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Pain Medicine  |   February 2002
Isoflurane and Nociception: Spinal α2AAdrenoceptors Mediate Antinociception while Supraspinal α1Adrenoceptors Mediate Pronociception
Author Affiliations & Notes
  • 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.
  • Mervyn Maze, M.B., Ch.B., F.R.C.P.
    #
  • * 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, ∥ 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.
Article Information
Pain Medicine
Pain Medicine   |   February 2002
Isoflurane and Nociception: Spinal α2AAdrenoceptors Mediate Antinociception while Supraspinal α1Adrenoceptors Mediate Pronociception
Anesthesiology 2 2002, Vol.96, 367-374. doi:
Anesthesiology 2 2002, Vol.96, 367-374. doi:
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 α2Badrenoceptors 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 α2-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 withdrawal thresholds. 5 We now show that saporin coupled to the antibody directed against dopamine β hydroxylase (DβH-saporin) lesioning of brainstem noradrenergic neurons, spinal cord transection, or supraspinally administered α1-adrenoceptor antagonists all enhanced isoflurane antinociception on the tail-flick assay, possibly by eliminating a noradrenergic-supraspinal α1-adrenoceptor–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 α2adrenoceptors, and such stimulation causes inhibition of nociceptive neurons in the deep dorsal horn. 8–10 We now show that a spinally administered α2-adrenoceptor antagonist inhibited isoflurane antinociception on the tail-flick assay, and this effect was lost after spinal transection. Using null mice for α2A-, α2B-, and α2C-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 α2Aadrenoceptor caused by a point mutation in its gene substituting aspartic acid by asparagine at amino acid residue #79; (2) α2A−/− null mice with a knockout of the α2A-adrenoceptor gene; (3) α2C−/− null mice with a knockout of the α2C-adrenoceptor gene; and (4) their wild-type controls. All of these strains were on a congenic C57BL/6J background. The α2B−/− null mice had a knockout of the α2B-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 α2A−/−, α2B−/−, α2C−/−, and D79N mice have been described previously. 11–13 
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 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.
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 ± 0.2 s, and after 30 min of isoflurane exposure, the mean latency increased to 5.1 ± 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.
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 ± 0.2 s, and after 30 min of isoflurane exposure, the mean latency increased to 5.1 ± 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.
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 ± 0.2 s, and after 30 min of isoflurane exposure, the mean latency increased to 5.1 ± 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.
×
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 β-hydroxylase-saporin (DβH-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 DβH-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 injected with DβH-saporin (3 μg/3 μl) or saline (3 μl) 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-μm-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-μl 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 μl 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-μl 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 isoflurane-induced 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.
The contribution of noradrenergic brainstem neurons to isoflurane-induced antinociception was determined by immunolesioning the noradrenergic neurons. Rats were injected with the immunotoxin DβH-saporin (3 μg/3 μl) 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 α1-adrenoceptor modulation of isoflurane-induced 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 α1-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 spinal-transected 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 μg/10 μl) 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 μg/ 10 μl) or vehicle on tail-flick latency response to isoflurane. Doses of prazosin in the range of 8–30 μg/μl have been previously reported to completely block the effects of endogenous and exogenous α1-adrenoceptor agonists when administered via  intrathecal and intracerebroventricular routes. 6,20,21 
To determine the dependence of isoflurane-induced antinociception on α2adrenoceptors, the α2-adrenoceptor antagonist yohimbine (2 mg/kg for intraperitoneal administration, 30 μg/10 μl for intrathecal and intracerebroventricular administration; Sigma Chemical) or vehicle (0.9% saline) was administered to intact and spinal-transected rats as described above for prazosin. Doses of yohimbine in the range of 8–30 μg/μl have been previously reported to completely block the effects of endogenous and exogenous α2-adrenoceptor agonists when administered via  intrathecal and intracerebroventricular routes. 6,21–24 
To identify the α2-adrenoceptor subtype mediating isoflurane antinociception, tail-flick testing was performed in α2A−/−, α2B−/−, α2C−/−, D79N mice, and in their respective wild-type controls. Baseline latencies 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.  DβH-saporin) t  tests. A repeated-measures 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:
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). Isoflurane-induced 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.
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 = 7 per cohort). The mean baseline tail-flick latencies in air were slightly less in the spinal-transected rats (2.7 ± 0.2 s) than in the sham-operated rats (3.5 ± 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 ± 1.0, 8.3 ± 0.8, and 9.7 ± 0.2 s, respectively; in sham-operated rats, they were 4.0 ± 0.1, 4.3 ± 0.3, and 5.4 ± 0.3 s, respectively). ##P  < 0.01, ###P  < 0.001 versus  sham surgery. (B  ) DβH-saporin lesioning of the bulbospinal noradrenergic neurons enhanced the isoflurane-induced antinociceptive effect on tail-flick assay by 110%versus  vehicle-treated rats (n = 8 per cohort). ##P  < 0.01 versus  vehicle.
