Analysis of the retinal capillary plexus layers in a murine model with diabetic retinopathy: effect of intravitreal injection of human CD34+ bone marrow stem cells

Kong Wa Cheung; Amirfarbod Yazdanyar; Christian Dolf; Whitney Cary; Nicholas Marsh-Armstrong; Jan A Nolta; Susanna S Park

doi:10.21037/atm-20-3930

Analysis of the retinal capillary plexus layers in a murine model with diabetic retinopathy: effect of intravitreal injection of human CD34⁺ bone marrow stem cells

Ann Transl Med. 2021 Aug;9(15):1273. doi: 10.21037/atm-20-3930.

Authors

Kong Wa Cheung¹, Amirfarbod Yazdanyar^{1

2}, Christian Dolf¹, Whitney Cary³, Nicholas Marsh-Armstrong¹, Jan A Nolta³, Susanna S Park¹

Affiliations

¹ Department of Ophthalmology & Vision Science, University of California Davis Eye Center, Sacramento, CA, USA.
² Department of Ophthalmology & Visual Sciences, SUNY Upstate, Syracuse, NY, USA.
³ Department of Internal Medicine, Stem Cell Program and Gene Therapy Center, University of California Davis, Sacramento, CA, USA.

Abstract

Background: Diabetic retinopathy is a retinal vasculopathy involving all three retinal capillary plexus layers. Since human CD34⁺ bone marrow stem cells (BMSCs) have the potential to promote revascularization of ischemic tissue, this study tests the hypothesis that intravitreal injection of human CD34⁺ BMSCs can have protective effects on all layers of the retinal vasculature in eyes with diabetic retinopathy.

Methods: Streptozotocin (STZ)-induced diabetic mice were injected intravitreally with 50,000 human CD34⁺ BMSCs or phosphate-buffered saline (PBS) into the right eye. Systemic immunosuppression with rapamycin and tacrolimus was started 5 days before the injection and maintained for study duration to prevent rejection of human cells. All mice were euthanized 4 weeks after intravitreal injection; both eyes were enucleated for retinal flat mount immunohistochemistry. The retinal vasculature was stained with Isolectin-GS-IB4. Confocal microscopy was used to image four circular areas of interest of retina, 1-mm diameter around the optic disc. Images of superficial, intermediate, and deep retinal capillary plexus layers within the areas of interest were obtained and analyzed using ImageJ software with the Vessel Analysis plugin to quantitate the retinal vascular density and vascular length density in the three plexus layers.

Results: Three distinct retinal capillary plexus layers were visualized and imaged using confocal microscopy. Eyes that received intravitreal injection of CD34⁺ BMSCs (N=9) had significantly higher vascular density and vascular length density in the superficial retinal capillary plexus when compared to the untreated contralateral eyes (N=9) or PBS treated control eyes (N=12; P values <0.05 using ANOVA followed by post-hoc tests). For the intermediate and deep plexus layers, the difference was not statistically significant.

Conclusions: The protective effect of intravitreal injection of the human CD34⁺ BMSCs on the superficial retinal capillary plexus layers is demonstrated using confocal microscopy in this murine model of diabetic retinopathy.

Keywords: Confocal microscopy; diabetic retinopathy; human CD34+ stem cells; retinal flat-mount; retinal vascular plexus.