Patterning in the regeneration of electroreceptors in the fin of Kryptopterus

The influence of the target tissue on afferent nerve regeneration was studied in the adult glass catfish, Kryptopterus. In this fish, electroreceptors in the anal fin are distributed in a characteristic pattern in the proximal part of the fin and are absent in the distal portion of the fin. We tested whether axons were more likely to induce electroreceptors in certain regions of fin epidermis than in others. We rotated fin transplants so that the location of the degenerating electroreceptors was altered with respect to the regenerating axons in the host tissue dorsal to the fin. The effects of these rotations were observed in the living animal with differential interference contrast optics over a period of 10 weeks. When transplants were reversed rostrocaudally, new electroreceptors formed in the caudal half of the interradial zone, where degenerating electroreceptors were at the time of transplantation. When transplants were rotated so that the dorsoventral and rostrocaudal axes were reversed, some new receptors formed in the old target site regions that were located in the caudal interradial zones (in the distal half of the graft with respect to the host). Regenerating axons reached these regions of the transplant by taking unusual routes around the electroreceptor-free regions of fin. Very few electroreceptors formed in the distal/caudal or proximal/caudal interradial quadrants of grafts where the original orientation of the tissue was maintained. We suggest that old target sites have a neurotropic influence on the regenerating afferent axons and discuss the possibility that the distal fin epidermis is not as permissive to electroreceptor formation as proximal fin epidermis.     

Functional aspects of the regeneration of unmyelinated axons in the rat saphenous nerve

Electrophysiological experiments have been carried out to investigate aspects of unmyelinated axon regeneration in a transected cutaneous nerve. Some comparisons with regeneration of myelinated axons in the same nerve have also been made.

By 3 months after injury approximately 80% of the unmyelinated axons that had survived in the proximal stump had regenerated into the distal stump. About the same proportion of myelinated axons had regrown into the distal stump by this time. With both groups of axons there was no marked increase in the amount of regeneration across the injury site with longer recovery times. Conduction velocities in the regenerated unmyelinated axons tended to be slower across the injury site than proximally; the proximal conduction velocities did not differ from those in control nerves. The unmyelinated axons seemed to take longer to resupply the skin than did the myelinated ones, but in both cases the extent of skin innervation had reached about 60% of control values by 6 months after the injury.     

Tuberous electroreceptor organs form in denervated regenerating skin of a weakly electric fish

Weakly electric fish use tuberous electroreceptor organs to detect their own electric fields. We investigated the role of innervation upon regeneration and differentiation of tuberous electroreceptor organs. The left, infraorbital, anterior lateral line nerve of brown ghosts (Apteronotus leptorhynchus) was sectioned, and the proximal stump was dipped in ricin to prevent regrowth. Immediately after denervation, a piece of cheek skin (∼0.5 cm²) was removed bilaterally to induce skin regeneration. After survival periods of 3, 4, or 5 weeks, regenerated skin from the left (denervated) and the right (reinnervated) sides was removed and processed for immunocytochemistry or electron microscopy. Tuberous electroreceptor organs were present in regenerated reinnervated, as well as regenerated denervated skin patches at all survival times. With increased time after skin removal, the number of fully differentiated organs increased in the reinnervated regenerated skin while the number of organs with degenerating receptor cells or entirely devoid of receptor cells increased in the denervated regenerated skin. These results suggest that innervation is not essential for tuberous electroreceptor organ development, but that it is necessary for complete sensory cell differentiation and long-term survival. © 1996 Wiley-Liss, Inc.     