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 = 7 per cohort). The mean baseline tail-flick latencies in air were slightly less in the spinal-transected rats (2.7 ± 0.2 s) than in the sham-operated rats (3.5 ± 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 ± 1.0, 8.3 ± 0.8, and 9.7 ± 0.2 s, respectively; in sham-operated rats, they were 4.0 ± 0.1, 4.3 ± 0.3, and 5.4 ± 0.3 s, respectively). ##P 
	< 0.01, ###P 
	< 0.001 versus 
	sham surgery. (B 
	) DβH-saporin lesioning of the bulbospinal noradrenergic neurons enhanced the isoflurane-induced antinociceptive effect on tail-flick assay by 110%versus 
	vehicle-treated rats (n = 8 per cohort). ##P 
	< 0.01 versus 
	vehicle.
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 = 7 per cohort). The mean baseline tail-flick latencies in air were slightly less in the spinal-transected rats (2.7 ± 0.2 s) than in the sham-operated rats (3.5 ± 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 ± 1.0, 8.3 ± 0.8, and 9.7 ± 0.2 s, respectively; in sham-operated rats, they were 4.0 ± 0.1, 4.3 ± 0.3, and 5.4 ± 0.3 s, respectively). ##P  < 0.01, ###P  < 0.001 versus  sham surgery. (B  ) DβH-saporin lesioning of the bulbospinal noradrenergic neurons enhanced the isoflurane-induced antinociceptive effect on tail-flick assay by 110%versus  vehicle-treated rats (n = 8 per cohort). ##P  < 0.01 versus  vehicle.
×
DβH-Saporin Treatment Destroyed Pontine Noradrenergic Neurons and Enhanced the Antinociceptive Effect of Isoflurane
Figure 2Billustrates that the antinociceptive effect of isoflurane (1.2% atm, 30 min) was significantly enhanced in the DβH-saporin–treated rats compared with saline-treated rats. There was no difference in baseline tail-flick latencies between control and immunolesioned rats.
Supraspinal α1Adrenoceptors Mediate Isoflurane-induced Pronociception
Systemic administration of the α1-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 spinal-transected rats (fig. 3B). When prazosin (30 μg/10 μl) was administered intrathecally or intracerebroventricularly in intact rats, it had no antinociceptive effect in air (fig. 3C). Intrathecal prazosin had no effect on isoflurane-induced antinociception, but intracerebroventricular prazosin enhanced isoflurane antinociception by 82% (fig. 3D).
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 = 8 per cohort). (B  ) Prazosin enhanced isoflurane (30-min exposure) antinociception in intact rats (vs.  vehicle, n = 8 per cohort) but had no effect in spinal-transected rats (n = 8 per cohort). (C  ) Compared with vehicle, intrathecal (IT) and intracerebroventricular (ICV) prazosin (30 μg/10 μl) 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 = 8 per cohort). *P  < 0.05, **P  < 0.01, ***P  < 0.001 versus  baseline latency; #P  < 0.05 versus  vehicle.
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 = 8 per cohort). (B 
	) Prazosin enhanced isoflurane (30-min exposure) antinociception in intact rats (vs. 
	vehicle, n = 8 per cohort) but had no effect in spinal-transected rats (n = 8 per cohort). (C 
	) Compared with vehicle, intrathecal (IT) and intracerebroventricular (ICV) prazosin (30 μg/10 μl) 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 = 8 per cohort). *P 
	< 0.05, **P 
	< 0.01, ***P 
	< 0.001 versus 
	baseline latency; #P 
	< 0.05 versus 
	vehicle.
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 = 8 per cohort). (B  ) Prazosin enhanced isoflurane (30-min exposure) antinociception in intact rats (vs.  vehicle, n = 8 per cohort) but had no effect in spinal-transected rats (n = 8 per cohort). (C  ) Compared with vehicle, intrathecal (IT) and intracerebroventricular (ICV) prazosin (30 μg/10 μl) 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 = 8 per cohort). *P  < 0.05, **P  < 0.01, ***P  < 0.001 versus  baseline latency; #P  < 0.05 versus  vehicle.
×
Spinal α2AAdrenoceptors Partially Mediate Isoflurane-induced Antinociception
Systemic administration of the α2-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 μg/10 μl) was administered intrathecally or intracerebroventricularly in intact rats, it had no effect on nociceptive latencies in air (fig. 4C). Figure 4Dillustrates that intrathecal yohimbine reduced isoflurane-induced antinociception by 72% (P  < 0.05), but intracerebroventricular yohimbine did not significantly reduce isoflurane antinociception (P  = 0.16).
