For detailed information, see
Reinhard et al., 2002
Following anesthesia (see animal documentation), the mice were intubated according to standard procedures (Brown et al. 1999) by inserting a 28 G i.v.-catheter (Sims Portex) 10 mm deep into the trachea. The animals were attached to the ventilator with the following settings:
Measurements were performed in duplicate except where noted, and the means were calculated and are available in the submitted data sets. Only the expiratory reserve volume [ERV] was measured once because it requires a full expiration which causes stress to the animal and induces atelectasis.
Briefly, measurements were performed using a computer-controlled piston-type servo ventilator. The ventilator provides for positive pressure ventilation of intubated mice and for defined respiratory maneuvers for lung function tests. Respiratory flow signals were given from movements of the respirator piston. Concentrations of the respiratory and the test gases, He and C18O, were measured by a magnetic sector field mass spectrometer. A miniaturized pressure transducer enables the measurement of airway opening pressure (Pao). A second pressure transducer, located at the end of a thin-walled, water-filled tube connected to an esophageal cannula allows monitoring of the esophageal pressure (Poe). The flow signal, Pao, Poe and gas concentration signals were continuously recorded on a multi- channel recorder. Additionally, during the lung function measurements all signals of interest were digitized and recorded on a PC.
Lung volumesInspiratory reserve capacity [IC] is the volume slowly inspired over 10 seconds from functional residual capacity [FRC] to a tracheal pressure of + 25 cm H2O. To account for differences in lung size between animals or strains, the duration of inspiration rather than the inspiratory flow rate was standardized during this and all other test maneuvers.
Expiratory reserve volume [ERV] is the volume slowly expired over 10 seconds from functional residual capacity [FRC] to a tracheal pressure of ~10 cm H2O.
Functional residual capacity [FRC] was determined by the helium dilution technique where a rebreathing volume of 80% IC labeled with 1% He in 21% O2, balance N2, was applied at a rate of 50 per minute for 15 cycles. The inspiratory and the mixed helium concentrations were determined by mass spectrometry and used for calculation of FRC.
The evaluation of inspiratory reserve capacity, expiratory reserve volume, and functional residual capacity allowed the calculation of:
vital capacity [VC] = IC + ERV
residual volume [RV] = FRC-ERV
Lung volumes related to body weight were calculated to account for differences in body size between mice; body weight was used in the calculation as a simple measure of body size.
ComplianceStatic compliance of the respiratory system [CRS] was determined from the linear portion of the pressure-volume curve obtained during a 6-second exhalation from TLC to almost RV. Static compliance of the lung [CL] was derived from the transpulmonary pressure-volume curve. Static compliance of the thorax [CTh] was determined accordingly.
Dynamic compliances of the respiratory system [C_dyn] were determined from the Pao-pressure differences at the points of flow reversal at breathing rates of 50, 90, and 130 breaths/min. During these measurements the tidal volume was set to 40% of IC for two subsequent breaths.
ResistanceRespiratory system resistance [R] was derived from the recordings of flow and Pao-pressure during the dynamic compliance maneuver at 130 breaths/min. The resistance was calculated from the change in airway pressure and flow at isovolumetric points (mid-inspiratory and mid-expiratory volume). Respiratory system resistance was corrected for the resistance of the cannula.
Helium expirogramSee Figure 1
Intrapulmonary gas mixing and series dead space volume were obtained from single-breath washin measurements. A test-gas containing breath (1% He in air) was applied over 3 seconds from relaxed expiratory level to TLC and back to slightly below FRC over 7 seconds without a breathhold. The alveolar slope of helium [slope] (change of partial pressure per unit of expired volume) was obtained by least squares fit of the gas tracing between 50% and 80% of expired volume (phase III) and normalized to the difference between inspired and mixed-expired partial pressures [n_slope].
Series dead space volumes were obtained from single-breath
washin measurements according to the conventional Fowler
method [VDF] and the Bohr method [VDB]. Due to the
experimental setup, dead space volume includes the volume of
the tracheal cannula (16 µl) and excludes the upper airways.
Pulmonary diffusing capacityDiffusing capacity for carbon monoxide [D_CO] was obtained from measurements of the rate of uptake of the indicator gas (C18O) and its transfer gradient, which is the partial pressure difference for C18O between the alveoli and the pulmonary capillary red cells. The single breathholding method as recommended by the European Respiratory Society adopted to the present experimental conditions was used (Cotes et al., 1993). The inspiration of test gas started from the relaxed expiratory level to TLC over a period of 3 seconds followed by a breathholding time of 3 seconds to ensure complete distribution of the tracer gas helium in the alveolar region of the lung. The diffusing capacity of carbon monoxide was determined using Equation 8 of Cotes et al. (1993). The diffusing capacity was related to the alveolar volume VA to assess its influence on the C18O transfer.
Alveolar volume [VA] was determined from the dilution of helium in the lung present in the breath of test gas. Equation 9 of Cotes et al. (1993) was applied for calculation.
Figure 1 - Helium expirogram