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.%Background Previous retinal vascular mounting only obtained part of retinal vessels for the study of retinal diseases,and thus it is difficult to comprehensively assess these diseases.So optimizing the trypsin digestion method to show the complete retinal capillary network is very important for the study of retinal diseases.Objective This study was to modify the preparing way of trypsin digested whole retinal capillary network and offer a basis for a quantitative analysis of cells and capillaries.Methods Thirty Wistar rats were divided into model group (20 rats) and control group (10 rats).Streptozotocinum(STZ) of 1.25％ dissolved in 0.05 mmol/L sodium citratehydrochloric acid buffer was intraperitoneally injected to establish diabetes models in the model group,and the equal volume of solvent was used in the same way in the control group.Eight months after injection,100 ml PBS was injected via ventriculus sinister and released via cut right atrium,and then 100 ml 4％ paraformaldehyde was injected into the ventriculus sinister.The rat retinas containing part of the optic nerve were entirely isolated,and digested by trypsin,and vitreous,inner limiting membrane and neural retinal tissue were removed.The whole retinal capillaries network was mounted on the slide.The ghost pericytes and acellular capillaries in the middle retinal area were identified and counted under the optical microscope.Results The retinal mount exhibited that the retinal vessels were stained by hematoxylin and periodic acid schiff.The vessels network presented with the entire type in shape with the radical central retinal arteries and veins and their branches.The capillary showed the shallow-and deep-layer networks between the small arteries and veins.Pericytes distributed and protruded vessel wall and formed the ghost cells without nuclei.The diameter of acellular capillaries was 20％ or more than that of near capillaries,and no cellular nuclei or ghost cell was found through the vessel.Conclusions The experimental technique for setting-up of cleaned vasculature and mounting vessels on glass microscopic slide provides intact vessels,which is helpful for the evaluation of retinal vascular morphology and quantitative analysis.
展开▼
