EXPERIMENTAL
Histochemical
NEUROLOGY
46, 345-354
(1975)
Changes in Innervated and Denervated Muscle Fibers Following Treatment With Bupivacaine (Marcain) E. C. B. HALL-CRAGGS Drpartmwt Gowrr
Skeletal
AND H. SINGH SEYAN
of Alkatomy, Uuiversity College Lofkdokr, Street, Loedokk, W.C.1.. E?kyland Rcccivrd
Scjtmkber
28,19i4
Widespread degenerative and regenerative changes were produced in the fast tibialis anterior and slow soleus muscles of rats by intramuscular injection of bupivacaine combined with hyaluronidase. The redifferentiation of muscle fiber types during the period of regeneration was followed in innervated and denervated muscles using histochemical methods. Evidence of differentiation was present in fast and slow innervated muscles 2 weeks after treatment and three fiber types could be recognized in the tibialis anterior at three weeks. By four weeks tibialis anterior and soleus showed a normal distribution of fiber types and associated differences in fiber caliber. Following denervation some enzyme activity was seen to appear in both muscles, but after four weeks fiber caliber remained markedly reduced and no differentiation of the enzyme activity of fibers was seen. It was concluded that a complete dedifferentiation of fibers had followed treatment with bupivacaine and that innervation was essential for the redifferentiation of fiber types.
INTRODUCTION The local anaesthetic drug bupivacaine, which was introduced into clinical practice by Telivuo (19), is known to produce changes in the underlying fibers when applied to the surface of skeletal muscles (17). These changes were first thought to be those of a denervation-like atrophy (12, 17) but Benoit and Belt (2) concluded that the drug had a specific myotoxic action. They described a rapid degenerative phase lasting approximately 48 hr during which fragmentation and phagocytosis of fiber cytoplasm occurred. This was followed by a regenerative phase which led to the resumption of normal fiber size after 32 days. These rapid degenerative and regenerative changes have since been confirmed and, by 345 Copyright 0 1975 by AcademicPress,Inc. AU rights of reproduction in any form reserved.
346
HALL-CRAGGS
AND
SEYAN
means of intramuscular injections combined with the enzyme hyaluronidase, have been extended to involve the whole muscle (9). This technique was devised to provide a suitable experimental model for a quantitative biochemical study of the development of the enzyme systems of fast and slow muscles. It seemed logical, therefore, to combine this with a histochemical study of the regenerating muscles. Only in this way can the metabolic development of the individual fiber types in a muscle be followed. In addition to this, a knowledge of the pattern of reestablishment of fiber types during the redifferentiation that follows treatment with bupivacaine promised a system in which the part played by innervation could be evaluated. MATERIAL
AND
METHODS
The experimental animals used in this study were female W&tar rats weighing ZOO-250 g. The tibialis anterior and soleus muscles were chosen as examples of fast and slow muscles, respectively. In one group of animals the tibialis anterior muscle received intramuscular injections of 0.5 ml of 0.5% bupivacaine 1 in 0.9% saline containing 15 IU of hyaluronidase on three consecutive days (9). In a second group of animals the initial injection was combined with denervation of the treated. muscle by excising 0.5 cm of the common peroneal nerve. Preliminary experiments showed that it was not possible consistently to involve the whole of the soleus using percutaneous intramuscular injections. However, it was found that, when a single intramuscular injection of O.ZS.,ml of the preparation used for tibialis anterior was made directly into the muscle after surgical exposure, the desired widespread change was produced. This technique was, therefore, applied to two further groups of rats, in one of which the nerve to the treated soleus was divided and the proximal stump embedded in the adjacent belly of gastrocnemius. All injections and surgical exposures were carried out under general anaesthesia using open ether or intraperitoneal chloral hydrate (400 mg/kg in a 4% solution). Animals from each of the four groups were killed 1, 2, 3, and 4 weeks after their initial day of treatment. The experimental and contralateral muscles were excised and placed in n-hexane cooled to -70 C in a mixture of acetone and solid carbon dioxide. Sections of 10 pm thickness were cut in a cryostat and stained with haemotoxylin and eosin or incubated to demonstrate menadione assisted alpha-glycerophosphate dehydrogenase ((Y GPDH) (4)) the diphosphopyridinenucleotide-tetrazolium reductase adenosine triphosphatase reaction (DPNH-TR) (4)) and alkali-stable (AM-ATPase) (6). 1 A Supply of Marcain was kindly made available by BDH
Pharmaceuticals
Ltd.
