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W. S. Lambert, T. A. Doser, J. C. Morrison, E. C. Johnson; Cellular Stretch and Interleukin-6 Exposure Produce Glaucoma-Like Gene Expression Responses in Primary Rat Astrocyte Cultures. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2792.
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In rats, initial injury due to elevated intraocular pressure (IOP) exposure results in dramatically increased optic nerve head (ONH) interleukin 6 (IL6) and leukemia inhibitory factor (LIF) mRNA levels. In this study we exposed cultured rat astrocytes to cellular stretch to determine if these responses could be modeled in vitro. Because microglia and other ONH cells may produce IL6 in the in vivo glaucomatous ONH, the effect of exogenous IL6 was also examined.
Two neonatal rat cortical astrocyte cell lines were grown on membranes and then exposed to an 8% equibiaxial stretch in serum-free culture. Gene expression levels were determined by qPCR after 2 to 48 hours in stretch conditions. This experiment was replicated in the presence of recombinant rat IL-6 with the inclusion of a 0 hour time point.
Stretch alone significantly increased IL6 mRNA expression at all time points examined, averaging 189% of matched, unstretched values (p<0.0001, n=18 for 2, 12 and 24 hour groups and n=32 for 48 hour groups). LIF mRNA levels were significantly increased to 171% of unstretched values at 2 hours of stretch, and then declined to within 25% of unstretched values at 24 hours (p<0.034). Stretch also resulted in a small but significant 33% decrease in GFAP mRNA at 48 hours (p<0.011). IL6 treatment of unstretched astrocytes significantly increased endogenous IL6 mRNA 96% (p<0.0004) and LIF mRNA 165% (p<0.0001) at 2 hours compared to the 0 hour time point (n=6 for 2-24 hours and n=12 for 0 and 48 hours). IL6 and LIF mRNA levels declined to within 30% and 40% of 0 hour values respectively at 12, 24 or 48 hours. GFAP expression was increased by IL6 treatment at all time points, reaching 694% of untreated values at 48 hours (p<0.003). Astrocyte responses to IL6 treatment were not significantly altered by simultaneous stretch exposure.
Exposure of astrocytes to 8% equibiaxial stretch or IL6 significantly increases IL6 and LIF mRNA levels, although the magnitude of increase is less than that produced by early, IOP-induced ONH injury. This study illustrates, in principle, that cultured astrocytes exposed to stretch and other in vitro conditions can mimic known ONH glaucomatous responses.
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