经巩膜外路至视网膜下腔移植视网膜细胞的实验研究

探讨视网膜光感受器细胞的移植方法及临床意义。方法将16只昆明鼠随机分为A组和B组,每组均8只鼠。于手术显微镜下,用特殊显微注射器穿过巩膜、脉络膜,在A组昆明鼠的视网膜下腔注入视网膜混合细胞,在B组昆明鼠的视网膜下腔注入纯光感受器细胞。于移植术后30、90及180 d摘除实验眼,于光镜下观察移植细胞在视网膜下腔生长的情况。结果大多数标本(13／15)HE染色显示视网膜细胞准确移植在受体眼的视网膜下腔,未见炎性细胞浸润和受体视网膜破坏;且移植到受体视网膜下腔的细胞在术后180 d仍存活。仅少数(2／15)标本可见受体视网膜结构破坏。移植的视网膜混合细胞均形成“玫瑰花”样结构,而移植的纯视网膜光感受器细胞则在视网膜下腔形成整齐的细胞层。结论经巩膜外路至视网膜下腔的显微注射法是较为理想的视网膜下腔注射给药和视网膜细胞移植方式。纯视网膜光感受器细胞移植后的生长状况和功能接近正常生理状态的视网膜组织结构,为临床治疗视网膜变性疾病提供了新途径。     

重组腺病毒介导的gfp-bcl-XL对视网膜变性鼠光感受器细胞保护作用的研究

目的 观察重组腺病毒rAd-gfp-bcl-XL治疗视网膜变性(retinal degeneration,RD)鼠的疗效。方法 40只RD鼠随机分成A、B组,每组20只,右眼为治疗眼,左眼为对照眼。A、B组治疗眼分别于RD鼠生后10d及22d视网膜下腔注射含绿色荧光蛋白基因gfp和抗凋亡基因bcl-XL的重组腺病毒颗粒;术后15、30d摘除治疗眼和对照眼行冰冻切片、透射电镜下观察、石蜡切片、HE染色并提取视网膜总RNA;行逆转录聚合酶链反应分析bcl-XL基因的mRNA表达水平;免疫组化分析治疗眼和对照眼的Bcl-XL蛋白水平。结果 治疗眼视网膜有较广泛的绿色荧光蛋白表达,说明重组腺病毒成功的将bcl-XL转染至视网膜光感受器细胞,且有bcl-XL表达;术后30d,A组鼠治疗眼和对照眼bcl-XL的mRNA表达水平及Bcl-XL蛋白含量有明显差异;透射电镜下可见治疗眼的内节段较对照眼完好;HE染色示治疗眼视网膜光感受器细胞层较对照眼厚。而B组鼠治疗眼和对照眼的视网膜光感受器细胞层的厚度无明显差异。结论 注射至早期RD鼠视网膜下腔中的rAd-gfp-bcl-XL能有效转染光感受器细胞并发挥抗凋亡作用,但对晚期RD鼠的疗效不明显。     

BMSCs联合ChABC对视网膜变性大鼠光感受器细胞凋亡的影响

目的：研究骨髓间充质干细胞（ bone mesenchymal stem cells, BMSCs ）联合硫酸软骨素酶（ chondroitinaseABC, ChABC）行视网膜下腔注射对碘酸钠诱导的视网膜变性大鼠光感受器细胞凋亡的影响。
　　方法：选取40只SD大鼠行腹腔注射碘酸钠（ NaIO3,30g／L,100mg／kg）造视网膜变性模型,分为A组不干预组,B组BMSCs注射组,C组BMSCs＋ChABC注射组,D组PBS注射组。造模后28d将ChABC处理或未处理的BMSCs注射入大鼠视网膜下腔,对照组注射PBS液,21 d后处死大鼠并取出眼球,行视网膜HE染色、视网膜细胞凋亡及免疫组化检测。
　　结果：B组凋亡率、外核层细胞数与A组、D组比较,差异均有统计学意义（P＜0．05）。 C组凋亡率、外核层细胞数与A组、D组比较,差异均有统计学意义（ P＜0．05）。 B组凋亡率、外核层细胞数与C组相比,差异无统计学意义（P＞0．05）。免疫组化显示BMSCs在眼内表达GFAP抗原。结论：BMSCs联合ChABC行视网膜下腔注射可缓解视网膜变性大鼠光感受器细胞的凋亡,延缓细胞数目的减少,从而保护视网膜光感受器细胞。     

