Influence of Anesthetics on the Incidence of Reperfusion-Induced Arrhythmias and Sudden Death in Rats

Abstract

Purpose: We compared the influence of pentobarbital (P, 60 mg/kg), urethane (U, 1.8 g/kg), and a contribution of diazepam with ketamine hydrochloride (D+K, 10 + 100 mg/kg) anesthesia on the incidence of ischemia/reperfusion-induced arrhythmias in rats. In anesthetized rats, myocardial ischemia was produced by a 6-min ligation of the left main coronary artery, followed by 5 min of reperfusion. The incidence of reperfusion-induced ventricular fibrillation (VF) and ventricular tachycardia (VT) was markedly decreased in the D+K-anesthetized animals compared with the P-anesthetized group (VF, 46 vs. 89%; VT, 64 vs. 94%). The mean blood pressure (MBP) before coronary ligation was significantly lower in U-anesthetized animals (72 ± 3.5 vs. 102 ± 4.1 and 108 ± 5.9 mm Hg in P and D+K, respectively). The MBP recovery was the best in the D+K-anesthetized group. In experimental work, pentobarbitone anesthesia may be recommended for studying arrhythmias, while the combination of diazepam and ketamine may be useful for hemodynamic investigations.

Restoration of the coronary blood flow after a brief ischemic period results in severe, life-threatening arrhythmias in experimental animals ⁽¹⁾. These arrhythmias can be consistently elicited in anesthetized rats and may represent arrhythmias developing after surgical reperfusion, thrombolysis, or spontaneous release of coronary vasospasm in humans. Most often they appear in the form of ventricular fibrillation, ventricular tachycardia, and premature ventricular beats. Reperfusion arrhythmias in anesthetized rats are easily reproducible, thus giving a good opportunity for examining the effects of different agents on these types of cardiac arrhythmias.

In rats the most severe reperfusion arrhythmias appear after 5-6 min of preceding ischemia. The mechanism leading to the development of these arrhythmias is still the subject of speculation; both reentry and increased automaticity can be responsible for the process ⁽²⁾. It is well established that the occlusion of the coronary artery creates a significant electrical inhomogeneity between the ischemic and nonischemic myocardium that seems to be rapidly and further exacerbated by reperfusion. This heterogeneity can be the electrophysiologic basis supporting the theory that reperfusion arrhythmias develop through reentry mechanisms. Other studies claimed that increased automaticity plays an important role in the formation of reperfusion arrhythmias ^(3,4). This contradiction seems to be resolved by a study from Kaplinsky et al. ⁽⁵⁾ who, on the basis of their experiments on greyhound dogs, suggested that reperfusion arrhythmias are of two types. The first type is reentry arrhythmias appearing within the first minute after the release of occlusion, with increased incidence of ventricular fibrillation. The second type is arrhythmias caused by enhanced automaticity, occurring between the second and seventh minutes after the onset of reperfusion, with a lower incidence of ventricular fibrillation.

An earlier study in our laboratory ⁽⁶⁾ showed that there is considerable difference between conscious and pentobarbitone-anesthetized animals in the survival rate and in the number of animals that developed irreversible ventricular fibrillation in response to acute coronary ligation. Thus the origin of some discrepancies in the literature concerning the results of reperfusion arrhythmia studies may also be found in the different anesthetization methods applied in those studies.

Little is known about the influence of different anesthetics on reperfusion-induced arrhythmias. Thus the aim of our experiments was to compare the influence of three anesthetic methods commonly used in cardiovascular studies (i.e., diazepam + ketamine hydrochloride, pentobarbitone, and urethane anesthesia) on reperfusion-induced arrhythmias in rats.

MATERIALS AND METHODS

Animals

This study was performed on male Sprague-Dawley CFY rats, weighing 300-350 g. The animals were housed six to a cage and allowed to have tap water and laboratory rat chow (Altromin, Gödöllö, Hungary) ad libitum until the experiment. The animals were handled according to a protocol reviewed and approved by the Ethical Committee for the Protection of Animals in Research of the Albert Szent-Györgyi Medical University, Szeged, Hungary.

Anesthetic agents

Pentobarbitone sodium (P; Ceva, Paris, France), 60 mg/kg in a volume of 2 ml/kg was administered intraperitoneally. Diazepam + ketamine hydrochloride (D+K; Richter, Budapest, Hungary), 10 and 100 mg/kg intraperitoneally, respectively, in a volume of 4 ml/kg. Urethane (U; Reanal, Budapest, Hungary) also was administered intraperitoneally (1.8 g/kg in a volume of 4 ml/kg).

