光动力法诱导大鼠视网膜新生血管动物模型的建立

目的:建立视网膜静脉阻塞诱导新生血管生成的动物模型,为视网膜新生血管性疾病的研究提供条件。方法:健康成年SD大鼠50只,常规麻醉后,尾静脉注射虎红,532nm激光光凝视网膜主要静脉,建立视网膜新生血管动物模型。光凝术后2wk,采用心内注射FITC-dextran,视网膜铺片法观察新生血管生成的情况。结果:光凝手术时,可见视网膜静脉内明显黑色血栓形成。光凝后第2d发现视网膜主要静脉迂曲粗大、可见栓塞引起出血。2wk后FITC-dextran心内灌注视网膜铺片显示光凝眼视网膜血管形态和分布发生明显改变,失去正常的放射状和网状结构,出现大量新生血管芽和新生血管枝,视网膜血管杂乱、荧光素渗漏。结论:成功的构建了光动力诱导视网膜静脉阻塞的动物模型,并使用FITC-dextran心内灌注视网膜铺片对视网膜血管进行造影,从整体显示视网膜血管和新生血管的形态。该模型稳定性和重复性好,是研究视网膜新生血管的较为理想的动物模型。     

改良的视网膜血管消化铺片联合共聚焦显微镜观察--视网膜血管的三维检查方法

目的探讨一种视网膜血管的三维检查方法。设计实验性研究。研究对象大鼠视网膜血管消化铺片。方法将大鼠深度麻醉后,行全身循环灌注,至眼球变苍白,再灌注4％多聚甲醛作血管内固定。取双眼以4％多聚甲醛固定24小时后,取出视网膜,改良视网膜血管消化铺片方法,采用胶原酶消化制备视网膜血管铺片,经免疫荧光染色后采用激光扫描共聚焦显微镜观察。主要指标视网膜血管铺片消化状况及三维结构的观察。结果胶原酶消化处理后可获得理想的、完整视网膜血管铺片, 通过激光扫描共聚焦显微镜扫描,可以观察到视网膜血管三维特征。结论改良的视网膜血管消化铺片联合共聚焦显微镜观察是一种有效的视网膜血管三维检查方法。     

完整视网膜毛细血管网消化铺片技术及细胞计数方法的改进

背景 以往视网膜血管消化铺片法仅能够获得部分视网膜血管,无法客观评价全视网膜血管的病理改变,因此进一步获得完整的视网膜血管网是相关疾病实验研究和临床研究的基础. 目的 在国外对全视网膜血管消化铺片技术的基础上进行改良,建立全视网膜血管的铺片方法. 方法 30只健康Wistar 大鼠随机数字表法分为模型组和对照组,模型组20只大鼠用溶于0.05 mmol/L柠檬酸缓冲液的质量分数1.25％链脲佐菌素（STZ）腹腔内注射法制作糖尿病模型,对照组10只大鼠以同样的方法注射等容量柠檬酸缓冲液.注射后8个月经左心室注射PBS 100 ml,剪开右心房使血液及PBS流出后注射质量分数4％多聚甲醛100 ml,摘除眼球,将完整剥离的大鼠视网膜先进行胰蛋白酶消化,然后剥离玻璃体及内界膜,去除视网膜神经组织,得到完整的视网膜毛细血管网,平铺于载玻片上,将视网膜毛细血管网以视盘为中心划为内、中、外3个区域,采用盲法在光学显微镜下判定和计数中间区域影周细胞及无细胞血管. 结果 视网膜铺片显示,大鼠的视网膜血管为全血管型,可见放射状的视网膜中央动脉与视网膜中央静脉主干及逐级分支结构、小动脉与小静脉之间表浅层和深层的毛细血管网.周细胞略突出于血管管壁,在其形成影周细胞时,原有的细胞核缺失.无细胞血管的管径为邻近毛细血管管径的20％以上,并且在整个血管上无细胞核和影周细胞.结论 全视网膜消化铺片技术能够提供实验所需的完整视网膜毛细血管网,配合细胞计数方法,能够较好地评价视网膜的形态改变,并为视网膜血管和管壁细胞的定量分析提供有利条件.     

