Age-dependent Bupivacaine-induced Muscle Toxicity during Continuous Peripheral Nerve Block in Rats

This study, including care of the animals involved, was conducted according to the official edict presented by the French Ministry of Agriculture (Paris, France), and the recommendations of the Declaration of Helsinki. Thus, these experiments were conducted in an authorized laboratory and under the supervision of an authorized researcher (K.N.-G.).

Plain bupivacaine hydrochloride, 0.25% (7.5 mm), was purchased from Astra Zeneca (Rueil-Malmaison, France) for rat administration. All other reagents were purchased from Sigma-Aldrich (Saint Louis, MO), with the exception of the ATP monitoring kit (ATP Bioluminescence Assay Kit HS II; Roche Diagnostics GmbH, Mannheim, Germany).

Experiments were conducted on young (3-week-old, 70- to 100-g) and adult (12-week-old, 200- to 240-g) male Wistar rats. Rats were housed in a regulated facility with a 12-h light/12-h dark cycle, were fed with chow, and were allowed free access to tap water. After anesthesia with intraperitoneal sodium pentobarbital (40 mg/kg) and subcutaneous injection of lidocaine (3 and 10 mg, respectively, in young and adult rats), a plexus catheter (Pajunk, Geisingen, Germany; 25 gauge, 0.8 mm OD, and 20 gauge, 0.9 mm OD, in young and adult rats, respectively) was surgically inserted under the inguinal ligament near the left femoral nerve sheath by an authorized and trained researcher (K.N.-G.). It was fixed with stitches on the quadriceps muscle, passed under the skin, and exited at the neck. Incisions were subsequently closed by suturing.

Animals were randomly divided into four different groups according to the type of perineural catheter injection of 1 ml/kg bupivacaine, 0.25% (bupivacaine groups), or saline (saline groups) and age (adult and young rats). Seven perineural injections 8 h apart were performed. They induced a decrease in pinprick sensation in the cutaneous distribution of the femoral nerve but not complete motor blockade in the first hour after each bupivacaine injection. Rats were killed by cervical dislocation 8 h after the last perineural injection, when the bupivacaine concentration in muscle was below the detection threshold (< 0.3 μg/g tissue), as previously published.23 

