ApoE基因缺失小鼠视网膜及Bruch膜组织形态观察

目的 探讨ApoE基因缺失(apolipoprotein E-deficient,ApoE-/-)小鼠视网膜及Bruch膜的组织形态改变.方法 随机将24只2月龄C57BL小鼠分为高脂组和普食组,每组12只,另用2月龄ApoE-/-小鼠12只作为普食组,喂养4个月后检测各组动物血浆总胆固醇(total cholesterol,TC)、低密度脂蛋白(low density lipoprotein,LDL)、甘油三脂(triglyceride,TG)含量,光镜和电镜下观察视网膜外核层(outer nuclear layer,ONL)细胞、视网膜色素上皮(retinal pigment epithelium,RPE)细胞和Bruch膜形态结构改变,半定量检测视网膜ONL、RPE细胞数和Bruch膜厚度.结果 6月龄ApoE-/-普食组小鼠、6月龄C57 BL高脂组小鼠、6月龄C57BL普食组小鼠血浆TC的含量分别为(9.72±0.41)mmol·L-1、(6.97±0.54) mmol·L-、(5.15±0.31)mmol·L-1,LDL的含量分别为(1.05±0.16)mmol·L-1、(1.25±0.10)mmol·L-1、(0.43±0.08)mmol·L-1,TG的含量分别为(0.78±0.16) mmol·L-1、(0.56±0.19)mmol·L-1、(0.53±0.17)mmol·L-1,ONL细胞核面积分别为(23 208.00±716.91)μm2、(24 269.00±1263.13) μm2、(26 952.00±2449.10) μm2,RPE细胞数目分别为10.20±0.83、11.70±0.62、13.80±1.01,Bruch膜厚度分别为(0.87±0.04) μm、(0.77±0.07) μm、(0.59±0.03) μm.TC:6月龄ApoE-/-普食组小鼠比6月龄C57BL高脂组和普食组明显增高(P＜0.001);LDL:6月龄ApoE-/-普食组小鼠比6月龄C57BL普食组明显增高(P ＜0.001);TG:6月龄ApoE-/-普食组小鼠比6月龄C57BL普食组明显增高(P＜0.05),比6月龄C57BL高脂组增高(P＞0.05).光镜下观察发现:6月龄ApoE-/-普食组小鼠视网膜ONL、RPE细胞排列紊乱、萎缩;半定量结果显示,6月龄ApoE-/-普食组小鼠比6月龄C57BL普食组视网膜RPE细胞明显减少(P＜0.001),ONL细胞数显著减少(P＜0.01),Bruch膜显著增厚(P＜0.001).6月龄ApoE-/-普食组小鼠比6月龄C57BL高脂组视网膜RPE细胞明显减少(P＜0.O1),ONL细胞数减少(P＞0.05),Bruch膜显著增厚(P＜0.05).电镜观察发现:6月龄ApoE-/-普食组小鼠RPE基底膜皱褶减少,胞质内线粒体轻度肿胀,可见核边聚现象,Bruch膜纤维结构不均匀,弹力层断裂,有空泡出现.结论 ApoE-/-普食组小鼠出现显著高脂血症,引起视网膜ONL、RPE及Bruch膜呈现AMD的病理改变,表明ApoE-/-与AMD具有明显相关性.     

