The scientific advice of Drs. Timothy McMahon and Siqing Shan is appreciated. The presenters thank Drs. David Kirsch and Patricia Steeg for the generous gift of the fluorescently labeled Mouse Sarcoma and metastatic MDAMB-231 cells, respectively. This work was funded in part by the U.S. Defense Advanced Research Projects Agency (DARPA) Prime Award Number N66001-10-C-2134, and in part by the Department of Radiation Oncology, Duke University Medical Center.

NOTA: Todos os procedimentos relacionados animais descritos neste protocolo ter sido previamente aprovado pela Institutional Animal Care e Use Comitê da Universidade Duke (DUIACUC). 1. Cancer Cultura de Células e Injeção <ol><li> Cultivar células cancerígenas metastáticas marcados com fluorescência (por exemplo, células de câncer de mama MDAMB231-GFP humanos, dom de Dr. Patricia Steeg, NCI, e células de sarcoma rato YFP marcadas, presente do Dr. David Kirsch, Duke University Medical Center, Department of Radiation Oncology) em meio de cultura apropriado (por exemplo, de Dulbecco Modified Eagle Médium (DMEM) com 10% de soro fetal bovino e 1% de penicilina / estreptavidina) a 37 ° C até cerca de 90% confluente. </li><li> Tripsinizar células, lave-as duas vezes com PBS, contar e depois injetá-las na veia da cauda de 10 semanas de idade ratos nus femininos anestesiados com isoflurano em 5 milhões de células por animal, usando uma seringa com uma agulha 27 G. Anesthes Cirúrgico nívelIA é verificado pela falta de reação aos pés pinch. </li></ol> 2. O controlo das metástases com MicroCT <ol><li> Examinar ratos uma vez por semana utilizando um micro-CT / micro-irradiador, para detectar a presença de metástases acima de aproximadamente 2 mm de diâmetro de diâmetro. O micro-CT é comissionado, como descrito anteriormente 12. <ol><li> Ratos sujeitos a 3% de isoflurano anestesia antes da imagem. Confirme anestesia profunda por toe pitada. </li><li> Após o início da anestesia, rápida transferência de ratos para o berço de imagem na câmara de imagem e se conectar através de um nariz-cone a uma mistura de isoflurano ao ar em 2,5-3%. Ajustar a posição do rato no suporte de uma maneira que a sua tórax é colocado no feixe de fotões do scanner MicroCT, utilizando os controlos de posição externos e delimitador do laser na base de imagens. Assegurar a porta para a câmara de imagem está fechada, para proteger o investigador dos raios gama. </li><li> Controlar a posição do animal usando novamentea câmera de vídeo a cores. Realize uma baixa resolução exame de imagem CT prazo, e usar a imagem resultante para ajustar o campo de visão para as dimensões xyz da cavidade torácica. </li><li> Imagem do tórax rato usando um filtro de 2 milímetros Al a 40 kVp, 2,5 mA, e 0,008 voxel às 7 FPS e devolver o animal para sua gaiola. Imagem de um animal deve ter não mais do que 15 ou 20 min. Não devolva um animal que passou por uma cirurgia para a companhia de outros animais até que esteja totalmente recuperado. Não deixe um animal sem supervisão até que ele recuperou a consciência suficiente para manter decúbito esternal. </li><li> Confirmar metástases pela aparência de objectos relativamente radio-opaco, que não pode ser explicado por vasos sanguíneos intratorácicas (Figura 1A) </li></ol></li></ol> Cirurgia 3. Window Câmara <ol><li> Anestesia, sinais vitais e de uma veia da cauda <ol><li> Escolha animais com presença de doença metastática. Injectar animal com um d intraperitonealose de 50 mg / kg de pentobarbital. Confirme anestesia de nível cirúrgico por toe pitada antes de prosseguir. Nota: Os protocolos de anestesia deve ser compensada com a respectiva configuração experimental. Pentobarbital foi escolhido aqui como um anestésico de ação longa, a fim de induzir a anestesia profunda para procedimentos demorados, além de oferecer a opção de re-dosagem fácil. No entanto, a perda de animais sobredosagem é um problema comum com anestesia de pentobarbital. Uma outra opção que preserva reflexos autonômicos a um grau maior do que o pentobarbital é cetamina, em combinação com o uso de sedativos como xilazina ou medetomidina, que no entanto só permite para um único ciclo de re-dosagem. </li><li> Raspar animais no lado do corpo que tem a doença metastática, e na área do pescoço, utilizando uma máquina de cortar. Limpe todos os restantes cabelo solto da pele. Depois de cabelo solto é removido, aplique pomada veterinária para os olhos, para evitar que sequem. NOTA: ratos pelados atímicos pode ter cabelo residual que seja necessária aremoção res antes de prosseguir com os procedimentos