This work was supported by National Institutes of Health grants HL087990 (G.L.).

Declaração de Ética: Adulto Masculino ratos Sprague-Dawley (pesando 330-380 g) foram utilizados neste protocolo. Este protocolo foi aprovado pelo Comitê de Cuidados e Uso de Animais Institucional (IACUC) na LSU Health Science Center, Shreveport e está em conformidade com o &quot;Guia para o Cuidado e Uso de Animais de Laboratório &#39;(oitava edição, da Academia Nacional de Ciências, 2011 ). 1 Preparação da modificação do tubo de PE-50 <ol><li> Segure um tubo de 30 centímetros de comprimento PE-50 acima de uma lareira a gás por mãos, gradualmente suavizar o tubo. Gentilmente esticar o tubo. </li><li> Selecione um ponto no tubo alongado utilizando um paquímetro digital (Figura 1), marcá-lo e cortar o tubo PE-50 modificado em um longo segmento de 25 cm com uma ponta longa 1 cm (diâmetro externo: ,30-,34 mm). </li></ol> 2 Preparação de homólogos Coágulos de sangue <ol><li> Anestesiar os ratos com isoflurano (5% para indução, 2-3% para manutenção) em 70% de N2O e 30% de O &lt;sub&gt; 2 por uma máscara facial antes da coleta de sangue. Confirme anestesia por uma pitada dedo do pé. </li><li> Realizar a canulação arterial femural de um rato dador com o método publicado em 4 e transferir sangue da artéria femoral directamente num 20 cm de comprimento de tubo PE-50. Colocar o tubo durante 2 horas à temperatura ambiente para coagular o sangue, e em seguida reter o tubo durante 22 horas a 4 ° C. Nota: A canulação é realizado utilizando uma técnica asséptica tal como descrito na etapa 3.1. O rato doador é recuperado para posterior amostragem. </li><li> Cortar o tubo de PE-50 a 50 mm e segmentos de expulsar o coágulo do tubo numa placa de Petri estéril contendo solução salina normal. </li><li> Transferir os 50 mm de comprimento de coágulo num comprimento de 60 mm de PE-10 e tubo de ligar cada extremidade do tubo de PE-10 a uma 20 cm de comprimento de tubo PE-50 ligado a uma seringa contendo ml de solução salina normal com 23 L de agulha (Figura 2) . </li><li> Deslocar o coágulo por movimento alternativo contínuo de uma seringa para a outra durante 5 minutos, o que é possível remover as células de sangue a partir deo coágulo e minimizar a fragilidade dos coágulos coagulado extravascular, sem interromper o núcleo de fibrina. </li><li> Cortar o coágulo em um segmento de 40 milímetros de comprimento e transferir o segmento de coágulo modificado num cateter PE-50, tal como descrito na etapa 1.2, em condições assépticas. Nota: Todos os tubos PE é esterilizado com óxido de etileno. </li></ol> 3. Embolic artéria cerebral média Oclusão (Figura 3A, B) <ol><li> Esterilizar todos os instrumentos cirúrgicos por autoclavagem (mínimo 121 ° C, 15 PSI, por 15 min). Higienizar a mesa de cirurgia e equipamentos cirúrgicos associados com 70% de etanol. </li><li> Anestesiar os ratos com isoflurano, conforme descrito no passo 2.1. Teste da profundidade da anestesia através da realização de uma ponta de aperto em ambos os pés traseiros, e qualquer movimento observado (retirada da pata), indica que o animal não seja suficientemente anestesiado para fazer a cirurgia. Aplique uma pequena quantidade de pomada veterinário em ambos os olhos para evitar a secura sob anestesia. Raspar a pele no pescoço e cabeça ventral reregiões com cortador elétrico para expor as áreas da pele. </li><li> Coloque o rato em decúbito dorsal sobre uma almofada de aquecimento. Insira uma sonda rectal, controlar e manter a temperatura do corpo entre 36,5-37,5 ° C, utilizando uma unidade de controlo cobertor homeotérmica. </li><li> Desinfetar a pele raspada e pele circundante com 10% de iodopovidona seguido por 70% de etanol. </li><li> Faça uma incisão na linha média 2 cm de comprimento no pescoço sob um microscópio de dissecação. Use retratores para expor o campo cirúrgico e dissecção da artéria carótida comum direita (CCA), artéria carótida externa (ECA), artéria carótida interna (ACI) e artéria pterigopalatina (PPA), isenta de nervos circundantes e fáscia (Figura 3A). Nota: Antes da cirurgia, mudar luvas estéreis se não houver assistente ajuda a preparar o animal. </li><li> Dissecar o CCA livre dos nervos que rodeiam (sem prejudicar o nervo vago) e coloque um estéril sutura 5-0 seda sob a artéria. Amarre um nó corrediço (# 1) e compreender a sutura usando uma pequena hemostat e puxe ensinado em direção ao corpo. </li><li> Dissecar o ECA e seus dois ramos, a artéria occipital (OA) e da artéria tireoidiana superior (STA). Coagular dois ramos usando uma