视神经远端切断后再生视网膜节细胞Bcl-2的表达

目的　成年金黄地鼠视神经远端切断后再生视网膜节细胞 (RGCs)Bcl 2的表达与再生的关系。方法　远端切断视神经并对接一段自体坐骨神经 ,玻璃体内注射CTx及 /或植入小段坐骨神经分支 (SN)。动物随机分为AG +CTx组 ;AG +SN组 ;AG +SN +CTx组 ,各组动物分别存活 4W ,用粒蓝 (GB)逆行标记和Bcl 2免疫荧光组织化学双标法观察再生的RGCs和Bcl 2表达。结果　再生RGCs胞质内有Bcl 2蛋白表达 ,玻璃体内给予CTx或植入SN组表达Bcl 2的再生RGCs分别为 32 2± 4 71和 2 9 4± 3 75个 ,约占其再生总数的 82 5 0 %及 80 96 % ,两组相比无显著性差异 (P >0 0 5 ) ;CTx与SN联用组Bcl 2阳性再生的RGCs为 15 1 8± 35 6 9个 ,占再生总数的 91 2 2 % ,与前两组相比 ,均有显著性差异 (P <0 0 5 )。结论　视神经远端切断后约 80 %的再生RGCs有Bcl 2表达 ,提示Bcl 2表达可能与节细胞再生有密切关系。     

P物质在再生视网膜节细胞中的表达及IBMX和CPT-cAMP 对其表达的影响

采用金黄地鼠视神经切断并缝接坐骨神经的再生实验模型,玻璃体内注射IBMX或/和CPT-cAMP,荧光金逆行标记再生的RGCs结合P物质免疫荧光组化双标法,研究外周神经缝接于视神经断端能否促进P物质阳性的视网膜节细胞(RGCs)再生及IBMX或/和CPT-cAMP处理对其再生的影响。实验结果:①术后四周,对照AG组每个视网膜 再生RGCs数1329±104,双标细胞平均数为45±5,占再生RGCs总数的3.4％;②AG+IBMX组每个视网膜再生RGCs数为2099±419,再生P物质阳性节细胞平均数为119±22,占再生RGCs总数的6.55％;③AG+cAMP组每个视网膜再生RGCs数为2048±133,再生P物质阳性节细胞平均数为127±37,占再生RGCs总数的6.15％;④AG+IB-MX+cAMP组每个视网膜再生RGCs数为4370±487,再生P物质阳性节细胞平均数为339±72,占再生RGCs总数的7.98％,与对照组的差异具有统计学意义。表明成年哺乳动物P物质阳性RGCs能再生,玻璃体内注射IBMX或/和CPT-cAMP可以促进该类RGCs再生。     

骨髓单个核细胞在周围神经修复中的应用

基础研究证实,多种细胞移植可以促进周围神经修复,其中来源丰富的骨髓单个核细胞,因具有取材过程简单、无交叉感染风险、无免疫排斥、可以自体移植等诸多优点,是目前重要的候选细胞之一。本文就近期有关骨髓单个核细胞的神经修复作用机制的研究、细胞植入修复受损周围神经的文献、以及与各种生物材料复合应用构建的组织工程化神经等方面最新进展进行综述,以期促进该领域基础向临床应用的转化。     

CTx促进远端视神经切断后视网膜节细胞轴突再生与c-Jun表达的关系

目的探讨霍乱毒素(CTx)促进成年金黄地鼠视神经远端切断后视网膜节细胞(RGCs)轴突再生与c-Jun的表达关系。方法远端切断视神经并对接一段自体坐骨神经,玻璃体内注射CTx及/或植入小段坐骨神经分支(SN)。动物随机分为AG CTx组;AG SN组;AG SN CTx组,各组动物分别存活4W,用荧光金(FG)逆行标记和c-Jun免疫荧光组织化学双标法观察轴突再生的RGCs内c-Jun表达情况。结果再生RGCs内有c-Jun蛋白表达,玻璃体内给予CTx或植入SN组RGCs表达c-Jun的再生RGCs分别为35·8±9·57和32·2±7·25个,约占其再生总数的94%及90%,两组相比无显著性差异(P>0·05);CTx与SN联用组c-Jun阳性再生的RGCs为150·2±43·92个,占再生总数的97%,与前两组相比,均有显著性差异(P<0·05)。结论视神经远端切断后约90%以上的再生RGCs有c-Jun表达,提示c-Jun表达与视神经远端受损后节细胞轴突再生密切相关,CTx及外周神经对RGCs轴突再生及c-Jun表达有协同促进作用。     

