Transcriptional responses in a mouse model of silicone wire embolization induced acute retinal artery Ischemia and Reperfusion

Acute retinal ischemia and ischemia-reperfusion injury are the primary causes of retinal neural cell death and vision loss in retinal artery occlusion (RAO). The absence of an accurate mouse model for simulating the retinal ischemic process has hindered progress in developing neuroprotective agents for RAO. We developed a unilateral pterygopalatine ophthalmic artery occlusion (UPOAO) mouse model using silicone wire embolization combined with carotid artery ligation. The survival of retinal ganglion cells and visual function were evaluated to determine the duration of ischemia. Immunofluorescence staining, optical coherence tomography, and haematoxylin and eosin staining were utilized to assess changes in major neural cell classes and retinal structure degeneration at two reperfusion durations. Transcriptomics was employed to investigate alterations in the pathological process of UPOAO following ischemia and reperfusion, highlighting transcriptomic differences between UPOAO and other retinal ischemia-reperfusion models. The UPOAO model successfully replicated the acute interruption of retinal blood supply observed in RAO. 60-minutes of ischemia led to significant loss of major retinal neural cells and visual function impairment. Notable thinning of the inner retinal layer, especially the ganglion cell layer, was evident post-UPOAO. Temporal transcriptome analysis revealed various pathophysiological processes related to immune cell migration, oxidative stress, and immune inflammation during the non-reperfusion and reperfusion periods. A pronounced increase in microglia within the retina and peripheral leukocytes accessing the retina was observed during reperfusion periods. Comparison of differentially expressed genes (DEGs) between the UPOAO and high intraocular pressure models revealed specific enrichments in lipid and steroid metabolism-related genes in the UPOAO model. The UPOAO model emerges as a novel tool for screening pathogenic genes and promoting further therapeutic research in RAO.

Methods
Retinal samples from UPOAO, HIOP, and UCCAO mice were extracted and promptly frozen in liquid nitrogen within 10 minutes of cervical dislocation. Total RNA extraction was performed according to the protocol outlined in the TRIzol Reagent manual (Life Technologies, CA, USA). RNA integrity and concentration were assessed using an Agilent 2,100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). The resulting RNA samples were then pooled and subjected to sequencing on the Illumina HiSeq3000 platform in a 150-bp paired-end read format. The raw RNA-sequencing (RNA-seq) reads were preprocessed and quantified using the featureCounts function in the SubReads package version 1.5.3, with default parameters.

RNA-seq analysis was conducted on samples from 5 mice in the non-perfusion group, 4 mice in the 3-days perfusion group, and 5 mice in the 7-days perfusion group of the UPOAO model. Additionally, retinas from both the 7-days HIOP experimental eyes and bilateral UCCAO eyes were collected (n=5) for analysis.