Coronary artery ligation and reperfusion

Acute coronary ligation and reperfusion was produced according to Kane et al. ⁽⁷⁾. After tracheal cannulation, thoracotomy was performed in the fourth intercostal space and the heart was exposed. A loose loop of 5-0 atraumatic silk (K 890 H; Ethicon, Edinburgh, England) was placed around the left main coronary artery, ∼2 mm from its origin. Both ends of the ligature were led out of the thoracic cavity through a flexible tube. The heart was set back in its place, and artificial respiration started immediately with 60 beats/min (Harvard rodent ventilator, model 683; Harvard Apparatus, South Natick, MA, U.S.A.).

Blood pressure was measured in the carotid artery by using a pressure transducer (Gould-Statham, P23ID; Hugo Sachs Electronik, March-Hugstetten, Germany) and was recorded on an oscillographic recorder (Watanabe, WTR 331; Hugo Sachs Electronik). The catheter was filled with isotonic saline containing heparin (500 IU/ml), but the animal was not heparinized. The standard ECG (lead II) was also recorded with the help of subcutaneous needle electrodes.

After stabilization of the blood pressure and heart rate that took ∼10 min, the loose loop was tightened and fixed by clamping on the silk, and thus local myocardial ischemia was produced. After 6 min of myocardial ischemia, the ligature was released and 5 min of reperfusion followed.

The incidence of arrhythmias was registered during both occlusion and reperfusion in accordance with the Lambeth conventions as ventricular tachycardia (VT), ventricular fibrillation (VF), and other types of arrhythmias including single extrasystoles, bigeminy, salvos, and bradycardia ⁽⁸⁾. The onset and duration of arrhythmias also were measured ⁽⁹⁾. An arrhythmia score was used to evaluate the incidence and duration of arrhythmias by giving a grade to each animal as follows: 0, no arrhythmias; 1, <10 s VT or other types of arrhythmias, no VF; 2, 11-30 s VT or other types of arrhythmias, no VF; 3, 31-90 s VT or other types of arrhythmias, no VF; 4, 91-180 s VT or other types of arrhythmias, or <10 s reversible VF, or both; 5, >180 s VT or other types of arrhythmias, or >10 s reversible VF, or both; 6, irreversible VF.

No attempt was made artificially to revert VF during ischemia or reperfusion. At the end of the experiments, the hearts were excised, and after tightening the ligation, they were perfused retrogradely with 10 ml isotonic saline and 2 ml of 96% ethanol through the aorta for determining the extent of the perfusable and nonperfusable areas ⁽⁹⁾. According to our previous investigations, using ethanol instead of formaldehyde for the retrograde perfusion does not change the perfusable areas significantly. The bright white area represented the perfusable area of the heart, whereas the color of the nonperfusable myocardium did not change. The hearts were cut along the epicardial borderline, and the wet weight of the nonperfusable myocardium was expressed as the percentage of the total weight of the ventricles. The weight of the nonperfusable myocardium was not significantly different among the three groups and varied between 37.7 ± 2.13% and 38.6 ± 1.77%. If the perfusion proved that the ligature was at an inadequate place (the whole heart could be perfused) and no change in the ECG (T-wave elevation, QRS distortion) and no decrease in blood pressure occurred on tightening of the ligature, the animal was excluded from the evaluation of the experiments. On the basis of these criteria, four animals were excluded.

Statistical evaluation

The incidence of arrhythmias was expressed as a percentage and compared by using the χ² method. All other parameters were expressed as mean ± standard error of the mean (SEM) and, after analysis of variance, compared by means of the modified t statistic of Wallenstein et al. ⁽¹⁰⁾.

RESULTS

Hemodynamic parameters

Urethane anesthesia resulted in a significantly lower mean blood pressure (MBP) at the end of the stabilization period that remained for the occlusion period as well (Table 1). During reperfusion, this significant difference in MBP was diminished. The decrease in blood pressure in D+K-anesthetized animals was significantly less expressed during coronary artery occlusion and during reperfusion compared with that in pentobarbitone-anesthetized rats (Table 1). There was a modest increase in the basal heart rate in the pentobarbitone-anesthetized group, but there was no significant difference in the heart rate response among the three groups during ischemia or reperfusion (Table 1).

