Partitioning of nasal and pulmonary resistance changes during noninvasive plethysmography in mice

Double-chamber plethysmography is a well established noninvasive method of assessing airflow obstruction in small lab animals. It allows measurement of the specific airway resistance (sRaw), which unlike enhanced pause (Penh), is a meaningful airway mechanics parameter. Since sRaw is measured in spontaneously breathing mice, a limitation of the method is the inability to exclude nasal resistance changes. We recently showed that mice are not truly obligate nasal breathers and that after nasal occlusion, nasally breathing mice can transition to an oral mode of breathing. We now show that it is experimentally possible to algebraically separate the average nasal and pulmonary (including laryngeal) components of total airway resistance change by a series of measurements made across groups of mice breathing nasally or orally, assuming that oral resistance remains constant. Using this approach, we show that nasal resistance change comprises one-half or more of the total resistance change during methacholine challenge. Inhibition of mucin secretion from airway goblet cells attenuates pulmonary but not nasal airway hyperresponsiveness (AHR), and nasal AHR in a murine model of rhinitis may be related to edema.     

Unrestrained acoustic plethysmograph for measuring specific airway resistance in mice.

An acoustic whole body plethysmograph was developed to estimate specific airway resistance (sRaw) in unrestrained mice. The plethysmograph uses acoustic principles to measure the thoracic breathing pattern and simultaneously measures the airflow entering and/or leaving the plethysmograph. Similarly to traditional methods utilizing a double-chamber plethysmograph, these measurements were combined to estimate sRaw. To evaluate the new system, we placed six conscious A/J mice individually in a whole body plethysmograph (Buxco System) for a 2-min exposure to aerosolized methacholine chloride dissolved in saline (0, 5, 10, and 20 mg/ml), which is known to increase sRaw in mice. Three minutes after exposure, the mice were transferred to the acoustic plethysmograph for 2 min for data collection. The mean baseline value of sRaw was 0.93+/-0.10 cmH2O.s. A dose-dependent increase in sRaw was shown, with an approximate tripling of sRaw at the highest dose. These results demonstrate the ability of the system to estimate sRaw based on plethysmograph airflow and acoustic amplitude.     

Acoustic plethysmography measures breathing in unrestrained neonatal mice.

Measurement of breathing volumes in neonatal mice is of growing importance in order to characterize the influence of development and genetic modifications on respiratory control to evaluate hypotheses concerned with human infant deficits that may affect sudden infant death syndrome, for example. Current techniques require undesirable physical constraints or incur possible artifacts specific to very small animals. We have examined the utility of a recently proposed approach using an acoustic resonance procedure that does not require undue physical constraint beyond placement in the acoustic plethysmograph. We show here that this approach can be applied to baby mice 5 days after birth and that it can be accurately calibrated. In addition, this approach should be useful to study unrestrained neonatal mice under conditions where body temperature approaches environmental temperature and barometric plethysmography cannot be used.     

Restrained whole body plethysmography for measure of strain-specific and allergen-induced airway responsiveness in conscious mice.

The mouse is the most extensively studied animal species in respiratory research, yet the technologies available to assess airway function in conscious mice are not universally accepted. We hypothesized that whole body plethysmography employing noninvasive restraint (RWBP) could be used to quantify specific airway resistance (sRaw-RWBP) and airway responsiveness in conscious mice. Methacholine responses were compared using sRaw-RWBP vs. airway resistance by the forced oscillation technique (Raw-FOT) in groups of C57, A/J, and BALB/c mice. sRaw-RWBP was also compared with sRaw derived from double chamber plethysmography (sRaw-DCP) in BALB/c. Finally, airway responsiveness following allergen challenge in BALB/c was measured using RWBP. sRaw-RWBP in C57, A/J, and BALB/c mice was 0.51 +/- 0.03, 0.68 +/- 0.03, and 0.63 +/- 0.05 cm/s, respectively. sRaw derived from Raw-FOT and functional residual capacity (Raw*functional residual capacity) was 0.095 cm/s, approximately one-fifth of sRaw-RWBP in C57 mice. The intra- and interanimal coefficients of variations were similar between sRaw-RWBP (6.8 and 20.1%) and Raw-FOT (3.4 and 20.1%, respectively). The order of airway responsiveness employing sRaw-RWBP was AJ > BALBc > C57 and for Raw-FOT was AJ > BALB/c = C57. There was no difference between the airway responsiveness assessed by RWBP vs. DCP; however, baseline sRaw-RWBP was significantly lower than sRaw-DCP. Allergen challenge caused a progressive decrease in the provocative concentration of methacholine that increased sRaw to 175% postsaline values based on sRaw-RWBP. In conclusion, the technique of RWBP was rapid, reproducible, and easy to perform. Airway responsiveness measured using RWBP, DCP, and FOT was equivalent. Allergen responses could be followed longitudinally, which may provide greater insight into the pathogenesis of chronic airway disease.     

