| Sign In to gain access to subscriptions and/or personal tools. |
X-ray Microanalysis of Elements in the Masticatory Muscle after Paresis of the Right Masseter
1 Department of Orthodontics, Correspondence: * corresponding author, harzer{at}rcs.urz.tu-dresden.de
Muscle activity and function appear to be related to ionic concentrations in the muscle. We investigated whether muscle paresis induced by injection of Botulinum toxin A (Botox) in 16-week-old pigs over a 56-day period is associated with ionic changes in the affected muscles. Tissue samples were taken from the masseter, temporalis, medial pterygoid, and geniohyoid muscles by a standardized method and used for energy-dispersive x-ray microanalysis in an environmental scanning electron microscope. The largest increase in Na+ was measured in the right and left sides of the masseter muscle in treated animals. Additionally, a significant elevation of Na+ was measured in the anterior part of the temporalis muscle and in the pterygoid muscle (P < 0.05). In temporalis and pterygoid muscles, an increase in sulfur in both sides of treated pigs’ heads was observed. Botox® has an indirect impact on ion concentrations, resulting in changes in muscle functional capacity and adaptive compensation of paretic muscle function by other muscles.
Key Words: masticatory muscle • Botulinum toxin • x-ray microanalysis • pigs.
Muscle contractility is mainly determined by the degree of the stimulus, the type of muscle fiber, and local factors such as ionic and ATP content. During contractions, extracellular sodium (Na+) is exchanged for intracellular potassium (K+). A decline in intracellular K+ and a doubling of extracellular K+ therefore occurs. This may lead to a reduction in force development, causing fatigue (Sjogaard, 1996). After muscle loading, changes in muscle pH, blood supply, and activity of the myosin ATPase may occur. In contrast to short load endurance, stress causes changes in muscle fiber composition to adapt to a modification of the myosin heavy-chains (MyHC). The newly formed isoforms of MyHC show a difference in energy consumption and energy production (Skorjanc et al., 1998). The changed energy production may have an influence on the content of bound elements (phosphorus and sulfur) in the muscle fibers. 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).
Animals and the Muscle Biopsy Process The experiments were carried out on 15 16-week-old pigs ("Deutsches Landschwein") randomized for the treatment. The protocol and all surgical procedures were approved by the Government Committee for Animal Research of the Federal State of Saxony (No. 24-9168.11-1-2002-1).
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.
Because of the different anatomical structures and fiber-course directions in the masseter and temporalis muscles, and due to the various functions of the masticatory muscle during chewing, muscle samples were taken from the masseter muscle (superficial, M1 = anterior, M2 = medial; and deep part, M3 = posterior) and from the temporalis muscle (TP1 = anterior, TP2 = posterior), as well as from the medial pterygoid (PM) and the geniohyoid (GH) muscles. Tissue samples were taken from both the injection (right) and the non-injection sides (left) of treated pigs, but from only the right side in the controls, because previous investigations had revealed no significant differences between the right and left sides of the masticatory muscles of healthy pigs (Gedrange et al., 2002). 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 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
Significant differences between muscle samples of control animals and respective muscles of the injection (right) sides of treated pigs as shown as * (Tables 1
Animal Characteristics The animals were in a good state of health throughout the eight-week treatment period. The body weight increased continuously and corresponding to age in both groups. In the pre-test, the control group weighed 32.1 kg ± 2.7 kg; after 8 wks, it weighed 48.7 kg ± 4.0 kg. The animals with paresis of the right masseter weighed, in the pre-test, 33.0 kg ± 2.7 kg, and after 8 wks of treatment, 47.7 kg ± 3.9 kg.
Changes in Muscle Ion Composition
The largest increase in Na+ was measured on the injection (right) and non-injection (left) sides of the masseter muscle in treated animals (Table 1  ). 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 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
The results show that EDX and ESEM facilitate the elemental analysis of small volumes of muscle tissue and provide information about the intracellular ionic environment, which, in turn, reflects changes or defects in the plasma membrane. Together with the study of ion composition, an analysis of ultrastructure is possible with an electron microscope (Roomans, 1988). 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.
The financial support of the Deutsche Forschungsgemeinschaft (German Research Council, GE 1154/2-1/2-2) is gratefully acknowledged. The authors thank R. Jung for skillful technical assistance. Received for publication July 21, 2004. Revision received July 13, 2005. Accepted for publication July 14, 2005.
Journal of Dental Research, Vol. 84, No. 11,
1026-1030 (2005) This article has been cited by other articles:
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
|
 |
|
Sign In to gain access to subscriptions and/or personal tools.
TOP
ABSTRACT
INTRODUCTION
