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.

Diffusion of Epidermal Growth Factor in Rat Brain Extracellular Space Measured by Integrative Optical Imaging

“…Neocortical slices were prepared from 145-to 180-g female Sprague-Dawley rats (postnatal day 40 -50; Taconic) as described previously (Nicholson and Tao 1993;Rice and Nicholson 1991). Animals were anesthetized with sodium pentobarbital (60 mg/kg ip) and decapitated by guillotine.…”

“…The OG514-EGF (6,600 M r ) was reconstituted to a final concentration of 4 g/ml (610 nM) with 90 g/ml unlabeled EGF (catalog E1257, Sigma) in phosphate buffered saline containing 0.1% BSA. Texas Red-labeled 3,000 M r dextran (TR-dex3; catalog D-3329, Molecular Probes) was used at a concentration of 1 mM in 154 mM NaCl, as described previously (Nicholson and Tao 1993). An additional 1 mM TR-dex3 solution containing 100 g/ml unlabeled EGF (catalog E1257, Sigma) was prepared to assess the influence of EGF on ECS structure.…”

“…Diffusion governs the movement of molecules through brain extracellular space (ECS) and determines, along with clearance (enzymatic breakdown or efflux across the blood-brain barrier) and cellular uptake, the distribution of molecules after their release into the ECS (Nicholson 2001;Nicholson and Syková 1998). Although the diffusion coefficients of small ions (Cserr et al 1991;Kume-Kick et al 2002;Lehmenkühler et al 1993;Nicholson and Phillips 1981;Pérez-Pinzón et al 1995) and macromolecules such as dextrans (Nicholson and Tao 1993) and albumins (Tao and Nicholson 1996) have been determined in brain tissue, there is no information about the diffusion characteristics of neurotrophic factors other than NGF (Stroh et al 2003).…”

“…The hindrance experienced by molecules as they travel through the porous brain ECS and encounter obstructions is described by , a dimensionless parameter formally defined as ϭ (D/D*) 1/2 where D is the diffusion coefficient of the molecule in a free medium (water or dilute agarose) and D* is the effective diffusion coefficient of the same molecule in brain (Nicholson 2001). Whereas ␣ is a property of the tissue, is sensitive to tissue properties such as geometry (Chen and Nicholson 2000;Hrabětová et al 2003;Kume-Kick et al 2002;Tao 1999) and extracellular matrix composition (Rusakov and Kullmann 1998;Vargová et al 2003;Voříšek et al 2002), as well as to binding (Berk et al 1997) and the physicochemical properties (size, shape, and charge) of the diffusing molecule (Nicholson and Tao 1993;Prokopová-Kubinová et al 2001;Tao and Nicholson 1996). In theory, diffusion analysis using appropriate small markers can isolate the effects of tissue properties on , because the influences of binding and the physicochemical properties of the diffusing molecule are negligible.…”

“…Accordingly, measurements with small ions such as tetramethylammonium have yielded ϳ 1.6 in most brain regions, both in vivo (Cserr et al 1991;Lehmenkühler et al 1993;Mazel et al 1998;Nicholson and Phillips 1981;Roitbak and Syková 1999) and in the rat brain slice preparation (Cragg et al 2001;Hrabětová and Nicholson 2000;Hrabětová et al 2002;Kume-Kick et al 2002;Pérez-Pinzón et al 1995;Rice and Nicholson 1991). Studies with dextran macromolecules have shown increases from about 1.8 for 3,000 and 10,000 M r dextrans to about 2.2 for 40,000 and 70,000 M r dextrans (Nicholson and Tao 1993), suggesting globular molecules experience an increase in hindrance above a critical size. To date, measurements with protein macromolecules have shown to be in the range of 2.1-2.5 (Stroh et al 2003;Tao and Nicholson 1996), although these studies have been limited to three albumin species (14,500 -66,000 M r ) and NGF (26,500 M r ).…”