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Departments of *Anesthesiology and Vascular Surgery, University of Heidelberg, Heidelberg; and Department of Neuropathology, University of Mainz, Mainz, Germany
Address correspondence and reprint requests to Bernhard M. Graf, MD, PhD, Department of Anesthesiology, University of Heidelberg, Im Neuenheimer Feld 110, D-69120 Heidelberg, Germany. Address e-mail to bernhard_graf{at}med.uni-heidelberg.de.
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Abstract |
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Introduction |
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IM injections of local anesthetics regularly result in striated muscle damage and myonecrosis, with a drug-specific and dose-dependent rate of toxicity (5). In this respect, tetracaine and procaine have been identified to produce the least, and bupivacaine the most severe muscle injury. Histologically, hypercontracted myofibrils become evident several minutes after injection, followed by lytic degeneration of striated muscle sarcoplasmic reticulum (SR), myocyte edema, and myonecrosis over the next few hours (6). Intriguingly, myoblasts, basal laminae, and connective tissue elements are not affected and, thus, tissue regeneration may occur within 2–4 wk. As a consequence, skeletal muscle damage after local anesthetic application has been considered to be entirely reversible.
Subcellular pathomechanisms of local anesthetic myotoxicity are still not completely revealed. However, excessively increased intracellular [Ca2+] levels have been shown to have the key role in myocyte injury (7). In this respect, bupivacaine and, to a smaller extent, ropivacaine both induce Ca2+ release from the SR, and simultaneously inhibit Ca2+ reuptake into the SR, resulting in persistently increased [Ca2+] levels (8–10). In contrast, less myotoxic drugs such as tetracaine may inhibit Ca2+ release, without affecting Ca2+ reuptake (11).
During recent years, catheter techniques for continuous application of long-acting local anesthetics have been well established in regional anesthesia. Therefore, in a previous study (12), we compared the acute myotoxic effects of bupivacaine and ropivacaine on skeletal muscle tissue using a porcine model of continuous peripheral neural blockade. In this clinically relevant setting, bupivacaine and ropivacaine both destroyed skeletal muscle fibers at the injection site. In comparison with bupivacaine, however, ropivacaine is characterized by a significantly slower rate of acute myotoxicity in equipotent concentrations (13). Additionally, bupivacaine, but not ropivacaine, induces apoptosis in adult myocytes in vivo. Based on these histological findings, our aim for the present study was to assess and to compare long term myotoxic effects of bupivacaine and ropivacaine on striated muscle tissue after continuous peripheral nerve blocks.
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Methods |
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After placing the catheters, 20 mL of bupivacaine (5 mg/mL), ropivacaine (7.5 mg/mL), and normal saline, respectively, were carefully injected over 5 min. Subsequently, bupivacaine (2.5 mg/mL), ropivacaine (3.75 mg/mL), and normal saline were continuously administered with a rate of 8 mL/h for a total period of 6 h.
Eight animals were treated with bupivacaine (left side) and ropivacaine (right side), respectively. For the control group, the remaining two animals were treated with normal saline on both sides.
Seven and 28 days after initial treatment, respectively, 5 animals at a time (1 control animal and 4 animals of the treatment group) received general anesthesia again. Intravitally, specimens of muscle tissue of equal size and volume (approximately 6 x 3 x 3 cm) were harvested from next to the femoral nerve sheaths, with the former tip region of each catheter located in the middle of each tissue block. For histological processing, the specimens from each injection site were subsequently fixed by immersion in 10% phosphate-buffered formalin (pH 7.5) for at least 12 h.
Using standard techniques, the dehydrated specimens were embedded in paraffin, cross-sectioned at 5 µm, and stained with Ehrlich hematoxylin and eosin (H&E) and von Kossa stain. Additionally, representative sections were exposed to an apoptosis-specific staining kit (indirect TUNEL labeling assay: In Situ Cell Death Detection Kit APTM; Roche Diagnostics, Germany) according to the manufacturer’s guidelines.
The muscle sections were evaluated by two independent and blinded examiners unaware of group or treatment. To assess the specific extent of skeletal muscle changes, eight cross-sections per injection site were randomly chosen (four H&E-stained sections and four von Kossa-stained sections). Per time point, 32 sections were examined in the bupivacaine group and in the ropivacaine group, respectively, and 16 sections in the control group.
According to Benoit et al. (7), the specimens were scored on a modified ordered scale as follows: 0 = no fiber damage, 1 = localized and/or sparsely scattered fiber destruction or damage limited in depth to 1 or 2 fibers, 2 = more extensive necrosis following major connective tissue planes and involving numerous muscle fascicles or destruction extending 3–5 fibers from the surface, and 3 = destruction of essentially the entire muscle mass or generalized damage/necrosis of more than 5 fibers’ depth.
Quantitative differences in striated muscle tissue changes among treatment groups were assessed using the 
2 test, and P values < 0.05 were considered significant. Agreement between examiners’ scorings yielded for the entire study a Kendall rank correlation coefficient of 0.95. All data are presented as mean ± sd.
