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RESEARCH REPORT |
1 Department of Orthodontics,
2 Department of Maxillo-Facial-Surgery, and
3 Department of Prosthetic Dentistry, School of Dentistry, Medical Faculty, Carl Gustav Carus, Technical University of Dresden, Fetscherstrasse 74, D-01307 Dresden, Germany; and
4 Institute of Pharmacology and Toxicology, Friedrich Schiller University, Jena, Germany;
* corresponding author, harzer{at}rcs.urz.tu-dresden.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: masticatory muscle • Botulinum toxin • x-ray microanalysis • pigs.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Stress or abnormal brain or spinal cord activity may lead to excessive or uncoordinated loading of muscles. This, in turn, may lead to non-physiological spasms of these muscles, often causing pain or changes in sweat gland function. Another kind of disturbance is insufficient muscle innervation. Small doses of Botulinum toxin (Botox®) injected into the muscles cause denervation. After paresis with Botox®, the activity of a muscle is reduced, at least in part (Westgaard and Lomo, 1988). The toxin thus paralyzes or weakens the injected muscle, leaving the other muscles unaffected in their function. Botox® binds to the nerve endings and blocks acetylcholine exocytosis from nerve endings that would otherwise give the muscle a signal to contract. The release of acetylcholine also induces small amounts of calcium to enter the cell. Further, it has been shown that muscle denervation results in a fall of resting membrane potential within only 3 days (Bray et al., 1976). This depolarization is believed to be caused by an alteration of ion movements across the fiber membranes, and possibly by a change in passive membrane permeability (Shabunova and Vyskocil, 1982).
Recently, we have shown that changes in jaw-closing kinetics due to endurance stress lead to differences in fiber types (Gedrange et al., 2001b). These changes in fiber composition are related to the content of bound and diffusible ions in the muscle.
From histological and morphological investigations, it is known that paresis causes muscle atrophy and changes in muscle fiber structure, with changes in ion content (Wroblewski et al., 1987). Thus, the ion composition of the muscles depends on muscle function. Therefore, it is important that the muscles show a typical composition. It is unclear whether jaw-opening and -closing after masseter muscle paresis cause disturbances in ion concentrations. Further, changes due to paresis have not been investigated in muscles treated with Botox®. Reports in the literature cite that Botox® acts only locally (Westgaard and Lomo, 1988). We hypothesized that Botox® has an influence not only on structural muscle changes, but also on the ion level in the affected muscle and surrounding masticatory muscles, which must work harder. Further, in cases of muscle atrophy, a shift of fiber type (from slow-twitch to fast-twitch) and changing bone morphology were observed. This interdependence between atrophy fiber shift and bone morphology could be important in muscular diseases, hemifacial hypertrophia, unilateral bruxism, and TMJ dysfunction. Unilateral paresis could also serve as a model for impairment of facial growth and development.
Thus, the aim of the present animal study was to examine the effects of both paresis of the right masseter muscle and chronic stress on the masticatory muscles, with no recovery phase. Paresis of the masseter muscle was induced by the injection of Botulinum toxin. The changes in ionic content in various muscle regions were evaluated by energy-dispersive x-ray microanalysis (EDX) in an environmental scanning electron microscope (ESEM).
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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When all pigs had been anesthetized, a 10-mL (100 IU) quantity of Botulinum toxin A (Botox®, Merz Pharma, Frankfurt, Germany) was injected into 10 defined regions of the exposed right masseter muscles of 7 pigs (Fig.
). For this, the muscle masseter was divided symmetrically into 10 regions, to allow for overall paresis. The defined area was calculated relative to anatomical landmarks between the zygomatic arch and the lower jaw margin. A 10-mL quantity of saline solution was injected into the same region of the 8 other pigs, serving as controls. Following the injection, the wound was monitored. The food consumption of the pigs was recorded during the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, and 8th wks. After 56 days, muscle biopsies were carried out on the animals under anesthesia without resuscitation. The animals were killed by an intravenous injection of T1 solution (B. Braun, Muenster, Germany).
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All 147 muscle samples (56 from controls and 91 from the treated group) were frozen in liquid nitrogen (–196°C). The samples were defrosted for x-ray analyses.
X-ray Microanalysis
The presence and distribution of ions in muscle tissues were analyzed by energy-dispersive x-ray microanalysis (EDX) in an environmental scanning electron microscope (ESEM) (ESEM XL30, Philips, Eindhoven, Netherlands) that included secondary electron, back-scattered electron- and x-ray detectors for ion measurement.
Quantitative x-ray microanalysis of biological specimens in a water vapor atmosphere is valid only if the sample is spread over a wide area, leading to mean elemental values for the entire preparation (Sigee and Gilpin, 1994). Therefore, 5 x 4 x 4-mm sections were mounted on a special carbon holder for x-ray microanalysis. Specimens were analyzed both in spot mode and over a full field of view (normally 900–1100 µm2). The intra- and extracellular surfaces of the muscle samples were measured and analyzed.
The specimen chamber pressure was 4 torr, with a water atmosphere of 45%. The temperature of the specimen stage in the ESEM was maintained at 8°C. The distance between detector and specimen was 14 mm. X-ray microanalysis was carried out with a 20-kV electron probe over 100 sec. The count rate was typically from 900 to 1100 cps. For ion assessment, the peak/background ratio was determined. The relative peak intensities were transformed to mmol/gwd (gram dry weight). The device has the ability to save the results as digital images in tagged image file format (TIFF). The video picture was used for determination of cell morphology.
Statistical Analysis
For statistical analysis, we applied an unpaired Student’s t test to evaluate differences in biochemical data between controls and treated animals (Gedrange et al., 2001a).
Significant differences between muscle samples of control animals and respective muscles of the injection (right) sides of treated pigs as shown as * (Tables 1,2), and significant differences between muscles of control pigs and the respective muscles of the non-injection (left) sides of treated pigs are shown as # (Tables 1,2). Significant differences between respective samples from the right and left sides in treated pigs are shown as + (Tables 1,2). Data with one symbol were evaluated at P < 0.05, the respective data marked with two symbols were evaluated at P < 0.01, and data with three symbols at P < 0.001.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Changes in Muscle Ion Composition
The effect of injection-side (right) masseter paresis on ion concentrations in the masticatory muscles investigated was significant in the masseter, temporalis, and pterygoid muscles (Tables 1
, 2, 3). Environmental scanning electron microscopy revealed no defects in the plasma membrane.
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). Additionally, a significant elevation of Na+ was measured in the anterior part of the temporalis muscle (TP1) and in the pterygoid muscle of treated pigs (P < 0.05) (Table 2). The Cl– content was enhanced only in the masseter muscles of treated pigs (Table 1). This effect was more pronounced in the masseter muscle treated with Botox® (P < 0.01) than in the contralateral muscle (P < 0.05). These differences in Cl– content measured between other treated and contralateral muscles failed to reach significance (Table 2).
A significant decrease in the K+ content was found in Botox®-treated masseter muscles (Table 1). This decrease was significant in comparison with controls (P < 0.01), as well as with the respective muscle of the contralateral side (P < 0.01).
The largest changes of all elements investigated were measured in phosphorus (P) content. In this case as well, however, changes were seen in the masseter muscle only. In treated animals, chronic stress caused a significant increase in P content in the masseter muscle (M1, M2) on the non-injection (left) side, and a decrease in the Botox®-treated masseter muscle (P < 0.05).
Enhanced sulfur (S) content was observed in almost all masticatory muscles examined, except in the geniohyoid muscles. Additionally, a distinct difference in sulfur content was found in the contralateral side of the masseter muscles of treated pigs compared with controls (P < 0.01). In comparison with controls, the increase in S content was not as pronounced as in Botox®-treated masseter muscles (P < 0.05). After the treatment, an increase in the S content of temporalis and pterygoid medial muscles was also observed on both sides. No changes were detected in the Ca++ and Mg++ concentrations in any of the muscles investigated (Tables 1, 2, 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The strongest changes in elemental muscle composition were measured in mobile ions such as potassium (K+), chloride (Cl–), and sodium (Na+). Electrical properties of the cell membranes depend on the selective distribution of these ions. It is the concentration difference of K+ across the muscle cell membrane that is mainly responsible for faster depolarization and, subsequently, faster contraction of the fiber. The concentration of K+ in the masseter muscle decreased as a result of treatment with Botox®. Also, in myopathy, a reduction in K+ content was seen (Edström et al., 1982). In our investigation, this K+ decrease was accompanied by a greater Na+ increase. Wroblewski et al.(1987) showed that muscle immobilization causes an increase in Na+ and Cl–. In the present investigation, a non-significant increase in Cl– was also measured.
Due to energy deficiency, ion shifts and changes in density of Na+/K+ pumps may occur in the muscle (Trump et al., 1979). For instance, endurance training has been shown to cause an increase in Na+/K+ pumps (Leivseth et al., 1992). This effect may be responsible for the changes in K+, Na+, and Cl– ions in contralateral muscles (TP1, TP2, PM). In the present investigation, an increase in Na+ and Cl– but no changes in the K+ content were observed in the muscles. According to our results, the amount of phosphorus (P) in the Botox®-treated masseter muscle was significantly lower than in the respective control muscle or on the contralateral side. The higher phosphorus content in the contralateral masseter may be partly associated with the high phosphorus level required for high metabolism of contractile and ion-transported proteins. It has been reported in the literature that trained muscles have a higher capillary density and a higher density of mitochondria than do paretic muscles (Skorjanc et al., 1998). The high content of released phosphorus is probably due to the high content of phosphocreatine. A rapid increase in the rate of adenosine 5'triphosphate (ATP) re-synthesis is required to support muscle contraction. The phosphorus changes suggest a relationship to the masseter’s role as the major muscle during masticatory activity. Furthermore, these changes may have resulted from some sort of compensatory activity in response to the Botox® injection. The increase of sulfur (S) in the muscles of treated pigs may be associated with changes in the content of the primary sulfur-containing amino acids, like cystine, cysteine, methionine, taurine, and the sulfur-containing tripeptide glutathione (GSH), which is composed of glutamic acid, cysteine, and glycine. In our investigation, the animals’ body weight increased continuously in line with their age. Thus, the nutrient supply was also sufficient in treated pigs, so the influence of nutrition on sulfur content is unlikely. However, the sulfur content of the muscle may also be influenced by Botox®. Sulfur may be associated with the protection of cell membranes by attenuating toxic substances and/or acting as an osmoregulator, and by providing optimal oxygen capacity (Keys and Zimmerman, 1999). The increase in sulfur was higher in untreated masseter muscles than in those treated with Botox® and in the controls. This may be due to a high metabolic rate. Endurance training involving increased metabolism is one source of the production of peroxides which cause oxidant damage. It has already been suggested, in previous investigations, that synthesis of glutathione may be partially responsible for the higher sulfur content in type I fibers (Gedrange et al., 2001b). In the present investigation, the degree of change in sulfur content was lower than in phosphorus, probably because GSH synthesized in the muscles is also released into the blood flow, since muscles can serve as an extra-hepatic GSH source (Kretzschmar and Muller, 1993).
No changes in calcium (Ca++) or magnesium (Mg++) content were found between controls and treated animals. The movement of calcium and magnesium into and out of cells supports the generation of nerve impulses and muscle contraction. However, low concentrations of calcium changes cannot be measured accurately with energy-dispersive x-ray microanalysis (Tylko et al., 1999). The masticatory muscles are among those with a low calcium content. Until now, high calcium levels have been found only in the stapedius muscle of guinea pigs (Wroblewski et al., 1981). Only in this muscle is the measurement of calcium changes possible (Wroblewski et al., 1981).
The higher magnesium content in controls and treated pigs underlines the importance of muscle function. This higher magnesium content was accompanied by a low calcium content, and this balance is controlled by parathyroid hormone. In muscles, magnesium acts as a calcium antagonist at the cell membrane level, which is necessary to maintain normal electrical potentials and to coordinate the muscle’s action (D’Angelo et al., 1992).
Changes in the ionic content after the application of Botox® suggest that the muscles are affected in a different way. The results show that a direct effect of Botox® on the ion content of muscles is unlikely. Botox® has an indirect impact on ion concentration, which results in changes of functional and energetic muscle capacity. Botox® has a very strong impact on muscle activity. For chewing properties to be maintained, the function of the paretic masseter muscle is taken over by other muscles. Thus, the investigation shows the effect of an imbalance in masseter function and suggests that an improvement of temporomandibular joint dysfunction or impairment of facial growth and development seems possible through balance in muscle function.
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ACKNOWLEDGMENTS |
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Received July 21, 2004; Last revision July 13, 2005; Accepted July 14, 2005
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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