Abstract
Nonarteritic anterior ischemic optic neuropathy (NAION) is a leading cause of sudden, painless vision loss in the elderly, yet no proven intervention exists. Ischemic preconditioning (IPC) is a promising neuroprotective strategy, but defining an effective clinical protocol remains a major challenge in fulfilling its translational potential. We recently discovered that 40 Hz flicker induces extracellular adenosine, a key neurochemical underpinning of IPC, in the visual pathway, suggesting a previously unexplored non-invasive IPC approach. Here, we demonstrated that 3-day 40 Hz flicker preconditioning significantly protected against NAION by reducing retinal ganglion cell loss, preserving ganglion cell layer structure, improving visual function, and attenuating microglial activation. Protection was strongest when ischemia occurred 12 hours after preconditioning, remained moderate at 24 hours, and persisted for at least 4 weeks. This effect was specific to preconditioning and flicker frequency-dependent (effective at 40 Hz, but not at 20 Hz or 80 Hz). Furthermore, neuroprotection by 40 Hz flicker was abolished by treatment with the equilibrative nucleoside transporter inhibitor dipyridamole and the A1 receptor antagonist DPCPX. These findings establish 40 Hz flicker as a non-invasive, adenosine-mediated IPC strategy, suggesting a potentially safe and translational approach for protecting against NAION and other ocular ischemic disorders.
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Data availability
All datasets generated and analyzed in this study are presented within the main figures and Supplementary materials. The numerical source data underlying each graph are provided in Supplementary Data. Correspondence and requests for materials should be addressed to Ying Gao or Jiangfan Chen.
Code availability
No custom computer code was used in this study. All data analyses were performed using standard software.
References
Bosley, T. M., Abu-Amero, K. K. & Ozand, P. T. Mitochondrial DNA nucleotide changes in non-arteritic ischemic optic neuropathy. Neurology 63, 1305–1308 (2004).
Abu-Amero, K. K. & Bosley, T. M. Increased relative mitochondrial DNA content in leucocytes of patients with NAION. Br. J. Ophthalmol. 90, 823–825 (2006).
Rizzo, J. F. 3rd. Unraveling the enigma of nonarteritic anterior ischemic optic neuropathy. J. Neuroophthalmol. 39, 529–544 (2019).
Liu, B., Yu, Y., Liu, W., Deng, T. & Xiang, D. Risk factors for non-arteritic anterior ischemic optic neuropathy: a large scale meta-analysis. Front. Med. 8, 618353 (2021).
Optic nerve decompression surgery for nonarteritic anterior ischemic optic neuropathy (NAION) is not effective and may be harmful. The Ischemic Optic Neuropathy Decompression Trial Research Group. JAMA 273, 625–632 (1995).
Saxena, R. et al. Steroids versus no steroids in nonarteritic anterior ischemic optic neuropathy: a randomized controlled trial. Ophthalmology 125, 1623–1627 (2018).
Brossard Barbosa, N., Donaldson, L. & Margolin, E. Asymptomatic fellow eye involvement in nonarteritic anterior ischemic optic neuropathy. J. Neuroophthalmol. 43, 82–85 (2023).
Murry, C. E., Jennings, R. B. & Reimer, K. A. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74, 1124–1136 (1986).
Meng, R. et al. Upper limb ischemic preconditioning prevents recurrent stroke in intracranial arterial stenosis. Neurology 79, 1853–1861 (2012).
Hausenloy, D. J. & Yellon, D. M. Ischaemic conditioning and reperfusion injury. Nat. Rev. Cardiol. 13, 193–209 (2016).
Keevil, H., Phillips, B. E. & England, T. J. Remote ischemic conditioning for stroke: a critical systematic review. Int J. Stroke 19, 271–279 (2024).
Sharma, D., Maslov, L. N., Singh, N. & Jaggi, A. S. Remote ischemic preconditioning-induced neuroprotection in cerebral ischemia-reperfusion injury: preclinical evidence and mechanisms. Eur. J. Pharm. 883, 173380 (2020).
