Effects of anti-VEGF agents on rat retinal Müller glial cells

PURPOSE: To evaluate the effects of an anti-rat vascular endothelial growth factor antibody (ARVA) and bevacizumab (Avastin) on rat retinal Müller glial cells (RMGCs) in vivo and in vitro. METHODS: Rat RMGCs were identified and cultivated, and were then treated with bevacizumab (0.1, 0.25, and 1 mg/ml), ARVA (0.1, 0.5, and 1 µg/ml), or 1 mg/ml of rat immunoglobulin G (IgG) for 12, 24, 48, and 72 h. The numbers of viable RMGCs were determined using a trypan blue dye exclusion assay and a methyl thiazolyl tetrazolium colorimetric assay. In the in vivo study, the rats received intravitreal injections of 5 µl bevacizumab (3.75 mg/ml), ARVA (15 µg/ml), and rat IgG (1 mg/ml). The electroretinogram was recorded. Seven days after the injections, histopathologic changes and glial fibrillary acidic protein expression of RMGCs in the retina were analyzed by immunohistochemistry with hematoxylin-eosin and fluorescent staining. RESULTS: After exposure to bevacizumab at various concentrations for various periods of time, the stained cell numbers and optical density values of mitochondrial dehydrogenase activity of RMGCs had no significant differences (p>0.05) from those of the control group and IgG medium. In the stained cells, ARVA demonstrated a dose-dependent increase. Compared with those treated for 12 and 24 h, the increase of stained cells treated with 0.5 and 1 µg/ml ARVA at 48 and 72 h was very significant (p<0.01). The optical densities of RMGCs exposed to 0.5 and 1 µg/ml of ARVA at 48 and 72 h were significantly lower than cells exposed to a fresh culture medium (p<0.01). The histology of both treated and control eyes after intravitreal injection was similar and showed no anatomic signs of toxicity. There were no obvious glial fibrillary acidic protein upregulations of RMGCs in all groups. The scotopic electroretinogram responses to flashes of light in the control and treated eyes had similar b-wave amplitudes. CONCLUSIONS: Intravitreal bevacizumab and ARVA had no short-term, direct retinal toxicity in rats. Bevacizumab exerts no inhibition on rat RMGCs, while ARVA at higher doses (over 0.5 µg/ml) may be harmful to the growth of RMGCs.