Experimental Neurology 255 (2014) 111

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Experimental Neurology
journal homepage: www.elsevier.com/locate/yexnr

Assessment of sensory thresholds and nociceptive ber growth after

sciatic nerve injury reveals the differential contribution of collateral
reinnervation and nerve regeneration to neuropathic pain
Stefano Cobianchi, Julia de Cruz, Xavier Navarro
Group of Neuroplasticity and Regeneration, Institute of Neurosciences and Department of Cell Biology, Physiology and Immunology, Universitat Autnoma de Barcelona, Bellaterra, and Centro de
Investigacin Biomdica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain

a r t i c l e

i n f o

Article history:
Received 25 November 2013
Revised 31 January 2014
Accepted 10 February 2014
Available online 16 February 2014
Keywords:
Collateral sprouting
Nerve regeneration
Skin innervation
Neuropathic pain
Hyperalgesia
Sensory nerve bers
Algesimetry test

a b s t r a c t
Following traumatic peripheral nerve injury reinnervation of denervated targets may be achieved by regeneration of injured axons and by collateral sprouting of neighbor undamaged axons. Experimental models commonly
use sciatic nerve injuries to assess nerve regeneration and neuropathic pain, but behavioral tests for evaluating
sensory recovery often disregard the pattern of hindpaw innervation. This may lead to confounding attribution
of recovery of sensory responses to improvement in sciatic nerve regeneration instead of collateral reinnervation
by the undamaged saphenous nerve. We used a standardized methodology to assess the separate contribution of
collateral and regenerative skin reinnervation on sensory responses. Section and suture of the sciatic nerve
induced loss of sensibility in the lateral and central areas of the injured paw, but nociceptive responses rapidly
recovered by expansion of the intact saphenous innervation territory. We used electronic Von Frey and Plantar
test devices to measure mechanical and thermal withdrawal thresholds in specic sites of the injured paw: lateral
site innervated by the sciatic nerve, medial site that remained innervated by the saphenous nerve, and central site
originally innervated by the sciatic nerve but affected by saphenous sprouting. After sciatic section, signs of early
hyperalgesia developed in medial and central paw areas due to saphenous sprouting and expansion. The
regenerating sciatic nerve bers reached the paw at 34 weeks and a late mechanical hyperalgesia was observed
at the lateral site. Immunohistochemical staining of sensory bers innervating the medial and lateral areas revealed a different pattern of skin reinnervation. Hypersensitivity in the intact saphenous nerve area was
paralleled by early ber sprout growth in the subepidermal plexus, but not entering the epidermis. On the
other side, late sciatic hyperalgesia was accompanied by gradual skin reinnervation after 4 weeks.
The standardization of algesimetry testing in sciatic nerve injury models, as proposed in this study, provides a
suitable model for studying in parallel neuropathic pain and sensory nerve regeneration processes. Our results
also indicate that collateral sprouting and axonal regeneration contribute differently in the initiation and maintenance of neuropathic pain.
2014 Elsevier Inc. All rights reserved.

Introduction
Peripheral nerve injuries induce functional decits caused by degeneration of injured axons, that can be recovered by two endogenous
mechanisms: the reinnervation of denervated targets by regeneration
of injured axons, and the reinnervation by collateral branching of undamaged axons (Navarro et al., 2007). Each of these mechanisms may
also contribute to the appearance of secondary effects, such as variations of sensory thresholds, hyperreexia and neuropathic pain. Despite
numerous studies on potential pain targets and involvement of hundreds of genes (LaCroix-Fralish et al., 2007), few basic discoveries
Corresponding author at: Facultat de Medicina, Universitat Autnoma de Barcelona,
E-08193 Bellaterra, Spain. Fax: +34 935812986.
E-mail address: xavier.navarro@uab.cat (X. Navarro).

http://dx.doi.org/10.1016/j.expneurol.2014.02.008
0014-4886/ 2014 Elsevier Inc. All rights reserved.

have been translated so far from rodent models into effective therapy
for neuropathic pain after nerve lesions. As recently observed (Mogil
et al., 2010), one possibility is the non-predictability of preclinical
animal pain models.
Several animal models of peripheral nerve injury reecting human
neuropathic pain syndromes have been developed and characterized.
These models mostly use injuries to the sciatic nerve, and behavioral
testing used to evaluate the recovery of sciatic sensory function typically
measures the hypersensitivity to touch stimuli by means of the Von Frey
test, and/or withdrawal responses to a heat source (Wood et al., 2011).
On the other hand, also studies investigating treatments for enhancing
nerve regeneration most frequently use the sciatic nerve of rodents
(Rodrguez et al., 2004) and algesimetry tests for assessment of sensory
recovery. However, in these studies the compensatory mechanism of
collateral reinnervation is often neglected, although it has been

