Schwann cell involvement in the neurological lesion of the dystonic mutant mouse: A nerve grafting study

Schwann cell function in the dystonic mutant mouse was studied by grafting peripheral nerve from normal into affected littermates of a C57/BL (Fa.) dt dystonic mouse colony and vice versa. In a control experiment, only unaffected animals of the colony were used, and nerve isografts were found to be ultrastructurally indistinguishable from normal nerve autografts. In addition, the isografts showed no features of the lymphocytic inflammatory rejection reaction observed in normal nerve allografts, and there was evidence that donor Schwann cells remained viable and were active in all the isografts examined. When nerve isografts from affected dystonic mutants were implanted into normal littermate nerves, the normal host axons regenerating through the grafted region acquired degenerative changes characteristic of naturally occurring dystonic peripheral nerve. These changes were not seen in the host axons regenerating either outside the dystonic graft regions, or more distally in the host nerve stumps. When normal nerve isografts were implanted into affected dystonic mutant nerves, the dystonic axons regenerated through the normal graft region and became normally myelinated. It is concluded that an underlying Schwann cell defect may be responsible for the abnormalities of the dystonic mouse peripheral neuropathy.     

Restless Legs Syndrome In Peripheral Neuropathy

The role of extracellular matrix (ECM) in the development and maintenance of the normal nervous tissue structure has been demonstrated. However, little is known about ECM modifications taking place in peripheral nerve pathology. This gap has prompted us to study the expression of several components of the peripheral nerve ECM in the major categories of human peripheral neuropathies. Normal and pathological sural nerve biopsies, including cases of CMT1, HNPP, CIDP and axonal neuropathies were studied by immunohistochemistry for the expression of collagen (COL) types I, III, IV, V, VI, laminin (LN), tenascin (TN), fibronectin (FN) and vitronectin (VN). The immunoreactivity of ECM proteins in pathologic nerves showed different patterns in relation to their distribution in normal nerves. Indeed, COL I and III, which are the main stromal components of the epineurium and endoneurium, respectively, showed increased expression in these compartments, generally in chronic neuropathies. COL IV, V, VI and LN localized mainly to basement membranes (BM) of Schwann cells, perineurial cells and blood vessels, showing increased deposition in the presence of BM redundancy. FN localized diffusely to the endoneurium and perineurium in control nerves and its expression increased in CIDP nerves. VN immunoreactivity was limited to several spots along the perineurium and epineurial vessels in normal nerve. However, in CIDP and in some cases of axonal neuropathy the VN signal was increased in the perineurium and appeared also in endoneurial vessels. Finally, TN was detected in the perineurium and the nodal/paranodal region in control nerve, while in some cases of CMT1 and CIDP it appeared to mark several internodes. These data, showing specific changes of the ECM components in peripheral neuropathies, suggest that a complex ECM remodeling is associated with mechanisms of nerve damage and repair. Supported by MURST-COFIN 98 (n°86) and by CNR 98 (n°03081 CT04).     

Comparison of different biogenic matrices seeded with cultured Schwann cells for bridging peripheral nerve defects

Tissue-engineering as laboratory based alternative to human autografts and allografts provides "custom made organs" cultured from patient's material. To overcome the limited donor nerve availability different biologic nerve grafts were engineered in a rat sciatic nerve model: cultured isogenic Schwann cells were implanted into acellular autologous matrices: veins, muscles, nerves, and epineurium tubes. Autologous nerve grafts, and the respective biogenic material without Schwann cells served as control. After 6 weeks regeneration was assessed clinically, histologically and morphometrically. The PCR analysis showed that the implanted Schwann cells remain within all the grafts. A good regeneration was noted in the muscle-Schwann cell-group, while regeneration quality in the other groups (with or without Schwann cells) was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. All venous and epineurium grafts had a more disorganized regeneration. Seemingly, the lack of endoneural tube like structures in vein grafts lead to impaired regeneration. And, apparently, the beneficial effects of implanted Schwann cells into a large luminal structure can only be demonstrated to a limited extent if endoneural like structures are lacking. A tube offers less area for Schwann cell adhesion and it is more likely to collapse. This underlines the role of the basal lamina, or at least an inner structure acting as scaffold in axonal regeneration. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biogenic graft with basal lamina tubes as pathway for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.     

