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Determination of the minimum alveolar concentration for halothane, isoflurane and sevoflurane in the gerbil I A Gómez de Segura, J Benito de la Víbora and A Criado Lab Anim 2009 43: 239 DOI: 10.1258/la.2008.006065 The online version of this article can be found at: http://lan.sagepub.com/content/43/3/239
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Original Article Determination of the minimum alveolar concentration for halothane, isoflurane and sevoflurane in the gerbil I A Go´mez de Segura1, J Benito de la Vı´bora1,2 and A Criado3 1
Department of Animal Medicine and Surgery, Veterinary Faculty, University Complutense of Madrid, Avda. Puerta de Hierro, 28040 Madrid, Spain; 2ECLAM Resident, Experimental Surgery, La Paz University Hospital, Paseo de la Castellana 261, 28046 Madrid, Spain; 3Laboratory Animal Science Department, GlaxoSmithKline, Verona, Italy Corresponding author: I A Go´mez de Segura. Email:
[email protected]
Abstract The present work determined the minimum alveolar concentrations (MAC) of halothane, isoflurane and sevoflurane in adult female gerbils (n ¼ 24). Animals were placed in a chamber for anaesthetic induction before performing tracheal intubation. The tracheal tube was connected to a non-rebreathing circuit with minimal dead space. Body temperature, blood pressure, heart and respiratory rates and end-tidal volatile anaesthetic levels were measured continuously. MAC was determined using a standard noxious stimulus (tail-clamp). All experiments were performed at the same time of the day, body temperature was maintained constant and blood–gas analysis was performed to confirm that values were within normal limits. The mean + SD MAC values were 1.06 + 0.11% halothane (n ¼ 8), 1.55 + 0.08% isoflurane (n ¼ 8) and 2.90 + 0.12% sevoflurane (n ¼ 7). Cardiovascular parameters at 1 MAC did not differ significantly among anaesthetics but the respiratory rate was significantly higher in the halothane group than in the isoflurane and sevoflurane groups. The SpO2 values recorded throughout anaesthesia and the pH and partial oxygen pressure values determined at the end of the study did not differ among the studied anaesthetics at 1 MAC. These data suggest that the MAC for halogenated inhalant anaesthetics in gerbils is lower than the average MAC values obtained in rats and mice.
Keywords: Inhalant anaesthetics, minimum alveolar concentration, gerbil, isoflurane, halothane, sevoflurane Laboratory Animals 2009; 43: 239– 242. DOI: 10.1258/la.2008.006065
The gerbil has become a common laboratory animal and is widely employed to investigate epilepsy, ageing or gastric cancer and is also a highly suitable reproducible animal model for cerebral ischaemia because it lacks a complete circle of Willis.1 Inhalant anaesthesia is commonly used to provide an adequate and safe level of anaesthesia in most species.2 However, appropriate use of these agents requires a clinical evaluation of their effects and available data on the proper use of inhalants in laboratory gerbil anaesthesia is limited.3,4 Minimum alveolar concentration (MAC) provides a reference value for anaesthetic potency that allows a comparison of effects among the different anaesthetics. MAC is defined as the concentration (at one atmosphere) that prevents movement in response to a standard noxious stimulus in 50% of subjects.5 MAC varies among species. Thus to provide the most appropriate level of anaesthesia during surgery requires a knowledge of MAC. The present study reports the MAC of the most common inhalant anaesthetics (halothane, isoflurane and sevoflurane) in the gerbil.
