Cellular, molecular, and developmental neuroscience 1
Evaluation of apoptotic pathways in dorsal root ganglion
neurons following peripheral nerve injury
Rebecca Wiberga,b, Liudmila N. Novikovaa and Paul J. Kinghama
Peripheral nerve injuries induce significant sensory
neuronal cell death in the dorsal root ganglia (DRG);
however, the role of specific apoptotic pathways is still
unclear. In this study, we performed peripheral nerve
transection on adult rats, after which the corresponding
DRGs were harvested at 7, 14, and 28 days after injury for
subsequent molecular analyses with quantitative reverse
transcription-PCR, western blotting, and
immunohistochemistry. Nerve injury led to increased levels
of caspase-3 mRNA and active caspase-3 protein in the
DRG. Increased expression of caspase-8, caspase-12,
caspase-7, and calpain suggested that both the extrinsic
and the endoplasmic reticulum (ER) stress-mediated
apoptotic pathways were activated. Phosphorylation of
protein kinase R-like ER kinase further implied the
involvement of ER-stress in the DRG. Phosphorylated
protein kinase R-like ER kinase was most commonly
Introduction
Peripheral nerve injury induces significant sensory neu-
ronal cell death in the dorsal root ganglia (DRG). In
contrast to spinal motoneurons, both neonatal and adult
primary sensory neurons in the DRG undergo cell death
even after distal peripheral nerve injury [1], which is also
the case for cranial motoneurons [2]. Moreover, cuta-
neous afferent neurons are more sensitive to injury-
induced degeneration in comparison with muscular
afferents [1,3,4]. Sensory neuronal cell death starts
shortly after nerve axotomy and experimentally it has
been shown that a significant cell loss is already evident
in the DRG 1 week after injury if repair is not carried out
[5]. Loss of sensory neurons increases with time, reaching
a value of 35–40% of the total neuronal cell population in
the DRG at 2 months after injury [5]. Despite advanced
microsurgical innovation, immediate epineural nerve
repair only mediates partial neuroprotection, reducing
the amount of neuronal cell loss from 21 to 5% 2 weeks
postoperatively [5], creating a need for an additional
pharmacological intervention.
Current data on the underlying mechanism behind
injury-induced cell death are conflicting, with uncer-
tainties with respect to the specific apoptotic pathways
involved. Apoptosis consists of several biochemical
events leading to characteristic morphological changes,
including membrane blebbing, cell shrinkage, nuclear
fragmentation, chromatin condensation, and DNA frag-
mentation, ultimately resulting in cell death because of
0959-4965 Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.
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associated with isolectin B4-positive neurons in the DRG
and this may provide an explanation for the increased
susceptibility of these neurons to die following nerve injury,
likely in part because of an activation of the ER-stress
response. NeuroReport 00:000–000 Copyright © 2018
Wolters Kluwer Health, Inc. All rights reserved.
NeuroReport 2018, 00:000–000
Keywords: apoptosis, endoplasmic reticulum, peripheral nerve injury,
sensory neurons
a
Department of Integrative Medical Biology, Section of Anatomy and bDepartment
of Surgical & Perioperative Sciences, Section of Hand and Plastic Surgery, Umeå
University, Umeå, Sweden
Correspondence to Rebecca Wiberg, PhD, Department of Integrative Medical
Biology, Section of Anatomy, Umeå University, SE-901 87 Umeå, Sweden
Tel: + 46 90 786 9754; fax: + 46 90 786 5480; e-mail: rebecka.wiberg@umu.se
