The role of laminin, a component of Schwann cell basal lamina, in rat sciatic nerve regeneration within antiserum-treated nerve grafts

Regeneration of the sciatic nerve in transplanted nerve grafts in which laminin was inactivated was examined electron microscopically. Nerve grafts for transplantation were obtained from close cloned donor Wistar rats; 1-cm nerve segments of the sciatic nerve were frozen and thawed to kill the Schwann cells. Control recipient rats received grafts treated with normal rabbit serum to repair the artificially-made complete defect of the right sciatic nerve, and the experimental group of rats received grafts doubly treated with normal serum and rabbit anti-laminin antiserum. In the control grafts regenerating axons grew almost completely through the inside of the basal lamina scaffolds (92%) and adhered to the structure, while in the anti-laminin antiserum treated grafts the axons were present outside (52%) and inside (48%) the scaffolds simultaneously. In this case, the adhesion of axons to the scaffolds was obscure. Axons were associated with and without Schwann cells both inside and outside the basal lamina scaffolds. No unassociated Schwann cells were observed. The maximal number of axons in a 2 mm portion of the antiserum-treated grafts was approximately 250 axons per 100 × 100 μm square and 520 in the control at 15 days. At 30 days, almost the same number of axons was found at the distal (8 mm) portion of both groups. The growth in the former was delayed for 3 days. These results indicate that regenerating peripheral nerve axons may enter the basal lamina scaffolds and grow well because of the neurotrophic function of laminin present at the inner side of Schwann cell basal lamina.     

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.     

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.     

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.     

Effect of allogeneic Schwann cell transplantation on peripheral nerve regeneration

Transplantation of allogeneic Schwann cells (SC) would make it feasible to reconstruct immediately peripheral nerve defects, compared to using autologous SC; however, this treatment modality has not been adequately evaluated. The aim of this study was to characterize and compare the effects of allogeneic versus syngeneic SC transplantation following peripheral nerve injury. Polyhydroxybutyrate conduits were used to bridge a 10-mm gap in the rat sciatic nerve. The conduits were filled with alginate hydrogel with or without cultured allogeneic or syngeneic genetically labeled SC, without the use of immunosuppressive therapy, and examined after 2, 3, and 6 weeks with 5-bromo-4-chloro-3-indoyl-beta-D-galactosidase chemical staining and immunohistochemistry to quantify SC migration into the conduit, axonal regeneration, the state of SC differentiation, and the expression of major histocompatibility complexes (MHC) I and II, as well as to quantify macrophage and B- and T-lymphocyte infiltration. Allogeneic SC were rejected by 6 weeks, whereas syngeneic SC could still be identified. Allogeneic and syngeneic SC equally enhanced the axonal regeneration distance but the quantity of axons was greater using syngeneic SC. The ingrowth of SC into the conduits containing allogeneic SC was similar to that observed in the presence of syngeneic SC, indicating the absence of deleterious immune response. SC continued to express phenotypic markers of nonmyelination and these were highest in conduits with allogeneic SC. Expression of MHC I and II was higher in the conduits with allogeneic SC at 3 weeks and without significant difference in the number of macrophages and lymphocytes, except at 6 weeks, when there was a larger number of lymphocytes using syngeneic SC. In conclusion, allogeneic SC enhanced axonal regeneration distance and did not induce a deleterious immune response. In a clinical setting the immediate availability of allogeneic SC for transplantation may compensate for the better outcome achieved by the use of autologous SC that require a longer preparation time in culture.     

Schwann cell endocytosis: a role in nerve regeneration?

Schwann cell plasma membrane vesicles have been shown to increase in numerical density after nerve injury but their function is unclear. In this study, ultrastructural tracers were micro-injected in vivo into crushed rat sciatic nerves after various time intervals to ascertain whether plasma membrane vesicles of Schwann cells are involved in the uptake and utilization of molecules from the endoneurium during axonal regeneration and remyelination. Horseradish peroxidase (HRP), a tracer of fluid-phase endocytosis, was taken up by macrophages and fibroblasts but remained external to Schwann cells throughout the study. After 14-16 days of crush injury, HRP was present within vessel lumina and in cytoplasmic vesicles of pericytes and vascular endothelia. Low-density lipoprotein-gold, which is primarily internalized by receptor-mediated endocytosis, and bovine serum albumin-gold, proposed as a tracer for fluid-phase endocytosis, were internalized by macrophages and fibroblasts but were not taken up by Schwann cells. Although Schwann cells formed pits in the plasma membrane and vesicles were evident in the cytoplasm, none of the tracers used were internalized by Schwann cells. It is suggested that Schwann cell plasmalemmal and cytoplasmic vesicles have a cellular role unrelated to endocytosis or alternatively the Schwann cell basal lamina may function as a diffusion barrier to the tracers employed.     

