Os autores gostariam de agradecer Keith Michel do MD Anderson pequena instalação de imagem animal para a sua assistência técnica com micro-tomografia computadorizada, Edward TH Yeh, MD, para a assistência cirúrgica e Rebecca Bartow, PhD, para a assistência editorial. Este trabalho foi financiado em parte pela American Heart Association.

1. Priming da bomba osmótica <ol><li> Use técnicas estéreis (por exemplo, luvas, câmara de fluxo laminar) na preparação das bombas. Remover a bomba e regulador de fluxo a partir da embalagem. Pesar a bomba de vazio e anotar o peso (mg). </li><li> Encher a bomba, lentamente, para evitar a criação de bolhas de ar, utilizando-se uma pequena seringa de 1 cc 27G e o tubo de enchimento de ponta romba. Quando a solução atinge a parte superior da bomba, parar o enchimento. </li><li> Limpar o excesso de solução e peso da bomba cheia. Para a maioria das soluções aquosas, o peso em mg é o mesmo que o volume em ul. A diferença no peso vazio contra cheia deve ser mais do que 90% do volume de enchimento significativo especificado. Se não, evacuar a bomba de seringa e com a recarga. Se tiver dificuldade técnica, por favor consulte a seção de solução de problemas das instruções de bomba em http://www.alzet.com/products/guide_to_use/filling.html </li><li> Use uma pinça para quebrar o branco flange do moderat fluxoou, tenha cuidado para não dobrar ou esmagar o tubo. Anexar um cateter P50 para cada extremidade do moderador, cobrindo cerca de 3-4 mm. Usando uma seringa para encher o cateter e anexado moderador e deixar a seringa fixada à parte distai do cateter. </li><li> Inserir o regulador de fluxo para dentro da bomba até que o cateter esteja nivelada com a superfície da bomba. Em seguida, remover a seringa a partir da extremidade distal do cateter. Coloque a bomba numa solução salina estéril a 0,9% ou em solução de PBS a 37 ° C incubadora durante um mínimo de 6 horas (de preferência S / N) para accionar a bomba, antes da implantação em que o rato. A taxa de entrega da bomba varia com o número do modelo (por exemplo, Modelo 1002 = 0,25 ul / h durante 14 dias). </li></ol> 2. Criação de isquemia dos membros posteriores Unilateral no Rato <ol><li> Anestesiar ratos com o conjunto entre 2-4% de isoflurano em 100% de O 2 a uma taxa de fluxo de 0,4 L / min e remover o cabelo (de preferência um dia antes da cirurgia) sobre a área do membro posterior e para trás com um depilatocreme ry. </li><li> Esterilizar todos os instrumentos cirúrgicos antes da cirurgia. Posicione o mouse na câmara de indução anestésica. Use as mesmas configurações de anestesia descritos na Seção 2.1. </li><li> Espere até que o mouse não é sensível a estímulos externos e, em seguida, colocá-lo na posição de bruços em cima da mesa de operação com um cone de nariz equipada que fornece um fluxo contínuo de isoflurano (1,5-2,0%). </li><li> Injectar a dose recomendada de anestésico (bupivacaína, 0,25% (&lt;1 mL / kg)) no local da incisão, tratar a pele com uma solução de iodo-povidona seguido por 70% de etanol. Adicione uma incisão de 4-6 mm na parte de trás (para o lado da coluna vertebral, na região escapular). Separe a membrana subcutâneo com uma pinça fina, em seguida, vire o mouse para a posição supina tratar a pele com uma solução de iodo-povidona seguido por 70% de etanol e fazer 0,8-1 cm incisão com tesoura micro-cirúrgicos acima do joelho em direção à coxa medial, seguindo o caminho da artéria femoral. </li><li> Tunnel até olado do torso com a pequena tubagem de revestimento (6-French) a partir da coxa medial a incisão nas costas. </li><li> Posicione o mouse na posição prona. Remover a bomba osmótica ferrado / cateter a partir da solução de PBS e guiar o cateter, através do tubo de revestimento, para a coxa. </li><li> Colocar a bomba dentro da parte traseira e fechar a incisão traseira com sutura prolene 6-0 (tratar a pele com uma solução de iodo-povidona seguido por 70% de etanol). Coloque a parte traseira do rato na posição supina; remover o tubo de revestimento, deixando o cateter exposto através da incisão na coxa. Fixe a perna com fita cirúrgica. </li><li> Separou-se a gordura do tecido subcutâneo para revelar a vasculatura. Para melhor visualizar a vasculatura, use um afastador para abrir a incisão. </li><li> Pierce a bainha