Alterations in lung mechanics in decorin-deficient mice

Decorin, a small leucine-rich proteoglycan with a widespread tissue distribution, is required for the normal fibrillogenesis of collagen in most tissues. Because collagen is important in determining the elastic behavior of the lung, we hypothesized that lung tissue mechanics would be altered in a mutant mouse in which the single decorin gene was abrogated by targeted deletion (Dcn-/-). Complex impedance of the respiratory system was measured in C57Bl/6 mice (Dcn-/- and Dcn+/+) using a small animal ventilator that delivers a volume signal with multiple frequencies to the trachea. A constant-phase model was fit to calculate airway resistance (R(aw)), tissue damping, and tissue elastance. Compliance of the respiratory system (C(rs)) was measured from a pressure volume curve during stepwise deflations. Lungs were excised, and parenchymal tissue strips were mounted in an organ bath for in vitro measurement of tissue impedance and quasistatic length-stress curves. In addition, pulmonary tissue was examined by immunohistochemistry and immunoblotting. In vivo, in the Dcn-/- mice, R(aw) was decreased and C(rs) was increased. Similarly, in vitro, length-stress curves showed increased compliance of the strips in the Dcn-/- mice. These alterations in lung tissue mechanical behavior in Dcn-/- mice support a critical role for decorin in the formation of the lung collagen network.     

Repeated invasive lung function measurements in intubated mice: an approach for longitudinal lung research

Invasive lung function measurements are useful tools to describe respiratory disease models in mice but only result in one time-point measurements because of tracheostomy. We explored if intubation may overcome the need for tracheostomy thereby allowing invasive lung function monitoring of individual mice over time. Repeated invasive lung function measurements with Scireq(?) - FlexiVent or Buxco(?) - Forced Pulmonary Maneuvers(?) were performed three times in BALB/c mice with intervals of 10 days. Each lung function assessment following intubation was compared with a similar measurement in age-matched tracheostomized mice, the golden standard in lung function measurements. Tracheostomy and intubation gave similar results for resistance, elastance and compliance of the whole respiratory system as assessed by Flexivent. Likewise, Forced Pulmonary Maneuvers used to measure lung volumes such as total lung capacity, functional residual capacity, forced expiratory volume in 0.1 s and forced vital capacity, resulted in identical outcomes for both airway approaches. No interaction was found between the procedures for any of the pulmonary function variables. The observed changes over time were rather related to animal growth than to repetitive intubation. Eighty percent of the animals survived three consecutive intubations, which were hampered by transient breathing difficulties, weight loss and neutrophilic bronchoalveolar lavage immediately postextubation. Repetitive invasive lung function measurements by intubation are feasible and reproducible in healthy mice and results are comparable to the standard method. This may open new perspectives for longitudinal research in animal models of respiratory diseases.     

Phenotyping Mouse Pulmonary Function In Vivo 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 are needed that can quantify the pathologic changes. Furthermore, to provide translational relevance to the mouse models, such measurements should be tests that can easily be done in both humans and mice. Unfortunately, in the present literature few phenotypic measurements of lung function have direct application to humans. One exception is the diffusing capacity for carbon monoxide, which is a measurement that is routinely done in humans. In the present report, we describe a means to quickly and simply measure this diffusing capacity in mice. The procedure involves brief lung inflation with tracer gases in an anesthetized mouse, followed by a 1 min gas analysis time. We have tested the ability of this method to detect several lung pathologies, including emphysema, fibrosis, acute lung injury, and influenza and fungal lung infections, as well as monitoring lung maturation in young pups. Results show significant decreases in all the lung pathologies, as well as an increase in the diffusing capacity with lung maturation. This measurement of lung diffusing capacity thus provides a pulmonary function test that has broad application with its ability to detect phenotypic structural changes with most of the existing pathologic lung models.     

The allergic mouse model of asthma: normal smooth muscle in an abnormal lung?

