Long-term changes in neurotrophic factor expression in distal nerve stump following denervation and reinnervation with motor or sensory nerve

Several factors have been proposed to account for poor motor recovery after prolonged denervation, including motor neuron cell death and incomplete or poor regeneration of motor fibers into the muscle. Both may result from failure of the muscle and the distal motor nerve stump to continue expression of neurotrophic factors following delayed muscle reinnervation. This study investigated whether regenerating motor or sensory axons modulate distal nerve neurotrophic factor expression. We found that transected distal tibial nerve up-regulated brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) mRNA, down-regulated neurotrophin-3 and ciliary neurotrophic factor mRNA, and that although these levels returned to normal with regeneration, the chronically denervated distal nerve stump continued to express these neurotrophic factors for at least 6 months following injury. A sensory nerve (the cutaneous saphenous nerve) sutured to distal tibial nerve lowered injury-induced BDNF and GDNF mRNA levels in distal stump, but repair with a mixed nerve (peroneal, containing muscle and cutaneous axons) was more effective. Repair with sensory or mixed nerves did not affect nerve growth factor or neurotrophin-3 expression. Thus, distal nerve contributed to a neurotrophic environment for nerve regeneration for at least 6 months, and sensory nerve repair helped normalize distal nerve neurotrophic factor mRNA expression following denervation. Furthermore, as BDNF and GDNF levels in distal stump increased following denervation and returned to control levels following reinnervation, their levels serve as markers for the status of regeneration by either motor or sensory nerve.     

The neurotrophin receptors, trkB and p75, differentially regulate motor axonal regeneration.

Neurotrophic factors that support neuronal survival are implicated in axonal regeneration after injury. Specifically, a strong role for BDNF in motor axonal regeneration has been suggested based on its pattern of expression after injury, as well as the expression of its receptors, trkB and p75. Despite considerable in vitro evidence, which demonstrate specific and distinct physiological responses elicited following trkB and p75 activation, relatively little is known about the function of these receptors in vivo. To investigate the roles of the trkB and p75 receptors in motor axonal regeneration, we have used a tibial (TIB)- common peroneal (CP) cross suture paradigm in p75 homozygous (-/-) knockout mice, trkB heterozygous (+/-) knockout mice, as well as in their wild-type controls. Contralateral intact TIB motoneurons, and axotomized TIB motoneurons that regenerated their axons 10 mm into the CP distal nerve stump were identified by fluorescent retrograde tracers and counted in the T11-L1 spinal segments. Regeneration was evaluated 2, 3, 4, 6, and 8 weeks after nerve repair. Compared to wild-type animals, there are significantly fewer intact TIB motoneurons in p75 (-/-), but not trkB (+/-) mice. The number of motoneurons that regenerated their axons was significantly increased in the p75 (-/-) knockout mice, but significantly attenuated in the trkB (+/-) mice compared to wild-type controls. These results suggest that p75 is important for motoneuronal survival during development, but p75 expression after injury serves to inhibit motor axonal regeneration. In addition, full expression of trkB is critical for complete axonal regeneration to proceed.     

The neurotrophin receptors,trkB and p75, differentially regulate motor axonal regeneration

Neurotrophic factors that support neuronal survival are implicated in axonal regeneration after injury. Specifically, a strong role for BDNF in motor axonal regeneration has been suggested based on its pattern of expression after injury, as well as the expression of its receptors, trkB and p75. Despite considerable in vitro evidence, which demonstrate specific and distinct physiological responses elicited following trkB and p75 activation, relatively little is known about the function of these receptors in vivo. To investigate the roles of the trkB and p75 receptors in motor axonal regeneration, we have used a tibial (TIB)‐ common peroneal (CP) cross suture paradigm in p75 homozygous (?/?) knockout mice, trkB heterozygous (+/?) knockout mice, as well as in their wild‐type controls. Contralateral intact TIB motoneurons, and axotomized TIB motoneurons that regenerated their axons 10 mm into the CP distal nerve stump were identified by fluorescent retrograde tracers and counted in the T11‐L1 spinal segments. Regeneration was evaluated 2, 3, 4, 6, and 8 weeks after nerve repair. Compared to wild‐type animals, there are significantly fewer intact TIB motoneurons in p75 (?/?), but not trkB (+/?) mice. The number of motoneurons that regenerated their axons was significantly increased in the p75 (?/?) knockout mice, but significantly attenuated in the trkB (+/?) mice compared to wild‐type controls. These results suggest that p75 is important for motoneuronal survival during development, but p75 expression after injury serves to inhibit motor axonal regeneration. In addition, full expression of trkB is critical for complete axonal regeneration to proceed. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 314–325, 2001     

Functional Recovery of Denervated Skeletal Muscle with Sensory or Mixed Nerve Protection: A Pilot Study

Functional recovery is usually poor following peripheral nerve injury when reinnervation is delayed. Early innervation by sensory nerve has been indicated to prevent atrophy of the denervated muscle. It is hypothesized that early protection with sensory axons is adequate to improve functional recovery of skeletal muscle following prolonged denervation of mixed nerve injury. In this study, four groups of rats received surgical denervation of the tibial nerve. The proximal and distal stumps of the tibial nerve were ligated in all animals except for those in the immediate repair group. The experimental groups underwent denervation with nerve protection of peroneal nerve (mixed protection) or sural nerve (sensory protection). The experimental and unprotected groups had a stage II surgery in which the trimmed proximal and distal tibial nerve stumps were sutured together. After 3 months of recovery, electrophysiological, histological and morphometric parameters were assessed. It was detected that the significant muscle atrophy and a good preserved structure of the muscle were observed in the unprotected and protective experimental groups, respectively. Significantly fewer numbers of regenerated myelinated axons were observed in the sensory-protected group. Enhanced recovery in the mixed protection group was indicated by the results of the muscle contraction force tests, regenerated myelinated fiber, and the results of the histological analysis. Our results suggest that early axons protection by mixed nerve may complement sensory axons which are required for promoting functional recovery of the denervated muscle natively innervated by mixed nerve.     

Effect of Delayed Peripheral Nerve Repair on Nerve Regeneration,Schwann Cell Function and Target Muscle Recovery

Despite advances in surgical techniques for peripheral nerve repair, functional restitution remains incomplete. The timing of surgery is one factor influencing the extent of recovery but it is not yet clearly defined how long a delay may be tolerated before repair becomes futile. In this study, rats underwent sciatic nerve transection before immediate (0) or 1, 3, or 6 months delayed repair with a nerve graft. Regeneration of spinal motoneurons, 13 weeks after nerve repair, was assessed using retrograde labeling. Nerve tissue was also collected from the proximal and distal stumps and from the nerve graft, together with the medial gastrocnemius (MG) muscles. A dramatic decline in the number of regenerating motoneurons and myelinated axons in the distal nerve stump was observed in the 3- and 6-months delayed groups. After 3 months delay, the axonal number in the proximal stump increased 2–3 folds, accompanied by a smaller axonal area. RT-PCR of distal nerve segments revealed a decline in Schwann cells (SC) markers, most notably in the 3 and 6 month delayed repair samples. There was also a progressive increase in fibrosis and proteoglycan scar markers in the distal nerve with increased delayed repair time. The yield of SC isolated from the distal nerve segments progressively fell with increased delay in repair time but cultured SC from all groups proliferated at similar rates. MG muscle at 3- and 6-months delay repair showed a significant decline in weight (61% and 27% compared with contra-lateral side). Muscle fiber atrophy and changes to neuromuscular junctions were observed with increased delayed repair time suggestive of progressively impaired reinnervation. This study demonstrates that one of the main limiting factors for nerve regeneration after delayed repair is the distal stump. The critical time point after which the outcome of regeneration becomes too poor appears to be 3-months.     

Nerve regeneration through side-to-side neurorrhaphy sites in a rat model: a new concept in peripheral nerve surgery

Despite great improvement and refinements in nerve repair techniques, there were still problems in repair of peripheral nerve injuries for which proximal stumps were not available. In these circumstances for which classic end-to-end neurorrhaphy was impossible, new treatment modalities, benefiting by an adjacent healthy nerve, have been under investigation to overcome this problem. Therefore, end-to-side nerve repair with its modifications came to view and axonal passages through this site were shown. Moreover, the results were unsatisfactory or necessitating sacrifice of another healthy nerve. Three groups, containing 10 rats each, were included in the study. First was the control group, with end-to-end repair of the peroneal nerve. Second was the end-to-side repair group, in which the distal stump of the peroneal nerve trunk was anastomosed to the lateral side of the tibial nerve. The third was the side-to-side repair group. In this technique, 1-mm diameter epineural windows, both from peroneal and tibial nerve trunks facing each other, were removed and side-to-side neurorrhaphy was performed. After 3 weeks, as the second step, the peroneal nerve was sectioned proximally. At 2, 4, 8, 12, 20, and 28 weeks, functional assessment of nerve regeneration was performed by using walking track analysis. The number of myelinated fibers and fiber diameters were measured and an electron microscopic evaluation was carried out. Statistically, both in morphometric and gait analysis, the differences in values between the groups were significant in favor of the control group, followed by the side-to-side group. The study showed that axonal passage was possible with side-to-side technique and the functional results were satisfactory and superior to the end-to-side technique. Continuous supply of neurotrophic factors from their target cells was the probable cause of superior functional return in side-to-side repair, because both joining nerves were intact and healthy during the anastomosis procedure and after 3 weeks. It was concluded that this technique could be indicated in salvage of nerves in cases for which any intermediate segments would be removed, as in tumor ablation surgery, harvesting of nerve grafts, or both.     

Cutaneous Collateral Axonal Sprouting Re-Innervates the Skin Component and Restores Sensation of Denervated Swine Osteomyocutaneous Alloflaps

Reconstructive transplantation such as extremity and face transplantation is a viable treatment option for select patients with devastating tissue loss. Sensorimotor recovery is a critical determinant of overall success of such transplants. Although motor function recovery has been extensively studied, mechanisms of sensory re-innervation are not well established. Recent clinical reports of face transplants confirm progressive sensory improvement even in cases where optimal repair of sensory nerves was not achieved. Two forms of sensory nerve regeneration are known. In regenerative sprouting, axonal outgrowth occurs from the transected nerve stump while in collateral sprouting, reinnervation of denervated tissue occurs through growth of uninjured axons into the denervated tissue. The latter mechanism may be more important in settings where transected sensory nerves cannot be re-apposed. In this study, denervated osteomyocutaneous alloflaps (hind- limb transplants) from Major Histocompatibility Complex (MHC)-defined MGH miniature swine were performed to specifically evaluate collateral axonal sprouting for cutaneous sensory re-innervation. The skin component of the flap was externalized and serial skin sections extending from native skin to the grafted flap were biopsied. In order to visualize regenerating axonal structures in the dermis and epidermis, 50um frozen sections were immunostained against axonal and Schwann cell markers. In all alloflaps, collateral axonal sprouts from adjacent recipient skin extended into the denervated skin component along the dermal-epidermal junction from the periphery towards the center. On day 100 post-transplant, regenerating sprouts reached 0.5 cm into the flap centripetally. Eight months following transplant, epidermal fibers were visualized 1.5 cm from the margin (rate of regeneration 0.06 mm per day). All animals had pinprick sensation in the periphery of the transplanted skin within 3 months post-transplant. Restoration of sensory input through collateral axonal sprouting can revive interaction with the environment; restore defense mechanisms and aid in cortical re-integration of vascularized composite allografts.     

Nerve interactions and regenerative processes occurring in newt limbs fused end-to-end

Peripheral nerve interactions and regenerative phenomena were studied in newt forelimbs fused end to end. After simple fusion, one or two spikelike structures regenerated at the plane of fusion in 88% of the cases. When one of the limbs was denervated at the time of fusion, no regeneration occurred from the plane of fusion. If the limbs were fused and one was amputated at the shoulder more than 10 days after fusion, regeneration from the amputation surface did not occur. When the limbs were reamputated 30 days later, regeneration of left limbs from the proximodistally reversed right limb stumps followed. If one of the limbs was denervated at the time of fusion, and amputation was subsequently carried out through the formerly denervated limb, regeneration always took place after the first amputation. On the basis of these results it is postulated that when regenerating nerves of opposite proximodistal polarity meet head-on, the majority of fibers, at least, do not grow into territories occupied by the other nerve. These results have also demonstrated that full limb regeneration can occur at a greater distance from the midline than the end of a normal limb. These experiments also provide a technique for artificially elongating peripheral nerves.     

