Os estudos atuais foram apoiadas pela United States National Institutes of Health concessão R01-HL092188, R01-DK083385 e R01HL098294, e Fundação para a Educação Anestesia e Pesquisa (FAER) Dá para HKE e um DFG (Deutsche Forschungsgemeinschaft) Bolsa de Investigação (GR2121/1- 1) e American Heart Association Grant para AG.

Observações gerais. Todas as operações devem ser realizada sob um microscópio de dissecação vertical (Leica) e usando um coagulador cirúrgico. Os camundongos dos grupos experimentais e simulação deve ser compensada de idade e peso para garantir a comparabilidade dos resultados. Administração de temperatura, pressão arterial anestesia, e fluido deve ser estável e monitorizados durante toda a experiência. 1. Anestesia e Cirurgia <ol><li> Camundongos C57BL / 6 deve estar dentro de uma faixa etária de 10 a 16 semanas e eles devem ser pareados por sexo nos grupos respectivos. </li><li> Anestesiar os ratos com pentobarbital sódico (70 mg / kg ip de peso corporal) e manter a anestesia com aprox. 10 mg de sódio / kg / h pentobarbital. A sobredosagem pode reduzir significativamente a pressão arterial e, portanto, pode alterar os resultados. </li><li> Ratinhos lugar sobre uma mesa de temperatura controlada aquecida (RT, Effenberg, Munique, Alemanha) com uma sonda do termómetro rectal ligado a um retorno térmicocontrolador para manter a temperatura corporal a 37 ° C. </li><li> Ratinhos posicionar numa posição supina sobre a mesa de cirurgia, com as extremidades superiores e inferiores ligados à mesa com fita removível. </li><li> Em caso de experiências de sobrevivência adequadas técnicas assépticas, incluindo o corte de cabelo, a desinfecção da pele com iodóforos (Betadine, por exemplo), tratamento de estéril de instrumentos autoclavadas, e utilizando máscaras um luvas esterilizadas para o cirurgião são necessários. </li></ol> 2. Portal Tríade Oclusão <ol><li> Cobrir a área de incisão com óleo mineral para evitar a inalação de cabelo do rato através do corte da pele e prevenindo assim o risco de alergias cabelo de rato. Realizar laparotomia mediana e incisão da linha alba até a extremidade superior de modo que você pode facilmente expor o fígado. Se possível realizar a incisão com um eléctrodo de coagulação (Erbe, ICC50, Tuebingen, Alemanha) para evitar o sangramento. </li><li> Coloque a tabela de aquecimento com o mouse em uma posição de que a cauda é direçãoted para o cirurgião. Essa posição lhe fornece a melhor configuração para localizar a tríade e colocar a sutura. </li><li> Deslocar o intestino, no lado direito, utilizando uma compressa de algodão molhado ponta para expor a tríade portal. </li><li> Ligeiramente mudar o lobo mediano e esquerdo em direção ao diafragma e do lobo direito para o intestino. Isso lhe dará uma visão clara da tríade portal acima da bifurcação da direita, mediana / esquerda lobos. </li><li> Verifique cuidadosamente a tríade portal e segure o lobo direito, com cuidado, com uma pinça branda. Isso lhe dará uma bela vista sobre a tríade portal esquerda. </li><li> Uma vez identificada visualmente lugar a agulha com a sutura (7/0 de sutura de nylon; Ethicon, Norderstedt, Alemanha) por meio de um suporte de agulha sob a esquerda da tríade portal, incluindo a artéria hepática, a veia hepática, e vias biliares a partir do lado esquerdo para o lado direito do mouse. </li><li> O ponto tem de ser cuidadosamente sob a tríade. A mais profunda do ponto pode ferir a veia vaca inferior que está localizado soba tríade. Portanto, manter os lobos hepáticos direito para baixo com a pinça para identificar a veia cava antes de colocar o ponto. </li><li> Tomar a ponta da agulha, no lado direito