Received 23 February 2018 accepted 20 March 2018
activation of cysteine-dependent aspartate-directed pro-
teases, the caspases. Three main different kinds of
apoptosis exist [6,7]: (i) the intrinsic apoptotic pathway,
which is regulated by the balance between proapoptotic
Bax and antiapoptotic Bcl-2, acting at the mitochondrial
level determining the membrane permeabilization, (ii)
the extrinsic apoptotic pathway, which is characterized by
the presence of death receptors (DRs), and (iii) apoptosis
mediated by endoplasmic reticulum (ER) stress, with
each pathway culminating in cleavage of the effector
caspase-3.
In this study, we used an animal model of peripheral
nerve injury to gain a better understanding of the apop-
totic mechanisms underlying sensory neuronal cell death
in the DRG. We have analyzed the expression of
caspase-3, caspase-8, and caspase-12 as respective med-
iators of the intrinsic, extrinsic, and ER-stress apoptotic
pathways.
Material and methods
Experimental animals and ethics statement
Adult (10–12 weeks old) female Sprague-Dawley rats
with a mean weight of 250 g were used in this study. The
animal husbandry was in accordance to the standards and
regulations provided by the National Institutes of Health
Guide for Care and Use of Laboratory Animals (NIH
publications no. 86–23, revised 1985) and the European
Communities Council Directive (86/609/EEC). All pro-
cedures were approved by the Northern Swedish
DOI: 10.1097/WNR.0000000000001031
 2 NeuroReport 2018, Vol 00 No 00
Regional Committee for Ethics in Animal Experiments
(DNR #A186-12). Surgery was performed aseptically
using general anesthesia consisting of a mixture of keta-
mine (Ketalar 50 mg/ml; Pfizer, Strängnäs, Sweden) and
xylazine (Rompun 20 mg/ml; Bayer Healthcare, Berlin,
Germany) by an intraperitoneal injection. The well-being
of the rats was observed throughout the experimental
period.
Surgical procedures and experimental groups
The animals were divided into four groups; sciatic nerve
transection without repair with 7 (n = 5), 14 (n = 5), and 28
(n = 5) days of survival, and a control group consisting of
unoperated animals (n = 9). A separate series of animals
was used for immunohistochemisty (n = 3) and for wes-
tern blotting (n = 4–6/group). The left sciatic nerve was
exposed by bluntly dividing the gluteal muscles of the
thigh. Under an operating microscope (Zeiss; Carl Zeiss,
Oberkochen, Germany), the nerve was transected at a
standardized distance from the spinal cord ∼ 5 mm
proximal of the sciatic nerve’s first nerve branch. The
nerve stumps were then capped to prevent distal rein-
nervation. Caps were made out of polyethylene tubes
and each nerve stump was introduced and anchored to
the cap using the 10-0 Ethilon suture. After surgery, the
wound was closed in layers: muscles with a 3-0 Silk
suture and the skin with a 3-0 Silk suture. The operated
animals were allowed to survive for 7, 14, and 28 days
before harvesting.
Tissue processing
At the end of the survival period, the rats were injected
terminally with an intraperitoneal overdose of sodium
pentobarbital (240 mg/kg; Apoteksbolaget, Stockholm,
Sweden). For reverse transcription-PCR (RT-PCR) and
western blotting, the L4, L5, and L6 DRGs from the
operated side were harvested and fast frozen in liquid
nitrogen. For immunohistochemistry, the animals were
transcardially perfused with Tyrode’s solution, followed
by 4% (w/v) paraformaldehyde in 0.1 M PBS (pH 7.4).
The L4, L5, and L6 DRGs were harvested, postfixed for
2–3 h, cryoprotected in 20 and 30% sucrose for 2–3 days,
