鸡胚视网膜基膜的免疫电镜的研究

扫描电镜观察到鸡胚视网膜基膜(RBM)上的菊花样结构,经免疫胶体金透射电镜证实为Müller胶质细胞的末端足,层粘连蛋白(laminin)分布在末端足的辐射臂及其周围的基膜上。培养的视网膜细胞,其神经纤维在RBM伸长,收缩了的Müller细胞末端足呈球状颗粒分布在神经纤维及其生长锥的上面和侧面。推测分布在Müller细胞末端足的辐射臂及其周围基膜上的层粘连蛋白在体内可能与神经纤维生长的导向有关。     

无蹼壁虎视网膜GFAP免疫阳性的分布和衰老相关性变化

运用免疫组织化学方法(ABC法)结合数学形态学技术观察了胶质纤维酸性蛋白(GFAP)在无蹼壁虎(Gekko swinhonis)视网膜的分布及与衰老相关性变化。结果显示,GFAP免疫阳性结果主要位于视网膜的Müller细胞内侧端的胞体、突起和膨大的终足,胞体外侧端呈弱阳性。比较成年组与老年组视网膜中央区GFAP免疫阳性形态学参数显示:老年组GFAP免疫阳性Müller细胞短径较成年组增粗;老年组GFAP免疫阳性细胞的光密度值较成年组增高;老年组视网膜各层GFAP免疫阳性面积占视网膜总面积的百分率较成年组增高。GFAP在无蹼壁虎视网膜的分布特点及年龄相关性变化,提示Müller细胞可能通过增加GFAP表达或减弱分解来参与视网膜衰老过程中的生理功能调节。     

牛蛙视网膜诱导型一氧化氮合酶免疫组化定位

用免疫组织化学方法研究了诱导型一氧化氮酶（iNOS)在牛蛙视网膜中的表达。结果显示,在正常状态视网膜中,无长突细胞呈弱阳性反应;节细胞层、双极细胞,水平细胞和光感受器内段呈阴性反应,在暗适应状态下,神经节细胞,内核层的无长突细胞呈强阳性反应;一些双极细胞,水平细胞和光感受器内段呈弱阳性反应,提示NO主要在暗适应状态下参与视网膜的信息传递过程。     

提前接受光照对小鼠视网膜TH和bFGF表达的影响

目的在通过手术使小鼠提前睁眼接受光照,并成功诱发近视的基础上,进一步研究TH和bFGF在视网膜中的表达变化,以探讨早产近视的发病机理.方法实验用新生第4d的C57BL/6J小鼠,通过手术分离单侧上、下眼睑,使之提前睁眼并接受正常光照,在第14d检查两眼屈光力,应用RT-PCR和免疫组化方法,检测视网膜中bFGF和TH的表达.结果提前接受光照能诱导形成-9.77±0.09D的相对近视.RT-PCR结果显示提前光照小鼠视网膜TH和bFGFmRNA含量明显减少(t值分别为4.316和12.189,P<0.01).免疫组织化学显示TH免疫阳性反应主要分布在节细胞层、内网层和内核层,bFGF免疫阳性反应主要分布在内网层及其两侧的部分节细胞和内核层细胞,另在内网层和视锥视杆层也有微弱表达.免疫组织化学染色显示提前光照小鼠视网膜中TH和bFGF的表达量明显下降.结论研究表明新生小鼠提前光照后,其视网膜TH和bFGF的表达都明显下降,提示TH和bFGF与早产近视的形成有密切关系.     

中国蛤蜊神经系统GABA和Glu的HPLC测定和免疫组化定位研究

采用邻苯二甲醛(OPA)和9-芴甲氧羰基(FMOC)联合衍生的HPLC法,测定了中国蛤蜊中枢神经系统中2种氨基酸类神经递质(Glu、GABA)的含量.结果表明:足、脑、脏神经节Glu的含量分别为4.77 μg/神经节、0.54μg/神经节、2.13 μg/神经节,GABA含量分别为11.65 μg/神经节、1.03 μg/神经节、4.89 μg/神经节.同时还运用免疫组织化学方法对2种氨基酸在中国蛤蜊中枢神经系统各神经节的分布进行了定位研究和形态学观察.结果显示:谷氨酸免疫阳性反应细胞分布于脑神经节腹侧和背外侧、脏神经节背外侧及后侧、足神经节偶见大型阳性反应细胞;γ-氨基丁酸阳性反应细胞则分布于脑神经节腹侧、脏神经节小细胞团和足神经节的外侧细胞层中.     

