Archs oral Biol. Vol. 32. No. II, pp. 833-841, 1987 ooo3-9949187 s3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright 0 1987 Pcrgamon Journals Ltd

ENZYME-HISTOCHEMICAL DIFFERENCES IN FIBRE-TYPE BETWEEN THE HUMAN MAJOR AND MINOR

ZYGOMATIC AND THE FIRST DORSAL INTEROSSEUS MUSCLES

P. STAL I P.-O. ERXSSON,‘~~ A. ERIKSSON’ and L.-E. THORNELL’

Departments of lAna;omy and *Clinical Oral Physiology and the ‘Institute of Forensic Medicine, University of UmeP, S-901 87 UmeP, Sweden

Summary-Human masticatory muscles, innervated by the trigeminal nerve, differ in tibre-type com- position from limb and trunk muscles, but the anterior and the posterior belly of the human digastric muscle, innervated by the trigeminal and facial nerves, respectively, do not. The major and minor zygomatic muscles from adult males, which originate from the second branchial arch and are supplied by the facial nerve, were analysed enzyme-histochemically and compared with the first dorsal interosseus hand muscle, which has spinal innervation and, like the masticatory and facial muscles, a large cortical representation. Both zygomatic muscles had a marked predominance of type II fibres, the minor one having the largest proportion (89.1 per cent) ever reported in human skeletal muscle. Besides type I, IIA, IIB. and a few type IIC fibres, there was a large group with an ATPase reaction at pH 4.6, between that of type IIA and type IIB, and termed IIAB. This tibre-type profile may reflect a special isomyosin composition. Type I and II tibres were of about equal diameter, corresponding to that of type I fibres in the masticatory muscles. Individual and intra-muscular variability in fibre size and shape was considerable. ‘Ilte unusually high frequency of type II fibres in the zygomatic muscles suggests that they have fast-contraction properties and relatively large motor units, and therefore are poorly adapted to finely-graded movements. The absence of muscle spindles supports this view. The hand muscle had a chequer-board pattern of type I, IIA and IIB fibres, similar to that of large limb and trunk muscles, with no difference between its two heads. Its predominance of type I fibres, and the occurrence of spindles indicates a capacity for finely-graded activity and resistance to fatique. These differences between the facial, masticatory, digastric and hand muscles imply that functional requirements are of greater importance for muscle differentiation than embryologic origin and nerve supply.

lNTRODtJCTION

Human jaw muscles have special fibre-type com- positions, different from limb and trunk muscles (Ringquist, 1974; Eriksson et al., 1982; Eriksson and Thomell, 1983). Thus, in masticatory muscles, inner- vated by the trigeminal nerve, there are significant differences in the proportion and size of both type I and the generally smaller typ II fibres (low and high ATPase activity at alkaline pH, respectively) not only between different muscles, but also within a muscle. Moreover, fibres with a moderate ATPase activity at pH 9.4, ATPase-IM fibres, are part of the normal population. In contrast, the anterior and posterior bellies of the digastric, innervated by the trigeminal and facial nerves respectively, have a histochemical enzyme profile similar to that of limb and trunk muscles, i.e. a mosaic pattern of type I and type II fibres of about equal diameter (Eriksson et al., 1982). As enzyme-histochemical characteristics are posi- tively correlated with contractile properties (reviewed by Burke, 1981; Schmalbruch, 1985). the enzyme profile of human jaw muscle suggests functional specialization, with functional demands rather than specific nerve supply primarily influencing the fibre-type pattern.

To gain further knowledge of the structural basis for normal oro-facial function, and of the influence of embryological origin and ‘innervation on the fibre-type composition, we have now investigated by

enzyme-histochemistry two facial muscles, the major and minor zygomatic muscles. Both originate from the second branchial arch and are supplied by the seventh cranial nerve (Hamilton, Boyd and Moss- man, 1957). We have compared these with the first dorsal interosseus muscle which, like jaw and facial muscles, has a large cortical representation (Chusid, 1976) and is involved in control of fine movements. This hand muscle has been used before as a reference in physiological studies of masticatory muscles (Yemm, 1977; Stalberg et al., 1986).

LMATERIALS AND METHODS

Muscle specimens

Specimens from the middle part of the major and minor zygomatic and first dorsal interosseus muscles of five healthy adult male cadavers (mean age 25.8, range 18-36) were examined.

Quantitative study was made on two to seven randomly-selected areas in one (first dorsal inter- osseus, subject no. 2) or two specimens from each muscle. The subjects had normal intermaxillary re- lations and a full or nearly full dentition in normal occlusion. In an extended study of the medial and lateral head of the first dorsal interosseus muscle, two more subjects (aged 36 and 41) were included. A total of seven hand muscles were analysed. For com- parison, samples from the masseter and the biceps brachii muscles were collected. The specimens were

08 22 ‘l--E 833

834 P. ST&. et al.

obtained one to three days post mortem, a delay which does not hamper reliable fibre typing (Eriksson et al., 1980). The investigation was approved by Socialstyrelsen, The National Board of Health and Welfare, Stockholm, Sweden.

Enzyme-histochemical methods

Specimens were mounted for transverse sectioning in OTC compound (Tissue Tekz, Miles Laboratories Naperville, Illinois, U.S.A.) and frozen in liquid propane chilled with liquid nitrogen. Serial cross sections, 1Opm thick, were cut in a cryostat micro- tome at -20°C and stained for the demonstration of myofibrillar ATPase (EC 3.6.1.3) at pH 9.4 after alkaline @H 9.4; Padykula and Herman, 1955) and acid (pH 4.6 and 4.3) pre-incubations (Brooke and Kaiser, 1970; Dubowitz, 1985). For the demonstra- tion of oxidative activity, the mitochondrial enzyme NADH-TR (nicotine amide adenine dinucleotide tet- razolium reductase, EC 1.6.99.3) was assayed (Scar- pelli, Hess and Pearse, 1958 as modified by Nystrc)m, 1968). In addition, cell borders and nuclei were assessed by a modified Gomori trichrome stain (Engel and Cunningham, 1963).

Fibre typing

This was based on staining for myofibrillar ATPase after alkaline and acid pre-incubations. At pH 9.4, lightly-stained fibres with low ATPase activity were termed type I, and darkly-stained fibres with high activity, type II. After acid pre-incubations, type II fibres were subdivided into type HA (unstained, inhibited reaction at both pH 4.6 and 4.3), type IIB fibres (unstained, inhibited reaction at pH 4.3) and type IIC fibres (also stained at pH 4.3; Dubowitz, 1985). Fibres with a staining in between that of type I and II at pH 9.4 were termed ATPase intermediate fibres (ATPase-IM; Ringquist, 1974). Muscle-spindle occurrence was also recorded.

Morphometric analyses

Sections stained for ATPase at pH 9.4 were posi- tioned in a co-ordinate system by means of the millimetre scales of a light-microscope (Leitz-Dialux- 20) stage. Co-ordinates were chosen randomly and two or more areas were photographed for each muscle (Table 1). The zygomatic muscles were classified and measured on photographs at 250 times magnification, and the first dorsal interosseus at 100 times magnification. All fibre-diameter measurements were done by the same person with a Zeiss TGZ 3 particle-size analyser. The equivalent-area diameter was measured: the diameter of a circle having the same area as the myofibre cross-sectional area. For each subject, the data from all samples were pooled. The percentage of overall cross-sectional area formed by various fibre types was also calculated.

Statistical analysis

The precision of estimation of fibre-type propor- tions was expressed as the standard error of the mean (SE). The variability of fibre-type proportion between subjects, and the variability in fibre diameter within and between subjects, were expressed by the standard deviation (SD). Analysis of variance was used to test the hypothesis of no difference in relative frequency

and diameter between different fibre types and be- tween different muscles. The null hypothesis was rejected at the 0.01 level of significance. Variation in fibre diameter for different fibre types was also ex- pressed as the coefficient of variation (CV = standard deviation x lOOO/mean fibre diameter).

RESULTS

Muscle jibre types

Zygomatic muscles. In both muscles, type I, IIA, IIB and IIC fibres were distinguished. However, the ATPase staining pattern of the type II fibres differed from that of the other muscles in having a wide and continuous spectrum of stainability from dark to light at pH 4.6. Type II fibres with moderate staining at pH 4.6, in between that of the light HA and the dark IIB fibres, were termed type IIAB (Plate Fig. 1). Further analysis of the sensitivity of type IIAB fibres to acid pre-incubations showed different degrees of strong staining at pH 4.7 and a reversed pattern at pH 4.5.