老年性黄斑变性所致大量视网膜下及玻璃体积血的手术治疗

目的 探讨老年性黄斑变性(age-related macular degeneration,AMD)所致大量视网膜下及玻璃体积血的手术治疗的方法及疗效。 方法 回顾分析14例手术前后经荧光素眼底血管造影（fundus fluorescein angiography ,FFA)确诊为AMD所致的大量视网膜下及玻璃体积血患者的14只患眼,行经睫状体平坦部玻璃体切割、单个小切口的视网膜切开、用平衡盐液进行视网膜下冲洗、气液交换、视网膜切口的眼内激光光凝、气体或硅油填充等手术治疗后,随访3～7个月的临床资料。 结果 14只患眼中2只患眼术后眼球萎缩,占14.3%;12只患眼视力均获得不同程度的提高,占85.7%;最好的矫正视力为0.2;12只患眼术后随访时间内视网膜保持平复,占85.7%;4只患眼术后7 d内出现前房泥沙样积血,占28.6%,行1～3次前房冲洗术。 结论 玻璃体视网膜手术能有效清除AMD所致的视网膜下及玻璃体积血,对预防和治疗视网膜下及玻璃体积血所致的前房泥沙样积血、血影细胞性青光眼及视网膜脱离等有积极意义。(中华眼底病杂志,2000,16:217-219)     

重组腺病毒介导的gfp-bcl-X_L对视网膜变性鼠光感受器细胞保护作用的研究

目的　观察重组腺病毒rAd gfp bcl XL治疗视网膜变性 (retinaldegeneration ,RD)鼠的疗效。方法　 40只RD鼠随机分成A、B组 ,每组 2 0只 ,右眼为治疗眼 ,左眼为对照眼。A、B组治疗眼分别于RD鼠生后 10d及 2 2d视网膜下腔注射含绿色荧光蛋白基因gfp和抗凋亡基因bcl XL 的重组腺病毒颗粒 ;术后 15、30d摘除治疗眼和对照眼行冰冻切片、透射电镜下观察、石蜡切片、HE染色并提取视网膜总RNA ;行逆转录聚合酶链反应分析bcl XL 基因的mRNA表达水平 ;免疫组化分析治疗眼和对照眼的Bcl XL 蛋白水平。结果　治疗眼视网膜有较广泛的绿色荧光蛋白表达 ,说明重组腺病毒成功的将bcl XL 转染至视网膜光感受器细胞 ,且有bcl XL 表达 ;术后 30d ,A组鼠治疗眼和对照眼bcl XL的mRNA表达水平及Bcl XL 蛋白含量有明显差异 ;透射电镜下可见治疗眼的内节段较对照眼完好 ;HE染色示治疗眼视网膜光感受器细胞层较对照眼厚。而B组鼠治疗眼和对照眼的视网膜光感受器细胞层的厚度无明显差异。结论　注射至早期RD鼠视网膜下腔中的rAd gfp bcl XL 能有效转染光感受器细胞并发挥抗凋亡作用 ,但对晚期RD鼠的疗效不明显。     

外源性可溶性促红细胞生成素受体对正常大鼠视网膜神经元存活的影响

目的观察玻璃体腔注射外源性可溶性促红细胞生成素受体（EPOsR）对视网膜神经元存活的影响。方法30只SD鼠随机分为正常对照组、PBS组、EPOsR 2ng组、EPOsR 20ng组、EPOsR 200ng组，每组6只（12只眼）。后4组玻璃体腔内分别注射5μL PBS，2、20、200ng EPOsR。注射前记录闪光视网膜电图（FERG），注射后3d再次进行FERG检查及视网膜神经元TUNEL凋亡原位检测，注射后7d进行光学显微镜和透射电镜检查。结果注药前后各组a波、b波的潜伏期及振幅差异无统计学意义（P〉0．05），震荡电位（OPs）的P4潜伏期和各子波总振幅差异亦无统计学意义（P〉0．05）；全层视网膜神经元TUNEL凋亡原位检测未见凋亡细胞；光学显微镜和透射电镜检查均未见明显的水肿、空泡状变性等组织病理学改变。结论玻璃体腔注射200ng以下剂量的外源性EPOsR不影响正常状态下视网膜神经元的存活。     

血管抑素玻璃体注射对糖尿病大鼠视网膜及虹膜血管渗漏的影响

目的 探讨血管抑素（Angiostatin）玻璃体腔注射对糖尿病大鼠视网膜、虹膜血管渗漏性的影响及其机理。方法 Brown Norway大鼠48只。静脉注射链唑霉素（Streptozotocin，STZ）建立糖尿病模型，随机分为3组（每组糖尿病鼠与正常鼠各8只）。甲组：右眼玻璃体腔注射磷酸盐缓冲生理盐水（Phosphate Buffered Saline，PBS）5山，左眼不注射；乙组、丙组：右眼玻璃体腔注射血管抑素7．5μg／5μl，左眼注射等量PBS；甲、乙组用埃文斯蓝微血管渗透性检测法检测视网膜和虹膜的血管渗透性。丙组用Western blot免疫印迹分析法检测视网膜血管内皮生长因子（Vascular Endothelial Growth Factor，VEGF）的表达。结果 糖尿病鼠较正常鼠的视网膜（P〈0．01）和虹膜（P〈0．05）的血管渗透性增加；糖尿病鼠血管抑素注射眼与对侧PBS注射眼相比，视网膜（P〈0．01）和虹膜（P〈0．05）血管渗透性降低；正常鼠注射血管抑素眼与对侧注射PBS眼相比，视网膜和虹膜血管渗透性无明显改变（P〉0．05）Western blot显示血管抑素显著降低了糖尿病鼠视网膜的VEGF水平，但对正常鼠无影响。结论 血管抑素可明显降低糖尿病鼠视网膜、虹膜血管渗透性，但对正常鼠影响不明显。血管抑素可下调糖尿病鼠视网膜VEGF的表达从而阻断其血管渗漏。因此预测血管抑素对糖尿病黄斑水肿、黄斑囊样水肿、葡萄膜炎等血管渗漏性疾病可能具有潜在的治疗价值。     

视黄酸对视网膜感光细胞增生作用的实验研究

目的 探讨外源性视黄酸是否能诱导成年大鼠视网膜感光细胞增生。方法 将32只成年健康Sprague-Dawley(SD)大鼠,每只体重约200－250g,随机平均分成4组。第1、2组：视网膜下腔注射视黄酸（retinoic acid,RA)5μl(0.001mol/L),第3、4组玻璃体腔注射RA 10μl（0.001mol/L)。其中第1、3组在注射RA前,通过结扎眼动脉,造成眼部缺血再灌注损伤的实验模型。对照组为5只健康SD大鼠,在不注射RA的条件下,制做短暂缺血再灌注损伤模型。实验眼于注射RA后2－4周取出眼球,进行HE和免疫组化染色,于光镜下检测分析。结果 第1组,于注射RA后第16天,视网膜下腔表达视杆细胞特异性标记抗体的视网膜感光细胞数量增多及内核层增厚;而第2组未发现任何细胞增生;第3、4组,玻璃体腔内注射RA后,无论在有无缺血再灌注损伤的条件下,均未见视网膜神经细胞数量增多。对照组在未注射RA的条件下,将组织学切片置光镜下观察,未见视网膜神经上皮层的形态学改变,也无感光细胞增生。结论 在缺血再灌注损伤的条件下,成年大鼠视网膜下腔注射外源性RA能够诱导视网膜感光细胞增生,这将为研究哺乳动物视网膜神经细胞再生提供新的思路。     

胚胎干细胞视网膜下移植实验研究初步报告

目的 探讨胚胎干细胞(embronic stem　cell,ESC)在视网膜下间隙分化发育的情况。 方法 ESC经体外悬浮培养4 d形成胚胎体(embryonic body,EB)备作移植。将ESC以及EB联合或不联合视黄酸(retinoic acid,RA)分别移植到SD大鼠玻璃体腔或视网膜下,在视网膜下间隙移植眼,于移植前结扎眼动脉40 s造成缺血再灌注损伤的实验模型。于移植后14～28 d摘除眼球,观察移植物分化发育的情况。 结果 ESC在SD大鼠玻璃体腔内迅速增生,形成非典型性增生团块;EB在缺血再灌注损伤眼视网膜下间隙经RA诱导能定向分化为视网膜感光细胞;而在无RA作用下向多极化分化发育。单独RA视网膜下间隙注射后导致神经视网膜上皮增生变厚。 结论 ESC源性的EB在缺血再灌注眼视网膜下间隙经RA诱导能向视网膜感光细胞分化。（中华眼底病杂志,2000,16：213-284）     

表达睫状神经营养因子的人胚肺成纤维细胞视网膜下间隙移植延缓RCS大鼠光感受器变性

目的以遗传性视网膜变性RCS大鼠为模型,探讨视网膜下间隙移植表达睫状神经营养因子(CNTF)的人胚肺(HFL)成纤维细胞治疗视网膜变性的可行性。方法通过脂质体将编码CNTF的表达质粒转入HFL成纤维细胞、MTX及ELISA筛选高水平表达CNTF的细胞克隆。手术显微镜直视下经角膜缘后1mm注射5μl含1×105个高水平表达CNTF的HFL成纤维细胞至4～5周龄RCS大鼠右眼视网膜下间隙,左眼不手术或注射PBS作为对照。分别于移植术后2、4、6、8、10、12及15周随机取1只鼠摘除眼球作光镜观察,随机取2只鼠摘除眼球作电镜观察。结果稳定转染CNTF的HFL成纤维细胞能高水平表达CNTF(91046.15pg/ml),7只经光镜观察的移植眼中的4只,其视网膜外颗粒层明显较对照眼厚,保存的光感受器数量明显较对照眼多(P≤0.002)。14只经电镜观察的移植眼中的10只,视网膜外颗粒层光感受器凋亡明显较对照眼少。在光感受器与视网膜色素上皮间堆积的盘膜碎片亦较少。宿主视网膜色素上皮细胞的形态保存较好,并可见吞噬小体。结论经过CNTF基因修饰的HFL成纤维细胞视网膜下间隙移植能够延缓RCS大鼠视网膜光感受器变性     

Retinal degeneration mutants in the mouse

The Jackson Laboratory, having the world's largest collection of mouse mutant stocks and genetically diverse inbred strains, is an ideal place to look for genetically determined eye variations and disorders. Through ophthalmoscopy, electroretinography and histology, we have discovered disorders affecting all aspects of the eye including the lid, cornea, iris, lens and retina, resulting in corneal disorders, cataracts, glaucoma and retinal degenerations. Mouse models of retinal degeneration have been investigated for many years in the hope of understanding the causes of photoreceptor cell death. Sixteen naturally occurring mouse mutants that manifest degeneration of photoreceptors in the retina with preservation of all other retinal cell types have been found: retinal degeneration (formerly rd, identical with rodless retina, r, now Pde6b(rd1)); Purkinje cell degeneration (pcd); nervous (nr); retinal degeneration slow (rds, now Prph(Rd2)); retinal degeneration 3 (rd3); motor neuron degeneration (mnd); retinal degeneration 4 (Rd4); retinal degeneration 5 (rd5, now tub); vitiligo (vit, now Mitf(mi-vit)); retinal degeneration 6 (rd6); retinal degeneration 7 (rd7, now Nr2e3(rd7)); neuronal ceroid lipofuscinosis (nclf); retinal degeneration 8 (rd8); retinal degeneration 9 (Rd9); retinal degeneration 10 (rd10, now Pde6b(rd10)); and cone photoreceptor function loss (cpfl1). In this report, we first review the genotypes and phenotypes of these mutants and second, list the mouse strains that carry each mutation. We will also provide detailed information about the cpfl1 mutation. The phenotypic characteristics of cpfl1 mice are similar to those observed in patients with complete achromatopsia (ACHM2, OMIM 216900) and the cpfl1 mutation is the first naturally-arising mutation in mice to cause cone-specific photoreceptor function loss. cpfl1 mice may provide a model for congenital achromatopsia in humans.     

An immune response after intraocular administration of an adenoviral vector containing a beta galactosidase reporter gene slows retinal degeneration in the rd mouse

BACKGROUND/AIMS: Retinal degenerations are a leading cause of blindness for which there are currently no effective treatments. This has stimulated interest in the investigation of gene therapy strategies for these diseases in a variety of animal models. A number of attempts have been made to prevent photoreceptor loss in the rd mouse model of retinal degeneration using adenoviral vectors containing either a copy of the missing functional gene or a gene encoding either a neurotrophic factor or an antiapoptotic factor. The authors have previously demonstrated that intraocular administration of an adenoviral vector containing a beta galactosidase gene (AV.LacZ) results in an immune response to viral gene products and beta galactosidase. Here the effect of the immune response on retinal degeneration is examined. METHODS: Juvenile rd mice were injected intravitreally with AV.LacZ and a proportion were depleted of either CD4+ or CD8+ T cells or both. Control animals were injected with PBS. The mice were sacrificed 10 and 20 days post-injection and their eyes embedded in paraffin wax and sectioned. RESULTS: 10 days after intravitreal injection of AV.LacZ, the outer nuclear layer contains an average of 2.5 rows compared with 1.5 in PBS injected animals (p<0.005). The protective effect of AV.LacZ is negated by immune suppression and does not extend beyond 20 days. CONCLUSION: An immune response to vector and transgene products is able to slow degeneration in the rd mouse. This phenomenon should be taken into account when analysing the degeneration in the rd mouse following gene transfer.     

原纤维蛋白-2(FBN2)抗体玻璃体内注射对小鼠视网膜变性的影响

目的　探讨原纤维蛋白-2（fibrillin-2,FBN2）抗体玻璃体内注射对小鼠视网膜变性的影响。方法　选取18只8周龄C57BL/6J小鼠,随机分为3组:正常对照组、PBS组、FBN2抗体组,每组6只。正常组小鼠不做任何处理,PBS组小鼠双眼玻璃体内注射4 μL PBS溶液,FBN2抗体组小鼠双眼玻璃体内注射4 μL FBN2抗体（0.2 g·L^-1）,每周注射1次,连续3周。利用眼底照相和视网膜电流图（ERG）分别检测3组小鼠眼底改变和视网膜功能。PAS染色法观察小鼠视网膜形态并测量眼球后极部视网膜、外核层以及内核层厚度,采用实时定量PCR和ELISA检测小鼠视网膜中FBN2 mRNA和蛋白的表达。结果　眼底照相显示:FBN2抗体组小鼠眼底出现明显渗出,黄白色似玻璃膜疣样沉积物以及色素沉着的病理改变,且随时间延长病理改变加重。ERG结果显示:每次注射后FBN2抗体组暗适应视杆细胞反应b波和暗适应混合细胞反应a波振幅均低于正常对照组和PBS组,差异均有统计学意义(均为P＜0.05),且两者振幅在抗体注射3次后均最低,分别为(13.28±3.41)μV和(21.67±8.81)μV;在注射2次和3次后,FBN2抗体组暗适应混合细胞反应b波振幅均低于正常对照组和PBS组,差异均有统计学意义（均为P＜0.05）,且该波振幅在抗体注射2次时最低为(59.12±18.00)μV;正常对照组和PBS组之间,各振幅差异均无统计学意义（均为P＞0.05）。PAS染色结果表明:FBN2抗体组视网膜厚度和外核层厚度 ［（129.33±15.38）μm、（23.39±3.93）μm］均低于正常对照组［(197.68±13.50）μm、（46.54±7.44）μm］和PBS组［（198.27±8.28）μm、（38.92±2.39）μm］,差异均有统计学意义（均为P＜0.05）;正常对照组和PBS组之间比较,差异均无统计学意义（均为P＞0.05）。ELISA检测显示:FBN2抗体组视网膜中FBN2蛋白的相对表达［(0.15±0.01）ng·mg^-1］低于正常组［(0.17±0.01）ng·mg^-1］和PBS组［(0.17±0.02）ng·mg^-1］ （均为P＜0.05）。PCR结果显示:FBN2抗体组视网膜中FBN2 mRNA的相对表达低于正对照组和PBS组。结论　玻璃体内注射FBN2抗体能够降低小鼠视网膜中FBN2蛋白的表达,引起小鼠发生视网膜变性 。     

Retinal degeneration 6 (rd6): a new mouse model for human retinitis punctata albescens

