In Situ Mouse Carotid Perfusion Model: Glucose and Cholesterol Transport in the Eye and Brain

Abstract

The in situ mouse brain perfusion method for measuring blood-brain barrier permeability was adapted to assess transport of solutes at the blood-brain and blood-eye barriers. The procedure was checked with radiolabeled markers in oxygenated bicarbonate-buffered fluid infused for 30 to 120 secs via a carotid artery. Vascular flow estimated with diazepam was 2.2-fold lower in the eye than in the brain. The vascular volume and the integrity markers sucrose and inulin indicated that a perfusion flow rate of 2.5 mL/min preserved the physical integrity of these organs. However, the brain vasculature integrity was more sensitive to acute perfusion pressure than the eye vasculature. The functional capacities of blood barriers were assessed with D-glucose; its transport followed Michaelis-Menten kinetics with an apparent K m of 7.6 mmol/L and a V max of 23 lmol/sec per g in the brain, and a K m of 22.9 mmol/L and a V max of 40 lmol/sec per g in the eye. The transport of cholesterol to the brain and eye was significantly enhanced by adding the Abca1 inhibitor probucol, suggesting an Abca1-mediated efflux at the mouse brain and eye blood barriers. Thus in situ carotid perfusion is suitable for elucidating transport processes at the blood-brain and blood-eye barriers.

Transport of Biogenic Amine Neurotransmitters at the Mouse Blood–Retina and Blood–Brain Barriers by Uptake1 and Uptake2

“…Surgical procedure and perfusion: We assessed the functional role of mediated carriers systems by measuring the transport of selected labeled compounds at the brain and eye of mice by in situ carotid perfusion, in which the blood is completely replaced by an artificial perfusion fluid for a very short time (Cattelotte et al, 2008;Smith, 1996;Takasato et al, 1984). Mice were anesthetized with ketamine-xylazine (140-8 mg/kg, intraperitoneally) and the appropriate vessels were ligated.…”

“…A catheter was inserted into the right common carotid artery and connected to the perfusion system. The thorax was opened, the heart was cut, and perfusion was started immediately at a constant flow rate (2.5 mL/ min) and pressure (Cattelotte et al, 2008;Takasato et al, 1984). Perfusion was terminated by decapitation and the brain and the right eye were removed from the skull; the brain was dissected out on a cold freezer pack, and the eye on dry ice.…”

“…To do this, we determined whether transport mechanisms are involved in the diffusion of selected biogenic amines at the mouse BBB and blood-eye barriers. We used in situ carotid perfusion (Cattelotte et al, 2008) because this enabled us to control the perfusion time and the composition of the luminal/vascular compartment. The perfusate replaces the blood and so allows the identification and exploration of the kinetics of transporters at the luminal (blood) side of membranes in vivo (Smith, 1996).…”

Clonidine Transport at the Mouse Blood—Brain Barrier by a New H+ Antiporter that Interacts with Addictive Drugs

“…Surgical procedure and perfusion: The transport of [ 3 H]clonidine into the mouse brain was measured by in situ brain perfusion (Cattelotte et al, 2008). Mice were usually anesthetized by injection with ketaminexylazine (140-8 mg/kg, intraperitoneally), but they were anesthetized with halothane (Belamont, Neuilly-sur-Seine, France) in some experiments.…”

“…We measured the transport of [ 3 H]clonidine at the luminal membrane of the endothelial cells that form the mouse BBB by in situ brain perfusion. This method can accurately determine BBB permeability and it is possible to control the composition of the vascular perfusate for a short time (Cattelotte et al, 2008;Smith, 1996;Takasato et al, 1984). We used exposure to various chemicals and ions, together with studies on transporter knockout (KO) mice, to identify the properties of clonidine transport at the mouse BBB.…”