N-亚硝基-N-甲基脲诱导视网膜变性小鼠模型的特征

目的探讨N-亚硝基-N-甲基脲（MNU）诱导的小鼠视网膜退行性病变的形态和电生理特征。方法实验研究。32只5周龄的成年C57/BL小鼠随机分为对照组和MNU造模组,分别腹腔注射0.9%氯化钠溶液和MNU（60 mg·kg-1）。在给药后3、7和14 d取视网膜,免疫荧光染色观察光感受器形态、氧化损伤情况和神经节细胞的数量。电镜观察细胞核、线粒体和带状突触（synaptic ribbon）的超微结构。Ganzfeld视网膜电图（ERG）检测暗适应最大混合反应。采用独立样本t检验进行数据分析。结果免疫荧光显示：注射MNU后外核层（ONL）的厚度逐渐减小,OPN1SW标记的光感受器细胞的外节（OS）逐渐损伤,GFAP标记的Müller细胞增生明显,ONL层硝基酪氨酸（Nitrotyrosine）着色增多提示MNU能导致氧化损伤。Brn-3a标记的神经节细胞数量减少不明显。扫描电子显微镜显示：MNU处理后,光感受器细胞的核呈浓染色,线粒体和带状突触消失。ERG结果显示：MNU处理后3 d最大混合反应的a波和b波振幅下降。结论MNU导致视网膜外核层和外丛状层的凋亡,电生理表现和视网膜色素变性疾病一致。     

腹腔注射MNU对大鼠视网膜结构与功能的影响

目的　探讨N 甲基 N 亚硝脲 (N methyl N nitrosourea,MNU)对大鼠视网膜的毒性作用。方法　生后 5 0d的雌性SD鼠 76只 ,随机抽取 4只为正常对照组 ,余 72只等分为 3组 ,分别按体重一次性腹腔注射MNU 5 0、4 0、30mg·kg-1。各组每次 4只分别于6、12、2 4、4 8h ,3、5d行闪光视网膜电图 (ERG)检查 ,并于造模 1、2、3、5、7、10d全麻后处死动物 ,取眼球做病理切片。结果　MNU 5 0、4 0、30mg·kg-1剂量组ERGa波消失时间分别为 6、12h ,3d ,b波消失时间分别是 12、2 4h ,5d。HE染色显示 :以中周部视网膜为观察对象 ,MNU 30mg·kg-1组第 3天开始出现外颗粒层结构改变 ,第 10天光感受器细胞显著减少 ,但未消失 ;MNU 4 0和 5 0mg·kg-1组第 1天即出现外颗粒层排列紊乱 ,外颗粒层消失时间前者在第 10天 ,后者在第 5天。所有模型切片结果 ,其神经节细胞层、内颗粒层以及色素上皮层与正常组相比均未见明显差异。结论　MNU能选择性地损伤视网膜光感受器细胞。     

枸杞子提取物对光诱导视网膜损伤的保护作用

目的　探索枸杞子提取物对人视网膜色素上皮（ARPE-19）细胞及C57BL/6J小鼠视网膜光诱导损伤的保护作用。方法　ARPE-19细胞分为正常细胞对照组,光诱导细胞损伤组,细胞低、中、高剂量组（0.1 g·L^-1、0.5 g·L^-1、1.0 g·L^-1枸杞子提取物+光诱导细胞损伤）,测定各组细胞活力以及细胞内活性氧（ROS）含量的变化。40只C57BL/6J小鼠随机分为正常动物对照组,光诱导动物损伤组,动物低、中、高剂量组（280 mg·kg^-1、370 mg·kg^-1、460 mg·kg^-1枸杞子提取物+光诱导动物损伤组）,每组8只。各剂量枸杞子提取物干预组小鼠在6周龄开始给予枸杞子提取物灌胃,8周后再给予10 000 lux光照射24 h;光照结束后ERG评估各组小鼠视网膜功能,OCT检测视网膜外核层（ONL）厚度,FFA检测视网膜血管渗漏情况,HE染色后对视网膜ONL细胞进行计数,同时检测血清中丙二醛（MDA）含量和超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）活性。结果　光诱导细胞损伤组ARPE-19细胞活力下降为正常细胞对照组的61.88%,细胞内ROS含量为正常细胞对照组的1.52倍;细胞低、中、高剂量组ARPE-19细胞活力较光诱导细胞损伤组均明显上升,细胞内ROS含量均明显下降,并均呈剂量依赖性（均为P＜0.05）。与动物对照组相比,光诱导动物损伤组小鼠ERG暗适应a波、b波振幅和明适应b波振幅均明显下降,动物低、中、高剂量组各波的振幅均得到不同程度改善。光诱导动物损伤组小鼠视网膜出现萎缩灶、血管渗漏和血管末端成珠样结构,视网膜ONL厚度变薄,为（52.18±4.23) μm,ONL每列细胞数明显减少,为（17.63±1.30）个;动物低、中、高剂量组小鼠视网膜病理改变及视网膜ONL厚度与ONL每列细胞数均得到不同程度改善,尤其动物高剂量组改善最为明显,ONL厚度为（59.72±2.76）μm, ONL每列细胞数为（20.00±1.51）个,与光诱导动物损伤组相比差异均有统计学意义（P=0.007、0.004）。相对于光诱导动物损伤组,动物低、中、高剂量组小鼠血清SOD、GSH-Px活性均明显提高,MDA含量均明显下降,且均呈剂量依赖性（均为P＜0.05）。结论　枸杞子提取物可以一定程度上保护视网膜免受光损伤。     

