Agradecemos a Hsia Ru-ching PhD, do Centro de Microscopia Eletrônica da Faculdade de Medicina da Universidade de Maryland, pelo seu suporte técnico no processamento das amostras de microscopia eletrônica de varredura.

Declaração de Ética: os protocolos para manejo de animais foram aprovados pelo Comitê Institucional de Cuidados e Uso Animal (IACUC) da Universidade de Maryland (número de protocolo 0416009) e conduzido de acordo com os padrões AAALAC-International. 1. Procedimento cirúrgico <ol><li> Técnica anestésica <ol><li> Esterilize todos os instrumentos utilizados na cirurgia de sobrevivência com esterilização a vapor a 121 ° C durante 30 min. </li><li> Induzamos a anestesia através de um tanque de indução com 100% de O2 e 2.5% de isoflurano administrado por vaporizador de precisão. Após a indução, interrompa o isoflurano e lave a câmara com O 2 . Manter anestesia com 1,5-2% de isoflurano através da máscara facial e 1 L / min O 2 por inalação. </li><li> Anexe a câmara de indução e a máscara facial a um agente de tratamento de carvão para adsorção de gás residual para proteger o pessoal. Assegurar um plano anestésico adequado por demonstraçãoQue não há resposta a estímulos nocivos (pitada de dedo do pé). </li><li> Crie um campo operacional consistindo em uma bandeja cirúrgica com almofada isotérmica para fornecer suporte térmico durante a cirurgia. Uma almofada isotérmica adicional proporcionará suporte térmico aos animais em sua gaiola de recuperação. </li></ol></li><li> Preparação Animal <ol><li> Realize a seguinte investigação sobre ratinhos C57BL / 6 machos de 10 a 12 semanas de idade. </li><li> Remova o cabelo na superfície abdominal ventral do animal do esterno para a região inguinal com um agente depilatório ou uma cortadora elétrica com uma lâmina número 40. <ol><li> Em caso de agente depilatório, aplique este composto na área cirúrgica por 2-3 min e depois remova-o com algodão. Utilizamos um composto comercialmente disponível de hidróxido de cálcio e hidróxido de sódio. </li></ol></li><li> Prepare a área sobre o ombro com 70% de álcool e injete subcutaneamente carprofen (5 mg / kg) com uma agulha de calibre 25 ou menor. esteO tratamento proporcionará analgesia pós-operatória para o animal. </li><li> Transfira o animal para o campo cirúrgico e posicione-se em recumbência dorsal. </li><li> Prepare o local cirúrgico esfregando 8-12% providone-iodo com um aplicador de algodão limpo ou uma gaze de algodão. Em seguida, enxágue a pele duas vezes com 70% de álcool. </li><li> Coloque o lubrificante ocular em ambos os olhos para reduzir a incidência de dessecação da córnea. Cubra o local cirúrgico com uma cobertura estéril. </li></ol></li><li> Técnica Operativa <ol><li> Faça uma mediana da incisão da laparotomia abdominal com aproximadamente 2-2,5 cm de comprimento com um bisturi começando imediatamente caudal no processo de xifóide e estendendo-se para a pélvis. </li><li> Mobilize o intestino delgado e o duodeno e reflita lateralmente à direita. Enrole uma tira de algodão estéril gasteado e embebido com solução salina estéril para injeção para permitir a embalagem das vísceras para melhorar a exposição. </li><li> Com o intestino delgado mobilizado para o lado direito doAbdômen, exponha o retroperitoneum e expõe a aorta abdominal da veia renal esquerda à bifurcação aórtica ( Figura 1 ). </li><li> Com um aplicador de ponta de algodão estéril, entregue 30 tritões consecutivos, cada cinco segundos de duração. </li><li> Remova a embalagem e permita que as vísceras voltem à sua posição nativa. </li><li> Faixa da fáscia com uma sutura de monofilamento absorvente 4-0 (polydioxanona). A pele é fechada com uma sutura de monofilamento não absorvível de 6-0 (nylon). </li></ol></li><li> Assistência de recuperação e pós-procedimento <ol><li> Após o procedimento, coloque o animal em uma gaiola de recuperação com roupa de cama limpa em uma almofada isotérmica para continuar o suporte térmico até que o animal possa se mexer normalmente. Não deixe o animal desacompanhado até demonstrar a capacidade de manter a decúbito esternal. </li><li> Monitore o animal a cada hora pelas primeiras 4 h após a cirurgia. Uma vez que o animal está ambulante normalmente retorna i<html> T para a sala de criação