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

Abstract

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.

Quantification of transient increase of the blood–brain barrier permeability to macromolecules by optimized focused ultrasound combined with microbubbles

“…Therefore, the BBB disruption assessed by the MRI and histological examination is more qualitative than quantitative. 23,24 To overcome aforementioned limitations, two-photon microscopy can be employed to quantitatively access the enhanced permeability of the cerebral vasculature after FUS. 25 In addition to micrometer spatial resolution, two-photon microscopy offers the advantage of deep tissue penetration, which is essential for the BBB permeability measurement.…”

“…The objective of the current study was to use our newly developed multiphoton microscopy 23 to more accurately assess the FUS-induced BBB permeability change in vivo. To accomplish this aim, MBs were generated and selected for the optimal size in inducing reversible BBB disruptions.…”

“…23,24 Briefly, the skull in the region of interest (ROI) was exposed by shaving off the hair and cutting away the skin and connective tissue. A section of the frontoparietal bone (either left or right) was carefully ground with a high speed micro-grinder (0-50,000 rpm, DLT 50KBU; Brasseler USA, Savannah, GA, USA) until a part of it (~4 mm ×6 mm) became soft and translucent.…”

“…The syringe was replaced by the one filled with TRITC-dextran-155 kDa solution (tracer) ( Figure 3B). The tracer solution was then introduced into the cerebral circulation via the carotid artery at a constant flow rate of ~3 mL/min 23,36 for 1 minute in an interval of 5 minutes. Simultaneously, the images of the ROI containing the microvessels and the surrounding brain tissue were captured for 1 minute at 5-minute intervals up to 25 minutes.…”

“…The images of 239 μm ×239 μm experimental protocol Figure 3C summarizes the experimental protocol for FUS effects on the BBB solute permeability. The MBs at a concentration of ~1.7×10 6 bubbles/mL were injected via rat carotid artery at a constant speed of ~3 mL/min, the normal blood flow velocity at the carotid artery; after 10-15 seconds, the MBs reach the microvascular bed ~100-200 μm below the pia mater, 23 and the FUS sonication was administrated for 1 minute. After sonication, the rat head was mounted into the multiphoton microscope, and the ROI with cerebral microvessels was found.…”