Esta publicação foi apoiada pela Pesquisa Clínica e Translacional (SCTR) Institute Carolina do Sul, com uma casa acadêmico na Universidade de Medicina da Carolina do Sul, NIH / NCATS Grant Número UL1 TR000062. Um apoio adicional foi fornecida pela VA prémio de mérito BX002327-01 de DRM. DJH foi apoiado pelo NIH / NCATS Grant Número TL1 TR000061 e pelo NIH Grant Número T32 GM008716. SEA foi apoiado pelo NIH Grant Número T32 HL07260.

Todos os procedimentos listados aqui foram aprovados pelo Comitê de Cuidado e Uso do animal Institucional na Universidade de Medicina da Carolina do Sul. As experiências aqui descritas são, experiências agudas não-sobrevivência. Como tal, não há uso de pomada e uma suíte de cirurgia estéril não é necessária. A eutanásia por exsanguinação é conseguida durante a colheita do coração. 1. Experimental Preparação <ol><li> Configure ou uma pressão constante ou fluxo constante de perfusão Langendorff Aparelho 4. </li><li> Prepare a 4 L de Krebs Henseleit modificado (em mM: NaCl 112, KCl 5, MgSO4 1,2, 1 K 2 HPO 4, 1,25 de CaCl2, 25 de NaHCO3, 11 de D-glicose, 0,2 de ácido octanóico, pH = 7,4). <ol><li> Faça 10x buffers de ações. <ol><li> Dissolve-se NaCl, KCl, MgSO4, e K 2 HPO 4 em 3 L de água ultrapura. </li><li> Dissolver CaCl2 em 500 ml de água ultrapura with 40 ml de 1 N de HCl. </li><li> Adiciona-se lentamente solução de CaCl2 ao tampão feito em 1.2.1.1. Adiciona-se água ultra-pura o suficiente para o total de 4 L. Deixar a agitar durante 1 h antes de se filtrar através de um filtro de 0,8 um. Armazenar a 4 ° C. </li><li> Dissolver NaHCO 3 em 4 L de água ultrapura. Deixar a agitar durante 1 h antes de se filtrar através de pelo filtro de 0,8 um. Armazenar a 4 ° C </li></ol></li><li> Combinar 400 mL de 10x tampão de Krebs Henseleit com 400 ml de 10x tampão de NaHCO3. Adicionar água ultrapura suficiente para totalizar 4 L. Dissolver glicose e ácido octanóico, sob agitação. Filtrar através de filtro de 0,8 um. </li></ol></li><li> Sistema de preparar-se para o coração. <ol><li> Adicionar 2 L de tampão de filtrado para um frasco de 4 L 38 colocado em cima da torneira de passagem ligado à cânula aórtica. Isto irá proporcionar uma pressão de perfusão de 70-80 mmHg para o coração, que é a pressão de perfusão recomendado 4. </li><li> Insira gás dispersão bubbler para o buffer na garrafa. </li><li> Execute o tampão através do sistema, tendo a certeza de eliminar quaisquer bolhas de dentro da tubulação. </li><li> Ligue gás (95% de O2 / 5% de CO 2), pelo menos, 30 min antes do coração está a ser colocado no sistema. Use uma sonda de fluxo de gás e analisador de gases de sangue para verificar a saturação de oxigênio. </li></ol></li><li> Preparar material cirúrgico e balão. <ol><li> Para a colheita de coração, prepare um par de tesouras cirúrgicas 6,75 &quot;, um par de 4.5&quot; tesouras, dois pares de hemostats, dois conjuntos de fórceps caramalt, duas seringas com agulhas de 27 G, 2 peças de 0-medidor fio de seda (cerca de 15 cm de comprimento), uma cânula farpado 1/16 polegadas, e uma cânula traqueal. </li><li> Para montar cânula traqueal, colar um pequeno pedaço de 1/16 de polegada de diâmetro tubo de plástico a um conector Y. </li><li> Para montar aparelhos balão: anexar uma agulha gavagem a uma peça de 1/16 de polegada tubulação, em seguida, anexar a tubulação para habitação transdutor de pressão. Uma pequena seringa ligada ao outro lado da pressão transducer habitação vai permitir que a inflação ea deflação do balão. <ol><li> Corte um quadrado de envoltório e local ponta de plástico de uma agulha gavage no centro. Envolva o envoltório de plástico em torno da agulha por sonda. </li><li> Encha o balão com soro fisiológico e amarrar com duas suturas. Inflar