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.     

银杏内酯B对N-甲基-N-亚硝脲诱导的大鼠视网膜变性的保护作用

目的观察不同剂量的银杏内酯B(GB)对N-甲基-N-亚硝脲(MNU)诱导的SD大鼠视网膜变性的保护作用。方法取出生后46d的SD大鼠86只,随机抽取6只为正常对照组;80只分为4大组,每组20只。随机分为模型对照组,GB大、中、小剂量组。各组每次4只分别于24h、48h、3、5、7d行右眼闪光视网膜电图(ERG)检查,并通过视网膜形态学分析测量中心视网膜的外视网膜厚度。结果ERG正常组a波振幅为(105·4±16·8)μV,b波振幅为(292·6±19·6)μV。模型对照组24h后,a波消失。GB各剂量治疗组a波消失时间分别为2、3、3d,小、中剂量组b波消失时间分别为5d和7d,大剂量组7d时b波振幅下降为正常的8%。中心视网膜厚度检查:正常组大鼠中心视网膜的外视网膜厚度为(98·4±1·8)μm。MNU处理7d模型对照组外视网膜厚度为(17·8±2·1)μm,而GB各剂量治疗组分别为(25·2±2·7)、(38·3±2·3)、(45·8±2·3)μm。结论GB对MNU诱导的实验性大鼠视网膜变性所致的大鼠视网膜光感受器细胞损伤和视功能损害具有保护作用,并且这种作用呈剂量依赖性。     

N-甲基-N亚硝脲诱导的大鼠视网膜损伤过程中光感受器间维生素A类结合蛋白和恢复蛋白的达达变化

观察N-甲基-N亚硝脲(MNU)诱导的大鼠视网膜损伤对光感受器间维生素A类结合蛋白(IRBP)和恢复蛋白(recoverin)在视网膜中的表达变化.方法雌性Sprague-Dawley大鼠30只随机分为正常对照组和MNU处理1、3、7、10d组,每组均为6只大鼠.不同时间MNU处理组大鼠按体重40 mg/kg给予一次性腹腔注射MNU;正常对照组大鼠腹腔注射生理盐水5 ml/kg.逆转录-聚合酶链反应(RT-PCR)和免疫荧光检测各组大鼠视网膜中IRBP和恢复蛋白的mRNA及蛋白表达.结果 RT-PCR检测结果显示,与正常对照组比较,不同时间MNU处理组IRBP的mRNA表达水平随MNU作用时间增加而逐渐降低,差异均有统计学意义(P＜0.01);恢复蛋白的mRNA表达水平随MNU作用时间增加而逐渐升高.MNU处理1d组恢复蛋白的mRNA表达水平与正常对照组比较,差异无统计学意义(P＞0.05);MNU处理组3、7、10d组恢复蛋白的mRNA表达水平与正常对照组比较,差异均有统计学意义(P＜0.05).免疫荧光显微镜观察结果显示,正常对照组大鼠视网膜IRBP主要表达在光感受器细胞内、外节;不同时间MNU处理组IRBP在视网膜各层均有表达并随MNU作用时间增加而逐渐降低.恢复蛋白阳性标记物主要见于视网膜内核层、内丛状层及节细胞层,随MNU作用时间增加恢复蛋白的表达逐渐增强.结论MNU可降低IRBP的表达,但增加恢复蛋白的表达.     

N-甲基-N-亚硝脲对猫视网膜光感受器细胞损害的研究

背景合理的视网膜色素变性（RP）动物模型的建立方法是进行RP干预和治疗的重要工具和手段。研究证实,N-甲基-N-亚硝脲（MNU）可造成哺乳动物光感受器细胞的选择性损害,但是否MNU可用于建立RP动物模型的报道较少。目的评估MNU对猫视网膜光感受器细胞的毒性损害作用。方法20只2岁龄的猫一次性股静脉注射MNU,并按照注射剂量的不同分为20、25、30、35、40mg／kgMNU组,每组4只。另外4只猫股静脉注射等量生理盐水作为对照组。MNU注射后每日观察实验猫的行为变化、瞳孔大小及对光反射情况,分别于注射后24h、72h、7d和14d用静脉注射过量的质量分数2％戊巴比妥钠处死实验猫,眼球摘除后制备视网膜切片进行组织病理学检查,评估不同剂量MNU对视网膜光感受器的影响。结果不同剂量MNU组的实验猫注射MNU7d后瞳孔散大,对光反射迟钝。MNU注射24h,猫视网膜光感受器细胞的损害以核固缩和排列紊乱为主,MNU注射72h,视网膜光感受器细胞的损害以外核层变薄和细胞碎裂为主,MNU注射7d后,40mg／kg注射组的实验猫均死亡,光感受器细胞层仅存在极少量细胞,MNU注射14d,视网膜光感受器细胞层均完全消失。各组视网膜损害程度与注射MNU剂量有关。结论MNU可以造成猫视网膜光感受器细胞的严重损害,MNU对视网膜光感受器的作用呈时间和剂量依赖性。     

视网膜内质网应激免疫组织化学抗体浓度选择的实验研究

目的:探索小鼠视网膜色素变性(RP)模型视网膜内质网应激免疫组织化学(IHC)染色抗体的最适浓度,为研究RP发病机制及干预措施提供相应的指标检测方法。方法:给予清洁级健康雄性C57BL/6J小鼠腹腔注射N-甲基-N-亚硝基脲(MNU,60mg/kg)制备RP小鼠模型,造模后第7d行视网膜电图(ERG)检测和苏木精-伊红(HE)染色验证造模成功,摘取眼球并采用IHC染色法检测视网膜内质网应激相关蛋白(IRE1、ATF6、PERK、GRP78、Caspase-12)表达情况。结果:IRE1、ATF6、PERK、GRP78、Caspase-12蛋白均在RP小鼠视网膜呈阳性表达,IRE1抗体浓度为1∶1000、ATF6抗体浓度为1∶500和1∶1000(此抗体两种浓度阳性表达无差异,P>0.05)、PERK抗体浓度为1∶1500、GRP78抗体浓度为1∶200、Caspase-12抗体浓度为1∶100时,阳性表达强度适宜,且与各自其余抗体浓度相应蛋白阳性表达均有差异(P<0.05)。结论:内质网应激相关蛋白抗体浓度IRE1为1∶1000、ATF6为1∶500和1∶1000、PERK...     

白蒺藜对光损伤小鼠光感受器细胞的保护作用

目的 探讨白蒺藜对小鼠视网膜光损伤的保护作用.方法 采用10 000 lux白炽光光照30 min建立视网膜光损伤模型,将BALB/c小鼠分为正常对照组、模型组和治疗组,每组各6只.光照前0.5h治疗组腹腔注射白蒺藜水煎液100 μL(生药100mg/20 g),正常对照组和模型组腹腔注射等量生理盐水.光照后3h、7d分别通过OCT检测小鼠视网膜形态学改变;造模后7d处死小鼠,取眼球固定、包埋、切片进行HE染色,视紫红质和短波敏感视蛋白(short wave-length sensitivity opsin,M-opsin)的免疫荧光化学染色,观察视网膜病理改变情况.结果 正常对照组内外核层结构清楚、界限明显,视网膜视杆细胞内外节排列整齐;模型组视网膜结构层次模糊且外核层变薄,与正常对照组差异显著;治疗组小鼠视网膜结构与正常对照组没有显著差异.与正常对照组比较,模型组视紫红质和M-opsin表达降低,治疗组显著提高了视紫红质和M-opsin的表达.模型组较正常对照组视网膜外核层厚度显著变薄(P＜0.05);治疗组较模型组能显著性预防小鼠视网膜外核层变薄(P＜0.05).结论 小鼠视网膜光损伤模型造模成功,白蒺藜对光损伤视网膜具有显著的保护作用.     

