Brain, Behavior, and Immunity 23 (2009) 474484

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Brain, Behavior, and Immunity

journal homepage: www.elsevier.com/locate/ybrbi

The role of IL-6 and IL-1b in painful perineural inammatory neuritis

Eli Eliav a,*, Rafael Benoliel b, Uri Herzberg c, Mythili Kalladka d, Michael Tal e
a

UMDNJ-New Jersey Dental School, Carmel Endowed Chair in Algesiology, Department of Diagnostic Sciences, Division of Orofacial Pain, 110 Bergen Street, Newark, NJ 07103, USA
Department of Oral Medicine, Faculty of Dental Medicine, The Hebrew University-Hadassah, P.O. Box 12272, Jerusalem 91120, Israel
Center for Biomaterials and Advanced Technologies, Johnson & Johnson Medical Device Group US Route 22W Somerville, NJ 08876-0151, USA
d
Department of Oral Medicine, Vydehi Institute of Dental Sciences and Research Center, # 82, EPIP Area, Whiteeld, Bangalore 560066, India
e
Department of Anatomy and Cell Biology, Faculty of Dental Medicine, The Hebrew University-Hadassah, P.O. Box 12272, Jerusalem 91120, Israel
b
c

a r t i c l e

i n f o

Article history:
Received 8 October 2008
Received in revised form 5 January 2009
Accepted 15 January 2009
Available online 29 January 2009
Keywords:
Neuritis
Neuropathic pain
Inammation
Cytokines
IL-6
IL-1b

a b s t r a c t
Inammation along a nerve trunk (perineural inammation), without detectable axonal damage, has
been shown to induce transient pain in the organ supplied by the nerve. The aims of the present study
were to study the role IL-6 and IL-1b, in pain induced by perineural inammation.
Methods: IL-6 and IL-1b secretion from rats sciatic nerves, L-5 Dorsal Root Ganglia (DRG), and the hind
paw skin, 3 and 8 days following exposure of the nerve to Complete Freunds Adjuvant (CFA), were measured using ELISA method. Hind paw tactile-allodynia, mechano-hyperalgesia, heat-allodynia and electrical detection thresholds were tested up to 8 days following the application of CFA, IL-6 or IL-1b adjacent
to the sciatic nerve trunk. Employing electrophysiological recording, saphenous nerve spontaneous activity, nerve trunk mechano-sensitivity and paw tactile detection threshold (determined by recording action
potential induced by the lowest mechanical stimulus) were assessed 3 and 8 days following exposure of
the nerve trunk to CFA, IL-6, or IL-1b.
Results: IL-6 and IL-1b secretion from the nerve was signicantly elevated on the 3rd day post-operation
(DPO). On the 8th DPO, IL-6 levels returned to baseline while IL-1b levels remained signicantly elevated.
The DRG cytokines level was increased on the 3rd and 8th DPOs, contralateral cytokines level was
increased on the 3rd DPO. The skin IL-6 level was increased bilaterally on the 3rd DPO and returned to
baseline on the 8th DPO. IL-1b levels increased in the affected side on the 3rd and bilaterally on the
8th DPO. Direct application of IL-6 or CFA on the sciatic nerve induced signicant hind paw tactile-allodynia from the 1st to 5th DPOs, reduced electrical detection threshold from the 1st to 3rd DPOs, mechano-hyperalgesia from 3rd to 5th DPOs and heat-allodynia on the 3rd DPO. Direct application of IL-1b
induced paw tactile and heat-allodynia on the 78th DPOs and mechano-hyperalgesia on the 58th DPOs.
Perineural inammation signicantly increased spontaneous activity myelinated bres 3 and 8 days following the application. Direct application of IL-6 induced elevation of spontaneous activity on the 3rd
while IL-1b on the 8th DPO.
Nerve mechano-sensitivity was signicantly increased on the 3rd day following exposure to CFA and IL-6
and on the 8th following CFA application. The rats paw lowest mechanical force necessary for induction
of action potential, was signicantly reduced 3 days following CFA application.
Conclusion: IL-6 and IL-1b play an important role in pain induced by perineural inammation. IL-6 activity is more prominent immediately following application (25th DPOs), while IL-1b, activity is more signicant in a later stage (58th DPOs).
2009 Elsevier Inc. All rights reserved.

1. Introduction
An inammatory reaction is an essential step in tissue and
nerve regeneration, yet it often contributes to the development
of acute and persistent chronic pain states (Lewin et al., 1994; Watkins et al., 1994; Sorkin et al., 1997; Boucher et al., 2000). Perineural inammation along a nerve trunk with no frank axonal damage
* Corresponding author. Fax: +1 973 972 3164.
E-mail address: eliavel@umdnj.edu (E. Eliav).
0889-1591/$ - see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbi.2009.01.012

is sufcient to induce pain in the organ innervated by the affected

nerve (the nerve distal end) (Eliav et al., 1999; Chacur et al., 2001;
Gazda et al., 2001; Benoliel et al., 2002). Hypersensitivity begins as
early as a few hours following the nerve exposure to the inammatory process and may last several days (Eliav and Gracely, 1998;
Eliav et al., 1999; Milligan et al., 2004). Pain is not limited to the
affected site: mirrorimage neuropathic pain may develop and is
probably associated with the potency of the immune or inammatory response (Gazda et al., 2001; Milligan et al., 2003, 2004; Kleinschnitz et al., 2005).

