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L. Jia, E.C. Johnson, W.O. Cepurna, S.L. Barber, J.C. Morrison; Rat Optic Nerve Head Mrna Responses to Elevated Iop or Optic Nerve Transection: Confirmation and Extension of Microarray Findings by QPCR . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1251.
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Purpose:Elevated intraocular pressure (IOP) is a major risk factor for glaucoma and the optic nerve head (ONH) is the most likely site of early injury in glaucoma. However, we know little of either the in vivo ONH molecular responses in response to IOP or their relationship to axonal degeneration. In this study, we use real time quantitative RT–PCR (qPCR) to verify selected changes in gene expression initially identified by microarray analysis of IOP–injured ONH and examine genes not included on the arrays. We extend this study to include analyses of ONH with focal IOP–induced injury and to compare these responses to those following optic nerve transection. Methods:Brown Norway rats (N= 39) received unilateral episcleral vein injections of hypertonic saline and IOP was measured on awake animals by TonoPen. Unilateral optic nerve transection was performed on 11 additional animals. At 5 weeks post–injection and 14 days post–transection, animals were sacrificed and ONH mRNA quantified relative to GAPDH using qPCR. Retrobulbar optic nerve injury was graded by masked observers. Data from elevated IOP ONH were grouped based on the optic nerve injury grade and compared to each other and to the transection group. Results:For periostin, tenascin–C, collagen VI, and fibulin–2 mRNA, qPCR confirmed the magnitude of increased expression in ONH severely injured by IOP elevation that were initially quantitated by microarray analysis. ON transection resulted in comparable mRNA increases in the first three, while fibulin–2 mRNA remained at fellow eye levels. For both fibulin–2 and tenascin–C, mRNA levels demonstrated a significant nonlinear relationship to IOP level (p<0.01), with the highest levels occurring in ONH with early focal optic nerve injury. In contrast, periostin, collagen VI and IV mRNA increased linearly with nerve injury due to elevated IOP (R2=0.51, 0.38 and 0.11, respectively). Aquaporin–4 mRNA was significantly decreased by both elevated IOP (R2=0.2) and transection (p<0.01). Connexin–43, TGFbeta (2), GAPDH and, surprisingly, GFAP (alpha, beta and gamma isoforms) mRNA levels were not significantly altered by either type of injury. Conclusions: This qPCR study confirms microarray analysis findings and provides evidence of unique patterns of changed gene expression in response to elevated IOP that differ from those seen following optic nerve transection. These sensitive molecular tools offer the potential for further identification of the specific cellular ONH events associated with glaucomatous axonal injury.
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