Nerve Growth Factor Promotes Regeneration of Sensory Axons into Adult Rat Spinal Cord

Injured adult mammalian axons are unable to regenerate spontaneously in the central nervous tissue. This study investigated in two adult rat models the effects of nerve growth factor (NGF) on the capacity of central primary sensory axons to regenerate back into the spinal cord. Sensory fibers were conditioned by transection of the peripheral nerve 1 week prior to the experiment and identified by anterograde tracing with cholera toxin B subunit injected in the sciatic nerve. In the first model, a predegenerated autologous peripheral nerve graft was implanted as a bridge for the transected sensory fibers into a resection gap in the dorsal columns at the tenth thoracic (T10) spinal cord segment. Vehicle or vehicle with purified mouse or recombinant human NGF was continuously infused for 2 weeks directly into the dorsal column at T9, 3 mm from the rostral border of the nerve graft. With vehicle infusion many ascending sensory axons had grown across the nerve bridge, but essentially none had grown back into the rostral cord. In sharp contrast, NGF promoted the reentry into the denervated dorsal columns of 51% of the sensory axons that had reached the rostral level of the nerve graft. Twenty-six percent had grown 2 mm into the spinal tissue and 10% had reached the NGF-infusion site at 3 mm from the nerve graft. A few fibers were found circling around, but not beyond, the infusion site, perhaps due to the chemoattractant action of NGF. In a second model, the fourth lumbar (L4) dorsal root was crushed 2 mm from its insertion point into the spinal cord and the dorsal roots L2, L3, L5, and L6 were transected. Vehicle or vehicle with purified mouse NGF was infused for 2 weeks directly into the lumbar spinal cord, 2.5 mm rostral to the transition zone of the crushed L4 root. With vehicle, only 6% of the regenerating fibers at the transition zone had crossed the root–spinal cord barrier, but not farther than 0.5 mm into the spinal tissue. With NGF, 18% of the fibers at the transition zone were found at 0.5 mm, 9% at 1.5 mm, and 5% at 2.5 mm (the infusion site) from the transition zone. The present results demonstrate that NGF can promote the regeneration of adult sensory fibers into the otherwise nonpermissive spinal cord white matter.     

Ensheathing glia transplants promote dorsal root regeneration and spinal reflex restitution after multiple lumbar rhizotomy

Previously, we have shown that transplants of olfactory bulb ensheathing cells promoted regeneration of transected dorsal roots into the spinal cord. In this study, we assessed the ability of regenerating axons to make functional connections in the cord. Dorsal roots L3 to L6 were sectioned close to their entrance into the spinal cord and reapposed after injecting a suspension of ensheathing cells into each dorsal root entry zone (Group G). Afferent regeneration into the cord and recovery of spinal reflexes were compared with animals that received no injection (Group S) or culture medium without cells (Group C). Electrophysiological tests, to measure nerve conduction and spinal reflexes (H response and withdrawal reflex) evoked by stimulation of afferents of the sciatic nerve, were performed. At 14 days after surgery, H response was found in only 1 of 7 rats of Group G, and withdrawal reflexes were absent from all animals. At 60 days, the H response reappeared in 7 of 10 rats of Group G, and 1 of 5 of each of Groups C and S. The withdrawal reflex recovered in 4 of 10 rats of Group G, but in none of Groups C and S. Immunohistochemical labeling for CALCITONIN GENE– RELATED PEPTIDE (CGRP) in rats of Group G showed immunoreactive fibers entering the dorsal horn from sectioned roots, although at lower density than in the contralateral side. In conclusion, transplanted ensheathing cells promoted central regeneration and functional reconnection of regenerating sensory afferents. Ann Neurol 1999;45:207–215     

Glial cell line-derived neurotrophic factor increases calcitonin gene-related peptide immunoreactivity in sensory and motoneurons in vivo

Calcitonin gene-related peptide (CGRP) is expressed at high levels in roughly 50% of spinal sensory neurons and plays a role in peripheral vasodilation as well as nociceptive signalling in the spinal cord. Spinal motoneurons express low levels of CGRP; motoneuronal CGRP is thought to be involved in end-plate plasticity and to have trophic effects on target muscle cells. As both sensory and motoneurons express receptors for glial cell line-derived neurotrophic factor (GDNF) we sought to determine whether CGRP was regulated by GDNF. Rats were treated intrathecally for 1-3 weeks with recombinant human GDNF or nerve growth factor (NGF) (12 microg/day) and dorsal root ganglia and spinal cords were stained for CGRP. The GDNF treatment not only increased CGRP immunoreactivity in both sensory and motoneurons but also resulted in hypertrophy of both populations. By combined in situ hybridization and immunohistochemistry we found that, in the dorsal root ganglia, CGRP was up-regulated specifically in neurons expressing GDNF but not NGF receptors following GDNF treatment. Despite the increase in CGRP in GDNF-treated rats, there was no increase in thermal or mechanical pain sensitivity, while NGF-treated animals showed significant decreases in pain thresholds. In motoneurons, GDNF increased the overall intensity of CGRP immunoreactivity but did not increase the number of immunopositive cells. As GDNF has been shown to promote the regeneration of both sensory and motor axons, and as CGRP appears to be involved in motoneuronal plasticity, we reason that at least some of the regenerative effects of GDNF are mediated through CGRP up-regulation.     

Nerve Growth Factor Treatment Increases Stimulus-evoked Release of Sensory Neuropeptides in the Rat Spinal Cord

In the adult rat, nerve growth factor (NGF) upregulates the nociceptive peptide substance P (SP) and calcitonin gene-related peptide (CGRP) in neuronal cell bodies of C fibres but the effects of NGF on release of these neuropeptides from the central afferent terminals have not been reported. Thus, this study compared rats treated with six doses of NGF over 2 weeks with controls and measured the release of the neuropeptides, SP and CGRP from the dorsum of the lumbar spinal cord in vitro. NGF (1.0 mg/kg s.c.) greatly increased basal and stimulus-evoked SP and CGRP release; at 0.1 mg/kg, NGF did not affect SP release but significantly increased CGRP basal outflow and evoked release. In a different set of experiments, topical application of NGF (100 ng/ml) to cords from naive rats did not increase stimulus-evoked release of SP or CGRP. These findings show marked stimulation by NGF treatment in vivo of release of sensory neuropeptides during stimulation of dorsal roots, albeit at relatively large doses of the neurotrophin.     

Spinal cord compression injury in adult rats initiates changes in dorsal horn remodeling that may correlate with development of neuropathic pain

Spinal cord injury commonly causes chronic, neuropathic pain. The mechanisms are poorly understood but may include structural plasticity within spinal and supraspinal circuits. Our aim was to determine whether structural remodeling within the dorsal horn rostral to an incomplete injury differs from a complete spinal cord transection. Four immunohistochemical populations of primary afferent C‐fibers, and descending catecholamine and serotonergic projections, were examined in segments T9–T12 at 2 and 12 weeks after a T13 clip‐compression injury in adult male rats. Dorsal root ganglia were also examined. Two weeks after injury, fibers immunoreactive for calcitonin gene‐related peptide (CGRP) or GDNF‐family receptors (GFRα1, GFRα2, GFRα3) showed distinct injury responses within the superficial dorsal horn. CGRP fibers decreased, but GFRα1, GFRα2 and GFRα3 fibers did not change. In contrast, all groups were decreased by 12 weeks after injury. Catecholamine fibers showed a decrease at 2 weeks followed by an increase in density at 12 weeks, whereas serotonergic fibers showed a decrease (restricted to deep dorsal horn) at 12 weeks. These results show that the dorsal horn of the spinal cord undergoes substantial structural plasticity rostral to a compression injury, with the most profound effect being a prolonged and possibly permanent loss of primary afferent fibers. This loss was more extensive and more prolonged than the loss that follows spinal cord transection. Our results provide further evidence that anatomical reorganization of sensory and nociceptive dorsal horn circuits rostral to an injury could factor in the development or maintenance of spinal cord injury pain. J. Comp. Neurol. 513:668–684, 2009. © 2009 Wiley‐Liss, Inc.     

Astrocyte-polymer implants promote regeneration of dorsal root fibers into the adult mammalian spinal cord.

To overcome obstacles to the regeneration of crushed dorsal root fibers at the dorsal root entry zone, we have employed specially designed Millipore implants coated with embryonic astrocytes to serve as a substrate for axonal growth. This strategy was successful in promoting the growth of crushed dorsal root axons into the grey matter of the adult mammalian spinal cord in a small number of animals. Fiber ingrowth into the spinal cord was closely associated with the surface of the polymer implant. In addition, unique terminal arbor malformations, not normally present, were seen in several animals. A consistent finding was the presence of a limited inflammatory response in regions immediately adjacent to the implant where axons penetrate the spinal cord. Our findings suggest that providing the dorsal root entry zone with an embryonic milieu can stimulate a limited amount of axonal regeneration into the adult mammalian spinal cord.     

Spinal entry route for ventral root afferent fibers in the cat

Twelve anesthetized and paralyzed cats were used to study the spinal entry routes of ventral root afferent fibers. In all animals, the spinal cord was transected at two different levels, L5 and S2. The L5 through S2 dorsal roots were cut bilaterally, making spinal cord segments L5-S2 neurally isolated from the body except for the L5-S2 ventral roots. From this preparation, a powerful excitation of the discharge rate of motor neurons and dorsal horn cells within the isolated spinal segments was observed after intraarterial injection of bradykinin (50 micrograms in 0.5 ml saline). This excitation of the spinal neurons can be considered the most convincing evidence of the potential physiologic role of the ventral root afferent fibers entering the spinal cord directly through the ventral root, because the apparent route of neuronal input from the periphery is through the ventral roots. However, additional control experiments conducted in the present study showed that the excitation persisted even after cutting all ventral roots within the isolated spinal segments, indicating that excitation was not mediated by the ventral roots. Furthermore, direct application of bradykinin on the dorsal surface of the spinal cord also increased the motoneuronal discharge rate, suggesting that excitation of spinal neurons produced by intraarterial injection of bradykinin is due to a direct action of bradykinin on the spinal cord. Thus, we provided an alternate explanation for the most convincing evidence indicating that physiologically important ventral root afferent fibers enter the spinal cord directly through the ventral root. Based on existing experimental evidence, it is likely that the majority of physiologically active ventral root afferent fibers travel distally toward the dorsal root ganglion and then enter the spinal cord through the dorsal root.     

Regeneration into the Spinal Cord of Transected Dorsal Root Axons Is Promoted by Ensheathing Glia Transplants

The permissivity of adult olfactory bulb to the ingrowth of olfactory axons could be due to the unique properties of ensheathing glia. To test whether these glial cells could be used to promote axonal regeneration in a spontaneously nonregenerating system, we transplanted suspensions of pure ensheathing cells into a rhizotomized spinal cord segment. Ensheathing cells were purified away from other cell types by immunoaffinity, using anti-p75 nerve growth factor receptor. After laminectomy at the lower thoracic level, the spinal cord was exposed and one dorsal root (T10) was completely transected at the cord entry point. The root stump was microsurgically anastomosed to the cord and a suspension of ensheathing cells was transplanted in the spinal cord at the dorsal root entry zone. Three weeks after transplantation, numerous regenerating dorsal root axons were observed reentering the spinal cord. Ingrowth of dorsal root axons was observed using Dil and antibodies against calcitonin gene-related peptide and growth-associated protein. Primary sensory afferents invaded laminae 1, 2, and 3, grew through laminae 4 and 5, and reached the dorsal grey commissure and lamina 4 of the contralateral side. We did not observe regenerating axons within the ipsilateral ventral horn and dorsal column. Transplanted ensheathing cells reached the same laminae as axons. Neither ensheathing cells nor regenerating axons invaded those laminae they did not inervate under normal circumstances. In conclusion, the regeneration of injured dorsal root axons into the adult spinal cord was possible after ensheathing glia transplantation. The use of ensheathing cells as stimulators of axonal growth might be generalized to other central nervous system injuries.     

