Differential effects of isoflurane and ketamine/inactin anesthesia on ...

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Nov 24, 2004 - Abstract We evaluated the effect of the inhalant anesthetic isoflurane and the injectable combination of anesthetics ketamine/inactin on cardiac ...
Basic Res Cardiol 100: 147 – 153 (2005) DOI 10.1007/s00395-004-0503-6

James R. Peña Beata M. Wolska

Received: 3 September 2003 Returned for 1. revision: 23 September 2003 1. Revision received: 20 September 2004 Returned for 2. revision: 7 October 2004 2. Revision received: 14 October 2004 Accepted: 18 October 2004 Published online: 24 November 2004

B. M. Wolska, Ph.D. (쾷) · J. R. Peña Center for Cardiovascular Research Department of Medicine Section of Cardiology (M/C 715) University of Illinois at Chicago 840 S. Wood St. Chicago, IL 60612 E-Mail: [email protected] B. M. Wolska Department of Physiology and Biophysics University of Illinois at Chicago Chicago, IL 60612

ORIGINAL CONTRIBUTION

Differential effects of isoflurane and ketamine/inactin anesthesia on cAMP and cardiac function in FVB/N mice during basal state and ␤-adrenergic stimulation

쮿 Abstract We evaluated the effect of the inhalant anesthetic isoflurane and the injectable combination of anesthetics ketamine/inactin on cardiac function by measuring left ventricular (LV) pressure in situ during control conditions and during ␤-adrenergic stimulation with isoproterenol (ISO). The control heart rate (HR) and the maximal rate of contraction were significantly higher in the isoflurane group, but there was no difference in the rate of relaxation. During the ISO (0.32 ng · g body wt–1·min–1) stimulation the developed pressure (DP) increased 9.8 ± 1.8% (n = 11) in the ketamine/inactin group and was unchanged in the isoflurane group. The HR increased 28.4 ± 4.8% (n = 11) in the ketamine/inactin group and only 3.4 ± 0.6% (n = 11) in the isoflurane group. The rate of contraction increased 103.2 ± 9.3% (n = 11) and 13.6 ± 4.6% (n = 11) in the ketamine/inactin and isoflurane groups, respectively. At this dose of ISO the rate of relaxation did not change significantly. In control conditions there was no difference in levels of cAMP between the groups (2.29 ± 0.25 pmol/mg protein (n = 5) in the ketamine/inactin group and 2.79 ± 0.35 pmol/mg protein (n = 6) in the isoflurane group). However, during the ISO stimulation the cAMP level increased only in the ketamine/ inactin group of animals (3.50 ± 0.30 pmol/mg protein; n = 5). This level was significantly higher than the level in the isoflurane group stimulated with ISO (2.22 ± 0.30 pmol/mg protein; n = 6). In summary, our results indicate that the anesthetics differ significantly in the extent of depression of the basal and ␤-adrenergic stimulated state with the second messenger cAMP playing a prominent role. 쮿 Key words In situ hemodynamic parameters – mouse heart

Introduction

BRC 503

Transgenic (TG) and knockout (KO) murine models are important tools for the study of in vivo mechanisms of cardiovascular disease. With the TG and KO models a gene thought to be involved in cardiac function, development, or pathogenesis can be ablated or modified and the resultant phenotype studied physiologically over a given period of time. The power and value of these models has increased with the timely advancement of the

technology used to accurately measure physiological parameters in small rodents. However, simulating the normal conscious physiological state, when the experimental protocol necessitates the use of anesthesia, remains problematic. Indeed, most in vivo murine studies report cardiac hemodynamic parameters that are well below the normal physiological range for mice [4, 5, 9, 17]. This obviously complicates and may even obscure the interpretation of data derived from such experiments, yielding false positives and negatives for important physiological phenotypes.

