房水抽吸联合激光房角光凝法建立大鼠慢性青光眼模型

目的建立一种新的大鼠慢性青光眼模型并观察相应的眼压变化和视网膜病理改变。方法 Wistar雌性大鼠80只,通过角巩膜缘前房穿刺抽吸房水使前房消失,使用多波长黄绿激光行右眼角膜缘（房角）360°光凝,角膜缘激光斑为60～80个,并光凝颞侧、颞上及颞下巩膜浅层静脉,每条静脉光凝3～4个斑点;左眼为对照眼。于术后1、3、7、14、21、28、35、42、49、56d用Tono-PenXL眼压计监测眼压。激光术后7、14、28、42、56d制作大鼠眼球视网膜铺片,行Nissl染色、视网膜神经节细胞（RGCs）定量检测。部分标本制备冰冻切片,行苏木精-伊红染色,光学显微镜下观察模型眼房角的病理变化。结果 73只大鼠实验眼眼压较术前明显升高。激光光凝术前大鼠眼压为（16.56±1.84）mmHg,术后1d眼压为（21.33±2.64）mmHg,术后7d模型眼眼压达峰值,为（30.31±3.22）mmHg,高眼压状态持续21d,28d时眼压为（17.08±1.36）mmHg。术后1～21d,眼压值与术前比较差异均有统计学意义（P〈0.05）,术后28d眼压值与术前相比差异无统计学意义（P〉0.05）。对照眼各时间点眼压间比较差异无统计学意义（P〉0.05）。组织病理学检测表明,实验眼前房角明显变窄,小梁网间隙压缩、变窄,部分闭锁、消失;少量梭形成纤维细胞聚集,小梁网组织致密、硬化,小梁细胞减少;部分虹膜卷曲、水肿、肥厚。术后28dRGCs计数实验眼为（56.67±1.64）个,对照眼为（67.56±6.13）个,二者比较差异有统计学意义（P〈0.05）;术后56d实验眼为（45.56±12.33）个,对照眼为（68.33±3.25）个,二者比较差异有统计学意义（P〈0.05）。结论房水抽吸联合激光房角光凝法是一种成功率高、操作简单的大鼠慢性青光眼模型制作方法 。     

两种方法建立大鼠慢性青光眼模型的对比研究

目的评价2种大鼠慢性青光眼模型的效果。方法通过单纯烙闭3条大鼠巩膜上静脉（单纯手术组,n=19）和烙闭巩膜上静脉同时给予5-氟尿嘧啶（5-fluorouracil,5-Fu）（手术给药组,n=23）2种方法制作大鼠慢性高眼压模型,观察术前和术后1d、3d、5d、1周、2周、1个月和2个月早晚眼压变化,并于术后1周、2周、1个月和2个月摘除眼球,光镜观察大鼠视网膜各层组织厚度变化,荧光金逆行标记视网膜神经节细胞（retinal ganglion cells,RGCs）并计数,TUNEL染色,比较2组模型检测指标。结果2组模型大鼠术后眼压均明显升高,随着高眼压时间持续延长,RGCs逐渐减少及视网膜神经纤维层逐渐变薄,剩余RGCs数量逐渐减少,但2组间差异无统计学意义（P〉0.05）。结论持续高眼压导致大鼠RGCs的病理改变过程及其结果符合青光眼视神经损伤的病理特点。单纯烙闭大鼠巩膜上静脉即可获得稳定有效的慢性青光眼模型。     

硅油填充眼硅油取出术后高眼压的临床研究

目的探讨硅油取出术后高眼压的原因及处理方法。方法2011年12月到2013年6月本院硅油填充眼93例（93只眼）,根据硅油取出术前眼压分为正常眼压组及青光眼组两组,观察各组硅油取出气体C,F。填充术后眼压变化,进行总结、分析,讨论术后高眼压的原因。结果正常眼压组硅油取出术后1周及2周的眼压,较硅油取出术前高,差异有统计学意义（P〈0．05）;而术后1个月、3个月及6个月眼压与硅油取出术前相比,其差异无统计学意义（P〉0．05）。而青光眼组硅油取出术后1周、2周、1个月、3个月及6个月眼压虽较硅油取出术前低,（P〈0．05）,但大多仍高于正常值,硅油取出术后高眼压者在正常眼压组占21．6％（11／51）,在青光眼组占54．8％（23／42）。结论硅油取出术后高眼压是术后常见并发症,故术后监测眼压是必要的,以便及时发现,运用降眼压药物或联合手术进行治疗。     