cirúrgicos. É muito importante para remover todo o cabelo completamente, pois pode interferir com os procedimentos cirúrgicos e de imagem. </li><li> Corrigir o animal numa posição supina sobre uma placa metálica que é colocado sobre uma almofada de aquecimento de 37 ° C a água circula. As extremidades dianteiras e traseiras são fixados na placa com fita. NOTA: É útil para controlar e registrar os sinais vitais, como batimentos cardíacos e oxigenação do sangue arterial, utilizando um oxímetro de pulso, ao longo de procedimentos cirúrgicos e experimentais. </li></ol></li><li> A intubação traqueal <ol><li> A fim de colocar um cateter para a ventilação do animal, em primeiro lugar fazer uma incisão cutânea transversal do colo do útero, seguido pela separação mediana ventral da musculatura longitudinal para a traqueia. </li><li> Use repetida acção de abertura-a-fecho com uma pinça afiada para criar uma passagem para o fio de sutura através do lado dorsal da traqueia. </li><li> Fazer uma pequena incisão na traqueia em tele lado ventral, não mais do que semi-circular, aproximadamente entre o segundo e terceiro anel traqueal. Deixar um tempo suficientemente longo de parte da traqueia exposta na superfície dorsal, para permitir a fixação do cateter traqueal. </li><li> Insira um 2,5 a 3,0 mm &quot;Y&quot; cânula traqueal na traquéia, e aperte com uma sutura de monofilamento 4-0. Assegurar a cânula está ligada a um ventilador de pressão cíclica, com uma garrafa ligada à conduta de validade que é preenchido com 6 cm de água, para manter a pressão pulmonar positiva. Fluxo de entrada de gás deve ser 100% de oxigénio, a menos que de outra forma desejada experimentalmente. </li><li> Inserir um cateter com uma agulha G 25-27, e preenchido com solução salina heparinizada em uma das veias da cauda do rato, e fixar com fita adesiva. Nota: certifique-patência do cateter na veia da cauda durante todo o procedimento por injecções sucessivas de uma pequena quantidade de soro fisiológico heparinizado na veia da cauda. Além disso, a intubação oro-traqueal, ou seja, a orientação de um traquealtubo através da boca do animal anestesiado e passado a laringe e a traqueia, é uma alternativa possível para o procedimento de traqueostomia que é descrito aqui. No entanto, este método requer treino e experiência especial, a fim de evitar danos para a traqueia, e também para impedir a canulação acidental do esófago. </li></ol></li><li> Aplicação da janela pulmonar <ol><li> Remover a pele do lado da caixa, onde a doença metastática é localizado, através da criação de uma incisão, e em seguida, retirar a pele utilizando uma dissecção romba. Nota: A pele pode ser excisado e removido subsequente ao descolamento </li><li> Prossiga dissecando as duas camadas de musculatura sobrepondo (peitoral, serratus, e grande dorsal), mas deixando os músculos intercostais intacta. Criar uma perfuração na cavidade torácica de aproximadamente 1,5 cm de diâmetro, através da remoção de porções de tipicamente duas nervuras adjacentes. Idealmente, localize a perfuração da região do sexto e sétimo rIBS. </li><li> Osteotomia: <ol><li> Para minimizar o sangramento e danos à superfície do pulmão, firmemente segurar a costela para ser cortado com uma pinça cirúrgica de dentes durante o corte. Utilizando uma tesoura cirúrgica, cortar o lado medial da primeira nervura, a um ângulo de aproximadamente 45 °, deixando o lado aguçado do osso remanescente nervura que aponta fora. </li><li> Posteriormente, o corte lateral do osso do reforço de uma forma semelhante, de novo deixando o lado aguçado do osso nervura que aponta para o exterior, para evitar danos na superfície do pulmão. </li><li> Repita o procedimento para a costela ao lado, em seguida, corte os músculos intercostais e remover a parte extirpada. Durante este procedimento, a manter a pressão pulmonar de forma que a interacção mecânica entre a superfície do pulmão e da caixa torácica é minimizado. Faça isso de forma adequada regulação da pressão inspiração no ventilador. </li></ol></li><li> Inserção da janela: <ol><li> Insira uma janela de pulmão feito à medida, que consiste em uma lamela que éligado a uma tomada de Plexiglas (Figura 1B). Anexar a janela para a tomada por meio de colagem, ou através da aplicação de uma quantidade muito pequena de gordura vácuo. Inserir a janela de uma forma que as metástases de superfície estão localizados perto do centro da janela. Se necessário, ajustar o furo inserido para trazer o micrometástases para o centro da janela através do alargamento do furo ligeiramente