This work was supported by NIH HL-10342

NOTA: Todos os protocolos de animais foram aprovados pelo Comitê Universidade Animal Care and Use a Johns Hopkins. 1. Preparação de animais <ol><li> Prepare ratos 6 C57BL / 6 de controle para a medição Dfco, anestesiando-los com cetamina e xilazina, conforme descrito no passo 2.3 abaixo. </li><li> Preparar todos os outros ratinhos com os diferentes patologias pulmonares mostrados na Tabela 1, utilizando o mesmo procedimento que para os controlos. Detalhes específicos necessários para estabelecer cada um destes modelos são encontrados nas referências relevantes. Os ratos de controlo e os dos outros grupos patológicos são todos 6-12 semanas de idade. </li></ol> 2. Medição do Fator de Difusão de monóxido de carbono (Dfco) <ol><li> Configure o módulo cromatógrafo a gás fornecido com o equipamento para medir os picos de nitrogênio, oxigênio, néon, e monóxido de carbono. Para este uso aplicação apenas os dados de néon e CO. NOTA: Este instrumento utiliza um s molecularescoluna IEVE com hélio como gás de transporte, com 12,00 mm filme, 320,00 mm ID e 10 m de comprimento. A coluna de cromatografia tem um volume de 0,8 ml, mas utilizou-se 2 ml para assegurar adequada compensação da tubagem de ligação com a amostra. </li><li> No início de cada dia experimental, antes de se efectuarem as medições das amostras a partir dos ratinhos, tomar uma amostra de 2 ml directamente a partir de um saco de mistura de gás que contém cerca de 0,5% Ne, 0,5% de CO, e equilibrar ar, e usar esta amostra para calibrar o cromatógrafo a gás. </li><li> Anestesiar ratos com cetamina (90 mg / kg) e xilazina (15 mg / kg), e confirmar a anestesia pela ausência do reflexo do movimento. Aplicar pomada veterinária sobre os olhos para evitar o ressecamento. Tracheostomize os ratos com uma cânula de agulha stub (18 g em adultos ou 20 G em muito jovem ratos). NOTA: O Dfco é completada em menos de 10 minutos após a anestesia e antes de qualquer ventilação mecânica ou outros procedimentos. </li><li> Em ratinhos superiores a 6 semanas de idade, usar uma seringa de 3 ml to retirar 0,8 ml de gás a partir do saco de mistura de gás. Ligar a seringa para a cânula traqueal e rapidamente inflar o pulmão. Usando um metrônomo, conte 9 seg, e então rapidamente retirar os 0,8 ml (no ar expirado). </li><li> Diluir esta retirados 0,8 ml ar exalado para 2 ml com ar ambiente, deixe-a descansar por pelo menos 15 segundos. Em seguida, injetar toda a amostra no cromatógrafo a gás para análise. </li><li> Ao analisar esta primeira amostra Dfco, inflar o pulmão do rato com um segundo de 0,8 ml do saco de mistura de gases, e, em seguida, processar esta amostra idêntica à primeira amostra. Calcular a média das duas medidas Dfco. NOTA: Para medições em ratos jovens como de 2 semanas de idade, usar um volume de 0,4 ml, 0,8 ml é desde um volume demasiado importante para fazer medições em pulmões de ratos muito jovens. É preferível utilizar o volume de 0,8 ml para os ratinhos mais velhos de 6 semanas, e que, se o volume de 0,4 ml é necessária para alguns ratinhos, que deve ser utilizado de forma consistente para todos os ratinhos no grupo a ser estudados. </li><li> Calcule Dfcocomo 1 - (CO 9 / CO c) / (Ne 9 / Ne c), onde C e 9 subscritos referem-se as concentrações dos gases de calibração injetados e os gases removidos após um 9 seg tempo apneia, respectivamente. </li><li> Analisar e comparar as diferenças com um one-way ANOVA e avaliar o nível de significância com correção de Tukey para comparações múltiplas em todos os ratos de coorte. Considere p &lt;0,05 como significativo. NOTA: Todos os ratos usados ​​aqui foram parte de estudos experimentais envolvendo várias medições posteriores de ventilação pulmonar, mecânica, lavagem pulmonar, ou histologia, que não são aqui relatados. Além disso, uma vez que o método é o mesmo em todos os modelos experimentais como foi feito acima nos ratinhos de controlo, somente os resultados de vários modelos patológicos são apresentados. Informação adicional sobre estes modelos é apresentada na tabela suplementar. </li><li> Eutanásia os animais por anestesia profundasobredosagem tic seguido por deslocamento cervical ou decapitação. Sempre que necessário, remover as células e / ou tecidos do pulmão dos ratos mortos para mais biológico ou processamento histológico e análise. </li></ol>

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Não há conflitos de interesse, e nada a divulgar.

