Este trabalho foi financiado pelo Programa da Comunidade Europeia Quadro (FP7/2007-2013), sob subvenção n ° 201024 e n ° 202213 (Network Curso Europeu). Financiamento adicional foi dada pelo für Bildung und Forschung Bundesministerium (Centro de Pesquisa para o curso de Berlim), ea Deutsche Forschungsgemeinschaft (Exzellenzcluster NEUROCURE). Os autores gostariam de agradecer a Mareike Thielke (Dep. de Neurologia Experimental, Charité de Berlim), pelo apoio durante a operação e Ludwig Elke (Serviços de Vídeo Charité) para produzir a animação.

Para garantir alta qualidade e reprodutibilidade, recomendamos a utilização de Procedimentos Operacionais Padrão (POP). Neste vídeo, SOPs publicado como desenvolvidos e utilizados em nosso laboratório são aplicadas 1. 1. Oclusão da artéria cerebral média <ol><li> Os ratos são anestesiados com um regime adequado anestésico em consultar com o pessoal veterinário. (Por exemplo, com a indução 1,5-2% isoflurano e manutenção com 1,0 - 1,5% do isoflurano em 03/02 N2O e 1 / 3 O2 usando um vaporizador). <ol><li> Temperatura corporal dos ratos é mantida a 36,5 ° C ± 0,5 ° C durante a cirurgia com uma placa de aquecimento. Um gabarito almofada de aquecimento controlado, que aquece de acordo com a temperatura retal do mouse, é altamente recomendado. </li><li> Desinfectar a pele e pêlo ao redor com um agente apropriado (por exemplo, 70% de álcool etílico) e seque-o depois. </li></ol></li><li> A incisão é feita na linha média do pescoço e os tecidos moles são separadas. </li><li> A artéria carótida comum esquerda (ACCE) é cuidadosamente dissecado livre de os nervos ao redor (sem prejudicar o nervo vago) e uma ligadura é feita através de 6.0/7.0 string. Cadeia de 5.0 também pode ser usado. </li><li> A artéria carótida externa (LECA) é separado e um segundo nó é feito. </li><li> Em seguida, a artéria carótida interna esquerda (ACIE) é isolado e um nó é preparado com um filamento 6.0. </li><li> Depois de obter boa visão da artéria carótida interna esquerda (ACIE) e da artéria pterigopalatina (LPA), ambas as artérias são cortadas, utilizando um clipe microvascular. </li><li> Um pequeno buraco é cortado na LCCA antes que ela se bifurca para o Leça e da ACIE. Um monofilamento feito de nylon revestido com 8,0 silício mistura endurecedor (veja abaixo) é então introduzida no ACIE, até que ele pára no clipe. Atenção tem de ser pago para não entrar na artéria occipital. (Figura 1) </li><li> As artérias cortadas são abertas enquanto o filamento é inserido no ACIE de ocluir a origem do TCE no círculo de Willis. </li><li> Terceiro nó na LICA está fechado para corrigir o filamento na posição. </li><li> Os ratos recebem 0,5 mL solução salina por via subcutânea, reposição de volume. Para alívio da dor, lidocaína gel é aplicada topicamente na ferida. </li><li> Se reperfusão destina-se, os ratos ficar por 30-90 min de oclusão em uma gaiola aquecida, a ferida pode ser fechada com um clipe de sutura de pequeno porte. Depois, uma segunda anestesia é realizada, o terceiro nó na ICA é momentânea e abriu o filamento retirada. </li><li> As suturas restantes são encurtados ea pele é adaptada com uma sutura cirúrgica. </li><li> Todos os animais recebem uma reposição segundo volume como descrito acima. </li><li> Animais são colocados em uma gaiola aquecida por duas horas para controlar a temperatura do corpo. <ol><li> Os animais devem ser verificadas diariamente após a cirurgia para sinais de desconforto. Os camundongos puderam mostrar alguma perda de peso pós-cirúrgico. Eles recebem comida amassada em uma placa de Petri, colocado no chão para promover a alimentação. A comida é substituída por dia durante sete dias. </li></ol></li></ol> 2. Sham Operação <ol><li> Para