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`
`Acta Neuropathol (1992) 83: 584 -589
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`� © Springer-Verlag 1992
`
`Comparison of behavior in muscle fiber regeneration
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`
`
`
`after bupivacaine hydrochloride-and acid
`
`anhydride-induced myonecrosis
`
`C.Akiyama1, 2, S. Kobayashi1• 3, and I. Nonaka1
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`
`
`
`1 National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187, Japan
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`2 Department of Pediatrics, Fukuoka University, School of Medicine,
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`
`
`3 Department of Pediatrics, Jichi Medical School,
`
`
`December 2 , 1991
`
`Received July 22, 1991/Revised, accepted
`
`
`
`Key words: Muscle necrosis -Regeneration -Fibrosis -
`
`Summary. We compared the morphologic characteris­
`
`
`
`lite cells, which play an important role in muscle fiber
`
`
`
`
`tics of muscle fiber necrosis and subsequent regenera­
`
`regeneration, is not yet known.
`
`
`
`
`tion after injury induced by intramuscular injections of
`One might ask what environmental factors are
`
`
`
`
`
`bupivacaine hydrochloride (BPVC) and a variety of
`
`
`necessary to induce phagocytosis, and what the fate of
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`
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`solutions at acid and alkaline pH (acetic anhydride,
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`
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`necrotic fibers may be if phagocytosis is less active. To
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`citric acid buffer, and sodium carbonate buffer). After
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`
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`answer these questions we injected various chemicals at
`
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`BPVC injection the necrotic muscle fibers were rapidly
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`
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`different pH into rat muscles and examined the behavior
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`invaded by phagocytic cells, followed by active regener­
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`
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`of necrotic and regenerative processes by means of
`
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`ation and very little fibrous scar formation. The regen­
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`muscle histochemistry with morphometric analysis and
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`erating muscle fibers increased rapidly in size and
`
`electron microscopy.We found that acid anhydride (AA)
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`
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`attained complete fiber type differentiation and
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`solution at a pH less than 4.0 caused extensive myone­
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`
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`regained their initial fiber diameter within 1 month.
`
`crosis, with no apparent phagocytic activity and poor
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`Both alkaline and acid solutions induced muscle fiber
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`
`regeneration. On the other hand, bupivacaine hydro­
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`
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`necrosis followed by regeneration. Fiber necrosis
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`chloride (BPVC; Marcaine), a local anesthetic, caused
`
`necrosis with marked phagocytic activity followed by
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`
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`induced by alkaline buffers and acetic anhydride solu­
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`
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`tions above pH 5.0 produced changes quite similar to
`
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`rapid muscle fiber regeneration [3, 5, 13-16, 20, 23]. We,
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`therefore, carried out a study comparing muscle degen­
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`
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`that induced by BPVC. However, injection with 0.1 M
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`
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`acetic anhydride at pH below 4.0 resulted in coagulative
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`
`erative and regenerative processes after injury with
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`necrosis of the injured muscle with very little phagocytic
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`BPVC and AA to identify factors which promote or
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`
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`infiltration with poor regenerative activity and dense
`
`inhibit muscle fiber regeneration.
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`
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`fibrous tissue scarring. Thus, pH 4.0 appears to be the
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`
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`critical pH determining the type of muscle injury and
`
`Materials and methods
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`
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`subsequent poor phagocytic and regenerative activities.
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`
`
`This model of acidic acetic anhydride injury may lead to
`Male Wistar rats weighing 200-250 g were used. The right soleus
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`the identification of factors which interfere with regen­
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`(red) and tibialis anterior (TA; white), muscles were surgically
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`eration and cause fibrous tissue scarring in human
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`exposed under general anesthesia and various chemical solutions,
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`muscular dystrophy.
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`
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`listed in Table 1, were injected directly into the muscles with a thin
`
`
`
`
`gauge needle. T he chemical solutions included 0.5 ml 0.5 % BPVC,
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`
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`0.1 M sodium carbonate at pH varying from 9.0 to 11.0, 0.1 M
`
`Bupivacaine -Acid anhydride
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`citric acid at pH 5.0-3.0, 0.1 M AA at pH 3.0, 0 .1 M AA solution
`
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`whose pH was adjusted between 3.0 and 6.0 with 0 .1 M NaOH, and
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`0.5 %-5 % AA in 0.85 % saline. The contralateral soleus and TA
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`muscles of each rat received saline in the same manner and served
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`as controls. In each trial, 2-3 rats were examined.
`Skeletal muscle fibers undergo necrosis after mechanical
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`To study the necrotizing process, the soleus muscles were taken
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`and chemical injuries including crush, ischemia, cold or
`from 2 each of 64 rats at 15 and 30 min, and 1, 2, 6, 12, 24, and 48 h
`
`
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`heat injuries, and injections of local anesthetics and
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`
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`aftertheinjectionsofBPVC, and0.1 MAAatpH3.0, 4.0 and5.0,
`
`
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`respectively. Each muscle was examined by histochemistry and
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`snake toxins [l, 8]. The necrotic fibers are then invaded
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`
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`electron microscopy. For electron microscopy, the soleus muscle
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`and cleared by phagocytic cells followed by an active
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`specimen was fixed in 0.1 M sodium cacodylate-buffered glutaral­
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`regenerative process. The mechanism to activate satel-
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`dehyde solution at pH 7.4 for 3-4 h. The tissue was washed in the
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`in same buffer solution, post-fixed in 1 %OsO4 and embedded
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`epoxy resin after dehydration in serial alcohol solutions.
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`see 1 above)
`(address Offprint requests to: C. Akiyama
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`Table 1. Myotoxic agents
`
`Chemicals
`
`pH
`
`Regeneration
`Myonecrosis
`Phagocytosis
`(10 days)
`(48 h)
`(48 h)
`
`585
`
`0.5 % bupivacaine hydrochloride
`
`6.5
`9.0, 11.0
`
`0.1 M sodium carbonate buffer
`5.0, 3.0
`
`0.1 M citric acid buffer
`3.0
`
`
`0.1 M acetic anhydride buffer
`
`
`0.1 M acetic anhydride + NaOH
`
`3.0
`4.0
`4.1
`4.3
`5.0
`
`0.5 % , 1 % , 3 % , 5 % , acetic anhydride
`3.0
`(<pH3.0)
`
`+++
`+
`+++
`+++
`
`+++
`+++
`++
`+
`+
`+++
`
`+++
`+++
`++
`
`++
`++
`+++
`
`F
`F
`M
`p
`
`p
`p
`M
`M
`F
`p
`
`
`
`
`
`
`
`
`
`-, none; +, localized; + +, moderate; + + +, marked; F, fair; M, moderate; P, poor
`
`
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`determine fiber types, 100-200 fibers in each muscle were analyzed
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`To study muscle fiber regeneration, the soleus and TA muscles
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`
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`in photographs from serial frozen sections printed at a final
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`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
`
`magnification of X 660.
`
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`AA at pH 3.0 (total = 70 rats). In 2-3 rats from each of the
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`
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`remaining groups listed in the Table 1, the treated muscles were
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`examined at 2 and 10 days after the injection.
`Results
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`
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`The soleus and TA muscles were immediately frozen in
`
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`
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`isopentane cooled in liquid nitrogen. Serial frozen sections were
`Muscle fiber necrosis
`
`
`stained with hematoxylin and eosin (H&E), modified Gomori
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`
`
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`trichrome, and a battery of histochemical methods including
`As shown in Table 1, various chemicals ( especially
`
`
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`
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`NADH-TR, routine ATPase and ATPase with preincubation at pH
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`BPVC and acidic solutions below pH 4.0) caused muscle
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`4.5 and 4.3, acid phosphatase, nonspecific esterase (NSE), and
`
`
`fiber necrosis. Immediately after BPVC injection,
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`
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`acridine orange (AO). To measure muscle fiber diameters and to
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`preserved, myofibrillar networks have disappeared and myonuclei
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`hydrochloride Fig. 1. Soleus muscles 48 h after bupivacaine
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`
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`were granulated (B). Note no macrophage invasion in extra-and
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`(BPVC) (A) and acid anhydride (AA) at pH 3.0 (B) injections.