Transgenic models to study disorders of respiratory control in newborn mice

Recent studies described the in vivo respiratory phenotype of mutant newborn mice with targeted deletions of genes involved in respiratory control development. Whole-body flow barometric plethysmography is the noninvasive method of choice for studying unrestrained newborn mice. The main characteristics of the early postnatal development of respiratory control in mice are reviewed, including available data on breathing patterns and on hypoxic and hypercapnic ventilatory responses. Mice are very immature at birth, and their instable breathing is similar to that of preterm infants. Breathing pattern abnormalities with prolonged apneas occur in newborn mice that lack genes involved in the development of rhythmogenesis. Some mutant newborn mice have blunted hypoxic and hypercapnic ventilatory responses whereas others exhibit impairments in responses to hypoxia or hypercapnia. Furthermore, combined studies in mutant newborn mice and in humans have helped to provide pathogenic information on genetically determined developmental disorders of respiratory control in humans.     

Effect of somatic growth, strain, and sex on double-chamber plethysmographic respiratory function values in healthy mice.

Double-chamber plethysmography has been recognized since 1979 as a reference technique to measure pulmonary function values in guinea pigs, but it has not gained attention for use in mice. Theoretically, however, this technique combines the advantages of single-chamber plethysmography with a quantitative assessment of flow and/or volume and a calculated resistance, the interpretation of which in terms of bronchoconstriction is not disputed. Here we show that, when appropriately preconditioned, mice are able to gradually grow accustomed to the apparatus and display extremely stable nasal and thoracoabdominal flow tracings. Overall, strain, sex, and somatic growth had a significant effect on pulmonary function values. The changes in specific airway resistance (sRaw) and enhanced pause (Penh) values were never in the same direction, indicating that they measure different things. The respiratory frequency was far higher in C57BL/6 compared with BALB/c mice. Peak flows, minute volume, specific tidal and minute volumes, and sRaw were also higher, but Penh was smaller. Males breathed at a higher frequency than females, leading to a higher minute volume. Nevertheless, the specific volumes were considerably higher among females. Penh was lower in males, whereas sRaw was identical in both sexes. Changes associated with somatic growth were rapid and important between 5 and 9 wk, then slowed down between 9 and 12-13 wk and became almost imperceptible after.     

Novel whole body plethysmography system for the continuous characterization of sleep and breathing in a mouse

Sleep is associated with marked alterations in ventilatory control that lead to perturbations in respiratory timing, breathing pattern, ventilation, pharyngeal collapsibility, and sleep-related breathing disorders (SRBD). Mouse models offer powerful insight into the pathogenesis of SRBD; however, methods for obtaining the full complement of continuous, high-fidelity respiratory, electroencephalographic (EEG), and electromyographic (EMG) signals in unrestrained mice during sleep and wake have not been developed. We adapted whole body plethysmography to record EEG, EMG, and respiratory signals continuously in unrestrained, unanesthetized mice. Whole body plethysmography tidal volume and airflow signals and a novel noninvasive surrogate for respiratory effort (respiratory movement signal) were validated against simultaneously measured gold standard signals. Compared with the gold standard, we validated 1) tidal volume (correlation, R(2) = 0.87, P < 0.001; and agreement within 1%, P < 0.001); 2) inspiratory airflow (correlation, R(2) = 0.92, P < 0.001; agreement within 4%, P < 0.001); 3) expiratory airflow (correlation, R(2) = 0.83, P < 0.001); and 4) respiratory movement signal (correlation, R(2) = 0.79-0.84, P < 0.001). The expiratory airflow signal, however, demonstrated a decrease in amplitude compared with the gold standard. Integrating respiratory and EEG/EMG signals, we fully characterized sleep and breathing patterns in conscious, unrestrained mice and demonstrated inspiratory flow limitation in a New Zealand Obese mouse. Our approach will facilitate studies of SRBD mechanisms in inbred mouse strains and offer a powerful platform to investigate the effects of environmental and pharmacological exposures on breathing disturbances during sleep and wakefulness.     

Hyperoxia-induced changes in mouse lung mechanics: forced oscillations vs. barometric plethysmography.