femoral membranoso delicadamente usando uma pinça fina pontas para acessar o feixe vascular. Dissecar e separar a artéria femoral da veia femoral e nervo perto da virilha, tomando cuidado para não causar exsangramento sivo. </li><li> Colocar duas suturas adjacentes prolene 8-0 para interromper a artéria femoral de 2-3 mm proximal à bifurcação femoral (a origem da femoral profunda e epigastrica artérias). </li><li> Corte o cateter para o comprimento adequado e coloque a ponta para a região ântero-lateral da coxa, prendendo-o com um 6-0 prolene sutura da fáscia superficial para garantir a entrega alvo da substância. Fechar a incisão perna com um 6-0 sutura. Administrar um final de injecção subcutânea de analgésico (buprenorfina, 0,1 mg / kg), tratar a pele com uma solução de iodo-povidona seguido por 70% de etanol e devolver o rato para o compartimento. Use uma lâmpada de aquecimento durante a recuperação. </li><li> Depois de um período de recuperação de 30 minutos, prepare o mouse para Doppler laser para confirmar a criação da isquemia dos membros posteriores. </li><li> Monitorizar os animais duas vezes por dia, durante 5 dias após a cirurgia. A mobilidade do animal será observada a ocorrência de dor, os resíduos na gaiola servirá como uma indiator de comer. Além disso, durante o período de monitor de determinar se o animal está sentindo dor ou desconforto e dar uma injeção subcutânea de buprenorfina (0,1 mg) duas vezes ao dia. </li></ol> 3. Imagem Preparação de Amostras <ol><li> Anestesiar o mouse como indicado na Seção 2.1. Transferir o mouse em cima da mesa de operação na posição supina. Injectar 1000 UI de heparina por via subcutânea para prevenir a coagulação na vasculatura. Enquanto o rato é ligado ao cone de protecção, efectuar uma incisão grande o suficiente para expor a cavidade torácica e o abdómen inferior. Mover os órgãos abdominais para o lado esquerdo do animal, para expor a aorta abdominal infra-renal (IAA). Separar cuidadosamente a aorta a partir da veia cava inferior. </li><li> Ligar o IAA proximal para reduzir o sangramento durante a inserção do cateter. Então, faça uma pequena incisão no IAA. Insira um 27 ½ G brusco da agulha (ligada a um cateter P10) no IAA e prenda-o com um 6-0. </li><li> Encha dois20cc seringas com solução salina heparinizada e 10% de formalina neutra tamponada (NBF). Encha uma seringa 5cc com um composto de borracha de silicone radiopaco contraste. Misture os três ingredientes do MV-composto: MV-diluente: MV agente de cura em relações de trabalho dos 1:1.25:0.5. Uma vez que a mistura (Microfil) tem apenas um tempo de trabalho de 20 minutos antes do agente de polimeriza, não é recomendado para adicionar o agente de cura até MV direita antes da injecção na vasculatura. </li><li> Fazer um corte no átrio direito e administrar uma dose excessiva de isoflurano para induzir a eutanásia e para permitir a drenagem do excesso de fluido. Fixe a seringa cheia de soro fisiológico para a ponta da agulha e começar a limpar a vasculatura por 5 min. Certifique-se de todo o sangue é liberado para fora dos vasos, desligue o isoflurano e desconecte o nosecone uma vez que a descarga começa. Em seguida, juntar a seringa cheia de formalina, o cateter descrito acima (3.2) e da pressão para perfundir um adicional de 5 min. Finalmente, o último perfundir seringa, com o contrasteagente, lentamente no ratinho durante 5 min. </li><li> Opcional: Fotografia do Microfil na vasculatura com uma câmara para garantir que apenas a estrutura arterial é perfundido. </li><li> Posicione o mouse em um recipiente com 10% de NBF e armazenar a 4 ° CO / N para a polimerização do agente de contraste. Após 24 horas, remover a pele na parte inferior do tronco (distai para a ligação da aorta abdominal inferior), utilizando ferramentas de dissecação, atualizar a NBF a 10%, e coloca o rato volta para o recipiente durante mais 4 dias à TA. </li><li> Dissecar os membros posteriores contralateral e isquêmico, e colocar as amostras em uma solução de ácido fórmico (Cal-Ex II) por 48 horas para facilitar a descalcificação do osso. Em seguida, lave as peças em água corrente por 1 hora e voltar para 10% NBF e prosseguir com Faxitron (sistema de raio-x usado para garantir o osso é descalcificado) e micro-tomografia computadorizada. </li></ol>

