Role of axon-deprived Schwann cells in perineurial regeneration in the rat sciatic nerve

The role of Schwann cells (SC) in perineurial regeneration after nerve injury has not yet been resolved. It was hypothesized that SC alone are able to induce at least partial morphological restoration of the destroyed orthotopic perineureum (PN). To test the hypothesis, a permanently denervated segment of the rat sciatic nerve was made acellular by freeze-thawing, except in its most proximal part where non-neuronal cells were left intact. Restoration of the frozen segment by these cells was examined by electron microscopy and immunohistochemistry of the SC marker, S-100 protein, 4 and 8 weeks after injury. The PN regenerated from undifferentiated fibroblast-like cells. In the presence of migrant SC without axons, regenerated cells in the place of the former PN were stacked in several layers and, in accordance with the hypothesis, partially expressed typical features of the perineurial cells (PC): pinocytotic vesicles, short fragments of basal lamina and tight junctions. Migrant SC induced formation of pseudo-minifascicles even in the epineurium. In these, SC organized the adjacent fibroblasts into a multilayered circular sheath, and induced their partial differentiation towards perineurial cells. Further experiments demonstrated that regenerating axons are required for complete morphological differentiation of the regenerated perineurial cells either in the orthotopic PN or in minifascicles.     

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 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.     

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.     

Molecular basis of interactions between regenerating adult rat thalamic axons and Schwann cells in peripheral nerve grafts II. Tenascin-C

Tenascin-C is a developmentally regulated extracellular matrix component. There is evidence that it may be involved in axon growth and regeneration in peripheral nerves. We have used in situ hybridization and immunocytochemistry to investigate the association of tenascin-C with central nervous system axons regenerating through a peripheral nerve autograft inserted into the thalamus of adult rats. Between 3 days and 4 weeks after implantation, tenascin-C immunoreactivity was increased in the grafts, first at the graft/brain interface, then in the endoneurium of the graft, and finally within the Schwann cell columns of the graft. By electron microscopy, reaction product was present around collagen fibrils and basal laminae in the endoneurium, but the heaviest deposits were found at the surface of regenerating thalamic axons within Schwann cell columns. Schwann cell surfaces were not associated with tenascin-C reaction product except where they faced the tenascin-rich basal lamina or were immediately opposite axons surrounded by tenascin-C. By 8 weeks after graft implantation tenascin-C in the endoneurium and around axons of the graft was decreased. In the brain parenchyma aroundthe proximal part of the graft, axonal sprouts associated with tenascin-C could not be identified earlier than 2 weeks after grafting and were sparse at this stage. Larger numbers of such axons were present at 8–13 weeks after grafting and were located predominantly where the glia limitans between brain and graft appeared to be incomplete, suggesting that the tenascin-C may have penetrated the brain parenchyma from the graft. By in situ hybridization, cells expressing tenascin-C mRNA (probably Schwann cells) appeared first at the brain/graft interface 3 days after grafting and thereafter were mainly located within the grafts. Lightly labelled cells containing tenascin-C mRNA (probably glial cells) were scattered in the thalamic parenchyma both ipsilateral and contralateral to the graft and a few heavily labelled cells were located very close to the tip of the graft. These results show that regenerating adult thalamic axons, unlike regenerating peripheral axons, become intimately associated with peripheral nerve graft-derived tenascin-C, suggesting that they express a tenascin-C receptor, as many neurons do during development, and that tenascin-C derived from Schwann cells may play a role in the regenerative growth of such axons through the grafts. © 1995 Wiley-Liss, Inc.     

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.     

Synergistic effects of micropatterned biodegradable conduits and Schwann cells on sciatic nerve regeneration

This paper describes a novel biodegradable conduit that provides a combination of physical, chemical and biological cues at the cellular level to facilitate peripheral nerve regeneration. The conduit consists of a porous poly(D,L-lactic acid) (PDLLA) tubular support structure with a micropatterned inner lumen. Schwann cells were pre-seeded into the lumen to provide additional trophic support. Conduits with micropatterned inner lumens pre-seeded with Schwann cells (MS) were fabricated and compared with three types of conduits used as controls: M (conduits with micropatterned inner lumens without pre-seeded Schwann cells), NS (conduits without micropatterned inner lumens pre-seeded with Schwann cells) and N (conduits without micropatterned inner lumens, without pre-seeded Schwann cells). The conduits were implanted in rats with 1 cm sciatic nerve transections and the regeneration and functional recovery were compared in the four different cases. The number or size of regenerated axons did not vary significantly among the different conduits. The time of recovery, and the sciatic function index, however, were significantly enhanced using the MS conduits, based on qualitative observations as well as quantitative measurements using walking track analysis. This demonstrates that biodegradable micropatterned conduits pre-seeded with Schwann cells that provide a combination of physical, chemical and biological guidance cues for regenerating axons at the cellular level offer a better alternative for repairing sciatic nerve transactions than conventional biodegradable conduits.     

