FIBRE TYPE REGIONALISATION
CHAPTER 5
ibre type regionalisation in lower hindlimb muscles of rabbit, rat and mouse: a comparative study
L.C.Wang and D.Kernell
Department of Medical Physiology, University of Groningen,
Postbus 196, 9700 AD Groningen, The Netherlands.
Submitted for publication, 2001
73
CHAPTER 5
Abstract
The topographical distribution of different fibre types was quantitatively determined
within muscles of the lower hindlimb in rabbits and mice. The results were compared
to those previously obtained, using the same new quantification methods, in homologous muscles of the rat. “Slow” type I fibres were identified and mapped out in crosssections stained for myofibrillar ATPase (in “slow” soleus done for type II fibres). At
the proximo-distal midlevel this was done for 11"fast” muscles in the rabbit and 7
“fast” muscles in the mouse. For 5 muscle species from each animal, more proximal
and distal levels were also included (extensor digitorum longus; flexor digitorum
and hallucis longus; gastrocnemius medialis; peroneus longus; tibialis anterior). All
the investigated “fast” muscles showed a significant degree of topographical eccentricity in the midlevel distribution of type I fibres. For most muscles, the direction of
this “vector regionalisation” of type I fibres was similar between the three animal
species. For homologous muscles, the degree of vector regionalisation was significantly different: mouse > rat > rabbit. The relative area of the region containing the
type I fibres, inversely related to the degree of “area regionalisation”, was also significantly different: mouse < rat < rabbit. Also within each animal species, muscles
with a marked degree of vector regionalisation tended to show a marked area
regionalisation. Proximo-distal differences in type I fibre density were observed in
all the three species of animals; also these patterns showed marked inter-species
differences. The findings demonstrate the general occurrence of, and systematic relationships between, different aspects of type I fibre regionalisation. The observed inter-species differences suggest that the expression of this phenomenon is adapted to
differing functional needs.
Key words: Myosin ATPase; Slow muscle fibres; Fibre type differentiation; Hindlimb
organisation.
Introduction
In the control of motor behaviour, most
muscles have to be used for rapid movements as well as for the prolonged maintenance of posture. Such different types
of motor activity are associated with different requirements for the power, speed
and endurance of the skeletal muscles.
Correspondingly, muscles contain fibres
with widely varying contractile and bio74
chemical properties, optimally suited for
different kinds of motor function. A major subdivision concerns that between
“fast” and “slow” fibres, and a coarse categorization along these lines can be made
using standard histochemical methods
(e.g. myofibrillar ATPase; Brooke & Kaiser, 1970; Burke, 1981).
Within muscles, there are not only
FIBRE TYPE REGIONALISATION
large differences between the properties
of individual muscle fibres, but there may
also exist marked regional variations in
the relative density of the various fibre
types (fibre type “regionalisation”; reviews: Armstrong, 1980; Kernell, 1998).
In an investigation of rat hindlimb muscles, we have recently developed a set of
simple methods for the quantitative description of this aspect of muscle organisation (Kernell & Wang, 2000; Wang &
Kernell, 2000; Wang & Kernell, 2001).
Most hindlimb muscles have a pinnate architecture, implying that their fibre type
composition may vary along the muscle
as well as within individual cross-sections. Within cross-sections, we made a
distinction between “area regionalisation”
and “vector regionalisation” of slow vs.
fast fibre types. Measures for area
regionalisation indicate the fraction of the
total cross-section area that contains the
target fibre type (typically the “slow” type
I fibres), and measures for vector
regionalisation denote the extent to which
the target fibres are eccentrically localised within the muscle section (including
the angular direction of this eccentricity).
In the rat lower hindlimb, most muscles are dominated by “fast” fibres (valid
for all except soleus), and their pattern of
“slow” type I fibre regionalisation was
found to be highly reproducible (Wang &
Kernell, 2001): all were vector-regionalised, most were also area-regionalised
and all investigated muscle species
showed a similar lengthwise pattern of
declining type I fibre density from proximal toward more distal levels (Wang &
Kernell, 2000). Furthermore, the direction
and degree of vector regionalisation was,
on average, related to the intra-limb position of the muscles: generally the type I
fibres tended to be accumulated toward
the limb centre and the degree of this
eccentricity was greater for superficial
muscles than in those situated closer to
the central core of the limb.
Comparative studies have shown
characteristic differences in fibre type
composition among homologous muscles
from different animal species, partly reflecting understandable differences in
motor requirements as dictated by, for
instance, motor behaviour (e.g. great
numbers of slow fibres in muscles of the
slow lori, Sickles & Pinkstaff, 1981; for
extensive comparative data, see also
Ariano et al. 1973; Armstrong, 1980). In
the present investigation we extend such
comparisons to include the phenomenon
of fibre type regionalisation, as seen in
homologous muscles of three species of
commonly used laboratory animals, adding new measurements from rabbits and
mice to those already collected in the rat
(cf. Wang & Kernell, 2000; 2001). Our
motivations for making this comparative
study were:
(1) from a theoretical point of view
we wished to know whether the
regionalisation characteristics of rat muscles reflect generalised aspects of muscle
organisation (e.g., as required, perhaps,
by differentiation processes in early development) or whether these characteristics varied significantly between homologous muscles of different species (e.g., reflecting, perhaps, different functional requirements).
(2) from a practical point of view,
knowledge concerning the regional distribution of different fibre types is important for the interpretation of results obtained by any kind of pointwise sampling
of their activity (e.g., electromyogram)
75
CHAPTER 5
and/or composition (e.g., biopsies, conventional histochemistry).
Methods
The new experimental measurements
were obtained from muscles of the lower
hindlimb in rabbits and mice. As far as
possible, the procedures were similar to
those described for rat muscle investigations in our preceding reports (Kernell &
Wang, 2000; Wang & Kernell, 2000;
2001).
Experimental material and histochemistry
Six adult female rabbits (HsdPoc:NZW,
1.42-2.14 kg) and 7 mice (C57BL/
6JolAHsD, 17.0-20.4 g) were used. Prior
to the dissection of the hindlimb muscles,
the animals were weighed (accuracy ± 10
g for the rabbit and ± 0.1 g for the mouse;
previously ± 1 g for the rat). Muscle dissections were performed under general
animal anaesthesia with pentobarbitone
(45 mg/kg i.v. for rabbit; 60 mg/kg i.p.
for mouse). Prior to their removal, the
posterior and/or lateral side of each muscle was labelled using water-insoluble
stains (blue, yellow). Thereafter, each
muscle was weighed and fixed by freezing in isopentane cooled by liquid nitrogen. The animals were killed by an
overdosis of pentobarbitone.
