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      Técnica histoquímica aplicada ao tecido ósseo desmineralizado e parafinado para o estudo do osteócito e suas conexões Translated title: Histochemical tecnique for osteocyte and its connections study in descalcified and paraffined bone tissue

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          Abstract

          O osteócito vem sendo alvo de pesquisas recentes. A avaliação in situ de sua morfologia, da atividade e das características de suas conexões é difícil, portanto é realizada apenas com técnicas avançadas. Devido à importância desse tipo celular na manutenção da matriz óssea, este estudo propõe uma técnica de coloração pela prata como alternativa para o estudo do osteócito e suas conexões em tecido ósseo desmineralizado e parafinado. Cortes de 4µm do fêmur de ratas foram desmineralizados, desparafinados em xilol e hidratados em concentrações decrescentes de álcool etílico (ETOH) e água miliQ. Para a impregnação foram utilizadas soluções de nitrato de prata a 50% e de ácido fórmico a 1% com 2% de gelatina microbiológica em estufa a 40ºC. Essa técnica permite visualizar facilmente as bordas lacunares dos osteócitos e suas conexões, proporcionando uma alternativa simples e eficaz para o estudo da morfologia desse tipo celular até então ainda não proposta com essa finalidade.

          Translated abstract

          The osteocyte has been the subject of recent research; however the in situ evaluation of its morphology, activity and connection characteristics is difficult and has been performed solely through advanced techniques. Due to the importance of this type of cell in the maintenance of the bone matrix, this study proposes a technique of silver staining as an alternative for the study of the osteocyte and its connections in demineralized and paraffined bone tissue. Cuts of 4µm from the femur of female rats were de-mineralized, deparaffined in xylol and hydrated in decreasing concentrations of ETOH and miliQ water. For the impregnation, solutions of silver nitrate at 50% and formic acid at 1% with 2% microbiological gel were used and incubated at 40ºC. This technique allows the easy visualizing of the osteocytes lacunae edges and its connections, offering a simple and efficient alternative to the study of this type of cellular morphology, so far not proposed with this purpose.

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          Most cited references10

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          Molecular events caused by mechanical stress in bone.

          The shape of bone changes as a result of bone remodeling corresponding to physical circumstances such as mechanical stress. The tissue which receives the loaded mechanical stress most efficiently is bone matrix. Recent studies revealed the function of osteocytes as mechanosensors in the early stage of bone remodeling. Loaded mechanical stress is converted to a series of biochemical reactions, and finally activates osteoclasts and osteoblasts to cause bone resorption and formation. Biochemical and molecular biological studies have recently resulted in the identification of the gene of which expression level is changed by mechanical stress. Nitric oxide (NO) and cAMP is secreted in response to mechanical stress in the immediate early stage. Genes encoding enzymes such as glutamate/aspartate transporter (GLAST), nitric oxide synthetase (NOS) and prostaglandin G/H synthetase (PGHS-2) are identified as mechanical stress-responsive. The expression level of IGF-I is enhanced under the control of PTH/PTHrP. The expression of c-fos is increased by loading of mechanical stress. AP1, a heterodimer of c-FOS/c-JUN, functions as a transcription factor of downstream gene(s). Elements including AP1 sites, cyclic AMP response elements (CRE) and shear stress response elements (SSRE) are found in the promoter region of mechanical stress-response genes. The enhanced expression of osteopontin (OPN) in the osteocytes of bone resorption sites was demonstrated by in situ hybridization and immunohistochemistry and transdifferentiation of chondrocytes with the abundant expression of BMP-2 and -4 in the process of distraction osteogenesis was observed.
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            Effects of mechanical strain on the function of Gap junctions in osteocytes are mediated through the prostaglandin EP2 receptor.

            Osteocytes embedded in the matrix of bone are thought to be mechanosensory cells that translate mechanical strain into biochemical signals that regulate bone modeling and remodeling. We have shown previously that fluid flow shear stress dramatically induces prostaglandin release and COX-2 mRNA expression in osteocyte-like MLO-Y4 cells, and that prostaglandin E2 (PGE2) released by these cells functions in an autocrine manner to regulate gap junction function and connexin 43 (Cx43) expression. Here we show that fluid flow regulates gap junctions through the PGE2 receptor EP2 activation of cAMP-dependent protein kinase A (PKA) signaling. The expression of the EP2 receptor, but not the subtypes EP1,EP3, and EP4, increased in response to fluid flow. Application of PGE2 or conditioned medium from fluid flow-treated cells to non-stressed MLO-Y4 cells increased expression of the EP2 receptor. The EP2 receptor antagonist, AH6809, suppressed the stimulatory effects of PGE2 and fluid flow-conditioned medium on the expression of the EP2 receptor, on Cx43 protein expression, and on gap junction-mediated intercellular coupling. In contrast, the EP2 receptor agonist butaprost, not the E1/E3 receptor agonist sulprostone, stimulated the expression of Cx43 and gap junction function. Fluid flow conditioned medium and PGE2 stimulated cAMP production and PKA activity suggesting that PGE2 released by mechanically stimulated cells is responsible for the activation of cAMP and PKA. The adenylate cyclase activators, forskolin and 8-bromo-cAMP, enhanced intercellular connectivity, the number of functional gap junctions, and Cx43 protein expression, whereas the PKA inhibitor, H89, inhibited the stimulatory effect of PGE2 on gap junctions. These studies suggest that the EP2 receptor mediates the effects of autocrine PGE2 on the osteocyte gap junction in response to fluid flow-induced shear stress. These data support the hypothesis that the EP2 receptor, cAMP, and PKA are critical components of the signaling cascade between mechanical strain and gap junction-mediated communication between osteocytes.
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              Improvement in the staining and in the visualization of the argyrophilic proteins of the nucleolar organizer region at the optical level

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                Author and article information

                Journal
                jbpml
                Jornal Brasileiro de Patologia e Medicina Laboratorial
                J. Bras. Patol. Med. Lab.
                Sociedade Brasileira de Patologia Clínica (Rio de Janeiro, RJ, Brazil )
                1676-2444
                1678-4774
                February 2006
                : 42
                : 1
                : 37-39
                Affiliations
                [02] orgnameUFMG orgdiv1Instituto de Ciências Biológicas orgdiv2Departamento de Patologia Geral
                [03] orgnameUFMG orgdiv1Escola de Veterinária orgdiv2Departamento de Clínica e Cirurgia
                [04] orgnameUFMG orgdiv1Escola de Veterinária orgdiv2Departamento de Clínica e Cirurgia
                [01] orgnameUniversidade Federal de Minas Gerais orgdiv1Escola de Veterinária orgdiv2Departamento de Clínica e Cirurgia
                Article
                S1676-24442006000100008 S1676-2444(06)04200108
                ac4be830-2460-4f71-af94-190186daecdf

                This work is licensed under a Creative Commons Attribution 4.0 International License.

                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 11, Pages: 3
                Product
                Product Information: website
                Categories
                Patologia

                Osteócito,Histoquímica,Osteocyte,Conexões,Osso,Histochemistry,Connections,Bone

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