E. Eliav et al. / Brain, Behavior, and Immunity 23 (2009) 474484

Perineural inammation induces spontaneous neurophysiological activity and nerve trunk mechanical sensitivity (Eliav
et al., 2001; Bove et al., 2003; Dilley et al., 2005), that may last
several days. This abnormal neural activity is known to play a
key role in triggering and maintaining neuropathic pain (Govrin-Lippmann and Devor, 1978; Kajander et al., 1992; Devor
et al., 1994; Tal and Eliav, 1996; Dilley and Bove, 2008; Hoffmann
et al., 2008).
The contribution of IL-1b, Tumour Necrosis Factor a (TNFa), and
IL-6 to neuropathic pain, has been studied using knock-out mice,
blocking antibodies or through direct cytokine application (Schwartz et al., 1994; Poole et al., 1995; Saeh-Garabedian et al.,
1995; Wagner and Myers, 1996; Woolf et al., 1997; Cunha et al.,
1999; Ignatowski et al., 1999; Boucher et al., 2000; Junger and Sorkin, 2000; Boucher and McMahon, 2001; Kleinschnitz et al., 2005;
Zelenka et al., 2005).
The aim of the present study was to investigate the role of IL-6
and IL-1b in a model of perineural inammation-induced pain
(Eliav et al., 1999).
IL-1b is an example of a potent proinammatory cytokine, and
IL-6 is an example of a pleiotropic cytokine with proinammatory
and probably regulatory activities.
IL-1b is produced primarily by monocytes and macrophages
(Wewers et al., 1997) but also by astrocytes, oligodendrocytes,
Schwann cells, T cells, broblasts and other cells. Amongst the
proinammatory cytokines, IL-1b is particularly known to modulate pain sensitivity. Following either peripheral or central exogenous administration, IL-1b usually produces hyperalgesia
(Watkins et al., 1994; Sommer et al., 1999; Tadano et al., 1999;
Falchi et al., 2001; Sachs et al., 2002). Moreover, treatment with
neutralizing antibodies to IL-1 receptor type I reduced the development of neuropathic pain in mice that underwent nerve constriction injury (Sommer et al., 1999) and blockade at the spinal
level prevented hyperalgesia induced by inammatory stimuli
(Samad et al., 2001; Sweitzer et al., 2002). Elevated levels of IL1 were detected following peripheral nerve injury and following
inammatory stimuli (Saeh-Garabedian et al., 1995; DeLeo
et al., 1997; Gazda et al., 2001; Shamash et al., 2002; Sweitzer
et al., 2002).
Similar to IL-1b, IL-6 has been studied extensively in association with nerve damage and pain and is thought to play an
important role in the genesis of neuropathic pain (Hirano,
1991a,b; DeLeo et al., 1996; Reichert et al., 1996; Murphy et al.,
1999).
IL-6 is a multifunctional cytokine, with proinammatory and
regulatory effects produced by a variety of tissues including neural
tissues and cell types such as T cells, B cells, macrophages, broblasts, microglia and astrocytes (Song and Kellum, 2005). In contrast to its proinammatory properties (Barton et al., 1996;
Barton, 1997), in other studies IL-6 reduced proinammatory cytokines synthesis, and increased glucocorticoid production (Tilg
et al., 1994; Ruzek et al., 1997; Hu and Xing, 1998).
IL-6 production is rapidly induced during acute inammation
associated with injury, infection, and neuronal death. In a mouse
study, IL-6 was detected as soon as 2 h following nerve transection
and lasted at least 21 days. IL-6 secretion was found to be strain
dependent and correlated to progressive Wallerian degeneration
strains (Reichert et al., 1996).
Not a lot is known about the role of IL-6 and IL-1b in pain induced by perineural inammation. To address this question, the
present set of experiments assessed IL-6 and IL-1b levels of secretion from a nerve exposed to a proinammatory agent, the related DRG and the related distal organ (skin). In addition the
effect of these cytokines directly applied to a nerve was studied
by means of pain-related behaviour and electrophysiological
measurements.

475

2. Materials and methods

The experiments were performed according to a protocol that
was approved by the Hebrew University in Jerusalem Animal Care
and Use Committee and followed the guidelines of the International Association for the Study of Pain (Zimmermann, 1983).
2.1. Experiment 1: Cytokine levels in perineural inammation
2.1.1. Animals and surgeries
Adult male SpragueDawley rats (Harlan, Israel) weighing 250
300 g, at time of surgery, were used. Under sodium pentobarbital
anaesthesia (45 mg/kg, IP; supplemented as necessary), the common sciatic nerves were exposed at mid-thigh level by blunt dissection through biceps femoris. A band (3 mm wide and 25 mm
long) of sterile haemostatic oxidized cellulose (OxycelTM; cotton
type; Parke-Davis & Co., Detroit, MI) was implanted adjacent to
the sciatic nerve. The Oxycel acts as a sponge and is placed loosely
in the vicinity of the nerve without causing nerve constriction or
damage. The Oxycel was saturated with 200 ll of a Complete Freunds Adjuvant (CFA). The control untreated (naive) group of rats
underwent only pentobarbital anaesthesia.
2.1.2. Cytokine Enzyme-Linked Immunosorbent Assay (ELISA)
Three and 8 days following the procedure, under pentobarbital
anaesthesia, sciatic nerves, L-5 DRGs and hind paw skin were harvested. From each CFA exposed nerve three 1 cm sections were collected: one section from the area adjacent to the CFA, a second
section 2 cm distal to the CFA exposed area and a third section
two cm proximal to the CFA application site. From the control
group and the CFA group contralateral side, 1 cm sciatic nerve sections were taken from the mid-thigh area. The L-5 DRGs were taken bilaterally from the CFA group and unilaterally from the
control group. Paw skin samples of approximately 2  2 mm from
the centre of the paw, a territory supplied by the sciatic nerve were
harvested from the affected and contralateral paws. The tissues
were weighed and placed separately in 2 ml medium containing:
89% Dulbeccos modied Eagles medium with D-Glucose
4500 mg/l, 10% Fetal Calf Serum, 1% PenicillinStreptomycin
Amphotericin B solution and the tissue was left in the medium
for 24 h. The medium was collected and the IL-6 or IL-1b levels
were assayed with a two site enzyme-linked immunoassay (ELISA,
R&D Systems Inc., MN, USA). A monoclonal specic rat antibody
was pre-coated onto a microplate for either IL-6 or IL-1b. Medium
collected from all tissues and standards (supplied by R&D) were
pipetted into the wells, any rat-tested cytokine is bound by the
immobilized antibody. After washing away unbound substances,
an enzyme-linked polyclonal antibody (specic for either rat IL-6
or IL-1b) was added to the wells. Following a wash to remove
any unbound antibody-enzyme reagent, a substrate solution was
added to the wells. The enzyme reaction yields a blue product that
turns yellow following exposure to a stop solution. The intensity
of the colour measured is proportional to the amount of the cytokine bound in the initial step. Using a curve plotted from standard
solutions, cytokine levels were calculated as: ng. (cytokine)/ml
(medium)/mg (tissue)/24 h.
2.2. Experiment 2: Cytokine application on the nerve
Adult male SpragueDawley rats (Harlan, Israel) weighing 250
300 g, at time of surgery, were used. Under sodium pentobarbital
anaesthesia (45 mg/kg, IP; supplemented as necessary), the common sciatic nerves were exposed at mid-thigh level by blunt dissection through biceps femoris. A band (3 mm wide and 25 mm
long) of sterile haemostatic oxidized cellulose (OxycelTM; cotton

