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Ultrastructural and biochemical changes of the medial pterygoid muscle induced by unilateral exodontia
Micron 39 (2008) 536–543 www.elsevier.com/locate/micron
Ultrastructural and biochemical changes of the medial pterygoid muscle induced by unilateral exodontia Emmanuel Bazan a, Joa˜o Paulo Mardegan Issa b, Ii-sei Watanabe c, Carlos Alberto Mandarim-de-Lacerda d, Elaine Aparecida Del Bel b, Mamie Mizusaki Iyomasa b,* a
Faculty of Medicine, Federal University of Sa˜o Paulo (UNIFESP), Sa˜o Paulo, Sa˜o Paulo, Brazil b Faculty of Dentistry, University of Sa˜o Paulo (USP), Ribeira˜o Preto, Sa˜o Paulo, Brazil c Institute of Biomedical Sciences (ICB), University of Sa˜o Paulo, Sa˜o Paulo, Sa˜o Paulo, Brazil d Biomedical Center, University of the State of Rio de Janeiro (UERJ), Rio de Janeiro, Rio de Janeiro, Brazil Received 5 June 2007; received in revised form 27 July 2007; accepted 30 July 2007
Abstract The aim of the present study was to investigate the histological, biochemical and ultrastructural effects of occlusal alteration induced by unilateral exodontia on medial pterygoid muscle in guinea pigs, Cavia porcellus. Thirty (n = 30) male guinea pigs (450 g) were divided into two groups: experimental-animals submitted to exodontia of the left upper molars, and sham-operated were used as control. The duration of the experimental period was 60 days. Medial pterygoid muscles from ipsilateral and contralateral side were analyzed by histological (n = 10), histochemical (n = 10), and ultrastructural (n = 10) methods. The data were submitted to statistical analysis. When the ipsilateral side was compared to the control group, it showed a significantly shorter neuromuscular spindle length (P < 0.05), lower oxidative metabolic activity, and microvessel constriction, in spite of the capillary volume and surface density were not significantly different (P > 0.05). In the contralateral side, the neuromuscular spindles showed significantly shorter length (P < 0.05), the fibers reflected a higher oxidative capacity, the blood capillaries showed endothelial cell emitting slender sprouting along the pre-existing capillary, and significantly higher blood capillary surface density, and volume density (Vv = 89% Mann–Whitney test, P < 0.05). This finding indicated a complex morphological and functional medial pterygoid muscle adaptation to occlusal alteration in this experimental model. Considering that neuromuscular spindles are responsible for the control of mandibular positioning and movements, the professional should consider if these changes interfere in the success of clinical procedures in medical field involving stomatognathic structures. # 2007 Elsevier Ltd. All rights reserved. Keywords: Medial pterygoid muscle; Transmission electron microscopy; Histochemistry; Neuromuscular spindle; Blood capillary
1. Introduction The stomatognathic or masticatory system consists of a set of structural arrangements that carry out common functions in integrated patterns along with the nervous, circulatory, endocrine, osseous and muscular systems. Masticatory muscles are dynamic elements that position or move the mandible in different directions according to their insertion and fiber orientation. A dysfunctional stomatognathic system induced by inadequate * Corresponding author at: Faculdade de Odontologia de Ribeira˜o Preto, Universidade de Sa˜o Paulo-USP, Departamento de Morfologia, Estomatologia e Fisiologia, Av. Cafe´ S/N, Monte Alegre, CEP 14040-904, Ribeira˜o Preto, SP, Brazil. Tel.: +55 16 36024094. E-mail address: [email protected] (M.M. Iyomasa). 0968-4328/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2007.07.006
muscular activity is clinically manifested by fatigue, pain, myospasm and myositis and frequently associated to occlusal interference (Christensen and Rassouli, 1995; Demir et al., 2005). In addition, muscular adaptation to adequate positioning seems to be relevant to the permanent correction of maxillomandibular malocclusion (Thuer et al., 1992) induced by adaptive processes in the neuromuscular masticatory system (Ingervall and Bitsanis, 1986). Nevertheless, controversial opinions still exist on specific roles of different muscular alterations appearing during treatment with functional orthodontic appliances (Eckardt et al., 2001). Unilateral occlusal alterations in animal experimental models have shown adaptation of the masseter muscle caused by unilateral exodontias (Maeda et al., 1990), by unilateral occlusal abrasion (Bani et al., 1999; Bani and Bergamini, 2002), or by insertion of an unilateral
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occlusal splint (Muller et al., 2000; Nishide et al., 2001). However, physiopathological mechanisms of muscular adaptation are still poorly understood and studies related to pathways to morphofunctional changes are scarce (Gendrage et al., 2003) in special on medial pterygoid muscle. The medial pterygoid muscle is a mandible elevator muscle, which along with the masseter muscle contributes to forward and lateral movements, but it is scarcely analyzed in conditions of occlusal alterations. The neuromuscular spindles are present in human and animals medial pterygoid muscles, usually located in deep areas of this muscle. The neuromuscular spindles are proprioceptors (Turker and Powers, 2001) that have the function to control mandibular movements (Kubota et al., 1980). In the masseter muscle, there are reports on degeneration of annulospiral endings in neuromuscular spindles due to soft diets in mice (Maeda et al., 1988), increase reflex in fusimotor activity during fatiguing muscle contraction (Ljubisavljevic and Anastasijevic, 1996), diameter reduction in both sides of intrafusal fibers by unilateral exodontia (Maeda et al., 1990) besides ultrastructural changes in neuromuscular spindles occurring through occlusal alterations (Santiwong et al., 2002; Bani and Bergamini, 2002). The morphological alterations of neuromuscular spindles in the medial pterygoid muscle may alert to the complex influence that a therapeutic or pathological occlusal modification induce on the stomatognathic system. Using histochemical reactions for myofibril ATPase and NADH (nicotine adenine dinucleotide), is possible to investigate, respectively, contraction velocity and metabolic capacity for ATP synthesis (Peter et al., 1972) in muscle fibers. It is known that the heavy chain protein subunit of myosin (MyHC) has ATPase activity, able to convert ATP to energy for muscular contraction (Weiss et al., 1999). Therefore, altered fibers type in the medial pterygoid muscle may contributed to clear controversial opinion that exist on the specific roles of different fiber muscle type appearing during orthodontic treatment. Other method also used in this investigation is electron microscopy analysis, since Bani and Bergamini (2002) revealed ultrastuctural alteration in the masseter muscle by occlusal alterations. The medial pterygoid muscle in guinea pig presents a trapezoidal shape with upper small base facing towards its origin and the lower large base faced towards the insertion area. This muscle showed partially separated in bundles by aponeuroses, and revealed the anterior (10.28 mm) and posterior (13.82 mm) limit of contralateral side larger then the ipsilateral one, following the unilateral exodontia (Mizusaki et al., 2004). This study aims to evaluate the ipsilateral and contralateral medial pterygoid muscle adaptation to changes, in occlusal alteration induced by unilateral removal of the left upper molars, in the guinea pig experimental-animal model. 2. Material and methods