血脂异常ApoE基因缺失小鼠视网膜色素上皮细胞胞浆内黑色素和脂褐素的改变

目的 探讨血脂异常载脂蛋白E(apolipoprotein E,ApoE)基因缺失(ApoE-/-)小鼠视网膜色素上皮(retinal pigment epithelium,RPE)细胞胞浆内黑色素和脂褐素的改变.方法 将24只2个月龄ApoE-/-小鼠随机分为高脂组和普食组,每组12只,另用12只2个月龄C57BL小鼠作为普食组,喂养4个月后检测其血浆总胆固醇(total plasma cholesterol,TC)、低密度脂蛋白(low density lipoprotein,LDL)、甘油三酯(plasma triglyceride,TG)含量,Masson三色、银氨液浸染法和醛品红法染色后光镜下观察视网膜光感受器细胞(photoreceptor cell,PRC)、RPE细胞、Bruch膜形态结构,半定量检测视网膜PRC数、RPE细胞数、Bruch 膜厚度、RPE细胞胞浆内黑色素和脂褐素含量,采用逐步引入-剔除法多元线性回归分析RPE细胞数与TC、LDL、TG含量的相关性及RPE细胞胞浆内黑色素、脂褐素含量与PRC数、RPE细胞数、Bruch膜厚度的相关性.结果ApoE-/-高脂组、ApoE-/-普食组、C57BL普食组小鼠血浆TC的含量分别为(13.262±0.458) mmol·L-1、(9.722±0.412) mmol·L-1、(5.150±0.308)mmol·L-1,LDL的含量分别为(2.030±0.186)mmol·L-1、(1.047±0.158)mmol·L-1、(0.427±0.080) mmol·L-1,TG的含量分别为(1.233±0.199) mmol·L-1、(0.777±0.161)mmol·L-1、(0.530±0.168) mmol · L-1,PRC细胞核面积分别为(20 135.000±1473.272) μm2、(23 208.000±716.913) μm2、(26 952.000±2449.104) μm2,RPE细胞数分别为8.900±1.148、10.200±0.835、13.800±1.016,Bruch膜厚度分别为(0.982±0.071) μm、(0.870±0.042) μm、(0.594±0.031) μm.ApoE-/-高脂组和普食组小鼠TC、LDL、TG含量较C57BL普食明显增高(P＜0.05或0.001).光镜下观察发现:ApoE-/-高脂组和ApoE-/-普食组小鼠视网膜PRC、RPE细胞排列紊乱、萎缩,RPE细胞内黑色素分布稀疏、面积小,脂褐素分布密集、范围广,Bruch膜粗细不等,胶原分叉,部分小鼠视网膜下可见玻璃膜疣形成;半定量结果显示,ApoE-/-高脂组和ApoE-/-普食组小鼠视网膜PRC细胞核面积、RPE细胞数较C57BL普食组明显减少(P＜0.01或0.001),Bruch膜显著增厚(P ＜0.001),RPE细胞内黑色素含量明显降低(P＜0.01或0.001),脂褐素含量明显增高(P ＜0.001).多元线性回归分析显示,RPE细胞数与TC、LDL、TG含量呈显著负相关(r=-0.911、-0.888、-0.669,P=0.000、0.00、0.001),黑色素含量与Bruch膜厚度呈明显负相关(r=-0.691,P =0.001),与RPE细胞数、PRC数呈明显正相关(r=0.745、0.408,P=0.000、0.047).脂褐素含量与Bruch膜厚度呈明显正相关(r =0.830,P=0.000),与RPE细胞数、PRC数呈负相关(r=-0.826、-0.340,P=0.000、0.084).结论 血脂异常ApoE-/-小鼠可出现明显高脂血症,引起视网膜RPE细胞损伤,细胞内黑色素减少、脂褐素增加,导致年龄相关性黄斑变性产生.     

健脾祛瘀法对血脂异常ApoE基因缺失小鼠色素上皮VEGF及VEGFR2的影响

目的：通过观察血脂异常ApoE缺失（apolipoprotein E-deficient,ApoE-/-）小鼠视网膜色素上皮（retinal pigment epithelium,RPE）组织及超微结构,探讨健脾祛瘀法对其RPE层VEGF/VEGFR2信号传导通路的影响。 方法：随机将36只2月龄ApoE-/-小鼠分为3组,即：普食组（A组）、高脂组（B组）、治疗组（C组）,每组动物12只,喂养5mo后检测其体质量、血脂、血流变,RPE层行光镜、免疫组织化学、透射电镜观察,Western-blot检测VEGF表达。 结果：7月龄ApoE-/-小鼠B组体质量、血脂、血流变指标均高于A组和C组,差异具有统计学意义（P<0.05）。A组RPE厚度明显高于B组和C组,差异具有统计学意义（P<0.05）,但B组与C组之间比较差异无统计学意义（P>0.05）。B组RPE层VEGF及VEGFR2染色面积及积分光密度均高于A组和C组,差异具有统计学意义（P<0.05）。B组视网膜VEGF蛋白的相对表达量明显高于A组和C组,差异具有统计学意义（P<0.05）。 结论:健脾祛瘀法对改善血脂异常ApoE-/-小鼠的血脂、血流变等生化指标有积极作用,并且提示对该AMD动物模型中RPE层VEGF/VEGFR2信号传导通路有一定调控作用。     

驻景丸加减方对干性年龄相关性黄斑变性模型小鼠视网膜的保护作用

目的　探讨驻景丸加减方对干性年龄相关性黄斑变性(AMD)模型小鼠视网膜的保护作用以及对核因子E2相关因子2 (Nrf2)通路和自噬的影响。方法　取6月龄健康雄性C57BL/6小鼠90只，将小鼠随机分为:年龄对照组(正常饮食)、溶媒对照组(高脂饮食+氢醌)、对照药物组(高脂饮食+氢醌+莱视盯)及中药组(高脂饮食+氢醌+驻景丸加减方，分低、中、高三个剂量组)6组，每组15只。莱视盯主要成分:叶黄素、β-胡萝卜素、葡萄糖酸锌 (每100 g含叶黄素 2.20 g、β-胡萝卜素 0.86 g、锌 1.50 g)。各组小鼠灌胃给药，每天1次，持续3个月。动物观察期满处死并摘取眼球，透射电镜观察视网膜组织，测量视网膜色素上皮(RPE)下沉积物面积及Bruch膜厚度，检测视网膜匀浆活性氧自由基(ROS)、丙二醛 (MDA) 含量，Western blot检测Nrf2、血红素加氧酶-1(HO-1)、醌氧化还原酶-1(NQO-1)、p62和LC3蛋白表达水平。结果　与年龄对照组相比，溶媒对照组RPE下沉积物面积显著增大、Bruch膜明显增厚，差异均有统计学意义(均为P<0.01)；与溶媒对照组相比，中药中、高剂量组RPE下沉积物面积减小、Bruch膜厚度变薄，差异均有统计学意义(均为P<0.01)；与溶媒对照组相比，中药低剂量组和对照药物组RPE下沉积物面积也均减小，差异均有统计学意义(均为P<0.05)，但Bruch膜厚度变化不明显。与溶媒对照组相比，中药中、高剂量组ROS、MDA含量显著降低 (均为P<0.01)，中药低剂量组和对照药物组ROS、MDA含量也均降低 (均为P<0.05)；与溶媒对照组相比，中药中、高剂量组细胞质Nrf2蛋白表达显著下调，细胞核Nrf2蛋白表达上调 (均为P<0.01)，中药低剂量组和对照药物组细胞质Nrf2蛋白表达下调，细胞核Nrf2蛋白表达上调 (均为P<0.05)；与溶媒对照组相比，中药中、高剂量组HO-1、NQO-1、p62和LC3 II蛋白表达均显著上调 (均为P<0.01)，中药低剂量组HO-1、p62和LC3 II蛋白表达均上调 (均为P<0.05)，对照药物组HO-1和LC3 II蛋白表达均上调 (均为P<0.05)。结论　驻景丸加减方可激活Nrf2通路，上调下游靶基因酶HO-1、NQO-1表达，快速清除ROS、MDA；增强自噬，加快清除氧化损伤的细胞器，对干性AMD模型小鼠视网膜有保护作用。     

MMP-2和PEDF在氧诱导小鼠视网膜新生血管中的表达

目的探讨基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)和色素上皮衍生因子(pigment epithelium derived factor,PEDF)在氧诱导小鼠视网膜新生血管中的表达和意义。方法随机选取80只健康C57BL/6J小鼠进行分组,取对照组(40只)和高氧组(40只)小鼠眼球作ADP酶视网膜铺片、病理切片及免疫组织化学法检测,观察视网膜血管的改变,计算视网膜新生血管突破内界膜的内皮细胞核数及检测MMP-2、PEDF蛋白的表达。结果高氧组视网膜大量新生血管形成;新生血管突破内界膜的内皮细胞核数为(32.45±1.34)个,与对照组(1.27±0.20)个相比差异有统计学意义(t=10.345,P<0.05)。高氧组与对照组相比,MMP-2蛋白在神经节细胞层、内丛状层、内核层和突破视网膜内界膜的新生血管中高表达(P<0.05);PEDF蛋白在神经纤维层、神经节细胞层、内丛状层、感光细胞层及视网膜色素上皮细胞层低表达(P<0.05)。两者表达呈显著负相关(r=-0.785,P<0.05)。结论 MMP-2和PEDF共同维持视网膜新生血管的形成。     

