玻璃体腔重复注射抗血管内皮生长因子单克隆抗体bevacizumab对糖尿病大鼠视网膜的毒性观察

目的 观察玻璃体腔重复注射抗血管内皮生长因子(VEGF)单克隆抗体bevacizumab(商品名Avastin)对糖尿病大鼠视网膜的毒性作用.方法 40只健康成年雄性Sprague-Dawley大鼠随机分为正常组(A组)和糖尿病组,分别为10、30只.糖尿病组采用链脲佐菌霉素(STZ)尾静脉注射方法制作糖尿病动物模型.随机选取10只作为糖尿病视网膜病变(DR)组(B组),不作任何处理;其余20只大鼠左眼为实验组(C组),玻璃体腔注射25 mg/ml的bevacizumab 3 μ1,共注射3次,每次间隔10 d;右眼为实验对照组(D组),不给予任何处理.末次注射后20 d,采用闪光视网膜电图(F-ERG)对各组大鼠行视网膜功能检测;溴化乙锭(EB)染色视网膜铺片,荧光显微镜观察各组大鼠视网膜血管变化情况;苏木精-伊红(HE)染色,光学显微镜观察大鼠视网膜形态学变化;免疫组织化学染色法观察大鼠视网膜各层Thy-1及VEGF阳性表达情况.结果 F-ERG检测显示,A、B、C、D 4组暗适应a、b波潜伏期,暗适应b波振幅及振荡电位(Ops)总振幅比较,差异均有统计学意义(F=33.165,36.162,19.955,23.243;P值均=0.000);A、B、C、D4组暗适应a波振幅比较,差异无统计学意义(F=0.097,P=0.961).荧光显微镜观察发现,A组大鼠视网膜血管走行良好,B组大鼠视网膜血管走行纡曲、扩张,C组大鼠视网膜血管走形规则、变细,D组大鼠视网膜可见微血管瘤.光学显微镜观察发现,A组大鼠视网膜层次结构整齐分明,B组大鼠视网膜各层细胞结构排列紊乱,C组大鼠视网膜各层细胞排列整齐,D组大鼠视网膜各层细胞排列不整齐.免疫组织化学染色发现,Thy-1阳性表达主要位于神经节细胞层(GCL),极少数位于内丛状层、内核层.VEGF阳性表达主要定位于GCL,少量位于神经纤维层、内核层及视网膜色素上皮层.结论 玻璃体腔重复注射bevacizumab对糖尿病大鼠视网膜有一定毒性作用.

Abstract：

Objective To observe the retinal toxicity of repeated intravitreal injection with bevacizumab(Avastin)in diabetic rats.Methods Forty male Sprague Dawley(SD)rats were randomly divided into normal group(Group A,10 rats)and diabetes mellitus group(30 rats).The rats in diabetes mellitus group were induced with streptozotocin injection for diabetic retinopathy model.And then randomly divided into diabetic retinopathy(DR)group(Group B,10 rats),the rats were not intervened;the left eyes of the other 20 rats were intravitreal injected with bevaeizumab 3 μ1(25 mg/m1)for 3 times as experimental group(Group C);the right eyes of the 20 rats were not intervened as experimental control group(Group D),20 days after last intravitreal injection,retinal function was measured by Flicker Electroretinogram (F-ERG);retinal vascular pattern was determined by fluorescence microscopy of ethidium bromide(EB)stained retinal flat mounts;retinal morphological changes were determined by light microscope on hematoxylin-eosin (HE) stained sections;Thy-1 and VEGF expression was measured by immunohistochemistry staining.Results F-ERG showed that-the differences of a-and b-waves-the b-wave amplitude and the Ops-wave amplitude in the implicit time between group A,B,C and D were significant (F=33.165,36.162,19.955,23.243;P=0.000);the differences of a-wave amplitude between group A,B,C and D was not significant(F=0.097,P=0v961).Retinal blood vessel pattern was normalin Group A;retinal vascular vessels were tortuous and irregularly expanded in Group B:retinal vascular vessels of Group C were regular and thinner than Group A;microaneurysm were showed in Group D.Light microscope displayed that the layers of the rat retina of Group A were regular,the retinal architectures of Group B were irregular,the retinal layers were regular in Group C,the retinal layers were irregular in Group D.Immunohistochemistry staining discovered that Thy-1 and VEGF were mainly expressed in ganglion cell layer(GCL).Conclusion Repeated intravitreal injection of bevacizumab iS toxic tO retina of diabetes mellitus rats.

Retinal toxicity of repeated intravitreal injection with bevacizumab in diabetic rats

Objective To observe the retinal toxicity of repeated intravitreal injection with bevacizumab(Avastin)in diabetic rats.Methods Forty male Sprague Dawley(SD)rats were randomly divided into normal group(Group A,10 rats)and diabetes mellitus group(30 rats).The rats in diabetes mellitus group were induced with streptozotocin injection for diabetic retinopathy model.And then randomly divided into diabetic retinopathy(DR)group(Group B,10 rats),the rats were not intervened;the left eyes of the other 20 rats were intravitreal injected with bevaeizumab 3 μ1(25 mg/m1)for 3 times as experimental group(Group C);the right eyes of the 20 rats were not intervened as experimental control group(Group D),20 days after last intravitreal injection,retinal function was measured by Flicker Electroretinogram (F-ERG);retinal vascular pattern was determined by fluorescence microscopy of ethidium bromide(EB)stained retinal flat mounts;retinal morphological changes were determined by light microscope on hematoxylin-eosin (HE) stained sections;Thy-1 and VEGF expression was measured by immunohistochemistry staining.Results F-ERG showed that-the differences of a-and b-waves-the b-wave amplitude and the Ops-wave amplitude in the implicit time between group A,B,C and D were significant (F=33.165,36.162,19.955,23.243;P=0.000);the differences of a-wave amplitude between group A,B,C and D was not significant(F=0.097,P=0v961).Retinal blood vessel pattern was normalin Group A;retinal vascular vessels were tortuous and irregularly expanded in Group B:retinal vascular vessels of Group C were regular and thinner than Group A;microaneurysm were showed in Group D.Light microscope displayed that the layers of the rat retina of Group A were regular,the retinal architectures of Group B were irregular,the retinal layers were regular in Group C,the retinal layers were irregular in Group D.Immunohistochemistry staining discovered that Thy-1 and VEGF were mainly expressed in ganglion cell layer(GCL).Conclusion Repeated intravitreal injection of bevacizumab iS toxic tO retina of diabetes mellitus rats.