Role of hypoxia-inducible factor-1α in preconditioning-induced protection of retinal ganglion cells in glaucoma

PURPOSE We recently demonstrated in a mouse model of glaucoma that endogenous epigenetic mechanisms can be activated by a repetitive hypoxic preconditioning (RHP) stimulus to provide robust retinal ganglion cell (RGC) protection. Although we also provided evidence that RHP prevents or delays the apoptotic demise of the RGC soma, the mechanisms responsible for signaling this epigenetic response, as well as the effectors of the glaucoma-tolerant phenotype at the somatic and axonal levels, remain unidentified. In the present study, we used conditional mutant mice lacking hypoxia-inducible factor-1α (HIF-1α) in RGCs (HIF-1α RGC-knockout [KO] mice) to test the hypothesis that RHP-mediated activation of this transcription factor in these cells protects them from glaucomatous injury. METHODS Adult HIF-1α RGC-KO mice, generated by mating floxed HIF-1α mice with math5-Cre mice, were used. Experimental glaucoma was induced unilaterally in the HIF-1α RGC-KO mice and matched wild-types by elevating the intraocular pressure to 16-20 mmHg for 3 consecutive weeks, secondary to episcleral vein ligation. Mice of each genotype were randomized to either an RHP protocol (six total exposures to systemic hypoxia [11% oxygen], interspersed over a 2-week period, completed 3 days before ligation surgery) or to an untreated group. RGC soma and axon injury was quantified with Neuronal Nuclei (NeuN) immunohistochemistry in retinal flat mounts and SMI32 immunohistochemistry in cross sections of the post-laminar optic nerve, respectively. RESULTS HIF-1α RGC-KO mice exhibited normal retinal function and morphology, and crosses of math5-Cre mice with floxed ROSA26 reporter mice confirmed Cre recombinase activity was confined to the RGC axons and soma. Untreated wild-type mice exhibited a 30±2% loss of RGC soma and a 31±3% loss of RGC axons after 3 weeks of intraocular hypertension (both p<0.05 versus fellow eye). The 90% and 81% improvement in soma and axon survival, respectively, observed in the wild-type mice treated with RHP (both p<0.05 versus the glaucoma eye in the untreated mice) was still observed to a near identical extent in the RHP-treated HIF-1α RGC-KO mice. RHP had no effect on the magnitude of intraocular pressure elevation in either the KO or wild-type groups, indicating that protection was realized in both genotypes in the face of ongoing intraocular hypertension. CONCLUSIONS These findings indicate that the robust, glaucomatous protection of the RGC soma and axons induced by RHP does not require HIF-1α-mediated transcription of survival genes and other adaptive responses within the RGCs themselves. Rather, we infer that RGC survival is augmented secondary to the activation of other hypoxia-sensitive transcription factors in RGCs and/or the action of diffusible HIF-1α target gene proteins released from neighboring retinal cells. Ideally, the involvement of such autocrine- and/or paracrine-based mechanisms would be confirmed in future studies, but distinct components of the integrated, pleiotropic, and multicellular basis of this endogenous epigenetic response may prove difficult to demonstrate experimentally, as we found in the present study.