Evidence for a systemic regulation of neurotrophin synthesis in response to peripheral nerve injury.

Up-regulation of neurotrophin synthesis is an important mechanism of peripheral nerve regeneration after injury. Neurotrophin expression is regulated by a complex series of events including cell interactions and multiple molecular stimuli. We have studied neurotrophin synthesis at 2 weeks time-point in a transvertebral model of unilateral or bilateral transection of sciatic nerve in rats. We have found that unilateral sciatic nerve transection results in the elevation of nerve growth factor (NGF) and NT-3, but not glial cell-line derived neurotrophic factor or brain-derived neural factor, in the uninjured nerve on the contralateral side, commonly considered as a control. Bilateral transection further increased NGF but not other neurotrophins in the nerve segment distal to the transection site, as compared to the unilateral injury. To further investigate the distinct role of NGF in regeneration and its potential for peripheral nerve repair, we transduced isogeneic Schwann cells with NGF-encoding lentivirus and transplanted the over-expressing cells into the distal segment of a transected nerve. Axonal regeneration was studied at 2 weeks time-point using pan-neuronal marker NF-200 and found to directly correlate with NGF levels in the regenerating nerve.

Ischemia and failed regeneration in chronic experimental neuromas.

Neuromas are generally considered to be swollen uniform collections of uncontrolled aberrantly sprouting axons. In early experimental neuromas, there are substantial rises in local blood flow associated with their formation, but human studies of chronic lesions have suggested that neuromas develop ischemia and become impediments to regeneration. The issue is important because traumatically severed human nerves are frequently considered for repair some time after injury, when neuroma formation has occurred. In this work, we examined local perfusion, axon penetration and other characteristics of long-term (6 month) experimental neuromas created by sciatic nerve transection and resection of the distal sciatic nerve and its branches. The scenario was designed to model prior transection in a human nerve, where late surgical reconnection might be contemplated. Local blood flow in the extrinsic plexus of neuromas, examined using a laser Doppler flowmetry probe, declined in distal portions of the stump to values considerably lower than observed in intact nerves. Intrinsic blood flow near the stump tip, examined using microelectrode hydrogen clearance polarography was highly nonuniform and included zones with very low perfusion. Correlated with these findings were nonuniform histological features with zones of absent axons and blood vessels, progressive distal disorganization, marked declines in distal axon penetration, nonremodelled microfascicles and persistent expression of 'regenerative' axon and Schwann cell markers. Uncontrolled axon sprouting was not a feature. Longstanding neuromas include zones of relative ischemia and limited axon penetration that develop in the absence of nerve trunk reconnection. These features would limit their suitability for later repair.

Accelerated axon outgrowth, guidance, and target reinnervation across nerve transection gaps following a brief electrical stimulation paradigm.

OBJECT: Regeneration of peripheral nerves is remarkably restrained across transection injuries, limiting recovery of function. Strategies to reverse this common and unfortunate outcome are limited. Remarkably, however, new evidence suggests that a brief extracellular electrical stimulation (ES), delivered at the time of injury, improves the regrowth of motor and sensory axons. METHODS: In this work, the authors explored and tested this ES paradigm, which was applied proximal to transected sciatic nerves in mice, and identified several novel and compelling impacts of the approach. Using thy-1 yellow fluorescent protein mice with fluorescent axons that allow serial in vivo tracking of regeneration, the morphological, electrophysiological, and behavioral indices of nerve regrowth were measured. RESULTS: The authors show that ES is associated with a 30%-50% improvement in several indices of regeneration: regrowth of axons and their partnered Schwann cells across transection sites, maturation of regenerated fibers in gaps spanning transection zones, and entry of axons into their muscle and cutaneous target zones. In parallel studies, the authors analyzed adult sensory neurons and their response to extracellular ES while plated on a novel microelectrode array construct designed to deliver the identical ES paradigm used in vivo. The ES accelerated neurite outgrowth, supporting the concept of a neuron-autonomous mechanism of action. CONCLUSIONS: Taken together, these results support a robust role for brief ES following peripheral nerve injuries in promoting regeneration. Electrical stimulation has a wider repertoire of impact than previously recognized, and its impact in vitro supports the hypothesis that a neuron-specific reprogrammed injury response is recruited by the ES protocol.

A long peripheral nerve autograft model in the sheep forelimb.

