**4. Chronobiology of anesthesia**

Anesthesia is often required in in vivo experiments to ensure comfort and to eliminate pain in animals. However, in small animals, the use of anesthesia can cause certain problems, and therefore, it is necessary to recognize the effect of anesthesia on the internal environment and to account for LD changes in the individual parameters of homeostasis. However, from experimental practice, we know that experiments are performed mostly during working hours (i.e., during light). Thus, if rats are synchronized to the light and dark modes corresponding to the annual season, experiments are performed in the light period of their regimen day (i.e., during their inactive period, when many physiological functions are inhibited). Experiments are, therefore, essentially performed on "sleeping" animals, and questions regarding function during the active part of their regimen day will remain. However, most methodologies do not specify the time of day at which the experiments are performed or the factors responsible for changes in the particular monitored parameters over time. Instead, they focus primarily on current mechanical and metabolic changes, often regardless of the functional status of the body systems over a 24 h period, which may be a problem from a chronobiological point of view [59, 60]. Animal adaptation should, therefore, be taken into account, particularly in in vivo experiments.

Normative data regarding arterial acid-base balance and plasma ion concentrations would help to identify healthy animals suitable for experiments [1], and there are studies that have examined the reliability of these data [61]. **Tables 1** and **2** summarize the ranges of some acid-base balance parameters and ion concentrations in arterial rat blood, which have been described in several published studies. However, the time at which the experiments were performed or the time of blood sampling for evaluation of blood gases, pH, bicarbonates, and some ions, or the synchronization of animals to the LD cycle, was not considered in the methodologies of these studies.

effect on biological functions [78]. Some have pointed to the temporal dependence of some anesthetic effects on the [78] circadian rhythm. For example, in locomotor activity, a phase shift of circadian rhythm occurred after administration of selected anesthetics, indicating its dependence on time. Pentobarbital injections induced both advanced and delayed phase shifts in the circadian rhythm of movement activity in SK mice; however, no phase shifts were observed in any circadian time with pentobarbital injections in C57BL mice. This suggests

**Author(s) (year of publication) pH pCO2**

Lewis et al. [62] 7.43 5.47 12.13 Pepelko and Dixon [63] 7.446–7.486 5.24–5.74 11.77–12.71

Hess et al. [66] 7.43–7.51 3.33–4.67 12.2–15.4 Dettmers et al. [67] 7.38–7.46 5.19–5.99 9.4–11 Chi et al. [68] 7.27–7.37 4.78–5.77 13.8–17 Ohoi and Takeo [69] — 4.66–5.32 13.3–17.3 Schultz et al. [70] 7.35–7.45 3.33–5.32 10.6–14.6 Sun and Wainwright [71] 7.40–7.45 4.64–5.32 11.3

Forkel et al. [72] 5.16–6.39 12.85–15.48

Luo et al. [75] — 5.58–6.08 10.37–12.19

Costa et al. [77] 138.9–141.1 4.74–4.86 6.72–7.38

**Authors (year of publication) Na+**

Menegon et al. [76] 142.1–143.9 3.6–3.8

Subramanian et al. [1] 140.7–145.6 3.08–4.02

Brun-Pascaud et al. [64] 7.45–7.49 4.2–4.99 11.26–12.72 24–27 Girard et al. [65] 7.46–7.47 4.57–4.71 12.72–13.02 25–25.8

Valenza et al. [73] 7.41–7.43 5.18–5.48 — 25.3–27.1 Subramanian et al. [1] 7.26–7.4 5.05–7.51 10.76–14.60 21.5–28.1 Peralta-Ramírez et al. [74] 7.2–7.46 5.62–6.20 — 23.2–25.8

**Range\* 7.369–7.452 4.75–5.298 10.75–14.184 23.8–26.78**

\*Ranges were calculated as the mean value from the lower and upper limits of the ranges reported in these studies.

**Table 1.** Values of pH, blood gases, and bicarbonate in the arterial blood of rats published in previous studies.

 **(mmol/l) K+**

Valenza et al. [73] 132.4–140 4.1–4.42 102.9–107.7

Peralta-Ramírez et al. [74] 134.6–137.3 3.93–4.25 1.23–1.29 104.4–108.1 **Range\* 137.4–140.7 3.86–4.21 ? 103.7–107.9**

\*Ranges were calculated as the mean value from the lower and upper limits of the ranges reported in these studies.

