视神经损伤后突触后密度蛋白-95在大鼠视网膜中的分布及表达研究

目的研究视神经损伤后突触后密度蛋白-95（PSD-95）在神经损伤后sD大鼠视网膜中的定位及表达的变化。方法采用钳夹法制作SD大鼠视神经损伤模型。分别于损伤后1d、3d、5d,7d及14d摘除眼球,以正常大鼠视网膜为对照,应用免疫组织化学方法和Westernblot法检测视神经损伤后PSD-95蛋白水平表达变化;免疫荧光双标技术检测PSD-95的细胞定位情况。结果在正常对照组中,视网膜外丛状层有大量的PSD-95存在。视神经损伤后,PSD-95在视网膜中的表达显著减少,损伤后1d,PSD-95在视网膜外层表达量最低,在损伤后7dPSD-95的表达恢复正常水平并维持到损伤后14d。免疫荧光双标染色结果显示视神经损伤后1dPSD-95与视杆细胞的标记物rhodopsin共定位减少,损伤后7d部分恢复。结论视神经钳夹伤导致了视网膜PSD-95表达量的减少和分布的改变,可能是视网膜神经元损伤后自我保护的一种反应。     

急性高眼压大鼠视网膜RhoA的分布及表达

目的:探讨急性高眼压大鼠视网膜RhoA的分布及表达变化。方法:将56只SD大鼠随机分成正常对照组、急性高眼压后1,3,7d共4组,用免疫组化和半定量RT-PCR方法检测大鼠视网膜组织中的RhoA的分布及表达情况。结果:正常及急性高眼压后1d,RhoA主要分布于视网膜神经纤维层及神经节细胞层;3d分布于神经节细胞层及内丛状层;7d分布于神经节细胞层、内丛状层、内核层及外丛状层。半定量RT-PCR提示:在正常大鼠视网膜组织中,RhoA mRNA仅有微量表达,急性高眼压后1,3,7d,RhoA表达均显著高于正常组(P<0.05),7d组表达量最高。结论:大鼠急性高眼压损伤后,视网膜RhoA蛋白的分布及表达显著增加,其在介导抑制性信号阻断轴突再生的抑制过程中发挥着重要作用。     

大鼠视神经损伤后视网膜小胶质细胞表达的初步观察

目的 研究大鼠视神经损伤后视网膜中小胶质细胞的表达情况.方法 实验研究.选取30只成年雌性健康SD大鼠,按照随机数字表法分为实验组和对照组各15只,分别用于细胞计数、免疫组织化学及免疫印迹实验.实验组在眼球后约1.5 ～2.0 mm处行右眼视神经鞘内切断术,术后5d于视神经断端处用荧光金逆行标记视网膜节细胞,手术后7d处死取材.对照组小鼠右眼行视神经切断术并标记,2d后处死取材.视网膜做铺片用于计数.用免疫组织化学法于视网膜切片上行小胶质细胞的表面标记物Iba-1染色,观察小胶质细胞的形态及数量,同时应用免疫印迹法检测视网膜内Iba-1蛋白含量的变化.两组间比较采用非配对student t-检验进行统计学分析.结果 对照组视网膜中有少量小胶质(Iba-1阳性)细胞表达,并呈非活化状态.视神经切断7d后小胶质细胞明显增多且呈半活化状态,免疫印迹结果显示损伤后Iba-1蛋白表达量明显增加到对照组的2.3倍(t=7.669,P=0.001).视视神经切断7d后节细胞数量为(1182±64)个/mm2,明显减少至对照组的51％(t=23.850,P＜0.01).结论 大鼠视神经损伤后小胶质细胞表达增多且呈部分激活状态,可能是视网膜受损后自我保护的表现之一.     

Rabbit retinal ganglion cell survival after optic nerve section and its effect on the inner plexiform layer

Structural modifications of the inner retina were studied after optic nerve section (ONS) in the rabbit. Retinal ganglion cells (RGC) were labelled by injection of Fast Blue into the optic nerve, and counted under fluorescent light in control retina and retina 7, 14, 21 and 26 days post-axotomy. Studies on retinal cross-sections were also performed. For this purpose, retinal sections were stained with haematoxylin-eosin and immunohystochemistry for alpha1 and beta2/beta3 sub-units of the GABA(A) receptors.One week after axotomy, there was no significant loss in the number of ganglion cells with respect to control counts (1086+/-173cellsmm(-2) in the visual streak and 119+/-46cellsmm(-2) in the periphery, mean+/-SD, n=5). At 14 days post-axotomy, 271+/-46cellsmm(-2) remained in the visual streak and 33+/-6cellsmm(-2) in the periphery, corresponding to a mean survival of 27%. The number of ganglion cells decreased further on the following days, reaching 7.54% 1 month after ONS. A significant reduction in the thickness of the inner plexiform and ganglion cell layers was also observed in retinal cross-sections. Immunocytochemical studies show a remarkable disorganization of the layer stratification in the inner plexiform layer (IPL). We conclude that after ONS, RGC death occurs mainly between 7 and 14 days post-axotomy and a progressive death up to 26 days, causing a decrease in the thickness of the IPL and subsequent disorganization of its layers.     

OPA1 expression in the human retina and optic nerve

Mutations in the optic atrophy type 1 (OPA1) gene give rise to human autosomal dominant optic atrophy. The purpose of this study is to investigate OPA1 protein expression in the human retina and optic nerve. A rabbit polyclonal antiserum was generated using a fusion protein covering amino acids 647 to 808 of the human OPA1 protein as the immunogenic antigen. Western blot and immunofluorescence staining were performed to examine OPA1 expression in the human retina and optic nerve. In human retina, we found that OPA1 expression was clearly present in retinal ganglion cells and photoreceptors. OPA1 immunoreactivity was also present in the nerve fiber layer, inner plexiform layer and outer plexiform layer. However, OPA1 protein was not detected in the choline acetyltransferase-positive, calretinin-positive, and calbindin-positive amacrine cells, nor in the calbindin-positive horizontal cells. In the human optic nerve, expression of OPA1 was present in the axonal tract that was labeled with neurofilament specific antibody. In conclusion, expression of OPA1 gene is present in the mitochondria-rich regions of the retina and optic nerve. This suggests that OPA1 protein might be involved in the functioning of the mitochondria that are present in both inner and outer retinal neurons.     

Developmental changes of aggrecan, versican and neurocan in the retina and optic nerve

We have used a monoclonal antibody to neurocan and specific polyclonal antibodies to the non-homologous glycosaminoglycan attachment regions of aggrecan and mRNA splice variants of versican to compare the localization and developmental changes of these structurally related hyaluronan-binding chondroitin sulfate proteoglycans in the rat retina and optic nerve. Staining for aggrecan and versican was first seen at embryonic day 16 in the optic nerve and retina, whereas neurocan was not detected in the embryonic eye. At postnatal day 0 (P0), beta-versican staining is largely confined to the inner plexiform layer whereas alpha-versican is also apparent in the neuroblastic layer. Both aggrecan and, much more weakly, neurocan immunoreactivity is present throughout the neonatal retina. At P9, aggrecan and versican immunoreactivity is most intense in the inner and outer plexiform and ganglion cell layers, accompanied by diffuse staining in the inner and outer nuclear layers. Aggrecan and alpha-versican are also present throughout the optic nerve and disk, whereas beta-versican and neurocan are confined to the laminar beams of the optic nerve. Between P0 and P9 there is a marked increase in beta-versican expression in the inner and outer nuclear layers and in the outer plexiform layer, whereas there is only weak staining of neurocan in the inner plexiform and ganglion cell layers of P9 retina. By 1 month postnatal the staining pattern of the fully differentiated retinal layers is essentially identical to that seen in the adult, where there is strong aggrecan and alpha-versican immunoreactivity in the retina and optic nerve, whereas beta-versican has essentially disappeared from the adult retina and, similarly to neurocan, is present only in the laminar beams of the optic nerve. The marked decrease of beta-versican in the retina is consistent with >90% decrease in its concentration in brain during postnatal development, suggesting that the developmental time-course for these proteoglycans in retina parallels that seen in other areas of the central nervous system.     

STZ诱导糖尿病视网膜FOXO1A的表达

目的探讨链脲霉素（streptozotocin,STZ）诱导糖尿病视网膜FOXO1A的表达及与糖尿病视网膜病变可能的相关性。方法建立STZ诱导的糖尿病大鼠模型,用免疫组织化学、Western blot方法检测对照组和糖尿病大鼠视网膜以及胰蛋白酶消化的视网膜微血管FOXO1A蛋白表达。结果免疫组化发现STZ诱导的糖尿病大鼠12周后视网膜及视网膜微血管FOXO1A免疫反应显著增加,主要分布在视网膜外网状层、内核层、内网状层、神经节细胞层及视网膜微血管壁。Western blot分析表明在糖尿病鼠8周和12周视网膜FOXO1A活性和表达较对照组显著增加,然而在糖尿病鼠4周,视网膜FOXO1A活性和表达较对照组减少。结论STZ诱导的大鼠慢性高血糖影响视网膜FOXO1A活性和表达,这可能与早期糖尿病视网膜神经细胞和微血管细胞的损伤有关。     

