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Biol. Pharm. Bull. 30(9) 1716—1720 (2007)

Vol. 30, No. 9

Comparison of Newly Developed Inhalation Anesthesia System and Intraperitoneal Anesthesia on the Hemodynamic State in Mice Yoshikazu MATSUDA,*, a Kazumasa OHSAKA,a Hidekazu YAMAMOTO,b Katsuhiko NATSUME,b Shirokazu HIRABAYASHI,c Masayoshi KOUNOIKE,d and Masaaki INOUEe a

Department of Integrative Physiology & Bio-System Control, Shinshu University School of Medicine; 3–1–1 Asahi, Matsumoto, Nagano 390–8621, Japan: b Natsume Seisakusho Co., Ltd.; 2–18–6 Yushima, Bunkyo-ku, Tokyo 113–8551, Japan: c Leaf International Inc.; 3–13–6 Hongo, Bunkyo-ku, Tokyo 113–0033, Japan: d Senko Medical Instrument Inc.; 3–23–13 Hongo, Bunkyo-ku, Tokyo 113–0033, Japan: and e S.K.I. Net. Inc.; 2–16–9 Yushima, Bunkyo-ku, Tokyo 113–0034, Japan. Received March 8, 2007; accepted June 6, 2007; published online June 7, 2007 KNI-472 is the first anesthetic system for mice and rats to incorporate a ventilator. It consists of a newly developed syringe pump-type vaporizer and gas monitor that can deliver accurate concentrations of anesthetic gas at an extremely low airflow. In this study, we compared the hemodynamic effects of isoflurane anesthesia using KNI-472 and intraperitoneal pentobarbital anesthesia. In the isoflurane anesthetic group, Institute of Cancer Research (ICR) mice were anesthetized with 5% isoflurane, followed by endotracheal intubation. Subsequently, they were ventilated mechanically, and anesthesia was maintained with 2% isoflurane for a 60-min period using KNI-472. In the pentobarbital anesthetic group, the ICR mice were anesthetized by an intraperitoneal injection of sodium pentobarbital (70 mg/kg). In isoflurane anesthesia, the heart rate (HR) and mean blood pressure (MBP) were stable. In contrast, in pentobarbital anesthesia, MBP decreased in the first stage after the initiation of anesthesia, after which it gradually increased. The intra-group variability in the estimated skin blood flow (SBF) was higher in the pentobarbital anesthesia than that in the isoflurane anesthesia. The PaO2 and PaCO2 values at 15 min after the initiation of pentobarbital anesthesia revealed hypoxia and hypercapnia compared with isoflurane anesthesia. In this study, isoflurane anesthesia using KNI-472, unlike pentobarbital anesthesia, did not induce changes in MBP, SBF, or blood gases. The changes induced by pentobarbital anesthesia were attributed to a change in the depth of anesthesia with time. These results indicate that inhalation anesthesia using KNI-472 is suitable in research on the hemodynamic state in mice. Key words

KNI-472; isoflurane; pentobarbital; hemodynamic status; blood gas

General anesthesia in laboratory animals is important from the viewpoint of not only animal welfare but also the reliability of pharmacological studies. The type of anesthetics used may vary depending on the type of experiment required or the study design. Some anesthetics have cardio- or renoprotective effects, which may be relevant in designing ischemiareperfusion protocols.1—3) Several studies have examined the influence of commonly used anesthetics on the short-term and noninvasive assessment of cardiac function via echocardiography.4—7) Zuurbier et al. reported that at similar surgical levels of anesthesia, the preferred anesthetic (isoflurane or ketamine–medetomidine–atropine) depends on the mouse strain used and whether monitoring of the blood pressure or heart rate is the focus of the study.8) Sodium pentobarbital and ketamine/xylazine have been widely used as anesthetics in mice.9) However, Chaves et al. reported that inhalation anesthetic agents such as halothane have significant advantages when compared with injectable agents such as ketamine/xylazine.4) It has been reported that when compared with other injectable anesthetics, such as pentobarbital and ketamine/xylazine, isoflurane anesthesia has a slight effect on the hemodynamic status of mice.10—13) Therefore, in animal experiments, it is important to consider the option of inhalation anesthesia. Indeed, the methodology of administering anesthesia changed markedly between the periods from 1990 to 1992 and from 2000 to 2002. Notably, there was an increase in the use of isoflurane and of injectable anesthetic combinations such as ketamine/ xylazine.14) However, since ketamine was classified as a narcotic drug in 2006, its use now poses difficulties in Japan. ∗ To whom correspondence should be addressed.

