cDNA Microarray Analysis of Gene Expression in Mouse Cornea After Pseudomonas aeruginosa Infection

Abstract

Abstract: : Purpose: The goal of the present study was to compare gene expression patterns of normal vs. Pseudomonas-infected corneas in resistant (cornea heals) vs. normal mice using cDNA microarray. Methods: Corneal scarification and topical delivery of P. aeruginosa (5 microliters; 1 X 10₆ CFU) was performed on the left eye of 40 BALB/c mice under anesthesia. The right cornea of each mouse served as a control. At 1 and 5 days p.i., total RNA was isolated from 20 corneas (10 normal and 10 infected) using Trizol. Poly A+ mRNA was isolated using a mRNA minikit (QIAGEN) according to the manufacturer's recommendations. Approximately 500 ng of mRNA was converted to cyanine-dye-labeled cDNA by a standard reverse-transcription reaction. One cDNA population was labeled with Cy3 dye and the other with Cy5 dye. Next, the two probe samples were mixed and simultaneously applied to a single microarray where they competitively hybridized to the arrayed cDNA molecules. High-density cDNA chips (Mouse GEM-5220, INCYTE Genomics) with over 9,000 known mouse genes and ESTs were used. In a separate set of experiments, Affymetrix murine U74A arrays also were used. Results: The mRNA expression levels of a number of genes (19 at 1 day p.i.; 34 at 5 day p.i.) were markedly higher (5-28X at 1 day and 5-63X at 5 days p.i.) in infected vs. normal cornea. The list of genes included: serum amyloid A3, lipocalin, serum proteinase inhibitors, laminins, IL-1, protease inhibitors, plasminogen activator, urokinase, annexin A3, members of the TNF superfamily (at 1 day p.i.). At 5 days p.i., although many of the same genes as above remained markedly higher, other genes including lactotransferrin, tenascin C, TIMP, P-selectin ligand and others were detected. Several genes such as Toll-like receptor 1 and LPS binding protein, caspases, and NF kappa B also were increased at about 2-4 fold at 5 days p.i. Conclusion: In addition to detection of genes encoding for proinflammatory cytokines such as IL-1 and TNF, expected to be elevated after bacterial infection, other genes have been revealed in the infected BALB/c mouse cornea that may provide clues to corneal healing after infection in this resistant mouse strain. Support: EY02986 (Suppl.) and P30EY04068.

Keywords: 417 gene/expression • 531 Pseudomonas • 449 keratitis