Anesth Analg 2006;102:1217-1222
© 2006 International Anesthesia Research Society
doi: 10.1213/01.ane.0000198634.25504.83
NEUROSURGICAL ANESTHESIA
Section Editor:
Adrian W. Gelb
Intrathecal Nicorandil and Small-Dose Morphine Can Induce Spastic Paraparesis After a Noninjurious Interval of Spinal Cord Ischemia in the Rat
Tatsuya Fuchigami, MD, PhD,
Manabu Kakinohana, MD, PhD,
Seiya Nakamura, MD, PhD,
Kenji Murata, MD, PhD, and
Kazuhiro Sugahara, MD, PhD
Department of Anesthesiology, University of the Ryukyus; Division of Anesthesiology, Okinawa Prefectural Nanbu Hospital, Okinawa, Japan
Address correspondence and reprint requests to Manabu Kakinohana, MD, PhD, Department of Anesthesiology, Faculty of Medicine, University of the Ryukyus, Okinwawa, 903-0215 Japan. Address e-mail to mnb-shk{at}ryukyu.ne.jp.
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Abstract
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We investigated the interaction between nicorandil, a K+ATP channel opener, and morphine on motor function after a noninjurious interval of spinal cord ischemia in the rat. Spinal ischemia was induced by aortic occlusion for 6 min with a balloon catheter in Sprague-Dawley rats. All animals received intrathecal (IT) injection of morphine (1–60 µg) 1 h after ischemia. In addition to IT injection of morphine, group M (control), group MN (combination of morphine and nicorandil), and group MNG (combination of morphine, nicorandil, and glibenclamide) received IT saline, nicorandil (10 µg), and both glibenclamide (10 µg) and nicorandil (10 µg) after 150 min of reperfusion, respectively. A quantal bioassay for the effect of IT morphine on neurological function after ischemia was performed to calculate 50% effective dose values (ED50) for inducing paraparesis at 3 h of reperfusion. The ED50 in group M and group MN was 15.1 ± 4.9 µg and 2.9 ± 1.0 µg of IT morphine, respectively (P < 0.05). In Group MNG, the dose-response curve shifted back to the right and the ED50 for inducing paraparesis was 11.6 ± 4.7 µg of IT morphine. The present study demonstrates that IT small-dose morphine combined with nicorandil induces spastic paraparesis after noninjurious interval of spinal cord ischemia in the rat.
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Introduction
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Opiates are commonly administered by intrathecal or epidural routes to produce analgesia during the postoperative period as well as to control chronic, otherwise unmanageable pain, such as in terminal cancer patients. Among the opiates, morphine is one of the most widely used despite frequently encountered side effects such as pruritus, respiratory depression, seizure, and/or occasional spinal toxicity (1). Under conditions of ischemia and reperfusion in the central nervous system, systemic administration of fentanyl was shown to exacerbate incomplete cerebral ischemia in the rat (2). Of particular importance is a clinical and experimental observation that neuraxial administration of morphine injected after transient aortic occlusion, as might occur during aortic aneurysm repair, triggers the development of spasticity and that this effect can be reversed by subsequent naloxone treatment (3). Kakinohana et al. (4) demonstrated in a dose-response study that the 50% effective dose (ED50) value of intrathecal morphine for inducing paraparesis after brief spinal ischemia was 16.1 µg, a value about 10 times larger than that for an antinociceptive effect. Nakamura et al. (5) also demonstrated, using a rat aortic occlusion model, that intrathecal morphine after aortic occlusion could induce spastic paraparesis and that this effect was enhanced by  -aminobutyric acid (GABA) antagonists through a synergistic interaction. We speculated that a smaller dose of intrathecal morphine after spinal ischemia could also induce spasticity in the hindlimb in the presence of some drugs that have a synergistic effect with morphine.
Many electrophysiological studies have demonstrated that agonists of µ-opioid receptors open K+ channels and bring about hyperpolarization in target neurons (6). The synergistic effect between morphine and a K+ATP channel opener on antinociception was shown in an animal model (7). In addition, K+ATP channel blockers also antagonize morphine-induced hyperthermia and hypermotility, as well as the exacerbation by morphine of bicuculline-induced convulsion (8). According to those data, it can be speculated that the K+ATP channel opener enhances the effect of morphine on spasticity that develops after brief aortic occlusion. The purpose of this study was to investigate the interaction between a K+ATP channel opener (nicorandil) and morphine on motor function after a noninjurious interval (6 min) of spinal cord ischemia in the rat and to clarify the mechanism of morphine-induced paraparesis.
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Methods
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These experimental animal studies were performed according to protocols approved by the Institutional Animal Care and Use Committee of University of the Ryukyus. Forty male Sprague-Dawley rats (300–400 g) were used. Intrathecal catheters were implanted according to a previously described method (9). After implantation, animals were allowed to recover for 5 days before induction of spinal ischemia. Rats showing motor weakness or signs of paresis upon recovery from anesthesia were immediately killed with IP injection of pentobarbital (100 mg/kg).
Details of the aortic occlusion model in the rat were previously reported (10). In brief, animals previously implanted with intrathecal catheters were anesthetized in a Plexiglas box with 5% halothane in room air. After anesthesia induction, rats were maintained with 1.0%–2.0% halothane delivered by an inhalation mask. A Teflon catheter (24-gauge) was inserted into the tail artery for monitoring of distal arterial blood pressure. For induction of spinal ischemia, the left femoral artery was isolated and a 2F Fogarty catheter was placed into the descending thoracic aorta so that the tip of the catheter reached the level of the left subclavian artery. To control the proximal arterial blood pressure (i.e., above the level of aortic occlusion) at 40 mm Hg during the period of aortic occlusion, a 20-gauge Teflon catheter connected to an external blood reservoir (38°C) was inserted into the left carotid artery. At the completion of all cannulations, heparin (200 U) was injected into the tail artery. To induce spinal ischemia, a balloon catheter was inflated with 0.05 mL of saline and the blood was allowed to flow into the external reservoir. The efficiency of occlusion was evidenced by an immediate and sustained loss of any detectable pulse pressure and a decrease in distal arterial blood pressure (i.e., below the level of aortic occlusion). After ischemia, the balloon was deflated and the blood was reinfused over a period of 60 s. Protamine sulfate (12 mg/kg) was administered subcutaneously. All arterial lines were then removed, incisions were closed, and animals were allowed to recover for 3 days. During this period, the recovery of motor function was assessed periodically.
During reperfusion, recovery of motor function was assessed according to a grading system. Motor function was quantified by assessments of ambulation and the placing/stepping reflex. For statistical purposes, ambulation (walking using hindlimbs) was graded as follows: 0, normal; 1, toes flat under the body when walking but ataxia present; 2, knuckle walking; 3, movement of lower extremities but unable to knuckle walk; and 4, no movement, drags hindlimbs. The placing/stepping reflex was assessed by dragging the dorsum of the hindpaw over the edge of a surface. This normally evokes a coordinated lifting and placing response (e.g., stepping) that was graded as follows: 0, normal; 1, weak; and 2, no stepping. A motor deficit index (MDI) was calculated for each rat at each time interval. The final MDI was the sum of the scores (walking using hindlimbs plus placing/stepping reflex). The MDI was calculated by observers without knowledge of the treatment group. For the quantal bioassay for the effect of intrathecal morphine on neurological function after 6 min of aortic occlusion, doses of intrathecal morphine were selected to span all grades of neurological function, ranging from "walk" (MDI: 0–4) to "paraparesis" (MDI: 5–6).
For investigation of the interaction between morphine and nicorandil, animals were divided into 3 groups according to intrathecal administration of drugs after short-term aortic occlusion (6 min). All animals received an intrathecal injection of morphine (1–60 µg) 1 h after reperfusion. In addition to the intrathecal injection of morphine, group M (control animals, n = 15) received intrathecal saline twice (10 µL) injected at 145 min and 150 min after reperfusion; group MN (combination of morphine and nicorandil, n = 10) received an intrathecal saline (10 µL) and nicorandil (10 µg) injected at 145 min and 150 min after reperfusion, respectively. Group MNG (combination of morphine, nicorandil, and glibenclamide, a K+ATP channel blocker, n = 15) received intrathecal glibenclamide (10 µg) 5 min before injection of intrathecal nicorandil (10 µg) at 150 min of reperfusion (Table 1).
The following drugs were used: morphine hydrochloride (Sankyou Pharm. Tokyo, Japan), nicorandil (Chugai Pharm. Tokyo, Japan), dimethyl sulfoxide (DMSO), and glibenclamide (Sigma, St. Louis, MO). Morphine hydrochloride and nicorandil were dissolved in 0.9% saline and glibenclamide was dissolved in DMSO. The vehicle (DMSO) for glibenclamide itself did not disturb the effect of morphine on neurological function after spinal ischemia (data not shown).
Statistical analysis of physiological data was performed by one-way analysis of variance followed by Fisher’s protected least-squares difference test. For analysis of neurological outcome in the individual treatment groups, significant overall values were obtained by quantal bioassay. Intrathecal morphine dose-response curves were constructed for each group (similar to the 50% lethal dose curves of pharmacological studies) (11). The computer calculated an ED50, which represents the dose of morphine that produced transient spastic paraparesis in 50% of animals within a group. Statistical significance was assessed by Student’s t-tests and adjusted for multiple comparisons with the Bonferroni correction (P > 0.05). Neurological exacerbation was considered present if a drug significantly decreased the ED50 in comparison with the control group. The quantal dose-response analysis method used in the present study has been published previously (11).
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Results
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During the preischemic and intraischemic periods, paravertebral muscle temperature ranged between 38.0°C and 38.5°C. Baseline distal arterial blood pressure ranged between 64 and 99 mm Hg and decreased to 0–7 mm Hg at the end of the 6-min aortic occlusion. No significant differences were detected among the experimental groups (Table 2).
The results of these experiments are presented in Tables 3 and 4 and are shown in Figure 1.
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Table 3. Effect of IT Morphine on Neurological Outcome in Rats Measured 3 h After Intrathecal Injection of Morphine
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Figure 1. Dose-response curve for the effect of intrathecal morphine on the probability of paraparesis. Beneath the curve, the normal (N)/paraparesis (P) data (• = individual rat in group M,  = in group MN, = in group MNG) versus the dose of morphine are displayed. The quantal bioassay assessment showed that the 50% effective dose (ED50) values for inducing paraparesis were 15.1, 2.93, and 11.6 µg in groups M, MN, and MNG, respectively, when behavioral analysis was assessed 3 h after intrathecal morphine administration. *Difference between group M and group MN is statistically significant (P < 0.05). #Difference between group MNG and group MN is statistically significant (P < 0.05). Data are presented as mean ± se. Group M = morphine/control; group MN = morphine and nicorandil; group MNG = morphine, nicorandil, and glibenclamide.
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All three groups received various dosages of intrathecal morphine 1 h after ischemia as described above. In group M, this series of experiments investigated the effects of intrathecal morphine when administered 60 min after reperfusion. Rats receiving more than 15 µg of intrathecal morphine developed spasticity of the hindlimbs and complete spastic paraplegia at 3 h of reperfusion. The ED50 for inducing paraparesis in this group, when behavioral analysis was assessed 3 h after intrathecal injection of morphine, was 15.1 ± 4.9 µg (mean ± se). In group MN, this series of experiments investigated the effects of intrathecal nicorandil (10 µg) when administered 150 min after reperfusion. Rats given more than 5 µg of intrathecal morphine had complete spasticity of the hindlimbs at 150 min after nicorandil treatment. In group MN, the dose-response curve shifted to the left and the ED50 was reduced significantly to 2.9 ± 1.0 µg compared with group M (P < 0.05). A pilot study found no effect of intrathecal nicorandil alone, even 100 µg, on any spasticity after spinal ischemia (n = 3, data not shown). In group MNG (glibenclamide 10 µg), the dose-response curve shifted back to the right and the ED50 for inducing paraparesis was 11.6 ± 4.7 µg, which was not significantly different from that in group M. Glibenclamide alone did not alter the effect of morphine on neurological function after spinal ischemia (n = 4, data not shown).
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Discussion
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The present study showed that administration of intrathecal nicorandil reduces the ED50 of intrathecal morphine required to induce paraparesis from 15.1 µg to 2.9 µg. This effect was not observed for pretreatment with glibenclamide, a K+ATP channel blocker. Therefore, it is suggested that intrathecal nicorandil enhances the effect of morphine through the K+ATP channels in inducing paraparesis after a noninjurious interval of spinal cord ischemia in rats. Taking these results into consideration, administration of even a small dose of neuraxial morphine may induce spastic paraparesis in the presence of a K+ATP channel opener after noninjurious spinal cord ischemia.
We previously reported that intrathecal administration of a large dose of morphine after transient aortic occlusion could be associated with a potent risk of paraparesis and the corresponding development of neurological dysfunction (3) in a dose-dependent manner (4). In the latter report (4), the ED50 value of intrathecal morphine for inducing paraparesis was reported to be 16.1 µg, which was approximately 10 times larger than that for an antinociceptive effect. It appears unreasonable in clinical situations that such a large dosage, one much larger than that needed for an antinociceptive effect, would be used. However, Nakamura et al. (5) also demonstrated using isobolographic analysis that in the presence of GABA receptor antagonists a much smaller dose of intrathecal morphine can induce spasticity of the hindlimbs after a noninjurious interval of spinal cord ischemia resulting from the synergism between morphine and GABA receptor antagonists. Taken together these results indicate that even a small dose of intrathecal morphine can increase spasticity after aortic occlusion when a drug having a synergistic effect on morphine antinociception is used. We should therefore be aware that the use of spinal morphine is not always safe and may produce devastating complications under some special conditions (for example, in combination with a K+ATP channel opener after spinal ischemia).
The mechanism of morphine-induced spasticity after spinal ischemia is not completely known. In general, opioid administration has a potent inhibitory effect on its target neurons in several brain and spinal cord lesions (12). It has been reported that systemic administration of morphine triggers neuronal excitation in the CA1 region in the hippocampus and that this excitation is likely mediated by the inhibition of GABAergic interneurons, which provide inhibitory input to pyramidal cells (13,14). Indeed, µ-opioid receptors are often found on GABAergic interneurons in the spinal cord, and an inhibitory effect of µ-opioid receptor activation on these neurons could be indirectly associated with excitatory effects of opioid agonists in vivo (15). In addition, a reduction in recurrent  -motoneuronal inhibition (mediated by Renshaw cells) after systemic injection of morphine was reported in spinal cord (16). Consistent with these observations, behavioral studies show that systemic or intrathecal administration of a relatively high dose of morphine triggers myoclonic seizures, loss of motor coordination, truncal rigidity (17,18) or the development of prominent but transient allodynia after intrathecal administration (19). A two- to threefold increase in brain concentration of µ-,  -, and  -opioid receptors was reported during the early reperfusion period after focal cerebral ischemia in the cat model (20), suggesting that sensitivity to morphine might increase in the central nervous system after ischemia. From these data, it can be speculated that the sensitivity to morphine after short-lasting spinal ischemia would increase in the spinal cord, and then GABAergic spinal-interneuron and/or Renshaw cell function would be inhibited by neuraxial morphine followed by a transient loss of spinal inhibition, which might result in the development of spasticity.
K+ATP channels have been widely identified in the central nervous system (21). These channels have been demonstrated to be involved in mediation of the antinociceptive action of morphine given intracerebroventricularly (22), systemically (23), and spinally (24). Several reports have suggested that K+ATP channel activity is closely connected with the analgesic action of morphine. K+ATP channel openers enhance opioid-induced analgesia (25–27), whereas K+ATP channel blockers inhibit morphine-induced analgesia (28–30). Because the order of potency obtained for the antagonism of morphine antinociception was identical to that obtained by a block of K+ATP channels in neurons (29), these channels would appear to play an important role in µ-opioid antinociception. In comparing the actions of K+ATP channel opener and morphine, activation K+ efflux leading to hyperpolarization of their target cells may be their pharmacologically common mechanisms (31,32). It is, therefore, reasonable that intrathecal nicorandil can enhance the effect of neuraxial morphine inducing paraparesis after aortic occlusion.
This study did not focus on histopathological changes in the spinal cord. Degeneration of -motor neurons was seen consistently in spinal cord taken from rats with morphine-induced spasticity even though this spasticity persisted for only 8 hours (3,4). Although it is likely that this spasticity should lead to the degeneration of -motor neurons in spinal cord even after a noninjurious interval of aortic occlusion, histopathological analysis for the spinal cord should be performed.
In conclusion, our study demonstrated that morphine-induced spastic paraparesis after a noninjurious interval of spinal ischemia could be enhanced by intrathecal nicorandil, a K+ channel opener, and that this enhancing effect was blocked by pretreatment with glibenclamide. The results indicate that neuraxial morphine, even at a small dose, can induce spastic paraparesis in the presence of a K+ channel opener. We should pay careful attention when using neuraxial morphine after thoracoabdominal aneurysm repair surgery.
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Footnotes
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Supported, in part, by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Tokyo, Japan. (No:14571454, 16591551)
Accepted for publication October 28, 2005.
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Abstract
Introduction
