Regenerating crayfish motor axons assimilate glial cells and sprout in cultured explants

Phasic and tonic motor nerves originating from crayfish abdominal ganglia, in 2-3-day-old cultured explants, display at their transected distal ends growth zones from which axonal sprouts arise. The subcellular morphology of this regenerative response was examined with thin serial-section electron microscopy and reveals two major remodeling features. First, the external sprouts that exit the nerve are a very small part of a much more massive sprouting response by individual axons comprising several orders of internal sprouts confined to the nerve. Both internal and external sprouts have a simple construction: a cytoskeleton of microtubules and populations of mitochondria, clear synaptic vesicles, membranous sacs, and extrasynaptic active zone dense bars, features reminiscent of motor nerve terminals. Close intermingling of the sprouts of several axons give rise to a neuropil-like arbor within the nerve. Thus, extensive sprouting is an intrinsic response of crayfish motor axons to transection. Second, an equally dramatic remodeling feature is the appearance of nuclei, which resemble those of adjacent glial cells, within the motor axons. These nuclei often appear where the adjoining membranes of the axon and glial cell are disrupted and where free-standing lengths of the double membrane are present. These images signify a breakdown of the dividing membranes and assimilation of the glial cell by the axon, the nucleus being the most visible sign of such assimilation. Thus, crayfish motor axons respond to transection by assimilating glial cells that may provide regulatory and trophic support for the sprouting response.     

Nerve compound action current (NCAC) measurements and morphometric analysis in the proximal segment after nerve transection and repair in a rabbit model

In the evaluation of nerve regeneration using magneto-neurography (MNG), the proximal segment showed a reproducible decrease in peak-peak amplitude of the nerve compound action current's (NCAC) of 60%. To explain these changes, morphometry of myelinated axons in the proximal segment is compared to the MNG signals. A standardised nerve transection and reconstruction was performed in rabbits. NCACs were measured approximately 5 cm proximal to the lesion from operated and control nerves after 12 weeks. Histological samples were taken from the same area of the nerve where the NCACs were obtained. Results showed a decrease of the peak-peak amplitude of the NCAC of 57% compared to the control. Conduction velocity decreased 15% (not significant). Morphometry elicited a decrease in larger (10-15 microm) axons (284 +/- 134 vs 82 +/- 55) and an increase in smaller (2-5 microm) axons (1445 +/- 360 vs 1921 +/- 393). A strong correlation existed between the decrease in amplitude and the decrease in larger axons (0.85). Peak-peak amplitude varies approximately with the square of the diameter axon. Therefore, because peak-peak amplitude is mainly dependent on the larger-diameter axons, the decrease in peak-peak amplitude of the NCACs may be explained by a decrease in numbers of 10-15-microm axons.     

Remodeling of nerves during claw reversal in adult snapping shrimps

Adult snapping shrimps, Alpheus heterochelis, undergo a reversal of their claw laterality following removal of the major claw, a process in which the existing minor claw transforms into a major and a new minor regenerates at the old major site. During such reversals the nerves to the ganglion are remodeled from one claw type to the other. Conversion of the nerves from the minor to the major type occurs within several days after removal of the contralateral major claw and involves the rapid addition of large numbers of sensory axons together with deletion of a few. Thus modeling of the nerves is essentially complete within the first intermolt in tandem with changes in the motoneurons but well ahead of changes in the muscle and external morphology. Conversion of the major nerves to the minor type is via massive degeneration of sensory axons during the first and second intermolts because of the loss of their peripherally located cell bodies. This is followed by proliferation of largely unmyelinated axons in the third intermolt, some of which become myelinated in the subsequent intermolts. Thus remodeling of the major nerves to minor, which is associated with the loss of a claw and the regeneration of a new minor claw, is a more traumatic and prolonged process compared to the remodeling of the minor nerves to major which is associated with the transformation of an existing claw.     

The numbers of unmyelinated and myelinated axons in normal and regenerated rat saphenous nerves

Counts have been made of the numbers of unmyelinated and myelinated axons in the proximal and distal stumps of regenerated rat saphenous nerves and from equivalent sites in normal nerves. In the proximal part of normal nerves there were averages of 1 045 myelinated axons and 4 160 unmyelinated ones. Regenerated nerves contained the same number of myelinated axons in their proximal stumps but there was a 40% reduction in the unmyelinated axon count. In the distal stumps of these nerves the myelinated axon count had increased by an average of 620; this comes about because some regenerated myelinated axons support more than one process in the distal stump. In contrast, the number of unmyelinated axons was reduced further, from a mean of 2 476 in the proximal stump to one of 2 219.

