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International Journal of Morphology

versión On-line ISSN 0717-9502

Int. J. Morphol. v.25 n.2 Temuco jun. 2007

http://dx.doi.org/10.4067/S0717-95022007000200008 

 

Int J. MorphoL, 25(2):295-299, 2007.

 

Histoenzimological Characterization of the Masseter Muscle, Superficial Bundle, in Guinea-Pigs After Malocclusion Induction

Caracterización Histoenzimológica de la Capa Superficial del Músculo Masetero, en Guinea-Pigs Después de Maloclusión Inducida

 

*Joáo Paulo Mardegan Issa; * Rodrigo Tiossi & **Mamie Mizusaki Iyomasa

* Graduate student, Faculty of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil.
**Professor, Faculty of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil.

Dirección para correspondencia


SUMMARY: The aim of this study was to analyze the histoenzymological alterations in the masseter muscle, superficial bundle, in guinea pigs induced by the occlusal alteration. The present study permitted to conclude that the masseter muscle, superficial bundle, in guinea-pigs was little sensible to chewing functional alteration in this period of time.

KEY WORDS: Masseter muscle; Histoenzymologic study, Chewing alteration.

RESUMEN: El objetivo de este estudio fue analisar las alteraciones mstoenzimológicas de la capa superficial del músculo maseter, inducidas por la alteración oclusal. Los resultados nos llevan a concluir que la capa superficial del referido músculor, en guinea-pigs, es poco sensible a la alteración funcional masticatoria.

PALABRAS CLAVE: Músculo masetero; Estudios histoenzimológicos; Alteración masticatoria.


INTRODUCTION

A reduction in the recruitment of muscle fibers due to a decreased masticatory load may alter the morphological and histoenzymological characteristics of the masticatory muscles. Craniofacial skeletal changes caused by different food consistencies (Luca etal., 2003) and altered proportions of muscle fibers in the masticatory muscles (Engstrom et al, 1986;Maedae?a/., 1987;Kiliaridise/'a/., 1988; Miyata etal, 1993; Sfondrini etal., 1996; Miehe etal, 1999) have previously been described, although the results of these studies are not necessarily consistent. Kiliaridis et al, revealed a reduced frequency and smaller size of type IIA fibers in the masseter of rats administered a soft diet than in those receiving a normal diet, but found no changes in the digastric muscle. Maeda et al, demonstrated that the fiber sizes of the masseter muscle and muscle spindles were significantly decreased in mice kept on a soft diet. Further, extraction of lateral teeth in combination with a soft diet has been reported to result in a shift in the muscle fiber composition towards the fastest fiber-type in rats (Miehe et al, 1999). However, Sfondrini et al, did not find any change in the masseter muscles but did find changes in the temporalis and digastric muscles in rats. However, these studies used rats or mice as experimental subjects, and all fiber-type transitions observed in these studies occurred within the subpopulation of fast (type II) fibers, rather than between slow (type I) and fast (type II) fibers, because in the jaw elevator muscles of rats and mice, the vast majority of the fibers are of type II (IIA and IIB), and only a few type I fibers are recognized, whereas, humans have a considerable proportion of type I fibers in the masseter muscles (Eriksson «fcThornell, 1983).

Thus, the aim of this study was to analyze the histoenzymological alterations in the masseter muscle, superficial bundle, in guinea pigs induced by the occlusal alteration in order to provide a better understanding of the effects of functional disorders on the structures of the stomatognathic system.

MATERIAL AND METHOD

This study used 10 adult male guinea pigs (Cavia porcellus), with an average weight of 450g, maintained at the University Animals House, under controlled temperature, with commercial chow and water available ad libitum. Half of these animals received the occlusal alteration induction and the other half was considered as control, only submitted to the surgical stress. This work was approved by the Ethics Committee on the Use of Animals in Experimentation of the University (CEUA).

Induction of occlusal alteration.The animals were anaesthetized with tribromoethanol (0.25g/kg body weight) and submitted to extraction of the upper molars at the left hemiarch, with animals receiving the antibiotic Pentabiotic (24000IU) as preoperative prophylaxis. After asepsis and disinfection of the surgical site with PVPI, the teeth extraction was performed with anatomical tweezers and hollenback 3 S. The extraction area was compressed to stop bleeding (Chompret maneuver). After surgery, the animals received anti-inflammatory and analgesic drugs (sodium diclofenac- 0.04mL/100g) and were maintained in appropriate cages for two months.

