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RESEARCH REPORT |
1 Department of Orthodontics, Technical University, Dresden, Germany;
2 Institute of Pathophysiology, Friedrich Schiller University, D-07740 Jena, Germany;
3 Institute of Anatomy, Technical University, Dresden, Germany; and
4 Institute of Laboratory Animal Science, Jena, Germany;
*corresponding author, rbau{at}mti-n.mti.uni-jena.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: muscle fiber types • capillarization • masticatory muscle blood flow • juvenile pigs
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, experimental studies have shown that at least during the initial phase of sagittal mandibular advancement, e.g., within a period of 4 wks, a marked conversion of muscle fibers occurred in masticatory muscles with a significant increase in the proportion of slow-twitch type I fibers (Gedrange et al., 2001a). This suggests an increased long-term activity of the respective regions of the masticatory muscles. Furthermore, these intramuscular alterations induced by sagittal mandibular advancement seem to be accompanied by increased oxidative stress, indicated by an altered intramuscular glutathione metabolism (Gedrange et al., 2001b). However, because type I muscle fibers are not only responsible for maintenance functions through increased fatigue resistance but also sensitive to oxygen shortages, a persisting maintenance of the remodeled masticatory muscle function needs an appropriate adaptation in blood supply. To clarify how an expected increase in oxygen and substrate demand of converted masticatory muscles is warranted, we determined the effects of sagittal mandibular advancement on regional blood flow and capillary density, together with assumed muscle fiber conversion, as shown previously (Gedrange et al., 2001a). It was hypothesized that an increase in type I fiber number may be accompanied by an increased capillary density and improved muscular blood flow.
Juvenile pigs were used because there are several craniofacial similarities with humans, e.g., both are omnivores and exhibit a comparable dentition, vertical occlusion, and stroke orientation, as well as relatively shortened faces (Ciochon et al., 1997). In addition, development dynamics and mature patterns of muscle fiber distribution and muscle function are comparable (Tuxen and Rostrup, 1993; Anapol and Herring, 2000). Therefore, the juvenile pig represents a valuable nonprimate model for studying morpho-functional aspects of corrective influences on malocclusion.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Procedures to Induce Long-term Lower Jaw Sagittal Advancement
The animals were initially anesthetized with ketamine hydrochloride (12 mg • kg-1 body weight, i.m.). After insertion of a venous catheter into an ear vein, anesthesia was maintained by propofol injection (Abbott, Ludwigshafen, Germany) (0.7 mg • kg-1 body weight, i.v.) as required. To induce sagittal advancement of the lower jaw, we fixed synthetic build-ups (Evicrol®, SPOFA-DENTAL, Prague, Czech Republic) to the upper and lower molars of 6 randomly selected animals and placed them at an oblique plane so that the lower jaw of the animal was bound to move forward during clenching (treated animals, group 2). The remaining 6 animals received a similar anesthesia but no build-ups and served as sham-operated controls (group 1). The duration of the surgical procedure was ca. 10-15 min. During the 1st, 2nd, and 3rd wks, the build-ups were checked and replaced as required. Treatment was continued for 28 days, during which time the animals were housed together with free access to water and nutrients.
Muscle Blood Flow Measurement
At the end of the four-week observation period, the animals were anesthetized with 1.5% isoflurane in nitrous oxide and oxygen (inspiration fraction of oxygen = 0.35-0.40). Additionally, all incision sites were infiltrated with 1% lidocaine (Jenapharm, Jena, Germany). The rectal temperature was maintained at 38°C by means of a heating pad and a temperature-controlled heating lamp. The urinary bladder was punctured and permanently drained (Cystofix, Minipaed®, Braun Melsungen AG, Melsungen, Germany). A central venous catheter was inserted through a branch of the left external jugular vein. A tracheotomy was then performed; the animals were paralyzed with pancuronium bromide (0.2 mg • kg-1 • h-1) and mechanically ventilated (Servo Ventilator 900C, Siemens-Elema, Solna, Sweden). The artificial ventilation was adjusted for measurement of normoxic and normocapnic blood gas values. A catheter was inserted into the left saphenous artery and advanced to the abdominal aorta for blood pressure monitoring and for withdrawal of arterial blood samples. A left thoracotomy was then performed through the 3rd intercostal space, and a catheter was inserted into the left atrium for injection of colored microspheres. All surgical wounds were closed, and the animals were allowed to recover from surgery for 1 hr.
