Early molecular response and microanatomical changes in the masseter muscle and mandibular head after botulinum toxin intervention in adult mice.

BACKGROUND: Masseter muscle paralysis induced by botulinum toxin type A (BoNTA) evokes subchondral bone loss in mandibular heads of adult rats and growing mice after 4 weeks. However, the primary cellular and molecular events leading to altered bone remodeling remain unexplored. Thus, the aim of the current work has been to assess the molecular response that precedes the early microanatomical changes in the masseter muscle and subchondral bone of the mandibular head in adult mice after BoNTA intervention. METHODS: A pre-clinical in vivo study was performed by a single intramuscular injection of 0.2 U BoNTA in the right masseter (experimental) of adult BALB/c mice. The contralateral masseter was injected with vehicle (control). Changes in mRNA levels of molecular markers of bone loss or muscle atrophy/regeneration were addressed by qPCR at day 2 or 7, respectively. mRNA levels of receptor activator of nuclear factor-κB ligand (RANKL) was assessed in mandibular heads, whilst mRNA levels of Atrogin-1/MAFbx, MuRF-1 and Myogenin were addressed in masseter muscles. In order to identify the early microanatomical changes at day 14, fiber diameters in transversal sections of masseter muscles were quantified, and histomorphometric analysis was used to determine the bone per tissue area and the trabecular thickness of subchondral bone of the mandibular heads. RESULTS: An increase of up to 4-fold in RANKL mRNA levels were detected in mandibular heads of the BoNTA-injected sides as early as 2 days after intervention. Moreover, a 4-6 fold increase in Atrogin-1/MAFbx and MuRF-1 and an up to 25 fold increase in Myogenin mRNA level were detected in masseter muscles 7 days after BoNTA injections. Masseter muscle mass, as well as individual muscle fiber diameter, were significantly reduced in BoNTA-injected side after 14 days post-intervention. At the same time, in the mandibular heads from the treated side, the subchondral bone loss was evinced by a significant reduction in bone per tissue area (-40%) and trabecular thickness (-55%). CONCLUSIONS: Our results show that masseter muscle paralysis induced by BoNTA leads to significant microanatomical changes by day 14, preceded by molecular changes as early as 2 days in bone, and 7 days in muscle. Therefore, masseter muscle atrophy and subchondral bone loss detected at 14 days are preceded by molecular responses that occur during the first week after BoNTA intervention.

Ultrastructural analysis of masseter and left medial pterigoid muscles, after unilateral mandibular molar extraction: experimental study / Análisis ultrastructural de los músculos masetero y pterigoideo medial izquierdo, después de la extracción molar mandibular unilateral: estudio experimental

Twelve adult male Wistar rats (220 g average weight) were divided in 3 experimental groups: GI -15. GII ­ 30 and GIII ­ 60 days, after mandibular molar extraction, with three experimental animals and one control per group. Qualitatively, ultrastructural changes of protein filaments from myofibrils of these muscles and ipsilateral to the extractions were observed. Ultrastructure asymmetry and disorganization of Z line and I band, in the experimental group GII, of Medial Pterigoid muscle (MPT) were observed. The temporomandibular dysfunction, stimulated by the unilateral extractions of mandibular molars in rats may lead to modifications in the Z line and I band, which showed to be sensitive to this dysfunction. Changes in the MPT muscle, probably related to its own functional characteristics and major participation in the dynamics of mastication, compared to Masseter were also observed. However, the muscular fibres seem to adapt to the new conditions along the experiment.

