Este trabalho foi financiado pela American Heart Association (EIA 0740129N) e T32 NIH HL90610.

<ol>
	<li>Goodwill, A. G., Frisbee, S. J., Stapleton, P. A., James, M. E., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Chronic+Anticholesterol+Therapy+on+Development+of+Microvascular+Rarefaction+in+the+Metabolic+Syndrome.">Impact of Chronic Anticholesterol Therapy on Development of Microvascular Rarefaction in the Metabolic Syndrome.</a> Microcirculation. , 1-18 (2009).</li><li>Goodwill, A. G., James, M. E., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+vascular+thromboxane+generation+impairs+dilation+of+skeletal+muscle+arterioles+of+obese+Zucker+rats+with+reduced+oxygen+tension.">Increased vascular thromboxane generation impairs dilation of skeletal muscle arterioles of obese Zucker rats with reduced oxygen tension.</a> Am. J. Physiol. Heart Circ. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+myogenic+responsiveness+of+skeletal+muscle+arterioles+with+juvenile+growth.">Increased myogenic responsiveness of skeletal muscle arterioles with juvenile growth.</a> Am. J. Physiol. Heart Circ. Physiol. 294, 2344-2351 (2008).</li><li>Dacey, R. G., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+rat+intracerebral+arterioles:+methods,+morphology,+and+reactivity.">A study of rat intracerebral arterioles: methods, morphology, and reactivity.</a> Am. J. Physiol. Heart Circ. Physiol. 243, H598-H606 (1982).</li><li>Fredricks, K. T., Liu, Y., Lombard, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+extraparenchymal+resistance+arteries+of+rat+skeletal+muscle+to+reduce+PO2.">Response of extraparenchymal resistance arteries of rat skeletal muscle to reduce PO2.</a> Am. J. Physiol. 267, H706-H715 (1994).</li><li>Durand, M. J., Raffai, G., Weinberg, B. D., Lombard, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin-(1-7)+and+low-dose+angiotensin+II+infusion+reverse+salt-induced+endothelial+dysfunction+via+different+mechanisms+in+rat+middle+cerebral+arteries.">Angiotensin-(1-7) and low-dose angiotensin II infusion reverse salt-induced endothelial dysfunction via different mechanisms in rat middle cerebral arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 299, H1024-H1033 (2010).</li><li>LeBlanc, A. J., Cumpston, J. L., Chen, B. T., Frazer, D., Castranova, V., Nurkiewicz, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+inhalation+impairs+endothelium-dependent+vasodilation+in+subepicardial+arterioles.">Nanoparticle inhalation impairs endothelium-dependent vasodilation in subepicardial arterioles.</a> J. Toxicol. Environ. Health A. 72, 1576-1584 (2009).</li><li>Jernigan, N. L., LaMarca, B., Speed, J., Galmiche, L., Granger, J. P., Drummond, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+salt+enhances+benzamil-sensitive+component+of+myogenic+constriction+in+mesenteric+arteries.">Dietary salt enhances benzamil-sensitive component of myogenic constriction in mesenteric arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 294, H409-H420 (2008).</li><li>Stapleton, P. A., Goodwill, A. G., James, M. E., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+mechanisms+of+endothelium-dependent+dilation+in+skeletal+muscle+arterioles+with+genetic+hypercholesterolemia.">Altered mechanisms of endothelium-dependent dilation in skeletal muscle arterioles with genetic hypercholesterolemia.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R1110-R1119 (2007).</li><li>Goodwill, A. G., Stapleton, P. A., James, M. E., d'Audiffret, A. C., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+arachidonic+acid-induced+thromboxane+generation+impairs+skeletal+muscle+arteriolar+dilation+with+genetic+dyslipidemia.">Increased arachidonic acid-induced thromboxane generation impairs skeletal muscle arteriolar dilation with genetic dyslipidemia.</a> Microcirculation. 15, 621-631 (2008).</li><li>Baumbach, G. L., Hadju, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+and+composition+of+cerebral+arterioles+in+renal+and+spontaneously+hypertensive+rats.">Mechanics and composition of cerebral arterioles in renal and spontaneously hypertensive rats.</a> Hypertension. 21, 816-826 (1993).</li><li>Uchida, E., Bohr, D. F., Hoobler, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+studying+isolated+resistance+vessel+from+rabbit+mesentery+and+brain+and+their+responses+to+drugs.">A method for studying isolated resistance vessel from rabbit mesentery and brain and their responses to drugs.</a> Circ. Res. 4, 525-536 (1967).</li><li>Davis, M. J., Kuo, L., Chilian, W. M., Muller, J. M. I. s. o. l. a. t. e. d., Barker, J. H., Anderson, G. L., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+23.">Chapter 23.</a> Isolated, perfused microvessels. In: Clinically Applied Microcirculation Research. 32, 435-456 (1995).</li><li>Lombard, J. H., Liu, Y., Fredricks, K. T., Bizub, D. M., Roman, R. J., Rusch, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+and+mechanical+responses+of+rat+middle+cerebral+arterieal+to+reduced+PO2+and+prostacyclin.">Electrical and mechanical responses of rat middle cerebral arterieal to reduced PO2 and prostacyclin.</a> Am. J. Physiol. 276, H509-H516 (1994).</li></ol>

