Cultured rat Schwann cells express low affinity receptors for nerve growth factor

Schwann cell cultures prepared from postnatal Sprague-Dawley rat sciatic nerves were used to demonstrate the presence of specific receptors for the beta-subunit of nerve growth factor (NGF) on rat Schwann cells. Indirect immunofluorescence microscopy with a monoclonal antineuronal NGF receptor (NGFR) antibody indicated that NGFR antigen was expressed on the surface of Schwann cells but not of endoneurial fibroblasts. Studies with 125I-NGF confirmed this distribution of NGFR in the cultures and showed that the Schwann cell NGFR had a single NGF binding affinity (Kd of 1.8 x 10(-9) M). 125I-NGF binding by the cultured Schwann cells increased with time in vitro, reaching a plateau level on the 4th day, but decreased with increasing age, reaching 40% of the neonatal value in Schwann cells isolated from 12-day-old rats. Treatment of the cultures with NGF did not alter Schwann cell phenotype, survival or proliferation.     

Transplantation of olfactory ensheathing cells or Schwann cells restores rapid and secure conduction across the transected spinal cord

Olfactory ensheathing cells (OECs) or Schwann cells were transplanted into the transected dorsal columns of the rat spinal cord to induce axonal regeneration. Electrophysiological recordings were obtained in an isolated spinal cord preparation. Without transplantation of cells, no impulse conduction was observed across the transection site; but following cell transplantation, impulse conduction was observed for over a centimeter beyond the lesion. Cell labelling indicated that the regenerated axons were derived from the appropriate neuronal source, and that donor cells migrated into the denervated host tract. As reported in previous studies, the number of regenerated axons was limited. Conduction velocity measurements and morphology indicated that the regenerated axons were myelinated, but conducted faster and had larger axon areas than normal axons. These results indicate that the regenerated spinal cord axons induced by cell transplantation provide a quantitatively limited but rapidly conducting new pathway across the transection site.     

以NgR为靶点治疗脊髓损伤的研究进展

NgR是最近发现并克隆的一种糖基磷脂酰肌醇（GPI）锚定膜蛋白，因其在髓磷脂抑制轴突再生信号转导过程中特殊的靶分子效应，日益受到重视。以NgR为靶点促进脊髓轴突再生的尝试为中枢神经系统（CNS）损伤的治疗提供了有益的启示。     

嗅鞘细胞移植后大鼠损伤脊髓神经生长因子的表达

目的 研究嗅鞘细胞移植对大鼠损伤脊髓内的神经生长因子(NGF)表达的影响,以从NGF角度探讨嗅鞘细胞移植修复大鼠脊髓损伤的机制.方法 48只SD大鼠用NYU -Ⅱ撞击机(10g-25 mm)损伤T10脊髓制作脊髓损伤(SCI)模型,随机分为嗅鞘细胞组、DMEM组各24只;另设立正常对照组6只.将GFP-嗅鞘细胞细胞悬液移植入嗅鞘细胞组大鼠损伤处,DMEM组用单纯的DMEM/F12液代替,正常对照组不做任何处理.移植术后1d、7d、14 d、21 d,用BBB评分法测定脊髓运动功能,RT - PCR方法比较各时间点NGF表达差异.移植术后第21天应用免疫组化比较嗅鞘细胞组、DMEM组、正常对照组大鼠脊髓损伤区NGF的表达差异.结果 移植后1d、7d、14 d、21 d,嗅鞘细胞移植组运动功能评分均高于同期DMEM组.NGF表达量在术后第1天最高,第7天达峰值,之后缓慢降低.移植后第21天,嗅鞘细胞移植组脊髓NGF表达量高于同期DMEM组和正常对照组.结论 嗅鞘细胞移植可上调损伤脊髓NGF的表达,从而促进了损伤脊髓的修复.     

