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G. R. Howell, R. T. Libby, A. Mehalow, A. F. Clark, S. W. M. John; An Extensive Microarray Study of DBA/2J: A Resource for Molecular Characterization of Glaucoma. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1554.
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The mechanisms leading to retinal ganglion cell (RGC) death and optic nerve (ON) degeneration in glaucoma are not well defined. DBA/2J (D2) mice develop an inherited glaucoma with hallmarks of human glaucoma. To help uncover the molecular pathophysiology of glaucoma, we performed a microarray analysis on tissues from D2 mice with varying degrees of glaucomatous damage.
Two strains were used, D2 and D2Gpnmb+ (genetically matched to D2 except for a wild type Gpnmb gene - this control strain does not develop glaucoma). Eyes were harvested at different time points and degree of glaucoma determined using a grading scale of ON damage. To enhance sensitivity, 10 biological replicates were processed for each glaucoma stage and controls. Retina and ON head were dissected free for each eye, and processed separately. RNAs were hybridized to Affymetrix arrays 430 v2, processed with standard tools and gene lists interrogated using a variety of software packages. Expression differences for genes of interest are being further assessed using high-throughput real time PCR (384 genes/run).
As ON damage increases, RGC specific genes (e.g. Pou4f2 and Nefl) decrease and genes associated with glial activation (e.g. Vim and Gfap) increase, validating the basic design. Analysis of eyes with mild glaucoma (compared to control) showed that 684 probes are significantly different (q≤0.05), including Lcn2 (10.2x). Analysis of Gene Ontology terms for all 684 probes reveals the most significant terms (p<0.01) are ‘cell adhesion’ (43 probes) and ‘defense response’ (63 probes).
Molecular changes from the initial response to high pressure, through to RGC cell loss, and ON remodeling in late disease are being revealed. Importantly, we can analyze expression changes in the retina and ONH separately for the same eye. This data set provides a resource for elucidating molecular events that underlie the earliest steps in the pathogenesis of glaucoma in response to IOP insult.
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