Enzyme-histochemical and morphological characteristics of muscle fibre types in the human buccinator and orbicularis oris

S3.00f0.00 0003-9969/90 Copyright 0 1990 Pergamon Press plc

Archsoral Bid. Vol. 35,No. 6,pp.449458, 1990 Printed in Great Britain. All rights reserved

ENZYME-HISTOCHEMICAL AND MORPHOLOGICAL CHARACTERISTICS OF MUSCLE FIBRE TYPES IN THE HUMAN BUCCINATOR AND ORBICULARIS ORIS P . STAL >'* P.-O. ERIKSSON,“’ A. ERIKSSON~ and L.-E. THORNELL’ Departments

of ‘Anatomy

and ‘Clinical Oral Physiology and the 31nstitute University of Umea, S-901 87 Umei, Sweden

(Received 28 September

1989; accepted 29 November

of Forensic

Medicine,

1989)

Summary--Human masticatory muscles, originating from the first branchial arch and innervated by the trigeminal nerve, have a fibre composition distinct from that of limb and trunk muscles. The zygomatic muscles, originating from the second branchial arch and innervated by the facial nerve, differ in fibre composition from either the masticatory or the limb and trunk muscles. To elucidate further the structural basis for function, and the influence of embryological origin and innervation on oro-facial muscles, the buccinator and orbicularis oris muscles, which originate from the second branchial arch and are innervated by the facial nerve, were investigated. Like the masticatory and zygomatic muscles, they have a large representation in the cerebral cortex. Both muscles were composed of type I, type IIA and a few type IIC libres of about equal diameter. However, the type I fibres had a different myofibrillar ATPase reaction from those in masticatory, zygomatic, limb and trunk muscles; this was a moderate to strong staining at pH 9.4, indicating a special isomyosin composition. Whereas the buccinator was composed of 53% type I libres, the or’oicularis oris had a 71% predominance of type II fibres. In both muscles, the mean fibre diameter and its marked intramuscular variability were similar to earlier findings in the zygomatic muscles. No muscle spindles were found. The large number of type I fibres in the buccinator implies a capacity for endurance during continuous work at relatively low levels of force. The predominance of type II fibres in the orbicularis oris indicates that it is built up of fast-twitch motor units, related to properties such as rapid acceleration and high speed during intermittent oro-facial movements. The similarities and differences in fibre-type composition between the facial, masticatory and limb muscles imply that specific functional demands are of greater importance for muscle differentiation than embryological origin and nerve supply.

Key words: enzyme-histochemistry,

fibre types, human

INTRODUCTION There is convincing evidence that the enzyme histochemical characteristics of muscle fibres correlate with their contractile: properties (reviewed by Burke, 1981; Schmalbruch. 1985). Thus, histochemically classified type I fibres belong to slow-twitch motor units and histochemically classified type II fibres belong to fast motor units. In human limb and trunk muscles, type I and type II fibres of about equal diameter are evenly distributed in a checker-board pattern, and the type II fibres can be subgrouped into type IIA, IIB and IIC (Dubowitz, 1985). Human masticatory muscles, in contrast, are characterized by many type I fibres of different sizes, small type II fibres and a relatively large proportion of type ATPase-IM (intermediately stained at pH 9.4) and IIC fibres (Ringqvist, 1974; Eriksson et al., 1981, 1982; Eriksson and Thornell, 1983). Such ATPase-IM and IIC fibres are ra.re or non-existent in adult limb

*Address correspondence to: P. Stal, Department of Anatomy, University of Umea, S-901 87 Umel, Sweden. nicotinamide adenine Abbreviations: NADH-TR, dinucleotide tetra-zolium reductase.

oa 35,-

449

jaw, muscle,

masticatory,

oro-facial.

muscles (MacDougall et al., 1980; Dubowitz, 1985). In an earlier investigation of the major and minor zygomatic muscles, we found that their fibre composition was different from that of either the masticatory or the limb and trunk muscles (Stil et al., 1987). Both zygomatic muscles had a marked predominance of type II fibres of about equal mean diameter and contained a large proportion of fibres with a staining intermediate between type IIA and type IIB, termed type IIAB. Moreover, intramuscular and inter-individual variability in fibre size and shape was considerable. We thus proposed that the enzyme profile of human jaw and facial muscles is probably related to their specialized functional demands (Eriksson et al., 1982; Eriksson and Thornell, 1983; St&l ef al., 1987). The orbicularis oris and the buccinator muscles, both originating from the second branchial arch, are innervated by the facial nerve and have a large cortical representation, just like the trigeminal nerve innervating masticatory muscles (Hamilton, Boyd and Mossman, 1957; Kandel and Schwartz, 1985). Our main objective was now to investigate the orbicularis oris and the buccinator to elucidate the structural basis for normal oro-facial function, and the influence of embryological origin and innervation

