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Weiye Li, Juan Wang, Jianfeng Shen, Jieping Zhang, Lixia Lu, Guo-Tong Xu; Comparison Of Pro-apoptotic Effects Of Avastin And Lucentis In Rat Primary Neuronal Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5318.
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To detect and compare the pro-apoptotic effects of Avastin and Lucentis by using a rat primary retinal neuronal cell culture.
Rat primary retinal neuronal (rPRN) cells were isolated from neuro-retinas of Sprague-Dawley rats on postnatal day (P) 1 and maintained in Neurobasal medium with B27 supplement. Cells were cultured for at least 7 days before experiments. rPRN cells were identified based on their morphology and expression of cell surface markers. The working concentrations are as follows: Avastin (2.5 mg/ml, 10 folds of the clinical concentration in the vitreous) and Lucentis (1 mg/ml, 10 folds of the clinical concentration in the vitreous). The rPRN cells were treated with Avastin or Lucentis for 48hrs, respectively, while H2O2 (0.8uM and 0.16uM) treated as positive control, and no treated as negative control. TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling), and MMOP (Mitochondrial Membrane Potential) were applied as apoptotic parameters. All Pairwise Multiple Comparison Procedures were used for statistic analysis. The biochemical and molecular mechanisms of neuron apoptosis by anti-VEGF agents were explored by using specific inhibitors of signaling pathways.
A mixed rPRN cells were obtained and identified to show the possession of characteristic morphology and specific cell markers of various retinal neurons. And then they were used for following comparative study. After Avastin or Lucentis treated for 48hrs, MMOP result, calculating the ratio of red to green fluorescent intensity, showed no statistic significance between treated groups (Avastin or Lucentis) and the negative control. But TUNEL assay showed Avastin caused 34.66%±10.75% cell apoptosis, while Lucentis caused 33.94%±14.56% cell apoptosis. The number of apoptotic cells significantly increased after either Avastin or Lucentis treatment (compared with 12.83%±9.88% cell apoptosis in the negative control group), although no significance between the two treatment groups (P=0.8925). The present data also indicate that both Erk and AKT pathways were involved in the pro-apoptotic effects of Avastin and Lucentis.
In rPRN cells, super-clinical concentrations of Avastin (2.5mg/ml) and Lucentis (1mg/ml) lead to neuron-apoptosis in vitro, although MMOP may not be sensitive enough to show this change. These findings suggest that a long-term and high-dose clinical application of anti-VEGF agents have potential side effect of neuronal apoptosis. The comparative study showed there was no significant difference regarding pro-apoptotic effects between Avastin and Lucentis, indicating whether these two anti-VEGF agents can be interchangeable merits further study.
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