Evaluation of regeneration of nerve and reinnervation of skeletal muscle in the hibernating ground squirrel

The retrograde transport of HRP was used to determine the status of axonal transport in the peroneal and sciatic nerves of hibernating and nonhibernating ground squirrels following crush of the peroneal nerve at 10 to 12 mm (SNS) or sciatic nerve at 33 to 35 mm (LNS) from its entrance into the extensor muscle. The ability of the proximal segment to reestablish axonal continuity and thus neuromuscular transmission was also studied. Two weeks to 3 months after nerve crush the extensor muscles were injected with HRP. We found that during hibernation no axonal transport across the site of crush was seen even after 3 months and that regeneration of the nerve during this period was minimal. Evidence of slight regeneration seen at 90 days could be due to periods of awaking of the animals during their natural hibernation cycle. In these animals HRP deposits were seen only in the nerve distal to crush, i.e., between crush site and muscle. In the nonhibernating squirrels, axoplasmic flow was reestablished at the site of injury as early as 2 weeks after crush, and HRP could be detected in the spinal cord in motoneurons of the ipsilateral ventral horn at spinal levels L₃ to L₅. In one hibernating animal the peroneal nerve was crushed at the distal site (SNS) and also the spinal cord was injured by dropping a weight. After nerve crush and the spinal cord injury the hibernating state could not be maintained and the animal stayed awake 22 days. The time course of regeneration of the nerve in that animal was similar to that seen in nonhibernating squirrels. After nerve crush in nonhibernating animals, reaction product was also found in sensory cell bodies of dorsal root ganglia as well as in terminals in the substantia gelatinosa of the spinal cord at the same levels. Thus, the axonal transport occurs in hibernating and non-hibernating squirrels in both sensory and motor nerve fibers. The extensor muscle fibers of the hibernating squirrels showed substantial membrane depolarization 90 days after crush. Action potentials from these fibers could be obtained from 15 to 35 days only through stimulating the nerve segment distal to the crush. Stimulation of the proximal nerve segment did not evoke muscle activity. These results demonstrate that nerve regeneration was nearly abolished during hibernation and that blockade of axonal transport continued across a region of nerve crush for the duration of the hibernating period.     

Reinnervation of muscular targets by nerve regeneration through guidance conduits

We established histopathologic and neurophysiologic approaches to examine whether different designs of polycaprolactone-engineered nerve conduits (hollow vs. laminated) could promote nerve regeneration as autologous grafts after transection of sciatic nerves. The assessments included morphometric analysis at the level of sciatic nerve, neuromuscular junction (NMJ) and gastrocnemius muscle, and nerve conduction studies on sciatic nerves. Six months after nerve grafting, the nerve fiber density in the hollow-conduit group was similar to that in the autologous-graft group; the laminated-conduit group only achieved approximately 20% of these values. The consequences of these differences were reflected in nerve growth into muscular targets; this was demonstrated by combined cholinesterase histochemistry for NMJ and immunohistochemistry for nerve fibers innervating NMJ with an axonal marker, protein gene product 9.5. Hollow conduits had similar index of NMJ innervation as autologous grafts; the values were higher than those of laminated conduits. Among the 3 groups there were same patterns of differences in the cross-sectional area of muscle fibers and amplitudes of compound muscle action potential. These results indicate that hollow conduits were as efficient as autologous grafts to facilitate nerve regeneration, and provide a multidisciplinary approach to quantitatively evaluate muscular reinnervation after nerve injury.     

