O protocolo de todo após o isolamento das células leva cerca de 2 horas. 1. Microsurgical Preparação <ol><li> Realizar o isolamento de células estaminais da medula óssea do dador e marcação fluorescente da subpopulação isolado de acordo com protocolos padronizados 9,12 aceitam as células repousar durante o tempo que o animal é realizada a operação. Para marcação fluorescente recomendamos CFDA (Carboxifluoresceína diacetate éster succinimidyl). </li><li> Anestesiar um rato macho pesando 20-25 g, com cetamina (75 mg / kg) e xilazina (2,5 mg / kg). Durante os procedimentos cirúrgicos e protocolos experimentais, anestesia deve ser mantida por suplementos (10% de injecção inicial, ip), conforme necessário (normalmente a cada 30-60 min). </li><li> Raspe suavemente a face anterior do escroto direito, bem como a coxa e virilha direita. Colete todos perdem o cabelo com um cotonete umedecido. Coloque o mouse lado ventral em um palco de visualização plexiglass e fixar a taxat usando cortina elástica. A fase é feito à medida, que consiste em uma placa de base e uma segunda placa da extremidade caudal da placa de base para a elevação de ambas as pernas e o escroto. Coloque a fase de uma almofada de aquecimento para manter a temperatura corporal a 37 ° C. Após a fixação no palco, coloque o animal sob um microscópio de operação e executar as seguintes etapas da operação por meio de 10 - a ampliação de 16 vezes. </li><li> Faça a incisão inicial usando uma tesoura de pele na coxa direita, logo anterior dos vasos femorais do joelho até a virilha. </li><li> Identificar a artéria femoral e segui-lo proximamente, mobilizando a artéria de tecidos moles circundantes. Identificar e cortou um grande ramo para o testículo esquerdo usando termocautério. Posteriormente colocar uma sutura de estadia superficialmente no testículo esquerdo. Tração suave vai facilitar ainda mais a mobilização proximal da artéria femoral. </li><li> Após a conclusão da mobilização proximal, identificar e cortar um grande ramo executando medially na coxa por termocautério. A partir de então ligar a artéria femoral, na parte distal da coxa direita. Tração suave na ligadura vai ajudar ainda mais a preparação. </li><li> Rode-se agora a atenção para a artéria femoral proximal e novamente colocar uma sutura em torno da artéria como proximalmente possível. Prepare um nó, mas não amarrá-lo para baixo ainda. Aplicar tração suave para a sutura e criar um kinking arterial. </li><li> Depois de estabelecer a dobras, inciso artéria é cuidadosamente usando uma microtesoura. Tome especial cuidado para não danificar a veia femoral. </li><li> Inserir um microcateter preparado (diâmetro interno 0,28 milímetro) através da incisão. O cateter deve ser deaired (cloreto de sódio a 0,9%, NaCl) e ligado a um inserção antes seringa de 1 ml. </li><li> Após a inserção com êxito do cateter, cuidadosamente solte o enroscamento, a fim de facilitar a passagem do cateter. Fluxo arterial deve ser imediatamente visível dentro do cateter. Delicadamente, avançar o cateter alguns milímetross além do kinking anterior. A partir de então, amarrar a ligadura preparado para fixação do cateter. </li><li> Lavar cuidadosamente o cateter (0,9% NaCl) e aspirado para confirmar a posição correta. Utilizar um pedaço de fita adesiva para prender ainda mais o cateter para a fase de operação. </li><li> Após a fixação adequada, colocar o cateter de lado para as seguintes etapas de preparação do músculo cremaster. Aplicar a lavagem regular e aspiração do cateter cada 5-10 min, a fim de impedir a formação de coágulos. </li><li> Vire a atenção agora para o escroto direito e fazer uma incisão, cortando a pele e fáscia acima do aspecto ventral do escroto direito. Tome cuidado para não tocar no tecido subjacente com quaisquer instrumentos. </li><li> Estender a incisão até a dobra inguinal e caudal para a extremidade distal do escroto. </li><li> Tecidos cuidadosamente separado subjacente suave e conjuntivo acima do saco cremaster, sem tocá-lo. Identificar a extremidade distal do cremaster, e colocar uma sutura de estadia