Diabetic retinopathy (DR) is the leading cause of blindness in adults. Early DR treatment prevents retinal damage and reduces the severity of DR complications. Therefore, early detection is paramount because it allows timely treatment before vision loss. The earliest clinical manifestation of DR is evenly distributed (diffuse) leakage of retinal blood vessels that can develop to focal leakage and other advanced DR complications. DR research relies heavily on rodent models. A characteristic of these DR models is diffuse vascular leakage without the other advanced retinal complications. This makes detection and quantification of DR in these models quite difficult. The Evans blue (EB) assay is sensitive enough to detect diffuse leakage, however, it is invasive and requires animal euthanasia. Therefore, non-invasive sensitive methods for longitudinal studies in the same animals during DR development and treatment are needed, but such methods currently do not exist. In this study, we evaluate the novel non-invasive approach of fluorescence angiography with dual fluorescence (FA-DF) to detect and quantify diffuse retinal vascular leakage in mouse models. FA-DF relies on ratiometric fluorescence measurements of two dyes of different molecular weights that escape the vasculature at different rates after injection. By analyzing the ratio between the dyes inside and outside blood vessels, retinal vascular leakage can be quantified sensitively without euthanizing the animal. Here, we aim to establish FA-DF as a non-invasive method to detect early retinal damage in DR research and further develop it for future use in the clinical setting.