476

E. Eliav et al. / Brain, Behavior, and Immunity 23 (2009) 474484

type; Parke-Davis & Co., Detroit, MI) was implanted adjacent to the
sciatic nerve. The Oxycel acts as a sponge and is placed loosely in
the vicinity of the nerve without causing nerve constriction or
damage. The Oxycel was saturated with 200 ll of a solution containing one of the following: 0.2 lg of IL-6, 0.4 lg of IL-1b, 200 ll
of CFA or 200 ll of saline. The quantity of cytokine applied was selected based on the levels detected in experiment 1 (i.e. the level
that was secreted within 24 h).
2.2.1. Behavioural assays
The rats mid-plantar hind paw (sciatic nerve territory) was
tested for tactile and heat-allodynia, electrical detection threshold,
and for tactile-hyperalgesia, prior to surgery and every other day
thereafter for 8 days, beginning the rst day post-operation
(DPO). Rats were habituated pre-operatively by allowing them
1015 min in the sensory-testing apparatus for ve consecutive
days. During this time the rats were tested in the hind paw area
with Von Frey hairs, a pin, heat and electrical stimuli and then returned to their cages. The examiner was unaware of the treatment
groups.
Heat-allodynia was assayed with the paw withdrawal test
(Bennett and Xie, 1988; Hargreaves et al., 1988). Rats are placed
on the glass oor of an elevated platform. A high intensity, movable radiant heat source, was placed underneath the glass and
aimed at the plantar surface of one hind paw. Care was taken
to initiate the test when the animal was at rest, not walking
and the hind paw was in contact with the glass oor of the test
apparatus. Stimulus onset activated a timer that was controlled
by a photocell. The hind paw withdrawal reex interrupted the
photocells light and automatically stopped the timer. Latencies
of the reex were measured from the onset of radiant heat until
hind paw withdrawal to the nearest 0.1 s. Each hind paw was
tested four times at intervals of 5 min. The light intensity was adjusted at the beginning of the experiment in order to produce
latencies of approximately 10 s and held constant thereafter.
We express the data as difference scores, computed by subtracting the latency of the control side from that of the treated side.
Negative difference scores thus indicate the presence of hypersensitivity; the normal difference score is approximately zero
(Bennett and Xie, 1988).
Mechano-hyperalgesia was assayed with the pin-prick test (Tal
and Bennett, 1994). The rat was placed on an elevated, perforated
oor and the tip of a 0.2 mm diameter blunted acupuncture needle
(Needle No. 3, Seirin, Japan) was pushed against the mid-plantar
hind paw, until the needle slightly bends (the skin was dimpled
but not penetrated). Under these conditions the bended needle exerts a mean force of 10.5 g as measured on a laboratory scale. The
duration of the pin-prick evoked nocifensive withdrawal reex was
timed with a stopwatch. Normal responses are of very small duration, too quick to time accurately by hand and were therefore arbitrarily assigned a duration of 0.5 s. The data are expressed as
difference scores, computed by subtracting the withdrawal duration of the control side from that of the treated side. Positive
difference scores thus indicate the presence of mechano-hypersensitivity; the normal difference score is zero.
Tactile-allodynia was tested with Von Frey hairs (Tal and Bennett, 1994) utilizing Semmes-Weinstein monolaments (Stoelting
Inc., Wood Dale, IL). This series includes monolaments sorted by
ranks expressing the log 10 of the force applied in milligrams. We
employed laments from 2.36 to 5.88 that apply a force of 0.02
60 g, respectively. With the rat placed on the perforated oor, the
monolaments were tested in order of increasing stiffness, with
each applied ve times at intervals of 14 s. to slightly different
loci of the mid-plantar hind paw. The rst hair to evoke at least
one withdrawal response was designated the threshold. We express the data as difference scores, computed by subtracting the

rank of the threshold of the control side from that of the treated
side. Negative difference scores thus indicate the presence of tactile-allodynia; the normal difference score is zero.
Electrical detection threshold (allodynia) was assessed using a
World Precision Instrument Electrical Stimulator A-365. With the
rat placed on a metal mesh, one electrode was attached to the grid
and the other (spherical, 1 mm diameter) gently placed on the rats
hind paw, when the rat was immobile a 100 Hz current was increased slowly (0.1 milliamperes per second) until the rat withdrew the hind paw. Data is expressed as the ratio of the
threshold in milliamperes from the treated side divided by that
from the contralateral side. Ratios lower than one, thus indicate
the presence of electrical allodynia; the normal ratio is one.
2.3. Experiment 3: Electrophysiology properties of cytokine application
on the nerve
Recordings were preformed 3 and 8 days following saphenous
nerve exposure either to CFA, Saline, IL-6, or IL-1b (n = 5 rats for
time point). The saphenous nerve was chosen as it is entirely sensory and therefore allows recordings with minimal background
activity. The nerve was exposed from the mid-calf to the ischium
and skin edges were used to form a pool that was lled with
warmed parafn oil (34 C). The perineurium of the nerve was incised at the site of recording about 35 mm proximal to the treatment site. Fine axon bundles (microlaments) were teased from
the nerve, disconnected centrally and placed on a single Ag/AgCl
recording electrode referenced to a nearby indifferent electrode.
The bundles remained in continuity peripherally. This permitted
detection of spontaneous discharge originating at the site of the
application or distally. Each strand was observed passively for
2 min. If in this period any spontaneous action potentials were observed, the period of observation was extended. For each strand we
monitored: the presence of spontaneous activity and spike response to nerve inammation site tactile stimulation with
5.88 log g monolament. Percentage of bres spontaneously active
and percentage of spike responses were calculated for each group,
3 and 8 days following the operation.
Teased microlaments differ in axon content, therefore, in order
to count the number of axons sampled in teased microlaments we
used the method described by Liu et al. (2000). Briey, we gradually increased the saphenous nerve stimulus intensity while noting
the number of all-or-non action potentials recruited to saturation.
Microlament counts were made from a random sample of 75
teased microlaments in seven intact rats. These contained a mean
of 5.2 6.8 A bres. The number of axons sampled per experiment
was estimated by multiplying these means by the number of
microlaments observed in each experiment.
The conduction velocity of spontaneously active axons in each
microlament was determined by dividing propagation distance
by the response latency to electrical stimulus pulses delivered to
the nerve mechano-sensitive site. The stimuli were square wave
pulses, 0.050.1 ms in duration, at 1 Hz at variable intensities up
to 20 mA.
For the cutaneous detection threshold (paw mechanical detection threshold) we identied rst the laments receptive eld,
by systematically applying supra-threshold tactile stimuli to the
rats paw; stimulus area that evoked recorded action potential
was considered as the laments receptive eld.
The force applied by lowest Semmes-Weinstein monolaments
lament (0.0260 g) that produced action potential was designated as the detection threshold.
The stimuli were employed in order of increasing stiffness, with
each applied ve times at intervals of 14 s. CFA, IL-1b and IL-6
groups were compared to rats that did not have any previous surgical procedure (Naive).

E. Eliav et al. / Brain, Behavior, and Immunity 23 (2009) 474484

2.4. Statistical analysis

Alpha (two-tailed) for signicance in all analyses was set at
0.05. Data was tabulated and analyzed using StatView 5 software
(SAS Institute Inc., NC, USA). Behavioural statistical analyses were
performed only on rats with data at all time points.
For tactile-allodynia, mechano-hyperalgesia, electrical thresholds, and heat-allodynia, time points of relevance were analyzed
with a factorial analysis of variance (ANOVA). Fishers PLSD procedure for pairwise comparisons was selected to examine differences
between individual groups. Within group data were compared
using a repeated measures ANOVA (RANOVA).
Percent of spontaneously active nerve bres was calculated at
days 3 and 8 following the procedure. Differences amongst groups
at these time points were analyzed with a factorial ANOVA followed by Fishers PLSD comparisons.
Levels of IL-6 and IL-1b were calculated at days 3 and 8 following the procedure. Differences amongst groups at these time points
were analyzed with a factorial ANOVA followed by Scheffs pairwise comparisons.

3. Results
All results in graphs and text are expressed as means standard
error of the mean (SEM).
3.1. Experiment 1: Cytokine levels following perineural inammation
3.1.1. Nerve (Fig. 1)
3.1.1.1. IL-6. Three days following the surgical procedure a signicant elevation in secreted IL-6 levels was found in the nerve segment adjacent to the CFA application (n = 10; 173.58 36.14 ng/
ml/mg/24 h; P = 0.04; ANOVA F4,18 = 3.95). compared to non-

477

manipulated control nerves (n = 4; 5.53 2.89 ng/ml/mg/24 h).

IL-6 levels were not elevated in the distal (n = 4; 70.04 10.35 ng/
ml/mg/24 h; P = 0.89) and the proximal (n = 4; 73.12 6.53 ng/ml/
mg/24; P = 0.87) segments or the contralateral nerves (n = 4;
45.47 2.96 ng/ml/mg/24 h; P = 0.97).
Eight days following the procedure IL-6 levels had been reduced
to normal levels in the segment adjacent to the CFA application
(n = 10; 17.30 3.09 ng/ml/mg/24 h; P = 0.22 in comparison with
non-manipulated control nerves; n = 4; 5.53 2.89 ng/ml/mg/
24 h).
3.1.1.2. IL-1b. Three days following the procedure the cytokine levels in the nerve segment adjacent to the inammation (n = 10;
473.55 99.87 ng/ml/mg/24 h; P = 0.006; ANOVA F6,23 = 4.10) was
signicantly elevated compared to the level assessed in the nonmanipulated control nerves (n = 4; 0.19 0.09 ng/ml/mg/24 h).
No signicant elevation was found, in the distal (n = 4; 272.55
60.26 ng/ml/mg/24 Hz; P = 0.092) the proximal (n = 4; 152.36
29.96 ng/ml/mg/24 h; P = 0.333) portions nor in the contralateral
nerves (n = 4; 131.66 3.48 ng/ml/mg/24 h; P = 0.40).
Eight days following the procedure the cytokine levels remained signicantly elevated in the nerve portion adjacent to the
inammation (n = 10; 356.70 55.78 ng/ml/mg/24 h; ANOVA
F4,18 = 5.85 P = 0.004) compare to the non-manipulated control
nerves.
3.1.2. DRG (Fig. 2)
3.1.2.1. IL-6. Three days following the CFA application, IL-6 was signicantly elevated in the ipsilateral DRG (n = 4; 156.64 19.62 ng/
ml/mg/24 h ANOVA F2,8 = 76.24) compared to the naive rats DRG
(n = 4; 3.18 0.18 ng/ml/mg/24 h, P < 0.001), and compared to
the contralateral DRG (n = 4; 30.61 0.57 ng/ml/mg/24 h,
P < 0.001). The contralateral DRGs IL-6 level was signicantly ele-

Fig. 1. IL-6 (A) and IL-1b (B) 24 h secretion levels from sciatic nerves exposed to CFA induced neuritis was quantied employing ELISA. IL-6 and IL-1b secretion were
signicantly () elevated on the 3rd day post-operation at the site subjected to CFA. On the 8th day, IL-6 level returned to baseline while IL-1b level remained signicantly
elevated. Data are presented as means SEM.

