Os autores agradecem ao técnico de prótese Nicola Lanfranconi para produzir o modelo de referência mestre eo Alicona Empresa por seu apoio contínuo com a melhoria do software de digitalização.

1. Preparação de Amostras / base <ol><li> Aplique uma base plana em todo o espécime. Coloque a amostra na mesa de digitalização. Orientar a superfície oclusal ao plano horizontal (figura 1). </li></ol> 2. Análise de Software <ol><li> Inicie o programa e, em seguida, o módulo de laboratório de medição (Figura 2) </li><li> Coloque a amostra no centro da mesa de digitalização. </li><li> Escolha a lente de ampliação correta. Para grandes objetos, como scans arco pleno, utilize o objetivo de 5X. </li><li> Mova a ótica de digitalização usando o mouse 3D até que a superfície da amostra é exibida na janela de visualização ao vivo (Figura 3). </li><li> Use o controle de sensor para ajustar a exposição eo contraste para obter parâmetros de digitalização ideais. Para superfícies de metal, utilizar uma exposição entre 400 e 800 ms e um contraste entre 0,3 e 0,8 (Figura 4). </li><li> Verifique a qualidade da imagem, verificando o botão&quot;Mostrar Qualidade de imagem Live Preview&quot;. </li><li> Defina os parâmetros corretos na seção &quot;Controle de Medição&quot;. O tipo de medição é &quot;3D Dataset&quot;; ImageField tipo é &quot;General ImageField&quot; (Figura 5). </li><li> Clique em &quot;New ImageField&quot;. </li><li> Definir o volume de digitalização. O software precisa das coordenadas delimitadoras do volume de medida. <ol><li> Mova a amostra para o maior eo menor nível de digitalização e clique em &quot;Adicionar Position&quot; em ambos os pontos. O valor de &quot;Z Range&quot; mostra a altura real do volume de varredura (Figura 6). </li><li> Mova a amostra para os boundings XY do volume de digitalização. Clique em &quot;Adicionar Position&quot; para definir o eixo X e os comprimentos do eixo Y. A dimensão do volume de digitalização é mostrada na aba &quot;informação&quot; e deve exceder as dimensões do espécime por 1 cm no eixo X e do eixo Y. </li></ol></li><li> Marque a opção &quot;Pontos&quot; número. O software é capaz de digitalização de 100 milhões de pontos da superfície em um &quot;ImageField&quot; digitalização. O número realde pontos de exceder esse limite. &quot;Dizimar&quot; o número do ponto de medida por um &quot;Downsampling Lateral&quot; do tamanho do ponto. <ol><li> Clique em &quot;Configurações avançadas&quot; (Figura 7). </li><li> Mova o controle deslizante &quot;Lateral Downsampling&quot; para a direita até o &quot;Pontos&quot; número é reduzido abaixo de 100 M (Figura 7). Downsampling reduz a resolução lateral dos pontos da superfície, o que resulta em maior tamanho do pixel do objecto digitalizado. Para garantir resultados da verificação ideais, a resolução vertical é aumentada. </li></ol></li><li> Clique em &quot;Iniciar medição&quot;. Isto irá iniciar o &quot;modo de visualização&quot;. O software executa uma pré-digitalização com as dimensões X e do eixo Y seleccionados. </li><li> Após a pré-digitalização estiver concluída, selecione a região de interesse. Isso ajuda a reduzir o tamanho do arquivo eo tempo de varredura (Figura 8). </li><li> Selecione todas as partes do pré-digitalização, exceto a amostra e dois campos de medição em todo o espécime, que contém a base plana (Figura 9). </li><li> Clique em &quot;Start&quot; para iniciar a digitalização. </li><li> Controle a varredura no &quot;ImageViewer&quot; usando o mouse e pressione o botão esquerdo do mouse (Figura 10). </li><li> Feche a janela e clique em &quot;Ver Pseudo Apenas Color&quot;. </li><li> Clique em &quot;Configurações&quot; e &quot;Pseudo colorir&quot; e escolha &quot;repetibilidade&quot;. </li><li> Defina o &quot;Max&quot;. valorizar a 0,2 e clique em &quot;Aplicar Range&quot;. </li><li> Controle a repetibilidade, deve ser igual para áreas com a mesma inclinação e material. Em particular, a base em torno do espécime deve exibir uma repetibilidade homogéneo (Figura 11). </li><li> Clique em &quot;banco de dados&quot; e salvar a digitalização para a pasta apropriada. </li><li> Exportar a digitalização para diferentes formatos de arquivos, se necessário. Clique em &quot;File/Export/3D dados como / ....&quot; O protocolo pode ser interrompido nesse ponto e continuou mais tarde. </li></ol> 3. Análise Diferença <ol><li> Use o &quot;Editor 3D&quot; para cortar a base. Esta área não vai ser utilizado para a diferençaanálise. </li><li> Para comparar e analisar os dois exames, inicie o software &quot;Medida de Diferença&quot; (Figura 12). </li><li> Escolha o segundo modelo para comparar. </li><li> Clique em &quot;Rough alinhamento automático&quot; para realizar uma primeira partida dos modelos. </li><li> Clique em &quot;Ajuste Manual&quot; e alinhar os modelos com rotação e que se deslocam até que eles estão na mesma orientação (Figura 13). </li><li> Clique em &quot;alinhamento automático&quot; e &quot;Aplicar&quot; para iniciar o melhor algoritmo de ajuste para o correspondente modelo ideal. </li><li> Clique em &quot;Diferenças&quot; para ver o mapa diferença dos dois modelos combinados. </li><li> Selecione um intervalo de cor adequada para exibir os desvios (Figura 14). Salve o desvio padrão visual, como uma imagem para análise. </li></ol> Clique em &quot;Estatísticas&quot; para exibir os valores estatísticos das diferenças. Escolha um tamanho de classe mM e salvar dados de histograma para um arquivo de texto para comparação estatística (Figura 15).

<ol>
	<li>Wettstein, F., Sailer, I., Roos, M., H&#228;mmerle, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+study+of+the+internal+gaps+of+zirconia+and+metal+frameworks+for+fixed+partial+dentures.">Clinical study of the internal gaps of zirconia and metal frameworks for fixed partial dentures.</a> Eur. J. Oral Sci. 116 (3), 272-279 (2008).</li><li>Persson, A. S., Oden, A., Andersson, M., Sandborgh-Englund, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digitization+of+simulated+clinical+dental+impressions:+virtual+three-dimensional+analysis+of+exactness.">Digitization of simulated clinical dental impressions: virtual three-dimensional analysis of exactness.</a> Dent. Mater. 25 (7), 929-936 (2009).</li><li>Del'Acqua, M. A., Arioli-Filho, J. N., Compagnoni, M. 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Mater. 19 (1), 19-24 (2003).</li><li>Mehl, A., Ender, A., M&#246;rmann, W., Attin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+testing+of+a+new+intraoral+3D+camera.">Accuracy testing of a new intraoral 3D camera.</a> International Journal of Computerized Dentistry. 12 (1), 11-28 (2009).</li><li>Fickl, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimensional+changes+of+the+ridge+contour+after+socket+preservation+and+buccal+overbuilding:+an+animal+study.">Dimensional changes of the ridge contour after socket preservation and buccal overbuilding: an animal study.</a> J. Clin. Periodontol. 36 (5), 442-448 (2009).</li><li>Vlaar, S. T., vander Zel, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+dental+digitizers.">Accuracy of dental digitizers.</a> Int. Dent. J. 56 (5), 301-309 (2006).</li><li>Ender, A., Mehl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+complete-arch+dental+impressions:+a+new+method+of+measuring+trueness+and+precision.">Accuracy of complete-arch dental impressions: a new method of measuring trueness and precision.</a> 109 (2), 121-128 (2013).</li><li>Ender, A., Mehl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+scanning+strategies+on+the+accuracy+of+digital+intraoral+scanning+systems.">Influence of scanning strategies on the accuracy of digital intraoral scanning systems.</a> Int. J. Comput. Dent. 16 (1), 11-21 (2013).</li><li>Ender, A., Mehl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full+arch+scans:+conventional+versus+digital+impressions--an+in-vitro+study.">Full arch scans: conventional versus digital impressions--an in-vitro study.</a> Int. J. Comput. Dent. 14 (1), 11-21 (2011).</li><li>Meer, W. J., Andriessen, F. S., Wismeijer, D., Ren, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+intra-oral+dental+scanners+in+the+digital+workflow+of+implantology.">Application of intra-oral dental scanners in the digital workflow of implantology.</a> PLoS One. 7 (8), (2012).</li></ol>

Os autores declaram que não têm concorrentes interesses financeiros ou outros conflitos de interesse.

Precisão é uma exigência básica em medicina dentária. O scanner de referência é capaz de digitalizar objetos pequenos e grandes com alto rigor e precisão. Com o método de digitalização ideal, mesmo em superfícies dentárias detalhadas morfológicas individuais podem ser digitalizadas com alta resolução e repetibilidade. Com os diferentes níveis de ampliação do scanner, estruturas morfológicas macro e micro pode ser adquirida. É possível digitalizar uma variedade de materiais modelo. <p class="jov...

Precisão é um grande interesse em muitos campos da medicina dentária. Substituindo tecido duro dental precisa de uma prótese de encaixe exato para garantir o funcionamento adequado e evitar destruir ainda mais a estrutura dental remanescente 1,2. Próteses parciais fixas e próteses totais são especialmente crítico para exata montagem em estruturas de apoio como dentes preparados ou implantes 3. É por isso que uma reprodução altamente preciso é necessário, em especial no domínio das impressões dentárias e fluxo de trabalho de laboratório dentário. No entanto, outros campos de tratamento dentário também se beneficiam de uma verdadeira e precisa resultado métrica, para verificar o sucesso do tratamento e avaliar novas estratégias de tratamento, por exemplo, aumento de tecidos moles e duros, erosão e abrasão monitoramento, tratamentos periodontais e tratamentos ortodônticos 4,5. Em muitos destes campos, os procedimentos de validação atuais são medidas lineares distância com pinças ou microscópios 6,7. Estes methods estão limitados a apenas alguns pontos de medição e informações limitado de três dimensões (3D) alterações de área de testes. Métodos mais recentes incluem a medição óptica de captura ou radiográfica de toda a superfície do objecto de teste 8,9. Aqui, a totalidade da superfície ou de volume é medido e apresentado como um objecto 3D no ecrã do computador. Medidas lineares são possíveis, bem como sobreposições de modelos de diferentes tempos de varredura. Com esta sobreposição, uma avaliação das alterações da superfície em cada ponto de verificação é possível. Isto permite a possibilidade de monitorizar uma área específica ou exibindo deformações em todos os três eixos de coordenadas. Além disso, as variações volumétricas pode ser medida 10. O ponto limitante com esses novos métodos é a precisão do scanner, usado para capturar o objeto de teste. Nenhuma das mudanças dentro da precisão do scanner de referência podem ser divididos em alterações do objeto de teste ou erros de digitalização. Precisão de digitalização é muitas vezes um valor dado pelo fabricantecante derivado de digitalização de objetos pequenos e calibrados 11. Este erro de digitalização mínima é diferente quando digitalizar objetos grandes, como a arcada dentária. Precisão consiste de veracidade e precisão. Veracidade é o desvio do objeto digitalizado a partir de sua geometria real. A precisão é o desvio entre as varreduras repetidas (ISO 5725-1). Neste estudo, um novo scanner óptico de referência, com base na variação técnica de exploração foco, foi introduzido para analisar amostras de único dente até modelos de arco pleno com maior precisão. Este scanner de referência foi utilizado como base para diversos estudos, comparando a precisão impressão dental a partir de técnicas convencionais e digitais 12-14 e para projectos concretos relativos oclusão dentária e abrasão de materiais odontológicos. O objetivo deste estudo foi o de fornecer informações básicas da precisão do scanner de referência e algumas possibilidades para utilizar este dispositivo no campo de pesquisa odontológica.

<table border="0" cellpadding="0" cellspacing="0" width="669">
	<tbody>
		<tr>
			<td>Reference model</td>
			<td></td>
			<td></td>
			<td>individual non-precious metal model, derived from a patient impression</td>
		</tr>
		<tr>
			<td>Araldit repair</td>
			<td>Huntsmen Advanced Material, Basel, Switzerland</td>
			<td></td>
			<td>used for making the base of the reference model</td>
		</tr>
		<tr>
			<td>CamBase</td>
			<td>Dentona, Dortmund, Germany</td>
			<td></td>
			<td>Type IV dental ston for pouring conventional impressions</td>
		</tr>
		<tr>
			<td>Identium</td>
			<td>Kettenbach, Eschenburg, Germany</td>
			<td></td>
			<td>Vinylsiloxanether impression material for conventional impression</td>
		</tr>
		<tr>
			<td>inEOS model holder</td>
			<td>Sirona Dental Systems, Bensheim, Germany</td>
			<td></td>
			<td>used for fixing stone models at the reference scanner</td>
		</tr>
		<tr>
			<td>Accutrans</td>
			<td>Coltene Whaledent, Altst&#228;tten, Switzerland</td>
			<td></td>
			<td>used for making the base of thestone models</td>
		</tr>
		<tr>
			<td>President putty</td>
			<td>Coltene Whaledent, Altst&#228;tten, Switzerland</td>
			<td></td>
			<td>mix with accutrans for betterstability of the base</td>
		</tr>
		<tr>
			<td>Alicona Infinite Focus</td>
			<td>Alicona Imaging, Graz, Austria</td>
			<td></td>
			<td>Reference scanner&#160;</td>
		</tr>
	</tbody>
</table>

accuracy in dental medicine, a new way to measure trueness and precision

Scanners de referência são usados ​​em medicina dentária para verificar uma série de procedimentos. O principal interesse é o de verificar os métodos de impressão como eles servem como uma base para restaurações dentárias. A limitação de corrente de muitos scanners de referência é a falta de precisão de digitalização de grandes objetos como arcos dentários completos, ou a possibilidade limitada para avaliar as superfícies detalhadas dente. Um novo scanner de referência, com base no foco variação técnica de varredura, foi avaliada com relação a mais alta precisão local e geral. Um protocolo de verificação específica foi testado para fazer a varredura da superfície do dente original impressões dentárias. Além disso, diferentes materiais do modelo foram verificados. Os resultados mostraram uma precisão de digitalização de alta do scanner de referência com um desvio médio de 5,3 ± 1,1 mm para a veracidade e 1,6 ± 0,6 mm para precisão em caso de exames de arcada completa. Métodos de impressão dental atual mostrou muito maior desvios (veracidade: 20,4 ± 2,2 mM, precisão: 12,5 ± 2,5 mm) than a precisão de varredura interna do scanner de referência. Objetos menores como superfície único dente pode ser digitalizado com uma precisão ainda maior, permitindo que o sistema para avaliar erosivo e abrasivo perda da superfície do dente. O scanner de referência pode ser usado para medir diferenças para um monte de campos de pesquisa odontológica. Os diferentes níveis de ampliação, combinadas com uma precisão geral e local elevada pode ser utilizado para avaliar as mudanças de dentes individuais ou restaurações até mudanças no arco cheias.

Figura 16A mostra o exame de uma amostra de gesso. Para verificar os parâmetros de digitalização ideais, controlar a repetibilidade para o material. Uma parte da amostra é digitalizada com diferentes configurações de contraste e brilho ea repetibilidade é verificada após cada varredura para encontrar as configurações de digitalização ideais. Superfícies metálicas apresentam os melhores resultados com um contraste mais baixo 1.0 e gesso ou resina materiais com um contraste superior. <strong...

Watch this Scientific Journal Video about Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision at JoVE.com

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Precisão é uma grande demanda na medicina dentária. Para verificar a exatidão, são necessários scanners de referência. Este artigo apresenta um novo scanner de referência com um método de varredura ajustado para adquirir uma ampla variedade de morfologias dentárias com alto rigor e precisão.

Precisão em Medicina Dentária, uma maneira nova de medir justeza e precisão

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

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Research

JoVE Journal

Medicine

13.3K visualizações.  University of Zürich.  Precisão é uma grande demanda na medicina dentária. Para verificar a exatidão, são necessários scanners de referência. Este artigo apresenta um novo scanner de referência com um método de varredura ajustado para adquirir uma ampla variedade de morfologias dentárias com alto rigor e precisão. 

Vídeo: Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 20081). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

Technique to Collect Fungiform Taste Papillae from Human Tongue

Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.

Técnica para coletar fungiformes (Taste) papilas da língua humana

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

Medicina

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

Ensaio de Point-of-Care rápida de eficácia anticoagulante enoxaparina em sangue total

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3&#160;trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

Construção de vapor Chambers usada para expor Ratos ao álcool durante o equivalente a todos os três trimestres de Desenvolvimento Humano

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Telemetria Intrauterine medir Rato contrátil Pressão<em&gt; In Vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

Um dispositivo simples de preparar rapidamente inteiras Mounts do mouse Intestino

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is important to standardize protocols to ensure proper analyses and interpretations. In our previous studies, we have measured boldness by monitoring a series of behaviors (time near surface, latency in transitions, number of transitions, and darts) in a 1.5 L trapezoidal tank. Likewise, we quantified aggression by observing bites, lateral displays, darts, and time near an inclined mirror in a rectangular 19 L tank. By dividing a 76 L tank into thirds, we also examined shoaling preferences. The shoaling assay is a highly customizable assay and can be tailored for specific hypotheses. However, protocols for this assay also must be standardized, yet flexible enough for customization. In previous studies, end chambers were either empty, contained 5 or 10 zebrafish, or 5 pearl danios (D. albolineatus). In the following manuscript, we present a detailed protocol and representative data that accompany successful applications of the protocol, which will allow for replication of behavioral syndrome experiments.

This manuscript describes the setup, implementation, and analysis of boldness, aggression, and shoaling in zebrafish and testing for the presence of a behavioral syndrome. A standardized approach for behavioral quantification will allow for easier comparison across studies. Modifications to this protocol are possible as each assay can be run individually.

Arrojo, agressão e Ensaios shoaling para Zebrafish Síndromes Comportamentais

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Abrir traqueostomia ácido gástrico aspiração Modelo murino de lesão pulmonar aguda Resultados em Maximal aguda não-letal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition leads to a better understanding of cariogenic and periopathogenic mechanisms. Microbial changes taking place in the oral cavity during childhood are of interest for several reasons. The evolution of the child oral microbiota and shifts in its composition need to be analyzed further to understand and possibly prevent the onset of disease. At the same time, advanced knowledge of the natural composition of oral biofilm is needed. Early stages of caries-free permanent dentition with healthy gums provide a widely unaffected subgingival habitat that can serve as an in situ baseline for studying features of oral health and disease. Analysis of children's oral biofilm during different stages in life is thus an important theme in the field. Modern molecular analysis methods can provide comprehensive information about the bacterial diversity of such biofilms. To enable microbiota data comparison, it is important to standardize each step in the procedure for molecular data generation. This procedure spans from clinical sampling, Next Generation Sequencing (NGS), bioinformatic data processing, to taxonomic interpretation. One of the most critical factors here is biofilm sampling. Sampling in children is even more challenging in particular due to limited space in subgingival areas. We thus focus on the use of paper points for subgingival sampling. This article provides a detailed protocol for oral biofilm sampling of the subgingival sulcus, the mucosa, and saliva in children.

Changes in the oral microbiome throughout childhood are of growing interest. Comparison of different microbiome studies reveals a lack of standardized sampling protocols. Limited space makes sampling the sound subgingival sulcus of children challenging. Paper point sampling is presented here in detail as the method of choice for this area.

Biofilme oral amostragem para análise de Microbiome em crianças saudáveis

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition ...

Conducting Maximal and Submaximal Endurance Exercise Testing to Measure Physiological and Biological Responses to Acute Exercise in Humans

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic responses to acute exercise are predominantly influenced by the duration and intensity of the exercise regimen. As exercise is increasingly thought of as a therapeutic treatment and/or diagnostic tool, it is important that standardizable methodologies be utilized to understand the variability and to increase the reproducibility of exercise outputs and measurements of responses to such regimens. To that end, we describe two different cycling exercise regimens that yield different physiologic outputs. In a maximal exercise test, exercise intensity is continually increased with a greater workload resulting in an increasing cardiopulmonary and metabolic response (heart rate, stroke volume, ventilation, oxygen consumption and carbon dioxide production). In contrast, during endurance exercise tests, the demand is increased from that at rest, but is raised to a fixed submaximal exercise intensity resulting in a cardiopulmonary and metabolic response that typically plateaus. Along with the protocols, we provide suggestions on measuring physiologic outputs that include, but are not limited to, heart rate, slow and forced vital capacity, gas exchange metrics, and blood pressure to enable the comparison of exercise outputs between studies. Biospecimens can then be sampled to assess cellular, protein, and/or gene expression responses. Overall, this approach can be easily adapted into both short- and long-term effects of two distinct exercise regimens.

To assess the influence of exercise intensity on physiologic and biologic responses, two different exercise testing protocols were utilized. Methods outlining exercise testing on a cycle ergometer as an incremental maximal oxygen consumption test and endurance, steady state submaximal endurance test are described.

Realização de exercícios de resistência máxima e submáxima testes para medir as respostas fisiológicas e biológicas ao exercício agudo em humanos

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic ...