<p class="jove_content"&gt; Os estudos atuais são suportados pelo National Heart, Lung, and Blood Institute Grant R01-HL0921, R01-DK083385 e R01HL098294 para HK Eltzschig, o 1K08HL102267-01 a T. Eckle e Fundação para a Educação Anestesia e Bolsas de Investigação para T. Eckle e HK Eltzschig, e da American Heart Association para Grant T. Eckle e Eltzschig HK e Deutsche Forschungsgemeinschaft bolsa de pesquisa (DFG) para M. Koeppen.</p

Observações gerais: Todas as operações devem ser realizadas em um microscópio de dissecação vertical (Olympus, SZX10 com Z-Axis Mensagem Crank com STU2 Fique lança Stand) e usando um coagulador cirúrgica (11). Os grupos experimentais devem ser combinados da melhor forma possível na idade e peso para garantir a comparabilidade dos resultados. Administração de temperatura, pressão arterial anestesia, e fluido deve ser estável por toda parte. 1. Anestesia e preparação traquéia <ol><li> Use camundongos C57BL / 6 que são pelo menos 10 semanas de idade e tem um corpo de 22-25 g. Induzir a anestesia com pentobarbital sódico na dose de um ip 70 mg / kg peso corporal (6). Manter a anestesia com cerca de 10 mg / kg / h de sódio pentobarbital. Seja cauteloso com overdose uma vez que este pode reduzir significativamente a pressão arterial. Re-dose de pentobarbital - mesmo depois de horas-pode levar a um aumento severo dos níveis plasmáticos. </li><li> Após indução da anestesia, os ratos segura em posição supina com as extremidades superiores e inferiores anexado à mesa com uma fita e uma sutura presa aos tornozelos. Faça o mesmo para a cabeça, usando os dentes. Restrição suficiente é importante para uma intubação de sucesso e uma cirurgia bem controlada. Antes da cirurgia, cobrir o mouse com óleo mineral para reduzir o risco de alergia cabelo mouse. A fim de assegurar que a temperatura corporal permanece estável cobrir os ratos com filme de alimentos disponíveis comercialmente. </li><li> Camundongos colocar em uma mesa aquecida com temperatura controlada (RT, Effenberg, Munique, Alemanha) com uma sonda termômetro retal ligado ao controlador de retorno térmico para manter a temperatura corporal a 37 ° C. </li><li> Expor a traquéia cirurgicamente. Dissecar laterais e dorsal da traquéia do tecido conjuntivo e coloque duas fio de seda 3,0 cirúrgico (aparelho Harvard, EUA) a cada 10 cm de comprimento por baixo da traquéia. As suturas devem ser de aproximadamente 1 cm. </li><li> Cuidadosamente inciso traquéia 3-4 mm abaixo da laringe entre duas cartilagens circular usando uma tesoura McPherson-Vannas (8 cm, lâmina reta; Instrumentos de Precisão World, EUA). Certifique-se de não causar um sangramento, já que isso pode confundir os parâmetros de resultados. </li><li> Realizar uma intubação traqueal com cânula de polietileno blunt (INSYTE 22g, Beckton Dickinson, EUA). Insira a ponta da cânula de polietileno em um ângulo de 85 graus na traquéia. Em seguida, incline a cânulas por isso está de acordo com a luz traqueal. Lentamente avanço do tubo mais abaixo da traquéia até a ponta da cânula desapareceu na abertura do tórax. Fixar o tubo nesta posição com o fio de seda dois cirúrgica colocada dorsal da traquéia (ver 1.4). </li></ol> 2. Técnica de ventilação mecânica da lesão pulmonar induzida <ol><li> Ligue o tubo a um ventilador. Para induzir a lesão pulmonar usamos uma pressão técnica de ventilação controlada usando um C 900 Servo da Siemens (DRE Veterinária, EUA). Animais serão ventiladas com pico de pressão inspiratória de 45 mbar, freqüência de 80 respirações / min e uma pressão positiva expiratória final de 0-3 mbar com uma FiO 2 = 1,0. A inspiração à relação de expiração deve ser de 1:1. Apesar do fato de que o C 900 Servo é construído como ventilador para os seres humanos, a sua utilização em um ambiente de ventilação pressão controlada excelentes trabalhos para a ventilação de camundongos. </li><li> Monitor de freqüência cardíaca com ECG (por exemplo, Hewlett Packard, B blingen, Alemanha). Certifique-se que a freqüência cardíaca não cai abaixo de 400. Deve-se ver uma mudança do eixo do coração para a direita, quando a ventilação mecânica é instituído como um sinal de aumento da pressão arterial pulmonar decorrente de um aumento na pressão intratorácica. Se o rato se desenvolve bradicardia, verifique a temperatura ea dose do anestésico / concentração. Xylacin / cetamina induz anestesia um coração coração de 250 / min e não é recomendada. </li><li> Aplique uma reposição de líquidos adequada. Uma infusão com solução salina 0,1 ml / hora através de um cateter arterial ou venosa deve ser realizada antes da ventilação e garantir o arquivamento venosa suficiente. Devido às pressões de ventilação alta o retorno venoso para o coração é prejudicada, o que pode levar a uma queda crítica da pressão arterial média. Além disso, uma solução salina em bolus de 500 mL pode ser dado ip antes da cirurgia. </li><li> Colocar um cateter na artéria carótida para a gravação contínua da pressão arterial (27). Fixe o braço para o corpo antes de começar a dissecção da