atribuída. O animal não será alojado na companhia de outro animal até que ele tenha se recuperado completamente. </li><li> Monitore o animal duas vezes ao dia durante as primeiras 72 h após a cirurgia e pelo menos 3 vezes por semana a partir de então. O monitoramento inclui a pesagem do animal três vezes por semana. </li><li> Administrar carpofen (5 mg / kg) por via subcutânea duas vezes ao dia durante as primeiras 72 h após a cirurgia. </li></ol></li></ol> 2. Procurement of Tissue <ol><li> Método de Eutanásia <ol><li> Eutanize os animais em pontos de tempo predeterminados. </li><li> Induzamos a anestesia através de um tanque de indução com 100% de O2 e 2.5% de isoflurano administrado por vaporizador de precisão. <ol><li> Para estudar a integridade do endotélio, administre o corante azul Evans para o animal. NOTA: Evans Blue é um corante azo com carga negativa com alta afinidade de ligação para a albumina e só pode manchar os vasos sanguíneos na ausência de um endotélio intactoS = &quot;xref&quot;&gt; 6. </li><li> Para estudos de integridade endotelial, no momento da eutanásia, perfusar os animais com 5 mL de 0,3% de corante Evans Blue seguido por 5 mL de PBS a pressão fisiológica durante 5 min. O animal é mantido em um plano cirúrgico de anestesia durante o procedimento de perfusão. </li></ol></li><li> Depois de atingir um plano anestésico profundo, abra o baú com esternotomia. Faça uma laceração no átrio direito para permitir a drenagem do sangue do animal e o acesso ao ventrículo esquerdo seja acessado com uma agulha 21 G e injete solução salina tamponada com fosfato (PBS) até que o efluente do átrio direito seja limpo. </li><li> Após a perfusão com PBS, entre no abdômen através de uma incisão na linha média. </li><li> Mais uma vez, mobilize o intestino delgado para o lado direito do abdômen, expondo a aorta da infra-renal, dissecando a aorta dos tecidos adjacentes e extrale-a da veia renal esquerda para a bifurcação aórtica. </li><li> Armazene a aorta excisada em um paraformald a 4%Solução de eído até o processamento do tecido. <ol><li> Para estudos de integridade endotelial, abra a aorta longitudinalmente e fixe-a para uma folha de cera expondo a totalidade da superfície luminal 6 . Realizar uma avaliação qualitativa da integridade endotelial pelo grau de coloração com corante Evans Blue 6 . </li><li> Para outros ensaios histológicos, separe a aorta transversalmente e incorpore-a em compostos óptimos de temperatura de corte (OCT) conforme ditado pelo método histológico a ser utilizado 7 . </li></ol></li></ol></li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+aortic+angioplasty+for+genomic+studies+of+neointimal+hyperplasia.">A mouse model of aortic angioplasty for genomic studies of neointimal hyperplasia.</a> J Vasc Res. 42 (4), 292-300 (2005).</li><li>Li, 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=Vascular+smooth+muscle+cells+of+recipient+origin+mediate+intimal+expansion+after+aortic+allotransplantation+in+mice.">Vascular smooth muscle cells of recipient origin mediate intimal expansion after aortic allotransplantation in mice.</a> Am J Path. 158 (6), 1943-1947 (2001).</li><li>Radu, M., Chernoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+assay+to+test+blood+vessel+permeability.">An in vivo assay to test blood vessel permeability.</a> J Vis Exp. (73), e50062 (2013).</li><li>Turbett, G. R., Sellner, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+optimal+cutting+temperature+compound+can+inhibit+amplification+by+polymerase+chain+reaction.">The use of optimal cutting temperature compound can inhibit amplification by polymerase chain reaction.</a> Diagn Mol Pathol. 6 (5), 298-303 (1997).</li><li>Puchtler, H., Waldrop, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+mechanism+of+Verhoeff's+elastica+stain:+a+convenient+stain+for+myelin+sheaths.">On the mechanism of Verhoeff's elastica stain: a convenient stain for myelin sheaths.</a> Histochem. 62 (3), 233-247 (1979).</li><li>Salic, A., Mitchison, T. 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Este trabalho foi financiado pelo Prêmio de Desenvolvimento de Carreira do Departamento de Assistentes de Veteranos (1IK2BX001553-01) (TSM) e a bolsa de estudos EJ Wylie da Cura Vascular (TSM).

