Este projeto é parcialmente financiado pelo NIH T32 GM008440 para Espen Walker, R01 NS27713 para William L.Young, P01 NS44155 para William L.Young e Hua Su, R21 NS070153 para Hua SU e pela American Heart Association AHA 10GRNT3130004 para Hua Su. Dr Ana Rodríguez-Hernández é apoiado por uma bolsa da &quot;Obra La social Caixa&quot;

1. Preparar o mouse <ol><li> Induzir a anestesia geral no rato com gás isoflurano. Administrar 0,15 ml de brupenorphine intraperitoneal para controle da dor. Antes de prosseguir, verifique se o nível de anestesia é satisfatória através da punção patas do rato. </li><li> Coloque o mouse em decúbito dorsal, com os quatro membros fixos por fita adesiva. Remover o cabelo do pescoço e parte superior do tórax, com uma tesoura. Por meio de injecção subcutânea, administrar 0,2-0,4 ml de solução salina a 0,9% para manter o rato hidratado durante o procedimento cirúrgico. </li><li> Prepare o campo operatório seguindo um método rigoroso estéril. A área da incisão na pele deve ser limpa com álcool 90%. </li></ol> 2. dissecção da artéria carótida comum ea veia jugular externa <ol><li> Faça uma incisão na linha média cervical horizontal em toda a área do pescoço inferior do mouse. Depois de aprofundar a ferida, elevar as glândulas salivares e dos tecidos moles do colo do útero, usando pelorAction sutura (Figura 1). Expor a veia jugular externa direita (EJV) lateralmente ao músculo esternocleidomastóideo (SCM). Esta etapa deve ser realizada sob o microscópio, pois tração excessiva sobre a veia pode danificá-lo e induzir a sua trombose. </li><li> Dissecar cuidadosamente o EJV direita ao longo do seu curso a partir da clavícula até a base do crânio. Normalmente pinça bipolar elétricos, coagular e dividir todas as ramificações para preparar um comprimento adequado para a colocação de clipe temporária depois e anastomose. </li><li> Lateral para a traquéia e medial para o SCM, explorar a artéria carótida comum (CCA). Ela deve ser cuidadosamente exposta a partir da clavícula apenas para além da sua bifurcação em artérias carótidas interna e externa. Durante essa etapa, deve ser dada atenção novamente para evitar tração excessiva ao EJV que mais tarde poderão comprometer a desobstrução da anastomose. </li></ol> 3. Preparar a anastomose <ol><li> Ligadura da CCA com 10-0 Nylon apenas proximal à sua bifurcação. Em seguida, aplique um clipe temporária do proximal CCA tão perto da clavícula possível. </li><li> Uma vez que o fluxo foi interrompido, transecto artéria logo abaixo da ligadura bifurcação e irrigar com soro fisiológico para lavar todo o sangue restante no interior do lúmen. Evite coagulação bipolar nesta etapa, uma vez que a lesão térmica para a parede da artéria poderia colocar o futuro anastomose em perigo. </li><li> A fim de melhorar a visibilidade das arestas do EJV, a parede medial é marcado com uma caneta de marcação azul ao longo do curso do venotomia planeado. Uma vez que o EJV é marcado, utilizar um fio de sutura 10-0 para ligar a extremidade distal como caudal quanto possível e aplicar um grampo vascular temporária na extremidade proximal como craniano como possíveis. </li><li> Com uma multa de 30 G agulha e seringa 0,5 ml, fazer uma abertura inicial sobre a área demarcada da EJV e imediatamente irrigar o lúmen com soro fisiológico para evitar a formação de trombos. Em seguida, estender a venotomia com a microtesouraaté que o comprimento é de cerca de 2-3 vezes o diâmetro do CCA. Evite violento alongamento e prestar atenção para manter a borda afiada e arrumado. </li><li> Aproximar a extremidade da CCA para o EJV. Adicione uma incisão lateral de corte no final do dador CCA para ajustar o diâmetro para o comprimento para o tamanho venotomia (Figura 2). </li></ol> 4. End-to-side Anastomosis <ol><li> Utilize uma sutura de nylon 11-0 monofilamento para a anastomose CCA-a-EJV ponta-a-lado. Sutura-se a parede medial da anastomose numa direcção craneo-caudal é o passo inicial. Cada ponto deve ser colocado a partir de fora para dentro da parede venosa primeira (Figura 3) e a partir de dentro para fora da parede arterial (Figura 4) próxima. Isto irá manter o nó na superfície exterior dos vasos anastomóticos em todos os momentos (Figura 5). Ou interrompido ou suturas contínuas podem ser utilizados, mas todas as suturas contínuas devem ser apertados como o passo final. </li><li> Once a parede medial foi suturada, repita o procedimento a partir do caudal para craniana com a parede lateral. Agora toda a costura deve ser colocado a partir de fora para dentro da parede arterial e primeiro a partir de dentro para fora da parede da veia próxima. Irrigação Saline vai ajudar a manter o lúmen da anastomose visível em todos os momentos durante o procedimento. </li><li> Depois de terminar todas as etapas da anastomose, a remover o grampo temporária da veia da primeira e da artéria próxima. O sangue arterial fluirá no EJV com pouca ou nenhuma exsudação da anastomose (ver vídeos modelo VH e modelo VH 2). O sangramento mínimo deve parar sem usar compressão de algodão sobre a anastomose. Isso deve ser evitado, de modo a prevenir a trombose da veia delicada. </li><li> Uma vez que o fluxo pulsátil através da anastomose e é confirmado e sem sangramento aparente é observada, irrigar o campo cirúrgico com solução salina e fechar a incisão cervical com uma sutura de nylon 6-0. Finalmente, administrar mais 0,15 ml de intrapbrupenorphine eritoneal para controle da dor pós-operatória e 0,2-0,4 ml de solução salina 0,9% subcutânea para reabastecer qualquer perda de sangue durante a cirurgia. </li></ol>

