Quantitative fluorescence microscopy provides high resolution imaging of passive diffusion and P-gp mediated efflux at the in vivo blood–brain barrier

Abstract

Quantitative fluorescent microscopy is an emerging technology that has provided significant insight into cellular dye accumulation, organelle function, and tissue physiology. However, historically dyes have only been used to qualitatively or semi-quantitatively (fold change) determine changes in blood–brain barrier (BBB) integrity. Herein, we present a novel method to calculate the blood to brain transfer rates of the dyes rhodamine 123 and Texas red across the in situ BBB. We observed that rhodamine 123 is subject to p-glycoprotein mediated efflux at the rat BBB and can be increased nearly 20-fold with p-glycoprotein inhibition. However, Texas Red appears to not be subject to MRP2 mediated efflux at the rat BBB, agreeing with literature reports suggesting MRP2 may lack functionality at the normal rat BBB. Lastly, we present data demonstrating that once dyes have crossed the BBB, diffusion of the dye molecule is not as instantaneous as has been previously suggested. We propose that future work can now be completed to (1) match BBB transfer coefficients to interstitial diffusion constants and (2) use dyes with specific affinities to cellular organelles or that have specific properties (e.g., subject to efflux transporters) to more fully understand BBB physiology.

Permeability across a novel microfluidic blood-tumor barrier model

“…Rhodamine 123 is subject to P-gp mediated efflux at both the BBB and the BTB. When rhodamine 123 and an inhibitor of P-gp are administered concurrently, dye accumulates in brain ~10- to 12-fold higher than in the absence of efflux inhibition [34]. Similarly, in this work, when Verapamil or Cyclosporine A was added to the outer chamber of the microfluidic device, Rhodamine 123 accumulation increased similar to in vivo reports [9].…”

“…1) in a novel microfluidic BBB and BTB model as validation to previously published literature [34]. Briefly in this model, endothelial cells are seeded in the outer compartments, while astrocytes (BBB) or brain seeding breast cancer cells (BTB) are seeded in the central compartment.…”

“…To determine if P-gp inhibitors alter the accumulation of P-gp sensitive fluorescent dye accumulation into the central compartment we perfused Rho123 in the absence and presence of P-gp inhibitors Cyclosporine A (10 mM), and Verapamil (50 mM)—concentrations that ensured maximal inhibition [34]. We qualitatively observed an increase in dye accumulation in the central compartment over the course of 90 min in both the BBB (Fig.…”

“…Some of the earliest work using dyes dates back to the nineteenth century, where Paul Ehrlich and Edwin Goldmann intravenously injected water-soluble dyes and observed that dyes did not have the ability to freely exchange between the vascular and brain parenchyma compartment (reviewed in [34]). Dyes have also been used as a tool to visualize and qualitatively measure disruption at the BBB [40–44] as well as the BTB [16, 34]. Passive permeability dyes are a simple way to compare rates of diffusion between different models in vivo and in vitro.…”

Physical blood-brain barrier disruption induced by focused ultrasound does not overcome the transporter-mediated efflux of erlotinib

“…Rhodamine-123 (MW = 380,8 g/ mol, LogP = 1.0 [76]) is a fluorescent ABCB1 substrate dye. Using in situ brain perfusion, it was shown that diffusion of rhodamine-123 across the BBB was increased up to 20-fold by ABCB1 inhibition in rats [77]. This showed the ability of Rhodamine-123 to cross the physical BBB and a predominant impact of ABCB1 in restricting the BBB permeation of this compound [76].…”