Caracterizamos os efeitos de um modelo de lesão aórtica murina que resulta em hiperplasia mediana e disfunção da barreira endotelial. O destacamento parcial da CE ao longo da íntima da aorta acompanhou a perda do contato celular e o aumento das protrusões celulares. Correspondentemente, a função de barreira endotelial foi significativamente prejudicada, o que estimulou as vias de sinalização sensíveis aos mitógenos, levando à proliferação de VSMCs e engrossamento da parede vascular. Os pontos fortes deste...

Restenosis Após procedimentos cardiovasculares (cirurgia de bypass, angioplastia ou stent) é um problema significativo que reduz a durabilidade desses procedimentos. Todos os procedimentos de revascularização estão repletos de reestenose. As estratégias atuais para prevenir a reestenose (stents de libertação de fármaco e balões revestidos de drogas) inibem a célula muscular muscular lisa vascular (VSMC) e a proliferação de células endoteliais (EC). Conseqüentemente, essas intervenções impedem a reestenose mediada por VSMC, mas também impedem a regeneração do endotélio. Sem um endotélio intacto, os pacientes devem estar em potentes agentes antiplaquetários para diminuir o risco de trombose in situ no risco de complicações hemorrágicas. Uma terapia ideal inibiria a proliferação de VSMC enquanto promovia a regeneração do endotélio. Assim, há uma necessidade de estudar simultaneamente a proliferação de VSMC e a função de barreira endotelial i n vivo . Atualmente, existemModelos de reestenose de mouse de al mouse 1 . Estes modelos incluem ligadura de carótida e lesão de fio arterial femoral 2 . Os modelos aórticos incluem posicionamento do stent 3 , lesão do balão 4 e aloenxerto aórtico 5 . Todos os modelos atuais são limitados. A ligação da carótida gera uma lesão neointimal mediada pelo fluxo e não possui lesão endotelial. Além disso, as artérias carotídeas e femorais têm muitas vezes menos camadas celulares que os vasos humanos, limitando seu valor de translação. A aorta do mouse, com aproximadamente 1,3 mm de diâmetro, é o único vaso que se aproxima de uma artéria humana (coronária) clinicamente relevante (3). Apesar do potencial de tradução dos modelos aórticos murinos da doença, os modelos atuais têm limitações. Esses modelos exigem habilidades micro-cirúrgicas avançadas e equipamentos especializados, como balões de angioplastia e stents. Aqui, apresentamosUma técnica nova e reprodutível para induzir simultaneamente a proliferação de VSMC e interromper a função de barreira endotelial.

<table border="0" cellpadding="0" cellspacing="0" width="920">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Ocular lubricant</td>
			<td style="width:160px;">Dechra</td>
			<td style="width:163px;">17033-211-38</td>
			<td style="width:247px;">Pharmaceutical agents</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Isoflurane</td>
			<td>VetOne</td>
			<td>502017</td>
			<td>Pharmaceutical agents</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carprofen</td>
			<td>Zoetis</td>
			<td>26357</td>
			<td>Pharmaceutical agents</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Precision vaporizer</td>
			<td>Summit Medical</td>
			<td>10675</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Charcoal scavenger</td>
			<td>Bickford Inc.</td>
			<td>80120</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Isothermal pad</td>
			<td>Harvard Apparatus</td>
			<td>50-7053-R</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Sterile cotton-tipped applicator</td>
			<td>Fisher Scientific</td>
			<td>23-400-124</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">4-0 absorbable monofilament suture&#160;</td>
			<td>Ethicon, Inc</td>
			<td>J310</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">5-0 non-absorbable monofilament suture</td>
			<td>Ethicon,Inc</td>
			<td>1666</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">21-gauge x 1 inch needle</td>
			<td>BD Biosciences</td>
			<td>305165</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">25-gauge x 1 inch&#160; needle</td>
			<td>BD Biosciences</td>
			<td>305125</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dry sterilizer</td>
			<td>Cellpoint&#160;</td>
			<td>7770</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-09</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adson forceps</td>
			<td>Fine Science Tools</td>
			<td>11006-12</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Dumont #5 fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Vannas Spring Scissors 3mm cutting edge</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Needle driver</td>
			<td>Fine Science Tools</td>
			<td>91201-13</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel handle #4</td>
			<td>Fine Science Tools</td>
			<td>10004-13</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel blades #10</td>
			<td>Fine Science Tools</td>
			<td>10010-00</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS&#160;</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td>Reagents for tissue processing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Evans Blue</td>
			<td>Sigma-Aldrich</td>
			<td>E2129</td>
			<td>Reagents for tissue processing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Reagents for tissue processing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Modeling wax</td>
			<td>Bego</td>
			<td>40001</td>
			<td>Reagents for tissue processing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OCT compound</td>
			<td>Tissue-Tek Sakura</td>
			<td>4583</td>
			<td>Reagents for tissue processing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mayer&#39;s hematoxylin solution</td>
			<td>Sigma-Aldrich</td>
			<td>MHS16</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eosin Y solution alcoholic&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>HT110316</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Elastin stain kit</td>
			<td>Sigma-Aldrich</td>
			<td style="width:163px;">HT25A</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Click-it Edu Alexa-488 Imaging Kit</td>
			<td>Invitrogen</td>
			<td>C10337</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Anti-Erk1/2 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4695</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Anti-phospho-Erk1/2 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4370</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Anti-p27kip1 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>3698</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Trichloroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T9159</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
	</tbody>
</table>

murine aortic crush injury: an efficient in vivo model of smooth muscle cell proliferation and endothelial function

A reconstrução arterial, seja a cirurgia de angioplastia ou de derivação, envolve trauma iatrogênico causando ruptura endotelial e proliferação de células musculares lisas vasculares (VSMC). Modelos murinos comuns estudam pequenos vasos como as artérias carótida e femoral. Aqui, descrevemos um sistema in vivo no qual tanto a proliferação de VSMC quanto a função de barreira endotelial podem ser avaliadas simultaneamente em um vaso grande. Estudamos a resposta aórtica infra-renal a lesão em camundongos C57BL / 6. A aorta foi ferida da veia renal esquerda para a bifurcação aórtica por 30 esmagamentos transmurais de 5 segundos de duração com um aplicador com ponta de algodão. As alterações morfológicas foram avaliadas com histologia convencional. A espessura da parede da aorta foi medida a partir da superfície luminal para adventitia. A integração de EdU e a coloração de contadores com DAPI e alfa-actina foram utilizadas para demonstrar a proliferação de VSMC. A ativação de ERK1 / 2, um conhecido moderador da formação de hiperplasia intimal, foi dissuadidoExtraído pela análise Western Blot. O efeito da inflamação foi determinado por imuno-histoquímica para células B, células T e macrófagos . As seções de face do endotélio foram visualizadas com microscopia eletrônica de varredura (SEM). A função de barreira endotelial foi determinada com coloração Evans Blue. A lesão transmural resultou em espessamento da parede aórtica. Esta lesão induziu a proliferação de VSMC, mais proeminente aos 3 dias após a lesão e a ativação precoce de ERK1 / 2 e diminuiu a expressão de p7 kip1. A lesão não resultou em aumento da infiltração de células B, células T ou macrófagos na parede do vaso. A lesão causou desnudação de células endoteliais parciais e perda de contato célula-célula. A lesão resultou em perda significativa da função de barreira endotelial, que retornou à linha de base após sete dias. O modelo de ferimento aórtico transmural transmural murino fornece um sistema eficiente para estudar simultaneamente a proliferação de VSMC e a função de barreira endotelial em um vaso grande.

