O trabalho deste manuscrito foi financiado pelo Dr. Neal pelo Programa Piloto do Instituto de Medicina Vascular em Hemostasia e Biologia Vascular (P3HVB) e pela AAST Research Fellowship. Este trabalho é apoiado por US National Institutes of Health concede 1 R35 GM119526-01 e UM1HL120877-01.

Os murganhos foram alojados de acordo com as orientações de cuidados com animais da Universidade de Pittsburgh (Pittsburgh, PA, EUA) e National Institutes of Health (NIH, Bethesda, MD, EUA) em condições livres de agentes patogénicos com ciclos claro-escuro de 12 h e livre acesso a Alimentação padrão e água. Todos os experimentos com animais foram aprovados e conduzidos de acordo com as diretrizes estabelecidas pelo Animal Research and Care Committee da Universidade de Pittsburgh. 1. Campo Cirúrgico e Configuração do Instrumento <ol><li> Esterilize todos os instrumentos cirúrgicos, sutura, gaze, aplicadores de ponta de algodão, tubulação e conectores de tubulação antes do procedimento. <ol><li> Esterilizar instrumentos cirúrgicos, sutura, gaze e aplicadores de ponta de algodão em uma autoclave. Esterilizar os conectores da tubulação e da tubulação com óxido de etileno. </li></ol></li><li> Campo cirúrgico <ol><li> Ligue uma almofada de aquecimento de circulação de água e ajuste para 37 ° C. Coloque um surgiCal azul pad em cima dela e, em seguida, um drapeado estéril em cima da almofada azul cirúrgica. </li><li> Abra todos os instrumentos esterilizados sobre a cortina estéril. Use luvas estéreis para evitar quebrar a esterilidade durante esta etapa. </li><li> Encha uma tigela de aço inoxidável com etanol a 70% e reserve. Isto será usado para limpar instrumentos entre animais. </li><li> Ligue o esterilizador de micro bolas e deixe aquecer até 150 ° C. Isto também será usado para limpar instrumentos entre animais. Se realizar a cirurgia em mais de 5 ratos, certifique-se de mudar os instrumentos para um novo conjunto estéril. </li></ol></li><li> Configuração do Transdutor <ol><li> Conecte um transdutor estéril, tubulação PE-50, duas agulhas 23G e macho-macho luer, e uma torneira de três vias. 6 </li><li> Calibrar e zerar o transdutor de acordo com as instruções do fabricante. </li></ol></li></ol> 2. Procedimento Cirúrgico de Laceração do Fígado <ol><li> Indução e posicionamento da anestesia <li> Injectar pentobarbital sódico por via intraperitoneal à dose de 70 mg / kg. A anestesia deve ter efeito entre 5-10 min; Avaliar a profundidade da anestesia com uma pitada de dedo do pé. Se o rato tiver resposta ao pinçamento do dedo do pé, é necessário tempo adicional ou anestesia. Se for necessária anestesia adicional durante o procedimento, suplementar pentobarbital de sódio. Não administrar suplementos em quantidades maiores do que 0,05 mL para evitar a sobredosagem. </li><li> Depois que o mouse está completamente sob anestesia, posicione o mouse em posição supina em uma placa cirúrgica. Segure os quatro membros do mouse na placa com fita adesiva. </li><li> Raspar o abdômen e as virilhas bilaterais com uma navalha. </li><li> Embeber gaze estéril com betadine e aplicar no abdómen e virilhas bilaterais para a cirurgia. Para experimentos de sobrevivência, prepare o abdómen e as virilhas com betadina seguido de etanol para um total de três ciclos de preparação. </li><li> Insira uma sonda de temperatura retal para monitorar a temperatura do núcleo durante todo o procedimento. Mantenha o cTemperatura entre 35-37 ° C. </li></ol></li><li> Artéria femoral e cânula venosa <ol><li> Para instalação de cateter venoso: preencher a tubagem de PE-10, uma agulha de 30 G e uma torneira de três vias com solução de Ringer lactato de um saco IV. </li><li> Para instalação do cateter arterial: preencher a tubagem de PE-10 e agulha 30G com solução salina heparinizada (diluição 1:10 de 1.000 U de heparina). Heparina-salina é necessária para evitar a coagulação. </li><li> Coloque o mouse sob um microscópio de dissecação. </li><li> Faça uma incisão longitudinal de 4-5 mm sobre o músculo da virilha usando tesoura de íris cirúrgica. Usando pinças Dumont agarrar o tecido adiposo conectado ao músculo adutor e puxar lateralmente para uma exposição limpa do feixe femoral. Não dissecar através do tecido adiposo, pois isso levará a lesão vascular. </li><li> Cuidadosamente dissecar o nervo longe da artéria e veia com a pinça Dumont. Há um bloco de gordura adjacente ao nervo. Pegue isso com um Dumont forcep umD puxar lateralmente; Isso