and frozen in liquid isopentane. Serial transverse 16-μm
thick sections were cut on a cryomicrotome (Leica
Instruments, Kista, Sweden), thaw-mounted in pairs onto
SuperFrost Plus slides, dried overnight at room tem-
perature, and stored at − 80°C before processing.
Quantitative reverse transcription-PCR
Total RNA was isolated from DRGs using a miRNeasy
mini kit (Qiagen, Sollentuna, Sweden) and the purified
RNA was quantified by determining the absorbance at
260 nm using a Nanodrop 2000/2000c spectrophotometer
(ThermoScientific, Gothenburg, Sweden). L4, L5, and
L6 DRGs from rats in each experimental group were
pooled, and 1–5 ng total RNA was converted to cDNA
using a First-Strand cDNA Synthesis Kit (BioRad iScript
Copyright r 2018 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
cDNA synthesis kit). Quantitative RT-PCR was subse-
quently performed using SsoFast EvaGreen Supermix
(BioRad, Solna, Sweden) according to the manufacturer’s
recommendations. Caspase-3: forward: 5′-GGACCTGT
GGACCTGAAAAA-3′ and reverse: 5′-AGTTCGGCTT
TCCAGTCAG-3′ primers with an annealing tempera-
ture (Ta) of 60.5°C, caspase-8: forward: 5′-CTGGGAAG
GATCGACGATTA-3′ and reverse: 5′-CATGTCCTGC
AGTTTTGATGG-3′ primers with Ta of 61°C, caspase-
12: forward: 5′-GGCAGACATACTGGTACTATTTG
G-3′ and reverse: 5′-GCTCAACACACATTCCTCATC
TGT-3′ primers with Ta of 61°C, caspase-7: forward:
5′-TACAAGATCCCGGTGGAAGCT-3′ and reverse:
5′-CTGGGTTCCTCCACGAATAAT-3′ primers with
Ta of 62°C, calpain: forward: 5′-TGCTCTGCCGAAGT
GTCACC-3′ and reverse: 5′-GCACTGGATGGCCT
GGAGTT-3′ primers with Ta of 66°C, and the reference
gene 18s: forward: 5′-TCAACTTTCGATGGTAGTC
GC-3′ and reverse: 5′-CCTCCAATGGATCCTCGTT
AA-3′ primers with Ta of 62°C were purchased from
Sigma (Poole, UK).
Western blotting
L4, L5, and L6 DRGs were dissected from nerve-
transected animals after 7, 14, and 28 days without
repair and also uninjured control animals. DRGs were
pooled in each group and prepared by homogenization in
protein lysis buffer. Whole-tissue lysates were analyzed
for protein content using a commercially available protein
assay kit (BioRad). The antibodies used were rabbit anti-
caspase-3 antibody (1 : 1000, Catalog Number 9662;
Cell Signaling Technology, BioNordika, Stockholm,
Sweden), mouse anti-caspase-7 antibody (1 : 500, Catalog
Number ab2301; Abcam, Cambridge, UK), rabbit anti-
caspase-12 antibody (1 : 500, Catalog Number ab62484;
Abcam), rabbit anti-caspase-8 antibody (1 : 1000, Catalog
Number NB100-56116; , Novus Biologicals, Abingdon,
UK), or rabbit anti-phospho-protein kinase R-like endo-
plasmic reticulum kinase (anti-phospho-PERK) (1 : 1000,
Catalog Number 3179; Cell Signaling Technology). Anti-
β-tubulin antibody (1 : 1000, Catalog Number ab6046;
Abcam) was used as the loading control antibody.
Immunohistochemistry
A 16-μm thick L4, L5, and L6 DRG sections were
blocked with normal serum, after which the following
primary antibodies were used: anti-active caspase-3
antibody (1 : 10, Catalog Number ab2302; Abcam), rab-
bit anti-phospho-PERK (1 : 200, Catalog Number sc-32577;
Santa Cruz Biotechnology, Heidelberg, Germany), and
monoclonal neurofilament H (1 : 400, Catalog Number
N0142; Sigma, Poole, UK). Primary antibodies were applied
for 2 h at room temperature. After rinsing in PBS, secondary
goat anti-mouse and goat anti-rabbit antibodies Alexa Fluor-
488 and Alexa Fluor-568 (1 : 300; Molecular Probes,
Invitrogen, ThermoFisher Scientific, Stockholm, Sweden)
were applied for 1 h at room temperature in the dark.
 Apoptotic pathways in DRG neurons Wiberg et al. 3