The sizes of Schwann cell units in the normal and regenerated nerves were also noted. Schwann cell units in the proximal and distal stumps of the regenerated nerves were smaller than those in the normal ones.

These changes associated with unmyelinated axons in regenerated nerves are likely to contribute to the sensory, vasomotor and sudomotor abnormalities that sometimes occur after peripheral nerve injury and regeneration.     

Dichotomy in phasic-tonic neuromuscular structure of crayfish inhibitory axons.

Crustacean muscles are unique in their innervation by both excitatory and inhibitory neurons; therefore, they exhibit polyneuronal and multiterminal innervation. Because excitatory motoneurons are broadly divided into phasic and tonic types, we hypothesized that inhibitory neurons would follow a similar dichotomy. The abdominal extensor muscles in crayfish are separated into parallel deep and superficial bundles; the former has fast muscle fibers innervated by phasic excitatory motoneurons, and the latter has slow fibers supplied by tonic excitatory motoneurons. Each muscle also is innervated by a single, separate inhibitory neuron that uses gamma-aminobutyric acid (GABA) as the inhibitory neurotransmitter. The pattern of axonal branching by the separate inhibitory axons in phasic and tonic abdominal extensor muscles was visualized with confocal microscopy in preparations labeled for GABA-like immunoreactivity. Initial observations indicated that the phasic muscle was covered by extensive GABAergic, filiform axon terminals, whereas innervation of the tonic muscle was comprised of more localized and varicose terminals. With quantitative analyses, we found that the phasic axon has a more highly branched nature than the tonic in first- and second-order branches. The phasic axon branches also were significantly longer than the tonic branches in the second- and third-order branches. Synaptic varicosities in the phasic branches were smaller and less frequent than those in the tonic branches. The fine structure of the inhibitory nerve terminals near synaptic contacts examined with thin-serial-section electron microscopy revealed distinct differences between the phasic system and the tonic system. The phasic terminals were smaller in cross-sectional area than the tonic terminals, and they had smaller synapses and fewer mitochondria. The presynaptic active zone dense bodies were similar in length and number between phasic and tonic synapses. However, their number per synaptic area was two-fold higher in phasic synapses compared with tonic synapses because of the smaller size of the phasic synapses. Thus, within the same neuromuscular system, inhibitory synaptic terminals revealed unique phasic and tonic identities similar to those observed for the excitatory axons.     

Axon resealing following transection takes longer in central axons than in peripheral axons: implications for axonal regeneration

Injury to axons in the CNS leads to little regenerative repair and loss of function. Conversely, injury to axons in the PNS results in vigorous regrowth of severed axons, usually with restoration of function. This difference is generally attributed to a CNS environment that either cannot support or actively inhibits regeneration and/or a failure of CNS neurons to survive axotomy. One of the earliest responses of neurons to axotomy is the resealing of cut axons. A delay in resealing could affect a neuron's ability to survive axotomy and to regenerate a new axon. In the present experiments, using a dye exclusion technique, we demonstrate that following transection of a peripheral sensory nerve, axons reseal within 8--10 h, whereas following optic nerve transection complete resealing does not occur for more than 20 h. These results show that resealing of cut axons in a CNS environment is significantly delayed compared with axons in the PNS and suggest that this could contribute to the failure of CNS neurons to regenerate following injury.     

Numbers of myelinated and unmyelinated axons in the dorsal, lateral, and ventral funiculi of the white matter of the S2 segment of cat spinal cord

The present work determines the numbers of myelinated and unmyelinated axons in the dorsal, lateral, and ventral funiculi of the S2 segment of the cat spinal cord. The major finding is that unmyelinated axons are almost as numerous as myelinated axons in these pathways. The myelinated axons tend to be distributed uniformly, although there is a slight concentration of these fibers in the dorsal part of the lateral funiculus. By contrast, the unmyelinated fibers, although found in significant numbers in all parts of these funiculi, concentrate in the dorsal part of the lateral funiculus and in the dorsal funiculus. Of particular note are the unmyelinated fibers in the dorsal funiculus, because it is highly likely that some of these are sensory. The findings in this study will serve as a basis for experimental studies to determine the numbers, locations, and types of unmyelinated fibers in the white matter of the mammalian cord.     

