大鼠生后视网膜发育过程中Nurr1与TH的相关关系研究

目的:观察核受体相关因子1(nuclear receptor-related factor 1,Nurr1)与多巴胺能神经细胞特异性标记物酪氨酸羟化酶(TH)在生后大鼠视网膜发育过程中的表达变化,探明Nurr1与视网膜多巴胺能神经元的相关关系。方法:石蜡包埋组织切片,免疫组织化学双重标记。结果:Nurr1在视网膜发育过程中的表达出现了显著的动态变化,Nurr1阳性产物主要表达在内核层细胞,生后3～7 d达到高峰,之后随着细胞的成熟阳性表达又逐渐减少,成熟的视网膜组织内仅见少量Nurr1阳性细胞,在视网膜神经细胞从幼稚到成熟的分化过程中仅见个别TH阳性细胞。Nurr1与TH的免疫组化双标结果显示两蛋白可以共表达在同一细胞中,但众多的Nurr1阳性细胞不表达TH。结论:Nurr1在大鼠视网膜多巴胺能神经细胞及非多巴胺能神经细胞从幼稚到成熟的分化过程中可能具有重要的调控作用。     

淀粉样β蛋白42在正常大鼠视网膜节细胞中的发育及表达

目的:探讨淀粉样β蛋白42(Aβ42)在正常大鼠视网膜节细胞巾的发育及表达.方法:SD大鼠分为正常见光组和避光饲养组.眼球做冷冻切片和视网膜裱片,采用免疫细胞化学方法检测Aβ42的表达.结果:出生后3、6、13 d组大鼠视网膜节细胞未见Aβ42表达,15 d(见光后)以后开始有表达,并逐渐增多,90 d组表达最多.其中15 d和25 d组在避光饲养下未见表达.结论:Aβ42在正常大鼠出生后第15日开始表达,并逐渐上调;在15 d后避光饲养动物的视网膜节细胞中未见表达,提示该蛋白在视网膜视觉功能的发育中可能发挥了重要作用.     

Nogo-A在小鼠胚胎新生视网膜节细胞及其轴突的表达

目的 研究小鼠胚胎阶段Nogo-A在视网膜节细胞(RGCs)及其轴突上的表达及时程变化. 方法 取不同发育阶段的小鼠胚胎,采用免疫荧光染色,以激光扫描共焦显微镜观察Nogo-A在视觉传导通路中的表达.并采用免疫双标染色确定视网膜中表达Nogo-A蛋白的细胞类型. 结果 在视网膜发育的早期阶段(E12),Nogo-A密集表达于具有放射状形态的细胞上,Nogo-A免疫阳性产物出现在胞质、胞膜以及轴突上.Nogo-A与Tuj-1双标染色显示,此阶段的视网膜中几乎所有RGCs及其轴突都表达有Nogo-A;在稍晚的发育阶段(E13),视网膜中表达Nogo-A的RGCs数量明显减少,且仅出现在节细胞层以外的室周带和睫状体边缘区.在视网膜的神经纤维层,大部分RGCs轴突不再表达Nogo-A,仅有少量视觉纤维为Nogo-A免疫阳性;RGCs的神经发生基本完成后(E15), 视网膜中几乎检测不到Nogo-A免疫阳性的细胞,但视网膜纤维层仍有少量表达Nogo-A的节细胞轴突.与之类似,视神经盘、视茎、视交叉和视束都观察到少量Nogo-A免疫阳性的轴突.值得注意的是,视束中表达Nogo-A的纤维集中位于表浅部位,而此处恰为新近到达轴突的通过部位. 结论 Nogo-A在视网膜RGCs以及轴突上表达的时程变化和位置特点提示,新生RGCs及其轴突表达Nogo-A,成熟后RGCs内Nogo-A的表达则下调.推测新生RGCs及其轴突中表达的Nogo-A可能与减少轴突分叉等细胞的内在功能有关.     

