作者：王启常，唐罗生 作者单位：(410011)中国湖南省长沙市,中南大学湘雅二医院眼科
【摘要】目的: 探讨灯盏细辛对外源性 VEGF诱导的大鼠早期视网膜血管病变的作用，并阐明其对视网膜内核层单个毛细血管管腔面积、内皮细胞面积和视网膜电图振荡电位的影响。方法: 大鼠48只随机分为正常对照组、VEGF组、VEGF+EBHM组和VEGF+NS组。VEGF+EBHM组每天ip灯盏细辛浸膏溶液。首次注射VEGF后2wk行荧光素眼底血管造影、视网膜电图振荡电位和组织学检查。结果: 注射VEGF实验眼，约10d后表现为眼底大血管呈结节样扩张，周围视网膜水肿。VEGF+EBHM组较VEGF组、VEGF+NS组轻。实验眼在视盘、视盘周围、后极部视网膜大血管出现高荧光，荧光素从曲张区中央开始消退。VEGF+EBHM组实验眼后极部血管局限性高荧光较VEGF组、VEGF+NS组弱、显示的范围小。VEGF组、VEGF+NS组实验眼视网膜内核层毛细血管管腔缩窄,血管内皮细胞肥大,与正常对照组比较,差异有显著性（P<0.05）。VEGF+EBHM组毛细血管管腔面积无减少，血管内皮细胞无肥大,与VEGF组比较，差异有显著性（P<0.05）。与正常对照组比较,差异无显著性（P>0.05）。VEGF组，VEGF+NS组实验眼视网膜电图振荡电位总波幅降低,与正常对照组比较,差异有显著性（P<0.05）。VEGF+EBHM组视网膜振荡电位总波幅没有变化，与VEGF组比较差异有显著性（P<0.05）。与正常对照组比较,差异无显著性（P>0.05）。结论:EBHM对VEGF诱导的大鼠早期视网膜血管病变有抑制作用。能抑制rrVEGF164诱导的大鼠早期视网膜内核层毛细血管管腔变窄、视网膜血管内皮细胞肥大。能抑制rrVEGF164诱导的大鼠早期视网膜电图振荡电位总波幅的降低。
【关键词】 视网膜毛细血管;视网膜血管病变,视网膜振荡电位;灯盏细辛
Inhibitory effect of erigeron breviscapus (vant.) handmazz on the early retinal vasculopathy induced by exogenous VEGF in rats
QiChang Wang, LuoSheng Tang
Department of Ophthalmology, the Second Xiangya Hospital, Central South University, Changsha 410011, Hunan Province, China
Correspondence to: QiChang Wang. Department of Ophthalmology, the Second Xiangya Hospital, Central South University, Changsha 410011, Hunan Province, China. [email protected]
AbstractAIM: To investigate the effect of EBHM on the early retinal vasculopathy induced by VEGF in rats and further clarify the influence of EBHM to the areas of single capillary lumen and endothelial cells in inner nuclear layer, and ERGOPs in the vasculopathy.METHODS: Fortyeight rats were randomly divided into four groups: normal group, VEGF group, VEGF+EBHM group, VEGF+NS group. Rats in VEGF+EBHM group were administrated intraperitoneally with EBHM solution. Fundus fluorescein angiography, ERGOPs and histological observation were performed 2 weeks after the first intravitral injection with VEGF. RESULTS: Dilated vessels were like nod in the eyes received VEGF, surrounded retinal edema 10 days after the first injection. This vasculopathy in the VEGF+EBHM group was less than that of in the VEGF group and VEGF+NS group. Limited hyperfluorescence occurred in optic disc, margin of optic disc, retinal vessels of posterior pole in the eyes received rrVEGF164 and began to disappear from the central of dilation. The tensity and range of fluolurence in rrVEGF164+EBHM group were less than those in VEGF group and VEGF+NS group. Smaller retinal capillary lumen and retinal endothelial cells hypertrophy in the eyes received VEGF in the VEGF group and VEGF+NS group. Significant difference was found, compared with normal group (P<0.05). There were no smaller lumen and endothelial cells hypertrophy in VEGF+EBHM group. Significant difference was found compared with VEGF group (P<0.05) and no significant difference was found compared with normal group (P>0。05). The differences of total amplitudes of ERGOPs in the eyes received VEGF were as same as this.CONCLUSION: EBHM can partially inhibit the diliation of vessels in the posterior pole in the early retinal vasculopathy induced by VEGF in rats. EBHM can inhibit the constriction of capillary lumen and capillary endothelial cells hypertrophy in inner layer and the decrease in the total of amplitudes of ERGOPs in the early retinal vasculopathy induced by rrVEGF164 in rats.
