孕酮对急性高眼压大鼠视网膜神经损伤保护作用的研究

目的：通过建立大鼠急性高眼压模型,应用孕酮干预,观察孕酮是否对视网膜神经细胞具有保护作用。

方法：Wistar大鼠140只,随机分为正常对照组(A组)20只、高眼压对照组(B组)60只、高眼压孕酮干预组(C组)60只。应用生理盐水前房灌注的方法建立大鼠急性高眼压模型,C组在高眼压后即刻予腹腔注射孕酮4.0mg/kg,以后于6,24h以及隔日行皮下注射,B组于相同时间注射等量的生理盐水。于高眼压后1,3,7d分别获取各组大鼠的视网膜组织。HE染色观察视网膜病理学的变化,用免疫组化染色、Western blot检测视网膜细胞凋亡基因Caspase-3蛋白的表达情况,TUNEL法检测细胞的凋亡程度,并对各组数据进行统计学分析。

结果：B组和C组的大鼠视网膜均发生水肿, C组水肿程度明显低于B组。免疫组化染色及Western blot相对定量检测,C组Caspase-3表达程度低于B组。Caspase-3蛋白主要表达于视网膜神经节细胞层和内核层,于高眼压后第1d强度最大,以后随时间的推移逐渐减少。TUNEL检测细胞凋亡,B组和C组均出现阳性细胞,主要位于视网膜的节细胞层和内核层,C组细胞凋亡数量明显低于B组。

结论：孕酮对大鼠视网膜急性高眼压损伤有保护作用。     

Cop-1诱导的自体免疫反应对慢性高眼压大鼠视网膜神经节细胞的保护作用

目的建立慢性高眼压大鼠Cop-1免疫模型,从形态学上观察Cop-1诱导的自体免疫对视网膜神经节细胞的影响。方法将动物分为正常组、高眼压组及高眼压免疫组。用巩膜浅层静脉烧烙法建立大鼠慢性高眼压模型。高眼压免疫组在建立高眼压模型后即刻于大鼠后肢皮下注射200μg Cop-1,2周及4周后重复注射200μg Cop-1。8周后,从心脏取血,离心取上清液,用ELISA检测Cop-1抗体效价。组织切片行常规HE染色,光镜下对节细胞层细胞数进行计数。以TUNEL染色检测视网膜神经节细胞凋亡。结果Cop-1免疫大鼠后8周,血清中Cop-1抗体呈阳性,效价高于1∶1000。正常组视网膜神经节细胞计数平均为(22.5±1.18)个,高眼压组视网膜神经节细胞计数平均为(15.5±0.85)个,高眼压免疫组为(20.5±1.43)个。统计学分析显示,高眼压免疫组及正常组视网膜神经节细胞数明显高于高眼压组(P<0.01),而正常组与高眼压免疫组之间视网膜神经节细胞数没有明显差异(P>0.05)。TUNEL染色显示高眼压组视网膜神经节细胞凋亡数明显高于另外两组。结论Cop-1诱导的自体免疫反应可以对高眼压状态下的视网膜神经节细胞起到保护作用,减少视网膜神经节细胞的凋亡。     

美金胺对急性高眼压大鼠视网膜神经节细胞保护作用的实验研究

目的探讨美金胺对急性高眼压大鼠视网膜神经节细胞（retinal ganglion cells，RGCs）的保护作用。方法以生理盐水在前房内加压灌注制作大鼠急性高眼压模型，然后腹腔内注射美金胺或生理盐水，分为美金胺治疗组和急性高眼压对照组各20只，另设健康对照组大鼠8只。采用免疫组织化学法和脱氧核苷酸末端转移酶介导的脱氧尿苷三磷酸末端标记法（terminal deoxynucleotidyl transferase mediated 2＇-deoxyuridine 5＇-triphosphate-biotin nick end labeling，TUNEL），检测大鼠RGCs caspase-3和凋亡细胞的表达。结果健康对照组无caspase-3与凋亡细胞表达。急性高眼压对照组在高眼压6h后视网膜出现凋亡细胞，逐渐增加至24h达高峰，后逐渐减少，72h时仅有少数凋亡细胞；caspase-3表达改变与凋亡细胞表达相似。美金胺治疗组的变化规律同急性高眼压对照组。急性高眼压6，24和72h后凋亡细胞与caspase-3的表达在3组间的差异均有统计学意义（P值均〈0．01）。结论细胞凋亡可能在视网膜急性高眼压损伤过程中起重要作用，美金胺对RGCs具有保护作用。     

