Name: a thrombotic stroke model based on transient cerebral hypoxia-ischemia
Uploaded: 2015-08-18T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia at JoVE.com

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><tbody><tr><td>adult male mice</td><td>Charles River</td><td>C57BL/6&amp;nbsp;</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% O&lt;sub&gt;2&lt;/sub&gt; balanced by 92.5% N&lt;sub&gt;2&lt;/sub&gt; tank</td><td>Airgas</td><td>UN1956</td><td>hypoxia system</td></tr><tr><td>Temperature Controller with heating lamp&amp;nbsp;</td><td>Cole Parmer&amp;nbsp;</td><td>EW-89000-10</td><td>temperature controllers</td></tr><tr><td>Rectal probe</td><td>Cole Parmer&amp;nbsp;</td><td>NCI-00141PG</td><td>temperature controllers</td></tr><tr><td>Dissecting microscope&amp;nbsp;</td><td>Olympus&amp;nbsp;</td><td>SZ40</td><td>surgical setup</td></tr><tr><td>Heat pump with warming pad</td><td>Gaymar&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>eye dryness prevention&amp;nbsp;</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>&lt;i&gt;in vivo&lt;/i&gt; TTC</td></tr><tr><td>2,3,5-triphenyltetrazolium chloride (TTC)&amp;nbsp;</td><td>Sigma</td><td>T8877</td><td>&lt;i&gt;in vivo&lt;/i&gt; TTC</td></tr><tr><td>Vibratome</td><td>Stoelting</td><td>51425</td><td>brain section for &lt;i&gt;in vivo&lt;/i&gt; TTC&amp;nbsp;</td></tr><tr><td>Digital microscope</td><td>Dino-Lite</td><td>AM2111</td><td>whole-brain 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><tr><td>Meloxicam SR</td><td>ZooPharm</td><td></td><td>NSAID analgesia</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 ...

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Research

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

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

O Modelo de encefalopatia hipóxico isquêmica Perinatal da Isquemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

Biologia

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Isquemia Photothrombotic: Um Modelo de lesão cortical fotoquímica Minimamente Invasiva e reprodutível para mouse Estudos curso

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

Medicina

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 ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

Transiente Artéria Cerebral Média Oclusão Modelo de AVC neonatal em ratos P10

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

Modelo de AVC fototrombótico enriquecido com fibrina e sensível ao tPA

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

Neurociência

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Modelagem de AVC em Camundongos: Lesões Corticais Focais por Fototrombose

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Modelagem de AVC em Camundongos: Oclusão da Artéria Cerebral Média Transitória através da Artéria Carótida Externa

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

Um modelo de isquemia cerebral transitória por oclusão da artéria cerebral média

Transient Middle Cerebral Artery Occlusion Model of Stroke

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models.
In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

Autor em destaque: Avaliando o dano isquêmico do AVC por meio do modelo de oclusão da artéria cerebral média 

This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.

Modelo de Oclusão Transitória da Artéria Cerebral Média do Acidente Vascular Encefálico

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies ...

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

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