孤眼青光眼术后白内障粘连性小瞳孔超声乳化术

目的探讨孤眼（另眼已盲且无法复明者）患者抗青光眼术后白内障粘连性小瞳孔超声乳化手术的临床效果。方法孤眼抗青光眼术后白内障粘连性小瞳孔43例（43眼）,进行超声乳化手术。比较分析术前、术后1周及术后3个月眼压及视力变化情况,及术后并发症。结果与术前相比,术后1周及3个月眼压≤21mmHg（1mmHg=0．133kPa）的比率提高,眼压〉21mmHg的比率及平均眼压均降低,差异具有统计学意义（P〈0．05）。与术前相比,术后1周及术后3个月视力均有所改善,其中术后1周视力〉0．5的比率及术后3个月视力0．3～0．5及〉0．5的比率均提高,术后3个月视力〈0．05的比率及视力0．05～0．25的比率均降低,差异具有统计学意义（P〈0．05）。全组43例术后均无严重并发症发生。结论超声乳化术治疗孤眼患者抗青光眼术后粘连性小瞳孔白内障手术风险较大,但经术前精心准备,手术细心操作及妥善的术后处理,术后眼压明显降低,视力明显提高,且无严重并发症发生,临床效果较好。     

两种植入物在非穿透性小梁手术中抗瘢痕化的临床观察

目的评价非穿透小梁手术中透明质酸钠生物胶和羊膜术后抗瘢痕化的作用。方法原发性开角型青光眼36例（60眼）随机分为A组和B组。A组非穿透性小梁手术联合透明质酸钠生物胶组18例（30眼）,B组非穿透性小梁手术联合羊膜组18例（30眼）,术后随访12～24个月。主要指标视力、眼压、结膜滤过泡、前房角、并发症。结果两组患者术后1周及术后6月最佳矫正视力比较,差异无统计学意义（F=0．647,0．735,P〉0．05）;术前两组眼压平均为（26．47±12．990）、（28．03±11．388）mmHg,差异无统计学意义（P〉0．05）,术后1周、6月两组眼压均较术前降低（P〈0．05）,术后1周1、6、12、24月平均眼压两组之间比较差异无统计学意义（F=0．01、0．093、1．104、0．788、0．039,P〉0．05）;术后1周、1月、6月、12月、24月滤过泡、手术成功率两组比较差异无统计学意义（P〉0．05）。结论非穿透小梁手术联合透明质酸钠生物胶和羊膜均可抑制术后瘢痕的形成,有效降低原发性开角型青光眼的眼压,两者疗效相当,但羊膜价格低廉、取材方便。     

原发性闭角型青光眼单侧急性发作对侧眼激光虹膜周边切除术后随访研究

背景对于有房角关闭高危因素的眼,激光虹膜周边切除术（LPI）是首选的治疗方法,但可疑原发性房角关闭患者行LPI术后2年内仍有28％发生房角关闭,因此有必要了解影响LPI术后疗效的相关因素。目的观察原发性闭角型青光眼（PACG）急性发作眼的对侧眼行LPI后眼压及房角变化情况,分析与手术疗效相关的影响因素。方法回顾性分析单侧急性发作史的PACG患者（≥40岁）87例87眼,其对侧眼接受LPI,于术后1周,3、6、9、12个月随访眼压,并在裂隙灯下使用压陷式Goldmann单面房角镜观察LPI后颞、鼻、上、下各象限房角开放情况并按Shaffer房角分级标准记录,同时观察周边同位性房角关闭（AAC）范围以及周边房角黏连范围,与术前值进行比较。手术成功标准为：术后眼压在未使用药物情况下保持在6～21mmHg;并未出现青光眼特征性视神经病变及相应的视野缺损;无需任何抗青光眼药物或手术治疗。对影响LPI成功率的因素进行Cox多因素逐步回归分析。结果共完成随访并纳入分析79例79眼,其中男33例（41．8％）,女46例（58．2％）,年龄（61．4±0．4）岁。手术前后6个组眼压差异的总体比较差异有统计学意义（F=4．056,P〈0．01）,LPI术后各时间点的眼压均较术前降低,差异有统计学意义（P〈0．01）。房角除下方之外各象限及平均Shaffer级别有所增加,LPI术后1周、3个月和6个月与术前比较差异均有统计学意义（P〈0．05）;术后1周和3个月AAC范围较术前缩小,差异均有统计学意义（P〈0．05）,而术后6、9、12个月的AAC范围与术前比较差异无统计学意义（P〉0．05）。术后1年LPI成功者61例（77．2％）,术前眼压、房角各象限及平均Shaffer级别、AAC范围在LPI手术成功与失败病例之间差异有统计学意义（P〈0．01）。Cox逐步回归分析发现,AAC范围与LPI生存率之间具有相关性（Wald=48．150,RR=1．963,P〈0．01）,而术前眼压、房角各象限及平均Shaffer级别、年龄、性别与LPI生存率无明显相关性（P〉0．05）。结论PACG急性发作眼的对侧眼行LPI后可使房角增宽、眼压降低,术后成功率与术前AAC的范围有关,提示在考虑术前ACC范围条件下,LPI可以更有效地预防房角关闭的发生。     