Immunomodulation by costimulation blockade inhibits rejection of nerve allografts

The aim of this study was to investigate if costimulation blockade could be used to modulate the immune response, to prevent rejection, and to stimulate regeneration into nerve allografts. Nerve allografts from Balb/C mice, and isogenic nerve grafts (isografts) from C57/BL6 mice, were used to bridge a 7-mm gap of the sciatic nerve in C57/BL6 mice. Allograft recipients were treated with either a triple treatment with anti-lymphocyte function antigen-1 (anti-LFA), anti-CD40 ligand (anti-CD40L), and cytotoxic T-lymphocyte antigen 4 immunoglobulin (anti-CTLA4Ig) or isotype antibodies (placebo) at postoperative days 0, 2, 4, and 6 (intraperitoneal). After 5 or 9 days, the nerve grafts, together with the proximal and the distal nerve segments, were evaluated by histology and immunocytochemistry for inflammatory cells [CD4-positive (CD4+) and CD8-positive (CD8+) staining cells] and axonal outgrowth (neurofilaments). The immune response was inhibited by costimulation blockade with less extensive inflammation and a lower number of CD4+ staining cells in triple-treated allografts at 9 days. The regeneration rate was significantly faster in isografts (0.75 mm/day) compared with allografts with placebo treatment (0.39 mm/day), but not when compared with triple-treated allografts (0.49 mm/day). At 9 days, the axons were significantly longer in nerve isografts than in nerve allografts, irrespective of treatment. Hence, costimulation blockade neither increased the regeneration rate nor the outgrowth length in triple-treated allografts. We conclude that costimulation blockade inhibits the immune response in nerve allografts without deterring early axonal outgrowth.     

Electrophysiological features in the distinction between hereditary demyelinating and chronic acquired demyelinating neuropathies

Axonal loss is a consistent and undesirable event even in primary demyelinating neuropathies, and is responsible for most disabling and permanent deficits. In fact, in several peripheral neuropathies the physiological ability of nerve to regenerate is lost or dramatically reduced. Schwann cells play a central role in nerve regeneration and the laminin‐laminin receptor‐cytoskeleton pathway is fundamental to control numerous Schwann cell functions and Schwann cell‐axon interaction. A model to reproduce the events involved in axonal degeneration and regeneration is recapitulated in the stereotyped model of crush injury of the sciatic nerve in rodents. With this tool, we investigated the efficiency of nerve regeneration in mice lacking the glial fibrillary acidic protein (GFAP) intermediate filament, a Schwann cell specific cytoskeleton constituent. The peripheral nerves of GFAP null mice normally develop and function, and we did not detect gross differences in laminin and laminin receptor expression. However, after injury, the GFAP null mice showed a delay in the early phase of nerve regeneration.     

Comparison of different biogenic matrices seeded with cultured Schwann cells for bridging peripheral nerve defects

Abstract

Tissue-engineering as laboratory based alternative to human autografts and allografts provides "custom made organs" cultured from patient's material. To overcome the limited donor nerve availability different biologic nerve grafts were engineered in a rat sciatic nerve model: cultured isogenic Schwann cells were implanted into acellular autologous matrices: veins, muscles, nerves, and epineurium tubes. Autologous nerve grafts, and the respective biogenic material without Schwann cells served as control. After 6 weeks regeneration was assessed clinically, histologically and morphometrically. The PCR analysis showed that the implanted Schwann cells remain within all the grafts. A good regeneration was noted in the muscle-Schwann cell-group, while regeneration quality in the other groups (with or without Schwann cells) was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. All venous and epineurium grafts had a more disorganized regeneration. Seemingly, the lack of endoneural tube like structures in vein grafts lead to impaired regeneration. And, apparently,the beneficial effects of implanted Schwann cells into a large luminal structure can only be demonstrated to a limited extent if endoneural like structures are lacking. A tube offers less area for Schwann cell adhesion and it is more likely to collapse. This underlines the role of the basal lamina, or at least an inner structure acting as scaffold in axonal regeneration. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biogenic graft with basal lamina tubes as pathway for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.     

Expression of type I and III collagen and laminin beta1 after rat sciatic nerve crush injury

Extracellular matrix changes are thought to be essential to the regeneration of peripheral nerves. The production of this matrix is believed to be regulated by interactions between axons and their supporting cells. In this study matrix production and cell proliferation were studied during rat sciatic nerve regeneration after a crush injury, and compared to that after rat sciatic nerve transection. Expression of proalpha1(I) and proalpha1(III) collagen and laminin beta1 mRNAs was followed in isolated endoneuria by Northern and in situ hybridization both proximally and distally to the site of either a crush injury or transection of rat sciatic nerve up to 18 weeks. Changes in the Schwann cell and fibroblast populations were monitored by morphometric analysis of endoneurial cross-sections immunostained for S-100 protein. The process of axonal regeneration was followed by Bielschowsky's silver staining. A crush injury initially resulted in increased expression of all mRNAs studied in the endoneurial cells. However, with progressing axonal regeneration the amount of collagen mRNAs returned to control levels, whereas the amount of laminin beta1 mRNA in the distal site of the crush remained elevated throughout the study period. The expression of type I collagen mRNA was enhanced after nerve transection injury compared to that after the crush injury. The epineurial fibroblasts actively expressed both type I and III collagen mRNAs after the injury. The proliferation of Schwann cells and the expression of collagen mRNAs are not, at least directly, related to the axonal regeneration. However, the long-lasting and strong expression of laminin beta1 mRNA after a nerve crush injury may be related to good axonal regeneration. The expression of type I collagen in the epineurium may lead to clinically well-recognized epineurial scarring and thus impede axonal regeneration.     

Immunohistochemical assessment of rat nerve isografts and immunosuppressed allografts

Objective: Autologous peripheral nerve grafts are commonly used clinically as a treatment for peripheral nerve injuries. However, in research using an autologous graft is not always feasible due to loss of function, which in many cases is assessed to determine the efficacy of the peripheral nerve graft. In addition, using allografts for research require the use of an immunosuppressant, which creates unwanted side effects and another variable within the experiment that can affect regeneration. The objective of this study was to analyze graft rejection in peripheral nerve grafts and the effects of cyclosporine A (CSA) on axonal regeneration.

Methods: Peripheral nerve grafts in inbred Lewis rats were compared with Sprague-Dawley (SD) rats to assess graft rejection, CSA side effects, immune responses, and regenerative capability. Macrophages and CD8+ cells were labeled to determine graft rejection, and neurofilaments were labeled to determine axonal regeneration.

Results: SD rats without CSA had significantly more macrophages and CD8+ cells compared to Lewis autografts, Lewis isografts, and SD allografts treated with CSA. Lewis autografts, Lewis isografts, and SD autografts had significantly more regenerated axons than SD rat allografts. Moreover, allografts in immunosuppressed SD rats had significantly less axons than Lewis rat autograft and isografts.

Discussion: Autografts have long been the gold standard for treating major nerve injuries and these data suggest that even though CSA is effective at reducing graft rejection, axon regeneration is still superior in autografts versus immunosuppressed allografts.     

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.     

Oxidized galectin-1 stimulates the migration of Schwann cells from both proximal and distal stumps of transected nerves and promotes axonal regeneration after peripheral nerve injury

Oxidized galectin-1 has recently been identified as a key factor that plays important roles in initial axonal growth in injured peripheral nerves. The aim of this study was to investigate the effects of oxidized galectin-1 on regeneration of rat spinal nerves using acellular autografts (containing no viable cells) and allografts (containing no cell membranes) with special attention to the relationship between axonal regeneration and Schwann cell migration. Immunohistochemically, endogenous galectin-1 was expressed in dorsal root ganglion (DRG) neurons, spinal cord motoneurons, and axons and Schwann cells in normal sciatic nerves. Administration of oxidized recombinant human galectin-1 (rh-gal-lox, 5 ng/ml) in autograft model promoted axonal regeneration from motoneurons as well as from DRG neurons; this was confirmed by a fluorogold tracer study (p < 0.05). Anti-rh-gal-1 antibody (30 microg/ml) strongly inhibited axonal regrowth (p < 0.05). Pretreatment of allografts with rh-gal-lox stimulated the migration of Schwann cells not only from proximal stumps but also from distal stumps into the grafts, resulting in accelerated axonal regeneration (p < 0.05). Moreover, Schwann cell migration preceded the axonal growth in the presence of exogenous rh-gal-lox in the grafts. These results strongly suggest that local administration of exogenous rh-gal-lox promotes the migration of Schwann cells followed by axonal regeneration from both motor and sensory neurons, resulting in acceleration of neuronal repair. This technique may also be of value in the repair of human nerves.     

