Name: measurement of the pressure-volume curve in mouse lungs
Uploaded: 2015-01-27T15:00:00.000+00:00
Duration: 9 min 49 s
Description: Watch this Scientific Journal Video about Measurement of the Pressure-volume Curve in Mouse Lungs at JoVE.com

This work has been supported by NIH HL-1034.

O Comitê de Cuidados e Uso de Animais da Universidade Johns Hopkins aprovado todos os protocolos de animais. 1. Equipamentos O sistema composto definido acima, pronta para medir a curva de PV é mostrado na Figura 1. <ol><li> Medição de volume: <ol><li> Gerar uma taxa constante de inflação e deflação, utilizando uma bomba de seringa com um interruptor que permite que o usuário para reverter rapidamente a bomba depois de atingir os limites de pressão. Para as curvas do mouse PV, use um muito levemente untada 5 ml seringa de vidro com o volume inicial (antes da inflação), fixado em 3 ml de ar. 3 ml é suficientemente grande para medir volumes em quase todas as curvas do mouse PV. </li><li> Medir o volume entregue pela bomba anexando um transformador diferencial linear para o alojamento da bomba, com uma pequena haste do sensor ligado ao êmbolo da seringa movendo. Nota: Um meio empíricos para corrigir para a compressão de gás no sistema é descrito sob o cu PVsecção de gravação rve. </li></ol></li><li> Medição de pressão: <ol><li> Usar um medidor de pressão barato padrão com um intervalo de 0-60 cm de H 2 O (0-1 psi). </li></ol></li><li> Gravação de medição: <ol><li> Para gravar a curva PV usar qualquer gravador digital com recursos de XY (por exemplo, PowerLab). Defina um canal para gravar o sinal de volume corrigido e outro canal para registrar a pressão transpulmonar (PTP), a fim de elaborar o gráfico da curva PV. Usar um pré-amplificador de ponte que liga ao Powerlab principal para medir a pressão. Calibrar o canal de pressão 0-40 cm H 2 O, e calibrar o canal de volume de 0-3 ml. </li></ol></li></ol> 2. Correção de Compressão de Gás Nota: Este é um passo inicial importante na configuração, uma vez que como a pressão aumenta, a diminuição do volume de gás, e, assim, o volume de ar fornecido para o rato irá ser cada vez menor do que o deslocamento do syrInge barril. <ol><li> Fechar a torneira que vai ligar o sistema de PV para os pulmões, para que o gás não possa deixar o sistema. Iniciar a infusão e observar se o canal Volume corrigido no gravador mostra todas as mudanças mensuráveis, como a pressão aumenta para cerca de 40 cm H 2 O. Se assim for, então correcta como nos passos seguintes. <ol><li> Correto para compressão de gás empiricamente subtraindo a partir da medição de deslocamento êmbolo (ou seja, o volume não corrigido) um termo proporcional à pressão de inflação. Faça isso em um canal Powerlab (chamado Vc) para mostrar o sinal de volume, menos um coeficiente vezes a pressão. </li><li> Determine o coeficiente na equação. Primeiro, faça uma estimativa inicial, vire o registro gráfico, e ligar a bomba. Uma vez que o tubo de inflação é selada, ajustar o coeficiente multiplicador de pressão para fazer o canal Vc ler zero como a pressão aumenta, 0-40 cm de H 2 O. Se ele vai para cima ou para baixo, basta ajustar o fator de correção até quepermanece estável ao longo da faixa de pressão. Este factor de correcção será sempre o mesmo, se as mesmas a partir de 3 ml de volume na seringa não é alterado. </li></ol></li></ol> 3. testes experimentais em camundongos <ol><li> Processo para a medição da curva de PV em ratinhos. Todos os protocolos de animais foram aprovados pelo Comitê de Johns Hopkins University Animal Care e Use. <ol><li> Anestesiar ratos (C57BL / 6 ratos às 6-12 semanas de idade) com cetamina (90 mg / kg) e xilazina (15 mg / kg), e confirmar a anestesia pela ausência do reflexo do movimento. Nota: A curva PV pode ser completada em ratos anestesiados, em menos de 10 min e é um procedimento terminal. </li><li> Tracheostomize os ratos com uma agulha 18 G stub cânula. Fazer isso através de uma pequena incisão na pele que recobre a traqueia, localizando a traqueia, em seguida, fazendo um pequeno corte na traqueia, onde o topo da agulha pode ser inserida. Fixe a cânula, amarrando com rosca. </li><li> Permitir que os ratos para respiração 100% de oxigênio para, pelo menos, 4 min. Isso pode ser feito através de respiração espontânea de um saco ou com um ventilador nominalmente definido com um volume corrente de 0,2 ml a 150 ciclos / min. </li><li> Fechar a cânula traqueal e permitir 3-4 min para o rato para absorver todo o oxigénio. Este procedimento de absorção de oxigénio resulta a morte dos animais e de uma desgaseificação quase completa do pulmão 11. Confirme morte do rato através da medição da cessação dos batimentos cardíacos com eletrodos de ECG ou observação direta. </li><li> Uma vez que a desgaseificação do pulmão está completa e o volume do pulmão é zero, começar inflar os pulmões com ar ambiente na bomba de seringa a uma taxa de 3 ml / min. Monitorar o traçado da pressão sobre o gravador digital, e quando chega a 35 cm de H 2 O, inverter a bomba. </li><li> Siga a curva de deflação até que a pressão atinge negativa de 10 cm H 2 O, época em que as vias aéreas entraram em colapso, prendendo o ar nos alvéolos que impedem uma maior redução de volume. Reverter imediatamente tele bombear novamente, permitindo que o pulmão de reinsuflação como as vias aéreas colapsadas abrir. Esta abertura heterogénea é normalmente aparente pelo membro inflação barulhento olhando para a parte inicial desta segunda inflação. </li><li> Quando a pressão novamente chega a 35 cm H 2 O, inverter a direção da bomba, e continuar a desinflar o pulmão até esta segunda parte deflação chega a 0 cm H 2 O. Em seguida, parar a bomba. </li><li> Veja o registro gráfico PowerLab de pressão e fluxo ea curva PV. Em seguida, analisar a curva PV para detectar alterações fenotípicas em parênquima pulmonar que ocorrem com diferentes patologias pulmonares. </li></ol></li></ol>

