**1. Introduction**

*In vivo* experimental animal models are often used to elucidate or, at least clarify, specific mechanisms and/or to identify interrelationships between monitored functions that cannot be observed directly in humans. The results of such studies are often approximated to preclinical or clinical research and, can thus, have a significant scientific impact on a more detailed understanding of the monitored system.

A specific feature of *in vivo* experimental animal models is the fact that experiments are usually performed with the animals under general anesthesia, in which homeostatic regulatory mechanisms are not removed and the animal responds to various interventions. Undoubtedly, this also applies to experiments in which changes in electrocardiographic (ECG) parameters are monitored after various interventions or after the administration of specific agents to assess the basis of the origin and development of heart rhythm disorders. However, different anesthetics may have varying impacts on myocardial electrophysiology. Thus, the extent to which ECG parameters

are altered from normal after anesthetic administration can become a confounder—if not a problem—even before assessing the effects of the intervention itself.

The second problem is that many published methodologies do not describe the synchronization of the animals to the light-dark (LD) cycle, mainly in studies based on rat models. The LD cycle is the strongest synchronizer for this type of laboratory animal, and it is known that all measurable cardiovascular parameters oscillate depending on the LD cycle. Moreover, even when this synchronization is described, the time of day at which the experiments are performed is often not reported. In common practice, experiments are performed during regular work hours (i.e., during the day); therefore, after synchronization of rats, for example, to the LD cycle (12 h: 12 h), these experiments are essentially being performed on "sleeping" animals during their naturally inactive period. The question then becomes, what are the oscillations of ECG parameter values during a 24-h period (i.e., spanning the light [inactive] and dark [active] period) in healthy, sexually mature rats?

Another possible problem in the correct evaluation of changes in myocardial electrophysiology in rats may be sex. Sex is not typically considered in *in vivo* cardiovascular and toxicological experiments involving rats, although this type of experimental model animal is commonly used to examine normal and pathological physiology. In the majority of experimental studies, only male rats are used; however, there is another sex (i.e., female) in which differences in the essence of functional systems and response(s) to the same interventions are different from males. The study of sex differences is also a driving force of development and, in many cases, the basis of health and medicine. However, there are opinions that the study of sex differences is ineffectual and does not merit extensive research [1]. One of the reasons why both sexes are not used in experiments is the simple fact that males and females are biologically different and these differences increase the range of variability. However, if sex differences are documented and accounted for in experimental studies, these must be respected. As such, future studies should address these questions and attempt to include females in experiments where possible.

This review aims to highlight the fact that there are differences in baseline or control values, which are, nevertheless, used as reference values in individual studies. However, they are impacted by the type of anesthesia used, and all the abovementioned confounders/problems can significantly affect the correct interpretation of the results obtained.

## **2. Evaluation of ECG parameters**

The methodologies of studies that performed *in vivo* rat cardiovascular or toxicological experiments were retrieved from a search of the Web of Science database for articles published mainly between 2000 and 2021; in total, 130 articles were retrieved. ECG parameters reported as baseline or control values were summarized and averages with ranges were calculated. Not all ECG parameters were described and evaluated in each study and, in some studies, two to three control values were reported. A relatively high number of studies described only changes in ECG parameters, in terms of lengthening and shortening, and these changes were directly indicated in graphs without reporting numerical baseline values.

Because each ECG parameter has diagnostic significance, we focused on commonly evaluated ECG parameters, including the following: heart rate (HR), atrial complex (PR interval, P wave duration, and P wave amplitude), and ventricular

complexes (QRS complex, QT and QTc interval duration, and R wave and T wave amplitude).

Tables consider studies (although there were only one or two), which also suggest a possible sex difference with regard to the LD cycle on the monitored parameter. The figures show the ranges of the monitored parameter from at least three baseline or control values.