MUSCLE
FIBER
DIFFERENTIATION
347
RESULTS The normal tibialis anterior muscle of the rat does not have an even distribution of fiber types throughout a cross-section. A larger superficial region is made up of fibers staining strongly for a GPDH and weakly for DPNH-TR (type A) among which are distributed fibers showing an intermediate reaction (type B). A smaller deeper region shows an additional fiber type of smaller caliber and staining strongly for DPNH-TR (type C). It was in the latter region that the redifferentiation of fiber types was followed. Soleus, on the other hand, is composed of a majority of type B fibers and a variable minority of type C fibers. These three fiber types were established by Stein and Padykula (18) and correspond, though not absolutely consistently, with the ap, p and (Y fibers (8) demonstrated by the AM-ATPase reaction (Figs. 1A and 1B). Sections stained with hematoxylin and eosin confirmed the morphological changes previously described and, by demonstrating the presence of central nuclei, provided evidence that fibers had in fact undergone a process of redifferentiation (2,9). One week after the administration of bupivacaine the fibers of tibialis anterior showed a reduction of caliber and had the appearance of young fibers with one or more central nuclei. No differentiation of fiber types could be seen and the overall staining reaction for LYGPDH and DPNHTR was weak. However, that for AM-ATPase was almost comparable to the response of the most darkly stained contralateral fibers (Fig. 1A). At two weeks an increase in fiber caliber was seen and at the same time a disparity in the caliber of individual fibers was apparent. This coincided with the demonstration of a wider spectrum of fibers when stained for DPNH-TR, the larger fibers being less densely stained and the smaller fibers being more so (Fig. 1C). Using the AM-ATPase reaction two fiber types could be clearly recognized and these corresponded to the (Y and a p fibers of Samaha et al. (15) (Fig. 1D). The third, or p, fiber was not seen until three weeks after the administration of bupivacaine when all reactions showed three fiber types to be present. After four weeks, apart from the presence of central nuclei in the majority of fibers, normal caliber and fiber type distribution had been restored (Fig. 2A and B). At one week after injection the soleus muscle showed a remarkably similar picture to that of tibialis anterior having a reduced staining intensity for the metabolic enzymes (Fig. 2C) but an increased homogeneous density for AM-ATPase. By two weeks a lightening of the staining intensity for AM-ATPase of the majority of fibers indicated some differentiation (Fig. 2D) which was not yet apparent for the enzyme (Y GPDH and DPNH-TR. The AM-ATPase reaction showed further differentiation after 3 weeks but it was not until 4 weeks after bupivacaine administration
348
HALL-CRAGGS
AND
SEYAN
FIG. 1. Times given are after first injection of bupivacaine. (A) Tibialis anterior. AM-ATPase. Contralateral control (above) showing types and experimental muscle (below) showing a strong reaction but no tion. (B) Soleus. AM-ATPase. Contralateral control showing two fiber Two weeks. Tibialis anterior. DPNH-TR. (D) Two weeks. Tibialis AM-ATPase. All photographs X120.
One week. three fiber differentiatypes. (C) anterior.
MUSCLE
FIBER
DIFFERENTIATION
;IG. 2. Times given are after first injection of bupivacaine. (A) Four weeks. Four weeks. Tibialis anterior. DPHN-TR. (D) Two weeks. Soleus. AM-ATPase. All
Til jialis anterior. AM-ATPase. (B) cc ) One week. Soleus. DPHN-TR. phcjtographs X120.