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	<li>Epstein, S. E., Fuchs, S., Zhou, Y. F., Baffour, R., Kornowski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+interventions+for+enhancing+collateral+development+by+administration+of+growth+factors:+basic+principles,+early+results+and+potential+hazards.">Therapeutic interventions for enhancing collateral development by administration of growth factors: basic principles, early results and potential hazards.</a> Cardiovasc. Res. 49, 532-542 (2001).</li><li>Esaki, J., Marui, A., Tabata, Y., Komeda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+release+systems+of+angiogenic+growth+factors+for+cardiovascular+diseases.">Controlled release systems of angiogenic growth factors for cardiovascular diseases.</a> Expert Opin. Drug Deliv. 4, 635-649 (2007).</li><li>Bentley, M. D., Ortiz, M. C., Ritman, E. L., Romero, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+microcomputed+tomography+to+study+microvasculature+in+small+rodents.">The use of microcomputed tomography to study microvasculature in small rodents.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, 1267-1279 (2002).</li><li>Golomb, B. A., Dang, T. T., Criqui, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+arterial+disease:+morbidity+and+mortality+implications.">Peripheral arterial disease: morbidity and mortality implications.</a> Circulation. 114, 688-699 (2006).</li><li>Hiatt, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+therapy+for+peripheral+arterial+disease+and+claudication.">Pharmacologic therapy for peripheral arterial disease and claudication.</a> J. Vasc. Surg. 36, 1283-1291 (2002).</li><li>Hirsch, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+arterial+disease+detection,+awareness,+and+treatment+in+primary+care.">Peripheral arterial disease detection, awareness, and treatment in primary care.</a> JAMA. 286, 1317-1324 (2001).</li><li>McDonald, D. M., Choyke, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+angiogenesis:+from+microscope+to+clinic.">Imaging of angiogenesis: from microscope to clinic.</a> Nat. Med. 9, 713-725 (2003).</li><li>Hirsch, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACC/AHA+Guidelines+for+the+Management+of+Patients+with+Peripheral+Arterial+Disease+(lower+extremity,+renal,+mesenteric,+and+abdominal+aortic):+a+collaborative+report+from+the+American+Associations+for+Vascular+Surgery/Society+for+Vascular+Surgery,+Society+for+Cardiovascular+Angiography+and+Interventions,+Society+for+Vascular+Medicine+and+Biology,+Society+of+Interventional+Radiology,+and+the+ACC/AHA+Task+Force+on+Practice+Guidelines+(writing+committee+to+develop+guidelines+for+the+management+of+patients+with+peripheral+arterial+disease)--summary+of+recommendations.">ACC/AHA Guidelines for the Management of Patients with Peripheral Arterial Disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Associations for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (writing committee to develop guidelines for the management of patients with peripheral arterial disease)--summary of recommendations.</a> J. Vasc. Interv. Radiol. 17, 1383-1397 (2006).</li><li>Zagorchev, L., Mulligan-Kehoe, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+of+vessels+in+mouse+models+of+disease.">Molecular imaging of vessels in mouse models of disease.</a> Eur. J. Radiol. 70, 305-311 (2009).</li><li>Losordo, D. W., Dimmeler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+angiogenesis+and+vasculogenesis+for+ischemic+disease.+Part+I:+angiogenic+cytokines.">Therapeutic angiogenesis and vasculogenesis for ischemic disease. Part I: angiogenic cytokines.</a> Circulation. 109, 2487-2491 (2004).</li><li>Waters, R. E., Terjung, R. L., Peters, K. G., Annex, B. 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Os autores declaram que não há conflito de interesse. Todos os procedimentos com animais foram conduzidos de acordo com as Diretrizes para o Cuidado e Uso de Animais de Laboratório e foram aprovados pelo Animal Care Institucional e Comitê de Uso (IACUC) da Universidade do Texas Health Science Center em Houston.