para o lado respectivo. NOTA: A doença metastática na superfície pleural podem ser identificados como pontos brancos claramente reconhecíveis ou as zonas das partes coloridas de outra forma saudáveis ​​rosa-salmão de superfície para o pulmão que aparecem predominantemente ao longo das fissuras. Enquanto micrometástases pode ocorrer em outras áreas do exterior pleural, as linhas de células investigadas quase sempre exibir tona micrometástases na área perfurada, uma vez que a doença metastática pode ser detectado radiologicamente. </li><li> Depois de inserir o soquete para a perfuração e criação de contato direto com a pleura visceral do lung, suturar as extremidades da moldura da janela para o músculo intercostal circundante, usando sutura de monofilamento 4.0 (Figura 1C). Use um ligeiro aumento de pressão inspiração no ventilador para ajudar ar residual para escapar e para criar um selo. Nota: A taxa de respiração de ratos pode variar muito, dependendo do estado de anestesia, o estado de excitação ou ansiedade, concentração de oxigênio do ar inspirado (FiO 2), etc. É recomendável ajustar a taxa de respiração entre 70 e 90 bpm. A pressão de inspiração deve ser ajustada com cuidado e não ser ajustado para mais de aprox. 8 centímetros H2O (0,6 mmHg), a fim de evitar danos à superfície do pulmão. </li></ol></li><li> Posicione o animal em um limitador projetada que é projetado para eliminar Z-direcional movimento (Figura 1D) em uma placa de aço que está posicionado em uma termostática (elétrica) cobertor de aquecimento, sob um microscópio de fluorescência. Controlar o corpo do animaltemperatura utilizando um termistor rectal. Ajuste os parafusos do restrainer animal, ea pressão inspiração no ventilador para conseguir o controle ideal de movimento lateral. Nota: A respiração natural em mamíferos envolve todos os três sentidos de circulação pulmonar e extensão peito: bilateral, dorso-ventral, e crânio-caudal. A fim de preservar o movimento de respiração natural, tanto quanto possível, é importante minimizar Z-direcional compressão na medida do necessário. Porque o sistema de retenção Z-dimensional tem o potencial para introduzir artefactos que possam afectar o fluxo de sangue e de outros parâmetros, é aconselhável manter as condições constantes durante série de medições repetidas no mesmo animal. </li></ol></li></ol> 4. Imaging and Measurement de Microcirculatory fluxo de sangue <ol><li> Coletar as células vermelhas do sangue através de punção cardíaca e rotulá-los com DII (1,1 = -dioctadecyl-3,3,3 = perclorato, 3 = -tetrametil-indocarbocianina), como descrito anteriormente &lt;sup&gt; 10. </li><li> Injectar 300 ul de hemácias marcadas na veia da cauda do rato antes da cirurgia câmara de janela é feito para evitar o efeito de primeira passagem de adesão para a janela de vidro. Eliminar quaisquer bolhas de ar na seringa ou cateter, como a introdução de ar na veia irá inibir quaisquer outras injecções. </li><li> Fluxo sanguíneo imagem com uma câmara CCD microscópio a -40 ° C chip de temperatura de arrefecimento, e aproximadamente 100X resolução geral (isto é, com uma objectiva de microscópio de 10x, 10x e pré-câmara ocular). Use padrão de filtro Rhodamine / TRITC sets (excitação 450-490 nm, emissão&gt; 515 nm). Grave a resolução e taxa de quadros real de pixel as sequências de imagens resultantes. Grave, pelo menos, 200 (idealmente em torno de 300) imagens por pilha, para garantir a análise de sucesso da velocidade do fluxo. </li><li> Reabastecer a perda de líquidos no animal através da injeção de aproximadamente 1 ml de solução salina IP cada hr. NOTA: As configurações experimentais que envolvem uma intervenção, por exemplo,uma droga que altera a velocidade do fluxo sanguíneo em uma metástase do câncer pulmonar, exige medições repetidas de velocidade do fluxo sanguíneo em câncer metastático pulmonar. Para estas experiências prolongadas, é importante para repor o animal com fluido suficiente. </li><li> Eutanásia dos animais por infusão de 3 ml de 3N de KCl na veia da cauda </li><li> Avaliar as pilhas de imagens usando um publicado, algoritmo de computador publicamente disponível baseado em Matlab que vai criar o fluxo de velocidade em tons de cinza e mapas de cor codificado para todo o fluxo de sangue traça 10,11. Posteriormente avaliar as imagens em tons de cinza resultantes usando software de análise de imagem comercial ou acessível ao público, tais como Imagem J, após limiarizar off valores que indicam nenhum movimento de células do sangue, ou seja, sem vasculatura ativa. </li></ol>