No presente trabalho, definimos uma nova métrica para quantificar a capacidade trocas gasosas do pulmão do rato. Esta métrica é análoga à capacidade de difusão, uma medida clínica comum que mede a função primária do pulmão, isto é, a sua capacidade para a troca de gás. A capacidade de difusão é a única medida funcional do pulmão que podem ser facilmente e rapidamente feito em ambos os ratos e os seres humanos. Para a detecção da doença de pulmão em camundongos, um objectivo importante é quantifica...

O mouse é agora o animal primário usado para modelar uma variedade de doenças pulmonares. Para estudar os mecanismos que underly tais patologias, são necessários métodos fenotípicos que podem quantificar a que as alterações patológicas. Embora existam muitos estudos com ratos, onde ventilação mecânica são medidos, essas medidas são geralmente relacionado com as avaliações padrão da função pulmonar normalmente feitos em seres humanos. Isso é lamentável, uma vez que a capacidade de executar as medidas equivalentes em camundongos e seres humanos podem facilitar a tradução dos resultados em modelos de ratos para doenças humanas. Uma das medidas mais comuns e facilmente feitas em seres humanos é a capacidade de difusão do monóxido de carbono (DLCO) 1,2, mas esta medida só foi raramente feito em modelos de ratos. Nesses estudos, onde foi relatado 3-7, não houve estudos de acompanhamento, em parte porque os procedimentos são frequentemente onerosos ou pode reqùire equipamentos complexos. Outra abordagem é a utilização de um método de CO reinalatório em um sistema de estado estacionário, o que tem a vantagem de ser capaz de medir a difusão de CO em ratos conscientes. No entanto, este método é muito complicado, e os resultados podem variar de acordo com o nível de ventilação do rato bem como de O 2 e CO 2 concentrações de 8,9. Essas dificuldades parecem ter impedido o uso rotineiro de capacidade de detectar patologias pulmonares em ratos difusão, apesar de suas várias vantagens. Para contornar os problemas com a medição da capacidade de difusão em camundongos, os detalhes de um meio simples para medi-lo em camundongos têm sido relatados recentemente 10. O procedimento elimina o difícil problema de amostragem de gás alveolar não contaminada por amostragem rapidamente um volume igual a todo o gás inspirado. Este procedimento resulta em uma medida muito reprodutível, o denominado factor de difusão para o monóxido de carbono (Dfco), que é sensível a uma série de pathologic alterações no fenótipo pulmonar. O Dfco é portanto calculada como 1 - (CO 9 / CO c) / (Ne 9 / Ne c), onde a e c 9 subscritos referem-se a concentrações de gases de calibração injectados e os gases eliminados após um tempo de 9 s apneia, respectivamente. Dfco é uma variável adimensional, o qual varia entre 0 e 1, com 1 reflectindo a absorção completa de todo o CO, e 0 reflectindo qualquer absorção de CO. Nesta apresentação, mostram como fazer a medição da capacidade de difusão, e como ela pode ser usada para documentar as alterações em quase todos os modelos existentes doença do pulmão do rato, incluindo enfisema, fibrose, lesão pulmonar aguda, e infecções virais e fúngicas.

<table><tbody><tr><td>Gas Chromatograph</td><td>Inficon</td><td>Micro GC Model 3000A</td><td>Agilent makes a comparable model</td></tr><tr><td>18 G Luer stub needle</td><td>Becton Dickenson</td><td></td><td>Several other possible vendors</td></tr><tr><td>3 ml plastic syringe</td><td>Becton Dickenson</td><td></td><td>Several other possible vendors</td></tr><tr><td>Polypropylene gas sample bags</td><td>SKC</td><td>1 or 2 L capacity works well</td><td>Other gas tight bags will work well</td></tr><tr><td>Gas tank, 0.3% Ne, 0.3% CO, balance air; (size ME)</td><td>Airgas, Inc</td><td>Z04 NI785ME3012</td><td>This is the standard mixture used for DLCO in humans</td></tr><tr><td>25 TCID50/mouse of influenza virus A/PR8 diluted in phosphate buffered saline.</td><td></td><td></td><td></td></tr><tr><td>Porcine pancreatic elastase</td><td>Elastin Products, Owensville, MO</td><td></td><td>5.4 U</td></tr><tr><td>Bleomycin</td><td>APP Pharmaceuticals, Schaumburg, IL</td><td></td><td>0.25 U</td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; LPS</td><td>Sigma L2880</td><td></td><td>3 &amp;mu;g/g body weight; O55:B5</td></tr><tr><td>&lt;i&gt;Aspergillus fumigatus &lt;/i&gt; (isolate Af293) conidia were collected from mature colonies grown on potato dextrose agar.</td><td></td><td></td><td></td></tr></tbody></table>

