Guidance of regenerating motor axons in vivo by gradients of diffusible peripheral nerve‐derived factors

During development of the central nervous system, neurons rely on target‐derived factors to guide their outgrowing processes. Several CNS target‐derived chemoattractive and repellant factors have been isolated and characterized, and their mechanism of action determined. For the peripheral nervous system, the results from numerous experiments suggest that during regeneration axons also respond to concentration gradients of target‐derived factors leading to an oriented outgrowth up the gradient to the denervated target in vivo. The results from in vitro experiments have shown that diffusible concentration gradients of factors released from a length of denervated peripheral nerve, composed predominantly of Schwann cells, direct the outgrowth of sensory and motor neuron growth cones over distances of several hundred microns. However, a conclusive demonstration of a chemoattractive influence of diffusible concentration gradients on regenerating adult motor axons in vivo has remained elusive. The present experiments show that concentration gradients of denervated peripheral nerve‐released factors direct the regeneration of adult motor axons in vivo, and that these gradients are effective over distances of more than 6.5 mm. Nonconditioned medium exerted no influence on the regenerating axons. Thus, results from in vivo experiments parallel those from in vitro experiments and indicate that isolated peripheral nerve‐released factors that are effective in vitro will play a similar role on sensory and motor axons in vivo. Finally, the results show that diffusible concentration gradients of target‐derived factors direct axon outgrowth both during both development and regeneration, as well as in vivo and in vitro. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 212–219, 2000     

The branchial arches and HGF are growth-promoting and chemoattractant for cranial motor axons

During development, cranial motor neurons extend their axons along distinct pathways into the periphery. For example, branchiomotor axons extend dorsally to leave the hindbrain via large dorsal exit points. They then grow in association with sensory ganglia, to their targets, the muscles of the branchial arches. We have investigated the possibility that pathway tissues might secrete diffusible chemorepellents or chemoattractants that guide cranial motor axons, using co-cultures in collagen gels. We found that explants of dorsal neural tube or hindbrain roof plate chemorepelled cranial motor axons, while explants of cranial sensory ganglia were weakly chemoattractive. Explants of branchial arch mesenchyme were strongly growth-promoting and chemoattractive for cranial motor axons. Enhanced and oriented axon outgrowth was also elicited by beads loaded with Hepatocyte Growth Factor (HGF); antibodies to this protein largely blocked the outgrowth and orientation effects of the branchial arch on motor axons. HGF was expressed in the branchial arches, whilst Met, which encodes an HGF receptor, was expressed by subpopulations of cranial motor neurons. Mice with targetted disruptions of HGF or Met showed defects in the navigation of hypoglossal motor axons into the branchial region. Branchial arch tissue may thus act as a target-derived factor that guides motor axons during development. This influence is likely to be mediated partly by Hepatocyte Growth Factor, although a component of branchial arch-mediated growth promotion and chemoattraction was not blocked by anti-HGF antibodies.     

The outgrowth response of the axons of developing and regenerating rat retinal ganglion cells in vitro to neurotrophin treatment

BDNF and NT-4 (but not NT-3 or CNTF) significantly enhanced the outgrowth of early embryonic and adult regenerating RGC axons when provided with a supportive substrate in vitro. BDNF and NT-4 treatment transiently increased RGC axon outgrowth from E15 rat retinas but not from retinas at older embryonic ages. The transient effect of BDNF and NT-4 and the inability of the neurotrophins to promote outgrowth from older embryonic retinal explants suggests a time frame of neurotrophin action and that other chemical factors (target-derived or otherwise) may be necessary for the continued maintenance of developing RGC axons. BDNF and NT-4 also enhanced the outgrowth of regenerating axons from adult retinal explants, but appeared to have a more subtle effect on axon outgrowth, in that the growth-promoting effects of BDNF and NT-4 appeared continuous throughout the incubation period. The suppression of RGC axon outgrowth from embryonic and adult retinae cultured in trkB-IgG-containing medium suggests that the response of developing and regenerating axons, to BDNF and NT-4 are likely to occur through trkB signalling.     

Changes in cytoskeletal protein synthesis following axon injury and during axon regeneration

Injury to the axons of facial motoneurons stimulates increases in the synthesis of actin, tubulins, and GAP-43, and decreases in the synthesis of neurofilament proteins: mRNA levels change correspondingly. In contrast to this robust response of peripheral neurons to axotomy, injured central nervous system neurons show either an attenuated response that is subsequently aborted (rubrospinal neurons) or overall decreases in cytoskeletal protein mRNA expression (corticospinal and retinal ganglion neurons). There is evidence that these changes in synthesis are regulated by a variety of factors, including loss of endoneurially or target-derived trophic factors, positive signals arising from the site of injury, changes in the intraaxonal turnover of proteins, and substitution of target-derived trophic support by factors produced by glial cells. It is concluded that there is, as yet, no coherent explanation for the upregulation or downregulation of any of the cytoskeletal proteins following axotomy or during regeneration. In considering the relevance of these changes in cytoskeletal protein synthesis to regeneration, it is emphasized that they are unlikely to be involved in the initial outgrowth of the injured axons, both because transit times between cell body and injury site are too long, and because sprouting can occur in isolated axons. Injuryinduced acceleration of the axonal transport of tubulin and actin in the proximal axon is likely to be more important in providing the cytoskeletal protein required for initial axonal outgrowth. Subsequently, the increased synthesis and transport velocity for actin and tubulin increase the delivery of these proteins to support the increased volume of the maturing regenerating axons. Reduction in neurofilament synthesis and changes in neurofilament phosphorylation may permit the increased transport velocity of the other cytoskeletal proteins. There is little direct evidence that alterations in cytoskeletal protein synthesis are necessary for successful regeneration, nor are they sufficient in the absence of a supportive environment. Nevertheless, the correlation that exists between a robust cell body response and successful regeneration suggests that an understanding of the regulation of cytoskeletal protein synthesis following axon injury must be a part of any successful strategy to improve the regenerative capacity of the central nervous system.     

Target-derived GFRalpha1 as an attractive guidance signal for developing sensory and sympathetic axons via activation of Cdk5

Immobilized and diffusible molecular cues regulate axon guidance during development. GFRalpha1, a GPI-anchored receptor for GDNF, is expressed as both membrane bound and secreted forms by accessory nerve cells and peripheral targets of developing sensory and sympathetic neurons during the period of target innervation. A relative deficit of GFRalpha1 in developing axons allows exogenous GFRalpha1 to capture GDNF and present it for recognition by axonal c-Ret receptors. Exogenous GFRalpha1 potentiates neurite outgrowth and acts as a long-range directional cue by creating positional information for c-Ret-expressing axons in the presence of a uniform concentration of GDNF. Soluble GFRalpha1 prolongs GDNF-mediated activation of cyclin-dependent kinase 5 (Cdk5), an event required for GFRalpha1-induced neurite outgrowth and axon guidance. Together with GDNF, target-derived GFRalpha1 can function in a non-cell-autonomous fashion as a chemoattractant cue with outgrowth promoting activity for peripheral neurons.     

Neurite outgrowth on cryostat sections of innervated and denervated skeletal muscle

To localize factors that guide axons reinnervating skeletal muscle, we cultured ciliary ganglion neurons on cryostat sections of innervated and denervated adult muscle. Neurons extended neurites on sections of muscle (and several other tissues), generally in close apposition to sectioned cell surfaces. Average neurite length was greater on sections of denervated than on sections of innervated muscle, supporting the existence of functionally important differences between innervated and denervated muscle fiber surfaces. Furthermore, outgrowth was greater on sections of denervated muscle cut from endplate-rich regions than on sections from endplate-free regions, suggesting that a neurite outgrowth-promoting factor is concentrated near synapses. Finally, 80% of the neurites that contacted original synaptic sites (which are known to be preferentially reinnervated by regenerating axons in vivo) terminated precisely at those contacts, thereby demonstrating a specific response to components concentrated at endplates. Together, these results support the hypothesis that denervated muscles use cell surface (membrane and matrix) molecules to inform regenerating axons of their state of innervation and proximity to synaptic sites.     

Influence of factors released from sciatic nerve on adult dorsal root ganglion neurons

Previous experiments have shown that medium conditioned (CM) by denervated peripheral nerve contains a process outgrowth promoting factor (s) for cultured adult frog dorsal root ganglion (DRG) neurons. The present experiments further characterize the influences of these factors on DRG neurons. The growth factors increases average process length by threefold, restricts the number of processes extended from four to two while simultaneously altering the morphology of those processes. Neurons with preexisting processes respond to the factors by significantly increasing the length of 35% of these processes. Only the newly elongated portions of preexisting processes have morphology typical of factor-induced processes, while the previously extended portions retain their original morphology. The number of processes of these neurons remains unchanged. Although composed of two population according to size, neurons in both populations are similarly influenced, suggesting that the factors influence neurons of all sensory modalities. To look at other possible influences of the nerve-released factors, a novel simple culture system has been developed in which concentration gradients of these factors can be established and maintained. The front of the outgrowth-promoting influence in these cultures could be followed over time (up to 9 days) as it affected the process length and morphology of neurons at increasing distances (up to 8 mm) from the source of the factors. The trophic factors may play important roles during regeneration in vivo by influencing the cytoskeletal organization in the cell body and growth cones to bring about a stabilization and consolidation of growth cone membrane of only a limited number of processes resulting in increasing the rate of process elongation. The factors may also serve to direct process outgrowth, which can be examined using the new culture system. 1994 John Wiley & Sons, Inc.     

Expression of NGF receptor and GAP-43 mRNA in DRG neurons during collateral sprouting and regeneration of dorsal cutaneous nerves

The collateral sprouting of intact sensory axons and the regenertion of damaged ones differ in a number of respects. Regeneration is triggered by axotomy-induced damage, probably involves the loss of a peripheral signal, and appears to occur independently of NGF, while collateral sprouting is evoked and sustained by an increase in a target-driven signal, namely NGF. New findings strengthen the distinction between these two phenomena. Nerve growth factor receptor (NGFR) mRNA is increased in undamaged DRG neurons whose axons are sprouting into denervated skin. This response is related to an increased availablity of target-derived NGF, a proposal supported by a number of findings including increased NGF mRNA in the denervated target. In contrast, we observed little or no change in the NGFR mRNA levels in regenerating neurons, consistent with the observations that NGF does not play a role in this process. However, increases in neuronal GAP-43 mRNA are found during both regeneration and collateral sprouting, a result in keeping with the proposal that GAP-43 is primarily associated with nerve growth, and the observation that GAP-43 expression is not especially influenced by NGF. 1994 John Wiley & Sons, Inc.     

Androgen regulation of axon growth and neurite extension in motoneurons

Androgens act on the CNS to affect motor function through interaction with a widespread distribution of intracellular androgen receptors (AR). This review highlights our work on androgens and process outgrowth in motoneurons, both in vitro and in vivo. The actions of androgens on motoneurons involve the generation of novel neuronal interactions that are mediated by the induction of androgen-dependent neurite or axonal outgrowth. Here, we summarize the experimental evidence for the androgenic regulation of the extension and regeneration of motoneuron neurites in vitro using cultured immortalized motoneurons, and axons in vivo using the hamster facial nerve crush paradigm. We place particular emphasis on the relevance of these effects to SBMA and peripheral nerve injuries.     

Okadaic Acid and Cultured Frog Sciatic Nerves: Potent Inhibition of Axonal Regeneration in Spite of Unaffected Schwann Cell Proliferation and Ganglionic Protein Synthesis

Abstract: Okadaic acid (OA) is a frequently used phosphatase inhibitor that by inhibiting dephosphorylation increases the net phosphorylation level in various systems. In the present study OA was used to assess the role of balanced phosphorylation-dephosphorylation reactions for successful regeneration of peripheral nerves. To achieve this, the effects of OA on phosphorylation levels, neurite outgrowth, injury-induced support cell proliferation, and neurofilament stability, respectively, were investigated in the in vitro regenerating, adult frog sciatic sensory nerve. OA at a moderate concentration (20 n M ) increased phosphorylation levels and almost completely inhibited the in vitro regeneration in a reversible way. The effect on regeneration was not due to induced neurofilament instability and was only seen when the drug was applied in the outgrowth region. The latter and the absence of effects on support cell proliferation indicate that OA acts locally at the level of newly formed axons. However, the inhibition of regeneration was not a consequence of reduced delivery of proteins by axonal transport, because this process in fact was increased by OA. Altogether, the study suggests that properly balanced phosphorylating-dephosphorylating reactions are critical for regeneration of peripheral nerves.     

VEGF-A and Semaphorin3A: Modulators of vascular sympathetic innervation

Sympathetic nerve activity regulates blood pressure by altering peripheral vascular resistance. Variations in vascular sympathetic innervation suggest that vascular-derived cues promote selective innervation of particular vessels during development. As axons extend towards peripheral targets, they migrate along arterial networks following gradients of guidance cues. Collective ratios of these gradients may determine whether axons grow towards and innervate vessels or continue past non-innervated vessels towards peripheral targets. Utilizing directed neurite outgrowth in a three-dimensional (3D) co-culture, we observed increased axon growth from superior cervical ganglion explants (SCG) towards innervated compared to non-innervated vessels, mediated in part by vascular endothelial growth factor (VEGF-A) and Semaphorin3A (Sema3A) which both signal via neuropilin-1 (Nrp1). Exogenous VEGF-A, delivered by high-expressing VEGF-A-LacZ vessels or by rhVEGF-A/alginate spheres, increased sympathetic neurite outgrowth while exogenous rhSema3A/Fc decreased neurite outgrowth. VEGF-A expression is similar between the innervated and non-innervated vessels examined. Sema3A expression is higher in non-innervated vessels. Spatial gradients of Sema3A and VEGF-A may promote differential Nrp1 binding. Vessels expressing high levels of Sema3A favor Nrp1-PlexinA1 signaling, producing chemorepulsive cues limiting sympathetic neurite outgrowth and vascular innervation; while low Sema3A expressing vessels favor Nrp1-VEGFR2 signaling providing chemoattractive cues for sympathetic neurite outgrowth and vascular innervation.     

Motor function recovery: deciphering a regenerative niche at the neuromuscular synapse

The coordinated movement of many organisms relies on efficient nerve–muscle communication at the neuromuscular junction (NMJ), a peripheral synapse composed of a presynaptic motor axon terminal, a postsynaptic muscle specialization, and non-myelinating terminal Schwann cells. NMJ dysfunctions are caused by traumatic spinal cord or peripheral nerve injuries as well as by severe motor pathologies. Compared to the central nervous system, the peripheral nervous system displays remarkable regenerating abilities; however, this capacity is limited by the denervation time frame and depends on the establishment of permissive regenerative niches. At the injury site, detailed information is available regarding the cells, molecules, and mechanisms involved in nerve regeneration and repair. However, a regenerative niche at the final functional step of peripheral motor innervation, i.e. at the mature neuromuscular synapse, has not been deciphered. In this review, we integrate classic and recent evidence describing the cells and molecules that could orchestrate a dynamic ecosystem to accomplish successful NMJ regeneration. We propose that such a regenerative niche must ensure at least two fundamental steps for successful NMJ regeneration: the proper arrival of incoming regenerating axons to denervated postsynaptic muscle domains, and the resilience of those postsynaptic domains, in morphological and functional terms. We here describe and combine the main cellular and molecular responses involved in each of these steps as potential targets to help successful NMJ regeneration.     

Promoting and directing axon outgrowth

Establishment of appropriate neuronal connections during development and regeneration requires the extension of processes that must then grow in the correct direction, find and recognize their targets, and make synapses with them. During development, embryonic neurons gradually establish central and peripheral connections in an evolving cellular environment in which neurotrophic factors are provided by supporting and target cells that promote neuronal survival, differentiation, and process outgrowth. Some cells also release neurotropic factors that direct the outgrowth of neuronal processes toward their targets. Following development the neurotrophic requirements of some adult neurons change so that, although they respond to neurotrophic factors, they no longer require exogenous neurotrophins to survive or to extend processes. Within the central nervous system (CNS), the ability of neurons to extend processes is eventually lost because of a change in their cellular environment from outgrowth permissive to inhibitory. Thus, neuronal connections that are lost in the adult CNS are rarely reestablished. In contrast, the environment of the adult peripheral nervous system fosters process outgrowth and synapse formation. This article discusses the neurotrophic requirements of embryonic and adult neurons, as well as the importance of neurotropic factors in directing the outgrowth of regenerating adult axons.     

GDNF and neurturin are target-derived factors essential for cranial parasympathetic neuron development

During development, parasympathetic ciliary ganglion neurons arise from the neural crest and establish synaptic contacts on smooth and striate muscle in the eye. The factors that promote the ciliary ganglion pioneer axons to grow toward their targets have yet to be determined. Here, we show that glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN) constitute target-derived factors for developing ciliary ganglion neurons. Both GDNF and NRTN are secreted from eye muscle located in the target and trajectory pathway of ciliary ganglion pioneer axons during the period of target innervation. After this period, however, the synthesis of GDNF declines markedly, while that of NRTN is maintained throughout the cell death period. Furthermore, both in vitro and in vivo function-blocking of GDNF at early embryonic ages almost entirely suppresses ciliary axon outgrowth. These results demonstrate that target-derived GDNF is necessary for ciliary ganglion neurons to innervate ciliary muscle in the eye. Since the down-regulation of GDNF in the eye is accompanied by down-regulation of GFRalpha1 and Ret, but not of GFRalpha2, in innervating ciliary ganglion neurons, the results also suggest that target-derived GDNF regulates the expression of its high-affinity coreceptors.     

Nerve Cross-Bridging to Enhance Nerve Regeneration in a Rat Model of Delayed Nerve Repair

There are currently no available options to promote nerve regeneration through chronically denervated distal nerve stumps. Here we used a rat model of delayed nerve repair asking of prior insertion of side-to-side cross-bridges between a donor tibial (TIB) nerve and a recipient denervated common peroneal (CP) nerve stump ameliorates poor nerve regeneration. First, numbers of retrogradely-labelled TIB neurons that grew axons into the nerve stump within three months, increased with the size of the perineurial windows opened in the TIB and CP nerves. Equal numbers of donor TIB axons regenerated into CP stumps either side of the cross-bridges, not being affected by target neurotrophic effects, or by removing the perineurium to insert 5-9 cross-bridges. Second, CP nerve stumps were coapted three months after inserting 0-9 cross-bridges and the number of 1) CP neurons that regenerated their axons within three months or 2) CP motor nerves that reinnervated the extensor digitorum longus (EDL) muscle within five months was determined by counting and motor unit number estimation (MUNE), respectively. We found that three but not more cross-bridges promoted the regeneration of axons and reinnervation of EDL muscle by all the CP motoneurons as compared to only 33% regenerating their axons when no cross-bridges were inserted. The same 3-fold increase in sensory nerve regeneration was found. In conclusion, side-to-side cross-bridges ameliorate poor regeneration after delayed nerve repair possibly by sustaining the growth-permissive state of denervated nerve stumps. Such autografts may be used in human repair surgery to improve outcomes after unavoidable delays.     

Optogenetic neuronal stimulation promotes axon outgrowth and myelination of motor neurons in a three-dimensional motor neuron–Schwann cell coculture model on a microfluidic biochip

Axonal regeneration and remyelination of peripheral motor neurons (MNs) are critical for restoring neuromuscular motor function after injury or peripheral neuropathy. We examined whether optogenetically mediated light stimulation (OMLS) could enhance the axon outgrowth and myelination of MNs using three-dimensional motor neuron–Schwann cell (MN–SC) coculture on a microfluidic biochip. The biochip was designed to allow SCs to interact with the axons of MNs, while preventing direct contact between SCs and the cell bodies of MNs. Following coculture with SCs on the microfluidic biochip, MNs were transfected with a light-sensitive channelrhodopsin gene. Transfected MNs subjected to repeated light stimulation (20 Hz, 1 hr) produced significantly longer axons than nontransfected MNs. OMLS of MNs greatly increased the number of myelin basic protein (MBP)-expressing SCs, promoting the initiation of myelination of MNs. Ultrastructurally, OMLS of MNs markedly enhanced the thickness of the compact myelin sheath around the MN axons such that the average thickness was closer to that of the theoretical estimates in vivo. Thus, the MN–SC coculture model on a microfluidic biochip augmented by OMLS of MNs is a feasible platform for studying the relationship of neuronal activity with regrowth and remyelination.     

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.     

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.     

The role of sensory input in motor neuron sprouting control

Primary sensory neurons project to motor neurons directly or through interneurons and affect their activity. In our previous paper we showed that intramuscular sprouting can be affected by changing the sensory synaptic input to motor neurons. In this work, motor axon sprouting within a peripheral nerve (extramuscular sprouting) was induced by nerve injury at such a distance from muscle so as not to allow nerve-muscle trophic interactions. Two different procedures were carried out: (1) sciatic nerve crush and (2) sciatic nerve crush with homosegmental ipsilateral L3-L5 dorsal rhizotomy. The number of regenerating motor axons innervating extensor digitorum longus muscle was determined by in vivo muscle tension recordings and an index of their individual conduction rate was obtained by in vitro intracellular recordings of excitatory postsynaptic end-plate potentials in muscle fibers. The main findings were: (1) there are more regenerated axons distally from the lesion than parent axons proximally to the lesion (sprouting at the lesion); (2) sprouting at the lesion was negatively affected by homosegmental ipsilateral dorsal rhizotomy; (3) the number of motor axons innervating extensor digitorum longus muscle extrafusal fibers counted proximally to the lesion increased following nerve injury and regeneration but this did not occur when sensory input was lost. A transient innervation of extrafusal fibers by &#110 motor neurons may explain the increase of motor axons counted proximally to the lesion.