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groups, experimental (n = 15) and control (n = 15). Animal groups were fed by pellets food, cabbage leaves, had ad libitum water and were kept in condition of controlled light (12 h light:12 h dark cycle) and temperature (24 1 8C). Only the experimental group was fed by powdered food during the 1st week. The Research Ethics Committee of the Federal University of Sa˜o Paulo/Hospital Sa˜o Paulo, Brazil approved all procedures in this study, compliance with international laws. The guinea pig experienced a decrease in body weight in the first 24 h after surgery. Thereafter, they gained weight at a rate parallel to the control animals. No post-operative complication could be observed. 2.2. Induction of occlusal alterations Animals were anesthetized with 2.5% tribromoethanol (Aldrich-0.25 g/kg, i.p.). Experimental group was submitted to exodontia of the upper molars and treated with a single dose of antibiotic (Veterinary pentabiotics-Fort Dodge 24,000 UI/kg, i.m.) and sodium diclofenac (10 mg/kg, anti-inflammatory effect). Control group, no build ups and served as sham-operated (this group was subjected to the same trauma during jaw opening of the surgical treatment). Sixty days after surgery the animals were sacrificed, both groups divided into three subgroups with five animals, which were processed for light and electron microscopy and for histochemical analysis, respectively. 2.3. Light microscopy Experimental and control subgroups (n = 5 in each) were anesthetized by 37.5% urethane (3 g/kg, Aldrich) and sacrificed by perfusion with intracardiac infusion of saline solution followed by 10% formaldehyde in phosphate buffer, pH 7.4. The dissection was designed as a lateral approach beginning with stripping of the masseter muscle of the zygomatic arch, which when pulled down exposes the lateral face of the mandible bone. It was cut down after sawed at condilo and first molar level exposing the medial pterygoid. This was dissected (left and right), and divided in two fragments by frontal section, to get cross-section of the muscle fiber, dehydrated in graded ethanol, passed through xylene and embedded in paraffin wax. Serial transverse sections, 6 mm thick of medial pterygoid muscle were stained with hematoxylin–eosin and the spindle muscle length analyzed under a photomicroscope (Leica DMRB, Germany) equipped with a digital camera (Olympus, DP111, USA). The sections number from initial extremity level to the end extremity, multiplied by the section thickness corresponding to the spindle muscle length. Ten spindle muscle measurements in each left and right side at both groups were recorded. All measurements were statistically compared intergroup variations using analysis of variance test (F), and the mean values of the five spindle muscle were compared using Tukey test.
2.1. Animals 2.4. Electron microscopy Thirty young male guinea pigs (Cavia porcellus) weighing on average 450 g housed in group of five animals per cage measuring 3300 cm2. They were randomly divided into two
Small fragments of different parts of each medial pterygoid muscle (left and right) were obtained from animals subgroups,
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experimental and control (n = 5 each), sacrificed as described above. The fragments were fixed for 1 h at 4 8C in cold 4% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, dehydrated in ascending concentration of ethanol, treated by propylene oxide and embedded in 812 Epoxi resin. Ultra thin sections were stained with 4% uranyl acetate and 0.4% lead citrate and examined by a JEOL 1010, 880Kv transmission electron microscope. Several micrographs were taken from each (right and left) fragments at magnification (4800 K 8000 K, 20,000 K, 30,000 K, 40,000 K and 60,000 K). This micrographs were randomly selected for qualitative analyze of the muscle fiber and capillary changes.
2.7. Statistical analysis Statistical analysis was performed using the Graph prism program, version 4.02 (San Diego, CA, USA). The nonparametric Mann–Whitney Rank Sum test for comparison two independent groups and the Wilcoxon Signed Rank test were used for the statistical analysis of vessel numbers and neuromuscular spindle length. 3. Results 3.1. Analysis of the medial pterygoid muscle in the control group
2.5. Fiber typing by enzyme reaction-histochemical method The subgroups of animals, experimental (n = 5) and control (n = 5) were sacrificed as well as described previously. The medial pterygoid muscle of each side (right and left) was excised from the mid portion, perpendicular to the main direction of the muscle fibers. Each specimen was mounted in an aluminum board with Tissue-tekR, optimal cutting temperature (OCT) compound, snap frozen in isopentane cooled by liquid nitrogen ( 150 8C) and kept at 80 8C until used. Serial transversal cross-sections, 10 mm thick, cut at 20 8C using a Leica cryostat microtome were stained to demonstrate myofibrillar ATPase activity after either alkaline (pH 10.2) or acid (pH 4.3–4.6) pre-incubations (Brooke and Kaiser, 1970). This method differentiates type I (light) fibers correlated to slow contraction capacity, and type II (dark) fibers correlated to fast contracting fibers. A mitochondrial enzyme, nicotine adenine dinucleotide tetrazolium reductase (NADH-TR) was used to demonstrate oxidative capacity (Novikoff et al., 1961). The serial sections stained for succinate dehydrogenase (SDH) following procedure described by Nachlas et al. (1957) demonstrated the muscle metabolic pattern. All serial section placed on slide glasses and stained were surveyed microscopically to qualitative analyze of fiber type. 2.6. Vessel stereological quantification Serial transversal cross-sections medial pterygoid muscle, 10 mm thick, cut at 20 8C using a Leica cryostat microtome with ATPase staining at pH 4.6 activity revealed the capillaries (Sta˚l et al., 1995). Fifteen randomly microscopic fields of the medial pterygoid muscles, from each subgroup animals sides (left and right), were used for vessel counting. Two parameters were stereologically analyzed using a cycloid arc test system connected to a video-microscope system (Mandarin de Lacerda, 2003): (a) Blood capillary volume density Vv [cs]: Vv = Pp/Pt, where Pp denoting partial points, corresponds to the number of points hitting blood capillaries [cs] and Pt is the total number of test points. (b) Blood capillary surface density Sv [cs]: Sv = 2I/Lt, where I is the number of blood capillaries intersections with cycloid arcs and Lt is the length of the test line based on the calibration system.
