In situ brain perfusion

Animals were anesthetized (100 mg/kg ketamine, 20 mg/kg xylazine, i.p.) and heparinized (10,000 U/kg, i.p.). Body temperature was maintained at 37 °C using a heating pad. The common carotid arteries were cannulated with silicone tubing connected to a perfusion circuit. The perfusate was an erythrocyte-free modified mammalian Ringer’s solution consisting of 117 mM NaCl, 4.7 mM KCl, 0.8 mM MgSO₄, 1.2 mM KH₂PO₄, 2.5 mM CaCl₂, 10 mM d-glucose, 3.9% (w/v) dextran (MW 75,000), and 1.0 g/l bovine serum albumin (type IV), pH 7.4, warmed to 37 °C and continuously oxygenated with 95% O₂/5% CO₂. Evan’s blue dye (55 mg/l) was added to the perfusate to serve as a visual marker of BBB integrity. Perfusion pressure and flow rate were maintained at 95–105 mmHg and 3.1 ml/min, respectively. Both jugular veins were severed to allow for drainage of the perfusate. Using a slow-drive syringe pump (0.5 ml/min per hemisphere; Harvard Apparatus, Holliston, MA), [³H]taurocholate (1.0 μCi/ml; 10 mM total concentration; PerkinElmer, Boston, MA) or [³H]atorvastatin (0.5 μCi/ml; 0.013 μM total concentration; American Radiolabeled Chemicals, St. Louis, MO) was added to the inflowing perfusate. For inhibition studies, animals were perfused with erythrocyte-free modified mammalian Ringer’s solution containing transport inhibitor [i.e., 100 μM estrone-3-sulfate (E3S) or fexofenadine (FEX)] for 10 min prior to perfusion with [³H]taurocholate or [³H]atorvastatin. After perfusion, the rat was decapitated and the brain was removed. Meninges and choroid plexus were excised and cerebral hemispheres were isolated and homogenized. At this time, TS2 tissue solubilizer (1 ml) was added and each sample was allowed to solubilize for 2 days at room temperature. To eliminate chemiluminescence, 100 μl of 30% glacial acetic acid was added, along with 2 ml Optiphase SuperMix liquid scintillation cocktail (PerkinElmer, Boston, MA). Samples were measured for radioactivity on a model 1450 liquid scintillation counter (PerkinElmer).

Results were reported as picomoles of radiolabeled drug per milligram of brain tissue (C; pmol/mg tissue), which is equal to the total amount of radioisotope in the brain (C_Brain; dpm/mg tissue) divided by the amount of radioisotope in the perfusate (C_Perfusate; dpm/pmol): C = C_Brain/C_Perfusate. In a previous study using modified Ringer's solution for perfusion, the brain vascular volume in rats was shown to range between 6 and 9 μl/g brain tissue [36]. Since brain tissue was processed immediately after perfusion with radiolabeled substrate, all uptake values required correction for brain vascular volume. This was accomplished by subtracting the average vascular volume (i.e., 8 μl/g brain tissue as calculated from data reported by Takasato and colleagues) from whole-brain uptake data obtained for [³H]taurocholate or [³H]atorvastatin.