As secções transversais da aorta embutidas em OCT foram seccionadas e coradas com hematoxilina e eosina, em seguida, foram coradas com mancha Verhoeff-Van Gieson (VVG) para identificar a lâmina elástica interna e externa 7 . Os danos causados ​​por esmagamento induziram o espessamento da parede aórtica em comparação com os aortas de animais tratados com procedimento simulado (laparotomia e mobilização do intestino delgado sozinho). A espessura da pare...

Watch this Scientific Journal Video about Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function at JoVE.com

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis após procedimentos cardiovasculares (cirurgia de bypass, angioplastia ou stent) é um problema significativo que reduz a durabilidade desses procedimentos. Uma terapia ideal inibiria a proliferação de células musculares lisas (VSMC) enquanto promovia a regeneração do endotélio. Descrevemos um modelo para avaliação simultânea da proliferação de VSMC e função endotelial in vivo.

Lesão por esmagamento aórtico murino: um eficiente<em&gt; In Vivo</em&gt; Modelo de proliferação de células musculares lisas e função endotelial

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

murine-aortic-crush-injury-an-efficient-vivo-model-smooth-muscle-cell

Research

JoVE Journal

Biology

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

8.3K visualizações.  Baltimore Veterans Affairs Medical Center.  Restenosis após procedimentos cardiovasculares (cirurgia de bypass, angioplastia ou stent) é um problema significativo que reduz a durabilidade desses procedimentos. Uma terapia ideal inibiria a proliferação de células musculares lisas (VSMC) enquanto promovia a regeneração do endotélio. Descrevemos um modelo para avaliação simultânea da proliferação de VSMC e função endotelial in vivo. 

Vídeo: Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

Focal Ca 2 + Detecção de transitórios no músculo liso

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Biologia

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

Explorando Arterial Músculo Liso KV7 função dos canais de potássio utilizando electrofisiologia patch clamp e Miografia Pressão

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

Avaliação da função muscular do músculo extensor longo dos dedos<em&gt; Ex vivo</em&gt; E músculo tibial anterior<em&gt; Em situ</em&gt; Em Ratos

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

A medição da função do músculo liso nas Isolado tecido de banho-aplicativos para Farmacologia Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Dois fótons de imagem de cálcio intracelular<sup&gt; 2+</sup&gt; Manuseio e produção de óxido nítrico no endotélio e músculo liso células de uma aorta de rato isolada

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Isolamento e cultura primária de células de camundongo aórtica endoteliais

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

Isolamento de Murino vasculares coronários células musculares lisas

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease processes. For these reasons, measuring proliferation is a common tool used to assess cell function. Cell proliferation can be measured simply by counting; however, this is an indirect means of measuring proliferation. One common means of directly detecting cells preparing to divide is by incorporation of labeled nucleoside analogs. These include the radioactive nucleoside analog 3H-thymidine plus non-radioactive nucleoside analogs such as 5-bromo-2&#8217; deoxyuridine (BrdU) and 5-ethynyl-2&#8242;-deoxyuridine (EdU). Incorporation of EdU is detected by click chemistry, which has several advantages when compared to BrdU. In this report, we provide a protocol for measuring proliferation by the incorporation of EdU. This protocol includes options for various readouts, along with the advantages and disadvantages of each. We also discuss places where the protocol can be optimized or altered to meet the specific needs of the experiment planned. Finally, we touch on the ways that this basic protocol can be modified for measuring other cell metabolites.

Proliferation is a critical part of cellular function, and a common readout used to assess potential toxicity of new drugs. Measuring proliferation is, therefore, a frequently used assay in cell biology. Here we present a simple, versatile method of measuring proliferation that can be used in adherent and non-adherent cells.

Medição da proliferação de células musculares vasculares lisas usando química clique

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease ...

In vivo Measurement of Knee Extensor Muscle Function in Mice

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

Medição in vivo da função muscular extensor do joelho em camundongos

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the ...

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

This paper describes an explant culture-based method for the isolation and culturing of primary, patient-specific human aortic smooth muscle cells and dermal fibroblasts. Furthermore, a novel method is presented for measuring cell contraction and subsequent analysis, which can be used to study patient-specific differences in these cells.

Isolamento de células musculares lisas aórticas específicas do paciente primário e medidas de contração em tempo real semiquantitativas in vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in ...