How much is there really? Why stereology is essential in lung morphometry

Quantitative data on lung structure are essential to set up structure-function models for assessing the functional performance of the lung or to make statistically valid comparisons in experimental morphology, physiology, or pathology. The methods of choice for microscopy-based lung morphometry are those of stereology, the science of quantitative characterization of irregular three-dimensional objects on the basis of measurements made on two-dimensional sections. From a practical perspective, stereology is an assumption-free set of methods of unbiased sampling with geometric probes, based on a solid mathematical foundation. Here, we discuss the pitfalls of lung morphometry and present solutions, from specimen preparation to the sampling scheme in multiple stages, for obtaining unbiased estimates of morphometric parameters such as volumes, surfaces, lengths, and numbers. This is demonstrated on various examples. Stereological methods are accurate, efficient, simple, and transparent; the precision of the estimates depends on the size and distribution of the sample. For obtaining quantitative data on lung structure at all microscopic levels, state-of-the-art stereology is the gold standard

Assessing recruitment of lung diffusing capacity in exercising guinea pigs with a rebreathing technique

Noninvasive techniques for assessing cardiopulmonary function in small animals are limited. We previously developed a rebreathing technique for measuring lung volume, pulmonary blood flow, diffusing capacity for carbon monoxide (DlCO) and its components, membrane diffusing capacity (DmCO) and pulmonary capillary blood volume (Vc), and septal volume, in conscious nonsedated guinea pigs at rest. Now we have extended this technique to study guinea pigs during voluntary treadmill exercise with a sealed respiratory mask attached to a body vest and a test gas mixture containing 0.5% SF6 or Ne, 0.3% CO, and 0.8% C2H2 in 40% or 98% O2. From rest to exercise, O2 uptake increased from 12.7 to 25.5 ml·min−1·kg−1 while pulmonary blood flow increased from 123 to 239 ml/kg. The measured DlCO, DmCO, and Vc increased linearly with respect to pulmonary blood flow as expected from alveolar microvascular recruitment; body mass-specific relationships were consistent with those in healthy human subjects and dogs studied with a similar technique. The results show that 1) cardiopulmonary interactions from rest to exercise can be measured noninvasively in guinea pigs, 2) guinea pigs exhibit patterns of exercise response and alveolar microvascular recruitment similar to those of larger species, and 3) the rebreathing technique is widely applicable to human (∼70 kg), dog (20–30 kg), and guinea pig (1–1.5 kg). In theory, this technique can be extended to even smaller animals provided that species-specific technical hurdles can be overcome

Lung diffusing capacity for nitric oxide measured by two commercial devices: a randomised crossover comparison in healthy adults

In Europe, two commercial devices are available to measure combined single-breath diffusing capacity of the lung for nitric oxide (D LNO) and carbon monoxide (D LCO) in one manoeuvre. Reference values were derived by pooling datasets from both devices, but agreement between devices has not been established. We conducted a randomised crossover trial in 35 healthy adults (age 40.0±15.5 years, 51% female) to compare D LNO (primary end-point) between MasterScreen™ (Vyaire Medical, Mettawa, IL, USA) and HypAir (Medisoft, Dinant, Belgium) devices during a single visit under controlled conditions. Linear mixed models were used adjusting for device and period as fixed effects and random intercept for each participant. Difference in D LNO between HypAir and MasterScreen was 24.0 mL·min-1·mmHg-1 (95% CI 21.7-26.3). There was no difference in D LCO (-0.03 mL·min-1·mmHg-1, 95% CI -0.57-0.12) between devices while alveolar volume (V A) was higher on HypAir compared to MasterScreen™ (0.48 L, 95% CI 0.45-0.52). Disparity in the estimation of V A and the rate of NO uptake (KNO=D LNO/V A) could explain the discrepancy in D LNO between devices. Disparity in the estimation of V A and the rate of CO uptake (KCO=D LCO/V A) per unit of V A offset each other resulting in negligible discrepancy in D LCO between devices. Differences in methods of expiratory gas sampling and sensor specifications between devices likely explain these observations. These findings have important implications for derivation of D LNO reference values and comparison of results across studies. Until this issue is resolved, reference values, established on the respective devices, should be used for test interpretation