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Mouse aversion to isoflurane versus carbon dioxide gas
Accepted Manuscript Title: Mouse aversion to isoflurane versus carbon dioxide gas Author: Carly M. Moody Daniel M. Weary PII: DOI: Reference:
S0168-1591(14)00114-2 http://dx.doi.org/doi:10.1016/j.applanim.2014.04.011 APPLAN 3897
To appear in:
APPLAN
Received date: Revised date: Accepted date:
3-10-2013 23-4-2014 28-4-2014
Please cite this article as: Moody, C.M., Weary, D.M.,Mouse aversion to isoflurane versus carbon dioxide gas, Applied Animal Behaviour Science (2014), http://dx.doi.org/10.1016/j.applanim.2014.04.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights Exposure to isoflurane via a vaporiser is more aversive to mice than carbon dioxide Mice are more willing to be exposed to isoflurane via a vaporiser than the drop method Mice find isoflurane re-exposure more aversive than initial exposure
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Title:
Mouse aversion to isoflurane versus carbon dioxide gas
Authors:
Carly M Moody* and Daniel M Weary
Institutional affiliations:
Animal Welfare Program, Faculty of Land and Food Systems,
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University of British Columbia, 2357 Main Mall, Vancouver,
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British Columbia, Canada V6T 1Z4
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*
Email: [email protected]
Co-author:
Email: [email protected]
Corresponding author:
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Abstract
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Isoflurane and carbon dioxide (CO2) gas are used for rodent euthanasia. This study compared
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mouse aversion to isoflurane versus gradual-fill CO2 gas, and compared two methods of
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isoflurane delivery: vaporiser and drop. Mouse acclimation to a light–dark apparatus was used to
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create a light aversion test based on an unconditioned preference for dark versus light areas. Mice
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chose between remaining in a dark compartment with rising concentration of one of three
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treatments (20% gradual-fill chamber vol/min of CO2, n=8; 5% isoflurane administered using a
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vaporiser set at 4 L/min oxygen flow, n=9; or 5% liquid isoflurane dropped on gauze, n=9), or
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escaping to a brightly lit compartment. On average (±S.E.) mice left the dark compartment after
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29.2 6.1 s in the isoflurane vaporiser treatment. Initial withdrawal time was lower for the CO2
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treatment (P=0.04), averaging 16.6 2.8 s, and lower still for the isoflurane drop treatment
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(P<0.001), averaging 2.9 0.79 s. Five of nine mice became recumbent in the dark compartment
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when exposed to the isoflurane vaporiser treatment compared to only two of nine mice during the
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drop treatment (P=0.3) and zero of eight mice during the CO2 method (P=0.03). The isoflurane
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concentrations rose more quickly using the drop versus the vaporiser method, likely explaining
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the increased willingness of mice to be exposed to isoflurane administered via a vaporiser
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machine. Re-exposure to isoflurane with the vaporiser was more aversive than initial exposure;
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only two of nine mice stayed in the dark compartment until recumbency. These results support
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the recommendation that mice with no previous exposure to isoflurane should be euthanised using
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isoflurane administered by a vaporiser rather than CO2 gas, and suggest that the drop method (as
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applied in the current study) is not a suitable alternative.
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Keywords: Euthanasia, vaporiser, drop method, recumbency, light-dark paradigm
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1. Introduction Current laboratory rodent euthanasia guidelines recommend using an inhalant anaesthetic over
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carbon dioxide gas (CO2) for rodent euthanasia (American Veterinary Medical Association, 2013;
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Canadian Council on Animal Care, 2010). Current evidence suggests isoflurane is less aversive to
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mice and rats than CO2 (Leach et al., 2002b; Leach et al., 2004; Makowska and Weary 2009;
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Wong et al., 2013) and other inhalant anaesthetics (Makowska et al., 2009). Isoflurane is a
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volatile liquid halogenated hydrocarbon. Generally, one of two methods can be used to administer
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isoflurane for euthanasia: a vaporiser machine or the drop method. When administering isoflurane
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using a vaporiser machine, a carrier gas and an anaesthetic waste gas scavenging system is
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required. Some animal users argue that the use of a vaporiser is unnecessary for rodent
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euthanasia. Vaporisers are intended to control the amount administered to reduce the risk of
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anaesthestic overdose, a feature of little value when the intention is to kill the animal. In addition,
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vaporiser machines can be costly to purchase and maintain, reducing accessibility for some users.
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The drop method involves placing liquid isoflurane on an absorbent material such as gauze, and
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placing this in a closed compartment. To our knowledge no studies have compared aversion to the
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drop versus vaporiser methods of isoflurane administration. In addition, many laboratories still
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use gradual-fill CO2 for euthanasia. Thus we tested mouse aversion to isoflurane administered by
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a vaporiser, isoflurane administered via the drop method, and gradual-fill CO2.
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The light-dark paradigm is a conflict-based anxiety test originally developed by Crawley and
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Goodwin (1980) to test anti-anxiety medications on mice. This paradigm uses the innate
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unconditioned preference for dark versus light areas and fear of open spaces in mice. The light-
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dark apparatus is composed of three compartments, a large light compartment, a small dark
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compartment and a middle compartment separating the light and dark areas. Acclimation to the
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apparatus changes this novel environment into a familiar one, therefore producing a light aversion
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test instead of testing anxiety (Matynia et al., 2012). This paradigm has been used to test rat
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aversion to CO2 versus isoflurane in rats (Wong et al., 2013).
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Using the light-dark box paradigm, we tested mouse aversion to three euthanasia methods: 1)
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20% gradual-fill chamber vol/min of CO2, 2) 5% isoflurane administered using a vaporiser set at
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4 L/min (40% chamber vol/min) oxygen (O2) flow, and 3) 5% isoflurane administered using the
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drop method. Mice were able to choose between remaining in a small dark compartment with a
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rising concentration of one of three treatments or escaping to a larger brightly lit compartment.
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Remaining in the small dark compartment with a rising concentration of test gas indicates that
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mice find the larger bright compartment more aversive than the test gas. Alternatively, leaving the
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small dark compartment with the test gas indicates that the test gas is more aversive than the large
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bright compartment. Initial exposure aversion was examined for all treatments. In addition, re-
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exposure aversion was examined for the isoflurane vaporiser treatment; mice commonly undergo
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surgical procedures using an isoflurane vaporiser machine, and re-exposure may be more aversive
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than initial exposure (Wong et al., 2013).
2. Materials and Methods
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2.1 Pre-trial
During the pre-trial we measured the rate at which isoflurane concentration increased within a
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compartment when using the vaporiser and drop treatments (Fig. 1b); the results allowed us to
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estimate the isoflurane concentrations that mice would be exposed to during the experiment. Use
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of a theoretical 20% gradual-fill CO2 curve (Fig. 1a), allowed us to estimate CO2 exposure
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concentrations during this experiment.
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An Innocage® disposable individually ventilated transparent mouse cage (Universal Euro Type
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II Long, Innovive Inc. San Diego, California, USA, 37.3 cm length x 23.4 cm width x 14.0 cm
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height, with 205 cm2 floor space) was used as the test cage. A Plexiglass lid with a centrally
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placed hole was placed on top of the cage during testing. A Capnomac Ultima™ (Datex Ohmeda
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Instrumentation Corporation, Helsinki, Finland) capnograph was used to measure the rising
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concentration of isoflurane in the cage, via a polyethylene/polyvinalchloride sampling line (Datex
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Ohmeda Instrument Corporation, Finland) inserted into a hole near the base of the anterior wall of
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the cage. Testing took place in Medical Block C at the University of British Columbia,
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Vancouver, Canada. For the isoflurane vaporiser treatment, 5% isoflurane (Baxter Corporation, Ontario, Canada)
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was administered via an Isotec 4 isoflurane vaporiser (Ohmeda, Steeton, West Yorkshire,
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England, UK) using 4 L/min (33% chamber vol/min) of room air as the carrier gas. The
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isoflurane drop treatment used wire mesh (Activ-wire mesh, Activa Products Inc., Marshall,
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TX, USA) shaped to create a rectangular apparatus (7 cm length x 3 cm width x 11 cm height),
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that was closed on all sides except the top, to allow a piece of 5.1 cm x 5.1 cm gauze
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(Professional Preference, Rafter 8 Products, Calgary, AB, Canada) opened length-wise for
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vertical placement down into the wire rectangular apparatus. The wire apparatus was placed at the
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end of the rectangular test cage, standing up against the cage wall. The volume of isoflurane
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required to provide a 5% concentration in the compartment was determined to be 4.6 mL using
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the universal gas law (PV=nRT) and a room temperature of 22 °C. A glass syringe was used to
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distribute the liquid isoflurane down the piece of gauze within the wire mesh apparatus.
