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1Cardiology Division, Department of Medicine, and Cardiovascular Research Institute, University of California; and 2Veterans Affairs Medical Center, San Francisco, California 94143-0124
Submitted 7 January 2003 ; accepted in final form 5 October 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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left ventricular hemodynamics; first derivative of left ventricular pressure; anesthesia; mouse physiology
To address this problem, we have developed methodology to assess LVP, its first derivative (dP/dt), heart rate (HR), LV chamber dimensions, and fractional shortening (FS) in conscious mice using a micromanometer catheter and transthoracic echocardiography. Using this technique, we have established normal values for physiological parameters in conscious mice, confirmed that commonly used anesthetic agents depress the LV inotropic state and HR acutely, and found that anesthesia-induced physiological depression persists for a variable time after recovery from anesthesia, particularly with ketamine and xylazine (K+X) anesthesia.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study was approved and performed in accordance with the guidelines of the Committee on Animal Research, University of California (San Francisco, CA). A total of 47 female C57BL/6 mice [mean body weight 21.5 g (18–25 g) and age 12 wk old] was included in this study.
Anesthesia
We employed two kinds of anesthesia, according to our study protocols (vide infra): an intraperitoneal injection of K+X or inhalation anesthesia with isoflurane (ISF) gas. In the mice anesthetized with K+X, a mixture of ketamine (80 mg/kg) and xylazine (5 mg/kg) was injected intraperitoneally. Mice anesthetized with ISF were placed in a box filled with 2 vol% ISF with 1 l/min oxygen flow for introduction of anesthesia. After induction, mice were intubated with polyethylene (PE)-50 tubing (Intramedics, Becton-Dickinson; Sparks, MD) and placed on a thermally controlled surgical table. Ventilation in K+X-anesthetized mice was with room air and with 1.5–2 vol% ISF with 1 l/min oxygen flow in ISF-anesthetized mice. In both groups, ventilation was done with a tidal volume of 500 µl at 110 cycles/min using a Harvard respirator (Harvard Apparatus; Holliston, MA).
Surgical Procedures
Under sterile conditions, the abdomen was opened from the xiphoid process to a left subaxillary level along the lower rib margin. The LV apex was exposed via a subdiaphragmatic incision, leaving the chest wall and sternum intact. The LV apex was sutured with 6-0 proline through a small incision in the diaphragm. By pulling on the LV apex suture gently, an apical stab wound was made with a 21-gauge needle, and PE-50 tubing filled with heparinized saline was inserted into the LV cavity. A ligature was tied around the PE-50 tubing 1 mm from its tip to check/prevent the tip of the tubing from entering the LV cavity by >1 mm. The tubing was fixed with a drop of cyanoacrylate adhesive (3M Vetbond Tissue Adhesive, 3M Animal Care Products; St. Paul, MN). Through a subcutaneous tunnel, the tubing curved leftward from the cardiac apex on the ventral surface of the mouse, around the left flank and on to the dorsal surface; the end of the tubing was exteriorized (
5 mm), and a metallic pin was inserted in the end of the tubing to act as a plug (Fig. 1). The diaphragm was sutured closed after suction of the chest cavity. Subcutaneous enrofloxacin (2.5 mg/kg) was administered immediately after skin closure. Once spontaneous respiration had resumed, animals were extubated, placed on a heating pad, and warmed with heat lamps. The mice were then allowed to recover in standard rodent cages with food and water ad libitum in a 12:12-h light-dark cycle. Postoperatively, mice were trained daily to lie quietly head first in a soft plastic cone (Mouse Decapicone, Braintree Scientific; Braintree, MA). The plastic cone is constructed of soft material and fit the animals snugly (Fig. 1). The animals became accustomed to these circumstances and did not struggle or move after a few days of training. The PE-50 tubing was flushed with 0.02 ml heparinized saline every day. The mice showed no signs of thrombotic embolization throughout the study.
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LV Pressure Measurements in Conscious Mice
Before every measurement of LV pressure (LVP), the micromanometer (1.4-Fr, model SPR 671, Millar Instruments; Houston, TX) was calibrated electronically in vitro by submerging its tip in warmed saline (37°C) and applying an external pressure of 100 mmHg.
Conscious animal experiments were performed in a silent air-conditioned room (22°C). At the designated times for pressure measurement (see Study Protocols), mice were laid naturally prone head first in the soft plastic cone, which was placed on a warm cushion (37°C) and fixed with adhesive tape. The exteriorized end of the PE-50 tubing was carefully pulled out via a slit made in the plastic cone (Fig. 1). The metallic pin was removed from its end, and a 1.4-Fr Millar catheter was advanced to the LV though the implanted tubing. There was minimal resistance as the catheter was inserted into the PE-50 tubing. Mild resistance was felt at the point of the tip of the PE-50 tubing, where the circumferential 6-0 prolene had been tied (see Surgical Procedures). We monitored the position of the tip by the length of catheter advanced and observation of the pressure waveform. After passing the mild resistance at the tip of the tubing, we were able to obtain a LV pressure waveform immediately (Fig. 2). This pressure was identified easily by its dynamic motion and reasonable systolic and diastolic pressure waveforms. After insertion of the catheter, we obtained stable hemodynamic variables (Fig. 2). In early cases we confirmed the position of the catheter by echocardiography for fear that we might advance the catheter too far and injure the LV. However, this did not happen. Particular attention was given to avoiding ambient noise and stimulation throughout the study in the conscious state.
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Analog inputs from the pressure transducer were amplified using an ARIA 1 (Millar Instruments) amplifier and digitized with a DAQ-Card-1200 (National Instruments; Austin, TX) analog-to-digital converter at a rate of 1,000 Hz. These data were analyzed and stored using Biobench data-acquisition software (National Instruments) on a microcomputer (Dell Computer; Round Rock, TX). Digital pressure data at microsecond intervals were transferred to an Excel sheet for analysis. LV dP/dt was defined as the average of nearest neighbors and LV end-diastolic pressure (LVEDP) as LVP at the time of the initial upstroke of dP/dt. LV maximal dP/dt (dP/dtmax) normalized to instantaneous developed pressure (LV dP/dtmax/IP) was determined as a minimally load-dependent measure. HR, peak LVP, minimal ventricular pressure, LV dP/dtmax, LV dP/dtmax/IP, LV minimal dP/dt (LV dP/dtmin), and LVEDP were calculated from the average of 10 consecutive beats.
Echocardiographic Examination
Transthoracic echocardiography was performed with a commercially available system (Acuson Sequoia c256, Acuson, Siemens; Mountain View, CA) using a 15-MHz linear array transducer. After the anterior chest was shaved, mice were inserted into the plastic cone and laid naturally prone in it. The cone was fixed with adhesive tape, and warm ultrasound transmission gel was filled between the chest and the cone, so that two-dimensional imaging could be obtained through the cone. Care was taken to avoid excessive pressure on the thorax, which can induce bradycardia.
Two-dimensional long-axis images of the LV were obtained at the plane of the aortic and mitral valves where the LV cavity is largest and visualization of the LV apex is adequate, and a short-axis image was recorded at the level of the papillary muscles. A two-dimensional guided M-mode echocardiogram was recorded through the anterior and posterior walls at a sweep speed of 200 mm/s. Images were acquired digitally and stored on a magnetooptical disk. All measurements were made from digital images captured on cine loops at the time of study with the use of a specialized software package (Acuson Sequoia). The LV end-diastolic dimension (LVEDD) and end-systolic dimension (LVESD) were determined as the largest and smallest dimensions of the LV, respectively, on M-mode echocardiogram, and FS was derived from following equation: FS = (LVEDD – LVESD)/LVEDD. The LV end-diastolic volume (LVEDV) was calculated using the following two-dimensional area-length method: LVEDV = (5/6)A x L, where A is the endocardial parasternal short-axis area at end diastole and L is the parasternal long-axis length.
Study Protocols
Subacute effects of anesthesia on LV function in conscious mice. LVP, LV dP/dt, and HR in the conscious condition were measured 3 days (0.5 wk) and 7 days (1 wk) after recovery from the PE-50 tubing surgery. Hemodynamic variables in conscious mice were compared depending on the type of anesthesia during the surgery: either K+X (n = 11) or ISF (n = 14).
