Quantitative morphometry of perifoveal capillary networks in the human retina

Purpose. To quantify the distribution and morphometric characteristics of capillary networks in the human perifovea. To determine correlations between the location of neuronal subcellular compartments and the morphometric features of regional capillary networks in the layered retina. Methods. The perifoveal region, located 2 mm nasal to the fovea, was studied in 17 human donor eyes. Novel micropipette technology was used to cannulate the central retinal artery and label the retinal microcirculation using a phalloidin perfusate. γ-synuclein, Goα, and parvalbumin antibodies were also used to co-localize the nerve fiber layer (NFL), retinal ganglion cell layer (RGCL), inner plexiform layer (IPL), and inner nuclear layer (INL). Confocal scanning laser microscopy was used for capillary imaging. Capillary diameter, capillary density, and capillary loop area measurements were compared between networks. Results. Four capillary networks were identified in the following retinal layers: (1) NFL, (2) RGCL and superficial portion of IPL, (3) deep portion of IPL and superficial portion of INL, and (4) deep portion of INL. Laminar configurations were present in NFL and deep INL networks. Remaining networks demonstrated three-dimensional configurations. Capillary density was greatest in the networks serving the IPL. Capillary loop area was smallest in the two innermost networks. There was no difference in capillary diameter between networks. Conclusions. Capillary networks in the human perifovea are morphometrically heterogeneous. Morphometric features of regional capillary networks in the layered retina may serve a critical role in supporting neuronal homeostasis. Improved knowledge of these features may be important for understanding pathogenic mechanisms underlying retinal vascular diseases.     

Morphometric and Microstructural Changes During Murine Retinal Development Characterized Using In Vivo Optical Coherence Tomography

PurposeThe purpose of this study was to develop an in vivo optical coherence tomography (OCT) system capable of imaging the developing mouse retina and its associated morphometric and microstructural changes.MethodsThirty-four wild-type mice (129S1/SvlmJ) were anesthetized and imaged between postnatal (P) day 7 and P21. OCT instrumentation was developed to optimize signal intensity and image quality. Semi-automatic segmentation tools were developed to quantify the retinal thickness of the nerve fiber layer (NFL), inner plexiform layer (IPL), inner nuclear layer (INL), and the outer retinal layers (ORL), in addition to the total retina. The retinal maturation was characterized by comparing layer thicknesses between consecutive time points.ResultsFrom P7 to P10, the IPL increased significantly, consistent with retinal synaptogenesis. From P10 to P12, the IPL and ORL also increased, which is coherent with synaptic connectivity and photoreceptor maturation. In contrast, during these periods, the INL decreased significantly, consistent with cellular densification and selective apoptotic “pruning” of the tissue during nuclear migration. Thereafter from P12 to P21, the INL continued to thin (significantly from P17 to P21) whereas the other layers remained unchanged. No time-dependent changes were observed in the NFL. Overall, changes in the total retina were attributed to those in the IPL, INL, and ORL. Regions of the retina adjacent to the optic nerve head were thinner than distal regions during maturation.ConclusionsChanges in retinal layer thickness are consistent with retinal developmental mechanisms. Accordingly, this report opens new horizons in using our system in the mouse to characterize longitudinally developmental digressions in models of human diseases.     

Expression of the gamma-aminobutyric acid (GABA) plasma membrane transporter-1 in monkey and human retina