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E. Eliav et al. / Brain, Behavior, and Immunity 23 (2009) 474484

Fig. 2. IL-6 (A), and IL-1b (B) 24 h secretion level from L-5 DRG, 3 and 8 days following sciatic nerve CFA induced neuritis were quantied employing ELISA. In the ipsilateral
DRG, levels of both cytokines were signicantly () elevated on the 3rd and 8th days. In the contralateral DRGs, IL-6 and IL-1b levels were signicantly elevated on the 3rd
DPO.

vated compared to the naive rats DRG (P = 0.017). Eight days following the procedure, although IL-6 levels in the ipsilateral DRG
decreased from the level of 3rd DPO, it remained signicantly elevated (n = 4; 37.40 3.17 ng/ml/mg/24 h ANOVA F2,8 = 153.59)
compared to the naive rats DRG (3.18. 0.18 ng/ml/mg/24 h,
P < 0.001) and the contralateral DRG (n = 4; 6.33 0.16 ng/ml/mg/
24 h, P < 0.001).
3.1.2.2. IL-1b. Three days following CFA application, IL-1b was signicantly elevated in the ipsilateral DRG (n = 4; 278.10
30.15 ng/ml/mg/24 h ANOVA F2,8 = 65.76, P < 0.001) compared to
the naive rats DRG (7.30 2.93 ng/ml/mg/24 h), and compared
to the contralateral DRG (69.11 1.04 ng/ml/mg/24 h P < 0.001).
The contralateral DRGs IL-1b level was signicantly elevated
compared to the level in the naive rats DRG (P = 0.034). Eight
days following the procedure, IL-1b was signicantly elevated in
the ipsilateral DRG (n = 4; 64.84 15.00 ng/ml/mg/24 h ANOVA
F2,8 = 11.19) compared to the naive rats DRG (7.298 2.927 ng/
ml/mg/24 h, P = 0.001), and compared to the contralateral DRG
(22.88 1.95, P = 0.009).
3.1.3. Paw skin (Fig. 3)
Signs of inammation such as swelling or colour changes were
not observed in paws of rats that were exposed to perineural
inammation, this is similar to our previous reports in the same
model (Eliav et al., 1999).
3.1.3.1. IL-6. Three days following the application IL-6 levels were
signicantly increased in the affected side (n = 6; 156.47 6.65 ng/
ml/mg/24 h ANOVA F2,8 = 94.76, P < 0.001) and in the contralateral
side (n = 6; 103.37 5.78 ng/ml/mg/24 h ANOVA F2,8 = 94.76,
P < 0.001) compared to the nave rats paw (n = 6; 27.40 3.14 ng/
ml/mg/24 h). On the 8th day following the application the IL-6 level
was not different from the nave rats secretion level.

The secretion from the proximal and distal portions of the affected nerve and from the contralateral nerves were elevated
compared to the secretion from nave nerves but was not
signicant.
3.1.3.2. IL-1b. Three days following the application IL-1b level was
signicantly elevated in the affected side (n = 6; 130.99 11.19 ng/
ml/mg/24 h ANOVA F2,8 = 5.98, P = 0.009) compared to the nave
rats paw (n = 6; 84.43 1.32 ng/ml/mg/24 h). On the 8th day the
IL-1b level in the affected side (n = 6; 140.56 8.39 ng/ml/mg/
24 h ANOVA F2,8 = 29.64, P < 0.001) and the contralateral (n = 6;
128.59 4.05 ng/ml/mg/24 h ANOVA F2,8 = 29.64, P < 0.001) side
were signicantly elevated compared to the naive rats paw level.
The secretion from the proximal and distal portions of the affected
nerve and from the contralateral nerves were elevated compared
to the secretion from nave nerves but was not signicant.
3.2. Experiment 2: Cytokines application on the nerve (Fig. 4)
3.2.1. Baseline measurements
The pre-treatment baseline scores were not signicantly different from the predicted normal values (difference scores of zero
for the mechanical and thermal behavioural tests and a ratio of
one for the electrical threshold). Moreover, there were no significant between-group differences for the baseline scores in each
behavioural modality. For all groups combined (with left and right
hind paws averaged together), baseline heat-evoked withdrawal
latencies were 9.9 0.12 s. The baseline pin-prick evoked withdrawal duration was 0.5 0.0 s. The baseline ratio of electricalevoked withdrawal threshold was 32.5 3.1 mA. The baseline
median Von Frey hair threshold was 5.07 log 10 mg; (10 g force).
No signicant changes were observed in the contralateral hind
paw compared to baseline in any of experimental groups or
modalities.

E. Eliav et al. / Brain, Behavior, and Immunity 23 (2009) 474484

479

(ANOVA F4,25 = 11.93 for day 3, F4,25 = 15.49 for day 5, F4,25 = 3.96
for day 7 and F4,25 = 4.79 for day 8. Fishers PLSD test P < 0.05).
3.2.4. Tactile-allodynia
Results are expressed as difference between the surgery side
paw and the contralateral side (difference log 10 g thresholds
SEM).
In the rst day following the procedure, the CFA ( 0.08
0.12 log 10 g) and IL-6 ( 0.63 0.13 log 10 g) groups difference
scores were signicantly lower compared to the IL-1b group.
Three days following the procedure, the CFA ( 1.08 0.19 log
10 g) and the IL-6 ( 0.88 0.23 log 10 g) groups difference scores
were signicantly lower compared to the saline control group.
The CFA group difference scores were signicantly lower compared
to the IL-1b group as well.
Five days following the procedure only the CFA group
( 1.08 0.19 log 10 g) difference scores were signicantly lower
compared to the saline group. Seven days following the procedure,
only the IL-1b group ( 0.50 0.23 log 10 g) difference scores were
signicantly lower compared to the saline group.
Eight days following the procedure the IL-1b group difference
score ( 0.51 0.09 log 10 g) were signicantly lower compared to
the IL-6 and saline groups. (1st day ANOVA F4,25 = 7.34, 2nd day:
ANOVA F4,25 = 14.37, 3rd day: ANOVA F4,25 = 9.41, 5th day: ANOVA
F4,25 = 4.63, 7th day: ANOVA F4,25 = 5.57, 8th day: ANOVA F4,25 =
10.04, Fishers PLSD test P < 0.05).

Fig. 3. IL-6 (A), and IL-1b (B) 24 h secretion level from hind paw skin, 3 and 8 days
following sciatic nerve CFA induced neuritis were quantied employing ELISA. In
the ipsilateral paw, level of both cytokines were signicantly () elevated on the 3rd
day while only IL-1b remained elevated on the 8th day. In the contralateral paws IL6 levels were signicantly elevated on the 3rd day while IL-1b level was elevated on
the 8th.

3.2.2. Heat-allodynia
Results are expressed as withdrawal latency difference scores
between the surgery and the contralateral side (seconds SEM).
The differences observed in the CFA ( 3.26 0.35 s) and IL-6
( 2.80 0.62 s) exposed groups, 3 days following the procedure
were signicantly lower compared with the baseline data. Seven
and 8 days following the procedure the difference scores observed
in rats exposed to IL-1b ( 4.62 1.18 and 4.77 1.58 s) were signicantly lower compared with IL-6 or saline exposed groups and
baseline values, (ANOVA F4,25 = 6.91 for 3 days; ANOVA F4,25 = 6.89
for 7 days; ANOVA F4,25 = 8.79 for 8 days, Fishers PLSD test
P < 0.05).
3.2.3. Mechano-hyperalgesia
Results are expressed as withdrawal duration difference scores
between the surgery side paw and the contralateral side (seconds
SEM).
Three and 5 days following the procedure the differences observed in CFA (4.58 0.63 and 3.75 0.40 s) and IL-6 (4.49 1.08
and 4.17 0.62 s) exposed groups, were signicantly higher compared with baseline and saline exposed groups.
Five, 7 and 8 days following the procedure, the difference scores
observed in the IL-1b group (5th day: 3.92 0.98 s, 7th day;
3.18 0.49 s, 8th day: 2.00 0.84 s) were signicantly higher than
the baseline and saline exposed groups. On days 7 and 8 they were
signicantly elevated compared to the CFA and IL-6 groups as well.

3.2.5. Electrical detection threshold

The results are expressed as the detection threshold ratio
(SEM) of the surgery side divided by the contralateral side.
On the rst day following the procedure the CFA (0.79 0.03)
and IL-6 (0.76 0.03) exposed groups ratios were signicantly lower compared to the baseline and the IL-1b or saline exposed groups.
Two and 3 days following the procedure the CFA (2nd day:
0.69 0.03, 3rd day: 0.64 0.02) and IL-6 (2nd day: 0.74 0.03,
3rd day: 0.70 0.03) groups ratios were signicantly lower compared to baseline and the saline exposed group. The CFA group ratio was signicantly lower compared to the IL-1b as well.
No signicant differences were found amongst the groups on
the 5th and 7th DPOs.
Eight days following the procedure the IL-1b exposed group ratio ( 0.503 0.23) was signicantly lower compared to the baseline results but only approached signicance compared to the
saline (P = 0.057) group.
(1st day ANOVA F4,25 = 19.91, 2nd day: ANOVA F4,25 = 8.85, 3rd
day: ANOVA F4,25 = 18.26, 8th day: ANOVA F4,25 = 3.55, Fishers
PLSD test P < 0.05).
3.3. Experiment 3: Electrophysiological results of CFA and cytokine
application on the nerve
3.3.1. Spontaneous activity (Fig. 5A)
Three days following the procedure, 11.56 1.5% from the CFA
exposed bres (total of 398 bres); 12.09 2.0% from the IL-6 exposed bres (total of 324 bres) were spontaneously active. Significantly lower spontaneous activity was measured in the IL-1b
exposed nerves: 2.62 1.4% (total of 255 bres), and the saline exposed nerves: 2.00 0.6% (total of 324 bres) (ANOVA F4,18 = 12.64,
Fishers PLSD test P < 0.05).
Eight days following the procedure 11.49 1.9% from the IL-b
exposed bres (total of 326 bres) and 7.75 1.9% of the CFA exposed bres (total of 264 bres) were spontaneously active. Significantly lower spontaneous activity was measured in the saline
exposed nerves: 0.97 0.64% (total of 321 bres) (ANOVA
F4,18 = 12.92, Fishers PLSD post hoc test P < 0.05).

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E. Eliav et al. / Brain, Behavior, and Immunity 23 (2009) 474484

Fig. 4. Hind paw heat-allodynia (A), mechano-hyperalgesia (B), tactile-alloynia (C) and electrical detection threshold (D) were assessed 1, 2, 3, 5, 7 and 8 days following
application of IL-6, IL-1b, CFA or Saline. IL-6 and CFA induced signicant () hind paw tactile-allodynia on days 15, electrical hypersensitivity on days 13, mechanohyperalgesia on days 35 and heat-allodynia only during day 3. Direct application of IL-1b induced paw tactile and heat-allodynia on days 7 and 8 and mechano-hyperalgesia
on days 58.

Fig. 5. Electrophysiological measurements from saphenous nerves exposed to CFA neuritis. (A) Spontaneous activity, (B) nerve trunk mechano-sensitivity, (C) paw tactile
detection threshold dened as the minimal force necessary to induce action potential. Signicantly increased activity was observed in the nerves exposed to CFA and IL-6 on
day 3, while in the nerves exposed to CFA and IL-1b on day 8. Signicantly increased mechano-sensitivity was observed in the nerves exposed to CFA and IL-6 on day 3, while
in the nerves exposed to CFA on day 8. The rats paw mechanical detection threshold was signicantly reduced 3 days following CFA application and 8 days following either
CFA or IL-6 application compared to naive rats.

E. Eliav et al. / Brain, Behavior, and Immunity 23 (2009) 474484

3.3.2. Nerve mechano-sensitivity (Fig. 5B)

Three days following the procedure, the percentage of the bres
responding to mechanical stimulus in the CFA group (10.12 3.36%
of 322 bres) was signicantly elevated compared to the saline exposed group (1.33 0.09% of 324 bres (ANOVA F4,18 = 5.89, Fishers PLSD test P < 0.05). The percentage of the bres responding
to mechanical stimulus in the IL-6 exposed group (6.98 1.11%
of 398 bres) was signicantly elevated compared to the saline
(P = 0.037) group. The percentage of the bres responding to
mechanical stimulus (5.12 0.83% of 255 bres) in the IL-1b group
was not signicantly elevated compared to the other groups.
Eight days following the procedure, the percentage of the bres
responding to mechanical stimulus in the CFA group: (10.32
3.07% of 264 bres) was signicantly elevated compared to the saline exposed group: (0.66 0.40% of 321 bres), and the IL-6 exposed group (1.24 0.7% total of 231 bres; ANOVA F4,18 = 6.12,
Fishers PLSD test P < 0.05). The percentage of the bres responding
to mechanical stimulus in the IL-1b group: (5.15 1.34% of 326 bres) was elevated, but not signicant.
3.3.3. Cutaneous mechano-sensitivity (Fig. 5C)
Three days following the procedure, CFA exposed nerves cutaneous detection threshold (0.92 0.153 g) was signicantly lower compared to IL-1b (4.65 0.712 g) and naive rats detection threshold
(3.71 0.334 g), ANOVA F4,11 = 11.66, Fishers PLSD test P < 0.05).
Eight days following the procedure CFA (1.66 0.194 g) and IL-6
(1.63 0.324 g) exposed nerves cutaneous detection threshold
was signicantly lower compared to naive rats detection threshold
(3.710 0.334 g).
3.3.4. Conduction velocity (Fig. 6)
Three days following the procedure the mean conduction velocity of the CFA exposed group spontaneously active bres was
14.86 1.332 m/s. A total of 47 bres were studied, 8% of the bres

481

conduction velocity was slower than 5 m/s, 45% 615 m/s, 30% 16
24 m/s and 17% faster than 25 m/s. The mean conduction velocity
of the IL-6 exposed spontaneously active bres was 29.56
1.23 m/s, where a total of 30 bres were observed: None of the bres conduction velocity was slower than 5 m/s, 37% 1624 m/s
and 63% faster than 25 m/s. The average conduction velocity of
the IL-1b exposed spontaneously active bres was 12.46
0.96 m/s, where a total of 22 bres were observed: None of the bres conduction velocity was slower than 5 m/s or faster than
24 m/s, 81% 615 m/s and 19% 1624 m/s.
Eight days following the procedure the average conduction
velocity of the CFA exposed group spontaneously active bres
was 15.86 1.69 m/s, a total of 22 bres were observed: 5% less
than 5 m/s, 41% 615 m/s, 36% 1624 m/s and 18% faster than
25 m/s. The average conduction velocity of the IL-6 exposed group
spontaneously active bres was signicantly (P < 0.0001) reduced
to 16.75 0.84 m/s, where a total of 21 bres were observed: None
of the bres conduction velocity was slower than 5 m/s, 33% were
615 m/s, 62% 1624 m/s and only 5% faster than 25 m/s.
The average conduction velocity of the IL-1b exposed group spontaneously active bres was signicantly elevated (P < 0.0001) to
25.80 1.01 m/s, where a total of 33 bres were observed: None of
the bres conduction velocity was slower than 5 m/s, 6% were 6
15 m/s, 15% 1624 m/s and 79% faster than 25 m/s.
4. Discussion
Previous studies have shown that perineural inammation with
no frank axonal damage is sufcient to produce pain and hypersensitivity in the nerves target organ (Eliav et al., 1999, 2001; Chacur
et al., 2001; Gazda et al., 2001; Benoliel et al., 2002; Bove et al.,
2003). A chronic, yet mild inammatory process that does not result in nerve damage as well as severe inammation that does give
rise to such injury can result in central sensitization and chronic
neuropathic pain (Gazda et al., 2001; Eliav et al., 2004; Milligan
et al., 2004).
The present set of experiments studied the contribution of IL-6
and IL-1b to perineural inammation related pain (neuritis). We
measured the levels of secreted cytokines from related nerves,
DRG and skin paw following perineural inammation. The role of
these cytokines was further evaluated by direct application on the
sciatic nerve trunk of IL-6 and IL-1b, followed by measurement of
pain-related behaviour of the rats paw and assessment of the related
nerves electrophysiological properties. The dose applied to the
nerve corresponded to the secreted levels from a nerve exposed to
an inammatory adjuvant (CFA) on the 3rd DPO (200 ng of IL-6
and 400 ng of IL-1b). It has been shown in previous studies that neuritis pain peaks between 2 and 5 days following CFA application and
resolves after 7 days (Eliav et al., 1999, 2001, 2004), therefore pain
behaviour was assessed up to 8 days following cytokine application.
For similar reasons, electrophysiological properties were recorded
on the 3rd and 8th days following the procedure.
4.1. Experiment 1 cytokine levels following perineural inammation

Fig. 6. The conduction velocity of spontaneously active axons in each microlament was determined by dividing propagation distance by the response latency to
electrical stimulus pulses delivered to the nerve mechano-sensitive site. Spontaneous activity induced by IL-6 and IL-1b, comprise of faster bres compared to the
activity induced by CFA.

Cytokines are present in the nerve area within 510 h following

nerve transection. Schwann cells secrete IL-1 and TNFa, which induce IL-6 secretion from local broblasts and Schwann cells. Three
to 4 days following the nerve transection further cytokine secretion is induced by macrophages that are recruited to the area (Reichert et al., 1996; Shamash et al., 2002). In the present study,
perineural inammation increased secretion of IL-6 and IL-1b not
only in the affected nerve vicinity but also in the DRG and the related paw skin. Moreover, signicant elevation was found in the
contralateral DRG (3 DPO) and the skin (IL-6 3 DPO and IL-1b 8

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E. Eliav et al. / Brain, Behavior, and Immunity 23 (2009) 474484

DPO), but not in the contralateral nerves. Previous studies that induced perineural inammation with a different inammatory
adjuvant (zymozen) and nerve injury studies, have demonstrated
increases in the contralateral nerve cytokines levels (Gazda et al.,
2001; Kleinschnitz et al., 2005). This discrepancy may be associated with the degree of injury or the severity of inammation; typically CFA induced neuritis does not result in frank nerve damage,
while zymosan-induced neuritis results in peripheral demyelination (Gazda et al., 2001; Kleinschnitz et al., 2005). Although the
CFA induced perineural inammation was not sufcient to induce
contralateral nerve cytokines secretion, the induction of some degree of central processing is supported by the elevation in contralateral DRG and skin cytokine levels (Wieseler-Frank et al., 2007).
It is well known that IL-6 and IL-1b play an important role in
neuropathic pain (Arruda et al., 1998, 2000; Granados-Soto et al.,
2001; Wolf et al., 2003; Bessler et al., 2006; Ma and Quirion,
2006), however the differential actions dependant upon the specic timing of appearance of these cytokines has not been previously reported. While IL-6 level followed the pain behaviour and
peaked on the 3rd DPO and returned to normal on the 8th DPO,
IL-1b level remained elevated on the 8th DPO in the absence of
detectable pain. This is true for all the three sites tested; the nerve,
DRG and related skin. This suggests a good association between
pain behaviour and IL-6 levels (Murphy et al., 1999). The cells
responsible for these cytokines were not investigated in this study,
this should be addressed in further research.
4.2. Experiment 2: Cytokines application on the nerve
IL-6 induced pain arose shortly after its application to the nerve
and lasted up to 5 days. In contrast, IL-1b application induced signicant pain from 5 days after its application and the pain lasted
throughout the experimental period; at least 8 days following
application (mechano-hyperalgesia 58 DOPs, heat and tactileallodynia 78 DPOs). A study that focused on IL-1b and TNFa demonstrated that sciatic nerve intraneural injection of IL-1b induced
mechano-allodynia after 6 days and heat-allodynia only after
9 days. In the same study TNFa injection produced tactile and
heat-allodynia earlier, at 3 and 4 days following the procedure
(Zelenka et al., 2005). Similar to the present study the cytokines
were in contact with the sciatic nerve trunk at the mid-thigh level
while the pain was assessed in the nerve target organ, the rats
paw. Consequently we can postulate that perineural IL-6 and TNFa,
initiate pain behaviour in the nerve distal end within 3 days while
IL-1b is active in pain generation later (69 days). The clinical signicance of this nding is not clear yet, however it may suggest
that various cytokines although present in the inammatory milieu
at the same time may contribute to pain and sensitivity in different
phases of the process. The immediate effect of IL-6 may suggest direct mechanisms in contrast to other cytokines that act indirectly
or by activation of other, downstream events.
IL-6 produced only brief heat-allodynia (on DPO 3) while IL-1b
produced signicant heat-allodynia that started on DPO 7 and was
still present on the 8th DPO, at the end of the experiment. This can
point towards selective cytokine effects on pain modalities. IL-1b
can signicantly affect heat-type pain while IL-6 may have only
limited effects on heat pain.
4.3. Experiment 3: Electrophysiology properties of cytokines
application on the nerve
Perineural inammation (neuritis) induced by CFA application
has been shown to produce nerve trunk mechano-sensitivity and
spontaneous activity, that typically correlates with pain behaviour
(Kajander et al., 1992; Tal and Eliav, 1996; Eliav et al., 2001, 2004;
Bove et al., 2003; Dilley et al., 2005). This correlation occurred fol-

lowing IL-6 and IL-1b application as well (Fig. 5A and B). IL-6, that
induced pain on the 35th DPOs, induced both mechano-sensitivity and spontaneous activity on the 3rd DPO but not on the 8th. IL1b application that induced pain on the 78th DPOs induced significant spontaneous activity on the 8th DPO but not on the 3rd. IL-1b
did not induce signicant mechano-sensitivity.
The percentage of mechano-sensitive and spontaneously active
bres varies amongst studies, this may be related to different methods, the assessment time following the procedure and the equipment utilized. Neuritis related neural spontaneous activity
comprises both C and A bres (Bove et al., 2003), most of the active
bres are thinly myelinated bres (Ad). Relying on the conduction
velocity to distinguish between the nerve types (Fig. 6), the spontaneous activity following CFA application in the present study was
not different. Three and 8 days following the application, 75% and
77% of the active bres, respectively, were Ad bres. IL-6 application
induced mechano-sensitivity and ectopic discharges on the 3rd
DPO, similar to the CFA application, however surprisingly 63% of
the active bres conduction velocity was faster than 30 m/s. Similar
patterns have been found following IL-1b application. IL-1b application induced signicant pain and ectopic discharge 8 days following
application, however 79% of the spontaneously active bres conduction velocity was faster than 30 m/s. The most painful phase following cytokine application was characterized with myelinated
nerves spontaneous activity. This data can be compared to a previous study (Tal and Eliav, 1996) on sham operated rats, where only
54% of the nerves conduction velocity was faster than 30 m/s 2
5 days following the procedure.
The cutaneous tactile detection threshold ndings may have
important clinical relevance (Fig. 5C). This experiment result shows
that nerve trunk exposure to perineural inammation produces
hypersensitivity in the nerve distal end (the rats paw in the present
study): less mechanical force is required to produce action potential.
Several studies demonstrated reduced mechanical detection threshold in conditions that presumably involve perineural inammation.
Third molar extraction studies revealed that the rst days following
the extraction are characterized by lingual and mental nerves dermatomes showing reduced mechanical and electrical detection
thresholds (Eliav and Gracely, 1998), and this hypersensitivity was
reversed by steroid treatment (Barron et al., 2004).
Joint-related temporomandibular disorders (TMD), probably
inducing auriculotemporal nerve perineural inammation revealed
reduced electrical detection threshold (Eliav et al., 2003) while
myogenic TMDs (either clinical or experimental) that does not involve inammation demonstrated mechanical, electrical and
vibrotactile elevated detection thresholds (Hollins et al., 1996;
Stohler et al., 2001; Eliav et al., 2003). Mechanical allodynia and
hypersensitivity have been shown to be present in patients suffering from osteoarthritis (Farrell et al., 2000a,b). Detection threshold
assessment of early oral malignant lesions, presumably involving
perineural inammation (Anneroth et al., 1986) demonstrated reduced electrical detection threshold in the nerves territory that
were exposed to the malignant process (Eliav et al., 2002). Paranasal sinusitis sensory assessment demonstrated reduced electrical
detection threshold of the skin overlying acute sinusitis (Benoliel
et al., 2006).
Perineural inammation induced with CFA, signicantly increased IL-6 and IL-1b secretion from the affected nerves, their related DRGs and the paw skin innervated by the nerve, induced pain
and hypersensitivity in the nerve target organ, spontaneous neural
activity, nerve trunk mechano-sensitivity and reduced the target
organ tactile detection threshold.
IL-6 secretion correlated with the pain behaviour induced by perineural inammation: elevated when the pain peaked and reduced
as the pain resolved. Il-1b secretion remained elevated after the resolution of the pain. Similar to perineural inammation induced by

E. Eliav et al. / Brain, Behavior, and Immunity 23 (2009) 474484

CFA application, application of IL-1b and IL-6 to the sciatic nerve

trunk vicinity produced pain in the nerve distal end (the rats paw),
spontaneous activity and nerve trunk mechano-sensitivity. IL-6
activity was more prominent immediately following application
(25 DPOs), while IL-1b, induced or maintained pain in a later stage
(58 DPOs). Most of the spontaneously active bres following perineural inammation were thinly myelinated Ad bres. Heat-allodynia induced by IL-1b was more robust than heat-allodynia
induced by IL-6. Both IL-1b and IL-6 contribute to neuropathic
inammatory pain, but in different phases of the process.
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