In the rabbit, all muscles traversing the
ankle joint were included in the study (n
= 12; names and abbreviations in Table
1). In the mouse, our labelling techniques
were less appropriate for the thinnest
muscles and the measurements were restricted to 8 relatively large ones (names
and abbreviations in Table 1). In both spe76
cies (as was also done earlier in the rat),
flexor digitorum and hallucis longus (FD)
was treated as one muscle.
Cross-sections from the proximodistal middle (“midlevel”) were analysed
in all muscles. For 5 selected muscle species, measurements were also made of
cross-sections obtained at different
proximo-distal levels (concerned ED, FD,
GM, PE, TA). These lengthwise
determinations were made at 7 equidistant levels for the rabbit (as was earlier
the case for the rat) and at 5 levels for the
mouse. Thus, the “midlevel” corresponded to level 4 in the rabbit (and rat)
and level 3 in the mouse.
At each analysed level, serial crosssections of 10 µm were cut in a cryostat
and stained for myofibrillar ATPase
(mATPase) after acid preincubation (acATPase, pH 4.3 - 4.5; Brooke & Kaiser,
1970) and after prefixation with
paraformaldehyde and alkaline
preincubation (alk-ATPase, pH 10.3; Guth
& Samaha, 1970; for details concerning
our staining procedures, Lind & Kernell,
1991). Fibres classified as type I were
consistently dark in ac-ATPase and light
in alk-ATPase, and the reverse staining
pattern was seen for the type II fibres. In
this investigation, we did not make any
distinction between the various subtypes
of type II fibre (e.g., types IIA, IIB etc.;
Brooke & Kaiser, 1970; Burke, 1981;
Lind & Kernell, 1991). Sections stained
for ac-ATPase were used for the various
quantitative determinations.
Measurements of fibre type regionalisation
In all muscles except soleus, fibre type
regionalisation was assessed by determin-
FIBRE TYPE REGIONALISATION
ing the distribution of type I fibres; in
soleus this was done for the type II fibres.
High-contrast photocopies were made of
the various cross-sections. In these photocopies, the muscle outline and the intramuscular position of each target fibre
was determined and digitised using a
graphic tablet, a PC and custom-made
software. On the graphic tablet, muscle
images were routinely positioned with
their posterior side at the top and their
lateral side toward the left.
The following parameters were calculated from the digitised measurements:
(1). muscle cross-section area
(MArea);
(2). muscle equivalent diameter
(EqD), calculated from MArea using the
formula for a circle;
(3). total number of target fibres
(typically type I fibres) within the section
(FibN);
(4). average target fibre density
within the section (FibD; fibres / mm2);
(5). the region containing the target
fibres (FRs), as determined using the “sector method” (for details, see Kernell &
Wang, 2000). The FRs measure was expressed as a percentage of the muscle
cross-section area and used for estimating the degree of “area regionalisation”,
a smaller FRs implying more
regionalisation. For fibres with a homogeneous distribution within the muscle
cross-section (i.e., no regionalisation),
FRs would approach 100% provided the
number of target fibres is very large. For
limited numbers of target fibres, a homogeneous fibre distribution would give a
target fibre region equal to CFR = 100 x (1
- FibN-0.5 )2. Thus, in order to demonstrate
the presence of an area regionalisation one
must show that FRs is significantly
smaller than CFR (Wang & Kernell, 2001).
(6). the length and direction of the
“target fibre vector” (Kernell & Wang,
2000), extending from the calculated centre of mass for the whole cross-section to
that for only the target fibres. The length
of this vector (VL) was expressed as a percentage of the muscle equivalent diameter, and the direction (VA) was given in
degrees (0º and 360º medial, 90º posterior, 180º lateral, 270º anterior). The relative length of the target fibre vector gives
a measure of the degree of “vector
regionalisation”, a larger VL implying
more regionalisation. For fibres with a
homogeneous distribution within the
muscle cross-section, VL would be zero
and VA unpredictable (“undefined”). In
order to demonstrate the presence of vector regionalisation one must show that VL
is significantly greater than zero and that
the standard deviation of VA is smaller
than ± 90º (i.e. full range less than half a
circle) (Wang & Kernell, 2001).
(7). a supplementary measure of the
degree of vector regionalisation (i.e. of
target fibre eccentricity), the “midline distribution pattern” (MLD): the percentage
of target fibres on the target-fibre-dominated side of a line drawn through the
middle of the cross-section (for details,
see Wang & Kernell, 2001). For fibres
with a homogeneous distribution within
the muscle cross-section, MLD would be
50%.