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2.2 Experiment
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2.2.1 Animals and Housing
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We used 30 male C57BL/6J mice housed at the University of British Columbia’s Centre
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for Disease Modeling, Vancouver, Canada. Male mice were used in the present study on the
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grounds that the light-dark paradigm was validated and developed using male mice and that
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responses may vary with female estrus cycle (Voikar et al., 2001). Mice were housed in groups of
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three or five in ventilated polysulfone type II long cages (Bioscape, Germany, 20.7 cm length x
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14.0 cm width x 36.5 cm height), using an individually ventilated cage system (Bio A.S. cage
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rack and blower system, Bioscape, Germany). All mice weighed between 22.4 to 28.3 g, and were
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2 months old at the time of testing. Mice were housed with a nest box, brown crinkle paper
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(Enviro-dri, Shepherd Specialty Papers Inc., Richland, MI, USA), one cotton nest square (Ancare,
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Bellmore, NY, USA), beta chip bedding (Nepco, Northeastern Products, Warrensburg, NY,
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USA), ad libitum access to food (Harlan 2918 Tekland Global Rodent Maintenance, Madison,
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WI, USA) and reverse osmosis chlorinated water, and kept under a reverse 12 h light:12 h (lights
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off at 07:00 h) dark cycle with light intensity ranging from 240 to 340 lux throughout the light
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phase. All animal procedures were approved by the University of British Columbia’s Animal
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Care Committee.
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All acclimation and testing trials took place in a darkened test room lit by a 35-watt lowpressure sodium vapour lamp (Master SOX PSG, Philips Lighting Canada, Markham, ON,
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Canada) undetectable to mice (McLennan and Taylor-Jeffs, 2004). All mice were placed in a
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biological safety cabinet within the test room 1 h before acclimation or testing trials started, to
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allow adjustment to the test room. In comparison to the pre-trial, no gas concentrations were
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measured during any acclimation or testing trials.
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The experimental apparatus consisted of a Plexiglas test box (67 cm x 20 cm x 25 cm; Fig. 2)
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divided into three compartments: 1) a light compartment (40 cm x 20 cm x 25 cm), 2) a dark
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compartment (20 cm x 20 cm x 25 cm), and 3) a middle buffer compartment (6 cm x 6 cm x 6
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cm). The middle compartment connected the light and dark compartments and contained a wire
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mesh (ActivTM-wire mesh, Activa Products Inc., Marshall, TX, USA) passageway (6 cm length x
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6 cm width x 6 cm height) to allow a mouse to pass between the light and dark compartments
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while minimizing time spent in this compartment. The openings to the wire mesh passageway of
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the middle compartment were covered with thin, overlapping black plastic strips with vertical
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slits, to minimize gas exchange between the compartments while allowing the mice to pass
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through. Holes (1 cm diameter, 32 on each side) drilled into the side of the middle compartment
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helped vent any gases entering this compartment. Black plastic was placed on all sides of the dark
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compartment, except the front from where video recording took place. The experimenter sat away
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from the testing apparatus and was hidden by a blind. Fresh beta chip bedding was placed in to
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the light (500 mL) and dark (300 mL) compartments between testing cages of mice. A lamp
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(Barometer work lamp, Ikea, China) with a 7-watt parabolic aluminized reflector 20, light-
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emitting diode (LED; Lightline, Brampton, ON, Canada) that did not heat up over time was used
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as the light source. The lamp head was directed downwards over the centre of the light
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compartment to minimize light diffusion into the other compartments. At the start of each trial, a
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lux meter (Traceable Dual-Range light meter, VWR international, Radnor, PA, USA) was used to
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measure light intensity in the light and dark compartments; measurements in the dark
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compartment did not exceed 3 lux and the darkest corner of the light compartment exceeded 700
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lux. Past studies using the light-dark paradigm have shown that mice find 500 lux aversive
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(Costall et al., 1989; Matynia et al., 2012).
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A Plexiglas lid with two holes (1.6 cm diameter), each centrally placed above the light and
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dark compartments, was custom made to fit the testing apparatus. Each of the three treatments
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tested required a unique equipment assembly. During the isoflurane vaporiser acclimation trials,
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the lid holes over the light and dark compartments allowed for the placement of separate gas lines
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connected to the same O2 tank (Praxair, Delta, BC, Canada) using an O2 flow meter (Western
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Medica, Westlake, OH, USA) to deliver 4 L/min of O2 into each compartment. During isoflurane
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vaporiser testing trials, the gas line delivering 4 L/min O2 to the dark compartment, was replaced
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with a gas line delivering a flow of isoflurane (Baxter Corporation, Mississauga, ON, Canada) via
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a isoflurane vaporiser machine (Highland Medical Equipment, Temecula, CA, USA) at 5% with 4
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L/min O2 as the carrier gas. The CO2 treatment acclimation trials used an identical set up as the
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isoflurane vaporiser acclimation trials, except the gas lines delivered 2 L/min (20% compartment
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vol/min flow rate) of O2 into each compartment. The CO2 treatment testing trials were similar to
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the acclimation trials, except gas flow into the dark compartment involved a switch from O2 to 2
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L/min of CO2 (Praxair, Delta, BC, Canada) measured using a CO2 flow meter (Western Medica,
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Westlake, OH, USA). For the isoflurane drop treatment trials, the two centrally placed holes over
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the light and dark compartment in the Plexiglas lid were covered with duct tape, as no gas lines
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were needed. Instead, wire mesh (Activ-wire mesh, Activa Products Inc., Marshall, TX, USA)
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was shaped to create a rectangular apparatus (11.5 cm length x 3 cm width x 24 cm height) that
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was closed on all sides except the top, to allow a piece of 10.2 cm x 10.2 cm gauze (Professional
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Preference, Rafter 8 Products, Calgary, AB, Canada) to be opened and placed down the mesh
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apparatus to increase surface area. The opened top of the rectangular wire mesh apparatus
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allowed liquid isoflurane to be distributed down the gauze during experimental testing; only
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during the testing trials was isoflurane used.
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2.2.3 Acclimation
Mice were individually assigned to one of three treatments: isoflurane vaporiser, isoflurane
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drop or CO2 gas. As well, the cages of mice were randomized with regards to the order of testing.
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Mice were individually acclimated to the test apparatus every other day for 6 d, totaling three
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acclimation trials each. Before the start of each acclimation trial, the light source above the light
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compartment was turned on and the lux meter was used to measure light intensity in the light and
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dark compartments. For both the isoflurane vaporiser and CO2 treatments, gas lines were placed
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into the holes over the dark and light compartments, delivering 4 L/min or 2 L/min of O2,
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respectively. For the isoflurane drop treatment, the holes in the Plexiglas lid were covered and the
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wire mesh apparatus was placed into the dark side against the end of the dark compartment. The
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start of the acclimation trials occurred when a mouse was placed into the light compartment. Our
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light source was aversive to the mice, as shown by mouse preference for the dark compartment.
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Mice frequently moved back and forth between compartments, spending more time in the dark
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compartment but rarely staying longer than 1.5 min. On this basis we used the criterion of 1
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continuous min in the dark compartment to signify preference, as mice are known to show high
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frequencies of entries and exits (Leach et al., 2002a). The trial ended after 20 min or when 1 min
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was spent continuously in the dark side, whichever occurred first. The test apparatus was cleaned with 70% alcohol between cages of mice and then aired out
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and wiped with water to decrease any smell or novelty. The apparatus was not cleaned between
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mice that shared the same home cage, following Hascoet and Bourin (1998). These authors
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suggest that mice are more influenced by light and dark areas within a light-dark box when using
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a soiled apparatus.
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Of 30 mice included in the study, four did not spend 1 min in the dark side during the acclimation trials and were thus removed from the study. A total of eight CO2 treatment mice and
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nine for each the isoflurane vaporiser and drop method treatments, moved on to the testing trials.
