In situ Rat Brain Perfusion Model for Delivery of CPT-Glu and Gd-DTPA Across the BBB

All protocols involving the use of animals, including in situ rat brain perfusion studies, were approved by the Institutional Animal Care and Use Committees (IACUC) at the University of Kansas. The in situ rat brain perfusion studies were done on adult male Sprague-Dawley rats weighing 250–350 g following similar methods described in previous studies.¹⁰ Brain delivery of CPT-Glu was done in the presence and absence of 1.0 mM HAV6 peptide. CPT-Glu (0.021 mmol/kg) was perfused in 10 mL of bicarbonate buffer (pH 7.4) containing 4.2 mM KCl, 1.5 mM CaCl₂, 0.9 mM MgCl₂, 128 mM NaCl, 2.4 mM NaH₂PO₄, and 24 mM NaHCO₃. Prior to the experiment, the perfusate was supplemented with D-glucose (6 mM) followed by filtration and oxygenation upon incubation under 95% O₂ and 5% CO₂ at 37°C. The rats were anaesthetized with a combination of ketamine (100 mg/kg) and xylazine (5 mg/kg) delivered intraperitoneally. Then, the left common carotid artery (LCCA) was cannulated with a polyethylene catheter (PE 50) containing heparinized saline (100 IU/mL). The left pteryopalatine, occipital, and superior thyroid arteries were ligated using surgical thread. A heat lamp was used to maintain the animal’s body temperature during the experiment.

Immediately after performing a heart-cut on the anesthetized rat, the LCCA was washed with saline delivered from a syringe pump (Model 341 B, Sage Instruments) at 5 mL/min for 10 sec. The brains were then perfused with 10 mL of 1.0 mM HAV6 in HCO₃⁻ buffer (pH 7.4) with 0.5 % Tween-20 at 5 mL/min. This was followed by perfusion of 10 mL of HAV6 (1.0 mM) and CPT-Glu (0.021 mmol/kg) in HCO₃⁻ buffer (pH 7.4) with 0.5 % Tween-20 at the same flow rate. Finally, a 10-sec post-perfusion wash with saline solution was delivered. The experiment was terminated by animal decapitation followed removal of the brain tissue. The whole brain was immediately stored in −80°C until further use. A similar study was done using only CPT-Glu as a control. The brains were first perfused with 10 mL of HCO₃⁻ buffer (pH 7.4) with 0.5 % Tween-20 at 5 mL/min. Then, 10 mL of CPT-Glu (0.021 mmol/kg) in HCO₃⁻ buffer (pH 7.4) with 0.5 % Tween-20 were subsequently perfused at the same flow rate. The samples were extracted and analyzed using LC-MS/MS as described above.

Similar to in situ brain perfusion of CPT-Glu, perfusion of Gd-DTPA in the presence and absence of HAV6 peptide was carried out followed by quantification of Gd-DTPA brain deposition using MRI. Briefly, the brains were perfused with 10 mL of 1.0 mM HAV6 in HCO₃⁻ buffer at pH 7.4 with 0.5 % Tween-20 at 5 mL/min. Then, a perfusion of 10 mL 0.6 mmol/kg Gd-DTPA (Magnevist, Berlex labs, NJ) in the presence of 1.0 mM HAV6 peptide in HCO₃⁻ buffer at pH 7.4 with 0.5 % Tween-20 was carried out at a flow rate of 5 mL/min. A 10-sec post-perfusion wash was delivered using saline solution. The amount of Gd-DTPA that penetrates across the BBB was determined from sacrificed animals at room temperature using a quantitative T₁ mapping MRI technique. Ex vivo MRI was performed using a quadrature volume coil in a 9.4 Tesla horizontal bore MR system equipped with a Agilent INOVA console (Agilent, Palo Alto, CA) and a 12-cm gradient insert (400 mT/m, 250 μs; Magnex Scientific, Abingdon, UK). T₁ mapping was performed using a modified Look-Locker sequence for multi-slice and multiple phase encodings per inversion pulse (TR/TE = 4/2 ms, FOV = 3 cm, slice thickness = 1 mm, matrix size = 128 × 128, flip angle = 20°, 22 inversion times from 40 – 5470 ms, 2 phasing encoding steps per inversion pulse, acquisition time = 8.5 min) and T₁ maps were generated using a program written in IDL (RSI, CO).¹⁶ R₁ (= 1/T₁) values were obtained from regions of interest (ROI) placed in olfactory bulbs, cortex, striatum, hippocampus, cerebellum, spinal cord, ventral, deep-rostral (mostly hypothalamus and pallidum), and deep-caudal (mostly midbrain) regions. The experiments were performed with n = 4 for each experimental group (control, vehicle + Gd-DTPA, and HAV peptide + Gd-DTPA).