Studies show that the sedative and analgesic effects of alpha2 adrenergic agonists decrease over time, which is a form of synaptic plasticity referred to as tolerance. Because both the N-methyl-D-aspartate (NMDA) receptor complex and nitric oxide synthase are pivotal for some forms of synaptic plasticity, their role in tolerance to the hypnotic and analgesic effects of alpha2 agonists was investigated.
After institutional approval, rats were made tolerant to the hypnotic or analgesic action of an alpha2 agonist, dexmedetomidine. The hypnotic response to dexmedetomidine was assessed by the duration of loss of righting reflex, and the analgesic response to dexmedetomidine was assessed by the tail-flick assay. In separate cohorts, either the NMDA receptors or nitric oxide synthase was antagonized by coadministration of MK-801, ketamine, or NO2-arginine, respectively, during induction of tolerance. In a separate series of experiments, after tolerance was induced, the hypnotic and analgesic responses to dexmedetomidine were assessed in the presence of acutely administered MK-801 or NO2-arginine.
Induction of tolerance to the hypnotic effect of dexmedetomidine is blocked by coadministration of MK-801, ketamine, and NO2-arginine. However, after tolerance developed, acute administration of MK-801, ketamine, or NO2-arginine did not prevent the expression of tolerance. Coadministration of MK-801 or NO2-arginine neither prevents the development nor reverses the expression of tolerance to the analgesic action of dexmedetomidine.
The underlying processes responsible for the development of tolerance to the hypnotic and analgesic actions of systemically administered alpha2 agonists were different, with only the sedative tolerance involving the NMDA receptor and nitric oxide synthase system.
IN the perioperative period, α2adrenergic agonists are efficacious for anxiolysis, 1preoperative sedation, 2and decreasing anesthetic requirements for volatile, 3opioid, 4and hypnotic agents. 5Both systemically and neuraxially administered α2agonists, such as clonidine and dexmedetomidine, alleviate pain in humans and in animal models. The α2agonists produce analgesia by a supraspinal 6as well as by a local spinal action. 7Unlike local anesthetics, α2agonists do not profoundly change motor or sensory function, and unlike opioids, they do not produce respiratory depression 8or induce drug-seeking behavior (i.e. , addiction). Because of these features, α2adrenergic agonists are attractive candidates for pain management and are effective for the reduction of postoperative pain 9and for pain relief during and after childbirth. 10,11
The potential of prolonged treatment with α2agonists for chronic pain states has undergone limited testing 12but seems to be very promising. 13Clinical studies addressing the duration of the analgesic effects of epidural administration of α2agonists after prolonged administration have yet to be performed, although α2agonists are now being advocated for prolonged periods of administration 14because of their potential benefit at each stage in the surgical patient’s perioperative care. However, biologically important adaptations to the immediate effects of α2agonists may lead to decreased drug effect over time; this is generally termed tolerance. Although tolerance to the sedative actions of clonidine rapidly develops and is considered desirable in the treatment of hypertension, it may lessen the effectiveness of α2agonists for chronic pain relief and prolonged sedation in the intensive care unit. 15Tolerance to the analgesic action of spinally administered α2agonists may be minimal because prolonged epidural administration of clonidine produced clinically useful analgesia for the treatment of chronic pain throughout the course of treatment. 10
Alterations in biologic responsiveness in the central nervous system—for example, long-term potentiation, central sensitization (“wind-up”), and tolerance—are referred to collectively as synaptic plasticity, and their molecular mechanisms may be similar despite diverse provocative settings. Previous studies showed that both the N -methyl-d-aspartate (NMDA) receptor complex 16and nitric oxide synthase (NOS) 17–19are pivotal for some forms of synaptic plasticity. Many studies have also shown that tolerance to opioids can be prevented by blocking the actions of the NMDA receptor 20–23and NOS. 24,25Because the second messenger systems activated by opioid and α2receptors are very similar, 26,27tolerance to α2adrenergic agonists may also be sensitive to the blockade of these same sites. From previous studies, we were also aware that it required less exposure to α2agonists to achieve hypnotic tolerance than to achieve analgesic tolerance, 28suggesting that these two forms of tolerance had different biologic substrates. To test for the involvement of the NMDA receptor and NOS in the mechanism of tolerance to the hypnotic and analgesic effects of α2adrenergic agonists, we undertook a series of studies to determine whether antagonism of these functional proteins could prevent the induction or the expression of this tolerance.
The experimental protocol was approved by the Animal Care and Use Committee of the Veterans Affairs Palo Alto Health Care System, Palo Alto, California. Male Sprague-Dawley rats (B&K, Fremont, CA) weighing 250–350 g were used. The rats were stratified to match the distribution of the weights in the groups as closely as possible. All tests were performed between 10 am and 6 pm. The number of animals for each experiment is listed in the figure legends.
Development of Tolerance
Rats were made tolerant to the anesthetic action of an α2agonist, dexmedetomidine, as previously described. 29Briefly, dexmedetomidine was chronically administered to rats using Alzet®osmotic minipumps (model 2002 or 1007D; Alza, Palo Alto CA), which discharge their contents at a mean pumping rate of 0.48 ± 0.02 μl/h. The pumps were inserted subcutaneously during isoflurane anesthesia in the dorsal thoracic region and loaded to administer 5 μg · kg−1· h−1for 7 days to induce hypnotic tolerance and 10 μg · kg−1· h−1for 14 days to induce analgesic tolerance. These dosing schedules were previously found to be optimal to generate a tolerant hypnotic or analgesic state. 28When MK-801 or Nω -nitro-l-arginine (NO2-arginine) was coadministered with dexmedetomidine, these compounds were included in the same pump. Previous studies 28have reported that the behavioral responses measured in sham surgery animals did not differ from rats implanted with pumps containing saline; therefore, the former were used. The appropriateness of substituting sham surgery for saline-filled pumps was also confirmed for analgesia studies. For the MK-801 and NO2-arginine hypnotic experiments, the pumps were removed 1 day before behavioral testing. This was to ensure that the effects observed were caused by the challenge dose of dexmedetomidine. Preliminary experiments found that removing the pumps did not qualitatively alter the results. In all other experiments, the pumps were not removed before testing.
Loss of Righting Reflex
Hypnotic response to dexmedetomidine, was defined by the loss of the rat’s righting reflex, and its duration was measured in minutes and referred to as sleep time. The duration of the loss of righting reflex was assessed as the time from the rat’s inability to right itself when placed on its back until the time when it spontaneously reverted completely to the prone position. The hypnotic response test was performed between 10 am and 6 pm as described previously. 29
Nociceptive Testing Procedures
Nociceptive response was assessed by the tail-flick response to a noxious thermal stimulus 40 min after administration of the dexmedetomidine challenge dose. A high-intensity light beam was focused on the tail, and the time for the rat to move its tail out of the light was recorded as tail-flick latency. This method has been described in the previous report. 6The latencies from three sites on the tail were averaged. A cut-off time of 10 s was predetermined to prevent tissue damage. Baseline measurements consisted of a set of three tail-flick determinations at 2-min intervals. Baseline tail-flick latencies ranged between 3 and 4 s.
The NOS inhibitor NO2-arginine (Sigma, St. Louis, MO) and the NMDA antagonists MK-801 (RBI, Natick, MA) and ketamine (Sigma) were diluted in normal saline and were acutely administered intraperitoneally or chronically by means of Alzet®osmotic minipumps (model 2002 or 1007D).
To minimize the number of pumps implanted into a rat, it was desirable to determine whether two drugs could be effectively administered in one pump. To test whether the concentrated solutions of MK-801 and NO2-arginine had an effect on the chemical stability of dexmedetomidine, MK-801 (9.6 mg/ml) or NO2-arginine (5 mg/ml) were mixed with dexmedetomidine (6 mg/ml) and incubated at 37°C for 14 days. These solutions were then diluted 40-fold to achieve a 150-μg/kg dose of dexmedetomidine, and these were injected into drug-naïve rats. The duration of loss of righting reflex was measured.
Loss of righting reflex and tail-flick data were analyzed using analysis of variance followed by post hoc Bonferroni tests or the Dunnett multiple comparison test or t test when appropriate.
Dose–Response Relation for Dexmedetomidine in Sham-treated and Chronically Treated Animals
The dose–response relations for the hypnotic and analgesic effect of dexmedetomidine are shown in figure 1. The hypnotic effect of dexmedetomidine dose dependently increased in sham-treated animals but was almost completely absent in rats treated for 7 days, even when large doses were administered (fig. 1A). A biphasic dose–response curve for this action of dexmedetomidine, with a maximal efficacy of approximately 300 μg/kg, has been previously shown and has been shown to be caused by a stimulatory action of dexmedetomidine mediated by activation of α1receptors. 30Exposure to dexmedetomidine for 14 days shifted the analgesic dose–response curve for dexmedetomidine approximately twofold and reduced the maximal effect (fig. 1B).
Prolonged Exposure of a Dexmedetomidine Solution to MK-801 or NO2-arginine Did Not Decrease Its Effectiveness in Causing Loss of Righting Reflex
Premixing of dexmedetomidine with MK-801 or NO2-arginine followed by incubation at 37°C for 14 days to simulate the situation of these drugs being coadministered by one pump did not decrease the effectiveness of dexmedetomidine to cause sleep (fig. 2). This shows that dexmedetomidine remains chemically intact after prolonged exposure to these agents and documents the validity of using a single pump to deliver two drugs in further experiments.
Prevention of the Induction of Tolerance to the Hypnotic Effects of Dexmedetomidine with the NMDA Receptor Antagonists MK-801 and Ketamine
Acute administration of MK-801 to drug-naïve animals did not affect sleep time (fig. 3A). When tolerance had developed, acute administration of MK-801 did not affect the expression of tolerance (fig. 3B). Chronic administration of MK-801 had no effect on the sleep time induced by acute administration of dexmedetomidine (fig. 3C), but coadministration of MK-801 with dexmedetomidine was able to prevent the development of tolerance (fig. 3D). In this experiment, the osmotic pumps were removed 1 day before behavioral testing.
Ketamine had a similar profile in that when acutely administered, it did not affect the sleep time caused by acute injection of dexmedetomidine (fig. 4A). Acute administration of 10 or 20 mg/kg ketamine could not reverse tolerance that was previously established (fig. 4B), but ketamine reversed tolerance to the sedative actions of dexmedetomidine when administered concurrently (fig. 4C). This same dose of ketamine had no effect by itself. In this experiment, the Alzet pumps were left in place for behavioral testing.
Prevention of the Induction of Tolerance to the Hypnotic Effects of Dexmedetomidine with an NO Synthase Inhibitor
When administered to drug-naïve animals, NO2-arginine increased sleep time only at high doses (fig. 5A). When tolerance had developed by 7-day administration of dexmedetomidine, acute administration of low doses of NO2-arginine that did not affect sleep time in drug-naïve animals did not reverse its expression (fig. 5B). When NO2-arginine was coadministered with dexmedetomidine, the induction of tolerance to the hypnotic effects of dexmedetomidine was attenuated (fig. 5C). Treatment with 1.25 μg · kg−1· h−1NO2-arginine only (last column) did not affect dexmedetomidine sleep time.
Lack of Prevention of the Induction of Tolerance to the Analgesic Effects of Dexmedetomidine with MK-801
Acute administration of MK-801 15 min before the challenge dose of dexmedetomidine suppressed its analgesic action at the two highest doses (fig. 6A). To determine whether MK-801 could affect the expression of tolerance, it was acutely administered to control and tolerant animals. A low dose of MK-801 that alone did not have any affect on dexmedetomidine-induced analgesia did not reverse the expression of tolerance (fig. 6B). A higher dose of MK-801 (400 μg/kg) that antagonized the analgesic action of dexmedetomidine in control animals was also unable to reverse tolerance. Coadministration of a dose of MK-801 that prevented the development of tolerance to the hypnotic effect of dexmedetomidine and was the maximum dose tolerated by the rat had no effect on the development of analgesic tolerance (fig. 6C). This dose of MK-801 had no effect on tail-flick latency when administered alone (data not shown).
Lack of Prevention of the Induction of Tolerance to the Analgesic Effects of Dexmedetomidine with NO2-arginine
Acute administration of NO2-arginine did not affect the analgesic action of dexmedetomidine (50 μg/kg intraperitoneally;fig. 7A). To determine whether NO2-arginine could affect the expression of tolerance, it was acutely administered to control and tolerant animals. NO2-arginine (1 and 20 mg/kg intraperitoneally) did not reverse the expression of tolerance (fig. 7B). Coadministration of a dose of 4 μg · kg−1· h−1NO2-arginine, a dose that prevented the development of tolerance to the hypnotic effect of dexmedetomidine, had no effect on dexmedetomidine-induced analgesia in control animals and had no effect on analgesic tolerance (fig. 7C). This dose of NO2-arginine had no effect on tail-flick latency when administered alone (data not shown). Increasing the dose of NO2-arginine further to 8 μg · kg−1· h−1was also ineffective in reversing tolerance.
This study shows that although NMDA receptors and NOS have a role in the development of tolerance to the hypnotic effect of dexmedetomidine, they do not seem to play a significant role in the development of analgesic tolerance. Neither NMDA receptor nor NOS inhibitors can affect the expression of hypnotic and analgesic tolerance. This indicates that there are likely multiple mechanisms by which behavioral tolerance to α2agonists is achieved. We have previously shown that acute administration of the L-type Ca2+channel antagonist nifedipine normalized the hypnotic response to dexmedetomidine in the α2-tolerant rats, but coadministration was ineffective in preventing the induction of this tolerance. 31
Our previous work has elucidated key sites for the intracellular signaling of the hypnotic and analgesic action of α2agonists. The locus ceruleus (LC) seems to be a pivotal site for producing α2-agonist–induced hypnosis. 32However, the analgesic effects of α2agonists are mediated spinally, supraspinally, and peripherally. 6,33,34The analgesic action of dexmedetomidine injected directly into the LC results in activation of α2adrenoceptors in the spinal cord because this analgesia can be blocked by intrathecal injection of the α2antagonist atipamezole and also by intrathecal administration of pertussis toxin, 6which ribosylates and thereby inactivates defined species of G proteins. Intrathecal administration of α2agonists, such as clonidine 35,36or dexmedetomidine, 6,37also produce analgesia. These data suggest that spinal α2adrenoceptors are a final common pathway in antinociception.
A possible reason for the differences in the development of tolerance for the two behavioral responses may be the fact that the anatomic sites at which the responses are transduced are not the same. Biochemical changes associated with the hypnotic tolerant state have been found in the LC. Our previous studies of the LC 38showed that tolerance to the hypnotic response was associated with changes in α2adrenoceptor affinity, ribosylation of guanine nucleotide regulatory proteins (G proteins) ex vivo , and a decreased sensitivity of forskolin-stimulated adenylyl cyclase to inhibition by dexmedetomidine. Although we assume that tolerance is dependent on these changes, they have not been established definitively. The fact that compared with hypnotic tolerance, analgesic tolerance is achieved only with extended administration of high doses of dexmedetomidine and has different pharmacology may be because of the multiple sites of analgesic action. Therefore, although supraspinally mediated processes may be attenuated, the signaling may still be intact at analgesic sites in the spinal cord and periphery.
We have characterized some of the biochemical changes associated with tolerance in the LC, but we have not yet characterized what transpires in the spinal cord, the final common pathway for analgesic mechanism. Profound tolerance to the analgesic action of intrathecally administered dexmedetomidine has been shown, 39indicating that changes in spinal cord play a role in analgesic tolerance. A full understanding of the process underlying α2analgesic tolerance will depend on a more detailed study of the biochemical processes involved.
The α2adrenoceptor system is thought to have many similarities with the opioid receptor system based on their similarities in physiologic actions in the LC 40,41and spinal cord 42,43and in their actions on nociceptive processing in the spinal cord. 44,45Experimental findings from models of opioid tolerance were initially hypothesized to be directly relevant to the situation with the α2adrenoceptor. However, in contrast to α2analgesic tolerance, development of opioid analgesic tolerance is sensitive to NMDA receptor antagonists 20–23and NOS inhibitors. 24,25,46Opioid tolerance develops, at least in part, at the spinal level because spinal administration of morphine is reversed by coadministration of MK-801 47,48and with the presumably selective neuronal NOS inhibitor, 7-nitroindazole. 48The lack of effect of NMDA and NOS inhibitors in α2analgesic tolerance indicates that the mechanisms underlying α2and opioid tolerance are likely different. This could explain why in some studies, no analgesic cross-tolerance between opioid and α2agonists was found, 49,50although cross-tolerance for opioid and α2agonists has been observed for peripheral antinociception. 51Others have found evidence that even in a cell culture system in which both opioids and α2agonists inhibit cAMP production, opioid and α2tolerance do not share common pathways. 27Therefore, even two drug classes apparently sharing a common second messenger system may have different mechanisms of tolerance.
Data from these studies with the pharmacologic probes (MK-801, ketamine, and NO2-arginine) strongly suggest that tolerance has at least two distinct phases (induction and expression) as is seen in other forms of synaptic plasticity (e.g. , long-term potentiation), and that the components of the signaling pathway follow in an orderly temporal sequence. The fact that the efficacy of α2agonists may decrease over time and be differentially affected by coadministration of other classes of drugs may also have clinical ramifications for intensive care unit sedation.
The authors thank the Medical Research Council, London, United Kingdom, and the National Institutes of Health, Bethesda, Maryland.