Agradecemos ao Dr. Kelly Stevens para os ensaios iniciais do protocolo, o Dr. Michael Simons, Hauch Kip Dr., e os membros de ambos os laboratórios para a discussão geral. Este trabalho é suportado por NIH concede HL087513 e P01 HL094374.

1. Os preparativos antes do início <ol><li> Encha cada lado do aparelho de perfusão com tampão de pressão vasodilatador (4mg / L Papaverina + 1 g / L adenosina em PBS) ou paraformaldeído a 4% (PFA) em PBS, respectivamente. </li><li> Prepara-se uma seringa de insulina 1/2cc (com um 29G permanentemente ligado ½ &quot;agulha), enchendo-a com 0,1 ml de 1:100 Heparina (estoque 5000U/ml) e dobrando a agulha para ~ ângulo de 120 graus com o bisel para cima. Fazer o mesmo com uma seringa de 1 ml (com uma agulha de 26G ½ &quot;) cheio com 0,3 ml de solução saturada de KCl. </li></ol> 2. A exposição do coração e da canulação da aorta <ol><li> Anestesiar o rato usando o anestésico de escolha. (Usamos uma overdose de uma mistura Cetamina / Xilazina:. Injecção IP de 130 mg de cetamina / kg e 8,8 Xilazina mg / kg em solução salina) </li><li> Pin o rato anestesiado no tabuleiro de dissecação, acima do lado ventral. Abra a cavidade abdominal por uma incisão na linha média e retrair a pelepara expor os órgãos. Mover os intestinos para um lado para expor uma região de veia cava posterior (PVC). </li><li> Injectar solução de heparina para o PVC. Como você extrair a agulha, cobrir o buraco da agulha com um aplicador com ponta de algodão para evitar vazamentos e mantenha-o por alguns segundos até que os coágulos de PVC de parede e vedações. Espere 2-3 minutos para a heparina para dispersar através da circulação do rato. </li><li> Dissecar o diafragma e caixa torácica para que você possa observar o coração batendo. Lentamente injetar solução de KCl no PVC até as prisões do coração. </li><li> Remova todos os órgãos abaixo do diafragma e impostos especiais de consumo a parte posterior do mouse, deixando a região anterior do diafragma intacta. Remover o diafragma, tomando cuidado para cortar o PVC perto do diafragma para que a porção proximal para o coração é fácil de localizar em passos subsequentes. </li><li> Localizar a extremidade cortada da aorta. Coloque uma longa duração de 6-0 fio de seda trançada debaixo da aorta alguns milímetros anteriores from da extremidade cortada de tal modo que a sutura é dobrada para trás sobre si mesmo. Corte este tempo de sutura na metade de forma que há 2 pedaços de sutura sob a aorta. Inserir o angiocateter para dentro da extremidade de corte da aorta (Fig. 1A, B) e amarrá cada sutura com um duplo nó para segurar o angiocateter no lugar e evitar qualquer contra-pressão no interior da aorta de vazar para fora. </li></ol> 3. Perfusão e injeção Microfil <ol><li> Ligue o angiocateter para o aparelho de perfusão de pressão (Fig. 2) e começar a perfusão dos vasos com tampão de vasodilatador (Fig. 1C) por bombagem do aparelho de perfusão a uma pressão de condução de 100-110 mmHg. Verifique que o buffer está irrigando através das coronárias, assegurando líquido está saindo do PVC. Continuar para perfundir durante pelo menos 3 minutos ou até que o fluido que sai do PVC é clara. (Continue com os passos seguintes enquanto perfusão). </li><li> Dissecar as costelas e costas pino (ou remover) a caixa torácica para expor o coração. Uma vez exposta, seja careful para não deixar o coração secar por espremedura gotas de tampão para o coração de um pedaço de tampão-embebida de gaze. Limpar o timo para expor o arco aórtico. Ligadura dos três principais ramos da aorta, utilizando fio de seda trançado 6-0 para garantir o fluido é desviado através das coronárias, em vez de através dessas embarcações maiores, de baixa resistência (Fig. 1D). </li><li> Perfundir o coração com fixador durante 15 minutos, em seguida, lavar com tampão de vasodilatação durante pelo menos 2 minutos. Enquanto isso, ligadura tanto Anterior veias cavas para evitar Microfil vaze para fora do coração após a injeção (Fig 1E). Suturas em volta da PVC e da aorta, mas não aperte-os até que após o enchimento. </li><li> Preparar o Microfil (tal como especificado na tabela de reagentes) e carregá-lo para uma seringa de 1 ml. Encher o tabuleiro de dissecção com água suficiente para cobrir o cateter (de modo a evitar a introdução de bolhas de ar quando se muda de tubagem de perfusão para a seringa Microfil). Desligue a perfusão apParatus a partir do cateter e ligar a seringa Microfil preparada. </li><li> Injectar a Microfil para a aorta até bom enchimento das artérias coronárias é evidente (Figs 1F, 3A): as artérias vai encher primeiro, e depois o Microfil irá &quot;derramar&quot; para os capilares como as ondas de tecido com a cor do Microfil. Uma vez no lado venoso, a natureza hidrofóbica do Microfil faz com que ele aparecem inicialmente como esferas independentes que emerge a partir dos vasos pequenos. Continue a injectar a Microfil até uma coluna contínua enche as veias. Enchimento completo será evidente quando o Microfil é contínuo dentro dos vasos, e saem através do PVC. </li><li> Após o enchimento está completa, rapidamente apertar as suturas que foram previamente colocadas em torno do PVC e aorta para evitar que a natureza elástica do tecido cardíaco de espremer o Microfil para fora dos vasos. </li><li> Cobrir o coração com gaze húmida (embebido em água a partir da bandeja de dissecação) Para evitar a sua secagem para fora, e deixar repousar durante aproximadamente 1 hora à temperatura ambiente até que a Microfil tem polimerizado. Evitar quaisquer pressões externas sobre o coração durante o processo de polimerização, tais como elevação ou rodando o coração, numa tentativa de obter uma vista precoce dos vasos cheios na parte de trás do coração. Isto pode apertar Microfil a partir de alguns vasos para uma área mais elástica do coração, causando quebras na Microfil. </li><li> Retire o coração e pós-consertá-lo em PFA 4% durante a noite a 4 ° C. Em seguida, armazenar em etanol a 70%, a 4 ° C. A vasculatura coração está agora pronto para imagiologia μCT. </li></ol> 4. Os resultados representativos Vasos que são efectivamente perfundidos por Microfil terá Microfil, contínua ininterrupta ao longo dos vasos (Fig. 3A). O grau de enchimento dos vasos coronários pode ser julgada pelo olho; veias são epicardially situado a 18, e pode ser facilmente observado (Figura 3A, ponta de seta), artérias,que são mais intramiocárdica 18, são também visíveis através da superfície do coração (Fig. 3A, seta). Enchimento capilar é também evidente, como o tecido cardíaco tem uma densidade muito elevada de capilares, e portanto, quando o preenchimento capilares, o tecido cardíaco irá limpar com a cor do Microfil (Fig. 3A, estrela). Assim, todas as redes vasculares que falharam para encher será perceptível devido à falta de Microfil (Fig. 3B, C). Descontinuidades na Microfil (asteriscos na Figura 3B), muitas vezes aparecem porque a natureza hidrofóbica da Microfil fará com que ele contraia em si mesmo e fazer com que &quot;quebra&quot; dentro de vasos cheios. Estes &quot;quebra&quot; pode ser reduzida se a pressão dentro dos vasos é mantida através de adequada tie-offs dos pontos de saída vasculares do coração. Outros descontinuidades pode ser causada por bolhas