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 spinal-transected rats in air. (B  ) Systemically administered yohimbine blocked isoflurane (30-min exposure) antinociception in intact, but not in spinal transected rats (n = 8 per cohort). (C  ) Compared with vehicle, intrathecal (IT) and intracerebroventricular (ICV) yohimbine (30 μg/10 μl) 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 = 8 per cohort). *P  < 0.05, **P  < 0.01, ***P  < 0.001 versus  baseline latency; #P  < 0.05 versus  vehicle.
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 spinal-transected rats in air. (B 
	) Systemically administered yohimbine blocked isoflurane (30-min exposure) antinociception in intact, but not in spinal transected rats (n = 8 per cohort). (C 
	) Compared with vehicle, intrathecal (IT) and intracerebroventricular (ICV) yohimbine (30 μg/10 μl) 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 = 8 per cohort). *P 
	< 0.05, **P 
	< 0.01, ***P 
	< 0.001 versus 
	baseline latency; #P 
	< 0.05 versus 
	vehicle.
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 spinal-transected rats in air. (B  ) Systemically administered yohimbine blocked isoflurane (30-min exposure) antinociception in intact, but not in spinal transected rats (n = 8 per cohort). (C  ) Compared with vehicle, intrathecal (IT) and intracerebroventricular (ICV) yohimbine (30 μg/10 μl) 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 = 8 per cohort). *P  < 0.05, **P  < 0.01, ***P  < 0.001 versus  baseline latency; #P  < 0.05 versus  vehicle.
×
The antinociceptive effects of isoflurane with the tail-flick assay in the α2A−/−, α2B−/−, α2C−/−, D79N mice, and in their respective wild-type controls are shown in figure 5. The isoflurane antinociceptive response was reduced by 54% in the α2A−/− mice and 62% in the D79N mice (vs.  wild-type controls;P  < 0.05), indicating that the α2A-adrenoceptor subtype partially mediates isoflurane antinociception. Although the isoflurane-induced antinociceptive effect in the α2B−/− mice was less than in the corresponding wild-type cohort, this was not significantly different (P  = 0.10).
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 α2A−/−, α2C−/− knockout mice (α2AKO and α2CKO), the D79N mutant mice with nonfunctional α2Aadrenoceptors, and in their genetically matched wild-type (WT) controls (on a C57BL/6J congenic background). The α2B−/− (α2BKO) mice were on a different genetic background (C57BL/6J × 129SvJ hybrid); therefore, they required their own genetically matched wild-type controls (wild-type for α2B). 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 α2Aknockout and the D79N mice had reduced isoflurane antinociception on the tail-flick assay compared with wild-type controls (11 ± 5 in α2Aknockout and 9 ± 3 in D79N vs.  24 ± 4% maximum possible effect in wild-type), indicating that the α2A-adrenoceptor subtype at least partially mediates isoflurane antinociception. The isoflurane antinociceptive responses (% maximum possible effect) in the other knockout and mutant mouse strains were:α2Cknockout, 22 ± 7; wild-type for α2B, 42 ± 7; α2Bknockout, 29 ± 4 (n = 16 for each group). #P  < 0.05 versus  respective wild-type control.
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 α2A−/−, α2C−/− knockout mice (α2AKO and α2CKO), the D79N mutant mice with nonfunctional α2Aadrenoceptors, and in their genetically matched wild-type (WT) controls (on a C57BL/6J congenic background). The α2B−/− (α2BKO) mice were on a different genetic background (C57BL/6J × 129SvJ hybrid); therefore, they required their own genetically matched wild-type controls (wild-type for α2B). 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 α2Aknockout and the D79N mice had reduced isoflurane antinociception on the tail-flick assay compared with wild-type controls (11 ± 5 in α2Aknockout and 9 ± 3 in D79N vs. 
	24 ± 4% maximum possible effect in wild-type), indicating that the α2A-adrenoceptor subtype at least partially mediates isoflurane antinociception. The isoflurane antinociceptive responses (% maximum possible effect) in the other knockout and mutant mouse strains were:α2Cknockout, 22 ± 7; wild-type for α2B, 42 ± 7; α2Bknockout, 29 ± 4 (n = 16 for each group). #P 
	< 0.05 versus 
	respective wild-type control.
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 α2A−/−, α2C−/− knockout mice (α2AKO and α2CKO), the D79N mutant mice with nonfunctional α2Aadrenoceptors, and in their genetically matched wild-type (WT) controls (on a C57BL/6J congenic background). The α2B−/− (α2BKO) mice were on a different genetic background (C57BL/6J × 129SvJ hybrid); therefore, they required their own genetically matched wild-type controls (wild-type for α2B). 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 α2Aknockout and the D79N mice had reduced isoflurane antinociception on the tail-flick assay compared with wild-type controls (11 ± 5 in α2Aknockout and 9 ± 3 in D79N vs.  24 ± 4% maximum possible effect in wild-type), indicating that the α2A-adrenoceptor subtype at least partially mediates isoflurane antinociception. The isoflurane antinociceptive responses (% maximum possible effect) in the other knockout and mutant mouse strains were:α2Cknockout, 22 ± 7; wild-type for α2B, 42 ± 7; α2Bknockout, 29 ± 4 (n = 16 for each group). #P  < 0.05 versus  respective wild-type control.
