The contributions of aquaporin‐4 to water exchange across the blood–brain barrier measured by filter‐exchange imaging

Purpose Water exchange across the blood–brain barrier ( WEX BBB ) is a promising biomarker for assessing the blood–brain barrier ( BBB) integrity. However, the physiological mechanisms governing WEX BBB remain unclear. This study was conducted to investigate the contribution of Na + /K + ‐ ATPase ( NKA ) on the luminal side of endothelial cells and aquaporin‐4 ( AQP4 ) to WEX BBB . Methods WEX BBB was measured using filter‐exchange imaging for BBB assessment ( FEXI ‐ BBB ) on rats, and data were fitted using an adapted two‐compartment crusher‐compensated exchange rate ( CCXR ) model. Test–retest reliability of the vascular water efflux rate constant ( k bo ) was assessed. Ouabain and 2‐(nicotinamide)‐1,3,4‐thiadiazole ( TGN ‐020) were administered to inhibit NKA on the luminal side of endothelial cells and AQP4 , respectively, to investigate their roles in WEX BBB measured by FEXI ‐ BBB . Results Fixing intravascular diffusivity in the two‐compartment CCXR model significantly improved estimation accuracy and precision of k bo and other parameters. The test–retest experiment demonstrated that this method had good reproducibility in measuring k bo (intraclass correlation coefficient = 0.79). Administering TGN ‐020, which inhibits AQP4 , significantly decreased k bo by 32% ( k bo = 3.07 ± 0.81 s −1 vs. 2.09 ± 1.10 s −1 , p < 0.05). However, the ouabain‐treated group showed no significant change in k bo compared with that of the control group (2.51 ± 0.58 s −1 vs. 2.37 ± 1.02 s −1 , p = 0.73) in the NKA inhibition experiment. Conclusions WEX BBB decreased by 32% after administering TGN ‐020, but no downward trend was noted after administering ouabain. Our findings indicate that AQP4 expression/function, but not NKA activity on the luminal side of endothelial cells, plays a significant role in regulating WEX BBB .