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

Abstract

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 approximately 31 nm. These findings have implications for the interstitial transport of molecules and drugs, and for modeling neurotransmitter diffusion during ectopic release and spillover.

Relative vascular permeability and vascularity across different regions of the rat nasal mucosa: implications for nasal physiology and drug delivery

“…The D value we measured for TR-BSA (Table 1) agreed well with protein correlation predictions for monomeric BSA (8.6–9.2 × 10 −7  cm 2 /s)30 as well as previous experimental measurements31. The experimental D and d H for TR-Dex70 (as well as the other two dextrans) were also well in line with prior estimates using nonlysine fixable dextrans28 as well as correlation predictions32. Taken together, the results confirmed the stability and monodisperse nature of all four tracers, yielding a 2.7–12.4 nm range of tracer d H to be used for nasal vascular permeability measurements (Table 1).…”

Probing the extracellular diffusion of antibodies in brain using in vivo integrative optical imaging and ex vivo fluorescence imaging

“…More flexibility in the IgG hinge region may allow certain IgGs to diffuse slightly better through the narrow brain ECS than expected based on the length of their hydrodynamic axes, e.g. studies with significantly larger dextrans have shown that conformational flexibility allows random coil polymers larger than the ECS width to extracellularly diffuse by processes such as reptation[44]. Another possible contributing factor to our AF488-IgG’s high λ value may be related to charge effects.…”

Diffusion in Brain Extracellular Space

“…The IOI method has been extended to encompass regions of anisotropic grey matter (417). Following an earlier study with the RTI-TMA method (314) (Section IV.A.5), the IOI method was used to make measurements in the ECS of the anisotropic molecular layer of the isolated turtle cerebellum using the small fluorophore Alexa Fluor 488 (AF, 0.547 kDa) and fluorophore-labeled dextrans, with molecular mass of 3, 75, 282 and 525 kDa (417) (Table 3).…”