Systemic ketamine causes anesthesia and profound analgesia in humans and animals (1,2). Although ketamine interacts with multiple binding sites (N-methyl-d-aspartate [NMDA] and non-NMDA glutamate receptors, nicotinic and muscarinic cholinergic receptors, and adrenergic and opioid receptors), it is generally believed that NMDA receptor antagonism accounts for most of its anesthetic and part of its analgesic effects (2), whereas some of ketamine’s analgesic effects are mediated through its agonistic effect at opioid receptors within the central nervous system (CNS) (3,4). Opioid and NMDA receptors and their endogenous ligands are found in large concentrations in areas of the CNS involved in the control of breathing (5,6). Recent studies have indicated the importance of both receptor systems in the central formation of breathing activity and respiratory plasticity. The endogenous opioid system has a modulatory role in respiratory rhythmogenesis (7,8), and the administration of exogenous opioids results in respiratory depression (8–11). NMDA receptors play a role in central CO2 chemoreception (12) and respiratory rhythmogenesis (13), are involved in central processing of afferent input from the carotid bodies (6), and play a role in short-term potentiation of breathing (14).
Few studies have assessed the involvement and contribution of the μ-opioid receptor (MOR) in generating ketamine’s respiratory effects (15). We examined the influence of S(+) ketamine on respiration in mice lacking exon 2 of the MOR gene and their wild-type (WT) littermates (8,16). We further assessed the involvement of the MOR in S(+) ketamine-induced antinociception. Our knockout model previously showed the importance of the μ-opioid system, but not the κ- and δ-opioid systems, in causing morphine-induced respiratory depression and the modulation of volatile anesthetic potency by the μ receptor (8).
There are two enantiomers of ketamine: S(+) and R(−). We chose to study the S(+) variant above the racemate because this is the more potent agonist at the NMDA and opioid receptors (1,2). Furthermore, studying a pure enantiomer rather than the racemic mixture has the advantage of a more selective pharmacodynamic and pharmacokinetic profile and hence less complex interactions with endogenous ligands and receptor systems (17).
Mice with a disruption of Exon 2 of the MOR gene (MOR−/−) and WT animals of the same mouse strain have been reported previously (8,16). All animals were 1:1 hybrids from the 129/SV and C57BL/6 mouse strains. Experiments were performed in adult mice of either sex (weight, 24–40 g) at ages 3 to 6 mo. The total number of animals used in this study was 33 (17 WT and 16 mutants). All experiments were conducted after approval of the protocol by the local Animal Ethics Committee. Animal care was in accordance with institutional guidelines.
Respiratory activity was measured by using whole body plethysmography with continuous flow of dry gases through the measurement and reference chambers (8). The measurement and reference chambers are see-through and have a volume of 600 mL each. The flow and composition of the gases were set by three mass flow controllers (Bronkhorst High Tec, Veenendaal, The Netherlands). The chambers were kept at room temperature (24°C–26°C). After an animal was placed in the chamber and after ample time for habituation, data acquisition started.
The animal in the chamber initially inhaled a hyperoxic gas mixture (25% oxygen, 0% CO2, and 75% nitrogen). After a data point was obtained, two increases in inspired CO2 concentration (3% and 5%) were applied to obtain data points for the steady-state hypercapnic ventilatory response (HCVR). The inhalation of these gas mixtures lasted for 7 min. When on-line analysis revealed that a ventilatory steady-state had not been reached, the duration of inhalation was extended. Tidal volume (Vt), breathing frequency (f), and minute ventilation (V = Vt ×f) were calculated per breath. These data were averaged over 50 breaths and stored for further analysis. A linear regression analysis was performed on the steady-state V-CO2 data. This slope of the curve (S) is the estimated ventilatory CO2 sensitivity (18).
Initially the response of intraperitoneal (IP) saline on respiration was determined, followed by a cumulative dose-response assessment of S(+) ketamine (Pfizer Nederland, Capelle a/d IJssel, The Netherlands). The following cumulative doses of S(+) ketamine were administered: 0 (saline), 10, 100, and 200 mg/kg IP. After the largest S(+) ketamine dose, the effect of naloxone 2 mg/kg IP was studied. Respiratory studies started 15 to 20 min after the injections. During the study, the researchers were blinded to the genotype of the animals. There were eight animals in each group.
