Acta Neuropathol (1992) 83: 584 - 589
`
`.__
`
`© Springer-Verlag 1992
`
`Comparison of behavior in muscle fiber regeneration
`after bupivacaine hydrochloride- and acid
`anhydride-induced myonecrosis
`C. Akiyama1, 2, S. Kobayashi1, 3, and I. Nonakal
`1 National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187, Japan
`2 Department of Pediatrics, Fukuoka University, School of Medicine,
`3 Department of Pediatrics, Jichi Medical School,
`
`Received July 22, 1991/Revised , accepted December 2, 1991
`
`Summary. We compared the morphologic characteris(cid:173)
`tics of muscle fiber necrosis and subsequent regenera(cid:173)
`tion after injury induced by intramuscular injections of
`bupivacaine hydrochloride (BPVC) and a variety of
`solutions at acid and alkaline pH (acetic anhydride,
`citric acid buffer, and sodium carbonate buffer). After
`BPVC injection the necrotic muscle fibers were rapidly
`invaded by phagocytic cells, followed by active regener(cid:173)
`ation and very little fibrous scar formation. The regen(cid:173)
`erating muscle fibers increased rapidly in size and
`attained complete
`fiber
`type differentiation and
`regained their initial fiber diameter within 1 month.
`Both alkaline and acid solutions induced muscle fiber
`necrosis followed by regeneration. Fiber necrosis
`induced by alkaline buffers and acetic anhydride solu(cid:173)
`tions above pH 5.0 produced changes quite similar to
`that induced by BPVC. However, injection with 0.1 M
`acetic anhydride at pH below 4.0 resulted in coagulative
`necrosis of the injured muscle with very little phagocytic
`infiltration with poor regenerative activity and dense
`fibrous tissue scarring. Thus, pH 4.0 appears to be the
`critical pH determining the type of muscle injury and
`subsequent poor pbagocytic and regenerative activities.
`This model of acidic acetic anhydride injury may lead to
`the identification of factors which interfere with regen(cid:173)
`eration and cause fibrous tissue scarring in human
`muscular dystrophy.
`
`Key words: Muscle necrosis - Regeneration - Fibrosis -
`Bupivacaine - Acid anhydride
`
`Skeletal muscle fibers undergo necrosis after mechanical
`and chemical injuries including crush, ischemia, cold or
`heat injuries, and injections of local anesthetics and
`snake toxins [1, 8]. The necrotic fibers are then invaded
`and cleared by phagocytic cells followed by an active
`regenerative process. The mechanism to activate satel-
`
`Offprint requests to: C. Akiyama (address see I above)
`
`lite cells, which play an important role in muscle fiber
`regeneration, is not yet known.
`One might ask what environmental factors are
`necessary to induce phagocytosis, and what the fate of
`necrotic fibers may be if phagocytosis is less active. To
`answer these questions we injected various chemicals at
`different pH into rat muscles and examined the behavior
`of necrotic and regenerative processes by means of
`muscle histochemistry with roorphometric analysis and
`electron microscopy.We found that acid anhydride (AA)
`solution at a pH less than 4.0 caused extensive myone(cid:173)
`crosis , with no apparent phagocytic activity and poor
`regeneration. On the other band , bupivacaine hydro(cid:173)
`chloride (BPVC; Marcaine), a local anesthetic, caused
`necrosis with marked phagocytic activity followed by
`rapid muscle fiber regeneration (3, 5, 13-16, 20, 23). We,
`therefore, carried out a study comparing muscle degen(cid:173)
`erative and regenerative processes after injury with
`BPVC and AA to identify factors which promote or
`inhibit muscle fiber regeneration.
`
`Materials and methods
`
`Male Wistar rats weighing 200- 250 g were used. The right soleus
`(red) and tibialis anterior (TA; white), muscles were surgically
`exposed under general anesthesia and various chemical solutions,
`listed in Table 1, were injected directly into the muscles with a thin
`gauge needle. The chemical solutions included 0.5 ml 0.5 % BPVC,
`0.1 M sodium carbonate at pH varying from 9.0 to 11.0, 0.1 M
`citric acid at pH 5.0- 3.0, 0.1 M AA at pH 3.0, 0.1 M AA solution
`whose pH was adjusted between 3.0 and 6.0 with 0.1 M NaOH, and
`0.5 % - 5 % AA in 0.85 % saline. The contralateral soleus and TA
`muscles of each rat received saline in the same manner and served
`as controls. In each trial, 2-3 rats were examined .
`To study the necrotizing process, the soleus muscles were taken
`from 2 each of 64 rats at 15 and 30 min, and 1, 2, 6, 12, 24, and 48 h
`after the injections of BPVC, and 0.1 M AA at pH 3.0, 4.0 and 5.0,
`respectively. Each muscle was examined by histochemistry and
`electron microscopy. For electron microscopy, the soleus muscle
`specimen was fixed in 0 .1 M sodium cacodylate-buffered glutaral(cid:173)
`dehyde solution at pH 7.4 for 3-4 h . The tissue was washed in the
`same buffer solution, post-fixed in 1 % OsO4 and embedded in
`epoxy resin after dehydration in serial alcohol solutions.
`
`FRESENIUS EXHIBIT 1056
`Page 1 of 6
`
`