S. Cobianchi et al. / Experimental Neurology 255 (2014) 111

extensively shown to strongly affect the recovery of sensibility (Devor

et al., 1979; Inbal et al., 1987). We particularly argue that measures of
sensibility obtained by algesimetry tests may be not properly conducted
in studies employing sciatic nerve injury models. Very often assessment
of sensory thresholds is made by grossly stimulating the injured
paw without consideration of the anatomical innervation territories,
and then concluding that recovery of sensory responses is only due to
an improvement in nerve regeneration rather than to collateral
reinnervation.
After sciatic nerve injuries, compensatory reinnervation of the
hindpaw denervated skin is mediated by collateral sprouting of the saphenous nerve (Devor and Govrin-Lippmann, 1979; Kingery and Vallin,
1989; Kingery et al., 1994), which normally provides innervation to the
most medial side of the rat paw, and partly overlaps with the tibial
nerve (Decosterd and Woolf, 2000). Indeed, the decrease of withdrawal
thresholds to mechanical and heat stimulation of medial and central
areas of the paw skin after sciatic nerve lesions has been related to saphenous sprouting (Kingery et al., 1994; Vallin and Kingery, 1991). This phenomenon causes early and enhanced responses to stimulation that can
be misinterpreted as the result of sciatic nerve ber regeneration.
Several studies have attempted to explain the occurrence of neuropathic pain symptoms related to neuroplastic changes in the innervation of the skin. Following sciatic nerve lesion, there is a loss of dermal
and epidermal sensory bers in the denervated area, with a course of
sciatic reinnervation dependent on the severity of the injury (Hsieh
et al., 2000; Navarro et al., 1995, 1997). At the same time, the spared
sensory bers rapidly increase in density (Ma and Bisby, 2000) with
peptidergic calcitonin gene-related peptide (CGRP) immunoreactive bers hyperinnervating the footpad skin (Duraku et al., 2012; Grelik et al.,
2005) compared with other types of bers (Duraku et al., 2013). Since
the sprouts produced by intact saphenous nerve bers are induced by
target-derived nerve growth factor (NGF) (Diamond et al., 1987;
Mendell et al., 1999), collateral reinnervation is mostly expected by
peptidergic nociceptors that express the TrkA receptor for NGF (Wolf
and Ma, 2007). The maintenance of neuropathic symptoms has been attributed to this altered pattern of reinnervation (Duraku et al., 2013), although it is still unknown how collateral sprouts growing through the
skin induce sensory changes before and after regeneration of the sciatic
nerve. Since the return of nociceptive responses anticipates the reinnervation of the epidermis, a different ber growth prole through dermis
and epidermis has also been suggested (Navarro et al., 1997; Verd and
Navarro, 1997), so nociceptors reinnervating sub-epidermal layers of
the skin may mediate early stimulus-dependent pain responses.
In this study we made a standardized evaluation of nociceptive reinnervation and changes in sensory thresholds differently induced by saphenous nerve sprouting and sciatic nerve regeneration. We show that
differentiation of the contribution of these two processes is essential for
the assessment of neuropathic pain. We used the sciatic nerve section
and suture repair model, which divides the rat paw into a saphenoussensitive and a sciatic-insensitive area, and stimulated specic skin
test sites. We eliminated the saphenous nerve at different times before
and after the return of sciatic reinnervation, and showed that stimulusinduced pain depends on the area of stimulation and has a time course
otherwise attributable to the two distinct pathways. We also characterized the time course of skin reinnervation by immunohistochemical labeling, and found that neuropathic pain is related to early changes in the
sub-epidermis differently induced by saphenous sprouting and sciatic
regeneration.
Materials and methods
Animals and surgery
Adult female SpragueDawley rats (240 30 g) were housed in
standard polyethylene cages (four per cage) and kept on standard laboratory food and water ad libitum with a lightdark cycle of 12 h. All

experimental procedures were approved by the Ethics Committee of

our institution, and followed the European Communities Council
Directive 86/609/EEC and the guidelines of the Committee for Research
and Ethical Issues of IASP (Zimmermann, 1983).
Rats were anesthetized with pentobarbital (40 mg/kg i.p.). The right
sciatic nerve was exposed at the mid thigh, transected at 92 mm from
the tip of the third toe, and repaired by epineurial sutures (100), maintaining the fascicular alignment of tibial, peroneal and sural branches.
The wound was closed in two layers and disinfected. Rats were kept
in a warm environment until complete recovery from anesthesia. They
were observed daily for behavioral signs indicative of pain, including
signs of autotomy of the injured limb.
In this study we used a total number of 21 rats. Nine rats were used
for sensory testing during the complete follow-up. Samples for immunohistochemical analysis were collected from the same animals euthanized the day after the last functional test. At two selected intervals
(14 and 28 days) after sciatic lesion, under isourane anesthesia, the
right saphenous nerve of other rats (6 at each time point) was exposed
by a small skin incision at the thigh, and transected. A fragment of
approx 10 mm was excised from the distal stump to ensure no regeneration. The wound was sutured and disinfected. The following day, the
animals were tested to verify complete loss of responses.
Assessment of skin nociceptive reinnervation
The progression of nociceptive reinnervation of the hindpaw was
assessed by means of the pinprick test (Navarro et al., 1994) performed
at different days post injury. Awake animals were gently kept in a cloth
with the injured paw facing upwards, and the plantar skin was stimulated with a blunt needle progressively from distal to proximal at distinct
sites (see Fig. 1B). Each site was stimulated twice, and responses were
recorded as positive only when clear pain reactions (as fast withdrawal
and vocalization) were triggered to the stimulation. Positive responses
were taken as sign of skin functional reinnervation. An index of
sprouting, calculated as the rate of responsive areas over the total
areas stimulated by pinpricking at different times, indicated the increasing contribution of saphenous sprouting to nociception. Statistical comparison was made with one-way ANOVA and Bonferroni post-hoc test.
The criterion for statistical signicance was p b 0.05.
Algesimetry tests for sensory threshold measurement
One week before surgery, all the animals were habituated to experimental devices for nociceptive baseline threshold measurement. The
algesimetry tests for mechanical and thermal stimuli were performed
on both hindpaws before and at 3, 7, 14, 21, 28, 40, 50 and 60 days
post-injury (dpi).
Sensibility to a normally non-noxious mechanical stimulus was
measured by means of an electronic Von Frey algesimeter (Bioseb,
Chaville, France). Rats were placed on a wire net platform in plastic
chambers 10 min before the experiment for habituation. We stimulated
three different regions of the hindpaw plantar surface: the lateral
(normally innervated by tibial and sural branches of the sciatic nerve),
the central (normally innervated by the tibial nerve) and the medial (covered by tibial and saphenous nerves) (Casals-Daz et al., 2009) (Fig. 1A).
For each region, we chose a specic test site on the skin at the outer
edge of the most proximal medial and lateral footpads, or at the center
of the foot (Fig. 2A). The medial and lateral test sites were chosen as selective saphenous and sciatic sensory territories respectively (Cobianchi
et al., 2013); the central test site was chosen as a non-selective test site.
The experimenter was blind regarding the objectives of the testing, but
trained in the correct execution of algesimetry tests. By using the electronic Von Frey algesimeter, a 0.4 mm non-noxious pointed probe was
gently applied to each test site, and then slowly increasing the pressure.
The threshold was expressed as the force (in grams) at which rats withdrew the paw in response to the stimulus. A cutoff force was set to 40 g

