Beneficial effects of benzodiazepine on masticatory muscle dysfunction induced by chronic stress and occlusal instability in an experimental animal study.

Psychological stress and occlusal alteration are important etiologic factors for temporomandibular/masticatory muscular disorders. In particular, the exact physiologic mechanism underlying the relation by occlusal alteration and temporomandibular disorders remains unclear. Our purpose was to test the hypothesis that benzodiazepine therapy is able to prevent metabolic and vascular changes in the medial pterygoid muscle of rats under chronic stress after 14 days of unilateral exodontia. Adult Wistar rats were submitted to unpredictable chronic mild stress (10 days) and/or unilateral exodontia and their plasma and medial pterygoid muscles were removed for analysis. A pre-treatment with diazepam was used to verify its effect on stress. The parameters evaluated included anxiety behavior, plasma levels of corticosterone, metabolic activity by succinate dehydrogenase, capillary density by laminin staining and ultrastructural findings by transmission electron microscopy. Occlusal instability induced anxiety-like behavior on elevated plus-maze test and diazepam administration blocked the appearance of this behavior. Unilateral exodontia promoted in the contralateral muscle an increase of oxidative fibers and capillaries and modification of sarcoplasmic reticulum. Chronic stress caused increased glycolytic metabolism, reduced capillary density and morphological changes in mitochondria on both sides. Association of both factors induced a glycolytic pattern in muscle and hemodynamic changes. Pharmacological manipulation with diazepam inhibited the changes in the medial pterygoid muscle after stress. Our results reveal a preventive benzodiazepine treatment for stress and occlusal instability conditions affecting masticatory muscle disorders. In addition, provide insights into the mechanisms by which chronic stress and exodontia might be involved in the pathophysiology of masticatory muscular dysfunctions.

Morphometric and high resolution scanning electron microscopy analysis of low-level laser therapy and latex protein (Hevea brasiliensis) administration following a crush injury of the sciatic nerve in rats.

This study evaluated the effect of low-level laser therapy (LLLT; 15 J/cm(2)) and a latex protein (F1) on a crush injury of the sciatic (ischiadicus) nerve. Seventy-two rats (male, 250 g) were divided into 6 groups: CG, control; EG, exposed nerve; IG, injured nerve without treatment; LG, injured nerve with LLLT; HG, injured nerve with F1; and LHG, injured nerve with LLLT and F1. After 4 or 8 weeks, the animals were euthanized and samples of the sciatic nerve were collected for morphometric and high-resolution scanning electron microscopy (HRSEM) analysis. After 4 weeks, the morphometry revealed improvements in the treated animals, and the HG appeared to be the most similar to the CG; after 8 weeks, the injured groups showed improvements compared to the previous period, and the results of the treatment groups were more similar to one another. At HRSEM after 4 weeks, the treated groups were similar and showed improvement compared to the IG; after 8 weeks, the LHG and HG had the best results. In conclusion, the treatments resulted in improvement after the nerve injury, and this recovery was time-dependent. In addition, the use of the F1 resulted in the best morphometric and ultrastructural findings.

Biological evaluation of the bone healing process after application of two potentially osteogenic proteins: an animal experimental model.

OBJECTIVE: The aim of this work was to analyse qualitatively and quantitatively the newly formed bone after insertion of rhBMP-2 and protein extracted from Hevea brasiliensis (P-1), associated or not with a carrier in critical bone defects created in Wistar rat calvarial bone, using histological and histomorphometrical analyses. MATERIALS AND METHODS: Eighty-four male Wistar rats were used, divided into two groups, according to the period of time until the sacrifice (2 and 6 weeks). Each one of these groups was subdivided into six groups with seven animals each, according to the treatments: (1) 5 µg of pure rhBMP-2, (2) 5 µg of rhBMP-2/monoolein gel, (3) pure monoolein gel, (4) 5 µg of pure P-1, (5) 5 µg of P-1/monoolein gel and (6) critical bone defect controls. The animals were euthanised and the calvarial bone tissue removed for histological and histomorphometrical analyses. RESULT AND CONCLUSION: The results showed an improvement in the bone healing process using the rhBMP-2 protein, associated or not with a material carrier in relation to the other groups, and this process demonstrated to be time dependent.

Evaluation of protective effect of a water-in-oil microemulsion incorporating quercetin against UVB-induced damage in hairless mice skin.

PURPOSE: In the present study, histological aspects were considered in order to evaluate the in vivo photoprotective effect of a w/o microemulsion containing quercetin against UVB irradiation-induced dermal damages. The toxicity in cell culture and the potential skin irritation resulting from topical application of this formulation were also investigated. METHODS: Mouse dorsal surfaces were treated topically with 300 mg of the unloaded and quercetin-loaded (0.3%, w/w) microemulsions before and after exposure to UVB (2.87 J/cm2) irradiation. The untreated control groups irradiated and non-irradiated were also evaluated. UVB-induced histopathological changes as well as the photoprotective effect of this formulation were evaluated considering the parameters of infiltration of inflammatory cells, epidermis thickening (basale and spinosum layers) and collagen and elastic fiber contents. The cytotoxicity of the reported formulation was evaluated in L929 mice fibroblasts by MTT assay and the skin irritation was investigated after topical application of both unloaded and quercetin-loaded microemulsions once a day for 15 days. RESULTS: The results demonstrated that the w/o microemulsion containing quercetin reduced the incidence of histological skin alterations, mainly the connective-tissue damage, induced by exposure to UVB irradiation, this allows the suggestion that protective effects of this formulation against UV-induced responses are not secondary to the interference of UV transmission (i.e., blocking the UVB radiation from being absorbed by the skin), as is usually done with UVB absorbers and sunscreens, but is instead due to different biological effects of this flavonoid. Furthermore, by evaluating the cytotoxic effect on L929 cells and histological aspects such as infiltration of inflammatory cells and epidermis thickness of hairless mice, the present study also demonstrated no toxicity of the proposed system. CONCLUSIONS: Therefore, based on these mouse models, a detailed characterization of the w/o microemulsion incorporating quercetin effects as a photochemoprotective agent on human skin is thus indicated.

Hyperhomocysteinemia induced by feeding rats diets rich in DL-homocysteine thiolactone promotes alterations on carotid reactivity independent of arterial structure.

We aimed to investigate whether hyperhomocysteinemia (HHcy) interferes on carotid vascular reactivity, and how morphological and functional aspects are related. With this purpose male Wistar rats received a solution of dl-homocysteine-thiolactone (1g/kg body weight/day) in the drinking water for 4, 15 and 30 days. Lipid profile, carotid artery-morphology and -responsiveness to acetylcholine, phenylephrine and endothelin-1 were analyzed. Similar increase on homocysteine plasmatic levels occurred in rats treated for 4, 15 and 30 days. High levels of serum cholesterol and triglycerides were observed after HHcy 30 days. Vascular reactivity experiments using standard muscle bath procedures showed that HHcy induced a time-dependent reduction on acetylcholine-induced-relaxation at 4, 15 and 30 days. HHcy enhanced the contractile response of endothelium-intact, but not denuded carotid rings to phenylephrine and endothelin-1, despite the treatment time. Morphometric analysis showed that intimal/medial area ratio was enhanced only at 30 days of HHcy, despite its reduced cell density. The major new finding of the present study is that it establishes a time-course relationship for the events involved on vascular effects associated with HHcy. We demonstrated that alterations on vascular responsiveness precede alterations on arterial structure. Based on such findings it is possible to suggest that vascular dysfunction occurs in early stages while alterations on vessel morphology take place in latest stages of HHcy.

Ultrastructure of motor nerve terminals in the anterior third of Wistar rat tongue.