PURPOSE: To characterize the genetics and phenotype of a new mouse mutant with retinal degeneration, rd6, that is associated with extensive, scattered, small white retinal dots seen ophthalmoscopically. METHODS: The phenotype was characterized using ophthalmoscopy, fundus photography, electroretinography, light microscopy, immunocytochemistry, and electron microscopy. Genetic characterization and linkage analysis studies were performed using standard methods. RESULTS: The inheritance pattern of rd6 is autosomal recessive. Linkage analysis mapped rd6 to mouse Chromosome 9 approximately 24 cM from the centromere, suggesting that the human homolog may be on chromosome 11q23. Ophthalmoscopic examination of mice homozygous for rd6 revealed discrete subretinal spots oriented in a regular pattern across the retina. The retinal spots appeared by 8 to 10 weeks of age and persisted through advanced stages of retinal degeneration. Histologic examination revealed large cells in the subretinal space, typically juxtaposed to the retinal pigment epithelium. The white dots seen on fundus examination corresponded both in distribution and size to these large cells. By 3 months of age, the cells were filled with membranous profiles, lipofuscin-like material, and pigment. These cells reacted strongly with an antibody directed against a mouse macrophage-associated antigen. Photoreceptor cells progressively degenerated with age, and an abnormal electroretinogram was initially detected between 1 and 2 months of age. CONCLUSIONS: The fundi of mice homozygous for rd6 exhibit phenotypic similarities to the human flecked retinal disorder retinitis punctata albescens. Thus, rd6/rd6 mice may be a model for understanding the etiology of this or similar disorders. The relationship between the aberrant subretinal cells and the concomitant photoreceptor degeneration remains to be established.     

Human melanopsin-AAV2/8 transfection to retina transiently restores visual function in rd1 mice

AIM: To explore whether ectopic expression of human melanopsin can effectively and safely restore visual function in rd1 mice. METHODS: Hematoxylin-eosin staining of retinal sections from rd1 mice was used to detect the thickness of the outer nuclear layer to determine the timing of surgery. We constructed a human melanopsin-AAV2/8 viral vector and injected it into the subretinal space of rd1 mice. The Phoenix Micron IV system was used to exclude the aborted injections, and immunohistochemistry was used to validate the ectopic expression of human melanopsin. Furthermore, visual electrophysiology and behavioral tests were used to detect visual function 30 and 45d after the injection. The structure of the retina was compared between the human melanopsin-injected group and phosphate buffer saline (PBS)-injected group. RESULTS: Retinas of rd1 mice lost almost all of their photoreceptors on postnatal day 28 (P28). We therefore injected the human melanopsin-adeno-associated virus (AAV) 2/8 viral vector into P30 rd1 mice. After excluding aborted injections, we used immunohistochemistry of the whole mount retina to confirm the ectopic expression of human melanopsin by co-expression of human melanopsin and YFP that was carried by a viral vector. At 30d post-injection, visual electrophysiology and the behavioral test significantly improved. However, restoration of vision disappeared 45d after human melanopsin injection. Notably, human melanopsin-injected mice did not show any structural differences in their retinas compared with PBS-injected mice. CONCLUSION: Ectopic expression of human melanopsin effectively and safely restores visual function in rd1 mice.     

Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate

PURPOSE: To characterize the potential of newborn retinal stem cells (RSCs) isolated from the radial glia population to integrate the retina, this study was conducted to investigate the fate of in vitro expanded RSCs transplanted into retinas devoid of photoreceptors (adult rd1 and old VPP mice and rhodopsin-mutated transgenic mice) or partially degenerated retina (adult VPP mice) retinas. METHODS: Populations of RSCs and progenitor cells were isolated either from DBA2J newborn mice and labeled with the red lipophilic fluorescent dye (PKH26) or from GFP (green fluorescent protein) transgenic mice. After expansion in EGF+FGF2 (epidermal growth factor+fibroblast growth factor), cells were transplanted intravitreally or subretinally into the eyes of adult wild-type, transgenic mice undergoing slow (VPP strain) or rapid (rd1 strain) retinal degeneration. RESULTS: Only limited migration and differentiation of the cells were observed in normal mice injected subretinally or in VPP and rd1 mice injected intravitreally. After subretinal injection in old VPP mice, transplanted cells massively migrated into the ganglion cell layer and, at 1 and 4 weeks after injection, harbored neuronal and glial markers expressed locally, such as beta-tubulin-III, NeuN, Brn3b, or glial fibrillary acidic protein (GFAP), with a marked preference for the glial phenotype. In adult VPP retinas, the grafted cells behaved similarly. Few grafted cells stayed in the degenerating outer nuclear layer (ONL). These cells were, in rare cases, positive for rhodopsin or recoverin, markers specific for photoreceptors and some bipolar cells. CONCLUSIONS: These results show that the grafted cells preferentially integrate into the GCL and IPL and express ganglion cell or glial markers, thus exhibiting migratory and differentiation preferences when injected subretinally. It also appears that the retina, whether partially degenerated or already degenerated, does not provide signals to induce massive differentiation of RSCs into photoreceptors. This observation suggests that a predifferentiation of RSCs into photoreceptors before transplantation may be necessary to obtain graft integration in the ONL.     

Intravitreal injection of fluorochrome-conjugated peanut agglutinin results in specific and reversible labeling of mammalian cones in vivo

PURPOSE: To investigate whether intravitreally injected peanut agglutinin (PNA) conjugated with a fluorochrome can specifically label retinal cones in vivo and to evaluate its clinical potential. METHODS: Fluorescein- or rhodamine-conjugated PNA (0.005%-0.5%) was intravitreally injected into anesthetized mouse, guinea pig, or monkey and retinas were removed at various intervals for fluorescence microscopy. Immunofluorescence and TUNEL assay were carried out to investigate whether PNA injection adversely affected other retinal neurons. Gross visual function was studied in a visual cliff test. The retina of an N-methyl, N-nitrosourea (MNU)-induced mouse model of retinal degeneration was stained with PNA to evaluate how spatiotemporal pattern of the staining would reflect the progression of degeneration. RESULTS: Intravitreally injected PNA resulted in specific labeling of cone outer and inner segments and cone pedicles within 30 minutes over the entire retina and in all tested species. The labeling was reversible; cones did not show any labeling 3 weeks after the injection but could be restained with PNA. TUNEL signal and expression pattern of several retinal proteins in PNA-injected mouse retina were indistinguishable from normal. Similarly, visual behavior of mouse 10 hours after the injection was normal. The pattern of PNA labeling in mice with MNU-induced retinal degeneration showed progressive disappearance of cones from the center to the periphery. CONCLUSIONS: Intravitreal injection of fluorochrome-conjugated PNA results in specific and reversible labeling of mammalian cones in vivo without causing any gross adverse effects. This novel method may eventually provide a clinical tool to examine diseased retina.     

Comparison of Electrical Stimulation Thresholds in Normal and Retinal Degenerated Mouse Retina

Purpose To compare the threshold for electrically elicited action potentials of retinal ganglion cells in normal mouse retina and photoreceptor degenerated (rd) mouse retina.Methods Microelectrode recordings were made from retinal ganglion cells of normal and rd mice. Mice with a genetically based retinal degeneration (rd mice) were grown to the age of 16 weeks, when light-evoked responses could no longer be recorded. A bare wire was placed in the vitreous to stimulate the retina with charge-balanced current pulses. The following pulse shapes were investigated: single, square biphasic pulse, single sine wave, and biphasic pulse trains.Results Normal mice had significantly lower stimulus thresholds than rd mice for all pulse shapes. In normal and rd mice, short pulses were more efficient with respect to total charge used, but required a higher current. In normal mice, sine wave stimulation was significantly more efficient than a biphasic pulse of the same duration. No difference was noted between sine wave and square wave stimulation in rd mice. Pulse trains offered little benefit over single pulses.Conclusion The amount of electrical charge required to elicit an action potential is dependent on the condition of the retina and the shape of the stimulus pulse used to deliver the charge. Jpn J Ophthalmol 2004;48:345–349 © Japanese Ophthalmological Society 2004     

地塞米松对玻璃体积血兔视网膜病理变化的影响

目的:观察地塞米松(dexamethasone,Dxm)对兔玻璃体积血后视网膜病理变化的影响。方法:白兔24只,随机分为实验对照组(A),积血组(B)和Dxm干预组(C),每组8只。A组玻璃体内注入生理盐水0.2mL,B组、C组玻璃体内注入自体血0.2mL;注入后1,24,48h,A和B组给予生理盐水3mL/kg,C组给予Dxm3mg/kg。分别于造模后3,7,14,21d随机处死每组中的2兔4眼观察视网膜病理变化。结果:A组所有时间点,B组3,7d,C组3,7,14d视网膜形态正常。B组14d神经节细胞、内丛状层水样变性;21d视网膜内界膜不完整,内、外核层细胞和光感受器内、外节水样变性。C组21d神经节细胞染色变淡,内丛状层、内核层水样变性。结论:玻璃体积血可引起视网膜细胞水样变性,Dxm可缓解玻璃体积血后视网膜水样变性。