Hyperoxia-induced lung damage was investigated via airway and respiratory tissue mechanics measurements with low-frequency forced oscillations (LFOT) and analysis of spontaneous breathing indexes by barometric whole body plethysmography (WBP). WBP was performed in the unrestrained awake mice kept in room air (n = 12) or in 100% oxygen for 24 (n = 9), 48 (n = 8), or 60 (n = 9) h, and the indexes, including enhanced pause (Penh) and peak inspiratory and expiratory flows, were determined. The mice were then anesthetized, paralyzed, and mechanically ventilated. Airway resistance, respiratory system resistance at breathing frequency, and tissue damping and elastance were identified from the LFOT impedance data by model fitting. The monotonous decrease in airway resistance during hyperoxia correlated best with the increasing peak expiratory flow. Respiratory system resistance and tissue damping and elastance were unchanged up to 48 h of exposure but were markedly elevated at 60 h, with associated decreases in peak inspiratory flow. Penh was increased at 24 h and sharply elevated at 60 h. These results indicate no adverse effect of hyperoxia on the airway mechanics in mice, whereas marked parenchymal damage develops by 60 h. The inconsistent relationships between LFOT parameters and WBP indexes suggest that the changes in the latter reflect alterations in the breathing pattern rather than in the mechanical properties. It is concluded that, in the presence of diffuse lung disease, Penh is inadequate for characterization of the mechanical status of the respiratory system.     

Unrestrained plethysmography is an unreliable measure of airway responsiveness in BALB/c and C57BL/6 mice.

There has been significant utilization of the technique described by Hamelmann et al. (Am J Respir Crit Care Med 156: 766-775, 1997) in which a parameter, enhanced pause (Penh), related to airways responsiveness is noninvasively measured by unrestrained plethysmography (UP). Investigating this technique, we sought to answer these questions: 1). How do changes in Penh compare with changes in traditional plethysmographic and lung mechanical parameters? 2). How do UP parameters perform in two different mouse strains? Awake immunized and control BALB/c (n = 16) and C57BL/6 (n = 14) mice were placed in the UP chamber and exposed to doses of aerosolized methacholine while the following parameters were measured at each concentration: inspiratory time (Ti), expiratory time (Te), total time (Ttot), Ti/Ttot, peak inspiratory pressure, peak expiratory pressure, Pause, Penh, tidal volume (Vt), Vt/Ti, Vt/Te, and Vt/Ttot. The next day, lung resistance (Rl) and compliance (Cl) were invasively measured in the same animals. For the BALB/c, the parameters with the highest magnitude of correlation coefficient vs. Rl are (in order) 1). Cl, 2). Pause and Penh, 3). parameters of breathing frequency (Te, Ttot, Ti), and 4). parameters related to Vt (inspiratory pressure, expiratory pressure). Flow parameters (Vt/Ttot, Vt/Te, Vt/Ti) and duty cycle parameters (Ti/Ttot) had insignificant correlations. This ordering is significantly different in C57BL/6 mice, in which the parameters with the largest correlations are 1). Cl, 2). parameters of breathing frequency, and 3). flow parameters. Pause, Penh, Vt, and duty cycle parameters had insignificant correlations. These data show that Penh is problematic in the sense that it is strain specific; it behaves very differently in BALB/c and C57BL/6 mice. We suggest that UP parameters largely originate as part of reflex control of breathing processes, rather than in the lung mechanics and conclude that it is inappropriate to use UP parameters in general, and Penh specifically, as substitute variables for invasive mechanical indexes such as Rl.     

A reevaluation of the validity of unrestrained plethysmography in mice.

Presently, unrestrained plethysmography is widely used to assess bronchial responsiveness in mice. An empirical quantity known as enhanced pause is derived from the plethysmographic box pressure [P(b)(t), where t is time] and assumed to be an index of bronchoconstriction. We show that P(b)(t) is determined largely by gas conditioning when normal mice breathe spontaneously inside a closed chamber in which the air is at ambient conditions. When the air in the chamber is heated and humidified to body conditions, the changes in P(b)(t) are reduced by about two-thirds. The remaining changes are thus due to gas compression and expansion within the lung and are amplified when the animals breathe through increased resistances. We show that the time integral of P(b)(t) over inspiration is accurately predicted by a term containing airway resistance, functional residual capacity, and tidal volume. We conclude that unrestrained plethysmography can be used to accurately characterize changes in airway resistance only if functional residual capacity and tidal volume are measured independently and the chamber gas is preconditioned to body temperature and humidity.     