puxa o nervo longe da artéria criando um plano para dissecção. Com outros Dumont forcep dissecar sem rodeios o tecido conjuntivo entre o nervo ea artéria. <ol><li> Não agarre o nervo durante esta parte da dissecção. </li></ol></li><li> Loop três suturas de seda 6-0 em torno da artéria e veia proximal ao femoral profunda decolar. <ol><li> Coloque a sutura 1 mais proximalmente e deixe solta. </li><li> Coloque a sutura 2 mais distalmente e amarre imediatamente. </li><li> Coloque a sutura 3 entre a sutura 1 e 2 e deixe solta. </li></ol></li><li> Faça uma arteriotomia na superfície ventral do vaso. O uso de tesoura microvascular é recomendado para fazer a arteriotomia para evitar a transecção do vaso. </li><li> Insira o cateter de três vias na artéria. Imediatamente amarrar Sutura 1 e 2 para fixar o cateter no lugar. </li><li> Conecte o cateter de três vias ao transdutor e colete os dados da pressão arterial basal. </li><li> Repita os passos 2.2.4 - 2.2.6 na virilha oposta. Canular a veia femoral de forma semelhante à artéria. Realizar uma venotomia na superfície ventral do vaso seguido de inserção do cateter. Este cateter pode ser utilizado para administração de fluido ou fármaco. </li></ol></li><li> Laceração do fígado <ol><li> Pré-pesar um tubo contendo 0,5 mL de PBS, três triângulos de absorção e um barco de pesagem por mouse. </li><li> Faça uma incisão da laparotomia da linha média ventral começando no processo xifóide e estendendo-se caudalmente para permitir a exposição do fígado completamente. </li><li> Insira um triângulo de absorção no abdômen contra a parede abdominal direita. Repita no lado esquerdo. <ol><li> Certifique-se que o triângulo de absorção está longe do fígado para evitar um efeito hemostático de embalagem após o fígado ser lacerado. </li></ol></li><li> Cuidadosamente pegue o lobo meio-esquerdo do fígado e lacerar 75% do lobo com uma tesoura de íris cirúrgica. Coloque o laceradoD num tubo contendo PBS. </li><li> Feche a parede abdominal com agrafos através de um aplicador de grampos. Segure a pele eo músculo em conjunto e disparar o grampo. Faça isso o mais rápido possível após a laceração do fígado para evitar a perda de sangue fora do abdômen. Em experimentos de sobrevivência, o abdômen é fechado em duas camadas. Uma sutura absorvível corrente para o músculo seguida por uma camada em execução de sutura não absorvível para a pele proporciona um fecho adequado. </li><li> Para ratinhos que sejam para tempos de sobrevivência superiores a 30 minutos, os cateteres femorais devem ser removidos, a artéria ea veia amarradas com a sutura 3 do passo 2.2.6. As virilhas bilaterais são então fechadas em duas camadas como descrito no passo anterior. </li><li> Após um período específico de interesse para hemorragia (30 min até 72 h), retire os grampos. Remova os triângulos de absorção e coloque em pesados ​​pesados ​​pré-pesados ​​correspondentes. Use triângulos de absorção adicionais para absorver qualquer sangue não absorvido. </li><li&gt; Pesar filtros de absorção para calcular a perda total de sangue. </li></ol></li><li> Cuidados pós-operatórios <ol><li> Deixar os ratos que devem ser sacrificados aos 30 min na placa cirúrgica e sob monitoração constante e sob anestesia completa até o momento do sacrifício. Os ratinhos são eutanasiados com uma combinação de punção cardíaca e uma sobredosagem de isoflurano inalado. </li><li> Coloque camundongos que são para pontos de tempo de sobrevivência mais longa em uma gaiola de recuperação em cima de uma almofada de aquecimento de circulação de água. Constantemente monitorar os ratos durante a recuperação e não deixar sem vigilância até que recuperar a consciência para manter a recumbência esternal. Retornar o mouse para gaiola espaço com outros ratos apenas uma vez que se recuperou da anestesia. </li><li> Administrar analgesia pós-operatória com 0,1 mg / kg de buprenex via injeção subcutânea uma vez acordado da anestesia e a cada 12 h após até o momento do sacrifício. </li><li> Permitir aos ratos acesso livre a comida e água depois de serem devolvidos aos seusGaiolas normais pós-operatórias. </li><li> No momento do sacrifício para ratinhos de sobrevivência, a anestesia é realizada com isoflurano inalado. Uma vez sob anestesia, o sangue é recolhido através de uma punção cardíaca direita, a perda de sangue é registada como descrito acima e finalmente a eutanásia é segurada com uma sobredosagem de isoflurano. </li></ol></li></ol>