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Results |
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After bupivacaine treatment, severe necrobiotic tissue changes were ubiquitously assessed throughout the tissue samples, and von Kossa stain revealed massive granular calcium depositions within destroyed and necrotic clusters of myocytes (Fig. 1A–D). However, obvious signs of fiber regeneration and a synchronous proliferation of mononuclear myoblasts (satellite cells) with formation and subsequent maturation of myotubes were also encountered. The topographical pattern of muscle fiber degeneration and regeneration showed concentrated activity along the surfaces of muscle fascicles, indicating the former path and direction of local anesthetic spreading within the muscles. Nevertheless, massive lesions in the center of the fiber bundles were also found, but were rare. In addition, within myoseptal and interstitial spaces, histological signs of an unspecific inflammatory response to initial tissue lesions were found, with dense populations of lymphocytes, plasma cells, macrophages, and a proliferation of fibroblasts. In contrast, neither vascular supply nor neural structures were visibly affected. The indirect TUNEL labeling assay, which was performed to detect characteristic DNA strand breaks, did not reveal any apoptotic events in the tissue sections.
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At this time point, ropivacaine-induced patterns of fiber damage qualitatively resembled histological tissue lesions caused by bupivacaine. Calcareous infiltrations of scattered necrotic areas were seen next to clusters of regenerating fibers, an invasion of inflammatory cells, and the moderate formation of granulation tissue within myoseptal spaces (Fig. 1E–H). Again, neither apoptotic myocytes nor histological lesions of vascular and neural structures were detected.
However, there was a significant quantitative difference between groups: In the ropivacaine group, slight to moderate changes in skeletal muscle fibers dominated (damage score 1.4 ± 0.5), whereas in the bupivacaine group, marked tissue lesions prevailed (damage score 2.4 ± 0.8, P < 0.01; Fig. 2A).
Twenty-eight days after application of normal saline, the tissue specimens obtained from control animals injected with only normal saline showed no histological changes and were similar to untreated muscles in every respect (damage score 0.0 ± 0.0).
After bupivacaine treatment, multiple calcareous infiltrations mainly at the surface of muscle fascicles were seen as remnants of initial tissue damage as well as an unspecific fibrous degeneration of myoseptal spaces and a moderate interfascicular spreading. In addition, clusters of regenerating myocytes with a persistence of central myonuclei and a moderate perivascular accumulation of inflammatory cells were encountered throughout the tissue specimen, whereas no apoptotic fibers became evident (Fig. 3A–D). Again, all nerves and vessels appeared to be visibly unaffected. Thus, at this time point, large areas of IM calcification and the persistence of central nuclei within regenerating myocytes were the only specific and consistently recognizable long term signs of former damage.
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Scattered calcific myonecrosis and the continuing formation of fibrous scar tissue at the surface of fascicles and within myosepta were also found in respective sections after ropivacaine treatment, and again, no apoptotic fibers could be detected. Although surrounded by an unusual dense sheath of connective tissue, the examined sections contained many regenerating myocytes of nearly normal size and shape, but some scattered areas of thin muscle fibers with centralized myonuclei remained (Fig. 3E–H).
Once more, this pattern of tissue damage qualitatively resembled bupivacaine-induced lesions, but quantitatively, ropivacaine-induced alterations in striated muscle tissue were significantly less pronounced (damage scores 1.1 ± 0.8 versus 2.2 ± 0.3, P < 0.01; Fig. 2B).
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Discussion |
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The clinical impact of local anesthetic myotoxicity has been debated (1,5). On the one hand, there is good experimental evidence that local anesthetics regularly cause marked lesions when injected into or adjacent to skeletal muscle tissue (1,6–8,14–20). On the other hand, many anesthesiologists do not consider local anesthetic myotoxicity a genuine clinical problem, because skeletal muscle injuries after the application of these drugs (i) remain clinically inapparent in most cases and (ii) are supposed to be reversible within several weeks (6,14–16). However, many case reports of myotoxic complications after local anesthetic administration have been published. In particular, the occurrence of clinically relevant myonecrosis and resulting muscular malfunction has been described after retrobulbar blocks, continuous peripheral blocks, infiltration of wound margins, and trigger point injections (2–5,17).
In this respect, we (12) recently examined the acute myotoxic effects of bupivacaine and ropivacaine and found that both drugs induced marked necrobiotic changes with a similar histological pattern. Compared with bupivacaine, tissue damage caused by ropivacaine was significantly less severe. Based on these findings, the present study was performed to histologically evaluate the long term myotoxic effects of both drugs, with special focus on structural reversibility of initial tissue changes, and on differences in myotoxic potencies. Again, nerve blocks were initiated with a bolus of either bupivacaine or ropivacaine, and maintained by the continuous application of both drugs for a total period of 6 hours (12). Seven and 28 days after the treatment, respectively, tissue specimens were harvested to assess the peak activity of fiber regeneration and the extent of reformation of "normal" skeletal muscle tissue (6,14,16,18–20). With regard to the relatively short duration of the initial infusion, however, this experimental approach might not entirely represent the current clinical practice. Nevertheless, we deliberately maintained this procedure in order to directly relate long term myotoxic effects to the acute tissue changes assessed in the previous study (12).