Doce ratas Wistar machos adultos (220 g de peso promedio) se dividieron en 3 grupos experimentales: GI - 15, GII - 30 y GIII - 60 días, después de una extracción molar mandibular. En cada grupo se dispusieron tres animales experimentales y un animal como control. Cualitativamente, se observaron cambios ultraestructurales de filamentos de proteínas de miofibrillas de estos músculos masticadores ipsilaterales a las extracciones. Se observó asimetría de la ultraestructura y desorganización de la línea Z y la banda I, en el músculo pterigoideo medial del grupo experimental GII, (MPT). La disfunción temporomandibular, estimulada por las extracciones unilaterales de los molares mandibulares en ratas, puede conducir a modificaciones en la línea Z y en la banda I, que mostraron ser sensibles a esta disfunción. Los cambios en el músculo pterigoideo medial, se debieron, probablemente, con sus propias características funcionales y una mayor participación en la dinámica de la masticación, en comparación con el músculo masetero. Sin embargo, las fibras musculares parecen adaptarse a las nuevas condiciones a lo largo del experimento.

Zymographic and ultrastructural evaluations after low-level laser irradiation on masseter muscle of HRS/J strain mice.

Low-level laser therapy (LLLT) has been widely used in the treatment of the stomatognathic system dysfunction; however, its biological effect remains poorly understood. This study evaluated the effect of LLLT (GaAlAs, 780 nm, 20 J/cm(2), 40 mW) on masseter muscle of HRS/J mice after different numbers of laser irradiations (three, six, and ten) for 20 s in alternate days. Three experimental groups were defined according to the number of laser irradiations and three control groups (n=5) were used. On the third day after the last irradiation, all animals were killed and the masseter muscle was removed and processed for the following analysis: (a) transmission electron microscopy, (b) zymography, (c) immunohistochemistry for vascular endothelial growth factor (VEGF) and VEGFR-2. The results showed: (a) with six laser applications, a dilation of T tubules, and sarcoplasmic reticulum cistern, increased pinocytosed vesicles in the endothelium; with ten laser applications, few pinocytic vesicles in the endothelium and condensed mitochondria. (b) Under the conditions of this study, the synthesis of other matrix metalloproteinases was not observed, only the MMP-2 and -9. (c) After ten laser irradiations, immunostaining was observed only for VEGFR-2. We conclude that after six laser applications, ultrastructural changes may facilitate the Ca(+2) transfer to cytosol and increase the fluid transport from one surface to another. The ultrastructural changes and no immunostaining for VEGF with ten applications may decrease the metabolic activity as well as damage the angiogenic process, suggesting that an effective number of laser applications may be less than ten, associating to this therapy a better cost-benefit.

Influence of laser therapy and muscle relaxant on the masseter muscle under occlusal wear: an ultrastructural study / Influencia de la terapia laser y relajante muscular en el músculo masetero bajo oclusión: un estudio ultraestructural

The aim of this study was to analyze the influence of low-intensity laser therapy and muscle relaxant in the characteristic ultra structural masseter muscle occlusal wear. Animals and Methods: 40 male Wistar rats were randomly divided into four groups: the control group (GI), occlusal wear (G-II), laser occlusal wear (G-III), and the muscle relaxant occlusal wear (G-IV). Under general anesthesia given intraperitoneally, animals in groups II, III and IV had unilateral amputation of upper and lower molar cusps to simulate an occlusal wear situation. The masseter muscle G-III received laser therapy (830nm, 4J/cm2, 40mW, f ~ 2mm) and the procedure was subsequently repeated every other day for 14/30 days. G-IV animals were treated with daily injection of dantrolene ® (2.5 mg / kg in 0.5 ml of H2O). From 24 hours after the elimination peak. The animals were euthanized with an overdose of anesthesia on days 14 and 30 after the removal of the cusps and the ipsilateral masseter muscle was excised and divided in two, one half was routinely processed for light microscopy and other for electron microscopy. There was no statistical difference between each experimental group and the control and between periods in each experimental group. However, the muscle fibers in the G-II showed the most pronounced changes. There is no causal relationship between muscles fibers injuries and occlusion and, despite signs of muscular tissue injury were more evident in the occlusal wear group. Results indicates a moderate action of laser therapy and muscle relaxants in skeletal muscle...