Materials and methods Animals All procedures were performed after approval from the Institutional Animal Care Committee, adhering to the European criteria for the protection of animals used for experimental and other scientific purposes (86/609/EEC). Twentyfour adult female Mongolian gerbils (Meriones unguiculatus) weighing 60.1 + 7.7 g were taken from the laboratory colony stock purchased from Janvier (Le Genest, Saint-Isle, France). Husbandry during experiment The animals were housed in groups in type IV Makrolon cages (Panlab, Barcelona, Spain) with a 12 h light:12 h dark cycle, relative humidity between 50% and 70%, and room temperature of 20 + 28C. Feeding Animals were fed a maintenance diet for rodents of food pellets (wheat 50%, corn 13.8%, vitamin A additive Laboratory Animals 2009; 43: 239– 242
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15,000 IU/kg, vitamin D3 1500 IU/kg, crude protein 15.4%, crude fat 2.9%, crude fibres 4.1%, ash 5.9%; Standard Commercial Diet, Panlab, Reus, Spain) and water provided ad libitum. Experimental procedure Animals were randomly assigned to receive halothane (n ¼ 8); isoflurane (n ¼ 8) or sevoflurane (n ¼ 8). Halothane was obtained from AstraZeneca (Fluothanew, Madrid, Spain) and isoflurane and sevoflurane from Abbott Laboratories (Foranew and Sevoranew, Madrid, Spain). All studies were performed during the morning starting at 09:00 h. Laboratory room temperature was 23.4 + 2.08C. Anaesthesia was induced in a chamber receiving oxygen 3 L/min containing 5% halothane, 5% isoflurane (Ohmeda Fluotec 3 and Isotec 3, BOC Health Care, Steeton, UK) or 8% sevoflurane (Vapor 2000, Dra¨ger, Lubeck, Germany). Once the animal became anaesthetized, usually within 2 – 3 min, tracheal intubation was performed using a 20-gauge polyethylene catheter (Abbott, Sligo, Ireland) with the animal positioned in sternal recumbency using a technique similar to that described for rats.6 After the correct position of the catheter was ascertained, animals were positioned in dorsal recumbency and connected to a non-rebreathing T-piece circuit with minimal dead space. Fresh gas flow to the T piece was adjusted to 0.75 L/min and the inspired concentration was reduced (approximately 1.5% halothane, 2% isoflurane and 2.5% sevoflurane). Once the gerbils were anaesthetized and instrumented, baseline monitoring data were recorded before placing the animals inside a zip-lock plastic bag. This was employed to minimize potential room air contamination of the fresh gas through the unsealed trachea, i.e. between the tracheal wall and the external part of the endotracheal tube since the tube was uncuffed. Also, the Ayre’s T piece was included in the plastic bag, so all fresh gas flow entered the lungs and also the bag maintaining the same anaesthetic concentration. Pulse rate (PR), respiratory rate (RR), blood oxygen saturation (SatO2), CO2 and volatile anaesthetic concentration (sampled from the endotracheal tube) were measured and recorded continuously (Capnomac Ultima, Datex-Ohmeda, Helsinki, Finland). The femoral artery was catheterized with PE50 tubing (Silastic, Dow Corning Corporation) after surgical cut-down. This access allowed arterial blood sampling and blood pressure measurement via a calibrated pressure transducer (Transpac IV, Abbott Laboratories, Abbott Park, IL, USA). Heart rate, systolic, diastolic and mean arterial blood pressures and electrocardiograph (needle electrodes placed on the left and right hindlimbs and the right forelimb) were recorded continuously (CM-8, Schiller, Switzerland). Arterial blood (0.3 mL, BD Preset, BD Diagnostics, Plymouth, UK) was collected for blood – gas analysis (Rapidlab860, Bayer) at the end of the study to ensure that values (at that time point) were within normal limits of pH (7.35 –7.45), partial oxygen pressure (PaO2 . 100 mmHg breathing oxygen 100%) and partial carbon dioxide pressure of (PaCO2 , 45 mmHg). Rectal temperature was monitored and kept between 378C and 388C by means of a circulating water warming
blanket (Heat Therapy Pump, Model TP-220, Gaymar, Orchand Park, NY, USA). Endotracheal tube gas sampling was used to measure the anaesthetic gas concentration for the MAC determination employing a fine 21 G needle with the tip located at the level of the luer cone. A similar method has been described in detail previously for rats and referred to as alveolar agent concentration, with the catheter located at the level of the carina.7 The proximal end of the needle was connected to a side-stream infrared analyser (Capnomac Ultima, Datex-Ohmeda, Helsinki, Finland), which was calibrated before each experiment using standard calibration gases provided by the manufacturer (Quick Cal Calibration Gas, Datex-Ohmeda, Helsinki, Finland). Values were taken in triplicate to ensure constant alveolar concentration. For this, four consecutive responses, i.e. positive– negative– positive –negative, or vice versa, allowed three MAC values when considering the average value between two consecutive, and different, responses. The final MAC value is obtained as the average value of the three individual MAC values previously obtained. MAC was determined by applying a standard noxious stimulus (tail-clamp), with a long haemostat (8 in. Rochester Dean Haemostatic forceps) clamped to the first ratchet lock on the tail for up to 60 s and less time if the gerbil moved. The tail was always stimulated proximal to a previous test site. A positive response was considered when a gross purposeful movement of the head, limbs and/or body was observed, whereas a negative response was considered the lack of movement or grimacing, swallowing, chewing or tail flick. When a negative response was seen, the alveolar agent concentration was then reduced in decrements of 0.1 –0.15% until the negative response became positive. After each step change in anaesthetic concentration delivered by the anaesthetic circuit, at least 15 min were allowed for equilibration before new gas samples were taken. The average time elapsed between two consecutive anaesthetic alveolar concentration determinations was approximately 30 min. MAC was considered to be the concentration midway between the highest concentration that permitted movement in response to the stimulus and the lowest concentration that prevented movement. Overall, the whole experiment lasted an average of 227 + 85 min. Finally, an arterial blood sample (0.3 mL) was collected from the femoral artery and a blood –gas analysis was performed to ensure that values were within normal limits. The gerbils were euthanized with an intravenous injection of potassium chloride (7.45%) while still deeply anaesthetized. Statistical procedures Data are expressed as mean + SD. All variables were tested to ensure that they fulfilled the requirements of normal distribution to allow the use of the analysis of variance (ANOVA) test. One-way ANOVA with linear contrast was performed. Post hoc Tukey test was used for multiple comparisons between groups. A P value , 0.05 was set to indicate statistical significance. The statistical analysis was
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performed with statistical software (SPSS for Windows 11, SPSS Inc, Chicago, IL, USA).
Results MAC of halothane, isoflurane and sevoflurane The obtained MAC values were: 1.06 + 0.11% halothane, 1.55 + 0.08% isoflurane and 2.90+0.12% sevoflurane. These data were obtained at similar body temperatures (Table 1). Cardiovascular and respiratory effects Overall, there were no significant differences between anaesthetics in their cardiovascular effects. The RR was significantly increased when halothane was given compared with the same animals receiving isoflurane or sevoflurane (P , 0.05). The pH value was within the normal range in the three groups. There were no significant differences for PaO2 and for SatO2 among groups. The PaCO2 and base excess values were not different among anaesthetics (Table 1). One animal became apnoeic and died due to airway obstruction by secretions from the sevoflurane group and was excluded from the analysis of the data.
Discussion MAC values allow an assessment of the potency of inhaled anaesthetics. Such values represent equipotent doses that permit pharmacological comparisons and give information on the effective and safe administration concentrations. They can facilitate comparison of anaesthetic effects among species.8 This is the first report of MAC values for common halogenated anaesthetics in the gerbil. Previous studies of the potency of these agents employed alternative methods. A recent evaluation of anaesthetic concentrations required to prevent response to interdigital fold clamping found a need for higher isoflurane and sevoflurane concentrations delivered through a nose cone.4 This can be explained by the employed method of measurement leading to an
overestimation of the required concentration, i.e. delivered vs. exhaled gases, since measurement of inspired gases provide a greater value than the use of end-tidal (alveolar) anaesthetic concentrations because of the uptake of the anaesthetic. This difference is reduced or minimized when appropriate equilibration times are provided. Previously, the effective dose-50 (ED50) of halothane of 1.32 vol% had been determined using a linear regression analysis of data from 45 animals.3 This latter value reflects a population value and although the variance may differ, the MAC value should not,9 so methodological differences may account for the difference observed with the MAC value found in our study. Since the availability of suitable anaesthetic gas analysers for very small rodents such as mice or gerbils is limited, purpose-made circuits and methods to overcome these differences are necessary. In the present study the animals were not only endotracheally intubated, but also placed inside a plastic bag to avoid room air entering the lungs through the unsealed trachea while gas samples were obtained from the endotracheal tube. The averaged MAC values measured in the gerbil were apparently higher than those found in rats or mice, i.e. about 15% higher, using a similar method10 – 12 but also when the same method was employed for isoflurane in rats.7 MAC values for the present study are close to those found in the hamster (halothane 1.15 vol%, isoflurane 1.62 vol% and sevoflurane 2.31 vol%)13 and, probably, in the cat.14 Not surprisingly, the difference in MAC values between rats and gerbils was similar for the three anaesthetics studied. The present study did not include a crossover design but a similar variation in response to these agents would be expected among individuals.14 The coefficient of variation, i.e. the variability in anaesthetic potency among individuals, was similar to that in other species (4.2 –10.1%).15 In conclusion, results from the present study indicate that the MAC values for halothane, isoflurane and sevoflurane in the gerbil are higher than in rats or mice, suggesting a lower potency. Inhalant anaesthetics can be potent cardiovascular and respiratory depressants and their effects may vary with the anaesthetic administered. However, at 1 MAC, the differences among the studied anaesthetics in the gerbils
Table 1 Cardiovascular and respiratory data at 1 MAC of halothane, isoflurane and sevoflurane (heart and respiratory rate, blood pressure, saturated O2 [SatO2] and temperature) and at the end of the study ( pH, partial oxygen pressure [PaO2], partial carbon dioxide pressure [PaCO2], base excess)
Heart rate (bpm) Systolic arterial pressure (mmHg) Mean arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Respiratory rate (bpm) SatO2 (%) Temperature (8C) pH PaO2 (mmHg) PaCO2 (mmHg) Base excess (mmol/L) Data are expressed as mean + SD Significantly different from halothane (P , 0.05)
Halothane
Isoflurane
Sevoflurane
332 + 52 86 + 15 75 + 14 66 + 16 140 + 21 100 + 0 37.2 + 0.2 7.41 + 0.06 222 + 62 26 + 3 27 + 3
288 + 29 79 + 20 68 + 22 61 + 23 104 + 13 100 + 0 37.8 + 0.4 7.39 + 0.04 279 + 63 29 + 3 28 + 3
308 + 14 79 + 9 65 + 9 58 + 15 92 + 8 100 + 0 37.3 + 0.2 7.43 + 0.03 259 + 85 29 + 3 25 + 4
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were limited, suggesting similar cardiorespiratory depressant actions although this does not necessarily determine the same response at different anaesthetic concentrations. Halothane did induce a higher RR compared with sevoflurane or isoflurane although with no consequences in carbon dioxide levels or pH in blood. Hypocapnia was observed with the three anaesthetics16 and while this may partially reflect a light anaesthetic level, it may also be a consequence of the study design, in which tachypnoea is commonly observed after tail clamping at approximately 1 MAC. However, the hypocapnia would be transient since pH, as well as PaO2 and SatO2, were within the normal range. In addition, RR was assessed before tail-clamping, suggesting a differentiated action of halothane on this parameter.
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6 Molthen RC. A simple, inexpensive, and effective light-carrying laryngoscopic blade for orotracheal intubation of rats. J Am Assoc Lab Anim Sci 2006;45:88 –93 7 Criado AB, Go´mez de Segura IA. Reduction of isoflurane MAC by fentanyl or remifentanil in rats. Vet Anaesth Analg 2003;30:250 –6 8 Docquier MA, Lavand’homme P, Ledermann C, Collet V, De Kock M. Can determining the minimum alveolar anesthetic concentration of volatile anesthetic be used as an objective tool to asses antinociception in animals. Anesth Analg 2003;97:1033 – 9 9 Paul M, Fisher DM. Are estimates of MAC reliable? Anesthesiology 2001;95:1362 – 70 10 Mazze RI, Rice SA, Baden JM. Halothane, isoflurane, and enflurane MAC in pregnant and non-pregnant female and male mice and rats. Anesthesiology 1985;62:339 –41 11 Orliaguet G, Vivien B, Langeron O, Bouhemad B, Coriat P, Riou B. Minimum alveolar concentration of volatile anaesthetics in rats during postnatal maturation. Anesthesiology 2001;95:734 –9 12 Steffey MA, Brosnan RJ, Steffey EP. Assessment of halothane and sevoflurane anesthesia in spontaneously breathing rats. Am J Vet Res 2003;64:470 –4 13 Vivien B, Hanouz JL, Gueugniaud PY, Lecarpentier Y, Coriat P. Minimum alveolar anesthetic concentration of volatile anesthetics in normal and cardiomyopathic hamsters. Anesth Analg 1999;88:489 –93 14 Barter LS, Ilkiw JE, Steffey EP, Pypendop BH, Imai A. Animal dependence of inhaled anaesthetic requirements in cats. Br J Anaesth 2004;92:275 –7 15 Valverde A, Morey TE, Herna´ndez J, Davies W. Validation of several types of noxious stimuli for use in determining the minimum alveolar concentration for inhalation anesthetics in dogs and rabbits. Am J Vet Res 2003;64:957 –62 16 Van Uitert RL, Levy DE. Regional brain blood flow in the conscious gerbil. Stroke 1978;9:67 –72 (Accepted 9 November 2008)