350
HALL-CRAGGS
AND
SEYAN
that the p fibers fully displayed their alkaline lability with a consequent reduction in staining intensity (Fig. 3A). By this time the two metabolic enzymes also showed a population of two fiber types. When the administration of bupivacaine was combined with denervation the picture in fast and slow muscles was again very similar at the end of one week. However, the fibers of both muscles had a much smaller caliber and stained only weakly for AM-ATF’ase (Fig. 3B). After two weeks both muscles had regained some enzyme activity (Fig. SC) and this was still present in tibialis anterior at the fourth week. At this time there was little change in caliber and although some variation in density of staining could be observed, no real evidence of fiber differentiation could be detected nor was the variation consistently correlated with fiber caliber as in the normal muscle (Fig. 3D). The soleus, however, when examined after four weeks appeared to have undergone a loss of fiber caliber and AM-ATPase activity although the fibers were still positive for NADH-TR and aGPDH. DISCUSSION Some information is available about the histochemical changes that folthe treatment of skeletal muscle with bupivacaine. Seven days after treatment Libelius et al. (12) reported atrophy and loss of succinic dehydrogenase activity in the two fiber types they defined. A ‘regression’ of the atrophy was visible on the tenth day with the reappearance of two fiber types and by the fifteenth day they recorded a normal staining intensity. However, an illustration of this stage shows that a substantial number of grossly atrophic fibers remained. Benoit and Belt (Z), using Altmann’s stain for mitochondria, again to determine two fiber types, concluded that the larger white (type A) fibers were more resistant to the drug. Both these reports stem from experiments in which the lesion was only partial. This may account for the appearance of normal fibers only two weeks after treatment. Observations made during preliminary experiments before widespread lesions were produced (9) confirm that type A fibers are frequently found to be unaffected although surrounded by groups of regenerating fibers of low metabolic enzyme activity. The more complete degeneration described in the present expriments allowed the affected fibers to be recognized confidently and at the end of the first week a homogeneous reduction in metabolic activity was evident. However, both fast and slow muscles gave a positive result when stained for AM-ATPase. This feature has been reported in developing and regenerating rat muscles by Askanas and Hee (1) and in the guinea pig by Riley ( 14), but Guth and Samaha (7) have shown that this positive histochemical reaction in neonatal muscle low
MUSCLE
FIBER
DIFFERENTIATION
FIG. 3. Times given are after first injection of bupivacaine. (A) Four weeks. Soleus. AM-ATPase X,120. (B) One week. Denervated tibia& anterior. AMATPase. X1.20. (C) Two weeks. Denervated soleus. AM-ATPase. X 190. (D) Four weeks. Denervated tibialis anterior. DPNH-TR. X190.
352
HALL-CRAGGS
AND
SEYAN
is not correlated with biochemical estimates of AM-ATPase activity or speed of contraction. Evidence of differentiation of fiber types was present in both muscles after two weeks but this was more pronounced in the fast muscle which by 3 weeks contained three distinct fiber types. This differentiation was most clearly demonstrated by the AM-ATPase reaction as the reaction is uniform and does not depend on mitochondrial morphology. Although no indication of the lineage of individual fibers can be gained, the order of differentiation is consistent with that described by Nystrijm (13) in developing kitten fast muscle. The progress of differentiation in the soleus appeared to lag when compared with the tibialis anterior but this may well be due to the fact that the predominant fiber type is the last to make its appearance when identified by the AM-ATPase reaction (i.e., the p or B fiber) and that the mitochondrial pattern of its component B and C fibers is not as distinctive as that of the A fibers seen in fast muscle. Nevertheless, at the end of 3 weeks, both muscles had re-established a fully differentiated fiber pattern as judged by the reactions used. It is also of interest that the distribution of types was similar to that of a normal muscle and that type grouping was very uncommonly seen. This suggests that little disturbance of the pattern of the neural element of the neuromuscular junction occurred