Reconhecemos Scott and White Hospital Auxílio à Pesquisa Programa de apoio financeiro e Texas A &amp; M Health Science Center College of Medicine Laboratório Integrado Imaging para o uso do microscópio confocal a laser. Reconhecemos o Sr. Glen Cryer para obter ajuda com a edição do manuscrito.

1. Plantio de células endoteliais <ol><li> Obter culturas primárias de RBMEC de adultos a partir de ratos Sprague Dawley (ou obtê-los comercialmente). </li><li> Cultivar RBMECs em 100 centímetros fibronectina (50 ug / ml) placas de petri revestidas usando o cérebro de rato meio de crescimento de células endoteliais. Mudar a forma de dois em dois dias, até que seja atingida a confluência. </li><li> Ao chegar a 80-90% de confluência, lavar suavemente as células em 5 ml de solução salina de fosfato tamponada (PBS), através de agitação. As células são, então, separado por expondo-os a um ml de solução quente de 0,25% tripsina ácido etilenodiaminotetracético (EDTA), equilibrado a 37 ° C. </li><li> Incubar as células a 37 ° C durante 2-5 min, até que as células são separadas e dispersas. NOTA: Toque na placa de cultura para separar as células. Ver as células ao microscópio para confirmar a separação completa de células da superfície do prato. </li><li> Adicionar 5 ml de meios completo numa placa de Petri a fim de neutralizar a tripsina.Pipeta o meio contendo células destacadas e recolhê-la para um tubo de centrífuga de 15 ml. . </li><li> Centrifuga-se o meio contendo as células endoteliais a 220 xg durante 5 min. </li><li> Aspirar o sobrenadante e preservar o pellet contendo cells.Suspend o sedimento em 3-5 ml fresco cérebro de rato meio de crescimento de células endoteliais por misturando suavemente para cima e para baixo com células pipeta será, em seguida, ser contados utilizando um contador de células automatizado ou um hemocitômetro. </li><li> Transferir a suspensão de células em fibronectina (50 ug / ml) pré-revestidas 8 sistema corrediça câmara bem estéril, 0,7 cm2 / poço com uma densidade de sementeira variando entre 10.000-15.000 células por poço. Crescer as células a 37 ° C até confluência é conseguido NOTA: Para a realização de transferências de Western ou outros ensaios, as células podem ser cultivadas em placas de 10 cm de cultura de células ou pratos especiais como exigido pela experiência. </li></ol> 2. oxigênio e glicose Privação-reoxigenação In VitroModelo NOTA: O protocolo a seguir foi adaptado de Zulueta et al. 1997; H Zhu et ai. 2012 8,9. <ol><li> Utilizar a hipóxia sistema de cultura celular para estudar os efeitos da