Development of a rat choroidal neovascularization model by overexpressing VEGF with and without treatment

Abstract

Choroidal neovascularisation (CNV) with the wet age-related macular degeneration (AMD) is a leading cause of vision loss in the developed countries. As the conventional treatment for CNV is not ideal, an efficient CNV animal model is needed to study new treatments for ocular diseases. Vascular endothelial growth factor (VEGF) is a key stimulator for CNV. The aim of this study was to develop a valid VEGF-induced CNV model and to test whether it can be used for investigation of new treatment options. The model was first developed by subretinal injection of HC Ad. VEGF vector, however, high retinal toxicity of the vector was observed in the HC Ad. EGFP control eyes. Therefore, AAV vector system was used in the latter experiments. 2x109 AAV-VEGF vector particles were subretinal injected into the eyes of female Long Evans rats. Fluorescein angiography (FA), indocyanine green angiography (ICG) and optical coherence tomography (OCT) were performed in all rats at different time points to confirm the best time point for this rat model. All the eyes were fixed for paraffin and EPON embedding and later investigated by light and electron microscopy (LM/EM) and immunohistochemistry (IHC). Additionally, 19 eyes were intravitreally injected with Avastin® (125μg / 5μl) 6 weeks after VEGF injection to test the effect of Avastin®, and 19 eyes were injected with AAV-VEGF vector simultaneously as untreated controls. All the eyes with CNV showed hyper-fluorescent CNV areas in FA/ICG and marked subretinal edema-like changes in OCT. 91% of AAV-VEGF transduced eyes presented with a fully grown CNV observable by angiography after 6 weeks’ duration of VEGF overexpression. No CNV eye showed spontaneous regression of the CNV within 9 weeks after AAV-VEGF vector injection. The retinal and CNV lesion thickness increased with time and showed a significant difference if compared between 6 and 9 weeks after VEGF transduction (ANOVA: p < 0.05). In the EM, newly formed blood vessels with fenestrations between Bruch’s membrane (BM) and retinal pigment epithelium (RPE) or between RPE cells, multi-layered RPE, loss of photoreceptors and collagen accumulation were observed, resembling human CNV. IHC verified VEGF overexpression (human VEGF), multi-layered RPE (RPE65), collagen accumulation (Masson trichrome staining), pericytes occurrence (alpha-smooth muscle actin (α-SMA) and neural/glial antigen 2 (NG2)), macrophages/ activated microglia (Iba1) and albumin occurrence in CNV areas. The thickness of retinal and CNV lesion decreased significantly one week after Avastin® treatment (t-test, P<0.05). The decreases were no longer significant at 3 weeks after treatment, but Avastin® still has a tendency to reduce the growth of CNV. The proportion of the VEGF positive CNV area decreased 1 week after Avastin® treatment. Based on the results, especially EM, this rat model resembles the whole process of human CNV. Avastin® tends to reduce CNV lesion thickness or at least to inhibit its growth in the short term in the rat CNV model. In conclusion, this CNV rat model developed by overexpression of AAV-VEGF vector is efficient for the investigation of new treatment options for CNV.