Local anaesthetics are known myotoxic drugs in clinical concentrations either in laboratory animals or in human [1-3]. All local anaesthetics previously studied have been proven to be toxic in a dose-dependent manner. Repeated doses or continuous infusion of the drug have greater effect on striated muscles . Simultaneously, various local anaesthetics seem not to behave in a uniform fashion, regarding myotoxic potency. Bupivacaine is considered to be the most myotoxic local anaesthetic, while procaine is believed to be the least . Nevertheless, the problem of possible local anaesthetic myotoxicity has been underestimated in clinical practice, partially because of the reversible muscular damage and partially because of the scattered reports of clinical obvious muscular impairment [1,4]. Although all local anaesthetics used in clinical practice have been studied, regarding their potential myotoxic effect, few data are available about the recently introduced local anaesthetic ropivacaine. The latter is the first pure S-enantiomer used in clinical practice and is characterized by lower cardiovascular and nervous system toxicity. Zink and colleagues  first investigated the possible myotoxic effect of ropivacaine and found that ropivacaine caused myotoxic damage in pigs striated muscles after continuous infusion. That study examined the potential destruction in tissues dissected and fixed 1 h after a continuous infusion had stopped.
In this study we evaluated the potential myotoxic effects of ropivacaine after single injection in rats under light and electron microscope. We compared the influence of two different concentrations used in clinical practice for wound infiltration. Furthermore, we investigated the time-course of tissue damage over a month period.
After approval of the Prefectural Laboratory Animal Care and Use Committee, we studied 128 male Wistar rats weighing 250-350 g. Rats were initially allocated to four groups. The first three groups received intramuscular injections with normal saline (Group S, n = 32), ropivacaine 0.75% (Group R75, n = 32) and ropivacaine 0.5% (Group R5, n = 32), respectively. The right tibialis anterior muscle was used for the intramuscular injections. The remaining group of rats received intramuscular puncture to right tibialis anterior muscle without any drug injection (Group N, n = 32).
Intramuscular puncture was performed via a 27-G needle in all groups. The needle penetrated the distal part of the muscle, reached the tibia perpendicularly and then was redirected longitudinally across the long axis of the muscle for 3 mm proximally. After aspiration, the solution was slowly injected and the needle was then withdrawn. The left tibialis anterior muscles of all animals did not receive any punctures and were used as control.
The solution of normal saline 0.9% (Braun® Melsungen AC) injected in Group S was preservative free. Commercial preparation of ropivacaine 0.75% (Narope Inc® Astra Zeneca AB, Södertälje, Sweden) was used for injection in R75 group. Ropivacaine 0.5% is not available in our country as a ready for use solution. The solution of ropivacaine 0.5% injected in the R5 group was prepared by mixing commercial available solution of ropivacaine 1% (Narope Inc® Astra Zeneca AB, Södertälje, Sweden) with normal saline 0.9% (Braun® Melsungen AC) in a ratio 1: 1. The injected volume for all regimens was 100 μL for each rat. This volume is not considered to harm the specific muscle, according to work of Foster and colleagues , who used larger volumes, without producing excessive intramuscular pressure. After completion of injections, rats were left to recover and then they were put in special Plexiglas cages with free access to food and water and 12 h light/12 h dark cycle.
Two days after intramuscular injections, eight rats from each group were sacrificed with lethal urethane dose. On day 4 after injection, another eight rats from each group were also sacrificed. Eight rats from each group were sacrificed 7 days after injections and the remaining eight rats from each of the four groups were sacrificed 30 days after injections. Animals' right and left tibialis anterior muscles were carefully dissected.
Samples from muscles were submitted to the Histology-Embryology Department for histologic and ultrastructural examination. For the electron microscope process, the samples were fixed in 10% neutral buffered formalin, embedded in paraffin, cut in sections of 5 μm thickness and stained according to the Haematoxylin-Eosin method. Samples obtained 7 days after injections were also examined under the transmission electron microscope (TEM). For the TEM process, the samples were immersed immediately after removing in 2.3% of glutaraldehyde in phosphate buffered saline (PBS) at pH = 7.4 for 3 h and then they were washed in PBS and post-fixed in 2% aqueous solution of osmium tetroxide for 1.5 h. The samples then were dehydrated through ascending grades of ethanol, infiltrated and embedded in Epon 812. Ultra thin sections were prepared using an ultracut Reichert-Jung microtome. The sections were stained with aqueous solutions of uranyl acetate and lead citrate and examined under a Jeol 2000 CX electron microscope.
