Stereological estimation of the total number of ventilatory units in mice lungs

We present an unbiased estimator of the total number of alveolar structures distal to the transition from a bronchiole to an alveolar duct system ('ventilatory units', VUs). In species without respiratory bronchioles, including mice, the number of VUs is equivalent to the number of acini. The acinus is a functional unit of gas exchange, defined as a parenchymal unit distal to a terminal bronchiole in which all airways contain alveoli and thus participate in gas exchange. The estimator combines two different estimators of the number of VUs: (1) an estimator derived from the Euler number of all the openings of the bronchial tree and (2) an estimator derived from direct counts of topological changes occurring at bronchiole-alveolar duct junctions. Combining the two estimators eliminates the requirement to be able to identify even vanishingly small pieces of bronchial tissue in physical disectors. We implemented the fractionator estimator in five adult mice lungs using physical fractionators with varying but known sampling fractions (Horvitz-Thompson estimator). We obtained total values of about 4200 VUs (CV = 0.05) in 21-day-old and 4480 (CV = 0.06) in 69-day-old animals. Being fractionator estimates, these total numbers are independent of shrinkage. The densities of VUs per unit volume of tissue (values corrected for tissue shrinkage) were similar in left and right lungs.