**Table 2.** Arterial plasma ion concentrations in the arterial blood of rats according to previously published studies.

 **(kPa) pO2**

 **(kPa) HCO3**

http://dx.doi.org/10.5772/intechopen.75174

Chronobiology of Acid-Base Balance under General Anesthesia in Rat Model

 **(mmol/l) Ca2+ (mmol/l) Cl<sup>−</sup>**

 **(mmol/l)**

**− (mmol/l)** 113

Although chronobiological studies investigating the interactions between general anesthesia and circadian rhythms are scarce, they all suggest that general anesthesia has a significant


What is the effect of anesthetics on ongoing ion-dependent processes? Evidence from voltage-clamp studies of individual nerve fibers suggests that, for example, molecules of local anesthetic interact with sodium channels directly from the inside of the nerve membrane. Anesthetics bind to sodium channels, which open during membrane depolarization and prevent normal sodium flow. Anesthetic molecules can separate from open channels, but not from channels that remain closed when the nerve is kept in the resting state. The "gate" properties, which regulate the opening and closing of sodium channels, are reversibly adjusted during anesthesia [57]. Despite the significant advances in chronobiological studies, the mechanisms of circadian regulation of ion channels remain largely unknown. By exploring and understanding the circadian regulation of the ion channel in detail, progress in the development of therapeutic effective strategies for the treatment of sleep disorders, cardiovascular diseases, and other diseases associated with circadian desynchronization [58]

Anesthesia is often required in in vivo experiments to ensure comfort and to eliminate pain in animals. However, in small animals, the use of anesthesia can cause certain problems, and therefore, it is necessary to recognize the effect of anesthesia on the internal environment and to account for LD changes in the individual parameters of homeostasis. However, from experimental practice, we know that experiments are performed mostly during working hours (i.e., during light). Thus, if rats are synchronized to the light and dark modes corresponding to the annual season, experiments are performed in the light period of their regimen day (i.e., during their inactive period, when many physiological functions are inhibited). Experiments are, therefore, essentially performed on "sleeping" animals, and questions regarding function during the active part of their regimen day will remain. However, most methodologies do not specify the time of day at which the experiments are performed or the factors responsible for changes in the particular monitored parameters over time. Instead, they focus primarily on current mechanical and metabolic changes, often regardless of the functional status of the body systems over a 24 h period, which may be a problem from a chronobiological point of view [59, 60]. Animal adaptation should, therefore, be taken into account, particularly in

Normative data regarding arterial acid-base balance and plasma ion concentrations would help to identify healthy animals suitable for experiments [1], and there are studies that have examined the reliability of these data [61]. **Tables 1** and **2** summarize the ranges of some acid-base balance parameters and ion concentrations in arterial rat blood, which have been described in several published studies. However, the time at which the experiments were performed or the time of blood sampling for evaluation of blood gases, pH, bicarbonates, and some ions, or the synchronization of animals to the LD cycle, was not considered in the

Although chronobiological studies investigating the interactions between general anesthesia and circadian rhythms are scarce, they all suggest that general anesthesia has a significant

will be developed.

in vivo experiments.

methodologies of these studies.

**4. Chronobiology of anesthesia**

112 Circadian Rhythm - Cellular and Molecular Mechanisms

\*Ranges were calculated as the mean value from the lower and upper limits of the ranges reported in these studies.

**Table 1.** Values of pH, blood gases, and bicarbonate in the arterial blood of rats published in previous studies.


**Table 2.** Arterial plasma ion concentrations in the arterial blood of rats according to previously published studies.

effect on biological functions [78]. Some have pointed to the temporal dependence of some anesthetic effects on the [78] circadian rhythm. For example, in locomotor activity, a phase shift of circadian rhythm occurred after administration of selected anesthetics, indicating its dependence on time. Pentobarbital injections induced both advanced and delayed phase shifts in the circadian rhythm of movement activity in SK mice; however, no phase shifts were observed in any circadian time with pentobarbital injections in C57BL mice. This suggests that differences in phase shifts after the use of pentobarbital are not quantitative but qualitative [79], and that pentobarbital-induced phase shifts are not the result of increasing levels of activity [80].

of possible circadian variations) and to determine whether there are differences between types of anesthesia after the immediate application of the most common anesthetics in in vivo rat experiments, pentobarbital (P), ketamine/xylazine (K/X), and zoletil (Z) in spontaneously

Chronobiology of Acid-Base Balance under General Anesthesia in Rat Model

http://dx.doi.org/10.5772/intechopen.75174

115

The present study conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH publication number 85–23, revised 1996). The study protocol was approved by the Ethics Committee of the Medical Faculty of Safarik University (Kosice, Slovak Republic) (permission numbers 2/05 and ŠVPS

The present study was performed using female Wistar rats (mean [±SD] weight 310 ± 20 g), 3–4 months of age after a 4-week adaptation to an LD cycle (12 h light:12 h dark [intensity of artificial illumination 80 Lux]; 40–60% humidity; cage temperature 24°C; two animals/cage; *ad libitum* access to food and water). The effect of the light period on the monitored parameters was examined after adaptation to an LD cycle, with the light period from 06:00 to 18:00 h. The effect of the dark period was monitored after adaptation to the inverse setting of the LD cycle

The animals were divided into one of three experimental groups according to anesthetic agent used (**Table 3**). Approximately 20 min after administration of anesthetic agent, the spontaneously breathing animals were fixed supine to an experimental table. pH and blood gases from blood samples obtained from the femoral artery were examined using a blood-gas analyzer

**Figure 1.** Scheme of adaptation to the light-dark (LD) cycle. Arrows indicate the time of the experiment. The experiments were performed once in each animal in the course of a single LD period (the first animal at 09:00 h and the second at 12:00 h).

(i.e., with the light period from 18:00 to 06:00 h) (**Figure 1**).

breathing rats.

SR: Ro-4234/15–221).

**6. Materials and methods**

In a study by Pang et al. [81], pentobarbital had no apparent effect on melatonin release and did not affect plasma levels of cerebral natriuretic peptide in rats, in which both hormones are at a relatively low level at 02:30 h [82]. Naguib et al. [83] described the effects of anesthesia on melatonin production. Anesthesia disrupts the circadian rhythm of melatonin, the major humoral transmitter of suprachiasmatic nuclei activities in the hypotalamus [84–86]. It appears that intravenous anesthetics with different behavioral profiles act on different and specific ligand-bound ion channels to create specific anesthetic behavior. Whether the anesthetic effect of melatonin is due to a direct effect on melatonin receptors remains largely unknown. Melatonin receptors, as such, are not commonly considered to be molecular targets for general anesthetic effects. However, there is evidence to suggest that the central effects of melatonin include at least partial facilitation of GABAergic transmission by modulation of GABA receptors [87–89]. In a study by Mihara et al. [90], pentobarbital demonstrated no effect on melatonin secretion or on movement activity, regardless of the time of dosing. On the other hand, in rats under general propofol anesthesia, the plasma concentration of melatonin decreased over the first 4 h after anesthesia induction and increased after 20 h. Thus, general propofol anesthesia abolishes the circadian rhythm of melatonin in rats adapted to an LD cycle [91].

Results of a study by Kana et al. [92], involving the inhalation anesthetic sevoflurane, reported that sevoflurane had the greatest efficacy in suppressing mPer2 expression (mPER2 acts as a positive rhythm transcription regulator in hypothalamic suprachiasmatic nuclei) in the morning. The investigators proposed that, in the morning, this biochemical reaction is inhibited by anesthesia, which can lead to suppression of mPer2 expression and effectively reflect circadian clocks. However, at the phase delay of movement cycle activation, sevoflurane acted independently of time.

Prudian et al. [93] and Pelissier et al. [94] reported a disrupting effect of ketamine on circadian rhythms; however, this effect was associated only with a modification of acrophase, amplitude or mesor, without loss of daily rhythmicity. To date, however, there is no literature evidence supporting the effect of general anesthesia on acid-base balance and ion concentration in arterial blood, depending on circadian rhythmicity or LD cycles. This highlights the fact that different anesthetics may have different effects on the circadian rhythms of many parameters.