After staining for NADH-TR the type I fibres had different degrees of moderate to very strong staining. Type II fibres had different degrees of moderately intense staining, with type IIA and IIAB having moderate to strong, and type IIB weak to strong intensity. In general, the type II fibres had a higher oxidative capacity than in the masseter and limb muscles (Plate Figs ID and 2D).

First dorsal interosseus muscle. Myofibrillar ATP- ase reaction at various pH demonstrated type I, type IIA, type IIB and a few type IIC and ATPase-IM fibres. In sections stained for NADH-TR, type I fibres had strong to very strong activity, and type II fibres different degrees of weak to strong activity (Fig. 2).

Masseter and biceps brachii muscles. Histochemical enzyme profiles were in accord with those previously reported (Eriksson et al., 1982; Eriksson and Thom- ell, 1983; Dubowitz, 1985). In the masseter, type I, comparatively small type IIB, type IIC, and ATPase- IM fibres were distributed in a heterogeneous pattern (Plate Fig. 3B). In the biceps brachii, type I, HA, IIB and rare IIC fibres of about the same diameters were distributed in a chequer-board pattern (Fig. 3).

Distribution, Relative Frequency and Diameter of Fibre Types

Zygomatic muscles

In general the fibre types were evenly intermingled over the muscle cross-section. The majority had a rounded rather than polygonal contour. Apart from individual variations, there was a marked intra- muscular variability in fibre diameter and shape (Plate Fig. 4). Variability in fibre diameter was es- pecially prominent in the minor zygomatic (see be- low). For both muscles, subject no. 2 had the lowest frequency of type I fibres, and subject no. 5 the highest (Table 1).

Major zygomatic muscle

Frequency. The type II fibre population (mean 74.9 per cent) was significantly larger than the type I (25.1 per cent). Type IIAB fibres (mean 48.1 per cent)

Tab

le

I. R

elat

ive

freq

uenc

y (p

er c

ent)

of

dif

fere

nt

fibr

e ty

pes

in t

he m

ajor

zy

gom

atic

(M

aj Z

), m

inor

zy

gom

atic

(M

in

Z)

and

the

firs

t do

rsal

in

tero

sseu

s (F

DI)

m

uscl

es

of f

ive

mal

e ad

ults

Subj

ect

no.

Maj

Z

Mitt

Z

FDI

Gro

up

valu

es

I 2 3 4 5 I 2 3 4 5 : 3 4 5

Typ

e I

Typ

e II

A

Typ

e II

AB

T

ype

IIB

T

ype

IIC

T

ype

AT

Pase

-IM

n Pe

r ce

nt

SE

Per

cent

SE

Pe

r ce

nt

SE

Per

cent

SE

Pe

r ce

nt

SE

Per

cent

SE

377

20.4

2.

08

22.0

2.

14

53.3

2.

57

3.7

0.97

0.

5 0.

37

229

19.2

2.

61

6.6

1.64

69

.9

3.04

I.

21

0.9

0.62

18

2 22

.0

3.08

20

.3

2.99

51

.6

3.71

:,:

255

27.5

2.

80

5.5

1.43

52

.2

3.13

14

:1

1.69

0.

5 0.

55

2.18

0.

8 0.

55

217

36.4

3.

27

SO.2

3.

40

13.4

2.

32

427

12.2

1.

58

19.4

I .

92

48.5

2.

42

19.9

I .

93

247

2.8

1.06

0

0 93

.5

1.57

3.

6 I.

19

211

12.3

2.

27

12.3

2.

27

63.5

3.

32

10.0

2.

07

1.9

0.94

43

9 6.

6 I.

19

12.3

1.

57

SO.8

2.

39

30.3

2.

20

348

20.4

2.

16

72.7

2.

39

6.9

1.36

47

3 63

.4

2.22

29

.4

2.10

5.

3 1.

03

0.2

0.21

1.

7 0.

59

230

32.6

3.

10

59.6

3.

24

4.3

1.35

3.

5 I.

21

641

54.8

1.

97

44.5

I.

96

0.5

0.27

0.

3 0.

22

879

70. I

1.

54

20. I

1.

35

4.2

0.68

5.

3 0.

76

0.2

0.16

64

2 75

.0

I.71

24

.8

1.70

0

0 0.

2 0.

16

0.2

0.15

i SD

f

SD

f SD

.f

SD

.?

SD

.f SD

Maj

Z

25.1

7.

1 20

.9

18.1

48

. I

20.8

5.

