Newly formed alveolar septa contain a double-layered capillary network. To optimize gas exchange, the two layers fuse to a single-layered capillary network during microvascular maturation. Because its timing is unknow in humans, microvascular maturation was stereologically estimated throughout postnatal human lung development. It is shown that maturation of the microvascular and alveolar septa takes place in parallel to alveolarization. At an age of 2–3 yr three-quarters of the septa are mature.

Professor Hans Jørgen G. Gundersen MD, DMSc (1943–2021) was a pioneering stereologist whose work has inspired and influenced researchers across the world for almost half a century. He was a charismatic character and one of the founding fathers of modern stereology, whose achievements and contributions are fondly remembered below by colleagues and co-workers. It was an enormous pleasure to be in his company and although future generation will miss this opportunity, his work will live on, to inspire and influence future generations of researchers.

The presence of bronchus-associated lymphoid tissue (BALT) and its size in humans largely depends upon age. It is detected in 35% of children less than 2 years of age, but absent in the healthy adult lung. Environmental gases or allergens may have an effect on the number of BALT. Lungs of rhesus macaque monkeys were screened by histology for the presence, size, and location of BALT after exposure to filtered air for 2, 6, 12, or 36 months or 12 and 36 months to ozone or 2, 12, or 36 months of house dust mite or a combination of ozone and house dust mite for 12 months. In the lungs of monkeys housed in filtered air for 2 months, no BALT was identified. After 6, 12, or 36 months, the number of BALT showed a significantly increased correlation with age in monkeys housed in filtered air. After 2 months of episodic house dust mite (HDM) exposure, no BALT was found. Monkeys exposed to HDM or HDM + ozone did not show a significant increase in BALT compared to monkeys housed in filtered air. However, monkeys exposed to ozone alone did show significant increases in BALT compared to all other groups. In particular, there were frequent accumulations of lymphocytes in the periarterial space of ozone exposed animals. In conclusion, BALT in rhesus monkeys housed under filtered air conditions is age-dependent. BALT significantly increased in monkeys exposed to ozone in comparison with monkeys exposed to HDM.

In 2011, a new classification of lung adenocarcinoma was published. Since then there have been multiple studies regarding observer variability in predominant subtype determination, with levels of agreement generally in the weak to moderate range. In the 2011 and subsequent WHO 2015 classification, a recommendation was also made to visually assess and record the percentage of each subtype in 5% increments. The present study was initiated to determine the reproducibility of such gestalt assessments and to compare these data to a formal morphometric assessment. Five experienced pathologists reviewed multiple single images of 25 adenocarcinomas, taken at 2× and 10×, and estimated the percentage of lepidic, acinar, papillary, micropapillary, and solid components in 5% increments. After 2 months all the pathologists again reviewed the same images presented to them in a different order. We found that there was poor reproducibility within observers at 2× power using a 5% evaluation, but that this improved using 10% or 25% cutoffs. Use of 10× magnification allowed weak to moderate reproducibility at 5% increments, and this was again improved using 10% or 25% cutoffs. Correlation with morphometric assessment was poor except for the papillary and micropapillary subtypes. Differences among pathologists were generally low except for the acinar and, to a lesser degree, lepidic subtypes, which showed a wide spread of data. When estimating tumor subtype proportions, use of a 10× objective, and utilization of 10% or preferably 25% cutoffs provides a greater degree of consistency than a 5% cutoff.

