游离锌离子在小鼠视网膜的定位研究

目的研究游离锌离子在小鼠视网膜的定位分布。方法应用ZnSe金属自显影技术(AMG)检测硒酸钠注射40 m in后小鼠视网膜内的锌离子。结果注射硒酸钠40 m in后发现游离锌离子主要分布于小鼠视网膜的色素上皮细胞层、光感受器的内节、外核层、外网层、内核层、内网层和神经节细胞层。在色素上皮细胞层、光感受器的内节和内核层与内网层交界处AMG阳性反应最为明显,在光感受器外节和神经纤维层几乎没有AMG阳性反应产物。结论小鼠视网膜内锌离子,在视网膜神经元视觉信息的传导和形成过程中可能起着重要作用。     

青光眼致病基因OPTN在大鼠光感受器细胞内的表达

目的观察OPTN在大鼠视网膜光感受器细胞内的表达及定位,探讨OPTN和NRL(neural retina leucine zipper)两蛋白相互的作用。方法制备正常Wistar大鼠的视网膜冷冻切片,用抗OPTN抗体、抗NRL抗体和抗CRX抗体分别进行多重免疫荧光染色。结果在外丛状层(OPL)、外核层(ONL)、光感受器细胞内节(IS)、神经节细胞层、内丛状层(IPL)和内核层(INL)中均观察到抗OPTN抗体不同强度的荧光信号。光感受器细胞内的荧光信号主要在细胞质内,而细胞核内未见染色,且具有视锥细胞特征的细胞未被抗OPTN抗体染色。结论 Optn(OPTN的大鼠同源基因)在除视锥细胞以外的光感受器细胞高表达。     

锌转运体ZNT7在小鼠视网膜的定位分布

目的研究锌转运体-7(zinc transporter 7,ZNT7)在小鼠视网膜的定位和分布。方法应用免疫组织化学技术检测CD-1小鼠视网膜内的ZNT7免疫反应产物的表达。结果ZNT7在小鼠视网膜内分布广泛,在神经节细胞和色素上皮细胞内ZNT7免疫阳性反应产物的表达最丰富,在无长突细胞和视神经纤维层中ZNT7免疫阳性反应产物为中等程度的表达,在内网层、外网层和光感受器外节中ZNT7免疫阳性染色较淡,在外核层和光感受器内节中ZNT7几乎没有表达。结论ZNT7可能在维持视网膜锌稳态过程中起到重要的作用。     

牛蛙视网膜内γ-氨基丁酸免疫阳性反应的分布

用免疫组织化学ABC法．研究了GABA免疫阳性反应在牛蛙视网膜的分布。证明光感受器内段（主要是视锥细胞）呈棕褐色的GABA反应;在外核层未见GABA标记的胞体,但在靠近外网层处偶见GABA标记的终末;在内核层,大量无长突细胞呈GABA反应阳性,并可鉴别出胞体染色较深和淡的两个亚群,一些双极细胞和个别水平细胞的胞体及它们的突起呈较弱的GABA反应阳性,偶见双极细胞轴突终末以膨体紧密贴附在GABA标记的无长突细胞上。在节细胞层,一些神经节细胞和散在的移位无长突细胞呈GABA反应阳性。此外．外网层和内网层均呈GABA反应阳性。上述结果表明,GABA广泛分布于牛蛙视网膜的各层,提示它在视觉信号的传递过程中发挥着重要作用。     

杞菊地黄汤防治MNU诱导大鼠视网膜变性的早期效应

目的:观察杞菊地黄汤在N-甲基-N-亚硝脲(MNU)诱导的SD大鼠视网膜变性早期的拮抗效应。 方法:生后46 d的雌性SD大鼠30只随机分为3组(n=10)：药物组以杞菊地黄汤灌胃，正常组和模型组以等量蒸馏水灌胃，每日1次，连续4 d。用药后第5 d，药物组和模型组大鼠皮下注射MNU 40 mg/kg，正常组皮下注射等量生理盐水做对照。注射后12 h，处死大鼠，每组大鼠的左眼（10眼）用于透射电镜观察；右眼（10眼）用于苏木素-伊红(hematoxylin eosin,HE)染色观察并测量分析视网膜全层及外核层厚度，部分眼球行TUNEL法染色并计算细胞凋亡百分率。 结果:光镜下各组大鼠视网膜组织学未见明显改变，视网膜全层及外核层厚度组间比较均无显著差异（P>0.05）。TUNEL法染色见模型组和药物组外核层存在细胞凋亡，正常组未见。电镜下正常组和药物组光感受器细胞大小和核染色质分布均匀；模型组光感受器细胞开始变小，核染色质开始浓缩, 外节膜盘疏松，膜型不完整。 结论:杞菊地黄汤通过抑制细胞凋亡等选择性拮抗MNU对大鼠视网膜光感受器细胞的早期损伤。     

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,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     

大鼠视网膜内髓鞘形成模型的建立与研究

目的研究体外培养的大鼠纹状体神经干细胞在植入同种视网膜后,向少突胶质细胞分化并产生髓鞘的过程,观察髓鞘形成对视网膜结构的可能影响,建立一种中枢神经髓鞘体内发育的新模型.方法将体外传代培养的胚胎纹状体神经干细胞植入新生大鼠的玻璃体腔内,在移植后不同时期观察视网膜内髓鞘的出现部位以及扩展趋势,利用不同染色方法对标本进行检验分析,利用透射电镜进行超微结构观察.结果移植细胞4周后部分视网膜内开始出现成束髓鞘,只分布于神经纤维层.髓鞘束出现的比例、分布面积和形态变化与移植后动物的存活时间相关.电镜观察可见节细胞轴突外包绕有结构正常的中枢神经样髓鞘.较厚的髓鞘束对视网膜节细胞的分布产生一定影响.结论大鼠纹状体神经干细胞可在同种视网膜内向成熟少突胶质细胞分化,视网膜神经纤维层具有促使髓鞘形成的作用;本实验可能为研究少突胶质细胞分化及髓鞘生成机制提供新的在体模型.     

硫酸软骨素酶联合脂肪间充质干细胞治疗大鼠视网膜变性北大核心

背景:有研究发现硫酸软骨素酶降解硫酸软骨素蛋白多糖能够促使视网膜上Müller细胞的移行,但硫酸软骨素酶降解硫酸软骨素蛋白多糖是否能促进脂肪间充质干细胞在视网膜变性大鼠视网膜的移行尚不明确。目的:探讨硫酸软骨素酶降解硫酸软骨素蛋白多糖对脂肪间充质干细胞治疗大鼠视网膜变性的影响。方法:分离并培养人脂肪间充质干细胞,建立视网膜变性大鼠模型,向视网膜变性大鼠视网膜下腔注射脂肪间充质干细胞+硫酸软骨素酶,观察移植后大鼠脂肪间充质干细胞迁移率和视网膜细胞凋亡情况。结果与结论:人脂肪间充质干细胞能够成功培养,Brd U对人脂肪间充质干细胞的标记率达90.0%以上。建模后7 d,视网膜外核层塌陷,光感受器细胞大量外节迸解,外核层贴附在Bruch’s膜上,视网膜呈拱桥样,中央视网膜和外周视网膜均受到损伤;正常大鼠视网膜各层清晰,光感受器细胞排列规律,视网膜色素上皮层完整。脂肪间充质干细胞+硫酸软骨素酶组脂肪间充质干细胞迁移率高于脂肪间充质干细胞组,且2组视网膜细胞凋亡率比较差异无显著性意义。表明硫酸软骨素酶降解硫酸软骨素蛋白多糖可提高人脂肪间充质干细胞在视网膜上的迁移能力。     

Rhodopsin和S-opsin在MNU诱导的大鼠视网膜损伤过程中的表达变化

目的:观察在N-甲基-N亚硝酸脲(MNU)诱导的大鼠视网膜外核层细胞损伤过程中视紫质(rhodopsin)和蓝光视蛋白(blue-sensitive opsin/S-opsin)在视网膜中的表达变化,分析其与MNU诱导的视网膜损伤的关系。方法:将30只SPF级50 d龄雌性SD大鼠随机分为正常对照组和MNU模型1、3、7、10 d组,每组各6只大鼠。模型组以腹腔注射MNU(40 mg/kg)建立MNU模型;正常对照组大鼠腹腔注射生理盐水(5 mL/kg)。右眼冰冻切片行苏木素-伊红(HE)染色判断视网膜损伤程度。通过逆转录-聚合酶链反应(RT-PCR)和免疫荧光检测各组视网膜中rhodopsin和S-opsin的mRNA及蛋白表达情况。结果:病理检测确定造模结果与以往实验一致,各组病理分级之间的差异显著(2=16.838,P0.01)。RT-PCR检测结果表明,与正常对照组相比,各MNU模型组rhodopsin和S-opsin的mRNA表达水平随MNU作用时间增加而逐渐降低且差异均显著(rhodopsin 1 d组P0.05;S-opsin 1 d组P0.01;3、7、10 d组rhodopsin和S-opsin均P0.01)。免疫荧光检测结果显示,在正常大鼠视网膜rhodopsin主要在光感受器细胞外段表达,MNU作用后rhodopsin主要在外核层表达,少量在内核层表达,并随MNU作用时间增加而逐渐表达降低。S-opsin在正常视网膜各层均有表达,在各模型组随MNU作用时间增加S-opsin的表达逐渐降低,在外核层和光感受器细胞内、外段尤为明显。结论:MNU诱导的视网膜外核层细胞损伤可降低rhodopsin和S-opsin的表达,并与MNU选择性引起光感受器细胞丧失有关。     

Ionotropic purinergic receptors P2X in frog and turtle retina: Glial and neuronal localization

Purinergic signaling is represented in both the peripheral and central nervous system (CNS), and in particular in the retina, which may be regarded as a part of the CNS. While purigenic signaling is relatively well studied in mammalian retinas, little is known about it in retinas of lower vertebrates. The aim of present study was to investigate, using immunocytochemistry, the distribution of purinoreceptors P2X in retinas of frog and turtle, which are appropriate models of the brain neuron-to-glia interactions. The results showed widespread expression of all seven ionotropic purinoreceptors (P2X1–P2X7) in both frog and turtle retinas. They were predominantly expressed in Müller cells, the principal glial cells in the retina. All structures typical of Müller cells: the outer and the inner limiting membranes, the cells bodies in the inner nuclear layer, the radial processes in the inner plexiform layer (IPL), and the so called endfeet (frog) or the orthogonal arrays of particles (turtle) in the ganglion cells layer were immunostained. Colocalizations between P2X1–P2X7 and the glial cell marker Vimentin proved that the immunostaining was in the Müller cells. In addition to the glial staining, neuronal staining was also seen as fine puncta in the inner plexiform layer and by small dots and patches in the outer plexiform layer. Some cell bodies of horizontal, amacrine and ganglion cells were also stained. The results obtained imply that the purinergic P2X receptors may significantly contribute to the neuron-to-glia signaling in retinas of the lower vertebrates.     

Ultramicroscopical immunolocalization of PAX6 in the adult chicken retina

Cell type-specific PAX6 protein expression was examined in all retinal layers of the normal chicken retina. The most intense PAX6 immunostaining was found in the ganglion cell and inner nuclear layers, and in lower amounts in the optic nerve fiber, the inner plexiform and the photoreceptor layers. PAX6 immunostaining was variable in terms of its subcellular localization, even within one cell. PAX6 immunostaining was mainly localized in nuclear heterochromatin of the ganglion cell and inner nuclear layers whereas in the outer nuclear layer, PAX6 immunostaining was only observed in the intercellular space and the cytoplasm. In photoreceptors, the myoid portion of the inner segment showed PAX6 immunostaining, but the ellipsoid portion and the outer segment did not. The ultrastructural distribution pattern of PAX6 in the adult chicken retina suggests that normal expression of PAX6 is variable even in subcellular structures in the same cell type.     

Microtubules in synapses of the retina

Summary Using a new method, microtubules can be seen running up to, and lying in close relationship with, the synaptic ribbons in the outer and inner plexiform layers of the frog retina. In the inner plexiform layer microtubules can be seen running up to the terminal membrane in the non-ribbon synapses. Unlike non-ribbon C.N.S. synapses (frog and rat) processed by the same method, there is no clear association between synaptic vesicles and microtubules in the approach regions.     

Dopamine- and adenosine-3':5'-monophosphate (cAMP)-regulated phosphoprotein of Mr 32,000 (DARPP-32) in the retina of cat, monkey and human.

The cellular localization of a dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32) was investigated in cat, monkey and human retina by immunohistochemistry. In cat, DARPP-32-immunoreactive cell bodies identified as Müller cells were demonstrated in the inner nuclear layer (INL) with processes closely surrounding the cell soma of photoreceptors in the outer nuclear layer. Some DARPP-32-IR cells were also seen in the nerve fiber layer (NFL) sending processes to the inner plexiform layer. In monkey and human retina, DARPP-32-IR cell bodies were also demonstrated in the INL, with few cells located in the NFL.     

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     

高血压大鼠视网膜组织Ca^2＋酸性磷酸酶组织化学的研究

目的：应用光镜定量酶组织化学方法对正常京都种大鼠（ＷＫＹ）和自发性高血压大鼠（ＳＨＲ）视网膜组织的Ｃａ＾２＋－酸性磷酸酶的分布和活性进行定量观察，结果：Ｃａ＾２＋酸性磷酸酶在ＷＫＹ视网膜组织的活性由强到弱依次为（Ｆ检验，Ｐ〈０．０５）；（１）杆锥细胞内节和外核层；（２）节细胞层；（３）内核层；（４）内网层和外网层；（５）杆锥细胞外节阴性，在ＳＨＲ视网膜组织中，各层Ｃａｓ＾２＋酸性磷酸酶活性下降，以