi
The action of 5Hz repetitive transcranial magnetic stimulation (rTMS) of the sensorimotor cortex on sensory, motor and
autonomic physiology in spinal cord injured people
P. Ellaway, C.J. Mathias, M. Craggs, A. Gall, A. Kuppuswamy, A. Balasubramaniam, R. Maksimovic
Behavioural assessment of a model of spinal root avulsion injury
D. Chew, T. Carlstedt, P. Shortland
Decrypting the psychologists mind: how to diagnose neuropathic pain?
P. Dokladal, M. Cook, H.J. van Hedel
Sensory testing near the level of a spinal cord contusion injury can be used to evaluate post-injury sensory changes in mice
E. Hoschouer, D. M. Basso, L.B. Jakeman
Conduction failure and recovery across spinal cord contusion injury in the adult rat
M.Davies, P. Sadeghi, S. McMahon, E. Bradbury
Interneurons and phrenic circuitry in the normal and injured spinal cord implications for respiratory plasticity
M.A. Lane, T.E. White, A.L. Jones, F. Hunsaker, B.E. OSteen, D.D. Fuller, P.J. Reier

10

Engineered expression of polysialic acid on Schwann cells enhances their survival and integration after transplantation into
the spinal cord
J. Luo, X. Bo, F. Gao, J. Yeh, P.M. Richardson, Y. Zhang
Olfactory ensheathing cells from human biopsies
Projected application in repair of brachial plexus cord avulsion injury
C. Kachramanoglou, S. Law, D. Li, Prof. Raisman, D. Choi
Olfactory ensheathing cells promote recovery from autonomic dysreflexia and modify preganglionic cell morphology in T4transected rats
T. Kalinck, A. Mackay-Sim, P. Carrive, P.M.E. Waite
Olfactory mucosa and its application in biomaterial
J. Kueh, D. Jenkin, S. Law, D. Li, B. Stevens, G. Raisman
Effect of olfactory ensheathing cells transplanted into the injured spinal cord on plasticity of sensorimotor pathways
T. Meng, T.C.P. Sardella, D.T. Scott, A. Toft, S.C. Barnett, J.S. Riddell
Axonal regeneration after delayed transplantation of olfactory ensheathing cells into spinal cord lesions
T.C.P. Sardella, A. Toft, T. Meng, G.M. Smith, S.C.Barnett, J.S.Riddell
AAV8 as a vector for the delivery of neurotrophic factor genes and RNA interference for the treatment of spinal cord injury
S.J. Jacques, M.R. Douglas, Z. Ahmed, M. Berry, A. Logan
Comparison of AAV serotypes for gene delivery to DRG neurons
M.R.J. Mason, R. Eggers, E.M.E. Ehlert, S. Hermening, A. Huseinovic, B. Blits, J. Verhaagen
Overcoming Tenascin-C Inhibition with Alpha9 Integrin
M.R. Andrews, S. Czvitkovich, E. Dassie, S. Schroter, B. Blits, J. Verhaagen, J.W. Fawcett
Promotion of axonal regeneration by over-expressing a NGF-soluble Nogo receptor fusion protein
X. Bo, D. Wu, P. Moshayedi, F. Gao, X. Zhang, W.L. Huang, V. King, J.V. Priestley, J. Yeh, Y. Zhang
Autologous bone marrow stromal cell therapy for the treatment of spinal cord injury: in vitro studies
K.T. Wright, W. El Masry, A. Osman, J. Trivedi, S. Roberts, W.E.B. Johnson
The Nucleoside Diphosphate Kinase, NM23H1, is Expressed by Human Bone Marrow Stromal Cells and Stimulates
Nerve Growth In Vitro
K.T. Wright, R. Seabright, A. Logan, F. Khanim, C.M. Bunce, W.E.B. Johnson
Neural cells and substrate mechanics: A biomedical view
P. Moshayedi, A. Christ, K. Franze, J. Fawcett, J. Guck

11

Spinal Research Network Meeting


Hilton Kensington, London
56 September 2008
The tenth Annual Research Network Meeting, held in
September in London, proved yet again, to be a great success.
The previous years joint meeting held in Switzerland left us
with a lot to live up to but our network format proved equal
to the challenge and the organising committee were to be
applauded for providing a fresh and stimulating series of
speaker sessions. Grant holders, students and invited
scientists alike participated in this two day meeting and
enjoyed an excellent mix of social and scientific interaction.

appropriately dealt with Chronic Spinal Cord Injury.


VIVIAN MUSHAHWAR began the session by describing
developments in intraspinal microstimulation (ISMS) for
standing and walking, a line of investigation that might offer
benefits to the chronic patient. The spinal cord is well suited
to microstimulation, she suggested, because stimulation was
removed from contracting muscle, delicate electrodes are
implanted within a bony protective spinal column and the
region of interest the lumbar/sacral enlargement, which is
relatively large in humans (5cm) contains many of the
important circuitry and control centres for natural joint
movement. She demonstrated that implantation of multiple
electrodes provided important control of complex
coordinated muscle function. Single electrode stimulation
from the implanted array elicit fairly large and strong single
joint movement which is both graded and natural in
contrast to peripheral nerve stimulation and its on-off
response. Mushahwars lab also demonstrated that multielectrode stimulation in anaesthetised cats could produce
synergistic coordinated multi-joint movement and standing
and weight-bearing stepping movement were also more
durable with ISMS versus direct muscle stimulation (32 min
versus 8 min, respectively). In order to understand why
ISMS and peripheral stimulation might result in different
outcomes the group analysed muscle fibres from animals
stimulated by the two methods. Biochemical and histological
analysis suggesting ISMS preferentially recruits activation of
non-fatigable muscle fibres whilst peripheral activation
recruits fatigable fibres. Mushahwar said, all these effects
cannot be the result of a simple recruitment of motoneurons
and admitted the mechanism(s) are still to be identified, but
the possibility that activation of afferent pathways (e.g.
priopioception) may contribute should not be ruled out.
How important is this mechanistic understanding to the
clinical design and application needs some consideration,
particularly, she said, when long-term placement and the
potential for stimulation-induced plasticity are taken into
account.

Professor John Priestley, the new Chairman of the


Scientific Committee, opened the meeting by welcoming
the 108 delegates before giving a review of Spinal Researchs
history and achievements including the establishment of
its studentship scheme and reminded us all of our mission
to resolve the non- or partial functioning of the spinal cord
after injury.
He reflected that it was easy to talk about the scientist,
the doctor and the patient being in this together but feared
it was likely scientists talked with scientists, clinicians to
clinician and patient to patient. It was his intention that one
of the purposes of the meeting was to foster better
communication between scientist and clinician.
He finished by focusing on Spinal Researchs future
strategy in the area of translational science. Recognising the
past decades had been mainly concerned with basic science
and the understanding of therapeutic targets and
mechanism, he stated that support of fundamental basic
science must continue but admitted there was a huge
amount of work we often dont think about. At the heart of
the Trusts Translational Initiative was recognising the need
to set clinically-relevant target profiles for emerging
treatments with milestone criteria that lead to the clinic and
that clinical trials should be intelligent and well-designed.
As he invited the audience to enjoy the meeting he left
two questions to consider; (i) what are the most promising
treatments for development? and (ii) how can we accelerate
clinical application of treatments?

Announcing its back to basics, PAUL REIER began by


suggesting that if we are to understand how to intervene we
must understand what circuits are involved, how neurons are
connected to each other, how they talk to one another and
how all this changes after injury. Reiers lab is interested in
respiratory function after SCI both as a model for study and
a therapeutic target. The model used is based on something
called the crossed-phrenic phenomenon and is seen as a
natural example of plasticity and compensatory response to
injury. The crossed-phrenic phenomenon is found in many
species and there is data to suggest it exists in humans also.
One of the advantages of the crossed-phrenic phenomenon
is, as Reier puts it, animals and humans breathe similarly,
which cannot be said of ambulation, and there are a variety

Summary of presentations at the meeting


DAY 1: SESSION 1
For decades researchers determined to tease apart the
underlying natural history of a spinal cord injury and
identify potential therapeutic targets. In doing so they
developed and refined experimental injury models and
mostly acute treatment paradigms. Studies seeking to
understand the progression of a disease have a tendency to
throw up targets that prevent rather than cure. Valuable as
this is, as was stated by John Priestley, Spinal Research has
an obligation to supporters to resolve paralysis as a result of
SCI not just prevent it and the first session of the meeting
12

of clinically-relevant outcomes that can be monitored e.g.


whole body plethysmography as well as amenability to
electrophysiological assessment. Two elements contribute to
the crossed-phrenic phenomenon. The first, a monosynaptic
connection between cells of the ventral respiratory column
and the phrenic motor neurons, provides respiratory drive
which Reier likened to a brainstem level pattern generator
for breathing. Complete hemisection results in loss of input
and respiratory drive that over time is reactivated by
the second component, a crossed-phrenic circuit. Using
plethysmography, Reier illustrated the effects of a high
cervical lesion; rapid, shallow breathing (panting) employed
to compensate and maintain blood gases. This altered postSCI respiratory pattern of increased frequency, lowered tidal
volume, persists over time and is sufficient to maintain
ventilation during normal activity but is impaired during
challenge. Reier wanted to know whether there was a parallel
increase in phrenic motor pool neuron activity associate with
high cervical lesions. There wasnt which led him to ask
whether there are other neurons within the circuitry that
contribute both positively and negatively. Using a
transneuronal tracer (pseudo rabies virus) injected into the
diaphragm they found evidence for labelling in a substantial
number of interneuron representing synaptic connections
between phrenic motoneurons and a population of cells few
had paid much attention to.

JIM GUEST finished the session with a reflective overview


of chronic SCI and the challenges faced when dealing with
and attempting to repair. Guest began by considering some
definitions of chronic SCI, suggesting these are not clear.
There are clinical definitions and biological definitions;
clinically it can be taken as meaning when neurological
function stabilises and the patient presents characteristic
reflexes of SCI, but there other ways we might define
chronic SCI, like the stage the efficacy of given biological
therapeutics falls to marginal levels more related to the
issue of a therapeutic window.
During a chronic SCI the focus is typically on the
chronic complications associated with SCI and maintenance
of function. As he prepared for the meeting he began to
appreciate the state of chronic SCI is itself a disease with
complications which he cited as including infection in the
urinary system, skin breakdown, osteoporosis, spasticity,
dysreflexia, excessive pain (in ~35% SCI subjects), but
there may be some ongoing damage to the spinal
cord; syringomyelia, cord tethering leading to progressive
spinal deformity.
The entire neural axis and many organ systems are
altered by SCI; the brain, the disconnected lumbar spinal
cord, even segments above the injury level, are all altered by
SCI. There is a chronically-elevated state of inflammation
which is both systemic and within the nervous system.

It raised the question, what do these by-pass circuits do?


Are they pre-existing or a result of sprouting? Double
labelling tracer studies from the ventral respiratory column
and from the diaphragm proved positive confirming these as
pre-existing circuits and ones that represent potential viable
targets for respiratory recovery.

Some important differences between acute and chronic


SCI exist. The Glial scar is already established in chronic
SCI which is likely to be an issue for local interventional
therapies. Growth-associated protein production is low and
extensive reorganisation and new synaptic connections have
occurred in response to denervation. This poses an
important question; How difficult will it be to displace these
new connections with restorative connections?.

Alluding to work not reported at the meeting he


suggested evidence for grey matter repair after SCI. Since
nature didnt put a continuous column of grey matter there for
no reason, he went on, why not provide pools of neuronal
cells that could replace or integrate with interneurons? A selfconfessed fan of neuronal replacement in repair, they
embarked, not seeking to restore normal function, but to see
the effects of transplants. Their approach was guided by work
carried out some years ago with Lyn Jakeman in which
regional microdissection of fetal cord tissue had a marked
effect on the fate and type of integration seen in graft. Using
regionally-defined grafts of lineage-restricted progenitor cells
(E14 fetal spinal cord tissue) they found clear differential
effects between grafts of ventral, dorsal or whole origin;
ventral-origin grafts resulting in a decrease in respiratory rate
to near normal levels, but showing little effect on phrenic
burst amplitude, whilst whole and dorsal-origin grafts
apparently reduced burst amplitude. Once again, retrograde
tracing studies showed these grafts to be connected to the
phrenic motor pool. Reier admits that much of this is
phenomenological and more work is needed to identify how
this is all integrated. Showering the audience with examples
of possible circuit pathways he concluded, The bottom line
is we are rediscovery interneurons, but they are key players
and maybe Nature has given us many fail safes, the key will be
how to recruit these cells.

Chronic SCI offers some advantages for clinical trials


stability of the neurological baseline, fewer acute care issues
and the family and patient have stabilised after the shock
and trauma of the injury. The lesion itself can be well
defined in terms of extent and severity by MRI. The
question of whether chronic SCI is associate with chronic
inflammatory disease remains, but multiple lines of evidence
exist that show SCI subjects have elevated c-reactive protein,
his own studies show cerebro-spinal fluid levels of TNF
increase, significant elevation of homocysteine levels (related
to cardiac disease) and there are issues related to sperm
quality. Guest therefore wonders whether this chronic
inflammatory state will affect future interventions.
Guest then spoke about his work in chronic
demyelination. In an anatomically incomplete injury some
of the clinical pathology and experimental data suggest a
proportion of axons are demyelinated and not repaired to a
functional state. While they remain patent between the cell
body and the synapse they may be recoverable to function.
This is a controversial issue, admitted Guest, that is being
examined by numerous laboratories at this time.

13

Miami possess a tissue bank containing over


200 cadaveric human spinal cords from SCI subjects from
which histopathology can be examined. He described cases
from the bank that had died at various time points post injury.
In one case (1 year post injury), there was extensive number
of axons that lacked myelin. In another (2 years post injury),
very extensive mesenchymal scarring could be seen around
nerve root and dura. Around cavity regions they observed
p0 myelin and some axons that had been demyelinated.

of receptors called the toll-like receptors able to recognise a


wide variety of liagnds. Exposure to these bacterial or viral
ligands causes massive up regulation of transcription in many
cell types, but what is interesting is that degeneration also
causes activation of the innate immune response in the
nervous system as a result of the peptide and lipid release
recognised by these receptors on microglia. Is this good
or bad?
Acute injury models such as nitric oxide injections
produce an acute injury and induce an inflammatory
response with increases in signalling cytokine production.
Various knockouts of TNFa IL1 to examine the role played.
TNFa-knockouts result in increased cell damage suggesting
a neuroprotective activity. Likewise, EtBr injections to the
white matter results in demyelination, myelin debris and
cell damage. When injury is accompanied by injection of
ligands for toll-like receptors which activate the innate
immune response there is better debris clearance and
reorganisation of tissue and recruitment of progenitor cells.

Identified Schwann cells that had entered the spinal cord


naturally and P0 positive regions but were not able to
identify remyelination of central axons categorically. In
another case, 10 years post injury, some axons were in
continuity across the injury site with junctions containing
proteolipo-protein and P0 which may represent repair by
Schwann cells arising from the peripheral nervous system.
This remains a controversial area with some believing there
is ongoing chronic demyelination, while others feel there to
be no evidence that spared axons that had been
demyelinated, persist as demyelinated.

After Sciatic nerve injury (a model of systemic injury)


neurons regenerate but in toll-like knockout mice IL1 is no
longer inducted and recovery is prevented or delayed.
Conversely, if you prime the innate response with toll-like
receptor ligands you can see improved recovery.
Glucocorticoid administration also prevents recovery by
attenuating the microglial response. Thus, in many injury
models the immune response contributes very positively to
recovery and regeneration.

One issue Guest was able to examine using bank tissue


was chronic retrograde ascending degeneration from the
lesion site axonal die-back. It may pose an impediment
to both regeneration and plasticity-promoting strategies.
An example from a case 7 years post injury showed that at
5 levels above the lesion they could not find evidence for
corticospinal tract axons and in the 152 cases examined they
found extensive die-back in all. It is pretty daunting to realise
that above a lumbar injury you may have as many as 17 segments
of CST die-back, he said, and when thinking about applying
restorative treatment it must be recognised that some of our
targets may have died back from the injury site to a very
significant extent.

Rivest examined the possible role of bone marrow derived


immune cells and resident immune cells. Using donor
chimeric animals (expressing GFP) the location and fate of
donor cells when given systemically to host animals can be
monitored. Donor cells infiltrate host CNS after central and
peripheral injuries and differentiate to microglial cells
exclusively. These infiltrating cells are different from resident
microglia in terms of receptor expression making their
phenotype more phagocytotic and therefore more effective,
he said. This is also seen in mouse models of Alzheimers with
host plaque infiltrated with donor microglia.

Guest also described clinical percutaneous endoscopic


surgical techniques they have been developing to place
shunt catheters (for syringomyelia) and may be useful for
minimally-invasive delivery of cell therapies.
He finished by describing a neurologically-stable
functionally impaired primate model of SCI in which they
are testing Schwann cell therapy. The injury is created using
a stereotactic guided radiofrequency insult to the medullary
pyramid (CST). The injury results in severe hemiplegia for
which they have developed behavioural tests hand pressure
tests and kinematic assessments. In data presented from one
animal which had been injured for 2.3 years prior to
transplantation and monitored for 6 months post treatment
they found significant recovery in weight supported
stepping. Histological examination is now being carried out
to determine whether there is evidence for survival and
remyelination by transplanted Schwann cells.

Irradiation induced immune deficiency also caused


accelerated onset and decrease survival in ALS models.
Stopping proliferation of macrophage also reduces
recovery after sciatic nerve injury. Macrophage are
responsible for release of many factors including
neurotrophins (e.g. NT-3) which have positive results.
Rivest suggests bone marrow derived progenitor cells are an
attractive source of cells useful for the repair of SCI and
other CNS diseases. Genetic modification may be required
to improve targeted response and efficacy.

DAY 1: SESSION 2
The Innate Immune System
SERGE RIVEST reminded us that when exposed to bacteria or
viral proteins there is a rapid innate immune response
because immunological cells (monocytes, macrophage,
neutrophils and microglial cells) possess very a specific family

SALVATORE CUZZOCREA shifted focus by looking at one


of the more damaging aspects of the immune response
inflammation. Why should there be a focus on
inflammation? One of the reasons for rehospitalisation for
SCI subjects is inflammatory disease in a number of
manifestations. The damaging secondary response following
14

SCI is characterised by many inflammatory-driven events.


To examine the role of inflammatory response in SCI
Cuzzocrea chose the compression injury model
(lamenectomy followed by clip compression on cord for
various times).

The groups strategy was initially to attempt to abolish the


apoptosis by using a Tat delivery system to introduce Bcl-xl
(or the BH4 domain) for 7 days. This treatment reduces
evidence for apoptosis but little or no effect on behavioural
outcomes (BBB or hypersensitivity to mechanical allodynia).

A number of agents exist that might be expected to


interfere with various elements of the inflammatory
response; administration of antioxidants, such as melatonin
(a natural neuronal free radical scavenger) and uric acid
(UA) might be expected to mop up reactive oxygen species
and reduce the damaging downstream effects such as lipid
peroxidation and nitrixc oxide production; PDTC modifies
IkB kinase thereby reducing activation of the transcription
factor NF-kB and inhibiting transcriptional activation of
iNOS, COX2, TNF-a, etc.; etanercept (a licensed dimeric
fusion protein consisting of the ligand binding region of
human TNFReceptor and human IgG1) competes for
TNF-a and inhibits receptor signalling. In addition to these
pharmacological approaches they looked at iNOS
knockouts to assess the effects of reduced nitric oxide
production and finally administration of 5-AIQ and 3-AB
to inhibit activation of the PARP suicide pathway.
Cuzzocrea presented NF-kB/DNA binding studies showing
reduction of binding following either melatonin, PDTC or
PARP inhibitor treatments.

Perez-Polo cautioned that indiscriminate rescue from


apoptosis would indeed spare neuronal and oligodenrocyte
populations but also spare activated astrocytes and microglia
resulting in an increase in inflammation its a mixed bag.
Perez-Polo suggests a more specific approach is preferable
and asks, what common inflammatory pathways are
triggered by SCI and, if inflammation can be blocked
specifically, will this be beneficial?
IL1 expression is clearly an early event, followed in time
by increases in TNFa, IL6 and some chemokines. Neurons
and oligodendrocytes possess receptors to IL1 and are thus
responsive to IL1 production and may constitute part of an
inflammatory cytokine cascade amenable to interference.
Initial thoughts were to use an IL1 receptor antagonist
(Kineret) to inhibit the cascade upstream of NF-kB and
therefore reduce downstream COX2/iNOS production etc.
and indeed, caspase-3 activity was reduced and neuronal cell
numbers increased with Kineret treatment. On analysis it
appeared Kineret protected GABAergic neurons in the
dorsal region of spinal cord which may be mechanistically
significant in developing a neuropathic pain state (i.e. loss
of these inhibitory neurons would result in increased
excitation following injury). To test this they looked at
central trunk sensitivity (girdling) as well as forelimb and
hindlimb sensitivity to non-noxious stimuli finding
improvements in treated groups. Locomotor scores (BBB)
also improved with IL-Ra treatments.

Treatment with etanercept also prevents TNF-a


expression by immunohistochemistry and IL-1. The result
of this is a reduction of iNOS expression. iNOS knockouts
increased activity of endothelial and neuronal isoforms of
NOS. Cross-talk between TNF and COX2 can also be
demonstrated by the reduction of COX2 expression following
etanercept treatment. Direct PARP inhibition or treatment
of agents to reduce oxidative stress (enteracept, PDTC, etc.)
results in reduced PARP activation in SCI models.

Looking at the effects of NF-kB they found increases in


iNOS and COX-2 early after injury. However, a variety of
stimuli cause distinct NF-kB subunit combinations that
exhibit differential binding affinities for multiple consensus
sequence combinations at 10 base pair promoter regions
(128 possibilities) this complex variation likely determines
specificity of NF-kB transcriptional regulation and suggests
targeting specific promoter regions might be highly selective
for inactivating NF-kB transcriptional effects (some of
which are positive, others that are negative). This general
approach is FDA approved and may be applicable to clinical
translation.

Cuzzocrea wanted to show that these treatments not


only had the potential to modulated key inflammatory
mediators but also resulted in beneficial effects presenting
histological evaluation under various sham, control and
treatment conditions. Tunel staining evident in control
tissue was not apparent in tissue from treated groups
suggesting apoptosis is a significant but preventable
mechanism for cell death after SCI.
Assessment of functional outcome for these treatments
also presented with general improvements seen as predicted,
though notably data presented without error bars.
Cuzzocrea believes combinations of these drugs given within
appropriately early therapeutic windows may offer
protection from some of the damaging secondary aspects of
SCI in patients.

Perez-Polo took decoy oligonucleotide sequences


complementary to COX-2 NF-kB promoter region (using
scrambled sequences as control) and injected into lesion site.
There is an improvement in BBB scores and pain responses
after treatment.

The session ended with a presentation from REGINO


PEREZ-POLO on anti-inflammation treatment of SCI.
Moderate contusion model injuries produce a decrease in
Bcl-xl/Bax ratio, an increase in caspace-3 and many other
classical apoptotic signatures, although there are differences
over those seen in the immune system, for example.

DAY 1: SESSION 3
Nathalie Rose Barr students were given an opportunity to
present their work in the final session of the day. Various
myelin associated ligands on oligodendrocytes (e.g. MAG,
Nogo and OMgp) along with astrocyte CSPGs interact with
neuronal receptors resulting in axonal growth arrest. The
15

various inhibitory intracellular signaling pathways converge


downstream on RhoA but further downstream there is the
ROCKII signaling molecule which has been targeted
pharmacologically. PHILIP DUFFY talked about his work
using ROCKII knockouts to investigate this target by
looking at anatomical and functional changes after
hemisection (affecting ascending and descending pathways)
and rhizotomy (sensory neurons) injuries. He found CST
regeneration up to but not caudal to the lesion in the
ROCKII -/- mouse when compared to heterozygous or
wildtype. There was evidence of Raphespinal axon
regeneration caudal to lesion but no functional recovery was
observed following this injury model. Sensory axon
regeneration was observed after rhizotomy and functional
recovery in the spontaneous exploration (rearing) test at day
57 post injury. Differences in behavioural outcomes
between these two injury models may represent differences
in regenerative susceptibility of sensory versus CST axons
in ROCKII knockouts although the severity of the
hemisection injury environment as compared to the
rhizotomy may pose an insurmountable hurdle to CST
axon regeneration and functional synaptogenesis.

(rTMS) can also induce lasting changes in cortical


excitability and has prompted investigations into the use of
rTMS as a potentially useful non-invasive therapy in
patients. The final student, NICK KING, described work he
had recently completed on the use of (rTMS) to increase
motor output to surviving spinal tracts in 10 chronic
incomplete SCI subjects in a randomized cross-over trial.
rTMS or sham was given over five consecutive days with a
washout period before cross-over. The rTMS protocol
involved paired pulses at 100ms intervals every 10s for 1hr
every day of treatment. In addition to functional (reaction
time and 9-hole pegboard test), neurological assessments
(ASIA motor and sensory scores) and neurophysiological
tests (MEP, cortical silent period using single pulse TMS,
electrical perceptual thresholds), EMG responses from the
thenar muscles were also recorded.
Functionally, reactions times did not differ between
treatment and sham groups. On the 9-hole pegboard test
the performance time in treated group was decreased and
was significantly different from sham at 1wk, 4wk and 8 wk
post treatment. ASIA motor scores were increased in the
treatment group although only by 10% and were not
significant. Sensory scores also increased becoming
significant at 1wk post treatment but not at other testing
time points.

MILAN MAKWANA spoke about the effects of Ras and


MEK1 on axonal plasticity and functional recovery after SCI.
Neurotrophins activate common signaling pathways and
numerous studies report enhanced axonal plasticity and
regeneration following neurotrophins or growth factor
application. Is it possible therefore to manipulate intracellular
signaling to activate the growth promoting effects of
neurotrophins? To address this Makwana used a constitutively
expressing Ras mouse with high expression levels of GTPRas.
Using the facial nerve injury model to examine axonal
sprouting response they found enhanced sprouting in the
facial nucleus in the Ras+ mouse and in a MEK1dn
(dominant negative) mouse as compared to wild-type. The
question was, would enhanced sprouting capacity in these
mutants provide improvements in functional outcome? To
address this anatomically, Mikwana looked at CST sprouting
after a C6 dorsal midline hemisection injury in the WT, Ras+,
MEK1dn and double mutant (DM) mice. Little difference
was observed in sprouting between these groups above the
lesion site, but caudal to injury DM and MEK1dn mice
showed significant enhancement in sprouting. Indeed, in the
DM mice labeling studies showed some evidence of axons
entering and crossing the lesion scar. Both the single as well
as the double mutant groups showed improved forepaw
functional recovery (rearing) as well as paw placement
following a C4 unilateral dorsal hemisection. Whilst no
correlation between the functional recovery and
neuroanatomical differenceswhere found in the CST between
the groups and control, the rubrospinal tract did display
enhanced sprouting below and above the lesion, particularly
in the DM. Therapeutically, these mutant studies would
appear to confirm intracellular signaling pathways as potential
targets for intervention.

No correlation was found between the changes in ASIA


sensory scores and the electrical perceptual threshold in SCI
subjects which was surprising according to King as this was
found in control subjects. And though not significant at most
assessment time points, it was suggested that rTMS may
increase cortical excitability and decrease cortical inhibition
leading King to speculate on this as a possible substrate for the
functional and neurological observations above.
Although some of the effects were modest using rTMS
it should perhaps be noted that the median time from injury
was approx. 10 years in this subject cohort: increasing the
treatment period or combining with motor training or
future interventions may yet prove helpful, though clearly
more work needs to be done before it can be established that
this technique as superior to simple task-specific training,
for example.
DAY 1: SESSION 4
Cell-based therapies for spinal cord injury
Olfactory ensheathing cells have gained and it must be said,
lost much support over the years but recent published
reviews concluded that OECs may have something to
offer. Beginning the session on cell-based therapies, JANE
ROSKAMS made it quite clear that human OECs are very, very,
very hard to grow in culture! But if we are to take advantage
of the properties of OECs in vivo we must strive to
mechanistically understand how they work. Unfortunately,
they dont like to passage, arent easily transfected and though
they can be identified using specific markers (e.g. S100b, p75)
many of the supplied antibodies also recognize human
fibroblasts from many different locations in the body. It is
therefore important to use combinations to ensure identity
of human OECs in such studies (Currie et al., 2008).

In incomplete SCI cortical reorganization takes place


and such plastic changes can be beneficial giving rise to
spontaneous functional recovery in human patients.
Application of repetitive transcranial magnetic stimulation
16

Can OECs promote regeneration of the corticospinal


tract? In order to address this Roskams lab, in work carried
out by Miranda Richter, wanted to look at CST neurons
that were mature rather than derived from neurons still
in a developmental phase in other words, post-targeted
CST neurons that would allow assessment of regeneration
in the absence of a strong influence from developmental
drive. Significant effort lead to successful culture of CST
neurons from post natal (P8) animals (Richter & Roskams,
2008) which they tested for neurite outgrowth and
branching (regeneration response) on a variety of cellular
substrates. Growth on astrocytes results in quite large
numbers of branched axons. Addition of CNTF resulted in
long processes and NT-3 stimulates more branching and
enhanced survival (over NGF and other controls). MAG
also produces an inhibitory response in these mature CST
neurons which is not seen if cultured from embryonic tissue.

WOLFRAM TETZLAFF presented work on neural crest


origin skin-derived progenitor cells (SKPs). Skin is an
abundant accessible tissue making such cells potentially
useful for autologous transplantation. SKPs can be grown
in FGF/EGF and can be pre-differentiated into Glial cells,
for example SKP-Schwann cells (SKP-SC) using neuregulin
and forskolin. Importantly, SKPs are found in adult humans
but grow slower.

When grown on OB OECs, neurons produce


ridiculously long neurites which is slightly unexpected, but
OECs with olfactory fibroblasts and fibroblasts alone will
also stimulate CST neurons.

Neuroprotection is a common feature regardless of the


type of cell transplanted but this is most significant with
SKP-SC. Clearly, it makes the choice of appropriate controls
important in such studies but the effects seen with various
controls are different. Tetzlaff suggests that the integration
seen with SKP-SC is because these cells dont make the
astrocytes too angry. Do axons grow? Tracing of the CST
was negative, but 5-HT and HT positive axons did grow
over time in SKP-SC compared with controls, but he could
not comment on whether they grew out of the SKP-SC
bridges citing difficulties in distinguishing truly regenerating
axons and those derived from spared tissue.

Tetzlaff transplanted rodent SKP-SC into rat spinal cord


contusion sites (OSU, moderate) at 7 days post injury
(800,000 cells per 5ul). Media controls showed cavitation in
this model. Cell-based controls such as nave SKPs or
Neuropheres either fill the cavity, but do not migrate into
host, or they distributed along the cavity wall, respectively.
SKP-SC fill cavity and integrate forming graft bridges and
little astrocyte demarcation.

When comparing axons (MAP2-positive) versus


dendrites (GAP43-positive) growth response to OECs they
found axonal outgrowth in preference to dendritic
outgrowth, which was not necessarily predicated but which
they were rather glad to see, she said. Was this due to
secreted factors or through physical contact? A dose response
to OEC conditioned-media which had never seen neurons
saw stimulated neurite outgrowth but at a fraction of the
extent seen when co-cultured. Conversely, growth on
plasma membrane fractions from OECs resulted in strong
axonal extension. So there must be something in OEC
plasma membrane that gives this stimulatory response
which must also be found in astrocytes, she suggested. What
is more, this plasma membrane magical cocktail contain
components that can overcome MAG inhibition in a dose
dependant way.

As might be expected, SKP-SC bridges stain heavily for


P0 a peripheral myelin marker with some evidence for
phenotypically-appropriate myelination. Approximately
35% SKP-SC survive, which myelinate demyelinated spared
host rim axons. Another feature found in SKP-SC
transplants is a massive stimulation of endogenous Schwann
cell infiltration into the host spared rim with about 50% of
the myelination coming from endogenous SC. But these
histological findings are not matched with robust behavioral
improvements (BBB, subscore and ladder test) during the
first 9 weeks, but there was a suggestion that at later time
points there was a difference in the open field scores, though
Tetzlaff readily admitted the experiment was terminated too
early to prove conclusive.

So there may be the possibility to exploit the membrane


component of OECs rather than the cells themselves
therapeutically to induce axonal extension over distance in
the CST. Work continues on identifying the components
in plasma membrane that are responsible for these effects.
Roskams finished with genuinely heartfelt thanks for the
support her lab has received from Spinal Research. Not only
did it allow the recruitment of a medical PhD with a lifelong
commitment to research, to the publication of papers
directly arising from the work but allowed collaborative
work to grow and the knock-on development and training
of careers that now exist around the world. She was willing
to admit that at one stage the Spinal Research grant was the
only one holding the group together and without that the
work could not have been completed sufficiently to allow
successful competition for further grants and is the direct
reason they are now looking for post docs. The audience
included a number of Trustees and staff from Spinal
Research and these few words were very much appreciated!

An important observation made by the Canadian team


was an alarming decrease in sensory thresholds found in
animals transplanted with nave SKPs flagging up concerns
about the choice of cells used in transplants and the
potential to modify sensory pathways giving rise to pain. To
underline this, Tetzlaff alluded to work done in
collaboration with Roskams showing impairment when
combining OEC cell transplants with BDNF. There is still
much to be learned as we attempt to translate basic science
to the clinic.
In an acute treatment of cervical injury paradigm SKPSC could also be seen to mediate functional benefits. The
acute setting is more hostile and cell survival is less,
nevertheless CATWALK (gait) analysis showed significant
17

improvement at late time points. Electrophysiological


assessment in this model showed the stimulus to elicit an
EMG response and the latency of response tending to
uninjured control levels in the treatment group. Other
cellular controls (nave SKP, nerveSC, fibroblasts) showed
improved electrophysiological efficacy in the rubrospinal
tract but to a lesser degree than SKP-SC group, once again
demonstrating many cell types elicit effect adding to the
difficulty of data interpretation.

OECs to augment this restricted regenerative capacity. One


such combination studied by the Glasgow group, involves a
peripheral conditioning lesion injury to peripheral nerve
stimulates cellular responses that in turn give rise to a more
vigorous regenerative response. OEC transplants in
combination with a conditioning lesion result in axon
regeneration into and beyond the lesion site but only where
OEC bridges appear; there is no evidence for axons in
regions devoid of OECs. The conditioning lesion appears
to boost both number and distance travelled by regenerating
axons but this effect is not restricted to OECs and can also
be seen with other cellular grafts (e.g. Schwann cells) so the
factors responsible may not be unique. Regardless, of
whether we can successfully achieve regeneration
substantially beyond the lesion site, the key is whether these
axons can make functionally useful reconnections once
there.

A clinical limitation of autologous SKP-SC transplants


will be the time required to harvest and culture these cells in
humans. To begin addressing this issue the group wanted to
see how well SKP-SC survived and whether they could
integrate after a chronic injury (5wks post injury). In a
thoracic contusion delayed transplantation, SKP-SC
integrated and bridged lesion site and attracted host axons.
There was massive recruitment of endogenous Schwann
cells and reduced cavity volume. Thus, even at this 5 week
time point post injury the scar is not yet so exclusive to
prevent integration into the host rim. Endogenous Schwann
cells are characterized by greater astrocytic reactivity than
seen regions containing SKP-SC. There was no significant
behavioural improvements in this model but once again the
experiment was terminated for histological examination
relatively early perhaps before such changes could manifest.

Examining whether cellular transplants could act via


neuroprotective or plasticity mechanisms, Riddell turned to
electrophysiological assessments. Stimulating dorsal roots
below the lesion they could assess sensory circuitry in the
region of the lesion and the effect of cellular transplants on
these. After peripheral stimulation, cord surface recordings
register a sharp afferent volley entering the cord followed by
a more diffuse (cord dorsum) potential representing synaptic
activity in the large number of collateral sprouts served
by the primary afferent. Injury causes a reduction in the
cord dorsum potential rostral and some distance (24 mm)
caudal to the region of root entry. The cord dorsum
potentials are significantly higher in transplanted OEC
animals and this correlates with increased sensory evoked
potentials in the sensorimotor cortex. But what of
mechanism? If sprouting or plasticity mechanisms are
envisaged then OECs may be inducing new sprouting to
compensate for the loss due to lesion. To separate this issue
from that of neuroprotection Riddell made us of cervical
lesions which fortuitously, in their experience, did not
result in OEC-filled lesion cavities but always resulted in
extended caudal distribution. If direct contact with OECs
in this region induces sprouting one would expect changes
in circuitry potentials in the region. Their results indicated
that cord dorsum potentials above the region are
significantly reduced as a result of axotomy but in both the
OEC and untreated groups a significant elevation of
potentials could be registered caudal to the injury site
suggesting a degree of spontaneous plasticity not however
related to OECs. This would seem to throw the emphasis
for the mechanism back on to neuroprotective rather than
regenerative/plasticity one.

The session returned to olfactory ensheathing cells when


JOHN RIDDELL considered the various ways OECs might
improve function after SCI; axonal regeneration,
neuroprotection, plasticity (sprouting &/or synaptic) and
remyelination of spared fibres. Support for OECs in
regeneration is polarized between those believing OECs
provide bridging sites across the lesion and support
regeneration across and beyond the lesion to recover
function, and others believing OECs support axonal entry,
but not extension beyond more in line with experience
using other cell types. Whilst OECs have, in the main, been
examined using the CST, Riddells group favour the
ascending dorsal column projection. Ascending fibres in the
dorsal column arise from DRG neurons entering via the
dorsal roots and bifurcating to send axons both rostrally and
caudally. These branches give off lateral collaterals
throughout their length, entering the grey matter to make
synaptic connections with spinal neurons. Some project the
full length of the spinal cord to terminate in the dorsal
column nuclei. Riddell studies regeneration of ascending
branches following wire knife lesion of the dorsal columns
between the L3/4. Lesions arising from this procedure form
cavities that elongate rostro-caudally. Lumbar lesions are
attractive, he says, because they are amenable to tracing via
the L4/5 spinal nerves (just below lesion) providing a
sensitive method for assessing axonal regeneration.

DAY 2: SESSION 1
Whilst identifying targets and developing therapeutics
against them is one major activity in creating clinical
treatments it is being able to deliver them safely and
compatibly within the clinical setting that often presents a
significant downstream obstacle in translation. This is a
particular challenge for drug delivery to the profoundly
delicate CNS protected by bony structures and physiological
barriers. MOLLY SHOICHET described some advances being
made in drug delivery technology for spinal cord injury

Transplanted OECs not only fill the cavity site created


after wire knife section but are found distributed r-c in OEC
tracts for quite substantial distances. Nevertheless,
regenerating dorsal column axons are mainly restricted to
the transplant site and very rarely are they found in these
bridging tracts of OECs. A number of groups are therefore
examining the potential of combinations of treatment with
18

repair using biomaterials. For compression injuries she


sought an injectable vehicle with certain design
characteristics: minimally invasive utilising biocompatible,
biodegradable materials non-adhesive to cells, providing
localised release in an injectable, fast gelling matrix capable
of dosage control. A mixture of hyaluronan:methylcellulose
(HAMC) provides a polymer mix that thins under pressure
and shearing forces found during injection (provided by
hyaluronan) and thermal gelling at body temperature (via
methyl cellulose). The HAMC polymer remains at the
injection site and degrades over the course of a few days.
Alone, it helped to reseal dura and reduce the extent of
inflammation and animals with compression injury scored
better with HAMC injection compared to control on the
BBB scale. As a test of effectiveness as a local delivery vehicle
for bioactive therapeutic agents EPO was administered either
by bolus intrathecal or intraperitoneal injection or with
HAMC local injection. HAMC delivery of EPO showed
reduced cavity volume and increased neuronal sparring
relative to conventional bolus administration routes. Further
studies continue with a focus on prolonged release of
neuroprotective and neuroregenerative factors and the more
challenging concept of combining therapeutic agents and
effecting different release rates.

throughput functional analysis of these 62 transcription


factors by 96-well plate automated image acquisition of
neurite outgrowth identified 10 new transcription factors.
Transcription factors Tsc22d3 and Rtel1, are downregulated following sciatic nerve crush and knock-down in
in vitro assay results in more outgrowth. Are these repressors
of the growth program? Things are not so clear cut. NFIL3
and Csrp3 are upregulated after sciatic nerve crush but
knock-down in in vitro assays results in enhanced neurite
growth. This apparent paradox was further confirmed when
NFIL3 was found to repress neurite outgrowth in cultured
DRGs and reduce regeneration-associated gene expression
(e.g. Gap43 and Arg1).
Verhaagen switched to a second project which will
certainly contribute to the debate on the utility of olfactory
ensheathing cells (OECs) in SCI repair. Evidence would
suggest OECs intermingle with scar tissue, promote
sprouting, tissue sparing and even angiogenesis. There are
also examples in the literature reporting functional recovery
but whether any of these features are unique to OECs or
mechanistically understood remains much debated. Very
much in keeping with themes he had developed earlier,
Verhaagen conceived of tackling these issues at the molecular
level, asking Are OECs a source of regeneration-promoting
molecules? Detergent-induced lesion of the olfactory
epithelium was followed by a time course microarray analysis
of the olfactory nerve layer (160 days post lesion).
Approximately 70% of the 819 genes regulated were upregulated as a result of this insult. Trend clustering and
gene-ontology analysis were performed with analysis centred
on genes encoding extracellular matrix and cell adhesion
molecules. Over the time course, clusters could be identified
as following either an up/down gene expression pattern or a
down/up. There was clear evidence for activation of the
complement and opsonin independent pathways. Many
regulated genes related to cytoskeletal activation and ROS
production hinting at active waste engulfment through
phagosome formation. Down regulation of genes in the
cholesterol pathway combined with upregulation of a
cholesterol efflux transporter would all likely increase the
levels of extracellular lipoproteins. Are these stimulants to
axonal growth, he asked. He summarised these data with a
proposal that following neuronal cell death OECs recognised
axonal debris that blocks axonal regeneration, there is
engulfment and waste management including clearance
and cycling of protein and lipid which in turn leaves a
permissive environment for axonal regrowth. 465 genes
where exclusively regulated during 6 to 30 days post insult of
which 88 were related to extracellular matrix or cell-adhesion
genes, including a set of genes associated with tumour
invasion. Perhaps the genes used in matrix remodelling
during tumour invasion where employed by OECs to
remodel matrix ready for axonal invasion. Interesting idea.

Biomaterials may also prove useful in creating a


permissive environment for axonal regeneration, particularly
in transection-type injuries. Porous biocompatible tubes can
be seeded, for example, with small molecules and/or cells to
aid regeneration, cell survival and control local differentiation.
JOOST VERHAAGEN took us to the opposite extreme of
drug discovery in his presentation on methods to improve
efficiency and effectiveness of identifying drugable targets.
Our knowledge of the molecular mechanisms that govern
neural repair is very incomplete, he said, but we can take the
view that proteins are the principle targets for drug discovery
and as such a comprehensive picture of the key proteins and
molecular pathways involved in, or failure of, neuronal repair
is of major importance. In this quest he described an
integrated 4-step model beginning with genomics to identify
target genes using microarray. Next, bioinformatics and
biocomputation to provide cluster, overrepresentation and
pathway analysis. Then cellomics, a set of medium
throughput bioassays (using interfering RNAs and viral
vector technology to establish proof of concept) and finally,
in vivo studies to establish therapeutic potential.
Verhaagen exemplified this concept by looking at one
project that aimed to identify the molecular master switch
that drive axonal regeneration. Dorsal root ganglion cells
are an ideal system to identify neuronally expressed genes
involved in successful regeneration as the central and
peripheral branches of DRG neurons differ in their response
to injury and capacity to regrow. As this is unlikely to be
due to differences in environmental support it should allow
comparative gene expression analysis in the same neuronal
cell population after lesions that give rise to successful
(peripheral) and unsuccessful (central) regeneration. Sixtytwo transcription factors showed differential regulation
following sciatic nerve and dorsal root crush. Medium

MICHEL KLIOT began a provocative talk announcing


the goal here is to take microsurgery to the cellular level by
embracing micro- and nanotechnology. The aim
admittedly most relevant to peripheral nerve repair is to
reconnect or splice acutely severely damaged axons that
would otherwise require regeneration to achieve functional
19

recovery. There are many challenges to repair even in the


periphery but if the nerve is quite dumb e.g. tibial nerve or
sciatic nerve, you can get reasonable, if variable, repair by
conventional surgical strategies. The new concept relies on
intervention before there has been axonal die back and distal
degeneration, removing the damaged region and
reconnecting the two ends employing technology that is
used in the production of hybridoma cells. The technique is
conceived in three stages; (i) trimming the damaged ends;
(ii) bring these together and; (iii) fuse the ends. Kliot
showed proof of concept that all these steps can be achieved
in vitro and later went on to demonstrate proof of concept
that stage 1 is achievable in vivo. Micromanipulation of
single axons allows cutting and trimming of single axons
with a microfabricated silicon-nitride micro-knives (radius
of curvature 20nm) then application of focal electrical
current bring cut ends in close proximity and fusion of
axonal membranes by local application of electrical fields
(c.f. hydridoma cells). The whole procedure is carried
out under high magnification (50100). Fused axons
have been shown to share contents once reconnected but
the next critical step is the demonstration of restored
electrophysiological function. Although feasible the
question remains how could you perform the necessary
manipulations on large numbers of damaged axons?
Additionally, all three stages need to be integrated first in
vitro then in vivo and finally show that successful
reconnection can be achieved in the more challenging in
vivo environment where movements from respiration and
tissue access will prove problematic. The technique will need
to be clinically acceptable and automated. Physiological
hurdles, such as rapid distal axon degeneration, would
suggest intervention needed acutely while techniques to
preserve these remain beyond us. This is a technology at the
Wright brother stage of development with significant
technical hurdles to overcome before it can ever be practical
in the peripheral setting let alone the CNS.

subclinical changes and that the zone of partial preservation


may prove a useful primary end point for sensory studies
after therapeutic interventions. [Note: Axon reflex flare
response was reduced below the lesion suggesting utility as
a model of autonomic dysreflexia]. Having established that
thermal perception thresholds at the border zone were
useful, repeatable and more sensitive than other measures
(e.g. psychophysic reports of sensation, VAS or McGill pain
questionnaires) CHEPs afforded a simple, non-invasive,
reliable and objective method to measure sensory function,
Anand suggested. CHEPs is complementary to other
quantitative sensory tests such as thermal, vibration and
touch but could also provide objective biomarkers for
disease-modifying strategies and symptom relief (e.g. pain).
A number of advantages were cited, including; it does not
require patient cooperation or intact motor pathways, it can
be performed on young children and babies, it selectively
activates A delta and C fibre evoked potentials. In addition,
it was suggested that it can detect early subclinical
dysfunction and neuropathies before other quantitative
sensory tests. Anand accepted that CHEPs was similar in
many respects to laser evoked potentials but was far safer
making it more flexible and applicable to face (an internal
control region in SCI) and hairy regions of the body.
Advances in brain fMRI have yet to be matched by spinal
fMRI methods. The sessions next speaker PATRICK STROMAN
spoke about improvements in spinal MRI imaging that
would perhaps allow this non-invasive technique to become
a practical clinical tool in the assessment of grey matter
function in spinal cords. The spinal cord is particularly
refractory to MRI because of the close proximity of
anatomical structures with large variations in tissue densities
which produce magnetic field artifacts. His work has
developed to a stage where many of these and other problems
have been overcome including modeling of spinal cord
motion within the spinal canal with each heart-beat, a
method for spatially normalizing the results from each
individual and automatic region labeling and region-ofinteresting masking of normalized data but systematic
variations across studies within individuals brought into
question issues about reliability. Was such variation due to
errors or did it reflect modulation of activity in the spinal cord
by emotion and/or cognitive functions? After all a volunteer
would perhaps be more anxious during the first series of
studies whilst struggling to stay wake during later studies, he
postulated. Using conditions designed to focus the subjects
attention either specifically on a changing sensory input (an
innocuous 15C cold stimulus to the hand) or distracted
using multiple choice-style puzzles during sensory thermal
tests he found that the variability in fMRI signals did not
reflect errors but demonstrated real differences in neuronal
function of the spinal cord under different emotional
conditions. These results showed that while care was needed
in interpretation they were nevertheless highly sensitive.
Further studies in healthy volunteers revealed areas of the
spinal cord where activity changes correlated regardless of the
timing and sequence of varying sensory input producing a
fMRI-derived functional connectivity map entirely consistent
with observed dependence on attention and anxiety, and with
previous studies of thermal responses in people after SCI.

DAY 2: SESSION 2
Beginning this session Chair Thomas Carlstedt pointing out
that there are essentially 2 methods of assessing SCI:
morphological and functional. As morphological assessment
is not possible in humans and is a terminal end point in
animals, there remains a great need for improved assessment
techniques, particularly as clinical assessment are not that
sophisticated. So, is there a third way?, he asked. The first
speaker, PRIVEEN ANAND, described a technique called
contact heat evoked potentials (CHEPs) in which a large
thermode placed on specific points on the skin rapidly
increases in temperature from 32C to 53C and back
again activating the vanilloid family of receptors including
the heat/capsaisin receptor (TRPV1). Heat activation is
recorded as evoked potentials on the scalp providing
objective information such as potential amplitudes and
latencies arising from small fibre (A delta, C fibre) circuitry.
The current interest in CHEPs was stimulated by earlier
findings carried out as part of Stage 1 of the Clinical
Initiative which examined a number of quantitative sensory
testing techniques in SCI subjects as well as pain measures
and axon reflex flare response. Their conclusion was that
thermal perception thresholds were the best at detecting
20

Stroman moved to describe a sexual function study where the


objective was to map activity in the lumbar and sacral regions
of the spinal cord to erotic visual stimuli, self-stimulation and
a combination of the two. The purpose is to determine the
effects of injury on sexual function and investigate how some
components of the function are preserved after trauma to the
cord. Data presented showed strong correlation between
fMRI response and level of physical arousal (in healthy
volunteers) after visual stimuli. Likewise, there was strong
correlation between fMRI responses and levels of physical
arousal after physical arousal.

