人胎视网膜神经肽Y免疫反应神经元的发育

本文用免疫细胞化学ＡＢＣ法,研究１５—３８周龄人胎视网膜神经肽Ｙ免疫反应（ＮｅｕｒｏｐｅｐｔｉｄｅＹｉｍｍｕｎｏｒｅｃｔｉｖｅ,ＮＰＹ－ＩＲ）神经元（以下称ＮＰＹ－ＩＲ细胞）的发育。结果表明：①胎龄１５周视网膜中央部已出现不同类型的ＮＰＹ－ＩＲ细胞：位于黄斑及其周围外核层的为ＮＰＹ－ＩＲ视锥细胞;位于内核层最内一列的为ＮＰＹ－ＩＲ无长突细胞位于节细胞层的可能为ＮＰＹ－ＩＲ移位无长突细胞或节细胞;内核层和节细胞层的ＮＰＹ－ＩＲ细胞的突起均分布在内网层的第１亚层。②胎龄２４周后,ＮＰＹ－ＩＲ视锥细胞完全消失。③随着视网膜的发育,内核层和节细胞层的ＮＰＹ－ＩＲ细胞数量增多,突起增粗增长,胞体分布由中央部扩展到周边部,其中内核层ＮＰＹ－ＩＲ细胞的密度呈现从中央部向周边部逐渐降低的分布方式,节细胞层ＮＰＹ－ＩＲ细胞则多数集中分布在视网膜的边缘和黄斑之间,形成较高密度的环状区。     

黑斑蛙视网膜3种神经内分泌细胞的免疫组织化学定位

目的研究神经肽Y(NPY)、五羟色胺(5-HT)和胰高血糖素(GLU)免疫阳性细胞在黑斑蛙(Rananigromaculata)视网膜上的组织学定位。方法应用过氧化物酶标记的链霉亲和素(SP法)免疫组织化学技术,并结合生物统计学分析。结果NPY细胞主要分布于内核层和节细胞层。内核层中出现两种阳性细胞,一种出现在第2、3亚层,常为多个细胞聚集;另一种出现在内侧,有突起伸入内网层。节细胞层阳性细胞分布较少,胞体有大小之分。5-HT细胞主要分布于内核层和节细胞层,位于内核层中邻近内网层一侧的阳性细胞有突起延伸入内网层。GLU细胞分布于外核层、内核层内侧以及节细胞层。结果 黑斑蛙视网膜上NPY、5-HT和GLU细胞的分布与其它物种有相似之处,也有其自身特点,符合其晨昏性生活习性。     

脑红蛋白在家兔视网膜中的分布

目的探讨脑红蛋白(NGB)在家兔视网膜的分布特征。方法选择健康成年家兔5只,利用免疫组织化学染色SP法,观察NGB在家兔视网膜中的分布情况。结果 NGB在家兔视网膜的视神经纤维层、节细胞层、内网状层、外网状层、光感受器内节段和色素上皮层中有强阳性表达,在视网膜的内核层有弱阳性表达,在视网膜的外核层和光感受器外节段中未见有阳性表达,内界膜、外界膜和视神经中亦发现有NGB阳性表达。家兔视网膜NGB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。结论除外核层和光感受器外节段外,NGB在家兔视网膜其它各层中均有表达,提示NGB在维持视网膜氧代谢平衡中发挥着重要作用。     

人胎视网膜含NOS神经元的发育

本文用一氧化氮合酶（ＮＯＳ）的组织化学方法对胎龄１５周至３６周的人胎视网膜含ＮＯＳ神经元的发育进行了研究。胚胎１５周视网膜颞侧半少部分神经元即有ＮＯＳ的表达,２０周视网膜含ＮＯＳ神经元数密度达峰值。大部分含ＮＯＳ神经元胞体位于内核层内带,只少部分位于节细胞层,其突起均分布于内同层,形成内同层的１、３、５亚层。含ＮＯＳ神经元的形态各异,依据其突起的多少分Ⅰ、Ⅱ、Ⅲ等三种类型,其中Ⅱ、Ⅲ型含ＮＯＳ神经元在２８周以后才开始出现,且愈近晚期胎龄,它们所占含ＮＯＳ神经元的数量比呈上升趋势。随视网膜发育成熟,含ＮＯＳ神经元胞体均面积呈不断增大的变化。本文结果显示：人胎视网膜内核层含ＮＯＳ神经元为无长突细胞,节细胞层的Ⅰ型含ＮＯＳ神经元为移位无长突细胞,推测它们在视网膜的发育过程中对内网层突触的形成与修饰可能起有重要作用。     

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。     

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.     

胚胎大鼠视网膜超微结构与Nestin表达的增龄变化

目的研究在体视网膜祖细胞分布及分化发育规律. 方法运用透射电镜观察E18(E:胚胎)视网膜超微结构,采用免疫组化了解Nestin在不同发育时期视网膜的表达变化.结果 E18视网膜超微结构:①在神经母细胞层中、外侧,大多数细胞核浆比例大,核呈纺锤形或椭圆形,染色较深,常染色质细小,异染色质少,胞质内含丰富核糖体及少量线粒体.②在近巩膜侧的神经母细胞层可见有丝分裂细胞.2. Nestin表达丰富的部位主要集中在E16和E18的神经母细胞层;P4和P7(P:出生后)发育期内网层和神经纤维层,但在P21,Nestin在视网膜包括睫状体边缘区均呈阴性.结论 1.E18视网膜祖细胞主要位于神经母细胞层.2. Nestin阳性细胞产生视网膜细胞的顺序是先产生节细胞,感光细胞和内核层细胞次之,最后为米勒细胞.     

环境无机汞对泥鳅视网膜S100表达影响

用含低浓度Hg2 的水饲养泥鳅(Misgurnus anguillicaudatus),研究Hg2 污染对视网膜中S100免疫反应阳性结构数量及分布的影响。用免疫组织化学ABC法标记S100免疫阳性(S100-IR)细胞,显微镜下观察S100免疫反应阳性细胞并计数。结果显示,处理组S100首先在神经节细胞层(GCL)、神经纤维层(NFL)表达,然后逐渐在其他各层表达。与对照组相比,处理组S100免疫反应阳性细胞数显著增多,且该变化与环境中无机汞处理时间成正相关;阳性细胞胞体明显膨胀,突起稠密粗大,S100阳性反应强。研究提示,Hg2 的神经毒害作用导致泥鳅视网膜中S100-IR细胞数的增多。推测Hg2 污染可能对视网膜有伤害性影响,而神经胶质细胞在视网膜修复过程中起了重要作用。     

胎儿下颌髁状突发育中软骨细胞凋亡及bcl-2的表达

目的 研究不同胎龄的胎儿下颌髁状突软骨发育中的细胞凋亡及bcl-2蛋白的表达。方法 32例胎儿下颌髁状突软骨按胎龄分为两组：A组（13-22周,17例）;B组（23-33周,15例）,采用原位末端标记法（TUNEL）及免疫组织化学法观察胎儿髁状突软骨发育不同时期细胞凋亡及bcl-2蛋白的表达,并对表达结果作定量分析。结果 两胎龄组中均有TUNEL及bcl-2阳性表达,凋亡细胞A组较B组多,凋亡细胞主要分布于增殖层及成软骨细胞层,肥大层较少;bcl-2阳性细胞主要分布于成软骨细胞层,其次为增殖层,肥大层阳性细胞明显减少,B组bcl-2阳性率高于A组。结论 细胞凋亡参与胎儿下颌髁状突软骨发育,bcl-2与髁状突软骨细胞分化及细胞凋亡有关。     

多巴胺受体1在皖西白鹅小脑皮质发育中的表达

采用普通染色及免疫组化SABC染色法研究皖西白鹅小脑皮质的发育和多巴胺受体1(DRD1)阳性细胞在其发育中的表达.结果表明,小脑皮质在胚龄13 d(E13)由外向内分为外颗粒层(EGL)、浦肯野细胞层(PCL)和内颗粒层(IGL),E19由外向内分为EGL、分子层(ML)、PCL和IGL.随发育天数的增加,EGL的厚度和细胞层次呈先升后降的变化趋势,细胞密度逐渐下降;ML厚度逐渐增大,在E24到E28时增值最大;浦肯野细胞(PC)在E13、E19、E24和E28时随胚龄增大逐渐增大,在E28后趋于稳定,细胞密度随着发育天数的增加逐渐下降,在小脑皮质发育中还发现有一部分PC呈多层排列,且细胞层次逐渐变少;IGL厚度呈先升后降的变化趋势,细胞密度呈上升趋势.外颗粒层和内颗粒层在E13、E19、E24和E28时有DRD1阳性细胞表达,分子层在E24、E28、日龄7 d(P7)和15d(P15)有阳性细胞表达,PC在所检测的6个时段均有阳性表达.研究表明,小脑皮质的发育主要与细胞增殖、迁移和凋亡有关,外颗粒层的逐渐消失是以细胞迁移和凋亡为主,多层PC逐渐退化成单层是与细胞凋亡和正常突触联系的建立有关;DRD1在皖西白鹅小脑皮质发育中对外颗粒层细胞和PC起着重要作用.     

Localization of glutamate in the human retina during early prenatal development

In this study we have localized glutamate (GLU) in fetal (14–25 weeks gestation, Wg) human retinas by immunohistochemistry. At 14 Wg, GLU-immunoreactivity (IR) was localized only in the central part of retina, showing a prominently labelled nerve fiber layero A few ganglion cells and displaced amacrine cells were very weakly labelled. At 17 Wg, GLU was localized conspicuously in many ganglion cells, displaced amacrine cells, some amacrine cells and the prospective photoreceptor cell bodies in the neuroepithelial layero With progressive development at 20 and 25 Wg, the IR for GLU was found additionally in the Müller cell endfeet, some bipolar cells as well as in the horizontal cells that were aligned in a row along the outer border of the inner nuclear layer of the central retinao The photoreceptor cell bodies in the outer nuclear layer were also prominently immunopositive for GLU. The developmental distribution of GLU in the human retina tends to indicate that it plays an important role in the differentiation and maturation of retinal neurons.     

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.     

四指马鲅视网膜早期发育的组织学研究

本文采用石蜡连续切片技术、H.E染色和显微测量法,对四指马鲅(Eleutheronema tetradactylum)早期发育过程中视网膜的结构、分化和形成过程以及视觉特性进行了研究。结果显示,受精后8 h54 min,视杯已经形成。初孵仔鱼视网膜没有分化。2日龄仔鱼可以清晰的辨认出色素上皮层、外核层、内核层和神经节细胞层。3日龄仔鱼内核层已经分化出水平细胞、双极细胞和无长突细胞。4日龄仔鱼视网膜10层结构完整。9日龄至14日龄,外核层胞核数目与神经节细胞数目的比值增大,视网膜会聚程度升高,是该鱼视觉特性发生变化的过渡期,这与其从浮游到浅海中下层和泥沙质海底活动的生态迁移相适应。在生长发育的早期阶段,其视网膜内核层水平细胞仅有1到2层,属于感光系统不甚发达的类型。该鱼在仔鱼浮游生活阶段,视敏度较高,视觉对其行为和摄食活动具有重要作用,适应生活于光照较充足的环境中,转入浅海中下层和泥沙质海底后,光敏度和视敏度均较差,视觉在其行为和摄食活动中不具有主要作用。     

Differential expression of syntaxin-1 and synaptophysin in the developing and adult human retina

Synaptophysin and syntaxin-1 are membrane proteins that associate with synaptic vesicles and presynaptic active zones at nerve endings, respectively. The former is known to be a good marker of synaptogenesis; this aspect, however, is not clear with syntaxin-1. In this study, the expression of both proteins was examined in the developing human retina and compared with their distribution in postnatal to adult retinas, by immunohistochemistry. In the inner plexiform layer, both were expressed simultaneously at 11–12 weeks of gestation, when synaptogenesis reportedly begins in the central retina. In the outer plexiform layer, however, the immunoreactivities were prominent by 16 weeks of gestation. Their expression in both plexiform layers followed a centre-to-periphery gradient. The immunoreactivities for both proteins were found in the immature photoreceptor, amacrine and ganglion cells; however, synaptophysin was differentially localized in bipolar cells and their axons, and syntaxin was present in some horizontal cells. In postnatal-to-adult retinas, synaptophysin immunoreactivity was prominent in photoreceptor terminals lying in the outer plexiform layer; on the contrary, syntaxin-1 was present in a thin immunoreactive band in this layer. In the inner plexiform layer, however, both were homogeneously distributed. Our study suggests that (i) syntaxin-1 appears in parallel with synapse formation; (ii) synaptogenesis in the human retina might follow a centre-to-periphery gradient; (iii) syntaxin-1 is likely to be absent from ribbon synapses of the outer plexiform layer, but may occur at presynaptic terminals of photoreceptor and horizontal cells, as is apparent from its localization in these cells, which is hitherto unreported for any vertebrate retina.     

Retinal neurogenesis: the formation of the initial central patch of postmitotic cells

We have investigated the relationship between the birthdate and the onset of differentiation of neurons in the embryonic zebrafish neural retina. Birthdates were established by a single injection of bromodeoxyuridine into embryos of closely spaced ages. Differentiation was revealed in the same embryos with a neuron-specific antibody, zn12. The first bromodeoxyuridine-negative (postmitotic) cells occupied the ganglion cell layer of ventronasal retina, where they formed a small cluster of 10 cells or less that included the first zn12-positive cells (neurons). New cells were recruited to both populations (bromodeoxyuridine-negative and zn12-positive) along the same front, similar to the unfolding of a fan, to produce a circular central patch of hundreds of cells in the ganglion cell layer about 9 h later. Thus the formation of this central patch, previously considered as the start of retinal neurogenesis, was actually a secondary event, with a developmental history of its own. The first neurons outside the ganglion cell layer also appeared in ventronasal retina, indicating that the ventronasal region was the site of initiation of all retinal neurogenesis. Within a column (a small cluster of neuroepithelial cells), postmitotic cells appeared first in the ganglion cell layer, then the inner nuclear layer, and then the outer nuclear layer, so cell birthday and cell fate were correlated within a column. The terminal mitoses occurred in three bursts separated by two 10-h intervals during which proliferation continued without terminal mitoses.     

Changing patterns of ganglion cell coupling and connexin expression during chick retinal development

We have used dye injection and immunolabeling to investigate the relationship between connexin (Cx) expression and dye coupling between ganglion cells (GCs) and other cells of the embryonic chick retina between embryonic days 5 and 14 (E5-14). At E5, GCs were usually coupled, via soma-somatic or dendro-somatic contacts, to only one or two other cells. Coupling increased with time until E11 when GCs were often coupled to more than a dozen other cells with somata in the ganglion cell layer (GCL) or inner nuclear layer (INL). These coupled clusters occupied large areas of the retina and coupling was via dendro-dendritic contacts. By E14, after the onset of synaptogenesis and at a time of marked cell death, dye coupling was markedly decreased with GCs coupled to three or four partners. At this time, coupling was usually to cells of the same morphology, whereas earlier coupling was heterogeneous. Between E5 and E11, GCs were sometimes coupled to cells of neuroepithelial morphology that spanned the thickness of the retina. The expression of Cx 26, 32, and 43 differed and their distribution changed during the period studied, showing correlation with events such as proliferation, migration, and synaptogenesis. These results suggest specific roles for gap junctions and Cx's during retinal development.     

Comparative immunolocalization of the plasma membrane calcium pump and calbindin D28K in chicken retina during embryonic development

The immunolocalization of the plasma membrane calcium pump (PMCA) was studied in 4-week-old chick retina in comparison with calbindin D28K (CaBP) immunostaining. We have demonstrated that the monoclonal anti-PMCA antibody SF10 from human erythrocyte plasma membrane cross-reacts with a Ca2+ pump epitope of the cells from the neural retina. The immunolocalization of both proteins was also studied during the embryonic development of the chicken retina. At age 4.5 days, the cells of the retina were faintly immunoreactive to PMCA and CaBP antibodies, but the lack of cellular aggregation and differentiation did not allow discrimination between the two proteins. A clear difference in the localization was seen from the tenth day of development through post-hatching with slight variation. PMCA localized mainly in the outer and inner plexiform layers, in some cells in the ganglion layer, in the nerve fiber layer and slightly in the photoreceptor cells. CaBP was intensely stained in cones, cone pedicles and some amacrine cells. The number of CaBP positive amacrine cells declined after hatching. A few ganglion cells and several nerve fibers were CaBP immunoreactive. The role of these proteins in the early stages of retinal development is unknown, but the results suggest that Ca2+ homeostasis in the retina is well regulated, probably to avoid excessive accumulation of Ca2+, which often leads to neurodegeneration.