Abstract
Objective
Local anesthetics may cause reversible skeletal muscle injury, which is most common with bupivacaine. Glutamate is present in most skeletal muscles and may regulate muscular metabolic pathways. The aim of study was to examine the changes in intramuscular glutamate level and the severity of myopathy after intramuscular administration of bupivacaine.
Methods
Twenty-four male Wistar rats were divided into four groups with six rats per group. A microdialysis probe was implanted in the right tibialis anterior (RTA) muscle in all rats. After equilibrating the microdialysis system for 2 hours, 0.2 mL of normal saline or 0.25%, 0.5% or 1% bupivacaine was injected into the RTA muscle and dialysate samples were collected every 30 minutes for 4 hours. Glutamate was measured by high-performance liquid chromatography. Three days later, the RTA muscle was dissected and the injection site was examined histologically to evaluate the severity of muscle damage on a four-point score, representing no damage to severe myonecrosis.
Results
Bupivacaine significantly increase the level of glutamate in a dosedependent manner in the RTA muscle. Histological assessment of the RTA muscle after bupivacaine administration revealed dose-dependent damage (mean score: normal saline, 0; 0.25% bupivacaine, 1.2 ± 0.6; 0.5% bupivacaine, 1.8 ± 0.7; 1% bupivacaine, 2.4 ± 0.8; p < 0.05).
Conclusion
A single intramuscular injection of bupivacaine induced dose-dependent increases in intramuscular glutamate levels and muscular damage.
Keywords
bupivacaine; drug toxicity; glutamates; muscles;
1. Introduction
Local anesthetics are myotoxic agents at clinical concentrations, and the potency of toxicity is drugspecific and dose-dependent.1−3 Bupivacaine is considered to be the most myotoxic local anesthetic. 1,2,4,5 In a laboratory setting, bupivacaineinduced myopathy has been established as an animal model to study muscle degeneration.6 However, the mechanisms involved in bupivacaine-induced myotoxicity are still unclear. An increased intracellular calcium concentration has been proposed to play a crucial role in the pathogenesis of bupivacaine- induced myotoxicity.7,8
Glutamate is a well-known excitatory neurotransmitter in the central nervous system and elevated glutamate concentrations are implicated in neurotoxicity. The role of glutamate in synaptic signaling has been the subject of a large number of studies. Glutamate signaling was recently demonstrated in non-neuronal tissues such as bone, pancreas and skin.9 In addition to the existence of glutamate receptors in the nervous system, glutamate receptors are also expressed on non-neuronal components, such as Schwann cells10 and keratinocytes. 11 These findings suggest that glutamate is a widespread transmitter and can influence cellular function in a number of different tissues. In skeletal muscle, glutamate may be involved in various metabolic pathways.12 Glutamate receptors have been discovered in the skeletal muscles of rats and mice13 and in myoblast cells.14 However, the physiological roles of the muscular glutamate receptors are still not understood. Therefore, the aim of the present study was to examine the changes of intramuscular glutamate levels and mus cular damage after intramuscular injection of bup vacaine in rats.
2. Methods
2.1. Animal models
Male Wistar rats were bought from the National Laboratory Animal Center, Taipei, Taiwan. The experimental protocol was approved by the Institutional Animal Care Committee of the National Defense Medical Center, Taipei, Taiwan. Twentyfour male Wistar rats (320−400 g) were used and allowed free access to food and water before the experiment. All experiments were carried out during the light time. An initial dose of sodium pentobarbital 60 mg/mL (0.14 mL/100 g body weight) was injected intraperitoneally for anesthesia and supplemental doses (0.015 mL/100 g body weight) were given every 30 minutes, if needed. Using a 20-G needle, the microdialysis probe linked to a PE-5 tube (two side holes at the dialysis membrane site) was placed in the right tibialis anterior (RTA) muscle of the rat, parallel with the muscle fibers. After equilibrating the microdialysis system, 0.2 mL of the study drug was injected through the PE-5 tube into the RTA muscle. Dialysate samples were collecte every 30 minutes for 4 hours. The rats were randomly divided into four groups (n = 6/group) and received an intramuscular administration of normal saline (control group) or an intramuscular administration of 0.25%, 0.50% or 1.0% bupivacaine (Sigma, St Louis, MI, USA) dissolved in normal saline.
2.2. Construction of microdialysis probe and dialysate samples collection
The microdialysis probes were constructed as previously described.15 In brief, the probe was constructed using two 5-cm PE-5 tubes (0.02 cm inner diameter, 0.036 cm outer diameter) and a 1.5-cm cuprophan hollow fiber (300 μm outer diameter, 200 μm inner diameter, 50 kDa molecular weight cut-off; Filtrai, AN 69-HF, Eicom Co., Kyoto, Japan), which were connected by two polycarbonate tubes (194 μm outer diameter, 102 μm inner diameter; 0.7 cm in length) with epoxy glue. Both ends of the dialysis probe were connected with a silastic tube. For drug administration, another 10-cm PE-5 tube with a blind end and two side holes over the dialysis membrane site was connected to the microdialysis probe. Figure 1 shows a schematic representation of the microdialysis system.
One of the external silastic tubes was connected to a syringe pump (CMA-100; CMA Microdialysis, Acton, MA, USA) to perfuse Ringer’s solution (8.6 mg/mL NaCl, 0.3 mg/mL KCl, 0.33 mg/mL CaCl2) at a flow rate of 5 μm/min, and the other was attached to a PE-10 tube for dialysate collection. After equilibration for 2 hours, two consecutive 30-minute dialysate samples were collected as the basal level, which was followed by the injection of normal saline or bupivacaine (0.25%, 0.5% and 1%). Dialysate was collected for 4 hours, with 30-minute intervals. During the in vitro measurements, the recovery rate of the dialysis probe was 45% at an infusion rate of 5 μm/min. All samples were collected in polypropylene tubes on ice, and stored at −80ºC until assayed. The concentrations of glutamate were measured by highperformance liquid chromatography (Agilent 1100; Agilent Tech nologies, Palo Alto, CA, USA) with a fluorescence detector set at 428 nm (Gilson model 121, which is a part of the Agilent 1100 set) as previously described.16
Download full-size image
2.3. Histopathological examination of muscles
Three days after saline or bupivacaine injection, the rats were sacrificed by intracardial injection of pentobarbital, and the RTA muscles were dissected at the injection sites. The muscle samples were immediately fixed in neutral formalin overnight and 4-μm-thick paraffin sections were prepared and stained with hematoxylin and eosin for light microscopy. The muscle sections were evaluated by an examiner unaware of group or treatment assignment. The samples were examined under a light microscope and the severity of damage was graded on a four-point scale, where 0 = no damage, 1 = mild muscle damage with localized inflammatory cells infiltration, 2 = moderate muscle cell damage with inflammatory cells infiltration, and 3 = severe destruction of the muscle mass with necrosis. To assess the specific extent of RTA muscle injury in each group, five cross-sections from one injection site were randomly chosen for histological examination and scoring. Thirty sections were examined per group.
2.4. Statistical analysis
Muscle damage is presented as the mean ± standard deviation. The differences in severity of muscle damage between the groups were analyzed using the Kruskal-Wallis test, and post hoc comparisons were performed using the Mann-Whitney U test. The percentage change from the basal glutamate level is presented as the mean ± standard error of the mean. Values at each time point for the four groups were analyzed by one-way analysis of variance, and post hoc multiple comparisons evaluated with unpaired two-tailed t tests. Values of p < 0.05 were considered statistically significant.
3. Results
The muscles obtained from the animals treated with normal saline showed no histological abnormality. In contrast, bupivacaine elicited significant dosedependent muscle damage (Figure 2 and Table 1). There was no change in the intramuscular glutamate level in rats treated with normal saline. In contrast,after the injection of 0.5% and 1% bupivacaine, the glutamate concentration was significantly elevated throughout the 4-hour study period (Figure 3). There was a mild increase in the glutamate concentration in the 0.25% bupivacaine group during the observation period (Figure 3).
Download full-size image
Download full-size image
4. Discussion
This study showed that bupivacaine caused skeletal muscle damage and an increase in glutamate levels in the RTA muscle in rats. These effects occurred in a dose-dependent manner.
Several lines of evidence from animal and human studies suggest that intramuscular glutamate is related to acute and chronic muscle pain.17−20 Intramuscular injection of glutamate may sensitize the sensory afferent fibers in the rat masseter muscle, which is mediated by the peripheral excitatory amino acid receptors.21 It has been reported that glutamate release in muscles may contribute to the sensation of muscle pain.19,20 Furthermore, direct injection of glutamate into the human masseter muscle evoked painful sensation.18,22 Bupivacaineinduced myopathy is well known to be a painful symptom. For example, Hogan et al1 reported a patient who received repeated interscalene brachial plexus block with 0.5% bupivacaine, and developed postoperative sternocleidomastoid muscle pain. In that patient, bupivacaine-induced myotoxicity was believed to be the underlying cause.
Benoit et al8 hypothesized that an increase in intracellular calcium plays a key role in local anesthetic- induced myotoxicity. An excessive calcium efflux from the sarcoplasmic reticulum (SR) was found after exposure to bupivacaine in pig muscle cells.23 Zink et al7 demonstrated that bupivacaine evokes calcium release from the SR and suppresses the calcium reuptake into the SR in mouse skeletal muscle fibers. The increased intracellular calcium concentration may trigger cellular degeneration and cell death. It has also been reported that intracellular calcium overload may induce mitochondria damage24 and, in fact, mitochondria play a key role in the pathogenesis of muscular cell death.25
In an in vitro study, Irwin et al26 showed that bupivacaine caused dose-dependent mitochondrial damage in rat skeletal muscle. Based on these findings, bupivacaine-induced muscle cell damage may be related to mitochondrial injury, which could be caused by elevated intracellular calcium levels. Lee et al14 demonstrated that exposure to glutamate increased the intracellular calcium level in C2C12 myoblast cells and that this effect occurred in a dose-dependent manner. Further more, they found that the increased intracellular calcium was due to calcium influx from the extracellular medium via N-methyl-D-aspartate receptors. In another study, Frank et al27 reported that glutamate-evoked calcium increase in C2C12 myoblast cells was due to the release of calcium from the intracellular stores. However, these results suggested that glutamate may increase intracellular calcium levels in muscle cells. Therefore, from the results of the present study, we believe that, in rat skeletal muscle, the increased glutamate release induced by bupivacaine may evoke an increase in the intracellular calcium level, and subsequently myopathologic changes.
In addition to myotoxicity, neurotoxicity is another form of local poisoning associated with local anesthetics. In terms of neurotoxicity, several possible mechanisms have been proposed, which include increased intracellular calcium concentrations,28 mitochondrial injury,29 increased glutamate concentrations, 30 and dissociation of calcium from the sodium channel.31 An increased intracellular calcium concentration,7,8 mitochondrial injury26 and dissociation of calcium from the sodium channel32 have also been proposed as possible mechanisms underlying local anesthetic-induced myotoxicity. Thus, we speculate that the mechanisms involved in local anesthetic-induced myotoxicity and neurotoxicity share a common pathway.
Tissue injury induced by the insertion of a microdialysis probe has been reported and it may induce the release of glutamate in muscles.33 Thus, the muscle must be restored to a steady state before sample collection, which involves a period of time for equilibration. Tegeder et al19 reported that the glutamate concentration was elevated for the first hour after inserting the microdialysis catheter in a human experimental muscle pain study and that the glutamate concentration returned to the baseline level within 2 hours. Therefore, to avoid similar confounding factors, in this study we allowed the microdialysis system to equilibrate for 2 hours before treatment and dialysate sample collection.
Local anesthetic-induced myotoxicity is not considered a major clinical problem because of the unapparent symptoms/signs and the reversibility of muscular damage.2,34 However, it is still a clinical problem because local anesthetics can induce muscle injury when they are administered directly in skeletal muscle or in tissue adjacent to skeletal muscle.1,3,5 Indeed, many clinical case reports have demonstrated muscle injury after local anesthetic injection for retro- and peribulbar blocks,35 trigger point infiltration for myofascial pain36 and peripheral nerve blocks,1 for example. Repeated injection appears to be a crucial factor involved in local anesthetic- induced myotoxicity.3,4 Thus, multistage oral surgery in which local anesthetics are injected into the tongue, and some treatments for myalgia or myofascial pain, in which repeated injections of local anesthetics are needed, must be managed with care.
In summary, a single injection of bupivacaine to rat skeletal muscle caused muscle damage and increased the intramuscular glutamate level in a dosedependent manner. Although the myotoxic effects of local anesthetics are still widely debated, further studies are needed to clarify whether there are long-term muscular effects after administration of these drugs.