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Department of Anesthesiology-Resuscitology Yamaguchi University School of Medicine, Yamaguchi, Japan
Address correspondence and reprint requests to Mishiya Matsumoto, MD, Department of Anesthesiology-Resuscitology, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi, 755-8505, Japan. Address e-mail to mishiya{at}yamaguchi-u.ac.jp.
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Abstract |
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-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, YM872, administered intrathecally, reduces neuronal injury caused by tetracaine. We first examined the effects of intrathecal YM872 10, 30, 100, or 300 µg in rabbits (n = 3 in each). YM872 produced reversible motor and sensory block in a dose-dependent manner. Then, we evaluated modulatory effects of YM872 (300 µg) on tetracaine-induced glutamate release and neuronal injury. Pretreatment of YM872 did not attenuate 1% or 2% tetracaine-induced increases in cerebrospinal fluid glutamate concentrations (n = 3 in each). For evaluation of neuronal injury, rabbits were assigned to 4 groups (n = 6 in each) and intrathecally received 1% tetracaine and saline (1%T), 1% tetracaine and YM872 (1%TY), 2% tetracaine and saline (2%T), or 2% tetracaine and YM872 (2%TY). The volume of saline, YM872, and tetracaine was 0.3 mL. Saline or YM872 was administered 30 min before tetracaine administration. Neurological and histopathological assessments were performed 1 wk after the administration. Two and 1 animals respectively, showed motor and sensory dysfunction in 1%T, whereas 5 animals showed both motor and sensory dysfunction in 2%T. YM872 improved 2% tetracaine-induced motor dysfunction and neuronal damage (chromatolytic neurons, identified by round-shaped cytoplasm with loss of Nissl substance from the central part of the cell and eccentric nuclei). In 2%TY, 3 animals showed normal motor function and 3 showed mild dysfunction (ability to hop, but not normally), whereas 4 animals showed moderate dysfunction (inability to hop) in 2%T (P = 0.042). Only 2 animals showed one chromatolytic neuron in 2%TY, whereas 5 animals showed 4–16 chromatolytic neurons in 2%T (P = 0.020). These results suggest that AMPA receptor activation is involved, at least in part, in the tetracaine-induced neurotoxicity in the spinal cord. |
Introduction |
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Gold et al. (4) and Johnson et al. (5) have suggested that the mechanism of neurotoxicity is local anesthetic-induced increase in intracellular Ca2+, leading to necrosis or apoptosis. Kitagawa et al. (6) have suggested that local anesthetics cause membrane disruption that results from the detergent nature of local anesthetics.
We have demonstrated, in rabbits, that intrathecal administration of large concentrations of local anesthetics increases glutamate concentrations in the cerebrospinal fluid (CSF) microdialysate and induces motor and sensory dysfunction (7–9). Characteristic histopathological findings in the animals with neurological dysfunction were central chromatolysis of the neuron and vacuolation that was confined within the dorsal funiculus in the spinal cord (7–9). There was no evidence of necrosis of the neurons in the gray matter of the spinal cord or dorsal ganglia. Central chromatolysis results from axonal injury near the cell body. Vacuolation of the dorsal funiculus is thought to be a result of Wallerian degeneration because the dorsal funiculus consists of primary afferent fibers without synapsing in the dorsal horn. The lack of necrosis in the gray matter neurons of the spinal cord or dorsal ganglia suggests that an increase in glutamate concentration in the CSF is not a result of necrosis of glutamatargic neurons. Therefore, we have hypothesized that excessive glutamate, released by large concentrations of intrathecal local anesthetics, leads to axonal injury. Because isolated dorsal columns of the spinal cord in rats were reported to be susceptible to excitotoxic injury via -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) but not N-methyl-d-aspartate (NMDA) receptors (10), we investigated the effect of an intrathecal AMPA receptor antagonist on neuronal injury caused by large concentrations of tetracaine in the present study.
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Methods |
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Experiment 1
To determine the appropriate dose of YM872 ([2,3-Dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl] acetic acid), an AMPA receptor antagonist (Astellas Pharma, Tokyo, Japan), for intrathecal administration to modulate neurotoxicity of tetracaine, we examined the effects of 4 doses of YM872 on motor and sensory function. Histopathological evaluation of the spinal cord was performed 1 wk after administration of YM872.
