Pharmacological AQP4 inhibition attenuates cerebral ischemia–reperfusion injury and modulates oxidative stress- and cell death-related signaling in mice

Cerebral ischemia–reperfusion (I/R) injury involves oxidative stress and changes in regulated cell death (RCD) pathways. Aquaporin-4 (AQP4), an astrocytic water channel, contributes to water homeostasis and has been implicated in edema, but whether its pharmacological inhibition alters injury-associated responses remains unclear. Male C57BL/6J mice underwent transient middle cerebral artery occlusion (tMCAO) and received AER-271, TGN-020, or vehicle. Neurological deficits, infarct volume, edema, protein expression, and subcellular localization were assessed using markers related to apoptosis (Bax, Bcl-2, cleaved caspase-3), necroptosis and pyroptosis (MLKL, GSDMD), autophagy (LC3-II, p62), ferroptosis-related defense (GPX4), and redox responses (HIF-1α, SOD1). I/R was accompanied by neurological impairment, infarction, edema, and changes in these markers, including increased Bax, cleaved caspase-3, MLKL, GSDMD, and HIF-1α; reduced Bcl-2, GPX4, and SOD1; and time-dependent changes in autophagy-related markers, with early LC3-II elevation followed by p62 accumulation. AER-271 treatment was associated with improved neurological outcomes and reduced infarct volume and edema. It was also associated with lower levels of apoptotic and lytic cell-death markers, altered autophagy-related marker profiles, normalization of redox-related markers, and reduced MLKL/GSDMD membrane redistribution. Similar changes were observed with TGN-020, although dosing regimens differed, potency was not assessed, and lack of a TGN-020-matched vehicle control limits definitive interpretation of these exploratory findings. These protein-level findings are correlative and do not establish causality. PANoptosome assembly, reactive oxygen species, lipid peroxidation, iron metabolism, autophagic flux, and perivascular AQP4 polarization were not assessed. Pharmacological AQP4 inhibition was associated with reduced I/R injury and changes in oxidative stress- and cell-death-related markers. These findings identify AQP4 inhibition as a candidate for further investigation, while causal links among AQP4, redox responses, autophagy and regulated cell death require direct validation in stroke.