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CARDIOVASCULAR ANESTHESIA

The Activation of Spinal N-Methyl-d-Aspartate Receptors May Contribute to Degeneration of Spinal Motor Neurons Induced by Neuraxial Morphine After a Noninjurious Interval of Spinal Cord Ischemia

Manabu Kakinohana, MD, PhD^*, Osamu Kakinohana, PhD, Jong Hun Jun, MD, PhD, Martin Marsala, MD, Kenneth J. Davison, MD, and Kazuhiro Sugahara, MD, PhD^*

*Department of Anesthesiology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan; Department of Anesthesiology, University of California, San Diego, California; Department of Anesthesiology, Hanyang University College of Medicine, Seoul, Korea; and Department of Anesthesiology, Massachusetts General Hospital, Boston, Massachusetts

Address correspondence and reprint requests to Manabu Kakinohana, MD, PhD, Department of Anesthesiology, University of the Ryukyus, 207 Uehara, Nishihara-cho, Okinawa, 903-0215, Japan. Address e-mail to mnb-shk{at}ryukyu.ne.jp.

	Abstract

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We investigated the relationship between the degeneration of spinal motor neurons and activation of N-methyl-d-aspartate (NMDA) receptors after neuraxial morphine following a noninjurious interval of aortic occlusion in rats. Spinal cord ischemia was induced by aortic occlusion for 6 min with a balloon catheter. In a microdialysis study, 10 µL of saline (group C; n = 8) or 30 µg of morphine (group M; n = 8) was injected intrathecally (IT) 0.5 h after reflow, and 30 µg of morphine (group SM; n = 8) or 10 µL of saline (group SC; n = 8) was injected IT 0.5 h after sham operation. Microdialysis samples were collected preischemia, before IT injection, and at 2, 4, 8, 24, and 48 h of reperfusion (after IT injection). Second, we investigated the effect of IT MK-801 (30 µg) on the histopathologic changes in the spinal cord after morphine-induced spastic paraparesis. After IT morphine, the cerebrospinal fluid (CSF) glutamate concentration was increased in group M relative to both baseline and group C (P < 0.05). This increase persisted for 8 hrs. IT MK-801 significantly reduced the number of dark-stained -motoneurons after morphine-induced spastic paraparesis compared with the saline group. These data indicate that IT morphine induces spastic paraparesis with a concomitant increase in CSF glutamate, which is involved in NMDA receptor activation. We suggest that opioids may be neurotoxic in the setting of spinal cord ischemia via NMDA receptor activation.

	Introduction

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Opiates are often injected into intrathecal (IT) or epidural spaces to produce analgesia in the postoperative period. IT morphine has been used for pain control after thoracoabdominal aortic aneurysm surgery (1) in which spinal cord ischemia could be a complication of aortic cross-clamping. In a previous case report (2), we showed that spastic paraparesis was induced by 4 mg of epidural morphine used for postoperative pain relief after thoracoabdominal aneurysm surgery. We demonstrated that neuraxial morphine, after a noninjurious interval of spinal cord ischemia in the rat, can induce transient spastic paraparesis and degeneration of selective -motoneurons in spinal cord (2,3). In addition, Fuchigami et al. (4) demonstrated that repetitive administration of IT morphine, after a noninjurious interval of spinal cord ischemia in rats, gave rise to irreversible paraplegia and selective -motoneuron death.

In the rat brain, large-dose alfentanil (5), fentanyl (6), and remifentanil (7) produce an increased metabolic rate in the limbic system, seizures, and histologic damage by activation of glutamate receptors (8). Neurological deficit, as a result of neuronal damage after reversible brain and spinal cord ischemia, is thought to be mediated in part by excessive excitatory amino acid (EAA) accumulation (9). Glutamate is believed to be the primary EAA neurotransmitter of the spinal cord, and direct excitotoxic effects by glutamate are thought to be mediated through excessive N-methyl-d-aspartate (NMDA) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor activation, leading to intracellular Ca²⁺ overload (10).