Nerve Regeneration in Predegenerated Basal Lamina Graft: The Effect of Duration of Predegeneration on Axonal Extension

We used predegenerated acellular grafts to bridge proximal and distal stumps of transected nerves and studied how the duration of predegeneration might affect axonal regeneration. Predegenerated acellular grafts were prepared by transecting the tibial nerve of donor rats and, after a period of degeneration, freeze-thawing a 40-mm long segment of the distal stump. Five degeneration periods were used: 0 days (for fresh grafts), 3 days, 1 week, 4 weeks, and 8 weeks. Fresh cellular grafts not treated with freeze-thawing were also used for comparison. Each graft was then transplanted to an isogeneic recipient rat, in which it was used to bridge the proximal stump of the transected left tibial nerve and the distal stamp of the transected right tibial nerve. Six weeks were allowed for the regeneration of axons in all grafts. The regeneration was then assessed by studying transverse sections of the grafts, to determine the maximum length that the axons had regenerated, and the packing density of axons (percentage of sampled areas occupied by axons). The results show that axons had grown to the maximum length in the 4-week predegenerated grafts, and had the highest packing density in the 1-week predegenerated grafts. Regeneration in the fresh acellular (0-day predegenerated) and 8-week predegenerated grafts, especially the latter, was poor. We examine the results with reference to time-dependent events of Wallerian degeneration and propose that there are beneficial effects of multiple factors on the grafts during the first 4 weeks of predegeneration, causing a slow but significant improvement in their capability to support axonal growth. The subsequent rapid deterioration of such capability may be related to structural changes in the extracellular scaffold.     

The role of Schwann cells and basal lamina tubes in the regeneration of axons through long lengths of freeze-killed nerve grafts

The ability of long acellular nerve grafts to support axonal regeneration was examined using inbred rats. Grafts (40 mm long) of tibial/plantar nerves were used either as live grafts or after freeze-drying to render the grafts acellular. The grafts were sutured to the proximal stump of severed tibial nerves in host animals which were then killed 1-12 weeks later. Axons rapidly regenerated through the living grafts but only extended 10-20 mm into the acellular grafts. This distance was achieved by 6 weeks and thereafter no significant further axonal extension occurred in the acellular grafts. A few naked axons lacking Schwann cell contact were identified in all acellular grafts, but became more numerous near the distal extent of axonal penetration into 6-12 week grafts. These axons contained large numbers of neurofilaments. When the distal 20 mm of 6 week acellular grafts (segments into which axons had not penetrated) were sutured to freshly severed tibial nerves, axons grew readily into the grafted tissue to a maximum distance of 9 mm. It is therefore likely that the limits to axonal regeneration through initially acellular grafts were set by factors intrinsic to the severed nerve. It is suggested that the limited migratory powers of Schwann cells may be one such factor. The concept that basal lamina tubes are not essential for axonal regeneration but may act as low resistance pathways for both axonal elongation and Schwann cell migration is discussed.     

Chondroitinase treatment increases the effective length of acellular nerve grafts

Acellular nerve allografts have been explored as an alternative to nerve autografting. It has long been recognized that there is a distinct limit to the effective length of conventional acellular nerve grafts, which must be overcome for many grafting applications. In rodent models nerve regeneration fails in acellular nerve grafts greater than 2 cm in length. In previous studies we found that nerve regeneration is markedly enhanced with acellular nerve grafts in which growth-inhibiting chondroitin sulfate proteoglycan was degraded by pretreatment with chondroitinase ABC (ChABC). Here, we tested if nerve regeneration can be achieved through 4-cm acellular nerve grafts pretreated with ChABC. Adult rats received bilateral sciatic nerve segmental resection and repair with a 4 cm, thermally acellularized, nerve graft treated with ChABC (ChABC graft) or vehicle-treated acellularized graft (Control graft). Nerve regeneration was examined 12 weeks after implantation. Our findings confirm that functional axonal regeneration fails in conventional long acellular grafts. In this condition we found very few axons in the distal host nerve, and there were marginal signs of sciatic nerve reinnervation in few (2/9) rats. This was accompanied by extensive structural disintegration of the distal graft and abundant retrograde axonal regeneration in the proximal nerve. In contrast, most (8/9) animals receiving nerve repair with ChABC grafts showed sciatic nerve reinnervation by direct nerve pinch testing. Histological examination revealed much better structural preservation and axonal growth throughout the ChABC grafts. Numerous axons were found in all but one (8/9) of the host distal nerves and many of these regenerated axons were myelinated. In addition, the amount of aberrant retrograde axonal growth (originating near the proximal suture line) was markedly reduced by repair with ChABC grafts. Based on these results we conclude that ChABC treatment substantially increases the effective length of acellular nerve grafts.     

The regeneration of axons through normal and reversed peripheral nerve grafts

The effect of proximo-distal orientation of peripheral nerve grafts upon axonal regeneration has been investigated using the sciatic nerve of the rat as a model. To test the hypothesis that the presence of nerve branches within a graft will cause misdirection of axons in normally oriented grafts but not in reversed grafts, all grafts studied contained branches. Qualitative electron microscopic examination of graft ultrastructure revealed no differences in nerve structure related to graft orientation. In most normally oriented grafts, branches persisted up to 12 months after surgery. These branches contained axons which terminated at the end of the branch. In all reverse oriented grafts, and in a small number of normally oriented ones, the branches could not be seen after two or more months of regeneration. Axons sprouting outside of the epineurium of the graft caused the branch to be incorporated into the nerve structure. Axon counts in the distal stump of grafted nerves after twelve months recovery revealed that normally oriented grafts with persistent branches led to poorer peripheral regeneration, especially of unmyelinated fibers. The results indicate that regeneration of axons to their peripheral targets may be facilitated by reversing the graft orientation.     

The medullary pacemaker nucleus is unnecessary for electroreceptor tuning plasticity in Sternopygus

Tuberous electroreceptors of the weakly electric fish Sternopygus macrurus are closely tuned to the frequency of electric organ discharge (EOD), which is determined by a medullary pacemaker nucleus (PMN). Previous studies have demonstrated that androgens lower the frequency of PMN discharge and concomitantly lower the best frequencies (BFs) of electroreceptors. In order to determine if the PMN serves as an internal reference for the hormone-mediated returning of electroreceptors, the PMN was lesioned and the change in mean BF was measured for dihydrotestosterone (DHT)-implanted or control animals. DHT-implanted fish showed the characteristic lowering of mean electroreceptor BF by approximately 25%, a significant change compared with controls (p less than 0.01, Mann-Whitney). This result indicates that the PMN is not necessary for the hormone-mediated shift of electroreceptor tuning. In a related study, the contribution of the PMN to the genesis of tuning in regenerating electroreceptors was examined by removing a patch of cheek skin from PMN-lesioned fish. Regenerating electroreceptors became sharply tuned to the previous EOD frequency by 6 weeks in the same fashion as regenerating receptors in intact fish. In addition, intact receptors from PMN-lesioned fish remained tuned for up to 160 d. Together, these results demonstrate that the pacemaker nucleus is unnecessary for the maintenance, development, or hormone-mediated shift of receptor tuning.     

The emergence of tuning in newly generated tuberous electroreceptors

Tuning curves of afferent electroreceptive fibers in the anterior lateral line nerve of the weakly electric fish, Sternopygus macrurus, indicate that the tuberous electroreceptors of each individual are well-tuned to its own electric organ discharge (EOD) frequency. In order to study how receptor tuning may develop, new receptor organs were induced to form in regenerating cheek skin, and their tuning properties were compared with those of intact receptors from the same fish. At 3 weeks after the onset of regeneration, new receptors of a given fish were broadly tuned with best frequencies (BFs) lower than that fish's EOD frequency and the BFs of its own intact tuberous receptors. Three weeks later, regenerated receptors of the same fish were indistinguishable from intact receptors in BF, although tuning curves were occasionally slightly broader than normal. To determine if the presence of an ongoing electric field is necessary for the genesis of proper tuning, receptors were allowed to regenerate in fish deprived of their EODs. At 6 weeks, tuning curves of these receptors also had BFs that were tuned similarly to intact receptors and to each individual's characteristic EOD frequency (determined by recordings of the pacemaker nucleus in the medulla). Thus, as regenerating receptors mature, they gradually become more sharply tuned and tuned to progressively higher frequencies until reaching the correct BF, which matches the EOD frequency; however, tuning to the appropriate EOD frequency occurs without reference to the ongoing electric field.     