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Testing took place during the dark cycle between 10:00 and 17:00 h, at an average (±S.D.)
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room temperature of 20.6 ± 0.5 °C. Experimental testing followed the acclimation procedure until
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a mouse spent 1 continuous min in the dark side, our criterion for preference. At this point, mice
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were tested for aversion to one of the three treatments in the dark compartment. For isoflurane
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vaporiser trials, the gas line delivering 4 L/min of O2 to the dark compartment was replaced with
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a gas line delivering 5% isoflurane via a vaporiser machine, with 4 L/min of O2 as the carrier gas.
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For CO2 trials, the gas line delivering 2 L/min of O2 to the dark compartment was replaced with a
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gas line delivering 2 L/min of CO2. For all isoflurane drop trials, the Plexiglas lid was opened just
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enough to deliver 3.7 mL isoflurane (5% volume as determined using the universal gas law:
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PV=nRT, with 20°C room temperature) onto the gauze within the wire mesh apparatus, and then
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the lid was closed.
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Trials ended when a mouse became recumbent in the dark compartment, or when 2 min were spent continuously in the light compartment indicating aversion to the gas treatment. Mice that
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stayed in the dark compartment until recumbency were transferred to an empty cage, identical to
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the home cage, placed on a heating pad, and allowed to recover before being returned to their
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home cage. Each mouse was tested only once, except those included in the vaporiser treatment.
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All the vaporiser treatment mice were re-tested once with the same procedure. Re-exposure
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occurred 1 week after initial exposure. These trials followed the same procedure as the initial test
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exposure.
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After completing these trials, all mice were euthanised using 5% isoflurane gas with 4 L/min of O2 as the carrier gas for anaesthetic induction, followed by 50% CO2 to complete the
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euthanasia procedure. Mice were then cervically dislocated as the secondary method of
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euthanasia.
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Both the pre-trial and the experimental trials were video recorded using a high definition camcorder (Model TM41P, Panasonic Corporation, Malaysia) in advanced coding high definition
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recording mode with 30 frames/s. Pre-trial videos were used to record capnograph readings every
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5 s. Testing trials were scored for: initial withdrawal time, recumbency, number of re-entries, and
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re-entry dwelling time in the dark compartment (Table 1). Only those mice that re-entered the
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dark compartment were used to analyse the number of re-entries and re-entry dwelling time.
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2.4 Statistical Analysis
The effect of treatment on initial withdrawal time, number of re-entries, and re-entry dwelling
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time was tested using ANOVA (SAS v. 9.3), with the difference between the two methods of
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isoflurane induction compared using one contrast statement, and the difference between the
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vaporizer method and the CO2 method compared using a second contrast. Mice that never left the
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dark compartment upon gas exposure were not included in the analysis for the number of re-visits
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or re-entry dwelling time. Differences in response to initial versus re-exposure with the vaporizer
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method were tested using a mixed model (SAS v. 9.3). For initial exposure trials, a Fisher exact
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test assessed the effect of treatment on the number of mice that became recumbent in the dark
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side. A Cochran-Mantel-Haenszel test was used to analyse the number of mice that became
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recumbent in the dark side for initial versus re-exposure isoflurane vaporiser trials. Means are
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reported ± S.E.
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3.Results
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3.1 Pre-trial
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The drop method of isoflurane administration resulted in a faster increase in concentration
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compared with the isoflurane vaporiser method (Fig. 1b). The target concentration of 5% was
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achieved after 5 min using the vaporiser versus 30 s when using the drop method.
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3.2.1 Initial Exposure
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Initial withdrawal time from the dark compartment was longer for mice exposed to isoflurane by the vaporiser machine compared with the drop (F1,18= 22.6, P<0.0001; Fig. 3a) and CO2
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methods (F1,17= 4.9, P= 0.04). Mice were also more likely to become recumbent in the dark
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compartment when isoflurane was delivered using the vaporiser versus drop method (F1,18= 7.0,
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P= 0.3; Table 2) or CO2 (F1,17= 8.0, P=0.03). Of the five mice that became recumbent during
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isoflurane vaporiser exposure, two never left the dark compartment after the isoflurane was
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turned on and three became recumbent upon re-entry. With the drop method, two mice became
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recumbent in the dark compartment upon re-entry. None of the eight CO2 treatment mice stayed
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in the dark compartment until recumbency upon initially leaving or re-entry.
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Mice exposed to the isoflurane vaporiser treatment more often re-entered the dark
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compartment after initially leaving, compared to either the drop method (F1,18= 7.5, P=0.013;
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Table 2) or the CO2 treatment (F1,17=8.5, P=0.008). The re-entry dwelling time in the dark
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compartment was greater in the vaporiser treatment versus both the drop (F1,12= 15.8, P=0.0012;
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Fig. 3b) and CO2 treatments (F1, 11= 19.7, P=0.0005).
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3.2.2 Re-exposure: Isoflurane vaporiser treatment
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The initial withdrawal time from the dark compartment decreased from a mean of 29.2 ± 6.1 s to 19.1 ± 5.0 s upon re-exposure to isoflurane (F1,8= 3.7, P=0.09; Fig. 4). Only two of nine mice
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became recumbent during re-exposure, compared to five of nine mice during initial exposure
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(Table 2). The two mice that became recumbent during re-exposure had also become recumbent
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during the initial exposure trials. The number of re-entries to the dark compartment decreased
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from a mean of 3.6 ± 1.0 during initial exposure to 1.0 ± 0.4 upon re-exposure (F1,6=5.8, P=0.05;
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Table 2). Re-entry dwelling time in the dark compartment decreased from 24.1 ± 4.2 s during
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initial exposure to 2.3 ± 0.9 s during re-exposure (F1,6=34.3, P=0.0011; Fig. 4).
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4. Discussion
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In the current study the vaporiser was at the highest setting possible (5%) combined with the
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highest flow rate of O2 (4 L/min); these settings are chosen to minimize the time between onset of
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aversion and insensibility when using the vaporiser (Makowska et al., 2009). During the pre-trial,
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the drop method isoflurane concentration rose to a maximum of 7.5% and then leveled out at 5%,
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likely due to sampling line placement within the cage. The results of the pre-trial showed that
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isoflurane concentration rises much more quickly when using the drop method versus the
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vaporiser. This difference means that mice spending the same amount of time in the dark
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compartment would be exposed to a higher total dosage of isoflurane in the drop versus vaporiser
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treatments. The initial withdrawal time from the dark compartment was 2.9 versus 29.2 s with the
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isoflurane drop and vaporiser treatments, respectively. At these times, the isoflurane
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concentration in the dark compartment would have been approximately 1 to 2%, suggesting that
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concentrations in excess of 1% are aversive to mice. Leach et al. (2002b) also reported that
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isoflurane was aversive at these concentrations.
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The number of re-entries to the dark side averaged one and four for the isoflurane drop and vaporiser treatments, respectively; this difference again may be attributed to the slower build up
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of isoflurane concentration using the vaporiser method. When mice chose to re-enter the dark side
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during the isoflurane drop treatment, the concentration of isoflurane was likely higher than at the
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equivalent time in the vaporiser treatment. This difference likely also explains the decreased re-
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entry dwelling time in the dark compartment with the drop treatment versus the vaporiser
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treatment.
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The initial withdrawal time for mice to leave the dark compartment was longer for the vaporiser treatment versus gradual-fill CO2. The concentration of CO2 likely exceeded 10% when
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mice chose to leave the dark compartment, based upon a theoretical 20% chamber vol/min
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gradual-fill CO2 curve. Previous studies have shown that CO2 concentrations ranging from 3-20%
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are aversive in mice and rats (Kirkden et al., 2008; Krohn et al., 2003; Makowska et al., 2009;
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Niel and Weary, 2007; Niel et al., 2008); the current results are consistent with these findings. In
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addition, concentrations of CO2 between 10-35% have been shown to cause fear responses in
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mice and rats (Concas et al., 1993; Niel and Weary, 2006; Ziemann et al., 2009); and a study by
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Ziemann et al. (2009) suggests that 10% CO2 acts an unconditioned fear stimulus. Thus, the
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aversion results of the current study build upon the abundance of existing work showing that CO2
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is aversive in rodents.
