实验性视网膜脱离的病理改变及细胞增生

目的:探讨实验性视网膜脱离的病理变化和细胞增生。方法:成年白化病兔32眼在间接检眼镜直视下,先抽取玻璃体液0.5mL软化眼球,用50nm玻璃微管在下方6:00赤道部部位刺入视网膜造成裂孔并注射生理盐水0.5mL于视网膜下间隙。术后0.5,1,3,6,24h;3,7d和14d摘除眼球作组织学观察并以免疫组织化学的方法检测视网膜下细胞增生。结果:32只兔眼术后均发生视网膜脱离,视网膜脱离范围3:00～9:00(髓腺下方视网膜均脱离)。自发性复位的时间在3～14d,组织学切片显示,3d可见视网膜色素上皮细胞积聚团,7dRPE细胞增生肥大呈巨噬细胞样改变,14dRPE多层化改变,并见巨噬细胞样RPE来源于单层的RPE,游离RPE层后,吸附于脱离的光感受器细胞外节;3d增生细胞核抗原即表达阳性,增生细胞表达角蛋白阳性。结论:在此模型中,RPE细胞表现出细胞增生,巨噬细胞样细胞也来源于RPE。     

增生性玻璃体视网膜病变发病机制的研究进展

增生性玻璃体视网膜病变(proliferative vitreoretinopathy，PVR)是在孔源性视网膜脱离或视网膜脱离复位术后，由于视网膜色素上皮细胞(RPE)及神经胶质细胞的增生和收缩，造成牵拉性视网膜脱离的病变。PVR的病理特征是细胞增生，RPE一旦开始增殖，就产生细胞生长因子，而这些细胞生长因子反过来又刺激细胞增殖。PVR的过程有多种细胞及因子的参与，参与PVR增生的细胞主要是RPE和视网膜神经胶质细胞；涉及PVR的生长因子有：PDGF、EGF、VEGF、TGF、FGF、IGF、HGF等。     

实验性视网膜脱离及复位后视网膜色素上皮细胞抗增殖性细胞核抗原的表达

目的 观察视网膜脱离和复位过程中抗增殖性细胞核抗原(proiferating cell nuclear antigen, PCNA)表达的状况，以评价其在接触抑制机制中的意义。 方法 对72只猫眼行晶状体 囊外摘除和玻璃体切除手术。3周后，利用微穿刺技术制作视网膜脱离和复位动物模型，在不同时间取眼球并制成组织切片。采用免疫组织化学方法，观察视网膜脱离后视网膜色素上皮(retinal pigment-epithelium, RPE)细胞PCNA的表达。光镜下确定PCNA阳性的RPE细胞并量化增殖反应。采用方差分析(ANOVA)方法 ，对脱离组的脱离区和非脱离区及复位组复位区RPE细胞层PCNA的表达水平进行比较。 结果 在视网膜脱离后24 h，脱离组脱离区视网膜RPE细胞层出现PCNA阳性细胞 ，5～6 d达到高峰，20 d后逐渐回落。在复位组复位区，PCNA标记的RPE细胞明显少于脱离组脱离区。脱离组非脱离区视网膜很少有PCNA标记的RPE细胞。3组PCNA标记的RPE细胞数比较，差异有显著性的意义。 结论 视网膜脱离诱导RPE细胞增殖，而视网膜复位后这种增殖受到抑制。表明视网膜复位过程中，接触抑制的机制在发挥作用。（中华眼底病杂志，2003，19：20-23）     

兔眼视网膜铁锈症的实验观察

在光镜和电镜下观察了25只兔眼植入铁异物1～30天的视网膜变化。结果表明,邻近铁异物的局部视网膜2天即发生变性坏死,7～30天坏死的部分由视网膜色素上皮细胞(RPE)和胶质细胞填充,形成视网膜重度萎缩。在间接铁锈症的轻度萎缩性视网膜病变中,导致感光细胞形态学改变的重要原因,可能是RPE吞噬功能异常增高。观察了RPE转变为吞噬细胞,向玻璃体侧移行的过程。     

兔眼IPE与RPE细胞膜表面K-NPPase酶组织化学的电镜图像分析

目的:为了探讨自体虹膜色素上皮细胞(IPE)能否代替视网膜色素上皮细胞(RPE)移植到视网膜下腔执行物质转运功能,对兔眼IPE与RPE细胞膜表面的Na ,K -ATPase采用K-NPPase酶组织化学技术进行定位和分析比较。方法:家兔12只,取IPE和RPE组织固定,配制孵育液后进行K-NPPase酶组化孵育,制成电镜标本,采集图像,利用Metamorph/DP10/BX51系统进行分析。随机选取IPE细胞膜以及RPE细胞顶端相邻K-NPPase颗粒两点间的距离进行测量。计算每组样本的均值±标准差(x±s),进行成组设计材料的t检验。结果:有色素组与无色素组汇总后的IPE细胞膜上K-NPPase颗粒的距离平均值40.16±1.19nm,RPE顶端微绒毛细胞膜上K-NPPase颗粒的距离平均值为41.07±1.78nm,两者之间的差异无显著性意义(0.5>P>0.2)。结论:兔眼IPE细胞膜表面与RPE细胞顶端微绒毛膜表面均有Na ,K -ATPase分布,采用K-NPPas酶组化电镜观察到的兔眼IPE细胞与RPE细胞可能具有相似的物质转运功能。     

兔孔源性视网膜脱离的多焦视网膜电图及超微结构改变

目的研究视网膜脱离后多焦视网膜电图和视网膜超微结构改变。方法对9只18眼健康有色素家兔,制造孔源性视网膜脱离模型,造模前及造模后检测多焦视网膜电图,并于透射电镜下观察视网膜超微结构改变。结果视网膜脱离后10 d,与造模前比较有色素家兔多焦视网膜电图的P1波振幅密度降低(P=0.013),P1波振幅降低(P=0.01),N1波幅值降低(P=0.053)。光镜下神经上皮层的颗粒层变薄,神经纤维层和内颗粒层可见空泡。透射电镜可见:视网膜色素上皮细胞表面纤毛完全消失,视网膜色素上皮细胞内颗粒减少,粗面内质网、线粒体嵴断裂,外颗粒层细胞排列紊乱,视细胞外段盘膜粗大,盘膜间隙增宽,内外丛状层空泡形成,神经节细胞、细胞器大部分消失,神经节细胞层可见细胞质有嵴性肿胀。结论孔源性视网膜脱离后10 d,视网膜全层即发生病理改变,多焦视网膜电图的P1波振幅密度降低,P1波振幅和N1波振幅降低。     

Cyclosporine玻璃体腔内注射对异种视网膜色素上皮移植的排异抑制作用

目的 观察兔眼视网膜下腔植入人胚眼视网膜色素上皮（retinal pigment epithe-lium,RPE）后不同时期的眼底和组织学改变。研究环胞菌素A（Cyclosporines,CsA）玻璃体腔内注射能否抑制人胚眼RPE在兔眼视网膜下腔中诱导的异种移植排异反应。方法 人胚眼色素上皮片和浓缩色素上皮细胞悬液植入36只兔眼的视网膜下腔,其中16眼为对照组,用于观察排异的自然转规。分别7d(8眼）和30d(8眼）后获取组织标本。另20眼为实验组。RPE移植后,每周一次玻璃体腔内注射CsA 1mg(12眼）或CsA0.1mg(8眼）。视网膜和视神经的毒性反应使用ERG进行检查。结果 人胚眼RPE片和浓缩的RPE细胞均能在视网膜下腔短期存活。移植的RPE与视细胞结合良好并显示吞噬功能。排异反应发生时间约在术后10～30d。对照组中7d的排异发生率为0/8;30d排异发生率为7/8。排异发生后荧光造影中移植区为高荧光区,组织切片中显示有大量的组织细胞积聚。CsA1mg组30d排异发生率为0/12,0.1mg组为5/8。ERG波幅的下降与CsA剂量和注射次数呈正相关。结论 异种RPE视网膜下腔移植在无免疫抑制剂的条件下,只能短期存活。CsA玻璃体腔中注射能抑制异种RPE移植的排异反应但易引起明显的视网膜毒性反应。     