PURPOSE: To determine the expression pattern of the predominant gamma-aminobutyric acid (GABA) plasma membrane transporter GAT-1 in Old World monkey (Macaca mulatta) and human retina. METHODS: GAT-1 was localized in retinal sections by using immunohistochemical techniques with fluorescence and confocal microscopy. Double-labeling studies were performed with the GAT-1 antibody using antibodies to GABA, vasoactive intestinal polypeptide (VIP), tyrosine hydroxylase (TH), and the bipolar cell marker Mab115A10. RESULTS: The pattern of GAT-1 immunostaining was similar in human and monkey retinas. Numerous small immunoreactive somata were in the inner nuclear layer (INL) and were present rarely in the inner plexiform layer (IPL) of all retinal regions. Medium GAT-1 somata were in the ganglion cell layer in the parafoveal and peripheral retinal regions. GAT-1 fibers were densely distributed throughout the IPL. Varicose processes, originating from both the IPL and somata in the INL, arborized in the outer plexiform layer (OPL), forming a sparse network in all retinal regions, except the fovea. Sparsely occurring GAT-1 processes were in the nerve fiber layer in parafoveal regions and near the optic nerve head but not in the optic nerve. In the INL, 99% of the GAT-1 somata contained GABA, and 66% of the GABA immunoreactive somata expressed GAT-1. GAT-1 immunoreactivity was in all VIP-containing cells, but it was absent in TH-immunoreactive amacrine cells and in Mab115A10 immunoreactive bipolar cells. CONCLUSIONS: GAT-1 in primate retinas is expressed by amacrine and displaced amacrine cells. The predominant expression of GAT-1 in the inner retina is consistent with the idea that GABA transporters influence neurotransmission and thus participate in visual information processing in the retina.     

Changes of GABA metabolic enzymes in acute retinal ischemia.

It is reported that GABA accumulates in Müller cells in ischemic and diabetic rat retina. To investigate the mechanism of GABA accumulation in Müller cells, we localized GABA and glutamate in ischemic rat retina and measured the activity of GAD and GABA-T, enzymes involved in GABA metabolism. Using general anesthesia, we incised the bulbar conjunctiva of the rat around the limbus and clamped the left optic nerve. A sham operation was performed on the right eyes. Ocular ischemia was sustained for 30, 60 and 90 minutes. Rat eyes were enucleated immediately after ischemia and prepared for immunohistochemistry and enzyme activity measurement. Glutamate-like immunoreactivity (Glu-IR) in the sham-operated rat retina was observed in all retinal layers, showing intense staining in the nerve fiber layer (NFL), ganglion cell layer (GCL), and inner plexiform layer (IPL). Glu-IR increased in the outer plexiform layer (OPL) and outer nuclear layer (ONL) in an ischemic time-dependent manner. GABA-like immunoreactivity (GABA-IR) in sham-operated rat retina was observed in NFL, GCL, IPL and inner nuclear layer (INL). When the ischemic time was extended, GABA-IR intensely stained Müller cells. GAD activity was not changed in ischemic rat retina as compared to normal rat retina, but GABA-T activity was significantly decreased in ischemic rat retina. These results suggested that glutamate was induced by ischemia and was converted to GABA by GAD activity. Increased GABA was not metabolized because GABA-T activity was decreased. GABA accumulation in Müller cells progressed during the change in activity of these metabolic enzymes.     

Quantification of retinal transneuronal degeneration in human glaucoma: a novel multiphoton-DAPI approach

PURPOSE: Glaucoma is presumed to result in the selective loss of retinal ganglion cells. In many neural systems, this loss would initiate a cascade of transneuronal degeneration. The quantification of changes in neuronal populations in the middle layers of the retina can be difficult with conventional histologic techniques. A method was developed based on multiphoton imaging of 4',6'-diamino-2-phenylindole (DAPI)-stained tissue to quantify neuron loss in postmortem human glaucomatous retinas. METHODS: Retinas from normal and glaucomatous eyes fixed in 4% paraformaldehyde were incubated at 4 degrees C overnight in DAPI solution. DAPI-labeled neurons at different levels of the retina were imaged by multiphoton confocal microscopy. Algorithms were developed for the automated identification of neurons in the retinal ganglion cell layer (RGCL), inner nucleus layer (INL), and outer nuclear layer (ONL). RESULTS: In glaucomatous retinas, the mean density of RGCs within 4 mm eccentricity was reduced by approximately 45%, with the greatest RGC loss occurring in a region that corresponds to the central 6 degrees to 14 degrees of vision. Significant neuron loss in the INL and ONL was also seen at 2 to 4 mm and 2 to 3 mm eccentricities, respectively. The ratios of neuron densities in the INL and ONL relative to the RGCL (INL/RGC and ONL/RGC, respectively) were found to increase significantly at 3 to 4 mm eccentricity. CONCLUSIONS: The data confirm that the greatest neuronal loss occurs in the RGCL in human glaucoma. Neuronal loss was also observed in the outer retinal layers (INL and ONL) that correlated spatially with changes in the RGCL. Further work is necessary to confirm whether these changes arise from transneuronal degeneration.     

Inhibition of cyclooxygenase-2 expression by zinc-chelator in retinal ischemia

The zinc ion (Zn2+) is abundant in neurons. However, excessive Zn2+ can induce neuronal cell death. This study examined the role of Zn2+ in transient retinal ischemia in adult male rats. The rats were sacrificed 4-24 h after retinal ischemia by high intra-ocular pressure, and the retinas were prepared for microscopic examination of retinal cell degeneration, and fluorescence microscopy using zinquin ethyl ester as the zinc ion-specific probe. Moreover, COX-2 expression was observed by Western blotting. In control retinas, there was a low Zn2+ concentration in the inner plexiform layer (IPL), a high Zn2+ concentration in the outer plexiform layer (OPL), and no detectable Zn2+ in either the ganglion cell layer (GCL) or the inner nuclear layer (INL). In contrast, in the retinas exposed to ischemia without the administration of the zinc ion chelators (Ca2+-EDTA and TPEN), Zn2+ deposits were found in the IPL and INL beginning 4 h after ischemia and degeneration of neurons was found in the GCL and INL. Less Zn2+ accumulation in the IPL and INL and less neuronal degeneration in the GCL and INL were found in the retinas treated with Ca2+-EDTA or TPEN before ischemia. Furthermore, the COX-2 protein levels increased 4-8 h after retinal ischemia, and chelation of zinc ion inhibited this effect. These results suggest that the accumulation of Zn2+ following an ischemic insult can cause retinal degeneration and induce abnormal COX-2 expression.     

Chronic bilateral common carotid artery occlusion: a model for ocular ischemic syndrome in the rat

Background Ocular ischemic syndrome is a devastating eye disease caused by severe carotid artery stenosis. The reduction of blood flow produced by bilateral common carotid artery occlusion (BCCAO) of rats for 7 days induces events related to gliosis with no evident histological damage. However, retinal degeneration and cellular death occur after 90 days of BCCAO. Our purpose has been to investigate the effects of BCCAO for 30 days in the retina of adult rats. Methods Adult Wistar rats were submitted to BCCAO or sham surgery. Both direct and consensual pupillary light reflexes were investigated before and after surgery, everyday for the first week and weekly for 30 days. After 1 month, eyes were enucleated and embedded in paraffin. The retinal ganglion cell (RGC) density and thickness of the internal plexiform (IPL), internal nuclear, outer plexiform, and outer nuclear layers were estimated. Results Four rats of the BCCAO group (50%) lost the direct pupillary reflex in both eyes, three rats (37%) lost this reflex in one eye, and only one (13%) maintained it in both eyes. RGC density (cells/mm) was diminished in the BCCAO group, and a significant decrease was found in the total retina and IPL thickness; however, no changes were evident in the other layers. BCCAO pupillary-reflex-negative rats presented with a significant decrease in total retinal thickness and retinal ganglion cell density compared with the sham group. Both BCCAO pupillary-reflex-positive) and -negative rats showed a decrease in IPL compared with the sham group. Conclusion This study demonstrates that BCCAO for 30 days induces functional and morphological damage to the retina with loss of the pupillary reflex and a decrease in IPL thickness and RGC number. We suggest that this protocol might be used as a model for ocular ischemic syndrome in the rat.     