unidade eletrocirúrgica veterinária. Coloque dois pedaços de estéreis 6-0 seda sob o ECA. </li><li> Separa-se as duas sedas colocados por baixo da artéria, uma peça para a cabeça (extremidade distal) e o outro na direcção do corpo. Amarre uma ligadura apertada (# 2) no lado mais próximo da cabeça, segure a seda com pequenas hemostatos e puxe ensinado em direção à cabeça. Prepare um nó frouxo (# 3) com a outra de seda para usar mais tarde. </li><li> Dissecar o ICA e PPA dos nervos circundantes (sem causar danos ao nervo vago). Amarre o PPA com um 6-0 (# 4). Faça um nó corrediço com 6-0 ao redor do ICA (# 5). Alternativamente, o clipe de ICA usando um clipe microvascular. </li><li> Corte um pequeno buraco (meia thru) na ECA entre o apertado (# 2) e soltos (# 3) ligaduras com uma tesoura primavera Vannas de estilo. Insira o modificada 50 PE-banheirae contendo coágulo de sangue na incisão e avançar para a bifurcação da CCA. Aperte a ligadura solta (# 3) em torno do lúmen apenas o suficiente para garantir a preservação da mobilidade do tubo em que habitam. </li><li> Corte o ECA no site do pequeno buraco para liberar o coto e posicionar o tronco abaixo da bifurcação da ECA e da ACI; isso vai permitir que o tubo modificado facilmente deslizar para o ICA. Abra o ICA e avançar suavemente o tubo a partir do lúmen da ECA na ICA até que a ponta do cateter atinge a origem da MCA (~ 17 mm da bifurcação). Nota: Antes de introduzir a ponta do tubo para o CEA, estéril a superfície exterior do tubo com etanol a 70%, e, em seguida, lava-se com solução salina normal estéril. </li><li> Injectar o coágulo através da modificação do cateter PE-50, juntamente com 10 ul de solução salina durante 10 segundos, utilizando uma seringa Hamilton de 100 l (Figura 3B). </li><li> Retirar o cateter da ECA cinco minutos mais tarde. Amarre o ECA e reabrir CCA. Suturar a incisão no pescoço. </li></ol> 4 Monitoramento Regional Cerebral Blood Flow (rCBF) <ol><li> Antes de oclusão MCA, fazer um 1,2 centímetros de comprimento incisão na linha média do couro cabeludo em expor o osso do crânio. Retire os tecidos no osso do crânio com um raspador dental e swaps de algodão estéril. Nota: Antes da cirurgia, a área do couro cabeludo exposto e pele circundante são desinfetados com 10% de iodopovidona seguido por 70% de etanol. </li><li> A broca de 1,5 mm de orifício de trépano diâmetro localizado a 2 mm posterior e 5 mm lateral ao bregma usando uma rebarba esférica de aço inoxidável de 0,7 milímetros (Figura 4). Nota: Mantenha a dura intacta. </li><li> Coloque a sonda de 0,5 milímetros acima da superfície da dura-máter. Monitorizar o rCBF a 0 (linha de base), 5, 15, 30, 60, 90, e 120 minutos após a embolização e continuar a 5, 15, 30, 45, e 60 min após a administração intravenosa de tPA. Depois da última medição de rCBF, a incisão no couro cabeludo é fechada por sutura. Nota: Antes da incisão no pescoço medimos rCBF como base antes MCA Occlusião. Oclusão pós MCA medimos rCBF após o fechamento da sutura de incisão no pescoço. Assim, a incisão do pescoço é mantida estéril. </li></ol> Cuidados 5. Pós-operatório <ol><li> Injectar 2,5 mL de solução salina por via subcutânea para evitar desidratação e injectar Buprenex (0,05 mg / kg, SC), imediatamente após a cirurgia a cada 6-12 horas e quando necessário para alívio da dor. </li><li> Pare de anestesia isoflurano. Colocar o rato numa câmara de recuperação veterinária 37 ° C (manter animais quente) e manter observação. Geralmente, leva 10 min para o rato para se recuperar da anestesia. Em seguida, coloque o animal em uma gaiola esterilizado, coloque um pouco de comida molhada em uma placa de Petri na gaiola, e voltar a gaiola para sala de esterilização animal. </li><li> 24 horas após o derrame, a eutanásia ratos com uma overdose de pentobarbital de sódio (100 mg / kg de peso corporal, IP). </li></ol> 6. Neurological Pontuação Deficit <ol><li> Execute o placar Bederson antes e 2 horas após a embolização. Use uma classificação de scale de 0-3 como descrito anteriormente 5: 0, levantando o rato pela cauda, ​​a animais que se estende para ambos os membros anteriores e apresentando o piso sem outros déficits (movimento normal); 1, elevando o rato pela cauda, ​​flexionar o membro anterior contralateral; 2, diminuição da resistência à pressão lateral (e membro anterior flexão), sem circular; 3, o mesmo comportamento de grau 2 com circulando. NOTA: Os ratos que mostram Bederson score = 0 (sem déficit) em 2 horas após a embolização são excluídos do estudo mais aprofundado. </li></ol>