We administered 0.3 mL of YM872 solution that contained 10, 30, 100, or 300 µg of YM872 into the lumbar subarachnoid space without general anesthesia. Each YM872 dose was studied 3 times. YM872 10 mg was dissolved in 0.97 mL of distilled water with 30 µL of 1N NaOH to adjust pH to 7.3–7.5 (12). Solutions of 10, 30, 100, and 300 µg per 0.3 mL were made using saline.
The region of the motor block was assessed at 2 levels: hindlimbs and forelimbs. The region of the sensory block was assessed at 4 levels: hindlimbs, lower thorax, high thorax, and forelimbs. The onset and recovery time for motor function were defined as the time when motor block of hindlimbs (inability to hop) developed and resolved, respectively. The onset and recovery time for sensory function were defined as the time when the analgesic effects developed and resolved at the thighs, respectively. An analgesia level was evaluated by seeking an aversive response to pinprick stimulation with a 23-gauge needle.
Rabbits were neurologically assessed daily until 1 wk after administration of YM872 by an observer unaware of the treatment groups. The hindlimb motor function was assessed using the 5-point grading scale proposed by Drummond and Moore (13): 4 = normal motor function; 3 = ability to draw legs under body and hop, but not normally; 2 = some lower extremity function with good antigravity strength but inability to draw legs under body and/or hop; 1 = poor lower extremity motor function, weak antigravity movement only; 0 = paraplegic with no lower extremity motor function. The score of the sensory function was assessed by the 3-point grading scale: 2 = normal; 1 = the region with diminished response present; 0 = the region with no response present (8).
After completion of the neurologic function scoring at 1 wk, the animals were reanesthetized and transcardiac perfusion and fixation were performed. The coronal sections of the spinal cord (8 µm) at L5 level were stained with hematoxylin and eosin. Histopathological changes of the spinal cord were assessed at a magnification of 40x–400x by an observer unaware of the treatment groups.
Experiment 2-A
To observe whether YM872 attenuates the increase in glutamate concentrations after tetracaine administration, we measured glutamate concentrations in the CSF by microdialysis. Under general anesthesia with isoflurane 1%, YM872 300 µg (0.3 mL) was intrathecally administered 30 min before intrathecal administration of 1% or 2% tetracaine (0.3 mL, n = 3 in each). The methods of measuring glutamate in the CSF were the same as those in our previous study (7–9).
Experiment 2-B
We intrathecally administered YM872 300 µg (0.3 mL) before tetracaine injection and investigated the effects of YM872 on the neurological and histopathological outcome after intrathecal administration of tetracaine (1% or 2%, 0.3 mL). Animals were mechanically ventilated under general anesthesia in Experiment 2-B because the combined use of YM872 and tetracaine may depress respiration.
The rabbits implanted with an intrathecal catheter were anesthetized in a plastic box with 5% sevoflurane in oxygen. An ear vein catheter was inserted for administration of drugs and lactated Ringer's solution (4 mL · kg–1 · h–1). Pentobarbital (20–30 mg) was administered for facilitation of intubation. After endotracheal intubation, mechanical ventilation was adjusted to maintain a Paco2 at 35–42 mm Hg (fraction of inspired oxygen 0.4, isoflurane 1%). After skin infiltration with 0.25% bupivacaine, a PE-60 catheter was inserted into the left femoral artery for measurement of arterial blood pressure and arterial blood gases. Esophageal temperature was monitored.
In our recent study (14), the cephalad analgesia level after administration of 0.3 mL of 1% and 2% tetracaine in awake rabbits was Th 9-12 and Th 3-11, respectively. Analgesic effects in all animals in the 1% group disappeared by 5 h after the tetracaine administration (14). However, in 3 of 6 animals in the 2% tetracaine group, sensory disturbance persisted until 7 days after the tetracaine administration (14). Based on those results, we selected 1% and 2% tetracaine to examine the effects of YM872 on the neurotoxicity of tetracaine in the present study.
Animals were randomly assigned to the following 4 groups (6 in each): 1% tetracaine and saline (1%T) group, 1% tetracaine and YM872 (1%TY) group, 2% tetracaine and saline (2%T) group, or 2% tetracaine and YM872 (2%TY) group. The 1%T and 2%T groups intrathecally received 0.3 mL of saline 30 min before intrathecal administration of 1% or 2% tetracaine. The 1%TY and 2%TY groups intrathecally received YM872 (300 µg) 30 min before intrathecal administration of 1% or 2% tetracaine. The dose of YM872 (300 µg) was selected based on the results of Experiment 1, which demonstrated that the largest dose of YM872 (300 µg) resulted in neither permanent neurological dysfunction nor histopathological damage. Tetracaine hydrochloride (Kyorin Pharmaceutical, Tokyo, Japan) was dissolved in saline. Tetracaine and YM872 were administered over 1 min.
Mean arterial blood pressure (MAP) was maintained more than 60 mm Hg by infusing phenylephrine after intrathecal administration of tetracaine. Anesthesia and MAP monitoring were continued until 150 min after the administration of tetracaine when phenylephrine was no longer required to maintain MAP. Then, the vascular catheter was removed and the incision was sutured. Extubation of the trachea was performed when adequate spontaneous ventilation occurred. Antibiotic (cephazolin 30 mg/kg, IM) was administered once daily.
Neurological and histopathological assessments were performed in the same manner as in Experiment 1. The degree of the spinal cord damage was assessed individually at the level of L4 to L7 for the chromatolytic change and vacuolation. The neurons with chromatolytic appearance were identified by round-shaped cytoplasm with loss of Nissl substance from the central part of the cell and eccentric nuclei. The chromatolytic neurons were counted in two sections at each level and averaged. The degree of vacuolation of the dorsal funiculus was assessed using a 4-point grading scale: 0 = no vacuolation; 1 = <10% area of the dorsal funiculus is vacuolated; 2 = 10%–50% area of the dorsal funiculus is vacuolated; 3 = more than 50% area of the dorsal funiculus is vacuolated.
Parametric data are presented as mean ± sd. Changes in physiological variables were compared with repeated-measures analysis of variance. The doses of phenylephrine were compared using factorial analysis of variance. The onset and recovery time for motor and sensory block were compared between the YM872 30 µg, YM872 100 µg, and YM872 300 µg groups using unpaired Student's t-test with Bonferroni corrections. The scores of the hindlimb motor function and cutaneous sensation, the number of chromatolytic neurons, and the degree of the vacuolation in the spinal cord were compared between the 1%T and 1%TY groups and between the 2%T and 2%TY groups using a nonparametric method (Mann-Whitney U-test). A Spearman's rank correlation test was applied to analyze the correlation of the number of chromatolytic neurons with the motor function score. P < 0.05 was considered statistically significant.
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Results |
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Experiment 2-A
The mean peak concentrations of glutamate after administration of 1% and 2% tetracaine were about 10-fold and 11-fold larger than baseline values, respectively (Fig. 1).
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Experiment 2-B
There were no significant differences in physiological variables among the four groups. In our preliminary study, intrathecal administration of 300 µg of YM872 decrease MAP to <60 mm Hg in 2 of 7 animals under general anesthesia with 1% isoflurane. In Experiment 2-B, however, only 1 animal (1%TY group) required phenylephrine to maintain MAP after intrathecal administration of YM872. When tetracaine was administered, all animals required phenylephrine to maintain MAP more than 60 mm Hg. The total doses of phenylephrine used after tetracaine administration in the 1%T, 1%TY, 2%T, and 2%TY groups were 0.52 ± 0.21, 0.50 ± 0.14, 0.34 ± 0.36, and 0.61 ± 0.29 mg, respectively. There were no significant differences among the groups.
At 1 wk after the administration of tetracaine, 2 animals showed mild motor dysfunction (score 3) and 1 animal showed mild sensory dysfunction (score 1) in the 1%T group, whereas 5 animals showed mild (score 3) to moderate (score 2) motor dysfunction and mild (score 1) to severe (score 0) sensory dysfunction in the 2%T group (Fig. 2). The hindlimb motor function score was significantly better in the 2%TY group compared with the 2%T group (P = 0.042), whereas there was no significant difference in the hindlimb motor function score between the 1%T and 1%TY group (Fig. 2–A). In the sensory function score, there was no significant difference between the 1%T and 1%TY groups or between the 2%T and 2%TY groups (Fig. 2–B).
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No chromatolytic neurons were observed in the 1%T or 1%TY groups. The total number of chromatolytic neurons at 4 levels (L4–7) was significantly smaller in the 2%TY group compared with the 2%T group (Fig. 3, P = 0.020). Although there was no correlation between the number of chromatolytic neurons and motor function score in the 2%T or 2%TY groups, all animals with moderate motor dysfunction (score 2) showed chromatolytic neurons. The vacuolation of the white matter was confined within the dorsal funiculus (Fig. 4). There were no significant differences in the degree of vacuolation in the dorsal funiculus between the 1%T and 1%TY groups or between the 2%T and 2%TY groups (Table 2). There was no inflammation or necrosis in the gray matter of the spinal cord or dorsal root ganglia.