Effects of unoprostone on phosphorylated extracellular signal-regulated kinase expression in endothelin-1-induced retinal and optic nerve damage

Endothelin-1 (ET-1), a potent vasoconstrictor peptide, has been implicated in the development of normal- and high-tension glaucoma. We investigated the effects of unoprostone on extracellular signal-regulated kinase (ERK) in ET-1-induced retinal ganglion cell (RGC) death and optic nerve injury. Our morphometric study showed that intravitreal injection of ET-1 led to cell loss in the RGC layer (RGCL) in 28 days. Western blot analysis showed decreased neurofilament (NF) protein in the optic nerve 28 days after ET-1 injection. In this in vivo model, increased phosphorylated ERK (p-ERK) was observed in the retina on 1 day and subsequently in the optic nerve from 7 days after ET-1 injection. Simultaneous injection of M1, as a metabolite of unoprostone, showed further increased p-ERK levels compared with ET-1 injection alone. Our morphometric study of flat-mount preparations stained with cresyl violet or retrograde labeling with a neuro-tracer and Western blot analysis of NF showed that inhibition of ERK phosphorylation led to acceleration of ET-1-induced RGC death and optic nerve damage. In addition, M1 significantly attenuated both RGC loss and the decrease in NF protein induced by ET-1. The protective effects of M1 were significantly inhibited by U0126, an ERK inhibitor. These results suggest that unoprostone has neuroprotective effects against ET-1-induced neuronal injury through ERK phosphorylation.     

Immunohistochemistry of the monkey retina with a monoclonal antibody against subunit V of cytochrome c oxidase.

A monoclonal antibody against subunit V of cytochrome c oxidase was newly prepared from purified bovine heart muscle enzyme. This antibody showed cross-reactivity against the bovine and the human cytochrome c oxidase on Western blot. Cytochrome c oxidase in paraffin embedded sections of monkey retinas and optic nerves was examined immunohistochemically using the monoclonal antibody. The inner segment of cone and rod photoreceptors, the inner nuclear layer and the inner plexiform layer showed marked reactivities. The optic nerve fiber axons also showed high reactivities, with more in the unmyelinated fibers peripheral to the lamina cribrosa. These immunohistochemical results using the antibody are mostly agreeable with previously reported histochemical localization of cytochrome c oxidase activity in the retina, but there were some noticeable differences in the plexiform layers.     

地塞米松对兔视神经钳夹伤后视网膜神经节细胞存活及Nogo-A表达的影响

目的观察地塞米松对家兔视神经钳夹伤后视网膜神经节细胞(retinal ganglion cell,RGC)存活的影响及损伤视神经、视网膜Nogo-A表达的变化。方法采用兔球后视神经钳夹伤模型,健康成年家兔分为正常对照组、损伤组、治疗组。分别于损伤后3d、7d、14d处死动物,观察单位面积RGC存活数量及损伤后Nogo-A在视神经、视网膜的表达变化。结果视神经损伤后,治疗组RGC的存活数高于损伤组及对照组(P<0.01)。对照组、损伤组损伤后3d、7d、14d视神经、视网膜Nogo-A表达增强,至损伤后7d达高峰;治疗组视神经、视网膜表达亦增强,各时间点均弱于损伤组、对照组,差异有显著统计学意义(P<0·01)。结论视神经损伤后,地塞米松能够增加RGC的存活数量,能够下调No-go-A的表达,这可能是地塞米松治疗作用机制之一。     

Up-regulation of cytochrome oxidase in the retina following optic nerve injury

The purpose of this study is to investigate the cytochrome oxidase (COX) activity in the retina and optic nerve following an optic nerve injury. The optic nerve crush of one eye was carried out in Balb/c mice. A semi-quantitative RT-PCR method was then adopted to evaluate the mRNA expression of cytochrome oxidase subunit 1 (COX1) in the retina after surgery. Up-regulation of COX1 mRNA in the retina was detected by RT-PCR at 24 hr following the optic nerve injury. Total retinal mitochondrial mass measured by fluorescent intensity of MitoTracker green was not altered following the injury. COX histochemistry performed on cryostat sections showed an elevated enzyme activity of COX in the retina and in the optic nerve. In the retina, elevation of the COX activity was observed in the retinal ganglion cell layer and the overlying nerve fibre layer. The increase of COX activity began from 24 hr after injury, peaked around day 3, and maintained up to 1 week after the operation. In the optic nerve, increase of COX activity was observed in regions distal to the crush line and distributed either randomly or in a cone shape. In conclusion, both the expression of COX1 mRNA in retina and the activity of COX in inner plexiform layer and retinal ganglion cell layer were elevated following optic nerve injury without affecting total retinal mitochondrial mass. These findings suggested that one of early responses in the retina and in the optic nerve after the optic nerve injury is to scale up the energy production.     