Samples examined under the light microscope were scored, based on damage severity on an ordered scale, according to the Benoit, Yagiela and Ferrell scoring system , from 0 to 3, with 0 corresponding to no damage, 1 corresponding to localized or sparsely scattered fibre destruction, 2 corresponding to extensive necrosis following major connective tissue planes and involving numerous fascicles, and 3 representing destruction of essentially the entire muscle mass. Kruskal-Wallis tests were conducted in order to evaluate the level of muscle damage caused 2, 4 and 7 days after injections. Moreover, pairwise group comparisons were made on days 2, 4 and 7 using U-test. Follow-up comparisons were conducted to evaluate the differences among groups. The Holm's Bonferroni method was used to control for Type I error at the 0.05 across all comparisons.
Light microscopy findings
The damage scores of the samples, according to Benoit scoring system, at 2, 4 and 7 days after injection are shown in Table 1. Statistical analysis using Kruskal- Wallis test revealed significant differences regarding damage scores among all groups except for N vs. S group (Table 2). In Group N samples, the mechanical trauma of the needle did not cause significant damage of the muscle. Samples obtained from muscles injected with normal saline (Group S) did not either show significant damage on any of the days observed (Fig. 1).
Two days after injections, extensive degeneration of the fibres was observed in the R75 group. The damaged area was infiltrated with phagocytic cells, which were removing the masses of debris from the necrotic fibres. Few fibres remained intact between the phagocytes. In samples obtained from muscles injected with ropivacaine 0.5% (Group R5), the infiltrated with phagocytes areas were significantly eliminated, in comparison to the R75 group corresponding to smaller damage scores (Table 1). According to statistical analysis, drug injection and muscle damage were found significantly related on day 2 (Table 3).
Four days after injections, the phagocyte infiltration persisted with the same expansion in the R75 group. At the same time, R5 group showed increased infiltration in comparison to 2 days group, but it was still less severe than R75 group. Significant differences among groups regarding damage scores were also found on day 4 (Table 3).
One week after the injection with ropivacaine 0.75% (Group R75), the infiltration persisted with the same intensity and few fibres remained intact (Fig. 2). At the same time, in the R5 group the infiltration not only persisted but it increased in comparison to 4 days (Table 1). Nevertheless, significant differences between Groups R75 and R5 were also found (Table 3).
One month later, the muscles from both groups injected with ropivacaine (R75 and R5 groups) had returned to normal. The appearance of muscle fibres in the control groups was also normal in all samples obtained on days 2, 4, 7 and 30.
One week after injection with ropivacaine 0.75%, muscle fibres showed severe morphologic changes. A variation of necrobiotic changes has been observed, ranging from slight intracellular oedema to total disintegration of cytoplasmic organelles and necrosis. Mostly, the sarcolema was intact even in the presence of severe cytoplasmic oedema. Mitochondria appeared oedematous or totally disintegrated. Limited local loss of myofibrils was observed in some fibres without any other destruction of the remaining sarcomers and the surrounding sarcoplasmic reticulum. A more serious effect of ropivacaine on myofibrils was the total disorientation, the disappearance of Z line and loss of the sarcomere and the surrounding sarcoplasmic reticulum architecture (Fig. 3). Findings from the group injected with ropivacaine 0.5% were similar to those of 0.75% injection, but the extension of the damage was eliminated. The cytoplasmic oedema decreased and the number of mitochondria in corresponding areas increased.
According to author's knowledge, there is only one study in the literature, regarding the potential myotoxic effect of ropivacaine. In this study, Zink and colleagues  compared ropivacaine and bupivacaine for their myotoxic effects in pig striated muscles after 6 h continuous local anaesthetic administration. Although ropivacaine seemed to be less toxic, compared to bupivacaine, Zink and co-workers found that both drugs induced muscle damage. These findings are consistent with our results, establishing the myotoxic effect of the newer local anaesthetic ropivacaine. Nevertheless, there are significant methodological differences between our study and Zink and colleagues. The latter investigated the myotoxic effect of ropivacaine after continuous infusion. In our study, we chose to examine the potential myotoxic effect of ropivacaine after single intramuscular injection, provided that local anaesthetic myotoxicity worsens after continuous infusion. Single intramuscular injection with local anaesthetics is frequently used in clinical practice during intraoperative infiltration of the surgical field. The last technique is an appealing method for postoperative pain relief, performed in ambulatory setting .
