Este trabalho foi apoiado por Grants-in-Aid for Scientific Research (KAKENHI) (18K09424 para Toshihide Kurihara e 20K18393 para Yukihiro Miwa) do Ministério da Educação, Cultura, Esportes, Ciência e Tecnologia (MEXT).

Todos os métodos aqui descritos foram aprovados pelo Comitê Institucional de Cuidados e Uso de Animais (IACUC) da Escola de Medicina da Universidade keio.
1. Preparação de instrumentos cirúrgicos e animais
<ol><li>Autoclave instrumentos cirúrgicos e mantê-los em 70% de álcool etílico. Antes de cada novo procedimento cirúrgico, limpe os instrumentos cirúrgicos cuidadosamente utilizando 70% de álcool etílico.</li>
	<li>Prepare camundongos BALB/cAJc1 machos (6 semanas de idade, 26-28 kg) em uma sala específica-livre de patógenos (SPF) para manter condições estéreis antes, durante e depois da cirurgia.</li>
</ol>2. Oclusão da artéria carótida comum bilateral transitória (tBCCAO)
<ol><li>Coloque um rato sob anestesia por injeção intraperitoneal com uma combinação de midazolam (40 μg/100 μL), medetomidina (7,5 μg/100 μL) e tartarato butorphanol (50 μg/100 μL), denominado "MMB", como descrito anteriormente8,9. Segure as peles traseiras do mouse para manter o rato longe de bater os olhos até que o rato esteja completamente anestesiado.<ol>
<li>Julgue a profundidade da anestesia beliscando o dedo do mouse até que ele não tenha resposta, do qual o método é comumente usado para verificar anestesia completa10. NOTA: Geralmente, menos de 5 min são necessários para que os ratos durmam. Receitas adequadas para anestesia geral podem ser diferentes pelas instituições.</li>
	</ol>
</li>
	<li>Aplique uma gota de 0,1% purificada solução de olírio de hialuronato de sódio aos olhos para evitar o ressecamento nos olhos sob anestesia.</li>
	<li>Coloque o mouse de costas e conserte as patas do mouse usando fitas adesivas.</li>
	<li>Desinfete a área do pescoço do rato usando 70% de álcool etílico antes da cirurgia. NOTA: O recorte adicional da pele não foi realizado, pois isso pode causar inflamação posterior da pele11,12.</li>
	<li>Realizar incisão sagital do pescoço com uma lâmina(Figura 1). NOTA: A incisão precisa ser feita na linha média entre o pescoço, esterno e traqueia.</li>
	<li>Separe as duas glândulas salivares cuidadosamente usando dois fórceps e mobilize-as para visualizar os CCAs subjacentes.</li>
	<li>Isole a CCA direita cuidadosamente dos respectivos nervos vagos e veias que acompanham sem prejudicar suas estruturas, e coloque duas suturas de seda 6-0 sob a CCA. Amarre os dois laços firmemente para bloquear o fluxo sanguíneo(Figura 1). NOTA: Durante o procedimento, pequenas veias podem ser danificadas. Se o sangramento for visto, é necessário limpar os CCAs claramente.</li>
	<li>Encontre a CCA esquerda cuidadosamente a partir dos respectivos nervos vagos e veias que acompanham sem prejudicar suas estruturas, e oclua a CCA esquerda por 2 segundos por um grampo(Figura 1). NOTA: É necessário colocar uma agulha de sutura de seda 6-0 sob a CCA esquerda para marcar um local para fixação.</li>
	<li>Após a reabertura da CCA esquerda, sutura feridas do pescoço por uma sutura de seda 6-0 e aplicam um pouco de antibiótico (50 μL) no pescoço para inibir infecção bacteriana. NOTA: Remova suavemente um grampo para evitar danificar a parede arterial ao reabrir a CCA esquerda.</li>
	<li>Injete 0,75 mg/kg de cloridrato de atipamezole intraperitoneally no camundongo para ajudar o rato a se recuperar de anestesia profunda rapidamente. Devolva o mouse para uma gaiola de rato com almofadas pré-aquecidas. NOTA: Não deixe o rato sair sozinho até que o rato recupere a consciência suficiente para manter a recumbência severa.</li>
	<li>Injete 0,4 mg/kg de tartarato butorphanol no mouse para o manejo da dor quando o rato acordar. NOTA: O protocolo pode ser pausado aqui. Como primeira dica para o sucesso do tBCCAO, pode-se observar a inclinação das pálpebras do mouse(Figura 2).</li>
	<li>Para eutanásia, injete 3x de mistura de MMB nos camundongos e sacrifique-os para experimentos.</li>
</ol>3. Observações gerais (taxas de sobrevivência e inclinação das pálpebras)
<ol><li>Após a cirurgia, verifique as taxas de sobrevivência de todas as causas de morte no dia 0 (após a cirurgia), 1, 3 e 7.</li>
	<li>Avalie a inclinação das pálpebras por uma escala de classificação de 4 pontos: 1 = sem inclinação, 2 = inclinação leve (~50%), 3 = inclinação severa (acima de 50%) e 4 = inclinação severa com descarga ocular.</li>
</ol>4. Perfusão sanguínea retinana
<ol><li>Injete 200 μL de FITC-dextran (25 mg/mL) no ventrículo esquerdo do camundongo, que é comumente usado para a observação da perfusão sanguínea nos vasos de retina do rato13,14.</li>
	<li>2 minutos após a circulação, enuclear os olhos e fixar em 4% paraformaldeído por 1 hora. As retinas foram cuidadosamente obtidas e montadas planas, como descrito anteriormente15, e examinadas através de um microscópio de fluorescência.</li>
	<li>Tire fotos das montagens inteiras da retina em 4x de ampliação e mescle em um único usando um analisador de mesclagem, descrito anteriormente16.</li>
	<li>Meça as áreas perfundidas através de uma ferramenta de análise de embarcações no software NIH Fiji/ImageJ.</li>
</ol>5. Mancha ocidental
<ol><li>3 e 6 horas após tBCCAO, obtenha os olhos dos ratos e transfira imediatamente para uma placa de Petri contendo PBS frio para isolar as retinas.</li>
	<li>Após o isolamento das retinas, realize manchas ocidentais, como descrito anteriormente9.</li>
	<li>Incubar com anticorpos para fator indutível de hipóxia-1α (HIF-1α; um marcador geral de hipóxia) e para β-Actin (um controle de carregamento interno) durante a noite seguido da incubação de anticorpos secundários conjugados pelo HRP. Visualize os sinais via chemiluminescência.</li>
</ol>6. PCR quantitativo (qPCR)
<ol><li>6, 12 e 24 horas após tBCCAO, processe as retinas obtidas para qPCR, como descrito anteriormente17.</li>
	<li>Execute qPCR via sistema PCR em tempo real. Os primers utilizados estão listados na Tabela 1. Calcule as alterações de dobra entre os níveis de diferentes transcrições pelo método ΔΔCT.</li>
</ol>7. Imunohistoquímica (IHC)
<ol><li>3 dias após tBCCAO, obtenha os olhos de ratos e incorpore em parafina.</li>
	<li>Corte os olhos embutidos na parafina por um microtome para obter as seções oculares.</li>
	<li>Desparafinar e manchar as seções oculares de 5 μm de espessura como descrito anteriormente13.</li>
	<li>Incubar com um anticorpo para proteína ácida fibrilar gliana (GFAP; um marcador confiável para astrócitos e células Müller na retina) durante a noite seguido pela incubação do anticorpo secundário Alexa Fluor 555-conjugado.</li>
	<li>Use DAPI (4′,6-diamidino-2-fenildole) para coloração do núcleo na retina. Visualize sinais através de um microscópio de fluorescência.</li>
	<li>Avalie a pontuação da morfologia por uma escala de classificação de 4 pontos, como descrito anteriormente13,18: 0 = sem sinal, 1 = poucos pés finais gliais positivos na camada de célula de gânglio (GCL), 2 = poucos processos rotulados que atingem de GCL à camada nuclear externa (ONL) e 3 = a maioria dos processos rotulados que atingem de GCL para ONL.</li>
</ol>8. Eletroretinografia (ERG)
<ol><li>3 e 7 dias após a tBCCAO, realize o ERG utilizando uma cúpula ganzfeld, sistema de aquisição e estimuladores led, como descrito anteriormente9.</li>
	<li>Após adaptação escura da noite para o dia, anestesia os ratos com uma combinação de MMB sob luz vermelha fraca.</li>
	<li>Use uma solução mista de 0,5% de trópico e 0,5% de fenilefrina para dilatar as pupilas.</li>
	<li>Coloque os eletrodos ativos na lente de contato e coloque o eletrodo de referência na boca.</li>
	<li>Obtenha respostas de ERG de ambos os olhos de cada animal.</li>
	<li>Registos scotopic registro sob adaptação escura com vários estímulos.</li>
	<li>Meça as amplitudes de uma onda da linha de base até o ponto mais baixo de uma onda.</li>
	<li>Meça as amplitudes da onda B do ponto mais baixo de uma onda até o pico da onda B.</li>
	<li>Mantenha todos os ratos aquecidos durante o procedimento usando almofadas de calor.</li>
</ol>9. Tomografia de coerência óptica (OCT)
<ol><li>2 semanas após tBCCAO, realize o OCT usando o sistema SD-OCT, como relatado anteriormente8,9.</li>
	<li>Para a medição, sujeitos camundongos à micrária por uma solução mista de 0,5% de tropamida e 0,5% fenilefrina, e à anestesia geral por uma mistura de MMB.</li>
	<li>Obtenha imagens de varredura B de fatias equatoriais de varreduras en-face.</li>
	<li>Examine as retinas a 0,2, 0,4 e 0,6 mm da cabeça do nervo óptico.</li>
	<li>Meça a espessura da retina da camada de fibra nervosa da retina (NFL) até a membrana limitante externa (ELM), e considere a média dos valores medidos como espessura da retina de um camundongo individual.</li>
	<li>Plote os resultados como diagramas de aranha.</li>
</ol>