Hausenloy, D. J. et al. Remote ischemic preconditioning and outcomes of cardiac surgery. N. Engl. J. Med. 373, 1408–1417 (2015).
Meybohm, P. et al. A multicenter trial of remote ischemic preconditioning for heart surgery. N. Engl. J. Med. 373, 1397–1407 (2015).
Chen, H. S. et al. Effect of remote ischemic conditioning vs usual care on neurologic function in patients with acute moderate ischemic stroke: the RICAMIS randomized clinical trial. JAMA 328, 627–636 (2022).
Hou, C. et al. Chronic remote ischaemic conditioning in patients with symptomatic intracranial atherosclerotic stenosis (the RICA trial): a multicentre, randomised, double-blind sham-controlled trial in China. Lancet Neurol. 21, 1089–1098 (2022).
Tsai, C. J. et al. Cerebral capillary blood flow upsurge during REM sleep is mediated by A2a receptors. Cell Rep. 36, 109558 (2021).
Sancho, M. et al. Adenosine signaling activates ATP-sensitive K(+) channels in endothelial cells and pericytes in CNS capillaries. Sci. Signal. 15, eabl5405 (2022).
Liu, Z. et al. Endothelial adenosine A2a receptor-mediated glycolysis is essential for pathological retinal angiogenesis. Nat. Commun. 8, 584 (2017).
Carlström, M., Wilcox, C. S. & Welch, W. J. Adenosine A2A receptor activation attenuates tubuloglomerular feedback responses by stimulation of endothelial nitric oxide synthase. Am. J. Physiol. Ren. Physiol. 300, F457–F464 (2011).
Paez, D. T. et al. Adenosine A(1) receptors and mitochondria: targets of remote ischemic preconditioning. Am. J. Physiol. Heart Circ. Physiol. 316, H743–H750 (2019).
Peart, J. N. & Headrick, J. P. Adenosinergic cardioprotection: multiple receptors, multiple pathways. Pharm. Ther. 114, 208–221 (2007).
Bookser, B. C. et al. Adenosine kinase inhibitors. 6. Synthesis, water solubility, and antinociceptive activity of 5-phenyl-7-(5-deoxy-beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidines substituted at C4 with glycinamides and related compounds. J. Med. Chem. 48, 7808–7820 (2005).
Heusch, G., Bøtker, H. E., Przyklenk, K., Redington, A. & Yellon, D. Remote ischemic conditioning. J. Am. Coll. Cardiol. 65, 177–195 (2015).
Sun, X. et al. 40 Hz light flickering facilitates the glymphatic flow via adenosine signaling in mice. Cell Discov. 10, 81 (2024).
Zhou, X. et al. 40 Hz light flickering promotes sleep through cortical adenosine signaling. Cell Res. 34, 214–231 (2024).
Iaccarino, H. F. et al. Gamma frequency entrainment attenuates amyloid load and modifies microglia. Nature 540, 230–235 (2016).
Zheng, L. et al. Rhythmic light flicker rescues hippocampal low gamma and protects ischemic neurons by enhancing presynaptic plasticity. Nat. Commun. 11, 3012 (2020).
Wang, W. et al. Gamma frequency entrainment rescues cognitive impairment by decreasing postsynaptic transmission after traumatic brain injury. CNS Neurosci. Ther. 29, 1142–1153 (2023).
Slater, B. J., Mehrabian, Z., Guo, Y., Hunter, A. & Bernstein, S. L. Rodent anterior ischemic optic neuropathy (rAION) induces regional retinal ganglion cell apoptosis with a unique temporal pattern. Investig. Ophthalmol. Vis. Sci. 49, 3671–3676 (2008).
Qin, Q. et al. Inhibiting multiple forms of cell death optimizes ganglion cells survival after retinal ischemia reperfusion injury. Cell Death Dis. 13, 507 (2022).
Wen, Y. T., Huang, T. L., Huang, S. P., Chang, C. H. & Tsai, R. K. Early applications of granulocyte colony-stimulating factor (G-CSF) can stabilize the blood-optic-nerve barrier and ameliorate inflammation in a rat model of anterior ischemic optic neuropathy (rAION). Dis. Model Mech. 9, 1193–1202 (2016).
Santiago, A. R. et al. Keep an eye on adenosine: its role in retinal inflammation. Pharm. Ther. 210, 107513 (2020).
Ostwald, P., Park, S. S., Toledano, A. Y. & Roth, S. Adenosine receptor blockade and nitric oxide synthase inhibition in the retina: impact upon post-ischemic hyperemia and the electroretinogram. Vis. Res. 37, 3453–3461 (1997).
Roth, S. et al. Concentrations of adenosine and its metabolites in the rat retina/choroid during reperfusion after ischemia. Curr. Eye Res. 16, 875–885 (1997).
Petrovic-Djergovic, D. et al. Tissue-resident ecto-5’ nucleotidase (CD73) regulates leukocyte trafficking in the ischemic brain. J. Immunol. 188, 2387–2398 (2012).
Schadlich, I. S. et al. Nt5e deficiency does not affect post-stroke inflammation and lesion size in a murine ischemia/reperfusion stroke model. iScience 25, 104470 (2022).
Ghiardi, G. J., Gidday, J. M. & Roth, S. The purine nucleoside adenosine in retinal ischemia-reperfusion injury. Vis. Res. 39, 2519–2535 (1999).
Braas, K. M., Zarbin, M. A. & Snyder, S. H. Endogenous adenosine and adenosine receptors localized to ganglion cells of the retina. Proc. Natl. Acad. Sci. USA 84, 3906–3910 (1987).
Soliño, M. et al. Adenosine A1 receptor: a neuroprotective target in light induced retinal degeneration. PLoS ONE 13, e0198838 (2018).
Winerdal, M. et al. Adenosine A1 receptors contribute to immune regulation after neonatal hypoxic ischemic brain injury. Purinergic Signal. 12, 89–101 (2016).
Olsson, T. et al. Deletion of the adenosine A1 receptor gene does not alter neuronal damage following ischaemia in vivo or in vitro. Eur. J. Neurosci. 20, 1197–1204 (2004).
Tauskela, J. S. et al. Elevated synaptic activity preconditions neurons against an in vitro model of ischemia. J. Biol. Chem. 283, 34667–34676 (2008).
Brown, G. C. Cell death by phagocytosis. Nat. Rev. Immunol. 24, 91–102 (2024).
Xu, T. et al. The roles of microglia and astrocytes in myelin phagocytosis in the central nervous system. J. Cereb. Blood Flow. Metab. 43, 325–340 (2023).
Madeira, M. H. et al. Selective A2A receptor antagonist prevents microglia-mediated neuroinflammation and protects retinal ganglion cells from high intraocular pressure-induced transient ischemic injury. Transl. Res. 169, 112–128 (2016).
Madeira, M. H. et al. Adenosine A2AR blockade prevents neuroinflammation-induced death of retinal ganglion cells caused by elevated pressure. J. Neuroinflammation 12, 115 (2015).
Boia, R. et al. Treatment with A(2A) receptor antagonist KW6002 and caffeine intake regulate microglia reactivity and protect retina against transient ischemic damage. Cell Death Dis. 8, e3065 (2017).
Solino, M. et al. Adenosine A2A receptor: a new neuroprotective target in light-induced retinal degeneration. Front. Pharm. 13, 840134 (2022).
Roth, S. et al. Preconditioning provides complete protection against retinal ischemic injury in rats. Investig. Ophthalmol. Vis. Sci. 39, 777–785 (1998).
Schneider, M., Tzanou, A., Uran, C. & Vinck, M. Cell-type-specific propagation of visual flicker. Cell Rep. 42, 112492 (2023).