Mice with allergically inflamed airways are widely used as animal models of asthma, but their relevance for human asthma is not understood. We, therefore, examined the time course of changes in respiratory input impedance during induced bronchoconstriction in BALB/c mice sensitized and challenged with ovalbumin. Our results indicate that bronchoconstriction in mice is accompanied by complete closure of substantial regions of the lung and that closure increases markedly when the lungs are allergically inflamed. With the aid of an anatomically accurate computational model of the mouse lung, we show that the hyperresponsiveness of mice with allergically inflamed airways can be explained entirely by a thickening of the airway mucosa and an increased propensity of the airways to close, without the involvement of any increase in the degree of airway smooth muscle shortening. This has implications for the pathophysiology of asthma and suggests that at least some types of asthma may benefit from therapies aimed at manipulating surface tension at the air-liquid interface in the lungs.     

Measuring the lung function in the mouse: the challenge of size

Measurement of the effects of drugs, mediators and infectious agents on various models of lung disease, as well as assessment of lung function in the intact mouse has the potential for significantly advancing our knowledge of lung disease. However, the small size of the mouse presents significant challenges for the assessment of lung function. Because of compromises made between precision and noninvasiveness, data obtained may have an uncertain bearing on the mechanical response of the lung. Nevertheless, considerable recent progress has been made in developing valid and useful measures of mouse lung function. These advances, resulting in our current ability to measure sophisticated indices of lung function in laboratory animals, are likely to lead to important insights into the mechanisms of lung disease.     

Psychological stress impairs hepatic blood flow via central CRF receptors in mice

Some previous works have further expanded the 'Brain-Gut axis', that is a bi-directional interaction between the gut and brain function, into a relationship of the brain with the liver. However, all the details of such brain-liver relationship were not fully understood because appropriate animal models had not been established yet. Here we developed a novel animal model, in which hepatic blood flow of conscious mice could be measured in real-time. In addition, using this model, we also demonstrated that exposure to psychological stress considerably reduced hepatic blood flow via central CRF receptors. Thus, this new model is considered to be a useful and promising tool for elucidating the precise effects of emotional factors on liver function.     

Invasive versus noninvasive measurement of allergic and cholinergic airway responsiveness in mice

Background

This study seeks to compare the ability of repeatable invasive and noninvasive lung function methods to assess allergen-specific and cholinergic airway responsiveness (AR) in intact, spontaneously breathing BALB/c mice.

Methods

Using noninvasive head-out body plethysmography and the decrease in tidal midexpiratory flow (EF₅₀), we determined early AR (EAR) to inhaled Aspergillus fumigatus antigens in conscious mice. These measurements were paralleled by invasive determination of pulmonary conductance (GL), dynamic compliance (Cdyn) and EF₅₀ in another group of anesthetized, orotracheally intubated mice.

Results

With both methods, allergic mice, sensitized and boosted with A. fumigatus, elicited allergen-specific EAR to A. fumigatus (p < 0.05 versus controls). Dose-response studies to aerosolized methacholine (MCh) were performed in the same animals 48 h later, showing that allergic mice relative to controls were distinctly more responsive (p < 0.05) and revealed acute airway inflammation as evidenced from increased eosinophils and lymphocytes in bronchoalveolar lavage.

Conclusion

We conclude that invasive and noninvasive pulmonary function tests are capable of detecting both allergen-specific and cholinergic AR in intact, allergic mice. The invasive determination of GL and Cdyn is superior in sensitivity, whereas the noninvasive EF₅₀ method is particularly appropriate for quick and repeatable screening of respiratory function in large numbers of conscious mice.     

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy,Intrabronchial Inoculation and In Vivo Sampling Techniques

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as models of pulmonary disease often resemble the situation of the human lung much better than mice do. Working with large animals also offers the opportunity to sample the same animal repeatedly over a certain course of time, which allows long-term studies without sacrificing the animals.The aim was to establish in vivo sampling methods for the use in a bovine model of a respiratory Chlamydia psittaci infection. Sampling should be performed at various time points in each animal during the study, and the samples should be suitable to study the host response, as well as the pathogen under experimental conditions.Bronchoscopy is a valuable diagnostic tool in human and veterinary medicine. It is a safe and minimally invasive procedure. This article describes the intrabronchial inoculation of calves as well as sampling methods for the lower respiratory tract. Videoendoscopic, intrabronchial inoculation leads to very consistent clinical and pathological findings in all inoculated animals and is, therefore, well-suited for use in models of infectious lung disease. The sampling methods described are bronchoalveolar lavage, bronchial brushing and transbronchial lung biopsy. All of these are valuable diagnostic tools in human medicine and could be adapted for experimental purposes to calves aged 6-8 weeks. The samples obtained were suitable for both pathogen detection and characterization of the severity of lung inflammation in the host.     

Methodology for the measurement of mucociliary function in the mouse by scintigraphy.

The objective of the study was to develop a scintigraphic method for measurement of airway mucociliary clearance in small laboratory rodents such as the mouse. Previous investigations have characterized the secretory cell types present in the mouse airway, but analysis of the mucus transport system has been limited to in vitro examination of tissue explants or invasive in vivo measures of a single airway, the trachea. Three methods were used to deposit insoluble, radioisotopic colloidal particles: oropharyngeal aspiration, intratracheal instillation, and nose-only aerosol inhalation. The initial distribution of particles within the lower respiratory tract was visualized by gamma-camera, and clearance of particles was followed intermittently over 6 h and at the conclusion, 24 h postdelivery. Subsets of mice underwent lavage for evidence of tissue inflammation, and others were restudied for reproducibility of the methods. The aspiration and instillation methods of delivery led to greater distributions of deposited activity within the lungs, i.e., approximately 60--80% of the total respiratory tract radioactivity, whereas the nose-only aerosol technique attained a distribution of 32% to the lungs. However, the aerosol technique maximized the fraction of particles that cleared the airway over a 24-h period, i.e, deposited onto airway epithelial surfaces and cleared by mucociliary function such that lung retention at 24 h averaged 57% for delivery by aerosol inhalation and > or =80% for the aspiration or intratracheal instillation techniques. Particle delivery methods did not cause lung inflammation/injury with use of inflammatory cells and chemoattractant cytokines as criteria. Scintigraphy can discern particle deposition and clearance from the lower respiratory tract in the mouse, is noninvasive and reproducible, and includes the capability for restudy and lung lavage when time course or chronic treatments are being considered.     

In vivo characterization of lung morphology and function in anesthetized free-breathing mice using micro-computed tomography.

Lung morphology and function in human subjects can be monitored with computed tomography (CT). Because many human respiratory diseases are routinely modeled in rodents, a means of monitoring the changes in the structure and function of the rodent lung is desired. High-resolution images of the rodent lung can be attained with specialized micro-CT equipment, which provides a means of monitoring rodent models of lung disease noninvasively with a clinically relevant method. Previous studies have shown respiratory-gated images of intubated and respirated mice. Although the image quality and resolution are sufficient in these studies to make quantitative measurements, these measurements of lung structure will depend on the settings of the ventilator and not on the respiratory mechanics of the individual animals. In addition, intubation and ventilation can have unnatural effects on the respiratory dynamics of the animal, because the airway pressure, tidal volume, and respiratory rate are selected by the operator. In these experiments, important information about the symptoms of the respiratory disease being studied may be missed because the respiration is forced to conform to the ventilator settings. In this study, we implement a method of respiratory-gated micro-CT for use with anesthetized free-breathing rodents. From the micro-CT images, quantitative analysis of the structure of the lungs of healthy unconscious mice was performed to obtain airway diameters, lung and airway volumes, and CT densities at end expiration and during inspiration. Because the animals were free breathing, we were able to calculate tidal volume (0.09 +/- 0.03 ml) and functional residual capacity (0.16 +/- 0.03 ml).     

Variation in senescent-dependent lung changes in inbred mouse strains.

Previous studies from our laboratories showed lung development differences between inbred strains of mice. In the present study, the C57BL/6J (B6) and DBA/2J (D2) strains were examined for senescent-dependent differences with respect to the lung structure and function. Specifically, we hypothesize that senescent changes in lung vary between strains due to identifiable gene expression differences. Quasi-static pressure-volume curves and respiratory impedance measurements were performed on 2- and 20-mo-old B6 and D2 mice. Lung volume at 30 cm H(2)O (V(30)) pressure was significantly (P < 0.01) increased with age in both strains, but the increase was proportionally greater in D2 (68%) than in B6 (40%) mice. In addition, decreased elastic recoil pressure at 50% of V(30) and a reduction in airway resistance as a function of positive end-expiratory pressure were observed in 20-mo-old D2 mice but not in B6 mice. Morphometric analysis of lung parenchyma showed significant decreases in elastic fiber content with age in both strains, but the collagen content was significantly (P < 0.01) increased with age in D2 but not B6 mice at 20 mo. Furthermore, using quantitative RT-PCR methods, gene expression differences between strains suggested that D2 mice significantly (P < 0.05) downregulated the expressions of elastin (Eln) and procollagen I, III, and VI (Col1a1, Col3a1, and Col6a3) in lung tissue at 20 mo of age. These age-dependent changes were accompanied by an increased gene expression in matrix metalloproteinase 9 (Mmp9) in D2 and an increase in tissue inhibitor of matrix metalloproteinase (Timp1 and Timp4) in B6 mice. In conclusion, the results from the present study demonstrate that lung mechanics of both strains show significant age-dependent changes. However, changes in D2 mice are accelerated relative to B6 mice. Moreover, gene expression differences appear to be involved in the strain-specific changes of lung mechanic properties.     

Transcutaneous measurement of renal function in conscious mice

Determination of glomerular filtration rate (GFR) in conscious mice is cumbersome for the experimenter and stressful for the animals. Here we report on a simple new technique allowing the transcutaneous measurement of GFR in conscious mice. This approach extends our previously developed technique for rats to mice. The technique relies on a miniaturized device equipped with an internal memory that permits the transcutaneous measurement of the elimination kinetics of the fluorescent renal marker FITC-sinistrin. This device is described and validated compared with FITC-sinistrin plasma clearance in healthy, unilaterally nephrectomized and pcy mice. In summary, we describe a technique allowing the measurement of renal function in freely moving mice independent of blood or urine sampling as well as of laboratory assays.     

Contribution of large airway to the input impedance of the respiratory system

To evaluate the contribution of the large airway to total respiratory impedance, we develop a one-dimensional model of pressure and flow in these airways by coupling conservation of mass and momentum equations with the geometric information obtained by the acoustic reflection technique. We use this model to calculate the impedance of the respiratory system distal to the carina from impedance data estimated at the airway opening by the forced oscillation technique. Simulations show that the real part of the impedance distal to the carina is uniformly decreased from the impedance at the airway opening, indicating a resistive loss, while the imaginary part is increased as a function of frequency. We estimate parameter values for a six-parameter two-compartment lung model and for a three-parameter reduction of this model before and after the application of the upper airway data to the impedance spectrum. Although compliance terms seem to be minimally affected by the manipulation of the data, resistance and inertance terms are influenced in a fashion that suggests that the resistive contribution of the upper airway to total respiratory impedance is significant. Furthermore it appears that the elastic nature of the walls of the upper airway also impact on estimates of total respiratory impedance at the airway opening.     

Estimation of alveolar pressure during forced oscillation of the respiratory system.

A method for obtaining a continuous estimate of alveolar pressure (PAlv) during periodic flow is described; it was developed to improve the precision of measurements of airway and respiratory tissue impedance using the improved resolution of relatively high-frequency (approximately 5 Hz) singlas. The respiratory system was modulated with a piston pump, and lung volume and the volume change due to compression and expansion of alveolar gas were measured plethysmorgraphically; these signals and an analog divider were used to obtain a continuous solution of Boyle's law during flow. The plethysmorgraph was of the "flow" type; with it volume changes at frequencies up to 10 Hz and with rates of change up to 6 l/s were measured without amplitude or phase distortion. The method permits control of frequency and flow amplitude during PAlv measurement and calibration of PAlv in the absence of an active chest wall. However, it is technically complex.     