phenotyping mouse pulmonary function in vivo with the lung diffusing capacity

O mouse é agora o animal primário usado para modelar uma variedade de doenças pulmonares. Para estudar os mecanismos que estão por trás dessas patologias, são necessários métodos fenotípicos que podem quantificar as alterações patológicas. Além disso, para proporcionar relevância translacional para os modelos de ratinho, tais medições devem ser testes que podem ser facilmente feitos em ambos os seres humanos e ratinhos. Infelizmente, no presente literatura algumas medições fenotípicas da função do pulmão têm aplicação directa em seres humanos. Uma exceção é a capacidade de difusão do monóxido de carbono, que é uma medida que é feito rotineiramente em seres humanos. No presente relatório, nós descrevemos um meio para medir rapidamente e simplesmente esta capacidade de difusão em camundongos. O procedimento envolve a inflação pulmonar breve com gases marcadores num rato anestesiado, seguido por um 1 min de tempo de análise de gás. Nós testamos a capacidade deste método para detectar várias patologias pulmonares, incluindo enfisema, fibrose, lesão pulmonar aguda e gripe einfecções pulmonares fúngicas, bem como a maturação pulmonar monitoramento em filhotes jovens. Os resultados mostram uma diminuição significativa em todas as patologias pulmonares, bem como um aumento da capacidade de difusão com a maturação pulmonar. Esta medida da capacidade de difusão pulmonar, portanto, fornece um teste de função pulmonar, que tem ampla aplicação com a sua capacidade de detectar mudanças estruturais fenotípicas com a maioria dos modelos de pulmão patológicas existentes.

A Figura 1 mostra as medições Dfco dos ratos adultos em grupos A, B, C, D, E e F. Houve uma redução significante com ambas as infecções por Aspergillus e da gripe, bem como diminuições significativas na fibrótica, enfisema, e aguda modelos de lesão do pulmão. A Figura 2 mostra as alterações do Grupo G de desenvolvimento em Dfco ao longo do tempo como a idade ratinhos 2-6 semanas. Houve um ligeiro mas significativo aumento no desenvolvimento pulmonar ao longo deste curso de ...

Watch this Scientific Journal Video about Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity at JoVE.com

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

Função Pulmonar Fenotipagem Rato<em&gt; In Vivo</em&gt; Com a capacidade pulmonar Diffusing

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods ...

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Research

JoVE Journal

Biology

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

10.6K visualizações.  Johns Hopkins University Bloomberg School of Public Health. We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

Vídeo: Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

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 ...

Medicina

Measurement of the Pressure-volume Curve in 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 phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

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 ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

Teste de tolerância à glicose intraperitoneal, medição da função pulmonar e a fixação do pulmão para estudar o impacto da obesidade e metabolismo prejudicado em resultados pulmonares

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

Imunologia e Infecção

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

Geração de um modelo de doença pulmonar obstrutiva crônica em ratos pela exposição repetida de ozônio

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

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 ...

Biologia

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

Avaliação da função respiratória em ratos conscientes por pletismografia dobro-câmara