北京鸭视网膜节细胞的大小、密度和分布

采用Nissl染色法、视神经溃变法和神经元逆行追踪标记辣根过氧化物酶(HRP)法,研究了北京鸭视网膜节细胞(RGCs)的大小和密度及其分布的变化。北京鸭RGCs形态多样,有圆形、椭圆形和多角形等,RGCs总数为1.3×106个(P0),RGCs平均密度为5370个/mm2(P0),在视网膜中央有一个偏向鼻侧的高密度区即中央高密度区(8860个/mm2),由中央区至周边部,细胞密度逐渐降低,颞侧周边部最低(3440个/mm2)。不同区域RGCs大小差异显著,中央区以小细胞为主(62.2±23.3μm2,P0),而周边部RGCs逐渐增大,颞侧周边部最大(P0:133.7±75.7μm2;P8:152.9±55.9μm2)。由此可见,伴随RGCs大小由中央区至周边部的递增而细胞密度呈现递减的变化,这种变化趋势在颞侧周边部最明显。与此同时,随日龄增长,RGCs总数和密度均递减而细胞大小递增。dACs是位于视网膜节细胞层的小神经元,细胞大小为23.7±4.0μm2     

视网膜神经节细胞的纯化和体外存活

We had used a specific anti-Thy 1.1 antibody binding method and a nylonmembrane sieve method to isolate and purify retinal ganglion cells from neonatal rats in order to compare the effect of tectal extract on these purified cells retinal ganglion cells. Isolated retinal cell suspension with retinal ganglion cells retrograde-prelabelled with Fast Blue were seeded on culture dishes coated with the specific anti-Thy 1.1 antibody for 30 minutes before nonadherent cells were removed. The percentage purity of the adherent retinal ganglion cells determined microscopically to be 95%. However, the percentage purity of the Fast Blue-labelled retinal ganglion cells recovered using the nylon membrane of pore size 15 microns was only 60 +/- 5%. Retinal ganglion cells purified by both methods could survive and grow into large, active neurons with neurite outgrowths in the presence of tectal extract. A MTT colorimetric microassay was used to quantify the survival growth activity of these purified retinal ganglion cells after culture for 24 hours. The result showed that the optical density ratio (+Te/-Te) of the retinal ganglion cells purified by anti-Thy 1.1 antibody binding method was 12.3 (0.111/0.009) and by the nylon membrane method was 6.4 (0.102/0.016), and the optical density ratio of the non-purified retinal cells was 3.8 (0.095/0.025), p less than 0.01 for all 3 sets of results. It was concluded that in the absence of other cells, the purified retinal ganglion cells responded specifically to the trophic activity in tectal extract, the purer the retinal ganglion cells and the clearer the effect.     

视神经损伤与再生的研究进展

人类拥有良好的视觉系统.视网膜神经节细胞(retinal ganglion cells, RGCs)连接眼球与大脑,损伤之后不能再生,最终可导致失明.视神经再生的困难部分归因于胶质瘢痕和髓磷脂中的抑制性分子以及RGCs轴突内在的再生能力不足.此外,视神经损伤之后RGCs会凋亡,使得再生更为困难.本文综述了视觉系统再生失败的原因,以及目前在修复方面所取得的一些成果,其中有些发现将来有望应用于临床,使受损伤的视神经达到有意义的再生.     

周围神经异体移植免疫机制的研究进展

周围神经异体移植由于移植物免疫原性的存在,移植后受体产生免疫排斥反应,造成移植失败。本文综述了移植物的免疫成分所在、SC及细胞因子在排斥反应中的作用、T淋巴细胞的激活途径及激活信号的转导过程,并讨论了减轻免疫排斥反应的治疗进展。     

脊髓损伤治疗中的细胞移植策略及预处理研究进展

急性脊髓损伤是骨科常见的严重疾患,伤后神经功能恢复及重建是近年来研究的热点,其中细胞移植的研究得到广泛的关注并取得较大的研究进展。本文介绍了细胞移植治疗脊髓损伤治疗的研究现状,其中对移植细胞的来源,移植的时机,移植的途径以及细胞移植存活的问题及应对策略做了重点阐述。同时对增加移植细胞存活率的预处理方法做了简要综述。     

Alterations in the morphology of ganglion cell dendrites in the adult rat retina after optic nerve transection and grafting of peripheral nerve segments

Summary Transected ganglion cell axons from the adult retina are capable of reinnervating their central targets by growing into transplanted peripheral nerve (PN) segments. Injury of the optic nerve causes various metabolic and morphological changes in the retinal ganglion cell (RGC) perikarya and in the dendrites. The present work examined the dendritic trees of those ganglion cells surviving axotomy and of those whose severed axons re-elongated in PN grafts to reach either the superior colliculus (SC), transplanted SC, or transplanted autologous thigh muscle. The elaboration of the dendritic trees was visualized by means of the strongly fluorescent carbocyanine dye DiI, which is taken up by axons and transported to the cell bodies and from there to the dendritic branches. Alternatively, retinofugal axons regrowing through PN grafts were anterogradely filled from the eye cup with rhodamine B-isothiocyanate. The transection of the optic nerve resulted in characteristic changes in the ganglion cell dendrites, particularly in the degeneration of most of the terminal and preterminal dendritic branches. This occurred within the first 1 to 2 weeks following axotomy. The different types of ganglion cells appear to vary in their sensitivity to axotomy, as reflected by a rapid degeneration of certain cell dendrites after severance of the optic nerve. The most vulnerable cells were those with small perikarya and small dendritic fields (type II), whereas larger cells with larger dendritic fields (type I and III) were slower to respond and less dramatically affected. Regrowth of the lesioned axons in peripheral nerve grafts and reconnection of the retina with various tissues did not result in a significant immediate recovery of ganglion cell dendrites, although it did prevent some axotomized cells from further progression toward posttraumatic cell death.     

Hepatocyte growth factor protects retinal ganglion cells by increasing neuronal survival and axonal regeneration in vitro and in vivo

Hepatocyte growth factor (HGF) is known to promote the survival and foster neuritic outgrowth of different subpopulations of CNS neurons during development. Together with its corresponding receptor c-mesenchymal-epithelial transition factor (Met), it is expressed in the developing and the adult murine, rat and human CNS. We have studied the role of HGF in paradigms of retinal ganglion cell (RGC) regeneration and cell death in vitro and in vivo. After application of recombinant HGF in vitro, survival of serum-deprived RGC-5 cells and of growth factor-deprived primary RGC was significantly increased. This was shown to be correlated to the phosphorylation of c-Met and subsequent activation of serine/threonine protein kinase Akt and MAPK downstream signalling pathways involved in neuronal survival. Furthermore, neurite outgrowth of primary RGC was stimulated by HGF. In vivo, c-Met expression in RGC was up-regulated after optic nerve axotomy lesion. Here, treatment with HGF significantly improved survival of axotomized RGC and enhanced axonal regeneration after optic nerve crush. Our data demonstrates that exogenously applied HGF has a neuroprotective and regeneration-promoting function for lesioned CNS neurons. We provide strong evidence that HGF may represent a trophic factor for adult CNS neurons, which may play a role as therapeutic target in the treatment of neurotraumatic and neurodegenerative CNS disorders.     

Exenatide prevents high-glucose-induced damage of retinal ganglion cells through a mitochondrial mechanism

Exenatide (exendin-4 analogue) is widely used in clinics and shows a neuroprotective effect. The main objectives of the present study were to prove that retinal ganglion cells (RGC-5) express GLP-1R, to ascertain whether exenatide prevents a high-glucose-induced RGC-5 impairment, to determine the appropriate concentration of exenatide to protect RGC-5 cells, and to explore the neuroprotective mechanisms of exenatide. Immunofluorescence and Western blot analyses demonstrated that RGC-5 cells express GLP-1R. We incubated RGC-5 cells with 25 mM glucose prior to incubation with either 25 mM glucose, 55 mM glucose (high), high glucose plus exenatide or high glucose plus a GLP-1R antagonist. The survival rates of the cells were measured by CCK-8, and cellular injury was detected by electron microscopy. There were statistical differences between the high-glucose group and the control group (P<0.05). Exenatide improved the survival rate of the cells and suppressed changes in the mitochondrial morphology. The optimum concentration of exenatide to protect the RGC-5 cells from high-glucose-induced RGC injury was 0.5 μg/ml, and this protective effect could be inhibited by exendin (9-39). To further study the mechanism underlying the beneficial effects of exenatide, the expression levels of cytochrome c, Bcl-2, Bax and caspase-3 were analysed by Western blot. The present study showed that treatment with exenatide significantly inhibited cytochrome c release and decreased the intracellular expression levels of Bax and caspase-3, whereas Bcl-2 was increased (P<0.05). These results suggested that GLP-1R activation can inhibit the cellular damage that is induced by high glucose. A mitochondrial mechanism might play a key role in the protective effect of exenatide on the RGC-5 cells, and exenatide might be beneficial for patients with diabetic retinopathy.     

Protection of exenatide for retinal ganglion cells with different glucose concentrations

Exendin-4 is a peptide resembling glucagon-like peptide-1 (GLP-1), which has protective effects on nerve cells. However, the effects of Exendin-4 on retinal ganglion cells (RGC) are still under clear. The purpose of the present study is to demonstrate that exenatide prevents high- or low-glucose-induced retinal ganglion cell impairment. We observed the expression of GLP-1R in RGC-5 cells by immunofluorescence and Western blot. To investigate the effect of exenatide on RGC-5 cells incubated different glucose concentrations, CCK-8 measured the survival rates and electron microscopy detected cellular injury. The expression levels of Bcl-2 and Bax were analyzed by immunocytochemistry and Western blot. Exenatide protects RGC-5 from high- or low-glucose-induced cellular injury and the optimum concentration was 0.5μg/ml. Exenatide can inhibit high- or low-glucose-induced mitochondrial changes. Exenatide protects RGC-5 from high- or low-glucose-induced Bax increased and Bcl-2 decreased. Furthermore, the protective effect of exenatide could be inhibited by Exendin (9-39). These findings indicate that exenatide shows a neuroprotective effect for different glucose concentrations-induced RGC-5 cells injury. Exenatide could protect RGC-5 cells from degeneration or death, which may protect retinal function and have a potential value for patients with diabetic retinopathy.     

Endoneurial mast cells in peripheral nerves of the armadillo dermis

Summary Electron microscopy of the dermis of the 9-banded armadillo, Dasypus novemcinctus, reveals an intimate relationship of mast cells to peripheral nerves. Mast cells are routinely found in the dermis in close proximity to nerves, and mast cells are also present within the perineurial cell sheath in the endoneurial space proper. These cells contain electron opaque granules and exhibit numerous surface folds as is characteristic of other species. The degranulation of the mast cells by injection of 0.5% aqueous trypan blue reveals sequential exocytosis of granules, similar to that described for the rat. Degranulation occurs in most dermal and endoneurial mast cells following a single intradermal injection of trypan blue. A small number of mast cells partially degranulate and a few exhibit no degranulation, suggesting a heterogeneous population. It is also noted that mitochondrial morphology differs in degranulating cells, in that they become swollen and vacuolated. The results of this study are discussed in light of previous results reported for other species, mainly at the light microscope level.Supported by institutional grant from the L.S.U. School of DentistryThe author thanks Ivis Insua and Angela Pepin for their technical and secretarial assistance and Garbis Kerimian for his photographic work     

Neuronal Nogo-A upregulation does not contribute to ER stress-associated apoptosis but participates in the regenerative response in the axotomized adult retina

Nogo-A, an axonal growth inhibitory protein known to be mostly present in CNS myelin, was upregulated in retinal ganglion cells (RGCs) after optic nerve injury in adult mice. Nogo-A increased concomitantly with the endoplasmic reticulum stress (ER stress) marker C/EBP homologous protein (CHOP), but CHOP immunostaining and the apoptosis marker annexin V did not co-localize with Nogo-A in individual RGC cell bodies, suggesting that injury-induced Nogo-A upregulation is not involved in axotomy-induced cell death. Silencing Nogo-A with an adeno-associated virus serotype 2 containing a short hairpin RNA (AAV2.shRNA-Nogo-A) or Nogo-A gene ablation in knock-out (KO) animals had little effect on the lesion-induced cell stress or death. On the other hand, Nogo-A overexpression mediated by AAV2.Nogo-A exacerbated RGC cell death after injury. Strikingly, however, injury-induced sprouting of the cut axons and the expression of growth-associated molecules were markedly reduced by AAV2.shRNA-Nogo-A. The axonal growth in the optic nerve activated by the intraocular injection of the inflammatory molecule Pam3Cys tended to be lower in Nogo-A KO mice than in WT mice. Nogo-A overexpression in RGCs in vivo or in the neuronal cell line F11 in vitro promoted regeneration, demonstrating a positive, cell-autonomous role for neuronal Nogo-A in the modulation of axonal regeneration.     

Gap-43 immunoreactivity and axon regeneration in retinal ganglion cells of the rat

Retinal ganglion cells (RGCs) in rats were retrogradely labeled with the fluorescent tracer Fluorogold (FG) and subjected to GAP-43 and c-JUN immunocytochemistry to identify those RGSs that are capable of regenerating an axon. After optic nerve section (ONS) and simultaneous application of FG to the nerve stump (group 1 experiments), GAP-43 immunoreactive RGCs (between 2 and 21 days after ONS) always represented a subfraction of both FG-labeled (i.e., surviving) RGCs and RGCs exhibiting c-JUN. GAP-43 immunoreactive RGCs represented 22% of RGCs normally present in rat retinae and 25% of surviving RGCs at 5 days after ONS but were reduced to 2% and 1%, which is 6% and 5% of survivors at 14 and 21 days, respectively. In animals that received a peripheral nerve (PN) graft after ONS (group 2 experiments), RGCs with regenerating axons were identified by FG application to the graft at 14 and 21 days. When examined at 21 and 28 days, all FG-labeled RGCs exhibited GAP-43 immunoreactivity, and FG/GAP-43-labeled RGCs were 3% and 2% of those resent in normal rat retinae. In relation to surviving. RGCs GAP-43 immunoreactive RGCs represented 10% at both time points. FG-/GAP-43 labeled RGCs also exhibited c-JUN, but c-JUN immunoreactive RGCs were at both time points at least twice as numerous a FG-/GAP-43-labeled RGCs. These data suggest that regenerating axons in PN grafts derive specifically from GAP-43 reexpressing RGCs. Appearance of GAP-43 immunoreactivity may therefore identify those RGCs that are capable of axonal regeneration or sprouting. 1994 John Wiley & Sons, Inc.     

Long-term in vivo and in vitro AAV-2-mediated RNA interference in rat retinal ganglion cells and cultured primary neurons

Viral vector-based expression of small interfering RNAs is a promising tool for gene regulation, both in cultured cells and in animal models. In this study, we analysed the ability of adeno-associated virus-2 to function as an RNAi vector in cultured primary hippocampal neurons in vitro and in retinal ganglion cells in vivo. We demonstrate a long-lasting, highly efficient, and specific down-regulation of gene expression in vivo and in vitro by the use of bicistronic vectors. This is the first evidence of a cell type-specific long-term (more than three-month-long) RNAi in the eye. Furthermore, our results constitute the prerequisite for the use of this technique in models of neurodegeneration and neuroregeneration in vivo and in vitro.