Cooperative roles of BDNF expression in neurons and Schwann cells are modulated by exercise to facilitate nerve regeneration

After peripheral nerve injury, neurotrophins play a key role in the regeneration of damaged axons that can be augmented by exercise, although the distinct roles played by neurons and Schwann cells are unclear. In this study, we evaluated the requirement for the neurotrophin, brain-derived neurotrophic factor (BDNF), in neurons and Schwann cells for the regeneration of peripheral axons after injury. Common fibular or tibial nerves in thy-1-YFP-H mice were cut bilaterally and repaired using a graft of the same nerve from transgenic mice lacking BDNF in Schwann cells (BDNF(-/-)) or wild-type mice (WT). Two weeks postrepair, axonal regeneration into BDNF(-/-) grafts was markedly less than WT grafts, emphasizing the importance of Schwann cell BDNF. Nerve regeneration was enhanced by treadmill training posttransection, regardless of the BDNF content of the nerve graft. We further tested the hypothesis that training-induced increases in BDNF in neurons allow regenerating axons to overcome a lack of BDNF expression in cells in the pathway through which they regenerate. Nerves in mice lacking BDNF in YFP(+) neurons (SLICK) were cut and repaired with BDNF(-/-) and WT nerves. SLICK axons lacking BDNF did not regenerate into grafts lacking Schwann cell BDNF. Treadmill training could not rescue the regeneration into BDNF(-/-) grafts if the neurons also lacked BDNF. Both Schwann cell- and neuron-derived BDNF are thus important for axon regeneration in cut peripheral nerves.     

BD™ PuraMatrix™ peptide hydrogel seeded with Schwann cells for peripheral nerve regeneration

This study investigated the effects of a membrane conduit filled with a synthetic matrix BD™ PuraMatrix™ peptide (BD) hydrogel and cultured Schwann cells on regeneration after peripheral nerve injury in adult rats.After sciatic axotomy, a 10 mm gap between the nerve stumps was bridged using ultrafiltration membrane conduits filled with BD hydrogel or BD hydrogel containing Schwann cells. In control experiments, the nerve defect was bridged using either membrane conduits with alginate/fibronectin hydrogel or autologous nerve graft. Axonal regeneration within the conduit was assessed at 3 weeks and regeneration of spinal motoneurons and recovery of muscle weight evaluated at 16 weeks postoperatively.Schwann cells survived in the BD hydrogel both in culture and after transplantation into the nerve defect. Regenerating axons grew significantly longer distances within the conduits filled with BD hydrogel when compared with the alginate/fibronectin hydrogel and alginate/fibronectin with Schwann cells. Addition of Schwann cells to the BD hydrogel considerably increased regeneration distance with axons crossing the injury gap and entering into the distal nerve stump. The conduits with BD hydrogel showed a linear alignment of nerve fibers and Schwann cells.The number of regenerating motoneurons and recovery of the weight of the gastrocnemius muscle was inferior in BD hydrogel and alginate/fibronectin groups compared with nerve grafting. Addition of Schwann cells did not improve regeneration of motoneurons or muscle recovery.The present results suggest that BD hydrogel with Schwann cells could be used within biosynthetic conduits to increase the rate of axonal regeneration across a nerve defect.     

Isolation of activated adult Schwann cells and a spontaneously immortal Schwann cell clone.

Successful mammalian peripheral nerve regeneration is dependent on activated Schwann cells. Schwann cells facilitate neuronal regrowth through the production of tropic cell membrane molecules, neurotrophins, and extracellular matrix components. To better understand Schwann cell function in the regenerating nerve, we have designed a method of isolating proliferating adult Schwann cells from the injured rat sciatic nerve. Relying on the mitotic signal that is present after a crush injury, we can obtain sufficient numbers of dividing Schwann cells within one week of initial culture. A spontaneously immortal Schwann cell clone (iSC) was observed in and isolated from one of these primary cultures. These cells were transformed at a time of maximal Schwann cell activation in response to injury. Both the primary Schwann cells and the iSC have been characterized as Schwann cells by morphology, immunohistochemistry and gene expression.     