Calibration of respiratory inductive plethysmography during quiet and active sleep in lambs

Respiratory inductive plethysmography provides a noninvasive method of measuring breathing patterns. Calibration of respiratory inductive plethysmography requires calculation of gain factors for ribcage and abdomen transducers utilizing 2 breathing patterns with different ribcage and abdomen contributions and tidal volume measured by either spirometry or integrated pneumotachography. The purpose of this study was to determine if respiratory inductive plethysmography can be calibrated to provide accurate measurements during quiet and active sleep in lambs. We used a least squares linear regression calibration technique with breaths selected from quiet sleep and active sleep to calculate gain factors in 6 tracheostomized lambs. Validation of gain factors was performed by comparing tidal volumes obtained simultaneously by respiratory inductive plethysmography and pneumotachography during quiet sleep and active sleep. Tidal volume differences between respiratory inductive plethysmography and pneumotachography on validation runs of 15 consecutive breaths each revealed 90% of validation breaths within +/- 20% during quiet sleep and 82% of validation breaths within +/- 20% during active sleep. These data provide evidence that respiratory inductive plethysmography can be calibrated to allow breathing pattern measurement during sleep.     

Pituitary adenylate cyclase-activating polypeptide maintains neonatal breathing but not metabolism during mild reductions in ambient temperature

Mild reductions in ambient temperature dramatically increase the mortality of neonatal mice deficient in pituitary adenylate cyclase-activating polypeptide (PACAP), with the majority of animals succumbing in the second postnatal week. During anesthesia-induced hypothermia, PACAP(-/-) mice at this age are also vulnerable to prolonged apneas and sudden death. From these observations, we hypothesized that before the onset of genotype-specific mortality and in the absence of anesthetic, the breathing of PACAP-deficient mice is more susceptible to mild reductions in ambient temperature than wild-type littermates. To test this hypothesis, we recorded breathing in one group of postnatal day 4 PACAP+/+, (+/-), and (-/-) neonates (using unrestrained, flow-through plethysmography) and metabolic rate in a separate group (using indirect calorimetry), both of which were exposed acutely to ambient temperatures slightly below (29 degrees C), slightly above (36 degrees C), or at thermoneutrality (32 degrees C). At 32 degrees C, the breathing frequency of PACAP(-/-) neonates was significantly less than PACAP+/+ littermates. Reducing the ambient temperature to 29 degrees C caused a significant suppression of tidal volume and ventilation in both PACAP+/- and (-/-) animals, while the tidal volume and ventilation of PACAP+/+ animals remained unchanged. Genotype had no effect on the ventilatory responses to ambient warming. At all three ambient temperatures, genotype had no influence on oxygen consumption or body temperature. These results suggest that during mild reductions in ambient temperature, PACAP is vital for the preservation of neonatal tidal volume and ventilation, but not for metabolic rate or body temperature.     

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.     

Detection of mediator-induced airway constriction by barometric plethysmography in mice

The barometric method has recently been employed to detect airway constriction in small animals. This study was designed to evaluate the barometric method to detect mediator-induced central and peripheral airway constriction in BALB/c mice. First, the central airway constrictor carbachol and the peripheral airway constrictor histamine were employed to induce airway constriction, which was detected by both the conventional body plethysmography and the barometric method in anesthetized mice. Second, bronchoconstriction induced by aerosolized carbachol or other mediators was detected with the barometric plethysmography in conscious, unrestrained mice. Carbachol inhalation caused about four-fold increase in pulmonary resistance (RL) and about two-fold increase in enhanced pause (Penh) in anesthetized mice. In contrast, in the same preparation, histamine aerosol induced a decrease in dynamic compliance (Cdyn), with no alteration in RL or Penh. In awake mice, carbachol and methacholine caused increases in Penh, frequency, and tidal volume (VT). On the other hand, histamine, histamine + bradykinin, and prostaglandin-D2 did not alter Penh but decreased VT in conscious mice. These data suggest that there was no sufficient evidence to indicate that Penh could be a good indicator of bronchoconstriction for the whole airways.     

