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International Journal of Morphology

versión On-line ISSN 0717-9502

Int. J. Morphol. v.21 n.4 Temuco  2003

http://dx.doi.org/10.4067/S0717-95022003000400006 

Int. J. Morphol., 21(4):291-298, 2003.

HIGH RESOLUTION SEM OF INTRINSIC MUSCLE FIBERS
OF ANTERIOR THIRD RET'S TONGUE

MEB DE LATA RESOLUCIÓN DE LAS FIBRAS DE LOS MÚSCULOS INTRÍNSICOS
DEL TERCIO ANTERIOR DE LA LENGUA DE RATAS

*Aracy A. Motoyama; *Ii-sei Watanabe & **Koichi Ogawa


MOTOYAMA, A. A.; WATANABE, I. & OGAWA, K. High-Resolution SEM of intrinsic muscle fibers of anterior third rat's tongue. Int. J. Morphol., 21(4):291-298, 2003.

SUMMARY: The characteristics of intrinsic muscle fibers in the anterior third of the adult rat tongue were studied employing light microscopy, SEM and high-resolution SEM (HRSEM) methods. For light microscopy, specimens were fixed in Bouin's solution and embedded in paraffin. In order to identify the muscle fibers and bundles of collagen fibers, frontal sections were stained with hematoxylin-eosin, azo-carmin and picro-sirius. The results showed that muscle fibers near the lamina propria are fixed with connective tissue, constituting several groups. The bundles of muscle fibers are arranged in longitudinal, vertical and transversal directions. For SEM, specimens were fixed in modified Karnovsky solution and freeze-fractured in liquid nitrogen revealed the collagen fibers and bundles of muscle fibers located in three-dimensional aspects. The samples treated in NaOH solution showed the original location of collagen fibers, constituting complex networks in three-dimensional SEM images. Under HRSEM, fractured specimens in DMSO revealed basement membrane of muscle cell containing sponge-like structures and networks of fine collagen fibrils. Cytoplasmic organelles, such as mitochondria with cristae mitochondriales and sarcoplasmic reticulum, were cleary seen in three-dimensional HRSEM images

KEY WORDS: 1. Muscle fibers; 2. Tongue; 3. Rat; 4. HRSEM; 5. Mitochondria; 6. Basement membrane.


INTRODUCTION

Several authors have studied connective tissue, muscle cells and other structures in the tongue muscle of rats (Orlandini, 1967; Sato et al., 1989), human fetuses (Bell, 1970; Barnwell et al., 1978a,b; Sato & Sato, 1992), Macaca fuscata-fuscata (Nakamura & Okada, 1992), Macaca fascicularis (DePaul & Abbs, 1996), and dogs (Ohtani et al., 1988). The rat tongue muscle spindle has also been studied (Smith, 1989).

Muscle fibers have been classified as red (rich), intermediate (moderate) or white, according to their size and the number of mitochondria present (Sato et al.). Based on histochemical features, they were classified as slow oxidative fiber, fast glycolytic fiber or fast glycolytic-oxidative fiber (Stein & Padykula, 1962; Henneman & Olson, 1965; Peter et al., 1972; Smith). The intrinsic muscle fibers of the tongue consist of numerous red and intermediate fibers. The majority of red fibers are found in the vertical and longitudinal bundles (Sato et al.). Using the ATPase method, muscle fibers of the tongue can be classified as type I (slow) or type IIA and IIB (fast). Of type II fibers, 99% were categorized as IIA (DePaul and Abbs). The anterior third of the tongue was composed almost completely of fast glycolytic-oxidative fibers (Smith).

The bundles of muscle fibers are surrounded by a connective tissue layer (perimysium), while each muscle fiber is covered by an endomysium, consisting of connective tissue, which is arranged in reticular fibers (MacConnachie et al., 1964; Nishimura et al., 1994). The connective tissues constitute each organ´s skeletal framework, which reflects the function of the organ. In 1987, Ohtani (1987) introduced a cell-maceration method with a low-temperature NaOH solution for SEM observations of connective tissue fiber arrangement. Ohtani et al. demonstrated that the collagen fibrillar network of the endomysium consists of two layers: the inner layer, made up of collagen fibrils running in two opposing helices along the muscle fibers, and the outer layer of bundles of collagen fibrils winding in longitudinal courses. The endomysium is a matrix that transfers tension among myofibrils during contraction and relaxation (Trotter & Purslow, 1992; Vasilev et al., 1995).

The present paper describes the morphological features of the intrinsic muscle fibers of the anterior third of the rat tongue revealed by light microscopy, SEM and HRSEM methods, evidencing the connective tissue and intracellular components of muscle cells.

