脉络膜血管平铺及CD105染色在大鼠脉络膜新生血管研究中的评价意义

Evaluation of RPE-choroid-sclera flat mounts and Endoglin in experimental choroidal neovascularization

目的评价异硫氰酸荧光素-右旋糖酐脉络膜血管平铺及CD105免疫组织化学染色作为分析指标在实验性脉络膜新生血管(choroidal neovascularization,CNV)治疗研究中的意义。方法激光光凝方式建立棕色挪威大鼠CNV模型,30只大鼠随机分为治疗组、生理盐水组、对照组,每组各10只。分别给予Endostatin 20μL(5 g.L-1)、生理盐水20μL视网膜下注射、对照组光凝后不做处理,观察期为14 d。在光凝后第7天及第14天行颈动脉灌注异硫氰酸荧光素-右旋糖酐标记CNV,眼球标本制成脉络膜铺片进行CNV定量分析,组织病理切片光镜、CD105及因子Ⅷ免疫组织化学染色观察,比较不同检测方法在评价血管生成抑制剂对CNV治疗效应中的意义。结果造模后第7天,光凝部位CNV呈一高荧光团;光凝后第14天,表现为形态各异的高荧光微血管网;脉络膜血管平铺CNV定量分析显示,光凝后第14天Endostatin组CNV面积小于生理盐水组及对照组,Endostatin组CNV面积为(15.58±5.32)×103μm2,生理盐水组及对照组CNV面积为(24.01±4.67)×103μm2、(25.27±5.08)×103μm2(F=307.351,P<0.001);CD105免疫组织化学显示,光凝后第14天生理盐水组及对照组光斑内CD105及因子Ⅷ呈强阳性表达,各组在视网膜内核层及神经纤维层内皮细胞均无CD105阳性表达;光镜观察造模后第7天激光损伤部位呈纺锤样外观,外核层排列紊乱,光感受器外节消失,RPE屏障破坏,光斑处CNV形成;光凝后第14天,光凝斑部位脉络膜增厚,内皮细胞及微血管增生,大量CNV形成;Endostatin组内皮细胞增生数量及新生血管明显少于生理盐水组和对照组。结论脉络膜血管平铺于CNV定量评估具有一定意义;与因子Ⅷ的检测相比,CD105对于CNV的定性评估更具特异性;Endostatin可以有效抑制CNV的形成。

Objective To discuss the significant of fluorescein isothiocyanate-dextran choroidal vessels tile preparation and CD105 immunohistochemistrical staining in experimental choroidal neovascularization （CNV）. Methods Experimental CNV models were induced by laser photocoagulation. Thirty ani- mals were divided randomly into treatment group given Endostatin 20 μL（5 g · L -1 ）, normal sodium group with 20 μL subretinal injection and control group, 10 cases in each group and observed for 14 days. The vasculature was labeled with FITC-dextran via carotid perfusion at 7 and 14 days after photocoagulation. The area of CNV at each rupture site was measured using high molecular weight FITC-dextran for high resolution angiography in RPE-choroid-sclera flat mounts. The sections were examined by light microscopy. Immunolocalization of Endoglin （ CD105 ） and factor Ⅷ on sections of CNV lesions was studied by im- munohistochemical evaluation. Results RPE-choroid-sclera Flat mounts demonstrated that CNV showed a high fluorophore at 7 days after laser and a high fluorescence network of microvessels at 14 days after laser, its extent and quan- tity increased dramatically. Quantitative analysis of CNV showed that CNV area of Endostatin group with （ 15.58 ±5.32） × 10^3μm2 was less than normal sodium group with （24.01 ±4.67）× 10^3μm2 and control group with （25.27 ±5.08） × 10^3μm2（F= 307. 351;P 〈0. 001 ）.Positive expression of CD105 and factor Ⅶ were found in light spot of normal sodium and control groups. CD105-positive cell were not detected in normal chorioretinal tissues. Light microscopy demonstrated that at 7 days after laser, lesion spot looked like spindle, the outer nuclear layer arranged disorder, photoreceptor outer segment vanished, the RPE barrier destroyed,micro blood vessels formed in the lesion. At 14 days after laser,the thickness of choroid in the lesion increased, endothelial cell and micro blood vessel proliferated, massive CNV formed. Hyperplasia endothelialis and neovas- cularization of Endostatin group were less than those of normal sodium group and control group. Conclusions These data suggested that RPE-choroid-sclera flat mounts and CD105 immunolocalization may provide a new approach for evaluation blood vessel growth in experimental choroidal neovaseularization. The development of CNV can be inhibited by injection of Endostatin, which suggest that Endostatin may be beuefical in treating CNV. [ Ree Adv Ophthalmol 2008 ;28 （ 8 ） ：573-577 ]