Gene expression patterns in hypoxic and post-hypoxic adult rat retina with special reference to the NMDA receptor and its interactome

PURPOSE: A gene expression analysis of hypoxic rat retina was undertaken to gain a deeper understanding of the possible molecular mechanisms that underlie hypoxia-induced retinal pathologies and identify possible therapeutic targets. METHODS: Rats were made severely hypoxic (6%–7% O(2)) for 3 h. Some rats were sacrificed at this time, and others were allowed to recover for 24 h under normoxic conditions. A focused oligonucleotide microarray of 1,178 genes, qRT–PCR of selected transcripts, and western analysis of hypoxia inducible factor-1α (HIF-1α) were used to compare retinas from the hypoxic and recovery groups to control animals that were not made hypoxic. SAM analysis was used to identify statistically significant changes in microarray data, and the bioinformatics programs GoMiner, Gene Set Enrichment Analysis (GSEA), and HiMAP were used to identify significant ontological categories and analyze the N-methyl-D-aspartate (NMDA) receptor interactome. RESULTS: HIF-1α protein, but not mRNA, was elevated up to 15-fold during hypoxia, beginning at 0.5 h, the shortest duration examined. Of the total of 1,178 genes examined by microarray, 119 were significantly upregulated following hypoxia. Of these, 86 were still significantly upregulated following recovery. However, 24 genes were significantly downregulated following hypoxia, with 12 still significantly downregulated after recovery. Of the 1035 genes that did not change with hypoxia, the expression of 36 genes was significantly changed after recovery. Ontological analyses showed significant upregulation of a large number of genes in the glutamate receptor family, including 3 of the 5 NMDA subunits. qRT–PCR analysis further corroborated these findings. Genes known to directly interact specifically with the NR1 subunit of the NMDA receptor were identified using HiMAP databases. GSEA analysis revealed that these genes were not affected by either hypoxia or altered after recovery. CONCLUSIONS: The identification of gene expression alterations as a function of hypoxia and recovery from hypoxia is important to understand the molecular mechanisms underlying retinal dysfunction associated with a variety of diseases. Gene changes were identified in hypoxic retina that could be linked to specific networks. Retinas recovering from hypoxia also showed distinct patterns of gene expression that were different from both normoxic control retinas and hypoxic retinas, indicating that hypoxia initiates a complex pattern of gene expression. Diseases of which hypoxia is a component may exhibit the several changes found here. Several potential therapeutic targets have been identified by our approach, including modulation of NMDA receptor expression and signaling, which until now have only been shown to play a role in responding to ischemia.