Na⁺-K⁺-2Cl^- cotransport inhibitor attenuates cerebral edema following experimental stroke via the perivascular pool of aquaporin-4

Introduction: The Na⁺-K⁺-2Cl^- cotransporter localized in the brain vascular endothelium has been shown to be important in the evolution of cerebral edema following experimental stroke. Previous in vivo studies have demonstrated that bumetanide, a selective Na ⁺-K⁺-2Cl^- cotransport inhibitor, attenuates ischemia-evoked cerebral edema. Recently, bumetanide has been shown to also inhibit water permeability via aquaporin-4 (AQP4) expressed in Xenopus laevis oocytes. We tested the hypothesis that the perivascular pool of AQP4 plays a significant role in the anti-edema effect of bumetanide by utilizing wild-type (WT) mice as well as mice with targeted disruption of α-syntrophin (α-Syn^-/-) that lack the perivascular pool of AQP4. Methods: Isoflurane-anesthetized adult male WT C57Bl6 and α-Syn^-/- mice were subjected to 90 min middle cerebral artery occlusion (MCAO) followed by 24 or 48 h of reperfusion. Adequacy of MCAO and reperfusion was monitored with laser-Doppler flowmetry over the ipsilateral parietal cortex. Infarct volume (tetrazolium staining), cerebral edema (wet-to-dry ratios), and AQP4 protein expression (immunoblotting) were determined in different treatment groups in separate sets of experiments. Results: Bumetanide significantly attenuated infarct volume and decreased ipsilateral hemispheric water content in WT mice compared to vehicle treatment. In α-Syn^-/- mice, bumetanide treatment had no effect on infarct volume or ischemia-evoked cerebral edema. Bumetanide-treated WT mice had a significant attenuation of AQP4 protein expression at 48 h post-MCAO compared to vehicle-treated WT mice. Conclusions: These data suggest that bumetanide exerts its neuroprotective and anti-edema effects partly via blockade of the perivascular pool of AQP4 and may have therapeutic potential for ischemic stroke in the clinical setting.