INTRODUCTION

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CARDIOVASCULAR DISEASES account for almost 50% of all deaths in the Western world (3). Coronary heart disease, i.e., myocardial infarction and sudden death, accounts for the largest proportion of this mortality. Much research activity is, therefore, conducted to reveal cardioprotective interventions to reduce cardiovascular mortality. One of the experimental preparations most often used for this purpose is the open-chest animal model. This model allows easy access to the heart for exact measurement of function, metabolism, and electrophysiological variables and their changes during various pathophysiological conditions, like regional myocardial ischemia. However, the heart in this model is exposed to an unphysiological environment. Exposure to room temperature and evaporation from the epicardial surface contribute to cardiac cooling. Because myocardial temperature influences both the occurrence of arrhythmias (13, 14) and development of necrosis during ischemia (1, 2, 5, 20), it is important to measure and control this parameter in experimental settings established to investigate mechanisms of cardiac damage related to ischemia and reperfusion.

Accordingly, much effort has been made to minimize temperature fluctuations in the open-chest animal model. One approach is to keep the exposed heart warm by the use of heating lamps. Heating lamps allow continuous access to the heart during the experiment. However, they do not shield the myocardium from convective air currents and subsequent temperature fluctuations. To reduce such temperature fluctuations, the chest with the exposed heart is often covered with transparent plastic film for insulation. However, insulation prevents easy access to the heart. Therefore, in an attempt both to reduce temperature fluctuations in the exposed heart and to maintain easy access to the heart, we designed and built a thermocage to be placed over the open thorax.

In the present paper, we present the open-chest pig model with the thermocage, and we compare data showing temperature changes, particularly in response to ischemia, in the anterior left ventricular (LV) wall of the exposed heart without any thoracic temperature-controlling device, after proper positioning of a heating lamp, and, eventually, after placement of the specially constructed thermocage. Pigs were used because they have few native collaterals (23), and, therefore, it was not necessary to incorporate collateral flow as a covariate in the comparison of temperature changes. Because both size of ischemic area and depth of myocardial temperature recordings may influence temperature changes during ischemia, comparisons of temperature changes with the heating lamp and the thermocage were performed on the same pigs. We restricted ischemia to a maximum of 10 min because myocardial temperature does not, according to pilot studies, change after this time. Additionally, myocardium subjected to 10 min of ischemia does not develop irreversible cell injury (16), and repeated recordings can be performed in the same heart without development of myocardial infarction.

	METHODS

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Animal preparation. Animals used in the present study were maintained and housed in accordance with the conditions set by the Norwegian Council for Animal Research. The investigation conformed with the "Guide for the Care and Use of Laboratory Animals" [DHEW Publication No. (NIH) 86-23, revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205].

Five domestic pigs of either sex (25.5-33.5 kg) were fasted overnight and anesthetized with pentobarbital sodium, initially 40 mg/kg body wt ip, followed by a sustaining infusion of 5-20 mg · kg¹ · h¹ iv pentobarbital sodium, according to the depth of anesthesia. The pigs were ventilated through a tracheostoma with 50% oxygen-50% air mixture by using a volume-regulated ventilator. A positive end-expiratory pressure of 5 cmH₂O was established. Ventilation frequency and volume were adjusted to keep PCO₂ and pH within normal ranges. Polyethylene catheters were placed in the right femoral vein for administration of drugs and fluids and in the right femoral artery for blood sampling and pressure recording. The heart was exposed through a midsternal split and suspended in a pericardial cradle. In two pigs, a 5- to 10-mm-long segment of left anterior descending (LAD) coronary artery, immediately distal to the first major branch, was dissected free to allow placement of a Mayfield clip for intermittent occlusions and a transit-time flow probe to measure coronary blood flow (CBF). Additionally, a 5-mm-long segment distal to the second major branch of LAD was dissected free to allow intermittent placement of a Mayfield clip. In three pigs, a 10- to 15-mm-long segment of LAD, distal to the first major branch, was dissected free to allow placement placement both of a hydraulic occluder and, distal to it, of a transit time flow probe. Body temperature was maintained at 38.0-39.0°C by using wrappings and a homeothermic blanket system unit (50-7103 Harvard Homeothermic Blanket System, South Natick, MA), with a thermistor probe placed in the abdomen. Urine was drained continuously through a cystostoma. After completion of the surgical preparation, all pigs were allowed a stabilization period of 30 min.

