During the past year, we investigated confocal retinal image to identify new anatomical features that can be used non-invasively to monitor retinal structure and disease progression. Using confocal scanning laser ophthalmoscope (cSLO) in imaging fundus of live mouse, we noticed dark-appearing dots approximately 10 to 15 um when imaged with the blue (488 nm) reflectance (BR) mode. These anatomical features appear in the inner retina below the level of the optic nerve fiber layer. When imaged with transgenic Cx3cr1-GFP mouse which microglia were fluorescently labelled, BR dark dots did not co-localize with the fluorescent signal indicating that they did not correspond to retinal microglia. When fluorescein angiography (FA) was used to reveal the structure of retinal blood vessels, BR dark dots co-localized well with intense fluorescent spots on FA. 3D structure of retinal vasculature reconstructed using Velocity 3D software from fluorescent tomography confirmed that fluorescent spots in FA images corresponded to connecting points of inner deeper plexus linking primary plexus and outer deeper plexus. In adult WT (C57816) mouse, BR dots were distributed with regular spacing in all four quadrants of the retina;mean dot densities were 908, 764, 855, 893 dots/mm2, (mean SD) for dorsal, ventral, nasal and temporal regions, respectively. Mouse models of retinal degeneration demonstrated lower densities of BR dots compared with WT mice. BR dot densities were 927, 325 and 505 dots/mm2, (mean SD) for WT (postnatal age 2 month) (n=8), rd1 (postnatal age 1 month) (n=2) and rd10 (postnatal age 3 month) (n=4), respectively. In conclusion, we discovered that the location, density, and distribution of connecting vessels in the retinal vasculature can be visualized non-invasively as dark dots on BR reflectance imaging. The number of dark dots decreased in mice with retinal degeneration, reflecting concurrent vascular degeneration in these models. As cSLO is a widely used diagnostic tool for retinal diseases, BR dots could represent a novel, non-invasive, imaging outcome measure that can be used to monitor vascular structure in normal retina and degeneration in retinal disease.
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