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).     

Measuring lung function in mice: the phenotyping uncertainty principle.

Measuring lung function in mice is essential for establishing the relevance of murine models to human lung disease. However, making such measurements presents particular technical challenges due to the small size of the animal, particularly with regard to the measurement of respiratory flows. In this review, we examine the various methods currently available for assessment of lung function in mice and contrast them in terms of a concept we call the phenotyping uncertainty principle; each method can be considered to lie somewhere along a continuum on which noninvasiveness must be traded off against experimental control and measurement precision. Unrestrained plethysmography in conscious mice represents the extreme of noninvasiveness and is highly convenient but provides respiratory measures that are so tenuously linked to respiratory mechanics that they cannot be considered as meaningful indicators of lung function. At the other extreme, the measurement of input impedance in anesthetized, paralyzed, tracheostomized mice is precise and specific but requires that an animal be studied under conditions far from natural. In between these two extremes lie methods that sacrifice some precision for a reduction in the level of invasiveness, a promising example being the measurement of transfer impedance in conscious, restrained mice. No method is optimal in all regards; therefore, the appropriate technique to use depends on the application.     

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.     

Effect of prolonged bed rest on lung volume in normal individuals

Pulmonary function was assessed in supine subjects before, during, and after three separate bed-rest studies of 11 and 12 days duration. Forced vital capacity (FVC) increased during bed rest in each subject. Total lung capacity (TLC) was measured by helium dilution in one bed-rest study and increased in each subject, while residual volume and functional residual capacity of the respiratory system did not change. No change in FVC was found in an ambulatory control group using identical measurement techniques. Maintaining base-line plasma volume during one bed rest by the use of exogenous estrogen did not prevent an increase in FVC, and decreasing plasma volume with diuretics in ambulatory subjects to the same degree as seen in the bed rests did not cause an increase in FVC. We conclude that prolonged bed rest results in a small significant increase in TLC and that this change is not dependent on alterations in plasma volume.     

Wistar大鼠肺功能参考值范围测定

目的建立Wistar大鼠肺功能各项指标的参考值。方法用创体描法小动物肺功能检测仪检测大鼠肺功能各项指标,根据肺功能指标检测结果,通过统计分析,确定其参考值范围。结果 Ri（吸气阻力）为1.81（0.94~4.10）cm H2O/（mL·s）,Re（呼气阻力）为1.83（0.71~3.57）cm H2O/（mL·s）,Cl（肺顺应性）为0.15（0.05~0.29）mL/cm H2O,MVV（最大通气量）为144.65（77.28~256.20）mL/min,FVC（用力肺活量）为8.49（5.82~12.70）mL,Fev0.2（第0.2秒用力呼气容积）为5.72（3.62~7.01）mL,Fev0.2/FVC（第0.2秒用力呼出容积占用力肺活量百分比）为8.12（39.14~85.28）%,FEF（25~75）%（用力中期呼气流速）为34.11（28.25~46.87）mL/min。PEF（用力最大呼气流速）为38.28（30.75~50.25）mL/min。结论 Wistar大鼠肺功能指标的参考值范围可为临床和科研工作以及未来制定国家标准和规范提供参考依据。     

Chronic rejection pathology after orthotopic lung transplantation in mice: the development of a murine BOS model and its drawbacks

Almost all animal models for chronic rejection (CR) after lung transplantation (LTx) fail to resemble the human situation. It was our attempt to develop a representative model of CR in mice. Orthotopic LTx was performed in allografts receiving daily immunosuppression with steroids and cyclosporine. Controls included isografts and mice only undergoing thoracotomy (SHAM). Allografts were sacrificed 2, 4, 6, 8, 10 or 12 weeks after LTx. Pulmonary function was measured repeatedly in the 12w allografts, isografts and SHAM mice. Histologically, all allografts demonstrated acute rejection (AR) around the blood vessels and airways two weeks after LTx. This decreased to 50-75% up to 10 weeks and was absent after 12 weeks. Obliterative bronchiolitis (OB) lesions were observed in 25-50% of the mice from 4-12 weeks. Isografts and lungs of SHAM mice were normal after 12 weeks. Pulmonary function measurements showed a decline in FEV(0.1), TLC and compliance in the allografts postoperatively (2 weeks) with a slow recovery over time. After this initial decline, lung function of allografts increased more than in isografts and SHAM mice indicating that pulmonary function measurement is not a good tool to diagnose CR in a mouse. We conclude that a true model for CR, with clear OB lesions in about one third of the animals, but without a decline in lung function, is possible. This model is an important step forward in the development of an ideal model for CR which will open new perspectives in unraveling CR pathogenesis and exploring new treatment options.     