神经生长因子对创伤性脊髓损伤大鼠膀胱功能和轴突损伤修复的作用研究

目的 探讨神经生长因子(Nerve growth factor, NGF)对创伤性脊髓损伤(Traumatic spinal cord injury, t-SCI)大鼠膀胱功能和脊髓神经轴突损伤修复的影响及其分子机制。方法 取30只雄性Sprague-Dawley(SD)大鼠,通过改良Allen’s击打法构建创伤性脊髓损伤模型,随机分为假手术组、损伤组和NGF组,每组各10只;采用血脑屏障(Blood-brain barrier, BBB)评分观察术前、术后大鼠的后肢运动功能;BL-420生物仪实验系统检测尿动力学;甲苯胺蓝染色吻合口远端截取的左侧腰6前根,计算有髓轴突数量;采用苏木精-伊红(Hematoxylin eosin, HE)染色大鼠膀胱组织;原位末端标记法(TdT-mediated dUTP nick and labeling, TUNEL)染色大鼠损伤严重的脊髓,观察脊髓神经细胞的凋亡率;蛋白免疫印迹法(Western blot)检测脊髓组织中原癌基因丝氨酸/苏氨酸蛋白激酶(proto-oncogene serine/threonine-protein kinase, R...     

Schwann cells differentiated from spheroid-forming cells of rat subcutaneous fat tissue myelinate axons in the spinal cord injury

In the present study, we found that nestin-expressing spheroid cells derived from multipotent adipose stem cells of subcutaneous fat tissue could efficiently differentiate into Schwann cells (SCs) in vitro based on expression of SC markers such as A2B5, GFAP, O4, p75, S100, Sox10, Krox-20, and L1. The induced SC is engrafted to spinal cord injury lesions and formed a peripheral nervous system (PNS)-type myelin sheath on central nervous system (CNS) axons. PNS-type myelin sheath formation in repaired tissue was confirmed by transplantation of both induced PKH26-labeled SC and induced EGFP-expressing SC generated from EGFP transgenic rats. In addition to direct participation as myelin sheath-forming SC in repaired tissue, the induced SC also expressed several neurotrophic factors, as did native SC, which may suggest an additional role for induced SC in stimulation of endogenous healing responses. Thus, spheroid-forming cells from subcutaneous fat tissue demonstrated rapid and efficient induction into SC, and such cells show therapeutic promise for repair of damage to the CNS and PNS.     

Synaptic development in the injured spinal cord cavity following co-transplantation of fetal spinal cord cells and autologous activated Schwann cells

Transplantation of activated transgenic Schwann cells or a fetal spinal cord cell suspension has been widely used to treat spinal cord injury. However, little is known regarding the effects of co-transplantation. In the present study, autologous Schwann cells in combination with a fetal spinal cord cell suspension were transplanted into adult Wistar rats with spinal cord injury, and newly generated axonal connections were observed ultrastructurally. Transmission electron microscopic observations showed that...     

Demyelination and Schwann cell responses adjacent to injury epicenter cavities following chronic human spinal cord injury

The natural history of post-traumatic demyelination and myelin repair in the human spinal cord is largely unknown and has remained a matter of speculation. A wealth of experimental studies indicate that mild to moderate contusive injuries to the mammalian spinal cord evolve into a cavity with a preserved rim of white matter in which a population of segmentally demyelinated axons persists. It is believed that such injured axons have abnormal conduction properties. Theoretically, such axons might show improved function if myelin repair occurred. Schwann cells can remyelinate axons affected by multiple sclerosis, but little evidence exists that such repair can occur spontaneously following traumatic human SCI. Therefore, it is important to determine if chronic demyelination is present following human spinal cord injury. There are no previous reports that have conclusively demonstrated demyelination in the human spinal cord following traumatic spinal cord injury using immunohistochemical techniques. Immunohistochemical methods were used to study the distribution of peripheral and central myelin proteins as well as axonal neurofilament at the injury epicenter in 13 postmortem chronically injured human spinal cords 1-22 years following injury. Of these seven could be assessed by our methods. We found that some axonal demyelination can be detected even a decade following human SCI and indirect evidence that invading Schwann cells contributed to restoration of myelin sheaths around some spinal axons.     

大鼠脊髓损伤后轴突病理变化与胶质瘢痕的关系

目的 观察脊髓损伤(SCI)后轴突变化及其与胶质瘢痕的关系.方法 应用Allen's法建立大鼠脊髓损伤模型,通过行为学评分、免疫荧光及神经束路示踪等观察SCI后轴突的病理变化,及其与胶质瘢痕的关系,并测量胶质瘢痕的厚度.结果 SCI后损伤处的轴突呈断裂、扭曲状,SCI后1 周损伤轴突呈再生趋势,2周时再生明显,与此相应动物运动功能逐渐恢复,4周时胶质瘢痕形成,再生的轴突被瘢痕阻挡.头尾侧胶质瘢痕厚度(107.00±20.12)μm大于两侧边厚度(69.92±24.37)μm.结论 SCI后轴突仍具有再生能力,但被胶质瘢痕所阻挡,瘢痕厚度的测量为将来去除胶质瘢痕提供了实验依据.     

Increase in nerve growth factor-like immunoreactivity and decrease in choline acetyltransferase following contusive spinal cord injury

We have previously described a graded spinal cord injury model in the rat. Mild contusive injury results in an initially severe functional deficit that is attenuated over time to reveal the mild chronic deficits that characterize this injury. In this study, we have shown that mild contusive injury also results in a significant decrease in choline acetyltransferase (ChAT) activity during the first week after injury. At 1 week ChAT activity is maximally reduced at the site of the contusion and is also significantly lowered throughout the spinal cord. ChAT activity then rebounds during the following 3 weeks, partially at the injury site where there is considerable loss of gray and white matter, and completely in rostral and caudal cord segments. The rebound in ChAT activity is temporally associated with the partial recovery of function. Further, the changes in ChAT activity after injury are mirrored by changes in nerve growth factor-like immunoreactivity (NGF-LI) as determined by a specific two-site ELISA. NGF-LI increases significantly after injury, reaching a maximum at 7 days after contusion and at the injury site. However, levels of NGF-LI are also significantly increased throughout the spinal cord. NGF-LI then decreases at 2 and 4 weeks as ChAT activity rebounds. Further experiments will be needed to examine the possibility of a role for NGF in promoting the recovery of function after spinal cord injury.     

Nerve growth factor and injured peripheral nerve regeneration

Nerve growth factor （NGF） exhibits many biological activities, such as supply of nutrients, neuroprotection, and the generation and rehabilitation of injured nerves. The neuroprotective and neurotrophic qualities of NGF are generally recognized. NGF may enhance axonal regeneration and myelination of peripheral nerves, as well as cooperatively promote functional recovery of injured nerves and limbs. The clinical efficacy of NGF and its therapeutic potentials are reviewed here. This paper also reviews the latest NGF research developments for repairing injured peripheral nerve, thereby providing scientific evidence for the appropriate clinical application of NGF.     

Continuous intrathecal fluid infusions elevate nerve growth factor levels and prevent functional deficits after spinal cord ischemia

Continuous intracerebroventricular or intrathecal infusions of neurotrophic factors have been reported to prevent neuronal degeneration, stimulate axonal sprouting and ameliorate behavioral deficits in various models of CNS injury and aging. In the present study, the ability of intrathecal infusions of recombinant human nerve growth factor (NGF) to reduce functional deficits following spinal cord ischemia was investigated. Adult rabbits underwent intrathecal cannulation and continuous infusions of either 300 microg/ml recombinant human NGF or artificial CSF (vehicle) at a rate of 143 microl/day for 7 days prior to induction of spinal cord ischemia. Continuous infusions were maintained after induction of ischemia. Four days later, both NGF-treated and vehicle-infused subjects showed a significant amelioration of functional motor deficits compared to lesioned, non-infused subjects (P<0.05). The average duration of tolerated ischemia increased from 23.4+/-1.8 min in lesioned, non-infused subjects to 35.5+/-3.1 min in lesioned, artificial CSF-infused subjects and 35.6+/-4.7 min in NGF-infused subjects (mean+/-S.E.M.). Significantly elevated NGF protein levels were attained within the spinal cords of both NGF-treated subjects and artificial CSF-infused subjects, although levels were substantially higher in NGF-treated subjects (9.8+/-3.8 ng/g in NGF-infused vs. 2.0+/-0.4 ng/g in vehicle-infused and only 0.4+/-0.2 ng/g in lesioned, non-infused animals). These findings indicate that the process of intrathecal cannulation and fluid infusion elicits alterations in the spinal cord environment that are neuroprotective, including spontaneous elevations in NGF levels.     

Rapid quantitative immunohistochemical assessment of human peripheral neuropathies using a monoclonal antibody against nerve growth factor receptor

Summary An analysis of nerve growth factor (NGF) receptor expression and density in human sural nerve biopsies was performed by immunocytochemistry with a murine monoclonal antibody against the human NGF receptor. Quantitative assessment of immunostaining density was made by histospectrophotometry on frozen sections. Although there was enhanced expression of NGF receptor within endoneurium in all patients with clinical neuropathies, expression was highest in nerves with axonal disease, consistent with the proposal that disruption of axon-Schwann cell interactions triggers the reexpression of the NGF receptor. These results with human nerves, together with previous studies with animal models, suggest that NGF and NGF receptor play important roles in the general response to neuronal injury.     

神经生长因子及其结合肌基膜管移植修复脊髓横断性损伤的组织学观察

目的 对应用神经生长因子（ＮＧＦ）及其结合肌基膜管（ＭＢＬ）修复脊髓横断性损伤进行组织学评价。方法 横断雌性家犬脊髓后,分为三组：（Ａ）ＭＢＬ移植结合注射ＮＧＦ组７只。（Ｂ）脊髓单纯横断注射ＮＧＦ组６只。（Ｃ）对照组切除脊髓０．５ｃｍ４只。六个月后用免疫且化学方法对神经轴突和胶质细胞网络框架结构,进行特异染色,并用图像分析方法对脊髓横断处的远近端横截面进行神经纤维数量对比。结果 各组远端神经纤维数     

Modulation of low-affinity nerve growth factor receptor in injured adult rat spinal cord motoneurons

Spinal and brainstem motoneurons of the adult rat reexpress low-affinity nerve growth factor receptor (LNGFR) and its mRNA after axotomy. We have previously reported the time courses of this reexpression after cut (no regeneration) or crush (followed by regeneration) of the sciatic nerve. We have shown that the length of the different phases of this reexpression (appearance, maintenance and disappearance) can vary according to the type of axotomy. With the present study we expand our previous data and describe and analyze the modulation the LNGFR expression in adult spinal cord motoneurons following different lesion paradigms. In one approach we have imposed three traumatic injuries that still allow regeneration of the sciatic nerve but with a different time course with respect to the crush injury (application of a silicone regeneration chamber, multiple crushes and delayed repair of ligated nerves). In a second approach, we have determined the capability of three toxic or metabolic injuries to induce LNGFR expression without any direct trauma of the nerve (experimental diabetogenesis, botulinum and alpha-bungarotoxin intoxication and 2,5-hexanedione intoxication). In a third approach, we have investigated the effect of the block of the axoplasmic transport on the LNGFR expression following different topical applications of vincristine combined with a nerve crush. The results we present are consistent with the idea that: (1) LNGFR immunoreactivity in adult motoneurons is expressed by motoneurons that are attending to an axonal outgrowth and not a generic signal of cellular damage or impairment of the motor function; (2) LNGFR expression in these motoneurons is related to and parallels the outgrowth process time frame, and (3) the signal/s that trigger and sustain this reexpression may be retrogradely transported from the periphery. © 1993 Wiley-Liss, Inc.     

A double-transgenic mouse used to track migrating Schwann cells and regenerating axons following engraftment of injured nerves

We propose that double-transgenic thy1-CFP(23)/S100-GFP mice whose Schwann cells constitutively express green fluorescent protein (GFP) and axons express cyan fluorescent protein (CFP) can be used to serially evaluate the temporal relationship between nerve regeneration and Schwann cell migration through acellular nerve grafts. Thy1-CFP(23)/S100-GFP and S100-GFP mice received non-fluorescing cold preserved nerve allografts from immunologically disparate donors. In vivo fluorescent imaging of these grafts was then performed at multiple points. The transected sciatic nerve was reconstructed with a 1-cm nerve allograft harvested from a Balb-C mouse and acellularized via 7 weeks of cold preservation prior to transplantation. The presence of regenerated axons and migrating Schwann cells was confirmed with confocal and electron microscopy on fixed tissue. Schwann cells migrated into the acellular graft (163+/-15 intensity units) from both proximal and distal stumps, and bridged the whole graft within 10 days (388+/-107 intensity units in the central 4-6 mm segment). Nerve regeneration lagged behind Schwann cell migration with 5 or 6 axons imaged traversing the proximal 4 mm of the graft under confocal microcopy within 10 days, and up to 21 labeled axons crossing the distal coaptation site by 15 days. Corroborative electron and light microscopy 5 mm into the graft demonstrated relatively narrow diameter myelinated (431+/-31) and unmyelinated (64+/-9) axons by 28 but not 10 days. Live imaging of the double-transgenic thy1-CFP(23)/S100-GFP murine line enabled serial assessment of Schwann cell-axonal relationships in traumatic nerve injuries reconstructed with acellular nerve allografts.     

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.     

自体骨髓基质干细胞动员联合神经生长因子及综合康复治疗脊髓损伤

目的 探讨自体骨髓基质干细胞(BMSCs)动员联合神经生长因子及综合康复治疗脊髓损伤(SCI)的临床疗效. 方法 选择河南省人民医院神经外科自2006年1月至2011年12月收治的SCI患者72例,根据患者意愿分为2组,对照组32例给予神经生长因子和综合康复治疗,治疗组40例在对照组基础上加用自体BMSCs动员治疗.治疗前后依据美国脊髓损伤学会(ASIA)评分、运动诱发电位(MEP)波幅及潜伏期的变化评价疗效,通过流式细胞术检测外周血细胞CD133+CD34+比例,MRI观察SCI治疗前后脊髓的形态学变化,同时观察患者不良反应. 结果 治疗组患者ASIA评分明显升高,MEP潜伏期明显缩短,波幅明显增高,外周血细胞CD133+ CD34+比例升高显著,与对照组比较差异均有统计学意义(P＜0.05).治疗及随访期间均未出现明显不良反应.结论 自体BMSCs动员联合神经生长因子及综合康复治疗可有效促进SCI的修复,显著改善患者的神经功能.     

小发夹RNA抑制Nogo基因表达促进脊髓损伤修复的实验研究

目的 利用RNA干涉（RNAi）技术使特定的基因（Nogo）沉默，探索脊髓损伤的治疗方法。方法 设计有小发夹结构的两条DNA序列，PCR扩增带有U6启动子的小发夹，腺病毒包装重组体，转染少突胶质细胞，采用Westernblot法分析Nogo-66的蛋白表达水平。结果成功地构建了靶向Nogo基因RNAi的带有U6启动子的小发夹重组体，Nogo-66蛋白表达水平明显下降。结论 靶向RNAi小发夹重组体经腺病毒包装后，成功转染少突胶质细胞并能有效抑制Nogo-66基因的表达。