视网膜内不同亚层对大分子物质的通透性差异研究

目的 了解视网膜内不同亚层对大分子物质的通透性差异.方法 将新鲜猪眼视网膜铺于自行设计的通透性装置上,将Carboxyfluorescein(相对分子质量为376)和相对分子质量为4 400、9 300、19 600、38 900、71 200、150 000的FITC-葡聚糖RPMI 1640溶液置于视网膜的一侧,1 h后,将视网膜取下进行冰冻切片,荧光显微镜下观察荧光分布情况,切片观察后固定并行苏木精-伊红染色.结果 Carboxyfluorescein和不同相对分子质量的葡聚糖在正常猪眼视网膜的分布是不同的.无论Carboxyfluorescein接触视网膜内层还是外层,视网膜的内核层和外核层最亮;接触视网膜内层,相对分子质量为4 400葡聚糖组内核层最亮,相对分子质量为9 300～71 200 葡聚糖组内核层暗淡而内核层以前较亮,相对分子质量为150 000葡聚糖组内丛状层以前部分较亮;接触视网膜外层,相对分子质量为4 400葡聚糖组视网膜全层均明亮,但外核层以外最亮,而相对分子质量为9 300～150 000葡聚糖组则仅外核层明亮,其余层均暗淡.结论 内核层和外核层是视网膜内主要的大分子通透屏障,大分子通透上限排序(由小到大)依次为外核层＜内核层＜内、外丛状层＜神经纤维层和内界膜.     

人胚胎九个月视网膜组织超微形态学观察

目的 观察人胚胎9个月时视网膜组织结构,探讨出生前期视网膜内神经元发育特征及意义.方法 取因外伤自愿终止妊娠的9个月人胚胎2例4个眼球.1例孕周为35周,1例为36周.胎龄根据孕妇的末次月经及测量胚胎坐高而确定.每只眼球于后极部定位取材视网膜组织片4个,常规电子显微镜标本处理程序后进行观察.扫描电子显微镜(SEM)和透射电子显微镜(TEM)共观察视网膜样本8个.结果 胚胎9个月时,外核层内含有5～6层感光细胞,外界膜外侧散在分布直径为2～3 μm的球形膜性结构.感光细胞内节段排列紧密,其间可见含粘连小带的外界膜,内含线粒体.其外侧可见纤毛.外节段较小,其内可见少量排列欠整的盘膜结构.感光细胞为多形核,常和(或)异染色质比例接近.感光细胞轴突分支少而细,与内核层细胞间联系较少,未见典型的"突触"结构.视网膜内核层含4～5层胞体.多种胞核常和(或)异染色质密度不均,部分为分叶核,核膜清晰.内网状层内各神经细胞分枝细小,神经突起间联系较少,少见典型的"突触"结构.视网膜神经节细胞(RGC)数量较少,胞膜完整,胞浆内可见大的细胞核和粗面内质网,核膜为连续的双层膜性结构,核内以常染色质为主.视网膜内界膜为双层膜性结构,其内侧可见排列整齐的神经纤维层,表面分布直径2～3μm的微孔,其间散在4～5 μm的较大孔隙.结论胚胎9个月时,人视网膜组织各层结构已经形成.但部分细胞结构和细胞联系尚未成熟,提示此期人视网膜发育仍处于发育重塑的重要时期.     

