230 Headache Medicine, v.3, n.4, p.198-235, Oct./Nov./Dec. 2012

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Fiber-type composition, fiber diameter and

capillary density of the human jaw muscles

Thatiana B. Guimarães

, Mariana B.F. Cabrini

, A. Wakamatsu

, Antônio S. Guimarães

, Suely K.N. Marie

Cirurgião dentista. Ambulatório de Disfunção Temporomandibular e Dor Orofacial/Unifesp,

Farmacêutica e Bioquímica/USP,

Neurologista/USP

Guimarães TB

, Cabrini MBF

, Wakamatsu A

, Guimarães AS

,Marie SKN. Fiber-type composition, fiber

diameter and capillary density of the human jaw muscles. Headache Medicine. 2012;3(4):222-5

Headache Medicine, v.3, n.4, p.198-235, Oct./Nov./Dec. 2012 231

Figure 1 – Imunohistochemical preparations on sequential sections of

masseter muscle of 3rd decade male with the antibodies against MyHC

slow (A) and fast (B), showing the double staining. C: single muscle

fiber manually teased from the masseter muscle of 4th decade male

prepared with antibody agaist MyHC slow demonstrating the presence

of a portion of the fiber stained and other without reactio, proving the

presence of hybrid fiber. D: temporalis muscle from 2nd decade male

prepared with antibody againt MyHCneonatal showing the presence

of neonatal fibers stained (in brown)

INTRODUCTION AND OBJECTIVES

Successful performance of daily oral activities, such

as biting, swallowing, chewing, and talking require a

synchronized function of jaw muscles. Precise position

control of the mandible combined with a change of jaw

muscle strength is feasible due to the complex architecture

of the involved muscles. The speed of muscle contraction

depends on the composition of the myosin heavy chain

(MyHC) isoform composition.

Muscle fibers have been classified according to the

differences in contraction speed, and propensity to

fatigability based on immunohistochemical findings.

Contraction velocity increases progressively from type I,

type IIA, type IIX to type IIB. The fiber-type composition

differs within different muscle groups (jaw-closers and jaw-

openers), different regions of the same muscle. It is likely

that specific function is reflected in specific fiber-type

composition.

Jaw-muscles notably present more hybrid fibers,

containing more than one MyHC isoform, than limb or

trunk muscles. These hybrid fibers have contractile

properties that differ from the pure fiber, and present

intermediate characteristics from each of the MyHC

isoforms they express. Thus, hybrid fiber expressing both

MyHC-I and IIA, for instance, will be faster than pure

MyHC-I fiber but slower than pure MyHC-IIA fiber.

During muscle maturation, developmental MyHC

isoforms (embryonic and neonatal) are replaced by adult

slow and fast MyHC isoforms in normal adult limb and

trunk muscle fibers. However, developmental MyHC

isoforms persist in some adult cranial muscles, including

the masseter and they may even increase in relative amount

with aging.

There are several differences between jaw muscles

and limb and trunk muscles. For example, the jaw muscles

contain many hybrid fibers, in contrast to limb and trunk

muscles. Many of these fibers co-express MyHC-neonatal.

Also, there is a difference in the fiber diameter between

the two fiber types. Type II fibers are larger than type I

fibers in limb and trunk muscles, while in jaw muscles the

opposite is observed. Another difference is the jaw muscle

fibers are 50% smaller than limb and trunk muscle fibers.

The aims of the present study were to determine: 1)

the fiber-type and fiber cross sectional area distribution in

masseter and temporal muscles through the aging process,

in both genders, 2) the proportion of hybrid and neonatal

fibers, and their capillary density along the nine decades

of life.

METHODS

We studied the differences of the fiber types in

masseter and temporalis muscles along the first to ninth

decades in both genders. Seventy-four (74) samples

were obtained from the Pathology Department of School

of Medicine of University of São Paulo, according to the

rules of the necropsy service, within an 8-18 hours post

mortem interval, of both genders, and from subjects in

the first to ninth decade of life. It included at least two

samples per decade per gender. Fragments of 2x2x1cm

were collected from deep and posterior portions of

masseter by extraoral access, and from medium and

superficial temporalis by superior access.

For statistical analysis, the samples were grouped into

three subgroups: young (0 to 25 years of age), adult (26

to 59 years of age) and old (60 and above years of

age). Comparisons between two groups was used the t

student test or the Mann-Whitney, and for three groups

used the ANOVA or the Kruskal-Wallis test. The significance

level was set at p<0.05.

ImmunohistochemistryImmunohistochemistry

Immunohistochemistry

The muscle specimens were mounted for transverse

sectioning and snap frozen in liquid nitrogen. Serial 6µm

thick cross-sections were prepared in a cryostat microtome

(MICROM HM 505E) at -25°C, and were kept stored in

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232 Headache Medicine, v.3, n.4, p.198-235, Oct./Nov./Dec. 2012

-80°C until used for histological, and immuno-

histochemical stainings.

Fiber-type classification was based on the staining

pattern for immunohistochemical myosin heavy chain slow

antibody (clone WB-MHCs, Novocastra, dilution 1:80),

and myosin heavy chain fast antibody (clone WB-MHCf

Novocastra, dilution 1:40). The presence of neonatal fiber

was determined by myosin heavy chain neonatal antibody

(cloneWB-MHCn, Novocastra, dilution 1:40), and the

capillary density with ulex europaeus aglutinin I (clone B-

1065 Vector, dilution 1:800) staining.

The cross-sections were photographed in 3 to 4 areas

using the Nikon-Eclipse E800. Classification of fiber type

was performed in at least 500 muscle fibers from each

muscle sample at 200 times magnification.

The histograms were obtained from a computer

program (CELL) developed specifically for cellular

morphometric studies for the myopathy laboratory of

the Department of Neurology at the University of São

Paulo.

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Figure 2 – Graphics of cross section area (A), hybrid fiber (B), capillary

density (C) showing the linear and tendency of the alterations along

the nine decades of age

Isolation of fixed single muscle fibersIsolation of fixed single muscle fibers

Isolation of fixed single muscle fibers

The isolation of fixed single muscle was prepared

from muscle biopsies obtained from one specimen from

a 39-year-old. The biopsy specimen was removed and

pinned to sylgard-coated dishes for fixation with 2%

paraphormaldeyde, followed by PBS rinses and fixation

in cold methanol. The permeabilized fibers were then

incubated with primary antibody overnight at 4°C, washed

in PBS, incubated with second antibody for 2h at room

temperature, washed and mounted in Vectashield (Vector

Laboratories, Burlingame, CA) on glass slides for analysis

by confocal microscopy.

RESULTS

Slow and fast muscle fiber distribution was similar in

both muscles in both genders. Hybrid fiber was observed

in all decades, and its frequency decreased significantly

(p<0.001) with aging in masseter.

Neonatal myosin expression was observed in all

decades; its expression was more frequent in masseter

(p=0.01), and males in temporalis (p=0.025).

Decrease of the cross-sectional area of fast and slow

fibers and decrease in capillary density were detected

with aging.

CONCLUSIONS

The jaw muscles proved to be highly unusual compared

to limb and trunk muscles. Masseter and temporalis muscles

contain many hybrid fibers, which numbers decrease with

aging, and numerous neonatal fibers in the majority of

samples over nine decades of age.

We also observed a decreased in the capillary density

and of the cross sectional area; together, all findings can

be related to the decrease in bite force with aging.

This muscles are essential components in the

stomatognatic system. Their complex architecture allows

them to execute several motor tasks. One of the structural

peculiarities is the presence of hybrid and neonatal fibers

in all decades.

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