Schwann cell delivery of neurotrophic factors for peripheral nerve regeneration

Current treatments of injured peripheral nerves often fail to mediate satisfactory functional recovery. For axonal regeneration, neurotrophic factors (NTFs) play a crucial role. Multiple NTFs and other growth‐promoting factors are secreted, amongst others, by Schwann cells (SCs), which also provide cellular guidance for regenerating axons. Therefore, delivery of NTFs and transplantation of autologous or genetically modified SCs with therapeutic protein expression have been proposed. This article reviews polymer‐based and cellular approaches for NTF delivery, with a focus on SCs and strategies to modulate SC gene expression. Polymer‐based NTF delivery has mostly resided on nerve conduits (NC). While NC have generally provided prolonged NTF release, their therapeutic effect has remained significantly below that achieved with autologous nerve grafts. Several studies demonstrated enhanced nerve regeneration using NC seeded with SCs. The SCs have sometimes been modified genetically using non‐viral or viral vectors. Whereas non‐viral vectors produced poor transgene delivery, adenoviral vectors mediated high transgene transduction efficiency of SCs. Further improvements of safety and transgene expression of adenoviral vector may lead to rapid translation of pre‐clinical research to clinical trials.     

Optic nerve regeneration within artificial Schwann cell graft in the adult rat

We investigate whether an artificial graft made by cultured Schwann cell, extracellular matrix (ECM) and trophic factors can provide the environment for the regeneration of retinal ganglion cell (RGC) axons in adult rats. Six kinds of artificial grafts were used: ECM (control); ECM and Schwann cells; ECM, Schwann cells and either nerve growth factor, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4); ECM, Schwann cells, BDNF and NT-4, combined with intravitreal injection of BDNF. The grafts were transplanted onto the transected optic nerve. RGC regeneration was evaluated by dil retrograde labeling, immunohistochemistry, and electron microscopy at 3 weeks post-operation. The degree of dil labeled RGC was approximately 2% for ECM alone, and 10% for ECM and Schwann cells (p < 0.01). The labeling increased to approximately 20% by administration of neurotrophins. The addition of intravitreous BDNF injection resulted in highest labeling percentage of 30%. Immunohistochemical study showed that axons were association with GAP-43 and cell adhesion molecules. Neurotrophin receptors (Trk-A and Trk-B) were detected in nerve fibers both in the retina and in the graft. Remyelination was seen by electron microscopic observation. These results demonstrate that the regeneration of RGC axons is induced with the use of cultured Schwann cells and ECM as promoting factors for regrowth. The degree of regeneration was significantly increased by neurotrophins in the grafts and in the vitreous.     

Neurotrophin-3 deficient Schwann cells impair nerve regeneration

Neurotrophin 3 (NT-3) is an important autocrine factor supporting Schwann cell (SC) survival and differentiation in the absence of axons. Prior studies have failed to define the explicit role of SC versus axon in NT-3 deficiency in relation to nerve regeneration and associated remyelination. In the paradigm we studied, using NT-3 heterozygous (NT3^+/−) knockout mice capable of survival into adult-life, the experimental design provided a model uniquely capable of differentiating SC/axon influences. In these studies we first identified a defect in nerve regeneration characterized by fewer SCs in the regenerating nerve fibers of crushed sciatic nerves of NT3^+/− mice. Subsequent experiments differentiated SC versus axonal influences as the culprit in defective nerve regeneration using sciatic nerve transplant paradigms. Results show an impairment in nerve regeneration in NT3^+/− mice with a retardation of the myelination process, and this defect is associated with decreased SC survival and an increase in the neurofilament packing density of regenerating axons. These observations indicate that NT3^+/− status of the SCs, but not of the axons, is responsible for impaired nerve regeneration and that NT-3 is essential for SC survival in early stages of regeneration-associated myelination in the adult peripheral nerve.     

Electroacupuncture and moxibustion promote regeneration of injured sciatic nerve through Schwann cell proliferation and nerve growth factor secretion