COZOOM (contiguous-slice zonal orthogonal multislice


imaging) which excites regions of reduced volume, reducing
artifacts and preserving spatial information. Water diffusionweighted measurements are collected on a mm scale resulting
in a measure of an apparent diffusion coefficients which
contain average 3D vector information on the direction and
strength of water diffusion in a given voxel. The resultant
(eigen) vector plots ultimately provide information on the
direction of fibres, for example and were shown to distinguish
between patients and controls and correlate with the level of
functional impairment. Future work will concentrate on
longitudinal studies to map changes in spectroscopic and fibre
integrity measures following an acute MS attack and an ISRTfunded exploration of DTI in spinal cord injury.

Stroman continues to increase the practicability of spinal


fMRI with newer developments that include automated
labeling of the spinal cord and brainstem and normalizing
images for subject to subject comparisons and detection of
cord motion whether cardiac-related or bulk motion,
eliminating the need for user input, bringing a reliable, robust
and user friendly standard clinical MRI protocol ever nearer.

DAY 2: SESSION 3
In a session focusing on translational science DAVID ALLAN,
Director of the Scottish National Spinal Injuries Unit
(Glasgow) offered his perspective as a clinician, a researcher
and a Trustee of ISRT. He began by asking how had Spinal
Research done over the past decade or so? To answer that
he reflected that medicine rarely cures things more often it
must be satisfied with modifying disease or alleviating
symptoms and for that reason the clinical service must look
from the perspective of the patient. We have to have elegant
research but the question is; can we translate that elegant
research into the patient? To exemplify his point, Allan
reviewed a case study of a patient involved in a road traffic
accident: they had a dislocation at L1/2 and complete
transection of the cord. The accident occurred in 1990
when the patient was stabilised surgically, received
rehabilitation, returned to university, married, had children
and returned to work. This, Allan said, would be deemed
a success. What is not a success, he proposed, is that nearly
2 decades later the clinical situation is exactly the same and
the treatment she would be offered today would be
identical. Mischievously he asked What have you guys be
doing? Before you could make out the pin dropping, he
readily admitted spinal cord injury presents particular
difficulties; there are great variations in level and severity of
injury giving rise to a spectrum of clinical presentations
which are not as easy to monitor and control as the research
setting. Nevertheless, ignoring the realities of the clinical
setting or failing to incorporate these into the research
planning will prove problematic.

The final presentation in the session continued with the


MRI theme as CLAUDIA WHEELER-KINGSHOTT described
techniques that are more pathologically specific to axonal
degeneration and repair. Two techniques were described, (i)
MRI spectroscopy which allows quantitative measurement of
metabolites which are themselves markers for axonal
dysfunction or reduced axonal density (e.g. N-acetyl-aspartate
(NAA)) or gliosis or inflammation (e.g. myo-inositol (m-Ins))
and (ii) diffusion tensor imaging (DTI) which measures water
molecule mobility in tissues theoretically yielding information
on structure (e.g. fractional anisotropy (FA) shown to be
sensitive to axonal degeneration and myelin breakdown). The
two techniques together are beginning to provide a tantalizing
glimpse into metabolic dysfunction and structural breakdown
during disease. The data presented was acquired from MS
patients and control subjects but the principles can equally
be applied to other spinal cord pathologies, it was suggested.
Data acquisition was performed during the acute phase in
relapsing MS patients in a 20 min procedure. One challenge
faced during data acquisition is placement of the voxel
(a defined volume from which signal acquired; in this case
~ 2.4ml) well within the spinal cord to reduce signal noise
from other non cord tissue. The 1H spectrum can readily
distinguish peaks associated with NAA and m-Ins in brain
tissue but corresponding spectra from spinal cord has a lower
signal to noise ratio as might be expected when one considers
the voxel placement will result in white, grey and CSF
contributions rather than white matter alone. Patients
exhibited significantly lower NAA concentrations than
control and a trend to higher concentrations of m-Ins
suggestive of increased gliosis and inflammatory response.
Moreover, level of functional impairment appeared to
correlate with metabolite levels making them potentially
useful biomarkers. Wheeler-Kingshott next described work
on diffusion tensor imaging MR of the spinal cord. DTI is
not easy in the spinal cord; issues common to all MRI
acquisitions include the need for high resolution in such a
small tissue volume, a pulsatile CSF flow requiring gating and
suppression and general motion sensitivity (note also
susceptibility to T2* effects). Improved imaging can be
achieved with appropriate pulse sequence techniques such as

He conceded that translation needs to be a partnership


accepting that for real translational progress the clinical
service itself must itself provide early reliable assessment
more difficult than you might think ideally assessing the
extent of injury and the potential for recovery at the
roadside. The clinical service also needs to provide a system
of clinical rehabilitation and care that will permit study of
experimental interventions and provide some form of long
term assessment follow up.
Allan considered models of translation; the concept of
bench-to-bedside is well know but bedside to bench where
patient clinical data inform basic research activity may have
something to offer. Spinal cord injury in far from unique in
struggling with translation and there remains a valley of
21

death between basic science and clinical research in many


medical disciplines, partly maintained by funding bodies
undervaluing or lacking experience of this phase of research.
For his part, Allan believes bringing the bench to the
bedside is the way forward, embedding a basic research
capacity within the clinical facility.

observed outcomes. A procedural control is currently part of


an ongoing extended study.
The title of the final presentation in the session was
Testing improved clinical, physiological and functional
assessments given by PETER ELLAWAY. Ellaway is one of the
original investigators of the Clinical Initiative which was
tasked with developing improved physiological tests to
supplement existing clinical and functional tests that are
available. Stage I of the Initiative identified a number of
improved physiological tests whilst Stage II examined the
relationship between clinical and functional changes to
physiological changes in sensorimotor and autonomic
function arising from non-invasive treatments, such as
weight assisted treadmill therapy (WAT), and sought to
validate these tests in SCI.

Continuing the clinical theme HOOSHANG SABERI


from the Department of Neurosurgery, Tehran University
of Medical Sciences presented preliminary results of a
Schwann cell transplantation study in chronic patients.
Saberi summarized a number of properties of Schwann cells
that supported the rationale for examining Schwann cell
transplants in patients including reports that they gave rise to
improvements in locomotor scores in various experimental
SCI models, that the are a terminally differentiated,
potentially autologous source of cell treatment. Nevertheless,
these pros are countered by observations that Schwann cells
favour sensory axon sprouting, induce astrogliosis and
provide an environment that act as an axon sink with limited
axon growth beyond the region of transplantation.

The Clinical Initiative presently comprises four groups


from London, Glasgow, Zurich and Edmonton, Canada.
Ellaway began by describing the work of his own group in
London. The London group chose two treatments through
which they hoped to induce changes in patient function; (i)
partial weight-supported treadmill therapy and; (ii) repetitive
transcranial magnetic stimulation (rTMS) applied to the
motor cortex. Magnetic stimulation originally used in a
single pulse mode to test the corticospinal pathway from the
motor cortex to peripheral muscles was discovered to tap
into the plasticity of the brain when given repetitively. A pilot
study carried out by Nick Davey using rTMS in SCI subjects
gave rise to functional improvements and some changes in
physiological parameters. These findings let to the present
study which employed, as treatment, a continuous train of
pulses at 5Hz for 2s, every 10s for a total of 900 pulses once
a day for 5 days (at 80% active motor threshold) in a single
blinded, randomized, cross-over trial using either sham or real
coil with a 2 week wash-out period. Subjects underwent a
battery of assessments including sensory (electrical perceptual
threshold (EPT)) and motor (motor evoked potentials and
threshold levels of single pulse TMS at rest and at 10% of
maximum voluntary contraction) and autonomic (e.g.
sympathetic skin response). The Action Research Arm Test
(ARAT) and peg-board tests were used to assess function.

Although in the actual clinical study used sural nerve


from the patient Human Schwann cells culture procedures
were optimized using sural nerves of brain-dead donors.
Culture procedure took between 4-6 weeks. Ethical approval
and informed consent was obtained for a 33 case beforeafter case series study. Safety was paramount; clean room
facilities and tissue screening required. Patients were
evaluated on eligibility criteria and underwent full
independent neurological evaluation. Patient selection
criteria included <50 years old with neurologically stable
contusion lesions above L1 (ASIA A-C). The lesion length
<20mm (by MRI) with no evidence for compression,
stenosis or tethering. Each patient received 6 months
physiotherapy including physical exercise, FES, ultrasonic
diathermy and infrared treatment. Patients were evaluated
by an independent team at 4 and 8 months post-transplant
(Saberi et al., Neurosci Lett 2008).
Transplantation followed a limited laminectomy, strinx
exposure and intramedullary transplantation by multiple
injections to rostral/caudal lesion end using image guidance.
Patients received post-operative care that included
antibiotics, prophylactic anticoagulants, progesterone and
insulin and were ambulated 24hrs post-op.

Clinically, rTMS did not produce changes in the ASIA


sensory or motor scores or level. They did find functional
improvement as measured by ARAT a series of defined
upper limb activities designed to test success and timing
parameters during manipulation of objects immediately
after rTMS although these improvements were not
sustained at 72hr and 120hr post treatment. In the pegboard test improvements were also seen but these did not
reach significance levels above sham.

This was principally a safety and feasibility study,


however sensory, motor, sphincter, sexual and FIM scores
were recorded. Saberi reported some changes in sensory and
motor scores as well as bladder sensation and control,
although none reached statistically significance. No change
was recorded in spasticity or measures of sexual function.
FIM scores changed from 84.1719.85 to 86.9620.8
(P=0.001).

The physiological measure EPT were measured by


placing a disc electrode on ASIA points on the body and
increasing the strength of stimulus and recording the lowest
strength of stimulus which can be perceived. These values can
be plotted quantitatively against normal values to assess
sensory processing. A number of case studies showed rTMS
treatment modifying the EPT, although not all patients
responded to treatment. The EPT has a number of merits as

Transient low grade fever, vomiting and headache


observed during first 24hrs and one case of transient
neurological decline. The procedure was clearly feasible and
safe in this group of patients, although the absence of a full
procedural control makes it difficult to comment on the
22

a clinical test; it is well tolerated, quick to perform, requires


little training, uses inexpensive equipment and is quantitative.
It also shows good repeatability (both inter- & intra-rater)
which is essential for any routine tests and validity is being
established for SCI.

with Bernie Conways study using robotic weight-assisted


treadmill therapy (Lokomat). Conways aim was to provide a
comprehensive evaluation of the physiological and functional
adaptations induced by locomotor training in incomplete
subjects. Their study took data from 11 acute and 5 chronic
SCI subjects who each received 1 week of pre-training
assessment, 6 weeks of Lokomat training and assessment
and a final week of post-training assessment. Assessment
comprised clinical and functional (ASIA and FIM),
locomotor (WISCII, video and foot contact for overground
parameters and Lokomat-derived data feedback), quantitative
sensory tests (vibration, EPT, temperature and monofilament
light touch) as well as sensory and motor electrophysiological
measures (time and frequency domain SEPs and MEPs).

The same coils used for rTMS treatment can be used in


single pulse mode to interrogate the voluntary controlled
pathways. Many parameters can be measures using this
technique including the threshold TMS needed to elicit a
motor evoked potential. One complication is that many
SCI subjects exhibit thresholds beyond the range of the
coils. However, under conditions of voluntary contraction
target (active) muscles exhibit lower thresholds and this
protocol was adopted in those patients with unattainably
high thresholds in the relaxed state. They found that rTMS
(treatment) invariably resulted in an increase in threshold
whether measured in the active or relaxed states and was
sustained for at least 5 days post rTMS treatment session.
An increase in threshold is somewhat surprising, he said,
but perhaps not entirely unexpected when you consider
sensory and motor integration making it difficult to predict
how the two cortexes communicate.

Patients who were able to tolerate a rapid reduction in


body weight support during training achieved better
functional outcomes. Acute subjects showed significant
changes in over ground parameters during the first three
weeks of training, though this was less marked in chronic
patients who nevertheless showed a general improvement in
quality of gait. These functional outcomes correlated with
Lokomat parameters.

Not all muscles responded, for example, the thenar and


first dorsal interosseous showed increased thresholds whilst
the wrist extensor muscle did not; which may relate to level
of innervation and injury. The merits of this physiological test
included; well tolerated and non-invasive, assesses voluntary
motor pathways and is quantitative. There are limitations, he
suggested; not quick, extensive training required, analysis not
straightforward, expensive equipment and repeatability not
well established. Nevertheless, Ellaways opinion was that this
technique was worth retaining and pursuing.

The Glasgow group are particularly interested in


examining somatosensory evoked potentials (SEPs).
Illustrative case data presented of an acute subject showed
SEP spectral changes indicative of increased sensory
processing as the subject went from pre-, mid- and posttraining. Such spectral changes were less apparent in chronic
patients. They have added to this a sophisticated frequency
domain analysis which allows analysis of how the spectral
shape changes after time post stimulation. Frequency domain
analysis suggests the stimulus causes a desynchroniziation of
the EEG activity in the brain as subjects go through training.
Their studies using single pulse TMS (test of motor function)
showed high variability and require further refinement.

Ellaway moved to discuss the work of the Edmonton


group led by Arthur Prochazka. Prochazka and colleagues
are developing a tele-supervised therapy on an automated
work station with functional electrical stimulation (FES)
of hand muscles in cervical SCI subjects and using this
treatment regime to evaluate the outcome for hand
function. The in-home tele-rehab system known as
ReJoyce uses a tooth click actuator to control an FES cuff
and a manipulandum designed to train the user in a full
range of upper limb movements relevant to tasks important
in daily living. Motivational feedback and data recording of
performance is through specifically designed video games
which are all relayed to the physiotherapist over the internet.
Subjects undergo 6 weeks on either ReJoyce training or a
standard rehab regime. Those using the ReJoyce system
showed better improvements on the ARAT exceeding levels
considered minimally clinically significant. Prochazka also
found some changes in single pulse TMS evoked thresholds
in the triceps and extensor carpi radialis longus, but not the
biceps or abductor digiti minimi. The electrical perceptual
threshold (EPT) did not change over the course of
treatment, however, the excellent repeatability observed
helps to validate this physiological technique.

Ellaway finally turned his attention to the work of the


group based in Zurich. The group led by Huub van Hedel
and Armin Curt looked at the spontaneous recovery of SCI
from a large cohort of patients accessible through EM-SCI
(European Multicentre study for clinical trials in human
SCI) as the test bed on which to evaluate the clinical
relevance of various functional and physiological tests.
Following patients from early injury for more than
12 months they collected data on functional, neurological
and measures of spinal conductivity, including EPT. Whilst
ASIA grades, motor and sensory scores, SCIM and WISCI
levels improved during the course of 50 weeks post injury
by contrast they observed relatively little change in
physiological variables such as SEPs and MEPs over this
extended period and tended to stabilize much earlier in the
recovery post injury. The implications of this are two-fold;
first, after therapeutic intervention physiological changes
may only be observed early in the post treatment phase and
second, evidence of changes in physiological parameters
may only signpost functional changes at a later stage. The
group concluded that (spontaneous) functional recovery
was not related to spinal conductivity (although Ellaway
himself would debate that point) and more likely due to

Ellaway briefly updated the delegates on the activities


of the two other groups in the Clinical Initiative beginning
23

compensation and neural plasticity, rather than regeneration


of spinal pathways.

severity, clinical management biases and non-uniform use


of outcome measure all will have bearing. So when making
the transition from rodent studies to human studies we will
encounter variability that exceeds those experienced in the
pre-clinical setting and the signal within the noise will have
to be that much greater.

In a study looking at 34 patients with cervical incomplete


SCI Curt and colleagues coupled EPT measurements with
dermatomal SSEPs (dSSEPs) to examine posterior spinal
column function and pain pathways. By extending the
stimulus from the EPT electrode into the noxious range they
could assess subjects ability to perceive touch and nonnoxious stimuli but unable

Practical issues also dictate compromises in therapeutic


window or routes of administration for which pre-clinical
data has not been collected.

Ellaway concluded with a summary of his view of the


current status of physiological tests being evaluated by the
Clinical Initiative. In terms of sensory systems, the electrical
perceptual threshold test (EPT) showed good repeatability
with validation in SCI on-going and was a useful adjunct to
standard quantitative sensory tests. Repeatability of
dermatomal SSEPs was less robust. For motor systems,
MEPs were showing limited repeatability and required
further validation. Although not presented tests for the
autonomic system, such as sympathetic skin response
showed good repeatability. In general, all tests require
further validation as clinical tools for the assessment of SCI.

It is quite clear we will need a robust effect at functional


and anatomical levels pre-clinically if we are to expect a
therapeutic effect in patients and that the pre-clinical data
set we strive to collect relevant to the human condition.
What candidate therapies fit the bill today, he asked.
Moderator of the session, Steve McMahon, put it another
way, asking delegates to suggest any candidates worth
progressing to the clinic? Few were offered from the floor.
One low risk approach might be to test pharmaceutical drugs
already in market, such as minocycline or statins for which
there has been some pre-clinical data to suggest positive effects
in SCI. This was a question of risk, said one delegate, going
so far as to say it was unethical to procrastinate for too long
about models etc. if there is little risk to the patient. There
was concern too that many behavioural studies are not
reported as blinded or randomized and there was a call on
authors to state, within publications, whether the reported
studies are blinded and, if not, for those reviewing grants and
papers to challenge authors as to why not.

The meeting came to a conclusion with a discussion


session on Priorities in Translational Research. Mark
Tuszynski began by raising issues faced when wishing to
translate pre-clinical discoveries to the clinic. He prefaced
this by suggesting that the many experimental approaches
adopted in the laboratory had elicited incremental growth
in axons. This, he said, represented much of the progress in
the field, and while recent experiments suggested combined
approaches can elicit more growth, even this was not
extensive. Such a status report should be considered within
the broader context of clinical translation; humans need
corticospinal tract (CST) axons for effective motor function,
yet there is not a large body of evidence from the literature
that this vital motor system can be stimulated. Furthermore,
the primate spinal cord has some fundamental differences in
size, organization and immune system when compared with
the rodent. More generally, 95% of medicines fail during
development because they lack safety, or there is an inability
to detect a therapeutic effect in patients. When considering
the challenge of developing treatments for acute SCI it has
to be recognized that ASIA A patients (those with the most
profound injury) improve in about 20% of cases and among
ASIA B patients this figure rises to 5075%, most likely due
to endogenous plasticity. Should a therapy targets an injury
at the acute stage the efficacy must exceed this spontaneous
recovery threshold to have a detectable benefit. Citing the
Novartis anti-Nogo mAb therapy currently in clinical trials,
Tuszynski pointed out that if this treatment targets plasticity
in spared systems these spontaneous recoverers are the ones
most like to respond and detecting improvement within this
cohort become an even greater challenge.

Another question from the floor accepted there might be


a need for replication of pre-clinical results, but should the
field be seeking to precisely repeat procedures or should
there be a level of repetition across different models and
species to verify robustness? The problem with imprecise
replication comes when it doesnt work, appeared to be the
consensus. Naomi Kleitman made the point that where
obvious controls where identified these should be included
first before extensive replication contracts are funded as this
often identifies false positives. She continued, stating that
of the 8 studies that have been funded to faithfully replicate
experiments (including, minocycline, erythropoietin, a
Nogo receptor inhibitors, chronic OEC) none were able to
replicate the behavioural findings of the original; some
where intermediate and some showed artifacts responsible
for behavioural responses. Echoing David Allans point on
the need to adopt bedside-to-bench translational thinking,
Kleitman concluded that valuable experience will be gained
through the current batch of trials, limited as they were in
terms of patient numbers, and the positive predictive value
of current animal models and the direction we may need to
take in the future.
Mike Craggs spoke of the priorities and needs of the
patient and the lack of relevance, in his view, of the current
animal models to assess autonomic dysfunction, such as
pelvic dysfunction, which ranks very highly with patients.
McMahon suggested that this might not be entirely a lack
of interest from the scientist and that reviewers and funders

Pre-clinical studies also tend towards conditions where


variables are reduced to a minimum surgical procedures,
personnel, in-bred strains, etc. but the human arena
presents a far greater diversity and for SCI in particular, the
age, gender and underlying mechanism of injury, level,
24

tests for hand function that make outcomes easier to assess,


but many patients put bladder/bowel and sexual function at
the top of their list of quality of life issues. So clinical trials
are not going in this direction but should the field be
pushing more in that direction, inviting comments from the
audience. Mike Craggs made the point that many of the
pelvic dysrelexia appear many weeks post injury and there
was an opportunity missed to look at what causes and how
we might prevent these aberrant reflexes. Even in the
absence of restoring voluntary control, being able to arrest
the development aberrant reflexes would at least ensure
future regenerative strategies would not result in an
encounter with and having to overcome dysfunctional
circuitry lower down, as the may be irreversible. Thomas
Carlstedt agreed and found many similarities between the
avulsed brachial plexus injury and conus injuries both
perhaps requiring relatively little CNS regeneration to
produce some function restoration.

may have a role to play in ensuring work within such areas


is not forgotten or undervalued when considering grant
applications. David Allan assumed some responsibility on
behalf of the clinical community for not articulating the
priorities and needs and complexities of the human
condition to the scientific community.
Discussion moved to the question of how much
improvement would be useful to the patient and we heard
from Spinal Researchs Chairman, John Hick, who believed
this obviously changed as patients moved further in time
from their injury; as one got older maintaining quality of life
became as important as improving it.
Mark Tuszynski offered the corollary to the patient
perspective by considering what independently quantifiable
change would be convincing to the regulatory authorities?
There appears to be no easy answer, particularly without
some gold standard against which to measure outcomes.

With rodent models presenting similar slow onset


aberrant reflexes and methods available to assess function, if
somewhat unfamiliar to most, this author got the feeling
that study into lower cord injuries may become a hot topic
for the future.

James Fawcett brought up the issue of at what level


should we do clinical trials. Initial clinical trials are mainly
concentrated on thoracic injuries for the very good reason
that if you make the injury worse it doesnt cause a huge
difference in the patients clinical state. There is a move
towards cervical injuries in part because there are functional

Mark Bacon Head of Research

25

Conference Delegates
David Allan
Praveen Anand
Patrick Anderson
Melissa Andrews
Amirthe Vernie Balasubramaniam
Xuenong Bo
Elizabeth Bradbury
Arthur Brown
Arthur Butt
William Cafferty
Thomas Carlstedt
Lucy Carter
Daniel Chew
Michael Craggs
Salvatore Cuzzocrea
Meirion Davies
Anoushka de Almeida
Volker Dietz FRCP
Petra Dokladal
Philip Duffy
Peter Ellaway
Vieri Failli
James Fawcett
Karim Fouad
Hans Frankel
Robin Franklin
Sujay Galen
Isabella Gavazzi
Guillermo Gracia Alias
James Guest
Jodie Hall
Noam Harel
Wenlong Huang
Hans Hultborn
Stephen Hunt
Kenneth Hunt
Ahmed Ibrahim
Steven Jacques
Lyn Jakeman
Nick Jeffery
Eustace Johnson
Carolina Kachramanoglou
Nick King
Peter Kirkwood
Naomi Kleitman
Michel Kliot
Annapoorna Kuppuswamy
Rob Labruyere
Michael Lane
Stuart Law
Roger Lemon
Daqing Li
Ying Li
Samantha Lloyd-Burton
Ann Logan
Juan Luo
Milan Makwana
Matthew Mason

SIU Glasgow
Imperial College London
University College London
University of Cambridge
Royal National Orthopaedic Hospital, Stanmore
Queen Mary University of London
Kings College London
John P Robarts Research Institute, Ontario
University of Portsmouth, Portsmouth, UK
Yale University School of Medicine
Royal National Orthopaedic Hospital, Stanmore
Kings College London
Barts and The London School of Medicine and Dentistry
Royal National Orthopaedic Hospital, Stanmore
University of Messina, Italy
Kings College London
The Institute of Neurology, London
University Hospital Balgrist, Zrich
Balgrist University Hospital
Kings College London
Imperial College London
Wings for Life
Cambridge University Brain Repair Centre
University of Alberta
SIU Stoke Mandeville
University of Cambridge
University of Strathclyde
Centre for Neuroscience Research, Kings College London
University of Cambridge
University of Miami
Queen Mary University of London
Yale University School of Medicine
Queen Mary University of London
University of Copenhagen
University College London
University of Glasgow
University College London
University of Birmingham
Ohio University
University of Cambridge
Oswestry SIU
University College London
Imperial College London
The Institute of Neurology, London
National Institute for Neurological Diseases and Stroke
University of Washington
Imperial College London
Balgrist University Hospital
University of Florida
University College London
The Institute of Neurology, London
University College London
University College London
University of British Columbia
University of Birmingham
Queen Mary University of London
University College London
Netherlands Institute for Neuroscience
26

Stephen McMahon
Tao Meng
Adina Michael-Titus
Fred Middleton
Lawrence Moon
Pouria Moshayedi
Vivian Mushahwar
Maria Cristina Ovejero-Boglione
Regino Perez-Polo
John Priestley
Geoffrey Raisman
Gennadij Raivich
Paul Reier
Heinz Redl
John Riddell
Serge Rivest
Jane Roskams
Hooshang Saberi
Thomas Sardella
Jan Schwab
Molly Shoichet
Peter Shortland
Patrick Stroman
Anba Soopramanien
Wolfram Tetzlaff
Andrew Toft
Mark Tuszynski
Huub van Hedel
Joost Verhaagen
Phil Waite
Difei Wang
Philippa Warren
Claudia Wheeler-Kingshott
Karina Wright
Mie Yamamoto
Yi Zhang
Binhai Zheng

Centre for Neuroscience, Kings College London


University of Glasgow
Queen Mary University of London
Royal National Orthopaedic Hospital, Stanmore
Kings College London
University of Cambridge
University of Alberta
Kings College London
University of Texas
Queen Mary University of London
The Institute of Neurology, London
University College London
University of Florida
Ludwig Boltzmann Institute, Austria
University of Glasgow
Lava University, Quebec City
University of British Columbia
Imam Khomeini Hospital
University of Glasgow
Wings for Life
University of Toronto
Barts and The London School of Medicine and Dentistry
Queens University, Canada
Salisbury SIU
University of British Columbia
University of Glasgow
University of California, San Diego
Balgrist University Hospital
Netherlands Institute for Neuroscience
University of NSW, Sydney
University of Cambridge
University of Cambridge
University College London
Oswestry SIU
University College London
Queen Mary University of London
University of California, San Diego

27

Strategy Grants
Grantholder

Location

Modulation of the glial response after spinal cord injury


Prof. A. Logan
University of Birmingham

Award

Grant term

140,971

2000, 3 years

Synthetic fibronectin conduits as guidance channels for directed regeneration within the spinal cord
Prof. J. Priestley
Queen Mary and Westfield College, London 135,292
2000, 3 years
Can olfactory epithelial ensheathing glia (OEG) successfully promote cross-species spinal cord tract regeneration?
Dr A.J. Roskams
University of British Columbia, Canada
C$245,565
2000, 3 years
Development of the therapeutic potential of a combined glial cell/biodegradable substrate in functional tissue repair
following chronic injury of the adult rat spinal cord
Dr E. Joosten
Maastricht University, The Netherlands
271,600
2001, 3 years
Restoration of spinal cord circuitry and function after nerve root injury in man
Mr T. Carlstedt
RNOH Stanmore, London
188,989
& Mr R. Birche

2002, 3 years

Mechanisms of autonomic dysreflexia following spinal cord injury


Dr AG Rabchevsky
University of Kentucky, USA
& Dr G. Smith

US$213,694

2002, 3 years

A practical combination of ex vivo and in vivo gene therapy for spinal injury
Dr A Blesch.
University of California San Diego, USA
US$170,765
& Prof. M. Tuszynski

2002, 3 years

The role of the ubiquitin pathway in the preservation of functional axons after spinal cord injury
Prof. V.H. Perry
University of Southampton
139,479
2002, 3 years
& Dr M. Coleman
Spinal axon sprouting: characterisation, manipulation and functional consequences
Dr M. Ramer
University of British Columbia, Canada
CAN$305,844
Genetic analysis of Nogo and Nogo receptor function in spinal cord regeneration
Prof. M. Tessier-Lavigne Stanford University, California, USA
US$486,398
(Grant finished because Prof. Tessier-Lavigne moved to Genentech)

2002, 3 years

2002, 3 years

Enhancing the role of propriospinal neurons in the recovery of motor function after spinal cord injury
Dr K. Fouad
University of Alberta, Canada
C$271,113
2003, 3 years
Synaptic connectivity in regenerating neurones of descending motor tracts
Dr P. Kirkwood
Institute of Neurology, London
206,274

2003, 3 years

Early selective blockade of intraspinal inflammation is neuroprotective and leads to improved motor, sensory and
autonomic outcomes
Dr D. Marsh
The John P. Robarts Research Institute,
C$68,500
2003, 2 years
Canada
Standing and stepping with intraspinal microstimulation after spinal cord injury
Dr V. Mushahwar
University of Alberta, Canada
C$135,000

2005, 3 years

Can human lamina propria olfactory ensheathing cells expand, migrate and stimulate rat SCI repair as well as mouse
OECs?
Dr A.J.I. Roskams
University of British Columbia, Canada
C$371,835
2005, 3 years
Neuroprotective strategies after spinal cord injury
Prof. W. Tetzlaff
University of British Columbia, Canada
28

C$194,928

2005, 2 years

Chemorepulsive axon guidance molecules in adult CNS axon regeneration failure: Class 3 semaphorins and
their receptors
Dr B. Zheng
University of California, San Diego, USA
US$286,955
2005, 3 years
Neuregulin growth factors in the repair of spinal cord axons by olfactory glia
Dr G. Raisman
NIMR Mill Hill, London
147,734

2005, 3 years

Repair of adult rat corticospinal tract by transplants of olfactory ensheathing cells


Dr G. Raisman
NIMR Mill Hill, London
1,460,901
Promoting axon regeneration in the injured spinal cord by RNAi-mediated knockdown of receptors for neurite
growth inhibitors
Prof. J. Verhaagen
Netherlands Inst. for Brain Research
299,108
2004, 3 years
& Dr S. Niclou
Development of functional magnetic resonance imaging for assessing human spinal cord injuries
Dr P.W. Stroman
Queen's University, Ontario, Canada
C$292,320
2005, 3 years
Role of microglia in spinal cord injury pain
Prof. S. McMahon
Kings College London

197,777

2005, 3 years

Improving cardiovascular function after spinal cord injury


Prof. P. Waite
University of New South Wales, Australia

Aus$499,500

2006, 3 years

Do experimental treatments for spinal cord injury induce functional plasticity in spared pathways?
Dr John Riddell
University of Glasgow
274,453
2008, 3 years
Rewiring the central nervous system following spinal cord injury using neurotrophins and rehabilitative training
Dr Karim Fouad
University of Alberta
C$395,555
2008, 3 years
Optimizing recovery by facilitating plasticity
Dr Lyn Jakeman
Ohio State University

US$228,918

2008, 2 years

Recent Awards/Approvals
Investigation into the conduction properties of surviving axons following chronic spinal cord contusion and whether
therapeutic intervention can restore normal function
Dr E. Bradbury
King's College London, Guy's Campus
251,862
2009, 3 years
Axonal Regeneration in the Chronically Injured Spinal Cord
Prof. M. Tuszynski
University of California

US$253,200

2008, 3 years

Comparative evaluation of surgical and pharmacological methods for removal of a mature scar in a chronic spinal
cord injury model and subsequent regeneration of stimulated sensory neurons through the treated wound
Prof. A. Logan
University of Birmingham
157,180
2009, 2.5 years
Locomotor training in chronic adult spinal cord injured rats: plasticity of interneurons and motoneurons
Dr R. Ichiyama
University of Leeds
97,174
2009, 2 years

Network Grants
Transplantation of fibroblasts genetically modified to secrete neurotrophic factors into spinal cord lesions in adult rats:
development of therapies for functional repair of acute and chronic spinal injuries
Prof. M. Murray
Medical College of Pennsylvania
$581,705
2000, 3 years
Hahnemann University

29

Generation and testing of rat and human ensheathing glia for spinal cord transplantation
Prof. P. Wood
The Miami Project, Florida, USA
US$278,783
2000, 3 years
& Prof. M. Bunge
Canine models of spinal cord injury:characterising and establishing the regeneration potential of canine olfactory
ensheathing cells
Dr N. Jeffery
University of Cambridge
184,765
2002, 3 years
Prof. R. Franklin

Clinical Initiative
Stage I
Development of procedures for assessment of functional and structural recovery following spinal cord injury in man
Prof. P. Ellaway et al.
Imperial College School of Medicine
785,916
2000, 3 years
& NSIC Stoke Mandeville
Stage II
Functional and Neuronal Recovery in incomplete SCI: Interproject of the ISRT (clinical initiative) and the European
Multicenterproject (EM-SCI) for monitoring motor recovery in human SCI
Dr A. Curt
University Hospital Balgrist, Switzerland
SFr491,100
20042008
& Prof. V. Dietz
Outcome evaluation of FES-assisted exercise therapy for hand function in quadriplegic people
Prof. A. Prochazka
University of Alberta, Canada
C$125,750
20042009
Treatments to aid recovery from spinal cord injury: testing improved clinical, physiological and functional assessments
Prof. P. Ellaway et al.
Imperial College School of Medicine
419,626
20052009
& Royal National Orthopaedic Hospital, London
Comprehensive evaluation of the physiological and functional adaptations induced by locomotor training in
incomplete spinal cord injured subjects
Prof. B. Conway
University of Strathclyde
246,732
20062009

Nathalie Rose Barr PhD Studentship


Supervisor

Student

Location

Grant term

Central neural regulation of autonomic and somatomotor function in human spinal cord injury
Prof. P. Ellaway,
Pietro Cariga
Imperial College School of
1999, 3 years
& Prof. C. Mathias
Medicine, London
Promotion of spinal cord regeneration by targeted neurotrophin gene transfer to ascending and descending neural
systems
Dr A. Logan
Elspeth Brown
Queen Elizabeth Medical
1998, 3 years
& Prof. M. Berry
Centre, Birmingham
Do OBECs have advantages over Schwann cells in their ability to mediate repair following transplantation into
astrocyte-containing areas of CNS damage?
Dr S. Barnett
Andras Lakatos
University of Glasgow
1998, 3 years
& Dr R. Franklin,
Comparison of the effectiveness of viral vectors and transfected cells for delivering neurotrophins to the injured
spinal cord
Prof. D.S. Latchman
Filitsa Groutsi
University College London
1998, 3 years

30

Promoting long-distance axonal regeneration and funtional reconnection using combined treatments for spinal
cord injury
Dr J. Riddell
Thomas Sardella
University of Glasgow
2005, 4 years
& Dr S. Barnett
Assessment of a novel chondroitinase-based strategy in promoting nerve regeneration and recovery of function after
spinal cord injury
Dr R. Keynes
Phillipa Warren
Cambridge Centre for Brain
2006, 4 years
Repair
& Prof. J.W. Fawcett
An investigation of the role of machinery intrinsic to the spinal cord in human movement
Dr J. Iles
Alima Ali
University of Oxford
1999, 4 years
Comparative characterisation of the signalling mechanisms activated by OECs and Schwann cells during growth and
migration into astrocyte-rich environments
Dr S. Barnett
Richard Fairless
University of Glasgow
2000, 4 years
& Dr M. Frame
The use of antisense connexin 43 as a neuroprotective agent following spinal cord injuries
Dr D. Becker
Michael Cronin
University College London
2001, 4 years
The response of adult NG2+ glial cells to spinal cord injury
Dr A. Butt
Paul Hubbard
Kings College London

2000, 4 years

Magnetic resonance imaging of transplanted glia


Dr R. Franklin
Mark Dunning

2001, 4 years

University of Cambridge

Mechanisms by which lens lesions promote axonal regeneration of central nervous system neurons
Dr D. Tonge
Aliza Panjwani
Kings College London
2001, 4 years
A neurophysiological study of residual supra-sacral sensory-motor pathways and their influence on sacral reflexes in
incomplete spinal cord injury
Prof. M. Craggs
Vernie Balasubramaniam
Royal National Orthopaedic
2002, 4 years
Hospital, University College
London,
Inflammation and regeneration of dorsal column fibres
Prof. P. Richardson
Sadashiv Karanth

The Royal London Hospital

2002, 3 years

Spinal tract regrowth after block of ephrin signalling


Prof. S. Bolsover
Jez Fabes

University College London

2002, 4 years

Electrophysiological study on corticospinal and reflex organisation in incomplete spinal cord injury
Dr B. Conway
Isam Izeldin
University of Strathclyde
2003, 3 years
Using small molecules to promote regeneration in the spinal cord
Prof. P. Doherty
Michelle Starkey
Kings College London

2002, 4 years

Role of RAS as an intracellular mediator of central axonal sprouting


Dr G. Raivich
Milan Makwana
University College London

2003, 4 years

Effects of chondroitinase treatment on axon and glial functions


Dr A. Butt
Maria Ovejero-Boglione
Kings College London

2003, 4 years

An investigation into the use of repetitive transcranial magnetic stimulation (rTMS) to improve functional recovery
after incomplete spinal cord injury in man
Dr N. Davey
Nick King
King Imperial College London 2004, 3 years
& Prof. P. Ellaway

31

Targeting Eph/Ephrin mediated inhibition at the damaged dorsal root entry zone (DREZ)
Dr I. Gavazzi
Philip Duffy
Kings College London
2004, 4 years
& Prof. J. Wood
siRNA knockdown of the p75/RhoA axon growth inhibitory pathway in DRG both in vitro and in vivo to promote
DRG neurite and dorsal column regeneration
Prof. A. Logan
Ruth Seabright
University of Birmingham
2004, 4 years
& Prof. M. Berry
Mechanisms involved in chronic pain after avulsion injury
Dr P. Shortland
Daniel Chew
Barts & The London School
& Prof. T. Carlstedt
of Medicine
Novel anti-inflammatory and neuroprotective strategies in spinal cord injury
Prof. J.V. Priestley
Jodie Hall
Queen Mary University
Dr A. Michael-Titus
of London
& Prof. V.H. Perry

2006, 4 years

2006, 4 years

The use of a YFP-expressing mouse in studies of spinal cord injury: mechanisms of chondroitinase-mediated repair
Dr E. Bradbury
Lucy Carter
Kings College London
2007, 3 years
& Prof. S. McMahon
Promoting spinal cord repair by genetic modification of Schwann cells to over-express PSA
Dr Y. Zhang
Lou Juan
Queen Mary University
2007, 3 years
& Dr X. Bo
of London
AAV8shRNA-RhoA and AAV8nt-3 transfection of dorsal root ganglion neurons (DRGN) in vivo mediates neuron
survival and disinhibited regeneration of dorsal column (DC) axons
Prof. A. Logan
Stephen Jacques
University of Birmingham
2007, 3 years
Spinal Cord Diffusion Imaging: Challenging Characterisation and Prognostic Value
Dr C.Wheeler-Kingshott
Torban Schneider
University College London

2008, 3 years

Non-integrating lentiviral expression of GMCSF to promote spinal cord regeneration


Prof. G. Raivich
Francia Acosta-Saltos
University College London

2009, 4 years

Recent Awards/Approvals
Promoting Neurological Recovery by Maximising Sensory-Motor Activation During Stepping and Walking:
development and assessment of robotics-assisted delivery platforms
Prof. K. Hunt, Prof. B. Conway, Dr H. van Hedel, Mr D. Allan
2009, 3 years
Physiological changes accompanying plasticity
Prof. J. Fawcett & Prof. S. McMahon

2009, 3 years

32

Reports by Nathalie Rose Barr PhD students


The use of a YFP-expressing mouse in studies of spinal cord injury: mechanisms of chondroitinase-mediated repair
Lucy Carter, Liz Bradbury and Stephen B. McMahon
Overcoming the molecular inhibitors that impede axonal regeneration following spinal cord injury
Philip Duffy, Isabella Gavazzi, William Cafferty and John Wood
Do omega-3 fatty acids modify inflammatory changes following a spinal cord compression injury?
Jodie C.E. Hall, J.V. Priestley, V.H. Perry and A. Michael-Titus
AAV8shRNA-RhoA and AAV8nt-3 transfection of dorsal root ganglion neurons (DRGN) in vivo mediates neuron survival and
disinhibited regeneration of dorsal column (DC) axons
Steven J. Jacques, Ann Logan, Martin Berry and Zubair Ahmed
Effects of injury and chondroitinase ABC treatment on neuro-glial morphology and interactions involved in CNS functioning,
particularly of the visual pathway
Maria Cristina Ovejero-Boglione and Arthur Butt
Promoting long-distance axonal regeneration and functional reconnection using combined treatments for spinal cord injury
Thomas Sardella, John S. Riddell and Susan C. Barnett

33

The use of a YFP-expressing mouse in studies of spinal


cord injury: mechanisms of chondroitinmiase-mediated
repair
Lucy Carter*, Dr Liz Bradbury and Prof. Stephen B. McMahon
Kings College London, UK
*PhD Student
INTRODUCTION

Council Directive of 24 November 1986 (86/609/EEC))


and sterile precautions were used throughout. Mice were
anaesthetised with Avertin mouse anaesthetic administered
i.p (0.5 ml/20 g). For intracerebroventricular (i.c.v)
cannulation, anaesthetised mice were placed into a
stereotaxic frame, the skull was exposed and a hole made
with a 25-gauge needle (coordinates; 0.5 mm from
Bregma, 1 mm lateral to the midline). A steel cannula
composed of an insertion end made of a 26-gauge needle
with a 90 bend at 2.5 mm and an exposed end, connected
to 30 mm long flexible silastic tubing (VWR, UK) was
inserted into the hole and secured into place using
cyanoacrylate gel (RS Components, UK). The skin over the
skull was sutured leaving only the tubing exposed. For
intrathecal (i.t) cannulation, a partial laminectomy was
performed at the T10 spinal level. Following application of
lignocaine, the dura was incised and 20 mm MicroRenathane tubing (Braintree Scientific, MA, USA) attached
to 40 mm silastic tubing was inserted 5 mm intrathecally
to lie just rostral to the T12 spinal segment and anchored to
the rostral vertebral bone with cyanoacrylate gel. The other
end was guided under the skin and externalised and secured
as above. Animals were placed on a heated blanket during
recovery before being returned to their home cage. One day
after cannulation surgery mice were re-anaesthetised with
Avertin, and received a dorsal column crush injury. Briefly,
mice were prepared for surgery by shaving and disinfecting
the skin of the back. Following skin incision, muscle tissue
was cleared from the dorsal processes of the lower thoracic
vertebrae and a laminectomy was performed to expose the
dorsal surface of the T12 spinal cord. A piece of gelfoam
soaked with lignocaine (0.2%) was placed on the spinal cord
for 2 mins, then the dura was opened. Lignocaine was
reapplied and the exposed dorsal columns were crushed to
a depth of 0.5 mm into the cord using a pair of fine forceps
(Fine Science Tools, Inc), which were held in place for
10 seconds before being removed. Muscle and skin layers
were sutured and mice were allowed to recover on a heated
blanket before being returned to their home cage. Control
mice underwent sham surgery, which was identical to the
above up to the laminectomy after which the muscle and
skin layers were sutured without lesion.

One important factor preventing growth and regeneration


in the injured adult mammalian CNS is the presence of
chondroitin sulphate proteoglycans (CSPGs) (Busch and
Silver, 2007;Galtrey and Fawcett, 2007). CSPGs comprise
a protein core with one or more chondroitin sulfate
glycosaminoglycan (CS-GAG) chains covalently attached.
At the site of CNS injury, CSPGs become upregulated
around the glial scar, where they are potent inhibitors of
axonal growth (Snow et al., 1990; Dou and Levine, 1994;
Smith-Thomas et al., 1994; Davies et al., 1999).
Degradation of the glycosaminoglycan (GAG) component
of CSPG molecules using chondroitinase ABC (ChABC)
renders these inhibitory substrates more permissive to
growth (McKeon et al., 1995; Zuo et al., 1998; Grimpe et
al., 2005) and, when delivered in vivo, enhances axon
regeneration (Moon et al., 2001; Bradbury et al., 2002;
Houle et al., 2006; Cafferty et al., 2007; Massey et al.,
2008), plasticity and sprouting (Barritt et al., 2006; Massey
et al., 2006; Cafferty et al., 2008) following CNS injury.
Crucially, improved functional recovery following ChABC
treatment has been demonstrated in several rodent models
of spinal cord injury (Bradbury et al., 2002; Caggiano et al.,
2005; Fouad et al., 2005), and more recently in spinal
injured cats (Tester and Howland, 2008). Thus, ChABC
treatment is emerging as one of the most promising
therapeutic strategies for the treatment of spinal cord injury.
Many studies have now documented the effects of
ChABC treatment on axonal growth and sprouting at the
level of the spinal cord. Therefore, in the present study we
assessed the response of the corticospinal neuron (CSN) cell
bodies to injury in the low thoracic spinal cord. We further
tested whether ChABC treatment could promote reparative
changes at the level of the cell body in layer V of the motor
cortex. Here we describe neuroprotection as a novel
mechanism underlying the reparative effects of ChABC
following spinal cord injury.
METHODS
Animals
YFP-H mice (B6.Cg-TgN(Thy1-YFP-H)2Jrs, Jackson
Laboratories, Bar Harbour, USA), described by Feng et al.,
(2000) were maintained as transgenic heterozygotes by
backcross to homozygous wildtype mice (C57 BL/6; Harlan
UK Ltd., Bicester, UK).

Delivery of ChABC
Immediately after the dorsal column lesion, mice received
either 6 l (10 U/ml) high-purity, protease-free ChABC
(Seikagaku Corporation, Japan) or penicillinase (Sigma,
same g protein delivered) followed by a 3 l saline flush.
Treatment was delivered as a slow bolus i.t. or i.c.v. injection
using a Hamilton syringe inserted into the externalised
cannula tubing, with tubing sealed after injection with

Spinal cord injury and cannulation surgery


All surgical procedures were performed in accordance with
UK Home Office regulations (European Communities
34

cyanoacrylate gel. Mice received further bolus injections on


days 2, 4, 6, 8 and 10 following dorsal column lesion, with
injections performed under isoflurane anaesthesia (Abbott
Laboratories Ltd; 5% induction and 2% maintenance).

Odyssey Infrared imaging system (LiCor Biosciences UK,


Cambridge). Membranes were stripped for 45 min at 50C
in a beta-mercaptoethanol stripping buffer, blocked again
in skimmed milk and re-probed for the non-phosphorylated
form of ERK1/2 (rabbit anti-p44/42 MAPK; 1:1000,
overnight; Cell Signalling). Densitometric band intensity
values for the phosphorylated and total form of ERK1/2
were normalised against the -III-tubulin loading control
for each sample and mean expression data was calculated
from 4 animals per treatment group.

Tissue processing
For histological analysis of cortical layer V neurons, mice
were deeply anaesthetised with pentobarbitone (80 mg/kg,
ip) and transcardially perfused with 10 ml heparinised saline
followed by 50 ml paraformaldehyde (4% in 0.1 M
phosphate buffer). Brains and spinal cords were dissected
and post-fixed in 4% paraformaldehyde (2 hours at 4C).
Tissue was then transferred to 20% sucrose (in 0.1M PB,
overnight at 4C) and blocked in OCT embedding
compound (BDH, Essex, UK) for cryostat sectioning.
Sagittal spinal cord sections (20 m) were thaw mounted
onto superfrost plus slides and coronal brain sections
(30 m) were collected and stored free-floating as above.
For Western blot analysis, following a rapid transcardial
saline flush, a 4 mm tissue pieces spanning the lesion site
were fresh dissected and snap frozen in liquid nitrogen.

RESULTS
Thoracic spinal cord injury leads to progressive atrophy,
of corticospinal neurons in the YFP-H mouse
To determine whether degenerative changes could be
observed in YFP-labelled pyramidal neurons in layer V of
the cortex following a thoracic dorsal column lesion, cell
atrophy and death were examined at several post-injury time
points. Layer V pyramidal neurons showed progressive
pathology following a T12 dorsal column lesion, with
evidence of cell atrophy apparent at 2 weeks post-injury and
the somata of many neurons appearing shrunken (Fig.
1C,D), compared to the large healthy pyramidal neurons
evident in uninjured mice (Fig. 1A,B,G). By four weeks
post-injury cell atrophy was severe, with a marked shrunken
appearance of cell soma (Fig. 1E,F,H). Quantification
revealed significant cell shrinkage in lesioned animals
compared to sham controls at 4 weeks after injury (Fig. 1I),
with a marked shift to the left in cumulative frequency
distribution (indicating smaller cell sizes) confirming
significant atrophy of layer V pyramidal neurons following
thoracic dorsal column injury (P < 0.001, KolmogorovSmirnov test; Fig. 1J). Cell counts confirmed that although
YFP-labelled CSNs underwent atrophy following thoracic
dorsal column lesion, there was no significant cell loss, with
mean cell numbers (within the region of interest) of 139
22, compared to 128 14 in sham controls and 4 week
injured mice respectively.

Analysis of cell size and survival


Cortical regions for analysis of CSN cell size were selected
according to the distribution of Fast Blue-labelled CSNs
retrogradely traced from the site of a T12 dorsal column
injury (data not shown). For analysis of cell size following
spinal cord injury, one section per animal corresponding to
Bregma 0.10 mm and Bregma 0.46 mm (Paxinos and
Franklin, 2001) were selected and a box (800 400 m)
was positioned over cortical layer V, 400 m from the
interhemispheric fissure. The profiles of all YFP-positive
layer V neurons within this area (which contained ~100 cells
per section) were then outlined using AxioVision analysis
package, by an investigator blinded to the treatment groups.
The diameter of each profile was recorded and a circular
area was then derived for each profile. Size/frequency
distributions were tabulated for each animal and a mean
distribution calculated for each treatment group. The total
YFP-positive cell count within the sampling area was also
recorded from one tissue section per animal and a mean cell
count determined for each group.

