2.3. In Situ Transcardiac Brain Perfusion to Assess BBB Transport

The transport of probe drugs across the BBB was assessed using an in situ transcardiac brain perfusion technique as previously described [24]. Surgical anesthesia of mice (n = 4–8 per genotype) was established using ketamine (133 mg/kg) and xylazine (10 mg/kg), and the mice were kept on a warm surgery pad throughout anesthesia. The thoracic cavity of the mice was cut open to expose the heart, the descending aorta was clamped to restrict blood flow to the peripheral organs, and both jugular veins were severed. Immediately thereafter, warm (37 °C) Krebs-ringer bicarbonate buffer containing the radioactive probe (0.05 µCi/mL) was injected into the left ventricle of the heart and maintained at a rate of 10 mL/min for 1 min controlled by a Harvard infusion pump (Harvard Apparatus, Holliston, MA, USA). After 1 min, the perfusion was stopped, and the brain was harvested and digested in 2 mL of Solvable™ at 70 °C for 6 h. A 200 µL aliquot of hydrogen peroxide (30% v/v) was added to neutralize the color. Ultima Gold scintillation cocktail (10 mL) was added to the brain sample and 2 mL of Ultima Gold scintillation cocktail was added to 100 µL of the radioactive perfusion fluid. Radioactivity in each sample was counted in a PerkinElmer 2800TR liquid scintillation analyzer (Waltham, MA, USA). The apparent brain distribution volume of the probe compound (brain:perfusate, mL/g) was calculated as previously described [24,25]. The vascular volume was quantified using brain:perfusate ratio of ¹⁴C-sucrose in C9 and their WT littermates, which was used for vascular volume subtraction for the calculation of apparent brain distribution volume of all other probes.