`

`Table 1. Myoto>..ic agents
`
`Chemicals
`
`0.5 % bupivacaine hydrochloride
`0.1 M sodium carbonate buffer
`0.1 M citric acid buffer
`0. 1 M acetic anhydride buffer
`0.1 M acetic anhydride + NaOH
`
`0.5 % , 1 % , 3 % , 5 % , acetic anhydride
`(<pH3.0)
`
`585
`
`pH
`
`6.5
`9.0, 11.0
`5.0, 3.0
`3.0
`
`3.0
`4.0
`4.1
`4.3
`5.0
`3.0
`
`Myonecrosis
`(48 h)
`
`Phagocytosis
`(48 h)
`
`Regeneration
`(10 days)
`
`++ +
`+
`+++
`++ +
`
`++ +
`++ +
`++
`+
`+
`++ +
`
`+++
`+++
`++
`
`++
`++
`+++
`
`F
`F
`M
`p
`
`p
`p
`M
`M
`F
`p
`
`- , none; +,localized;+ + , moderate;++ +, marked; F, fair; M, moderate; P, poor
`
`To study muscle fiber regeneration, the soleus and TA muscles
`were taken from 5 animals each at 2, 3, 7, 10, 15, 20, and 30 days
`after injection from the two groups treated with BPVC and 0.1 M
`AA at pH 3.0 (total = 70 rats). In 2- 3 rats from each of the
`remaining groups listed in the Tobie 1, the treated muscles were
`examined at 2 and 10 days after the injection.
`The soleus and TA muscles were immediately frozen in
`isopenrane cooled in liquid nitrogen. Serial frozen sections were
`stained with hematoxylin and eosin (H&E), modified Gomori
`trichrome, and a battery of histochemical methods including
`NADH -TR, routine ATPase and ATPase with preincubation at pH
`4.5 and 4.3, acid phosphatase, nonspecific esterase (NSE), and
`acridine orange (AO). To measure muscle fiber diameters and to
`
`determine fiber types, 100-200 fibers in each muscle were analyzed
`in photographs from serial frozen sections printed at a final
`magnification of x 660.
`
`Results
`
`Muscle fiber necrosis
`
`As shown in Table 1, various chemicals (especially
`BPVC and acidic solutions below pH 4.0) caused muscle
`fiber necrosis. Immediately after BPVC injection,
`
`Fig. 1. Soleus muscles 48 h after bupivacaine hydrochloride
`(BPVC) (A) and acid anl1ydride (AA) at pH 3.0 (B) injections.
`Numerous necrotic fibers are invaded by phagocytic cells iu the
`BPVC-treated muscle (A). Although the contour of fibers is
`
`preserved, myofibrillar networks have disappeared and myonuclei
`were granulated (B). Note no macrophage invasion in extra- and
`intracellular spaces (B). A, B H&E, x 240
`
`FRESENIUS EXHIBIT 1056
`Page 2 of 6
`
`

`

`586
`almost all the muscle fibers in the treated soleus and TA
`underwent massive necrosis characterized by opaque
`fibers and empty sarcolemmal tubes, with foci of hyper(cid:173)
`contraction bands on longitudinal sections. The necrotic
`fibers were invaded by acid phosphatase-positive macro(cid:173)
`phages within 12 h after the injection. At 24 and 48 h,
`the necrotic fibers and the extracellular space were filled
`with numerous mononuclear phagocytic cells as well as
`some polymorpbonuclear leukocytes (Fig. l A).
`
`Fig. 2. An electron micrograph of a necrotic fiber 15 min after AA
`at pH 5.0 injection. The myofibrils are disrupted and organelJes
`swollen. T he sarcolemma has focally disappeared. x 24000
`
`Almost all the muscle fibers treated with dtric acid
`and sodium carbonate buffers, A A and AA buffers
`showed extensive necrosis. The appearance of the
`necrotic fibers treated with citric and sodium carbonate
`buffers was similar to those seen in BPVC-treated soleus
`muscles.
`On the other hand, in the muscle injected with AA
`below pH 4.0 and AA buffer at pH 3.0 the necrotic
`muscle fibers had faintly staining cytoplasm with loss of
`the intermyofibrillar networks and myonuclei. Muscle
`fiber necrosis after 0.5 % , 1 % , 3 % , and 5 % AA
`injections had a similar appearance. In the muscles
`treated with such strong acidic solutions, the contour of
`necrotic fibers remained unchanged with little pbagocyt(cid:173)
`ic activity even 24 and 48 h after injection (Fig. lB). The
`blood vesseles and peripheral nerve bundles in the
`interstitium were not clearly identifiable.
`
`Electron microscopy of necrotic fibers
`
`Fifteen minutes after BPVCand 0.1 MAA at pH 4.0 and
`5.0 injections, electron microscopy revealed disruption
`of the myofibrils with the formation of hypercontraction
`bands, and the sarcoplasmic reticulum appeared to be
`dilated (Fig. 2). There was focal loss of plasmalemma.
`A t 2 h , myofibrils were fragmented at the level of the
`Z-line (Fig. 3A) . The basal lamina and :satellite cells
`were well preserved at all stages. At 48 h, many
`phagocytic cells were found in the extra- and intracellu(cid:173)
`lar spaces, and satellite cells were activated.
`
`Fig. 3. Soleus muscle 2 h after AA at pH 5.0 (A) and at 3.0 (B) injections. Myofibrils are fragmented from a selective Z.line dissolutio n
`(A). The myofibrils are fragmented at va1ious levels of the sarcomeres including the Z.line, A and I bands (B). A, B x 6 000
`
`FRESENIUS EXHIBIT 1056
`Page 3 of 6
`
`

`

`In the muscle treated with O .1 M AA at pH 3. 0, there
`was disruption of both the basal lamina and plasma
`membrane 15 min after the injection. TI1ere was degen(cid:173)
`eration of both muscle fibers and satellite cell nuclei with
`loss of chromatin granules (Fig. 4). At 2 h, the myofi(cid:173)
`brils were fragmented at different levels of the myofibrils
`
`Soleus (red)
`
`60 I '6
`
`--·------·
`
`Untreated
`
`- - - - -
`
`60
`
`30
`
`587
`
`Tibialis anterior (white)
`
`---------
`---
`
`--------
`--
`
`A
`
`7 10
`
`15
`
`30
`
`7 10
`
`15
`
`30
`
`60
`
`.I
`]
`u::
`
`'O
`
`60
`
`-~z-:
`....
`------- - 30
`
`~
`
`- - - - - - -
`
`_..,....-----
`------
`----
`
`Fig. 4. Soleus muscle 15 min after AA at pH 3.0 injection. The
`basal lamina and plasma membrane have disappeared. Satellite
`cells are also degenerated. X 12 000
`
`B
`
`7 10
`
`15
`
`7 10
`30
`Days after injection
`
`15
`
`30
`
`Fig. S. Although the regenerating fibers (--0-) treated with BPVC
`(A) rapidly recovered and regained their initial size in both soleus
`(red) and tibialis anterior (white) muscles, when treated wit h AA
`solutio n at pH 3.0 (B) they showed markedly delayed recovery.
`One standard deviation of the mean control (untreated) fiber
`diameter (....._) is indicated by dotted lines
`
`4
`
`•
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`114
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`Fig. 6 A, B. At 30 days after BPVC injection. Centrally placed nuclei are seen in almost all of the regenerating fibers in both soleus (A) and
`tibialis anterior (B) muscles. H&E, x 240
`
`'>.,,.
`; ~ ....
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`
`FRESENIUS EXHIBIT 1056
`Page 4 of 6
`
`

`

`588
`including the Z-line, A and I bands (Fig. 3B). Even 48 h
`after the injection, no phagocyticcells had appeared and
`the fragmente d myofibrils remained unchanged from
`those at 2 b.
`Muscle fiber degeneration in the soleus muscles
`treated with these three different chemicals is summa(cid:173)
`rized in Table l.
`
`Muscle fiber regeneration
`
`In BPVC-treated TA and soleus muscles, small caliber
`regenerating fibers with basophilic cytoplasm and cen(cid:173)
`trally placed vesicular nuclei appeared in a pool of
`mononuclear cells 72 h after the injection on H&E.
`After 7 days, numerous regenerating fibers charac(cid:173)
`terized by basophilic cytoplasm, vesicular nuclei and
`prominent nucleoli were clearly identifiable. The regen(cid:173)
`erating fibers regained their initial diameter within
`30 days , but the recovery of the white muscle was
`slightly slower than that of the red muscle (Fig. 5). At
`30 days, almost all of the regenerating fibers still
`retained their centrally placed nuclei (Fig. 6).
`As expected from the behavior of myonecrosis, there
`was a marked delay in muscle fiber regeneration in
`necrotic muscles treated with strong AA solution below
`pH 4.0. Seven days after the injection, there were only a
`few small fibers with histologic characteristics of regen(cid:173)
`erating fibers and numerous necrotic fibers with very
`little phagocytic activity remained. Fifteen days after
`AA injection, regenerating fibers were still of small
`caliber and they were embedded in the fibrotic tissue
`scar (Fig. 7) .
`
`Histochemical characteristics of regenerating fibers
`
`The histochemical characteristics of regenerating fibers
`in soleus and TA muscles treated with BPVC are
`
`Fig. 7. Soleus muscle 30 days after AA at pH 3.0 injection . The
`regenerating fibers are small with variation in fiber size. Note
`increased fibrous tissue proliferation. H&E, x 240
`
`Soleus (red)
`
`Tiblalis anterior (wMe)
`
`Basophllia
`
`Central
`nuclei
`
`ATPase
`
`Acridine
`orange
`
`Nonspecific
`esterase
`
`7
`
`15
`
`7
`30
`Days after injection
`
`15
`
`30
`
`Fig. 8. Except for slightly delayed muscle fiber type differentiation
`on ATPase staining, there are no histochemical differences in
`regenerating fibers between white and red muscle
`
`summarized in Fig, 8. At 3 days, small regenerating
`fibers already showed strong orange fluorescence with
`AO staining, and high enzyme activities for NSE, acid
`phosphatase and NADH-TR. AO-positive fibers were
`no longer found at 15 days. The activities of acid
`phosphatase and NSE returned to normal by 15 days.
`Regenerating fibers initially had the characteristics of
`undifferentiated type 2C fibers by ATPase staining, and
`they began to differentiate at 10 days in TA and at
`15 days in soleus. Fiber type differentiation was com(cid:173)
`pleted by 20 days in TA and by 30 days in soleus
`(Fig. 8).
`
`Discussion
`
`There have been a number of reports indicating that
`regenerative activity of muscle fibers following necrosis
`is best studied in BPVC-induced muscle injury, because
`BPVC preferentially damages the muscle membrane but
`spares the basement membrane, blood vessels and
`peripheral nerves [3, 20, 23]. Red muscle fibers are
`more susceptible to BPVC damage than white [3]. The
`regenerating fibers are almost uniform in size, and the
`total regenerative process is readily reproducible (20].
`To determine the histologic profiles of necrotic and
`regenerating fibers after myonecrosis in both red and
`white muscles, we injected BPVC into the rat soleus
`(red) and TA (white) muscles. The behavior of muscle
`fiber regeneration in the present study is quite similar to
`that seen in chicken muscle treated with BPVC [21].
`To identify the factors which reduce regenerative
`capacity we injected various alkaline and acid solutions
`into the rat muscles. Except for strong acidic solution&
`including AA below pH 4.0 and AA buffer solution at
`pH 3.0, all solutions induced myonecrosis with phago(cid:173)
`cytosis followed by rapid regeneration, as seen in
`BPVC-treated muscles. Our study confirmed that 0.1 M
`AA below pH 4.0 induced myonecrosis, but interfered
`with factors which stimulate phagocytic activity. Less
`active phagocytosis appeared to inhibit muscle fiber
`regeneration, whereas massive phagocytosis in BPVC(cid:173)
`injured muscle induced rapid fiber regeneration, sug-
`
`FRESENIUS EXHIBIT 1056
`Page 5 of 6
`
`

`

`gesting that persistence of necrotic material inhibits the
`following regeneration [12].
`The basal lamina and satellite cells in muscles treated
`with BPVC and AA at pH 5.0 were well preserved, with
`preferential damage to the muscle membrane and
`dissolution of the myofibrils at the Z-line, probably
`induced by a certain proteolytic process. On the other
`hand , in muscles treated with strong acidic solution
`below pH 4.0, all intracellular organelles, satellite cells,
`and blood vessels were damaged, and myofibrils were
`disrupted at various levels of the sarcomere. The process
`of muscle fiber damage induced by the strong acidic
`solution is not proteolytic in nature but causes coagula(cid:173)
`tion of the protein. Following coagulative necrosis of
`muscle fibers, phagocytosis is limited, regenerative
`activity is reduced, and dense interstitial fibrosis fol(cid:173)
`lows. The necrotizing process in BPVC- and pH 5.0
`AA-injured muscles was similar to that seen in various
`muscle diseases including progressive muscular dystro(cid:173)
`phy [4, 6, 9, 19, 22).
`Muscle fiber regeneration in muscle treated with
`alcohol/oil emulsion was slower than that in BPVC(cid:173)
`treated muscles [10], fib rous tissue was increased in
`amount, and most muscle fibers in the regenerating
`region were still small 6 weeks after injury. Fibrous tissue
`proliferation also occurs after crush injury (18], after
`repeated injections of chlorpromazine [7] and even local
`anesthetics [2, 5]. Thus, delayed regenerative velocity
`may induce interstitial fibrous tissue proliferation or
`vice versa.
`In progressive muscular dystrophy, the disease pro(cid:173)
`gresses without interruption despite an active regenera(cid:173)
`tive process because muscle fiber degeneration is not
`fully compensated by muscle fiber regeneration. Certain
`environmental factors, including interstitial fibrosis
`which impairs the blood supply to individual regenerat(cid:173)
`ing fibers, are probably responsible for reducing the
`regenerative capacity [17]. As suggested from the
`present study, the slow regenerative velocity might
`induce further interstitial fibrosis. This vicious cycle
`seems to be responsible for progression in progressive
`muscular dystrophy.
`
`References
`
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`Nerve 4:234-245
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`repeated injections of mepivacaine (Carbocaine) in the rat.
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`3. Benoit PW, Belt WD (1970) Destruction and regeneration of
`skeletal muscle after treatment with a local anesthetic, bupi(cid:173)
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`
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`