Aqui apresentamos um método para a droga / substância entrega osmótica em um modelo do rato de isquemia dos membros posteriores. Além disso, descrevemos uma técnica de fundição em que temos usado Microfil para produzir uma reconstrução 3D para análise da rede vascular. O grau ou a gravidade da isquemia varia de acordo com a qual a ligadura / excisão arterial é feita. Criamos uma ligação dupla na artéria femoral comum proximal à origem da femoral profunda e epigastrica artéri...

A doença arterial periférica (DAP) é uma doença que provoca aterosclerótica fornecimento insuficiente de sangue nas pernas 4. Ela afeta 8-12.000.000 americanos e tratamentos médicos atuais oferecem apenas um alívio limitado 5,6. Novos agentes terapêuticos que melhoram a circulação do sangue nas pernas não só restringir a progressão da doença, mas também melhorar a qualidade de vida. A incidência de PAD é maior em pessoas com idade superior a 50 anos, a terapia farmacológica para local é uma modalidade de tratamento mais desejável, porque a redução da função renal e hepática frequentemente observada em pacientes mais velhos podem diminuir o metabolismo de drogas e aumentar os efeitos colaterais com a administração sistêmica. Assim, criamos um modelo de mouse PAD para examinar se os agentes administrados localmente aliviar a isquemia dos membros posteriores, promovendo a angiogênese ea remodelação microvascular. Especificamente, utilizou-se uma bomba osmótica cateterizada para fornecer continuamente o agente terapêutico para oisquêmico músculo da coxa de ratos. Usando o nosso sistema de entrega, fomos capazes de manter as concentrações ideais da droga no ambiente local; esta abordagem permite a bioatividade de drogas adequado, evita possíveis efeitos colaterais sistêmicos, e supera a desvantagem de acesso local da droga limitado associados com a administração sistêmica. Adicionalmente, para avaliar se os agentes localmente administrados promover revascularização, usamos fundição avançada e técnicas de imagem de alta definição que permitem a quantificação de alterações na microcirculação. Colectivamente, a combinação das metodologias utilizadas neste artigo de vídeo é útil em estudos pré-clínicos para auxiliar na compreensão revascularização farmacologicamente induzida em pacientes com DAP 7-9.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent/Material</td>
		 </tr>
		 <tr>
			<td>Surgical tools</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Puritan sterile cotton swabs</td>
			<td>Fisher Scientific</td>
			<td>22-029-499</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Betadine (povidone-iodine)</td>
			<td>Fisher Scientific</td>
			<td>19-065534</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>70% Alcohol pads</td>
			<td>Fisher Scientific</td>
			<td>NC9926371</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Phosphate buffered saline</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>6-0 prolene suture</td>
			<td>Cardinal Health</td>
			<td>8709</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>8-0 prolene suture</td>
			<td>Cardinal Health</td>
			<td>2775</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Depilatory cream</td>
			<td>Nair</td>
			<td></td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Osmotic pump</td>
			<td>ALZET</td>
			<td>1002</td>
			<td>Type: Tool, 14 day release</td>
		 </tr>
		 <tr>
			<td>Vinyl catheter</td>
			<td>ALZET</td>
			<td>7760</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Heparinized saline (0.9%)</td>
			<td>Baxter</td>
			<td>2B0944</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Neutral buffered formalin</td>
			<td>Richard-Allan Scientific</td>
			<td>5705</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Microfil (silicone rubber contrast agent)</td>
			<td>Flowtech</td>
			<td>MV-112</td>
			<td>Type Reagent, Microfil White</td>
		 </tr>
		 <tr>
			<td>Cal-Ex II (formic acid solution)</td>
			<td>Fisher Scientific</td>
			<td>CS511-1D</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Buprenex</td>
			<td>CIII</td>
			<td>7571</td>
			<td>Type: Analgesic</td>
		 </tr>
		 <tr>
			<td>Bupivicaine</td>
			<td>Hospira, Inc.</td>
			<td>381</td>
			<td>Type: Analgesic</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>Dissecting microscope</td>
			<td>Carl Zeiss Microimaging</td>
			<td>Zeiss Stemi 2000-C</td>
			<td>Type:Equipment</td>
		 </tr>
		 <tr>
			<td>Laser Doppler perfusion imager</td>
			<td>Perimed Inc.</td>
			<td>Periscan PIM3 </td>
			<td>Type:Equipment</td>
		 </tr>
		 <tr>
			<td>Micro-CT imaging system</td>
			<td>GE Healthcare</td>
			<td>Explore Locus SP </td>
			<td>Type:Equipment</td>
		 </tr>
		</tbody>
 </table>

osmotic drug delivery to ischemic hindlimbs and perfusion of vasculature with microfil for micro-computed tomography imaging

A investigação pré-clínica em modelos animais de doença arterial periférica desempenha um papel vital em testar a eficácia de agentes terapêuticos destinados a estimular a microcirculação. A escolha do método de entrega destes agentes é importante, porque a via de administração afecta profundamente a bioactividade e a eficácia destes agentes de 1,2. Neste artigo, vamos demonstrar como administrar localmente uma substância hindlimbs isquêmicos usando uma bomba osmótica cateterização. Esta bomba pode fornecer um volume fixo de solução aquosa continuamente durante um período de tempo atribuído. Nós também apresentamos o nosso modelo do rato de isquemia dos membros posteriores unilateral induzida por ligadura da artéria femoral comum proximal à origem da femoral profunda e epigastrica artérias do membro posterior esquerdo. Por fim, descreve-se a punção em vivo e ligadura da aorta abdominal infra-renal e perfusão da vasculatura hindlimb com Microfil, um agente de elenco radiopaque silicone. Microfil pode perfundir e encher todo o leito vascular (arterial e venosa), e porque já ligado a principal conduta vascular para a saída, o agente pode ser retida na vasculatura para futura imagiologia ex vivo com a utilização de pequenas amostras de micro-CT 3 .

A animação demonstra a inserção de bomba osmótica e cirurgia da isquemia dos membros posteriores detalhados no protocolo. Figura 1 mostra imagens laser Doppler de perfusão do membro posterior, o que confirma a isquemia. Após o vazamento vascular com Microfil, uma imagem da rede microvascular-CT 3D mostra que Microfil pode preencher efectivamente os vasos (Figura 2A), mas que as descontinuidades podem ocorrer (Figura 2B), devido a vários factores processuais ...

Watch this Scientific Journal Video about Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging at JoVE.com

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Mostramos aqui a<em&gt; In vivo</em&gt; Inserção de uma bomba osmótica para a entrega local de drogas constante e a criação de isquemia dos membros posteriores em modelo de ratinho. Além disso, a vasculatura do membro posterior é perfundido com Microfil, um agente radiopaco de silicone, para preparar micro-tomografia computadorizada (micro-CT).

Osmótica Drug Delivery para hindlimbs isquêmicos e perfusão da vasculatura com Microfil para Micro-tomografia computadorizada

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

osmotic-drug-delivery-to-ischemic-hindlimbs-perfusion-vasculature

Research

JoVE Journal

Medicine

12.2K visualizações.  The Texas Heart Institute at St. Luke's Episcopal Hospital. Mostramos aqui a In vivo Inserção de uma bomba osmótica para a entrega local de drogas constante e a criação de isquemia dos membros posteriores em modelo de ratinho. Além disso, a vasculatura do membro posterior é perfundido com Microfil, um agente radiopaco de silicone, para preparar micro-tomografia computadorizada (micro-CT).

Vídeo: Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT) imaging.

Perfusão retrógrada e enchimento da vasculatura coronariana do rato como preparação para Micro tomografia computadorizada

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

Medicina

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Avaliação da viabilidade de gordura humana injecção em ratinhos nu com Micro-Tomografia Computadorizada

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

Usando tomografia Micro-computadas para a avaliação do desenvolvimento do tumor e Acompanhamento da resposta ao tratamento em um modelo do rato do cancro do pulmão

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Drug Delivery intraluminal para o Rato Fístula Arteriovenosa endotélio

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Combinada Near-infrared imagens fluorescentes e tomografia Micro-computado para Diretamente Visualizando Cerebral tromboêmbolos

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Método de entrega directa segmentos intra-hepática Usando um modelo de rato Liver Hilar Grampo

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Método otimizado de LC-MS/MS para a análise do elevado-throughput de amostras clínicas de Ivacaftor, seus principais metabolitos e Lumacaftor em fluidos biológicos de pacientes de fibrose cística

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Melhor método para o estabelecimento de uma In Vitro barreira hemato - encefálica modelo baseado em células endoteliais do cérebro de suínos

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

Técnicas de preparação de tecidos para imagens de tomografia microcomputante aprimorada de contraste de grandes modelos cardíacos de mamíferos com doença crônica

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.