视神经挫伤后视网膜形态学和Bcl-2/Bax表达

目的 研究大鼠视神经夹挫伤后视网膜神经节细胞（RGC）形态学改变及Bcl-2／Bax蛋白表达的变化，为了解视神经损伤的病理机制提供一定的依据。方法 建立大鼠视神经夹挫伤动物模型，伤后1d、3d、5d、7d、9d、2周、4周处死，HE染色观察RGC的动态变化，免疫组化方法检测RGC表达Bcl-2及Bax的水平。结果 视神经伤后RGC数目严重下降，2周内RGC快速减少，2周以后缓慢减少；伤后Bcl-2及Bax表达随时间而有不同程度的增加，Bax对损伤的反应较Bcl-2稍晚，两者表达均呈现先升后降的趋势，并维持一定的时间。Bcl-2和Bax蛋白表达比与RGC存活数目有一定的相关性。结论 视神经损伤后RGC数目减少是其视功能下降的病理基础之一，Bcl-2和Bax在RGC死亡机制中起重要作用，Bcl-2／Bax比率与RGC的减少呈一定的相关性。     

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.     

过氧化物酶体增生物激活受体γ在鼠眼球中的分布

背景过氧化物酶体增生物激活受体γ（PPAR-γ）是一类由配体激活的核转录因子,是潜在的抗炎、抗纤维增生、抗新生血管形成及神经保护因子,其在动物和人体组织中的生理病理功能是目前的研究热点之一,PPARγ与眼科疾病的研究受到关注。目的研究PPARγ在眼部不同组织细胞中的表达,为PPARγ激动剂在眼科疾病治疗中的应用提供参考依据。方法取SPF级C57BL／6J小鼠6只及SD大鼠1只,用质量分数3％水合氯醛麻醉处死后立即摘除眼球,采用Western blot法检测小鼠角膜、晶状体和视网膜组织中PPARγ蛋白的表达;采用免疫组织化学和免疫荧光化学法检测PPARγ在小鼠角膜、晶状体、视网膜、睫状体及视神经组织中的表达及定位。结果Western blot法检测表明,PPARγ在小鼠角膜、晶状体、视网膜中均呈阳性表达。免疫组织化学和免疫荧光化学法检测显示,PPARγ在角膜组织中主要表达于上皮层,以基底细胞染色最强,而角膜内皮及基质细胞上仅有弱表达。PPARγ在晶状体中主要表达于上皮细胞和浅皮质层;在视网膜组织中,PPARγ主要表达于视网膜节细胞层、内丛状层、外丛状层和内核层,此外PPARγ在SD大鼠睫状体组织中主要表达于无色素上皮。免疫荧光化学法检测显示,其在视网膜中与Muller细胞标志物谷氨酰胺合成酶（GS）共定位表达明显;PPARγ在视神经组织中的表达与星形胶质细胞标志物胶质纤维酸性蛋白（GFAP）共定位表达明显。结论PPARγ广泛分布于眼不同组织中并呈特异性表达,该结果为相关眼科疾病的靶向治疗提供了依据。     

Expression of glycosaminoglycans during development of the rat retina

PURPOSE: To investigate the spatiotemporal expression of glycosaminoglycans during development of the rat retina. METHODS: Hyaluronan and sulfated glycosaminoglycans, including chondroitin sulfate, heparan sulfate and keratan sulfate were detected using biotinylated hyaluronan binding protein, immunohistochemical analysis, respectively, in the rat retina at various stages of development. RESULTS: Hyaluronan was expressed in the nerve fiber layer, inner plexiform layer and outer plexiform layer during early postnatal stages (postnatal day 1-14; P1-P14) and was undetectable after P21. In contrast, hyaluronan was faintly observed in the photoreceptor layer on P7, and gradually increased up to P49. The spatiotemporal expression pattern of chondroitin sulfate was similar to that of hyaluronan. Heparan sulfate was also detected in the nerve fiber layer, inner plexiform layer and outer plexiform layer during early postnatal stages (P1-P14). In addition, heparan sulfate was expressed in the inner limiting membrane during all stages of development. Keratan sulfate was not detected in the retina at any stage of development. CONCLUSIONS: Hyaluronan, chondroitin sulfate and heparan sulfate are expressed in nerve fiber-rich layers during early postnatal stages and may regulate neurite outgrowth. In adulthood, both hyaluronan and chondroitin sulfate are expressed in the photoreceptor layer and may consist of the interphotoreceptor matrix. In addition, heparan sulfate is expressed in the inner limiting membrane throughout the various stages of development and may be associated with the structure of the inner limiting membrane.     

Blocking LINGO-1 function promotes retinal ganglion cell survival following ocular hypertension and optic nerve transection

PURPOSE: LINGO-1 is a functional member of the Nogo66 receptor (NgR1)/p75 and NgR1/TROY signaling complexes that prevent axonal regeneration through RhoA in the central nervous system. LINGO-1 also promotes cell death after neuronal injury and spinal cord injury. The authors sought to examine whether blocking LINGO-1 function with LINGO-1 antagonists promotes retinal ganglion cell (RGC) survival after ocular hypertension and optic nerve transection. METHODS: An experimental ocular hypertension model was induced in adult rats using an argon laser to photocoagulate the episcleral and limbal veins. LINGO-1 expression in the retinas was investigated using immunohistochemistry and Western blotting. Soluble LINGO-1 protein (LINGO-1-Fc) and anti-LINGO-1 mAb 1A7 were injected into the vitreous body to examine their effects on RGC survival after ocular hypertension and optic nerve transection. Signal transduction pathways mediating neuroprotective LINGO-1-Fc effects were characterized using Western blotting and specific kinase inhibitors. RESULTS: LINGO-1 was expressed in RGCs and up-regulated after intraocular pressure elevation. Blocking LINGO-1 function with LINGO-1 antagonists, LINGO-1-Fc and 1A7 significantly reduced RGC loss 2 and 4 weeks after ocular hypertension and also promoted RGC survival after optic nerve transection. LINGO-1-Fc treatment blocked the RhoA, JNK pathway and promoted Akt activation. LINGO-1-Fc induced Akt phosphorylation, and the survival effect of LINGO-1 antagonists was abolished by Akt phosphorylation inhibitor. CONCLUSIONS: The authors demonstrated that blocking LINGO-1 function with LINGO-1 antagonists rescues RGCs from cell death after ocular hypertension and optic nerve transection. They also delineated the RhoA and PI-3K/Akt pathways as the predominant mediator of LINGO-1-Fc neuroprotection in this paradigm of RGC death.     

Nogo-A mRNA在视神经损伤后视网膜中的表达和分布

目的 定位检测Nogo-A mRNA在成年大鼠正常视网膜和视神经损伤后视网膜中的表达和分布。方法 将90只成年大鼠随机分为2组，有眼为对照组，左眼为实验组。实验组在眼球后2mm用视神经损伤夹夹持9s，根据处死时间不同分别于夹伤后1、3、7d取出大鼠眼球，沿其长轴制作视网膜冰冻切片。应用原位杂交方法检测视网膜中Nogo-A mRNA的表达和定位。将切片图像输入图像分析仪进行处理。结果 视网膜中可见Nogo-A mRNA在不同细胞层中的表达：视网膜神经节细胞层、内颗粒层、外颗粒层中可见Nogo-A mRNA表达，其中以视网膜神经节细胞层为最多。夹伤组中1、3、7d组分别与对照组比较，阳性细胞染色面积差异有极显著统计学意义（t=3．82，4．21，4．07；P〈0．01），A值差异有非常显著统计学意义（t=3．90，4．52，3．78；P〈0．01）。结论 Nogo-A mRNA在视神经损伤后视网膜中的表达明显增多，在抑制视神经损伤后轴突再乍的机制中可能起重要作用。     

Intrinsic activation of PI3K/Akt signaling pathway and its neuroprotective effect against retinal injury

PURPOSE: The aim of this study was to determine whether the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway can function as a neuroprotective pathway following induced retinal injury. METHODS: The activation of Akt was assessed by immunoblot analysis, and the role of PI3K/Akt pathway was evaluated by TUNEL staining and counting the number of retrogradely-labeled retinal ganglion cells (RGCs) in the whole retina at 168 h after injury with or without PI3K specific inhibitor, LY294002. RESULTS: Akt was induced within one hr and reached a maximum 6 hrs after optic nerve clamping. The activation was observed in the RGC layer including RGCs, the inner plexiform layer, inner nuclear layer, and in the photoreceptor outer segments. The number of surviving RGCs was decreased significantly 168 hrs after injury. LY294002 partially inhibited the activation of Akt, and significantly decreased the number of surviving RGCs as compared with that of injury alone. CONCLUSIONS: These results indicate that the PI3K/Akt signaling pathway is activated intrinsically and has a neuroprotective effect on injured RGCs.