SUPPLEMENTAL MATERIAL
Whole body plethysmography
Ventilatory parameters were recorded in a whole body plethesmograph by the barometric method
described and validated in the rat (1). The mice were placed in a rectangular Plexiglas chamber and
were restrained for measurement of ventilatory parameters and rectal temperature using a Plexiglas
cylinder (internal diameter: 2.5 cm, adjustable length up to 10 cm) (Harvard Apparatus, Holliston,
MA). This chamber was connected to a reference chamber of the same size by a high resistance leak to
minimize the effect of pressure changes in the experimental room. The animal chamber was flushed
continuously with humidified air during the recording periods. The inlet and outlet tubes were
temporarily clamped and pressure changes associated with each breath were recorded with a
deferential pressure transducer (Validyne MP, 45 ± 3 cmH2O, Northridge, CA), connected to the
animal and reference chambers. Whole body plethysmography requires the simultaneous
measurements of pressure, as well as ambient and rectal temperatures. The spirogram was recorded
and stored on a computer with an acquisition data card (PCI-DAS1000, Dipsi, Chatillon, France) using
respiratory acquisition software (Acquis 1 software, CNRS, GIF-sur-Yvette, France) for analysis offline.
This technique was daily validated with a series of leak tests (leak was signaled by a diminution of the
signal amplitude exceeding 33% in 5 s) (2) and a series of calibrations performed by 10 injections of
100 µl of air into the chamber. The quantification threshold corresponded to a minimum air volume
injection of 11 µl. Within the range of tested volumes (11 to 250 µl), measurements were linear. The
mean coefficient of intra-day variability (four series of 5 measurements carried out the same day) was
1.3 ± 0.2%. The mean coefficient of inter-day variability (25 measurements carried out on 3 different
days) was 1.7 ± 0.1%. We verified that the mean CO2 measured using an Ohmeda 5250 RGM
capnograph (rebreathing test) during clamping periods did not exceed 0.6% of the air contained in the
chamber.
During experimentation, the first measurement was performed after a 60 min-period of
accommodation, while the animal was quiet and not in deep or rapid eye movement sleep which can
be roughly estimated from their behavior, the response to noise, and the pattern of breathing. Then, the
animal was gently removed from the chamber for ip injection, and replaced in the chamber for the
remaining measurements. Ventilation was recorded at -30, -15, -5, 5, 10, 15, 20, 30, 40, 60, 80, 120
min, each record lasting about 60 s. The following parameters were measured: the tidal volume (V T),
the inspiratory time (TI), the expiratory time (TE), and the respiratory cycle duration (TTOT = TI + TE).
Additional parameters were calculated: the respiratory frequency (f), and the minute ventilation (VE =
VT x f). At the end of experimentation, rats were euthanized using a carbon dioxide chamber.
In Situ Brain Perfusion
Surgery and perfusion. Buprenorphine (BUP) and norbuprenorphine (N-BUP) transport at the bloodbrain barrier (BBB) was measured in mice by in situ brain perfusion, in which the blood is completely
replaced by an artificial perfusion fluid for a short time (3). Mice were anesthetized with ketamine
(140 mg/kg ip) + xylazine (8 mg/kg, ip). Briefly, the right common carotid artery was exposed and the
external carotid artery was ligated at the level of the bifurcation of the common and internal carotid
arteries. The right common carotid artery was catheterized and the catheter connected to a syringe
containing the perfusion fluid placed in an infusion pump. The thorax was opened, the heart was cut,
and perfusion was started immediately (flow rate = 2.5 ml/min). The perfusion fluid consisted of
bicarbonate-buffered physiological saline containing 128 mM NaCl, 24 mM NaHCO3, 4.2 mM KCl,
2.4 mM NaH2PO4, 1.5 mM CaCl2, 0.9 mM MgCl2, and 9 mM D-glucose. The solution was gassed
with 95% O2-5% CO2 for pH control (7.4) and warmed to 37°C in a water bath. The compounds
including [3H]-BUP (0.4 µCi/ml; 5 nM) or N-BUP (15 µM) were added to the perfusion fluid with or
without PSC833 (5 µM). [14C]-sucrose (0.1 µCi/ml) added in the perfusion fluid was used as a
vascular space marker. Perfusion times were 30 s for [3H]-BUP and 180 s for N-BUP (to gain
sufficient analytical detection due to the expected low BBB extraction of N-BUP). Brain perfusion
was terminated by decapitating the mouse at these selected times.
The brain was removed from the skull and dissected out on ice. The right cerebral hemisphere and
aliquots of the perfusion fluid were placed in tarred vials and weighed. To measure BUP transport,
radioactive samples were digested in 2 ml of Solvable (Perkin Elmer) at 50°C and mixed with 9 ml of
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Ultima GoldXR (Perkin Elmer). Dual label counting was performed in a Packard Tri-Carb 1900TR.
To measure N-BUP transport, hydrochloric acid was added (2 ml/g of brain tissue), and the mixture
was homogenized using sonication and stored at -80°C before determination of N-BUP concentrations
by gas chromatography-mass spectrometry (GC-MS).
Calculation of the BBB transport parameters. Two parameters express the transport of a compound at
the BBB: the apparent brain volume of distribution (Vbrain, µl/g) and the brain transport parameter Kin
(µl/s/g) also called brain clearance. The linear time course of brain accumulation for BUP and N-BUP
up to 180 s (data not shown) was assessed and it was found that the perfusion times were short enough
to ensure that the distribution of the compounds was kinetically governed only by the transport
processes acting at the luminal side of the BBB (4). Transport parameter calculations were corrected
for brain vascular contamination, as previously described (3,4).
The brain vascular volume Vv (µl/g) was calculated using the distribution of [14C]-sucrose, which does
not measurably cross intact BBB in a short time: Vv = Xv / Cv, where Xv (dpm/g) is the [14C]-sucrose
measured in the right hemisphere and Cv (dpm/µl) is the concentration of [14C]-sucrose in the
perfusion fluid. The physiological Vv values for short perfusion times do not exceed 20 µl/g (3). If the
Vv was above the normal value (>20 µl/g) for a mouse experiment, the transport parameter was not
calculated and the experiment was discarded.
The Vbrain was calculated using the amount in the right hemisphere corrected for vascular
contamination: Vbrain = (Xtot - Vv.Cperf) / Cperf; Kin = Vbrain / T, where Cperf is BUP or N-BUP
concentrations in the perfusion fluid, Xtot the total BUP or N-BUP in the tissue sample (vascular +
extravascular), and T the perfusion time.
The coefficient of brain extraction was calculated by the ratio of the K in of the studied compound to
diazepam Kin (42.3 µl/s/g) (3,4), which is considered as a vascular flow marker.
BUP and N-BUP GC/MS assay
The frozen brain and perfusion fluid samples (-80°C) were thawed at room temperature for 15 min,
and then 1 ml of sample was transferred to polypropylene tube. Two milliliters acetate buffer (0.1 M;
pH 5.0) and 0.1 ml of internal standard stock solution (1 µg/ml) of d4-BUP and d3-N-BUP were
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added. The mixture was vortexed and then loaded onto a Clean Screen® solid phase extraction column
(UCT, Bristol, PA, USA) that was preconditioned with 3 ml methanol and 3 ml sterile water, and then
equilibrated with 2 ml 0.1M acetate buffer. The mixture was added to the column. The column was
washed with 2 ml sterile water and 3 ml 0.1N acetate buffer and 3 ml methanol. The column was then
dried under vacuum for 10 min. The analytes were collected in a 5 ml glass tube by elution with 3 ml
dichloromethane, isopropyl alcohol and ammonia solution 25% fresh mixture (78/20/2). The solvent
was then evaporated under a gentle stream of nitrogen. The residue was reconstituted with 20 µl ethyl
acetate and 20 µl BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) with 1% TMS (trimethylsilane),
vortexed briefly and transferred to an autosampler vial insert for GC/MS analysis. The Thermo Focus
DSQ II GC/MS system was used. The system was equipped with an Uptibond® UB5 premium column
(30 m x 0.25 mm x 0.25 µm). The instrument was programmed (room 200°C to 220°C at 30°C/min
and held for 3 min before being programmed to 390°C at 15°C/min and held for 17 min, for a total
analysis time of 20 min). The transfer line temperature was maintained at 280°C. One microliter of
derivatized extract was injected. The injection port temperature was held at 250°C and operated in the
pulsed splitless mode. The instrument utilized electron impact ionization and was operated in the
selected ion monitoring (SIM) mode. Ions with m/z 468 (N-BUP-TMS), 450 (BUP-TMS), 454 (d4BUP-TMS), and 471 (d3-N-BUP-TMS) were monitored.
REFERENCES
1. Bartlett D Jr, Tenney SM: Control of breathing in experimental anemia. Respir Physiol 1970; 10:
384-395
2. Bonora M, Bernaudin JF, Guernier C, et al: Ventilatory responses to hypercapnia and hypoxia in
conscious cystic fibrosis knockout mice Cftr-/-. Pediatr Res 2004; 55: 738-746
3. Dagenais C, Rousselle C, Pollack GM, et al: Development of an in situ mouse brain perfusion
model and its application to mdr1a P-glycoprotein deficient mice. J Cereb Blood Flow Metab
2000; 20: 381-386
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4. Cattelotte J, André P, Ouellet M, et al: In situ mouse carotid perfusion model: glucose and
cholesterol transport in the eye and brain. J Cereb Blood Flow Metab 2008; 28: 1449-1459
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