S. Cobianchi et al. / Experimental Neurology 255 (2014) 111

Fig. 1. Reinnervation of rat plantar skin after sciatic nerve section and repair. (A) The rat hind paw plantar skin is innervated by saphenous, tibial and sural nerves (tibial and sural are
branches of the sciatic nerve), covering distinct territories shown in the gure in different gray shadows, with overlapping territories in white. (B) Schema indicating the different
areas of the plantar surface that were stimulated by pinpricking to assess functional innervation. Nomenclature of the considered areas according to Kennedy et al. (1988). (C) Mapping
of responses to pinprick in foot skin areas at different days post-injury (dpi), represented in a gray scale ranking from 0% responsive rats (white) to 100% responsive rats (black), shows the
progression of nociceptive reinnervation from medial to central and lateral areas and toes. (D) Mapping of responses to pinprick after cutting the saphenous nerve at 2 different times
(16 and 29 dpi) reveals the contribution of saphenous collateral sprouting to hindpaw skin reinnervation before and after sciatic nerve regeneration. (E) An index of sprouting, calculated
as the rate of responsive areas over the total areas stimulated by pinpricking at different times after sciatic nerve injury, indicates the increasing contribution of saphenous sprouting to
nociception (one-way ANOVA, Bonferroni post-hoc comparison; ***p b 0.001, 3 dpi vs. 28, 50 and 60 dpi; ### p b 0.001, 15 and 29 dpi vs. 14 and 28 dpi respectively).

at which stimulus lifted the paw with no response. Mechanical nociceptive threshold was taken as the mean of three measurements per test
site, with 5 min interval between each measurement.
Thermal sensibility was assessed by means of a Plantar test
algesimeter (Ugo Basile, Comerio, Italy). Rats were placed into a plastic
box with an elevated plexiglass oor. The beam (6 mm diameter) of a
lamp was focused on the hindpaw plantar surface, and pointed to the
same test sites as above. We paid attention to avoid cross-stimulation
of distinct skin territories; particularly, in the medial and lateral test
sites, we pointed the stimulating beam to not affect beyond the skin
borders of and footpads as showed in Fig. 2A. Intensity was set to
low power (40 mW/cm2) with a heating rate of 1 C/s, to induce activation of unmyelinated bers (Le Bars et al., 2001). A cutoff time of stimuli
was set at 20 s to prevent tissue damage. Heat pain threshold was

calculated as the mean of three trials per test site, with 10 min resting
period between each trial, and expressed as the latency (in seconds)
of paw withdrawal response.
Particular attention was paid during the execution of tests to avoid
stimulating the same paw consecutively, and when the animal was
not in a balanced posture. Data are presented as mean SEM. Statistical
comparisons for nociceptive thresholds were made with repeated measures ANOVA and Bonferroni post-hoc test. The criterion for statistical
signicance was p b 0.05.
Immunohistochemistry of paw skin innervation
Plantar pads corresponding to the medial and lateral algesimetry
test sites (named as and in Fig. 1B) were removed from both