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	<li>Bederson, J. B., Wiestler, O. D., Br&#252;stle, O., Roth, P., Frick, R., Ya&#351;argil, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+venous+hypertension+and+the+effects+of+venous+outflow+obstruction+in+a+rat+model+of+arteriovenous+fistula.">Intracranial venous hypertension and the effects of venous outflow obstruction in a rat model of arteriovenous fistula.</a> Neurosurgery. 29, 341-350 (1991).</li><li>Gao, P., Zhu, Y., Ling, F., Shen, F., Lee, B., Gabriel, R. A., Hao, Q., Yang, G. Y., Su, H., Young, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonischemic+cerebral+venous+hypertension+promotes+a+pro-angiogenic+stage+through+HIF-1+downstream+genes+and+leukocyte-derived+MMP-9.">Nonischemic cerebral venous hypertension promotes a pro-angiogenic stage through HIF-1 downstream genes and leukocyte-derived MMP-9.</a> J. Cereb. Blood Flow Metab. 29, 1482-1490 (2009).</li><li>Kim, H., Su, H., Weinsheimer, S., Pawlikowska, L., Brain Young, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=arteriovenous+malformation+pathogenesis:+a+response-to-injury+paradigm.">arteriovenous malformation pathogenesis: a response-to-injury paradigm.</a> Acta Neurochir. Suppl. 111, 83-92 (2011).</li><li>Lawton, M. T., Arnold, C. M., Kim, Y. J., Bogarin, E. A., Stewart, C. L., Wulfstat, A. A., Derugin, N., Deen, D., Young, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+arteriopathy+in+the+transgenic+arteriovenous+fistula+model.">Radiation arteriopathy in the transgenic arteriovenous fistula model.</a> Neurosurgery. 62, 1129-1138 (2008).</li><li>Lawton, M. T., Stewart, C. L., Wulfstat, A. A., Derugin, N., Hashimoto, T., Young, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transgenic+arteriovenous+fistula+in+the+rat:+an+experimental+model+of+gene+therapy+for+brain+arteriovenous+malformations.">The transgenic arteriovenous fistula in the rat: an experimental model of gene therapy for brain arteriovenous malformations.</a> Neurosurgery. 54, 1463-1471 (2004).</li><li>Schaller, B., Graf, R., Sanada, Y., Tolnay, M., Rosner, G., Wienhard, K., Heiss, W., D, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+changes+after+occlusion+of+the+posterior+superior+sagittal+sinus:+an+experimental+PET+study+in+cats.">Hemodynamic changes after occlusion of the posterior superior sagittal sinus: an experimental PET study in cats.</a> AJNR Am J Neuroradiol. 24, 1876-1880 (2003).</li><li>Zhu, Y., Lawton, M. T., Du, R., Shwe, Y., Chen, Y., Shen, F., Young, W. L., Yang, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+hypoxia-inducible+factor-1+and+vascular+endothelial+growth+factor+in+response+to+venous+hypertension.">Expression of hypoxia-inducible factor-1 and vascular endothelial growth factor in response to venous hypertension.</a> Neurosurgery. 59, 687-696 (2006).</li><li>Herman, J. M., Spetzler, R. F., Bederson, J. B., Kurbat, J. M., Zabramski, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genesis+of+a+dural+arteriovenous+malformation+in+a+rat+model.">Genesis of a dural arteriovenous malformation in a rat model.