de ar no interior da Microfil. Para evitar a introdução de ar, verifique se o angiocateter está totalmente submerso na água quando se muda da perfusão ap<html> Paratus à seringa Microfil. Se uma bolha de ar é introduzida, que muitas vezes podem ser removidos de forma simples, continuando a perfusão Microfil até que a bolha foi empurrada através e para fora dos vasos coronários. Redes vasculares podem não encher completamente se uma porção do leito vascular é bloqueado (Fig. 3B, seta). Enquanto Heparina inibe a formação de coágulos sanguíneos, bloqueios ocasionais pode ainda ocorrer devido à perfusão Heparina incompleta antes de se iniciar o processo, ou devido a outros factores desconhecidos. Se ocorre um bloqueio, não existe, a nosso conhecimento, nenhum método para desalojar o bloqueio para completar o enchimento vascular. Enchimento incompleto pode também resultar se a pressão muito pouco é utilizado durante o enchimento, como a Microfil não será forçado em todos os leitos vasculares e as redes de capilares (Fig. 3C). Inversamente, a pressão em excesso pode causar a capilares para rebentar e extravasamento Microfil para o tecido circundante (Fig. 3D). files/ftp_upload/3740/3740fig1.jpg &quot;alt =&quot; Figura 1 &quot;/&gt; Figura 1. Visão geral do sistema de perfusão Microfil. (A) A aorta eo PVC são cortadas a aproximadamente o nível do diafragma. (B) da aorta ascendente é canulada com um angiocateter. (C) tampão Vasodilatação é perfundido através dos vasos, impulsionadas pelo aparelho pressão de perfusão (não ilustrado), enquanto (D) os três principais ramos fora do arco aórtico são ligadas. (E) PFA a 4% é perfundido através das coronárias, enquanto ambas as Cavas Anterior Veia são ligados. (F) utilizando uma seringa, a Microfil é perfundido através das coronárias até que seja observado que sai do PVC. <img src="/files/ftp_upload/3740/3740fig2.jpg" alt="A Figura 2"/> Figura 2. Aparelho de perfusão. Dois balões de Erlenmeyer, cada um preenchido com um tampão de vasodilatação ou 4% PFA, são unidas e pressurizado através de tubos ligados às suas sidearms. O sistema é pressurizado por meio de bombeamento manual dobolbo, e um medidor de pressão está ligada a um dos frascos para permitir o controlo e manutenção da pressão. Pequenos tubos estendem-se através rolhas de borracha e para baixo para dentro do fluido em cada frasco. Pressão de entrada das bombas armas brancas o fluido de cada balão para fora estes tubos de menor diâmetro. Os tubos então fundir a uma torneira de passagem que apenas permite que o fluido flua a partir de um balão de cada vez. <img src="/files/ftp_upload/3740/3740fig3.jpg" alt="Figura 3"/> Figura 3. Experimente corações microparticulada. (A) Os navios que são preenchidos bem terá poucos (se houver) quebras na Microfil, eo tecido do coração será cor matizada do Microfil devido aos capilares cheios (estrela, e comparar com C). Ambas as artérias (seta - artéria descendente anterior esquerda) e veias (ponta de seta - Veia coronária esquerda) são visíveis através da superfície do coração. (B) Um coração com quebras na Microfil (asteriscos), bem como os bloqueios em alguns vasos que impedia Mic completarofil penetração. Os vasos bloqueados permanecem vermelho (seta), tal como o sangue não foi purgada durante o processo de perfusão. (C) Um coração com os vasos que foram preenchidos de forma incompleta. Note o tecido não foi tomada sobre a cor amarela da Microfil, indicando a Microfil não penetrar na capilares. (D) Um coração onde o rebentamento de capilares durante o enchimento, fazendo com que o Microfil a vazar para o tecido circundante (seta).