It is unknown whether selective -motoneuron death, induced by neuraxial morphine after a short period of spinal cord ischemia, would be associated with excessive release of EAAs from the spinal cord. In this study, we used the IT loop microdialysis technique (11), which is sufficiently robust to permit continuous or repetitive dialysate collection from the lumbar IT space at intervals for 2 days in unanesthetized and unrestricted rats to characterize the time-dependent release of glutamate following IT administration of morphine after a noninjurious interval of spinal ischemia. In addition, we investigated the effect of MK-801, a NMDA receptor antagonist, on the degeneration of spinal motor neurons induced by neuraxial morphine after a noninjurious interval of spinal cord ischemia in rats.

	Methods

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All studies were performed in accordance with a protocol approved by the Animal Subjects Committee of University of the Ryukyus. To place the IT and microdialysis catheters, we used a technique similar to that previously described for the placement of IT catheters (11,12). Male Sprague-Dawley rats (320–380 g) were anesthetized in an acrylic box with 5% isoflurane in an oxygen and room air mixture (1:1). After anesthetic induction, rats were maintained with 2.5% isoflurane delivered by mask, and the back of the head and neck was shaved. The animals were then placed in a stereotaxic headholder with the head flexed forward. A midline incision was made on the back of the neck. The muscle was freed at the point of attachment to the skull and retracted with a flat elevator, thus exposing the cisternal membrane. The membrane was opened with a stab blade and modestly retracted with a small dural hook. The loop portion of the catheter was inserted through the cisternal opening and passed 9 cm caudal into the IT space. This placed the dialysis membrane at the L3 to L5 spinal segments. After that, an IT catheter was placed for IT drug injection. To accomplish this, an 8.5-cm length of polyethylene (PE)-10 tubing that previously had been stretched to reduce its diameter was inserted after the loop catheter was positioned. The wounds were closed with sutures, and anesthesia was discontinued. Unless otherwise stated, all animals were allowed to recover for a minimum of 5 days before experimentation. Rats that showed motor weakness or signs of paresis upon recovery from anesthesia were euthanized.

On the fifth postimplantation day, rats were anesthetized in an acrylic box with 5% isoflurane in an oxygen and room air mixture (1:1), and anesthesia was maintained with 1.5%–2.5% isoflurane delivered by mask. Rats were prepared for the induction of reversible spinal cord ischemia by a previously described technique (13). To control paravertebral muscle temperature (38.0°C–38.3°C) during anesthesia, a thermocouple was placed into those muscles. Warmed water (38.5°C) was perfused at 100 mL/min through a heat exchanger constructed from metal tubing, and the heat exchanger was placed on the back in a subcutaneous tunnel (T4 to S2). Subsequently, a 2F Fogarty catheter was placed into the descending thoracic aorta through the left femoral artery so that the tip of the balloon catheter reached the level of the left subclavian artery. To control proximal arterial blood pressure at 40 mm Hg during aortic occlusion, the left carotid artery was cannulated with a 22G Teflon catheter, and blood was allowed to flow into an external reservoir during aortic occlusion. Immediately after the completion of arterial cannulation, all animals received 200 U of heparin injected into the tail artery. To induce spinal cord ischemia, the balloon was inflated with 0.05 mL of saline. At 6 min after the induction of transient spinal cord ischemia, the balloon was deflated, and blood was reinfused over 60 s. All catheters were then removed, the wounds were sutured, anesthesia was discontinued, and all animals were allowed to recover. In sham-operated animals, the experimental procedure described above was performed; however, the balloon catheter was not inflated, and hypotension was not induced by withdrawal of blood.

Study 1: Morphine-Induced Paraparesis and Glutamate Release from the Spinal Cord
The animals were assigned to one of the following four groups according to the injection administered 30 min after recovery from anesthesia (n = 8 per group): group C (control), IT saline injection (10 µL); group M, IT injection of morphine (30 µg); group SM, sham-operated and IT injection of morphine (30 µg); and group SC, sham-operated and IT injection of saline (10 µL).