El objetivo del estudio fue analizar la influencia de la terapia láser de baja intensidad y del relajante muscular sobre las características ultraestructurales del músculo masetero en el desgaste oclusal. 40 ratas macho Wistar, se dividieron al azar en cuatro grupos: grupo de control (GI), desgaste oclusal (G-II), laserterapia desgaste oclusal (G-III), y relajante muscular desgaste oclusal (G-IV). Bajo anestesia general por vía intraperitoneal, los animales de los grupos II, III y IV sufrieron amputación unilateral de las cúspides de los molares superiores e inferiores para simular una situación de desgaste oclusal. El músculo masetero del G-III recibió la terapia con laser (830nm, 4J/cm2, 40mW, f ~ 2mm) después del procedimiento el cual se repitió durante 14/30 días. Los animales del G-IV fueron tratados con una inyección diaria de Dantroleno® (2,5 mg/Kg en 0,5 ml de H2O). Los animales fueron sacrificados con una sobredosis de anestesia general en los días 14 y 30. Después de la remoción de las cúspides el músculo masetero ipsilateral se extirpó y se dividió en dos, una mitad fue procesada para microscopía de luz y la otra para microscopía electrónica. No hubo diferencias estadísticamente significativas entre cada grupo experimental y el control, así como, entre los períodos en cada grupo experimental. Sin embargo, las fibras musculares en el G-II mostraron los cambios más pronunciados. En conclusión no existe relación causal entre las lesiones de las fibras musculares y la oclusión, a pesar que los signos de lesión de los tejidos musculares fue más evidente en el grupo con desgaste oclusal. Los resultados indican una acción moderada ejercida por la terapia láser y de los relajantes musculares sobre el músculo esquelético...

Effects of low-level laser irradiation in ultrastructural morphology, and immunoexpression of VEGF and VEGFR-2 of rat masseter muscle.

The present study evaluates by ultrastructural and immunohistochemical methods, the possible changes on muscular tissue affected by LLLI during a treatment, for example, in cases of temporomandibular joint disorders. Sixty male Wistar rats divided into 6 groups (n=10) received ten laser irradiations, with different energy densities (groups I-0; II-0.5; III-1.0; IV-2.5; V-5.0; and VI-20 J/cm(2)). Muscles were removed and processed for transmission electron microscopic and immunohistochemical (VEGF and VEGFR-2) analyses. Captured photomicrographs of immunohistochemistry and transmission electron microscopy were evaluated. It was observed in the irradiated muscles, mitochondria of different shapes and sizes, with increased plasticity evidenced by organelles in fusion, division and the presence of elongated structures with characteristics of mitochondria, proximity with the dilated sarcoplasmatic reticulum, suggesting organelles with large amounts of energy, and the presence of cytoplasmic protrusions in the capillaries with high dosages. All studied groups showed immunostainings for both markers (VEGF and VEGFR-2), but in general those who received higher doses also showed the markings more pronounced, suggesting dose-dependent biomodulation. It was concluded that the LLLI was able to modify the ultrastructural characteristics and immunohistochemical pattern of VEGF and VEGFR-2 in the masseter muscle of rats.

Masseter muscle inserted in the temporomandibular joint discocapsular complex, a case report

The presence of muscle insertions in the temporomandibular joint disc have a great importance in the dynamic joint. This article presents a case of bilateral insertion of deep fascicle of the masseter muscle in the temporomandibular joint capsule and disc in a spain corpse, describes the microscopic and macroscopic appearance of variation and a brief review of the functional implications.

La presencia de inserciones musculares en el disco de la articulación temporomandibular tiene gran importancia en la dinámica de la articulación. En este artículo se presenta un caso de la inserción bilateral del fascículo profundo del músculo masétero en la cápsula y disco de la articulación temporomandibular en un cadáver español, se describe el aspecto macroscópico y microscópico de la variación y se realiza una breve revisión de las implicancias funcionales.