P. SrdL et al.

450

on the fibre-type composition. Very little is known about the fibre-type composition of these muscles (Schwarting et al., 1982, 1984; Haapak, Burggasser and Grubet, 1988). The findings should also be of value as a baseline in evaluating changes in various neuromuscular diseases. MATERIAL Muscle

AND METHOD

specimens

Samples from the superior and inferior portions of the left orbicularis oris and buccinator were taken post mortem from 5 previously healthy adult males (mean age 31 yr, range 15-41). The orbicularis oris specimens were from the intermediate part between the angulus oris and the midline. The buccinator specimens were from the anterior and middle parts of the upper and lower portions of the muscle. The quantitative study was based on the examination of 2 specimens from each muscle portion, except for the orbicularis oris superior in subject No. 2 and orbicutaris oris inferior of subjects Nos 2 and 3, where the analysis was based on one sample from each portion. The subjects had a normal craniofacial morphology and a complete or nearly complete dentition in normal occlusion. Samples from the masseter, the major zygomatic and the biceps brachii muscles were used for comparison. The specimens were obtained l-3 days post mortem, a delay which does not hamper reliable fibre typing (Eriksson ef al., 1980). The investigation was approved by Socialstyrelsen, The National Board of Health and Welfare, Stockholm, Sweden.

muscular and myo-tendinous junctions Bormiolo and SchialIino, 1977).

(Pierobon-

Fibre typing

Classification of fibre types was based on the staining for myofibrillar ATPase after alkaline and acid pre-incubations. Fibres with a low ATPase activity, lightly stained after alkaline pre-incubations at pH 9.4 and or pH 10.3, were termed type I, and fibres with a high activity, darkly stained, were termed type II. After acid pre-incubations, the type II fibre group was divided into type HA (unstained, inhibited reaction, at both pH 4.6 and 4.3), type IIB (unstained, inhibited reaction, at pH 4.3) and type IIC fibres (slightly stained at pH 4.3; Dubowitz, 1985). Morphometric analysis

Sections stained for ATPase at pH 10.3 were positioned in a co-ordinate system by means of the millimetre scales of a light-microscope (Olympus Vanox, model AH-2) stage. Co-ordinates were chosen randomly and 3-6 areas were photographed in each muscle portion. Classification and measurement of fibres was performed on photographs at a magnification of 250 times. All fibre-diameter measurements were done by the same person with a Zeiss TGZ 3 particle-size analyser. The equivalent-area diameter was measured; this being the diameter of a circle having the same area as the myofibre cross-sectional area (Clancy and Herlihy, 1979). For each subject and muscle portion, the data from all samples were pooled. The relative overall cross-sectional area taken up by various fibre types was also calculated.

Enzyme histochemical methods

Statistical analysis

The muscle specimens were mounted for transverse sectioning in OTC compound (Tissue TekH Miles Laboratories. Naperville, IL, U.S.A.) and frozen in liquid propane chilled with liquid nitrogen. With a cryostat microtome, 10 pm serial cross-sections were cut at -20°C. The sections were stained to demonstrate myofibrillar ATPase (EC 3.6.1.3) at pH 9.4 after alkaline (pH 10.3 and 9.4) and acid (pH 4.6, 4.5 and 4.3) pre-incubations (Padykula and Herman, 1955; Brooke and Kaiser, 1970; Dubowitz, 1985). To demonstrate the oxidative activity, a mitochond~al (EC 1.6.99.3), was assayed enzyme, NADH-TR (Nystrom, 196X).A modified Gomori trichrome stain was used to assess the cell borders and nuclei (Engel and Cunningham, 1963). To evaluate the possibility that the marked variability in fibre diameter was related to a tapering of the fibres when they fused to connective tissue within the muscles, longitudinal serial sections were cut and stained by the van Giesson method (Dubowitz, 1985). These sections were also stained to demonstrate acetylcholinesterase

The precision ofestimation of fibre-type proportions was expressed as the standard error of the mean (SE). The variability of fibre-type proportions and fibre diameters for fibre types and muscle portions was 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 types of fibre and between the different muscle portions and muscles. The null hypothesis was rejected at the 0.01 level of significance. The variation in fibre diameter for different fibre types was also expressed as the coefficient of variation (CV = standard deviation x lOOO/mean fibre diameter; Dubowitz, 1985).

(Koelle

and Friedenwald,

1949) and so detect myo-

RESULTS

fibre types Control-masseter, major zygomatic and biceps brachii. The myofibrillar ATPase stainings at differ-

hfuscle

ent pH values were in accord with those reported by Dubowitz (1985) and St&l et al. (1987) (Plate Fig. 1).

Plate 1 Fig. I. Serial cross-sections of the biceps brachii muscle stained for the demonstration of myofibrillar ATPase at pH 10.3 (A), 9.4 (B), 4.7 (C), 4.6 (D), 4.3 (E) and for NADH-TR (F). Fibre types I, IIA and IIB have been labelled. x 230. Fig. 2. Serial cross-sections of the buccinator muscIe stained for the demonstration of myofibrillar ATPase at pH 10.3 (A), 9.4 (B), 4.7 (C), 4.6 (D), 4.3 (E) and for NADH-TR (F). Fibre types I and IIA have been labelled. x 230.

Fibre composition in human oro-facial muscles

451

452

P.

ST.&L

et

Plate 2

a/.

Fibre composition Table

in human

oro-facial

453

muscles

frequency (%) of different fibre types in the superior (Bucc sup) and inferior (Bucc inf) portion of the buccinator muscle

1. Relative

-

Type IIA

Type I Bucc sup

inf

Subject No.

n

%

%

SE

1 2 3 4 5

198 228 441 257 233

49.0 72.8 64.2 87.5 34.3

3.56 2.95 2.29 2.06 3.12

51.0 27.2 35.8 11.3 65.7

3.56 2.95 2.29 2.06 3.12

I

234 306 370 332 244

27.8 43.8 40.5 72.3 41.0

2.93 2.84 2.56 2.46 3.15

72.2 55.6 59.5 26.8 58.6

2.93 2.85 2.56 2.43 3.16

2 3 4 5 Group values (n = 5) Bucl: sup Buc~c inf Buc’cinator muscle

SE

Type IIC %

SE

1.2

0.67

0.7

0.46

0.9 0.4

0.52 0.4

x

SD

.?

SD

.?

SD

61.6 45.1 53.4

20.7 16.4 17.2

38.2 54.5 46.4

21.0 16.8 17.7

0.2 0.4 0.4

0.5 0.4 0.45

Buccinator and orbicularis oris. In both muscles, type I, IIA and a few IIC fibres were distinguished. However, the type I iibres had a different myofibrillar ATPase reaction to those of the masseter and limb muscles; this was moderate to dark staining at pH 9.4. At pH 10.3 all type I fibres were lightly stained (Plate Figs 2 and 3). For NADH-TR, ,the type I fibres showed a wide spectrum of staining, from moderate to very strong, and the type IIA fibres showed different degrees of moderately intense staining. In general, these type II fibres stained more intensely than those in the masseter and limb muscles. Distribution, relative frequency and diameter of jbre types

The different fibre types were, in general, evenly intermingled over the cross-sections. However, in some areas there were clusters, mainly composed of type I fibres. Besides the inter-individual variation, there was a marked intramuscular variability in fibre diameter and shape. In most sections there were also areas with a complicated fibre geometry showing various degrees of both cross- and horizontallyorientated fibres (Plate Fig. 4). Frequency. In the superior portion, type I fibres predominated in 3 :subjects (Nos 2, 3 and 4, range 64.2-87.5%) whilst in the inferior portion, a type I fibre predominance ‘was observed only in one subject (No. 4, 72.3%). Only occasional type IIC fibres were

found (mean 0.4%). There was no significant difference in the frequency of the different fibre types between the superior and inferior parts (Table 1). For subject 4, both the inferior and the superior part had the highest frequency of type I fibres. Diameter. In the superior portion, the mean values for the type I and the type IIA fibres were 33.4 and 43.7 pm, respectively; the corresponding values for the inferior portion were 33.0 and 38.1 pm. When the fibre diameters of the two portions were pooled, the mean diameter for the type I and the type IIA fibres was 33.2 and 41.0 pm, respectively; this was not a signifciant difference (Table 2). The largest mean diameter was in type IIA fibres of subject 4 (63.1 pm) and the smallest in type I fibres of subject 3 (26.2 pm). Relative cross-sectional area. The type I fibres occupied 51.7% of the estimated cross-sectional area; the type IIA and type IIC fibres occupied 46.9 and 0.7%, respectively (Text Fig. 6). The orbicularis oris superior and inferior muscles

The different fibre types in both the superior and inferior portions were, in general, evenly intermingled over the cross-section. Also, this muscle had areas with clusters of fibres, mainly type I. In most samples the majority of the fibres had a rounded rather than a polygonal contour. Both muscles had large intramuscular and inter-individual variations in fibre diameter and shape, and the geometry of the fibre orientation was in places very complicated. Single

Plate 2 Fig. 3. Serial cross-sections of the orbicularis oris muscle stained for the demonstration of myofibrillar ATPase at pH 10.3 (A), 9.4 (B), 4.7 (C), 4.6 (D), 4.3 (E) and for NADH-TR (F). Fibre types I and IIA have been labelled. x230. Fig. 4. Cross-section of the buccinator muscle stained for ATPase, pH 4.6. Note the variability in distribution and size of muscle fibres and the presence of both cross-sectioned and horizontally fibres. To the left in this figure a large fascicle with mainly type I fibres (dark stained) is seen To the right, another large fascicle contains a mixture of type I (dark) and type II (light) fibres arrow). x 60. Fig. 5. Cross-section

oris muscle. Note the marked intramuscular variability in fibre distribution of muscle fibres in the connective tissue layer the oral mucosa (arrows). ATPase at pH4.6. x60.

of the orbicularis

diameter and shape. Also, note the irregular beneath

staining, oriented (arrow). (double

P. STAL et al.

454

Orb Oris

O.?-

per cent

Fig. 6. Percentage of overall muscle buccinator (bucc) and orbicularis

fIbre cross-sectional area occupied by different fibre types in the oris (orb oris) muscles. Mean fibre area from 5 adult males.

fibre type. According to Dubowitz (1985), a CV higher than 250 in limb and trunk muscles suggests abnormal variability in fibre size. In both the superior and inferior portions of the orbicularis oris, all fibre types had values higher than 250, whereas in the buccinator muscle this was the case only for type I fibres (Table 5).

fibres or groups of fibres were scattered in the connective tissue surrounding the muscle bundles in the lip (Plate Fig. 5). Frequency. In both the superior (mean 71.8%) and inferior (mean 67.0%) portions, the type IIA fibre population was significantly larger than the type I. In one subject (No. 4, superior portion) a reversed pattern was found, with a predominance of type I fibres (mean 57.6%). There was no significant difference in fibre-type frequency between the superior and inferior orbicularis oris (Table 3). Diumeter. The mean diameter of the type I, IIA and IIC fibres (28.4-32.8 pm) did not differ significantly within or between the superior and inferior portions (Table 4). Relative cross-sectional area. The type IIA fibres occupied 74.2% of the total cross-sectional area; the type I and IIC fibres occupied 25.3 and 0.5%, respectively (Text Fig. 6).

Myo-muscular and myo-tendinous junctions

The localization of cholinesterase activity in the muscle fibres suggested the presence of both myomuscular and myo-tendinous junctions in both muscles. Muscle spindles Muscle spindles were not observed in any buccinator or orbicularis oris muscle.

Variability in jibre diameter

Comparative frequency and diameter of Jibre types in various muscles

The variability of fibre diameter, expressed as the coefficient of variation, CV, was estimated for each

These basic comparative data from this and earlier studies are shown in Tables 6 and 7.

Table 2. Diameter

@m) of fibre types in the superior the buccinator Type Subject No.

I

Bucc sup

2 3 4 5

1

Bucc inf

2 3 4 5 Group

values (n = 5)

Bucc sup Bucc inf Buccinator

muscle

(Bucc sup) and inferior muscle

1

(Bucc inf) portion

Type HA

Type IIC

n

x

SD

x

SD

198 228 441 257 233

33.6 35.6 26.2 38.8 32.9

8.7 9.0 6.7 10.9 8.0

39.9 42.4 30.4 63.1 42.9

11.6 11.5 8.2 12.6 9.6

334 306 370 332 244

36.5 31.0 32.0 36.0 29.4

8.6 6.7 6.0 8.5 8.5

40.8 34.1 33.5 40.2 42.0

9.3 7.3 6.4 9.3 11.0

40.0 40.0

10.0

.?