Study 2: The Effect of MK-801 on Histopathologic Changes in the Spinal Cord After IT Morphine Injection
The animals were assigned to one of the following two groups according to IT injection 1 h after IT morphine (30 µg) at 30 min of reperfusion (n = 6 per group): control, IT saline injection (10 µL) 1 h after IT morphine; or MK-801, IT injection of MK-801 (30 µg) 1 h after IT morphine. Histopathologic analysis of the spinal cord was performed 48 h after spinal cord ischemia.

During reperfusion, the recovery of motor function was quantified by assessment of ambulation and placing and stepping responses (2,14). Ambulation (walking with the lower extremities) 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; or 4, no movement, drags lower extremities. The placing/stepping reflex was assessed by dragging the dorsum of the hindpaw over the edge of a surface. This normally evokes a coordinating lifting and placing response (e.g., stepping), which was graded as follows: 0, normal; 1, weak; or 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 (ambulation plus placing/stepping reflex).

To initiate dialysis, one of the externalized PE-10 connections was attached to a 60-cm length of PE-50 tubing (inflow), and the other arm was attached to a 90-cm length of PE-10 (outflow). A syringe pump (Harvard Compact Infusion Pump, Model 975, with a 10-mL plastic syringe) was connected, and the dialysis tubing was perfused with artificial cerebrospinal fluid (ACSF) containing 151.1 mM Na⁺, 2.6 mM K⁺, 0.9 mM Mg²⁺, 1.3 mM Ca²⁺, 122.7 mM Cl^–, 21 mM HCO₃^–, 2.5 mM HPO₄^–, and 3.5 mM dextrose. The ACSF was bubbled by CO₂, with a final pH of 7.4. After a 30-min washout, the microdialysis samples were collected as follows: two baseline samples (30 min each) before ischemia, followed by samples collected after 30 min of reperfusion (before IT injection) and at 2, 4, 8, 24, and 48 h of reperfusion.

Dialysate samples were collected on ice and frozen at –70°C until analysis for glutamate. Analysis was accomplished by the phenyl isothiocyanate derivatization procedure with a Waters high-performance liquid chromatograph with a reverse-phase C18 column (3.9 x 300 mm; 4-µm particle) and an ultraviolet detector. The glutamate content was measured from single 25-µL aliquots. Methionine sulfone was added to the glutamate sample and used as an internal standard. Sensitivity was 5–10 pmol per injection. Amino acid peak heights were initially normalized to the methionine sulfone peak and then quantified on the basis of a linear relationship between the peak height and amounts of corresponding standards. All values are expressed as picomoles per 25-µL tube. External standards were run daily.

At the end of the survival period, rats were killed with pentobarbital (100 mg/kg intraperitoneally) and phenytoin (25 mg/kg intraperitoneally). The rats were then transcardially perfused with 100 mL of heparinized saline followed by 150 mL of 4% paraformaldehyde in phosphate buffer (pH 7.4). Twenty-four hours later, the spinal cords were removed and postfixed in the same fixative for 2–14 days. The L3, L4, and L5 spinal nerves and the L3, L4, and L5 spinal segments were then dissected and cryoprotected in 30% sucrose solution. Frozen transverse sections (20–30 µm) were prepared and stained by the Kluver-Barrera or Nissl method. For systematic analysis, 10 representative sections taken from segments L3 to L5 (a total of 30 sections from each spinal cord) were coded for each animal and then subjected to a systematic examination. The number of normal-appearing and dark-stained -motoneurons was counted by the observer without knowledge of the treatment group (MK).

Drugs were purchased as follows: morphine sulfate from Dainihon Pharm (Tokyo, Japan) and MK-801 from Research Biochemicals (Natick, MA). Drugs were dissolved in saline for injections. Doses for spinal delivery were delivered in a volume of 10 µL. Each IT injection was followed by an injection of 10 µL of saline to flush the catheter.

Results of MDI are expressed as the median. CSF glutamate concentrations are expressed as mean ± sd. Statistical analyses of physiologic data were performed by one-way analysis of variance (ANOVA) for multiple comparisons followed by the Dunnett post hoc test. For analysis of neurological outcome in the individual groups, significant overall values were obtained by the Friedman test followed by Wilcoxon's signed rank test. Specific comparisons between experimental groups at individual time points after reflow were made with the Kruskal-Wallis test followed by the Tukey-Kramer test. Comparison of CSF glutamate levels was made by one-way repeated-measures ANOVA followed by the Dunnett test, and the difference among groups at each time was examined by one-way ANOVA followed by Fisher's least squares difference test. The Pearson's correlation coefficients (r) between the area under the curve for glutamate (AUC_GLU) and the percentage of dark-stained -motoneurons were determined. A P value of <0.05 was considered significant. Statistical analyses were performed with SPSS 8.0.1 for Windows (SPSS Institute, Chicago, IL).

	Results

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Study 1
During the pre-, intra-, and postischemic periods, there were no significant differences in any variable among the groups except for the intraischemic distal blood pressure in the sham-operation groups (groups SM and SC) (Table 1). In group S (IT saline), all animals had modest and transient motor weakness (median MDI of 3) 30 min after reperfusion (before IT injection), followed by gradual recovery over 24–48 h of reflow. During the initial 8 h of reperfusion, the motor deficit was manifested as the presence of ataxia (but with a preserved ability to walk) and partial weakness in the placing/stepping reflex. At 48 h, no significant motor deficit was seen, and all animals had complete recovery. In group M, IT injection of morphine (30 µg) resulted in a gradual development of spasticity and a nearly complete loss of the ability of animals to stand, walk, or step. Significant morphine-induced spasticity was observed in the initial 4 h after morphine administration (P < 0.01) and persisted for 8 h. MDI in group M was worse than that of group S at 2, 4, and 8 h after reflow. Between 24 and 48 h after reflow, most animals in group M regained motor function. In the sham-operated rats, IT injection of morphine (group SM) or saline (group SC) had no significant effect on neurological function, and all animals ambulated normally after recovery from anesthesia (Table 2).

The CSF glutamate concentration at 30 min after short-lasting (6 min) spinal ischemia did not change significantly compared with baseline in all groups. Also, there was no significant difference among groups. After IT injection of morphine, a significant increase in the CSF glutamate concentration in group M was observed, and this persisted for 8 h in comparison with baseline and group S at each point. Such an increase in the CSF glutamate concentration was not observed 24 and 48 h after reperfusion: the level of each group was comparable with baseline (Table 3).

Systematic histopathologic analysis of the L3 to L5 spinal segments at the end of 48 h of survival showed only a few dark-stained -motoneurons in group S (Fig. 1c) and no detectable neurodegenerative changes in groups SM (Fig. 1d) or SC. In contrast, in group M (Fig. 1, a and b), histopathologic analysis of spinal cords revealed the occasional presence of dark-stained -motoneurons. There were significantly more degenerative -motoneurons in group M than in groups S, SM, and SC (Table 4). There was a significant linear correlation between the AUC_GLU and the number of dark-stained -motoneurons (r² = 0.432; P = 0.006) in groups S and M (Fig. 2).

View larger version (151K): [in this window] [in a new window]   Figure 1. a and b, Light microphotographs of a transverse (20-µm) section taken from the L4 spinal segment of an animal subjected to 6 min of spinal cord ischemia and 48 h of reperfusion (group M). This animal was injected with 30 µg of intrathecal (IT) morphine 30 min after the onset of reperfusion. Dark-stained motoneurons (arrows) were observed in the ventral horn (original magnification, 10x [a] and 20x [b]). c, Light microphotograph of a transverse (20-µm) section taken from the L3 spinal segment of an animal subjected to 6 min of spinal cord ischemia 48 h after reperfusion. This animal received 10 µL of IT saline at 30 min after the onset of reperfusion (group C). Although a few dark-stained -motoneurons can be seen in only two rats, normally appearing -motoneurons and interneurons were observed in most animals (original magnification, 20x). d, Light microphotograph of a transverse (20-µm) section taken from the L3 spinal segment of an animal subjected to sham operation and 30 µg of IT morphine 30 min after the operation (group SM). Normal-appearing -motoneurons and interneurons can be observed (original magnification, 20x).