On some tissue sections, isolectin B4 (IB4)-FITC (1 : 10; Fig. 1

Sigma) was applied. Images were captured using a Nikon
DXM1200 digital camera (Nikon Instruments, Europe,
Amsterdam, The Netherlands).

Statistical analysis
To determine the statistical difference between groups,
one-way analysis of variance complemented by Bonferroni’s
test (GraphPad Prism; GraphPad Software Inc., La Jolla,
California, USA) was used. Statistical significance was set
as a P value less than 0.05. Nonsignificant differences are
reported (P = NS).

Results
Peripheral nerve injury activates caspase-3 in the dorsal
root ganglia
Analyses by quantitative RT-PCR showed that at 7, 14,
and 28 days following nerve transection, the expression
of caspase-3 was significantly (P < 0.05) increased
3.33 ± 0.24-, 3.24 ± 0.11-, and 2.23 ± 0.28-fold, respec-
tively, in comparison with the control (Fig. 1a). Western
blotting showed that nerve injury induced an increased
expression of full-length caspase-3 protein at 7 and
14 days following nerve transection, but we could not
detect the cleaved caspase-3 fragments (Fig. 1b).
However, using immunohistochemistry, we did observe
active caspase-3 in a small number of neurons (Fig. 1c).

Both extrinsic and endoplasmic reticulum-stress

pathways are activated by peripheral nerve injury
Caspase-8, which is a member of the extrinsic apoptotic
pathway, was increased 1.95 ± 0.20-fold (P < 0.05),
3.35 ± 0.24-fold (P < 0.05), and 1.36 ± 0.11-fold (P = NS)
in comparison with the control 7, 14, and 28 days,
respectively, following nerve transection (Fig. 2a).
Western blotting showed that nerve injury induced
caspase-8 protein processing with increased levels of
cleaved intermediate fragment caspase-8 at 14 days fol-
lowing nerve transection, but we could not detect the
active large (17–21 kDa) and small (10–13 kDa) caspase-8
fragments (Fig. 2b). At 7, 14, and 28 days following nerve
transection, the gene expression of caspase-12 was
increased 3.63 ± 0.28-, 2.72 ± 0.51-, and 3.19 ± 0.70-fold,
respectively, in comparison with the control (Fig. 3a; all
P < 0.05). The increased expression of caspase-12 was
paralleled by an increased expression of calpain, a known
activator of caspase-12 [8]. At 7, 14, and 28 days following
nerve transection, the expression of calpain was increased
2.62 ± 0.17-, 1.91 ± 0.10-, and 1.42 ± 0.05-fold, respec-
tively, in comparison with the control (Fig. 3b; all
P < 0.05). In addition to calpain, the increased expression
of caspase-12 was paralleled by an increased expression
of caspase-7, which is another known activator of caspase-
12. At 7, 14, and 28 days following nerve transection, the
expression of caspase-7 was increased 2.29 ± 0.15-fold
(P < 0.05), 2.39 ± 0.25-fold (P < 0.05), and 1.63 ± 0.30-fold
(P = NS), respectively, in comparison with the control
Expression of caspase-3 in dorsal root ganglia (DRG) after nerve injury.
(a) Quantitative reverse transcription-PCR analysis of caspase-3
expression in ipsilateral L4, L5, and L6 DRGs at 7, 14, and 28 days after
peripheral nerve injury. Significant differences are expressed relative to the
control (normal uninjured DRGs); *P < 0.05; n = 5 animals per
experimental group. (b) Western blot analysis of caspase-3 expression at
7, 14, and 28 days after nerve transection and in control (con) lysates
prepared from pooled DRGs from n = 4 animals per experimental group.
β-Tubulin was used as a loading control. (c) Representative
immunohistochemistry of active caspase-3 expression (red) 14 days after
peripheral nerve injury (arrow indicates cell with fragmented nucleus).
Scale bar = 40 μm. Staining was repeated in three individual animals.
(Fig. 3c). Western blotting showed that the increased
caspase-12 gene expression was associated with activa-
tion of the enzyme. The 38 kDa active fragment of
caspase-12 was increased at 7, 14, and 28 days following
nerve transection (Fig. 3d). The increased caspase-7 gene
expression also correlated with activation of the enzyme,
with increased protein levels observed at 7 and 14 days
following nerve transection (Fig. 3d).

