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PAIN MEDICINE

Etomidate Depresses Lumbar Dorsal Horn Neuronal Responses to Noxious Thermal Stimulation in Rats

Toshihiko Mitsuyo, MD, Joseph F. Antognini, MD^*, and Earl Carstens, PhD

*Department of Anesthesiology and Pain Medicine, University of California, Davis, Section of Neurobiology, Physiology and Behavior, University of California, Davis, Department of Anesthesiology, Ehime University, Matsuyama, Japan

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.

	Abstract

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Etomidate is a widely used IV anesthetic, but little is known about its analgesic properties, in particular, its effects on spinal cord neuronal responses to noxious stimuli. We hypothesized that etomidate would depress lumbar neuronal responses to noxious heat. Rats (n = 15) were anesthetized with isoflurane (1.2%) and laminectomy was performed to record single unit activity. Lumbar neuronal responses to noxious thermal (52°C, 12 s) stimulation of the hindpaw were recorded before and every 2 min (up to 13 min postinjection) after administration of etomidate. The responses at peak effect of etomidate (as a percentage of the control response) were 63% ± 16%, 63% ± 16%, 38% ± 25%, 36% ± 30%, and 41% ± 26% for the 0.125, 0.25, 0.5, 1 and 2 mg/kg doses, respectively. The responses quickly recovered, usually by the 10-min period postinjection. Similar responses were obtained in decerebrate, isoflurane-free rats administered etomidate and in isoflurane-anesthetized rats administered propofol. These data demonstrate that etomidate depresses spinal cord neuronal responses to noxious stimulation and is a possible mechanism by which this drug might produce analgesia.

	Introduction

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Etomidate is a sedative/hypnotic anesthetic drug widely used for induction of anesthesia. It has a rapid onset of action and provides cardiovascular stability with less effect on the respiratory drive as compared with other induction drugs, such as methohexital (1). Etomidate acts at the -amino butyric acid (GABA_A) receptor to enhance the effect of GABA, thereby increasing inhibition (2,3). In addition to its hypnotic effect, etomidate is reported to provide analgesia (4), although this is not established and universally accepted (5); little work has been done to determine analgesic actions of etomidate. Zhang et al. (6) reported that etomidate enhanced GABA-mediated currents in mechanically dissociated dorsal horn neurons in vitro, an inhibitory action that could depress nociceptive transmission. Whether this action is important vis-à-vis analgesia is unclear, as the effect of etomidate on spinal neuronal responses to noxious stimulation in vivo is unknown. In the present study we tested the hypothesis that etomidate would depress these responses. We also tested the effects of propofol for comparison.

	Methods

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The University of California, Davis animal care and use committee approved the study. Acute terminal experiments were conducted on adult male Sprague-Dawley rats (400–600 g). Rats were placed in a plastic chamber and anesthesia was induced with 4%–5% isoflurane (Minrad, Buffalo, NY). Each rat was then placed on mask anesthesia (2.0% isoflurane), and tracheotomy was performed to insert a 14-gauge catheter into the trachea. Each animal was mechanically ventilated with isoflurane mixed in 100% O₂. A jugular vein was cannulated for Ringer's solution administration and drug administration. In most animals a carotid artery was cannulated for measuring mean arterial blood pressure (MAP) (model PB-240; Puritan-Bennett, Hazelwood, MO). Rectal temperature was maintained at 37°C-39°C using a heating lamp. End-tidal CO₂ (maintained at 30–45 mm Hg) and anesthetic concentrations were monitored with a calibrated anesthetic agent analyzer (Ohmeda Rascal II analyzer; Datex-Ohmeda, Helsinki, Finland).

A midline incision was made over the lower back, and the lumbar enlargement of the spinal cord was exposed via a laminectomy (7). The superficial muscle and tendons along the spine were removed to permit placement of vertebral clamps rostral and caudal to the laminectomy. The rat was then placed into a stereotaxic frame (D. Kopf Instruments, Tujunga, CA) and secured using the vertebral clamps and ear bars. The dura was removed, and warm agar was poured over the spinal cord. A tungsten microelectrode (resistance 8–11 MOhm; FHC, Bowdoinham, ME) was advanced into the dorsal horn (5-µm increments) using a hydraulic microdrive (D. Kopf Instruments). Action potentials were amplified (Model P50 amplifier; Grass, Braintree, MA) and filtered with 300 and 3000 Hz cutoffs. The signal was fed to a computer and recorded using Chart5 (AD Instruments, Colorado Springs, CO).