In this group, the neuromuscular spindles in the medial pterygoid muscle had average lengths of 1.082 0.12 and 0.972 0.17 mm, for the right and left sides, respectively, a difference not statistically significant (P > 0.05). ATPase myofibrillar activity in the middle of the lateral muscle portion indicated the presence of heterogeneously colored type II muscular fibers but scarce type I fibers. Concerning oxidative enzyme reactivity (NADH reaction), the normal aspects of the muscle showed abundant fibers with a rough product in the subsarcolemmal region (Fig. 1A and B), fibers slight colored and the strong reactive to NADH were few, looked like the SDH reactivity. Ultrastructurally, one type of fiber revealed spherical or eggshaped mitochondria, mainly located in the subsarcolemmal region, near the nucleus (Fig. 2A), besides some rare mitochondria isolated between myofibrils; the other type showed only scarce small-sized mitochondria dispersed between the myofibrils. Both types had spherical lipid inclusions, averaging 0.5–1 mm in diameter and vesicles. In control group, the blood capillaries normal aspect showed broad lumen, revealing endothelial cells with abundant pinocytic vesicles (Fig. 3A and B). 3.2. Analysis of the medial pterygoid muscle in the experimental group 3.2.1. Ipsilateral muscle to the molar exodontia Animals of the experimental group model adopted in this study, showed neuromuscular spindles in the ipsilateral pterygoid muscle significantly shorter than the ones in the control left side, measuring 0.556 0.12 mm in length (P < 0.05), 60 days after the occlusal changes induced by unilateral removal of upper molars. Type II fibers, homogeneously colored were revealed by ATPase myofibrillar reaction. Tests for reactivity to NADH revealed scarce fibers with a rough product in the subsarcolemmal region, and more slight colored fibers (Fig. 1C), similar to SDH reactivity. Experimental left side medial pterygoid muscle examined by electron microscope showed scarce small-sized mitochondria in small fibers, and over large spherical lipid inclusions having diameters between 0.8 and 1.8 mm (Fig. 2B). Under higher magnification electron micrograph, some fibers showed small and less concentrated varied-shaped and irregular borders
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Fig. 1. Photomicrographs of NADH staining from medial pterygoid muscle left (A) and right (B) side of the control group showing abundant fibers with reaction product localized in the subsarcolemmal region (arrow), and scarce light colored fiber (curve arrow) and dark fiber with reaction product uniformly distributed (head arrow). Experimental group showed predominance of light colored fiber (head arrow) on left side (C) and predominance of fibers with the reaction product localized in the subsarcolemmal region (arrow) on right side (D) (original magnification 20).
mitochondria located in the subsarcolemmal region, glycogen accumulation (Fig. 2C), and large lipid inclusions. Blood capillaries between muscle fibers were with a greatly narrowed lumen and irregular walls (Fig. 2B). The endothelial cell showed scarce pinocytic vesicles and caveolae (Fig. 3C), and elongated vacuoles completely surrounded by cytoplasm (Fig. 2C) under higher magnification electron micrograph analysis. Capillary volume and surface density in the ipsilateral muscle were not significantly different from those of control animals (Figs. 4 and 5) (P > 0.05).
cytoplasmic lipid inclusions measuring between 0.33 and 0.55 mm (Fig. 2D). The blood capillaries showed endothelial cell with intraluminal protrusions and numerous pinocytotic vesicles (Fig. 3D) emitting slender sprouting along the preexisting capillary (Fig. 2D) under electron micrograph analysis. Capillary surface density (Sv) and volume density (Vv = 89%) stereological analysis of the contralateral side showed a significant increase when compared to control group (P < 0.05) (Figs. 4 and 5). 4. Discussion
3.2.2. Contralateral muscle to molar exodontia Neuromuscular spindles of the contralateral medial pterygoid muscle to the exodontia measured 0.596 0.14 mm in length, and were significantly different when compared to control animals (P < 0.05). Muscles that were submitted to alkaline myofibrillar ATPase showed predominance of type II fibers, with heterogeneous staining and large diameters. Reactivity to NADH and SDH revealed predominant fibers with a rough product broadly distributed in the subsarcolemmal region, and less slight colored fibers (Fig. 1D). Ultrastructurally, in the fibers were identified spherical or egg-shaped mitochondria distributed among myofibrils, and densely grouped under the subsarcolemmal region. In addition, muscle fibers showed small-sized
In the masticatory system the medial pterygoid muscle plays a role not only in raising the mandible but also in moving this bone from left to right, but is strongly associated with the grinding movement (Abe et al., 1997), and suited to produce large molar bite force (Hannam and Wood, 1989). The mandibular movement in guinea pigs comprises bilateral and unilateral excursion (Byrd, 1981), which resembles the masticatory movement of humans. Therefore, guinea pig is an important experimental model to analyze masticatory muscle adaptation following occlusal alteration. In our experimental protocols 60 days after unilateral exodontia in guinea pigs, the neuromuscular spindle was significantly shorter in both sides of the medial pterygoid muscle when
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Fig. 2. Medial pterygoid muscle electron micrograph. Control group muscle fiber showing nucleus (n), spherical mitochondria at the subsarcolemmal region and open lumina (*) blood capillary (A) (original magnification 8000, bar = 1 mm). Left side of the experimental group showing small fibers with nucleus (n) and blood capillary with a greatly narrowed lumen (*) (B) (original magnification 4800, bar = 2 mm). Higher magnification of the left side muscle in experimental group showing less concentrated varied-shaped mitochondria (m) under subsarcolemmal region and glycogen (arrow) accumulation (C) (original magnification 30,000, bar = 200 nm). Experimental group, right side showing large number of mitochondria (m) at the subsarcolemmal region next to the fiber nucleus (n) and endothelial cell sprouting (arrow) (D) (original magnification 8000, bar = 1 mm).
