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Fiber type composition of monkey forearm muscle.

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THE ANATOMICAL RECORD 211:403-409 (1985)
Fiber Type Composition of Monkey Forearm Muscle
JOAN S. McINTOSH, MARGARETA RINGQVIST, AND EDWARD M. SCHMIDT
Laboratory of Neural Control, National Institute of Neurological and Communicative
Disorders and Stroke, Bethesda, MD 20205
ABSTRACT
Histochemical staining methods were applied to selected superficial
forearm muscles of Macaca mulatta monkeys. The muscles were analyzed with
regard to relative percentage distribution of different fiber types. In extensor carpi
radialis brevis, extensor carpi radialis longus, and palmaris longus there was a n
even dispersion of each fiber type from the superficial to the deep part of the muscle.
Extensor digiti communis showed a slightly higher percentage of type I fibers and
correspondingly lower percentage of type I1 fibers in its central as compared to its
superficial area. Three muscles, bracioradialis, extensor carpi ulnaris, and flexor
carpi radialis, displayed marked differences between their superficial and deep
areas. All of them contained a higher proportion of type I fibers (and correspondingly
lower percentage of type I1 fibers) in their deep parts than in their superficial areas.
Flexor carpi ulnaris (FCU) differed from the other muscles studied in that it
showed distinctly different fiber proportions on either side of a central tendon. While
the ulnar head of FCU was dominated by type I1 fibers (71% compared to 27% type
I fibers), the humeral head contained a larger proportion of type I fibers (58%vs.
40% type I1 fibers). This difference in fiber type distribution suggests different
functional demands for the two heads of FCU, with the possibility of more sustained
activity in the humeral head.
There are several studies presenting evidence that
histochemical characteristics of muscle fibers are correlated with physiological properties (e.g., Edstrom and
Kugelberg, 1968; Burke et al., 1973; Dum and Kennedy,
1980). In interpreting results from functional experiments, knowledge about fiber type composition is
essential.
While leg muscles have been extensively studied histochemically in both human and animals, there is more
limited information on the histochemical composition of
arm muscles and of forearm muscles in particular. Brachioradialis (BR) was one of the muscles included in a
study of the comparison of red and white voluntary
skeletal muscles (Beatty et al., 1966). The flexor carpi
radialis (FCR) muscle has been thoroughly investigated
in the cat (Gonyea and Ericson, 1977);the extensor digiti
communis (EDC) and BR muscles were included in a
study on 36 human muscles (Johnson et al., 1973). Recently, human extensors carpi radialis longus (ECRL),
brevis (ECRB), and BR muscles were studied (FuglMeyer et al., 1982). However, there is little data on
monkey forearm muscles, especially for species that are
often used in experiments on motor control. The lack of
information on the fiber composition of monkey forearm
muscles prompted this study. A preliminary report has
been given elsewhere (McIntosh et al., 1980).
analyzed. In the flexor group, FCR, flexor carpi ulnaris
(FCU), and palmaris longus (PL) were studied. The relative location of these muscles or their tendons in a
forearm cross section is shown in Figure 1.
The animals used in this study were obtained from
other investigators after they completed their experiment. These experiments did not influence muscle fiber
type distribution. One or both arms were removed from
deeply anesthetized (pentobarbital sodium) animals prior
to perfusion fixation. All muscles were dissected in toto
and tied to metal splints in a slightly stretched position.
The muscles were frozen by immersion in isopentane
cooled with liquid nitrogen and stored in a freezer a t
-70°C until processed. Blocks were cut from the largest
girth of the muscle belly of each frozen muscle and serial
cross sections were cut at 10 pm on a cryostat microtome
a t -20°C.
Staining
Sections were stained with a n oxidative stain, NADHTR (Novikoff et al., 19611, and for ATPase a t pH 9.4.
