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Morphometric aspects of the facial and skeletal muscles in fetuses
G Model
PEDOT-7539; No. of Pages 5 International Journal of Pediatric Otorhinolaryngology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Morphometric aspects of the facial and skeletal muscles in fetuses Hiroshi Moriyama a,*, Kaori Amano b, Masahiro Itoh c, George Matsumura b, Naruhito Otsuka a a
Department of Anatomy, Showa University School of Medicine, 5-8, Hatanodai 1, Shinagawa-ku, Tokyo 142-8555, Japan Department of Anatomy, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka-shi, Tokyo 181-8611, Japan c Department of Anatomy, Tokyo Medical University, 1-1, Shinjuku 6, Shinjuku-ku, Tokyo 160-8402, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 February 2015 Accepted 7 April 2015 Available online xxx
Objectives: There are few research reports providing a comparison of the muscle fiber morphometry between human fetuses and adults. Data on fetal and adult muscle fibers would be valuable in understanding muscle development and a variety of muscle diseases. This study investigated human muscle fiber growth to clarify the difference between the facial muscles and other skeletal muscles. Methods: The materials were obtained from three male fetuses (6-month-old) and 11 Japanese male cadavers aged 43–86 years (average: 71.8). Human buccinator muscles (facial muscles), masseter and biceps brachii muscles (skeletal muscles) were resected. We counted the muscle fibers and measured their transverse area. We also calculated the number of muscle fibers per mm2 (NMF) and the average transverse area of the muscle fibers (TAMFs). Results: The average of the NMF of the buccinator, masseter and biceps brachii muscles in fetuses had, respectively, 19, 37, and 22 times as many fibers as those in adults. The average fetus/adult ratios of the TAMF of the buccinator, masseter and biceps brachii muscles were 4.0%, 2.4%, 4.1%, respectively. Conclusions: The average NMF for all kinds of muscles decreased after birth; however, the peak in lifespan or decreases with the aging process tended to vary with the kind of muscles examined. The average TAMF for all kinds of muscles enlarged after birth. We considered that the enlargement of the TAMF was connected with the emergence of fetal movements and functional demands after birth. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Buccinator muscle Masseter muscle Biceps brachii muscle Muscle fiber Morphometry Fetus
1. Introduction
2. Materials and methods
Much of the previous work concerned with muscle fiber growth has been carried out on animal fetuses [1] or on human after birth [2]. These materials were muscles from the torso or extremities [3], but no details of the facial muscles have been available in textbooks. Furthermore, there are few research reports providing a comparison of muscle fiber morphometry between human fetuses and adults. Understanding the morphometry of human fetal and adult muscle fibers would be valuable in understanding muscle development and a variety of muscle diseases. We used facial and other skeletal muscles from human fetuses and adults, and performed a morphometric analysis of muscle fibers. This study closely investigated human muscle fiber growth to clarify the difference between the facial muscles and other skeletal muscles.
Human buccinator muscles (facial muscles), masseter and biceps brachii muscles (skeletal muscles) were resected with skins and connective tissues. The materials were obtained from three male fetuses (6-month-old) from the Japanese fetal cadaver collection of the Department of Anatomy, Kyorin University School of Medicine, Japan. The ages were determined by measuring the crown-rump length (CRL) of the materials. To determine the approximate age of the fetus, we used the ‘‘Classification of Shimamura’’ [4]. This chart converts the CRL of a Japanese fetus into its corresponding age in terms of months of gestation. The other adult materials were obtained from 11 Japanese male cadavers aged 43–86 years (average: 71.8). All the fetuses were donated with the surviving families’ consent, and all the cadavers were donated with the individuals’ consent. We proceeded to perform this research in accordance with the law concerning autopsies and the preservation of corpses, and concerning their donation for medical and dental education. In no case was there a history of neuromuscular disease such as myopathy or facial palsy,
* Corresponding author. Tel.: +81 3 3784 8107; fax: +81 3 5498 7800. E-mail address: [email protected] (H. Moriyama). http://dx.doi.org/10.1016/j.ijporl.2015.04.009 0165-5876/ß 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: H. Moriyama, et al., Morphometric aspects of the facial and skeletal muscles in fetuses, Int. J. Pediatr. Otorhinolaryngol. (2015), http://dx.doi.org/10.1016/j.ijporl.2015.04.009
G Model
PEDOT-7539; No. of Pages 5 H. Moriyama et al. / International Journal of Pediatric Otorhinolaryngology xxx (2015) xxx–xxx
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or of treatment with toxic agents or irradiation therapy (both the parents of the fetuses and the cadavers). Moreover, each parent or cadaver had 20 teeth or more (mastication capacity standard), and they supported themselves in daily life (biceps brachii capacity standard). The causes of death did not directly or indirectly influence the muscular or nervous system, so the muscles were considered to be normal. The preparation of sections involved fixation, washing, dehydration, embedding, and sectioning, as described in our previous report [5]. All the cadavers were fixed with a 10% solution of formalin (3.7% formaldehyde) within 24 h of postmortem. After resecting the muscle, a 10% solution of formalin (3.7% formaldehyde) was used for immersion for at least a week. The solution was changed once in the first 30–60 min, and again later if desired. The formalin-fixed materials were then transferred, without washing, to the secondary fixative to be held at room temperature for two weeks. If the solution became turbid or precipitated it was changed. After this, the fixation was continued at 37 8C for an additional week. The volume of fixative used was at least ten times the volume of the specimens. In this process, materials had been fixed with pins at four corners of the board. After washing, dehydration, and celloidin embedding, we cut sections 15-mm thick and stained them with hematoxylin and eosin (H&E).
Fig. 2. A high-power view of the muscle fibers in buccinator muscle staining with H&E. 6-Month-old male fetus; scale bar = 100 mm.
Germany), and a computer (Precision 530, Dell, Austin, TX, USA) with analyzing system software (analySIS 3.0, Soft Imaging System, Mu¨nster, Germany) for storing data on-line, calculations, and statistical analyses.
2.1. Morphometry 3. Results We observed the microscopic section at low power, and covered the entire area of the distributed muscle fibers in the section by moving the eyepiece grid vertically and horizontally as described in our previous report [6]. We confirmed that we could distinguish muscle fiber structures from other tissues with both a computer and the naked eye in every grid. We counted the muscle fibers and measured the transverse area of the muscle fibers in a square eyepiece grid at high power (Figs. 1 and 2), and then calculated the number of muscle fiber per mm2 (NMF) and the average transverse area of the muscle fibers (TAMFs). To avoid duplicate counts, we counted and measured all muscle fibers on the side of the grid that did not come into contact with the other grids. In the case of grids adjacent to the other grids, we counted and measured only the muscle fibers on the lower right side of the grid, not those on the upper left side. We used a microscope in a transmitted light mode (BX50, Olympus, Tokyo, Japan) equipped with a high-resolution digital camera (ColorView12, Soft Imaging System, Mu¨nster, Germany), a motorized XYZ stage (Ma¨rzha¨user, Wetzlar-Steindorf, Germany), a stage controller (Ma¨rzha¨user, Wetzlar-Steindorf,
3.1. Number of muscle fibers per mm2 (NMF) We counted the muscle fibers and calculated the number of muscle fibers per mm2 (NMF). Fetuses had a higher NMF than did adults in three kinds of muscle (Fig. 3). The average NMF for the buccinator, masseter and biceps brachii muscles in the three fetuses were 22,849 4798 (mean SD, and so on), 43,874 13,162, 26,173 7852, respectively. As for the adults, the average NMF for the buccinator, masseter and biceps brachii muscles in the 11 cadavers were 1177 248, 1184 506, 1180 460, respectively. The average NMF for the buccinator, masseter and biceps brachii muscles in fetuses showed, respectively, 19, 37, and 22 times as many as those in adults. 3.2. The average transverse area of the muscle fibers (TAMFs) We calculated the average transverse area of the muscle fibers (TAMFs). Fetuses had a smaller TAMF than did adults in three kinds of muscle (Fig. 4). The average TAMFs for buccinator, masseter and biceps brachii muscles in the three fetuses were 18.1 4.5 (mean SD, and so on), 14.5 3.1, 29.3 10.3 mm2, respectively. As to adults, the average TAMF for the buccinator, masseter and biceps brachii muscles in the 11 cadavers were 448.1 108.7, 602.8 230.4, 722.9 301.6, respectively. The average fetus/adult ratios of the TAMF for the buccinator, masseter and biceps brachii muscles were 4.0%, 2.4%, 4.1%, respectively. 4. Discussion
Fig. 1. A high-power view of the muscle fibers in buccinator muscle staining with H&E. 43-Year-old man; scale bar = 100 mm.
The number of muscle fibers appears to be genetically determined, showing no significant change after birth in most animals [1,7]. It is evident that the number of muscle fibers in a given muscle increases before birth until the genetically determined number is attained. Stickland’s investigation of human fetuses showed that at the same timing as myotube appearance being lost, at around 6-months of age, the number of muscle fibers per unit area changed from an increasing trend to a marked decrease [8]. Reports as described above indicated that there is an initial slow increase in the total numbers of muscle fibers up to
Please cite this article in press as: H. Moriyama, et al., Morphometric aspects of the facial and skeletal muscles in fetuses, Int. J. Pediatr. Otorhinolaryngol. (2015), http://dx.doi.org/10.1016/j.ijporl.2015.04.009
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Fig. 3. Comparison of the number of muscle fibers per mm2 between adults and fetuses. Red bars indicate the number of muscle fibers per mm2 in fetuses. Blue bars indicate the number of muscle fibers per mm2 in adults. Each value is the mean SD. Each italic number with times indicates the ratio of fetus/adult. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
around 3 or 4-months of age, followed by a very rapid increase up to around 6-months of age, after which the number reaches its peak in life-span at around birth. The number of muscle fibers per unit area, on the other hand, changes from an increasing trend to a marked decrease at around 6-months of age [8]. Researchers reported that this distinct change must be due partly to a slowing down in the decrease of intercellular space and to the commencement of a more rapid muscle fiber hypertrophy phase [8–10]. We supposed that the NMF reached its peak in life-span at around 6month-old for the reports mentioned above. We compared the data of 6-month-old fetuses with those of adults. Our data showed that the fetus had a higher NMF than did the adult, however, the ratio of the adult/fetus NMF depended on the muscle kinds (Fig. 3). The average of the NMF for the buccinator, masseter and biceps brachii muscles in fetuses showed, respectively, 19, 37, and 22 times as many as that in adults. Joubert reported that different
muscles developed at varying rates at the cellular level [11]. It is one of the items of evidence that the NMF reaches its peak in lifespan with variance depending on the kind of muscles. We also considered that all three individual muscles indicate their peak in the NMF in life-span at around 6-month-old, however, the decrease of the NMF with the aging process varies with the kind of muscles. It is known from several studies on animals that muscle fibers can be affected by many factors such as age [7], exercise [12] and level of nutrition [13]. With regard to muscle fiber size, our data showed that the fetus had a smaller TAMF than did the adult (Fig. 4). Researchers reported that there is an enlargement of the muscle fiber as animals mature [1,14,15]. Rowe and Goldspink reported that 129/ Re strain mouse showed an enlargement of the muscle fiber diameter until the age of three to nine weeks, varying with the kind of skeletal muscles [1]. As for human skeletal muscle, researchers
Fig. 4. Comparison of the average transverse area of muscle fibers between adults and fetuses. Red bars indicate the average transverse area of muscle fibers (mm2) in fetuses. Blue bars indicate the average transverse area of muscle fibers (mm2) in adults. Each value is the mean SD. Each italic number indicates the percentage of fetus/adult. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: H. Moriyama, et al., Morphometric aspects of the facial and skeletal muscles in fetuses, Int. J. Pediatr. Otorhinolaryngol. (2015), http://dx.doi.org/10.1016/j.ijporl.2015.04.009
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reported that muscle fiber size showed a significant reduction with age [2]. They used the vastus lateralis muscles from physically healthy men between 15 and 83 years of age, and they also reported that the average reduction in the muscle fiber size from 20 to 80 years is 26%. Vogler and Bove also reported that the rate of muscle fiber growth in infants and children varied with age and muscle [16]. We supposed that fetal movements and behavior were one of the factors behind the fetus/adult ratio of the TAMF varying with the kind of muscles (Fig. 4). Isolated arm movements without other parts moving were observed in fetuses from eight weeks, and hand-face contact observed from 10 weeks [17]. The biceps brachii muscle takes part in these movements. Sucking (indicating that the fetus is drinking amniotic fluid) was observed from 12 weeks [17], and the buccinator muscle takes part in sucking. Moreover, yawn-like movement that prolonged wide opening of the jaws followed by quick closure often with retroflexion of the head and sometimes elevation of the arms was observed from around 14 or 15 weeks [17]. The masseter muscle takes part in this yawn-like movement. Goldspink reported that the size of muscle fibers could be affected by exercise [12]. The average fetus/adult ratio of the TAMF of biceps brachii, buccinator, and masseter muscles showed 4.1%, 4.0%, 2.4%, respectively in this study. Therefore, our results showed that the order of the size of muscle fibers in three kinds of muscles agreed with the order of the emergence of the fetal movements that their muscles took part in. Next, we compared the developmental characteristics among the three kinds of muscles. Mammalian skeletal muscle cells are known to derive from the paraxial mesoderm adjacent to the neural tube. The paraxial mesoderm in the body forms clear segmental structures, which are referred to as somites. The forelimb, including the biceps brachii muscle of the chick, is derived from somites 15–20 [18]. On the other hand, the head paraxial mesoderm is composed of seven incompletely segmented mesenchymal structures, referred to as the somitomeres. Chick/ quail graft transplantation experiments have demonstrated the developmental origins of chick head muscles [18,19]. Most of the head muscles originate from the somitomeres, the buccinator and masseter muscles are derived from somitomeres 6 and 4, respectively [18,20]. To relate the development of the muscle to the development of function, it is necessary to study the progression of myogenesis and synaptogenesis. During synaptogenesis, the expression level, distribution, and subunit composition of the nicotinic acetylcholine receptor (nAChR) are drastically changed. In the development of the mouse masseter muscle, the differentiation of myoblasts occurs most actively between embryonic day (ED) 13 and birth, and the active maturation of myofibers begins at around ED15 and continues after the shift of feeding behavior from suckling to biting at around four weeks of age [21]. Moreover, the nAChR switch and elimination begin at around ED17 and one week of age, respectively, and they are nearly complete after the shift of feeding behavior at around three to four weeks of age [21]. During the development of the mouse limb muscle, differentiation of myoblasts occurs most prominently between ED13 and birth, and the active maturation of myofibers is initiated at around ED15 and ends at around two weeks of age [21]. Furthermore, the nAChR switch and elimination occur most prominently between birth and two weeks of age [22,23]. Considering the circumstances mentioned above, the myogenesis and synaptogenesis of masseter muscle are delayed in comparison with limb muscles, and the maturation of myofibers and synaptogenesis, such as the nAChR switch and elimination, still continue after the shift in feeding behavior from suckling to biting at approximately three weeks of age. Our data in fetuses showed the average of the TAMF of the masseter muscle (14.5 mm2; indicated mean, and so on) smaller than the biceps brachii muscle (29.3 mm2) (Fig. 4). As there is an enlargement of the muscle fiber
as the animals mature [1,14,15], we suppose that muscle fiber size tends to depend on the myogenesis and synaptogenesis of the muscles. Moreover, the average of the TAMF of masseter muscle in fetuses (14.5 mm2) was also smaller than the buccinator muscle (18.1 mm2) (Fig. 4). This difference in the progress of myogenesis and synaptogenesis between the masseter and buccinator muscles seems to depend on the difference in the functional demands of each type of muscle. Since the masseter muscle mainly functions in the biting movement of the jaw, the completion of myogenesis and synaptogenesis is probably unnecessary until biting movements begin. On the other hand, myogenesis and synaptogenesis in the buccinator muscle seems to progress more quickly to meet functional demands such as suckling and swallowing of milk immediately after birth. 5. Conclusions The average of the NMF of every buccinator, masseter and biceps brachii muscles decreased after birth, however, its peak in life-span or the decrease of that with the aging process tended to vary with the kind of muscles. With regard to the average of the TAMF, that of every buccinator, masseter and biceps brachii muscles enlarged after birth. We considered that the enlargement of the average TAMF was closely connected with the emergence of the fetal movements that their muscles took part in, and was also connected with functional demands after birth such as suckling, swallowing of milk, and biting movements. Conflicts of interest We declare that we have no conflicts of interest. Acknowledgements We thank Ms. Ikuko Moriyama for assistance in preparing the manuscript. This work was supported by a Grant-in-aid for Scientific Research B14370007 from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] R.W.D. Rowe, G. Goldspink, Muscle fibre growth in five different muscles in both sexes of mice. I. Normal mice, J. Anat. 104 (1969) 519–530. [2] J. Lexell, C.C. Taylor, M. Sjo¨stro¨m, What are the causes of ageing atrophy? Total number, size and proportion of different fiber types studied in whole vastus lateralis muscle from 15- to 83-year-old men, J. Neurol. Sci. 84 (1988) 275–294. [3] A.W. Racca, A.E. Beck, V.S. Rao, G.V. Flint, S.D. Lundy, D.E. Born, et al., Contractility and kinetics of human fetal and human adult skeletal muscle, J. Physiol. 591 (2013) 3049–3061. [4] A. Shimamura, Age determination and physical measurement of Japanese embryo, Jpn. J. Legal. Med. 11 (1957) 795–811. [5] H. Moriyama, K. Shimada, N. Goto, Morphometric analysis of neurons in ganglia: geniculate, submandibular, cervical spinal and superior cervical, Okajimas Folia Anat. Jpn. 72 (1995) 185–190. [6] H. Moriyama, K. Shimada, M. Itoh, T. Takahashi, N. Otsuka, Morphometric analysis of the inferior alveolar nerve fails to demonstrate sexual dimorphism, J. Oral Maxillofac. Surg. 65 (2007) 1555–1561. [7] N.C. Stickland, G. Goldspink, A possible indicator muscle for the fibre content and growth characteristics of porcine muscle, Anim. Prod. 16 (1973) 135–146. [8] N.C. Stickland, Muscle development in the human fetus as exemplified by m. sartorius: a quantitative study, J. Anat. 132 (1981) 557–579. [9] J.W. Dickerson, E.M. Widdowson, Chemical changes in skeletal muscle during development, Biochem. J. 74 (1960) 247–257. [10] E.M. Widdowson, Changes in the extracellular compartment of muscle and skin during normal and retarded development, Bibl. Nutr. Dieta 13 (1969) 60–68. [11] D.M. Joubert, A study of pre-natal growth and development in sheep, J. Agric. Sci. 47 (1956) 382–428. [12] G. Goldspink, The combined effects of exercise and reduced food intake on skeletal muscle fibres, J. Cell. Physiol. 63 (1964) 209–216. [13] N.C. Stickland, E.M. Widdowson, G. Goldspink, Effects of severe energy and protein deficiencies on the fibres and nuclei in skeletal muscle of pigs, Br. J. Nutr. 34 (1975) 421–428.
Please cite this article in press as: H. Moriyama, et al., Morphometric aspects of the facial and skeletal muscles in fetuses, Int. J. Pediatr. Otorhinolaryngol. (2015), http://dx.doi.org/10.1016/j.ijporl.2015.04.009
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PEDOT-7539; No. of Pages 5 H. Moriyama et al. / International Journal of Pediatric Otorhinolaryngology xxx (2015) xxx–xxx [14] J.J. Chiakulas, J.E. Pauly, A study of postnatal growth of skeletal muscle in the rat, Anat. Rec. 152 (1965) 55–61. [15] M. Ontell, R.F. Dunn, Neonatal muscle growth: a quantitative study, Am. J. Anat. 152 (1978) 539–555. [16] C. Vogler, K.E. Bove, Morphology of skeletal muscle in children. An assessment of normal growth and differentiation, Arch. Pathol. Lab. Med. 109 (1985) 238–242. [17] J.I. de Vries, G.H. Visser, H.F. Prechtl, The emergence of fetal behaviour. II. Quantitative aspects, Early Hum. Dev. 12 (1985) 99–120. [18] B. Christ, C.P. Ordahl, Early stages of chick somite development, Anat. Embryol. 191 (1995) 381–396. [19] X. Borue, D.M. Noden, Normal and aberrant craniofacial myogenesis by grafted trunk somitic and segmental plate mesoderm, Development 131 (2004) 3967–3980.
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[20] D.M. Noden, The embryonic origins of avian cephalic and cervical muscles and associated connective tissues, Am. J. Anat. 168 (1983) 257–276. [21] A. Yamane, T. Saito, Y. Nakagawa, Y. Ohnuki, Y. Saeki, Changes in mRNA expression of nicotinic acetylcholine receptor subunits during embryonic development of mouse masseter muscle, Zoolog. Sci. 19 (2002) 207–213. [22] A.C. Missias, G.C. Chu, B.J. Klocke, J.R. Sanes, J.P. Merlie, Maturation of the acetylcholine receptor in skeletal muscle: regulation of the nAChR gamma-toepsilon switch, Dev. Biol. 179 (1996) 223–238. [23] M.N. Zoubine, J.Y. Ma, I.V. Smirnova, B.A. Citron, B.W. Festoff, A molecular mechanism for synapse elimination: novel inhibition of locally generated thrombin delays synapse loss in neonatal mouse muscle, Dev. Biol. 179 (1996) 447–457.
Please cite this article in press as: H. Moriyama, et al., Morphometric aspects of the facial and skeletal muscles in fetuses, Int. J. Pediatr. Otorhinolaryngol. (2015), http://dx.doi.org/10.1016/j.ijporl.2015.04.009
PEDOT-7539; No. of Pages 5 International Journal of Pediatric Otorhinolaryngology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Morphometric aspects of the facial and skeletal muscles in fetuses Hiroshi Moriyama a,*, Kaori Amano b, Masahiro Itoh c, George Matsumura b, Naruhito Otsuka a a
Department of Anatomy, Showa University School of Medicine, 5-8, Hatanodai 1, Shinagawa-ku, Tokyo 142-8555, Japan Department of Anatomy, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka-shi, Tokyo 181-8611, Japan c Department of Anatomy, Tokyo Medical University, 1-1, Shinjuku 6, Shinjuku-ku, Tokyo 160-8402, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 February 2015 Accepted 7 April 2015 Available online xxx
Objectives: There are few research reports providing a comparison of the muscle fiber morphometry between human fetuses and adults. Data on fetal and adult muscle fibers would be valuable in understanding muscle development and a variety of muscle diseases. This study investigated human muscle fiber growth to clarify the difference between the facial muscles and other skeletal muscles. Methods: The materials were obtained from three male fetuses (6-month-old) and 11 Japanese male cadavers aged 43–86 years (average: 71.8). Human buccinator muscles (facial muscles), masseter and biceps brachii muscles (skeletal muscles) were resected. We counted the muscle fibers and measured their transverse area. We also calculated the number of muscle fibers per mm2 (NMF) and the average transverse area of the muscle fibers (TAMFs). Results: The average of the NMF of the buccinator, masseter and biceps brachii muscles in fetuses had, respectively, 19, 37, and 22 times as many fibers as those in adults. The average fetus/adult ratios of the TAMF of the buccinator, masseter and biceps brachii muscles were 4.0%, 2.4%, 4.1%, respectively. Conclusions: The average NMF for all kinds of muscles decreased after birth; however, the peak in lifespan or decreases with the aging process tended to vary with the kind of muscles examined. The average TAMF for all kinds of muscles enlarged after birth. We considered that the enlargement of the TAMF was connected with the emergence of fetal movements and functional demands after birth. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Buccinator muscle Masseter muscle Biceps brachii muscle Muscle fiber Morphometry Fetus
1. Introduction
2. Materials and methods
Much of the previous work concerned with muscle fiber growth has been carried out on animal fetuses [1] or on human after birth [2]. These materials were muscles from the torso or extremities [3], but no details of the facial muscles have been available in textbooks. Furthermore, there are few research reports providing a comparison of muscle fiber morphometry between human fetuses and adults. Understanding the morphometry of human fetal and adult muscle fibers would be valuable in understanding muscle development and a variety of muscle diseases. We used facial and other skeletal muscles from human fetuses and adults, and performed a morphometric analysis of muscle fibers. This study closely investigated human muscle fiber growth to clarify the difference between the facial muscles and other skeletal muscles.
Human buccinator muscles (facial muscles), masseter and biceps brachii muscles (skeletal muscles) were resected with skins and connective tissues. The materials were obtained from three male fetuses (6-month-old) from the Japanese fetal cadaver collection of the Department of Anatomy, Kyorin University School of Medicine, Japan. The ages were determined by measuring the crown-rump length (CRL) of the materials. To determine the approximate age of the fetus, we used the ‘‘Classification of Shimamura’’ [4]. This chart converts the CRL of a Japanese fetus into its corresponding age in terms of months of gestation. The other adult materials were obtained from 11 Japanese male cadavers aged 43–86 years (average: 71.8). All the fetuses were donated with the surviving families’ consent, and all the cadavers were donated with the individuals’ consent. We proceeded to perform this research in accordance with the law concerning autopsies and the preservation of corpses, and concerning their donation for medical and dental education. In no case was there a history of neuromuscular disease such as myopathy or facial palsy,
* Corresponding author. Tel.: +81 3 3784 8107; fax: +81 3 5498 7800. E-mail address: [email protected] (H. Moriyama). http://dx.doi.org/10.1016/j.ijporl.2015.04.009 0165-5876/ß 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: H. Moriyama, et al., Morphometric aspects of the facial and skeletal muscles in fetuses, Int. J. Pediatr. Otorhinolaryngol. (2015), http://dx.doi.org/10.1016/j.ijporl.2015.04.009
G Model
PEDOT-7539; No. of Pages 5 H. Moriyama et al. / International Journal of Pediatric Otorhinolaryngology xxx (2015) xxx–xxx
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or of treatment with toxic agents or irradiation therapy (both the parents of the fetuses and the cadavers). Moreover, each parent or cadaver had 20 teeth or more (mastication capacity standard), and they supported themselves in daily life (biceps brachii capacity standard). The causes of death did not directly or indirectly influence the muscular or nervous system, so the muscles were considered to be normal. The preparation of sections involved fixation, washing, dehydration, embedding, and sectioning, as described in our previous report [5]. All the cadavers were fixed with a 10% solution of formalin (3.7% formaldehyde) within 24 h of postmortem. After resecting the muscle, a 10% solution of formalin (3.7% formaldehyde) was used for immersion for at least a week. The solution was changed once in the first 30–60 min, and again later if desired. The formalin-fixed materials were then transferred, without washing, to the secondary fixative to be held at room temperature for two weeks. If the solution became turbid or precipitated it was changed. After this, the fixation was continued at 37 8C for an additional week. The volume of fixative used was at least ten times the volume of the specimens. In this process, materials had been fixed with pins at four corners of the board. After washing, dehydration, and celloidin embedding, we cut sections 15-mm thick and stained them with hematoxylin and eosin (H&E).
Fig. 2. A high-power view of the muscle fibers in buccinator muscle staining with H&E. 6-Month-old male fetus; scale bar = 100 mm.
Germany), and a computer (Precision 530, Dell, Austin, TX, USA) with analyzing system software (analySIS 3.0, Soft Imaging System, Mu¨nster, Germany) for storing data on-line, calculations, and statistical analyses.
2.1. Morphometry 3. Results We observed the microscopic section at low power, and covered the entire area of the distributed muscle fibers in the section by moving the eyepiece grid vertically and horizontally as described in our previous report [6]. We confirmed that we could distinguish muscle fiber structures from other tissues with both a computer and the naked eye in every grid. We counted the muscle fibers and measured the transverse area of the muscle fibers in a square eyepiece grid at high power (Figs. 1 and 2), and then calculated the number of muscle fiber per mm2 (NMF) and the average transverse area of the muscle fibers (TAMFs). To avoid duplicate counts, we counted and measured all muscle fibers on the side of the grid that did not come into contact with the other grids. In the case of grids adjacent to the other grids, we counted and measured only the muscle fibers on the lower right side of the grid, not those on the upper left side. We used a microscope in a transmitted light mode (BX50, Olympus, Tokyo, Japan) equipped with a high-resolution digital camera (ColorView12, Soft Imaging System, Mu¨nster, Germany), a motorized XYZ stage (Ma¨rzha¨user, Wetzlar-Steindorf, Germany), a stage controller (Ma¨rzha¨user, Wetzlar-Steindorf,
3.1. Number of muscle fibers per mm2 (NMF) We counted the muscle fibers and calculated the number of muscle fibers per mm2 (NMF). Fetuses had a higher NMF than did adults in three kinds of muscle (Fig. 3). The average NMF for the buccinator, masseter and biceps brachii muscles in the three fetuses were 22,849 4798 (mean SD, and so on), 43,874 13,162, 26,173 7852, respectively. As for the adults, the average NMF for the buccinator, masseter and biceps brachii muscles in the 11 cadavers were 1177 248, 1184 506, 1180 460, respectively. The average NMF for the buccinator, masseter and biceps brachii muscles in fetuses showed, respectively, 19, 37, and 22 times as many as those in adults. 3.2. The average transverse area of the muscle fibers (TAMFs) We calculated the average transverse area of the muscle fibers (TAMFs). Fetuses had a smaller TAMF than did adults in three kinds of muscle (Fig. 4). The average TAMFs for buccinator, masseter and biceps brachii muscles in the three fetuses were 18.1 4.5 (mean SD, and so on), 14.5 3.1, 29.3 10.3 mm2, respectively. As to adults, the average TAMF for the buccinator, masseter and biceps brachii muscles in the 11 cadavers were 448.1 108.7, 602.8 230.4, 722.9 301.6, respectively. The average fetus/adult ratios of the TAMF for the buccinator, masseter and biceps brachii muscles were 4.0%, 2.4%, 4.1%, respectively. 4. Discussion
Fig. 1. A high-power view of the muscle fibers in buccinator muscle staining with H&E. 43-Year-old man; scale bar = 100 mm.
The number of muscle fibers appears to be genetically determined, showing no significant change after birth in most animals [1,7]. It is evident that the number of muscle fibers in a given muscle increases before birth until the genetically determined number is attained. Stickland’s investigation of human fetuses showed that at the same timing as myotube appearance being lost, at around 6-months of age, the number of muscle fibers per unit area changed from an increasing trend to a marked decrease [8]. Reports as described above indicated that there is an initial slow increase in the total numbers of muscle fibers up to
Please cite this article in press as: H. Moriyama, et al., Morphometric aspects of the facial and skeletal muscles in fetuses, Int. J. Pediatr. Otorhinolaryngol. (2015), http://dx.doi.org/10.1016/j.ijporl.2015.04.009
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Fig. 3. Comparison of the number of muscle fibers per mm2 between adults and fetuses. Red bars indicate the number of muscle fibers per mm2 in fetuses. Blue bars indicate the number of muscle fibers per mm2 in adults. Each value is the mean SD. Each italic number with times indicates the ratio of fetus/adult. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
around 3 or 4-months of age, followed by a very rapid increase up to around 6-months of age, after which the number reaches its peak in life-span at around birth. The number of muscle fibers per unit area, on the other hand, changes from an increasing trend to a marked decrease at around 6-months of age [8]. Researchers reported that this distinct change must be due partly to a slowing down in the decrease of intercellular space and to the commencement of a more rapid muscle fiber hypertrophy phase [8–10]. We supposed that the NMF reached its peak in life-span at around 6month-old for the reports mentioned above. We compared the data of 6-month-old fetuses with those of adults. Our data showed that the fetus had a higher NMF than did the adult, however, the ratio of the adult/fetus NMF depended on the muscle kinds (Fig. 3). The average of the NMF for the buccinator, masseter and biceps brachii muscles in fetuses showed, respectively, 19, 37, and 22 times as many as that in adults. Joubert reported that different
muscles developed at varying rates at the cellular level [11]. It is one of the items of evidence that the NMF reaches its peak in lifespan with variance depending on the kind of muscles. We also considered that all three individual muscles indicate their peak in the NMF in life-span at around 6-month-old, however, the decrease of the NMF with the aging process varies with the kind of muscles. It is known from several studies on animals that muscle fibers can be affected by many factors such as age [7], exercise [12] and level of nutrition [13]. With regard to muscle fiber size, our data showed that the fetus had a smaller TAMF than did the adult (Fig. 4). Researchers reported that there is an enlargement of the muscle fiber as animals mature [1,14,15]. Rowe and Goldspink reported that 129/ Re strain mouse showed an enlargement of the muscle fiber diameter until the age of three to nine weeks, varying with the kind of skeletal muscles [1]. As for human skeletal muscle, researchers
Fig. 4. Comparison of the average transverse area of muscle fibers between adults and fetuses. Red bars indicate the average transverse area of muscle fibers (mm2) in fetuses. Blue bars indicate the average transverse area of muscle fibers (mm2) in adults. Each value is the mean SD. Each italic number indicates the percentage of fetus/adult. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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reported that muscle fiber size showed a significant reduction with age [2]. They used the vastus lateralis muscles from physically healthy men between 15 and 83 years of age, and they also reported that the average reduction in the muscle fiber size from 20 to 80 years is 26%. Vogler and Bove also reported that the rate of muscle fiber growth in infants and children varied with age and muscle [16]. We supposed that fetal movements and behavior were one of the factors behind the fetus/adult ratio of the TAMF varying with the kind of muscles (Fig. 4). Isolated arm movements without other parts moving were observed in fetuses from eight weeks, and hand-face contact observed from 10 weeks [17]. The biceps brachii muscle takes part in these movements. Sucking (indicating that the fetus is drinking amniotic fluid) was observed from 12 weeks [17], and the buccinator muscle takes part in sucking. Moreover, yawn-like movement that prolonged wide opening of the jaws followed by quick closure often with retroflexion of the head and sometimes elevation of the arms was observed from around 14 or 15 weeks [17]. The masseter muscle takes part in this yawn-like movement. Goldspink reported that the size of muscle fibers could be affected by exercise [12]. The average fetus/adult ratio of the TAMF of biceps brachii, buccinator, and masseter muscles showed 4.1%, 4.0%, 2.4%, respectively in this study. Therefore, our results showed that the order of the size of muscle fibers in three kinds of muscles agreed with the order of the emergence of the fetal movements that their muscles took part in. Next, we compared the developmental characteristics among the three kinds of muscles. Mammalian skeletal muscle cells are known to derive from the paraxial mesoderm adjacent to the neural tube. The paraxial mesoderm in the body forms clear segmental structures, which are referred to as somites. The forelimb, including the biceps brachii muscle of the chick, is derived from somites 15–20 [18]. On the other hand, the head paraxial mesoderm is composed of seven incompletely segmented mesenchymal structures, referred to as the somitomeres. Chick/ quail graft transplantation experiments have demonstrated the developmental origins of chick head muscles [18,19]. Most of the head muscles originate from the somitomeres, the buccinator and masseter muscles are derived from somitomeres 6 and 4, respectively [18,20]. To relate the development of the muscle to the development of function, it is necessary to study the progression of myogenesis and synaptogenesis. During synaptogenesis, the expression level, distribution, and subunit composition of the nicotinic acetylcholine receptor (nAChR) are drastically changed. In the development of the mouse masseter muscle, the differentiation of myoblasts occurs most actively between embryonic day (ED) 13 and birth, and the active maturation of myofibers begins at around ED15 and continues after the shift of feeding behavior from suckling to biting at around four weeks of age [21]. Moreover, the nAChR switch and elimination begin at around ED17 and one week of age, respectively, and they are nearly complete after the shift of feeding behavior at around three to four weeks of age [21]. During the development of the mouse limb muscle, differentiation of myoblasts occurs most prominently between ED13 and birth, and the active maturation of myofibers is initiated at around ED15 and ends at around two weeks of age [21]. Furthermore, the nAChR switch and elimination occur most prominently between birth and two weeks of age [22,23]. Considering the circumstances mentioned above, the myogenesis and synaptogenesis of masseter muscle are delayed in comparison with limb muscles, and the maturation of myofibers and synaptogenesis, such as the nAChR switch and elimination, still continue after the shift in feeding behavior from suckling to biting at approximately three weeks of age. Our data in fetuses showed the average of the TAMF of the masseter muscle (14.5 mm2; indicated mean, and so on) smaller than the biceps brachii muscle (29.3 mm2) (Fig. 4). As there is an enlargement of the muscle fiber
as the animals mature [1,14,15], we suppose that muscle fiber size tends to depend on the myogenesis and synaptogenesis of the muscles. Moreover, the average of the TAMF of masseter muscle in fetuses (14.5 mm2) was also smaller than the buccinator muscle (18.1 mm2) (Fig. 4). This difference in the progress of myogenesis and synaptogenesis between the masseter and buccinator muscles seems to depend on the difference in the functional demands of each type of muscle. Since the masseter muscle mainly functions in the biting movement of the jaw, the completion of myogenesis and synaptogenesis is probably unnecessary until biting movements begin. On the other hand, myogenesis and synaptogenesis in the buccinator muscle seems to progress more quickly to meet functional demands such as suckling and swallowing of milk immediately after birth. 5. Conclusions The average of the NMF of every buccinator, masseter and biceps brachii muscles decreased after birth, however, its peak in life-span or the decrease of that with the aging process tended to vary with the kind of muscles. With regard to the average of the TAMF, that of every buccinator, masseter and biceps brachii muscles enlarged after birth. We considered that the enlargement of the average TAMF was closely connected with the emergence of the fetal movements that their muscles took part in, and was also connected with functional demands after birth such as suckling, swallowing of milk, and biting movements. Conflicts of interest We declare that we have no conflicts of interest. Acknowledgements We thank Ms. Ikuko Moriyama for assistance in preparing the manuscript. This work was supported by a Grant-in-aid for Scientific Research B14370007 from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] R.W.D. Rowe, G. Goldspink, Muscle fibre growth in five different muscles in both sexes of mice. I. Normal mice, J. Anat. 104 (1969) 519–530. [2] J. Lexell, C.C. Taylor, M. Sjo¨stro¨m, What are the causes of ageing atrophy? Total number, size and proportion of different fiber types studied in whole vastus lateralis muscle from 15- to 83-year-old men, J. Neurol. Sci. 84 (1988) 275–294. [3] A.W. Racca, A.E. Beck, V.S. Rao, G.V. Flint, S.D. Lundy, D.E. Born, et al., Contractility and kinetics of human fetal and human adult skeletal muscle, J. Physiol. 591 (2013) 3049–3061. [4] A. Shimamura, Age determination and physical measurement of Japanese embryo, Jpn. J. Legal. Med. 11 (1957) 795–811. [5] H. Moriyama, K. Shimada, N. Goto, Morphometric analysis of neurons in ganglia: geniculate, submandibular, cervical spinal and superior cervical, Okajimas Folia Anat. Jpn. 72 (1995) 185–190. [6] H. Moriyama, K. Shimada, M. Itoh, T. Takahashi, N. Otsuka, Morphometric analysis of the inferior alveolar nerve fails to demonstrate sexual dimorphism, J. Oral Maxillofac. Surg. 65 (2007) 1555–1561. [7] N.C. Stickland, G. Goldspink, A possible indicator muscle for the fibre content and growth characteristics of porcine muscle, Anim. Prod. 16 (1973) 135–146. [8] N.C. Stickland, Muscle development in the human fetus as exemplified by m. sartorius: a quantitative study, J. Anat. 132 (1981) 557–579. [9] J.W. Dickerson, E.M. Widdowson, Chemical changes in skeletal muscle during development, Biochem. J. 74 (1960) 247–257. [10] E.M. Widdowson, Changes in the extracellular compartment of muscle and skin during normal and retarded development, Bibl. Nutr. Dieta 13 (1969) 60–68. [11] D.M. Joubert, A study of pre-natal growth and development in sheep, J. Agric. Sci. 47 (1956) 382–428. [12] G. Goldspink, The combined effects of exercise and reduced food intake on skeletal muscle fibres, J. Cell. Physiol. 63 (1964) 209–216. [13] N.C. Stickland, E.M. Widdowson, G. Goldspink, Effects of severe energy and protein deficiencies on the fibres and nuclei in skeletal muscle of pigs, Br. J. Nutr. 34 (1975) 421–428.
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Please cite this article in press as: H. Moriyama, et al., Morphometric aspects of the facial and skeletal muscles in fetuses, Int. J. Pediatr. Otorhinolaryngol. (2015), http://dx.doi.org/10.1016/j.ijporl.2015.04.009