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 4 NeuroReport 2018, Vol 00 No 00
Fig. 2
Expression of caspase-8 in dorsal root ganglia (DRG) after nerve injury.
(a) Quantitative RT-PCR analysis of caspase-8 expression in ipsilateral
L4, L5, and L6 DRGs at 7, 14, and 28 days after peripheral nerve injury.
Significant differences are expressed relative to the control (normal
uninjured DRGs); *P < 0.05; NS, n = 5 animals per experimental group.
(b) Western blot analysis of caspase-8 showing precursor and cleaved
intermediate fragment caspase-8 at 7, 14, and 28 days after nerve
transection and in control (con) lysates prepared from pooled DRGs
from n = 4 animals per experimental group. β-Tubulin was used as a
loading control.
The ER-stress response is mediated by three different
ER membrane receptors, activating transcription factor
6, inositol-requiring enzyme 1, and PERK [9] and we
showed that the phosphorylation of PERK was increased
at 7 and 14 days following nerve transection (Fig. 4a).
Analysis of the DRGs by immunohistochemistry indi-
cated that phospho-PERK expression was mainly loca-
lized to IB4-positive neurons in control tissue (Fig. 4b).
Discussion
Current data on the underlying mechanism behind
injury-induced DRG neuronal cell death are conflicting,
with uncertainties as to which apoptotic pathways are
involved. Previous research has suggested that apoptosis
in the DRG is mediated by the intrinsic pathway [10,11],
with the role of other apoptotic pathways being largely
overlooked.
The intrinsic pathway is regulated by the balance
between proapoptotic Bax and antiapoptotic Bcl-2. When
the equilibrium points towards Bax, the formation of
pores take place in the mitochondrial membrane, allow-
ing the release of cytochrome C [6,7]. Once released into
the cytosol, cytochrome C interacts with apoptotic pro-
tease activating factor 1 and procaspase-9, forming the
apoptosome, resulting in caspase-3 activation and cell
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death [6,7]. Several kinds of cellular stress are known to
initiate the intrinsic pathway including for example oxi-
dative stress and depletion of neurotrophic factors [6,7].
The extrinsic pathway is characterized by the presence of
DRs, which are activated by corresponding ligands. So
far, eight different DRs have been identified in humans,
Fas (CD95) being the most described DR. Upon FasL-
mediated activation of Fas, Fas-associated protein with
death domain recruits procaspase-8 and procaspase-10,
forming a death-inducing signaling complex (DISC)
[7,12]. Procaspase-8 is then autoproteolytically cleaved at
the DISC, generating an active protease, which in turn
activates the downstream executioner caspase-3 [7,12].
Type I cells generate high levels of DISC formation and
increased amounts of active caspase-8, which results in
direct activation of the downstream effector caspase-3
[7,12]. Type II cells, however, generate low levels of
DISC formation and require an amplification loop
through caspase-8-mediated processing of Bid to trun-
cated Bid, inducing the release of cytochrome C from
the mitochondria with subsequent activation of caspase-3
[7,12].
In addition, apoptosis can be induced by the ER. The
ER regulates the intracellular calcium (Ca2 +) home-
ostasis in addition to serving as the cellular site of newly
synthesized secretory and membrane proteins, which
must be properly folded and post-translationally modified
before exit from the organelle. Proper protein folding and
modification requires molecular chaperone proteins as
well as an ER environment conducive for these reactions.
When ER luminal conditions are altered or chaperone
capacity is overwhelmed, so-called ER stress, the cell
activates signaling cascades to restore a favorable folding
environment through activation of three different ER
membrane receptors: activating transcription factor 6,
inositol-requiring enzyme 1, and PERK [9]. The pro-
tective mechanisms include translational attenuation to
limit further accumulation of misfolded proteins, tran-
scriptional activation of genes encoding ER-resident
chaperones such as BiP/GRP78 and GRP94, and ER-
associated degradation, which serves to reduce the stress
and thereby restore the folding capacity by directing
misfolded proteins in the ER back into the cytosol for
degradation by the 26s proteasome [9]. If these protec-
tive mechanisms are not sufficient, apoptosis will take
place through activation of caspase-12 [13].
ER stress is known to be induced in response to spinal
cord injury [14]. In this study, we showed that caspase-12
mRNA was significantly upregulated in sensory neurons
following nerve transection, as well as calpain and
caspase-7. Furthermore, phosphorylation of PERK was
upregulated, indicating that the ER might be involved in
the injury-induced apoptosis following peripheral nerve
injury. Bax and Bak associate not only with mitochondria
membranes but also the ER [15], and mouse fibroblasts
 Apoptotic pathways in DRG neurons Wiberg et al. 5

Fig. 3

Expression of caspase-12, calpain, and caspase-7 in dorsal root ganglia (DRG) after nerve injury. Quantitative RT-PCR analysis was carried out on
ipsilateral L4, L5, and L6 DRGs to determine the expression of (a) caspase-12, (b) calpain, and (c) caspase-7 at 7, 14, and 28 days after peripheral
nerve injury. Significant differences are expressed relative to the control (normal uninjured DRGs); *P < 0.05; NS, n = 5 animals per experimental
group. (d) Western blot of caspase-12 and caspase-7 expression at 7, 14, and 28 days after peripheral nerve injury and in control (con) lysates
prepared from pooled DRGs from n = 4 animals per experimental group. β-Tubulin was used as a loading control.

lacking both Bax and Bak (Bax− /–/Bak–/–) are resistant to
apoptosis induced by thapsigargin, tunicamycin, and
brefeldin A [16]. During ER stress, Bax and Bak undergo
conformational changes and oligomerization in the ER
membrane [16], releasing Ca2 + from the ER into the
cytoplasm [16]. The increase in the Ca2 + concentration
in the cytosol activates m-calpain, which cleaves and
activates procaspase-12 [8]. Activated caspase-12 then
cleaves and activates procaspase-9, which in turn acti-
vates the downstream caspase cascade including caspase-
3 [13]. Calpain-deficient mouse fibroblasts have reduced
ER stress-induced caspase-12 activation and are resistant
to ER stress-associated apoptosis [17].
Furthermore, caspase-7 translocates from the cytosol to
the cytoplasmic side of the ER membrane in response to
ER stress, where it cleaves the prodomain to generate
active caspase-12, resulting in increased cell death. In
addition, cotransfection of cells with caspase-12 and the
catalytic mutant of caspase-7 not only blocks the cleavage
of caspase-12, but it also attenuates cell death [18]. It
should be noted that although caspase-12 is activated
during ER stress, the involvement of caspase-12 in ER
stress-induced apoptosis is still controversial. Some stu-
dies show that fibroblasts from caspase-12− / − mice are
resistant to apoptosis in response to ER stress [19],
whereas others claim that caspase-12 does not affect the
survival rate, but rather the degree of inflammation [20].
Furthermore, Sanges et al. [21] have proposed that ER
stress-induced apoptosis is mediated by calpain, but not
by caspases, on the basis of the observation that calpain
inhibitors, but not a pan-caspase inhibitor, block tunicamycin-
induced and thapsigargin-induced apoptosis.
Adult DRGs comprise a heterogenous pool of sensory
neurons that can be divided into subpopulations according
to several parameters including neuronal size, neu-
rochemistry, trophic requirements, and sensory modality
[22]. It is possible to distinguish between small unmyeli-
nated afferents and large myelinated afferents; the former
type comprises two populations of cells differentiated by
their potential to synthesize neuropeptides: peptidergic
afferents, which have a predominantly nociceptive function
and identified by their immunoreactivity for calcitonin
gene-related peptide, and nonpeptidergic afferents,
which are exclusively nociceptive and identified by their

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 6 NeuroReport 2018, Vol 00 No 00
Fig. 4
(a) Western blot for phosphorylated protein kinase R-like endoplasmic
reticulum kinase (PERK) in control tissue (con) and 7, 14, and 28 days
after peripheral nerve injury. Lysates were prepared from pooled dorsal
root ganglia from n = 4 animals per experimental group. β-Tubulin was
used as a loading control. (b) Immunohistochemistry of neurofilament
(NF)-H-positive and isolectin B4 (IB4)-positive control dorsal root
ganglion neurons together with phospho-PERK. Arrows show NF-H-
negative neurons that are positive for phospho-PERK. The asterisk
shows an IB4 neuron that is negative for phospho-PERK. Scale
bar = 40 μm. Staining was repeated in three individual animals.
binding of IB4. Myelinated afferents innervate peripheral
mechanoreceptors and proprioceptors, and are identified
by their binding of the high-molecular-weight neurofila-
ment H. Specific sensory neuronal subpopulations show
contrasting responses to peripheral nerve injury, as shown
by the axotomy-induced death of many cutaneous sensory
neurons, whereas muscular sensory afferents survive an
identical insult [1,3,4]. Furthermore, the proportion of
IB4-positive neurons decreases in axotomized DRGs [23].
Reid et al. [10] reported that medial gastrocnemius neu-
rons markedly downregulate caspase-3 mRNA in response
to axotomy, whereas sural neurons upregulate its gene
expression at 1 week after injury, which was hypothesized
to be attributable to the differing IB4-positive composition
of the subpopulations described. In this study, we found
an upregulation of phospho-PERK following axotomy that
was mainly restricted to IB4-positive neurons, raising the
possibility that the increased susceptibly observed in these
cells is because of activation of ER stress.
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The various apoptotic pathways interact. In parallel with
caspase-12 activation, ER stress triggers caspase-8 activation,
resulting in cytochrome C/caspase-9 activation through Bid
processing [24]. We observed an increased expression of
caspase-8 following sciatic nerve transection, indicating that
the extrinsic pathway might also be involved in the injury-
induced apoptosis following peripheral nerve injury.
In addition to our results suggesting the involvement of
caspases 3, 8, and 12 in injury-induced sensory neuron
cell death, Vignesvara et al. [25] observed significantly
elevated levels of cleaved caspase-2, compared with
cleaved caspase-3, following sciatic nerve transection.
Furthermore, siRNA-mediated downregulation of caspase-2
protected 50% of DRG neurons from apoptosis after serum
withdrawal, whereas downregulation of caspase-3 had no
effect on DRG neuron survival.
Conclusion
As adult DRGs comprise a heterogenous pool of sensory
neurons, it can be assumed that several apoptotic path-
ways are involved in the injury-induced cell death. We
speculate that the intrinsic and extrinsic apoptotic path-
ways, together with the ER-stress response, are involved.
Acknowledgements
This research was funded by Umeå University
(Insamlingsstiftelsen) and Västerbotten County Council.
Conflicts of interest
There are no conflicts of interest.
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