枸杞多糖对糖尿病大鼠视网膜病理改变及其VEGF表达的影响

糖尿病视网膜病变（DR）的主要病理基础是缺氧和炎症引起的新生血管形成,导致血-视网膜屏障（BRB）的破坏,而血管内皮生长因子（VEGF）是主要的血管生成因子.研究证实,枸杞多糖（LBP）具有保护细胞抗氧化损伤的作用,但其在眼科疾病的应用研究较少. 目的 观察LBP对不同时期糖尿病大鼠视网膜的病理改变及VEGF表达的影响. 方法 取SPF级健康雄性SD大鼠117只,以随机数字表法将动物随机分为正常对照组、糖尿病组和LBP组.糖尿病组和LBP组大鼠一次性腹腔内注射55 mg/kg链脲佐菌素（STZ）柠檬酸缓冲液诱导1型糖尿病大鼠模型,正常对照组注射等体积的柠檬酸钠-柠檬酸缓冲液.造模成功后LBP组大鼠每日以250 mg/kg LBP定时定量灌胃,分别于成模后4、10、16周取各组大鼠行伊文思蓝（EB）染色视网膜铺片,动态观察视网膜血管的形态改变;以45 mg/kg EB由大鼠右颈静脉缓慢注入,循环2h后将质量分数1％多聚甲醛由左心室灌注,循环3 min后摘除眼球并分离视网膜,用标准曲线法检测视网膜中EB质量分数.采用免疫组织化学法和实时荧光定量PCR（real-time PCR）法分别检测视网膜中VEGF蛋白及其mRNA的表达. 结果 EB视网膜铺片显示,造模后4、10、16周,LBP组大鼠视网膜血管的走行、管径及渗漏等形态学改变均明显轻于糖尿病组大鼠.造模后4、10、16周,LBP组大鼠视网膜中EB的质量分数分别为（12.17±1.55）、（16.46±1.60）和（19.55± 1.49） mg/g,均明显低于同时期糖尿病组大鼠的（15.76±1.90）、（21.61±2.05）和（26.30±2.28） mg/g,差异均有统计学意义（P＜0.05）.免疫组织化学染色结果显示,造模后4、10、16周,大鼠视网膜中VEGF蛋白的表达以视网膜神经节细胞（RGCs）层为主,LBP组大鼠各时间点VEGF蛋白的染色明显弱于糖尿病组.免疫组织化学法检测表明,造模后4、10、16周LBP组大鼠VEGF蛋白在视网膜中的表达量分别为0.234±0.011、0.331±0.023和0.536±0.031,均明显低于同期糖尿病组大鼠的0.281±0.018、0.533±0.055和0.765±0.075,差异均有统计学意义（P＜0.05）.Real-time PCR显示,造模后4、10、16周LBP组VEGF mRNA的相对表达量分别为0.157±0.013、0.505±0.114和1.577±0.074,均低于同期糖尿病组的0.235±0.209、1.043±0.084和2.446±0.061,差异均有统计学意义（P＜0.05）. 结论 LBP可以减轻糖尿病造成的视网膜血管形态改变,减少血管壁的渗漏,从而保护BRB功能.     

