Early Gene Expression Changes in the Retinal Ganglion Cell Layer of a Rat Glaucoma Model

The progressive loss of vision in glaucoma patients has been attributed to degeneration of retinal ganglion cells (RGCs) and their axons and the eventual death of these cells. As intraocular pressure (IOP) remains the best established risk factor for glaucoma, therapy for this blinding disease continues to rely heavily on lowering pressure by medical and surgical measures. Although pressure reduction has been effective in preventing onset of glaucoma and delaying its progression,1,2 some patients do not respond well or are intolerant of these treatments, whereas others continue to experience vision loss despite significant reduction in IOP. Recently, neuroprotective strategies other than pressure lowering, including supplementation of neurotrophins and prevention of caspase activation and apoptosis, have been proposed and are being investigated as new goals for glaucoma therapy.3,4 Effective neuroprotection, aimed at salvaging functional RGCs and their axons before they are committed to die, requires early intervention and targeting of upstream events. These early changes may involve increased susceptibility of RGCs to elevated IOP, which could explain progressive vision loss in patients despite lowered eye pressure. Conversely, they may be protective mechanisms that could be therapeutically enhanced. Identification of the early critical molecular events in RGCs would add to our understanding of the nature of glaucomatous injury and provide potential targets for neuroprotective strategies. The primary injury in glaucoma is now widely believed to be axonal at the lamina in the optic nerve head.5–7 However, the nature of the injury and the initial molecular events occurring in injured RGCs are largely unknown. Studies have described early morphologic changes in RGCs,8–10 suggesting that structural and possibly functional changes occur well in advance of their death. Also, pathways involved in RGC soma degeneration, although not well characterized, have been suggested to be distinct from those in axon degeneration in glaucoma.11 To examine global changes in gene expression, several studies have applied genomewide analysis of microarrays to the retina of rodent and primate glaucoma models as well as the DBA/2J mice.12–17 These studies have found significant regulation of gene expression in the retina and implicate multiple processes in glaucomatous injury, including neuroinflammation, glia activation, apoptosis, and cytoskeleton-based processes. However, because RGCs represent only a small fraction of the total retinal cell population, in these studies performed on whole retina samples, many responses specific to RGCs may have been masked. In an effort to identify changes more specific to RGCs, Wang et al.18 studied global gene expressions in individual RGCs captured by laser in experimental glaucoma and reported extensive expression changes in genes associated with multiple signaling pathways and apoptosis. Like the whole-retina studies, however, this study did not specifically target the events of early injury. Using a rodent glaucoma model in which chronic IOP elevation is achieved by injection of hypertonic saline into episcleral veins,19 we have recently demonstrated that detection of RGC responses can be greatly enhanced by analyzing just the RGC layer (GCL) compared with the whole retina.20 Successfully isolating the GCL from the whole retina by laser capture microdissection (LCM) and applying cDNA microarray analysis to the GCL mRNA, we identified a significantly larger number of genes with altered expression in the GCL compared with those in whole retinas of eyes with comparable injury. That report, which focused on responses in eyes with extensive nerve injury, represents only part of a larger study in which we examined the GCL in eyes with all grades of optic nerve injury. Here, we present the complete analysis, but mainly concentrate on early-injury responses that, as mentioned previously, are likely to be important for understanding the cellular mechanisms of glaucoma and identifying their therapeutic implications.