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Um modelo é apresentado que é viável a imagem muda em microcirculação outros processos dinâmicos em metástases pulmonares de ratos, o fluxo de sangue e, através de microscopia intravital e análise de fluxo sangüíneo computacional. Embora existam outros métodos para efectuar microscopia em pulmões expostos em caixas torácicas abertas de roedores, este modelo também é o primeiro a metástases pulmonares de imagem através de uma perfuração da parede torácica, num ambiente de tórax fechado. Usando este ...

The lung is one of the most important target organs of metastatic disease, and because this condition is difficult to treat successfully with chemo- and radiation therapy, a cure is still rare1,2. Specific pathophysiological and microcirculatory features of solid primary and metastatic tumors, such as microregional hypoxia, diffusion limitation and inefficient tumor vasculature, greatly contribute to their resistance to anticancer treatment3,4. Due to the microscopic scale and dynamic nature of parameters such as microvascular blood flow, intravital microscopy of the tumor in the living animal has become a very important research tool in the field5. While intravital microscopy models have been applied to tumors in different organ sites, including the metastatic lung within an open rib cage, no protocol has been developed yet for the research of pulmonary metastases in a physiologically preserving, closed-chest environment6,7. Such an endeavor is particularly hampered by the necessity to surgically access the rib cage without affecting the overall function of the lung7-9. Recently, a method was introduced to image pulmonary microcirculatory blood flow in a close-chest setting in live rats, using fluorescence intravital microscopy10. This protocol enables the systematic quantification of blood flow velocity from injected, fluorescently labeled red blood cells, using computerized analysis, while keeping the animal physiologically stable and preserving the integrity of the lung11. In this present study, it is shown how this technology can be modified to image and quantify microcirculatory blood flow in tail vein-inoculated pulmonary metastases on the pleural surface in the immunocompromised rat. This model is also the first one to study metastatic lung tumors in a closed-chest intravital microscopy setting.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Athymic nude rats</td>
			<td style="width:71px;">Charles River</td>
			<td style="width:119px;">Strain code 316</td>
			<td style="width:167px;">Female 10 week-old athymic nude rats</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micro-CT/micro-Irradiator&#160;</td>
			<td style="width:71px;">Precision X-ray Inc.</td>
			<td style="width:119px;">Xrad 225Cx</td>
			<td>Use MicroCT to detect metastases</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DiI (1,1=-dioctadecyl-3,3,3=,3=-tetramethyl-indocarbocyanine perchlorate)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">468495-100MG</td>
			<td>Mix 100 ul packed red blood cells with 100 ul of 0.5 mg/ml DiI in 200 proof ethanol, 2 ml of 5% dextrose solution in water, and fill up to a 10-ml final volume with saline</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Rodent ventilator</td>
			<td style="width:71px;">Kent Scientific</td>
			<td style="width:119px;">TOPO Small Animal Ventilator</td>
			<td>Device is important to maintain positive lung pressure after application of pneumothorax</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss Axioskop fluorescence microscope upright</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Microscope for intravital imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Andor CCD camera</td>
			<td style="width:71px;">Andor</td>
			<td style="width:119px;">iXonEM 885</td>
			<td>CCD camera for live imaging of blood flow</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pulse oximeter</td>
			<td style="width:71px;">StarrLife</td>
			<td style="width:119px;">MouseOx</td>
			<td>Pulse oximeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescence microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Fluorescence microscope</td>
		</tr>
	</tbody>
</table>