com uma pinça e puxá-la suavemente para fora da área do fígado sem ferir o fígado. </li><li> Anexar a cada extremidade da sutura um tubo Eppendorf (aproximadamente 3 g, cheio de água) e colocar tanto a sutura termina sobre suportes de sutura. Coloque o suporte de sutura distalmente da costura de modo a que a sutura não interfere com a tríade direita. </li><li> Ao aplicar os pesos da tríade será imediatamente ocluída, causando a interrupção do fornecimento de sangue para os lobos esquerdo e mediano do fígado. Oclusão bem sucedida pode ser facilmente confirmado por inspeção visual de branqueamento pálida dos lobos isquêmicos. Em contraste, a mudança de cor imediatamente desaparece quando os pesos de suspensão são libertadas a partir dos pólos e do fígado é reperfundido. Este modelo de isquemia parcial hepática evita congestionamento mesentérica, preservando bloofluxo d para os lobos do fígado direito. </li><li> Substituir o intestino para a cavidade abdominal. Mantenha o fígado e intestino quente com um cotonete úmido embebido em água a 37,0 ° C. Além disso para manter a temperatura corporal estável cobrir os ratinhos com o envoltório de alimentos comercialmente disponível depois de terminar a cirurgia. </li><li> Execute isquemia, conforme exigido por protocolo experimental. Depois de um lançamento definida isquemia tempo os pesos e reperfusão dos lobos hepáticos médio e esquerdo começará imediatamente. Esta técnica permite a produção de isquemia reversível em ratinhos. Se não houver tempo de isquemia mais será realizado cuidadosamente remover a sutura. </li><li> Adicionar aproximadamente 400 ul de cloreto de sódio para a cavidade abdominal 15 minutos antes e imediatamente após a reperfusão. Continuar a substituir fluido a cada 30 minutos, numa quantidade de 100 ul para compensar a perda de fluido durante o tempo de reperfusão. </li><li> Para os experimentos de sobrevivência da ferida cirúrgica é fechada com sutura contínua da parede muscular e da pele with uma sutura 4/0. </li></ol> 3. Alterações na perfusão de fígado Os parâmetros de resultados a seguir são recomendados para determinar fígado isquemia hepática seguinte: Determinar as enzimas hepáticas (por exemplo, ALT, AST) e histologia hepática (coloração H &amp; E) de isquemia e reperfusão hepática seguinte 3,9. <img alt="A Figura 1" src="/files/ftp_upload/2550/2550fig1.jpg" /> A Figura 1 Modelo de uma suspensão do sistema de peso-para isquemia do fígado:. Um sistema de suspensão de peso foi estabelecida para induzir isquemia do fígado do lobo esquerdo e mediana 3. (A) Por conseguinte lobo caudado e direita foram cuidadosamente separadas do lobo esquerdo. O lobo direito foi, então, ligeiramente deslocado para ver claramente a tríade portal acima da bifurcação da direita, lobos mediano e esquerdo. Uma sutura de nylon 8,0 foi colocada sob a tríade hepático esquerdo. (B) No final de cada sutura um peso pequeno (3 g Eppendorf TUbes cheio com água) foi ligado e aplicada. Isquemia hepática é visível por uma mudança de cor do lobo esquerdo e mediana de vermelho para pálido. Este projeto experimental permite a oclusão precisa e reperfusão da tríade hepática esquerda, aplicando e liberando a carga de peso. <img alt="A Figura 2" src="/files/ftp_upload/2550/2550fig2.jpg" /> Figura 2. Efeito de diferentes tempos de isquemia do fígado em alanina (ALT) e aspartato (AST) aminotransferase em ratinhos. Parcial portal isquemia tríade foi induzida como indicado (0 - 60 min de isquemia). Após 2 horas de reperfusão, ALT (A) e AST (B) foram medidos. Os resultados são expressos como médias ± SE de 6 a 7 ratos / grupo. * P &lt;0,05 comparado com 0 min de isquemia (sham controle).

<ol>
	<li>Rook, M., Rand, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictors+of+long-term+outcome+after+liver+transplant.">Predictors of long-term outcome after liver transplant.</a> Curr. Opin. Organ. Transplant. 16, 499-504 (2011).</li><li>Henrion, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia/reperfusion+injury+of+the+liver:+pathophysiologic+hypotheses+and+potential+relevance+to+human+hypoxic+hepatitis.">Ischemia/reperfusion injury of the liver: pathophysiologic hypotheses and potential relevance to human hypoxic hepatitis.</a> Acta. Gastroenterol. Belg. 63, 336-347 (2000).</li><li>Hart, M. L., Much, C., Kohler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+hanging-weight+system+for+liver+ischemic+preconditioning+in+mice.">Use of a hanging-weight system for liver ischemic preconditioning in mice.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 294, 1431-1440 (2008).</li><li>Schneider, M., Geyte, K., Fraisl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+or+silencing+of+the+PHD1+prolyl+hydroxylase+protects+livers+of+mice+against+ischemia/reperfusion+injury.">Loss or silencing of the PHD1 prolyl hydroxylase protects livers of mice against ischemia/reperfusion injury.</a> Gastroenterology. 138, 1143-1154 (2010).</li><li>Coriat, R., Leconte, M., Kavian, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mangafodipir+Protects+against+Hepatic+Ischemia-Reperfusion+Injury+in+Mice.">Mangafodipir Protects against Hepatic Ischemia-Reperfusion Injury in Mice.</a> PLoS One. 6, e27005 (2011).</li><li>Sakai, N., Van Sweringen, H. L., Schuster, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+activator+of+NF-kappaB+ligand+(RANKL)+protects+against+hepatic+ischemia/reperfusion+injury+in+mice.">Receptor activator of NF-kappaB ligand (RANKL) protects against hepatic ischemia/reperfusion injury in mice.</a> Hepatology. , (2011).</li><li>Zhang, J., Hu, W., Xing, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Protective+Role+of+CD59+and+Pathogenic+Role+of+Complement+in+Hepatic+Ischemia+and+Reperfusion+Injury.">The Protective Role of CD59 and Pathogenic Role of Complement in Hepatic Ischemia and Reperfusion Injury.</a> Am. J. Pathol. 179, 2876-2884 (2011).</li><li>Beldi, G., Banz, Y., Kroemer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+CD39+on+natural+killer+cells+attenuates+hepatic+ischemia/reperfusion+injury+in+mice.">Deletion of CD39 on natural killer cells attenuates hepatic ischemia/reperfusion injury in mice.</a> Hepatology. 51, 1702-1711 (2010).</li><li>Hart, M. L., Much, C., Gorzolla, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+adenosine+production+by+ecto-5'-nucleotidase+protects+during+murine+hepatic+ischemic+preconditioning.">Extracellular adenosine production by ecto-5'-nucleotidase protects during murine hepatic ischemic preconditioning.</a> Gastroenterology. 135, 1739-1750 (2008).</li><li>Beraza, N., Ludde, T., Assmus, U., Roskams, T., Vander Borght, S., Trautwein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte-specific+IKK+gamma/NEMO+expression+determines+the+degree+of+liver+injury.">Hepatocyte-specific IKK gamma/NEMO expression determines the degree of liver injury.</a> Gastroenterology. 132, 2504-2517 (2007).</li><li>Lentsch, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+and+function+of+hepatocyte+NF-kappaB+in+postischemic+liver+injury.">Activation and function of hepatocyte NF-kappaB in postischemic liver injury.</a> Hepatology. 42, 216-218 (2005).</li><li>Luedde, T., Assmus, U., Wustefeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+IKK2+in+hepatocytes+does+not+sensitize+these+cells+to+TNF-induced+apoptosis+but+protects+from+ischemia/reperfusion+injury.">Deletion of IKK2 in hepatocytes does not sensitize these cells to TNF-induced apoptosis but protects from ischemia/reperfusion injury.</a> J. Clin. Invest. 115, 849-859 (2005).</li><li>Kapitsinou, P. P., Liu, Q., Unger, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+HIF-2+regulates+erythropoietic+responses+to+hypoxia+in+renal+anemia.">Hepatic HIF-2 regulates erythropoietic responses to hypoxia in renal anemia.</a> Blood. , (2010).</li></ol>

Não há conflitos de interesse declarados.

O presente estudo descreve uma técnica de realização de isquemia num modelo murino, utilizando uma suspensão do sistema de peso para a oclusão da esquerda tríade hepática portal. Embora seja um método fácil para aprender as armadilhas que se seguem devem ser tidos em conta. Um erro muito comum no início é um ponto de profundidade, que inclui a tríade portal e da veia cava. A taxa de respiração e de coração irá abrandar no prazo de 15 minutos, devido ao aumento da resistência e da diminuição no retorno do sangue ao coração, resultando em morte precoce um dos ratinhos. A melhor maneira de impedir que o incluindo da veia cava é localizar a veia cava antes do ponto mantendo os lobos direito para baixo com um fórceps. Outra questão importante é que os pesos não são pesados ​​o que pode resultar em muito estresse sobre o julgamento portal esquerda e direita que também interrompe o fluxo sanguíneo para os lobos direito. Por conseguinte, a quantidade de água na frascos Eppendorf deve ser ajustada para o peso do rato eo sdetentores RÓXIMOS deve ser colocada distalmente a partir da localização do ponto. No lado oposto, se os pesos são a baixa pressão sanguínea não é completamente interrompido. Ambos os erros são facilmente detectáveis ​​por uma mudança de cor adicional para pálido do lobo direito ou mudança de cor insuficiente dos lobos mediano e esquerdo, respectivamente. Por conseguinte, a mudança de cor hepática após a aplicação dos pesos deve ser controlada a cada 10 minutos. Outra complicação comum cirúrgica é o ferindo do fígado com a agulha. Por conseguinte, os lobos direito deve ser mantido para baixo com um fórceps brandas de modo que a ponta da agulha pode ser facilmente vistos e tomado com um suporte de agulha. Se estas armadilhas no início são tidos em conta a suspensão do sistema de peso-para isquemia hepática fornece uma lesão altamente reprodutível devido à isquemia, minimizando a variabilidade de danos no fígado associado com aperto da tríade portal. Ao utilizar uma suspensão do sistema de peso, a tríade portal é apenas manipulado uma vez ao longo de todoprocedimento cirúrgico, causando significativamente menos danos para os lobos hepáticos. Além disso, nenhum congestão hepática ou intestinal ocorre com esta técnica. Trauma mais tecido ocorre por remoção e substituição de braçadeiras de navios, especialmente com reaplicação do grampo durante os ciclos de isquemia múltiplos como durante o pré-condicionamento isquêmico. O uso de pesos que são penduradas em um local remoto a partir do tecido do fígado, de acordo com as nossas observações, proporciona a vantagem de oclusão fiável, evitando trauma dos tecidos devido à manipulação dos lobos hepáticos por reaplicação de uma braçadeira. Tomados em conjunto, o presente estudo fornece viabilidade do sistema de enforcamento peso para oclusão da tríade portal durante a isquemia, minimizando a variabilidade e limitações associadas com aperto. Assim, esta técnica pode ser de interesse para outros pesquisadores que consideram estudando isquemia hepática.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Pentobarbital sódico (Plus Fatal) </td><td> Vortech Farmacêutica Ls, Ltd </td><td> VPL 9372 </td><td> 4 mg / mL em solução salina </td></tr><tr><td> Sutura, prolene 8,0 </td><td> Ethicon, EUA </td><td> M8739 </td><td> reutilizável </td></tr><tr><td> microscópio de dissecção </td><td> Leica </td><td> n / a </td><td> considerar o trabalho à distância generosa </td></tr><tr><td> Tabela Aquecimento </td><td> Rt, Effenberger, Alemanha </td><td> n / a </td><td> único fornecedor e único </td></tr><tr><td> Os instrumentos cirúrgicos </td><td> WPI, Dumont </td><td></td><td> Fórceps, porta-agulhas </td></tr><tr><td> Coagulador </td><td> Erbe, ICC50, Tuebingen, Alemanha </td><td> n / a </td><td> Diferentes provedores de coagulators </td></tr></tbody></table> Tabela 1. Reagentes específicos e equipamentos.

use of a hanging-weight system for liver ischemia in mice

Lesão hepática aguda por isquemia pode ocorrer durante diversos procedimentos clínicos de transplante de fígado, por exemplo, a ressecção do tumor hepático ou reparação trauma e pode resultar em insuficiência hepática, que tem uma alta taxa de mortalidade 1-2. Portanto estudos murino de isquemia hepática têm se tornado um importante campo de pesquisa, proporcionando a oportunidade de utilizar estudos farmacológicos e genéticos 3-9. Especificamente, os camundongos com deleção condicionais tecido específico de um gene (CRE, Flox sistema) fornecer insights sobre o papel de proteínas em tecidos específicos 10-13. Devido à dificuldade técnica associada com manualmente aperto da tríade portal em ratos, foi realizada uma avaliação sistemática utilizando uma suspensão do sistema de peso-para o portal de oclusão tríade que foi previamente descrito 3. Ao utilizar uma suspensão do sistema de peso que se coloca um sutura em torno do ramo esquerdo da tríade portal sem causar qualquer dano para os lobos hepáticos, uma vez que também amais finos disponíveis braçadeiras pode causar danos no tecido hepático, devido à localização perto do tecido do fígado para os vasos. Além disso, o ramo direito da tríade hepática ainda é perfundido, portanto, nenhuma congestão intestinal ocorre com esta técnica como o fluxo sanguíneo para os lobos hepáticos direito é preservado. Além disso, a tríade portal é apenas uma vez manipulado durante todo o procedimento cirúrgico. Como resultado, os procedimentos de procura de pré-condicionamento, com tempos curtos de isquemia e reperfusão, pode ser facilmente executada. Uma avaliação sistemática do modelo, realizando isquemia diferente e os tempos de reperfusão revelaram uma estreita correlação do tempo de isquemia hepática com lesão hepática medida pela alanina (ALT) e aspartato (AST) aminotransferases níveis séricos 3,9. Tomados em conjunto, esses estudos confirmam lesão hepática altamente reprodutível quando se utiliza o sistema de enforcamento peso para isquemia e reperfusão hepática intermitente. Assim, esta técnica pode ser útil para o interesse outros investigadoresed em estudos de fígado isquemia em ratinhos. Por conseguinte, o clipe de vídeo fornece uma descrição passo-a-passo detalhada desta técnica.

Watch this Scientific Journal Video about Use of a Hanging-weight System for Liver Ischemia in Mice at JoVE.com

Use of a Hanging-weight System for Liver Ischemia in Mice

Nós estabelecemos um novo modelo murino de um sistema de peso pendurado por isquemia hepática. Esta técnica pode ser útil para futuras investigações de isquemia em modelos murinos hepáticas.

A utilização de uma suspensão do sistema de peso para isquemia hepática em ratos

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and ...

use-of-a-hanging-weight-system-for-liver-ischemia-in-mice

Research

JoVE Journal

Medicine

15.8K visualizações.  University of Colorado, Denver. Nós estabelecemos um novo modelo murino de um sistema de peso pendurado por isquemia hepática. Esta técnica pode ser útil para futuras investigações de isquemia em modelos murinos hepáticas.

Vídeo: Use of a Hanging-weight System for Liver Ischemia in Mice

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important 7, 14, 30, 39. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.
Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP 39. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.
We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds39. In addition to infarct staining, we tested cardiac troponin I (cTnI)
as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction6, 8, 30, 40, 41. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.

A murine model for myocardial ischemia and ischemic preconditioning is an important tool study cardioprotective mechanisms in vivo. Here, we report an easy applicable in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion.

Uso de um Sistema de Peso de suspensão para oclusão da artéria coronária em ratos

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. ...

Medicina

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

Uso de um sistema de suspensão de peso para oclusão da artéria renal isolada

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Uso de Modelo Animal de Sepse para avaliar novas terapias à base de plantas

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusões artéria carótida para farmacocinéticas e farmacodinâmicas Análise de taxanos em Ratos

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Fototrombose induzida por isquemia focal como um Modelo de Lesão Medular em Ratos

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

Use of a Low-flow Digital Anesthesia System for Mice and Rats

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers utilize a syringe pump to directly administer volatile anesthetics into a gas stream. Unlike a traditional vaporizer, it can be used at very low flow rates, making it ideal for use on mice and rats. 
The equipment's capability to use low flow rates could result in a substantial cost savings due to the reduced need for anesthetic agents, compressed gas, and charcoal scavenging filters1. A lower flow rate means less waste of anesthetic gas and likely reduces the risk of anesthetic exposure to laboratory personnel. Thus, the high levels of precision and safety associated with direct injection vaporizers, along with a reduced need for anesthetic agents, compressed gas, and charcoal filters are beneficial for research requiring small animal anesthesia. 
The goal of this protocol is to demonstrate the use of a syringe-driven direct injection vaporizer as part of a digital, low-flow anesthesia system. The direct injection vaporizer is capable of accurately delivering anesthesia at very low flow rates compared to a traditional vaporizer, making it a promising alternative for controlled gas anesthetic delivery to rodents.

Here, we present a protocol to more safely and efficiently administer anesthetic gas to mice using a digital, low flow anesthesia system utilizing a syringe-driven direct injection vaporizer.

O uso de um baixo fluxo Sistema de Anestesia Digital para camundongos e ratos

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Guiada por ultra-som intracardíaca injeção de células-tronco mesenquimais humanas para aumentar Homing para o intestino para uso em modelos murino de doenças intestinais inflamatórias Experimental

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Murine Precision-Cut Liver Slices como um modelo ex vivo de biologia hepática

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

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

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

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

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

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

Transplante de fígado em camundongo e análise da perviedade do aloenxerto

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality images to be obtained of micro-vasculature and are high-fidelity tools in the field of organ transplantation. In this model of orthotopic liver transplantation (OLT) in mice, microCT affords the opportunity to evaluate allograft anastomosis in real time and has the added benefit of not having to sacrifice study animals. The choice of contrast, as well as image acquisition settings, create a high-definition image, which gives researchers invaluable information. This allows for evaluation of the technical aspects of the procedure as well as potentially evaluating different therapeutics over an extended duration of time. In this protocol, we detail an OLT model in mice in a stepwise fashion and finally describe a microCT protocol that can give high-quality images, which aid researchers in in-depth analysis of solid organ transplantation. We provide a step-by-step guide for liver transplantation in a mouse, as well as briefly discuss a protocol for evaluating the patency of the graft through microCT angiography.

Autor em destaque: Aprimorando a pesquisa de transplante por meio de angiografia MicroCT em modelos murinos 

In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans.

Transplante Ortotópico de Fígado em Camundongos Utilizando Angiotomografia Microcomputadorizada

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality ...