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.