This study was supported by the NIH grant NS074559 (to C. K.). We thank all collaborators who contributed to our research articles that the present methodology report is based upon.

Este protocolo é aprovado pelo cuidado e uso Comitê Institucional Animal (IACUC) da Universidade de Emory e segue os Institutos Nacionais de Saúde Guia para Cuidados e Uso de Animais de Laboratório. 1. Setup <ol><li> Prepare o leito cirúrgico na almofada de aquecimento ligado com bomba de calor a 37 ° C durante pelo menos 15 minutos antes da cirurgia. Coloque um rolo de pescoço usando o barril de 3 ml seringa no leito cirúrgico. Prepara-se o gás a anestesia com isoflurano a 2% em ar medicinal. </li><li> Prepare fórceps autoclavada, tesouras, porta-agulhas, micro pinça hemostática, cotonetes e suturas. Prepare a cola de tecido e pomada. </li><li> Configure o sistema de hipóxia e controladores de temperatura com lâmpada de aquecimento e sonda retal. Prepare gás hipoxia com 2% de isoflurano em 7,5% de O2 em relação 92,5% de N2. </li><li> Uma hora antes da cirurgia, os ratos são analgesized por injecção subcutânea de uma de libertação lenta O meloxicam (4,0 mg / kg). </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 2. Transient-hipoxia cerebral isquemia (Figura 1B) <ol><li> Anestesiar 10-13 semanas de idade C57BL masculino / 6 ratos pesando 22 a 30 g na câmara de indução anestésica com 3% de isoflurano até que o animal está respondendo a uma compressão de pé, e depois remover o cabelo no pescoço direita usando um barbeador eletrônico. </li><li> Coloque ratinhos no leito cirúrgico conectado com isoflurano a 2% em ar medicinal a um caudal de 2 L / min. Forelimbs seguros estendeu ao longo do rolo pescoço em lados usando fita adesiva médica. </li><li> Limpe o local da cirurgia para a incisão com betadine seguido de álcool e, em seguida, cotonetes. </li><li> Sob microscópio de dissecação, fazer uma incisão 0,5 centímetros direita-cervical utilizando uma pinça reta e uma tesoura micro cerca de 0,2 cm lateral da pele na linha média. </li><li> Utilizar um par de fórceps finos serrilhada para separar a fáscia e tecido para expor a artéria carótida comum direita (RCCA). Cuidadosamente separar a RCCA do nervo vagal, usando um par de fórceps finos lisas. </li><li> Vivo nó dois pré-cortada 5-0 sutura de seda (destacável) na RCCA, e depois costurar a pele usando 4-0 sutura de monofilamento de nylon (Figura 1C). </li><li> Aplicar pomada olho em ambos os olhos para prevenir o ressecamento. </li><li> Transferir rapidamente os ratos a hipoxia sistema e colocá nariz e boca na máscara facial com 2% de isoflurano em 7,5% de O 2 a uma taxa de fluxo de 0,5-1 L / min durante 30 min. <ol><li> Durante a hipóxia, utilizar controladores de temperatura com lâmpadas de aquecimento para controlar a temperatura rectal em 37,5 ± 0,5 ° C. Monitorar a freqüência respiratória em 80-120 ciclos / min. A manutenção da temperatura corporal acima de 37 ° C durante a hipóxia é importante para criar enfarte cerebral consistente. Frequência respiratória baixa geralmente acontece depois de 20 min hipóxia. Remova a máscara e permitir que o suprimento de ar normal se a freqüência respiratória cair abaixo de 40. Isso leva 1-2 min e não conta para a duração hipóxia 30 min. </li></ol></li><li> Depois de hipoxia, transferircamundongos para um leito cirúrgico e liberar as duas suturas de RCCA. Fechar a ferida usando cola de tecido e, em seguida, retornar os ratos para a jaula. Excluir os animais se ambos dois nós ao vivo são inesperadamente libertado depois hipoxia. </li><li> Monitorar os ratos para 5-10 min para se recuperar de hipóxia e anestesia. Coloque o alimento molhado na gaiola e devolvê-lo à facilidade de cuidado animal. Nota: Os animais que apresentem leve a grave comportamento circular às 24 h após Thi estão correlacionados com infração cérebro. A maioria dos animais com sintomas de apreensão morrer antes do ponto de tempo de 24 horas depois de Thi. </li></ol> 3. Laser Speckle Contrast Imaging Nota: Embora isso não é um