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Two prototypic anesthetic regimens currently used in murine studies are the injectables and the inhalants. Most of the anesthetics in these two categories have been shown to cause significant cardiac depression [1, 8, 21, 25, 28). The level of cardiac depression depends not only on the anesthetic agents used but also on the strain of mouse studied and may vary even within the same strain of mice [21]. The mechanism of this depression is still unclear, but may involve multiple cardiac targets [7, 10], which may in part explain the difference in the spectrum of action and degree of cardiac depression among the anesthetics. That the sympathetic system may play a dominant role in the mechanism of cardiac depression is likely given that mouse basal cardiac function is predominantly under sympathetic control [3, 11]. The simultaneous growth of molecular biology and transgenic technology has led to numerous in vivo murine transgenic models that now require the development of methodologies to accurately measure cardiac function. Critical to this development is the assessment and critique of the benefits and limitations of the anesthetics currently available. To this end, we have designed experiments to examine and compare the effects of the injectable anesthetic combination ketamine/inactin and the inhalant anesthetic isoflurane on basal and ␤-adrenergic stimulated cardiac function in normal FVB/N mice, since they are the most common strain used for transgene overexpression. Moreover, although it has been recently shown that conscious FVB mice have a high basal sympathetic and low parasympathetic activity and do not respond to bolus injection of ␤-agonist, isoproterenol (ISO) [23], it is unknown whether anesthetics (ketamine/inactin vs. isoflurane) alter the state of the autonomic nervous system and as a result effects the response to ␤-adrenergic stimulation. The assessment of cardiac function was performed by closed-chest left ventricular (LV) catheterization in normal mice. In addition, we sought to determine whether the second messenger cAMP may be involved in the complex mechanisms that underlie the diverse effects of anesthesia on cardiac function. Our results indicate that the anesthetics differ significantly in the extent of depression of basal and ␤adrenergic stimulated state with the second messenger cAMP playing a prominent role.

Materials and methods 쮿 Experimental animals Twenty-nine male and female adult FVB/N mice weighing 22–34 g were used for hemodynamic experiments and eleven for measurement of cyclic adenosine monophosphate concentration. The mice were housed in cages

at 24 ˚C with a 12 : 12 hour light-dark cycles and with free access to food and water, in full compliance with the Public Health Service animal welfare policy and the American Association for the Accreditation of Laboratory Animal Care. An animal research protocol was fully approved by the University of Illinois/Chicago Animal Care and Use Committee and experiments were performed according to these guidelines.

쮿 Experimental protocol A set of mice was anesthetized by intraperitoneal injection of 50 µg/g body weight of ketamine and 100 µg/g body weight of thiobutabarbital sodium (Inactin, Research Biochemical International, Natick, MA) [4, 17]. Supplemental doses of anesthesia (1/3 of initial dose) was given after the initial dose as needed to maintain adequate depth of anesthesia, as determined by spontaneous movement and maximal response to toe pinch [4, 17]. Another set of mice was anesthetized with the use of a vaporizer (Vapomatic, A.M. Bickford, INC., Wales Center, NY) delivering 1.5% isoflurane (Abbott Laboratories, Chicago, IL). In each set the mice were intubated while in the supine position and ventilated with 100 % oxygen (tidal volume 0.3 mL; rate 125 breaths/min; respiratory/ expiratory ratio 1; positive end-expiratory pressure (PEEP) was between 5 and 10 cm H2O) at a flow rate of 0.8 L/min via an animal ventilator (Columbus Instruments, Columbus, OH). The depth of anesthesia with each anesthetic was comparable as determined by the pedal withdrawal reflex. Every twenty minutes throughout the experimental protocol, the right or left hind limb was stretched by pulling the fascia in between the toes and subsequently pinching it with a blunt forcep. When a sharp withdrawal of the hind limb was observed after the fascia was pinched, it was assumed that the surgical anesthesia had diminished and an additional IP bolus of the anesthetic mixture was given. The toe pinch was repeated to determine whether the anesthetic dose was sufficient to eliminate the reflex. The total number of times that the animal demonstrated a pedal withdrawal reflex during the experiment was used as an index of anesthesia level [28]. The body temperature was maintained at 37 ˚C with a thermally controlled warming plate and monitored throughout the experiment using a rectal temperature probe. To gain access to the left ventricle, the right carotid artery was isolated and the distal end ligated [4]. The artery was then cannulated with a Millar 1.4 F Mikro-Tip transducer (model SPR-671, Millar, Houston, Texas). With the continuous online pressure signal serving as a guide, the transducer was advanced retrogradely down the right carotid, into the aorta, through the aortic valve, and into the left ventricle (LV). When a stable left ventricular waveform was displayed, the transducer was

J. R. Peña and B. M. Wolska Anesthesia and cardiac function in mice

secured in place with suture. As recommended by the manufacturer, the transducer was calibrated, before each experiment, in a sealed chamber containing warm saline at 20 and 200 mmHg. At the termination of each experiment, the transducer was immediately rebalanced in warm saline to check for drift. To gain venous access, the right femoral vein was catheterized with a stretched piece of PE-10 polyethylene tubing. The free end of the tubing was connected to a 250 µl Hamilton glass syringe mounted on an infusion pump (Harvard Apparatus, Holliston, MA). All of the surgical incisions were covered with saline-soaked gauze to minimize body fluid evaporation. Post-surgical cardiac dynamics were stable for 30 minutes before commencement of the experimental protocol. Basal and ␤-adrenergic stimulated cardiac dynamics were measured for each anesthetic group. To analyze the myocardial response to ␤-adrenergic simulation, we infused, via the femoral venous line, isoproterenol (ISO 0.32 and 0.64 ng · g body wt–1·min–1). The infusion vehicle consisted of 0.9% saline with 10 U/mL heparin added to prevent clotting of the venous line. During the experimental infusion, the animals’ hearts were excised and immediately freeze clamped in liquid N2 for the measurement of cAMP. The raw data signal were amplified on the internal amplifier in a WindowGraf Chart Recorder (Gould Instrument Systems, Valley View, OH), recorded at 2,000 Hz, and analyzed using the Left Ventricular Pressure Module of the Po-ne-mah Digital Acquisition Analysis and Archive System software (Gould Instrument Systems) on a personal computer. This program performs calculation of heart rate (HR), LV end-diastolic pressure (LVEDP), LV systolic pressure (LVSP), developed pressure (DP), maximal rate of pressure increase over time (dP/dtmax), and maximal rate of pressure decrease over time (dP/dtmin). The left ventricular pressure parameters were calculated every five seconds and the raw data transferred to an Excel (Microsoft, Seattle) file for further calculations. A continuous 15-minute interval, immediately prior to ISO infusion, was used to obtain the mean baseline values for each animal. After obtaining stable recordings during infusion of ISO, a continuous 5-minute infusion interval was used to obtain mean values for ␤-adrenergic stimulation. In addition, to examine the impact of infusing a volume of 0.1 µL/g body weight over 3 min on cardiac function, we infused the vehicle alone (0.9 % saline) and found no effect.

쮿 Measurement of cyclic adenosine monophosphate concentration Hearts were excised from mice that were under the anesthetic isoflurane or ketamine/inactin with or without ISO. The hearts were excised and immediately freeze clamped in liquid N2 and stored at –80 °C until use. The

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hearts were homogenized in 10 volumes of Hank’s balanced salt solution (without calcium and magnesium) and containing 5 mM EDTA with a motor driven tissue homogenizer. The homogenates were centrifuged at 1000 g for 10 min at 4 °C. A portion of the homogenate was used to measure the amount of total protein by the Bradford method (Bio-Rad, Hercules, CA). Cyclic AMP was extracted from the supernatant fractions by the solid-phase extraction method using Amprep Sax columns (Amersham Pharmacia, Piscataway, NJ). The concentrations of cAMP were measured by the Biotrak cAMP enzyme immunoassay system (Amersham Pharmacia, Piscataway, NJ).

쮿 Data computation and statistical analysis All results were presented as mean ± SE. The significance of difference between the means was evaluated by oneway ANOVA or ANOVA for repeated measurements followed by the Student-Newman-Keuls test. A value of P ≤ 0.05 was the criterion for significance.

