Anesth Analg 2003;97:1663-1666
© 2003 International Anesthesia Research Society
ANESTHETIC PHARMACOLOGY
Orexin A Elicits Arousal Electroencephalography Without Sympathetic Cardiovascular Activation in Isoflurane-Anesthetized Rats
Yoshikazu Yasuda, MD PhD*,
Akiko Takeda, MD*,
Satoru Fukuda, MD PhD*,
Hisato Suzuki, MD*,
Masayuki Ishimoto, MD*,
Yoshiteru Mori, MD*,
Hirotake Eguchi, MD*,
Ritsuko Saitoh, MD*,
Hideyoshi Fujihara, MD PhD*,
Kazumasa Honda, PhD
, and
Takashi Higuchi, MD PhD
Departments of *Anesthesiology & Reanimatology and
Physiology, Fukui Medical University, Fukui, Japan
Address correspondence and reprint requests to Satoru Fukuda, MD, PhD, Department of Anesthesiology and Reanimatology, Fukui Medical University, 23-3 Shimoaizuki, Matsuoka-cho, Yoshida-gun, Fukui 910-1193, Japan. Address e-mail to fukudas{at}fmsrsa.fukui-med.ac.jp
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Abstract
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We studied the effects of intracerebroventricular injection of the novel neuropeptide orexin A on electroencephalogram (EEG) and autonomic nervous system activity in rats under isoflurane anesthesia. The administration of orexin A changed burst suppression patterns to arousal patterns on the EEG at 1.0 minimum alveolar anesthetic concentration (MAC) isoflurane and decreased the burst suppression ratio at 1.5 MAC isoflurane. However, orexin A did not influence the heart rate or mean arterial blood pressure at either isoflurane concentration. These findings demonstrated that orexin A elicited anesthetic arousal under isoflurane anesthesia in terms of EEG pattern without sympathetic cardiovascular activation in the rat.
IMPLICATIONS: The novel neuropeptides orexins induce arousal associated with activation of the sympathetic nervous system in conscious rats. It is not known whether orexins affect the electroencephalogram (EEG), autonomic nerve activity, or both under anesthesia. Orexin A induced EEG arousal without sympathetic cardiovascular activation in the isoflurane-anesthetized rat. Orexin A might influence the depth of anesthesia.
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Introduction
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The novel neuropeptides orexin/hypocretin were isolated and identified directly from the lateral hypothalamus of the rat (1). Anatomical and histochemical studies have demonstrated the presence of orexin-immunoreactive neurons within the hypothalamus, and orexin neurons project to several areas, including those important for arousal in close relation to sympathetic activation (2,3). Intracerebroventricular (ICV) injection of orexin A increased arousal and sympathetic nervous system activity in conscious rats (4–6). The findings suggest that in conscious animals, orexin may play an important role in the maintenance of wakefulness and the regulation of the autonomic nervous system. However, it is not known whether orexin influences the depth of inhaled anesthesia in relation to sympathetic activation. Thus, we investigated the effects of lateral ICV injection of orexin A on electroencephalogram (EEG), heart rate (HR), and mean arterial blood pressure (MAP) under isoflurane anesthesia in the rat.
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Methods
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This study was approved by the Animal Investigation Committee of Fukui Medical University. Eleven male Wistar rats weighing 300–450 g were used. They were maintained in a light-controlled (lights on between 6:00 AM and 6:00 PM), air-conditioned room. Rodent chow pellets and tap water were provided ad libitum. The rats were tracheally intubated and ventilated via a respirator under 3.0% isoflurane (2.5 minimum alveolar concentration; MAC) (7). Blood pressure (BP) was monitored through an arterial catheter inserted into the femoral artery, and fluid and drugs were administered through a venous catheter inserted into the femoral vein. Muscle paralysis was induced by a 1-mg IV bolus injection of vecuronium and repeated as necessary. After placing the animal in a stereotaxic frame in a prone position, a burr hole was drilled in the skull 0.8 mm posterior and 1.5 mm lateral to the bregma. A guide cannula (0.4-mm inner diameter/0.7-mm outer diameter) was inserted into the left lateral cerebroventricle through the burr hole and was fixed to the skull with two screws and dental cement. The electrocardiogram (ECG) was monitored via a pair of needle electrodes inserted into the subcutaneous tissues.
EEG signals were obtained from a pair of needle electrodes inserted into the temporal muscle on each side of the head. Rectal and pericranial temperatures were maintained at 36.5°C–37.5°C by heating pad, and PaCO2 was kept at 35–45 mm Hg. The inspired isoflurane concentration was maintained at 1.2% (n = 6) or 1.8% (n = 5) (1.0 and 1.5 MAC) for 30 min. After stabilization of EEG, ECG, HR, and BP, 5 µL of 1 nmol orexin A (Peptide Institute, Osaka, Japan) was injected (over 1 min) into the left lateral cerebroventricle through the infusion cannula (0.15-mm inner diameter/0.35-mm outer diameter) connected to a 5-µL Hamilton microsyringe. The EEG and ECG signals were sampled at a rate of 200/s by the PowerLab® system (ADInstruments Pty. Ltd., Castle Hill, New South Wales, Australia) before and 2, 10, 30, 60, and 120 min after orexin A.