View larger version (14K): [in this window] [in a new window]   Figure 2. Linear association between the area under the curve (AUC) of the cerebrospinal fluid glutamate level (AUCGLU) and the percentage of dark-stained -motoneurons (r2 = 0.432; P = 0.006) in groups C (saline) and M (morphine).

Study 2
During all of the experiments, body temperature (paravertebral muscle temperature) ranged from 37.3°C to 38.2°C. The baseline distal arterial pressure was 84 ± 10 mm Hg and decreased to 3 ± 1 mm Hg at the end of 6 min of aortic occlusion in all of the experiments. No significant differences among experimental groups were detected in the physiological data (Table 5).

Histopathologic analysis revealed the occasional presence of dark-staining -motoneurons in the control group but few neurodegenerative changes of -motoneurons in the MK-801 group (Fig. 3). There was a significant difference in the number of dark-staining -motoneurons between the control and the MK-801 groups (Table 6).

View larger version (133K): [in this window] [in a new window]   Figure 3. a and b, Light microphotographs of a transverse (20-µm) section taken from the L3 spinal segment of an animal subjected to 6 min of spinal cord ischemia and injected with intrathecal (IT) morphine (at 30 min of reperfusion) and saline (at 1 h of IT morphine) (control group). Some dark-stained -motoneurons (arrows) localized in the ventral horn (VH) can be seen. c and d, Light microphotographs of a transverse (20-µm) section taken from the L3 spinal segment of an animal subjected to 6 min of spinal cord ischemia, IT morphine (at 30 min of reperfusion), and MK-801 (at 1 h after IT morphine) (MK-801 group). Normal-appearing -motoneurons and interneurons were observed.

	Discussion

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Rats that received IT morphine after a short interval of aortic occlusion gradually developed spasticity and a nearly complete loss of ability to stand, walk, or step. Additionally, the peak concentration of glutamate in CSF after IT morphine after a noninjurious interval of spinal ischemia significantly increased (approximately 55%) over baseline during reperfusion. Of importance to us, histopathologic analysis of the spinal cord revealed degeneration of -motoneurons only in rats with a significant increase in the glutamate concentration in CSF and the neuroprotective effect of MK-801 (noncompetitive NMDA receptor antagonist) against the degeneration of -motoneurons after IT morphine injection after a noninjurious interval of spinal cord ischemia.

Our recent article (2) was the first clinical and experimental report of paraparesis induced by neuraxial morphine after a noninjurious interval of aortic occlusion. In our previous reports (2–4), we speculated on several mechanisms by which IT morphine can induce spasticity after a noninjurious interval of spinal ischemia. The first is increased sensitivity to morphine in the ischemic spinal cord. Ting et al. (15) demonstrated a twofold to threefold increase in binding sites of brain µ, , and agonists during the early reperfusion period after temporary focal cerebral ischemia in the cat. From this report, it can be considered that through an increase in the concentration of spinal opioid receptors during the reperfusion period, sensitivity to morphine should be increased by an ischemic insult to the spinal cord. Second, -aminobutyric acid (GABA) or glycinergic interneurons may be blocked by morphine. With respect to the interaction between morphine and GABA or glycinergic interneurons, it was reported that opiate alkaloids, including morphine, appeared to inhibit GABA and glycinergic interneuronal function in the spinal cord (16,17). From these results and suggestions, it can be speculated that the increase in sensitivity to opioids after a spinal ischemic insult might enhance the effect of IT morphine and that this might block the inhibitory input (GABA, glycinergic, or both) to motoneurons, thus leading to increased spasticity in the hind limb.

