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Exp. Anim. 56(2), 131–137, 2007

NARCOBIT—A Newly Developed Inhalational Anesthesia System for Mice Yoshikazu MATSUDA1), Kazumasa OHSAKA1), Hidekazu YAMAMOTO2), Kouzou JIYOURAKU2), Katsuhiko NATSUME2), Shirokazu HIRABAYASHI3), Masayoshi KOUNOIKE4), and Masaaki INOUE5)

1)Department

of Integrative Physiology & Bio-System Control, Shinshu University School of Medicine, 3–1–1 Asahi, Matsumoto, Nagano 390-8621, 2)Natsume Seisakusho Co., Ltd., 2–18–6 Yushima, Bunkyo-ku, Tokyo 113-8551, 3)Leaf International Inc., 3–13–6 Hongo, Bunkyo-ku, Tokyo 113-0033, 4)Senko Medical Instrument Inc., 3–23–13 Hongo, Bunkyo-ku, Tokyo 113-0033, and 5)S.K.I. Net. Inc., 2–16–9 Yushima, Bunkyo-ku, Tokyo 113-0034, Japan

Abstract: NARCOBIT is the first anesthetic system for mice and rats to incorporate a ventilator. Therefore, it is expected to improve the reliability of mice and rat experiments by accurately controlling and maintaining the depth of anesthesia. In this study, we used NARCOBIT for inducing inhalational anesthesia in mice and evaluated the changes in their hemodynamic parameters. ICR mice were anesthetized with 5% isoflurane and room air, followed by endotracheal intubation. Subsequently, they were mechanically ventilated, and anesthesia was maintained by 2% isoflurane for a 60-min period (maintenance state) using NARCOBIT. In study 1, the heart rate (HR) and mean arterial blood pressure (MAP) were measured. The skin blood flow (SBF) from the hind legs was continuously measured during the maintenance state. Subsequently, the concentration-dependent effects of isoflurane on MAP were examined. In study 2, blood samples were obtained from the abdominal aorta for blood gas analysis. The HR and MAP decreased after anesthesia but were stable during the maintenance state. Decreased MAP and concentration-dependent effects of isoflurane were observed. The SBF increased slightly during the maintenance state but this increase was insignificant. The blood gas analysis showed neither hypoxia nor hypercapnia. Since the use of NARCOBIT enables the anesthetic concentration of isoflurane to be easily changed, a suitable anesthesia depth can be obtained for experimental purposes. Therefore, we conclude that NARCOBIT can be used for providing inhalational anesthesia to mice. Key words: blood gas analysis, isoflurane, mean arterial pressure, NARCOBIT, skin blood flow from the hind leg

(Received 10 August 2006 / Accepted 4 December 2006) Address corresponding: Y. Matsuda, Department of Integrative Physiology & Bio-System Control, Shinshu University School of Medicine, 3–1–1 Asahi, Matsumoto, Nagano 390-8621, Japan

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Introduction From the viewpoint of the welfare of laboratory animals, it is important to use anesthesia. Sodium pentobarbital and ketamine-xylazine have been used widely as anesthetics for mice [1]. Chaves et al. reported that inhalational anesthetics agents such as halothane have significant advantages when compared with injectable agents such as ketamine/xylazine [2]. Moreover, it has been reported that when compared with other injectable anesthetics, isoflurane anesthesia has a slight influence on the hemodynamic status in mice [5, 7, 9, 10]. Therefore, volatile anesthetics such as isoflurane are suitable for anesthesia of mice. It is possible to use an inhalational anesthetic instrument for humans and animals that are bigger than dogs and cats; however, in the case of animals that are as small as mice, the usefulness and accuracy of such instruments are not satisfactory since these animals have very low tidal flow rates. Anesthetic instruments and ventilators for mice are individually available, but since different manufacturers have designed them on the basis of different concepts, their combined use becomes rather complicated. NARCOBIT is the first anesthetic system that has been specifically designed for mice in which a ventilator has been incorporated (Fig. 1). The newly developed syringe pump type vaporizer and anesthetic gas monitor allow the delivery of accurate concentrations of anesthetic gas at very low airflows. In this system, the concentration of the anesthetic gas is continuously measured by an infrared absorption sensor (IRMA OR, PHASEIN AB, Sweden). It is possible to measure the concentrations of isoflurane, sevoflurane, and halothane, and the percentage of inaccuracy in the measurement is ± 0.15% ABS. Therefore, NARCOBIT improves the reliability of animal experiments by accurately controlling and maintaining the depth of anesthesia at the required level. The ventilator incorporated in NARCOBIT is of the constant flow, time-cycled, pressure limited type. When the airway pressure exceeds the high airway pressure alarm level for any reason, the ventilator switches from the inspiratory phase to the expiratory phase. However, so long as the high airway pressure alarm is not activated, the inspiratory time is constant at a preset level, leading to the following equation. Tidal volume = flow rate × inspiratory time.