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The number of visits and re-entry dwelling time were both lower for mice exposed to CO2
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compared to vaporised isoflurane, suggesting that initial exposure to CO2 is more aversive than
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initial exposure to vaporised isoflurane. None of the mice exposed to CO2 stayed in the dark
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compartment until recumbency, compared to about half of the mice tested with the isoflurane
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vaporiser treatment. It is possible mice that stayed in the dark compartment until recumbency may
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have become too sedated to escape the gas-filling compartment even if they wanted to. However,
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no mice stayed until recumbency with CO2, suggesting that induction with isoflurane is less
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aversive than induction with CO2. In accordance with past aversion work (Leach et al., 2002b;
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Leach et al., 2004; Makowska et al., 2009; Makowska and Weary, 2009; Wong et al., 2013), the
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results of this study indicate that mice find CO2 more aversive than isoflurane.
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Our results suggests that re-exposure to isoflurane is more aversive than initial exposure. A previous study (Makowska et al., 2009) using an approach-avoidance paradigm failed to find
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learned aversion to isoflurane in mice, but a more recent study using the light-dark paradigm
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(Wong et al., 2013) found learned aversion in rats re-exposed to isoflurane. These contradictory
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results suggest that more work is needed to understand learned aversion associated with re-
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exposure to isoflurane in rodents. As well, perhaps the protocols involving repeated exposure to
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isoflurane should be re-considered.
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In agreement with current literature, the current study results suggest that mice tolerate exposure to concentrations of isoflurane that cause anaesthesia (delivered by a vaporiser) but not
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with CO2 gas. Mice always chose to leave the dark compartment and enter the larger brightly lit
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compartment before CO2 concentrations reached those that could render a mouse insensible.
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Although this study showed that isoflurane was less aversive than CO2, the treatment was still
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aversive as indicated by some mice choosing to leave the dark compartment. Isoflurane has a
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pungent odour (Flecknell, 2009) and is known to cause eye irritation and irritation to upper
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airway mucosa (Cervin and Lindberg,1998; Doi and Ikeda, 1993); these effects are likely
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pronounced at higher concentrations, such as those experienced in the drop method treatment of
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the current study.
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5. Conclusion
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The results of the current study correspond with other recent results indicating that mouse
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euthanasia using isoflurane administered by a vaporiser is a humane alternative to the use of
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gradual-fill CO2. Some animal users without access to a vaporiser may wish to use the drop
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method, but our results suggest that the drop method (as tested in the current study) should be
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avoided. Re-exposure to isoflurane administered with a vaporiser was more aversive than initial
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exposure, suggesting that re-exposure to isoflurane should be avoided.
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We thank Jurgen Pehlke for building the light-dark box apparatus and Warren Riley from
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Innovive Inc. (USA) for donating the test cage used in the pre-trial. We also thank Devina Wong
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for input into experimental design and Bernard Macleod and Tim Fung for help with the pre-trial.
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We are grateful to Joanna Makowska for review of this manuscript and many conversations
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regarding rodent euthanasia. This research was funded by a Discover grant to DMW from the
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Natural Sciences and Engineering Research Council of Canada.
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American Veterinary Medical Association. 2013. Guidelines for the euthanasia of animals: 2013
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Canadian Council on Animal Care. 2010. CCAC guidelines on: euthanasia of animals used in science. http://www.ccac.ca/Documents/Standards/Guidelines/Euthanasia.pdf
Cervin, A., Linderberg, S. 1998. Changes in mucociliary activity may be used to investigate the
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Concas, A., Sanna, E., Cuccheddu, T., Paola, M. 1993. Carbon dioxide inhalation, stress and
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anxiogenic drugs reduce the function of GABAA receptor complex in the rat brain. Prog.
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Costall, B., Jones, B.J., Kelly, M.E., Naylor, R.J., Tomkins, D.M. 1989. Exploration of mice in a black and white box: validation as a model of anxiety. Pharmacol. Biochem. Behav. 32:777-
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Crawley, J.N., Goodwin, F.K. 1980. Preliminary report of a simple animal behaviour for the anxiolytic effects of benzodiazepines. Pharmacol. Biochem. Behav. 13:167-170. Doi, M., Ikeda, K. 1993. Airway irritation produced by volatile anaesthetics during brief
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Flecknell, P. 2009. Laboratory animal anaesthesia, third ed. Academic Press, UK. p54.
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Hascoet, M., Bourin, M. 1998. A new approach to the light/dark procedure in mice. Pharamcol.
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Kirkden, R.D., Niel, L., Lee, G., Makowska, I.J., Pfaffinger, M.J., Weary, D.M. 2008. The
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validity of using an approach-avoidance test to measure the strength of aversion to carbon
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dioxide in rats. Appl. Anim. Behav. Sci. 114:216-234.
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Krohn, T.C., Hansen, A.K., Dragsted, N. 2003. The impact of low levels of carbon dioxide on rats. Lab. Anim. 37:94-99.
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Leach, M.C., Bowell, V.A., Allan, T.F., Morton, D.B. 2004. Measurement of aversion to
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determine humane methods of anaesthesia and euthanasia. Anim. Welfare. 13:S77-S86
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Leach, M.C., Bowell, V.A., Allan, T.F., Morton, D.B. 2002a. Aversion to gaseous euthanasia agents in rats and mice. Comp. Med. 52:249-257.
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Leach, M.C., Bowell, V.A, Allan, T.F., Morton, D.B. 2002b. Degrees of aversion shown by rats
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and mice to different concentrations of inhalational anaesthetics. Vet. Rec. 150:808-815.
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euthanasia for laboratory mice. Appl. Anim. Behav. Sci. 121:230-235.
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Makowska, I.J., Vickers, L., Mancell, J., Weary, D.M. 2009. Evaluating methods of gas
Makowska, I.J., Weary, D.M. 2009. Rat aversion to induction with inhalant anaesthetics. Appl.
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Matynia, A., Parikh, S., Chen, B., Kim, P., McNeill, D.S., Nusinowitz, S., Evans, C., Gorin, M.B. 2012. Intrinsically photosensitive retinal ganglion cells are the primary but not exclusive
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circuit for light aversion. Exp. Eye Res. 105:60-69.
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McLennan, I. S., Taylor-Jeffs, J. 2004. The use of sodium lamps to brightly illuminate mouse houses during their dark phases. Lab. Anim. 38(4):384-92. Niel, L., Stewart, S.A., Weary, D.M. 2008. Effect of flow rate on aversion to gradual-fill carbon
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dioxide exposure in rats. Appl. Anim. Behav. Sci. 109:77-84.
Niel, L, Weary, D.M. 2007. Rats avoid exposure to carbon dioxide and argon. Appl. Anim. Behav. Sci. 107:100-109.
Niel, L., Weary, D.M. 2006. Behavioural responses of rats to gradual-fill carbon dioxide euthanasia and reduced oxygen concentrations. Appl. Anim. Behav. Sci. 100:295-308.
Voikar, V., Koks, S., Vasar, E., Rauvala, H. 2001. Strain and gender differences in the behavior of mouse lines commonly used in transgenic studies. Wong, D., Makowska, I.J., Weary, D.M. 2013. Rat aversion to isoflurane versus carbon dioxide. Biol. Lett. 9:20121000.
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Ziemann, A.E., Allen, J.E., Dahdaleh, N.S., Drebot, I.I., Coryell, M., Wunsch, A.M., Lynch,
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C.M., Faraci, F.M., Howard, M.A., Welsh, M.J., Wemmie, J.A. 2009. The amygdala is a
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chemosensor that detects carbon dioxide and acidosis to elicit fear behavior. Cell. 139:1012-
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1021.
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Fig. 1. Rising concentrations of test gases during a) CO2 administered at 20% chamber vol/min
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gradual-fill (based on gas fill equations), and b) isoflurane administered either with a vaporizer
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(using a 5% concentration and 4 L/min O2 as the carrier gas) or the drop method (measured using
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a capnograph).
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Fig. 2. Diagram of the light-dark experimental apparatus
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Fig. 3. Mean (± S.E.) initial withdrawal time for: a) mice to leave the dark compartment after
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initial exposure to CO2 (n=8), isoflurane vaporiser (n=9) and isoflurane drop (n=9), and b) re-
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entry dwelling time spent in the dark compartment after first leaving when exposed to CO2 (n=5),
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isoflurane via the vaporiser (n=7) or the drop method (n=6).
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Fig. 4. Mean (± S.E.) initial withdrawal time during initial (n=8) and re-exposure (n=8) to the
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isoflurane vaporiser treatment, as well as re-entry dwelling time during initial (n=7) and re-
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exposure (n=7).
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Table 1
Table 1. Behaviours used to assess mice in the light-dark box. Definition
Initial withdrawal Recumbency
Initial time exposed to isoflurane or CO2 before choosing to leave the dark compartment Head resting on floor with loss of muscle tone for 5 s
Re-entries
Number of times a mouse chose to re-enter the dark side after first leaving
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Re-entry dwelling Time spent in the dark compartment after initially leaving
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Table 2
Table 2. The number of mice that became recumbent in the dark compartment relative to the number of mice in each treatment, and mean (±S.E.) number of times mice re-entered the dark compartment.
0/8
2/9
5/9
II. Re-entries
1.0 ± 0.3
1.2 ± 0.4
3.6 ± 1.0
2/9
1.0 ± 0.4
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Isoflurane Drop Isoflurane Vaporiser Isoflurane Vaporiser Initial Exposure Re-exposure
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S0168-1591(14)00114-2 http://dx.doi.org/doi:10.1016/j.applanim.2014.04.011 APPLAN 3897
To appear in:
APPLAN
Received date: Revised date: Accepted date:
3-10-2013 23-4-2014 28-4-2014
Please cite this article as: Moody, C.M., Weary, D.M.,Mouse aversion to isoflurane versus carbon dioxide gas, Applied Animal Behaviour Science (2014), http://dx.doi.org/10.1016/j.applanim.2014.04.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights Exposure to isoflurane via a vaporiser is more aversive to mice than carbon dioxide Mice are more willing to be exposed to isoflurane via a vaporiser than the drop method Mice find isoflurane re-exposure more aversive than initial exposure
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Title:
Mouse aversion to isoflurane versus carbon dioxide gas
Authors:
Carly M Moody* and Daniel M Weary
Institutional affiliations:
Animal Welfare Program, Faculty of Land and Food Systems,
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University of British Columbia, 2357 Main Mall, Vancouver,
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British Columbia, Canada V6T 1Z4
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*
Email: [email protected]
Co-author:
Email: [email protected]
Corresponding author:
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Abstract
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Isoflurane and carbon dioxide (CO2) gas are used for rodent euthanasia. This study compared
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mouse aversion to isoflurane versus gradual-fill CO2 gas, and compared two methods of
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isoflurane delivery: vaporiser and drop. Mouse acclimation to a light–dark apparatus was used to
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create a light aversion test based on an unconditioned preference for dark versus light areas. Mice
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chose between remaining in a dark compartment with rising concentration of one of three
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treatments (20% gradual-fill chamber vol/min of CO2, n=8; 5% isoflurane administered using a
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vaporiser set at 4 L/min oxygen flow, n=9; or 5% liquid isoflurane dropped on gauze, n=9), or
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escaping to a brightly lit compartment. On average (±S.E.) mice left the dark compartment after
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29.2 6.1 s in the isoflurane vaporiser treatment. Initial withdrawal time was lower for the CO2
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treatment (P=0.04), averaging 16.6 2.8 s, and lower still for the isoflurane drop treatment
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(P<0.001), averaging 2.9 0.79 s. Five of nine mice became recumbent in the dark compartment
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when exposed to the isoflurane vaporiser treatment compared to only two of nine mice during the
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drop treatment (P=0.3) and zero of eight mice during the CO2 method (P=0.03). The isoflurane
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concentrations rose more quickly using the drop versus the vaporiser method, likely explaining
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the increased willingness of mice to be exposed to isoflurane administered via a vaporiser
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machine. Re-exposure to isoflurane with the vaporiser was more aversive than initial exposure;
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only two of nine mice stayed in the dark compartment until recumbency. These results support
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the recommendation that mice with no previous exposure to isoflurane should be euthanised using
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isoflurane administered by a vaporiser rather than CO2 gas, and suggest that the drop method (as
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applied in the current study) is not a suitable alternative.
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Keywords: Euthanasia, vaporiser, drop method, recumbency, light-dark paradigm
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1. Introduction Current laboratory rodent euthanasia guidelines recommend using an inhalant anaesthetic over
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carbon dioxide gas (CO2) for rodent euthanasia (American Veterinary Medical Association, 2013;
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Canadian Council on Animal Care, 2010). Current evidence suggests isoflurane is less aversive to
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mice and rats than CO2 (Leach et al., 2002b; Leach et al., 2004; Makowska and Weary 2009;
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Wong et al., 2013) and other inhalant anaesthetics (Makowska et al., 2009). Isoflurane is a
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volatile liquid halogenated hydrocarbon. Generally, one of two methods can be used to administer
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isoflurane for euthanasia: a vaporiser machine or the drop method. When administering isoflurane
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using a vaporiser machine, a carrier gas and an anaesthetic waste gas scavenging system is
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required. Some animal users argue that the use of a vaporiser is unnecessary for rodent
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euthanasia. Vaporisers are intended to control the amount administered to reduce the risk of
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anaesthestic overdose, a feature of little value when the intention is to kill the animal. In addition,
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vaporiser machines can be costly to purchase and maintain, reducing accessibility for some users.
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The drop method involves placing liquid isoflurane on an absorbent material such as gauze, and
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placing this in a closed compartment. To our knowledge no studies have compared aversion to the
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drop versus vaporiser methods of isoflurane administration. In addition, many laboratories still
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use gradual-fill CO2 for euthanasia. Thus we tested mouse aversion to isoflurane administered by
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a vaporiser, isoflurane administered via the drop method, and gradual-fill CO2.
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The light-dark paradigm is a conflict-based anxiety test originally developed by Crawley and
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Goodwin (1980) to test anti-anxiety medications on mice. This paradigm uses the innate
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unconditioned preference for dark versus light areas and fear of open spaces in mice. The light-
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dark apparatus is composed of three compartments, a large light compartment, a small dark
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compartment and a middle compartment separating the light and dark areas. Acclimation to the
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apparatus changes this novel environment into a familiar one, therefore producing a light aversion
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test instead of testing anxiety (Matynia et al., 2012). This paradigm has been used to test rat
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aversion to CO2 versus isoflurane in rats (Wong et al., 2013).
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Using the light-dark box paradigm, we tested mouse aversion to three euthanasia methods: 1)
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20% gradual-fill chamber vol/min of CO2, 2) 5% isoflurane administered using a vaporiser set at
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4 L/min (40% chamber vol/min) oxygen (O2) flow, and 3) 5% isoflurane administered using the
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drop method. Mice were able to choose between remaining in a small dark compartment with a
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rising concentration of one of three treatments or escaping to a larger brightly lit compartment.
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Remaining in the small dark compartment with a rising concentration of test gas indicates that
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mice find the larger bright compartment more aversive than the test gas. Alternatively, leaving the
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small dark compartment with the test gas indicates that the test gas is more aversive than the large
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bright compartment. Initial exposure aversion was examined for all treatments. In addition, re-
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exposure aversion was examined for the isoflurane vaporiser treatment; mice commonly undergo
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surgical procedures using an isoflurane vaporiser machine, and re-exposure may be more aversive
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than initial exposure (Wong et al., 2013).
2. Materials and Methods
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2.1 Pre-trial
During the pre-trial we measured the rate at which isoflurane concentration increased within a
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compartment when using the vaporiser and drop treatments (Fig. 1b); the results allowed us to
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estimate the isoflurane concentrations that mice would be exposed to during the experiment. Use
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of a theoretical 20% gradual-fill CO2 curve (Fig. 1a), allowed us to estimate CO2 exposure
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concentrations during this experiment.
