低氧诱导因子1α在急性高眼压大鼠视网膜中的表达

目的了解低氧诱导因子1α(hypoxia-inducible factor-1alpha,HIF-1α)在急性高眼压大鼠视网膜中的表达,并探讨其在急性青光眼损伤中的作用。方法 80只Wistar大鼠随机分为对照组、穿刺组、加压后2h、12h、1d、3d、7d、14d组。对照组不做任何处理,穿刺组只进行前房穿刺不加压,加压各组前房灌注加压法制作急性高眼压模型。HE染色观察视网膜的形态学变化,免疫组织化学方法和RT-PCR方法观察HIF-1α蛋白和mRNA在视网膜中的表达情况。结果急性高眼压损伤后1d,光学显微镜下可见视网膜高度水肿,7d时水肿基本消失,14d时视网膜各层结构紊乱,明显萎缩变薄。HIF-1α蛋白和mRNA在加压后2h时表达增多(HIF-1α蛋白IOD值0.54±0.06,mRNA相对表达量1.5162±0.1199),12h达到高峰(HIF-1α蛋白IOD值1.73±0.10,mRNA相对表达量2.9041±0.1690),随时间推移表达逐渐减少,14d时降至最低(HIF-1α蛋白IOD值0.52±0.03,mRNA相对表达量1.2847±0.0434),但仍高于对照组(HIF-1α蛋白IOD值0.25±0.04,mRNA相对表达量1.0009±0.0480),差异均有统计学意义(均为P<0.05)。结论大鼠视网膜的HIF-1α在急性高眼压后表达增加,该因子在急性青光眼发病机制中具有重要作用。     

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。     

活血化瘀方剂对急性高眼压大鼠视网膜HIF-1α表达的影响

目的观察活血化瘀方剂对大鼠视网膜急性高眼压损伤的保护作用及对低氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)表达的影响。方法 140只清洁级Wistar大鼠随机分为对照组10只、穿刺组10只、加压组60只和治疗组60只。前房灌注加压法制作急性高眼压模型维持60min(眼压至66mmHg,1kPa=7.5mmHg)。加压组分别于造模后2h、12h、1d、3d、7d、14d处死动物,治疗组在造模后即刻给予活血化瘀方剂4mL灌胃,并分别在造模后2h、12h、1d、3d、7d、14d处死动物。通过HE染色观察视网膜的形态学变化,免疫组织化学方法和实时定量逆转录聚合酶链反应(Real-TimeqRT-PCR)方法检测HIF-1α在各组视网膜中的表达情况。结果造模加压后1d,光学显微镜下可见视网膜高度水肿,7d时水肿基本消失,14d时视网膜各层结构紊乱,明显萎缩变薄。治疗组造模加压后2h、12h视网膜形态学变化与加压组同一时间点无明显差别,其他各时间点视网膜损伤程度均较加压组同一时间点轻。免疫组织化学染色及Real-Time qRT-PCR结果显示:造模加压后2 h HIF-1α的表达即增加(IOD0.54±0.06,mRNA1.5162±0.1199),12h达到高峰(IOD1.73±0.10,mRNA2.9041±0.1690),随着时间推移表达逐渐减少,14d时降至最低(IOD0.52±0.03,mRNA1.2847±0.0434),但仍高于对照组(IOD0.25±0.04,mR-NA1.0009±0.0480,P<0.05);治疗组该因子的表达变化趋势与加压组相同,但除造模后2h、12h外,治疗组各时间点HIF-1α的表达量均比加压组同一时间点低,差异均有统计学意义(均为P<0.05)。结论活血化瘀方剂对急性高眼压造成的视网膜损伤有保护作用,这种保护作用可能与其减少视网膜内源性HIF-1α的表达有关。     

急性高眼压后大鼠视网膜谷氨酰胺合成酶mRNA表达的变化

目的观察急性高眼压后大鼠视网膜谷氨酰胺合成酶（GS）mRNA的表达变化。方法前房灌注加压至110mmHg，维持60min。提取视网膜总RNA，以RT—PCR半定量GSmRNA。结果大鼠前房加压解除后4h，GSmRNA的表达较对照眼开始升高（P〈0．05），至24h达高峰（P〈0．01），36h降低（P〈0．05），72h（P〉0．05）恢复至正常水平。结论急性高眼压后大鼠视网膜GSmRNA的表达呈现明显升高过程，可能引起GS表达上调，促进谷氨酸转化为谷氨酰胺，从而减轻视网膜神经节细胞（RGCs）的损伤。     