Additionally, since no appropriate instrument for the inhalation anesthesia of mice was available, this technique was performed only in a limited number of laboratories. Thus, the development of a system that can perform the inhalation anesthesia easily was desired. KNI-472, a newly developed anesthetic system that incorporates a ventilator, is the first anesthetic system designed specifically for mice and rats (Fig. 1). We performed inhalation anesthesia with isoflurane using KNI-472 and observed that the hemodynamic parameters in mice were stable.15) We believe that this stability using KNI-472 was the result of maintaining a constant depth of anesthesia by strictly controlling the concentration of the anesthetic.15) Therefore, inhalation anesthesia using KNI-472 is an important candidate for a method of general anesthesia. However, there are no results available with which to compare the inhalation anesthesia using KNI-472 with the other widely used anesthesia methods. In this study, we evaluated the difference between the hemodynamic parameters obtained after administering the widely used intraperitoneal pentobarbital anesthesia with those obtained after administering inhalation anesthesia with isoflurane using KNI-472. We believe that the data obtained in this study could provide a basis for anesthetic regimens in future pharmacological studies. MATERIALS AND METHODS This study was performed in accordance with the institutional guidelines of Shinshu University and was approved by

e-mail: [email protected]

© 2007 Pharmaceutical Society of Japan

September 2007

Fig. 1. Photograph of the Newly Developed Inhalation Anesthesia System (KNI-472) and Experiment Scenery KNI-472 is a compact and useful inhalation anesthesia system specially designed for small animals. Air is used as carrier gas and an air pump is included in the system. Gas flow is accurately measured and controlled by a mass flow controller. In place of conventional wick type vaporizer, a syringe pump is used for injecting the calculated volume of anesthetic agent (isoflurane, sevoflurane and halothane) in the vaporizing chamber. A constant flow, time-cycled pressure limited ventilator is integrated in the system.

the Animal Care and Use Committee. Nine-week-old Institute of Cancer Research (ICR) mice (Japan SLC Inc., Japan) were purchased for 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 environmental temperature was set at 22
2 °C. General Anesthesia with Isoflurane or Pentobarbital Ten male ICR mice (10-week-old) weighing 32
1 g were used. In the isoflurane group, anesthesia was induced by placing the mice in an anesthesia induction chamber (KN1010; Natsume Seisakusho Co., Ltd., Japan) containing 5% isoflurane (Forane; Abbott Japan Co., Ltd., Japan) and room air; this was followed by endotracheal intubation of the mice. Subsequently, the mice were mechanically ventilated and the anesthesia was maintained for a 60-min period (anesthetic maintenance state) using 2% isoflurane (respiratory rate, 90 bpm; tidal volume, 1.9—2.0 ml). The ventilation conditions were determined from reference values10) and a preliminary experiment. Mechanical ventilation and the maintenance of anesthesia were performed using KNI-472 (NARCOBIT; Natsume Seisakusho Co., Ltd., Japan). In the pentobarbital group, the ICR mice were intraperitoneally anesthetized with 70 mg/kg sodium pentobarbital (Nembutal; Dainippon-Sumitomo Seiyaku Co., Ltd., Japan). In the preliminary experiment, the dose of pentobarbital at which the righting reflex disappeared in 60 min or more was determined to be 70 mg/kg in order to perform pentobarbital anesthesia under the same conditions as those for isoflurane anesthesia. In this study, the time of disappearance of the righting reflex in pentobarbital anesthesia was 72.2
5.6 min. All experiments were conducted from 10:00 a.m. to 4:00