</a> J. Neurosurg. 83, 539-545 (1995).</li><li>Terada, T., Higashida, R. T., Halbach, V. V., Dowd, C. F., Tsuura, M., Komai, N., Wilson, C. B., Hieshima, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+acquired+arteriovenous+fistulas+in+rats+due+to+venous+hypertension.">Development of acquired arteriovenous fistulas in rats due to venous hypertension.</a> J. Neurosurg. 80, 884-889 (1994).</li><li>Yassari, R., Sayama, T., Jahromi, B. S., Aihara, Y., Stoodley, M., Macdonald, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiographic,+hemodynamic+and+histological+characterization+of+an+arteriovenous+fistula+in+rats.">Angiographic, hemodynamic and histological characterization of an arteriovenous fistula in rats.</a> Acta Neurochir (Wien). 146, 495-504 (2004).</li><li>Solheim, O., Skeidsvoll, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+global+amnesia+may+be+caused+by+cerebral+vein+thrombosis.">Transient global amnesia may be caused by cerebral vein thrombosis.</a> Med. Hypotheses. 65, 1142-1149 (2005).</li><li>Yang, B., Shergill, U., Fu, A. A., Knudsen, B., Misra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+arteriovenous+fistula+model.">The mouse arteriovenous fistula model.</a> J Vasc Interv Radiol. 20, 946-950 (2009).</li><li>Choi, E. J., Choi, E. J., Walker, E. J., Shen, F., Oh, S. P., Arthur, H. M., Young, W. L., Su, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+Homozygous+Endothelial+Deletion+of+Eng+with+VEGF+Stimulation+Is+Sufficient+to+Cause+Cerebrovascular+Dysplasia+in+the+Adult+Mouse.">Minimal Homozygous Endothelial Deletion of Eng with VEGF Stimulation Is Sufficient to Cause Cerebrovascular Dysplasia in the Adult Mouse.</a> Cerebrovascular diseases. 33, 540-547 (2012).</li><li>Hao, Q., Su, H., Marchuk, D. A., Rola, R., Wang, Y., Liu, W., Young, W. L., Yang, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+tissue+perfusion+promotes+capillary+dysplasia+in+the+ALK1-deficient+mouse+brain+following+VEGF+stimulation.">Increased tissue perfusion promotes capillary dysplasia in the ALK1-deficient mouse brain following VEGF stimulation.</a> Am. J. Physiol. Heart Circ. Physiol. 295, H2250-H2256 (2008).</li><li>Su, H., Hao, Q., Shen, F., Zhu, Y., Lee, C. Z., Young, W. L., Yang, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+cerebral+microvascular+dysplasia+model+in+rodents.">Development of a cerebral microvascular dysplasia model in rodents.</a> Acta Neurochir. Suppl. 105, 185-189 (2008).</li><li>Walker, E. J., Su, H., Shen, F., Degos, V., Jun, K., Young, W. 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Os procedimentos experimentais com animais de laboratório foram aprovados pelo Comitê de Cuidados e Uso de Animal Institucional da Universidade da Califórnia, em San Francisco (UCSF). Os autores não têm potenciais conflitos de interesse relacionados com os medicamentos e materiais utilizados neste procedimento.

Hipertensão venosa cerebral sustentada foi intimamente relacionada com as manifestações clínicas mais graves e mau prognóstico em pacientes com FAV dural e MAVs cerebrais 3. Estes efeitos da CVH têm sido amplamente estudado em modelos de ratos 1,2,8. Um modelo equivalente no rato permitiria o uso de animais geneticamente modificados que acabaria por permitir a análise das vias moleculares envolvidas na patogênese da hipertensão venosa e sua relação com a FAV dural e AVM cérebro. <p ...

Os modelos animais de hipertensão venosa cerebral provaram ser um instrumento fundamental para a compreensão da fisiopatologia das malformações arteriovenosas cerebrais e fístulas arteriovenosas 1-7. O mais utilizado é o modelo de rato criado através de uma fístula artificial entre a artéria carótida comum (ACC) e a veia jugular externa (EJV), o que provoca uma hipertensão venosa cerebral consistente (CVH) no rato 1,8-10. Modelos de ratos equivalentes, abrindo a possibilidade de utilizar diferentes estirpes de ratinhos transgénicos, que permitiria o estudo adicional sobre patofisiologia não só mas também terapias genéticas potenciais para estas doenças cerebrovasculares. Além disso, a construção do experimento, podem também ser ainda adaptado para o estudo de outras doenças cerebrovasculares relacionadas com a hipertensão venosa, tais como enxaqueca, amnésia global transitória, cegueira monocular transiente, etc. 11 No entanto, tentativas anteriores para construir estes modelos de ratos hav demonstrou as dificuldades com a permeabilidade da fístula devido à anatomia diminutivo 5,12. Aqui, descrevemos nosso protocolo passo-a-passo para a anastomose bem sucedida do murino CCA e EJV que se traduz em uma fístula patente a longo prazo e uma hipertensão venosa durável no mouse.

<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>10-0 Sterile Microsuture</td>
			<td>Arosurgical Ic.</td>
			<td>VT5A010Q10</td>
		</tr>
		<tr>
			<td>11-0 Sterile Microsuture</td>
			<td>Arosurgical Ic</td>
			<td>VT4A00N07</td>
		</tr>
		<tr>
			<td>DUROTIP Scissors</td>
			<td>Aesculap</td>
			<td>BC210R</td>
		</tr>
		<tr>
			<td>Micro-Adson Tissue Forceps</td>
			<td>Aesculap</td>
			<td>BD510R</td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>Aesculap</td>
			<td>OC496R</td>
		</tr>
		<tr>
			<td>Micro Forceps #5 Jewelers</td>
			<td>Aesculap</td>
			<td>BD331R</td>
		</tr>
		<tr>
			<td>Angled Jewelers Forceps</td>
			<td>Aesculap</td>
			<td>BD329R</td>
		</tr>
		<tr>
			<td>Micro Suture Forceps</td>
			<td>Aesculap</td>
			<td>BD338R</td>
		</tr>
		<tr>
			<td>DUROGRIP Needle Holder</td>
			<td>Aesculap</td>
			<td>BM009R</td>
		</tr>
	</tbody>
</table>

adult mouse venous hypertension model: common carotid artery to external jugular vein anastomosis.

O entendimento da fisiopatologia das malformações arteriovenosas cerebrais e fístulas arteriovenosas melhorou graças a modelos animais. Um modelo de rato criando uma fístula artificial entre a artéria carótida comum (ACC) e da veia jugular externa (EJV) tem sido amplamente descrita e comprovada tecnicamente viável. Esta construção provoca uma hipertensão venosa cerebral consistente (CVH), e, portanto, tem ajudado a estudar a contribuição de hipertensão venosa para a formação, os sintomas clínicos, e prognóstico de MAVs cerebrais e FAV dural. Modelos de ratos equivalentes foram apenas pouco descrita e têm mostrado problemas com estenose da fístula. Um modelo murino estabelecida permitirá o estudo da fisiopatologia, não só mas também terapias genéticas potenciais para estas doenças cerebrovasculares. Nós apresentamos um modelo de fístula arteriovenosa, que produz uma hipertensão venosa intracraniana durável no mouse. Microsurgical anastomose of o murino CCA e EJV pode ser difícil devido à anatomia diminuto e freqüentemente resultam em uma fístula não-patente. Neste protocolo passo-a-passo que enfrentar todos os desafios importantes encontrados durante este procedimento. Evitando a retracção excessiva da veia durante a exposição, utilizando suturas 11-0 10-0 em vez de, e fazendo uma anastomose cuidadosamente planeada extremo-a-lado são alguns dos passos críticos. Embora este método requer técnicas de microcirurgia avançada e uma curva de aprendizagem mais longo que o equivalente no rato, ele pode ser desenvolvido de forma consistente. Este romance modelo foi concebido para integrar técnicas de camundongos transgênicos com um sistema experimental previamente bem estabelecida, que tem se mostrado útil para estudar AVMs cerebrais e FAV dural. Ao abrir a possibilidade de utilizar ratinhos transgénicos, um espectro mais amplo de modelos válidos, podem ser alcançados e tratamentos genéticos podem também ser testados. A construção experimental também pode ser ainda adaptado para o estudo de otsuas doenças cerebrovasculares relacionados com hipertensão venosa, como enxaqueca, a amnésia global transitória, cegueira monocular transitória, etc.

Um resultado positivo do modelo é uma fístula arteriovenosa patente que induz hipertensão venosa no cérebro murino. Para validar o modelo, inicialmente medido a pressão venosa intracraniana no seio sagital dos ratos em 2, 3 e 4 semanas após a cirurgia. 6 ratinhos foram atribuídos diferentes para cada grupo de tempo. A pressão do seio foi de 8,8 ± 1,2 mm Hg no grupo medido de duas semanas após a cirurgia. Nos 6 ratos medidos 3 semanas após a cirurgia, a pressão do seio foi de 4,7 ± 1,4 mmHg. Finalmente, os r...

Watch this Scientific Journal Video about Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis. at JoVE.com

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

Descreve-se um método para a criação de um modelo de confiança de hipertensão venosa cerebral no rato adulto. Este modelo tem sido amplamente descrita e testada em ratos. Este novo homólogo nos ratinhos abre a possibilidade de utilização de animais geneticamente modificados e, assim, alarga a aplicação do modelo.

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 ...

adult-mouse-venous-hypertension-model-common-carotid-artery-to

Research

JoVE Journal

Medicine

14.0K visualizações.  University of California, San Francisco.  Descreve-se um método para a criação de um modelo de confiança de hipertensão venosa cerebral no rato adulto. Este modelo tem sido amplamente descrita e testada em ratos. Este novo homólogo nos ratinhos abre a possibilidade de utilização de animais geneticamente modificados e, assim, alarga a aplicação do modelo. 

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

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Treinar um técnica microcirúrgica sofisticado: Interposição de enxerto de veia jugular externa na artéria carótida comum em ratos

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

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 ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

Cateterismo da artéria carótida e jugular para executar as medidas hemodinâmicas, infusões e Sangue de amostragem em um Rat Consciente Modelo

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Férrico Thrombosis Modelo rato na artéria carótida e Mesentery Vessel induzida por Chloride

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

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, ...

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, ...

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

Imagens de ressonância magnética fase contraste na artéria carótida comum de rato

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

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 ...