<ol>
	<li>Chang, R., Cardenas, J. C., Wade, C. E., Holcomb, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+understanding+of+trauma-induced+coagulopathy.">Advances in the understanding of trauma-induced coagulopathy.</a> Blood. 128 (8), 1043-1049 (2016).</li><li>Kutcher, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+paradigm+shift+in+trauma+resuscitation:+evaluation+of+evolving+massive+transfusion+practices.">A paradigm shift in trauma resuscitation: evaluation of evolving massive transfusion practices.</a> JAMA surgery. 148 (9), 834-840 (2013).</li><li>Tsukamoto, T., Pape, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+for+Trauma+Research.">Animal Models for Trauma Research.</a> Shock. 31 (1), 3-10 (2009).</li><li>Darwiche, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudofracture:+an+acute+peripheral+tissue+trauma+model.">Pseudofracture: an acute peripheral tissue trauma model.</a> J Vis Exp. (50), (2011).</li><li>Lomas-Niera, J. L., Perl, M., Chung, C. -. S., Ayala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shock+and+Hemorrhage:+an+Overview+of+Animal+Models.">Shock and Hemorrhage: an Overview of Animal Models.</a> Shock. 24, 33-39 (2005).</li><li>Kohut, L. K., Darwiche, S. S., Brumfield, J. M., Frank, A. M., Billiar, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixed+volume+or+fixed+pressure:+a+murine+model+of+hemorrhagic+shock.">Fixed volume or fixed pressure: a murine model of hemorrhagic shock.</a> J Vis Exp. (52), (2011).</li><li>Matsuoka, T., Hildreth, J., Wisner, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+injury+as+a+model+of+uncontrolled+hemorrhagic+shock:+resuscitation+with+different+hypertonic+regimens.">Liver injury as a model of uncontrolled hemorrhagic shock: resuscitation with different hypertonic regimens.</a> J Trauma. 39 (4), 674-680 (1995).</li><li>Komachi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+and+Robust+Multilayered+Poly(lactic+acid)+Nanosheets+for+Hemostatic+Dressing+in+Liver+Injury+Model.">Adhesive and Robust Multilayered Poly(lactic acid) Nanosheets for Hemostatic Dressing in Liver Injury Model.</a> J. Biomed. Mater. Res. Part B Appl. Biomater. , (2016).</li><li>Orfanos, N. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+antioxidants+on+a+porcine+model+of+liver+hemorrhage.">The effects of antioxidants on a porcine model of liver hemorrhage.</a> J Trauma Acute Care Surg. 80 (6), 964-971 (2016).</li><li>Morgan, C. E., Prakash, V. S., Vercammen, J. M., Pritts, T., Kibbe, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+4+different+rat+models+of+uncontrolled+hemorrhage.">Development and validation of 4 different rat models of uncontrolled hemorrhage.</a> JAMA Surgery. 150 (4), 316-324 (2015).</li><li>Rosselli, D. D., Brainard, B. M., Schmiedt, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+a+topical+bovine-derived+thrombin+solution+as+a+hemostatic+agent+in+a+rodent+model+of+hepatic+injury.">Efficacy of a topical bovine-derived thrombin solution as a hemostatic agent in a rodent model of hepatic injury.</a> Can J Vet Res. 14 (14), 303-308 (2015).</li><li>Sheppard, F. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Nonhuman+Primate+(Rhesus+Macaque)+Model+of+Uncontrolled+Traumatic+Liver+Hemorrhage.">Development of a Nonhuman Primate (Rhesus Macaque) Model of Uncontrolled Traumatic Liver Hemorrhage.</a> Shock. 44, 114-122 (2015).</li><li>Nemzek-Hamlin, J. A., Hwang, H., Hampel, J. A., Yu, B., Raghavendran, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+murine+model+of+blunt+hepatic+trauma.">Development of a murine model of blunt hepatic trauma.</a> Comp Med. 63 (5), 398-408 (2013).</li><li>Vogel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-derived+HMGB1+is+a+critical+mediator+of+thrombosis.">Platelet-derived HMGB1 is a critical mediator of thrombosis.</a> J Clin Invest. 125 (12), (2015).</li><li>Modery-Pawlowski, C. L., Tian, L. L., Ravikumar, M., Wong, T. L., Sen Gupta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+hemostatic+capabilities+of+a+functionally+integrated+platelet-mimetic+liposomal+nanoconstruct.">In vitro and in vivo hemostatic capabilities of a functionally integrated platelet-mimetic liposomal nanoconstruct.</a> Biomaterials. 34 (12), 3031-3041 (2013).</li></ol>