S. Cobianchi et al. / Experimental Neurology 255 (2014) 111

S. Cobianchi et al. / Experimental Neurology 255 (2014) 111

hindpaws of rats euthanized at 3, 8, 16, 29 and 60 days after sciatic

nerve injury. Cryotome sections 60 m thick were processed together
as previously described (Navarro et al., 1997; Verd and Navarro,
1997). We used antibodies against protein gene product 9.5 (PGP, rabbit, 1:1000, ABDserotec) and calcitonin gene-related peptide (CGRP,
goat, 1:500, Abcam) to label all the nerve bers and the peptidergic bers, respectively. Immunolabeling against growth-associated protein
43 (GAP-43, rabbit, 1:1000, Millipore) was performed in order to visualize the growing axons. Four sections of four samples per group were
used for quantication of skin innervation. Images were captured with
a Zeiss LSM 700 confocal microscope, and analyzed using ImageJ software (NIH, Bethesda, USA). To avoid measuring degenerating bers,
that still show some fading immunoreactivity (Navarro et al., 1997),
we selected only nerve bers with immunoreactivity above the threshold of the completely denervated lateral footpad at 3 dpi. Intraepidermal nerve bers (IENFs) were counted under an epiuorescence
microscope in a footpad epidermis eld of 500 m adjacent to the
algesimetry test site (Fig. 3A). The mean length of sub-epidermal
nerve bers (SENFs) was measured on reconstructed images of the entire footpad section. Data were analyzed using two-way ANOVA and
Tukey post-hoc test. The criterion for statistical signicance was p
b 0.05.
Results
Mapping of plantar skin nociceptive reinnervation
The rat plantar hindpaw skin is normally innervated by saphenous,
tibial and sural nerves (tibial and sural are branches of the sciatic
nerve), with overlapping territories between them (Fig. 1A). Transection of the sciatic nerve divides the rat paw into a saphenous-sensitive
(medial) and a sciatic-insensitive (lateral) area. Suture repair permitted
the sciatic nerve to regenerate and to reinnervate the paw skin after
34 weeks. Pinprick stimuli were applied at 17 different plantar areas
(Fig. 1B). For each area, we plotted the mean percentage of rats with
positive responses over time after sciatic nerve injury (Fig. 1C). At
3 dpi, all the rats had positive responses only in the medial side of the
sole and toes 1 and 2, which are normally innervated by saphenous
and tibial nerves. A few rats had also positive responses in and A footpads, also partially innervated by the saphenous nerve. Stimulation of
these medial footpads induced withdrawal response in most of the
rats at 7 and 14 dpi, whereas new areas in the central medial sole and
B footpad became gradually responsive. At 21 dpi almost all the rats
had responses in the medial half of the plantar surface except toe 3.
One week later, central lateral areas, D and footpads and toe 3 became
responsive in some animals, and at days 5060 almost all the sciatic
nerve territory was reinnervated, except for toe 5 and the most lateral
area, corresponding to the sural nerve territory, in some rats.
To analyze the separate contribution on nociceptive responses of
collateral and regenerative reinnervation of the skin, we transected
the saphenous nerve at two different times, and repeated the mapping
test the following day. As shown in Fig. 1D, the entire plantar skin was
unresponsive after cutting the saphenous nerve at 14 dpi, demonstrating that it exclusively mediated the early nociceptive responses after
sciatic nerve injury. However, when cutting the saphenous nerve at
28 dpi, we found that ankle, central areas and lateral footpads remained
responsive in most of the rats the day after (Fig. 1D). This can be attributed to the arrival of newly regenerated axons from the sciatic nerve

between 3 and 4 weeks after injury. Notably, at 4 weeks, positive responses in distal areas that are solely innervated by the tibial nerve in
the normal condition, such as the B footpad and the toe 3, completely
disappeared after saphenous cut.
In order to assess the contribution of collateral sprouting from the
saphenous nerve to nociception before and after reinnervation by the
sciatic nerve, a composite score was calculated as the ratio of
responding versus total stimulated areas (Fig. 1E). The proportion of
responsive areas signicantly increased from about 23% at 3 dpi to
53% at 28 dpi (p b 0.001), to 73% at 50 dpi (p b 0.001) and to 84% at
60 dpi (p b 0.001). Interestingly, the elimination of the saphenous
nerve at 29 dpi still left 22% areas responding to pinprick (p b 0.001
vs. 28 dpi), implying that at 28 dpi the saphenous nerve was mediating
innervation of about 30% of the skin area, a larger surface than that at
3 dpi.
Nociceptive thresholds measured at different skin innervation territories
Fig. 2 shows sensory thresholds measured at three different test sites
(Fig. 2A) as mentioned above. Withdrawal responses to Von Frey mechanical stimulation in normal paws were found at 3031 g. After sciatic
nerve injury, no withdrawal responses were found at 3, 7 and 14 dpi in
the sciatic lateral test site (Fig. 2B, top charts). Since muscles proximal to
the knee in the injured leg have spared innervation and allow withdrawal of the paw when innervated skin areas are stimulated, the lack
of withdrawal response is attributable to the absence of sensation to
painful stimuli. At 21 dpi responses to stimuli reappeared in a few rats
but at higher force than in the contralateral intact paw (p b 0.001). At
later times, the recovery of sensitivity progressed, but with abnormal
responses below the normal threshold (p b 0.001 at 28, 40, 50 and
60 dpi), reecting a state of mechanical hyperalgesia similar to that reported after reinnervation in sciatic nerve crush and spared nerve injury
models (Casals-Daz et al., 2009; Vogelaar et al., 2004).
Withdrawal responses to heat stimulation had a latency around 14 s
in the intact hindpaw. As observed for mechanical stimuli, in the denervated paw there were no responses to thermal stimuli applied at the
sciatic lateral region at 3, 7 and 14 dpi. At 21 dpi recovery of sensitivity started in some rats, but at longer than normal withdrawal latency (p b 0.001). Nociceptive thermal threshold of the injured paw
progressively normalized by 4 weeks. Unlike the mechanical response,
no thermal hyperalgesia developed until the end of follow-up at 60 dpi.
The time course from hypoesthesia to hyperalgesia observed at the
lateral side of the paw reects the expected course of denervation and
regeneration of the injured sciatic nerve. However, compared with the
lateral site, responses to thermal and mechanical stimuli applied
at the central test site (Fig. 2B, mid charts), used in the study as
non-specic site, started to recover already at 3 (p b 0.001) and 7
dpi (p b 0.001) respectively, and they were similar to contralateral
values after 23 weeks. Then, early responses recorded at central
areas of the paw may be erroneously attributed to reinnervation by sciatic regeneration, whereas as demonstrated in the mapping tests, they
are due to collateral reinnervation from the saphenous nerve. To conrm this point, we tested the rats at the central site after cutting the saphenous nerve. We observed at 14 dpi a complete loss of responses, and
at 28 dpi a signicant increase of mechanical and thermal thresholds
(Fig. 2C), demonstrating that saphenous nerve mediated the changes
in sensory thresholds even if stimuli were delivered to sciatic central
areas.