ChABC promotes neuroprotection of corticospinal


projection neurons following spinal cord injury in the
YFP-H mouse
To determine whether degenerative changes in YFP-labelled
pyramidal neurons could be reversed following degradation
of CSPGs, cell atrophy was examined at four weeks postinjury in mice subjected to a thoracic dorsal column lesion
and treated with penicillinase (a control enzyme) or
ChABC, delivered via i.c.v or i.t. infusion. At Bregma
0.10 mm cell atrophy was marked at 4 weeks post-injury
in animals treated with penicillinase (Fig. 2A,B,F). In
contrast, following i.c.v ChABC treatment, many more
neurons with large normal-looking somata were apparent
(Fig. 2C,D,G). A cell size distribution analysis revealed
significant cell shrinkage following injury and penicillinase
treatment compared to uninjured sham controls (Fig. 2H),
with a significant leftward shift in cumulative frequency
distribution confirming significant cell atrophy (Fig. 2I; P
< 0.001, lesion plus penicillinase compared to sham controls
Kolmogorov-Smirnov). Interestingly, i.c.v delivery of
ChABC following thoracic dorsal column injury led to a

Western blotting
Samples were homogenised in ice-cold lysis buffer and left
under agitation for 2hr on ice before centrifugation and
collection of the supernatant. The protein concentration of
lysates was estimated using a colorimetric protein assay and
15 g of total protein from each sample was electrophoresed
in loading buffer across a 10% acrylamide gel (2 hours at
120 mV). Proteins were transferred to a nitrocellulose
membrane using a semi-dry transfer method (45 min,
15 mV). Membranes were then blocked in skimmed milk
(10%, 1hr) prior to incubation with primary antibodies,
rabbit anti-phospho(thr202/tyr204)p44/42 MAPK (1:1000,
overnight; Cell Signalling) and mouse anti-beta-III tubulin
(1:1000, overnight; Promega, Maddison, WI). Membranes
were washed and then incubated with secondary antibodies
(Donkey anti- rabbit IRDye 800, goat anti-mouse IRDye
680, 1 hour; LiCor Biosciences) and visualised using
35

Fig. 2. ChABC treatment, via two distinct methods of


delivery promotes neuroprotection of corticospinal
projection neurons following spinal cord injury in the
YFP-H mouse. In coronal sections through the YFP-H sensorimotor
cortex at Bregma 0.10 mm (AG), representative low (A,C) and
high power (B,D) images show many more large healthy YFP-labelled
somata in the sensorimotor cortex following ChABC treatment (C,D)
as compared with penicllinase treated animals (A,B). Higher
magnification images highlight the shift in cell size from large healthy
cell somata in nave (uninjured) animals (E, arrows) to severely
atrophied cells in injured animals treated with penicillinase (F,
arrowheads). Following injury and ChABC treatment (G) atrophy is
apparent in only a small number of neurons (arrowheads), with the
majority retaining normal cell sizes (arrows). Histogram (H) and
cumulative frequency (I) plots confirm YFP-labelled CSNs in
penicllinase treated animals are significantly reduced in area compared
with sham operated animals (P < 0.05 Kolmogorov-Smirnov test).
ChABC treatment results in complete rescue of CSNs from injury
induced atrophy at Bregma 0.10 mm, with the cell size distribution
not significantly different from sham (P > 0.05, Kolmogorov-Smirnov
test). Intrathecal and intracerebroventricular treatment with ChABC
promote equivalent protection of lesioned CSNs (I). Cell size frequency
distribution histograms display mean frequency SEM, n = 4 per
treatment group. Scale bar = 500 m (A,C) and 250 m (B,D) and
20 m (EG).

Fig.1. Spinal cord injury induces progressive atrophy of


corticospinal neurons in the YFP-H mouse. Representative low
(A,C,E) and high power (B,D,F) images of coronal sections through
the YFP-H mouse sensorimotor cortex at Bregma 0.46 mm show
injury induced changes in YFP-labelled layer V pyramidal neurons. In
naive (uninjured) animals (A,B) many large healthy pyramidal
somata are apparent; arrows denote region of interest. Following
thoracic dorsal column lesion, progressive atrophy of these cells is
observed at two (C,D) and four (E,F) weeks post injury. Higher
magnification images (G,H) highlight the striking contrast between
large healthy somata in nave YFP-H mouse cortex (G, arrows) and
severely atrophied somata at 4 weeks following spinal cord injury (H,
arrow heads). Cell size distribution histogram (I) and cumulative
frequency (J) plots demonstrate that YFP-labelled pyramidal neurons
have undergone significant shrinkage at four weeks post lesion, with
a significant leftward shift in cell size distribution towards smaller cell
areas, compared to sham controls (Two sample Kolmogorov Smirnov
test P < 0.001). Scale bars = 500 m (A,C,E) and 250 m (B,D,F)
and 25 m (G,H).

distribution curve perfectly aligned with that of the i.c.v.


ChABC treatment group and the uninjured sham controls,
(Fig. 2I; lesion plus i.t ChABC significantly different from
lesion plus penicillinase, P < 0.001 and not significantly
different from lesion plus i.c.v. ChABC or sham, P = 0.9,
Kolmogorov-Smirnov).

complete reversal of cell atrophy, with the cumulative


frequency curve closely aligning with that of the uninjured
sham group (Fig. 2I; lesion plus i.c.v. ChABC significantly
different from lesion plus i.c.v. penicillinase, P < 0.001, and
not significantly different from sham, P = 0.76,
Kolmogorov-Smirnov).

Extensive digestion of spinal cord CSPGs by both i.c.v.


and i.t. ChABC delivery
Intracerebroventricular delivery of ChABC can degrade
CSPGs throughout the neuraxis (Garcia-Alias et al., 2008)
including CSPGs in the cerebral cortex close to corticospinal

Strikingly, the neuroprotective effects following i.t.


ChABC treatment were virtually identical to those observed
following i.c.v treatment, with the cell size distribution
revealing fewer small diameter cells and many more cells of
larger diameter (Fig. 2H) and the cumulative frequency
36

ChABC treatment is associated with increased MAPK


signalling at the spinal cord injury site

projection neurons in layer V (data not shown). In contrast,


following i.t. delivery, the limited rostral dispersal of ChABC
within CSF resulted in limited digestions of CSPGs in the
sensorimotor cortex. However, these two methods of
ChABC delivery resulted in similar levels of digestion in the
spinal cord, suggesting the effects of ChABC on promoting
neuroprotection of lesioned CSNs may be mediated at the
injury site in the spinal cord, rather than at the cell body.

We investigated whether ChABC treatment could promote


signalling changes in vivo by examining the expression and
activation state of several distinct signalling pathways at the
site of spinal cord injury four weeks post-injury (a time
point where neuroprotection and regenerative sprouting was
observed) using Western blot analysis (Fig. 4). Extracellular
signal regulated kinases 1 and 2 (ERK1/2, p44/42 MAPK)
play an important role in cell survival, growth and
differentiation in response to trophic factors and are
activated upon threonine and tyrosine phosphorylation by
a single upstream protein kinase, ERK kinase (MEK).
ChABC treatment via both i.c.v. and i.t. delivery promoted
a significant increase in the phosphorylation of ERK1 (p44
MAPK) at the lesion site, as compared with penicillinasetreated controls (Fig. 4A,C; normalised band intensity
SEM, penicillinase 0.80 0.10; i.c.v., ChABC 1.55 0.13,
P = 0.004; i.t. ChABC 1.30 0.12, P = 0.04; One way
ANOVA with block design, Tukey post-hoc analysis). The
total expression of ERK1 in ChABC-treated animals was
not significantly different to controls at four weeks post
injury (Fig. 4B; penicillinase 4.42 1.17; i.c.v. ChABC
4.58 0.81; i.t. ChABC 4.97 1.12, P > 0.05) and no
significant differences in the expression or phosphorylation
state of ERK2 (p42 MAPK) were observed (Fig. 4A;
densitometry not shown).

ChABC treatment promotes growth of injured axons


in the spinal cord of the YFP-H mouse
Both descending and ascending axon projections were clearly
labelled in sagittal sections of uninjured YFP-H mouse spinal
cord (Fig. 3A). Four weeks after a dorsal column injury there
was a clear disruption of both the descending corticospinal
tract and the ascending projection in the dorsal columns, with
classic retraction bulbs apparent at the severed axon endings
and no evidence of regrowth into the injury site (Fig. 3B). In
the spinal cord of lesioned animals treated with penicillinase
there was no evidence of growth of YFP-labelled axons into
the lesion site (Fig. 3C,D), with the central lesion core
virtually devoid of YFP-labelled fibres. In contrast, following
i.t. (Fig. 3E,F) and i.c.v. treatment with ChABC, significantly
more fine fibres were observed growing into the lesion site,
within the central core of necrotic tissue which is known to
be highly inhibitory and refractory to regrowth. Thus, in vivo
ChABC treatment following spinal cord injury in the YFP-H
mouse leads to a robust sprouting response of injured axons
at the lesion site, which could have a retrograde effect on cell
body survival in the cortex.

Fig. 3. ChABC treatment promotes regeneration of YFP labelled fibres. Sagittal section of the nave (A) and injured (BF) YFP-H mouse
spinal cord shows that crush injury to the dorsal columns (B) disrupts the main descending CST projection (long arrows) and the ascending dorsal
column projection (arrowheads), leaving the ventral tracts intact (short arrows). At the injury epicentre low (C,E) and high (D,F) power images
show that the lesion epicentre is devoid of YFP-labelled fibres in penicillinase treated animals (C,D). In contrast, following i.t. ChABC treatment,
numerous small YFP-labelled fibres are apparent (E,F) indicating regenerative growth of lesioned YFP-labelled axons into the modified scar.
Scale bars = 500 m (A,B), 100 m (C,E) and 20 m (D,F).
37

Fig. 4. ChABC treatment promotes ERK1 signalling at the site of spinal cord injury. Four weeks after dorsal column injury, the expression
and activation state MAPK signalling pathways was evaluated at the site of injury. Representative Western blots (A) and densitometric
quantification (B,C) are shown for p44/42 MAPK. Treatment with ChABC using either i.c.v or i.t. delivery promoted a significant increase
in the phosphorylation of p44 MAPK (ERK1) after spinal cord injury as compared with penicillinase treated controls (C), but did not alter the
total expression level of the kinase (B). Densitometric values for target proteins or phosphoproteins were normalised against a -III- tubulin
loading control for each sample. Bar graphs represent mean normalised densitometric reading SEM of 4 animals per group. * P < 0.05, ** P
< 0.01 One way ANOVA with block design using Tukey post-hoc analysis.

CONCLUSION
In the present study, we have used the YFP-H transgenic
mouse as a novel tool to study degenerative changes and
repair following spinal cord injury. Using the intrinsic highintensity labelling of layer V corticospinal neurons provided
by this line we have shown that corticospinal neurons
undergo progressive atrophy following thoracic spinal cord
injury and provided evidence that degradation of CSPGs by
ChABC restores soma size to that of uninjured controls. We
also demonstrate that ChABC administered to the spinal cord
injury site has neuroprotective effects equivalent to i.c.v.
ChABC treatment, which also degrades CSPGs around the
cell bodies. At the spinal cord injury site we report that
ChABC treatment resulted in enhanced signalling via the
intracellular kinase ERK1 and rendered the lesion epicentre
more permissive to the in-growth of lesioned fibres. Thus,
this work demonstrates that modification of the extracellular
matrix at the spinal cord injury site promotes a protective
response in injured projection neurons and represents a novel
mechanism underlying ChABC-mediated repair.

injury of the rat spinal cord. J. Neurotrauma 22:226239.


Davies S.J., Goucher D.R., Doller C., Silver J. (1999)
Robust regeneration of adult sensory axons in degenerating
white matter of the adult rat spinal cord. J. Neurosci.
19:58105822.
Dou C.L., Levine J.M. (1994) Inhibition of neurite growth
by the NG2 chondroitin sulfate proteoglycan. J. Neurosci.
14:76167628.
Feng G., Mellor R.H., Bernstein M., Keller-Peck C.,
Nguyen Q.T., Wallace M., Nerbonne J.M., Lichtman J.W.,
Sanes J.R. (2000) Imaging neuronal subsets in transgenic
mice expressing multiple spectral variants of GFP. Neuron
28:4151.
Fouad K., Schnell L., Bunge M.B., Schwab M.E., Liebscher
T., Pearse D.D. (2005) Combining Schwann cell bridges
and olfactory-ensheathing glia grafts with chondroitinase
promotes locomotor recovery after complete transection of
the spinal cord. J. Neurosci. 25:11691178.
Galtrey C.M., Fawcett J.W. (2007) The role of chondroitin
sulfate proteoglycans in regeneration and plasticity in the
central nervous system. Brain Res. Rev. 54:118.
Garcia-Alias G., Lin R., Akrimi S.F., Story D., Bradbury
E.J., Fawcett J.W. (2008) Therapeutic time window for the
application of chondroitinase ABC after spinal cord injury.
Exp. Neurol. 210:331338.
Grimpe B., Pressman Y., Lupa M.D., Horn K.P., Bunge
M.B., Silver J. (2005) The role of proteoglycans in Schwann
cell/astrocyte interactions and in regeneration failure at
PNS/CNS interfaces. Mol. Cell Neurosci. 28:1829.
Houle J.D., Tom V.J, Mayes D., Wagoner G., Phillips N.,
Silver J. (2006) Combining an autologous peripheral
nervous system bridge and matrix modification by
chondroitinase allows robust, functional regeneration
beyond a hemisection lesion of the adult rat spinal cord. J.
Neurosci. 26:74057415.
Massey J.M., Amps J., Viapiano M.S., Matthews R.T.,
Wagoner M.R., Whitaker C.M., Alilain W., Yonkof A.L.,
Khalyfa A., Cooper N.G., Silver J., Onifer S.M. (2008)
Increased chondroitin sulfate proteoglycan expression in
denervated brainstem targets following spinal cord injury
creates a barrier to axonal regeneration overcome by
chondroitinase ABC and neurotrophin-3. Exp. Neurol.
209:426445.

REFERENCES
Barritt A.W., Davies M., Marchand F., Hartley R., Grist J.,
Yip P., McMahon S.B., Bradbury E.J. (2006) Chondroitinase
ABC promotes sprouting of intact and injured spinal systems
after spinal cord injury. J. Neurosci. 26:1085610867.
Bradbury E.J., Moon L.D., Popat R.J., King V.R., Bennett
G.S., Patel P.N., Fawcett J.W., McMahon S.B. (2002)
Chondroitinase ABC promotes functional recovery after
spinal cord injury. Nature 416:636640.
Busch S.A., Silver J. (2007) The role of extracellular matrix
in CNS regeneration. Curr. Opin. Neurobiol. 17:120127.
Cafferty W.B., Bradbury E.J., Lidierth M., Jones M., Duffy
P.J., Pezet S., McMahon S.B. (2008) Chondroitinase ABCmediated plasticity of spinal sensory function. J. Neurosci.
28:1199812009.
Cafferty W.B., Yang S.H., Duffy P.J., Li S., Strittmatter
S.M. (2007) Functional axonal regeneration through
astrocytic scar genetically modified to digest chondroitin
sulfate proteoglycans. J. Neurosci. 27:21762185.
Caggiano A.O., Zimber M.P., Ganguly A., Blight A.R.,
Gruskin E.A. (2005) Chondroitinase ABCI improves
locomotion and bladder function following contusion
38

improves basic and skilled locomotion in spinal cord injured


cats. Exp. Neurol. 209:483496.
Zuo J., Neubauer D., Dyess K., Ferguson T.A., Muir D.
(1998) Degradation of chondroitin sulfate proteoglycan
enhances the neurite-promoting potential of spinal cord
tissue. Exp. Neurol. 154:654662.

Massey J.M., Hubscher C.H., Wagoner M.R., Decker J.A.,


Amps J., Silver J., Onifer S.M. (2006) Chondroitinase ABC
digestion of the perineuronal net promotes functional
collateral sprouting in the cuneate nucleus after cervical
spinal cord injury. J. Neurosci. 26:44064414.
McKeon R.J., Hoke A., Silver J. (1995) Injury-induced
proteoglycans inhibit the potential for laminin-mediated
axon growth on astrocytic scars. Exp. Neurol. 136:3243.
Moon L.D., Asher R.A., Rhodes K.E., Fawcett J.W. (2001)
Regeneration of CNS axons back to their target following
treatment of adult rat brain with chondroitinase ABC. Nat.
Neurosci. 4:465466.
Paxinos G., Franklin K.B.J. (2001) The Mouse Brain in
Stereotaxic Coordinates. San Diego, CA: Academic Press.
Smith-Thomas L.C., Fok-Seang J., Stevens J., Du J.S., Muir
E., Faissner A., Geller H.M., Rogers J.H., Fawcett J.W.
(1994) An inhibitor of neurite outgrowth produced by
astrocytes. J. Cell Sci. 107 ( Pt 6):16871695.
Snow D.M., Lemmon V., Carrino D.A., Caplan A.I., Silver
J. (1990) Sulfated proteoglycans in astroglial barriers inhibit
neurite outgrowth in vitro. Exp. Neurol. 109:111130.
Tester N.J., Howland D.R. (2008) Chondroitinase ABC

FUTURE PLANS
Studies currently underway and planned for the coming
year will continue to use the YFP-H transgenic mouse and
focus on anatomical and molecular outcomes following
spinal cord injury and Chondroitinase ABC treatment.
Following on from work described here, it is hoped that
primary neuronal cultures established from adult YFP-H
mice (see previous progress report) may be used to assess the
effects of CSPGs and scar-associated factors on the
behaviour of corticospinal neurons in vitro.
Further to this, the response of other spinal cord
systems, suich as ascending sensory projections and regions
de-innervated following spinal injury will be assessed in vivo
using the intrinsic neuronal labeling provided by the mouse.

39

Overcoming the molecular inhibitors that impede axonal


regeneration following spinal cord injury
Philip Duffy*, Isabella Gavazzi, William Cafferty and John Wood
Kings College London, UK. isabella.gavazzi@kcl.ac.uk
*PhD Student
INTRODUCTION

All surgical procedures and post-operative care was


performed in accordance with the guidelines of the Yale
University Animal Care Policy.

Functional recovery from neurological trauma and


pathologies, such as spinal cord injury, multiple sclerosis,
stroke, and traumatic brain injury is limited by the failure
of CNS axons to grow through an inhibitory environment.

Behavioural Testing: RockII/, rockII+/ and rockII+/+


mice that underwent sham lesion or dorsal rhizotomy were
assessed using the spontaneous exploration and thermal
withdrawal tests. Baselines for spontaneous exploration were
obtained pre-injury to assess preferred fore limb use during
normal exploratory activity. No pre-training was required
in this test, but the recording of baselines doubled as
habituation for the animals. Mice were placed in a clear
Perspex cylinder (300 mm in height, 80 mm in diameter)
for the duration of 15 rears, or 3 minutes, whichever was
sooner. A mirror was placed at an angle behind the cylinder
so that the fore paws were visible at all times. The testing
was recorded (Canon digital video camera recorder, Elura
2MC) and scored at a later date. Three baselines on three
consecutive days were obtained prior to lesion. The
following behaviors were scored: (1) Paw used to push off
the table when rearing; (2) paw used for weight support
during a rear, scored an either independent use of the right,
left or both paws for weight support on the wall of the
cylinder during a rear, or to regain balance while moving
laterally in a vertical position along the wall of the cylinder;
and (3) paw used for weight support when landing
following a rear. Animals were tested post injury on days 1,
2, 3, 5, 7, 14, 21 and 28.

Molecular analysis of the inhibitors present in the


injured CNS have focused on two classes of proteins: the
myelin-associated inhibitors Nogo-A, myelin-associated
glycoprotein (MAG), oligodendrocyte myelin glycoprotein,
and ephrin-B3; and members of the chondroitin sulphate
proteoglycan (CSPG) present in areas of reactive gliosis such
as at the glial scar which forms at the site of CNS lesion
following damage. We have designed experiments to target
these inhibitors either genetically to enhance the growth of
axons into the spinal cord after rhizotomy.
Dorsal root lesion (rhizotomy) damages sensory neurons
as they enter the spinal cord. After retraction and
degeneration these axons are prevented from re-entering the
spinal cord due to the formation of a glial scar at the dorsal
root entry zone (DREZ). The damaged DREZ is rich in
glial cells that express axon-growth inhibitors. We assessed
the impact of negating the inhibitory effects of Rho kinase
ROCK II signaling on the regeneration of sensory neurons
after rhizotomy. Mice null mutant for the signaling kinase
ROCKII (Rho-associated Kinase II), illustrated significant
restitution of sensory function and regeneration of damaged
sensory neurons after cervical rhizotomy.

The thermal withdrawal test is a measure of nociceptive


sensory function and can be used to measure functional
recovery of nociceptive neurons. The forepaws of each
animal were dipped into a 52C water bath, and the time
taken for them to withdraw their paws was recorded.
Animals were tested three times before surgery and on days
1, 2, 3, 5, 7, 14, 21 and 28 post injury.

METHODS
Mice rhizotomy: Adult (46 months) female rockII/ (n=9),
rockII+/ (n=9) or rockII+/+ (n=9) littermates were deeply
anesthetized with intraperitoneal ketamine (100 mg/kg) and
xylazine (15 mg/kg). A hemi-laminectomy was performed
to expose the lateral portion of spinal cord corresponding
to the C4-T1 spinal levels. The dura matter overlying dorsal
roots C5-C8 was pierced just caudal to each individual root,
fine (Dumont #5) forceps were introduced subdurally
between the DREZ and DRG, and the left dorsal roots were
crushed by squeezing the forceps closed for 5 seconds.
Translucence of injured root demonstrated complete
interruption at the time of injury, and histological studies in
control groups confirmed complete injury at follow-up.
Sham animals (n=6) received hemi-laminectomy without
root crush. Muscle and skin were sutured with 4.0 vicryl.

Tissue Processing: Sections of rockII/, rockII+/ and


rockII+/+ spinal cord were processed to detect reactive
astrocytes (Glial Fibrillary Acidic Protein, GFAP 1:10,000
Dako, UK), regenerating axons (small proline-rich protein
1A, SPRR1A 1:2,500, Strittmatter Laboratory), nociceptive
neurons (calcitonin gene-related peptide, CGRP 1:1,000,
Sigma), and cholera toxin 1:2,500, List Biological
Laboratories, Campbell, CA, US).
Assessment of axon regeneration: The average number of
CTB, N52, CGRP and SPRR1A-expressing regenerating
axons was counted in 5 randomly selected sections containing
the DREZ from each animal. The number of regenerating
axons was counted. The number of axons crossing a line
perpendicular to the entering dorsal root was reported

Axonal tracing: Three days before perfusion, animals


received a 1 l microinjection of a 1% solution of cholera
toxin -subunit (CTB) (List Biological Laboratories,
Campbell, CA) in their left median nerve.

40

Following rhizotomy of rockII/, rockII+/ and rockII+/+


mice, regenerating CGRP-positive fibres were seen to cross
the injured DREZ (50% GFAP) in significantly greater
numbers in rockII/ animals than in rockII+/ and rockII+/+
groups (Figure 2B,C,E,F,G) (9.71.8 for rockII/ versus
7.61.4 for rockII+/ and 7.52.1 for rockII+/+ p<0.05,
ANOVA). This trend was also present in regions past the
DREZ, with significantly more CGRP-positive axons
present in the CNS (100% GFAP) in rockII/ mice than
in rockII+/ and rockII+/+ mice (5.81.5 for rockII/ versus
1.21.4 for rockII+/ and 1.11.5 for rockII+/+ , p<0.01
ANOVA Figure 4.3.9 G).

as a function of location at the PNS (0% GFAP


immunoreactivity) or CNS (100% GFAP immunoreactivity)
boundary at the DREZ. The number of axons present at the
DREZ were also counted, and assigned the term 50% GFAP.
RESULTS
RockII/ mice exhibit sensory axon regeneration following
rhizotomy: CTB-labelled large diameter myelinated fibres
were observed crossing the DREZ in significantly greater
numbers in rockII/ mice than in rockII+/+ and rockII+/
mice (Figure 1B,C,E,F,G). Quantification of axon numbers
between groups revealed more axons reached the DREZ
(50% GFAP) in the rockII/ group than in rockII+/ and
rockII+/+ groups (Figure 1G) (9.91.78 versus 5.61.6 and
5.71.5, p<0.01 ANOVA). More CTB-labelled axons also
passed through the damaged DREZ and into the CNS
(100% GFAP) in rockII/ group (5.42.34 for rockII/
versus 0.850.69 for rockII+/ and 0.752.3 for rockII+/+
p<0.01, ANOVA, Figure 1G).

SPRR1A-positive axons were significantly more


numerous in the rockII/ group at the injured DREZ (50%
GFAP Figure 3E,F,G. 15.51.9 for rockII/ versus
13.22.3 for rockII+/ and 11.53.1 for rockII+/+, p<0.05,
one-way ANOVA). In areas of the spinal cord past the
lesioned DREZ in the CNS (100% GFAP), there was a

Fig. 1. ROCK II gene deletion enhances mechanosensory neuron regeneration after dorsal rhizotomy. (AF) Photomicrographs
illustrate high power transverse sections of cervical spinal cord from rockII+/+ (A,B,C) and rockII/ mice (D,E,F) that had undergone unilateral
rhizotomy at C5-C8 28 days previously. CTB-immunoreactive axons can be seen in the dorsal root in rockII+/+ mice (B,C), but few axons
penetrate the DREZ and enter the spinal cord (C). In contrast, significant numbers of CTB-immunoreactive axons can be seen growing up to
and past the DREZ and entering the spinal cord in rockII/ mice (E,F). Scale bar 100 m. G, Quantification of the number of CTBimmunoreactive axons at the injured root. X-axis demarcates PNS from CNS by GFAP antibody labelling; 0% GFAP-immunoreactivity refers
to the PNS, 50% refers to the DREZ and 100% refers to the CNS. RockII-/- animals (white bar) have significantly more axons entering the
spinal cord compared with rockII+/+ (black bar) mice and rockII+/ animals (gray) (data represent meanSEM p<0.01, 1 WAY ANOVA).
41

rockII+/, and 827% for rockII+/+). Use of a single fore limb


for weight support during rearing occurred less frequently
(Figures 4A and B, for the left fore limb 94% for rockII/,
83% for rockII+/ and 93% for rockII+/+ and for the right
fore limb; 117% for rockII/, 75% for rockII+/ and
97% for rockII+/+ mice).

significantly greater number of SPRR1A-expressing axons


in rockII/ animals than in rockII+/ and rockII+/+
(11.53.04 for rockII/ versus 3.11.8 for rockII+/ and
2.51.4 for rockII+/+, p<0.01, 1-WAY ANOVA, Figure 3G).
RockII/ mice recover more rapidly after rhizotomy than
rockII+/ and rockII+/+ mice in the spontaneous exploration test:
Mice that underwent sham operation or dorsal rhizotomy
were assessed using the spontaneous exploration test.
During baselines, mice predominantly used both fore limbs
together for weight support during exploration of the
cylinder (Figure 4C, 8012% for rockII/, 858% for

Following rhizotomy, there was a marked change in the


relative use of the injured fore limb for weight support (Figure
4B). The percentage of rears using the injured left fore limb
for support decreased significantly in comparison baseline
(31% for rockII/, 41% for rockII+/, 31.5% for rockII+/+,

Fig. 2. Deletion of the ROCK II gene promotes nociceptive axon regeneration after dorsal rhizotomy. (AF) Photomicrographs
illustrate high power transverse sections of ipsilateral cervical spinal cord from rockII+/+ (AC) and rockII/ (DF) 28 days after dorsal rhizotomy.
GFAP-immunoreactive astrocytes bulge into the PNS in both rockII+/+ (A,C) and rockII/ (D,F) mice. Regenerating CGRP-immunoreactive
axons abruptly stop growing when they reach the damaged DREZ in rockII+/+ mice (B,C). CGRP-immunoreactive axons traverse the damaged
DREZ in rockII/ mice (E,F) and re-enter the spinal cord. G, Quantification of the number of CGRP-immunoreactive axons at the injured
root. CGRP-positive axons traverse the damaged DREZ in significantly greater numbers in rockII/ mice (white bar) than in rockII+/+ (black
bar) and rockII+/ mice (gray bar). 0% GFAP-immunoreactivity refers to the PNS, 50% refers to the DREZ and 100% refers to the CNS. (data
represent meanSEM p<0.01 1 WAY ANOVA).
42

Fig. 3. ROCK II gene deletion promotes regeneration of sensory neurons after dorsal rhizotomy. (AF) Photomicrographs illustrate
high power transverse sections of cervical spinal cord from adult rockII+/+ (A,B,C) and rockII/ (D,E,F) mice which underwent rhizotomy from
C5-C8 28 days previously. SPRR1A axons can be seen in the dorsal root of rockII+/+ (B) mice, but few axons penetrate the DREZ (E) and enter
the spinal cord. In contrast, significant numbers of SPRR1A axons can be seen growing up to and past the DREZ and entering the spinal cord
(E,F) in rockII/ mice. Scale bar, 100 m. G, Quantification of the number of SPRR1A immunoreactive axons at the injured DREZ reveals
significantly more SPRR1A-expressing axons are present crossing the injured DREZ in rockII/ mice (white bar) than in rockII+/+ (black bar)
and rockII+/ animals. X-axis demarcates PNS from CNS by GFAP staining, 0% GFAP-immunoreactivity refers to the PNS, 50% GFAPimmunoreactivity refers to the DREZ, 100% GFAP-immunoreactivity refers to the CNS. Data represent meanSEM p<0.01 1 WAY ANOVA.

in comparison to baselines of 94% for rockII/, 83% for


rockII+/ and 93% for rockII+/+, p<0.01, 1 WAY ANOVA).

throughout the testing period. The percentage of rears using


both fore limbs together for support significantly decreased
compared to baseline (Figure 4C, 643% for rockII/,
6314% for rockII+/ and 6514% for rockII+/+ p<0.05,
one-way ANOVA). This pattern was maintained
throughout the testing period of up to 4 weeks. Analysis
revealed significant differences compared to baselines at
every time point post lesion for use of uninjured right fore
limbs (p<0.01, 1 Way ANOVA, Figure 4.3.11 A).

The percentage use of the uninjured right fore limb


significantly increased after injury in comparison to baseline
(Figure 4A, 339% for rockII/, 3314% for rockII+/,
3211% for rockII+/+ in comparison to baselines of 117%
for rockII/, 75% for rockII+/, and 97% for rockII+/+,
p<0.05, 1 WAY ANOVA). This difference was maintained
43

Fig. 4. Analysis of rockII-/- rockII+/- and rockII+/+ paw preference during spontaneous exploration following rhizotomy. (A)
Percentage use of the uninjured fore limb for weight support during spontaneous exploration increased in rockII+/+ mice (black bars), rockII+/
(gray bars) and rockII/ (white bars) mice immediately after lesion. This trend was evident until 21 days post injury. RockII/ (white) mice
exhibited a significant decrease in percentage use of the uninjured paw by day 5 post lesion (DataSEM, 1 WAY ANOVA, p<0.01). (B)
Percentage use of the injured fore limb for weight support during spontaneous exploration following rhizotomy. RockII+/+ (black bars), rockII+/
(gray bars) and rockII/ (white bars) mice exhibited significantly decreased use of the injured fore limb during a rear immediately after the injury
(asterisk). RockII/ mice had significantly increased use of the injured paw by Day 5 post lesion (DataSEM, 1 WAY ANOVA, p<0.01). This
trend was also evident at 7 days post lesion (DataSEM, 1 WAY ANOVA, p<0.01). RockII+/ and rockII+/+ groups were not significantly
difference after these time points to each other or baseline scores. (C) RockII+/+ (black bars), rockII+/ (gray bars) and rockII/ (white bars) mice
showed significantly decreased use of both paws for weight support during spontaneous exploration following injury (DataSEM, 1 WAY ANOVA,
p<0.05). This decrease in use of both paws was present at each time point tested.
44

Use of the injured fore limb was significantly different


to baseline until day 5 post lesion, where the rockII/ injured
fore limb use was not significantly different from baseline
(74% in comparison to baseline of 97% Figure 4B).
RockII+/+ and rockII+/ groups returned to levels not
significantly different to baseline by 14 days post lesion
(rockII+/+ day 14, 83%, in comparison to 93% at baseline.
RockII+/ day 14, 74%, in comparison to 83% at baseline,
p>0.05, Figure 4B).

after injury (asterisk). Thermal sensation began to return by


the second day. However, by the 5th day post surgery,
rockII/, rockII+/ and rockII+/+ groups displayed decreased
withdrawal times, with withdrawal times significantly lower
to baseline and sham animals (rockII/; 6.144.6, rockII+/;
6.54.6, rockII+/+; 4.31.3 in comparison to sham;
14.30.9 p<0.05, ANOVA, double asterisk). This trend
continued until end of testing period 28 days post injury
(Figure 5).

RockII/ animals displayed a significant reduction in


compensatory use of the uninjured fore limb in comparison
to rockII+/ and rockII+/+ by the 5th day post injury (334%
for rockII/, 425% for rockII+/ and 405% for rockII+/+,
and p<0.01, ANOVA, Figure 4A). This trend was also
present at the following testing period at 7 days post injury
(29%5 for rockII/, 43%6 for rockII+/ and 42%8 for
rockII+/+, p<0.01 ANOVA, Figure 4A), after which time
rockII+/ and rockII+/+ groups had recovered to an extent not
significantly different to rockII/ animals (Figure 4A).

CONCLUSION
Dorsal rhizotomy results in primary afferent degeneration
and retraction from the crush site (Sengelaub et al., 1997;
Belyantseva and Lewin, 1999). Regeneration following
rhizotomy of sensory axons has previously been shown after
administration of neurotrophic factors (Ramer et al., 2000;
Ramer et al., 2002). The effects of these growth factors are
diminished by the presence of an inhibitory myelin-rich
environment (Mukhopadhyay et al., 1994). Myelin
inhibitors including Nogo, MAG and OMgp prevent axon
growth through signalling through nogo receptor 1
(Fournier et al., 2001; Liu et al., 2002a; Wang et al., 2002b),
nogo receptor 2 (Venkatesh et al., 2005), or in a complex
with the p75 neurotrophin receptor (Wang et al., 2002a) or
in a signalling complex with the tumour necrosis factor
TROY (Park et al., 2005; Shao et al., 2005). This leads to
rockII and RhoA activation further downstream. RhoA
activation leads to actin depolymerisation and growth cone
collapse (Jalink et al., 1994; Gallo and Letourneau, 2004).
For this reason we assessed the rockII kinase signalling on
sensory axon regeneration following rhizotomy. RockII/
mice underwent rhizotomy lesions and anatomical and
functional recovery was analysed.

There were no significant differences between baselines


and post injury fore limb usage at any time point in the
other two parameters of the spontaneous exploration test
analysed, those of use of landing paw and push paw from
the surface (p>0.05, 1 WAY ANOVA, data not shown).
RockII/ animals do not exhibit an increased rate of
recovery in thermal withdrawal thresholds after rhizotomy:
RockII/ animals did not display enhanced recovery in
thermal withdrawal thresholds following rhizotomy
(DataSEM, 1 WAY ANOVA). Retraction of the injured
fore limb from a water bath set to 52C was absent 24 hours

Fig. 5. RockII-/- mice do not recover thermal sensation more rapidly than rockII+/- and rockII+/+ mice following rhizotomy.
Immediately after the lesion until 3 days post lesion, withdrawal latency was significantly elevated in rockII+/+ (black line), rockII+/ (gray line)
and rockII/ (brown line) mice compared with sham animals (blue line, asterisk). Injured groups recovered thermal sensation by the 5th day
post lesion, and were decreased in comparison to baseline throughout the remainder of the testing period (Data represent meanSEM withdrawal
time p<0.05, 1 WAY ANOVA).
45

When placed in a cylinder, mice will spontaneously rear


to a standing position to explore using either one or both of
its fore paws (Schallert et al., 2000). Preferred paw usage for
weight bearing and exploration are thought to be indicative
of functionality of the fore limb (Soblosky et al., 1996). It
has previously been shown that uninjured, freely behaving
rats use each limb separately for weight support
approximately 25% of the time, with the animal using both
limbs together simultaneously the remaining 50% of the
time (Liu et al., 1999). This is in contrast with a more recent
study (Starkey et al., 2005) where uninjured mice were
observed to use both fore paws for weight support the
majority of the time (~80%), with the remainder divided
up equally between use of each fore paw individually. This
test does not appear to be affected by repeated testing
(Schallert et al. 2000) and is conserved across strains (Webb
et al., 2004). This test can be used to compare paw usage
preference and recovery of normal limb function after a
sensory lesion such as the rhizotomy. RockII/ animals
recovered use of the injured fore limb more rapidly in the
spontaneous exploration test than rockII+/ and rockII+/+
littermates following quadruple cervical root rhizotomy
(Figure 4B). RockII/ animals show a trend towards
recovery of the injured forepaw in the spontaneous
exploration test starting 2 days post lesion, becoming
significant by 5 days. The spontaneous exploration test has
been used extensively in the past as a measurement of
forelimb asymmetries after injury (Schallert et al., 2000;
Soblosky et al., 2001; Baskin et al., 2003; Starkey et al.,
2005; Wells et al., 2005). Other studies have reported that
forepaw use in rats returned to control levels within
24 weeks of lesion (Napieralski et al., 1998; Liu et al.,
1999). However, in mice with a traumatic brain injury, the
asymmetry of fore limb use did not recover to baseline levels
for 5 months post injury (Baskin et al., 2003). The results
for use of the injured for limb following rhizotomy in the
rockII experiment show a similar trend to those seen by
Baskin and colleagues.

display regeneration of nociceptive axons, identified by


CGRP immunohistochemistry (Figure 2). Rhizotomy is
known to induce terminal sprouting of spared afferents
(Sengelaub et al., 1997; Belyantseva and Lewin, 1999;
Darian-Smith, 2004) and of serotinergic neurons (Wang et
al., 1991b, a). It is possible that deletion of the ROCK II
gene may have led to enhanced intraspinal reorganisation
of intact primary terminals from adjacent spared dorsal
roots which led to the early restoration of sensory function.
It is also possible that there was a learning effect associated
with the test, and animals learned to withdraw the fore limb
from the water before the point was reached where it
became uncomfortable.
CTB- and CGRP-immunoreactive fibre regeneration was
observed through the damaged DREZ in rockII/ mice
(Figure 1 and Figure 2). CTB-immunoreactive fibres are
responsible for mechanoceptive and proprioceptive function.
Their regeneration is correlated with recovery in the
spontaneous exploration test in the rockII/ group, implying
that these axons made functional synapses with their contacts
within the spinal cord. CGRP is widely distributed
throughout the CNS and is believed to have a function in
nociception and modulation of the autonomic and
endrocrine systems (Zaidi et al., 1990). In the CNS, CGRP
expression is present in lamina I and lamina II outer where
primary afferent fibres terminate. Laminae V and X also
contain abundant CGRP immuno-reactive fibres (Rosenfeld
et al., 1983). In the dorsal root ganglia, CGRP
immunoreactivity is observed in most of the small and some
of the intermediate sized cells (Gibson et al., 1984). Following
rhizotomy, CGRP levels in the CNS are greatly reduced
(Chung et al., 1988), and CGRP-expressing axons fail to
traverse the damaged DREZ in wild type mice (Ramer et al.,
2000; Cafferty et al., 2007). CGRP expressing fibres were
detected crossing the injured DREZ (Figure 2) in rockII/
mice. Lack of rockII expression likely attenuates or prevents
RhoA-mediated growth cone collapse of CTB- and CGRPexpressing regenerating neurons at the injured DREZ.

In order to assess the contribution of ROCK IIdependent axon regeneration to mouse performance, the
thermal withdrawal test (Ramer et al., 2004a; Cafferty et
al., 2007) was performed. This test is a measure of the
nociceptive sensory capacity of mice after sensory injury.
Mice had their injured fore paw immersed in water set to 52
degrees celsius. Both rockII/ and rockII+/+ animals
exhibited an initial significant increase in withdrawal
latencies to thermal stimulus, followed by significant
reduction to levels below that of pre-surgical baselines by
the 5th day post injury (Figure 5). The decrease in
withdrawal times could be the result of local plastic changes
by spared afferents which has been linked in the past to the
development of neuropathic pain (Bruce et al., 2002).
Intraspinal reorganisation (Darian-Smith, 2004) and the
emergence of thermal hyperalgesia (Abad et al., 1989;
Ramer et al., 2004a) are known to occur after dorsal root
injury, and these factors may have been the origin for the
recovery in nociceptive function observed in wild type mice
in our behavioral tests. The increase in withdrawal latencies
was not mediated by regeneration since this phenomenon
was also observed in rockII+/+ animals, which did not

The small proline-rich repeat protein 1A (SPRR1A) is a


regeneration-associated gene (RAG) not expressed in
uninjured mouse neurons during development or adulthood,
but is induced over 60-fold in adult mice after peripheral
nerve damage (Bonilla et al., 2002). The level of SPRR1A
induction due to axotomy is extremely high during successful
regeneration and its overexpression in vitro produces large
increases in neurite extension rates (Bonilla et al., 2002).
SPRR1A expression shifts adult sensory neurons from a
branching to an elongating mode of growth, and its
suppression decreases the outgrowth potential of
preconditioned adult neurons (Bonilla et al., 2002). DRGs
and spinal motor neurons both express SPRR1A after
peripheral axonal injury. In contrast, centrally axotomised
DRG neurons have a minor increase in SPRR1A expression
and have a modest capability for axonal regeneration (Bonilla
et al., 2002). Following rhizotomy in the rockII/ mouse,
SPRR1A expression is induced rhizotomy, and possibly due
to mechanical damage of tracing the median nerve. Traction
on the ventral root during surgery may also potentially have
initiated expression on motor neurons. These mechanical
46

interventions led to SPRR1A gene induction within intact


motor neurons of the same segmental level and DRG neurons
of adjacent spinal levels of the lesion site. However, SPRR1A
immunoreactivity present in the deeper laminae of the spinal
cord is easily distinguished from the SPRR1A-positive fibres
visible in the injured dorsal root. Assessment of animals
4 weeks after injury revealed that regenerated SPRR1Aexpressing axons were present past the damaged DREZ and
in the CNS spinal cord in the ROCK II knockout group
(Figure 3). In rockII+/+ mice, no SPRR1A-positive fibres
crossed the DREZ into the CNS (Figure 3A, G). Myelinderived inhibitors NogoA, MAG and OMgp hinder the
growth of axons through interaction with nogo receptor 1
(Fournier et al., 2001; Liu et al., 2002a), which in turn act
with the p75 receptor (Wang et al., 2002a) to activate RhoA
(Yamashita et al., 2002). This RhoA activation leads to actin
depolymerisation and growth cone collapse (Jalink et al.,
1994; Gallo and Letourneau, 2004). RhoA is highly activated
at the lesion site following SCI (Dubreuil et al., 2003), and
antagonism of RhoA or its downstream effector, ROCK
enhances both regeneration and recovery after SCI (Dergham
et al., 2002; Fournier et al., 2003). Inhibition of ROCK
signalling has been observed previously to attenuate cold
hyperalgesia and enhance plasticity (Ramer et al., 2004), but
the experiment in this thesis is the first to describe functional
regeneration across the DREZ following rhizotomy in
a rockII/.

astrocytic scar genetically modified to digest chondroitin


sulfate proteoglycans. J. Neurosci. 27:21762185.
Chung K., Lee W.T., Carlton S.M. (1988) The effects of
dorsal rhizotomy and spinal cord isolation on calcitonin
gene-related peptide-labeled terminals in the rat lumbar
dorsal horn. Neurosci. Lett. 90:2732.
Darian-Smith C. (2004) Primary afferent terminal
sprouting after a cervical dorsal rootlet section in the
macaque monkey. J. Comp. Neurol. 470:134150.
Dergham P., Ellezam B., Essagian C., Avedissian H., Lubell
W.D., McKerracher L. (2002) Rho signaling pathway
targeted to promote spinal cord repair. J. Neurosci. 22:6570
6577.
Dubreuil C.I., Winton M.J., McKerracher L. (2003) Rho
activation patterns after spinal cord injury and the role of
activated Rho in apoptosis in the central nervous system. J.
Cell Biol. 162:233243.
Fournier A.E., GrandPre T., Strittmatter S.M. (2001)
Identification of a receptor mediating Nogo-66 inhibition
of axonal regeneration. Nature 409:341346.
Gallo G., Letourneau P.C. (2004) Regulation of growth cone
actin filaments by guidance cues. J. Neurobiol. 58:92102.
Gibson S.J., Polak J.M., Bloom S.R., Sabate I.M., Mulderry
P.M., Ghatei M.A., McGregor G.P., Morrison J.F., Kelly
J.S., Evans R.M., et al. (1984) Calcitonin gene-related
peptide immunoreactivity in the spinal cord of man and of
eight other species. J. Neurosci. 4:31013111.
Jalink K., van Corven E.J., Hengeveld T., Morii N.,
Narumiya S., Moolenaar W.H. (1994) Inhibition of
lysophosphatidate- and thrombin-induced neurite
retraction and neuronal cell rounding by ADP ribosylation
of the small GTP-binding protein Rho. J. Cell Biol.
126:801810.
Liu Y., Kim D., Himes B.T., Chow S.Y., Schallert T.,
Murray M., Tessler A., Fischer I. (1999) Transplants of
fibroblasts genetically modified to express BDNF promote
regeneration of adult rat rubrospinal axons and recovery of
forelimb function. J. Neurosci. 19:43704387.
Liu B.P., Fournier A., GrandPre T., Strittmatter S.M.
(2002a) Myelin-associated glycoprotein as a functional
ligand for the Nogo-66 receptor. Science 297:11901193.
Mukhopadhyay G., Doherty P., Walsh F.S., Crocker P.R.,
Filbin M.T. (1994) A novel role for myelin-associated
glycoprotein as an inhibitor of axonal regeneration. Neuron
13:757767.
Napieralski J.A., Banks R.J., Chesselet M.F. (1998) Motor
and somatosensory deficits following uni- and bilateral
lesions of the cortex induced by aspiration or
thermocoagulation in the adult rat. Exp. Neurol. 154:8088.
Park J.B., Yiu G., Kaneko S., Wang J., Chang J., He X.L.,
Garcia K.C., He Z. (2005) A TNF receptor family member,
TROY, is a coreceptor with Nogo receptor in mediating the
inhibitory activity of myelin inhibitors. Neuron 45:345351.
Ramer M.S., Priestley J.V., McMahon S.B. (2000)
Functional regeneration of sensory axons into the adult
spinal cord. Nature 403:312316.
Ramer M.S., Bishop T., Dockery P., Mobarak M.S.,
OLeary D., Fraher J.P., Priestley J.V., McMahon S.B.
(2002) Neurotrophin-3-mediated regeneration and recovery
of proprioception following dorsal rhizotomy. Mol. Cell
Neurosci. 19:239249.