Nerve Growth Factor- and Neurotrophin-3-Releasing Guidance Channels Promote Regeneration of the Transected Rat Dorsal Root

Dorsal roots have a limited regeneration capacity after transection. To improve nerve regeneration, the growth-promoting effects of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) were evaluated. The proteins were continuously released by synthetic nerve guidance channels bridging a 4-mm gap in the transected dorsal root. Four weeks after lesion, the regenerated nerve cables were analyzed for the presence of myelinated and unmyelinated axons. While BDNF showed a limited effect on axonal regeneration (863 +/- 39 axons/regenerated nerve, n = 6), NGF (1843 +/- 482) and NT-3 (1495 +/- 449) powerfully promoted regeneration of myelinated axons compared to channels releasing the control protein bovine serum albumin (293 +/- 39). In addition, NGF, but not BDNF nor NT-3, had a potent effect on the regeneration of unmyelinated axons (NGF, 55 +/- 1.4; BDNF, 4 +/- 0.3; NT-3, 4.7 +/- 0.3 axons/100 microm(2); n = 6). The present study suggests that synthetic nerve guidance channels slowly and continuously releasing the neurotrophins NGF and NT-3 can overcome the limited regeneration of transected dorsal root.     

Developmental regulation and possible alternative cleavage of presenilin 1 in the rat retina

Injured dorsal root axons fail to regenerate into the adult spinal cord, leading to permanent sensory loss. We investigated the ability of intrathecal neurotrophin-3 (NT3) to promote axonal regeneration across the dorsal root entry zone (DREZ) and functional recovery in adult rats. Quantitative electron microscopy showed robust penetration of CNS tissue by regenerating sensory axons treated with NT3 at 1 and 2 weeks postrhizotomy. Light and electron microscopical anterograde tracing experiments showed that these axons reentered appropriate and ectopic laminae of the dorsal horn, where they formed vesicle-filled synaptic buttons. Cord dorsum potential recordings confirmed that these were functional. In behavioral studies, NT3-treated (but not untreated or vehicle-treated) rats regained proprioception. Recovery depended on NT3-mediated sensory regeneration: preventing regeneration by root excision prevented recovery. NT3 treatment allows sensory axons to overcome inhibition present at the DREZ and may thus serve to promote functional recovery following dorsal root avulsions in humans.     

Regenerative effect of human recombinant NGF on capsaicin-lesioned sensory neurons in the adult rat

Nerve growth factor (NGF) has the ability to increase the content of peptide transmitter in intact primary sensory afferents of the adult rat. We have previously shown that NGF can also induce a refill of peptide transmitters in capsaicin-depleted peptidergic nerve terminals of the rat paw skin upon intraplantar injection. The present study was aimed at investigating the neurochemical, immunohistochemical and functional recovery of peripheral and central terminals of capsaicin-lesioned afferents following administration of recombinant human NGF-β (rhNGF-β). The systemic capsaicin treatment in adult rats by 50 mg/kg s.c. (day 0) was followed by intraplantar rhNGF-β injections (4 μg each) into one hind paw on days 1, 2, 3, 5, 6 and by the analysis on day 8. The content of the marker peptide calcitonin gene-related peptide (CGRP) showed a 100% NGF-induced recovery in the peripheral (sciatic nerve) and central axons (lumbar dorsal roots) on the side of the NGF treatment and also in the contralateral sciatic nerve and lumbar dorsal roots. In the terminals of the hind paw skin, the recovery of the CGRP content, as measured by radioimmunoassay, was 100% in the plantar and 80% in the dorsal skin ipsilaterally, and 55% in the dorsal and plantar hind paw skin contralaterally. In the lumbar dorsal spinal cord, CGRP content recovered by 85% bilaterally. The morphological appearance of the sensory nerve terminals was visualized by CGRP-immunohistochemistry. In the paw skin, the CGRP-immunoreactive (CGRP-IR) nerve endings were restricted to a fragmentary subepidermal plexus after the capsaicin treatment, whereas the subsequent NGF treatment caused a bilateral recovery of the subepidermal plexus and an intact reinnervation of the epidermis and blood vessels with free nerve terminals. The capsaicin-induced fragmentation of the CGRP terminal plexus in laminae I and II of the lumbar spinal dorsal horn was also markedly repaired on both sides by the intraplantar NGF injections. The NGF treatment caused the CGRP nerve terminals in the spinal cord to regain their ability of releasing transmitter upon capsaicin stimulation as shown in tissue slice superfusion experiments. These results show that within one week, rhNGF-β can induce a complete reinnervation of skin and spinal cord with intact CGRP-IR nerve terminals after an acute capsaicin lesion.     

HSV-mediated gene transfer of C3 transferase inhibits Rho to promote axonal regeneration

Although surgical re-implantation of spinal roots may improve recovery of proximal motor function after cervical root avulsion, recovery of sensory function necessary for fine motor coordination of the hand has been difficult to achieve, in large part because of failure of regeneration of axons into the spinal cord. In order to enhance regeneration, we constructed a non-replicating herpes simplex virus (HSV)-vector carrying the gene coding for bacterial C3 transferase (C3t). Subcutaneous inoculation of the vector into the skin of the forepaw 1 week after a dorsal C5-T1 rhizotomy resulted in expression of C3t in dorsal root ganglion (DRG) neurons and inhibition of Rho GTPase activity, resulting in extensive axonal regeneration into the spinal cord that correlated with improved sensory-motor coordination of the forepaw.     

A comparison of the regeneration potential of dorsal root fibers into gray or white matter of the adult rat spinal cord.

To assess the role of white matter inhibition as a barrier to neurite outgrowth in vivo, we unilaterally transected three consecutive lumbar dorsal roots (L4-L6), incised the spinal cord, and transplanted the peripheral stump of L4 either medially onto the white matter of the dorsal columns or laterally, just superficial to the gray matter of the dorsal horn at the level of L5. Three weeks to seven months later, the translocated root was retransected, and its central stump was anterogradely labeled with HRP. The staining pattern demonstrated that regenerating sensory axons had entered the spinal cord from both medially and laterally placed roots. Axonal staining from medially placed dorsal roots (onto the white matter of the dorsal columns) was sparse and limited to the white matter. Staining of laterally placed roots revealed a small subpopulation of regenerating axons which had entered the gray matter and formed terminal arbors. Successful axonal regeneration into the gray matter, albeit minimal, was associated with a localized and limited inflammatory response near the sites of axonal ingrowth.     

Extensive sprouting of sensory afferents and hyperalgesia induced by conditional expression of nerve growth factor in the adult spinal cord.

Genetic transfer of growth-promoting molecules was proposed as a potential strategy to modify the nonpermissive nature of the adult CNS to induce axonal regeneration. To evaluate whether overexpression of neurotrophins or cellular adhesion molecules would effect axonal plasticity, adenoviruses encoding fibroblast growth factor-2 (FGF-2/Adts), nerve growth factor (NGF/Adts), neurotrophin-3, and the cell adhesion molecules N-cadherin and L1 were injected into the dorsal horn of the adult spinal cord. Transgene expression was primarily localized to astrocytes in the dorsal horn and motor neurons within the ventral horn. Overexpression of these factors, with the exception of NGF/Adts, failed to increase axonal sprouting. Eight days after NGF/Adts injections, axonal sprouting within the dorsal horn was apparent, and after 4 weeks, extensive spouting was observed throughout the entire dorsal horn, extending into the ventral horn and the white matter of the lateral funiculus. These axons were identified primarily as a subpopulation of nociceptive fibers expressing calcitonin gene-related peptide and substance-P. Behavioral analysis revealed thermal hyperalgesia and perturbation of accurate paw placement on grid-walking tasks for both FGF-2- and NGF-treated animals. These results indicate that the administration of growth-promoting molecules can induce robust axonal plasticity of normal adult primary sensory neurons into areas of transgene expression, causing significant alterations in behavioral responses. This observation also indicates that gene transfer protocols that aim to reconstruct diseased or injured pathways should also be designed to prevent the sprouting of the normal circuitry from adjacent unaffected neurons.     

SSeCKS immunolabeling in rat primary sensory neurons

SSeCKS (src suppressed C kinase substrate) is a protein kinase C substrate that may play a role in tumor suppression. Recently described in fibroblasts, testes and mesangial cells, SSeCKS may have a function in the control of cell signaling and cytoskeletal arrangement. To investigate the distribution of SSeCKS throughout the nervous system, representative sections of brain, spinal cord and dorsal root ganglia were processed using immunofluorescence. Labeling of central axonal collaterals of primary sensory neurons was observed in the dorsal horn at all spinal levels. SSeCKS-immunoreactivity was also observed in the cerebellum, medulla and sensory ganglia (including trigeminal ganglia). The pattern and distribution of anti-SSeCKS labeling in dorsal root ganglia and the dorsal horn of the spinal cord was similar to that observed for other markers of small primary sensory neurons. Therefore, the coexistence of SSeCKS with substance P, CGRP and acid phosphatase was examined in sections of sensory ganglia, spinal cord and medulla using double immunofluorescent labeling for SSeCKS and substance P/CGRP or sequential SSeCKS immunofluorescence and acid phosphatase/fluoride-resistant acid phosphatase enzyme histochemistry. A small portion of the SSeCKS-labeled cell bodies appeared to represent a subpopulation of substance P (4.8%) and CGRP (4.7%) containing neurons, while 45.0% contained fluoride-resistant acid phosphatase reactivity. These results indicate that SSeCKS has a restricted distribution within the nervous system and that expression of this protein may reflect the specific signaling requirements of a distinct population of nociceptive sensory neurons.     

Neurotrophins promote regeneration of sensory axons in the adult rat spinal cord

We have investigated the effects of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) on the intraspinal regeneration of anterogradely labeled axotomized ascending primary sensory fibers in the adult rat. These fibers were allowed to grow across a predegenerated peripheral nerve graft and back into the thoracic spinal cord. In control animals that had been infused with vehicle for two weeks into the dorsal column, 3 mm rostral to the nerve graft, essentially no fibers had extended from the nerve graft back into the spinal cord. The number of sensory fibers in the rostral end of the nerve graft was not significantly different between control and neurotrophin-infused animals. With infusion of NGF, 37+/-2% of the fibers at the rostral end of the graft had grown up to 0.5 mm into the dorsal column white matter, 30+/-2% up to 1 mm, 19+/-3% up to 2 mm and 8+/-2% up to 3 mm, i.e., the infusion site. With infusion of NT-3, sensory fiber outgrowth was similar to that seen with NGF, but with BDNF fewer fibers reached farther distances into the cord. Infusion of a mixture of all three neurotrophins did not increase the number of regenerating sensory fibers above that seen after infusion of the individual neurotrophins. These findings suggest that injured ascending sensory axons are responsive to all three neurotrophins and confirm our previous findings that neurotrophic factors can promote regeneration in the adult central nervous system.     

Sensory nerves in adult rats regenerate and restore sensory function to the skin independently of endogenous NGF.

We have investigated the possible roles of NGF, and of impulse activity, in the regeneration of sensory nerves. Unexpectedly, the ability of crushed axons to regrow and to restore functional recovery of three sensory modalities in adult rat skin (A alpha-mediated touch, A delta-mediated mechanonociception, and C-fiber-mediated heat nociception) was totally unaffected by anti-NGF treatment. This finding applied even when the anti-NGF dosage was almost eight times that which entirely blocked collateral sprouting of the undamaged axons of both classes of nociceptive nerves (the A alpha-axons do not sprout in adult animals). In the same anti-NGF-treated animal, regeneration would proceed normally on the one side, while collateral sprouting was prevented on the other. Light microscopic and EM examination revealed that in the denervated skin the regenerating axons utilized the same dermal perineurial pathways followed by collaterally sprouting axons. Regeneration within these antibody-accessible pathways progressed normally during anti-NGF treatment, extending 1-2 cm beyond the former field borders, that is, into territory whose invasion by collaterally sprouting axons was totally blocked. The blood-nerve barrier is absent within the degenerating peripheral nerve trunk, a putative NGF source for regenerating fibers but not for sprouting ones. The NGF-independent regeneration was also found to be unaffected when putative spinal cord sources of NGF were eliminated by dorsal root excision. Anti-NGF treatment also failed to block regeneration across 4 mm excision gaps in the nerve trunk. The daily anti-NGF regime continued to be effective for at least 8 weeks, at which time newly evoked collateral sprouting could still be blocked. Exogenous NGF, in doses that evoke collateral sprouting de novo in normal skin, failed to influence regeneration. Finally, an electrical stimulus regime, which markedly reduces the latency of collateral sprouting, failed to affect the time to arrival of regenerating axons at the skin, or the rate of their arborization in it. We conclude that, in striking contrast to their collateral sprouting, the regeneration of nociceptive axons occurs independently of endogenous NGF and is unaffected by impulse activity. These findings further support the proposal that these two growth behaviors have basically different biological functions in the organism.     

Olfactory ensheathing cell grafts have minimal influence on regeneration at the dorsal root entry zone following rhizotomy

The effectiveness of grafts of olfactory ensheathing cells (OECs) as a means of promoting functional reconnection of regenerating primary afferent fibers was investigated following dorsal root injury. Adult rats were subjected to dorsal root section and reanastomosis and at the same operation a suspension of purified OECs was injected at the dorsal root entry zone and/or into the sectioned dorsal root. Regeneration of dorsal root fibers was then assessed after a survival period ranging from 1 to 6 months. In 11 animals, electrophysiology was used to look for evidence of functional reconnection of regenerating dorsal root fibers. However, electrical stimulation of lesioned dorsal roots failed to evoke detectable cord dorsum or field potentials within the spinal cord of any of the animals examined, indicating that reconnection of regenerating fibers with spinal cord neurones had not occurred. In a further 11 rats, immunocytochemical labeling and biotin dextran tracing of afferent fibers in the lesioned roots was used to determine whether regenerating fibers were able to grow into the spinal cord in the presence of an OEC graft. Although a few afferent fibers could be seen to extend for a limited distance into the spinal cord, similar minimal in-growth was seen in control animals that had not been injected with OECs. We therefore conclude that OEC grafts are of little or no advantage in promoting the in-growth of regenerating afferent fibers at the dorsal root entry zone following rhizotomy.     

Comparison of dorsal and ventral spinal root regeneration through semipermeable guidance channels.

Semipermeable guidance channels have been shown to support nerve regeneration in the peripheral nervous system (PNS) possibly through interactions with the wound healing environment. This study quantitatively assesses the ability of such channels to support regeneration in the PNS segment of the spinal roots across a 4 mm gap and compares the resultant dorsal and ventral root regeneration. Acrylic copolymer guidance channels with a molecular weight (Mw) cutoff of 50,000 Da were used in a transected rat spinal root model. Cohorts of 23 animals (11 ventral, 12 dorsal) were examined at four weeks; 6 animals (3 ventral, 3 dorsal) at ten weeks; and 10 animals (5 ventral, 5 dorsal) at twenty-four weeks post-implantation. Both the dorsal and ventral roots were able to regenerate across the gap within the semipermeable channel. At all time periods, the regenerated dorsal roots contained fewer myelinated axons than found in the contralateral control root and consisted of an abundance of collagenous tissue. In contrast, by ten weeks the regenerated ventral roots contained twice the contralateral control number of myelinated axons and were composed predominantly of large, myelinated axons. At twenty-four weeks the number of unmyelinated axons was also quantified, with the regenerated dorsal root containing only one-fifth of the control number and the regenerated ventral root containing more than four times the control. Due to the proximity of the dorsal root lesion to the axonal cell bodies, the dorsal root ganglion (DRG) neuronal cell loss was investigated at four weeks post-implantation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.