In a separate set of animals, we tested the stability of the respiratory model. To assess the within-day variability, three to four CO2 responses (without any intervention or drug administration) were obtained at 30-min intervals in six mice (three mutants and three WT). To assess between-day variability, single CO2 responses on four different days, 2 wk apart, were obtained in 15 animals (8 mutants, 7 WT).
To measure the antinociceptive effect of S(+) ketamine, two nociceptive tests were performed: the tail-immersion test and the hotplate test. For the tail-immersion test, the tails of the mice were immersed −2 cm in water of 54°C, and the latency time to a rapid tail flick was recorded. The cutoff time for this test was 15 s to prevent tissue damage to the mouse’s tail. For the hotplate test, mice were placed on a rectangular metal plate heated to 52°C. The antinociceptive response was the latency time to jumping, hind paw licking, or vocalization, with a cutoff of 30 s to prevent tissue damage to the mouse’s paws. After a positive response, the mouse was removed from the metal surface. The tail-immersion test preceded the hotplate test with 30 s between tests (19).
After baseline values were obtained, the effect of normal saline was assessed, followed by a cumulative dose-response assessment. The following doses of S(+) ketamine were administered: 0, 50, 100, and 200 mg/kg IP. Nociceptive studies started 15 to 20 min after the injections. During the study, the researchers were blinded to the genotype of the animals. There were eight animals in each group. To correct for individual differences in baseline latencies, the data were converted to percentage of maximum possible effect or percentage analgesia by using the following equation:MATH
A one-way analysis of variance (ANOVA) was performed on V, Vt, and f at 5% inspired CO2 and on the slope of the HCVR to test for treatment effects in animals of the same genotype (treatment levels in the respiratory studies were 0, 10, 100, and 200 mg/kg ketamine, naloxone; in the analgesia studies, the levels were 0, 50, 100, and 200 mg/kg ketamine). Post hoc analysis was by the least significant difference test. Differences between genotype were tested with two-way ANOVA (with factors treatment and genotype). A significant genotype effect was assumed when the interactive term treatment × genotype was significant. P values <0.05 were considered significant. Values reported are mean ± sem.
Our respiratory animal model showed high stability: the within-day coefficient of variation of the slopes of the CO2 responses was 8% (range, 4.0%–10.0%) in both genotypes, and the between-day variability was 17.5% (range, 4.4%–24.0%) in WT animals and 17.8% (7.7%–27.1%) in mutant mice.
In both genotypes, 100 and 200 mg/kg S(+) ketamine caused dose-dependent respiratory depression (Table 1). However, there were important differences between the two genotypes. In Figure 1, a live recording of the typical effects of saline (0 mg/kg) and 200 mg/kg S(+) ketamine are shown for a WT mouse and a MOR−/− mouse. Note that at 200 mg/kg S(+) ketamine, relative to the MOR−/− mouse, the WT mouse showed smaller Vts and longer periods of apnea.
The effect of S(+) ketamine on the S of the HCVR, minute V, and Vt differed between the genotypes, with greater depression seen in WT mice (two-way ANOVA;Table 1 and Fig. 2). At the largest dose tested, S(+) ketamine caused a reduction in the slope of the HCVR by 93% ± 15% and 49% ± 6% in WT and mutant mice (Fig. 2), respectively. This indicates the abolishment of CO2 responsiveness in WT animals. Two-hundred milligrams per kilogram of S(+) ketamine decreased minute V at a fixed inspired CO2 concentration of 5% by 80% and 40% in WT and MOR−/− mice, respectively, and Vt by 60% and 40% (Fig. 2), respectively. S(+) ketamine had no effect on f in MOR−/− mice (Fig. 2). In WT mice, S(+) ketamine reduced the breathing rate at 200 mg/kg only (reduction ∼50%). In both genotypes, episodes of apnea were apparent at 100 and 200 mg/kg S(+) ketamine. However, relative to the mutant mice, the occurrence of apneic episodes was more frequent and the duration longer, both by a factor of 3 to 4, in WT mice. The severe respiratory depression seen in the WT animals precluded the testing of S(+) ketamine at doses larger than 200 mg/kg.
Naloxone, given after the largest S(+)-ketamine dose, had an effect in WT animals only, causing an increase in ventilation, Vt, f, and S of the HCVR (Table 1 and Fig. 2). The increase in WT animals was such that the respiratory variables in both genotypes of mice did not differ (Student’s t-test, P > 0.05). After naloxone, apneic episodes were still observed in both genotypes, although in WT mice the frequency and duration were reduced to levels observed in the MOR−/− mice.
Baseline latencies for the tail-immersion test were 2.6 ± 0.4 s in WT and 3.0 ± 0.3 s in MOR−/− mice (not significantly different), and for the hotplate test they were 8.0 ± 2.0 s in WT and 5.0 ± 1.2 s in MOR−/− mice (not significant). In both genotypes, S(+) ketamine produced a dose-dependent increase in latencies in the hotplate test, with latencies in MOR−/− mice smaller compared with those observed in WT animals (Fig. 3; 200 mg/kg, P < 0.05). In contrast to WT mice, MOR−/− mice displayed no ketamine-induced antinociception in the tail-immersion test, with latencies after 50, 100, and 200 mg/kg S(+) ketamine not different from those after saline (Fig. 3).
By using a knockout mouse model, we examined the influence of the MOR system on S(+) ketamine-induced respiratory depression and antinociception. The results of the respiratory and antinociceptive studies are in agreement, showing greater respiratory depression and antinociception in mice with active MORs compared with mutant mice (Figs. 2 and 3). Furthermore, relative to animals without active MORs, animals with active MORs displayed more frequent and longer periods of apnea after S(+) ketamine (Fig. 1). The mouse strain we used has been the focus of multiple studies showing the importance of the MOR in morphine spinal and supraspinal analgesia, reward, physical dependence, respiratory depression, and immunosuppression (8,16,20). Our current data imply the importance of the μ-opioid system in at least part of S(+) ketamine-induced respiratory depression and spinal and supraspinal antinociception.
The S(+) ketamine-induced respiratory depression observed in our study was caused by depression of Vt in both genotypes (with greater depression in WT animals) and a reduction in f in WT animals only. Naloxone, at a dose that blocks μ, κ and δ receptors (21) and given after the largest S(+) ketamine dose, had no effect on any of the measured respiratory variables in MOR−/− mice, whereas in WT animals it increased f to baseline and increased the S of the HCVR and Vt to values observed in the knockout mice (Fig. 2).
The differences in respiratory responses with S(+) ketamine in WT and MOR−/− mice may be related to 1) possible compensatory mechanisms in the knockout mice related to the absence of the gene throughout development, 2) differences in S(+) ketamine pharmacokinetics in the two genotypes, or 3) the presence and absence of the MOR gene product. The fact that respiration did not differ between genotypes under control conditions (i.e., without any active drug administered), our previous observations that morphine had no effect on respiration in the exon 2 MOR−/− mice (8), and the finding that both genotypes showed a similar increase in slope of the CO2 response upon the administration of the opioid antagonist naloxone (8) indicate the absence of relevant phenotypic compensations important to the control of breathing in the mice with a knocked out MOR gene. There are no reasons to assume a difference in S(+) ketamine pharmacokinetics in the two genotypes. Altogether, the difference in respiratory responses observed in this study is best explained by an effect of S(+) ketamine via the MOR system. This may be a direct effect at the MOR or an indirect effect via the release of endogenous opioid peptides acting at the opioid receptors. In vitro opiate binding assays indicate that ketamine binds stereospecifically to opiate receptors as an agonist, with two to three times higher binding of the S(+) isomer (3,4). In vivo studies indicate that ketamine displaced [3H]etorphine, a potent opioid, from various areas in the CNS, including the brainstem and thalamic regions (4). NMDA and MORs coincide in various areas of the CNS involved in the control of breathing, such as the nucleus tractus solitarius and the locus ceruleus (22). Studies on the interaction of exogenous opioids and NMDA-receptor antagonists indicate synergy in antinociception related to presynaptic effects of opioids (causing reduced glutamate release) and postsynaptic effects of NMDA-receptor antagonists (23,24), respectively. A similar mechanism may be responsible for the profound respiratory depression caused by ketamine in our WT animals.
The opioid-specific effect of S(+) ketamine on Vt is surprising, taking into account that in the same mouse strain morphine exhibits an effect on f without affecting Vt (8). However, some studies indicate an effect of opioids on Vt, especially when consciousness is lost (11). Also, in our mice the opioid-specific reduction in Vt may be dependent on the CNS arousal state. Although we did not measure the CNS arousal state of the mice with objective measures (such as the electroencephalogram), the mice seemed sedated, as observed by reduced motility, but the righting reflex remained intact. The occurrence of apneic episodes in both genotypes is specific to NMDA-receptor blockade and is also associated with loss of consciousness (25). We relate the observation of more frequent and longer duration of apneas in WT compared with MOR−/− mice to (direct or indirect) μ-receptor activation or μ-NMDA receptor interaction in areas of the brain involved in respiratory rhythmogenesis (for example, the pre-Bötzinger complex) (7).
The absence of a stimulatory effect of naloxone after 200 mg/kg S(+) ketamine on any of the measured respiratory responses in the mutant mice indicates the lack of involvement of other opioid receptors in mediating ketamine’s respiratory effects. This stands in contrast to earlier findings of the involvement of δ-opioid receptors in mediating ketamine’s central effects in dogs (15). It is possible that these differences are related to species differences or the need for active μ receptors in the generation of respiratory depression from δ receptors (26). Note, however, that our previous findings of a naloxone-induced increase in the S of the HCVR in both genotypes indicate intact δ-opioid receptor-related respiratory activity (8).
The stimulatory effect of naloxone after 200 mg/kg S(+) ketamine in the WT but not the MOR−/− genotype may be clinically important, because it indicates that μ-opioid-specific respiratory depression by ketamine can be reversed by naloxone. Whether the large doses tested in this study relate directly to humans remains unknown. Note, however, that there are large potency differences in the effect of opioids in humans and rodents (for example, the potency of IP morphine respiratory depression in our mouse strain is approximately 1:500–1:800 relative to IV morphine respiratory depression in humans) (8–10). Until the issue of ketamine’s potency difference between humans and our mice strain has been examined, our findings should be regarded as phenomenologic.
The tail-immersion and the hotplate tests involve different nociceptive reflexes (see Ref. 19 and references cited therein): the response observed in the tail-immersion test is regarded as a spinal reflex and hence is appropriate for detection of spinally-mediated antinociception; the hotplate test is considered supraspinal in that it requires an intact CNS and hence is appropriate for detection of supraspinally-mediated antinociception. The absence of S(+) ketamine-induced antinociceptive effect in the tail-immersion test in MOR−/− mice (Fig. 3) is in agreement with a previous study that found no increase in tail-flick latencies by ketamine (up to 160 mg/kg) in rats pretreated with 10 mg/kg naloxone (3). It emphasizes the importance of intact MORs but not that of other receptors in mediating ketamine’s antinociceptive effects at the spinal level. At the supraspinal level, some non-μ-opioid-mediated analgesia by ketamine is observed, although at 200 mg/kg, it accounts for only 40% of S(+) ketamine’s effect in the hotplate test (Fig. 3). These findings are in agreement with the results of our respiratory study and suggest that S(+) ketamine affects ventilatory control and hotplate antinociception at supraspinal sites via similar molecular mechanisms and pathways involving NMDA and MORs (see above). Furthermore, also quantitatively, the effects of S(+) ketamine on ventilatory control and supraspinal antinociception were comparable in WT mice: the dose that caused a 50% reduction of the S of the HCVR was 112 ± 17 mg/kg, and the dose that caused 50% supraspinal analgesia was 120 ± 9 mg/kg (Figs. 2 and 3).
Finally, some methodologic issues deserve further comment. The background oxygen concentration was set at 25% in our respiratory studies. This was done to offset the occurrence of hypoxia caused by overt hypoventilation or atelectasis. Furthermore, we remained uninformed on the arterial or end-tidal CO2 tensions in the animals. As a consequence, the reduction of the S of the relationship between V and inspired CO2 may be related to ketamine-induced depression of respiratory neurons or a reduction in metabolic rate (8). However, because ketamine does not decrease metabolic rate (27), the latter mechanism does not play a role in explaining the reduction in S of the HCVR by ketamine.
In summary, by using a μ-opioid knockout mouse model, we observed that at supraspinal sites S(+) ketamine interacts with the MOR system. This interaction contributes significantly to S(+) ketamine-induced respiratory depression and supraspinal antinociception.
1. Hirota K, Lambert DG. Ketamine: its mechanism(s) of action and unusual clinical uses. Br J Anaesth 1996; 77: 441–4.
2. Kohrs R, Durieux ME. Ketamine: teaching an old drug new tricks. Anesth Analg 1998; 87: 1186–93.
3. Smith DJ, Pekoe GM, Martin LL, Coalgate B. The interaction of ketamine with the opiate receptor. Life Sci 1980; 26: 789–95.
4. Finck AD, Ngai SH. A possible mechanism of ketamine-induced analgesia. Anesthesiology 1982; 56: 291–7.
5. Santiago TV, Edelman NH. Opioids and breathing. J Appl Physiol 1985; 59: 1675–85.
6. Chitravanashi VC, Sapru NH. NMDA as well as non-NMDA receptors in phrenic nucleus mediate respiratory effects of carotid chemoreflexes. Am J Physiol 1997; 271: R302–10.
7. Gray PA, Rekling JC, Bocchiaro CM, Feldman JL. Modulation of respiratory frequency by peptidergic input to rhythmogenic neurons in the preBötzinger complex. Science 1999; 286: 1566–8.
8. Dahan A, Sarton E, Teppema LJ, et al. Anesthetic potency and influence of morphine and sevoflurane on respiration in μ-opioid receptor knockout mice. Anesthesiology 2001; 94: 824–32.
9. Dahan A, Sarton E, Teppema LJ, Olievier CN. Sex-related differences in influence of morphine on ventilatory control in humans. Anesthesiology 1998; 88: 903–13.
10. Sarton E, Teppema L, Dahan A. Sex differences in morphine-induced ventilatory depression reside within the peripheral chemoreflex loop. Anesthesiology 1999; 90: 1329–38.
11. Dahan A, Nieuwenhuijs D, Olofsen E, et al. Response surface modeling of alfentanil-sevoflurane interaction on cardiorespiratory control and bispectral index. Anesthesiology 2001; 94: 982–91.
12. Nattie EE, Li A. Fluorescence location of RVLM kainate microinjections that alter the control of breathing. J Appl Physiol 1990; 68: 1157–68.
13. Greer JJ, Smith JC, Feldman JL. Role of excitatory amino acids in the generation and transmission of respiratory drive in the neonatal rat. J Physiol (Lond) 1991; 437: 727–49.
14. Poon CS, Siniaia MS, Young DL, Eldridge FL. Short-term potentiation of carotid chemoreflex: an NMDAR neural integrator. Neuroreport 1999; 10: 2261–5.
15. Latasch L, Freye E. Opioid receptors-mediated respiratory effects and antinociception after S(+)-ketamine. Acta Anaesthesiol Belg 1993; 44: 93–102.
16. Matthes HWD, Maldonado R, Simonin F, et al. Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the μ-opioid-receptor gene. Nature 1996; 383: 819–23.
17. Joo G, Horvath G, Klimscha W, et al. The effects of ketamine and its enantiomers on the morphine- or dexmedetomidine-induced antinociception after intrathecal administration in rats. Anesthesiology 2000; 93: 231–41.
18. Dahan A, DeGoede J, Berkenboch A, Olievier I. The influence of oxygen on the ventilatory response to carbon dioxide in man. J Physiol (Lond) 1990; 428: 485–99.
19. South SM, Smith MT. Apparent insensitivity of the hotplate latency test for detection of antinociception following intraperitoneal, intravenous or intracerebroventricular M6G administration to rats. J Pharmacol Exp Ther 1998; 286: 1326–32.
20. Gavériaux-Ruff C, Matthes HMW, Peluso J, Kieffer BL. Absence of morphine immunosuppression in mice lacking the mu-opioid receptor gene. Proc Natl Acad Sci U S A 1998; 95: 6326–30.
21. Lord JAH, Waterfield AA, Hughes J, Kosterlitz HW. Endogenous opioid peptides: multiple agonists and receptors. Nature 1977; 267: 495–9.
22. Oleskevich S, Clements JD, Williams JT. Opioid-glutamate interactions in rat locus coeruleus neurons. J Neurosci 1993; 70: 931–7.
23. Chapman V, Dickenson AH. The combination of NMDA antagonism and morphine produces profound antinociception in the rat dorsal horn. Brain Res 1992; 573: 321–3.
24. Dickenson AH. NMDA receptor antagonists: interaction with opioids. Acta Anaesthesiol Scand 1997; 41: 112–5.
25. Cassus-Soulanis S, Foutz AS, Denavit-Saubié M. Involvement of NMDA receptors in inspiratory termination in rodents: effects of wakefulness. Brain Res 1995; 679: 25–33.
26. Matthes HWD, Smadja C, Valverde O, et al. Activity of δ-opioid activity is partially reduced, whereas activity of the κ-receptor is maintained in mice lacking the μ-receptor. J Neurosci 1998; 18: 7285–95.
27. Dawson B, Michenfelder JD, Theye RA. Effects of ketamine on canine cerebral blood flow and metabolism: modification by prior administration of thiopental. Anesth Analg 1971; 50: 443–7.