SD

63.3

11.5

30.0

.?

SD

Range

_?

SD

Range

X

SD

Range

33.4 33.0 33.2

4.6 3.1 3.2

I O-70

43.7 38.1 41.0

11.9 4.0 5.6

10-90 l&80 l&90

63.3 36.7 50.0

5.8 23.5

5&80 3&50 3&80

I@-60 l&70

of

Fibre composition in human oro-facial muscles

455

Table 3. Relative frequency (%) of different fibre types in the superior (Orb oris sup) and inferior (Orb oris inf) portion of the orbicularis oris muscle

n

%

SE

%

SE

2 3 4 5

449 414 401 238 449

15.6 23.9 23.9 57.6 18.0

1.71 2.10 2.13 3.21 1.82

84.4 75.6 76.1 41.6 81.3

1.71 2.11 2.13 3.20 1.84

1 2 3 4 5

476 427 312 393 454

30.0 26.7 49.4 33.1 23.1

2.10 2.14 2.84 2.38 1.98

69.5 73.3 50.0 66.4 76.0

2.11 2.14 2.84 2.39 2.01

1

Orb oris sup

Orb oris inf

Type IIC

Type IIA

Type I Subject No.

Group values (n = 5) Orb oris sup Orb oris inf Orbicularis oris muscle

%

SE

0.5

0.34

0.8 0.7

0.59 0.38

0.4

0.30

0.6 0.5 0.9

0.45 0.36 0.44

2

SD

P

SD

z

SD

27.8 32.5

17.0 10.2

71.8 67.0

17.3 10.2

0.4 0.5

0.4 0.3

28.3

10.5

69.4

17.8

0.5

0.3

Table 4. Diameter (pm) of fibre types in the superior (Orb oris sup) and inferior (Orb oris inf) oortion of the orbicularis oris muscle

n

k

SD

2

SD

449 414 401 238 449

26.9 24.3 27.9 33.9 29.0

9.6 7.4 6.8 7.3 7.2

25.7 27.3 25.0 39.0 33.2

9.4 7.2 5.9 8.3 9.3

1 2 3 4 5

416 427 312 393 454

22.9 26.7 30.8 29.0 30.7

6.2 7.6 6.6 9.0 8.6

28.8 29.8 29.7 30.7 34.2

8.2 7.7 6.2 8.6 8.2

Orb oris inf

Orb oris sup Orb oris inf Orbicukis oris muscle

Type IIC

1 2 3 4 5

Orb oris sup

Group values (n = 5)

Type IIA

Type1

Subject No.

z

SD

25.0

7.1

40.0 33.3

5.8

25.0

7.1

35.0 20.0 35.0

7.1 14.1 5.8

,?

SD

Range

_?

SD

Range

X

SD

Range

28.4 28.2

3.5 4.4

10-50 lo-60

30.1 30.6

6.0 2.1

1060 lo-70

32.8 28.8

7.5 7.5

20-40 2040

28.3

2.9

1060

30.4

3.1

lo-70

30.8

4.8

2&40

Table 5. The variability of fibre diameter, expressed as the coefficient of variation, of fibre types in the human superior (sup) and inferior (inf) portion of buccinator (Bucc), orbicularis oris (Orb oris), major zygomatic (Zyg maj), minor zygomatic (Zyg min) and the first dorsal interosseus (FDI) muscles Type I Muscles Bucc: sup inf Orb oris: sup inf Zyg maj* Zyg mint FDI*

.u

SD

Type IIA .?

SD

259 232

43 38

245 228

54 43

269 271 237 283 186

38 38 47 136 35

266 254 189 278 175

40 39 47 37 28

*From St&l (1987).

Type IIAB

Type IIB

X

SD

:

SD

188 286

38 65

194 320 154

69 108 31

456

P. ST.&Let

al.

Table 6. Relative frequency (%) of different fibre types in the buccinator (Bucc), orbicularis oris (Orb oris), major zygomatic (Zyg maj), minor zygomatic (Zyg min), first dorsal interosseus (FDI), masseter and biceps brachii muscles Type IIA

Type I Bucc Orb oris Zyg maj* Zyg min* Masseteri FDI* Biceps? *From

x

SD

xSD

53.4 28.3 25.1 10.9 62.5 59.2 41.8

17.2 17.0 7.1 6.7 10.0 16.7 8.5

46.4 71.8 20.9 23.4 2.1 35.7 25.8

StHl (1987). tFrom

Eriksson

17.7 17.3 18.1 28.5 4.7 16.2 8.1

Type IIAB

Type IIB

Type IIC

X

i

SD

P

SD

P

SD

5.3 12.4 8.8 2.6 12.4

0.4 0.5 0.5 0.4 2.7 1.9 0.5

0.5 0.3 0.3 0.8 2.6 2.4

6.0 0.5

5.6 0.7

48.1 52.6

SD

20.8 31.2

5.4 12.8 26.7 2.8 31.9

Type ATPase-IM

(1982).

DISCUSSION

We show that the fibres of human buccinator and orbicularis oris muscles have specific enzyme-histochemical characteristics, different from those of mast&tory, and limb and trunk muscles. Both muscles were composed of type I, type IIA and a few type IIC fibres. However, their type I fibres were more alkaline-stable than those of limb and masticatory muscles, expressed as moderate to strong myosin ATPase staining at pH 9.4 and a light staining at pH 10.3. The orbicularis oris had a marked predominance of type II flbres, similar to the zygomatic muscles (S&l er al., 1987), whereas in the buccinator type I fibres predominated. As the isomyosin form and the rate of ATP hydrolysis are correlated (Bar&try, 1967), these histochemical profiles of the buccinator and orbicularis oris suggest that the isomyosin composition and the functional properties are also specific. We have earlier shown that the anterior and posterior bellies of the human digastric muscle, innervated by the trigeminal and facial nerves respectively, both have an enzyme histochemical profile similar to that of limb and trunk muscles (Eriksson et al., 1982). However, the masticatory muscles, originating from the first branchial arch and innervated by the trigeminal nerve, and the zygomatic muscles, originating from the second branchial arch and innervated by the facial nerve, differ from each other in fibre-type composition as well as from limb and trunk muscles (Eriksson et al., 1981; Eriksson and Thornell, 1983; Stll et al., 1987). Taken together, our earlier and present findings imply that functional demands are of greater importance for muscle differentiation than embryological origin and nerve supply. Haapak et at. (I 988) also reported a high frequency of type I fibres in the buccinator muscle. As type I fibres belong to slow-twitch motor units, which are resistant to fatigue (Burke, 1981; Schmalbruch, 1985), the type I fibre pattern of this muscle implies a large capacity for endurance during continuous work at relatively low levels of force, This is also in line with the findings by Blanton, Briggs and Perkins (1970) of a continuous electromyographic activity pattern for the buccinator during swallowing, blowing, sucking, chewing and various lip and mandibular movements. We found no statistically significant difference in fibre-type frequency between the upper and lower parts of the bu~inator. However, there was marked intramuscular variability for both portions, as well as

apparent inter-individual differences. The intramuscular differences indicate that the motor units in various parts of this muscle have different functional tasks. This interpretation is supported by the electromyographic study by Charles, Isley and Basmaijan (1973), who found that various parts of the buccinator muscle could act inde~ndently of each other. From studies in limb muscles, it is known that type II muscle fibres make up fast-twitch motor units, which are large and involved in rapid acceleration and relatively fast and coarse activity (Burke, 1981). The orbicularis oris, with its predominance of type II fibres, is involved in fine motor oro-facial functions and has a large representation in the motor cortical area (Kandle and Schwartz, 1985). It may, therefore, be more meaningful to compare the physiology of the orbicularis oris with that of the extra-ocular muscles, which are known to be involved in very accurate movements. Extra-ocular muscle has a predominance of type II fibres with a small mean diameter (Hoogenraad, Jennekens and Tank, 1979) and few fibres per motor unit (Christensen, 1959). Furthermore, Schmalbruch (1985) has suggested that the extra-ocular muscles, when compared with the limb muscles, appear to be better suited to force gradation because their fibres develop less tension. Further studies are needed to discover the motor unit size of the orbicularis oris muscle. Our finding of a predominance of type II fibres and a lack of type IIB fibres in the orbicularis oris differs from that of Schwarting et nt. (1982), who recorded equal proportions of type I and II tibres and a low frequency of type IIB fibres. This discrepancy may be partly explained by the fact that our data were based on autopsy specimens from subjects aged 1541 years, whereas Schwarting studied biopsies from patients undergoing facial surgery, aged 14-89 years. It must also be remembered that because of the marked intramuscular variability that we now demonstrate, small biopsies would probably not be representative of the muscle as a whole. Like the buccinator, the orbicularis oris showed no statistically significant difference in fibre-type frequency between its superior and inferior portions but a marked intramuscular and inter-individual variation in fibre pattern. This variability in morphology is in line with electrophysiological findings of a marked variation in electromyographic activity within and between orbicularis oris muscles (Miilier, 1966; Leanderson and Lindblom, 1972; Kelman and Gutehouse, 1975).

Fibre composition in human oro-facial muscles

‘? w

09’0 wd

\9f? bW

451

From anatomical and electrophysiological studies of perioral muscles, Blair and Smith (1986) pointed out the difficulty of achieving a reliable recording from single muscles or from a homogeneous population of muscle fibres in this region. The fact that fibres of different muscle groups and different fibre orientation interdigitate in the oro-facial region and that the muscles mainly attach to soft tissue and lack a well-defined fascia, implies that data from electrophysiological analyses of these muscles must be critically interpreted (Blair and Smith, 1986). From earlier investigations we know that the type II fibres of the human major and minor zygomatic muscles (Stil er al., 1987) and the extra-ocular muscles (Hoogenraad et al., 1979), have a higher oxidative capacity than limb muscles. The type II fibre population of the orbicularis oris and buccinator also showed a strong oxidative enzyme activity. Therefore, a large capacity for aerobic metabolism seems to be a normal property of the type II fibres in the human facial muscles. The oxidative enzyme staining pattern of the type II fibres may be related to their frequent use in oral functions such as chewing, speech and emotional expression. Muscle fibre diameter is related to the use made of the motor units. Whereas exercise may increase the cross-sectional area, disuse will decrease it (reviewed by Saltin and Gollnick, 1983). Both the orbicularis oris and the buccinator had a considerable intramuscular and inter-individual variability in fibre diameter. As expressed by the coefficient of variation (CV), all the fibre types in the buccinator showed a large variation, and in the orbicularis oris, all the fibre types exceeded the limit for abnormal variability in size proposed for the limb and trunk muscles (Dubowitz, 1985). Abnormal CV values for fibre diameter have been reported for the major and minor zygomatic and in all masticatory muscles (St51 et al., 1987). As our specimens were from normal subjects, it seems that our abnormal CV values have to be regarded as a normal feature rather than representing a pathological process; and they indicate specific morphological and functional properties of these muscles. In an earlier investigation of the zygomatic muscles, we found no evidence to suggest that their marked variability in fibre diameter was due to tapering fibres fusing to connective tissue within the muscle (Stal et al., 1987). In the present study, we also included an analysis to test whether tapering fibres could explain the extreme variation in fibre diameter and found no evidence to support this possibility. Acknowledgements-We wish to thank MS Inga Johansson for skilful technical assistance. Financial support was provided by the Swedish Medical Research Council (3934 and 6874) and the Swedish Dental Society. REFERENCES Barany M. (1967) ATPase activity of myosin correlated with speed of muscle shortening. J. gen. Physiol. 50, 197-218. Blair C. and Smith A. (1986) Emg recording in human lip muscles: can single muscles be isolated. J. Speech Hear. Res. 29, 256266. Blanton P. L., Briggs N. L. and Perkins R. C. (1970) Electromyographic analysis of the buccinator muscle. J. dent. Res. 49, 389-394.

458

P.

STAL

Brooke M. H. and Kaiser K. K. (1970) Muscle fibre types: how many and what kind? Archs Neural. Psychiat., Chicago 23, 369-379.

Burke R. E. (1981) Motor units: anatomy, physiology and functional organization. In: Handbook of Physiology. Section I, The Nervous System. Vol. II. Motor Control (Edited by Brooks V. B.) pp. 345422. American Physiological Society, Washington, D.C. Charles L., Isley J. R. and Basmaijan J. V. (1973) El~tromyography of the human cheeks and lips. Anat. Rec. 176, 143-148. Christensen E. (1959) Topography of terminal motor innervation in striated muscles from stillborn infant. Am. J. phys. Med. 38, 40114015.

Clancy M. J. and Hearlihy P. D. (1979) Muscle fibre sizes and their frequency distributions. Meet. Sue. Exp. Bioi. Bevast. Abstr. Dubowitz V. (1985) Muscle Biopsy-A Practical Approach, 2nd edn, pp. 34--102. Bailliere Tindall, London. Engel W. K. and Cunningham G. C. (1963) Rapid examination of muscle tissue-An improved trichrome method for fresh-frozen biopsy sections. Xevrotogy, Minneap. 13, 219-226.

Eriksson P.-O. and Thornell L.-E. (1983) Histochemical and morphological muscle fibre characteristics of the human masseter, the medial pterygoid and the temporal muscles. Archs oral Biol. 28, 781-795.

Eriksson P.-O., Eriksson A., Ringqvist M. and Thornell L.-E. (1980) The reliability of histochemical fibre typing of human necropsy muscles. Hjstochem~stry 65, 193-205.

Eriksson P.-O., Eriksson A., Ringqvist M. and Thornell L.-E. (1981) Special histochemical muscle-fibre characteristics of the human lateral pterygoid. Archs oral Eiol. 26, 495-507.

Eriksson P.-O., Eriksson A., Ringqvist M. and Thornell L.-E. (1982) Histochemical fibre composition of the human digastric muscle. Archs oral Biof. 27, 207215.

Haapak W., Burggasser G. and Gruber H. (1988) Histochemical characteristics of human mimic muscles. J. neurol. Sci. 83, 25-35. Hamilton W. J., Boyd J. D. and Mossman H. W. (1957) Human Embryology, Chap. f4, 2nd edn, pp. 3.55-364. Heffer, Cambridge. Hoogenraad T. V., Jennekens F. G. I. and Tank E. W. (1979) Histochemica! fibre types in human extraocular muscles. an investigation of inferior oblique muscle. Acta neuroparhol. (Berl.) 45, 73-78.

et

al.

Kandel E. R. I. and Schwartz J. H. (1985) Principles qf Neural Science, 2nd edn, pp. 6-10. Elsevier, New York. Kelman A. W. and Gutehouse S. (1975) A study of the electromyographic activity of the muscle orbicularis oris. Folia Phoniat. 27, 177-189. Koelle G. B. and Friedenwald J. S. (1949) Histochemical method for localisation of cholinesterase activity. Proc. Sot. exp. Biol. Med. 70, 617622.

Leanderson R. and Lindblom B. E. F. (1972) Muscle activation of labial speech gestures. Acta oto~aryngoi. (Stockh.) 73, 362-373.

MacDougall J. D., Elder G. C. B., Sale D. G., Moroz J. R. and Sutton J. R. (1980) Effects of strength training and immobilization on human muscle fibres. Eur. 1. appl. Physiol. 43, 25-34.

Miiller E. (1966) The chewing apparatus: an electromyographic study of the action of the muscles of mastication and its correlation to facial morphology, Acta physiol. stand. Suppl. 280 69, l-229. Nystriim B. (1968) Histochemistry of developing cat muscles. Acta neural. stand, 44, 405439. Padykula J. A. and Herman E. (1955) The specificity of the histochemical method for adenosine t~phosphatase. J. Histochem. Cytochem. 3, 170-183. Pierobon-Bormiolo S. and Schiaffino S. (1977) Myomuscular junctions in reinnervated rat skeletal muscles. J. Anat. 124, 359-370.

Ringqvist M. (1974) Fibre types in human masticatory muscles. Relation to function. Stand. J. dent. Res. 82, 333-335. Saltin B. and Gollnick P. D. (1983) Skeletal muscle adaptability; significance for metabolism and performance. In: Handbook of Physiology, Section 10, Chap. 19. Skeletal Muscle. ,.. DD. 579-589. Williams & Wilkins. Baltimore. MD. Schmalbruch H. (1985) Skeletal muscle. in: Handbook of Microscopic Anatomy (Edited by Oksche A. and Vollrath L.) Part 6, Vol. 2. Springer, Berlin. Schwarting M., Schriider E., Stennert H. H. and Goebel H. H. (1982) Enzymehistochemical and histographic data on normal human facial muscles. Oto-Rhino-LaryngoL 44, 51-59.

Schwarting M., SchrGder E., Stennert H. H. and Goebel H. H. (1984) Morphology of denervated human facial muscles. Oto-Rb~o-Larvn~ol. 46, 248-256. St&i P., Eriksson P.-O., &&son A. and Thomeli L.-E. (1987) Enzyme-histochemical differences in fibre type between the human major and minor zygomatic and the first dorsal interossesus muscle. Archs orai Biol. 32, 833-841.