睫状体光凝联合前房穿刺术对新生血管性青光眼的止痛效果观察

目的分析新生血管性青光眼半导体激光经巩膜睫状体光凝术后眼痛症状的成因及联合前房穿刺术后对减轻眼痛症状的效果。方法 35例（35眼）青光眼以半导体激光经巩膜的睫状体光凝联合前房穿刺术。术后随访眼压及疼痛6个月以上。结果术前平均眼压（56.73±21.62）mmHg,术后第2周平均眼压（28.36±11.01）mmHg（t=8.099,P=0.001）。术前轻度疼痛5眼,重度疼痛22眼,剧痛8眼;术后第2周无痛22眼,轻度疼痛11眼,重度疼痛2眼;术后1月只有1眼仍有轻度疼痛,2月后疼痛消失。结论新生血管性青光眼半导体激光经巩膜睫状体光凝联合前房穿刺术可以有效地减轻术后疼痛。     

高度近视眼白内障术后眼压变化的观察分析

目的分析治疗高度近视合并白内障患者超声乳化术后眼压变化。方法对单纯老年性白内障51例（55眼）（A组）,与同期进行的高度近视合并白内障21例（33眼）（B组）均行超声乳化吸出联合人工晶状体植入术,观察术前术后视力（裸眼及矫正视力）、眼压、并发症、眼底。结果术后视力：A组〉0．3者占90．91％,B组〉0．3者占60．61％,均获得较好的疗效,但两者之间有较大的差异。A、B两组术前眼压均在正常范围,两组间差异无统计学意义（P〉0．05）。A组术后1d、1周、1月的眼压与术前相比差异均无统计学意义（P〉0．05）。B组术后1d的眼压与术前相比差异有统计学意义（P〈0．05）。B组术后1d的眼压与术后1周眼压差异无统计学意义（P〉0．05）,但与B组术后1月眼压相比前者明显高于后者,差异有统计学意义（P〈0．05）。同时分别比较B组术后1d、1周、1个月的眼压与A组术后1d、1周、1个月的眼压,差异均无统计学意义（P〉0．05）。结论超声乳化吸出术治疗高度近视合并白内障可获得良好的手术效果,但要求熟练的手术技巧。超声乳化术可能是高度近视合并白内障术后继发开角型青光眼的一个诱因。手术前后监测眼压变化,及时降眼压治疗是必要的。     