The role of Schwann cells in the regeneration of peripheral nerve axons through muscle basal lamina grafts

Evacuated muscle is a possible substitute for nerve autografts in the repair of damaged peripheral nerves. Previous experiments have shown that killed or evacuated muscle grafts are as effective as nerve autografts for bridging gaps of up to 4 cm between proximal and distal nerve stumps. Evacuated muscle grafts are made of extracellular matrix components, which are good substrates for axon growth in vitro. However, experiments in vivo have generally demonstrated that live Schwann cells are essential for successful axon regeneration. In the present experiments we have used immunohistochemical techniques with anti-S100 and anti-neurofilament antibodies to visualize axon growth and Schwann cell migration into muscle grafts over the first 10 days following grafting. We only saw axons growing into grafts accompanied by Schwann cells, and most though not all Schwann cells were associated with axons. Schwann cell migration from the proximal stump in association with axons was much faster and more extensive than from the distal stump. We examined muscle grafts over the first 20 days after grafting by electron microscopy. Regenerating axons were always associated with Schwann cells, which were mostly in the basal lamina-lined tubes left by the evacuated myofibrils. A comparison between evacuated muscle grafts and grafts in which the muscle had been killed but not evacuated revealed that 7 days after grafting there were more than twice as many regenerated axons in and distal to the evacuated grafts, but that by 20 days the numbers of axons were similar in the two groups.     

Time course of structural changes in regenerating electroreceptors of a weakly electric fish

We examined the regenerating electroreceptors of the weakly electric fish Sternopygus by light and electron microscopy to search for possible structural correlates of known physiological changes that occur during regeneration (Zakon: J. Neurosci. 6(11):3297-3308, 1986) and to compare them with developing electroreceptors in larval fish (Vischer: Brain Behav. Evol. 33:223-236). Nine days after removal of a patch of cheek skin, new skin had filled the wound and undifferentiated precursor cell clusters were located in the epidermis just above the dermis. Nerve fibers were present near most, but not all, cell clusters. A few recognizable tuberous and ampullary precursor organs were seen at this time. Tuberous organs were composed of a group of large cells surrounded by smaller cells without a lumen and showed the beginning of a cellular plug. Ampullary organs appeared as a ball of cells with a small lumen opening into a nascent canal. Degenerating cells were found within organs, and sometimes entire organs degenerated. These were not innervated. By 2 weeks the large cells of the tuberous organ were developing into sensory cells, while the smaller cells were forming the capsule wall and the underlying basal cells. The characteristic tuberous organ canal filled with loosely packed epidermal cells was evident. The sensory cells of the ampullary organs were visible within the epithelial layer at the base of the lumen, and the large synaptic discs were beginning to form. The sensory cells and postsynaptic terminals contained numerous vesicles. The presynaptic vesicles, which appear in normal receptor cells, remained throughout regeneration and presumably underlie transmitter release. The postsynaptic vesicles appeared transiently in large numbers but declined to adult values by 4 weeks. We presume that these may serve a trophic role. By 3 weeks, organs generally appeared mature and began dividing into daughter organs. The formation of individual receptor organs during regeneration is similar to that observed in development. Receptor organs continued dividing until the appropriate number of organs per afferent was reached for the size of the fish. Although the organization of the receptors appeared generally normal, there were a few anomalies. Some afferents sent sprouts into the epidermis, and, as a result of such sprouting, some of these afferents innervated multiple organs over a greater distance than normal. This was first seen early in regeneration and persisted for as long as 5 months.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechano-sensory organ regeneration in adults: the zebrafish lateral line as a model

In this report, we present a study of regeneration of the lateral line, a collection of mechano-sensory organ, in the adult zebrafish caudal fin. As all neuromasts are innervated by axon fibers, neuronal regeneration is a key issue in the regenerating process. We first show that support cells from the last neuromast adjacent to the amputation plane divide and migrate to colonize the blastema in order to reform the missing part of the lateral line. We then show that nerve re-growth takes place later than neuromast progenitor cell migration. We also provide evidence that new growth cones form at the amputation plane and subsequently follow the migrating placode-like structure to re-innervate regenerated neuromasts as they differentiate. Altogether, our observations indicate that caudal lateral line regeneration is not a mere recapitulation of the ontogenic process.     

Neomycin damage and regeneration of hair cells in both mechanoreceptor and electroreceptor lateral line organs of the larval Siberian sturgeon (Acipenser baerii)

The lateral line found in some amphibians and fishes has two distinctive classes of sensory organs: mechanoreceptors (neuromasts) and electroreceptors (ampullary organs). Hair cells in neuromasts can be damaged by aminoglycoside antibiotics and they will regenerate rapidly afterward. Aminoglycoside sensitivity and the capacity for regeneration have not been investigated in ampullary organs. We treated Siberian sturgeon (Acipenser baerii) larvae with neomycin and observed loss and regeneration of sensory hair cells in both organs by labeling with DASPEI and scanning electron microscopy (SEM). The numbers of sensory hair cells in both organs were reduced to the lowest levels at 6 hours posttreatment (hpt). New sensory hair cells began to appear at 12 hpt and were regenerated completely in 7 days. To reveal the possible mechanism for ampullary hair cell regeneration, we analyzed cell proliferation and the expression of neural placodal gene eya1 during regeneration. Both cell proliferation and eya1 expression were concentrated in peripheral mantle cells and both increased to the highest level at 12 hpt, which is consistent with the time course for regeneration of the ampullary hair cells. Furthermore, we used Texas Red‐conjugated gentamicin in an uptake assay following pretreatment with a cation channel blocker (amiloride) and found that entry of the antibiotic was suppressed in both organs. Together, our results indicate that ampullary hair cells in Siberian sturgeon larvae can be damaged by neomycin exposure and they can regenerate rapidly. We suggest that the mechanisms for aminoglycoside uptake and hair cell regeneration are conserved for mechanoreceptors and electroreceptors. J. Comp. Neurol. 524:1443–1456, 2016. © 2015 Wiley Periodicals, Inc.     

Development of the lateral line system in the shovelnose sturgeon

The lateral line systems of aquatic amphibians and all chondrichthyan and osteichthyan fish present a similar array of mechanoreceptors. However, electroreceptors, the second major component of the lateral line system, have clearly undergone more significant evolutionary change. Chondrichthyans and non-neopterygian fish possess primitive ampullary organ electroreceptors, whereas significantly different 'new' ampullary organs and tuberous electroreceptors are found in a few groups of teleosts (mormyrids, gymnotids and some catfish). The pairing of mechano- and electroreceptors in the lateral line system, as well as the morphologically and physiologically distinct electroreceptors of teleosts have inspired several recent studies on the origin and evolution of the lateral line receptors. We described the development of the lateral line system in sturgeon (Scaphirhynchus platorynchus) as part of an outgroup analysis of lateral line development in three taxa: vertebrates that have both mechanoreceptive neuromasts and primitive electroreceptors; neopterygian fish that only have mechanoreceptors; and teleosts that have re-evolved new electroreceptors. Development in Scaphirhynchus was consistent with previously studied taxa in that the lateral line system developed from a series of six dorsolateral placodes. Interestingly, we found that the octaval placode was bound rostrally and caudally by large placodal fields, out of which the six lateral line placodes arose. This finding supports recent suggestions for a common placodal primordium for all placodes. Each of the six placodes gave rise to the lateral line nerves before elongating into sensory ridges, which contained neuromast primordia. The ampullary organ fields of Scaphirhynchus arose from the lateral zones of the anterodorsal, anteroventral, otic and supratemporal sensory ridges, which is also consistent with recently studied taxa. Comparisons of the lateral line system of Scaphirhynchus and close relatives, Acipenser and Polyodon, indicate that variation in some aspects of lateral line receptor numbers and distribution are related to changes in head morphology and feeding strategy, whereas other changes, such as a reduction in receptor number without a change in placode field size, indicate changes in placode development.     