4 5.

3 0.

5 0.

3 M

in Z

10

.9

6.7

23.4

28

.5

52.6

31

.2

12.8

12

.4

0.4

0.8

FDI

59.2

16

.7

35.7

16

.2

2.8

2.6

I.9

2.4

0.5

0.7

Mam

eter

+ 62

.5

10.0

2.

1 4.

7 26

.7

8.8

2.1

2.6

6.0

5.6

Bic

eps*

41

.8

8.5

25.8

8.

1 31

.9

12.4

0.

5

Mea

n va

lue

(a),

SE

and

SD

. n

=

Tot

al

num

ber

of f

ibre

s.

*Fro

m

Eri

ksso

n (1

982)

.

Subj

ect

no.

Tab

le

2. D

iam

eter

(c

m)

of t

ibre

ty

pes

in t

he m

ajor

zy

gom

atic

(M

aj

Z),

m

inor

zy

gom

atic

(M

in

Z)

and

the

firs

t do

rsal

in

tero

sseu

s (F

DI)

Typ

e 1

Typ

e II

A

Typ

e B

AB

T

ype

IIB

T

ype

IIC

T

ype

AT

Pase

-IM

n f

SD

.f SD

.f

SD

,?

SD

.f SD

f

SD

Maj

Z

I 37

7 43

.5

9.1

46.6

8.

3 47

.3

7.4

47.1

12

.0

40.0

14

.1

2 22

9 39

.5

12.2

35

.3

5.2

39.0

6.

6 33

.8

5.2

35.0

7.

1 3

182

37.7

8.

6 38

.1

10.0

46

.5

11.1

49

.0

5.7

50.0

4

255

27.8

5.

1 27

.9

4.3

32.6

7.

2 38

.6

9.6

40.0

14

.1

5 21

7 39

.9

10.4

43

.7

9.1

48.3

7.

6 M

in 2

I

427

34.8

12

.4

37.8

9.

9 38

.8

12.0

38

.7

11.5

2

247

42.9

7.

6 41

.6

11.3

41

.1

9.3

3 21

1 28

.1

8.0

45.4

12

.1

41.9

12

.0

51.9

14

.7

32.5

5.

0 4

439

16.6

8.

1 22

.8

7.6

26.1

9.

7 22

.0

10.5

5

348

31.8

7.

0 35

.0

8.8

33.8

6.

5 FD

I I

473

65.1

12

.4

88.3

12

.5

86.4

10

.4

80.0

48

.8

11.3

2

230

62.4

7.

9 69

.7

11.6

61

.0

11.0

65

.0

9.3

3 64

1 65

.0

12.5

72

.5

12.7

76

.7

5.8

65.0

7.

1 4

879

54.9

11

.4

85.0

18

.6

80.8

13

.2

72.8

17

.5

70.0

42

.4

5 64

2 66

.6

14.4

78

.4

13.8

80

.0

70.0

Gro

up

valu

es

i SD

R

ange

.f

SD

Ran

ge

.f SD

R

ange

.?

SD

R

ange

.f

SD

Ran

ge

,f SD

R

ange

Maj

2

37.6

6.

2 l&

60

38.3

7.

3 IO

-70

42.7

6.

8 20

-80

42.1

7.

2 20

-70

41.2

6.

3 30

-60

Min

2

30.8

9.

7 iw

xt

35.3

9.

4 IO

-60

36.4

6.

6 l&

90

38.4

12

.4

I@-9

0 32

.5

3tG

io

FDI

62.8

4.

7 20

-110

78

.8

7.9

20-1

20

76.0

13

.3

4&10

0 74

.9

6.3

40-1

00

63.4

10

.1

30-1

00

Mas

.set

er*

43.9

4.

8 It

HO

39

.1

IO-6

0 24

.5

3.6

I@60

29

.1

4.6

IO-5

0 31

.2

3.8

IO-6

0 B

icep

s*

66.2

9.

5 4t

HO

8 76

.4

15.3

40

-120

71

.1

15.2

40

-110

67

.8

40-8

0

Mea

n va

lue

(a),

SD

and

ra

nge.

n

= T

otal

nu

mbe

rs

of t

ibre

s m

easu

red.

*F

rom

E

riks

son

(198

2).

Fibrc composition in human facial and hand muscles 837

predominated in four subjects (range 51.6-69.9 per cent), and type IIA in one ( no. 5, 50.2 per cent). The mean frequency of type IIA fibres was 20.9 per cent and of type IIB, 5.4 per cent. Only occasional type IIC fibres were found (mean 0.5 per cent; Table 1).

Diameter. Mean values for type I, IIA, IIAB and type IIB fibres were 37.6, 38.3, 42.7 and 42.1 pm, respectively, with no statistically significant differences (Table 2). However, in all subjects, type I fibres had the smallest mean diameter.

Relative cross-sectional area. Mean values for the estimated overall cross-sectional area were largest for the type IIAB fibres, 51.3 per cent; type I, IIA, IIB and IIC fibres occupied 21.5, 19.9, 6.8 and 0.5 per cent, respectively (Text Fig. 5).

Minor zygomatic muscle

Frequency. The type II fibre population (mean 89.1 i per cent) was significantly larger than the type I. Type IIAB fibres (mean 52.6 per cent) predominated in the same four subjects (range 48.5-93.5 per cent) in which the major muscle had a predominance of this type, and the type IIA in one (no. 5, 72.7 per cent). Type IIA, IIB and type I fibre proportions were 23.4, 12.8 and 10.9 per cent, respectively. Type IIC fibres (mean 0.4 per cent) were found in only one subject (no. 3, Table 1).

Diameter. Type I, IIA, IIAB and IIB fibre di- ameters ranged between 30.8 and 38.4pm without any statistically significant differences (Table 2). The lowest mean value was for type I fibres.

Relative cross-sectional area. The type IIAB fibres occupied the largest area, 54.7 per cent, with the type I, IIA, IIB and IIC fibres occupying 7.9, 23.9, 13.2 and 0.2 per cent, respectively (Fig. 5).

First dorsal interosseus

Both heads of the muscle had a chequer-board pattern of type I and type II fibres.

Frequency. Type I fibres (mean 59.2 per cent)

Maj Z Min Z ._ . . .:. .,., .:._ 6.8 ;:~::::.:~;~,;;:~~;~;;.

per :_. : : :.I.. ? P?,-?, .,. ,_ CUM :: ,.,. ;,. ‘_ :_. ;‘:,. ;,

; : : :; ,’ Q ; _. _‘_., 0.2

Q5 2,. 5 . . . . $.y.:_::.:.:. w

Per Percent 19.9 ‘. ipw cent?

cent cent

.$qqP

Fig. 5. Percentage of overall muscle fibre cross-sectional area occupied by different tibre types in the major zygomatic (Maj Z), minor zygornatic (Min Z) and the first dorsal interosseus (FDI) muscles. Mean fibre area from five adult

male muscles.

significantly outnumbered the others. Type IIA fibre frequency (35.7 per cent) was significantly greater than that of type IIB (2.8 per cent), IIC (I .9 per cent) and ATPase-IM fibres (0.5 per cent; Table 1).

Diameter. Mean diameter did not differ significantly between the type I (62.8pm), IIA (78.7 pm), IIB (76.0pm), IIC (74.9 pm) and ATPase-IM fibres (63.4 pm; Table 2).

Relative cross-sectional area. Type I fibres occupied 39 per cent and type IIA, 36.5 per cent of the estimated cross-sectional area. The corresponding values for type IIB, type IIC and ATPase-IM fibres were 14.9, 7.9 and 1.7 per cent, respectively (Fig. 5).

There was no significant difference in fibre-type frequency or diameter between the two heads of this muscle.

Variability in jibre diameter

To further evaluate this, the coefficient of vari- ation, CV, was estimated for each fibre type in the facial and hand muscles (Table 3). According to Dubowitx (1985), a CV higher than 250 in limb and trunk muscles demonstrates abnormal variability in fibre size. In the minor zygomatic muscle, all fibre types had values higher than 250, whereas in the major, only type I fibres had a high value (237). For type IIAB fibres, the difference between the two facial muscles was statistically significant. In the first dorsal interosseus muscle, CV values ranged between 154 and 186.

Muscle-spindle occurrence

No muscle spindles were observed in the two zygomatic muscles; all first dorsal interosseus muscles contained two to seven spindles.