RockII/ animals thus display a sensory neuron


regenerative phenotype in this lesion model. The next step
will be to assess the relative contribution of different
inhibitory molecules such as Nogo, MAG and OMgp on
neurite retraction and inhibition of outgrowth as a result of
signalling through the ROCKII pathway. This can be
achieved through in vitro culture experiments of rockII+/+
and rockII/ neurons with these inhibitory molecules as
substrates. In addition, possible synergistic outgrowth due
to ROCK II ablation and RhoA inhibition can be measured
with the administration to these cultures of the RhoA
inhibitor, Y27632.
REFERENCES
Abad F., Feria M., Boada J. (1989) Chronic amitriptyline
decreases autotomy following dorsal rhizotomy in rats.
Neurosci. Lett. 99:187190.
Baskin Y.K., Dietrich W.D., Green E.J. (2003) Two effective
behavioral tasks for evaluating sensorimotor dysfunction
following traumatic brain injury in mice. J. Neurosci.
Methods 129:8793.
Belyantseva I.A., Lewin G.R. (1999) Stability and plasticity
of primary afferent projections following nerve regeneration
and central degeneration. Eur. J. Neurosci. 11:457468.
Bonilla I.E., Tanabe K., Strittmatter S.M. (2002) Small
proline-rich repeat protein 1A is expressed by axotomized
neurons and promotes axonal outgrowth. J. Neurosci.
22:13031315.
Bruce J.C., Oatway M.A., Weaver L.C. (2002) Chronic
pain after clip-compression injury of the rat spinal cord.
Exp. Neurol. 178:3348
Cafferty W.B., Yang S.H., Duffy P.J., Li S., Strittmatter
S.M. (2007) Functional axonal regeneration through
47

Ramer L.M., Borisoff J.F., Ramer M.S. (2004a) Rho-kinase


inhibition enhances axonal plasticity and attenuates cold
hyperalgesia after dorsal rhizotomy. J. Neurosci. 24:10796
10805.
Rosenfeld M.G., Mermod J.J., Amara S.G., Swanson L.W.,
Sawchenko P.E., Rivier J., Vale W.W., Evans R.M. (1983)
Production of a novel neuropeptide encoded by the
calcitonin gene via tissue-specific RNA processing. Nature
304:129135.
Schallert T., Fleming S.M., Leasure J.L., Tillerson J.L.,
Bland S.T. (2000) CNS plasticity and assessment of
forelimb sensorimotor outcome in unilateral rat models of
stroke, cortical ablation, parkinsonism and spinal cord
injury. Neuropharmacology 39:777787.
Sengelaub D.R., Muja N., Mills A.C., Myers W.A.,
Churchill J.D., Garraghty P.E. (1997) Denervation-induced
sprouting of intact peripheral afferents into the cuneate
nucleus of adult rats. Brain Res. 769:256262.
Shao Z., Browning J.L., Lee X., Scott M.L., ShulgaMorskaya S., Allaire N., Thill G., Levesque M., Sah D.,
McCoy J.M., Murray B., Jung V., Pepinsky R.B., Mi S.
(2005) TAJ/TROY, an orphan TNF receptor family
member, binds Nogo-66 receptor 1 and regulates axonal
regeneration. Neuron 45:353359.
Soblosky J.S., Matthews M.A., Davidson J.F., Tabor S.L.,
Carey M.E. (1996a) Traumatic brain injury of the forelimb
and hindlimb sensorimotor areas in the rat: physiological,
histological and behavioral correlates. Behav. Brain Res.
79:7992.
Starkey M.L., Barritt A.W., Yip P.K., Davies M., Hamers F.P.,
McMahon S.B., Bradbury E.J. (2005) Assessing behavioural

function following a pyramidotomy lesion of the


corticospinal tract in adult mice. Exp. Neurol. 195:524539.
Venkatesh K., Chivatakarn O., Lee H., Joshi P.S., Kantor
D.B., Newman B.A., Mage R., Rader C., Giger R.J. (2005)
The Nogo-66 receptor homolog NgR2 is a sialic aciddependent receptor selective for myelin-associated
glycoprotein. J. Neurosci. 25:808822.
Wang S.D., Goldberger M.E., Murray M. (1991a) Plasticity
of spinal systems after unilateral lumbosacral dorsal
rhizotomy in the adult rat. J. Comp. Neurol. 304:555568.
Wang S.D., Goldberger M.E., Murray M. (1991b) Normal
development and the effects of early rhizotomy on spinal
systems in the rat. Brain Res. Dev. Brain Res. 64:5769.
Wang K.C., Kim J.A., Sivasankaran R., Segal R., He Z.
(2002a) P75 interacts with the Nogo receptor as a coreceptor for Nogo, MAG and OMgp. Nature 420:7478.
Wang K.C., Koprivica V., Kim J.A., Sivasankaran R., Guo
Y., Neve R.L., He Z. (2002b) Oligodendrocyte-myelin
glycoprotein is a Nogo receptor ligand that inhibits neurite
outgrowth. Nature 417:941944.
Wells J.E., Biernaskie J., Szymanska A., Larsen P.H., Yong
V.W., Corbett D. (2005) Matrix metalloproteinase (MMP)12 expression has a negative impact on sensorimotor
function following intracerebral haemorrhage in mice. Eur
J. Neurosci. 21:187196.
Yamashita T., Tohyama M. (2003) The p75 receptor acts as
a displacement factor that releases Rho from Rho-GDI. Nat.
Neurosci. 6:461467.
Zaidi M., Moonga B.S., Bevis P.J., Bascal Z.A., Breimer
L.H. (1990) The calcitonin gene peptides: biology and
clinical relevance. Crit. Rev Clin. Lab. Sci. 28:109174.

48

Novel anti-inflammatory and neuroprotective strategies in


spinal cord injury
Do omega-3 fatty acids modify inflammatory changes following a spinal cord compression injury?
Jodie C.E. Hall1*, J.V. Priestley1, V.H. Perry2 and A. Michael-Titus1
1Neuroscience Centre, ICMS, Barts and The London, Queen Mary University of London, London E1 2AT.
j.v.priestley@qmul.ac.uk
2Southampton Neuroscience Group, School of Biological Sciences and School of Medicine, University of Southampton,
Southampton SO16 7PX
*PhD Student
INTRODUCTION

in improved locomotion, decreased lipid and protein


oxidation and reduced levels of cyclooxygenase-2 (King et
al., 2006, Huang et al., 2007b).

Following spinal cord injury (SCI), there is an inflammatory


response which involves the innate immune system. The
cells responsible for the innate immune response are
principally the white blood cells (leukocytes) and microglia,
the resident macrophages of the CNS (Trivedi et al., 2006).
These cells release cytokines such as tumour necrosis factor
(TNF-) and interleukin-1 (IL-1), chemokines and
metalloproteases (MMPs), which aid in loosening the
extracellular matrix and recruiting additional immune cells
to the site of injury (Profyris et al., 2004). After SCI, the
inflammatory response in the spinal cord is considered to
drive further death and degeneration of nerve fibres above
and below the lesion site (Popovich et al., 1997; Dusart and
Schwab, 1994). This represents a window of opportunity
for pharmacological intervention with the use of antiinflammatory treatments.

The use of n-3 PUFAs as treatment after SCI will be


relatively easy to transfer to the clinic, due to their safety
and minimal toxicity, but it would be extremely beneficial
to have precise knowledge of the time course of entry of
various inflammatory cells into the injured spinal cord and
activation of resident spinal cord cells such as microglia, and
of their many actions, destructive or beneficial, that affect
neural tissue in order to develop second generation, more
selective treatments. Therefore, the first aim of this study is:
to characterize, using immunohistochemistry, inflammatory
markers in the spinal cord following compression
SCI. Next, to test the interaction of n-3 PUFAs such as
DHA on the time course, extent and outcome of the
inflammatory response.

Apart from the inflammatory response at the site of SCI,


there is also a significant systemic influence from increased
levels of inflammatory cytokines such as IL-1, IL-6 and
TNF- in the circulation, which are collectively termed
acute-phase proteins (APPs) and are secreted by hepatocytes
in the liver. The changes that are influenced by these APPs,
such as leukocyte mobilization, fever, and changes in serum
levels of glucocorticoids and cytokines, are part of the acutephase response (APR) (Parham, 2000; Campbell et al.,
2003). After SCI in the rat, the production of certain
chemokine APPs has been found to enhance the response to
injury, increasing leukocyte recruitment. Blocking this
response using chemokine antagonists has been shown to
lead to a corresponding decrease in the numbers of
leukocytes recruited to the injured spinal cord and a
reduction of damage to the blood brain barrier. Although
the hepatic chemokine response may not be specific to CNS
injury, it opens the possibility for a new target for
therapeutic intervention (Campbell et al., 2005).

METHODS
Surgery and injection of DHA
A compression was performed at vertebral level T12 as
described by Huang et al. 2006a on adult female Sprague
Dawley rats (220250 g). Briefly, a 50 g weight was placed
for 5 min on the exposed dura. In groups receiving treatment,
a bolus i.v. injection of DHA (250 nmol/kg), or saline
was administered 30 min after SCI. Animals were sacrificed
at several time points following injury using CO2
inhalation and perfused intracardially with saline followed
by 4% paraformaldehyde. Tissue was post fixed for 2 hours,
cryoprotected in 20% sucrose and 5 mm segments containing
the lesion site or equivalent level (nave/laminectomy groups)
were processed for immunohistochemistry.
Immunohistochemistry & quantification
15 m transverse spinal cord and 10 m liver cryostat sections
were labelled with the following primary antibodies: JT-1
(neutrophils, 1:1000, H. Perry), OX42 (microglia/
macrophages, 1:100, Serotec), Iba1 (microglia/macrophages,
1:500, Wako) ED1 (macrophages, 1:100, Serotec). Sections
were incubated overnight, washed and incubated with
secondary antibodies tagged with either fluorescein
isothiocyanate or tetra-rhodamine isothiocyanate (both
1:400), or amplified with ABC and revealed with diaminobenzidine (DAB). Neutrophils in spinal cord were
quantified by counting all labelled cells within the field of
view (x400) in the dorsal horns (DH), ventral horns (VH),

Omega 3 polyunsaturated fatty acids (n-3 PUFAs), such


as docosahexaenoic acid (DHA), are essential for the brain
and appear to be extremely effective treatments for several
conditions such as heart disease, cancer and depression.
Furthermore, they are used to treat CNS and inflammatory
conditions such as Alzheimers disease, multiple sclerosis and
rheumatoid arthritis. Work in our laboratory in the rat has
demonstrated that administering one bolus intravenous
injection of DHA within 3 hours of compression SCI results

49

dorsal columns (DC) and ventrolateral white matter


(VLWM). In the liver, three random areas were selected under
the 20 objective and labelled cells counted. ED1
immunoreactivity of macrophages/microglia were analysed
in the same areas using Q-Win software. A 1000 800 pixel
measuring frame was placed over the area of interest and a
binary image representing areas of ED1 immunoreactivity
was created. Areas covered by large tears or holes were
eliminated from the total area measured. Immunoreactivity
was expressed as the % of the area of the measuring frame
that contained signal (field detected area, FDA). All treatment
groups were kept blind until after the values were measured.
Regions from at least 3 sections per animal were counted or
measured and data expressed as means S.E.M. An outlier
was identified in the DHA group while quantifying
neutrophils in the 1 d spinal cord. Therefore, this animal was
excluded from the epicentre analysis.

Gaithersburg, Fig. 3a). Detection antibody (MSD,


Gaithersburg) and Read Buffer (MSD, Gaithersburg) were
added to the plate with PBS washes in between each step.
The plate was then read and the output signal given in units
of counts of light measured by a charge-coupled device
camera SECTORTM Imager SI2400 (MSD, Gaithersburg).
A standard curve was calculated for each cytokine and
sample signal levels converted to g/ml. All data were
analysed using Students t test.
RESULTS
Compression SCI induces distinct neuroinflammatory
reactions
Quantification of neutrophils using the JT1 antibody in the
epicentre after compression SCI revealed that there was an
influx of neutrophils to the dorsal horn and dorsal columns
within 4 hours, spreading to the ventral horns within 1 day,
the response reaching a peak at 1 day (Fig. 1a). Only 1 or 2
neutrophils were found 7, 14, 21 and 28 days after SCI and
in laminectomy control tissue. Using the ED1 antibody,
quantitative analysis of the spinal cord revealed only 1 or 2
labeled cells infiltrating the injured epicenter within the first
24 hours (Fig. 1b). By 7 days and up to 28 days many ED1
labeled macrophages were distributed throughout the grey
and white matter covering 712% of the measured fields.
Overall, comparatively fewer macrophages were seen in the
ventrolateral white matter. Microglia in uninjured
laminectomy spinal cord showed characteristic long, ramified

Cytokine and chemokine analysis


Nave, laminectomised or injured animals were given a
lethal overdose of pentobarbitone 4 hours after SCI, the
spinal cord tissue was quickly removed and frozen on dry ice
and kept at 80C. 5 mm epicentre samples were then
homogenised at 4C in HEPES buffer with protease
inhibitors. After centrifugation (12 000 g, 15 min, 4C) 25 l
of supernatant and standards were incubated with agitation
on a 7-plex cytokine array plate coated with antibodies
against IFN-, IL-1, IL-4, IL-5, IL8, IL-13, TNF (MSD,

Fig. 1. Quantification of inflammatory cells in the spinal cord epicentre following compression SCI. (a) JT1 neutrophils infiltrated
the dorsal horn and dorsal columns 4 hours after SCI, spreading to the ventral horns within 1 day. By 7 days they were largely absent from the
epicentre. (b) ED1 macrophages were not visible in the epicentre until 7 days after SCI. Their numbers remained elevated until 28 days. Results
are expressed as mean SEM of the number of animals indicated in brackets. (c) Microglia (white arrow, Iba1, green; OX42, red; co localisation
in yellow) in laminectomy spinal cord. (d) Microglia (Iba1, green; ED1, red) in the spinal cord epicentre 14 days and (e) 28 days after SCI.
White arrow points to cells co localised with Iba1 and ED1 (d), or Iba1 and OX42 (e).
50

processes (Fig. 1c). After SCI, microglia increased in size and


displayed heterogeneous phenotypes: some were Iba1+/ED1
or Iba1+/ED1+ (arrowhead, Fig. 1d); whereas others were
Iba1+/OX42+, Iba/OX42+ or Iba1+/OX42 (Fig. 1e).

recruitment, although further work needs to be done in order


to accurately quantify neutrophil infiltration.
REFERENCES
Campbell S.J., Hughes P.M., Iredale J.P., Wilcockson D.C.,
Waters S., Docagne F., Perry V.H., Anthony D.C. (2003)
CINC-1 is an acute-phase protein induced by focal brain
injury causing leukocyte mobilization and liver injury.
FASEB J. 17:11681170.
Campbell S.J., Perry V.H., Pitossi F.J., Butchart A.G.,
Chertoff M., Waters S., Dempster R., Anthony D.C.
(2005) Central nervous system injury triggers hepatic CC
and CXC chemokine expression that is associated with
leukocyte mobilization and recruitment to both the central
nervous system and the liver. Am. J. Pathol. 166:14871497.
Dusart I., Schwab M.E. (1994) Secondary cell death and
the inflammatory reaction after dorsal hemisection of the
rat spinal cord. Eur. J. Neurosci. 6:712724.
King, V.R. et al. (2006). Omega-3 fatty acids improve
recovery, whereas omega-6 fatty acids worsen outcome, after
spinal cord Injury in the adult rat. J. Neurosci. 26 (17):
46724680.
Huang W.L. et al. (2007a). The characteristics of neuronal
injury in a static compression model of spinal cord injury in
adult rats. Eur. J. Neurosci. 25 (2):36272.
Huang, W.L. et al. (2007b). A combination of intravenous
and dietary docosahexaenoic acid significantly improves
outcome after spinal cord injury. Brain.
Parham P. (2000) The Immune System. London: Current
Trends/Garland.
Popovich P.G., Wei P., Stokes B.T. (1997) Cellular
inflammatory response after spinal cord injury in SpragueDawley and Lewis rats. J. Comp. Neurol. 377:443464.
Profyris C., Cheema S.S., Zang D., Azari M.F., Boyle K.,
Petratos S. (2004) Degenerative and regenerative
mechanisms governing spinal cord injury. Neurobiol. Dis.
15:415436.

DHA affects infiltration of neutrophils into some areas


of the epicentre but not into the liver
4 hours after injury and following treatment with DHA or
saline given 30 min after SCI, fewer neutrophils were found
in all quantified areas of the spinal cord epicentre in the DHA
treated group, although this only reached significance in the
dorsal columns (55% less, *p<0.05, Fig. 2a). At 1 day, fewer
neutrophils were found in the dorsal columns (31% less, Fig.
2b), the ventrolateral white matter (37%) and significantly
less in the ventral horns (51% less, *p<0.05) in the DHA
animals compared to the vehicle group. A small number of
neutrophils were found in the liver of uninjured animals and
this amount tripled 4 hours and 1 day after SCI. However,
there was no difference in the number of neutrophils recruited
to the liver at the same time points after laminectomy, SCI or
treatment with DHA or saline (Fig. 2c,d).
IL-1, TNF- and KC/GRO/CINC levels increase in
the epicentre 4 hours after SCI
Following SCI, there was a significant increase in the proinflammatory cytokines Il-1 (9 fold), TNF- (30 fold) and
KC/GRO/CINC (13 fold) 4 hours in the epicentre
compared to laminectomy or nave animals (Fig. 3bd).
CONCLUSION
Following compression SCI, there is a coordinated
inflammatory reaction in the epicentre involving cytokines,
neutrophils and a heterogenous population of macrophages/
microglia. There is also increased recruitment of neutrophils
to the liver after laminectomy alone and spinal cord injury.
There is some indication that DHA affects neutrophil

Fig. 2. DHA decreased neutrophil recruitment to some areas of the epicentre. (a) There were fewer neutrophils in the DHA treated group
in the dorsal columns (*p<0.05) at 4 hours and a trend towards fewer in all other areas of the spinal cord epicenter. (b) At 1 day, the number
of neutrophils in the epicentre quadrupled in the saline group compared to 4 hours and there were fewer neutrophils in the ventral horn of the
DHA treated group. In the liver, there was no difference in the number of recruited neutrophils at (c) 4 hours and (d) 1 day. Nave numbers
are represented by the red dotted line. Control = untreated. Results are mean SEM number of animals in brackets.
51

Fig. 3. Measurement of cytokines in the e picentre after compression SCI. (a) 25 l of sample was analysed on a 7 plex cytokine array
plate. (bd) There were significant increases of Il-1 (nine fold, b), TNF- (30 fold, c) and IL-8 (13 fold, d) in the SCI (n=3) compared to
the control (n=3: 2 laminectomy + 1 nave) group. Results are mean SEM number of animals in brackets.*p<0.05, **p<0.01.

FUTURE PLANS
In order to complete the study of acute inflammatory
events, the effects of DHA on four of the main cytokines
that have been found to be involved in the progression of
the inflammatory response after SCI (IL-1, TNF-, IL-6
and KC/GRO/CINC) will be analysed in the epicentre,
plasma and liver 4 hours and 1 day after compression SCI.

Trivedi A., Olivas A.D., Noble-Haeusslein L.J. (2006)


Inflammation and Spinal Cord Injury: Infiltrating
Leukocytes as Determinants of Injury and Repair Processes.
Clin. Neurosci. Res. 6:283292.
Hall J.C., Priestley J.V., Perry V.H., Michael-Titus A.
(2008) The effects of omega-3 fatty acids on early
inflammatory events after spinal cord injury in the rat.
London: Spinal Research Annual Meeting and William
Harvey Research Day, Barts and The London.
Hall J.C., Michael-Titus A. (2008) The inflammatory
response and locomotor recovery following a spinal
compression injury. P79. Dublin: UCD International
Neuroimmunology Symposium.
Hall J.C., Perry V.H., Priestley J.V. (2007) Systemic and
local inflammatory changes following a spinal cord
compression injury. Zurich: Spinal Research Annual
Meeting and Spinal Research Christmas supporter
reception. November 2007.

To investigate chronic inflammatory events, we will


study the effects of the DHA bolus injection combined with
chronic exposure to DHA in the diet on changes in
microglia/macrophages by measuring specific markers and
signalling molecules that give some indication of their
function. For example, microglia expressing iNOS or TNFare thought to display a damaging phenotype, whereas
microglia expressing TGF- are said to display a beneficial
phenotype. If the composition of this population is altered
following treatment with DHA, it might contribute to
different outcomes after SCI.

52

AAV8shRNA-RhoA and AAV8nt-3 transfection of dorsal


root ganglion neurons (DRGN) in vivo mediates neuron
survival and disinhibited regeneration of dorsal column
(DC) axons
Steven J. Jacques*, Ann Logan, Martin Berry, Zubair Ahmed
University of Birmingham, Birmingham, UK. a.logan@bham.ac.uk
*PhD Student
INTRODUCTION

of this neurotrophin has not been associated with


hyperalgesia, presumably due to sprouting of nociceptive
DRGN or sympathetic terminals within the DRG (P. J.
Dyck et al., 1997). Our group has previously produced and
evaluated shRNARhoA and demonstrated effective RNA and
protein knockdown in DRGN (unpublished data).

The inability of the central nervous system (CNS) to


regenerate axons following injury is a well-recognised
phenomenon, which may be explained by a number of
potentially maladaptive components of the CNS injury
response e.g. reviewed in (A. Sandvig et al., 2004). For
example, lack of trophic support, significant neuronal death
and the presence of axon growth inhibitory ligands (AGIL) all
contribute to the lack of axon regeneration seen post-injury.

Hypothesis
Simultaneous AAV8shRNA-RhoA and AAV8NT-3
transfection of dorsal root ganglion neurones (DRGN) in vivo
mediates neurone survival and disinhibited regeneration of
DC axons.

This project will use the dorsal column (DC) injury


model to examine the effects of axonal regenerationpromoting treatments. In brief, axons of the DC are a subset
of the central projections of neurones lying in the dorsal
root ganglia (DRG). These neurones, the dorsal root
ganglion neurones (DRGN), convey proprioceptive and
certain cutaneous modalities. When the central axons of
DRGN are transected, they fail to regenerate their axons
and an appreciable number of them apoptose or undergo
atrophic changes (Y. A. Chelyshev et al., 2005). A subset of
DRGN, whose axons project to the DC express the
neurotrophin receptor TrkC, and are therefore responsive
to the trophic effects of neurotrophin-3 (NT-3) (C. Chen et
al., 1996).

Overall aims of the project


Construct and evaluate AAV8 vectors encoding NT-3
and shRNARhoA
Evaluate the effects of these vectors in vitro
Inject these viruses into L4/L5 DRG in vivo after DC
transection and examine their effects on regeneration
of the central axons of DRGN
Specific aims addressed this year
1. Development of a method to measure DRGN neurite
outgrowth in vitro.
2. Construction of plasmids for production of AAV8
encoding NT-3.
3. Evaluation of the biological activity of FLAG tagged
NT-3.
4. Development of an in vitro DRGN axon growth
inhibitory assay.
Evaluation of AAV8GFP as an axonal tracer in the CNS in
vivo using DRGN projection tracts as a model.

Nogo, myelin-associated-glycoprotein (MAG) and


oligodendrocytes-myelin-glycoprotein (OMgp) all exert their
effects as AGIL via the non-signalling NgR receptor. Alone,
NgR is unable to transduce a signal so is found in association
with leucine-rich repeat and Ig domain containing
(LINGO-1) along with either p75NTR or TNFRSF
expressed on the mouse embryo (TROY). Successful
activation of this signalling complex by AGIL results in an
intracellular signalling cascade, eventually leading to RhoA
mediated collapse of the actin cytoskeleton, arresting further
growth (reviewed in (A. Sandvig et al., 2004).

METHODS
The spinal column of an adult Sprague Dawley rat (150
250 g) was removed as a crude section. After removing the
soft tissue of the spine, the pedicles of each vertebra were
cut and the entire dorsal segment removed to expose the
spinal cord and associated DRG. The DRG from the L2-L5
levels were removed under direct vision and incubated in
collagenase for two hours. Following this, DRG were
triturated in Neurobasal medium, layered on 15% BSA and
spun at 120 g. The supernatant was removed and the
resulting cell pellet, enriched for DRGN resuspended in
medium containing soybean trypsin inhibitor and DNase.
DRGN were counted and plated in 8 well chamber slides
coated in poly-D-lysine with or without laminin at
500 DRGN/well.

It is increasingly recognised that combinatorial strategies


offer a powerful way to stimulate growth of axons and
overcome the barriers to their effective regeneration (A. Logan
et al., 2006). The main aim of this project is to examine
regeneration of DC axons in the spinal cord after delivery of
recombinant adeno-associated viruses (AAV8) containing
NT-3 and an RNA-interference construct designed to knock
down RhoA (shRNARhoA).
NT-3 supports the survival and axonal growth of DRGN
both in explants and in vivo (e.g. (B. Blits et al., 2003)). It
was decided to target NT-3 responsive DRGN since delivery
53

COS-1 cell culture

buffer. Samples were removed at all stages of purification


for subsequent analysis by SDS-PAGE and ELISA.

COS-1 cells were maintained in T75 tissue culture flasks in


DMEM medium, supplemented with 10% FBS and 1%
penicillin/streptomycin. They were passaged every 24 days
using 0.05% trypsin. When plating for subsequent
transfection, COS-1 cells were trypsinised and seeded onto
6-well tissue culture plates at 500 000 cells/well in 2 ml
DMEM. They were left for 24 hours before transfection.

Injection of DRGs with AAV8GFP in vivo


Adult male Sprague Dawley rats (150250 g) were operated
upon by Professor Martin Berry in accordance with the
regulations of the UK Animal Act 1986.
Anaesthesia was induced with 4% isoflurane and
maintained at 2% throughout the procedure. Buprenorphine,
at a dose of 0.03 mg/kg was used for analgesia, given at the
start of the procedure and twice daily for a further 2 days
following surgery as required.

Transfection of COS-1 cells with Lipofectamine 2000


Having seeded the cells as described above, the medium was
replaced with DMEM alone. 500 l of a mixture containing
10 l Lipofectamine 2000 and 4 g of DNA was added to
each well and left on, at 37C in 5% CO2 for five hours.
After this, medium was replaced with complete DMEM
containing FBS and antibiotics. Cells were allowed to grow
for 6 days following transfection with addition of 1 ml
complete DMEM after 3 days.

Aseptic conditions were used throughout. The animal


was placed on a heat pad in a custom-made stereotactic
apparatus allowing the whole animal to be moved through
all degrees of freedom, ensuring that the spine is kept
straight at all times. A 2 cm incision was made in the
midline over the lumbar region and held open with a
retractor. The ligamentous insertions of erector spinae were
visualised and a small mark made on the contralateral side
at the level of L4. A 2 cm paramedian incision was made,
with L4 as its midpoint, around 1 mm to the left of the
spinous processes down to the articulating surfaces of the
facet joints. Ligamentous attachments to the articular
surfaces were severed, followed by further blunt dissection
to reveal the lateral processes which were removed to reveal
the underlying DRG. Haemorrhage was stopped using
Spongistan gel foam.

SDS-PAGE and Western blotting


Samples were boiled in 1 loading buffer for four minutes
before being loaded onto a 12% Tris-glycine gel. The gel
was run at 125 V for 1 hour 50 min followed by either
Coomassie or silver staining or transfer onto a
nitrocellulose membrane over 2 hours at 25 V. After
blocking, the membranes were stained with anti-FLAG M2
antibody at a dilution of 1:1000 and NT3 antibody at a
dilution of 1:200. Bands were visualized by exposure onto
Kodak Biomax light film, using HRP-conjugated secondary
antibodies followed by application of ECL according to the
manufacturers instructions.

10 l of a solution of AAV8GFP diluted in sterile PBS


was injected into each DRG using a glass microelectrode
attached to a 20 ml syringe containing air. Injection was
deemed successful if the DRG was observed to swell. The
animal was closed using catgut for the muscle layer and skin
staples. Animals were observed closely post-op until they
made a recovery, and then checked daily for any signs of
autophagia or paralysis.

ELISA
ELISAs were performed according to the detailed protocol
provided by Promega in their Emax Immunoassay kits.
Briefly, wells of a 96 well plate were coated with a primary
antibody overnight at 4C in carbonate coating buffer of
appropriate pH. Following this, wells were washed in TBST
and samples added at appropriate dilutions and incubated
with vigorous shaking for 6 hours. Well were then washed
again and a second primary antibody added which was
incubated overnight. The wells were washed and an HRP
conjugated secondary antibody was applied to the wells and
incubated for 2.5 hours. After a final wash, TMB One was
added to each well giving a blue coloured product. After
15 min the reaction was stopped by addition of HCl,
resulting in a yellow final product being formed. The
absorbance of each well was assessed using a plate reader set
at 450 nm. Concentrations in unknown samples were
determined by comparison with a standard curve prepared
on the same plate as the standards.

RESULTS
1. Development of a method to quantify neurite
outgrowth in vitro
Classically, quantification of neurite outgrowth has relied
upon measurement of the length of individual neurites. For
example, longest neurite per neurone, or mean length of
neurites across multiple neurones. However, this approach
is almost impossible to put into practice when the neurite
growth is dense, as is often the case in cultures of DRGN
(Figure 1). Initial results from dissociated DRG cultures
ran into such problems with dense neuritic growth, and
this necessitated the development of an alternative approach
to quantification which took advantage of image analysis
technology.

Immunoaffinity chromatography of NT3-FLAG


Conditioned media from COS-1 cell transfections with
NT3-FLAG containing constructs were collected and
centrifuged to remove suspended cells. This conditioned
medium was passed through a polypropylene column
containing 100 l of anti-FLAG M2 affinity beads. The
beads were washed and the protein eluted using a low-pH

Image analysis can be used to extract numerical


parameters from an image such as a light micrograph. These
numbers are typically relatively easy for a human operator to
measure manually, such as length of neurites or cell counts.
However, image analysis approaches offer a number of
54

advantages over human operators, including lower


susceptibility to bias, speed, accuracy and reproducibility. It
must be borne in mind, however, that image analysis is also
a highly complex field with numerous potential pitfalls and
many caveats. The following account describes attempts to
develop a method of quantification of dense neurite
outgrowth using a combination of image analysis software
and spreadsheet technology.

is pretty much constant overall, this is an accurate measure


for total neurite length. In order to standardise the total
neurite length the total number of intersections must be
divided by the number of neurones in the image to give a
figure proportional to the total neurite length per neurone.
Hence, the measure which is derived is a surrogate for the
actual total neurite length per neurone.
The Weibel grid is a useful and oft quoted method of
estimation of neurite outgrowth. However, it is very time
consuming (some experiments can take days to quantify)
and prone to bias. Hence an automated version was
developed based on the above mentioned principles but
using image analysis software, as described below.
An image is loaded into a suitable graphical package,
and cell bodies are completely blacked out using a
paintbrush tool. This is the only manual intervention in the
process, and is quite resistant to bias since the process is
simple and discrete find a green blob and make it black.
The image is then saved and reloaded into ImagePro, an
image analysis package. In ImagePro, the first operation
which is done is to convert the image to greyscale, only
extracting the green channel (the colour of FITC stained
neurites). Next, the background is flattened, ensuring that
any variations are evened out, so that the background
becomes uniform. This is necessary to establish at a later
stage the intensity of background pixels.

Fig. 1. Fluorescence micrograph of DRGN (3-tubulin, FITC (green))


and non-neuronal cells (DAPI (blue)) in vitro at 100 magnification.
Note dense DRGN neuritic growth, presenting a significant challenge
to quantification by manual means (100 magnification).

Next, a bitmap of the image is created. A bitmap is


basically a spreadsheet showing the intensity of values of all
the pixels in an image, allowing mathematical operations to
be done on them. This bitmap is exported to Microsoft
Excel for further processing. It must be noted that not the
whole bitmap of the image is exported due to limitations in
the software used. Actually, every seventh pixel is sampled by
the software providing data representative of the whole
picture. In Excel, the background pixel value is first
established, which is mode of the values in the bitmap, since
the flatten filter ensured that the background is even. This
background value will be the most frequently encountered
value in the spreadsheet.

The initial step in any image analysis is to capture the


image. In this case, all images were of cultures of dissociated
lumbar DRGs grown on 8-well chamber slides and stained
for III-tubulin using a standard immunocytochemistry
protocol, which did not vary between experiments. Images
were taken at 100 magnification using an Axioplan
2 microscope, fitted with an Axiocam HRc camera and
running AxioVision v4.6 software. Regardless of the method
of quantification, it is vital when capturing images that the
same exposure settings are used within an experiment, and
ideally across all experiments, a constraint not always
possible due to subtle differences in staining between
experiments. The reason for such rigour is that different
exposure settings lead to very different values for the same
item being photographed. Also, allowing the experimenter
to alter the exposure between pictures can leads to the
introduction of bias into the results.

Having established the background value, a position has


been reached to decide a threshold, above which a pixel is said
not to belong to a neurite. This has been arbitrarily set at 10%
above the background, although strictly it should probably
be two to three standard deviations above the background
based on statistical theory. The latter was not used for two
main reasons: (i) addition of the standard deviation to the
mode is not valid, as standard deviation applies to the mean;
(ii) it is very difficult to establish the mean of the background
since it would need to be isolated from the image, and this
itself would require a threshold value to be set.

Before going on to describe how image analysis was


applied to this problem it is necessary to describe the simple
method which provided some inspiration. One way to
measure neurite outgrowth uses a method developed by
Weibel in the 1960s for quantification of lung tissue, but
which can be easily adapted for use in neurones grown in vitro
(E.R. Weibel and D.M. Gomez, 1962). A Weibel grid is
placed over the image of interest, the number of intersections
between neurites and grid lines being proportional to the total
length of all the neurites in the picture. Intersections between
cell bodies and grid lines are ignored. This method makes the
assumption that the width of neurites is constant, measuring
the total area of neurites in the picture. Since neurite width

Thus, with threshold set, the number of pixels above


threshold level is counted. This will give the total number of
neurite pixels which, as described above, is proportional to
neurite length. Dividing this figure by the number of
neurones in the picture estimates the mean length of
neurites per neurone.
55

Fig. 2. Automated method of assessing neurite outgrowth by the Weibel grid method (A) compares favourably with automated assessment of area
covered by neurites (B). Note that relative orders of magnitude are well maintained and standard error is reduced in (B). Fields of view taken
at 100 magnification.

The above method has been incorporated into a macro


on the ImagePro software which, when run in conjunction
with Excel, is able to do all of the above having simply
opened the appropriate image file. This enables results to
be gathered in around half a day, a job which would
previously have taken two days to complete. The automated
method was validated by comparison with the Weibel grid
method in estimating neurite outgrowth, using data from a
colleagues experiment (Figure 2).

To produce an AAV8 virus containing both FLAGtagged and un-tagged NT-3 the human NT-3 gene was
cloned into pAAVIRES-hrGFP at the MCS, creating
pAAVIRES-hrGFP-NT3-STOP and pAAVIRES-hrGFP-NT3 (FLAGtag). pAAVIRES-hrGFP-NT3-STOP produces non-FLAG-tagged
NT-3 due to the incorporation of a stop codon distal to the
NT-3 coding region but proximal to the FLAG tag
sequence. Also, the above described pAAVRC was modified
by removing the AAV2 rep and cap sequences and replacing
them with those from AAV8. Acknowledgments to Dr Mike
Douglas (MNG, University of Birmingham) for performing
the above cloning steps.

In conclusion, a method has been developed for the semiautomated quantification of dense neurite outgrowth in
dissociated cultures of dorsal root ganglia. It provides
comparable results to an established quantification technique,
but is much faster and more convenient. However, the result
it provides is still only a surrogate measure of neurite
outgrowth, which may prove problematic in comparison with
results reported in the literature. Thus, in certain
circumstances, it may be necessary to grow DRGN in
conditions hostile to growth in order to reduce relative neurite
outgrowth, thereby enabling quantification of growth
parameters manually, which is discussed later on.

3. Evaluation of the biological activity of FLAG-tagged


NT-3
Before going on to produce AAV8 using pAAVIRES-hrGFPNT3 (FLAG tag) it is necessary to confirm that the addition
of the FLAG tag does not alter the biological activity of
the expressed NT-3. In order to do this, COS cells were
transfected with pAAVIRES-hrGFP-NT3 (FLAG tag) to produce
FLAG-tagged NT3 (NT3-FLAG) which was then subjected
to Western blotting and quantification by ELISA followed by
assessment of biological activity in a DRGN survival assay.

2. Construction of plasmids for production of AAV8


encoding NT-3
In order to produce AAV8 in vitro, three plasmids are cotransfected into 293 cells using the CaCl2 method. These
consist of a helper plasmid (pAAVHelper) containing genes
vital for production of assembled viral particles, a plasmid
containing the genes encoding the appropriate capsid
proteins (pAAVRC) and a plasmid containing the viral
genome spanned by inverted-terminal-repeats (ITRs),
designated pAAVIRES-hrGFP (Figure 3). The ITR-containing
plasmid also possesses a multiple-cloning-site (MCS) with
a 3 FLAG sequence downstream of this, allowing for the
insertion of a gene of interest which will be expressed in a
FLAG-tagged form. These plasmids are commercially
available in the AAV Helper Free System (Stratagene).

Transfection of COS cells


Transfection of COS cells using lipofectamine and 2 g
plasmid (pAAVhrGFP, pAAVIRES-hrGFP-NT3-STOP or
pAAVIRES-hrGFP-NT3) per well of a six well plate produced a
high proportion of GFP expressing cells (90100%) (Figure
4). Four transfections were performed. One well was
transfected with lipofectamine alone as a control
(designated Untransfected); a second well was transfected
with pAAVhrGFP and the third and fourth wells were
transfected with pAAVIRES-hrGFP-NT3-STOP and pAAVIREShrGFP-NT3 (FLAG tag) respectively. Having grown
transfected cells on for six days, the conditioned medium
(~3 ml in total per well) was removed and passed through a
0.22 m filter to remove any contaminating cells prior to
further analysis.
56

added and serial 1:2 dilutions


made of them. An HRPbound tertiary antibody
provided the colorimetric
output which was read on a
plate reader at 450 nm. The
concentration of NT-3 in
each well was determined by
comparison with standards
followed by correction for
dilution factors. It was found
that no NT-3 was present in
supernatant from untransfected cells and cells
transfected with GFP alone.
Hence, COS-1 cells do not
produce NT-3. Transfection
with NT-3 and NT-3-FLAG
containing
constructs
resulted in production of
high levels of NT-3, around
the 60 ng/ml level. This provided a concentration of NT-3 for
use in an assay for biological activity.

Fig. 3. The plasmids necessary for production of AAV in vitro. Note


that to make AAV8, pAAV-RC was manipulated to contain AAV8
rep and cap genes.

Fig. 4. Lipofectamine and 2 g pAAVhrGFP in a 6-well plate provides


an excellent transfection rate in COS cells, assessed by GFP expression.
(A) Phase contrast image, (B) fluorescent image (FITC); 100
magnification.

SDS-PAGE and Western blotting on conditioned


medium
Whole conditioned medium from the above transfection was
loaded into a standard SDS-PAGE gel, transferred to a
nitrocellulose membrane and probed using an antibody
against the FLAG tag. This demonstrated a bright band in
the lane loaded with supernatant from conditioned medium
from COS cells transfected with pAAVIRES-hrGFP-NT3 (FLAG
tag) at the predicted molecular mass of NT3-FLAG (~17kDa)
(Figure 5). The other, less bright bands in the blot are
probably a result of non-specific binding by the secondary.
Thus it has been qualitatively demonstrated that NT3-FLAG
was produced in relatively high concentrations by COS cells
transfected with pAAVIRES-hrGFP-NT3 (FLAG tag).

Fig. 5. FLAG-tagged NT-3 is produced in large quantities, producing


a bright band on Western blot on transfected COS cell conditioned
medium when probed with an antibody against the FLAG tag.
Abbreviations explained in text.

Evaluation of biological activity of NT3-FLAG


To assess biological activity, whole supernatant was applied
to dissociated DRG cultures at 50% concentration (in each
well of culture, 150 l growth medium with 150 l COStransfection supernatant). The result was surprising, and is
shown in Figure 6. Although this experiment only used dorsal
root ganglia from one animal, it was convincing that DRGN
neurite growth was not supported by the addition of any NT3 containing supernatants. One reason for this may be that
the baseline growth was too high, not allowing the effect of
exogenous NT-3 to become apparent. This is a common
problem in DRG cultures since they have a high level of nonneuronal cells (including Schwann cells, satellite cells and
fibroblasts) which are known to secrete neurotrophic factors,
leading to high levels of growth even in untreated cells. It is
possible to overcome this by using mitotic inhibitors such as
5-fluoro-2-deoxyuridine (5-FDU).

ELISA on conditioned medium


The next step was to attempt to quantify the concentration
of NT3-FLAG in whole supernatant in order to assess
whether this was physiologically relevant. This was achieved
by use of a commercially available ELISA kit from Promega,
which is sensitive in the pg/ml range. Briefly, standard curves
were set up in a 96 well plate using recombinant
NT-3 in serial 1:2 dilutions. Alongside this, samples were
57

using this method has been performed with encouraging


results. Six 15 cm dishes of COS cells, transfected with a
plasmid containing NT-3-FLAG were grown for six days, as
before and then the conditioned medium harvested and
filtered. It was then run through an immunoaffinity
chromatography column loaded with a commercially
available gel coated with antibodies against the FLAG
peptide. The tagged protein was eluted using a low pH
solution, and the resulting eluates and subsequent washes
run on an SDS-PAGE gel, followed by silver staining for
total protein. This demonstrated a faint but definite band in
the fraction collected following the first elution (Figure 9).
Thus, proof of principle of NT3-FLAG purification was
demonstrated, but larger quantities of protein needed to be
made to allow in vitro bioactivity to be assessed.
Fig. 6.When whole conditioned medium from transfected COS cells
was applied to adult rat DRG cultures at a 1:2 dilution, no significant
effect was observed on the amount of neurite outgrowth, as measured
by the automated percentage neurite coverage method. (One-way
ANOVA, p=0.090). Abbreviations explained in text.

Another possibility was that whole COS supernatant


contained a large amount of toxic by-products of cellular
metabolism, especially given that the cells were grown
without change of medium for six days. Thus, these waste
products could be counteracting the effect of any growth
promotion caused by NT-3. This problem was partially
overcome by concentrating the supernatant by a factor of 10
using a microconcentrator with a molecular weight cut-off at
10 kDa. Thus, toxic metabolites usually with low molecular
weights would pass through, but NT-3 (~13 kDa) and NT3-FLAG (~17 kDa) would be retained. Concentrated
supernatant was added to cells in increasing doses as shown
in Figure 7. No effect was seen on growth at any of the doses.
These negative results suggested that DRGN neurite
outgrowth may be the wrong assay for evaluating NT-3
bioactivity. It was thus decided to develop a simpler bioassay,
firstly using recombinant NT-3.

Fig. 7. Dose response curves of growth elicited by COS cell conditioned


medium following transfection of COS cells with plasmids. No clear
effect on neurite outgrowth was demonstrated in any of the groups
compared with control. The only significant difference detected was
that there was less growth following addition of conditioned medium
from pAAV-hrGFP than that from untransfected COS cells (two-way
ANOVA with Games-Howell post-hoc test gave p=0.005).

DRGN are robust, and will grow well with minimal cell
death when placed on a substrate of poly-D-lysine (PDL) and
laminin. One way to unmask the effect of a trophic factor is
to make conditions for cells hostile, so that they become
relatively more dependent upon exogenous survival and/or
growth signals. This was achieved with DRGN by removing
the laminin from their substrate, which seems to result in
around eight times less survival with no trophic support.
When DRGN were grown on a substrate of PDL alone, with
added recombinant human NT-3 (rhNT-3), twice as many
survived at a concentration of 10 ng/ml compared with
control (Figure 8). This was a real effect, repeated using tissue
cultured from four separate animals, with a significant
difference between control and maximal effect.
At the time of writing this report NT-3-FLAG was
being purified using immunoaffinity chromatography on
supernatant from a large scale transfection of COS-1 cells in
order to make a large batch for testing in vitro as described
above. A smaller scale attempt at purification of NT-3-FLAG

Fig. 8. rhNT-3 supports the survival of DRGN grown on PDL alone


at a concentration of 10 ng/ml. (One-way ANOVA, Games-Howell
post-hoc test p=0.013).

58

Fig. 9. Silver stain on samples from immunoaffinity chromatography


purification of NT3-FLAG. A band corresponding to the predicted
mass of NT3-FLAG came out in the first elution (bold arrow), with
the lighter rhNT3 clearly visible (regular arrow).

A second batch of conditioned medium from transfection of


COS cells was produced and purified using the above
method, and elution was carried out using a FLAG peptide
followed by dialysis. Unfortunately, NT-3 was not detected
in the purified conditioned medium. This may be explained
by the fact that a very low transfection efficiency was
observed. A further batch of conditioned medium has been
made by transfecting COS cells using Lipofectamine 2000,
a method which is known to work well. The results of this
purification await quantification.
4. Development of an in vitro axon growth inhibitory
assay
During the coming year it will become necessary to evaluate
the capacity of a short hairpin RNA against RhoA
(shRNARhoA) to disinhibit the growth of DRGN in
the presence of an inhibitory environment. Thus, in the
first place a reliable method of inhibiting DRGN must
be developed.

Fig. 10. Dose-response curves for increasing concentrations of NogoP4 peptide applied to DRGN in culture. 5-FDU was used to inhibit
the proliferation of non-neuronal cells. (A) Survival of DRGN at
varying concentrations of Nogo-P4. Note that diminished survival
due to Nogo-P4 begins at a concentration of 46 M. (B) Complete
inhibition of neurite outgrowth is achieved at a concentration of
46 M. Also note an apparent stimulation of outgrowth at 4 M and
11 M, the significance of which is discussed in the text.

In order to inhibit the growth of neurones in vitro it is


necessary to deliver inhibitory ligands at appropriate
concentrations. This has been achieved in our group by
using a preparation of rat CNS myelin, derived from adult
Sprague-Dawley rat brains. This is low in lipid and contains
all of the known inhibitors of neurite outgrowth (Z. Ahmed
et al., 2006). However, these myelin preparations also
contain significant levels of FGF-2, which interferes with
the inhibitory activity of the CNS myelin. Also, there have
been considerable difficulties in replicating results using
these CNS myelin preparations presumably due to
variations in concentrations of contaminating growth
factors. Thus, it was decided to try a recombinant inhibitory
ligand in this case, a fragment of the Nogo protein (NogoP4). A preliminary experiment (Figure 10) gave promising
results, demonstrating complete inhibition of neurite
outgrowth from adult DRGN at 46 M. However, this
concentration also caused 60% cell death compared with
control. It is planned in further experiments to try
intermediate concentrations with multiple replicates in
order to determine the concentration of Nogo-P4
which causes maximal inhibition in the context of
minimal mortality.

An interesting feature of the result described above is the


apparent stimulation of growth at moderate concentrations
of Nogo-P4. It is already recognised that the effects of
certain guidance molecules, and indeed the AGIL known
as myelin-associated glycoprotein (MAG), is altered by the
relative levels of cAMP within the cell (M.T. Filbin, 2006).
Therefore, further study is needed to determine exactly the
dose-response characteristics of NOGO-P4.
5. Evaluation of AAV8GFP as an axonal tracer in the
CNS in vivo using DRGN projection tracts as a model
Before injecting AAV8 containing NT-3 in vivo it is necessary
to understand the distribution of the delivered NT-3 in the
nervous system. This will enable an understanding of which
tracts contain NT-3, whether transynaptic transport of virus
occurs and also what the transfection efficiency of the virus is
in the DRG. Since all of the NT-3 containing constructs
contain hrGFP, expression of GFP will be used as a surrogate
for expression of other expressed proteins.

59

AAV8 is known to be tropic for DRGN, although the


most effective serotype for these neurones remains
contentious, with some groups finding AAV5 to give the
highest transfection of DRGN ((B. Storek et al., 2008);
Joost Verhaagen, personal communication; unpublished
data from our group). A series of injections to the L4/L5
DRG of adult male Sprague-Dawley rats has been
performed with the aim of assessing transfection efficiency
and the distribution of AAV8GFP in the CNS. Preliminary
results indicate that DRGN are transfected and that axons
are labelled in the dorsal root following injection of
AAV8GFP. Full quantification of the transfection efficiency
will be performed in due course, as well as a characterisation
of the distribution of the virus in the sensory pathways.

Dyck P.J., Peroutka S., Rask C., Burton E., Baker M.K.,
Lehman K.A., Gillen D.A., Hokanson J.L., OBrien P.C.
(1997) Intradermal recombinant human nerve growth
factor induces pressure allodynia and lowered heat-pain
threshold in humans. Neurology 48:501505.
Filbin M.T. (2006) Recapitulate evelopment to promote
axonal regeneration: good or bad approach? Philos Trans R
Soc Lond B Biol Sci 361:15651574.
Logan A., Ahmed Z., Baird A., Gonzalez A.M., Berry M.
(2006) Neurotrophic factor synergy is required for neuronal
survival and disinhibited axon regeneration after CNS
injury. Brain 129:490502.
Sandvig A., Berry M., Barrett L.B., Butt A., Logan A.
(2004) Myelin-, reactive glia-, and scar-derived CNS axon
growth inhibitors: expression, receptor signaling, and
correlation with axon regeneration. GLIA 46:225251.
Storek B., Reinhardt M., Wang C., Janssen W.G.M., Harder
N.M., Banck M.S., Morrison J.H., Beutler A.S. (2008)
Sensory neuron targeting by self-complementary AAV8 via
lumbar puncture for chronic pain. Proceedings of the
National Academy of Sciences of the United States of
America 105:10551060.
Weibel E.R., Gomez D.M. (1962) A Principle For
Counting Tissue Structures On Random Sections. J. Appl.
Physiol. 17:343-8.

CONCLUSION
As a result of the work carried out this year, I am now in a
position to produce an AAV8 virus containing NT-3 for in
vitro and in vivo delivery, and to determine the biological
activity of the expressed growth factor. The foundation has
been laid for a DRGN growth inhibitory assay which will be
useful also for bioactivity studies, but which has itself
produced interesting results concerning the pharmacology
of the NOGO-P4 peptide. Dense neuritic outgrowth is now
measurable using a semi-automated unbiased system.
Finally, insights have been gained into the distribution of
AAV8 in the CNS after DRG injection.

PUBLICATIONS
Poster presented at September 2008 ISRT network meeting
in London entitled AAV8 as a vector for the delivery of
neurotrophic factor genes and RNA interference for the
treatment of spinal cord injury

REFERENCES
Ahmed Z., Mazibrada G., Seabright R.J., Dent R.G., Berry
M., Logan A. (2006) TACE-induced cleavage of NgR and
p75NTR in dorsal root ganglion cultures disinhibits
outgrowth and promotes branching of neurites in the
presence of inhibitory CNS myelin. FASEB Journal
20:19391941.
Blits B., Oudega M., Boer G.J., Bartlett Bunge M., Verhaagen
J. (2003) Adeno-associated viral vector-mediated
neurotrophin gene transfer in the injured adult rat spinal cord
improves hind-limb function. Neuroscience 118:271281.
Chelyshev Y.A., Raginov I.S., Guseva D.S., Masgutov R.F.
(2005) Survival and phenotypic characteristics of
axotomized neurons in spinal ganglia. Neuroscience &
Behavioral Physiology 35:457460.
Chen C., Zhou X.F., Rush R.A. (1996) Neurotrophin-3
and trkC-immunoreactive neurons in rat dorsal root ganglia
correlate by distribution and morphology. Neurochemical
Research 21:809814.

Currently writing a review examining the relevance of


neurotrophic factors to DRGN. Intend to submit to the
Open Neuroscience Journal within the next 6 months.
FUTURE PLANS
This work will pave the way for a number of future studies
to be performed over the course of the coming year. This
includes a full evaluation of axonal tracing with AAV8GFP.
Also, the effects of AAV8 containing NT-3 and shRNARhoA
will be evaluated by injecting in vivo followed by culturing
the transfected DRGs and looking for NT-3 expression and
RhoA knockdown. If NT-3 is produced in appreciable
quantities and RhoA knocked down sufficiently then the
effect of these viruses on DC injury can be examined.

