1 Department of Anesthesiology, Jichi Medical School, Tochigi, Japan. 2 Division of Organ Replacement Research, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan. 3 Department of Urology, Jichi Medical School, Tochigi, Japan. 4 Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
*Corresponding author. E-mail: eijikoba{at}jichi.ac.jp
Accepted for publication: December 27, 2003
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Abstract |
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Methods. In the present study, daily variations in the hypnotic effect of ketamine were determined in wild-type mice and NMDA 1 knockout (KO) mice.
Results. The effect of ketamine had a definite daily variation in wild-type mice. No significant difference in blood concentration was observed at different dosing times (10:00 and 22:00). In NMDA receptor 1 KO mice, the hypnotic effect of ketamine was weaker than in wild-type mice and there was no dependence on the time of administration. Significant pharmacokinetic differences were not observed between wild-type and KO mice.
Conclusions. The enhanced hypnotic effect in the active phase of the circadian cycle is likely a result of changes with the time of day in the susceptibility of the central nervous system to ketamine. Knockout of the NMDA receptor 1 subunit gene markedly reduced the effect of ketamine, and eliminated the time-dependent sensitivity to ketamine.
Br J Anaesth 2004; 92: 85964
Keywords: mouse; pharmacology, ketamine; receptors, ketamine
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Introduction |
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Some i.v. anaesthetics are believed to act in an agent-specific fashion, by activating or inhibiting synaptic transmission or both.8 Ketamine is a widely used anaesthetic that exerts its depressant effect by reducing neuronal excitation via the N-methyl-D-aspartate (NMDA) receptor as a non-competitive antagonist and mediating sympathetic responses. The NMDA receptor channel is formed from at least two families of subunits, the 14 (NR2AD) and
(NR1). The anaesthetic effect of ketamine is therefore believed to correlate with the circadian rhythms of neural activities; previous studies have demonstrated that the pharmacological response to ketamine shows daily variation.10 11 However, although in vitro studies showed
14 subunits are involved in the effect of ketamine,12 the in vivo contribution of the NMDA receptor
1 subunit to this chronopharmacological phenomenon is unclear.
In the present study we examined the chronopharmacological effect of ketamine and the resulting pharmacokinetics in wild-type mice. We then investigated the hypnotic effect of ketamine in NMDA receptor 1 subunit knockout (KO) mice at different times of the day.
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Materials and methods |
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Measurement of daily variation of water intake in wild-type and NMDA receptor 1 subunit KO mice
Water intake of experimental wild-type and NMDA receptor 1 subunit KO mice was measured every 6 h for 3 days (n=6 in each). Water was provided in 15-ml test tubes via stainless steel ball-bearing stopper tubes (DM-G1; OHara, Tokyo, Japan). These animals were made familiar with this environment for 7 days. Over a period of 3 days the tubes were weighed four times daily, at 01:00, 07:00, 13:00, and 19:00.
Measurement of the effect of ketamine dosing at different times of the day
Ketamine (Sankyo Co., Ltd, Tokyo, Japan) diluted in saline was administered intraperitoneally. The volume injected was standardized to 0.2 ml·animal1 in each experiment. The hypnotic effect was evaluated as ketamine-induced sleep times using the duration of loss of the righting reflex (LORR) after injection of 200 mg·kg1 ketamine. Each mouse received a single dose of ketamine. LORR was defined as having occurred when the mouse failed to right itself for at least 10 s after being placed on its back. Recovery from the LORR was defined as having occurred when the mouse spontaneously righted itself.18 During anaesthesia the animals were kept warm on a plate heated to 38°C. To examine the daily variation of effect, ketamine was injected at 04:00, 10:00, 16:00, and 22:00 in wild-type mice (n=12 in each group). To analyse the effect of the dosing time in KO mice, ketamine was injected at 10:00 and 22:00 (n=12). The investigators were blinded to the experimental groups.
Measurement of blood plasma concentrations of ketamine and cytochrome P450 enzymes in liver
To compare the pharmacokinetics of drug administrations at 10:00 and 22:00, the blood concentration of ketamine was measured in wild-type mice. Mice were killed at each time point, and blood samples were obtained at the time of the i.p. injection and at 10, 20, 40, and 60 min after i.p. injection of ketamine 200 mg·kg1 at 10:00 and 22:00 (n=8 in each group). The concentration of ketamine in plasma was determined by high-performance liquid chromatography.19
Since ketamine is metabolized by cytochrome P450s and then eliminated,1921 we investigated the activity of total CYPs and isoenzymes (CYP2B6, CYP2C8/9, and CYP3A) in wild-type and KO mice to examine if the hepatic metabolism in KO mice was different from that of wild-type mice. Activity of total and isoenzymes of CYPs in the liver of wild-type and KO mice were measured by high-performance liquid chromatography (n=5 in each group as described).1921 Mice were killed at 22:00 and live samples were obtained.
Statistical analysis
Statistical analyses were performed using t-test or analysis of variance (ANOVA). Laboratory data are expressed as the mean (SD). Single cosinor method was used to analyse the circadian rhythm.22 23 This method involves fitting to a curve of predefined period by the method of least squares.22 23 A value of P<0.05 was considered statistically significant. For analysis of dosing time-dependent effect of ketamine in wild-type and KO mice, a value of P<0.0125 was considered statistically significant by Bonferroni correction.
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Results |
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Discussion |
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In wild-type mice, ketamine administered during the active phase-induced longer sleep, which we take as a surrogate measure of the hypnotic effect of ketamine, than in the inactive phase. The pattern of daily variation in the chronopharmacological effect of ketamine is similar to that of circadian locomotor activity, corresponding to water intake. Because changes in drug effectiveness based on pharmacokinetics depend on the time of administration,30 31 it is possible that pharmacokinetic rhythms contribute to variations in the response to drugs. We accordingly investigated blood plasma concentrations of ketamine at the time of administration and at 10, 20, 40, and 60 min after administration in both the active and inactive phases in wild-type mice. There was no significant dosing time-dependent difference in plasma pharmacokinetics between the phases. However, sleep time differed significantly between the two wild-type groups, and most mice given an injection at 10:00 were already awake 60 min later. These findings suggest that the enhanced effect of ketamine in the active phase is more likely a result of the enhanced susceptibility or sensitivity of the central nervous system rather than the result of differences in pharmacokinetics in wild-type mice.
It has been suggested that the anaesthetic effects of ketamine are mediated through block of NMDA receptor channels, which are a subtype of glutamate receptors.32 33 NMDA receptors play an important role in excitatory neurotransmission.9 They are ligand-gated cation channels with high Ca2+ permeability, and are a combination of a (NR1) subunit and any one of the
14 (NR2AD) subunits.9 Analysis of recombinant receptors expressed in Xenopus oocytes indicates that ketamine blocks the four
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channels to a similar extent.12 To determine the contribution of the NMDA receptor
1 subunit to the hypnotic effect of ketamine in vivo, we examined ketamine-induced sleep times using LORR and compared the hypnotic effect of ketamine in wild-type mice and in mutant mice lacking the NMDA receptor
1 subunit. In these KO mice, no abnormal development or abnormal motor function has been reported under normal conditions.13 1517 Our results showed that the effect of ketamine on the NMDA receptor
1 subunit KO mice was significantly weaker at the time of day that ketamine was most effective in wild-type mice. Because the levels of CYPs and the blood concentration of ketamine after i.p. injection were not different from those in wild-type mice at 22:00, the difference in hypnotic effectiveness could be a result of sensitivity differences of the CNS resulting from disruption of the NMDA receptor
1 subunit gene, rather than pharmacokinetic differences. Consequently, the data suggest that the hypnotic effect of ketamine involves an NMDA receptor channel, comprising the
1 subunit, and NMDA receptor
1 subunit contributes to the dosing time-dependent effect of ketamine.
As NMDA receptor antagonist inhibited the light-induced phase shift of locomotor activity rhythm, it might be possible that NMDA receptors mediate photic entrainment of the biological clock in the SCN.3437 It has been shown that glutamate also increases the amplitude of the circadian clock-associated gene products Per1 and Per2; either or both of these may be critical for photic phase shifts.17 38 39 Thus, although the similar circadian patterns of water consumption in NMDA receptor 1 subunit KO mice and wild-type mice suggest that abrogation of the dosing time-dependent hypnotic effect of ketamine in KO mice was caused by the NMDA receptor
1 subunit related hypnotic pathway rather than a circadian pathway, anaesthetic drugs such as ketamine or knocking out the NMDA receptor
1 subunit might modify the circadian oscillation of the clock genes to change their expression in the SCN in a dosing time-dependent fashion.
To conclude, we have shown that the hypnotic action of ketamine has a marked dosing time-dependent effect. This might be the result of daily variations in anaesthetic sensitivity in the CNS rather than pharmacokinetic variation. Our findings also indicate that the NMDA receptor 1 subunit is involved in the anaesthetic effect of ketamine, and plays a major role in the induction of the dosing time dependency.
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Acknowledgements |
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