脯氨酰羟化酶在糖尿病大鼠视网膜的表达及其意义

目的 观察探讨糖尿病(DM)大鼠视网膜组织中脯氨酰羟化酶(PHD-2)的表达及意义.方法 雄性Wistar大鼠108只,随机分为DM模型组和正常对照组,分别为60、48只.对DM模型组大鼠进行一次性腹腔注射1％链脲霉素枸橼酸(STZ)溶液建模,正常对照组腹腔注射等量的构橼酸缓冲液.造模后1、3、6个月,荧光显微镜下观察大鼠视网膜血管分布形态.造模后1～6个月,采用伊凡思蓝(EB)灌注视网膜铺片检测视网膜组织内EB浓度;免疫组织化学染色法观察大鼠视网膜PHD-2阳性染色的分布特点;蛋白质免疫印迹法(Western blot)检测大鼠视网膜中PHD-2、缺氧诱导因子-1α(HIF-1α)及血管内皮生长因子(VEGF)的表达.结果 荧光显微镜观察结果显示,正常对照组大鼠视网膜血管走行规则,浅深层血管形态清晰;DM模型组大鼠视网膜血管渗漏持续存在.造模后1～6个月,DM模型组大鼠视网膜血管外组织内EB含量较正常对照组明显增加,差异有统计学意义(t=3.810,2.722,2.845,2.342,2.456,3.823;P＜0.05).免疫组织化学染色结果显示,DM模型组及正常对照组大鼠视网膜中均可见PHD-2阳性染色,主要分布于视网膜内核层、神经节细胞层及血管壁.Western blot检测结果显示,DM模型组大鼠视网膜中PHD-2表达在造模后1、2个月时较正常对照组下降(t=16.230,16.390;P＜0.05);HIF-1a表达在造模后1、2、3个月时较正常对照组明显升高(t=27.073,36.709,10.176; P＜0.05);VEGF表达在造模后1～6个月均较正常对照组升高(t=13.547,31.984,21.897,8.912,9.019,14.046;P＜0.05).结论 PHD-2在DM大鼠视网膜组织中有丰富表达.PHD-2可能在糖尿病视网膜病变发生发展中有一定的调控作用,其作用途径及机制与VEGF作用通路有关.     

视网膜消化铺片的免疫荧光染色探讨

目的：探讨视网膜消化铺片联合免疫荧光染色技术的可行性。方法：正常Wistar大鼠和链尿佐菌素（Streptozotocin,STZ)诱导的糖尿病2周大鼠各5只。经心脏灌注后，取右眼视网膜作胰酶消化铺片，并在铺片上进行血管细胞粘附分子－1（Vascular cell adhesion molecule-1,VCAM-1)单克隆抗体免疫荧光染色。结果；大鼠视网膜消化铺片可以清晰看到视网膜血管走行、分布、正常大鼠未见VCAM－1阳性表达，糖尿病2周组视网膜血管壁见VCAM－1阳性细胞表达。结论：视网膜血管铺片联合免疫荧光染色技术是可行的。     

雌激素对视网膜血管渗漏的影响

目的探讨用去势方法研究雌激素影响眼部疾病的可行性以及雌激素对视网膜血管渗漏的影响。方法健康成熟的雌性SD大鼠22只，随机分为实验组和对照组。实验组雌性SD大鼠用去势方法降低体内雌激素水平；对照组用假去势方法，不影响雌激素水平。手术效果用阴道上皮涂片方法证实。用光动力诱导方法建立视网膜静脉阻塞模型，用分光光度计测量视网膜伊文思蓝渗漏量。结果实验组大鼠阴道上皮成熟值降低明显（t=21.008，P=0.000），对照组大鼠成熟值变化不明显（t=0.319，P=0.756）。实验组大鼠视网膜平均伊文思蓝渗漏量为（25.503 0±4.378 47）ng/mg，对照组大鼠视网膜平均伊文思蓝渗漏量为（17.830 0±4.265 69） ng/mg。2组之间差异有统计学意义（t=3.969 36，P=0.001）。结论去势方法是研究雌激素在眼部疾病作用的可靠模型，雌激素水平降低时视网膜血管的渗漏增加。(中华眼底病杂志,2005,21:174-176)     

Evans蓝灌注血管造影对改良型高氧诱导小鼠视网膜新生血管形态观察

目的 验证Evans蓝灌注血管造影对小鼠视网膜新生血管形态观察的可行性.方法 C57BL/6J小鼠于生后7 d置于含体积分数（75±2）%氧气的密闭氧箱内,每日加食换水替换母鼠,5 d后返回正常大气环境,制作高氧诱导的小鼠视网膜新生血管动物模型.于生后15 d、17d、19 d、21 d、24 d、30 d麻醉小鼠后行质量分数2%Evans蓝上腔静脉灌注,5 min后处死,摘取眼球,固定后视网膜铺片,荧光显微镜下观察、照相.结果 Evans蓝灌注血管造影可以清楚地显示小鼠视网膜深浅2层血管网及中间的连接血管,并能动态反映高氧诱导的小鼠视网膜形成无灌注区、血管扩张迂曲及渗漏、新生血管及血管瘤的形成.后期血管增殖反应慢慢消退这一过程也能呈现.显影清晰、完全,方法简便、可重复性高.结论 Evans蓝灌注血管造影可准确、动态反映小鼠视网膜新生血管病变形态特点,且价格低廉、简单易行,为视网膜新生血管性眼病的预防与治疗提供广阔的研究前景.     

尾静脉注射骨髓细胞对糖尿病小鼠视网膜新生血管化的治疗作用

目的:研究尾静脉注射骨髓细胞对于链脲佐菌素(strepto-zotocin,STZ)诱导的糖尿病(diabetes mellitus,DM)小鼠视网膜振荡电位(oscillatory potentials,OPs)和视网膜新生血管密度的影响。方法:40只C57BL/6J小鼠随机分成正常对照(NC)组和DM造模组。DM造模组给予多性腹腔注射,造模成功后再随机分为DM对照组和DM骨髓治疗组,饲养16wk,罗兰视觉电生理仪检测其OPs波,视网膜铺片联合免疫荧光染色检测视网膜新生血管密度。DM骨髓治疗组给予小鼠骨髓细胞悬液尾静脉注射,注射2wk和8wk后再次分别检测OPs波及视网膜新生血管密度。结果:高血糖的影响使得DM小鼠造模成功后OPs波振幅呈明显降低趋势,OPs峰潜时明显延长,与对照组小鼠各时期相比有显著差异(P<0.05)。骨髓治疗2wk和8wk后即小鼠26wk及32wk时,DM骨髓治疗组较DM组OPs波幅明显升高,OPs峰潜时明显缩短,有显著差异(P<0.05)。而26wk及32wk时NC组及DM骨髓治疗两组OPs振幅及峰潜时相比无显著差异(P>0.05)。视网膜铺片正常对照组血管发育成熟,血管网结构清晰,DM对照组血管迂曲阻塞,大量结构异常新生血管,丧失了血管网结构,DM骨髓治疗组见血管网结构清晰,仍有少部分深层血管阻塞,形态接近正常小鼠。结论:骨髓细胞移植可以有效抑制视网膜新生血管形成。     

聚乙烯亚胺修饰的荧光素钠纳米颗粒在荧光素眼底血管造影术的应用和安全性

目的　探讨聚乙烯亚胺修饰的荧光素钠（PEI-NHAc-FS）纳米颗粒（nanoparticles,NPs）在荧光素眼底血管造影术的应用和安全性。方法　选取90只健康棕色雄性挪威大鼠作为实验动物。将1 mL 荧光素钠（200 g·L^-1）加入10 mol的1-乙基-（3-二甲基氨基丙基）碳酰二亚胺盐酸盐（EDC）混合物中搅拌30 min,再加入10 mol的N-羟基琥珀酰亚胺搅拌3 h。然后将该溶液加入到5 mL聚乙烯亚胺（polyethyleneimine,PEI）（76 g·L^-1）中,剧烈搅拌3 d获得PEI-NH₂-FS。将2 mL三乙胺加入到PEI-NH₂-FS原液中,30 min后再将1 mL的乙酸酐加入混合物中搅拌24 h,合成PEI-NHAc-FS。使用核磁共振波谱仪和紫外可见光吸收光谱观察PEI-NHAc-FS的结构,并对合成的NPs结构进行表征;CCK-8检测其细胞毒性。选取30只大鼠,根据荧光素钠和PEI-NHAc-FS NPs的浓度（5 g·L^-1、10 g·L^-1、50 g·L^-1）将大鼠分为6组,每组5只,分别于注射前及注射后5 min、20 min、30 min和60 min进行眼底摄像,采集大鼠眼底血管荧光素图像。用激光诱导大鼠脉络膜新生血管模型后,取10只大鼠,随机分为10 g·L^-1荧光素钠组和PEI-NHAc-FS NPs组,每组5只,分别于注射前及注射后5 min、20 min、30 min和60 min通过眼底血管造影成像观察病灶处渗漏。另取40只大鼠,随机分为10 g·L^-1荧光素钠组和PEI-NHAc-FS NPs组,每组20只,于注射后10 min、20 min、30 min和60 min进行荧光冰冻切片。将10只大鼠随机分为对照组（注射生理盐水）和PEI-NHAc-FS NPs组,每组5只,行组织切片HE染色和视网膜电图检测。采用HE染色和视网膜电图观察PEI-NHAc-FS NPs在体内的生物安全性。结果　通过核磁共振和紫外可见光吸收光谱观察到荧光素钠与PEI成功耦合。发射荧光光谱显示,游离荧光素钠和PEI-NHAc-FS NPs的最大发射波长分别出现在546 nm和544 nm处。CCK-8检测结果表明,不同浓度的荧光素钠和PEI-NHAc-FS NPs处理人脐静脉内皮细胞12 h、24 h后,细胞活性差异无统计学意义（P>0.05）。即使用10 μmol·L^-1 PEI-NHAc-FS NPs处理24 h和未处理之间细胞存活率差异亦无统计学意义（P>0.05）。在5 g·L^-1游离荧光素钠组和PEI-NHAc-FS NPs组中,均仅显影视网膜主要血管,不能用于荧光素眼底血管造影的诊断。而在10 g·L^-1游离荧光素钠组和PEI-NHAc-FS NPs组可清晰地显示视网膜主要血管及微血管的结构,且在注射后30 min时血管荧光强度基本相同。PEI-NHAc-FS NPs组在60 min时荧光强度已基本消失,而游离荧光素钠组仍保持较强的荧光强度。10 g·L^-1游离荧光素钠组和PEI-NHAc-FS NPs组荧光素眼底血管造影结果显示,经尾静脉注射造影剂后,视网膜血管内可见荧光成像;同时病灶部位可见荧光渗漏。游离荧光素钠组在视网膜组织中荧光较强,对视网膜组织有较强的吸附和渗透作用,而PEI-NHAc-FS NPs组在视网膜组织中荧光较弱,对视网膜组织吸附较弱。HE染色和视网膜电图对造影剂进行体内生物安全性分析结果显示,PEI-NHAc-FS NPs安全性较好。结论　PEI-NHAc-FS NPs能够安全、有效地用于荧光素眼底血管造影。     

Choroidal absorption angiography with patent blue V.

The vital dye patent blue V is used as a negative contrast dye in order to obtain information about the vascular filling events of the choroid in relation to the retinal circulation and the papilla employing intravenous injection and serial photography. Patent blue is bound to plasma proteins to a limited degree and the angiograms therefore do not only picture the choroidal vessels but also reveal the filling of the choriocapillaris and the transcapillary exchange. It is demonstrated by microdensitometry that the patent blue leakage and subsequent reabsorption is rapid, and that the choroidal dilution curve almost parallels that of the retinal vessels. Our measurements might indicate a diffusion of patent blue from the choroid to the papillary tissue.     

Study of the Influence of Angiostatin Intravitreal Injection on Vascular Leakage in Retina and Iris of the Experimental Diabetic Rats

The breakdown of the blood-retinal barrier or increased vascular permeability is an early complication of diabetes and a major cause of diabetic macular edema[1,2]. It is found that the increase of retinal vascular permeabil- ity precedes the appearance of clinical retinopathy at early stages of diabetic retinopa- thy[3,4]. Diabetic macular edema is a major cause of vision loss in diabetic patients. Although the pathogenic mechanism on the breakdown of the blood-retinal barrier and the increas…     

Mechanism of angiostatin induced reduction of vascular leakage in retina and iris of rats with retinopathy of prematurity

AIM: To study the effect of an intravitreal injection of angiostatin on vascular leakage in the retina and iris of oxygen-induced retinopathy of prematurity (ROP). METHODS: Brown Norway rats at postnatal day 7 (P7) were exposed to hyperoxia (750mL/L O₂) for 5 days (P7-12) and then returned to normoxia to induce retinopathy. Angiostatin was reconstituted in sterile Phosphate Buffered Saline (PBS) and diluted to desired different concentrations. Angiostatin solution was injected into the vitreous of the right eye of the ROP rats at P14 and the age-matched normal rats through pars plana using a glass capillary, and the left eye received the same volume of sterile PBS as the control. Vascular permeability was quantified at 1, 2 and 3 days after the injection by measuring albumin leakage from blood vessels into the retina and iris using the Evans blue method and normalized by total protein concentrations. The expression of vascular endothelial growth factor (VEGF) in retina was evaluated using the Western Blot analysis and immunohis- tochemistry 24 hours following the injection. RESULTS: ROP rats showed significant increases of vascular permeability in the retina and iris (P <0.01). Angiostatin reduces vascular permeability in a dose-dependent manner in the retina of ROP rats. The reduction showed a time course trend. Angiostatin injection reduced retinal vascular permeability by approximately 1.5 and 2-fold at P15 (P <0.05) and P16 (P < 0.01), respectively. Angiostatin injection significantly reduced VEGF levels in the retina of ROP rats but did not affect retinal VEGF levels in normal rats. CONCLUSION: Angiostatin significantly decreases patholog- ical vascular permeability in the retina and iris of ROP rats but not in normal rats. Angiostatin down-regulates VEGF expression in retina of ROP rats. These results suggest that angiostatin may have a therapeutic potential in the treatment of ROP and other diseases with vascular leakage.     

Systemic and periocular deliveries of plasminogen kringle 5 reduce vascular leakage in rat models of oxygen-induced retinopathy and diabetes

PURPOSE: Increased retinal vascular permeability is a common complication of diabetes and a major cause of vision loss in diabetic patients. The current study is to determine the effect of plasminogen kringle 5 (K5) on vascular leakage via systemic and periocular deliveries. METHODS: Oxygen-induced retinopathy (OIR) was generated by exposing newborn rats to 75% oxygen. Diabetes was induced in adult rats by injection of streptozotocin (STZ). Retinal vascular permeability was measured by the Evans blue-albumin leakage method. RESULTS: Subcutaneous, intraperitoneal, subconjunctival, and retrobulbar injections and topical eyedrop application of K5 significantly reduced retinal vascular permeability in both the OIR and STZ-diabetic rat models. Compared with the periocular deliveries, systemic administration requires higher doses of K5. K5 deliveries downregulated VEGF expression in the retina. CONCLUSIONS: K5 can reduce retinal vascular permeability through systemic and periocular deliveries. These delivery routes of K5 have therapeutic potential in the treatment of vascular leakage.     

糖尿病大鼠视网膜黏附连接蛋白表达的变化

目的:探讨链脲佐菌素(streptozotocin,STZ)诱导的糖尿病视网膜黏附连接VE-cadherin和β-catenin的表达。方法:SD大鼠50只,随机分为正常对照组(Con)、糖尿病组(DM)。腹腔注射STZ建立DM模型,2,5,8wk处死动物。用伊凡思蓝方法检测视网膜的渗透性;用免疫组织化学和Western blot方法检测各组大鼠视网膜VE-cadherin和β-catenin的表达情况。结果:造模后2,5,8wk大鼠视网膜血管渗透性增加。免疫组化分析证实DM大鼠视网膜VE-cadherin主要表达在视网膜血管上,β-catetin主要表达在视网膜外界膜、外核层、外网状层、内网状层和内界膜。Western blot分析证明随着DM视网膜病变的发生发展,视网膜VE-cadherin表达量显著减少,2,5,8wk组与Con组两两比较均有差异(P<0.05);而β-catetin表达量显著增加,2,5,8wk组与Con组两两比较差异有统计学意义(P<0.05)。结论:STZ诱导的DM大鼠VE-cadherin表达量减少而β-catetin表达量显著增加,两者成反比。     

伊凡思蓝在测定血视网膜屏障 破坏中的作用

目的探讨能否应用伊凡思蓝(Evans blue, EB)作为示踪物定量诊断血视网膜屏障破坏(blood retinal barrier breakdown, BRBB)。方法 实验动物选用雄性Sprague-Dawley大鼠。首先应用EB测定由血管内皮生长因子(vascular endothelial growth factor, VEGF)引起的视网膜血管渗漏。然后用EB测 量1周糖尿病导致的BRBB。以甲酰胺提取视网膜中的EB，用分光光度计测量提取液中的EB浓度。最后根据视网膜重量标准化视网膜内的EB含量。结果VEGF 处理眼视网膜内的EB含量显著高于未处理眼视网膜内的EB含量(P<0.0001)。早期糖尿病大鼠视网膜内的EB含量亦显著高于正常对照组视网膜内的EB含量(P<0.05)。结论EB是一种可以定量测定BRBB的敏感示踪物。(中华眼底病杂志,2001,17:221-223)     

眼前节内眼模拟手术诱发血眼屏障破坏的大鼠动物模型

目的 建立眼前节内眼模拟手术诱发血眼屏障破坏的大鼠动物模型。方法 清洁级健康成年雄性Sprague-Dawley大鼠150只,随机分为对照组和模型组,每组75只。按1 ml/kg的剂量腹腔注射盐酸氯胺酮 盐酸甲苯噻嗪混合液麻醉大鼠。磷酸盐缓冲液灌注袋连接三通管。三通管一端连接24G静脉留置针,手术显微镜下在3点时钟位从角巩缘前透明角膜30°斜行穿刺入前房,退出针头,留置套管;另一端连接24G静脉留置针套管,与测压计相连测量大鼠眼压。大鼠眼压波动于0～12 mm Hg（1 mm Hg=0.133 kPa）之间,波动30 次/min,重复60 次。采用氧氟沙星滴眼液滴眼。建模后第1、2、3、5、7天,采用免疫组织化学法检测大鼠白蛋白;定量检测大鼠房水、视网膜中伊凡思蓝（EB）浓度。结果 免疫组织化学染色结果显示,建模后第1、2、3、5、7天对照组白蛋白阳性染色均局限于虹膜和视网膜血管内,脉络膜弥漫性着色。建模后第1天,模型组白蛋白阳性染色主要位于虹膜和视网膜神经层血管周围;建模后第2、3天,阳性染色扩散到虹膜和视网膜全层;建模后第5、7天,阳性染色主要局限于虹膜和视网膜血管内。模型组房水中EB浓度在建模后第1、2、3、5天,均较对照组高（t=25.781,37.433,25.150,19.171;P＜0.01）;建模后第7天,与对照组接近（t=1.303,P=0.209）。模型组视网膜EB浓度在建模后第1、2、3天,均较对照组高（t=11.997,14.622,23.014;P＜0.01）;建模后第5、7天,与对照组接近（t=2.027,0.756;P=0.058,0.459）。结论 通过模拟眼前节内眼手术损伤因素,可建立内眼手术诱发的血眼屏障破坏的大鼠动物模型。     

血管抑素降低ROP幼鼠视网膜及虹膜血管渗漏性的作用机制研究

目的：探讨血管抑素（angiostatin）玻璃体注射对氧诱导的早产儿视网膜病变（retinopathy of prematurity，ROP）的视网膜及虹膜血管渗漏的作用及其机制。方法：将出生7d（P7）的Brown Norway鼠置高氧环境（750mL／L O2）5d后再置正常氧环境诱导ROP，建立ROP动物模型，并以年龄相匹配的正常鼠作为正常对照。所有ROP鼠（P14）及正常鼠均右眼玻璃体腔注射血管抑素，左眼注射相同剂量的PBS（磷酸盐缓冲生理盐水）作为对照。用Evans蓝微血管渗透性检测法及总蛋白标准化分别于注射后1，2和3d检测视网膜和虹膜的血管渗透性；用Western blot蛋白印迹分析和免疫组化方法检测注射24h后血管内皮生长因子（vascular endothelial growth factor，VEGF）在视网膜的表达。结果：ROP鼠视网膜及虹膜的血管渗透性明显增加（P〈0．01）；中剂量（3．75ug/眼）和高剂量（7．5ug/眼）血管抑素降低ROP鼠视网膜血管渗透性（P〈0．05，P〈0．01），而低剂量组（1．88ug／眼）没有引起明显改变，呈现剂量依赖型；三种不同剂量的血管抑素玻璃体注射后ROP鼠的虹膜均未发生明显的血管渗透性的改变。血管抑素注射后第1d和第2d视网膜血管渗透性明显降低（P〈0．05。P〈0．01），而第3d无明显降低，其作用呈现出时间进程。Western blot蛋白印迹和免疫组化分析表明血管抑素显著降低了ROP鼠视网膜的VEGF水平，但对正常鼠无影响。结论：血管抑素可以降低ROP鼠视网膜的病理性血管渗漏，其血管渗透性下降可能与血管抑素下调VEGF的表达有关。血管抑素可能对ROP等其他视网膜血管渗漏性疾病具有潜在的治疗作用。     