Hemodynamic measurements. A microtip pressure-transducer catheter (Millar Instruments, Houston, TX) was introduced into the LV through the right carotid artery for measurements of LV pressure and the contractility parameter, the maximal positive value of the first derivative of LV pressure (LVdP/dt_max). An electromagnetic flow probe was placed on the ascending aorta and connected to a square-wave electromagnetic flowmeter (model 376; Nycotron, Drammen, Norway). Arterial blood pressure was measured by a Statham pressure transducer (model P23 Gb; Gould Instruments, Hato Rey, Puerto Rico). CBF was measured by transit-time flowmetry (T208; Transonic Systems).

Hemodynamic variables were continuously recorded on an eight-channel galvanometric recorder (model 7758 B; Hewlett-Packard, Medical Products Group, Andover, MA). Immediately before each LAD occlusion, when higher resolution of data was required, the output of the recorder was sampled at 100 Hz, and the signals were transformed by an analog-to-digital converter and stored on floppy disks. Computer samplings of hemodynamic variables were obtained at end expiration, and values from four to six consecutive beats were averaged by the computer.

Experimental procedure. To evaluate temperature changes in the anterior LV wall during regional ischemia, all pigs underwent 8-11 LAD occlusions of 1.5- to 10-min duration. At least four of these occlusions were of 5-min duration. In two of the pigs, both proximal and distal LAD occlusions were performed, in random order, to clarify whether the size of area at risk influenced myocardial temperature changes during ischemia. In the remaining three pigs, LAD occlusions were performed, also in random order, 1) with no thoracic heating equipment, 2) with the heating lamp, and 3) with the thermocage placed over the open chest of the pig. In one of the pigs, equipped with the thermocage, myocardial temperature measurements were performed at multiple points within the wall. Room temperature was kept between 22 and 27°C.

In the regions subjected to ischemia, at least one thermocouple was placed to record changes just beneath the epicardial connective tissue, and another was placed ~5 mm deep into the myocardium. Total coronary occlusion was achieved either by inflating a hydraulic occluder (within 5 s) or by application of a Mayfield clip and was verified by a decline in CBF to zero. Occlusions were only induced during stable CBF. To reduce accidental air currents during the experiments, all doors and windows to the operating theater were closed, and movements of staff members were reduced to a minimum. If ventricular fibrillation occurred, the heart was given one or, if necessary, more direct-current countershocks (15-30 J). If electroconversion was successfully achieved within 1 min, the animal was allowed to continue the experimental protocol. At the end of the experiment, the animals were killed by intracardial KCl injection.

Equipment to control myocardial temperature. To maintain normal cardiac temperature during ischemia, a 150-W heating lamp (Philips IP 150 R) was used. At intervals, the lamp was placed 40-45 cm above the heart, so that epicardial temperature became close to midmyocardial temperature. The effects of the lamp at that distance were recorded by several mercury thermometers placed on a table covered with white cotton sheets. As illustrated in Fig. 1, the central temperature 45 cm below the lamp, i.e., at the same level as the heart, was equal to normal temperature for the pig (6), but the heating effect decreased sharply 5-10 cm away from the center.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Temperatures on a horizontal table covered with white cotton sheets 45 cm below a heating lamp (150 W). Temperatures recorded at given radial distances from the center are presented. Maximal (max) temperature was recorded in central beam underneath center of the lamp. Room temperature was 24°C.