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An Innocage® disposable individually ventilated transparent mouse cage (Universal Euro Type
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II Long, Innovive Inc. San Diego, California, USA, 37.3 cm length x 23.4 cm width x 14.0 cm
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height, with 205 cm2 floor space) was used as the test cage. A Plexiglass lid with a centrally
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placed hole was placed on top of the cage during testing. A Capnomac Ultima™ (Datex Ohmeda
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Instrumentation Corporation, Helsinki, Finland) capnograph was used to measure the rising
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concentration of isoflurane in the cage, via a polyethylene/polyvinalchloride sampling line (Datex
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Ohmeda Instrument Corporation, Finland) inserted into a hole near the base of the anterior wall of
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the cage. Testing took place in Medical Block C at the University of British Columbia,
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Vancouver, Canada. For the isoflurane vaporiser treatment, 5% isoflurane (Baxter Corporation, Ontario, Canada)
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was administered via an Isotec 4 isoflurane vaporiser (Ohmeda, Steeton, West Yorkshire,
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England, UK) using 4 L/min (33% chamber vol/min) of room air as the carrier gas. The
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isoflurane drop treatment used wire mesh (Activ-wire mesh, Activa Products Inc., Marshall,
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TX, USA) shaped to create a rectangular apparatus (7 cm length x 3 cm width x 11 cm height),
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that was closed on all sides except the top, to allow a piece of 5.1 cm x 5.1 cm gauze
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(Professional Preference, Rafter 8 Products, Calgary, AB, Canada) opened length-wise for
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vertical placement down into the wire rectangular apparatus. The wire apparatus was placed at the
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end of the rectangular test cage, standing up against the cage wall. The volume of isoflurane
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required to provide a 5% concentration in the compartment was determined to be 4.6 mL using
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the universal gas law (PV=nRT) and a room temperature of 22 °C. A glass syringe was used to
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distribute the liquid isoflurane down the piece of gauze within the wire mesh apparatus.
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2.2 Experiment
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2.2.1 Animals and Housing
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We used 30 male C57BL/6J mice housed at the University of British Columbia’s Centre
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for Disease Modeling, Vancouver, Canada. Male mice were used in the present study on the
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grounds that the light-dark paradigm was validated and developed using male mice and that
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responses may vary with female estrus cycle (Voikar et al., 2001). Mice were housed in groups of
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three or five in ventilated polysulfone type II long cages (Bioscape, Germany, 20.7 cm length x
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14.0 cm width x 36.5 cm height), using an individually ventilated cage system (Bio A.S. cage
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rack and blower system, Bioscape, Germany). All mice weighed between 22.4 to 28.3 g, and were
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2 months old at the time of testing. Mice were housed with a nest box, brown crinkle paper
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(Enviro-dri, Shepherd Specialty Papers Inc., Richland, MI, USA), one cotton nest square (Ancare,
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Bellmore, NY, USA), beta chip bedding (Nepco, Northeastern Products, Warrensburg, NY,
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USA), ad libitum access to food (Harlan 2918 Tekland Global Rodent Maintenance, Madison,
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WI, USA) and reverse osmosis chlorinated water, and kept under a reverse 12 h light:12 h (lights
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off at 07:00 h) dark cycle with light intensity ranging from 240 to 340 lux throughout the light
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phase. All animal procedures were approved by the University of British Columbia’s Animal
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Care Committee.
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All acclimation and testing trials took place in a darkened test room lit by a 35-watt lowpressure sodium vapour lamp (Master SOX PSG, Philips Lighting Canada, Markham, ON,
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Canada) undetectable to mice (McLennan and Taylor-Jeffs, 2004). All mice were placed in a
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biological safety cabinet within the test room 1 h before acclimation or testing trials started, to
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allow adjustment to the test room. In comparison to the pre-trial, no gas concentrations were
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measured during any acclimation or testing trials.
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The experimental apparatus consisted of a Plexiglas test box (67 cm x 20 cm x 25 cm; Fig. 2)
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divided into three compartments: 1) a light compartment (40 cm x 20 cm x 25 cm), 2) a dark
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compartment (20 cm x 20 cm x 25 cm), and 3) a middle buffer compartment (6 cm x 6 cm x 6
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cm). The middle compartment connected the light and dark compartments and contained a wire
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mesh (ActivTM-wire mesh, Activa Products Inc., Marshall, TX, USA) passageway (6 cm length x
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6 cm width x 6 cm height) to allow a mouse to pass between the light and dark compartments
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while minimizing time spent in this compartment. The openings to the wire mesh passageway of
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the middle compartment were covered with thin, overlapping black plastic strips with vertical
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slits, to minimize gas exchange between the compartments while allowing the mice to pass
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through. Holes (1 cm diameter, 32 on each side) drilled into the side of the middle compartment
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helped vent any gases entering this compartment. Black plastic was placed on all sides of the dark
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compartment, except the front from where video recording took place. The experimenter sat away
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from the testing apparatus and was hidden by a blind. Fresh beta chip bedding was placed in to
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the light (500 mL) and dark (300 mL) compartments between testing cages of mice. A lamp
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(Barometer work lamp, Ikea, China) with a 7-watt parabolic aluminized reflector 20, light-
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emitting diode (LED; Lightline, Brampton, ON, Canada) that did not heat up over time was used
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as the light source. The lamp head was directed downwards over the centre of the light
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compartment to minimize light diffusion into the other compartments. At the start of each trial, a
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lux meter (Traceable Dual-Range light meter, VWR international, Radnor, PA, USA) was used to
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measure light intensity in the light and dark compartments; measurements in the dark
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compartment did not exceed 3 lux and the darkest corner of the light compartment exceeded 700
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lux. Past studies using the light-dark paradigm have shown that mice find 500 lux aversive
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(Costall et al., 1989; Matynia et al., 2012).
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A Plexiglas lid with two holes (1.6 cm diameter), each centrally placed above the light and
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dark compartments, was custom made to fit the testing apparatus. Each of the three treatments
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tested required a unique equipment assembly. During the isoflurane vaporiser acclimation trials,
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the lid holes over the light and dark compartments allowed for the placement of separate gas lines
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connected to the same O2 tank (Praxair, Delta, BC, Canada) using an O2 flow meter (Western
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Medica, Westlake, OH, USA) to deliver 4 L/min of O2 into each compartment. During isoflurane
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vaporiser testing trials, the gas line delivering 4 L/min O2 to the dark compartment, was replaced
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with a gas line delivering a flow of isoflurane (Baxter Corporation, Mississauga, ON, Canada) via
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a isoflurane vaporiser machine (Highland Medical Equipment, Temecula, CA, USA) at 5% with 4
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L/min O2 as the carrier gas. The CO2 treatment acclimation trials used an identical set up as the
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isoflurane vaporiser acclimation trials, except the gas lines delivered 2 L/min (20% compartment
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vol/min flow rate) of O2 into each compartment. The CO2 treatment testing trials were similar to
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the acclimation trials, except gas flow into the dark compartment involved a switch from O2 to 2
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L/min of CO2 (Praxair, Delta, BC, Canada) measured using a CO2 flow meter (Western Medica,
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Westlake, OH, USA). For the isoflurane drop treatment trials, the two centrally placed holes over
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the light and dark compartment in the Plexiglas lid were covered with duct tape, as no gas lines
166
were needed. Instead, wire mesh (Activ-wire mesh, Activa Products Inc., Marshall, TX, USA)
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was shaped to create a rectangular apparatus (11.5 cm length x 3 cm width x 24 cm height) that
168
was closed on all sides except the top, to allow a piece of 10.2 cm x 10.2 cm gauze (Professional
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Preference, Rafter 8 Products, Calgary, AB, Canada) to be opened and placed down the mesh
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apparatus to increase surface area. The opened top of the rectangular wire mesh apparatus
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allowed liquid isoflurane to be distributed down the gauze during experimental testing; only
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during the testing trials was isoflurane used.
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2.2.3 Acclimation
Mice were individually assigned to one of three treatments: isoflurane vaporiser, isoflurane
176
drop or CO2 gas. As well, the cages of mice were randomized with regards to the order of testing.
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Mice were individually acclimated to the test apparatus every other day for 6 d, totaling three
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acclimation trials each. Before the start of each acclimation trial, the light source above the light
179
compartment was turned on and the lux meter was used to measure light intensity in the light and
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dark compartments. For both the isoflurane vaporiser and CO2 treatments, gas lines were placed
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into the holes over the dark and light compartments, delivering 4 L/min or 2 L/min of O2,
182
respectively. For the isoflurane drop treatment, the holes in the Plexiglas lid were covered and the
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wire mesh apparatus was placed into the dark side against the end of the dark compartment. The
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start of the acclimation trials occurred when a mouse was placed into the light compartment. Our
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light source was aversive to the mice, as shown by mouse preference for the dark compartment.
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Mice frequently moved back and forth between compartments, spending more time in the dark
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compartment but rarely staying longer than 1.5 min. On this basis we used the criterion of 1
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continuous min in the dark compartment to signify preference, as mice are known to show high
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frequencies of entries and exits (Leach et al., 2002a). The trial ended after 20 min or when 1 min
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was spent continuously in the dark side, whichever occurred first. The test apparatus was cleaned with 70% alcohol between cages of mice and then aired out
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and wiped with water to decrease any smell or novelty. The apparatus was not cleaned between
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mice that shared the same home cage, following Hascoet and Bourin (1998). These authors
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suggest that mice are more influenced by light and dark areas within a light-dark box when using
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a soiled apparatus.
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Of 30 mice included in the study, four did not spend 1 min in the dark side during the acclimation trials and were thus removed from the study. A total of eight CO2 treatment mice and
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nine for each the isoflurane vaporiser and drop method treatments, moved on to the testing trials.
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Testing took place during the dark cycle between 10:00 and 17:00 h, at an average (±S.D.)
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room temperature of 20.6 ± 0.5 °C. Experimental testing followed the acclimation procedure until
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a mouse spent 1 continuous min in the dark side, our criterion for preference. At this point, mice
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were tested for aversion to one of the three treatments in the dark compartment. For isoflurane
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vaporiser trials, the gas line delivering 4 L/min of O2 to the dark compartment was replaced with
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a gas line delivering 5% isoflurane via a vaporiser machine, with 4 L/min of O2 as the carrier gas.
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For CO2 trials, the gas line delivering 2 L/min of O2 to the dark compartment was replaced with a
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gas line delivering 2 L/min of CO2. For all isoflurane drop trials, the Plexiglas lid was opened just
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enough to deliver 3.7 mL isoflurane (5% volume as determined using the universal gas law:
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PV=nRT, with 20°C room temperature) onto the gauze within the wire mesh apparatus, and then
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the lid was closed.
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Trials ended when a mouse became recumbent in the dark compartment, or when 2 min were spent continuously in the light compartment indicating aversion to the gas treatment. Mice that
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stayed in the dark compartment until recumbency were transferred to an empty cage, identical to
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the home cage, placed on a heating pad, and allowed to recover before being returned to their
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home cage. Each mouse was tested only once, except those included in the vaporiser treatment.
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All the vaporiser treatment mice were re-tested once with the same procedure. Re-exposure
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occurred 1 week after initial exposure. These trials followed the same procedure as the initial test
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exposure.
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After completing these trials, all mice were euthanised using 5% isoflurane gas with 4 L/min of O2 as the carrier gas for anaesthetic induction, followed by 50% CO2 to complete the
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euthanasia procedure. Mice were then cervically dislocated as the secondary method of
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euthanasia.
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Both the pre-trial and the experimental trials were video recorded using a high definition camcorder (Model TM41P, Panasonic Corporation, Malaysia) in advanced coding high definition
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recording mode with 30 frames/s. Pre-trial videos were used to record capnograph readings every
229
5 s. Testing trials were scored for: initial withdrawal time, recumbency, number of re-entries, and
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re-entry dwelling time in the dark compartment (Table 1). Only those mice that re-entered the
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dark compartment were used to analyse the number of re-entries and re-entry dwelling time.
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2.4 Statistical Analysis
The effect of treatment on initial withdrawal time, number of re-entries, and re-entry dwelling
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time was tested using ANOVA (SAS v. 9.3), with the difference between the two methods of
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isoflurane induction compared using one contrast statement, and the difference between the
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vaporizer method and the CO2 method compared using a second contrast. Mice that never left the
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dark compartment upon gas exposure were not included in the analysis for the number of re-visits
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or re-entry dwelling time. Differences in response to initial versus re-exposure with the vaporizer
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method were tested using a mixed model (SAS v. 9.3). For initial exposure trials, a Fisher exact
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test assessed the effect of treatment on the number of mice that became recumbent in the dark
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side. A Cochran-Mantel-Haenszel test was used to analyse the number of mice that became
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recumbent in the dark side for initial versus re-exposure isoflurane vaporiser trials. Means are
244
reported ± S.E.
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3.Results
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3.1 Pre-trial
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The drop method of isoflurane administration resulted in a faster increase in concentration
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compared with the isoflurane vaporiser method (Fig. 1b). The target concentration of 5% was
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achieved after 5 min using the vaporiser versus 30 s when using the drop method.
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3.2.1 Initial Exposure
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Initial withdrawal time from the dark compartment was longer for mice exposed to isoflurane by the vaporiser machine compared with the drop (F1,18= 22.6, P<0.0001; Fig. 3a) and CO2
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methods (F1,17= 4.9, P= 0.04). Mice were also more likely to become recumbent in the dark
257
compartment when isoflurane was delivered using the vaporiser versus drop method (F1,18= 7.0,
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P= 0.3; Table 2) or CO2 (F1,17= 8.0, P=0.03). Of the five mice that became recumbent during
259
isoflurane vaporiser exposure, two never left the dark compartment after the isoflurane was
260
turned on and three became recumbent upon re-entry. With the drop method, two mice became
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recumbent in the dark compartment upon re-entry. None of the eight CO2 treatment mice stayed
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in the dark compartment until recumbency upon initially leaving or re-entry.
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Mice exposed to the isoflurane vaporiser treatment more often re-entered the dark
264
compartment after initially leaving, compared to either the drop method (F1,18= 7.5, P=0.013;
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Table 2) or the CO2 treatment (F1,17=8.5, P=0.008). The re-entry dwelling time in the dark
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compartment was greater in the vaporiser treatment versus both the drop (F1,12= 15.8, P=0.0012;
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Fig. 3b) and CO2 treatments (F1, 11= 19.7, P=0.0005).
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3.2.2 Re-exposure: Isoflurane vaporiser treatment
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The initial withdrawal time from the dark compartment decreased from a mean of 29.2 ± 6.1 s to 19.1 ± 5.0 s upon re-exposure to isoflurane (F1,8= 3.7, P=0.09; Fig. 4). Only two of nine mice
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became recumbent during re-exposure, compared to five of nine mice during initial exposure
273
(Table 2). The two mice that became recumbent during re-exposure had also become recumbent
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during the initial exposure trials. The number of re-entries to the dark compartment decreased
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from a mean of 3.6 ± 1.0 during initial exposure to 1.0 ± 0.4 upon re-exposure (F1,6=5.8, P=0.05;
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Table 2). Re-entry dwelling time in the dark compartment decreased from 24.1 ± 4.2 s during
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initial exposure to 2.3 ± 0.9 s during re-exposure (F1,6=34.3, P=0.0011; Fig. 4).
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4. Discussion
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In the current study the vaporiser was at the highest setting possible (5%) combined with the
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highest flow rate of O2 (4 L/min); these settings are chosen to minimize the time between onset of
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aversion and insensibility when using the vaporiser (Makowska et al., 2009). During the pre-trial,
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the drop method isoflurane concentration rose to a maximum of 7.5% and then leveled out at 5%,
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likely due to sampling line placement within the cage. The results of the pre-trial showed that
285
isoflurane concentration rises much more quickly when using the drop method versus the
286
vaporiser. This difference means that mice spending the same amount of time in the dark
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compartment would be exposed to a higher total dosage of isoflurane in the drop versus vaporiser
288
treatments. The initial withdrawal time from the dark compartment was 2.9 versus 29.2 s with the
289
isoflurane drop and vaporiser treatments, respectively. At these times, the isoflurane
290
concentration in the dark compartment would have been approximately 1 to 2%, suggesting that
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concentrations in excess of 1% are aversive to mice. Leach et al. (2002b) also reported that
292
isoflurane was aversive at these concentrations.
293
The number of re-entries to the dark side averaged one and four for the isoflurane drop and vaporiser treatments, respectively; this difference again may be attributed to the slower build up
295
of isoflurane concentration using the vaporiser method. When mice chose to re-enter the dark side
296
during the isoflurane drop treatment, the concentration of isoflurane was likely higher than at the
297
equivalent time in the vaporiser treatment. This difference likely also explains the decreased re-
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entry dwelling time in the dark compartment with the drop treatment versus the vaporiser
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treatment.
