Mechanisms of enhancement of neurite regeneration in vitro following a conditioning sciatic nerve lesion

To examine the mechanisms responsible for the more rapid nerve regeneration observed after a previous (conditioning) nerve injury, adult rats were subjected to a midthigh sciatic nerve transection by using one of three protocols designed to facilitate or restrict nerve regeneration: 1) ligation, in which transected axons were prevented from regenerating; 2) cut, in which transected axons were permitted to extend into peripheral target tissue but were separated from the denervated peripheral nerve stump; and 3) crush, in which axons could regenerate normally through the denervated distal nerve tract. The affected dorsal root ganglia (DRG) were subsequently removed, dissociated, and cultured for up to 3 days, and the timing of neurite initiation, rate of outgrowth, and arborization pattern of previously injured neurons were compared with control DRG. Our results indicate that conditioning lesions have at least four distinct and differentially regulated effects on neuronal morphogenesis: 1) conditioning lesions promote earlier neurite initiation, 2) prior nerve injury decreases the ability of neurons to extend long neurites following a second axotomy, 3) exposure to the environment of a denervated peripheral nerve stimulates greater initial rates of neurite outgrowth, and 4) conditioning lesions reduces initial neuritic branching frequency, resulting in straighter neurites whose growth cones extend further distances from their cell bodies. The primary effect of all conditioning lesions on cultured DRG neurons appeared to be to advance the timing of morphogenesis, resulting in conditioning-lesioned neurons that exhibited characteristics consistent with control neurons that had been cultured for an additional day or more. A secondary effect of conditioning lesions on neurite outgrowth rates was dependent on the local environment of the axons prior to culturing. J. Comp. Neurol 391:11–29, 1998. © 1998 Wiley-Liss, Inc.     

Electromagnetic fields influence NGF activity and levels following sciatic nerve transection

Pulsed electromagnetic fields (PEMF) have been shown to increase the rate of nerve regeneration. Transient post‐transection loss of target‐derived nerve growth factor (NGF) is one mechanism proposed to signal induction of early nerve regenerative events. We tested the hypothesis that PEMF alter levels of NGF activity and protein in injured nerve and/or dorsal root ganglia (DRG) during the first stages of regeneration (6–72 hr). Rats with a transection injury to the midthigh portion of the sciatic nerve on one side were exposed to PEMF or sham control PEMF for 4 hr/day for different time periods. NGF‐like activity was determined in DRG, in 5‐mm nerve segments proximal and distal to the transection site and in a corresponding 5‐mm segment of the contralateral nonoperated nerve. NGF‐like activity of coded tissue samples was measured in a blinded fashion using the chick DRG sensory neuron bioassay. Overall, PEMF caused a significant decrease in NGF‐like activity in nerve tissue (P < 0.02, repeated measures analysis of variance, ANOVA) with decreases evident in proximal, distal, and contralateral nonoperated nerve. Unexpectedly, transection was also found to cause a significant (P = 0.001) 2‐fold increase in DRG NGF‐like activity between 6 and 24 hr postinjury in contralateral but not ipsilateral DRG. PEMF also reduced NGF‐like activity in DRG, although this decrease did not reach statistical significance. Assessment of the same nerve and DRG samples using ELISA and NGF‐specific antibodies confirmed an overall significant (P < 0.001) decrease in NGF levels in PEMF‐treated nerve tissue, while no decrease was detected in DRG or in nerve samples harvested from PEMF‐treated uninjured rats. These findings demonstrate that PEMF can affect growth factor activity and levels, and raise the possibility that PEMF might promote nerve regeneration by amplifying the early postinjury decline in NGF activity. J. Neurosci. Res. 55:230–237, 1999. Published 1999 Wiley‐Liss, Inc.     

Neuronal age influences the response to neurite outgrowth inhibitory activity in the central and peripheral nervous systems.

