Addition of cultured Schwann cells to tendon autografts and freeze-thawed muscle grafts improves peripheral nerve regeneration

The effects of addition of Schwann cells on peripheral nerve regeneration through a novel graft material-the tendon autograft-and a conventional freeze-thawed muscle graft, were studied in the rat sciatic nerve. Adult Schwann cell cultures were established from predegenerated nerves. The Schwann cells were added to the autologous grafts by coculture (tendon autograft) or injection (freeze-thawed muscle graft). Both graft types supported adherence of the added Schwann cells. Addition of cultured Schwann cells to the two different graft models improved regeneration by increasing the rate of axonal outgrowth as compared with similar grafts without added cells.     

Supplementation of acellular nerve grafts with skin derived precursor cells promotes peripheral nerve regeneration

Introduction of autologous stem cells into the site of a nerve injury presents a promising therapy to promote axonal regeneration and remyelination following peripheral nerve damage. Given their documented ability to differentiate into Schwann cells (SCs) in vitro, we hypothesized that skin-derived precursor cells (SKPs) could represent a clinically-relevant source of transplantable cells that would enhance nerve regeneration following peripheral nerve injury. In this study, we examined the potential for SKP-derived Schwann cells (SKP–SCs) or nerve-derived SCs to improve nerve regeneration across a 12 mm gap created in the sciatic nerve of Lewis rats bridged by a freeze-thawed nerve graft. Immunohistology after 4 weeks showed survival of both cell types and early regeneration in SKP seeded grafts was comparable to those seeded with SCs. Histomorphometrical and electrophysiological measurements of cell-treated nerve segments after 8 weeks survival all showed significant improvement as compared to diluent controls. A possible mechanistic explanation for the observed results of improved regenerative outcomes lies in SKP–SCs' ability to secrete bioactive neurotrophins. We therefore conclude that SKPs represent an easily accessible, autologous source of stem cells for transplantation therapies which act as functional Schwann cells and show great promise in improving regeneration following nerve injury.     

A Schwann cell matrix component of neuromuscular junctions and peripheral nerves

Molecules localized to the synapse are potential contributors to processes unique to this specialized region, such as synapse formation and maintenance and synaptic transmission. We used an immunohistochemical strategy to uncover such molecules by generating antibodies that selectively stain synaptic regions and then using the antibodies to analyse their antigens. In this study, we utilized a monoclonal antibody, mAb 6D7, to identify and characterize an antigen concentrated at frog neuromuscular junctions and in peripheral nerves. In adult muscle, immunoelectron microscopy indicates that the antigen is located in the extracellular matrix around perisynaptic Schwann cells at the neuromuscular junction and in association with myelinated and nonmyelinated axons in peripheral nerves. The maintenance of the mAb 6D7 epitope is innervation-dependent but is muscle-independent; it disappears from the synaptic region within 2 weeks after denervation, but persists after muscle damage when the nerve is left intact. mAb 6D7 immunolabelling is also detected at the neuromuscular junction in developing tadpoles. Biochemical analyses of nerve extracts indicate that mAb 6D7 recognizes a glycoprotein of 127 kDa with both N- and O-linked carbohydrate moieties. Taken together, the results suggest that the antigen recognized by mAb 6D7 may be a novel component of the synaptic extracellular matrix overlying the terminal Schwann cell. The innervation-sensitivity of the epitope at the neuromuscular junction suggests a function in the interactions between nerves and Schwann cells.     