Reinnervation of denervated muscle by transplantation of fetal spinal cord to transected sciatic nerve in the rat

When motor neurons in the spinal cord are destroyed, regeneration of motor axons and muscle reinnervation cannot be expected. We attempted reinnervation of the denervated muscle, i.e. motor unit reconstruction, using transplantation of the fetal spinal cord to the peripheral nerve. The sciatic nerve of an adult rat was resected for 20 mm, and a cavity was prepared using an autologous femoral vein at the distal stump of the nerve. The fetal spinal cord was then transplanted into the venous cavity. After 3–6 months, no voluntary muscle contraction was observed due to the absence of communication with the central nervous system. However, reinnervation of the muscles via the sciatic nerve by the transplanted spinal neurons was demonstrated electrophysiologically and histochemically. This suggested that a motor unit can be reconstructed by fetal spinal cord transplantation even if the original motor neurons in the spinal cord are not available.     

Schwann cells transduced with a lentiviral vector encoding Fgf‐2 promote motor neuron regeneration following sciatic nerve injury

Fibroblast growth factor 2 (FGF‐2) is a trophic factor expressed by glial cells and different neuronal populations. Addition of FGF‐2 to spinal cord and dorsal root ganglia (DRG) explants demonstrated that FGF‐2 specifically increases motor neuron axonal growth. To further explore the potential capability of FGF‐2 to promote axon regeneration, we produced a lentiviral vector (LV) to overexpress FGF‐2 (LV‐FGF2) in the injured rat peripheral nerve. Cultured Schwann cells transduced with FGF‐2 and added to collagen matrix embedding spinal cord or DRG explants significantly increased motor but not sensory neurite outgrowth. LV‐FGF2 was as effective as direct addition of the trophic factor to promote motor axon growth in vitro. Direct injection of LV‐FGF2 into the rat sciatic nerve resulted in increased expression of FGF‐2, which was localized in the basal lamina of Schwann cells. To investigate the in vivo effect of FGF‐2 overexpression on axonal regeneration after nerve injury, Schwann cells transduced with LV‐FGF2 were grafted in a silicone tube used to repair the resected rat sciatic nerve. Electrophysiological tests conducted for up to 2 months after injury revealed accelerated and more marked reinnervation of hindlimb muscles in the animals treated with LV‐FGF2, with an increase in the number of motor and sensory neurons that reached the distal tibial nerve at the end of follow‐up. GLIA 2014;62:1736–1746     

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.     

Immediate electrical stimulation enhances regeneration and reinnervation and modulates spinal plastic changes after sciatic nerve injury and repair

We have studied whether electrical stimulation immediately after nerve injury may enhance axonal regeneration and modulate plastic changes at the spinal cord level underlying the appearance of hyperreflexia. Two groups of adult rats were subjected to sciatic nerve section followed by suture repair. One group (ES) received electrical stimulation (3 V, 0.1 ms at 20 Hz) for 1 h after injury. A second group served as control (C). Nerve conduction, H reflex, motor evoked potentials, and algesimetry tests were performed at 1, 3, 5, 7 and 9 weeks after surgery, to assess muscle reinnervation and changes in excitability of spinal cord circuitry. The electrophysiological results showed higher levels of reinnervation, and histological results a significantly higher number of regenerated myelinated fibers in the distal tibial nerve in group ES in comparison with group C. The monosynaptic H reflex was facilitated in the injured limb, to a higher degree in group C than in group ES. The amplitudes of motor evoked potentials were similar in both groups, although the MEP/M ratio was increased in group C compared to group ES, indicating mild central motor hyperexcitability. Immunohistochemical labeling of sensory afferents in the spinal cord dorsal horn showed prevention of the reduction in expression of substance P at one month postlesion in group ES. In conclusion, brief electrical stimulation applied after sciatic nerve injury promotes axonal regeneration over a long distance and reduces facilitation of spinal motor responses.     

Craniocerebral injury promotes the repair of peripheral nerve injury

The increase in neurotrophic factors after craniocerebral injury has been shown to promote fracture healing. Moreover, neurotrophic factors play a key role in the regeneration and repair of peripheral nerve. However, whether craniocerebral injury alters the repair of peripheral nerve injuries remains poorly understood. Rat injury models were established by transecting the left sciatic nerve and using a free-fall device to induce craniocerebral injury. Compared with sciatic nerve injury alone after 6–12 weeks, rats with combined sciatic and craniocerebral injuries showed decreased sciatic functional index, increased recovery of gastrocnemius muscle wet weight, recovery of sciatic nerve ganglia and corresponding spinal cord segment neuron morphologies, and increased numbers of horseradish peroxidase-labeled cells. These results indicate that craniocerebral injury promotes the repair of peripheral nerve injury.     

Chronic spinal cord transection does not affect peripheral nerve regeneration

Spinal cord transection is known to cause progressive changes in motor neurons and hind limb muscles. In the present study, regeneration of the peroneal nerve was examined in rats 25 weeks after a T9 spinal cord transection. Successful regeneration and innervation of the target muscle was observed after crush injury to the nerve in the spinal cord transected animals. It is concluded that the ability of peripheral nerve to regenerate remains preserved after spinal cord injury.     

Morphological changes in spinal motor neurons giving rise to long-term regenerated sciatic nerve axons

Morphological properties of rat spinal motor neurons were examined 14-16 months following unilateral sciatic nerve crush and compared to the properties observed in neurons contralateral to injury and in cord segments from age-matched control rats. Regenerated and control motor neurons were identified by retrograde labelling with HRP applied to sciatic nerves distal to the site of crush or at a comparable location in control nerves. Many of the experimental motor neurons were enlarged and had thickened dendritic processess compared to the finer dendrites seen in control cells. Mean cell area ipsilateral to the crush lesions was larger than mean control cell area (P-value less than 0.001) despite representation of all control cell areas in the regenerated population. These data suggest that persistent or continued morphological abnormalities occur in mammalian motor neurons following simple sciatic crush injury when examined at extended times beyond the period of axonal regeneration and clinical recovery.     

Modulation of serotonin 5-HT3 receptor expression in injured adult rat spinal cord motoneurons

The effects of sciatic nerve lesions on the expression of serotonin 5-HT3 receptor (5-HT3R) alpha subunit in motoneurons of the spinal cord was investigated by semi-quantitative immunohistochemistry. Following sciatic nerve crush, a significant reduction in density of staining in motoneurons was observed in longitudinal sections of the ventral horn at 3 and 15 days on the lesioned side when compared to the contralateral side (p<0.01). At 30 days after crush, after completion of sciatic nerve regeneration and reinnervation of peripheral targets, intensity of staining had returned to normal. Conversely, after sciatic nerve cut, a lesion that does not allow for target reinnervation, highly significant reductions were observed at 3, 15, 30 and 45 days. These results suggest a role for functional contacts with muscular targets in the maintenance of 5-HT3R expression in spinal motoneurons.     

Interfacing peripheral nerve with macro-sieve electrodes following spinal cord injury

Macro-sieve electrodes were implanted in the sciatic nerve of five adult male Lewis rats following spinal cord injury to assess the ability of the macro-sieve electrode to interface regenerated peripheral nerve fibers post-spinal cord injury. Each spinal cord injury was performed via right lateral hemisection of the cord at the T_(9–10) site. Five months post-implantation, the ability of the macro-sieve electrode to interface the regenerated nerve was assessed by stimulating through the macro-sieve electrode and recording both electromyography signals and evoked muscle force from distal musculature. Electromyography measurements were recorded from the tibialis anterior and gastrocnemius muscles, while evoked muscle force measurements were recorded from the tibialis anterior, extensor digitorum longus, and gastrocnemius muscles. The macro-sieve electrode and regenerated sciatic nerve were then explanted for histological evaluation. Successful sciatic nerve regeneration across the macro-sieve electrode interface following spinal cord injury was seen in all five animals. Recorded electromyography signals and muscle force recordings obtained through macro-sieve electrode stimulation confirm the ability of the macro-sieve electrode to successfully recruit distal musculature in this injury model. Taken together, these results demonstrate the macro-sieve electrode as a viable interface for peripheral nerve stimulation in the context of spinal cord injury.     

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.     

The distribution of C-Fos protein immunolabeled cells in the spinal cord of the rat after electrical and noxious thermal stimulation following sciatic nerve crush, or transection and repair

The distribution of stimulus evoked Fos protein-like immunoreactivity in spinal cord neurons was studied in adult rats at different survival times after sciatic nerve crush or transection and epineural repair. Fos protein-like immunoreactivity was induced either by electrical stimulation of the sciatic nerve central to the injury, at C-fiber strength, at 21, 39, and 92 days post-lesion, or by noxious heat applied to the skin of the hind paw 92 days post-lesion. The contralateral uninjured side served as control. The results with electrical stimulation showed, with some exceptions, that the distribution of c-fos expressing cells in the spinal cord on the normal and on the previously injured side were similar after both crush and transection with repair. The main finding was an up-regulation of the number of Fos protein immunoreactive neurons in the inner portion of Rexed's lamina II. The results following heat stimulation 92 days post-lesion showed a decrease in the number of labeled neurons in most laminae after both types of injury. This was more pronounced in cases with sciatic nerve transection with repair compared to cases with crush. The results indicate time-dependent alterations in the distribution of stimulus evoked c-fos expression in spinal cord neurons during regeneration after nerve injury. Furthermore, the results from heat stimulation may indicate a slower and perhaps more incomplete restoration process after transection with repair than after crush.     

Biomimetic chitosan scaffolds with long-term controlled release of nerve growth factor repairs 20-mm-long sciatic nerve defects in rats

Although autogenous nerve transplantation is the gold standard for treating peripheral nerve defects of considerable length,it still has some shortcomings,such as insufficient donors and secondary injury.Composite chitosan scaffolds loaded with controlled release of nerve growth factor can promote neuronal survival and axonal regeneration after short-segment sciatic nerve defects.However,the effects on extended nerve defects remain poorly understood.In this study,we used chitosan scaffolds loaded with nerve growth factor for 8 weeks to repair long-segment(20 mm)sciatic nerve defects in adult rats.The results showed that treatment markedly promoted the recovery of motor and sensory functions.The regenerated sciatic nerve not only reconnected with neurons but neural circuits with the central nervous system were also reconstructed.In addition,the regenerated sciatic nerve reconnected the motor endplate with the target muscle.Therefore,this novel biomimetic scaffold can promote the regeneration of extended sciatic nerve defects and reconstruct functional circuits.This provides a promising method for the clinical treatment of extended peripheral nerve injury.This study was approved by the Animal Ethics Committee of Capital Medical University,China(approval No.AEEI-2017-033)on March 21,2017.     

Reinnervation of the gastrocnemius muscle by the contralateral S1 nerve root

Functional regeneration after transposition of a ventral nerve root was established in the adult cat. Reconstruction of the ventral root, using microsurgical methods, directed the right S1 ventral nerve root to innervate the left gastrocnemius muscle. Stimulus-induced unit responses were recorded from the left gastrocnemius muscle 5 to 8 months after the root cross, demonstrating the reestablishment of neuromuscular connections. The innervation of the left gastrocnemius muscle by neurons in the right ventral horn of the spinal cord was verified by injecting horseradish peroxidase into the muscle. Horseradish peroxidase reaction product was located in alpha and gamma motor neurons in the right S1 segment of the spinal cord. Computer-assisted determination of the soma area of the labeled neurons was compared with a normal S1 innervation of the gastrocnemius muscle. Analysis of the percentage of cells of a given soma area demonstrated an overall decrease in soma area in the operated animals. Because ventral root reconstruction can result in innervation of a foreign muscle, studies such as this may encourage repair or reconstruction of nerve roots to gain some functional recovery after spinal cord or nerve root injuries.     

Axonal regeneration induced by blockade of glial inhibitors coupled with activation of intrinsic neuronal growth pathways

Several pharmacological approaches to promote neural repair and recovery after CNS injury have been identified. Blockade of either astrocyte-derived chondroitin sulfate proteoglycans (CSPGs) or oligodendrocyte-derived NogoReceptor (NgR1) ligands reduces extrinsic inhibition of axonal growth, though combined blockade of these distinct pathways has not been tested. The intrinsic growth potential of adult mammalian neurons can be promoted by several pathways, including pre-conditioning injury for dorsal root ganglion (DRG) neurons and macrophage activation for retinal ganglion cells (RGCs). Singly, pharmacological interventions have restricted efficacy without foreign cells, mechanical scaffolds or viral gene therapy. Here, we examined combinations of pharmacological approaches and assessed the degree of axonal regeneration. After mouse optic nerve crush injury, NgR1-/- neurons regenerate RGC axons as extensively as do zymosan-injected, macrophage-activated WT mice. Synergistic enhancement of regeneration is achieved by combining these interventions in zymosan-injected NgR1-/- mice. In rats with a spinal dorsal column crush injury, a preconditioning peripheral sciatic nerve axotomy, or NgR1(310)ecto-Fc decoy protein treatment or ChondroitinaseABC (ChABC) treatment independently support similar degrees of regeneration by ascending primary afferent fibers into the vicinity of the injury site. Treatment with two of these three interventions does not significantly enhance the degree of axonal regeneration. In contrast, triple therapy combining NgR1 decoy, ChABC and preconditioning, allows axons to regenerate millimeters past the spinal cord injury site. The benefit of a pre-conditioning injury is most robust, but a peripheral nerve injury coincident with, or 3days after, spinal cord injury also synergizes with NgR1 decoy and ChABC. Thus, maximal axonal regeneration and neural repair are achieved by combining independently effective pharmacological approaches.     

Acute treatment with pulsed electromagnetic fields and its effect on fast axonal transport in normal and regenerating nerve

The mechanism whereby low-frequency electromagnetic fields accelerate axonal regrowth and regeneration of peripheral nerve after crush lesion is not known. One candidate is an alteration in axonal transport. In this study we exposed unoperated rats for 15 min/day, and rats that had undergone a crush lesion of the sciatic nerve, for 1 hr/day for 2 days, to 2-Hz pulsed electromagnetic fields. To label fast transported proteins, [³H]-proline was microinjected into the spinal cord, and the sciatic nerves were removed 2, 3.5, and 5 hr later. The rates of fast axonal transport were obtained for animals in all groups by counting sequential 2-mm segments of nerves. The following transport rates were found: in unoperated normal sciatic nerve not exposed to PEMF, 373 ± 14 mm/day; in unoperated normal nerve exposed to PEMF, 383 ± 14 mm/day; in sham crush nerves not exposed to PEMF, 379 ± 19 mm/day; in sham crush nerve exposed to PEMF, 385 ± 17 mm/day; in crushed nerves not exposed to PEMF, 393 ± 16 mm/ day; and in crushed nerves exposed to PEMF, 392 ± 15 mm/day. The results of these experiments indicate that (1) a crush injury to the sciatic nerve does not alter the rate of fast axonal transport, and (2) low-frequency pulsed electromagnetic fields do not alter fast axonal transport rates in operated (crush) or unoperated sciatic nerves. © 1995 Wiley-Liss, Inc.     

A blind controlled trial of adrenocorticotropin and cerebral gangliosides in nerve regeneration in the rat

Rapid axonal regeneration and successful reinnervation of muscle and sensory end organs are the requisites for effective recovery from nerve lesions. Axonal regeneration has been reportedly accelerated by cerebral gangliosides and ACTH. We measured the rate of regeneration of sensory axons in rats after sciatic nerve crush by using the pinch test. We found no difference between rats treated with ACTH or gangliosides and the control group.     

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.