METHODS: Both eyes of 48 rats received a series of 20 spots of laser irradiation (659nm,360mW,50μm,0.05s). The rats were randomly divided into three groups: the control group (16 rats), traditional Chinese medicine (TCM) group (16 rats), TA group (16 rats). The control group received 9g/L physiological saline by gastric gavage, the TCM group were administrated liangxuehuayu decoction drugs by gastric gavage, and TA group underwent an intravitreous injection with 5μL (0.2mg) of TA. Fundus fluorescein angiography(FFA), histopathologic examination, Ⅷ straining cells by immunohistochemistry and high molecular weight FITCDextran (MW 2×106) for high resolution angiography in RPEchoroidsclera flat mounts were performed on 7,14,21 and 28 days after photocoagulation. Group in all three time points, respectively, were randomly selected to do a rat choroidal vascular ray Shop.
RESULTS: FFA showed that in control group the fluorescein leakage in grave sc ne, the fluorescein leakage were less in TCM group than in control group, and in TA group the scar staining were greatly bigger than the control group. Histologically，the CNV membrane was observed beneath the retina and the factor Ⅷ positive cells were seen．CNV thickness was firstly appeared on day 7 after photocoagulation, reaching the peak on day 21 in control group, and decreased on 28 days. In TCM group, CNV thickness was reaching the peak on day 14, and decreased greatly on 21 days, increased again on 28 days. And in TA group, the CNV thickness was getting thinner from 7 to 14 days, get thinnest on 14 days, then the CNV thickness was getting thicker. The staining intensity of Ⅷ in cellular plasma showed that in control group the staining intensity was reaching the peak on day 14, and then decreased greatly. In TCM group the staining intensity was reaching the peak on day 7, and then decreased greatly. In TA group the staining intensity was reaching the peak on day 14, but the degree was less than the control group, then decreased greatly. Morphologic and quantitative analysis of flat mounts and histologic sections showed that in control group CNV firstly appeared on day 7 after photocoagulation and peaked in day 28, in TCM group CNV peaked on 14 days, and decreased significantly on 21 days, then increased on 28 days. In TA group, CNV was getting bigger from 7 to 14 days, but the scars after photocoagulation were obvious on 21,28 days.
CONCLUSION: Intravitreal triamcinolone acetonide (TA) inhibit experimental CNV induced by laser trauma in a rat as a model, the scar was obvious. The liangxue huayu decoction can suppress experimental CNV, and the scar was lesser.
KEYWORDS: liangxuehuayu；choroidal neovascularization；triamcinolone acetonide；traditional chinese medicine；animal
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。我们发现凉血化瘀中药对年龄相关性黄斑变性和中心性渗出性脉络膜视网膜病变的CNV均有良好的治疗效果[1，2]，故应用659nm氪激光光凝棕色挪威（Brown Norway, BN）大鼠视网膜建立CNV模型，并分别给予凉血化瘀中药灌胃和曲安奈德玻璃体腔注射治疗，观察并比较凉血化瘀中药及曲安奈德（triamcinolone acetonide，TA）对CNV生成及变化的影响。
1材料和方法
1.1材料 清洁级BN大鼠48只，9～11周龄，体质量200～250g，雄性（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。凉血化瘀中药：由姜黄、三七粉、丹参、川芎、枸杞子、生黄芪等组成（除三七粉来自武汉天紫红药业外，其余药品均来自北京燕北制药厂），由中国中医科学院眼科医院中药房负责煎制。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，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 (FITC，上海联合利华公司)；TA 40g/L（昆明积大制药有限公司）。
1.2方法 实验前100g/L水合氯醛溶液3.5mL/kg行大鼠ip，双眼散瞳，用659nm氪激光（功率360mW，直径50μm，曝光时间0.05s）环绕视盘光凝2排（20个光斑），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。按随机数字表将大鼠分为空白对照组、中药组和TA组3组，每组16只。空白对照组自动物模型制作后1d开始给予生理盐水1.5mL灌胃， 2次/d；中药组给予凉血化瘀的中药1.25g/kg灌胃，2次/d；TA组在动物模型制作后1d给予曲安耐德5μL（0.2mg）玻璃体腔内注射。
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各取3只大鼠，行100g/L荧光素钠(1mL/kg) ip，取造影晚期像（FFA>5min）观察CNV形成渗漏情况。计算机图像分析系统计算各时间点CNV的荧光素渗漏积分面积。
1.2.2病理学检查 大鼠于FFA检查后2d过量麻醉处死，立即摘除眼球，置于40g/L多聚甲醛磷酸盐缓冲液固定24h，经梯度乙醇脱水后石蜡包埋，平行于角膜至视盘的矢状位连续4μm切片切至激光斑处。常规HE染色。CNV厚度判定通过光镜200倍视野下取连续切片中CNV区域内色素上皮层至视网膜内最高点的最大距离，每个时间点取20张切片进行测量，取平均值。Ⅷ因子相关抗原免疫组织化学检测按试剂盒说明书进行，采用SP法染色，AEC显色，经苏木素复染后封片。兔抗人Ⅷ因子多克隆抗体的工作浓度为1∶50，着色部位为胞质呈红色染色。通过半定量测定阳性反应物吸光度来反映CNV形成程度。
1.2.3脉络膜血管铺片检查 分别于光凝后7，14，21和28d 3组各取1只大鼠，麻醉后参照Edelman等[3]的血管灌注技术，用FITCD乳酸盐林格氏液20mL(内含FITCD 100mg，明胶2g)缓慢灌流固定大鼠，灌流结束后，摘除眼球，避光保存于40g/L的多聚甲醛中1h后去除眼前节，揭除神经视网膜层，获得视网膜色素上皮层（RPE）脉络膜巩膜复合体，做4～6条放射状切开后放置于载玻片上。利用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荧光显微镜)以488nm激光激发RPE脉络膜巩膜复合体，收集530nm荧光信号，观察脉络膜巩膜铺片上CNV的形态并测定面积。图像记录软件为Laser Scanning Microscope Fluoview Version 4.3，面积测量软件为ImagePro Plus 5.0。
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