In this study the following key observations were made: 1) The distribution of CGRP-IR fibers in the epidermis of the footpads is lower compared to that of the glabrous skin of the foot sole. 2) Denervation of the central foot sole area by the SNI procedure results in a prominent and persistent mechanical hypersensitivity of the lateral and medial sides of the foot sole and in heat and cold hyperalgesia and allodynia. 3) The central area of the foot sole including the footpads re-innervates with CGRP-IR fibers from the lateral and medial sides.
Neuropathic pain and density of CGRP innervation of the epidermis
Our study shows an almost complete loss of CGRP-IR fibers in the center area, following an SNI procedure, whereas the density of the medial part of the foot sole remains unchanged until 5 weeks PO when an increase in density was noted. At the lateral side of the foot, the density of CGRP-IR fibers initially decreased (at 2 weeks PO) but recovered at 5 weeks PO and increased at 10 weeks. Nevertheless, by 2 weeks PO mechanical hypersensitivity at both edges of the foot sole was noted. Because electrophysiological changes accompanying neuropathic pain behavior have also been reported to occur in uninjured fibers as early as one week PO in partial peripheral nerve injury models
[24, 25], it seems likely that electrophysiological and behavioral changes precede detectable changes in morphology. Interestingly, it has also been shown that after a ventral rhizotomy, which results in degeneration of myelinated fibers that in turn leads to neuropathic pain behavior in the rat. The postulated mechanism is that through Wallerian degeneration reacting Schwann cells produces factors that diffuse to nearby uninjured unmyelinated C-fibers and alter their properties
. In addition, degeneration of myelinated motor fibers leads to mitogenic changes to nonmyelinating uninjured Schwann cells with intact axons, which in turn may play a significant role in the development of neuropathic pain
[27, 28]. However, it is also possible that other subtypes of sensory fibers may show morphological changes that correlate more precisely with the mechanical hypersensitivity. However, preliminary data from our group shows that the peptidergic Substance P, non-peptidergic P2X3 and myelinated NF-200 fibers do not show hyper-innervation of the epidermis in SNI-rats as compared to sham. More specifically the non-peptidergic P2X3 fibers exhibit almost no re-innervation as described in an earlier study
. However to discuss this in more detail is out of the scope of the current study.
The sural nerve-innervated lateral aspect of the foot sole has a more severely increased mechanical pain threshold compared to the medial side (innervated by the saphenous nerve). Although agreeing with previous results
, this seems at odds with the observed initial lateral reduction in density of CGRP-IR fibers. The latter effect indicates that there may be considerable spatial overlap in innervation of the tibial and sural nerves. We suggest that the observed medio-lateral differences in sensitivity at 2 weeks PO could be related to the fact that the saphenous nerve is mostly derived from the L3 dorsal root ganglion where there is less interaction between injured and non-injured neuronal cell bodies (Figure
10) as compared to the L4-L6 ganglia from which the sural nerve is derived
. This may lead to a delayed increase of CGRP-IR epidermal fibers on the sural side compared to the saphenous side, while the sural side has a lower mechanical threshold.
Although the SNI procedure leads to a virtually complete absence of CGRP-IR epidermal fibers in the center area of the foot sole, only a marginally increased mechanical withdrawal threshold nor a complete absence of the withdrawal reflex were noted. This could be due to stimulation of the hypersensitive lateral or medial region of the foot sole, which is inevitable when applying forces exceeding 40 g. At 5 and 10 weeks PO the observed re-innervation of epidermal CGRP-IR in the center area is in accordance with a return of the mechanical withdrawal threshold to control levels.
The increase in epidermal CGRP-IR fibers might be an increase in expression levels of CGRP and not an increase of fibers. It is known that after peripheral nerve injury the uninjured DRG neurons, adjacent to injured neurons, increase CGRP expression through the p38 mitogen activated protein kinase (MAPK) pathway
[30, 31], consequently leading to increased peripheral transport and filling of nerve terminals in already existing peripheral but undetected in control conditions. However, the increased density of CGRP-IR fibers in the sural (lateral) and saphenous (medial) territories found at 5 weeks PO, is in accordance with a previous study where, in the SNI model, an increased density was observed in small biopsies of the lateral hind paw area using PGP 9.5, which is a marker for all types of nerve fibers
. Therefore the collateral sprouting of CGRP-IR fibers in the SNI model seems more plausible. Other studies, using the CCI neuropathic model, which also leads to thermal and mechanical hypersensitivity
, have reported both normal as well as inverse correlations between neuropathic pain and density of nociceptive fibers
[7, 8, 10, 11]. From these studies it appears that the occurrence and severity of neuropathy in CCI models is highly variable
[33–37], which may relate to the varying results in re-innervation patterns in hind paw glabrous skin. The variability in CCI results is likely to be related to inconsistent tightness of the ligatures, which may result in a variable percentage of permanently damaged axons
. The CCI procedure also has an inflammatory component, evoking intraneural edema, also resulting in variable constriction
[14, 15]. Therefore, it is uncertain if the changes in skin innervation are due to injured, uninjured or a combination of these two groups of fibers.
In the SNI model, the injury consists of a complete transection of nerve fibers, which are subsequently prevented from regrowing by the ligation as was substantiated by the lack of Evans Blue extravasation at 10 weeks PO in SNI rats. Therefore, the re-innervation patterns of epidermal CGRP-IR fibers in the SNI model must originate from uninjured fibers located in adjacent areas. As such, we propose that the SNI model has distinct advantages over the CCI model for studying re-innervation of denervated skin by undamaged axons.
Effect of SNI on epidermal thickness
Our results show that, in contrast to previous reports
[18, 19, 38], there is an inverse correlation between epidermal thickness and density of intra-epidermal fibers. Peptidergic epidermal fibers have been suggested to promote proliferation of keratinocytes and maintenance of skin tropism
[39, 40]. It should be pointed out, however, that the rats in these studies did not suffer chronic neuropathic pain as is the case in the SNI model. Keratinocytes not only produce CGRP-β (whereas fibers mostly contain CGRP-α), likely to have an autocrine/paracrine role in epidermal homeostasis
, but also release β-endorphin, which, by binding to μ-opioid receptors located on CGRP containing sensory endings, seems to ameliorate heat-pain induced withdrawal
. Although CGRP-β is normally expressed at low levels and rather heterogeneously among epidermal keratinocytes, chronic pain conditions induces an increased and more homogeneous expression
. Therefore, we suggest that the combination of chronic pain, increased densities of CGRP-IR fibers and potentially also of increased CGRP-β release by non-proliferating keratinocytes are instrumental in preventing epidermal thickening.