Estimation of alveolar number in the lung has traditionally been done by assuming a geometric shape and counting alveolar profiles in single, independent sections. In this study, we used the unbiased disector principle to estimate the Euler characteristic (and thereby the number) of alveolar openings in rat lungs and rhesus monkey lung lobes and to obtain robust estimates of average alveolar volume. The estimator of total alveolar number was based on systematic, uniformly random sampling using the fractionator sampling design. The number of alveoli in the rat lung ranged from 17.3 ⅐ 10 6 to 24.6 ⅐ 10 6 , with a mean of 20.1 ⅐ 10 6 . The average number of alveoli in the two left lung lobes in the monkey ranged from 48.8 ⅐ 10 6 to 67.1 ⅐ 10 6 with a mean of 57.7 ⅐ 10 6 . The coefficient of error due to stereological sampling was of the order of 0.06 in both rats and monkeys and the biological variation (coefficient of variance between individuals) was 0.15 in rat and 0.13 in monkey (left lobe, only). Between subdivisions (left/right in rat and cranial/caudal in monkey) there was an increase in variation, most markedly in the rat. With age (2Ϫ13 years) the alveolar volume increased 3-fold (as did parenchymal volume) in monkeys, but the alveolar number was unchanged. This study illustrates that use of the Euler characteristic and fractionator sampling is a robust and efficient, unbiased principle for the estimation of total alveolar number in the lung or in well-defined parts of it. Anat Rec Part A 274A: 216 -226, 2004.

Alveolarization in humans and nonhuman primates begins during prenatal development. Advances in stereological counting techniques allow accurate assessment of alveolar number; however, these techniques have not been applied to the developing human lung. Based on the recent American Thoracic Society guidelines for stereology, lungs from human autopsies, ages 2 mo to 15 yr, were fractionated and isometric uniform randomly sampled to count the number of alveoli. The number of alveoli was compared with age, weight, and height as well as growth between right and left lungs. The number of alveoli in the human lung increased exponentially during the first 2 yr of life but continued to increase albeit at a reduced rate through adolescence. Alveolar numbers also correlated with the indirect radial alveolar count technique. Growth curves for human alveolarization were compared using historical data of nonhuman primates and rats. The alveolar growth rate in nonhuman primates was nearly identical to the human growth curve. Rats were significantly different, showing a more pronounced exponential growth during the first 20 days of life. This evidence indicates that the human lung may be more plastic than originally thought, with alveolarization occurring well into adolescence. The first 20 days of life in rats implies a growth curve that may relate more to prenatal growth in humans. The data suggest that nonhuman primates are a better laboratory model for studies of human postnatal lung growth than rats.

Increased mucus production in asthma is an important cause of airflow obstruction during severe exacerbations. To better understand the changes in airway epithelium that lead to increased mucus production, ovalbumin-sensitized and -challenged mice were used. The phenotype of the epithelium was dramatically altered, resulting in increased numbers of mucous cells, predominantly in the proximal airways. However, the total numbers of epithelial cells per unit area of basement membrane did not change. A 75% decrease in Clara cells and a 25% decrease in ciliated cells were completely compensated for by an increase in mucous cells. Consequently, by day 22, 70% of the total epithelial cell population in the proximal airways was mucous cells. Electron microscopy illustrated that Clara cells were undergoing metaplasia to mucous cells. Conversely, epithelial proliferation, detected with 5-chloro-2-deoxyuridine immunohistochemistry, was most marked in the distal airways. Using ethidium homodimer cell labeling to evaluate necrosis and terminal dUTP nick-end labeling immunohistochemistry to evaluate apoptosis, this proliferation was accompanied by negligible cell death. In conclusion, epithelial cell death did not appear to be the stimulus driving epithelial proliferation and the increase in mucous cell numbers was primarily a result of Clara cell metaplasia. (Am J

Hyde DM, Blozis SA, Avdalovic MV, Putney LF, Dettorre R, Quesenberry NJ, Singh P, Tyler NK. Alveoli increase in number but not size from birth to adulthood in rhesus monkeys.

Mouse gammadelta T cells produce IL-17 in response to lung injury and are required for an organized inflammatory response and epithelial repair. The lack of gammadelta T cells correlates with increased inflammation and fibrosis.

SummaryBackground-Accumulation of immune cell populations and their cytokine products within tracheobronchial airways contributes to the pathogenesis of allergic asthma. It has been postulated that peripheral regions of the lung play a more significant role than proximal airways with regard to inflammatory events and airflow obstruction.

Rationale: Changes in the density of bronchial vessels have been proposed as a part of airway remodeling that occurs in chronic asthma. Objectives: Using an established nonhuman primate model of chronic allergic asthma, we evaluated changes in vascular density as well as the contribution of bronchial epithelium to produce vascular endothelial growth factor (VEGF). Methods: Eight juvenile rhesus macaques were divided into two groups of four. One group was exposed to 11 cycles of aerosolized house dust mite allergen (HDMA), whereas the other was exposed to filtered air. Bronchial wall vasculature was identified using an immunohistochemical approach, and vascular density was quantified stereologically. A semiquantitative polymerase chain reaction approach was used to estimate VEGF splice variant gene expression at discrete airway generations. Cell culture of primary tracheal epithelial cells with varying concentrations of HDMA was used to quantify the direct contribution of the epithelium to VEGF production. Results: Bronchial vascular density was increased at mid-to lower airway generations, which was independent of changes in the interstitial compartment. The VEGF 121 splice variant was significantly increased at lower airway generations. VEGF protein increased in a dose-dependant fashion in vitro primarily by an increase in VEGF 121 gene expression. Conclusion: This study highlights that increased vascular density in an animal model of chronic allergic asthma is airway generation specific and associated with a unique increase of VEGF splice variant gene expression. Airway epithelium is the likely source for increased VEGF.

Acute lung injury induced by reactive oxygen gases such as ozone (O(3)) is focal and site-selective. To define patterns of acute epithelial injury along intrapulmonary airways, we developed a new analytic approach incorporating labeling of permeable cells, airway microdissection, and laser scanning confocal microscopy, and applied it to isolated perfused rat lungs where ventilation and breathing pattern could be controlled. After exposure to O(3) (0, 0.25, 0.5, or 1.0 ppm), lungs were lavaged to assess lactate dehydrogenase (LDH) and protein, or infused with the permeability marker ethidium homodimer-1 (EthD-1) via tracheal cannula, gently lavaged, and fixed by airway infusion. The airway tree of the right middle lobe was exposed by microdissection of the axial pathway down to the terminal bronchioles; the dissection was incubated with a second nuclear dye, YOPRO-1, to label all nuclei; and whole mounts were examined by confocal microscopy. Abundance of EthD-1-positive (injured) cells was estimated as the number per epithelial volume using stereology on Z-series of projected images. For ozone concentrations of 1.0 ppm, lavage fluid LDH and total protein did not increase over controls. Exposure produced a concentration- dependent but nonhomogeneous increase in the abundance of EthD-1-labeled cells in proximal and distal conducting airways both in the main pathway, including terminal bronchioles, and in side branches. Overall, the highest EthD-1 labeling occurred in the side branches of the most proximal part of the airway tree at 1 ppm with the adjacent axial pathway airway having approximately one-third the labeling density. Density of EthD-1-labeled cells was lowest in terminal bronchioles at all O(3) doses. For the model we used, identification of injured epithelial cells by differential permeability and laser confocal microscopy appeared to be highly sensitive and permitted mapping of acute cytotoxicity throughout the airway tree and quantitative comparisons of sites with different branching histories and potential dosimetry rates.