Fig. 1. Photograph of NARCOBIT. NARCOBIT is an inhalational anesthesia system for small laboratory animals in which a newly developed vaporizer, respiratory gas monitor and ventilator are incorporated. Air flow rate (A), concentration of anesthetics (B), ventilation rate (C), upper limit of peak inspiratory pressure (D), tidal volume (E), and peak inspiratory pressure (F) are shown.

In NARCOBIT, the flow rate is controlled by a mass flow controller with the percentage of inaccuracy being ± 1.5% FS; this implies that at a respiratory rate of 90 bpm, the error in measurement of the tidal volume is within ± 0.17 ml. In this study, we evaluated the effects of isoflurane anesthesia on the mean arterial blood pressure (MAP), heart rate (HR), and skin blood flow (SBF) from the hind leg in mice by using NARCOBIT. Further, blood gas analysis was performed while the mice were in the isoflurane anesthesia maintenance state. Materials and Methods This study was performed in accordance with the institutional guidelines of Shinshu University and was approved by the Animal Care and Use Committee. Nineweek-old ICR mice (Japan SLC. Inc., Japan) were used in this study. The animals were housed under standard laboratory conditions before and after the study. The mice were acclimated 1 week prior to the experiment. Water and food were provided ad libitum, and the envi-

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ronmental temperature was set at 22°C ± 2°C. Study 1 Five male ICR mice (ten-week-old) weighing 32 ± 1 g were used in study 1. Anesthesia was induced by placing the mice in an anesthesia induction chamber (KN-1010; Natsume Seisakusho Co., Ltd. Japan) filled with 5% isoflurane (Forane; Abbott Japan Co., Ltd. Japan) and room air, followed by endotracheal intubation of the mice. Subsequently, the mice were mechanically ventilated and the anesthesia was maintained for a 60-min period (maintenance state) using 2% isoflurane (respiratory rate, 90 bpm; tidal volume, 1.9–2.0 ml). The ventilation conditions were determined from reference values [5] and a preliminary experiment. Mechanical ventilation and anesthesia maintenance were performed using NARCOBIT (KN472; Natsume Seisakusho Co., Ltd. Japan). The MAP and HR were measured at the tail artery using a noninvasive blood pressure monitor (BP-98A; Softron, Japan). Both these parameters were measured in the conscious state (pre) and at 0, 5, 15, 30, and 60 min after the initiation of the maintenance state. The MAP and HR were measured 3 times at each time point, and the values were expressed as an average of the 3 measurements. The SBF from the hind leg was continuously measured over a 60-min period during the maintenance state by a Laser Doppler Perfusion Imager (PeriScan PIM II; Integral, Japan). The measurement of SBF at each time point (0, 5, 15, 30, 45, and 60 min) was the 5-min averages as calculated by the a personal computer (Windows XP system). After the maintenance state of anesthesia, the concentration-dependent effects of isoflurane (at 1%, 2%, and 4%) were examined as an index of MAP. Study 2 Five male ICR mice (ten-week-old) weighing 32 ± 1 g were used in study 2. Prior to endotracheal intubation, the mice were anesthetized with 5% isoflurane. Subsequently, they were mechanically ventilated and the anesthetized condition was maintained for a 15-min period using 2% isoflurane. Both mechanical ventilation and anesthesia maintenance were performed using NARCOBIT. The anesthesia methods used in this study were the same as those described in study 1. Arterial blood was sampled from the abdominal aorta, and the

blood gases were analyzed using an automatic blood gas analyzer (pHOX Basic; NovaBiochemical, Japan). During the entire period of studies 1 and 2, the anesthetized mice were placed on a mat to keep them warm and to prevent hypothermia. Statistical analysis The results are expressed as mean ± standard error (SE). To compare the anesthetic effect, the relative differences between the average values obtained during the conscious resting state and those obtained during the anesthetized state were calculated. Statistical significance was determined by analysis of variance (ANOVA) followed by the Dunnet test, and p values below 0.05 were considered to be significant. Results Table 1 summarizes the average hemodynamic values obtained in this study when the mice were in the conscious resting state. The average resting values of the HR and MAP were 635.8 ± 30.1 bpm and 76.8 ± 1.8 mmHg, respectively. Figures 2 and 3 show the average changes in the HR and MAP of mice over a 60-min period during the 2% isoflurane anesthesia maintenance state using NARCOBIT. The data are expressed relative to the average resting values obtained during the conscious condition. Compared with the average resting values of HR and MAP of animals in the conscious resting state, isoflurane significantly reduced HR and MAP of the animals by 14% and 31%, respectively, at the initiation of the maintenance state (0 min). However, HR and MAP during the maintenance state (at 0, 5, 15, 30, and 60 min) were stable, and no significant differences were observed between these values and those obtained at the start of the maintenance state. Figure 4 shows the average changes in the SBF in mice induced during the 2% isoflurane anesthesia maintenance state using NARCOBIT. Compared with the average resting values of the animals at the initiation of the maintenance state (0 min), SBF was slightly increased in the maintenance state at 30, 45, and 60 min. However, this increase was not significant in comparison with that observed at the initiation of the maintenance state. The concentration-dependent effects of isoflurane on MAP are shown in Fig. 5. Compared with the average

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Table 1. Baseline heart rate (HR) and the mean arterial blood pressure (MAP) before inhalational anesthesia with isoflurane in ICR mice No. of Mice

5 635.8 ± 30.1 76.8 ± 1.8

HR (bpm) MAP (mmHg) Values indicate the mean ± SE.

Fig. 3. Effect of isoflurane inhalational anesthesia on the mean arterial blood pressure (MAP) of ICR. Values indicate the mean ± SE and are expressed relative to the resting value obtained during the conscious condition. *P < 0.05: significant differences when compared with the conscious condition. NS: No significant differences when compared with the values at the initiation of the anesthesia maintenance state (0 min).

Fig. 2. Effect of isoflurane inhalational anesthesia on the heart rate (HR) of ICR mice. Values indicate the mean ± SE and are expressed relative to the resting values obtained during the conscious condition. *P < 0.05: significant differences when compared with the conscious condition. NS: No significant differences when compared with the values at the initiation of the anesthesia maintenance state (0 min).

resting values of the animals in the conscious resting state, the MAP values under the effect of 1%, 2%, and 4% isoflurane were 70.9% ± 2.9%, 63.6% ± 2.2%, and 43.7% ± 3.0%, respectively. The concentration-dependent effect was significant in the maintenance state. The results of blood gas analysis 15 min after the initiation of the maintenance state are listed in Table 2. The blood gas analysis showed neither hypoxia nor hypercapnia (blood pH: 7.45 ± 0.02; PaO2: 98.6 ± 3.8 mmHg; PaCO2: 25.5 ± 1.2 mmHg). Discussion This is the first study to report the mechanical ventilation of mice under inhalational anesthesia using a ventilator/inhalational anesthesia system (NARCOBIT). Anesthesia is often required for experimental interventions and phenotypic evaluations in mice. However, in experiments involving mice, anesthetic accidents such

Fig. 4. Effect of isoflurane inhalational anesthesia on the skin blood flow (SBF) from the hind legs of ICR mice. Values indicate the mean ± SE.

as death and unexpected hypotension occur. The type of anesthetic used may have a significant impact on the hemodynamic parameters, and a wide range of anesthetic regimens has been used in mice with dose regimens differing across laboratories depending on strain differences, experiences, and institutional regulations [8, 14]. Anesthetic instruments and ventilators for mice are individually available, but since different manufacturers have designed them on the basis of different concepts, their combined use becomes rather complicated. NARCOBIT is the first anesthetic system for mice to incorporate a ventilator. It improves the reliability of animal experiments by accurately controlling and maintaining the depth of anesthesia at the required level. Therefore, it is expected that the use of NARCOBIT in animal experiments will enhance the

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Fig. 5. Concentration-dependent effects of isoflurane inhalational anesthesia on the mean arterial blood pressure (MAP) of ICR mice. Values indicate the mean ± SE. *P < 0.05 and **P < 0.01: significant differences when compared with 1% isoflurane.

Table 2. Blood gas analysis in ICR mice under isoflurane inhalational anesthesia with NARCOBIT No. of Mice pH PaO2 (mmHg) PaCO2 (mmHg)

5 7.45 ± 0.02 98.6 ± 3.8 25.4 ± 1.2

Values indicate the mean ± SE. The arterial blood sample was obtained from the abdominal aorta 15 min after the initiation of the maintenance state.

accuracy of experiments. In this study, mice were anesthetized using an inhalational anesthetic that was administered by NARCOBIT, and their hemodynamic parameters were evaluated. Isoflurane has been widely used as an inhalational anesthetic in recent studies; therefore, isoflurane was used in this study. The mice were anesthetized with 2% isoflurane using NARCOBIT, and HR, MAP and SBP were stable during the 60-min anesthetic maintenance state. The MAP response changed promptly when the isoflurane concentration was varied, and this change was concentration dependent. Additionally, the blood gas analysis showed neither hypoxia nor hypercapnia at 15 min after the initiation of the maintenance state. Since NARCOBIT enables the anesthetic concentration of isoflurane to be easily changed, a suitable depth of anesthesia can be obtained for experimental purposes. Based on our results, we conclude that NARCOBIT can be used to provide inhalational anesthesia to mice.

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Janssen et al. reported that in mice, isoflurane has fewer systemic hemodynamic effects than pentobarbital anesthetics. They also reported that when compared with the resting conscious resting state, the cardiac index was only slightly decreased during anesthesia with the volatile anesthetic isoflurane [5]. Szczesny et al. also reported that inhalation anesthesia with isoflurane is useful for experimental studies in mice for the following reasons: (1) simplicity of anesthetic administration, (2) rapid anesthesia induction, (3) easy control of the depth of anesthesia, (4) low percentage of complications, and (5) stable MAP and HR over a long observation period [12]. These reports suggest that isoflurane is a useful anesthetic for animal experiments. Initially, however, there were no instruments that could be used to easily administer isoflurane anesthesia. Therefore, inhalational anesthesia for mice was carried out in only a few laboratories. In this study, we showed the ease of use anesthesia management by NARCOBIT, and we believe that this system, which employs an anesthesia instrument/ventilator to administer anesthesia, has immense future potential. Previously reported methods of inhalational anesthesia involved the use of a nasal cone (mask) [2, 11]. However, the use of a nasal cone (mask) involves spontaneous breathing, and even when injection anesthesia is not used, the process involves risks such as hypoxia and hypercapnia. In this study, a stable hemodynamic state could be achieved despite endotracheal intubation and the use of a fixed anesthesia concentration under mechanical ventilation. Based on the results of the blood gas analysis, anesthetized mice also showed neither hypoxia nor hypercapnia. Therefore, we consider NARCOBIT is useful for anesthesia maintenance under conditions of mechanical ventilation and endotracheal intubation. Zuurbier et al. reported that during isoflurane anesthesia (1.5%–2%), MAP and HR reduced to –80 mmHg and 600 bpm, respectively. These values correspond to the steady-state values observed in Swiss and C57BL6 mice anesthetized for 3 h [14]. This report shows that the safety of inhalational anesthesia during prolonged anesthesia is not strain-specific. Moreover, it is expected that there is prompt weaning from inhalational anesthesia, and the cardiovascular load on the mice during the experiment can be mitigated. In the present

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study, although anesthesia was maintained for 60 min, we consider that a longer duration of anesthesia maintenance is also possible with NARCOBIT, of similar length to that reported by Zuurbier et al. Comparable observations have also been made in rats [3]. This may explain why the outcome of various surgical interventions is generally more successful when isoflurane anesthesia is used than when non-volatile anesthetics are used. Control of the depth of anesthesia is much easier with inhalation anesthesia than with injectable anesthetics. We consider that inhalation anesthesia using NARCOBIT can also be applied to rat experiments. Janssen et al. reported that a relatively high systemic blood flow during isoflurane anesthesia preserves peripheral organ perfusion during surgical intervention [5]. In the present study, the SBF increased slightly during the anesthesia maintenance state. Although the details have not been examined, we hypothesize that the vasodilatory action of isoflurane [6] and the effect of keeping the animals warm were responsible for the increased blood flow to the hind leg. Additionally, it is possible that this slight increase in SBF also contributed to the recovery from anesthesia. The exact pharmacological mechanism of anesthetics remains unknown. Recently, it has been reported that several pharmacological actions of isoflurane and related volatile anesthetics, such as a protective preconditioning-like effect on myocardial tissues [4] and the serotonergic system, may be altered significantly after inhalational anesthesia [13]. NARCOBIT is equipped with a newly developed syringe pump type vaporizer, and a calculated volume of the anesthetic is injected to the evaporation chamber through which room air flows at a controlled rate. Since NARCOBIT is excellent for regulating the concentration of an anesthetic, it will be useful for studying the dose-dependent actions of an anesthetic drug, or the distribution of an anesthetic in the body, etc., and could be used to clarify the new pharmacological mechanism of an anesthetic drug. In summary, a constant anesthetic depth can be achieved by inhalational anesthesia with isoflurane using NARCOBIT because HR and MAP remain stable during the anesthetic maintenance state. SBF increased slightly during the maintenance state. Decreased MAP and concentration-dependent effects of isoflurane were

observed. The mice recovered rapidly after the experiment. The blood gas analysis showed neither hypoxia nor hypercapnia. Since the use of NARCOBIT enables the anesthetic concentration of isoflurane to be easily changed, a suitable anesthesia depth can be obtained for experimental purposes. Therefore, we conclude that NARCOBIT can be used to provide inhalational anesthesia to mice. Acknowledgments We thank M. Kobayashi (Softron Corp.), Y. Nagaoka (Integral Corp.), and H. Hayashida (Nova Biomedical Corp.) for their excellent technical assistance. References 1. Bauer, J.A. and Fung, H.L. 1991. Concurrent hydrazine administration prevents nitroglycerin-induced hemodynamic tolerance in experimental heart failure. Circulation 84: 35– 39. 2. Chaves, A.A., Wienstein, D.M., and Bauer, J.A. 2001. Noninvasive echocardiographic studies in mice influences of anesthetic regimen. Life Sci. 69: 213–222. 3. Debaene, B., Goldfarb, G., Braillon, A., Jolis, P., and Lebree, D. 1990. Effects of ketamine, halothane, enflurane and isofulurane on systemic and splanchic hemodyanamics in normovolemic and hypovolemic cirrhotic rats. Anesthesiology 73: 118–124. 4. Hu, G., Vasiliauskas, T., Salem, M.R., Rhone, D.P., and Crystal, G.J. 2003. Neutrophils pretreated with volatile anesthetics lose ability to cause cardiac dysfunction. Anesthesiology 98: 712–718. 5. Janssen, B.J.A., Celle, T.D., Debets, J.J., Brouns, A.E., Callahan, M.F., and Smith, T.L. 2004. Effects of anesthetics on systemic hemodynamics in mice. Am. J. Physiol. Heart. Circ. Physiol. 285: H1618–H1624. 6. Kehl, F., Shen, H., Moreno, C., Farber, N.E., Roman, R.J., Kampine, J.P., and Hudetz, A.G. 2002. Isoflurane-induced cerebral hyperemia is partially mediated by nitric oxide and epoxyicosatrienoic acids in mice in vivo. Anesthesiology 97: 1528–1533. 7. Lorenz, J.N. 2002. A practical guide to evaluating cardiovascular, renal, and pulmonary function in mice. Am. J. Physiol. Regul. Integr. Comp. Physiol. 282: R1565– R1582. 8. Rao, S. and Verkman, A.S. 1999. Analysis of organ physiology in transgenic mice. Am. J. Physiol. Cell Physiol. 33: 328–333. 9. Rohrer, D.K., Schauble, E.H., Desai, K.H., Kobilka, B.K., and Bernstein, D. 1998. Alterations in dynamic heart rate control in the β1-adrenergic receptor knockout mouse. Am. J. Physiol. Heart Circ. Physiol. 274: H1184–H1193. 10. Roth, D.M., Swaney, J.S., Dalton, N.D., Gilpin, E.A., and

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inhalatory isoflurane in different strains of mice—the hemodynamic effects. Lab. Anim. 38: 64–69. 13. Whittington, R.A. and Virag, L. 2006. Isoflurane decreases extracellular serotonin in mouse hippocampus. Anesth. Analg. 103: 92–98. 14. Zuurbier, C.J., Emos, V.M., and Ince, C. 2002. Hemodynamics of anesthetized ventilated mouse model: Aspects of anesthetics, fluid support, and strain. Am. J. Physiol. Heart C. 282: H2099–H2105.