Regeneration of central and peripheral synaptic connections in the locomotor system of the pteropod molluscClione limacina

The neural network underlying rhythmic wing movements in the molluscClione limacina is well-studied. Two different groups of motoneurons innervate two distinct groups of wing muscles. The locomotor rhythm generated in the left and right pedal ganglia is synchronized by interneurons. When the axons of the locomotor motoneurons are crushed, numerous fine neurites sprout towards the denervated muscles and reach them in 8–15 days. At this stage motoneurons project to and synapse on not only correct but equally incorrect muscle targets. After 2 weeks of regeneration the number of incorrect neurites and synaptic connections begins to decrease and following 1.5–2 months all incorrect connections are eliminated, incorrect axons are withdrawn and the behavioral deficit is compensated. In this study the regeneration of interneurons and the growth profiles of inter- and motoneurons were also studiedin vitro. Two individually isolated pedal ganglia were co-cultured in three different configurations: a) the wing nerve stump from one ganglion was fixed against the commissural stump from another ganglion; b) the wing nerve stumps were fixed against each other; c) the commissural stumps were fixed against each other. Under the above experimental conditions we found that the interneurons were able to cross only the contact between two commissural stumps, and in this case found their original targets, restored correct connections and synchronized the rhythm in two pedal ganglia. In contrast, motoneurons were able to cross all types of contacts.     

Fate of 3H-thymidine labelled myogenic cells in regeneration of muscle isografts

Summary Intact and denervated extensor digitorum longus (EDL) muscles of 20-day-old inbred Lewis-Wistar rats were labelled with ³H-thymidine. Ninety minutes after the injection of the isotope 4.0% of the nuclei were labelled in the intact (i.e. innervated) and 9.6% in the muscles, denervated 3 days before administration of the isotope. The labelled EDL muscles were grafted into the bed of the previously removed EDL muscles of inbred animals and these isografts were studied 30 days later.In the EDL muscles, regenerated from innervated isografts only occasionally labelled endothelial cells were found whereas in the muscles regenerated from denervated isografts also parenchymal muscle nuclei were regularly labelled. The incidence of labelled nuclei in the regenerated EDL muscles was, however, about 20 times lower than in the donor EDL muscles. The present experiments provide a direct proof of utilization of donor satellite cell nuclei for regeneration in grafted muscle tissue. With respect to the low incidence of labelled nuclei in regenerated EDL muscles, other sources of cells apparently also contribute to the regeneration process.     

Epineurial Window Is More Efficient in Attracting Axons than Simple Coaptation in a Sutureless (Cyanoacrylate-Bound) Model of End-to-Side Nerve Repair in the Rat Upper Limb: Functional and Morphometric Evidences and Review of the Literature

End-to-side nerve coaptation brings regenerating axons from the donor to the recipient nerve. Several techniques have been used to perform coaptation: microsurgical sutures with and without opening a window into the epi(peri)neurial connective tissue; among these, window techniques have been proven more effective in inducing axonal regeneration. The authors developed a sutureless model of end-to-side coaptation in the rat upper limb. In 19 adult Wistar rats, the median and the ulnar nerves of the left arm were approached from the axillary region, the median nerve transected and the proximal stump sutured to the pectoral muscle to prevent regeneration. Animals were then randomly divided in two experimental groups (7 animals each, 5 animals acting as control): Group 1: the distal stump of the transected median nerve was fixed to the ulnar nerve by applying cyanoacrylate solution; Group 2: a small epineurial window was opened into the epineurium of the ulnar nerve, caring to avoid damage to the nerve fibres; the distal stump of the transected median nerve was then fixed to the ulnar nerve by applying cyanoacrylate solution. The grasping test for functional evaluation was repeated every 10–11 weeks starting from week-15, up to the sacrifice (week 36). At week 36, the animals were sacrificed and the regenerated nerves harvested and processed for morphological investigations (high-resolution light microscopy as well as stereological and morphometrical analysis). This study shows that a) cyanoacrylate in end-to-side coaptation produces scarless axon regeneration without toxic effects; b) axonal regeneration and myelination occur even without opening an epineurial window, but c) the window is related to a larger number of regenerating fibres, especially myelinated and mature, and better functional outcomes.     

Degeneration and Regeneration in Crustacean Neuromuscular Systems

Crayfish motor neurons seem to repair damage to peripheral axonsby selective fusion of outgrowing proximal stumps with severeddistal processes that can survive morphologically and physiologicallyintact for over 200 days. Survival of isolated motor and CNSgiant axons is associated with much hypertrophy of their glialsheath. The severed stumps of peripheral sensory neurons oftendegenerate within 21 days and their glial sheath does not hypertrophy.Denervation and immobilization produce relatively little changein the morphology and physiology of the opener muscle, whereastenotomy produces much atrophy within 30-60 days. Crayfish motor and CNS giant neurons show no capability forregenerating ablated cell bodies, whereas peripheral sensorysomata regenerate after limb autotomy. An entire opener musclecan be replaced after limb autotomy but the organism shows littleor no ability to redifferentiate an entire muscle in the absenceof body part regeneration. However, a few opener muscle fiberscan be regenerated if the bulk of the muscle mass remains intact.The significance of all these findings are interpreted withrespect to the developmental capabilities and environmentaladaptations of the crayfish together with the evolution of regenerativeabilities in anthropods and vertebrates.     

Laminin B1 and Collagen Type IV Gene Expression in Transected Peripheral Nerve: Reinnervation Compared to Denervation

The expression of B1 laminin and type IV collagen was followed in the microsurgically isolated endoneurium of transected rat sciatic nerves from 3 days until 8 weeks. Northern hybridizations revealed that after nerve transection the proximal stumps of denervated, as well as freely regenerating, nerves showed a markedly increased expression of laminin and type IV collagen which lasted from 3 days up to 8 weeks. In the distal stumps, close to the site of transection (2-7 mm), the expression of laminin, and to a certain extent that of type IV collagen, seemed to be enhanced if free axonal reinnervation was allowed. Further distally (10-15 mm), the patterns of B1 laminin and type IV collagen expression were similar in both experimental groups, so that an increased expression was noticed during the first 2 weeks. The present results suggest that laminin and type IV collagen gene expression is markedly different in different parts of transected rat sciatic nerve. During peripheral nerve regeneration, there is a long-lasting basement membrane gene expression in the proximal stump. In the distal part of the transected nerve, the axonal reinnervation possibly up-regulates, but is not essential for, the expression of B1 laminin and type IV collagen.     

Collateral sprouting occurs following end-to-side neurorrhaphy

Recent evidence supports the use of end-to-side neurorrhaphy for the treatment of certain peripheral nerve disorders. However, the mechanism by which nerves regenerate following this procedure is still unclear. To address this question, the authors designed a new end-to-side coaptation model in rats in which the donor nerves were uninjured. The regenerated axons at the coaptation site were observed directly using fluorescent dye as the neural tracer. The sciatic nerve from adult Wistar rats was transplanted between the left and right median nerves. Fifteen rats were divided into three groups. In group I, the donor (right median) nerve was sutured end to side to the divided grafted nerve using a noninjury technique. In group II, the aponeurosis of the spinal muscles was harvested and the sciatic and right median nerves were coapted end to side noninjuriously by wrapping them in the excised aponeurosis. In group III, a perineurial window was created and a partial neurectomy was carried out at the suture site, after which the sciatic and right median nerves were sutured end to side. Sixty days after the operation, nerve regeneration was evaluated by recording action potentials in the grafted nerve, by performing electromyography in the flexor muscles in the forearm, and by histological examination. The grafted nerves were fixed and sectioned, the number of regenerated nerve fibers was counted, and axonal diameters were measured. Fluorescent dye crystal was used, in conjunction with confocal microscopy, to observe the regenerated axons at the co-aptation site. The results showed that nerve regeneration had occurred in the animals, as determined electrophysiologically and histologically. Both the right and left flexor muscles of the forearm contracted simultaneously as a result of indirect electric stimulation of the grafted nerve, which suggests that the regenerated nerve was physiologically connected with the donor nerve. Nerve fiber counts did not show any differences among groups (p > 0.05), but axonal diameters were significantly greater in group III than in the other two groups. Fluorescent dye staining revealed the presence of regenerated nerve fibers beyond the coaptation site. In group III, the regenerating nerves were observed within the whole section of the coaptation site and collateral sprouting was found to occur even at a site distal to the suture. From these results, the authors conclude that in end-to-side neurorrhaphy, nerve regeneration occurs by collateral sprouting from the donor nerve.     

Consequences of slow Wallerian degeneration for regenerating motor and sensory axons.

The time course of Wallerian degeneration in the tibial and saphenous nerves was compared in Balb/c mice and mice of the C57BL/Ola strain (Lunn et al., 1989). Axons, particularly myelinated ones, in nerves of C57BL/Ola mice are very slow to degenerate, many still being present 3 weeks after axotomy. Nuclear numbers in the distal stump peak much later and do not reach the levels found in Balb/c mice; debris removal is very slow, and Schwann cell numbers only rise slightly above normal levels in the long term. Regeneration was investigated electrophysiologically and by electron microscopy (EM). Myelinated sensory axons regenerated slowly and incompletely compared with motor ones which were only slightly slowed after nerve crush (although they were significantly hindered after nerve section). Total myelinated axon numbers were still some 20% less than normal even after 200 days in sensory nerves. Even after all axons had degenerated in C57BL/Ola mice, regeneration rates of neither myelinated nor unmyelinated sensory axons reached those achieved in Balb/c mice. It is concluded that while regeneration can eventually proceed slowly when Wallerian degeneration is much delayed, the usual rapid time course of Wallerian degeneration is necessary if axons, particularly sensory ones, are to regenerate at optimal rates and to maximum extent. While local obstruction to axon growth probably impedes the early phase of regeneration in C57BL/Ola mice, it seems possible that a lack of adequate early signals affects regeneration permanently by minimizing the cell body reaction to injury.     

Enhancement of peripheral nerve regeneration due to treadmill training and electrical stimulation is dependent on androgen receptor signaling

Moderate exercise in the form of treadmill training and brief electrical nerve stimulation both enhance axon regeneration after peripheral nerve injury. Different regimens of exercise are required to enhance axon regeneration in male and female mice (Wood et al.: Dev Neurobiol 72 (2012) 688–698), and androgens are suspected to be involved. We treated mice with the androgen receptor blocker, flutamide, during either exercise or electrical stimulation, to evaluate the role of androgen receptor signaling in these activity‐based methods of enhancing axon regeneration. The common fibular (CF) and tibial (TIB) nerves of thy‐1‐YFP‐H mice, in which axons in peripheral nerves are marked by yellow fluorescent protein (YFP), were transected and repaired using CF and TIB nerve grafts harvested from non‐fluorescent donor mice. Silastic capsules filled with flutamide were implanted subcutaneously to release the drug continuously. Exercised mice were treadmill trained 5 days/week for 2 weeks, starting on the third day post‐transection. For electrical stimulation, the sciatic nerve was stimulated continuously for 1 h prior to nerve transection. After 2 weeks, lengths of YFP+ profiles of regenerating axons were measured from harvested nerves. Both exercise and electrical stimulation enhanced axon regeneration, but this enhancement was blocked completely by flutamide treatments. Signaling through androgen receptors is necessary for the enhancing effects of treadmill exercise or electrical stimulation on axon regeneration in cut peripheral nerves. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 531–540, 2014     

Neural Stem Cells Enhance Nerve Regeneration after Sciatic Nerve Injury in Rats

With the development of tissue engineering and the shortage of autologous nerve grafts in nerve reconstruction, cell transplantation in a conduit is an alternative strategy to improve nerve regeneration. The present study evaluated the effects and mechanism of brain-derived neural stem cells (NSCs) on sciatic nerve injury in rats. At the transection of the sciatic nerve, a 10-mm gap between the nerve stumps was bridged with a silicon conduit filled with 5?×?10⁵ NSCs. In control experiments, the conduit was filled with nerve growth factor (NGF) or normal saline (NS). The functional and morphological properties of regenerated nerves were investigated, and expression of hepatocyte growth factor (HGF) and NGF was measured. One week later, there was no connection through the conduit. Four or eight weeks later, fibrous connections were evident between the proximal and distal segments. Motor function was revealed by measurement of the sciatic functional index (SFI) and sciatic nerve conduction velocity (NCV). Functional recovery in the NSC and NGF groups was significantly more advanced than that in the NS group. NSCs showed significant improvement in axon myelination of the regenerated nerves. Expression of NGF and HGF in the injured sciatic nerve was significantly lower in the NS group than in the NSCs and NGF groups. These results and other advantages of NSCs, such as ease of harvest and relative abundance, suggest that NSCs could be used clinically to enhance peripheral nerve repair.     

Visualizing Peripheral Nerve Regeneration by Whole Mount Staining

Peripheral nerve trauma triggers a well characterised sequence of events both proximal and distal to the site of injury. Axons distal to the injury degenerate, Schwann cells convert to a repair supportive phenotype and macrophages enter the nerve to clear myelin and axonal debris. Following these events, axons must regrow through the distal part of the nerve, re-innervate and finally are re-myelinated by Schwann cells. For nerve crush injuries (axonotmesis), in which the integrity of the nerve is maintained, repair may be relatively effective whereas for nerve transection (neurotmesis) repair will likely be very poor as few axons may be able to cross between the two parts of the severed nerve, across the newly generated nerve bridge, to enter the distal stump and regenerate. Analysing axon growth and the cell-cell interactions that occur following both nerve crush and cut injuries has largely been carried out by staining sections of nerve tissue, but this has the obvious disadvantage that it is not possible to follow the paths of regenerating axons in three dimensions within the nerve trunk or nerve bridge. To try and solve this problem, we describe the development and use of a novel whole mount staining protocol that allows the analysis of axonal regeneration, Schwann cell-axon interaction and re-vascularisation of the repairing nerve following nerve cut and crush injuries.     

Deletion of p75NTR impairs regeneration of peripheral nerves in mice

AimsAfter peripheral nerve injury, p75NTR was upregulated in Schwann cells of the Wallerian degenerative nerves and in motor neurons but down-regulated in the injured sensory neurons. As p75NTR in neurons mediates signals of both neurotrophins and inhibitory factors, it is regarded as a therapeutic target for the treatment of neurodegeneration. However, its physiological function in the nerve regeneration is not fully understood. In the present study, we aimed to examine the role of p75NTR in the regeneration of peripheral nerves.Main methodsIn p75NTR knockout mice (exon III deletion), the sciatic nerves and facial nerves on one side were crushed and regenerating neurons in the facial nuclei and in the dorsal root ganglia were labelled by Fast Blue. The regenerating fibres in the sciatic nerve were also labelled by an anterograde tracer and by immunohistochemistry.Key findingsThe results showed that the axonal growth of injured axons in the sciatic nerve of p75NTR mutant mice was significantly retarded. The number of regenerated neurons in the dorsal root ganglia and in the facial nuclei in p75NTR mutant mice was significantly reduced. Immunohistochemical staining of regenerating axons also showed the reduction in nerve regeneration in p75NTR mutant mice.SignificanceOur data suggest that p75NTR plays an important role in the regeneration of injured peripheral nerves.     

High-Frequency Electrical Stimulation Can Be a Complementary Therapy to Promote Nerve Regeneration in Diabetic Rats

The purpose of this study was to evaluate whether 1 mA of percutaneous electrical stimulation (ES) at 0, 2, 20, or 200 Hz augments regeneration between the proximal and distal nerve stumps in streptozotocin diabetic rats. A10-mm gap was made in the diabetic rat sciatic nerve by suturing the stumps into silicone rubber tubes. Normal animals were used as the controls. Starting 1 week after transection, ES was applied between the cathode placed at the distal stump and the anode at the proximal stump every other day for 3 weeks. At 4 weeks after surgery, the normal controls and the groups receiving ES at 20, and 200 Hz had a higher success percentage of regeneration compared to the ES groups at 0 and 2 Hz. In addition, quantitative histology of the successfully regenerated nerves revealed that the groups receiving ES at a higher frequency, especially at 200 Hz, had a more mature structure with more myelinated fibers compared to those in the lower-frequency ES groups. Similarly, electrophysiology in the ES group at 200 Hz showed significantly shorter latency, larger amplitude, larger area of evoked muscle action potentials and faster conduction velocity compared to other groups. Immunohistochemical staining showed that ES at a higher frequency could significantly promote calcitonin gene-related peptide expression in lamina I-II regions in the dorsal horn and recruit a higher number of macrophages in the diabetic distal sciatic nerve. The macrophages were found that they could stimulate the secretion of nerve growth factor, platelet-derived growth factor, and transforming growth factor-β in dissected sciatic nerve segments. The ES at a higher frequency could also increase cutaneous blood flow in the ipsilateral hindpaw to the injury. These results indicated that a high-frequency ES could be necessary to heal severed diabetic peripheral nerve with a long gap to be repaired.