Zink and colleagues  evaluated muscle destruction in tissues dissected and fixed only 1 h after the infusion had stopped. In these samples, ropivacaine application, examined under light microscope, led to focal fibre damage in combination with interstitial and myoseptal oedema. Fibre damage ranged from fibres with confluent vacuoles and condensed, disintegrated myofibrils to entirely necrotic cells. Regarding time-course of muscle damage, these findings revealed that ropivacaine myotoxicity is developed as soon as in minutes, as has been proved for bupivacaine [8,9]. On the other hand, our samples examined under the light microscope showed a different pattern of damage, consisting of extensive degeneration of the fibres, infiltration with phagocytic cells removing the masses of debris from the necrotic fibres. Differences between our findings and Zing and colleagues observations may be due to the different timing in samples dissection, as we chose to examine ropivacaine myotoxic effects as early as 2 days after injections and not earlier as Zink did. Nevertheless, our findings on days 2, 4 and 30 after ropivacaine injection were similar to that observed by Foster and colleagues after bupivacaine injection , although less massive, showing similarities between the two local anaesthetics in damage time-course.
Samples injected with ropivacaine 0.75% and dissected 2, 4 and 7 days after injection showed massive macrophage infiltration with few remained intact fibres between the phagocytes. Macrophage infiltration is an indirect marker of muscle fibre disintegration, as the extension of infiltration shows the number of necrotic fibres. By using this indirect marker of muscle fibre destruction, we observed that samples obtained from muscles injected with ropivacaine 0.5% showed disintegration to a lesser extend than ropivacaine 0.75% samples. These differences between R75 and R5 groups were observed either on 2 days samples or on 4 and 7 days samples. Consequently, ropivacaine myotoxicity seems to be dose-dependent. Nevertheless, there were differences regarding the time-course of maximum damage observed between the two different ropivacaine concentrations studied. Ropivacaine 0.75% cause maximum muscle damage as early as day 2 after injection. This maximum muscle damage remained constant up to 7 days after injection. Ropivacaine 0.5% caused less severe damage on days 2, 4 and 7 after injection. Nevertheless, six out of eight samples injected with ropivacaine 0.5% were scored with damage score = 2 on day 7. This observation means that on day 7, light microscopy process revealed that ropivacaine 0.5% caused the most extensive damage few days later than ropivacaine 0.75% did. Despite this fact, statistical analysis revealed significant difference between R75 and R5 on days 2, 4 and 7, showing that dose-dependent differences in muscle damage are prominent until the complete muscle regeneration.
Based on our preliminary study (unpublished observations), we decided to examine under the TEM, samples dissected only 7 days after injection and not earlier because that was the time of maximum damage observed under electron microscope for muscles injected with ropivacaine 0.5%. Muscles injected with ropivacaine 0.75% seemed to be disintegrated to the same degree on days 2, 4 and 7. Consequently, 7 days after injection is the time of the maximum morphological lesions observed under light microscope for ropivacaine 0.75% and ropivacaine 0.5%. Although morphological changes in samples infiltrated with ropivacaine 0.75% were more intense compared to those observed in ropivacaine 0.5%, muscle fibres from both groups showed mitochondrial and myofibril destruction, intracellular oedema, local loss of myofibrils and dilation of sarcoplasmic reticulum, while the sarcolemma is preserved. These findings are consistent with previous studies, where muscle fibres were examined under the electron microscope after injection with bupivacaine or ropivacaine [5,10].
Although several authors have investigated the possible mechanisms involved in local anaesthetic myotoxicity, few data are known about ropivacaine mechanism of action. It is clear that neither mechanical trauma nor fluid volume injected is responsible for muscle destruction. Groups N and S samples showed significantly lesser muscle damage compared to R75 and R5 group, indicating local anaesthetic involvement in muscle tissue destruction. The latter observation has also been confirmed by previous studies [2,11,12]. Benoit and colleagues  have proposed that an increase of sarcoplasmic Ca2+ is responsible for cellular death.