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.     

Effects of prematurity and intrauterine growth on respiratory health and lung function in childhood.

OBJECTIVE--To determine whether birth weight and gestational age are associated with respiratory illness and lung function in children aged 5-11 years. DESIGN--Cross sectional analysis of parent reported birth weight, gestational age, and respiratory symptoms; parental smoking and social conditions; forced vital capacity (FVC), forced expiratory volume in one second (FEV1), forced expiratory rates between 25% and 75% and 75% and 85% (FEF25-75 and FEF75-85), and height. SETTING--Primary schools in England and Scotland in 1990. SUBJECTS--5573 children aged 5-11 (63.3% of eligible children) had respiratory symptoms analysed and 2036 children (67.1% of eligible children) had lung function measured. MAIN OUTCOME MEASURES--Symptoms of asthma, bronchitis, occasional and frequent wheeze, cough first thing in the morning, and cough at any other time and lung function. RESULTS--Birth weight adjusted for gestational age was significantly associated with all lung function measurements, except FEF25-75. The association remained for FVC (b = 0.475, 95% confidence interval 0.181 to 0.769) and FEV1 (b = 0.502, 0.204 to 0.800) after adjustment for gestational age, parental smoking, and social factors. FEF75-85 was the only lung function related to gestational age. Respiratory symptoms, especially wheeze most days (adjusted odds ratio 0.9, 0.84 to 0.97) were significantly associated with prematurity. Every extra week of gestation reduced the risk of severe wheeze by about 10%. CONCLUSIONS--Lung function is affected mainly by intrauterine environment while respiratory illness, especially wheezing, in childhood is related to prematurity.     

Microscopic wire guide-based orotracheal mouse intubation: description, evaluation and comparison with transillumination

Airway access is needed for a number of experimental animal models, and the majority of animal research is based on mouse models. Anatomical conditions in mice are small, and the narrow glottic opening allows intubation only with a subtle technique. We therefore developed a microscopic endotracheal intubation method with a wire guide technique in mice anaesthetized with halothane in oxygen. The mouse is hung perpendicularly with its incisors on a thread fixed on a vertical plate. The tongue is placed with a pair of forceps between the left hand's thumb and forefinger and slightly pulled, while the neck and thorax are positioned using the third and fourth fingers. By doing so, the neck can be slightly stretched, which allows optimal visualization of the larynx and the vocal cords. To ensure a safe intubation, a fine wire guide is placed under vision between the vocal cords and advanced about 5 mm into the trachea. An intravenous 22G x 1 in. plastic or Teflon catheter is guided over this wire. In a series of 41 mice, between 21 and 38 g, the success rate for the first intubation attempt was >95%. Certainty of the judgement procedure was 100% and success rate was higher using the described method when compared with a transillumination method in a further series. The technique is safe, less invasive than tracheostomy and suitable for controlled ventilation and pulmonary substance application.     

Application of carbon monoxide diffusing capacity in the mouse lung

In the past decade the mouse has become the primary animal model of a variety of lung diseases. To assess various mechanisms underlying such pathologies, it is essential to make functional measurements that can reflect the developing pathology. In this regard, the diffusing capacity for carbon monoxide is a variable that directly reflects structural changes in the lung. Although measurement of single-breath diffusing capacity of the lung for carbon monoxide (DL(CO)) has also been previously reported in mice by a number of investigators, a number of technical issues have precluded routine and widespread use of this metric in mouse models. In the present report, we describe a means to quickly and simply measure a dimensionless variable closely related to the DL(CO) in mice, termed a diffusion factor for carbon monoxide (DF(CO)). The DF(CO) procedure involves a 9-s lung inflation with tracer gases in an anesthetized mouse, followed by a 1-min gas analysis time. We have tested the approach with two common models of lung pathology, elastase-induced emphysema and bleomycin-induced fibrosis. Results show a significant 15% reduction in DF(CO) in emphysema, and a 41% reduction in the fibrosis model. Repeat measurements within a mouse were found to be highly reproducible. This pulmonary function test can thus be used to detect structural changes with these pathological models. The method can also be used to measure changes in pulmonary blood volume, since the uptake of CO is highly dependent on this variable in addition to the gas exchange surface area.     

