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Department of Anesthesiology and Pain Medicine, Section of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, California
Address correspondence and reprint requests to Joseph F. Antognini, MD, Department of Anesthesiology TB-170, University of California, Davis, Davis, CA 95616. Address e-mail to jfantognini{at}ucdavis.edu.
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Abstract |
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Introduction |
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As part of our approach, we have used electrical microstimulation of the midbrain reticular formation (MRF), an area critical to cerebral arousal and consciousness (5,6). Moruzzi and Magoun (7) first reported that microstimulation of this area resulted in EEG arousal, and their work laid the groundwork for further studies on the importance of the reticular formation to arousal. Although many of the animals used in that study were anesthetized with chloralose, little work has since been performed to determine how anesthetics alter EEG responses to electrical microstimulation of the MRF and pontine reticular formation. Isoflurane and halothane are two commonly used anesthetics that have disparate EEG effects, with isoflurane generally resulting in more EEG depression than halothane. In the present study, we hypothesized that isoflurane would cause greater depression of the EEG activation elicited by electrical microstimulation of the reticular formation, as compared with halothane.
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Methods |
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Four small stainless steel screws were inserted into the skull for EEG measurement. A ground electrode was placed in the midline over the anterior skull 15 mm anterior to the interaural line and 2 were inserted (on either side) 5 mm lateral to midline and 5–7 mm rostral to the interaural line. The reference electrode was placed over the midline 2–3 mm caudal to the interaural line. Cables were attached to the screws and the EEG signals were downloaded to a Bispectral index (BIS) monitor (A-1050; Aspect Medical, Newton, MA) at 256 samples/s. The filters were set at 2–70 Hz. Impedance was <5000 ohms. The BIS monitor calculated the median edge frequency (MEF) and spectral edge (95%) frequency (SEF) every 5 s and these were downloaded to computer hard drive. The BIS monitor used a "rolling" average of the previous 30-s period. In addition, the raw EEG was recorded onto a computer hard drive using Chart version 5.1 (ADInstruments, Colorado Springs, CO).
The rats were placed into a stereotaxic frame (D. Kopf, Tujunga, CA). Two bipolar stimulating electrodes (FHC, Bowdoinham, ME) were attached to an electrode holder 1 mm apart in the rostral caudal axis and lowered into the brain. We used two electrodes to increase the chance to elicit EEG activation. The areas targeted were the MRF (2 mm lateral to midline, 2 mm rostral to the interaural line, and 6–7 mm below the brain surface) and the pontomesencephalic tegmental area (2 mm lateral to midline, 2 mm rostral to the interaural line, and 7–8 mm below the brain surface). The stimulating sites were confirmed histologically. Pancuronium was administered (0.2–0.3 mg/kg every 1–2 h) to minimize electromyographic artifacts in the EEG recording.
A crossover experimental paradigm was used. The anesthetic concentration was adjusted to either 0.8 or 1.2 minimum alveolar concentration (MAC) (order alternated experiment to experiment) and maintained for at least 15 min. Population MAC values were assumed for halothane (1%) and isoflurane (1.2%) based on previous studies from our laboratory (9,10). Electrical stimuli consisted of 2-s trains of 100-µs square wave pulses delivered at 300 Hz, at current intensities of 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mA. Two to 3 min elapsed between each stimulus train, permitting return to the baseline EEG pattern. The order of the current intensities was randomized. The same paradigm was used to deliver electrical stimuli through the other electrode. In addition, we separately determined EEG responses to supramaximal stimulation of the tail using 2 methods: electrical current (60 mA, 50 Hz for 30 s) via percutaneous platinum needle electrodes placed into the proximal tail and a hemostat clamp applied to the tail for 30 s. The anesthetic concentration was switched and after 15–20 min equilibration the stimulating paradigm was repeated. The anesthetic was then changed (from halothane to isoflurane, or vice versa, counterbalanced across experiments) in 10 animals. After approximately 45 min equilibration, the stimulation paradigm was repeated at 0.8 and 1.2 MAC as described above.
On completion of the study the rats were killed with excess anesthetic and IV potassium chloride. An electrolytic lesion was made by delivering current (6–8 V, 30–60 s) through each of the electrodes, after which the brain was removed. The brains were fixed in 10% buffered formalin and sectioned several days later and counterstained with Neutral Red. Electrolytic lesions were examined under the light microscope and compiled on representative brain sections taken from the atlas of Paxinos and Watson (11).
Data are presented as mean and standard deviation. The EEG parameters were averaged for the 30-s period before microstimulation and the second 30-s period after microstimulation. The EEG responses were nearly identical between the rostrally and caudally placed electrodes so the data from both electrodes were pooled. The EEG data (MEF and SEF) were compared before and after stimulation. The values for each anesthetic were compared using repeated-measures analysis of variance; post hoc testing was performed using the Student-Newman-Keuls test. To compare the 0.8 and 1.2 MAC post-stimulus values for each anesthetic we used an area under the curve analysis whereby the MEF and SEF values for each stimulus current were summed and evaluated using a paired Student's t-test. For the 1.2 MAC isoflurane EEG data, the burst suppression ratio was calculated as the period of isoelectricity divided by the total EEG period evaluated, which was 30 s. The MAP was analyzed using analysis of variance followed by the Student-Newman-Keuls test or with the paired Student's t-test to compare MAP responses at 0.8 and 1.2 MAC. A P < 0.05 was considered significant.
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Results |
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Summary data are shown in Figure 2. Electrical microstimulation in the midbrain resulted in EEG activation as demonstrated by the increased SEF and MEF at 0.8 and 1.2 MAC halothane and 0.8 MAC isoflurane. The post-stimulus MEF and SEF for all currents combined were greater at 0.8 MAC as compared with values at 1.2 MAC for both halothane and isoflurane (P < 0.01). The prestimulus SEF at 0.8 MAC was significantly greater compared with the value at 1.2 MAC for halothane (P < 0.05) but not isoflurane. Electrical stimulation of the tail and the tail clamp generally were as effective in causing EEG activation when compared to the electrical microstimulation of the MRF. At 1.2 MAC isoflurane, however, electrical microstimulation did not change SEF and MEF (Fig. 2) but decreased the amount of EEG isoelectricity occurring during burst suppression. At 1.2 MAC isoflurane, the prestimulus burst suppression ratio was 24% ± 19%, whereas the post-stimulus ratio (average of all post-stimuli values) was 8% ± 7%, P < 0.007. The suppression ratios for each stimulus are shown in Figure 3. Burst suppression rarely occurred during 0.8 MAC isoflurane, with the average period of isoelectricity representing only 1% of the EEG activity. Burst suppression was not observed during halothane anesthesia.
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The MAP increased as a result of electrical microstimulation, with the peak occurring at about 0.5 mA (Fig. 4). The MAP for all currents combined was greater at 0.8 MAC as compared with the values at 1.2 MAC for both isoflurane and halothane (Fig. 4). The stimulus sites were located in the reticular formation, extending from the rostral midbrain to the rostral pons, including the area surrounding the pedunculopontine tegmental nucleus (Fig. 5).
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Discussion |
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When increased from 0.8 to 1.2 MAC, isoflurane ablates EEG activation that follows noxious stimulation (9). Is this effect primarily the result of an action in the brain, or is action in the spinal cord partly responsible? Our prior work suggests that this blunting effect is in large part because of action in the spinal cord (1,2). For example, addition of isoflurane to the torso (and hence spinal cord) ablates EEG activation in animals that have approximately 1 MAC isoflurane administered to the brain (1). With respect to the blunting of EEG activation, how important is isoflurane's and halothane's action in the brain? In the present study we addressed this question by applying a stimulus to the reticular formation that essentially bypasses the spinal cord. Had isoflurane and halothane prevented EEG activation, we could reasonably conclude that their action in the brain was significant. As this was not the case, however, the present study provides further evidence that isoflurane and halothane can depress EEG activity partly via action in subcortical structures such as the spinal cord.