Psoas muscle was quickly dissected adjacent to the femoral nerve, with the former tip region of the catheter located in the middle of the tissue block, and placed in a normoxic (i.e. , equilibrated with air), cooled (4°C) relaxing solution (solution 1: 10 mm EGTA, 3 mm Mg²⁺, 20 mm taurine, 0.5 mm dithiothreitol, 5 mm ATP, 15 mm phosphocreatine, 20 mm imidazole, and 0.1 m K⁺2-[N-morpholino]ethane sulfonic acid, pH 7.2). To assess mitochondrial respiration, we used a permeabilized muscle fiber technique.24Bundles of 2- to 5-mg fibers were excised from the surface of the psoas and then permeabilized in solution 1 with 50 μg/ml saponin added. The bundle was then washed twice for 10 min each time in solution 2 (10 mm EGTA, 3 mm Mg²⁺, 20 mm taurine, 0.5 mm dithiothreitol, 3 mm phosphate, 1 mg/ml fatty acid–free bovine serum albumin, 20 mm imidazole, and 0.1 m K⁺2-[N-morpholino]ethane sulfonic acid, pH 7.2) to remove saponin. All procedures were performed at 4°C with extensive stirring. The success of the permeabilization procedure was estimated by determining the activity of the cytosolic lactate dehydrogenase, and the mitochondrial citrate synthase in the medium. After 15–20 min of permeabilization, more than 60% of the cytosolic lactate dehydrogenase was found in the external medium, and the mitochondrial citrate synthase activity in the medium remained below 5%.24,25The oxygen consumption rate was measured polarographically at 30°C using a Clark-type electrode (Strathkelvin Instruments, Glasgow, United Kingdom) connected to a personal computer that displayed on-line the respiration rate value (949 Oxygen System; Strathkelvin Instruments). Oxygen solubility in the medium was considered to be equal to 450 nmol O/ml. For each measurement, a 2-ml oxygraph chamber was filled with one bundle of fibers in solution 2 with 10 mm malate plus 10 mm glutamate, 10 mm malate plus 10 mm pyruvate (substrates of complex I), or succinate plus complex I inhibitor rotenone (1 mg/ml dimethyl sulfoxide and ethanol 1:1) as substrates; 50 μm di(adenosine 5′)-pentaphosphate, 20 μm EDTA, and 1 mm iodoacetate were also added to the cuvette to inhibit extramitochondrial ATP synthesis (via  the glycolysis or the adenylate kinase) and ATP hydrolysis.26After 5 min, adenosine diphosphate was added to a final concentration of 1 mm to initiate state 3 respiration (concomitant with ATP synthesis) under saturating conditions.27State 4 respiration was recorded after addition of 70 μm atractyloside (adenine nucleotide translocator inhibitor) and 1 μm oligomycin (ATPase inhibitor). After the measurements, fibers were removed, dried on a precision wipe, and weighed. Respiration was expressed as ng atom O · min⁻¹· mg wet weight⁻¹of the muscle fiber. Concomitant ATP synthesis measurements were performed by bioluminescence measurements (luciferin–luciferase system) of the ATP produced after addition of 1 mm adenosine diphosphate. At various time intervals after addition of adenosine diphosphate, 10-μl aliquots were withdrawn from the oxygraph chamber, quenched in 100 μl dimethyl sulfoxide, and diluted in 5 ml ice-cold distilled water. Standardization was performed with known quantities of ATP measured under the same conditions. ATP synthesis rate was expressed as nmol ATP · min⁻¹· mg wet weight⁻¹of the muscle fiber.26This allowed for calculation of the efficiency of ATP production (ATP/O ratio).

For the enzymatic measurements, approximately 60 mg of psoas muscle was minced and homogenized with a glass Potter homogenizer, in ice-cold medium (10% wt/vol) containing 225 mm mannitol, 75 mm sucrose, 10 mm Tris-HCl, and 0.10 mm EDTA, pH 7.2. The homogenate was then centrifuged for 20 min at 650 g . The supernatant was collected, and the protein concentration was determined.28Enzymatic activity of citrate synthase was assessed using previously described spectrophotometric procedures on a SAFAS UVmc2 (SAFAS, Monaco, France) and was expressed in nanomoles of substrate transformed per minute and per milligram of protein. Citrate synthase activity was measured as described by Srere29in the presence of 4% Triton (vol/vol) by monitoring at 412 nm wavelength at 30°C the formation of thionitrobenzoate dianion from the reaction of coenzyme A and 5,5′-dithiobis(2-nitrobenzoic acid).

The muscle sample (20–40 mg) was initially fixed with 3.5% solution of glutaraldehyde in 0.1 m phosphate buffer (pH 7.4).30,31The samples were fixed at 4°C for 24 h until postfixation. The minced tissue samples were postfixed by using 2% osmium tetroxide. After postfixation, the samples were subsequently dehydrated by using a graded series of ethanol washes (30, 50, 75, 95, and 100%). The tissue samples were longitudinally oriented and embedded in epoxy resin (Spurr). Three sample blocks were obtained from each muscle biopsy sample. Thin sections (80–90 nm) were obtained initially and placed on 200-mesh copper grids for electron microscopy. The sections on each grid were stained by using 1.5% uranyl acetate in 70% ethanol, rinsed with distilled water, and then stained with 0.1% lead citrate. Sections were viewed on a Hitachi 7100 electron microscope (Tokyo, Japan) operated at 75 kV in the Centre de Ressources en Microscopie Electronique, Montpellier, France. A representative section on each grid was viewed at ×5,000 magnification, and micrographs were taken of each fiber.

The samples were evaluated by two independent and blind examiners unaware of group or treatment. To access the specific extent of skeletal muscle changes, 100 cross sections per injection site were performed, and 15 cross sections among them were randomly chosen.

Each viable muscle fiber was analyzed for structural muscle damage. Hypercontracted fibers were excluded from the analysis, because the cause of associated damage was unable to be determined.32A viable muscle fiber was defined as a longitudinally oriented fiber with minimum visible length of 200 μm and minimal muscle contraction. Each fiber was analyzed individually for the extent of damage, and all fibers per subject were assessed for the extent of fiber disruption. The percentage of fibers exhibiting myofibrillar disruption was then calculated for each subject. A disrupted fiber was defined as any fiber containing disruptions in the normal myofibrillar banding pattern. Specifically, any fibers exhibiting Z-line streaming or M-band disruption, as well as disruption of the myofilament structure within sarcomeres, were classified as damaged. An area of disruption occupying one to two adjacent myofibrils and/or one to two continuous sarcomeres was classified as a “focal” disruption.30,31An area of disruption encompassing 3–10 adjacent myofibrils and/or 3–10 continuous sarcomeres was termed “moderate” disruption, and an area of disruption covering more than 10 adjacent myofibrils and/or continuous sarcomeres was considered “extreme.”

For mitochondrial respiration and citrate synthase activity, quantitative data were reported as a median [25th and 75th percentiles] because of a nonnormal distribution. Data from the four groups was then compared using a Kruskal–Wallis test. Mann–Whitney tests were performed by comparing young saline group versus  young bupivacaine group, young saline group versus  adult saline group, and young bupivacaine group versus  adult bupivacaine group (with P < 0.0166 as significant according to the Bonferroni correction).

Ultrastructural damage in psoas muscle among treatment and age groups was assessed by using the chi-square test, and P  values less than 0.05 were considered significant. Agreement between the scorings of the two examiners yielded a Kendall rank correlation coefficient of 0.95 for the microscopic study.

Tests were performed using Sigmastat 3.1 (Systat Software Inc., San Jose, CA).

Forty-eight rats were included, and no self-mutilation after catheter placement was observed. The catheters were inserted into the perimysium connective tissue, and between muscle fibers without destruction, to reach the vicinity of the femoral nerve where bupivacaine was released. No catheter displacement was observed, and no rat was excluded from the analysis.

Measurements of coupled oxygen consumption rate and the corresponding ATP synthesis were performed in permeabilized fibers using glutamate or pyruvate plus malate, or succinate as substrates (tables 1–3). In the saline groups, the level of ATP synthesis with the three substrates was significantly higher in young rats than in adult rats. Bupivacaine induced a significant decrease in adenosine diphosphate–stimulated oxygen consumption, along with a significant inhibitory effect on ATP synthesis in both groups. The efficiency of oxidative phosphorylation (ATP/O ratio) was also reduced. No significant difference was observed between the young and the adult rats treated with bupivacaine. In addition, in saline groups, citrate synthase activity was significantly higher in young rats than in adult rats (531 [496–549] and 361 [337–366] ng nmol substrate · min⁻¹· mg protein⁻¹, respectively; P = 0.002, Mann–Whitney post hoc  test). Bupivacaine induced a significant decrease in citrate synthase activity in young rats (440 [428–457] nmol substrate · min⁻¹· mg protein⁻¹; P = 0.002 vs.  saline young rats, Mann–Whitney post hoc  test) and adult rats (278 [269–323] nmol substrate · min⁻¹· mg protein⁻¹; P = 0.009 vs.  saline adult rats, Mann–Whitney post hoc  test).

Electron micrographs of muscle sections taken from adult and young rats were obtained after treatment with saline solution (figs. 1A and B, respectively) or bupivacaine (figs. 1C and D). More precisely, muscle damage was primarily absent, focal (1–2 sarcomeres; fig. 1B), or moderate (3–10 sarcomeres; fig. 1C) in both young and adults rats in the saline groups. Bupivacaine groups revealed a wide range of abnormalities, ranging from absent to focal, moderate, and extreme (fig. 2). Extreme muscle damage (> 10 damaged sarcomeres) was seen in bupivacaine groups only (figs. 1D and 2). Extreme muscle damage was significantly more frequent in the young bupivacaine group than in the adult bupivacaine group (56% vs.  19%, respectively; P = 0.000016, chi-square test).