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None of the authors have any financial interests that would be in conflict with the material presented in this paper.

Neste papel um método reprodutível simples tem sido descrito para medir em ratinhos um método clássico de fenotipagem elasticidade pulmonar, a curva total PV pulmão. Essas curvas foram fundamentais para a descoberta de surfactante pulmonar e sua importância no fornecimento de estabilidade de pulmão. Aqui mostra-se como a curva PV também é útil ao proporcionar um meio para medir diversas variáveis ​​relacionadas com a elasticidade pulmonar no pulmão adulto do mouse. Houve mudanças muito significativas em...

O rato é agora o modelo animal primária de uma variedade de doenças pulmonares. Em modelos de enfisema ou fibrose, as alterações fenotípicas essenciais são melhor avaliadas medindo as alterações na elasticidade do pulmão. Embora existam muitas maneiras de medir a elasticidade, o método clássico é que a curva de pressão-volume total (PV) medido a partir do volume residual (VR) a capacidade pulmonar total (CPT). Essa medição foi feita no pulmão adulto de quase todas as espécies de mamíferos que remonta quase 100 anos 1-3. Tais curvas PV também desempenhou um papel importante na descoberta e compreensão da função do surfactante pulmonar em desenvolvimento pulmonar fetal 4-7. Apesar de a importância da curva de PV como uma medição do fenótipo do pulmão, não houve nenhuma maneira padronizada de efectuar esta medição. Foi feito simplesmente inflar e desinflar o pulmão com passos discretos (esperando um tempo variável de equilíbrio após cada) ou com bombas quepode continuamente inflar e desinflar o pulmão. A curva PV é feito muitas vezes ao longo de um intervalo de volume entre zero e alguma capacidade pulmonar definir pelo usuário, mas o tempo de duração de cada ciclo de volume pressão relatada por diferentes laboratórios tem sido extremamente variável, variando de alguns segundos de 8 a 2 hr. Alguns pesquisadores se referem a esta curva PV pulmonar total como estático ou quasiestático, mas estes são termos qualitativos que oferecem pouco de visão, e eles não são utilizados aqui. Além disso, a curva de PV não tem sido amplamente relatado no ratinho, apesar do facto de que pode fornecer informação útil sobre os efeitos de alterações estruturais macroscópicos nos pulmões. Várias questões resultaram em variabilidade na aquisição da curva PV incluindo: 1) a taxa de inflação e deflação; 2) as excursões de pressão para a inflação e deflação; e 3) os meios para determinar uma medida absoluta do volume pulmonar. No método aqui presente, uma taxa de 3 ml / min foi escolhido como um compromise, não sendo demasiado curto para refletir a elasticidade dinâmica associada à ventilação normal e não é demasiado lenta para fazer a medição impraticável, particularmente quando se estuda grandes grupos. Uma vez que uma capacidade pulmonar total nominal em um / 6 rato saudável C57BL é da ordem de 1,2 ml 9, esta taxa normalmente permite que dois completo fechado PV loops para ser feito em cerca de 1,5 min. Na literatura extensas onde as curvas PV foram relatados, a pressão de enchimento pico utilizado tem sido extremamente variável, variando de tão baixo como 20 a mais de 40 cm de H 2 O. Parte dessa variabilidade pode estar relacionada à espécie, mas o objetivo principal de estabelecer o limite de pressão superior para curvas PV é inflar o pulmão a capacidade pulmonar total (CPT), ou volume pulmonar máxima. A TLC em seres humanos é definida pelo esforço voluntária máxima de um indivíduo pode fazer, mas, infelizmente, esta nunca poderá ser duplicada em qualquer modelo animal. Assim, o volume máximo em curvas PV experimentais é dissuadirminado por uma pressão máxima arbitrariamente definida pelo investigador. O objectivo é estabelecer uma pressão onde a curva PV é plana, mas, infelizmente, o membro de inflação de uma curva PV pulmão de mamífero é nunca plana. Assim, a maioria dos investigadores definir uma pressão onde a curva começa a inflação para achatar substancialmente, tipicamente de 30 cm H 2 O. No rato, no entanto, a curva PV é ainda mais complexo, com uma corcunda dupla no membro inflação, e onde esta parte da inflação é muitas vezes continua a aumentar acentuadamente a 30 cm H 2 O 10, portanto 30 não é um bom ponto final para a curva PV. Por esta razão, é utilizado 35 centímetros H2O como o limite da pressão para a curva de rato PV, que é uma pressão à qual os membros de enchimento de todas as estirpes que examinamos começam a achatar. Uma vez que a própria curva PV é muito linear, o aparecimento de um loop PV irá depender do volume a partir de onde a curva começa. Alguns ventiladores comerciais permitem que os usuários façam grandes laços PV, a partir de FRC, mas se o volume da FRC é desconhecido, então é possível interpretar tais mudanças na curva de PV com qualquer patologia, uma vez que essas modificações podem simplesmente resultar de uma alteração no volume de partida, e não alterações estruturais no pulmão. Assim, sem uma medição de volume absoluto, curvas PV são quase impossíveis de interpretar e, portanto, têm pouca utilidade. Apesar de, há várias maneiras de medir volumes pulmonares, estes são muitas vezes complicado e requer equipamento especial. Na abordagem simples descrita aqui, a curva PV começa no volume zero após um processo de desgaseificação in vivo. Em resumo, este trabalho demonstra um método simples para padronizar medição curva PV de pulmão no pulmão do rato, e define várias métricas que podem ser calculados a partir da curva que estão ligados à estrutura do pulmão. A curva PV, portanto, fornece um teste de função pulmonar, que tem aplicação direta em ser capaz de detectar mudanças estruturais fenotípicas em camundongos com commem patologias pulmonares como enfisema e fibrose.

<table><tbody><tr><td>Syringe pump</td><td>Harvard Apparatus</td><td>55-2226</td><td>Infuse/withdraw syringe pump</td></tr><tr><td>Pump 22 reversing switch</td><td>Harvard Apparatus</td><td>552217</td><td>Included with pump</td></tr><tr><td>Linear displacement transformer</td><td>Trans-Tek, Inc.</td><td>0244-0000</td><td></td></tr><tr><td>5 ml glass syringe</td><td>Becton Dickenson</td><td></td><td>Several other possible vendors</td></tr><tr><td>Digital recorder</td><td>ADInstruments</td><td>PL3504</td><td>Several other possible vendors</td></tr><tr><td>Bridge amp signal conditioner</td><td>ADInstruments</td><td>FE221</td><td></td></tr><tr><td>Gas tank, 100% oxygen</td><td>Airgas, Inc</td><td></td><td>Any supplier or hospital source will work</td></tr><tr><td>Pressure transducer: 0 - 1 psi&amp;nbsp;mV output</td><td>Omega Engineering</td><td>PX-137</td><td>Range &amp;asymp; 0 - 60 cm H&lt;sub&gt;2&lt;/sub&gt;O</td></tr></tbody></table>

measurement of the pressure-volume curve in mouse lungs

Nas últimas décadas, o ratinho tornou-se o modelo animal primária de uma variedade de doenças pulmonares. Em modelos de enfisema ou fibrose, as alterações fenotípicas essenciais são melhor avaliada por medição das alterações na elasticidade do pulmão. Para melhor compreender os mecanismos específicos subjacentes a essas patologias em camundongos, é essencial para fazer medições funcionais que podem refletir a patologia em desenvolvimento. Embora existam muitas maneiras de medir a elasticidade, o método clássico é o do pulmão pressão-volume total (PV) curva feito ao longo de toda a gama de volumes pulmonares. Essa medição foi feita no pulmão adulto de quase todas as espécies de mamíferos que remonta quase 100 anos, e essas curvas PV também desempenhou um papel importante na descoberta e compreensão da função do surfactante pulmonar em desenvolvimento pulmonar fetal. Infelizmente, tais curvas totais PV não têm sido amplamente relatado na rato, apesar do fato de que eles podem fornecer informações úteis sobre o macroscOPIC efeitos de mudanças estruturais no pulmão. Embora curvas PV parciais para avaliar apenas as variações no volume do pulmão são, por vezes relatados, sem uma medida de volume absoluto, a natureza não-linear da curva total PV faz com que essas parciais muito difícil de interpretar. No presente estudo, descrevemos uma maneira padronizada para medir a curva total PV. Estamos, então, testaram a capacidade dessas curvas para detectar mudanças na estrutura do pulmão do rato em duas patologias pulmonares comum, enfisema e fibrose. Os resultados mostraram alterações significativas em diversas variáveis ​​consistentes com as mudanças estruturais esperadas com estas patologias. Esta medição da curva de PV de pulmão em ratinhos proporciona assim um meio simples para controlar a progressão das alterações fisiopatológicas ao longo do tempo e o efeito potencial de procedimentos terapêuticos.

