Austin Sun1, Joanne Wang2. 1. Department of Pharmaceutics, University of Washington, H272 Health Sciences Building, Seattle, WA, 98195-7610, USA. 2. Department of Pharmaceutics, University of Washington, H272 Health Sciences Building, Seattle, WA, 98195-7610, USA. jowang@uw.edu.
Abstract
PURPOSE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.
PURPOSE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.
Authors: Xiaomei Chen; Richard F Keep; Yan Liang; Hao-Jie Zhu; Margareta Hammarlund-Udenaes; Yongjun Hu; David E Smith Journal: Biochem Pharmacol Date: 2017-02-10 Impact factor: 5.858
Authors: Catherine Chiu; Miles C Miller; Ilias N Caralopoulos; Michael S Worden; Thomas Brinker; Zachary N Gordon; Conrad E Johanson; Gerald D Silverberg Journal: Fluids Barriers CNS Date: 2012-01-23