The nerve terminals of intrinsic muscular fibers of the tongue of adult wistar rats was studied by using silver impregnation techniques, transmission electron microscopy (TEM), and high resolution scanning electron microscopy (HRSEM) to observe the nerve fibers and their terminals. Silver impregnation was done according to Winkelman and Schmit, 1957. For TEM, small blocks were fixed in modified Karnovsky solution, postfixed in 1% buffered osmium tetroxide solution, and embedded in Spurr resin. For HRSEM, the parts were fixed in 2% osmium tetroxide solution with 1/15 M sodium phosphate buffer (pH 7.4) at 4 degrees C for 2 h, according to the technique described by Tanaka, 1989. Thick myelinated nerve bundles were histologically observed among the muscular fibers. The intrafusal nerve fiber presented a tortuous pathway with punctiform terminal axons in clusters contacting the surface of sarcolemma. Several myelinated nerve fibers involved by collagen fibers of the endoneurium were observed in HRSEM in three-dimensional aspects. The concentric lamellae of the myelin sheath and the axoplasm containing neurofilaments interspersed among the mitochondria were also noted. In TEM, myofibrils, mitochondria, rough endoplasmic reticulum, Golgi's apparatus, and glycogen granules were observed in sarcoplasm. It is also noted that the sarcomeres constituted by myofilaments with their A, I, and H bands and the electron dense Z lines. In areas adjacent to muscular fibers, there were myelinated and unmyelinated nerve fibers involved by endoneurium and perineurium. In the region of the neuromuscular junction, the contact with the sarcolemma of the muscular cell occurs forming several terminal buttons and showing numerous evaginations of the cell membrane. In the terminal button, mitochondria and numerous synaptic vesicles were observed.

Quercetin in w/o microemulsion: in vitro and in vivo skin penetration and efficacy against UVB-induced skin damages evaluated in vivo.

The present study evaluated the potential of a w/o microemulsion as a topical carrier system for delivery of the antioxidant quercetin. Topical and transdermal delivery of quercetin were evaluated in vitro using porcine ear skin mounted on a Franz diffusion cell and in vivo on hairless-skin mice. Skin irritation by topical application of the microemulsion containing quercetin, and the protective effect of the formulation on UVB-induced decrease of endogenous reduced glutathione levels and increase of cutaneous proteinase secretion/activity were also investigated. The w/o microemulsion increased the penetration of quercetin into the stratum corneum and epidermis plus dermis at 3, 6, 9 and 12h post-application in vitro and in vivo at 6h post-application. No transdermal delivery of quercetin occurred. By evaluating established endpoints of skin irritation (erythema formation, epidermis thickening and infiltration of inflammatory cells), the study demonstrated that the daily application of the w/o microemulsion for up to 2 days did not cause skin irritation. W/o microemulsion containing quercetin significantly prevented the UVB irradiation-induced GSH depletion and secretion/activity of metalloproteinases.

Glândula submandibular de ratos com envelhecimento: observações ao microscópio eletrônico de varredura de alta resolução / Submandibular gland of rats with ageing: observations with high resolution scanning electron microscopy

As características tridimensionais dos componentes intracelulares de células acinares e de ductos foram reveladas usando o método ósmio-DMSO-ósmio. As amostras foram maceradas em solução de tetróxido de ósmio diluído após a fratura na solução de dimetil sulfoxido. As lamelas do retículo endoplasmático granular são reveladas entremeadas por várias mitocôndrias. As lamelas do retículo endoplasmático granular são localizados ao redor dos núcleos na porção basal e estas estruturas são observadas em imagens tridimensionais de microscopia eletrônica de alta resolução.

The three-dimensional characteristics of the intracellular components of acinar and ductal cells were revealed using the osmium-DMSO-osmium method. The samples were macerated in diluted osmium after fractured in DMSO solution. The stacks of the rough endoplasmic reticulum are revealed intermingling by several mitochondria. The lamellae of the rough endoplasmic reticulum are located around the nuclei at basal portion and these structures are shown in three-dimensional HRSEM images.

New role of the trigeminal nerve as a neuronal pathway signaling brain in acute periodontitis: participation of local prostaglandins.