`
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`intracellular spaces H&E, x 240
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`Numerous necrotic fibers are invaded by phagocytic cells in the
`(B). A, B
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`BPVC-treated muscle (A). Although the contour of fibers is
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`almost all the muscle fibers in the treated soleus and TA
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`Almost all the muscle fibers treated with citric acid
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`underwent massive necrosis characterized by opaque
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`and sodium carbonate buffers, AA and AA buffers
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`fibers and empty sarcolemmal tubes, with foci of hyper­
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`showed extensive necrosis. The appearance of the
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`necrotic fibers treated with citric and sodium carbonate
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`contraction bands on longitudinal sections. The necrotic
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`buffers was similar to those seen in BPVC-treated soleus
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`fibers were invaded by acid phosphatase-positive macro­
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`phages within 12 h after the injection. At 24 and 48 h,
`muscles.
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`the necrotic fibers and the extracellular space were filled
`On the other hand, in the muscle injected with AA
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`with numerous mononuclear phagocytic cells as well as
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`below pH 4.0 and AA buffer at pH 3.0 the necrotic
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`some polymorphonuclear leukocytes (Fig. lA).
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`muscle fibers had faintly staining cytoplasm with loss of
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`the intermyofibrillar networks and myonuclei. Muscle
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`fiber necrosis after 0.5 % , 1 % , 3 % , and 5 % AA
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`injections had a similar appearance. In the muscles
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`treated with such strong acidic solutions, the contour of
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`necrotic fibers remained unchanged with little phagocyt­
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`ic activity even 24 and 48 h after injection (Fig. lB). The
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`blood vesseles and peripheral nerve bundles in the
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`interstitium were not clearly identifiable.
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`Electron microscopy of necrotic fibers
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`Fifteen minutes after BPVC and 0.1 MAA at pH 4.0 and
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`5.0 injections, electron microscopy revealed disruption
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`of the myofibrils with the formation of hypercontraction
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`bands, and the sarcoplasmic reticulum appeared to be
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`dilated (Fig. 2). There was focal loss of plasmalemma.
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`At 2 h, myofibrils were fragmented at the level of the
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`Z-line (Fig. 3A). The basal lamina and.satellite cells
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`were well preserved at all stages. At 48 h, many
`fiber 15 min after AA Fig. 2. An electron micrograph of a necrotic
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`phagocytic cells were found in the extra-and intracellu­
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`at pH 5.0 injection. The myofibrils are disrupted and organelles
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`lar spaces, and satellite cells were activated.
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`
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`swollen. The sarcolemma has focally disappeared. x 24 000
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`Z-line dissolution from a selective Myofibrils are fragmented Fig. 3. Soleus muscle 2 h after AA at pH 5.0 (A) and at 3.0 (B) injections.
`
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`
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`including the Z-line, of the sarcomeres at various levels (A). The myofibrils are fragmented A and I bands (B). A, B x 6000
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`In the muscle treated with O .1 M AA at pH 3. 0, there
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`
`Soleus (red)
`
`was disruption of both the basal lamina and plasma
`60
`
`
`There was degen­membrane 15 min after the injection.
`�
`eration of both muscle
`
`
`fibers and satellite cell nuclei with
`
`
`
`loss of chromatin granules (Fig. 4). At 2 h, the myofi­
`'6
`�
`
`
`
`
`brils were fragmented at different levels of the myofibrils
`iI: 30
`
`587
`
`Tibialis
`
`anterior (white)
`
`A
`
`7 10 15
`
`30
`
`7 10 15
`
`30
`
`60
`
`60
`
`---
`-----
`-
`--
`---------
`---------------------
`-----------
`30
`30
`iI:
`
`---
`
`B
`
`7 10 15
`
`7 10 15
`30
`Days after injection
`
`30
`
`Fig. 5. Although the regenerating fibers (-0-) treated with BPVC
`
`
`
`
`
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`(A)rapidly recovered and regained their initial size in both soleus
`
`
`
`
`
`(red) and tibialis anterior (white) muscles, when treated with AA
`
`
`solution at pH 3.0 (B) they showed markedly delayed recovery.
`Fig. 4. Soleus muscle 15 min after AA at pH 3.0 injection. The
`
`
`
`
`
`One standard deviation of the mean control (untreated) fiber
`basal lamina and plasma membrane have disappeared. Satellite
`
`
`cells are also degenerated. X 12 000
`
`
`diameter (-•-) is indicated by dotted lines
`
`�
`
`•
`
`._
`'
`
`,,
`
`•
`� .
`....
`
`,
`
`I!.
`
`;
`
`'
`
`.
`
`B
`
`.. '
`•.
`
`I- '
`
`,,
`
`.
`
`�-
`'
`'
`
`,.
`
`.r
`
`
`
`
`
`
`
`
`
`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
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`tibialis anterior (B) muscles. H&E, x 240
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`588
`including the Z-line, A and I bands (Fig. 3B). Even 48 h
`
`
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`
`
`
`after the injection, no phagocytic cells had appeared and
`
`
`
`the fragmented myofibrils remained unchanged from
`Basophilia
`those at 2 h.
`Central
`Muscle fiber degeneration in the soleus muscles
`
`nuclei
`
`
`
`treated with these three different chemicals is summa­
`ATPase
`rized in Table 1.
`
`Soleus (red)
`
`
`
`Muscle fiber regeneration
`
`Acridine
`orange
`
`Nonspecific
`esterase
`
`
`
`
`
`Tibialis anterior (wh�e)
`
`7 15
`
`30
`
`
`Days after injection
`
`7 15
`
`30
`
`In BPVC-treated TA and soleus muscles, small caliber
`
`
`
`
`
`
`regenerating fibers with basophilic cytoplasm and cen­
`Fig. 8. Except for slightly delayed muscle fiber type differentiation
`
`
`
`
`
`
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`trally placed vesicular nuclei appeared in a pool of
`
`
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`on ATPase staining, there are no histochemical differences in
`
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`mononuclear cells 72 h after the injection on H&E.
`
`
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`regenerating fibers between white and red muscle
`
`
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`After 7 days, numerous regenerating fibers charac­
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`
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`
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`terized by basophilic cytoplasm, vesicular nuclei and
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`
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`prominent nucleoli were clearly identifiable. T he regen­
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`summarized in Fig. 8. At 3 days, small regenerating
`
`
`
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`erating fibers regained their initial diameter within
`
`
`
`
`fibers already showed strong orange fluorescence with
`
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`AO staining, and high enzyme activities for NSE, acid
`
`30 days, but the recovery of the white muscle was
`
`
`slightly slower than that of the red muscle (Fig. 5). At
`
`
`phosphatase and NADH-TR. AO-positive fibers were
`
`no longer found at 15 days. The activities of acid
`
`
`30 days, almost all of the regenerating fibers still
`
`
`
`
`retained their centrally placed nuclei (Fig. 6).
`
`
`phosphatase and NSE returned to normal by 15 days.
`
`
`
`As expected from the behavior of myonecrosis, there
`
`
`
`
`Regenerating fibers initially had the characteristics of
`
`was a marked delay in muscle fiber regeneration in
`
`
`undifferentiated type 2C fibers by ATPase staining, and
`
`
`
`
`
`necrotic muscles treated with strong AA solution below
`
`they began to differentiate at 10 days in TA and at
`
`pH 4.0. Seven days after the injection, there were only a
`
`
`15 days in soleus. Fiber type differentiation was com­
`
`few small fibers with histologic characteristics of regen­
`
`pleted by 20 days in TA and by 30 days in soleus
`
`
`
`
`
`erating fibers and numerous necrotic fibers with very
`(Fig. 8).
`
`
`
`
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`little phagocytic activity remained. Fifteen days after
`
`
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`AA injection, regenerating fibers were still of small
`
`
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`caliber and they were embedded in the fibrotic tissue
`scar (Fig. 7) .