姜黄素对慢性高眼压大鼠视网膜神经节细胞凋亡的影响及机制

目的：探讨姜黄素对慢性高眼压大鼠视网膜神经节细胞(RGCs)凋亡的影响及机制。

方法：将21只SD大鼠随机分为3组,每组7只,高眼压模型组和姜黄素治疗组大鼠通过烧灼巩膜上静脉法建立慢性高眼压模型,假手术组大鼠仅剪开球结膜,不烧灼巩膜上静脉; 姜黄素治疗组给予4mL/kg姜黄素灌胃,假手术组和高眼压模型组给予4mL/kg纯水灌胃,连续3wk。造模后3wk,采用HE染色观察各组大鼠视网膜组织形态病理变化、RGCs数量及神经节细胞层(GCL)厚度; 采用TUNEL染色观察各组大鼠RGCs和视网膜细胞凋亡情况; 采用实时荧光定量PCR、免疫组织化学染色和Western blot法检测各组大鼠视网膜谷氨酰半胱氨酸连接酶调节亚基(GCLM)与血红素加氧酶-1(HO-1)的表达水平。

结果：与假手术组相比,高眼压模型组和姜黄素治疗组大鼠视网膜组织形态紊乱,RGCs数量减少,GCL变薄,RGCs和视网膜细胞凋亡率均升高,GCLM和HO-1表达量均升高; 与高眼压模型组相比,姜黄素治疗组大鼠视网膜组织形态基本正常,RGCs数量增多,GCL增厚,RGCs和视网膜细胞凋亡率均降低,GCLM和HO-1表达量均升高。

结论：姜黄素在慢性高眼压大鼠模型中可通过上调抗氧化基因GCLM与HO-1的表达抑制RGCs凋亡。     

兔实验性高眼压视网膜神经节细胞的凋亡

目的 探讨实验性高眼压免眼中是否存在视网膜神经节细胞的凋亡。方法 血细胞注射法诱导青光眼建立大白兔血细胞诱导的高眼压实验动物模型。通过制作视网膜组织超薄切片，透射电镜鉴定视网膜神经节细胞(RGCS)凋亡的存在。制备视网膜细胞悬液，利用流式细胞仪检测高眼压兔眼视网膜神经节细胞凋亡的数量。结果 实验组血细胞注射后眼压有不同程度的升高，透射电镜下可见典型的RGCs凋亡阳性细胞存在，经流式细胞仪检测凋亡率为O.76％，～O．93％。结论血细胞诱导青光眼可得到良好的慢性高眼压实验动物模型，实验性高眼压兔眼中有视网膜神经节细胞的凋亡缔朐稠亡旱免高眼RGCs损伤；流式细胞术     

经瞳孔温热疗法对急性高眼压大鼠视网膜神经节细胞的保护作用

目的研究低阈值经瞳孔温热疗法（TTT）对急性高眼压大鼠视网膜神经节细胞（RGC）是否具有保护作用。设计实验研究。研究对象BN大鼠。方法采用810nm二极管激光机对10只大鼠视网膜进行热刺激，照射光斑1．2mm，能量50mW，照射时间20s，干预后3d光镜下观察视网膜形态结构的改变，免疫组化方法检测HSP70、HSP27在视网膜组织表达。采用上述激光参数，照射视网膜后3d，制作急性高眼压模型（TTT＋I/R组，n=10），采用TUNEL法检测RGC层细胞凋亡数量，及计数高倍镜下RGC层细胞数，与未干预的急性高眼压模型组（I/R组，n=10）、单纯TTT干预组（TTT组）及正常对照组（n=6）进行比较。主要指标免疫组化染色RGC细胞数及RGC层细胞凋亡数。结果采用低阈值TTT可诱导BN大鼠视网膜神经节细胞HSP70及HSP27表达，且光镜下未出现明显视网膜脉络膜形态的改变。TTT＋I／R组RGC层细胞凋亡数量明显少于I／R组（P=0．048），且前者RGC层细胞数量明显多于后者（辟0．016）；TTT组与正常对照组比较RGC层细胞凋亡数量无显著性差异（P=0．882），但RGC层细胞数明显少于正常对照组（P=0．001）。结论低阈值TTT可诱导BN大鼠视网膜HSP70、HSP27表达，并在急性高眼压损伤下对大鼠RGC凋亡具有抑制作用。（眼科，2007,16：48—51）     

