Abstract
Purpose :
Retinal light injury models can be useful in understanding aspects of retinal degeneration and retinal oxidative stress. We recently developed the FCD-LIRD model of light-induced retinal degeneration and demonstrated some retinal recovery from the original damage. In the current study, our aim was to perform cell-type specific analyses using single cell RNA sequencing of the acute and subacute retinal responses to light injury using this FCD-LIRD model.
Methods :
C57BL/6J mice (4m old) were exposed to FCD-LIRD. Eyes were collected from control mice and from mice that had been exposed to light injury 4 h, 48h and 5d prior. Retinas were peeled off from the posterior eyecups and 2 mm posterior retina, centered on the optic disc spot, was cut out using a trephine. These retinal samples were then prepared for retinal single-cell suspensions using a Papain Dissociation System. Droplet-based single cell RNA-seq of dissociated retinal cells was performed with the GemCode Single Cell Platform (10X Genomics, Pleasanton). Libraries were sequenced on an Illumina NextSeq 500. Cell Ranger 3.0.0 (10X Genomics) was used to process and analyze the raw sequencing data. Seurat R package (v3.0.0) was used for downstream gene expression analysis. Cell clusters were then identified and named on the basis of known gene markers specific to various cell types found in the retina. Differential gene expression, pathway analyses and trajectory analyses of each cluster were performed, using Seurat and Ingenuity Pathway Analysis software.
Results :
Our analysis generated 23 clusters with a resolution of 0.5. In addition to confirming previously published gene markers for retinal cell types, we were able to identify new ones. Using both unbiased and marker-based methods for data processing we identified genes that were significantly altered in each cell type after light injury. Using trajectory analysis we were able to identify several differentiation fates, including some suggestive of activation of pathways for cellular recovery.
Conclusions :
Photo-oxidative/photo-inflammatory retinal injury leads to acute and subacute cell type-specific responses. A better understanding of these responses may be helpful in identifying therapeutic approaches to minimize retinal damage and maximize recovery after exposure to injury.
This is a 2021 ARVO Annual Meeting abstract.