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubbles+Targeted+to+Intercellular+Adhesion+Molecule-1+Bind+to+Activated+Coronary+Artery+Endothelial+Cells.">Microbubbles Targeted to Intercellular Adhesion Molecule-1 Bind to Activated Coronary Artery Endothelial Cells.</a> Circulation. 98, 1-5 (1998).</li><li>Wei, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Myocardial+Blood+Flow+With+Ultrasound-Induced+Destruction+of+Microbubbles+Administered+as+a+Constant+Venous+Infusion.">Quantification of Myocardial Blood Flow With Ultrasound-Induced Destruction of Microbubbles Administered as a Constant Venous Infusion.</a> Circulation. 97, 473-483 (1998).</li><li>Beckmann, N., Stirnimann, R., Bochelen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Magnetic+Resonance+Angiography+of+the+Mouse+Brain:+Application+to+Murine+Focal+Cerebral+Ischemia+Models.">High-Resolution Magnetic Resonance Angiography of the Mouse Brain: Application to Murine Focal Cerebral Ischemia Models.</a> Journal of Magnetic Resonance. 140, 442-450 (1999).</li><li>Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+MR+angiography+of+intratumoral+vasculature+using+a+novel+macromolecular+MR+contrast+agent.">3D MR angiography of intratumoral vasculature using a novel macromolecular MR contrast agent.</a> Magnetic Resonance in Medicine. 46, 579-585 (2001).</li><li>Nezafat, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B1-insensitive+T2+preparation+for+improved+coronary+magnetic+resonance+angiography+at+3+T.">B1-insensitive T2 preparation for improved coronary magnetic resonance angiography at 3 T.</a> Magn. Reson. Med. 55, 858-864 (2006).</li><li>Wagner, S., Helisch, A., Ziegelhoeffer, T., Bachmann, G., Schaper, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+angiography+of+collateral+vessels+in+a+murine+femoral+artery+ligation+model.">Magnetic resonance angiography of collateral vessels in a murine femoral artery ligation model.</a> NMR in Biomedicine. 17, 21-27 (2004).</li><li>Cochet, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+MR+angiography+and+velocity+measurement+in+mice+coronary+arteries+at+9.4+T:+assessment+of+coronary+flow+velocity+reserve.">In vivo MR angiography and velocity measurement in mice coronary arteries at 9.4 T: assessment of coronary flow velocity reserve.</a> Radiology. , 254-441 (2010).</li></ol>

Os ratos foram tratados com métodos aprovados pelo Institutional Animal Care e do Comitê Utilização da Universidade de Washington e de acordo com o Guia para o Cuidado e Uso de Animais de Laboratório publicado pelo National Institutes of Health EUA (NIH Publication No. 85-23, revista em 1996).

Tecido cardíaco tem uma procura muito elevada metabólica, e, portanto, requer um fornecimento constante de nutrientes e oxigénio a partir do sangue entregue pela vasculatura coronária. Doenças dos vasos coronários, que diminuem a função coronária devido a estenose do vaso e bloqueio, podem levar à hipóxia tecidual e isquemia, e colocar em risco pacientes acometidos de infarto do miocárdio e um dano irreparável ao músculo do coração. Uma melhor compreensão do estado de doença destes navios é necessári...

<html><table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Seringas de 1 ml </td><td> <a href="http://www.bd.com/" target="_blank">Becton Dickinson</a> </td><td> BD-309602 </td><td></td></tr><tr><td> 1/2cc seringas de insulina com agulhas fixas ½ 29G &quot; </td><td> <a href="http://www.bd.com/" target="_blank">Becton Dickinson</a> </td><td> BD-309306 </td><td></td></tr><tr><td> 2 &quot;x 2&quot; gaze </td><td> Med101store.com </td><td> SKU 2208 </td><td></td></tr><tr><td> 24G ¾ &quot;Angiocath cateter IV </td><td> <a href="http://www.bd.com/" target="_blank">Becton Dickinson</a> </td><td> BD-381112 </td><td></td></tr><tr><td> 26G ½ &quot;bitola agulhas </td><td>lank &quot;&gt; Becton Dickinson </td><td> BD-305111 </td><td></td></tr><tr><td> A adenosina </td><td> <a href="http://www.sigmaaldrich.com/" target="_blank">Sigma</a> </td><td> A9251 </td><td> 1g / L em tampão PBS durante a vasodilatação (com Papaverina) </td></tr><tr><td> Angled Pinça Graefe </td><td> <a href="http://www.finescience.com/" target="_blank">Belas Ferramentas Ciência</a> </td><td> 11052-10 </td><td></td></tr><tr><td> Com ponta de algodão aplicadores: 6 &quot;não-estéril </td><td> <a href="http://www.cardinal.com/" target="_blank">Cardinal Health</a> </td><td> C15055-006 </td><td></td></tr><tr><td> Curvas tesoura cirúrgica </td><td> <a href="http://www.finescience.com/" target="_blank">Belas Ferramentas Ciência</a> </td><td> 14085-09 </td><td></td></tr><tr><td> Dissecando estereoscópio e fonte de luz </td><td> <a href="http://www.nikon.com/" target="_blank">Nikon</a> </td><td> NA </td><td> NA </td></tr><tr><td> Bandeja de dissecação, 11.5 x 7.5 polegadas </td><td> <a href="http://www.coleparmer.com/" target="_blank">Cole-Parmer</a> </td><td> YO-10.915-12 </td><td> Cheio de alcatrão para fixando-se o mouse </td></tr><tr><td> Belas curvas Pinças </td><td> <a href="http://www.aesculapusa.com/" target="_blank">Aesculap</a> </td><td> FD281R </td><td> Precisa de dois </td></tr><tr><td> Heparina, 5000 estoque U / ml </td><td> <a href="http://www.apppharma.com/" target="_blank">APP Pharmaceuticals LLC</a> </td><td> NDC 63323-047-10 </td><td> 1:100 de diluição em água </td></tr><tr><td> KCl </td><td> <a href="http://www.fishersci.com/" target="_blank">Pescador</a> </td><td> P217 </td><td> Solução saturada em H2O </td></tr><tr><td> A cetamina (Ketaset), 100 ações mg / ml </td><td> Fort Dodge, Overland Park, KS, EUA </td><td> NDC 0856-2013-01 </td><td> Misturados, como peso do corpo 130 mg / kg, com xilazina em 0,9% salinhar </td></tr><tr><td> Microfil </td><td> <a href="http://www.flowtech-inc.com/" target="_blank">Fluxo de Tecnologia</a> </td><td> MV-122 (amarelo). Outras opções de cores também estão disponíveis. </td><td> Misturar 1:1 em peso, com 10% em volume de agente de cura. Prepare-se imediatamente antes da injecção, e vortex para garantir que é bem misturado </td></tr><tr><td> Não estéril Sutura: 6-0, trançada de seda </td><td> <a href="http://www.harvardapparatus.com/" target="_blank">Harvard Apparatus</a> </td><td> 723287 </td><td></td></tr><tr><td> Papaverina </td><td> <a href="http://www.americanregent.com/" target="_blank">Americana Regent Inc.</a> </td><td> NDC 0517-4010-01 </td><td> 4mg / L em tampão PBS durante a vasodilatação (com adenosina) </td></tr><tr><td> Paraformaldeído </td><td> <a href="http://www.sigmaaldrich.com/" target="_blank">Sigma</a> </td><td> P6148 </td><td> Preparado como solução a 4% </td></tr><tr><td> Aparelho de perfusão </td><td></td><td></td><td> Ver a figura 2 </td></tr><tr><td> Tesoura Primavera </td><td> <a href="http://www.finescience.com/" target="_blank">Belas Ferramentas Ciência</a> </td><td> 15018-10 </td><td></td></tr><tr><td> Xilazina (Anased), 20 ações mg / gl </td><td> <a href="http://www.lloydlab.com/" target="_blank">Lloyd Labs</a> </td><td> NADA # 139-236 </td><td> Misturados, como peso corporal 8,8 mg / kg, com cetamina em solução salina a 0,9% </td></tr></tbody></table>

retrograde perfusion and filling of mouse coronary vasculature as preparation for micro computed tomography imaging