神经生长因子-β、脑源性神经营养因子、胶质细胞源性神经营养因子对体外压力作用下视网膜神经细胞的保护作用

高眼压是青光眼最主要的致病因素，青光眼的视野丧失是节细胞死亡的结果，供应视网膜的神经营养因子被剥夺、细胞内离子的失衡等参与了细胞的损害过程。神经生长因子(NGF)、脑源性神经营养因子(BDNF)、胶质细胞源性神经营养因子(GDNF)、睫状节神经营养因子(CNTF)等可减少实验性高眼压缺血、视神经切断后的视网膜神经节细胞的死亡。为     

补阳还五汤加减对高眼压大鼠视网膜神经节细胞的保护作用

目的 探讨补阳还五汤加减对高眼压大鼠模型视网膜神经节细胞的保护作用.方法 90只SD大鼠随机分为假手术组(n=30)和高眼压模型组(n=60).造模成功大鼠56只被随机分为治疗组(n=28)和对照组(n=28).造模后4周,模型治疗组给予补阳还五汤加减浓缩剂灌胃,其余2组灌服等剂量生理盐水,每组随机选取10只动物经上丘注射50 μL体积分数10% DiI.用药后4周行视网膜神经节细胞的TUNEL染色、DiI阳性细胞计数、节细胞凋亡率的检测.结果 假手术组视网膜无TUNEL阳性细胞,模型治疗组及模型对照组视网膜均存在明显的TUNEL阳性细胞.假手术组视网膜神经节细胞数为1 685.49±583.47,对照组为405.61±229.88,而治疗组为1 069.74±387.53.治疗组明显多于对照组而少于假手术组.假手术组视网膜神经节细胞凋亡率为(7.32±6.89)%,对照组为(41.23±8.76)%,治疗组为(23.12±10.11)%.治疗组明显低于对照组而高于假手术组.结论 补阳还五汤加减可通过抑制青光眼大鼠视网膜神经节细胞凋亡而发挥视神经保护作用.     

雌激素对慢性高眼压兔视网膜损伤的保护作用

目的 探讨雌激素对慢性高眼压兔视网膜损伤的保护作用.方法 18只成年健康新西兰雌性大白兔随机分为去势组和假去势组.去势组切除两侧卵巢,假去势组仅切除卵巢周围部分脂肪组织.利用前房注射复方卡波姆建立兔慢性高眼压模型.采用末端脱氧核酸转移酶介导的脱氧三磷酸尿苷缺口末端标记法(TUNEL)检测视网膜组织中凋亡细胞的表达;免疫组织化学方法检测B细胞淋巴瘤/白血病(bcl)-2、bax蛋白的表达情况;应用计算机图像分析系统对检测结果进行分析.结果 造模后4、6、8周,去势组较假去势组视网膜神经节细胞数目明显减少,视神经纤维层和内核层变薄;TUNEL阳性细胞表达均较假去势组高(t=3.285,4.012,3.624;P值均<0.01);视网膜bcl-2表达均较假去势组低(t=4.256,3.867,3.459;P值均<0.01);bax阳性细胞表达情况均较假去势组高(t=3.211,3.625,3.253;P值均<0.01).bcl-2的表达与TUNEL的表达呈负相关,凋亡细胞随着bcl-2的表达增多而减少,bcl-2抑制了凋亡的发生.bax的表达与TUNEL的表达呈正相关,凋亡细胞随着bax的表达增多而增多,bax诱导了凋亡的发生.结论 雌激素对慢性高眼压兔视网膜损伤有神经保护作用,这一保护作用主要是通过减少视网膜组织中的凋亡细胞而实现的.     

VEGF对大鼠慢性高眼压视网膜神经节细胞的作用

目的：探讨血管内皮生长因子（vascular endothelium growth factor, VEGF）对大鼠视网膜色素上皮生长因子（pigment epithelium-derived factor, PEDF）表达的影响及VEGF对慢性高眼压条件下神经节细胞（retinal ganglion cells, RGCs）的保护作用和可能途径。方法：雌性SD大鼠30只,随机分为高眼压＋VEGF组A(包括A3d,A14d)、高眼压＋安慰剂组B(包括B3d,B14d)和正常＋VEGF组C(包括C3d,C14d),A、B组模型制作应用巩膜浅层静脉烧烙法建立慢性高眼压模型,C组只剪开球结膜,不烧烙巩膜浅静脉。A、C组在模型建立后即刻用10μL微注射器于大鼠角膜缘后2mm处刺入玻璃体腔,抽出玻璃体2μL,再向玻璃体腔内注射2μL（0.05μg/μL）重组大鼠VEGF,B组同法注入等量的去离子水。在3d和14d后处死动物,取眼球,冰冻切片,免疫组织化学法观察视网膜PEDF的表达,用TUNEL染色检测各组视网膜神经节细胞的凋亡,免疫荧光双标观察PEDF染色阳性的细胞是何细胞。结果：术后眼压明显升高(P<0.05),术后3d和14d的眼压无显著性差异。视网膜PEDF染色阳性细胞,B组多于A组,A组多于C组;TUNEL荧光染色显示VEGF高眼压组RGCs的凋亡明显的少于高眼压组。 结论：玻璃体腔注射VEGF可减少高眼压视网膜神经节细胞的凋亡。     

Changes of the Retina and Intrinsic Survival Signals in a Rat Model of Glaucoma following Brinzolamide and Travoprost Treatments

Purpose: The purpose of this study is to examine the changes of the retina and the intrinsic survival signals of the retina by brinzolamide (Azopt?) and travoprost (Travatan?) in a rat model of chronic ocular hypertension. Methods: Chronic ocular hypertension was induced by cauterization of three episcleral veins. Terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labeling (TUNEL) staining was performed and the expression of glial fibrillary acidic protein (GFAP) was examined to evaluate changes of retinal ganglion cell (RGC) apoptosis and glial cell activation. Western blot analyses of the bcl-2 family and extracellular signal-regulated kinases (ERK) were done to identify changes of the intrinsic survival signaling pathway in the retina. Results: GFAP expression and TUNEL staining revealed significant decreases in RGC apoptosis and glial cell activation after brinzolamide and travoprost administration; bcl-2 and bcl-xL expression were significantly increased after intraocular pressure elevation and it was further increased with brinzolamide and travoprost treatment. This enhancement of survival signaling may have contributed to the decrease in RGC apoptosis. However, the role of ERK signaling was not significant. Conclusions: Decrease in retinal damage and increased intrinsic survival signals suggests the neuroprotective potential of brinzolamide and travoprost in an animal model of chronic ocular hypertension, but further studies are required.     

雌激素对慢性高眼压兔视网膜损伤的保护作用

目的 探讨雌激素对慢性高眼压兔视网膜损伤的保护作用.方法 18只成年健康新西兰雌性大白兔随机分为去势组和假去势组.去势组切除两侧卵巢,假去势组仅切除卵巢周围部分脂肪组织.利用前房注射复方卡波姆建立兔慢性高眼压模型.采用末端脱氧核酸转移酶介导的脱氧三磷酸尿苷缺口末端标记法(TUNEL)检测视网膜组织中凋亡细胞的表达;免疫组织化学方法检测B细胞淋巴瘤/白血病(bcl)-2、bax蛋白的表达情况;应用计算机图像分析系统对检测结果进行分析.结果 造模后4、6、8周,去势组较假去势组视网膜神经节细胞数目明显减少,视神经纤维层和内核层变薄;TUNEL阳性细胞表达均较假去势组高(t=3.285,4.012,3.624;P值均<0.01);视网膜bcl-2表达均较假去势组低(t=4.256,3.867,3.459;P值均<0.01);bax阳性细胞表达情况均较假去势组高(t=3.211,3.625,3.253;P值均<0.01).bcl-2的表达与TUNEL的表达呈负相关,凋亡细胞随着bcl-2的表达增多而减少,bcl-2抑制了凋亡的发生.bax的表达与TUNEL的表达呈正相关,凋亡细胞随着bax的表达增多而增多,bax诱导了凋亡的发生.结论 雌激素对慢性高眼压兔视网膜损伤有神经保护作用,这一保护作用主要是通过减少视网膜组织中的凋亡细胞而实现的.     

雌激素对慢性高眼压兔视网膜损伤的保护作用

目的 探讨雌激素对慢性高眼压兔视网膜损伤的保护作用.方法 18只成年健康新西兰雌性大白兔随机分为去势组和假去势组.去势组切除两侧卵巢,假去势组仅切除卵巢周围部分脂肪组织.利用前房注射复方卡波姆建立兔慢性高眼压模型.采用末端脱氧核酸转移酶介导的脱氧三磷酸尿苷缺口末端标记法(TUNEL)检测视网膜组织中凋亡细胞的表达;免疫组织化学方法检测B细胞淋巴瘤/白血病(bcl)-2、bax蛋白的表达情况;应用计算机图像分析系统对检测结果进行分析.结果 造模后4、6、8周,去势组较假去势组视网膜神经节细胞数目明显减少,视神经纤维层和内核层变薄;TUNEL阳性细胞表达均较假去势组高(t=3.285,4.012,3.624;P值均<0.01);视网膜bcl-2表达均较假去势组低(t=4.256,3.867,3.459;P值均<0.01);bax阳性细胞表达情况均较假去势组高(t=3.211,3.625,3.253;P值均<0.01).bcl-2的表达与TUNEL的表达呈负相关,凋亡细胞随着bcl-2的表达增多而减少,bcl-2抑制了凋亡的发生.bax的表达与TUNEL的表达呈正相关,凋亡细胞随着bax的表达增多而增多,bax诱导了凋亡的发生.结论 雌激素对慢性高眼压兔视网膜损伤有神经保护作用,这一保护作用主要是通过减少视网膜组织中的凋亡细胞而实现的.     

Neutralizing tumor necrosis factor-alpha activity suppresses activation of infiltrating macrophages in experimental autoimmune uveoretinitis

PURPOSE: During experimental autoimmune uveoretinitis (EAU), infiltrating macrophages become activated to express nitric oxide synthase (NOS)-2 and generate nitric oxide (NO). The current study was designed to determine whether neutralizing TNF activity with a soluble fusion protein of TNFp55 receptor (sTNFr-IgG) inhibits macrophage activation, thereby contributing to reduced tissue damage observed with such treatment. METHODS: EAU was induced in Lewis rats by active immunization with soluble retinal extract (RE) and pertussis toxin (intraperitoneally), and animals were treated on days 6 and 8 after immunization with either sTNFr-IgG or human (hu)IgG. Disease course and severity were noted clinically, and eyes were enucleated for histologic scoring, including TUNEL immunofluorescence, at various stages of disease. Infiltrating retinal macrophages were isolated through a density gradient and subsequently phenotyped by flow cytometry, analyzed for ability to produce nitrite, either spontaneously or after cytokine stimulation, and assayed by PCR for cytokine gene expression. RESULTS: Neutralizing TNF activity suppressed tissue damage without impeding myeloid cell infiltrate. Moreover, with sTNFr-IgG treatment, infiltrating macrophages demonstrated reduced nitrite production at the height of disease, and the level of apoptosis within the retina of both ED1(+) cells and resident cells was reduced. PCR analysis demonstrated a significant increase in TGF beta signal and absent or low TNF signal throughout the disease course after treatment with sTNFr-IgG. CONCLUSIONS: sTNFr-IgG successfully suppresses retinal damage and impairs macrophage activation but not trafficking during EAU. sTNFr-IgG-mediated suppression of NO production results in reduced levels of apoptosis of inflammatory cells and reduction in photoreceptor damage.     