Axonal regeneration is abortive in the central nervous system (CNS) of adult mammals, but readily occurs in the injured peripheral nervous system (PNS). Recent experiments indicate an important role for both intrinsic neuronal features and extrinsic substrate properties in determining the propensity for axonal regrowth. In particular, certain components of adult mammalian CNS myelin have been shown to exert a strong inhibitory influence on neurite outgrowth. To determine whether the potent neurite outgrowth inhibitory activity found in CNS myelin may also be present in PNS myelin and to study the influence of neuronal age on neurite outgrowth, we used a cryoculture assay in which dissociated rat dorsal root ganglion (DRG) neurons of different ages were challenged to extend neurites on fractionated myelin and cryostat sections from the PNS (sciatic nerve and myelin-free degenerated sciatic nerve) and CNS (optic nerve) of adult rats. The CNS environment of the optic nerve did not support E17 to P8 DRG neurite adhesion or outgrowth. E17 DRG neurons, unlike their older counterparts, however, were able to attach and extend neurites onto normal sciatic nerve and onto purified PNS myelin. In contrast, a vigorous neurite outgrowth response from all the ages tested was observed on the myelin-free degenerated sciatic nerve. These results indicate that PNS myelin is a potent inhibitor of neurite outgrowth and that DRG neuronal age plays an important role in determining the propensity for neurite outgrowth and regenerative response on inhibitory PNS and CNS substrata.     

Immediate anti‐tumor necrosis factor‐α (etanercept) therapy enhances axonal regeneration after sciatic nerve crush

Peripheral nerve regeneration begins immediately after injury. Understanding the mechanisms by which early modulators of axonal degeneration regulate neurite outgrowth may affect the development of new strategies to promote nerve repair. Tumor necrosis factor‐α (TNF‐α) plays a crucial role in the initiation of degenerative cascades after peripheral nerve injury. Here we demonstrate using real‐time Taqman quantitative RT‐PCR that, during the time course (days 1–60) of sciatic nerve crush, TNF‐α mRNA expression is induced at 1 day and returned to baseline at 5 days after injury in nerve and the corresponding dorsal root ganglia (DRG). Immediate therapy with the TNF‐α antagonist etanercept (fusion protein of TNFRII and human IgG), administered systemically (i.p.) and locally (epineurially) after nerve crush injury, enhanced the rate of axonal regeneration, as determined by nerve pinch test and increased number of characteristic clusters of regenerating nerve fibers distal to nerve crush segments. These fibers were immunoreactive for growth associated protein‐43 (GAP‐43) and etanercept, detected by anti‐human IgG immunofluorescence. Increased GAP‐43 expression was found in the injured nerve and in the corresponding DRG and ventral spinal cord after systemic etanercept compared with vehicle treatments. This study established that immediate therapy with TNF‐α antagonist supports axonal regeneration after peripheral nerve injury. © 2009 Wiley‐Liss, Inc.     

Neurite-promoting factors and extracellular matrix components accumulating in vivo within nerve regeneration chambers

The outgrowth of neurites from cultured neurons can be induced by the extracellular matrix glycoproteins, fibronectin and laminin, and by polyornithine-binding neurite-promoting factors (NPFs) derived from culture media conditioned by Schwann, or other cultured cells. We have examined the occurrence of fibronectin, laminin and NPFs during peripheral nerve regeneration in vivo. A previously established model of peripheral nerve regeneration was used in which a transected rat sciatic nerve regenerates through a silicone chamber bridging a 10 mm interstump gap. The distribution of fibronectin and laminin during regeneration was assessed by indirect immunofluorescence. Seven days after nerve transection the regenerating structure within the chamber consisted primarily of a fibrous matrix which stained with anti-fibronectin but not anti-laminin. At 14 days, cellular outgrowths from the proximal and distal stumps (along which neurites grow) had entered the fibronectin-containing matrix, consistent with a role of fibronectin in promoting cell migration. Within these outgrowths non-vascular as well as vascular cell stained with anti-fibronectin and anti-laminin. Wihtin the degenerated distal nerve segment, cells characteristics of Bungner bands (rows of Schwann cells along which regenerating neurites extend) stained with anti-fibronectin and laminin. The fluid surrounding the regenerating nerve was found to contain NPF activity for cultured ciliary ganglia neurons which markedly increased during the period of neurite growth into the chamber. In previous studies using this particular neurite-promoting assay, laminin but to a much lesser extent fibronectin also promoted neurite outgrowth. Affinity-purified anti-laminin antibody failed to block chamber fluid NPF activity while completely blocking the neurite-promoting activity of laminin. These two results suggested that chamber fluid NPF activity did not consist of individual molecules of either fibronectin or laminin. The spatial and temporal distribution of insoluble fibronectin and laminin and the temporal correlation between chamber fluid NPF accumulation and neurite outgrowth support the possibility that these agents influence regenerative events including axonal elongation in vivo.     

