Enlarged extracellular space of aquaporin-4–deficient mice does not enhance diffusion of Alexa Fluor 488 or dextran polymers

Aquaporin-4 (AQP4) water channels expressed on glia have been implicated in maintaining the volume of extracellular space (ECS). A previous diffusion study employing small cation tetramethylammonium and a real-time iontophoretic (RTI) method demonstrated an increase of about 25% in the ECS volume fraction (α) in the neocortex of AQP4^−/− mice compared to AQP4^+/+ mice but no change in the hindrance imposed to diffusing molecules (tortuosity λ). In contrast, other diffusion studies employing large molecules (dextran polymers) and a fluorescence recovery after photobleaching (FRAP) method measured a decrease of about 10%–20% in λ in the neocortex of AQP4^−/− mice. These conflicting findings on λ would imply that large molecules diffuse more readily in the enlarged ECS of AQP4^−/− mice than in wild type but small molecules do not. To test this hypothesis, we used integrative optical imaging (IOI) to measure tortuosity with a small Alexa Fluor 488 (molecular weight MW] 547, λ_AF) and two large dextran polymers (MW 3000, λ_dex3 and MW 75,000, λ_dex75) in the in vitro neocortex of AQP4^+/+ and AQP4^−/− mice. We found that λ_AF=1.59, λ_dex3=1.76 and λ_dex75=2.30 obtained in AQP4^−/− mice were not significantly different from λ_AF=1.61, λ_dex3=1.76, and λ_dex75=2.33 in AQP4^+/+ mice. These IOI results demonstrate that λ measured with small and large molecules each remain unchanged in the enlarged ECS of AQP4^−/− mice compared to values in AQP4^+/+ mice. Further analysis suggests that the FRAP method yields diffusion parameters not directly comparable with those obtained by IOI or RTI methods. Our findings have implications for the role of glial AQP4 in maintaining the ECS structure.