MATERIAL AND METHODS

For light microscopy, two adult Wistar rats, weighing 200 to 250g, were used. The animals were anesthetized by intraperitoneal injection of 30mg/kg pentobarbital sodium, sacrificed and the tongues were excised carefully. Specimens were fixed in Bouin's solution for 48h at room temperature, then rinsed in water for 72h and dehydrated in graded ethanol series and embedded in paraffin. Frontal sections (5mm in thickness) were cut in a microtome (Young) and stained with hematoxilyn-eosin and azo-carmim to show the muscle fibers and connective tissue. The sections stained with picro-sirius permitted the identification of endomysium and perimysium collagen fiber bundles by polarized light microscopy according to the Junqueira et al.(1978).

For SEM, four adult rats were anesthetized by intraperitoneal injection of 30mg/kg pentobarbital sodium and the thoraxes of the animals were opened to expose the heart. A needle was inserted into the left ventricle for perfusion with 0.9% physiological saline solution and with modified Karnovsky solution containing 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1M sodium phosphate buffer (pH 7.4). The tongues were fixed in the same solution for 24h at 4°C, then rinsed in distilled water. After fracturing in liquid nitrogen, some specimens were treated in 10% NaOH solution for three to five days at room temperature (Ohtani, 1987). The specimens were rinsed in distilled water, post-fixed in 1% osmium tetroxide for 2h at 4°C and immersed for 1h in 2% tannic acid solution. Dehydration was made in a graded ethanol series, and critical-point in a Balzers CPD-030 apparatus using liquid CO2. The samples were mounted on metal stubs, coated with gold and examined under a scanning electron microscopy JEOL, JSM-6,100 at 10 kV.

For HRSEM, samples from two rats were prepared using the DMSO method (Tanaka, 1980; 1989, Tanaka &
Mitsushima, 1984; Watanabe et al., 1992). Small tissue samples (3mm in length) were removed from the animals and fixed in 2% buffered osmium tetroxide solution for 2h at 4C. The tissues were rinsed with distilled water and then successively immersed in 12.5%, 25% and 50% DMSO solution for 30min each. The specimens were frozen on a metal plate, chilled with liquid nitrogen. They were then split in a freeze-fracture apparatus (TF-2, Eiko Engineering Co. Ltd., Japan), using a razor blade and hammer. The split pieces were immediately placed in 50% DMSO and thawed at room temperature. The tissues were rinsed with distilled water and post-fixed in 2% osmium tetroxide solution for 2h at 4°C. The specimens were again washed in distilled water and treated with 1% tannic acid solution for 2h at room temperature They were then dried in an Eiko ID-2 critical-point dryer, coated with palladium-gold using the BIO-RAD-SEM Coating System (Japan) and observed with an HRSEM (Hitachi, S-900) at 10 kV at Department of Anatomy, Fukuoka University School of Medicine, Japan.

RESULTS

In the anterior third of the rat tongue, the vertical, longitudinal and transverse muscle fibers and collagen fibers were observed. The histological sections showed a superficially stratified keratinized epithelium and the connective tissue layer of the lamina propria (Fig.1). The adjacent area contained muscle fibers arranged in vertical and longitudinal directions. Transverse bundles of muscular fibers were observed in the deep layer (Fig. 1).

Collagen fibers were observed covering the muscle fibers (perimysium). A thin layer of collagen fibers surrounding each individual muscle fiber composing the endomysium (Figs. 1 and 2) was also noted. At high magnification, the peripheral nuclei of the muscle cells were seen (Fig. 2).

Polarized light microscopy examination showed the presence of the muscle fiber bundles separated by collagen fibers and connective tissue of the lamina propria (Fig. 3). The muscle cells were surrounded by intensive refractive connective tissue delimiting spaces of various sizes (Fig. 4).


Fig 1 Light microscopy showing the muscle fiber bundles of the anterior third of the rat tongue. Collagen fibers in the lamina propria (\) and muscle fiber groupings are seen (arrows). Azo carmin X88
Fig 2 Muscle cells containing periphery nuclei are clearly shown. (arrows). HE X585
Fig 3 Polarized light microscopy view showing collagen fiber bundles surrounding the muscle fibers (perimysium) (arrow). Picro-sirius X220.
Fig 4 Polarized light microscopy view showing a network of variable spaces (\) and revealing collagen fibers in the endomysium (arrows). Picro-sirius X585.

