This work was supported by the American Heart Association Scientist Development Grant and the Foundation for Anesthesia Education and Research (MLJ). We would like to thank Talaignair N. Venkatraman PhD for his assistance with magnetic resonance imaging.

Declaração de Ética: Este protocolo foi aprovado pelo Comitê da Universidade Duke Institutional Animal Care e Use e segue todas as diretrizes para o uso ético de animais. 1. Preparação de Equipamento <ol><li> Autoclave os instrumentos cirúrgicos antes da cirurgia. </li><li> Desinfectar o aparelho estereotáxico com etanol a 70%. </li><li> Ligue banho de água e manter a temperatura da água a 42 ° C. </li><li> Dissolver tipo IV-S colagenase de Clostridium, em solução salina normal com uma concentração de 0,075 L por 0,4 ul. </li></ol> 2. Injeção Modelo colagenase <ol><li> Pesar o mouse. </li><li> Anestesiar o mouse em uma câmara de indução com 5% de isoflurano em 30% O 2/70% N2. Anestesia adequada é sinalizado após cerca de 2 min, quando respirations rato ter abrandado a 1 por segundo. </li><li> Entubar a traqueia com um G 30 mm 20 cateter intravenoso. </li><li> Conectar o cateter para umventilador roedor e ventilar mecanicamente os pulmões com 1,6% de isoflurano em 30% O 2/70% N2 a uma taxa de 105 ciclos por minuto, com um volume corrente fornecido de 0,75 ml .. </li><li> Raspar o couro cabeludo com um barbeador eletrônico. Uma vez que o mouse é anestesiado e entubado, movê-lo para uma estação de trabalho diferente para fazer a barba e, em seguida, voltou para o banco cirúrgico. </li><li> Fixe a cabeça em um quadro estereotáxico, e nivelar a cabeça com sutura tanto coronal e sagital, como pontos de referência. </li><li> Aplicar pomada oftálmica para os olhos. </li><li> Insira uma sonda de temperatura retal. Manter a temperatura rectal em 37,0 ± 0,2 ° C usando um colchão de água em circulação substrutura. </li><li> Limpe a área cirúrgica com betadine seguido com etanol 70% e repetir 3 vezes. </li><li> Faça uma incisão no couro cabeludo um centímetro da linha média e limpe periósteo lateralmente com um aplicador com ponta de algodão estéril para expor bregma. </li><li> Exercício 1 mm de diâmetro burr hole 2,2 milímetros deixou latral para bregma com uma broca refrigerado a água. </li><li> Gire colagenase frasco 5 vezes, em seguida, lave a seringa de 0,5 mL com agulha 25 G (anexo ao quadro estereotáxico) com 0,5 mL solução de colagenase 5 vezes (Deixar de 0,5 ml de solução de colagenase em seringa após a última lavagem). </li><li> Alinhe a ponta da agulha com orifício de trepanação, em seguida, expulsar 0,1 mL da seringa e limpe a agulha de bisel com navalha descartar. </li><li> Usando um micromanipulador, avançar a agulhas 3 mm de profundidade ao córtex e deixar imóvel por 30 seg. </li><li> Injetar 0,4 mL mais de 90 seg. </li><li> Diminuir isoflurano a 1% e deixar agulha imóvel por 5 min. </li><li> Retire a agulha lentamente. </li><li> Aplicar 1-2 gotas de bupivacaína a 0,25% por via subcutânea e suturar a pele. </li><li> Desligue o isoflurano vaporizador e retire do mouse a partir do quadro estereotáxico. </li><li> Permitir mouse para recuperar a ventilação espontânea com posterior extubação traqueal. </li><li> Retorno do mouse para uma gaiola limpa e permitir o acesso livre paraalimentos e água. </li></ol> 3. Autólogo Modelo Injeção de sangue <ol><li> Siga os passos 2,1-2,11 para o modelo de injeção de colagenase. </li><li> Desenhar 50 ul de solução salina normal estéril numa seringa de 30 mL, G 50. </li><li> Conecte a seringa microlitro com um tubo PE10 70 centímetros. </li><li> Expulse todo o soro fisiológico do microlitro seringa no tubo PE10 completamente a tubulação de ar-DE. </li><li> Puxar o êmbolo da seringa para fora microlitro de 1 mM a fazer uma bolha de ar na abertura distal do aparelho de seringa de tubo PE10-microlitro para evitar a mistura de solução salina e de sangue durante procedimentos posteriores. </li><li> Seque a região da artéria caudal centro distal do rato com etanol a 70%, e cortar a artéria com uma lâmina de 0,5 a 1 cm para a ponta da cauda. </li><li> Recolha de 40 mL de sangue a partir da cauda cortada em o aparelho de seringa de tubo PE10-microlitro. Nota: que a heparina não é usado na agulha, tubo ou um rato. </li><li> Fixe a seringa microlitro na injebomba cção. </li><li> Ligue a porção de cânula metálica de uma agulha de 27 G para a extremidade do tubo PE10, e proteger a agulha a um micromanipulador na armação estereotáxica. </li><li> Expulse 2 l de sangue de 27 G agulha e limpe a agulha de bisel com navalha descartar. </li><li> Alinhe a ponta da agulha com orifício de trepanação e inserir as agulhas 3 mm de profundidade ao córtex. </li><li> Injectar 35 mL de sangue autólogo, a uma taxa de 2 mL por minuto. </li><li> Diminuir isoflurano a 1% e deixar agulha imóvel por 10 min. </li><li> Retire a agulha mais de 30 seg. </li><li> Aplicar 1-2 gotas de bupivacaína a 0,25% por via subcutânea e suturar a pele. </li><li> Desligue o isoflurano vaporizador e retire do mouse a partir do quadro estereotáxico. </li><li> Permitir mouse para recuperar a ventilação espontânea com extubação subseqüente. </li><li> Retorno do mouse para uma gaiola limpa e permitir o acesso livre à comida e água. </li></ol> 4. Sham Operação <ol><li> Siga os mesmos procedimentos para a colagenase injectiomodelo n, exceto sem injeção após a inserção da agulha. </li></ol> 5. Cuidados pós-cirurgia <ol><li> Injectar 0,5 mL de uma solução salina normal subcutaneamente na noite do procedimento cirúrgico na parte de trás do pescoço do animal. </li><li> Fornecer comida amolecida com água e comida de gel em pequenas chávenas de plástico colocadas sobre o chão da gaiola. Substitua os alimentos por dia durante 7 dias. </li><li> Verifique se há perda de peso, cicatrização de feridas, e sinais de desconforto por dia durante 7 dias. </li><li> Se são requeridos intervalos de recuperação superiores a 7 dias, a remoção da sutura pode ser realizada sob anestesia leve inalado (aproximadamente 1% de isoflurano em 30% O 2/70% N 2), se necessário. </li></ol>

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Lei, Sheng, Wang, Lascola, Warner, and Laskowitz have no conflicts of interest to declare. James received grant funding by American Heart Association, National Institutes of Health, and Cephalogics.

Apesar emergente pesquisa pré-clínica e grandes ensaios clínicos resultantes para prometendo terapêutica 15-18, não há intervenções farmacológicas demonstraram para melhorar o resultado na ICH e cuidados permanece em grande parte de suporte. As listas de possíveis terapias podem ser gerados pelas tecnologias de alto rendimento, como o trabalho transcriptomic e proteômica. Embora estas tecnologias continuam a avançar o nosso conhecimento de potenciais alvos terapêuticos, para frente e para trás t...

Hemorragia intracerebral (ICH) é uma forma relativamente comum de doença cerebrovascular, com cerca de 40-50% dos pacientes que sofrem de morrer dentro de 30 dias 1. Infelizmente, pouca melhoria foi feita na taxa de mortalidade nos últimos 20 anos 2. Os relatórios dos Institutos Nacionais de Saúde 3 e diretrizes da American Heart Association 4 sublinhou a importância do desenvolvimento de modelos clinicamente relevantes de ICH para ampliar a compreensão da fisiopatologia e desenvolver alvos para novas abordagens terapêuticas. Existem diversos modelos de imitar ICH humano 5. Como compreensão da patofisiologia da ICH amadurece, tornou-se evidente que uma variedade de modelos podem ser utilizados para examinar diferentes aspectos da doença. Modelos utilizados anteriormente incluem murino angiopatia amilóide 6, inserção microbalão intraparenquimatosa e inflação 7, eo sangue arterial diretainfiltração de 8,9. Lobar de angiopatia amilóide foi modelada com a utilização de ratos transgénicos e representa um subtipo ICH distinta. Modelos microbalão imitar efeito de massa aguda de formação de hematoma, mas não conseguem capturar a resposta celular do cérebro para a presença de sangue. Finalmente, a infiltração do sangue arterial direta sujeita o cérebro a pressão arterial da artéria femoral. Assim, este modelo reproduz pressão arterial ea presença de sangue, mas não submeta o cérebro a lesão microvascular de ruptura de pequenos vasos de sangue. Além disso, este modelo tem inerentemente uma alta variabilidade. Curiosamente, ratos espontaneamente hipertensos 10 desenvolver ICH espontânea à medida que envelhecem. O estudo destes animais após o desenvolvimento de HIC pode imitar a doença na presença de um dos principais co-morbidades que predispõem os seres humanos para HIC. Embora existam esses outros modelos, injeção intrastriatal de Clostridial colagenase 11 ou injeção instrastiatal de umtodo utologous sangue 12 são, atualmente, os dois modelos mais comuns utilizados na pesquisa pré-clínica ICH. ICH selecção do modelo deve ser feita com base no objectivo de questão experimental, incluindo a seleção de espécies e método de induzir a formação de hematoma. Por exemplo, os porcos são grandes animais com volumes relativamente grandes de substância branca do cérebro, em comparação com ratinhos. Assim, os modelos de suínos são adequados para estudar a fisiopatologia da substância branca após ICH. Em contraste, os cérebros de roedores são matéria em grande parte cinza, mas sistemas de transgênicos fazem roedores útil para avaliar os mecanismos moleculares de lesão e recuperação após ICH. Cada modelo tem seus pontos fortes e fracos (Tabela 1) inerentes, que devem ser cuidadosamente considerados antes da experimentação. Os seguintes protocolos de demonstrar o sangue e injecção colagenase modelos autólogas em ratinhos. Estes modelos têm cada foram traduzidos a partir de modelos desenvolvidos originalmente em ratos13,14 e permitir a utilização de tecnologia transgénica largamente disponível para explorar mecanismos moleculares associadas com a morte celular após a ICH. Ambos representam distintamente diferentes mecanismos de lesão do ICH humano, e ambos têm resultado esperado distintamente diferentes em termos de medidas comportamentais e histológicas. Assim, certas hipóteses podem prestar-se a um modelo sobre o outro, mas muitas ideias podem exigir validação em ambos os modelos. Tabela 1. Comparação das características de injeção de sangue autólogo modelos hemorragia intracerebral colagenase-e. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td style="width:197px;"></td><td style="width:80px;"> Colagenase Injeção </td><td style="width:66px;"> Injeção de sangue </td></tr><tr><td style="width:197px;"> Fácil de Usar </td><td style="width:80px;"> + + + </td><td style="width:66px;"> + + </td></tr><tr&gt; <td style="width:197px;"> Reprodutibilidade </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> Controle de Hemorragia Tamanho </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + + </td></tr><tr><td style="width:197px;"> Refluxo de sangue </td><td style="width:80px;"> + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> Simula Doenças Humanas </td><td style="width:80px;"> + </td><td style="width:66px;"> - </td></tr><tr><td style="width:197px;"> Simplicidade </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> Uso em múltiplas espécies </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr></tbody></table>

<table border="0" cellpadding="0" cellspacing="0" width="416">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Name</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalog Number</td>
			<td style="width:88px;">Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Stereotactic frame</td>
			<td style="width:88px;">Stoelting Co.</td>
			<td style="width:88px;">51603</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Probe holder with corner clamp</td>
			<td style="width:88px;">Stoelting Co.</td>
			<td style="width:88px;">51631</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Mini grinder</td>
			<td style="width:88px;">Power Glide</td>
			<td style="width:88px;">Model 60100002</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">0.5 &#181;l Hamilton syringe</td>
			<td style="width:88px;">Hamilton Co.</td>
			<td style="width:88px;">86259</td>
			<td style="width:88px;">25 gauge needle</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">50 &#181;l Hamilton syringe</td>
			<td style="width:88px;">Hamilton Co</td>
			<td style="width:88px;">7637-01</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">26G Hamilton needle</td>
			<td style="width:88px;">Hamilton Co</td>
			<td style="width:88px;">7804-03</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Syringe pump</td>
			<td style="width:88px;">KD Scientific</td>
			<td style="width:88px;">Model 100</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Heat therapy water pump</td>
			<td style="width:88px;">Gaymar Industries, Inc.</td>
			<td style="width:88px;">Model# TP650</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Circulating waterbed</td>
			<td style="width:88px;">CMS Tool &#38; Die, Inc.</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Rodent ventilator</td>
			<td style="width:88px;">Harvard Apparatus</td>
			<td style="width:88px;">Model 683</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Isoflurane vaporizer</td>
			<td style="width:88px;">Drager</td>
			<td style="width:88px;">Vapor 19.1</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Air flowmeter</td>
			<td style="width:88px;">Cole Parmer</td>
			<td style="width:88px;">Model PMR1-010295</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Induction chamber</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
			<td style="width:88px;">Self made</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Otoscope</td>
			<td style="width:88px;">Welch Allyn</td>
			<td style="width:88px;">22820</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">intravenous catheter</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">381534</td>
			<td style="width:88px;">20-gauge, 1.16 inch Insyte-W</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Isoflurane</td>
			<td style="width:88px;">Baxter Healthcare Corporation</td>
			<td style="width:88px;">NDC10019-360-69</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Collagenase Type IV-S</td>
			<td style="width:88px;">Sigma</td>
			<td style="width:88px;">C1889</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Polyethylene tubing PE20</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">427406</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Polyethylene tubing PE10</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">427401</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">30G 1 inch needle</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">305128</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">27G 1 1/4 inch needle</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">305136</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Surgical scissors</td>
			<td style="width:88px;">Miltex</td>
			<td style="width:88px;">21-539</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Forceps</td>
			<td style="width:88px;">Miltex</td>
			<td style="width:88px;">17-307</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Needle holder</td>
			<td style="width:88px;">Boboz</td>
			<td style="width:88px;">RS-7840</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Monofilament suture</td>
			<td style="width:88px;">Ethicon</td>
			<td style="width:88px;">8698</td>
			<td style="width:88px;">Size 5-0</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Indicating controller</td>
			<td style="width:88px;">YSI</td>
			<td style="width:88px;">73ATD</td>
			<td style="width:88px;"></td>
		</tr>
	</tbody>
</table>

