Anesth Analg 2003;97:238-243
© 2003 International Anesthesia Research Society
NEUROSURGICAL ANESTHESIA
The Electroencephalographic Effects of IV Anesthetic Doses of Melatonin: Comparative Studies with Thiopental and Propofol
Mohamed Naguib, MB BCh, MSc, FFARCSI, MD,
Phillip G. Schmid, III, MD, and
Max T. Baker, PhD
Department of Anesthesia, University of Iowa College of Medicine, Iowa City, Iowa
Address correspondence and reprint requests to Mohamed Naguib, MB, BCh, MSc, FFARCSI, MD, Department of Anesthesia, University of Iowa College of Medicine, 200 Hawkins Dr., 6JCP, Iowa City, IA 52242-1009. Address e-mail to mohamed-naguib{at}uiowa.edu
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Abstract
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We have demonstrated that large-dose IV melatonin can exert hypnotic effects similar to those caused by thiopental and propofol. In this study, we compared the electroencephalographic (EEG) effects of melatonin with those of thiopental and propofol. Sprague-Dawley rats were assigned to receive equipotent bolus doses of thiopental (23.8 mg/kg), propofol (14.9 mg/kg), or melatonin (312 mg/kg). EEG effects were recorded at periodic intervals over 10 minutes. Of eight processed EEG variables analyzed, only relative total power (rTP), relative spectral edge 95% (rSE95), and relative approximate entropy (rAE) were altered by all drugs compared with their control vehicles. Drug administration decreased the values relative to baseline, with subsequent return toward baseline during the 10-min time course. Thiopental significantly increased rTP, whereas propofol and melatonin did not. All drugs significantly decreased rSE95. However, the time course of peak effect and duration differed for each, with melatonin exhibiting a slower onset and a more sustained EEG effect. All drugs significantly decreased rAE, with similar time courses for thiopental and propofol and a slower onset/longer duration for melatonin. Melatonin produced effects on processed EEG variables similar to those of thiopental and propofol, specifically a decrease in the rSE95 and a decrease in the rAE.
IMPLICATIONS: Anesthetic doses of melatonin produced effects on processed electroencephalographic variables similar to those of thiopental and propofol.
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Introduction
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The pineal hormone melatonin (N-acetyl-5-methoxytryptamine) has several putative functions, including regulation of circadian rhythms (1), anticonvulsant effects (2), and antioxidant activity (3). Exogenous melatonin, which is often administered orally, exerts a number of beneficial effects. It facilitates the onset and improves the quality of sleep (4), alleviates preoperative anxiety, and produces sedation (5,6).
Recently, we have demonstrated that IV melatonin can exert an anesthetic action with both hypnotic and antinociceptive effects in the rat (7). The electroencephalographic (EEG) effects of melatonin at anesthetic doses have not been reported. The following studies determined the EEG effects of IV-administered melatonin in rats and compared them with those of two other commonly used rapidly acting IV anesthetics: thiopental and propofol.
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Methods
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The following investigations were performed under a protocol approved by the University of Iowa Animal Care and Use Committee. All experiments were performed in male Sprague-Dawley rats (300–350 g) (Harlan, Indianapolis, IN). Rats were maintained on a 12-h light/dark cycle with free access to food and water. All surgical procedures were performed under aseptic conditions in nonfasting rats anesthetized with isoflurane. The right jugular vein was cannulated with a heparinized (20 U/mL), saline-filled (PE-50) catheter. The free end of the tubing was tunneled subcutaneously to exit posterior to the occiput, trimmed to remove excess length, and capped. The skin and subcutaneous tissues over the dorsum of the head were removed to expose the skull. Two 1-mm-diameter stainless-steel screws were implanted unilaterally over the right or left frontoparietal cortex between the bregma and lambda sutures, approximately 2 mm lateral to the midline and at least 2 mm apart. The dura and deeper brain parenchyma were not disrupted. The screws were cemented in place with cyanoacrylate glue to further minimize movement and associated EEG artifacts. After recovery from anesthesia, the animals were placed in individual plastic cages and were returned to the Animal Care Unit for routine care. Each subsequent day, laboratory personnel inspected and weighed the rats and flushed the catheters with 0.2 mL of heparinized saline. Animals that failed to resume weight gain after 3–5 days were not used for subsequent experiments. Studies were done 5–7 days after surgery.