artéria. A artéria carótida é exposta através de dissecção romba da musculatura paratraqueal. Após a exposição ainda mais cuidado e evitar de qualquer trauma tecidual (particular do nervo vago), um cateter é inserido dentro do vaso usando duas suturas e uma braçadeira pequena (37). Isto irá expor um longo segmento da artéria. Coloque uma ligadura no final da artéria carótida. Anexar um maior aperto para o final da sutura para obter ª tensão ou fixarsutura e para a mesa usando fita. Coloque outro sutura ao redor da artéria e dissecar a artéria até o fim distal. Aqui, colocar uma braçadeira de pequeno porte. Use uma tesoura para cortar micro de uma pequena abertura na diagonal dentro da artéria. Segure a abertura com uma pinça fina (Dumont, WPI) e avançar o cateter de tamanho apropriado com as mãos / fórceps. Faça um nó com o seu segundo e segura de sutura da artéria. Solte a braçadeira e avançar o cateter ainda mais. Fixe o cateter com vários nós e fita. Alternativamente, o cateter de artéria carótida pode ser realizado no final do experimento para coletar amostras de sangue arterial para gasometria. </li></ol> 3. Recuperação de amostras de tecido Após 3 horas de ventilação mecânica, serão colhidas amostras para avaliar a extensão da lesão pulmonar. Recomendamos coletar lavado brochnoalveolar (BAL), sangue arterial e no tecido pulmonar. <ol><li> Obter LBA, no final do experimento. Depois de aprofundamento da anestesia, lave o tubo traqueal com 1 ml solução salina tamponada com fosfato (PBS). O fluido deve permanecer na traquéia / pulmão por três segundos antes que ele é a recuperação através da seringa conectada. O LBA é snap-congelados em nitrogênio líquido e armazenadas a -80 ° C para análise posterior. Estar ciente de que o volume pode ser recuperado significativamente menor do que 1 ml. </li><li> Realizar a análise de gases sanguíneos, no final do experimento. A fim de fazer isso, uma incisão deve ser feita logo abaixo do esterno. Segure o esterno com uma pinça e extensão da incisão ao longo da caixa torácica. Em seguida, o diafragma é incisão nas bordas e é cortado a partir as costelas. Agora há uma visão aberta para o mais baixo da abertura do tórax. Levantar o esterno até com uma pinça e abrir o tórax por cortes longos no lado direito e esquerdo (como laterais possível) para que a parede do tórax anterior completa está ligado-alas. Isto deve ser feito, enquanto o animal ainda é experimental ventilação mecânica. O ventrículo esquerdo é perfurado usando um 27 ½ G agulha e análise do sangue arterial é realizada utilizando o Sistema i-STAT (Abbott, EUA). Se a gasometria arterial deve ser realizada, uma BAL não pode ser obtida, uma vez que este será um fator de confusão significativa para os resultados. </li><li> Em alternativa ao método acima, as amostras de sangue arterial podem ser coletadas através do cateter de artéria carótida. No entanto, isto deve ser feito antes do LBA é obtido. </li><li> Especiais de consumo os pulmões em bloco, puxando-se o coração e cortar a traquéia. Ter um pedaço de tecido pronto para absorver o sangue, de modo que o sítio cirúrgico é visível. Puxe o coração na direção do abdômen e corte cuidadosamente ao longo da espinha para mobilizar todos os órgãos do tórax. Corte a aorta, remover os órgãos torácicos e coloque-os sobre uma mesa limpa cirúrgico. </li><li> Cortar o coração e os vasos principais da amostra de tecido. Certifique-se que não há tecido do timo ainda ligado ao pulmão. Separar os pulmões com uma tesoura e coloque em tubos individuais e encaixe-congelante. Armazenar a -80 ° C para análise posterior. </li></ol> 4. Medição de lesão pulmonar Recomendamos a utilização dos parâmetros seguintes resultados para avaliar a extensão da lesão pulmonar: Realizar um ELISA albumina (Bethyl Laboratories, EUA) e um mieloperoxidase (MPO) ELISA (Hycult Biotecnologia, EUA) para avaliar a extensão da disfunção de barreira e a quantidade de inflamatória células no LBA. Realizar um ELISA MPO também formam o tecido pulmonar. Se wet-a-seco proporção deve ser medida não obtemos LBA e da circulação pulmonar não é lavada (ver 3.3). Medir o peso dos pulmões após a excisão. Em seguida, os pulmões são liofilizadas por 48 h e no tecido pulmonar é medido novamente. Então a relação de wet-a-seco é medido em mg de água por mg de tecido seco (5). <img src="/files/ftp_upload/2525/2525fig1.jpg" alt="Figura 1" /> Figura 1. Teor de proteínas na BAL em resposta a VILI. Camundongos foram anestesiados com pentobarbital, ventilação mecânica foi instituída e os ratos foram ventilados com pressão controlada configurações (pressão inspiratória de 45 mbar, positiva ao final da exspiratory pressão de 3 mbar, 100% inspirado concentração de oxigênio). Após 0, 1, 2 e 3 horas de ventilação BAL foi colhida eo teor de proteína foi quantificado através de um ensaio de ácido bicinchoninic (ensaio BCA). A mudança relativa do teor de proteínas é mostrado normalizado para 0 horas de ventilação (n = 4 por grupo, * indica p &lt;0,05 comparado ao grupo controle, média ± SEM)