Bupivacaine, the most myotoxic of the local anaesthetic studied, seemed to enhance Ca2+ efflux and to inhibit Ca2+ reuptake from sarcoplasmic reticulum. All local anaesthetics seemed to enhance Ca2+ permeability through sarcoplasmic reticulum and this action has been correlated to local anaesthetic lipophilicity . Nevertheless, it is possible that bupivacaine is able to specifically enhance Ryanodine receptor activity and to block Ca2+ reuptake from sarcoplasmic reticulum leading to a significant muscle damage [13-15]. On the other hand, tetracaine, an ester local anaesthetic with lipophilicity that resembles that of bupivacaine, is less potent myotoxic, than bupivacaine, possibly because tetracaine inhibits Ryanodine receptor activity . Although similarities in morphologic appearance under light and electron microscope between bupivacaine and ropivacaine could correspond to common histopathologic mechanisms of cellular damage, further studies are necessary in order to investigate the specific mechanisms involved in ropivacaine myotoxicity.
We compared two different concentrations of ropivacaine, the ropivacaine 0.75% solution with the ropivacaine 0.5% solution. We injected tibialis anterior muscle with 100 μL of solution. This volume is not considered to cause pressure damage to muscle. Foster and colleagues  used 200 μL of the solution without leading to pressure damage. Similarly, we mixed ropivacaine 1% with preservative-free normal saline 0.9% in order to prepare the ropivacaine 0.5% solution. The potential presence of preservatives in the solution could cause muscle damage, which would be independent of the local anaesthetic action.
In conclusion, this experimental study has revealed the severe myotoxic effect of ropivacaine after single intramuscular injection. Ropivacaine myotoxicity is dose-dependent, as has been shown for other local anaesthetics studied. Use of high-dose solutions of ropivacaine 0.75% will lead to extensive muscle damage, compared to less concentrated solutions. Additionally, the use of lower dose of ropivacaine caused the maximum muscle damage later in the study period. This myotoxic effect seemed to be fully reversible after a 4-week period and this fact is significant regarding the use of this local anaesthetic for wound infiltration or nerve blockades. Although reversible, this extensive muscle damage could occasionally be clinicallysignificant .
1. Hogan Q, Dotson R, Erickson S, Kettler R, Hogan K. Local anesthetic myotoxicity: A case and review. Anesthesiology
2. Foster AH, Carlson BM. Myotoxicity of local anesthetics and regeneration of the damaged muscle fibers. Anesth Analg
3. Zink W, Graf BM. Local anesthetic myotoxicity. Reg Anesth Pain Med
4. Gómez-Arnau JI, Yanguela J, Gonzalez A et al
. Anaesthesia-related diplopia after cataract surgery. Br J Anaesth
5. Zink W, Seif C, Bohl JRE et al
. The acute myotoxic effects of bupivacaine and ropivacaine
after continuous peripheral nerve blockades. Anesth Analog
6. Benoit PW, Yagiela JA, Fort NF. Pharmacological correlation between local anesthetic-induced myotoxicity and disturbances of intracellular Ca2+
distribution. Toxicol Appl Pharmacol
7. Petterson N, Berggren P, Larsson M, Westman B, Hahn RG. Pain relief by wound infiltration with bupivacaine or high-dose ropivacaine
after inguinal hernia repair. Reg Anesth Pain Med
8. Benoit PW, Belt WD. Destruction and regeneration of skeletal muscle after treatment with a local anaesthetic, bupivacaine (Marcaine). J Anat
9. Hall-Craggs ECB. Early ultrastructural changes in skeletal muscle exposed to local anesthetic bupivacaine (Marcaine). Br J Exp Pathol
10. Kytta J, Heinon E, Rosenberg PH et al
. Effects of repeated bupivacaine administration on sciatic nerve and surrounding muscle tissue in rats. Acta Anaesthesiol Scand
11. Basson MD, Carlson BM. Myotoxicity of single and repeated injections of Mepivacaine (carbocaine) in the rat
. Anesth Analg
12. Pere P, Watanabe H, Pitkanen M, Wahlstrom T, Rosenberg PH. Local myotoxicity of bupivacaine in rabbits after continuous supraclavicular bracial plexus block. Reg Anesth
13. Komai H, Lokuta AJ. Interaction of bupivacaine and tetracaine with the sarcoplasmic reticulum Ca2+
release channel of skeletal and cardiac muscles
14. Zink W, Graf BM, Sinner B, Martin E, Fink RHA, Kunst G. Differential effects of bupivacaine on intracellular Ca2+
15. Takahashi S. Local anaesthetic bupivacaine alters function of sarcoplasmic reticulum and sarcolemmal vesicles from rabbit masseter muscle. Pharmacol Toxicol