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THE ANATOMICAL RECORD 254:521–533 (1999)
Long-term Regeneration of Fast
and Slow Murine Skeletal Muscles
after Induced Injury by ACL Myotoxin
Isolated from Agkistrodon contortrix
laticinctus (Broad-banded
Copperhead) Venom
TANIA DE FÁTIMA SALVINI,1* CLÁUDIO CÉSAR MORINI,1
HELOISA SOBREIRO SELISTRE DE ARAÚJO,2
AND CHARLOTTE LEDBETTER OWNBY3
1Laboratório de Neurociências, Departamento de Fisioterapia,
Universidade Federal de São Carlos, 13565-905 São Carlos, São Paulo, Brazil
2Departamento de Ciências Fisiológicas, Universidade Federal
de São Carlos, 13565-905 São Carlos, São Paulo, Brazil
3Department of Anatomy, Pathology, and Pharmacology, Oklahoma State University,
Stillwater, Oklahoma 74078-2007
ABSTRACT
The aim of the present work was to analyze the regenerated muscle
types I and II fibers of the soleus and gastrocnemius muscles of mice, 8
months after damage induced by ACL myotoxin (ACLMT). Animals received
5 mg/kg of ACLMT into the subcutaneous lateral region of the right hind
limb, near the Achilles tendon; contralateral muscles received saline.
Longitudinal and cross sections (10 µm) of frozen muscle tissue were
evaluated. Eight months after ACLMT injection, both muscle types I and II
fibers of soleus and gastrocnemius muscles still showed centralized nuclei
and small regenerated fibers. Compared with the left muscle, the incidence
of type I fibers increased in the right muscle (21% ⫾ 03% versus 12% ⫾ 06%,
P ⫽ 0.009), whereas type II fibers decreased (78% ⫾ 02% versus 88% ⫾ 06%,
P ⫽ 0.01). The incidence of type IIC fibers was normal. These results confirm
that ACLMT induced muscle type fiber transformation from type II to type I,
through type IIC. The area analysis of types I and II fibers of the
gastrocnemius revealed that injured right muscles have a higher percentage
of small fibers in both types I and II fibers (0–1,500 µm2) than left muscles,
which have larger normal type I and II fibers (1,500–3,500 µm2). These
results indicate that ACLMT can be used as an excellent model to study the
rearrangement of motor units and the transformation of muscle fiber types
during regeneration. Anat Rec 254:521–533, 1999. r 1999 Wiley-Liss, Inc.
Key words: muscle injury; muscle regeneration; gastrocnemius muscle;
soleus muscle; ACL myotoxin
Skeletal muscle fibers are injured after exposure to
several kinds of snake venom (Ownby, 1990). Damage in
skeletal muscles frequently leads to degeneration and
subsequent regeneration of muscle fibers (Schmalbruch,
1976; Mauro, 1979), usually by repair of surviving fiber
r 1999 WILEY-LISS, INC.
*Correspondence to: Tania F. Salvini, Departamento de Fisioterapia, Universidade Federal de São Carlos, CEP: 13565-905, São
Carlos, SP, Brazil. Fax: 0055-16/261-2081.
E-mail: tania@power.ufscar.br
Received 14 July 1998; Accepted 4 December 1998
522
SALVINI ET AL.
Fig. 1. Cross section of the right gastrocnemius muscle 8 months after ACLMT injection (toluidine blue
staining). Note the difference in the diameter of the muscle fibers and the abundant presence of muscle fibers
with a centralized nucleus (arrowheads) and small regenerated split fibers (asterisks). Scale bar ⫽ 40 µm.
fragments and by the development of new fibers. Mononucleate satellite cells, which are located between the
plasma membrane and basal lamina, are the source of new
muscle fibers (Snow, 1977, 1978). New myonuclei can come
only from mononuclear myoblasts, which are descendants
of satellite cells in mature muscles (Mauro et al., 1970;
Holtzer et al., 1975).
Injured skeletal muscle regenerates rapidly, forming
myotubules by the end of 3 days, functionally reinnervated
muscle fibers by days 4–5, and fully repaired fibers after
21–28 days (Schmalbruch, 1976; Grubb et al., 1991; Wernig
et al., 1991a,b; Morini et al., 1998). Although the process of
muscle fiber regeneration has been thoroughly studied,
questions remain to be answered, especially concerning
the regeneration process of different types of muscle fibers
months after induced injury.
Previous reports from our laboratory showed that ACL
myotoxin (ACLMT) purified from the venom of the broadbanded copperhead (Agkistrodon contortrix laticinctus)
is an excellent model to induce a homogeneous site of
muscle injury in both soleus and gastrocnemius muscles
of mice (Morini et al., 1998). ACLMT was first isolated
by Johnson and Ownby (1993), and it was determined to be
a Lys49 type II phospholipase A2 (PLA2) (Selistre de
Araujo et al., 1996). Members of this class of PLA2 have no
or very low phospholipase activity despite high myotoxic
activity.
The myotoxic effect of ACLMT was evaluated 3 hr and 3
and 21 days after subcutaneous injection of the toxin in the
soleus and gastrocnemius muscles of mice (Morini et al.,
1998). ACLMT injured both muscle type I and type II fibers
in the soleus and gastrocnemius muscles. Twenty-one days
after ACLMT injection, both muscles were completely
regenerated, and there were many muscle fibers with
centralized nuclei, split fibers, and clusters of newly
regenerated muscle fibers. Muscle fiber type transformation was also observed, with a significant increase in the
incidence of type IIC and a decreased incidence of type II
fibers. Although ACLMT is known for its myotoxic activity,
these results indicate that it can also be used as a model to
induce rearrangement of the motor units and a change in
muscle fiber types.
These data stimulated new questions about the morphologic characteristics of regenerated types I and II fibers
several months after damage induced by ACLMT. Could
there be significant differences between types I and II
Fig. 2. Longitudinal sections of the right gastrocnemius muscle 8
months after ACLMT injection (toluidine blue staining). (A) Muscle fibers
with a small diameter (asterisk) and a centralized nucleus (arrowhead).
(B) Presence of branched fibers (arrow) and a centralized nuclei (arrowheads). (C) A row of centralized nucleus along the muscle fiber (arrowheads). Scale bar ⫽ 20 µm.
REGENERATION OF FAST AND SLOW MURINE MUSCLES
Figure 2.
523
524
SALVINI ET AL.
Fig. 3. Serial cross sections in the deep (A–C) and the superficial
regions (D–F) of the right gastrocnemius muscle. Note that both regions of
the muscle show split fibers (asterisks) and a centralized nucleus
(arrowheads). In the deep region of the muscle, there are split fibers and
centralized nuclei in type I and type II fibers. Some of the type I fibers are
indicated by an asterisk. (B: ac-mATPase, pH 4.3; C: alc-mATPase, pH
10.3). In the superficial region of the muscle, where there are exclusively
type II fibers (E: ac-mATPase, pH 4.3), it is possible to note a split fiber
(asterisk, D–F) with an AChE reaction (arrowhead, F). Scale bar ⫽ 20 µm.
REGENERATION OF FAST AND SLOW MURINE MUSCLES
Fig. 4. Serial cross sections in the deep region of the right gastrocnemius muscle 8 months after ACLMT injection (A,C–F). (A) Toluidine blue
staining shows skeletal muscle fibers with a centralized nucleus (arrowheads). (C) Note a small type I split fiber (A, star) with AChE reaction (C,
star). Type I muscle fiber shows an intense reaction after ac-mATPase at
pH 4.3 (E) but no reaction after alc-mATPase at pH 10.3 (F). (D) The split
525
type I fiber (star) has lower SDH activity. When A, E and F are compared, it
is possible to identify the presence of a centralized nucleus in both type I
and II fibers. B: Left control gastrocnemius after toluidine blue staining
shows a normal morphologic aspect, with the nucleus located in the
periphery of the muscle fibers (arrowhead). Scale bar ⫽ 20 µm.
526
SALVINI ET AL.
soleus and gastrocnemius muscles of mice, 8 months after
induced damage by ACLMT.