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD39/ectonucleoside+triphosphate+diphosphohydrolase+1+provides+myocardial+protection+during+cardiac+ischemia/reperfusion+injury.">CD39/ectonucleoside triphosphate diphosphohydrolase 1 provides myocardial protection during cardiac ischemia/reperfusion injury.</a> Circulation. 116, 1784-1794 (2007).</li><li>Kuhlicke, J., Frick, J. S., Morote-Garcia, J. C., Rosenberger, P., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+Inducible+Factor+(HIF)-1+Coordinates+Induction+of+Toll-Like+Receptors+TLR2+and+TLR6+during+Hypoxia.">Hypoxia Inducible Factor (HIF)-1 Coordinates Induction of Toll-Like Receptors TLR2 and TLR6 during Hypoxia.</a> PLoS ONE. 2, e1364-e1364 (2007).</li><li>Morote-Garcia, J. C., Rosenberger, P., Kuhlicke, J., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1-dependent+repression+of+adenosine+kinase+attenuates+hypoxia-induced+vascular+leak.">HIF-1-dependent repression of adenosine kinase attenuates hypoxia-induced vascular leak.</a> Blood. 111, 5571-5580 (2008).</li><li>Morote-Garcia, J. C., Rosenberger, P., Nivillac, N. M., Coe, I. R., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor-dependent+repression+of+equilibrative+nucleoside+transporter+2+attenuates+mucosal+inflammation+during+intestinal+hypoxia.">Hypoxia-inducible factor-dependent repression of equilibrative nucleoside transporter 2 attenuates mucosal inflammation during intestinal hypoxia.</a> Gastroenterology. 136, 607-618 (2009).</li><li>Reutershan, J., Vollmer, I., Stark, S., Wagner, R., Ngamsri, K. C., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine+and+inflammation:+CD39+and+CD73+are+critical+mediators+in+LPS-induced+PMN+trafficking+into+the+lungs.">Adenosine and inflammation: CD39 and CD73 are critical mediators in LPS-induced PMN trafficking into the lungs.</a> FASEB J. 23, 473-482 (2009).</li><li>Schingnitz, U., Hartmann, K., Macmanus, C. F., Eckle, T., Zug, S., Colgan, S. P., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+through+the+A2B+adenosine+receptor+dampens+endotoxin-induced+acute+lung+injury.">Signaling through the A2B adenosine receptor dampens endotoxin-induced acute lung injury.</a> J Immunol. 184, 5271-5279 (2010).</li><li>Thompson, L. F., Eltzschig, H. K., Ibla, J. C., Van De Wiele, C. J., Resta, R., Morote-Garcia, J. C., Colgan, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crucial+role+for+ecto-5'-nucleotidase+(CD73)+in+vascular+leakage+during+hypoxia.">Crucial role for ecto-5'-nucleotidase (CD73) in vascular leakage during hypoxia.</a> J. Exp. Med. 200, 1395-1405 (2004).</li><li>Aherne, C. M., Kewley, E. M., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+resurgence+of+A2B+adenosine+receptor+signaling.">The resurgence of A2B adenosine receptor signaling.</a> Biochim Biophys Acta. , (2010).</li><li>Eckle, T., Koeppen, M., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+extracellular+adenosine+in+acute+lung+injury.">Role of extracellular adenosine in acute lung injury.</a> Physiology (Bethesda). 24, 298-306 (2009).</li><li>Koeppen, M., Feil, R., Siegl, D., Feil, S., Hofmann, F., Pohl, U., de Wit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cGMP-dependent+protein+kinase+mediates+NO-+but+not+acetylcholine-induced+dilations+in+resistance+vessels+in+vivo.">cGMP-dependent protein kinase mediates NO- but not acetylcholine-induced dilations in resistance vessels in vivo.</a> Hypertension. 44, 952-955 (2004).</li></ol>

