40 Hz flicker preconditioning protects nonarteritic anterior ischemic optic neuropathy via adenosine signaling

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.