山茱萸多糖对衰老小鼠视网膜相关因子表达的影响

目的 观察山茱萸多糖对D-半乳糖致衰老小鼠视网膜组织相关因子表达的影响.方法 昆明小鼠90只,随机分为3组,每组30只,其中给药组:100 mg·kg-1·d-1皮下注射D-半乳糖45 d后,按照0.4 g·kg-1体质量经胃给予山茱萸多糖,连续给药30 d;衰老组:100 mg·kg-1·d-1皮下注射D-半乳糖45 d后,经胃给予相当于给药组药量体积的温开水连续30 d:对照组:每日上午侧腹部皮下注射生理盐水,剂量参照衰老组D-半乳糖给药量,45 d后经胃给予相当于给药组药量体积的温开水连续30 d.于末次给药后次日处死小鼠,测定小鼠视网膜组织超氧化物歧化酶(total superoxide dismutase,T-SOD)含量及总抗氧化能力(total antioxidant capacity,T-AOC),RT-PCR法测定小鼠视网膜组织SIRT1、p53 mRNA的表达.结果 对照组视网膜组织中的T-SOD和T-AOC分别为(341.27±10.22)U·mgprot-1和(287.40±10.70)U·mgprot-1,明显高于衰老组的(66.92±22.74)U·mgprot-1和(104.36±6.76)U·mgprot-1及给药组的(78.61±27.70)U·mgprot-1和(209.44±11.11)U· mgprot-1(均为P＜0.05),而给药组与衰老组相比差异均无统计学意义(均为P＞0.05).与对照组小鼠视网膜组织中SIRT1、p53 mRNA相对表达量(0.64±0.10、0.12±0.03)相比,衰老组及给药组的SIRTl mRNA表达下降,相对表达量分别为0.13±0.05、0.24±0.07,p53 mRNA的表达上升,相对表达量分别为0.82±0.06、0.54±0.16,给药组与衰老组相比差异均无统计学意义(均为P＞0.05).结论 随着机体的衰老,SIRT1在视网膜组织中的表达减少,p53在视网膜组织中的表达增多;山茱萸多糖尚不能有效地提高D-半乳糖致衰老小鼠视网膜组织中SIRT1基因的表达.     

尾静脉注射骨髓间充质干细胞对小鼠视网膜激光损伤后细胞凋亡的影响

目的 观察尾静脉注射骨髓间充质干细胞（MSCs）对小鼠视网膜激光损伤后细胞凋亡的影响。 方法 体外培养绿色荧光蛋白（GFP）标记的C57BL/6小鼠骨髓间充质干细胞（GFP-MSCs）。135只C57BL/6小鼠随机分为正常对照组、损伤对照组和MSCs治疗组,分别为15、60、60只。采用激光光凝方式建立视网膜激光损伤模型。激光光凝后1 d,MSCs治疗组小鼠尾静脉注射0.2 ml GFP-MSCs细胞悬液（含MSCs 1×10⁶个）,损伤对照组小鼠尾静脉注射等体积磷酸盐缓冲液。分别于激光光凝后3、7、14、21 d,处死各组小鼠并摘除眼球。采用苏木精-伊红（HE）染色观察视网膜组织形态,并用图像处理软件测量激光斑直径和外核层光感受器细胞核完全缺损区域直径;原位末端标记（TUNEL）法检测细胞凋亡情况;荧光显微镜观察MSCs治疗组GFP-MSCs的迁移情况。 结果 光学显微镜观察发现,正常对照组视网膜组织结构完整;损伤对照组及MSCs治疗组均可见视网膜组织结构损伤,但MSCs治疗组较损伤对照组损伤程度减轻。激光光凝后3 d,损伤对照组与MSCs治疗组激光斑直径比较,差异无统计学意义（t=0.964,P＞0.05）;MSCs治疗组外核层细胞核完全缺损区域直径小于损伤对照组,差异有统计学意义（t=5.769,P＜0.05）。激光光凝后7、14、21 d,MSCs治疗组激光斑直径（t=5.180、5.417、2.381）和外核层细胞核完全缺损区域直径（t=3.530、3.224、3.162）均小于损伤对照组,差异均有统计学意义（P＜0.05）。 激光光凝后3、7、14、21 d,正常对照组小鼠视网膜内均未见凋亡细胞;损伤对照组及MSCs治疗组小鼠损伤部位视网膜内可见凋亡细胞;MSCs治疗组平均细胞凋亡数均小于损伤对照组,差异均有统计学意义（t=11.142、7.479、6.678、3.953,P＜0.05）。荧光显微镜观察发现,激光光凝后3、7、14、21 d均未见MSCs治疗组GFP-MSCs大量向视网膜内迁移;仅于激光光凝后7、14 d时在视网膜下及脉络膜新生血管处有少量迁移。结论 尾静脉注射MSCs能有效限制小鼠视网膜激光损伤范围及抑制细胞凋亡。     