To lessen both convective and evaporative heat loss, the thermocage was used. Briefly, it consists of double Plexiglas (~1-cm space between) formed into a top plate and a front and a back wall (Fig. 2). The space between the Plexiglas was connected to a heating bath (M3 LAUDA, Dr. R. Wobser; KG, D-6970 Lauda-Königshofen Postf. 1251, Germany). The two open sides were closed with wet towels, plastic film, and finally an insulating coat. Coat sleeves made it possible to place the hands within the cage without a drop in temperature or humidity. Both a thermometer and a hygrometer (34- 1549 Clas Ohlson, Oslo, Norway) were placed inside the box.

View larger version (148K): [in this window] [in a new window]   Fig. 2.   Photograph of thermocage that was placed across the open chest of pigs. Tubings supplied warm water for circulation. Open sides were closed with wet towels, plastic films, and an insulating coat (not shown), completely enclosing the open thorax.

Temperature measurements. To measure temperature changes, thermocouples, made of insulated constantan and copper wire (OD 0.075 and 0.09 mm, respectively), soldered together, and sealed within a polyethylene tube (ID 0.25 mm, OD 0.75 mm), were inserted into the myocardium and connected to a reference thermocouple in the pig's abdomen. The junction between copper and constantan generates 40 µV/°K. By connecting the constantan part in the myocardium to the constantan part in the abdomen and connecting the two copper ends to a recorder, the circuit would generate 40 µV/°C temperature difference between the two elements. The voltage differences between the cardiac thermocouples and the reference thermocouple in the pig's abdomen were therefore recorded by means of a direct-current amplifier and a six-channel recorder (Multicorder, model MC6621, Graphtec, Tokyo, Japan). The actual temperatures recorded by the thermocouples were obtained by help of a mercury thermometer positioned at the same place as the reference thermocouple. Room temperature and the temperature within the thermobox were measured with electronic thermometers (Microprocessor Digital Thermometer, model 819, Tegam, Geneva, OH). Simultaneous temperature measurements in a waterbath with all thermometers revealed maximal temperature differences of <0.25°C for temperatures between 35 and 42°C.

Ischemic area. The size of the ischemic area was estimated in pigs that underwent both proximal and distal LAD occlusions. After the pigs were killed, 10 ml of 0.1 M sodium phosphate buffer containing 2% (wt/vol) triphenyltetrazolium chloride were injected into the LAD at the distal occlusion site, after the artery just proximal to the injection site had been tied off. Then, a 10-ml suspension of 2-mg zinc-cadmium sulfide particles (1-10 µm in diameter; Duke Scientific, Palo Alto, CA) per milliliter isotonic saline was injected into the LAD at the proximal occlusion site, after the artery just proximal to this injection site had been tied off. The heart was then excised, both atria were removed, and the ventricles were moulded in 2% agarose at 37°C and cooled in a refrigerator. After the agarose gelled, the ventricles were cut into ~7-mm-thick slices. The slices were cleaned for agarose, weighed, and placed between two glass slides to a uniform thickness. The ventricular area, the area distal to the distal occlusion site (tetrazolium positive), and the area distal to the proximal occlusion site (tetrazolium positive + fluorescent under ultraviolet light) were traced directly onto acetate sheets and planimetered on a digitizing tablet (Videoplan 2, Kontron Electronics, Germany). The weights of the ischemic areas were then calculated by multiplying the fractions of the ischemic areas for each slice with the slice weight and summing the products. Total weight of the ischemic areas was expressed as a percentage of total ventricular weight. Finally, the area perfused with triphenyltetrazolium chloride was carefully examined for any pale regions denoting necrosis.

Statistical analysis. For comparison of hemodynamic data over time, temperature change over time, and transmyocardial temperature differences, a paired t-test was used. A one-way repeated-measures ANOVA was used to compare temperature fluctuations at baseline with no heating equipment, the lamp, and the thermocage. To test the relationship between hemodynamic variables and myocardial temperature, Pearson product moment correlation coefficient (r) was calculated. A value of P < 0.05 was considered statistically significant (two tailed). All values are presented as means ± SD. Because of continuous fluctuations in myocardial temperature, average values were obtained during 0.5- to 1-min intervals.

	RESULTS

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Baseline myocardial temperature fluctuations. In the uncovered open-chest model, we recorded continuous temperature fluctuations in the myocardium of the LV wall (Fig. 3). These fluctuations were more pronounced just beneath the epicardial connective tissue than 5 mm deep into the myocardium and varied between 0.002 and 0.3 Hz. By use of the thermocage, the peak fluctuation during a 4-min period was reduced to approximately one-tenth (Table 1) (Fig. 3). Actually, during shorter time periods, only thermal oscillations with peak amplitude of ~0.005°C and synchronous with the ventilation were observed.

View larger version (111K): [in this window] [in a new window]   Fig. 3.   Recordings showing differences in myocardial and intra-abdominal temperatures (°C) of exposed heart when no thoracic heating equipment (A), or when heating lamp (B) or thermocage (C) were used. Temperature changes by left anterior descending (LAD) coronary artery occlusion in both midmyocardium and epicardium are shown. Recording pens were separated by 5 mm on the recorder; accordingly, deflections are separated by 20 s, with paper speed of 15 mm/min for data presentation. Intra-abdominal temperature was set to zero. Both heating lamp and thermocage prevent cardiac cooling, but only thermocage reduces fluctuations in myocardium to a minimum.

Baseline myocardial temperature. In the open-chest model with no thoracic heating equipment, the temperature just beneath the epicardial connective tissue was 1.89 ± 0.55°C below the intra-abdominal temperature (P < 0.05). At a myocardial depth of 5 mm, it was 0.61 ± 0.25°C warmer than at the epicardial surface (P < 0.05) but still as much as 1.28 ± 0.33°C below the intra-abdominal temperature (P < 0.05). Both the heating lamp and the thermocage prevented the difference between myocardial and intra-abdominal temperatures (Fig. 3).

Figure 4 shows the effect of the thermocage on both myocardial temperature and temperature fluctuations. Furthermore, Fig. 4 illustrates that work with one hand within the thermocage can be performed without any significant decrease in temperature. When using the thermocage, which minimized temperature fluctuations due to air currents within the operating theater, we found that myocardial temperature in the midwall was 0.37 ± 0.05°C higher than that of LV cavity blood.

View larger version (52K): [in this window] [in a new window]   Fig. 4.   Myocardial temperature changes when a hand is put into thermocage and when thermocage is removed and replaced. Intra-abdominal temperature was set to zero.

Myocardial temperature during ischemia. When no thoracic heating equipment was used during a 5-min LAD occlusion, myocardial temperature decreased significantly in the ischemic region (Fig. 3). Just beneath the epicardial connective tissue, the temperature decreased by 2.10 ± 1.01°C (P < 0.05) and at 5-mm depth by 1.86 ± 1.02°C (P < 0.05). Both the heating lamp and the thermocage prevented this decrease in temperature (Fig. 3). Actually, the thermocage enabled us to observe a reproducible temperature increase during each of the LAD occlusions for the first 60 ± 9 s of ischemia, amounting to between 0.04 and 0.16°C, depending on the position of the thermoelement. This increase in temperature occurred both when the temperature within the thermocage was 0.5°C above and when it was 0.5°C below the intra-abdominal temperature. Sometimes a slight increase in myocardial temperature was also noticed during ischemia when the heating lamp was used. However, due to the large temperature fluctuations in the uncovered myocardium, precise temperature measurements were difficult to obtain.

In the two pigs in which the size of the ischemic area was varied, proximal LAD occlusions induced a larger decline in temperature, measured centrally in the ischemic area that constituted 18 ± 8% of the ventricular weight, than did distal LAD occlusions that caused ischemia in only 5 ± 3% of the ventricular weight. This observation is presented in Fig. 5. The decline in temperature by repeated distal or by repeated proximal LAD occlusions was highly reproducible within each animal. No sign of necrosis was found in the area that was subjected to ischemia by both proximal and distal LAD occlusions.