Local anesthetics (e.g. , bupivacaine) are widely used for postoperative regional analgesia in children, even though some studies have clearly demonstrated a potential muscle toxicity risk in adults.9,33,34Our results show that such a risk is increased in young rats, because we observed more severe alterations in mitochondrial oxidative capacities and a broader alteration of muscle ultrastructure.

First, we observed that the main metabolic fluxes, i.e. , oxygen consumption rate or ATP synthesis rate, are higher (factor of 2) in young rats as compared with adults, when expressed per mass of tissue. This was explained by a higher content of mitochondria in the young muscle, as evidenced by the corresponding twice higher activity of citrate synthase. Recently, Benard et al.  35evidenced a good correlation between citrate synthase activity and the expression level of respiratory chain complexes I and III, as well as mitochondrial transcription factor A, in five different rat tissues. This suggests that citrate synthase activity could be coordinated with the expression level of respiratory chain complexes and be closely linked to the content of mitochondrial respirasomes. Similar observations were reported in the study of Marcinek et al. ,36which compared the rate of mitochondrial ATP synthesis and oxygen uptake in 30-day-old and 7-month-old mice, using magnetic resonance spectroscopy. They found a significant increase in ATP synthesis rate in younger muscle. Likewise, the analysis of mitochondrial DNA copy number and cytochrome c  oxidase gene expression in rat and in human skeletal muscle showed a substantial decline with aging.37–39More precise analysis of human skeletal muscle from individuals of different ages (5 days old and 52 yr old) provided transcriptional evidence for an age-related decline in metabolic capacity, with a decreased expression of the genes encoding proteins involved in mitochondrial bioenergetics, including ATP synthase.39Therefore, the higher oxidative phosphorylation activity of young rat muscle observed in our study could be due to a higher content of mitochondria (suggested by the significant increase in citrate synthase activity per mass of tissue proteins) and embedded oxidative phosphorylation complexes.

In our study, we also confirmed the inhibitory effect of bupivacaine on mitochondrial respiration and linked-ATP synthesis, as observed in previous work.14Remarkably, we observed here a greater inhibition of OXPHOS in young rats, with respect to the relative effect of bupivacaine. For example, the rate of mitochondrial ATP synthesis was reduced by 75% in the young rats (using pyruvate–malate as substrates), whereas it was decreased by 55% in the adult rats. This could be explained by the initial difference of the respiratory steady state between adult and young rats, because the metabolic control analysis predicts that the impact of a given inhibition of a mitochondrial enzyme activity on the overall flux of energy production depends on this steady state.18,40Accordingly, the higher state of ATP production and oxygen consumption measured in young rat muscle suggests that OXPHOS delivers a larger part of muscle energy, so a deficiency of mitochondrial ATP synthesis might lead to more severe consequences. This is in accord with the increased level of muscle ultrastructural alterations observed in young rats.

The mechanisms of bupivacaine-induced myotoxicity might be similar in young and adult individuals, because both can be explained by the impairment of mitochondrial function. In vitro , it is now well established that bupivacaine uncouples oxidative phosphorylation on isolated mitochondria and further inhibits complex I activity.13,16The mechanisms of bupivacaine uncoupling have been extensively investigated.13,16,41–44In a physiologic rat model of iterative exposure to local anesthetics, bupivacaine-induced myotoxicity is somewhat different, with a decrease in ATP synthesis accompanied by a reduction in the activity of all respiratory chain complexes I–IV and of citrate synthase activity.14,23In the same way, iterative injections of bupivacaine could induce a decrease of mitochondrial content in muscles. Recent morphologic studies highlight the close link between ATP concentration and mitochondrial network organization.45,46ATP deprivation leads to mitochondrial network fragmentation and can intervene early in the mediation of mitochondrial network reorganization. In our study, we could not access the mitochondrial network, but morphologic mitochondrial damages were observed in both bupivacaine groups coupled with ultrastructural muscle alterations. However, the muscle fibers exhibiting severe damage were exclusively seen in bupivacaine groups, suggesting that such severe lesions are drug dependent and that the hypothesis of mechanical fiber damage due to the applied fluid volume could be ruled out. Moreover, substantial quantitative differences in damaged fibers have been described when the toxic effects of bupivacaine and ropivacaine are compared on porcine skeletal muscle tissue.47The lower toxic effects of ropivacaine compared with bupivacaine have also been described in human neuronal cells.48Therefore, both the biochemical properties of local anesthetics and the age of the animals influence bupivacaine-induced myotoxicity.

Age-related differences in drug-induced mitochondrial bioenergetic toxicity in young tissue or cells, after bupivacaine exposure, have not been reported so far, whereas they have been after exposure to methylmercury.49The latter induced strong inhibition both of state 3 mitochondrial respiration and ATP synthesis, with mitochondrial structural abnormalities characterized by disorganized cristae.50Moreover, the toxic effects of methylmercury on mitochondrial function are age dependent,49with greater decrease in mitochondrial metabolic function in young (postnatal day 21) rats than in adult (10- to 14-week-old) rats. In that study, the higher sensitivity of young rats to methylmercury contamination was attributed to lower basal activities in reactive oxygen species–detoxifying mechanisms. Such difference could also participate toward the stronger toxicity of bupivacaine in young individuals, because bupivacaine also triggers hydroxyl radical formation.51Moreover, recent studies on the investigation of insulin resistance or statin-induced myotoxicity have shown a significant correlation between rodent and human muscle such that our results obtained on rats could have significance for humans.52,53In the same way, bioenergetics (especially ATP synthesis) and reactive oxygen species–detoxifying mechanisms seem to be similar in rodent and human skeletal muscle.52,54Furthermore, local anesthetic–induced myotoxicity could be overlooked clinically in children, and the rare case report does not mean that muscle pain and dysfunction do not happen. Our results highlight a larger toxicity in young rats, suggesting the lack of protective mechanisms, or the higher dependence of muscle on OXPHOS to produce energy. However, it can be proposed that (1) myotoxicity may well occur clinically in children but is not reported because of the difficulty of its detection or the absence of investigation; or (2) myotoxicity is characterized by a concentration-dependent effect, i.e. , local anesthetic blocks in children have been performed with concentrations lower than 0.5%, potentially less toxic. Therefore, in the absence of supplemental data, clinical practice in children should not be changed, except that practitioners could be more vigilant regarding toxicity, particularly in situations where higher concentrations are needed to perform analgesia.

In conclusion, this study shows that iterative injections of bupivacaine induce a decrease in mitochondrial ATP synthesis and citrate synthase synthesis, suggesting a reduction of mitochondrial respiratory chain content, and muscle fibers exhibiting severe damages in rat. Moreover, young rats exhibit a higher rate of citrate synthase activity and ATP synthesis than adult rats. This study demonstrates that young age can enhance local anesthetic-induced myotoxicity. The clinical impact of our work remains to be evaluated in practice, and the need for a clinical evaluation of bupivacaine myotoxicity in young patients remains to be defined.

The authors thank Chantal Cazevieille, M.Sc. (Technician, Institut National de la Santé et de la Recherche Médicale ERI 25, Montpellier, France), for her technical assistance and for interpreting the data regarding ultrastructural evaluation. They also thank Françoise Masson, M.D. (Research Assistant, Pôle Anesthésie Réanimation, Centre Hospitalier Universitaire Bordeaux, Bordeaux, France), for statistical support and Ray Cooke, Ph.D. (Assistant Research, Département des langues vivantes, Université Victor Segalen Bordeaux 2, Bordeaux, France), for proofreading the manuscript.