Os autores não têm nada a revelar.

No estudo, mostramos que o tBCCAO, utilizando suturas simples e um grampo, poderia induzir isquemia de retina e disfunção retinista. Além disso, demonstramos que nosso protocolo atual para o desenvolvimento de um modelo de camundongo de isquemia de retina é mais fácil e rápido em comparação com outros protocolos anteriores para o desenvolvimento de modelos de lesão isquêmica de retina2,3,7.
...

A retina é um tecido neurossensorial para função visual. Uma vez que uma quantidade substancial de oxigênio é necessária para a função visual, a retina é conhecida como um dos tecidos mais exigentes de oxigênio no corpo1. A retina é suscetível a doenças vasculares, pois o oxigênio é entregue através dos vasos sanguíneos. Vários tipos de doenças vasculares, como a retinopatia diabética e a oclusão dos vasos sanguíneos da retina (veias ou artérias), podem induzir isquemia da retina. Para investigar mecanismos patológicos de isquemia da retina, são considerados necessários modelos experimentais reprodutíveis e clinicamente relevantes de isquemia de retina. A oclusão da artéria cerebral média (MCAO) por inserção de um filamento intraluminal é o método mais utilizado para o desenvolvimento de modelos de roedores in vivo de isquemia cerebral experimental2,3. Devido à proximidade da artéria oftalmológica (OpA) com a MCA, os modelos de MCAO também são utilizados simultaneamente para entender a fisiopatologia da isquemia da retina4,5,6. Para induzir isquemia cerebral junto com isquemia de retina, filamentos longos são tipicamente inseridos através da incisão da artéria carótida comum (CCA) ou da artéria carótida externa (ECA). Esses métodos são difíceis de realizar, requerem muito tempo para completar a cirurgia (mais de 60 minutos para um rato), e levam a altas variabilidades nos desfechos após a cirurgia7. É importante desenvolver um modelo melhor para melhorar essas preocupações.
Neste estudo, bastamos usar a oclusão bilateral de CCA transitória curta (tBCCAO) com agulhas e um grampo para induzir isquemia de retina em camundongos e analisamos resultados típicos de lesões isquêmicas na retina. Neste vídeo, vamos dar uma demonstração do procedimento tBCCAO.