Detection of allergen-induced airway hyperresponsiveness in isolated mouse lungs

Airway hyperresponsiveness (AHR) is a hallmark of bronchial asthma. Important features of this exaggerated response to bronchoconstrictive stimuli have mostly been investigated in vivo in intact animals or in vitro in isolated tracheal or bronchial tissues. Both approaches have important advantages but also certain limitations. Therefore, the aim of our study was to develop an ex vivo model of isolated lungs from sensitized mice for the investigation of airway responsiveness (AR). BALB/c mice were sensitized by intraperitoneal ovalbumin (Ova) and subsequently challenged by Ova inhalation. In vivo AR was measured in unrestrained animals by whole body plethysmography after stimulation with aerosolized methacholine (MCh) with determination of enhanced pause (P(enh)). Twenty-four hours after each P(enh) measurement, airway resistance was continuously registered in isolated, perfused, and ventilated lungs on stimulation with inhaled or intravascular MCh or nebulized Ova. In a subset of experiments, in vivo AR was additionally measured in orotracheally intubated, spontaneously breathing mice 24 h after P(enh) measurement, and lungs were isolated further 24 h later. Isolated lungs of allergen-sensitized and -challenged mice showed increased AR after MCh inhalation or infusion as well as after specific provocation with aerosolized allergen. AR was increased on days 2 and 5 after Ova challenge and had returned to baseline on day 9. AHR in isolated lungs after aerosolized or intravascular MCh strongly correlated with in vivo AR. Pretreatment of isolated lungs with the beta(2)-agonist fenoterol diminished AR. In conclusion, this model provides new opportunities to investigate mechanisms of AHR as well as pharmacological interventions on an intact organ level.     

Neural control of breathing: insights from genetic mouse models.

Recent studies described the in vivo ventilatory phenotype of mutant newborn mice with targeted deletions of genes involved in the organization and development of the respiratory-neuron network. Whole body flow barometric plethysmography is the noninvasive method of choice for studying unrestrained newborn mice. Breathing-pattern abnormalities with apneas occur in mutant newborn mice that lack genes involved in the development and modulation of rhythmogenesis. Studies of deficits in ventilatory responses to hypercapnia and/or hypoxia helped to identify genes involved in chemosensitivity to oxygen and carbon dioxide. Combined studies in mutant newborn mice and in humans have shed light on the pathogenesis of genetically determined respiratory-control abnormalities such as congenital central hypoventilation syndrome, Rett syndrome, and Prader-Willi syndrome. The development of mouse models has opened up the field of research into new treatments for respiratory-control disorders in humans.     

Tidal midexpiratory flow as a measure of airway hyperresponsiveness in allergic mice

A method for the noninvasive measurement of airway responsiveness was validated in allergic BALB/c mice. With head-out body plethysmography and the decrease in tidal midexpiratory flow (EF(50)) as an indicator of airway obstruction, responses to inhaled methacholine (MCh) and the allergen ovalbumin were measured in conscious mice. Allergen-sensitized and -challenged mice developed airway hyperresponsiveness as measured by EF(50) to aerosolized MCh compared with that in control animals. This response was associated with increased allergen-specific IgE and IgG1 production, increased levels of interleukin-4 and interleukin-5 in bronchoalveolar lavage fluid and eosinophilic lung inflammation. Ovalbumin aerosol challenge elicited no acute bronchoconstriction but resulted in a significant decline in EF(50) baseline values 24 h after challenge in allergic mice. The decline in EF(50) to MCh challenge correlated closely with simultaneous decreases in pulmonary conductance and dynamic compliance. The decrease in EF(50) was partly inhibited by pretreatment with the inhaled beta(2)-agonist salbutamol. We conclude that measurement of EF(50) to inhaled bronchoconstrictors by head-out body plethysmography is a valid measure of airway hyperresponsiveness in mice.     

Effects of covert subject actions on percent body fat by air-displacement plethsymography

Air-displacement plethysmography (ADP) is used for estimation of body composition, however, some individuals, such as athletes in weight classification sports, may use covert methods during ADP testing to alter their apparent percent body fat. The purpose of this study was to examine the effect of covert subject actions on percent body fat measured by ADP. Subjects underwent body composition analysis in the Bod Pod following the standard procedure using the manufacturer's guidelines. The subjects then underwent 8 more measurements while performing the following intentional manipulations: 4 breathing patterns altering lung volume, foot movement to disrupt air, hand cupping to trap air, and heat and cold exposure before entering the chamber. Increasing and decreasing lung volume during thoracic volume measurement and during body density measurement altered the percent body fat assessment (p < 0.001). High lung volume during thoracic gas measures overestimated fat by 3.7 ± 2.1 percentage points. Lowered lung volume during body volume measures overestimated body fat by an additional 2.2 ± 2.1 percentage points. The heat and cold exposure, tapping, and cupping treatments provided similar estimates of percent body fat when compared with the standard condition. These results demonstrate the subjects were able to covertly change their estimated ADP body composition value by altering breathing when compared with the standard condition. We recommend that sports conditioning coaches, athletic trainers, and technicians administering ADP should be aware of the potential effects of these covert actions. The individual responsible for administering ADP should remain vigilant during testing to detect deliberate altered breathing patterns by athletes in an effort to gain a competitive advantage by manipulating their body composition assessment.     

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.     

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.