de OGD-R em RBMECs (ver Figura 1). Configuração e calibração do sistema de cultura celular hipoxia antes de começar a experiência, de acordo com as instruções do fabricante. </li><li> Retirar as lâminas de câmara confluente (passo 1.8) a partir do 37 ° C incubadora. Substituir o meio completo na corrediça câmara com desoxigenado, não glicose, Meio de Eagle Modificado por Dulbecco (DMEM) e colocados em câmara de hipoxia de 95%, N 2 e CO 2 a 5% durante 2 h a 37 ° C, para representar OGD condição. </li><li> Mover as células de volta para a incubadora com 95% de O2, 5% de CO2 a 37 ° C e fornecidas com células endoteliais do cérebro de rato meio completo fresco e incuba-se durante mais 1 h a 37 ° C. NOTA: Este passo representa uma situação reoxigenação. </li><li> Use os slides câmara para localização de imunofluorescência e rotulagem rhodamine phalloidin (ver secção 3). </li></ol> 3. Localização por imunofluorescência de ZO-1 e F-actina Etiquetagem Usando rodamina faloidina <ol><li> Expor as lâminas de câmaras contendo as monocamadas RBMEC a 100 ul de meio Opti-MEM / soro reduzida / poço durante 1 h. Lavam-se as lâminas de câmara 3 vezes em 100 ul de solução salina tamponada com fosfato (PBS, pH 7,0-7,2). </li><li> Fixar as células utilizando 100 uL de paraformaldeído a 4% em PBS (pH 7,0-7,2) durante 15 minutos e lava-se as lâminas de câmara por mais 3 vezes em PBS (pH 7,0-7,2). </li><li> Permeabilizar as células utilizando 100 ul de 0,5% de Triton X-100 em PBS, (pH 7,0-7,2) durante mais 15 min. Bloquear com 100 ul de 2% de albumina de soro bovino (BSA) em PBS durante uma hora. Após este passo, quer células mancha / etiqueta quer para ZO-1 ou f- actina. <ol><li> Para imunofluorescência staining incubar as células com um anticorpo anti-coelho primário contra ZO-1 em 1: 150, preparado em 2% de BSA-PBS, durante O / N a 4 ° C. Lavam-se as células três vezes em PBS, (pH 7,0-7,2). Incubar com 100 ul de fluoresceína isotiocianato (FITC) tagged anticorpo secundário anti-coelho durante 1 h à TA. </li><li> Para a etiquetagem Rodamina Faloidina, seguindo bloqueio, expor as células a 100 ul de rodamina faloidina na diluição 1:50, preparado em 2% de BSA-PBS, durante 20 min. </li><li> Lavam-se as células de coloração por imunofluorescência e rodamina faloidina rotulagem em PBS, (pH 7,0-7,2). Monte as câmaras que usam meios de montagem contendo reagente anti-fade com DAPI. </li></ol></li><li> Visualizar as células utilizando uma lente de imersão em água e X 60 células são digitalizadas num plano óptico sob um microscópio confocal. </li></ol>