A magnetic evaluation of peripheral nerve regeneration: I. The discrepancy between magnetic and histologic data from the proximal segment

Histologic techniques can quantify the number of axons in a nerve, but give no information about electrical conductibility. The number of functional myelinated neuronal units in a nerve can be quantified based on a magnetic recording technique. When studying reconstructed peripheral nerves a significant difference between the results found with these two techniques can be observed. A comparison was made between the long-term changes in the number of histologically and magnetoneurophysiologically measured neuronal units proximal to a nerve reconstruction. This study was performed on 6 New Zealand White rabbits, 20 weeks after the peroneal nerve had been reconstructed. The contralateral nerves were used as a control. Histologic examination demonstrates a statistically significant decrease of approximately 5% in the number of myelinated fibers. The magnetoneurophysiological results demonstrate a decrease which is estimated to be caused by the loss of approximately 50% of the functional myelinated neuronal units in the nerve. Therefore we conclude that of the initially available myelinated neuronal units, 5% degenerate completely, 45% are vital but lose their signal conducting capability, and the remaining 50% are vital and continue to conduct signals. Apparently, only this latter group of 50% of the initially available functional neuronal units appears to remain available for functional recovery. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:739–749, 1998.     

Effects of sciatic nerve regeneration on axonal populations in tributary nerves

The present study tests 2 hypotheses: (1) that the numbers of axons that regenerate into a tributary nerve are in part dependent on the type of lesion used to transect the axons in the parent nerve; and (2) that the numbers of axons that regenerate will be different in different tributary nerves. Axons were counted in the sural nerve and the nerve to the medial gastrocnemius muscle 8 weeks following crush, simple transection, transection with removal of 4 mm and transection with removal of 8 mm of the sciatic nerve in the rat. The counts of myelinated and unmyelinated axons are presented in the text. If axon numbers in the 2 nerves are normalized, the proportion of regenerated to normal myelinated axon numbers are approximately the same in the 2 nerves, with more regenerated axons than normal following crush, simple transection, or 4 mm gap transection and fewer following 8 mm gap transection. The unmyelinated axons behave differently. In the nerve to the medial gastrocnemius muscle, the numbers of unmyelinated axons are greater than or equal to the normal numbers following our various surgical paradigms whereas in the sural nerve there are always fewer unmyelinated axons than normal. These findings indicate that the above hypotheses are correct for the nerves tested in the rat.     

Myelinated fiber regeneration after crush injury is retarded in sciatic nerves of aging mice

To compare nerve regeneration in young adult and aging mice, the right sciatic nerves of 6- and 24-month-old mice were crushed at the sciatic notch. Two weeks later, both groups of mice were perfused with an aldehyde solution, and, after additional fixation, the sciatic nerves were processed so that the transverse sections of each nerve subsequently studied by light and electron microscopy included the entire posterior tibial fascicle 5 mm distal to the crush site. The same level was sectioned in unoperated contralateral nerves; these nerves served as controls. Electron micrographs and the Bioquant Image Analysis System IV were used to measure areas of posterior tibial fascicles and count the number of myelinated axons, the number of unmyelinated axons, and their frequency in Schwann cell units. In aging mice, the total number of regenerating myelinated axons was significantly reduced, but totals of regenerating unmyelinated axons in aging and young adults did not differ significantly. In aging mice, the frequency of Schwann cells that contained a single unmyelinated axon was greater, suggesting that before myelination began, Schwann cell ensheathment of axons also was slowed. After axotomy by a crush injury, the area of the posterior tibial fascicle was less than that in young adults and the distal disintegration of myelin sheath remnants also appeared to be retarded. The results indicate that responses of neurons, axons, and Schwann cells could be important in slowing the regeneration of myelinated fibers found in sciatic nerves from aging mice.     

The response of optic tract glia during regeneration of the goldfish visual system. I. Biosynthetic activity within different glial populations after transection of retinal ganglion cell axons

We monitored biosynthetic activity of optic tract glia during regeneration of retinal ganglion cell axons in the goldfish and found that the greatest level of incorporated [3H]thymidine and [3H]leucine occurred in glia by 10-15 days after axotomy. During this period there was a marked increase in the number of oligodendroglia and multipotential glia near the site of injury with no change occurring in the astroglial population. Electron microscopic autoradiography showed that oligodendroglia and multipotential cells incorporated 5-7-fold more thymidine than did cells of intact control preparations. Though all glial cell types incorporated more [3H]leucine during axonal regeneration, oligodendroglia and multipotential cells together accounted for more than 90% of measured radioactivity. In order to characterize glial-stimulating events specific to axonal regeneration, we produced axonal degeneration in the optic tract by removal of the retina. Optic tract glia during axonal degeneration incorporated less amino acid when compared to glia associated with regenerating axons. The degenerating optic tract also had less 2',3'-cyclic nucleotide 3'-phosphohydrolase, an enzyme produced by oligodendroglia, than that found in the regenerating visual system. Our results suggest that in response to ganglion cell axotomy oligodendroglia and multipotential glia of the goldfish optic tract proliferate. Moreover, regenerating axons provide one type of stimulant for glial protein biosynthesis.     