60

Effects of chondroitinase ABC treatment on axon and glial


functions
Effects of injury and chondroitinase ABC treatment on neuro-glial morphology and interactions involved in CNS
functioning, particularly of the visual pathway
Maria Cristina Ovejero-Boglione*, Arthur Butt
Kings College London, UK. arthur.butt@port.ac.uk
*PhD student
INTRODUCTION

treatment overcomes the major obstacles to functional


recovery in CNS regeneration, namely (1) neuronal loss
following injury, (2) cavitation at the lesion site, (3) axon
growth through the glial scar, (4) directed axon growth along
a restructured glial cytoarchitecture, and (5) re-establishment
of synaptic connections at the target. The third major aim of
this project, therefore, was to address these issues by
examining the actions of ChABC on the injured rodent optic
pathway using an optic tract cavitation lesion model (Jen and
Lund, 1981; Jen et al., 1989) to determine whether filling the
cavitation with a gelfoam matrix containing ChABC can, in
the long-term (A) affect RGC survival, (B) bridge the
cavitation, (C) facilitate directional axon regrowth to the
targets in the SC, and (D) alter the glial injury response.

The bacterial enzyme chondroitinase ABC (ChABC) has


been effectively used to dismantle the potent AGI barrier
presented by the injury scar and promote functional CNS
regeneration (Moon et al., 2001; Krekoski et al., 2001;
Bradbury et al., 2002; Zuo et al., 2002; Tropea et al., 2003;
Yick et al., 2003; Grimpe and Silver, 2004). ChABC
treatment mainly removes the AGI activity of the CSPGs
expressed by astrocytes and NG2-glia at the glial scar.
However, CSPGs expressed by astrocytes and NG2-glia have
multiple functions in cell-cell interactions, the importance
of which are only poorly understood. It is known that
CSPGs play roles in stabilising the structure of neuronal
synapses and axonal nodes of Ranvier, which are contacted
by the processes of both astrocytes and NG2-glia (Butt et al.,
2002). Therefore, it is possible that ChABC may have
adverse effects on cell-cell interactions, particularly for NG2glia, which are defined by their expression of the NG2
CSPG. This is highly relevant, because it is our hypothesis
that NG2-glia are involved in axonal growth and guidance to
the normal CNS target, as well as contributing to synaptic
connectivity and stabilization, and may therefore be required
to achieve sustainable long-term re-establishment of axonal
connections with the normal target. The optic pathway was
used as a simple CNS model of unidirectional regeneration
in order to interpret observations that are likely to be made
in the more complex CNS white matter of the spinal cord.
It is assumed that the interactions of neuronal elements with
cells along the injured optic pathway to achieve regeneration
to the correct brain targets are likely to resemble the
interactions taking place in the spinal cord. A primary aim
of this project was to determine the acute effects of ChABC
on the normal uninjured optic nerve that remains in the
proximities of the injury site. Additionally, OT cavitation
lesion was used as a practical model of CNS injury to enable
the study of true regeneration, instead of just sprouting and
plasticity ending in reconnection to pathways with a CNS
target different from the original target. The acute effects of
ChABC were investigated for the first time using this injury
model and applying ChABC in a gelfoam matrix that
resembles the naturally existing extracellular matrix.

METHODS
Adult Wistar and Sprague-Dawley rats and GFAP-GFP
mice were used.
1. Nissl staining of RGC
RGCs were examined in retinal whole-mounts using Nissl
staining to study three experimental groups: (1) normal
control rats; (2) rats with left OT cavitation; (3) left OT
cavitation with implantation of ChABC embedded in gelfoam. Retinae were examined at 42 dpl, and RGC cell
density within the ganglion cell layer was determined by
counting cells with somal diameter larger than 6 m.
2. Anterograde HRP retinocollicular path tracing
HRP was injected intraocularly to anterogradely label RGC
and their retinocollicular projections in four experimental
groups: (1) normal control rats (n=6); (2) rats with left OT
cavitation (n=3); (3) left OT cavitation with implantation
of ChABC embedded in gel-foam (n=6). At 41 dpl, HRP
was injected intraocularly and 24 hours later, at 42 dpl, the
SC was fixed in PFA plus glutaraldehyde, paraffin wax
embedded and 6 m sections cut in the vertical plane.
Sections were reacted to visualise the HRP, and examined
by DIC microscopy (Zeiss). The area of HRP-labelled
retinocollicular projections was measured in a minimum of
3 sections in each experimental group, using Axiovision
acquisition and analysis software.

Clearly, it was an important objective of this project to


address the fact that in the cases where ChABC promotes
CNS regeneration, directed axon growth was limited and
functional recovery was due primarily to plasticity, which
comprises the growth of collateral branches and the
establishment of new synaptic connections (Bradbury et al.,
2002). In fact, it still remains unresolved whether ChABC

3. Immunohistochemistry
Immunolabeling for NG2 and GFAP was used to examine
the time-course of the glial injury response along the visual
system in four experimental groups: (1) normal rats (n=6);
(2) left OT cavitation alone, analysed at 5 dpl (n=6), 21 dpl

61

(n=5), and 42 dpl (n=6); (3) left OT cavitation with


implantation of ChABC embedded in gel-foam, analysed
at 5 dpl (n=6), 21 dpl (n=6), and 42 dpl (n=6). Optic
pathways were fixed in PFA and paraffin wax-embedded,
and sections cut at 6 m. Fluorescence and ABC
immunolabeling were performed, and in the latter case
sections were counterstained with hematoxylin. Interactions
between glial cells were observed using fluorescence
microscopy with the ApoTome device (Zeiss) and
colocalization was measured using a Volocity Acquisition
software (Improvision). Sections were examined using DIC,
fluorescence or confocal microscopy (Zeiss). Cell counts of
NG2-glia and astrocytes in the SC were performed in a
minimum of 2 FOV in 3 sections (n>6).

organotypic cultures optic nerves attached to the chiasm


were compared between 1 hour and 24 hours incubated
either in normal medium or in medium containing ChABC,
before degenerative changes were observed in controls. The
nerves were subsequently detached from the chiasm for
impalements to measure the RMP of glial cells, life imaging
of axonal integrity and axonal CAP measurements.
Optic nerves from normal mice were incubated for
24 hours in normal medium or medium containing
ChABC, and nerves were then either placed in a brain slice
chamber and continuously perfused with aCSF for
measurement of the CAP (Fig. 1), or in a chamber for live
cell imaging of axonal integrity (Fig. 2); in the latter case,
axons were anterogradely labeled by intraocular injection of
LRD 24 hours prior to optic nerve isolation. Following
incubation in ChABC for 24 hours, there was a 50 + 3.1%
(n=5) reduction in CAP amplitude (Fig. 1B) compared to
controls (Fig. 1A), which displayed a 20 + 2.5% (n=4)
reduction in CAP in nerves incubated in medium with PBS
(analysis in Fig. 1C) as compared to normal nerve CAP
measured 1 hour after isolation.

4. Measurement of glial RMP and intracellular dye


injection
Optic nerves from GFAP-GFP mice were incubated in vitro
for 1 hour or 24 hours in either normal medium (n=5) or
in medium containing ChABC (n=5). Nerves were then
placed in a brain slice chamber and continuously perfused
with aCSF for electrophysiological measurement of RMP
and intracellular dye-filling with the fluorescent dye LRD.
At the end of the experiment, nerves were fixed in 4% PFA
for 30 min and whole-mounted in methyl salicylate, for
examination under a Provis (Zeiss) fluorescence microscope.
5. Measurement of axonal CAP
Optic pathways from GFAP-GFP mice were incubated in
vitro for 24 hours in either normal medium (n=4) or in
medium containing ChABC (n=5). After 24 hours, nerves
were placed in a brain slice chamber and constantly perfused
with aCSF, and the CAP was measured using suction
electrodes.
6. Live Imaging
Optic nerves from GFAP-GFP mice and from normal mice
were used following intraocular LRD injection to
anterogradely label axons. Nerves attached at the chiasm
were incubated in normal medium (n=5) or medium
containing ChABC (n=5) and imaged on a Zeiss
fluorescence microscope for a period of at least 4 hours.
7. Statistical analysis
Cell density was compared between multiple groups using
Kruskal-Wallis test at 95% interval, while pairs of groups
were compared using the relevant post-hoc analysis, onetailed Mann-Whitney unpaired non-parametric t-test.

Fig. 1. Electrophysiological recordings of the ON CAP. The CAP


was measured in optic nerves incubated for 24hours in normal
medium (A), or in medium containing ChABC (B). There was a
marked reduction in CAP amplitude in nerves treated with ChABC.
(C) Bar graph illustrating mean (+ SEM) CAP amplitude from optic
nerves incubated in control medium (filled bars; n=4) or medium
containing ChABC (open bars; n=5), *p<0.05 (Mann-Whitney test).

RESULTS
Strictly confidential (unpublished data).

To examine axonal integrity following ChABC


treatment, LRD was injected intraocularly to anterogradely
label RGC axons, and nerves were isolated 24 hours later and
immediately incubated in medium containing ChABC
(n=5). Live cell imaging was performed, and axonal integrity
did not alter between 0 hours (Fig. 2A) and 4 hours (Fig.
2B). Interestingly, exhudates of Rhodamine can be observed
as large red dots immediately after incubation in medium
(Fig. 24A) and following addition of active ChABC (Fig.

1. Acute effects of ChABC on normal ON function


The effects of ChABC on the normal optic pathway were
thoroughly investigated to gain an insight into the safety of its
use to normal pathways surrounding the injured pathway.
An ex-vivo model was developed in order to examine the
effects of ChABC on the optic nerve. In this model,
62

2B), indicating that the dye is lost during incubation in


medium. Due to technical difficulties including medium
evaporation and the gradual loss of fluorescence, longer
periods of live cell imaging were not possible.

isolated for sectioning and analysis. Serial sections of the


optic regions of the SC were used to create a 3D
reconstruction of the optic SC (Fig. 4). HRP labeling was
represented by the grey-black shading on the striped
unlabelled SC. In controls (n=6), HRP reaction product
was distributed across the entire optic layers of the
contralateral (left) SC (Fig. 4A). The small pale area
observed in the medial SC in controls corresponds to the
damage performed during HRP injection into the lower
temporal retina to confirm normal topographic
retinocollicular connections at the SC target.

Fig. 2. Effects of ChABC on axonal integrity in the ON. RGC


axons were anterogradely labelled by intraocular injection of LRD
(red) 24 hours prior to optic nerve isolation. Optic nerves were
maintained in culture medium containing ChABC and images were
captured at the beginning of the experiment, 0 hours (A), and then
every 15 min until 4 hours (B). Although some oscillations were
observed in the direction of axons, there was no change in axonal
integrity during this period. Scale bar: 100 m.

Fig. 3. Acute effects of OT cavitation lesion before and after


ChABC treatment on NG2-glia in the rostral OTs, OC and
caudal ONs. (A) NG2 glial rows can be seen in the OC decussating
predominantly in the direction of uninjured axons at 5 dpl following
OT cavitation lesion. Note the disappearance of the caudal half of the
ON corresponding to the injured OT. (B) NG2-glial distribution
along the entire white matter of the optic pathway was studied
following acute OT cavitation lesion and treatment with ChABC at
5 dpl. Note the thinning of the injured OT and corresponding ON
and the distribution of NG2-glia along normally decussated OC
axons. riot: rostral injured optic tract, ruOT: rostral uninjured OT,
OC: optic chiasm, cON: caudal optic nerve. Scale bar: 100 m.

Therefore, acute ChABC treatment had adverse but not


detrimental effects on axons, as it caused a significant
decrease in ON CAP amplitude (Fig. 1) but the ON
remained viable (Fig. 2).
2. Acute retrograde effects of OT cavitation lesion before
and after ChABC treatment on neuron-glial relations at
the proximal OT, the OC and the caudal ON
The acute effects of ChABC on the injured optic pathway
were thoroughly investigated.
Only 5 days post OT cavitation lesion (Fig. 3A) the white
matter of the optic pathway underwent major changes. The
caudal half of the ON corresponding to the injured OT was
completely degenerated. NG2-glial rows in the OC ran along
axons decussating towards the uninjured OT and overtook
the OC area corresponding to the decussating axons that
could no longer extend from the degenerated caudal ON
towards the rostral remainders of the injured OT (Fig. 3A).

Fig. 4. Three-dimensional reconstruction of retinocollicular


axonal projections terminating at the SC before and after OT
cavitation lesion without or with ChABC treatment. 3D
reconstructions of the optical area of the SC were created from serial
sections obtained rostro-caudally in (A) uninjured controls and (B)
following OT cavitation lesion without or (C) with ChABC
treatment. Note that the grey-black shading represents HRP labeling,
as compared with HRP labeling illustrated in A. Note the small pale
area in controls, corresponding to the damage performed during HRP
injection into the lower temporal retina to confirm normal topographic
retinocollicular connections at the SC target. Scale bar: 200 m.

Notably, the ON was observed to survive OT cavitation


lesion following acute ChABC treatment (Fig. 3B). At 5 dpl,
following ChABC treatment, NG2-glia distributed normally
along the entire white matter fibres of the optic pathway (Fig.
3B). Shrinkage of the injured OT and corresponding ON
was observed (Fig. 3B). However, axons and accompanying
NG2-glial rows decussated normally at the OC (Fig. 3B).

At 42 days post left OT cavitation lesion (n=6), no HRP


reaction product was observed in the retinocollicular
projections at the SC, as OT cavitation lesion invariably
resulted in the complete loss of HRP labeling in all the
rostral and caudal sections of the optic SC (Fig. 4B).
Moreover, in cases that received unilateral left OT cavitation
lesion combined with intracranial implantation of heatinactivated ChABC in a gel-foam, again, no retinocollicular
projection was observed (data not shown).

3. Long-term effects of OT cavitation lesion before and


after ChABC treatment compared to the normal HRP
retinocollicular projections to the SC
To investigate whether ChABC promoted directed
regeneration of RGC axons to their targets in the SC,
retinocollicular axonal projections to the SC were also
examined using HRP anterograde labeling. In all cases,
HRP was injected intraocularly, and 24 hours later SCs were
63

In 4 of the 6 rats that received OT cavitation and


ChABC treatment (n=6) anterograde labeling failed, but in
2 of the rats HRP reaction product was observed along a
band covering half of the stratum griseum superficialis
(SGS) and one fifth of the stratum opticus (SO) of the left
SC, with the thickest region of this band concentrating in
the centre of these nuclei. A 3D reconstruction of the SC
images was created to show the rostro-caudal deepness of
regeneration in the SC, which was restricted to the rostral
half of the SC at 42 days post OT lesion and ChABC
treatment (Fig. 4C), compared to the normal uninjured SC
(Fig. 4A). In the normal retina a small pale area lacking
HRP labeling appeared in the rostral-medial part of the
optic superior colliculus (Fig. 4A), which corresponded to a
needle damage made on the lower temporal retina to study
whether the pattern of labeling at the target indicated that
pathway regeneration (after ChABC treatment) was
topographic. Since there was no labeling in the medial third
of the OT injured and ChABC treated SC, it was not
possible to confirm whether the pattern of labeling at the
target indicated topographic regeneration (Fig. 4C).

outstanding results obtained for the effects of chondroitinase


application to the normal and injured CNS. Further results
can be consulted in the book produced for the entire
PhD thesis.

Fig. 5. Progressive long-term changes in NG2-glial morphology


in the SC before and after OT cavitation lesion without and
with ChABC treatment. (A,B,C) Photomicrographs showing NG2glia (NG2) in the SC in (A) uninjured controls and at (B) 21 dpl and
(C) 42 dpl following OT cavitation lesion treated with ChABC. (D)
Quantification of NG2-glial numbers in the SC was performed for the
normal uninjured SC and at 42 dpl without and with ChABC
treatment. (B) Note the distinguished increase in the density of NG2glial processes at 21 dpl following lesion and ChABC treatment
compared to controls. (C,D) Although NG2-glial counts largely
increased compared to controls, (C) the long processes of most of the
NG2-glia were lost, and the few NG2-glia that were seen extending
processes had one long single process or double well-ramified processes.
Scale bar: 100 m.

4. Progressive long-term effects of OT cavitation lesion


treated with ChABC on NG2-glial functions in the SC
target
In order to assess the effects of ChABC on NG2-glial cells
(NG2 antibody) after complete transection of the OT,
immunoreactivity was determined for reactive NG2-glia at
the SC target of the normal and regenerated optic pathway
(Fig. 5). The pattern of NG2 immunoreactivity was studied
in uninjured controls (Fig. 5A) and at 21 dpl (Fig. 5B) and
42 dpl after OT lesion and ChABC treatment (Fig. 5C).
Interestingly, at 42 days post ChABC treatment (Fig. 5C,
quantified in 5D), NG2-glial density was significantly
higher than the normal uninjured counterparts (Fig. 5A,
quantified in 5D), but much lower than NG2-glial density
following OT cavitation lesion alone (not illustrated here,
but quantified in 5D). Additionally, the reactivity of NG2glia decreased the longer the ChABC treatment lasted,
following comparisons between early (21 dpl) and late
(42 dpl) long-term ChABC treatment (respectively Fig. 5B
and 5C). In fact, a marked increase was observed in the
density of NG2-glial processes at 21 dpl following ChABC
treatment (Fig. 5B) compared to the uninjured SC (Fig.
5A). Although NG2-glial counts appeared to increased at
42 dpl (Fig. 5C) compared to the uninjured SC (Fig. 5A)
and the SC at 21 dpl (Fig. 5B), the long processes of most
of the NG2-glia were lost, and the few NG2-glia that were
seen extending processes had either one very long single
process or double well ramified processes (Fig. 5C). The
changes in NG2-glial morphology that were observed after
42 dpl were major following OT cavitation lesion (with
inactive ChABC treatment), where NG2-glial counts were
tremendously large and the number of processes was very
low (data not shown), the latter effect was similar to that
observed following ChABC treatment (Fig. 5C).

CONCLUSION
The evidence presented here has demonstrated that acute
ChABC treatment largely diminishes normal axons and glial
functions, but does not damage their integrity. Additionally,
acute ChABC treatment on the injured CNS pathway
causes substantial improvements by rescuing axons and glia
from major degenerative consequences. In the long-term, a
single application of ChABC at the time of CNS cavitation
lesion, which is probably the worst type of injury that occurs
in the CNS, resulted in targeted regeneration, however this
only occurred in approximately 33% rodents. Furthermore,
after long-term treatment with ChABC, functional recovery
of neuronal and glial elements at the target of the injured
pathway is unlikely due to major loss of NG2-glial
processes, and therefore, their interaction with neuronal
elements. Nonetheless, glial morphology and density are
substantially recovered after long-term ChABC treatment,
compared to long-term OT cavitation lesion without active
ChABC treatment. The results in this report suggest that
ChABC alone may not be an effective treatment to promote
recovery of normal functions in neuronal and glial elements,
however ChABC treatment may form the bases to an
effective combination of treatments that could result in
sustainable long-term CNS regeneration.

These results were selected from numerous observations


and data obtained after experimentation using one of two
CNS injury models that were investigated during the length
of this doctorate project, in order to represent the most
64

Tropea D., Caleo M., Maffei L. (2003). Synergistic effects


of brain-derived neurotrophic factor and chondroitinase
ABC on retinal fiber sprouting after denervation of the
superior colliculus in adult rats. J. Neurosci. 23: 70347044.
Yick L.W., Cheung P.T., So K.F., Wu W. (2003). Axonal
regeneration of Clarke's neurons beyond the spinal cord
injury scar after treatment with chondroitinase ABC. Exp.
Neurol. 182:160168.
Zuo J., Neubauer D., Graham J., Krekoski C.A., Ferguson
T.A., Muir D. (2002). Regeneration of axons after nerve
transection repair is enhanced by degradation of chondroitin
sulfate proteoglycans. Exp. Neurol. 176: 221228.

REFERENCES
Bradbury E.J., Moon L.D., Popat R.J., King V.R., Bennett
G.S., Patel P.N., Fawcett J.W., McMahon S.B. (2002).
Chondroitinase ABC promotes functional recovery after
spinal cord injury. Nature 416:636640.
Butt A.M., Kiff J., Hubbard P., Berry M. (2002).
Synantocytes: new functions for novel NG2 expressing glia.
J. Neurocytol. 31: 551565.
Grimpe B., Silver J. (2004). A novel DNA enzyme reduces
glycosaminoglycan chains in the glial scar and allows
microtransplanted dorsal root ganglia axons to regenerate
beyond lesions in the spinal cord. J Neurosci. 24:13931397.
Jen L.S., Lund R.D. (1981). Experimentally induced
enlargement of the uncrossed retinotectal pathway in rats.
Brain Res. 211:3757.
Jen L.S., Cheung Y.M., Chow K.L. (1989). The origin and
terminal arbors of individual uncrossed retinogeniculate
fibers in rabbits. Neuroscience 29:479493.
Krekoski C.A., Neubauer D., Zuo J., Muir D. (2001).
Axonal regeneration into acellular nerve grafts is enhanced
by degradation of chondroitin sulfate proteoglycans. J.
Neurosci. 21:62066213.
Moon L.D., Asher R.A., Rhodes K.E., Fawcett J.W. (2001).
Regeneration of CNS axons back to their target following
treatment of adult rat brain with chondroitinase ABC. Nat.
Neurosci. 4: 465466.

PUBLICATIONS
06/09/07 08/09/07 Conference on Spinal Cord Repair
organised by the International Spinal Research Trust
(ISRT), Christopher and Dana Reeve Foundation (CDRF)
and Swiss Institute for Research in Paraplegia (IFP) in
Kartause Ittingen, Zurich, Switzerland.
16/11/0722/11/07 Oral presentation for Kings
representation in Nankai University was prepared and given
in Beijing, China, at the 10th Challenge Scientific Cup
competition. An award was won for this presentation.

65

Promoting long-distance axonal regeneration and


functional reconnection using combined treatments for
spinal cord injury
Thomas Sardella*, John S. Riddell, Susan C. Barnett
University of Glasgow, Scotland, UK
*PhD Student
INTRODUCTION

METHODS

Spinal cord injury leads to the loss of communication


between the brain and the spinal cord below the level of the
lesion. This may result in loss of sensation and of control of
movement, which is normally permanent because the spinal
cord has very little capacity for repair. One of the main
strategies for repair is to restore communication by
encouraging regenerating axons across the injury site and
functional reconnection with neurons on the other side.

Fig.1 provides a summary of the timing of the experimental


procedures carried out.
Olfactory bulb OECs and sciatic nerve Schwann cells of
seven day old fisher rats were purified by fluorescenceactivated cell sorting (FACS, by selecting for GalC- and O4+
cells) and partial trypsinization respectively. OECs and
Schwann cells were then cultured and transduced by a
lentiviral vector to express enhanced green fluorescent protein
(eGFP). These cell populations used for transplantation
were highly pure: immunocytochemistry coupled with
fluorescence microscopy showed that 98100% of the OECs
and 94100% of the Schwann cells were positive to the
P75 antibody.

Olfactory ensheathing cells (OECs) are amongst the best


candidates for cell transplantation in the nervous system.
These specialized glial cells are found in the olfactory system
and are associated with the physiological restoration of
neuronal circuitry in the first cranial nerve of the adult
(Barnett & Riddell, 2007). There is conflicting evidence on
the extent to which OECs promote long distance axonal
regeneration when transplanted alone (Li et al., 1997;
Ruitenberg et al., 2005). In previous work from our
laboratory we have found that regeneration of the ascending
branches of sensory fibres following acute transplantation
of OECs is largely confined to the transplant site (Toft et
al. 2007).
In this project we aimed to investigate the potential of
boosting this limited regeneration by using combined
treatments. In the first stage of this project transplants were
performed acutely following a spinal cord injury after a
conditioning lesion of the sciatic nerve (a procedure which
appears to prime DRG axons for growth; Richardson & Issa,
1984; Neumann & Woolf, 1999; Neumann et al. 2005).
Under these conditions, we showed that significant numbers
of dorsal column fibres extended beyond the lesion, though
only where a continuous track of transplanted cells extended
rostrally from the transplant site as described in last
years report.

Fig. 1. Timing of experimental procedures.

Conditioning lesions of the peripheral processes of the


L4 and L5 dorsal root ganglia were made by sectioning the
sciatic nerve. In the acute transplantation paradigm, the
conditioning lesions were performed once or twice at
different time points before and/or after the spinal cord
lesion to determine which would enhance most effectively
axonal regeneration. Three groups of animals have been
studied: one with a single conditioning lesion made 2 weeks
before a dorsal column lesion, another with double
conditioning lesions performed two weeks prior and
concurrently with the central lesion, and a third with double
lesions made 3 days before and 7 days after the central
lesion. In rats that received a delayed transplantation,
the conditioning lesion was performed at the time of
the transplant.

Since the last report we have started to investigate


whether Schwann cell transplants are also capable of
supporting regeneration beyond a lesion or whether this
property is unique to OECs
Any clinical use of OEC transplants will, however, likely
involve a delay between injury and treatment, and since a
glial scar will develop and the regenerative response of the
axotomised fibres may subside, it is not clear to what extent
regeneration will occur. We have therefore also started the
investigation of axonal regeneration following a delayed
transplant procedure.

In adult fisher rats, transverse lesions of the dorsal


columns were made close to the L3-L4 border using a wire
66

knife device. The wire knife makes a highly reproducible,


neat bilateral lesion of the complete dorsal columns and
therefore interrupts most or all of the ascending branches
of sensory neurons in the L4 and L5 dorsal root ganglia
(DRG). Cells are transplanted immediately following
lesioning (acute transplantation) or at a separate operation
performed 4 weeks later (delayed transplantation). OECs
(typically 1.01.5106 cells) are transplanted into the lesion
by injection from a glass micropipette using brief pressure
pulses. Both in acute and delayed transplantation, in
addition to animals with peripheral conditioning lesions,
nave animals were transplanted with OECs following
dorsal column lesions, to act as controls.

typically the OECs were distributed throughout the lesion


cavity, often leaving small gaps in the labeling.
In a high proportion of animals, OECs could also be
seen isolated or in dense broad tracts outside the lesion
(46/47; 98%), rostrally (44/47; 94%), caudally (45/47;
96%) both (43/47; 92%). In many animals (32/47; 68%)
these cells formed virtually continuous tracts leading away
from the lesion site and extending rostrally, often for more
than 2 mm.
Axonal regeneration within the transplant
In both conditioning lesion and control animals six weeks
after the wire knife injury numerous BDA labeled axons
could be seen to cross the caudal border of the lesion and to
regenerate within the transplanted lesion site. In all animals,
labeled fibres were observed within the transplant area.

Four weeks after the cell transplantation the left L4 and


L5 spinal nerves were injected with the neurotracer biotin
dextran amine (BDA) to label the sensory fibres entering
through the L4 and L5 spinal roots. Because of the
possibility of anastomoses between the L4 and L3 dorsal
roots, as a precaution, the left L3 spinal root was cut at the
time of the injury to minimize the possibility of labeled
non-injured fibres entering rostrally to the lesion.

Axonal regeneration beyond the lesion


Quantitative analysis of BDA labeled fibres rostral to the
transplanted lesion has so far been performed on 14 control
animals and 18 animals with conditioning lesions.

Two weeks following the tracer injections animals are


sacrificed by perfusion fixation and the spinal cord removed
and post-fixed. The lesion site was cut into parasagittal
sections and the sections treated with fluorescence
histochemestry techniques. Anti-Nf200, anti-GFAP and
anti-GFP primary antibodies followed by appropriate
secondary antibodies conjugated to fluorophores were used
to label respectively the axons, the astrocytes (lesion
boundaries) and the OECs. BDA was visualized using a
fluorophore conjugated to avidin. The sections were then
examined by fluorescence and confocal microscopy. Analysis
was performed also using computer software (confocal
assistant, adobe photoshop, ImageJ-NeuronJ).

Particularly in conditioning lesion animals, a proportion


of the BDA labeled fibres which had entered the transplant
within the lesion could be seen to exit the lesion at the
rostral border. Labeled axons were seen rostral to the lesion
in both the white matter and grey matter, often occupying
the central canal space. Interestingly, most fibres of control
and conditioning lesioned animals were within a region
containing OECs, suggesting that the presence of OEC is
necessary for the fibres to regenerate above the lesion.
Regenerating fibres might not require direct contact with
OEC, as evidence of tight association between GFP
expressing OEC and BDA labeled fibres was seen only
occasionally. None the less, BDA fibres direction closely
mimics the direction of OECs extension.

RESULTS
Acute transplantation
Distribution of transplanted cells
The distribution of GFP labeled OECs has been determined
in all the 47 transplanted animals. Fig.2 shows confocal
images illustrating typical examples of sections from control
(A+B) and conditioning lesion animals (C+D).

The number of regenerating fibres of conditioning


lesioned animals declined progressively with distance rostral
to the lesion consistent with these being regenerating fibres
over varying distances. Further more, the aberrant location
and the unusual direction of these fibres, the presence of
end bulb enlargements and frequent loops, strongly suggests
that these are regenerated fibres rather than spared fibres.
Quantitative data on the numbers of regenerating axons
detected above the lesion is shown in Table.1; data on the
distance they reach above the lesion is shown in Fig.3 and
confocal images illustrating regenerating fibres are shown
in Fig.4.
Delayed transplantation
Distribution of transplanted cells
Surprisingly, the distribution of OECs following delayed
transplantation was similar to that after an acute transplant.
Fig.5 shows confocal images illustrating typical examples of
sections of control (C,D,E), of conditioning lesion animals
(F) and of non transplanted rats (A,B). In successfully
injected animals, cells not only filled the lesion site, but

Fig. 2. Examples of GFP labeled OECs distribution 6 weeks after


injury (A+B control animals, C+D conditioning lesion animals). Red
is BDA, green is GFP, blue is GFAP. The boxed area in D is shown at
higher magnification in Fig 3. Scale bar 200 m.

In all animals GFP expressing olfactory ensheathing cells


were found within the lesion site defined by GFAP labeling;
67

were also distributed both rostrally and caudally in the


surrounding spinal cord in 8 of the 14 animals analysed.
OECs where seen isolated or clustered in broad tracts
outside of the lesion (14/19; 74%), rostrally (13/19; 68%),
caudally (10/19; 53%) both (9/19; 47%). In a proportion
of animals (8/19; 42%) these cells formed virtually
continuous tracts leading away from the lesion site and
extending rostrally.

Fig. 5. Confocal images of dorsal column lesions. A and B, appearance


of non transplanted lesion 4 and 10 weeks after lesioning, respectively.
Note the expansion of the lesion cavity. CF, lesion sites transplanted
with OECs 4 weeks after spinal cord lesion without (CE) and with
(F) a conditioning lesion. Note the wide spread distribution of OECs.
OECs green , GFAP blue. Scale bars=200 m.

Fig. 3. Numbers of fibres at different distances rostral to the lesion.


Symbols for data from control and conditioning lesioned animals are
as shown in the key legend. Error bars are indicating the standard
error. *: t test <0.05.

Fig. 4. Regeneration of dorsal column fibres into an acute OEC


transplant. A, low power scan of the transplanted lesion showing the
distribution of OECs. B, high power scan of the boxed area indicated
in A. Regenerating fibres in the lesion transplant (C) and reaching
the lesion boundary (D). E, presence of regenerating fibres within the
track of OECs 0.51.0 mm rostral to the lesion. BDA red, GFP green,
GFAP blue. All scale bars 100 m, except in A, 100 m.

Fig. 6. Ingrowth of Neurofilament labeled fibres into a delayed OEC


transplant. A shows the distribution of cells into the lesion site and B
shows in greater detail the ingrowth of regenerating fibres. Nf200 red,
GFP green, GFAP blue. Scale bars=100 m.

Axonal regeneration beyond the lesion


In order to determine whether dorsal column fibres were
among those that regenerate into a delayed OEC transplant
and to determine whether regenerating fibres remained
within the transplant or were able to regenerate across the
lesion, we used the tract-tracer BDA to label dorsal column
fibres entering the spinal cord below the lesion. Regenerating
dorsal column fibres could be seen entering OEC transplants
in 5 of the seven animals examined up to now. This shows
that dorsal column fibres retain the ability to regenerate into
an OEC transplant even when the transplant is made four
weeks after the lesion. Neither the glial scarring that develops
during the intervening period, nor a reduction in the intrinsic
growth response of the axotomised dorsal column axons
therefore prevents regeneration. However, as with acute
transplants, regenerating axons were mainly confined to the
OEC transplant, even though OECs could be seen rostral to
the lesion (see Table 1).

Axonal regeneration within the transplant


Following acute transplants of OECs, regenerating axons
grow into the transplant in profusion and can be detected
using immunolabelling for neurofilament. In order to test
whether regenerating axons are still able to grow into OECs
transplanted into a dorsal column lesion four weeks after the
injury, immunocytochemistry for neurofilament 200 was
carried out on sections of the lesion site six weeks after
delayed transplantation (Fig.6).
Numerous neurofilament immunolabelled axons were
detected within OEC transplants in all animals investigated.
This shows that regenerating axons retain the ability to grow
into transplants even when these are made four weeks after
the original spinal cord lesion.

68

No of animals with fibres rostral to lesion


Average of fibres 200 rostral
Average No of fibres 2001800 rostral

Acute Control
7/14
9
4

Acute Conditioning lesion


12/18
22
14

Delayed Control
4/5
5
2

Table 1. Quantification of fibres located rostral to the lesion (measurements taken at intervals of 200 , starting at 200 and ending at
1800 rostral to lesion).

Effect of delayed conditioning lesion

after the original injury. Furthermore, preliminary evidence


suggests that peripheral conditioning lesions may be
effective in promoting bridging regeneration even when
performed four weeks after the original dorsal column lesion.

To determine whether a delayed conditioning lesion


would result in bridging regeneration, sciatic nerve section
was performed at the same time as the delayed transplant
operation. Of the five animals so far prepared in this way,
one had transplanted OECs which were distributed rostrally
while the other four did not. Bridging regeneration was seen
only in the one animal with rostrally distributed OECs.
These preliminary results suggest that peripheral
conditioning lesions may be effective in promoting
bridging regeneration when combined with OECs even
when they are performed four weeks after the original dorsal
column lesion.

REFERENCES
Barnett S.C., Riddell J.S. (2007) Nat. Clin. Pract. Neurol. 3,
152161.
Li Y., Field P.M., Raisman G. (1997). Science 277, 20002.
Neumann S., Woolf C.J. (1999). Neuron. 23: 8391.
Neumann S., Skinner K., Basbaum A.I. (2005). PNAS 102,
1684852.
Richardson P., Issa V. (1984). Nature 309, 7913.
Ruitenberg M.J., Levison D.B., Voon Lee S., Verhaagen J.,
Harvey A.R., Plant G.W. (2005). Brain 128, 83953.
Steward O.,Zheng B., Tessier-Lavigne M. (2003). The
Journal of Comparative Neurology 459:18.
Toft A., Scott D.T., Barnett S.C., Riddell J.S. (2007). Brain
130:97084

CONCLUSION
Conditioning lesions appear to facilitate the regeneration
of sensory axons through an OEC transplant and beyond
the lesion in acute conditions of transplantation. The
regeneration can be observed in the white and grey matter,
in the central canal and specifically in areas containing
OECs which have spread rostral to the lesion.

FUTURE PLANS
Final year research plans
The data we have already collected from Schwann cell
transplanted animals shows that these cells distribute within
and beyond the lesion very similarly to OECs. Quantification
of BDA labeled fibres rostral to the lesion shows similar if not
greater axonal regeneration support of Schwann cells as
compared to OECs when coupled with conditioning lesions.

Moving a step closer to the clinic we have started to study


the effects of delayed transplantation after a spinal cord
injury. The results of this study show that when OECs are
transplanted four weeks after a dorsal column lesion, the cells
fill the lesion cavity but also become distributed both
rostrally and caudally within the spinal cord in some animals.
Neurofilament labeling shows that fibres regenerate
successfully and profusely into delayed transplants. Tracing
with BDA shows that ascending dorsal column fibres remain
confined mainly to the transplant, as seen in acute
transplantation. This shows that regeneration can be
successful even when transplants are performed four weeks

During this final year I shall complete the experimental


work described above, write up and discuss my thesis and
prepare the work for publication in pear reviewed scientific
journals.

69

Project Grant Reports


Promoting axon regeneration in the injured spinal cord by RNAi-mediated knockdown of receptors for neurite growth inhibitors
B. Blits, E. Ehlert and Joost Verhaagen
Role of microglia in spinal cord injury pain
Fabien Marchand, Elizabeth Bradbury and Stephen McMahon
Standing and Stepping with Intraspinal Microstimulation after Spinal Cord Injury
Vivian K. Mushahwar
Can Human Lamina Propria Olfactory Ensheathing Cells Expand, Migrate and Stimulate Rat SCI Repair as well as Mouse OECs?
Jane Roskams
Development of functional magnetic resonance imaging for assessing human spinal cord injuries
P.W. Stroman, R. Smith, R. Pokrupa and K. Smith
Improving cardiovascular function after spinal cord injury
P. Waite, P. Carrive and A. Mackay-Sim
Comprehensive evaluation of the physiological and functional adaptations induced by locomotor training in incomplete spinal cord
injured subjects
Bernie Conway, Ken Hunt, David Allan, Sujay Galen and Celia Caton

70

Promoting axon regeneration in the injured spinal cord


by RNAi-mediated knockdown of receptors for neurite
growth inhibitors
B. Blits, E. Ehlert and Joost Verhaagen
Department of Neuroregeneration, Netherlands Institute for Neuroscience
INTRODUCTION

astrocyte to a meningeal cell substrate is enhanced by


antibody-mediated blockade of the Npn-2 receptor (Shearer
et al., 2003). The growth-inhibitory properties of Sema3A
were further described when a selective inhibitor of Sema3A
was used to enhance regenerative responses and functional
recovery after spinal cord injury (Kaneko et al., 2006). An
attempt to block or remove semaphorin receptor expression
in the injured spinal neuron to promote regeneration
through the lesion core has not been performed yet.

The lesion environment of the injured spinal cord


constitutes a major impediment to regenerating axons
(reviewed in Schwab and Bartholdi, 1996, Silver and Miller,
2004, Niclou et al., 2006). The inhibitory environment at
the lesion site is a major obstacle for regenerating axons.
During the last two decades several neurite growth
inhibitors expressed in and around the lesion area have been
identified. These include scar-derived inhibitory factors such
as chondroitin sulphate proteoglycans (CSPGs) and secreted
semaphorins as well as the myelin-derived inhibitors
NogoA, myelin-associated glycoprotein (MAG) and
oligodendrocyte myelin glycoprotein (OMgp) (Asher et al.,
2001, De Wit et al., 2001, Filbin, 2003) Enzymatic removal
of CSPGs from a CNS lesion site leads to enhanced axon
growth into the scar and limited growth across the lesion
site, combined with a certain degree of functional recovery
(Moon et al., 2001, Bradbury et al., 2002, reviewed in
Fawcett, 2006). An attempt to collectively remove the other
two major types of inhibitory factors, semaphorins and
myelin-derived inhibitors and/or their receptors in a rat
spinal cord lesion model has not been reported yet.

The myelin inhibitors NogoA, MAG and OMgp are


potent neurite growth inhibitors in vitro and signal through
a common receptor complex composed of the Nogo66
receptor (NgR) and the low affinity neurotrophin receptor
p75 (McGee and Strittmatter, 2003). Additional NogoA
receptors may be involved but have not been identified
(Oertle et al., 2003). The group of Schwab (Schnell and
Schwab, 1990, Bayere et al., 2002) has shown that
antibodies against NogoA induce extensive nerve sprouting
and improve functional recovery in spinal cord lesioned rats
and primates (Freund at al., 2006). Similarly, a peptide
(NEP140) that prevents binding of Nogo66 to NgR
enhances regenerative growth in a spinal cord injury model
both after intrathecal or systemic delivery GrandPre et al.,
2002, Li and Strittmatter, 2003). Both approaches however
do not block the inhibitory effect of MAG and OMgp and
no intervention has been reported so far that unequivocally
removes the effect of all 3 myelin inhibitors.

Secreted semaphorins are expressed by meningeal


fibroblasts invading the lesion site after traumatic brain and
spinal cord injury (Pasterkamp et al., 1998, 1999, 2001, De
Winter et al., 2002). Semaphorins are potent
chemorepellents that guide axons during development and
their induction after CNS injury suggests that they
contribute to the inhibition of axon growth in the lesion
core. The receptor for secreted semaphorins is composed of
a neuropilin binding subunit (neuropilin-1 or neuropilin-2)
and a plexinA signaling subunit (plexin A1-A4). These
receptor components are expressed in neurons of the mature
nervous system and remain expressed following axonal
injury of spinal tract neurons e.g. in corticospinal tract and
rubrospinal tract (RST) neurons (De Winter et al., 2002,
Reza et al., 1999). Layer 5 cortical neurons strongly express
neuropilin-1 (Npn-1) and neuropilin-2 (Npn-2), while
rubrospinal neurons in the red nucleus express high levels of
Npn-2 but no Npn-1. Similarly, the signaling component
plexinA1 and the intracellular signaling molecule CRMP2
are present in cortical and rubrospinal neurons (De Winter
et al., 2002). Moreover, following injury of the RST, plexin
A1 and 4 remain expressed, whereas A2 is upregulated and
A3 undetectable in the red nucleus (Spinelli et al., 2007).
Thus the two major descending motor tracts in the spinal
cord are potentially sensitive to meningeal cell-derived
semaphorin activity in the lesion core. Recently, we have
shown that axon outgrowth is considerably improved when
neurons are cultured on semaphorin3A-deficient meningeal
cells (Niclou et al., 2003) and that axon crossing from an

In addition to expression of Npn-2 and class A Plexins,


rubrospinal neurons express NgR (Hunt et al., 2002,
Kobayashi et al., 1997), thus their regenerating axons are
potentially sensitive to both scar-derived semaphorins and
myelin inhibitors. Using viral vectors, these injured neurons
can be genetically manipulated at the level of the cell body
to deliver therapeutic molecules (for review, see Blits and
Bunge, 2006).
Aim of the project
The original first aim of this project was to locally knockdown Npn-2 and NgR in neurons of the injured RST using
viral vector-derived expression of small interfering RNAs
(shRNA). We have extended this aim, to studies on the knockdown of NP1 and NP2 in dorsal root ganglion neurons.
Furthermore we have started work on an alternative approach
to neutralize semaphorins, namely the use of semaphorinreceptor bodies (NP1 and NP2 bodies). The reasons for these
additional experimental approaches are motivated below and
specifically described in results of this report.
Most mature neurons that give rise to the long
descending pathways of the spinal cord, including
71

rubrospinal neurons, undergo severe atrophy upon spinal


axotomy. Deprivation of target-derived neurotrophic factors
is thought to be a main reason for lesion-induced atrophy.
Delivery of brain derived neurotrophic factor (BDNF) to
the cell soma of injured rubrospinal neurons delivered either
via minipump or via viral vectors has been shown to
overcome atrophy even when applied 18 months post- injury
(Kobayashi et al., 1997, Kwon et al., 2002, Ruitenberg et al.,
2004). Furthermore, BDNF application is able to activate
the expression of regeneration-associated genes and to
enhance axon growth into a permissive environment
(Kobayashi et al., 1997). These findings provide the basis for
the second aim of the project namely to combine viral
vector-mediated delivery of BDNF with application of
shRNAs against receptors for inhibitory factors developed in
the first aim. We hypothesize this combinatorial approach
will stimulate the intrinsic growth potential at the level of
the cell body while at the same time desensitizing the growth
cone for inhibitory molecules at the lesion site. A similar
strategy combining NgR blockade with stimulation of the
growth program of retinal ganglion cells has been shown to
be necessary to achieve regeneration through the injured
optic nerve (Fisher et al., 2004). By simultaneously
neutralizing more than one growth inhibitory pathway and
promoting the growth program with BDNF, the regenerative
response is hypothesized to be more robust.

injection into the L4 and L5 DRG or by stereotactic


infusion in the red nucleus prior.
Histology: Immunohistochemistry and in situ
hybridization. Co-expression of GFP in transduced
rubrospinal neurons and DRG neurons will allow to
identify siRNA-expressing neurons and their axons. In situ
hybridization and immunohistochemistry on rubrospinal
neurons and DRG neurons has been carried out to show
the loss of receptor NP1 and NP2 mRNA expression.
RESULTS
Part 1: Selection and characterization of NP2 shRNA
sequences.
To generate vectors that contain shRNA sequences that
efficiently target NP2, shRNAs were designed and cloned
into pSuper (Brumelkamp et al., 2002). As a primary
screening method, the resulting vectors were co-transfected
in HEK293T cells together with a myc-Npn-2 expression
cassette containing vector. Two days after transfection, mycNpn-2 expression was analyzed by Western blotting. The
Npn-2 expression was quantified and revealed knockdown
efficiencies of 20 to 75% with different shRNAs. Seven out
of the 11 tested shRNA sequences were successfully
knocking-down NP2 (knock-down >70%) using the cotransfection screening method.

METHOD
Small interfering RNAs. siRNA is a relatively new tool to
silence gene expression in a sequence-specific manner. We
used the pSUPER plasmid carrying the H1 RNA-polymerase
III promoter (Brummelkamp et al. 2002) to drive expression
of small hairpin RNAs complementary to the gene of interest
to knock down expression of NP1, NP2. The efficiency of
protein knockdown was analysed by qPCR and Western blot.
Functional siRNAs were cloned in lentiviral (LV) or AAV
vector expressing the marker green fluorescent protein (GFP).
Infection with this viral vector in vitro or in vivo, was used to
stably express siRNAs. Transduced neurons could be
identified by GFP fluorescence.

To validate the knockdown efficiency on endogenous


expression levels of Npn-2, we transfected these seven
shRNA sequences in F11 cells, a cell line derived from of rat
embryonal dorsal root ganglion and mouse neuroblastoma
cells. Two days after transfection total RNA was prepared
and Np2 mRNA expression was determined using
quantitative PCR. We observed residual Np2 expression
ranging from 18.45.9 % indicating efficient knockdown.

Bioassays: collapse, repulsion and neurite outgrowth


assay. The growth cone collapse assay, the neurite repulsion
and outgrowth assay were used as bioassays to test the
sensitivity of neurons to neurite growth inhibitory
molecules following shRNA-mediated knock-down of NP1
or NP2.
Viral vectors: generation and application in the nervous
system. The in vitro experiments have been performed with
lentiviral vector (LV)-driven siRNA expression since these
vectors efficiently transduce neuronal cell lines and cultured
primary neurons. In contrast, various adeno-associated viral
vectors (AAV1 to 6 and AAV8) were used for the in vivo
experiments as they are more efficient than LV in
transducing neurons in vivo. Furthermore AAV has been
used successfully in our lab to deliver growth factors to
intact and injured rubrospinal neurons. All AAV vectors
were produced by transient transfection of 293T cells and
purified using a iodoxanol gradient. AAV vectors delivering
GFP or the appropriate siRNAs were injected by direct

Fig. 1. Efficient lentivirus mediated Npn-2 knockdown in F11 cells.


F11 cells were infected with a lentivirus expressing GFP and a control
shRNA or shRNA directed against Npn-2. Two days after infection
Npn-2 mRNA expression was knocked down to 7.8 1.1 % and 13.3
1.0 % (mean SEM) (* p<0.005).

Since our goal is to achieve viral vector mediated


knockdown of Npn-2 in vivo, we examined whether the
selected shRNAs could also efficiently knock down Npn-2
using viral vector-mediated transduction. To this end, ten
72

shRNA sequences were cloned into a lentiviral plasmid


expressing both GFP and the shRNA and 10 lentiviral
vectors were produced. F11 cells were transduced and total
RNA was isolated after 2 days. Only 2 out of ten shRNA
sequences successfully reduced Npn-2 mRNA expression to
7.8 1.1 % and 13.3 1.0 % (mean SEM) (Fig.1). The
most likely reason for this is that a viral vector gives lower
levels of shRNA-expression per cell then a transfection with
an expression plasmid. Thus, only the most effective shRNA
sequences will result in knock-down of the target gene after
viral-vector mediated shRNA transfer.

AAV2 vectors containing each of the two effective


shRNAs were produced and used to determine whether we
would be able to knock down the expression of Npn-2 in
vivo. Rats (n=4 per group) were injected in the red nucleus
with an AAV-2 vector encoding these two shRNAs. Similar
to the lentiviral construct, the expression cassette also
contained CMV-GFP to allow recognition of transduced
cells. At 4 weeks post-injection, GFP positive neurons were
observed scattered throughout the nucleus. However, in situ
hybridization for Npn-2 mRNA revealed no clear difference
in NP2 mRNA expression between transduced and nontransduced neurons or between neurons transduced with a
control AAV2-GFP vector and an AAV2-shRNA-NP2
vector. Based on the relatively low level of transgene
expression following AAV2-mediated gene transfer in the
red nucleus we concluded that a higher transduction
efficiency, for instance using an alternative AAV serotype,
would be required to knock-down NP-2 (see part 5 below).

Part 2: in vivo knock-down of NP2 with shRNA


The two lentiviral vectors encoding the effective NP2shRNA sequences were used to examine whether
knock-down of NP2 could be achieved in neurons of the
red nucleus. Rats (n=4 per group) were stereotactically
injected with a lentiviral vector encoding an NP2-shRNA or
a control shRNA. Animals were sacrificed 1, 2 and 4 weeks
after the injection and processed for immunohistochemistry
(to detect GFP) and in-situ hybridisation (to detect NP2
mRNA). Based on GFP-labelling the LV-vector transduced
both astroglia cells as well as some rubrospinal neurons.
Unexpectedly we observed an adverse tissue response in the
nucleus with increased in time and was apparent in all
animals at 4 weeks. Although the observed tissue damage
appeared to be clearly related to the expression of the
shRNA we could not exclude the possibility that the
lentiviral vector itself or and immunological response against
GFP contributes to this phenomenon. We therefore decided
to reclone the shRNA expression cassettes into adenoassociated viral vector plasmids. AAV vectors preferentially
transduce neurons and may therefore be a much more
effective and specific viral vector system for knock-down of
a gene in neurons then LV.