operações de farsa o filamento é inserida para ocluir TCE e retirados imediatamente para permitir reperfusão instantâneas (1,8). A operação subseqüente é idêntico ao que realizou sobre os animais submetidos a isquemia cerebral (1,9-1,14), incluindo uma anestesia segundo. </li></ol> 3. Preparação do Filamento <ol><li> Esterilidade do filamento deve ser considerada. O uso de equipamentos esterilizados, bem como um manejo adequado do filamento depois é um pré-requisito para uma cirurgia estéril. Desinfecção do filamento é difícil, pois muitos dos métodos de esterilização comum pode piorar a qualidade do filamento. No entanto métodos tais como a radiação, por exemplo, com a luz UV ou raios γ, ou esterilização química, por exemplo, com gases altamente reativos, como óxido de etileno, são aplicáveis. </li><li> 8,0 filamento de nylon é cortado em pedaços de 11 milímetros sob o microscópio </li><li> A ponta filamento deve ser revestida completamente e uniformemente ao longo de um comprimento de 8 mm com uma mistura de endurecedor Xantopren M Mucosa e NF Activator Optosil </li></ol> 4. Resultados representante Dependendo da duração da restrição do fluxo sanguíneo, motor diferente e resultado déficits comportamentais. Ambos após 30 e 60 min de isquemia cerebral, animais na maioria dos casos apresentam resistência diminuiu para empurrar lateral e circulando devido a perturbações na locomoção. Milder manifestar lesões como uma posição flexor na frente limbs. Estes sinais facilmente observável pode ser usado como uma pontuação básica para o sucesso da operação. 2 Morfologicamente a lesão pode ser avaliada usando histologia ou Ressonância Magnética (MRI). Sessenta minutos de oclusão da artéria cerebral média produz pannecrosis tecido em uma área, incluindo tanto o striatum eo neocórtex, enquanto 30 minutos de isquemia causar a morte celular neuronal principalmente limitada para o estriado. 3 (Figura 2) Em termos de volume de infarto, esperamos um desvio padrão inferior a 30% em um conjunto de operações. Mortalidade depende do tempo de oclusão com cerca de 5% após 30 min de isquemia e 10 - 20% após 60 min. Outra possibilidade minimamente invasivo, é o uso de Laser Doppler fluxometria (FDL) durante a operação, o que permite um controle direto de seu sucesso. Em um animal individual, redução de 10-20% dos valores preocclusion indica claramente a indução bem-sucedida de isquemia cerebral focal 4 No entanto, LDF não pode ser usada como um método para comparações interindividuais, pois LDF só pode medir mudanças quantitativas (em percentagem) de sangue. fluxo dentro de um volume pequeno e limitado o tecido da amostra. Não dá informações sobre a extensão espacial da redução do fluxo de sangue. 5 Existem vários testes para avaliar aspectos comportamentais após um AVC, incluindo análise da marcha 6,7, Rotarod 8, teste Pólo 9,10, teste de remoção de adesivo 11,12, teste escadaria 13,14, teste escada degrau 15,16 e labirinto aquático de Morris 17. Em todos esses testes, os ratos submetidos a MCAo executar menos sucesso do que os animais controle. <img src="/files/ftp_upload/2423/2423fig1.jpg" alt="Figura 1" /> Figura 1. Esquema da arquitetura do navio que irrigam o cérebro (retratado no fundo) no mouse. Cepas diferentes podem apresentar variações, por exemplo a artéria occipital, por vezes, sai da artéria carótida interna. <img src="/files/ftp_upload/2423/2423fig2.jpg" alt="Figura 2" /> Figura 2. Ilustração esquemática de tamanhos de lesão típica depois de momentos diferentes de reperfusão no modelo MCAo proximal. No meio, o curso típico da atividade funcional e do fluxo sanguíneo cerebral após MCAo são retratados. (MCAo: cerebral média de oclusão da artéria, LDF: Laser medição de fluxo Doppler)