The following drugs were used in the study: thiopental (Abbott Laboratories, Northern Chicago, IL), propofol emulsion (AstraZeneca Pharmaceuticals, Wilmington, DE), and melatonin (Sigma Chemical Co., St. Louis, MO). Thiopental and propofol were dissolved in saline and IntralipidTM, respectively. Melatonin was dissolved in a mixture comprising 25% vol/vol propylene glycol (Fisher Chemicals, Fair Lawn, NJ) and 25% vol/vol 1-methyl-2-pyrrolidinone (Aldrich Chemicals, Milwaukee, WI) in sterile water. Sufficiently large concentrations of melatonin could not be achieved in conventional solvents such as 2-hydroxypropyl-ß-cyclodextrin, propylene glycol, or dimethyl sulfoxide.
Two EEG electrodes were connected to the screws of a conscious rat confined in an open-topped 10 x 15 x 4 in. (width x length x height) plastic chamber, which provided adequate room for free movement. The EEG electrodes were connected to a Grass 79DEEG/Polygraph data-recording system (Grass Instrument Co., Quincy, MA). After amplification, the EEG data signals were fed to an Omega CIO-DAS16JR A/D board (Omega Engineering, Inc., Stamford, CT). Signals were acquired and processed with a custom program written in TestPoint data-acquisition software (Capital Equipment Corp., Billerica, MA). This program acquired and recorded the millivolt EEG signals at a rate of 256 Hz during 4-s epochs. Animals were not restrained during the study; however, every attempt was made to record only during periods when the animal was resting, to minimize noise in the EEG signal. After the connection of EEG recording leads and drug syringes, each animal was allowed to rest undisturbed for approximately 10 min to minimize any arousal brought on by the initial handling.
Three baseline EEGs were recorded for each animal. Equipotent doses of thiopental (23.8 mg/kg), propofol (14.9 mg/kg), or melatonin (312 mg/kg) or the vehicles for these drugs were administered via the internal jugular venous catheter. These doses represented the 95% effective dose as calculated for the loss of righting reflex (7). The doses were administered in a volume of 0.3–0.4 mL for propofol and 0.6 mL for the rest of the drugs and vehicles. The EEG was recorded at 1, 3, 5, and 10 min after the injection. Some of the test animals were aroused by the injections, and their movement precluded adequate EEG acquisition at one or two time points. If the EEG could not be adequately recorded at three time points, the animal was excluded from the study; seven animals were excluded in this manner.
Each EEG was subsequently processed within the custom TestPoint software by applying a Hamming window function and then a fast Fourier transformation for subsequent spectral analysis (8). Low- and high-frequency filtering with cutoffs of 0.5 and 60 Hz, respectively, were applied. The total power (TP) of the signal strength versus frequency data set was determined, along with median power frequency (MPF), 95% spectral edge frequency (SE95), and fractional power ("fractional" meaning fraction of TP) within each of the four conventional EEG frequency bands: 
(0–4 Hz), 
(4–8 Hz), 
(8–13 Hz), and ß (>13 Hz). For example, fractional power (FDP) equals absolute power (DP) divided by TP (i.e., FDP = DP/TP). Because the raw signal voltages differed substantially between animals because of variations in screw position, electrode to screw contact, and circuit impedance, TP and fractional frequency band power values were converted to relative power values expressed as a fraction of baseline (i.e., "relative" is relative to baseline). The baseline for these variables was the mean of the three recordings made before any drug or vehicle administration. These relative power values, then, became relative TP (rTP), relative FDP, relative fractional power, relative fractional power, and relative fractional ß power. On the basis of prior work in halothane-anesthetized animals (9), relative changes in frequency were shown to correlate better with MAC; therefore, the MPF and SE95 values were also converted to relative values (as fraction of baseline) and were designated relative MPF and relative SEF95, respectively. Approximate entropy (AE) is a new statistical variable derived from the Kolmogorov-Sinai entropy formula that quantifies the amount of regularity in data. The AE quantifies the predictability of subsequent amplitude values of the EEG on the basis of the knowledge of the previous amplitude values. For the AE determinations, the raw EEG data captured via the TestPoint software were processed with the AE algorithm of Pincus et al. (10) by using custom software written in FORTRAN. On the basis of the work of Bruhn et al. (11), we used m = 2, a filter factor of r = 0.2, and n = 1024. As for all other processed EEG variables, the absolute AE values were converted to relative values by dividing by the predrug baseline AE.