Embora o procedimento para as curvas PV é demonstrado no vídeo apenas para controlo de ratos saudáveis, foi examinada a capacidade da curva PV para detectar alterações funcionais e patológicos em ratinhos com duas patologias comuns diferentes, enfisema e fibrose. Os detalhes destes modelos tradicionais descrito em outros lugares 12,13. Muito brevemente, após anestesia com isoflurano a 3% foi enfisema causado por 3 ou 6 L elastase pancreática porcina instilado na traqueia e estudou 3 semanas mais tarde...

Watch this Scientific Journal Video about Measurement of the Pressure-volume Curve in Mouse Lungs at JoVE.com

Measurement of the Pressure-volume Curve in Mouse Lungs

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

Medição da curva pressão-volume no mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

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The Kinetic Theory of Gases

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18.5K visualizações.  Johns Hopkins University. Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung’s structural changes with developing pathology.

Vídeo: Measurement of the Pressure-volume Curve in Mouse Lungs

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Ventilação a pressão controlada para induzir lesão pulmonar aguda em ratos

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Medicina

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

Sistólica do ventrículo direito Medição de Pressão em combinação com colheita de amostras de tecido pulmonar e imunológica em ratos

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

Imunologia e Infecção

A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), representing the gas-exchange capacity of the lung, is a critical criterion to assess respiratory function. However, observer bias can significantly influence measured values for lung morphological parameters. The protocol that we describe here minimizes variations during measurements of two morphological parameters used for ISA calculation: internal lung volume (ILV) and mean linear intercept (MLI). Using ISA as a morphometric and functional parameter to determine the outcome of alveolar regeneration in both pneumonectomy (PNX) and prosthesis implantation mouse models, we found that the increased ISA following PNX treatment was significantly blocked by implantation of a prosthesis into the thoracic cavity1. The ability to accurately quantify ISA is not only expected to improve the reliability and reproducibility of lung function studies in injured-induced alveolar regeneration models, but also to promote mechanistic discoveries of multiple pulmonary diseases.

Internal lung surface area (ISA) is a critical criterion for assessing lung morphology and physiology in lung diseases and injury-induced alveolar regeneration. We describe here a standardized method that can minimize the measurement bias for ISA in both lung pneumonectomy and prosthesis implantation mouse models.

Um Método Padronizado para Medição da Superfície de Pulmão Interna via Neumonectomia de Mouse e Implante de Prótese

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), ...

Biologia do Desenvolvimento

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Um método simples de intubação Lung Rato

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 &#181;m in diameter3.
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 &#181;m inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 &#181;m which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 &#181;m which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2 and the response fades after 30-40 min at hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological phenomenon by which at alveolar hypoxia lung perfusion is matched to ventilation. The major vascular segment contributing to HPV is the intra-acinar artery. Here, we describe our protocol for the analysis of HPV of murine pulmonary vessels with diameters of 20-100 &mu;m.

Análise videomorfométrica da vasoconstrição pulmonar hipoxica de artérias intra-pulmonares usando fatias pulmonares cortadas por precisão de murina

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

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 ...

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

Caracterização do isolado, ventilado, Lung Mouse e Instrumentados perfundidos com fluxo pulsátil

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

Biologia

Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in na&iuml;ve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).

Avaliação da mecânica respiratória em ratos usando a técnica de oscilações forçadas

The  forced oscillation technique (FOT) is a powerful, integrative and translational  tool permitting the experimental assessment of lung function in mice ...

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung&rsquo;s structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.

Presented here is a useful protocol for lung fixation that creates a stable condition for histological evaluate of lung specimens from a mouse model of emphysema. The main advantage of this model is that it can fix many lungs with the same constant pressure without lung collapse or deflation.

Fixação pulmonar pressão constante para avaliação de enfisema em camundongos

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

Medir a função respiratória em ratos usando Unrestrained pletismografia de corpo inteiro

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...