×
Discussion
In attempting to elucidate the mechanisms for the analgesic effect of anesthetic agents, it is important to obviate any effect that loss of consciousness can exert on the antinociceptive assay. We previously reported dissociation between N2O-evoked antinociception and N2O-induced 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 1illustrates 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 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 tail-flick 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 tail-flick 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 DβH-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 isoflurane (fig. 2B). Furthermore, the systemic administration of the α1-adrenoceptor antagonist prazosin also enhanced the antinociceptive effect of isoflurane, and this effect was lost after spinal transection (fig. 3B), suggesting that activation of α1adrenoceptors evoke the pronociceptive effects of isoflurane via  descending spinal pathways. Pronociceptive actions have been attributed to both supraspinal 6,7 and spinal 7,23,29 α1adrenoceptors 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 α1adrenoceptors 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 α2-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 α2adrenoceptors 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 α2adrenoceptors mediate an isoflurane-evoked descending spinal antinociceptive effect, similar to N2O-evoked analgesia. 2 
Pharmacologic evidence in rats 30,31 and studies using D79N mice with dysfunctional α2Aadrenoceptors 32–34 have established that the α2Asubtype mediates the antinociceptive response to α2-adrenoceptor agonists. Now we observe that mice deficient in the α2A-adrenoceptor subtype or D79N mice with dysfunctional α2Aadrenoceptors showed reduced isoflurane-induced antinociception, indicating that the spinal α2A-adrenoceptor subtype mediates an antinociceptive response to isoflurane.
We described a noradrenergic-supraspinal α1-adrenoceptor pathway that mediates the pronociceptive effect of isoflurane. In addition, we observed a noradrenergic-spinal α2-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 α1-adrenoceptor pronociceptive and α2-adrenoceptor 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 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 α1-adrenoceptor–mediated descending pronociceptive mechanism; (2) a noradrenergic neuron-spinal α2-adrenoceptor–mediated descending antinociceptive mechanism; and (3) an intrinsic nonadrenergic spinal mechanism of analgesia.
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 α1adrenoceptors 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 α2Aadrenoceptors 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.
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 α1adrenoceptors 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 α2Aadrenoceptors 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.
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 α1adrenoceptors 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 α2Aadrenoceptors 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.
×
Our interpretation of these data are that the α1-adrenoceptor–mediated descending pronociceptive effect of isoflurane is much greater than its α2-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.
References
Sawamura S, Kingery WS, Davies MF, Agashe G, Clark JD, Kobilka BK, Hashimoto T, Maze M: Antinociceptive action of nitrous oxide is mediated by stimulation of noradrenergic neurons in the brainstem and activation of alpha2B adrenoceptors. J Neurosci 2000; 20: 9242–51Sawamura, S Kingery, WS Davies, MF Agashe, G Clark, JD Kobilka, BK Hashimoto, T Maze, M
Guo TZ, Poree L, Golden W, Stein W, Fujinaga J, Maze M: Antinociceptive response to nitrous oxide is mediated by supraspinal opiate and spinal alpha 2 adrenergic receptors in the rat. A nesthesiology 1996; 85: 846–52Guo, TZ Poree, L Golden, W Stein, W Fujinaga, J Maze, M
Zhang C, Davies MF, Guo TZ, Maze M: The analgesic action of nitrous oxide is dependent on the release of norepinephrine in the dorsal horn of the spinal cord. A nesthesiology 1999; 91: 1401–7Zhang, C Davies, MF Guo, TZ Maze, M
Borges M, Antognini JF: Does the brain influence somatic responses to noxious stimuli during isoflurane anesthesia? A nesthesiology 1994; 81: 1511–5Borges, M Antognini, JF
Zhang Y, Eger EI, Dutton RC, Sonner JM: Inhaled anesthetics have hyperalgesic effects at 0.1 minimum alveolar anesthetic concentration. Anesth Analg 2000; 91: 462–6Zhang, Y Eger, EI Dutton, RC Sonner, JM
Zhang C, Guo YQ, Qiao J-T, Dafney N: Locus coeruleus modulates thalamic nociceptive reponses via adrenoceptors. Brain Res 1998; 784: 116–22Zhang, C Guo, YQ Qiao, J-T Dafney, N
Hirakawa N, Tershner SA, Fields HL, Manning BH: Bi-directional changes in affective state elicited by manipulation of medullary pain-modulatory ciruitry. Neuroscience 2000; 100: 861–71Hirakawa, N Tershner, SA Fields, HL Manning, BH
Kingery WS, Davies MF, Maze M: Molecular mechanisms for the analgesic properties of alpha-2 adrenergic agonists, Molecular Neurobiology of Pain. Edited by Borsook D. Seattle, IASP Press, 1997, pp 275–304
Byrum CE, Stornetta R, Guyenet PG: Noradrenergic projections to all regions of the spinal cord arise almost entirely from the dorsolateral pontine catecholamine cell groups the locus coeruleus (LC), A5 and A7 that all play a role in antinociception. Brain Res 1984; 303: 15–29Byrum, CE Stornetta, R Guyenet, PG
Willis WD, Westlund KN: Neuroanatomy of the pain system and of the pathways that modulate pain. J Clin Neurophysiol 1997; 14: 2–31Willis, WD Westlund, KN
Altman JD, Trendelenburg AU, MacMillan L, Bernstein D, Limbird L, Starke K, Kobilka BK, Hein L: Abnormal regulation of the sympathetic nervous system in alpha2A-adrenergic receptor knockout mice. Mol Pharmacol 1999; 56: 154–61Altman, JD Trendelenburg, AU MacMillan, L Bernstein, D Limbird, L Starke, K Kobilka, BK Hein, L
Link RE, Desai K, Hein L, Stevens ME, Chruscinski A, Bernstein D, Barsh GS, Kobilka BK: Cardiovascular regulation in mice lacking alpha 2-adrenergic receptor subtypes B and C. Science 1996; 273: 803–5Link, RE Desai, K Hein, L Stevens, ME Chruscinski, A Bernstein, D Barsh, GS Kobilka, BK
MacMillan LB, Hein L, Smith MS, Piascik MT, Limbird LE: Central hypotensive effects of the alpha2A-adrenergic receptor subtype. Science 1996; 273: 801–3MacMillan, LB Hein, L Smith, MS Piascik, MT Limbird, LE
Stevens VC, Dolan WM, Gibbons RT, White A, Eger EI, Miller RD, DeJong RH, Elashoff RM: Minimum alveolar concentrations (MAC) of isoflurane with and without nitrous oxide in patients of various ages. A nesthesiology 1975; 42: 197–200Stevens, VC Dolan, WM Gibbons, RT White, A Eger, EI Miller, RD DeJong, RH Elashoff, RM
Vitez TS, White PF, Eger EI: Effects of hypothermia on halothane MAC and isoflurane MAC in the rat. A nesthesiology 1974; 41: 80–1Vitez, TS White, PF Eger, EI
Martin WJ, Gupta NK, Loo CM, Rohde DS, Basbaum AI: Differential effects of neurotoxic destruction of descending noradrenergic pathways on acute and perisistent nociceptive processing. Pain 1999; 80: 57–65Martin, WJ Gupta, NK Loo, CM Rohde, DS Basbaum, AI
Wrenn CC, Picklo MJ, Lappi DA, Robertson D, Wiley RG: Central noradrenergic lesioning using anti-DBH-saporin: Anatomical findings. Brain Res 1996; 740: 175–84Wrenn, CC Picklo, MJ Lappi, DA Robertson, D Wiley, RG
Mestre C, Pelissier T, Fialip J, Wilcox G, Eschalier A: A method to perform direct transcutaneous intrathecal injection in rats. J Pharmacol Toxicol Meth 1994; 32: 197–200Mestre, C Pelissier, T Fialip, J Wilcox, G Eschalier, A
Guo TZ, Tinklenberg J, Oliker R, Maze M: Central alpha 1-adrenoceptor stimulation functionally antagonizes the hypnotic response to dexmedetomidine, an alpha 2-adrenoceptor agonist. A nesthesiology 1991; 75: 252–6Guo, TZ Tinklenberg, J Oliker, R Maze, M
Fang F, Proudfit HK: Antinociception produced by microinjection of morphine in the rat periaqueductal gray is enhanced in the foot, but not the tail, by intrathecal injection of alpha1-adrenoceptor antagonists. Brain Res 1998; 790: 14–24Fang, F Proudfit, HK
Howe JR, Wang JY, Yaksh TL: Selective antagonism of the antinociceptive effect of intrathecally applied alpha adrenergic agonists by intrathecal prazosin and intrathecal yohimbine. J Pharmacol Exp Ther 1983; 224: 552–8Howe, JR Wang, JY Yaksh, TL
Solomon RE, Brody MJ, Gebhart GF: Pharmacological characterization of alpha adrenoceptors involved in the antinociceptive and cardiovascular effects of intrathecally administered clonidine. J Pharmacol Exp Ther 1989; 251: 27–38Solomon, RE Brody, MJ Gebhart, GF
Nuseir K, Proudfit HK: Bidirectional modulation of nociception by gaba neurons in the dorsolateral pontine tegmentum that tonically inhibit spinally projecting noradrenergic A7 neurons. Neuroscience 2000; 96: 773–83Nuseir, K Proudfit, HK
Gyires K, Ronai AZ, Mullner K, Furst S: Intracerebroventricular injection of clonidine releases beta-endorphin to induce mucosal protection in the rat. Neuropharmacology 2000; 39: 961–8Gyires, K Ronai, AZ Mullner, K Furst, S
Deady JE, Koblin DD, Eger EI, Heavner JE, D'Aoust B: Anesthetic potencies and the unitary theory of narcosis. Anesth Analg 1981; 60: 380–4Deady, JE Koblin, DD Eger, EI Heavner, JE D'Aoust, B
Russell GB, Schwentker MC, Graybeal JM: Preservation of neurogenic motor-evoked potentials during isoflurane electroencephalographic burst suppression in rats. Spine 1994; 23: 2632–6Russell, GB Schwentker, MC Graybeal, JM
King BS, Rampil IJ: Anesthetic depression of spinal motor neurons may contribute to lack of movement in response to noxious stimuli. A nesthesiology 1994; 81: 1484–92King, BS Rampil, IJ
Haghighi SS: Influence of isoflurane anesthesia on motor evoked potentials elicited by transcortical, brainstem, and spinal root stimulation. Neurol Res 1998; 20: 555–8Haghighi, SS
Holden JE, Schwartz EJ, Proudfit HK: Microinjection of morphine in the A7 catecholamine cell group produces opposing effects on nociception that are mediate by alpha1- and alpha2-adrenoceptors. Neuroscience 1999; 91: 979–90Holden, JE Schwartz, EJ Proudfit, HK
Millan MJ: Evidence that an alpha2A-adrenoceptor subtype mediates antinociception in mice. Eur J Pharmacol 1992; 215: 355–6Millan, MJ
Millan MJ, Bervoets K, Rivet J-M, Widdowson P, Renouard A, LeMarouille-Girardon S, Gobert A: Multiple alpha-2 adrenergic receptor subtypes. II. Evidence for a role of rat alpha-2A adrenergic receptors in the control of nociception, motor behavior and hippocampal synthesis of noradrenaline. J Pharmacol Exp Ther 1994; 270: 958–72Millan, MJ Bervoets, K Rivet, J-M Widdowson, P Renouard, A LeMarouille-Girardon, S Gobert, A
Lakhlani PP, MacMillan LB, Guo TZ, McCool BA, Lovinger DM, Maze M, Limbird LE: Substitution of a mutant alpha 2A-adrenergic receptor via “hit and run” gene targeting reveals the role of this subtype in sedative, analgesic, and anesthetic-sparing responses in vivo. Proc Natl Acad Sci U S A 1997; 94: 9950–5Lakhlani, PP MacMillan, LB Guo, TZ McCool, BA Lovinger, DM Maze, M Limbird, LE
Hunter JC, Fontana DJ, Hedley LR, Jasper JR, Lewis R, Link RE, Secchi R, Sutton J, Eglen RM: Assessment of the role of alpha2-adrenoceptor subtypes in the antinociceptive, sedative and hypothermic actions of dexmedetomidine in transgenic mice. Br J Pharmacol 1997; 122: 1339–44Hunter, JC Fontana, DJ Hedley, LR Jasper, JR Lewis, R Link, RE Secchi, R Sutton, J Eglen, RM
Stone LS, MacMillan LB, Kitto KF, Limbird LE, Wilcox GL: The alpha2A adrenergic receptor subtype mediates spinal analgesia evoked by alpha2 agonists and is necessary for spinal adrenergic-opioid synergy. J Neurosci 1997; 17: 7157–65Stone, LS MacMillan, LB Kitto, KF Limbird, LE Wilcox, GL
Budai D, Harasawa I, Fields HL: Midbrain periaqueductal gray (PAG) inhibits nociceptive inputs to sacral dorsal horn nociceptive neurons through alpha2-adrenergic receptors. J Neurophysiol 1998; 80: 2244–54Budai, D Harasawa, I Fields, HL
Rampil IJ, Mason P, Singh H: Anesthetic potency (MAC) is independent of forebrain structures in the rat. A nesthesiology 1993; 78: 707–12Rampil, IJ Mason, P Singh, H
Antognini JF, Schwartz K: Exaggerated anesthetic requirements in the preferentially anesthetized brain. A nesthesiology 1993; 79: 1244–9Antognini, JF Schwartz, K
Rampil IJ: Anesthetic potency is not altered after hypothermic spinal cord transection in rats. A nesthesiology 1994; 80: 606–10Rampil, IJ
Savola MKT, Woodley SJ, Maze M, Kendig JJ: Isoflurane and an alpha2-adrenoceptor agonist suppress nociceptive neurotransmission in neonatal rat spinal cord. A nesthesiology 1991; 75: 489–98Savola, MKT Woodley, SJ Maze, M Kendig, JJ
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 ± 0.2 s, and after 30 min of isoflurane exposure, the mean latency increased to 5.1 ± 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.