Data were analyzed by using two-way analysis of variance with repeated measures. Comparisons between each drug and its vehicle were done by using the Mann-Whitney U-test, the Student-Newman-Keuls multiple range test, or Kruskal-Wallis test for multiple comparisons, where appropriate. For multiple comparisons in the Kruskal-Wallis test, the null hypothesis was rejected if ZSTAT was larger than the critical value ZC:
where PHI is the cumulative standard normal distribution function, ALPHA is the desired overall significance level, and K is the number of groups compared. All statistical analyses were performed with the BMDP Dynamic statistical package (University of California Press, Berkeley, CA). Unless otherwise specified, results were expressed as means ± SEM and were considered significant when P < 0.05.
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Results
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Studies were successful in 47 animals (9 thiopental, 6 propofol, 9 melatonin, 9 saline, 7 Intralipid, and 7 melatonin vehicle). Each animal that received a hypnotic drug rapidly lost consciousness, as demonstrated by a loss of righting reflex. No animal died during or after drug injection, and no animal became apneic or developed gross changes in respiration, nor did any animal show evidence of cyanosis. Although our study considered only the first 10 min of drug effects, all animals that lost consciousness ultimately appeared to regain full consciousness with no overt residual behavioral abnormalities.
Sample EEGs are shown in Figure 1 and show that gross changes in the EEG caused by either drug or vehicle are not easily discernible. Analysis of a multitude of processed EEG variables revealed that rTP, relative SE95 (rSE95), and relative AE (rAE) were the only variables consistently altered by all drugs as compared with their respective vehicles. Neither normal saline nor Intralipid had any significant effects on these three EEG variables at any time after bolus injection. The vehicle for melatonin given by bolus injection transiently affected all three of these EEG variables at 1 min, with complete resolution by 3 min (P < 0.05).
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Figure 1. Sample electroencephalographic (EEG) recordings at baseline (before drug or vehicle) and 5 min after drug or vehicle administration.
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Bolus administration of 23.8 mg/kg of thiopental was associated with a significant increase in rTP when compared with its vehicle (Fig. 2). This increase was evident within 1 min, peaked at 3 min, and resolved by 10 min. Equipotent doses of propofol (14.9 mg/kg) or melatonin (310 mg/kg) did not affect rTP compared with their respective vehicles. Thiopental also significantly decreased the rSE95 at all time points after drug administration, with a peak effect at 1 min postinjection and a slow return toward baseline rSE95 thereafter (Fig. 3). An equipotent dose of propofol also significantly decreased rSE95 at 3 and 5 min, with a peak effect at 3 min and resolution of effect by 10 min (Fig. 3). Melatonin depressed rSE95 after injection, although this depression was statistically significant only at 5 min postinjection (Fig. 3). Thiopental and propofol significantly decreased rAE at 1, 3, and 5 min, with a peak effect at 1 min and a steady return to baseline rAE by 10 min (Fig. 4). The rAE was likewise depressed by melatonin, with significant depression noted at 5 and 10 min postinjection (Fig. 4).