Acute effects of anesthesia on LV function in conscious mice. Three days from the surgical implantation of PE-50 tubing in the LV apex, LVP, LV dP/dt, and HR were measured in conscious mice as described above. After completion of baseline measurements in the conscious mice, gas anesthesia with ISF (1.5 vol% at 1 l/min oxygen flow) was induced and maintained via a facial mask. LVP during gas anesthesia was measured for 30 min. After hemodynamic stability was ensured, LVP and LV dP/dt were obtained during gas anesthesia. The gas was then removed, and the mice were allowed to recover over 30 min. Recovery from the gas anesthesia was confirmed by the activity of the mice and recognition that HR and peak LVP were stable and back to a level above 90% of those obtained in the conscious state. Next, a mixture of ketamine hydrochloride (80 mg/kg) and xylazine (5 mg/kg) was injected intraperitoneally, and LVP was measured and monitored for 30 min. These doses of K+X or ISF were chosen because they have been used by other investigators in mice (10, 15, 20). We excluded these mice from our hemodynamic analysis at 1 wk for concern that the anesthesia with K+X might still have an effect (see RESULTS).
An additional group of five mice underwent surgical implantation of the LV tubing and were allowed to recover for 1 wk before being tested in the conscious state to determine the effects of K+X on LV function. In these mice, LV function was tested before and after an intraperitoneal injection of K+X (as above) and again 0.5 wk later to assess any persistent effects of K+X on LV function.
Feasibility of Our Model
Echocardiographic examinations were done before surgery and on the same day as LVP measurements to investigate the effect of LV tubing implantation on LV cardiac function and the subacute effect of anesthetizing agents.
Statistical Analysis
All values in this study are reported as means ± SD. One-way ANOVA for repeated measures was used to analyze the data. Comparisons between the hemodynamic data obtained in ISF and K+X mice were made with unpaired t-tests. Statistical significance was defined as P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Table 2 shows LV variables in conscious mice after recovery from surgical implantation of the LV tubing. HR, LV dP/dtmax, LV dP/dtmax/IP, and LV dP/dtmin were significantly reduced in conscious mice 0.5 wk after surgery with K+X [K+X(all)] compared with those with ISF. Because the first six surgical implantations of LV tubing were done with K+X anesthesia in this study, it was possible that an inadequate surgical experience might be responsible for the slow recovery of cardiac function in that group after surgery. Therefore, we evaluated cardiac function in an additional five mice where K+X was used as the operative anesthesia at the end of the study [K+X(Late)], when the surgical technique was the same or more skillful than that with ISF. Cardiac function in the K+X(Late) group was also worse than that with ISF at 0.5 wk after surgery. To determine whether the depression of LV function 0.5 wk after surgery (Table 2) in mice that had undergone surgery with K+X was due to persistent effects of K+X alone or persistent effects only when K+X was used in association with major surgery, we did additional experiments. In five mice that were recovered for 1 wk after surgery and had normal baseline LV function, K+X produced acute depression of LV function, but LV function recovered completely by 3 days later, in contrast to the observations detailed in Table 2. The fact that K+X injection 1 wk after surgery did not depress cardiac function 0.5 wk after the injection suggested that the K+X-related depression in LV function 0.5 wk after surgery was associated with the combination of K+X together with the surgical procedure.
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Although there were no significant differences in hemodynamic variables between 0.5 and 1 wk in ISF mice, LV dP/dtmax, LV dP/dtmax/IP, and LV dP/dtmin 1 wk after surgery in K+X mice increased significantly compared with those 0.5 wk after surgery. There were no differences in LV variables at 1 wk in conscious mice between the two groups.
