Phenotyping Mouse Pulmonary Function <em>In Vivo</em> with the Lung Diffusing Capacity

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 ...

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 ...

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer ...

Johns Hopkins University Bloomberg School of Public Health

Johns Hopkins University

A weak estrogenicity of puerarin on reproductive organs was addressed in female rats. In short-term treatment, immature ovariectomized rats were injected with 0.7 mg/kg BW/day of puerarin, for 14 days. Puerarin did not increase uterus weights, endometrium and myometrium areas, and the percent of cornified cells (%Co), but it increased the number of uterine glands. In long-term treatment, mature rats were injected with 7.0mg/kg BW/day of puerarin for 140 days. Puerarin did not increase uterus weights, endometrium and myometrium areas, and the number of uterine glands, but a significant increase in the %Co was observed from day 98 onwards.

Puerarin exhibits weak estrogenic activity in female rats.

Asthma is a chronic airway disorder characterized by recurrent attacks of breathlessness and wheezing, affecting 300 million people around the world (available at: www.who.int). To date, genetic factors associated with asthma susceptibility have been unable to explain the full etiology of asthma. Recent studies have demonstrated that the epigenetic disruption of gene expression plays an equally important role in the development of asthma through interaction with our environment. We sensitized 6-week-old C57BL/6J mice with house-dust-mite (HDM) extracts intraperitoneally followed by 5 weeks of exposure to HDM challenges (three times a week) intratracheally. HDM-exposed mice showed an increase in airway hyper-responsiveness (AHR) and inflammation together with structural remodeling of the airways. We applied methylated DNA immunoprecipitation-next generation sequencing (MeDIP-seq) for profiling of DNA methylation changes in the lungs in response to HDM. We observed about 20 million reads by a single-run of massive parallel sequencing. We performed bioinformatics and pathway analysis on the raw sequencing data to identify differentially methylated candidate genes in HDM-exposed mice. Specifically, we have revealed that the transforming growth factor beta signaling pathway is epigenetically modulated by chronic exposure to HDM. Here, we demonstrated that a specific allergen may play a role in AHR through an epigenetic mechanism by disrupting the expression of genes in lungs that might be involved in airway inflammation and remodeling. Our findings provide new insights into the potential mechanisms by which environmental allergens induce allergic asthma and such insights may assist in the development of novel preventive and therapeutic options for this debilitative disease.

Alterations of the lung methylome in allergic airway hyper-responsiveness.

Chronic idiopathic pulmonary fibrosis (IPF) is a progressive, fatal, and untreatable disease with unclear etiology. There are few models of this chronic pathology, and although delivery of bleomycin to induce acute lung injury is the most common animal model of pulmonary fibrosis, there is considerable uncertainty about whether this acute injury resolves in those animals that survive. In this report, we have systematically followed groups of mice for up to 6 months following a single insult of bleomycin. We assessed changes in lung function and pathology over this time course, with measurements of the diffusion capacity for carbon monoxide, lung mechanics, quantitative stereology, and collagen. Our results show that, while there is some repair over this extended time course, the injury in the lung never fully resolves. This persistent degree of fibrosis may have similarities to many features of human IPF. Thus, these chronic fibrotic changes in mouse lungs could be a useful model to evaluate potential therapeutic interventions to accelerate repair and possible treat this debilitating disease.

A mouse model of chronic idiopathic pulmonary fibrosis.