Retrograde regeneration following neurotmesis of the ulnar nerve

A 41-year-old woman experienced a gunshot wound to the forearm with neurotmesis of the ulnar nerve. Surgery 9 months later revealed a neuroma-in-continuity in the midforearm. Intraoperative nerve stimulation failed to elicit direct nerve responses or motor responses from the first dorsal interosseous (FDI) and abductor digiti minimi (ADM) muscles. However, neurotonic discharges in response to mechanical irritation of the neuroma were recorded in the FDI, but not the ADM. Surprisingly, after resecting the ulnar nerve distal to the neuroma, neurotonic discharges were still elicited in the FDI following perturbation of the neuroma. Moreover, neurotonic discharges were elicited during ulnar nerve resection 2 cm proximal to the neuroma. No anastomoses or anomalous branches were noted. The findings suggest that regenerating fibers did not reach the FDI through the distal nerve segment. Rather, we speculate that nerve fibers regenerating at random, or impeded by scar tissue, contacted the proximal nerve portion, at which point growth became polarized in a retrograde direction. Retrograde regeneration may have proceeded to a branch point in the forearm (possibly an undetected anomalous branch or fibrous adhesion), where growth of regenerating fibers extended outward into surrounding damaged tissue planes before redirecting distally to reach the FDI.     

Accurate reinnervation of motor end plates after disruption of sheath cells and muscle fibers

After injury, regenerating motor axons grow back to form neuromuscular junctions at the original synaptic sites on muscle fibers. The pathways they grow along consist of basement membrane, Schwann cells, and perineurium that remained after degeneration of the original axons. All the factors necessary for directing axons to the original synaptic sites persist in muscles even after disruption of myofibers. The aim of the present experiments was to determine whether structural integrity of nerve sheath cells is required for precise reinnervation in the presence and absence of muscle fiber targets. The region of innervation of the cutaneous pectoris muscle of the frog was briefly frozen to eliminate all living cells from neuromuscular junctions, intramuscular nerve bundles, and from a 1-3-mm length of the nerve trunk. Only extracellular matrices persisted within the frozen region of muscle and nerve. These consisted of the basement membrane sheaths of myofibers, of Schwann cells, and of perineurial cells and the small fragments of disrupted cells that were bound to them. In some preparations new muscle fibers developed within the basement membrane sheaths. Regenerating axons grew through the naked basement membrane sheaths of original Schwann cells, formed numerous branches, and contacted the myofibers precisely at the original synaptic sites. By 5 weeks 75% of the original synaptic sites became reinnervated; the terminals were indistinguishable from those at normal neuromuscular junctions. In contrast, preparations in which all muscle fibers were prevented from regenerating far fewer synaptic sites became reinnervated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Meta analysis of olfactory ensheathing cell transplantation promoting functional recovery of motor nerves in rats with complete spinal cord transection

OBJECTIVE:To evaluate the effects of olfactory ensheathing cell transplantation on functional recovery of rats with complete spinal cord transection.DATA SOURCES: A computer-based online search of Medline(1989–2013),Embase(1989–2013),Cochrane library(1989–2013),Chinese Biomedical Literature Database(1989–2013),China National Knowledge Infrastructure(1989–2013),VIP(1989–2013),Wanfang databases(1989–2013) and Chinese Clinical Trial Register was conducted to collect randomized controlled trial data regarding olfactory ensheathing cell transplantation for the treatment of complete spinal cord transection in rats.SELECTION CRITERIA: Randomized controlled trials investigating olfactory ensheathing cell transplantation and other transplantation methods for promoting neurological functional recovery of rats with complete spinal cord transection were included in the analysis.Meta analysis was conducted using Rev Man 4.2.2 software.MAIN OUTCOME MEASURES: Basso,Beattie and Bresnahan scores of rats with complete spinal cord transection were evaluated in this study.RESULTS: Six randomized controlled trials with high quality methodology were included.Meta analysis showed that Basso,Beattie and Bresnahan scores were significantly higher in the olfactory ensheathing cell transplantation group compared with the control group(WMD = 3.16,95% CI(1.68,4.65); P 0.00001).CONCLUSION: Experimental studies have shown that olfactory ensheathing cell transplantation can promote the functional recovery of motor nerves in rats with complete spinal cord transection.     