`Discussion
`
`There have been a number of reports indicating that
`
`
`
`
`
`regenerative activity of muscle fibers following necrosis
`
`
`Histochemical characteristics of regenerating fibers
`
`
`
`
`is best studied in BPVC-induced muscle injury, because
`
`
`
`The histochemical characteristics of regenerating fibers
`
`
`
`
`BPVC preferentially damages the muscle membrane but
`
`
`in soleus and TA muscles treated with BPVC are
`
`
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`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
`
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`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 solutions
`
`
`including AA below pH 4.0 and AA buffer solution at
`
`
`pH 3.0, all solutions induced myonecrosis with phago­
`
`
`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
`
`
`Fig. 7. Soleus muscle 30 days after AA at pH 3.0 injection. The
`
`
`
`
`regeneration, whereas massive phagocytosis in BPVC­
`
`
`
`regenerating fibers are small with variation in fiber size. Note
`
`
`
`injured muscle induced rapid fiber regeneration, sug-
`
`
`
`increased fibrous tissue proliferation. H&E, x 240
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`589
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`gesting that persistence of necrotic material inhibits the
`
`""1-. Bonilb E. Schotland DL, Waka\·arna Y ( 1978) Duchenne
`
`
`
`
`dystrophy: focal alterations in the distribution of concanavalin
`
`
`following regeneration [12].
`
`
`A binding sites at the muscle cell surface. Ann Neural
`
`
`The basal lamina and satellite cells in muscles treated
`4:117-123
`with BPVC and AA at pH 5.0 were well preserved
`, with
`5.Bradley WG (1979) Muscle fiber splitting. In: Mauro A (ed)
`
`
`
`
`preferential damage to the muscle membrane and
`
`
`Muscle regeneration. Raven Press. New York, pp215-232
`
`
`
`dissolution of the myofibrils at the Z-line, probably
`
`
`
`
`6.Bradley WG, Fulthorpe JJ (1978) Studies of sarcolemmal
`
`
`
`
`induced by a certain proteolytic process. On the other
`
`
`
`
`integrit) in myopathic muscle. l\curolugy 28:670-677
`
`
`
`7 Brumback RA, Empting L. Susag \TE. Staton RD (1982)
`
`hand, in muscles treated with strong acidic solution
`
`
`
`
`l\1usde fibrnsis associated with intrammcular chlorpromazine
`
`
`
`below pH 4.0, all intracellular organelles. satellite cells,
`
`
`
`admmistration. A preliminary report. J Phann Phannacol
`
`
`and blood vessels were damaged. and myofibrils were
`3-.\:526-528
`
`
`
`
`disrupted at various levels of the '.'-arcomere. The process
`8.Carlson B.\1 (1973) The regeneration .if skeletal muscle - a
`
`
`of muscle fiber damage induced by the strong acidic
`
`revirn. Am J Anat 137:119-!S0
`
`solution is not proteolytic in nature but causes coagula­
`9.Carpenter S, Karpati G (1979) Duchenne muscular dystrophy.
`
`
`
`
`
`
`
`
`
`
`Plasma membrane loss initiates muscle cell necrosis unless it is
`
`
`tion of the protein. Following coagulative necrosis of
`
`repaired. Brain 102:147-161
`
`muscle fibers, phagocytosis is limited, regenerative
`
`
`10.Dhoot GK, Perry SV (1982) Changes in the forms of the
`
`
`
`is reduced, and dense interstitial fibrosis fol­
`activity
`
`
`components of the troponin complex during regeneration of
`
`
`lows. The necrotizing process in BPVC-and pH 5.0
`
`
`injured skeletal muscle. Muscle Nerve 5:39-47
`
`
`AA-injured mu,-cles was similar to that seen in various
`
`
`11.Grim .\L Rcrabkova L, Carlson B1\1 ( 1988) A test for muscle
`
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` PFIZER, INC. v. NOVO NORDISK A/S - IPR2020-01252, Ex. 1056, p. 6 of 6
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