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Modification of Bupivacaine-Induced Myotoxicity with Dantrolene and Caffeine In Vitro

Plank, Christoph MD*; Hofmann, Petra MD*; Gruber, Michael PhD*; Bollwein, Gabriele*; Graf, Bernhard M. MD, MSc*; Zink, Wolfgang MD; Metterlein, Thomas MD*

doi: 10.1213/ANE.0000000000000988
Anesthetic Pharmacology: Research Report

BACKGROUND: Local anesthetics, especially bupivacaine, have myotoxic effects in clinically used concentrations and context. Detailed mechanisms of these effects are unknown, but an increase in intracellular calcium levels is suspected to be the most important trigger. Dantrolene and caffeine modify cellular calcium release from the sarcoplasmic reticulum. The aim of our study was to investigate the effect of dantrolene and caffeine on bupivacaine-induced myotoxicity in vitro.

METHODS: A cell culture model of primary muscle cells of BALB/c AnNCrl mice was established. Cells were incubated simultaneously with increasing concentrations of bupivacaine, dantrolene, and caffeine. The fraction of dead cells was calculated after staining with propidium iodide and analysis by flow cytometry. The half-maximal inhibitory concentration of bupivacaine was calculated for each concentration. Group differences were determined by using 1-way analysis of variances with subsequent post hoc 1-way Dunnett t test.

RESULTS: Both dantrolene and caffeine alone had no effect on muscle cell survival. Increasing concentrations of bupivacaine caused increasing cell death. Dantrolene dose-dependently reduced the fraction of necrotic cells, whereas caffeine dose-dependently increased the fraction of dead cells.

CONCLUSIONS: Dantrolene attenuated, and caffeine enhanced, bupivacaine-induced myotoxicity, presumably by modifying sarcoplasmic calcium release. This indicates that intracellular calcium release is an important factor for local anesthetic–induced cell death.

Published ahead of print September 29, 2015

From the *Department of Anesthesiology, University Hospital Regensburg, Regensburg, Germany; and Department of Anesthesiology and Intensive Care Medicine, Klinikum Ludwigshafen, Ludwigshafen, Germany.

Accepted for publication July 20, 2015.

Published ahead of print September 29, 2015

Funding: None.

The authors declare no conflicts of interest.

Address correspondence to Christoph Plank, MD, Department of Anesthesiology, University Hospital Regensburg, 93053 Regensburg, Germany. Address e-mail to christoph.plank@ukr.de.

Local anesthetics (LAs) are widely used, despite their myotoxic properties. Although their systemic effects have been investigated, their local toxic effects on neurons or muscular tissue, although discussed by Brun1 as early as 1959, are now attracting clinical interest and gaining attention in current research. In the meantime, several case reports on LA-induced tissue damage, with or without persistent patient-based handicaps such as diplopia, have been published.2–5 All recently used LAs have myotoxic effects, with bupivacaine causing the most severe structural long-term damage in clinically used concentrations.1,6–13

Various in vitro models have demonstrated influence of both LAs in terms of concentration and time of exposure.14,15 Furthermore, we could previously show various sensitivities of different cell culture types. This can be explained by structural differences in the sarcoplasmic reticulum (SR) due to different cell differentiation.

The exact details of LA’s toxic mechanisms are still unknown. Apart from apoptotic effects and influence on mitochondrial energy production, a direct influence on intracellular calcium levels has been examined.14,16–19 Previous investigations suggest an influence of LAs on both the release of calcium from the SR—similar to other myotoxic substances (volatile anesthetics, statins, fluoroquinolones)—and the reuptake of calcium into the SR.15,20,21 An acutely elevated intracellular calcium level triggers cellular energy consumption, leading to a gap between energy demand and supply, finally resulting in cell breakdown. Furthermore, a chronically mild elevation of intracellular calcium induces apoptosis via mitochondrial pathways.16–18,22 If increased intracellular calcium levels are responsible for LA-induced myotoxic effects, a modification of the intracellular calcium homeostasis would alter these effects.

Several agents are capable of influencing intracellular calcium levels. Most of them modify sarcoplasmic calcium release via the type 1 ryanodine receptor. Dantrolene lowers the intracellular calcium level by reducing the opening probability of type 1 ryanodine receptor. Protective effects of dantrolene on muscle cells have also been noted in research on the toxicology of volatile anesthetics and statins. And further cytoprotective effects of dantrolene have been described in models of LA-induced neuronal cell death.23–25 Caffeine nonspecifically increases the permeability of sarcoplasmic membranes and induces an increase in the intracellular calcium.26,27

Although the application of dantrolene or caffeine modifies calcium levels, there is still the question of whether this also leads to a modification of LA-induced myotoxicity. If so, this would support the hypothesis of intracellular calcium as a relevant factor of LA toxicity. The aim of the current study was to investigate the impact of dantrolene and caffeine on the myotoxicity of bupivacaine in a previously established cell culture model.

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METHODS

Figure 1

Figure 1

With the consent of the local committee for Laboratory Animal Care, a cell culture model of primary murine muscle cells was established. Muscle fibers (Mm. extensor digitorum longus, Mm. soleus, and Mm. tibialis anterior) of BALB/c AnNCrl mice (Charles River Laboratories, Sulzfeld, Germany) were extracted and processed according to a standardized protocol described earlier.14 After establishing a monolayer of mice myoblasts, cells were differentiated into myotubes.14 The differentiation process was controlled by successive microscopy (Fig. 1). Cells were disseminated on 24-well plates (Schubert & Weiss, Munich, Germany).

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Pilot Testing

Probes of differentiated cells were incubated for 2 hours with bupivacaine (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) in a concentration of 7.9 mM (2500 ppm) based on phosphate-buffered saline (PBS) (Sigma-Aldrich Chemie GmbH). For control, there were further probes without LA. pH-metry (Knick GmbH & Co. KG, Berlin, Germany) was used to maintain physiologic conditions.

After incubation, cells were washed with PBS, harvested with 0.1% trypsin-EDTA (Sigma-Aldrich Chemie GmbH), and resuspended with PBS. After a recovery time of 2, 4, 16, 24, 48, and 72 hours, both propidium iodide (PI) (Invitrogen GmbH, Karlsruhe, Germany) and annexin V (Becton Dickinson, Heidelberg, Germany) were added (each 5 μg/well) to tag necrotic and apoptotic cells for following flow cytometry with FACSCalibur™ (Becton Dickinson). For each sample, the total numbers of cells and the numbers of annexin- and propidium-positive cells were counted. Fractions of vital, only annexin–positive cells, annexin- and PI-positive cells, and only PI-positive cells were calculated. There were 8 independent samples (wells) within every group of recovery time with bupivacaine and another 8 independent samples within every group for control without LA.

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Main Testing

After interpretation of the pilot testing results, main testing was modified and only PI was further used to identify cellular damage. Differentiated cells were incubated for 2 hours simultaneously with dantrolene in concentrations of 60, 120, and 180 μM, or caffeine in concentrations of 2, 4, 6, and 8 mM; and bupivacaine hydrochloride (all Sigma-Aldrich Chemie GmbH) in concentrations of 1.6, 3.2, 5.5, and 7.9 mM (500, 1000, 1750, and 2500 ppm). All solutions were based on PBS again with pH-metry used to maintain physiologic conditions. Additional samples without the agents mentioned previously were analyzed for control. There were 6 independent samples (wells) for each substance combination.

After incubation, cells were washed with PBS, harvested with 0.1% trypsin, and resuspended with PBS. Only PI was added (5 μg/well) to tag necrotic cells for following flow cytometry after 2 hours recovery time. For each sample, the total number of cells and the number of necrotic cells were counted. The fraction of necrotic cells was calculated. For all caffeine and dantrolene concentrations, the half maximal inhibitory concentration of bupivacaine (IC50) was determined by using a predefined pharmacodynamic model of Phoenix® Nonlinear Mixed Effects Modeling (Pharsight Certara, St. Louis, MO). IC50 describes the bupivacaine concentration at which half of the treated cells survive.

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Statistics and Modeling

Statistical analyses were done with Microsoft Excel 2010 (Microsoft Corporation, Redmond, WA) and IBM SPSS Statistics version 22 (IBM Deutschland GmbH, Ehningen, Germany). Normal distribution of the residuals was tested by using the Lilliefors test; equality of variances was checked by using the Brown-Forsythe statistics. Group differences were determined by using 1-way analysis of variances with subsequent post hoc 1-way Dunnett t test. A P value ≤0.05 was assumed as statistically significant. Phoenix 64 Nonlinear Mixed Effects Modeling 1.3 (Pharsight Certara) was used to calculate a naive-pooled sigmoid Emax model with baseline effect parameter (E = E0 + (Emax × Cγ)/(Cγ + EC50y), where C is the bupivacaine concentration; E, E0, Emax are the fractions of PI-positive cells. An additive residual error was estimated for each fitting, with the concentrations of dantrolene or caffeine as continuous covariates. Simulations with the derived fixed effects were calculated in the range from 0 to 7.9 mM (2500 ppm) bupivacaine on 25 points.

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RESULTS

Pilot Testing

Table 1

Table 1

Figure 2

Figure 2

First, we investigated whether measurement of PI-positive cells is adequate for interpretation of LA-induced myotoxicity and as acceptable as an annexin/PI-combination. Complete data from 8 independent samples for all recovery times in both the bupivacaine and the control group were obtained. Fractions of vital, annexin-positive cells, annexin- and PI-positive cells, only PI-positive cells, and the fraction of all PI-positive cells are presented in Table 1. There is a constant fraction of vital cells within the control group. Within the bupivacaine-treated cells, distribution of necrosis and apoptosis seems to depend on recovery time and therefore on time of performing flow cytometry. After 2 hours of recovery, the number of only annexin–positive cells is still low (<20%) within both the bupivacaine and the control group. The number of only annexin–positive cells increases with a longer period of recovery within the bupivacaine samples (Fig. 2).

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Main Testing

Complete data were obtained from all independent samples. There were 6 samples for each combination of bupivacaine and dantrolene and also 6 independent samples for each combination of bupivacaine and caffeine. Descriptive statistics are given in Tables 2 and 3 as means ± SD. Normal distribution and equality of variances were given with all P ≥ 0.14. Increasing concentrations of bupivacaine led to an increasing fraction of necrotic muscle cells (Table 2). Both dantrolene (P = 0.451) and caffeine (P = 0.319) alone had no significant effect on primary muscle cell survival by themselves within the used concentrations (Table 3). Dantrolene dose-dependently reduced the fraction of necrotic cells as shown in Figure 3, with statistical significance (P = 0.029) at a bupivacaine concentration of 5.5 mM (1750 ppm) and a dantrolene concentration ≥120 μM (Table 3). Caffeine dose-dependently increases the fraction of necrotic muscle cells at concentrations ≥4 mM (Fig. 3; Table 3). Statistically significance was seen at a bupivacaine concentration of 3.2 mM (1000 ppm; P < 0.001).

Table 2

Table 2

Table 3

Table 3

Figure 3

Figure 3

Figure 4

Figure 4

Pharmacodynamic calculation of the IC50 of bupivacaine shows an increased concentration of bupivacaine with dantrolene added and a reduced concentration with caffeine (Table 4; Fig. 4).

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DISCUSSION

The serious systemic side effects of LAs, such as neuro- and cardiotoxicity, have been investigated for many years, although their incidence is rather low.28–30 Local toxic effects are known from histopathologic examinations, but there is still little knowledge about causative pathophysiology.1,8,10,14,15,31

Because of unknown tissue concentrations, variable resorption, and a generally high tissue regeneration, the clinical relevance of LA-induced myotoxicity remains uncertain. However, because regional anesthesia is being used in more and more clinical settings, there is increasing interest in local tissue damage. The clinically relevant toxic effects of LAs, such as persistent diplopia after retrobulbar blocks, have been described frequently.2–4,32 Furthermore, LA-induced tissue damage seems to be a clinically relevant problem in orthopedic surgery. Damage of articular cartilage and intervertebral disk cell viability by continuous LA application for pain management has been reported.5,33,34

Although all LAs have myotoxic properties, bupivacaine was chosen because of its severe effect on muscle cells, even in clinically used concentrations.13 Nevertheless, all other LAs provoke similar damage in higher concentrations or during longer exposures.14

Cell culture models are useful for investigating the direct effects of agents on cells, although there has been little consideration of physiologic parameters, such as resorption, distribution, and degradation. The cell culture model used by our department works with primary muscle cells, which are more sensitive to toxic effects than commonly used and commercially available immortalized cell lines. This is possibly due to a lower amount of SR.14 Our survival rates (Tables 1 and 2) demonstrate the reproducible high quality of the model in a robust experimental setting.

