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Global Gene Expression Changes in Rat Retinal Ganglion Cells in Experimental Glaucoma

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Glaucoma Global Gene Expression Changes in Rat Retinal Ganglion Cells in Experimental Glaucoma Dan Yi Wang,1 Arjun Ray,1 Kathryn Rodgers,1 Ceren Ergorul,1 Bradley T. Hyman,2 Wei Huang,1 and Cynthia L. Grosskreutz1 PURPOSE. Intraocular pressure (IOP) is an important risk factor laucoma is the second leading cause of blindness world- in glaucoma. Gene expression changes were studied in glau- Gwide.1 It is a neurodegenerative disease that is character- comatous rat retinal ganglion cells (RGCs) to elucidate altered ized by the slow and progressive degeneration of retinal gan- transcriptional pathways. glion cells (RGCs) and their axons, leading to atrophy of the optic nerve and loss of vision. Elevated intraocular pressure METHODS. RGCs were back-labeled with Fluorogold. Unilateral (IOP) is the leading risk factor for loss of RGCs and develop- IOP elevation was produced by injection of hypertonic saline ment of optic nerve atrophy. It is now clear that RGCs die by into the episcleral veins. Laser capture microdissection (LCM) apoptosis in glaucoma.2–5 However, the trigger of the apopto- was used to capture an equal number of RGCs from normal and sis is still unknown. To successfully target the mechanisms, we glaucomatous retinal sections. RNA was extracted and ampli- must to understand the molecular signaling networks in RGC fied, labeled, and hybridized to rat genome microarrays, and death. We used laser capture microdissection (LCM) to specif- data analysis was performed. After selected microarray data ically capture RGCs, and microarray technology, an accepted were confirmed by RT-qPCR and immunohistochemistry, bio- and powerful tool for large-scale gene expression profiling, to logical pathway analyses were performed. elucidate a global view of gene interaction networks in RGCs. RESULTS. Significant changes were found in the expression of To date, several gene expression studies have been per- 905 genes, with 330 genes increasing and 575 genes decreas- formed on the retina as a whole, to catalog changes in tran- ing in glaucomatous RGCs. Multiple cellular pathways were scription that accompany glaucoma. The mRNA expression involved. Ingenuity pathway analysis demonstrated significant patterns in whole retina of several animal models of glaucoma, 6 7 changes in cardiac ␤-adrenergic signaling, interferon signaling, including DBA/2J mice, cynomolgus macaque monkeys, 8–11 12 glutamate receptor signaling, cAMP-mediated signaling, che- rat, and C57B16/J mice have been investigated. These mokine signaling, 14-3-3-mediated signaling, and G-protein- studies revealed up- and downregulation of many genes in coupled receptor signaling. Gene set enrichment analysis response to elevated IOP. However, the outcome and interpre- showed that the genes involved in apoptotic pathways were tation of these microarray studies can be strongly affected by enriched in glaucomatous RGCs. The prosurvival gene Stat3 inherent tissue heterogenicity, as the retina is a complex tissue was upregulated in response to elevated IOP, and immunohis- composed of neuronal, glial, and vascular cell types. The RGCs tochemistry confirmed that Stat3 and phosphorylated-Stat3 lev- comprise only 1% or less of the retinal cells. It is therefore difficult to causally link changes in gene expression patterns els were increased in RGCs in experimental glaucoma. In from whole retina to specific RGC gene expression, the asso- addition, the expression of several prosurvival genes normally ciated pathways and subsequent physiological effects. In addi- expressed in RGCs was decreased. tion, site-specific, pathology-related gene expression changes CONCLUSIONS. There are extensive changes in gene expression can be diluted by the gene expression profile of other cell in glaucomatous RGCs involving multiple molecular pathways, types in a whole retinal homogenate and thus not be readily including prosurvival and prodeath genes. The alteration in the detectable. Previous efforts to identify the complete picture of balance between prosurvival and prodeath may contribute to retinal gene expression may thus have missed important RGC RGC death in glaucoma. (Invest Ophthalmol Vis Sci. 2010;51: genes, due to the low relative frequency of RGCs and, hence, 4084–4095) DOI:10.1167/iovs.09-4864 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- 1 From the Department of Ophthalmology, Harvard Medical School, genome regulation of RGCs under conditions of elevated IOP. Massachusetts Eye and Ear Infirmary, Boston, Massachusetts; and the We hypothesized that gene expression would change in glau- 2MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts. comatous RGCs compared with normal RGCs in a rat model of Supported by the Massachusetts Lions Eye Research Fund, experimental glaucoma. To test our hypothesis, we applied National Eye Institute Grant R01-EY13399, the Cannistraro Fund, LCM to capture an equal number of glaucomatous RGCs and the Margolis fund, and National Eye Institute Core Facility Grant normal RGCs and used microarray analysis to identify the genes EY014104. and functional gene classes most altered in expression in the Submitted for publication November 4, 2009; revised January 22 rat RGCs after exposure to experimentally elevated pressure. and February 26, 2010; accepted March 3, 2010. Then, we used real-time quantitative PCR (RT-qPCR) to validate Disclosure: D.Y. Wang, None; A. Ray, None; K. Rodgers, None; selected changes in gene expression identified by microarray C. Ergorul, None; B.T. Hyman, None; W. Huang, None; C.L. analysis. The JAK/STAT signaling pathway, especially the Stat3 Grosskreutz, None protein, has been implicated in neurodegenerative and neuro- Corresponding author: Cynthia L. Grosskreutz, Department of 14 Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear protective mechanisms. The phosphorylation of Stat3 is Infirmary, 243 Charles Street, Boston, MA 02114; known to activate Stat3 and has been shown to be protective [email protected]. of RGCs after ischemic insult.15–17 The increased transcript Investigative Ophthalmology & Visual Science, August 2010, Vol. 51, No. 8 4084 Copyright © Association for Research in Vision and Ophthalmology Downloaded from jov.arvojournals.org on 10/01/2021 IOVS, August 2010, Vol. 51, No. 8 RGC Gene Expression Changes in Glaucoma 4085 expression of the prosurvival gene, signal transducer, and Estimation of total RGC number in each retina was performed with activator of transcription 3 (Stat3) was confirmed at the protein statistically unbiased stereological protocols by using an optical dissec- level in RGCs by immunohistochemistry (IHC). In addition, tor and a fractionator, as previously described.3,19 Fluorogold-positive Stat3 phosphorylation was seen in RGCs after IOP elevation. cells were immunostained with an anti-Fluorogold antibody (1:200, Fluorochrome LLC) and counted (C.A.S.T. system; ver. 2.3.1.2; Olym- pus, Albertslund, Denmark). METHODS Optic nerves were assessed in saline-injected eyes and fellow eyes. All corresponding optic nerve sections were taken from approximately Study Design 2 mm posterior to the globe, fixed in glutaraldehyde, embedded in plastic, stained with toluidine blue, and evaluated by light microscopy. The study was performed in five adult male Brown Norway rats. RGCs Optic nerve damage was graded from 1 (no injury) to 5 (degeneration were back-labeled with Fluorogold. Seven days later, unilateral eleva- involving the total nerve area) by four masked observers who used a tion of IOP was produced in one eye by injecting hypertonic saline into modification of the method described by Morrison.3,18 episcleral veins. The fellow eye served as the control. After 10 days of elevated IOP, the rats were killed, and the eyes were processed for LCM. First, to confirm the enrichment of RGCs, the gene expression of LCM of Fluorogold-Labeled RGCs glial fibrillary acidic protein (Gfap, an astrocyte marker) and thymus For LCM, a commercial system (PixCell IIe; Arcturus, Mountain View, cell antigen 1, theta (Thy1, an RGC marker) was compared between CA) was used, as previously described.3,20 Briefly, the tissue sections captured RGCs and captured ganglion cell layer (GCL) from normal were dehydrated by serial immersions in 75%, 95%, and 100% ethanol eyes. Next, two thousand RGCs were captured from each normal eye for 2 minutes, followed by a 2-minute immersion in xylene. Tissue and each eye with elevated IOP in three rats. Gene expression in the sections were then air-dried for 2 to 5 minutes at room temperature. A glaucomatous RGCs was compared with that in the fellow normal cleanup pad (Cap-Sure; Arcturus) was used to remove any extra tissue RGCs by gene microarray (Rat GeneChip 230.2; Affymetrix, Santa on the slide. RGCs were identified by the presence of Fluorogold Clara, CA). Finally, an equal number of RGCs were captured from five back-labeling (excitation filter, 330–380 nm; barrier filter, 400 nm). An rats, including the three rats from the microarray experiments. RT- equal number of RGCs from each eye were captured onto single caps qPCR was used to verify selected changes in gene expression initially (Cap-Sure MacroLCM Caps; Arcturus), by using the following parame- identified by microarray analysis. Retinal tissue sections were also used ters:
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