a slightly estender o músculo. Ressecar agora tecido mole remanescente sob o saco muscular, puxando suavemente o músculo caudal e anteriormente. </li><li> Após a separação completa do tecido conjuntivo, abra o saco cremaster em um ponto na borda cranial com uma tesoura. Estender a incisão desta abertura para baixo a extremidade distal do músculo. </li><li> Selar a pequena embarcação que liga o testículo eo cremaster usando termocautério, depois cortar as conexões restantes entre o músculo eo testículo com uma tesoura. </li><li> Deixe de lado o testículo, fora do campo de microscopia. </li><li> O cremaster abriu agora está na horizontal no palco. Coloque quatro 6-0 estadia prolene nas bordas do tecido e corrigi-los com caimento elástico, a fim de espalhar o tecido do músculo cremaster. De agora em diante continuamente humedecer o tecido com solução salina tamponada com fosfato (PBS) utilizando uma seringa de 1 ml (Figura 1). </li><li> Permitir agora a preparação para descansar por 15 min. Posteriormente injetar0,05-0,1 ml de 1% de Rodamina-dextrano através da artéria femoral esquerda para o realce do contraste da microvasculatura. Se algum quimiocinas ou agentes inflamatórios estão previstas para aplicação, você deve agora topicamente umedecer o músculo com o respectivo reagente usando uma seringa de 1 ml. No caso de uma experiência de controlo, continuar humedecendo com PBS. </li></ol> 2. Microscopia intravital <ol><li> Manter o rato fixado na fase de operação e mover-se sob um microscópio de fluorescência modificado para epi-iluminação e ligada a uma câmara CCD (charge-coupled device) da câmara de vídeo. Tome especial cuidado para não deslocar o cateter de artéria femoral. </li><li> Realizar experimentos usando uma objetiva de imersão em água. </li><li> Depois de ajustar o objetivo de visualização suficiente da microcirculação cremaster e antes da injeção das células, estocasticamente definir seis vênulas pós-capilares, para posterior análise da aderência de células-tronco firme. </li><li> Administrar as células estaminais marcado com fluorescentes,Dissolveu-se em 200 ul de PBS, a 0,4 x 10 6 culas por injecção, com um total de cinco injecções consecutivas, utilizando o cateter da artéria femoral e uma seringa de 1 ml. Agitar a amostra de células antes da primeira injecção. </li><li> Células estaminais da ficha de rolamento imediatamente após a injecção das células, definindo, assim, &quot;rolar&quot; como uma redução de mais de 50% da velocidade da célula ao longo do revestimento endotelial em combinação com típico celular &quot;vara e solte&quot; movimentos. Realizar contagem de células-tronco rolantes e todos as células-tronco que passam a uma distância pré-definida venular durante 1 min de observação após cada injeção única. A quantidade de rolamento de células estaminais é expressa como percentagem de todas as células que passam. Não conte células exibindo fenômenos amarrar breves aleatórios como células de rolamento. </li><li> Após a última injecção de células determinar o número de células estaminais, firmemente aderentes por contagem e expressar a quantidade em relação à superfície endotelial de th calculadoe seis vênulas predefinidos (diâmetro x comprimento x π) como células aderentes / mm 2. Adesão firme é considerado quando há movimento celular é detectável por 30 seg. </li><li> Após a conclusão da microscopia intravital remova cuidadosamente o músculo cremaster e armazená-lo em correspondência para posterior análise (imunohistoquímica e biologia molecular). Sacrificar o animal por deslocamento cervical. A análise de microscopia intravital gravações para a quantificação de células estaminais de rolamento e adesão celular endotelial firmes, bem como a hemodinâmica microvasculares, tais como a velocidade do sangue de células vermelhas (mm / s), o comprimento do vaso (mm), o diâmetro dos vasos (mM), a taxa de partes de parede (seg -1); deve ser realizada off-line por um investigador cego para o respectivo tratamento do tecido experimental utilizando um software de processamento de imagem. </li></ol>

<ol>
	<li>Madlambayan, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=marrow+stem+and+progenitor+cell+contribution+to+neovasculogenesis+is+dependent+on+model+system+with+SDF-1+as+a+permissive+trigger.">marrow stem and progenitor cell contribution to neovasculogenesis is dependent on model system with SDF-1 as a permissive trigger.</a> Blood. 114, 4310-4319 (2009).</li><li>Lapidot, T., Dar, A., Kollet, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+stem+cells+find+their+way+home?.">How do stem cells find their way home?.</a> Blood. 106, 1901-1910 (2005).</li><li>Lehr, H. A., Vollmar, B., Vajkoczy, P., 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=Intravital+fluorescence+microscopy+for+the+study+of+leukocyte+interaction+with+platelets+and+endothelial+cells.">Intravital fluorescence microscopy for the study of leukocyte interaction with platelets and endothelial cells.</a> Methods Enzymol. 300, 462-481 (1999).</li><li>Gavins, F. N., Chatterjee, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+for+the+study+of+mouse+microcirculation+in+anti-inflammatory+drug+research:+focus+on+the+mesentery+and+cremaster+preparations.">Intravital microscopy for the study of mouse microcirculation in anti-inflammatory drug research: focus on the mesentery and cremaster preparations.</a> J. Pharmacol. Toxicol. Methods. 49, 1-14 (2004).</li><li>Leister, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peritoneal+cavity+chamber+for+intravital+microscopy+of+the+liver+under+conditions+of+pneumoperitoneum.">A peritoneal cavity chamber for intravital microscopy of the liver under conditions of pneumoperitoneum.</a> Surg. Endosc. 17, 939-942 (2003).</li><li>Sriramarao, P., Anderson, W., Wolitzky, B. A., Broide, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+bone+marrow-derived+mast+cells+roll+on+P-selectin+under+conditions+of+flow+in+vivo.">Mouse bone marrow-derived mast cells roll on P-selectin under conditions of flow in vivo.</a> Lab Invest. 74, 634-643 (1996).</li><li>Baez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open+cremaster+muscle+preparation+for+the+study+of+blood+vessels+by+in+vivo+microscopy.">An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.</a> Microvasc. Res. 5, 384-394 (1973).</li><li>Bagher, P., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+cremaster+muscle+preparation+for+intravital+imaging+of+the+microcirculation.">The mouse cremaster muscle preparation for intravital imaging of the microcirculation.</a> J. Vis. Exp. , e2874 (2011).</li><li>Kaminski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+NOS+is+required+for+SDF-1alpha/CXCR4-mediated+peripheral+endothelial+adhesion+of+c-kit++bone+marrow+stem+cells.">Endothelial NOS is required for SDF-1alpha/CXCR4-mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells.</a> Lab Invest. 88, 58-69 (2008).</li><li>Furlani, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HMGB-1+induces+c-kit++cell+microvascular+rolling+and+adhesion+via+both+toll-like+receptor-2+and+toll-like+receptor-4+of+endothelial+cells.">HMGB-1 induces c-kit+ cell microvascular rolling and adhesion via both toll-like receptor-2 and toll-like receptor-4 of endothelial cells.</a> J. Cell Mol. Med. 16, 1094-1105 (2012).</li><li>Furlani, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+intravascular+administration+of+mesenchymal+stem+cells+safe?+Mesenchymal+stem+cells+and+intravital+microscopy.">Is the intravascular administration of mesenchymal stem cells safe? Mesenchymal stem cells and intravital microscopy.</a> Microvasc. Res. 77, 370-376 (2009).</li><li>Donndorf, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysing+migratory+properties+of+human+CD+133(+)+stem+cells+in+vivo+after+intraoperative+sternal+bone+marrow+isolation.">Analysing migratory properties of human CD 133(+) stem cells in vivo after intraoperative sternal bone marrow isolation.</a> Cell Transplant. , (2012).</li></ol>

