Identifying motor and sensory myelinated axons in rabbit peripheral nerves by histochemical staining for carbonic anhydrase and cholinesterase activities

Carbonic anhydrase (CA) and cholinesterase (CE) histochemical staining of rabbit spinal nerve roots and dorsal root ganglia demonstrated that among the reactive myelinated axons, with minor exceptions, sensory axons were CA positive and CE negative whereas motor axons were CA negative and CE positive. The high specificity was achieved by adjusting reaction conditions to stain subpopulations of myelinated axons selectively while leaving 50% or so unstained. Fixation with glutaraldehyde appeared necessary for achieving selectivity. Following sciatic nerve transection, the reciprocal staining pattern persisted in damaged axons and their regenerating processes which formed neuromas within the proximal nerve stump. Within the neuromas, CA-stained sensory processes were elaborated earlier and in greater numbers than CE-stained regenerating motor processes. The present results indicate that histochemical axon typing can be exploited to reveal heterogeneous responses of motor and sensory axons to injury.     

Rapid growth of regenerating axons across the segments of sciatic nerve devoid of Schwann cells

The characteristic response of Schwann cells (SC) accompanies peripheral nerve injury and regeneration. To elucidate their role, the question of whether or not regenerating axons can elongate across the segments of a peripheral nerve devoid of SC was investigated. Rat sciatic nerve was crushed so that the continuity of SC basal laminae was not interrupted. A segment about 15 mm long distal to the crush was either repeatedly frozen/thawed to eliminate SC or scalded by moist heat which, in addition, denatured the proteins in the SC basal laminae, too. Both sensory and motor axons grew rapidly across the frozen/thawed segment of the nerve. Their rate of elongation was reduced by only 30% in comparison to control crushed nerves. SC were not present along the path of growing axons adhering tightly to the bare SC basal laminae. The rate of elongation of regenerating sensory and motor axons in scalded nerve segments was eight times lower than in control crushed nerves. SC were present in that part of the scalded region that had been invaded by the regenerating axons but no further distally. These results suggest that acellular basal laminae of SC provide very good, although not optimal, conditions for elongation of regenerating sensory and motor axons. If biochemical integrity of the basal lamina is destroyed, the regenerating axons must be accompanied or preceded by viable SC. and axon elongation rate is significantly reduced.     

Altered expression of pp60c-src induced by peripheral nerve injury.

The normal src protein (pp60c-src) is localized principally in the nerve growth cone of developing neurons and declines to low levels with synaptic maturation. To determine whether pp60c-src is reexpressed in regenerating axons, its expression was studied by immunoblotting and immunocytochemical analyses in adult chicken sciatic nerve following nerve crush injury. pp60c-src expression was found to increase during nerve repair with a temporal and spatial pattern consistent with a localization in regenerating axons. At the crush site, pp60c-src increased to maximal levels 7 days postinjury, increasing fivefold relative to 0 day nerve. In the nerve segment distal to the injury, the maximal increase in pp60c-src was sevenfold and occurred between 11 and 21 days postinjury. Immunoperoxidase staining revealed pp60c-src in regenerating axons and certain nonneuronal cells at the site of nerve repair. pp60c-src was induced in both motor and sensory neurons, as shown by increased pp60c-src immunoreactivity in their cell bodies located in the spinal cord and dorsal root ganglion. Phosphotyrosine-modified proteins that were potential targets of pp60c-src increased following nerve crush, and were localized to outgrowing neurites as well as to nonneuronal cells. These results suggest that pp60c-src is a common component of cellular mechanisms regulating growth cone migration in both regenerating and developing axons.     

Selective reinnervation of distal motor stumps by peripheral motor axons

Random matching of regenerating axons with Schwann tubes in the distal nerve stump is thought to contribute to the often poor results of peripheral nerve repair. Motor axons would be led to sensory end organs and sensory axons to motor end plates; both would remain functionless. However, the ability of regenerating axons to differentiate between sensory and motor environments has not been adequately examined. The experiments reported here evaluated the behavior of regenerating motor axons when given equal access to distal sensory and motor nerve stumps across an unstructured gap. "Y"-shape silicon chambers were implanted within the rat femoral nerve with the proximal motor branch as axon source in the base of the Y. The distal sensory and motor branches served as targets in the branches of the Y, and were placed 2 or 5 mm from the axon source. After 2 months for axon regeneration, horseradish peroxidase was used to label the motoneurons projecting axons into either the motor or the sensory stump. Equal numbers of motoneurons were labeled from the sensory and motor stumps at 2 mm, but significantly more motoneurons were labeled from the motor stump at 5 mm. (P = 0.016). This finding is consistent with selective reinnervation of the motor stump. Augmentation of this phenomenon to produce specific reunion of individual motor axons could dramatically improve the results of nerve suture.     

Preferential reinnervation of motor nerves by regenerating motor axons

Regeneration of axons into inappropriate distal nerve branches may adversely affect functional recovery after peripheral nerve suture. The degree to which motor axons reinnervate sensory nerves, and vice versa, has not been determined. In these experiments, HRP is used to quantify the sensory and motor neurons that reinnervate sensory and motor branches of the rat femoral nerve after proximal severance and repair. Motoneurons preferentially reinnervate the motor branch in juveniles and adults, even if the repair is intentionally misaligned or a gap is imposed between proximal and distal stumps. A specific interaction thus occurs between regenerating motor axons and the Schwann cell tubes that lead to the motor branch. This interaction is independent of mechanical axon alignment.     

Specificity in regenerative outgrowth and target reinnervation by mammalian peripheral axons

There are indications that specific factors are present in the distal stump of transected nerves which preferentially attract axons of the corresponding proximal stump into the distal nerve stumps. However, the impact of these factors is unclear, since there is abundant evidence that numerous regenerating motor and sensory axons are topographically misdirected after nerve transection and repair. Topographic reinnervation is improved after fascicular repair of fasciculated nerves, and quite precise after nerve crush. The latter may not be true, however, for non-myelinated axons, which show a high degree of aberrant growth even after crush. In contrast, regenerative outgrowth appears to be topographically specific after neonatal nerve transection. Reinnervation of muscle fibers appears to be unspecific in adult mammals, but specific after neonatal injury under certain circumstances. Some preference for reinnervation of the appropriate sensory receptors seems to exist although this preference does not preclude reinnervation of receptors by 'foreign' sensory fibers. In conclusion, incorrect topographic and target reinnervation commonly occurs after peripheral regeneration in adult mammals, and most certainly explains some of the functional disturbances after peripheral nerve lesions. Topographic regeneration appears to be better after nerve injury in developing mammals indicating that mechanisms from the developmental period may persist and aid in accurate regenerative outgrowth.     

The specificity of re-innervation by identified sensory and motor neurons in the leech

Re-innervation of skin and muscle by identified sensory and motor neurons in segmental ganglia of the leech was studied using physiological techniques. After lesions of peripheral nerves, sensory axons which re-innervated the skin always regained sensitivity to their original stimulus modality (touch, pressure or noxious stimuli). Motor neurons invariably re-innervated the appropriate type of body wall muscle, such as longitudinal or circular muscle layers. Both sensory and motor axons usually returned to the appropriate region of the body wall (dorsal, lateral, or ventral) when regenerating after a nerve crush or cut. This capacity was lost, however, when growth along old nerve branches was prevented by evulsing long segments of the nerve. Re-innervation usually occurred by way of growth of new axons all the way the periphery, but in a few cases reconnection with the surviving distal segment of the original axon had taken place. The specificity of reinnervation can be accounted for by a combination of selective growth along appropriate nerve branches and specific interactions with target tissues.     

Preconditioning selective ventral root injury promotes plasticity of ascending sensory neurons in the injured spinal cord of adult rats – possible roles of brain-derived neurotrophic factor, TrkB and p75 neurotrophin receptor

