糖尿病大鼠血-视网膜屏障功能评估中伊文思蓝灌注视网膜铺片技术的改良和应用  

Modification and application of Evans blue-perfused retinal wholemount method in blood-retinal barrier evaluation of diabetic rats

作　　者：董蒙[1] 陈松[1] 段红涛[1] 王月欣[1] 孔佳慧[1] 李泽东[1] 王昀[1] 

机构地区：[1]天津医科大学眼科临床学院 天津市眼科医院 天津市眼科学与视觉科学重点实验室 天津市眼科研究所,300020

出　　处：《中华实验眼科杂志》2015年第7期606-609,共4页Chinese Journal Of Experimental Ophthalmology

基　　金：天津市科技计划项目（13ZCZDSY01500）

摘　　要：背景 伊文思蓝（EB）灌注的大鼠视网膜铺片是实验研究过程中观察血-视网膜屏障（BRB）功能的常用方法,但以往视网膜标本经固定液处理后透光性差,仅能用激光扫描共焦显微镜进行观察,对实验设备要求较高. 目的 探讨EB灌注糖尿病（DM）大鼠视网膜后在PBS中制作透明视网膜标本,并用普通荧光显微镜观察BRB功能技术的可行性. 方法 应用随机数字表法将40只SD大鼠随机分为对照组、DM1个月组、DM 3个月组和DM 6个月组.将质量分数2％链脲佐菌素（STZ）溶于0.05 mmol/L枸橼酸缓冲液,以60 mg/kg的剂量一次性腹腔内注射建立大鼠DM模型,对照组大鼠腹腔内注射等容量枸橼酸缓冲液.分别于造模后1、3、6个月按45 mg/kg的剂量经大鼠股静脉注射30 g/L的EB溶液,注射后15 min摘除双侧眼球,立即置于PBS中完整剥离视网膜,将视网膜放射状切开并平铺于载玻片上,避光晾至完全透明,封片,置于荧光显微镜下观察并拍照.应用IPP6.0软件分析图像,测量EB渗漏区面积与视网膜面积的百分比. 结果 对照组大鼠视网膜血管形态和走行正常,EB在波长546 nm激发光下呈红色荧光,均匀充盈于血管腔.DM 1个月组大鼠视网膜背景荧光较对照组大鼠略增强;DM 3个月组大鼠视网膜背景荧光进一步增强,视网膜血管呈现节段性扩张,可见明显的红色高荧光区;DM 6个月组大鼠视网膜血管粗细不均,部分走行迂曲,可见片状低灌注区及大量高荧光区.对照组、DM 1个月组、DM 3个月组和DM 6个月组EB渗漏区面积百分比分别为（0.05±0.02）％、（0.27±0.06）％、（1.17±0.1 8）％和（4.77±0.66）％,总体比较差异有统计学意义（F=795.800,P＜0.001）,其中DM 3个月组和DM 6个月组大鼠视网膜EB渗漏区面积百分比值明湿高于对照组,差异均有统计学意义（q＆#39;=10.338、43.475,均P＜0.001）. 结论 改良的EB灌注视网膜铺片技术操作简单,普通荧光显Background Retina fixed flat-mount perfused by Evans blue （EB） is a common method for the evaluation of blood-retinal barrier （BRB）.However,previous method is inconvenient for some laboratories because the retinal specimen can not be observed by gereral microscope rather than confocal laser scanning microscope after the fixation.Objective This study was to modify the preparing way of flat-mounted retina in order to obtain transparent specimen for the observation of rat retinal vessels and the evaluation of leakage under the ordinary fluorescence microscope.Methods Forty male SD rats were divided into the control group,diabetes mellitus （DM） 1-month group,DM 3-month group and DM 6-month group according to the random number table.Streptozotocinum （STZ） of 2% dissolved in 0.05 mmol/L sodium citrate-hydrochloric acid buffer was intraperitoneally injected in SD rats to establish DM models,and the equal volume of solvent was injected in the same way in the control rats.One month,three months and six months after injection,EB of 30 g/L was injected via rat femoral vein in the dose of 45 mg/kg.Fifteen minutes after injection of EB,the rats were sacrificed and the retinas were isolated and cut radially to prepare the flat-mounted retinas in PBS immediately and then were dried till the specimens were transparent.The specimens were examined under the fluorescence microscope.The percentage of EB leakage was quantitatively calculated by IPP 6.0 software.All procedures were performed following approval of the institutional animal care and use committee of Tianjin Medical University.Results The retina morphology was normal in the control group,and EB filled the vessels,exhibiting the red fluorescence under the fluorescence microscope.Compared with the control group,retinal background fluorescence was enhanced slightly in the DM 1-month group,and focal leakage of the EB from capillaries and focal dilated vessels were found in the DM 3-month group,further,vascular caliber inequality,retinal hypoperfusion area and

关 键 词：视网膜铺片 糖尿病视网膜病变 动物模型 SPRAGUE-DAWLEY大鼠 血-视网膜屏障 伊文思蓝 

分 类 号：R774.1[医药卫生—眼科] R587.2[医药卫生—临床医学]