We decided to study NP2 knock-down in an additional


model system often used for regeneration studies, namely the
rat dorsal root ganglia (DRG). An advantage of the DRG is
that DRG-neurons have a more uniform expression of NP-2 as
compared to red nucleus neurons. Moreover, we could benefit
from results obtained by our colleague Matthew Mason who
demonstrated that a single injection of AAV-5 encoding GFP
into adult rat DRG resulted in high levels of transgene
expression in approximately 5080% of DRG-neurons. We
therefore cross packaged our NP2 targeting shRNA vectors into
AAV-5 particles and injected this vector into the L4 and L5
DRG of 3 adult female Wistar rats. 3 Weeks after injection
NP2 expression was analysed by in situ hybridisation. One of
the two shRNA sequences was able to significantly reduce the
proportion NP2 expressing cells (Figure 2) while the second
shRNA was not effective. It is not clear why only one of the two
shRNA sequences was effective in vivo.

Fig. 2. AAV5 mediated knockdown of Npn-2 in DRG in vivo. AAV5 particles expressing GFP and a control shRNA (a,b,c) or shRNA directed
against Npn-2 (d,e,f) were injected in the L4 and L5 DRG of 3 adult female wistar rats. Three Weeks after injection Npn-2 mRNA was
detected by in situ hybridisation (red: a,c,d,f). In the same section GFP was detected with immunohistochemistry (green: a,b,d,e). In the control
animals the percentage of npn-2 expressing cells among the GFP positive cells was 37.2 6.59 %. In the animals expressing shRNA the percentage
of npn-2 expressing cells is significantly lower (16.45 4.96 %, mean SEM, * p<0.03).
73

Fig. 3. Efficient lentivirus mediated Npn-1 knock-down in F11 cells. F11 cells were infected with a lentiviral vector expressing GFP and a control
shRNA or shRNA directed against NP1. Np1 mRNA expression was reduced to 31.4 1.8 % and 17.5 3.4 %. (a). Western blot quantification
(b,c) showed a similar knockdown efficiency 39.2 9.7 % and 5.7 2.6 % (mean SEM) (* <0.05, ** p<0.005).

Part 3: Selection and characterization of Npn-1 shRNA


sequences.
Following a similar strategy as described above, we have
developed two lentiviral vectors expressing shRNAs that
knock down Np1 mRNA expression to 31.4 1.8 % and
17.5 3.4 % in F11 cells (Fig 3a). Western blot analysis
revealed that NP1 protein expression was reduced to 39.2
9.7 % and 5.7 2.6 % (fig. 3b and 3c).

red nucleus (Fig. 4). As shown previously, the traditionally


used serotype AAV-2 drives transgene expression in scattered
neurons throughout the red nucleus. This is sufficient in
situations where the expression of a secreted molecule such
as BDNF is required (Ruitenberg et al., 2004). However, for
shRNA-mediated experiments at the level of the cell body, it
is critical that the majority of, if not all, neurons is
transduced to efficiently knock-down the protein of interest
in a large proportion of neurons. When we injected titermatched batches of AAV of serotypes 16 and 8, AAV-1 and
8 transduced much more neurons in the red nucleus as
compared to AAV-2 (Figure 5). We also confirmed that the
lentiviral vector transduced both glial and neuronal cells and
would therefore not be the vector of choice for shRNA
delivery to neurons into the nucleus (see also part 2 above).

Part 4: LV-shRNA-mediated knock-down of NP1 in


F11 cells leads to increase outgrowth of these cells on
human neural scar tissue containing sema3A
Human neural scar tissue that is neurosurgically removed
from infants with severe brachial plexus injuries prior to
reconstructive surgery contains high levels of sema3A. F11
cells grown on sections of human scar tissue were
significantly inhibited in their neurite outgrowth response.
The LV-shRNA-NP1 vector was employed to test whether
knock-down of NP1 would lead to enhanced neurite
outgrowth of F11 cells on human neural scar tissue. F11 cells
transduced with LV-shRNA-NP1 and transduced with
control vector were plated on 20um thick fresly frozen
neuroma tissue. Neurite outgrowth was stimulated with
forskolin and 48 hours after plating cells were fixed and
neurite outgrowth was measured. F11 cells with NP1 knockdown did extend significantly longer neurites on human scar
tissue. This demonstrates that NP1 knock-down could be a
potential strategy to (partially) overcome the inhibitory
activity of sema3A in scar tissue. This part of the project has
recently been published in Tannemaat et al. 2007.
Part 5: Optimization of transduction of the red
nucleus using seven AAV serotypes.
So far, gene transfer into the red nucleus has been successfully
performed with recombinant AAV vectors of serotype 2. It is
becoming increasingly clear that other serotypes may have
differential and more favorable transduction properties for
various sets of neurons. Since we did not observe knockdown of NP2 following injection of AAV2 vectors encoding
shRNA targeting NP2 (see part 2 above) we decided to study
the transduction properties of 7 AAV serotypes (16 and 8)
and a lentiviral vector encoding GFP after injection into the

Fig. 4. Overview of the experiment. Crosspackaged rAAV stocks


(serotypes 16 and 8) encoding GFP were titermatched at
51011 GC/ml and 1 l was injected into the red nucleus (RN). Also
in this experiment, a group of animals received injection of a lentiviral
vector with the same expression cassette. At 1 week and 1 month postinjection, transduction efficiency and specificity of the red nucleus was
determined. Moreover, the number of GFP positive rubrospinal tract
fibers were measured in the spinal cord at the cervical level 3 at
1 month postinjection. RN, red nucleus; cg, central grey; RST,
rubrospinal tract.

74

Fig. 5. Correlation between transduction efficiency of AAV-transduced RN neurons and labeling of RST fibers at C3. A strong correlation was
found (R2=0.96) between the proportion of cell bodies transduced and the number of fibers at the C3 level in the spinal cord in AAV-injected
animals. The LV-injected animal did not fit the plotted trend line, probably because LV vectors transduce many other cells as well. These data
suggest that AAV-1 vectors are most optimal to express shRNA to knock down receptor expression and study its effect on nerve fiber regeneration.

Part 6: AAV1-mediated overexpression of shRNA in


the red nucleus results in an adverse tissue response
and neuronal degeneration
The results described above demonstrate that AAV1 is the
most efficient vector for transduction of the red nucleus.
Therefore AAV1 vectors were generated encoding GFP, a
control shRNA and two independent NP2-shRNAs. These
vectors were stereotactically injected in the red nucleus of rats
(n=4 per group). Animals were sacrificed at 4 weeks after the
injection and processed for GFP-immunohistochemistry and
in-situ hybridization for NP2 mRNA. In the red nucleus of
animals injected with AAV1 vectors encoding a control
shRNA or a NP2-shRNA an adverse tissue response was
clearly visible while in animal injected with AAV1-GFP
control vector transduced neurons had a healthy appearance
(Figure 6A and inserts).
High magnification photomicrographs demonstrate
vacuolar structures in neurons transduced with an AAV1
vector encoding shRNA (Figure 6B). These vacuolar
structure are not present in AAV1-GFP transduced neurons
(Figure 6C). The toxic effects of shRNA have recently also
been observed and described by others in the brain
(McBride et al. 2008) and in the liver (Grimm, 2006)

Fig. 6. shRNA induced toxicity in the red nucleus. AAV1 particles


expressing GFP and a control shRNA (a,b) or GFP alone (c) were
injected in the red nucleus of adult female rats. Injection of AAV1
particles expressing GFP and a control shRNA results in a reduction
of rubrospinal motor neurons and small diameter cell infiltration as
shown in cresyl violet staining (a). Insets show enlargements of the
injected and contralateral side (a, a) High magnification images of
GFP epifluorescence show vacuolar structures in neurons transduced
with an AAV1 vector encoding shRNA (b). These vacuolar structure
are not present in AAV1-GFP transduced neurons (C).
75

Part 7: NP1 receptor bodies as an alternative strategy


for shRNA.

2. The inhibition of neurite outgrowth of F11 cells grown


on human scar tissue could be partially overcome by
shRNA-mediated knock-down of NP1 demonstrating
that interference with semaphorin-neuropilin signaling
may be an effective strategy to promote regeneration.
3. Unexpectedly, viral vector-mediated expression of
shRNA in the red nucleus resulted in an adverse tissue
response and neuronal degeneration. Toxic effects of
shRNA have recently also been documented by others
(McBride et al. 2008, Grimm, 2006) and have important
implications for the therapeutic development of RNA
interference. Solutions for this problem are now
discussed in the shRNA community and in future studies
we will test a number of potential solutions.
4. AAV5-mediated NP2-shRNA expression in the DRG
was successful with one shRNA sequence but failed
with another shRNA sequence. It is not clear what the
explanation for this is but this demonstrates that
shRNAs that are effective in cultured cells may not
always be effective in vivo.
5. The observation that AAV5-mediated expression of
NP2-shRNA in DRG was successful and did not induce
toxicity, while in contrast, the same shRNA sequence did
induce toxicity in red nucleus neurons suggests that
different populations of neurons respond differently to
the same shRNA sequence. This may be due to
differential expression of components of the miRNA
processing pathway in different sets of neurons.
6. NP-1 receptor-bodies effectively neutralize sema3A and
may provide a valuable and power alternative approach
to overcome inhibition by semaphorins.

In view of the problems encountered with the application of


shRNA in an in vivo setting we decided that it was necessary
to adopt an alternative strategy to neutralize class 3
semaphorins. To this end we have developed NP-1 receptor
bodies. Receptor bodies are secreted receptors that interact
and neutralize the ligand. Lentiviral vectors containing an
Fc fragment fused to the extracellular domain of Npn-1,
under the transcriptional control of the CMV promoter were
produced and secretion of the Npn-1 bodies after infection
was verified using Western blotting. The biological activity of
the Npn-1 bodies was tested in DRG outgrowth assays in
the presence of Sema3A. The repulsive effect of Sema3A
could effectively be abolished by co-expressing NP1 receptor
bodies (Figure 7). Moreover, we have developed Npn-2
receptor bodies to desensitize neurons to additional members
of class 3 semaphorins. These sequences encoding Npn-2
bodies will be cloned into a lentiviral vector. We plan to use
the viral vector-mediated shRNA following rubrospinal tract
lesion at the level of the cell bodies in the red nucleus,
whereas the receptor bodies will be produced by genetically
modified primary Schwann cells that serve as biological
minipumps at the level of the injury site.
CONCLUSION
1. Effective knock-down of the semaphorin receptors NP1
and NP2 with shRNA was achieved in non-neuronal
cells and in a neuronal cell line (F11 cells). Knock-down
was demonstrated following transfection of these cells
with expression plasmids as well as after transduction
with lentiviral vectors.

Fig. 7. Neuropilin-1 receptor bodies neutralize semaphorin3a neurite repulsion. A sema3a expressing cell clump repulses DRG neurite outgrowth
(a). Following infection of these sema3A expressing cells with a lentiviral vector encoding NP1 bodies Sema3a repulsion is abolished (b).
Quantification of neurite outgrowth in C further documents this effect. Normally outgrowth in quadrants 1 and 3 is similar and the ration
between outgrowth in 1 and 3 approximates 1.0. After exposure of the DRG to sema3A secreting cells outgrowth in segment 1 well be absent as
fibers are repulsed resulting in a significantly higher relative repulsion. Co-expression of sema3A and GFP has no effect on repulsion, in contrast
co-expression of sema3A and the NP1-body results in near normal outgrowth in quadrant 1.
76

REFERENCES
Asher R.A., Morgenstern D.A., Moon L.D., Fawcett J.W.
Chondroitin
sulphate
proteoglycans:
inhibitory
components of the glial scar. Prog. Brain Res. 132, 611619
(2001).
Blits B., Bunge M.B. Direct gene therapy for repair of the
spinal cord. J. Neurotrauma 23: 508520 (2006).
Bradbury E.J. et al. Chondroitinase ABC promotes
functional recovery after spinal cord injury. Nature 416,
636640 (2002).
Brummelkamp T.R., Bernards R., Agami R. A system for
stable expression of short interfering RNAs in mammalian
cells. Science 296, 550553 (2002).
De Winter F. et al. Injury-induced class 3 semaphorin
expression in the rat spinal cord. Exp. Neurol. 175, 6175
(2002).
De Wit J., Verhaagen J. Role of semaphorins in the adult
nervous system. Prog. Neurobiol. 71, 249267 (2003).
Fawcett J. Overcoming Inhibition in the damaged spinal
cord. J. Neurotrauma 23: 371383 (2006).
Fischer D., He Z., Benowitz L.I. Counteracting the Nogo
receptor enhances optic nerve regeneration if retinal
ganglion cells are in an active growth state. J. Neurosci. 24,
16461651 (2004).
Filbin M.T. Myelin-associated inhibitors of axonal
regeneration in the adult mammalian CNS. Nat. Rev.
Neurosci. 4, 703713 (2003).
Freund P., Schmidlin E., Wannier T., Bloch J., Mir A.,
Schwab M.E., Rouiller, E. Nogo-A-s pecific antibody
treatment enhances sprouting and functional recovery
after cervical lesion in adult primates. Nat. Med. 12: 790
792. (2006).
GrandPre T., Li S., Strittmatter S.M. Nogo-66 receptor
antagonist peptide promotes axonal regeneration. Nature
417, 547551 (2002).
Grimm D. Fatality in mice due to oversaturation of cellular
miRNA/short hairpin pathways. Nature 441: 537541
(2006)
Hamers F.T.P., Koopmans G.C., Joosten, E.A.J. Catwalkassisted gait analysis in the assessment of spinal cord injury.
J. Neurotrauma 23: 537548 (2006).
Higuchi H., Yamashita T., Yoshikawa H., Tohyama M.
Functional inhibition of the p75 receptor using a small
interfering RNA. Biochem. Biophys. Res. Commun. 301,
804809 (2003).
Hunt D., Mason M.R., Campbell G., Coffin R., Anderson
P.N. Nogo receptor mRNA expression in intact and
regenerating CNS neurons. Mol. Cell Neurosci. 20, 537552
(2002).
Kaneko S et al. A selective Sema3A inhibitor enhances
regenerative responses and functional recovery of the injured
spinal cord. Nat. Med. 12:13809 (2006)
Kobayashi N.R. et al. BDNF and NT-4/5 prevent atrophy
of rat rubrospinal neurons after cervical axotomy, stimulate
GAP-43 and Talpha1-tubulin mRNA expression, and
promote axonal regeneration. J. Neurosci. 17, 95839595
(1997).
Kwon B.K. et al. Survival and regeneration of rubrospinal
neurons 1 year after spinal cord injury. Proc. Natl. Acad. Sci.
USA 99, 32463251 (2002).

Li S., Strittmatter S.M. Delayed systemic Nogo-66 receptor


antagonist promotes recovery from spinal cord injury. J.
Neurosci. 23, 42194227 (2003).
McGee A.W.,& Strittmatter S.M. The Nogo-66 receptor:
focusing myelin inhibition of axon regeneration. Trends
Neurosci. 26, 193198 (2003).
McBride J.L., Boudreau R.L., Harper S.Q., Davidson B.L.
Artificial miRNAs mitigate shRNA-mediated toxicity in the
brain: Implications for the therapeutic development of
RNAi. PNAS 105: 58685873 (2008)
Moon L.D., Asher R.A., Rhodes K.E., Fawcett J.W.
Regeneration of CNS axons back to their target following
treatment of adult rat brain with chondroitinase ABC. Nat.
Neurosci. 4, 465466 (2001).
Niclou S.P., Franssen E.H., Ehlert E.M., Taniguchi M,
Verhaagen J. Meningeal cell-derived semaphorin 3A inhibits
neurite outgrowth. Mol. Cell Neurosci. 24, 902912 (2003).
Niclou S.P., Ehlert E.M.E., Verhaagen J. Chemorepellent
axon guidance molecules in spinal cord injury. J.
Neurotrauma 23: 409421 (2006).
Oertle T. et al. Nogo-A inhibits neurite outgrowth and cell
spreading with three discrete regions. J. Neurosci. 23, 5393
5406 (2003).
Pasterkamp R.J., De Winter F., Holtmaat A.J., Verhaagen J.
Evidence for a role of the chemorepellent semaphorin III
and its receptor neuropilin-1 in the regeneration of primary
olfactory axons. J. Neurosci. 18, 99629976 (1998).
Pasterkamp R.J. et al. Expression of the gene encoding the
chemorepellent semaphorin III is induced in the fibroblast
component of neural scar tissue formed following injuries of
adult but not neonatal CNS. Mol. Cell Neurosci. 13, 143
166 (1999).
Pasterkamp R.J., Anderson P.N., Verhaagen J. Peripheral
nerve injury fails to induce growth of lesioned ascending
dorsal column axons into spinal cord scar tissue expressing
the axon repellent Semaphorin3A. Eur. J. Neurosci. 13, 457
471 (2001).
Reza J.N., Gavazzi I., Cohen J. Neuropilin-1 is expressed
on adult mammalian dorsal root ganglion neurons and
mediates semaphorin3a/collapsin-1-induced growth cone
collapse by small diameter sensory afferents. Mol. Cell
Neurosci. 14, 317326 (1999).
Ruitenberg M.J. et al. Adeno-associated viral vectormediated gene transfer of brain-derived neurotrophic factor
reverses atrophy of rubrospinal neurons following both
acute and chronic spinal cord injury. Neurobiol. Dis. 15,
394406 (2004).
Schwab M.E., Bartholdi D. Degeneration and regeneration
of axons in the lesioned spinal cord. Physiol. Rev. 76, 319
370 (1996).
Shearer M.C. et al. The astrocyte/meningeal cell interface
is a barrier to neurite outgrowth which can be overcome by
manipulation of inhibitory molecules or axonal signalling
pathways. Mol. Cell Neurosci. 24, 913925 (2003).
Silver J., Miller J.H. Regeneration beyond the glial scar. Nat.
Rev. Neurosci. 5, 146156 (2004).
Spinelli E.J. Class A plexin expression in axotomized
rubrospinal and facial motoneurons. Neuroscience 144,
126677 (2007)
Tannemaat M.R., Korecka J., Ehlert E.M.E., Mason M.J.,
Van Duinen S.G., Boer G.J., Malessy M.J.A., Verhaagen J.
77

as a result of work performed in the context of the ISRTgrant. The following future experiments will be performed
and are mostly aimed at solving the issues with toxicity and
effectiveness of shRNA. First, we will adopt the strategy of
McBride et al. (2008) and embed the shRNAs that we have
developed into a naturally occurring microRNA context.
McBride et al. have convincingly shown that this reduces
or even mitigates shRNA toxicity. We are currently
communicating with the group of Beverly Davidson about
the various technical aspects of this approach. Second, we
will execute an experiment in which we will use the AAV5NP2-shRNA vector to knock-down NP2 in DRG neurons
after a lesion. With this vector knock-down was successful in
vivo in DRG with intact ascending fibers and we are of the
opinion that a lesion experiment may yield important results.
Third, after the issues with the application of shRNA have
been resolved we will execute a combination-treatment
experiment in which the effect of simultaneous knock-down
of NP1 and NP2 and overexpression of BDNF is
investigated. Finally, the alternative and shRNA-independent
approach, i.e. the application of NP1 and NP2 receptor
bodies at the siteof the lesion, will also be actively pursued.

Human neuroma contains increased levels of semaphorin3A,


which surrounds nerve fibers and reduces neurite extension in
vitro. J. Neuroscience 26: 14260264 (2007)
Tannemaat M.R., Korecka J., Ehlert E.M.E., Mason M.J.,
Van Duinen S.G., Boer G.J., Malessy M.J.A., Verhaagen J.
Human neuroma contains increased levels of semaphorin3A,
which surrounds nerve fibers and reduces neurite extension in
vitro. J. Neuroscience 26: 14260264 (2007)
Pasterkamp R.J., Verhaagen J. Semaphorins in axon
regeneration: developmental guidance molecules gone
wrong? Phil. Trans. R. Soc. 361: 14991511 (2006)
Several publications are anticipated to be submitted in the
coming years that build on the results obtained in this grant.
The ISRT will be acknowledged in these paper.
FUTURE PLANS
Due to the problems that we have encountered with the in
vivo application of shRNA (as documented in this final
report) we have not yet achieved the final objectives of the
ISRT-funded grant proposal. Fortunately, we have secured
funding in the form of a European FP-7 grant that will
enable us to build on the results and the experience gained

78

Role of microglia in spinal cord injury pain


Fabien Marchand, Elizabeth Bradbury, Stephen McMahon
Centre of Age Related Disease, Kings College London, UK
INTRODUCTION

RESULTS

Pain following spinal cord injury severely compromises the


quality of life in nearly 70% of patients. When rated by
patients, its impact is only surpassed by loss of movement,
sexual function and bladder/bowel function (WinderstromNoga et al., 1999). Several types of pain are commonly seen
with different characteristics; among these, neuropathic pain
is frequent (38% of those reporting pain), the most
debilitating, and described as severe or excruciating pain. It
is also the most difficult to treat (Yzierski, 2000). Despite
the prevalence and severity of this type of pain, it has been
investigated much less than pain resulting from injury to
peripheral tissues. As a result, the mechanisms underlying
spinal injury pain are still poorly understood. Recent studies
have demonstrated a major role of microglial activation in
neuropathic pain development after peripheral nerve injury
(Watkins and Maier, 2003). In that case, a cascade of events
within microglia includes the de novo expression of ATP
receptors, activation of p38MAPKinase and release of painproducing cytokines (Jin et al., 2003; Tsuda et al., 2003;
Inoue et al., 2004). While there is widespread activation of
microglia after spinal cord injury, their contribution to
spinal cord injury pain is unknown.

Paper 1. Marchand F., Tsantoulas C., Singh D., Grist J.,


Clark A.K., Bradbury E.J., McMahon S.B. Effects of
Etanercept and Minocycline in a rat model of spinal cord
injury. Eur. J. Pain. 2008 Oct 10.
This study was a direct test of the major hypothesis
set out in the original application (from the application:
aim (1) is to determine whether microglial activation is
a key mechanism underlying spinal cord injury pain.). We
confirmed using the microglial inhibitor minocycline could
prevent the emergence of neuropathic pain behavior in rats
subjected to experimental spinal cord injury. The same
study also revealed that a critical mediator of pain under
these conditions was TNF released within the damaged
spinal cord.
Paper 2. Marchand F., Perretti M., McMahon S.B. Role of
the immune system in chronic pain. Nat. Rev. Neurosci.
2005 Jul;6(7):52132.
This invited review of the role of the immune system in
neuropathic states was published in a high impact journal
and has been much quoted (ISI web of knowledge finds 138
citations). It is likely to have significantly promoted our
ideas and hypotheses about the role of microglial cells
amongst the scientific community.

Thus, the principal aim of this project grant was to test


the hypothesis that spinal cells contribute to neuropathic
pain, particularly in the context of spinal cord injury.
METHODS
We undertook a number of experimental studies as part of this
grant. All of these were performed in the rat. The general
thrust of the work was to study both experimental models of
spinal cord injury to asses the role of glial cells, or other, more
general models, of neuropathic pain, in order to identify novel
mechanisms that might contribute to such pain states. Spinal
cord injury was induced in rats either as traumatic and
controlled lesions (section of specific spinal tracts) or with a
commercial contusion device (an Infinite Horizon impactor).
Pain related behavior was assessed in these models using
traditional techniques, that is, either the degree of stimulation
to induce vocalization or that necessary to promote
coordinated withdrawal reflexes by the animal. Other
techniques anatomical, immunohistochemical, behavioural,
were used as necessary to test hypotheses at different times.
The principle ways of assessing the role of glial cells (the central
hypothesis posited) was to either examine microglial responses
under different conditions (using immunohistochemistry,
Western blotting, anatomy) or behavioral outcomes in the
presence or absence of glial inhibitors.

Paper 3. Thacker M.A., Clark A.K., Bishop T., Grist J., Yip
P.K., Moon L.D., Thompson S.W., Marchand F.,
McMahon S.B. CCL2 is a key mediator of microglia
activation in neuropathic pain states. Eur. J Pain. 2008
This paper provided evidence for the importance of one
particular chemokine, CCL2, in the activation of spinal glial
cells in neuropathic pain states. The findings suggest that
antagonists of the CCL2 receptor may be of some benefit in
treating spinal cord injury pain.
Paper 4. Clark A.K., Yip P.K., Grist J., Gentry C., Staniland
A.A., Marchand F., Dehvari M., Wotherspoon G., Winter J.,
Ullah J., Bevan S., Malcangio M. Inhibition of spinal
microglial cathepsin S for the reversal of neuropathic pain.
Proc. Natl. Acad. Sci. USA. 2007 Jun 19;104(25):1065560.
This study, also published in a high profile journal,
demonstrated the importance of a previously unrecognized
enzyme synthesized within spinal microglia, in the
generation of neuropathic pain. It also provided a unifying
hypothesis about the role of fractalkine in these states.

A brief description of each study is given in section 4


below, presented as a description of the 8 original research
papers and three review articles produced directly as a result
of the ISRT grant with the ISRT fellow as an author.

Paper 5. Wodarski R., Clark A.K., Grist J., Marchand F.,


Malcangio M. Gabapentin reverses microglial activation in
the spinal cord of streptozotocin-induced diabetic rats. Eur.
J. Pain. 2008. Oct. 30 [Epub ahead of print]
79

This study demonstrated the potential use of an existing


analgesic drug, gabapentin, in blocking microglial responses
in neuropathic pain states.

four of which are in the premiership of scientific journals.


The original hypothesis posited was demonstrated to be true
and numerous aspects of the underlying contributory
mechanism were elucidated. This has proved a competitive
field and several publications from the laboratory of Dr
Hains were published during the period of the grant which
reinforced our original hypothesis (Hains et al., 2005; Hains
and Waxman 2006; Zhao et al.,2007). The findings of our
work and that of others all support the contention that
interventions aimed at limiting glial responses are likely to
be of benefit in treating spinal cord injury pain, especially if
implemented early.

Paper 6 and 7. Pezet S., Marchand F., DMello R., Grist J.,
Clark A.K., Malcangio M., Dickenson A.H., Williams R.J.,
McMahon S.B. Phosphatidylinositol 3-kinase is a key
mediator of central sensitization in painful inflammatory
conditions. J. Neurosci. 2008 Apr. 16;28(16):426170.
And Clark A.K., DAquisto F., Gentry C., Marchand F.,
McMahon S.B., Malcangio M. Rapid co-release of
interleukin 1beta and caspase 1 in spinal cord inflammation.
J. Neurochem. 2006 Nov.;99(3):86880. Epub. 2006 Aug. 29

REFERENCES
Hains B.C., Saab C.Y., Waxman S.G. (2005) Changes in
electrophysiological properties and sodium channel Nav1.3
expression in thalamic neurons after spinal cord injury.
Brain. 2005 Oct.;128(Pt 10):235971. Epub. 2005 Aug. 18.
Hains B.C., Waxman S.G.. (2006) Activated microglia
contribute to the maintenance of chronic pain after spinal
cord injury. J. Neurosci. 26(16):430817.
Inoue K., Tsuda M., Koizumi S.. ATP- and adenosinemediated signaling in the central nervous system: chronic
pain and microglia: involvement of the ATP receptor P2X4.
J. Pharmacol. Sci. 2004 ;94(2):1124.
Jin S.X., Zhuang Z.Y., Woolf C.J., Ji R.R.. p38 mitogenactivated protein kinase is activated after a spinal nerve
ligation in spinal cord microglia and dorsal root ganglion
neurons and contributes to the generation of neuropathic
pain. J. Neurosci. 2003 15;23(10):401722.
Tsuda M., Shigemoto-Mogami Y., Koizumi S., Mizokoshi
A., Kohsaka S., Salter M.W., Inoue K. P2X4 receptors
induced in spinal microglia gate tactile allodynia after nerve
injury. Nature. 2003 14;424(6950):77883.
Watkins L.R., Maier S.F. Glia: a novel drug discovery target
for clinical pain. Nat. Rev. Drug Discov. 2003 2(12):97385.
Widerstrom-Noga E.G., Felipe-Cuervo E., Broton J.G.,
Duncan R.C., Yezierski R.P. Perceived difficulty in dealing
with consequences of spinal cord injury. Arch. Phys. Med.
Rehabil. 1999;80(5):5806.
Yzierski R.P. Pain following spinal cord injury:
pathophysiology and central mechanisms. In: Sandkulher
J., Bromm B., Gehbart G.F., editors. Progress in Brain
Research, vol. 129. Elsevier, 2000. pp. 429449.
Zhao P., Waxman S.G., Hains B.C.. (2007) Modulation of
thalamic nociceptive processing after spinal cord injury
through remote activation of thalamic microglia by cysteine
cysteine chemokine ligand 21. J. Neurosci. 27(33):8893902.

These two studies were somewhat tangential to the main


thrust of the original grant proposal, but they arose as
collaborations exploiting skills and knowledge that was
gained during the pursuit of the grant proposal. They
demonstrate the synergy possible in science and the value
of opportunistic collaborations. Both papers are in high
impact journals and examine the mechanisms contributing
to centrally generated pain.
Paper 8 and 9. Thacker M.A., Clark A.K., Marchand F.,
McMahon S.B. Pathophysiology of peripheral neuropathic
pain: immune cells and molecules. Anesth. Analg. 2007
Sep.;105(3):83847.
McMahon S.B., Cafferty W.B., Marchand F. Immune
and glial cell factors as pain mediators and modulators. Exp.
Neurol. 2005 Apr;192(2):44462.
Two review articles invited on the basis of our work on
role of spinal glial in neuropathic pain.
Paper 10 and 11. Barritt A.W., Davies M., Marchand F.,
Hartley R., Grist J., Yip P., McMahon S.B., Bradbury E.J.
Chondroitinase ABC promotes sprouting of intact and
injured spinal systems after spinal cord injury. J. Neurosci.
2006 Oct. 18;26(42):1085667.
And Bordet T., Buisson B., Michaud M., Abitbol J.L.,
Marchand F., Grist J., Andriambeloson E., Malcangio M.,
Pruss R.M. Specific antinociceptive activity of cholest-4-en3-one,oxime (TRO19622) in experimental models of
painful diabetic and chemotherapy-induced neuropathy.
J. Pharmacol. Exp. Ther. 2008 May 20.
Two further papers the former in a very high profile
journal emerging from collaborations made possible by
the skills gained from the ISRT grant. The work does not
directly address spinal-cord injury pain but examined a
possible therapeutic intervention in neuropathic pain states
in general.

FUTURE PLANS
We will continue to explore the mechanisms of spinal cord
injury pain, building on the data obtained with this ISRT
grant. Our current efforts are focused on the role of P2X
receptors on microglia. Based on our preclinical data, we are
trying to establish a European network of preclinical and
clinical laboratories who will be able to test out some of the
hypotheses in small clinician-led trials. The consortium is
being coordinated by Dr N. Finnerup in Denmark.

CONCLUSION
This was a very successful and productive period of research
supported by an ISRT grant. It has generated a total of
11 original publications (and two further in preparation),
80

Standing and Stepping with Intraspinal Microstimulation


after Spinal Cord Injury
Vivian K. Mushahwar
Department of Biomedical Engineering, University of Alberta Canada. vivian.mushahwar@ualberta.ca
INTRODUCTION

combination of rehabilitation interventions, which could


include ISMS, will ultimately lead to the best functional
recovery after SCI.

The overall goal of this project is to evaluate the long-term


stability and functionality of intraspinal microstimulation
(ISMS) in restoring standing and over-ground stepping after
spinal cord injury (SCI). Intraspinal microstimulation is a
novel neuroprosthetic approach for restoring motor function
after SCI, head trauma, or stroke. It entails implanting very
fine wires in the spinal cord and delivering electrical stimuli
through these wires to generate functional limb movements.
Previous findings in intact animals with chronically
implanted microwires suggested that ISMS in the
lumbosacral region of the cord (the region that controls leg
movements which is only ~5cm-long in humans) may be safe
and feasible. Initial experiments in animals with complete
SCI showed that ISMS activates inherent neuronal networks
and is capable of inducing single joint movements and
coordinated multi-joint synergies. The current project is
designed to investigate the efficacy of ISMS in restoring stable
standing and over-ground walking after SCI, and to examine
certain aspects of neuronal and muscular plasticity induced by
long-term ISMS. It will assess the pre-clinical feasibility of
ISMS and provide the basis for the development of a
preliminary clinical trial in suitable subjects. If successful, we
anticipate that ISMS will allow people with SCI a greater
degree of leg mobility which will in turn increase functional
independence, cardiovascular fitness, bone density and
muscle health. By the end of the funding period, we expect to
have evaluated ISMS as a means for restoring functional leg
movements, and to provide detailed parameters regarding the
number and configuration of implanted microwires, and the
best stimulation parameters for generating stable weightbearing standing and over-ground stepping.

ISRT SUPPORTED STUDIES, RESULTS, AND


CONCLUSIONS
In 2007, we a) continued our investigations in understanding
the mechanism and sites of actions of ISMS, b) continued
our work in assessing the long-term effects of ISMS on
muscle, and c) initiated studies related to understanding the
effects of long-term ISMS on neural plasticity.
a) Mechanism and sites of action of intraspinal
microstimulation
Our results to date demonstrate that ISMS through
individual microwires generates graded single joint
movements as well as coordinated multi-joint synergies such
as whole limb extensor, flexor, forward and backward
movements. Phasic ISMS through as few as 4 microwires in
each side of the of the cord in adult cats with complete
thoracic SCI produces near-normal in-place stepping of the
paralyzed hindlimbs that is fully weight-bearing during
stance and has ample foot clearance during swing. Moreover,
tonic, subthreshold ISMS through a small group of microwires
placed bilaterally in the ventral horn in cats with SCI
produces rhythmically alternating locomotor-like patterns
of the hindlimbs involving the full orchestration of the
locomotor networks within the cord. These exciting results
raised questions regarding the mechanisms of action of
ISMS. More specifically, we asked: how does the focally
applied ISMS recruit distally located motoneuron pools in
the lumbosacral cord to produce multi-joint synergies and
locomotor rhythms? We hypothesized that in addition to
recruiting directly motoneurons in the close vicinity of the
microwire tips, ISMS activates axons in passage that are
composed of both projections of afferent fibers in the ventral
horn and propriospinal neurons. Afferent projections,
especially those of muscle sensors such as spinal afferents,
make extensive synapses in the ventral horn, synapsing on
motoneurons within a pool as well as neurons of synergistic
pools. Therefore, they form reflex pathways that produce
synergies. Propriospinal neurons are neurons that originate
and terminate within the spinal cord and their projections
serve to interconnect various segments within the cord to
coordinate the activation of distally located neurons.

Intraspinal microstimulation is unique among its


implanted neuroprosthetic counterparts in a number of ways.
1) It requires a one-time surgical procedure for microwire
implantation in a small region of the spinal cord. 2) It accesses
neuronal networks within the cord not readily activated by
peripheral electrical stimulation system. 3) It activates
motoneurons trans-synaptically and recruits motor units and
muscle fibers in a near-normal physiological order, in contrast
to peripheral nerve stimulation. 4) It provides specific input
to locomotor-related networks in the ventral horn resulting in
the generation of weight-bearing stepping. This comes in
contrast to epidural stimulation which activates sensory
inputs of multiple modalities that may not be specific to
locomotion, and evokes non-weight bearing movements of
the legs.

To determine the extent to which ISMS activates these


fibers in passage, we conducted acute experiments in which
recordings from cells located throughout the lumbosacral
enlargement (3 cm long) in adult cats were obtained during
ISMS. We particularly characterized the effect of ISMS on
the firing rate of various cells and identified the location of
these cells relative to the location of the ISMS microwire

If proven to be feasible and efficacious, ISMS could be


used in conjunction with other rehabilitation interventions
such as regeneration, pharmacological agents and bodyweight supported treadmill locomotor training. A
81

I/IIA muscle fibers at 3 motor threshold in rat quadriceps


muscles. This suggested that ISMS activates motoneurons
trans-synaptically and thereby recruits them in a nearnormal order based on their size. Given this result from
acute experiments, we hypothesized that long-term ISMS
after SCI would be capable of preserving the general
distribution of muscle fibre types while long-term peripheral
stimulation will result in transforming the muscle to a
predominantly slow phenotype. To test this hypothesis, we
examined the muscle transformation that occurs following
chronic application of these stimulation paradigms in
spinalized, female Sprague-Dawley rats.

tips. We conducted these experiments in two preparations:


animals with intact afferent input to the spinal cord and
animals that had been unilaterally deafferented by cutting
the dorsal roots in the lumbosacral enlargement. These two
preparations allowed us to determine the differential effects
of ISMS in activating afferent and propriospinal projections.
In the animals with intact afferent input (n=6) we
recorded from a total of 119 cells but only 47% of those
showed changes in their firing rate during ISMS (100 A,
100 s, monophasic, monopolar, 2 Hz). Of the cells that
did respond to ISMS, 85% experienced a significant
increase in their firing rate while 15% showed a significant
decrease in firing rate. The cells that responded to ISMS
were located 1.010.0 mm rostrally or caudally from the
microwire tip. Not surprisingly, the deafferented animals
(n=3) showed a general suppression of overall cellular
activity and fewer cells could be recorded from (total =
22 cells). However, of these cells, 25% experienced a change
in their firing rate due to ISMS, with 67% having an
increase in firing rate and 33% a decrease. The cells were
located 1.06.0 mm from the ISMS microwire tip.

Twelve (12) adult rats were completely spinalized at the


T8 level and 2 weeks later assigned to one of two groups: 1)
nerve cuff stimulation (NCS) of the femoral nerve, and 2)
ISMS in the quadriceps region of the spinal cord.
In the NCS group, a 2-lead nerve cuff was implanted
around the femoral nerve of one leg and the leads were
tunneled under the skin where they terminated in an acrylic
cap secured to the rats head. In the ISMS group, 2 microwires
were implanted in each side of the spinal cord, and their tips
targeted the quadriceps motoneuron pool in the ventral horn
of the lumbar enlargement (stimulation was applied through
wires in one side of the cord only). A multi-stranded stainless
steel wire was placed underneath the skin nearby to serve as
the return electrode. The microwires were anchored to a
spinous process proximal to the site of implantation and
subsequently tunneled along with the return electrode under
the skin to terminate in an acrylic headpiece. One week after
recovery from implantation electrical stimulation ensued in
both groups to activate the quadriceps muscles in one
leg. Stimulation in the NCS group consisted of trains of
50 pulses/sec delivered in a bipolar fashion while in the ISMS
group, stimulation consisted of monopolar pulses delivered
at a rate of 25 pulses/sec through each microwire in one side
of the cord. The stimuli through the two wires were
interleaved such that 50 pulses/sec were received by the
muscle resulting in fused contractions. For both NCS and
ISMS, the stimuli were biphasic, charged-balanced pulses,
pulse amplitude was held at 5 motor threshold, and pulse
width was 200 s. The animals were stimulated 4 hours per
day, for 30 days.

These results demonstrated that even though ISMS


directly activates motoneurons in the close vicinity of the
microwire tips, the effect of the focally applied stimulus is
amplified by activation of fibers in passage. These fibers in
passage likely involve both afferent and propriospinal
projections and act to coordinate the activation of synergistic
motoneuron pools. Therefore, through the activation of these
pathways, ISMS is capable of producing coordinated, multijoint synergies and rhythmic locomotor-like patterns with
only a few microwires implanted in each side of the spinal
cord. Moreover, the stimulation of these fibers leads to the
activation of motoneurons indirectly (or trans-synaptically),
thereby recruiting them in a normal order based on the size
principle and reducing the rate of muscle fatigue often seen
with peripheral electrical stimulation systems.
These findings improved our understanding of the
mechanisms of action of ISMS. They also have significant
implications to other central system electrical stimulation
approaches such as epidural stimulation of the spinal cord
and deep brain stimulation.
Recent publications resulting from this work:
1. R. Calixto and V.K. Mushahwar, Understanding the
Mechanisms of Action of Intraspinal Microstimulation,
12th Annual Conference of the International Functional
Electrical Stimulation Society, Philadelphia, PA,
November 2007.
Abstract was selected as a finalist in the student competition
2. A full manuscript expected to be submitted in
September

Following chronic electrical stimulation, functional


force recordings were obtained during a terminal
experiment to investigate the transformation in muscle
properties due to SCI and long-term electrical stimulation.
The quadriceps muscles of the unstimulated, contralateral
leg were used as control. All force records during the
terminal experiments were obtained by stimulating the
muscles through nerve cuff electrodes implanted around the
femoral nerve. In the NCS group, the same cuff in the
experimental leg was used while a new cuff was implanted
in the contralateral leg. In the ISMS group, cuffs were
implanted on both femoral nerves to conduct the testing.
Functional force recordings consisted of isometric twitch
responses, whole muscle sag tests and tetanic fatigue tests.
The quadriceps muscles were then extracted bilaterally and
frozen for subsequent immunohistochemical analyses.

b) Effect of long-term ISMS on muscle fiber-type


transformation
We previously established that nerve cuff stimulation
preferentially recruits fatigable type-IID/IIB muscle fibers
while ISMS preferentially recruits fatigue-resistant type
82

Functional force recordings have now been obtained from


all animals and immunohistochemical assessments are
currently underway. Contrary to our hypothesis, initial
analyses of functional data suggest that long-term ISMS
applied in the manner described above generates similar force
responses as those seen with long-term NCS. If these results
hold upon the completion of the analyses, we would conclude
that recruitment order of motoneurons is not the only factor
contributing to the preservation of the distribution of fibre
types in a muscle after SCI. Two additional factors would
need to be taken into consideration: 1) the loading
characteristics of the muscle during the contractions induced
by electrical stimulation, and 2) the pattern of activation used.
In our experiments, the evoked muscle contractions were not
loaded, i.e., the muscles were not required to contract against
a load placed across the tibia. Also, the pattern of stimulation
was monotonic consisting of repeated one-sec-on, one-secoff trains of stimulation at a fixed stimulation frequency. The
effects of these two factors on muscle properties with longterm ISMS will be assessed in future studies.

for the same duration as the ISMS group. After perfusion,


the region of the cord corresponding to that implanted with
microwires in the ISMS group was extracted.
To date, the spinal cords from all animals have been
extracted and histological analysis of the tissue will soon
ensue. The analysis will focus on assessing: 1) the effect of
long-term ISMS on the general features of the surrounding
tissue in the spinal cord, and 2) reorganizations of neuronal
circuits in the region surrounding the ISMS implant.
Therefore, the histological analyses will consist of the
following:

2007 publications resulting from this work:


1. J.A. Bamford, C.T. Putman and V.K. Mushahwar, The
effects of chronic intraspinal microstimulation or nervecuff stimulation on spinalized rat quadriceps muscle,
Society for Neuroscience 37th Annual Meeting, San
Diego, CA, November 2007.
2. J.A. Bamford, C.T. Putman and V.K. Mushahwar,
Muscle recruitment properties of intraspinal
microstimulation, 12th Annual Conference of the
International Functional Electrical Stimulation Society,
Philadelphia, PA, November 2007.
3. A full manuscript is currently in preparation and
expected to be submitted by December (2008)

NeuN to determine the presence and extent of


neuronal death around the tips of microwires
GFAP to assess the astrocytic scar and astrocytic
processes around the site of microwire implantation
IBA1 to determine whether activated microglia and
macrophages are still present around the site of
implantation; their presence would signify the existence
of an ongoing chronic inflammatory process either due
to micromotion on the implant or due to the electrical
stimulation itself
MAP2 to evaluate the extent of dendritic
arborizations in the vicinity of the implanted wires
Synaptophysin to quantify the number of general
synaptic terminals on neurons in the vicinity of the
implanted microwires

The SCI control group will provide a baseline of neuronal


features expected to be seen after chronic SCI. The unstimulated side of the spinal cord in the ISMS group will
serve as a control with passive (i.e., non-stimulated)
microwire implant.
We expect to see a very small region of astrocytic
encapsulation surrounding each microwire in which no
neurons will be present. Based on preliminary results from
experiments in cats, we do not expect to find activated
microglia or macrophages around the site of microwire
implantation. From the small, but clear reductions in motor
threshold over time, we do expect to find increased dendritic
arborizations in the region surrounding the implant as well
as increased synaptic terminals on neurons in the vicinity.

c) Effect of long-term ISMS on Neuronal


Reorganizations within the Spinal Cord
In addition to investigating the plasticity induced by longterm ISMS in paralyzed muscles, we initiated studies as part
of this grant focusing on assessing the general reorganizations
of neuronal networks induced by this form of stimulation.
These studies we conducted in adult rats with complete
transection of the spinal cord.

2007 publications resulting from this work:


None, experiments in animals have recently been completed.
Tissue analyses will begin shortly. A full manuscript is
expected to be submitted by December (2008).

Ten (10) adult female Sprague Dawley rats were used.


All rats received a complete SCI at the T8 level and divided
into two groups: 1) chronic ISMS (n = 6), and 2) SCI
control (n = 4). Two weeks later, the rats in the ISMS group
were implanted with ISMS microwires bilaterally targeting
the quadriceps region of the cord (same as the ISMS group
described in b above). One week following recovery from the
implant, one side of the spinal cord was stimulated through
the implanted microwires (5 motor threshold, 4 hours per
day) for 30 days. Changes in motor threshold were
monitored throughout the period of stimulation. At the end
of the experiment, the animals were deeply anesthetized and
perfused through heart with a formaldehyde fixative
solution. The region of the spinal cord containing ISMS
implant was extracted and preserved for subsequent
histological analysis. The SCI control group was maintained

PLANS FOR COMING YEAR:


Complete functional and histological assessments of the
effect of long-term ISMS on muscle fiber-type
transformation in chronically spinal rats.
Complete histological analyses of spinal cord tissue to
assess the neuronal reorganizations induced by longterm ISMS.

83

Can Human Lamina Propria Olfactory Ensheathing Cells


Expand, Migrate and Stimulate Rat SCI Repair as well as
Mouse OECs?
Jane Roskams
University of British Columbia, Canada. roskams@zoology.ubc.ca
INTRODUCTION

received by dozens of investigators currently trying to


culture and identify different types of human glia. These
results are now detailed in a submitted manuscript in
(Currie et al. 2008).

The Main Aim of this grant is to develop a paradigm for


expanding Olfactory Ensheathing Cells from biopsy and
post-mortem samples of human Lamina Propria (LPOECs), and test their ability to migrate and promote
regeneration in vitro, then in vivo. Specifically, we will test
(1) If biopsies from patients of different ages/medical
conditions yield OECs with different capabilities of
expansion (2) If human OECs can migrate and stimulate
differential axonal outgrowth using the same pathways as
rodent OECs; (3) If transplantation of expanded human
OECs can stimulate regeneration in an acute or delayed rat
contusion model. This project has three different
components, which have proceeded in parallel. All have
resulted in papers that are either published, or are currently
under review.

(II) Biopsy-Derived human OECs in vitro: In our


previous reports, we outlined two main approaches we have
used based on the work of others (Rawson lab, Monell and
F. Feron) explant-based culture, and dissociated culture.
Both have yielded mixed populations of cells that include
some OECs. Using the antibodies that give specific staining
in vivo as a guide, we have been able to identify some OECs
in these cultures, and determine that explant-derived
cultures that have been gently dissociated yield OECs more
consistently. These are, however, still mixed cultures
containing, at most 610% OECs, and most of our efforts
this year have been aimed at enhancing the yield and
increasing their purification (enrichment).

AIM ONE
(I) Olfactory Biopsies: The sinus surgeon with whom we
have collaborated Dr Amin Javer has tested two independent
parameters for biopsy sampling. (1) Site of biopsy and
likelihood to produce tissue containing nerves and
ensheathments (tested by us). (2) Potential changes in sense
of olfaction after biopsy and surgery, compared with similar
patients, undergoing surgery alone.

Increasing the Yield of Human OECs: The majority of


contaminating cells detected in our cultures are fibroblasts,
but dissociated cultures also contained basal cell colonies,
which, in mice, have the capacity to produce multiple cell
types, including glia. To reduce the number of fibroblasts,
and enrich for OECs we: (1) Spent 6 months collaborating
with Dr Jamie Piret, a stem cell biologist at UBC, to test if
combinatorial factors in a bioreactor may enhance human
OEC or basal cell expansion. Unfortunately, because of the
low percentage representation of OECs in our cultures
overall, these experiments did not yield sufficient cells for
comprehensive analysis, and the fibroblasts expanded more
rapidly. (2) In parallel, in growing phenotypically older
OECs from mice and rats, we found we could increase their
expansion if we plate them on dilute monolayers of early
passage neonatal mouse OECs. This approach proved to be
the most effective way to help in the post-culture survival of
human OECs. (3) In the last year of the project, a great deal
of effort has been put into reversibly immortalizing our
human OE cultures, to attempt to expand them in greater
quantities, to allow us to specifically select OECs. Although
we have generated a virus driving telomerase (as outlined in
our original proposal), this virallymediated approach did
not produce as significant an increase in expansion as we
originally expected, even in enhancing the expansion of
human fibroblasts. All our subsequent work has been
performed with a different retrovirus produced by a UKbased company Re-Neuron, where c-myc is reversibly
inducible with tamoxifen, and which incorporates stringent
selection criteria. We also developed an approach to get
efficient transduction of GFP into human cells, using a
Lentivirus:GFP vector generously provided to us by Dr.
Fabio Rossi (Biomedical Research Centre, UBC).

