Profiling miRNAs in a hyperglycemic and hypoinsulinemic Ins2Akita mouse model

Abstract

Purpose: Diabetic retinopathy (DR) is a leading cause of blindness worldwide involving neoangiogenesis and blood retinal barrier breakdown. Our goal is to identify novel genetic targets involved in DR. microRNAs (miRNAs) are non-coding RNAs that regulate gene expression and modulate several pathologic diseases. The purpose of this study is to characterize the in vivo perturbations of retinal miRNAs in a hyperglycemic and hypoinsulinemic mouse model. Here, we characterize the vascular and macroscopic anatomic properties of the Ins2Akita mouse and then profiled the miRNA abundance longitudinally.

Methods: Heterozygous male Ins2Akita with a mutation in the insulin 2 gene, were age matched with C57BL6/J mice. We analyzed vascular permeability by Evans blue extravasation, identified retinal vascular macroscopic lesions by fundus photography and fluorescein angiography, and surveyed for functional visual impairment by electroretinography (ERG). We measured miRNA abundance in the retina and choroid/retinal pigmented epithelium (RPE) at several time points using multiplexed libraries of Ins2Akita and wild-type mice at 1 and 3 months of age. We identified cell-type specificity of miRNAs in fresh frozen retina tissue sections using a modified in situ hybridization (ISH) procedure.

Results: Morphological changes and fluorescein angiogram abnormalities were identified in the retinas of Ins2Akita at 1 and 3 months. We observed substantially increased vascular permeability in the Ins2Akita retina at 3 months. However, no functional changes were observed by ERG for up to 3 months of age. miRNA profiles were generated for Ins2Akita and control mice (1 and 3 months), which showed substantial down regulation of several miRNAs including miR-126 and miR-143 in the retina and choroid/RPE. ISH studies showed cell-type specific staining in several retinal layers in wild-type retina tissue sections.

Conclusions: We further characterized the Ins2Akita mouse model of hyperglycemia and hypoinsulinemia. We found that despite substantial macroscopic morphological changes in the fundus and increase in retinal vascular permeability of Ins2Akita mice, no functional change was yet detectable by ERG at 3 months. Several miRNAs are deregulated in the Ins2Akita mouse and several miRNAs are cell type specific. The miRNAs identified in our screen offer potential targets for genetic modulation of hyperglycemic induced pathology in the eye.

Keywords: 498 diabetes • 688 retina • 499 diabetic retinopathy