compared to the control group. In similar conditions, but in the masseter muscle, Maeda et al. (1990) verified decrease of intrafusal fibers diameter, in both sides. Also, there are reports of neuromuscular spindles alteration due to soft diets in mice (Maeda et al., 1988), and arising from occlusal alterations (Santiwong et al., 2002; Bani and Bergamini, 2002). Maeda et al. (1990) suggested a crossing pathway of sensorial stimuli, from the extracted molar to the contralateral motor neuron to explain the alteration phenomena in neuromuscular spindles. The afferent pathways from jaw-muscle spindles have projection areas to trigeminal motor nucleus (Luo and Dessem, 1999; Luo et al., 2001) and to hypoglossal motoneurons (Zhang et al., 2003) coordinating masticatory muscle stretch reflexes (Luo et al., 1995), and oral motor behaviors (Zhang et al., 2003). Therefore, the neuromuscular spindle morphologic abnormalities caused by occlusal alteration in medial pterygoid muscle may play significant alteration on masticatory function, since this muscle activity was associated with early masticatory closing cycle. Notwithstanding all these studies, alterations in neuromuscular spindles still need explanation. The medial pterygoid muscle in control group were characterized by predominance of heterogeneous type II fibers and scarce type I ones, following the alkaline myofibrillar ATPase activity. This fact reflects a fast contraction of this muscle, similarly to masseter muscle normal characteristic in the same animal, reported by Suzuki (1977) and Tuxen and Kirkeby (1990). However, different fiber types in the guinea
pig’s medial pterygoid muscle were reported by Masuda et al. (1974), which muscle fiber stained darkly with succinic dehydrogenase and myosin ATPase. The differences findings may be explained by the masticatory muscle characteristics, that has a heterogeneous architectural design. In many instances the masticatory muscle is considered an anatomically and functionally uniform structure, difficulting to compare the muscle fiber type. Similarities between the contralateral medial pterygoid muscles, in the experimental and control groups, revealed by ATPase reaction suggest that contraction velocity was not much altered in the experimental group. However, following bilateral extraction of lateral teeth, Miehe et al. (1999) observed that the medial pterygoid muscle fiber composition shifted in favor to IIb fibers, but toward the end of the time period (126 days), shifting occurred back to the original composition. Then, regarding the contraction velocity, the 60 day observed time period can lead to adaptations of the contralateral medial pterygoid muscles fiber type, in the experimental groups, which period may be sufficient to back to the original composition. The serial transversally cut of the medial pterygoid muscle fibers in the control group showed abundant reaction products localized in the subsarcolemmal region following NADHdiaphorase reaction. This metabolic capacity on type II fiber characterizes the fast oxidative glycolitic fiber (Peter et al., 1972), indicating that this bundles fibers contract fastly and is fatigue resistant. The ipsilateral side of the medial pterygoid
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Fig. 3. Blood capillary electron micrograph. Control group showing endothelial cell nucleus (n) of blood capillary showing caveolae (thick arrow) and pinocytic vesicles (arrow) (A) (original magnification 40,000, bar = 200 nm). Higher magnification of the control group blood capillary showing abundant pinocytic vesicles (arrow) in the endothelial cell and capillary lumen (*) (original magnification 60,000, bar = 100 nm) (B). The experimental group left side muscle blood capillary lumen (*) with erythrocyte (e) and scarce pinocytic vesicles in the endothelial cell. (original magnification 30,000, bar = 200 nm) (C). Experimental group right side blood capillary lumen (*) showing abundant pinocytic vesicles in the endothelial cell (original magnification 20,000, bar = 200 nm) (D).
muscle, in the experimental group, light colored fibers reflecting lower oxidative capacity and less fatigue resistance. This side does not have antagonist teeth, thus does not need to exert a large force by the medial pterygoid muscle during mastication. Such features probably resulted in a reduced activity level of the masticatory musculature and consequently its hypofunction adaptation. This characteristic was confirmed
morphologically when fibers with less concentrated mitochondria in the subsarcolemmal region, and glycogen presence were shown by transmission electron microscopy. The present study showed predominance of fibers contained reaction products localized in the subsarcolemmal region in the contralateral medial pterygoid muscle, which could be considered to be performing a higher resistance exercise when chewing and grinding food than the ipsilateral. The SDH staining density of fibers confirmed the contralateral and ipsilateral muscle metabolic pattern. However, the contralateral masseter muscle
Fig. 4. Blood capillary surface density (Sv) in the medial pterygoid muscle. The experimental contralateral muscle (*) showed significantly higher blood capillary surface density than the control group (*Mann–Whitney test, P = 0.03). Each column represented the mean (S.E.M.).
Fig. 5. Blood capillary volume density (Vv) in the medial pterygoid muscle. The experimental contralateral muscle (*) showed significantly higher blood capillary volume density (Vv = 89%) than the control group (*Mann–Whitney test, P = 0.03). Each column represented the mean (S.E.M.).
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was only slightly affected or even unaffected following (14 and 26 days) stressing trigger induced by cusps of the molar amputation in rats (Bani et al., 1999). These differences may be explained by the task specific performed in multifasciculated muscle, which force of muscle is distributed heterogeneously throughout the muscle (van Eijden et al., 1996). In the experimental model used in this study, the higher blood capillary surface and volume density, the numerous pinocytotic vesicles and slender sprouting of endothelial cell seen under electron micrograph, could be related to the increased oxidative capacity of the fibers in the contralateral medial pterygoid muscle. This fact is according to the wellestablished correlation between capillary density and muscle oxidative capacity (Cebasek et al., 2005). It is known that growth factors like vascular endothelial growth factor (VEGF) are the metabolic stimulus for angiogenesis (Gavin and Wagner, 2001; Brown and Hudlicka, 2003), therefore, muscular overwork in the contralateral medial pterygoid muscle could to bring about by the vascular endothelial growth factor, increasing the endothelial cell sprouting and consequently blood capillary surface and volume density. In the ipsilateral muscle, in spite of blood capillary volume and surface density were not significantly different from those of control animals, narrowed lumen and irregular walls were prominent feature under electron microscopy analysis, according to Bani et al. (1999), when unilateral occlusal abrasion was the stressing trigger on masseter muscle. In hypofunctional mastication this vasoconstriction suggests that an ischemic injury is an event in the onset of the muscle tissue damage. It was concluded that a complex adaptation of the medial pterygoid muscle, to the chronic unilateral mastication takes place after 60 days, at morphological, biochemical and ultrastructural levels. On the ipsilateral side, the hypofunction leads to shorter neuromuscular spindle, lower oxidative metabolic activity, and microvessel constriction. On the contralateral side, the hyperfunction leads to shorter neuromuscular spindle alterations, higher oxidative capacity of the fibers, higher blood capillary surface and volume density, endothelial cell sprouting and numerous endothelial pinocytotic vesicles. Considering that neuromuscular spindles are proprioceptors responsible for the control of mandibular positioning and movements, their morphologic abnormalities suggested masticatory functional alterations in the experimental model adopted in this study. Although, alterations in neuromuscular spindle, in fiber metabolism, and in blood vessel parameter of masticatory muscle, do not interfere in the animal survival, investigators should consider if these changes interfere in the success of clinical procedures in medical field involving stomatognathic structures. Further researches considering heterogeneous architectural design of masticatory muscle should be conducted in order to better understand the correlation between occlusal alteration and task specific of the multifasciculated muscle. Acknowledgement We are grateful to FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo) for financial support.
