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measure anesthetic potency using the minimum alveolar anesthetic concentration (MAC), which is defined as the alveolar concentration of anesthetic that ...
䡵 LABORATORY INVESTIGATIONS Anesthesiology 2002; 96:367–74

© 2002 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc.

Isoflurane and Nociception Spinal ␣2A Adrenoceptors Mediate Antinociception while Supraspinal ␣1 Adrenoceptors Mediate Pronociception Wade S. Kingery, M.D.,* Geeta S. Agashe, M.D.,† Tian Z. Guo, M.D.,† Shigehito Sawamura, M.D.,‡ M. Frances Davies, Ph.D.,† J. David Clark, M.D., Ph.D.,§ Brian K. Kobilka, M.D.,储 Mervyn Maze, M.B., Ch.B., F.R.C.P.#

Background: The authors recently established that the analgesic actions of the inhalation anesthetic nitrous oxide were mediated by noradrenergic bulbospinal neurons and spinal ␣2B adrenoceptors. They now determined whether noradrenergic brainstem nuclei and descending spinal pathways are responsible for the antinociceptive actions of the inhalation anesthetic isoflurane, and which ␣ adrenoceptors mediate this effect. Methods: After selective lesioning of noradrenergic nuclei by intracerebroventricular application of the mitochondrial toxin saporin coupled to the antibody directed against dopamine ␤ hydroxylase (D␤H-saporin), the antinociceptive action of isoflurane was determined. Antagonists for the ␣1 and ␣2 adrenoceptors were injected at spinal and supraspinal sites in intact and spinally transected rats to identify the noradrenergic pathways mediating isoflurane antinociception. Null mice for each of the three ␣2-adrenoceptor subtypes (␣2A, ␣2B, and ␣2C) and their wild-type cohorts were tested for their antinociceptive response to isoflurane. Results: Both D␤H-saporin treatment and chronic spinal transection enhanced the antinociceptive effects of isoflurane. The ␣1-adrenoceptor antagonist prazosin also enhanced isoflurane antinociception at a supraspinal site of action. The ␣2-adrenoceptor antagonist yohimbine inhibited isoflurane antinociception, and this effect was mediated by spinal ␣2 adrenoceptors. Null mice for the ␣2A-adrenoceptor subtype showed a reduced antinociceptive response to isoflurane. Conclusions: The authors suggest that, at clinically effective concentrations, isoflurane can modulate nociception via three different mechanisms: (1) a pronociceptive effect requiring descending spinal pathways, brainstem noradrenergic nuclei, and supraspinal ␣1 adrenoceptors; (2) an antinociceptive effect requiring descending noradrenergic neurons and spinal ␣2A adrenoceptors; and (3) an antinociceptive effect mediated within the spinal cord for which no role for adrenergic mechanism has been found.

THE mechanisms and pathways of anesthetic action are unknown. Part of the difficulty in determining how anesthetics transduce their effects is that the state of anesthesia encompasses a syndrome of “behaviors,” including analgesia (pain relief), hypnosis–sedation, amnesia (loss of memory), and muscle relaxation, and the effects of an anesthetic agent on each of these behaviors may have a unique mechanism of action.1 Nearly all behavioral investigations of volatile anesthetic mechanisms measure anesthetic potency using the minimum alveolar anesthetic concentration (MAC), which is defined as the alveolar concentration of anesthetic that prevents movement in 50% of subjects in response to a painful stimulus. Which of the behavioral effect or effects (analgesia, hypnosis, or muscle relaxation) contributes to MAC is not fully understood. To better define the mechanism of anesthetic action, we sought to deal with each element of the behavioral response separately. This approach was predicated by our finding that the hypnotic and analgesic responses to the anesthetic agent nitrous oxide (N2O) are mediated at different sites by different signaling pathways.1 We previously demonstrated that noradrenergic brainstem nuclei and ␣2B adrenoceptors play a pivotal role in the analgesic, but not the hypnotic effects of N2O.1 N2O exposure activated noradrenergic brainstem neurons with descending spinal projections, which increased the release of norepinephrine in the spinal cord and evoked an analgesic response that was blocked by a spinally administered ␣2-adrenoceptor antagonist, evidence supporting the hypothesis that noradrenergic bulbospinal neurons mediate N2O analgesic action.1–3 The neuropharmacologic basis of N2O-evoked analgesia and hypnosis clearly differ, indicating a need to dissect the mechanisms of action for an anesthetic agent by examining each component of the anesthetic state. Using the methods established in our previous investigations with N2O, we examined the noradrenergic mechanisms and pathways responsible for the analgesic effects of isoflurane on the tail-flick assay in rats. The mechanisms mediating the anesthetic actions of isoflurane are unknown. When isoflurane is selectively administered to just the brain and brainstem, it has a pronociceptive effect as measured by MAC,4 and concentrations of isoflurane below anesthetic threshold also have a pronociceptive effect on hind-paw radiant heat