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M., Prass, K., Meisel, A., Dirnagl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+nervous+system+injury-induced+immune+deficiency+syndrome.">Central nervous system injury-induced immune deficiency syndrome.</a> Nat Rev Neurosci. 6, 775-786 (2005).</li><li>Engel, O., Meisel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+Infection+Before+and+After+Stroke:+Investigating+New+Targets.">Models of Infection Before and After Stroke: Investigating New Targets.</a> Infect Disord Drug Targets. 9, (2010).</li><li>Dirnagl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bench+to+bedside:+the+quest+for+quality+in+experimental+stroke+research.">Bench to bedside: the quest for quality in experimental stroke research.</a> J Cereb Blood Flow Metab. 26, 1465-1478 (2006).</li><li>Macleod, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Good+laboratory+practice:+preventing+introduction+of+bias+at+the+bench.">Good laboratory practice: preventing introduction of bias at the bench.</a> Stroke. 40, 50-52 (2009).</li><li>Group, S. T. A. I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+standards+regarding+preclinical+neuroprotective+and+restorative+drug+development.">Recommendations for standards regarding preclinical neuroprotective and restorative drug development.</a> Stroke. 30, 2752-2758 (1999).</li></ol>

Experimentos em animais foram realizados de acordo com as diretrizes e regulamentos estabelecidos pela Landesamt fuer Gesundheit und Soziales, Berlim, Alemanha.

