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From the Department of Anesthesiology, The University of Tokyo, Faculty of Medicine, Tokyo, Japan.
Address correspondence to: Dr. Tomoki Nishiyama, 3-2-6-603, Kawaguchi, Kawaguchi-shi, Saitama, 332-0015, Japan. Phone: 81-3- 5800-8668; Fax: 81-3-5800-9655; E-mail: nishit-tky{at}umin.ac.jp
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Abstract |
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Methods: One hundred seventy-six (n = 8 animals per dose escalation) male Sprague-Dawley rats were instrumented with lumbar intrathecal catheters. Tail withdrawal in response to thermal stimulation, or paw flinching and shaking in response to sc hind paw formalin injection were compared following intrathecal injection of midazolam (1, 3, 10, 30, or 100 µg in 10 µL) or ip administration (3, 30, 300, or 3,000 µg in 300 µL). Saline 10 µL or 300 µL was used as a control. Behavioural side effects and motor disturbance were also examined.
Results: Intrathecal administration of midazolam increased tail flick latency dose dependently (P < 0.05) with a 50% effective dose (ED50) of 1.60 µg, whereas ip administration did not increase latency. Both intrathecal and ip routes of administration decreased the number of paw flinches in both phases 1 and 2 of the formalin test (P < 0.05). The ED50s were 1.26 µg [confidence interval (CI), 0.35–3.18 µg], (phase 1) and 1.20 µg (CI, 0.29–3.71 µg), (phase 2) with intrathecal administration, and 11.6 µg (CI, 2.5–19.3 µg), (phase 1) and 52.2 µg (CI, 18.3–102.7 µg), (phase 2) with ip administration.
Conclusion: Systemically administered midazolam induced antinociception for inflammatory pain only, while intrathecal administration elicited antinociceptive effects on both acute thermal and inflammatory-induced pain.
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Introduction |
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Recent studies have shown that midazolam produces an analgesic action through the benzodiazepine/
-aminobutyric acid (GABA)A receptor complex in the spinal cord.1,2 However, intraperitoneally administered midazolam may be associated with hyperalgesia,3 while it potentiates isoflurane-induced antinociception at doses where no effect is seen with midazolam alone.4 Therefore, it is uncertain as to whether systemically administered midazolam is analgesic or hyperalgesic. As a result of this controversy, the antinociceptive effects of systemically and intrathecally administered midazolam were investigated on two different types of pain in a rat model. We hypothesized that systemic administration of midazolam would elicit analgesic effects, while the response would be less intense in comparison with intrathecal administration of this relatively short-acting benzodiazepine.
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Methods |
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Midazolam (Sigma, St. Louis, MO, USA) was dissolved in normal saline to achieve solutions of 1, 3, 10, 30, and 100 µg in 10 µL for intrathecal administration, or 3, 30, 300, and 3,000 µg in 300 µL for ip administration. Normal saline was used as a control. A microsyringe was used for intrathecal injection and after each injection, the catheter was flushed with normal saline 10 µL to clear the catheter dead space (8.5 ± 0.6 µL). A 1-mL syringe with a 23-G needle was used for ip injection.
The tail flick test was performed with the Tail-Flick Analgesia Meter (MK-330A; Muromachi Kikai Co. Ltd., Tokyo, Japan). Rats were placed in a clear plastic cage with their tails extending through a slot located at the rear of the cage. Thermal stimulation was given by a beam of high-intensity light focused on the tail 2 to 3 cm proximal to the end. The time between the start of the stimulation and tail withdrawal was measured as the tail flick latency. The cut-off time in the absence of a response was set to 14 sec to prevent tissue injury. The tests were performed at five, ten, 15, 30, 60, 90, and 120 min after drug administration in the ip group, adding 180 and 240 min in the intrathecal group.
The formalin test was performed ten minutes after drug administration. Fifty microlitres of 5% formalin was injected subcutaneously into the dorsal surface of the right hind paw with a 30-G needle. Immediately after injection, the rat was placed in an open clear plastic chamber, and its flinching or shaking paw response was observed at five minutes intervals for a period of one hour. The number of flinches was counted for one minute. Usually two phases were observed: phase 1 for the first six minutes after injection, and phase 2 beginning after about ten minutes, with an interval of no flinches between phases.
Behavioural side effects were examined and judged as present or absent in rats for the tail flick test, measured at the same time as the tail flick assessment. Agitation was judged as spontaneous irritable movement, vocalization, or both. Allodynia was judged as escape, vocalization or both, induced by lightly stroking the flank of the rat with a small probe. The placing or stepping reflex was evoked by drawing the dorsum of either hind paw across the edge of the table. Normally, rats try to put the paw ahead into a position to walk. The righting reflex was assessed by placing the rat horizontally with its back on the table. Normally, rats twist their bodies to an upright position immediately. Flaccidity was judged as muscle weakness by placing the forepaw 3 to 5 cm higher than the hind paw. Normally, rats will walk up using the hind paw. The pinna and corneal reflexes were examined with a paper string. When a string is placed in the auditory canal or gently applied to the cornea, rats normally shake their heads. Abnormal ambulation was judged as asymmetrical movement in walking.
The tail flick data are presented as the percentage of maximum possible effect, shown as (post-drug latency – pre-drug latency at time 0) x 100 / (cut-off time – pre-drug latency at time 0). The effective dose in 50% of animals (ED50) was calculated using the maximum effects in the tail flick test and the area under the curve in the formalin test. Other data are shown as mean ± SD or 95% confidence interval (CI). Statistical analysis was performed with one way factorial analysis of variance followed by the Student Neuman Keuls test as a post hoc analysis for the dose response data. A P value < 0.05 was considered to be statistically significant.
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Results |
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, 2). The ED50 as determined by tail flick latency was 1.60 µg (CI, 0.45–3.02 µg) with intrathecal administration, but the ED50 was not obtained by tail flick latency following ip administration.
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, 4). The ED50s were 1.26 µg (CI, 0.35–3.18 µg), (phase 1) and 1.20 µg (CI, 0.29–3.71 µg), (phase 2) with intrathecal administration, and 11.6 µg (CI, 2.5–19.3 µg), (phase 1) and 52.2 µg (CI, 18.3–102.7 µg), (phase 2) with ip administration.
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). No sedative effects were observed in any animal.
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Discussion |
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Several issues are raised by these interesting observations. Although motor disturbance was evident with both intrathecal and ip administration of high dose midazolam, the rats were still able to produce vigorous tail flick responses and paw flinches, indicating that the motor disturbance did not interfere with the animal’s ability to respond to a noxious stimulus, while some evidence of muscle weakness was apparent.
The tail flick response is mediated by A
and C fibres at the level of the spinal cord, whereas behavioural responses in the formalin test are mediated by both the spinal and supraspinal sites. The phase 1 response of the formalin test is caused by the direct stimulation of nociceptors by formalin or tissue damage, and is thought to be an acute pain reaction.5 This reflects activity that is prominent in Aß, A and highthreshold C nociceptor afferent fibres. The phase 2 response is caused by subsequent inflammation after formalin injection and central sensitization related to C-fibre activity.6 It reflects activity in mechanically insensitive afferent fibres and activity of A and C fibres.7 The present results suggest that intraperitoneally administered midazolam has few effects at the level of the spinal cord while eliciting some antinociceptive effects in the periphery and/or the brain. In contrast, intrathecal administration of the drug provides spinally-mediated analgesia.
Yanez et al. reported that intrathecally administered midazolam produces dose-dependent antinociception on thermally induced pain.2 Midazolam administered into the subarachnoid space of the rat could produce potent analgesia that was antagonized by flumazenil. 1 A segmental effect of intrathecal midazolam was demonstrated using transcutaneous electrical stimulation. 8 Enhancement of presynaptic inhibition might be a possible mechanism for the action of midazolam, because benzodiazepines are known to increase GABA transmission via their specific binding site co-located with the GABAA receptor in the spinal cord.9 As shown in these previous studies and corroborated by the present results, it is clear that intrathecally administered midazolam has antinociceptive effects through the benzodiazepine/GABAA receptor complex in the spinal cord.
In a previous study using the rat tail flick test, 1–10 mg·kg–1 (about 300–3000 µg) of ip midazolam was shown to produce a hyperalgesic effect, which could be blocked by the benzodiazepine receptor antagonist flumazenil. In contrast, intrathecal midazolam in doses of 10–100 µg produces antinociception.3 Tatsuo et al. also reported that ip injection of midazolam 10 mg·kg–1 induced a significant decrease of tail flick latency and produced a long-lasting nociceptive effect in the formalin test, characterizing a hyperalgesic effect.10 However, in the present study, we did not observe a hyperalgesic response. The only important difference between studies was the species of the rat. We used Sprague Dawley rats, while Niv et al.3 used albino rats and Tatsuo et al.10 used Wistar rats. In addition, Niv et al.3 observed sedation with the doses that achieved hyperalgesia. Therefore, variations in responses may be, in part, species-specific. Alternatively, sedation might be a confounding factor in interpretation of the tail flick response.
When midazolam was injected intracerebroventricularly, it reduced the antinociceptive effects of morphine as measured by the tail flick test.11 Midazolam potentiates morphine action at the spinal level, while an antagonistic effect can be seen at the supraspinal level.12 Benzodiazepines produce a clinically significant antagonism of opioid analgesia.13 Systemically administered midazolam attenuates the antinociceptive effects of morphine, probably by inhibiting the descending inhibitory system.14 The GABAA receptor is involved in modulating supraspinal actions of opioid receptor occupancy.15 Therefore, benzodiazepines may modulate the antinociceptive effect of opioids in the brain by means of physiologic mechanisms that are distinct from the effects of benzodiazepines in the spinal cord. This mechanism is different from hyperalgesia.
It has been suggested that midazolam in conjunction with morphine will suppress both peripheral and central sensitization and enhance the effects of preemptive analgesia. Midazolam iv has been shown to decrease anesthetic requirements in enflurane-anesthetized dogs.16 Therefore, midazolam might have antinociceptive effects in both the periphery and the brain.
Intravenous midazolam suppresses noxiouslyevoked activity of spinal wide dynamic range neurons with maximum effect at a dose of 1 mg·kg–1 that is reversible by a benzodiazepine antagonist.17 Systemically administered midazolam reduces A fibre-evoked responses of the neurons of the dorsal horn of the spinal cord, and also reduces the C fibremediated activity in a spinal nerve ligation model of neuropathic pain, most likely acting at spinally located benzodiazepine receptors.18 Therefore, systemically administered midazolam may have actions at the level of the spinal cord. The present formalin data support these findings.
Intraperitoneal midazolam had an ED50 which was five times greater in phase 2 compared to phase 1, while intrathecal midazolam was equally effective for both phases 1 and 2. These results suggest that systemically administered midazolam was less effective for central sensitization than the nociceptive response in the periphery.
The present study used ip injection in rats. Therefore, these results cannot be extrapolated to clinical practice for patients receiving iv or im midazolam injections. Furthermore, although the formalin test was used to mimic postoperative pain, the nature and intensity of noxious stimulation with the formalin test may not be comparable to the varying sites and intensities of acute postoperative pain. Therefore, follow-up clinical trials are warranted to confirm the analgesic properties of systemically-administered midazolam.
In conclusion, systemically administered midazolam induced antinociception for inflammatory pain only, while intrathecal administration elicited antinociceptive effects on both acute thermal and inflammatory- induced pain. When given in relatively low and non-sedating doses, systemically administered midazolam might be a useful adjunct for postoperative pain management.
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Footnotes |
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Accepted for publication June 12, 2006. Revision accepted July 10, 2006.
Competing interests: None declared.
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References |
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2 Yanez A, Sabbe MB, Stevens CW, Yaksh TL. Interaction of midazolam and morphine in the spinal cord of the rat. Neuropharmacology 1990; 29: 359–64.[Medline]
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