## **3. Prognostic significance of changes in HR in arrhythmogenesis**

HR is an easily measurable parameter of cardiac activity, and alterations in HR can have a direct effect on the cardiovascular system. Caetano and Alves [2] reported that increased resting HR is an independent predictor of cardiovascular and overall mortality in the general population. Thus, the occurrence of arrhythmias is often associated with baseline HR, which has prognostic significance. In a review article titled "Arrhythmias and heart rate: Mechanisms and significance of a relationship", Zaza et al. [3] describe, in detail, the mechanisms influencing arrhythmogenesis according to HR, in which the authors focused on several factors related mainly to electrical stability of the myocardium. HR also reflects autonomic balance, which also affects myocardial stability. The prognostic significance of the relationship between arrhythmias and HR may vary depending on the substrate present in a specific case and should be considered. In rats, electrical stability of the heart has been shown to be greatest at increased HRs in the dark (i.e., active) part of the regimen day, when myocardial vulnerability to ventricular arrhythmias decreases [4].

It has been found that tachycardia may provide greater electrical stability to the myocardium; however, if an abnormal substrate is present, it may trigger arrhythmia [5]. Severe bradycardia, in contrast, can trigger life-threatening arrhythmias, thus reflecting its destabilizing effect on repolarization. Zaza et al. [3] remained cautious, arguing that, from a mechanistic perspective in assessing the relationship between HR and arrhythmias, the question should be "what is the appropriate sinus rate for autonomic balance?" and not "what is the high (or low) heart rate?" Thus, it can be assumed that baseline HR in *in vivo* cardiovascular studies can significantly affect the results obtained during experimentation. The considerations mentioned above are also generally valid for rats. However, it is interesting that the effect of some interventions on HR is monitored and the impact of this change on myocardial electrophysiology is not further analyzed [6–8].

#### **3.1 Telemetry and HR**

To establish reference values for HR, as well as other ECG parameters, logically, the most suitable method is using telemetry studies, in which rats are not placed under general anesthesia and ECG can be recorded continuously throughout the day. Telemetry studies help to reveal very important information about fluctuations in myocardial electrophysiological parameters during the day. Currently, however, relatively few telemetry studies have analyzed ECG parameters in rats under *in vivo* conditions, and did not address circadian dependence and the dependence on sex.

Sex can also be a confounder. Nevertheless, several experimental rat studies [9] did not report any sex differences in heart repolarization, or that there is little clear evidence supporting sex differences in ventricular repolarizations *per se*, in which there is only a short estrous cycle lasting only 4 days [10]. Although no sex differences were found in the repolarization of isolated ventricular myocytes, they were

associated with excitation and contraction [11]. Sex differences were not found in APD90 between isolated ventricular myocytes, in external K+ currents, Ipk and Isus, in internal rectification current IK1, or ICa [11, 12]. While less information is available from animal models, sex differences in the ionic basis of the effective refractory period in the atria and atrioventricular node may also contribute to sex differences in the incidence of atrial fibrillation and supraventricular tachycardias. Nevertheless, the physiological significance of sex differences has yet to be fully determined; as such, further studies are needed to clarify the basic mechanisms.

Baseline HR analysis from telemetry studies involving non-anesthetized rats, in which a chronobiological approach was applied, indicates that there is a circadian rhythm in HR among rats, with a higher HR during the active (i.e., dark) period of the regimen day and not only in males [13–17] but also in females [15, 18]. If HR exhibits circadian fluctuations, then when it is evaluated, it can be problematic.

The question is whether there are also sex differences in single-lighted periods. Telemetry studies have revealed that among females, HR values are lower in both light periods (**Table 1**). The averaged results of baseline HR values indicate that sex differences are exhibited in both the light and dark periods of the rat regimen day; however, more experimental studies are needed to confirm these data. In female rats, changes in HR depended on the LD cycle; however, LD differences were modified by the anesthetic used [19, 20]. Although the adaptation of animals to the LD cycle was described in the Methods sections, it is not clear from the methodologies whether the values of the presented HRs were average values from the entire 24-h period, or the current baseline value only from certain time intervals before the intervention itself when the measurements were performed or recorded.