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We do not have any potential conflicting interests to disclose.

Neste estudo, foi demonstrado um método padrão para a realização de um modelo de acidente vascular cerebral embólico MCAO em ratos, em que a origem da MCA é tapada por um coágulo rico em fibrina. A principal vantagem deste modelo é: a oclusão da haste de MCA com um coágulo de sangue ricos em fibrina, é semelhante ao derrame tromboembólico em humanos, o modelo de acidente vascular cerebral embólico é adequado para a realização de investigação pré-clínica da terapêutica fibrinolítica, e que este mode...

A correction was made to <a href="/video/51956">Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats</a>. The institution information was updated.
The institution "Louisiana State University Health Science Center" was changed to "Louisiana State University Health Science Center, Shreveport".

A correction was made to <a href="/video/51956">Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats</a>. The institution information was updated.

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

O AVC é a terceira principal causa de morte nos Estados Unidos, mas as opções de tratamento para o AVC agudo permanecem limitados. No momento, a perfusão intravenosa de ativador do plasminogênio tecidual recombinante (tPA) para dissolver os coágulos de sangue é a terapêutica mais eficaz para AVC isquêmico agudo. No entanto, o uso de tPA é limitada pela sua estreita janela terapêutica e pelo aumento do risco de hemorragia intracerebral. Portanto, um modelo de acidente vascular cerebral adequado para pesquisa trombolítica é urgentemente necessária. A artéria cerebral média (MCA) é na maioria das vezes a artéria oclusa em acidente vascular cerebral em seres humanos. Focada em esta artéria, muitos modelos animais de acidente vascular cerebral isquêmico foram estabelecidas. Actualmente, dois tipos principais de modelos de isquemia focal de roedores por oclusão da MCA foram desenvolvidos: sutura modelo MCAO e embólico modelo MCAO. Embora os modelos de MCAO via a técnica de sutura intraluminal, têm sido amplamente utilizados em investigação orientada para o mecanismo de acidente vascular cerebral, estes modelos não fazer suturasacidente vascular cerebral humano mímico, como até 80% dos acidentes vasculares cerebrais humanos são causadas por trombose ou embolia. No entanto, o modelo de acidente vascular cerebral embólico usando coágulos sanguíneos imita traço humano e é considerado adequado para o estudo do trombolítico. Este modelo embólico foi desenvolvido pela primeira vez em ratos por Overgaard et al. 1 em 1992 e caracteriza-se por Zhang et al., Em 1997, 2 e por Dinapoli et al., Em 2006, 3. Embora embólico OACM ganha cada vez mais atenção, há problemas técnicos enfrentados por muitos laboratórios. Neste artigo, vamos demonstrar um método padrão para a produção de embólico OACM com coágulos de sangue homólogo no rato adulto, que pode desenvolver um infarto do previsível dentro do território da ACM. As técnicas apresentadas neste artigo deve ajudar os pesquisadores a superar problemas técnicos para o estabelecimento deste modelo para a pesquisa derrame.

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			<td height="24" style="height:24px;width:232px;">1ml syringe</td>
			<td style="width:135px;">Consumable</td>
			<td style="width:237px;">Becton, Dickinson and Company</td>
			<td style="width:135px;">309659</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">23G needle</td>
			<td style="width:135px;">Consumable</td>
			<td style="width:237px;">Becton, Dickinson and Company</td>
			<td style="width:135px;">305143</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:232px;">30G needle</td>
			<td style="width:135px;">Consumable</td>
			<td style="width:237px;">Becton, Dickinson and Company</td>
			<td style="width:135px;">305106</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:232px;">PE-50 tubing</td>
			<td style="width:135px;">Consumable</td>
			<td style="width:237px;">Becton, Dickinson and Company</td>
			<td style="width:135px;">427517</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:232px;">PE-10 tubing</td>
			<td style="width:135px;">Consumable</td>
			<td style="width:237px;">Becton, Dickinson and Company</td>
			<td style="width:135px;">427400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">6-0 Silk suture</td>
			<td style="width:135px;">Consumable</td>
			<td>Harvard apparatus</td>
			<td style="width:135px;">723287</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:232px;">5-0 Silk suture</td>
			<td style="width:135px;">Consumable</td>
			<td>Harvard Apparatus</td>
			<td>517607</td>
		</tr>
	</tbody>
</table>

embolic middle cerebral artery occlusion (mcao) for ischemic stroke with homologous blood clots in rats

Terapia clínica, com uso de trombolítico ativador do plasminogênio tecidual recombinante (tPA) continua a ser o tratamento mais eficaz para AVC isquêmico agudo. No entanto, o uso de tPA é limitada pela sua estreita janela terapêutica e pelo aumento do risco de transformação hemorrágica. Há uma necessidade urgente de desenvolver modelos tempos adequados para estudar novos agentes e estratégias de trombolíticos para o tratamento de acidente vascular cerebral isquêmico. Actualmente, dois tipos principais de modelos isquémicos tempos têm sido desenvolvidos em ratos e ratinhos: intraluminal MCAO sutura e embólico MCAO. Embora os modelos de MCAO via a técnica de sutura intraluminal, têm sido amplamente utilizados em investigação orientada para o mecanismo de acidente vascular cerebral, estes modelos de sutura não mimetizar a situação clínica e não são adequados para estudos trombolíticos. Entre esses modelos, o modelo OACM embólico imita AVC isquêmico humano e é adequado para a investigação pré-clínica de terapia trombolítica. Este modelo embólico foi desenvolvido pela primeira vez em ratos por Overgaard et al. 1 em 1992 e caracteriza-se por Zhang et al., em 1997 2. Embora embólico OACM ganha cada vez mais atenção, existem problemas técnicos enfrentados por muitos laboratórios. Para atender às necessidades crescentes de pesquisa trombolítica, apresentamos um modelo altamente reprodutível de embólico OACM no rato, que pode desenvolver um volume de infarto previsível dentro do território da ACM. Em resumo, um tubo de PE-50 modificado é avançada suavemente a partir da artéria carótida externa (ECA) para dentro do lúmen da artéria carótida interna (ICA), até que a ponta do cateter atinge a origem da MCA. Através do cateter, um coágulo de sangue homólogo único é colocado na origem da MCA. Para identificar o sucesso de oclusão MCA, o fluxo sanguíneo cerebral regional foi monitorizada, défices neurológicos e os volumes de enfarte foi medida. As técnicas apresentadas neste artigo deve ajudar os pesquisadores a superar problemas técnicos para o estabelecimento deste modelo para a pesquisa derrame. &lt;/ P&gt;

A fluxometria de Doppler a laser (LDF) foi utilizado para monitorizar rCBF durante a indução da isquémia cerebral 6,7. Muitos laboratórios, incluindo o nosso laboratório tem usado rCBF para identificar os animais com oclusão MCA bem sucedido, mas os limites de linha de base variou entre os laboratórios que estão relacionados com o local de medição. A sonda do LDF está posicionado a 2 mm posterior a 5 mm lateral ao bregma como descrito anteriormente 6. rCBF foi monitorizada nos tempos 0, ...

Watch this Scientific Journal Video about Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats at JoVE.com

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 ...

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Research

JoVE Journal

Medicine

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

26.8K visualizações.  Louisiana State University Health Science Center, Shreveport. 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. 

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

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

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Modelo do rato de Oclusão da Artéria Cerebral Média

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

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 ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Utilizando uma janela craniana para Visualizar a artéria cerebral média Durante endotelina 1 Induzida por oclusão da artéria cerebral média

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

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 ...

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 ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Realizando Permanente Distal Oriente cerebral com carótida comum Oclusão da Artéria em ratos idosos para estudar Cortical Isquemia com Deficiência Sustentada

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.