<ol>
	<li>Eltzschig, H. K., Eckle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia+and+reperfusion--from+mechanism+to+translation.">Ischemia and reperfusion--from mechanism to translation.</a> Nat Med. 17 (11), 1391-1401 (2011).</li><li>Kalogeris, T., Baines, C. P., Krenz, M., Korthuis, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+of+ischemia/reperfusion+injury.">Cell biology of ischemia/reperfusion injury.</a> Int Rev Cell Mol Biol. 298, 229-317 (2012).</li><li>Yang, X., 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=Lycium+barbarum+polysaccharides+reduce+intestinal+ischemia/reperfusion+injuries+in+rats.">Lycium barbarum polysaccharides reduce intestinal ischemia/reperfusion injuries in rats.</a> Chem Biol Interact. 204 (3), 166-172 (2013).</li><li>Kaur, C., Ling, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+brain+barrier+in+hypoxic-ischemic+conditions.">Blood brain barrier in hypoxic-ischemic conditions.</a> Curr Neurovasc Res. 5 (1), 71-81 (2008).</li><li>Khatri, R., McKinney, A. M., Swenson, B., Janardhan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier,+reperfusion+injury,+and+hemorrhagic+transformation+in+acute+ischemic+stroke.">Blood-brain barrier, reperfusion injury, and hemorrhagic transformation in acute ischemic stroke.</a> Neurology. 79 (13), S52-S57 (2012).</li><li>Hossmann, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+for+the+investigation+of+brain+ischemia.">Experimental models for the investigation of brain ischemia.</a> Cardiovasc Res. 39, 106-120 (1998).</li><li>Ogawa, S., Gerlach, H., Esposito, C., Pasagian-Macaulay, A., Brett, J., Stern, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+modulates+the+barrier+and+coagulant+function+of+cultured+bovineendothelium.+Increased+monolayer+permeability+and+induction+of+procoagulant+properties.">Hypoxia modulates the barrier and coagulant function of cultured bovineendothelium. Increased monolayer permeability and induction of procoagulant properties.</a> J Clin Invest. 85 (4), 1090-108 (1990).</li><li>Zulueta, J. J., Sawhney, R., Yu, F. S., Cote, C. C., Hassoun, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+generation+of+reactive+oxygen+species+in+endothelial+cellsexposed+to+anoxia-reoxygenation.">Intracellular generation of reactive oxygen species in endothelial cellsexposed to anoxia-reoxygenation.</a> Am J Physiol. 272 (5 Pt 1), L897-L902 (1997).</li><li>Zhu, H., 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=Baicalin+reduces+the+permeability+of+the+blood-brain+barrier+during+hypoxia+in+vitro+by+increasing+the+expression+of+tight+junction+proteins+in+brain+microvascular+endothelial+cells.">Baicalin reduces the permeability of the blood-brain barrier during hypoxia in vitro by increasing the expression of tight junction proteins in brain microvascular endothelial cells.</a> J Ethnopharmacol. 141 (2), 714-720 (2012).</li><li>Abramov, A. Y., Scorziello, A., Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+distinct+mechanisms+generate+oxygen+free+radicals+in+neurons+and+contribute+to+cell+death+during+anoxia+and+reoxygenation.">Three distinct mechanisms generate oxygen free radicals in neurons and contribute to cell death during anoxia and reoxygenation.</a> J Neurosci. 27 (5), 1129-1138 (2007).</li><li>Gundimeda, U., 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=Green+tea+polyphenols+precondition+against+cell+death+induced+by+oxygen-glucose+deprivation+via+stimulation+of+laminin+receptor,+generation+of+reactive+oxygen+species,+and+activation+of+protein+kinase+Cepsilon.">Green tea polyphenols precondition against cell death induced by oxygen-glucose deprivation via stimulation of laminin receptor, generation of reactive oxygen species, and activation of protein kinase Cepsilon.</a> J Biol Chem. 287 (41), 34694-34708 (2012).</li><li>Mehta, S. L., Manhas, N., Raghubir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+targets+in+cerebral+ischemia+for+developing+novel+therapeutics.">Molecular targets in cerebral ischemia for developing novel therapeutics.</a> Brain Res Rev. 54 (1), 34-66 (2007).</li><li>Alluri, H., 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=Reactive+Oxygen+Species-Caspase-3+Relationship+in+Mediating+Blood-Brain+Barrier+Endothelial+Cell+Hyperpermeability+Following+Oxygen-Glucose+Deprivation+and+Reoxygenation.">Reactive Oxygen Species-Caspase-3 Relationship in Mediating Blood-Brain Barrier Endothelial Cell Hyperpermeability Following Oxygen-Glucose Deprivation and Reoxygenation.</a> Microcirculation. 21 (2), 187-195 (1111).</li><li>Sun, H., Breslin, J. W., Zhu, J., Yuan, S. Y., Wu, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+and+ROCK+signaling+in+VEGF-induced+microvascular+endothelial+hyperpermeability.">Rho and ROCK signaling in VEGF-induced microvascular endothelial hyperpermeability.</a> Microcirculation. 13 (3), 237-247 (2006).</li><li>Doggett, T. M., Breslin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+actin+cytoskeleton+in+live+endothelial+cells+expressing+GFP+actin.">Study of the actin cytoskeleton in live endothelial cells expressing GFP actin.</a> J Vis Exp. (57), (2011).</li></ol>