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The initial withdrawal time for mice to leave the dark compartment was longer for the vaporiser treatment versus gradual-fill CO2. The concentration of CO2 likely exceeded 10% when
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mice chose to leave the dark compartment, based upon a theoretical 20% chamber vol/min
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gradual-fill CO2 curve. Previous studies have shown that CO2 concentrations ranging from 3-20%
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are aversive in mice and rats (Kirkden et al., 2008; Krohn et al., 2003; Makowska et al., 2009;
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Niel and Weary, 2007; Niel et al., 2008); the current results are consistent with these findings. In
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addition, concentrations of CO2 between 10-35% have been shown to cause fear responses in
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mice and rats (Concas et al., 1993; Niel and Weary, 2006; Ziemann et al., 2009); and a study by
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Ziemann et al. (2009) suggests that 10% CO2 acts an unconditioned fear stimulus. Thus, the
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aversion results of the current study build upon the abundance of existing work showing that CO2
310
is aversive in rodents.
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The number of visits and re-entry dwelling time were both lower for mice exposed to CO2
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compared to vaporised isoflurane, suggesting that initial exposure to CO2 is more aversive than
313
initial exposure to vaporised isoflurane. None of the mice exposed to CO2 stayed in the dark
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compartment until recumbency, compared to about half of the mice tested with the isoflurane
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vaporiser treatment. It is possible mice that stayed in the dark compartment until recumbency may
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have become too sedated to escape the gas-filling compartment even if they wanted to. However,
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no mice stayed until recumbency with CO2, suggesting that induction with isoflurane is less
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aversive than induction with CO2. In accordance with past aversion work (Leach et al., 2002b;
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Leach et al., 2004; Makowska et al., 2009; Makowska and Weary, 2009; Wong et al., 2013), the
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results of this study indicate that mice find CO2 more aversive than isoflurane.
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Our results suggests that re-exposure to isoflurane is more aversive than initial exposure. A previous study (Makowska et al., 2009) using an approach-avoidance paradigm failed to find
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learned aversion to isoflurane in mice, but a more recent study using the light-dark paradigm
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(Wong et al., 2013) found learned aversion in rats re-exposed to isoflurane. These contradictory
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results suggest that more work is needed to understand learned aversion associated with re-
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exposure to isoflurane in rodents. As well, perhaps the protocols involving repeated exposure to
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isoflurane should be re-considered.
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In agreement with current literature, the current study results suggest that mice tolerate exposure to concentrations of isoflurane that cause anaesthesia (delivered by a vaporiser) but not
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with CO2 gas. Mice always chose to leave the dark compartment and enter the larger brightly lit
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compartment before CO2 concentrations reached those that could render a mouse insensible.
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Although this study showed that isoflurane was less aversive than CO2, the treatment was still
333
aversive as indicated by some mice choosing to leave the dark compartment. Isoflurane has a
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pungent odour (Flecknell, 2009) and is known to cause eye irritation and irritation to upper
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airway mucosa (Cervin and Lindberg,1998; Doi and Ikeda, 1993); these effects are likely
336
pronounced at higher concentrations, such as those experienced in the drop method treatment of
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the current study.
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5. Conclusion
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The results of the current study correspond with other recent results indicating that mouse
341
euthanasia using isoflurane administered by a vaporiser is a humane alternative to the use of
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gradual-fill CO2. Some animal users without access to a vaporiser may wish to use the drop
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method, but our results suggest that the drop method (as tested in the current study) should be
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avoided. Re-exposure to isoflurane administered with a vaporiser was more aversive than initial
345
exposure, suggesting that re-exposure to isoflurane should be avoided.
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346 Acknowledgements
We thank Jurgen Pehlke for building the light-dark box apparatus and Warren Riley from
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Innovive Inc. (USA) for donating the test cage used in the pre-trial. We also thank Devina Wong
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for input into experimental design and Bernard Macleod and Tim Fung for help with the pre-trial.
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We are grateful to Joanna Makowska for review of this manuscript and many conversations
352
regarding rodent euthanasia. This research was funded by a Discover grant to DMW from the
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Natural Sciences and Engineering Research Council of Canada.
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American Veterinary Medical Association. 2013. Guidelines for the euthanasia of animals: 2013
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Canadian Council on Animal Care. 2010. CCAC guidelines on: euthanasia of animals used in science. http://www.ccac.ca/Documents/Standards/Guidelines/Euthanasia.pdf
Cervin, A., Linderberg, S. 1998. Changes in mucociliary activity may be used to investigate the
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Hascoet, M., Bourin, M. 1998. A new approach to the light/dark procedure in mice. Pharamcol.
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Krohn, T.C., Hansen, A.K., Dragsted, N. 2003. The impact of low levels of carbon dioxide on rats. Lab. Anim. 37:94-99.
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Leach, M.C., Bowell, V.A., Allan, T.F., Morton, D.B. 2004. Measurement of aversion to
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Leach, M.C., Bowell, V.A., Allan, T.F., Morton, D.B. 2002a. Aversion to gaseous euthanasia agents in rats and mice. Comp. Med. 52:249-257.
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Leach, M.C., Bowell, V.A, Allan, T.F., Morton, D.B. 2002b. Degrees of aversion shown by rats
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McLennan, I. S., Taylor-Jeffs, J. 2004. The use of sodium lamps to brightly illuminate mouse houses during their dark phases. Lab. Anim. 38(4):384-92. Niel, L., Stewart, S.A., Weary, D.M. 2008. Effect of flow rate on aversion to gradual-fill carbon
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Niel, L., Weary, D.M. 2006. Behavioural responses of rats to gradual-fill carbon dioxide euthanasia and reduced oxygen concentrations. Appl. Anim. Behav. Sci. 100:295-308.
Voikar, V., Koks, S., Vasar, E., Rauvala, H. 2001. Strain and gender differences in the behavior of mouse lines commonly used in transgenic studies. Wong, D., Makowska, I.J., Weary, D.M. 2013. Rat aversion to isoflurane versus carbon dioxide. Biol. Lett. 9:20121000.
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Ziemann, A.E., Allen, J.E., Dahdaleh, N.S., Drebot, I.I., Coryell, M., Wunsch, A.M., Lynch,
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C.M., Faraci, F.M., Howard, M.A., Welsh, M.J., Wemmie, J.A. 2009. The amygdala is a
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1021.
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Fig. 1. Rising concentrations of test gases during a) CO2 administered at 20% chamber vol/min
412
gradual-fill (based on gas fill equations), and b) isoflurane administered either with a vaporizer
413
(using a 5% concentration and 4 L/min O2 as the carrier gas) or the drop method (measured using
414
a capnograph).
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Fig. 2. Diagram of the light-dark experimental apparatus
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Fig. 3. Mean (± S.E.) initial withdrawal time for: a) mice to leave the dark compartment after
419
initial exposure to CO2 (n=8), isoflurane vaporiser (n=9) and isoflurane drop (n=9), and b) re-
420
entry dwelling time spent in the dark compartment after first leaving when exposed to CO2 (n=5),
421
isoflurane via the vaporiser (n=7) or the drop method (n=6).
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Fig. 4. Mean (± S.E.) initial withdrawal time during initial (n=8) and re-exposure (n=8) to the
423
isoflurane vaporiser treatment, as well as re-entry dwelling time during initial (n=7) and re-
424
exposure (n=7).
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Table 1
Table 1. Behaviours used to assess mice in the light-dark box. Definition
Initial withdrawal Recumbency
Initial time exposed to isoflurane or CO2 before choosing to leave the dark compartment Head resting on floor with loss of muscle tone for 5 s
Re-entries
Number of times a mouse chose to re-enter the dark side after first leaving
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Re-entry dwelling Time spent in the dark compartment after initially leaving
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Table 2
Table 2. The number of mice that became recumbent in the dark compartment relative to the number of mice in each treatment, and mean (±S.E.) number of times mice re-entered the dark compartment.
0/8
2/9
5/9
II. Re-entries
1.0 ± 0.3
1.2 ± 0.4
3.6 ± 1.0
2/9
1.0 ± 0.4
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Isoflurane Drop Isoflurane Vaporiser Isoflurane Vaporiser Initial Exposure Re-exposure
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Fig. 1
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Fig. 4
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