Then, the regional blood flow was measured with colored microspheres. The application of this method in piglets has been validated and described in detail (Bauer et al., 1996; Walter et al., 1997). Briefly, yellow microspheres (3 x 106 in 1 mL saline and 0.01% Tween 80) were injected within 10-15 sec into the left atrium (Dye Trak, Triton Technology, San Diego, CA, USA). During injection, an arterial reference sample was withdrawn from the abdominal aorta at a rate of 3.53 mL • min-1 in 120 sec, starting 15 sec before microsphere injection (syringe pump SP210iw, World Precision Instruments Inc., Sarasota, FL, USA). After microsphere injection, an arterial blood sample was taken for measurement of oxygen saturation, blood hemoglobin, and blood gases (OSM2, ABL 50, Radiometer, Copenhagen, Denmark). The animals were then killed by intravenous injection of saturated potassium chloride solution, and masticatory muscles were taken and carefully isolated. Specimens of the right masticatory muscles were taken for histological evaluation.
The left and remaining right masticatory muscles were used for muscle blood flow measurement. Three regions of the masseter muscle were differentiated, i.e., the anterior (M1) and medial (M2) regions of the superficial part and the posterior region of the profound part (M3), and were dissected from animals of both groups. Additionally, the anterior (TP1) and posterior (TP2) parts of the temporal muscle together with samples of the medial pterygoid muscle (PM) and the geniohyoid muscle (GH) were investigated. Muscle and arterial reference blood samples were digested in 4 mol potassium chloride containing 4% Tween 80 and filtered through an 8-µm-pore polyester-membrane filter. We recovered the dye from the microspheres by adding 150 µL N,N-dimethylformamide and measured the absorption spectra of the dye solution with a UV/VIS spectrophotometer (Model 7500, Beckman Instruments, Fullerton, CA, USA). We used specific absorbencies of the individual dyes to quantify the colored microspheres (MISS software package; Triton Technology, San Diego, CA, USA) of each tissue sample. We calculated regional muscle blood flows (mL • min-1 • 100 g-1) using the equation:
(flow ratetissue = number of CMStissue • (flow ratereference/number of CMSreference)
Immunohistochemistry
Two cross-sections of muscle tissue were taken for immunohistochemical analysis, one for immunohistochemical determination of myosin heavy chain [(Narusawa et al., 1987); mouse monoclonal anti-skeletal myosin heavy-chain type I antibody, Clone NOQ 7.5.4D; Sigma, Deisenhofen, Germany; dilution 1:20] and the other for immunohistochemical identification of capillaries [(Song et al., 1997); mouse monoclonal anti-caveolin-1; CAV-1 antibody; Transduction Laboratories, distributed by Dianova, Hamburg, Germany; dilution, 1:20].
Specimens were sectioned (thickness 8 µm) at 20°C by means of a microtome (model 2055, Leica, Bensheim, Germany). Serial sections were collected on poly-L-lysine-coated glass slides, dried, and stored at room temperature, and then processed in parallel. Cross-sections were pre-incubated in TRIS-buffered saline and then exposed to 3.0% hydrogen peroxide in 60% methanol-40% buffered saline for 20 min at room temperature. To block nonspecific binding of antibodies, we incubated the cross-sections in 0.1 M TRIS-buffered saline containing 1% bovine serum albumin and 10% normal serum for 4 hrs at room temperature.
Primary antibodies were applied at 37°C for 45 min. After being washed in TRIS-buffered saline for 10 min with biotinylated secondary antibodies, the serial sections were detected by incubation for 30 min with a commercially available antimouse goat antibody (Dako, Glostrup, Denmark; dilution, 1:400). Sections were then incubated for 60 min with avidin-biotinylated enzyme complex solution (Vectastain kit, Vector, Burlingame, CA, USA). The color was developed with diaminobenzidine (Aldrich Chemical Company, Inc., Milwaukee, WI, USA), and the sections were counterstained with hematoxylin. As negative controls, the primary antibodies were replaced by TRIS-buffered saline or non-relevant monoclonal IgG.