Differential regulation of S100 beta and mRNAs coding for S100-like proteins (42A and 42C) during development and after lesion of rat sciatic nerve

The changes in the levels of S100 beta (a protein that stimulates neurite extension and neuronal survival) and 42A and 42C (S100-like proteins whose mRNAs are induced in PC12 cells by nerve growth factor) during development and after rat sciatic nerve lesions were analyzed. S100 beta, 42A, and 42C mRNAs showed differential regulation during development. S100 beta mRNA was present both in sciatic nerve and brain, and increased more than 11-fold during the first 3 wk of nerve postnatal development. 42A and 42C mRNAs were essentially restricted to sciatic nerve, with little found in either embryonic or adult brain. The levels of 42C and 42A mRNAs in sciatic nerve increased 4- and 14-fold, respectively, by postnatal day 23 compared to postnatal day 2. 42A, 42C, and S100 beta mRNAs also showed a differential regulation during sciatic nerve degeneration and regeneration. Axotomized and control sciatic nerves were examined by Northern blots at various times after a crush or cut injury. 42A and 42C mRNA levels increased rapidly in the distal segment of axotomized nerve, remained two- to five-fold higher than controls at day 14 after injury but returned to control levels by 40 days. In contrast, S100 beta mRNA showed a three-fold decrease in the axotomized nerve between days 1 and 3 after injury, and slowly returned towards control levels over the next few weeks. The decrease in S100 beta mRNA was reflected by a corresponding decrease in S100 beta protein levels. The induction of 42A and 42C mRNAs and repression of S100 beta mRNA remained if nerve regeneration was prevented.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Thrombospondin expression in nerve regeneration I. comparison of sciatic nerve crush, transection, and long-term denervation

Patterns of expression of the extracellular matrix molecule thrombospondin (TSP) were examined during peripheral nerve regeneration following sciatic nerve crush or transection. In noninjured nerve, was present in the axoplasm, Schwann cells, endoneurium, and perineurium of the adult mouse sciatic nerve. Following nerve crush or nerve transection, levels of TSP rapidly increased distal to the trauma site. Elevated levels of TSP were present distal to regenerating axons, while expression gradually returned to normal proximal to the regenerating axons. When reinnervation was blocked, TSP levels remained high in the endoneurium in excess of 30 days, but TSP was absent by 60 days. Following reanastomosis of the proximal and distal segments after 60 days of denervation, TSP was re-expressed in the distal nerve stump. These results indicate that TSP, which is involved in neuronal migrations in the embryo and neurite outgrowth in vitro, appears to play a role in axonal regeneration in the adult peripheral nervous system.     

Behaviour of DRG sensory neurites at the intact and injured adult rat dorsal root entry zone: postnatal neurites become paralysed, whilst injury improves the growth of embryonic neurites.

The dorsal root entry zone is a PNS-CNS junction between Schwann cells and astrocytes, defining the site where dorsal root ganglia (DRG) axons enter the adult mammalian spinal cord. Following dorsal root injury (rhizotomy), DRG axons regenerate within the PNS environment of the root but stop at the DREZ and fail to re-enter the spinal cord. We have used an in vitro model to compare how neurites growing from embryonic (E13) and postnatal (P0 and adult) DRG neurons behave at the uninjured and rhizotomized adult rat DREZ. We find that both freshly dissected and conditioned-lesioned postnatal DRG neurons seldom grow neurites across cryosections of the uninjured or rhizotomized DREZ. However, embryonic DRG neurons more readily grow neurites across cryosections of the uninjured and 7-day post-lesion (dpl) DREZ and are dramatically better able to cross the 21 dpl DREZ. This enhanced growth was abolished by co-incubation with a function-blocking antiserum to beta1-integrin receptors, whilst immunoreactivity for some beta1-integrin ligands (tenascin-C and fibronectin) increased at the DREZ by 21 dpl, suggesting that beta1-integrin ligands may stimulate the growth of embryonic neurites across the 21 dpl DREZ. Fluorescence time-lapse video-microscopy was used to record the behaviour of dye-labelled postnatal DRG neurites as they encounter the uninjured adult DREZ in vitro. Neurites rarely turned around at the DREZ, but instead became paralysed. Of a variety of chemical modifications to uninjured DREZ cryosections, only treatment with methanol, chloroform, or the protease inhibitor D-phe-pro-arg chloromethylketone hydrochloride (PPACK, 100 microM) caused any increase in the proportion of postnatal neurites which crossed the DREZ.     

Sciatic nerve regeneration across gaps within silicone chambers: long-term effects of NGF and consideration of axonal branching

We examined whether the short-term beneficial effects of nerve growth factor (NGF) upon regeneration are sustained over a prolonged period of time across 8-mm gaps within silicone chambers. Rat sciatic nerve regeneration both with and without NGF was examined after 10 weeks. Myelinated counts from the regenerated sciatic and distal tributary nerves were correlated to the numbers of motor and sensory neurons retrogradely labeled with horseradish peroxidase (HRP) applied distal to the regenerated segment. Regenerated sciatic and sural nerves were examined ultrastructurally for morphological analysis. Both regenerated groups by 10 weeks achieved essentially complete counts of myelinated axons in the distal tributary nerves and the regenerated segment of the sciatic nerve compared to the uninjured controls. There were similar numbers of retrogradely labeled sensory and motor neurons in the dorsal root ganglia (DRG) and lumbar spinal cord of both groups and, surprisingly, of the uninjured normal control group. Ultrastructural analysis demonstrated no difference in the distribution of axonal diameters or myelin thickness between the regenerated groups. In evaluating regeneration in experimental silicone chamber models, it is important to determine such parameters as the percentage of neurons that grow across the gap and the incidence of axonal sprouting. One can then make accurate assessments of experimental perturbations and predict whether they improve the naturally occurring regeneration through chambers. These results must ultimately be compared with equivalent determinations in the uninjured nerve. At 10 weeks there was essentially complete regeneration of both the NGF and control regenerative groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regeneration of axons from adult rat retinal ganglion cells on cultured Schwann cells is not dependent on basal lamina.

