Evaluation of Large Organic Anion Transport at the Blood‐CSF Barrier by Quantitative Fluorescence Microscopy

Abstract

The blood-cerebrospinal fluid barrier (BCSFB), formed by the epithelial cells of the choroid plexus, expresses several multispecific membrane transporters that actively remove drugs, toxins, and metabolic wastes from the brain. Transporter-mediated clearance by the BCSFB typically occurs as a two-step process, where uptake transporters on the apical membrane extracts drug from the CSF into the choroid plexus cells, and efflux transporters on the basolateral membrane extrude intracellular drug into the blood. These transporters can be determinants of local cerebrospinal fluid (CSF) drug concentrations, impacting drug efficacy, local toxicity, or drug interaction predictions within the CNS. However, transporter activity at the BCSFB is understudied and remains poorly characterized. The objective of this study is to develop and utilize a quantitative fluorescent microscopy approach to evaluate organic anion transport mediated by organic anion transporting peptide (Oatp) at the murine BCSFB. Confocal imaging was used to observe time-dependent uptake of several large organic anion fluorescent probes in live choroid plexus tissue isolated from adult C57BL/6 mice. Image segmentation was performed using ImageJ to individually quantify intracellular choroid plexus cell and blood capillary accumulation. The fluorescent probes fluorescein-methotrexate (FL-MTX), fluorescein-cyclicAMP (Fluo-cAMP), and sulforhodamine101 (SR101) all demonstrated rapid transepithelial transport across the BCSFB from the CSF into the blood, reaching a peak accumulation within 15 minutes. Choroid plexus cell accumulation remains minimal, suggesting apical uptake as the rate limiting step for transepithelial BCSFB transport of large organic anions. FL-MTX, Fluo-cAMP, and SR101 accumulation were abolished by rifampin or bromosulfophthalein inhibition. Both rifampin and bromosulfophthalein are classical OATP/Oatp inhibitors, indicating functional contribution of OATP/Oatps for apical uptake at the BCSFB. MK571, a potent MRP inhibitor, increased choroid plexus cell accumulation and reduced blood capillary accumulation, consistent with MRP function at the basolateral membrane. This work suggests that Oatps at the murine BCSFB are capable of actively and rapidly removing a range of large organic anion compounds from the CSF, and that apical uptake is the rate limiting step for transport of large organic anions across the BCSFB.