脑红蛋白在绵羊视网膜中的分布

目的:探讨脑红蛋白在绵羊视网膜的分布特征。方法:利用免疫组织化学显色SP法,观察脑红蛋白在健康成年绵羊视网膜中的分布情况。结果:脑红蛋白在绵羊视网膜的视神经纤维层、内网状层、外网状层和光感受器内节段中有强阳性表达,在视网膜的内核层和节细胞层有弱阳性表达,在视网膜外核层、光感受器外节段和色素上皮层中未见有阳性表达,内界膜、外界膜和视神经中亦有脑红蛋白阳性表达。绵羊视网膜脑红蛋白阳性表达的细胞类型主要有节细胞、双极细胞和光感受器细胞,其中节细胞的阳性表达定位于细胞质,胞核中未见表达。结论;除外核层、光感受器外节段和色素上皮层外,脑红蛋白在绵羊视网膜其他各层中均有表达,提示脑红蛋白在维持视网膜中氧平衡状态时发挥重要作用。     

大鼠视神经切断后视网膜双极细胞PKC-α和recoverin的表达

为了探讨视神经切断后视网膜内部是否存在突触可塑性改变,本实验采用大鼠视神经切断模型,通过免疫组织化学方法检测视神经切断后视网膜双极细胞PKC-α和recoverin的表达变化。结果显示:正常视网膜中,PKC-α和recoverin阳性产物主要见于视网膜内核层、内网层及节细胞层,另外外核层也可见少量recoverin阳性细胞。视神经切断后3d,大鼠视网膜内网层高倍镜下可见PKC-α和recoverin免疫阳性终末的数量开始增加,14d时增至最高,21d、28d呈现逐渐减少的趋势。本研究结果提示视神经切断后视网膜双极细胞与节细胞之间的突触可能存在早期增生,后期溃变的可塑性变化。     

兔视网膜中P物质样免疫反应神经元的发育

本实验用免疫细胞化学ABC法,研究了成年、新生和生后兔视网膜中P物质(SP)样免疫反应神经元的定位和发育。结果表明,成年兔视网膜SP样免疫反应细胞胞体位于内核层和节细胞层,胞突分布在内网层的第1、3、5亚层,偶见于视神经纤维层。细胞密度以视纹最高,从视纹向背腹视网膜边缘区密度渐变小。在新生兔视网膜已有SP阳性胞体和胞突出现,胞体主要位于节细胞层,突起在内网层第5亚层,但未形成连续网层,在第1亚层很少,第3亚层未见SP阳性突起。SP阳性细胞密度从新生到生后第4天增加,生后第6天到第12天细胞密度渐下降。生后第12天SP阳性胞体主要位于内核层。生后第20天,SP阳性细胞的形态、密度与分布已接近成年水平。上述结果提示,在兔视网膜中SP样免疫反应胞体和突起在生前已出现,生后继续发育,到生后20天后其形态发育已接近成熟。     

大鼠视网膜发育过程中细胞增殖与凋亡的形态学

目的:观察视网膜发育过程中增殖细胞核抗原(PCNA)的表达变化及细胞凋亡状态。方法:14~20 d胎鼠(E14-20 d)、生后0~15 d幼鼠(P0-15)及成年鼠(P36),取出眼球组织,免疫组织化学显色及原位末端标记(TUNEL)观察PCNA的表达和细胞凋亡变化。结果:发育早期E14~P7,可观察到有较强PCNA阳性表达,P9时PCNA阳性细胞数目明显减少,P15开始未见明显PCNA阳性表达。E18可观察到凋亡细胞,随着发育的进行,凋亡细胞逐渐增多,P7时凋亡细胞数目最多,之后又逐渐减少,成熟的视网膜组织内未见明显凋亡细胞。结论:在视网膜神经细胞发育过程中,细胞的增殖和凋亡呈现了有序的动态变化,正是这种有序的增殖和凋亡的平衡才使视网膜最终发育成了正常的组织结构及功能。     

碱性成纤维细胞生长因子对谷氨酸毒性损伤豚鼠视网膜内突触小泡蛋白表达的影响

目的:探讨过量谷氨酸毒性损伤豚鼠视网膜内突触小泡蛋白(SYP)的表达及碱性成纤维细胞生长因子(bFGF)对其表达的影响。方法:豚鼠随机分成谷氨酸钠损伤组、对照组和bFGF治疗组。采用免疫组织化学方法和图像分析技术,对各组豚鼠视网膜内SYP免疫反应产物的表达进行检测。结果:对照组豚鼠视网膜内SYP的表达为强阳性,主要定位于外网状层和内网状层,损伤组视网膜相应区域内阳性反应产物的光密度值明显低于对照组,bFGF治疗后SYP的阳性表达明显高于损伤组,且与对照组相比,无显著性差异。结论:bFGF可抑制过量谷氨酸钠诱发的视网膜内SYP的表达减少,上调SYP的表达,对视神经损伤有显著地促功能修复作用。     

促红细胞生成素对视网膜色素变性的RCS大鼠的作用

为观察促红细胞生成素对视网膜色素变性的RCS大鼠的作用,探讨其对视网膜变性神经元保护的可能机制。我们把出生后雄性RCS大鼠48只,随机分为给药组和对照组。RCS大鼠给药组从生后5d开始腹腔注射重组人促红细胞生成素(rhEPO),每5d注射一次,剂量为4000IU/kg。RCS大鼠对照组注射生理盐水,剂量同上。HE染色和TUNEL检测观察rhEPO对视网膜色素变性神经元的保护作用。应用免疫组织化学方法检测caspase2蛋白的表达。结果显示:(1)RCS大鼠给药组20d,25d,感光细胞的数目与对照组大鼠相比明显增加(P<0.05);(2)RCS大鼠给药组25dTUNEL检测阳性细胞数目与对照组相比,差异有统计学意义(P<0.05);(3)生后25d到40d,RCS大鼠给药组和对照组在节细胞层和内核层观察到caspase2阳性染色,RCS大鼠给药组在20d和25d内核层caspase2阳性细胞数目多于对照组(P<0.05)。结果提示:在RCS大鼠视网膜变性的早期,rhEPO对神经元起保护作用,可以使感光细胞得到更多存留;rhEPO对视网膜变性神经元的早期保护作用可能是通过抑制凋亡的方式进行的;caspase2蛋白在视网膜的一过性高表达提示其参与了视网膜感光细胞的凋亡过程,rhEPO可减少其早期的表达,对早期变性的神经元发挥保护作用。     

正常和视神经损伤后Nogo(N-18)在金黄地鼠视网膜的表达

目的 观察Nogo-A蛋白在金黄地鼠视神经受损后不同时间点视网膜各层的表达，探讨Nogo-A蛋白的分布。方法 采用眶内眼球后2mm视神经切断术，动物分别存活3d、5d、7d后，取眼球固定、冰冻后做水平切片，分别进行Nogo(N—18)抗体的免疫组织化学染色，显微镜观察Nogo-A蛋白的表达。结果 在视网膜各层均有不同程度的Nogo-A蛋白表达；存活3d、5d、7d后在节细胞层表达强烈，其中3d的反应最明显；随着存活时间的延长，Nogo—A蛋白表达的节细胞层细胞数量逐渐下降。结论 Nogo-A蛋白并非神经胶质所特有的分泌物质；视网膜Nogo-A蛋白表达的分布范围和表达程度与视神经受损后的时间相关。     

Immunohistochemical localization of protein kinase C-alpha in the retina of pigs during postnatal development

The cellular localization and protein expression level of protein kinase C (PKC)-alpha was examined in pig retina at different ages. Western blot analysis detected PKC-alpha in the retinas of 3-day-old piglets and indicated significantly increased expression in 6-month-old young adult and 2-year-old adult pigs. Immunohistochemistry of 3-day-old retinas revealed intense PKC-alpha reactivity in the inner plexiform and inner nuclear cell layers, weak reactivity in the ganglion cell layer, and few positive cells in the outer nuclear cell layer. The cellular localization of PKC-alpha in the adult retina was similar, with staining more intense than that in neonates. PKC-alpha was co-localized in some glial fibrillary acidic protein-positive cells and glutamine synthetase-positive cells in the retina. This study demonstrates that the protein level of retinal PKC-alpha is increased with maturation and suggests that PKC-alpha plays a role in signal transduction pathways for postnatal development in porcine retina.     

Immunohistochemical localization of protein kinase C (PKC) beta I in the pig retina during postnatal development

In order to investigate the expression of protein kinase C (PKC) beta I in the retinas of pigs during postnatal development, we analyzed retinas sampled from 3-day-old and 6-month-old pigs by Western blotting and immunohistochemistry. Western blot analysis detected the expression of PKC beta I in the retinas of 3-day-old piglets and it was increased significantly in the retinas of 6-month-old adult pigs. Immunohistochemical staining showed PKC beta I in the retinas of both groups. Immunohistochemistry of 3-day-old retinas revealed weak PKC beta I reactivity in the ganglion cell layer, inner plexiform layer, inner nuclear cell layer, outer plexiform layer and rod and cone cell layer. In the 6-month-old pig retina, the cellular localization of PKC beta I immunostaining was similar to that of the 3-day-old retina, where PKC beta I was localized in some glial fibrillary acidic protein-positive cells, glutamine synthetase-positive cells, parvalbumin-positive cells, and PKC alpha-positive cells in the retina. This is the first study to show the expression and cellular localization of PKC beta I in the retina of pigs with development, and these results suggest that PKC beta I, in accordance with PKC alpha, plays important roles in signal transduction pathways in the pig retina with development.     

急性高眼压后大鼠视网膜谷氨酰胺合成酶的表达变化

为了探索谷氨酰胺合成酶在青光眼视网膜中的表达变化及其可能作用,本实验用急性高眼压模型,结合免疫组织化学染色和Western blot检测了急性高眼压后大鼠视网膜中谷氨酰胺合成酶的表达。结果显示:正常视网膜中,谷氨酰胺合成酶免疫组织化学染色主要见于Muller细胞胞体;0 d组中,Muller细胞胞体染色稍淡,而内网层中表达增加,且呈明显的点状分布; 1d组、3 d组中Muller细胞胞体染色进一步变淡,但内网层中呈现弥散染色。至再灌第7 d、14 d,Muller细胞胞体又出现浓的染色。平均灰度值显示:与正常组相比,0 d组中谷氨酰胺合成酶表达有增加但差异无显著性;1 d组中表达显著增加,3 d组、7 d组表达逐渐减少,至第14 d时基本恢复正常。Western blot显示谷氨酰胺合成酶为一分子量约为45 kD的单一蛋白带,与其它组相比,1 d组中表达显著增加。提示:急性高眼压导致的视网膜缺血再灌早期,Muller细胞中谷氨酰胺合成酶的快速重新分布和表达上调可能加速了胞外谷氨酸的代谢,对缺血再灌条件下的视网膜特别是节细胞起到保护作用。     

Synapsin I expression in the rat retina during postnatal development

Summary The expression of the synapsin I gene was studied during postnatal development of the rat retina at the mRNA and protein levels. In situ hybridization histochemistry showed that synapsin I mRNA was expressed already in nerve cells in the ganglion cell layer of the neonatal retina, while it appeared in neurons of the inner nuclear layer from postnatal day 4 onward. Maximal expression of synapsin I mRNA was observed at P12 in ganglion cells and in neurons of the inner nuclear layer followed by moderate expression in the adult. At the protein level a shift of synapsin I appearance was observed from cytoplasmic to terminal localization during retinal development by immunohistochemistry. In early stages (P4 and P8), synapsin I was seen in neurons of the ganglion cell layer and in neurons of the developing inner nuclear layer as well as in the developing inner plexiform layer. In the developing outer plexiform layer synapsin I was localized only in horizontal cells and in their processes. Its early appearance at P4 indicated the early maturation of this cell type. A shift and strong increase of labelling to the plexiform layers at P12 indicated the localization of synapsin I in synaptic terminals. The inner plexiform layer exhibited a characteristic stratified pattern. Photoreceptor cells never exhibited synapsin I mRNA or synapsin I protein throughout development.Abbreviations GCL ganglion cell layer - INB inner neuroblast layer - INL inner nuclear layer - IPL inner plexiform layer - ONB outer neuroblast layer - ONL outer nuclear layer - OPL outer plexiform layer     

Expression profile of heat shock protein 108 during retinal development in the chick

In the developing chick retina, heat shock protein 108 (HSP108), which exhibits transferrin binding activity, has been demonstrated at the mRNA level, while transferrin shows two expression peaks. Here, we investigated the expression profile of HSP108 in the developing chick retina at the protein level. The localization of HSP108 in embryonic days 15 (E15), E18, and postnatal day 2 (P2) chick retina was examined immunohistochemically using monoclonal antibody 9G10 specific for chick HSP108, while the expression levels of HSP108 in developing chick retina from E12 to P2 and adult were measured by Western blot analysis. HSP108 was expressed in the ganglion cell layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, inner segments of photoreceptors and retinal pigment epithelium. Two peaks of HSP108 expression were found at around E13 and E18, respectively. Since the two HSP108 peaks appeared to be correlated with the transferrin expression peaks during retinal development, HSP108 may be associated with iron metabolism during the development of the retina.     

大鼠,金黄地鼠和家兔视网膜内一氧化氮合酶分布的比较

用ＮＡＤＰＨ黄递酶组织化学染色法观察了正常成年大鼠、金黄地鼠和家兔视网膜内一氧化氮合酶的分布，并比较了３种不同动物的区别。结果显示，在视网膜内ＮＯＳ阳性神经元主要为分布于内核层的无长突细胞、节细胞层的移位无长突细胞和少数节细胞，不同种类动物的视网膜内，ＮＯＳ阳性细胞的配布、密度和细胞形态均有差异。大鼠视网膜内ＮＯＳ阳性细胞多尾于内核层无长突细胞和节细胞层移位无长细胞，偶见于视网膜节细胞。金黄地鼠视     

视神经切断术后视网膜内信号转导子与转录激活子STAT3蛋白的表达变化

目的 探讨视神经损伤后，信号转导子与转录激活子(STAT3)蛋白在视网膜内表达和分布的变化。方法 免疫细胞化学、Westetn blot及计算机图像分析技术。结果 视神经切断术后，视网膜内STAT3表达水平上调，术后1d达到高峰，并出现核转位增多现象。5d后STAT3水平逐渐下降至正常。结论 Janus激酶一信号转导子与转录激活子(JAK-STAT)信号转导途径可能通过STAT3蛋白的活化，参与视神经切断后视网膜内的病理改变过程。     

Expression and cell localization of brain-derived neurotrophic factor and TrkB during zebrafish retinal development

Brain‐derived neurotrophic factor (BDNF) signaling through TrkB regulates different aspects of neuronal development, including survival, axonal and dendritic growth, and synapse formation. Despite recent advances in our understanding of the functional significance of BDNF and TrkB in the retina, the cell types in the retina that express BDNF and TrkB, and the variations in their levels of expression during development, remain poorly defined. The goal of the present study is to determine the age‐dependent changes in the levels of expression and localization of BDNF and TrkB in the zebrafish retina. Zebrafish retinas from 10 days post‐fertilization (dpf) to 180 dpf were used to perform PCR, Western blot and immunohistochemistry. Both BDNF and TrkB mRNAs, and BDNF and full‐length TrkB proteins were detected at all ages sampled. The localization of these proteins in the retina was very similar at all time points studied. BDNF immunoreactivity was found in the outer nuclear layer, the outer plexiform layer and the inner plexiform layer, whereas TrkB immunoreactivity was observed in the inner plexiform layer and, to a lesser extent, in the ganglion cell layer. These results demonstrate that the pattern of expression of BDNF and TrkB in the retina of zebrafish remains unchanged during postembryonic development and adult life. Because TrkB expression in retina did not change with age, cells expressing TrkB may potentially be able to respond during the entire lifespan of zebrafish to BDNF either exogenously administered or endogenously produced, acting through paracrine mechanisms.