The ability of sciatic nerve grafts to support in vivo regeneration of retinal ganglion cell axons in the adult rat raises the question of which peripheral nerve constituents may be required to promote this unexpected central regenerative response. Prime candidates for this role include the surface of the Schwann cell and components of extracellular matrix present in peripheral nerve trunks. To determine the relative importance of Schwann cells and their basal lamina in promoting retinal ganglion cell axon regeneration in the mammalian visual system, we have used an in vitro model. This approach allowed analysis of the abilities of defined peripheral nerve constituents to promote in vitro outgrowth of neurites from explants of adult rat retina harvested 7 to 10 days after in vivo optic nerve crush. Neurite outgrowth was assessed by neurofilament immunofluorescence after 3 to 20 days in vitro. Culture substrata, consisting of isolated Schwann cells (SC), Schwann cells with their assembled extracellular matrix (SC + ECM), or isolated extracellular matrix from which the Schwann cells had been removed (ECM), were prepared by first co-culturing rat Schwann cells with embryonic dorsal root ganglion neurites on a layer of type I collagen, and then manipulating the cultures to produce the desired substrata. Type I collagen alone did not support neurite growth from adult rat retina. SC and SC + ECM supported regeneration of axons from retinal explants at average growth rates of 18 and 30 microns/h, respectively. Isolated ECM was a poor substrate for retinal neurite growth; the few neurites that gained access to this material grew at rates averaging less than 3 microns/h. These observations suggest that regeneration of adult mammalian retinal ganglion cell axons through peripheral nerve grafts (in vivo) is primarily dependent on neurite-promoting factors present on the surface of Schwann cells and does not require organized extracellular matrix.     

Differentiation of mesenchymal stem cells to support peripheral nerve regeneration in a rat model

Mesenchymal stem cells (MSCs) support axon regeneration across artificial nerve bridges but their differentiative capacity and ability to promote nerve regeneration remains unclear. In this study, MSCs isolated from bone marrow of Sprague–Dawley rats were characterized by plastic adherence and pluripotency towards mesodermal lineages. Isolated undifferentiated MSCs (uMSCs) were stimulated towards a Schwann cell (SC) phenotype using specific growth factors, and cell marker analysis was performed to verify SC phenotype in vitro. Differentiation resulted in temporally dependent positive immunocytochemical staining for the SC markers, glial fibrillary acidic protein (GFAP), S100, and nerve growth factor receptor (NGFR), with maximal marker expression achieved after 6 days of treatment with differentiation media. Quantitative analysis demonstrated that ~ 50% of differentiated MSCs (dMSCs) have a SC phenotype. Using an indirect co-culture system, we compared the ability of dorsal root ganglion (DRG) cells to extend neurites in indirect contact with uMSCs and dMSCs as compared to SCs. The mean values of the longest length of the DRG neurites were the same for the dMSCs and SCs and significantly higher than the uMSC and DRG mono-culture systems (p < 0.05). In vivo, compared to an empty conduit, dMSC seeded collagen nerve conduits resulted in a greater number of sciatic motoneurons regenerating axons through the conduit into the distal nerve stump. We conclude that bone marrow-derived MSCs differentiate into a SC-phenotype that expresses SC markers transiently and sufficiently to support limited neurite outgrowth in vitro and axonal regeneration equivalent to that of SCs in vitro and in vivo. The nerve autograft remains the most effective conduit for supporting regeneration across nerve gaps.     

Electrical stimulation of intact peripheral sensory axons in rats promotes outgrowth of their central projections

A lesion of a peripheral nerve before a second injury (conditioning lesion, CL), enhances peripheral and central regeneration of dorsal root ganglion (DRG) axons. This effect is mediated by elevated neuronal cAMP. Here we wanted to investigate whether electrical stimulation (ES) of an intact nerve, which has been shown to accelerate peripheral axon outgrowth, is also effective in promoting axon regeneration of injured DRG axons in vitro and of the central DRG axons in vivo and, whether this effect is mediated by elevation of cAMP. For the in vitro assay, the intact sciatic nerve of adult rats was stimulated at 20 Hz for 1 h, 7 days before harvest and primary culture of DRG neurons on a growth permissive substrate. In the in vivo study, the central axons of the lumbosacral DRGs were cut in the Th8 dorsal column, and the sciatic nerve was either cut or left intact, and subjected to 1 h ES at 20 Hz or 200 Hz. In vitro, ES increased neurite outgrowth 4-fold as compared to non-stimulated DRG neurons. In vivo, ES at 20 Hz significantly increased axon outgrowth into the central lesion site as compared to the Sham control. The 20 Hz ES was as effective as the CL in increasing axon outgrowth into the lesion site but not in promoting axonal elongation even though 20 Hz ES increased intracellular cAMP levels in DRG neurons as effectively as the CL. Thus elevation of cAMP may account for the central axonal outgrowth after ES and a CL.     