Os autores não têm interesses financeiros concorrentes para declarar.

O modelo de laceração de fígado murino aqui descrito fornece um modelo confiável e consistente de hemorragia não controlada. Este modelo é simples de executar, mas há etapas importantes que exigem consideração meticulosa. A parte mais desafiadora tecnicamente do modelo é a canulação dos vasos femorais para monitorização hemodinâmica e administração de fluido / fármaco. Cuidado deve ser tomado durante a dissecção do nervo e da arteriotomia / venotomia. É importante não tocar o nervo durante a dissec...

O trauma é a principal causa de morte e incapacidade entre os jovens em todo o mundo. 1 A hemorragia não controlada continua a ser uma das principais causas de mortalidade entre os pacientes com traumatismos gravemente feridos. 2 O tratamento do paciente com trauma hemorrágico é duplo: controle do sangramento cirúrgico e ressuscitação e reposição do sangue perdido. Modelos animais de choque hemorrágico têm sido a pedra angular na pesquisa de trauma e podem ser utilizados na avaliação da fisiopatologia e tratamento de choque traumático / hemorrágico. 3 , 4 O choque em modelos animais pode ser conseguido amplamente por dois métodos: hemorragia controlada e hemorragia descontrolada. 5 , 6 A hemorragia controlada é realizada pela remoção de um volume fixo de sangue ou por remoção de sangue para atingir determinada pressão sanguínea (pressão fixa). Enquanto oOs modelos são úteis na avaliação dos mecanismos e das alterações imunes no choque hemorrágico, não são aplicáveis ​​ao teste de agentes hemostáticos e não imitam o cenário clínico de hemorragia após trauma. Nesse sentido, procurou-se desenvolver um modelo de hemorragia descontrolada que nos permitisse testar alterações hemostáticas e agentes pró-coagulantes em um modelo murino. O fígado é uma opção atraente para a hemorragia descontrolada, em parte por causa do fornecimento de sangue dupla para o fígado e é um dos mais comumente lesionado órgãos intrabdominais, tanto em trauma fechado e penetrante. Dada a alta relevância clínica, o fígado tem sido utilizado como um modelo de hemorragia não controlada, mais comumente em modelos de ratos e porcinos, mas recentemente também em primatas. 7 , 8 , 9 , 10 , 11 , 12 </suModelos murinos também incorporaram lesão hepática, como um modelo de esmagamento ou trauma fechado; Contudo, estes modelos não resultam em choque hemorrágico secundário à lesão hepática. 13 , 14 Os modelos de ratos e porcinos de hemorragia hepática descontrolada, embora valiosos na observação de práticas de ressuscitação e monitorização hemodinâmica, são menos vantajosos do que um modelo murino por várias razões, tais como custo, número de animais utilizados e, sobretudo, a falta relativa de linhas transgénicas disponíveis para análise De sinalização celular e molecular específica. O presente modelo de murino compartilha semelhanças importantes com modelos de hemorragia hepática existentes, incluindo laceração hepática padronizada, quantificação de perda de sangue, monitorização hemodinâmica e capacidade para realizar a análise de sobrevivência. Muitos modelos existentes incorporam apenas alguns destes aspectos, enquanto o nosso modelo foi desenvolvido para medir muitas das variações fisiológicasBles simultaneamente e em múltiplos ratos. Além disso, o desenvolvimento de um modelo murino abre a porta a investigações além da reanimação e a mecanismos fisiopatológicos maiores em hemorragia descontrolada com o potencial de um modelo de baixo custo e alto rendimento usando técnicas moleculares avançadas.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>SS/45 dumonts</td>
			<td>Fine Science Tools</td>
			<td>11203-25</td>
		</tr>
		<tr>
			<td>surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14068-12</td>
		</tr>
		<tr>
			<td>hemostats</td>
			<td>Fine Science Tools</td>
			<td>13009-12</td>
		</tr>
		<tr>
			<td>microscissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
		</tr>
		<tr>
			<td>0.8mm curved forceps</td>
			<td>Fine Science Tools</td>
			<td>11009-13</td>
		</tr>
		<tr>
			<td>suture reel 6-0</td>
			<td>Fine Science Tools</td>
			<td>18020-60</td>
		</tr>
		<tr>
			<td>suture 4-0 silk w/ needle</td>
			<td>Owens Minor</td>
			<td>K188H</td>
		</tr>
		<tr>
			<td>gauze 4x4</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>cotton-tip applicator</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>30G needle</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>23G needle</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>10cc syringe</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>50cc conical tube</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>1cc syringe w/ 25G needle</td>
			<td>Fisher Scientific</td>
			<td>14-826-88</td>
		</tr>
		<tr>
			<td>Polyethylene 10 tubing 100`(PE-10)</td>
			<td>Fisher Scientific</td>
			<td>14-170-12P</td>
		</tr>
		<tr>
			<td>Polyethylene 50 tubing 100`(PE-50)</td>
			<td>Fisher Scientific</td>
			<td>14-170-12B</td>
		</tr>
		<tr>
			<td>3-way stopcock</td>
			<td>Fisher Scientific</td>
			<td>NC9779127</td>
		</tr>
		<tr>
			<td>surgical blue pad</td>
			<td>Fisher Scientific</td>
			<td>50-7105</td>
		</tr>
		<tr>
			<td>Sterile Field dressings</td>
			<td>Fisher Scientific</td>
			<td>NC9517505</td>
		</tr>
		<tr>
			<td>tape rolls 1&#34;</td>
			<td>Corporate Express</td>
			<td>MMM26001</td>
		</tr>
		<tr>
			<td>straight side wide mouth jars</td>
			<td>VWR</td>
			<td>159000-058</td>
		</tr>
		<tr>
			<td>stainless steel tray 8&#34; x 11&#34;</td>
			<td>VWR</td>
			<td>62687-049</td>
		</tr>
		<tr>
			<td>male-male leur lock 3-way</td>
			<td>VWR</td>
			<td>20068-909</td>
		</tr>
		<tr>
			<td>sterilization pouch 3&#34;x8&#34;</td>
			<td>VWR</td>
			<td>24008</td>
		</tr>
		<tr>
			<td>sterilization pouch 5&#34;x10&#34;</td>
			<td>VWR</td>
			<td>24010</td>
		</tr>
		<tr>
			<td>absorption triangles</td>
			<td>Fine Science Tools</td>
			<td>18105-03</td>
		</tr>
		<tr>
			<td>7mm wound clip applier</td>
			<td>Fisher Scientific</td>
			<td>E0522687</td>
		</tr>
		<tr>
			<td>1000 7mm wound clips</td>
			<td>Fisher Scientific</td>
			<td>E0522687</td>
		</tr>
		<tr>
			<td>betadine (4oz)</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>sterile gloves</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>eppendorfs&#160;</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>1/2cc Lo-Dose insulin syringe</td>
			<td>Fisher Scientific</td>
			<td>12-826-79</td>
		</tr>
		<tr>
			<td>small weigh boat</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>lactated ringers</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>hepranized saline solution (.1&#181; hep + 9.9&#181;NaCl)</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>phosphate buffered saline&#160;</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>pentobarbital&#160;</td>
			<td colspan="2">can be purchased through any global vendor</td>
		</tr>
		<tr>
			<td>Wild M650 microscope w/ boom stand</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Digi-Med BPA-400 analyzer &#38; systems integrator</td>
			<td>Micro-Med</td>
			<td>SYS-400</td>
		</tr>
		<tr>
			<td>TXD-310 (Digi-Med Transducer)&#160;</td>
			<td>Micro-Med</td>
			<td>TXD-300</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Dell</td>
			<td></td>
		</tr>
		<tr>
			<td>microbead instrument sterilizer</td>
			<td>VWR</td>
			<td>11156-002</td>
		</tr>
		<tr>
			<td>Oster A5 clippers w. size 40 blade</td>
			<td>VWR</td>
			<td>10749-020</td>
		</tr>
		<tr>
			<td>circulating heating pad 18x26</td>
			<td>Harvard</td>
			<td>py872-5272</td>
		</tr>
		<tr>
			<td>rectal thermometer</td>
			<td>Kent Scientific</td>
			<td>RET-3</td>
		</tr>
	</tbody>
</table>

uncontrolled hemorrhagic shock modeled via liver laceration in mice with real time hemodynamic monitoring