Fig. 2. Sensory thresholds measured with algesimetry tests. (A) Assessment of sensory thresholds was conducted by applying mechanical (Von Frey test) and thermal (Plantar test) stimuli
to a medial and a lateral test site, corresponding to selective saphenous and sciatic nerve territories, or to the center of the foot. (B) Mechanical and thermal thresholds measured on the
lateral sciatic, medial saphenous and central test sites of the hindpaw (*p b 0.05; **p b 0.01; ***p b 0.001, injured vs. contralateral paw). Sciatic nerve injury induced loss of sensitivity at
lateral site (top charts) until 21 dpi when rats started to respond due to nerve regeneration, and subsequently developed mechanical but not thermal hyperalgesia. However, responses to
stimuli applied at the central test site (mid charts) started to recover already at 7 dpi, approaching normal values. Early responses recorded at central areas of the paw may be erroneously
attributed to reinnervation by sciatic regeneration. Thresholds measured at the medial site in the saphenous territory (bottom charts) were signicantly decreased after sciatic nerve injury, revealing marked mechanical and milder thermal hyperalgesia. (C) Mechanical and thermal thresholds in the central test site measured before and after resection of the saphenous
nerve at 14 or at 28 dpi (arrows) demonstrated that the saphenous nerve mediated the changes in sensory thresholds even if stimuli were delivered to the central paw site.

S. Cobianchi et al. / Experimental Neurology 255 (2014) 111

Fig. 3. Sensory innervation of footpad epidermis. (A) Epidermis (E), sub-epidermal plexus (SEp), sweat glands (SG) and algesimetry test site (ts) are represented on a rat footpad section
(scale bar = 100 m). In microscope images, the test site can be easily identied as adjacent to a concavity that the footpad forms with the adjacent skin. (B) Representative images of PGPand CGRP-immunoreactive bers in the epidermis of a normal footpad and of the medial and lateral test sites taken at 8, 29 and 60 days after sciatic nerve section and repair. Note extensive
epidermal denervation, followed by clear reinnervation at 60 dpi. Scale bar = 50 m. (C) Counts of PGP- and CGRP-immunoreactive intra-epidermal nerve bers (IENFs) at 3, 8, 16, 29 and
60 dpi show that these bers are almost completely lost in medial and lateral sites between 3 and 29 dpi. IENFs return to around normal values at 60 dpi, but CGRP-immunoreactive bers
were higher than normal in the medial site (*p b 0.05; **p b 0.01; ***p b 0.001, injured vs. contralateral paw).