Neurite-promoting factors and extracellular matrix components accumulating in vivo within nerve regeneration chambers

The outgrowth of neurites from cultured neurons can be induced by the extracellular matrix glycoproteins, fibronectin and laminin, and by polyornithine-binding neurite-promoting factors (NPFs) derived from culture media conditioned by Schwann, or other cultured cells. We have examined the occurrence of fibronectin, laminin and NPFs during peripheral nerve regeneration in vivo. A previously established model of peripheral nerve regeneration was used in which a transected rat sciatic nerve regenerates through a silicone chamber bridging a 10 mm interstump gap. The distribution of fibronectin and laminin during regeneration was assessed by indirect immunofluorescence. Seven days after nerve transection the regenerating structure within the chamber consisted primarily of a fibrous matrix which stained with anti-fibronectin but not anti-laminin. At 14 days, cellular outgrowths from the proximal and distal stumps (along which neurites grow) had entered the fibronectin-containing matrix, consistent with a role of fibronectin in promoting cell migration. Within these outgrowths non-vascular as well as vascular cell stained with anti-fibronectin and anti-laminin. Wihtin the degenerated distal nerve segment, cells characteristics of Bungner bands (rows of Schwann cells along which regenerating neurites extend) stained with anti-fibronectin and laminin. The fluid surrounding the regenerating nerve was found to contain NPF activity for cultured ciliary ganglia neurons which markedly increased during the period of neurite growth into the chamber. In previous studies using this particular neurite-promoting assay, laminin but to a much lesser extent fibronectin also promoted neurite outgrowth. Affinity-purified anti-laminin antibody failed to block chamber fluid NPF activity while completely blocking the neurite-promoting activity of laminin. These two results suggested that chamber fluid NPF activity did not consist of individual molecules of either fibronectin or laminin. The spatial and temporal distribution of insoluble fibronectin and laminin and the temporal correlation between chamber fluid NPF accumulation and neurite outgrowth support the possibility that these agents influence regenerative events including axonal elongation in vivo.     

Failure of host axons to regenerate through a once successful but later rejected long nerve allograft

Host axons will regenerate through a long nerve allograft in an immunologically tolerant rat. However, if tolerance is abolished, rejection occurs and allogeneic cells (e.g., Schwann, vascular, perineurial, etc.) as well as regenerated host axons disappear from the allograft. Because following tolerance abolition host axons begin to regenerate into the connective tissue remnants of the rejected nerve allografts, the extent of this renewed axonal growth was investigated. It was found that in a tolerance-abolished rat, host axons only regrew into the proximal 1 cm of a 4-cm allograft which in a fully tolerant recipient would have had numerous allogeneic Schwann cell-myelinated axons throughout its length. It is concluded that viable allogeneic cells (i.e., Schwann, fibroblast, and vascular) together with their connective tissue matrix provide the best way to aid host nerve fiber regeneration through a long nerve allograft.     