A hemorragia não controlada é uma causa importante de óbitos evitáveis ​​entre pacientes com trauma. Desenvolvemos um modelo murino de hemorragia descontrolada através de uma lacera- ção hepática que resulta em perda sanguínea consistente, alterações hemodinâmicas e sobrevida. Os ratos sofrem uma ressecção padronizada do lobo médio esquerdo do fígado. Eles são autorizados a sangrar sem intervenção mecânica. Os agentes hemostáticos podem ser administrados como terapia de pré-tratamento ou de resgate, dependendo do interesse do investigador. Durante o tempo de hemorragia, é realizada monitorização hemodinâmica em tempo real através de uma linha arterial femoral esquerda. Os ratinhos são então sacrificados, a perda de sangue é quantificada, o sangue é recolhido para análise posterior e os órgãos são colhidos para análise da lesão. O desenho experimental é descrito para permitir o teste simultâneo de múltiplos animais. Hemorragia hepática como modelo de hemorragia não controlada existeNa literatura, principalmente em modelos de ratos e suínos. Alguns destes modelos utilizam monitorização hemodinâmica ou quantificam a perda de sangue mas não têm consistência. O presente modelo incorpora a quantificação de perda sangüínea, monitoramento hemodinâmico em tempo real em um modelo murino que oferece a vantagem de usar linhas transgênicas e um mecanismo de alto rendimento para investigar os mecanismos fisiopatológicos em hemorragia não controlada.

O modelo de laceração hepática resulta em perda de sangue reproduzível e consistente em ratos. A Figura 1A demonstra o peso consistente de fígado lacerado que pode ser obtido com um desvio padrão de apenas 0,02 g. Esta consistência no peso de fígado lacerado permite a capacidade de reproduzir o modelo entre camundongos e em diferentes configurações experimentais, tais como diferentes protocolos de ressuscitação. Além disso, o peso reproduzível do fígado la...

Watch this Scientific Journal Video about Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring at JoVE.com

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

A hemorragia descontrolada, uma importante causa de mortalidade entre os pacientes com trauma, pode ser modelada utilizando uma laceração hepática padrão em um modelo murino. Este modelo resulta em perda de sangue consistente, sobrevivência e permite o teste de agentes hemostáticos. Este artigo fornece o processo passo a passo para executar este modelo valioso.

Choque hemorrágico não controlado modelado via laceração hepática em ratos com monitorização hemodinâmica em tempo real

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via ...

uncontrolled-hemorrhagic-shock-modeled-via-liver-laceration-mice-with

Research

JoVE Journal

Medicine

8.5K visualizações.  University of Pittsburgh.  A hemorragia descontrolada, uma importante causa de mortalidade entre os pacientes com trauma, pode ser modelada utilizando uma laceração hepática padrão em um modelo murino. Este modelo resulta em perda de sangue consistente, sobrevivência e permite o teste de agentes hemostáticos. Este artigo fornece o processo passo a passo para executar este modelo valioso. 