藏红花素与灯盏细辛对慢性高眼压模型大鼠视神经保护作用的比较

背景 青光眼视神经损害的主要机制是视觉神经元的凋亡和视网膜血液供应的减少,灯盏细辛已证实对高眼压大鼠视网膜神经节细胞(RGCs)和视神经有保护作用.研究发现,藏红花提取物具有抗炎、抑制神经元凋亡和调节血流等作用,但其能否保护青光眼患者的RGCs尚不清楚. 目的 探讨藏红花素对慢性高眼压模型大鼠视神经的保护作用.方法 采用随机数字表法将32只SD大鼠随机分为假手术组、模型对照组、灯盏细辛组、藏红花素组,每组8只,均以右眼为实验眼.模型对照组、灯盏细辛组、藏红花素组大鼠采用巩膜静脉烧灼法建立慢性高眼压模型;假手术组大鼠仅剪开结膜,灯盏细辛组和藏红花素组大鼠于术前30 rmin和术后每日分别腹腔内注射灯盏细辛注射液150 mg/kg(0.5 ml)和藏红花素20 mg/kg(0.5 ml),共给药4周,假手术组和模型对照组大鼠以同样的方式注射0.5 ml生理盐水.各组大鼠均于术前和术后1d、3d、1周、2周、3周、4周测量眼压.术后4周制备大鼠眼球及视神经标本,采用苏木精-伊红染色法测定视网膜厚度;采用TUNEL染色法计数RGCs凋亡数量;采用透射电子显微镜观察各组大鼠视神经轴突超微结构的变化;采用Western blot法检测视网膜中bcl-2和bax蛋白的表达.结果 模型对照组、灯盏细辛组和藏红花素组大鼠造模和干预后不同时间点眼压均明显高于假手术组,造模后不同时间点大鼠的眼压值均明显高于造模前,各组大鼠在造模前后不同时间点眼压值的变化差异均有统计学意义(F分组=169.079,P=0.000;F时间=50.505,P=0.000).假手术组、模型对照组、灯盏细辛组和藏红花素组大鼠视网膜厚度分别为(192.72±4.28)、(165.15±3.89)、(177.75±3.35)和(182.48±4.12) μm,藏红花素组大鼠视网膜厚度值明显低于假手术组而高于模型对照组和灯盏细辛组,差异均有统计学意义(均P＜0.05).假手术组、模型对照组、灯盏细辛组和藏红花素组大鼠RGCs凋亡率分别为(2.58±1.33)％、(42.10±4.71)％、(28.34±2.96)％和(19.95±2.93)％,藏红花素组大鼠RGCs凋亡率明显低于模型对照组和灯盏细辛组,差异均有统计学意义(均P＜0.05).藏红花素组大鼠视神经有髓纤维数量和bcl-2/bax值均明显高于模型对照组和灯盏细辛组,差异均有统计学意义(均P＜0.05).结论 藏红花素可抑制RGCs凋亡和视神经纤维的变性,对慢性高眼压大鼠视网膜神经细胞发挥保护作用,其对视神经的保护作用强于灯盏细辛.     

慢性高眼压大鼠模型视网膜中Brn-3a表达的动态变化

背景Brn-3a是最近发现的一种视网膜神经节细胞（RGCs）特异性标记物。高眼压的视功能损害主要与RGCs损伤有关,但高眼压大鼠视网膜损伤与Brn-3a表达的关系尚不清楚。目的观察慢性高眼压大鼠不同时期眼压、视网膜的形态学和Brn-3a表达的变化。方法将35只健康成年SD大鼠用随机数字表法随机分为正常对照组5只和模型组30只,术前测量眼压。模型组大鼠一侧眼用Shareef—Sharma手术方法建立慢性高眼压模型,对侧眼只切开结膜,不烙闭巩膜表层静脉作为伪手术眼。按照术后不同处理时间随机将模型组分为术后1、3、5、7、14、28d组共6个亚组,每组5只。分别于术前及术后30min,1、3、7、14、28d用Tono—Pen接触式眼压笔测量双眼眼压。各模型组于术后各相应时间点与正常对照组分别取5只大鼠过量麻醉处死,制作视网膜石蜡切片行常规组织病理学检查,评价视网膜形态变化,用甲苯胺蓝染色法计数RGCs,采用免疫组织化学法检测各时间点各组大鼠Brn-3a在RGCs中的表达。结果高眼压模型组大鼠术后各时间点眼压均明显高于术前,差异有统计学意义（F=95．631,P=0．001）,术后28d时眼压是正常对照组大鼠眼压的1．59倍。与伪手术眼相比,模型眼术后各时间点眼压均明显升高,差异均有统计学意义（q=18．418、15．261、10．987、6．931、4．975、2．962,P〈0．05）。正常对照组RGCs数量为（29．08±1．98）个／高倍视野,造模后3、5、7、14、28d组大鼠RGCs计数逐渐下降,差异均有统计学意义（t=5．943、8．034、15．023、17．004、19．371,P〈0．05）。免疫组织化学染色表明,随着造模时间的延长,各组Brn-3a阳性RGCs数量逐渐下降,差异有统计学意义（F=127．583,P=0．000）。结论采用Shareef—Sharma法可成功建立大鼠慢性高眼压动物模型,其眼压为中等程度升高;高眼压持续时间越长,RGCs的丢失越多。Brn-3a仅在RGCs层表达,随眼压持续时间的增加,Brn-3a的表达减少。     

Dorzolamide and timolol saves retinal ganglion cells in glaucomatous adult rats.

PURPOSE: This study was designed to evaluate the effects of a dorzolamide-timolol combination or dorzolamide on retinal ganglion cell (RGC) density and intraocular pressure (IOP) in glaucomatous eyes of adult rats. METHODS: Glaucoma was induced in the right eye of adult Wistar rats by episcleral venous occlusion. One experimental group was administered dorzolamide 2%-timolol 0.5% combination eye drops, while the other experimental group was administered dorzolamide 2% eye drops. Control groups had surgery without drug administration. Drug application was initiated either 2 weeks before surgery (Group A), from the day of surgery (Group B), 2 weeks after surgery (Group C), or 4 weeks after surgery (Group D). RGCs were labeled by intratectal Fluorogold injections and counted from flat-mount preparations, and IOP was measured using Tonopen. RESULTS: Both dorzolamide-timolol combination and dorzolamide, when applied topically, significantly reduced IOP and improved RGC densities in experimental eyes when compared to control eyes. Earlier initiation, as well as longer duration of drug application, resulted in higher RGC densities. CONCLUSIONS: Topical application of a dorzolamide-timolol combination or dorzolamide saved RGCs to a significant extent and reduced IOP in glaucomatous rat eyes.     

睫状神经营养因子对大鼠急性高眼压眼视网膜神经节细胞的保护作用

背景 青光眼可以引起视网膜神经节细胞(RGCs)凋亡,据报道睫状神经营养因子(CNTF)对外伤性视神经损伤有修复作用,其是否对青光眼视神经病变有保护作用尚少见报道. 目的 观察CNTF对大鼠急性高眼压眼RGCs的保护作用.方法 24只Wistar大鼠双眼采用眼前房平衡盐液加压灌注法建立大鼠急性高眼压模型,造模前2d左眼玻璃体内注入0.5μg CNTF 5μl,右眼以同样的方法注射磷酸钠溶液5μl,另取3只正常大鼠作为正常对照.造模后1、3、7、14 d过量麻醉法处死动物并摘除眼球,制备视网膜组织学切片后采用苏木精-伊红染色法进行形态学观察,光学显微镜下计数RGCs数目;采用免疫组织化学染色法观察RGCs层谷氨酸的表达情况.结果 正常对照组大鼠视网膜各层排列整齐,细胞边界清晰;模型对照组大鼠RGCs细胞膜、细胞核均发现异常改变,有细胞空泡样变;CNTF治疗组大鼠造模后变性的RGCs数量少.与模型对照组比较,造模后3、7、14 d CNTF治疗组RGCs数目明显增加,差异均有统计学意义(均P=0.000).免疫组织化学染色表明,造模后3～7d,CNTF治疗组RGCs层谷氨酸阳性细胞数分别为(5.50±1.04)个／3个高倍视野和(6.00±1.41) 个/3个高倍视野,明显低于模型对照组的(9.00±2.91)个／3个高倍视野和(10.83±1.94)个／3个高倍视野,差异均有统计学意义(均P=0.000),而造模后1d和14 d两组间谷氨酸阳性细胞数的差异均无统计学意义(P=0.578、0.180).结论 CNTF能够下调急性高眼压眼谷氨酸在RGCs中的表达,从而对RGCs提供保护作用.     

鼠青光眼模型视网膜神经节细胞中热休克蛋白27表达的研究

目的探讨热休克蛋白27(HSP27)在鼠青光眼模型视网膜神经节细胞(RGCs)中的表达及其与血清中相应抗体的关系。方法将49只Wistar大鼠随机分为高眼压组(32只鼠)、sham对照(假手术)组(12只鼠)及正常对照组(5只鼠)。电凝鼠巩膜表面至少3组静脉及角膜缘周围血管,减少房水静脉回流,建立鼠青光眼模型。分别于术后1、2、3、4及8周分别处死大鼠。处死前测眼压,抽血2ml,供酶联免疫吸附测定使用,分析视网膜组织内HSP27蛋白抗原产生的血清中相应抗体水平。同时对鼠眼视网膜组织进行石蜡切片,采用免疫组化法检测RGCsHSP27的表达及分布情况,并对检测结果进行统计学分析。结果高眼压组右眼术后眼压明显升高,术后3d眼压(27.52±6.63)mmHg(1mmHg=0.133kPa),术后1周眼压(31.42±6.18)mmHg,此后眼压基本稳定。术后各时间点高眼压组右眼眼压与其术前、左眼及sham组右眼和左眼比较,差异有统计学意义(P<0.01)。血清中抗HSP27抗体滴度在术后1周缓慢升高,2、3周达高峰,此后逐渐下降至接近正常水平。术后2周和3周,高眼压组血清中HSP27抗体含量与sham对照组和正常对照组比较,差异有统计学意义(P<0.01和P<0.05)。RGCs中HSP27阳性表达率在术后各时间点,高眼压组右眼与左眼、sham对照组右眼和左眼及正常对照组右眼和左眼比较,差异有统计学意义(P<0.01)。RGCs中HSP27阳性表达随着眼压升高及高眼压持续时间延长逐渐增强,且视网膜神经纤维层中也出现较明显的HSP27的阳性表达。结论内源性HSP27表达增强可能在青光眼视神经病变中具有重要作用。     

Characterization of retinal damage in the episcleral vein cauterization rat glaucoma model

Episcleral vein cauterization (EVC) is used in rats to generate a glaucoma model with high intraocular pressure (IOP). The long-term retinal damage in this glaucoma model, however, has not been accurately quantified. We report the location and amount of retinal ganglion cell (RGC) damage caused by (EVC) induced IOP elevation in two rat strains. IOP was raised in one eye of Wistar (N = 5) and Brown-Norway(B-N)(N = 7) rats by EVC and monitored monthly until IOP in contralateral eyes equalized at 5 months post-surgery. Animals were maintained for 3.5-4.5 additional months. B-N rats (N = 7) that had no EVC served as controls for this strain. Scotopic flash ERGs were recorded at baseline and just prior to euthanasia. Automated counts of all retrogradely labeled RGCs in retinal flat-mounts were determined and compared between contralateral eyes. RGC density maps were constructed and RGC size distribution was determined. Oscillatory potentials in the group of eyes which had elevated IOP were decreased at the time of euthanasia, when IOP had returned to normal. The group of normal B-N rats had similar RGC counts between contralateral eyes. In the experimental group the mean number of RGCs was not significantly different between control and experimental eyes, but 1 of 5 Wistar and 2 of 7 B-N experimental eyes had at least 30% fewer RGCs than contralateral control eyes. Total retinal area in B-N experimental eyes was higher compared to contralateral eyes. Cumulative IOP exposure of the experimental eyes was modestly correlated with RGC loss while oscillatory potentials appeared to be inversely related to RGC loss. In retinas with extensive (> 30% RGC loss) but not complete damage, smaller cells were preserved better than larger ones. The above results indicate that RGC loss in both Wistar and B-N strains is variable after a prolonged elevation of IOP via EVC. Such variability despite equivalent IOP levels and ERG abnormalities, suggests unknown factors that can protect IOP-stressed RGCs. Identification and enhancement of such factors could prove useful for glaucoma therapy.     

Detection of early neuron degeneration and accompanying microglial responses in the retina of a rat model of glaucoma

PURPOSE: To characterize the early reaction of retinal ganglion cells (RGCs) in a rat model of glaucoma using in vivo imaging and to examine the involvement of retinal microglia in glaucomatous neuropathy. METHODS: Glaucoma was induced in adult female Sprague-Dawley rats by cauterizing two episcleral veins, which resulted in a 1.6-fold increase in intraocular pressure (IOP). Retinal ganglion cells were retrogradely labeled with the fluorescent dye, 4-[didecylaminostyryl]-N-methyl-pyridinium-iodide (4-Di-10ASP) and monitored in vivo after elevation of IOP using fluorescence microscopy imaging. The number of RGCs was quantified on retinal flatmounts. Dying RGCs were surrounded by activated microglia that became visible after taking up the fluorescent debris. Immunocytochemistry was conducted to characterize further the ganglion cells and microglia. RESULTS: Cauterizing two of the four episcleral veins resulted in a consistent increase of IOP to 25.3 +/- 2.0 mm Hg, as measured with a handheld tonometer. IOP remained high for at least 3 months in glaucomatous eyes. The earliest sign of RGC death was detected in anesthetized animals 20 hours after induction of glaucoma. RGCs continued to decrease in number over time, with 40% of RGCs having degenerated after 2.5 months. Fundoscopic examination of the optic nerve head revealed cupping 2 months after induction of glaucoma. In addition, microglia were detected on retinal flatmounts as early as 72 hours after induction. Activated microglia and RGCs were also identified immunocytochemically, with an antibody against ionized calcium-binding adaptor molecule (Iba)-1 and an antibody specific to the 200-kDa subunit of the neurofilament protein, respectively. CONCLUSIONS: Occlusion of episcleral veins is a reproducible method that mimics human glaucoma, with chronically elevated IOP-induced RGC loss. This study shows that in vivo imaging permits the detection of ganglion cells in the living animal in the early stages of the disease and highlights the importance of in vivo imaging in understanding ophthalmic disorders such as glaucoma. Secondly, activation of intraretinal microglia coincides with degeneration of RGCs in glaucoma.     

TLR-4在大鼠慢性高眼压模型视网膜中的表达

】背景TLR-4是一种天然免疫受体,在免疫性中枢神经系统损伤的修复中发挥重要作用,但在青光眼视神经损伤中的具体作用机制不详。目的从基因和蛋白质水平研究正常大鼠及慢性高眼压模型大鼠视网膜组织中TLR4的表达情况,探讨高眼压视网膜神经节细胞（RGCs）损伤的免疫机制及TLR-4在RGCs修复中的可能作用。方法选择d～5周龄的雄性清洁级纯种SD大鼠150只,用烧烙3条巩膜静脉联合术中应用丝裂霉素的方法建立大鼠慢性高眼压模型。Tono—penⅡ型笔式眼压计测量造模前、造模后2h,1、3、7、14、28、56d大鼠眼压。上述各时间点分别取5只高眼压组及空白对照组大鼠视网膜行免疫组织化学染色,观察视网膜TLR4蛋白的表达情况。应用逆转录聚合酶链反应（RT．PCR）法及Westernblot法检测各组视网膜组织中TLR-4mRNA及蛋白质的表达。结果实验组手术后眼压明显升高,与空白对照组比较差异有统计学意义（P〈0．01）。免疫组织化学检测结果显示造模后早期TLR--4在视网膜组织中的表达开始增加,表达量均高于空白对照组（P〈O．05～0．01）;RT—PCR检测表明,大鼠慢性高眼压模型眼造模后各时间点TLR-4mRNA在视网膜的表达量明显高于空白对照组（P〈0．05～0．01）;WesternNot检测显示TLR4在造模后早期视网膜组织中即有表达,结果显示组间差异有统计学意义（P〈0．05～0．01）。结论TLR-4在大鼠慢性高眼压模型眼视网膜中的表达明显上调,提示其在大鼠慢性高眼压视神经退行性病变中发挥免疫调节作用。     

鼠青光眼模型中热休克蛋白27的表达及其作用

目的:探讨热休克蛋白27(HSP27)在鼠青光眼模型视网膜神经节细胞(RGCs)中的表达以及眼压对抗HSP27自身抗体的影响。方法:使用SPSS12.0软件将55只Wistar大鼠随机分为高眼压组(25只鼠)、sham对照(假手术)组(25只鼠)及正常对照组(5只鼠)。采用电凝鼠巩膜表面至少3组静脉及角膜缘周围血管,建立鼠青光眼模型。采用免疫组化和酶联免疫吸附测定(ELISA)方法分别检测术后1,2,3,4及8wk视网膜中RGCs以及神经纤维层(RNFL)HSP27的表达、分布以及血清中抗HSP27抗体水平。结果:随着眼压升高及高眼压持续时间延长,高眼压组右眼RGCs中HSP27表达逐渐增强,与其左眼、sham对照组右、左眼和正常对照组右、左眼比较,差异均有统计学意义(P<0.001),且RNFL中也出现HSP27的表达。高眼压组血清中抗HSP27抗体水平在术后1wk轻度升高(P>0.05),随着眼压升高及高眼压持续至术后8wk,血清中HSP27抗体水平逐渐升高并稳定于较高水平,与sham对照组和正常对照组比较,差异有统计学意义(P<0.05)。结论:内源性HSP27表达增强可能在青光眼视神经病变中具有重要作用。     

Correlation between retinal ganglion cell death and chronically developing inherited glaucoma in a new rat mutant