Evans blue staining to detect deep blood vessels in peripheral retina for observing retinal pathology in early-stage diabetic rats

AIM: To observe and compare the statistical significance of superficial and deep vascular leakage in the pathological changes of the diabetic rats retina after the Evans blue (EB) perfusion, and utilize the modified whole-retina spreading method to make the slides while protecting the periphery of the retina. METHODS: The Sprague-Dawley (SD) rats were randomly divided into 6 groups. Each group named as the normal groups for 4, 8, and 12wk and the diabetic groups for 4, 8, and 12wk. The EB was injected into the cardiovascular system of the rats at the different time points. The retina of each group was obtained for observation. RESULTS: The superficial vascular leakage was found in all 6 groups. The size of leakage area of superficial retinal blood vessels was (0.54±0.23)%, (0.65±0.11)%, and (0.58±0.10)% in normal group. No notable leakage was found in the deep blood vessels [(0.03±0.04)%, (0.03±0.05)%, and (0.03±0.05)%]. The deep retinal vascular leakage was found in the peripheral retina of diabetic rats. The size of leakage area of superficial retinal blood vessels in diabetic group were (0.53±0.22)%, (0.69±0.16)%, and (0.52±0.11)%. The leakage areas of deep blood vessels were (0.54±0.50)%, (1.42±0.16)%, and (1.80±0.07)% at 4, 8, and 12wk, respectively. There was a statistically difference of the leakage area between the 8th week and the 4th week of diabetes group (P=0.003). The statistically significant difference between the diabetes and the control groups was noted at 4wk and 8wk (P<0.001). CONCLUSION: The main retinal pathological changes of early-stage diabetic rats are the vascular leakage of the periphery of deep retina. Diabetic rats modeled after 8wk have semi-quantitative statistical difference compared with the normal rats, thus early intervention treatment research can start at this time point.     

Characterization of azurocidin as a permeability factor in the retina: involvement in VEGF-induced and early diabetic blood-retinal barrier breakdown

PURPOSE: Azurocidin, released by neutrophils during leukocyte-endothelial interaction, is a main cause of neutrophil-evoked vascular leakage. Its role in the retina, however, is unknown. METHODS: Brown Norway rats received intravitreal injections of azurocidin and vehicle control. Blood-retinal barrier (BRB) breakdown was quantified using the Evans blue (EB) dye technique 1, 3, and 24 hours after intravitreal injection. To block azurocidin, aprotinin was injected intravenously before the intravitreal injections. To investigate whether azurocidin plays a role in vascular endothelial growth factor (VEGF)-induced BRB breakdown, rats were treated intravenously with aprotinin, followed by intravitreal injection of VEGF(164). BRB breakdown was quantified 24 hours later. To investigate whether azurocidin may mediate BRB breakdown in early diabetes, aprotinin or vehicle was injected intravenously each day for 2 weeks to streptozotocin-induced diabetic rats, and BRB breakdown was quantified. RESULTS: Intravitreal injection of azurocidin (20 microg) induced a 6.8-fold increase in vascular permeability compared with control at 1-3 hours (P < 0.05), a 2.7-fold increase at 3 to 5 hours (P < 0.01), and a 1.7-fold increase at 24 hours (P < 0.05). Aprotinin inhibited azurocidin-induced BRB breakdown by more than 95% (P < 0.05). Furthermore, treatment with aprotinin significantly suppressed VEGF-induced BRB breakdown by 93% (P < 0.05) and BRB breakdown in early experimental diabetes by 40.6% (P < 0.05). CONCLUSIONS: Azurocidin increases retinal vascular permeability and is effectively blocked by aprotinin. The inhibition of VEGF-induced and early diabetic BRB breakdown with aprotinin indicates that azurocidin may be an important mediator of leukocyte-dependent BRB breakdown secondary to VEGF. Azurocidin may become a new therapeutic target in the treatment of retinal vascular leakage, such as during diabetic retinopathy.