Global gene expression changes in rat retinal ganglion cells in experimental glaucoma.

Glaucoma is the second leading cause of blindness worldwide.1 It is a neurodegenerative disease that is characterized by the slow and progressive degeneration of retinal ganglion cells (RGCs) and their axons, leading to atrophy of the optic nerve and loss of vision. Elevated intraocular pressure (IOP) is the leading risk factor for loss of RGCs and development of optic nerve atrophy. It is now clear that RGCs die by apoptosis in glaucoma.2–5 However, the trigger of the apoptosis is still unknown. To successfully target the mechanisms, we must to understand the molecular signaling networks in RGC death. We used laser capture microdissection (LCM) to specifically capture RGCs, and microarray technology, an accepted and powerful tool for large-scale gene expression profiling, to elucidate a global view of gene interaction networks in RGCs. To date, several gene expression studies have been performed on the retina as a whole, to catalog changes in transcription that accompany glaucoma. The mRNA expression patterns in whole retina of several animal models of glaucoma, including DBA/2J mice,6 cynomolgus macaque monkeys,7 rat,8–11 and C57B16/J mice12 have been investigated. These studies revealed up- and downregulation of many genes in response to elevated IOP. However, the outcome and interpretation of these microarray studies can be strongly affected by inherent tissue heterogenicity, as the retina is a complex tissue composed of neuronal, glial, and vascular cell types. The RGCs comprise only 1% or less of the retinal cells. It is therefore difficult to causally link changes in gene expression patterns from whole retina to specific RGC gene expression, the associated pathways and subsequent physiological effects. In addition, site-specific, pathology-related gene expression changes can be diluted by the gene expression profile of other cell types in a whole retinal homogenate and thus not be readily detectable. Previous efforts to identify the complete picture of retinal gene expression may thus have missed important RGC genes, due to the low relative frequency of RGCs and, hence, their mRNA transcripts. LCM overcomes such obstacles.13 LCM allows morphologic preservation of tissue and the laser does not damage macromolecules such as DNA, RNA, or proteins. In the present study, we sought to investigate the whole-genome regulation of RGCs under conditions of elevated IOP. We hypothesized that gene expression would change in glaucomatous RGCs compared with normal RGCs in a rat model of experimental glaucoma. To test our hypothesis, we applied LCM to capture an equal number of glaucomatous RGCs and normal RGCs and used microarray analysis to identify the genes and functional gene classes most altered in expression in the rat RGCs after exposure to experimentally elevated pressure. Then, we used real-time quantitative PCR (RT-qPCR) to validate selected changes in gene expression identified by microarray analysis. The JAK/STAT signaling pathway, especially the Stat3 protein, has been implicated in neurodegenerative and neuroprotective mechanisms.14 The phosphorylation of Stat3 is known to activate Stat3 and has been shown to be protective of RGCs after ischemic insult.15–17 The increased transcript expression of the prosurvival gene, signal transducer, and activator of transcription 3 (Stat3) was confirmed at the protein level in RGCs by immunohistochemistry (IHC). In addition, Stat3 phosphorylation was seen in RGCs after IOP elevation.