TABLE 1: Hemodynamic parameters during ischemia and reperfusion in pentobarbitone-, diazepam + ketamine-, and urethane-anesthetized rats

The pressure-rate index (PRI) is the product of mean blood pressure and heart rate (mm Hg/min × 1,000) and corresponds to the oxygen and energy demand of the myocardium. The PRI was significantly lower in the urethane-anesthetized rats before occlusion compared with pentobarbitone-anesthetized animals, and during ischemia, it was significantly higher in D+K-anesthetized rats than in the pentobarbitone-anesthetized group (Table 1).

Arrhythmias during occlusion

The arrhythmias during coronary artery ligation occurred in the sixth minute in all groups. During the 6-min occlusion period, five animals died in the pentobarbitone-anesthetized group, whereas in the other two groups, all animals survived occlusion (Table 2). The incidence of VT and VF was significantly less in both the D+K- and urethane-anesthetized groups compared the pentobarbitone-anesthetized animals (Table 2). The incidence of other types of arrhythmias, including sporadic extrasystoles, bigeminy, and salvos, was significantly higher in the pentobarbitone-anesthetized animals than in the urethane-anesthetized group. The arrhythmia scores of the pentobarbitone-anesthetized animals were markedly higher during occlusion than those of the animals in the other two groups (Table 2).

TABLE 2: The survival rate, incidence of arrhythmias, and the arrhythmia scores during ischemia and reperfusion in pentobarbitone-, diazepam + ketamine-, and urethane-anesthetized rats

In the D+K- and urethane-anesthetized groups, the arrhythmias during myocardial ischemia appeared later than in the pentobarbitone group (Table 3). The duration of the period characterized by different arrhythmias during ischemia was shorter both in the D+K- and urethane-anesthetized groups than in the pentobarbitone-anesthetized group (Table 3).

TABLE 3: The appearance, duration, and length of arrhythmic attacks during ischemia and reperfusion in pentobarbitone-, diazepam + ketamine-, and urethane-anesthetized rats

Arrhythmias during reperfusion

Reperfusion-induced arrhythmias appeared within 10-30 s after the release of the ligature and were more severe than those developing during the 6 min of ischemia. In the D+K-anesthetized group, the incidences of VF and VT during rerperfusion were significantly lower than those in pentobarbitone- or urethane-anesthetized animals (Table 2). As a result, the survival rate during reperfusion was significantly better in the D+K-anesthetized group, and the arrhythmia score was also significantly smaller than in the other two groups (Table 2). There was no significant difference in the duration of reperfusion arrhythmias in the surviving animals in the three groups. However, VF did not develop in the D+K- and urethane-anesthetized surviving animals in contrast to the pentobarbitone-anesthetized animals (Table 3).

DISCUSSION

The experimental induction of reperfusion arrhythmias is a well-accepted and useful tool for measuring the efficacy of potentially antiarrhythmic drugs, because these arrhythmias can be reproduced under precise conditions in both in vitro and in vivo experiments. The severity of reperfusion arrhythmias depends on the time course of the preceding coronary occlusion ⁽²⁾. Our intention was to produce serious and reproducible arrhythmias during reperfusion. The 6 min of preceding coronary occlusion in our study provided severe and rapidly appearing arrhythmias after the release of the coronary ligation, although the ischemic period was not long enough for a significant amount of severe ischemic arrhythmias to develop ⁽¹¹⁾.

The majority of the in vivo experiments investigating arrhythmias during occlusion and reperfusion are performed on anesthetized animals; thus it seems to be important to consider not only the analgesic and anesthetic effects of the anesthetic but also the general depressant action on the autonomic nervous system.

In cardiovascular experimental work, pentobarbitone, urethane, diazepam, and ketamine are widely used anesthetic agents. Few data can be found in the literature concerning the effects of these anesthetic agents on reperfusion-induced arrhythmias. In our experiments, we compared the effects of three anesthetic methods (i.e., urethane, diazepam, and ketamine combination, and pentobarbitone) on ischemia- and reperfusion-induced arrhythmias in rats.

Sodium pentobarbitone has well-known autonomic reflex-decreasing and vagolytic effects, of which the most prominent manifestation is its vagolytic action on the heart (i.e., the increase in heart rate). In our experiments, we also found a moderate increase in the basal heart rate of sodium pentobarbitone-anesthetized animals. It is also a membrane depressant on myocytes and reduces the responsiveness of these cells to sympathetic stimulation, thus having some kind of antiarrhythmic effect ⁽¹²⁾. Indeed, Siegmund et al. ⁽⁶⁾ demonstrated that during pentobarbitone anesthesia, compared with conscious animals, a significantly lower number of animals developed irreversible VF during the first 20 min of myocardial infarction. Dawson et al. ⁽¹³⁾ showed that pentobarbitone affects the VF threshold in dogs, thus influencing the vulnerability of the myocardium to VF.

In our experiments comparing three different types of anesthesia, however, the incidence of VF and VT was the highest in the pentobarbitone-anesthetized animals during both occlusion and reperfusion. Hunt and Ross ⁽¹⁴⁾ found that pentobarbitone prolonged the QT interval without changing ventricular refractoriness in dogs, whereas Saarnivaara and Lindgren ⁽¹⁵⁾ found that thiopental, another barbiturate, also prolongs the QT interval during clinical induction of anesthesia. This mechanism possibly predisposes the myocardium to the development of serious ventricular dysrhythmias.

Diazepam is commonly administered for sedation to patients with acute cardiac ischemia. Some studies have reported that the beneficial effects of diazepam lie not only in its anxiolytic action but it also has direct cardiac effects by reducing contractility and increasing myocardial blood flow in both in vivo and isolated heart investigations ^(16,17), thereby improving the prospects of the ischemic myocardium to survive. The identification of peripheral benzodiazepine receptors in the heart seems to support this theory ⁽¹⁸⁾. Diazepam have been found to exert antiischemic effects in patients with documented coronary artery disease ⁽¹⁹⁾. Diazepam in combination with ketamine is widely used for anesthetic purposes in human interventions as well. Diazepam has been found to block the positive inotropic and chronotropic effects of ketamine with excellent analgesic and amnesic effects, and is thereby suggested as an alternative anesthesia in open-heart surgery ⁽²⁰⁾. Our findings (i.e., the hemodynamic parameters of diazepam + ketamine-anesthetized animals were the most impressive during ischemia and at the end of the reperfusion period) correspond with the beneficial hemodynamic effects of diazepam on the ischemic myocardium as outlined in the literature. Moreover, the combination of diazepam and ketamine induced remarkably fewer and less severe arrhythmias in our study. The incidence of VF and VT was significantly lower in D+K-anesthetized rats than in the pentobarbitone-anesthetized animals, resulting in significantly increased survival rate.

Urethane is reported to have a marked depressive effect on the cardiovascular system ^(21-23). Indeed, we found that the blood pressure and the PRI was significantly lower in the urethane-anesthetized animals than in the pentobarbitone- or in the diazepam + ketamine-anaesthetized rats. This effect of urethane on the cardiovascular system may have great importance not only for investigators measuring hemodynamic parameters but also for those who study arrhythmias in anesthetized animals. Goes et al. ⁽²⁴⁾ found that the duration of reperfusion arrhythmias after 10-min occlusion significantly increased in isolated rat hearts taken from urethane-anesthetized animals compared with hearts taken from pentobarbitone-anesthetized animals. In our experiments, the duration of these arrhythmias in urethane-anesthetized rats did not differ significantly from that in pentobarbitone-anesthetized animals. However, the length of VF and the total length of arrhythmic attacks during reperfusion in the surviving pentobarbitone-anesthetized animals was significantly longer compared with the urethane-anesthetized animals. In our investigations, there was no significant difference in the incidence of reperfusion arrhythmias between the pentobarbitone- and urethane-anesthetized groups that corresponds to the findings of the in vitro study by Goes et al. ⁽²⁴⁾.

In conclusion, because the combination of diazepam and ketamine has a remarkable activity in decreasing the incidence of ischemia- and reperfusion-induced arrhythmias, this type of anesthesia may be recommended mainly for surgical interventions in experiments in which avoiding ischemia- and reperfusion-induced arrhythmias is desirable (i.e., hemodynamic investigations during myocardial infarction in animal experiments). Pentobarbitone anesthesia has advantages in cardiovascular experimental work for studying arrhythmias and potentially antiarrhythmic drugs during occlusion and reperfusion, because it allows the development of severe ventricular arrhythmias, providing the opportunity for the investigated drugs to diminish dysrhythmias themselves. The hemodynamic parameters remain sufficiently stable throughout pentobarbitone anesthesia, whereas we must be aware of the cardiovascular depressive effects of anesthesia when applying urethane for experimental purposes.

Acknowledgment: This work was supported by the Hungarian National Research Fund (OTKA grant T 5270) and Ministry of Welfare (ETT grant T06521). We thank Mrs. Zsuzsa Åbrahám-Kovács and Mrs. Mária Györffy-Kosztka for their skillful technical assistance.