Trophic influence of the distal nerve segment on GABAA receptor expression in axotomized adult sensory neurons

The depolarizing action of gamma-aminobutyric acid (GABA), or the GABAA receptor agonist muscimol, on rat dorsal root (L4 and L5) fibers is attenuated following transection, but not crush, of the sciatic nerve. Following discrete nerve crush, axons actively regenerate and contact both the distal nerve segment and the peripheral target tissues. The aim of the present study was to distinguish between these two regions as possible sources of trophic support for retrograde maintenance of dorsal root GABA receptor sensitivity. A surgical procedure was employed to permit a delimited segment of axonal regeneration while prohibiting reestablishment of end organ innervation; the sciatic nerve was crushed and a ligature was placed 3 cm distal to the crush site. Under these conditions, the injury-induced decrement in the dorsal root GABA response, observed between 12 and 21 postoperative days, was significantly attenuated relative to that of ligated nerves, in which regeneration into the distal stump does not occur. The data suggest that nerve transection by ligation restricts trophic support for maintenance of GABA receptor expression in dorsal root ganglion (DRG) neurons. Furthermore, during regeneration the denervated distal nerve segment assumes a neurotrophic role in the maintenance of dorsal root GABA sensitivity, consistent with the hypothesis that growth factors derived from reactive Schwann cells may positively regulate the expression of receptors on axotomized sensory neurons.     

Diminished dorsal root GABA sensitivity following chronic peripheral nerve injury

The depolarizing effect of gamma-aminobutyric acid (GABA) on rat lumbar dorsal roots was studied in a sucrose gap chamber following axotomy or crush injury of the sciatic nerve or dorsal root. The mean depolarization elicited by GABA on normal dorsal roots (3.96 +/- 0.71 mV, N = 14) was significantly reduced following chronic sciatic axotomy (2.02 +/- 0.99 mV, N = 15). Chronic sciatic crush injury had no significant effect on dorsal root GABA sensitivity. The amplitudes of the dorsal root compound action potentials were the same from rats with normal and injured sciatic nerves, indicating that axons proximal to the sciatic nerve lesion did not undergo appreciable degeneration. A marked loss of dorsal root GABA sensitivity was also seen following dorsal root axotomy or crush injury (1.02 +/- 0.98 mV (N = 10) and 0.69 +/- 0.70 mV (N = 9), respectively). These results indicate that GABA sensitivity of dorsal roots is attenuated following peripheral nerve lesions in which regeneration and functional reconnection with peripheral targets are prevented. Previous work indicates that the primary afferent depolarization is reduced under similar conditions. The reduction in GABA sensitivity of dorsal root fibers described here may have a contributory role in the reduced primary afferent depolarization that follows peripheral nerve transection, which has pathophysiologic implications in chronic pain syndromes.     

Timing of c-jun protein induction in lumbar dorsal root ganglia after sciatic nerve transection varies with lesion distance

In situ hybridization and immunohistochemical studies have shown that sciatic nerve crush or transection induces upregulation of the immediate early gene c-jun mRNA and protein in lumbar dorsal root ganglion neurons. Here we have used enhanced chemiluminescent (ECL) immunoblotting as a sensitive and quantitative way of measuring the time course of c-jun protein induction following sciatic nerve transection at two distances from the L4 and L5 dorsal root ganglia. c-Jun protein was first detected within 3 h of proximal sciatic nerve transection and within 6 h of distal nerve transection. These results indicate substantially earlier increases in c-jun protein after nerve injury than previously reported, which can be attributed to the sensitivity of this detection method. The earlier induction of c-jun after proximal as compared to distal nerve transection supports the hypothesis that the c-jun response to sciatic nerve injury involves a distance-dependent signalling mechanism. © 1997 Elsevier Science B.V. All rights reserved.     