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Figure 2. The effect of bolus injection of equipotent doses of thiopental (23.8 mg/kg), propofol (14.9 mg/kg), or melatonin (312 mg/kg) or their vehicles on the relative total power. Relative total power = absolute total power at a given time/absolute total power at baseline (Time 0). *P < 0.05 versus the respective vehicle.
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Figure 3. The effect of bolus injection of equipotent doses of thiopental (23.8 mg/kg), propofol (14.9 mg/kg), or melatonin (312 mg/kg) or their vehicles on the relative 95% spectral edge frequency (SE95). Relative SE95 = absolute SE95 at a given time/absolute SE95 at baseline (Time 0). *P < 0.05 versus the respective vehicle.
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Figure 4. The effect of bolus injection of equipotent doses of thiopental (23.8 mg/kg), propofol (14.9 mg/kg), or melatonin (312 mg/kg) or their vehicles on the relative approximate entropy. Relative approximate entropy = absolute approximate entropy at a given time/absolute approximate entropy at baseline (Time 0). *P < 0.05 versus the respective vehicle.
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Discussion
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Numerous processed EEG variables have been used to examine anesthetic effects in the time (12,13) and frequency (14–16) domains. More recently, anesthetic effects on higher-order spectra, such as the bispectrum (14), have been investigated and used to develop monitors that are purportedly clinically useful for assessing anesthetic depth (17). Nonlinear analysis methodologies may better characterize the EEG (18,19). Anesthetic effects on both the AE and Shannon entropy of the EEG (11,20), which quantify the amount of regularity in the EEG, have been reported. For this investigation, we chose to analyze the EEG by using both linear and nonlinear system techniques, choosing variables that correlate with the dose of a variety of volatile and IV drugs.
Although smaller doses of melatonin can be administered orally without the use of a solvent, the administration of large doses by IV methods is hampered because of the insolubility of melatonin in conventional IV drug vehicles. IV formulations of melatonin with ethanol (21), dimethyl sulfoxide (22), or Tween 80 (23) have been studied. However, these vehicles can exert toxic effects on the central nervous system (CNS). Methyl pyrrolidinone has the capacity to solubilize large quantities of melatonin while apparently having minimal CNS effects. Although the vehicle itself had a short-lived EEG effect in this study, the effect of melatonin was readily discriminated from the vehicle effect.
Our investigation confirms previous documented qualitative anesthetic effects on the EEG (16) and constitutes the first report of the anesthetic effects of melatonin on the EEG. Our results show that all three drugs used in the study produce depression of SE95 and AE in a similar fashion, although the time course for melatonin was somewhat longer. Unlike this investigation, most previous studies of the relationship of anesthetic drug and/or dose to EEG have associated the EEG changes with onset or emergence from anesthesia in a qualitative sense or have compared EEG variables with drug plasma concentration (24) rather than following the time course after a single injection. The EEG does not change in a linear or monotonic fashion with changing anesthetic depth, nor do all anesthetics produce similar EEG patterns (25). Kuizenga et al. (16) reported that there was no consistent relationship between the time of occurrence of the peak EEG effect or the value of the EEG variable and the moment of loss of consciousness during the induction of anesthesia with thiopental, propofol, etomidate, midazolam, or sevoflurane. Todd (25) stated that "it is possible to have identical values of the spectral edge frequency at widely different points on the continuum between awake and deeply anesthetized or at different points with different agents." This observation is true of every single-variable measure derived from the power spectrum of the EEG (SEF, TP, frequency/band ratios, and others) (25). Whether this is true for AE remains to be determined. Our data document, in a qualitative sense, the effects of melatonin on processed EEG variables in comparison to the effects of thiopental and propofol and verify that the melatonin vehicle did not produce these effects. Melatonin produced effects on processed EEG variables similar to those of thiopental and propofol, specifically, a decrease in the SE95 and a decrease in AE.
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Accepted for publication February 21, 2003.
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Abstract
Introduction