MATERIALS AND METHODS
ACL Myotoxin
ACLMT was purified from the crude venom of the
broad-banded copperhead (Agkistrodon contortrix laticinctus), as previously described (Johnson and Ownby, 1993).
Briefly, this process consists of fractionation of crude
venom by anion exchange chromatography followed by
final purification using cation exchange chromatography.
Animal Care and Experimental Groups
Eight male mice (white Swiss), weighing 30–35 g, were
used. The animals were housed in groups in standard
plastic cages in an animal room under controlled environmental conditions (12 hr dark/12 hr light cycle; temperature 22.5°C). Mice received standard food and had access
to food and water ad libitum. All animals were given one
dose of ACLMT (5 mg/kg) into the subcutaneous lateral
region of the right hind limb, near the Achilles tendon. The
injection was made in the distal to proximal direction in
the middle line between the insertion of the Achilles
tendon and the distal surface of the lateral malleolus of
fibula. A similar region of the contralateral left muscle was
injected with saline and used as a control.
Histology and Histochemistry
Fig. 5. Schematic representation of serial cross sections along 210
µm obtained in the deep region of the right gastrocnemius muscle 8
months after ACLMT injection. There are fibers with a centralized nucleus
(fibers 1 and 2) along the 210 µm. Split fibers are also present (stars).
Scale bar ⫽ 40 µm.
fibers in both soleus (slow twitch) and gastrocnemius (fast
twitch) muscles of mice several months after such an
induced injury?
The purpose of the experiments reported here was to
contribute to the knowledge of the longtime regeneration
characteristics of muscle type fibers after injury induced
by a snake venom toxin. The primary aim was to analyze
the regenerated skeletal muscle types I and II fibers of
Eight months after ACLMT injection, the animals were
weighed under deep ethyl ether anesthesia. Afterward,
right and left gastrocnemius and soleus muscles were
removed, weighed, immediately frozen in melting isopentane, and stored in a freezer at ⫺56°C. Frozen muscles
were cut through the proximal to distal region using a
cryostat (10-µm cross sections). Alternate serial sections
were obtained in the middle region of both muscles to
evaluate all muscle fibers of both soleus and gastrocnemius muscles. Histologic cross sections were stained with
1% toluidine blue / 1% borax or for acid phosphatase
(AcPase; Lojda et al., 1976), myofibrillar ATPase activity
(mATPase) after alkali (alc-mATP, pH 10.3; Guth and
Samaha, 1969 and Butler and Cosmos, 1981) or acid
pre-incubations (ac-mATP, pH 4.3; Brooke and Kaiser,
1970), succinate dehydrogenase (SDH; Nachlas et al.,
1957), and acetylcholinesterase (AChE; Karnovsky and
Roots, 1964). Longitudinal sections (10 µm), which were
stained with toluidine blue, were obtained from two right
soleus and gastrocnemius muscles.
The incidence of damaged fibers was evaluated using
videoprint montages of single serial cross sections of the
middle region of muscles stained with toluidine blue or
submitted to mATPase reactions (pH 4.3 and 10.3). This
region was chosen because it contains the highest number
of muscle fibers. All damaged fibers of the serial cross
sections were identified. Furthermore, serial cross sections
submitted to mATPase (pH 4.3 and 10.3) were used to
Fig. 6. Schematic representation of serial cross sections along 420
µm obtained from the superficial region of the gastrocnemius muscle 8
months after ACLMT injection. There are muscle fibers with centralized
nuclei in all sections evaluated (arrowheads). Note also the presence of
split fibers. Scale bar ⫽ 80 µm.
REGENERATION OF FAST AND SLOW MURINE MUSCLES
Figure 6.