*Rat Electrocardiography and General Anesthesia DOI: http://dx.doi.org/10.5772/intechopen.104928*


*Data presented as average heart rate (beats/min) (range); (n, number of baseline or control values from which heart rate was evaluated). Not specified—the methodology did not specify the lighted period when the experiments were performed.*

#### **Table 1.**

*Heart rate under individual types of anesthesia according to sex and light cycle (light [inactive]) versus dark [active]).*

#### **3.2 General anesthesia and HR**

The question is what are the reference values for HR in the rat under normal circumstances? Based on the values reported in **Table 1**, is clear that HR varies depending on the type of general anesthesia, which can be problematic in evaluating changes in HR after an intervention. Other factors, in addition to general anesthesia, that may directly or indirectly affect the initial HR can be the methodology used to determine HR, the time of day (or part of the rat regimen day) at which the experiments are performed, or the fact that the majority of ECGs are evaluated only in male rats; as such, there is little-to-no information about HR in females.

Evaluation of HR in telemetry studies involving male rats [21–31] reported a mean HR of 347 beats/min, with a range of 303 beats/min up to 362 beats/min without taking into account the evaluation methodologies and the time of day the experiments were performed.

If we consider that the average HR value with the range reported in telemetry studies involving male rats is our desired reference value, then a slightly increased average HR in pentobarbital (approximately 28 beats/min.) [32–51], and urethane anesthesia (approximately 32 beats/min) [52–60]. In female rats under pentobarbital anesthesia, baseline HR values were reported in only one study, depending on the LD cycle [20]. Even with pentobarbital anesthesia, although nonsignificant, there were LD differences. In female Wistar rats, pentobarbital probably only modifies circadian rhythms, but does not disturb them. Thiopental anesthesia [31, 61–71] did not alter HR from the mean HR reported in telemetry studies.

A significant tachycardic effect was found under isoflurane (approximately 62 beats/min) [72–75], desflurane (approximately 95 beats/min) [72], and chloralose (approximately 72 beats/min) [76, 77] anesthesia in male rats.

Under ketamine/xylazine anesthesia [45, 78–92], HR was drastically reduced in males and reduced values were also recorded in females [93, 94]. In females have been preserved significant LD differences [19].

The effect of phenobarbital [95], ketamine/medetomidine [96], ketamine/midazolam [97], and ketamine/diazepam [96, 98] on anesthesia could not be assessed as valid because there was only one study.

Although ether is no longer used to induce general anesthesia, some works used this type of light anesthesia needed to perform ECG recordings [99–104]. However, ether anesthesia had virtually no effect on HR. One study describing HR in isolated rat hearts did not reveal any significant deviation, in terms of tachycardia or bradycardia [105]. Interesting differences were also found between young and old rats under tribromoethanol anesthesia, where higher values prevailed in older rats (405 ± 11 beats/ min vs. 381 ± 1 beats/min) [106]. Unfortunately, these comparisons are only from males and without a description of the adaptation of the animals to the LD cycle.

From **Table 1** and **Figure 1**, it is evident that for different types of general anesthesia, baseline or control HR values can differ significantly compared to the mean baseline HR from telemetry studies, which can logically be considered as a reference value. There is very little information about HR in females and almost none of the studies took circadian fluctuations into account.

#### **Figure 1.**

*Distribution of average values and ranges of heart rate (HR) from telemetry studies and under different types of general anesthesia in rat males without taking into account the light periods of the rat regimen day when the experiments were performed. Only HR ranges from at least three studies where HR has been evaluated are shown in the figure. Telemetry studies (n = 16), pentobarbital anesthesia (n = 22), thiopental anesthesia (n = 13), ketamine/xylazine anesthesia (n = 18), isoflurane anesthesia (n = 5), ether anesthesia (n = 6), urethane anesthesia (n = 14). (n—number of baseline or control values from which heart rate [HR] was evaluated).*