Results We evaluated the effects of the inhalant anesthetic isoflurane and the injectable combination of anesthetics ketamine/inactin on cardiac function by measuring LV pressure in situ during control conditions (without ISO) and during ␤-adrenergic stimulation (with ISO). The basal hemodynamic parameters in mice anesthetized with isoflurane or ketamine/inactin are summarized in Table 1. There was no significant difference in LVEDP and DP between the isoflurane and ketamine/inactin groups. However, the basal HR was significantly higher in the isoflurane group compared with ketamine/inactin group. Also the maximal rate of contraction (dP/dtmax) was significantly higher in the isoflurane group, but there was no difference in the rate of relaxation (dP/dtmin). Next we compared the effect of ␤-adrenergic agonist ISO (0.32 ng·g body wt–1·min–1) on the hemodynamic parameters in the isoflurane and ketamine/inactin group of animals (Table 1). We have chosen this dose of ISO since it was previously shown [4] that this dose has maximal effects on the hemodynamic parameters in FVB/N mice under ketamine/inactin anesthesia. Moreover, our preliminary experiments showed that lower doses of ISO (0.01 – 0.16 ng · g body wt–1· min–1) were without any effects on the hemodynamic parameters in FVB/N mice under isoflurane anesthesia (data not shown). Figure 1 shows the % change in DP, HR, dP/dtmax and dP/dtmin in both groups of mice during ISO stimulation. The response to ISO was significantly different in the ketamine/inactin group when compared to the isoflurane

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Table 1 Hemodynamic parameters during basal (control) state and isoproterenol (ISO) stimulation Group

Treatment

LVEDP (mm Hg)

DP (mm Hg)

HR (bpm)

dP/dtmax (mm Hg/s)

dP/dtmin (mm Hg/s)

Ketamine/ Inactin

Control (n = 11)

2.6 앐 2.2

73.2 앐 2.0

460 앐 14

5860 앐 340

5752 앐 231

ISO (n = 11)

1.5 앐 1.4

80.5 앐 1.71

585 앐 131

11695 앐 4661

6261 앐 145

Control (n = 11)

2.8 앐 1.8

75.8 앐 2.6

555 앐 102

8022 앐 4202

6547 앐 297

ISO (n = 11)

1.8 앐 1.1

75.7 앐 2.2

574 앐 11

9038 앐 4882

6173 앐 292

Isoflurane

Values are means 앐 SE. LVEDP left ventricular end-diastolic pressure; DP developed pressure; HR heart rate; dP/dtmax maximal rate of pressure increase over time; dP/dtmin maximal rate of pressure decrease over time 1 Significant difference within group 2 Significant difference between groups

Fig. 1 Effects of isoproterenol (ISO) on changes in developed pressure (DP), heart rate (HR) and rates of contraction (dP/dtmax) and relaxation (dP/dtmin) in hearts of mice anesthetized using ketamine/inactin (n = 11) or isoflurane (n = 11). Values are presented as means 앐 SE. *Significant difference from ketamine/inactin

Fig. 2 Effects of ketamine/inactin (n = 5) and isoflurane (n = 6) on the whole heart cyclic adenosine monophosphate (cAMP) level induced by isoproterenol (ISO). Values are presented as means 앐 SE. *Significant difference from control value. 쏆 Significant difference from ketamine/inactin with ISO

group. The DP increased 9.8 ± 1.8% (n = 11) in the ketamine/inactin group and was unchanged in the isoflurane group. The HR increased 28.4 ± 4.8% (n = 11) in the ketamine/inactin group and only 3.4 ± 0.6% (n = 11) in the isoflurane group. The rate of contraction increased 103.2 ± 9.3% (n = 11) and 13.6 ± 4.6% (n = 11) in the ketamine/ inactin and isoflurane groups, respectively. At this dose of ISO the rate of relaxation did not change significantly in either anesthetic group. In the next series of experiments (n = 7) we used a higher dose of ISO (0.64 ng·g body wt–1·min–1) to prove that isoflurane truly blunts the response to ISO and was not due to a shift in the dose response curve. We found that the response to this higher dose of ISO was not significantly different from the lower dose of ISO. The HR increased 2.9 ± 1.0% and 3.7 ± 2.0% at lower and higher doses of ISO, respectively. The dP/dtmax increased 15.9 ± 5.0% at the lower dose of ISO and 25.4 ± 10.6% at the higher dose of ISO. At both doses of ISO neither DP nor dP/dtmin changed significantly. To test whether the reduced response to ISO in the isoflurane group was due to lack of activation of PKA, we measured the cAMP levels in hearts from the ketamine/ inactin and the isoflurane groups in the basal state and during ISO (0.32 ng·g body wt–1·min–1) stimulation (Fig. 2). There was no difference in levels of cAMP in basal state between both groups (2.29 ± 0.25 pmol/mg protein (n = 5) in the ketamine/inactin group vs. 2.79 ± 0.35 pmol/mg protein (n = 6) in the isoflurane group). However, during the ISO stimulation the cAMP level increased only in the ketamine/inactin group of animals (3.50 ± 0.30 pmol/mg protein; n = 5). This level was significantly higher than the level in the isoflurane group stimulated with ISO (2.22 ± 0.30 pmol/mg protein; n = 6).