Evaluation Before Thoracotomy

When a thoracotomy is being considered, the physician must first determine whether the lesion is potentially resectable. However, an equally important decision is whether the patient can tolerate pulmonary resection if there are other serious underlying medical problems. In any patient with signs or symptoms of pulmonary disease, a spirogram, arterial blood gas study and electrocardiogram should be done as part of the routine preoperative evaluation. The detection of abnormal pulmonary function should prompt preoperative institution of respiratory care to optimize maximally a patient''s cardiorespiratory status before operation. If any of several indicators of a high risk of postoperative cardiopulmonary problems are found, split pulmonary function studies should be done to determine whether the lung remaining after resection will have adequate ventilation and perfusion. Xenon radiospirometry is preferable to bronchospirometry and right heart catheterization because of its noninvasiveness. Resection should not be undertaken in the presence of a predicted postoperative forced expiratory volume in one second (FEV₁) less than 0.8 liter, an arterial carbon dioxide partial pressure (PaCO₂) greater than 45 mm of mercury or Xenon scans which show poor ventilation/perfusion (˙V/˙Q) matchup in what would be the remaining lung after resection. A case is reported which shows the value of this approach to preoperative evaluation in determining the risk of postoperative problems.     

Reliability of Transcutaneous Measurement of Renal Function in Various Strains of Conscious Mice

Measuring renal function in laboratory animals using blood and/or urine sampling is not only labor-intensive but puts also a strain on the animal. Several approaches for fluorescence based transcutaneous measurement of the glomerular filtration rate (GFR) in laboratory animals have been developed. They allow the measurement of GFR based on the elimination kinetics of fluorescent exogenous markers. None of the studies dealt with the reproducibility of the measurements in the same animals. Therefore, the reproducibility of a transcutaneous GFR assessment method was investigated using the fluorescent renal marker FITC-Sinistrin in conscious mice in the present study. We performed two transcutaneous GFR measurements within three days in five groups of mice (Balb/c, C57BL/6, SV129, NMRI at 3–4 months of age, and a group of 24 months old C57BL/6). Data were evaluated regarding day-to-day reproducibility as well as intra- and inter-strain variability of GFR and the impact of age on these parameters. No significant differences between the two subsequent GFR measurements were detected. Fastest elimination for FITC-Sinistrin was detected in Balb/c with significant differences to C57BL/6 and SV129 mice. GFR decreased significantly with age in C57BL/6 mice. Evaluation of GFR in cohorts of young and old C57BL/6 mice from the same supplier showed high consistency of GFR values between groups. Our study shows that the investigated technique is a highly reproducible and reliable method for repeated GFR measurements in conscious mice. This gentle method is easily used even in old mice and can be used to monitor the age-related decline in GFR.     

Emergent behavior of regional heterogeneity in the lung and its effects on respiratory impedance

The ability to maintain adequate gas exchange depends on the relatively homogeneous distribution of inhaled gas throughout the lung. Structural alterations associated with many respiratory diseases may significantly depress this function during tidal breathing. These alterations frequently occur in a heterogeneous manner due to complex, emergent interactions among the many constitutive elements of the airways and parenchyma, resulting in unique signature changes in the mechanical impedance spectrum of the lungs and total respiratory system as measured by forced oscillations techniques (FOT). When such impedance spectra are characterized by appropriate inverse models, one may obtain functional insight into derangements in global respiratory mechanics. In this review, we provide an overview of the impact of structural heterogeneity with respect to dynamic lung function. Recent studies linking functional impedance measurements to the structural heterogeneity observed in acute lung injury, asthma, and chronic obstructive pulmonary disease are highlighted, as well as current approaches for the modeling and interpretation of impedance. Finally, we discuss the potential diagnostic role of FOT in the context of therapeutic interventions.     

Anesthetic inhibition in ischemic and nonischemic murine heart: comparison with conscious echocardiographic approach

It is well known that the level of anesthesia obtained by intraperitoneal injection is variable and may alter cardiac function. In this study, we compared the effects of different anesthetics on cardiac function with the conscious state using high-resolution two-dimensional echocardiography in nonischemic and ischemic mice. Eighty-four mice were tested before and after surgery with ligation of the coronary artery. All 84 mice were studied in the conscious state and under high-dose intraperitoneal anesthesia. Twenty-two of 84 mice were studied under low-dose intraperitoneal anesthesia. Another 22 mice were also studied under gas anesthesia and spontaneous breathing. Experiments in the conscious state were performed by two investigators before the administration of anesthesia: one investigator held the animal and the transducer and the other operated the ultrasound equipment. Left ventricular systolic function was measured, and measurements obtained after surgery were compared with infarcted areas assessed by histological staining. Results showed that both high- and low-dose intraperitoneal anesthesia significantly reduced heart rates and left ventricular contractility in both pre- and postsurgical mice as opposed to conscious mice (P < 0.01). There were significantly higher correlation coefficients between mean fractional area change (FAC) and infarcted area in conscious state compared with high-dose intraperitoneal anesthesia (P < 0.05). The correlation coefficient between FAC and infarcted area during gas anesthesia was also significantly higher compared with high-dose intraperitoneal anesthesia (P < 0.05). In conclusion, conscious experiments or the use of gas anesthesia is preferred for echocardiographic assessment of cardiac function in mice because intraperitoneal injection significantly induces a significant reduction in heart rate and left ventricular systolic function.     

Airway resistance due to alveolar gas compression measured by barometric plethysmography in mice.

We developed a method for measuring airway resistance (R(aw)) in mice that does not require a measurement of airway flow. An analysis of R(aw) induced by alveolar gas compression showed the following relationship for an animal breathing spontaneously in a closed box: R(aw) = A(bt)V(b)/[V(t) (V(e) + 0.5V(t))]. Here A(bt) is the area under the box pressure-time curve during inspiration or expiration, V(b) is box volume, V(t) is tidal volume, and V(e) is functional residual capacity (FRC). In anesthetized and conscious unrestrained mice, from experiments with both room temperature box air and body temperature humidified box air, the contributions of gas compression to the box pressure amplitude were 15 and 31% of those due to the temperature-humidity difference between box and alveolar gas. We corrected the measured A(bt) and V(t) for temperature-humidity and gas compression effects, respectively, using a sinusoidal analysis. In anesthetized mice, R(aw) averaged 4.3 cmH(2)O.ml(-1).s, fourfold greater than pulmonary resistance measured by conventional methods. In conscious mice with an assumed FRC equal to that measured in the anesthetized mice, the corrected R(aw) at room temperature averaged 1.9 cmH(2)O.ml(-1).s. In both conscious mice and anesthetized mice, exposure to aerosolized methacholine with room temperature box air significantly increased R(aw) by around eightfold. Here we assumed that in the conscious mice both V(t) and FRC remained constant. In both conscious and anesthetized mice, body temperature humidified box air reduced the methacholine-induced increase in R(aw) observed at room temperature. The method using the increase in A(bt) with bronchoconstriction provides a conservative estimate for the increase in R(aw) in conscious mice.     

Respiratory system dynamical mechanical properties: modeling in time and frequency domain

The mechanical properties of the respiratory system are important determinants of its function and can be severely compromised in disease. The assessment of respiratory system mechanical properties is thus essential in the management of some disorders as well as in the evaluation of respiratory system adaptations in response to an acute or chronic process. Most often, lungs and chest wall are treated as a linear dynamic system that can be expressed with differential equations, allowing determination of the system’s parameters, which will reflect the mechanical properties. However, different models that encompass nonlinear characteristics and also multicompartments have been used in several approaches and most specifically in mechanically ventilated patients with acute lung injury. Additionally, the input impedance over a range of frequencies can be assessed with a convenient excitation method allowing the identification of the mechanical characteristics of the central and peripheral airways as well as lung periphery impedance. With the evolution of computational power, the airway pressure and flow can be recorded and stored for hours, and hence continuous monitoring of the respiratory system mechanical properties is already available in some mechanical ventilators. This review aims to describe some of the most frequently used models for the assessment of the respiratory system mechanical properties in both time and frequency domain.