title: Hindered diffusion of high molecular weight compounds in brain extracellular microenvironment measured with integrative optical imaging.
abstract: This paper describes the theory of an integrative optical imaging system and its application to the analysis of the diffusion of 3-, 10-, 40-, and 70-kDa fluorescent dextran molecules in agarose gel and brain extracellular microenvironment. The method uses a precisely defined source of fluorescent molecules pressure ejected from a micropipette, and a detailed theory of the intensity contributions from out-of-focus molecules in a three-dimensional medium to a two-dimensional image. Dextrans tagged with either tetramethylrhodamine or Texas Red were ejected into 0.3% agarose gel or rat cortical slices maintained in a perfused chamber at 34 degrees C and imaged using a compound epifluorescent microscope with a 10 x water-immersion objective. About 20 images were taken at 2-10-s intervals, recorded with a cooled CCD camera, then transferred to a 486 PC for quantitative analysis. The diffusion coefficient in agarose gel, D, and the apparent diffusion coefficient, D*, in brain tissue were determined by fitting an integral expression relating the measured two-dimensional image intensity to the theoretical three-dimensional dextran concentration. The measurements in dilute agarose gel provided a reference value of D and validated the method. Values of the tortuosity, lambda = (D/D*)1/2, for the 3- and 10-kDa dextrans were 1.70 and 1.63, respectively, which were consistent with previous values derived from tetramethylammonium measurements in cortex. Tortuosities for the 40- and 70-kDa dextrans had significantly larger values of 2.16 and 2.25, respectively. This suggests that the extracellular space may have local constrictions that hinder the diffusion of molecules above a critical size that lies in the range of many neurotrophic compounds.
authors.affiliation: Department of Physiology and Biophysics, New York University Medical Center, New York 10016.
authors.forename: C
authors.initials: C
authors.lastname: Nicholson
authors.forename: L
authors.initials: L
authors.lastname: Tao
mesh_terms: D000818:Animals; D001704:Biopolymers / Q000493:pharmacokinetics*; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D003911:Dextrans / Q000493:pharmacokinetics*; D004058:Diffusion; D005110:Extracellular Space / Q000378:metabolism*; D005260:Female; D008297:Male; D008433:Mathematics; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008959:Models, Neurological; D008962:Models, Theoretical; D008970:Molecular Weight; D051381:Rats; D017207:Rats, Sprague-Dawley; D012685:Sepharose
chemical_list: D001704:Biopolymers; D003911:Dextrans; D012685:Sepharose