Results: Using guided imaging to biopsy has increased


the success rate of finding a patch of olfactory epithelium
from which to take a suitable biopsy. Dr Javer has confirmed
three different locations within the superior turbinates that
are the best sites for this. In total, we have received biopsies
from almost 60 different patients. None of the patients
sampled in these three areas have demonstrated any
significant reduction in olfactory sensitivity 3 months after
surgery. For a selection of the larger biopsy samples, we have
taken a small sample of each biopsy to test using
immunohistochemistry against both glial and neuronal
antigens found in axons and ensheathing glia of the olfactory
nerve. This approach has served two purposes to test
efficacy of biopsy, and also to establish which antibodies may
be the most specific and suitable for determining the purity
of OECs in vitro. Some of these results (from 6 different
patients) are included in a manuscript currently under review
(Currie et al., 2008).
It is important to note that several commercially
available antibodies used by others to mark OECs, Schwann
cells and other cells of CNS/PNS origin, are not as specific
as they should be in our hands and also recognize antigens
found in human fibroblasts. Some of this work was
presented at the 2006 SFN meeting, and was very well
84

AIM TWO
Migration and differential axon sprouting induced by
human OECs

Overall Progress: We made considerable progress in


every aim of this project, which resulted in a series of
publications that have made a significant impact on our
understanding of how OECs may be applied in the context
of spinal cord injury. Equally important are a series of findings
pinpointing their limitations in different lesion settings. The
main limitation we envisage is in being to generate enough
human OECs from small samples, with a degree of purity
strong enough to pass the regulatory agencies stipulations for
cellular therapeutics, to make these cells effective in a human
SCI setting. Our results would caution against the feasibility
of using limited amounts of biopsy material in the generation
of human OECs and argue for exploring other sources (donor
tissue, especially from immunologically-matched younger
sources or fetuses) in pursuing this avenue in the future.

While we worked on improving the yield of human OECs,


we developed two sets of assays to test different batches of
cells for their migration, and ability to stimulate differential
outgrowth. We used these assays to demonstrate significant
differences between Lamina propria and olfactory bulbderived OECs in vitro (Richter et al., 2005), extended this to
the cross-comparative phenotypic analysis of OECs and
Schwann cells (Richter and Roskams 2007). In order to assess
the outgrowth of functionally important tracts, we developed
an assay employing Thy1:YFP mice (gift, Josh Sanes) which
express YFP in subsets of cortical and subcortical neurons.
This technique allowed us to test mature corticospinal
neurons for their ability to extend axons in response to OECs,
or OEC-conditioned media, and to differentially assay
dendritic sprouting and axonal outgrowth. This novel assay is
now the focus of a manuscript currently under review (Richter
and Roskams 08). Erin Currie, the research associate employed
on our ISRT project, used this corticospinal assay to test
baseline outgrowth on human fibroblasts, in anticipation of
developing a significant population of more highly purified
human OEC preparations.

Personnel during this period:


1. Erin Currie, Research Associate. Jan. 2007Dec. 2007.
Erins time spent on this project was dedicated to
general lab duties in our OEC projects. Erin worked
side-by-side with Adele Vincent, concentrating on the
expansion of human OECs, while Adele worked on
assay development and in vivo analyses, in collaboration
with the Tetzlaff lab.
2. Miranda Richter, graduate student: Funded by her
own studentship, her salary was topped up from this
grant, March 2007Jan. 2008.
3. Adele Vincent, arrived in February, 2006, and began
working on this project immediately. Originally
supported by this grant, but was topped up from July
2007Jan. 2008, as she received a fellowship which
began in July 2007.

AIM THREE
Human OECs into a rat spinal contusion model
While we worked on the two largely in vitro assays outlined
above for migration of human OECs and CNS outgrowth
assays, we worked on two distinct lines of investigation for
OEC transplantation, outlined below (1). In collaboration
with Mark Tuczynski, we tested the efficacy of different
modes of introduction of cells (OECs versus MSCs), and
their differential effect on the remodeling of the lesion site,
and axon extension, following a wire knife lesion of the
dorsal columns. We found that the complete rostro-caudal
dispersion of either OECs or MSCs depends on the speed,
volume and mode of introduction, coupled with placement
of cells to create an initial pressure effect of injection that
will take a percentage of injected cells directly to the lesion
site. Rearrangement in the rostro-caudal plane, by limited
local migration, occurs thereafter. These results were
published in the last year, (Lu et al.), in J. Neuroscience. (2)
We concurrently worked with a PDF fellow in the Tetzlaff
lab, Frederic Bretzner, to perform T10 contusion lesions,
and transplanted OECs into these rats to assess initial
behavioral changes (e.g. BBB, catwalk). Frederic has used a
number of combined approaches with OECs BDNF cell
body treatment, OECs, rock and rho inhibitors. He
presented some of his initial findings (indicating that OECs
plus BDNF treatment were no more effective than either
strategy alone), at SFN last year. Two independent
manuscripts, outlining both behavioral and anatomical
outcomes following the employment of combination
strategies (OECs with BDNF and other treatments),
surprisingly indicated a reduced efficacy in the recovery of
some behavioral parameters when used in combination.
One of these manuscripts is in press (E.J. Neurosci.), and
the other is currently under review.

PUBLICATIONS
1. Richter M.W., Roskams A.J. Olfactory ensheathing cell
transplantation following spinal cord injury: Hype or
hope? Exp. Neurol. 2007 Jun. 30;
2. *Edmund Au, *Miranda W. Richter, *#Wolfram
Tetzlaff, **Ruedi Aebersold, ***E. Helene Sage, and *A.
Jane Roskams, SPARC is Secreted by Olfactory
Ensheathing Cells to Stimulate Neurite Outgrowth and
CNS Repair, J. Neurosci. 2007 Jul. 4;27(27):720821
3. Paul Lu, Hong Yang, Maya Culbertson, Lori Graham, A.
Jane Roskams and Mark H. Tuszynski. Olfactory
Ensheathing Cells Exhibit Minimal Migratory and
Axonal Growth Promoting Properties After Spinal Cord
Injury. J. Neurosci. 2006 Oct. 25;26(43):1112030.
4. *Richter M., *Au E., Liu J., Tetzlaff W. and Roskams
A.J. Lamina Propria And Olfactory Bulb Ensheathing
Cells Exhibit Differential Integration, Migration, And
Promote Differential Axon Sprouting In The Lesioned
Spinal Cord. J. Neurosci. 2005 Nov. 16;25(46):10700
11 ** Chosen as a Highlight in the Journal
5. Oswald Steward PhD1, Kelli Sharp1, Edmund Au2,
Gowri Selvan PhD1, Anthony Hadden MD1, Maura
Hofstadter PhD1, Jane Roskams PhD2A Re-assessment
of the Consequences of Delayed Transplantation of
Olfactory Ensheathing Cells Following Complete
Spinal Cord Transection In Rats. Exp. Neurol. 2006
Apr.;198(2):48399. Epub. 2006 Feb 21.
85

6. Frederic Bretzner, Jie Liu, Tigran Bajgoric, Erin Currie,


A. Jane Roskams, and Wolfram Tetzlaff* Undesired
effects of a combinatorial treatment for spinal cord
injury: Transplantation of olfactory ensheathing cells
with BDNF treatment of rubrospinal neurons E.J.
Neuroscience (in press)
7. Currie E., Vincent A. and Roskams A.J. Human
Olfactory Ensheathing Cells: Morphology, and
antigenicity in vivo and in vitro. Glia (under review,
Jul. 08)

8. Richter M.W., and Roskams A.J. Corticospinal


Neurons Respond Differentially to Neurotrophins and
Myelin Associated Glycoprotein in vitro, J. Neurosci.
Res. (under review, Jun. 08)

86

Development of functional magnetic resonance imaging


for assessing human spinal cord injuries
P.W. Stroman, R. Smith, R. Pokrupa and K. Smith
Queens University, Ontario, Canada
INTRODUCTION

Analysis:

The purpose of this research program is to develop noninvasive imaging techniques based on magnetic resonance
imaging (MRI), in order to map function in the human
spinal cord, and to be able to assess the effects of spinal cord
trauma in individuals. Mapping of neuronal function by
means of functional MRI (fMRI) in the spinal cord (spinal
fMRI) presents specific challenges that are not encountered
in fMRI the brain, due to the proximity of the spinal cord
to bony structures in the spine, and the small cross-sectional
dimensions of the spinal cord. Nonetheless, the research
program I have been carrying out for the past 11 years has
demonstrated that spinal fMRI can produce highly sensitive
and reliable maps of function in the human spinal cord.
Significant advances have been realized in the past 3 years as
a direct result of this ISRT funded research.

1. Characterization of spinal cord motion within the


spinal canal, including variations between cervical,
thoracic and lumbar regions, and methods to
compensate for this motion in spinal fMRI analysis.
2. Automated labelling of the spinal cord, CSF, and
vertebral bodies, in MRI data to support objective
automated analysis.
Acquisition methods/hardware:
3. Development of a device to provide automated delivery
of thermal-sensory stimuli for spinal fMRI in a clinical
setting.
Interpretation of results, and neuroanatomical studies:
4. Study of responses to a range of innocuous stimuli and
study-related emotional/cognitive influences.
5. Study of the effects of attention focus on activity
patterns in the cervical cord and brainstem.

At the current state of development, the key challenges


now being faced include:
1. few researchers are working on developing spinal fMRI,
although the number is growing, and so progress is not
as rapid as that of brain fMRI
2. there is no "gold-standard" method of detecting the
spatial distribution of function in the human spinal
cord, that can be used to confirm the accuracy of the
results obtained with spinal fMRI
3. there is relatively little published data available on the
functional neuroanatomy of the healthy human spinal
cord and so it must be inferred from animal studies and
the effects of trauma or disease
4. to be useable in a clinical setting, the spinal fMRI
method (both data acquisition and analysis) has to
be easy to use, require relatively little additional time
in the MRI system, and be as user-independent
as possible

METHODS
All of the studies in the past year were carried out with
human volunteers in a 3 tesla, clinical-style MRI system at
Queens University in Kingston, Ontario, Canada. This is a
research-dedicated system that is identical to that used in
many hospitals, without any modification. Functional MRI
studies involve repeated imaging of a region of interest,
every few seconds, for several minutes in order to describe
a time-course of changes. During this time-course the
volunteer experiences sensations, or performs tasks, in
repeated blocks typically of 30 to 60 seconds duration,
interleaved with baseline conditions for comparison. Any
tissue changes that occur consistently during the stimulation
blocks, but not during the baseline conditions are reflected
by corresponding, albeit very subtle, changes in the images.
Image changes that occur consistently with each stimulation
block are therefore inferred to reflect neuronal activity. For
the purposes of the studies described above, stimuli included
thermal changes, or light touch or brush sensations. These
were applied with a Medoc TSA-II thermal sensory analyzer,
or manually with Von-Frey hairs or with artists brushes,
respectively. In addition, cognitive variations, such as
attention, distraction, empathy, etc, were applied by means
of visual cues or audio-visual presentations. These were
projected onto a rear-projection screen outside the head-end
of the MRI system and the volunteers viewed the screen via
a mirror placed in front of their eyes, as they lay inside the
MRI system. Audio components of the presentations were
delivered through MR-compatible headphones.

These challenges are being overcome in the present research


as the body of published work on spinal fMRI is growing,
more researchers are beginning to investigate the method.
The results obtained to date are yielding highly consistent
findings that are supported by our current knowledge of
the functional neuroanatomy of the spinal cord. Now,
however, we are able to map the three-dimensional extent of
spinal cord function in awake humans who can
simultaneously report their sensations. This is providing
new information about the function of the healthy intact
human spinal cord.
The research projects undertaken in the past year to
further the development of spinal fMRI as a practical
clinical tool for spinal cord assessment include:

The resulting image data were analyzed using custommade software I have written in MatLab, and modified as
needed for each study.
87

RESULTS

Toward this end, I have developed a Self-Timed


Automated stimulus DeliverY System (STADYS, pronounced
status) that provides thermal stimulation at 44C with four
MR-compatible thermodes. The four temporal patterns of
heating and passive cooling are linearly independent, making
it possible to distinguish the four response patterns with a
general linear model analysis. The design concept is so that an
MR technologist need only position the four thermodes on
dermatomes above and below the level of injury, on the right
and left sides of the body, when positioning the patient for
MRI. At some point during the scanning session, the
technologist will initiate a preset imaging sequence to acquire
a 7 minute long time series of images, and at the start of the
sequence will simply press the go button on the STADYS.
No other action is required by the technologist. Given that
the temporal patterns of stimulation are thus fixed for all
fMRI acquisitions, the analysis parameters do not need to be
changed, and the analysis can therefore be essentially
automated. Each component of this method have been tested
in a research setting, and in the coming year the integrated
process will be tested extensively on patients who are research
volunteers.

Spinal Cord Motion Compensation


Results obtained from this project in previous years have
demonstrated that a key source of noise in spinal fMRI data
arises from the motion of the spinal cord within the spinal
canal, driven by the flow of CSF with each heart-beat. In the
past year we have succeeded in developing analysis methods
to compensate for this motion, resulting in considerably
higher sensitivity and reliability of the maps of activity
obtained in the cord. Another important finding has been
that the spinal cord motion depends on the rostral-caudal
position within the spine. The motion is greatest in the
cervical regions, diminishes more caudally, and is negligible
in lumbar and sacral regions. This has important
implications for our ability to determine the impact of spinal
cord motion on our results. For example, we have started
studies using diffusion-weighted MRI in order to investigate
the impact of spinal cord motion on measured water selfdiffusion rates. This will enhance our ability to obtain images
depicting structural changes at the cellular level, because
these changes can influence water self-diffusion rates.
Automated Feature Labeling
Automated labeling of the spinal cord, CSF, and vertebrae
has been achieved by means of a neural net algorithm. This
method was first developed for various tasks such as reading
characters in postal codes for the US Postal Service, and we
have now applied it to the spinal cord, with an example
shown in Figure 1. The advantage of this for spinal fMRI
analysis is that now we can automatically detect changes in
cord position or flexion over the course of an fMRI study.
Moreover, we can label the edge of the spinal cord, as
needed for the spatial normalization procedure developed
in previous years. This will eventually lead to almost
completely automated analysis software.

Investigation of fMRI Study-Related Emotional/Cognitive


Influences
The influence of the initial anxiety/fear/alertness of being in
an MRI system, which then wanes over time into
boredom/sleepiness was investigated in 15 healthy volunteers
by carrying out spinal fMRI at 5 different innocuous
stimulation temperatures (29, 25, 22, 18 and 15C). The
order of the experiments was varied systematically so that
every combination of temperature and order was applied an
equal number of times across the 15 volunteers. Activity in
the ipsilateral dorsal gray matter (dGM), in response to
innocuous thermal stimuli, reflects the net result of sensory
input to the cord, and modulation from the descending
analgesia system (via the rostral ventromedial medulla,
RVM) and from affective (emotional) components of
sensation (via the locus coeruleus, LC). Similarly, activity in
the ipsilateral vGM depends on that in the dGM plus
descending modulation from affective components (via the
PRF). In contrast, contralateral ventral gray matter (vGM)
activity reflects local input from the ipsilateral dGM.
Descending modulation of activity in the contralateral vGM
via the reticular formation cannot be ruled out, but was not
observed with 1529C stimuli used in this study.

Fig. 1. An example of the results output by


the automated feature labelling method,
applied to a single mid-line sagittal slice
from a spinal fMRI data set. In this
example, the spinal cord within the center
region of the image has been labelled red,
and the vertebrae labelled blue, and the
cerebellum is labelled green. The CSF
is not labelled in this image, for clarity.
Further refinements of the method
are underway.

The order of the experiments is linked to systematic


decreases over time in the volunteers levels of stress and
anxiety, and increasing boredom. Group fMRI results
demonstrated significant dependences on the order of the
experiments, most notably in the LC. These results also
demonstrated significant dependences on the stimulus
temperatures in the observed regions of activity in the
brainstem and cervical spinal cord, showing both inhibition
of pain and motor responses with lower temperatures, and
apparent sensory facilitation with stimulation at the
warmest temperature, 29C.

Development of a Stimulation Device for Clinical Spinal


fMRI (STADYS)
A fundamental challenge that is foreseen for clinical
applications of spinal fMRI, is that clinical MRI facilities
do not typically have the space available, or the personnel
time available, to set up complicated stimulation devices in
order to augment standard clinical scans with fMRI. In
order to be practical, particularly in early stages until its
clinical value becomes established, it must be easy to use,
and place minimal extra burden on the MR Technologists.

This study reveals, for the first time, the complete


network of neuronal function in the cervical spinal cord
88

cord and brainstem. This includes afferent sensory input,


and descending modulation via the opiate analgesia system
(PAG and RVM), affective components of pain (thalamus,
locus coeruleus), and regulation of motor responses (reticular
formation) (1). The magnitudes of the signal intensity
changes are inferred to reflect the input to an area (2). Both
positive and negative areas of activity are therefore to be
expected based on the neuroanatomy, corresponding to
increased or decreased descending modulatory input in
response to the subjects attention focus. Significantly greater
input was observed with distraction, particularly in the
vicinity of the PAG, RVM, and in the ipsilateral dGM at
around C6. This implies that greater descending input to the
cervical spinal cord from the analgesia system plays an
important role in altering perceived sensations, with changes
in attention focus.

resulting from innocuous thermal sensory input and


descending modulation, in human volunteers who can
simultaneously report their sensations. The results of this
study will enable accurate interpretation of spinal fMRI
results, including the potential to identify altered ascending
and descending pathways as a result of disease or trauma to
the cord. They also demonstrate the potential of spinal fMRI
to provide a view of spinal cord function in humans without
the complications of invasive procedures or anesthetics. This
view reveals anatomical and functional details of distributed
networks in the human brainstem and spinal cord that
produce a net sensory response, in unprecedented detail, and
has implications for the future study of conditions such as
neuropathic pain and regeneration after spinal cord injury.
Investigation of the Effects of Attention Focus on Activity
Patterns in the Cervical Cord and Brainstem
Descending modulation of responses was investigated in this
study by modulating each participants attention focus. This
was done by providing written instructions to focus on the
sensation and provide intermittent ratings of the discomfort
caused by a cold thermal stimulus, or by having him/her
answer mentally-challenging multiple-choice questions.
Responses were recorded by means of eye-tracking and
instructing the participant to fix their gaze on the chosen
answer on the display.

The results of this study demonstrate that attention focus


significantly influences activity detected by means of spinal
fMRI in the cervical spinal cord during innocuous thermal
sensory stimulation. This has implications for the use of
spinal fMRI for research or clinical evaluation, because, if
not controlled, it is a potential source of significant intertrial variability. Alternatively, by intentionally varying the
subjects attention focus between repeated studies, it may be
possible to discriminate ascending and descending input
pathways, thereby revealing important features of normal
spinal cord function, as well as the effects of trauma
or disease.

The areas of activity that were identified (Figure 2) are


consistent with the known anatomical regions involved with
pain and temperature responses across the cervical spinal

Fig. 2. Comparison of the activity detected in regions of the midbrain, rostral medulla, and C6 spinal cord segment, in response to a thermal
sensory stimulus applied to the right hand, with participants either focussing their attention on the sensation (attending) or focussing their
attention on answering mentally-challenging multiple-choice questions (distracted). Each image frame represents a 1 mm thick transverse slice
through the region, and adjacent frames are contiguous to demonstrate the 3D extent of the active regions. Images are also in radiological
orientation, with the dorsal aspect of the body toward the bottom of the frame, and the right side of the body toward the left side of the frame.
89

CONCLUSION

Spinal fMRI investigation of human spinal cord function


over a range of innocuous thermal sensory stimuli and
study-related emotional influences P.W. Stroman Submitted
to Magnetic Resonance Imaging (Nov. 15, 2008)
Tactile sensory transmission in the human spinal cord and
brainstem N.F. Ghazni, C.C. Cahill, P.W. Stroman Submitted
to American Journal of Neuroradiology (May, 2008)
Development and validation of retrospective spinal cord
motion time-course estimates (RESPITE) for spin echo
spinal fMRI: Improved sensitivity and specificity by means
of a motion-compensating general linear model analysis
C.R. Figley, P.W. Stroman. NeuroImage (in press, available
online doi: 10.1016/j.neuroimage.2008.08.040)
Investigation of cardiac-related motion in the lowerthoracic, lumbar, and sacral spinal cord: Implications for
imaging human spinal cord structure and function C.R.
Figley, D. Yau, P.W. Stroman. American Journal of
Neuroradiology 29:14501454 (2008).
Spatial normalization and motion correction for functional
magnetic resonance imaging of the spinal cord and
brainstem P.W. Stroman*, C.R. Figley, C.M. Cahill.
Magnetic Resonance Imaging 26: 809814 (2008).
Magnetic resonance imaging of neuronal and glial swelling
as an indicator of function in cerebral tissue slices P.W.
Stroman*, A.S. Lee, K.K. Pitchers, R.D. Andrew. Magnetic
Resonance in Medicine 59: 700706 (2008).
Investigation of Human Cervical and Upper Thoracic
Spinal Cord Motion: Implications for Imaging Spinal Cord
Structure and Function C.R. Figley, P.W. Stroman. Magnetic
Resonance in Medicine 58: 185189 (2007).
Detection of the neuronal activity occurring caudal to the
site of spinal cord injury that is elicited during lower limb
movement tasks J. Kornelsen, P.W. Stroman. Spinal Cord
45: 485490 (2007).
Discrimination of errors from neuronal activity in
functional magnetic resonance imaging in the human spinal
cord by means of general linear model analysis P.W.
Stroman. Magnetic Resonance in Medicine 56: 452456,
(2006)

The research in the past year completes key steps in the


analysis to compensate for dominant sources of noise, and
to therefore make the results more reliable and more
sensitive. At the same time, this has improved our
understanding for other MRI methods as well, such as DTI
and anatomical imaging. Key steps have also been
completed toward making the analysis as fully automated
and user-independent as possible. These include the design
and fabrication of the STADYS, the definition of fixed
stimulation paradigms that are useful for assessing both
ascending and descending pathways and motor reflex
responses to thermal stimuli, and software for automated
image labelling and definition of reference points for
normalization.
Investigations of key neuroanatomical features have
revealed unprecedented detail of normal human spinal cord
function, which will influence how we can use spinal fMRI
and how we interpret the results. These results have revealed
function in the cervical spinal cord in response to thermal
sensory stimulation, and how it is modified by descending
control from brainstem structures, including the opiate
analgesia system, and motor control from the reticular
formation. Affective components of the responses were also
identified, both on relation to changes in anxiety/attention/
alertness over the time span of each study as the participants
lay in the MRI system, and in response to systematic
changes in attention focus. These results demonstrate the
high sensitivity and reliability of the results to neuronal
activity in the spinal cord and brainstem, and also
demonstrate important information to be taken into
consideration for the interpretation of spinal fMRI results.
The areas of activity observed in the spinal cord depend on
emotional factors and attention focus, and by controlling
these factors we can both improve the consistency of results
and discriminate ascending and descending pathways.
The work completed in the past year therefore represents
significant advances toward developing spinal fMRI into a
practical and reliable clinical assessment tool. These
advances are equally useful for research applications, because
of the demonstrated sensitivity and accuracy, and the factors
that have been identified as affecting the image quality, such
as spinal cord motion.

CONFERENCE PRESENTATIONS:
Development of Functional Magnetic Resonance Imaging
for Assessing Human Spinal Cord Injuries P. W. Stroman
International Spinal Research Trust, Network Meeting
London, England, Sept 45, 2008. (invited speaker)
Functional Magnetic Resonance Imaging of the Human
Spinal Cord: Progress Towards a Clinical Tool P.W. Stroman,
C.R. Figley, N. Foad-Ghazni, C. Nahanni, N. Kozyrev Spinal
Cord: Function, Repair and Rehabilitation after injury
Montreal, QC, June 2526, 2008. (invited speaker)
The Enigma of Intermediate and Ventral Spinal Cord
Activity with Thermal Sensory Stimulation: A Spinal FMRI
Investigation P.W. Stroman, C.R. Figley, N. Foad Ghazni,
N. Kozyrev International Society for Magnetic Resonance in
Medicine, 16th Annual meeting, Toronto, Ontario,
Canada, May 39, 2008
Examination of Cardiac-Related Motion in the Lower
Thoracic and Lumbar Spinal Cord D. Yau, C.R. Figley, C.
Nahanni, P.W. Stroman International Society for Magnetic
Resonance in Medicine, 16th Annual meeting, Toronto,
Ontario, Canada, May 39, 2008

REFERENCES
1. S. Hochman. Spinal Cord 17(22), R950-R955 (2007).
2. N.K. Logothetis, et al., Nature 412(6843), 150 (2001).
Peer-Reviewed Journal Papers Arising from the Project
The principal author is listed first, and the project team
leader is listed last (unless the project team leader is also the
principal author). The project team leader is therefore
marked with an asterisk (*). Researchers, technicians, and
trainees under my supervision are underlined.
Non-invasive observation of cervical spinal cord activity in
children by functional MRI during cold thermal stimulation
J. Lawrence, J. Kornelsen, P.W. Stroman Submitted to
Journal of Pediatrics (Oct. 31, 2008)
90

Altered spinal cord and brainstem activation in response to


peripheral sensitization to sensory stimuli: a spinal fMRI
study N.F. Ghazni1, C.M. Cahill, C.F. Pukall, N. Kozyrev,
P.W. Stroman
International Society for Magnetic Resonance in Medicine,
16th Annual meeting, Toronto, Ontario, Canada, May 3
9, 2008
Physiological noise reduction in spinal fMRI from a singlestage, motion compensating GLM approach C.R. Figley,
P.W. Stroman International Society for Magnetic Resonance
in Medicine, 16th Annual meeting, Toronto, Ontario,
Canada,, May 39, 2008
Magnetic resonance imaging of neuronal and glial swelling
as an indicator of function in cerebral tissue slices P. W.
Stroman*, A.S. Lee, K.K. Pitchers, R.D. Andrew; Program
No. 535.3. 2007 Neuroscience Meeting Planner. San Diego,
CA: Society for Neuroscience, 2007
Functional MRI of touch and brush sensation in human
spinal cord and brainstem after peripheral sensitization N.
Foad Ghazni, C.M. Cahill, C.F. Pukall, P.W. Stroman
Program No. 285.7. 2007 Neuroscience Meeting Planner.
San Diego, CA: Society for Neuroscience, 2007
Functional MRI of the Human Spinal Cord P.W. Stroman
American Spinal Injury Association, Tampa, Florida, May
30June 1, 2007 (invited speaker)
Functional MRI of the brainstem and spinal cord of
children based on SEEP contrast P. W. Stroman*, J.
Lawrence, J. Kornelsen International Society for Magnetic
Resonance in Medicine, 15th Annual meeting, Berlin,
Germany, May 1925, 2007, p. 1967.
Functional Magnetic Resonance Imaging of Cortical Tissue
Slices by means of Signal Enhancement by Extravascular
water Protons (SEEP) Contrast P. W. Stroman*, R.D.
Andrew International Society for Magnetic Resonance in
Medicine, 15th Annual meeting, Berlin, Germany, May 19
25, 2007, p. 1966.
A new method of motion-compensated spinal fMRI:
Identifying human spinal cord function with increased
sensitivity and reproducibility C.R. Figley, P.W. Stroman
International Society for Magnetic Resonance in Medicine,
15th Annual meeting, Berlin, Germany, May 1925, 2007
Development of a Novel Method for Motion-Compensated
Spinal fMRI C.R. Figley, P.W. Stroman Imaging Network of
Ontario, Toronto, Ontario, March 2325, 2007
Functional magnetic resonance imaging of light touch and
brush sensations in the human spinal cord and brainstem
N.F. Ghazni, C.M. Cahill, P. . Stroman Imaging Network of
Ontario, Toronto, Ontario, March 2325, 2007
Functional Imaging of the Spinal Cord P.W Stroman.
American Society of Spine Radiology. Marco Island,
Florida. February 2125, 2007. (invited speaker)
Development of Spinal fMRI towards a practical tool for
assessing spinal cord injury P.W. Stroman*, C.R. Figley, J.
Kornelsen, C. Cahill, K. Smith, R. Pokrupa International
Spinal Research Trust, 9th Research Network Meeting,
London UK, 12 Sept 2006
Characterization of Spinal Cord Motion: A Source of Errors
in Spinal fMRI? C. R. Figley, P.W. Stroman International
Society for Magnetic Resonance in Medicine, 14th Annual
meeting, Seattle, Washington, May 49, 2006
Functional Magnetic Resonance Imaging of the Human

Spinal Cord and Brainstem During Heat Stimulation P.W.


Stroman*, C.M. Cahill International Society for Magnetic
Resonance in Medicine, 14th Annual meeting, Seattle,
Washington, May 49, 2006
Improved Sensitivity of Spinal fMRI by Using Physiological
Recordings in General Linear Model Analysis P.W. Stroman
International Society for Magnetic Resonance in Medicine,
14th Annual meeting, Seattle, Washington, May 49, 2006
Advances In Functional MRI Of The Spinal Cord P.W.
Stroman American Society for Neuroradiology, April 2006,
San Diego (Invited Speaker)
Investigation of neuronal activity of the injured spinal cord
during lower limb movement with spinal fMRI. J.
Kornelsen, P.W. Stroman Society for Neuroscience,
Washington DC, Nov. 1216, 2005.
INVITED PRESENTATIONS (Presentations that did
not result in a published abstract and, therefore, are
not listed above):
Mapping Neuronal Activity in the Human Spinal Cord
with fMRI P.W. Stroman Vanderbilt University, Nashville,
TN, Feb. 1st, 2008.
Mapping Neuronal Activity in the Human Spinal Cord
with fMRI P.W. Stroman Cincinnati Childrens Hospital,
Cincinnati, OH, Jan. 31st, 2008.
Noninvasive MR imaging of Spinal Cord Injury and Repair
P.W. Stroman, Session Chair, International Symposium of
Neuro-regeneration, Asilomar Conference Center, Pacific
Grove, CA, Dec. 59, 2007.
Mapping Neuronal Activity in the Human Spinal Cord
with fMRI P.W. Stroman University of Alabama at
Birmingham, Radiology Grand Rounds, April 20th, 2007.
Recent advances in functional magnetic resonance imaging
of the human spinal cord P. W. Stroman Dept of Radiology,
CHUM, Hopital Notre-Dame, Montreal, De.c 5th, 2006.
Is there a change in water proton density associated with
functional magnetic resonance imaging? P.W. Stroman III
Krakow-Winnipeg Workshop on Magnetic Resonance and
Molecular Imaging, Krakow Poland, Sept. 2023, 2005.
Imaging of neuronal function in the human spinal cord
with fMRI J. Kornelsen and P.W. Stroman Spinal Research,
Research Network Meeting, London UK, Sept. 23, 2005
Functional MRI of the human spinal cord: Methods and
applications P. . Stroman* McConnel Brain Imaging Center,
Montreal Neurological Institute, Montreal QC, Jan. 10th,
2005.
FUTURE PLANS
In keeping with the original plans for this project, the
coming year will focus on collecting spinal fMRI data from
people who have had a spinal cord injury. We will use the
STADYS to collect data from as many people as we can with
spinal cord injury, as well as a group of healthy control
subjects as a reference. These results will be analyzed as a
group relative to the standard clinical assessments of
function, and characteristics of the extent and location of
the injury. Figure 3 shows an example of one image of an
fMRI data set from a volunteer with a spinal cord injury at
C5. An implant for fixing the spine is apparent as a signal
void, with screws penetrating into the C4 and C6 vertebral
91

bodies. This essentially represents the worst-case scenario


for presenting challenges for MRI, and demonstrates the
effectiveness of the method I have developed.

motion correction, and compensation for spinal cord


movement within the spinal canal, including self-checking
of the effectiveness of this compensation. Once this
development is complete, the only input required for spinal
fMRI analysis, using the STADYS, will be the name and
location of the image data files to analyze. The output will
be in two forms, one spatially normalized for easy
comparison with reference data sets, and the other in the
original anatomical format.

A parallel line of work will continue with the


development and testing of computer algorithms to
automatically label the spinal cord and vertebrae in each
person for which we collect image data. This software will
be integrated into the existing analysis I have developed over
the past several years, in order to make the spatial
normalization process entirely automated. The automatic
labelling will also be used to detect subtle spinal cord
motion during the fMRI time-series and this will be
compared with the predicted motion as characterized
relative to the cardiac cycle. The outcome will be fully
automated analysis that includes spatial normalization, bulk

If successful, by the end of the coming year we will have


produced all of the components needed to use spinal fMRI
as a practical clinical tool, including data to demonstrate its
usefulness for clinical assessments, and it will be ready for
clinical trials.

92

Improving cardiovascular function after spinal cord injury


P. Waite, P. Carrive and A. Mackay-Sim
University of New South Wales, Australia
INTRODUCTION

changes in sympathetic preganglionic neurons both


above and below the level of transection. In addition
we have characterised the temperature changes that are
associated with AD, as a basis for testing the effects of
OECs on thermal regulation.
2. We have tested OECs in a delayed transplantation
model. Injection of the cells one week after the spinal
injury has been found to be ineffective in reducing AD.
Delayed transplantation of OECs also had no effect on
resting cardiovascular parameters.

Cardiovascular problems after spinal cord injury have


received relatively little attention from researchers,
compared with paralysis and loss of sensation. This is despite
their obvious clinical relevance, in terms of morbidity and
mortality. Our study aims to address this imbalance, by
better characterizing the central and peripheral mechanisms
involved in cardiovascular abnormalities and trialing
potential interventions such as cellular transplantation.
Cardiovascular dysfunction is particularly common after
cervical or upper thoracic injuries (Teasell et al., 2000).
Abnormalities include postural hypotension, loss of diurnal
rhythms and autonomic dysreflexia (AD). Episodes of
dysreflexia are commonly triggered by noxious stimuli below
the injury level, such as bladder or bowel distension. Such
episodes are associated with a throbbing headache, sweating
and flushing above the injury, vasoconstriction in the lower
limbs, and can be life-threatening. While several studies have
shown that both central and peripheral mechanisms are
involved in the development of AD, our understanding is still
relatively limited and prevention is not yet available.

To gain a better understanding of the peripheral changes that


occur in the vasculature after spinal cord injury, changes in
blood flow in vessels above and below the injury level have been
recorded by Doppler-flow probes, both before and after
ganglionic blockade. In response to bolus injections of
phenylephrine, spinal cord injury causes widespread changes in
conductance in vessels above as well as below the level of injury.
METHODS
1a Central effects of OECs
Nine weeks after spinal cord transection at T4, 6 animals with
OEC transplants and 4 controls (media only), were deeply
anaesthetized, perfused with 4% paraformaldehyde and
segments T2-T6 of their spinal cord harvested, postfixed and
cryoprotected. Tissue samples were cut horizontally on a
cryostat and the injury site assessed for general features of
scarring and cavitation. Sympathetic preganglionic neurons
(SPNs) in the intermediolateral columns were stained for
NADPH-diaphorase. SPN morphology was evaluated by a
researcher blind to the treatment. In each animal, 10 SPNs
located rostral (T2-T3) and 10 SPNs located caudal (T5-T6)
to the injury site with clearly distinguishable perikarya and
dendritic trees were selected and measured with ImageJ
software. Only SPNs from nucleus intermediolateralis pars
principalis and pars funicularis were considered (Petras and
Cummings, 1972). Three morphological features were
recorded: area of perikaryon, sum of lengths of all visible
processes and number of primary dendrites.

This study uses a rodent model of spinal injury, with a


transection of the cord at T4 to further our understanding of
the basic mechanisms involved in AD and to trial potential
therapeutic interventions, such as olfactory ensheathing cell
(OEC) transplantation. OECs normally guide the growth of
olfactory processes into the brain (Raisman, 2001). Several
studies have reported that these cells can also support regrowth
of spinal axons, after transplantation into the damaged spinal
cord, and can lead to improved motor function such as in
breathing and locomotion. Our group has also shown that they
can reduce the duration of AD, although their mechanism of
action for this effect is not yet established. The original grant
proposal aimed to address the following 3 questions:
1. What changes in central connectivity underlie the
improvement in cardiovascular function seen after
OEC treatment of the completely transected spinal
cord?
2. Can improvements in cardiovascular function still
occur with delayed transplantation of OECs, for
relevance in treating previous cord injuries?
3. Can similar improvements in cardiovascular function
occur after partial contusion injuries of the cord, which
are the more common injuries seen in humans?

Comparison of morphometric features of SPNs rostral


and caudal to the injury in the OEC-treated and control
animals was carried out with a linear mixed model
(MIXED, SPSS 15.0). This model has been previously used
in other studies of treatment effects dealing with multiple
measurements in subjects (Khan et al., 2006; Santovena et
al., 2007). Measurements were grouped according to the
subject (= animal) and restricted maximum likelihood
analysis (REML) was performed. Post-hoc comparison was
done with Mann-Whitney U tests.

The past year has seen substantial progress on Questions 1


and 2. Additional studies of peripheral vascular changes after
spinal cord injury have provided further insights into the
basic mechanisms involved in autonomic dysreflexia.
1. We have established that the reduced duration of
autonomic dysreflexia seen after OECs is not associated
with regeneration across the transection site. Rather,
the transplantation of OECs leads to morphological

1b Surface temperature changes during autonomic dysreflexia


Adult male AAW rats (n=5) were surgically implanted with
radiotelemetric probes to allow cardiovascular recording in
93

unrestrained, conscious animals. After a 2 week recovery


period the rats were transected at the T4 level. Colorectal
distension was used to trigger AD before, and for 6 weeks
following, transection surgery. During AD recording, an
infrared camera was set to make automatic temperature
readings minute. Following 8 minutes of baseline temperature
and cardiovascular recording, the colorectal balloon was
inflated for a period of 1 minute. The cardiovascular and
temperature recordings were continued until baseline values
were reestablished. The surface temperature of various parts
of each rats body, including the tail, was calculated using
thermal imaging software. Statistical analysis of the
cardiovascular and temperature responses to colorectal
distension from animals before (intact) and each week after
SCI were made using repeated measure ANOVA.

that the morphometric features of control SPNs did not


differ between the rostral and caudal segments. However, in
OEC-treated animals all 3 morphometric features were
higher in rostral segments. Analysis showed that there was
no significant effect of treatment on any of the features
(p > 0.4). Except for soma size (p < 0.001), there was no
effect of rostro-caudal position of the SPNs either (p >
0.05). However, the analysis revealed marked interactions
between treatment and position for all 3 features (soma size:
p = 0.047; dendritic length: p = 0.02; number of primary
dendrites: p = 0.007). This indicated that the difference
between SPNs located rostrally and caudally to spinal cord
injury (SCI) was affected by the OEC treatment.
Fig. 1. Morphometric features
of SPNs. A, example of an SPN
stained for NADPH-diaphorase
in horizontal section through
nucleus intermediolateralis.
Comparison of soma size (B),
dendritic length (C) and
number of primary dendrites
(D) in SPNs rostral and caudal
to the injury site in OECtreated (black) and control
(grey) animals. For all analysed
features, a marked interaction
between position and treatment
was found. Graphs demonstrate
differences uncovered by posthoc analyses (* p < 0.05,
p = 0.055).

2. Effects of delayed OEC transplantation


Rats (n=13) were chronically implanted with radiotelemetric
probes for the measurement of mean arterial pressure (MAP)
and heart rate (HR). After a 2 week recovery period the rats
were transected at the T4 level. One week after the transection,
a suspension of GFP-OECs (0.5 l of 50,000 cells/l) in
culture medium (DMEM/F12 supplemented with 10% fetal
calf serum) was injected into each spinal cord (0.7 mm lateral
and 0.7 mm deep) 1 mm rostral and caudal to the injury site.
Control animals received fibroblasts at similar concentrations.
Colorectal distension was carried out at 4, 6 and 8 weeks after
the spinal cord injury. After 8 weeks the animals were perfused
with 4% paraformaldehyde and the spinal cord removed and
sectioned for visualization of OECs.
3. Peripheral vascular changes after spinal transection
Intact (n=7) and T4 transected (6 weeks post-injury, n=7)
animals underwent a terminal experiment under
pentobarbitone anesthesia. MAP and HR were recoded in
response to bolus injections of adrenergic agonists.
Ultrasonic Doppler-flow probes were placed on arteries
above (common carotid and brachial) and below (renal and
femoral) the T4 transection level and vascular conductance
(blood flow/MAP) was calculated.
The response to the alpha1-adrenergic agonist
phenylephrine (10 microg/kg in 0.3 ml heparinized
saline, infusion rate 0.1 ml/s) was recorded before and
after ganglionic blockade with chlorisondamine (0.6 mg/kg
iv). After this, dose response curves of the vascular
conductance responses were complied for 10 different
doses of phenylephrine (0.03100 microg/kg iv). Finally,
methoxamine (alpha-adrenergic agonist) injections were
given. As with the phenylephrine responses, a large dose
(100 microg/kg) was initially administered, followed by six
doses (1300 microg/kg iv) in randomized order.

Post-hoc analysis with Mann-Whitney U-test showed that


there was no difference in the morphometric features of SPNs
rostral and caudal to the injury in control animals (rostral vs
caudal; soma size: 355 33 microm2 vs 328 26 microm2,
p > 0.4; dendritic length: 339 12 microm vs 359 72 m,
p > 0.9; number of primary dendrites: 3.06 0.15 vs 3.24
0.22, p > 0.6).Yet, significant or close to significant differences
between SPNs rostral and caudal to SCI in OEC-treated
animals were found (rostral vs caudal; soma size: 381
17 microm2 vs. 279 27 microm2, p = 0.016; dendritic
length: 428 35 microm vs. 312 40 microm, p = 0.055;

RESULTS
1a Central effects of OECs
Scar tissue extended mediolaterally completely across the
transection site with no normal tissue or nerve fibres
traversing between rostral and distal cord. NADPH staining
revealed SPNs in the intermediolateral column with a
typical appearance shown in figure 1A. Figures 1B-D show
94

Fig. 2. Thermographic images showing the surface temperature of a spinally transected rat (T4 level, 5 weeks post-transection) before (A) and
during (B) an episode of autonomic dysreflexia. Comparison of these images shows that the mid-tail temperature decreased by 1.7C during
autonomic dysreflexia.

number of primary dendrites: 3.43 0.12 vs. 2.88 0.13,


p = 0.012, respectively). The post-hoc analysis also revealed an
effect of treatment on two of the SPN morphometric features.
In SPNs caudal to the injury, somata of the cells were
significantly smaller in the OEC-treated animals than in the
control animals (p < 0.05). In SPNs rostral to SCI, the
summative dendritic length of SPNs of OEC-treated animals
exceeded that of SPNs of control animals (p < 0.05)

response gradually increased, to be significantly larger


(30 mmHg) from week 46 post-injury (p=0.0003). The
surface temperature of the tail decreased during AD in SCI
animals (Figure 2) and at weeks 45 post-injury was also
significantly greater than that seen in animals prior to injury
(p=0.006). This result suggests that during autonomic
dysreflexia the tail vasculature undergoes constriction leading
to decreased temperature dissipation from the skin surface.

1b. Surface temperature changes during autonomic


dysreflexia
Intact animals experienced a 10 mmHg increase in blood
pressure in response to colorectal distension. After SCI this

2. Effects of delayed OEC transplantation


The cardiovascular baseline values are displayed in Fig. 3A,
B. 2 weeks after spinal cord transection, the MAP
was significantly lower, and HR significantly higher, than

Fig. 3. Cardiovascular parameters in T4 transected animals with transplantation of OECs (red) or fibroblasts (blue) one week after the transection
injury. A, B: Baseline mean arterial pressure (MAP) and Heart Rate (HR) before and 2, 4, 6 and 8 weeks after transection. C, D: Changes in
MAP and HR evoked by colorectal distension for 1 minute at time = 0.
95

pre-transection levels, as found in previous studies. Basal


MAP persisted at lower than normal levels for 8 weeks. No
significant differences were found between OECs and
Fibroblast transplanted groups.

rostral to the injury site. Nine weeks after OEC


transplantation, the soma size of the SPNs caudal to
the SCI was reduced and the total dendritic length of
the SPNs rostral to the SCI increased, compared to the
SPNs of control animals. While this effect might play
a role in shortening of the recovery from autonomic
dysreflexia, the molecular basis of the OECs effect on
the morphology of SPNs needs to be investigated.
Thermal imaging is a non-invasive technique that can
be used in studies investigating the autonomic effects of
spinal cord injury. One application of the imaging is
measurement of the level of cutaneous blood flow of
spinalised animals (and patients) at rest and during
autonomic dysreflexia to understand which vascular
beds are involved in the response.
2. Our results show that delayed transplantation of OECs
does not shorten the duration of autonomic dysreflexia,
unlike the result after acute transplantation. This is
unlikely to be due to poor OEC survival as preliminary
results suggest survival is also poor after acute
transplantation.
3. The presence of enhanced responses to phenylephrine
in blood vessels above and below injury level in T4
transected animals suggests that widespread vascular
changes occur following high level transection. Perhaps
spinal cord injury leads to altered function, or numbers,
of the NET1 transporter, throughout the whole body,
leading to decreased adrenergic agonist removal and
thus prolonged vasoconstriction. This mechanism may
play a role in the development of autonomic dysreflexia
following high level cord injuries.

Cardiovascular responses to colorectal distension 8 weeks


following SCI for OECs and Fibroblasts transplanted animals
are displayed in Fig. 3C, D. In both groups, colorectal
distension caused an increase in MAP accompanied by a
bradycardia (Fig. 1C, D) with no significant differences
between groups. Similarly, no significant differences were
found between the cardiovascular responses produced by
colorectal distension in OECs and Fibroblast groups at 4 and
6 weeks after spinal cord transection (data not shown).
Survival of OECs was assessed at 8 weeks post
transection. GFP expression in OECs was confirmed prior to
transplantation. At 8 weeks OECs were seen in only 2 out of
8 animals. In these cases OECs were longitudinal oriented
close to the injury site. However, no OECs expressing GFP
could be seen within the injury site or in the other 6 animals.
3. Peripheral vascular changes after spinal transection
The response to phenylephrine was enhanced (both in
magnitude and duration) in blood vessels of SCI compared
to intact animals, even following ganglionic blockade
(Figure 4, p<0.035). The enhanced response was present
not only in blood vessels below the lesion (renal and
femoral), but also those above (carotid and brachial), i.e.
those with intact supraspinal control. The phenylephrine
dose response curves of the SCI animals did not show any
signs of supersensitivity to the agonist which could explain
the hyper-responsiveness of the blood vessels.

FUTURE PLANS
For 2008 we will focus on the following studies:
1. Mechanisms of OEC action As noted above our lab has
shown that transplantation of OECs can modify
sympathetic preganglionic neurons above and below a
transection lesion. In this study, we will evaluate the
effect of OECs on the morphology of sympathetic
preganglionic neurones in normal animals, to better
understand their mode of action.
2. Temperature regulation We will use thermography and
skin blood flow to compare skin temperature and
vascular function in normal animals with those after
spinal cord transection. We will evaluate temperatures at
rest and following a cold challenge and assess whether
transplantation of OECs can improve thermal
regulation.
3. Treadmill training and dysreflexia Treadmill training is a
commonly used rehabilitative strategy which can
improve motor function in the lower limbs in patients
after spinal injury. However, the effects of such training
on autonomic dysreflexia have not been evaluated. Here
we aim to assess whether a similar training regimen in
spinalised rats can improve cardiovascular function and
reduce autonomic dysreflexia.

Fig. 4. The change in mean arterial pressure (MAP) seen in intact and
spinally transected animals (T4 level) in response to phenylephrine
(10 microg/kg) injection. The injection was given at time = 0.

Responses to methoxamine did not differ between the


transected and intact animals (p>0.1). The dose response
curves of the two groups to methoxamine injection were also
similar. The absence of exaggerated responses to methoxamine
following spinal transection suggests that the mechanism
causing the hyper-responsiveness to phenylephrine is related to
a phenylephrine dependent action. It is known that
phenylephrine is a substrate of the norepinephrine uptake
transporter NET1, whilst methoxamine is not.
CONCLUSION
1. Outcomes of this study suggest that OECs transplanted
into injured spinal cord change the morphology of
SPNs in the intermediolateral columns caudal and
96

Publications and Presentations from this work

Kalinck T., Mackay-Sim A., Carrive P. and Waite P.M.E.