Prior collateral sprouting enhances elongation rate of sensory axons regenerating through acellular distal segment of a crushed peripheral nerve

Regenerating axons in crushed peripheral nerves grow through their distal nerve segments even in the absence of Schwann cell support, but their elongation rate is reduced by 30%. We examined whether prior exposure of sensory neurons to trophic factors achieved either by collateral sprouting or regeneration after conditioning lesion could enhance subsequent regeneration of their axons after crush, and compensate for loss of cell support. Collateral sprouting of the peroneal cutaneous sensory axons in the rat was evoked by transection of adjacent peripheral nerves in the hind leg. The segment of the peroneal nerve distal to the crush was made acellular by repeated freezing. Sensory axon elongation rate during regeneration was measured by the nerve pinch test. Prior axonal sprouting for two weeks increased the elongation rate of sensory axons through the acellular distal nerve segment back to normal value observed in control crushed nerves. The number of axons in the acellular distal segment at a fixed distance from the crush site was about 50% greater in sprouting than in control non-sprouting nerves. However, prior sprouting caused no further increase of axon elongation rate in control crushed nerves. Prior collateral sprouting, therefore, could in some respect compensate for loss of cell support in the distal nerve segment after crush lesion. This suggests that loss of cell-produced trophic factors is probably responsible for slower elongation rate through the acellular distal nerve segment. Surprisingly, prior conditioning lesion caused no enhancement of elongation rate of the sensory axons regenerating in the absence of cell support.     

Insulin as an in vivo growth factor

Insulin peptide has been identified to promote regeneration of axons in culture and in some in vivo model systems. Such actions have been linked to direct actions of insulin, or to cross occupation of closely linked IGF-1 receptors. In this work, we examined insulin support of peripheral nerve regenerative events in mice. Systemic insulin administration accelerated the reinnervation of foot interosseous endplates by motor axons after sciatic nerve transection and enhanced recovery of functional mouse hindpaw function. Similarly, insulin accelerated the regeneration-related maturation of myelinated fibers regrowing beyond a sciatic nerve crush injury. That such benefits might occur through direct signaling on axons was supported by immunohistochemical studies of expression with an antibody directed to the beta insulin receptor (IR) subunit. The proportion of sensory neurons expressing IRbeta increased ipsilateral to a similar sciatic crush injury in the L4 and L5 dorsal root ganglia. Insulin receptors, although widely expressed in axons, were also preferentially and intensely expressed on axons regrowing just beyond a peripheral nerve crush injury zone. The findings indicate that insulin imparts a substantial impact on regenerating peripheral nerve axons through upregulation of its expression following injury. Although the findings do not exclude insulin coactivating IGF-1 receptors during regeneration, its own receptors are present and available for action on injured nerves.     

Intrinsic versus extrinsic factors in determining the regeneration of the central processes of rat dorsal root ganglion neurons: The influence of a peripheral nerve graft

The relative contribution of intrinsic growth capacity versus extrinsic growth-promoting factors in determining the capacity of transected dorsal root axons to regenerate long distances was studied. L4 dorsal root axons regenerating into 4-cm peripheral nerve grafts on transected dorsal roots were counted. Few dorsal root myelinated axons regenerated to the distal end of the grafts by 10 weeks unless the sciatic nerve was also crushed. Regeneration of unmyelinated axons was also increased by peripheral lesions. Crush or transection of the dorsal roots without grafting did not alter GAP-43 mRNA expression in L4 dorsal root ganglion (DRG) cells. Grafting a peripheral nerve onto the cut end of an L4 dorsal root doubled the number of DRG cells expressing high levels of GAP-43 mRNA after a delay of several weeks. Peripheral nerve crush at the time of nerve grafting resulted in a very rapid rise in GAP-43 mRNA expression, which then declined to a steady level, twice that of controls, by 7 weeks. Thus, the rapid increase in the number of DRG neurons expressing high levels of GAP-43 mRNA after peripheral but not central axotomy correlates with the regeneration of central axons through nerve grafts. Because GAP-43 mRNA is slowly upregulated in a subpopulation of sensory neurons in response to exposure of their central axons to a peripheral nerve environment, environments favourable for axonal growth may act by increasing the intrinsic growth response of neurons. Lack of intrinsic growth capacity may contribute to the failure of dorsal root axons to regenerate into the spinal cord. © 1996 Wiley-Liss, Inc.     