Nerve regeneration through biodegradable polyester tubes

One approach to repair of transected nerves is to attempt extrinsic guidance of axons across the gaps. We inserted the proximal and distal stumps of severed mouse sciatic nerves into opposite ends of biodegradable polyester tubes. The nerves and ensheathing tubes were examined after postoperative survival times of as long as 2 years. Myelinated fiber number in each successfully regenerated nerve was measured and correlated with the tube's residual lumen size. In selected regenerated nerves axonal sizes and myelin sheath widths were sampled and compared with control values. Swelling and deformation of tube walls occurred in nearly all tubes. Successful regeneration was obtained through more than half of the implants, and was more probable in tubes with larger initial lumens. Myelinated fiber number in regenerated nerves ranged from 231 to 3561 (normally 3900 to 4200); larger values again were found in tubes with larger initial lumens. Mean axonal areas in regenerated nerves were roughly half of normal, though myelin sheaths became appropriately thick. We concluded that the more biodegradable a tube, the more likely it was to incur distortion and luminal narrowing. Tube composition per se seemed of importance mainly as it related to maintenance of adequate luminal size over the length of the degrading tubes; luminal adequacy, not tube composition, seemed paramount in determining the extent of nerve regeneration.     

Properties of cutaneous mechanosensitive afferents during the early stages of regeneration in man

The technique of percutaneous microneurography was used to record single unit activity from 75 regenerated primary afferents innervating the glabrous skin of the human hand. Thirteen patients were studied, who had suffered complete transection, with subsequent suture or graft, of the median or ulnar nerves. The recordings were obtained from 7 to 23 months postoperatively (early regeneration). Three types of mechanoreceptive afferents (RA, SAI, SAII) and many deep units of unknown origin were found. No regenerated PC units could be identified. The reinnervated receptors were predominantly located in the palm and proximal fingers, comparable to those found 3 years or more postoperatively (late regeneration). Response thresholds and in general, discharge and receptive field characteristics of the majority of afferents were largely comparable to late regeneration and normal. The properties of SAII units were like normal in all respects. However, several distinct abnormalities were encountered early during regeneration: multiple receptive fields innervated by a single afferent (2/9 RA and 2/9 SAI), unusually small or large receptive fields (RA and SAI), pronounced fatigue on repetitive stimulation (7/15 SAI, 4/6 deep). Responses of reinnervated skin to sustained and repeated indentations were found to be similar to those of normal skin, and therefore, could not account for the abnormal discharge behavior. It is suggested that the transitional properties of regenerating afferents reflect unstable axon-end organ connections and immature axonal properties. Both factors would contribute to the slow course of sensory recovery, making prognosis on tactile recovery unpredictable.     

Nerve growth factor-hypersecreting Schwann cell grafts augment and guide spinal cord axonal growth and remyelinate central nervous system axons in a phenotypically appropriate manner that correlates with expression of L1.

Schwann cells contribute to efficient axonal regeneration after peripheral nerve injury and, when grafted to the central nervous system (CNS), also support a modest degree of central axonal regeneration. This study examined (1) whether Schwann cells grafted to the CNS exhibit normal patterns of differentiation and association with spinal axons and what signals putatively modulate these interactions, and (2) whether Schwann cells overexpressing neurotrophic factors enhance axonal regeneration. Thus, primary Schwann cells were transduced to hypersecrete human nerve growth factor (NGF) and were grafted to spinal cord injury sites in adult rats. Comparisons were made to nontransfected Schwann cells. From 3 days to 6 months later, grafted Schwann cells exhibited a phenotypic and temporal course of differentiation that matched patterns normally observed after peripheral nerve injury. Schwann cells spontaneously aligned into regular spatial arrays within the cord, appropriately remyelinated coerulospinal axons that regenerated into grafts, and appropriately ensheathed but did not myelinate sensory axons extending into grafts. Coordinate expression of the cell adhesion molecule L1 on Schwann cells and axons correlated with establishment of appropriate patterns of axon-Schwann cell ensheathment. Transduction of Schwann cells to overexpress NGF robustly increased axonal growth but did not otherwise alter the nature of interactions with growing axons. These findings suggest that signals expressed on Schwann cells that modulate peripheral axonal regeneration and myelination are also recognized in the CNS and that the modification of Schwann cells to overexpress growth factors significantly augments their capacity to support extensive axonal growth in models of CNS injury.     