Two modifications of the routine ATPase method were
used. The first modification was sequential preincubation at pH 10.3 and pH 4.6 (10.314.6) (Groenneroed et al.,
1977). This preincubation procedure was carried out as
follows before staining a t pH 9.4: 1)fixation for 5 min in
2% formaldehyde, pH 7.6, 4°C; 2) rinse in 100 mM Tris
MATERIALS AND METHODS
HCl, pH 7.8, with 18 mM CaC1,; 3) preincubation for 10
Histochemical staining methods were applied to su- min at pH 10.3 in 100 mM 221 buffer in 50 mM CaC12,
perficial forearm muscles from six adult Macaca mulatta room temperature; 4) Tris rinse (same as in step 2); 5)
monkeys of either sex. In the extensor group, BR, ECRB,
Received April 3, 1984; accepted November 9, 1984
ECRL, extensor carpi ulnaris (ECU), and EDC were
0 1985 ALAN R. LISS, INC
404
J.S. MCINTOSH, M. RINGQVIST, AND E.M. SCHMIDT
FCR
PL -
Fig. 1. Relative locations of wrist muscles of a Mucuca rnulutta monkey at a midforearm cross
section drawn from a photograph. At this level the tendons of ECRB, ECRL, and PL are seen.
The boxes shown in brachioradialis (BR) illustrate the areas where fiber counts were obtained.
Box A1 is the superficial portion, A2 the central, and A3 the deepest area of the muscle. h,
Humeral head; u, ulnar head.
FIBER TYPE OF MONKEY FOREARM MUSCLES
405
preincubation for 5 min at pH 4.6 in barbital acetate
buffer (Dubowitz and Brooke, 1973) at room temperature; 6) Tris rinse (same as in step 2); 7) incubation for
20 min at pH 9.4 in ATPase incubation medium (Dubowitz and Brooke, 1973). The second modification was
preincubation in barbital acetate buffer at pH 4.35 for
3.5 min (for procedure see Dubowitz and Brooke, 1973).
Fiber Type Distribution
A
B
The relative proportions of different types of fibers
within the muscles studied were obtained from photomicrographs of cross sections stained for ATPase after
preincubation at pH 10.314.6. The typing was consistently checked by comparing the same fibers in serial
sections stained for ATPase after preincubation at pH
4.35 and by the NADH-TR method. The muscle cross
sections, except those of FCU, were arbitrarily divided
into different areas as illustrated in Figure 1. Three
areas were studied, A1 representing the superficial, A2
the central, and A3 the deepest area of the muscle section. An area contained about 150 fibers. All technically
acceptable fibers were counted in each area. As for FCU,
a pilot study had demonstrated that although there was
a clear difference in the proportions of type I and type I1
fibers between the humeral (FCUh) and ulnar (FCUu)
heads of the muscle, within each head there was a n even
distribution throughout from superficial to the deep
area. Hence, FCU was analyzed only in the central area
(A21 of the humeral and ulnar heads, respectively.
Fiber typing
Figure 2 illustrates the results of the three staining
procedures used in this study. On the basis of ATPase
staining at pH 9.4 after sequential preincubation at pH
10.314.6, the darkest stained fibers of Figure 2A were
termed type IIB, moderately stained fibers type IIA, and
fibers with no or very weak staining type I. Fibers with
a staining intensity between type IIB and type IIA or
between type IIA and type I were termed “unclassified”
and grouped together. After preincubation a t pH 4.35
for 3.5 minutes, type I fibers stained darkly, type IIB
moderately, and type IIA showed weak staining, as seen
in Figure 2B. Extending the preincubation a t pH 4.35
to 5 min produced a strong reaction in type I and no
reaction in most of the type I1 fibers. Occasional type I1
fibers still showed a moderate reaction. With the NADHTR method type I stained darkly, type IIA moderately,
and type IIB weakly, as illustrated in Figure 2C. Unclassified fibers showed various levels of reaction intermediate between type I and type IIA and between type
IIA and type IIB for ATPase at pH 4.35 as well as with
the NADH-TR method.
Statistical method
The methodological error of a single mean value for
the percentage of a given type of fiber was determined
by counting on two separate occasions all fibers in a
defined area (about 100 fibers) for each of seven arbitrar-
c
Fig. 2. Serial cross sections from brachioradialis (Mucucu muluttu)
showing the staining patterns of type I, type IIA, and type IIB fibers.
A) ATPase staining at pH 9.4 after sequential preincubation at pH
10.3 and pH 4.6.B) ATPase staining at pH 9.4 after preincubation at
pH 4.35.C) NADH-TR. Calibration 100 p m .
J.S. MCINTOSH, M. RINGQVIST, AND E.M. SCHMIDT
406
TABLE 1.Distribution of fiber types in three different areas of forearm muscles
-
X
ECU
A1
A2
A3
EDC
A1
A2
A3
ECRB
A1
A2
A3
ECRL
A1
A2
A3
BR
A1
A2
A3
FCR
A1
A2
A3
PL
A1
A2
A3
FCUu
A2
FCUh
A2
Type 1
Range
-
X
Type IIA
Range
Type IIB
Range
-
X
31-54
23-501**]
16-48
29.8
27.7
24.5
21-46
21-37
16-31
46.8
38.3
33.6
24-33
17-29
24.3
23.8
26.6
20-27
21-26
22-31
53.5
47.7
49.0
23.4
25.3
24.1
18-41
15-34
19-40
28.0
22.7
27.9
21-36
8-37
13-40
46.9
51.1
47.6
26-60
38-63
24-68
24.3
27.9
23.9
18-32
15-41
12-38
21.9
20.4
21.6
12-32
11-29
14-32
53.0
50.5
53.4
35-65
32-66
31-74
9.0
15.6
22.3
1;I;;lj
19.3
20.1
24.0
8-31
7-30
16-34
71.4
63.1
52.9
65-77
50-81
40-64
18.0
29.0
27.5
;;:::I*]*
25-30
23.5
27.2
24.8
16-36
14-34
17-37
57.0
43.5
47.0
;;:El*]*
33-58
25.7
27.5
23.7
13-52
15-53
8-51
27.4
27.3
28.0
24-31
19-40
17-37
45.4
44.7
47.6
20-59
25-66
24-65
26.9
16-41
22.0
8-33
48.8
31-607
58.2
41-81
20.2
7-30
21.9
33.2
41.2
15-29
22-501**]
27-68
21.8
27.8
24.0
14-30
**
.I***
I***
-
-
7-33
*
1
***
I***
Mean values, (X) and ranges for relative percentage distribution of various fiber types in monkey forearm muscles.
A1 is superficial area, A2 is central area, and A3 is deep area of the muscle.
Level of significance for differences between different areas (Al-A2, Al-A3, and A2-A3, respectively): *P < 0.05;
**P < o.oi; ***P < 0.001.
ily chosen cross sections. Estimated as the coefficient of
variation, the methodological error was 15% for type I,
8%for type IIA, and 9% for type IIB.
Differences between the superficial and central areas
(Al-AZ), superficial and deep (Al-A3), and central and
deep areas (A2-A3), respectively, were evaluated by tests
of paired observations of mean values for the various
types of fibers in the given areas. As for FCU, values
from the humeral and ulnar heads of the muscle were
paired. Use of the t-test instead of ANOVA is not vitiated
since all three possible two-way comparisons were made.
Inflation of the true significance level is minimal.
RESULTS
Table 1 shows values for the relative percentage distribution of the various fiber types in the superficial
(Al), central (AZ), and deep areas (A3).
The variation in the percentages of type I1 fibers was
due mainly to the variation in the frequency of type IIB
fibers. The relative percentage distribution of type IIA
fibers did not differ significantly in the different areas
studied. Unclassified fibers generally accounted for less
than 2% of the total number of fibers counted in each
area.
There were generally large interanimal variations in
the percentage distribution of each fiber type, which is
evident in the wide ranges of the means shown in Table
1.ECRB, ECRL, and PL showed a n even distribution of
the various fiber types throughout, from the superficial
to the deep area, as seen in PL muscle in Figure 3A. On
a n average there were 25% type I, 25% type IIA, and
50% type IIB fibers. EDC contained a slightly higher
percentage of type I fibers in its central than in its
superficial area (P < 0.05). The percentages of type Ill3
fibers were correspondingly lower (P < 0.05). The other
muscles showed more variation in the proportions of
type I and type IIB fibers in the different areas. ECU,
BR, and FCR had a higher percentage of type I fibers in
their central and deep areas than in their superficial
regions a s illustrated in Figure 3B,C for FCR. For BR
the difference between the superficial and deep areas
was highly significant (P < 0.001). The relative mean
percentage distribution of type I fibers in the deep area
of ECU was 41%. The range for mean percentage distribution of type I fibers in the deep area was 22-28% for
muscle other than ECU. BR contained low relative percentages of type I fibers in its superficial (9%)and central areas (16%). Ranges for corresponding values for
407
FIBER TYPE OF MONKEY FOREARM MUSCLES
muscles other than BR and FCU were 18-26% and 2533%, respectively. The relative percentages of type IIA
and ID3 fibers combined in BR were correspondingly
higher in the superficial and central areas (91 and 83%,
respectively). Ranges for type I1 fibers of muscles other
than BR and FCU in the superficial and central areas
were 73-81% and 66-74%, respectively.
FCU, divided by a tendon, showed distinctly different
proportions of fiber types on either side of the tendon
(Table 1, Fig. 4).The relative proportion of type I fibers
was 58% in the humeral head and 27% in the ulnar
head. Corresponding values for type I1 fibers were 40
and 71%, respectively.
DISCUSSION
A
B
C
The monkey forearm muscles studied, except for the
humeral head of FCU and the deep area of ECU, contained a large proportion of type I1 fibers (average 75%,
of which type IIB constituted approximately 50%)indicating that they are well adapted for strong or rapid
contractions. EDC and BR showed higher percentages
of type I1 fibers than corresponding human muscles
(Johnson et al., 1973). The proportions of type I1 fibers
in monkey FCR also seem to be larger than in cat FCR
(Gonyea and Ericson, 1977). In a recent study, FuglMeyer et al. (1982) demonstrated significant differences
in fiber type proportions between the wrist extensors
ECRL and ECRB in human. Thus ECRB, whose function is mainly postural (Fugl-Meyer et al., 1982) showed
a significantly higher proportion of type I fibers than
the ECRL and BR muscles. In the present study ECRL
and ECRB contained similar proportions of type I and
type I1 fibers; both were characterized by a high content
of type I1 fibers. BR, a prime mover for elbow flexion,
particularly when the movement is performed quickly
or against resistance (Rasch and Burke, 1978), had a
predominance of type I1 fibers (59%) in human (FuglMeyer et al., 1982). In monkey the proportion of type I1
fibers was even higher (91, 83, and 77% in the superficial, central, and deep area, respectively).
FCU, divided by a tendon, differed from the other
muscles studied with its distinctly different proportions
of type I and type I1 fibers in its ulnar and humeral
heads. While the mean relative percentage of type I
fibers in the ulnar head of FCU was 27 and about the
same as in the other muscles studied, it was as high as
58 in the humeral head. In line with observations by
Ariano et al. (1973) that there generally is constancy in
fiber type population within a given muscle of different
species, McIntosh, Ringqvist, and Schmidt (unpublished
observations, 1981)also noted that monkey and cat FCU
showed a similar fiber type pattern, although the difference between the two portions was not as distinct in cat
as in monkey FCU. This organization of FCU suggests
different functions of the two parts-the ulnar head having a capacity for fast acceleration and speed in wrist
flexion, the humeral head being equipped for endurance
during continuous work. Moreover Gonyea et al. (1981)
PL
FCR
FCR
CENTRAL
SUPERFICIAL
-
Fig. 3. Palmaris longus (PL) is representative of muscles with a
uniform mosaic pattern of type I, IIA, and IIB fibers found throughout
the cross section. Flexor carpi radialis VCR) is representative of the
group of muscles showing a difference in muscle fiber distribution
between central and superficial sections of the muscle (ATPase staining at pH 4.35, calibration 100 pm).
FCU
HUMERAL HEAD
ULNAR HEAD
Fig. 4. The top of the figure is a cross section of flexor carpi ulnaris
(FCU) from Macaca nulatta, ATPase staining at pH 4.35. Higher
power photomicrographs are shown of the humeral head (left portion)
and ulnar head (right portion) of the muscle. In the humeral portion
there is a larger proportion of type I fibers, while in the ulnar portion
there is a prevalance of type I1 fibers. Calibration 1mm for low power
and 100 pm for high power photomicrographs.
have reported on the cat FCU as having a fiber type
distribution corresponding to what we found for the
Macaca mulatta FCU muscle.
-
parison of red and white voluntary skeletal muscles of several
species of primates. J. Histochem. Cytochem., 14.590-600.
Burke, R.E., D.N. Levine, P. Tsairis, and F.E. Zajac (1973) Physiological types and histochemical profiles in motor units of the cat gastrocnemius. J. Physiol., 243t723-748.
Dubowitz, V., and M.H. Brooke (1973) Muscle Biopsy: A Modern ApLITERATURE CITED
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FIBER TYPE OF MONKEY FOREARM MUSCLES
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Fugl-Meyer, A.R., A. Eriksson, M. Sjostrom, and G. Soderstrom (1982)
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Gonyea, W.J., S.A. Marushia, and J.A. Dixon (1981) Morphological
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Groenneroed, O., H.A. Dahl, and 0. Vaage (1977) Easy typing of hu-
409
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