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.
In addition, Stat3 phosphorylation was seen in RGCs after IOP elevation.
The study was performed in five adult male Brown Norway rats. Seven days later, unilateral elevation of IOP was produced in one eye by injecting hypertonic saline into episcleral veins. After 10 days of elevated IOP, the rats were killed, and the eyes were processed for LCM.
These studies revealed up- and downregulation of many genes in response to elevated IOP.Gene expression in the glaucomatous RGCs was compared with that in the fellow normal RGCs by gene microarray (Rat Gene Chip 230.2; Affymetrix, Santa Clara, CA).Finally, an equal number of RGCs were captured from five rats, including the three rats from the microarray experiments.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.
Xl dating Albertslund
Laser capture microdissection (LCM) was used to capture an equal number of RGCs from normal and glaucomatous retinal sections.RNA was extracted and amplified, labeled, and hybridized to rat genome microarrays, and data analysis was performed.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.In addition, the expression of several prosurvival genes normally expressed in RGCs was decreased.
There are extensive changes in gene expression in glaucomatous RGCs involving multiple molecular pathways, including prosurvival and prodeath genes.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-q PCR) to validate selected changes in gene expression identified by microarray analysis.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.