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Evaluation of Blood-CSF Barrier Transport by Quantitative Real Time Fluorescence Microscopy



Transporters at the blood-cerebrospinal fluid (CSF) barrier (BCSFB) play active roles in removing drugs and toxins from the CSF. The goal of this study is to develop a fluorescence microscopy approach to quantitatively study the transepithelial transport processes at the murine BCSFB in real time.


Choroid plexus (CP) tissues were isolated from mouse lateral ventricles and incubated with anionic (fluorescein-methotrexate, 8-fluorescein-cAMP) or cationic (IDT307) fluorescent probes. The CSF-to-blood transport was imaged and quantified using compartmental segmentation and digital image analysis. Real time images were captured and analyzed to obtain kinetic information and identify the rate-limiting step. The effect of transporter inhibitors was also evaluated.


The transport processes of fluorescent probes can be captured and analyzed digitally. The intra- and inter- animal variability were 20.4% and 25.7%, respectively. Real time analysis showed distinct transport kinetics and rate-limiting step for anionic and cationic probes. A CP efflux index was proposed to distinguish between transepithelial flux and intracellular accumulation. Rifampin and MK571 decreased the overall transepithelial transport of anionic probes by more than 90%, indicating a possible involvement of organic anion transporting polypeptides (Oatps) and multidrug resistance-associated proteins (Mrps).


A CP isolation method was described, and a quantitative fluorescence imaging approach was developed to evaluate CSF-to-blood transport in mouse CP. The method is consistent, reproducible, and capable of tracking real time transepithelial transport with temporal and spatial resolution. The approach can be used to evaluate transport mechanisms, assess tissue drug accumulation, and assay potential drug-drug interactions at the BCSFB.

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Data Availability

The datasets generated during the current study are available from the corresponding author upon reasonable request.



Blood-cerebrospinal fluid barrier


Choroid plexus

CPE cells:

Choroid plexus epithelial cells


Choroid plexus efflux index


Cerebrospinal fluid


Differential image contrast


Fluorescein methotrexate


Fluorescein cyclic AMP


Multidrug resistance associated protein


Organic anion transporting polypeptide


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The authors would like to thank Nathaniel Peters at the UW Keck Microscopy Center for his help and insights in confocal microscopy practice.


This work was supported in part by the National Institutes of Health grant R21 AG071827, Faculty Innovation Fund, and the Elmer M. Plein Endowment Research Fund from the School of Pharmacy, University of Washington.

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Original study conception and design was completed by Joanne Wang and Austin Sun. Experiments were performed by Austin Sun, and data analysis was performed by Austin Sun and Joanne Wang. The initial draft of the manuscript was written by Austin Sun and critically revised by Joanne Wang.

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Correspondence to Joanne Wang.

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Sun, A., Wang, J. Evaluation of Blood-CSF Barrier Transport by Quantitative Real Time Fluorescence Microscopy. Pharm Res 39, 1469–1480 (2022).

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  • choroid plexus
  • drug transport
  • quantitative fluorescence microscopy
  • blood-CSF barrier