S. Cobianchi et al. / Experimental Neurology 255 (2014) 111

On the other hand, the nociceptive thresholds measured in the saphenous territory were dramatically decreased after sciatic nerve injury
(Fig. 2B, bottom charts). Withdrawal threshold to mechanical stimulation in the medial site progressively decreased to about 25% of contralateral values from 3 dpi and remained so during the two month
follow-up (p b 0.001 at 3, 7, 14, 21, 28, 40, 50 and 60 dpi). The measurement of heat sensitivity revealed a similar but lower decrease of withdrawal latency in the medial region of the injured paw, by 35% of
normal, at 21 dpi. Compared to the contralateral paw, heat threshold
was signicantly lower at 14 (p b 0.001), 21 (p b 0.01), 28 (p b 0.001)
and 50 dpi (p b 0.001), and gradually recovered to near normal at
60 dpi.
Taken together, the algesimetry results show that the rst pain responses are mediated by the saphenous nerve, that rapidly induced mechanical and thermal hyperalgesia in the saphenous normal territory
and then gradually sensitized contiguous sciatic areas, as observed at
the central test site. Responses mediated by the regenerated sciatic
nerve appeared 34 weeks after injury and produced a late mechanical
hyperalgesia.
Plantar skin reinnervation
We showed that the section and suture of sciatic nerve induces opposite changes in nociceptive threshold, followed by a different sensory
recovery, if thresholds are measured in different test sites where sciatic
and saphenous nerves selectively cater for skin innervation. Then we
wanted to analyze if the observed changes were paralleled by changes
in the bers innervating the specic medial and lateral footpad test
sites. Fig. 3A shows representative footpad epidermal and subepidermal territories and test site area evaluated for ber innervation.
The test site can be easily identied as adjacent to a concavity that the
footpad forms with the at skin. Immunohistochemical images of PGP
immunoreactive proles showed important changes after sciatic nerve
injury (Fig. 3B). The number of IENFs was markedly reduced in the epidermis of both lateral and medial skin samples until 29 dpi (p b 0.001 at
3, 8, 16 and 29 dpi). The epidermis appeared thinner and numerous PGP
positive Langerhans cells were found in both medial and lateral sites, as
previously reported (Duraku et al., 2013; Stankovic et al., 1999). The
rst regenerating axons were found from day 29, but only a few reached
the epidermis and reformed less corpuscular endings than normal, as
previously described (Navarro et al., 1997). At 60 dpi nerve bers extensively reinnervated medial and lateral sites of the paw, but with a less
organized pattern than that in the control skin. The subpopulation of
CGRP immunoreactive proles was also signicantly reduced after injury (medial site: p b 0.01 at 3 dpi, p b 0.001 at 8 dpi, p b 0.05 at 16 and
29 dpi; lateral site: p b 0.01 at 3, 8 and 16 dpi, p b 0.05 at 29 dpi)
(Fig. 3B). CGRP staining evidenced a more complex pattern of reinnervation at two months after injury in the medial site compared with
the lateral site. Counts of IENFs (Fig. 3C) show that PGP- and CGRP- positive bers almost disappeared from both medial and lateral epidermis
during the rst month. However, the skin was well reinnervated at
60 dpi, when the mean number of peptidergic IENFs was even signicantly higher than normal in the medial site (p b 0.05). This result indicates that nociceptive epidermal hyper-reinnervation may be involved
in the maintenance of hypersensitivity at long time from the injury
(Duraku et al., 2013).
Since the progression of IENF innervation did not reect the early
changes of sensory thresholds observed at the medial site with
algesimetry tests, we hypothesized that sprouting bers that initially
grow in deep skin layers at the overlapping territory and reach the
sub-epidermal plexus rather than entering the epidermis, may be able
to respond to stimulation. We observed peptidergic SENFs elongating
from medial test site to the medial footpad dermis and along the basal
membrane during the rst days following injury (Fig. 4A), whereas in
the lateral footpad SENFs immunoreactivity almost disappeared during
the rst weeks, and was back to normal levels at 60 dpi (Fig. 4B). When

Fig. 4. Sensory innervation of footpad sub-epidermis. (A) Representative images of PGPand CGRP-immunoreactive bers in the sub-epidermal skin layer (SENFs) of the medial
test site, at 3, 8 and 29 days after sciatic nerve injury, show bers elongating through
the sub-epidermal plexus which are longer with time. Scale bar = 100 m. The PGPand CGRP-positive ber area in the subepidermal plexus of the medial footpad linearly
increased with time, whereas in the lateral footpad SENFs immunoreactivity almost disappeared during the rst weeks and reappeared near to normal after 29 dpi (*p b 0.05; **p
b 0.01; ***p b 0.001, injured vs. contralateral paw). (B) Measurement of the length of
PGP- and CGRP-positive proles at 3, 8, 16, 29 and 60 dpi. Medial SENFs progressively
elongated until 29 dpi, and CGRP positive SENFs even exceeded the normal length at 29
dpi (*p b 0.05; **p b 0.01; ***p b 0.001, injured vs. contralateral paw).

analyzing the immunoreactive prole length (Fig. 4C) in the subepidermal plexus of the medial pad, we found an increase with time; SENFs
were signicantly shorter at 3 dpi (p b 0.01, PGP; p b 0.05, CGRP) but
rapidly elongated until 29 dpi (p b 0.05 CGRP). CGRP positive SENFs signicantly exceeded the normal length at 29 dpi, indicating an abnormal

S. Cobianchi et al. / Experimental Neurology 255 (2014) 111

Fig. 5. Growth phenotype of peptidergic reinnervating bers. (A) Representative images of double-labeled CGRP- and GAP43-positive bers in the normal and in the injured skin of the
medial test site at 8, 29 and 60 days post-injury show a growing ber phenotype after injury. Scale bar = 100 m. (B) Plots of the rate of CGRP/GAP43-immunoreactive SENFs in medial and
lateral skin test sites at different times after injury demonstrate that active ber growth is different in timing at the medial and lateral sites (*p b 0.05, **p b 0.01, ***p b 0.001, injured vs.
contralateral paw).