Discussion The major finding of the present study is the lack of hemodynamic and cAMP responses to ␤-adrenergic

J. R. Peña and B. M. Wolska Anesthesia and cardiac function in mice

stimulation with the use of isoflurane but not ketamine/ inactin anesthesia in FVB/N mice. We found that in FVB/N mice not only the basal hemodynamic parameters differ between mice anesthetized using ketamine/inactin and isoflurane, but even more significantly there is a differential response to the selective ␤-agonist, ISO. We also observed a similar lack of hemodynamic response to ISO in C57BL/J mice anesthetized with isoflurane (Wolska, unpublished observation). The basal HRs in anesthetized mice (Table 1) were lower than those reported in conscious mice [2, 14, 23, 27). Similar HR values were seen in Swiss, CD-1, BalbC and C57Bl6 mice given the anesthetic regimen ketaminemedetomidine-atropine or isoflurane [28]. In contrast, lower HRs was seen with the anesthetic regimen xylazineketamine in Swiss mice [8]. The anesthetic regimen used in this study, inactin/ketamine, appears to have similar effects on HR as the ketamine-medetomidine-atropine regimen [28]. Moreover, the HR was less depressed in mice anesthetized with isoflurane compared to the ketamine/inactin group. The primary advantage of isoflurane is that it preserves sympathetic vasomotor activity, has minimal cardio-depressive effects, and permits precise control of the anesthetic plane [16, 21]. Our data show that the HR of the isoflurane group was about 560 bpm, which is in the lower range of reported resting HRs in conscious mice, indicating minimal depression [2, 3, 12, 14, 27]. However, during ␤-adrenergic stimulation the HR increased only to 574 bpm, which is significantly below that reported for exercise or ␤-adrenergic stimulated HR of 750–850 bpm in conscious mice [3, 12, 23, 27]. It appears that under isoflurane anesthesia there is relatively small depression of basal HR but a lack of response to ␤-adrenergic stimulation. In mice under ketamine/inactin the basal HR was 460 bpm indicating significant depression, and yet the mice responded to ␤adrenergic stimulation to yield HRs in the normal basal physiological range. This is consistent with previous work using ketamine/inactin [4]. Interestingly the 28% increase in HR with ␤-adrenergic stimulation observed in this work corresponds to the documented 30% maximal HR above resting rates [18]. The maximal HR reserve differs among species and is considerably smaller in small mammals such as mice [15]. Moreover, the maximal HR reserve may also be different among different strains of mice. For example, it has been recently shown indirectly that FVB mice have higher basal sympathetic and lower basal parasympathetic activity than C57Bl6 mice and they respond differently to ␤-stimulation and ␤-blockade [23]. Bolus injection of ISO did not change the HR in conscious FVB mice but increased the HR in conscious C57Bl6 mice [23]. This was most likely due to the higher baseline HR in the FVB mice compared to C57Bl6 mice and therefore greater HR reserve in C57Bl6 mice. In our experiments we observed a significant increase in HR with ISO with inactin/keta-

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mine anesthesia, but not with isoflurane anesthesia. This was most likely partially due to the lower basal HR and greater HR reserve in the inactin/ketamine group compared to isoflurane group. However, during ␤-adrenergic stimulation the HRs recorded in anesthetized mice were still significantly lower than that reported in conscious mice suggesting that maximal HR is significantly depressed by both anesthetics. The rates of contraction and relaxation in anesthetized mice were also lower than that predicted based on hemodynamic parameters in other mammalian species in the conscious state. It has been observed that the ratio of dP/dtmax to HR is well conserved between mammalian species and varies between 25 and 30 [15]. Therefore, for mice with a HR of 550 bpm the dP/dtmax should be between 13,750 and 16,000 mmHg/s. These values predicted for dP/dtmax have been seen in mice awakening from anesthesia [18]. Others have suggested that under maximal dobutamine stimulation dP/dtmax can be as high as 30,000 mmHg/s at a HR of 700 bpm [16]. Our value of about 8000 mmHg/s for basal dP/dtmax under isoflurane demonstrates substantial depression and a dP/dtmax below the normal physiologic range. An even greater depression in the dP/dtmax was seen with mice under ketamine/inactin. As in the case with HR, the dP/dtmax in mice under isoflurane showed a small response, about 13% increase, to ISO (0.32 ng·g body wt–1·min–1). Moreover, the response observed at 0.64 ng·g body wt–1·min–1 of ISO was not significantly different. In contrast mice under ketamine/inactin displayed a substantial response, a 103% increase, to 0.32 ng·g body wt–1·min–1 of ISO. It is apparent from our results that isoflurane almost completely eliminates the response to ␤-adrenergic stimulation. Myocardial ␤-adrenergic receptors mediate an increase in HR, the force of heart contraction, the rate of cardiac activation and relaxation in response to ISO. Stimulation of the ␤-adrenergic receptors results in the activation of adenylate cyclase via the stimulatory G-protein Gs to trigger formation of cAMP. A major function of cAMP is to activate cAMP-dependent protein kinase (PKA), which in turn phosphorylates protein targets involved in cardiovascular regulation. This critical rise in cAMP levels in response to ␤-adrenergic stimulation was not observed in mice under isoflurane but did occur in mice under ketamine/inactin. This explains the attenuation of ␤-adrenergic stimulation by isoflurane and provides a mechanism for this observation. The step at which isoflurane inhibits ␤-adrenergic stimulation could not be determined from our in situ experiments and it was not a goal of our studies, but probably precedes the activation of adenylate cyclase. Studies done in isolated vascular smooth muscle of rat aorta suggested that isoflurane interferes with ␤-adrenoreceptor-mediated responses at a point after the agonist receptor binding but before adenylate cyclase activation

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[26]. However, work done on rat subepicardial coronary arteries demonstrated that isoflurane attenuates cAMPdependent vasodilation at a step distal to adenylate cyclase and cAMP phosphodiesterase is not involved in this effect [19]. In contrast, Schotten et al. reported no effect of isoflurane on ␤-adrenergic responsiveness in isolated rat papillary muscle [22]. These conflicting reports may reflect the multiple effects of isoflurane on cardiovascular physiology indicating significant difference in the response to anesthetics between in vivo and in vitro situations. Moreover, it has been shown that in vitro isoflurane alters myofibrillar responsiveness to Ca2+ [13] and cellular Ca2+ control [6]. Isoflurane also may have direct effects on PKA [20] and PKC [24]. Our results indicate that in vivo the diverse effects may act in concert but ␤-adrenergic suppression is the dominant effect. In summary, although the manipulation of the mouse genome has produced many TG and KO mouse models

with important cardiovascular phenotypes, the size and unique physiology of the mouse pose many challenges to both invasive and noninvasive assessment of cardiovascular function. We have found that independent of the mouse strain, age and gender, not only the basal hemodynamic parameters but also the degree of response to ␤-stimulation strongly depends on the anesthetic used. With some anesthetics such as isoflurane the typical positive inotropic effect of ISO was significantly blunted and the effect on cAMP level was absent. Therefore, to properly interpret murine cardiovascular data it is critical to understand the multiple side effects of anesthesia in use.

Acknowledgments This research was supported by NIH research grant RO1 HL-64209. B.M.W. is an Established Investigator of the American Heart Association.

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