Sacrifice and animals' perfusión. After two months, the animals were anaesthetized with urethane 37.5% (0.4mL/ lOOg body weight) and submitted to perfusión. This procedure involves an intracardiac infusion of saline solution (lOOmL) followed by 10% formalin and paraformaldehyde 4% in phosphate buffer 0.2M (1 OOmL). The superficial bundles of the masseter muscles were removed for histoloenzymological processing.

Specimen preparation for histoenzymological analysis. After two months, the animals were anaesthetized with urethane overdose (3g/kg body weight). The superficial bundles of the masseter muscle were carefully removed, immediately weighted following Scherle (1970) method, and then made the orthogonal cleavages, which were destined to histoenzymological study. The masseter muscle samples were washed in physiological saline and immersed in N-hexane at -70°C for 1 minute. After this period of time the material was stored in a freezer at -80°C, until transferred to a cryostat microtome at -20°C, where the 10 µm thick cuts were made. Some cuts were dyed in hematoxylin-eosin and others submitted to reactions for the following enzymes: succinate dehydrogenase (Nachlas et al, 1957), nicotinamide adenine dinucleotide diaphorase (Novikoff et al, 1961), and myofibril ATPase at pH 9.4 after pre-incubation in alkaline medium (pH 10.4) and in acid medium (pH 4.6) (Guth & Samaria, 1969). Based on enzyme reactivity, the fibers were catalogued according to Peter et al. (1972) nomenclature in: Slow Oxydative (SO), fast glycolitic (FG) and fast oxydative glycolitic (FOG).

RESULTS

The reactions with ATPase after alkaline pre-incubation at pH 10.4 of the masseter muscle, superficial bundle, in the control group showed intermediate reactivity fibers although with a different intensity of reaction among them, beside low reactivity fiber (Fig. 1a). On both sides, the fibers showed low or intermediate reactivity on the superficial region from the middle portion of the muscle. After acid (pH 4.6) pre-incubations in depth of the muscle some high reactivity small fibers were observed (Fig. 1b).

The reaction with NADH and SDH evidenced in the control group a similar way the fibers with intermediate reactivity varying in a small quantity the reactivity degree (Figs, 1c and 1d); however, it is observed, depth in the muscle, scarce fibers with intense reactivity (Fig. 1d). No significant difference was observed between the left and the right sides.

In the guinea pigs submitted to surgery, the masseter muscle on the right side, after processed by ATPase with alkaline pre-incubation, revealed intermediate activity fibers on the superficial region from the middle portion of the masseter muscle and some with high reactivity spread as mosaic in a highly restricted area, deeper in the muscle after acid pre-incubation (Figs, 1e and 1f).

The reaction with NADH and SDH evidenced in a similar way the fibers with intermediate reactivity varying, in a small quantity, the reactivity degree in the superficial region from the middle portion of the right masseter muscle (Figs, 1g and 1h), in the occlusal altered group.

The middle portion of the left masseter muscle, animals submitted to surgery, it was found on the superficial region, fibers with intermediate reactivity after alkaline pre-incubation (Fig. li) and in depth, some with high reactivity showing different intensity (Fig. 1j) after acid (pH 4.6) pre-incubation. As judged from the reaction for NADH and SDH, the reactivity of the fibers decreased (Figs. 1k and 1l).


Fig. 1a. Photomicrograph showing fibers with intermediate reactivity (arrow) and with low reactivity (little arrow) in the superficial region, middle portion, of the masseter muscle, in both sides of the control group (x62.5 of original magnification). ATPase reaction. pH 10.4
Fig. 1b. Photomicrograph showing smalls fibers with intermediate reactivity (large arrow) and scarce fibers with high reactivity (short arrow) in the depth region of the right masseter muscle (x125 of original magnification). ATPase reaction. pH 4.6
Fig. 1c. Photomicrograph showing fibers with intermediate reactivity (arrow) in the superficial portion of the right masseter muscle, control group (x62.5 of original magnification). SDH reaction.
Fig. 1d. Photomicrograph showing the depth portion of the right masseter muscle, control group, with intermediate reactivity of the fibers (long arrow) and scarce fibers with intense reactivity (short arrow) (x62.5 of original magnification). SDH reaction.
Fig. 1le. Photomicrograph showing fibers with intermediate reactivity (arrow) in the middle portion, superficial region, of the right masseter muscle, occlusal altered group (x500 of original magnification). ATPase reaction. pH 10.4
Fig. If. Photomicrograph showing fibers with intermediate reactivity (arrow) and high reactivity, in the depth region, middle portion, of the right masseter muscle of the occlusal altered group (x62.5 of original magnification). ATPase reaction. pH 4.6
Fig. 1g. Photomicrograph showing fibers with intermediate reactivity, in the middle portion, superficial region of the right masseter muscle, occlusal altered group (x125 of original magnification). SDH reaction.
Fig. 1h. Photomicrograph showing a high magnification of anterior view showing the different levels of the fibers reactivity (x500 of original magnification). SDH reaction.
Fig. 1i. Photomicrograph showing fibers with intermediate reactivity on the superficial region, middle portion of the left masseter muscle, occlusal altered group (x125 of original magnification). ATPase reaction. pH 10.4
Fig. 1j. Photomicrograph showing fibers with intermediate reactivity (thick arrow) and high reactivity (thin arrow), in the depth region, middle portion of the left masseter muscle, occlusal altered group (x250 original magnification). ATPase reaction. pH 4.6
Fig. 1k. Photomicrograph showing fibers with intermediate reactivity on the superficial region, middle portion, of the left masseter muscle, occlusal altered group (x125 of original magnification). SDH reaction.
Fig. 1l. Photomicrograph showing fibers with intermediate reactivity (arrow) and scarce fibers with high reactivity, in the depth region, middle portion, of the left masseter muscle, occlusal altered group (x500 of original magnification). SDH reaction.

DISCUSSION

The aim of this study was to show the histoenzymological alterations in masseter muscle, superficial bundle, in guinea-pigs (Caviaporcellus), after occlusal alteration.

Considering the masticatory strength adjustment, not only the direction of the fibers and the diameter of the masticatory muscles section are determinant factors, but also the fibers composition is important (He et al., 2004). Through histoenzymological methods, different kinds of muscular fibers can be distinguished according to its enzymatic content. Using reactions for myofibril ATPase on pH 9.4, the type I fibers present clear coloration, which indicates low ATPase activity and slow contraction time, while the type IIA fibers, of dark coloration, indicate high ATPase activity correlating to fast contraction time (Eriksson & Thornell and Bakke, 1993). The type II fibers are subdivided into type IIA, mitochondria rich, and type IIB, mitochondria poor. The type I fibers are responsible for functional maintenance, they do not quickly fatigue, are not sensitive to the lack of oxygen, have an important amount of myoglobin and are surrounded by thin capillary, this fiber type requires an abundant ATP supply and have an intense aerobic metabolism. The type II fibers have few myoglobin, high ATPase activity and obtains its energy through glycolytic way (Miehe etal).

The types of fibers that characterize the masseter muscle of a guinea pig described in the literature are type IIA (Rowlerson etal, 1988; Ohnuki etal, 2000); type IIB (Ohnuki et al.) and type I (Rowlerson et al). The histoenzymological methods used in various studies show a remarkable difference in the composition of masseter fiber types between humans and animals. The most common aspect of the human masseter muscle is the presence of type I fiber, in animals, is the presence of type IIA fiber, despite the presence of other types of fibers in different proportions in both species (Tuxen & Kirkeby, 1990; Tuxen & Rostrup, 1993).

Regarding the operated guinea pigs, the reactions with ATPase after acid and alkaline pre-incubation, NADH and SDH did not present significant differences on the superficial region of the right and left masseter muscle when compared to the control group. However, the high reactivity fibers located in the depth of left masseter muscle showed between themselves modification on diameters and intensity of reaction, and some lower oxidative capacities in the reaction for NADH and SDH, when were compared to the right muscle. Alteration in the composition of the muscular fibers from one kind to another suggest the ability to adapt themselves to muscle hyperactivity, fatigue resistance and to develop signs and symptoms of mandibular dysfunction (Eriksson & Thornell), which is an adaptive response to the new functional demand (Ohnuki et al, 2000).

The present study permitted to conclude that the masseter muscle, superficial bundle, in guinea-pigs was little sensible to chewing functional alteration in this period of time.

 

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This study was supported by FAPESP (01/01142-6).

Received: 02-12-2006 Accepted: 20-01-2007

Correspondence to:

Dr. Joao Paulo Mardegan Issa
Rua Garibaldi, 806, ap-601
CEP 010-170
Bairro Centro
Ribeirao Preto- SP,
BRASIL
E-mail: jpmissa@forp.usp.br

 

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