(2007) Transplants of olfactory ensheathing cells promote
recovery of cardiovascular functions after autonomic
dysreflexia in rats with high spinal cord injury 7th IBRO
World Congress of Neuroscience, Melbourne, Australia.
July 2007
Cloutier F., Lauschke J., Carrive P., Waite P.M.E. (2008)
Potentiation of the hypertensive effect induced by
intracerebroventricular injection of tachykinin NK-3 agonist
senktide following high thoracic spinal cord injury. Australian
Neuroscience Society, Hobart, Australia. January 2008

Papers
Laird A.S., Carrive P., Waite, P.M.E. (2006) Cardiovascular
and temperature changes in spinal cord injured rats at rest
and during automatic dysreflexia. J. Physiology (London)
577 (2): 53948
Laird A.S., Waite P.M.E., Carrive P. (2008) Peripheral
changes above and below injury level lead to prolonged
vascular responses high spinal cord injury. Am. J. Phys: Heart
and Circulatory 294 (2): H78592
Abstracts
Laird A.S., Carrive P., Waite P.M.E. (2007) Treadmill
training after SCI leads to spinal morphological changes
and accelerated development of autonomic dysreflexia.
5th Congress of the International Society of Autonomic
Neuroscience, Kyoto, Japan. October 2007
Laird A.S., Waite P.M.E., Carrive P. (2007) Chronic spinal
cord injury results in vascular hyper-responsiveness both
above and below the level of injury. 7th IBRO World
Congress of Neuroscience, Melbourne. 7, 239. July 2007
Kalinck T., Mackay-Sim A., Carrive P., Waite P.M.E.
(2008) Olfactory ensheathing cells alter morphology of
sympathetic preganglionic neurons and ameliorate
autonomic dysreflexia in a T4-transected rat spinal cord.
6th FENS Forum of European Neuroscience, Geneva,
Switzerland. July 2008
Kalinck T., Mackay-Sim A., Carrive P., Waite P.M.E.
(2007) Acute transplantation of olfactory ensheathing cells
promotes recovery from autonomic dysreflexia in T4 spinal
cord transection. International Symposium on Nerve
Regeneration 2007, Asilomar, CA, USA. December 2007
Kalinck T., Mackay-Sim A., Carrive P., Waite P.M.E.
(2007) Effects of olfactory ensheathing cell transplantation
on cardiovascular disturbances following high spinal cord
injury in rat. Acta Physiologica 2007, Vol 191, Suppl. 658:
OW0829. September 2007

REFERENCES
Khan M.K., Mamou F., Schipper M.J., May K.S., Kwitny
A., Warnat A., Bolton B., Nair B.M., Kariapper M.S.,
Miller M., Brewer G., Normolle D., Merajver S.D., Teknos
T.N. (2006). Combination tetrathiomolybdate and
radiation therapy in a mouse model of head and neck
squamous cell carcinoma. Arch. Otolaryngol. Head Neck
Surg. 132(3): 3338.
Petras J.M., J.F. Cummings (1972). Autonomic neurons in
the spinal cord of the Rhesus monkey: a correlation of the
findings of cytoarchitectonics and sympathectomy with
fiber degeneration following dorsal rhizotomy. J. Comp.
Neurol. 146(2): 189218.
Raisman G. (2001). Olfactory ensheathing cells another
miracle cure for spinal cord injury? Nat. Rev. Neurosci. 2(5):
36975.
Santovena A., Garcia J.T., Farina J.B., Llabres M. (2007).
A linear mixed-effects statistical model for in-vivo evaluation
of recombinant human growth hormone implants
in hypophysectomized rats. J. Pharm. Pharmacol. 59(9):
13137.
Teasell R.W., Arnold J.M., Krassioukov A., Delaney G.A.
(2000). Cardiovascular consequences of loss of supraspinal
control of the sympathetic nervous system after spinal cord
injury. Arch. Phys. Med. Rehabil. 81(4): 50616.

97

Comprehensive evaluation of the physiological and


functional adaptations induced by locomotor training in
incomplete spinal cord injured subjects
Bernie Conway, Ken Hunt, David Allan1, Sujay Galen, Celia Caton
Bioengineering Unit, University of Strathclyde & 1Queen Elizabeth National Spinal Injuries Unit, Southern General
Hospital, Glasgow.
measure changes in spinal cord physiology. The functional
gains seen with Lokomat training along with some trends
observed in the physiological tests are highlighted in this
report together with aspects of the on-going pooled analysis
of our testing programme.

INTRODUCTION
A comprehensive evaluation of the recovery of spinal cord
function is required to study the efficacy of new and
emerging therapeutic interventions that are being proposed
in the repair of spinal cord. It is hoped that identifying these
evaluation tools and establishing its validity and reliability
will enable these new and emerging therapeutic interventions
to be translated into clinical practice. The clinical initiative
phase I for this purpose identified a battery of physiological
and functional measures that could be used to study recovery
in spinal cord injury (P.H. Ellaway et al., 2004), either as a
result of natural history or through novel interventions. The
objective of phase II of the clinical initiative was to utilize
these physiological and functional tests to monitor changes
during an intervention thats most likely to induce
adaptations within the injured spinal cord.

METHODS
Fourteen Subjects have successfully completed the study
(12 Males and 2 Females) out of a target of 20 subjects. The
subjects were either classified as Chronic (> 6 months from
injury, n=5) or Acute (< 6 months from injury, n=9). A
single subject repeated measures study design was adopted
to monitor the functional changes induced by locomotor
training using the Lokomat. Each subject participated in the
study for a total period of 8 weeks (Figure 1). Lokomat
training was provided for an hour each day to each subject
from Week 2 to Week 7 (a total of 6 weeks). Assessments
were performed during Week 1 (Pre-Intervention), Week 5
(Mid-Intervention) and Week 8 (Post-Intervention). The
assessments performed included the battery of tests that were
recommended by Clinical Initiative Phase I and additionally
electrophysiological tests such as Somatosensory Evoked
Potentials (SEP) were added to perform a more robust
assessment of the integrity of the sensory pathways such as
the dorsal column.

Locomotor training using Body Weight Supported


Treadmill Training (BWSTT) has been used as a successful
intervention in incomplete spinal cord injury (E.C. FieldFote, 2001; N.J. Postans et al., 2004; A.L. Hicks et al.,
2005). The use of robotic assistance during BWSTT
has recently emerged as an alternative to traditional methods
of therapist assisted or FES assisted locomotor training
and is hypothesized to be a more efficient method of
delivering locomotor training (T.G. Hornby et al., 2005).
The Lokomat (Hocoma AG, Switzerland ) is a robotic
Driven Gait Orthosis (DGO) used for locomotor training
that incorporates a motorized treadmill and Body Weight
Support system. The lokomat can be programmed to offer
100% guidance to the subject in moving their lower limb
during BWSTT. This ensures that the subject receives an
intensive repetitive training with an almost consistent lower
limb kinematics. The consistent lower limb movement
produced by the Lokomat results in sensory feedback
through phasic afferent signals that induce stepping in ISCI
patients (S.J. Harkema, 2008). This afferent feedback
along with its interaction with other neuronal pools that
may exist such as the central pattern generators (CPG)
in the spinal cord, and input from the supra spinal centers
through the descending pathways is suggested to produce
recovery of locomotion in ISCI patients (B.H. Dobkin,
1999; V. Dietz, 2008). It is hypothesized that this
will enable the subject to learn the movement pattern
(M. Wirz et al., 2001) and later be able to reproduce it
during over ground walking.

Fig. 1. A prospective single-subject study design. The yellow area


represents the assessment phase. Assessments included both neurophysiological and Functional tests. The blue area represents the
Lokomat training phase. Lokomat training was for an hour everyday
five days a week.

A complete list of all the assessments that were


performed are as follows:
Clinical neurological assessment
ASIA classification
Light Touch Score
Pin Prick Score
Upper Limb Motor Score
Lower Limb Motor Score
Functional Independence Measure (FIM)

Therefore the objective of this study is to measure the


functional improvements in locomotion associated with
Lokomat training and in parallel examine the utility of a
comprehensive battery of tests that have the potential to
98

Sensory assessment

The value obtained as the asymmetry index is a


dimensionless number. A value of zero indicates good
symmetry between the right and left foot. A positive value
indicates a longer stance phase and a shorter swing phase
on the left compared to the right. A negative value indicates
a longer stance phase and a shorter swing phase on the right
compared to the left. The subjects were asked to walk over
a 6 meter walkway while these recordings were performed.

Quantitative sensory testing (QST)

Vibratory Perceptual Threshold (VPT)


Electrical Perceptual Threshold (EPT)
Warmth Sensation (WS)
Cold Sensation (CS)
Light Touch Monofilaments

Electrophysiological Sensory Assessment

The SEP measurements were performed with the subjects


in a supine position and the evoked responses were recorded
using a Neuroscan system with Synamps. Recordings from
25 Scalp locations (1020 system) were taken using an
EasyCap with Sliver/Sliver Chloride sintered electrodes. SEPs
were elicited by 4 Hz stimulation (0.5 ms square wave pulse)
from a constant current stimulator (Digitimer DS7A)
delivered to the posterior tibial nerve via a 20 mm fixed bar
electrode (VIASYS Healthcare). The cathode was placed
midway between the Achilles tendon and the medial
malleolus with the anode placed distally. The EEG data was
band-pass filtered (52000 Hz), with an amplifier gain of
1000, and sampled at 10000 Hz. The intensity of the pulse
was set at twice motor threshold. A total of 999 pulses were
taken and recorded for later analysis. SEPs were also recorded
through stimulation of bilateral median and ulnar nerves in
the upper limb. A frequency domain analysis was also
performed using EEGLABS to study stimulus linked changes
in the power of the EEG spectrum. These Event Related
Spectral Pertubations (ERSP), which are usually observed as
Event related synchronsation (ERS) or Event related
desynchronisation (ERD), are more robust in detecting non
phase locked EEG events that cannot be picked up by
traditional averaging techniques.

Somatosensory Evoked Potentials (SEP)

SEPs responses were recorded through stimulation of


bilateral Median and Ulnar nerves in the upper limb
and Posterior Tibial nerve in the lower limb.
Time Domain Analysis Latency and Amplitude
Frequency domain analysis (ERD/ERS)

Electrophysiological Motor Assessment


Motor Evoked Potentials (MEP) using TMS
MEPs were recorded in the upper and lower limb
bilaterally in the key ASIA muscles
MEPs were recorded in bilateral erector spinae muscles
at the level of the lesion, one level above and two levels
below.
Time Domain analysis Latency and Amplitude.
Locomotor assessment on the Lokomat
Evaluation of Lokomat assisted walking
Video analysis of Gait
Evaluation of Lower limb stiffness using assessment
tools on the Lokomat
Evaluation of Lower Limb Strength using assessment
tools on the Lokomat
EMG activity of key lower limb muscles during
lokomat assisted walking (coherence analysis)
All assessments were performed at the Queen Elizabeth
National Spinal Injuries Unit (QENSIU), Glasgow and
ethical approval was obtained from the NHS local ethics
committee and the University of Strathclyde ethics
committee. The clinical neurological assessments were
perfromed before and after the Lokomat training, and so
were the other functional tests such as the FIM and WISCI
II assessments. In subjects who were able to walk either
before or during the course of the study, their ambulatory
status was assessed using the WISCI II scale and a further
detailed gait analysis was performed. Video analysis of gait
included both the saggital and coronal views, and the
quality of gait was assessed using the Ranchos observational
gait analysis method which enabled to score gait deviation.
An instrumented insole was also used to record various
temporal gait parameters and foot contact pattern. An
asymmetry index was also calculated from this data using
the following algorithm.

In addition to the SEP, quantitative sensory tests were


also used to assess the integrity of the sensory pathways.
Light touch sensation was assessed using Semmes-Weinstein
monofilaments. The perception of hot and cold sensation
were objectively assessed using the Medoc thermal analyzer.
The electrical perceptual threshold was assessed using a
Digitimer DS 7A electrical stimulator and the vibratory
thresholds were assessed using a neurothesiometer. The
sensory thresholds were recorded across all dermatomes
from C3-S2 and the protocols that were followed were as
described in the literature (P.H. Ellaway et al., 2004; A.
Nicotra and P.H. Ellaway, 2006; G. Savic et al., 2006).
A pooled analysis of the data was performed in some
outcome measures that were deemed appropriate. Repeated
measures ANOVA was performed to asses the effect of the
intervention over time. Post hoc pair wise comparisons were
made to compare changes in the dependent variable
between various study phases. SPSS (version 16) was used to
perform all statistical analysis.
RESULTS
All subjects (Average age : 49.3 years) who participated in the
study so far went on to complete the study. The details of the
patients who participated in the study are provided in Table 1,
(see p.84). Compliance to the training regime (30 sessions) for
the 14 subjects who have completed the study was high and

If L = % of stance phase on the Left % of swing phase on the Left


If R = % of stance phase on the Right % of swing phase on the
Right
Asymmetry Index = (2(LR)/(L+R)) 100
99

averaged 94% with the majority of subjects missing fewer than


3 sessions over the 6 week training period. The reasons for
missing a session were all unrelated to the use of the Lokomat.
Analysis is ongoing and a data mining exercise will be
conducted on all data sets to establish statistical relationships
once our target recruitment level has been reached.

The SEP responses presented in this report were obtained


from below the level of lesion through stimulation of the
posterior tibial nerve at the ankles. Results from other
stimulation sites above the level of lesion are not presented in
this report. Traditional averaging techniques have been used
to study the short latency components of the SEP using Brain
Electrical Source Analysis (BESA) software whilst a timefrequency representation of the SEP was computed using
EEGLAB to investigate late and non-phase locked event
related potentials. Short latency components of the SEP in
normal subjects are generally stable with good repeatability.
For the patient group the latency of the early SEP
components (P1,N1,P2,N2) were delayed in comparison to
normals (Figure 3). Effects on latency associated over the time
course of training were more commonly observed in the acute
patient group. The example shown in Figure 4A of SEPs
recorded in an acute patient (P3) being the most striking seen
so far show changes in SEP short latency following the mid
intervention assessments which is maintained through the
post intervention assessments. In contrast with a chronic
patient (P7) illustrated in Figure 4B who showed no change
in SEP short latency. Interestingly, for the acute patients a
pooled analysis suggests that the major changes in SEP appear
over the first 3 week of training, which is a similar trend
observed in the gait results, thereafter amplitude changes may
dominate. The averages also suggest marked changes in the
late components of the SEPs and this is largely confirmed by
time-frequency analysis which shows changes in patterns of
Event Related Synchronisation (ERS) and Event Related
Desynchronisation (ERD) over the course of Lokomat
training. In the example shown in Figure 5 no clear evidence
of short latency changes in the SEP (Figure 4B) were evident
suggesting that measures of ERS/ERD can reveal changes in
evoked potentials not evident with standard averaging.

Fig. 2. The WISCI-II Scores recorded in all subjects Pre and Post
Intervention. A score of zero indicates that the subject is non-ambulant
and a maximum score of 20 indicates that the subject was able to
ambulate independently.

The main clinical measure used in this study was the


Standard Neurological Classification of Spinal Cord Injury
(ASIA), which helped classify the type and level of injury. The
Patients capacity to ambulate was assessed using the Walking
Index in Spinal Cord Injury (WISCI II). Five acute patients
were non-ambulant at the start of the study, and three of these
five patients subsequently went on to become ambulant by
the end of the study (Figure 2). As shown in Figure 2 acute
patients seem to have made more gains in their WISCI II
scores compared to the chronic patients. A moderate to strong
correlation was found between the change in the WISCI II
score and the patients ability to tolerate reduced body weight
support (calculated as the slope of the best fit line denoting
reduction in body weight support) during the treadmill
training, (Spearmans Rho R= 0.746 p=0.002). Among the
acute patients there was a moderate but significant correlation
between the change in BWS and the WISCI-II score post
intervention (Spearmans Rho R=0.774 p=0.014). The
spatio-temporal parameters of gait recorded in four chronic
subjects and five acute subjects were analyzed separately in
each of these groups. Four acute subjects and one chronic
subject were not included the analysis because they were
either unable to walk 6 meters (n=3) or they required an
orthosis to ambulate (n=2). The analysis of the pooled data
showed that the acute patients made significant gains
(p<0.05) in walking speed, stride length and cadence during
the first three weeks of lokomat training (Pre-mid assessment)
(Table 2b, see p.103), with the exception of walking speed
which also improved significantly during the last three weeks
of training (Mid-Post assessment). There was a significant
increase (P<0.05) between the Pre and Post measures in
walking speed, stride length and cadence and a significant
decrease (p=0.006) in double support which indicates a better
balance (Table 2a, see p.103). The chronic patients showed
moderate gains in all the spatio-temporal parameters, and
perhaps interestingly their walking speed and cadence
decreased during the first three weeks of training (Pre-Mid
Assessment). There was a trend towards a better symmetry of
gait in all patients (Tables 2A and 2B, see p.103).

Fig. 3. Pooled latencies of early SEP components are shown in healthy


subjects and patients. The early SEP components are shown along the
x axis. The Latency in ms is plotted along the y axis.

The MEP responses were recorded in bilateral erector


spinae muscles in all subjects. MEPs were recorded at the
level of the lesion, one level above and two levels below. The
variability of the MEP responses were quite large as seen in
the analyses of the pooled data of both chronic and acute
subjects (n=10) (Figure 6). The area of the MEP responses
slightly increased below the level of lesion following the
6 week Lokomat training. However no significant
differences were found between the pre and post measures
both in chronic and acute subjects.
100

these QST measures are believed to reflect the integrity of


the dorsal columns their relationship with the short latency
posterior tibial nerve SEP has been examined. Using the
Standard Deviation (SD) observed in the SEP recording of
healthy subjects, the various components in the SEP
response in the patients were classified as Normal (within 3
SD of the mean), Impaired (out with 3 SD of the mean) and
absent (could not be detected). The mean EPT (Figure 7A)
and VPT (Figure 7B) scores recorded at the L5 dermatome
in acute and chronic patients are presented grouped
according to the pre-Lokomat P1 SEP latency classification.

Fig. 4A. SEP recorded in an acute subject (P3) showing changes in the
short latencies. The pre mid and post lokomat sep traces are shown in
black red and blue respectively.

Fig. 7A. Mean electrical perceptual threshold prior to Lokomat


training at L5 dermatome below the level of lesion in chronic (in red)
and acute (in purple) patients compared to pre Lokomat training
P1 Latency.

Fig. 4B. SEP recorded in a chronic subject (P7) showing no clear


change in the short latencies. The pre mid and post lokomat sep traces
are shown in black red and blue respectively.

Fig. 5. An ERS/ERD analysis in a chronic subject (P7) SEP recording


during various study phases are presented. The vertical red dotted line
indicates the time when the stimulus was delivered (Event). Areas
shown in red indicate synchronisation and areas shown in dark blue
indicate desynchronisation.
Fig. 7B. Mean vibration perceptual threshold prior to Lokomat
training at L5 dermatome below the level of lesion in chronic (in red)
and acute (in purple) patients compared to pre Lokomat training
P1 Latency.

The control data of EPT and VPT values were recorded


at the L5 dermatome in healthy subjects (Figures 7A&B).
The pooled analyses showed a similar behaviour in the
relationship between EPT and VPT and P1 SEP classification
in acute cases. The EPT and VPT values are highest where
abnormal SEPs exist and reduce with reductions in the
abnormality ascribed to the SEP. However, in chronic subjects
this relation can be observed for VPT but is less evident for
EPT. This difference in SEP relations to perceptual thresholds
requires further investigation but may reflect features of
long term adaptive changes to mechanisms associated with
sensory perception.

Fig. 6. MEP responses in the erector spinae muscle have been presented
as MEP area (mvms). The MEP responses Pre-Intervention are displayed
in blue and the responses Post-Intervention are displayed in green.

The Electrical Perceptual Threshold (EPT) and


Vibration Perceptual Threshold (VPT), were recorded as part
of a battery of QSTs that were performed on all subjects. As
101

CONCLUSION

Harkema S.J. (2008) Plasticity of interneuronal networks


of the functionally isolated human spinal cord. Brain
Research Reviews 57:255264.
Hicks A.L., Adams M.M., Ginis K.M., Giangregorio L.,
Latimer A., Phillips S.M., McCartney N. (2005) Long-term
body-weight-supported treadmill training and subsequent
follow-up in persons with chronic SCI: effects on functional
walking ability and measures of subjective well-being. Spinal
Cord 43:291298.
Hornby T.G., Zemon D.H., Campbell D. (2005) Roboticassisted, body-weight-supported treadmill training in
individuals following motor incomplete spinal cord injury.
Phys. Ther. 85:5266.
Nicotra A., Ellaway P.H. (2006) Thermal perception
thresholds: assessing the level of human spinal cord injury.
Spinal Cord 44:617624.
Postans N.J., Hasler J.P., Granat M.H., Maxwell D.J.
(2004) Functional electric stimulation to augment partial
weight-bearing supported treadmill training for patients
with acute incomplete spinal cord injury: A pilot study.
Arch. Phys. Med. Rehabil. 85:604610.
Savic G., Bergstrom E., Frankel H.L., Jamous M.A.,
Ellaway P.H., Davey N.J. (2006) Perceptual threshold to
cutaneous electrical stimulation in patients with spinal cord
injury. Spinal Cord 44:560566.
Wirz M., Colombo G., Dietz V. (2001) Long term effects
of locomotor training in spinal humans. J. Neurol.
Neurosurg. Psychiatry 71:9396.

The project so far has been successful in achieving the goals


that were set, and can be summarized as follows

A total of sixteen patients have been recruited so far out


of a target of 20 patients. Fourteen patients have
completed the study and two are currently undergoing
training, and no patients have dropped out of the study.

Functional gains were made by most patients who


participated in the study which is evident from the gait
and WISCI II results. Its clear that acute patients
benefit the most in the first three weeks of training
compared to chronic patients. This finding can be
useful in planning future research projects and also in
clinical decision making with regards to Lokomat
training.
Correlations between the functional outcomes (WISCI
II scores) and the patients ability to tolerate reduction
in BWS may be used as an indicator of the benefits a
patient may receive as a result of lokomat training, and
can useful in determining the duration of lokomat
training.
Gait analysis offers a more robust assessment of
functional recovery in comparison to the assessment
provided by the WISCI II scale, and can be easily
performed in a clinical environment using
instrumented insoles.
The neurophysiological tests such as the SEP have
shown differences in some subjects. The ERS/ERD
tests have been shown to be useful in assessing the
function of ascending pathways in the spinal cord even
when the SEPs are absent, or no marked differences in
the short latency measurements. Therefore SEP
measurements along with ERS/ERD tests have proved
to be valuable tools in assessing recovery in spinal cord
function and its sensory pathways.
The results of this study also suggest close relationships
between the EPT and VPT values and SEP responses in
acute subjects and there longitudinal study may reveal
aspects of adaptive processes that occur post injury.

FUTURE PLANS
We expect the work in the coming year to include the
following
The remaining subjects will be recruited to achieve the
target recruitment number.
Data analysis needs to be performed in all the
remaining SEP data including the ERS/ERD tests
Data Analysis of other outcome measures eg. MEP in
upper and lower limbs, scoring of gait deviations using
observational gait analysis.
A follow-up study of a subset of our subjects is being
planned to ascertain if the functional gains and
associated physiological changes have been maintained
on the long term.
A data mining exercise is also planned once all the
outcome measures have been analysed to perform
various correlation analyses.
The validity and reliability of outcome measures that
have been identified as valuable will be established in
healthy subjects and patients.

REFERENCES
Dietz V. (2008) Body weight supported gait training: from
laboratory to clinical setting. Brain Res. Bull 76:459463.
Dobkin B.H. (1999) An overview of treadmill locomotor
training with partial body weight support: A
neurophysiologically sound approach whose time has come
for randomized clinical trials. Neurorehabilitation and
Neural Repair 13:157165.
Ellaway P.H., Anand P., Bergstrom E.M.K., Catley M.,
Davey N.J., Frankel H.L., Jamous A., Mathias C., Nicotra
A., Savic G., Short D., Theodorou S. (2004) Towards
improved clinical and physiological assessments of recovery
in spinal cord injury: a clinical initiative. Spinal Cord
42:325337.
Field-Fote E.C. (2001) Combined use of body weight
support, functional electric stimulation, and treadmill
training to improve walking ability in individuals with
chronic incomplete spinal cord injury. Arch. Phys. Med.
Rehabil 82:818824.
102

Subject

Age (years)

Sex

Acute/
Chronic

Time since
injury (weeks)

Syndrome

Neurological
Level

P1

26

Chronic

84

Brown-Sequard

P2

49

Acute

13

P3

61

Acute

P4

30

P5

48

P6

ASIA Grade
Pre

Post

C45

Anterior Cord

T8

20

Central Cord

C3

Chronic

44

Anterior Cord

L1

Acute

Anterior Cord

T11

61

Chronic

26

Central Cord

C56

P7

59

Chronic

169

Central Cord

C45

P8

59

Acute

14

Central Cord

C4

P9

58

Acute

Anterior Cord

T11

P10

30

Acute

18

Central Cord

C4

P11

63

Acute

Central Cord

C56

P12

42

Acute

Anterior Cord

T9

P13

57

Acute

13

Anterior Cord

T2

P14

53

Chronic

110

Central Cord

C5

Table 1. Details of the subjects who have completed the study.

Chronic (n=4)

Table 2A
Pre-Mid
Mean
Change

Mid-Post

Cl-lower Cl-upper p-value

Mean
Change

Mean
Change

Cl-lower Cl-upper p-value

13.16

0.171

3.32

6.30

12.93

0.352

0.03

0.19

0.13

0.580

0.12

0.62

0.86

0.642

0.06

14.00

0.25

0.402

0.08

0.10

0.26

0.268

3.03

5.14

11.20

0.323

1.97

6.64

10.58

0.519

13.22

39.11

12.68

0.203

3.80

64.78

57.19

0.856

1.41

12.32

9.49

0.708

4.73

Double Support

0.15

0.64

0.94

0.588

Stride Length

0.02

0.20

0.24

0.820

1.05

11.35

11.35

0.765

9.42

59.32

59.32

0.692

Asymmetry Index

Cl-lower Cl-upper p-value


3.69

Walking Speed

Cadence

Pre-Post

Table 2A. Gait Outcomes in Chronic subjects. CI = 95% confidence Intervals.

Acute (n=5)

Table 2B
Pre-Mid
Mean
Change

Mid-Post

Cl-lower Cl-upper p-value

Mean
Change

Pre-Post

Cl-lower Cl-upper p-value

Mean
Change

Cl-lower Cl-upper p-value

Walking Speed

23.99

3.93

44.06

0.029

4.14

73

7.55

0.028

28.13

8.48

47.79

0.016

Double Support

0.16

0.41

0.09

0.114

0.09

0.18

0.054

0.22

0.29

0.14

0.006

Stride Length
Cadence
Asymmetry Index

0.61

0.05

1.18

0.039

0.06

0.04

0.15

0.171

0.67

0.13

1.22

0.027

16.44

0.82

32.07

0.043

2.2

0.86

5.27

0.117

18.64

3.4

33.88

0.027

3.57

10.35

17.49

0.385

6.74

33.02

46.5

0.662

29.77

39.41

98.95

0.205

Table 2B. Gait Outcomes in Acute subjects. Significant differences have been highlighted in grey. CI = 95% confidence Intervals.

103

ISRT Grantholders
Mr David Allan
Queen Elizabeth National Spinal Injuries Unit
South Glasgow University Hospitals NHS Trust
1345 Govan Road
Glasgow G51 4TF
Tel: 0141 201 2555
Fax: 0141 201 2991

Prof. S. Bolsover
Department of Physiology
University College London
Gower Street
London WC1E 6BT
Tel: 020 7679 6564
Fax: 020 7387 6368
Email: s.bolsover@ucl.ac.uk

Prof. P. Anderson
Department of Anatomy & Developmental Biology
University College London
Gower Street
London WC1E 6BT
Tel: 020 7419 3300
Fax: 020 7380 7349
Email: ucgapna@ucl.ac.uk

Dr E. Bradbury
Centre for Neuroscience Research
Kings College London
Hodgkin Building
Guys Campus
London SE1 1UL
Tel: 020 7848 6192
Fax: 020 7848 6165
Email: elizabeth.bradbury@kcl.ac.uk

Prof. S. Barnett
Department of Medical Oncology
Glasgow University
CRC Beatson Laboratories
Garscube Estate
Switchback Road
Glasgow G61 1BD
Tel: 0141 330 4353
Fax: 0141 330 4127
Email: s.barnett@clinmed.gla.ac.uk

Dr A. Butt
Centre for Neuroscience Research
Kings College London
Hodgkin Building, Guys Campus
London Bridge
London SE1 1UL
Tel: 020 7848 6263
Fax: 020 7848 6569
Email: arthur.butt@kcl.ac.uk

Prof. M. Berry
Department of Medicine
Wolfson Research Laboratories
Edgbaston
Birmingham B15 2TH
Tel: 0121 627 2334
Fax: 0121 472 0499
Email: m.berry@bham.ac.uk

Mr T. Carlstedt
Royal National Orthopaedic Hospital Trust
Brockley Hill
Stanmore
Middlesex HA7 4LP
Tel: 020 8954 2300
Fax: 020 8420 6582
Email: thomascarlstedt@aol.com

Mr R. Birche
Royal National Orthopaedic Hospital Trust
Brockley Hill
Stanmore
Middlesex HA7 4LP
Tel: 020 8909 5803
Fax: 020 8420 6582

Prof. B. Conway
Bioengineering Unit
University of Strathclyde
106 Rotten Row
Glasgow G4 0NW
Tel: 0141 548 3316
Fax: 0141 552 6098
Email: b.a.conway@strath.ac.uk

Dr X. Bo
Neuroscience Centre
Medical Sciences Building
St Bartholemews & the Royal London School of Medicine
& Dentistry
Queen Mary University of London
Mile End Road
London E1 4NS
Tel: 020 7882 2294
Fax: 020 7882 7726
Email: x.bo@qmul.ac.uk

Prof. M. Craggs
Spinal Research Centre
Royal National Orthopaedic Hospital Trust
Brockley Hill
Stanmore
Middlesex HA7 4LP
Tel/Fax: 020 8909 5343
Email: michael.craggs@ucl.ac.uk

104

Dr A. Curt
ICORD (International Collaboration on Repair
Discoveries)
The University of British Columbia
Vancouver, British Columbia V6T 1Z4
Email: curt@cord.ubc.ca

Dr K. Fouad
379 Corbett Hall
Faculty of Rehabilitation Medicine
University of Alberta
Edmonton
Alberta T6G 2G4
Canada
Tel: 00 1 780 492 5971
Fax: 00 1 780 4921626
Email: karim.fouad@ualberta.ca

Prof. P. Doherty
MRC Centre for Developmental Neurobiology
Kings College London
New Hunts House
London SE1 1UL
Tel: 020 7848 6811
Fax: 020 7848 6816
Email: patrick.doherty@kcl.ac.uk

Dr I. Gavazzi
Centre for Neuroscience Research
Kings College London
Hodgkin Building
Guys Campus, London Bridge
London SE1 1UL
Tel: 020 7848 6270
Fax: 020 7848 6165
Email: isabella.gavazzi@kcl.ac.uk

Prof. P. Ellaway
Department of Sensorimotor Systems
Division of Neuroscience & Psychological Medicine
Imperial College School of Medicine
Charing Cross Campus
St Dunstons Road
London W6 8RF
Tel: 020 8846 7293
Fax: 020 8846 7338
Email: p.ellaway@imperial.ac.uk

Prof. R. Keynes
Department of Anatomy
Downing Street
Cambridge CB2 3DY
Email: rjk10@cam.ac.uk

Dr Manuel Enrquez-Denton
Sobell Department for Motor Neuroscience and
Movement Disorders
Institute of Neurology
University College London
Queen Square
London WC1N 3BG
Tel: 020 7837 3611
Fax: 020 7813 3107
Email: mdenton@ion.ucl.ac.uk

Dr P. Kirkwood
Sobell Department of Neurophysiology
The Institute of Neurology
Queen Square
London WC1N 3BG
Tel: 020 7837 3611
Fax: 020 7813 3107
Email: pkirkwoo@ion.ucl.ac.uk
Prof. A. Logan
Department of Clinical Chemistry
Wolfson Research Laboratories
University of Birmingham
Edgbaston
Birmingham B15 2TH
Tel: 0121 627 2268
Fax: 0121 472 0499
Email: a.logan@bham.ac.uk

Prof. Dr med V. Dietz FRCP


University Hospital Balgrist
Forchstrasse 340
8008 Zurich
Switzerland
Tel: 0041 1 386 39 01
Fax: 0041 1 386 39 09
Email: volker.dietz@balgrist.ch

Prof. S. McMahon
Centre for Neuroscience Research
Kings College London
Hodgkin Building
Guys Campus
London SE1 1UL
Tel: 020 7848 6270
Fax: 020 7848 6165
Email: stephen.mcmahon@kcl.ac.uk

Prof. J. Fawcett
University of Cambridge Centre for Brain Repair
Robinson Way
Cambridge CB2 2PY
Tel: 01223 331188
Fax: 01223 331174
Email: jf108@cam.ac.uk

105

Dr A. Michael-Titus
Neuroscience Centre
St Barts & Royal London Sch Medicine & Dentistry
Queen Mary, University of London
4 Newark Street, Whitechapel
London E1 2AT
Tel: 020 7882 2290
Fax: 020 7882 2180
Email: a.t.michael-titus@qmul.ac.uk

Dr G. Raivich
Perinatal Brain Repair Group
Department of Obstetrics and Gynaecology
Department of Anatomy
University College London
8696 Chenies Mews
London WC1E 6HX
Tel: 020 7679 6068
Fax: 020 7383 7429
Email: g.raivich@ucl.ac.uk

Dr V. Mushawar
University of Alberta
Department of Biomedical Engineering
University Centre for Neuroscience
513 Heritage Medical Research Centre
Edmonton, Alberta T6G 2S2
Email: vivian.mushahwar@ualberta.ca

Dr John Riddell
Division of Neuroscience and Biomedical Systems
Institute of Biomedical and Life Sciences
University of Glasgow
Glasgow G12 8QQ United Kingdom
Tel: 0141 330 4495
Fax: 0141 330 4100
Email: j.s.riddell@bio.gla.ac.uk

Prof. H. Perry
CNS Inflammation Group
School of Biological Sciences
Biomedical Sciences Building
University of Southampton
Southampton
Hampshire SO16 7PX
Tel: 023 8059 5931
Fax: 023 8059 2711
Email: perry@walt.ccs.fau.edu

Dr J. Roskams
Department of Zoology
University of British Columbia
6270 University Boulevard
Vancouver, British Columbia V6T 1Z4
Canada
Tel: 001 604 827 5080
Fax: 001 604 827 5085
Email: roskams@zoology.ubc.ca

Prof. J. Priestley
Neuroscience Centre
Medical Sciences Building
St Bartholemews & the Royal London School of
Medicine & Dentistry
Queen Mary University of London
Mile End Road
London E1 4NS
Tel: 020 7882 6343
Fax: 020 7882 7726
Email: j.v.priestley@qmul.ac.uk

Dr P. Shortland
Neuroscience Centre
Institute of Cell & Molecular Sciences
Barts & The London School of Medicine & Dentistry
4 Newark Street, Whitechapel
London E1 2AT
Tel: 020 7882 2295
Email: p.j.shortland@qmul.ac.uk
Dr P. Stroman
Department of Diagnostic Radiology
c/o Centre for Neuroscience Studies
231 Botterell Hall
Queens University
Kingston, Ontario K7L 3N6
Tel: 001 613 5333245
Fax: 001 613 5336840
Email: stromanp@post.queensu.ca

Prof. A. Prochazka
Division of Neuroscience
507 HMRC
University of Alberta
Edmonton, AB T6G 2S2
Canada
Tel: 001 780 492 3783
Fax: 001 780 492 1617
Email: arthur.prochazka@ualberta.ca

Prof. W. Tetzlaff
ICORD
University of British Columbia
Biol.Sci. Building RM 2465
6270 University Boulevard
Vancouver
BC V6T 1Z4
Tel: 001 604 822 1675
Fax: 001 604 822 2924
Email: tetzlaff@cord.ubc.ca

Prof. G. Raisman
Chair of Neural Regeneration
Director, Spinal Repair Unit
Institute of Neurology, UCL
Queen Square
London WC1N 3BG
Tel: 020 7676 2172 (direct)
Fax: 020 7676 2174
Email: g.raisman@ion.ucl.ac.uk

106

Prof. J. Verhaagen
Meibergdreef 33
1105 Az Amsterdam
The Netherlands
Tel: 0031 20 55665500
Fax: 0031 20 6961006
Email: j.verhaagen@nih.knaw.nl

Dr Y. Zhang
Neuroscience Centre
Medical Sciences Building
St Bartholemews & the Royal London School of
Medicine & Dentistry
Queen Mary University of London
Mile End Road
London E1 4NS
Tel: 020 7882 7256
Fax: 020 7882 7726

Prof. P. Waite
Neural Injury Research Unit
University of New South Wales
School of Medical Sciences
Randwick
Sydney, New South Wales 2052
Australia
Tel: 6129 3852475
Fax: 6129 3852809
Email: p.waite@unsw.edu.au

Dr B. Zheng
Department of Neurosciences
University of California San Diego
9500 Gilman Drive, MC0691
Leichtag Building, Rm. 3A12
La Jolla, CA 92093-0691
Tel: 001 858 534 5807
Fax: 001 858 822 1021
Email: binhai@ucsd.edu

107

ISRT Scientific Committee


The Scientific Committee is an international body of non-remunerated eminent scientists and clinicians who advise the
Trustees on research matters. The membership of the Committee reflects a diverse range of expertise from molecular and
cell biology to neurosurgery and clinical practice. The Scientific Committee is called upon as part of the grant review
process and their input instrumental in developing our strategic approach to research funding. They also provide advice
on other matters, such as in response to specific enquiries from the press or official bodies.
Chairman
Prof. J. Priestley PhD MA DPhil
Neuroscience Centre
Medical Sciences Building
St Bartholomews & the Royal London School of Medicine
& Dentistry
Queen Mary University of London
Mile End Road
London E1 4NS
Tel: 020 7882 6343
Fax: 020 7882 7726
Email: j.v.priestley@qmul.ac.uk

Prof. P. Ellaway PhD


Department of Sensorimotor Systems
Division of Neuroscience & Psychological Medicine
Imperial College School of Medicine
Charing Cross Campus
St Dunstons Road
London W6 8RF
Tel: 020 8846 7293
Fax: 020 8846 7338
Email: p.ellaway@imperial.ac.uk
Prof. J. Fawcett PhD FRCP
Centre for Brain Repair
Cambridge University
Forvie Site, Robinson Way
Cambridge CB2 2PY
Tel: 01223 331 188
Fax: 01223 331 174
Email: jf108@cam.ac.uk

Prof. S. Barnett PhD


Division of Clinical Neuroscience
University of Glasgow
Glasgow Biomedical Research Centre,
Room 4B/17, 120 University Avenue,
Glasgow G12 8TA
Tel: 0141 330 8409/4353
Email: s.barnett@clinmed.gla.ac.uk

Dr J. Guest PhD FRSC


University of Miami
Department of Neurological Surgery
Lois Pope LIFE Center
Miami
Florida 33136
USA
Tel: 00 1 305 575 7059
Fax: 00 1 305 575 3337
Email: jguest@med.miami.edu

Mr T. Carlstedt MD PhD FRCS


Royal National Orthopaedic Hospital Trust
Brockley Hill
Stanmore
Middlesex HA7 4LP
Tel: 020 8954 2300
Fax: 020 8420 6582
Email: thomascarlstedt@aol.com
Prof. M. Craggs
Spinal Research Centre
Royal National Orthopaedic Hospital Trust
Brockley Hill
Stanmore
Middlesex HA7 4LP
Tel/Fax: 020 8909 5343
Email: michael.craggs@ucl.ac.uk

Prof. S. Hunt PhD FMedSci


Department of Anatomy & Developmental Biology
Medawar Building
University College London
Gower Street
London WC1E 6BT
Tel: 020 7679 1332
Fax: 020 7383 0929
Email: hunt@ucl.ac.uk

Prof. Dr V. Dietz MD FRCP


Spinal Cord Injury Centre
University Hospital Balgrist
Forchstrasse 340
CH-8008 Zurich
Switzerland
Tel: 00 41 1 386 39 01
Fax: 00 41 1 386 39 09
Email: dietz@balgrist.unizh.ch

Prof. R. Lemon PhD FMedSci


Sobell Department of Neurophysiology
The Institute of Neurology
Queen Square
London WC1N 3BG
Tel: 020 7837 3611
Fax: 020 7813 3107
Email: rlemon@ion.ucl.ac.uk

108

Prof. A. Logan
Department of Clinical Chemistry
Wolfson Research Laboratories
University of Birmingham
Edgbaston
Birmingham B15 2TH
Tel: 0121 627 2268
Fax: 0121 472 0499
Email: a.logan@bham.ac.uk

Prof. J. Silver PhD


Professor of Neurosciences
Case Western Reserve University
10900 Euclid Avenue
Cleveland
Ohio 441064975
USA
Tel: 00 1 216 368 6251
Fax: 00 1 216 368 4650
Email: jxs10@po.cwru.edu

Prof. S. McMahon PhD FMedSci


Centre for Neuroscience Research
Kings College London
Hodgkin Building
Guys Campus
London SE1 1UL
Tel: 020 7848 6270
Fax: 020 7848 6165
Email: stephen.mcmahon@kcl.ac.uk

Prof. J. Verhaagen
Netherlands Institute for Brain Research
Meibergdreef 33
1105 Az Amsterdam
The Netherlands
Tel: 00 31 20 55665500
Fax: 00 31 20 6961006
Email: j.verhaagen@nih.knaw.nl

Prof. G. Raisman DM DPhil FRS


Chair of Neural Regeneration
Director, Spinal Repair Unit
Institute of Neurology, UCL
Queen Square
London WC1N 3BG
Tel: 020 7676 2172
Fax: 020 7676 2174
Email: g.raisman@ion.ucl.ac.uk

Dr G. Raivich
Perinatal Brain Repair Group
Department of Obstetrics and Gynaecology
Department of Anatomy
University College London
8696 Chenies Mews
London WC1E 6HX
Tel: 020 7679 6068
Fax: 020 7383 7429
Email: g.raivich@ucl.ac.uk

Prof. P. Richardson PhD


Academic Unit of Neurosurgery
Royal London Hospital
Whitechapel
London E1 1BB
Tel: 020 7377 7000
Fax: 020 7377 7021
Email: p.richardson@qmw.ac.uk

109

International Campaign for Cures of Spinal Cord Injury


Paralysis (ICCP)
An alliance between the Christopher and Dana Reeve
Foundation, Institut pour la Recherche sur la Molle pinire
et lncephale, Japan Spinal Cord Foundation, International
Spinal Research Trust, Miami Project to Cure Paralysis, Neil
Sachse Foundation, Paralyzed Veterans of America, Rick
Hansen Foundation, Spinal Cure Australia and Wings for Life.
The ICCP is a body of affiliated, not-for-profit
organisations that are working to fund research into cures
for paralysis caused by spinal cord injury. The mission of
the ICCP coalition is to expedite the discovery of cures for
spinal cord injury paralysis.
With this in mind organisations that promote spinal cord
research determined how their collaborative efforts might
further hasten progress. On the 12th of May 1998 in
Charlottesville Virginia they signed a Statement of Intent,
and formed the International Campaign for Cures of Spinal
Cord Injury Paralysis (ICCP).
New members are welcome and the Japan Spinal Cord
Foundation joined the ICCP in 2004. The organisations
meet regularly and produce a multipurpose, general
information package that outlines current research and
statistics on the worldwide prevalence of spinal cord injury.
ICCP member organisations are credited with funding
research that has realised many significant discoveries,
brought scientists new optimism, and significantly changed
the outlook for people who have a spinal cord injury. The
question today is not if effective treatments and cures will be
found, it is a question of when. One of the latest ICCP
initiatives has been the development of a series of guideline
papers on clinical trial design and implementation.

110

ICCP objectives

5. To promote links and communication between


laboratories, scientists, clinicians and other relevant
organisations.
6. To promote heightened communication between
fundraising groups and encourage shared utilisation of
resources and expertise.

1. To attract the best scientists, researchers and clinicians


to the area of nerve regeneration and repair in the CNS,
particularly those who are newly graduating, and
encourage their career commitment to spinal cord
research.
2. To promote public support for the development of
effective treatments and cures by highlighting the
individual vulnerability to injury and the benefits of
cures to present and future generations.

7. To encourage the development of internationally


accepted strategies and priorities for spinal cord injury
research.
8. To evaluate the progress and success of the Campaign
against concrete, measurable outcomes and report
progress.

3. To promote government financial support for spinal


cord research by highlighting the economic cost of
lifetime care following injury.
4. To consider conducting collaborative awareness and
fundraising campaigns to promote the global nature of
spinal cord injury and paralysis cure research.

Information on the global impact of spinal cord injury is included on the ICCP website
(http://www.campaignforcure.org), and a downloadable information pack is available.
Also on the website is a searchable database that provides details of the grants awarded by all member organisations, and
includes links to the websites of all member organisations and opportunities for applying for research grants.
More information on the background and aims of the ICCP is included in
Adams, M. and Cavanagh, J.F.R. (2004) International campaign for cures of spinal cord injury paralysis (ICCP):
another step forward for spinal cord injury research. Spinal Cord 42, 273280.
111

ICCP Spinal Cord Injury Clinical Trials Guidelines


The number of potential cellular-based and pharmaceutical
drug treatments aimed at repairing a spinal cord injury has
increased dramatically over the past few years. Some clinical
trials have already started, many involving drastic invasive
surgery, and several more are planned for the near future.
As the numbers of potential treatments and trials continues
to increase, there is concern that currently there is no
international standard that ensures trials are carried out
consistently and as safely as possible.

The degree and level of injury, timing of clinical


intervention, and the statical power needed to achieve
a valid outcome

Determining appropriate clinical outcome measures for


each type of intervention

Patient selection criteria (inclusion/exclusion) and


ethics
Controlling potential confounding variables
(standardisation of adjunct treatments) and the
organisation of multi-centre trials

It is crucial that there is an effective, international forum


where all aspects of clinical trial design can be discussed,
including topics such as the strength of preclinical data,
participant inclusion criteria, trial design, trial management,
trial duration, validity of outcome measures and
interpretation of results. Enhanced communication between
groups conducting trials and information sharing will
benefit all, including investigators who conduct clinical
trials and patients who participate in them.

The initial workshop brought together researchers and


clinicians from around the world, all of whom are currently
involved in clinical trials in spinal cord injury treatments.
One consistent point of concern was the standard of
examination before and after the treatment. There are many
methods of assessing patients, from measures of muscle
strength to quality-of-life questionnaires. Spinal Researchs
Clinical Initiative was praised as taking a lead in developing
new, accurate and reliable methods of measuring small
changes in either sensation or muscle function that would
be applicable to most clinical trials.

With this in mind, Spinal Research co-sponsored a unique,


important scientific workshop that took place in Vancouver,
Canada in February 2004 under the auspices of the ICCP.
Consequently, three additional study groups met to consider
the particular practical and ethical issues that are associated
with, or peculiar to, clinical trials in spinal cord injury. One
significant outcome from the Workshop was the initiation
of a series of panel meetings with specialists in the field over
a period of 18 months to discuss in detail several of the
issues that must be dealt with before taking potential
therapies to clinical trial. These issues include:

The deliberations and the subsequent conclusions drawn


from this series of meetings culminated in the publication of
five guideline papers, one of which is intended as a summary
document for patients thinking of participating in clinical
trials. The summary document is available for download on
our website homepage or directly (www.spinal-research.org/
downloads/Experimental_Treatment_for_SCI.pdf ).

Fawcett, J.W., Curt, A., Steeves, J.D. et al. (2006) Guidelines for the conduct of clinical trials for spinal cord injury as
developed by the ICCP panel: spontaneous recovery after spinal cord injury and statistical power needed for therapeutic
clinical trials. Spinal Cord 45, 190205.
Lammertse, D., Tuszynski, M.H., Steeves, J.D. et al. (2006) Guidelines for the conduct of clinical trials for spinal cord injury
as developed by the ICCP panel: clinical trial design. Spinal Cord 45, 232242.
Steeves, J.D., Lammertse ,D., Curt, A. et al. (2006) Guidelines for the conduct of clinical trials for spinal cord injury (SCI)
as developed by the ICCP panel: clinical trial outcome measures. Spinal Cord 45, 206221.
Tuszynski, M.H., Steeves, J.D., Fawcett, J.W. et al. (2006) Guidelines for the conduct of clinical trials for spinal cord injury
as developed by the ICCP Panel: clinical trial inclusion/exclusion criteria and ethics. Spinal Cord 45, 222231.
112

ICCP Participating Organisations


Further information and links to members websites are included on the ICCP website http://www.campaignforcure.org
Dr Mark Bacon
Head of Research
International Spinal Research Trust
Bramley Business Centre
Bramley, Guildford, GU5 0AZ
UK
Tel: +44 (0) 1483 898786
Fax: +44 (0) 1483 898763
Email: research@spinal-research.org
http://www.spinal-research.org

Prof. John Steeves


ICORD
University of British Columbia
Vancouver, BC V6T 1Z4
Canada
Tel: +1 604 822 1849
Fax: +1 604 822 9486
Email: steeves@icord.org
http://www.rickhansen.org
Mr David Prast
Spinal Cure Australia
PO Box 131
Artarmon NSW 1570
Australia
Tel: +61 2 9660 1040
Fax: +61 2 9660 4494
Email: david@spinalcure.org.au
http://www.spinalcure.org.au

Ms Susan Howley
Executive VP & Director of Research
Christopher Reeve Paralysis Association
500 Morris Avenue
Springfield, NJ 07081 USA
Tel: +1 973 379 2690
Fax: +1 973 912 9433
Email: showley@crpf.org
http://www.christopherreeve.com

Mr Neil Sachse
Neil Sachse Foundation
141 Ifould Street
Adelaide SA 5000
Australia
Tel: +61 8 8227 1777
Fax: +61 8 8232 4311
http://www.nsf.org.au

Dr Alain Privat
Institut pour la Recherche sur la Molle pinire
Universit de Montpellier II, CC106
Place Eugne Bataillon
34095 Montpellier Cedex 05
France
Tel: +33 4 67 14 33 86
Fax: +33 4 67 14 33 18
Email: u336@crit.univ-montp2.fr
http://www.irme.org

Wings for Life


Frstenallee 4
5020 Salzburg
Austria
Tel: +43 662 6582 4206
Fax: +43 662 6582 5062
http://www.wingsforlife.com

Dr Tamio Hirose
Japan Spinal Cord Foundation
4716 Sumiyosi-cho
Fuchu-city
Tokyo 183-0034
Japan
Tel: 00 81 42 366 5153
Email: jscf@jscf.org
http://www.jscf.org

Beth Goldsmith
Executive Director
The Craig H. Neilsen Foundation
16633 Ventura Blvd., Suite 1050
Encino, CA 91436
Tel: +1 818 8177616
Fax: +1 818 9957099
Email: beth@chnfoundation.org

Mr Thomas Stripling
Paralyzed Veterans of America
801 18th St., NW
Washington, DC 20006
USA
Tel: +1 202 416 7668
Fax: +1 202 416 7641
Email: toms@pva.org
http://www.pva.org

The IRP Foundation


54, avenue Dapples
Case postale 655
CH-1001 Lausanne
Tel: +41 (0) 21 614 7777
Fax: +41 (0) 21 614 7778
Email: info@irp.ch
http://www.irp.ch

113