Confocal microscopy analysis of the retinal vascular layers in a murine model of diabetic retinopathy: Effect of intravitreal injection of human CD34+ Bone Marrow Stem Cells

Abstract

Purpose : Human CD34+ bone marrow stem cells (hBMSCs) have the potential to promote revascularization of ischemic tissue. Our previous work showed that intravitreal injection of CD34+ hBMSCs has protective effects on the retinal vasculature in murine eyes with diabetic retinopathy. This study used confocal microscopy to evaluate further the effect of the CD34+ hMBSCs on the vascular density in the various retinal capillary plexus layers in this murine model of diabetic retinopathy.

Methods : Streptozotocin-induced diabetic mice were injected intravitreally with 50,000 CD34+ hBMSCs or PBS (phosphate-buffered saline) into the right eye. The left eye received no injection. Systemic immunosuppression with tacrolimus and rafamycin was applied 5 days before the injection and maintained for 4 more weeks to prevent rejection. All mice were euthanized 4 weeks after intravitreal injection and both eyes were enucleated for retinal flat mount immunohistochemistry. The retinal vasculature was stained with Isolectin-GS-IB4. Confocal microscopy was used to image 4 circular 1.0cm² areas of interest of retina around the optic disc. Images of superficial, intermediate, and deep retinal capillary plexus layers in the areas of interest were obtained and analyzed using ImageJ software with the Vessel Analysis plugin to quantitate the retinal vascular density and length in the three retinal vascular plexus layers.

Results : Using confocal microscopy, the three distinct retinal capillary plexus layers could be visualized and imaged. Eyes that received CD34+ hBMSCs intravitreal injection (N=9) had significantly higher vascular density and vascular length in all three capillary plexus layers when compared to the untreated contralateral eyes (N=9) or PBS treated eyes (N=13; p values < 0.05 using two-tailed t-test).

Conclusions : Confocal microscopy could be used to visualize the three retinal capillary layers in a murine model of diabetic retinopathy. Intravitreal injection of CD34+ hBMSCs appears to have a protective effect on the retinal vasculature in all three retinal capillary plexus layers in this model.

This is a 2020 ARVO Annual Meeting abstract.