Vídeo: Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

Fixed Volume or Fixed Pressure: A Murine Model of Hemorrhagic Shock

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, 2, 3, 4, 5 These signaling events can also lead to sepsis and/or multiple organ dysfunction (MOD). 6, 7, 8, 9 It is critical then to investigate the causes of suppressed immune function and detrimental signaling cascades in order to develop more effective ways to help patients who suffer from traumatic injuries. 10 This fixed pressure Hemorrhagic Shock (HS) procedure, although technically challenging, is an excellent resource for investigation of these pathophysiologic conditions. 11, 12, 13 Advances in the assessment of biological systems, i.e. Systems Biology have enabled the scientific community to further understand complex physiologic networks and cellular communication patterns. 14 Hemorrhagic Shock has proven to be a vital tool for unveiling these cellular communication patterns as they relate to immune function. 15, 16, 17, 18 This procedure can be mastered! This procedure can also be used as either a fixed volume or fixed pressure approach. We adapted this technique in the murine model to enhance research in innate and adaptive immune function. 19, 20, 21 Due to their small size HS in mice presents unique challenges. However due to the many available mouse strains, this species represents an unparalleled resource for the study of the biologic responses. The HS model is an important model for studying cellular communication patterns and the responses of systems such as hormonal and inflammatory mediator systems, and danger signals, i.e. DAMP and PAMP upregulation as it elicits distinct responses that differ from other forms of shock. 22, 23, 24, 25 The development of transgenic murine strains and the induction of biologic agents to inhibit specific signaling have presented valuable opportunities to further elucidate our understanding of the up and down regulation of signal transduction after severe blood loss, i.e. HS and trauma 26, 27, 28, 29, 30. 
There are numerous resuscitation methods (R) in association with HS and trauma. 31, 32, 33, 34 A fixed volume resuscitation method of solely lactated ringer solution (LR), equal to three times the shed blood volume, is used in this model to study endogenous mechanisms such as remote organ injury and systemic inflammation. 35, 36, 38 This method of resuscitation is proven to be effective in evaluating the effects of HS and trauma 38, 39.

The Hemorrhagic Shock model has been a reliable and reproducible resource facilitating the identification and understanding of signaling cascades associated with inflammation and end-organ damage after trauma. This article provides a step-by-step description of surgical and mechanical aspects associated with the Hemorrhagic Shock experimental procedure in mice.

Volume fixo ou de pressão fixa: um modelo murino de choque hemorrágico

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, ...

Medicina

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Controlada por laceração do colo do útero em camundongos

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

Em tempo real de Citotoxicidade em Sangue Total Humano

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

Monitoramento da sistêmicas e hepática Hemodinâmica em Ratos

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Análise em tempo real de pressão-volume de infarto agudo do miocárdio em ratos

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Complete and Partial Aortic Occlusion for the Treatment of Hemorrhagic Shock in Swine

Hemorrhage remains the leading cause of preventable deaths in trauma. Endovascular management of non-compressible torso hemorrhage has been at the forefront of trauma care in recent years. Since complete aortic occlusion presents serious concerns, the concept of partial aortic occlusion has gained a growing attention. Here, we present a large animal model of hemorrhagic shock to investigate the effects of a novel partial aortic balloon occlusion catheter and compare it with a catheter that works on the principles of complete aortic occlusion. Swine are anesthetized and instrumented in order to conduct controlled fixed-volume hemorrhage, and hemodynamic and physiological parameters are monitored. Following hemorrhage, aortic balloon occlusion catheters are inserted and inflated in the supraceliac aorta for 60 min, during which the animals receive whole-blood resuscitation as 20% of the total blood volume (TBV). Following balloon deflation, the animals are monitored in a critical care setting for 4 h, during which they receive fluid resuscitation and vasopressors as needed. The partial aortic balloon occlusion demonstrated improved distal mean arterial pressures (MAPs) during the balloon inflation, decreased markers of ischemia, and decreased fluid resuscitation and vasopressor use. As swine physiology and homeostatic responses following hemorrhage have been well-documented and are like those in humans, a swine hemorrhagic shock model can be used to test various treatment strategies. In addition to treating hemorrhage, aortic balloon occlusion catheters have become popular for their role in cardiac arrest, cardiac and vascular surgery, and other high-risk elective surgical procedures.

Here, we present a protocol demonstrating a hemorrhagic shock model in swine that uses aortic occlusion as a bridge to definitive care in trauma. This model has application in testing a wide range of surgical and pharmacological therapeutic strategies.

Oclusão da aorta total e parcial para o tratamento do choque hemorrágico em suínos

Hemorrhage remains the leading cause of preventable deaths in trauma. Endovascular management of non-compressible torso hemorrhage has been at the ...

Measurement of the Hepatic Venous Pressure Gradient and Transjugular Liver Biopsy