Morphometric Characteristics of Muscle Tissue Cross-sections
The histological preparations were scanned at 3.2-fold enlargement with 600 pixels’ resolution, stored on a personal computer, and quantified off-line by automatic evaluation of marked capillaries and fiber areas (Leica, Cambridge, England).
The fiber ratio was measured from the surfaces of type I fibers (stained) and type II fibers (unstained). Capillary density was defined as the number of capillaries per millimeter squared. The number of capillaries was counted for all fibers in the field of vision. For fibers in the periphery of the field cut by the frame of the vision field, an area corresponding to one-half of the area of the marginal fiber cross-section was added. In addition, fiber density (fiber number per millimeter squared) was determined from the anterior region of the superficial part of the masseter muscle (M1), and the capillary per muscle fiber ratio was calculated in both groups.
Statistical Analyses
Data are reported as means ± SD. Comparisons between untreated and treated animals were made with unpaired t tests. Linear correlation was estimated between capillary density and regional blood flow of corresponding regions of masticatory muscles by means of the Pearson Product Moment Correlation coefficient. Differences were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Furthermore, there were similar physiologic parameters in both groups during blood flow measurement of the masticatory muscles (Table).
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), whereas chronic lower jaw protrusion induced a marked blood flow increase in the anterior and medial regions of the superficial part of the masseter and in the medial pterygoid muscle (P < 0.05). The remaining regions of the masticatory muscles studied showed no significant blood flow alterations after chronic protrusion. Furthermore, regional distributions of capillary density as well as type I fibers in masticatory muscles were quite similar in untreated juvenile pigs (Fig. 1). After chronic lower jaw protrusion, a marked increase in capillary density was found in all regions of masticatory muscles with an increased blood flow as well as in the posterior part of the temporal muscle (Fig 1, P < 0.05). The amount of type I fibers of masticatory muscles was increased in the same regions, which was reflected in an increased capillary density (Fig 1, P < 0.05). However, total muscle fiber density was similar in all muscles of treated and untreated animals. Consequently, the capillary-to-fiber ratio, estimated in M1, increased markedly (group 1, 0.6 + 0.13; group 2, 1.3 + 0.25, P < 0.01).
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, r = 0.89, P < 0.01).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Combined characterization of simultaneously increased numbers of type I fibers, increased capillary density, and enhanced blood flow in distinct regions of the masticatory muscles resulting from a certain period of artificial mandibular protrusion suggest a coordinated structural and functional change in these muscle regions. However, the causal mechanisms responsible for morphological and functional changes in masticatory muscles during the induction of lower jaw sagittal advancement have yet to be studied. Findings reported from skeletal muscles have shown that augmented conversion to slow-twitch, "fatigue-resistant" type I fibers is influenced in vivo by impulse activity as determined by the nerve (Salmons and Sreter, 1976), by the level of physical activity (Salmons and Henriksson, 1981), and by passive stretch (Goldspink et al., 1992). During fast-to-slow transformation, genes encoding slow isoforms of myosin heavy- and light-chains as well as genes encoding enzymes involved in the oxidative metabolism are up-regulated (Pette and Vrbova, 1992). Recently, it has been shown that an intracellular signaling pathway dependent on calcineurin is crucially involved in the control of fiber-type specific gene expression in skeletal muscles (Chin et al., 1998; Olson and Williams, 2000). Interestingly, the temporal behavior of chronic muscle activity appears to be responsible for the effectiveness of fast-to-slow transformation of contractile proteins. It has been shown that initiation of a fast-to-slow transformation process requires a minimum duration of the activation period used during each stimulation cycle, and the total number of stimuli applied is not the decisive parameter for the fiber type switch, which is caused by a nuclear accumulation of specific transcription factors (Kubis et al., 2002). However, calcineurin alone is not sufficient to mediate the complete transformation, because mitochondrial and cytosolic enzymes of energy metabolism are not regulated by this pathway (Meissner et al., 2001).