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p.m. Moreover, each anesthesia group was formed and examined randomly. Evaluating Mean Blood Pressure, Heart Rate, and Skin Blood Flow The heart rate (HR), mean blood pressure (MBP), and skin blood flow (SBF) were simultaneously measured in the same animal (Fig. 1). The HR and MBP 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 anesthetic maintenance state. The HR and MBP were measured 3 times at each time point, and the values were expressed as an average of the 3 measurements. The SBF in the hind leg was continuously measured over a 60-min period during the anesthetic maintenance state by using a Laser Doppler Perfusion Imager (PeriScan PIM II; Integral, Japan). The SBF values calculated at each time point (0, 5, 15, 30, 45, 60 min) were the averages of the values recorded over 5 min; these averages were calculated using a personal computer (Windows XP system). The variation in SBF estimates were compared using coefficients of variation (CV%), which were calculated as standard deviation/mean100%. Blood Gas Analysis Ten male ICR mice (10-week-old) weighing 32
1 g were used. In the isoflurane group, the mice were anesthetized with 5% isoflurane prior to endotracheal intubation. Subsequently, they were mechanically ventilated and the anesthetized condition was maintained for a 15-min period using 2% isoflurane. Both the mechanical ventilation and maintenance of anesthesia were performed using KNI-472. The methods used in administering anesthesia in this study were the same as those described previously. On the other hand, in the pentobarbital group, the ICR mice were anesthetized with 70 mg/kg sodium pentobarbital. The arterial blood was sampled from the abdominal aorta, and the blood gases were analyzed using an automatic blood gas analyzer (pHOX Basic; Nova Biomedical, Japan). During the entire period of the study, the anesthetized mice were placed on a mat to keep them warm and to prevent hypothermia. Statistical Analysis The results are expressed as means
standard error (S.E.). In order to compare the effects of the anesthetics, the relative differences between the average values obtained during the conscious resting state and those obtained during the anesthetic maintenance state were calculated. Statistical significance was determined by analysis of variance (ANOVA) followed by the Dunnet test, and p values less than 0.05 were considered to be significant. RESULTS Figure 2 shows the average changes in the HR of mice over a 60-min period during isoflurane or pentobarbital anesthesia. The data are expressed relative to the average resting values obtained during the conscious condition. Compared with the average resting values of HR in the conscious resting state, the HR values in the isoflurane or pentobarbital anesthesia were significantly reduced by 14.3% and 9.7%, respectively, at the initiation of the anesthetic maintenance state (0 min). However, the HR during the anesthetic maintenance state was stable, and no significant differences were observed between the HR values in isoflurane and pentobarbital anesthesia.

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Fig. 2. The Influence of Isoflurane and Pentobarbital Anesthesia on the Heart Rate (HR) in ICR Mice Values indicate the mean
S.E. (n5) and are expressed relative to the resting values obtained during the conscious condition. The values for the conscious condition are 635.8
30.1 bpm in the isoflurane anesthetic group and 628.6
20.7 bpm in the pentobarbital anesthetic group.

Vol. 30, No. 9

Fig. 4. The Influence of Isoflurane and Pentobarbital Anesthesia on the Skin Blood Flow (SBF) in the Hind Leg of the ICR Mice Values indicate the mean
S.E. (n5).

Fig. 3. The Influence of Isoflurane and Pentobarbital Anesthesia on the Mean Arterial Blood Pressure (MBP) in ICR Mice Values indicate the mean
S.E. (n5) and are expressed relative to the resting values obtained during the conscious condition. The values for the conscious condition are 76.8
1.8 mmHg in the isoflurane anesthetic group and 80.2
3.1 mmHg in the pentobarbital anesthetic group. ∗ p0.05 indicates a significant difference when compared with the isoflurane anesthesia.

