Abstract
Purpose :
While increasing evidence from patients and animal models suggests that early diabetic retinal neurodegeneration may precede the retinal vascular abnormalities that currently define diabetic retinopathy (DR), the underlying pathophysiology and relevant cell types affected in early diabetic retinal disease (DRD) remain poorly understood. In order to identify molecular pathways dysregulated in specific retinal neuronal cell types in early DRD, we performed single-nuclei RNA sequencing of the retina in diabetic mice at two time points following disease onset.
Methods :
To induce diabetes, male C57BL/6J mice were treated with five daily intraperitoneal injections of 60mg/kg streptozotocin (STZ) or vehicle. Animals with random glucose >250 mg/dL were considered diabetic and the early retinal phenotype was confirmed at 6 weeks after treatment using optical coherence tomography and dark-adapted electroretinogram. At either 6 or 36 weeks following STZ treatment, retinas from control and diabetic mice (n=8 mice per group per time point) were dissected and separated into two batches for methanol fixation, homogenization, and nuclei preparation. Nuclei were subjected to sequential combinatorial labeling using HiFseq and library sequencing was performed using the Illumina NovaSeq6000 S4 platform.
Results :
After preprocessing, we selected cells with >300, <2,000 UMIs (unique molecular identifiers) and <5% mitochondrial counts, then normalized the count matrix in the Seurat package. In total, 18,328 and 3,643 cells were identified at the 6- and 36-week time points, respectively. Unsupervised cell-type annotation analyses yielded 6 clusters representing all retinal neuronal cell types, though relative proportions differed from expected likely due to the use of nuclear rather than whole cell transcripts. The most highly differentially-expressed genes were dysregulated across multiple cell types at the early time point. Integration of gene regulatory networks and our dataset to infer transcription factor activity for each cell suggested unique transcriptional programs at different stages of DRD.
Conclusions :
Single-nuclei RNA sequencing of the retina in the STZ-induced diabetes mouse model reveals patterns of gene dysregulation in retinal neurons that may underlie the pathogenesis of early diabetic retinal neurodegeneration and contribute to subsequent progression of DR.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.