Metabolic changes in masseter muscle of rats submitted to acute stress associated with exodontia

Clinical evidence has shown that stress may be associated with alterations in masticatory muscle functions. Morphological changes in masticatory muscles induced by occlusal alterations and associated with emotional stress are still lacking in the literature. The objective of this study was to evaluate the influence of acute stress on metabolic activity and oxidative stress of masseter muscles of rats subjected to occlusal modification through morphological and histochemical analyses. In this study, adult Wistar rats were divided into 4 groups: a group with extraction and acute stress (E+A); group with extraction and without stress (E+C); group without extraction and with acute stress (NO+A); and control group without both extraction and stress (NO+C). Masseter muscles were analyzed by Succinate Dehydrogenase (SDH), Nicotinamide Adenine Dinucleotide Diaphorase (NADH) and Reactive Oxygen Species (ROS) techniques. Statistical analyses and two-way ANOVA were applied, followed by Tukey-Kramer tests. In the SDH test, the E+C, E+A and NO+A groups showed a decrease in high desidrogenase activities fibers (P < 0.05), compared to the NO+C group. In the NADH test, there was no difference among the different groups. In the ROS test, in contrast, E+A, E+C and NO+A groups showed a decrease in ROS expression, compared to NO+C groups (P < 0.05). Modified dental occlusion and acute stress - which are important and prevalent problems that affect the general population - are important etiologic factors in metabolic plasticity and ROS levels of masseter muscles106FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2011/00856-7; 2011/15209-7; 2011/18889-

Morphophysiological effects of chronic stress and exodontia in the masseter muscle of rats

O estresse parece favorecer a hiperalgesia e alodinia, podendo estar associados à alteração da função muscular mastigatória. Alterações morfofisiológicas em músculos da mastigação induzidos pela alteração oclusal associado ao estresse crônico ainda são escassas na literatura. Este estudo investigou os efeitos do estresse crônico repetido em músculo masseter superficial e profundo de ratos submetidos ou não à exodontia unilateral no ganho do peso dos animais, nas alterações morfológicas (HE, MET), histoquímicas (NADH, SDH e ROS), imunoistoquímicas (laminina e CD31), atividade de MMP-2, -9 e infiltração de neutrófilos (MPO). Vinte ratos (machos, 200g) foram alocados em quatro grupos (n=5): controle (GC), exodontia unilateral (GM), estresse crônico repetido (GE), exodontia associado ao estresse crônico repetido (GME). GE e GME foram submetidos a 10 dias de protocolo de estresse crônico repetido (2 horas diárias) a partir do 14º dia após a exodontia. Houve uma diminuição significativa no ganho de peso dos animais GE e GME. Não foram observadas alterações nos níveis de MMPs e na infiltração de neutrófilos no feixe superficial dos diferentes grupos. GE, GM e GME demostraram alterações morfofisiológicas, ultraestruturais e histoquímicas no feixe profundo, com características específicas e distintas de GC; GE apresentou as maiores alterações. Conclui-se que a exodontia e sua associação ao estresse foram responsáveis por discretas alterações morfofisiológicas no músculo masseter de ratos, contudo o estresse crônico repetido causou modificações morfofisiológicas e ultraestruturais significantes, sendo responsável também pela alteração no peso dos animais.Stress seems to favor the hyperalgesia and allodynia, which may be related with altered masticatory muscle function. Morphological and physiological changes in the masticatory muscles induced by occlusal alteration associated with chronic stress are still scarce in the literature. This study investigated the effects of repeated chronic stress in superficial and deep masseter muscle of rats with or without the extraction unilateral weight gain of animals and morphological changes (HE MET), histochemical (NADH, SDH and ROS), immunohistochemical (laminin and CD31), MMP-2, -9 activities and neutrophil infiltration (MPO). Twenty rats (male, 200g) were allocated into four groups (n=5): control group (CG), unilateral exodontia (MG), repeated chronic stress (EG), extodontia and repeated chronic stress (MEG). EG and MEG were submitted to 10 days of repeated chronic stress protocol, 2 hours daily, from the 14th day after the extraction. There was a significant decrease in weight gain of animals EG and MEG. No changes were observed in the levels of MMPs and neutrophil infiltration among different groups. EG, MG and MEG have shown morphophisyological, ultrastructural and histochemical changes with specific characteristics and distinct GC GE presenting the higher changes. We conclude that the exodontia and its association to stress were responsible for discrete morphophysiological changes in the masseter muscle of rats, however repeated chronic stress caused significant morphophysiological and ultrastructural changes, being also responsible for change in weight of the animals

