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Diffusion of Flexible Random-Coil Dextran Polymers Measured in Anisotropic Brain Extracellular Space by Integrative Optical Imaging

Fanrong Xiao ^*, Charles Nicholson , Jan Hrabe  ^* and Sabina Hrabtová ^* 

^* Department of Anatomy and Cell Biology, The Robert F. Furchgott Center for Neural and Behavioral Science, State University of New York, Downstate Medical Center, Brooklyn, New York; Department of Physiology and Neuroscience, New York University School of Medicine, New York, New York; and Center for Advanced Brain Imaging, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, New York

Correspondence: Address reprint requests to Dr. Sabina Hrabtová, Tel.: 718-221-5392; E-mail: sabina.hrabetova{at}downstate.edu.

There are a limited number of methods available to quantify the extracellular diffusion of macromolecules in an anisotropic brain region, e.g., an area containing numerous aligned fibers where diffusion is faster along the fibers than across. We applied the integrative optical imaging method to measure diffusion of the fluorophore Alexa Fluor 488 (molecular weight (MW) 547) and fluorophore-labeled flexible random-coil dextran polymers (dex3, MW 3000; dex75, MW 75,000; dex282, MW 282,000; dex525, MW 525,000) in the extracellular space (ECS) of the anisotropic molecular layer of the isolated turtle cerebellum. For all molecules, two-dimensional images acquired an elliptical shape with major and minor axes oriented along and across, respectively, the unmyelinated parallel fibers. The effective diffusion coefficients, D*_major and D*_minor, decreased with molecular size. The diffusion anisotropy ratio (DAR = D*_major/D*_minor) increased for Alexa Fluor 488 through dex75 but then unexpectedly reached a plateau. We argue that dex282 and dex525 approach the ECS width and deform to diffuse. In support of this concept, scaling theory shows the diffusion behavior of dex282 and dex525 to be consistent with transition to a reptation regime, and estimates the average ECS width at 31 nm. These findings have implications for the interstitial transport of molecules and drugs, and for modeling neurotransmitter diffusion during ectopic release and spillover.