葡萄糖调节蛋白在急性高眼压大鼠视网膜中的表达及其意义

背景 青光眼导致的视网膜神经节细胞(RGCs)进行性死亡是导致患者视功能损害的主要病理基础,研究表明内质网应激(ERS)参与此过程,葡萄糖调节蛋白78( GRP78)是内质网中的特异性标志物,检测GRP78在高眼压诱导视网膜中的表达对青光眼患者视神经功能保护机制的研究具有重要意义. 目的 检测GRP78在大鼠急性高眼压后不同时期视网膜中的表达情况,探讨ERS在急性青光眼损伤中的作用.方法 56只Wistar大鼠采用随机数字表法分为正常对照组、前房穿刺组、急性高眼压12h组及急性高眼压1、3、7、14 d组,每组8只眼.急性高眼压模型的制作采用前房穿刺灌注生理盐水法,眼压提高至66 mmHg.分别于造模后12h及1、3、7、14 d用过量麻醉法处死大鼠.苏木精-伊红染色法观察各组大鼠视网膜的病理形态学改变;免疫组织化学法检测视网膜中的GRP78蛋白表达的变化;实时定量逆转录聚合酶链反应(RTPCR)法检测视网膜中GRP78 mRNA的表达变化. 结果 正常对照组大鼠视网膜各层细胞排列整齐,急性高眼压后12h视网膜水肿增厚、细胞核肿胀,1d时视网膜形态学异常改变达到高峰,3d后水肿减轻,视网膜变薄.免疫组织化学染色显示正常对照组、前房穿刺组大鼠RGCs层和内核层GRP78蛋白呈弱阳性表达,A值分别为0.195±0.006、0.196±0.005,急性高眼压12h组大鼠GRP78在视网膜的表达明显增强,A值为0.293±0.011,造模后1d时GRP78表达强度达到高峰,A值为0.499±0.039,造模后3d时GRP78表达明显降低,A值为0.268±0.017,与正常对照组大鼠比较差异均有统计学意义(t=0.098、0.304、0.073,P＜0.05),但造模后7d和14 d时GRP78在大鼠视网膜RGCs层和内核层中的表达量接近正常,与正常对照组比较差异均无统计学意义( t=0.002、0.001,P＞0.05).实时定量RT-PCR检测表明,GRP78 mRNA在正常对照组大鼠视网膜的相对表达量( 2-△△CT)为1.011±0.013,眼压升高12 h快速上调,为1.536±0.145,1 d时达高峰,为2.141±0.171,3d时迅速降低,为1.420±0.212,与正常对照组比较差异均有统计学意义(t=0.525、1.130、0.409,P＜0.05),而造模后7d及14 d GRP78 mRNA在大鼠视网膜的表达量明显下降,与正常对照组比较差异均无统计学意义(t=0.020.0.004,P＞0.05).结论 GRP78参与大鼠急性高眼压后视网膜损伤的发生机制,提示通过对ERS过程进行干预可能达到保护急性高眼压眼视神经功能的目的.     

兔实验性急性高眼压模型视网膜谷氨酸的变化

目的 研究兔实验性急性高眼压状态下视网膜谷氨酸含量的变化。探讨谷氨酸在青光眼高神经损害中的作用。方法 应用邻苯二甲醛衍生法、高效液相色谱仪测定视网膜谷氨酸的含量,将１８只大白兔分成３组,其中２个组１个正常对照组,每组均６只兔（６只眼）。实验１组：每只兔随机选取１只眼行生理盐水前房内高压灌注,６０ｍｍＨｇ（１ｍｍＨｇ＝０．１３３ｋＰａ）持续４ｈ。另１只眼生理盐水前房内灌注,方法同前,量压力为２０ｍｍ     

急性高眼压状态视网膜组织学和酶组织化学实验研究

本文用酶组织化学方法观察了急性高眼压动物模型视网膜与能量代谢有关的酶活性变化,发现琥珀酸脱氢酶、磷酸化酶、三磷酸腺苷酶活性降低，而乳酸脱氢酶及葡萄糖-6-磷酸酶活性增加，提示高眼压后视网膜发生的能量代谢障碍，可能是造成功能和结构损伤的重要原因之一。 （中华眼底病杂志，1993，9：141-144）     

T、B淋巴细胞联合免疫缺陷对急性高眼压小鼠视网膜神经细胞的影响

目的研究T、B淋巴细胞联合缺陷对急性高眼压小鼠视网膜神经细胞的影响。方法选取重度联合免疫缺陷（SCID）小鼠和野生型C57BL／6小鼠各16只。2种小鼠分别随机取6只不做任何处理作为正常对照组,剩余10只作为模型组。采用前房穿刺的方法建立缺血一再灌注模型,每只小鼠取右眼为实验眼,左眼为模型对照眼。通过荧光金逆行标记技术,观察并计数再灌注后21d存活的视网膜神经节细胞（RGCs）;同时进行视网膜切片苏木精一伊红染色,观察再灌注后21d视网膜形态并测量内核层厚度。结果正常对照组SCID小鼠和C57BL／6小鼠的RGCs形态和数量、视网膜结构及厚度均无明显差异。视网膜缺血一再灌注损伤后21d,SCID小鼠RGCs的存活率为91％±5％,C57BL／6小鼠RGCs的存活率为78％±5％,二者比较差异有统计学意义（P=0．003）;SCID小鼠实验眼内核层厚度为（33．52±2．13）μm,模型对照眼为（34．06±3．00）μm,二者比较差异无统计学意义（P〉0．05）;C57BL／6小鼠实验眼内核层厚度为（22．44±1．70）μm,模型对照眼为（31．06±3．75）μm,二者比较差异有统计学意义（P=0．004）。结论急性高眼压模型中,T、B淋巴细胞联合免疫缺陷小鼠RGCs的存活率较高,视网膜损伤明显轻于野生型C57BL／6小鼠。     

慢性高眼压兔模型视网膜中生长相关蛋白-43表达的变化

目的:研究持续高眼压状态下视网膜中生长相关蛋白-4(GAP-43)表达的变化。方法:兔眼前房注射复方卡波姆溶液制成慢性高眼压模型(n=21),用免疫组织化学方法观察在高眼压不同时间段视网膜中GAP-43的表达变化。结果:正常兔眼视网膜内丛状层中存在GAP-43的表达。持续性高眼压7d后内丛状层中的GAP-43免疫反应产物密度增加,且在节细胞和神经纤维层也出现阳性产物,与对照组比较有显著差异(P<0.05)。高眼压14d时阳性反应产物与7d时相比有所下降,但仍高于正常对照组(P<0.05);21d时基本恢复至正常对照组水平(P>0.05)。结论:慢性高眼压时视神经、视网膜结构受损,导致视网膜中GAP-43短暂升高。     

Necrostatin-1对急性高眼压大鼠模型中视网膜神经节细胞程序性坏死的抑制作用

目的:探讨程序性坏死特异性抑制剂necrostatin-1(Nec-1)对急性高眼压大鼠模型中视网膜神经节细胞(RGCs)程序性坏死的抑制作用。方法:采用随机数表法将24只雄性SD大鼠随机分为正常对照组、模型对照组、Nec-1处理组和阴性对照组,每组6只。其中,正常对照组大鼠不做任何处理;其余3个组大鼠左眼采用前房灌注...     

Effect of erythropoietin on the expression of HIF-1 and iNOS in retina in chronic ocular hypertension rats

AIM: To research the effect of erythropoietin (EPO) to the HIF-1\iNOS signal transduction path in retina in chronic ocular hypertension rat. METHODS: One hundred and twenty Wistar rats were divided into 12 groups randomly. Two episcleral veins were coagulated unilaterally in rats with electric coagulator to establish the glaucoma model. PT-PCR and Western Blot analysis were used to examine the expression of Caspase-9 genes in retina. And the changes of ERG-b wave before and after were detected using EPO. RESULTS: In EPO drug treatment group, the amplitude of ERG-b wave of retina restored remarkably. There was significant difference between two groups (P＜0.05). The expressions of HIF-1\iNOS mRNA and protein in EPO drug treatment group were weakened remarkably. It was statistically different compared with the non-drug treatment group. CONCLUSION: One of protect mechanisms of EPO to injured retina caused by chronic intraocular hypertension is through HIF-1\iNOS signal conduct path.     

金丝桃素对兔急性高眼压视网膜的保护作用

目的 探讨蛋白激酶C抑制剂金丝桃素对兔急性高眼压视网膜谷氨酸含量的影响以及对视网膜的保护作用.方法 将新西兰大白兔随机分成处理组、对照组和正常组.处理组向玻璃体腔内注射0.1 mL的金丝桃素,对照组向玻璃体腔内注射0.1 mL生理盐水,48 h后经前房灌注升高眼压达100 mmHg(1 kPa=7.5 mmHg),持续90 min,24 h后取材.正常组不做上述处理直接取材.高效液相色谱法检测视网膜谷氨酸含量;光镜、电镜观察视网膜超微结构的变化,并采用医学图像分析系统计数视网膜神经节细胞(retinal ganglion cells,RGC)、测量视网膜总厚度及内层厚度;TUNEL法检测视网膜细胞的凋亡.结果 处理组、对照组和正常组视网膜谷氨酸含量分别为(56.6±2.1)×10-6 mmol·g-1、(84.1±2.7)×10-6 mmol·g-1和(17.1±2.3)×10-6 mmol·g-1,处理组视网膜谷氨酸含量明显低于对照组(P＜0.05);3组长450 μm视网膜片段的RGC数分别为(22.30±0.83)、(15.50±0.56)和(25.50±0.67),处理组和正常组RGC数均明显高于对照组(P＜0.05),而RGC层及内核层内凋亡的细胞数明显少于对照组.结论 金丝桃素能减缓兔急性高眼压视网膜谷氨酸水平的升高、减轻RGC的凋亡,对视网膜具有一定的保护作用.     

Proteomic analysis of rat retina in a steroid-induced ocular hypertension model: Potential vulnerability to oxidative stress

Purpose To investigate global protein expression profiles in the retinas of normal and glucocorticoid-induced ocular hypertensive rats by proteomic analysis. Methods Ocular hypertension was induced by topical application of dexamethasone (DEX) for 4 weeks. Age-matched untreated rats served as controls. Intraocular pressure (IOP) was monitored by an electronic tonometer. Retinal protein expression profiling was carried out by two-dimensional fluorescence difference gel electrophoresis (2-D DIGE). Proteins were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Results In DEX-treated rats, average IOP was elevated significantly compared with controls. With DEX treatment, levels of four proteins were altered, as revealed by 2-D DIGE and MALDI-TOF mass spectrometry: apolipoprotein A1 (apoA1), a lipid-binding protein, upregulated 1.9-fold, P < 0.05; alpha A crystallin (CRYAA), a molecular chaperone, downregulated 2.7-fold, P < 0.01; superoxide dismutase 1 (SOD1), an antioxidant enzyme, downregulated 2.3-fold, P < 0.05; and triosephosphate isomerase 1 (TPI1), a glycolytic enzyme, downregulated 2.3-fold, P < 0.01. Conclusions Downregulation of CRYAA, SOD1, and TPI1, observed here after a short period of DEX-induced ocular hypertension, may be involved in the onset of neural damage in steroid-induced glaucoma.     

Expression of Nogo-A on the retina in rat model with chronic ocular hypertension

AIM: To study the expressive variation of Nogo-A on rat retina in the process of chronic ocular hypertension. METHODS: Thirty-six healthy adult male Wistars were randomly divided into control group (6 rats) and chronic hypertension group (30 rats). Chronic hypertension was created by cauterizing the superficial scleral veins. Immunohistochemistry technique was used to evaluate the expressive varieties of Nogo-A at different time points during the course of chronic ocular hypertension. RESULTS: The success of the model was indicated by over 40% of increase in the IOP as compared with normal rats. Compared with control group, as time passed chronic hypertension group gradually had detectable morphology changes in the retina. At the 21st day of chronic ocular hypertension, retinas became thinner and the quantity of retinal ganglion cells (RGC) decreased (P<0.05). Assoicated with the morphological changes, the expression of Nogo-A was strongly increased (P<0.05). CONCLUSION: Myelin associated protein Nogo-A plays a part in the process of chronic ocular hypertension.     

Expression and role of autophagy related protein p62 and LC3 in the retina in a rat model of acute ocular hypertension

AIM: To investigate the expression and possible role of the autophagy related protein p62 and LC3 in the retina based on a rat model of acute ocular hypertension.METHODS: Fifty rats were randomized into five groups: control group A, B, C, and D. Groups A to D all received normal saline perfusion into the anterior chamber with pressure of 80 mm Hg for one hour, and retina tissue was obtained at 6, 12, 24 and 48 h after perfusion respectively, to investigate the activation of autophagy following ischemiareperfusion. The distribution and semi-quantification of autophagy related protein p62 and LC3 in the retina were detected using immunohistochemistry technique. The expression level of these two proteins was evaluated using Western blot.RESULTS: The number of retinal ganglion cells(RGCs) decreased with increasing reperfusion time, and significant reduction in the retinal thickness was observed 48 h after perfusion. In normal adult rats, LC3 protein was mainly expressed in the ganglion cell layer(GCL), and p62 protein was expressed in the nerve fiber layer(NFL), GCL, inner plexiform layer(IPL), inner nuclear layer(INL) and outer plexiform layer(OPL). In comparison to the control group, the expression level of LC3-II was higher in all the experimental groups(P<0.05), with the peak expression at 12 h after reperfusion. Additionally, the expression level of p62 was higher in all the experimental groups than the control(P<0.05, except for group A), with the peak level occurred 24 h after reperfusion. CONCLUSION: Both p62 and LC3 show low level and uneven expression in the retina of normal adult rats. Acute ocular hypertension can lead to upregulation of LC3-II and p62 expression in the retina. Autophagy flux is damaged 12 h after reperfusion, potentially resulting in further loss of RGCs.