Hu, H. et al. Noninvasive light flicker stimulation promotes optic nerve regeneration by activating microglia and enhancing neural plasticity in zebrafish. Investig. Ophthalmol. Vis. Sci. 65, 3 (2024).
Seydyousefi, M. et al. Exogenous adenosine facilitates neuroprotection and functional recovery following cerebral ischemia in rats. Brain Res. Bull. 153, 250–256 (2019).
Héron, A. et al. Effects of an A1 adenosine receptor agonist on the neurochemical, behavioral and histological consequences of ischemia. Brain Res. 641, 217–224 (1994).
Von Lubitz, D. K. et al. Chronic administration of selective adenosine A1 receptor agonist or antagonist in cerebral ischemia. Eur. J. Pharm. 256, 161–167 (1994).
Agarwal, P. & Agarwal, R. Tackling retinal ganglion cell apoptosis in glaucoma: role of adenosine receptors. Expert Opin. Ther. Targets 25, 585–596 (2021).
Matherne, G. P., Linden, J., Byford, A. M., Gauthier, N. S. & Headrick, J. P. Transgenic A1 adenosine receptor overexpression increases myocardial resistance to ischemia. Proc. Natl. Acad. Sci. USA 94, 6541–6546 (1997).
Melani, A. et al. Ecto-ATPase inhibition: ATP and adenosine release under physiological and ischemic in vivo conditions in the rat striatum. Exp. Neurol. 233, 193–204 (2012).
Rudolphi, K. A. & Schubert, P. Modulation of neuronal and glial cell function by adenosine and neuroprotection in vascular dementia. Behav. Brain Res. 83, 123–128 (1997).
Coelho, J. E. et al. Hypoxia-induced desensitization and internalization of adenosine A1 receptors in the rat hippocampus. Neuroscience 138, 1195–1203 (2006).
Hamil, N. E., Cock, H. R. & Walker, M. C. Acute down-regulation of adenosine A(1) receptor activity in status epilepticus. Epilepsia 53, 177–188 (2012).
Ho, J. K., Stanford, M. P., Shariati, M. A., Dalal, R. & Liao, Y. J. Optical coherence tomography study of experimental anterior ischemic optic neuropathy and histologic confirmation. Investig. Ophthalmol. Vis. Sci. 54, 5981–5988 (2013).
Guo, Y., Mehrabian, Z. & Bernstein, S. L. The rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION). J. Vis. Exp. 117, 54504 (2016).
Acknowledgements
This work was supported by Science & Technology Initiative STI2030-Major Project Grant No.2021ZD0203400 (to J.-F.C.), Key project of National Natural Science Foundation of China Grant No.82430045 (to J.-F.C.), National Natural Science Foundation of China Grant No.81600991 (to Y.G.), Scientific Research Starting Foundation of Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health) Grant No. OJQDSP2022007 (to J.-F.C.), Scientific Research Starting Foundation of Wenzhou Medical University Grant No. QTJ12003 (to J.-F.C.).
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L.S., J.C., and Y.G. conceived the study and designed the experiments. J.C. and Y.G. provided financial support and supervised the work. L.S. established the NAION models. L.S. and M.J. performed UPLC experiments. L.S., R.L., L.-J. H., L.-B. H., Y.W., and X.H. performed OCT, VEP and retinal fundus experiments. L.S., R.L., and T.S. performed immunofluorescence staining and microscopy imaging. L.-J.H. and J.L. performed Western blot analyses. L.S., L.-J. H., M.J., J.L., Y.W., Y.H., and Z.S. analyzed the data and prepared the figures. L.S., J.C., and Y.G. drafted the manuscript. All authors contributed to manuscript revision and approved the final version.
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Su, L., Lu, R., Huang, L. et al. 40 Hz flicker preconditioning protects nonarteritic anterior ischemic optic neuropathy via adenosine signaling. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09591-1
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DOI: https://doi.org/10.1038/s42003-026-09591-1