Using electroacupuncture and moxibustion to treat peripheral nerve injury is highly efficient with low side effects. However, the electroacupuncture-and moxibustion-based mechanisms underlying nerve repair are still unclear. Here, in vivo and in vitro experiments uncovered one mechanism through which electroacupuncture and moxibustion affect regeneration after peripheral nerve injury. We first established rat models of sciatic nerve injury using neurotomy. Rats were treated with electroacupuncture or moxibustion at acupoints Huantiao(GB30) and Zusanli(ST36). Each treatment lasted 15 minutes, and treatments were given six times a week for 4 consecutive weeks. Behavioral testing was used to determine the sciatic functional index. We used electrophysiological detection to measure sciatic nerve conduction velocity and performed hematoxylin-eosin staining to determine any changes in the gastrocnemius muscle. We used immunohistochemistry to observe changes in the expression of S100—a specific marker for Schwann cells—and an enzyme-linked immunosorbent assay to detect serum level of nerve growth factor. Results showed that compared with the model-only group, sciatic functional index, recovery rate of conduction velocity, diameter recovery of the gastrocnemius muscle fibers, number of S100-immunoreactive cells, and level of nerve growth factor were greater in the electroacupuncture and moxibustion groups. The efficacy did not differ between treatment groups. The serum from treated rats was collected and used to stimulate Schwann cells cultured in vitro. Results showed that the viability of Schwann cells was much higher in the treatment groups than in the model group at 3 and 5 days after treatment. These findings indicate that electroacupuncture and moxibustion promoted nerve regeneration and functional recovery; its mechanism might be associated with the enhancement of Schwann cell proliferation and upregulation of nerve growth factor.     

Phenomenon of Schwann cell apoptosis in a case of congenital hypomyelinating neuropathy with basal lamina onion bulb formation

We analyzed a sural nerve biopsy of a child with congenital hypomyelinating neuropathy. A lack of normally myelinated fibres, abnormal architecture of premyelin fibres and basal lamina onion bulbs were identified. The most prominent pathological finding was the appearance of significant death of Schwann cells with apoptotic morphology. This new finding may suggest that abnormal premyelin fibres are susceptible to death and that their disappearance is responsible for empty basal lamina onion bulb formation.     

Peripheral nerve regeneration through nerve guides seeded with adult Schwann cells

A.D. Ansselin, T. Fink and D.F. Davey (1997) Neuropathology and Applied Neurobiology 23 , 387–398
Peripheral nerve regeneration through nerve guides seeded with adult Schwann cells
This study tested the usefulness of Schwann cells in the repair of a severed nerve with a biosynthetic bridge or guide. Reinforced collagen nerve guides were used to bridge an 18 mm gap in the sciatic nerve of 21 young adult rats. The animals were divided into three groups and the guides were filled with: (i) more than 0.5 × 10⁶ cultured syngeneic adult Schwann cells (group L, n = 12); (ii) less than 0.5 × 10⁶ Schwann cells (Group S, n = 6); and (iii) phosphate buffered saline (control, n = 3). Schwann cells were pre-labelled with Hoechst dye. Regeneration was assessed functionally and histologically at 1, 2, 3 and 6+ months after surgery. Group L animals showed numerous regenerated axons surrounded by implanted Schwann cells within the first month. The total number of myelinated fibres (12.5 × 10³) remained above normal unoperated values (7 × 10³) in long-term animals. Regenerated axons were found in Group S in the third month, but no Hoechst labelled cells were found. The number of myelinated fibres (3.9 × 10³) remained below normal values in long-term animals. Control guides failed to support axonal regeneration. Functional recovery was evident at week 20 (Group L) and week 30 (Group S) after surgery, with no difference in function between the two groups by the end of the study. Supplementing guides with Schwann cells enhances regeneration of peripheral axons over a distance normally prohibitive. This effect is greatest in the early stages of regeneration (1–3 months) and is dependent on the number of cells implanted.     

Extracellular fluid conditioned during peripheral nerve regeneration stimulates Schwann cell adhesion, migration and proliferation

Schwann cell movement and proliferation occur during peripheral nerve regeneration and remyelination. We asked whether soluble factors promoting these activities were present in fluid surrounding rat sciatic nerves regenerating across a 10-mm gap bridged by a silicone tube. In this model, regenerated and remyelinated axons extend across the gap by 28 days following nerve transection and tube implantation. Fluid conditioned by cells participating in nerve regeneration (RCF) was assayed for its ability to promote Schwann cell adhesion, migration and proliferation in vitro. RCFs collected at post-transectional days 1-28 were equally effective in promoting Schwann cell-substratum adhesion. In contrast, the motility-promoting activity of RCF was minimal at 1-2 days following nerve-transection, peaked at 7 days and remained elevated through 21 days. The RCF peak response was 87-fold greater than control. Schwann cell proliferative activity of RCF exhibited peaks of activity at 1 and 14 days post-transection. The biological potency of this fluid for each activity assayed in vitro correlated well with the behavior of Schwann cells chronicled during nerve repair in vivo. These findings suggest that soluble factors promoting Schwann cell adhesion, migration, and proliferation accumulate extracellularly during peripheral nerve regeneration and remyelination.     

Chronic Schwann cell denervation and the presence of a sensory nerve reduce motor axonal regeneration

Motor axonal regeneration is compromised by chronic distal nerve stump denervation, induced by delayed repair or prolonged regeneration distance, suggesting that the pathway for regeneration is progressively impaired with time and/or distance. In the present experiments, we tested the impacts of (i) chronic distal sensory nerve stump denervation on axonal regeneration and (ii) sensory or motor innervation of a nerve graft on the ability of motoneurons to regenerate their axons from the opposite end of the graft. Using the motor and sensory branches of rat femoral nerve and application of neuroanatomical tracers, we evaluated the numbers of regenerated femoral motoneurons and nerve fibers when motoneurons regenerated (i) into freshly cut and 2-month chronically denervated distal sensory nerve stump, (ii) alone into a 4-cm-long distally ligated sensory autograft (MGL) and, (iii) concurrently as sensory (MGS) or motor (MGM) nerves regenerated into the same autograft from the opposite end. We found that all (315 +/- 24: mean +/- SE) the femoral motoneurons regenerated into a freshly cut distal sensory nerve stump as compared to 254 +/- 20 after 2 months of chronic denervation. Under the MGL condition, 151 +/- 5 motoneurons regenerated, which was not significantly different from the MGM group (134 +/- 13) but was significantly reduced to 99 +/- 2 in the MGS group (P < 0.05). The number of regenerated nerve fibers was 1522 +/- 81 in the MGL group, 888 +/- 18 in the MGM group, and 516 +/- 44 in the MGS group, although the high number of nerve fibers in the MGL group was due partly to the elaboration of multiple sprouts. Nerve fiber number and myelination were reduced in the MGS group and increased in the MGM group. These results demonstrate that both chronic denervation and the presence of sensory nerve axons reduced desired motor axonal regeneration into sensory pathways. A common mechanism may involve reduced responsiveness of sensory Schwann cells within the nerve graft or chronically denervated distal nerve stump to regenerating motor axons. The findings confirm that motor regeneration is optimized by avoiding even short-term denervation. They also imply that repairing pure motor nerves (without their cutaneous sensory components) to distal nerve stumps should be considered clinically when motor recovery is the main desired outcome.     

Coincidence of Schwann cell-derived basal lamina development and loss of regenerative specificity of spinal motoneurons

The Schwann cell-derived basal lamina forms a tube around single peripheral axons or small groups of axons that is continuous from the spinal cord to the target. In bullfrog tadpoles (Rana catesbeiana), motor axons transected at early developmental stages regenerate to the appropriate hindlimb region. In the present paper, we found that at these stages Schwann tubes are absent by morphological criteria, and individual axons are separated only by occasinal extensions of support cells. At stages when axons no longer regenerate to the correct hindlimb region, every axon is encased in a basal lamina tube. Schwann tubes persist in the distal stump after nerve transection, and regenerating axons grow within these tubes. These findings are consistent with previous results showing that the errors regenerating axons make in older animals are not random, but depend upon the course of the denervated Schwann tubes to which they ahve acces. In order to determine whether formation of the Schwann tube itself or interaction of its molecular constituents with growing axons was associated with loss of regenerative specificity, the expression during development of two major constituents of the basal lamina, laminin and heparan sulfate proteoglycan, was investigated. Immunoreactivity to both constituents was present both before and after the transition from specific to nonspecific regeneration, indicating that their expression per se was not sufficient to limit regnerative specificity. These data support the hypothesis that the physical constraint imposed by the Schwann cell-derived basal lamina prevents regenerative specificity. © 1993 Wiley-Liss, Inc.     

Effects of short- and long-term Schwann cell denervation on peripheral nerve regeneration, myelination, and size

Poor functional recovery after peripheral nerve injury has been generally attributed to inability of denervated muscles to accept reinnervation and recover from denervation atrophy. However, deterioration of the Schwann cell environment may play a more vital role. This study was undertaken to evaluate the effects of chronic denervation on the capacity of Schwann cells in the distal nerve stump to support axonal regeneration and to remyelinate regenerated axons. We used a delayed cross-suture anastomosis technique in which the common peroneal (CP) nerve in the rat was denervated for 0-24 weeks before cross-suture of the freshly axotomized tibial (TIB) and chronically denervated CP nerve stumps. Motor neurons were backlabeled with either fluoro-ruby or fluorogold 12 months later, to identify and count TIB motor neurons that regenerated axons into chronically denervated CP nerve stumps. Number, size, and myelination of regenerated sensory and motor axons were determined using light and electron microscopy. We found that short-term denervation of < or =4 weeks did not affect axonal regeneration but more prolonged denervation profoundly reduced the numbers of backlabeled motor neurons and axons in the distal nerve stump. Yet, atrophic Schwann cells retained their capacity to remyelinate regenerated axons. In fact, the axons were larger and well myelinated by long-term chronically denervated Schwann cells. These findings demonstrate a progressive inability of chronically denervated Schwann cells to support axonal regeneration and yet a sustained capacity to remyelinate the axons which do regenerate. Thus, axonal interaction can effectively switch the nonmyelinating phenotype of atrophic Schwann cells back into the myelinating phenotype.