View larger version (75K): [in this window] [in a new window]   Fig. 5.   Myocardial temperature changes by a distal (A) and a proximal (B) LAD occlusion in open-chest pig model without any thoracic heating equipment. Intra-abdominal temperature was set to zero. Both epicardial and midmyocardial recordings were obtained. Epicardial temperature was lower than midmyocardial temperature.

Epicardial exsiccation. In the uncovered open-chest model, there was a visible exsiccation of the epicardial surface during ischemia. Epicardial exsiccation, identified as a dry surface with poor reflection of light, was especially pronounced when the heating lamp was used to maintain myocardial temperature during ischemia. The thermocage prevented epicardial exsiccation during ischemia, as a relative humidity >90% was easily obtained within the cage.

CBF and myocardial temperature. Blood gases and hemodynamic variables recorded at start and end of the experimental procedure, lasting on average 5 h 42 min ± 2 h 42 min, are presented in Table 2. Whereas the blood gases were kept stable by respiratory adjustment, LV systolic pressure fell by 22 ± 16 mmHg (P < 0.05), LVdP/dt_max by 26 ± 18% (P < 0.05), aortic flow by 27 ± 16% (P < 0.05), and CBF by 29 ± 12% (P < 0.05). No significant changes were found in heart rate and mean arterial pressure.

In all experiments, CBF decreased slowly in the course of the experimental protocol. When no thoracic heating equipment was used, the decrease in CBF was accompanied by an increased difference between the intra-abdominal and myocardial temperatures. Accordingly, a significant correlation was found between CBF and the difference between myocardial and intra-abdominal temperatures. Data from one experiment are shown in Fig. 6. The influence of CBF on myocardial temperature was also detected during reactive hyperemia with no thoracic heating equipment, when myocardial temperature approached the intra-abdominal temperature (Fig. 3). With the thermocage applied, midmyocardial temperature, on the contrary, declined during postischemic hyperemia (Fig. 3). No significant correlation was found between the rate-pressure product (heart rate multiplied by mean arterial pressure) and the difference between myocardial and intra-abdominal temperatures.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Coronary blood flow (CBF) as %flow at start of experiment, plotted against difference between intra-abdominal and myocardial temperatures (°C) in open-chest pig model without any thoracic heating equipment. Data were obtained immediately before each occlusion in 1 pig. Note both lower temperature and larger variation just beneath epicardial connective tissue () than at 5-mm myocardial depth (). CBF correlates with myocardial temperature, both at epicardial surface and at 5-mm depth (r = 0.80 and 0.94, respectively; P < 0.05 for both). Regression lines for both depths are shown.

	DISCUSSION

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The present study demonstrates that cardiac cooling, both at baseline and during LAD occlusions in the open-chest pig model, can be avoided by the use of either a heating lamp or a thermocage. However, only the thermocage prevents epicardial exsiccation and reduces fluctuations in myocardial temperature to a minimum.

In an experimental model, designed to find potential cardioprotective interventions against ischemia and reperfusion damage, one should ideally control all factors that might influence myocardial damage. In the open-chest animal model, determinants of infarct size, such as duration of ischemia, the size of area at risk, collateral blood flow, and myocardial oxygen demand are well known (8, 10, 11, 15, 17-19). However, only recently have myocardial temperature variations in the normothermic range been shown to greately influence infarct size development. In the open-chest pig model, we found a significant difference between the epicardial and intra-abdominal temperatures, when no thoracic heating equipment was used, both at baseline and during ischemia. The magnitude of this difference showed a large variability, both within pigs and between pigs, depending on the position of the myocardial thermocouples, CBF, and the size of the ischemic area. Our study emphasizes the importance of measuring and controlling temperature in the myocardium and not relying only on rectal temperature. As myocardial temperature influences both occurrence of arrhythmias (13, 14) and development of necrosis during ischemia (1, 2, 5, 20), this aspect is most important in studies designed to reveal cardioprotective mechanisms against ischemia and reperfusion damage.

When transmyocardial temperature gradients, as observed in the present study, are not taken into consideration, both misleading and confusing conclusions may be drawn. With regard to arrhythmias, duration of cardiac action potentials depends on myocardial temperature (7); myocardial temperature gradients may induce dispersion of refractoriness and, thereby, the occurrence of artificial arrhythmias. Furthermore, transmyocardial temperature gradients in the open-chest model may result in small epi/endo infarct size ratios and, thereby, lead to erroneous conclusions about how infarction actually evolves in the intact organism. The influence of temperature on infarction in the open-chest pig model has been studied by Duncker et al. (2). These reasearchers occluded the LAD for 45 min at different temperatures and found, by linear regression analysis, that 20% of the area at risk became infarcted for each 1°C increase in body core temperature.

By using both the heating lamp and the thermocage, we were able to simulate the closed-chest situation with regard to temperature, with an intrathoracic temperature close to rectal temperature (21). The heating lamp resulted, however, in a significant epicardial exsiccation during ischemia, an unphysiological side effect with unknown effects. Furthermore, correct use of the heating lamp required measurement of temperature within the epicardium. The thermocage secured a stable cardiac environment with regard to both temperature and humidity. Thus evaporative heat loss was almost completly avoided. Furthermore, it minimized myocardial temperature fluctuations and thereby allowed precise temperature recordings. As the water-heated side and top panels prevented condensation, continuous inspection of the heart was possible. Inspection is often necessary when manipulation on the heart is performed or when easy verification of ischemia should be obtained. Both the heating lamp and the thermocage allowed manipulation of the heart without significant changes in myocardial temperature.

Methodological consideration. By using a paired design, it was unnecessary to incorporate both size of ischemic area and position of thermoelements as covariates when comparing temperature changes during the three different interventions. Furthermore, the experimental design enabled us to detect significant differences by use of only a few animals. The use of pigs, with few native collaterals, secured an almost identical degree of ischemia during repetitive occlusions. Therefore, temperature differences due to collateral flow were avoided. Due to the paired design, it was possible to keep the animal preparation essentially stable during the experimental protocol. The homeothermic heating system allowed us to keep the intra-abdominal temperature within a narrow range. However, we recorded a decrease in LV systolic pressure, LVdP/dt_max, aortic flow, and CBF during the experiment. The decrease in CBF, probably representing an adjustment to both a decreased energy demand in stunned myocardium (4, 12) and a decreased global cardiac work requirement, influenced the myocardial temperature during periods with no thoracic heating equipment. To compensate for the hemodynamic changes, the different interventions were performed in random order, and only the median value from each intervention was used.

It is well known that both the occurrence of ventricular fibrillation and the duration of reactive hyperemia increase with increasing occlusion time (9, 22). Accordingly, to limit the occurrence of ventricular fibrillation and to reduce the total duration of experiments, the reproducibility of temperature changes induced by ischemia was mainly evaluated by short occlusion periods. However, to confirm stable myocardial temperature during ischemia with the heating equipment, longer lasting occlusions were also performed. The longer lasting occlusions were restricted to a maximum of 10 min, because myocardium subjected to 10 min of ischemia does not develop irreversible cell injury (16), and repeated recordings can be performed in the same heart without development of myocardial infarction. Examination of epicardial exsiccation was only determined by visual inspection. Accordingly, we did not further quantify the extent of epicardial exsiccation. However, this might be necessary when studies are performed to examine physiological consequences of epicardial exsiccation in the open-chest model.

In conclusion, to control myocardial temperature fluctuations and avoid transmyocardial temperature gradients in the open-chest pig model, either a thermocage or a heating lamp may be used. Both the thermocage and the lamp allow continuous inspection and manipulation of the heart, but only the thermocage prevents epicardial exsiccation and almost completely prevents all unphysiological temperature fluctuations.