Figure 3 shows the average changes in the MBP of mice over a 60-min period during the isoflurane or pentobarbital anesthesia. The data are expressed relative to the average resting values obtained during the conscious condition. Compared with the average resting values of MBP in the conscious resting state, the MBP values in isoflurane or pentobarbital anesthesia significantly decreased by 30.7% and 51.3%, respectively, at 0 min. In pentobarbital anesthesia, MBP markedly decreased at 0, 5, and 15 min after the initiation of the anesthetic maintenance state (48.7
4.1%, 44.3
3.3%, and 44.9
3.6%, respectively); these changes were significantly different from those observed in isoflurane anesthesia. This decrease in MBP, which occurred after the initiation of pentobarbital anesthesia, gradually increased. Figure 4 shows the average changes induced in SBF during isoflurane or pentobarbital anesthesia. Compared with the average resting values of SBF at 0 min, the SBF values in the anesthetic maintenance state were not significantly different in either group. However, when the individual data were evaluated, the variation in the SBF values in pentobarbital anesthesia was remarkable as compared to that in isoflurane anesthesia (Fig. 5). Table 1 compares the intra-group variability in the measured SBF values between isoflurane and pentobarbital anesthesia. The CV% was determined from the standard deviation and mean of the population following

Fig. 5. Individual Data on the Influence of Isoflurane and Pentobarbital Anesthesia on the Skin Blood Flow (SBF) in the ICR Mice Table 1. The Coefficient of Variation (CV%) in Skin Blood Flow (SBF) after Anesthesia with Isoflurane and Pentobarbital Time after anesthetic maintenance state (min) Group 5 Isoflurane Pentobarbital

7.5 12.6

15

30

45

60

4.2 16.2

8.1 17.7

11.7 28.5

10.0 29.1

Values represent the mean (of 5 mice/group) CV% calculated as described in Materials and Methods.

each treatment. The CV% in pentobarbital anesthesia were between 12.6—29.1% and those in isoflurane anesthesia were between 4.2—11.7%. The CV% values were higher in pentobarbital anesthesia than those in isoflurane anesthesia (from 1.7- to 2.9-fold). The results of blood gas analysis 15 min after the initiation of the anesthetic maintenance state are shown in Fig. 6. Neither hypoxia nor hypercapnia was observed in isoflurane anesthesia. Pentobarbital anesthesia was associated with increased acidosis of the blood as compared with isoflurane anesthesia. Moreover, a significant increase in PaCO2 and a

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Fig. 6. The Influence of Isoflurane and Pentobarbital Anesthesia on the Arterial Blood Analysis in ICR Mice Values indicate the mean
S.E. (n5). ∗ p0.05 and ∗∗ p0.01 indicate significant differences when compared with isoflurane anesthesia.

significant decrease in PaO2 were observed in pentobarbital anesthesia as compared with isoflurane anesthesia. DISCUSSION This is the first report that compared our newly developed inhalation anesthesia system (KIN-472) with the other widely used anesthesia methods. We studied the hemodynamic effects of isoflurane anesthesia using KNI-472 and pentobarbital anesthesia, since pentobarbital is a well-established and commonly used injectable anesthesia in rodents,16) and isoflurane inhalation has been useful in mice according to recent reports.14) In isoflurane anesthesia, MBP and the variation in SBF were comparatively more stable than in pentobarbital anesthesia. Further, the data obtained in isoflurane anesthesia was sufficient for interpreting the results of the blood gas analysis as compared to those obtained in pentobarbital anesthesia. Thus, we think that isoflurane anesthesia using KNI-472 is suitable to be used in pharmacological experiments in which the hemodynamic state of mice. Further, we believe that the selection of anesthetics can have a profound effect on the estimation of hemodynamic parameters in mice. Anesthesia is often required for experimental interventions and phenotypic evaluations in mice. However, in such experiments, anesthetic accidents such as death and unexpected hypotension can 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, previous experiences, and institutional regulations.17,18) Janssen et al. reported that in mice, isoflurane exhibits fewer systemic hemodynamic effects than pentobarbital anesthetics. They also reported that as compared to