High blood pressure,antihypertensive medication and lung function in a general adult population

Background

Several studies showed that blood pressure and lung function are associated. Additionally, a potential effect of antihypertensive medication, especially beta-blockers, on lung function has been discussed. However, side effects of beta-blockers have been investigated mainly in patients with already reduced lung function. Thus, aim of this analysis is to determine whether hypertension and antihypertensive medication have an adverse effect on lung function in a general adult population.

Methods

Within the population-based KORA F4 study 1319 adults aged 40-65 years performed lung function tests and blood pressure measurements. Additionally, information on anthropometric measurements, medical history and use of antihypertensive medication was available. Multivariable regression models were applied to study the association between blood pressure, antihypertensive medication and lung function.

Results

High blood pressure as well as antihypertensive medication were associated with lower forced expiratory volume in one second (p = 0.02 respectively p = 0.05; R²: 0.65) and forced vital capacity values (p = 0.01 respectively p = 0.05, R²: 0.73). Furthermore, a detailed analysis of antihypertensive medication pointed out that only the use of beta-blockers was associated with reduced lung function, whereas other antihypertensive medication had no effect on lung function. The adverse effect of beta-blockers was significant for forced vital capacity (p = 0.04; R²: 0.65), while the association with forced expiratory volume in one second showed a trend toward significance (p = 0.07; R²: 0.73). In the same model high blood pressure was associated with reduced forced vital capacity (p = 0.01) and forced expiratory volume in one second (p = 0.03) values, too.

Conclusion

Our analysis indicates that both high blood pressure and the use of beta-blockers, but not the use of other antihypertensive medication, are associated with reduced lung function in a general adult population.     

Pulmonary and ventilatory responses to pregnancy, immersion, and exercise

To examine the effects of pregnancy, immersion, and exercise during immersion on pulmonary function and ventilation, 12 women were studied at 15, 25, and 35 wk of pregnancy and 8-10 wk postpartum. Pulmonary function and ventilation were measured under three experimental conditions: after 20 min of rest on land (LR), after 20 min of rest during immersion to the level of the xiphoid (IR), and after 20 min of exercise during immersion at 60% of predicted maximal capacity (IE). Forced vital capacity remained relatively constant, except for a decrease at 15 wk, for the duration of pregnancy. Expiratory reserve volume decreased with a change in the pregnancy status and with the duration of pregnancy. However, the forced vital capacity was maintained by an increase in the inspiratory capacity during pregnancy. Forced expiratory volume for 1 s, expressed as percent of forced vital capacity, did not differ significantly between conditions or as a result of pregnancy. Forced vital capacity was lower during the IR trial compared with LR and IE trials. The decreased forced vital capacity of the IR trials was mediated by a decrease in the expiratory reserve volume. Whereas the inspiratory capacity increased during IR and IE compared with LR, the increase was not large enough to offset the decrease in the expiratory reserve volume. Resting immersion resulted in a significant decrease in maximal voluntary ventilation as did pregnancy. Pregnancy resulted in significant increases in minute ventilation (VE), which were related to increases in the O2 consumption.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Superiority of PC-SOD to other anti-COPD drugs for elastase-induced emphysema and alteration in lung mechanics and respiratory function in mice

Bronchodilators (such as ipratropium bromide), steroids (such as fluticasone propionate), and newly developed anti-inflammatory drugs (such as roflumilast) are used for patients with chronic obstructive pulmonary disease (COPD). We recently reported that lecithinized superoxide dismutase (PC-SOD) confers a protective effect in mouse models of COPD. We here examined the therapeutic effect of the combined administration of PC-SOD with ipratropium bromide on pulmonary emphysema and compared the effect of PC-SOD to other types of drugs. The severity of emphysema in mice was assessed by various criteria. Lung mechanics (elastance) and respiratory function (ratio of forced expiratory volume in the first 0.05 s to forced vital capacity) were assessed. Administration of PC-SOD by inhalation suppressed elastase-induced pulmonary emphysema, alteration of lung mechanics, and respiratory dysfunction. The concomitant intratracheal administration of ipratropium bromide did not alter the ameliorating effects of PC-SOD. Administration of ipratropium bromide, fluticasone propionate, or roflumilast alone did not suppress the elastase-induced increase in the pulmonary level of superoxide anion, pulmonary inflammatory response, pulmonary emphysema, alteration of lung mechanics, or respiratory dysfunction as effectively as did PC-SOD. PC-SOD, but not the other drugs, showed a therapeutic effect even when the drug was administered after the development of emphysema. PC-SOD also suppressed the cigarette smoke-induced pulmonary inflammatory response and increase in airway resistance. Based on these results, we consider that the inhalation of PC-SOD would be therapeutically beneficial for COPD.     