Regeneration of primary afferent neurons containing substance P-like immunoreactivity

We compared changes in levels of substance P-like immunoreactivity (SPLI) in L4-6 dorsal root ganglia (DRG), L4-6 dorsal roots, sciatic nerve, tibial nerve and hind foot skin in rats following resection or crush injury of the sciatic nerve. The initial depletion of SPLI, which occurred in all areas sampled, was similar after either type of lesion. In DRG and dorsal roots, recovery to control values occurred in SPLI levels 35-45 days after sciatic crush, but not after resection. In sciatic nerve proximal to the injury, a partial recovery in SPLI content to about 60% of control occurred following crush injury, but not following resection. Distal to the injury, tibial nerve levels recovered rapidly following crush injury, consistent with the previously observed rapid regeneration of SPLI-containing axons. After resection, no recovery was observed until after 35 days, when it appeared that some axons succeeded in crossing the resection zone and regaining the distal nerve stump. Delayed and poor recovery of SPLI levels was observed in foot skin, even after crush injury. This correlated with the poor recovery of the plasma extravasation reaction, a functional index of SP-innervation of skin. In contrast, reinnervation by high-threshold mechanoreceptors was more rapid and complete, in agreement with a previous study. We conclude that although SPLI-containing axons regenerate rapidly, they appear to reinnervate skin less successfully than other afferents. Axon regeneration is associated with a recovery of SPLI levels which fell after axotomy: no recovery occurs if regeneration is prevented. Recovery was almost complete in DRG and roots, but incomplete in sciatic nerve. This peptide transmitter in afferent neurons thus behaves in a similar fashion to previously studied low-molecular weight transmitters and related materials in efferent neurons. Since recovery of SPLI levels begins before there is evidence for target reinnervation, it seems that axon regeneration is a sufficient condition for reversal of some axotomy-induced changes in these neurons. Further studies on substance P synthesis and on the response of individual DRG neurons to axotomy and regeneration will be required to explain fully the discrepancy between partial recovery of SPLI levels in sciatic nerve and full recovery in DRG and dorsal roots.     

Peripheral nerve regeneration is delayed in neuropilin 2-deficient mice

Peripheral nerve transection or crush induces expression of class 3 semaphorins by epineurial and perineurial cells at the injury site and of the neuropilins neuropilin-1 and neuropilin-2 by Schwann and perineurial cells in the nerve segment distal to the injury. Neuropilin-dependent class 3 semaphorin signaling guides axons during neural development, but the significance of this signaling system for regeneration of adult peripheral nerves is not known. To test the hypothesis that neuropilin-2 facilitates peripheral-nerve axonal regeneration, we crushed sciatic nerves of adult neuropilin-2-deficient and littermate control mice. Axonal regeneration through the crush site and into the distal nerve segment, repression by the regenerating axons of Schwann cell p75 neurotrophin receptor expression, remyelination of the regenerating axons, and recovery of normal gait were all significantly slower in the neuropilin-2-deficient mice than in the control mice. Thus, neuropilin-2 facilitates peripheral-nerve axonal regeneration.     

Amino acid incorporation into rat spinal cord and brain after simultaneous and interval sciatic nerve lesions

The incorporation of [³H]lysine into the trichloracetic acid (TCA) precipitable protein and soluble fractions of brain and spinal cord of rats subjected to immediate transection distal and crush proximal or transection distal, two week interval, and crush proximal of the left sciatic nerve has been studied at 5, 11, and 18 days postoperative. In addition, the regeneration of the nerve fibers was measured. A greater rate of (P < 0.05) nerve regeneration was noted 11 and 18 days after interval sciatic nerve lesion compared to the simultaneous lesion. In the amino acid uptake studies, the overall trend of [³H]lysine into protein in brain and spinal cord showed a peak uptake occurring at 11 days for both simultaneous (S) and interval sciatic lesioned animals, although the effect was most pronounced after interval sciatic lesion. The 11-day interval lesioned group showed the highest uptake of amino acid precursor into protein of all groups averaged over all samples. After interval sciatic lesions, the uptake of amino acid into protein showed the relationship, 11 day group > 18 day group > 5 day group. However, no significant differences in uptake into protein in spinal cord and brain were seen between the simultaneous lesion sciatic animals over postoperative days.In brain, differences between the [³H]lysine incorporation into protein of the left (L) and right (R) cortical hemispheres was noted. The incorporation into somatomotor cortex showed a L > R difference which was most pronounced after simultaneous sciatic nerve lesion. Interestingly, the largest R > L difference was in occipital cortex after interval lesions of sciatic nerve.In spinal cord, the protein radioactivity of the L5–6 and S1–4 segments which include most of the affected neurons was greater on the right (unlesioned) side than on the left (sciatic crush) side over all lesioned animals. This effect was most pronounced after interval sciatic lesion. The alteration of central nervous system incorporation of lysine after peripheral nerve (sciatic) lesion and regeneration are discussed.     

Axonal regeneration in dorsal spinal roots is accelerated by peripheral axonal transection

Regeneration of crushed axons in rat dorsal spinal roots was measured to investigate the transganglionic influence of an additional peripheral axonal injury. The right sciatic nerve was cut at the hip and the left sciatic nerve was left intact. One week later, both fifth lumbar dorsal roots were crushed and subsequently, regeneration in the two roots was assessed with one of two anatomical techniques. By anterograde tracing with horseradish peroxidase, the maximal rate of axonal regrowth towards the spinal cord was estimated to be 1.0 mm/day on the left and 3.1 mm/day on the right. Eighteen days after crush injury, new, thinly myelinated fibers in the root between crush site and spinal cord were 5-10 times more abundant ipsilateral to the sciatic nerve transection. The central axons of primary sensory neurons regenerate more quickly if the corresponding peripheral axons are also injured.     

A deficiency of axonal regeneration in C57BL/6J mice

Axonal regeneration within peripheral nerves and dorsal spinal roots was investigated in inbred strains of mice with known differences in macrophage recruitment and inflammatory functions. During the second week after sciatic nerve crush, counts of regenerating newly myelinated fibres were significantly lower in C57BL/6J mice than in 4 other strains. After dorsal root crush with or without concomitant sciatic nerve transection to enhance regeneration, fibre counts in roots of C57BL/6J were one-fifth of those in A/J mice. Axonal regeneration is subnormal in C57BL/6J mice but this defect appears not to be linked to known deficiencies in macrophage function.     

Differential Regulation of mRNAs for GDNF and its Receptors Ret and GDNFRα After Sciatic Nerve Lesion in the Mouse

Glial cell line-derived neurotrophic factor (GDNF), first characterized for its effect on dopamine uptake in central dopaminergic neurons, appears to be a powerful neurotrophic factor for motor neurons. GDNF has recently been shown to signal through a multisubunit receptor. This receptor is composed of a ligand-binding subunit, called GDNF receptor α (GDNFRα), and a signalling tyrosine kinase subunit, Ret. To gain further insight into GDNF function, we investigated the expression of GDNF and its receptors after nerve lesion in adult mice. Analysis of expression in muscle, nerve and spinal cord by RNase protection assay and in situ hydridization revealed that, in adult non-lesioned mice, GDNF mRNA was expressed in the nerve and GDNFRα mRNA in the nerve and the spinal cord, while the expression of Ret was restricted to spinal cord motor neurons. After a sciatic nerve crush a rapid increase in GDNF mRNA was observed in the distal part of the nerve and a delayed elevation in the muscle, while GDNFRα mRNA was up-regulated in the distal part of the sciatic nerve but not in proximal nerve or spinal cord. The lesion also induced a rapid increase in Ret mRNA expression, but the increase was observed only in spinal cord motor neurons and in dorsal root ganglion neurons. A pattern of expression of GDNF and its receptors similar to that seen after lesion in the adult was detected during embryonic development. Administration of GDNF enhanced sciatic nerve regeneration measured by the nerve pinch test. Taken together, these results suggest that GDNF has an important role during regeneration after nerve damage in the adult.     

GAP-43 mRNA in Rat Spinal Cord and Dorsal Root Ganglia Neurons: Developmental Changes and Re-expression Following Peripheral Nerve Injury

The expression of growth-associated protein GAP-43 mRNA in spinal cord and dorsal root ganglion (DRG) neurons has been studied using an enzyme linked in situ hybridization technique in neonatal and adult rats. High levels of GAP-43 mRNA are present at birth in the majority of spinal cord neurons and in all dorsal root ganglion cells. This persists until postnatal day 7 and then declines progressively to near adult levels (with low levels of mRNA in spinal cord motor neurons and 2000–3000 DRG cells expressing high levels) at postnatal day 21. A re-expression of GAP-43 mRNA in adult rats is apparent, both in sciatic motor neurons and the majority of L4 and L5 dorsal root ganglion cells, 1 day after sciatic nerve section. High levels of the GAP-43 mRNA in the axotomized spinal motor neurons persist for at least 2 weeks but decline 5 weeks after sciatic nerve section, with the mRNA virtually undetectable after 10 weeks. The initial changes after sciatic nerve crush are similar, but by 5 weeks GAP-43 mRNA in the sciatic motor neurons has declined to control levels. In DRG cells, after both sciatic nerve section or crush, GAP-43 mRNA re-expression persists much longer than in motor neurons. There was no re-expression of GAP-43 mRNA in the dorsal horn of the spinal cord after peripheral nerve lesions. Our study demonstrates a similar developmental regulation in spinal cord and DRG neurons of GAP-43 mRNA. We show moreover that failure of re-innervation does not result in a maintenance of GAP-43 mRNA in axotomized motor neurons.     

Lack of effects of transforming growth factor-alpha gene knockout on peripheral nerve regeneration may result from compensatory mechanisms.

Transforming growth factor-alpha (TGF-alpha), previously identified as a major member of the epidermal growth factor (EGF) family of growth factors, plays a role in proliferation, differentiation, and survival of neuronal and glial precursors and is implicated in development of the nervous system. However, its roles in nerve injury-induced responses remain obscure. The current study examined roles of endogenous TGF-alpha in peripheral nerve regeneration using sciatic nerve injury models with TGF-alpha knockout mice. Three weeks after a sciatic nerve crush, no significant differences were found between TGF-alpha wild-type and mutant mice in the number of retrogradely labeled L5 dorsal root ganglion (DRG) sensory neurons and L5 spinal cord motor neurons and in the morphology of myelinated regenerating nerve fibers, indicating that TGF-alpha is not essential for sensory and motor nerve regeneration. To assess a possible functional redundancy among TGF-alpha-related ligands in response to a nerve injury, mRNA expression of the EGF family was analyzed by RT-PCR in L4/L5 DRG pools and distal degenerating sciatic nerve segments after sciatic nerve ligation. Prior to and 1 day after ligation, there was a higher level of EGF-R mRNA in DRGs and in nerve in TGF-alpha null mice compared to wild types, and there was an induction of ligand amphiregulin mRNA in DRGs in mutant mice in place of the TGF-alpha upregulation present in wild types. These results indicate that TGF-alpha gene knockout does not affect peripheral nerve regeneration, probably due to a functional redundancy within the EGF family through a compensatory expression mechanism at both the receptor and ligand levels in TGF-alpha knockout mice.     

Pulsed electrical stimulation protects neurons in the dorsal root and anterior horn of the spinal cord after peripheral nerve injury

Most studies on peripheral nerve injury have focused on repair at the site of injury, but very few have examined the effects of repair strategies on the more proximal neuronal cell bodies. In this study, an approximately 10-mm-long nerve segment from the ischial tuberosity in the rat was transected and its proximal and distal ends were inverted and sutured. The spinal cord was subjected to pulsed electrical stimulation at T10 and L3, at a current of 6.5 m A and a stimulation frequency of 15 Hz, 15 minutes per session, twice a day for 56 days. After pulsed electrical stimulation, the number of neurons in the dorsal root ganglion and anterior horn was increased in rats with sciatic nerve injury. The number of myelinated nerve fibers was increased in the sciatic nerve. The ultrastructure of neurons in the dorsal root ganglion and spinal cord was noticeably improved. Conduction velocity of the sciatic nerve was also increased. These results show that pulsed electrical stimulation protects sensory neurons in the dorsal root ganglia as well as motor neurons in the anterior horn of the spinal cord after peripheral nerve injury, and that it promotes the regeneration of peripheral nerve fibers.     

Light and electron microscopic localization of B-50 (GAP43) in the rat spinal cord during transganglionic degenerative atrophy and regeneration.

Crush or transection of a peripheral nerve is known to induce transganglionic degenerative atrophy (TDA) in the segmentally related, ipsilateral Rolando substance of the spinal cord. When the lost peripheral connectivity is reestablished, the consecutive regenerative synaptoneogenesis results in restoration of the circuitry in the formerly deteriorated upper dorsal horn. Enhanced expression of the growth-associated protein (GAP43) B-50 occurs during neuronal differentiation, axon outgrowth, and peripheral nerve regeneration. This study documents changes in immunocytochemical distribution of B-50 in the regions of the lumbar spinal cord which are segmentally related to the axotomized sciatic nerve. At the light microscopic level, a weak B-50 immunoreactivity (BIR) is present in the neuropil of the upper dorsal horn of control animals. After unilateral transection and ligation of the sciatic nerve, BIR increased in the ipsilateral upper dorsal horn at 17 days postinjury, but decreased again after 24 days with respect to the contralateral side. Differences between effects of crush and transection were prominent in combined crush-cut experiments as well (i.e., after unilateral crush and contralateral transection and ligation of the sciatic nerve). Electron microscopic studies show that in the uninjured and injured spinal cord, BIR is detected in axons and axon terminals, but not all are stained. After transection of the sciatic nerve, BIR is found in afflicted primary sensory axon terminals, including those contacting substantia gelatinosa neurons and in axon terminals undergoing glial phagocytosis. The localization of BIR seen after crushing the sciatic nerve is similar. However, at 24 days after crush, BIR is detected also in axonal growth cones. In the ventral horn of control animals, synaptic boutons impinging upon motor neurons exhibited weak BIR. At 17 days after unilateral transection of the sciatic nerve, the pericellular BIR surrounding motor neurons is decreased at the ipsilateral with respect to the contralateral side, whereas 24 days after crush injury it increased considerably. Our results show that peripheral nerve injury inducing TDA also affects BIR distribution in the spinal gray matter. Successful regeneration of the peripheral nerve after crush lesion is associated with enhanced expression of B-50 in growth cones of sprouting central axons. The neuroplastic response of B-50 is in line with a function of B-50 in axonal sprouting and reactive synaptogenesis.     

Blockade of transient receptor potential cation channel subfamily V member 1 promotes regeneration after sciatic nerve injury

The transient receptor potential cation channel subfamily V member 1(TRPV1) provides the sensation of pain(nociception). However, it remains unknown whether TRPV1 is activated after peripheral nerve injury, or whether activation of TRPV1 affects neural regeneration. In the present study, we established rat models of unilateral sciatic nerve crush injury, with or without pretreatment with AMG517(300 mg/kg), a TRPV1 antagonist, injected subcutaneously into the ipsilateral paw 60 minutes before injury. At 1 and 2 weeks after injury, we performed immunofluorescence staining of the sciatic nerve at the center of injury, at 0.3 cm proximal and distal to the injury site, and in the dorsal root ganglia. Our results showed that Wallerian degeneration occurred distal to the injury site, and neurite outgrowth and Schwann cell regeneration occurred proximal to the injury. The number of regenerating myelinated and unmyelinated nerve clusters was greater in the AMG517-pretreated rats than in the vehicle-treated group, most notably 2 weeks after injury. TRPV1 expression in the injured sciatic nerve and ipsilateral dorsal root ganglia was markedly greater than on the contralateral side. Pretreatment with AMG517 blocked this effect. These data indicate that TRPV1 is activated or overexpressed after sciatic nerve crush injury, and that blockade of TRPV1 may accelerate regeneration of the injured sciatic nerve.