Visualização da vasculatura está se tornando cada vez mais importante para compreender muitos estados diferentes da doença. Enquanto existem várias técnicas para geração de imagens vasculatura, poucos são capazes de visualizar a rede vascular como um todo, enquanto se estende a uma resolução que inclui o menor 1,2 navios. Além disso, muitas técnicas de fundição vasculares destruir o tecido circundante, impedindo posterior análise da amostra 3-5. Um método que contorna essas questões é micro-tomografia computadorizada (μCT). μCT imagem pode digitalizar com resoluções menos de 10 mícrons, é capaz de produzir reconstruções 3D da rede vascular, e deixa o tecido intacto para posterior análise (por exemplo, histologia e morfometria) 6-11. Contudo, os navios de imagem por métodos ex vivo μCT exige que os navios ser preenchido com um composto radiopaco. Como tal, a representação precisa da vasculatura produzido por μCT imagem está dependenterecheio fiável e completa dos vasos. Neste protocolo, descrevem uma técnica para encher vasos coronarianos de rato em preparação para a imagem μCT. Duas técnicas predominam existem para o enchimento da vasculatura coronária: in vivo através de canulação e retrógrada de perfusão da aorta (ou uma ramificação fora do arco aórtico) 12-14, ou ex vivo através de uma perfusão de Langendorff 15-17 sistema. Aqui nós descrevemos um método in vivo canulação aórtica, que foi projetado especificamente para garantir o preenchimento de todos os navios. Usamos um composto radiopaco de baixa viscosidade que pode chamado Microfil perfundir através dos navios mais pequenos para preencher todos os capilares, assim como ambos os lados arteriais e venosas da rede vascular. Os vasos são perfundidos com tampão utilizando um sistema de perfusão pressurizado e, em seguida cheio com Microfil. Para garantir que Microfil enche os pequenos vasos de maior resistência, nós ligadura dos ramos grandes emanating da aorta, que desvia a Microfil nas coronárias. Uma vez que o enchimento é completa, para evitar que a natureza elástica do tecido cardíaco de espremer Microfil fora de alguns vasos, nós ligadura acessíveis principais pontos de saída vasculares imediatamente após o enchimento. Portanto, a nossa técnica é otimizado para o preenchimento completo e máximo de retenção do agente de enchimento, permitindo a visualização da rede vascular coronária completa - artérias, capilares e veias iguais.

Watch this Scientific Journal Video about Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging at JoVE.com

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

Visualização dos vasos coronários é fundamental para avançar na compreensão das doenças cardiovasculares. Descrevemos aqui um método para a perfusão vasculatura coronária murino com uma borracha de silicone radiopaco (Microfil), em preparação para imagiologia Tomografia micro-computadorizada (μCT).

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 ...

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JoVE Journal

Medicine

32.3K visualizações.  University of Washington. Visualização dos vasos coronários é fundamental para avançar na compreensão das doenças cardiovasculares. Descrevemos aqui um método para a perfusão vasculatura coronária murino com uma borracha de silicone radiopaco (Microfil), em preparação para imagiologia Tomografia micro-computadorizada (μCT).

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

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

Segmentação e medição de volumes de gordura em modelos de obesidade em roedores usando raios-X Tomografia Computadorizada

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Medicina

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

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

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 ...

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 ...

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 ...