527
⫺
⫺
⫺
⫺
01 (0.2%)
0.0
02 (0.5%)
01 (0.2%)
0.2% ⫾ 0.2
428
491
404
465
447 ⫾ 39
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
288 (51%)
353 (52%)
270 (50%)
231 (56%)
52% ⫾ 3**
561
669
552
411
548 ⫾ 106
1
2
3
4
X ⫾ SD
⫺
⫺
⫺
⫺
6,868
6,136
5,750
4,286
5,760 ⫾ 1,086
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
1
2
3
4
X ⫾ SD
1,548 (21%)
1,184 (17%)
1,329 (20%)
1,216 (18%)
19% ⫾ 1.6*
7,548
7,055
6,590
6,748
6,985 ⫾ 422
Animal
*P ⫽ 0.00009 (paired Student’s t test).
**P ⫽ 0.00001 (paired Student’s t test).
aSplit fiber: ⫹⫹⫹, frequent; ⫺, rarely observed.
Animal
Split
fibera
Centralized
nucleus
19 (0.3%)
07 (0.1%)
07 (0.1%)
12 (0.3%)
0.2% ⫾ 0.09
Centralized
nucleus
Left muscle
Number
of fibers
Split
fibera
Centralized
nucleus
Right muscle (ACLMT)
Number
of fibers
Soleus
Left muscle
Number
of fibers
Split
fibera
Eight months after the injection of ACLMT, the right
gastrocnemius muscles contained small regenerated muscle
fibers, large numbers of centralized nuclei, and split fibers
(Fig. 1). Longitudinal sections showed that centralized
nuclei were arranged as isolated nuclei or together in a
row (Fig. 2). Longitudinal sections also showed that some
fibers branched (Fig. 2B) and, in some of them, the
branches reunited at varying distances. Muscle fibers with
centralized nuclei and split fibers were identified in both
deep (Fig. 3A–C) and superficial (Fig. 3D–F) regions of the
muscle. The deep region of the gastrocnemius muscle of
mice is composed of types I and II fibers (Fig. 3A–C),
whereas the superficial region contains exclusively type II
fibers (Fig. 3D–F). Several small regenerated and differentiated type II (Fig. 3D–F) and type I (Fig. 4A,C–F) fibers
showed positive AChE reactions. Both types I and II fibers
had signs of previous injury, such as a centralized nucleus
and split fibers. Serial cross sections taken in the proximal, middle, and distal regions of the muscle had these
signs in all regions. The left gastrocnemius had normal
morphologic features in all regions analyzed (Fig. 4B).
Using serial cross sections, bundles of muscle fibers of both
the deep and superficial regions of the right gastrocnemius
were photographed and reconstructed along 210 and 420
µm, respectively (Figs. 5 and 6). Split fibers were present
in both deep and superficial regions of the muscle. This
schematic representation revealed that all muscle fibers
showed at least a centralized nucleus, and some fibers
contained a centralized nucleus in all sections evaluated.
The number of muscle fibers with one or more centralized nuclei was assessed applying previously established
criteria (Wernig et al., 1991a) to one series of cross sections
through the medial region of muscle. The incidence of
fibers with a centralized nucleus was significantly increased in the right muscle (19% ⫾ 1.6% versus 0.2% ⫾
Centralized
nucleus
Gastrocnemius Muscle
Right muscle (ACLMT)
The average weight of the animals was 76 ⫾ 4 g. There
was no difference between the average weights of the right
and left gastrocnemius muscles (0.26 g ⫾ 0.02 g versus
0.26 g ⫾ 0.02 g, respectively) or the right and left soleus
muscles (0.016 g ⫾ 0.002 g versus 0.016 g ⫾ 0.002 g,
respectively) 8 months after ACLMT injection.
Number
of fibers
RESULTS
Body, Soleus, and Gastrocnemius Weights
Gastrocnemius
identify the incidence of muscle fiber types (I, II, IIC) in the
deep region of the gastrocnemius. Each split muscle fiber
was counted as one fiber. Muscle fiber types were quantified only in the deep region of the muscle because the
superficial region showed the exclusive presence of type II
fibers. To locate the deep region of the gastrocnemius
muscle, the distance from the deep to the superficial region
of each cross section was measured by light microscopy,
and the middle point of the cross section was identified.