Echocardiographic Examination
In both ISF- and K+X-anesthetized mice, echocardiographic examination showed geometrical deformity with small but significant increases in LVEDD and decreases in long-axis L 1 wk after LV tubing surgery (Table 3). Because alterations in the LV shape could be due to the tubing surgery and suturing of the apex, LVEDV was calculated by a two-dimensional area-length method. LVEDV was decreased at 0.5 wk (along with body weight) but recovered by 1 wk after surgery, when body weight had returned to its value before the surgery. LVEDV 0.5 wk after surgery was decreased without any change in LVEDD due to a shape change in which L was decreased. The reduction in LVEDV was accompanied by a mild but significant decrease in LV dP/dtmax/IP and peak aortic flow velocity compared with those 1 wk after the surgery. A significant reduction in body weight after surgery was observed at 0.5 wk (Fig. 5) in both K+X and ISF mice. This loss of body weight had recovered 1 wk after surgery, suggesting that dehydration (due to loss of blood and/or extracellular fluid during surgery and anorexia during recovery) might be a factor in the reduction in LVEDV 0.5 wk after surgery. Interestingly, there was a greater body weight reduction in K+X mice than in ISF mice at 0.5 wk after surgery (Fig. 5; P = 0.006), suggesting longer term anorexia in this group. To clarify whether the change in LV geometry occurred as a result of the implantation of PE-50 tubing, we also examined the LV size for 2 wk before and after surgery using echocardiography in five PE-50-implanted and five sham-operated mice (Fig. 6). Both surgeries were done as usual, but LVP measurements were not attempted throughout the study. In these animals, LVEDV responded similarly in both groups, with a decrease at 0.5 wk after surgery. LVEDV returned to the presurgical level by 1 wk after surgery in both groups and remained unchanged after that. In sham-operated mice, the change in LVEDD was parallel to that in LVEDV, but long-axis L was unchanged throughout the observation period. In the mice that underwent apical suturing, apical stab wound, and placement of PE-50 tubing in the LV through its apex, LVEDD did not change at 0.5 wk but increased at 1 wk. Restoration in LVEDV after 1 wk postsurgery was accomplished by an enlarged LVEDD, in accordance with the shortened LV long axis. Histological examination in three mice showed no myocardial damage throughout the heart except where the suture was tied at the apex, where there was replacement fibrosis.
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Aortic Pressure
In four mice, the Millar catheter could be advanced easily from the LV apex past the aortic valve into the ascending aorta. In these conscious mice, systolic aortic pressure was 121 ± 8 mmHg, diastolic aortic pressure was 68 ± 8 mmHg, and mean aortic pressure was 97 ± 6 mmHg.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Acute Effects of Anesthesia
During anesthesia, LV hemodynamics were altered substantially, suggesting depressed myocardial contractility. LV dP/dtmax was depressed by 57 ± 14% with K+X and 25 ± 8% with ISF. LVEDP rose 139 ± 352% with K+X and 143 ± 247% with ISF. Similar changes were seen in peak LVP and LV dP/dtmin. There are a few studies in the literature that focus specifically on the effect of anesthetics on LV function in mice (14, 17, 22). The acute effects of anesthesia in these studies, however, were evaluated with echocardiography and a tail-cuff blood pressure-measuring system. No study on the effect of anesthetics on directly measured LVP has been reported previously, to our knowledge. This study adds to our understanding of the acute effects of anesthesia on directly measured LVP in mice by directly comparing the effects of different anesthetics on measurements of LV and other hemodynamic parameters made in the same mice in the conscious state.
Persistent Effects of Anesthesia
In addition to identifying a marked acute depressant effect of anesthesia with either K+X or ISF on LV contractile function and HR, we observed what may be a persistent effect of K+X anesthesia for up to 0.5 wk after surgery. As seen in Table 2, LV dP/dtmax was 20% lower in K+X versus ISF mice at 0.5 wk after surgery; HR was also slightly depressed but could not account for the difference in LV dP/dtmax (see below). This persistent depression of function with K+X was not due solely to the anesthesia but rather to its use in association with the surgical procedure, because a separate cohort of mice studied after full recovery (1 wk) from surgery showed an acute but not persistent depression of LV function post-K+X. Thus hypovolemia due to intraoperative fluid loss and postoperative anorexia might have resulted in delayed excretion of K+X, which is excreted primarily by the kidneys in rodents (1). It is also reported that xylazine prolongs the plasma half-life of ketamine and significantly delays formation of the primary metabolite of ketamine. Under these conditions, recovery from the surgery in K+X mice might be prolonged. ISF, which is cleared rapidly by the lungs, would not be expected to persist in the circulation beyond the operative day. In our study, the reduction in body weight 0.5 wk after surgery was significantly greater in K+X mice than in ISF mice; this reduction suggests a prolonged anorexia due to delayed recovery in K+X mice compared with ISF mice. It has been reported that either acute fasting or chronic caloric restriction produces body weight loss (negative energy balance) and reduces HR and mean arterial pressure through the modification of autonomic balance in mice (21). Therefore, fasting due to delayed recovery in K+X mice might be responsible for the depressed peak LVP and HR.
Desai et al. (3) assessed the effect of anesthesia on cardiovascular function in conscious mice using cannulated blood pressure and reported that the effect of gas anesthesia (1–2% methoxyflurane) and surgery was no longer detected 12 h after the cessation of anesthesia. However, Janssen et al. (9) reported that hemodynamic stabilization was obtained after 5 days after surgery (anesthesia with pentobarbital) using a telemetry blood pressure-measuring system, suggesting that the effect of surgery and anesthesia had an influence on the cardiovascular system 4 days after surgery. The possibility is proposed that a difference in the extent of surgery could be responsible for the different results in these studies. The surgical procedures of the latter study (9) were more invasive than those of the former study (3) (laparotomy vs. cannulation). In our study, we employed more invasive surgery (thoracotomy and laparotomy), and recovery from the surgery in our study might be expected to be more prolonged. Moreover, we evaluated cardiac function with directly measured LVP using a high-fidelity micromanometer, which made it possible to assess LV function more precisely than measures of peripheral arterial pressure. Therefore, it is difficult to compare our results with those previously reported because of differences in the severity of the surgery and different methods for assessment of LV function. In addition to the quantitative description of the effect of anesthesia on LV hemodynamics in mice, we also show for the first time the very long duration of postanesthetic effects on LV hemodynamics, a finding that has important implications for the design of experiments involving invasive surgery and genetically altered mice.
Limitations of the Study
Some limitations of this study and the new technique presented need to be pointed out. First, the LV cavity underwent a geometrical shape change from oval to more spherical (increased LVEDD, decreased L) after the surgery, with LV FS remaining normal. This finding may be explained as an effect of the tubing in the LV, the apical suturing, or other unknown factors. Myocardial damage is unlikely to explain the enlargement of the LV cavity, because histological examination showed only focal myocardial damage at the apex. A second limitation is that LVP could not be measured continuously while the mice were unrestricted or exercising but required mild restraint in a soft plastic cone and insertion of the micromanometer catheter. Development of permanently implanted manometers and the use of telemetry, as in larger animals, would be an ideal next step. A third limitation is that we did not make more frequent (e.g., daily) measurements of LV hemodynamics in the week after surgery and thus cannot define more precisely the duration of depressed function with different anesthetic agents.
In summary, we have developed a technique for repeated measurement of LV hemodynamics in conscious mice. We demonstrate that normal LV dP/dtmax is much higher than in most previous studies and that in some circumstances the effect of anesthesia (particularly with K+X) is associated with depressed LV function and HR for days after its administration.
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ACKNOWLEDGMENTS |
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This study was supported in part by a grant from the Wayne and Gladys Valley Foundation (to W. Grossman) and by National Heart, Lung, and Blood Institute Grants HL-31113 and HL-54890 (to P. C. Simpson).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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1A/C- and 1B-adrenergic receptors are required for physiological cardiac hypertrophy in the double-knockout mouse. J Clin Invest 111: 1783–1791, 2003.[CrossRef][Web of Science][Medline]
1-adrenergic receptor knockout mouse. Am J Physiol Heart Circ Physiol 274: H1184–H1193, 1998.. Circ Res 82: 416–423, 1998.This article has been cited by other articles:
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  D. T. McCloskey, S. Turcato, G.-Y. Wang, L. Turnbull, B.-Q. Zhu, T. Bambino, A. P. Nguyen, D. H. Lovett, R. A. Nissenson, J. S. Karliner, et al. Expression of a Gi-coupled receptor in the heart causes impaired Ca2+ handling, myofilament injury, and dilated cardiomyopathy Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H205 - H212. [Abstract] [Full Text] [PDF]
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D. T. McCloskey, L. Turnbull, P. M. Swigart, A. C. Zambon, S. Turcato, S. Joho, W. Grossman, B. R. Conklin, P. C. Simpson, and A. J. Baker Cardiac transgenesis with the tetracycline transactivator changes myocardial function and gene expression Physiol Genomics, June 16, 2005; 22(1): 118 - 126. [Abstract] [Full Text] [PDF]
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 T. Tokudome, T. Horio, I. Kishimoto, T. Soeki, K. Mori, Y. Kawano, M. Kohno, D. L. Garbers, K. Nakao, and K. Kangawa Calcineurin-Nuclear Factor of Activated T Cells Pathway-Dependent Cardiac Remodeling in Mice Deficient in Guanylyl Cyclase A, a Receptor for Atrial and Brain Natriuretic Peptides Circulation, June 14, 2005; 111(23): 3095 - 3104. [Abstract] [Full Text] [PDF]
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