Our findings demonstrate that bupivacaine and ropivacaine both induced marked focal degeneration of skeletal muscle tissue, followed by a remarkable rate of regeneration of the damaged myocytes (21). However, substantial quantitative differences in tissue lesion became evident as long term myotoxic effects of bupivacaine significantly exceeded those of ropivacaine. Except for degree, the histological patterns and the time course of tissue damage and regeneration were qualitatively very similar: After 7 days, myocyte degeneration and fragmentation were encountered throughout the entire tissue specimens, and synchronous regeneration subsequently reestablished almost normal-appearing striated myocytes within the following days. Nevertheless, we postulate that ultrastructural tissue changes are not completely reversible in our model (22): Next to focal scar tissue formation, clusters of calcareous infiltrations were encountered as obvious remnants of initial tissue damage. Thus, despite a rapid rate of myocyte regeneration, these results indicate that bupivacaine and ropivacaine in clinical concentrations may induce persistent lesions in mammalian skeletal muscle tissue because these marked calcifications are not likely to be totally cleared (23,24).
These findings partly contradict the results of numerous experimental studies on long term myotoxic effects: In these examinations, the histopathological changes and the time course of skeletal muscle injury and regeneration after local anesthetic administration also appeared to be rather uniform and not drug-specific, but calcific myonecrosis has never been described (7,14,16,19,20,25). The main reason for this obvious discrepancy might be the use of different animal species (mainly mice and rats) and experimental protocols (e.g., subcutaneous injections). Thus, it remains unclear whether these findings can be transferred to a clinically relevant setting in humans. In our opinion, a porcine model of peripheral nerve blockade seems to be a more appropriate approach to this topic. Nevertheless, it remains speculative whether calcific myonecrosis is specific for porcine skeletal muscle tissue, but there is reason to suspect that the reactions of human muscles exposed to similar doses of local anesthetics would not greatly differ from those reported here (6,22,26).
The present study was designed to compare the long term myotoxic effects of local anesthetics rather than to reveal specific mechanisms of myotoxicity. However, in control animals, muscle fibers underlying the area of injection remained visibly unaffected and normal in all respects. This indicates that fluid volumes per se are not responsible for subsequent tissue alterations and, consequently, mechanical reasons for fiber damage can be excluded (12). Furthermore, we previously showed that apoptosis is involved in acute myocyte damage after the application of bupivacaine, but not of ropivacaine. However, in the present study, apoptotic myocytes were not detected, which reconfirms our previous postulation that pathways of cell death other than apoptosis are quantitatively much more important in bupivacaine-induced tissue damage in vivo (7,12,22). As membrane active drugs, local anesthetics perturb both internal and external membrane systems of striated myocytes, and thus may cause a variety of changes in Ca2+-homeostasis and permeability. Because of the profound impact of Ca2+ on cellular survival and structural integrity, Benoit et al. (7) were the first to propose that local anesthetic myotoxicity may be the result of an intracellular increase in free Ca2+. Recent studies revealed that bupivacaine, as well as ropivacaine, in clinical concentrations not only induce Ca2+ release from the SR, but also inhibit Ca2+ reuptake into the SR, which consequently results in massively increased intracellular Ca2+ levels (8–10,27). However, bupivacaine’s effects on intracellular Ca2+ homeostasis are markedly stronger than those of ropivacaine in equivalent concentrations (27). Because of its pronounced lipophilicity, bupivacaine rapidly accumulates in the myoplasm, and thus may induce an immediate increase in intracellular Ca2+ in skeletal muscle fibers. In contrast, the less lipophilic ropivacaine slowly perturbs the sarcolemmal barrier, and has only moderate effects on intracellular Ca2+ levels. Consequently, these mechanisms may explain the similarities as well as the differences in bupivacaine’s and ropivacaine’s long term myotoxic effects, which basically seem to be determined by the respective extent of initial tissue damage (12). Furthermore, the increase in free intracellular Ca2+ may also be involved in the formation of irreversible calcification within necrotic areas (22–24).
In conclusion, we found that in a clinically relevant setting, bupivacaine and ropivacaine in equipotent concentrations induce multiple calcareous infiltrations in skeletal muscle tissue within four weeks. Despite an impressive rate of fiber regeneration, these calcific lesions are not presumed to be entirely reversible. In addition, long term myotoxic effects of bupivacaine significantly exceed those of ropivacaine, which reconfirms bupivacaine’s exceptional rate of myotoxicty. Because the functional impact of these findings is still undetermined, clinical investigations are warranted to evaluate the incidence of calcific myonecrosis in humans after application of these drugs, and to assess the influence of local anesthetic-induced tissue lesions on skeletal muscle performance.
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Footnotes |
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Presented, in part, at the German Congress of Anesthesiology (DAC) in Nürnberg, Germany, June 19–22, 2004, and at the Annual Meeting of the American Society of Anesthesiologists in Las Vegas, NV, October 23–27, 2004.
Accepted for publication December 22, 2004.
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P < 0.01 in comparison with the ropivacaine group.