Laminin-coated multifilament entubulation,combined with Schwann cells and glial cell line-derived neurotrophic factor,promotes unidirectional axonal regeneration in a rat model of thoracic spinal cord hemisection

Biomaterial bridging provides physical substrates to guide axonal growth across the lesion. To achieve efficient directional guidance, combinatory strategies using permissive matrix, cells and trophic factors are necessary. In the present study, we evaluated permissive effect of poly (acrylonitrile-co-vinyl chloride) guidance channels filled by different densities of laminin-precoated unidirectional polypropylene filaments combined with Schwann cells, and glial cell line-derived neurotrophic factor for axonal regeneration through a T10 hemisected spinal cord gap in adult rats. We found that channels with filaments significantly reduced the lesion cavity, astrocytic gliosis, and inflammatory responses at the graft-host boundaries. The laminin coated low density filament provided the most favorable directional guidance for axonal regeneration which was enhanced by co-grafting of Schwann cells and glial cell line-derived neurotrophic factor. These results demonstrate that the combinatorial strategy of filament-filled guiding scaffold, adhesive molecular laminin, Schwann cells, and glial cell line-derived neurotrophic factor, provides optimal topographical cues in stimulating directional axonal regeneration following spinal cord injury. This study was approved by Indiana University Institutional Animal Care and Use Committees (IACUC #:11011) on October 29, 2015.Key Words: axonal regeneration, extracellular molecule, filament density, hemisection, laminin, neurotrophic factor, Schwann cell, spinal cord injury, thoracic, transplantation

Chinese Library Classification No. R453; R741; R364.5     

The perineurium modifies the effects of phenol and glycerol in rat sciatic nerve

Endoneurial cell response and type of nerve fibre damage were studied after perineural injections of 7% phenol-aqua and pure glycerol. Our previous studies have shown that phenol and glycerol induce different types of nerve fibre degeneration after intraneural injections: phenol dissolves axons and Schwann cells inside the basal lamina tubes but glycerol breaks them down into cellular flakes. The current study investigated whether the difference in type of endoneurial damage also appears after perineural application and how the perineurium affects the effect of these neurolytic agents. Rat sciatic nerves were treated with perineural injections of 7% phenol-aqua or pure glycerol and were followed up to 6 months. The results support the previous findings that perineural phenol injection induces damage that covers almost the whole endoneurium, but glycerol injection results in minor subperineurial damage. An ultrastructural study showed that the endoneurial effects are much milder after perineural injection than after intraneural injections. Phenol-induced nerve fibre dissolving was only rarely seen and the nerve fibre damage appeared similar to that after regular Wallerian degeneration in both groups. Axonal regeneration began within 2 weeks of the injections. Endoneurial macrophages were numerous in the damaged area in many individual nerves even at 3–6 months in both groups, which may indicate impaired phagocytotic activity. Regenerating axonal sprouts were seen first at 1 week post injection and Schwann cells proliferated within 2 weeks in both groups. However, the number of axonal sprouts was higher (P=0.002) and the size of the sprouts appeared larger after glycerol injection at 4 weeks post injection. The present study shows that the effects of extraneurally applied neurolytic agents phenol and glycerol are modified by the perineurium. Phenol readily penetrates the perineurium, but glycerol causes only subperineurial damage. The type of damage is rather similar to regular Wallerian degeneration in both groups and the endoneurial effects differ from those seen after intraneural injections.     

Fresh and predegenerate nerve allografts and isografts in trembler mice

In order to investigate whether Schwann cell or myelin was the principal antigen responsible for nerve graft reiection, fresh nerve grafts and those in which myelin had been previously allowed to degenerate (predegenerate grafts) from both isogeneic BALB/c and allogeneic C57/B1 mice were inserted into trembler BALB/c mice. Schwann cells within nerve allografts from C57/B1 mice were rejected, whether or not the grafts contained myelin. Nerve isografts from normal BALB/c animals produced normally myelinated trembler axons within the grafted segments, and across these segments conduction velocity was restored towards the normal value. It is concluded that Schwann cells, not myelin, constitute the principlal antigen within nerve allografts and it is Schwann-cell rejection that limits the sucessful use of nerve allografts.     