The presence of burst suppression during 1.2 MAC isoflurane anesthesia, and its absence during 1.2 MAC halothane anesthesia, is consistent with other evidence that isoflurane causes more EEG depression than halothane (12–14). In a prior study we (9) reported burst suppression during 1.2 MAC isoflurane anesthesia that was minimally altered by repetitive stimulation that produced EEG activation at 0.8 MAC isoflurane and which normally produces neuronal windup. We did not observe burst suppression during halothane anesthesia. In the present study, electrical microstimulation of the reticular formation, as well as noxious stimulation applied to the tail, significantly decreased the period of isoelectricity during 1.2 MAC isoflurane, indicating that even under deep anesthesia the brain remains responsive to noxious stimulation applied to the periphery and to electrical microstimulation of the reticular formation.
In the present study the burst suppression in isoflurane-anesthetized rats complicated EEG analysis, inasmuch as the SEF and MEF do not accurately reflect the EEG changes (15). That is, although the SEF and MEF were unchanged after electrical microstimulation, the period of EEG isoelectricity decreased from approximately 20% to as little as 1%–2% at the greater electrical currents. Thus, comparing isoflurane and halothane at 1.2 MAC was difficult. Some have argued that SEF and MEF can be linearly corrected for the amount of burst suppression (15). For example, if 50% of the EEG is isoelectric, then the SEF and MEF can be decreased 50%. Although this is a convenient method to handle the data, the choice of linear adjustment is arbitrary. We chose to report the unadjusted SEF and MEF as well as the burst suppression ratio.
The EEG pattern (low amplitude, high frequency) resulting from electrical microstimulation of the reticular formation is normally associated with arousal. In humans in a persistent vegetative state, prolonged microstimulation of deep brain structures, including the reticular formation, can increase arousal (16). Evoked potentials are enhanced after electrical microstimulation of the reticular formation (17). Furthermore, electrical stimulation in the reticular formation causes behavioral arousal in awake animals (18) and is associated with enhanced learning (19). However, depending on the method and site of stimulation, central nervous system depression can also occur. For example, stimulation of the thalamus for 30–60 seconds at 4–8 Hz, instead of faster frequencies, such as the 50–300 Hz described by Moruzzi and Magoun (7), can produce EEG slowing and induce sleep (20). The mechanism is unclear but is possibly related to activation of sleep-promoting areas of the brain, such as the ventrolateral preoptic nucleus and the tuberomammilary nucleus (21).
The reticular formation as a site of anesthetic-induced unconsciousness has received varying attention over the years. Anesthetics such as isoflurane differentially alter neuronal activity, with both excitation and depression occurring (22,23). In addition, neural activity, as indirectly determined using positron emission tomography, is depressed at halothane and isoflurane concentrations associated with unconsciousness (24,25). Infusion of volatile anesthetics into the reticular formation causes unconsciousness (26). In addition, IV anesthetics such as propofol and pentobarbital appear to act, at least in part, on subcortical structures to produce unconsciousness. Devor and Zalkind (27) reported that microinjections of pentobarbital in the mesopontine tegmental area produced anesthesia, while Nelson et al. (21) reported that propofol and pentobarbital act in the tuberomammillary nucleus to produce anesthesia. Collectively, these data suggest that anesthetic action in subcortical structures, including the reticular formation, might be important to anesthetic-induced unconsciousness. Others have argued that anesthetics act in the cerebral cortex to produce unconsciousness (28), in part because cortical neurons, as compared with thalamic (and other subcortical) neurons, appear to be more sensitive to anesthetics. The present data and previously published data (29) indicate, however, that at sub-MAC anesthetic concentrations, the cortex remains responsive to electrical stimulation in the reticular formation as well as to peripheral stimulation.
In summary, we found that electrical microstimulation of the reticular formation resulted in EEG activation that was affected differently by isoflurane and halothane. In the transition from 0.8 MAC to 1.2 MAC, halothane depressed the SEF and MEF response to microstimulation, whereas for isoflurane the amount of burst suppression was decreased. During halothane and isoflurane anesthesia the cortex and subcortical structures remain responsive to electrical stimulation, although the response is depressed.
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Footnotes |
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Supported, in part, by National Institutes of Health grants GM 57970, GM61283, and P01-GM47818
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