processo essencial do modelo Thi, um salpico de laser sistema de imagiologia de contraste bidimensional 16 pode ser utilizado para caracterizar as alterações do fluxo sanguíneo cerebral (CBF) durante ou após a hipóxia-isquémia transiente. Para documentar as alterações da CBF sob Thi, ficha imediatamente após a step 2.6. Alternativamente, para comparar a recuperação CBF após insulto Thi, estes procedimentos podem ser realizados seguindo o passo 2.10. <ol><li> Inserir um rato anestesiado em decúbito ventral e executar a 1 cm de comprimento incisão na linha média no couro cabeludo com o crânio exposto, mas sem abrir. </li><li> Monitorar CBF em ambos os hemisférios cerebrais sob um imager fluxo de sangue de acordo com o protocolo do fabricante e começar a gravar o fluxo sanguíneo cerebral imediatamente após a cirurgia CCAO (passo 2.6). Continue por 50 min. </li><li> Mostrar imagem CBF com unidades arbitrárias em uma paleta de 16 cores e analisar em tempo real as regiões selecionadas usando o software MoorFLPI seguindo as instruções do fabricante (Figura 2). </li><li> Depois de gravar a imagem CBF, feche o couro cabeludo com cola de tecido e devolver o animal para a jaula. </li></ol> Administração 4. tPA <ol><li> Injectar animais na veia da cauda com o solvente ou 10 mg / kg de tPA recombinante (220-300 &amp;# 956; ul de 1 mg / m tPA) a 0,5, 1, 4 ou mais horas após tCCAo hipoxia (Figura 4). </li></ol> 5. Dano Cerebral Detecção com vários diferentes opções Nota: Para a coleta de amostras de cérebro, eutanásia os ratos de 1, 4 ou 24 horas após Thi. <ol><li> Execute quantificação do volume de enfarte in vivo por cloreto de 2,3,5-Trifeniltetrazólio método (TTC) em 24 horas após Thi insulto como descrito anterior. 17 <ol><li> Injectar animais por via intraperitoneal com solução de manitol 1,4 M (~ 0,1 ml / g de peso corporal) 30 min antes da perfusão transcardial. Ratinhos perfundir transcardial com PBS, seguido por 10 ml de 2% de TTC. </li><li> Remover o cérebro de animais com instrumentos cirúrgicos, depois de 10 minutos e colocar em paraformaldeído a 4% para a fixação durante a noite e em secção de 1 mm de espessura com um vibratome. </li><li> Tire uma série de quatro lâminas do cérebro segmentado por microscópio digital e quantify o volume de enfarte como a razão da área de enfarte (área branca do lado direito) para a área do hemisfério não danificado, contralateral utilizando o software ImageJ. </li></ol></li><li> Como alternativa, realizar a formação de trombose por imunofluorescência em 1 hora após Thi insulto. <ol><li> Congelar o cérebro fixado em composto outubro e os cérebros na seção 12 mm de espessura utilizando um criostato. </li><li> Incubar as lâminas do cérebro com anticorpo de coelho anti-f ibrinogénio (1: 100) seguido de anticorpo de cabra anti-coelho Alexa Fluro 488 corante (1: 200) para observar a fluorescência num microscópio fluorescente. </li></ol></li><li> Como alternativa, executar obstrução do vaso por injecção na veia da cauda de 100 ul 2% corante azul de Evans às 4 horas após Thi insulto. <ol><li> Euthanize os ratos e rapidamente cortar a cabeça para remover cérebros em paraformaldeído a 4% após injeção de azul de Evans. Nota: Demora 5-10 min para Evans azul circulação com cor azul de ambos previsão e pélvicos. </li><li> Sectiem cérebros fixados a espessura de 100 um utilizando um micrótomo deslizante e observar a fluorescência usando um filtro de emissão de 680 nm num microscópio fluorescente. </li></ol></li></ol>

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O AVC é um importante problema de saúde de crescente importância para qualquer sociedade com o envelhecimento da população. Globalmente, o AVC é a segunda principal causa de morte com um número estimado de 5,9 milhões de eventos fatais em 2010, o equivalente a 11,1% de todas as mortes 18. O AVC é também a terceira maior causa de anos de vida ajustados por incapacidade (DALYs) perdidos globalmente em 2010, elevando-se a partir da quinta posição em 1990 19. Esses dados epidemiológicos ap...