实验性视网膜脱离模型中视网膜色素上皮细胞黏附分子的表达

目的观察大鼠视网膜脱离及复位状态下视网膜色素上皮（RPE）细胞黏附分子的表达变化，探讨其与增生性玻璃体视网膜病变（PVR）发生的关系。方法通过视网膜下注射透明质酸钠的方法制造视网膜脱离的动物模型。在不同的时间点摘除眼球，制作冰冻切片，进行免疫组织化学及免疫荧光染色，比较RPE细胞上几种黏附分子，包括神经钙粘素、上皮钙粘素、整合素α5、整合素β1，以及纤维连接蛋白（FN）在正常视网膜、脱离的视网膜、复位的视网膜中的表达情况。结果几种黏附分子在脱离区的RPE细胞上的表达均明显高于正常视网膜以及脱离后复位的视网膜。结论视网膜脱离会导致RPE细胞上几种重要的黏附分子的表达增加，视网膜复位可逆转此种变化。视网膜脱离可能是PVR发生的始动因素。     

虹膜色素上皮移植

自体虹膜色素上皮容易获得，能够吞噬光感受器外节盘膜，使得用它替代视网膜色素上皮细胞移植到视网膜下腔以治疗视网膜色素上皮变性疾病成为可能。对IPE和视网膜色素上皮(RPE)细胞的功能进行了比较，并对IPE细胞的培养方法，用IPE细胞移植治疗老年性黄斑变性和视网膜色素变性等技术进行了综述。     

兔视网膜色素上皮细胞移植的实验研究

目的：探索用改良的二步酶法制备视网膜色素上皮（RPE）细胞悬液，经改良的内路法，不经玻璃体切割的情况下进行视网膜下间隙的移植，方法：用酶1（含220kU/L透明质酸酶和0．1％的胰蛋白酶）松解视网膜神经上皮细胞与RPE细胞之间的联接，再用酶II（含0．25％胰蛋白酶）从Bruch膜上离散RPE细胞，制成细胞悬液，用特制的注射针头，通过人为造成的局限性视网膜脱离区将其移植入受体视网膜下间隙，结果：术后2周供体RPE细胞层呈单层排列于受体视网膜下间隙，并存活，未见明显炎症反应，电镜观察，术后7周供体RPE细胞位于受体Bruch膜上并与受体感光细胞外节盘模型成镶嵌关系。结论：改良的内路法可以将供体RPE细胞成功移植在受体眼视网膜下间隙，并至少存活7周，改良的内路法渴望为视网膜移植的实验室研究提供一条可供借鉴的途径。     

肝细胞生长因子诱导实验性视网膜脱离的病理机制研究

目的 观察肝细胞生长因子（HGF）对视网膜色素上皮（RPE）细胞屏障功能的影响以及RPE内过度表达HGF导致视网膜脱离（RD）的病理机制。 方法 编码HGF（AdCMV.HGF）、绿色荧光蛋白（Ad CMV.GFP）的E1/E3缺失的腺病毒载体，以5×10⁴ 噬斑形成单位（pfu）/眼注射到成年有色兔的视网膜下。检查注射后3、7、14、28 d时的眼底及组织病理变化,利用免疫组织化学和酶联免疫吸附试验（ELISA）方法检测HGF在视网膜和玻璃体的表达水平。 结果 对照组注射Ad CMV.GFP眼显示GFP几乎仅表达于PRE单核细胞层，AdCMV.HGF注射眼在注射点处的PRE细胞出现强的HGF免疫阳性反应。玻璃体内HGF的表达水平在注射7 d后达到最高峰、28 d后降低到基础水平。在HGF的表达期内AdCMV.HGF注射眼出现慢性RD和脉络膜慢性炎症。在RD区域，视网膜下的空间内可见增生性的RPE细胞，部分实验兔眼还产生多层的细胞膜结构。 结论 RPE内过度表达的HGF能引发慢性浆液性RD，同时伴有视网膜下RPE增生。提示HGF可能作为治疗RD的作用靶点。(中华眼底病杂志，2007，23：193-197)     

复杂视网膜脱离视网膜下膜超微结构研究

目的研究复杂视网膜脱离病例中视网膜下膜的超微结构特征,探讨视网膜下膜的细胞成分。方法对21例复杂孔源性视网膜脱离伴视网膜下膜者行玻璃体切割术加视网膜下膜切除术,将所获得的视网膜下膜经处理后于光镜下选择细胞较密集处做超薄切片,染色后行透射电镜观察并摄影。结果复杂视网膜脱离视网膜下膜中,色素上皮细胞多呈散在分布,未见明显基底膜供细胞附着,在条索或片状膜当中的色素上皮细胞形态存在变化。神经胶质细胞胞浆内有较丰富的细胞器和直径约10nm的中间型微丝形成。视网膜下膜的细胞间质含有大量胶原纤维。成纤维细胞形状不规则,活跃,胞浆中见大量直径4～6nm微丝。结论视网膜下膜主要由视网膜色素上皮细胞、神经胶质细胞、成纤维细胞和胶原纤维组成。视网膜下膜中色素上皮细胞、神经胶质细胞和成纤维细胞有转化与增殖能力。     

N-cadherin expression in a rat model of retinal detachment and reattachment

PURPOSE: To observe the changes in N-cadherin expression in the retina after experimental retinal detachment (RD) and reattachment in the rat and to explore the role N-cadherin might play after RD. METHODS: Forty rat retinas were detached by transscleral injection of 1.4% sodium hyaluronate into the subretinal space. The eyes were enucleated at different time intervals (n = 5), followed by fixation, embedding, and sectioning. The differences in N-cadherin expression in the normal retina, detached retina, and spontaneously reattached retina were determined. Furthermore, an N-cadherin antagonist was injected in combination with 1.4% sodium hyaluronate into the subretinal space in another 10 eyes, in an attempt to demonstrate the role N-cadherin plays after RD. RESULTS: N-cadherin was not expressed in the RPE layer of the normal rat retina. After RD, intense immunolabeling of N-cadherin was seen in the RPE cells, the photoreceptors, and the outer limiting membrane (OLM). An increasing number of cytokeratin (CK)-positive cells likely to be RPE cells was found attached to the outer surface of the detached neural retina. Where the retina was reattached, the N-cadherin immunolabeling rapidly decreased. In eyes treated with an N-cadherin antagonist, the retinas appeared thinner than that in eyes without treatment, and the photoreceptor nuclei showed significantly loss. Moreover, CK-positive cells attached to the outer surface of the detached retina were markedly fewer in number. CONCLUSIONS: Increased expression of N-cadherin in the RPE cells, the photoreceptor cells, and the OLM of the retina after RD may contribute to RPE cell migration and photoreceptor survival. These changes could be reversed by retinal reattachment.     

Hepatocyte growth factor and its role in the pathogenesis of retinal detachment

PURPOSE: Hepatocyte growth factor (HGF) regulates barrier function of retinal pigment epithelial (RPE) cells. The purpose of this study was to determine whether overexpression of HGF in the RPE induces retinal detachment (RD). METHODS: E1/E3-deleted adenoviral vectors encoding HGF (Ad CMV.HGF), green fluorescent protein (Ad CMV.GFP), or connective tissue growth factor (AdCMV.CTGF) were injected subretinally in adult pigmented rabbits (5 x 10(4) plaque-forming units [pfu]/eye). Animals were observed for up to 28 days with fundus photography. HGF expression in the retina and vitreous was determined using immunohistochemistry, and ELISA. Histopathologic examinations were performed with light and electron microscopy. RESULTS: Control eyes injected with AdCMV.GFP showed GFP expression almost exclusively in the RPE monolayer. Eyes injected with AdCMV.HGF showed strong HGF immunopositivity in RPE cells at the injection site. Elevated HGF levels were found in the vitreous peaking at postinjection day 7, diminishing to baseline by postinjection day 28. Eyes injected with AdCMV.HGF developed chronic RD and chronic inflammation in the choroid within the time frame of HGF expression. Groups of proliferating RPE cells were seen in the subretinal space in the region of the RD, and in some cases multilayered cellular membranes developed. No RD and minimal morphologic changes were seen in the eyes injected with AdCMV.GFP or AdCMV.CTGF. CONCLUSIONS: Overexpression of HGF in RPE induces chronic, serous RD with subretinal proliferation of RPE. This work provides insight into the pathogenesis of RD and suggests that HGF should be further investigated as a target for therapeutic intervention in RD.     

Macular recovery after retinal detachment

Macular recovery after surgery for retinal detachment (RD) depends on preoperative and postoperative predictive factors. Preoperative visual acuity is the main preoperative factor correlating positively with good macular recovery. Preoperative factors, which influence macular recovery negatively, include duration of macular detachment, height of macular detachment and vitreomacular traction. Postoperative factors, which influence macular recovery negatively, include cystoid macular oedema, epiretinal membranes, retinal folds, subretinal retinal pigment epithelium (RPE) migration and persistent subretinal fluid on optical coherence tomography (OCT). According to the latest available data, a detached macula has to be reattached within 5 days to optimize functional recovery. However, new therapeutic options such as exposure to hyperoxia or different growth factors may help to improve the final visual outcome in the presence of an already detached macula.     

实验性视网膜脱离复位后的视网膜细胞超微结构观察

目的 研究视网膜脱离复位后视网膜超微结构改变,以探讨视功能恢复障碍的机制。方法 12只灰兔通过在视网膜下注射透明酸钠的方法建立视网膜脱离模型。用透射电镜观察术后1,2,3,4周的视网膜。结果 视网膜复位早期光感受器外节段缺失、断裂。内节、视细胞核、双极细胞、神经节细胞均可见胞浆水肿,线粒体肿胀和嵴断裂。复位3周后,视网膜细胞结构基本正常,但仍有部分外节变短,神经纤维空洞存在。结论 视网膜复位后不仅光感受器,而且视网膜其他细胞和神经纤维均有不可逆的改变,这些改变导致视功能恢复不良。     

兔视网膜脱离后视网膜神经节细胞及微血管的形态学观察

目的 观察视网膜脱离( retinal detachment,RD)后视网膜神经节细胞及微血管的形态学变化,以探讨RD后视功能损伤机制.方法 36只青紫兰灰兔左眼经玻璃体内注入透明质酸酶(10U· mL-1)液化玻璃体,抽吸液化玻璃体并以此液流冲击视网膜造成RD模型,右眼作为对照组.根据RD后不同时间分组,分别为RD后6h组、1d组、3d组、7d组、14 d组、28 d组,每组6只兔,每组均于光镜及透射电镜下观察视网膜情况.结果 光镜下观察,与对照组相比,RD后6h组视网膜神经节细胞略肿胀,胞浆着色浅,胞浆中部的Nissl小体消失,仅在细胞周边部有少量残余;1d组视网膜神经节细胞肿胀,神经纤维水肿加重;3 d组视网膜神经节细胞胞周空隙增大,胞体缩小,胞核偏位;7 d组大部分视网膜神经节细胞萎缩、死亡,细胞核固缩;14 d组视网膜神经节细胞数量明显减少;28 d组仪有极少量视网膜神经节细胞存在,神经纤维层明显变薄萎缩.电镜下观察,与对照组相比,视网膜神经节细胞线粒体RD后6h组即出现肿胀,嵴变短;14 d组肿胀线粒体破裂,数量减少;28 d组细胞固缩,难以找到线粒体;RD后视网膜微血管光镜下未见明显改变,但电镜下显示损伤明显.主要表现在基底膜逐渐肿胀、增厚,形态不规则;另外,血管内皮细胞、周细胞内出现空泡,线粒体肿胀、嵴变短甚至空泡样变.结论 随着RD时间的延长,视网膜微血管基底膜逐渐增厚、视网膜神经节细胞缺氧性损伤逐渐加重,可能是RD后视功能损伤机制之一.     

Short-term study of retinal pigment epithelium sheet transplants onto Bruch's membrane

The purpose of this study is to investigate the survival and behaviour of retinal pigment epithelium sheets transplanted onto hydraulically debrided Bruch's membrane. Uncultured retinal pigment epithelium sheets obtained from male cats and sandwiched between two gelatin sheets were transplanted onto the tapetal area of female cats after native retinal pigment epithelium was debrided. For controls, the gelatin carrier was transplanted after debridement. Each transplant or control specimen was analyzed histologically and immunohistochemically. Transplanted male retinal pigment epithelial cells were identified by in situ labelling of the cat Y chromosome. Over half of the transplants appeared as retinal pigment epithelium multilayers in the subretinal space. Retinal pigment epithelium pigment dispersion into the subretinal space was seen in most of the transplants, and retinal pigment epithelium pigment infiltration into the neural retina was seen in all 7-day survival transplants. A few condensed darkly stained retinal pigment epithelium nuclei and Terminal Transferase dUTP Nick End Labelling-positive retinal pigment epithelium cells were observed in all transplants. Cellular retinaldehyde-binding protein was present up to day-7 in most transplanted RPE cells. In both transplant and control specimens, the antibody against the Ki-67 nuclear antigen labelled a few retinal pigment epithelium cells at day-3. Terminal Transferase dUTP Nick End Labelling-positive outer nuclear layer nuclei were most frequently observed at day-1 but were much less frequent at day-3 in both transplants and controls. The survival and effectiveness of retinal pigment epithelium sheet transplants appeared similar to the retinal pigment epithelium microaggregates transplants conducted previously in this model.