automated measurement of microcirculatory blood flow velocity in pulmonary metastases of rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

A vasculatura em tumores sólidos é conhecido por diferir significativamente de suprimento de sangue normal, mostrando maiores graus de tortuosidade e maiores distâncias intervasculares 13. Por conseguinte, as faixas de fluxo de sangue em metástases de cancro da mama e do sarcoma pulmonares experimentais têm formas irregulares e grandes lacunas intervasculares (Figura 2A, dois painéis inferiores) em comparação com a microcirculação pulmonar normal (Figura 2A, painel ...

Watch this Scientific Journal Video about Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats at JoVE.com

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Automated Medição da microcirculação sanguínea velocidade na Pulmonar Metástases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

automated-measurement-microcirculatory-blood-flow-velocity-pulmonary

Research

JoVE Journal

Medicine

8.4K visualizações.  Duke University Medical Center. A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Vídeo: Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

Na medição in vivo da camada superficial do rato pulmonar endotelial

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Medicina

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

Automáticos de medição de enfisema pulmonar e das vias aéreas pequenas Reformas em cigarro expostas ao fumo Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH.

Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension.

O aumento de artéria pulmonar fluxo pulsátil melhora Hypoxic Hipertensão Pulmonar em Leitões

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

Transcutaneous Microcirculatory Imaging in Preterm Neonates

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest capillaries, arterioles and venules. The magnifying effect visualizes the flow pattern of erythrocytes through these vessels.
It's non-invasive character makes it suitable to apply in (preterm) neonates, even in cardiorespiratory unstable patients. In adults and children, MI is mainly performed sublingually, but this is not possible in preterm infants as these cannot cooperate and the size of the probe is problematic. In preterm infants, MI is therefore performed transcutaneously. Their thin skin makes it possible to obtain high quality images of peripheral microcirculation. 
In this manuscript we will demonstrate the method of transcutaneous MI in preterm infants. We will focus on the different techniques and provide tips to optimize image quality. The highlights of software settings, safety and offline analysis are also addressed.

Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.

Transcutânea Microcirculatory Imaging no recém-nascidos pré-termo

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

Um Modelo de Cardiologia remodela Através constrição da Aorta Abdominal em Ratos

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

Gravação simultânea de eletrocardiografia e sangue invasiva pressão medida em ratos

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Avaliação da reserva de fluxo coronariano após reperfusão de isquemia miocárdica em ratos

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

Teste de hemocompatibilidade de implantes de contato com o sangue em um modelo de loop de fluxo imitando o fluxo sanguíneo humano

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.