血脂异常对C57BL小鼠晶状体的影响

目的探讨实验性血脂异常对C57BL小鼠晶状体组织结构的影响。方法将48只3月龄雄性C57BL小鼠随机分为3月龄普食组、8月龄普食组和8月龄高脂组，前两组普通饲料喂养，第3组高脂饲料喂养。各组动物按时处死后做血液生化指标检测，同时摘取眼球做光镜和电镜病理切片观察。结果8月龄高脂组小鼠的体重明显高于3月龄普食组和8月龄普食组（P〈0．01），8月龄高脂组小鼠的血脂、血液流变学指标和血清丙二醛显著高于3月龄普食组和8月龄普食组（P〈0．01），8月龄高脂组小鼠晶状体中央前囊膜厚度高于3月龄普食组和8月龄普食组（P〈0．01）。结论血脂异常能够加速C57BL小鼠的晶状体改变，其改变与白内障的发生密切相关。     

高脂诱导肥胖小鼠的视网膜变性及其与氧化应激的关系

目的:探讨高脂饮食建立的C57BL/6小鼠肥胖模型的视网膜变性的超微结构改变及其与氧化应激的关系。方法:高脂饲料喂养19wk后,小鼠分为肥胖抵抗(DIO-R)组和肥胖倾向(DIO)组,同时对照组小鼠给予基础饲料。应用光镜、透射电镜及TUNEL法检测3组小鼠视网膜超微结构的改变及凋亡情况,并应用生化方法检测视网膜的氧化和抗氧化指标。结果:与对照组及DIO-R组比较,DIO组小鼠视网膜及外核层(ONL)变薄(P<0.01);感光细胞出现凋亡及坏死,其外段视盘膜排列紊乱,部分溶解、断裂,神经节细胞体积变小,细胞质分布紊乱,核染色质固缩;TUNEL法检测结果显示,在ONL层可见较多的凋亡细胞,其细胞凋亡率显著升高(P<0.01)。与对照组比较,DIO组小鼠视网膜匀浆丙二醛(MDA)含量明显升高(P<0.01),超氧化物歧化酶(SOD)活力显著下降(P<0.05)。结论:高脂诱导的肥胖可导致C57BL/6小鼠视网膜变性及细胞凋亡,其机制可能与氧化应激有一定关系。     

Captopril抑制鼠视网膜新生血管的形成(英文)

目的:探讨Captopril对鼠视网膜新生血管形成的抑制作用。方法:将60只7d龄小鼠随机分为对照组(30只)和治疗组(30只),置于体积分数750±50mL/L高氧环境下饲养5d后回到正常空气环境中饲养,出氧箱后治疗组每天1次玻璃体腔内注射2.7mL/kg Captopril,对照组注射9g/L的氯化钠注射液2.7mL/kg,连续5d。两组小鼠均于17d处死并摘除眼球,采用ADP酶视网膜铺片、HE染色及免疫组织化学法分别观察视网膜血管的改变、计数视网膜新生血管内皮细胞核数及检测MMP-2、PEDF蛋白的表达。结果:治疗组与对照组相比视网膜血管分布规则、分支良好、新生血管密度减少,且突破视网膜内界膜的血管内皮细胞核数目明显减少(P<0.05);治疗组MMP-2染色较对照组减弱,PEDF染色较对照组增强。结论:玻璃体腔内注射2.7mL/kg Captopril能够有效抑制高氧诱导下的小鼠视网膜新生血管形成, Captopril有望成为防治血管增生性视网膜病变的一种有效的方法。     

高氧诱导小鼠视网膜新生血管模型中端粒酶逆转录酶的表达变化

目的 研究高氧诱导视网膜新生血管模型中小鼠端粒酶逆转录酶(TERT)基因表达水平是否有变化,为进一步研究视网膜新生血管疾病的预防和治疗提供新的靶点.方法 实验研究.选取7 d龄C57BL/6J新生小鼠32只,分高氧组和对照组,每组16只.高氧组小鼠以密闭氧箱内以75%±2%氧浓度饲养5 d后置于正常氧浓度环境中,正常对照组小鼠于正常氧环境中饲养.于小鼠生后12、14及19 d时分别取高氧组和对照组小鼠各2只(4只眼),经尾静脉行2%伊文思蓝溶液灌注并做视网膜铺片,荧光显微镜下观察视网膜新生血管的形成情况.高氧模型组和正常对照组生后19 d幼鼠3只,行HE染色,光学显微镜下观察视网膜血管形态,观察突破内界膜的内皮细胞核数.取生后19 d高氧组和对照组小鼠,分别取其视网膜组织并提取总RNA,反转录成cDNA后行反转录PCR,2%琼脂糖凝胶电泳并照相.提取视网膜总RNA,反转录成cDNA后(同RT-PCR),配制荧光定量实时PCR反应体系(总计20μl),在60℃检测荧光信号,分析图像.分别取高氧模型组和正常对照组P19小鼠行眼球切片,常规处理后TERT抗体孵育37℃ 60 min,HRP酶标二抗孵育30 min,DAB显色,中性胶封片,镜下观察并照相.结果高氧诱导模型小鼠牛后12 d眼底后极部出现大片无灌注区,生后14 d眼底后极部出现新牛血管迂曲、渗漏等视网膜血管病变.生后17～19 d视网膜新生血管形成达到高峰.正常小鼠视网膜组织切片HE染色基本看不剑突出内界膜的血管芽及血管管腔,内界膜下视网膜内的血管内皮细胞核散在分布、数量较少;高氧组见大量突出内界膜伸向玻璃体腔的血管管腔及血管芽,内界膜下视网膜内也有大量血管内皮细胞增生.19 d高氧模型组小鼠视网膜TERT及bFGF mRNA表达较同日龄正常对照组小鼠明显提高,二者差异有统计学意义(F=8.575,5.667;P＜0.05).生后19 d实时PCR检测高氧模型组小鼠视网膜TERT mRNA表达较同日龄正常对照组小鼠明显上调,差异有统计学意义(F=173.104,P＜0.05).生后19 d高氧诱导小鼠视网膜新生血管模型中视网膜新生血管TERT表达阳性,同日龄对照组新生小鼠视网膜血管TERT表达阴性.结论高氧诱导视网膜新生血管小鼠模型中端粒酶逆转录酶和新生血管形成相关因子表达水平明显上调,可能会成为视网膜新生血管疾病预防和治疗的新靶点.(中华眼科杂志,2009,45:199-205)     

肝硬化患者黄斑区视网膜和脉络膜厚度的变化

目的：分析肝硬化患者和健康人的黄斑区视网膜厚度、视网膜体积和脉络膜厚度的差异,初步探讨肝硬化患者视网膜和脉络膜的改变。

方法：采用横断面观察研究。将2015-01/2018-03我院肝硬化患者84例168眼和健康人50例100眼(正常对照组)纳入研究,其中肝硬化患者包括肝硬化代偿期组34例68眼,肝硬化失代偿期组50例100眼。采用OCT对所有受检眼测量黄斑区视网膜厚度和体积。采用EDI-OCT行脉络膜厚度扫描,以Bruch膜强反射线到脉络膜巩膜交界面强反射线的垂直距离作为脉络膜厚度。分析三组受检眼黄斑区视网膜厚度、视网膜体积和脉络膜厚度的差异。

结果：三组受检者黄斑区各象限视网膜厚度和视网膜体积无差异(P>0.05)。三组中心凹下脉络膜厚度分别为338.50±70.44、357.00±89.16、319.53±74.37μm(P>0.05)。三组间中心凹鼻侧脉络膜厚度有差异(P<0.05); 肝硬化失代偿组与正常对照组比较,鼻侧脉络膜偏厚(P<0.05)。

结论：肝硬化失代偿期患者较健康者中心凹鼻侧脉络膜偏厚,肝硬化患者与健康人黄斑区视网膜厚度、视网膜体积无明显差异。     

小鼠视网膜急性谷氨酸损伤后囊泡膜与质膜谷氨酸转运体的表达

目的:探讨不同类型囊泡膜和质膜谷氨酸转运体在小鼠视网膜急性损伤后的表达变化及其可能性作用。方法:C57BL/6小鼠78只,随机分为3组:正常对照组6只,实验对照组36只(玻璃体内注射生理盐水),实验组36只(玻璃体内注射谷氨酸)。分别于注射后0.5,3,6,12,24,72h各处死6只,其中3只鼠的眼球用于冰冻切片,以免疫细胞化学法检测各时间点视网膜内Ⅰ型和Ⅱ型囊泡膜谷氨酸转运体(VGluT1和VGluT2)及3种质膜谷氨酸转运体:谷氨酸/天门冬氨酸转运体(glutamate/aspartate transporter,GLAST)、谷氨酸转运体-1(glutamate transporter-1,GLT-1)及兴奋性谷氨酸转运体-1(excitatory amino acid transporter-1,EAAC-1)的表达情况。另外3只鼠的眼球用于RNA抽提,以半定量RT-PCR检测各谷氨酸转运体mRNA的水平变化。结果:与实验对照组相比,实验组小鼠视网膜兴奋性损伤12h内,VGluT1和VGluT2的表达水平和分布情况均未看到明显变化;24～72h,虽然VGluT1的表达仍未发生改变,但VGluT2却明显地减少。GLAST,GLT-1及EAAC-1的表达在谷氨酸损伤3～12h均有明显增高,但在24～72h它们的表达水平开始逐渐降低。半定量RT-PCR结果与免疫细胞化学结果一致。结论:视网膜急性损伤12h内囊泡膜谷氨酸转运体表达没有发生变化,而质膜谷氨酸转运体表达明显升高。     

Effects of cholesterol and apolipoprotein E on retinal abnormalities in ApoE-deficient mice.

PURPOSE. To examine the pathologic changes in the retina of apolipoprotein E (apoE)-deficient mice fed a high-cholesterol diet. METHODS. ApoE-deficient mice (ApoE) were maintained on either regular mouse chow (ApoE-R) or a high-cholesterol diet (ApoE-C) for 25 weeks. Age-matched control C57BL/6J mice (C57) were also maintained on either regular mouse chow (C57-R) or a cholesterol-containing diet (C57-C). Retinal function was assessed by dark-adapted electroretinography (ERG). The eyes were embedded, sectioned, and analyzed by histologic and immunohistochemical methods, as well as by light and transmission electron microscopy. RESULTS. After the 25-week feeding period, ERG tracings of ApoE-C mice revealed significant increases of a- and b-wave implicit times when compared with the C57-R group of mice. In addition, there were reductions in oscillatory potential (OP) amplitudes in the ApoE-C group. However, a- and b-wave amplitudes appeared to be unchanged among the four groups of mice. Light microscopic examination of the retinas showed that compared with control C57-R mice, ApoE-C mice had significantly lower cell numbers in the inner and outer nuclear layers (85.1% +/- 4.6%, P < 0.05 and 81.4% +/- 3.7%, P < 0.01 of C57-R controls, respectively). Transmission electron microscopy of apoE-deficient mice revealed cells of the inner nuclear layer with condensation of nuclear chromatin and perinuclear vacuolization in focal areas. Bruch's membrane was also found to be thicker, and its elastic lamina appeared disorganized and discontinuous. Immunohistochemistry demonstrated diminished or no immunoreactivity for carbonic anhydrase II and calretinin in the retinal layers of apoE-deficient mice. CONCLUSIONS. Overall, there were increasing abnormalities of retinal function and cellular morphology among the four groups of mice in the order of C57-R < C57-C < ApoE-R < ApoE-C. These findings suggest that apoE and/or cholesterol play an important role in retinal function.     

Apolipoprotein E modulates establishment of HSV-1 latency and survival in a mouse ocular model

PURPOSE: To evaluate and compare the neuroinvasiveness and neurovirulence after ocular HSV-1 infection in ApoE knockout (ApoE-/-) and control C57BL/6 (ApoE+/+) mice. METHODS: Age-matched (14 weeks of age) C57BL/6J (ApoE+/+) female mice and female ApoE knockout (ApoE-/-) mice were inoculated by corneal scarification with HSV-1 strain 17Syn+. Analysis of HSV-1 replication in the mouse cornea was assessed through infectious virus assays of ocular (tear film) swabs at 1 to 5 days postinoculation (PI), slit-lamp examination (SLE) of corneas at PI days 1 to 7, and survival of infected mice. The contribution of apoE to the efficient establishment of latency was measured by real-time PCR quantitation of the latent viral genome in the trigeminal ganglia (TG) of infected mice. RESULTS: These studies showed that HSV-1 strain 17Syn+ replicates efficiently in the eyes, regardless of the host ApoE genotype. Neither the scoring of corneal pathology via SLE nor the infectious virus assay of the tear film resulted in any statistical differences between ApoE knockout (-/-) mice or the C57BL/6 (ApoE+/+) mice. In mice latently infected with HSV-1, our real-time PCR data showed significantly lower viral copy numbers of HSV-1 DNA in ApoE knockout (ApoE-/-) mice compared with C57BL/6 (ApoE+/+) mice. C57BL/6 (ApoE+/+) mice are more susceptible to the neurovirulence of HSV-1 strain 17Syn+ than female ApoE knockout (-/-) mice, as demonstrated by the fact that 50% (7/14) of the female C57BL/6 (ApoE+/+) mice inoculated with 17Syn+ died, as opposed to none (0/14) of the age- and sex-matched ApoE knockout mice. CONCLUSIONS: These data indicate that age (14 weeks) and sex-matched (female) wild mice with an ApoE null background (ApoE-/-) are more resistant and less efficient in the establishment of latency compared with ApoE+/+ mice in the C57BL/6 background.     

