Parasympathetic nerve fibers invade the upper dermis following sensory denervation of the rat lower lip skin

The sympathetic division of the autonomic nervous system is known to play a role in the genesis of neuropathic pain. In the skin of the rat lower lip (hairy skin), sympathetic and parasympathetic fibers normally innervate the same blood vessels in the lower dermis but do not occur in the upper dermis. However, we have shown that sympathetic fiber migration into the upper dermis occurs following mental nerve lesions (Ruocco et al. [2000] J. Comp. Neurol. 422:287-296). As sensory denervation has a dramatic effect on sympathetic fiber innervation patterns in the rat lower lip skin, we decided to investigate the possible changes in the other autonomic fiber type in the skin-the parasympathetic fiber. Sensory denervation of the rat lower lip was achieved by bilateral transection of the mental nerve, and animals were allowed to recover for 1-8 weeks. Lower lip tissue was processed for double-labeling light microscopic immunocytochemistry (ICC), using antibodies against substance P (SP), which labels a subpopulation of peptidergic sensory fibers, and against the vesicular acetycholine transporter (VAChT), as a marker for parasympathetic fibers. In sham-operated rats, SP-immunoreactive (IR) sensory fibers were found in the epidermis and upper and lower dermal regions, whereas VAChT-IR fibers were confined to the lower dermis. Mental nerve lesions induced the gradual disappearance of SP-IR fibers from all skin layers accompanied by the progressive migration of VAChT-IR fibers into the upper dermis. Cholinergic fiber migration was evident by the second week post surgery, and the ectopic innervation of the upper dermis by these fibers persisted even at the last time point studied (8 weeks) when SP-IR fibers have completely regrown. VAChT-IR fibers were observed in the upper dermis, well above the opening of the sebaceous glands into the hair follicles. These results show that considerable changes occur in the innervation patterns of parasympathetic fibers following mental nerve lesions.     

Delayed reinnervation by nonpeptidergic nociceptive afferents of the glabrous skin of the rat hindpaw in a neuropathic pain model

Painful peripheral neuropathies have been associated with a reorganization of skin innervation. However, the detailed changes in skin innervation by the different afferent fiber types following a neuropathic nerve injury have never been characterized in an animal model of neuropathic pain. Our objective was to thoroughly characterize such changes in the thick skin of the foot in a well-established rat model of neuropathic pain, namely, the chronic constriction injury (CCI) of the sciatic nerve. We used the immunofluorescence detection of calcitonin gene-related peptide (CGRP), purinergic receptor P2X3, and NF200 as markers of peptidergic nociceptive fibers, nonpeptidergic nociceptive C fibers, and myelinated afferents, respectively. We observed that CCI-operated animals developed significant mechanical allodynia and hyperalgesia as well as thermal hyperalgesia. At 3 days following nerve injury, all CCI-operated animals had a significant decrease in the density of fibers immunoreactive (IR) for CGRP, P2X3, and NF200 within the upper dermis. A recovery of CGRP-IR fibers occurred within 4 weeks of nerve injury and sprouting above control levels was observed at 16 weeks. However, the myelinated (NF200-IR) fibers never recovered to control levels within a period of 16 weeks following nerve injury. Interestingly, the P2X3-IR fibers took considerably more time to recover than the CGRP-IR fibers following the initial loss. A loss in P2X3-IR fibers persisted to 16 weeks and recovered to levels above that of control at 1.5 years following nerve injury. Further studies are required to clarify the relevance of these innervation changes for neuropathic pain generation and maintenance.     

Peripheral nerve injury leads to the establishment of a novel pattern of sympathetic fibre innervation in the rat skin

Peripheral nerve injury has been shown to result in sympathetic fibre sprouting around dorsal root ganglia (DRG) neurons. It has been suggested that this anomalous sympathetic fibre innervation of the DRG plays a role in neuropathic pain. Other studies have suggested an interaction between sympathetic and sensory fibres more peripherally. To date, no anatomical study of these possible interactions in the terminal fields of sensory and sympathetic fibres has been performed; therefore, the authors set out to study them in the rat lower lip after bilateral lesions of a sensory nerve, the mental nerve (MN). Immunocytochemistry for both substance P (SP) and dopamine-beta-hydroxylase (DbetaH) was performed. Within the first week post-MN lesions, the SP-immunoreactive (IR) fibres had degenerated almost completely, whereas DbetaH-IR fibres were found in the upper dermis, an area from which they normally are absent. These DbetaH-IR fibres were present in the upper dermis at all postsurgery times studied (1, 2, 3, 4, 6, and 8 weeks). It is noteworthy that, although, by week 6 post-MN lesions, SP-IR fibre reinnervation of the lower lip was occurring, the DbetaH-IR fibres still were present in the upper dermis. Quantification revealed that the migration and branching of the DbetaH-IR fibres into the upper dermis occurred gradually and was most significant at 4 weeks post-MN lesions, as demonstrated by the fact that the DbetaH-IR fibres were found 169.6 +/- 91.4 microm away from the surface of the skin compared with 407.1 +/- 78.4 microm away in sham-operated animals. These findings suggest that the ectopic innervation of the upper dermis by sympathetic fibres may be important in the genesis of neuropathic pain through the interactions of sympathetic and SP-containing sensory fibres.     

Sympathetic sprouting in the dorsal root ganglia of the injured peripheral nerve in a rat neuropathic pain model

The extent of the sprouting of sympathetic postganglionic fibers in the dorsal root ganglion (DRG) and the peripheral nerves was examined in neuropathic rats at different postoperative times. After the L5 and L6 spinal nerves were ligated on one side, three different pain behavior tests (representing mechanical allodynia, cold allodynia, ongoing pain exacerbated by cold stress) were performed at various time intervals. The sympathetic postganglionic fibers were visualized by immunostaining with antibodies to tyrosine hydroxylase (TH). In the neuropathic rats, all three pain behaviors were fully developed within 3 days after the surgery, maintained up to 2 weeks, and then started to decline gradually afterward. At 20 weeks after neuropathic surgery, pain behaviors were reduced significantly compared to the peak response, but were still higher than the presurgery levels. Sympathectomy, performed 4 days after neuropathic surgery, almost completely abolished the signs of mechanical allodynia and ongoing pain behaviors, and it reduced the behaviors of cold allodynia to approximately half. The numerical density of sympathetic fibers in the DRG of an injured segment was significantly higher at 1, 4, and 20 weeks after neuropathic surgery as compared to the normal, suggesting that there is sprouting of sympathetic fibers in the DRG after peripheral nerve injury. Sprouting of sympathetic fibers in the DRG was extensive as early as 2 days after the spinal nerve ligation, and the sprouted fibers were almost completely eliminated after sympathectomy. The data suggest that sympathetic innervation of the DRG may play an important role in the development and maintenance of sympathetically maintained neuropathic pain. © 1996 Wiley-Liss, Inc.     

Autonomic fibre sprouting and changes in nociceptive sensory innervation in the rat lower lip skin following chronic constriction injury

In this study we used immunocytochemistry to investigate whether autonomic fibres sprouted in the skin of the lower lip in a rat model of neuropathic pain. We used a bilateral chronic constriction injury (CCI) of the mental nerve (MN), a branch of the trigeminal nerve. In this model, we also studied the accompanying changes in peptidergic [calcitonin gene-related peptide (CGRP)-immunoreactive] sensory fibres, as well as in trkA receptor immunoreactivity in the sensory nerves. Autonomic (sympathetic and parasympathetic) fibre sprouting was first observed 1 week post-injury with a peak in the number of sprouted fibres occurring at 4 and 6 weeks post-CCI. CGRP-IR fibres almost disappeared at 2 weeks post-CCI, but quickly sprouted, leading to a significant peak above sham levels 4 weeks post-injury. trkA receptor expression was found to be up-regulated in small cutaneous nerves 4 weeks post-CCI, returning to sham levels by 8 weeks post-CCI. There was no sympathetic fibre sprouting in the trigeminal ganglion following CCI. At 4 weeks post-CCI, rats displayed spontaneous, directed grooming to the area innervated by the MN that was not seen in sham animals, which we interpreted as a sign of spontaneous pain or dysesthesiae. Collectively, our findings indicate that as a result of autonomic sprouting due to CCI of the MN, remaining intact nociceptive fibres may potentially develop sensitivity to sympathetic and parasympathetic stimulation, which may have a role in the generation of abnormal pain following nerve injury.     