balão para garantir que não haja vazamentos. O volume de solução salina dentro do balão inflado deve ser de aproximadamente 200 ul, mas pode variar dependendo do tamanho do rato. </li><li> Como alternativa, use um berço dedo de látex no lugar do plástico. Coloque agulha gavage dentro dedo berço, encha com solução salina e amarrar. </li></ol></li></ol></li></ol> 2. Colheita do coração <ol><li> Contenha um rato Sprague Dawley utilizando um decapicone ou outro dispositivo de segurança manual. Pesar rato. </li><li> Administrar cetamina / mistura de xilazina (0,85 mg / kg de cetamina e 0,15 mg / kg de xilazina) através de injecção intraperitoneal para anestesiar ratos. Esta dose de anestésico irá fornecer suficientes para anestesia20-40 min, mas o procedimento deve ser concluído o mais rapidamente possível após a anestesia é confirmada. </li><li> Confirme grau adequado de anestesia pelo teste toe pitada reflexo. </li><li> Traqueotomia <ol><li> Retirar pele de meados de forepaw à base de mandíbula, utilizando hemostats para elevar pele e tesoura para fazer incisão. Manter a pele elevada para separá-lo do músculo subjacente, permitindo que um corte ao longo da pele e removê-lo. </li><li> Usando hemostats, levante o tecido glandular e fazer uma incisão horizontal na fáscia inferior ao tecido glandular. Use hemostats para espalhar incisão aberta, tomando cuidado para não nick as veias jugulares. </li><li> Usando pinças hemostáticas, músculo sobrejacente pitada traqueia, e usando uma tesoura fazer uma incisão transversal no músculo logo abaixo da ponta das pinças hemostáticas. Usando pinças hemostáticas, espalhar tecido para além de revelar traqueia. </li><li> Insira com cuidado hemostats sob traquéia, limpando fáscia enquanto progredindo suavemente avançar e recuar hemostats. </li><li>Uma vez hemostats são colocados com sucesso por trás da traquéia, beliscar 0-0 sutura de seda com hemostats e hemisect a traqueia com um pequeno par, de tesoura afiada. </li><li> Insira cânula traqueal na traquéia hemisected, puxe sutura por trás da traquéia, e amarrar a sutura ao redor da traqueia canulada, ancorando a sutura com o Y-virilha da cânula com um nó cirurgiões. </li><li> Usando uma agulha de 27 G, injectar 1.000 U / kg de heparina na veia jugular e permitir a circular durante 30 seg. </li></ol></li><li> Toracotomia <ol><li> Retirar pele de meados de abdômen para região mid-pata dianteira por compressão com hemostats e corte com tesoura. </li><li> Adicione uma incisão transversal ¾ de polegada na parede muscular abdominal, imediatamente abaixo do diafragma. </li><li> Faça uma incisão vertical até a parede a parede abdominal e torácica, cortando ao longo do esterno. </li><li> Corte o diafragma volta bilateralmente, em seguida, espalhar caixa torácica para revelar coração, ribcage braçadeira com hemostats para garantir que ele permanece spread. </li&gt; <li> Usando hemostats, remova gentilmente timo, expondo a aorta ascendente. </li><li> Tease hemostats através de loop aórtica, avançar e recuar hemostats e gentilmente espalhando fascia para permitir ponta de hemostats completamente. </li><li> Use hemostats para puxar 0-0 sutura de seda por trás da aorta ascendente, amarre sutura em um meio-quadrado nó frouxo. </li><li> Virar a torneira para iniciar o fluxo de tampão de perfusão, hemisect aorta e imediatamente inserir uma cânula para lúmen da aorta. Rapidamente apertar um nó meia-quadrado e completar o nó, apertando bem. </li><li> Use a tesoura para dissecar coração longe dos grandes vasos, remova coração do peito e fixe a coluna de perfusão. </li><li> Corte o excesso de tecido pulmonar e permitir que o coração se equilibrar em coluna por cerca de 15 min antes da inserção de balão. Usar uma