Exodontia‐induced muscular hypofunction by itself or associated to chronic stress impairs masseter muscle morphology and its mitochondrial function

Stress is associated with orofacial pain sensitivity and is qualified as a temporomandibular disorder risk factor. During stressful periods, painful thresholds of masticatory muscles in individuals suffering muscle facial pain are significantly lower than in controls, but the exact physiologic mechanism underlying this relation remains unclear. Our hypothesis is that chronic unpredictable stress and masticatory hypofunction induce morphologic and metabolic masseter muscle changes in rats. For test this hypothesis, adult Wistar rats were submitted to chronic unpredictable stress and/or exodontia of left molars and the left masseter muscle was removed for analysis. The parameters evaluated included ultrastructure, oxidative level, metabolism activity and morphological analysis in this muscle. Our data show by histological analysis, that stress and exodontia promoted a variation on diameters and also angled contours in masseter fibers. The masticatory hypofunction increased oxidative metabolism as well as decreased reactive species of oxygen in masseter muscle. The ultrastructural analysis of muscle fibers showed disruption of the sarcoplasmic reticulum cisterns in certain regions of the fiber in stress group, and the disappearance of the sarcoplasmic reticulum membrane in group with association of stress and exodontia. Our findings clarify mechanisms by which chronic stress and masticatory hypofunction might be involved in the pathophysiology of muscular dysfunctions. Masticatory hypofunction influenced oxidative stress and induced oxidative metabolism on masseter muscle, as well as altered its fiber morphology. Chronic stress presented malefic effect on masseter morphology at micro and ultra structurally. When both stimuli were applied, there were atrophic fibers and a complete mitochondrial derangement825530537FAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulo2015/03053-3; 2012/22128-6Art, science, microscopy and ED

Application of a Low-Level Laser Therapy and the Purified Protein from Natural Latex (Hevea brasiliensis) in the Controlled Crush Injury of the Sciatic Nerve of Rats: A Morphological, Quantitative, and Ultrastructural Study

This study analyzed the effects of a low-level laser therapy (LLLT, 15 J/cm2, 780 nm wavelength) and the natural latex protein (P1, 0.1%) in sciatic nerve after crush injury (15 Kgf, axonotmesis) in rats. Sixty rats (male, 250 g) were allocated into the 6 groups (n=10): CG—control group; EG—nerve exposed; IG—injured nerve without treatment; LG—crushed nerve treated with LLLT; PG—injured nerve treated with P1; and LPG—injured nerve treated with LLLT and P1. After 4 or 8 weeks, the nerve samples were processed for morphological, histological quantification and ultrastructural analysis. After 4 weeks, the myelin density and morphological characteristics improved in groups LG, PG, and LPG compared to IG. After 8 weeks, PG, and LPG were similar to CG and the capillary density was higher in the LG, PG, and LPG. In the ultrastructural analysis the PG and LPG had characteristics that were similar to the CG. The application of LLLT and/or P1 improved the recovery from the nerve crush injury, and in the long term, the P1 protein was the better treatment used, since only the application of LLLT has not reached the same results, and these treatments applied together did not potentiate the recovery

Metabolic Changes in Masseter Muscle of Rats Submitted to Acute Stress Associated with Exodontia.

Clinical evidence has shown that stress may be associated with alterations in masticatory muscle functions. Morphological changes in masticatory muscles induced by occlusal alterations and associated with emotional stress are still lacking in the literature. The objective of this study was to evaluate the influence of acute stress on metabolic activity and oxidative stress of masseter muscles of rats subjected to occlusal modification through morphological and histochemical analyses. In this study, adult Wistar rats were divided into 4 groups: a group with extraction and acute stress (E+A); group with extraction and without stress (E+C); group without extraction and with acute stress (NO+A); and control group without both extraction and stress (NO+C). Masseter muscles were analyzed by Succinate Dehydrogenase (SDH), Nicotinamide Adenine Dinucleotide Diaphorase (NADH) and Reactive Oxygen Species (ROS) techniques. Statistical analyses and two-way ANOVA were applied, followed by Tukey-Kramer tests. In the SDH test, the E+C, E+A and NO+A groups showed a decrease in high desidrogenase activities fibers (P < 0.05), compared to the NO+C group. In the NADH test, there was no difference among the different groups. In the ROS test, in contrast, E+A, E+C and NO+A groups showed a decrease in ROS expression, compared to NO+C groups (P < 0.05). Modified dental occlusion and acute stress--which are important and prevalent problems that affect the general population--are important etiologic factors in metabolic plasticity and ROS levels of masseter muscles

An in situ gelling liquid crystalline system based on monoglycerides and polyethylenimine for local delivery of siRNAs

The development of delivery systems able to complex and release siRNA into the cytosol is essential for therapeutic use of siRNA. Among the delivery systems, local delivery has advantages over systemic administration. In this study, we developed and characterized non-viral carriers to deliver siRNA locally, based on polyethylenimine (PEI) as gene carrier, and a self-assembling drug delivery system that forms a gel in situ. Liquid crystalline formulations composed of monoglycerides (MO), PEI, propylene glycol (PG) and 0.1 M Tris buffer pH 6.5 were developed and characterized by polarized light microscopy, Small Angle X-ray Scattering (SAXS), for their ability to form inverted type liquid crystalline phases (LC2) in contact with excess water, water absorption capacity, ability to complex with siRNA and siRNA release. In addition, gel formation in vivo was determined by subcutaneous injection of the formulations in mice. In water excess, precursor fluid formulations rapidly transformed into a viscous liquid crystalline phase. The presence of PEI influences the liquid crystalline structure of the LC2 formed and was crucial for complexing siRNA. The siRNA was released from the crystalline phase complexed with PEI. The release rate was dependent on the rate of water uptake. The formulation containing MO/PEI/PG/Tris buffer at 7.85:0.65:76.5:15 (w/w/w/w) complexed with 10 μM of siRNA, characterized as a mixture of cubic phase (diamond-type) and inverted hexagonal phase (after contact with excess water), showed sustained release for 7 days in vitro. In mice, in situ gel formation occurred after subcutaneous injection of the formulations, and the gels were degraded in 30 days. Initially a mild inflammatory process occurred in the tissue surrounding the gel; but after 14 days the tissue appeared normal. Taken together, this work demonstrates the rational development of an in situ gelling formulation for local release of siRNA

Quantification of Reactive Oxygen Species (ROS) in different group of rats.

<p>Panel A, photomicrographs with fluorescent labeled for ROS of masseter muscle of rats. 200x magnification. Magnification bars: 50 μm. Panels B, C and D means de fluorescence intensity of ROS in differente groups. Fig 3B: P < 0.05, NO+C group <i>vs</i> E+C, NO+A and E+A groups; and P < 0.05, E+A group <i>vs</i> E+C group (Tukey-Kramer test).</p

Demonstration of Nicotinamide Adenine Dinucleotide Diaphorase (NADH) in different groups of rats.

<p>Panel A, photomicrographs of the masseter muscle of rats. 200x magnification. Magnification bars: 100 μm. Panels B, C and D means the percentage of Light Fibers (B), Intermediate Fibers (C) and Dark Fibers (D) in different groups.</p