Não há conflitos de interesse declarados.

O protocolo apresentado descreve a canulação remoção de isolamento, e uma dupla de microvasos músculo esquelético, embora esta técnica geral, pode ser facilmente aplicada a maior parte dos tecidos. Para o manuscrito atual, o termo &quot;arteríola&quot; tem sido usado pelos autores para descrever um navio resistência variando entre 70-120 m de diâmetro em tom de descanso ativo, que é também um contributo importante para a regulação da resistência de perfusão de um órgão ou de tecido. <p class="jove_...

<table border="1"><tbody><tr><td> Reagentes e Equipamentos </td><td> Companhia </td><td> Comentários / Catálogo # </td></tr><tr><td> Câmara navio </td><td> Personalizado </td><td> Dave Eick (MCW) </td></tr><tr><td> Banho de água aquecida circulação </td><td> PolyScience e Haake </td><td> Haake DC 10 </td></tr><tr><td> Pipetas </td><td> Frederick Haer &amp; Co. </td><td> Tubulação capilar 2,0 milímetros OD x 1,0 mm ID (27-33-1) </td></tr><tr><td> Monitor de Pressão </td><td> Instrumentos de precisão do mundo </td><td></td></tr><tr><td> Reservatório de Água Jacketed </td><td> Personalizado </td><td></td></tr><tr><td> Fonte de luz externa </td><td> Instrumentos de precisão do mundo </td><td> Novaflex </td></tr><tr><td> Extrator pipeta </td><td> Microdados Instruments </td><td> PMP102 micropipeta Puller </td></tr><tr><td> Comple completomento de instrumentos cirúrgicos </td><td> Belas Ferramentas Ciência </td><td> Dumont </td></tr><tr><td> Ultra pinça fina </td><td> Belas Ferramentas Ciência </td><td> Inox # 5 </td></tr><tr><td> Fio de sutura de seda </td><td> Ethilon </td><td> # 10-0 ou 9-0 </td></tr><tr><td> Microscópio estéreo </td><td> Olimpo </td><td> Olympus SZ-11 </td></tr><tr><td> Analógicas Calipers Vídeo </td><td> Boeckeler </td><td> Via Controller (Via-100) </td></tr><tr><td> Câmera de alta resolução analógica </td><td> Panasonic </td><td> GP-MF 602 </td></tr><tr><td> Tanque de oxigênio </td><td> Regional </td><td> Balanço de nitrogênio de 21% e 5% CO 2 balanço de nitrogênio </td></tr><tr><td> Tubulação </td><td> Tygon </td><td></td></tr><tr><td> Escorra bomba </td><td> Cole Parmer Instrument Co. </td><td></td></tr><tr><td> Modificados PSS Rato </td><td> Ver receita abaixo </td><td></td&gt; </tr><tr><td> PSS relaxante Van Breemen de </td><td> Ver receita abaixo </td><td></td></tr></tbody></table> Tabela 1. Uma lista dos componentes principais de configuração isolado de microvasos estação apresentados nas Figuras. <table border="1"><tbody><tr><td> Receita PSS Modificado Rato </td><td> Para fazer dois litros de PSS </td><td> 20X estoque Salt (2L) </td><td> 20X