<table>
	<tbody>
		<tr>
			<td>Atipamezole hydrochloride</td>
			<td>Zenoaq</td>
			<td>Antisedan</td>
			<td>For anti-anesthesia</td>
		</tr>
		<tr>
			<td>Applied Biosystems 7500 Fast</td>
			<td>Applied Biosystems</td>
			<td>-</td>
			<td>For qPCR</td>
		</tr>
		<tr>
			<td>Butorphanol tartrate</td>
			<td>Meiji Seika Pharma</td>
			<td>Vetorphale</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>BZ-II Analyzer</td>
			<td>KEYENCE</td>
			<td>-</td>
			<td>For an image merge</td>
		</tr>
		<tr>
			<td>BALB/cAJc1</td>
			<td>CLEA</td>
			<td>-</td>
			<td>Mouse strain</td>
		</tr>
		<tr>
			<td>&#946;-Actin (8H10D10) Mouse mAb</td>
			<td>CST</td>
			<td>3700</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>Clamp Forcep</td>
			<td>World Precision Instruments</td>
			<td>WPI 500451</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Dumont forceps #5</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>DAPI solution</td>
			<td>Dojindo</td>
			<td>340-07971</td>
			<td>For IHC</td>
		</tr>
		<tr>
			<td>Envisu SD-OCT system</td>
			<td>Leica</td>
			<td>R4310</td>
			<td>For OCT</td>
		</tr>
		<tr>
			<td>FITC-dextran</td>
			<td>Merk</td>
			<td>FD2000S</td>
			<td>For retinal blood perfusion</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>KEYENCE</td>
			<td>BZ-9000</td>
			<td>For fluorescence detection</td>
		</tr>
		<tr>
			<td>Gatifloxacin hydrate</td>
			<td>Senju Pharmaceutical</td>
			<td>Gachifuro</td>
			<td>For anti-bacterial infection</td>
		</tr>
		<tr>
			<td>GFAP Monoclonal Antibody (2.2B10)</td>
			<td>Thermo</td>
			<td>13-0300</td>
			<td>For IHC</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Marukan</td>
			<td>RH-200</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>HIF-1&#945; (D1S7W) XP Rabbit mAb</td>
			<td>CST</td>
			<td>36169</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>ImageQuant LAS 4000 mini</td>
			<td>GE Healthcare</td>
			<td>-</td>
			<td>For chemiluminescence</td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td>Sandoz K.K</td>
			<td>SANDOZ</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Microtome Tissue-Tek TEC 6</td>
			<td>Sakura</td>
			<td>-</td>
			<td>For sectioning</td>
		</tr>
		<tr>
			<td>Medetomidine</td>
			<td>Orion Corporation</td>
			<td>Domitor</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Handaya</td>
			<td>HS-2307</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>PuREC</td>
			<td>MAYO Corporation</td>
			<td>-</td>
			<td>For ERG</td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>Fine Science Tools</td>
			<td>91460-11</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Sodium hyaluronate</td>
			<td>Santen Pharmaceutical</td>
			<td>Hyalein</td>
			<td>For eye lubrication</td>
		</tr>
		<tr>
			<td>Tropicamide/Penylephrine hydrochloride</td>
			<td>Santen Pharmaceutical</td>
			<td>Mydrin-P</td>
			<td>For mydriasis</td>
		</tr>
		<tr>
			<td>6-0 silk suture</td>
			<td>Natsume</td>
			<td>E12-60N2</td>
			<td>For surgery</td>
		</tr>
	</tbody>
</table>

a murine model of ischemic retinal injury induced by transient bilateral common carotid artery occlusion

Diversas doenças vasculares, como retinopatia diabética, oclusão de veias da retina ou artérias e síndrome isquêmica ocular podem levar à isquemia da retina. Para investigar mecanismos patológicos da isquemia da retina, são necessários desenvolver modelos experimentais relevantes. Anatomicamente, um dos principais vasos de abastecimento sanguíneo da retina é a artéria oftalmômica (OpA) e a OpA é originária da artéria carótida interna da artéria carótida comum (CCA). Assim, a interrupção da CCA poderia efetivamente causar isquemia retinista. Aqui, estabelecemos um modelo de camundongo de isquemia de retina por oclusão da artéria carótida comum transitória (tBCCAO) para amarrar a CCA direita com suturas de seda 6-0 e ocluir a CCA esquerda transitoriamente por 2 segundos através de um grampo, e mostrou que o tBCCAO poderia induzir isquemia aguda de retina levando à disfunção da retina. O método atual reduz a dependência de instrumentos cirúrgicos usando apenas agulhas cirúrgicas e um grampo, encurta o tempo de oclusão para minimizar a morte inesperada de animais, o que é frequentemente visto em modelos de camundongos de oclusão da artéria cerebral média, e mantém a reprodutibilidade de achados isquêmicos comuns da retina. O modelo pode ser utilizado para investigar a fisiopatologia de retinopatias isquêmicas em camundongos e ainda pode ser usado para triagem in vivo de medicamentos.

Após a circulação sistêmica do FITC-dextran por 2 minutos, foram examinadas vasculaturas de retina das retinas esquerda e direita nos camundongos operados por sham e camundongos operados por tBCCAO(Figura Suplementar 1). FITC-dextran era totalmente visível nas duas retinas nos camundongos operados por farsa e na retina esquerda nos camundongos operados por tBCCAO, enquanto era parcialmente detectável na retina direita nos camundongos operados por tBCCAO.
Após tBCCAO, fo...

Watch this Scientific Journal Video about A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion at JoVE.com

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Aqui, descrevemos um modelo de camundongo de isquemia de retina por oclusão da artéria carótida comum bilateral transitória usando suturas simples e um grampo. Este modelo pode ser útil para entender os mecanismos patológicos da isquemia da retina causada por anormalidades cardiovasculares.

Um modelo murino de lesão isquêmica da retina induzida pela oclusão da artéria da carótida comum bilateral transitória

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

a-murine-model-ischemic-retinal-injury-induced-transient-bilateral

Research

JoVE Journal

Medicine

7.4K visualizações.  Keio University School of Medicine. Aqui, descrevemos um modelo de camundongo de isquemia de retina por oclusão da artéria carótida comum bilateral transitória usando suturas simples e um grampo. Este modelo pode ser útil para entender os mecanismos patológicos da isquemia da retina causada por anormalidades cardiovasculares.

Vídeo: A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

Um modelo de Disturbed Aterosclerose Flow-induzido em rato artéria carótida pela ligadura parcial e um método simples de isolamento de RNA a partir do endotélio Carótidas

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

Medicina

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 ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

Adulto Rato venoso Hipertensão Modelo: artéria carótida comum para External Jugular Vein anastomose.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

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, ...

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 ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

Na Vivo Transferência de genes para o endotélio da artéria carótida comum de coelho

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Um modelo de coelho da expressão do Transgene durável na veia Jugular para enxertos de interposição da artéria carótida comum

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

Trombose da artéria carótida canino induzida por cloreto férrico: Um modelo Animal grande de lesão Vascular

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

Oclusão de artéria Cerebral média, permitindo a reperfusão através de reparação de artéria carótida comum em ratos

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...