This microdialysis study with a loop microdialysis catheter in the subarachnoid space has a number of specific advantages, including the reduction of the effects of surgical stress and the permission of continuous or repetitive dialysate collection in unanesthetized and unrestricted rats (11). Therefore, behavioral assessment during reperfusion in unanesthetized and unrestricted animals can be performed simultaneously with the collection of dialysate. The mean CSF glutamate concentration (approximately 30 pmol/25 µL) observed in intact rats in this study is consistent with data in a previous study (18) that used the loop microdialysis technique in rats. Our microdialysis study showed that the concentration of glutamate in CSF after IT morphine in group M significantly increased (approximately 55%) over baseline and returned to the baseline, corresponding to a transient motor dysfunction. These data suggest that the time-course changes of glutamate concentration in CSF may be related to onset of spasticity and its recovery to baseline levels. Earlier studies demonstrated that there is a selective degeneration of small and medium interneurons (GABAergic, glycinergic, or both) typically localized between laminae V and VII after periods of transient spinal ischemia sufficient to produce spastic paraplegia (13,19,20). In previous studies (21,22), it has been shown that spinal administration of strychnine (a glycine receptor antagonist) or bicuculline (a GABA_A receptor antagonist) is associated with an increase in spinal CSF glutamate release and the development of allodynia, which is effectively blocked by NMDA receptor antagonists. These data support, although not directly, our speculation that the increased glutamate concentration in CSF after IT morphine may be associated with blocking of the inhibitory system (GABAergic, glycinergic, or both) by activating opioid receptors after spinal ischemia.

A significant increase in the spinal extracellular concentration of glutamate, as measured by intraparenchymal (approximately 200%–300% of baseline) (23) or IT (approximately 40%–50% of baseline) (24) microdialysis, was reported during an injurious interval of aortic occlusion in rats. Thus, the morphine-induced increase in glutamate concentration (about 55% above baseline) appears to be similar to that observed during an injurious interval of spinal cord ischemia. Our previous reports (2–4) demonstrated that histopathologic damage to -motoneurons can be induced by a single IT morphine injection after six minutes of aortic occlusion and also that repetitive administration of IT morphine after six minutes of spinal cord ischemia can induce irreversible paraplegia with the loss of -motoneurons. From those data, however, it was not clarified whether this selective damage to -motoneurons was associated directly with excessive accumulation of glutamate in the CSF after IT injection of morphine. This study showed that the CSF glutamate concentration after IT morphine administration may have something to do with degeneration of - motoneurons (Fig. 2). It was reported that acute excitotoxicity in spinal motoneurons is mediated by the activation of glutamate receptors (25–28). In Study 2, it was found that IT treatment with MK-801 (a noncompetitive NMDA receptor antagonist) decreased the number of degenerative -motoneurons in spinal cord taken from rats with morphine-induced paraplegia (Table 6). Thus, it appears likely that persistent accumulation of glutamate in CSF followed by IT morphine after a noninjurious interval of spinal ischemia may play an important role in the damage to spinal -motoneurons through activation of NMDA receptors.

A single injection of IT morphine induced transient spastic paraplegia, and this motor dysfunction was reversed at 24–48 hours after spinal cord ischemia. This duration corresponded to the typical time course of spinally-administered morphine in the rat (29). Although this motor dysfunction was not persistent, histopathologic analysis revealed selective degeneration of some -motoneurons. In our previous study (4), repetitive IT morphine after six minutes of aortic occlusion induced irreversible paraplegia with degenerative changes of most -motoneurons. These results suggest that a persistent activation of spinal opioid receptors might induce irreversible damage in spinal -motoneurons, even after a noninjurious interval of aortic occlusion. In the clinical setting, postoperative analgesia is sometimes performed by neuraxial morphine infusion after thoracoabdominal aneurysm repair surgery. In that case, patients are often sedated such that motor function cannot be fully assessed in the early postoperative period. Considering our data, we would like to emphasize that all anesthesiologists should be aware of the possibility of morphine-induced spasticity after thoracic aorta surgery.

	Conclusion

Top Abstract Introduction Methods Results Discussion Conclusion References

 
IT morphine administration was associated with transient spastic paraparesis in rats subjected to a noninjurious interval of spinal cord ischemia. Increased CSF concentrations of glutamate, corresponding to a gradual development of spasticity, were associated with the degenerative changes of -motoneurons induced by activation of the NMDA receptor. Although, behaviorally, there was no evidence of a persistent neurological dysfunction in the rats, our current results suggest that the effects of morphine in clinical scenarios need to be carefully assessed.

	Footnotes

 
This study was supported in part by a grant-in-aid for scientific research (No. 14571454).

Accepted for publication July 27, 2004.

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