The fate of cryopreserved nerve isografts and allografts in normal and immunosuppressed rats

Donor Schwann cells, perineurial cells, and vasculature are known to survive in grafts of peripheral nerve. In the present study, we attempted to cryopreserve nerve to determine whether these cellular components of nerve would survive after transplantation and support host axonal regeneration through the graft. Four-centimeter lengths of peroneal nerves were removed from inbred adult American Cancer Institute (ACI) rats and placed into vials that contained a cryoprotective mixture of dimethyl sulfoxide and formamide (DF) at room temperature. Each vial with nerves in DF was cooled at a rate of 1–1.5°C/minute down to –40°C at which point the vials were plunged into liquid nitrogen at –196°C. After 5 weeks of storage, the nerves were thawed and DF removed. Some of the cryopreserved-thawed ACI nerves were transplanted as isografts into the legs of ACI rats. Other ACI nerves were used as allografts and inserted into immunologically normal Fischer (FR) rats that were untreated or were immunosuppressed with the drug Cyclosporin A (Cy-A). At surgery, only one end of the nerve graft was joined to the cut proximal end of the peroneal nerve of the host. The cellular elements of ACI grafts were present at 5 weeks in grafts removed from ACI rats and FR rats treated with Cy-A. Non-immunosuppressed FR rats rejected ACI nerves as did FR rats in whom Cy-A was stopped after 5 weeks of treatment. All surviving ACI grafts underwent Wallerian degeneration and consisted of columns of Schwann cells, which in their proximal portion were associated with regenerating host axons. The donor perineurial sheath and vasculature were also present in surviving grafts. ACI isografts only were examined 20 weeks postoperatively. All normal tissue components survived in these older grafts and contained regenerated and myelinated host axons throughout their 4 cm lengths. These results demonstrated that the cellular elements of nerve can be cryopreserved, and after transplantation, survive and function. Because nerves survived after prolonged cryopreservation, it seems feasible to establish a nerve bank from which grafts can be withdrawn to repair gaps in injured nerves. However, cryopreserved nerves used as allografts remain immunogenic and require immunosuppression for their survival. Published in 1993 by Wiley-Liss, Inc.     

The influence of cultured Schwann cells on regeneration through acellular basal lamina grafts

Acellular basal lamina grafts have been shown to be less immunogenic in comparison to cellular grafts, but possess a limited potential for supporting axonal regeneration through them. The present study describes the effect of cultured Schwann cells on enhancing regeneration through acellular grafts. 2 cm long acellular grafts, and in vitro Schwann cell populated acellular grafts were used to repair a surgically created gap in the host peroneal nerve. The transplants were analyzed at 1, 2, 4 and 8 weeks to determine their ability to support axonal regeneration. Host axonal regeneration through Schwann cell cocultured acellular grafts occurred rapidly and was significantly better as compared to non-cultured acellular grafts. The results demonstrate a beneficial effect of Schwann cell culture pretreatment on regeneration through acellular grafts and an improved recovery of the target muscle. The procedure of first preparing acellular grafts with subsequent coculture with Schwann cells offers a novel approach for the repair of injured nervous tissue.     

Long-term endoneurial changes after nerve transection

Summary Long-term endoneurial changes in the distal stump of transected rat sciatic nerve were examined from 8 to 50 weeks after nerve transection. The morphological alterations were followed both in nerves which were allowed to regenerate and in nerves in which regeneration was prevented by suturing. The nerves prevented from regenerating showed markedly atrophied Schwann cell columns after 20 weeks and a disappearance of some Schwann cell columns after 30 weeks. The surrounding endoneurial fibroblast-like cells gradually lost their delicate cytoplasmic extensions and formed rough fascicles around numerous shrunken Schwann cell columns or around areas from which Schwann cells had apparently disappeared. Inside the fascicles, the Schwann cell loss was replaced by collagen fibrils or occasionally, by a dense accumulation of microfibrils. The loss of endoneurial cytoplasmic processes continued up to 50 weeks, leaving behind patches of thin fibrils around numerous shrunken Schwann cell columns or around collagenous areas where Schwann cells were lost. The endoneurial matrix showed presence of thin 25- to 30-nm collagen fibrils close to shrunken Schwann cell columns up to 50 weeks but in areas with advanced degeneration a shift towards regular 50- to 60-nm collagen fibrils occurred. The degenerated areas resembled those described in Renaut bodies and neurofibromas. Despite suturing of transected nerves to prevent sprouting, occasional regenerating sprouts were noted in the Schwann cell columns. These axons were surrounded in a sheath-like fashion by pre-existing endoneurial cell fascicles covered by a basal lamina. In the reinnervating nerves the endoneurial space gradually lost its compartmentized structures consisting of collagen fibrils and endoneurial fibroblast-like cells. After 20 weeks the endoneurial cells were inconspicuous and the extracelluar matrix consisted mainly of 50- to 60-nm collagen fibrils. During axonal growth and maturation, Schwann cells containing unmyelinated axons surrounded large, myelinated axons in a collar-like fashion. Close to these collars of Schwann cells, thin 25- to 30-nm collagen fibrils were noted in focal areas, even after 50 weeks. Occasionally, numerous clusters of regenerating axonal sprouts were noted in the perineurium. These were surrounded by multiple layers of cells possessing basal lamina. The present results show that after nerve transection the distal stump of the severed nerve shows dynamic changes in the endoneurial space, especially in nerves where reinnervation is prevented. The endoneurial fascicles around occasional axonal sprouts in sutured nerves, representing possibly a delayed type of regeneration, show that axons have a strong ability to grow but on the other hand endoneurial structures are unable to respond normally to axonal growth after advanced degeneration.     

Stimulation of Schwann cell proliferation and axonal regeneration by FK 506

PURPOSE: Nerve allografts are highly antigenic and, thus, require the continuous use of immunosuppressive drugs. FK 506 was found to pre-vent rejection successfully. However, clinically neurotoxic complications have been noted in the central and peripheral nervous system although an increased rate of axonal regeneration has also been shown after nerve crush experiments. To investigate whether a possible regeneration pro-moting potency of FK 506 is determined via an influence on Schwann cells, Schwann cells were cultured from the sciatic nerve of the rat. METHODS: The effect of 100 micro M FK 506 administered daily on these cultures was assessed microscopically over a period of seven days and compared to an untreated control group of cultures. Additionally, the changes in intracellular calcium were recorded using a laser scanning microscope. In vivo regeneration of autologous rat sciatic nerve grafts was assessed clinically, histologically and morphometrically after two and six weeks. The animals received a daily administration of 0.6 mg FK 506/kg body weight, the control received saline. RESULTS: In vitro FK 506 increased the Schwann cell number in culture significantly compared to non treated cultures, while the fibrocyte population was decreased. FK 506 caused a transient increase of intracellular calcium levels in cultured cells. In vivo, a significantly higher axon count was observed in the FK 506 treated grafts after two weeks regeneration compared with controls. Good regeneration was noted in all grafts after six weeks regeneration. CONCLUSIONS: The increased axon counts and decreased myelin debris in the FK 506 grafts after two weeks indicate an accelerated Wallerian degeneration and increased axon sprouting into the graft initially. The inhibition of calcineurin activity is not the mediator of the neurotrophic effect. FK 506 promotes axonal regeneration through binding to FKBP-12. The increase of intracellular calcium may induce Schwann cell pro-liferation via calmodulin. The therapeutic relevance for autologous nerve grafting, however, has to be defined in further studies.     

Schwann cells synthesize alpha7beta1 integrin which is dispensable for peripheral nerve development and myelination

Defects in laminins or laminin receptors are responsible for various neuromuscular disorders, including peripheral neuropathies. Interactions between Schwann cells and their basal lamina are fundamental to peripheral nerve development and successful myelination. Selected laminins are expressed in the endoneurium, and their receptors are developmentally regulated during peripheral nerve formation. Loss-of-function mutations have confirmed the importance and the role of some of these molecules. Here we show for the first time that another laminin receptor, alpha7beta1 integrin, previously described only in neurons, is also expressed in Schwann cells. The expression of alpha7 appears postnatally, such that alpha7beta1 is the last laminin receptor expressed by differentiating Schwann cells. Genetic inactivation of the alpha7 subunit in mice does not affect peripheral nerve formation or the expression of other laminin receptors. Of note, alpha7beta1 is not necessary for basal lamina formation and myelination. Nonetheless, these data taken together with the previous demonstration of impaired axonal regrowth in alpha7-null mice suggest a possible Schwann cell-autonomous role for alpha7 in nerve regeneration.