米诺环素对视网膜色素变性小鼠光感受器细胞的保护作用

目的 观察米诺环素对视网膜色素变性（RP）的rd小鼠［C3H/HeN (Pde6brd-/rd-)］RP过程的影响。方法 40只新生rd小鼠随机分为10组，5组为实验组，5组为对照组，每组4只小鼠。实验组，出生后每日腹腔注射米诺环素22.5 mg/kg；对照组，出生后每日腹腔注射生理盐水10 ml/kg。在出生后1、7、14、21、28 d各处死一组实验组和对照组小鼠，取眼球做组织学观察并行凋亡细胞检测，并对两组视网膜光感受器细胞数、外核层厚度以及凋亡细胞数目进行统计分析。结果 （1）rd小鼠出生后7 d光感受器细胞开始凋亡，14 d达高峰，28 d光感受器细胞完全消失；（2）出生后7 d实验组光感受器细胞数目和外核层厚度与对照组比较差异无统计学意义；（3）出生后14、21 d实验组光感受器细胞数目和外核层厚度多于对照组相应时间点，差异有统计学意义（14 d：t=-3.03、P=0.016，t=-4.469，P=0.004；21d: t=-8.782、P＜0.001，t=-3.497、P=0.004）；（4）出生后7、14 d实验组外核层凋亡细胞数目少于对照组相应时间点，差异有统计学意义（t=-3.497、P=0.004，t=-8.782、P＜0.0001）。结论 米诺环素在rd小鼠RP早期可以延缓光感受器细胞丢失，但不能完全阻止RP的发生。     

Caspase-3 inhibitor rescues N -methyl- N -nitrosourea-induced retinal degeneration in Sprague-Dawley rats

The effect of a caspase-3 inhibitor on N -methyl- N -nitrosourea (MNU)-induced retinal degeneration was investigated. Sixty mg kg(-1)MNU was given intraperitoneally to 50 day old female Sprague-Dawley rats, and 4000 ng Ac-DEVD-CHO, a caspase-3 inhibitor, was injected intravitreally twice at 0 and 10 hr after MNU. In both peripheral and central retina, an apoptotic index of the photoreceptor cells 24 hr after MNU treatment was calculated by TUNEL labeling, and retinal damage 7 days after MNU treatment was evaluated from retinal thickness and a retinal damage ratio (length of damaged retina : whole retinal length). In MNU-treated rats, the TUNEL index 24 hr post-MNU was 79.5% in the peripheral and 83.7% in the central retina, while the Ac-DEVD-CHO injection significantly reduced it to 59.7 and 71.8%, respectively. Total retinal thickness 7 days after MNU was 38 microm in the peripheral and 75 microm in the central retina. Ac-DEVD-CHO injection increased these values to 72 and 77 microm, respectively. The retinal damage ratio 7 days after MNU was 98.5%. Ac-DEVD-CHO injection significantly reduced this value to 54.4%. The use of a caspase-3 inhibitor was effective in the suppression of MNU-induced retinal apoptosis and may be a therapeutic intervention in human retinitis pigmentosa.     

Diurnal rhythm in the human rod ERG: retinitis pigmentosa

Rod ERGs were measured at three times of day over an extensive range of retinal illuminances in six light-entrained patients with autosomal recessive or isolate forms of retinitis pigmentosa and at five times of day in six light-entrained normal volunteers. B-wave amplitude versus retinal illuminance functions from each time of day were described by determining the parameters of the best-fit Naka-Rushton function. Results from normal subjects showed that rod ERG threshold (defined as the log retinal illuminance necessary to elicit a 2.0 microV response) was elevated 1 1/2 hr after daily light-onset due to both an increase in log k (semi-saturation constant) and a decrease in log Vmax (maximum rod amplitude). The magnitude of the threshold elevation 1 1/2 hr after light-onset was comparable in patients with retinitis pigmentosa and normal subjects. Whereas thresholds returned to pre-light exposure levels rapidly during the light-phase of the daily cycle in normal subjects, thresholds continued to rise in patients with retinitis pigmentosa due primarily to a further increase in log k. These findings are consistent with abnormal rod photoreceptor disc renewal mechanisms in retinitis pigmentosa.     

Biochemical characterization of retinal protein and phospholipid synthesis in mice exposed transplacentally to N-methyl-N-nitrosourea

Transplacental exposure to the DNA alkylating agent N-methyl-N-nitrosourea on day 16 of gestation in CD-1 albino mice induces a degeneration of the retina, the severity of which depends upon the dosage level of the drug. A 1 mg kg-1 dose provokes a progressive retinal degeneration in the offspring which begins at about 4-6 weeks of age and is characterized by gradual thinning of the retinal layers. A 15 mg kg-1 dosage of MNU provokes severe retinal dysplasia characterized morphologically by rosettes in the outer nuclear layer and loss of rod outer segments (ROS). In the present biochemical experiments, retinal protein synthesis was examined in mice 2-, 4-, and 6 weeks of age exposed to 1 mg kg-1 MNU and 2- and 5 weeks of age exposed to 15 mg kg-1 MNU. Phospholipid synthesis was examined in mice 2-, 4-, 6- and 12 weeks of age exposed to 1 mg kg-1 MNU and at 2 weeks in mice exposed to 15 mg kg-1 MNU. Retinas were incubated for 2 hr at 37 degrees C in media supplemented with either [3H]leucine for protein synthesis studies or [3H]glycerol for phospholipid synthesis experiments. Aliquots of crude ROS and the retinal debris were taken for protein determination, scintillation counting, SDS-PAGE separation of labeled opsin, phosphorus determination and TLC separation of phospholipids. Results indicated that mice exposed to 1 mg kg-1 MNU did not differ significantly from age-matched controls in these measurements, whereas mice exposed to 15 mg kg-1 MNU were significantly different from controls. These results suggest that even as early as 2 weeks of age protein and lipid metabolism are adversely affected in mice exposed to the higher dose of the alkylating agent at a critical time in retinal development, but general protein and lipid synthesis is not affected in animals exposed to 1 mg kg-1 MNU at least up to 12 weeks of age. These studies suggest further investigation of more subtle derangement in the retinal function in animals exposed to low levels of MNU.     

新型Micron Ⅳ视网膜影像系统在三种小鼠疾病模型中的应用

目的 观察新型Micron Ⅳ视网膜影像系统检查在3种小鼠疾病模型中的应用效果。方法 健康雄性C57BL/6J小鼠24只。分别建立氧诱导视网膜病变（OIR）模型组、N-甲基-N-亚硝脲（MNU）模型组、N-甲基-D-天冬氨酸（NMDA）模型组。应用Micron Ⅳ视网膜影像系统对3种模型组小鼠行眼底彩色照相;OIR模型组、MNU模型组小鼠行荧光素眼底血管造影（FFA）、光相干断层扫描(OCT)检查;MNU模型组、NMDA模型组小鼠行视网膜电图（ERG）检查。结果 OIR模型组小鼠眼底视网膜血管高度扩张、纡曲,走形僵直;FFA检查可见形态异常、分布紊乱的新生血管丛,荧光渗漏致玻璃体混浊;OCT检查可见视网膜前纤维血管组织及纡曲扩张血管后方粗大的阴影暗区。MNU模型组小鼠眼底视盘呈蜡黄色萎缩,视网膜无明显色素性改变;FFA检查可见视网膜血管轻微变细;OCT检查可见视网膜厚度和结构变化不明显;ERG检查结果 显示,a、b波潜伏期变化不大,建模型后2、3 d最大混合反应a波振幅分别为（15.38±4.36）、（13.78±5.52） μV,明显下降。NMDA模型组小鼠眼底视网膜呈苍白色改变,血管痉挛纡曲;ERG检查结果 显示,a、b波潜伏期变化不大,建模型12、24 h最大混合反应b波振幅分别为（72.28±7.18）、（65.35±9.18） μV,明显下降。结论 Micron Ⅳ视网膜影像系统能实时、无创观察视网膜结构和功能。     

Disease course of patients with X-linked retinitis pigmentosa due to RPGR gene mutations

PURPOSE: To measure the rates of visual acuity, visual field, and ERG loss in patients with X-linked retinitis pigmentosa due to RPGR mutations and to determine whether these rates differ from those of patients with dominant retinitis pigmentosa due to RHO mutations. METHODS: Snellen visual acuities, Goldmann visual field areas (V4e white test light), and 30 Hz (cone) full-field ERG amplitudes were recorded for an average of 9.8 years in 113 patients with RPGR mutations. After censoring data to eliminate ceiling and floor effects, we used longitudinal regression to estimate mean rates of change and to compare these rates with those of a previously studied cohort of 134 patients with dominant retinitis pigmentosa due to RHO mutations, who were followed for an average of 8.9 years. Survival analysis was used to compare the age distribution of legal blindness in these two groups. To explain group differences in visual acuity, optical coherence tomograms were recorded in some patients to visualize central retinal structure. RESULTS: Mean annual exponential rates of decline for the patients with RPGR mutations were 4.0% for visual acuity, 4.7% for visual field area, and 7.1% for ERG amplitude. Each of these rates was significantly different from zero (P < 0.001). The rates of visual acuity and visual field loss were significantly faster than the corresponding rates in the RHO patients (1.6%, P < 0.001 and 2.9%, P = 0.002, respectively), whereas the rate of ERG amplitude loss was comparable to that in the RHO patients (7.7%, P = 0.39). The median age of legal blindness was 32 years younger in the RPGR patients than in the RHO patients, due primarily to loss of visual acuity rather than to loss of visual field. Loss of acuity in RPGR patients appeared to be associated with foveal thinning. CONCLUSIONS: Patients with X-linked retinitis pigmentosa due to RPGR mutations lose visual acuity and visual field more rapidly than do patients with dominant retinitis pigmentosa due to RHO mutations.     

P23H rhodopsin transgenic rat: correlation of retinal function with histopathology

PURPOSE: To correlate retinal functional changes with structural changes in P23H rhodopsin transgenic rats as a model of autosomal dominant retinitis pigmentosa. METHODS: P23H heterozygote (lines 1 and 3) and Sprague-Dawley control rats were studied at 4 to 29 weeks by retinal histology, electroretinogram (ERG), and a-wave transduction modeling. RESULTS: Both line 1 (faster degeneration) and line 3 (slower degeneration) showed progressive rod outer segment (ROS) shortening and outer nuclear layer (ONL) cell loss with age. ERG b-wave maximum amplitude (Vb(max)) decreased with age, but b-wave threshold remained constant within each line despite progressive ONL thinning and ROS shortening. The only exception was in line 1 at 29 weeks, which showed a slight threshold change relative to earlier ages. Va(max) and a-wave threshold changed more rapidly and were more sensitive than the b-wave in reflecting histologic degeneration. Va(max) was linearly proportional to the product of (ROS x ONL) across a two log unit range of data combined from both lines. The photopic b-wave was normal for both lines until the ONL thinned beyond 50%. Phototransduction sensitivity was normal for both lines, and dark-adaptation recovery after bleaching rhodopsin was normal. CONCLUSIONS: The P23H transgenic rat has a slow rod degeneration with initially normal cone function, consistent with clinical findings of P23H patients. However, the normal bleach recovery and the normal phototransduction sensitivity in this rat model are different from human P23H disease. a-Wave measures were more sensitive than the b-wave for tracking changes. b-Wave threshold was inexplicably poor for tracking degeneration. Although line 1 degenerated faster than line 3, the functional-structural correlates were the same. The tight linear relationship between saturated a-wave amplitude and the product of (ROS x ONL) indicates that the density of cGMP-gated channels per unit ROS plasma membrane area remains constant over a wide range of degenerations.