Submodality-Selective Hyperalgesia Adjacent to Partially Injured Sciatic Nerve in the Rat is Dependent on Capsaicin-Sensitive Afferent Fibers and Independent of Collateral Sprouting or a Dorsal Root Reflex

We studied submodality dependence of sensory changes produced by unilateral ligation of the sciatic or the saphenous nerve in the rat. We focused especially on sensory changes in the skin area adjacent to the innervation area of the injured nerve. Moreover, we examined the roles of capsaicin-sensitive nociceptive fibers, collateral sprouting and a dorsal root reflex in sensory changes observed behaviorally. Assessment of sensory changes was performed by a pattern of behavioral tests: hot-plate test and hindlimb withdrawal responses induced by radiant heat, hot-water bath, innocuous mechanical stimuli, and noxious mechanical stimuli. In one group, the saphenous nerve ipsilateral to the sciatic ligation was topically treated with capsaicin (1%) at the time of the surgery. A proximal stump of a saphenous nerve strand was orthodromically stimulated to induce a dorsal root reflex (an antidromic volley) in nociceptive fibers of the saphenous nerve trunk. For visualization of plasma extravasation induced by a dorsal root reflex, a dye-labeling (Evans blue) technique was used. A collateral sprouting of nociceptive fibers of the uninjured saphenous nerve was evaluated by determining the plasma extravasation response induced by antidromic stimulation of the saphenous nerve. Three and 10 days following the sciatic constriction injury, the hindlimb withdrawal threshold evoked by noxious mechanical stimulation of the medial side of the paw (the innervation are of the intact saphenous nerve) was significantly decreased. There was no corresponding thermal hyperalgesia adjacent to the injured sciatic nerve. Chronic constriction of the saphenous nerve did not produce any significant hyper- or hypoalgesia to mechanical or thermal stimulation of the uninjured sciatic nerve area. Topical treatment of the ipsilateral (intact) saphenous nerve at the time of the sciatic nerve ligation completely prevented the development of mechanical hyperalgesia in the medial side of the paw (the innervation area of the saphenous nerve). No dorsal root reflex in nociceptive fibers mediating the adjacent hyperalgesia could be evoked. No collateral sprouting of the uninjured nociceptive fibers of the saphenous nerve was observed. The results indicate that the constriction injury of the sciatic nerve produced a selective hyperalgesia to mechanical stimulation in the innervation area of the neighboring saphenous nerve. At the peripheral level, the mechanical hyperalgesia adjacent to the innervation area of the injured nerve was mediated by capsaicin-sensitive nociceptive fibers. Collateral sprouting of nociceptive fibers from the uninjured to the injured innervation area did not contribute to the present sensory findings. The sciatic nerve injury did not induce a dorsal root reflex in nociceptive fibers innervating the hyperalgesic saphenous nerve area.     

Normal and injury-induced sympathetic innervation of rat dorsal root ganglia increases with age

In rats, partial injury to a peripheral nerve often leads to sympathetically maintained pain (SMP). In humans, this condition is especially apparent in the elderly. Nerve injury also causes perivascular sympathetic axons to sprout into the dorsal root ganglion (DRG), forming a possible anatomical substrate for SMP. Here, we describe the effects of chronic sciatic nerve constriction injury (CCI) in young (3 months) and old (16 months) rats on neuropathic pain behavior and on sympathetic sprouting in DRG. Behavioral tests assessed changes in thermal allodynia and hyperalgesia and in mechanical allodynia. We found that 1) sympathetic innervation of the DRG increased naturally with age, forming pericellular baskets mainly around large DRG neurons, and that sympathetic fibers were often associated with myelinated sensory axons; 2) sympathetic fiber density following CCI was also greater in old than in young rats; and 3) in old rats, thermal allodynia was less pronounced than in young rats, whereas thermal hyperalgesia and mechanical allodynia were more pronounced. These results highlight the possibility that sympathetic sprouting in the DRG is responsible for the sympathetic generation or maintenance of pain, especially in the elderly. J. Comp. Neurol. 394:38–47, 1998. © 1998 Wiley-Liss, Inc.     