Single units were sought (at 1.2% isoflurane) and isolated by applying innocuous mechanical stimulation to the plantar surface of the ipsilateral hindpaw. Units that had receptive field areas corresponding to the L4–6 spinal cord were chosen. Only one unit was recorded in each animal, except in one case in which two discernable action potentials were recorded simultaneously. The units were classified as either wide-dynamic range (WDR), in that they exhibited increasing responses to innocuous touch through noxious pressure and pinch, or nociceptive-specific, in that a response was evoked only with noxious stimulation.

Once a single unit was isolated, responses to noxious thermal stimulation were recorded. Pancuronium (GensiaSicor, Irvine, CA) was administered IV (0.2–0.3 mg/kg every 1–2 h). A Peltier thermode (NTE-2A, Physitemp, Clifton, NJ) was placed onto the receptive field of the hindpaw to deliver a noxious heat stimulus (52°C, 12 s, from an adapting temperature of 35°C, increase time 12°C/s). Control responses to 3 heat stimuli (separated by at least 2-min intervals) were first recorded. Etomidate (Ben Venue Labs, Bedford, OH) was then administered IV (0.2 mg/mL in 20% propylene glycol) and neuronal responses determined 1 min after injection and every 2 min thereafter, up to 13 min postinjection. In the initial experiments we sought to determine the dose range that appeared to produce the minimal and maximal effects. After determining that etomidate 0.5–1 mg/kg produced a maximal effect, most responses were determined at these doses in the subsequent studies. In these experiments, etomidate was usually administered in ascending dosage, with at least 30 min elapsing between doses to permit dissipation of the effects of the prior dose. The dose range used (0.125 to 2 mg/kg) encompasses the dose range that produces loss of the righting reflex and loss of motor responses to noxious tail clamping (8). In 4 rats we administered 0.2–0.4 mL of 35% propylene glycol (Sigma, St. Louis, MO) as a vehicle control for the largest etomidate doses (1–2 mg/kg). We then recorded neuronal responses to noxious heat as described above. At the end of the experiment the rats were euthanized with IV pentobarbital.

Because we used isoflurane as the background anesthetic, we also determined etomidate's effects in rats that had undergone decerebration and had the isoflurane removed. In brief, the rats (n = 6) were prepared as described above except that the remaining carotid artery was ligated. In addition, a craniotomy was performed and a mid-collicular decerebration performed by passing a spatula between the colliculi and aspirating the cerebral contents anterior to the transection. Isoflurane was discontinued and after a 60-min recovery period each rat was placed into the frame and lumbar spinal neurons sought. We tested the effect of etomidate on neuronal responses to thermal stimulation at 0.25, 0.5, and 1 mg/kg, with at least 30 min between doses.

To facilitate comparison, the effects of propofol (Abbott Labs, Chicago, IL) were determined in 10 rats anesthetized with isoflurane and prepared as described for intact rats in the etomidate group. The doses of propofol were 1 and 5 mg/kg, with at least 30 min between doses. This dose range encompasses the range that produces loss of the righting reflex and loss of motor responses to noxious tail clamping (9).

The number of action potentials was counted for the 60-s period after onset of each noxious heat stimulus. The 3 responses before injection of etomidate were averaged and compared with the average of the 3 responses immediately after etomidate injection (time 1, 3, and 5 min) and the average of the three subsequent responses (time 7, 9, and 11 min). These averages were compared using analysis of variance. Post hoc testing was performed using the Student-Newman-Keuls test. In addition, we determined the peak effect on the neuronal response by comparing the smallest response postinjection to the average of the three control responses (paired Student's t-test). The depression of neuronal responses by etomidate in the decerebrate group was compared with the depression by the respective etomidate dose in the intact rats using an unpaired Student's t-test. Likewise, the depression by propofol was compared with the depression by etomidate at equipotent doses (0.125 and 1 mg/kg) using the unpaired Student's t-test. Significance was assumed at P < 0.05.

	Results

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In intact rats anesthetized with isoflurane, 16 WDR neurons were recorded in 15 rats. The mean depth of recording sites was 484 ± 144 µm below the cord surface. Etomidate injection resulted in a dose-dependent depression of neuronal responses to noxious heat. Figure 1 shows an individual example of depression of neuronal responses by etomidate. The smallest dose (0.125 mg/kg) did not appreciably affect the response (upper right-hand trace), but the 0.5 mg/kg and 1 mg/kg doses caused significant depression (middle and lower right-hand traces, respectively). The greatest depression usually occurred 1 min postinjection of etomidate. The responses at peak effect of etomidate (as a percentage of the control response) were 63% ± 16%, 63% ± 16%, 38% ± 25%, 36% ± 30%, and 41% ± 26% for the 0.125, 0.25, 0.5, 1, and 2 mg/kg doses, respectively. The data are summarized in Figure 2. Figure 2A plots the averaged normalized responses at each time point post-etomidate, and Figure 2B-F plots responses before (control, filled bars) and at 1–5 min (open bars) or 7–11 min (hatched bars) post-etomidate at each respective dose. The peak effect of etomidate was achieved at 0.5 mg/kg, and larger doses resulted in prolongation of the depressant effect, as shown by the failure to recover completely in the 7–11 min period after etomidate injection.

View larger version (28K): [in this window] [in a new window]   Figure 1. Shown are examples of action potentials recorded from lumbar spinal cord in a rat anesthetized with isoflurane before (Control) and Post-Etomidate. Application of a noxious thermal stimulus (52°C, black bar) to the receptive field on the hindpaw caused the neuron to increase its firing rate; IV administration of etomidate (0.125 mg/kg) did not significantly alter the neuronal response evoked 1 min after injection. Injection of etomidate at 0.5 mg/kg and 1 mg/kg significantly depressed the neuronal response to noxious heat 1 min after injection. Each tracing is 60 s long.

View larger version (37K): [in this window] [in a new window]   Figure 2. Shown are summary data (mean, standard deviation) for responses to noxious thermal stimulation before and after etomidate. A, the three control responses and the responses after etomidate (expressed as a percentage of the average of the three control responses). Note that the peak depression was reached at the 0.5 mg/kg dose (#P < 0.05 compared with control). For clarity the standard deviation bars have been deleted on every other mean. Panels B-F show the averages of the 3 control responses and the responses at minutes 1–5 and minutes 7–11 after etomidate. *P < 0.05 compared with control. Panel G shows responses (as percentage of control) in decerebrate animals without baseline isoflurane; *P < 0.05 depression by 0.25 mg/kg dose is significantly greater than depression caused by same dose in the intact animals in Panel A. Panel H shows data (as percent of control) from intact rats anesthetized with isoflurane and administered propofol; *P < 0.05 compared with control.

In the decerebrate, isoflurane-free group, we studied 6 WDR neurons in 6 rats, with a recording depth of 780 ± 190 µm. Etomidate at 0.25 mg/kg resulted in more neuronal depression (to approximately 40% of control) as compared with the response in the intact rats anesthetized with isoflurane (approximately 70% of control); however, the effect of etomidate at 0.5 and 1 mg/kg in the decerebrate rats was not different from the responses in the intact animals at the same doses (Fig. 2G).

We studied 10 WDR neurons in 10 rats anesthetized with isoflurane and administered propofol (mean recording depth 594 ± 241 µm). The 1 mg/kg dose of propofol did not produce consistent neuronal depression, but the 5 mg/kg dose depressed neuronal responses to approximately 40% of control (Fig. 2H), similar to that resulting from the larger doses (e.g., 1 mg/kg) of etomidate.

Control administration of propylene glycol vehicle did not affect neuronal responses (681 ± 249 impulses preinjection; 653 ± 247 impulses postinjection, n = 4; P > 0.05). The MAP decreased slightly after etomidate (e.g., for the 1 mg/kg dose: 106 ± 26 mm Hg preinjection to 80 ± 17 mm Hg 30–60 s postinjection; paired Student's t-test, P < 0.01). The MAP usually recovered by 1–2 min. Smaller doses resulted in smaller changes in MAP. The lowest postinjection MAP in any animal in the intact group with any dose was 61 mm Hg. Decerebration resulted in hypotension that recovered (>50–60 mm Hg) by the end of the 60-min stabilization period.

	Discussion

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The main findings of the present study are that etomidate depressed lumbar neuronal responses to noxious heat and that there appeared to be a ceiling effect. Maximal depression was achieved at 0.5 mg/kg and doses larger than that did not cause larger peak depression, although these larger doses had a more long-lasting depressant effect. These data are consistent with the hypothesis that etomidate possesses analgesic properties.

Etomidate is useful as an induction drug, especially in the setting of preexisting altered hemodynamics, as it is associated with minimal cardiovascular compromise, such as decreasing MAP (10). In addition, etomidate is often used for sedation during short procedures, including those outside the operating room, such as emergency room procedures (11). Thus, it would be beneficial to use a drug that has analgesic properties. Although depression of neuronal responses as presently demonstrated is consistent with this goal, it is unclear whether the neuronal depression is achieved at doses that would still permit consciousness. The etomidate dose that produces loss of the righting reflex in rats (0.86 mg/kg) (8), however, is larger than the doses that produced neuronal depression in the present study, suggesting that etomidate might produce analgesia at doses that permit consciousness.

Etomidate has been shown to cause central nervous system depression. For example, etomidate depresses the electroencephalogram (12). However, not all central nervous system sites are depressed by etomidate, as the H-reflex is minimally affected by etomidate administration as compared with other anesthetics such as halothane (13). Interestingly, the somatosensory evoked potential can sometimes be enhanced by administration of etomidate (14). For example, during spine surgery somatosensory evoked potentials are used to determine whether the spinal cord is damaged. If the cortical signal is low, etomidate can be given as an infusion to enhance the amplitude, which thereby permits easier detection of spinal cord injury. In light of the present data, this suggests that etomidate could depress spinal neuronal responses to noxious thermal stimulation while there is enhancement of evoked potential signals that travel in pathways separate from those involved in thermal nociception. For example, etomidate's enhancement of evoked potentials could occur at the cortical level.

Etomidate is thought to exert its action at the GABA_A receptor by enhancing the effect of GABA (2,3). Jurd et al. (2) showed that mutation of the ß-3 subunit almost completely eliminated the anesthetic action of etomidate, demonstrating that the GABA_A receptor is by far the most important site of action for etomidate. Furthermore, etomidate binds in a specific way at the GABA_A receptor, as the R(+) isomer is nearly 20 times more potent than the S(–) isomer (15). Other sites, however, could also be involved, such as the glycine receptor (16), although some potassium channels and the acetylcholine receptor are minimally affected by etomidate (17,18). We also cannot exclude an action on peripheral receptors, such as the transient receptor potential channels. Nonetheless, there are convincing data suggesting that etomidate's anesthetic action occurs predominantly, if not exclusively, at the GABA_A receptor.

Compared with other induction drugs, etomidate produced similar depression of spinal neuronal responses to noxious stimulation. For example, the depression by propofol in the present study was similar to that caused by etomidate. In addition, we found that propofol and thiopental, when administered to isoflurane-anesthetized goats, resulted in 50%–75% depression of lumbar neuronal responses to noxious mechanical stimulation (19,20).

There are several limitations to the present study. First, etomidate was studied with a baseline of isoflurane anesthesia. We cannot exclude the possibility that there was interaction between the two anesthetics (i.e., enhanced depression); however, the responses in the isoflurane-free group were similar to those in the intact rats. Indeed, the more depressive effect of the 0.25 mg/kg dose of etomidate in the decerebrate rats argues against enhancement of neuronal depression by the baseline isoflurane anesthesia. Second, we did not examine responses to mechanical stimulation, as the paradigm we used did not permit testing of neuronal responses to mechanical stimulation while maintaining consistent application (every 2 minutes) of the thermal stimulus to the receptive field on the hindpaw. We cannot exclude the possibility that responses to mechanical stimulation would have differed from those determined using thermal stimulation. Third, there was a transient decrease in MAP, but this was not likely to have impacted the results. The lowest MAP was still acceptable for recording purposes. In addition, the MAP decrease was small and lasted only 30–60 seconds, whereas the depression lasted several minutes.

In summary, we found that etomidate depressed dorsal horn neuronal responses to noxious heat. This depression was maximal at 0.5 mg/kg, as larger doses did not enhance it. Propofol at equipotent doses caused similar depression. The data are consistent with the notion that etomidate has analgesic actions.

The authors thank Emigdio Bravo for his technical assistance.

	Footnotes

 
Accepted for publication December 1, 2005.

Supported, in part, by National Institutes of Health grants GM 57970, GM61283 and P01-GM47818.

	References

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Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999