The EEG data were reviewed by using a software analysis program (Focus 2000®; MEGIS Software GmbH, Gräfelfing, Germany). The EEG power for 30 s was calculated for selected frequency bands: 
, 0.5–4 Hz; 
, 4–8 Hz; 
, 8–13 Hz; ß, 13–30 Hz; and 
, 30–50 Hz. Relative powers were calculated by dividing the absolute amplitude within a given frequency range by corresponding measures of total amplitude. If the EEG was exhibiting a burst suppression pattern, the burst suppression ratio (BSR = percentage of isoelectric EEG) for 60 s was measured. At the end of the experiment, pontamine sky blue (1 µL) was injected to verify the correct placement of the ICV cannula tip.
All values were expressed as mean ± SD. One-way analyses of variance followed by the least significant difference test for multiple comparisons were used for the comparisons of BSR on EEG, HR, and MAP before and after orexin A. Differences were considered significant at P < 0.05.
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Results
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The burst and suppression patterns were observed on EEG under 1.0 and 1.5 MAC isoflurane anesthesia (n = 11) (Fig. 1). The BSR at 1.0 MAC isoflurane was smaller than that at 1.5 MAC isoflurane. Orexin A injection changed the burst and suppression patterns to arousal patterns in the EEG within 2 min at 1.0 MAC isoflurane. In some animals, these changes in EEG did not return to the preinjected level, even at 2 h after orexin A. The relative power spectra of EEGs at 2, 10, 30, 60, and 120 min after orexin A at 1.0 MAC isoflurane are shown in Table 1. The ratio of plus powers to total power was >70%, and the power, which represents cortical arousal (8), was almost 1%. At 1.5 MAC isoflurane, orexin A significantly decreased BSR (Table 2).
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Figure 1. Typical tracings of a electroencephalogram (EEG) before and after lateral cerebroventricular injection of orexin A. Basal EEG traces showed burst and suppression patterns. Injection of orexin A changed a burst and suppression pattern to an arousal pattern of the EEG within 2 min at 1.0 minimum alveolar anesthetic concentration (MAC) isoflurane. At 1.5 MAC isoflurane, orexin A decreased the burst suppression ratio.
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Table 1. Relative Power of Electroencephalogram After Cerebroventricular Injection of Orexin A at 1.0 Minimum Alveolar Anesthetic Concentration Isoflurane Anesthesia
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Table 2. Electroencephalogram Burst Suppression Ratio (BSR) Under 1.0 and 1.5 Minimum Alveolar Anesthetic Concentration (MAC) Isoflurane Anesthesia
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Basal MAP at 1.0 MAC isoflurane was significantly more than that at 1.5 MAC isoflurane. However, there was no difference in basal HR between 1.0 and 1.5 MAC isoflurane before orexin A. Orexin A did not significantly increase the HR or MAP at either isoflurane concentration (Table 3).
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Table 3. Changes in Heart Rate (HR) and Mean Arterial Blood Pressure (MAP) Under 1.0 and 1.5 Minimum Alveolar Anesthetic Concentration (MAC) Isoflurane Anesthesia
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Discussion
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This study is the first to demonstrate that ICV injection of orexin A elicited the arousal EEG under isoflurane anesthesia. Several studies have reported on the neuronal arousal system under anesthesia. In human, IV injections of the acetylcholine esterase inhibitor physostigmine reversed the unconsciousness state induced by propofol (9). Under pentobarbital anesthesia, ICV administration of histamine caused a dose-related decrease in narcosis duration in rats (10). These findings suggest involvement of either the cholinergic or histaminergic neuronal system in the regulation of the arousal system under anesthesia. This study suggests that, in addition to the cholinergic and histaminergic neuronal systems, the orexinergic neuronal system may be a third arousal system during anesthesia.
In conscious animals, ICV injection of orexin A provoked increases in HR and MAP (4,6). Sato-Suzuki et al. (11) reported that microinjections of orexin A into the rat paraventricular nucleus induced increases in HR, but not in BP, under pentobarbital anesthesia. In our study, neither HR nor MAP was increased by ICV injection of orexin A. The disparity between the previous report and ours may be due to use of the different anesthetics or different injection sites. Our study revealed that isoflurane may act differentially on the cortical arousal system and sympathetic cardiovascular activation system evoked by orexin A.
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Acknowledgments
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Supported in part by a Grant-in-Aid for Science Research (15390472) from the Ministry of Education, Science, Sports and Culture, Japan.
The authors would like to thank Prof. David S. Warner for his advice and Drs. Chukwuma O. Okure and Alison Douglas for editing this manuscript.
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References
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Accepted for publication July 10, 2003.
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Abstract
Introduction