Fast-to-slow fiber remodeling of the masticatory muscles also encompasses an increase in capillary density in identical muscle regions (Fig. 1
, P < 0.05). Such an augmentation of exchange area between microcirculatory bed and slow-twitch contractile fibers is presumably caused by angiogenesis. It has been shown that adaptation in the size of the capillary bed by endurance training is due to an increasing capillary number rather than to the degree of capillary tortuosity (Poole et al., 1989). Local increases in blood pressure and endothelial injury have been suggested to be important factors initiating the formation of new capillaries. Angiogenesis during fast-to-slow fiber remodeling has been related to an increased expression of vascular endothelial growth factor (Brown et al., 1995). In stretched muscles, endothelial cell proliferation and abluminal sprouting gave rise to new capillaries, with basement membrane loss only at sprout tips. These distinct mechanisms appear to be additive, since both forms of capillary growth occurred in chronically stimulated muscles (Egginton et al., 2001).
We determined the regional distribution of capillary density in masticatory muscles and found a similar distribution for slow-twitch muscle fibers. Furthermore, the time course of capillarization and conversion in the oxidative metabolism during remodeling is obviously quite different. As shown in skeletal muscles, angiogenesis occurred just a few days after the beginning of low-frequency muscle stimulation and reached a plateau after 2 wks. During this period, vascular endothelial growth factor activity was markedly enhanced. However, citrate synthase activity, a marker of aerobic-oxidative metabolic potential, was first registered after 1 wk and increased gradually throughout the seven-week observation period, indicating that increases in capillarization precede increases in the aerobic-oxidative potential of the energy metabolism (Skorjanc et al., 1998).
Capillary density and capillary-to-fiber ratio in masticatory muscles have not been determined before. Nevertheless, similar values in the size of the capillary bed were reported in the canine gracilis muscle, with a similar percentage of type I fibers under normal conditions and a comparable conversion index after chronic stimulation (Hudlicka et al., 1987). The strong linear correlation between capillary density and regional blood flow of masticatory muscles (Fig. 2, r = 0.89, P < 0.01) indicates that the regional perfusion rate of the microcirculatory bed is a function of the number of microvessels. Therefore, the angiogenesis induced during chronic sagittal advancement of the lower jaw led to an increase in capillary surface area as a prerequisite of increased striated muscle aerobic capacity (Hepple, 2000).
In conclusion, we have shown that chronic lower jaw sagittal advancement in the juvenile pig induced an orchestrated remodeling of distinct parts of masticatory muscles with transformation of fast, fatigable muscles toward slower, fatigue-resistant ones, together with an increase in size of the capillary bed and an associated increase in regional blood flow. These changes suggest a complex muscle adaptation for long-term, fatigue-resistant activity during the early corrective period of mandibular-maxillary malocclusion.
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ACKNOWLEDGMENTS |
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Received October 23, 2002; Last revision April 24, 2003; Accepted April 29, 2003
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Bauer R, Walter B, Wurker E, Kluge H, Zwiener U (1996). Colored microsphere technique as a new method for quantitative-multiple estimation of regional hepatic and portal blood flow. Exp Toxicol Pathol 48:415–420.[ISI][Medline]
Brown MD, Hudlicka O, Makki RF, Weiss JB (1995). Low-molecular-mass endothelial cell-stimulating angiogenic factor in relation to capillary growth induced in rat skeletal muscle by low-frequency electrical stimulation. Int J Microcirc Clin Exp 15:111–116.[ISI][Medline]
Chin ER, Olson EN, Richardson JA, Yang Q, Humphries C, Shelton JM, et al. (1998). A calcineurin-dependent transcriptional pathway controls skeletal muscle fiber type. Genes Dev 12:2499–2509.
Ciochon RL, Nisbett RA, Corruccini RS (1997). Dietary consistency and craniofacial development related to masticatory function in minipigs. J Craniofac Genet Dev Biol 17:96–102.[ISI][Medline]
Eckardt L, Gebert E, Harzer W (2001). Tensor analytical evaluation of the effects of a skeletonized activator in the treatment of Class II, Division1 patients. J Orofac Orthop 62:337–349.[Medline]
Egginton S, Zhou AL, Brown MD, Hudlicka O (2001). Unorthodox angiogenesis in skeletal muscle. Cardiovasc Res 49:634–646.
Gedrange T, Luck O, Hesske G, Buttner C, Seibel P, Harzer W (2001a). Differential expression of myosin heavy-chain mRNA in muscles of mastication during functional advancement of the mandible in pigs. Arch Oral Biol 46:215–220.[ISI][Medline]
Gedrange T, Lupp A, Walter B, Harzer W, Bauer R (2001b). Oxidative state and histological changes in muscles of mastication after conditioning training. Exp Toxicol Pathol 53:89–96.[ISI][Medline]
Goldspink G, Scutt A, Loughna PT, Wells DJ, Jaenicke T, Gerlach GF (1992). Gene expression in skeletal muscle in response to stretch and force generation. Am J Physiol 262:R356–R363.
Hepple RT (2000). Skeletal muscle: microcirculatory adaptation to metabolic demand. Med Sci Sports Exerc 32:117–123.[ISI][Medline]
Hudlicka O, Hoppeler H, Uhlmann E (1987). Relationship between the size of the capillary bed and oxidative capacity in various cat skeletal muscles. Pflügers Arch 410:369–375.[ISI][Medline]
Ingervall B, Bitsanis E (1986). Function of masticatory muscles during the initial phase of activator treatment. Eur J Orthod 8:172–184.
Kubis HP, Scheibe RJ, Meissner JD, Hornung G, Gros G (2002). Fast-to-slow transformation and nuclear import/export kinetics of the transcription factor NFATc1 during electrostimulation of rabbit muscle cells in culture. J Physiol 541:835–847.
Lefaucheur L, Edom F, Ecolan P, Butler-Browne GS (1995). Pattern of muscle fiber type formation in the pig. Dev Dyn 203:27–41.[ISI][Medline]
Lefaucheur L, Ecolan P, Plantard L, Gueguen N (2002). New insights into muscle fiber types in the pig. J Histochem Cytochem 50:719–730.
Meissner JD, Gros G, Scheibe RJ, Scholz M, Kubis HP (2001). Calcineurin regulates slow myosin, but not fast myosin or metabolic enzymes, during fast-to-slow transformation in rabbit skeletal muscle cell culture. J Physiol 533:215–226.
Narusawa M, Fitzsimons RB, Izumo S, Nadal-Ginard B, Rubinstein NA, Kelly AM (1987). Slow myosin in developing rat skeletal muscle. J Cell Biol 104:447–459.
Olson EN, Williams RS (2000). Calcineurin signaling and muscle remodeling. Cell 101:689–692.[ISI][Medline]
Pette D, Vrbova G (1992). Adaptation of mammalian skeletal muscle fibers to chronic electrical stimulation. Rev Physiol Biochem Pharmacol 120:115–202.[ISI][Medline]
Poole DC, Mathieu-Costello O, West JB (1989). Capillary tortuosity in rat soleus muscle is not affected by endurance training. Am J Physiol 256:H1110–H1116.
Salmons S, Henriksson J (1981). The adaptive response of skeletal muscle to increased use. Muscle Nerve 4:94–105.[ISI][Medline]
Salmons S, Sreter FA (1976). Significance of impulse activity in the transformation of skeletal muscle type. Nature 263:30–34.[Medline]
Skorjanc D, Jaschinski F, Heine G, Pette D (1998). Sequential increases in capillarization and mitochondrial enzymes in low-frequency-stimulated rabbit muscle. Am J Physiol 274:C810–C818.
Song KS, Tang Z, Li S, Lisanti MP (1997). Mutational analysis of the properties of caveolin-1. A novel role for the C-terminal domain in mediating homo-typic caveolin-caveolin interactions. J Biol Chem 272:4398–4403.
Suzuki A, Cassens RG (1980). A histochemical study of myofiber types in muscle of the growing pig. J Anim Sci 51:1449–1461.
Thuer U, Ingervall B, Burgin W, Demisch A (1992). No posterior mandibular displacement in Angle Class II, division2 malocclusion as revealed with electromyography and sirognathography. Eur J Orthod 14:162–171.
Tuxen A, Rostrup E (1993). Histochemical characterization of pig masseter muscle: an animal model. Scand J Dent Res 101:57–61.[ISI][Medline]
Walter B, Bauer R, Gaser E, Zwiener U (1997). Validation of the multiple colored microsphere technique for regional blood flow measurements in newborn piglets. Basic Res Cardiol 92:191–200.[ISI][Medline]
Watted N, Witt E, Kenn W (2001). The temporomandibular joint and the disc-condyle relationship after functional orthopaedic treatment: a magnetic resonance imaging study. Eur J Orthod 23:683–693.
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