Reliability of the mouse model of choroidal neovascularization induced by laser photocoagulation

Abstract Purpose: We attempted to reproduce published studies that evaluated whether the following factors influence the choroidal neovascularization (CNV) induced by laser photocoagulation in murine retinas: small interfering RNA (siRNA), cobra venom factor, the complement factors C3 and C5, and complement receptor C5aR. In addition, we explored whether laser-induced CNV in mice is influenced by the vendor of origin of the animals or by the scientist administering the laser. Methods: Experimental procedures for standardizing the measurement of laser-induced CNV were developed and validated with oral doses of pazopanib, a low molecular weight (LMW) vascular endothelial growth factor (VEGF) receptor kinase inhibitor. Reagents or genotypes reported by others to influence CNV in this model were assessed utilizing our standard procedures, with dose regimens closely mimicking those used in respective publications. Retrospective analyses of control or placebo mice in many experiments were done to evaluate whether the CNV area induced by laser photocoagulation varied according to vendor, laser operator, or year that the experiment was conducted. Results: We developed methods that reproducibly showed that oral doses of pazopanib dose-dependently reduced CNV. The administration of the following agents did not have a substantial impact on the choroidal neovascularization induced by laser burns in mice: small interfering RNA (siRNA), a LMW inhibitor of the C5a receptor (PMX53), or cobra venom factor. JAX mice lacking either C3 or C5 had increased neovascularization compared to non-littermate JAX wild-type controls. Taconic mice lacking C3 had reduced CNV compared to non-littermate Taconic wild type control mice. A retrospective analysis of vehicle-treated wild-type C57BL/6 mice used as controls across 132 experiments conducted from 2007 - 2010 revealed that mice purchased from the Jackson Labs or from Charles River produced less neovascularization than mice from Taconic. There was no significant difference in choroidal neovascularization between 4 different scientists administering laser burns to wild-type mice from one vendor. A further retrospective analysis of vehicle-treated mice from Taconic revealed similar average CNV areas in 2007 – 2010 but comparatively smaller average CNV areas in 2011 and 2012. Conclusions: We were not able to reproduce results from four publications using the laser-induced CNV model in mice. This non-reproducibility could be related to unidentified variations in the responsiveness of mice derived from different vendors or differences in reagents, laser applications, blood vessel measurements, or techniques of compound administration. Alternatively, the discrepancy between our results compared to other published studies could be due to published studies having type 1 errors because of insufficient power, no pre-determined inclusion and exclusion criteria, no or insufficient masking, lack of a positive controls, or lack of confirmation studies. We present our recommended methods for conducting experiments with the mouse laser-induced CNV model to enhance reproducibility and minimize investigator bias