Funding for the production of this manuscript was provided by Nottingham Trent University. The authors wish to thank the University yard manager, Anna Gregory for the use of her horses and provision of fecal samples for use in the protocol. Thanks also to Chester Zoo Wildlife Endocrinology Laboratory for use of their facilities.

declaração de ética: os procedimentos que envolvem temas de amostragem de campo e animais foram aprovados pela Escola de Animal, Rural e Ciência Ambiental (ARES) na Nottingham Trent University. 1. Recolha de amostras fecais NOTA: Devem ser usadas luvas ao manusear amostras fecais e metanol. Se existe uma forte suspeita de que um animal pode estar sofrendo de uma doença zoonótica, roupas de proteção, como também deve ser usado um jaleco. <ol><li> Recolha das amostras fecais, o mais rapidamente possível (dentro de minutos a algumas horas) após a defecação e colocá-los dentro de um saco de amostra. De três a cinco subamostras de qualquer amostra defecated com um peso total de aproximadamente 10 g. Rotular o saco com o tempo e a data da colheita, identificação individual e congelar a amostra a -20 ° C até à análise. </li><li> Para minimizar o erro, congelar as amostras a -20 ° C até o estudo estar completa, em seguida, reunir as amostras para análise em conjunto como um único lote. &lt;/ Li&gt; </ol> 2. Fecal Hormone Extração <ol><li> Deixar as amostras fecais para descongelar à temperatura ambiente. </li><li> Certifique-se de que as amostras são homogeneizadas por mistura manualmente cada amostra em seu próprio saco de amostra. </li><li> Para cada amostra, pesar 0.50 (± 0,003 g) de material fecal numa microbalança de um barco de pesagem, em seguida, transferir as fezes para um frasco de vidro de 8 mL. Use um novo pesar barco para cada amostra e limpar a pinça com 30% de metanol entre cada amostra. </li><li> Combinar a 0,5 g de material fecal, com 5 ml de 90% de metanol: água, e agitar O / N num agitador orbital, à temperatura ambiente. </li><li> Na manhã seguinte, vortex a amostra e, em seguida, centrifuga-se durante 20 min a 600 g. </li><li> Decantar a fracção de metanol num tubo de vidro de 16 x 125 mm2 e colocar num suporte num banho de água regulado para uma temperatura de 37 ° C. Evaporar a seco usando ar em uma câmara de exaustão. Lavar o frasco de extração e coloque o sedimento fecal restante em um saco de resíduos clínicos para a eliminação. </li><li> Re-suspender os extractos fecais em 100%, 1 ml de metanol e transferir para um tubo de polipropileno de 12 x 75 mm2. Imediatamente PAC e armazenar a -20 ° C até análise. </li></ol> 3. Análise Hormona <ol><li> Realizar a análise com uma enzima ligada-imunoensaio (EIA), utilizando soro policlonal corticosterona CJM006. NOTA:. Um segundo EIA foi testado (Cortisol R4866 anti-soro, mas este não conseguiu alcançar a etapa de validação bioquímica (ver secção 4.1) O anti-soro policlonal corticosterona CJM006 e Cortisol R4866 anti-soro e do respectivo HRP de são fornecidos por CJ Munro, da Universidade da Califórnia, Davis, CA. </li><li> Diluir o anti-soro a 1: 15000 em tampão de revestimento (NaHCO3 0,05 M, pH 9,6) utilizando uma pipeta repetitivo e uma ponta de pipeta 50 ul, carregar uma placa de microtitulação de 96 poços com 50 ul / poço. Cobrir a placa com um vedante de placa e incubar O / N a 4 ° C. Deixar dois poços não revestidos para representar as cavidades de ligação não específica (NSB). </li><li> Imediatamente antes da utilização lavar as placas de microtítulo cinco vezes utilizando um lavador de placas de microtitulação automático (solução de lavagem; 0,15 M de NaCl, 0,05% de Tween 20). </li><li> Carregar as placas com 50 ul por poço, durante a NSB de e poços [tampão EIA (0,1 M NaPO4, NaCl 0,149, 0,1% de albumina de soro de bovino, pH 7,0) somente] &quot;zero&quot;, padrões (corticosterona, 3,9 - 1.000 pg / bem) ou amostras de extracto fecal (apropriadamente diluído em 1:20 tampão EIA, ver secção 4.1). Normas e zeros deve ser executado em triplicado e quadriplicado, respectivamente. Diluídas amostras de extracto fecais e NSB de deve ser executado em duplicado. </li><li> Siga este imediatamente com a adição de 50 ul / cavidade do rótulo ᵻ rábano conjugado com peroxidase diluído em tampão EIA para 1: 70000. </li><li> Incubar as placas de microtitulação no escuro durante 2 horas à temperatura ambiente. </li><li> Lavam-se as placas de microtitulação com uma solução de lavagem 5 vezes e incuba-se as placas à TA no escuro com 100 ul / WELl de substrato RT [0,4 mM de 2,2&#39;-azino-di- sal (ácido 3-etilbenztiazolino sulfónico) de diamónio, 1,6 mM de H 2 O 2, citrato 0,05 M, pH 4,0]. Prepara-se o substrato imediatamente antes do uso. </li><li> Usar um espectrofotômetro no comprimento de onda de 405 nm e ler as placas de microtítulo. A incubação das placas é considerada completa quando a densidade óptica dos poços de zero atinge 0,8 a 1,0. </li><li> Para assegurar a reprodutibilidade, utilizar controlos [normas EIA ou uma amostra biológica diluída em tampão EIA para se ligar a cerca de 30% (padrão EIA), 50% (uma amostra biológica, por exemplo, piscina de cavalo doméstico extracto fecal) e 70% (padrão EIA) ] para demonstrar uma intra- e inter-ensaio coeficientes de variação de &lt;10% e &lt;15%, respectivamente. </li></ol> 4. Enzyme Linked-imunoensaio: Validação para as espécies Estudo e Sexo <ol><li> Biochemical Validation (Paralelismo) <ol><li> Usando tampão EIA, execute uma diluição em série em pool fecal adicionalct, de preferência proveniente de amostras suspeitas de alta e baixa concentração de hormônio de vários indivíduos (intervalo: puro, 1: 2 a 1: 8192) e executado no EIA como descrito na seção 3. NOTA: Um paralelismo é considerada alcançada quando as diluições em série de extractos fecais produzir uma curva de deslocamento paralelo com os resultados da curva de regressão linear e padrão demonstram R2&gt; 0,90, uma inclinação&gt; 0,50 e p &lt;0,05. O extracto fecal diluídos em série que liga-se a cerca de 50% na curva padrão deve ser considerada a diluição ideal para executar todos os extractos fecais (por exemplo, 1:20). Não há nenhuma interferência considerado no ensaio, quando os resultados de regressão linear demonstram R2&gt; 0,90 e p &lt;0,05. </li></ol></li><li> Biochemical Validation (Interferência) <ol><li> Pico de 200 ul de padrões diluídos em série (zero, 3,9 a 1000 ng / poço de corticosterona) com 200 ul do extracto fecal apropriadamente diluído reunidas [diluído a 50% de ligação determinadas por ParalleliSM (01:20)] e executado no EIA como na secção 3. A seguir trama análise EIA a concentração observada menos o fundo (concentração da amostra zero) em função da concentração esperada. NOTA: Se a adição de extrato fecal reunidas diluída com padrões não altera significativamente os resultados da regressão quantidade esperada e lineares produz R 2&gt; 0,90, uma encosta perto de 1,0 e p &lt;0,05. Nenhuma interferência com o EIA foi alcançado. </li></ol></li><li> Validação Biológica: NOTA: A demonstração de que o EIA escolhido tem a capacidade de medir as mudanças biologicamente relevantes da hormona de interesse [por exemplo, o aumento da concentração de glicocorticóides na sequência de um farmacológico (ACTH é muitas vezes um procedimento licenciado) ou desafio biológico (por exemplo, psicológica e / ou ambiental; mais provável que seja um acontecimento desregulada oportunista)]. <ol><li> Selecione, extrair e analisar amostras fecais para as concentrações de metabólitos de glicocorticóidesusando o processo descrito nas secções 1-3 antes e depois de um evento desafiador. NOTA: Um evento desafiador será espécies específicas. Um método de exemplo é fornecido abaixo para o cavalo doméstico. Exemplos completos de eventos desafiadores para os cavalos têm sido publicados anteriormente, incluindo o uso de gestão 9 e maneio procedimentos 16. Um aumento significativo nas concentrações de metabolitos de glucocorticóides após o evento desafiador devem ser observadas e confirmadas por meio de análise estatística. Esta deve incorporar, onde apropriado, o efeito de medidas repetidas, data da amostra e individuais. validação biológico é então considerado alcançado. </li></ol></li></ol>

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The authors have nothing to disclose.

análise de corticosterona fecal fornece um meio de avaliar os padrões de longo prazo da atividade adrenal em cavalos. A natureza não-invasiva do método supera os efeitos de confusão de outros métodos de amostragem utilizados para avaliar a atividade adrenal incluindo saliva e no plasma análise 9. Além disso, a técnica tem uma vantagem não-invasivo claro se estudando cavalos livres que vão. Há vários pontos-chave para discussão em relação a este método e seu uso ad...

O método descrito pretende analisar concentrações de corticosterona em fezes equina, a fim de fornecer uma avaliação não-invasiva da actividade supra-renal. Medir a atividade do hipotálamo-pituitária-adrenal (HPA) é uma abordagem aceite para estudar a resposta a situações potencialmente aversivas em ambas as espécies em cativeiro e domésticos. A técnica de referência e o método mais amplamente utilizado é o uso de um plasma sanguíneo no entanto, métodos alternativos, tais como a análise fecal foram desenvolvidas de modo a ultrapassar a tensão induzida por amostragem de sangue em si e permitem que a capacidade de monitorar as espécies que vão livre. Durante uma situação de aversivo, homeostase fisiológica é interrompido. O hipotálamo no cérebro libera hormônio liberador de corticotrofina (CRH), que atua na glândula pituitária e estimula a liberação de hormônio adrenocorticotrófico (ACTH). ACTH entra na corrente sanguínea e estimula o córtex adrenal a secretar speciede s glicocorticóides específicos (CG). Os glicocorticóides estão intimamente ligados a eventos estressantes ao invés de ser consistentemente produzidos em todos os estados de energia elevada, portanto, eles são muitas vezes medidos em preferência sobre outros de estresse ligado hormônios 2. Os glucocorticóides são responsáveis ​​por diversos efeitos adaptativos em cavalos. A energia é rapidamente mobilizados a partir de locais de armazenagem no corpo sob a forma de ácidos gordos e de glucose, o consumo de oxigénio é aumentada, a função sensorial é reforçada 3 e o fluxo sanguíneo é reduzido em áreas não necessárias para o movimento 4. Além de atuar como um mecanismo de enfrentamento, o aumento induzido stress na glicocorticóides também pode ajudar a preparar o animal para a próxima estressor 5. Avaliando os níveis de hormona no plasma e na saliva envolve a medição da hormona em circulação real no entanto, a medição dos metabolitos nas fezes medidas do produto final metabólico da hormona. esteróides circulantes são catabolized na lIver antes da excreção para a bílis onde sofrem mais alterações facilitados pelas atividades enzimáticas da flora bacteriana intestinal dentro da faixa 6. Portanto, imunoensaios dirigidos a glucocorticóides no sangue pode não ser adequado para análise de metabolitos fecais 7 glucocorticóides. Como recolha de fezes pode ser levada a cabo sem perturbar o cavalo, a análise de fezes para corticosterona, tem sido amplamente utilizado para monitorizar a actividade HPA num número de circunstâncias. Corticosterona elevada nas fezes de cavalos tem sido relatada em resposta a situações potencialmente aversivas, nomeadamente durante o tratamento veterinário pós-operatório 8 e em habitação restritiva 9. Amostragem fecal reflete um nível de glicocorticóides agrupada ao longo do tempo, em vez de o ponto de amostragem de tempo oferecido pelo plasma e saliva tornando-o adequado para o acompanhamento a longo prazo, crónica ou padrões sazonais 10. Devido à não-invasivanatureza do método, as amostras podem ser colhidas repetidamente para um indivíduo sem a necessidade de captura ou de contenção 11. No entanto, as espécies de tempo específico trânsito intestinal deve ser levado em conta no planejamento de um protocolo de amostragem. Em cavalos, tempo de trânsito intestinal é de cerca de 18 h, por conseguinte, 12, resposta adrenal e metabolitos subsequentes de corticosterona pode ser detectado nas fezes um dia após a activação inicial do eixo HPA. Ao utilizar técnicas de imunoensaio não-invasivos uma validação cuidadosa para a espécie a ser investigada é essencial 13. Além disso, as diferenças de sexo na excreção de metabolitos da hormona foram relatados provavelmente devido a diferenças na taxa metabólica e o tipo de metabólito excretado corticosterona em várias espécies, incluindo ratos, 14 e 15 galinhas. Foi, portanto, importante como parte deste método que o ensaio foi validado para uso em cavalos domésticos masculinos e femininos, como está detalhado no the protocolo. Essa diferença no metabolismo hormonal entre os sexos tem consequências para a qualidade dos dados, no entanto, raramente é abordada e incluído como parte da validação do ensaio. Este método não-invasivo permite a avaliação de longo prazo da atividade adrenal em cavalos domésticos. Os detalhes do protocolo tanto a validação do ensaio e da técnica de ensaio em si.

<table>
	<tbody>
		<tr>
			<td>Corticosterone antibody &#38; HRP kit</td>
			<td>Coralie Munro - UC Davis</td>
			<td>NA</td>
			<td>No longer available through UC Davis - please see Arbor Assays</td>
		</tr>
		<tr>
			<td>Cortisol antibody &#38; HRP kit</td>
			<td>Coralie Munro - UC Davis</td>
			<td>NA</td>
			<td>No longer available through UC Davis - please see Arbor Assays</td>
		</tr>
		<tr>
			<td>Corticosterone synthetic standard hormone</td>
			<td>Sigma Aldrich</td>
			<td>50-23-7</td>
			<td>Harnful if ingested or with skin contact. Use in fume cupboard</td>
		</tr>
		<tr>
			<td>Cortisol synthetic standard hormone</td>
			<td>Sigma Aldrich</td>
			<td>15087-01-1</td>
			<td>Harnful if ingested or with skin contact. Use in fume cupboard</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma Aldrich</td>
			<td>67-56-1</td>
			<td>Irritant. Use in fume cupboard</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>144-55-8</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Sodium Carbonate Anhydrous</td>
			<td>Sigma Aldrich</td>
			<td>497-19-8</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic</td>
			<td>Sigma Aldrich</td>
			<td>7558-79-4</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic</td>
			<td>Sigma Aldrich</td>
			<td>10049-21-5</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>9048-46-8</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich</td>
			<td>9005-64-5</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Sigma Aldrich</td>
			<td>77-92-9</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>ABTS</td>
			<td>Sigma Aldrich</td>
			<td>30931-67-0</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide 30%</td>
			<td>Sigma Aldrich</td>
			<td>7722-84-1</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>7647-14-5</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Buffer capsules - pH 4</td>
			<td>VWR</td>
			<td>332732B</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer capsules - pH 7</td>
			<td>VWR</td>
			<td>332742D</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer capsules - pH 10</td>
			<td>VWR</td>
			<td>332762H</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma Aldrich</td>
			<td>435570</td>
			<td>Irritant. Use in fume cupboard</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>S5881</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Fisher Scientific</td>
			<td>BFS-525-010A</td>
			<td></td>
		</tr>
		<tr>
			<td>Air compressor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer +printer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fridge-freezer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drying apparatus</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>+tubing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flammable liquid storagecabinet</td>
			<td>VWR</td>
			<td>649-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fume cupboard</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hot-plate stirrer</td>
			<td>VWR</td>
			<td>640-282</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate washer</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Research&#174; pipettes - multipack option 2</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 1000ul</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 200ul</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 20ul</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Repeater pipette</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette filler</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Progen Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Hilsonic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Warm water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water purification system</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fecal glucocorticoid analysis: non-invasive adrenal monitoring in equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

cavalos domésticos (n = 16, 8 éguas, 8 capões), com uma idade média de 15 anos (± 3) foram agrupados de acordo com o sexo e submetido a quatro projetos de habitação com níveis crescentes de isolamento social (n = 4 cavalo / tratamento). Habitação 1 envolveu cavalos que vivem em um ambiente de rebanho, simulando intimamente seu habitat natural. Habitação 2 envolvidos cavalos que vivem em pares em um celeiro interior. Habitação 3 cavalos envolvidos alojado sozinho em estábul...

Watch this Scientific Journal Video about Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids at JoVE.com

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal Análise de glicocorticóides: Non-invasive Monitoring Adrenal em eqüídeos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

fecal-glucocorticoid-analysis-non-invasive-adrenal-monitoring

Research

JoVE Journal

Biology

9.6K visualizações.  Nottingham Trent University. Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Vídeo: Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Mouse Adrenal Chromaffin Cell Isolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex.  The digestion of the medulla into single chromaffin cells is then demonstrated.

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.

Rato de isolamento de células Adrenal Cromafim

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

Biologia

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Rotulagem e hESCs hMSCs com óxido de ferro nanopartículas para Não-invasiva em Rastreamento vivo com ressonância magnética

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Rotulagem células-tronco com corantes fluorescentes para detecção não invasiva com imagens ópticas

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Não-invasivo 3D Visualization com Sub-micron Resolução Usando Síncrotron-X-ray-tomografia

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

A reversível, método não-invasivo para medições de Resistência das Vias Aéreas e Amostragem Líquido da Lavagem Broncoalveolar em Ratos

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A longo prazo de Teste de toxicidade letal com o crustáceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Análise Genética rápida de epitelial-mesenquimal sinalização durante a regeneração do cabelo

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

A Triagem procedimento químico para Glucocorticoid Sinalização com um Sistema Reporter Zebrafish Zergling luciferase

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal welfare guidelines, non-invasive techniques should be preferred whenever applicable. In our group, we work on different species of song birds in the lab and in the field, and we successfully apply non-invasive methods to sex and individually mark chicks. This paper presents a comprehensive non-invasive tool-box. Sexing birds prior to the expression of secondary sexual traits requires the collection of DNA-bearing material for PCR. We established a quick and easy method to sex birds of any age (post hatching) by extracting DNA from buccal swabs. Results can be obtained within 3 hours. For individual marking chick's down feathers are trimmed in specific patterns allowing fast identification within the hatching order. This set of methods is easily applicable in a standard equipped lab and especially suitable for working in the field as no special equipment is required for sampling and storage. Handling of chicks is minimized and marking and sexing techniques are non-invasive thereby supporting the RRR-principle of animal welfare guidelines.

This protocol provides a convenient set of methods, which enables extremely fast, easy, non-invasive, reliable and low-cost, molecular sex determination of birds and their non-invasive, quick, safe and easily recognizable marking shortly after hatching. Only limited handling of chicks is required. This convenient toolbox of methods complies entirely with the RRR-guidelines.

Quem é Quem? Métodos não-invasivos para Individualmente Sexo e Mark altriciais Chicks

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

Ensaio de absorção de LDL colesterol usando célula viva análise com célula de imagem monitoramento de integridade

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...