Here we provide a detailed protocol describing the clinical procedure of hepatic venous pressure gradient (HVPG) measurement in patients with advanced chronic liver disease followed by an instruction for transjugular biopsy. Under local anesthesia and ultrasound guidance, a catheter introducer sheath is placed in the right internal jugular vein. Using&#160;fluoroscopic guidance, a balloon catheter is advanced into the inferior vena cava (IVC) and inserted into a large hepatic vein. Correct and sufficient wedge position of the catheter is ensured by injecting contrast media while the balloon is blocking the outflow of the cannulated hepatic vein. After calibrating the external pressure transducer, continuous pressure recordings are obtained with triplicate recordings of the wedged hepatic venous pressure (WHVP) and free hepatic venous pressure (FHVP). The difference between FHVP and WHVP is referred to as HVPG, with values &#8805;10 mm Hg indicating clinically significant portal hypertension (CSPH). Before removing the catheter, pressure readings obtained in the IVC at the same level, as well as the right atrial pressure are recorded.
Finally, a transjugular liver biopsy can be obtained via the same vascular route. Different systems are available; however, core biopsy needles are preferred over aspiration needles, especially for cirrhotic livers. Again, under fluoroscopic guidance a biopsy needle introducer sheath is advanced into an hepatic vein. Next, the transjugular biopsy needle is gently advanced through the introducer sheath: (i) in case of aspiration biopsy, the needle is advanced into the liver parenchyma under aspiration and then removed quickly, or (ii) in case of a core biopsy, the cutting-mechanism is triggered inside the parenchyma. Several separate passages can be safely performed to obtain sufficient liver specimens via transjugular biopsy. In experienced hands, the combination of these procedures takes about 30-45 min.

Here, we present a protocol for measurement of hepatic venous pressure gradient (HVPG),the gold standard to diagnose clinically significant portal hypertension. Moreover, we describe how to perform a transjugular liver biopsy within the same session.

Medição do Gradiente de Pressão Venosa Hepática e Biópsia hepática transjugular

Here we provide a detailed protocol describing the clinical procedure of hepatic venous pressure gradient (HVPG) measurement in patients with advanced ...

Standardized Hemorrhagic Shock Induction Guided by Cerebral Oximetry and Extended Hemodynamic Monitoring in Pigs

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an insufficient oxygen supply and irreversible organ failure. The brain exerts only limited compensation capacities and is particularly at high risk of severe hypoxic damage.This article demonstrates the reproducible induction of life-threatening hemorrhagic shock in a porcine model by means of calculated blood withdrawal. We titrate shock induction guided by near-infrared spectroscopy&#160;and extended hemodynamic monitoring to display systemic circulatory failure, as well as cerebral microcirculatory oxygen depletion. In comparison to similar models that primarily focus on predefined removal volumes for shock induction, this approach highlights a titration by means of the resulting failure of macro- and microcirculation.

Hemorrhagic shock is a severe complication in seriously injured patients, which leads to life-threatening oxygen undersupply. We present a standardized method to induce hemorrhagic shock via blood withdrawal in pigs that is guided by hemodynamics and microcirculatory cerebral oxygenation.

Indução de choque hemorrágico padronizado guiada por oximetria cerebral e Monitorização hemodinâmica estendida em suínos

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an ...

Real-Time Monitoring of Neurocritical Patients with Diffuse Optical Spectroscopies

Neurophysiological monitoring is an important goal in the treatment of neurocritical patients, as it may prevent secondary damage and directly impact morbidity and mortality rates. However, there is currently a lack of suitable non-invasive, real-time technologies for continuous monitoring of cerebral physiology at the bedside. Diffuse optical techniques have been proposed as a potential tool for bedside measurements of cerebral blood flow and cerebral oxygenation in case of neurocritical patients. Diffuse optical spectroscopies have been previously explored to monitor patients in several clinical scenarios ranging from neonatal monitoring to cerebrovascular interventions in adults. However, the feasibility of the technique to aid clinicians by providing real-time information at the bedside remains largely unaddressed. Here, we report the translation of a diffuse optical system for continuous real-time monitoring of cerebral blood flow, cerebral oxygenation, and cerebral oxygen metabolism during intensive care. The real-time feature of the instrument could enable treatment strategies based on patient-specific cerebral physiology rather than relying on surrogate metrics, such as arterial blood pressure. By providing real-time information on the cerebral circulation at different time scales with relatively cheap and portable instrumentation, this approach may be especially useful in low-budget hospitals, in remote areas and for&#160;monitoring in open fields (e.g., defense and sports).

Presented here is a protocol for non-invasively monitoring cerebral hemodynamics of neurocritical patients in real-time and at the bedside using diffuse optics. Specifically, the proposed protocol uses a hybrid diffuse optical systems to detect and display real-time information on cerebral oxygenation, cerebral blood flow and cerebral metabolism.

Monitoramento em Tempo Real de Pacientes Neurocríticos com Espectroscopias Ópticas Difusas

Neurophysiological monitoring is an important goal in the treatment of neurocritical patients, as it may prevent secondary damage and directly impact ...

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, ...