The authors would like to thank Hans-Joachim Schurek for invaluable scientific advice. The authors would like to thank Monique Carrel and Mich&#232;le Heidemeyer for excellent technical assistance, David Penton Ribas and Nourdine Faresse for a critical reading of the manuscript and Carsten Wagner and J&#252;rg Biber for the NaPi-2a antibody. This work was supported by the Swiss National Centre for Competence in Research "Kidney.CH" and by a project grant (310030_143929/1) from the Swiss National Science Foundation.

Todos os procedimentos que envolvem animais descritos neste manuscrito foram conduzidos de acordo com a lei suíça e aprovado pela administração veterinária do Cantão de Zurique, Suíça. 1. Preparação Tampão <ol><li> Preparar soluções 1 - 4 e a solução de hormônio antidiurético (ADH) (Tabela 1). </li><li> Prepara-se o tampão de diálise (Tabela 1). NOTA: Este é o tampão utilizado como o tampão de diálise durante a perfusão. Mais tarde, os eritrócitos serão diluídas neste tampão para formar o perfusato final. </li><li> preparação de eritrócitos. <ol><li> Dilui-se 250 ml de concentrado de eritrócitos humana (material testado obtido a partir do banco de sangue local) para 500 ml com tampão de diálise. Centrifugar a 2000 xg durante 8 minutos. Remover o tampão, tomando cuidado para não remover quaisquer eritrócitos. 3x. </li></ol></li><li> Prepara-se o (albumina de soro de bovino; BSA) de albumina de tampão. <ol><li> Em 200 ml de dialysitampão s, dissolver 44 g de BSA usando uma barra de agitação. Filtra-se a solução com papel de filtro. </li></ol></li><li> Prepara-se o perfusado. <ol><li> Filtrar os eritrócitos a partir do passo 1.3.1 através de papel de filtro para o tampão de BSA. Encha até um volume total de 800 ml com tampão de diálise. NOTA: Este é o perfusato final. O hematócrito agora deve situar-se entre 8 e 12%. O perfusato pode ser armazenado durante até 12 h a 4 ° C. </li></ol></li></ol> 2. Início de Diálise e Oxigenação <ol><li> Ligar o banho de água em torno do reservatório maior tampão, menor do reservatório tampão, e a câmara de humidade (uma pequena câmara, de parede dupla trazida a 37 ° C e 100% de humidade para realizar mais tarde, o rim) a 37 ° C (Figura 2) . </li><li> Encher o reservatório de tampão de maior com o tampão de diálise e o reservatório menor com o perfusato. </li><li> Ligue a 5% de CO 2/95% O 2 afluxo de gás para o tampão de diálise. </li><li> Ligar diálise contínua do perfusado contra o tampão de diálise. Tome o cuidado de usar tubos de diálise baixo fluxo. Vá para a Etapa 3. </li></ol><img alt="Figura 2" src="/files/ftp_upload/54712/54712fig2.jpg" /> Figura 2: Desenho esquemático do circuito de perfusão. Esquema mostra os principais componentes do circuito de perfusão e a direcção do fluxo tampão. Todos os componentes rodeados por azul escuro são mantidos a 37 ° C com um banho de água / termostato. 1: tampão de diálise de pelo menos 3 vezes o volume de tampão de perfusão é continuamente borbulhada com 95% de O2 / 5% de CO 2. 2: tampão de diálise e tampão de perfusão são continuamente dialisada contra o outro num tubo de diálise por uma bomba de rolos. 3: througho constante Devido a esta diálise, o tampão de perfusão é enriquecida com 9% de O2 / 5% de CO 2 e de electrólitos são mantidos níveisperfusão ut. 4: Uma bomba de cilindro impele o tampão de perfusão para o rim. 5: Uma Windkessel remove as ondas peristálticas e funciona como uma armadilha de bolhas. 6: transdutor de pressão (ligados a 4. (bomba de roletes) para manter a pressão contínua, permitindo o fluxo alternando livremente). 7: Durante a perfusão, o rim permanece numa câmara húmida para 100% de humidade atmosférica e 37 ° C Temperatura de rim. <a href="http://ecsource.jove.com/files/ftp_upload/54712/54712fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> 3. Cirúrgica Procedimento Parte 1 (para um diagrama de todas as ligaduras, consulte a Figura 3) Nota: Executar todas as ligaduras usando 5-0 fio cirúrgico. <ol><li> Anestesiar um rato por injecção intraperitoneal (10 mL / g de peso corporal, de 20 mg / ml de cetamina e 1 mg / ml de xilazina dissolvido em 0,9% de NaCl). Confirmar profundidade suficiente de AnesthAIAS por testar a ausência de reflexos traseira pés. </li><li> Corrigir o rato em decúbito dorsal na câmara húmida. Proteger os olhos com pomada veterinário. Coloque uma seringa de 1 ml abaixo da coluna vertebral para elevar os vasos lombares. </li><li> Realizar uma laparotomia mediana da crista púbica para a abertura do esterno primeira a pele, em seguida, os músculos abdominais, com uma tesoura. </li><li> Remover o intestino e colocá-lo no lado esquerdo da lateral do mouse a partir do abdômen. </li><li> Libertar a bexiga de tecido conjuntivo e explorar ambos os ureteres e uretra. </li><li> Coloque uma ligadura em torno do ureter esquerdo (ligadura I). Fecha-o. </li><li> Colocar uma ligadura em torno da uretra (ligadura II). Fecha-o. </li><li> Coloque um &quot;laço&quot; ligadura em torno de toda a bexiga (ligadura III). </li><li> Faça uma incisão na bexiga 1 mm. </li><li> Canular a abertura com dois centímetros PE 50 tubos. </li><li> Fechar ligadura III em torno do tubo. </li><li> Cortar o uréter esquerdo e uretra distai das ligaduras. o bladder agora está ligado ao ureter direito apenas e movimentando-se livremente. </li><li> Desmarque a aorta abdominal de tecido conjuntivo e gordura. </li><li> Coloque uma ligadura meados de aorta abdominal (ligadura IV). </li><li> Colocar uma ligadura em torno da aorta abaixo do diafragma entre a artéria mesentérica superior e o tronco celíaca (V ligadura). </li><li> Colocar uma ligadura à volta da artéria mesentérica superior (VI ligadura). </li><li> Coloque uma ligadura da aorta imediatamente abaixo do direito e acima da artéria renal esquerda (VII ligadura). </li><li> Coloque uma ligadura em torno do pacote de caudal veia (veia) (VIII ligadura). Vá para a Etapa 4. </li></ol><img alt="Figura 3" src="/files/ftp_upload/54712/54712fig3.jpg" /> Figura 3: Desenho esquemático das ligaduras colocadas durante a cirurgia. Vista do abdómen aberto após a laparotomia. O intestino é movido para fora para a esquerda. L e R indicam o rim esquerdo eo direito. olinhas pretas mostram a área da respectiva laqueação. Ligaduras primeira colocação e depois fechada, na sequência indicada no texto. X marca a localização da incisão para canulação da aorta. <a href="http://ecsource.jove.com/files/ftp_upload/54712/54712fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> 4. Priming do circuito de perfusão <ol><li> Inicie a bomba rotativa e encher o tubo com perfusato. Tome cuidado para esvaziar todas as bolhas de ar. </li><li> Encha o dispositivo Windkessel a cerca de nível médio com perfusato. </li><li> Calibra-se o transdutor de pressão até 0 mm de Hg quando toda a tubagem é cheio e o fluxo é 0. Manter a agulha de perfusão, a nível renal durante este tempo. </li><li> Manter o fluxo em um nível mínimo constante (0,6 ml / min) e vá para a Etapa 5. </li></ol> 5. Surgical Procedimento Parte 2 <ol><li> Coloque um grampo entre ligadura IV ea ramificação da rena esquerdal artéria. </li><li> Faça uma pequena incisão no caudal aorta de ligadura IV, tomando cuidado para não cortar a parede dorsal. </li><li> Dilatar a abertura da aorta com um vaso dilatador. </li><li> Canular a aorta, com uma agulha (2 cm de comprimento, puxado PE 50), empurrando a ponta apenas para o grampo. </li><li> Abra o grampo. </li><li> Empurrar a ponta da agulha cranialmente até atingir a junção da artéria renal direita e da aorta. </li><li> Fechar ligadura VII. </li><li> Fechar ligadura IV. </li><li> Abra o peito com uma tesoura dissecando o diafragma. Com um único corte, separe a aorta, a veia cava, coração e nervos vegetativos. Com este passo, o animal foi sacrificado através de exsanguinação sob anestesia profunda rápida contínua. </li><li> Iniciar o controle da pressão da bomba de perfusão. Manter a pressão média entre 80 e 100 mmHg. </li><li> Fechar V. ligadura </li><li> Fechar ligadura VI. </li><li> Fechar ligadura VIII. </li><li> Livre do rim direito de tissu conjuntivoe e sua incorporação na cápsula adiposo com uma tesoura. </li><li> Corte a aorta proximal à ligadura V. </li><li> Corte da artéria mesentérica superior distalmente à ligadura VI. </li><li> Cortar o feixe vascular-sustentável rim para fora, tomando cuidado para não cortar os próprios vasos. </li><li> Cortar o fígado na ligação para o rim. Cuidar para libertar o rim, mas deixa uma pequena parte do aderente fígado a ele, de modo que a veia cava é mantida aberta por ela. </li><li> Pegue o pacote de rim para fora do mouse. Remover o rato da câmara úmida. </li><li> Coloque um &quot;laço&quot; ligadura à volta da ligação de fígado e rim (IX ligadura). </li><li> Canular a veia cava com uma linha venosa (dois centímetros PE 50). </li><li> Fechar ligadura IX. fluxo venoso através do cateter venoso deve começar imediatamente. </li><li> Feche a câmara úmida. </li></ol> 6. As análises a jusante <ol><li> Durante a hora seguinte, monitorar continuamente o fluxo sanguíneo e intravasculpressão de ar 15. Recolhe fluxo venoso, que pode ser utilizado para análise de, por exemplo, a libertação de renina renal 7. Coleta de urina para análise da concentração de eletrólitos e taxa de filtração glomerular 14. Após 1 hora de perfusão, os rins podem ser snap-congelados por western blot ou ser fixado para a imagem latente aproxima 16. </li></ol>

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The authors have no competing financial interests and nothing else to disclose.

O mouse rim isolado perfundido é uma ferramenta para o estudo da função renal em um ambiente controlado ex vivo, durante 1 hora, fazendo a ponte entre experiências in vivo em animais intactos, o que pode ser falho pelo impacto de vários fatores sistêmicos e experimentos in vitro em segmentos dos néfrons isolados ou células cultivadas, o que necessariamente negligenciam o impacto da estrutura de órgãos intactos na função. Há, ao conhecimento dos autores, nenhuma técnica alternativ...

A perfusão isolada de órgãos tem sido objecto de um esforço contínuo entre os fisiologistas por muitas décadas 1. A técnica permite que a função do órgão, sem influências sistêmicas, tais como pressão arterial, hormônios ou nervos, a ser estudado. Carl Eduard Loebell é considerado para ser o primeiro a ter descrito a perfusão bem sucedida de um rim isolado, em 1849 2. Desde então, o aparelho de perfusão foi submetido refinamento significativa. Frey e Gruber introduzido um pulmão artificial para bombas de oxigenação e pulsátil para perfusão contínua 2. Enquanto os primeiros pesquisadores estudaram principalmente os rins de mamíferos de grande porte, ou seja, porcos e cães 2 3 -o primeiro relatório do uso de rins de ratos, por Weiss et al. , Foi um marco no estudo da perfusão-mamífero-órgão pequeno 4. Schurek et ai. relataram a necessidade de adição de eritrócitos de mamífero para o perfusato se tubular renal suficienteoxigenação deveria ser alcançado 5. Crítico para experiências a longo prazo foi a introdução de diálise contínua do tampão pelo mesmo grupo de pesquisa 6. Em 2003, Schweda et al. foram os primeiros a relatar um rim do rato funcional isolado perfundido (MIPK) 7, posteriormente refinado por Rahgozar et al. 18 e Lindell et ai. 14. Embora tecnicamente mais exigente do que o de rato perfundido isolado de rim, o uso do MIPK carrega a vantagem de permitir o uso de uma grande variedade de ratinhos geneticamente alterados. Este artigo apresenta os dados sobre o método dos autores para perfusão isolados de rato rins durante 1 h. O método permite a avaliação contínua da taxa renal fluxo, resistência vascular, liberação de hormônio, gasometria, análise de urina, bem como a aplicação de medicamentos. Seguindo o procedimento, rins podem ser processadas para análise molecular e bioquímica, ser fixo por microscopia, outransplantado para um rato receptor (Figura 1). <img alt="figura 1" src="/files/ftp_upload/54712/54712fig1.jpg" /> Figura 1: Visão geral do Possível entrada / saída para o rim isolado perfundido. BGA: Hemogasometria. <a href="http://ecsource.jove.com/files/ftp_upload/54712/54712fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Esta técnica provavelmente vai receber uma atenção crescente nos próximos anos, como muitas aplicações inovadoras estão sendo discutidas com o alvorecer da perfusão renal normotérmica prolongado antes do transplante (com ou sem a aplicação de anti-rejeição ou drogas edição do genoma) 8, 9, 10 , 11, a bioengenharia dos rins inteiros de andaimes descelularizados 12, bem como a aplicação de doses elevadas de corantes fluorescentes para a imagem latente multiphoton 13 </sup&gt;. Também é um modelo ideal com o qual se estudar o papel de genes específicos durante a lesão renal aguda 14. Um protocolo de passo-a-passo é dado para permitir que outros laboratórios para realizar isolado rato perfusão renal com sucesso. Em primeiro lugar, a composição e preparação do buffer é especificado. Em seguida, a cirurgia é descrito em pormenor e os passos críticos são mostrados. Em terceiro lugar, os dados são apresentados que são representativos de uma preparação bem sucedida: o fluxo sanguíneo renal, resistência vascular, taxa de filtração glomerular, e eletrólitos fraccionada medições funcionais de viabilidade e eletrônica de transmissão micrografias da morfologia dos diferentes segmentos dos néfrons de Rins todos excreção- fixado após 1 hora de perfusão.