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.     

Short-term hypoxic exposure at rest and during exercise reduces lung water in healthy humans.

Hypoxia and hypoxic exercise increase pulmonary arterial pressure, cause pulmonary capillary recruitment, and may influence the ability of the lungs to regulate fluid. To examine the influence of hypoxia, alone and combined with exercise, on lung fluid balance, we studied 25 healthy subjects after 17-h exposure to 12.5% inspired oxygen (barometric pressure = 732 mmHg) and sequentially after exercise to exhaustion on a cycle ergometer with 12.5% inspired oxygen. We also studied subjects after a rapid saline infusion (30 ml/kg over 15 min) to demonstrate the sensitivity of our techniques to detect changes in lung water. Pulmonary capillary blood volume (Vc) and alveolar-capillary conductance (D(M)) were determined by measuring the diffusing capacity of the lungs for carbon monoxide and nitric oxide. Lung tissue volume and density were assessed using computed tomography. Lung water was estimated by subtracting measures of Vc from computed tomography lung tissue volume. Pulmonary function [forced vital capacity (FVC), forced expiratory volume after 1 s (FEV(1)), and forced expiratory flow at 50% of vital capacity (FEF(50))] was also assessed. Saline infusion caused an increase in Vc (42%), tissue volume (9%), and lung water (11%), and a decrease in D(M) (11%) and pulmonary function (FVC = -12 +/- 9%, FEV(1) = -17 +/- 10%, FEF(50) = -20 +/- 13%). Hypoxia and hypoxic exercise resulted in increases in Vc (43 +/- 19 and 51 +/- 16%), D(M) (7 +/- 4 and 19 +/- 6%), and pulmonary function (FVC = 9 +/- 6 and 4 +/- 3%, FEV(1) = 5 +/- 2 and 4 +/- 3%, FEF(50) = 4 +/- 2 and 12 +/- 5%) and decreases in lung density and lung water (-84 +/- 24 and -103 +/- 20 ml vs. baseline). These data suggest that 17 h of hypoxic exposure at rest or with exercise resulted in a decrease in lung water in healthy humans.     

Trachea and lung dimensions in nonsmoking twins: morphological and functional studies

To compare genetic and environmental factors that determine lung function and dimensions, chest radiographs and pulmonary function were measured in 17 pairs of nonsmoking twin adolescent boys (12 monozygotic pairs and 5 dizygotic pairs). Genetic factors dominated in tracheal width and lung dimensions (height, width, and apicofissural and fissurodiaphragmatic distances) at residual volume. Genetic factors also affected forced vital capacity, functional residual capacity, forced expiratory volume in 1 s, maximum expiratory flow at 25% vital capacity, and maximum flow at 50% vital capacity-to-forced vital capacity ratio. Peak expiratory flow correlated with tracheal width at residual volume. Age correlated with lung dimensions (width and depth) but not with tracheal width. These results indicate that genetic factors determine the dimensions and function of central airways, peripheral airways, and lung parenchyma in adolescent males. The effects of genetic factors on some functional measurements (airway resistance, closing volume-to-vital capacity ratio, and phase III in single-breath N2 washout) may be masked because of poor reproducibility of the tests.     

Respiratory function in lambs after in utero treatment of lung hypoplasia by tracheal obstruction.

Tracheal obstruction (TO) stimulates growth of hypoplastic lungs in the fetus, but there is little knowledge of subsequent postnatal respiratory function. We have determined the effectiveness of TO in fetal sheep with existing lung hypoplasia in restoring postnatal respiratory function. Lung hypoplasia was induced by lung liquid drainage from 112 days of gestation to term ( approximately 148 days). We used an untreated group (ULH), a treated group (TLH) in which the trachea was obstructed for 10 days, and a control group. ULH lambs died within 4 h of birth. TLH lambs were hypoxic for the first week and were hypercapic at 2 days. Pulmonary diffusing capacity, gas volumes, and respiratory compliances were not different between control and TLH lambs. Minute ventilation was not different between the two groups; however, tidal volumes were lower and respiratory frequencies were higher in TLH lambs than in controls for 2 wk after birth. We conclude that 10 days of TO in the presence of initial lung hypoplasia prevents death at birth and returns most aspects of pulmonary function to normal by 1-2 wk after birth.