during the initial degenerative phase. Histochemical demonstration of acetylcholinesterase followed by maceration and teasing has shown that after bupivacaine treatment the distribution of motor end-plates was unchanged and less than 2% of fibers could be found to receive dual innervation (unpublished observation). To this can be added the fact that miniature end-plate potentials were recorded by Jirmanova’ and Thesleff (11) only three days after the application of bupivacaine to surface fibers. As far as can be judged from previous work on the differentiation of developing muscle, fibers caused to degenerate by bupivacaine, subsequently recapitulate the normal process of differentiation. As experimental models, muscles so treated have an advantage in that the fibers behave synchronously over a fairly well defined time course and development is not gradual and random as in the normal animal (3). Denervation has long been known to interfere with the normal process of muscle development (20). Engel and Karpati (5) in a histochemical study of developing rat limb muscles found severe impairment of maturation with the persistence of many myotubular forms when sciatic neurectomy was performed in neonatal animals. They also suggested that type B and C fibers were still able to develop into mature forms. In a similar study using electron microscopy ( 16) the typical ultrastructural characteristics of fiber types were not found. These findings have since been confirmed following neurectomy three days prior to birth (10). Jirmanova and
MUSCLE
FIBER
DIFFERENTIATION
353
Thesleff (11) have made some ultrastructural observations following denervation and bupivacaine treatment of rat fast muscle. They found that the initial stages of regeneration proceeded normally but a delay was apparent at the fifth day and at the fifteenth fibers had not developed beyond the stage of young fibers with myofibrils out of register and an immature sarcoplasmic reticulum and T-system. In the experiments described here, denervation was again found to have a profound effect on the regeneration of fibers following treatment with bupivacaine. Unlike the innervated fibers, denervated fibers showed IittIe AM-ATPase activity at 1 week and although some enzyme activity was regained later, no sign of any differentiation of fiber types was observed after 4 weeks. At this time the fibers of soleus were probably entering a new phase of denervation atrophy. It seems clear, therefore, that the degeneration produced by treatment of muscle fibers with bupivacaine involves a complete dedifferentiation and that innervation is essential for subsequent redifferentiation from a pool of myoblasts possibly derived from the original differentiated fibers. REFERENCES 1. ASKANAS, V., and HEE, D. 1973. Histochemistry of cultured, embryonic and regenerating rat muscle. J. Histochem. Cytochem 21 : 78.5793. 2. BENOIT, P. W. and BELT, W. D. 1970. Destruction and regeneration of skeletal muscle after treatment with a local anaesthetic bupivacaine (Marcaine). J. Anat. 107: 547-556. 3. DUBOVITZ, V. 1965. Enzyme histochemistry of skeletal muscle. I. Developing animal muscle. J. Ne~rol. Newosurg. Psychiat. 28: 516-519. 4. ENGEL, W. K., and BROOKE M. H. 1966. Muscle biopsy as a clinical aid, pp. 99146. In “Neurological Diagnostic Techniques” W. S. Fields [Ed.]. Thomas, Springfield. 5. ENGEL, W. K., and KARPATI, A. 1968. Impaired skeletal muscle maturation following neonatal neurectomy. Dew. Biol. 17: 713-723. 6. GUTH, L. and SAMAHA, F. J. 1970. Procedure for the histochemical demonstration of actomyosin ATPase. Exp. Nczrrol. 28: 365-367. 7. GUTH, L. and SAMAHA, F. J. 1972. Erroneous interpretations which may result from application of the “myofibrillar ATPase” histochemical procedure to developing muscle. Exp. Nrurol. 34: 465-475. 8. GUTH, L. and YELLIN, H. 1971. The dynamic nature of the so-called “fiber-types” of ,mammalian skeletal muscle. Exp. Nrztrol. 31 : 277-300. 9. HALL-CRAGGS, E. C. B. 1974. Rapid degeneration and regeneration of a whole skeletal muscle following treatment with bupivacaine (Marcaine). Exp. Neural. 43 : 349-358. 10. HANZLIKOV& V. and SCHIAFFINO, S. 1973. Studies on the effect of denervation in developing muscle III. Diversification of myofibrillar structure and origin of the heterogeneity of muscle fiber types. Z. Zellforsch. 147: 75-85. 11, JIRMANOVA, I. and THESLEFF, S. 1972. Ultrastructural study of experimental muscle degeneration and regeneration in the adult rat. 2. Zellforsch. 131 : 77-97.