A morphological correlate of target recognition by regenerating motor axons in the cockroach.

A specific cell recognition process during regeneration of severed axons of identified cockroach motor neurons eventually leads to the reformation of the original innervation pattern of target muscles in the leg. This occurs even though, at early times after nerve crush, the multiple branches of each regenerating axon grow into both appropriate and inappropriate muscles. In this study, we sought to examine whether there are any structural differences between regenerating axon branches in appropriate and inappropriate muscles that could lead to an understanding of why only those in inappropriate muscles are eliminated. A neuron subset-specific monoclonal antibody, NSS-2A, which labels the inhibitory motor neurons, was used to make their axon branches visible at various times after nerve crush. In inappropriate muscles, these axons grow primarily parallel to the muscle fibers and are later eliminated. In the appropriate muscles, these axon branches initially also grow parallel to the muscle fibers, but subsequently grow many interstitial collaterals. The formation of the collateral branches is a morphological correlate of the specific interaction of a neuron with its appropriate muscle. The simultaneous occurrence of axonal elimination and collateral sprouting supports the idea that the two processes are causally related, as suggested by the sibling neurite bias hypothesis.     

Short-term observations of the regenerative potential of injured proximal sensory nerves crossed with distal motor nerves

Motor nerves and sensory nerves conduct signals in different directions and function in different ways.In the surgical treatment of peripheral nerve injuries,the best prognosis is obtained by keeping the motor and sensory nerves separated and repairing the nerves using the suture method.However,the clinical consequences of connections between sensory and motor nerves currently remain unknown.In this study,we analyzed the anatomical structure of the rat femoral nerve,and observed the motor and sensory branches of the femoral nerve in the quadriceps femoris.After ligation of the nerves,the proximal end of the sensory nerve was connected with the distal end of the motor nerve,followed by observation of the changes in the newly-formed regenerated nerve fibers.Acetylcholinesterase staining was used to distinguish between the myelinated and unmyelinated motor and sensory nerves.Denervated muscle and newly formed nerves were compared in terms of morphology,electrophysiology and histochemistry.At 8 weeks after connection,no motor nerve fibers were observed on either side of the nerve conduit and the number of nerve fibers increased at the proximal end.The proportion of newly-formed motor and sensory fibers was different on both sides of the conduit.The area occupied by autonomic nerves in the proximal regenerative nerve was limited,but no distinct myelin sheath was visible in the distal nerve.These results confirm that sensory and motor nerves cannot be effectively connected.Moreover,the change of target organ at the distal end affects the type of nerves at the proximal end.     

Electrophysiological evaluation of conduction in the most proximal motor root segment

Root conduction time (RCT), defined as the time difference between M-wave latency by cervical magnetic stimulation (CMS) and the total peripheral motor conduction time calculated from the shortest F-wave latency, was investigated in patients with inflammatory demyelinating neuropathies (IDP) and amyotrophic lateral sclerosis (ALS). The minimal threshold for CMS also was studied. In the IDP patients, conduction in the proximal motor root segment was considered abnormal in 52% by the RCT and in 47% by the minimal threshold for CMS, whereas both were normal in 85% of the ALS patients. These findings suggest that the RCT and minimal threshold for CMS might be additional parameters for evaluating motor nerve conduction in IDP.     

Growth hormone treatment enhances the functional recovery of sciatic nerves after transection and repair

Introduction: Although nerves can spontaneously regenerate in the peripheral nervous system without treatment, functional recovery is generally poor, and thus there is a need for strategies to improve nerve regeneration. Methods: The left sciatic nerve of adult rats was transected and immediately repaired by epineurial sutures. Rats were then assigned to one of two experimental groups treated with either growth hormone (GH) or saline for 8 weeks. Sciatic nerve regeneration was estimated by histological evaluation, nerve conduction tests, and rotarod and treadmill performance. Results: GH‐treated rats showed increased cellularity at the lesion site together with more abundant immunoreactive axons and Schwann cells. Compound muscle action potential (CMAP) amplitude was also higher in these animals, and CMAP latency was significantly lower. Treadmill performance increased in rats receiving GH. Conclusion: GH enhanced the functional recovery of the damaged nerves, thus supporting the use of GH treatment, alone or combined with other therapeutic approaches, in promoting nerve repair. Muscle Nerve, 2012