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Discussion |
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Central chromatolysis of the neuron and vacuolation that was confined within the dorsal funiculus in the lumbar spinal cord suggest that nerve root injury is the main feature of tetracaine-induced neurotoxicity. Li and Stys (10) have demonstrated in an in vitro study using isolated dorsal columns of the rat that astrocytes, oligodendrocytes, and, in particular, the myelin sheath are the primary targets for excitotoxic injury via AMPA but not NMDA receptors. If this is the case in in vivo models, it may be possible that astrocytes, oligodendrocytes, and myelin sheaths around motor and sensory nerve fibers near the nerve root entry zone are damaged via AMPA receptor-mediated glutamate toxicity. In a previous study (14), we reported that nerve fibers myelinated by oligodendrocytes at the nerve root entry zone are highly vulnerable to a large concentration of tetracaine. Similar findings have been reported in a rat model (18).
YM872 is a selective, potent, and highly water soluble AMPA receptor antagonist. The affinity of YM872 for AMPA receptors is similar to that of 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F) quinoxaline (NBQX) (19). YM872 binds only weakly to high-affinity kainate receptors and does not bind significantly to the glutamate binding site or the strychnine-insensitive glycine binding site of NMDA receptors (19). Nishiyama et al. (20) reported in rats that the intrathecal administration of YM872 10 µg produced a disturbance of placing and stepping for 30 minutes and a disturbance of the righting reflex and flaccidity of hindlimbs for 60 minutes after injection and that those effects were reversible in 120 minutes. Although they did not examine histopathology of the spinal cord, the neurobehavioral results are consistent with the present results.
We selected 300 µg of YM872 in the present study because this dose appeared to be the maximum tolerable dose, as motor and sensory block reached the lower cervical level in Experiment 1. In our previous studies, the glutamate concentration in the CSF microdialysate returned to <5 µM within 60 minutes after 2% tetracaine administration (7–9). Therefore, it is likely that AMPA receptors were sufficiently blocked while glutamate concentrations in the CSF were at the toxic level. However, YM 872 did not completely eliminate the neuronal injury caused by tetracaine. YM872 improved neither sensory dysfunction nor the vacuolation of the dorsal funiculus. The discrepancy in the effect of YM872 on the motor and sensory system might be explained by the difference in the vulnerability to tetracaine because dorsal roots have been reported to be more severely damaged than ventral roots after intrathecal administration of tetracaine in rats (18). The findings that dorsal roots are more vulnerable than ventral roots have been confirmed in our recent study (14). Other mechanisms including a local anesthetics-induced increase in intracellular Ca2+ that leads to necrosis or apoptosis (4,5) and membrane disruption that results from the detergent nature of local anesthetics (6) may also be involved. The possibility cannot be excluded that other glutamate receptors including kainate or metabotropic glutamate receptors play a role in the neurotoxicity of tetracaine.
The present study has some limitations. First, we did not assess the cauda equina. However, the cauda equina does not appear to be sensitive to the neurotoxicity of tetracaine in this model (14). Finding little injury to the cauda equina suggests a major difference between the model used in the present study and the clinical cases of local anesthetic injury, which generally involve cauda equina injury. In the rabbit model, nerve fibers myelinated by oligodendrocytes at the nerve root entry zone are highly vulnerable (14). Thus, the effects of an AMPA receptor antagonist on the nerve root entry zone should be evaluated in the future investigation. Second, the number of animals in each group was small. Third, as the dose of YM872 was large, it might be necessary to exclude nonspecific effects of YM872, despite high selectivity of YM872 to an AMPA receptor. Fourth, the partial neutralization of tetracaine solutions by the YM872 solution might affect the results because pH of the YM872 solution is 7.3–7.5, whereas pH of saline is 6.5–6.6.
In summary, we demonstrated that the intrathecal administration of an AMPA receptor antagonist, YM872, reduced the neurological and histopathological injury caused by intrathecal administration of tetracaine. These results suggest that AMPA receptor activation is involved, at least in part, in tetracaine-induced neurotoxicity in the spinal cord.
The authors thank Astellas Pharma Inc. for the supply of YM872.
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