Impact of phlebotomy induced anemia on whole retinal transcriptome at P15 and P20 in the rat 50/10 OIR model

Phlebotomy-induced-anemia (PIA), which induces tissue hypoxia and angiogenesis, occurs universally among infants at risk for severe retinopathy of prematurity (ROP). We hypothesized that PIA exacerbates pathologic retinal neovascularization in ROP. Methods: We induced PIA to a hematocrit of 18% among rats undergoing the established 50/10 oxygen-induced retinopathy (OIR) model. Rats were euthanized at P15 and P20, during the avascular and neovascular phases of OIR, respectively. Whole retinal transcriptomes were compared to non-PIA controls. Results: RNA sequencing showed dampened pathways of angiogenesis, inflammation, and neural development in anemic OIR females at P20. Conclusion: PIA dampened transcriptomic pathways central to retinal vascular and neural development in neonatal rats. These data suggest PIA provides a protective effect from ROP. Samples for RNA isolation were selected from 4-8 pups/group that were 50% male, 50% female/ group and weight-matched within the group. RNA was extracted from single whole retina using RNAqueous RNA Isolation Kits (Ambion, Waltham, MA). RNA concentration and purity were measured as previously described19. The University of Minnesota Genomic Center performed qualification analysis (RIN score > 8.0), coded library construction, and next generation sequencing, as previously described19. Differentially expressed genes (DEGs) between RA versus RA+PIA pups and OIR versus OIR+PIA pups were identified by the EdgeR package (negative binomial feature in CLCGWB, Qiagen, Redwood City, CA) using raw read counts and a corrected-false discovery rate < 0.05. Additionally, P20 groups (n=8/group) were divided by sex (4 male, 4 female) and DEGs calculated between OIR versus OIR+PIA females and OIR versus OIR+PIA males.