Morphological alterations in the masseter muscle induced by unilateral extraction of the upper molars in guinea pigs (Cavia porcellus)

El objetivo del presente estudio fue analizar los aspectos morfológicos del sistema estomatognático y su relación con la mala oclusión. Con tal propósito, determinamos las alteraciones morfológicas que ocurren en el músculo masetero de cobayas, Cavia porcellus después de la inducción de mala oclusión a través de la extracción de los molares superiores del lado izquierdo. Los animales (n=20) fueron divididos en dos grupos (control y experimental), los cuales fueron sometidos a análisis biométricos, angioarquitecturales macroscópicos y al microscopio de luz. Los resultados obtenidos permitieron concluir que, después de 60 días de extracción dental unilateral hubo reducción en el ancho y en el grosor del músculo masetero del grupo experimental. Microscópicamente, se observaron fibras musculares de variados diámetros, de forma redondeada y poligonal, fibras musculares alteradas con un núcleo central, además de fibras en degeneración y macrófagos evidentes en la parte profunda del músculo.

Actividad de fosfatasa acida en tejidos de camundongos inmunes de germenes y convencionales / Acid phosphatase activity in tissues of germ-free mice and in conventional ones

La relación que existe entre la flora microbiana y la actividad de la fosfatasa ßcida (FAc) en los tejidos animales es poco conocida y su acción sobre los tejidos orales de animales inmunes de gÚrmenes (GF) es poco estudiada. El objetivo de este trabajo es verificar la influencia de la flora indÝgena sobre la actividad de FAc en el masetero, lengua, riñón e hÝgado de animales GF y convencionales (CV). Fueron seleccionados camundongos con 45 dÝas, machos y hembras: 10 GF y 10 CV. Para el estudio fueron removidos el masetero, la lengua, el riñón y el hÝgado y posteriormente teñidos histoquÝmicamente para la actividad de FAc. Anßlisis de los resultados mostraron una reacción enzimßtica fuertemente positiva en los animales CV, en cuanto que los tejidos de los animales GF se presentó francamente positiva. Este estudio sugiere que la microbiota indÝgena es importante para la reaccion de FAc en los tejidos de los animales estudiados

Hipertrofia maseterina. Estudio ultraestructural / Masseter muscle hypertrophy. Ultrastructural study

El presente trabajo estudia la hipertrofia maseterina desde el punto de vista ultraestructural del músculo masetero con hipertrofia. Para el mismo se toma una muestra de los músculos que presentan esta condición en 13 pacientes que acudieron a la Facultad de Odontología de la UCV. Las muestras de los pacientes con hipertrofia maseterina diagnosticada clínicamente, se observaron al miscroscopio electrónico. En el estudio se reportan hallazgos que hacen inferir que se está en presencia de una seudohipertrofia más que una hipertrofia verdadera. Se presentan los resultados parciales de esta investigación como aporte al estudio ultraestructural de los músculos masticatorios creando nuevas rutas en la investigación, en este renglón

Aspectos morfológicos do músculo masseter do coelho após simpatectomia cervical / Morphological aspects of masseter muscle of rabbits after cervical sympatectomy

Este trabalho teve como objetivos analisar o músculo masseter do coelho (O. cuniculus), após a retirada do gânglio simpático cervical superior direito, quanto aos aspectos histológicos, histoquímicos, imuno-histoquímicos e ultraestruturais. Foram utilizados 2 grupos de animais: 30 com simpatectomia e 10 sem simpatectomia - grupo controle. Os coelhos controles foram sacrificados 6, 12, 22 e 32 meses após o início do experimento, e os com simpatectomia, 3, 6, 9, 12, 15 e 18 meses após a retirada do gânglio simpático. Após o sacrifício, foram retirados os músculos masseteres direito e esquerdo dos animais com simpatectomia e controles; foi estudada a porçäo superficial do músculo, subdividida em 3 regiöes: próxima à inserçäo proximal - "anterior", regiäo mediana e próxima à inserçäo distal - "posterior". Os fragmentos foram congelados em n-hexana, e cortes histológicos foram submetidos às seguintes coloraçöes e reaçöes: HE, Tricrômico de Gomori, PAS, SDH, NADH-TR, ATPase miofibrilar (mATPase), Esterase Inespecífica e Imunohistoquímica, utilizando-se anticorpos específicos para a miosina "fast", (tipo IIa) e "slow", (tipo I). Outros fragmentos musculares foram coletados para a análise ultra-estrutural. O músculo masseter dos grupos controles, apresentou aspectos morfológicos similares, em ambos os lados: as fibras musculares mostraram morfologia geral preservada, à microscopia óptica e eletrônica; distribuiçäo em mosaico; frequência maior de fibras tipo IIa, seguida por fibras do tipo I, e menor frequência de fibras IIb. Nos grupos com simpatectomia, foram observadas lesöes nas fibras musculares, nos animais dos grupos de 6, 12 e em maior grau, no grupo de 18 meses. Em geral, houve predominíio de fibras tipo I, nos grupos com 3, 6, 9, 12 e 15 meses; no grupo de 18 meses, houve predomínio de fibras tipo IIa, sendo observada frequência maior de fibras IIb, em relaçäo aos demais grupos, com ou sem simpatectomia.

Ultraestructura muscular en la miositis osificante / Muscular ultrastructure in myositis ossificans

Para la presente investigación se estudió una biopsia del músculo masetero derecho perteneciente a un paciente con diagnóstico clínico de miositis osificante. Las alteraciones observadas incluyeron una atrofia muscular con daños a los sistemas sarcotubular y contráctil, la presencia en el sarcoplasma de numerosas estructuras lisosomales, así como mitocondrias de aspecto pleomórfico y de gran densidad electrónica. Asociadas a estas últimas se observaron cuerpos filamentosos. Como consecuencia de la atrofia, las fibras presentaron numerosos plegamientos y fibrillas de colágeno. Cabe destacar la presencia de complejos de Golgi en áreas subsarcolémicas. El presente trabajo extiende el conocimiento en la caracterización histopatológica de la miositis osificante

Peculiar characteristics of the insertion area of the masseter muscle in rats: scanning electron microscope study

Estruturas de superfície óssea correspondentes à área de inserçäo do músculo masseter foram estudadas em 20 ratos, albinus Wistar. As peças foram fixadas em soluçäo de Karnovsky modificada durante 12 horas e tratadas segundo a técnica de LESTER et alii. Os espécimes foram examinados ao microscópio eletrônico de varredura JEOL, JSM-P15. Os resultados mostraram que a área de inserçäo do músculo de ratos apresenta uma superfície lisa, revestida por espessos feixes de fibras colágenas calcificadas. Nas áreas próximas ao bordo inferior da mandíbula observaram-se áreas com características de reabsorçäo óssea e forames vasculares. Estes medem de 30 a 50 µm de diâmetro e que distam entre si de 200 a 460 µm. Também, observaram-se lacunas de osteócitos medindo de 18 a 25 µm de diâmetro, as quais säo rodeadas de feixes de fibras colágenas de disposiçäo lon gitudinal

Características peculiares da area de origem do músculo masseter, em ratos: estudo ao microscópio electrônico de varredura / Characteristics of the origin of the masseter muscle in mouses: electronic microscopy scanning study

Estruturas da superfície óssea correspondentes a área de origem do músculo masseter foram estudadas em 20 ratos, albinus Wistar. As peças foram fixadas em soluçäo de Karnovsky modificada durante 12 horas e tratadas segundo a técnica de LESTER et al. (1981) E GARCIA (1986). Os espécimes foram examinados ao microscópico eletrônico de varredura. Os resultados mostram que a área de origem do músculo masseter de ratos apresentam uma superfície lisa revestida por espessos feixes de fibras colágenas calcificadas. Os forames vasculares medem de 1,0 a 35 micrômetros de diâmetro e a distância entre eles varia de 10 a 20 micrômetros. Observou-se que a superfície lisa contém feixes de fibras de colágenas dispostos longitudinalmente säo entremeados de pequenos espaços de 0,5 a 1,0 micrômetro de comprimento. Cada forame vascular é rodeado por feixes de fibras colágenas dispostas circularmente