Glaucoma is a progressive optic neuropathy with characteristic optic disc changes, retinal ganglion cell loss and progressive visual field defects. Elevated intraocular pressure is considered to be a major risk factor in glaucomatous neuropathy. This study aimed to characterize and document a new chronic glaucoma model in the rat with respect to the effect of elevated intraocular pressure on overall retinal dysfunction and retinal ganglion cell loss, and to elucidate the possible mechanisms underlying this cell loss. Intraocular pressure (IOP) was measured in rats using a Tonopen. RGCs were retrogradely labeled with the fluorescent dye, 4-[didecylaminostyryl]-N-methyl-pyridinium-iodide (4-Di-10 ASP) and quantified on retinal flat mounts using fluorescence microscopy. The optic nerve head was examined fundoscopically. Changes in the histological appearance of the whole eyes was studied in paraffin sections, and immunohistochemistry was carried out on cryostat sections. The levels of mRNA for several genes were compared between control and glaucomatous retinae using semi-quantitative RT-PCR. Mutant animals are affected with either a unilateral or bilateral enlargement of the globes having an IOP that ranged from 25 to 45 mmHg, as compared to control values of 12-16 mmHg. The IOP of glaucomatous eyes increased significantly with age to attain a value of 35+/-7.3 at 1.5 years. Concomitant with the rise in IOP, the number of labeled RGCs continued to decrease in number with age. A total of 1887+/-117RGC mm(-2) could be labeled in wild-type control and juvenile mutant pre-glaucomatous retinas, whereas this number dropped to 92+/-26RGC mm(-2) at 1.5 years. Ophthalmoscopy revealed atrophied optic nerve heads in the affected eyes. The pars plicata and the pars plana of the ciliary body of glaucomatous eyes were hypertrophied and elongated, respectively. The anterior chamber was narrow and the irido-corneal angle open in glaucoma eyes. The mRNA of glial-fibrillary-acidic protein, endothelin-1, STAT-3 and STAT-6 increased in the retinas correlating with the severity and duration of the disease. Changes in the expression of GFAP and endothelin-1 could be confirmed using immunohistochemistry. This model may help to address several fundamental issues in the pathogenesis of glaucoma and aid in the development of neuroprotective strategies.     

Translimbal laser photocoagulation to the trabecular meshwork as a model of glaucoma in rats

PURPOSE: To develop and characterize a model of pressure-induced optic neuropathy in rats. METHODS: Experimental glaucoma was induced unilaterally in 174 Wistar rats, using a diode laser with wavelength of 532 nm aimed at the trabecular meshwork and episcleral veins (combination treatment group) or only at the trabecular meshwork (trabecular group) through the external limbus. Intraocular pressure (IOP) was measured by a tonometer in rats under ketamine-xylazine anesthesia. Possible retinal vascular compromise was evaluated by repeated fundus examinations and by histology. The degree of retinal ganglion cell (RGC) loss was assessed by a masked, semiautomated counting of optic nerve axons. Effects of laser treatment on anterior ocular structures and retina were judged by light microscopy. RESULTS: After the laser treatment, IOP was increased in all eyes to higher than the normal mean IOP of 19.4 +/- 2.1 mm Hg (270 eyes). Peak IOP was 49.0 +/- 6.1 mm Hg (n = 108) in the combination group that was treated by a laser setting of 0.7 seconds and 0.4 W and 34.0 +/- 5.7 mm Hg (n = 46) in the trabecular group. Mean IOP after 6 weeks was 25.5 +/- 2.9 mm Hg in glaucomatous eyes in the combination group compared with 22.0 +/- 1.8 mm Hg in the trabecular group. IOP in the glaucomatous eyes was typically higher than in the control eyes for at least 3 weeks. In the combination group, RGC loss was 16.1% +/- 14.4% at 1 week (n = 8, P = 0.01), 59.7% +/- 25.7% at 6 weeks (n = 88, P < 0.001), and 70.9% +/- 23.6% at 9 weeks (n = 12, P < 0.001). The trabecular group had mean axonal loss of 19.1% +/- 14.0% at 3 weeks (n = 9, P = 0.004) and 24.3% +/- 20.2% at 6 weeks (n = 25, P < 0.001), increasing to 48.4% +/- 32.8% at 9 weeks (n = 12, P < 0.001). Laser treatment led to closure of intertrabecular spaces and the major outflow channel. The retina and choroid were normal by ophthalmoscopy at all times after treatment. Light microscopic examination showed only loss of RGCs and their nerve fibers. CONCLUSIONS: Increased IOP caused by a laser injury to the trabecular meshwork represents a useful and efficient model of experimental glaucoma in rats.