DISCUSSION

Both zygomatic muscles had a marked prudom- inance of type II muscle fibres; no human muscle has been reported to have a type II fibre content as high as we found in the zygomatic minor. Furthermore, the zygomatic muscles contained a special type II fibre, here termed type IIAB, with ATPase staining at pH 4.6, in between type IIA and IIB. This histo- chemical enzyme profile differs from that of human masticatory and digastric muscles (Eriksson et al., 1981, 1982; Eriksson and Thomell, 1983), and from that of limb and trunk muscles (Johnsson et al., 1973; Hiiggmark and Thorstensson, 1979; Dubowitz, 1985).

When found in human limb muscles, the type IIAB fibre is held to represent a transitional state related to the amount and type of usage (Andersen and Hen- riksson, 1977; Ingjer, 1979; Staron, Hikida and Hag- erman, 1983). However, the large number of type IIAB fibres in the zygomatic muscles suggests that these are part of the normal population, and for permanent use, rather than undergoing trans- formation. The high proportion of type IIAB fibres may reflect a special isomyosin structure for the zygomatic muscles; biochemical investigations in progress indicate that this is so.

The predominance of type II fibres in the zygo- matic muscles is in line with the findings of Schwart- ing et al. (1982). However, in their biopsies from the

838 P. ST.&L et al.

Table 3. The coefficient of variation (CV) of fibre diameter of fibre types in the major zygomatic (Maj Z), minor zygomatic (Min Z). the first dorsal interosseus (FDI)

muscles and human jaw muscles

Type I Type IIA Type IIAB Type IIB

Muscles f l SD i SD .f SD f SD

Maj Z 237 47 189 47 188 38 194 69 Min 2 FDI Massetert:

Superficial portion Anterior part Posterior part

Temporalt: Superficial portion Anterior part Posterior part

Medial pterygoidt: Anterior part Posterior part

Digastrict:

283 136 278 37 286 65 320 108 186 35 175 28 154 31

222 50 270 114 219 60 294 50

258 I8 307 69 242 47 315 96

260 27 242 34 268 48 251 30

Anterior belly 177 27 167 20 105 34 Posterior belly 187 17 170 33 200 35

*Mean and SD values calculated for five adult males. tcalculated from data given in Eriksson and Thomell (1983) and Eriksson er al.

(1982).

zygomatic major muscle they demonstrated, by acid pre-incubations at pH 4.5 and 4.3, type IIA but no type IIB fibres. Our type II subtyping, which reveals type IIB fibres in both zygomatic muscles, was made after pre-incubation at pH 4.6 and 4.3, which are routine stainings (Dubowitz, 1985). At pH 4.5, we found an inhibited enzyme reaction for a portion (type IIAB) of the type II fibre population.

Type II fibres in limb muscles belong to large, high-threshold motor units which contract quickly in brief bursts and are poorly adapted to finely-graded movements (Burke, 1981; Schmalbruch, 1985). The unusually large proportion of type II fibres in the zygomatic muscles therefore suggests that these muscles have fast contraction properties, in agree- ment with the observation that the zygomatic major fires in intermittent bursts (Basmajian, 1978). The absence of muscle spindles supports the view that the zygomatic muscles are not primarily involved in fine motor control. However, the extraocular muscles, active in fine eye movements, are composed mainly of type II fibres. It is frequently stated that these muscles contain few fibres per motor unit. Whether this is correct has been contested by Schmalbruch (1985), who also postulates that, compared with the motor units in limb muscles, the extraocular muscles are better suited to force gradation because the tension in them is small due to the smallness of their fibres. The frequency modulation of force is probably more effective in fast fibres with high tetanic-fusion frequency than in slow fibres with low frequency (Schmalbruch, 1985). The functional significance of the human zygomatic muscle fibre-type profile needs to he further evaluated.

Van Boxtel et al. (1983) found much longer endur- ance in the facial muscles, including the zygomatic major, than in the masseter, in electromyographic studies during static contraction. We found high

oxidative-enzyme activity in type II fibres, indicating a high resistance to fatigue. However, if fibre-type composition is taken as the basis for deducing the fatigue resistance, the masseter, with a predominance of type I fibres (Eriksson, 1982; Eriksson and Thom- ell, 1983) known to have the highest oxidative capac- ity (Burke, 1981), would be best adapted to resist fatique. One explanation of Van Boxtel’s results may be that the zygomatic muscles lack a firm insertion, which may hamper static contraction and the devel- opment of high tension necessary for obstruction of the blood circulation and fatiguability.

