Abstract
Purpose :
The purpose of this study is to determine the functional role of Decorin (Dcn) using CRISPR-Cas9 knockout of Dcn in a murine model.We hypothesize that Dcn provides a conducive extracellular matrix scaffold required for the development of retinal microvasculature and its loss may be detrimental to the retina.
Methods :
This study utilized Dcn knockout mice developed at the Medical College of Wisconsin Transgenic facility. The mice were divided into three groups: Wild type (WT), heterozygous (Dcn+/-), and knock-out (Dcn-/-). At postnatal day 90, the mice were euthanized and underwent bilateral whole-globe enucleation. The eyes were fixed in 4% paraformaldehyde, washed with 1x PBS, and the retinas were dissected. Once the retinas were harvested, they were further fixed in cold methanol, permeabilized with 1% Triton X-100, and stained with isolectin GS-IB4 overnight before mounting on to slides. The retinas were then imaged using a Keyence BZ-X810 fluorescent microscope. Images taken included two 20x z-stacks, one taken in the peripheral retina and another in the central retina. From the z-stacks, three images were selected, one from each vascular plexus – superficial, intermediate and deep (SVP, IVP, and DVP). The images were then run through the program AngioTool to measure vascular characteristics and statistical analysis was performed using GraphPad Prism.
Results :
Our results support our hypothesis that Dcn is necessary for retinal microvasculature development, particularly in the peripheral retina. In the DVP of the peripheral retina, there were significant differences between WT and Dcn+/- (p-value = 0.0381) and WT and Dcn-/- (p-value = 0.017) in Vessel Percentage Area, WT and Dcn-/- (p-value = 0.0204) in Total Vessel Length, and between WT and Dcn-/- (p-value = 0.0257) in Lacunarity. In the SVP of the peripheral retina, there was significant difference between WT and Dcn+/-(p-value = 0.0031), and WT and Dcn-/- (p-value = 0.061) in Total Number of Endpoints. In the central retina, there were also significant differences between WT and Dcn+/- in Total Number of Endpoints in the IVP (p-value = 0.0281) and DVP (p-value 0.0231). The remaining plexuses of both the peripheral and central retina did not yield significant results.
Conclusions :
Dcn plays a significant role in the development of retinal microvasculature and its loss results in aberrant microvasculature in the DVP of the peripheral retina.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.