Retinoschisin is a peripheral membrane protein with affinity for anionic phospholipids and affected by divalent cations

PURPOSE: Retinoschisin (RS) is a retina-specific, secreted protein implicated in X-linked juvenile retinoschisis and essential for the structural and functional integrity of the retina. This biochemical characterization and ultrastructural localization of RS in intact murine retina was performed to further understanding of the molecular basis of its function. METHODS: Subcellular fractions and fractions enriched in photoreceptor inner and outer segments were prepared from mouse retina by differential or density gradient ultracentrifugation. Immunoblot analysis was used to assess the expression of RS in various subcellular compartments and its fractionation into soluble phase on treatment of retinal cell membranes with several solubilizing reagents. RS-lipid interactions were evaluated by a protein-lipid overlay assay that used wild-type and mutant forms of RS discoidin domain glutathione S-transferase (GST) fusion proteins. The subcellular localization of RS in mouse retina was visualized by pre-embedding immunogold electron microscopy. Ultrastructure was evaluated by transmission electron microscopy. RESULTS: RS was intimately associated with cell membranes of the retina. It was found to cluster on the outer leaflet of the plasma membrane of the photoreceptor inner segments, which synthesize and secrete it. It was released from the membrane at high pH, which is characteristic of a peripheral membrane protein. It was extracted from the membrane by the nonionic detergent NP-40, together with glycerophospholipids. Protein-lipid overlay assays indicated a preferential interaction between RS and anioic phospholipids. Extraction of RS from the membrane was inhibited by divalent cations. Photoreceptor inner segment morphology was markedly affected in RS(-)(/y) mice, which failed to express RS protein. CONCLUSIONS: RS in intact retina is a peripheral membrane protein. Although distributed over the two membrane faces, RS is associated primarily with the outer leaflet of the inner segment plasma membrane through anionic phospholipids and divalent cations. RS's localization in photoreceptors and its biochemical properties suggest a functional role locally, at the site of secretion and membrane adhesion, in maintaining the photoreceptor inner segment stability and architecture.     

Structural consequences after intravitreal bevacizumab injection without increasing apoptotic cell death in a retinopathy of prematurity mouse model

Purpose: To evaluate the effect of different bevacizumab concentrations on retinal endothelial cell proliferation, retinal structures and apoptotic activity after intravitreal injection in a retinopathy of prematurity (ROP) mouse model. Methods: A total of 35 of C57BL/J6 mice were exposed to 75 ± 2% oxygen from postnatal day 7 to postnatal day 12. On day 12, 10 mice (group C) were injected with 2.5 μg intravitreal bevacizumab (IVB), 11 mice (group D) were injected with 1.25 μg IVB, and 14 mice (group E) were injected with 0.625 μg IVB in one eye. The contralateral eyes were injected with isotonic saline (control group = group B). Four nonexposed mice served as negative controls (group A). Neovascularization was quantified by counting the endothelial cell proliferation on the vitreal side of the inner limiting membrane of the retina. Histological and ultrastructural changes were examined by light and electron microscopy. Terminal deoxynucleotidyl transferase deoxy‐UTP‐nick end labelling (TUNEL) was used to detect apoptosis. Results: The endothelial cell count per histological section was lower in groups C (p < 0.0001), D (p < 0.0001) and E (p < 0.0001) compared with the control group B. Histological evaluation showed no retinal toxicity in any group. Electron microscopy revealed hyperoxia‐induced mitochondrial dysmorphology in group B. Mitochondrial dysmorphology displayed dose‐dependent gradual increase in IVB‐injected eyes. Intravitreal bevacizumab induced no significant increase in apoptotic cell death. Conclusion: Bevacizumab suppresses endothelial cell proliferation in a ROP mouse model. In addition to hyperoxia‐induced mitochondrial dysmorphology of C57BL/J6 retina, morphological findings implicate further mitochondrial vulnerability because of bevacizumab without increase in apoptotic cell death.     

Evidence for pigment epithelium-derived factor receptors in the neural retina.