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the cardiac index in the resting conscious state, that during anesthesia using the volatile anesthetic isoflurane decreased only slightly.10) Szczesny et al. also reported that inhalation anesthesia with isoflurane is useful for experimental studies on 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 MBP and HR over a long observation period.19) These reports suggest that isoflurane is a useful anesthetic for animal experiments. However, instruments for administering inhalation anesthesia easily have not been previously available. Therefore, inhalation anesthesia for mice has been performed only in a few laboratories, and general anesthesia has been performed experimentally by simple injection anesthesia. For this reason, we developed a new inhalation anesthesia system (KNI-472) and observed that the hemodynamic parameters were stable during anesthesia.15) We think that the administration of inhalation anesthesia with high accuracy is achievable in experiments on small animals. Therefore, we believe that inhalation anesthesia using KNI-472 is an important candidate for a method of general anesthesia. In this study, although no difference was observed in the HR values of both isoflurane and pentobarbital anesthesia, a significant difference in MBP was observed in the early stages. This can be attributed to a change in the depth of pentobarbital anesthesia with time. On the other hand, Janssen et al. reported that both HR and stroke index (SI) decreased in pentobarbital anesthesia.10) They inferred that these decreases may be related to the direct negative inotropic action of pentobarbital.2) Therefore, we believe that the negative inotropic effect of pentobarbital has also influenced the present data. The intra-group variability in SBF was higher in pentobarbital anesthesia than in isoflurane anesthesia. It is well known that changes in HR and/or MBP affect SBF. Therefore, it is important to control the depth of anesthesia for stable HR, MBP, and SBF. Clearly, controlling the depth of anesthesia is considerably easier with inhalated than with injected anesthetics. On the other hand, the relatively high systemic blood flow during isoflurane anesthesia preserves peripheral organ perfusion during surgical interventions. In addition, isoflurane induces an increase in the regional cerebral cortical blood flow in mice.20) Therefore, a stable SBF in isoflurane anesthesia may be related to the control of anesthetic concentration and increase in the systemic blood flow. Moreover, we think that a change in the depth of pentobarbital anesthesia influences the variation in SBF. Alternatively, the well-known parasympatholytic effect of pentobarbital21) may also be related to the variation in SBF. In previously reported methods of inhalation anesthesia, a nasal cone was used.4,22) However, this involves spontaneous breathing and poses the risks of hypoxia and hypercapnia. In this study, a stable hemodynamic state in isoflurane anesthesia could be achieved despite endotracheal intubation and the use of a fixed anesthesia concentration under mechanical ventilation. Indeed, the results of the blood gas analysis further revealed that the mice anesthetized with isoflurane exhibited neither hypoxia nor hypercapnia as compared with those anesthetized with pentobarbital. Therefore, we believe that mechanical ventilation is also required for stabilizing the

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hemodynamic state. We also established a methodology that involves simple endotracheal intubation in mice, which in turn facilitates mechanical ventilation. On the other hand, it is believed that mechanical ventilation is also useful in pentobarbital anesthesia; however, since the depth of anesthesia changes with time, an arrangement of mechanical ventilation conditions is difficult to achieve. In this study, we demonstrated that the hemodynamic state was more stable in isoflurane anesthesia using KNI-472 than in pentobarbital anesthesia. When performing the experiments, it is necessary to consider that the depth of anesthesia may also affect humoral factors such as cortisol, catecholamine, and immune responses. Thus, these results reveal the importance of selecting an appropriate anesthetic drug and a suitable anesthesia methodology in pharmacological experiments. We believe that the new anesthetization method involving the use of KNI-472 is an important candidate for a general anesthesia method in pharmacological experiments. In summary, there were no changes in the MBP, SBF, or blood gas parameters in isoflurane anesthesia using KIN-472, unlike in pentobarbital anesthesia. The changes in the MBP, PaO2, and PaCO2 parameters observed in pentobarbital anesthesia were considered to be a result of the changes in the depth of anesthesia with time. These results indicate the importance of selecting an anesthesia methodology according to the purpose of an experiment in pharmacological studies. The inhalation anesthesia using KIN-472 is an important candidate for a method of general anesthesia in 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) Lee H. T., Krichevsky I. E., Xu H., Ota-Setlik A., D’Agati V. D., Emala C. V., Am. J. Physiol. Renal Physiol., 286, F111—F119 (2004).

Vol. 30, No. 9 2) Oguchi T., Kashimoto S., Yamaguchi T., Nakamura T., Kumazawa T., J. Pharmacol. Toxicol. Methods, 29, 37—43 (1993). 3) Van Der Linden P. J., Daper A., Trenchant A., De Hert S. G., Anesthesiology, 99, 516—517 (2003). 4) Chaves A. A., Wienstein D. M., Bauer J. A., Life Sci., 69, 213—222 (2001). 5) Hart C. Y., Burnett J. C., Jr., Redfield M. M., Am. J. Physiol. Heart Circ. Physiol., 281, H1938—H1945 (2001). 6) Kiatchoosakun S., Kirkpatrick D., Hoit B. D., Comp. Med., 51, 26—29 (2001). 7) Yang X. P., Liu Y. H., Rhaleb N. E., Kurihara N., Kim H. E., Carretero O. A., Am. J. Physiol. Heart Circ. Physiol., 277, H1967—H1974 (1999). 8) Zuurbier C., Emons V. M., Ince C., Am. J. Physiol. Heart Circ. Physiol., 282, H2099—H2105 (2002). 9) Bauer J. A., Fung H. L., Circulation, 84, 35—39 (1991). 10) Janssen B. J. A., Celle T. D., Debets J. J., Brouns A. E., Callahan M. F., Smith T. L., Am. J. Physiol. Heart Circ. Physiol., 285, H1618—H1624 (2004). 11) Lorenz J. N., Am. J. Physiol. Regul. Integr. Comp. Physiol., 282, R1565—R1582 (2002). 12) Rohrer D. K., Schauble E. H., Desai K. H., Kobilka B. K., Bernstein D., Am. J. Physiol. Heart Circ. Physiol., 274, H1184—H1193 (1998). 13) Roth D. M., Swaney J. S., Dalton N. D., Gilpin E. A., Ross J., Jr., Am. J. Physiol. Heart Circ. Physiol., 282, H2134—H2140 (2002). 14) Richardson C. A., Flecknell P. A., Altern. Lab. Anim., 33, 119—127 (2005). 15) Matsuda Y., Ohsaka K., Yamamoto H., Jiyouraku K., Natsume K., Hirabayashi S., Kounoike M., Inoue M., Exp. Anim., 55, 131—137 (2007). 16) Kawahara Y., Tanonaka K., Daicho T., Nawa M., Olikawa R., Nasa Y., Takeo S., J. Pharmacol. Sci., 99, 95—104 (2005). 17) Rao S., Verkman A. S., Am. J. Physiol. Cell Physiol., 33, 328—333 (1999). 18) Zuurbier C. J., Emos V. M., Ince C., Am. J. Physiol. Heart Circ. Physiol., 282, H2099—H2105 (2002). 19) Szczesny G., Veihelmann A., Massberg S., Nolte D., Messmer K., Lab. Anim., 38, 64—69 (2004). 20) Kehl F., Shen H., Moreno C., Farber N. E., Roman R. J., Kampine J. P., Hudetz A. G., Anesthesiology, 97, 1528—1533 (2002). 21) Murthy V. S., Zagar M. E., Vollmer R. R., Schmidt D. H., Eur. J. Pharmacol., 84, 41—50 (1982). 22) Sahbaie P., Madanlou S., Gharagozlou P., Clark J. D., Lameh J., Delorey T. M., Anesth. Analg., 103, 620—625 (2006).