In the official joint policy document of the American Thoracic Society and European Respiratory Society (Hsia et al., Am J Respir Crit Care Med 181:394-418), the need for proper stereologic assessment of lungs was emphasized. In this document it was emphasized that for the quantitative analysis of lung histologic sections, one of the most robust and reliable methods is point and intercept counting (Knudsen et al., J Appl Physiol 108:412-421). One of the practical aspects of this method is how many points or intercepts are needed. The answer to this question has been considered from a theoretical perspective, and it depends on the relative magnitudes of the methodological and biologic variabilities. Although it is generally accepted that in a normal lung, one needs only 100-200 points to sufficiently lower the methodological variability, given the increased variability often seen in experimental emphysematous lung injury, the requisite number of points of intercepts has not been evaluated. In this study, we examined this question by focusing on some of the relevant sampling levels in mice with extensive elastase-induced emphysema. Using fixed samples of tissue blocks, we varied the number of sampling points or intercepts from about 25 to 1,000 in control and emphysematous lungs. Our results show that, at the sampling levels investigated, even with the increased heterogeneity in the lung tissue damage caused by elastase, the number of sampling points needed to detect changes is similar to what is needed for control mice. Anat Rec, 2014. © 2014 Wiley Periodicals, Inc.

Effect of Point Sampling Density in Quantifying Mouse Lung Emphysema.

Telomere syndromes have their most common manifestation in lung disease that is recognized as idiopathic pulmonary fibrosis and emphysema. In both conditions, there is loss of alveolar integrity, but the underlying mechanisms are not known. We tested the capacity of alveolar epithelial and stromal cells from mice with short telomeres to support alveolar organoid colony formation and found that type 2 alveolar epithelial cells (AEC2s), the stem cell-containing population, were limiting. When telomere dysfunction was induced in adult AEC2s by conditional deletion of the shelterin component telomeric repeat-binding factor 2, cells survived but remained dormant and showed all the hallmarks of cellular senescence. Telomere dysfunction in AEC2s triggered an immune response, and this was associated with AEC2-derived up-regulation of cytokine signaling pathways that are known to provoke inflammation in the lung. Mice uniformly died after challenge with bleomycin, underscoring an essential role for telomere function in AEC2s for alveolar repair. Our data show that alveoloar progenitor senescence is sufficient to recapitulate the regenerative defects, inflammatory responses, and susceptibility to injury that are characteristic of telomere-mediated lung disease. They suggest alveolar stem cell failure is a driver of telomere-mediated lung disease and that efforts to reverse it may be clinically beneficial.

Telomere dysfunction causes alveolar stem cell failure.

Emphysema, one of the major components of COPD, is characterized by the progressive and irreversible loss of alveolar lung tissue. Even though more than 80% of COPD cases are associated with cigarette smoking, only a relatively small proportion of smokers develop emphysema, suggesting a potential role for genetic factors in determining individual susceptibility to emphysema. Although strain-dependent effects have been shown in animal models of emphysema, the molecular basis underlying this intrinsic susceptibility is not fully understood. In this present study, we investigated emphysema development using the elastase-induced experimental emphysema model in two commonly used mouse strains, C57BL/6J and BALB/cJ. The results demonstrate that mice with different genetic backgrounds show disparate susceptibility to the development of emphysema. BALB/cJ mice were found to be much more sensitive than C57BL/6J to elastase insults in both a dose-dependent and time-dependent manner, as measured by significantly higher mortality, greater body weight loss, greater decline in lung function, and a greater loss of alveolar tissue. The more susceptible BALB/cJ strain also showed the persistence of inflammatory cells in the lung, especially macrophages and lymphocytes. A comparative gene expression analysis following elastase-induced injury showed BALB/cJ mice had elevated levels of il17A mRNA and a number of classically (M1)- and alternatively (M2)- activated macrophages genes, whereas the C57BL/6J mice demonstrated augmented levels of IFN-γ. These findings suggest a possible role for these cellular and molecular mediators in modulating the severity emphysema, and highlight the possibility that they might contribute to the heterogeneity observed in clinical emphysema outcomes.

Nathachit Limjunyawong

Department of Environmental Health Sciences

Johns Hopkins University Bloomberg School of Public Health

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

Measurement of the Pressure-volume Curve in Mouse Lungs

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

Nathachit Limjunyawong

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Environmental Health Sciences

Johns Hopkins University Bloomberg School of Public Health

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

Measurement of the Pressure-volume Curve in Mouse Lungs

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

Department of Environmental Health Sciences