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12. LIBELIUS, R., SONESSON, B., STAMENOVI~, B. A. and THESLEFF, S. 1970. Denervation-like changes in skeletal muscle after treatment with a local anaesthetic. J. Anat. 106 : 297309. 13. NYSTR~M, B. 1966. Succinic dehydrogenase in developing cat leg muscles. Nature (London) 212 : 954-5. 14. RILEY, D. A. 1973. Histochemical changes in ATPase activity during regeneration of adult skeletal muscle fibers. Exp. Neural. 41: 690-704. 15. SAMAHA, F. J., GUTH, L. and ALBERS, R. W. 1970. Phenotypic differences between the actomyosin ATPase of the three fiber types of mammalian skeletal muscle. Exg. Neural. 26 : 120-125. 16. SHAFIQ, S. A., ASIEDU, S. A. and MILHORAT, A. T. 1972. Effect of neonatal neurectomy on differentiation of fiber types in rat skeletal muscle. Exp. Neurol. 35 : 52+540. 17. SOKOLL, M. D., SONESSON, B. and THESLEFF, S. 1968. Denervation changes produced in an innervated skeletal muscle by long-continued treatment with a local anaesthetic. Eur. J. Pharmacol. 4: 179-187. 18. STEIN, J. M. and PADYKULA, H. A. 1962. Histochemical classification of individual skeletal muscle fibres of the rat. Amer. J. Anat. 110: 103-123. 19. TELIVUO, L. 1963. A new long-acting local anaesthetic solution for pain relief after thoracotomy. Ann. Chir. Gynaecol. Fenn. 52: 513-520. 20. ZELENA, J. 1962. The effect of denervation on muscle development, pp. 103-126. In “The Denervated Muscle.” E. Gutmann [Ed.]. Publishing House of the Czechoslovak Academy of Sciences, Prague.
Histochemical
NEUROLOGY
46, 345-354
(1975)
Changes in Innervated and Denervated Muscle Fibers Following Treatment With Bupivacaine (Marcain) E. C. B. HALL-CRAGGS Drpartmwt Gowrr
Skeletal
AND H. SINGH SEYAN
of Alkatomy, Uuiversity College Lofkdokr, Street, Loedokk, W.C.1.. E?kyland Rcccivrd
Scjtmkber
28,19i4
Widespread degenerative and regenerative changes were produced in the fast tibialis anterior and slow soleus muscles of rats by intramuscular injection of bupivacaine combined with hyaluronidase. The redifferentiation of muscle fiber types during the period of regeneration was followed in innervated and denervated muscles using histochemical methods. Evidence of differentiation was present in fast and slow innervated muscles 2 weeks after treatment and three fiber types could be recognized in the tibialis anterior at three weeks. By four weeks tibialis anterior and soleus showed a normal distribution of fiber types and associated differences in fiber caliber. Following denervation some enzyme activity was seen to appear in both muscles, but after four weeks fiber caliber remained markedly reduced and no differentiation of the enzyme activity of fibers was seen. It was concluded that a complete dedifferentiation of fibers had followed treatment with bupivacaine and that innervation was essential for the redifferentiation of fiber types.
INTRODUCTION The local anaesthetic drug bupivacaine, which was introduced into clinical practice by Telivuo (19), is known to produce changes in the underlying fibers when applied to the surface of skeletal muscles (17). These changes were first thought to be those of a denervation-like atrophy (12, 17) but Benoit and Belt (2) concluded that the drug had a specific myotoxic action. They described a rapid degenerative phase lasting approximately 48 hr during which fragmentation and phagocytosis of fiber cytoplasm occurred. This was followed by a regenerative phase which led to the resumption of normal fiber size after 32 days. These rapid degenerative and regenerative changes have since been confirmed and, by 345 Copyright 0 1975 by AcademicPress,Inc. AU rights of reproduction in any form reserved.
346
HALL-CRAGGS
AND
SEYAN
means of intramuscular injections combined with the enzyme hyaluronidase, have been extended to involve the whole muscle (9). This technique was devised to provide a suitable experimental model for a quantitative biochemical study of the development of the enzyme systems of fast and slow muscles. It seemed logical, therefore, to combine this with a histochemical study of the regenerating muscles. Only in this way can the metabolic development of the individual fiber types in a muscle be followed. In addition to this, a knowledge of the pattern of reestablishment of fiber types during the redifferentiation that follows treatment with bupivacaine promised a system in which the part played by innervation could be evaluated. MATERIAL
AND
METHODS
The experimental animals used in this study were female W&tar rats weighing ZOO-250 g. The tibialis anterior and soleus muscles were chosen as examples of fast and slow muscles, respectively. In one group of animals the tibialis anterior muscle received intramuscular injections of 0.5 ml of 0.5% bupivacaine 1 in 0.9% saline containing 15 IU of hyaluronidase on three consecutive days (9). In a second group of animals the initial injection was combined with denervation of the treated. muscle by excising 0.5 cm of the common peroneal nerve. Preliminary experiments showed that it was not possible consistently to involve the whole of the soleus using percutaneous intramuscular injections. However, it was found that, when a single intramuscular injection of O.ZS.,ml of the preparation used for tibialis anterior was made directly into the muscle after surgical exposure, the desired widespread change was produced. This technique was, therefore, applied to two further groups of rats, in one of which the nerve to the treated soleus was divided and the proximal stump embedded in the adjacent belly of gastrocnemius. All injections and surgical exposures were carried out under general anaesthesia using open ether or intraperitoneal chloral hydrate (400 mg/kg in a 4% solution). Animals from each of the four groups were killed 1, 2, 3, and 4 weeks after their initial day of treatment. The experimental and contralateral muscles were excised and placed in n-hexane cooled to -70 C in a mixture of acetone and solid carbon dioxide. Sections of 10 pm thickness were cut in a cryostat and stained with haemotoxylin and eosin or incubated to demonstrate menadione assisted alpha-glycerophosphate dehydrogenase ((Y GPDH) (4)) the diphosphopyridinenucleotide-tetrazolium reductase adenosine triphosphatase reaction (DPNH-TR) (4)) and alkali-stable (AM-ATPase) (6). 1 A Supply of Marcain was kindly made available by BDH
Pharmaceuticals
Ltd.
MUSCLE
FIBER
DIFFERENTIATION
347
RESULTS The normal tibialis anterior muscle of the rat does not have an even distribution of fiber types throughout a cross-section. A larger superficial region is made up of fibers staining strongly for a GPDH and weakly for DPNH-TR (type A) among which are distributed fibers showing an intermediate reaction (type B). A smaller deeper region shows an additional fiber type of smaller caliber and staining strongly for DPNH-TR (type C). It was in the latter region that the redifferentiation of fiber types was followed. Soleus, on the other hand, is composed of a majority of type B fibers and a variable minority of type C fibers. These three fiber types were established by Stein and Padykula (18) and correspond, though not absolutely consistently, with the ap, p and (Y fibers (8) demonstrated by the AM-ATPase reaction (Figs. 1A and 1B). Sections stained with hematoxylin and eosin confirmed the morphological changes previously described and, by demonstrating the presence of central nuclei, provided evidence that fibers had in fact undergone a process of redifferentiation (2,9). One week after the administration of bupivacaine the fibers of tibialis anterior showed a reduction of caliber and had the appearance of young fibers with one or more central nuclei. No differentiation of fiber types could be seen and the overall staining reaction for LYGPDH and DPNHTR was weak. However, that for AM-ATPase was almost comparable to the response of the most darkly stained contralateral fibers (Fig. 1A). At two weeks an increase in fiber caliber was seen and at the same time a disparity in the caliber of individual fibers was apparent. This coincided with the demonstration of a wider spectrum of fibers when stained for DPNH-TR, the larger fibers being less densely stained and the smaller fibers being more so (Fig. 1C). Using the AM-ATPase reaction two fiber types could be clearly recognized and these corresponded to the (Y and a p fibers of Samaha et al. (15) (Fig. 1D). The third, or p, fiber was not seen until three weeks after the administration of bupivacaine when all reactions showed three fiber types to be present. After four weeks, apart from the presence of central nuclei in the majority of fibers, normal caliber and fiber type distribution had been restored (Fig. 2A and B). At one week after injection the soleus muscle showed a remarkably similar picture to that of tibialis anterior having a reduced staining intensity for the metabolic enzymes (Fig. 2C) but an increased homogeneous density for AM-ATPase. By two weeks a lightening of the staining intensity for AM-ATPase of the majority of fibers indicated some differentiation (Fig. 2D) which was not yet apparent for the enzyme (Y GPDH and DPNH-TR. The AM-ATPase reaction showed further differentiation after 3 weeks but it was not until 4 weeks after bupivacaine administration
348
HALL-CRAGGS
AND
SEYAN
FIG. 1. Times given are after first injection of bupivacaine. (A) Tibialis anterior. AM-ATPase. Contralateral control (above) showing types and experimental muscle (below) showing a strong reaction but no tion. (B) Soleus. AM-ATPase. Contralateral control showing two fiber Two weeks. Tibialis anterior. DPNH-TR. (D) Two weeks. Tibialis AM-ATPase. All photographs X120.
One week. three fiber differentiatypes. (C) anterior.
MUSCLE
FIBER
DIFFERENTIATION
;IG. 2. Times given are after first injection of bupivacaine. (A) Four weeks. Four weeks. Tibialis anterior. DPHN-TR. (D) Two weeks. Soleus. AM-ATPase. All
Til jialis anterior. AM-ATPase. (B) cc ) One week. Soleus. DPHN-TR. phcjtographs X120.
350
HALL-CRAGGS
AND
SEYAN
that the p fibers fully displayed their alkaline lability with a consequent reduction in staining intensity (Fig. 3A). By this time the two metabolic enzymes also showed a population of two fiber types. When the administration of bupivacaine was combined with denervation the picture in fast and slow muscles was again very similar at the end of one week. However, the fibers of both muscles had a much smaller caliber and stained only weakly for AM-ATF’ase (Fig. 3B). After two weeks both muscles had regained some enzyme activity (Fig. SC) and this was still present in tibialis anterior at the fourth week. At this time there was little change in caliber and although some variation in density of staining could be observed, no real evidence of fiber differentiation could be detected nor was the variation consistently correlated with fiber caliber as in the normal muscle (Fig. 3D). The soleus, however, when examined after four weeks appeared to have undergone a loss of fiber caliber and AM-ATPase activity although the fibers were still positive for NADH-TR and aGPDH. DISCUSSION Some information is available about the histochemical changes that folthe treatment of skeletal muscle with bupivacaine. Seven days after treatment Libelius et al. (12) reported atrophy and loss of succinic dehydrogenase activity in the two fiber types they defined. A ‘regression’ of the atrophy was visible on the tenth day with the reappearance of two fiber types and by the fifteenth day they recorded a normal staining intensity. However, an illustration of this stage shows that a substantial number of grossly atrophic fibers remained. Benoit and Belt (Z), using Altmann’s stain for mitochondria, again to determine two fiber types, concluded that the larger white (type A) fibers were more resistant to the drug. Both these reports stem from experiments in which the lesion was only partial. This may account for the appearance of normal fibers only two weeks after treatment. Observations made during preliminary experiments before widespread lesions were produced (9) confirm that type A fibers are frequently found to be unaffected although surrounded by groups of regenerating fibers of low metabolic enzyme activity. The more complete degeneration described in the present expriments allowed the affected fibers to be recognized confidently and at the end of the first week a homogeneous reduction in metabolic activity was evident. However, both fast and slow muscles gave a positive result when stained for AM-ATPase. This feature has been reported in developing and regenerating rat muscles by Askanas and Hee (1) and in the guinea pig by Riley ( 14), but Guth and Samaha (7) have shown that this positive histochemical reaction in neonatal muscle low
MUSCLE
FIBER
DIFFERENTIATION
FIG. 3. Times given are after first injection of bupivacaine. (A) Four weeks. Soleus. AM-ATPase X,120. (B) One week. Denervated tibia& anterior. AMATPase. X1.20. (C) Two weeks. Denervated soleus. AM-ATPase. X 190. (D) Four weeks. Denervated tibialis anterior. DPNH-TR. X190.
352
HALL-CRAGGS
AND
SEYAN
is not correlated with biochemical estimates of AM-ATPase activity or speed of contraction. Evidence of differentiation of fiber types was present in both muscles after two weeks but this was more pronounced in the fast muscle which by 3 weeks contained three distinct fiber types. This differentiation was most clearly demonstrated by the AM-ATPase reaction as the reaction is uniform and does not depend on mitochondrial morphology. Although no indication of the lineage of individual fibers can be gained, the order of differentiation is consistent with that described by Nystrijm (13) in developing kitten fast muscle. The progress of differentiation in the soleus appeared to lag when compared with the tibialis anterior but this may well be due to the fact that the predominant fiber type is the last to make its appearance when identified by the AM-ATPase reaction (i.e., the p or B fiber) and that the mitochondrial pattern of its component B and C fibers is not as distinctive as that of the A fibers seen in fast muscle. Nevertheless, at the end of 3 weeks, both muscles had re-established a fully differentiated fiber pattern as judged by the reactions used. It is also of interest that the distribution of types was similar to that of a normal muscle and that type grouping was very uncommonly seen. This