Characterization of Schwann cells from normal nerves and from neurofibromas in the bicolour damselfish

Summary Schwann cells are an important component of neurofibromas, one of the primary lesions encountered in neurofibromatosis type 1 in man. A central question in studies of neurofibromatosis type 1 has been whether the Schwann cells present in these tumours are intrinsically abnormal or exhibit abnormal phenotypes in response to stimuli from other cell types in these tumours. Damselfish neurofibromatosis is a naturally occurring disease in a species of marine fish, the bicolour damselfish, that is being developed as an animal model of neurofibromatosis type 1. Affected fish exhibit multiple neurofibromas and neurofibrosarcomas (malignant schwannomas). The present study compares the morphology, antigen expression and proliferative capacityin vitro of Schwann cells derived from peripheral nerves of normal, healthy fish with cells isolated from both spontaneously occurring and experimentally induced neurofibromas. Schwann cells from normal nerves expressed S100 antigens but not fibronectin or glial fibrillary acidic protein antigens and were similar in morphology and proliferative capacity to Schwann cells isolated from mammalian peripheral nerves. Tumour-derived cultures contained variable proportions (27–79%) of S100-positive cells that were identified as Schwann cells based on this feature. These tumour-derived Schwann cells exhibited a different morphology than normal Schwann cells, usually exhibited an increased reactivity to anti-S100 antibodies and were able to proliferate in vitro without added mitogens. Repeated subculturing of tumour-derived cultures led to the production of six cell lines all of which were composed exclusively of Schwann cells as indicated by S100 expression. These findings show that Schwann cells are an important component of tumours in Damselfish neurofibromatosis and that these cells are morphologically and physiologically altered in this disease. Observations of cell lines also suggest that tumour-derived Schwann cells are intrinsically abnormal and that this phenotype is not a result of stimuli from other cell types in the tumours.     

自体神经桥接陈旧性周围神经缺损后雪旺细胞的组织学观察

目的:探索长时间失神经损伤后的雪旺细胞的促神经再生功能。方法:切除成年雌性SD大鼠左侧坐骨神经4 mm,分别饲养3、6个月后,修剪近、远侧神经断端制成不同持续时间的大鼠坐骨神经10 mm陈旧性缺损模型。实验组切取对侧正常坐骨神经桥接缺损,对照组缺损模型制备完成后不予任何修复。各组动物术后再饲养3个月取材,标本进行神经三色染色、免疫荧光染色,电镜等组织学方法观察。结果:各组损伤近端神经结构无明显差异,实验组桥接物段可观察到粗细不等的有髓神经纤维。电镜下,实验组远侧断端皆可观察到髓鞘形成良好的有髓神经纤维和存活的雪旺细胞。结论:长时间失神经损伤的雪旺细胞仍能在一定时间内存活并维持其促神经再生功能。     

Anatomic basis of vascularised nerve grafts: the blood supply of peripheral nerves

The present study was carried out on 30 cadavers (5 fresh, 20 preserved adult and 5 fresh stillborn) following injection of red latex through the subclavian and common iliac arteries. The blood supply to the peripheral nerves was studied in general, together with the vascular pedicles to the ulnar, saphenous, sural, deep and superficial peroneal nerves, and the superficial branch of the radial nerve. The nutrient arteries supplying the peripheral nerves came from either the adjacent axial artery or the fasciocutaneous or muscular arteries. They formed anastomotic channels in the epineurium and penetrated it to form a continuous longitudinal artery. Based on the presence of absence of dominant arterial pedicles, five patterns of blood-supply to the nerves could be identified. I: no dominant arterial pedicle; II: only one dominant artery (e.g. artery with a diameter more than 0.8 mm and accompanying the nerve for most of its length); III: only one dominant vessel that divided into ascending and descending branches to supply the nerve; IV: multiple dominant pedicles; V: multiple dominant arterial pedicles forming a continuous artery that accompanied the nerve. The arterial pedicles to the ulnar, saphenous and deep peroneal nerves and the superficial branch of the radial n. had mean diameters of over 0.8 mm, thus being suitable for microvascular anastomosis. Those to the sural nerve were not present in two thirds of the dissected cadavers. In 10% of the cadavers the superficial peroneal nerve had an arterial pedicle that accompanied the nerve for less than two cm with a mean diameter less than 0.8 mm. The ulnar nerve could be very suitable as a donor vascularized nerve graft as it had a dominant vascular pedicle in all the cases studied; however, its use should be restricted to C8 and T1 root damage of the brachial plexus. The superficial branch of the radial n. might be suitable for vascularized nerve grafting, but this is difficult in practice since the radial artery is a major limb artery. The saphenous nerve had a dominant arterial pedicles in all the cadavers dissected and could be the most suitable as a donor vascularized nerve graft, unlike the sural nerve which did not have a dominant arterial pedicle in two-thirds of the specimens. The deep and superficial peroneal nerves may also be unsuitable since the former is accompanied by a major limb vessel while the latter had a dominant vascular pedicle that accompanied the nerve for only a short distance in 10% of the dissected cadavers.     

Potential of Schwann cells from unmyelinated nerves to produce myelin: a quantitative ultrastructural and radiographic study

Summary In adult mice, most nerve fibres in the cervical sympathetic trunk (CST) are unmyelinated whereas a large proportion of sural nerve fibres are myelinated. This study of nerve grafts in syngeneic mice was designed to determine if Schwann cells originating from the unmyelinated CST would produce myelin when in contact with regenerating axons of the sural nerve. Quantitative microscopy of tritiated thymidine-labelled CST segments grafted to unlabelled sural nerve stumps revealed that, one month after grafting, previously unmyelinated grafts contained many myelinated fibres. By phase and electron microscope radioautography, nearly 40% of the myelin-producing cells in the reinnervated graft were shown to have originated in the unmyelinated CST. These findings indicate that Schwann cells originating from unmyelinated fibres are able to differentiate into myelin producing cells.     

许旺细胞及小肠黏膜下层复合生长因子缓释微球修复周围神经缺损

背景：前期实验已初步证实许旺细胞复合小肠黏膜下层及碱性成纤维细胞生长因子构建的人工神经具有体外神经活性、趋化性。 目的：观察许旺细胞及小肠黏膜下层复合碱性成纤维细胞生长因子缓释微球修复周围神经缺损后神经传导的再通情况。 方法：制作SD大鼠坐骨神经缺损模型,随机分组：实验组以许旺细胞及小肠黏膜下层复合碱性成纤维细胞生长因子缓释微球修复,阳性对照组以许旺细胞及小肠黏膜下层复合游离碱性成纤维细胞生长因子修复,阴性对照组以许旺细胞及小肠黏膜下层修复,空白对照组以自体神经修复。 结果与结论：术后16周实验组再生神经纤维数目,DiI示踪标记的阳性神经元数量、S-100及神经细丝蛋白的阳性表达率、髓鞘及再生轴突的超微结构恢复、神经传导速度及复合动作电位的改善均优于阳性对照组与阴性对照组(P < 0.05)。表明许旺细胞复合小肠黏膜下层及碱性成纤维细胞生长因子缓释微球构建的人工神经可重建坐骨神经缺损后的神经传导通路。     

以化学去细胞法处理同种异体神经与周围静脉伴行修复面神经缺损

背景：有研究表明化学去细胞法处理的同种异体神经修复面神经缺损可以取得较好的修复效果。 目的：在化学去细胞法处理同种异体神经的基础上探索一种修复面神经缺损更有效的修复方式。  方法：将新西兰大白兔随机分为2组,实验组制备面神经颊支缺损动物模型,采用经化学去细胞的同种异体腓肠神经进行移植,且与伴行静脉行外膜缝合;对照组在同样位置的远近端分别切断面神经,但不破坏被切断神经与周围组织的正常解剖关系,再切断处行外膜缝合桥接。 结果与结论：修复后3个月两组兔均存活,面部表情基本对称,胡须活动正常,神经移植处未见明显瘢痕及神经瘤形成。电镜观察结果显示,实验组与对照组右侧面神经颊支传导速度,移植体远端吻合口附近5.0 mm段有髓神经纤维数量,靶肌肉运动终板计数均差异无显著性意义(P > 0.05)。结果证实,化学去细胞同种异体神经与周围静脉伴行修复家兔面神经缺损的方法可以达到与自体面神经原位移植相似的修复效果。     

Observations on the migratory behaviour of Schwann cells from adult peripheral nerve expiant cultures

Summary The migration of Schwann cells from adult sciatic nerve explant cultures has been examined by time-lapse photomicrography. Analysis of Schwann cell migratory behaviour indicates that the initial outwandering by individual Schwann cells was random. Although chance cell-cell contacts resulted in temporary immobilization of pairs of cells, stable multicellular structures did not form during this initial phase. As local cell densities increased, Schwann cells assembled networks within which Schwann cell movement continued to be observed.A second form of Schwann cell outgrowth was observed from degenerating fibres in which arrays of highly oriented Schwann cells migrated away from their basal laminai tubes onto the culture dish.These observations of Schwann cell random migration, network self-assembly and coordinated extratubal migration are considered to highlight aspects of Schwann cell behaviour, independent of axonal influences, which may have relevance to their role in peripheral nerve repair following nerve section.     

Schwann cells of the olfactory nerves contain glial fibrillary acidic protein and resemble astrocytes

—Two antisera to glial fibrillary acidic protein from human brain and an antiserum to a 49 k dalton glial filament protein from human brain detected a cross-reacting antigen in the Schwann cells of the olfactory and vomeronasal nerves. The antigen was demonstrated at light- and electron-microscope levels. It was found throughout the cytoplasm and in association with cytoplasmic filaments of olfactory nerve Schwann cells in intact tissue and in Schwann cells grown in vitro.This observation, together with observations on the ultrastructure of olfactory nerve Schwann cells, relates them to central astroglia and to glial cells of the myenteric plexus, rather than to Schwann cells of other peripheral nerves. The unusual properties of olfactory nerve Schwann cells are of interest in relation to the regenerative abilities of the olfactory nerves.     

Electrical pulse stimulation of cultured skeletal muscle cells as a model for in vitro exercise – possibilities and limitations

The beneficial health‐related effects of exercise are well recognized, and numerous studies have investigated underlying mechanism using various in vivo and in vitro models. Although electrical pulse stimulation (EPS) for the induction of muscle contraction has been used for quite some time, its application on cultured skeletal muscle cells of animal or human origin as a model of in vitro exercise is a more recent development. In this review, we compare in vivo exercise and in vitro EPS with regard to effects on signalling, expression level and metabolism. We provide a comprehensive overview of different EPS protocols and their applications, discuss technical aspects of this model including critical controls and the importance of a proper maintenance procedure and finally discuss the limitations of the EPS model.     

Glycyrrhizic acid promotes sciatic nerves recovery in type 1 diabetic rats and protects Schwann cells from high glucose-induced cytotoxicity

The present study aims to investigate the therapeutic effect and mechanism of glycyrrhizic acid (GA) in diabetic peripheral neuropathy (DPN). GA significantly mitigated nerve conduction velocity (NCV) deficit and morphological abnormality and reduced high-mobility group box-1 (HMGB1) expression in the sciatic nerves of diabetic rats independent of blood glucose and body weight. Notably, GA alleviated the increase of HMGB1 and the decrease of cell viability in high glucose-stimulated RSC96 cells. Furthermore, GA obviously reduced the concentration of inflammatory cytokines in the sciatic nerves of diabetic rats and supernatants of high glucose-exposed RSC96 cells, then restored the decreased expression levels of nerve growth factor (NGF) and neuritin-1, and the increased expression levels of cleaved caspase-3 and neuron-specific enolase. Additionally, GA markedly inhibited receptor for advanced glycation end products (RAGE) expression, p38MAPK phosphorylation, and the nuclear translocation of NF-κBp65 in diabetic rats and high glucose-exposed RSC96 cells. The promotional effect of high glucose in RSC96 cells was diminished following Hmgb1 siRNA treatment. Our findings indicate that GA may exert neuroprotection on DPN by suppressing HMGB1, which lead to extenuation of inflammation response, balance of NGF, neuritin-1 and caspase-3, as well as inactivation of RAGE/p38MAPK/NF-κBp65 signaling pathway.     

Regeneration of peripheral nerves by transplanted sphere of human mesenchymal stem cells derived from embryonic stem cells

In cell therapy, the most important factor for therapeutic efficacy is the stable supply of cells with best engraftment efficiency. To meet this requirement, we have developed a culture strategy such as three-dimensional sphere of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) in serum-free medium. To investigate the in?vivo therapeutic efficacy of hESC-MSC spheres in nerve injury model, we transected the sciatic nerve in athymic nude mice and created a 2-mm gap. Transplantation of hESC-MSC as sphere repaired the injured nerve significantly better than transplantation of hESC-MSC as suspended single cells in regard to 1) nerve conduction (sphere; 28.81?±?3.55 vs. single cells; 18.04?±?2.10, p?相似文献   

去细胞肌肉生物支架与人脐带间充质干细胞的相容性

背景：去细胞肌肉生物支架联合人脐带间充质干细胞移植将是治疗脊髓损伤的一项重要措施。但两者是否具有良好的相容性,人脐带间充质干细胞能否在去细胞肌肉生物支架中长期存活并均匀分布,尚未得到证实。 目的：观察大鼠去细胞肌肉生物支架与人脐带间充质干细胞的相容性。 方法：改良化学法制备大鼠去细胞肌肉生物支架,将第3代人脐带间充质干细胞Hoechest33342荧光标记后分为3组进行实验,细胞+支架组、细胞+支架大鼠体内组和单纯细胞组。分别应用苏木精-伊红、Masson染色方法观察去细胞肌肉生物支架的组织形态,以荧光倒置相差显微镜和扫描电镜观察人脐带间充质干细胞的吸附和生长情况。 结果与结论：人脐带间充质干细胞与去细胞肌肉生物支架充分附着,生长增殖活跃,细胞在支架内分布均匀。细胞+支架体内组与细胞+支架组相比在移植后1-7 d人脐带间充质干细胞数量差异无显著性意义(P > 0.05),在移植14 d细胞+支架体内组人脐带间充质干细胞数量大于细胞+支架组(P < 0.05)。提示去细胞肌肉生物支架与人脐带间充质干细胞有较好的相容性,体内环境更有利于细胞增殖和两者融合。     

Embryonic Schwann cell development: the biology of Schwann cell precursors and early Schwann cells

The cellular events leading to the generation of Schwann cells from the neural crest have recently been clarified and it is now possible to outline a relatively simple model of the Schwann cell lineage in the rat and mouse. Neural crest cells have to undergo 3 main developmental transitions to become mature Schwann cells. These are the formation of Schwann cell precursors from crest cells, the formation of immature Schwann cells from precursors and, lastly, the postnatal and reversible generation of non-myelin- and myelin-forming Schwann cells. Axonal signals involving neuregulins are important regulators of these events, in particular of the survival, proliferation and differentiation of Schwann cell precursors.     

An electron microscope study of quantitative relationships between axon and Schwann cell sheath in myelinated fibres of peripheral nerves

Summary The quantitative relationships between the crossectional area of the Schwann cell sheath (myelin included) and that of its related axon were studied by electron microscopy in the nerve fibres of the spinal roots of lizard (Lacerta muralis). In both ventral and dorsal roots the cross-sectional area of the Schwann cell sheath (myelin included) was found to be directly proportional to that of its related axon (correlation coefficients between 0.88 and 0.92). The ratio between the cross-sectional area of the Schwann cell sheath (myelin included) and that of its related axon tends to diminish as the cross-sectional area of the latter increases. Thus, under normal conditions, in myelinated fibres of the spinal roots of the lizard a quantitative balance exists between the nerve tissue and its associated glial tissue. This result agrees with those previously obtained in the spinal ganglia of the lizard, gecko, cat and rabbit. Some of the mechanisms probably involved in the control of the quantitative balance between nerve tissue and its associated glial tissue in peripheral nerves are presented and discussed.