转基因鼠晶状体特异性表达的制瘤素对眼发育的影响

目的 研究晶状体特异性表达的制瘤素（oncostatin M ，OSM )对眼发育的影响。 方法 将去除部分序列的小鼠OSM cDNA［661碱基对（base pair,bp)］ 连接到αA-晶状体蛋白 （αA-crystallin）启动子上，然后用显微注射的方法将其导入单细胞胚胎内，建立转基因鼠。常规组织学及免疫组织化学方法对转基因鼠进行特性鉴定，末端脱氧核苷酸转移酶介导的脱氧尿嘧啶末端标记（terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling,TUNEL ）试验检测细胞凋亡，原位杂交检测caspase-3-mRNA的表达，兔抗活化的caspase-3 抗体检测活化的caspase-3蛋白质。 结果 胚胎期14.5、17.5 d，转基因蛋白OSM特异性表达于晶状体纤维细胞内，从胚胎期17.5 d开始，转基因鼠视网膜开始发生变性，出生时，50％以上的视网膜细胞丢失。TUNEL试验显示转基因鼠视网膜细胞凋亡。转基因鼠视网膜细胞中caspase-3被激活。 结论 晶状体特异性的OSM表达激活caspase-3，导致了眼的异常发育、细胞凋亡及广泛的视网膜变性。 （中华眼底病杂志，2003，19：201-268）     

UV irradiation causes multiple cellular changes in cultured human retinal pigment epithelium cells

Background The retinal pigment epithelium maybe causally involved in the development and progression of age-related macula degeneration; however, the mechanisms leading to the development of age-related macula degeneration remain largely unknown. The purpose of this study was to examine cellular changes in the retinal pigment epithelium induced by direct irradiation with UV light in culture.Methods Retinal pigment epithelium cells from post-mortem human retinas were used to obtain dissociated cultures with cells retaining the ability to differentiate in vitro. These cells were cultured over several days to weeks. The UV radiation (UV-A and UV-B) occurred under sterile conditions with a 100 HBO/mercury bulb attached to a dissecting microscope, delivering co-axial illumination. The time dependence of irradiation effects was analysed using morphometric, immunohistochemical, functional and apoptosis-detecting techniques.Results Vital and proliferating retinal pigment epithelium cell cultures could be prepared consistently. The cells showed tissue-specific morphologies in vitro for several days to weeks. Pigment epithelium-derived factor was detected in these cells using immunocytochemistry and Western blots. The UV irradiation but not white light resulted in measurable alterations of cell shape and size. The irradiated cells showed partial swelling and shrinkage reminiscent of progressing apoptotic degeneration. TUNEL staining revealed that apoptosis was induced by UV light, but not detectably by white light. The phagocytosis of fluorescent micro-particles diminished after irradiation. These effects were dependent on the duration of irradiation.Conclusions Cultures of retinal pigment epithelium are suitable and sensitive models to study cell damage and may contribute to unravelling the pathogenetic mechanisms of retinal degeneration.     

Vascular damage in a mouse model of diabetic retinopathy: relation to neuronal and glial changes

PURPOSE: Lack of information about the development of diabetic retinopathy in mice has greatly hindered the use of genetic mouse models for the study of disease mechanisms and the development of therapeutic strategies. The objective of this study was to characterize the occurrence and pathologic progression of diabetic retinopathy in C57Bl/6J mice. METHODS: Diabetes was induced with five consecutive injections of streptozotocin (STZ). The retinas were collected at different time points (2 weeks to 22 months) after the induction of diabetes and examined by using molecular, histologic, and immunohistochemical techniques and morphometric analysis. RESULTS: There was transient induction of cell apoptosis and caspase-3 activation in retinal neurons of C57Bl/6 mice within days of diabetes induction. Glial fibrillary acidic protein (GFAP), a marker of glial activation, likewise was transiently upregulated, seemingly in astrocytes but not in Müller cells. These abnormalities quickly returned to normal; ultimately, no detectable loss of retinal ganglion cells (RGCs) was noted by any of three independent methods (number of cells in ganglion cell layer of retinal cross-sections, retrograde labeling of retinal ganglion cells with fluorescent dye, or TUNEL staining) after up to a 1-year duration of diabetes. Despite this apparent lack of evidence for progressive damage in neurons and glial cells, diabetic mice developed vascular disease characteristic of the early stage of diabetic retinopathy beginning at 6 months after the onset of disease. The vascular damage-formation of acellular capillaries and pericyte ghosts-continued to increase through the 18 months examined. CONCLUSIONS: Diabetic C57Bl/6J mice develop capillary lesion that are characteristic of the early stages of diabetic retinopathy in patients. The data suggest that diabetes-induced degeneration of retinal capillaries can develop independent of neuronal loss or chronic GFAP upregulation in glial cells.     

Attenuated glial reactions and photoreceptor degeneration after retinal detachment in mice deficient in glial fibrillary acidic protein and vimentin

PURPOSE: To characterize the reactions of retinal glial cells (astrocytes and Müller cells) to retinal injury in mice that lack glial fibrillary acidic protein (GFAP) and vimentin (GFAP-/-Vim-/-) and to determine the role of glial cells in retinal detachment (RD)-induced photoreceptor degeneration. METHODS: RD was induced by subretinal injection of sodium hyaluronate in adult wild-type (WT) and GFAP-/-Vim-/- mice. Astroglial reaction and subsequent monocyte recruitment were quantified by measuring extracellular signal-regulated kinase (Erk) and c-fos activation and the level of expression of chemokine monocyte chemoattractant protein (MCP)-1 and by counting monocytes/microglia in the detached retinas. Immunohistochemistry, immunoblotting, real-time quantitative polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) were used. RD-induced photoreceptor degeneration was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and measurement of outer nuclear layer (ONL) thickness. RESULTS: RD-induced reactive gliosis, characterized by GFAP and vimentin upregulation, Erk and c-fos activation, MCP-1 induction, and increased monocyte recruitment in WT mice. Absence of GFAP and vimentin effectively attenuated reactive responses of retinal glial cells and monocyte infiltration. As a result, detached retinas of GFAP-/-Vim-/- mice exhibited significantly reduced numbers of TUNEL-positive photoreceptor cells and increased ONL thickness compared with those of WT mice. CONCLUSIONS: The absence of GFAP and vimentin attenuates RD-induced reactive gliosis and, subsequently, limits photoreceptor degeneration. Results of this study indicate that reactive retinal glial cells contribute critically to retinal damage induced by RD and provide a new avenue for limiting photoreceptor degeneration associated with RD and other retinal diseases or damage.     

Immunohistochemical characterisation of surgically excised choroidal neovascularisation in age-related macular degeneration

AIM: The objective of the study was to gather further information about the pathogenesis of choroidal neovascularisations (CNV), which is still not clearly understood, and to establish criteria for making decisions on a appropriate therapy. Immunohistochemical characteristation should allow a more comprehensive evaluation of cellular components of the membranes and their functional role. PATIENTS AND METHODS: In 29 patients (16 women, 13 men) with age-related macular degeneration ranging in age from 46 to 91 years (mean age, 76.4 years), CNV were excised by pars-plana vitrectomy. Sections were stained with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) and examined by light microscopy. For the immunohistochemical characterisation of the surgical specimens the following anti-genetic determinants were used: glial fibrillary acid protein (GFAP) for glial cells, synaptophysin for neuronal cells, neuron-specific enolase (NSE) for neuronal and neuroectodermal cells, CD 31 for endothelial cells and pancytokeratin (KL1) for cells of the retinal pigment epithelium (RPE). Cells undergoing apoptosis were labeled with the TUNEL technique. RESULTS: 22 (76%) surgical specimens showed TUNEL positive cells in the connective tissue, vascular endothelium and retinal pigment epithelium. Positive immunostaining of neuronal antigenetic determinants was found for glial fibrillary acid protein in 22 patients (76%), for synaptophysin in 28 patients (97%) and for neuron-specific enolase in 21 patients (72%) CNV. The epithelial marker KL1 was positive in 28 patients (97%) and the endothelial marker CD 31 in 20 patients (69%). CONCLUSION: The immunohistochemical analyses of CNV showed that in the majority of cases during the excision of choroidal neovascularizations in addition to scar tissue and connective tissue also parts of the native retinal pigment epithelium and of the neurosensory retina are removed which is only partly visible with standard staining techniques. These findings suggest that the mostly not satisfying postoperative results are partly due to the damage of neuronal cells and a partial loss of the retinal pigment epithelium. Apoptosis as a regulating mechanism in choroidal neovascularization. The variable appearence of apoptosis suggests that it is possibly related to the degree of activity of CNV.