Fentanylâœfluanisoneâœmidaz
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REPORTS Fentanyl–fluanisone–midazolam Combination Results in More Stable Hemodynamics than does Urethane–α-chloralose and 2,2,2-tribromoethanol in Mice WILLEKE M.C. JONG, MSC,1 COERT J. ZUURBIER, MSC, PHD,3 ROBBERT J. DE WINTER, MSC, MD, PHD,1* DANYEL A. F. VAN DEN HEUVEL,3 PIETER H. REITSMA, PHD,2 HUGO TEN CATE, MD, PHD,2 AND CAN INCE, PHD3 Near-physiologic hemodynamic conditions for several hours were needed to study cardiovascular physiology in a murine model. We compared two commonly used anesthetic treatments, urethane–α-chloralose (U–αCh; 968 mg U and 65 mg αCh/kg) and 2,2,2- tribromoethanol (TBE; 435 mg/kg) and fentanyl–fluanisone–midazolam (FFM; 3.313 mg fentanyl, 104.8 mg fluanisone, and 52.42 mg midazolam/kg) with respect to mean arterial blood pressure (MAP) and heart rate (HR) for 100 min at similar levels of surgical anesthesia. Assessed every 10 to 15 min, the U–αCh+TBE group maintained a significantly (P < 0.001) lower mean MAP (49 ± 4 mmHg) than did the FFM group (78 ± 5 mmHg). Mean HR in the U–αCh+TBE group significantly (P < 0.001) increased from 308 ± 34 bpm at the beginning to 477 ± 43 bpm at the end of the experiment. In comparison, the FFM group showed a stable HR of 431 ± 37 bpm. The MAP and HR of the U–αCh+TBE group were extremely unstable, with sudden and unpredictable changes in MAP when examined at 1-min intervals. The results of our study show that U–αCh+TBE anesthesia should not be used in murine models in which stable, near-physiologic hemodynamics are needed for cardiovascular studies. The growing availability of genetically altered mice has accel- The opiate fentanyl takes effect rapidly but is relatively short- erated the development of new techniques to study acting. It causes a profound depression of the nervous system, cardiovascular physiology. Measurement of changes in physi- including the respiratory centers in the brain stem, leading to ologic parameters during a cardiovascular intervention study respiratory depression and bradycardia (9, 13). Fluanisone is a requires controlled anesthesia. General anesthesia can be in- neuroleptic of the butyrophenone group. This agent potenti- duced by several volatile, intraperitoneal (i.p.) or intravenous ates the analgesia provided by fentanyl but counteracts its emetic (i.v.) anesthetics. The choice of an anesthetic agent or combina- side effect and partially antagonizes the respiratory depression tion of agents is limited by its side effects, such as cardiac and (9, 13). Midazolam is a benzodiazepine and produces general respiratory depression. Particularly in cardiovascular interven- anesthesia with sufficient muscle relaxation for major surgery tion studies, stable blood pressures and heart rates are crucial. when used in combination with fentanyl and fluanisone. This Anesthetic treatments with urethane (i.e., ethyl carbamate) effect is synergistic (9). If midazolam is used alone, it has a very alone (1-3) or in combination with α-chloralose (4) yield mini- broad safety margin and a short induction time (14). mal cardiovascular and respiratory system depression, and both Advances in the cardiovascular intervention studies in mice at drugs have been advised for use in cardiovascular studies (5, 6). our laboratory prompted the question whether FFM was a good In some countries, however, the use of urethane is prohibited in alternative to using U–αCh+TBE for the reduction of pain in survival studies because of its carcinogenic potential for both anesthetized animals. Here we describe the direct comparison mice and humans (7). After urethane administration, mice fre- of these anesthetic treatments with respect to blood pressure quently develop pulmonary adenomas early in life (7). (BP) and heart rate at similar levels of surgical anesthesia. Supple- The anesthetic 2,2,2-tribromoethanol (TBE) is administered mental doses of anesthetics were administered at signs of a pedal to produce surgical anesthesia (8), with good skeletal muscle withdrawal reflex and a concomitant temporal increase in BP relaxation and only a moderate degree of respiratory depres- even if the mean arterial blood pressure (MAP) was low. Our sion (9). In addition, it is popular now for use in transgenic mice results show that FFM anesthesia is preferable to using U– (10). However, TBE is reported to cause a high rate of post- αCh+TBE for cardiovascular studies in mice. operative fatalities associated with abdominal adhesions, peritonitis, and intestinal disturbances such as diarrhea or even Materials and Methods complete obstruction. These problems are exacerbated when Animals. The Animal Studies Ethics Committee of the Uni- TBE is exposed to light or not used promptly after preparation. versity of Amsterdam approved all experiments. The 13 male Further, TBE must be stored at 4°C (8, 11). SW mice used (Harlan Sprague Dawley, Inc., Horst, The Neth- Flecknell and Mitchell introduced the use of the anesthetic erlands) weighed 25 to 35 g and were housed in open-topped mixture fentanyl–fluanisone–midazolam (FFM) in 1984 (12). cages at constant temperature (21 to 22°C), humidity (50% ± 10%), and light cycle (12:12-h dark:light). Rodent chow (AM-II Department of Cardiology, Cardiovascular Research Institute Amsterdam,1 Laboratory for 10 mm, Hope Farms, Woerden, The Netherlands) and drinking 2 Experimental Internal Medicine, and Department of Anesthesiology, Cardiovascular Re- water were available ad libitum. search Institute Amsterdam,3 Academic Medical Center, University of Amsterdam, P.O. Box 22660, 1100 DD Amsterdam, The Netherlands Preparation of anesthetic mixtures. To prepare the U–αCh *Corresponding author: Dept. of Cardiology, Room B2-137, Academic Medical Center, mixture, we first dissolved α-chloralose (Sigma, St. Louis, Mo.) University of Amsterdam, P.O. Box 22660, 1100 DD Amsterdam, The Netherlands in normal saline at 60°C to a final concentration of 10 mg/ml. 28 CONTEMPORARY TOPICS © 2002 by the American Association for Laboratory Animal Science Volume 41, No. 3 / May 2002 We then dissolved urethane (Sigma)by adding the α-chloralose To determine HR, the recorder speed was increased for 2 to 3 solution to a final concentration of 150 mg/ml. sec every 15 min, and the systolic blood pressure peaks were The TBE (Sigma) stock solution was prepared by adding 25 counted. A Y-tube (Y210, Endomed, Didam, The Netherlands) ml 2-methyl-2-butanol (tertiary amyl alcohol; Sigma Aldrich) was inserted directly into the trachea via tracheotomy and con- to a 25-g bottle of dry TBE. A 1:40 dilution in normal saline nected to a small-animal ventilator (CIV101, Columbus resulted in the 2.5% working solution for in vivo use. Congo Instruments, Columbus, Ohio) to ventilate the mouse artificially. red (Sigma) with a transition interval from pH 3.0 (blue) to In light of pilot data (not shown), we decided to ventilate with pH 5.0 (red) was added as an indicator of intact TBE (15), which warmed and humidified air containing 40% O2 at tidal volume was safe for use only as long as the pH had not changed (pH of 0.3 to 0.5 ml, inspiration time/expiration time of 0.25 to 0.30, must be > 5.0). and 100 to 120 breaths per minute. The FFM mixture was prepared by dissolving 1 ml Hypnorm At the end of the experiment, the mice were killed by cervical (Janssen Pharmaceutica, Beerse, Belgium) containing fentanyl dislocation while still anesthetized. and fluanisone and 1 ml Dormicum (Roche, Mijdrecht, The Statistical analysis. In the analysis, we used the averaged MAP Netherlands) containing midazolam in 2 ml sterile water. The tracings for successive 10-min intervals (the “mean MAP”) re- final concentrations were 0.079 mg/ml fentanyl citrate, 2.5 mg/ sulting (together with the MAP at 0 min) in 11 data points per ml fluanisone, and 1.25 mg midazolam/ml (12). mouse during the recording time of 100 min. Study design. The group of seven mice received U–αCh+TBE, To analyze the effect of an anesthetic treatment on the re- and the group of six mice received FFM in random order. peated (averaged) measurements of BP and HR, we set up a (i) Induction. To induce U–αCh anesthesia, each mouse in mixed linear model. Using mixed model methodology, we can that group initially was given 300 mg TBE/kg (15) i.p. A few incorporate the correlation (covariance structure) between dif- minutes later, each received 600 mg U/kg and 40 mg αCh/kg ferent measurements over time from the same animal. We i.p. To induce FFM anesthesia, each mouse received 0.553 mg/ modeled whether measurements close in time are more corre- kg fentanyl citrate, 17.5 mg/kg fluanisone, and 8.75 mg lated than measurements farther apart by imposing a first-order midazolam/kg BW (7 ml FFM/kg) i.p. autoregressive pattern of covariance (18). Time was treated as a (ii) Control and maintenance. A constant infusion of Hartmann continuous variable, and in a sequential analysis, the need for solution (NPBI, Emmer Compascuum, The Netherlands; con- higher-order polynomials of time was examined. centration per liter: 7 g NaCl, 0.4 g KCl, 3.0 g Na lactate, 0.2 g We tested for the effect of treatment and treatment by time CaCl2) or sterile 0.9% saline (Braun, Melsungen, Germany) was interaction. Furthermore, we estimated the differences in mean applied at a rate of 7 ml/kg hourly via the tail vein (9). Extra scores between the groups and identified the 95% confidence boluses of Hartmann solution or pasteurized 4% plasma pro- intervals. P values below 0.05 were considered significant. All tein solution (i.e., gepasteuriseerde proteïne oplossing [GPO]; mixed models were completed by using SAS software version Central Laboratory for Blood Research, Amsterdam, The Neth- 6.12 with the “proc mixed” procedure (19).