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W. Huang, A. Dobberfuhl, M. Ingelsson, T. Filippopoulos, C.L. Grosskreutz; Detection of gene expression changes of caspase 8 and Thy1 in rat retinal ganglion cells after optic nerve crush using laser capture microdissection (LCM) with real–time PCR . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2160.
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Purpose: Retina ganglion cells (RGC) die predominantly by apoptosis after optic nerve crush. Caspase–8 is an upstream activator of the caspase cascade during apoptosis. Thy1 mRNA is a RGC cell–specific gene. The aim of this study was to 1) explore the possibility that the change of caspase–8 and Thy1 gene expression might be involved or changed during RGC death after optic nerve crush in the rat 2) apply the technique of LCM coupled with real–time PCR to study diseases of the optic nerve and RGC. Method: RGC were retrogradely labeled from both superior colliculi with stereotactic injections of 3% Fluorogold in 3 male Brown Norway Rats. Seven days later, the right optic nerves were crushed approximately 2.5–3.0mm posterior to the globe and the left used as control. Five days later the animals were sacrificed and transcardially perfused with 4% PFA. Cryosections (12µm) were prepared. Similar numbers of RGC from control and crushed retinas were captured using LCM under epi–fluorescent microscopy. RNA was extracted from captured cells, DNAse–treated, and reverse–transcribed. Expression of caspase 8 and Thy1 mRNA was determined by quantitative real–time RT–PCR and GAPDH was used as the internal control to standardize expressed message from captured cells. Results: After ONC, caspase 8 mRNA could be detected using 50 captured RGC cells in all cases, while only one out of 3 control eyes had detectable levels of Caspase 8 m–RNA from 50 captured cells. Caspase 8 m–RNA was >20 fold higher in the crushed eyes. Thy1 mRNA levels decreased 5 days after ONC to <50% of control. Conclusion: The level of caspase 8 mRNA was significantly up–regulated after crush injury. Thy1 mRNA level decreased at the same time point consistent with previous reports using whole retina. LCM with quantitative real–time PCR is a powerful approach for the study of gene expression in RGC.
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