Cultured peripheral nervous system cells support peripheral nerve regeneration through tubes in the absence of distal nerve stump

Axons of a cut peripheral nerve will grow across a gap (less than or equal to 10 mm in adult rodents) formed when the proximal and distal stumps are placed at opposite ends of an impermeable, inert tube, but will not grow to the end of a blind-ended tube in the absence of the distal stump [Williams et al, 1984]. Work reported here demonstrates that cultured peripheral nervous system (PNS) cells suspended in a collagen matrix will provide an effective milieu that directs and supports axonal regeneration from a severed nerve into a blind-ended tube in the absence of a distal stump. Adult mouse sciatic nerves were cut and the proximal stumps were inserted into close-ended tubes that contained either a collagen matrix containing dissociated cells from embryonic mouse dorsal root ganglia (DRG), a collagen matrix saturated with medium conditioned by cultured DRG cells, or a collagen matrix saturated with fresh medium. In all three cases cellular cables formed that ran the full length of the tubes, but myelinated and unmyelinated axons regenerated the length of the tubes only when cultured cells had been added. The critical factor in influencing axonal regeneration through the length of the tubes was the presence of cultured cells, since collagen alone or collagen saturated with conditioned medium did not support axonal regrowth even though cells had migrated into the chambers from the proximal stumps in all cases. Ordered structure was not a requisite for axonal growth, since the cultures consisted of random arrays of dissociated cells.     

Spatiotemporal progress of nerve regeneration in a tendon autograft used for bridging a peripheral nerve defect

We have previously shown that a tendon autograft from the rat tail can support regeneration across a gap in the continuity of the rat sciatic nerve. In this study, we characterized the spatiotemporal progress of regeneration in such a graft bridging a 10-mm defect in the sciatic nerve of the rat. Regeneration was assessed 7, 10, 14, or 18 days postoperatively, by immunocytochemistry for axons, Schwann cells, and macrophages and histochemistry for blood vessels. Axonal regrowth into the grafts showed an initial delay period of 6.8 days, whereafter axons grew at a rate of 1.0 mm/day. Schwann cells grew into the grafts from both the proximal and distal nerve segments, proximally just ahead of the axonal front. Macrophages were initially preferentially located at the periphery of the grafts, but gradually increased inside the grafts. Blood vessels entered the grafts from both the proximal and distal aspects of the severed nerve. The onset of vascularization appeared to coincide with axonal regeneration into the grafts.     

Altered expression of pp60c-src induced by peripheral nerve injury.

The normal src protein (pp60c-src) is localized principally in the nerve growth cone of developing neurons and declines to low levels with synaptic maturation. To determine whether pp60c-src is reexpressed in regenerating axons, its expression was studied by immunoblotting and immunocytochemical analyses in adult chicken sciatic nerve following nerve crush injury. pp60c-src expression was found to increase during nerve repair with a temporal and spatial pattern consistent with a localization in regenerating axons. At the crush site, pp60c-src increased to maximal levels 7 days postinjury, increasing fivefold relative to 0 day nerve. In the nerve segment distal to the injury, the maximal increase in pp60c-src was sevenfold and occurred between 11 and 21 days postinjury. Immunoperoxidase staining revealed pp60c-src in regenerating axons and certain nonneuronal cells at the site of nerve repair. pp60c-src was induced in both motor and sensory neurons, as shown by increased pp60c-src immunoreactivity in their cell bodies located in the spinal cord and dorsal root ganglion. Phosphotyrosine-modified proteins that were potential targets of pp60c-src increased following nerve crush, and were localized to outgrowing neurites as well as to nonneuronal cells. These results suggest that pp60c-src is a common component of cellular mechanisms regulating growth cone migration in both regenerating and developing axons.     

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.     

Effect of lumbar 5 ventral root transection on pain behaviors: a novel rat model for neuropathic pain without axotomy of primary sensory neurons

A peripheral nerve injury often causes neuropathic pain but the underlying mechanisms remain obscure. Several established animal models of peripheral neuropathic pain have greatly advanced our understanding of the diverse mechanisms of neuropathic pain. A common feature of these models is primary sensory neuron injury and the commingle of intact axons with degenerating axons in the sciatic nerve. Here we investigated whether neuropathic pain could be induced without sensory neuron injury following exposure of their peripheral axons to the milieu of Wallerian degeneration. We developed a unilateral lumbar 5 ventral root transection (L5 VRT) model in adult rats, in which L5 ventral root fibers entering the sciatic nerve were sectioned in the spinal canal. This model differs from previous ones in that DRG neurons and their afferents are kept uninjured and intact afferents expose to products of degenerating efferent ventral root fibers in the sciatic nerve and the denervated muscles. We found that the L5 VRT produced rapid (24 h after transection), robust and prolonged (56 days) bilateral mechanical allodynia, to a similar extent to that in rats with L5 spinal nerve transection (L5 SNT), cold allodynia and short-term thermal hyperalgesia (14 days). Furthermore, L5 VRT led to significant inflammation as demonstrated by infiltration of ED-1-positive monocytes/macrophages in the DRG, sciatic nerve and muscle fibers. These findings demonstrated that L5 VRT produced behavioral signs of neuropathic pain with high mechanical sensitivity and thermal responsiveness, and suggested that neuropathic pain can be induced without damage to sensory neurons. We propose that neuropathic pain in this model may be mediated by primed intact sensory neurons, which run through the milieu of Wallerian degeneration and inflammation after nerve injury. The L5 VRT model manifests the complex regional pain syndrome in some human patients, and it may provide an additional dimension to dissect out the mechanisms underlying neuropathic pain.     

Morphometric analysis of the peripheral neuropathy of AIDS

A morphometric study of the peripheral nervous system at autopsy was undertaken in 11 AIDS patients and 10 controls. The left L4, L5, and S1 dorsal root ganglia (DRG) and samples of the sciatic nerve at the buttock, tibial nerve at the knee, and sural nerve at the ankle were collected. Indices of neuronal/axonal degeneration and of segmental demyelination/remyelination were measured at each level. The small number of cases and evidence of neuropathy in a number of the control cases resulted in statistical significance for only a limited number of comparisons. Nodules of Nageotte in the DRG were increased fivefold in AIDS cases compared with controls, and axonal degeneration in single-teased nerve fibers was increased 9-fold in the sciatic nerve, 28-fold in the tibial nerve, and 12-fold in the sural nerve. The ratios of AIDS to controls for the density of remaining DRG neurons and large myelinated axons were reduced to 0.71 in the DRG, 0.84 in the sciatic nerve, 0.84 in the tibial nerve, and 0.66 in the sural nerve. Axonal regeneration in single-teased nerve fibers was increased threefold at the sciatic nerve level in AIDS, but was markedly reduced at distal levels. Acute segmental demyelination in single-teased nerve fibers was present to a greater extent than in controls at all levels of the peripheral nerves in the AIDS cases. Remyelinating fibers were increased compared with controls only in the proximal sciatic nerve. No case showed the changes of cytomegalovirus infection. In a parallel immunohistochemical study of these AIDS peripheral nerves, T-cell and macrophage infiltration, with cytokine expression, was demonstrated. The pathological process in the neuropathy of terminal AIDS appears to be a multifocal immunologically mediated inflammatory disease, with increased density of macrophages and T cells at all levels of the peripheral nervous system, producing segmental demyelination and axonal degeneration. Reparative processes (axonal regeneration and remyelination) occurred only at the most proximal levels of the nerves. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:1188–1195, 1998.