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BJ Tripathi, YY Xing, RC Tripathi, VA Pakalnis, KV Chalam; Expression of -Interferon mRNA by Astrocytes . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4056.
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Purpose: To investigate whether astrocytes express the mRNA for γ-interferon, which is known to induce expression of nitric oxide synthase-2 by these cells. Methods: Astrocytes, cultured from the optic nerve of adult porcine eyes and plated onto the plastic substrate of a specially designed pressure chamber, were exposed to either an acute increase in hydrostatic pressure from 15 mm Hg to 40 mm Hg and sustained for 24 hrs, or to a gradual stepwise elevation of pressure from 15 mm Hg to 40 mm Hg over a period of 72 hrs. Astrocytes exposed to 15 mm Hg served as controls. In another set of experiments, confluent cultures of astrocytes were incubated for 6 hrs to 96 hrs in medium that contained tumor necrosis factor (TNF)-α at concentrations of 4, 8, 12, 16, or 20 ng/ml. Total RNA was extracted, reverse transcribed and amplified using primer pairs for γ-interferon and for GAPDH, as internal control. The PCR products were separated on 2% ethidium bromide gels for analysis. Results: Using primers specific for GAPDH, a single PCR-amplified product with the expected size of 258 bp was obtained from astrocytes exposed to increased hydrostatic pressure or incubated in TNF-α. The mRNA for γ-interferon was not detectable in astrocytes exposed to an acute or a gradually raised hydrostatic pressure from 15 mm Hg to 40 mm Hg or in control cells maintained at 15 mm Hg. Astrocytes incubated in TNF-α at concentrations of 4 to 20 ng/ml for 6 hrs up to 4 days did not express mRNA for γ-interferon. Conclusion: The excessive production of nitric oxide through the nitric oxide synthase-2 pathway by reactive astrocytes is believed have an important role in neurotoxicity of retinal ganglion cell axons in the optic nerve. Our results show that γ-interferon is neither expressed constitutively by astrocytes nor induced in these cells on exposure to hydrostatic pressure or TNF-α. The expression of γ-interferon by retinal ganglion cells in eyes with early glaucoma, which we have demonstrated previously, may be an initiating or contributing factor in the synthesis of nitric oxide by astrocytes.
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