Todos os experimentos com animais, realizados para este manuscrito ter sido aprovado pelo comitê de cuidados com os animais e uso local. Os autores declaram que não têm interesses financeiros concorrentes.

<html> Microscopia de fluorescência intravital permite a visualização direta e análise quantitativa de interações célula-tronco do endotélio. O modelo do músculo cremaster é particularmente útil, já que a exposição de microcirurgia e técnicas de visualização intravital têm sido bem estabelecidos durante a investigação sobre as interacções de leucócitos-endotélio e os vários componentes químicos podem ser selectivamente aplicado ao tecido alvo através de uma simples técnica de superfusão. Devi...

O objetivo do presente artigo é descrever a técnica de microscopia intravital (IVM), aplicada sobre a microcirculação cremaster rato para observação direta e análise de medula óssea migração de células-tronco periférica. Regeneração de tecidos e órgãos O conceito atual de células-tronco com base envolve o homing de medula óssea células-tronco derivadas do tecido lesado 1. Um passo crucial para a migração de células-tronco de sucesso inclui tronco interação célula com o endotélio local dentro do órgão lesionado seguido de migração transendotelial e, eventualmente, o enxerto de órgãos 2. Análise intravital destas células-tronco - as interações de células endoteliais formam um parâmetro ideal para a quantificação de uma resposta regenerativa com células-tronco com base em diferentes contextos fisiopatológicos in vivo. Além disso, parece concebível, que, no futuro, a análise intravital da capacidade migratória das células estaminais p específicoopulations dentro de ambientes microcirculação padronizadas, pode ser aplicado antes avanço dessas populações para mais testes clínicos. Microscopia intravital foi inicialmente desenvolvido para a observação e quantificação da interacção das células de leucócitos-células endoteliais in vivo, no campo da pesquisa inflamatória 3. As primeiras gravações de sucesso de estudos de microscopia intravital foram relatados por Cohnheim já no século 19, estudando línguas e mesentério de rã sob um microscópio de luz 4. Desde a sua primeira utilização IVM sofreu um desenvolvimento técnico enorme e hoje certos componentes essenciais formar os pré-requisitos para a realização de MIV quantitativa: (i) as preparações de tecidos que permitam o acesso óptico, (ii) as sondas moleculares que podem ser detectados por um microscópio, (iii ) um microscópio ligado a um sistema de detecção e (iv) a partir de sistemas de análise de computador que é capaz de extrair os parâmetros de interesse a partir dos dados de imagemset 4. Uma grande variedade de preparações de tecido tenha sido introduzida para estudos IVM incluindo os mesentério e no fígado do rato e na ratazana 5, as câmaras de dobras cutâneas dorsal de rato e hamster 6, a orelha de coelho e a bolsa jugal do hamster, para citar alguns. No entanto, a seguir vamos nos concentrar no músculo cremaster mouse, o que representa um tecido ideal para observações intravital, como são possíveis preparação e visualização por um procedimento cirúrgico bem padronizado, e em geral não ocorrem problemas de artefatos de movimento. A preparação do músculo cremaster aberto foi realizada pela primeira vez em 1970 por Baez e colegas 7. Originalmente descrito para ratos, tem sido adotado com sucesso também para o mouse 8. Após estudos anteriores haviam se concentrado principalmente nas interações leucócitos com a parede do vaso, o nosso próprio grupo introduziu recentemente a preparação do músculo cremaster mouse como um valuable ferramenta para a visualização direta e análise quantitativa de interações célula-tronco do endotélio dentro de um microambiente definida 9. Vários subpopulações de células estaminais têm sido estudada utilizando este modelo, incluindo murino células estaminais da medula óssea 133 + c-kit + de medula óssea em células estaminais e células estaminais mesenquimais, assim como humana CD 10-12. A seguir ao isolamento de células de medula óssea do dador e marcação fluorescente para visualização, as células estaminais são aplicadas selectivamente na microcirculação cremaster utilizando uma injecção arterial através da artéria femoral, evitando-se assim qualquer aprisionamento de células dentro de órgãos remotos. Além disso, o modelo do músculo cremaster é particularmente útil uma vez que várias quimiocinas potencialmente medeiam a migração de células estaminais local dentro das respectivas configurações, pode ser topicamente aplicada ao tecido alvo através de uma simples técnica de superfusão.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Carboxy-fluorescein diacetate succinimidylester (CFDA)</td>
			<td>Invitrogen, Carlsbad, CA, USA</td>
			<td>C1157</td>
		</tr>
		<tr>
			<td>Rhodamine 6 G (1%)</td>
			<td>Sigma-Aldrich, Munich, Germany</td>
			<td>83697</td>
		</tr>
		<tr>
			<td>Ketamin (Ketanest)</td>
			<td>Pfizer, Berlin, Germany</td>
			<td>not available</td>
		</tr>
		<tr>
			<td>Xylacin (Rompun)</td>
			<td>Bayer, Leverkusen, Germany</td>
			<td>not available</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate - buffered saline (PBS)</td>
			<td>PAN Biotech, Aidenbach, Germany</td>
			<td>P04-36500</td>
		</tr>
	</tbody>
</table>

intravital microscopy of the microcirculation in the mouse cremaster muscle for the analysis of peripheral stem cell migration

Na era de protocolos de aplicação intravascular de células no contexto da terapia celular regenerativa, os mecanismos subjacentes à migração de células-tronco para nonmarrow tecido não foram completamente esclarecidas. Descrevemos aqui a técnica de microscopia intravital aplicada a microcirculação cremaster rato para a análise de medula óssea a migração de células estaminais periféricas in vivo. Microscopia intravital do cremaster M. tenha sido previamente introduzido no campo da pesquisa inflamatória por observação directa de interacção de leucócitos com o endotélio vascular. Desde tronco periférica suficiente e migração de células progenitoras inclui passos iniciais semelhantes de rolando e adesão firme ao endotélio forro é concebível aplicar o modelo cremaster M. para a observação e quantificação da interação de células-tronco administrados intravasculary com o endotélio. Como vários componentes químicos pode ser aplicada selectivamente ao alvo tquestão por meio de técnicas de superfusão simples, é possível estabelecer um requisito essencial do microambiente, para o recrutamento de células-tronco inicial para ocorrer em um organismo vivo fora da medula óssea.

Em geral, a interação de injetado diretamente as células-tronco ou progenitoras com o endotélio vascular dentro da microcirculação músculo cremaster é um evento raro e ocorre exclusivamente em vênulas pós-capilares (diâmetro: 30-80 mm). Devido à fluorescência rotulagem de rolamento e as células-tronco, firmemente aderentes pode ser quantificada claramente na separação de leucócitos circulantes endógenos nas vénulas observados (Figura 2). As condições da ...

Watch this Scientific Journal Video about Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration at JoVE.com

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

Microscopia intravital do cremaster microcirculação rato M. oferece um único e bem padronizados modelo in vivo para a análise de medula óssea a migração de células estaminais periféricas.

Microscopia intravital da Microcirculação no músculo Rato Cremaster de Análise de Migração Stem Peripheral celular

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

intravital-microscopy-microcirculation-mouse-cremaster-muscle-for

Research

JoVE Journal

Biology

15.1K visualizações.  University Rostock.  Microscopia intravital do cremaster microcirculação rato M. oferece um único e bem padronizados modelo in vivo para a análise de medula óssea a migração de células estaminais periféricas. 

Vídeo: Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

O Mouse Preparação Muscle Cremaster for Imaging intravital da Microcirculação

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Biologia

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission1 by delivering neurotransmitters and neuropeptides onto neurons reproducibly2. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation3 has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.
A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain6. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be &tilde; 1 &mu;m or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.
Fabrication of micropipettes is facilitated with sophisticated electronic pullers8. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). 
The present protocol demonstrates microiontophoresis of acetylcholine (ACh+ Cl-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40BAC-GCaMP2 transgenic mice9 enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

Microiontophoresis e Micromanipulação para imagens de fluorescência intravital da Microcirculação

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental  preparation. ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Geração de camundongos derivadas de células-tronco pluripotentes induzidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Isolamento de Blood-derivado do navio Multipotentes Precursores do músculo-esquelético humano

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Identificação dos fatores chave que regula a auto-renovação e diferenciação em células precursoras hematopoéticas EML por Análise RNA-seqüenciamento

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

Visualização e quantificação da Camada de livre-Cell nas arteríolas do músculo Rat Cremaster

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Análise do Gap Junction-dependente Transferência de miARN com 3D-FRAP Microscopia

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Intravital Subcellular Microscopy of the Mammary Gland

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676&#160;or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs &#8805; 0.7 &#181;m. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 &#181;m or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

<meta charset="utf8"></head><body>Microscopia Subcelular Intravital da Glândula Mamária

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

Microscopia Subcelular Intravital da Glândula Mamária

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory ...

Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment failure is mainly due to the invasiveness of GBM cells, which speaks for the need for a better understanding of GBM motile properties. To investigate the molecular mechanism supporting GBM invasion, new physiological models enabling in-depth characterization of protein dynamics during invasion are required. These observations would pave the way to the discovery of novel targets to block tumor infiltration and improve patient outcomes. This paper reports how an orthotopic xenograft of GBM cells in the zebrafish brain permits subcellular intravital live imaging. Focusing on microtubules (MTs), we describe a procedure for MT labeling in GBM cells, microinjecting GBM cells in the transparent brain of 3 days post fertilization (dpf) zebrafish larvae, intravital imaging of MTs in the disseminating xenografts, altering MT dynamics to assess their role during GBM invasion, and analyzing the acquired data.

We report a technique permitting live imaging of microtubule dynamics in glioblastoma (GBM) cells invading a vertebrate brain tissue. Coupling the orthotopic injection of fluorescently tagged GBM cells into a transparent zebrafish brain with high-resolution intravital imaging allows the measurement of cytoskeleton dynamics during in situ cancer invasion.

Imagem ao vivo da dinâmica dos microtúbulos em células de glioblastoma invadindo o cérebro do peixe-zebra

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment ...