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

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 (Dx, Dy) 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 70kDa 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.