Freshly isolated retinal capillaries to determine efflux transporter function at the inner BRB

Lipopolysaccharide-Induced Functional Alteration of P-glycoprotein in the Ex Vivo Rat Inner Blood–Retinal Barrier

“…In the present study, alteration of P-gp transport activity at the inner BRB by LPS treatment was assessed using freshly prepared rat retinal capillaries with NBD-CSA [ 7 ]. Our previous study showed that the luminal accumulation of the NBD-CSA in the retinal capillaries was decreased by 43% in the presence of 100 µM quinidine, a well-known substrate of P-gp with a Km of 5.4 µM [ 7 , 34 ], indicating that luminal NBD-CSA accumulation in the retinal capillaries is an index of P-gp activities at the rat inner BRB. Under LPS treatment at 1 ng/mL LPS ( Figure 1 ), the NBD-CSA accumulation in the lumens of the retinal capillaries was significantly attenuated by 47%.…”

“…L-NMMA was purchased from Dojindo (Kumamoto, Japan). NBD-CSA was synthesized following our previous report by Tajima et al [ 7 ]. All other reagents were commercially obtained.…”

“…Retinal capillaries were freshly isolated by density gradient centrifugation and serial filtration with nylon-meshes from the rat retinae as described previously [ 7 ]. The prepared retinal capillaries were suspended on ice with the isolation buffer, which consisted of 137 mM NaCl, 10 mM D-glucose, 8.1 mM Na 2 HPO 4 , 2.7 mM KCl, 2.0 mM sodium pyruvate, 1.8 mM CaCl 2 , 1.5 mM KH 2 PO 4 , 0.65 mM MgCl 2 , and 1% BSA at pH 7.4.…”

“…In bovine retinal endothelial cells, it is suggested that intracellular accumulation of the other substrate of P-gp, taxol, is markedly increased by P-gp-4008, a P-gp-selective antagonist [ 6 ]. Furthermore, Tajima et al have demonstrated the ex vivo retina-to-blood transport mediated by P-gp using freshly prepared rat retinal capillaries with a fluorescent P-gp substrate, 7-nitro-2,1,3-benzoxadiazole-cyclosporin A (NBD-CSA) [ 7 ]. These cumulative lines of evidence show the importance of P-gp at the inner BRB to understand the retinal substance distribution.…”

“…The purpose of the present study was to clarify the mechanisms of altering P-gp activity at the inner BRB by LPS. We previously established a confocal imaging-based ex vivo assay to evaluate P-gp activity at the inner BRB using freshly isolated retinal capillaries from rats [ 7 ]. By using this method, we examined the P-gp activity at the inner BRB under LPS-treated conditions.…”