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Y. Zhu, L. Zhang, J. M. Gidday; Glaucoma-Induced Apoptosis of Retinal Ganglion Cells Prevented by Hypoxic Preconditioning Prior to IOP Elevation: A Model of "Glaucoma Tolerance". Invest. Ophthalmol. Vis. Sci. 2008;49(13):5512.
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© ARVO (1962-2015); The Authors (2016-present)
Several endpoint measures in experimental glaucoma models support the hypothesis that retinal ganglion cells (RGCs) die by apoptosis in response to intraocular hypertension. We showed previously in mice that repetitive hypoxic preconditioning (RHP) promotes a long-lasting (one-month) period of retinal ischemic tolerance (IOVS 48: 1735-1743, 2007), and when given prior to chronic elevation of intraocular pressure by episcleral vein ligation, blocks RGC loss as measured 3 wks later by both brn-3b immunostaining and optic nerve axon density measurements (Schmidt J.F. et al., ARVO 2005 #1328). We used the latter model of "glaucoma tolerance" in the present study to determine if the protection of RGCs by RHP resulted from a reduction in apoptotic cell death.
Episcleral veins of one eye of adult male C57Bl/6 mice were ligated to obtain a sustained increase in IOP (from 10.1±1.4 [baseline] to 22.2±1.3 mmHg [3wks]; n=16; TonoLab) between 1 day and 3 wks later. Some animals received a two-week RHP protocol involving six, 2-h exposures (every other day) to systemic hypoxia (11% oxygen) that finished 3 days prior to vein ligation. A third group only received RHP without subsequent vein ligation, and a fourth nonpreconditioned, nonligated group served as sham controls. After 3 wks intraocular hypertension, eyes were enucleated and retinae were either flash frozen for quantitative protein analyses by immunoblotting and cellular localization by immunohistochemistry.
After 3 wks of intraocular hypertension in nonpreconditioned animals, the bcl-2/bax ratio was decreased (p<0.05; n=5) below baseline, and cleaved caspase-9 protein levels were increased (p<0.05; n=6). However, when RHP preceded intraocular hypertension, the bcl2/bax ratio was increased above baseline (p<0.05; n=5) and caspase-9 protein expression was reduced (p<0.05; n=6) to levels lower than controls. Increases in the number of RGCs immunopositive for caspase-3, caspase-9, and TUNEL in nonpreconditioned mice were all robustly abrogated by prior RHP treatment (n=3).
The ability of prior RHP to attenuate or completely block these pro-apoptotic changes in RGCs following intraocular hypertension, in conjunction with our previous results demonstrating RGC preservation following RHP, suggest that RHP activates endogenous survival mechanisms in RGCs that can prevent apoptotic cell death following experimental glaucoma. The molecular basis of this intrinsic "glaucoma tolerance" to apoptotic cell death is unknown, but represents a new paradigm for promoting RGC protection in glaucoma.
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