depósito (2L) </td></tr><tr><td> NaCl </td><td></td><td> 278,0 g </td><td></td></tr><tr><td> KCl </td><td></td><td> 14,0 g </td><td></td></tr><tr><td> MgSO4-7H 2 ó </td><td></td><td> 11,5 g </td><td></td></tr><tr><td> CaCl2-H2O </td><td></td><td> 9,4 g </td><td></td></tr><tr> &lt;td&gt; de NaHCO3 </td><td></td><td></td><td> 80,8 g </td></tr><tr><td> EDTA </td><td></td><td></td><td> 0,4 g </td></tr><tr><td> NaH 2 PO 4 </td><td> 0,28 g </td><td></td><td></td></tr><tr><td> Glicose </td><td> 1,98 g </td><td></td><td></td></tr><tr><td> 20x estoque Sal </td><td> 100 mL </td><td></td><td></td></tr><tr><td> 20x depósito </td><td> 100 mL </td><td></td><td></td></tr><tr><td> Água destilada </td><td> 1800 mL </td><td></td><td></td></tr></tbody></table> Tabela 2. Receita para solução de sal de padrão fisiológico (PSS) utilizado nos protocolos microvascular tumoral isolados. Comentários sobre Receita: Faça 2 L de Banco de sal e 2 L de depósito. Estes cum ser refrigerado quando não estiverem sendo usados, mas agitá-los bem e, muitas vezes antes de preparar PSS. Os ingredientes adicionais são adicionados no momento da preparação de PSS finais. <table border="1"><tbody><tr><td> PSS relaxante Van Breemen de </td><td> Para fazer 2 litros de PSS </td><td> 20X estoque Salt (1L) </td><td> 20X depósito (1L) </td></tr><tr><td> NaCl </td><td></td><td> 107,4 g </td><td></td></tr><tr><td> KCl </td><td></td><td> 7,0 g </td><td></td></tr><tr><td> MgSO4-7H 2 ó </td><td></td><td> 5,76 g </td><td></td></tr><tr><td> MgCl 2-6H 2 ó </td><td></td><td> 81,32 g </td><td></td></tr><tr><td> De NaHCO3 </td><td></td><td></td><td> 40,4 g </td></tr><tr><td> EDTA </td><td></td><td></td><td> 0,2 g </td></tr><tr><td> EGTA </td><td></td><td></td><td> 15,22 </td></tr><tr><td> NaH 2 PO 4 </td><td> 0,28 g </td><td></td><td></td></tr><tr><td> Glicose </td><td> 1,98 g </td><td></td><td></td></tr><tr><td> 20x estoque Sal </td><td> 100 mL </td><td></td><td></td></tr><tr><td> 20x depósito </td><td> 100 mL </td><td></td><td></td></tr><tr><td> Água destilada </td><td> 1800 mL </td><td></td><td></td></tr></tbody></table> Tabela 3. Receita para solução Van Breemen do sal relaxante fisiológica (PSS) utilizado nos protocolos microvascular tumoral isolados sob condições de tom activa zero. Comentários sobre Receita: Faça 1 L de Banco de sal pornd L 1 de depósito. Estes podem ser refrigerado quando não estiverem sendo usados, mas agitá-los bem e, muitas vezes antes de preparar PSS. Os ingredientes adicionais são adicionados no momento da preparação de finais PSS relaxantes.

the ex vivo isolated skeletal microvessel preparation for investigation of vascular reactivity

A preparação de microvasos isolado é uma preparação ex vivo que permite a análise das diferentes contribuições de factores que o diâmetro dos vasos de controlo, e assim, a resistência de perfusão 1-5. Esta é uma preparação clássica experimental que foi, em grande medida, inicialmente descrita por Uchida et al. De 15 há várias décadas. Esta descrição inicial serviu de base para as técnicas que foi amplamente modificado e melhorado, principalmente no laboratório do Dr. Brian Duling da Universidade de Virginia 6-8, e nós apresentamos uma abordagem atual nas páginas seguintes. Esta preparação irá referir-se especificamente à arteríola gracilis num rato como o microvasos de escolha, mas a preparação de base pode facilmente ser aplicado aos recipientes isolados a partir de quase qualquer outro tecido ou órgão em várias espécies 9-13. Mecânico (ie, dimensional) alterações no microvasos isolado pode ser facilmente avaliadaem resposta a uma ampla gama de fisiológico embora ex (por exemplo, pressão, hipóxia intravascular, ou cisalhamento) ou desafios farmacológicos, e pode fornecer uma visão em elementos mecanicistas compreendendo respostas integrados em um intacto, in vivo de tecidos,. O significado deste método é que ele permite uma manipulação fácil das influências sobre a regulação integrado de diâmetro microvascular, enquanto também permite que para o controlo de muitas das contribuições a partir de outras fontes, incluindo a pressão intravascular (miogênica), inervação autonômica, hemodinâmica ( por exemplo, estresse, cisalhamento), disfunção endotelial, estímulos dependentes ou independentes, hormonais e influências parenquimatosas, para fornecer uma lista parcial. Sob condições experimentais adequadas e com metas apropriadas, este pode servir como uma vantagem sobre in vivo ou in situ de tecidos de órgãos / preparações, que não permitem prontamente para o controlo fácil do mais amplos variáveis ​​sistémicas. A malimitação jor desta preparação é essencialmente a conseqüência de suas forças. Por definição, o comportamento destes navios está sendo estudada sob condições em que muitos dos contribuintes mais significativos para a regulação da resistência vascular tenham sido removidos, incluindo neural, humoral, metabólicas, etc Como tal, o investigador deve ser advertido para evitar o excesso de interpretação e extrapolação dos dados que são coletados utilizando esta preparação. A outra área significativa de preocupação em relação a esta preparação é que ele pode ser muito fácil de danificar os componentes celulares, tais como o revestimento endotelial ou o músculo liso vascular, tal fonte que variável de erro pode ser introduzido. É fortemente recomendada que o investigador indivíduo utilizar medições adequadas para assegurar a qualidade da preparação, tanto no início da experiência e periodicamente durante todo o curso de um protocolo.

Watch this Scientific Journal Video about The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity at JoVE.com

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

Um<em&gt; Ex vivo</emPreparação&gt; é descrita para o isolamento das maiores grácil arteríolas musculares de resistência para interrogatório de ambas as respostas vasculares aos estímulos vasoativos e na avaliação de propriedades estruturais básicas via mecânica da parede passivos.

O Ex vivo Preparação microvasos isolada esquelético de Investigação da reatividade vascular

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

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JoVE Journal

Biology

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

10.6K visualizações.  West Virginia University. Um Ex vivo é descrita para o isolamento das maiores grácil arteríolas musculares de resistência para interrogatório de ambas as respostas vasculares aos estímulos vasoativos e na avaliação de propriedades estruturais básicas via mecânica da parede passivos.

Vídeo: The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

Medição in vivo Micro-circulação no músculo esquelético por Intra-vital Microscopia

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Biologia

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Método para a Cultura de Embriões início pintainho ex vivo (Nova Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentivirus mediada Durante Knockdown Ex Vivo Eritropoese de Células-Tronco Hematopoéticas

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Um Sistema de<em&gt; Ex vivo</em&gt; A cultura de Pâncreas embrionárias

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

Avaliação ex vivo de Fatigabilidade Contratilidade e Alternans em músculos esqueléticos isolados

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Método de Cultura para o Rato coração embrionário

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

Quantificar Único microvasos Permeabilidade em perfusão isolada-Sangue Rat Lung Preparação

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Medidas de contratilidade no isolado músculos papilares de Investigação de Cardiac inotropia em Ratos

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

A proteína c-Fos Detecção imuno: Uma ferramenta útil como um marcador de vias centrais envolvidos em respostas fisiológicas específicas<em&gt; In Vivo</em&gt; e<em&gt; Ex Vivo</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.