Preconditioning sciatic nerve injury enhances axonal regeneration of ascending sensory neurons after spinal cord injury. A key question is whether direct injury of sensory nerves is necessary for the enhanced regeneration. The lumbar 5 ventral root transection (L5 VRT) model, a model of selective motor nerve injury, provides a useful tool to address this question. Here we examined the effects of a preconditioning L5 VRT on the regeneration after a subsequent dorsal column transection (DCT) in adult Sprague–Dawley rats. We found that L5 VRT 1 week before DCT increased the number of Fast Blue (FB)-labeled neurons in the L5 dorsal root ganglia (DRG) and promoted sprouting/regenerating axons to grow into the glial scar. L5 VRT also induced a dramatic upregulation of expression of brain-derived neurotrophic factor (BDNF) in the preconditioned DRG and in the injured spinal cord. Moreover, almost all of the FB-labeled sprouting/regenerating neurons expressed BDNF, and approximately 55% of these neurons were surrounded by p75 neurotrophin receptor-positive glial cells. This combined injury led to an increase in the number of BDNF- and TrkB-immunoreactive nerve fibers in the dorsal column caudal to the lesion site. Taken together, these findings demonstrate that L5 VRT promotes sprouting/regeneration of ascending sensory neurons, indicating that sensory axotomy may not be essential for the plasticity of injured dorsal column axons. Thus, the sensory neurons could be preprimed in the regenerative milieu of Wallerian degeneration and neuroinflammation, which might alter the expression of neurotrophic factors and their receptors, facilitating sprouting/regeneration of ascending sensory neurons.     

Progressive degeneration of motor nerve terminals in GAD mutant mouse with hereditary sensory axonopathy

The evolution of motor nerve degeneration was examined in gracile axonal dystrophy (GAD) mutant mice, which develop initial sensory ataxia and subsequent motor paresis. Using the anterior gracilis (AG) muscle, which is innervated at two discrete and well-separated end-plate zones, we demonstrated that axonal degeneration occurred first at motor nerve terminals in the distal end-plate zone, and then extended gradually from the distal to the more proximal parts of affected axons in the intramuscular nerve trunk. In contrast to the degeneration in the distal zone, active degeneration was less marked in the proximal endplate zone and, furthermore, most terminal axons had begun to produce regenerating sprouts. Ventral horn cells were histologically normal, even at advanced stages. These results indicate that, as previously observed in sensory nerves, dying back degeneration progresses later in the lower motor neuron system, even within one muscle. The mechanism(s) influencing the activation of axonal regeneration are discussed. This mutant mouse will be a useful model for the study of regenerating phenomena in dying back degeneration of genetically compromised motor neurons, as well as for the study of the pathogenesis of hereditary sensory and motor neuropathies in man.     

Long-term consequences of impaired regeneration on facial motoneurons in the C57BL/Ola mouse

Peripheral nerves of the C57BL/Ola mouse mutant undergo markedly slowed Wallerian degeneration following injury. This is associated with impaired regeneration of both sensory and motor axons. Following a crush lesion of the facial nerve, there was no cell loss in facial nuclei of normal (C57BL/6J) adult mice, but 40% cell loss occurred in Ola mice and the survivors increased in size during the period when functional reinnervation was established. These results are interpreted as a result, first, of prolonged deprivation of target-derived trophic factor in the slowly regenerating Ola motoneurons and second, increased peripheral field size of the survivors. Within the regenerated facial nerve, there was marked heterogeneity of myelinated fibre size in Ola mice. Some Ola axons, both proximal and distal to the lesion site, had areas over twice as great as the largest 6J axons when measured 1 year following injury. A population of small diameter fibres, not observed in 6J nerves, persisted distal to the crush site in Ola nerves, and this was associated with an increase in the total number of myelinated axons in the distal nerve: on average, each parent Ola axon retained three persistent daughter axons. The delayed Wallerian degeneration in Ola mice not only impairs immediate axon regrowth, but also results in a breakdown of the normal mechanisms which regulate axon number and size in regenerating nerve.     

Neurite-promoting influences of proliferating Schwann cells and target-tissues are not prerequisite for rapid axonal elongation after nerve crush

An important role in peripheral nerve regeneration has been ascribed to humoral trophic and tropic agents arising from the nonneuronal cells in the distal nerve stump and the denervated targets. In order to estimate their contribution to axonal elongation after crush injury to the rat sciatic nerve, an in vivo model was designed in which local cellular and target-derived influences were eliminated by 1) freeze-thawing of a long nerve segment distal to the crush site and 2) cutting the nerve far distally to the crush site, but within the frozen-thawed segment, and deflecting the frozen-thawed nerve stump in the opposite direction from its natural course. The sensory and motor axon elongation rate was estimated from the results of the nerve pinch test and choline acetyltransferase distribution along the nerve segment distal to the crush. The elongation rate of regenerating axons in deflected nerve segments, either non-treated or frozen-thawed, was close in magnitude to that obtained when target-derived influences were not eliminated. Neurotropism of axonal targets is therefore of little importance for axon elongation after nerve crush. In the absence of Schwann cells along the axonal path in frozen-thawed nerve segments, the elongation rate of both sensory and motor axons declined by about 40%. This implies that interactions between viable Schwann cells and growth cones of regenerating axons are not prerequisite for rapid axon elongation when Schwann cell basal lamina constitutes the growth substratum. Nevertheless, Schwann cells in Bungner bands possibly enhance the axon elongation rate by humoral or cell surface-mediated mechanisms.     

Selective reinnervation of somatotopically appropriate muscles after facial nerve transection and regeneration in the neonatal rat

The organization of the facial motor nucleus (FMN) has been examined after transection and regeneration of the facial nerve (FN) in neonatal and adult rats. In one series of experiments, horseradish peroxidase (HRP) was applied bilaterally to the superior or inferior buccal ramus 5 months after neonatal FN transection. In another series of experiments, wheat germ agglutinin-horseradish peroxidase conjugate was injected in selected vibrissae follicular muscles on both sides in animals surviving 5 months after FN transection at the neonatal or adult stage. The number and distribution of HRP-labeled cell bodies in the FMN after regeneration was compared with the contralateral side. On the uninjured side, labeled neurons were somatotopically organized. Ipsilateral to nerve injury the number of labeled cells was markedly reduced after neonatal nerve transection, but somatotopy was preserved. However, after nerve lesion at the adult stage, no significant loss of motoneurons occurred, but motor nucleus somatotopy was not maintained. Two alternative principal explanations are proposed for the re-establishment of the normal somatotopy after neonatal injury: that regenerating axons grow in a random fashion but inappropriate connections are subsequently eliminated or that regenerating axons of surviving neurons immediately follow a pathway leading to the appropriate muscle.     

Insulin as an in vivo growth factor

Insulin peptide has been identified to promote regeneration of axons in culture and in some in vivo model systems. Such actions have been linked to direct actions of insulin, or to cross occupation of closely linked IGF-1 receptors. In this work, we examined insulin support of peripheral nerve regenerative events in mice. Systemic insulin administration accelerated the reinnervation of foot interosseous endplates by motor axons after sciatic nerve transection and enhanced recovery of functional mouse hindpaw function. Similarly, insulin accelerated the regeneration-related maturation of myelinated fibers regrowing beyond a sciatic nerve crush injury. That such benefits might occur through direct signaling on axons was supported by immunohistochemical studies of expression with an antibody directed to the beta insulin receptor (IR) subunit. The proportion of sensory neurons expressing IRbeta increased ipsilateral to a similar sciatic crush injury in the L4 and L5 dorsal root ganglia. Insulin receptors, although widely expressed in axons, were also preferentially and intensely expressed on axons regrowing just beyond a peripheral nerve crush injury zone. The findings indicate that insulin imparts a substantial impact on regenerating peripheral nerve axons through upregulation of its expression following injury. Although the findings do not exclude insulin coactivating IGF-1 receptors during regeneration, its own receptors are present and available for action on injured nerves.     

Guiding adult Mammalian sensory axons during regeneration

Misdirection of axons after nerve injury impairs successful regeneration of adult neurons. Investigations of axon guidance in development have provided an understanding of pathfinding, but their relevance to regenerating adult axons is unclear. We investigated adult mammalian axon guidance during regeneration after peripheral nerve injury and focused on the effects of the prototypic guidance molecule nerve growth factor (NGF). Adult rat sensory neurons from dorsal root ganglia that expressed the NGF receptor tropomyosin-related kinase A (trkA) were presented with a point source of NGF in vitro. Naive trkA neurons had no net turning response to NGF, but if they had been preconditioned by a peripheral nerve transection in vivo before culturing, their growth cones were attracted toward the NGF gradient. A laminin substrate was required for this behavior and an anti-trkA antibody interrupted turning. These data demonstrate that injured adult mammalian axons can be guided as they regenerate. Moreover, despite the downregulation of trkA mRNA and protein levels within the dorsal root ganglion after injury, sensory neurons retain and increase trkA protein at the injury site where the regenerating axons are found. This may enhance the axonal response to NGF and allow guidance along an NGF gradient created in vivo in the distal nerve stump.     

Sensory axon targeting is increased by NGF gene therapy within the lesioned adult femoral nerve

Even though peripheral nerves regenerate well, axons are often misrouted and reinnervate inappropriate distal pathways post-injury. Misrouting most likely occurs at branch points where regenerating axons make choices. Here, we show that the accuracy of sensory axon reinnervation is enhanced by overexpression of the guidance molecule nerve growth factor (NGF) distal to the bifurcation. We used the femoral nerve as a model, which contains both sensory and motor axons that intermingle in the parent trunk and distally segregate into the saphenous (SB) and motor branches (MB). Transection of the parent trunk resulted in misrouting of axon reinnervation to SB and MB. To enhance sensory axon targeting, recombinant adenovirus encoding NGF was injected along the SB close to the bifurcation 1 week post-injury. The accuracy of axon reinnervation was assessed by retrograde tracing at 3 or 8 weeks after nerve injury. NGF overexpression significantly increased the accuracy of SB axon reinnervation to the appropriate nerve branch, in a manner independent of enhancing axon regeneration. This novel finding provides in vivo evidence that gradient expression of neurotrophin can be used to enhance targeting of distal peripheral pathways to increase axon regeneration into the appropriate nerve branch.     

Misdirection of regenerating motor axons after nerve injury and repair in the rat sciatic nerve model

Misdirection of regenerating axons is one of the factors that can explain the poor results often found after nerve injury and repair. In this study, we quantified the degree of misdirection and the effect on recovery of function after different types of nerve injury and repair in the rat sciatic nerve model; crush injury, direct coaptation, and autograft repair. Sequential tracing with retrograde labeling of the peroneal nerve before and 8 weeks after nerve injury and repair was performed to quantify the accuracy of motor axon regeneration. Digital video analysis of ankle motion was used to investigate the recovery of function. In addition, serial compound action potential recordings and nerve and muscle morphometry were performed. In our study, accuracy of motor axon regeneration was found to be limited; only 71% (± 4.9%) of the peroneal motoneurons were correctly directed 2 months after sciatic crush injury, 42% (± 4.2%) after direct coaptation, and 25% (± 6.6%) after autograft repair. Recovery of ankle motion was incomplete after all types of nerve injury and repair and demonstrated a disturbed balance of ankle plantar and dorsiflexion. The number of motoneurons from which axons had regenerated was not significantly different from normal. The number of myelinated axons was significantly increased distal to the site of injury. Misdirection of regenerating motor axons is a major factor in the poor recovery of nerves that innervate different muscles. The results of this study can be used as basis for developing new nerve repair techniques that may improve the accuracy of regeneration.     

Developmentally regulated changes in femoral nerve regeneration in the mouse and rat

The attractive influence of muscle on regenerating motor neuron axons is well-known. Less is known, however, about the intrinsic abilities of different nerve pathways to support these axons prior to end-organ contact. The age at which a nerve injury is sustained is also known to affect the relationship between regenerating motor axons and muscle. The femoral nerve model, with its distinct muscle and cutaneous pathways, is ideal to study intrinsic pathway properties because the influence of end-organs can easily be removed surgically. However, recent results using this model in adult mice are at odds with the same model in neonatal rats. To reconcile these discrepancies, we used the femoral nerve model to examine possible age differences in intrinsic pathway support for regenerating motor neurons in the mouse and rat. Rat motor neurons showed a preference to regenerate into the muscle pathway after axotomy at 3 weeks of age, but this preference was lost after axotomy at 6 weeks of age. Interestingly, mouse motor neurons showed no pathway preference after axotomy at 3 weeks of age but developed one for the cutaneous pathway after axotomy at 6 weeks of age. These results suggest that in the absence of end-organ contact there is no general preference for motor neurons to project to the muscle pathway.     

Guidance of regenerating motor axons in larval and juvenile bullfrogs

The segmental distribution of regenerating bullfrog motor axons was mapped in advanced tadpoles and juvenile frogs by stimulating selected muscle nerves and recording from the distal ends of the 3 lumbar ventral roots (VRs) that innervate the hindlimb. When motoneurons were axotomized by VR transection, they reestablished their original innervation fields, rarely, if ever, growing beyond the territory normally supplied by their spinal segment. However, when motoneurons were axotomized in the spinal nerves at the level of the hindlimb plexus, some of them regenerated into limb nerves that lay outside the axons' normal segmental boundaries, and many regenerated into the medial femoral cutaneous nerve, a pathway normally limited to sensory axons. These observations suggest that the ultimate destinations of regenerating axons are largely determined by structures the axons encounter as they penetrate the distal nerve stumps. Thus, axons regenerating from a severed VR grow into that root's own distal stump and reinnervate the hindlimb in a manner that is segmentally appropriate; axons transected near the plexus have access to the pathways of sensory, as well as motor, axons in all 3 lumbar segments, and establish innervation fields that are inappropriate for their segment of origin and their motor function.     

The L2/HNK-1 Carbohydrate Epitope is Involved in the Preferential Outgrowth of Motor Neurons on Ventral Roots and Motor Nerves

Based on the observation that in adult mice the carbohydrate epitope L2/HNK-1 is detectable on Schwann cells in ventral spinal roots, but only scarcely in dorsal roots (Martini et al., Dev. Biol., 129, 330 - 338, 1988), the possibility was investigated that the carbohydrate is involved in the outgrowth of regenerating motor neuron axons on peripheral nerve substrates expressing the epitope. To monitor whether the L2 carbohydrate remains present during the time periods in which regenerating axons penetrate the denervated distal nerve stumps, the expression of L2 in motor and sensory branches of the femoral nerve was investigated in normal animals and after a crush lesion. During the first two postoperative weeks, L2 immunoreactivity remained high in the myelinating Schwann cells of the motor branch, whereas L2 immunoreactivity was virtually absent in the sensory branch. In a first experimental approach, cryosections of ventral and dorsal spinal roots and of motor and sensory nerves of adult rats and mice were used as substrates for neurite outgrowth. Neurites of motor neurons from chicken embryos were approximately 35% longer after 30 h of maintenance on ventral roots than on dorsal roots. Neurites from sensory neurons had the same length on dorsal as on ventral motors and were as long as neurites from motor neurons grown on dorsal roots. L2 antibodies reduced neurite outgrowth of motor neurons on ventral roots but not on dorsal roots. Neurite outgrowth of sensory neurons on both roots was not altered by the antibodies. Neurite outgrowth of motor neurons on a mixture of the extracellular matrix glycoprotein laminin and the L2 carbohydrate-carrying glycolipid was significantly higher than on the laminin substrate mixture with GD1b ganglioside or sulphatide. L2 antibodies reduced neurite outgrowth of motor neurons by 50% on the L2 glycolipid, but not on GD1b or sulphatide. These observations indicate that the L2 carbohydrate promotes neurite outgrowth of motor neurons in vitro and may thus contribute to the preferential reinnervation of motor nerves by regenerating motor axons in vivo.     

Directional specificity of regenerating primary sensory neurons after peripheral nerve crush or transection and epineurial suture A sequential double-labeling study in the rat

Clinical and experimental observations have demonstrated that peripheral nerve transection generally results in lasting disturbed sensory discrimination whereas nerve crush is followed by more or less complete functional restoration. This has been explained by an increased misdirection of regenerating fibers after transection as compared to crush injury. In the present study, sequential double-labeling was used to investigate the relative proportions of peripherally misdirected sensory fibers in the sural and tibial nerve branches after crush or transection of the parent sciatic nerve in the rat. Control experiments showed that 0.21% ± 0.12 (mean ± S.D.) of all labeled tibial and sural neurons normally send axons to both nerves. After sciatic nerve crush or transection, 1.31% ± 0.78 and 3.79% ± 3.01, respectively, of all labeled tibial and sural axons were double-labeled indicating previously sural axons now having an axon in the tibial. Statistically significant differences in the percentages of bidirectional sciatic sensory neurons were found between the normal controls and after crush injury (P < 0.01) or transection injury (P < 0.001), respectively, but not between transection and crush (P > 0.05). The results indicate that the number of sensory neurons having an axon in two peripheral nerves is normally very small, that a substantial number of sensory axons become misdirected after both crush and transection with resuture, and that the number of misdirected fibers in the major sciatic branches after these types of injury is similar.