Abstract
Purpose :
Vascularization of the eye is a temporally controlled process that is essential for proper retinal neurogenesis, maturation, and maintenance. Reciprocally, retinal neurons regulate the retinal vascular landscape, as mice lacking retinal ganglion cells (RGCs) do not properly develop retinal vasculature. Previously, a light-dependent melanopsin (OPN4)-mediated response pathway has been shown to be required for normal retinal vasculature development, implicating a role for the OPN4-expressing intrinsically photosensitive RGCs (ipRGCs). The goal of our study was to test the hypothesis that the ipRGC itself is required for normal development and maturation of retinal vasculature.
Methods :
We used Opn4DTA/DTA mice, in which ipRGCs are genetically ablated during early post-natal development. At P27.5, mutant mice (n=1) and wildtype controls (n=2) were euthanized, enucleated, and dissected for retinal extraction. Whole mount retinas were stained with CD31 (vascular endothelial cell marker) antiserum and imaged using laser confocal microscopy (Zeiss LSM 800) at 20x magnification. Retinal whole mount images were obtained by stitching individual 652x652 µm ROI tiles. Images were then segmented using VolumeCut (MATLAB). Each vascular plexus was binarized (AdaptiveThreshold; ImageJ), skeletonized (ImageJ), and analyzed for branch and junction densities.
Results :
Our preliminary data suggests ipRGCs are physically required for the maturation of the superficial vascular plexus (SVP), but not the intermediate or deep plexuses. Compared to control mice, Opn4DTA/DTA mice had an increase in both branch density and junction density in the SVP (control (mean +/- sem): 391.18 +/- 9.51 branches/mm2, 197.69 +/- 5.39 junctions/mm2; mutant: 657.49 branches/mm2, 337.56 junctions/mm2).
Conclusions :
This preliminary work suggests that ipRGCs themselves may be required for normal maturation of retinal vasculature. Studying the role of ipRGCs in retinal vascularization of the mouse has a potential for high translational value in increasing understanding of numerous human conditions affected by pathological vascularization and/or angiogenesis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.