Trombólise e recanalização é a terapia mais eficaz de AVC isquêmico agudo na prática clínica 1. No entanto, a maioria da investigação pré-clínica foi realizada a neuroprotecção num modelo transiente mecânico obstrução (oclusão da artéria cerebral média sutura intraluminal) que produz uma recuperação rápida de fluxo sanguíneo cerebral após a remoção da oclusão vascular e mostra pouca ou nenhuma benefícios por tPA trombólise. Tem sido sugerido que a escolha duvidosa de modelos de acidente vascular cerebral contribui, pelo menos em parte, da dificuldade em traduzir terapia neuroprotectora para pacientes 2,3. Assim, há um crescente apelo para o emprego de modelos de AVC tromboembólica tPA-responsivos em pesquisa pré-clínica, mas tais modelos também têm problemas técnicos (ver Discussão) 4-7. Aqui nós descrevemos um novo modelo de acidente vascular cerebral trombótico com base em (ITU) insulto hipóxico-isquêmica transitória unilateral e suas respostas à terapia tPA intravenosa 8. O modelo de acidente vascular cerebral Thi foi desenvolvido com base no procedimento de Levine (ligadura permanente da artéria carótida comum unilateral seguida por exposição a hipoxia transitória numa câmara) que foi inventado para experiências com ratos adultos em 1960 9. O procedimento Levine inicial desapareceu na obscuridade porque ele só produziu danos cerebrais variável, mas o mesmo insulto causado neuropatologia consistente em filhotes de roedores quando foi re-introduzido por Robert Vannucci e seus colegas como um modelo de neonatal encefalopatia hipóxico-isquêmica (HIE) em 1981 10. Nos últimos anos, alguns investigadores re-adaptado ao modelo Levine-Vannucci para ratinhos adultos por ajustamento da temperatura na câmara hipóxica 11. É plausível que as lesões cerebrais inconsistentes no procedimento Levine original pode surgir a partir de temperaturas flutuantes do corpo de roedores adultos na câmara hipóxica. Para testar esta hipótese, nós modificamos o procedimento Levine pela administração de gás hipóxicoatravés de uma máscara facial, enquanto se mantinha a temperatura interna a 37 ° roedor C na mesa cirúrgica 12. Como esperado, o controlo rigoroso da temperatura corporal aumentou significativamente a reprodutibilidade da patologia cerebral induzida por HI. O insulto HI também provoca a coagulação, a autofagia, e grisalhos e branco-matéria lesão 13. Outros pesquisadores também usaram o modelo HI para investigar pós-AVC respostas inflamatórias 14. Uma característica única do modelo de acidente vascular cerebral HI é que ele segue de perto tríade de Virchow de formação de trombos, incluindo a estase do fluxo de sangue, lesão endotelial (por exemplo, devido ao estresse oxidativo induzido por HI), e hipercoagulabilidade (ativação plaquetária induzida pelo HI) ( Figura 1A) 15. Como tal, o modelo HI pode capturar alguns mecanismos fisiopatológicos relevantes para AVC isquêmico no mundo real. Com esta ideia em mente, nós refinou ainda mais o modelo HI com ligadura reversível da ONUartéria carótida comum ilateral (portanto, para criar um insulto HI transitória), e testou suas respostas ao tPA trombólise com ou sem edaravona. Edaravona é um captador de radicais livres já aprovado no Japão para tratar AVC isquêmico dentro de 24 horas do início 9. As nossas experiências mostraram que tão breve quanto 30 min transiente HI desencadeia enfarte trombótico, e que o tratamento com tPA-edaravona combinada confere benefícios sinérgicos 8. Aqui nós descrevemos os procedimentos cirúrgicos detalhadas e considerações metodológicas do modelo Thi, que podem ser utilizados para otimizar tratamentos reperfusão de AVC isquêmico agudo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>adult male mice</td>