suggests that little disturbance of the pattern of the neural element of the neuromuscular junction occurred during the initial degenerative phase. Histochemical demonstration of acetylcholinesterase followed by maceration and teasing has shown that after bupivacaine treatment the distribution of motor end-plates was unchanged and less than 2% of fibers could be found to receive dual innervation (unpublished observation). To this can be added the fact that miniature end-plate potentials were recorded by Jirmanova’ and Thesleff (11) only three days after the application of bupivacaine to surface fibers. As far as can be judged from previous work on the differentiation of developing muscle, fibers caused to degenerate by bupivacaine, subsequently recapitulate the normal process of differentiation. As experimental models, muscles so treated have an advantage in that the fibers behave synchronously over a fairly well defined time course and development is not gradual and random as in the normal animal (3). Denervation has long been known to interfere with the normal process of muscle development (20). Engel and Karpati (5) in a histochemical study of developing rat limb muscles found severe impairment of maturation with the persistence of many myotubular forms when sciatic neurectomy was performed in neonatal animals. They also suggested that type B and C fibers were still able to develop into mature forms. In a similar study using electron microscopy ( 16) the typical ultrastructural characteristics of fiber types were not found. These findings have since been confirmed following neurectomy three days prior to birth (10). Jirmanova and
MUSCLE
FIBER
DIFFERENTIATION
353
Thesleff (11) have made some ultrastructural observations following denervation and bupivacaine treatment of rat fast muscle. They found that the initial stages of regeneration proceeded normally but a delay was apparent at the fifth day and at the fifteenth fibers had not developed beyond the stage of young fibers with myofibrils out of register and an immature sarcoplasmic reticulum and T-system. In the experiments described here, denervation was again found to have a profound effect on the regeneration of fibers following treatment with bupivacaine. Unlike the innervated fibers, denervated fibers showed IittIe AM-ATPase activity at 1 week and although some enzyme activity was regained later, no sign of any differentiation of fiber types was observed after 4 weeks. At this time the fibers of soleus were probably entering a new phase of denervation atrophy. It seems clear, therefore, that the degeneration produced by treatment of muscle fibers with bupivacaine involves a complete dedifferentiation and that innervation is essential for subsequent redifferentiation from a pool of myoblasts possibly derived from the original differentiated fibers. REFERENCES 1. ASKANAS, V., and HEE, D. 1973. Histochemistry of cultured, embryonic and regenerating rat muscle. J. Histochem. Cytochem 21 : 78.5793. 2. BENOIT, P. W. and BELT, W. D. 1970. Destruction and regeneration of skeletal muscle after treatment with a local anaesthetic bupivacaine (Marcaine). J. Anat. 107: 547-556. 3. DUBOVITZ, V. 1965. Enzyme histochemistry of skeletal muscle. I. Developing animal muscle. J. Ne~rol. Newosurg. Psychiat. 28: 516-519. 4. ENGEL, W. K., and BROOKE M. H. 1966. Muscle biopsy as a clinical aid, pp. 99146. In “Neurological Diagnostic Techniques” W. S. Fields [Ed.]. Thomas, Springfield. 5. ENGEL, W. K., and KARPATI, A. 1968. Impaired skeletal muscle maturation following neonatal neurectomy. Dew. Biol. 17: 713-723. 6. GUTH, L. and SAMAHA, F. J. 1970. Procedure for the histochemical demonstration of actomyosin ATPase. Exp. Nczrrol. 28: 365-367. 7. GUTH, L. and SAMAHA, F. J. 1972. Erroneous interpretations which may result from application of the “myofibrillar ATPase” histochemical procedure to developing muscle. Exp. Nrurol. 34: 465-475. 8. GUTH, L. and YELLIN, H. 1971. The dynamic nature of the so-called “fiber-types” of ,mammalian skeletal muscle. Exp. Nrztrol. 31 : 277-300. 9. HALL-CRAGGS, E. C. B. 1974. Rapid degeneration and regeneration of a whole skeletal muscle following treatment with bupivacaine (Marcaine). Exp. Neural. 43 : 349-358. 10. HANZLIKOV& V. and SCHIAFFINO, S. 1973. Studies on the effect of denervation in developing muscle III. Diversification of myofibrillar structure and origin of the heterogeneity of muscle fiber types. Z. Zellforsch. 147: 75-85. 11, JIRMANOVA, I. and THESLEFF, S. 1972. Ultrastructural study of experimental muscle degeneration and regeneration in the adult rat. 2. Zellforsch. 131 : 77-97.
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