人胚胎九个月视网膜组织超微形态学观察

目的 观察人胚胎9个月时视网膜组织结构,探讨出生前期视网膜内神经元发育特征及意义.方法 取因外伤自愿终止妊娠的9个月人胚胎2例4个眼球.1例孕周为35周,1例为36周.胎龄根据孕妇的末次月经及测量胚胎坐高而确定.每只眼球于后极部定位取材视网膜组织片4个,常规电子显微镜标本处理程序后进行观察.扫描电子显微镜(SEM)和透射电子显微镜(TEM)共观察视网膜样本8个.结果 胚胎9个月时,外核层内含有5～6层感光细胞,外界膜外侧散在分布直径为2～3 μm的球形膜性结构.感光细胞内节段排列紧密,其间可见含粘连小带的外界膜,内含线粒体.其外侧可见纤毛.外节段较小,其内可见少量排列欠整的盘膜结构.感光细胞为多形核,常和(或)异染色质比例接近.感光细胞轴突分支少而细,与内核层细胞间联系较少,未见典型的"突触"结构.视网膜内核层含4～5层胞体.多种胞核常和(或)异染色质密度不均,部分为分叶核,核膜清晰.内网状层内各神经细胞分枝细小,神经突起间联系较少,少见典型的"突触"结构.视网膜神经节细胞(RGC)数量较少,胞膜完整,胞浆内可见大的细胞核和粗面内质网,核膜为连续的双层膜性结构,核内以常染色质为主.视网膜内界膜为双层膜性结构,其内侧可见排列整齐的神经纤维层,表面分布直径2～3μm的微孔,其间散在4～5 μm的较大孔隙.结论胚胎9个月时,人视网膜组织各层结构已经形成.但部分细胞结构和细胞联系尚未成熟,提示此期人视网膜发育仍处于发育重塑的重要时期.     

Presumptive catecholaminergic ganglion cells in the pigeon retina

An antiserum directed against tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of dopamine, was used to study the pigeon retina. Labeled cells were observed in both the inner nuclear layer (INL) and ganglion cell layer (GCL). Two populations of TH-immunoreactive neurons were observed in the INL. Some of these cells were 7-10 microns in diameter and gave rise to processes that arborized in three layers of the inner plexiform layer (IPL). These cells appeared similar to the dopaminergic amacrine cells described previously (Marc, 1988). Other labeled cells in the INL were 12-20 microns in diameter and were recognizable as a previously described subpopulation of TH-immunoreactive displaced ganglion cells (Britto et al., 1988). A population of labeled cells was observed in the GCL. Counts of these cells in two retinae revealed 5000 and 7000 cells, respectively. They ranged in size from 8-15 microns in diameter in the central retina and from 8-20 microns in diameter in the peripheral retina. The density of labeled cells was highest in the central retina and red field and lowest in the retinal periphery. The difference in cell size and cell density as a function of eccentricity is characteristic of the total population of ganglion cells in the avian retina (Ehrlich, 1981; Hayes, 1982). Some of the TH-positive cells in the GCL could be classified as ganglion cells for two reasons: (1) The axons of many of the TH-positive cells in the GCL were TH-immunoreactive as well and could be followed to the optic nerve head. (2) The injection of rhodamine-labeled microspheres into the nucleus geniculatus lateralis, pars ventralis (GLv), resulted in the retrograde labeling of many of the TH-positive cells in the contralateral retina.     

电子烟对小鼠视网膜组织及超微结构影响的研究

目的:研究电子烟对小鼠视网膜组织及超微结构的影响以及可能的相关机制。方法:将18只8周龄雄性c57BL小鼠随机分为对照组(6只),0mg尼古丁组(6只)和12mg尼古丁组(6只),分别用HE染色观察视网膜各层结构的改变,在透射电子显微镜下观察视网膜色素上皮(RPE)细胞超微结构的改变,通过免疫荧光染色观察视网膜中Tuj1、8-OHdG的表达情况。结果:与对照组相比,实验组(0mg和12mg尼古丁组)视网膜全层、视神经纤维层和内丛状层厚度均显著减少(P<0.01),但实验组间均无明显差异(P>0.05)。RPE细胞顶部仅见零星微绒毛,残存微绒毛长度变短。实验组中节细胞层、视神经纤维层和内丛状层可观察到Tuj1表达减少,但是神经节细胞数目无显著变化(P>0.05)。0mg和12mg尼古丁组中节细胞层、内核层内观察到8-OHdG表达增加。结论:电子烟可对小鼠视网膜造成损伤,其损伤可能是由氧化应激反应造成的。     

Immunolocalization of TRPC channel subunits 1 and 4 in the chicken retina

In the vertebrate retina, multiple cell types express G protein-coupled receptors linked to the IP3 signaling pathway. The signaling engendered by activation of this pathway can involve activation of calcium permeable transient receptor potential (TRP) channels. To begin to understand the role of these channels in the retina, we undertake an immunocytochemical localization of two TRP channel subunits. Polyclonal antibodies raised against mammalian TRPC1 and TRPC4 are used to localize the expression of these proteins in sections of the adult chicken retina. Western blot analysis indicates that these antibodies recognize avian TRPC1 and TRPC4. TRPC1 labeling is almost completely confined to the inner plexiform layer (IPL) where it labels a subset of processes that ramify in three broad stripes. Occasionally, cell bodies are labeled. These can be found in the inner nuclear layer (INL) proximal to the IPL, the IPL, and the ganglion cell layer (GCL). Double-labeling experiments using a polyclonal antibody that recognizes brain nitric oxide synthase (bNOS) in the chicken indicate that many of the TRPC1-positive processes and cell bodies also express bNOS. Labeling with the TRPC4 antibody was much more widespread with some degree of labeling found in all layers of the retina. TRPC4 immunoreactivity was found in the photoreceptor layer, in the outer plexiform layer (OPL), in radially oriented cells in the INL, diffusely in the IPL, and in vertically oriented elements below the GCL. Double-labeling experiments with a monoclonal antibody raised against vimentin indicate that the TRPC4-positive structures in the INL and below the GCL are Müller cells. Thus, TRPC1 and TRPC4 subunits have unique expression patterns in the adult chicken retina. The distributions of these two subunits indicate that different retinal cell types express TRP channels containing different subunits.     

Developmental expression profile of the optic atrophy gene product: OPA1 is not localized exclusively in the mammalian retinal ganglion cell layer

PURPOSE: Autosomal dominant optic atrophy (ADOA) is characterized by primary degeneration of retinal ganglion cells and atrophy of the optic nerve. The OPA1 gene encodes a 960-amino-acid protein. In the current study the temporal and spatial localization of OPA1 were examined in developing and adult murine ocular tissues and the adult human eye. Because the Bst/+ mouse has been postulated as a model of ADOA, the mOPA1 expression in the Bst/+ retina was also examined. METHODS: A polyclonal antibody generated against a C-terminal peptide of OPA1 was used to assess by immunohistochemistry the expression of mOPA1 in the wild-type embryonic and postnatal mouse ocular tissues and the Bst/+ retina. Western blot analyses of total proteins from a panel of adult human tissues were used to examine the expression of human OPA1, and spatial localization was assessed by immunohistochemistry. RESULTS: The ocular expression of mOPA1 begins at E15 in the inner retina in a location corresponding to that of the subsequently developing ganglion cell layer (GCL) and peaks between postnatal day (P)0 and P1 in the retina and the optic nerve. There is a sharp decline in mOPA1 expression after P2, but it is expressed at a basal level until at least P12 in the GCL, inner plexiform layer (IPL), and inner nuclear layer (INL) of the retina as well as in the optic nerve. In the adult Bst/+ retina, mOPA1 is strongly expressed in the GCL and IPL and weakly in the INL. In the adult human eye, OPA1 is expressed in the GCL, IPL, INL, and outer plexiform layer (OPL) of the retina and in the optic nerve, where it is observed only in the myelinated region. CONCLUSIONS: OPA1 is not restricted to the GCL of the mammalian retina, and its expression extends into the IPL, INL, and OPL. OPA1 is distinctly expressed in the myelinated region beyond the lamina cribrosa in the human optic nerve, whereas its expression is weaker in the mouse optic nerve. In the Bst/+ mouse retina, despite the structural defects, mOPA1 expression is comparable to that observed in the wild-type adult mouse retina. These observations suggest a wider role for OPA1 than previously anticipated.     

人胎视网膜血管发生方式的研究

目的：观察人胎视网膜血管的形成方式。方法：收集13～38周胎儿视网膜86例，免疫组织化学染色(ABC)法，光镜观察。结果：在胎儿12—13周时，间充质的梭形细胞从视盘处进入视网膜，并向锯齿缘迁移，同时分化、增殖为内皮细胞索，此索经管道化和改建成为节细胞层内的血管。第26周，节细胞层内已生成血管“出芽”，朝神经纤维层和内核层生长，分别在神经纤维层内和内核层的内、外缘各形成一层毛细血管网。结论：足月胎儿视网膜基本具有四层血管，分别由两种不同方式生成。 （中华眼底病杂志，1996，12：88-90）     

Immunohistochemical localization of GABAA receptors in the retina of the new world primate Saimiri sciureus

A large population of amacrine cells in the retina are thought to use GABA as an inhibitory neurotransmitter in their synaptic interactions within the inner plexiform layer. However, little is known about their synaptic targets; the neurons that express the receptors for GABA have not been clearly identified. Recently, the GABAA receptor has been isolated and antibodies have been raised against it. These antibodies have proven useful for the immunocytochemical localization of the receptor, and two brief reports describing the distribution of GABAA receptor immunoreactivity in the retina have appeared (Richards et al., 1987; Mariani et al., 1987). We used a monoclonal antibody (62-3G1) against the GABAA receptor to study the retina of the New World primate Saimiri sciureus. Labeled somata were found in the inner nuclear layer (INL) and ganglion cell layer (GCL). The staining was confined to what appeared to be the cell's plasmalemma and small cytoplasmic granules. Most of the labeled neurons in the INL had small somata (5-7 microns in diameter) located at the vitreal edge of the layer. They arborized in two laminae (approximately 2 and 4) of inner plexiform layer (IPL). Ventral to the optic disc (2.5 mm) they comprised 29% of the cells present. A few of the labeled neurons appeared to be interplexiform cells or flat bipolar cells, with labeled processes that extended into both the IPL and the inner half of the outer plexiform layer. In the GCL, the labeled somata were among the largest present (13-20 microns in diameter), and 2.5 mm ventral to the optic disc they made up 15% of the cells present. Experiments in which immunoreactive somata were retrogradely labeled following the injection of fluorescent tracers into the optic tract provided a conclusive demonstration that some of the immunoreactive somata were ganglion cells. The antibody often labeled their axons in the optic fiber layer. This suggests that the GABAA receptors are transported anterogradely to the retinal terminal fields. The dendrites of the immunoreactive ganglion cells extended into the 2 laminae of labeled processes in the IPL, and their primary dendritic arbors were, at any given eccentricity, quite similar in appearance. This homogeneity suggests that they comprise a particular subset of the ganglion cells. Sections simultaneously labeled with the monoclonal antibody against the GABAA receptor and antisera against either L-glutamic acid decarboxylase (GAD) or GABA revealed that the GAD/GABA was distributed much more widely in the IPL than the GABAA receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

