Retinal Ganglion Cells Toxicity Of Repeated Intravitreal Injection Of Bevacizumab In Diabetic Rats Eyes

Song Chen

Objective: To observe the changes of retinal ganglion cells after repeated intravitreal injection of bevacizumab in diabetic rats eyes, discuss the possible side effect of bevacizumab treating for diabetic retinopathy, and provide the basic rationale treatment for diseases about diabetic retinopathy and other retinal neovascularization.

Method: 1.Fifty male Sprague-Dawley rats aged 12-14 weeks, weighed approximately 250g, 10 rats as normal control group(GroupA) and the left 40 rats were selected as diabetes mellitus groups by random. Diabetic rats were made by streptozocin. After the model of diabetic retinopathy was tested by Evans blue(EB) staining angiograthy, 10 of diabetic rats as diabetes mellitus control group(GroupB) by random. 3ul(25mg/ml) bevacizumab was injected into the vitreous in the left eye of each rat of the left 30 diabetic rats three times, interval time were 10 days ,as experimental group (GroupC),and the right eyes were given no treatment,as experimental control group(GroupD).

2. The eyes were removed in different groups, after last intravitreal injection for group C 10 days. Histopathology were taken to examine the pathological changes of the retina in different groups. EB staining angiography were used to assess the retinal vascular pattern, Hematoxylin-eosin(HE) staining was used to observed the morphological changes of the retina, immunohistochemistry staining was used to observe the masculine staining in the rat retina. Then we analyzed the pictures using Image-Pro Plus 6.0. Mean integrated optic density (IOD) was used as the main statistic index.

3. The results were input the spss11.5 statistic software. Different treatment groups were analyzed by one-way ANOVA. Then using SNK test to compare each group, P<0.05 was regarded as the standard of statistical significance.

Result: 1. EB staining angiography
Vascular morphology and distribution were changed in the retina of diabetic rats and lose the normal distribution pattern. Retinal vascular was disorder. Retinal neovascularization, fluorescein leakage and microaneurysms was showed in the fluorescence image of retina in diabetic rats. Retinal vascular was thinner than normal after intravitreal injection of bevacizumab in the left eyes of diabetic rats three times, the right eyes were similar to normal.

2. HE staining
HE staining displayed that the layers of the rat retina of Group A were regular, the retinal ganglion cell were regular, arranged equally,the nuclears stained deep violet, the photoreceptor cells were regular, the boundary was sharp.Proliferative retinal vascular endothelial cell can be found from the HE staining in Group B. Retinal ganglion cells was less than normal after intravitreal injection of bevacizumab in the left eyes of diabetic rats three times, the right eyes were similar to normal eyes.

3. Immunohistochemistry staining
Thy-1 mainly located in the ganglion cell layer (GCL) and a weak postivity in the inner plexiform layer (IPL) and inner nuclear layer (INL) . The difference of mean IOD of Thy-1 among every group had statistical significance(F=25.819,P=0.000). Futher more, we compared each group with SNK test, the difference between group B and ACC and D were significantiPƒ0.05),. the difference between C and A were significantiPƒ0.05).

Conclusion: In our reseach, we used a method of intravitreal injection to study the retinal toxicity of repeated intravitreal injection of bevacizumab in diabetic ratsL eyes. And observed the expression of Thy-1 by the way of immunohistochemistry staining. We found the DM rats expression of Thy-1 was less than normal. After repeated intravitreal injection of bevacizumab for DM rats, the expression of Thy-1 was less than normal in the injected eyes; the expression of Thy-1 were similar to normal in the no injected eyes. We concluded that DM damaged to RGCs, repeated intravitreal injection of bevacizumab was toxic to RGCs, and was effect to the fellow eye.

Keywords: bevacizumab diabetic retinopathy retinal ganglion cells toxicity