猫视网膜年龄相关的形态学变化

取老年猫(12龄,3～3．5kg)和青年猫(1～3龄,2～2．5kg)各4只的视网膜,经4％多聚甲醛处理后,用H．E．染色以显示视网膜结构,Nissl染色显示神经节细胞,免疫组织化学ABC法染色以显示星形胶质细胞特征性标志物胶质纤维酸性蛋白(GFAP)的阳性反应细胞的分布。显微镜下观察测量视网膜厚度,计数神经节细胞、GFAP免疫反应阳性细胞数。与青年猫比较,老年猫视网膜总厚度以及外核层、外网状层、内核层和内网状层厚度均显著减小;神经节细胞层的细胞密度显著下降;GFAP免疫反应阳性细胞显著增加,GFAP阳性细胞阳性反应强,胞体明显膨胀,突起稠密粗大。推测在衰老过程中视网膜细胞有神经元丢失现象,可能是造成视觉功能衰退的重要原因之一;视网膜星形胶质细胞的功能增强可能会延缓衰老。     

Cx43在人胚胎早期食管上皮组织中的表达

目的:探讨间隙连接蛋白Cx43在人胚胎早期食管上皮组织中的表达规律.方法:应用免疫组织化学SABC法检测第2、3、4三个月龄段人胚胎食管上皮层Cx43蛋白的表达.结果:第2～4个月龄段,食管上皮层均有Cx43蛋白阳性的细胞分布.第2个月胚龄段,Cx43蛋白在人胚胎食管上皮基底层呈强阳性表达,由基底层向管腔面,细胞阳性强度逐渐降低;第3个月胎龄段,Cx43蛋白阳性细胞在食管上皮层广泛分布;第4个月胎龄段,Cx43蛋白在食管上皮层近基底面部分细胞呈弱阳性表达.结论:Cx43蛋白在人胚胎早期食管上皮层细胞的生长发育过程中起重要的作用.     

大鼠视网膜中HAP1的免疫组织化学定位及不同光照条件对HAP1表达的影响

目的探讨亨廷顿蛋白相关蛋白1(huntingtin associated protein 1, HAP1)是否存在于视网膜内及是否与视觉有关.方法对正常大鼠眼球壁用ABC法进行免疫组织化学染色,观察HAP1在视网膜中的定位;用半定量免疫印迹方法(Western blotting)检测不同光照条件对大鼠视网膜中HAP1表达的影响.结果 HAP1较广泛地分布在大鼠视网膜各层,但以内核层及外核层中免疫反应较强,阳性反应产物主要定位在节细胞层和内核层/外核层中部分细胞胞体内;其余各层中,HAP1免疫反应较弱,阳性产物呈弥散分布,未见明显的阳性胞体.在连续处于黑暗环境中72小时大鼠视网膜中,HAP1表达较常规光照动物明显减少,而连续光照72h大鼠视网膜内HAP1表达无明显变化.结论 HAP1在视网膜中的存在及不同光照条件对视网膜HAP1表达的影响表明,HAP1可能与视觉活动有关.     

谷氨酸受体在实验性青光眼视网膜细胞损伤中的作用

青光眼是第二大致盲性眼病,为不可逆致盲的最主要原因。视网膜神经节细胞损伤和死亡是青光眼所致视功能损害的根本原因。在青光眼视神经损伤的众多病理过程中,谷氨酸受体功能的改变是导致神经节细胞凋亡的重要因素。本研究组在大鼠慢性高眼压实验性青光眼模型上,围绕这一主题开展了一系列研究。研究结果表明,一方面,高眼压导致的众多信号变化通过直接调控谷氨酸的NMDA和AMPA受体功能参与神经节细胞的凋亡过程;另一方面,高眼压导致的细胞外谷氨酸集聚激活Müller细胞上的Ⅰ型代谢型谷氨酸受体(group Ⅰ metabotropic glutamate receptors,mGluRI),经下调细胞膜的Kir4.1钾通道引发Müller细胞的胶质化激活,进而导致神经节细胞的凋亡。结合这些结果,本文综述了有关谷氨酸受体在实验性青光眼视网膜细胞损伤中的作用及机制的若干研究进展。     