The nerve regenerative microenvironment: Early behavior and partnership of axons and Schwann cells

This review will address new ideas, including several from our laboratory, on the role of local molecules and signaling within the microenvironment of injured peripheral nerve trunks. These include the concepts of axon-Schwann cell (SC) outgrowth partnership such as the secretion of local molecules that may facilitate or inhibit regenerative activity and the role of directional cues secreted by the SCs to guide regrowing axons. Several specific themes along these lines are explored: (i) a role for peptidergic axon synthesis and signaling to SCs; (ii) the expression of molecular regeneration brakes in regenerating axons, specifically activated RHOA GTPase; (iii) the concept of misdirected axon outgrowth, focusing on the prototypic NGF and local TrkA interaction in adult regrowth; (iv) the role of extracellular basement membrane constituents such as laminin, RGD/fibronectin and their integrin receptors. We show that these different themes play an important but not exclusive role in determining regenerative success. Collectively, these individual findings help us appreciate the many facets of regenerative success which depend on the surrounding environment, the expressed receptors, and the internal state of the growing axon.     

Different effects of astrocytes and Schwann cells on regenerating retinal axons

Following a crush injury of the optic nerve in adult rats, the axons of retinal ganglion cells, stimulated to regenerate by a lens injury and growing within the optic nerve, are associated predominantly with astrocytes: they remain of small diameter (0.1-0.5 microm) and unmyelinated for > or = 2 months after the operation. In contrast, when the optic nerve is cut and a segment of a peripheral nerve is grafted to the ocular stump of the optic nerve, the regenerating retinal axons are associated predominantly with Schwann cells: they are of larger diameter than in the previous experiment and include unmyelinated axons (0.2-2.5 microm) and myelinated axons (mean diameter 2.3 microm). Thus, the grafted peripheral nerve, and presumably its Schwann cells, stimulate enlargement of the regenerating retinal axons leading to partial myelination, whereas the injured optic nerve itself, and presumably its astrocytes, does not. The result points to a marked difference of peripheral (Schwann cells) and central (astrocytes) glia in their effect on regenerating retinal axons.     

Hyperglycaemia inhibits Schwann cell proliferation and migration and restricts regeneration of axons and Schwann cells from adult murine DRG

Poorly-controlled hyperglycaemia reduces peripheral nerve regeneration in diabetes through ill-understood mechanisms. Apoptosis is one proposed primary response. We examined how hyperglycaemia affects regeneration of axons and Schwann cells (SC) from cultured adult mouse Dorsal Root Ganglia (DRG) to separate cell-autonomous responses from systemic influences. Hyperglycaemia reduced neurite growth rate by 20-30% without altering growth cone density, indicating neuronal apoptosis was negligible. Moderate hyperglycaemia also profoundly retarded SC migration from DRG explants. This effect was independent of neuritogenesis and was reversible, indicating that SC had not died. In purified SC, even mild hyperglycaemia inhibited neuregulin-beta1-induced bromodeoxyuridine-incorporation and phosphorylation of retinoblastoma protein, indicating a block at the G1-S boundary. Moreover, migration of purified SC was inhibited by >90%. Thus, SC proliferation and migration, and axon regeneration from DRG neurons, are impaired by hyperglycaemia cell autonomously, while apoptosis is negligible. Impairment of these functions over time may exacerbate nerve injury-related diabetic neuropathy.     

FK506 enhances regeneration of axons across long peripheral nerve gaps repaired with collagen guides seeded with allogeneic Schwann cells

We assessed the effects of FK506 administration on regeneration after a 6-mm gap repair with a collagen guide seeded with allogeneic Schwann cells (SCs) in the mouse sciatic nerve. SCs were isolated from predegenerated adult sciatic nerves and expanded in culture using a defined medium, before being seeded in the collagen guide embedded in Matrigel. Functional reinnervation was evaluated by noninvasive methods to determine recovery of motor, sensory, and autonomic functions in the hindpaw over 4 months postoperation. Histological analysis of the regenerated nerves was performed at the end of the study. Using simple collagen guides for tubulization repair, treatment with an immunosuppressant dose of FK506 (5 mg/kg/day) resulted in significant improvement of the onset and the degree of reinnervation. While the introduction of allogeneic SCs did not improve regeneration versus a collagen guide filled only with Matrigel, treatment with FK506 allowed for successful regeneration in all the mice and for significant improvement in the levels of functional recovery. Compared with the untreated group, there was greater survival of transplanted pre-labeled SCs in the FK506-treated animals. Morphologically, the best nerve regeneration (in terms of nerve caliber and numbers of myelinated axons) was obtained with SC-seeded guides from FK506-treated animals. Thus, FK506 should be considered as adjunct therapy for various types of tubulization repair.     

Optic axons regenerate into sciatic nerve isografts only in the presence of Schwann cells

Optic axons regenerate into normal but not acellular peripheral nerve (PN) grafts. The first axons penetrate the PN graft before 5 days and grow inside the basal lamina tubes amongst the Schwann cells. By 30 days, 4% of the surviving retinal ganglion cells (RGC) regenerate axons for at least 10 mm into the PN graft. Laminin rich basal lamina tubes persist in the acellular PN transplants but only a few axons penetrate the most proximal parts of the tubes by 5 days and none grow farther into the graft by 30 days. RGC counts demonstrate that 34% of the normal RGC population survive 30 days after anastomosing a normal PN to the transected optic nerve. After anastomosing acellular PN grafts, 25% of RGCs survive compared with 10% after optic nerve section. These findings demonstrate that laminin does not promote regeneration of axons and that Schwann cells play the primary role of offering trophic support and even a substrate for growth. RGC survival is also enhanced by PN grafts even when Schwann cells are absent. This latter result suggests that RGC survival is promoted by a trophic substance released from axons and/or Schwann cells in the PN grafts which survives the thawing/freezing procedure (used to kill the Schwann cells) and is active in the grafts in the immediate post operative period.     

聚四氟乙烯膜管内植入许旺细胞与神经生长因子桥接面神经的比较

背景：在周围神经修复领域中，神经营养与趋化理论得到普遍关注，随之各种神经再生室及许旺细胞营养因子应用的实验增多，但尚缺乏深入系统的报道。 目的：以聚四氟乙烯膜管作为神经再生室，观察分别植入许旺细胞与神经生长因子后对桥接面神经缺损的修复效果。 设计、时间及地点：随机对照动物实验，于2008-01在山东大学动物实验中心完成。 材料：新西兰纯种白兔24只，随机分为许旺细胞组、神经生长因子组、模型对照组，8只/组。聚四氟乙烯膜由上海塑料研究所制备，厚度0.15 mm，微孔径10~30 μm。神经生长因子为烟台北方制药有限公司产品。 方法：3组兔显露右侧面神经颊支，切除8 mm建立面神经缺损模型。将造模时切下的神经段于镜下剥除外膜，胰蛋白酶与胶原蛋白酶联合消化，L-多聚赖氨酸纯化后获得纯度较高且具有活性的许旺细胞悬液。将聚四氟乙烯膜管套缝固定于各组缺损区神经的两断端上，使神经缺损两断端在管内相距10 mm，许旺细胞组吸取自体许旺细胞悬液注入膜管内，组织黏接剂封闭膜管两端口；神经生长因子组吸取神经生长因子注入膜管内；膜管对照组不进行任何干预。 主要观察指标：采用四导生理记录仪检查修复后面神经传导速度，苏木精-伊红染色观察神经纤维再生程度。 结果：细胞修复后6，12周，许旺细胞组面神经传导速度>神经生长因子组>膜管对照组（F=72.319，F =106.134，P均< 0.01）。细胞修复后12周，许旺细胞组神经干粗而直，走向顺畅，可见于再生室膜的微孔结构中，许旺细胞包绕神经纤维；神经生长因子组新生神经纤维较少，可见发育成熟的许旺细胞增多；单纯膜管组再生室内神经纤维较少。 结论：许旺细胞或神经生长因子植入聚四氟乙烯膜管内均可促进面神经恢复，但前者修复效果明显优于后者。     

Local control of axonal properties by Schwann cells: neurofilaments and axonal transport in homologous and heterologous nerve grafts.

A number of axonal properties, including slow axonal transport and neurofilament phosphorylation, are altered in a mutant mouse strain with a Schwann cell deficiency, the Trembler. The Trembler phenotype is associated with poor myelination and reduced axonal caliber in the peripheral nervous system, but the genetic lesion has not yet been identified. To determine whether changes in axonal properties resulted from a direct action of Schwann cells on the axon, a segment of sciatic nerve from myelin-deficient Trembler mouse was grafted into the sciatic nerve of a normal mouse and normal axons were allowed to regenerate. Normal axons surrounded by Trembler Schwann cells are reduced in diameter, but resume their original diameter distal to the graft. Neurofilament transport was also affected locally in sciatic nerves with Trembler grafts into normal nerve. The velocity of neurofilament transport was not significantly different from controls in portions of the nerve proximal to the Trembler graft, but there was a reduction in neurofilament transport rates upon entering the Trembler graft. This was accompanied by an increase in the ratio of neurofilament over tubulin in the case of the Trembler graft, suggesting both a slowing of the neurofilament and an increase in the rate of tubulin transport. Using heterologous grafts of Trembler nerve segments into wildtype nerves, Schwann cells were shown to locally influence axonal caliber, neurofilament organization, and slow axonal transport. These observations emphasize the importance of glial cells in modulating neuronal structure and functions, as well as focusing attention on the role of glia in the etiology of neuropathologies that alter the neuronal environment.     

Rat Schwann cells in bioresorbable nerve guides to promote and accelerate axonal regeneration

A micro-structured, biodegradable, semipermeable hollow nerve guide implant was developed to bridge nerve lesions. Quantitative comparison of cell migration and axonal growth using time lapse video recording in vitro revealed that axons grow eight times faster than neuritotrophic Schwann cells migrate. To accelerate regeneration, purified Schwann cells are best injected into nerve guides before implantation. Nerve guides made from resorbable poly-lactide-co-glycolide support Schwann cell attachment, cell survival, and axonal outgrowth in vitro. The therapeutic concept aims at the development of an 'intelligent neuroprosthesis' that first mediates regeneration and then disappears.     

Hyperbaric oxygen treatment has different effects on nerve regeneration in acellular nerve and muscle grafts

Effects of hyperbaric oxygen treatment (HBO) on nerve regeneration in acellular nerve and muscle grafts were investigated in rats. Nerve and muscle grafts were made acellular by freeze-thawing and the obtained grafts were used to bridge a 10-mm gap in the sciatic nerve on the left and right sides, respectively. Rats were treated with HBO (100% oxygen for 90 minutes at 2.5 atmospheres absolute pressure ATA) twice a day for 7 days. Axonal outgrowth, Schwann cell migration and invasion of macrophages were examined 10 days after the graft procedure by staining neurofilaments, S-100 proteins and the macrophage antibodies ED1 and ED2, respectively. Axonal outgrowth and Schwann cell migration in acellular nerve grafts were superior to that found in the acellular muscle grafts. However, there was no difference between HBO-treated and nontreated rats in acellular nerve grafts. Such a difference was found in acellular muscle grafts concerning both axonal outgrowth and Schwann cell migration from the proximal nerve end. No differences in the content of macrophages or neovascularization (alkaline phosphatase staining) in either of the grafts and treatments were seen. It is concluded that there is a differential effect of HBO-treatment in acellular nerve and muscle grafts and that HBO-treatment has no effect on the regeneration process in acellular nerve grafts, in contrast to fresh cellular nerve grafts where a beneficial effect has previously been reported.     

脐带间充质干细胞与许旺细胞共移植修复大鼠坐骨神经缺损

Previous research has demonstrated that cotransplantation of umbilical cord mesenchymal stem cells (UCMSCs) and Schwann cells (SCs) can repair spinal nerve injury, but few studies have investigated their use in peripheral nerve regeneration. In the present study, we cotransplanted UCMSCs and SCs to repair 5-mm left sciatic nerve defects in rats, and compared the effects of UCMSCs + SCs transplantation with UCMSCs or SCs transplantation alone. After UCMSCs + SCs transplantation, nerve conduction velocity of the left sciatic nerve and gait were both improved. Retrograde tracing analysis demonstrated that the mean count of fluorogold-labeled neurons, as well as the mean axon count and axon density, were significantly greater in the left sciatic nerve after UCMSCs + SCs transplantation, compared with UCMSCs or SCs transplantation alone. Improvements in conduction velocity and increased sheath thickness in the left sciatic nerve were similar after UCMSCs transplantation and UCMSCs + SCs transplantation. These findings suggest that UCMSCs transplantation can promote the repair of sciatic nerve defects to some extent, but that combined UCMSCs + SCs transplantation has a significantly greater regenerative effect.