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Research Papers

Quantification of Blood-Brain Barrier Solute Permeability and Brain Transport by Multiphoton Microscopy

[+] Author and Article Information
Lingyan Shi, Min Zeng

Department of Biomedical Engineering,
The City College of the City
University of New York,
160 Convent Avenue,
New York, NY 10031

Yi Sun

Department of Electrical Engineering,
The City College of the City
University of New York,
160 Convent Avenue,
New York, NY 10031

Bingmei M. Fu

Department of Biomedical Engineering,
The City College of the City
University of New York,
160 Convent Avenue,
New York, NY 10031
e-mail: fu@ccny.cuny.edu

1Corresponding author.

Contributed by the Bioengineering Division of ASME for publication in the Journal of Biomechanical Engineering. Manuscript received May 24, 2013; final manuscript received September 23, 2013; accepted manuscript posted November 5, 2013; published online February 13, 2014. Assoc. Editor: Mohammad Mofrad.

J Biomech Eng 136(3), 031005 (Feb 13, 2014) (9 pages) Paper No: BIO-13-1240; doi: 10.1115/1.4025892 History: Received May 24, 2013; Revised September 23, 2013; Accepted November 05, 2013

Development of an optimal systemic drug delivery strategy to the brain will require noninvasive or minimally invasive methods to quantify the permeability of the cerebral microvessel wall or blood-brain barrier (BBB) to various therapeutic agents and to measure their transport in the brain tissue. To address this problem, we used laser-scanning multiphoton microscopy to determine BBB permeability to solutes (P) and effective solute diffusion coefficients (Deff) in rat brain tissue 100–250 μm below the pia mater. The cerebral microcirculation was observed through a section of frontoparietal bone thinned with a microgrinder. Sodium fluorescein, fluorescein isothiocyanate (FITC)-dextrans, or Alexa Fluor 488-immunoglobulin G (IgG) in 1% bovine serum albumin (BSA) mammalian Ringer's solution was injected into the cerebral circulation via the ipsilateral carotid artery by a syringe pump at a constant rate of ∼3 ml/min. P and Deff were determined from the rate of tissue solute accumulation and the radial concentration gradient around individual microvessels in the brain tissue. The mean apparent permeability P values for sodium fluorescein (molecular weight (MW) 376 Da), dextran-4k, -20k, -40k, -70k, and IgG (MW ∼160 kDa) were 14.6, 6.2, 1.8, 1.4, 1.3, and 0.54 × 10−7 cm/s, respectively. These P values were not significantly different from those of rat pial microvessels for the same-sized solutes (Yuan et al., 2009, “Non-Invasive Measurement of Solute Permeability in Cerebral Microvessels of the Rat,” Microvasc. Res., 77(2), pp. 166–73), except for the small solute sodium fluorescein, suggesting that pial microvessels can be a good model for studying BBB transport of relatively large solutes. The mean Deff values were 33.2, 4.4, 1.3, 0.89, 0.59, and 0.47 × 10−7 cm2/s, respectively, for sodium fluorescein, dextran-4k, -20k, -40k, -70k, and IgG. The corresponding mean ratio of Deff to the free diffusion coefficient Dfree, Deff/Dfree, were 0.46, 0.19, 0.12, 0.12, 0.11, and 0.11 for these solutes. While there is a significant difference in Deff/Dfree between small (e.g., sodium fluorescein) and larger solutes, there is no significant difference in Deff/Dfree between solutes with molecular weights from 20,000 to 160,000 Da, suggesting that the relative resistance of the brain tissue to macromolecular solutes is similar over a wide size range. The quantitative transport parameters measured from this study can be used to develop better strategies for brain drug delivery.

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Figures

Grahic Jump Location
Fig. 1

Illustration of the scanning region and the orientation of a microvessel in the brain tissue using multiphoton microscopy to determine the blood-brain barrier solute permeability and brain-tissue diffusion coefficient. x-z is the cross-sectional plane of the microvessel and y is the axial direction of the vessel. The ROI (scanning region) has a size of ∼200 μm × 8 μm × 100 μm (x, y, z).

Grahic Jump Location
Fig. 2

In vitro calibration experiments to determine the linear range of fluorescence intensity versus concentration under the same instrumental settings in the in vivo experiments. (a) Sodium fluorescein, (b) FITC-dextran-20k, and (c) IgG.

Grahic Jump Location
Fig. 3

Determination of microvessel solute permeability P using multiphoton microscopy in rat brain. (a) A cross-sectional image (x-z) showing the fluorescence dye (dex-20k) filling the microvessel lumen and spreading into the surrounding tissue. The ROI for determining P is enclosed by the dashed line. Its circumference was chosen as 10–20 μm from the vessel perimeter to avoid contamination from the adjacent vessels. (b) Total fluorescence intensity in the ROI (a) as a function of perfusion time. Fluorescence intensity in this figure is proportional to the total mass of solute accumulated in the measuring region surrounding the microvessel. The slope of regression line over the initial linear accumulation is used for the estimation of permeability P = 1/I0*(dI/dt)0* r/2. r is the radius of the vessel.

Grahic Jump Location
Fig. 4

Determination of effective solute diffusion coefficient Deff in rat brain tissue. (a) Eight straight lines were drawn from the center of the vessel lumen. The averaged fluorescence intensity along these eight directions is plotted from the vessel wall to ∼20 μm far from the wall at different times, the green lines shown in (b). Exp 25 sec, exp 50 sec, and exp 75 sec are the measured intensity profiles from the experiments at 25 s, 50 s, and 75 s, respectively. The black lines are the best-fitting model predictions when the proper Deff is chosen. The best-fitting value of Deff/Dfree is 0.145 in this run of experiments for dex-20k. Model 25 sec, model 50 sec, and model 75 sec are the predicted intensity profiles by the theoretical model at 25 s, 50 s, and 75 s, respectively.

Grahic Jump Location
Fig. 5

Apparent permeability P as a function of solute Stokes radius. P of individual microvessels (○) and mean values ± SD (▪) are plotted.

Grahic Jump Location
Fig. 6

Comparison of solute diffusive permeability Pd for rat cerebral microvessels (▪ current study) and that for rat pial microvessels (◊ [27]) and (○ [10]). Values are mean ± SD (*p = 0.016).

Grahic Jump Location
Fig. 7

Effective solute diffusion coefficient Deff plotted as a function of solute Stokes radius. Deff measured from the current in vivo study (▪) are also compared with those from previous in vivo and ex vivo studies. Values are mean ± SD.

Grahic Jump Location
Fig. 8

Comparison of the original (top left) and corrected (top right) images for the wavefront distortion and scattering and the effect of the image distortions on determining the solute brain tissue diffusion coefficient Deff (bottom). In the bottom plot, the green lines are the curves of the averaged intensity over eight directions versus time (see Fig. 4(b)) from the original images, the red lines are from the corrected images, and the black lines are the model predictions with the best-fitting Deff/Dfree. Model 25 sec, model 50 sec, and model 75 sec are the predicted intensity profiles by the theoretical model at 25 s, 50 s, and 75 s, respectively. Exp 25 sec and exp 25 sec (corrected), exp 50 sec and exp 50 sec (corrected), and exp 75 sec and exp 75 sec (corrected) are the intensity profiles determined out of the original and corrected images from the experiments at 25 s, 50 s, and 75 s, respectively.

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