intrastriatal injection of autologous blood or clostridial collagenase as murine models of intracerebral hemorrhage

Hemorragia intracerebral (ICH) é uma forma comum de doença vascular cerebral e está associada a significativa morbidade e mortalidade. A falta de tratamento eficaz e fracasso de grandes ensaios clínicos destinados a hemostasia e remoção do coágulo demonstrar a necessidade de mais-driven mecanismo de investigação do ICH. Esta pesquisa pode ser realizada através da estrutura fornecida pelos modelos pré-clínicos. Dois modelos murinos em uso popular incluem intrastriatal (gânglios basais) ou injeção de sangue autólogo todo ou colagenase clostridiana. Uma vez que, cada modelo representa distintamente diferentes características fisiopatológicas relacionadas com HIC, o uso de um determinado modelo pode ser seleccionado com base no que os aspectos da doença está a ser estudado. Por exemplo, a injeção de sangue autólogo representa com mais precisão a resposta do cérebro à presença de sangue intraparenquimatosa, e pode replicar mais de perto hemorragia lobar. Clostridial injeção de colagenase representa com mais precisão as sruptura do vaso shopping e hematoma evolução característica de hemorragias profundas. Assim, cada modelo resulta na formação diferente hematoma, resposta neuroinflammatory, desenvolvimento de edema cerebral, e os resultados neurocomportamentais. Robustez de uma intervenção terapêutica pretendida pode ser melhor avaliada por ambos os modelos. Neste protocolo, a indução da ICH usando ambos os modelos, a demonstração de pós-operatório imediato de lesão, e técnicas de primeiros cuidados pós-operatórios são demonstrados. Ambos os modelos resultar em lesões reprodutíveis, volumes hematoma e déficits neurocomportamentais. Devido à heterogeneidade do ICH humana, vários modelos pré-clínicos são necessários para explorar completamente os mecanismos fisiopatológicos e testar estratégias terapêuticas potenciais.

Devido às diferenças na formação de hematoma (Figura 1), de virada ipsilateral é mostrado imediatamente após o despertar para o sangue autólogo camundongos injetados e dentro de 2-4 horas após a injeção de colagenase, como a expansão do hematoma ocorre (Figura 2). Ausência de viragem ipsilateral deve causar preocupação para a ausência de lesões significativas. No primeiro dia pós lesão, os ratos de ambos os modelos devem demonstrar déficits neurológicos significativo...

Watch this Scientific Journal Video about Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage at JoVE.com

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

Injection intraestriatal de sangue autólogo ou Clostridial colagenase como modelos murinos de hemorragia intracerebral

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective ...

intrastriatal-injection-autologous-blood-or-clostridial-collagenase

Research

JoVE Journal

Medicine

14.6K visualizações.  Duke University. Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase. 

Vídeo: Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

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

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Ligadura da artéria coronária e injeção intramiocárdica em um modelo murino de infarto

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

Hemorragia modelagem intracerebral em camundongos: Injeção de sangue autólogo ou colagenase bacteriana

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Um modelo murino de hemorragia subaracnóide

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

Estereológica e Citometria de Fluxo Caracterização de Leucócitos subpopulações em modelos de transitórias ou permanentes Cerebral Isquemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

O coelho Modelo Blood-shunt para o Estudo da aguda e tardia Sequelas de hemorragia subaracnóidea: Aspectos Técnicos

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

Férricos induzida pelo cloreto modelos murinos Trombose

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

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 ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

Nervo guiadas estimulador injeção de células-tronco autólogas perto do nervo laríngeo recorrente esquerdo equina

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

Punção da veia subclávia como um método alternativo de coleta de amostra de sangue em ratos

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...