KEYWORDS: retinal capillary; retinal vasculopathy; OPs; EBHM
0引言
近年来，药物防治视网膜血管病变越来越被人们所重视，体现出副作用少、疗效确切等特点。中药治疗视网膜血管病变，报道甚少。我们采用外源性血管内皮生长因子（vascular endothelial growth factor, VEGF)诱导的大鼠早期视网膜血管病变作为动物模型，探讨灯盏细辛[erigeron breviscapus (vant.) handmazz, EBHM]的抑制作用，为中药治疗视网膜血管病变提供理论依据。
1材料和方法
1.1材料
SpragueDaweley大鼠48只(中南大学湘雅二医院动物实验中心提供,雌雄各半,体质量200～240g),随机分为4组,每组12只:正常对照组不作眼内注射。VEGF组实验眼(右眼)玻璃体腔注射浓度为100mg/L rrVEGF164 4μL，隔天1次，共4次。自身对照眼(左眼)，玻璃体腔注射等量的0.1mol/L PBS，注射频率与实验眼一致。VEGF+EBHM组于玻璃体腔注射当天开始，按150mg/kg，60g/L灯盏细辛(EBHM)浸膏ip，1次/d。VEGF+NS组于玻璃体腔注射当天开始，按EBHM计量，生理盐水ip，1次/d。VEGF+EBHM组,VEGF+NS组实验眼和自身对照眼玻璃体腔注射与VEGF组相同。每组于2wk散瞳后直接眼底镜观察眼底,随机选取各6只作眼底荧光血管造影、视网膜电图振荡电位(electroretinographic oscillatory potentials, ERGOPs)检查后，再随机选取各6只处死行视网膜组织学切片、透射电镜检查。Topcon眼底照相机，视觉电生理诊断仪，正方测试格，玻璃毛细管，重组大鼠血管内皮生长因子（rrVEGF164，Sigma公司）， FITCdextran（Sigma公司）。60g/L灯盏细辛浸膏(湘雅制药有限公司)。
1.2方法
大鼠充分散瞳后，30g/L戊巴比妥钠溶液1mL/kg ip麻醉。结膜囊表面麻醉，眼科手术显微镜下用30#BD针头于背侧角膜缘后1mm处经平坦部刺穿眼球壁后立即出针，角膜缘行前房穿刺，消毒剪刀将自制玻璃体腔微量注射针针尖剪去，暴露开口。10μL加样移液器抽取rrVEGF164 或PBS 4μL注于消毒的点样纸上。自制玻璃体腔微量注射针立即吸取。沿预先穿刺好的针孔处以45°～50°的角度进入眼内并缓慢注射，停留约15s后拔针。术眼涂眼膏，术后3d 3g/L诺氟沙星滴眼液滴眼，2次/d。注射rrVEGF164或PBS前后，出现玻璃体出血者不纳入实验对象。散瞳后，ip麻醉大鼠，结膜囊表面麻醉。暗适应30min，暗红光下安放3个电极，小号银针柄端连接于电极线末端后，角膜电极插于上方周边部角膜基质层，参考电极置于两眼连线的中点皮下，地电极置于同侧耳根皮下，黑色胶纸遮盖未检查眼。参考国际临床视觉电生理学会制定的视觉电生理标准化方案（1994年），设定刺激参数,双眼分别记录视网膜电图振荡电位。散瞳后眼底荧光血管造影，鼠尾静脉注射50g/L FITCdextran 1.0mL，固定动物，注射造影剂后5s，各个方位眼底照相，每次拍片间隔5～10s，持续3min，选择性拍片直至注射造影剂50min。剥离视网膜，常规电镜制片，每只大鼠每眼随机观察2个铜网，每个铜网随机照相2张。同一组、同一眼别的样本分散，使各组样本容量达24张。对视网膜内核层单个毛细血管管腔面积、血管内皮细胞面积进行形态计量，采用正方测试格计数法测量视网膜内核层单个毛细血管管腔面积、血管内皮细胞的面积。
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