Effects of an anti-VEGF-A monoclonal antibody on laser-induced choroidal neovascularization in mice: Optimizing methods to quantify vascular changes
Effects of an anti-VEGF-A monoclonal antibody on laser-induced choroidal neovascularization in mice: Optimizing methods to quantify vascular changes
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DOI：
10.1167/iovs.07-1194
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发表时间：
2008-03-01
影响因子：
4.4
通讯作者：
Ferrara, Napoleone
中科院分区：
文献类型：
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作者：
Campa, Claudio;Kasman, Ian;Ferrara, Napoleone
目的.本研究的目的是评价检测和定量实验诱导的脉络膜新生血管（CNV）和抗VEGF-A单克隆抗体诱导的CNV血管变化的不同方法。用532 nm半导体激光诱导C57 BL/6小鼠脉络膜新生血管形成。激光照射后10天，采用以下方法检测新生血管：异硫氰酸荧光素葡聚糖高分辨率血管造影，生物素化异凝集素、兔抗NG 2、大鼠抗CD 31、兔抗VWF、大鼠抗CD 105、兔抗IV型胶原、大鼠抗ICAM-2、兔抗结蛋白和大鼠抗MECA 32免疫组织化学;和活体内注射荧光素标记的番茄凝集素。为了验证这些染色方法在定量治疗的CNV中的有效性，作者在激光诱导CNV并用抗VEGF全抗体（G6-31）治疗后将这些技术中最有效的应用于三组小鼠。异硫氰酸黄绿素-葡聚糖血管造影、大鼠抗ICAM-2免疫染色和番茄凝集素活体注射是识别脉络膜新生血管的最有效方法。每种方法在CNV区域均检测到一定量的非特异性荧光。当使用异硫氰酸荧光素-葡聚糖时，这种荧光显得更强。番茄凝集素注射法和大鼠抗ICAM-2免疫组化法能较好地反映抗血管生成药物的作用。由于易于执行，低背景荧光，和新血管的详细可视化，番茄凝集素的活体注射，然后基于阈值荧光的定量是测量CNV和抗VEGF-A单克隆抗体诱导的小鼠血管变化的最佳技术。
PURPOSE. The purpose of this study was to evaluate different methods of detecting and quantifying experimentally induced choroidal neovascularization (CNV) and vascular changes induced on CNV by an anti-VEGF-A monoclonal antibody.METHODS. Choroidal neovascularization was induced by 532-nm diode laser in C57BL/6 mice. Ten days after the laser, the following methods were used to detect the new vessels: high-resolution angiography with fluorescein isothiocyanatedextran; immunohistochemistry with biotinylated isolectin, rabbit anti-NG2, rat anti-CD31, rabbit anti-VWF, rat ani-CD105, rabbit anti-collagen IV, rat anti-ICAM-2, rabbit anti desmin, and rat anti-MECA 32; and intravital injection of fluorescein-labeled Lycopersicon esculentum (tomato) lectin. To verify the validity of these staining methods in the quantification of treated CNV, the authors applied the most effective of these techniques to three groups of mice after laser induction of CNV and treatment with an anti-VEGF full antibody (G6-31).RESULTS. Fluorescein isothiocyanate-dextran angiography, rat anti-ICAM-2 immunostaining, and tomato lectin intravital injection resulted in the most effective means of identifying choroidal neovascularization. A certain amount of nonspecific fluorescence was detected in the area of CNV for each method. This fluorescence appeared more intense when fluorescein isothiocyanate-dextran was used. Tomato lectin injection and rat anti-ICAM-2 immunostaining were the methods that better recorded the antiangiogenic drug effect.CONCLUSIONS. Because of easy execution, low background fluorescence, and detailed visualization of new vessels, intravital injection of tomato lectin followed by a quantification based on threshold fluorescence represents the best technique for measuring CNV and the vascular changes induced by anti-VEGF-A monoclonal antibody in mice.