Only muscle fibers of the deep region were analyzed, also
using videoprint montages. The muscle fiber types were
evaluated using computer software (Vinspec) connected to
a microscope. Chronic signs of muscle fiber injury were
identified by the presence of split fibers and centralized
nuclei, without acute signs of damage, such as necrosis,
basophilia, and cellular infiltration (Carpenter and Karpati,
1984, Wernig et al., 1991a, Morini et al., 1998).
Split
fibera
SALVINI ET AL.
TABLE 1. Incidence of centralized nucleus and split fibers in the right and left gastrocnemius
and soleus muscles 8 months after ACLMT injection
528
529
REGENERATION OF FAST AND SLOW MURINE MUSCLES
TABLE 2. Incidence of skeletal muscle type fibers (I, II, IIC) in the right and left
gastrocnemius 8 months after ACLMT injection
Gastrocnemius
Right muscle (ACLMT)
Left muscle
Animal
Type I
Type II
Type IIC
Type I
Type II
Type IIC
1
2
3
4
X ⫾ SD
468 (19%)
686 (24%)
471 (18%)
634 (21%)
21% ⫾ 03*
1,923 (79%)
2,206 (76%)
2,095 (81%)
2,331 (77%)
78% ⫾ 02**
46 (1.8%)
18 (0.6%)
19 (0.7%)
44 (1.5%)
1.2% ⫾ 0.6***
130 (09%)
548 (20%)
152 (10%)
152 (8%)
12% ⫾ 06
1,269 (90%)
2,117 (79%)
1,386 (90%)
1,698 (92%)
88% ⫾ 06
04 (0.2%)
12 (0.4%)
00 (0.0%)
02 (0.1%)
0.2% ⫾ 0.2
*P ⫽ 0.009 (paired Student’s t test).
**P ⫽ 0.01 (paired Student’s t test).
***P ⫽ 0.02 (paired Student’s t test).
0.09%, P ⫽ 0.00009, paired Student’s t test; Table 1). The
degree of injury is probably underestimated in these
studies because of its segmental occurrence and the evaluation at only one level. When more levels along the length
of the muscles are evaluated, the total estimate of damaged fibers increases (see Figs. 5 and 6). The presence of
split fibers was also high in the injured muscle but rare in
the left control gastrocnemius (Table 1). The right gastrocnemius muscle always showed an increased number of
muscle fibers when compared with the left one (Table 1).
It was interesting that 8 months after ACLMT injection,
there is still a significant change in the incidence of types I
and II muscle fibers (Table 2). The incidence of type I fibers
increased in the right muscle (21% ⫾ 03% versus 12% ⫾
06%, P ⫽ 0.009, paired Student’s t test; Table 2), whereas
that of type II decreased (78% ⫾ 02% versus 88% ⫾ 06%,
P ⫽ 0.01, paired Student’s t test; Table 2) compared with
the left one. Although there was also an increased percentage of type IIC fibers in the right muscle (1.2% ⫾ 06%
versus 0.2% ⫾ 02%, P ⫽ 0.02, paired Student’s t test; Table
2), this percentage is still in the normal range for mamma–an skeletal muscles.
The area analysis of types I and II muscle fibers of the
gastrocnemius revealed that injured muscles on the right
have a higher percentage of small regenerated fibers of
both types I and II (0–1,500 µm2, Fig. 7) than the control
left muscles, which have predominantly larger normal
fibers (1,500–3,500 µm2; Fig. 7). Larger fibers observed in
the right muscles (1,750–4,000 µm2) were probably recovered fibers, whereas the smaller fibers were regenerated
fibers.
Soleus Muscle
Morphologic patterns similar to the longitudinal and
cross sections of the right gastrocnemius were observed in
the skeletal muscle fibers of the right soleus 8 months after
ACLMT injection. All regions of the muscle showed large
numbers of fibers with a centralized nucleus and split
fibers (Fig. 8A). Despite the presence of a centralized
nucleus, most of the fibers were differentiated into type I or
type II (Fig. 8C,D). Type IIC fibers were rare. The control
left soleus presented a normal morphologic aspect (Fig.
8B). When the soleus muscle fibers were analyzed to
quantify the presence of centralized nuclei using only a
serial cross section, it was observed that 52% ⫾ 3% of the
right muscles had a centralized nucleus, while the left
muscle had 0.2% ⫾ 0.2% (P ⫽ 0.00001, paired student’s t
test; Table 1).
DISCUSSION
We previously reported (Morini et al., 1998) a significant
decrease in the percentage of type II fibers and an increase
in the percentage of type IIC fibers in the deep region of the
murine gastrocnemius 21 days after damage induced by
ACLMT. There was no change in the superficial region of
the muscle, which contained only regenerated type II
fibers. These results led us to suggest that there was a
change in muscle fiber type in the deep region of gastrocnemius from type II to type I, through type IIC. Twenty-one
days after ACLMT injection, 17% of muscle fibers identified in the deep region of the gastrocnemius were type IIC
(Morini et al., 1998), whereas 8 months later the incidence
of type IIC was 1.2% (see Table 2). This result, associated
with an increased percentage of type I and decreased
percentage of type II, confirms the hypothesis that ACLMT
induced muscle fiber type transformation from type II to
type I, through type IIC. The results reported here also
indicate similar morphologic characteristics in both types I
and II muscle fibers of regenerated soleus and gastrocnemius murine muscles, 8 months after induced injury by
ACLMT.
Despite their metabolic and physiologic differences, both
muscles had significant numbers of muscle fibers with one
or more centralized nuclei and split fibers in all regions
(proximal, middle, and distal) evaluated. It is also interesting that 8 months after induced damaged, there was a
large number of small regenerated types I and II muscle
fibers with centralized nuclei. This suggests that despite
the differentiation that took place in the small regenerated
new fibers, hypertrophy and migration of the centralized
nucleus to the periphery of muscle fibers did not accompany this process.
Mouse soleus muscle contains an approximately equal
number of slow-twitch (type I) and fast-twitch (type IIA)
fibers (Lewis et al., 1982; Desypris and Parry, 1990;
Wernig et al., 1991a), whereas the gastrocnemius muscle
contains predominantly type II muscle fibers (Morini et al.
1998). In addition, the gastrocnemius muscle has two
distinct regions, which can be identified by mATPase—a
deep region composed of types I and II fibers and a
superficial region composed of types IIA and IIB fibers only
(Armstrong and Phelps, 1984; Morini et al., 1998).
No fibrosis was observed in the evaluated muscles,
indicating that, in general, all muscle fibers regenerated
after induced injury by ACLMT. This observation confirms
the efficiency of ACLMT as a model to induce muscle injury
530
SALVINI ET AL.
Fig. 7. Average area (µm2) of types I and II fibers in the right (ACLMT) and left (control) gastrocnemius
muscle 8 months after ACLMT injection.
REGENERATION OF FAST AND SLOW MURINE MUSCLES
531
Fig. 8. Right soleus muscle 8 months after ACLMT injection
(A: toluidine blue; C: alc-mATPase at pH10.3; D: SDH). Serial cross
sections show that muscle fibers with a centralized nucleus (arrowheads,
A) could be type I or type II (type II fibers are indicated by asterisks, A, C,
D). (B) Left soleus muscle (toluidine blue) shows a normal morphologic
aspect, with the nucleus located at the periphery of the muscle fibers
(arrowheads). Scale bar ⫽ 20 µm.
and muscle regeneration in slow- and fast-twitch skeletal
muscles. Fibrosis is common after muscle necrosis and can
be induced by different procedures, such as ischemia
(Ownby et al., 1990), several types of myopathies (for
review see Engel and Banker, 1986), and periodic contusions (Minamoto et al., 1999). The presence of fibrosis, a
centralized nucleus, and split fibers were also observed in
the soleus muscle of rat 6 months after induced injury by
injection of Ringer solution at 60–70°C (Schmalbruch,
1976).
Serial cross sections obtained from soleus and gastrocnemius muscles showed that both types I and II fibers had
centralized nuclei 8 months after induced injury by ACLMT.
It is important to note that all muscle fibers evaluated by
serial cross sections in the deep and superficial regions of
the gastrocnemius had centralized nuclei (see Figs. 5 and
6). The presence of a centralized nucleus and split fibers
are well described in several conditions, such as denervated muscles (Lu et al., 1997; Rodrigues and Schmalbruch, 1995), regenerated muscles after induced injury by
toxins (Davis et al., 1991; Morini et al., 1998) and running
exercise (Wernig et al., 1991a), muscle graft (Carlson and
Faulkner, 1983), and after muscle contusion (Minamoto et
al., 1999).
The proportion of type IIC muscle fibers is higher than
normal after muscle injury because actively regenerating
and recently regenerated fibers are histochemically type
IIC, and this type of fiber is considered to be in the process
of changing from one fiber type to another (Pette and
Staron, 1990; Wernig et al., 1991a, b). Such change probably occurs after denervation and re-enervation, when a
muscle fiber becomes connected to a new motoneuron of a
different type (Wernig et al., 1991b). Another possibility is
that the temporary disconnection of muscle from nerve
results in the reprogramming of the motor neurons (Davis
et al., 1991).
Type IIC fibers are classified as undifferentiated and are
usually rare, but not absent, in the normal muscles of
adult mammals (Pette and Staron, 1990). This is in
agreement with the low incidence of type IIC fibers
532
SALVINI ET AL.
observed in the present study in the right and left murine
gastrocnemius, thus indicating a low degree of ongoing
changes in both muscles. An increased incidence of types
IIC and I fibers and a decrease of type II fibers were also
identified in mouse soleus muscle after muscle damage
induced by running exercise, freezing, or overuse (Wernig
et al., 1991a,b). Predominance of type I fibers in regenerated muscles were found in the soleus rat muscle 56 days
after local injection of the crude venom of Notechis scutatus (Davis et al., 1991).
Our hypothesis for the transformation process of fiber
types is that in the presence of muscle damage or muscle
paralysis, the motor neuron terminals responsible for the
innervation of type I muscle fibers produce axonal sprouts
faster than motor neurons of type II motor units (Brown et
al., 1981; Desypris and Parry, 1990). This difference in the
speed of production of axonal sprouts between fast and
slow motor units was also observed in the skeletal muscles
of mice (Duchen, 1970; Brown et al., 1980). It could explain
why only the deep region of gastrocnemius showed an
increased incidence of type IIC muscle fibers 21 days after
damage by ACLMT injection (Morini et al., 1998) and an
increased incidence of type I fibers 8 months after induced
injury by ACLMT. Perhaps it occurs because only this
region of the muscle has type I motor units. The absence of
type IIC fibers in the superficial region of the gastrocnemius, where there are only type II motor units, confirms
this hypothesis. The increased number of type I fibers and
the presence of AChE activity in the small regenerated
fibers of the gastrocnemius muscle 8 months after ACLMT
injection suggest that the injury produced by ACLMT
induced axonal sprouts and rearrangement of the motor
units. The probable mechanism for the rearrangement of
motor units after injury is denervation of the necrotized
muscle fibers and subsequent re-enervation of regenerated
muscle fibers by axonal sprouting (Brown et al., 1981;
Wernig et al., 1991a,b).
In conclusion, the results of this work indicate that
ACLMT induced muscle injury in types I and II fibers in
both soleus and gastrocnemius murine muscles; chronic
signs of previous injury, such as centralized nuclei, split
fibers, and small regenerated fibers, are seen 8 months
after ACLMT injection. Moreover, the regenerated characteristics of both types I and II fibers are similar, and
muscle injury induced muscle type fiber transformation
from type II to type I, through type IIC. Thus, ACLMT can
be considered an excellent model for inducing muscle
damage and muscle regeneration in both fast- and slowtwitch muscles of mice, stimulating the rearrangement of
the motor units and transformation of muscle fiber types.
ACKNOWLEDGMENTS
C.C. Morini was the recipient of a Master Fellowship
from Coordenacão de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). This work was
supported by Conselho Nacional de Ensino i Pesquisa
(CNPq) and FAPESP (Brazilian agencies). Tereza F.F.
Piassi was the recipient of a technical fellowship from
CNPq, and we thank her for technical assistance.
LITERATURE CITED
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