**6. Materials and methods**

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

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

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

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

The specific objective of the present in vivo study is to investigate chronobiological aspects of the status of acid-base balance and plasma ion concentrations in arterial blood (i.e., existence

activity [80].

114 Circadian Rhythm - Cellular and Molecular Mechanisms

an LD cycle [91].

independently of time.

parameters.

**5. Aims**

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 SR: Ro-4234/15–221).

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

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).


**Table 3.** Experimental groups.

(ABL 800 Flex, Radiometer Medical, Copenhagen, Denmark) in the Department of Laboratory Medicine, Faculty Hospital Louis Pasteur in Kosice. The depth of anesthesia was estimated according to whether painful stimuli evoked noticeable motor or cardiovascular responses.

#### **6.1. Statistical analysis**

The data were analyzed using GraphPad InStat (GraphPad Software, USA) and presented as mean ± SD. ANOVA was used to detect significant differences within a single end point. The Tukey-Kramer test was used to identify significant differences between groups; p < 0.05 was considered to be statistically significant. The experiments were performed over the course of an entire year, and the results were averaged independent of season and estrous cycle.
