Isso funciona foi apoiada através de subsídios da Natural Sciences and Engineering Research Council of Canada (RGPIN 238382-2011) e Canadian Institutes of Health Research (MOP115076). Heather Paul foi apoiada por uma Ciências Naturais e do Conselho de Investigação do Canadá Engenharia de Bolsas de Pós-graduação e uma bolsa Alberta inova Health Solutions. Megan Hallam foi apoiado por uma bolsa de estudos em Ciências Naturais e Engenharia do Conselho de Pesquisa de Pós-Graduação, um Scholarship Frederick Banting e Charles Best Canadá graduação, e Prêmio de Crianças Alberta Hospital Research Institute Formação em Genética, Desenvolvimento Infantil e Saúde.

Este protocolo foi aprovado pelo Comitê da Universidade de Calgary Animal Care e conformado com o Guia para o Cuidado e Uso de Animais de Laboratório. 1. Dam Separe Offspring <ol><li> Separa-se a barragem de sua prole para um mínimo de 5 min antes da ordenha 12. NOTA: A represa pode ser ordenhadas até 5-6 horas após a separação 1,6,13, no entanto períodos de separação mais do que 4 horas podem alterar a composição do leite 14. Enquanto tempo de separação não parece afetar coleção volume de leite 12, é aconselhável que um tempo de separação consistentes ser mantida durante todo o estudo. A composição do leite pode mudar ao longo da lactação 15, portanto, devem ser feitas tentativas para manter o dia da coleta de leite consistente. Máxima produção de leite é sugerida para ocorrer no dia 14 de lactação 12. </li><li> Usando uma câmara de aquecimento, garantir os filhotes são capazes de manter temperatu do corpo adequadare sem a presença da mãe durante a duração do procedimento de ordenha. NOTA: No estudo apresentado a seguir, ordenha foi realizada ao desmame, quando as barragens eram aproximadamente 22 semanas de idade e The Offspring 21 dias de idade, portanto, nenhuma câmara de aquecimento foi utilizado. </li></ol> 2. Set-up e Preparação <ol><li> Colete todos os materiais necessários para o procedimento de ordenha. NOTA: Todos os materiais pode ser encontrada na tabela de materiais e equipamento. </li><li> Coloque uma almofada de aquecimento no banco onde a ordenha terá lugar e cobrir a almofada com um banco absorvente sob-pad. </li><li> Configure o sistema anestésico. Assegure-se que o sistema tem de oxigénio e isoflurano antes do início suficiente. Fixar o anestésico máscara que irá ser utilizada para a indução da anestesia inicial para a máquina. Coloque a máscara que irá ser utilizado para a manutenção da anestesia nas proximidades se for diferente do que a máscara de anestesia inicial. </li><li> Coloque uma agulha 25 G para uma seringa de 1 ml para ocitocinainjecção utilizando uma técnica asséptica. </li><li> Ligue a almofada de aquecimento para que a temperatura do corpo materno é mantida durante o processo de ordenha. NOTA: Monitorar a temperatura da almofada de aquecimento para assegurar a almofada não se torna muito quente e causar queimaduras. Em alternativa, usar uma fonte de calor, tal como uma mesa de operações aquecida, que pode ser ajustado para uma temperatura específica. </li></ol> 3. anestesiar o Dam Usando Isoflurane <ol><li> Abra o tanque de oxigênio e virar o fluxo de 1 L (1000 cc) por min. Ligue o fluxo de isoflurano e ajustado para 5%. CUIDADO: Evite inalação direta de anestésico e evitar a acumulação de vapores anestésicos. </li><li> Anestesiar a barragem. </li><li> Mudar para a máscara de manutenção, se necessário, colocando o supino barragem no banco almofada absorvente. Confirme anesthetization por falta de pedal reflexo. </li><li> Reduzir o fluxo de isoflurano a 2-3% para manutenção da anestesia. Monitorar continuamente barragem durante todo o processo para garantir depressão da respiração não ocorre. NOTA: Uma vez sob anestesia, os olhos da represa devem ser protegidos com um lubrificante de olho estéril para evitar que os olhos sequem ou tornar-se riscado. </li></ol> 4. A oxitocina injeção <ol><li> Garantir a ocitocina (20 USP Unidades / ml) não passou da data de validade. Desinfetar o frasco de ocitocina com um álcool estéril limpar / álcool de limpeza pad. </li><li> Utilizando uma técnica asséptica, elaborar 2 UI (0,1 ml) de ocitocina para a seringa. Utilize uma nova agulha e seringa para cada barragem que vai ser ordenhadas. NOTA: A oxitocina doses variam geralmente de injecções individuais de 1 a 5 IU 1,6,12,16. Alternativamente, uma dose de 4 UI / kg de peso corporal pode ser utilizado 12. Uma dose única de 2 Ul pode ser repetida uma vez, se a ordenha for encontrada dificuldade. </li><li> Injectar a oxitocina por via intraperitoneal. Insira a agulha no quadrante inferior direito do abdômen com a agulha apontando para a cabeça, em um ângulode 15-30 °, cerca de 0,5 cm de profundidade. </li><li> Puxe o êmbolo para garantir pressão negativa antes da injeção. Se qualquer líquido (sangue, urina, conteúdo intestinal, etc.) é aspirada para a seringa, a agulha e remover tentar a injecção com uma agulha e seringa. Se nenhum fluido é aspirado injetar a oxitocina e descarte a agulha e seringa imediatamente em um recipiente de risco biológico. </li><li> Aguarde cerca de 5-15 min para a ocitocina para estimular a descida do leite. </li></ol> 5. Preparação de Sites de ordenha <ol><li> Escolha os sites / tetos de leite serão coletadas. O leite pode ser recolhida a partir de qualquer tetina 12. </li><li> Remova suavemente a pele ao redor dos tetos de ser ordenhadas com os trimmers, como a pele pode causar dificuldade na coleta de amostra devido à absorção da do leite. Seja gentil - a pele ao redor dos bicos é extremamente sensível e pode ser seca e, como tal, é suscetível a arranhões e lágrimas. </li><li> Esterilização do i tetinaNão é necessário, mas opcionalmente, limpar a tetina com água morna após a pele é removida. Preparar pelo menos dois locais como mais do que um sítio possa ser necessária para a recolha de leite. NOTA: Se a análise do leite inclui perfis de microbiana, a área de tetina pode exigir a esterilização com iodo 5. </li></ol> Coleção 6. Leite <ol><li> Espremer suavemente a base da teta, expulsando o leite manualmente para recolha. NOTA: Se a análise do leite inclui perfis de microbiana, as primeiras gotas de leite devem ser descartados. </li><li> Recolher as gotas de leite para um tubo capilar, o enchimento do tubo capilar. Tubos capilares, que podem acomodar volumes maiores (por exemplo, 50 ul) facilitar o processo. NOTA: Se houver dificuldade na coleta de leite é encontrado, uma segunda dose de oxitocina pode ser administrado. Recomenda-se a dose não deve exceder 4 IU total. </li><li> Repartir o leite a partir do tubo capilar para um tubo de microcentrífuga esterilizadotocar a extremidade do tubo que foi utilizado para desenhar a partir do leite da teta para o lado do tubo de microcentrífuga - observar o leite a ser desenhada para fora do tubo capilar por meio de acção capilar. NOTA: &#39;funda para fora &quot;do leite que não é puxado para dentro do tubo, usando uma agulha de 18 G ligada a uma seringa de 1 ml. </li><li> Monitorar continuamente a barragem para sinais de dor ou depressão respiratória e ajustar o fluxo de isoflurano em conformidade. </li><li> Continuar a recolher o leite como descrito nesta seção até que o leite suficiente tiver sido coletado para a análise de leite escolhido. Para a determinação da concentração de proteína e crematócrito descrito abaixo, recolher 0,25 ml de leite. NOTA: Use um local de ordenha diferente se a descida do leite diminui ou o local escolhido não expulsar leite suficiente. Um máximo de cerca de 2,5 ml de leite por animais pode ser recolhida 17, ou até 0,5 ml por tetina. Os autores recomendam que o tempo total sob anestesia ser limitado a aproximadamente 45-60 min, oR 45 min de ordenha. </li><li> Recolha do leite para um tubo de micro-hematócrito para medição crematócrito a partir de amostras de leite fresco, e vedar a extremidade do tubo com vedante argila. Rotular o tubo de microhematócrito com o ID barragem e armazenar a amostra de leite na posição vertical. </li><li> Quando a ordenha é completa, desligue o fluxo de isoflurano e oxigênio. Retire a máscara da barragem e continuará a acompanhar barragem até acordado. Recomenda-se que se não totalmente consciente, a barragem deve ser colocada sobre uma almofada absorvente banco durante o período de recuperação, ao invés de diretamente na cama gaiola, para evitar que a roupa de cama de ser aspirado ou coçar os olhos da represa durante a recuperação. NOTA: Não deixe a barragem autônoma até que tenha recuperado a consciência suficiente para manter recumbancy esternal. </li><li> Se não forem necessárias análises adicionais, congelar o leite à temperatura de -80 ° C. Outros têm sugerido que o leite possa ser armazenado durante até 3 horas a 4 ° C ou 5 meses a -20 ° C 18. </li></ol> 7. Medição crematócrito <ol><li> Estimar o teor de gordura do leite por meio do cálculo do crematócrito leite (a percentagem de creme na amostra de leite) 19. NOTA: As medições com leite humano demonstraram que o leite fresco ou congelado pode ser utilizado para uma medição crematócrito, no entanto leite fresco é mais altamente correlacionados (r = 0,92 versus R = 0,90) com a concentração lipídica 11. O uso de leite fresco ou congelado para medições crematócrito deve ser mantido consistente em todo o estudo, como o leite descongelado está associado a uma pequena diminuição no crematócrito valores 11. </li><li> Para o leite fresco, recolher uma amostra de leite da teta em um tubo de microhematócrito; preencher pelo menos ¾ cheio (cerca de 15-20 mL). Alternativamente, extrair leite fresco a partir da amostra recolhida para dentro do tubo capilar depois de misturar bem. Selar o fim com selante de barro. </li><li> Colocar o tubo capilar para hematócrito da fieira, com aextremidade vedada apontando para o exterior, garantindo a centrífuga é equilibrada. </li><li> Comece o spin hematócrito (120 segundos a 13.700 xg). NOTA: O tempo da rotação ou velocidade pode mudar, dependendo do modelo de centrífuga utilizada. </li><li> Remover o tubo da centrifugadora depois da rotação é completo e realizar as medições para calcular o crematócrito. Observar separação da amostra numa camada de creme e uma camada clara. </li><li> Medir e registar o comprimento total de fluido no tubo e o comprimento da gordura (creme) camada usando compassos de calibre ou uma régua. NOTA: O crematócrito é expressa como a percentagem da camada de creme na amostra de leite a 19, calculada como (comprimento da camada de nata / comprimento total da coluna de leite) x 100 (Figura 1A). Calcular os valores de concentração e de energia a partir da medição de gordura crematócrito como se segue: concentração de gordura (g / L) = (crematócrito (%) - 0,59) /0.146 (Figura 1B) 19; Valor energético (kcai / L) = 290 + (* crematócrito 66,8 (%) (Figura 1C) 19. </li></ol> 8. Proteína Concentração Determinação <ol><li> Utilizando albumina de soro bovino como padrão de proteína, determinar a concentração total de proteína do leite, utilizando um ensaio de proteína padrão, tal como ensaio de proteínas de Lowry um 6. NOTA: A diluição do leite pode ser necessária para as medições de proteínas de leite a cair dentro da curva padrão do ensaio. </li></ol>

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Os autores não têm conflitos de interesse a declarar. Todos os experimentos com animais foram realizados em conformidade com os protocolos do CCAC aprovado.

Investigations into maternal milk components have increased as interest in early life development research rises. As the sole source of nutrition during the neonatal period, the bioactive compounds in milk are essential for ideal growth and development, especially in the context of intestinal and immune health3. The method presented here is a simple, non-invasive method of collecting milk from the lactating rat in amounts sufficient for downstream analysis, such as oligosaccharide profiling6. The me...

O leite é a única fonte de nutrição para mamíferos recém-nascidos, fornecendo energia e nutrientes para o crescimento e desenvolvimento infantil 1,2. Quando o leite é constituído principalmente por células, lípidos e proteína 1, que também contém uma pletora de compostos bioactivos que modulam o desenvolvimento vida precoce da descendência, incluindo enzimas, hidratos de carbono, hormonas, anticorpos, factores de crescimento, citocinas, exossomas, microvesículas, e pequenos RNAs tais como microARN 1,2. O papel fundamental do leite materno na criação da prole imunitário e saúde intestinal 3, juntamente com evidências de que crianças amamentadas são menos suscetíveis à doença 2, destaca a importância de identificar os constituintes do leite associados a processos de doenças no início da vida e os mecanismos moleculares envolvidos em suas ações. O rato é o desenvolvimento de um modelo popular para investigar o efeito de vários nutricional, fisiológico e intervenções químicas no iníciodesenvolvimento -life 4. A análise do leite do rato podem, portanto, proporcionar novos insights sobre a saúde materna e prole. Avanços científicos atuais fornecem agora aumentar as oportunidades para investigações aprofundadas sobre os efeitos de constituintes do leite específicos na saúde e na doença. Por exemplo, a sequenciação de leite perfis bacterianas elucidou o seu papel na colonização intestinal precoce do intestino infantil 5, análise de espectrometria de massa de oligossacarídeos do leite forneceram uma visão sobre a alteração dos perfis de oligossacarídeos do leite via dieta materna 6 e profunda seqüenciamento de microRNA secretada em os glóbulos de gordura do leite materno destaca possíveis papéis na transcrição do gene, o metabolismo e a função imunológica 7. Modelos de ratos representam um dos organismos modelo mais populares usados ​​em estudos materno 8,9. Uma vantagem é seus períodos de gestação e aleitamento curtas, durando apenas approximatEly 21 dias cada; Por conseguinte, o tempo total a partir do início da gravidez até lactação representa um curto período de tempo em que os dados valiosos podem ser gerados. Quanto maior o tamanho de ratos em comparação com os ratinhos, no contexto de recolha do leite, pode proporcionar uma vantagem significativa no que diz respeito ao volume de leite e facilidade de recolha do leite; produção de leite no rato, por exemplo, parece ser dependente do peso corporal total com os ratos mais pesados ​​que produzem mais leite 10. Aqui, uma descrição geral para a coleta manual de leite de ratas lactantes é fornecido. Este protocolo requer um equipamento mínimo, é não-invasiva e de baixo custo, e podem ser usadas para recolher os volumes adequados de leite para análises adicionais a jusante. Em breve, a barragem é anestesiados com isoflurano, a descida do leite é estimulada pela oxitocina, e de leite são recolhidas em tubos capilares por meio de extracção manual do leite. Finalmente, como dois principais componentes de leite são proteínas e gordura, uma breve description de estimar o teor de gordura do leite por meio de medidas crematócrito 11 e quantificação da concentração total de proteínas usando um ensaio de proteína padrão é apresentada.

<table border="0" cellpadding="0" cellspacing="0" width="829">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Equipment - Milking</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml syringes</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">309602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ul Microdispenser Capillary Tubes</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">21-169D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxytocin (20 USP Units/ml)</td>
			<td style="width:191px;">Bimeda-MTC</td>
			<td style="width:119px;">1OXY015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">PPC Vet Isoflurane Inhalation Anesthetic, 250 ml</td>
			<td style="width:191px;">Fresenius Kabi</td>
			<td style="width:119px;">M60302</td>
			<td>Used on the order of a veterinarian</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Alcohol Prep Pad</td>
			<td style="width:191px;">Dukal</td>
			<td style="width:119px;">853</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Absorbent Bench Underpad</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">82020-845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Maxi-Therm Hyper/Hypothermia Blanket</td>
			<td style="width:191px;">Cincinnati Sub-Zero</td>
			<td style="width:119px;">274</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Rodent Anesthesia Machine with Vaporizer</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;"></td>
			<td>Subject to individual laboratory needs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">Animal Masks</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;">50100/50102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge Tubes</td>
			<td style="width:191px;">Axygen</td>
			<td style="width:119px;">MCT-060-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChroMini Professional Trimmer</td>
			<td style="width:191px;">Wahl</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Equipment - Creamatocrit</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StatSpin SafeCrit Plastic Microhematocrit Tubes (Untreated)</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">22-274-914</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Critoseal Capillary Tube Sealant Tray</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">470161-478</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StatSpin CritSpin Microhematocrit Centrifuge</td>
			<td style="width:191px;">Beckman Coulter, Inc</td>
			<td style="width:119px;">X00-004999-001</td>
			<td></td>
		</tr>
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</table>

milk collection in the rat using capillary tubes and estimation of milk fat content by creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also contains a vast number of bioactive compounds that greatly affect the development of the neonate. The analysis of milk components will help elucidate key factors that link maternal metabolism and health with offspring growth and development. The laboratory rat represents a popular model organism for maternal studies, and rat milk can be used to examine the effect of various maternal physiological, nutritional, and pharmacological interventions on milk components, which may then impact offspring health. Here a simple method of manually collecting milk from the lactating rat that can be performed by a single investigator, does not require specialized vacuum or suction equipment, and provides sufficient milk for subsequent downstream analysis is described. A method for estimating the fat content of milk by measuring the percentage of cream within the milk sample, known as the creamatocrit, is also presented. These methods can ultimately be used to increase insight into maternal-child health and to elucidate maternal factors that are involved in proper growth and development of offspring.

O leite foi recolhido como descrito no desmame de fêmeas Wistar (cerca de 22 semanas de idade, pesando 350 a 400 g) que consumiram um controlo (AIN-93G, n = 5), elevado teor de proteínas (40% de caseína p / p, n = 5) , ou alta fibra pré-biótica (21,6% p / p, 1: 1 razão de oligofrutose e inulina, n = 4) dieta durante a gravidez e lactação. A dose de oxitocina foi de 2 UI. O leite foi recolhido utilizando tubos capilares, e um tubo foi centrifugado usando um spinner hematócrito para determinar crematócrito <stro...

Watch this Scientific Journal Video about Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit at JoVE.com

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Coleta de Leite no Rato Usando Capilar Tubos e Estimativa da gordura do leite por conteúdo crematócrito

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also ...

milk-collection-rat-using-capillary-tubes-estimation-milk-fat-content

Research

JoVE Journal

Biology

12.2K visualizações.  University of Calgary. Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Vídeo: Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A Neuronal e astrocitária Ensaio Co-Cultura de Análise de Conteúdo de Alta de neurotoxicidade

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biologia

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Isolamento de fibroblastos de rato por Portal<em&gt; In situ</em&gt; Perfusão de fígado

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Imagem Funcional de gordura marrom em ratos com FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk Collection Methods for Mice and Reeves&#039; Muntjac Deer

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

Métodos de coleta de leite para Ratos e Reeves Muntjac cervos

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

Coleta e análise de<em&gt; Arabidopsis</em&gt; Floema exsudados Usando o método EDTA facilitada

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Redox Cellular Profiling Usando Microscopia de Alto Conteúdo

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

Determinação de ácidos sialic em amostras de fígado e leite de tipo selvagem e<em&gt; CMAH</em&gt; Ratinhos de eliminação.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans.&#160;The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis.

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

The present protocol describes the induction of autophagy in the Drosophila melanogaster larval fat body via nutrient depletion and analyzes changes in autophagy using transgenic fly strains.

Análise da Autofagia Induzida pela Inanição no Corpo Gorduroso de Larvas de Drosophila melanogaster

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. ...

Quantificação da infiltração gordurosa do músculo esquelético via descelularização

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Autor em destaque: Quantificação baseada em descelularização da infiltração gordurosa do músculo esquelético  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.