* Assistant Professor, Department of Functional Restoration, Stanford University and Physical Medicine and Rehabilitation Service, Veterans Affairs Palo Alto Health Care System. † Research Associate, ‡ Postdoctoral Fellow, Department of Anesthesiology, 储 Associate Professor, Howard Hughes Medical Institute, Stanford University. § Third Assistant Professor, Department of Anesthesiology, Stanford University, and Anesthesiology Service, Veterans Affairs Palo Alto Health Care System. # Professor, Magill Department of Anaesthetics, Imperial College School of Medicine, London, United Kingdom. Received from the Department of Anesthesia, Stanford University, Stanford, California. All experiments were performed at the VA Palo Alto Health Care System, Palo Alto, CA. Submitted for publication May 11, 2001. Accepted for publication August 29, 2001. Supported by the Department of Veterans Affairs, Washington, DC, and grant No. 30232 from the National Institutes of General Medical Sciences, Bethesda, Maryland, and the Medical Research Council, London, United Kingdom. Address reprint requests to Dr. Maze: Magill Department of Anaesthetics, Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9NH, United Kingdom. Address electronic mail to: [email protected]. Individual article reprints may be purchased through the Journal Web site, www.anesthesiology.org.

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368 withdrawal thresholds.5 We now show that saporin coupled to the antibody directed against dopamine ␤ hydroxylase (D␤H-saporin) lesioning of brainstem noradrenergic neurons, spinal cord transection, or supraspinally administered ␣1-adrenoceptor antagonists all enhanced isoflurane antinociception on the tail-flick assay, possibly by eliminating a noradrenergic-supraspinal ␣1adrenoceptor–mediated pronociceptive effect.6,7 Noradrenergic projections to all regions of the spinal cord arise almost entirely from the dorsolateral brainstem catecholamine cell groups A5, the locus coeruleus, and the A7. Electrical or chemical stimulation in the dorsolateral pons produces analgesic effects mediated by spinal ␣2 adrenoceptors, and such stimulation causes inhibition of nociceptive neurons in the deep dorsal horn.8 –10 We now show that a spinally administered ␣2-adrenoceptor antagonist inhibited isoflurane antinociception on the tail-flick assay, and this effect was lost after spinal transection. Using null mice for ␣2A-, ␣2B-, and ␣2C-adrenoceptor subtypes, we also identify the subtype involved in mediating the noradrenergic antinociceptive action of isoflurane. In addition to the noradrenergic mechanisms that we identified as mediators of the antinociceptive and pronociceptive actions of isoflurane, there appears to be an intrinsic nonadrenergic spinal antinociceptive effect of isoflurane.

Methods Animals These experiments were reviewed and approved by the Subcommittee on Animal Studies (Veterans Affairs Palo Alto Health Care System, Palo Alto, CA) and were in accordance with the provisions of the Animal Welfare Act, the Public Health Service (PHS) Guide for the Care and Use of Laboratory Animals, and Veterans Affairs Policy. All neuroablative and immunolesioning experiments were performed in adult male Sprague-Dawley rats (240 –260 g) obtained from B&K Universal (Fremont, CA). Additional behavioral studies were performed in adult (20 –30 g) male mice. Various genetically engineered mice strains were examined, including the following: (1) D79N mice with a nonfunctioning ␣2A adrenoceptor caused by a point mutation in its gene substituting aspartic acid by asparagine at amino acid residue #79; (2) ␣2A⫺/⫺ null mice with a knockout of the ␣2A-adrenoceptor gene; (3) ␣2C⫺/⫺ null mice with a knockout of the ␣2C-adrenoceptor gene; and (4) their wild-type controls. All of these strains were on a congenic C57BL/6J background. The ␣2B⫺/⫺ null mice had a knockout of the ␣2B-adrenoceptor gene on a hybrid C57BL/6J and 129SvJ background; as their controls we used generationally matched wild-type mice on the same hybrid background (C57BL/6J ⫻ 129SvJ). Production of the ␣2A⫺/⫺, ␣2B⫺/⫺, ␣2C⫺/⫺, and D79N mice have been described previously.11–13 Anesthesiology, V 96, No 2, Feb 2002

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Fig. 1. Time-course study examining isoflurane-induced antinociceptive effect on tail-flick assay in rats. Baseline tail-flick latencies were determined in air, and then the tail-flick and loss of righting reflex (LORR) assays were tested after 5, 10, 15, 30, and 90 min of exposure to isoflurane (1.2% atm). All rats had LORR at 5 min, at which time no antinociceptive effect had developed. The mean baseline tail-flick latency was 3.5 ⴞ 0.2 s, and after 30 min of isoflurane exposure, the mean latency increased to 5.1 ⴞ 0.4 s. These data indicate that the antinociceptive and hypnotic effects of isoflurane can be independently measured using the tail-flick and LORR assays. *P < 0.05 versus baseline latency.

Behavioral Testing All behavioral testing was performed in a blinded manner, and the experimental groups were tested simultaneously to ensure identical gas exposure conditions. Using a heating blanket, the tail temperatures were maintained within 0.5°C of 30°C. Tail-flick latencies were determined from the mean of three (in rats) or two (in mice) consecutive latencies using a tail-flick apparatus (Columbus Instruments, Columbus, OH). A different patch of the middle (rat) or distal (mice) third of the tail was exposed to the light beam each time to minimize the risk of tissue damage. The same light stimulus intensity was used for all experiments in a given strain, having been preset at an intensity that elicited a mean latency of 3.5 s in room air. Latency measurements were taken only when the rat or mouse was calmly resting while being gently held under a towel, and a cutoff time of 10 s was used to prevent tissue injury. The loss of righting reflex was assessed by placing the rat on its back and determining if the animal could right itself within 60 s. Gas Exposures Behavioral studies were performed in a Plexiglas chamber large enough to contain the tail-flick device and equipped with rubber flap iris diaphragm air seals. Antinociceptive testing was always performed after 30-min isoflurane exposure because we had demonstrated a maximal antinociceptive effect in mice and rats at this time interval (fig. 1). Fresh gas flow (rate varied between 3–10 l/min) was introduced into the chambers via an inflow port; two fans were used to achieve adequate mixing within the chamber, and gases were purged by

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vacuum. Oxygen concentration in the chamber was maintained at 30% atmospheres (atm), and isoflurane at 1.2% atm (for intact rats), which is the approximate MAC concentration of isoflurane for both rats and humans.14,15 Because the 1.2% concentration of isoflurane did not have a significant antinociceptive effect on the tail-flick assay in the C57BL/6J mice, a 1.7% isoflurane concentration was used in the mice. Control exposure was with room air. An airway gas monitor (Model 254; Datex, Helsinki, Finland) was used to continuously monitor the concentrations of isoflurane, oxygen, and carbon dioxide in the chamber, and flow rates were adjusted to maintain the desired concentrations. Temperature in the chamber was controlled by a heating blanket, and the tail and rectal temperatures were monitored before each behavioral test. Rectal temperature was maintained within 0.5° of 36.5°C. Surgery The rats were anesthetized with sodium pentobarbital (50 mg/kg, administered intraperitoneally), and a laminectomy was performed at T7–T8. The spinous processes and lamina were removed to expose a circular region of dura approximately 5 mm in diameter. The spinal column was stabilized and the spinal cord completely transected between T7 and T8. Postoperatively, the rats were injected subcutaneously with 5 ml saline and intramuscularly with 3 mg/kg enrofloxacin daily for the next 2 days. Sham-operated rats underwent the same surgical procedure without laminectomy or spinal transection. Postoperative care included daily manual bladder emptying, soft bedding on the cage floor that was changed daily, with food and water supplied on the floor of the cage to ensure accessibility. Animals were also weighed and washed daily. The rats were monitored for postoperative complications, and there was no evidence of wound or bladder infection, skin breakdown, or excessive weight loss. Drug effects on tail-flick latencies were assessed at 6 and 7 days after surgery (crossover design), and then all rats were immediately killed. Immunolesioning The antidopamine ␤-hydroxylase-saporin (D␤H-saporin) immunotoxin (Advanced Targeting Systems, San Diego, CA) is injected intracerebroventricularly and permanently destroys noradrenergic neurons in the locus coeruleus and the A5 and A7 brainstem nuclei over 14 days.16,17 After D␤H-saporin treatment, the tyrosine hydroxylase–positive noradrenergic neurons in the locus coeruleus completely disappear; residual staining is observed in 29% of the A5 and 26% of the A7 noradrenergic neurons.1 Rats were anesthetized with intraperitoneal injection of pentobarbital (50 mg/kg). While the skull was fixed in a stereotaxic apparatus, the animal was Anesthesiology, V 96, No 2, Feb 2002

369 injected with D␤H-saporin (3 ␮g/3 ␮l) or saline (3 ␮l) into the lateral ventricle as previously described.1 Immunohistochemistry After nociceptive testing, all rats were anesthetized with pentobarbital, transcardially perfused, decapitated and the brain removed, fixed, cryoprotected, sliced into 40-␮m-thick sections, and every third section of the brainstem (from caudal periaqueductal gray to rostral medulla) was retained for immunohistochemical analysis. Sections were stained using antibodies for tyrosine hydroxylase as previously described,1 and tyrosine hydroxylase–positive neurons in the A5, locus coeruleus, and A7 nuclei were counted, and the aggregate for all sections for each nucleus in each rat was derived. The investigator performing the counting was blinded to the treatment. Injection Techniques Intrathecal injections were performed using a modified single percutaneous injection technique.18 Rats were lightly anesthetized with isoflurane, and then a 27-gauge needle was inserted at the L5–L6 intervertebral space. Intrathecal placement was confirmed by a slight tail twitch. All rats were injected with a 10-␮l volume of either drug or vehicle and, after allowing 20 s for the injectant to disperse, the needle was slowly withdrawn. While developing this technique we injected 20 ␮l of 5% lidocaine intrathecally in six rats, which consistently caused a transient hind-paw paralysis. To perform intracerebroventricular injections, the rats were cannulated stereotaxically (Plastics One Inc., Roanoke, VA) in the left lateral ventricle as previously described.19 Rats were injected with a 10-␮l volume of either drug or vehicle over a 2-min period using a Harvard 22 infusion pump (Harvard Apparatus Inc., South Natick, MA). At the completion of the behavioral experiment, the correct placement of the cannula was confirmed histologically in each rat with Evans blue dye. Experimental Protocols To demonstrate that isoflurane-induced prolongation of tail-flick latency was not caused by loss of consciousness, the time course for the development of isofluraneinduced hypnosis and antinociception were established. Loss of righting reflex and tail-flick latencies were measured in rats during exposure to air and then after 5, 10, 15, 30, and 90 min of isoflurane (1.2% atm) exposure. To determine whether isoflurane-induced antinociception was mediated at a spinal or supraspinal level, rats underwent either spinal transection or sham surgery. Six days later, baseline tail-flick latencies were determined, and after 30 min exposure to isoflurane, the tail-flick latencies were repeated. Three different concentrations of isoflurane were tested on consecutive days: 0.8, 1.0, and 1.2% atm.

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The contribution of noradrenergic brainstem neurons to isoflurane-induced antinociception was determined by immunolesioning the noradrenergic neurons. Rats were injected with the immunotoxin D␤H-saporin (3 ␮g/3 ␮l) or with saline (administered intracerebroventricularly). Fifteen days after the intracerebroventricular injection, the baseline tail-flick latencies were determined, and after a 30-min exposure to either isoflurane or air, the tail-flick latencies were repeated. After the tail-flick measurement, the rat was anesthetized, and the brain was harvested and immunostained for tyrosine hydroxylase to evaluate the efficacy of noradrenergic lesioning. To evaluate ␣1-adrenoceptor modulation of isofluraneinduced antinociception and to determine the site of this action, intact and spinal-transected rats were tested for baseline tail-flick latencies and then injected with either the ␣1-adrenoceptor antagonist prazosin (2 mg/kg administered intraperitoneally; Sigma Chemical, St. Louis, MO) or vehicle (20% 2 hydroxypropyl-b-cyclodextrin in sterile water; RBI, Natick, MA). After 55 min in air, the tail-flick latencies were repeated. The next day the same procedure was repeated, except that 25 min after the injection of prazosin or saline, the rats were exposed to isoflurane (1.2% atm for intact rats, 0.8% atm for spinaltransected rats). After 30 min of isoflurane exposure, the tail-flick latencies were repeated. Another cohort of rats was tested for baseline tail-flick latencies and then intrathecally injected with either prazosin (30 ␮g/10 ␮l) or vehicle. Ten minutes after injection, the rats were exposed to either isoflurane (1.2% atm) or air for 30 min, and the tail-flick testing was repeated. This protocol was also used in another cohort of rats to examine the effects of intracerebroventricular injections of prazosin (30 ␮g/ 10 ␮l) or vehicle on tail-flick latency response to isoflurane. Doses of prazosin in the range of 8–30 ␮g/␮l have been previously reported to completely block the effects of endogenous and exogenous ␣1-adrenoceptor agonists when administered via intrathecal and intracerebroventricular routes.6,20,21 To determine the dependence of isoflurane-induced antinociception on ␣2 adrenoceptors, the ␣2-adrenoceptor antagonist yohimbine (2 mg/kg for intraperitoneal administration, 30 ␮g/10 ␮l for intrathecal and intracerebroventricular administration; Sigma Chemical) or vehicle (0.9% saline) was administered to intact and spinaltransected rats as described above for prazosin. Doses of yohimbine in the range of 8 –30 ␮g/␮l have been previously reported to completely block the effects of endogenous and exogenous ␣2-adrenoceptor agonists when administered via intrathecal and intracerebroventricular routes.6,21–24 To identify the ␣2-adrenoceptor subtype mediating isoflurane antinociception, tail-flick testing was performed in ␣2A⫺/⫺, ␣2B⫺/⫺, ␣2C⫺/⫺, D79N mice, and in their respective wild-type controls. Baseline latencies Anesthesiology, V 96, No 2, Feb 2002

were determined in the gas chamber during room-air conditions; thereafter the mice were removed from the chamber, which was then equilibrated with the test gas mixture (1.7% atm of isoflurane). After 30 min, the mice were placed back in the gas chamber and exposed to the test gas mixture for 30 min. The nociceptive testing was then repeated. Statistical Analysis All data are presented as mean ⫾ SEM, and differences are considered significant at P ⬍ 0.05. Tail-flick latencies were compared using paired (air vs. isoflurane) and unpaired (saline vs. D␤H-saporin) t tests. A repeatedmeasures analysis of variance was used to test for the development of isoflurane antinociception over time, and a paired t test was used to test for latency differences from baseline. A one-way analysis of variance was performed on the tail-flick latencies when comparing groups of mice, and an unpaired t test was used to test for contrasts. Isoflurane antinociceptive effects for the tail-flick assay are shown as the percentage of the maximum possible effect: %MPE ⫽ 共关post-gas latency ⫺ baseline latency兴/关cut-off latency ⫺ baseline latency兴兲 ⫻ 100.

Results Hypnotic and Antinociceptive Effects of Isoflurane Are Temporally Uncoupled After a 5-min exposure to isoflurane (1.2% atm), all rats had loss of righting reflex, but there was no significant effect on tail-flick antinociception (fig. 1). Isofluraneinduced antinociception for tail flick gradually developed over 30 min, achieving significance after 10 min. Spinal Cord Transection Enhanced the Antinociceptive Effect of Isoflurane After spinal cord transection, the antinociceptive effect of isoflurane was dramatically enhanced compared with sham-operated control rats (fig. 2A). When the rats were exposed to a 0.8% atm concentration of isoflurane for 30 min, there was a 770% increase in antinociceptive effect after spinal cord transection. A smaller increase in antinociceptive effect after spinal transection was observed with a 1.0% concentration (580%) and a 1.2% concentration (250%) of isoflurane. D␤H-Saporin Treatment Destroyed Pontine Noradrenergic Neurons and Enhanced the Antinociceptive Effect of Isoflurane Figure 2B illustrates that the antinociceptive effect of isoflurane (1.2% atm, 30 min) was significantly enhanced in the D␤H-saporin–treated rats compared with saline-

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Fig. 2. (A) Three different isoflurane concentrations (0.8, 1.0, and 1.2% atm; 30-min exposure) were tested in sham-operated and spinal cord–transected rats (n ⴝ 7 per cohort). The mean baseline tail-flick latencies in air were slightly less in the spinaltransected rats (2.7 ⴞ 0.2 s) than in the sham-operated rats (3.5 ⴞ 0.2 s, in air). The antinociceptive effect of isoflurane on the tail-flick assay was greatly enhanced by spinal cord transection (after 0.8, 1.0, and 1.2% atm isoflurane exposure, the latencies in spinal rats were 6.9 ⴞ 1.0, 8.3 ⴞ 0.8, and 9.7 ⴞ 0.2 s, respectively; in sham-operated rats, they were 4.0 ⴞ 0.1, 4.3 ⴞ 0.3, and 5.4 ⴞ 0.3 s, respectively). ##P < 0.01, ###P < 0.001 versus sham surgery. (B) D␤H-saporin lesioning of the bulbospinal noradrenergic neurons enhanced the isoflurane-induced antinociceptive effect on tail-flick assay by 110% versus vehicle-treated rats (n ⴝ 8 per cohort). ##P < 0.01 versus vehicle.

treated rats. There was no difference in baseline tail-flick latencies between control and immunolesioned rats. Supraspinal ␣1 Adrenoceptors Mediate Isofluraneinduced Pronociception Systemic administration of the ␣1-adrenoceptor antagonist prazosin (2 mg/kg, administered intraperitoneally) had no effect on nociceptive thresholds in the intact or spinal-transected rats when tested in air (fig. 3A). Prazosin enhanced isoflurane-induced antinociception by 132% in the intact rats but had no effect in the spinaltransected rats (fig. 3B). When prazosin (30 ␮g/10 ␮l) was administered intrathecally or intracerebroventricularly in intact rats, it had no antinociceptive effect in air (fig. 3C). Intrathecal prazosin had no effect on isofluraneinduced antinociception, but intracerebroventricular prazosin enhanced isoflurane antinociception by 82% (fig. 3D). Spinal ␣2A Adrenoceptors Partially Mediate Isoflurane-induced Antinociception Systemic administration of the ␣2-adrenoceptor antagonist yohimbine (2 mg/kg, administered intraperitoneally) reduced tail-flick latencies in the intact rats when tested in air (⫺14% maximum possible effect) but had no effect in the spinal-transected animals (fig. 4A). Systemic yohimbine completely blocked isoflurane-induced antinociception in the intact rats but had no effect in spinal-transected rats (fig. 4B). When yohimbine (30 ␮g/10 ␮l) was administered intrathecally or intracAnesthesiology, V 96, No 2, Feb 2002

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Fig. 3. (A) Compared with vehicle, systemically administered prazosin (2 mg/kg, administered intraperitoneally) had no effect on tail-flick latencies in intact and spinal cord–transected rats when tested in room air (n ⴝ 8 per cohort). (B) Prazosin enhanced isoflurane (30-min exposure) antinociception in intact rats (vs. vehicle, n ⴝ 8 per cohort) but had no effect in spinal-transected rats (n ⴝ 8 per cohort). (C) Compared with vehicle, intrathecal (IT) and intracerebroventricular (ICV) prazosin (30 ␮g/10 ␮l) had no effect on tail-flick latencies in intact rats in room air. (D) Intrathecal prazosin had no effect on isoflurane (30-min exposure) antinociception in intact rats, but intracerebroventricular prazosin enhanced the antinociceptive effect of isoflurane (n ⴝ 8 per cohort). *P < 0.05, **P < 0.01, ***P < 0.001 versus baseline latency; #P < 0.05 versus vehicle.

erebroventricularly in intact rats, it had no effect on nociceptive latencies in air (fig. 4C). Figure 4D illustrates that intrathecal yohimbine reduced isoflurane-induced antinociception by 72% (P ⬍ 0.05), but intracerebroventricular yohimbine did not significantly reduce isoflurane antinociception (P ⫽ 0.16). The antinociceptive effects of isoflurane with the tailflick assay in the ␣2A⫺/⫺, ␣2B⫺/⫺, ␣2C⫺/⫺, D79N mice, and in their respective wild-type controls are shown in figure 5. The isoflurane antinociceptive response was reduced by 54% in the ␣2A⫺/⫺ mice and 62% in the D79N mice (vs. wild-type controls; P ⬍ 0.05), indicating that the ␣2A-adrenoceptor subtype partially mediates isoflurane antinociception. Although the isoflurane-induced antinociceptive effect in the ␣2B⫺/⫺ mice was less than in the corresponding wild-type cohort, this was not significantly different (P ⫽ 0.10).

Discussion In attempting to elucidate the mechanisms for the analgesic effect of anesthetic agents, it is important to

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Fig. 4. (A) Compared with vehicle injection, systemically administered yohimbine (2 mg/kg, administered intraperitoneally) had a modest pronociceptive effect on tail-flick latencies when intact rats were tested in room air but had no effect on spinaltransected rats in air. (B) Systemically administered yohimbine blocked isoflurane (30-min exposure) antinociception in intact, but not in spinal transected rats (n ⴝ 8 per cohort). (C) Compared with vehicle, intrathecal (IT) and intracerebroventricular (ICV) yohimbine (30 ␮g/10 ␮l) had no effect on tail-flick latencies in intact rats in room air. (D) Intrathecal yohimbine reduced but did not completely block isoflurane (30-min exposure) antinociception in intact rats. Intracerebroventricular yohimbine had no significant effect on the antinociceptive effect of isoflurane (n ⴝ 8 per cohort). *P < 0.05, **P < 0.01, ***P < 0.001 versus baseline latency; #P < 0.05 versus vehicle.

obviate any effect that loss of consciousness can exert on the antinociceptive assay. We previously reported dissociation between N2O-evoked antinociception and N2Oinduced hypnosis since these occurred at different concentrations; this enabled us to distinguish the mechanism underlying these effects in experimental paradigms. However, for isoflurane, the hypnotic effect develops at a lower concentration than the antinociceptive effect.25 To separate these two effects, we used the radiant heat tail-flick assay, which measures the latency of a spinal withdrawal reflex to noxious heat, which is independent of hypnotic-induced decrement in purposeful movement. Figure 1 illustrates that continuous isoflurane inhalation (1.2% atm) gradually increased the tail-flick latency over a 30-min period. Numerous studies have shown that isoflurane, at concentrations up to 2.2% atm, has no effect on spinal or peripheral nerve evoked compound motor nerve action potential amplitudes or compound muscle action potential amplitudes,26 –28 thereby negating motor paralysis Anesthesiology, V 96, No 2, Feb 2002

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Fig. 5. Antinociceptive effect of isoflurane in genetically modified mice. This figure illustrates the antinociceptive effects of isoflurane (30 min, 1.7% atm concentration) on the tail-flick assay in the ␣2Aⴚ/ⴚ, ␣2Cⴚ/ⴚ knockout mice (␣2A KO and ␣2C KO), the D79N mutant mice with nonfunctional ␣2A adrenoceptors, and in their genetically matched wild-type (WT) controls (on a C57BL/6J congenic background). The ␣2Bⴚ/ⴚ (␣2B KO) mice were on a different genetic background (C57BL/6J ⴛ 129SvJ hybrid); therefore, they required their own genetically matched wild-type controls (wild-type for ␣2B). There were no differences in baseline tail-flick latencies between the various mouse strains (data not shown). All strains of mice had significant isoflurane antinociception. Only the ␣2A knockout and the D79N mice had reduced isoflurane antinociception on the tailflick assay compared with wild-type controls (11 ⴞ 5 in ␣2A knockout and 9 ⴞ 3 in D79N vs. 24 ⴞ 4% maximum possible effect in wild-type), indicating that the ␣2A-adrenoceptor subtype at least partially mediates isoflurane antinociception. The isoflurane antinociceptive responses (% maximum possible effect) in the other knockout and mutant mouse strains were: ␣2C knockout, 22 ⴞ 7; wild-type for ␣2B, 42 ⴞ 7; ␣2B knockout, 29 ⴞ 4 (n ⴝ 16 for each group). #P < 0.05 versus respective wild-type control.

as a mechanism for change in latency. The inhibitory effect of isoflurane on tail-flick latencies was not a result of the hypnotic–sedative effects of isoflurane, because the tailflick latency was unchanged after 5-min exposure to isoflurane, at which time all the rats were unconscious and unable to right themselves when laid on their backs. Furthermore, after complete spinal cord transection, the tailflick withdrawal response was intact, and the isoflurane analgesic effect was greatly enhanced compared with sham-operated controls (fig. 2A). Although the tail-flick response is clearly a spinal reflex, it is modulated by brainstem neurons with descending spinal projections that facilitate (decrease the latency) and inhibit (increase the latency) nociceptive processing in the spinal cord. The enhanced isoflurane antinociceptive effect we observed after spinal transection suggests that isoflurane evoked a descending pronociceptive effect on the tail-flick assay. Spinal transection blocked the descending facilitory effect of isoflurane, thus unmasking the intrinsic spinal antinociceptive effect of isoflurane on the tail-flick reflex. Using D␤H-saporin to selectively destroy the brainstem noradrenergic neurons, we observed an enhanced isoflurane antinociceptive effect, indicating that these neurons contribute to the pronociceptive effect of isoflu-

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Fig. 6. Model illustrating the proposed neuronal pathways that mediate the bidirectional effects on nociception produced by isoflurane. (A) Isoflurane pronociceptive effect is mediated by activation of pontine noradrenergic neurons (PNN), causing the release norepinephrine from brainstem projections, which act on supraspinal ␣1 adrenoceptors to activate descending spinal pathways that facilitate the tail-flick reflex in the spinal cord. (B) Isoflurane antinociceptive effect is partially mediated by activation of PNN, causing the release norepinephrine from spinal cord projections, which act on spinal ␣2A adrenoceptors to inhibit the tail-flick reflex in the spinal cord. (C) Isoflurane antinociceptive effect is also partially mediated by a direct inhibitory effect on spinal neurons that does not require PNN or intact spinal pathways.

rane (fig. 2B). Furthermore, the systemic administration of the ␣1-adrenoceptor antagonist prazosin also enhanced the antinociceptive effect of isoflurane, and this effect was lost after spinal transection (fig. 3B), suggesting that activation of ␣1 adrenoceptors evoke the pronociceptive effects of isoflurane via descending spinal pathways. Pronociceptive actions have been attributed to both supraspinal6,7 and spinal7,23,29 ␣1 adrenoceptors when activated by endogenous or exogenous adrenergic ligands. We demonstrated that supraspinally administered prazosin enhanced isoflurane-induced antinociception, but no effect was observed with the spinal administration of prazosin (fig. 3D), indicating that supraspinal ␣1 adrenoceptors mediate the isoflurane pronociceptive effect. Although the predominant effect of isoflurane activation of the brainstem noradrenergic neurons is clearly pronociceptive, there is also an antinociceptive effect. Systemic administration of the ␣2-adrenoceptor antagonist yohimbine inhibited the antinociceptive effect of isoflurane, and this effect was lost after spinal transection (fig. 4B), suggesting that norepinephrine released by brainstem neurons acted on ␣2 adrenoceptors to evoke an antinociceptive effect via descending spinal pathways. Furthermore, spinally administered yohimbine reduced isoflurane-induced antinociception, but no effect was observed with the supraspinal administration of yohimbine or in the presence of spinal transection (fig. 4D), indicating that spinal ␣2 adrenoceptors mediate an isoflurane-evoked descending spinal antinociceptive effect, similar to N2O-evoked analgesia.2 Pharmacologic evidence in rats30,31 and studies using D79N mice with dysfunctional ␣2A adrenoceptors32–34 have established that the ␣2A subtype mediates the antinociceptive response to ␣2-adrenoceptor agonists. Now we observe that mice deficient in the ␣2A-adrenoceptor subtype or D79N mice with dysfunctional ␣2A adrenoceptors showed reduced isoflurane-induced antinociception, indicating that the spinal ␣2A-adrenocepAnesthesiology, V 96, No 2, Feb 2002

tor subtype mediates an antinociceptive response to isoflurane. We described a noradrenergic-supraspinal ␣1-adrenoceptor pathway that mediates the pronociceptive effect of isoflurane. In addition, we observed a noradrenergic-spinal ␣2-adrenoceptor pathway mediating an antinociceptive isoflurane effect. These opposing isoflurane effects are consistent with behavioral and electrophysiologic evidence that stimulating the pontine noradrenergic neurons simultaneously activates ␣1-adrenoceptor pronociceptive and ␣2adrenoceptor antinociceptive pathways.6,23,29,35 It has been reported that low concentrations of isoflurane have a pronociceptive effect on hind-paw withdrawal latencies in rats.5 When the isoflurane concentration is selectively lowered in brain, the systemic isoflurane concentration required for MAC is reduced by 43% in goats.4 These investigators concluded that a 1-MAC isoflurane concentration in the brain actually increased the systemic MAC requirement, which could indicate a supraspinal isoflurane pronociceptive effect. The enhanced antinociceptive action of isoflurane after spinal cord transection is a novel finding, but several other lines of investigation support the hypothesis that a component of isoflurane-induced antinociception is generated intrinsically within the spinal cord neurons by a mechanism that is independent of cortical inhibition or descending spinal pathways. Isoflurane tail clamp MAC was unchanged in rats undergoing forebrain aspiration, indicating that isoflurane anesthetic action is primarily mediated in the midbrain or lower.36 Furthermore, when only the brain of the goat is perfused with isoflurane, the concentration required for MAC is increased 140%, another indication that isoflurane-induced MAC has an intrinsic spinal mechanism.37 In another study, 1 h after a spinal cord freeze lesion, the isoflurane MAC (measured by hind-paw withdrawal after tail clamp) modestly changed from 1.26 to 1.03% atm.38 Isoflurane also concentration-dependently (0.2–1.3%) inhibited the dorsal root evoked slow ventral root response in isolated

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neonatal rat spinal cord, electrophysiologic evidence of an isoflurane-evoked intrinsic spinal mechanism of nociceptive inhibition.39 In summary, the data from this investigation supports the hypothesis that, at clinically relevant concentrations, there are at least three concurrent components whereby nociception is modulated by isoflurane (fig. 6): (1) a noradrenergic neuron-supraspinal ␣1-adrenoceptor–mediated descending pronociceptive mechanism; (2) a noradrenergic neuron-spinal ␣2-adrenoceptor–mediated descending antinociceptive mechanism; and (3) an intrinsic nonadrenergic spinal mechanism of analgesia. Our interpretation of these data are that the ␣1-adrenoceptor–mediated descending pronociceptive effect of isoflurane is much greater than its ␣2-adrenoceptor–mediated descending antinociceptive effect; thus, the combined descending spinal effect is predominantly pronociceptive. Normally, the aggregate descending spinal pronociceptive effect of isoflurane is concealed by the more potent intrinsic spinal cord antinociceptive action of isoflurane, which has no adrenergic basis. Noradrenergic lesioning or spinal cord transection eliminates the aggregate descending spinal pronociceptive effect, thus appearing to enhance the intrinsic spinal isoflurane antinociceptive action. The complexity of these interactions suggests that the mechanisms and sites of anesthetic action will not be easily defined. The authors thank Rekha R. Rapaka, B.S. (Undergraduate Research Student, Biological Sciences, Stanford University, Stanford, CA), and Rebecca K. Berquist, B.A. (Undergraduate Research Student, Human Biology, Stanford University, Stanford, CA), for their invaluable technical assistance; and Lee E. Limbird, Ph.D. (Professor, Department of Pharmacology, Vanderbilt University, Nashville, TN), for providing us with the D79N transgenic mice.

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