			<td>Charles River</td>
			<td>C57BL/6&#160;</td>
			<td>10~13 weeks old (22~30 g)</td>
		</tr>
		<tr>
			<td>Mobile Laboratory Animal Anesthesia System</td>
			<td>VetEquip</td>
			<td>901807</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Medical air (Compressed) air tank</td>
			<td>Airgas</td>
			<td>UN1002</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Healthcare</td>
			<td>NDC 66794-013-25</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Multi-Station Lab Animal AnesthesiaSystem</td>
			<td>Surgivet</td>
			<td>V703501</td>
			<td>hypoxia system</td>
		</tr>
		<tr>
			<td>7.5% O2 balanced by 92.5% N2 tank</td>
			<td>Airgas</td>
			<td>UN1956</td>
			<td>hypoxia system</td>
		</tr>
		<tr>
			<td>Temperature Controller with heating lamp&#160;</td>
			<td>Cole Parmer&#160;</td>
			<td>EW-89000-10</td>
			<td>temperature controllers</td>
		</tr>
		<tr>
			<td>Rectal probe</td>
			<td>Cole Parmer&#160;</td>
			<td>NCI-00141PG</td>
			<td>temperature controllers</td>
		</tr>
		<tr>
			<td>Dissecting microscope&#160;</td>
			<td>Olympus&#160;</td>
			<td>SZ40</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Heat pump with warming pad</td>
			<td>Gaymar&#160;</td>
			<td>TP700</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Fine curved forceps (serrated)</td>
			<td>FST</td>
			<td>11370-31</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>Fine curved forceps (smooth)</td>
			<td>FST</td>
			<td>11373-12</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>micro scissors</td>
			<td>FST</td>
			<td>15000-03</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>micro needle holders</td>
			<td>FST</td>
			<td>12060-01</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>Halsted-Mosquito hemostats</td>
			<td>FST</td>
			<td>13008-12</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>5-0 silk suture&#160;</td>
			<td>Harvard Apparatus</td>
			<td>624143</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>4-0 Nylon monofilament suture</td>
			<td>LOOK</td>
			<td>766B</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>Tissue glue</td>
			<td>Abbott Laboratories</td>
			<td>NC9855218</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>Puralube Vet ointment</td>
			<td>Fisher</td>
			<td>NC0138063&#160;</td>
			<td>eye dryness prevention&#160;</td>
		</tr>
		<tr>
			<td>MoorFLPI-2 blood flow imager</td>
			<td>Moor</td>
			<td>780-nm laser source</td>
			<td>Laser Speckle Contrast Imaging</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma</td>
			<td>M4125</td>
			<td>in-vivo TTC</td>
		</tr>
		<tr>
			<td>2,3,5-triphenyltetrazolium chloride (TTC)&#160;</td>
			<td>Sigma</td>
			<td>T8877</td>
			<td>in-vivo TTC</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Stoelting</td>
			<td>51425</td>
			<td>brain section for in-vivo TTC&#160;</td>
		</tr>
		<tr>
			<td>Digital microscope</td>
			<td>Dino-Lite</td>
			<td>AM2111</td>
			<td>whole-braina imaging</td>
		</tr>
		<tr>
			<td>O.C.T compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-rabbit Alexa Fluro 488</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Vibratome</td>
			<td>ultrapro 5000</td>
			<td>brain section for IHC</td>
		</tr>
		<tr>
			<td>Evans blue</td>
			<td>Sigma</td>
			<td>E2129</td>
			<td>Detecting vascular perfusion</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Electron Microscopy Sciences</td>
			<td>5000</td>
			<td>brain section for histology</td>
		</tr>
		<tr>
			<td>Avertin (2, 2, 2-Tribromoethanol)</td>
			<td>Sigma</td>
			<td>T48402</td>
			<td>euthanasia</td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Olympus</td>
			<td>DP73</td>
			<td></td>
		</tr>
	</tbody>
</table>

a thrombotic stroke model based on transient cerebral hypoxia-ischemia

Pesquisa derrame tem sofrido muitos contratempos em traduzir terapias neuroprotetoras para a prática clínica. Em contraste, a terapia do mundo real (tPA trombólise) raramente produz benefícios em modelos experimentais à base de oclusão mecânica, os quais dominam a investigação pré-clínica acidente vascular cerebral. Esta divisão entre o banco e cabeceira sugere a necessidade de empregar modelos tPA-responsivos em pesquisa pré-clínica acidente vascular cerebral. Para este fim, um modelo de acidente vascular cerebral trombótico simples e de tPA-reactivo é inventado e descrito aqui. Este modelo consiste de oclusão transiente da artéria carótida comum unilateral e entrega de 7,5% de oxigénio através de uma máscara facial em ratos adultos, durante 30 min, enquanto se mantinha a temperatura rectal dos animais a 37,5 ± 0,5 ° C. A ligadura reversível da artéria carótida unilateral ou hipóxia cada suprimido o fluxo sanguíneo cerebral apenas transitoriamente, a combinação de ambos os insultos causada duradoura défices de reperfusão, a fibrina e a deposição de plaquetas, e grande INFARCT no território fornecido pelo artéria cerebral média. Importante, a injecção de tPA recombinante a 0,5, 1, ou 4 h pós-THI (10 mg / kg) da veia da cauda proporcionou uma redução tempo-dependente do tamanho do enfarte e mortalidade. Este novo modelo de acidente vascular cerebral é simples e pode ser padronizado em laboratórios para comparar os resultados experimentais. Além disso, induz a trombose sem craniectomia ou a introdução de êmbolos pré-formadas. Tendo em conta estes méritos exclusivos, o modelo Thi é uma adição útil para o repertório de pesquisa do curso pré-clínico.

Bidimensional imagiologia de contraste salpico do laser (LSCI) 16 foi utilizado para comparar as alterações do fluxo sanguíneo cerebral (CBF) por oclusão da carótida 30 minutos transiente unilateral (tCCAO), 30 min de exposição a hipoxia (7,5% de oxigénio), e 30 min unilateral carótida ligadura sob hipóxia (ITU). Esta experiência revelou que tCCAO sob normoxia suprimiu a CBF no hemisfério carótida ligado a ~ 50% do valor da linha de base, o qual se recuperou rapidamente para acima de 85% após a ...

Watch this Scientific Journal Video about A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia at JoVE.com

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Um Modelo de acidente vascular cerebral trombótico Baseado em Transient isquemia-hipoxia cerebral

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

a-thrombotic-stroke-model-based-on-transient-cerebral-hypoxia-ischemia

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JoVE Journal

Medicine

14.7K visualizações.  The Emory University School of Medicine. Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients. 

Vídeo: A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Curso de modelagem em Ratos - Oclusão da Artéria Cerebral Média com o modelo de Filamento

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Medicina

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Intraluminal artéria cerebral média Oclusão Model (MCAO) para Acidente Vascular Cerebral Isquêmico com Laser Doppler Fluxometria Orientação em Ratos

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

Oclusão bilateral artéria carótida comum como um estímulo pré-condicionamento adequados para induzir a tolerância isquêmica início da Isquemia Cerebral Focal

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Endotelina-1 Induzida Médio modelo de oclusão da artéria cerebral para AVC isquêmico com Laser Orientação Fluxometria Doppler em ratos

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

Sistema otimizado para Monitorização de perfusão cerebral no modelo do rato do curso de oclusão da artéria cerebral média intraluminal

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

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 ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxico Estimulação do Microslices cérebro como um<em&gt; In vitro</em&gt; Modelo de AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Curso de modelagem em Ratos: Coagulação permanente da artéria distal Cerebral Média

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

Embolic artéria cerebral média Oclusão (MCAO) para Acidente Vascular Cerebral Isquêmico com homólogos Coágulos de sangue em ratos

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Simultânea de imagem PET / MRI Durante Rato isquemia-hipoxia cerebral

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...