Defective Schwann cell function in canine inheritedHypertrophic Neuropathy

Summary Segments of peripheral nerve from dogs with canine inherited hypertrophic neuropathy (CIHN) were transplanted to the transected sciatic nerve of immuno incompetent mice. Regenerating mouse axons penetrated the grafts and were myelimated by dog Schwann cells. In grafts collected 3 or more months after transplantation, filamentous or granular material, identical to that occurring in nerves of affected dogs, accumulated in myelinating Schwann cells. Demyelinated fibers were only rarely found in grafted segments of affected nerve. Neither filamentous accumulations nor demyelination were observed in grafts of control canine nerve.These results indicate that CIHN is associated with a defect in Schwann cell function, and the abnormal accumulations of filaments suggest that the defect may be in the cytoskeleton. The rarity of demyelination in grafts suggests that some factor in addition to the Schwann cell defect is required to precipitate myelin destruction.Supported by NIH Biomedical Research Support Grant 2507-RR05462-20 and by the Medical Research Council of Canada     

Pathology of roots, spinal cord and brainstem in syringomyelia-like syndrome of Tangier disease.

We report here a post-mortem examination of a 46-year-old patient who died after a 23-year-long syringomyelia-like syndrome of Tangier disease. The L5 dorsal root and the superficial peroneal nerve showed fiber loss and lipid vacuole accumulation in Schwann cell cytoplasm. The L5 ventral root had moderate fiber loss without lipid vacuoles. In the cervical roots, fiber loss was intense and there were no foamy Schwann cells. Motor neuron loss was severe in the cervical spinal cord and the facial nerve nucleus and slight at the lumbar level. Under electron microscopy, some neurons of the lower spinal cord showed atypical inclusions. These data suggest that an unknown metabolic defect is responsible for a primary neuronopathy. Lipid accumulation in Schwann cells, resulting from fiber degeneration is probably transient, accounting for the absence of foamy cells in regions with longstanding involvement.     

Syngeneic Schwann cells derived from adult nerves seeded in semipermeable guidance channels enhance peripheral nerve regeneration.

At present, clinical strategies to repair injured peripheral nerve concentrate on efforts to attain primary suture of the cut nerve ends. If this is not possible, autografts are used to unite the separated nerve segments. Both strategies are based on the recognition that the Schwann cells resident in the peripheral nerve trunk play a crucial role in the regenerative process. Neither strategy may be feasible, however, in extensive or multiple injuries because the amount of autograft material is limited, and allografts are subject to immune rejection. Artificially produced nerve bridges constructed of autologous Schwann cells seeded in guidance channels could be used to overcome these limitations. In the present experiments, the potential of Schwann cells derived from adult nerves and seeded in permselective guidance channels to promote neurite regeneration across an 8 mm nerve gap was evaluated in transected rat sciatic nerves. Immunological sequalae were evaluated by comparing Schwann cells from syngeneic and heterologous rat strains. Schwann cells from either adult outbred (Sprague-Dawley, CD) rats or inbred (Fisher, F) rats were suspended in a Matrigel solution at a density of 80 x 10(6) cells/ml (CD) or 40, 80, or 120 x 10(6) cells/ml (F-40, F-80, and F-120 channels, respectively). Channels containing Schwann cells were compared to sciatic nerve autografts, empty channels, or channels filled with Matrigel alone. One day after seeding permselective synthetic guidance channels with a Schwann cell suspension, a central cable of Schwann cells oriented along the axis of the tube was formed due to syneresis of the hydrogel. By 3 weeks postimplantation, regenerating axons had grown into all channels and autografts. Sciatic nerve autografts supported extensive regeneration, containing 4-5 x 10(4) myelinated axons at the graft midpoint. The ability of channels containing syngeneic Schwann cells to foster regeneration was dependent on the Schwann cell seeding density. At the channel's midpoint, the myelinated axon population in F-120 tubes was intermediate between that in sciatic nerve autografts and F-80 channels, and was significantly higher than in F-40 or control channels. The nerve cable in Schwann cell-containing tubes consisted of larger, more organotypic fascicles than acellular control channels. In contrast, heterologous (CD) Schwann cells elicited a strong immune reaction that impeded nerve regeneration. The present study shows that cultured adult syngeneic Schwann cells seeded in permselective synthetic guidance channels support extensive peripheral nerve regeneration.(ABSTRACT TRUNCATED AT 400 WORDS)     

Further development of reconstructive and cell tissue-engineering technology for treatment of complete peripheral nerve injury in rats