Painful dysaesthesias following peripheral nerve injury: a clinical and electrophysiological study

Thirty-three patients with complete median, ulnar or digital nerve transections were studied 4 months to 13 years subsequent to suture or nerve grafting. In all cases, sensory disturbances, in terms of paraesthesia or hypaesthesia, were encountered. Painful or unpleasant symptoms, allodynia or hyperpathia, were observed most frequently in patients with poor recovery. The clinical findings and the patients' subjective complaints were correlated to microneurographic single fibre recordings of regenerated cutaneous mechanoreceptors. In more than 80% of the recordings, discharge properties of regenerated receptors, thresholds and a variety of other electrophysiological data were similar or equal to normal controls. Less than 20% of the receptors exhibited atypical properties suggesting defective steady-state regeneration. The ratio of rapidly adapting (RA-units) to slowly adapting mechanoreceptors (SA-units) was inverse in relation to normals. The density of regenerated RA-receptors was higher in the proximal than in the distal part of the reinnervated area. This paralleled the clinical finding of reduced sensory discrimination in these cases and suggests that SA-units may regenerate preferentially. In painful conditions no single fibres could be recorded, reflecting the relative paucity of fibres and probably the atrophy of the nerve. The results of the microstimulation experiments, although less reliable, revealed some evidence that the central processing of regenerated units is abnormal. Clinical and electrophysiological data supported this concept of central changes underlying some of the phenomena observed during peripheral nerve regeneration.     

Immature optic nerve glia of rat do not promote axonal regeneration when transplanted into a peripheral nerve

The ability of immature central nervous system (CNS) glia to promote axonal regeneration was studied by grafting segments of embryonic and neonatal rat optic nerves into the sciatic nerves of adult rats. Unexpectedly, very few axons regenerated through these grafts. The majority of the axons bypassed the grafts and were associated with Schwann cells. These results were similar to those obtained with grafts of adult rat optic nerves. The failure of immature CNS glia to promote axonal regeneration under these conditions suggests that they may be less effective than Schwann cells in promoting the regeneration and growth of axons.     

Are Schwann cells essential for axonal regeneration into muscle autografts?

When axons regenerate through frozen–thawed (FT) muscle grafts, they are accompanied by co–migrating Schwann cells derived from the nerve stumps. Although acellular, FT muscle grafts contain an internal scaffold of basal laminae rich in components capable of supporting neurite outgrowth in vitro such as laminin and fibronectin: it is not known whether Schwann cells are essential for axonal regrowth within these grafts. In this paper we test the hypothesis that sarcolemmal basal laminae will support axonal regeneration in the absence of Schwann cells. Two groups of 12 adult Wistar rats were used. All rats received a 0.5 cm FT muscle graft, and 12 rats also received a subperineurial injection of the anti–mi to tic agent mitomycin C (400 μg/ml in physiological saline) prior to grafting. Previous studies have shown that this dose effectively depresses cell proliferation within the endoneurium for 3–4 weeks [17, 18, 28]. Rats were killed ( n = 3) 1, 2, 3 or 4 weeks later. The spatio–temporal sequence of axonal regeneration into the grafts was assessed histologically, by immunofluorescence using antibodies against GAP–43; S–100; RT97; laminin and macrophages (EDI), and by transmission electron microscopy. Outgrowth of almost all axons from the mitomycin C–treated proximal stumps was delayed for up to 3 weeks, after which time vigorous regeneration occurred into the persisting tubes of sarcolemmal basal lamina. All axons regenerating within the grafts (irrespective of mitomycin C–treatment) were accompanied by co–migrating Schwann cells. The results suggest that Schwann cells play an important role in axonal regeneration across FT muscle autografts and that sarcolemmal basal laminae alone are insufficient to support axonal regeneration.