Neutrophils are central players in inflammation and the innate immune response to invading microbial pathogens. At any given time, the majority of neutrophils are sequestered in the microvasculature of the lung, a location that is well suited for them to respond immediately to inflammatory stimuli originating from the external environment. They sequester in the lung capillaries through unique hemodynamic and geometric properties of the pulmonary microvasculature, as well as changes in the rheological properties of neutrophils during activation. In contrast to the systemic circulation, the branching pulmonary microvasculature has the capacity to shunt blood flow away from inflamed areas through vasoconstriction that acts to limit the extent of neutrophil emigration into the airspaces. Due to these unique features of the pulmonary circulation, neutrophil migration in acute pulmonary inflammation is not always dependent on the typical multistep process of selectin-mediated rolling that transitions to chemokine-activated integrin-dependent arrest on immunoglobulin-like cell-adhesion molecules (Ig-CAMs). Neutrophils can cause extensive damage to the lung in a variety of infectious and toxicological inflammatory disorders. This is accomplished by their production of reactive oxygen species (ROS), release of granule components such as proteolytic enzymes and cationic proteins and excretion of neutrophil extracellular traps (NETs) upon activation. Experimental models suggest that repetitive pulmonary inflammation and accompanying epithelial injury exacerbate the inflammatory cascade and enhance the extent of pulmonary inflammation and epithelial injury in subsequent cycles. However, there is also evidence that neutrophils play an important role in removing injured pulmonary cells to enhance repair of the lung. The mechanisms utilized by neutrophils to target injured cells and selectively kill them or cause their junctional detachment in the absence of parenchymal derangement are just beginning to be understood. Clearly this beneficial action of the neutrophils occurs only in mild toxicological injury to the lung since it appears to be a dose-dependent response with mild injury resulting in low-to-moderate neutrophil emigration, while severe injury is associated with robust neutrophil recruitment, degranulation, and reactive oxygen elaboration. Under these conditions of widespread and severe injury, the main function of neutrophils appears to be removal of dead cells and debris and stimulation of the remaining viable cells to spread and proliferate in order to promote reepithelialization of the lung, the first step to organ restoration.

High-profile investigations have focused on alveolar regeneration after lung injury, notably expansion of the alveolar epithelial type 2 cell (AEC2) population (1–4). Investigations of AEC2 expansion, particularly mechanistic investigations using pharmacologic or genetic manipulation, require an accurate, unbiased method of quantitation to ensure valid comparison between experimental groups. AEC2 expansion is typically assessed by flow cytometry of cells recoverable from a lung digest (1–3, 5–7) or by counting cell profiles in arbitrary fields of immunostained lung sections (planimetry) (1, 2, 4, 7). AEC2 expansion is usually measured as the percentage of AEC2s that are actively cycling, as determined by incorporation of nucleoside analogs (1–7) or AEC2 number as a percentage of a larger population, for example, total lung cells (1, 2, 5); rarely is absolute AEC2 number assessed (1, 7). These methods have not been validated and have important theoretical limitations. Flow cytometric analysis may be limited by incomplete and variable recovery of cells. Planimetric analysis may be biased by tissue inflation and shrinkage, cell loss during sectioning, the overrepresentation of larger cells in two-dimensional sections, and nonrandom sampling, which is particularly problematic for lung injury with a patchy distribution (8–10). Evaluating AEC2 expansion as the percentage of actively cycling cells is limited by the duration of the pulse and ascertains S phase, not cell division. Expressing the number of actively cycling cells as a percentage of total AEC2s, or total AEC2 number as a percentage of a larger population, may be confounded by changes in the denominator. Epithelial regeneration requires increased absolute AEC2 numbers to replace cells lost during injury. Stereology is an unbiased approach recommended by the American Thoracic Society for the quantitation of cell number (10) but is rarely used to assess AEC2 expansion. Here, we employed stereology as the “gold standard” method for quantifying AEC2 expansion during repair after lung injury. We measured the absolute number of AEC2s, the key parameter for alveolar regeneration, and the absolute number and percentage of actively cycling AEC2s. The results were compared with flow cytometric and planimetric analyses.

The alveolar epithelium consists of squamous alveolar type (AT) I and cuboidal ATII cells. ATI cells cover 95-98% of the alveolar surface, thereby playing a critical role in barrier integrity, and are extremely thin, thus permitting efficient gas exchange. During lung injury, ATI cells die, resulting in increased epithelial permeability. ATII cells re-epithelialize the alveolar surface via proliferation and transdifferentiation into ATI cells. Transdifferentiation is characterized by down-regulation of ATII cell markers, up-regulation of ATI cell markers, and cell spreading, resulting in a change in morphology from cuboidal to squamous, thus restoring normal alveolar architecture and function. The mechanisms underlying ATII to ATI cell transdifferentiation have not been well studied in vivo. A prerequisite for mechanistic investigation is a rigorous, unbiased method to quantitate this process. Here, we used SPCCreERT2; mTmG mice, in which ATII cells and their progeny express green fluorescent protein (GFP), and applied stereologic techniques to measure transdifferentiation during repair after injury induced by LPS. Transdifferentiation was quantitated as the percent of alveolar surface area covered by ATII-derived (GFP1) cells expressing ATI, but not ATII, cell markers. Using this methodology, the time course and magnitude of transdifferentiation during repair was determined. We found that ATI cell loss and epithelial permeability occurred by Day 4, and ATII to ATI cell transdifferentiation began by Day 7 and continued until Day 16. Notably, transdifferentiation and barrier restoration are temporally correlated. This methodology can be applied to investigate the molecular mechanisms underlying transdifferentiation, ultimately revealing novel therapeutic targets to accelerate repair after lung injury.

Early life is a critical period for the progressive establishment of immunity in response to environmental stimuli; the impact of airborne challenges on this process is not well defined. In a longitudinal fashion, we determined the effect of episodic house dust mite (HDM) aerosol and ozone inhalation, both separately and combined, on peripheral blood immune cell phenotypes and cytokine expression from 4 to 25weeks of age in an infant rhesus monkey model of childhood development. Immune profiles in peripheral blood were compared with lung lavage at 25weeks of age. Independent of exposure, peripheral blood cell counts fluctuated with chronologic age of animals, while IFNγ and IL-4 mRNA levels increased over time in a linear fashion. At 12weeks of age, total WBC, lymphocyte numbers, FoxP3 mRNA and IL-12 mRNA were dramatically reduced relative to earlier time points, but increased to a steady state with age. Exposure effects were observed for monocyte numbers, as well as CCR3, FoxP3, and IL-12 mRNA levels in peripheral blood. Significant differences in cell surface marker and cytokine expression were detected following in vitro HDM or PMA/ionomycin stimulation of PBMC isolated from animals exposed to either HDM or ozone. Lavage revealed a mixed immune phenotype of FoxP3, IFNγ and eosinophilia in association with combined HDM plus ozone exposure, which was not observed in blood. Collectively, our findings show that airborne challenges during postnatal development elicit measureable cell and cytokine changes in peripheral blood over time, but exposure-induced immune profiles are not mirrored in the lung.

ABSTRACTThe aging lung is associated with increased susceptibility to chronic inflammatory diseases such as chronic obstructive pulmonary disease where females have been reported to be more susceptible than males. The changes in reproductive hormones due to aging may directly or indirectly affect lung structure and function and little is known on the mechanism of these changes. Twenty female rhesus macaques were divided into four groups. Ovariectomy (OVX) was performed on eight animals with three receiving estrogen/progesterone therapy (HRT) and five animals given implants containing vehicle. The remaining 12 animals represented control groups of ages 10–14 years (n = 6) and ages 20–24 (n = 6). A design‐based stereological method was employed to estimate the number of alveoli in the right middle lung lobe along with hormone analysis for possible correlation. A significant decrease was found in the number of alveoli in the vehicle OVX animals compared to intact younger adult females (P < 0.001). A significant increase in alveoli between OVX vehicle animals and those on HRT was also found (P < 0.0001). There was difference in the number of alveoli between younger adult animals and animals on HRT. The loss of ovaries and hormones had a significant effect on alveolar lung morphology. This result mimics what is seen in the aging process and could contribute to gender differences reported in the elderly. Hormone replacement, as reported here, could possibly slow the loss of alveoli due to the aging process or aid in alveolar regeneration through direct or indirect mechanisms. Anat Rec, 299:973–978, 2016. © 2016 Wiley Periodicals, Inc.

Structural and functional complexities of the mammalian lung evolved to meet a unique set of challenges, namely, the provision of efficient delivery of inspired air to all lung units within a confined thoracic space, to build a large gas exchange surface associated with minimal barrier thickness and a microvascular network to accommodate the entire right ventricular cardiac output while withstanding cyclic mechanical stresses that increase several folds from rest to exercise. Intricate regulatory mechanisms at every level ensure that the dynamic capacities of ventilation, perfusion, diffusion, and chemical binding to hemoglobin are commensurate with usual metabolic demands and periodic extreme needs for activity and survival. This article reviews the structural design of mammalian and human lung, its functional challenges, limitations, and potential for adaptation. We discuss (i) the evolutionary origin of alveolar lungs and its advantages and compromises, (ii) structural determinants of alveolar gas exchange, including architecture of conducting bronchovascular trees that converge in gas exchange units, (iii) the challenges of matching ventilation, perfusion, and diffusion and tissue-erythrocyte and thoracopulmonary interactions. The notion of erythrocytes as an integral component of the gas exchanger is emphasized. We further discuss the signals, sources, and limits of structural plasticity of the lung in alveolar hypoxia and following a loss of lung units, and the promise and caveats of interventions aimed at augmenting endogenous adaptive responses. Our objective is to understand how individual components are matched at multiple levels to optimize organ function in the face of physiological demands or pathological constraints.

Bone morphogenetic protein (BMP) signaling is important for correct lung morphogenesis, and there is evidence of BMP signaling reactivation in lung diseases. However, little is known about BMP signaling patterns in healthy airway homeostasis and inflammatory airway disease and during epithelial repair. In this study, a rhesus macaque (Macaca mulatta) model of allergic airway disease was used to investigate BMP signaling throughout the airways in health, disease, and regeneration. Stereologic quantification of immunofluorescent images was used to determine the expression of BMP receptor (BMPR) Ia and phosphorylated SMAD (pSMAD) 1/5/8 in the airway epithelium. A pSMAD 1/5/8 expression gradient was found along the airways of healthy juvenile rhesus macaques (n = 3, P < 0.005). Membrane-localized BMPRIa expression was also present in the epithelium of the healthy animals. After exposure to house dust mite allergen and ozone, significant down-regulation of nuclear pSMAD 1/5/8 occurs in the epithelium. When the animals were provided with a recovery period in filtered air, proliferating cell nuclear antigen, pSMAD 1/5/8, and membrane-localized BMPRIa expression were significantly increased in the epithelium of conducting airways (P < 0.005). Furthermore, in the asthmatic airways, altered BMPRIa localization was evident. Because of the elevated eosinophil presence in these airways, we investigated the effect of eosinophil-derived proteins on BMPRIa trafficking in epithelial cells. Eosinophil-derived proteins (eosinophil-derived neurotoxin, eosinophil peroxidase, and major basic protein) induced transient nuclear translocation of membrane-bound BMPRIa. This work mapping SMAD signaling in the airways of nonhuman primates highlights a potential mechanistic relationship between inflammatory mediators and BMP signaling and provides evidence that basal expression of the BMP signaling pathway may be important for maintaining healthy airways.

This chapter focuses on the epithelium of the distal conducting airways, or bronchioles, with emphasis on the area of transition between conducting airways and the gas-exchange area. The bronchiolar region has a substantial variation from one species of mammal to the next. This variability includes the microenvironment in which epithelial cells are found, particularly the zone of transition between bronchiolar, non-gas exchange regions and the alveolar gas-exchange regions. There is also considerable interspecies variability in the general composition of the bronchiolar epithelium. The principal alterations involve the distribution, number, and percentage of the population that is nonciliated cells. In addition, there is substantial variability in the composition and differentiated expression of the nonciliated epithelial population in this region.

In rhesus macaques, previous studies have shown that episodic exposure to allergen alone or combined with ozone inhalation during the first 6months of life results in a condition with many of the hallmarks of asthma. This exposure regimen results in altered development of the distal airways and parenchyma (Avdalovic et al., 2012). We hypothesized that the observed alterations in the lung parenchyma would be permanent following a long-term recovery in filtered air (FA) housing. Forty-eight infant rhesus macaques (30days old) sensitized to house dust mite (HDM) were treated with two week cycles of FA, house dust mite allergen (HDMA), ozone (O3) or HDMA/ozone (HDMA+O3) for five months. At the end of the five months, six animals from each group were necropsied. The other six animals in each group were allowed to recover in FA for 30 more months at which time they were necropsied. Design-based stereology was used to estimate volumes of lung components, number of alveoli, size of alveoli, distribution of alveolar volumes, interalveolar capillary density. After 30months of recovery, monkeys exposed to HDMA, in either group, had significantly more alveoli than filtered air. These alveoli also had higher capillary densities as compared with FA controls. These results indicate that early life exposure to HDMA alone or HDMA+O3 alters the development process in the lung alveoli.

Background Hypertrophic cardiomyopathy ( HCM ) is the abnormal thickening of the ventricles and an increase in cardiac mass. Methods Pedigree analyses involving 108 rhesus macaques with pronounced HCM were performed. Results Analyses revealed a strong genetic predisposition to this disease. Conclusions Macaques are ideal for investigating HCM because of their marked similarity to humans genetically, physiologically, and anatomically.

Purpose/Aim: Angiogenesis is a central component of normal wound healing but it has not been fully characterized in lung repair following acute inflammatory injury. The current literature lacks vital information pertaining to the extent, timing, and location of this process. This information is necessary for examining mechanisms that drive normal lung repair in resolving acute inflammatory injury. The goal of our study was to formally characterize lung angiogenesis over a time course of bleomycin-induced lung injury. Materials and Methods: Female C57BL/6 mice age 8-12 weeks were treated with a single dose of intratracheal bleomycin. Total lung endothelial cells were quantified with flow cytometry 0, 7, 14, 21, and 28 days following bleomycin administration, and endothelial cell replication was assessed using bromodeoxyuridine (BrdU) incorporation. Results: Endothelial cell replication was maximal 14 days after bleomycin administration, while total lung endothelial cells peaked at day 21. Tissue analysis with stereology was performed to measure total lung vascular surface area in bleomycin at day 21 relative to controls and demonstrated a trend toward increased vasculature in the bleomycin group. Conclusions: Angiogenesis begins shortly after injury in the bleomycin model and leads to an expansion in the lung endothelial cell population that peaks at day 21. This study offers the first longitudinal examination of angiogenesis following acute inflammatory lung injury induced by bleomycin. Information provided in this study will be vital for further investigating mechanisms of angiogenesis in both normal and abnormal lung repair.

Thickening of the basement membrane zone (BMZ) is a characteristic feature of airway remodeling in the lungs of asthmatics. However the significance of a thickened BMZ in the pathology of the asthmatic airway is not known. In this review we show that the columnar epithelium is linked to the reticular BMZ through the supracellular anchoring network. We discuss the evidence that changes in the width of the BMZ in control airways are part of a supracellular anchoring mechanism for increasing the strength of attachment between the airway epithelium and the extracellular matrix (ECM). We then review the effects of asthma on this anchoring mechanism. We conclude that both thickening of the BMZ and sloughing of columnar epithelium (creola bodies) in asthma represent abnormalities in the supracellular anchoring network attaching the airway epithelium to the ECM. Future research directed toward studying the regulation and development of the supracellular anchoring network may help better understand sloughing of columnar epithelium and the significance of reticular BMZ thickening in the asthmatic airway.

This chapter focuses on the normal aging of lungs in various mammalian species such as mouse, rat, and dog. Various studies have demonstrated that lung aging in mice is associated with decreases in specific functional and structural parameters. These include increases in the phagocytic cell populations present in the lung airspaces, but decreased ability to engulf foreign particles. Decreases in antioxidant defense systems have also been noted in the lungs of aging mice. From a structural perspective, hyperinflation of the lungs and increases in interalveolar pore size and number are key features of the lung aging process in mice. The dynamics of lung growth, development, and aging in the rat is a continuous process that involves every tissue compartment of the lungs. Significant changes in aging adult rats are primarily within alveolar type II cells and the noncellular portions of the interstitium. Changes in cell number, size, and function associated with aging are likely to impact on lung physiology, metabolism, and immunity. Such changes could significantly alter the normal functions of the lung and its susceptibility to injury. Aging of canine lungs has many similarities to that seen in human lungs, but the aging process in dogs occurs over a shorter time frame than in humans. The accumulation of dust-laden macrophages as well as lumenal enlargement of alveolar ducts are hallmarks of this aging process. Changes in the relative velocity of mucus flow in the trachea with aging also parallels changes observed in the aging human trachea.