Innervation of footpads of normal and mutant mice lacking sweat glands

Footpads of normal adult mice are innervated by sympathetic and sensory fibers. The sympathetic fibers associated with sweat glands contain acetylcholinesterase and immunoreactivity for vasoactive intestinal peptide. Although catecholamine histofluorescence is absent, the gland innervation exhibits immunoreactivity for tyrosine hydroxylase. A distinct population of sympathetic fibers, which possess catecholamines and neuropeptide Y as well as tyrosinehydroxylase immunoreactivity, innervates blood vessels. Sensory fibers containing immunoreactivity for substance P and calcitonin gene-related peptide course beneath the epidermis and some form endings in it. Treatment of neonatal mice with the adrenergic neurotoxin, 6-hydroxydopamine, results in loss of sympathetic innervation of sweat glands and blood vessels, permits growth of sensory axons into sweat glands, but does not alter the peptidergic sensory innervation of the dermis and epidermis. Three mouse mutations, Tabby (Ta), crinkled (cr), and downless (dl), disrupt the interactions between the mesenchyme and epidermis that are required for normal development of specific epidermal derivatives, including sweat glands. The sympathetic innervation of blood vessels and sensory innervation of footpad skin of the three mutant mice that lack sweat glands is indistinguishable from normal. The sympathetic fibers that normally innervate sweat glands, however, are not present. These results indicate that in the absence of their normal target, the sympathetic fibers that innervate sweat glands are lacking. Furthermore, they suggest that, although sensory fibers may sprout into sympathetic targets in the footpad, the domains occupied by sensory fibers are not normally accessible to sympathetic axons. © 1994 Wiley-Liss, Inc.     

Skin blood vessels are simultaneously innervated by sensory,sympathetic, and parasympathetic fibers

Despite the known major role of skin blood vessel innervation in blood flow control, particularly in disease, little information on the co-innervation of blood vessels by sensory and autonomic fibers and the relationships of these fibers to one another is available. To fill this gap, we performed a light and electron microscopic analysis of the innervation of skin vessels by sensory and autonomic fibers by using the rat and monkey lower lips as a model. In rats, double-labeling immunocytochemistry revealed that combinations of fibers immunoreactive for substance P (SP) and dopamine-beta-hydroxylase (DbetaH), SP and vesicular acetylcholine transporter (VAChT), as well as DbetaH and VAChT occurred only around blood vessels in the lower dermis. All fiber types travelled in parallel and in close proximity to one another. In the upper dermis, blood vessels were innervated by SP-containing fibers only. Although nerve terminals displayed synaptic vesicles, synaptic specializations were never observed, suggesting that, in this territory, these fibers do not establish synaptic contacts. Quantification of the distance between the various immunoreactive terminals and their presumptive targets (smooth muscle cells and endothelial cells) revealed that both sympathetic and parasympathetic fibers were significantly closer to the endothelial cell layer and smooth muscle cells compared with sensory fibers. In monkeys, double-labeling immunocytochemistry was performed for SP-DbetaH and SP-VAChT only. The results obtained are similar to those found in rats; however, the fiber density was greater in monkeys. Our findings suggest that the regulation of skin microcirculation might be the result of the coordinated functions of sensory and autonomic fibers.     