The authors have nothing to disclose.

OGD-R como um modelo in vitro para a lesão de isquemia-reperfusão tem sido bem estabelecido para o estudo de neurónios 10,11. Existem também estudos que mostram o efeito de OGD em células endoteliais cerebrais e alterações na permeabilidade e TJ integridade 9. No entanto, o nosso estudo mostra o efeito de OGD, bem como reoxigenação, que é uma representação mais próxima da lesão de reperfusão isquémica em condições in vivo que ocorrem após acidente vascular cereb...

A isquemia-reperfusão (IR) é encontrada lesão a ser a causa frequente de diversas complicações debilitantes e mortes associadas com acidente vascular cerebral, enfarte do miocárdio, trauma, doença vascular periférica e lesão cerebral traumática 1,2. IR lesão em vasos cerebrais é uma lesão secundária precoce levando a inflamação e edema 3. Uma das complicações graves que ocorrem como um resultado de stress oxidativo e metabólica seguinte inflamação é a perda de equilíbrio homeostático que conduz à formação de radicais livres, alterações na barreira sangue-cérebro (BBB) ​​junções apertadas (TJs) e permeabilidade microvascular 4,5. Actualmente, em modelos in vivo utilizados para estudar os efeitos do dano por IR na certificação incluem a oclusão da artéria cerebral média (MCAO), microembolia, e animais transgénicos ou knockout. No entanto, cada um tem os seus inconvenientes e limitações como discutido por Hossmann 6. Modelo MCAO é usado para estudar o effecst de estresse redox, mudanças nas comunicações de junção da certificação e as interações entre o cérebro e as células do sistema imunológico. No entanto, eles apresentam vários desafios técnicos, tais como a necessidade de procedimentos microcirúrgicos precisas e as dificuldades das mesmas. Microembolismo instantaneamente quebra da BBB, enquanto o uso de animais transgénicos ou knockout para estudar a isquemia cerebral pode ter desafios como influências dependente de genes moleculares na formação de enfarte, alterações na anatomia vascular e diferentes pesos corporais 6. Portanto, modelos in vitro de isquemia ter encontrado um interesse crescente nos últimos tempos, principalmente devido à sua aplicabilidade na realização de estudos sobre os mecanismos de drogas. No entanto, os resultados de estudos in vitro podem não representar plenamente um estudo in vivo e deve ser interpretado com cautela 6. Counteractive efeito de baixas concentrações de oxigênio em monocamadas de células endoteliais e de permeabilidade microvascular ter sidoestudados por Ogawa 7. Células endoteliais microvasculares cérebro de rato (RBMECs) foram utilizados para desenvolver in vitro a certificação. A privação de oxigênio-glucose e reoxigenação técnica (OGD-R) apresentado neste protocolo foi adaptado de estudos de Zulueta et al e Zhu et al 8,9. Nós expostas células endoteliais cerebrais a OGD-R, colocando-os numa câmara de hipoxia / anoxia contendo 0% de O2, 5% de CO 2 e 95% de N2. As células foram depois avaliadas para alterações na integridade e formação de fibras de stress TJ usando localização imunofluorescência e marcação com rodamina-faloidina respectivamente. Imunofluorescência para zonula occludens-1 (ZO-1) é realizada para determinar a integridade TJ, como ZO-1 é um importante membrana andaimes proteína ligada TJ. Rotulagem rodamina faloidina determina a actina filamentosa (F-actina) no citoesqueleto da célula e é uma clara indicação de formação de fibras de stress de actina em células endoteliais. &lt;p class = &quot;jove_content&quot;&gt; O objetivo deste método é fornecer uma visão sobre o desenvolvimento de OGD-R como um modelo no IR vitro para estudar a certificação células endoteliais integridade TJ e formação de fibras de stress f-actina. Os resultados irão fornecer informações sobre o destino de proteína TJ, ZO-1 e formação de fibras de stress seguinte OGD-R. Compreender essas relações será uma oportunidade para determinar os mecanismos moleculares subjacentes que são acionados seguintes OGD-R e desenvolver potenciais estratégias terapêuticas para melhorar a interrupção certificação após o tratamento OGD-R.

<table border="0" cellpadding="0" cellspacing="0" width="917">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Name of Material/ Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:129px;">Catalog Number</td>
			<td style="width:251px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Proox model 110</td>
			<td style="width:167px;">Biospherix</td>
			<td style="width:129px;">Model 110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">DMEM, no glucose</td>
			<td style="width:167px;">Gibco, Life technologies</td>
			<td style="width:129px;">11966-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Rhodamine Phalloidin</td>
			<td style="width:167px;">Life technologies</td>
			<td style="width:129px;">R415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">ZO-1 Rabbit Polyclonal Antibody</td>
			<td style="width:167px;">Life technologies</td>
			<td align="right" style="width:129px;">617300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc Lab Tek II-CC 8&#160;well sterile, glass&#160;slides&#160;</td>
			<td style="width:167px;">Thermo scientific</td>
			<td align="right" style="width:129px;">177402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC-tagged anti-rabbit secondary antibody&#160;</td>
			<td style="width:167px;">Santa cruz</td>
			<td style="width:129px;">sc-2090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">DPBS 1X</td>
			<td style="width:167px;">Thermo scientific</td>
			<td style="width:129px;">SH 30028.03</td>
			<td>Any other PBS available can be used</td>
		</tr>
	</tbody>
</table>

oxygen-glucose deprivation and reoxygenation as an in vitro ischemia-reperfusion injury model for studying blood-brain barrier dysfunction

Isquemia e reperfusão (IR) lesão é conhecido por contribuir significativamente para a morbidade e mortalidade associadas com acidentes vasculares cerebrais isquêmicos. Acidentes vasculares cerebrais isquêmicos são responsáveis ​​por 80% de todos os acidentes vasculares cerebrais. Uma causa comum de lesão de IR é o rápido ingresso de fluidos após uma oclusão crónica / aguda de sangue, os nutrientes, oxigénio ao tecido provocando a formação de radicais livres. Acidente vascular cerebral isquémico é seguido pela barreira sangue-cérebro (BBB) ​​e disfunção vasogénico edema cerebral. Estruturalmente, as junções apertadas (TJS) entre as células endoteliais desempenham um papel importante na manutenção da integridade da barreira sangue-cérebro (BBB). IR lesão é uma lesão secundária precoce conduz a uma resposta não específica, inflamatória. Estresse oxidativo e inflamação metabólica seguinte desencadeia dano cerebral secundário, incluindo a certificação permeabilidade e perturbação da junção apertado integridade (TJ). Nosso protocolo apresenta in vitro &lt;/ Em&gt; exemplo de privação de oxigênio-glucose e reoxigenação (OGD-R) em células endoteliais cérebro de rato integridade TJ e formação de fibras de stress. Actualmente, vários experimental em modelos in vivo são utilizados para estudar os efeitos da lesão de IR; no entanto eles têm várias limitações, como os desafios técnicos na realização de cirurgias, gene influências moleculares dependentes e dificuldade em estudar as relações mecanicistas. No entanto, em modelos in vitro pode ajudar a superar muitas destas limitações. O protocolo apresentado pode ser usado para estudar os mecanismos moleculares e várias relações mecanísticos para proporcionar potenciais estratégias terapêuticas. No entanto, os resultados de estudos in vitro pode ser diferente da padrão em estudos in vivo e deve ser interpretado com cautela.

Células cultivadas em pré-revestidas fibronectina lâminas de câmara Nunc II foram submetidos a OGD-R, colocando em um modelo de 110 câmara Biospherix ProOx. Após sujeitar as células a OGD-R, que foram processados ​​para ZO-1 coloração juncional utilizando a técnica de imunofluorescência, conforme mostrado na Figura 2 e montagem do citoesqueleto indicando a formação de fibras de stress de actina F usando etiqueta mancha rodamina faloidina como mostrado na Figura 3. As c?...

Watch this Scientific Journal Video about Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction at JoVE.com

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Oxigênio-Glucose Privação e reoxigenação como um<em&gt; In Vitro</em&gt; Isquemia e reperfusão Lesão modelo para estudar Disfunção Barreira Hematoencefálica

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

oxygen-glucose-deprivation-reoxygenation-as-an-vitro-ischemia

Research

JoVE Journal

Medicine

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

17.8K visualizações.  Texas A&M University Health Science Center College of Medicine. Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells. 

Vídeo: Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

Cardiac Arrest normotérmica e Ressuscitação Cardiopulmonar: um modelo do rato de isquemia-reperfusão

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

Medicina

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Transplante de Células-tronco em um In vitro Modelo de isquemia / reperfusão Simulated

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

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 ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Estique em células cerebrais endoteliais microvasculares (cEnd) como um<em&gt; In Vitro</em&gt; Modelo de lesão cerebral traumática do cérebro Barreira Hemato

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

Criação de um<em&gt; In Vitro</em&gt; Modelo de Rato barreira hemato-encefálica (BBB): A Focus on BBB impermeabilidade e Transportes Receptor mediada

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

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, ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Melhor método para o estabelecimento de uma In Vitro barreira hemato - encefálica modelo baseado em células endoteliais do cérebro de suínos

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

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 ...

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, ...