BACKGROUND: Autologous nerve grafts remain the only proven means of bridging lengthy gaps in peripheral nerve. However, there is very little literature on a reliable long (> 5 cm) nerve autograft animal model. OBJECTIVE: To establish a reproducible long nerve gap and autograft animal model that is clinically relevant but not cost prohibitive. METHODS: The extent of nerve regeneration and electrophysiological recovery after segmental repair of a long nerve defect was evaluated with a sheep model. Thirteen Suffolk sheep were used. An 18-cm segment of radial sensory nerve was harvested from the forelimb, trimmed, divided into 2 equal segments of 7 cm each, and microsurgically repaired to a surgically created defect of 5 cm in the median nerve within the same forelimb. Electrophysiological studies were performed on 6 sheep at 6 months and 6 sheep at 9 months. Samples of the grafted segments were obtained for histology, immunohistochemistry, and morphometric analyses. Electric studies were also performed on an uninjured median nerve of a control animal in tissue that was similarly harvested and processed. RESULTS: At 6 and 9 months, all sheep had recordable robust nerve action potentials. Nerve conduction velocity and amplitude were slightly decreased compared with control, but the difference was statistically insignificant. Histomorphometric assessment demonstrated that the autografts contained a large number of regenerating axons through graft fascicles in all animals. CONCLUSION: The median nerve in the sheep forelimb is a reproducible and reliable model for assessing regeneration through long peripheral nerve grafts.

Motoneuron survival after chronic and sequential peripheral nerve injuries in the rat.

OBJECT: Surgical repair of peripheral nerves following chronic nerve injury is associated with poor axonal regeneration and outcome. An underlying possibility is that chronic injuries may increase motoneuron cell death. The hypothesis that substantial motoneuron death follows chronic and sequential nerve injuries was tested in adult rats in this study. METHODS: Thirty adult male Lewis rats underwent bilateral multistage surgeries. At initial surgery, Fast Blue (FB) tracer was injected at a nerve-crush injury site in the right control femoral motor nerve. The left femoral motor nerve was transected at the same level and either capped to prevent regeneration (Group 1), or repaired to allow axonal regeneration and reinnervation of the target quadriceps muscle (Group 2) (15 rats in each group). After 8 weeks in 6 rats/group, the left femoral nerve was cut and exposed to FB just proximal to prior nerve capping or repair and the rats were evaluated for FB-labeled motoneuron counts bilaterally in the spinal cord (this was considered survival after initial injury). In the remaining 9 animals/group, the left nerve was recut (sequential injury), exposed to FB, and repaired to a fresh distal saphenous nerve stump to permit axonal regeneration. Following another 6 weeks, Fluoro-Gold, a second retrograde tracer, was applied to the cut distal saphenous nerve. This allowed us to evaluate the number of motoneurons that survived (maintained FB labeling) and the number of motoneurons that survived but that also regenerated axons (double labeled with FB and Fluoro-Gold). RESULTS: A mean number of 350 and 392 FB-labeled motoneurons were found after 8 weeks of nerve injury on the right and the left sides, respectively. This indicated no significant cell death due to initial nerve injury alone. A similar number (mean 390) of motoneurons were counted at final end point at 14 weeks, indicating no significant cell death after sequential and chronic nerve injury. However, only 50% (mean 180) of the surviving motoneurons were double labeled, indicating that only half of the population regenerated their axons. CONCLUSIONS: The hypothesis that significant motoneuron cell death occurs after chronic and or sequential nerve injury was rejected. Despite cell survival, only 50% of motoneurons are capable of exhibiting a regenerative response, consistent with our previous findings of reduced regeneration after chronic axotomy.

Prolonged target deprivation reduces the capacity of injured motoneurons to regenerate.

OBJECTIVE: To investigate whether or not it is the frustrated growth state (no axon growth) that reduces regenerative capacity or the inability of axotomized motoneurons to remake muscle connections (axon growth-no muscle contact) that accounts for poor regenerative capacity of chronically axotomized motoneurons. METHODS: We chronically axotomized rat femoral motoneurons for 2 months by cutting the nerve and either capping the proximal nerve to prevent axon regeneration (Group 1, no axon growth for 2 mo) or encouraging axon regeneration but not target reinnervation by suture to the distal stump of cut saphenous nerve (Group 2, axon growth with no muscle contact). In the control fresh axotomy group (axon growth with muscle contact), femoral nerve stumps were resutured immediately. Two months later, the femoral nerve was recut and sutured immediately to encourage regeneration in a freshly cut saphenous nerve stump for 6 weeks. Regenerating axons in the saphenous nerve were back-labeled with fluorogold for enumeration of the femoral motoneurons that regenerated their axons into the distal nerve stump. RESULTS: We found that significantly fewer chronically axotomized motoneurons regenerated their axons than freshly axotomized motoneurons that regenerated their axons to reform nerve-muscle connections in the same length of time. The number of motoneurons that regenerated their axons was reduced in both the conditions of no axon growth and axon growth with no muscle contact; thus chronic axotomy for a 2-month period reduced regenerative success irrespective of whether the motoneurons were prevented from regenerating or encouraged to regenerate their axons in that same period of time. CONCLUSION: Axonal regeneration does not protect motoneurons from the negative effects of prolonged axotomy on regenerative capacity. It is the period of chronic axotomy, in which motoneurons remain without target nerve-muscle connection, and not simply a state of frustrated growth that accounts for the reduced regenerative capacity of those neurons.

Regeneration into protected and chronically denervated peripheral nerve stumps.

OBJECTIVE: Delayed repair of peripheral nerve injuries often results in poor motor functional recovery. This may be a result of the deterioration or loss of endoneurial pathways in the distal nerve stump before motor axons can regenerate into the stump. METHODS: Using the rat femoral nerve, we protected distal endoneurial pathways of the saphenous nerve with either cross-suture of the quadriceps motor nerve (Group A) or resuture of the saphenous nerve (Group B) to compare later motor regeneration into the "protected" saphenous nerve pathway to chronic denervation and "unprotected" saphenous nerve (Group C). A total of 60 rats, 20 per group, were operated on. After this protection (or lack thereof) for 8 weeks, the motor branch of the femoral nerve was cut and sutured to the distal saphenous nerve to allow motor regeneration into protected and unprotected saphenous nerve stumps. The quantitative assessment of axonal regeneration was performed after 6 weeks by use of nerve sampling for axon counts and retrogradely labeled motor neuron counts. RESULTS: Significantly more myelinated axons innervated the motor (A) than the sensory (B) and no-protection (C) groups. There were significantly more retrogradely labeled femoral motor neurons in Group A than in the unprotected group (C). CONCLUSION: We conclude that even 2 months of denervation of the distal nerve pathway is deleterious to regeneration and that protection of the pathway improves subsequent reinnervation and regeneration. Moreover, if the desired regeneration is motor, protection of the distal nerve pathway by a motor nerve conditions is better than a sensory nerve.

Histological and magnetic resonance analysis of sciatic nerves in the tellurium model of neuropathy.

Ingestion of tellurium (Te), a toxic element, produces paralysis of the hind limbs in weanling rats that is due to temporary, segmental demyelination of the sciatic nerves bilaterally. Weanling rats were fed a 1.1% elemental Te diet and sacrificed at various time points for histological and magnetic resonance (MR) analysis of the sciatic nerves. No controls exhibited impairments of the hind limbs, whereas Te-treated animals became progressively impaired with increased Te exposure. Toluidine blue-stained nerve sections of Te-treated animals showed widened endoneurial spaces, disrupted myelin sheaths, swollen Schwann cells, and a few instances of axonal degeneration. Te decreased healthy myelin by 68% and increased percent extracellular matrix by 45% on day 7. MR experiments showed a decrease in the area of the short T2 component, an increase in average T1, and an increase in the position of the intermediate T2 component in Te-treated nerves. The correlation coefficient for healthy myelin and average T1 was 0.88 and that for healthy myelin and the area underneath the short T2 component was 0.77. The area of the short T2 component has been postulated as the best measure of the process of demyelination.

Mu opioid receptors and analgesia at the site of a peripheral nerve injury.

Opioid ligands may exert antinociception through receptors expressed on peripheral afferent axons. Whether local opioid receptors might attenuate neuropathic pain is uncertain. In this work, we examined the function and expression of local mu opioid receptors (MORs) associated with the chronic constriction injury (CCI) model of sciatic neuropathic pain in rats. Low-dose morphine or its carrier were percutaneously superfused over the CCI site with the injector blinded to the identity of the injectate. Morphine, but not its carrier, and not equimolar systemic doses of morphine reversed thermal hyperalgesia in a dose-related, naloxone-sensitive fashion. Moreover, analgesia was conferred at both 48 hours and 14 days after CCI, times associated with very different stages of nerve repair. Equimolar local DAGO ([D-Ala2, N-Me-Phe4, Gly5-(ol)] enkephalin), a selective MOR ligand, provided similar analgesia. Local morphine also attenuated mechanical allodynia. MOR protein was expressed in axonal endbulbs of Cajal just proximal to the injury site, in aberrantly regenerating small axons in the epineurial sheath around the CCI site and in residual small axons distal to the CCI lesion. Sensory neurons ipsilateral to CCI had an increase in the proportion of neurons expressing MOR. We suggest that local MOR expressed in axons may be exploited to modulate some forms of neuropathic pain.