Pretesting results are consistent with previous studies16–19 and show both apoptotic and directly necrotic mechanisms to be coexistent. Measurement by tagging cells with annexin V and PI is adequate for summarizing both these effects. However, the timing of flow cytometry is a relevant factor. Focusing on early measurement, PI can be used as a single marker for cell death.

Bupivacaine causes a dose-dependent necrosis of muscle cells measured by the fraction of propidium-positive cells in flow cytometry. This confirms previously reported results.14 In clinically used concentrations, a relevant portion of cells is already necrotic. In higher concentrations, only <10% of all cells remain intact.

Myocytes have large intracellular calcium stores within the SR. The myotoxic effects of various other substances can be explained by an alteration of intracellular calcium levels. An acute disruption of the intracellular calcium homeostasis leads to muscle cell breakdown and stimulates apoptotic pathways. A similar pathomechanism is discussed for LAs.15,22,35 Dantrolene, a potent inhibitor of cellular calcium release from the SR,20,24 had by itself no influence on survival of primary muscle cells in our study. In combination with bupivacaine, dantrolene leads to significantly improved survival rates and an increased IC50 concentration of bupivacaine. A lower intracellular calcium level seems to be protective against toxic LA effects. The calcium-liberating effects of caffeine, however, lead to reduced cell survival rates. The IC50 concentration is lowered, indicating an enhanced toxic effect of caffeine. The exposure to caffeine itself did not alter cell survival rates in our study.

Statistical significance was seen only in lower bupivacaine concentrations, although absolute data (Table 3) indicate an overall trend. Higher LA doses have limited statistically significance because only <10% of all cells remain vital, and maximal LA concentration was further limited by the solubility of bupivacaine. This fact is reflected in the enormous ranges of the 95% confidence interval and the differing base IC50 values of dantrolene and caffeine in the IC50 model (Table 4). Furthermore, the concentration range of dantrolene in our setting was probably not wide enough to show a maximal effect.

Table 4

Table 4

The additive toxic effects of caffeine and the protective effects of dantrolene indicate an important role for intracellular calcium on LA-induced myotoxic effects. LAs apparently increase intracellular calcium levels.15 If this is augmented with other calcium-liberating substances, cell death increases. If the LA-induced calcium release is impeded by dantrolene, cell survival is increased. Studies with direct intracellular calcium measurements show increased calcium levels after exposition to LAs. There is still the question of whether this is the trigger for cell death or only a result of the harming mechanism. Our data show an alteration of cellular death rates after modification of the intracellular calcium level, suggesting that calcium peaks are not only a result, but also probably one of the factors contributing to necrosis and/or apoptosis. Further studies are needed to confirm the exact role of intracellular calcium, especially by focusing on subcellular calcium peaks, as well as on intracellular calcium transport.

Our results indicate that dantrolene might have protective effects on LA-induced myotoxicity. In a previous clinical study, dantrolene had protective effects on neuronal tissue.23 It has yet to be proven whether dantrolene can also protect muscle tissue within in vivo conditions.

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CONCLUSIONS

Calcium levels seem to play an important role in cell survival of bupivacaine-induced cell death within the investigated concentration range (1.6–7.9 mM). Dantrolene attenuated, and caffeine enhanced, bupivacaine-induced myotoxicity, presumably by modifying sarcoplasmic calcium release. This indicates that intracellular calcium release from the SR is an important factor of LA-induced cell death and the underlying damaging intracellular processes. Further research is needed to quantify the clinical impact, to investigate the exact biochemical mechanisms, and to find a scope for protection.

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DISCLOSURES

Name: Christoph Plank, MD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Christoph Plank has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Petra Hofmann, MD.

Contribution: This author helped design the study and conduct the study.

Attestation: Petra Hofmann has seen the original study data and approved the final manuscript.

Name: Michael Gruber, PhD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Michael Gruber has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Gabriele Bollwein.

Contribution: This author helped conduct the study.

Attestation: Gabriele Bollwein has seen the original study data and approved the final manuscript.

Name: Bernhard M. Graf, MD, MSc.

Contribution: This author helped design the study.

Attestation: Bernhard M. Graf has seen the original study data and approved the final manuscript.

Name: Wolfgang Zink, MD.

Contribution: This author helped design the study and conduct the study.

Attestation: Wolfgang Zink has seen the original study data and approved the final manuscript.

Name: Thomas Metterlein, MD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Thomas Metterlein has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

This manuscript was handled by: Markus W. Hollmann, MD, PhD, DEAA.

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