S. Cobianchi et al. / Experimental Neurology 255 (2014) 111

prolongation and perhaps a difculty to cross the basal membrane to

enter the epidermis. At 60 dpi, both PGP and CGRP labeled SENFs
were found with similar length to normal, suggesting retraction after reinnervation by sciatic nerve bers. In contrast, in the lateral pad, the
SENFs almost completely disappeared, indicating degeneration, and
then reappeared with normal length in samples taken at 29 and 60
dpi due to the arrival of sciatic regenerating axons. This result demonstrates that, at least during the rst 2 weeks after sciatic injury, SENFs
elongating through the medial skin identify sprouting saphenous axons.
Growing state of reinnervating bers
We examined the proportion of CGRP-positive bers co-labeled
with GAP43 in order to characterize their growing state in the skin
(Fig. 5). Only a few CGRP nociceptive bers expressed GAP43 in the
normal skin (Fig. 5A). However, after sciatic nerve section most spared
SENFs at the medial site showed positive GAP43 staining, with a significantly increased proportion of GAP43 positive bers at 3 (p b 0.01),
8 (p b 0.001), 16 (p b 0.001) and 29 dpi (p b 0.05) than in the contralateral intact paw (Fig. 5B), suggesting a sprouting phenotype. At 60 dpi
the proportion of bers expressing GAP43 was lower, but still higher
than normal. In contrast, in the lateral pad of the paw GAP43 expression
was not found distinctly until 29 dpi. After 29 dpi, when CGRP positive
axons newly entered the skin, lateral skin samples showed signicant
colabeling (p b 0.001 at 29 dpi; p b 0.05 at 60 dpi). These observations
evidence that the injured paw skin underwent a differential pattern of
reinnervation by early sprouting of saphenous and late regeneration
of sciatic nerve bers.
Discussion
After sciatic nerve injuries, algesimetry tests are used to assess sensitivity to mechanical and thermal stimuli, but usually the stimuli are indiscriminately applied to the plantar foot skin, and the improvement of
sensory function is often related to recovery or changes of the sciatic
nerve innervation. The results of this study show that sensory assessment can be misleading in the evaluation of sciatic nerve regeneration
if saphenous and sciatic territories are not distinguished in the injured
paw. Different sensory stimuli have been applied in different ways
and on different areas of the foot for assessment of nerve regeneration
and functional reinnervation (Casals-Daz et al., 2009; Nichols et al.,
2005; Vogelaar et al., 2004). In the most common experimental model
used, i.e. the sciatic nerve in rodents, the generally accepted rule was
that the medial aspect of the paw should be avoided, as the saphenous
nerve partially supplies this region (De Koning et al., 1986; Devor
et al., 1979). However, in this way the information on the development
of collateral hyperalgesia may be missed. In this study we selectively detected the changes in response to stimulation applied to specic sites in
the paw, and assessed in parallel sciatic nerve regeneration and saphenous nerve sprouting effects on pain thresholds. The medial side of the
paw remained sensitive due to sparing of saphenous bers after sciatic
transection, and showed marked hyperalgesia from a few days postlesion,
consistent with adjacent neuropathic pain (Kingery and Vallin, 1989). In
contrast, the lateral side was anesthetic until reinnervation by regenerating
sciatic axons, which started by the fourth week, and then it became quickly
hyperalgesic (Cobianchi et al., 2013). Interestingly, stimuli applied to the
center of the paw showed mild recovery of responses from the rst week
and a slow progression of threshold decrease to below normal levels,
which were attributable rst to sprouting extension of the saphenous
nerve and later to the arrival of regenerating sciatic axons.
The development and the resolution of hyperalgesia are dependent
on the injury model and the reinnervation of end-organs. Neurotmesis
of the sciatic nerve with distal stump excision causes that a large area
of the hindpaw remains unresponsive, but the saphenous mediated
hyperalgesia is sustained over weeks/months (Attal et al., 1994;
Casals-Daz et al., 2009; Kingery et al., 1994). In contrast, after nerve

crush or section repaired lesions, regeneration of sciatic axons allows

for regaining skin innervation and initiates the resolution of
hyperalgesia (Casals-Daz et al., 2009; Kingery et al., 1994). However,
areas of hyperalgesia and hypoalgesia may coexist in the affected
hindpaw, due to variable extension of saphenous sensory bers
sprouting and incomplete sciatic nerve regeneration. For this reason
the assessment of somatosensory function mediated by the sciatic
nerve after injury should be made in areas of the paw that are not affected by sprouting of the intact saphenous nerve, i.e. the lateral aspects of
plantar skin and the lateral toes (Wood et al., 2011).
Our results clearly demonstrate that after sciatic nerve injury a primary source of early hyperalgesia is due to the saphenous nerve.
Sprouting of the saphenous nerve following sciatic denervation has
been well documented, allowing functional extension of the saphenous
territory for nociceptive and autonomic bers (Devor et al., 1979;
Kennedy et al., 1988; Kingery and Vallin, 1989; Markus et al., 1984).
Saphenous nerve mediated mechanical hyperalgesia was long-lasting
and remained after return of sciatic regenerating axons, whereas saphenous heat hyperalgesia gradually returned to normal values concomitantly to reappearance of responses at the sciatic territory from
4 weeks. Other studies found that adjacent hyperalgesia was more pronounced to mechanical than to heat stimulation (Kauppila and Xu,
1996; Mansikka and Pertovaara, 1997), and resolution or reduction of
saphenous-mediated hyperalgesia coincident with sciatic nerve reinnervation (Kingery et al., 1994; Kinnman et al., 1992). Nonetheless,
the hyperexcitability of the saphenous neurons could reect chronic
sensitization of its pathway, including spinal cord circuits (LaMotte
et al., 1989; Markus et al., 1984; Sotgiu and Biella, 1995), contributing
to maintain the hyperalgesia affecting more mechanical than thermal
sensitive neurons.
Collateral sprouting is a characteristic of normal undamaged nerves
in response to elimination of other axons within the same target tissues.
It is well known that skin denervation induces growth of collateral
sprouts from neighbor uninjured axons (Diamond and Foerster, 1992;
Diamond et al., 1987; Mendell et al., 1999), which is promoted by the
secretion of neurotrophins by the denervated tissues (Diamond et al.,
1992; Gloster and Diamond, 1992). Langerhans cell activation may
also participate in intact nerve ber sprouting or sensitization. For instance, Langerhans cells are in close apposition with epidermal bers,
and when activated they release pro-inammatory mediators, such as
nitric oxide and cytokines tumor necrosis factor and interleukin-1
(Andersson et al., 2004; Hsieh et al., 1996; Qureshi et al., 1996). This
compensatory reinnervation allows for partial restoration of autonomic
and nociceptive functions after nerve damage (Ahcan et al., 1998; Inbal
et al., 1987). Small myelinated and unmyelinated bers show stronger
capacity to collaterally sprout and enlarge their territory of innervation
than that of large myelinated bers (Nixon et al., 1984; Doucette and
Diamond, 1987; Jackson and Diamond, 1984; Kennedy et al., 1988;
Gloster and Diamond, 1992), thus underlying increased nociceptive
sensitivity that may be a protective mechanism for the previously denervated skin. Collateral reinnervation by saphenous nociceptive bers
rapidly extends to overlapping and contiguous areas previously innervated by the sciatic nerve (Devor et al., 1979; Kingery and Vallin,
1989; Kinnman et el., 1992). This explains the early recovery of sensitivity in the central areas of the paw, that disappeared after sectioning the
saphenous nerve at 14 dpi. These observations point out that the site of
sensory testing is crucial for assessing neuropathic pain and sensory
recovery after nerve lesions because of the different pattern of reinnervation. We also found that saphenous peptidergic bers showed early
sprouts in the sub-epidermal plexus expressing GAP43 much earlier in
time than returning sciatic regenerative axons, demonstrating that
signs of neuropathic pain are associated to remodeling of sensory innervation in dermal layers of the skin.
Sciatic nerve repair is a model of injury clinically relevant for the
study of peripheral nerve regeneration (Rodrguez et al., 2004), which
allows reinnervation differently from models of partial sciatic lesion

10

S. Cobianchi et al. / Experimental Neurology 255 (2014) 111

(Grelik et al., 2005; Ma and Bisby, 2000) in which sprouting and regenerative responses may be mixed. The reinnervation of the skin by axons
regenerating after sciatic nerve section and suture repair started after
4 weeks, progressing from proximal to distal areas of the paw and gradually reaching sub-epidermal and epidermal layers up to the
hyperreinnervation of the skin at 2 months. Sciatic reinnervation induced mechanical hyperalgesia in the lateral side of the paw, that
reverted slowly over time (Lancelotta et al., 2003; Vogelaar et al.,
2004). However, we found in parallel a higher proportion of CGRP bers
only in the saphenous territory, suggesting that skin nociceptive
hyperreinnervation is not necessary to induce neuropathic phenotype
after regeneration of peripheral nerves. This observation raises the possibility that different models of injury may induce a dishomogeneous
distribution of receptors and sensory ber types due to different contributions of collateral and regenerative reinnervation.
Changes occurring in the primary afferent neurons alter the processing of nociceptive information following nerve injury. Peptidergic and
non-peptidergic bers play different roles in the development and
maintenance of skin sensitization (Taylor et al., 2012). Previous works
(Duraku et al., 2012, 2013; Grelik et al., 2005) showed a prominent
role of peptidergic but not non-peptidergic bers in reinnervating the
skin after peripheral nerve injury. Most peptidergic bers terminate in
the more supercial layers of the epidermis (Zylka et al., 2005), and convey sensory information to specic laminae of the spinal cord dorsal
horn (Molliver et al., 1995). We observed that epidermal and subepidermal layers have different patterns of reinnervation in relation
with neuropathic abnormal responses. Epidermal reinnervation was
not correlated with the signicant decrease of withdrawal thresholds
(Duraku et al., 2013). However, we found that peptidergic bers early
growing through the dermis anticipated epidermal reinnervation to
trigger hyperalgesic responses. As previously hypothesized (Verd
and Navarro, 1997), the fact that pain responses started earlier than
nerve bers entered the epidermis may be explained by the activation
of afferent bers in the subepidermal plexus. To prove that active ber
growth is concomitant to hyperalgesic responses, we used GAP43 as a
marker of axonal growth (Oestreicher et al., 1997). GAP43 is expressed
in unmyelinated bers of dorsal root ganglion following nerve injury
(Coggeshall et al., 1991; Jaken et al., 2011; Woolf et al., 1988). We
found that saphenous peptidergic bers rapidly elongated through the
subepidermal plexus after sciatic nerve injury. After one month, sciatic
regenerating axons enter the skin and highly express GAP43 until
2 months, showing that collateral sprouting gradually extends in overlapping territory and is later substituted by growth of regenerative
axons.
In conclusion, peripheral nerve injuries induce intact nerve collateral
sprouting, that is the cause of early recovery of sensory responses and
neuropathic pain symptoms. The outcome of sensory testing depends
on the site of stimulation, and landmarks of denervated and adjacent innervated skin should be used when performing algesimetry tests, together with histological analysis, for parallel investigation of the
sprouting and regenerative capacities of sensory neurons. These processes are complementary in functional recovery, but contribute differently in the initiation and maintenance of neuropathic pain.
Acknowledgments
This research was supported by a grant FP7-278612 (BIOHYBRID)
from the EU and funds of RETIC TERCEL from the Instituto de Salud Carlos III of Spain. The authors are grateful for the technical help of Mnica
Espejo, Jessica Jaramillo and Marta Morell.
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