Statistics
Whenever applicable, mean values in the
text are given ± SD. Pearson correlation
coefficients were calculated for analysing
the degree of co-variation between different variables. Differences in properties
77
CHAPTER 5
Table 1. General muscle properties
A. Rabbit
Muscle N
Mwt (mg)
MArea (mm2)
EqD (mm)
FibN
FibD (mm-2)
GL
6
4893±782
GM
6
3040±428
127.4±20.6
12.7±1.1
3645.2±1094.9
28.5±7.1
72.0±8.8
9.6±0.6
3300.8±780.4
47.1±17.0
PL
6
2967±421
69.4±4.1
9.4±0.3
3024.0±815.9
43.9±13.2
FD
6
2693±356
49.4±8.2
7.9±0.7
2199.0±620.3
45.5±14.3
TA
6
1980±398
46.0±10.2
7.6±0.9
679.5±171.4
15.5±5.8
ED
6
1726±268
37.1±5.2
6.9±0.5
859.8±322.2
24.3±12.5
SO
6(4)
963±135
25.9±3.0
5.7±0.3
396.5 (58, 735)
17.8 (2.4, 33.3)
PB
6
523±89
9.6±1.6
3.5±0.3
196.5±62.5
20.3±5.5
TP
6(1)
461±74
16.6±3.3
4.6±0.4
153.8±160.0
10.3±12.5
PE
6
374±51
12.1±2.7
3.9±0.5
307.3±122.9
26.8±12.1
D5
6
304±41
14.4±1.9
4.3±0.3
206.7±53.7
14.6±4.2
D4
6
298±52
10.6±1.7
3.7±0.3
86.3±74.0
8.4±7.8
B. Mouse
Muscle
N
Mwt (mg)
MArea (mm2)
EqD (mm)
FibN
FibD (mm-2)
GL
6
52.7±10.7
6.21±0.74
2.81±0.17
65.83±31.80
10.33±4.24
GM
7
33.7±4.7
3.73±0.43
2.17±0.13
102.71±21.61
27.91±6.94
PL
6
13.5±0.8
1.08±0.08
1.17±0.05
15.67±3.72
14.59±3.61
FD
7
24.4±3.2
2.84±0.43
1.90±0.14
64.86±16.50
23.51±7.62
TA
7(1)
34.7±4.1
4.00±0.42
2.25±0.12
5.50±6.38
1.36±1.54
ED
7(2)
7.9±1.0
1.00±0.29
1.12±0.16
13.00±6.38
10.96±8.19
SO
4
8.0±1.0
0.86±0.13
1.04±0.08
305.75±20.93
361.51±64.41
PE
5
8.3±1.2
0.80±0.14
1.00±0.09
15.20±8.35
19.40±10.72
Means±SD. For rabbit soleus (SO), only 2 of the 6 muscles had any target fibres (type II); hence, for
averages of FibN and FibD the scatter of the data is indicated by value ranges instead of by SD values.
General abbreviations: N, number of measured muscles in each group; numbers in parenthesis indicate
the number of muscles (if any) that did not contain any target fibres; Mwt, muscle weight; MArea,
muscle cross-section area; EqD, equivalent muscle diameter; FibN, number of target fibres (type II for
SO, type I for all other muscles); FibD, target fibre density (fibres per mm2). Muscle abbreviations: GL,
gastrocnemius lateralis; GM, gastrocnemius medialis; PL, plantaris; FD, flexor digitorum & hallucis
longus; TA, tibialis anterior; ED, extensor digitorum longus; SO, soleus; PB, peroneus brevis; TP, tibialis
posterior; PE, peroneus longus; D5, peroneus digiti 5; D4, peroneus digiti 4. For rabbits (section A)
muscles are placed in order of size accoding to their weight (Mwt). For mouse (section B), muscles are
placed in the same order as that for the rabbit.
78
FIBRE TYPE REGIONALISATION
between different groups of muscles were
analysed using standard t test procedures.
Calculations were made using Excel
(Microsoft) or the software package
SYSTAT. Cases with P < 0.05 were considered statistically significant.
Results
Rabbit muscles
General properties.
General properties of rabbit muscles are
listed in Table 1A. The muscles have been
Fig. 1. Target fibre distribution in rabbit hindlimb muscles. Digitised midlevel sections from all the 12
analyzed hindlimb muscles, all from one animal (rabbit 4). Muscle abbreviations as in Table 1. Type I
fibres indicated in all muscles except soleus (SO, type II fibres). All sections shown at about the same
absolute size (calibration bars 1 mm) and with the same rotational position (posterior up, medial to the
right). Muscles arranged in same order as Table 1 A.
79
CHAPTER 5
Table 2. Regionalisation parameters at midlevel of muscles
A. Rabbit
Muscle
GL
GM
CFR (%)
FRs (%)
VL (%)
MLD (%)
VA (degrees)
96.6
86.4±6.4#
12.4±3.7
63.7±5.4
249±8
96.5
83.9±9.1
24.1±7.6
74.2±9.1
279±7
#
PL
96.3
95.0±3.9
4.8±3.8
55.8±5.8
202±35
FD
95.7
89.7±4.1#
6.4±3.4
54.7±3.9
196±30
TA
92.3
63.8±9.2#
11.9±2.8
70.3±6.3
29±15
ED
93.0
91.7±3.0
3.1±1.9
55±2.7
385±41
SO
84.1
39.6(5.0, 74.2)
23.8(9.4, 38.2)
83(66.0, 100)
210(202, 217)
PB
85.8
81.0±5.8
6.0±3.6
60.9±3.3
243±76
TP
80.1
42.8±25.5#
8.3±5.1
59.8±13.0
60±39
PE
88.1
82.6±7.2
15.7±4.3
68.6±3.4
362±8
D5
86.3
81.1±4.8
7.4±4.0
58.8±5.8
373±26
D4
75.8
43.8±26.4#
16.5±11.6
76.1±15.2
290±28
#
#
B. Mouse
Muscle
CFR (%)
GM
81.0
GL
PL
FD
TA
ED
75.2
55.3
76.3
22.6
50.8
FRs (%)
VL (%)
MLD (%)
VA (degrees)
18.1±5.1
28.7±7.7
97.1±4.6
239±14
83.6±7.6
239±21
5.9±1.8#
15.8±8.8
33.0±6.5
#
#
#
19.0±6.0
17.2±3.4
41.1±5.7
5.7±4.8#
8.6±5.9
29.9±3.2
31.0±9.2
#
SO
88.9
85.7±2.0
PE
51.6
14.0±6.7#
#
100.0±0
92.2±6.5
100.0±0
95.8±4.9
341±14
305±37
57±16
338±7
4.4±3.6
57.3±6.1
302±63
23.9±6.0
94.6±7.4
299±21
Mean±SD (range of values for rabbit soleus, SO). Abbreviations: CFR, fibre region correction factor, i.e.
percentage of muscle area (MArea) expected to be covered by target fibres in case of a uniform
distribution; FRs, target fibre region calculated by “sector method” (% of MArea); VL, length of target
fibre vector (% of EqD); MLD, midline distribution (% of target fibres on “target side” of middle of
muscle section); VA, angle of target fibre vector (degrees). Target fibres were type II in soleus (SO) and
type I in all other muscles. Number of muscles per group and muscle abbreviations as in Table 1.
Statistics (t tests, significance for P<0.05 or better): For “fast” muscles (all except SO), all VL values
were significantly greater than zero and all MLD values significantly greater than 50%. FRs values
which were significantly smaller than CFR are labelled with#.
80
FIBRE TYPE REGIONALISATION
ordered according to their weight (Mwt),
from the heavy gastrocnemius lateralis
(GL, 4893 mg) to the tiny peroneus
digitorum iv (D4, 298 mg). Midlevel
measures of size, such as the midlevel
cross-section area (MArea) and midlevel
equivalent diameter (EqD) were ranked
in much the same way and showed a high
degree of correlation to muscle weight
(Mwt vs. EqD, r=0.990, P<0.001). The
mean overall density of type I fibres
(FibD) also varied considerably across the
11 “fast” muscles, ranging from 47.1 fibres/mm 2 for gastrocnemius medialis
(GM) down to 8.4 fibres/mm2 for peroneus digitorum iv (D4). As is evident
from Table 1A, the highest type I fibre
densities were seen in large muscles (FibD
vs. EqD, r=0.629, n=11, P<0.05). In the
relatively small tibialis posterior, type I
fibres were even lacking completely in 1
of the 6 rabbits. In 4 of the 6 rabbits, soleus contained no type II fibres; hence,
with regard to this muscle no reliable statistical evaluations could be made concerning its average target fibre
regionalisation.
Table 3. Correlations between proximodistal level and type I fiber distribution
A. Rabbit
Muscle
FibD
FD
0.827
GM
ns
FRs
VL
VA
(-0.738)
ns
0.904
ns
ns
ns
ns
ns
-0.859
-0.931
0.875
-0.852
ns
ns
Muscle
FibD
FRs
VL
VA
FD
ns
ns
-0.986
-0.921
TA
ED
PE
ns
ns
-0.886
ns
B. Mouse
GM
TA
ED
PE
-0.989
0.889
-0.911
-0.999
-0.959
ns
ns
-0.956
0.950
ns
ns
ns
-0.895
ns
(0.862)
(-0.808)
Correlation coefficients calculated for average values of each parameter vs. proximo-distal level (n=7
for rabbit; n=5 for mouse). Abbreviations as in Tables 1 and 2. Statistics: ns: not significant (P>0.1);
values in parenthesis: 0.1>P>0.05; all other values: P<0.05 or better. Muscles arranged in same order as
in Tables 1-2.
81
CHAPTER 5
Midlevel regionalisation.
Figure 1 shows the distribution of target
fibres within midlevel cross-sections for
all the investigated muscles of one rabbit
(taken from one of the four animals showing no type II fibre in soleus), again with
the muscles ordered according to weight.
Statistical data on target fibre regionalisation are listed in Table 2A.
As is seen in Fig.1, most of the muscles had a less than complete coverage of
type I fibres across their cross-section, i.e.
most muscles showed some evidence for
an area regionalisation. Statistically, this
was true for all the “fast” muscles except
ED, PB and PL (Table 2A, comparisons
of FRs vs. CFR). The average sizes of the
latter three muscles varied over a wide
range (weights 523 - 2967 mg) and they
had a relatively high overall density of
type I fibres (20.3 - 43.9 fibres / mm2;
Table 1A).
The degree of intra-muscular type I
fibre eccentricity, as quantified using the
vector length parameter (VL), varied remarkably over an eight-fold range, from
about 3 % for extensor digitorum longus
(ED) up to about 24 % for gastrocnemius
medialis (GM). The mean value of VL
was in all “fast” muscles significantly
greater than zero (t test, P < 0.05) and the
angle of the vector (VA) had a standard
deviation of 76ο or less (mostly 41ο or less,
Table 2A). Hence, all the fast muscles
showed a significant degree of “vector
regionalisation” (cf. Wang & Kernell,
2001). Accordingly, the supplementary
measure of asymmetrical target fibre dis-
Fig. 2. Target fibre distribution in mouse hindlimb muscles. Digitised midlevel sections from all the 8
analysed hindlimb muscles, all from one animal (mouse 6). Muscle abbreviations as in Table 1. Type I
fibres indicated in all muscles except soleus (SO, type II fibres). All sections shown at about the same
absolute size (calibration bars 1 mm) and with the same rotational position (posterior up, medial to the
right). Muscles arranged in same order as Table 1 B. This mouse had no type I fibres in ED and only one
type I fibre in TA.
82
FIBRE TYPE REGIONALISATION
tribution (MLD) also consistently showed
a mean value significantly greater than
that expected for a random fibre distribution (i.e., greater than 50% in all “fast”
muscles). As expected, a high correlation
was found between the two eccentricity
parameters (VL vs. MLD; r=0.900, n=11,
P<0.001).
There was a suggested tendency for
muscles with a high degree of vector
regionalisation also to show a relatively
marked degree of area regionalisation.
Thus, for the five “fast” muscles with the
smallest VL-values, the type I fibre region averaged 87.7±6.3 %, whereas for
the remaining six muscles the corresponding value was 67.2±20.2 % (t test, P <
0.06).
Lengthwise regionalisation.
In only 3 out of the 5 studied muscle species was there a significant correlation
between the overall density of type I fibres and the proximo-distal analysis level
(Table 3A, Fig.3 A, C). In two of these
muscles (FD, PE), type I fibres became
slightly more common distally than proximally, and for one muscle (TA) the opposite was true and the differences were
more pronounced.
Within cross-sections, the degree of
vector regionalisation was not systematically different at different proximo-distal
levels. In 2 of the 5 muscle species (PE,
TA), the area regionalisation became more
pronounced distally than proximally (Table 3A, Fig.3E).
The direction of vector regionalisation
(VA, vector angle) showed relatively
marked proximo-distal shifts for FD (increasing) and GM (decreasing toward
distal; Fig.4C).
Mouse muscles
General properties.
As stated above, only 8 relatively large
hindlimb muscles of the mouse were included in this study. Their general properties are listed in Table 1B. Muscle
weights ranged from 52.7 mg for GL
down to 7.9 mg for ED. Also in these
cases the weights of the muscles were
highly correlated to their midlevel dimensions (Mwt vs. EqD, r=0.990, P<0.001).
One of the 7 examined mice did not have
type I fibres at any proximo-distal level
of its TA muscle; in two other mice, type
I fibres were completely absent from the
ED muscles. For the 6 TA muscles which
did contain type I fibres, the midlevel
number and density of such fibres was
very low (mean 1.36 fibres per mm2, Table 1B). For all the other “fast” muscles,
the density of type I fibres ranged between
about 10-11 fibres per mm2 for GL and
ED up to about 28 fibres per mm2 for GM.
As is evident from Table 1B, type I fibre
density showed no significant co-variation with “fast” muscle size (FibD vs.
EqD, r = -0.15, P>0.7, n=7).
Soleus muscles of the mouse consistently had a rather high density of type II
fibre (361.51 per mm2).
Midlevel regionalisation.
Figure 2 shows the distribution of target
fibres within midlevel cross-sections for
all the investigated muscles species of one
mouse (taken from one of the two animals lacking type I fibres in ED), with
the muscles ordered as in Tables 1-2. Statistical data on target fibre regionalisation
are listed in Table 2B.
As is seen in Fig.2, the type I fibres (if
present) covered only a restricted portion
83
CHAPTER 5
Fig. 3. Mean longitudinal changes in type I fibre density and area regionalisation for muscles of rabbits
(left-side graphs) and mice (right-side graphs). Error bars show ±SE (A, B, E, F; n as in Table 1) Measurements plotted vs. proximo-distal level. In panel F, data for ED and TA were not included; at many of
the proximo-distal levels these two muscles had too few type I fibres for appropriate calculations of type
I fibre regions. Different symbols for each species of muscle (abbreviations as in Table 1). A, B Type I
fibre density (parameter FibD; number of type I fibre per mm2 cross-section area). C-D Normalised
values for type I fibre density (%): FibD values expressed as percentage of maximum mean value for
each muscle. E-F Type I fibre region (parameter FRs), given as percentage of total cross-section area.
of the cross-section area in the 7 “fast”
muscles. Correspondingly, all these muscles showed a significant degree of “area
regionalisation”, i.e. a type I fibre region
(FRs) smaller than the corresponding control value (CFR; Table 2B). There was
84
some suggestion that the degree of area
regionalisation might be less pronounced
(i.e., larger FRs) in “fast” muscles with a
relatively high type I fibre density (FRs
vs. FibD, r=0.736, n=7, 0.05<P<0.1). In
the slow soleus, the type II fibres showed
FIBRE TYPE REGIONALISATION
Fig. 4. Mean direction of type I fibre regionalisation at different proximo-distal levels (A, C rabbit; B,
D mouse). Y-values give means ± SE for the direction (degrees) of the “type I fibre vector” (parameter
VA). Abbreviations as in Table 1. A-B Data for ED, PE and TA muscles. C-D Data for FD and GM
muscles (cf. Tables 2-3).
a significant (but low) degree of area
regionalisation (Table 2B).
The degree of intra-muscular target
fibre eccentricity, as quantified using the
vector length parameter (VL), was in all
“fast” muscles significantly greater than
zero (t test, P < 0.01 or better) and the
angle of the vector (VA) had a standard
deviation of 37ο or less (Table 2B). Hence,
all the investigated “fast” mouse muscles
showed a significant degree of vector
regionalisation (cf. Wang & Kernell,
2001). Accordingly, the supplementary
measure of asymmetrical target fibre distribution (MLD) also consistently showed
a mean value significantly greater than
that expected for a random fibre distribution (i.e., greater than 50% in all “fast”
muscles), and a high correlation was
found between the two eccentricity parameters (“fast” muscles, VL vs. MLD;
r=0.82, n=7, P<0.05).
In the mouse “fast” muscles, the degree of midlevel area regionalisation
(FRs) was significantly correlated to the
degree of type I fibre eccentricity (i.e. FRs
vs. VL or MLD; r=-0.769, -0.922 respectively, P<0.05).
Lengthwise distribution of properties.
Among the five “fast” mouse muscles that
were studied in a lengthwise direction, the
85
CHAPTER 5
Table 4. Statistical comparisons between mouse, rat and rabbit with regard to
type I fibre density and regionalization
Parameter
Mouse vs. Rat
Rat vs. Rabbit Mouse vs. Rabbit
Sign. of difference (paired t test)
Type I fibre density (FibD)
Type I fibre region (FRs)
Vector angle (VA)
Vector length (VL)
Midlline distribution pattern (MLD)
(<0.08)
<0.001
ns
(<0.06)
<0.03
ns
<0.01
ns
<0.01
<0.001
<0.001
<0.001
ns
<0.01
<0.001
Correlation coefficient
Type I fibre density (FibD)
Type I fibre region (FRs)
Vector angle (VA)
Vector length (VL)
Midlline distribution pattern (MLD)
ns
ns
0.96
ns
0.83
ns
ns
0.94
ns
ns
0.81
ns
0.82
ns
ns
Comparisons between mean values for all the 7 “fast” muscles that had been investigated in each one of
the three animal species (i.e. ED, FD, GL, GM, PE, PL, TA; cf. data plotted in Fig.5). Upper part of
table: P values obtained in paired t tests. Lower part: correlation coefficients. Values reaching statistical
significance (P<0.05) shown bold.
type I fibre density was typically systematically different between different
proximo-distal levels, although the direction of the change differed between the
muscles. In ED, GM and PE densities
showed a pronounced decrease from
proximal to distal, in FD the type I fibre
density had a maximum at midlevel and
decreased in both directions, and in TA a
slight but significant increase of type I
fibre density was seen from proximal toward distal (Table 3B, Fig.3 B, D).
Within cross-sections, the degree of
vector regionalisation was not consistently varying with proximo-distal level:
toward distal it decreased for FD, increased for GM and showed no significant change for TA, PE and ED (Table
3B). Similarly, for area regionalisation no
significant proximo-distal change was
noted for TA, and ED (few type I fibres
present), whereas the regionalisation was
86
more pronounced distally than proximally
for GM and PE, and in FD the type I fibre
region had its maximum close to the
midlevel and decreased in both directions
(Table 3B, Fig. 3F).
The direction of vector regionalisation
(VA, vector angle) showed relatively
moderate proximo-distal shifts for FD and
GM (both decreasing toward distal;
Fig.4D, Table 3B).
Comparisons between muscles of rabbits,
mice and rats
In comparisons between the muscles of
different animal species, we will relate the
new data presented above to our previously published measurements for the rat
(Figs.5-6, Table 4), which were obtained
and processed using the same methods
(Wang & Kernell 2000; 2001).
FIBRE TYPE REGIONALISATION
Fig. 5. Comparisons between “fast” muscles of rabbits, rats and mice with regard to parameters of type
I fibre regionalisation. Data included for all the 7 muscles that were studied in each one of the 3 animal
species (ED, FD, GL, GM, PE, PL, TA). In each panel, mean values for rabbit, rat and mouse are plotted
vs. those of the rat. Rat data joined by straight lines (i.e., corresponding to a unity line of x = y). A Type
I fibre region (%, FRs), B Angle of the type I fibre vector (degrees, VA). C Length of the type I fibre
vector (%, VL). D Midline distribution parameter, a supplementary measure of type I fibre eccentricity
(%, MLD). For statistics, see Table 4.
A. Type I fibre regionalisation
Midlevel regionalisation: similarities.
(1). In all the three animal species, the
type I fibres of “fast” muscles all showed
a significant degree of eccentricity in their
midlevel distribution, i.e., vector length
was in all cases significantly greater than
zero (Table 2). Furthermore, this eccentricity had a reasonably reproducible direction; the standard deviation for the
vector angle was always <90º and mostly
<40º. Thus, according to our criteria
(Wang & Kernell, 2001), all the investigated “fast” hindlimb muscles of rabbits,
rats and mice showed a significant degree
of “vector regionalization” (cf. Wang &
Kernell, 2001).
(2). In all the three animal species, the
area regionalisation tended to be more
pronounced for muscles with a marked
degree of type I fibre eccentricity (large
VL value) than in those with a less eccentric fibre distribution. This was most
evident in rats and mice: a significant
negative correlation was found between
the vector length (VL) and the type I fibre region (parameter FRs). In rabbits, the
type I fibre region (FRs) showed a suggested tendency (P < 0.06) to be larger
87
CHAPTER 5
Fig. 6. Relationship between type I fibre region (%, FRs) and type I fibre density (fibres / mm2, FibD) in
muscle cross-sections from rabbit (open circles), rat (crosses) and mouse (filled triangles). Includes data
for cross-sections from all proximo-distal levels in the five longitudinally studied muscles (ED, FD,
GM, PE, TA). Separate regression line drawn for data from each animal species. All correlations statistically significant (P<0.01 or better; n = 35 for rabbit, 35 for rat, 25 for mouse).
for muscles with a small than in those with
a large type I fibre vector (VL).
(3). The intra-muscular direction of the
type I fibre eccentricity (i.e., the angle of
the vector regionalisation, parameter VA)
was similar in all the three animal species. Thus, across the 7 “fast” muscles
studied in all the three species, the VA
values were highly correlated and there
were no differences from one species to
another (Fig.5B, Table 4). In the rat, the
type I fibres were generally accumulated
in a direction toward the centre of the
limb; the results of Fig.5B suggest that
this is also generally the case for mouse
and rabbit hindlimb muscles.
The inter-species similarity of vector
angles was not universal: for two of the
smaller muscles, the vector angle was
markedly different between rabbits and
rats (muscles not studied in mice, hence
88
not represented in Fig.5). Thus, for peroneus brevis (PB) the vector angle was
243º in the rabbit and 65º in the rat. For
tibialis posterior, it was 60º in the rabbit
and 271º in the rat. The reasons behind
this peculiar “mirror-like” reversion of
eccentricity between the two species are
obscure.
(4). In the rat, we had found that the
degree of vector regionalisation (VL)
tended to be more marked for muscles
lying far from the limb centre than for
those placed in a deeper, more central
position. If this were also the case in the
mouse and rabbit, one might expect a significant correlation between the respective “eccentricity” parameters (VL, MLD)
across the 3 animal species. Such a correlation was indeed observed for comparisons between rat and mouse but not between rat and rabbit (Table 4; cf. Fig.5 C,
FIBRE TYPE REGIONALISATION
D). Further and more direct investigations
are needed for establishing whether, in the
rabbit, the degree of type I fibre eccentricity is related to the intra-limb position
of the muscles.
(5). In all the three animal species, a
positive correlation was found between
the overall density of type I fibres and the
relative size of the type I fibre region
(Fig.6). Thus, within each species, the
area regionalisation tended to be less pronounced in muscles rich in type I fibres
than in those that were more dominated
by type II fibres.
Midlevel regionalisation: systematic differences.
(1). The relative size of the type I fibre region (FRs) was significantly different between the three animal species and
ranked such that rabbit > rat > mouse
(Fig.5A, Table 4). As is demonstrated in
Fig.6, these differences were evident also
between muscles showing the same overall density of type I fibres: the three regression lines in Fig.6 are shifted along
the y-axis. Some of the rat muscles (PB,
PD) and some of the rabbit muscles (ED,
PB, and PL) actually failed to show a significant average degree of area
regionalisation (Table 2A; Wang &
Kernell, 2001).
(2). The relative degree of vector
regionalisation (VL, MLD) was significantly different between the three animal
species and ranked such that rabbit < rat
< mouse (Fig.5 C, D, Table 4). Thus, species with a large degree of vector
regionalisation tended to have a small type
I fibre region (i.e., a high degree of area
regionalisation). These inter-species differences were in accordance with the finding that, also within each animal species,
the muscles with a large degree of type I
fibre eccentricity tended to show small
type I fibre areas (cf. Table 2; Wang &
Kernell, 2001).
Lengthwise regionalisation: similarities.
(1). In the rat, the lengthwise
organisation of type I fibres showed a very
consistent behaviour for all the 5 “fast”
muscles studied from this point of view:
the overall density of type I fibres
decreased from proximal toward distal
(Wang & Kernell, 2000). With regard to
this behaviour only a partial similarity was
observed between the three species. A
proximo-distal decline in type I fibre
density was indeed seen for 3 of the 5
studied muscles in mice (ED, GM, PE,
Fig.3D). In the rabbit, this was seen in
only 1 of the studied 5 muscles (TA,
Fig.3C).
(2). In all the three animal species,
lengthwise shifts in the degree of area
regionalisation tended to occur such that
the type I fibre region (FRs) became
smaller distally than proximally (Fig.3E,
F). However, in mouse and rabbit several
muscles failed to show any significant
proximo-distal changes of FRs (Table 3).
(3). In all the three animal species,
several muscle species showed systematic proximo-distal variations in the angular direction of the vector regionalisation. For gastrocnemius medialis (GM)
this behaviour was consistent across all
the three species: a progressive decrease
of vector angle occurred from proximal
toward distal (Fig.4 C, D; Wang &
Kernell, 2000). Such apparent shifts in the
direction of type I fibre accumulation
might (partly) be a consequence of the
manner in which the individual fibres fit
into the pinnate structure of these mus89
CHAPTER 5
cles (cf. measurements of muscle architecture in Wang & Kernell, 2000).
Lengthwise regionalisation: differences.
(1). As was already noted in the previous section, several muscles of the mouse
and rabbit failed to demonstrate the
proximo-distal decline of type I fibre density seen for all these hindlimb muscles
in the rat. In some of the muscles of rabbit and mouse, the type I fibre density
even showed a (moderate) increase from
proximal toward more distal levels (FD
and PE in rabbit; TA in mouse; Table 3).
(2). In the rat, the vector regionalisation became progressively more pronounced at more distal levels. Such
proximo-distal differences were not typically seen in the mouse and rabbit muscles (Table 3).
B. General muscle properties and composition
(1). Muscle vs. body weight. In comparison to rats and mice, rabbits had a very
large plantaris muscle (PL), rather moderate gastrocnemii (GM, GL), and a relatively small tibialis anterior (TA). For rabbit, rat and mouse the average relation
between muscle and body weight was
0.164, 0.121 and 0.082 % for PL, 0.437,
0.622 and 0.500 % for GM+GL and 0.108,
0.216 and 0.204 % for TA (t test, P < 0.05
or better for all inter-species differences).
Such findings suggest the presence of differences in the kinesiological roles of
these various muscles between the three
species. The plantaris muscle might, for
instance, be of particular importance for
the hopping behaviour in the rabbit.
90
(2). Midlevel type I fibre density. In
homologous muscles, the overall density
of type I fibres was significantly and
markedly lower in mice than in rabbits
(cf. Figs. 1 and 2; Tables 1, 4). Surprisingly, however, a wide overlap of type I
fibre density was noted between rabbit
and rat (no significant difference, Table
4; see also Fig.6). Among the 7 species
of “fast” muscle shown in Fig.5, the highest type I fibre density was actually seen
in a muscle of the rat, the peroneus longus (mean 56.5 ± 25.5 per mm2; Wang &
Kernell, 2001).
Discussion
General presence and patterns of fibre
type regionalisation
This study is an extension of our previous investigations on fibre type regionalisation in rat muscles. Those studies
revealed that, within the rat lower
hindlimb, fibre type regionalisation is not
restricted to only a small number of large
and conspicuous muscles, but it is a highly
significant and general feature of intraand inter-muscular organisation (Wang &
Kernell, 2000; 2001). In the present investigation, this general conclusion is
shown to be valid for yet two other species of common laboratory animals, rabbits and mice. In each of the three animal
species, all the fast hindlimb muscles were
significantly vector-regionalised (Figs.1,
2, Table 2) and in each species many of
the muscles also showed a heterogeneous
distribution of type I fibres in the lengthwise direction (Table 3, Fig.3). Such findings have significant practical implications: when sampling the activity or properties of hindlimb muscles, one always
FIBRE TYPE REGIONALISATION
has to keep track of the intra-muscular site
from which the observations were obtained. Our present findings provide topographical information of importance as a
background for such sampling activities.
The findings are also of interest with regard to the possible theoretical and functional implications of fibre type
regionalisation, which will be further
commented upon below.
With regard to pointwise samplings of
fibre properties and muscle activity, the
area regionalisation represents the most
obvious source of possibly erroneous interpretations: in most of the “fast”-mixed
muscles of mice and rats and in many of
the rabbit ones, the slow type I fibres are
completely lacking from some muscle
regions (Figs.1-2). Interestingly, the three
animal species showed systematic differences in their area-regionalisation: even
for muscles with the same mean density
of type I fibres, the relative size of the
type I fibre region was smaller in mice
than in rats and smaller in rats than in rabbits (Figs.1, 2, 5-6, Table 4). For a limited number of forelimb muscles, a greater
degree of area regionalisation in rats than
in rabbits was noted also by Fuentes et al.
(1998).
The general phenomenon of fibre type
regionalisation has been noted in earlier
studies of various limb muscles in several different species (e.g. amphibian,
Rowlerson & Spurway, 1988; turtle,
Laidlaw et al., 1995; mouse, Parry &
Wilkinson., 1990; rat, Pullen, 1977 a, b;
Armstrong & Phelps., 1984; cat, Gordon
& Phillips, 1953; English :& Letbetter,
1982; Chanaud et al., 1991; Rafuse &
Gordon, 1996; rabbit, Lobley et al., 1977;
Lexell et al., 1994; dog, Armstrong et al.,
1982; cattle, Totland & Kryvi., 1991;
Brandstetter et al., 1997; primates, Sickles & Pinkstaff., 1981; Acosta & Roy,
1987; humans, Johnson et al., 1973;
Henriksson-Larsen et al., 1983; Lexell et
al., 1983; reviews: Armstrong, 1980;
Kernell, 1998). However, in our present
study we have, for the first time, used new
quantitative methods and procedures of
regionalisation assessment for direct inter-species comparisons of fibre type
regionalisation. In some cases, our conclusions differ from those of earlier reports concerning corresponding muscles;
such differences might perhaps largely
reflect differences in methodology. For
instance, in muscles showing no significant area regionalisation, the existence of
a significant degree of vector regionalisation may be difficult to assess without
our present type of quantitative measurements (cf. Fig.1). Such factors might explain why a largely homogeneous fibre
type distribution was previously reported
for the rabbit’s PL and ED (Lobley et al.,
1977) while our measurements revealed
the presence of a small but significant
degree of vector regionalisation in both
muscles (Table 2A). Other factors (differences in animal weight and gender?)
might be needed to explain the fact that
we found no significant lengthwise
regionalisation in the rabbit ED (Table
3A) while a moderate degree of proximodistal increase of type I fibre frequency
was noted for the same muscle by Lexell
et al. (1994). Furthermore, in our cases
the rabbit’s TA showed a distal decrease
of type I fibre density (Fig.3 A, C) which
was not noted by Lexell et al. (1994). The
observations of the latter authors were,
however, restricted to muscle portions
within 15 mm from the midlevel. For the
rabbit TA muscles of Fig.3 (A, C), the
91
CHAPTER 5
distal decrease of type I fibre density was
only prominent at the two most distal levels, which were situated at about 16 - 24
mm below the midlevel. In larger rabbits,
like those used by Lexell et al. (1994),
the two most distal analysis levels would
have been even further remote from the
muscular midlevel.
Theoretical and functional implications
It is as yet unknown why fibre types are
regionalised within muscles and which the
functional implications are of such arrangements. Below, some of the possible
alternative (and not mutually exclusive)
hypotheses are discussed in the light of
the present findings.
Stereotyped patterns vs. inter-species differences: how generalised are the mechanisms underlying fibre type regionalisation?
The two main aspects of regionalisation
are potentially independent: muscles
might, for instance, conceivably have a
high degree of area- and a low degree of
vector-regionalisation (e.g. a restricted
type I fibre region with a perfectly central localisation) or vice versa. Still, in all
the three animal species, muscles with a
high degree of area regionalisation also
tended to display a high degree of vectorregionalisation. Thus, it seems likely that
there are causal links between these two
aspects of regionalisation. A strong “regionalising influence” apparently tends to
cause the target fibres to be markedly
clumped together in a “regionalising” direction. In the rat, these influences were
largely directed toward the limb centre
and stronger for superficially placed muscles than for those close to the limb core
92
(Wang & Kernell, 2001). This is probably also largely true for corresponding
muscles in the mouse and rabbit. For homologous muscles across the three species, the direction of vector regionalisation was co-varying in a similar way
(Fig.5B) and, at least for mouse vs. rat,
this was true also for the degree of eccentricity (MLD parameter, Table 4). Thus,
in all the three animal species, general
transverse aspects of regionalisation
might have been primarily caused (e.g.
during embryological development) by a
gradient of a “slow morphogen” originating from deep within the limb (or alternatively, a gradient of “fast morphogen”
originating from the surface) (Condon et
al. 1990).
Although the present number of compared animal species is limited, it is intriguing to note that some aspects of the
muscle composition and regionalisation
seemed to co-vary with animal size. The
relative size of the type I fibre region was
ranked mouse < rat < rabbit and the opposite was true for the relative degree of
eccentricity (parameters VL and MLD;
Figs.5-6, Table 4). Humans seem, roughly,
to fit into this ranking scheme: we are
heavier than rabbits and type I and II fibres are typically seen within all muscle
regions, but often in systematically varying proportions (e.g., Johnson et al., 1973;
Lexell et al., 1983; Henriksson-Larsen et
al., 1983), i.e. human limb muscles seem
often to display signs of vector
regionalisation but, apparently, seldom or
never any area regionalisation.
Intuitively, one might expect the relative size of the type I fibre region to grow
in direct relation to the overall density of
the type I fibres. However, although these
two organisational aspects were indeed
FIBRE TYPE REGIONALISATION
correlated (Fig.6), there was apparently
not a direct causal linkage between them:
also for muscles with the same density of
type I fibres the type I fibre regions were
ranked rabbit > rat > mouse. Furthermore,
our results do not show the existence of
simple and consistent relationships between animal size and type I fibre density: for a group of homologous muscles,
mice generally had low densities of type
I fibres while no significant differences
in this respect were found between rats
and rabbits (Table 4).
With regard to the longitudinal aspects
of fibre type regionalisation, species differences were clearly present (Table 3,
Fig.3). Only in the rat was a consistent
proximo-distal decrease of type I fibre
density seen for all the five muscle species that were studied from this point of
view. Three of the muscle species behaved
similarly in rats and mice (ED, GM, and
PE) whereas only one of the muscles
showed a proximo-distal type I fibre decrease in the rabbit (TA) (Table 3). Thus,
for this aspect of regionalisation, there
was no strong evidence for a generalised
scheme.
On the whole, the various inter-species differences in type I fibre
regionalisation (Figs.3, 5-6; Tables 3-4)
seem too large to support a view that regionalised fibre type distributions in
hindlimb muscles might mainly reflect
embryological strategies for building
mixed-composition muscles (i.e. that regionalised type I fibres might represent
“vestiges of the original muscle primordium”; cf. discussions of Narusawa et al.
1987; Wang & Kernell, 2000; Wang &
Kernell, 2001). Hence, fibre type
regionalisation is likely to represent a
property giving some kind of functional
advantage for the animal.
Regional muscle organisation and functional requirements.
Several, not mutually exclusive, factors
have been mentioned in previous publications that might often make it functionally advantageous to keep the slow type I
fibres accumulated in deep rather than in
more superficial muscle regions. These
factors include:
(1). Conservation of heat: also under
relatively resting conditions the type I fibres are well circulated and a superficial
localisation would then, in warm-blooded
animals, lead to relatively large heat losses
(Loeb, 1987).
(2). Thermal balance and dynamic
adaptation of speed and power: during
strong shortening contractions the central
portions of a muscle would be expected
to become more heated-up than superficial (subcutaneous) muscle regions. Muscle fibres increase their shortening speed
and power as they become warmer (e.g.,
Bergh & Ekblom, 1979; Ranatunga, 1982;
Sargeant, 1987). Thus, the contribution of
the slow type I fibres to intense shortening contractions of a muscle might become more effective if they were localised in deep rather than in more superficial muscle portions (Wang & Kernell,
2000).
(3). Biomechanical advantage: due to
architectural features of the limb a superficial intralimb position might, for instance, confer biomechanical advantages
for shortening contraction of fast fibres
(e.g. providing longer moment arms).
Further experimental studies are
needed for analysing the potential importance of these various possibilities. The
present results provide a starting point for
such an analysis in rabbits, rats and mice.
93
CHAPTER 5
Due to their greater surface / volume ratio, thermal factors (points 1-2) would be
expected to be more critical in small than
in larger animals. Therefore, keeping type
I regions restricted (and deep) might provide greater functional gains for mice and
rats than for rabbits. For similar reasons,
particularly in small animals like rats and
mice, proximal deep limb portions might
show significantly less heat loss and, perhaps, a more efficient activity-dependent
heating-up than that of more distal deep
limb portions (small distal muscle mass,
more remote from central body “core”).
If so, this might motivate the common
accumulation of type I fibres proximally
rather than more distally within many of
the mouse and rat muscles. For the rabbit
with its larger muscles, such proximodistal differences in fibre type organisation might have smaller functional effects
and, hence, be less essential to implement.
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