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:

• ventilation rates: 120-130 breaths/min
• tidal volume: 280-325 microliters
• endotracheal peak inspiratory pressure: ~ 10 cm H₂O
• CO₂ and O₂: physiological endexpiratory concentrations

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.

Experimental setup
A detailed description of the experimental setup is available in Schulz et al. (2002).

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.

Notes:
Some measurements are not available in the project data set. All data may be accessed in the supplemental data set. Both available here.

Lung volumes

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:

total lung capacity [TLC] = IC + FRC
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.

Lung volume ratios were calculated to determine allometric relationships between different sized lungs.

Compliance

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.

Specific compliances [ratio_Cdyn_TLC] were calculated based on TLC to take different lung sizes into consideration.

Resistance

Specific resistance [SR] was calculated as R times TLC.

Helium expirogram

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 capacity

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