【摘要】 目的：评价氪激光诱导棕色挪威（brown norway, BN）大鼠脉络膜新生血管（choroidal neovascularization, CNV）模型的可行性。方法：BN大鼠32只一眼行氪激光（659nm）眼底光凝，另一眼作空白对照。激光功率、光凝斑直径和曝光时间分别为360mW、50μm及0.05s。分别在光凝后7，14，21，28d随机选取7只大鼠行眼底照像、荧光素眼底血管造影（FFA）检查。随机选取1只大鼠行脉络膜血管铺片检查。眼球标本行组织病理切片光镜、免疫组织化学染色观察。结果：光凝后7d，CNV开始形成，21d达到高峰，14～28d CNV无明显变化。7～21d，FFA显示造影晚期荧光素渗漏及光镜下CNV厚度逐渐增加，28d时可能因瘢痕开始形成，故FFA渗漏和CNV厚度开始减少。结论：氪激光诱导BN大鼠的CNV模型是一种较为理想的模型，FFA及CNV厚度分析是CNV定量研究的有效手段。
【关键词】 氪激光；脉络膜新生血管；动物模型
Establishment and quantitative analysis of animal model of choroidal neovascularization
XueJing Li, YouZhi Tang, HuiJuan Wang, Jun Feng, Li Zhang
Foundation item: National Natural Science Foundation of China(No.30472229)
Beijing Eye Hospital of China Academy of Chinese Medical Sciences, Beijing 100040, China
AbstractAIM: To investigate the feasibility of Krypton laserinduced choroidal neovasculation (CNV) model in the Brown Norway rats.METHODS: One eye of 32 rats received a series of 20 spots of laser irradiation (659nm, 360mW, 50μm, 0.05s), the other eye were as controls. CNV was evaluated by fundus fluorescein angiography (FFA), high molecular weight FITCDextran (MW 2×106) for high resolution angiography in RPEchoroidsclera flat mounts, and histopathologic examination was performed in 7, 14, 21 and 28 days after photocoagulation. RESULTS: CNV was firstly appeared on day 7 after photocoagulation, reaching the peak on day 21, no significant progress occurred in 1428 days. The fluorescein leakage and the thickness of laserinduced CNV were increased from day 7 to day 21, were decreased in day 28. CONCLUSION: The Krypton laser induced model of CNV in the pigment rats may be useful, and the quantitative analysis of FFA and the thickness of CNV are useful for in vivo studies of angiogenesis and its modulation via various therapy.
KEYWORDS: Krypton laser; choroidal neovasculation; animal model
0引言
脉络膜新生血管（choroidal neovascularization, CNV）又称视网膜下新生血管（subretinal neovascularization, SRNV），是导致年龄相关性黄斑变性、中心性渗出性脉络膜视网膜病变和高度近视等多种眼底疾病视力丧失的主要原因。探讨CNV的发病机制与有效治疗是目前国内外学者的研究热点。我们应用659nm氪激光光凝棕色挪威（brown norway, BN）大鼠视网膜，旨在建立一种可行的CNV模型，并采用几种常见的评价CNV变化的观察手段对该模型进行评估，以探讨CNV生成及变化情况。
1材料和方法
1.1材料
清洁级BN大鼠32只，9～11周龄，体质量200～250g，雄性，由北京维通利华实验动物有限公司提供。实验前双眼前节和眼底检查均正常。盐酸奥布卡因滴眼液（商品名倍诺喜，日本参天制药株式会社），复方托吡卡胺滴眼液(北京双鹤现代医药技术有限责任公司)，100g/L水合氯醛溶液(解放军总医院)；10g/L甲基纤维素滴眼液(解放军总医院)；200g/L荧光素钠注射液 (广西梧州制药股份有限公司)；兔抗Ⅷ因子多克隆抗体(福州迈新公司)；SP试剂盒及AEC显色试剂盒（福州迈新公司）；异硫银酸荧光素葡聚糖(上海联合利华公司)。氪激光机(美国Coherent公司，型号为Novua 2000)；共焦激光眼底血管造影仪(德国Heideberg公司)；光学显微镜 (德国Leica公司，MPS 30)；激光共焦荧光显微镜(美国Bio Rad公司，MRA/2型，配置日本Nikon公司E600荧光显撇镜)。
1.2方法
实验前 15min大鼠100g/L水合氯醛溶液3.5mL/kg行ip，双眼复方托吡卡胺散瞳。大鼠实验眼结膜囊内滴适量倍诺喜、10g/L甲基纤维素后，眼前放置一53.00D的接触镜，用波长为659nm氪激光光凝视网膜，光斑能量输出功率360mW，直径50μm，曝光时间0.05s，距视盘1.5～2.0PD处、避开血管、环绕视盘光凝2排，以光凝后有气泡产生或伴有轻度出血（有时伴有轻响）标志击破Bruch膜，记为有效点。共20个激光斑。按随机数字分为氪激光光凝视网膜后7，14，21和28d共4组，每组8只大鼠。右眼为实验眼，左眼为对照眼。
1.2.1荧光素眼底血管造影
麻醉等准备工作同前。分别于光凝后7，14, 21和28d，将100g/L荧光素钠1mL/kg ip，行眼底荧光血管造影(fundus fluorescence angiography，FFA )检查。
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