活体免疫染色评估眼底新生血管实验研究

Assessment of retinal and choroidal neovascularization by in vivo immunostaining

目的探寻更好地对视网膜新生血管(RNV)和脉络膜新生血管(CNV)及其渗漏作定性和定量分析的方法。方法13只小鼠随机分为RNV评估组(n=7)和CNV评估组(n=6)。①RNV评估:取3只小鼠作为正常对照组,其余4只制作成未成熟视网膜病变(ROP)模型,分别于出生后第16天双眼内注射FITC标记的抗体或未经FITC标记的抗体以及空白组,12 h后直接视网膜铺片或用带荧光的二抗孵育后再铺片镜检。②CNV渗漏评估:激光诱导6只小鼠(12眼)制作CNV模型,随机平分为两组,分别于光凝后第7和14天眼内注射非荧光标记的抗体,12 h后腹腔内注射荧光素钠,并定量分析CNV面积及其渗漏面积。结果FITC标记抗体组的视网膜表现出高荧光、高清晰度的视网膜结构血管和RNV,而非FITC标记抗体组的视网膜只选择性染色RNV,几乎无干扰荧光背景,可观察到细微变化,利于应用图像软件对RNV图片进行定性和定量分析。应用SPOT图像软件把CNV染色的图片与CNV渗漏荧光的图片进行叠加,能够明确区分CNV及其渗漏面积;第14天与第7天CNV面积及其渗漏面积比较差异有统计学意义(P<0.05)。结论使用活体免疫染色技术可以方便地观察视网膜血管细微结构,并精准地定性和定量分析RNV和CNV渗漏。

Objective To explore the optimal method of qualitative and quantitative assessment of retinal neovascularization （RNV）, ehoroidal neovascularization （CNV） and leakage from CNV. Methods Thirteen mice were randomly divided into RNV assessment group （n = 7） and CNV assessment group （ n = 6）. For RNV assessment, 3 miee were served as normal controls, and the other 4 were made into models of retinopathy of prematurity （ ROP）. At postnatal d16, mice in normal control group and 3 ROP model mice received intraocular injection with FITC labeled or unlabeled antibody and were sacrificed 12 h later. The retinas were fiat mounted （FITC labeled antibody） or incubated with secondary antibody （unlabeled antibody） and then fiat mounted. The other ROP model mouse was served as blank control. For assessment of CNV and leakage from CNV, 6 mice （12 eyes） were induced into models of CNV, and were randomly divided into two groups. Seven and 14 d after model establishment, mice were intraocularly injected with unlabeled antibody, and fluorescein sodium were intraperitoneally injected 12 h later. The area of CNV and that of CNV leakage were calculated. Results Retinas from eyes injected with FITC labeled antibody exhibited good resolution of uhrastructure of RNV, while retinas from eyes injected with unlabeled antibody showed selective staining of RNV with no background, greatly facilitating identification and quantification of RNV. Merging images of stained CNV with images of fluorescein sodium leakage from CNV revealed double labeled CNV surrounded by fluorescein that had leaked into surrounding tissues. The mean area of CNV and its leakage of d14 were significantly different from those of d 7 （ P 〈 0. 05）. Conclusion In vivo immunostaining allows detailed struetural analysis of retinal vessels and facilitate identification and quantification of RNV and leakage of CNV.