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Perfusion Circuit:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Moist chamber 834/8</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2901</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Cannular with basket and side port</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2947</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Thermostat TC120-ST5&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-4544</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">ISM 827/230V Roller Pump Reglo Analogue</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-0114</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Reservoir jacketed for buffer solution 1L</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3438</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Reservoir jacketed for buffer solution 0.5L</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3436</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Pressure Transducer APT300&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3862</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">TAM-D Plugsys Transducer</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-1793</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">SCP Plugsys servo controller</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2806</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Windkessel&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3717</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">HSE-USB data acquisition</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Low-Flux Dialysator Diacap Polysulfone</td>
			<td style="width:173px;">B.Braun</td>
			<td align="right" style="width:119px;">7203525</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:447px;">PE-Tubing for aorta cannulation 1.19mm I.D. x 1.70mm O.D.</td>
			<td style="width:173px;">Scientific Commodities Inc.</td>
			<td style="width:119px;">BB31695-PE/8</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:447px;">Name</td>
			<td style="width:173px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Buffer reagents:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Aminoplasmal 10%</td>
			<td style="width:173px;">B.Braun</td>
			<td align="right" style="width:119px;">134518064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Sodium pyruvate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2256-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">L-Glutamic acid monosodium salt hydrate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1626-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">L-(-)-Malic acid sodium salt</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">M1125-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Sodium-L-Lactate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">L7022-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">alpha-Ketoglutaric acid sodium salt</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">K1875-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">31434-1KG-R</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">NaHCO3</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761-5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">60130-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Urea</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">U5378-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Creatinine</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4255-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td style="width:173px;">Roche</td>
			<td align="right" style="width:119px;">10835242001</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgCl2 * 6H2O</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">M2393-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">D-Glucose</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8270-1KG</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">CaCl2 * 6H2O&#160;</td>
			<td style="width:173px;">Riedel-de-Ha&#235;n</td>
			<td align="right" style="width:119px;">12074</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">NaH2PO4</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9638-500G</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Na2HPO4</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S0876-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Antidiuretic Hormone dDAVP</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">V2013-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">FITC-Inulin</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Filter used for erythrocyte filtration</td>
			<td>Macherey-Nagel</td>
			<td style="width:119px;">MN 615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">BGA Analysis:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">ABL 80 flex</td>
			<td style="width:173px;">Radiometer Medical ApS</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Electron Microscope:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Philips CM100 TEM</td>
			<td style="width:173px;">FEI</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

the mouse isolated perfused kidney technique

O mouse rim isolado perfundido (MIPK) é uma técnica para manter um rim do rato em condições ex vivo perfundidos e funcionais durante 1 h. Este é um pré-requisito para o estudo da fisiologia do órgão isolado e para muitas aplicações inovadoras que possam ser possíveis no futuro, incluindo decellularization perfusão de bioengenharia nos rins ou a administração de anti-rejeição ou drogas edição do genoma em altas doses para preparar o rim para transplante. Durante o tempo da perfusão, o rim pode ser manipulada, a função renal pode ser avaliada, e vários produtos farmacêuticos administrados. Após o procedimento, o rim transplantado ou pode ser processado para a biologia molecular, bioquímica, ou microscopia. Este artigo descreve o perfusado e da técnica cirúrgica necessária para a perfusão ex-vivo de rins de rato. Os detalhes do aparelho de perfusão são dados e os dados são apresentados mostrando o vESPONSABILIDADE da preparação do rim: fluxo sangüíneo renal, resistência vascular, e urina dados como funcionais, micrografias eletrônicas de transmissão de diferentes segmentos dos néfrons como leituras morfológicas e Western blot de proteínas de transporte de diferentes segmentos dos néfrons como leitura molecular.

Com o método descrito, rins isolados do rato podem permanecer viáveis ​​durante pelo menos 1 h. Foi testada a viabilidade dos tecidos depois de 1 hora de perfusão contínua com funcional (fluxo sanguíneo renal e resistência vascular, gasometria do fluxo venoso, taxa de filtração glomerular, urinário fraccionada Na + e K + excreção, e osmolaridade urinária) e morfológico (eletrônica de transmissão microscopia, TEM) em quatro métodos rins de tipo se...

Watch this Scientific Journal Video about The Mouse Isolated Perfused Kidney Technique at JoVE.com

The Mouse Isolated Perfused Kidney Technique

O mouse rim isolado perfundido (MIPK) é uma técnica para manter um rim do rato em condições ex vivo perfundidos e funcionais durante 1 h. Os tampões e técnica cirúrgica encontram-se descritos em pormenor.

A técnica de rim perfundidos rato isolado

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

the-mouse-isolated-perfused-kidney-technique

Research

JoVE Journal

Biology

21.5K visualizações.  University of Zürich.  O mouse rim isolado perfundido (MIPK) é uma técnica para manter um rim do rato em condições ex vivo perfundidos e funcionais durante 1 h. Os tampões e técnica cirúrgica encontram-se descritos em pormenor. 

Vídeo: The Mouse Isolated Perfused Kidney Technique

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

Chip baseado em Cultura de Células tridimensional em perfundidos Micro-biorreatores

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Biologia

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Mapeamento óptica de corações perfundidos Langendorff-Rat

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo de imagens e tratamentos terapêuticos em um modelo de camundongo ortotópico de câncer de ovário

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Isolamento e cultura de células da zona nefrogênica do rim do rato embrionárias

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

Caracterização do isolado, ventilado, Lung Mouse e Instrumentados perfundidos com fluxo pulsátil

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

Transplante de células diretamente no Rim de Adulto Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Dissecção do rim Zebrafish Adulto

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

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

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

Geração de camundongos derivadas de células-tronco pluripotentes induzidas

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

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Parâmetro Multi-Medição da abertura dos poros transição de permeabilidade em mitocôndrias isoladas de coração Rato

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

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

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

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

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

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