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Ultrastructural and biochemical changes of the medial pterygoid muscle induced by unilateral exodontia Emmanuel Bazan a, Joa˜o Paulo Mardegan Issa b, Ii-sei Watanabe c, Carlos Alberto Mandarim-de-Lacerda d, Elaine Aparecida Del Bel b, Mamie Mizusaki Iyomasa b,* a
Faculty of Medicine, Federal University of Sa˜o Paulo (UNIFESP), Sa˜o Paulo, Sa˜o Paulo, Brazil b Faculty of Dentistry, University of Sa˜o Paulo (USP), Ribeira˜o Preto, Sa˜o Paulo, Brazil c Institute of Biomedical Sciences (ICB), University of Sa˜o Paulo, Sa˜o Paulo, Sa˜o Paulo, Brazil d Biomedical Center, University of the State of Rio de Janeiro (UERJ), Rio de Janeiro, Rio de Janeiro, Brazil Received 5 June 2007; received in revised form 27 July 2007; accepted 30 July 2007
Abstract The aim of the present study was to investigate the histological, biochemical and ultrastructural effects of occlusal alteration induced by unilateral exodontia on medial pterygoid muscle in guinea pigs, Cavia porcellus. Thirty (n = 30) male guinea pigs (450 g) were divided into two groups: experimental-animals submitted to exodontia of the left upper molars, and sham-operated were used as control. The duration of the experimental period was 60 days. Medial pterygoid muscles from ipsilateral and contralateral side were analyzed by histological (n = 10), histochemical (n = 10), and ultrastructural (n = 10) methods. The data were submitted to statistical analysis. When the ipsilateral side was compared to the control group, it showed a significantly shorter neuromuscular spindle length (P < 0.05), lower oxidative metabolic activity, and microvessel constriction, in spite of the capillary volume and surface density were not significantly different (P > 0.05). In the contralateral side, the neuromuscular spindles showed significantly shorter length (P < 0.05), the fibers reflected a higher oxidative capacity, the blood capillaries showed endothelial cell emitting slender sprouting along the pre-existing capillary, and significantly higher blood capillary surface density, and volume density (Vv = 89% Mann–Whitney test, P < 0.05). This finding indicated a complex morphological and functional medial pterygoid muscle adaptation to occlusal alteration in this experimental model. Considering that neuromuscular spindles are responsible for the control of mandibular positioning and movements, the professional should consider if these changes interfere in the success of clinical procedures in medical field involving stomatognathic structures. # 2007 Elsevier Ltd. All rights reserved. Keywords: Medial pterygoid muscle; Transmission electron microscopy; Histochemistry; Neuromuscular spindle; Blood capillary
1. Introduction The stomatognathic or masticatory system consists of a set of structural arrangements that carry out common functions in integrated patterns along with the nervous, circulatory, endocrine, osseous and muscular systems. Masticatory muscles are dynamic elements that position or move the mandible in different directions according to their insertion and fiber orientation. A dysfunctional stomatognathic system induced by inadequate * Corresponding author at: Faculdade de Odontologia de Ribeira˜o Preto, Universidade de Sa˜o Paulo-USP, Departamento de Morfologia, Estomatologia e Fisiologia, Av. Cafe´ S/N, Monte Alegre, CEP 14040-904, Ribeira˜o Preto, SP, Brazil. Tel.: +55 16 36024094. E-mail address: [email protected] (M.M. Iyomasa). 0968-4328/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2007.07.006
muscular activity is clinically manifested by fatigue, pain, myospasm and myositis and frequently associated to occlusal interference (Christensen and Rassouli, 1995; Demir et al., 2005). In addition, muscular adaptation to adequate positioning seems to be relevant to the permanent correction of maxillomandibular malocclusion (Thuer et al., 1992) induced by adaptive processes in the neuromuscular masticatory system (Ingervall and Bitsanis, 1986). Nevertheless, controversial opinions still exist on specific roles of different muscular alterations appearing during treatment with functional orthodontic appliances (Eckardt et al., 2001). Unilateral occlusal alterations in animal experimental models have shown adaptation of the masseter muscle caused by unilateral exodontias (Maeda et al., 1990), by unilateral occlusal abrasion (Bani et al., 1999; Bani and Bergamini, 2002), or by insertion of an unilateral
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occlusal splint (Muller et al., 2000; Nishide et al., 2001). However, physiopathological mechanisms of muscular adaptation are still poorly understood and studies related to pathways to morphofunctional changes are scarce (Gendrage et al., 2003) in special on medial pterygoid muscle. The medial pterygoid muscle is a mandible elevator muscle, which along with the masseter muscle contributes to forward and lateral movements, but it is scarcely analyzed in conditions of occlusal alterations. The neuromuscular spindles are present in human and animals medial pterygoid muscles, usually located in deep areas of this muscle. The neuromuscular spindles are proprioceptors (Turker and Powers, 2001) that have the function to control mandibular movements (Kubota et al., 1980). In the masseter muscle, there are reports on degeneration of annulospiral endings in neuromuscular spindles due to soft diets in mice (Maeda et al., 1988), increase reflex in fusimotor activity during fatiguing muscle contraction (Ljubisavljevic and Anastasijevic, 1996), diameter reduction in both sides of intrafusal fibers by unilateral exodontia (Maeda et al., 1990) besides ultrastructural changes in neuromuscular spindles occurring through occlusal alterations (Santiwong et al., 2002; Bani and Bergamini, 2002). The morphological alterations of neuromuscular spindles in the medial pterygoid muscle may alert to the complex influence that a therapeutic or pathological occlusal modification induce on the stomatognathic system. Using histochemical reactions for myofibril ATPase and NADH (nicotine adenine dinucleotide), is possible to investigate, respectively, contraction velocity and metabolic capacity for ATP synthesis (Peter et al., 1972) in muscle fibers. It is known that the heavy chain protein subunit of myosin (MyHC) has ATPase activity, able to convert ATP to energy for muscular contraction (Weiss et al., 1999). Therefore, altered fibers type in the medial pterygoid muscle may contributed to clear controversial opinion that exist on the specific roles of different fiber muscle type appearing during orthodontic treatment. Other method also used in this investigation is electron microscopy analysis, since Bani and Bergamini (2002) revealed ultrastuctural alteration in the masseter muscle by occlusal alterations. The medial pterygoid muscle in guinea pig presents a trapezoidal shape with upper small base facing towards its origin and the lower large base faced towards the insertion area. This muscle showed partially separated in bundles by aponeuroses, and revealed the anterior (10.28 mm) and posterior (13.82 mm) limit of contralateral side larger then the ipsilateral one, following the unilateral exodontia (Mizusaki et al., 2004). This study aims to evaluate the ipsilateral and contralateral medial pterygoid muscle adaptation to changes, in occlusal alteration induced by unilateral removal of the left upper molars, in the guinea pig experimental-animal model. 2. Material and methods
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groups, experimental (n = 15) and control (n = 15). Animal groups were fed by pellets food, cabbage leaves, had ad libitum water and were kept in condition of controlled light (12 h light:12 h dark cycle) and temperature (24 1 8C). Only the experimental group was fed by powdered food during the 1st week. The Research Ethics Committee of the Federal University of Sa˜o Paulo/Hospital Sa˜o Paulo, Brazil approved all procedures in this study, compliance with international laws. The guinea pig experienced a decrease in body weight in the first 24 h after surgery. Thereafter, they gained weight at a rate parallel to the control animals. No post-operative complication could be observed. 2.2. Induction of occlusal alterations Animals were anesthetized with 2.5% tribromoethanol (Aldrich-0.25 g/kg, i.p.). Experimental group was submitted to exodontia of the upper molars and treated with a single dose of antibiotic (Veterinary pentabiotics-Fort Dodge 24,000 UI/kg, i.m.) and sodium diclofenac (10 mg/kg, anti-inflammatory effect). Control group, no build ups and served as sham-operated (this group was subjected to the same trauma during jaw opening of the surgical treatment). Sixty days after surgery the animals were sacrificed, both groups divided into three subgroups with five animals, which were processed for light and electron microscopy and for histochemical analysis, respectively. 2.3. Light microscopy Experimental and control subgroups (n = 5 in each) were anesthetized by 37.5% urethane (3 g/kg, Aldrich) and sacrificed by perfusion with intracardiac infusion of saline solution followed by 10% formaldehyde in phosphate buffer, pH 7.4. The dissection was designed as a lateral approach beginning with stripping of the masseter muscle of the zygomatic arch, which when pulled down exposes the lateral face of the mandible bone. It was cut down after sawed at condilo and first molar level exposing the medial pterygoid. This was dissected (left and right), and divided in two fragments by frontal section, to get cross-section of the muscle fiber, dehydrated in graded ethanol, passed through xylene and embedded in paraffin wax. Serial transverse sections, 6 mm thick of medial pterygoid muscle were stained with hematoxylin–eosin and the spindle muscle length analyzed under a photomicroscope (Leica DMRB, Germany) equipped with a digital camera (Olympus, DP111, USA). The sections number from initial extremity level to the end extremity, multiplied by the section thickness corresponding to the spindle muscle length. Ten spindle muscle measurements in each left and right side at both groups were recorded. All measurements were statistically compared intergroup variations using analysis of variance test (F), and the mean values of the five spindle muscle were compared using Tukey test.
2.1. Animals 2.4. Electron microscopy Thirty young male guinea pigs (Cavia porcellus) weighing on average 450 g housed in group of five animals per cage measuring 3300 cm2. They were randomly divided into two
Small fragments of different parts of each medial pterygoid muscle (left and right) were obtained from animals subgroups,
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experimental and control (n = 5 each), sacrificed as described above. The fragments were fixed for 1 h at 4 8C in cold 4% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, dehydrated in ascending concentration of ethanol, treated by propylene oxide and embedded in 812 Epoxi resin. Ultra thin sections were stained with 4% uranyl acetate and 0.4% lead citrate and examined by a JEOL 1010, 880Kv transmission electron microscope. Several micrographs were taken from each (right and left) fragments at magnification (4800 K 8000 K, 20,000 K, 30,000 K, 40,000 K and 60,000 K). This micrographs were randomly selected for qualitative analyze of the muscle fiber and capillary changes.
2.7. Statistical analysis Statistical analysis was performed using the Graph prism program, version 4.02 (San Diego, CA, USA). The nonparametric Mann–Whitney Rank Sum test for comparison two independent groups and the Wilcoxon Signed Rank test were used for the statistical analysis of vessel numbers and neuromuscular spindle length. 3. Results 3.1. Analysis of the medial pterygoid muscle in the control group
2.5. Fiber typing by enzyme reaction-histochemical method The subgroups of animals, experimental (n = 5) and control (n = 5) were sacrificed as well as described previously. The medial pterygoid muscle of each side (right and left) was excised from the mid portion, perpendicular to the main direction of the muscle fibers. Each specimen was mounted in an aluminum board with Tissue-tekR, optimal cutting temperature (OCT) compound, snap frozen in isopentane cooled by liquid nitrogen ( 150 8C) and kept at 80 8C until used. Serial transversal cross-sections, 10 mm thick, cut at 20 8C using a Leica cryostat microtome were stained to demonstrate myofibrillar ATPase activity after either alkaline (pH 10.2) or acid (pH 4.3–4.6) pre-incubations (Brooke and Kaiser, 1970). This method differentiates type I (light) fibers correlated to slow contraction capacity, and type II (dark) fibers correlated to fast contracting fibers. A mitochondrial enzyme, nicotine adenine dinucleotide tetrazolium reductase (NADH-TR) was used to demonstrate oxidative capacity (Novikoff et al., 1961). The serial sections stained for succinate dehydrogenase (SDH) following procedure described by Nachlas et al. (1957) demonstrated the muscle metabolic pattern. All serial section placed on slide glasses and stained were surveyed microscopically to qualitative analyze of fiber type. 2.6. Vessel stereological quantification Serial transversal cross-sections medial pterygoid muscle, 10 mm thick, cut at 20 8C using a Leica cryostat microtome with ATPase staining at pH 4.6 activity revealed the capillaries (Sta˚l et al., 1995). Fifteen randomly microscopic fields of the medial pterygoid muscles, from each subgroup animals sides (left and right), were used for vessel counting. Two parameters were stereologically analyzed using a cycloid arc test system connected to a video-microscope system (Mandarin de Lacerda, 2003): (a) Blood capillary volume density Vv [cs]: Vv = Pp/Pt, where Pp denoting partial points, corresponds to the number of points hitting blood capillaries [cs] and Pt is the total number of test points. (b) Blood capillary surface density Sv [cs]: Sv = 2I/Lt, where I is the number of blood capillaries intersections with cycloid arcs and Lt is the length of the test line based on the calibration system.
In this group, the neuromuscular spindles in the medial pterygoid muscle had average lengths of 1.082 0.12 and 0.972 0.17 mm, for the right and left sides, respectively, a difference not statistically significant (P > 0.05). ATPase myofibrillar activity in the middle of the lateral muscle portion indicated the presence of heterogeneously colored type II muscular fibers but scarce type I fibers. Concerning oxidative enzyme reactivity (NADH reaction), the normal aspects of the muscle showed abundant fibers with a rough product in the subsarcolemmal region (Fig. 1A and B), fibers slight colored and the strong reactive to NADH were few, looked like the SDH reactivity. Ultrastructurally, one type of fiber revealed spherical or eggshaped mitochondria, mainly located in the subsarcolemmal region, near the nucleus (Fig. 2A), besides some rare mitochondria isolated between myofibrils; the other type showed only scarce small-sized mitochondria dispersed between the myofibrils. Both types had spherical lipid inclusions, averaging 0.5–1 mm in diameter and vesicles. In control group, the blood capillaries normal aspect showed broad lumen, revealing endothelial cells with abundant pinocytic vesicles (Fig. 3A and B). 3.2. Analysis of the medial pterygoid muscle in the experimental group 3.2.1. Ipsilateral muscle to the molar exodontia Animals of the experimental group model adopted in this study, showed neuromuscular spindles in the ipsilateral pterygoid muscle significantly shorter than the ones in the control left side, measuring 0.556 0.12 mm in length (P < 0.05), 60 days after the occlusal changes induced by unilateral removal of upper molars. Type II fibers, homogeneously colored were revealed by ATPase myofibrillar reaction. Tests for reactivity to NADH revealed scarce fibers with a rough product in the subsarcolemmal region, and more slight colored fibers (Fig. 1C), similar to SDH reactivity. Experimental left side medial pterygoid muscle examined by electron microscope showed scarce small-sized mitochondria in small fibers, and over large spherical lipid inclusions having diameters between 0.8 and 1.8 mm (Fig. 2B). Under higher magnification electron micrograph, some fibers showed small and less concentrated varied-shaped and irregular borders
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Fig. 1. Photomicrographs of NADH staining from medial pterygoid muscle left (A) and right (B) side of the control group showing abundant fibers with reaction product localized in the subsarcolemmal region (arrow), and scarce light colored fiber (curve arrow) and dark fiber with reaction product uniformly distributed (head arrow). Experimental group showed predominance of light colored fiber (head arrow) on left side (C) and predominance of fibers with the reaction product localized in the subsarcolemmal region (arrow) on right side (D) (original magnification 20).
mitochondria located in the subsarcolemmal region, glycogen accumulation (Fig. 2C), and large lipid inclusions. Blood capillaries between muscle fibers were with a greatly narrowed lumen and irregular walls (Fig. 2B). The endothelial cell showed scarce pinocytic vesicles and caveolae (Fig. 3C), and elongated vacuoles completely surrounded by cytoplasm (Fig. 2C) under higher magnification electron micrograph analysis. Capillary volume and surface density in the ipsilateral muscle were not significantly different from those of control animals (Figs. 4 and 5) (P > 0.05).
cytoplasmic lipid inclusions measuring between 0.33 and 0.55 mm (Fig. 2D). The blood capillaries showed endothelial cell with intraluminal protrusions and numerous pinocytotic vesicles (Fig. 3D) emitting slender sprouting along the preexisting capillary (Fig. 2D) under electron micrograph analysis. Capillary surface density (Sv) and volume density (Vv = 89%) stereological analysis of the contralateral side showed a significant increase when compared to control group (P < 0.05) (Figs. 4 and 5). 4. Discussion
3.2.2. Contralateral muscle to molar exodontia Neuromuscular spindles of the contralateral medial pterygoid muscle to the exodontia measured 0.596 0.14 mm in length, and were significantly different when compared to control animals (P < 0.05). Muscles that were submitted to alkaline myofibrillar ATPase showed predominance of type II fibers, with heterogeneous staining and large diameters. Reactivity to NADH and SDH revealed predominant fibers with a rough product broadly distributed in the subsarcolemmal region, and less slight colored fibers (Fig. 1D). Ultrastructurally, in the fibers were identified spherical or egg-shaped mitochondria distributed among myofibrils, and densely grouped under the subsarcolemmal region. In addition, muscle fibers showed small-sized
In the masticatory system the medial pterygoid muscle plays a role not only in raising the mandible but also in moving this bone from left to right, but is strongly associated with the grinding movement (Abe et al., 1997), and suited to produce large molar bite force (Hannam and Wood, 1989). The mandibular movement in guinea pigs comprises bilateral and unilateral excursion (Byrd, 1981), which resembles the masticatory movement of humans. Therefore, guinea pig is an important experimental model to analyze masticatory muscle adaptation following occlusal alteration. In our experimental protocols 60 days after unilateral exodontia in guinea pigs, the neuromuscular spindle was significantly shorter in both sides of the medial pterygoid muscle when
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Fig. 2. Medial pterygoid muscle electron micrograph. Control group muscle fiber showing nucleus (n), spherical mitochondria at the subsarcolemmal region and open lumina (*) blood capillary (A) (original magnification 8000, bar = 1 mm). Left side of the experimental group showing small fibers with nucleus (n) and blood capillary with a greatly narrowed lumen (*) (B) (original magnification 4800, bar = 2 mm). Higher magnification of the left side muscle in experimental group showing less concentrated varied-shaped mitochondria (m) under subsarcolemmal region and glycogen (arrow) accumulation (C) (original magnification 30,000, bar = 200 nm). Experimental group, right side showing large number of mitochondria (m) at the subsarcolemmal region next to the fiber nucleus (n) and endothelial cell sprouting (arrow) (D) (original magnification 8000, bar = 1 mm).
compared to the control group. In similar conditions, but in the masseter muscle, Maeda et al. (1990) verified decrease of intrafusal fibers diameter, in both sides. Also, there are reports of neuromuscular spindles alteration due to soft diets in mice (Maeda et al., 1988), and arising from occlusal alterations (Santiwong et al., 2002; Bani and Bergamini, 2002). Maeda et al. (1990) suggested a crossing pathway of sensorial stimuli, from the extracted molar to the contralateral motor neuron to explain the alteration phenomena in neuromuscular spindles. The afferent pathways from jaw-muscle spindles have projection areas to trigeminal motor nucleus (Luo and Dessem, 1999; Luo et al., 2001) and to hypoglossal motoneurons (Zhang et al., 2003) coordinating masticatory muscle stretch reflexes (Luo et al., 1995), and oral motor behaviors (Zhang et al., 2003). Therefore, the neuromuscular spindle morphologic abnormalities caused by occlusal alteration in medial pterygoid muscle may play significant alteration on masticatory function, since this muscle activity was associated with early masticatory closing cycle. Notwithstanding all these studies, alterations in neuromuscular spindles still need explanation. The medial pterygoid muscle in control group were characterized by predominance of heterogeneous type II fibers and scarce type I ones, following the alkaline myofibrillar ATPase activity. This fact reflects a fast contraction of this muscle, similarly to masseter muscle normal characteristic in the same animal, reported by Suzuki (1977) and Tuxen and Kirkeby (1990). However, different fiber types in the guinea
pig’s medial pterygoid muscle were reported by Masuda et al. (1974), which muscle fiber stained darkly with succinic dehydrogenase and myosin ATPase. The differences findings may be explained by the masticatory muscle characteristics, that has a heterogeneous architectural design. In many instances the masticatory muscle is considered an anatomically and functionally uniform structure, difficulting to compare the muscle fiber type. Similarities between the contralateral medial pterygoid muscles, in the experimental and control groups, revealed by ATPase reaction suggest that contraction velocity was not much altered in the experimental group. However, following bilateral extraction of lateral teeth, Miehe et al. (1999) observed that the medial pterygoid muscle fiber composition shifted in favor to IIb fibers, but toward the end of the time period (126 days), shifting occurred back to the original composition. Then, regarding the contraction velocity, the 60 day observed time period can lead to adaptations of the contralateral medial pterygoid muscles fiber type, in the experimental groups, which period may be sufficient to back to the original composition. The serial transversally cut of the medial pterygoid muscle fibers in the control group showed abundant reaction products localized in the subsarcolemmal region following NADHdiaphorase reaction. This metabolic capacity on type II fiber characterizes the fast oxidative glycolitic fiber (Peter et al., 1972), indicating that this bundles fibers contract fastly and is fatigue resistant. The ipsilateral side of the medial pterygoid
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Fig. 3. Blood capillary electron micrograph. Control group showing endothelial cell nucleus (n) of blood capillary showing caveolae (thick arrow) and pinocytic vesicles (arrow) (A) (original magnification 40,000, bar = 200 nm). Higher magnification of the control group blood capillary showing abundant pinocytic vesicles (arrow) in the endothelial cell and capillary lumen (*) (original magnification 60,000, bar = 100 nm) (B). The experimental group left side muscle blood capillary lumen (*) with erythrocyte (e) and scarce pinocytic vesicles in the endothelial cell. (original magnification 30,000, bar = 200 nm) (C). Experimental group right side blood capillary lumen (*) showing abundant pinocytic vesicles in the endothelial cell (original magnification 20,000, bar = 200 nm) (D).
muscle, in the experimental group, light colored fibers reflecting lower oxidative capacity and less fatigue resistance. This side does not have antagonist teeth, thus does not need to exert a large force by the medial pterygoid muscle during mastication. Such features probably resulted in a reduced activity level of the masticatory musculature and consequently its hypofunction adaptation. This characteristic was confirmed
morphologically when fibers with less concentrated mitochondria in the subsarcolemmal region, and glycogen presence were shown by transmission electron microscopy. The present study showed predominance of fibers contained reaction products localized in the subsarcolemmal region in the contralateral medial pterygoid muscle, which could be considered to be performing a higher resistance exercise when chewing and grinding food than the ipsilateral. The SDH staining density of fibers confirmed the contralateral and ipsilateral muscle metabolic pattern. However, the contralateral masseter muscle
Fig. 4. Blood capillary surface density (Sv) in the medial pterygoid muscle. The experimental contralateral muscle (*) showed significantly higher blood capillary surface density than the control group (*Mann–Whitney test, P = 0.03). Each column represented the mean (S.E.M.).
Fig. 5. Blood capillary volume density (Vv) in the medial pterygoid muscle. The experimental contralateral muscle (*) showed significantly higher blood capillary volume density (Vv = 89%) than the control group (*Mann–Whitney test, P = 0.03). Each column represented the mean (S.E.M.).
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was only slightly affected or even unaffected following (14 and 26 days) stressing trigger induced by cusps of the molar amputation in rats (Bani et al., 1999). These differences may be explained by the task specific performed in multifasciculated muscle, which force of muscle is distributed heterogeneously throughout the muscle (van Eijden et al., 1996). In the experimental model used in this study, the higher blood capillary surface and volume density, the numerous pinocytotic vesicles and slender sprouting of endothelial cell seen under electron micrograph, could be related to the increased oxidative capacity of the fibers in the contralateral medial pterygoid muscle. This fact is according to the wellestablished correlation between capillary density and muscle oxidative capacity (Cebasek et al., 2005). It is known that growth factors like vascular endothelial growth factor (VEGF) are the metabolic stimulus for angiogenesis (Gavin and Wagner, 2001; Brown and Hudlicka, 2003), therefore, muscular overwork in the contralateral medial pterygoid muscle could to bring about by the vascular endothelial growth factor, increasing the endothelial cell sprouting and consequently blood capillary surface and volume density. In the ipsilateral muscle, in spite of blood capillary volume and surface density were not significantly different from those of control animals, narrowed lumen and irregular walls were prominent feature under electron microscopy analysis, according to Bani et al. (1999), when unilateral occlusal abrasion was the stressing trigger on masseter muscle. In hypofunctional mastication this vasoconstriction suggests that an ischemic injury is an event in the onset of the muscle tissue damage. It was concluded that a complex adaptation of the medial pterygoid muscle, to the chronic unilateral mastication takes place after 60 days, at morphological, biochemical and ultrastructural levels. On the ipsilateral side, the hypofunction leads to shorter neuromuscular spindle, lower oxidative metabolic activity, and microvessel constriction. On the contralateral side, the hyperfunction leads to shorter neuromuscular spindle alterations, higher oxidative capacity of the fibers, higher blood capillary surface and volume density, endothelial cell sprouting and numerous endothelial pinocytotic vesicles. Considering that neuromuscular spindles are proprioceptors responsible for the control of mandibular positioning and movements, their morphologic abnormalities suggested masticatory functional alterations in the experimental model adopted in this study. Although, alterations in neuromuscular spindle, in fiber metabolism, and in blood vessel parameter of masticatory muscle, do not interfere in the animal survival, investigators should consider if these changes interfere in the success of clinical procedures in medical field involving stomatognathic structures. Further researches considering heterogeneous architectural design of masticatory muscle should be conducted in order to better understand the correlation between occlusal alteration and task specific of the multifasciculated muscle. Acknowledgement We are grateful to FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo) for financial support.
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