O presente estudo descreve uma técnica de realizar ventilador da lesão pulmonar induzida em camundongos. Este modelo demonstra lesão altamente reprodutíveis, devido à ventilação de alta pressão. Investigadores que consideram estudar lesão pulmonar aguda em ratos podem beneficiar deste modelo.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Pentobarbital sódico (Plus Fatal) </td><td> Vortech Pharmaceutical Ls, Ltd, </td><td> VPL 9372 </td><td> 4mg/ml em solução salina </td></tr><tr><td> INSYTE 22 G </td><td> Beckton Dickinson </td><td> n / a </td><td></td></tr><tr><td> Sutura, de seda 4,0 </td><td> Harvard Apparatus </td><td> 517698 </td><td></td></tr><tr><td> Sutura, Prolene 8,0 </td><td> Ethicon, EUA </td><td> M8739 </td><td> reutilizáveis </td></tr><tr><td> Siemens 900 ° C </td><td> DRE Veterinária, EUA </td><td> # 336 </td><td> remodelado </td></tr><tr><td> microscópio de dissecação (SZX10) </td><td> Olimpo </td><td> n / a </td><td> considerar a distância de trabalho generosa </td></tr><tr><td> Tabela de aquecimento </td><td> Rt, Effenberger, Alemanha </td><td> n / a </td><td> único fornecedor e único </td></tr><tr><td> Dispositivo de pressão arterial </td><td> Cyber-Sense, Inc </td><td> BPM02 </td><td></td></tr><tr><td> STAT I </td><td> Abbott </td><td> n / a </td><td></td></tr></tbody></table>