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 ± 0.2 s, and after 30 min of isoflurane exposure, the mean latency increased to 5.1 ± 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.
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 ± 0.2 s, and after 30 min of isoflurane exposure, the mean latency increased to 5.1 ± 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.
×
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 = 7 per cohort). The mean baseline tail-flick latencies in air were slightly less in the spinal-transected rats (2.7 ± 0.2 s) than in the sham-operated rats (3.5 ± 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 ± 1.0, 8.3 ± 0.8, and 9.7 ± 0.2 s, respectively; in sham-operated rats, they were 4.0 ± 0.1, 4.3 ± 0.3, and 5.4 ± 0.3 s, respectively). ##P  < 0.01, ###P  < 0.001 versus  sham surgery. (B  ) DβH-saporin lesioning of the bulbospinal noradrenergic neurons enhanced the isoflurane-induced antinociceptive effect on tail-flick assay by 110%versus  vehicle-treated rats (n = 8 per cohort). ##P  < 0.01 versus  vehicle.
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 = 7 per cohort). The mean baseline tail-flick latencies in air were slightly less in the spinal-transected rats (2.7 ± 0.2 s) than in the sham-operated rats (3.5 ± 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 ± 1.0, 8.3 ± 0.8, and 9.7 ± 0.2 s, respectively; in sham-operated rats, they were 4.0 ± 0.1, 4.3 ± 0.3, and 5.4 ± 0.3 s, respectively). ##P 
	< 0.01, ###P 
	< 0.001 versus 
	sham surgery. (B 
	) DβH-saporin lesioning of the bulbospinal noradrenergic neurons enhanced the isoflurane-induced antinociceptive effect on tail-flick assay by 110%versus 
	vehicle-treated rats (n = 8 per cohort). ##P 
	< 0.01 versus 
	vehicle.
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 = 7 per cohort). The mean baseline tail-flick latencies in air were slightly less in the spinal-transected rats (2.7 ± 0.2 s) than in the sham-operated rats (3.5 ± 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 ± 1.0, 8.3 ± 0.8, and 9.7 ± 0.2 s, respectively; in sham-operated rats, they were 4.0 ± 0.1, 4.3 ± 0.3, and 5.4 ± 0.3 s, respectively). ##P  < 0.01, ###P  < 0.001 versus  sham surgery. (B  ) DβH-saporin lesioning of the bulbospinal noradrenergic neurons enhanced the isoflurane-induced antinociceptive effect on tail-flick assay by 110%versus  vehicle-treated rats (n = 8 per cohort). ##P  < 0.01 versus  vehicle.
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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 = 8 per cohort). (B  ) Prazosin enhanced isoflurane (30-min exposure) antinociception in intact rats (vs.  vehicle, n = 8 per cohort) but had no effect in spinal-transected rats (n = 8 per cohort). (C  ) Compared with vehicle, intrathecal (IT) and intracerebroventricular (ICV) prazosin (30 μg/10 μl) 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 = 8 per cohort). *P  < 0.05, **P  < 0.01, ***P  < 0.001 versus  baseline latency; #P  < 0.05 versus  vehicle.
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 = 8 per cohort). (B 
	) Prazosin enhanced isoflurane (30-min exposure) antinociception in intact rats (vs. 
	vehicle, n = 8 per cohort) but had no effect in spinal-transected rats (n = 8 per cohort). (C 
	) Compared with vehicle, intrathecal (IT) and intracerebroventricular (ICV) prazosin (30 μg/10 μl) 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 = 8 per cohort). *P 
	< 0.05, **P 
	< 0.01, ***P 
	< 0.001 versus 
	baseline latency; #P 
	< 0.05 versus 
	vehicle.
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 = 8 per cohort). (B  ) Prazosin enhanced isoflurane (30-min exposure) antinociception in intact rats (vs.  vehicle, n = 8 per cohort) but had no effect in spinal-transected rats (n = 8 per cohort). (C  ) Compared with vehicle, intrathecal (IT) and intracerebroventricular (ICV) prazosin (30 μg/10 μl) 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 = 8 per cohort). *P  < 0.05, **P  < 0.01, ***P  < 0.001 versus  baseline latency; #P  < 0.05 versus  vehicle.
×
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 spinal-transected rats in air. (B  ) Systemically administered yohimbine blocked isoflurane (30-min exposure) antinociception in intact, but not in spinal transected rats (n = 8 per cohort). (C  ) Compared with vehicle, intrathecal (IT) and intracerebroventricular (ICV) yohimbine (30 μg/10 μl) 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 = 8 per cohort). *P  < 0.05, **P  < 0.01, ***P  < 0.001 versus  baseline latency; #P  < 0.05 versus  vehicle.
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 spinal-transected rats in air. (B 
	) Systemically administered yohimbine blocked isoflurane (30-min exposure) antinociception in intact, but not in spinal transected rats (n = 8 per cohort). (C 
	) Compared with vehicle, intrathecal (IT) and intracerebroventricular (ICV) yohimbine (30 μg/10 μl) 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 = 8 per cohort). *P 
	< 0.05, **P 
	< 0.01, ***P 
	< 0.001 versus 
	baseline latency; #P 
	< 0.05 versus 
	vehicle.
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 spinal-transected rats in air. (B  ) Systemically administered yohimbine blocked isoflurane (30-min exposure) antinociception in intact, but not in spinal transected rats (n = 8 per cohort). (C  ) Compared with vehicle, intrathecal (IT) and intracerebroventricular (ICV) yohimbine (30 μg/10 μl) 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 = 8 per cohort). *P  < 0.05, **P  < 0.01, ***P  < 0.001 versus  baseline latency; #P  < 0.05 versus  vehicle.
×
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 α2A−/−, α2C−/− knockout mice (α2AKO and α2CKO), the D79N mutant mice with nonfunctional α2Aadrenoceptors, and in their genetically matched wild-type (WT) controls (on a C57BL/6J congenic background). The α2B−/− (α2BKO) mice were on a different genetic background (C57BL/6J × 129SvJ hybrid); therefore, they required their own genetically matched wild-type controls (wild-type for α2B). 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 α2Aknockout and the D79N mice had reduced isoflurane antinociception on the tail-flick assay compared with wild-type controls (11 ± 5 in α2Aknockout and 9 ± 3 in D79N vs.  24 ± 4% maximum possible effect in wild-type), indicating that the α2A-adrenoceptor subtype at least partially mediates isoflurane antinociception. The isoflurane antinociceptive responses (% maximum possible effect) in the other knockout and mutant mouse strains were:α2Cknockout, 22 ± 7; wild-type for α2B, 42 ± 7; α2Bknockout, 29 ± 4 (n = 16 for each group). #P  < 0.05 versus  respective wild-type control.
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 α2A−/−, α2C−/− knockout mice (α2AKO and α2CKO), the D79N mutant mice with nonfunctional α2Aadrenoceptors, and in their genetically matched wild-type (WT) controls (on a C57BL/6J congenic background). The α2B−/− (α2BKO) mice were on a different genetic background (C57BL/6J × 129SvJ hybrid); therefore, they required their own genetically matched wild-type controls (wild-type for α2B). 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 α2Aknockout and the D79N mice had reduced isoflurane antinociception on the tail-flick assay compared with wild-type controls (11 ± 5 in α2Aknockout and 9 ± 3 in D79N vs. 
	24 ± 4% maximum possible effect in wild-type), indicating that the α2A-adrenoceptor subtype at least partially mediates isoflurane antinociception. The isoflurane antinociceptive responses (% maximum possible effect) in the other knockout and mutant mouse strains were:α2Cknockout, 22 ± 7; wild-type for α2B, 42 ± 7; α2Bknockout, 29 ± 4 (n = 16 for each group). #P 
	< 0.05 versus 
	respective wild-type control.
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 α2A−/−, α2C−/− knockout mice (α2AKO and α2CKO), the D79N mutant mice with nonfunctional α2Aadrenoceptors, and in their genetically matched wild-type (WT) controls (on a C57BL/6J congenic background). The α2B−/− (α2BKO) mice were on a different genetic background (C57BL/6J × 129SvJ hybrid); therefore, they required their own genetically matched wild-type controls (wild-type for α2B). 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 α2Aknockout and the D79N mice had reduced isoflurane antinociception on the tail-flick assay compared with wild-type controls (11 ± 5 in α2Aknockout and 9 ± 3 in D79N vs.  24 ± 4% maximum possible effect in wild-type), indicating that the α2A-adrenoceptor subtype at least partially mediates isoflurane antinociception. The isoflurane antinociceptive responses (% maximum possible effect) in the other knockout and mutant mouse strains were:α2Cknockout, 22 ± 7; wild-type for α2B, 42 ± 7; α2Bknockout, 29 ± 4 (n = 16 for each group). #P  < 0.05 versus  respective wild-type control.
×
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 α1adrenoceptors 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 α2Aadrenoceptors 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.
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 α1adrenoceptors 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 α2Aadrenoceptors 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.
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 α1adrenoceptors 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 α2Aadrenoceptors 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.
×