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Extracellular Space Diffusion in Central Nervous System: Anisotropic Diffusion Measured by Elliptical Surface Photobleaching

Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143

Correspondence: Address reprint requests to Alan S. Verkman, MD, PhD, 1246 Health Sciences East Tower, Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0521. Tel.: 415-476-8530; Fax: 415-665-3847; E-mail: verkman{at}itsa.ucsf.edu; http://www.ucsf.edu/verklab.

Diffusion in the extracellular space (ECS) is crucial for normal central nervous system physiology. The determinants of ECS diffusion include viscous interactions with extracellular matrix/plasma membranes ("viscosity") and ECS geometry ("tortuosity"). To resolve viscosity versus tortuosity effects, we measured direction-dependent (anisotropic) diffusion in ECS in mouse spinal cord by photobleaching using an elliptical spot produced by a cylindrical lens in the excitation path. Anisotropic diffusion slowed fluorescence recovery when the long axis of the ellipse was parallel versus perpendicular to the direction of faster diffusion. A mathematical model was constructed to deduce diffusion coefficients (D_x, D_y) from fluorescence recovery measured for parallel and perpendicular orientations of the long axis of the ellipse. Elliptical spot photobleaching was validated by photobleaching aqueous-phase fluorophores on a diffraction grating, where diffusion is one-dimensional. Measurement of the diffusion of 70 kDa FITC-dextran in spinal cord in living mice indicated that viscosity slows diffusion by 1.8-fold compared with its diffusion in solution. ECS geometry hinders diffusion across (but not along) axonal fibers in spinal cord further by approximately fivefold. In cerebral cortex, however, 50% of the hindrance to ECS diffusion comes from viscosity and 50% from tortuosity. We suggest that the extracellular matrix might have evolved to facilitate rather than hinder diffusion even for large molecules.

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