Comparative analysis of single-cell and single-nucleus RNA-sequencing in a rabbit model of induced proliferative vitreoretinopathy

Abstract

Purpose : Proliferative vitreoretinopathy (PVR) is the most common cause of failure of retinal reattachment surgery. There is no available pharmacologic treatment for PVR. The molecular pathways involved in the initiation and progression of PVR are poorly understood. In this study, we sought to identify the genes and transcriptional networks involved in PVR progression.

Methods : All animal procedures were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Johns Hopkins Animal Care and Use Committee. PVR was induced unilaterally in specific pathogen free Dutch Belted rabbits by vitrectomy, retinotomy, retinal detachment, platelet-rich plasma injection, and cryotherapy. At different time points after PVR induction, extracted retinas were dissociated into either single cells or single nuclei. Samples were processed to assess the transcriptional profile using single cell or single nucleus RNA sequencing (sc-RNAseq or sn-RNAseq). Fellow eyes were used as non-PVR controls.

Results : Retinas (N=9) were harvested at time points ranging from four hours to 30 days after PVR induction. We profiled >40,000 single cells and nuclei in this study. Retinal pigment epithelium cells, glial cells, and neural retinal cell types were identified in the sc-RNAseq and sn-RNAseq data sets using selected marker genes. The data identified several candidate genes involved in PVR progression, including inflammatory response and cellular proliferation genes. Pseudotime analysis was performed to examine progression from control conditions to early and late stage PVR.

Conclusions : Our high throughput analysis identified several genes that were differentially expressed between control and disease states. SnRNA-seq may have an advantage over scRNA-seq as it can reduce dissociation-induced cellular stress responses. These studies support snRNA-seq and sc-RNAseq as useful tools to study transcriptional networks in animal models of PVR and to identify novel molecular targets for potential pharmacologic modulation.

This is a 2021 ARVO Annual Meeting abstract.