pressure controlled ventilation to induce acute lung injury in mice

Modelos murinos são amplamente utilizados para investigar lesões agudas de diferentes sistemas dos órgãos (1-34). Lesão pulmonar aguda (LPA), que ocorre com a ventilação mecânica prolongada, contribui para a morbidade e mortalidade da doença crítica, e estudos sobre novos alvos farmacológicos ou genéticos são áreas de intensa investigação (1-3, 5, 8, 26, 30, 33 -36). ALI é definido pelo início agudo da doença, o que leva a não-cardíacas edema pulmonar e insuficiência pulmonar subseqüente de troca gasosa (36). Nós desenvolvemos um modelo murino de ALI usando uma ventilação com pressão controlada para induzir o ventilador da lesão pulmonar induzida (2). Para este fim, os camundongos C57BL / 6 são anestesiados e uma traqueostomia é realizada seguida de indução da LPA através de ventilação mecânica. Ratos são ventilados em um ambiente com pressão controlada com um pico de pressão inspiratória de 45 mbar por 1-3 horas. Como parâmetros de resultado, edema pulmonar (wet-a-seco ratio), conteúdo líquido broncoalveolar albumina, fluido broncoalveolar e conteúdo mieloperoxidase pulmonar tecidos e as trocas gasosas pulmonares são avaliados (2). Usando esta técnica nós poderíamos mostrar que suficientemente induz inflamação pulmonar aguda e pode distinguir entre diferentes grupos de tratamento ou genótipos (1-3, 5). Portanto, esta técnica pode ser útil para pesquisadores que buscam mecanismos moleculares envolvidos na ALI usando uma abordagem genética em camundongos com o gene-alvo exclusão.

Watch this Scientific Journal Video about Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice at JoVE.com

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Um modelo murino para o ventilador lesão pulmonar induzida é uma ferramenta importante para estudar uma lesão pulmonar aguda<em&gt; In vivo</em&gt;. Aqui, nós relatamos uma aplicáveis ​​fácil<em&gt; In situ</em&gt; Modelo de lesão pulmonar aguda de alta pressão de ventilação mecânica para induzir insuficiência aguda do pulmão.

Ventilação a pressão controlada para induzir lesão pulmonar aguda em ratos

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

pressure-controlled-ventilation-to-induce-acute-lung-injury-in-mice

Research

JoVE Journal

Medicine

18.2K visualizações.  University of Colorado. Um modelo murino para o ventilador lesão pulmonar induzida é uma ferramenta importante para estudar uma lesão pulmonar aguda In vivo. Aqui, nós relatamos uma aplicáveis ​​fácil In situ Modelo de lesão pulmonar aguda de alta pressão de ventilação mecânica para induzir insuficiência aguda do pulmão.

Vídeo: Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

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 ...

Medicina

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

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

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

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

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

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Controlado Modelo Impact Cortical para lesão cerebral traumática

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

O cateterismo cardíaco em camundongos para medir o volume Pressão do relacionamento: Investigando o efeito Bowditch

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Abrir traqueostomia ácido gástrico aspiração Modelo murino de lesão pulmonar aguda Resultados em Maximal aguda não-letal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

Lesão baseada no balão para induzir a hiperplasia Myointimal da Aorta Abdominal de Mouse

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Um modelo de peito fechado para induzir a constrição da aorta transversa em ratos

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

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 ...

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung&rsquo;s structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.

Presented here is a useful protocol for lung fixation that creates a stable condition for histological evaluate of lung specimens from a mouse model of emphysema. The main advantage of this model is that it can fix many lungs with the same constant pressure without lung collapse or deflation.

Fixação pulmonar pressão constante para avaliação de enfisema em camundongos

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Induzindo lesão pulmonar aguda em camundongos por instilação de lipopolissacárido intratraqueal direto

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...