O modelo de transitória, MCA proximal da oclusão 18,19 apresentadas aqui imita um dos tipos mais comuns de AVC isquêmico em pacientes. Baseado em 20 vezes reperfusão variável, o modelo oferece diferentes graus de danos que variam de ataque isquêmico transitório (TIA) para grandes infartos, incluindo grandes partes do hemisfério isquêmica. Isso permite que o pesquisador a estudar mecanismos fisiopatológicos diferentes após o AVC. 20,21 Cirurgia pode ser realizada em um curto período de tempo e produz lesões altamente reprodutível. No entanto, isto requer um rigoroso controle de fatores de confusão. 22 pequenas diferenças na técnica de operação podem ser responsáveis ​​por efeitos diferentes sobre o infarto. 23,24 Além disso, devido a variações na anatomia vascular cerebral, diferentes linhagens de camundongos mostram um resultado diferente. 25,26 Corpo a temperatura afeta a danos neurológicos, com hipotermia levando a lesões menores 27 e hipertermia para mais graves déficits. 28 Por conseguinte controle de temperatura, e manutenção é altamente relevante neste modelo. 29 Além disso, a pressão arterial e gases sanguíneos são importantes fatores de confusão de resultados ea necessidade de ser monitorado. 30,31 O uso de métodos rápidos e mínima invasiva (não-invasivo de medição da pressão arterial, adequado e fácil locais de recolha acessível sangue) é recomendada. A escolha do anestésico também é altamente importante, pois alguns podem ter efeitos neuroprotetores e / ou ser vasodilatadores, como por exemplo isoflurano. 32 Por conseguinte, a exposição a anestesia deve ser tão curto quanto possível e padronizados. Excluímos animais que se submeteram à cirurgia por mais de 15 min. Raspar o sítio cirúrgico produz microabrasions e inflamação e libera fragmentos de cabelo. Isto pode aumentar ainda mais a inflamação e promover a infecção local, que possa ter impacto na fisiopatologia acidente vascular cerebral. Condições de habitação, especialmente o uso de enriquecimento, pode afetar o resultado acidente vascular cerebral e deve ser padronizada e descrita em relatórios de pesquisa. 6,33,34 O uso de enriquecimento ambiental e seus efeitos sobre a reprodutibilidade é uma questão de discussão. Confundidor 35 Outra importante é a risco para a infecção induzida por acidente vascular cerebral, especialmente depois de tempos mais longos de isquemia 36, o que leva a morbidade adicional e aumento da mortalidade. 37,38 Como as infecções tornam-se sintomáticas em torno de 3 a 5 dias, isto tem consequências importantes para o longo prazo de acompanhamento em tais modelos. Para produzir resultados relevantes para o desenvolvimento de estratégias de tratamento da novela para a normalização acidente vascular cerebral e controle de qualidade é muito importante na investigação translacional pré-golpe 39 &quot;Boas Práticas de Laboratório&quot; 40,41 mandatos.: <ol><li> uma descrição adequada e pormenorizada dos animais utilizados; </li><li> cálculo amostral e informação sobre o tamanho do efeito esperado; </li><li> critérios de inclusão e exclusão, criada antes do estudo; </li><li> randomização da alocação em grupos; </li><li> ocultação da alocação no que diz respeito aos investigadores; </li><li> relatórios animais excluídos da análise; </li><li> avaliação cega dos resultados; </li><li> relatórios potenciais conflitos de interesse e de financiamento do estudo. </li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Surgical scissors (skin cut)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>14028-10</td>
<td>&nbsp;</td>
<tr>
<td>2 Dumont forceps (Medical #5 straight tip)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11253-20</td>
<td>&nbsp;</td>
<tr>
<td>Spring scissors (according to Vannas)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15000-00</td>
<td>&nbsp;</td>
<tr>
<td>Applying forceps for micro clamps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>00072-14</td>
<td>&nbsp;</td>
<tr>
<td>Micro vascular clamp (e.g. S&amp;G B1-V)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>00396-01</td>
<td>Also Serrefine possible</td>
</tr>
<tr>
<td>2 Hemostats according to Hartmann</td>
<td>&nbsp;</td>
<td>FST</td>
<td>13002-10</td>
<td>&nbsp;</td>
<tr>
<td>Needle holder (according Olsen-Hegar or other)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>12002-14</td>
<td>&nbsp;</td>
<tr>
<td>Standard anatomical forceps (for wound closure)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11000-12</td>
<td>&nbsp;</td>
<tr>
<td>5/0 sutures </td>
<td>&nbsp;</td>
<td>Suprama</td>
<td>&nbsp;</td>
<td>for vascular ligatures</td>
</tr>
<tr>
<td>6/0 sutures </td>
<td>&nbsp;</td>
<td>Suprama</td>
<td>&nbsp;</td>
<td>for skin suture, 5/0 also possible</td>
</tr>
<tr>
<td>Lidocaine (e.g. Xylocain Gel 2%)</td>
<td>&nbsp;</td>
<td>Astra Zeneca</td>
<td>&nbsp;</td>
<td>or other local pain relief</td>
</tr>
<tr>
<td>Dissecting microscope (stereo microscope), magnification 6x to max. 40x</td>
<td>&nbsp;</td>
<td>Leica Zeiss</td>
<td>MZ6 Stemi2000C</td>
<td>&nbsp;</td>
<tr>
<td>Cold light source</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>KL1500</td>
<td>&nbsp;</td>
<tr>
<td>Temperature feedback controlled heating pad system</td>
<td>&nbsp;</td>
<td>FST</td>
<td>21052-00</td>
<td>With mouse pad and small probe</td>
</tr>
<tr>
<td>Anaethesia system for isoflurane</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane</td>
<td>&nbsp;</td>
<td>Abott</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Veterinary Recovery Chamber</td>
<td>&nbsp;</td>
<td>Peco Services</td>
<td>V1200</td>
<td>Heated cage</td>
</tr>
<tr>
<td>8.0 nylon filament</td>
<td>&nbsp;</td>
<td>Suprama</td>
<td>TEL181005</td>
<td>for coating</td>
</tr>
<tr>
<td>Scalpel</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For cutting filament in pieces</td>
</tr>
<tr>
<td>Ruler</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>To cut correct length of filament</td>
</tr>
<tr>
<td>Xantopren M Mucosa</td>
<td>&nbsp;</td>
<td>Heraeus Kulzer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Activator Universal Plus</td>
<td>&nbsp;</td>
<td>Heraeus Kulzer</td>
<td>&nbsp;</td>
<td>Optosil - Xantopren</td>
</tr>
<tr>
<td>2 Dumont forceps (Medical #5 straight tip)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11253-20</td>
<td>&nbsp;</td>
<tr>
<td>A soft underlay for storing and grasping the uncoated filaments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>e.g. swabs, foam, &#x2026;</td>
</tr>
<tr>
<td>Receptacle for new build filaments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>e.g. petri dish, flexible foam,&#x2026;</td>
</tr>		</tbody>
		</table>
		</div>