大鼠p75NTR荧光真核表达载体的构建及其在HEK293细胞中的表达

目的:构建大鼠p75神经营养素受体(p75 neurotrophin receptor,p75NTR)cDNA序列的绿色荧光真核表达载体并鉴定其在人胚肾293(human embryo kidney 293,HEK293)细胞中的表达.方法:采用PCR方法从含野生型大鼠p75NTR的pDC316-RP75质粒中扩增目的片段,经EcoRⅠ和SaⅡ双酶切,定向克隆于pEGFP-N1质粒中,构建绿色荧光真核表达栽体pEGFP-N1-RP75,经酶切及测序鉴定后,通过脂质体转染HEK293细胞,激光共聚焦及免疫组织化学法鉴定大鼠p75NTR的表达.结果:重组质粒经酶切鉴定和序列分析证实含有大鼠p75NTR的编码序列,转染后经激光共聚焦显微镜及免疫组织化学染色观察表明重组质粒能够在HEK293细胞中表达出具有活性的大鼠p75NTR片段.结论:大鼠p75NTR绿色荧光真核表达栽体构建成功并可在HEK293细胞中表达,为进一步研究奠定了基础.     

Expression of beta-amyloid precursor and Bcl-2 proto-oncogene proteins in rat retinas after intravitreal injection of aminoadipic acid.

In order to investigate the role of glia in relation to factors that affect the expression of beta-amyloid precursor protein (betaAPP) and B cell lymphoma oncogene protein (Bcl-2) in the central nervous tissue, the patterns of expression of betaAPP and Bcl-2 in developing and mature rat retinas were studied immunocytochemically after intravitreal injection of alpha-aminoadipic acid (alpha-AAA), a glutamate analogue and gliotoxin that is known to cause injury of retinal Müller glial cells. In normal developing retinas, betaAPP and Bcl-2 were expressed primarily but transiently in a small number of neurons in the ganglion cell layer during the first postnatal week. Immunoreactivity of betaAPP and Bcl-2 appeared in the endfeet and proximal part of the radial processes of Müller glial cells from the second postnatal week onwards. In rats that received intravitreal injection of alpha-AAA at birth, there was a loss of immunoreactivity to vimentin, and a delayed expressed on betaAPP or Bcl-2 in Muller glial cells until 3-5 weeks post-injection. Immunoreactive neurons were also observed in the inner retina especially in the ganglion cell layer from 5 to 35 days after injection. A significant reduction in numerical density of cells with large somata in the ganglion cell layer was observed in the neonatally injected retinas at P56, which was accompanied by an increased immunostaining in radial processes of Müller glial cells. In contrast, no detectable changes in the expression of betaAPP and Bcl-2 were observed in retina that received alpha-AAA as adults. These results indicate that the gliotoxin alpha-AAA has long lasting effects on the expression of betaAPP and Bcl-2 in Müller glial cells as well as neurons in the developing but not mature retinas. The loss of vimentin and delayed expression of betaAPP and Bcl-2 in developing Müller glial cells suggests that the metabolic integrity of Müller cells was temporarily compromised, which may have adverse effects on developing neurons that are vulnerable or dependent on trophic support from the Müller glial cells.     

Expression of a novel Müller glia specific antigen during development and after optic nerve lesion

To generate monoclonal antibodies, immunogen fractions were purified from embryonic chick retinae by temperature-induced detergent-phase separation employing Triton X-114. Under reducing conditions, the monoclonal antibody (mAb) 2M6 identifies a protein doublet at 40 and 46 x 10(3) Mr, which appears to form disulfide-coupled multimers. The 2M6 antigen is regulated developmentally during retinal histogenesis and its expression correlates with Müller glial cell differentiation. Isolated glial endfeet and retinal glial cells in vitro were found to be 2M6-positive, identified with the aid of the general glia marker mAb R5. mAb 2M6 does not bind to any other glial cell type in the CNS as judged from immunohistochemical data. Cell-type specificity was further substantiated by employing retinal explant and single cell cultures on laminin in conjunction with two novel neuron-specific monoclonal antibodies. MAb 2M6 does not bind either to neurites or to neuronal cell bodies. Incubation of retinal cells in vitro with bromodeoxyuridine (BrdU) and subsequent immunodouble labelling with mAb 2M6 and anti-BrdU reveal that mitotic Müller cells can also express the 2M6 antigen. To investigate whether Müller cell differentiation depends on interactions with earlier differentiating ganglion cells, transections of early embryonic optic nerves in vivo were performed. This operation eliminates ganglion cells. Müller cell development and 2M6 antigen expression were not affected, suggesting a ganglion-cell-independent differentiation process. If, however, the optic nerve of juvenile chicken was crushed to induce a transient degeneration/regeneration process in the retina, a significant increase of 2M6 immunoreactivity became evident. These data are in line with the hypothesis that Müller glial cells, in contrast to other distinct glial cell types, might facilitate neural regeneration.     

Nerve growth factor inhibits osmotic swelling of rat retinal glial (Müller) and bipolar cells by inducing glial cytokine release

Osmotic swelling of neurons and glial cells contributes to the development of retinal edema and neurodegeneration. We show that nerve growth factor (NGF) inhibits the swelling of glial (Müller) and bipolar cells in rat retinal slices induced by barium‐containing hypoosmotic solution. NGF also reduced Müller and bipolar cell swelling in the post‐ischemic retina. On the other hand, NGF prevented the swelling of freshly isolated Müller cells, but not of isolated bipolar cells, suggesting that NGF induces a release of factors from Müller cells that inhibit bipolar cell swelling in retinal slices. The inhibitory effect of NGF on Müller cell swelling was mediated by activation of TrkA (the receptor tyrosine kinase A), but not p75^NTR, and was prevented by blockers of metabotropic glutamate, P2Y₁, adenosine A₁, and fibroblast growth factor receptors. Basic fibroblast growth factor fully inhibited the swelling of freshly isolated Müller cells, but only partially the swelling of isolated bipolar cells. In addition, glial cell line‐derived neurotrophic factor and transforming growth factor‐β1, but not epidermal growth factor and platelet‐derived growth factor, reduced the swelling of bipolar cells. Both Müller and bipolar cells displayed TrkA immunoreactivity, while Müller cells were also immunostained for p75^NTR and NGF. The data suggest that the neuroprotective effect of NGF in the retina is in part mediated by prevention of the cytotoxic glial and bipolar cell swelling.

     

The role of cell adhesion molecules in neurite outgrowth on Müller cells

The roles of neural cell adhesion molecule (NCAM), L1, N-cadherin, and integrin in neurite outgrowth on various substrates were studied. Antibodies against these cell surface molecules were added to explants of chick retina and the neurites from retinal ganglion cells were examined for effects of the antibodies on neurite length and fasciculation. On laminin, an anti-integrin antibody completely inhibited neurite outgrowth. The same antibody did not inhibit neurite outgrowth on polylysine or Müller cells. Antibodies to NCAM, L1, and N-cadherin did not significantly inhibit neurite outgrowth on laminin but produced significant inhibition on Müller cells. The inhibition of neurite outgrowth on glia by anti-L1 antibodies supports the hypothesis that L1 is capable of acting in a heterophilic binding mechanism. On laminin, both anti-N-cadherin and anti-L1 caused defasciculation of neurites from retinal ganglion cells, while anti-NCAM did not. None of these antibodies produced defasciculation on Müller cells. The results indicate that these three cell adhesion molecules may be very important in interactions with glia as axons grow from the retina to the tectum and may be less important in axon-axon interactions along this pathway. No evidence was found supporting the role of integrins in axon growth on Müller cells.     

Nitric oxide synthase immunoreactivity in the developing and adult human retina

Nitric oxide synthase (NOS) catalyzes the formation of nitric oxide (NO) from L-arginine. In this study, the cellular localization of neuronal NOS (nNOS) activity in the human retina since fetal development was examined by immunohistochemistry. No detectable staining in the fetal retina was present at 14 weeks of gestation (wg), the earliest age group examined. A centro-peripheral gradient of development of nNOS immunoreactivity was evident at 16–17 wg, with the midperipheral retina showing nNOS immunoreactivity in most of the cell types and the inner plexiform layer while the peripheral part demonstrated moderate immunoreactivity only in the ganglion cell layer and photoreceptor precursors. A transient increase in nNOS immunoreactivity in the ganglion cells and Müller cell endfeet between 18–19 and 24–25 wg was observed at the time when programmed cell death in the ganglion cell layer, loss of optic nerve fibres as well as increase in glutamate immunoreactivity and parvalbumin (a calcium binding protein) immunoreactivity in the ganglion cells was reported. These observations indicate that programmed cell death of ganglion cells in the retina may be linked to glutamate toxicity and NO activity, as also suggested by others in the retina and cerebral cortex. The presence of nNOS immunoreactivity in the photoreceptors from 16–17 weeks of fetal life to adulthood indicates other functions, besides their involvement in photoreceptor function of transduction and information processing.     

Distribution of glial fibrillary acidic protein in normal and gliotic human retina

The distribution of glial fibrillary acidic protein (GFAP) in normal human retina and in retinae with gliosis due to different diseases was studied by immunohistochemical methods. In normal retina, an evident GFAP-positivity is encountered only in the nerve fiber and ganglion cell layers; Müller cells do not stain. In retinal gliosis, together with an enhanced positivity of the perivascular and accessory glia, a strong staining for GFAP is observed in Müller cells, which extends from the inner to the outer limiting layers. A correlation between the intensity of immunohistochemical glial staining, its anatomical localization and the degree of retinal changes is suggested.     

Atoh7 promotes retinal Müller cell differentiation into retinal ganglion cells

Glaucoma is one of the leading eye diseases due to the death of retinal ganglion cells. Increasing evidence suggests that retinal Müller cells exhibit the characteristics of retinal progenitor cells and can differentiate to neurons in injured retinas under certain conditions. However, the number of ganglion cells differentiated from retinal Müller cells falls far short of therapeutic needs. This study aimed to promote the differentiation of retinal Müller cells into ganglion cells by introducing Atoh7 into the stem cells dedifferentiated from retinal Müller cells. Rat retinal Müller cells were isolated and dedifferentiated into stem cells, which were transfected with PEGFP-N1 or PEGFP-N1-Atoh7 vector, and then further induced to differentiate into ganglion cells. The proportion of ganglion cells differentiated from Atoh7-tranfected stem cells was significantly higher than that of control transfected or untransfected cells. In summary, Atoh7 promotes the differentiation of retinal Müller cells into retinal ganglion cells. This may open a new avenue for gene therapy of glaucoma by promoting optic nerve regeneration.     

Comparative study of glial fibrillary acidic protein (GFAP)-like immunoreactivity in the retina of some representative vertebrates.

A polyclonal glial fibrillary acidic protein (GFAP) antiserum was used to study the distribution of GFAP-like immunoreactivity in the retina of adult vertebrates (teleosts, amphibians, reptiles, birds and mammals). GFAP-positive Müller cells were demonstrated in all the species studied, although with different degrees and patterns of immunoreactivity. In nonmammalian vertebrates, Müller cells were the only immunoreactive retinal elements. The staining was located throughout the retina of the species examined, with the exception of the rabbit, which exhibited regional variability in the expression of GFAP. The data indicate that GFAP expression in retinal Müller cells is a common feature of a wide variety of adult vertebrate species.     

Role of Müller glia in neuroprotection and regeneration in the retina

Glial cells are thought to protect neurons from various neurological insults. When there is injury to retina, Müller cells, which are the predominant glial element in the retina, undergo significant morphological, cellular and molecular changes. Some of these changes reflect Müller cell involvement in protecting the retina from further damage. Müller cells express growth factors, neurotransmitter transporters and antioxidant agents that could have an important role in preventing excitotoxic damage to retinal neurons. Moreover, Müller cells contact to endothelial cells to facilitate the neovascularization process during hypoxic conditions. Finally, recent studies have pointed to a role of Müller cells in retina regeneration after damage, dedifferentiating to progenitor cells and then giving rise to different neuronal cell types. In this article we will review the role of Müller glia in neuroprotection and regeneration after damage in the retina.