In this work we evaluated the efficacy of biodegradable composite co-polymer guiding neurotube, based on tissue-engineering technology, for the treatment of complete peripheral nerve injury where the nerve defect is significant. The right sciatic nerve of 12 three-month-old rats was completely transected and peripheral nerve segment was removed. A 2.2-cm biodegradable co-polymer neurotube containing viscous gel (NVR-N-Gel) with survival factors, neuroprotective agents and Schwann cells was placed between the proximal and the distal parts of the transected nerve for reconnection a 2-cm nerve defect. The proximal and distal parts of the nerve were fixed into the neurotube using 10-0 sutures. Ultrasound observation showed growth of the axons into the composite neurotube 2 months after the surgery. Electrophysiological study indicated compound muscle action potentials in nine out of 12 rats, 2-4 months after peripheral nerve reconstructive surgery. The postoperative follow-up (up to 4 months) on the operated rats that underwent peripheral nerve reconstruction using composite co-polymer neurotube, showed beginning of re-establishment of active foot movements. The tube was dissolved and nerve showed complete reconnection. Histological observation of the nerve showed growth of myelinated axons into the site where a 2-cm nerve defect replaced by composite co-polymer neurotube and into the distal part of the nerve. In CONCLUSION: (1) an innovative composite neurotube for reconstruction of significant loss of peripheral nerve segment is described; (2) a viscous gel, containing survival factors, neuroprotective agents and Schwann cells served as a regenerative environment for repair. Further investigations of this reconstructive procedure are being conducted.     

Lysosomal abnormalities in cultured schwann cells from a patient with peripheral neuropathy and continuous muscle fiber activity

Schwann cell cultures were established from a sural nerve containing large membrane-bound vacuoles in its Schwann cells, obtained from a patient with neuropathy and continuous muscle fiber activity. Cultured Schwann cells contained many large membrane-bound vacuoles, presumably lysosomes, resembling those present in the biopsied nerve. The acid phosphatase reaction was excessive in the patient's cultured Schwann cells but practically negative in normal cultured Schwann cells. This study indicates that the patient's neuropathy is primary dysschwannian with abnormal lysosomes as a major abnormality.     

去细胞同种异体神经支架种植许旺细胞的体外培养

摘要 背景：前期的研究中分别探讨了使用复合酶消化法及差速贴壁法对许旺细胞培养的影响以及聚氧乙烯辛烷基酚(Triton X-100)制备同种异体神经支架。 目的：通过化学萃取法制备同种异体神经支架,并将培养的许旺细胞与支架进行体外结合培养。 方法：取Wistar大鼠双侧坐骨神经,运用30 g/L三硝基甲苯和40 g/L脱氧胆酸钠分别萃取1,2和3次,在萃取神经和未萃取神经的中段取材,行苏木精-伊红染色,S-100及Laminin免疫组织化学染色及透射电镜检测。取SD胎鼠坐骨神经及臂从神经,使用复合酶消化,差速贴壁及Arab-c抑制成纤维细胞生长的培养方法获得大量高纯度的许旺细胞。再把许旺细胞注入同种异体神经支架内,并行透射电镜及扫描电镜观察。 结果与结论：用三硝基甲苯和脱氧胆酸钠萃取后,坐骨神经内细胞和髓鞘被清除,神经基底膜被保留。电镜下可见萃取后的神经由空的神经基底膜管和管之间的胶原纤维构成。萃取次数增加后,神经内残留的S-100蛋白显著减少,但反复萃取后支架结构受破坏。去细胞同种异体神经支架适合许旺细胞体外生长,并有迁移排成行的特性。结果提示,用三硝基甲苯和脱氧胆酸钠萃取2次可去除细胞而保留神经基底膜管,是制备具有仿生结构神经支架的理想方法。神经支架种植许旺细胞后可能成为一种理想的神经缺损修复材料。     

Schwann cells stimulated to proliferate in the absence of neurons retain full functional capability

Schwann cells from neonatal rat sciatic nerve can be maintained and grown in culture in the absence of neurons. We are interested in substantially expanding such cultures for use in the study of Schwann cells, their growth responses, and their interactions with neurons. However, it was important to determine if expanded cell populations retained their distinguishing biological properties and their ability to differentiate when recombined with neurons. Therefore, we have compared the functional properties of extensively expanded populations of sciatic nerve Schwann cells to those of embryonic dorsal root ganglion (DRG) Schwann cells that had been briefly expanded in vitro in the continuous presence of ganglion neurons. Sciatic nerve Schwann cells were cultured and purified according to the methods of Brockes et al. (1979). A combination of crude glial growth factor and forskolin was found to act synergistically in providing maximal stimulation of Schwann cell proliferation. Sciatic nerve Schwann cells that were continuously expanded for at least 2 months were compared to Schwann cells derived from fetal dorsal root ganglia. The results indicate that the complement of secreted proteins from both cell populations, either in isolation or recombined with neurons, was essentially identical; both cell populations expressed the cell-surface antigens laminin and Ran 1 (217C antibody); after seeding onto DRG neurons, both cell populations associated with neuronal processes with the same time course; and under identical nutrient conditions, both cell populations were observed to exhibit a comparable capacity for myelination of DRG axons in vitro. Thus, methods used to establish primary cultures of rat sciatic nerve Schwann cells and to expand secondary cultures in vitro in the absence of neurons preserve basic Schwann cell functions.     

Pre-degenerated peripheral nerves co-cultured with bone marrow-derived cells：a new technique for harvesting high-purity Schwann cells

Schwann cells play an important role in the peripheral nervous system, especially in nerve repair following injury, so artificial nerve regeneration requires an effective technique for obtaining purified Schwann cells. In vivo and in vitro pre-degeneration of peripheral nerves have been shown to obtain high-purity Schwann cells. We believed that in vitro pre-degeneration was simple and controllable, and available for the clinic. Thus, we co-cultured the crushed sciatic nerves with bone marrow-derived cells in vitro. Results demonstrated that, 3 hours after injury, a large number of mononuclear cells moved to the crushed nerves and a large number of bone marrow-derived cells infiltrated the nerve segments. These changes promoted the degradation of the nerve segments, and the dedifferentiation and proliferation of Schwann cells. Neural cell adhesion molecule and glial fibrillary acidic protein expression were detected in the crushed nerves. Schwann cell yield was 9.08 ± 2.01 × 104/mg. The purity of primary cultured Schwann cells was 88.4 ± 5.79%. These indicate a successful new method for obtaining Schwann cells of high purity and yield from adult crushed sciatic nerve using bone marrow-derived cells.     

Local production of serum amyloid a is implicated in the induction of macrophage chemoattractants in Schwann cells during wallerian degeneration of peripheral nerves

The elevation of serum levels of serum amyloid A (SAA) has been regarded as an acute reactive response following inflammation and various types of injuries. SAA from the liver and extrahepatic tissues plays an immunomodulatory role in a variety of pathophysiological conditions. Inflammatory cytokines in the peripheral nerves have been implicated in the Wallerian degeneration of peripheral nerves after injury and in certain types of inflammatory neuropathies. In the present study, we found that a sciatic nerve axotomy could induce an increase of SAA1 and SAA3 mRNA expression in sciatic nerves. Immunohistochemical staining showed that Schwann cells are the primary sources of SAA production after nerve injury. In addition, interleukin‐6‐null mice, but not tumor necrosis factor‐α‐null mice showed a defect in the production of SAA1 in sciatic nerve following injury. Dexamethasone treatment enhanced the expression and secretion of SAA1 and SAA3 in sciatic nerve explants cultures, suggesting that interleukin‐6 and corticosteroids might be major regulators for SAA production in Schwann cells following injury. Moreover, the stimulation of Schwann cells with SAA1 elicited the production of the macrophage chemoattractants, Ccl2 and Ccl3, in part through a G‐protein coupled receptor. Our findings suggest that locally produced SAA might play an important role in Wallerian degeneration after peripheral nerve injury. © 2012 Wiley Periodicals, Inc.     

Erythropoietin reduces Schwann cell TNF-alpha, Wallerian degeneration and pain-related behaviors after peripheral nerve injury

Chronic sciatic nerve constriction injury (CCI) induces Wallerian degeneration and exaggerated pain-like behaviors. These effects are mediated in large part by pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha). In this study, we demonstrate that systemically administered recombinant human erythropoietin (rhEpo) facilitates recovery from chronic neuropathic pain associated with CCI in rats. Because TNF-alpha has been implicated in the development of pain-related behaviors, we measured TNF-alpha mRNA at the nerve injury site. Systemically or locally administered rhEpo decreased TNF-alpha mRNA, compared with that observed in untreated animals. RhEpo also significantly (P < 0.05) decreased axonal degeneration. Immunohistochemistry of CCI nerve showed abundant TNF-alpha in Schwann cells, axoplasm and macrophages. In rhEpo-treated animals, TNF-alpha immunopositivity was decreased selectively in Schwann cells. These results suggest a model in which rhEpo counteracts the effects of TNF-alpha in CCI by blocking expression of TNF-alpha in Schwann cells. To further test this model, we studied primary Schwann cell cultures. RhEpo inhibited TNF-alpha expression in response to lipopolysaccharide, supporting the conclusions of our in vivo CCI experiments. In addition, rhEpo directly counteracted Schwann cell death induced by exogenously added TNF-alphain vitro. These results indicated that rhEpo regulates TNF-alpha by multiple mechanisms; rhEpo regulates TNF-alpha mRNA expression by Schwann cells but also may directly counteract TNF-alpha signaling pathways that lead to injury, chronic pain and/or death.