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

Hyperpolarized 129Xe Lung MRI and Spectroscopy in Mechanically Ventilated Mice

Hyperpolarized (HP) xenon-129 (129Xe) is an inhaled magnetic resonance imaging (MRI) contrast agent with unique spectral and physical properties that can be exploited to quantify pulmonary physiology, including ventilation, restricted diffusion (alveolar-airspace size), and gas exchange. In humans, it has been used to evaluate disease severity and progression in a variety of pulmonary disorders and is approved for clinical use in the United States and United Kingdom. Beyond its clinical applications, the ability of 129Xe MRI to noninvasively assess pulmonary pathophysiology and provide spatially resolved information is valuable for preclinical research. Among animal models, mice are the most widely used due to the accessibility of genetically modified disease models. Here, 129Xe MRI is promising as a minimally invasive, radiation-free, and sensitive technique to longitudinally monitor lung disease progression and therapy response (e.g., in drug discovery). This technique can extend to preclinical applications by incorporating an MRI-triggered, free-breathing apparatus or mechanical ventilator to deliver gas. Here, we describe the steps and provide checklists to ensure robust data collection and analysis, including creating a thermally polarized xenon gas phantom for quality control, optimizing polarization, animal handling (sedation, intubation, ventilation, and care for mice), and protocols for ventilation, restricted diffusion, and gas exchange data. While preclinical 129Xe MRI can be applied in various animal models (e.g., rats, pigs, sheep), this protocol focuses on mice due to the challenges posed by their small anatomy, which are balanced by their affordability and the availability of many disease models.

Hyperpolarized xenon MRI can quantify regional lung microstructure (air-space dimensions) and physiology (ventilation and gas exchange) in translational research and clinical care. Although challenging, it can provide comparable pulmonary insights in preclinical studies. This protocol describes the infrastructure and procedures needed to perform routine xenon lung MRI in mice.

Ressonância magnética e espectroscopia de pulmão hiperpolarizada 129Xe em camundongos ventilados mecanicamente

Hyperpolarized (HP) xenon-129 (129Xe) is an inhaled magnetic resonance imaging (MRI) contrast agent with unique spectral and physical properties that can ...

Bioengenharia

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 ...

In vivo Evaluation of Mucociliary Clearance in Mice

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a metachronal, synchronal fashion, these multiple, motile, actin-based organelles generate a cephalad fluid flow clearing the respiratory tract of inhaled pollutants and pathogens. With increasing environmental pollution, novel viral pathogens and emerging multi-drug resistant bacteria, cilia generated mucociliary clearance (MCC) is essential for maintaining lung health. MCC is also depressed in multiple congenital disorders like primary ciliary dyskinesia, cystic fibrosis as well as acquired disorders like chronic obstructive pulmonary disease. All these disorders have established, in some case multiple, mouse models. In this publication, we detail a method using a small amount of radioactivity and dual-modality SPECT/CT imaging to accurately and reproducibly measure MCC in mice in vivo. The method allows for recovery of mice after imaging, making serial measurements possible, and testing potential therapeutics longitudinally over time. The data in wild-type mice demonstrates the reproducibility of the MCC measurement as long as adequate attention to detail is paid, and the protocol strictly adhered to.

In this publication, we describe protocols for assessing airway mucociliary clearance (MCC) in mice in vivo utilizing dual-modality radionuclide imaging. This protocol is designed for a single photon emission computed tomography (SPECT) and computed tomography (CT) acquisition protocol using mouse whole body (MWB) collimators in a dual SPECT/CT system.

Avaliação in vivo de liberação mucociliaria em camundongos

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a ...

Evaluation of Lung Function in an Ex vivo Murine Model

Respiratory mechanics are a key area of study in defining and treating lung pathologies by assessing functional lung capacity. Lung mechanics can be evaluated through various lung maneuvers that involve different oscillatory waveforms. When applied to the lungs, these maneuvers measure multiple variables, such as pressure, volume, and flow, based on the response to the waveforms. These signals are then computed and analyzed to determine parameters such as hysterisivity, resistance, compliance, tissue damping, and tissue elastance, providing a detailed assessment of overall lung function. The analysis of respiratory mechanics is particularly important in evaluating donor lungs for lung transplantation. The present protocol is the first of its kind, offering a comprehensive and reproducible stepwise method for assessing respiratory mechanics using an ex vivo murine model. It includes details on the selected animal model, lung recovery, storage and preservation, and experimentation using a forced oscillation technique-based system. Additionally, it outlines data analysis, clinical significance, and the applications of the forced oscillation technique in studying an ex vivo model.

The present protocol describes a stepwise method for analyzing the respiratory mechanics of an ex vivo murine model using the forced oscillation technique (FOT).

Avaliação da Função Pulmonar em um Modelo Murino Ex vivo

Respiratory mechanics are a key area of study in defining and treating lung pathologies by assessing functional lung capacity. Lung mechanics can be ...

Função Pulmonar Fenotipagem Rato

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