taxa de fluxo normal do tampão por meio de coração de 10-20 ml / min. </li></ol></li></ol> 3. Langendorff de perfusão e isquemia-reperfusão Lesão <ol><li> Colocação de pré LVssure balão <ol><li> Desinflar o balão ao rolar em forma de cone e vire torneira para garantir balão continua a ser esvaziado. </li><li> Excise átrio esquerdo com um pequeno par de tesouras para expor o acesso à válvula mitral. </li><li> Insira balão através do átrio esquerdo e da válvula mitral em ventrículo esquerdo. </li><li> Inicie o software de monitoramento de pressão (LabChart Pro). </li><li> Encher o balão através da abertura de válvula e suavemente o aumento da quantidade de solução salina em balão até que a pressão diastólica leitura de micromanómetro excede zero. </li><li> Para verificar a resposta comprimento-tensão, inflar o balão lentamente a uma pressão diastólica de 75 mmHg, desinflar lentamente, repetir uma vez. </li><li> Regule a pressão diastólica de 10 mm Hg através da modulação da quantidade de solução salina no balão. Vire torneira para selar balão a este nível de inflação, e manter sistema de pressão fechado entre balão LV e transdutor de pressão. </li><li> Lower coração em câmara câmara ea tampa aquecida com plástico. </li></ol></li><li> De Anúnciosministração de isquemia-reperfusão Lesão <ol><li> Ligue o fundo da câmara de aquecimento e câmara de permitir a encher-se com tampão de Effused. </li><li> Quando o buffer tenha submergido coração, virar a torneira para parar o fluxo de tampão para o coração, induzindo isquemia global. Tempo de isquemia pode variar de acordo com os objetivos do experimento. </li><li> Após 30 min de isquemia, virar a torneira para restaurar o fluxo de tampão para o coração, o início da reperfusão. </li><li> Permitir reperfusão do coração durante 1 hora antes de terminar o experimento. Use tempos de reperfusão variáveis, dependendo dos objetivos do experimento. </li></ol></li><li> Rescisão de Experiência e de colheita de Tissue <ol><li> Usando a porta lateral de torneira, administrar 10 ml de solução salina tamponada com fosfato a 4 ° C para o coração a uma taxa de 10 ml / min para parar o coração. </li><li> Remover coração da coluna perfusão, aparar restante tecido atrial. </li><li> Coloque coração em uma matriz de tecido corte de aço inoxidável, cubra com Parafilm, e colocar a -80 ° C durante 8 minutos, ou até que o coração tem uma consistência como marshmallows. </li><li> Remova o bloco do freezer e coloque razorblades em cortar matriz de cortar o coração em fatias de 2 mm. Blocos de tecido também pode ser comprado com outros que 2 mm tamanhos fatia. </li><li> Remover fatias um de cada vez e soltar em nitrogênio líquido a piscar congelar por estudos bioquímicos. Reter a fatia ventricular terceira a partir do ápice para a coloração do enfarte. </li><li> Remover o tecido a partir de azoto líquido e armazenar a -80 ° C até à utilização em experiências bioquímicas. </li></ol></li><li> Enfarte Coloração <ol><li> Incubar a terceira fatia ventricular em 10 mL de 1% w / v de cloreto de trifeniltetrazólio (TTC) a 37 ° C durante 15 min. Nota: A incubação no TTC pode ser realizada durante 15 ou 30 min, sem qualquer alteração na eficácia 15. </li><li> Transferir fatia de tecido de TTC a 10% de formalina tamponada e deixa-se incubar à temperatura ambiente durante a noite. Para melhores resultados, ouvirts deve ser retirado de formalina e fotografada dentro de 24 horas 4. </li><li> Fotografia manchado fatia e uso de software como o ImageJ para estimar o tamanho do infarto de acordo com o protocolo do fabricante. Tirar fotografias, logo que possível após a sua fixação, como o corante vai desaparecer com o tempo. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

O coração perfundido isolado de rato pode ser utilizado com êxito para estudar o efeito da intervenção farmacológica no pré-condicionamento cardíaco em lesão de reperfusão de isquemia 9. No entanto, existem alguns passos essenciais para o procedimento que deve ser normalizados, a fim de assegurar a reprodutibilidade dos resultados. A manutenção de uma temperatura de 37,4 ° C no interior do sistema é crítica, já que mesmo a hipotermia e hipertermia pode causar pré-condicionamento cardíaco <su...

A elucidação dos eventos subjacentes à resposta cardíaca tanto para isquemia e reperfusão são essencial para melhorar o tratamento de enfarte 1 e procedimentos cirúrgicos cardíacos do miocárdio que necessitam pinçamento aórtico 2. Embora os modelos in vivo de lesão de reperfusão de isquemia permitir análise de ponto final muito úteis, eles não são tão eficazes para estudar os efeitos funcionais da isquemia lesão de reperfusão aguda em tempo real. Além disso, em modelos de isquemia reperfusão in vivo geralmente produzem uma variabilidade significativa da dimensão do enfarte e a entrega directa de drogas para o coração no momento da reperfusão é um desafio. A utilização de um sistema de coração de Langendorff isolado para estudar a lesão por isquemia reperfusão permite a avaliação funcional em tempo real dos tratamentos farmacológicos, área uniforme do tecido enfartado e entrega instantânea de fármaco directamente ao miocárdio. Descrita pela primeira vez by Oscar Langendorff em 1895 3, o coração de Langendorff isolado é um modelo robusto para o estudo da lesão de reperfusão de isquemia, tendo sido utilizado na pesquisa de isquemia reperfusão durante os últimos 40 anos 4,5. Aqui, algumas modificações são feitas para aperfeiçoar o coração isolado para análise funcional. Em situ canulação da aorta, enquanto o coração está a bater garante que o coração não experimentar o pré-condicionamento isquémico, que iria alterar os resultados dos ensaios isquemia reperfusão 6. Para facilitar isto, uma traqueotomia é efectuada, permitindo a ventilação e garantir a estabilidade fisiológica do rato durante a cirurgia. O coração é depois ligado a uma coluna em espiral com camisa de água de vidro através da qual tampão de Krebs Henseleit é entregue através de perfusão retrógrada directamente para a aorta. Um balão cheio de soro fisiológico é inserido no ventrículo esquerdo e ligado a um transdutor de pressão, que permite a medição das pressões em tempo real a partir do um ventrículod cálculo de vários parâmetros funcionais. Na conclusão do experimento, o coração é lavada com solução salina fria para prender contração e flash congelado em nitrogênio líquido para permitir a análise a jusante dos níveis de DNA, RNA e proteínas. Assim modificado, o coração de Langendorff perfundido serve como um sistema eficaz para a monitorização directa do efeito fisiológico de intervenções farmacológicas, em qualquer momento de forma aguda durante a lesão por isquemia-reperfusão.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma Aldrich</td>
			<td align="right">203726</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>Sigma Aldrich</td>
			<td>RES20765-A7</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C8106</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Octanoic Acid</td>
			<td>Sigma Aldrich</td>
			<td>C2875</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3,5-triphenyltetrazolium chloride</td>
			<td>Sigma Aldrich</td>
			<td>T8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical Pressure Transducer</td>
			<td>MEMSCAP</td>
			<td>SP844</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Peristaltic Pump</td>
			<td>Cole Parmer</td>
			<td>EW-07521-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Easy Load Pump Head</td>
			<td>Cole Parmer</td>
			<td>EW-07518-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated circulating water bath</td>
			<td>Lauda</td>
			<td>M3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing Flow Module</td>
			<td>Transonic</td>
			<td>TS410</td>
			<td></td>
		</tr>
		<tr>
			<td>Modular Research Console</td>
			<td>Transonic</td>
			<td>T402</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline flow sensor</td>
			<td>Transonic</td>
			<td>ME2PXN</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLab Data Acquisition Device</td>
			<td>AD Instruments</td>
			<td>PL3508</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart data acquisition software</td>
			<td>AD Instruments</td>
			<td>MLU60/8</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction and assessment of ischemia-reperfusion injury in langendorff-perfused rat hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

O aparelho de balão do ventrículo esquerdo permite a monitorização em tempo real da pressão desenvolvida pela entidade ventrículo esquerdo (Figura 1). Como descrito anteriormente 7, este traçado da pressão pode ser usado para calcular muitos dos parâmetros da função ventricular. Estes cálculos podem ser realizados na fase de linha de base, bem como a fase de reperfusão, em média, em traços múltiplos dentro de cada grupo, e comparadas a fim de determinar se a intervenção farm...

Watch this Scientific Journal Video about Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts at JoVE.com

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

Indução e Avaliação de isquemia-reperfusão em Langendorff-perfundidos Rato Corações

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

induction-assessment-ischemia-reperfusion-injury-langendorff-perfused

Research

JoVE Journal

Medicine

22.6K visualizações.  Medical University of South Carolina. The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

Vídeo: Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

Real-time Digital Imaging de leucócito-endotélio Interação em isquemia-reperfusão (IRI) do músculo cremaster de rato

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

Medicina

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite tissue. Ischemia reperfusion injury (IRI) is a known cause of partial free flap failure and has no effective treatment. Establishing a laboratory model of this injury can prove costly both financially as larger mammals are conventionally used and in the expertise required by the technical difficulty of these procedures typically requires employing an experienced microsurgeon. This publication and video demonstrate the effective use of a model of IRI in rats which does not require microsurgical expertise. This procedure is an in situ model of a transverse abdominis myocutaneous (TRAM) flap where atraumatic clamps are utilized to reproduce the ischemia-reperfusion injury associated with this surgery. A laser Doppler Imaging (LDI) scanner is employed to assess flap perfusion and the image processing software, Image J to assess percentage area skin survival as a primary outcome measure of injury.

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is widely employed in reconstructive surgery to restore form and function following oncological resection and trauma. Preconditioning this tissue prior to surgery may improve outcome. This article describes an in situ transverse rectus abdominis myocutaneous flap (TRAM) in rats as a means for testing preconditioning strategies.

In situ transversal reto abdominal miocutâneo: um modelo de rato de miocutâneo Isquemia Traumatismo por Reperfusão

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Isquemia-reperfusão modelo de lesão renal aguda e Pós Lesão fibrose em camundongos

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Modelo murino de isquemia-reperfusão

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Avaliação da reserva de fluxo coronariano após reperfusão de isquemia miocárdica em ratos

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Um modelo pré-clínico do rato para o estudo da lesão da Isquemia-reperfusion na microcirurgia reconstrutiva

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Estudos de Optocardiografia e Eletrofisiologia de Ex Vivo Langendorff-perfundido Corações

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

Entrega intramiocárdica atrasada de células-tronco após lesão de reperfusão de Isquemia em um modelo murino

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Lesão por Isquemia-Reperfusão de Órgãos simulando alterações hemodinâmicas no modelo de transplante de fígado de rato

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

Avaliação em tempo real da microperfusão da medula espinhal em um modelo suíno de isquemia/reperfusão

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...