As expressed by the coefficient of variation (CV), diameter variability was considerable in both zygo- matic muscles, but was most prominent in the minor one. Here all fibre types exceeded the limit for abnormal variability in size which, for limb and trunk muscles, is proposed to indicate a pathological pro- cess (Dubowitz, 1985). Abnormal CV values for fibre diameter are also found in the masticatory but not in the digastric muscles of normal subjects (Table 3). It is well known that muscle fibre diameter is related to the use of the motor units. Whereas exercise may increase the cross-sectional area, disuse will decrease it (reviewed by Saltin and Gollnick, 1983). As our specimens were from normal subjects, the marked intra- as well as intermuscular variation indicate specific functional properties for the human facial muscles rather than a pathological process.

To evaluate the possibility that the marked di- ameter variability was related to tapering of fibres when they are fused to connective or tendinous tissue, we analysed (data not shown) longitudinally- sectioned zygomatic muscle fibres. In addition to the staining described above, these longitudinal sections were stained by the van Gieson method (routine connective-tissue staining; Dubowitz, l985), and for acetylcholinesterase (Koelle and Friedenwald, 1949),

Fibre composition in human facial and hand muscles 839

to detect myomuscular and myotendinous junctions (Pierobon-Bormiolo and SchiafBno, 1977). NO evi- dence of tapering fibres was found.

The zygomatic muscles had both similarities and differences in histochemical enzyme profile when compared with the digastric. The similarity is the predominance of type II fibres; the difference is that they had a special myosin ATPase reactivity with numerous type IIAB fibres, and a more irregular pattern of fibre size and shape. These three muscles have different patterns of representation in the brain stem. The zygomatic muscles are represented in the facial nucleus (Szentagothai, 1948; Shohara and Sakai, 1983), the anterior digastric in the ventro- medial division of the trigeminal nucleus (accessory trigeminal motor nucleus; Szentagothai, 1949; Miz- uno et al., 1981; Rokx, Jiich and Willigen, 1985) and the posterior digastric in the accessory facial nucleus (Szentagothai, 1948; Matsuda et al., 1979). The myo- topical arrangement of motor neurones, together with the histochemical enzyme profile, imply that functional requirements, i.e. specific movements, are of greater importance for muscle differentiation than origin and nerve supply.

In contrast to the zygomatic muscles, the first dorsal interosseus was similar in fibre-type profile to large limb and trunk muscles (Dubowitz, 1985), and contained spindles. Mimer-Brown, Stein and Yemm (1973) proposed that in this muscle the number of small, slow motor units with low-twitch tension greatly exceeds the number of units with high-twitch tension and rapid contraction. This is in line with our finding of a predominance of type I fibres, suggesting an ability for fine, continuous motor activity. Fur- thermore, a macro-EMG technique (Stalberg et al., 1986) has shown that low-threshold motor units in this muscle are somewhat smaller than motor units in large arm and leg muscles, and of about the same size as those in the masseter and temporal muscles (St&l- berg ef al., 1986). The similarity in fibre-type profile between the two portions of the hand muscle suggests fairly equal functional capacities.

Acknowledgements-We wish to thank MS Inga Johansson and MS Lena Siiderberg for skilful technical assistance and Dr Barbara Kay Grove for English revision. Financial support was provided by the Swedish Medical Research Council (3934 and 6874) and the Swedish Dental Society.

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Plate 1

Fig. I. Serial cross-sections from the major xygomatic muscle stained for the demonstration of myofibrillar ATPase at pH 9.4 (A), pH 4.6 (B), pH 4.3 (C) and for NADH-TR (D). Fibrc types I, IIA, IIAB and

IIB have been labelled. x 200

Fig. 2. Serial cross-sections from the first dorsal interosseus muscle stained for ATPase at pH 9.4 (A), pH 4.6 (B). pH 4.3 (C) and for NADH-TR (D). Fibre types I, HA. IIB and IIC have bum labelled. x 200

Fig. 3. Cross-sections from the major xygomatic (A). the masseter (B), the biceps brachii (C) and the first dorsal interosseus (D) muscles of the same subject stained for myofibrillar ATPase at pH 9.4. Note the

differences in fibre diameter between the facial, masticatory and limb muscles. x 200

Fig. 4. Different areas (A, B and C) within the same cross-section from the major ygomatic muscle demonstrating the considerable variability in shape, size and packing of fibres. ATPase at pH 9.4. x 256

_--. . ._-- _. -- Plats I