色素上皮衍生因子基因修饰的脐带间充质干细胞对糖尿病大鼠视网膜组织病理改变的影响

目的　观察玻璃体内注射色素上皮衍生因子基因修饰的人脐带间充质干细胞（pigment epithelial-derived factor gene-modified human umbilical cord mesenchymal stem cells,PEDF-MSCs）对糖尿病大鼠视网膜组织病理结构改变的影响。方法　组织块培养法获取人脐带间充质干细胞（human umbilical cord mesenchymal stem cells,hUCMSCs）。获取的hUCMSCs表达CD105、CD73和CD90,不表达CD34、CD45、CD11b、CD19和HLA-DR。用慢病毒载体以感染复数值为50对其转染。利用链脲佐菌素腹腔注射途径诱导糖尿病大鼠模型,实验动物分为四组:正常对照组（N 组）、实验对照组糖尿病注射PBS组（D1组）、糖尿病注射hUCMSCs治疗组（D2组）、糖尿病注射PEDF-MSCs治疗组（D3组）。造模成功后3个月进行干预治疗,N组不予特殊治疗。玻璃体内注射2周后利用荧光显微镜观察PEDF-MSCs在大鼠眼内表达情况。干预治疗2周后进行HE染色观察各层视网膜结构,及各组视网膜总厚度变化。结果　玻璃体内注射后2周,荧光显微镜下观察到D2组大鼠玻璃体内簇状排列的红色荧光,视网膜中未发现明显红色荧光;D3组大鼠玻璃体内簇状排列的绿色荧光,视网膜中未发现明显绿色荧光。HE染色显示,N组大鼠的视网膜各层结构完整,层次清晰,细胞排列整齐,染色均匀;D1组大鼠视网膜神经纤维层（nerve fiber layer,NFL）出现明显水肿、血管扩张,内丛状层（innerplexiform layer,IPL）结构疏松,内核层（inner nuclear layer,INL）细胞排列紊乱;D2组NFL水肿减轻;D3组NFL水肿明显减轻,INL与外核层（outer nuclear layer,ONL）层细胞排列规整。视网膜总厚度N组为（103.82±4.15）μm、D1组为（138.86±4.71）μm、D2组为（131.17±3.89）μm、D3组为（112.24±4.22）μm;各组间差异均有统计学意义（均为P＜0.05）。结论　PEDF-MSCs可较长时间在糖尿病大鼠玻璃体内存活且持续表达。玻璃体内注射 PEDF-MSCs能明显改善糖尿病大鼠视网膜损伤,减轻视网膜水肿。     

Cholinoceptive neurons in the retina of the chick: an immunohistochemical study of the nicotinic acetylcholine receptors

Monoclonal antibodies directed against nicotinic acetylcholine receptors (nAChRs) were used to identify and characterize cholinoceptive neurons in the chick retina. Two monoclonal antibodies (mAbs), mAb 210 and mAb 270, stained many neurons in both the inner nuclear layer (INL) and ganglion cell layer (GCL). A class of large labeled cells in the inner INL were positioned at the INL/IPL (inner plexiform layer) border and resembled displaced ganglion cells (DGCs). Their identity was confirmed with injections of rhodamine-labeled microspheres into the ventral tectum and nucleus of the basal optic root (nBOR). Four days after the injection, large nAChR-positive neurons in the inner INL were labeled with beads. The distribution of these cells matched that reported for DGCs in the chicken and pigeon (Reiner et al., 1979; Fite et al., 1981). Many smaller cells in the INL also exhibited nAChR immunoreactivity. These cells were not retrogradely labeled after bead injections into retinal recipient areas. Their processes entered IPL where they arborized in a band comprised of the inner leaflet of lamina 1 and all of lamina 2. In some instances, a process continued inward to lamina 4. These neurons were tentatively identified as amacrine cells because of their position and branching pattern. Approximately 12-18% of the cells in the GCL exhibited nAChR immunoreactivity. Many of these cells could be classified as ganglion cells as their axons were also labeled following exposure to nAChR antibodies. Their distribution mirrored that of all ganglion cells with a higher density of cells in the central retina than in the periphery (Ehrlich, 1981). A "double label" technique was used to compare the distribution of nAChR-positive neurons with that of the choline acetyltransferase-positive (ChAT), cholinergic neurons in the chick retina. The two antigens were visualized with two different fluorophores: FITC and RITC. We were unable to find any cells in either the INL or GCL that exhibited both ChAT- and nAChR-like immunoreactivity. The nAChR-positive cells and the ChAT-positive cells both arborized in two bands within the IPL. The patterns were in perfect register in the inner IPL (lamina 4). But, in the outer IPL, the nAChR-positive dendrites were observed in the inner leaflet of lamina 1 and in all of lamina 2 while the ChAT-positive dendrites did not extend into the innermost portion of lamina 2.