PURPOSE: The neurotrophic activity of pigment epithelium-derived factor (PEDF), an extracellular factor present in the retina, is mediated by binding to cell-surface receptors in responsive cell cultures. In the present study, the expression of PEDF receptors in native neural retinas from adult steers was examined. METHODS: Binding reactions were performed with (125)I-PEDF and fluoresceinated PEDF using plasma membranes, detergent-soluble membrane proteins, or cryosections of retina from adult bovine eyes. Radioligand-binding and competition analyses were performed with a computer-assisted program. Ligand blot analysis of detergent-soluble membrane proteins was performed with (125)I-PEDF followed by autoradiography. Ligand-affinity column chromatography of detergent-soluble membrane proteins was performed with PEDF-coupled resin followed by SDS-PAGE. Binding of fluoresceinated PEDF to retina cryosections was detected by confocal microscopy. RESULTS: Radioligand-binding assays showed that (125)I-PEDF bound in a specific and saturable fashion to one class of sites on retina membranes (K(d) = 2.5-6.5 nM; maximum binding [B(max)] = 1-48 x 10(10) sites/retina). A peptide of 44 amino acids (44-mer), identified as the receptor-binding region of PEDF, competed efficiently for (125)I-PEDF binding to retina membranes with kinetics similar to the full-length PEDF. Ligand blot analysis and ligand-affinity chromatography revealed a specific and high-affinity PEDF-binding protein of approximately 85 kDa in retina plasma membranes. Confocal microscopy showed that fluorescein-conjugated PEDF stained exclusively the inner segments of photoreceptors and cells of the ganglion cell layer in retinal cryosections. CONCLUSIONS: Altogether, these data conclusively demonstrate the existence of PEDF receptors discretely distributed on the surface of cells from the adult neural retina of bovine eyes. Furthermore, they provide evidence for the direct action of PEDF on photoreceptor and ganglion cell neurons and an anatomic basis for studies to assess PEDF neurotrophic effects on the adult retina.     

Antioxidant effects of vitamins C and E, multivitamin-mineral complex and flavonoids in a model of retinal oxidative stress: the ApoE-deficient mouse

The aim of the current study was to investigate the biochemical changes in the plasma and retina of apolipoprotein E deficient (apoE-/-) mice supplemented with various antioxidants. Ten wild type (WT-Con, C57BL/6) and 10 apoE-/- (AE-Con) mice received drinking water. Another 40 apoE-/- animals were divided into four groups of 10 mice each and received either chromocarbe diethylamine (AE-CD, 50mg/kg), cyaninosides chloride (AE-CC, 50mg/kg), multivitamin complex (AE-MC, 50mg/kg), or vitamins C and E (AE-CE, 100mg/kg and 200IU/kg). Cholesterol, triglycerides, and lipid peroxidation (thiobarbituric acid reactive substances [TBARS]) were measured in plasma, and TBARS and nitric oxide metabolites (NOx) concentration were determined in retinal homogenates. Transmission electron microscopy was performed to examine the retinal ultrastructure. AE-Con mice had significantly (P<0.05) increased oxidative stress in the plasma and retina with augmented production of retinal NOx compared with WT-Con mice. Retinal TBARS decreased in the AE-MC and AE-CE animals compared with the AE-Con group (P<0.05 and P<0.01, respectively). Only AE-CE treatment significantly (P<0.01) lowered retinal NOx. Morphologic retinal changes in the AE-Con group decreased in the AE-CE and AE-MC groups. There were no significant changes in the biochemical and structural parameters in the AE-CD and AE-CC groups. AE-Con mice had increased systemic and retinal oxidative stress compared with WT-Con animals. Vitamins C and E and the multivitamin-mineral complex reduced oxidative stress and ultrastructural retinal changes in this murine model of hypercholesterolemia.     

视黄酸视网膜下腔注射未能逆转鼠视网膜变性

目的：探讨视网膜下腔注射视黄酸对视网膜变性发展的阻断作用。方法：14d龄视网膜色素变性鼠(retinal degeneration mouse，Rd鼠)各8只视网膜下腔分别注射10^-3，mol／L浓度视黄酸和磷酸盐缓冲液(phosphate buffered saline，PBS)，术后1个月对注射眼进行病理取材。并取7、14、28d和3个月龄Rd鼠各3只作正常对照。结果：视网膜下腔注射视黄酸和PBS，1个月后均可见部分眼视网膜内核层细胞增殖，视网膜下腔注射视黄酸较注射PBS未见有明显差异。结论：通过视网膜下腔注射视黄酸并不能使已经变性的视网膜恢复正常或阻断视网膜变性的发展。