modeling stroke in mice - middle cerebral artery occlusion with the filament model

AVC está entre as causas mais freqüentes de morte e incapacidade de adultos, especialmente em países altamente desenvolvidos. No entanto, as opções de tratamento, até à data são muito limitados. Para atender a necessidade de novas abordagens terapêuticas, a pesquisa derrame experimental freqüentemente emprega modelos de roedores de isquemia cerebral focal. Maioria dos pesquisadores usa oclusão permanente ou transitória da artéria cerebral média (MCA) em camundongos ou ratos. Oclusão proximal da artéria cerebral média (MCA), através da técnica do fio intraluminal (chamado de filamento ou modelo de sutura) é provavelmente o modelo mais utilizado em pesquisas curso experimental. O modelo MCAO intraluminal oferece a vantagem de induzir isquemia transitória ou permanente reprodutível do território da ACM de forma relativamente não-invasivo. Abordagens intraluminal interromper o fluxo de sangue de todo o território desta artéria. Filamento de oclusão proximal, portanto, prisões fluxo para as artérias lenticulo-estriado, que fornecem os gânglios basais. Filamento de oclusão dos resultados MCA em lesões reprodutível no córtex e estriado e pode ser permanente ou transitória. Em contraste, os modelos de indução distal (à ramificação das artérias lenticulo-estriado) MCA oclusão tipicamente poupar o striatum e envolvem principalmente o neocórtex. Além disso, estes modelos exigem craniotomia. No modelo demonstrado neste artigo, um filamento de silício revestido é introduzido na artéria carótida comum e avançado ao longo da artéria carótida interna no Círculo de Willis, onde bloqueia a origem da artéria cerebral média. Em pacientes, oclusão da artéria cerebral média estão entre as causas mais comuns de AVC isquêmico. Desde diferentes intervalos isquêmicos podem ser escolhidos livremente neste modelo, dependendo do ponto de tempo de reperfusão, as lesões de isquemia com diferentes graus de severidade podem ser produzidos. Reperfusão pela remoção do filamento oclusão pelo menos parcialmente modelos a restauração do fluxo sangüíneo após espontâneo ou terapêutico de lise (tPA) de um coágulo tromboembólicos em seres humanos. Neste vídeo iremos apresentar a técnica básica, bem como as armadilhas e fatores de confusão que pode limitar o valor preditivo deste modelo.

Watch this Scientific Journal Video about Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model at JoVE.com

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

Filamentosas de oclusão da artéria cerebral média é um modelo comum para o estudo de AVC isquêmico em ratos.

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 ...

modeling-stroke-mice-middle-cerebral-artery-occlusion-with-filament

Research

JoVE Journal

Medicine

91.4K visualizações.  Center for Stroke Research Berlin, Charité Universitätsmedizin. Filamentosas de oclusão da artéria cerebral média é um modelo comum para o estudo de AVC isquêmico em ratos.

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

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 ...

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 ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

A aplicação de ligadura da artéria Cerebral Média Permanente no mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

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 ...

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 ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Quantificação de Proteção Neurovascular sequência repetitiva Hypoxic Pré-condicionamento e Transient Oriente oclusão da artéria cerebral em ratos

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

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, ...