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C.L. Grosskreutz, W. Huang, A. Dobberfuhl, T. Filippopoulos, J. Fileta; Upregulation of Caspase 8 and 9 mRNA Levels in Retinal Ganglion Cells (RGC) in Experimental Glaucoma . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1254.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have previously shown that caspase 8 transcription is elevated in RGC 8 days after optic nerve crush. The purpose of this study was to use laser capture microdissection (LCM) to isolate RGC coupled with real–time PCR to test the hypothesis that gene expression of initiator caspases would be elevated during RGC death in experimental glaucoma. Methods: RGC were backlabeled with Fluorogold (FG). One week later, the intraocular pressure (IOP) of the OS eyes was elevated by injecting 1.9M saline into the episcleral veins in male Brown–Norway rats (250–300 gm). IOP was measured every other day with the TonoPen. Following 10 days of elevated IOP, the retinas were either removed for protein isolation or fixed and sectioned for LCM. Similar numbers of FG backlabeled RGC from control and experimental retinas were captured. Expression of caspase 8, caspase 9 and Thy1.1 mRNA was determined by real–time PCR. Gene changes in photoreceptors were used as internal controls. Protein level changes of procaspase 8, procaspase 9 and cleaved caspase 9 were determined by Western blot. Results: After IOP was elevated for ten days, the mRNA levels increased for caspase 8 (3.3±1.0 fold, n=6, p<0.05) and caspase 9 (1.9±0.6 fold, n=6, p<0.05) and decreased for Thy1.1 (0.5±0.2, n=6, p<0.05) in RGC in glaucoma eyes. No change in caspase 8 or caspase 9 mRNA was seen in photoreceptors in glaucoma eyes and no Thy1.1 mRNA was detected in photoreceptors in any eyes. A significant increase in procaspase 9 protein (1.5±0.4 fold, n=21, p<0.05) and cleaved caspase 9 protein (4.4±0.3 fold, n=21, p<0.05) was observed in glaucoma eyes. Procaspase 8 levels decreased but this change was not statistically significant (0.7±0.4, n=21,p>0.05). Conclusions: These data show that changes of caspase 8, caspase 9, and Thy1.1 mRNA occur specifically in RGC in experimental glaucoma. This supports the hypothesis that a coordinated program of transcriptional regulation of apoptosis related genes occurs in RGC in glaucoma.
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