The fractured surface shows an epithelial layer, connective tissue of the lamina propria, and the directions and insertions of the adjacent muscle fibers (Fig. 5). In fractured frontal sections, the three-dimensional aspect of the muscle fibers surrounded by dense connective tissue can be seen (Fig. 6). Transversal fractured muscle fibers were observed with elongated form and the endomysium colagens fibers network surrounding the muscle basal lamina (Fig. 7). At high magnification, the interior of each muscle fiber presented fibers connected to the cell membrane, and a longitudinal arrangement of myofibrils was observed (Fig. 8).

The collagen fiber network that covers the muscle fibers form trabeculae delineating the original spaces of each muscle fiber. The three-dimensional network of collagen fibers that surround each muscle fiber (endomysium) not only serves as a framework, but also connects the adjacent muscle fibers. This gives the whole a honeycomb-like appearance (Fig. 9). High magnification shows a layer of reticular fibers in the small blood vessel walls (Fig. 10).


Fig. 5. General view of fractured rat tongue surface showing an epithelial layer (\), connective tissue (arrows) and muscle fiber bundles disposed in several directions. X42.

Fig. 6. Three-dimensional SEM image of fractured muscle fibers (large arrows) and capillaries (small arrows). X515.
Fig. 7. Transversal fractured shows the muscle fibers surrounded by bundles of collagen fibers. X2.000.
Fig. 8. Image under SEM of fractured muscle fiber surface, revealing the longitudinal disposition of myofilaments. X3.100.
Fig. 9. Images under SEM of specimens treated with NaOH solution. Note original disposition of transversal collagen fiber sections in the endomysium (arrows). X210.
Fig. 10. Images under SEM of specimens treated with NaOH solution, showing collagen fiber bundles disposition in capillary walls (\) X1.240.

The general aspect of the longitudinal fractured surface of muscle cells presents bundles of myofilaments (Fig. 11) which reveal intermingled mitochondria (Fig. 12). The external muscle cell surface revealed a smooth area in the basement membrane (Fig. 13). The reticular collagen fibrils were noted on the surface of the basal lamina (Fig. 14). The HRSEM images revealed a sponge-like structures containing fine collagen fibrils (Fig. 15). Numerous mitochondria were seen in the muscle cell cytoplasm (Fig. 16). At high magnification, mitochondria were observed to have a column-like disposition (Fig. 17) between the myofilaments bundles (Fig. 18). In HRSEM images, the cristae mitochondriales were cleary seen (Fig. 19).


Fig. 11. Muscle fiber surface freeze-cracked with DMSO. Image under HRSEM reveals the intracellular structures arranged in parallel directions. X.800.
Fig. 12. Sarcoplasm freeze-cracked with DMSO. Mitochondria (small arrows) are vertically arranged between the bundles of myofilaments (large arrows). X5,800.
Fig. 13. Muscle fiber surface freeze-cracked with DMSO and macerated in diluted solution of osmium. Shows the internal cytoplasmic structures and the basement membrane (\) containing a network of collagen fibers. X16.200.
Fig. 14. Basement membrane displaying a meshwork structure with thin and thick collagen fibrils in a three-dimensional SEM image (arrow). X18.000.
Fig. 15. At high magnification, the basement membrane shows a sponge-like structure and a few collagen fibrils (arrows). X110.000.
Fig. 16. Image under HRSEM reveals a number of large and small mitochondria (M). X18.000.


Fig. 17. Muscle cell surface freeze-cracked with DMSO. Mitochondria (\) are visible in column-like alignment. X32.400.
Fig. 18. Muscle cell surface freeze-cracked with DMSO. Observe the three-dimensional arrangement of myofilaments bundles. X32.400.
F


Fig. 19. At high magnification. cristae mitocondriales are projected from the internal layers of the membrane The lamellae of cristae mitochondriales are clearly visible (arrows). X81.000.

DISCUSSION

Ours results demonstrated the location of muscle fibers bundles in the anterior third of the rat tongue. The bundles were either isolated or in groups and were located adjacent to the dense connective tissue layer of the lamina propria. These fibers are very closely related the mucous layer, as previously established by Bell. The numerous muscle fiber bundles present in longitudinal, vertical and transversal direction were different in that the number of fibers was variable. The number of vertical muscle fiber bundles was relatively small. Variation in groups of muscle fibers and in type of skeletal muscle fibers have also been reported by several authors (Stein & Padykula; Henneman & Olson; Padykula & Gauthier, 1967; Peter et al.).

The muscle cell nuclei present themselves as round or cylindrical in shape and are located at the periphery, forming a bulge in the sarcolemma (MacConnachie et al.; Enesco & Puddy, 1964).

The tongue presents a complex distribution of connective tissue (endomysium and perimysium), which is basically composed of collagen fibers. These collagen fibers play an essential role in determining the shape and the mechanic and viscoelastic proprieties of the connective tissue (Vasilev et al.). Ours results show that the endomysium contains bundles of collagen fibers constituting an interlacing network, with thin nerves and capillaries. This framework was composed of types I and III collagen fibers. Type I collagen presented a yellow, orange or red color, while type III collagen appeared green. However, precise characterization of type III collagen is difficult because it is always accompanied by type I (Junqueira et al.). The collagen fiber network consists of filaments of 20 to 50nm in diameter (Mauro & Adams, 1961; Boyde & Willians, 1968; Ishikawa et al ., 1982; Watanabe et al., 1992; Vasilev et al.).

In HRSEM images, the sarcolemma lamina was seen to be adhering to the sarcoplasm and the collagen fiber network of the endomysium was observed to be in close proximity to the sponge-like structure of the basal lamina.

The endomysial collagen fibers observed in HRSEM presented a three-dimen sional network external to the sarcolemma basal lamina. We can suggest that this collagen fiber network assists in the uniform distribution of tensile forces during contraction and relaxation, thereby preventing muscle fatigue (Vasilev et al., 1995).

Our data also demonstrates the disposition of sarcoplasmic components such as mitochondria, myofilaments and sarcoplasmic reticulum. The mitochondria were located longitudinal along the myofilaments, and presented a round or oval shape (Lea and Rollenberg, 1989; Lea et al., 1994). The inner and outer mitochondrial membranes and the cristae mitochondriales were either lamellar or tubular in shape (Tanaka). An intermembranous space separated the outer membrane from the inner membrane, and a central lumen continous with that space was apparent (Lea & Rollenberg). The mitochondrial cristae were observed either running from one side to another along the membrane or fusing with other cristae along the way while moving in a posterior direction (Tanaka, 1980; Watanabe et al.). At high magnifications, many spherical-shaped particles were observed attached to the surface of the mitochondrial cristae, corresponding to the inner membrane, which contains ATPase (Tanaka, 1989).

ACKNOWLEDGMENTS: This paper was supported by CAPES, a Brazilian research foundation.


MOTOYAMA, A. A.; WATANABE, I. & OGAWA, K. Microscopía electrónica de barrido de alta resolución de las fibras de los músculos intrínsecos del tercio anterior de la lengua de ratas. Int. J. Morphol., 21(4):291-298, 2003.

SUMMARY: Las características de las fibras musculares del tercio anterior de la lengua de la rata adulta fueron estudiadas em- pleando microscopio de luz, MEB y métodos de alta resolución de MEB (ARMEB). Para la miscroscopía de luz, los especímenes fueron fijados en solución de Bouin e incluidos en parafina. Con el propósito de identificar las fibras musculares y los haces de fibras colágenas, cortes frontales fueron teñidos con hematoxilia-eosina, azo-carmin y picro-sirius. Los resultados mostraron que las fibras musculares cerca de la lámina propia están fijos con tejido conectivo, constituyendo varios grupos. Los haces de fibras musculares están dispuestos en direcciones longitudinal, vertical y transversal. Para la MEB, los especímenes fueron fijados en solución de Karnovsky modificada y fracturadas en frío en nitrógeno líquido revelándose las fibras colágenas y fascículos de fibras musculares dispuestas en tres dimensiones. Las muestras tratadas en solución de NaOH mostraron la original localización de las fibras colágenass, constituyendo complejas redes en imágenes tridimensionales de MEB. A través de ARMEB, los especímenes fracturados en DMSO revelaron la membrana basal de la célula muscular conteniendo estructuras tipo esponja y finas redes de fibras colágenas. Organelos citoplasmáticos, tales como mitocondrias con sus crestas y retículo sarcoplasmático, fueron claramente observados en imágenes tridimensionales con ARMEB.

KEY WORDS: 1. Fibras musculares; 2. Lengua; 3. Rata; 4. ARMEB; 5. Mitocondria; 6. Membrana basal.


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Correspondence to:
Prof. Dr. Ii-sei Watanabe
Department of Anatomy
Institute of Biomedical Sciences
University of São Paulo
Av. Prof. Lineu Prestes, 2415.
CEP 05508-000
São Paulo, BRAZIL

Tel. 3091-7386. Fax: 3091-7366.

E-mail: watanabe@icb.usp.br.

Received : 06-10-2003
Accepted: 10-11-2003


* Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.

** Department of Anatomy, Fukuoka University School of Medicine, Japan.

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