The systemic induction of cytokines and prostaglandins plays a key role in the development of fever. However, whether fever is triggered by local injection of lipopolysaccharide (LPS) and the involvement of locally produced prostaglandins in periodontal tissue has never been assessed. Thus, we tested the hypothesis that the trigeminal nerve is a neuronal pathway that signals the brain during acute periodontitis, and this response involves prostaglandin induction. Rats were given a gingival intra-pouch injection of sterile saline or Escherichia coli lipopolysaccharide, at doses of 10 and 100 microg/kg. Some animals were pre-treated with the local anesthetic mepivacaine or had the peripheral branches of the trigeminal nerves transected. Another group of animals were pre-treated (locally or systemically) with the nonselective inhibitor of cyclooxygenases diclofenac. Body core temperature (T (b)) was measured by means of biotelemetry before and after injections. LPS elicited a dose-dependent increase in T (b), which was abolished by mepivacaine, bilateral transection of the peripheral branches of the trigeminal nerve, or local treatment with diclofenac. The results indicate that there is an activation of periodontal nerves to induce fever by LPS. It also shows that local formation of prostaglandins plays a role in fever development. Moreover, immunohistochemistry detected c-fos expression in the subnucleus caudalis of spinal trigeminal nucleus and in the preoptic area of the hypothalamus 2 and 3 h after LPS injection, further confirming the role of trigeminal nerve signaling brain in acute periodontitis.

Morphology of the submandibular gland of the gerbil meriones unguiculatus: a macroscopic and light microscopy study

La morfología de la glándula submandibulardel gerbil fue estudiada macro y microscópicamente. Diez gerbiles fueron anestesiados, perfundidos e inyectados con formalina al 10 por ciento. La región cervical anterior de cada animal fue disecada para efectuar el análisis macroscópico de la glándula submandibular. Para el análisis histológico, las piezas fueron fijadas en la misma solución e incluidas en parafina para los procesos de rutina. Cortes seriados de 6-um fueron teñidos con hematoxilina-eosina y picrosirius, para análisis al microscopio de luz. Los resultados mostraron que la glándula submandibular de gerbil presentaba una forma ovoidea y localizada en la región submandibular, midiendo aproximadamente 9.0 mm de longitud y 5.9 mm de ancho. Histológicamente, cada acino estaba constituido por cuatro tipos de células dispuesta irregularmente. El sistema de ductos constaba de cuatro segmentos distintos: ductos intercalar, granuloso, estriado y excretor

Morphologic study of the masseter muscle of gerbil: meriones unguiculatus

El daño de los músculos masticadores relacionados con desórdenes cráneomandibulares son aspectos comunes en la clínica. Es importante contar con datos morfológicos de estos músculos en el sistema estomatognático. El propósito de este trábajo es mostrar la morfología del músculo masetero del gerbil Meriones unguiculatus, desde el nivel macroscópico hasta el de microscopía electrónica. En la observación macroscópica fueron utilizados 5 animales que fueron perfundidos con sulución de formalina al 100 por ciento. Las disecciones fueron fotografiadas. Posteriormente, los músculos fueron removidos e sumergidos en la solución de formalina, por unos pocos días. Las muestras fueron procesadas a través de técnicas histológicas convencionales y sumergidas en parafina. Cortes seriados de 6 um fueron teñidos con H. E. analizados y fotografiados con un fotomicroscopio JENAMED. Nuestros resultados mostraron que el músculo masetero presentaba 5 fascículos, los cuales se mezclaban en la porción media, y algunos husos estaban fusionados con la cápsula externa de ellos mismos, pareciendo husos gigantes

Scanning electron microscopy of the rat tongue mucosa with special attention to bacteria on epithelial cell membranes / Microscopia eletrônica de varredura da mucosa da língua de ratos com enfoque em bactérias em menbranas de células epiteliais

The authors examined the filiform and fungiform papillae surfaces of rat tongue by scanning electron microscopy showing the numerous groupings of bacteria on the epithelial cell membranes. The fungiform papillae were round in shape and presented few bacteria. The epithelial cell of filiform papillae revealed numerous streptococci. The grouping of the bacteria are attached on the epithelial cell membrane, demonstrating three-dimensional SEM images

High resolution scanning electron microscopy study of the bacteria adhesion in the epithelial cell membranes of the third anterior part of young rat tongue mucosa

Los autores relatan la presencia de bacterias sobre la superficie de las células epiteliales el tercio anterior de la mucosa lingual de ratas jóvenes a través del microscopio de barrido de alta resolución. Las células epiteliales de las papilas filiformes y fungiformes revelaron numerosos grupos de bacterias. Estas bacterias están fijadas sobre la membrana de las células epiteliales claramente visualizadas en imágenes tridimensionales

Characteristics of the palatine mucosa of the calomys callosus: light and sem study

Se estudiaron las características de la superficie e interfase del tejido epitelio-conjuntivo de la mucosa palatina del paladar duro del Calomys callosus, a través del microscopio de luz y de la microscopía electrónica de barrido. Seis ejemplares fueron fijados en soluciones de Bouin y de Kamovsky modificada. Los resultados mostraron que las regiones anterior y media presentan pliegues transversales y superficies planas adyacentes. La superficie mostraba un epitelio escamoso queratinizado. La lámina propia de la mucosa palatina presentaba abundante tejido conjuntivo papilar de variadas formas. Próximo a la región de los dientes, el tejido conjuntivo papilar era más abundante. El pliegue transversal medía entre 150 µm y 300 µm de diámetro. La superficie de la mucosa palatina del paladar duro del Calomys callosus presentaba células epiteliales poligonales. Algunas células descamativas se notaban claramente. La superficie de la interfase del tejido epitelio-conjuntivo presentaba abundante tejido conjuntivo papilar primario y secundario. La red de gruesos haces de fibras colágenas se observaban en cada papila, mostrando imágenes tridimensionales

Scanning electron microscopic study of the epithelial cells and surface of interface epithelium-connective tisue of the palatine mucosa of nine-banded armadillo, dasypus novemcintus

Las características de las células epiteliales y de la superficie de interfase del tejido conectivo-epitelial de la mucosa palatina del armadillo (dasypus novemcintus), se estudiaron usando el microscopio electrónico de barrido. La mucosa del paladar duro está cubierta por epitelio queratinizado escamoso estratificado, que tiene algunas células epiteliales poligonales en proceso de escamación. La mucosa palatina mostró proyecciones puntiagudas de alturas diferentes, con diámetros de 100-200 µm. La lámina propia revela abundante tejido conjuntivo papilar digitiforme, que contiene un entramado de fibras colágenas. Fueron observadas numerosas coberturas de forma circular o elíptica de los conductos salivales de glándula. La superficie de las células epiteliales presentó gran cantidad de micropliegues

Scanning electron microscopic study of the epithelial cells and the epithelium-connective tissue interface of the inferior surface of macaca fuscata tongue mucosa

The characteristics of the epithelial cells and the epithelium-connective tissue interface of the inferior surface of Macaca fuscata tongue mucosa were described employing a scanning electron microscope. The specimens were fixed in buffered modified Karnovsky solution and after that with or without treatment with NaOH, were postfixed in osmium tetroxide. They were dehydrated with acetone, critical point dried with Hitachi, HCP-2, covered with gold ions in a Polaron coating device and examined in a Hitachi S-800, scanning electron microscope. The inferior surface of Macaca fuscata tongue mucosa presents numerous microridges. These layers are constituted by polygonal epithelial cells, containing numerous microplicae. These are isolated or grouped, with circular, elongated or pointed shapes. In NaOH treated specimens we noted the existence of numerous connective tissue papillae of several forms and sizes. In the intermediate phase of corrosion, the deep layer of epithelial cells can be seen. Small foramina of ducts of salivary glands are found on the connective tissue surface, and they are surrounded by a circular disposition of collagen fibrils.