Effects of endogenous neurotrophins on sympathetic sprouting in the dorsal root ganglia and allodynia following spinal nerve injury

Peripheral nerve injury is often complicated by a chronic pain syndrome that is difficult to treat. In animal models of peripheral nerve injury, sympathetic nerve terminals in the dorsal root ganglia (DRG) sprout to form baskets around large diameter neurons, an anatomical change that has been implicated in the induction of neuropathic pain. In the present study, we have investigated whether neurotrophins derived from peripheral sources play any roles in sympathetic sprouting and neuropathic pain in a rat model of peripheral nerve injury. After transection of the left lumbar (L) 5 spinal nerve, antisera specific to neurotrophins were injected intraperitoneally twice a week for 2 weeks. The foot withdrawal response to von Frey hairs was examined on days 1, 3, 7, 10, and 14 postlesion. After completion of behavioral tests, sympathetic sprouting in DRG was examined by tyrosine hydroxylase (TH) immunohistochemistry. The number of TH-immunoreactive (ir) fibers and baskets around large neurons within the lesioned DRG was dramatically increased in the rats treated with control normal sheep serum. Antisera specific to nerve growth factor (NGF), neurotrophin-3 (NT3), and brain-derived neurotrophic factor (BDNF) significantly reduced the sympathetic sprouting and the formation of baskets. L5 spinal nerve lesion induced a significant increase in foot withdrawal responses to von Frey hair stimuli, which was attenuated by treatment of antisera to neurotrophins with a different time sequential. The effect of BDNF antiserum occurred earlier and lasted longer than those of NGF and NT3 antisera. These results implicate that peripherally derived neurotrophins are involved in the induction of sympathetic sprouting and neuropathic pain following peripheral nerve injury.     

Paclitaxel- and vincristine-evoked painful peripheral neuropathies: loss of epidermal innervation and activation of Langerhans cells

Experimental painful peripheral neuropathies produced by the chemotherapeutic drugs, paclitaxel and vincristine, are produced by relatively low doses that do not cause axonal degeneration in peripheral nerve. Using quantitative immunolabeling with the PGP9.5 antibody, we have investigated whether these painful neuropathies might be associated with degeneration that is confined to the region of the sensory fiber's receptor terminals in the skin. Because complete and partial nerve transections are known to cause an increase in PGP9.5 in epidermal Langerhans cells (LCs), we also examined whether this effect occurs in chemotherapy-treated animals. At the time of peak pain severity, rats with paclitaxel- and vincristine-evoked painful peripheral neuropathies had a significant decrease (24% and 44%, respectively) in the number of intraepidermal nerve fibers (IENF) in the hind paw glabrous skin and an increase (217% and 121%, respectively) in the number of PGP9.5-positive LCs, relative to control. However, neither loss of IENF nor an increase in PGP9.5-positive LCs was found in rats with a painful peripheral neuropathy evoked by the anti-HIV agent, 2',3'-dideoxycytidine. We also confirmed that there is a decrease in IENF and an increase in PGP9.5-positive LCs in rats with neuropathic pain following a partial nerve injury (CCI model) and in rats with a complete sciatic nerve transection. Partial degeneration of the intraepidermal innervation suggests mechanisms that might produce chemotherapy-evoked neuropathic pain, and activation of cutaneous LCs suggests possible neuroimmune interactions that might also have a role.     

Complete recovery by nerve growth factor of neuropeptide content and function in capsaicin-impaired sensory neurons

In the present study the ability of nerve growth factor (NGF) to facilitate the recovery of peptidergic primary sensory C-fibers after an acute capsaicin treatment (50 mg/kg s.c.) was investigated in adult rats. NGF (4 μg 1 /day for 3 days) was injected into the plantar of one hind paw starting 24 h after the capsaicin treatment. Without NGF, there was a significant reduction of calcitonin gene-related peptide (CGRP) and substance P content of the paw skin and the sciatic nerve. CGRP and substance P levels were completely replenished in the NGF-treated paw skin and in the innervating sciatic nerve they even increased over control levels as determined 40 h after the last injection of NGF. CGRP levels also recovered in the contralateral paw and sciatic nerve, but no recovery was observed in other tissues such as the front paw, the auricle, or the urinary bladder. Mustard oil-induced neurogenic plasma extravasation, taken as a functional parameter for peptidergic primary sensory C-fibers, was significantly decreased after the capsaicin treatment and showed a complete recovery by NGF in the injected paw as well as in the contralateral paw skin. These results show that NGF not only was able to reverse the decrease of transmitter content caused by capsaicin but also restored the peripheral function of primary afferent neurons.     

Unilateral nerve injury produces bilateral loss of distal innervation

There are no known anatomical connections between neurons that innervate homologous right and left body parts. Nevertheless, some patients develop bilateral abnormalities after unilateral injury, a phenomenon often unrecognized and not yet characterized. Therefore, we examined in rats the effects of ligating and cutting one tibial nerve on sensory function and on density of innervation in hind paws contralaterally as well as ipsilaterally to the injury, at times between 1 day and 5 months after surgery. Punches removed from tibial- or sural-innervated planter paw skin were immunolabeled to quantitate epidermal nerve endings. Naive and sham-operated rats provided controls. Axotomized rats had near-total loss of PGP9.5(+) innervation within ipsilateral tibial-innervated skin at all time-points. Adjacent ipsilateral sural-innervated skin had persistent hyperalgesia without denervation, and robust axonal sprouting at 5 months after surgery. Contralesional hind paws lost 54% of innervation in tibial-innervated epidermis starting 1 week after surgery and persisting throughout. Contralesional sural-innervated skin had neither neurite loss nor sprouting. These results imply that unilateral nerve injury can cause profound, long lasting, nerve-branch-specific loss of distal innervation contralaterally as well as ipsilaterally. They discredit the practice of using tissues contralateral to an injury to provide normative controls and suggest the possibility of rapid, transmedian postinjury signals between homologous mirror-image neurons.     

Differences in Sympathetic Innervation of Mouse DRG Following Proximal or Distal Nerve Lesions

Nerve injury leads to novel sympathetic innervation of the dorsal root ganglion (DRG). We have hypothesized previously that the degenerating nerve increases the sympathetic sprouting in the DRG and pain after chronic sciatic constriction injury (CCI) by virtue of its influence on sensory and sympathetic axons spared by the injury. However, L5 spinal nerve ligation and transection (SNL) results in the complete isolation of the L5 DRG from the degenerating stump, yet sympathetic axons invade the ganglion, and sympathetically dependent pain develops. We investigated the role of Wallerian degeneration in both sympathetic sprouting and neuropathic pain in these two models of painful peripheral neuropathy by comparing responses of normal C57B1/6J and C57B1/Wld^smice in which degeneration is impaired. After CCI, Wld^smice, unlike 6J mice, did not develop thermal or mechanoallodynia or sympathetic innervation of the L5 DRG. After SNL, both strains developed mechanoallodynia and sympathetic sprouts in L5, but only 6J mice developed thermal allodynia. Observation of the origins of the invading sympathetic axons revealed that after CCI, sympathetics innervating blood vessels and dura (probably intact) sprouted into the ganglion, but after SNL sympathetics (probably axotomized) invaded from the injured spinal nerve. Based on these findings, we hypothesize that there are two mechanisms for sympathetic sprouting into DRG, differentially dependent on Wallerian degeneration. Analysis of pain behavior in these animals reveals that (i) mechanoallodynia and sympathetic innervation of the DRG tend to coincide and (ii) thermal allodynia and Wallerian degeneration, but not sympathetic innervation of the DRG tend to coincide.     

Number and diameter distribution of myelinated afferent fibers innervating the paws of the cat and monkey.

The number and the diameter distribution of the myelinated cutaneous fibers innervating fore and hind paw were histologically examined in the cat and monkey. In five cats, the superficial peroneal nerve innervating the dorsal surface of the hind paw and the superficial plantar nerve innervating the palmar surface were composed of 2668–2950 fibers, 40–44% of which were group II fibers. On the other hand, the superficial radial nerve innervating the dorsal surface of the fore paw and the sensory branch of median nerve innervating the palmar surface were composed of 3270–3680 fibers, of which 67–72% were group II fibers. Therefore, it was found that the cutaneous fore paw nerves contained more group II fibers than the hind paw cutaneous nerves. In five monkeys the percentages of group II fibers composing the superficial peroneal nerve and superficial plantar nerve (2893–3374 fibers) were the same as those of the hind paw nerves in the cat. On the other hand, the percentages of group II fibers of the median nerve (4173–4472 fibers) were 75–78%, about 10% larger than those of the superficial radial nerve (3596–3821 fibers) whose values were 66–69%. Therefore, in the monkey, the forepaw nerves innervating the palmar surface also contain more group II afferent fibers compared to the ones innervating the dorsal one (hairy skin).     

Cutaneous nerve terminal degeneration in painful mononeuropathy

Nociceptive nerves innervate the skin and play an important role in the generation of neuropathic pain. However, it remains elusive whether and how nociceptive nerve terminals degenerate in neuropathic pain conditions. To address this issue, we investigated cutaneous innervation in a model of painful mononeuropathy, the chronic constriction injury (CCI). The hind paws of rats were immunocytochemically stained with a pan-axonal marker, protein gene product 9.5 (PGP 9.5). Within 2 days after CCI, rats exhibited thermal hyperalgesia, and there was a partial depletion of epidermal nerves. The extent of reduction in epidermal nerves after CCI was variable with an epidermal nerve density of 3.65 +/- 1.97 fibers/mm (compared to 15.39 +/- 1.58 fibers/mm on the control side, P < 0.02). There was a mild but concomitant increase in PGP 9.5 (+) Langerhans cells in the epidermis of the skin with CCI (10.19 +/- 1.99 vs 7.75 +/- 1.36 cells/mm, P < 0.05). In the skin denervated by tight ligation of the sciatic nerve, epidermal nerves were completely depleted (0 fibers/mm vs. 12.26 +/- 1.44 fibers/mm on the control side, P < 0.001). Animals with tight ligation of the sciatic nerve exhibited thermal anesthesia. These findings suggest that the epidermis is partially denervated in CCI, and that a partial injury of nerves is correlated with the development of neuropathic pain.     

Involvement of the peripheral sensory and sympathetic nervous system in the vascular endothelial expression of ICAM-1 and the recruitment of opioid-containing immune cells to inhibit inflammatory pain

Endogenous opioids are known to be released within certain brain areas following stressful stimuli. Recently, it was shown that also leukocytes are a potential source of endogenously released opioid peptides following stress. They activate sensory neuron opioid receptors and result in the inhibition of local inflammatory pain. An important prerequisite for the recruitment of such leukocytes is the expression of intracellular adhesion molecule-1 (ICAM-1) in blood vessels of inflamed tissue. Here, we investigated the contribution of peripheral sensory and/or sympathetic nerves to the enhanced expression of ICAM-1 simultaneously with the increased recruitment of opioid peptide-containing leukocytes to promote the inhibition of inflammatory pain. Selective degeneration of either peripheral sensory or sympathetic nerve fibers by their respective neurotoxins, capsaicin or 6-hydroxydopamime, significantly reduced the subcutaneous immigration of β-endorphin- (END-) and met-enkephalin- (ENK-)-containing polymorphonuclear leukocytes (PMN) (in the early phase) and mononuclear cells (in the late phase) during painful Freund’s complete adjuvant (FCA) rat hind paw inflammation. In contrast, this treatment did not alter the percentage of opioid peptide-containing leukocytes in the circulation. Calcitonin gene-related peptide- (CGRP-) and tyrosine hydroxylase- (TH-) immunoreactive (IR) nerve fibers were in close contact to ICAM-1 IR blood vessels within inflamed subcutaneous tissue. The selective degeneration of sensory or sympathetic nerve fibers attenuated the enhanced expression of vascular endothelial ICAM-1 after intraplantar (i.pl.) FCA and abolished endogenous opioid peptide-mediated peripheral analgesia. Our results suggest that, during localized inflammatory pain, peripheral sensory and sympathetic nerve fibers augment the expression of vascular endothelial ICAM-1 simultaneously with the increased recruitment of opioid peptide-containing leukocytes which consequently promotes the endogenous opioid peptide-mediated inhibition of inflammatory pain. They support existing evidence about a close link between the nervous and the immune system.     

Characterization of chronic constriction of the saphenous nerve, a model of neuropathic pain in mice showing rapid molecular and electrophysiological changes

Neuropathic pain is one of the most inextricable problems encountered in clinics, because few facts are known about its etiology. Nerve injury often leads to allodynia and hyperalgesia, which are symptoms of neuropathic pain. The aim of this study was to understand some molecular and electrophysiological mechanisms of neuropathic pain after chronic constriction of the saphenous nerve (CCS) in mice. After surgery, CCS mice displayed significant allodynia and hyperalgesia, which were sensitive to acute systemic injection of morphine (4 mg/kg), gabapentin (50 mg/kg), amitriptyline (10 mg/kg), and the cannabinoid agonist WIN 55,212-2 (5 mg/kg). These behavioral changes were accompanied after surgery by an increase of c-Fos expression and by an overexpression of mu-opioid and cannabinoid CB1 and CB2 receptors in the spinal cord and the dorsal hind paw skin. In combination with the skin-nerve preparation, this model showed a decrease in functional receptive fields downstream to the injury and the apparition of A-fiber ectopic discharges. In conclusion, CCS injury induced behavioral, molecular, and electrophysiological rearrangements that might help us in better understanding the peripheral mechanisms of neuropathic pain. This model takes advantage of the possible use in the future of genetically modified mice and of an exclusively sensory nerve for a comprehensive study of peripheral mechanisms of neuropathic pain.     

Regional differences in epidermal thickness and behavioral response following partial denervation of the rat paw

In order to investigate the relation between changes in epidermal thickness and pain-related behavior in different rat hind paw areas we performed partial denervation using two experimental models of neuropathic pain injury: spinal nerve ligation (SNL) and chronic constriction injury (CCI). Denervation produced significant epidermal thinning in both models. The most pronounced thinning was observed in the lateral paw region of the SNL group. Both models showed increase in hyperalgesic response during the postoperative period and no behavioral differences between testing regions. Our study showed that partial denervation induces regional differences in epidermal thinning but not in pain-related behavior.     

Chronic constriction injury of sciatic nerve induces the up-regulation of descending inhibitory noradrenergic innervation to the lumbar dorsal horn of mice

Peripheral nerve injury in rodents results in hypersensitivity to mechanical and thermal stimuli accompanied by reduced antinociceptive efficacy of opioids and, in some models, sensitivity to sympathetic blockade. 2-Adrenergic receptor agonists increase in potency and efficacy after nerve injury in rodents and effectively relieve neuropathic pain in humans who do not get pain relief from opioids. However, the underlying mechanisms are unclear. It has been well known that the major noradrenergic innervation of the spinal dorsal horn originates from the locus coeruleus nucleus (LC) in the brainstem. Therefore, the aim of this study is to examine whether peripheral nerve injury that causes neuropathic pain modulates the noradrenergic innervation to the lumbar dorsal horn, in order to determine the possible anatomical substrates underlying increased potency and efficacy of noradrenergic receptor agonists in alleviating neuropathic pain. At 2 weeks after chronic constriction injury (CCI) of the sciatic nerve, a remarkable increase in tyrosine-hydroxylase (TH) and dopamine β-hydroxylase (DβH) immunoreactive (IR) axonal terminals was observed in the ipsilateral L4–L6 dorsal horn. Consistently, greater numbers of both TH- and DβH-IR neurons were detected in the ipsilateral LC. Interestingly, in the lower lumbar and upper sacral spinal dorsal horn, numerous TH-IR neurons were observed in the superficial dorsal horn (primarily lamina I). CCI of the sciatic nerve did not change the number of these TH-IR cells. These findings suggest that augmented descending inhibitory noradrenergic innervation to the dorsal horn could be one of the mechanisms underlying the increased effectiveness in the anti-allodynic effect elicited by 2-adrenergic receptor agonists.