Identifying Coronary Artery Calcification on Non-gated Computed Tomography Scans

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed tomography (CT) scans with a high correlation with gated cardiac CT scans. This standardized protocol takes a step-wise approach to not only optimizing an image for the identification of calcification but also to distinguishing CAC from other common causes of calcification in the cardiac silhouette. Recognition of CAC on non-gated CT scans helps to identify a very powerful prognostic factor that can influence therapeutic interventions or downstream diagnostic testing without requiring a gated cardiac scan. These non-gated CT scans are often acquired as part of the routine care of the patient, and this data is readily available without another dose of&#160;ionizing radiation. This protocol allows for the precise and accurate extraction of this data for the purposes of retrospective data analysis in clinical research studies, but also in the clinical evaluation and management of patients.

Here, we present a protocol to reliably and systematically identify coronary artery calcification (CAC) on non-gated computed tomography (CT) scans of the chest or abdomen. CAC provides an objective measure of coronary artery disease for both research and clinical purposes.

Identificando a calcificação arterial coronariana na não-bloqueadas computado exames de tomografia computadorizada

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed ...

Continuous Blood Sampling in Small Animal Positron Emission Tomography/Computed Tomography Enables the Measurement of the Arterial Input Function

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal blood time-activity concentration also known as arterial input function (AIF) is a key point, especially for the characterization of animal disease models and the introduction of newly developed radiotracers. The knowledge of radiotracer availability in the blood helps to interpret PET/CT-derived data of tissue activity. For this purpose, online blood sampling during the PET/CT imaging is advisable to measure the AIF. In contrast to manual blood sampling and image-derived approaches, continuous online blood sampling has several advantages. Besides the minimized blood loss, there is an improved resolution and a superior accuracy for the blood activity measurement. However, the major drawback of online blood sampling is the costly and time-consuming preparation to catheterize the femoral vessels of the animal. Here, we describe an easy and complete workflow for catheterization and continuous blood sampling during small animal PET/CT imaging and compared it to manual blood sampling and an image-derived approach. Using this highly-standardized workflow, the determination of the fluorodeoxyglucose ([18F]FDG) AIF is demonstrated. Further, this procedure can be applied to any radiotracer in combination with different animal models to create fundamental knowledge of tracer kinetic and model characteristics. This allows a more precise evaluation of the behavior of pharmaceuticals, both for diagnostic and therapeutic approaches in the preclinical research of oncological, neurodegenerative and myocardial diseases.

Here a protocol for continuous blood sampling during PET/CT imaging of rats to measure the arterial input function (AIF) is described. The catheterization, the calibration and setup of the system and the data analysis of the blood radioactivity are demonstrated. The generated data provide input parameters for subsequent bio-kinetic modeling.

A amostragem contínua do sangue no tomography de emissão de Positron animal pequeno/tomography computado permite a medida da função de entrada arterial

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal ...

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.

DUCT: Fundição de Resina Dupla seguida de Tomografia Microcomputada para Análise hepática 3D

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the ...

Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ function. Clarifying the mechanism of blood vessel formation is therefore extremely useful for elucidating developmental processes and pathological mechanisms. Murine hind-limb vessels are often used as a model for physiological and pathological angiogenesis. Evaluation is mainly performed via a two-dimensional method using tissue sections. However, methods for evaluating three-dimensional (3D) vascular morphology are particularly limited. This paper introduces a method for visualizing murine hind-limbs using computed tomography (CT). Radiation-opaque resin is injected through the descending aorta, and whole vessels are filled with dye. By adjusting the time of dye injection, arterial-specific filling is also possible, and samples can be obtained with any micro-X-ray CT device. This contrast method provides a basic technique for the 3D evaluation of murine blood vessels in the lower extremities. Furthermore, this method can be used to visualize all blood vessels below the diaphragm and evaluate blood vessels in the abdominal organs.

Here, we describe a visualization and quantification method for murine hind-limb vessels using micro-X-ray computed tomography.

Preparação amostral para visualização tridimensional baseada em tomografia computadorizada de vasos de membros traseiros murinos

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ ...