Elderly Female Rhesus Macaques Preserve Lung Alveoli With Estrogen/Progesterone Therapy.

The 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.

Growth of alveoli during postnatal development in humans based on stereological estimation.

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.

Accelerated structural decrements in the aging female rhesus macaque lung compared with males.

Aging is associated with morphometric changes in the lung that lead to decreased lung function. The nonhuman primate lung has been shown to have similar architectural, morphological, and developmental patterns to that of humans. We hypothesized that the lungs of rhesus monkeys age in a pattern similar to human lungs. Thirty-four rhesus monkeys from the California National Primate Research Center were euthanized, necropsied, and the whole lungs sampled. Stereological analysis was performed to assess the morphological changes associated with age. The number of alveoli declined significantly from age 9 to 33 yr with a greater decline in females compared with males. Lungs of females contained roughly 20% more alveoli at age 9 yr than males, but by ∼30 yr of age, females had 30% fewer alveoli than males. The volume of alveolar air also showed a significant linear decrease in females relative to age, while males did not. The number-weighted mean volume of alveoli showed a significant positive correlation with age in females but not in males. The volume of alveolar duct showed a significant positive correlation with age in females, but not in males. Structural decrements due to aging in the lung were increased in the female compared with male rhesus monkey.

Ozone exposure during the early postnatal period alters the timing and pattern of alveolar growth and development in nonhuman primates.

Exposure to oxidant air pollutants in early childhood, with ozone as the key oxidant, has been linked to significant decrements in pulmonary function in young adults and exacerbation of airway remodeling in asthma. Development of lung parenchyma in rhesus monkeys is rapid during the first 2 years of life (comparable to the first 6 years in humans). Our hypothesis is that ozone inhalation during infancy alters alveolar morphogenesis. We exposed infant rhesus monkeys biweekly to 5, 8 hr/day, cycles of 0.5 ppm ozone with or without house dust mite allergen from 1 to 3 or 1 to 6 months of age. Monkeys were necropsied at 3 and 6 months of age. A morphometric approach was used to quantify changes in alveolar volume and number, the distribution of alveolar size, and capillary surface density per alveolar septa. Quantitative real time PCR was used to measure the relative difference in gene expression over time. Monkeys exposed to ozone alone or ozone combined with allergen had statistically larger alveoli that were less in number at 3 months of age. Alveolar capillary surface density was also decreased in the ozone exposed groups at 3 months of age. At 6 months of age, the alveolar number was similar between treatment groups and was associated with a significant rise in alveolar number from 3 to 6 months of age in the ozone exposed groups. This increase in alveolar number was not associated with any significant increase in microvascular growth as measured by morphometry or changes in angiogenic gene expression. Inhalation of ozone during infancy alters the appearance and timing of alveolar growth and maturation. Understanding the mechanism involved with this altered alveolar growth may provide insight into the parenchymal injury and repair process that is involved with chronic lung diseases such as severe asthma and COPD.

In utero and postnatal exposure to environmental tobacco smoke (ETS) alters alveolar and respiratory bronchiole (RB) growth and development in infant monkeys.

UNLABELLED: The direct effect of environmental tobacco smoke (ETS) exposure in utero on the development of the lung parenchyma is not known. We used design-based stereologic methods to evaluate in utero and postnatal ETS exposure on alveolar and respiratory bronchiole (RB) development in the rhesus macaque. METHODS: Timed-pregnant rhesus macaques and their offspring were exposed to filtered air or various amounts of ETS during the prenatal and postnatal period. The left cranial lobe from necropsied infants was evaluated by design-based stereological methods and general pathological review. RESULTS: Infants in the in utero and six-month ETS groups had an 18% and 17% relative decrease, respectively, in alveolar number and a 57% and 33% increase, respectively, in alveolar size compared to filtered air (FA) monkeys. Lung volume positively correlated with alveolar number in the FA and six-month ETS group and negatively correlated in the in utero ETS group. The distribution of alveolar size was much more variable in the in utero group. Overall, RB volume was significantly increased in the six-month ETS group (p < .04). CONCLUSIONS: Taken together, these results indicate that in utero and postnatal ETS exposure is associated with altered parenchymal lung development.

IL-17 producing gammadelta T cells are required for a controlled inflammatory response after bleomycin-induced lung injury.

BACKGROUND: gammadelta T cells play a key role in the regulation of inflammatory responses in epithelial tissue, and in adaptive immunity, as gammadelta T cell deficient mice have a severely impaired capacity to clear lung pathogens. gammadelta T cells regulate the initial inflammatory response to microbial invasion and thereby protect against tissue injury. Here we examined the response of gammadelta T cells to lung injury induced by bleomycin, in an effort to study the inflammatory response in the absence of any adaptive immune response to a pathogen. RESULTS: After lung injury by bleomycin, we localized the gammadelta T cells to the lung lesions. gammadelta T cells were the predominant source of IL-17 (as detected by flow cytometry and real-time PCR). Moreover, gammadelta T cell knockout mice showed a significant reduction in cellular infiltration into the airways, reduced expression of IL-6 in the lung, and a significant delay in epithelial repair. CONCLUSION: 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.

Alveoli increase in number but not size from birth to adulthood in rhesus monkeys.

Postnatal developmental stages of lung parenchyma in rhesus monkeys is about one-third that of humans. Alveoli in humans are reported to be formed up to 8 yr of age. We used design-based stereological methods to estimate the number of alveoli (N(alv)) in male and female rhesus monkeys over the first 7 yr of life. Twenty-six rhesus monkeys (13 males ranging in age from 4 to 1,920 days and lung volumes from 41.7 to 602 cm(3), 13 females ranging in age from 22 to 2,675 days and lung volumes from 43.5 to 380 cm(3)) were necropsied and lungs fixed, isotropically oriented, fractionated, sampled, embedded, and sectioned for alveolar counting. Parenchymal, alveolar, alveolar duct core air, and interalveolar septal tissue volumes increased rapidly during the first 2 yr with slowed growth from 2 to 7 yr. The rate of change was greater in males than females. N(alv) also showed consistent growth throughout the study, with increases in N(alv) best predicted by increases in lung volume. However, mean alveolar volume showed little relationship with age, lung volume, or body weight but was larger in females and showed a greater size distribution than in males. Alveoli increase in number but not volume throughout postnatal development in rhesus monkeys.

Vascular remodeling is airway generation-specific in a primate model of chronic asthma.

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 VEGF121 splice variant was significantly increased at lower airway generations. VEGF protein increased in a dose-dependant fashion in vitro primarily by an increase in VEGF121 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.

Effect of rapid shallow breathing on the distribution of 18O-labeled ozone reaction product in the respiratory tract of the rat.

We examined the effect of breathing pattern on ozone reaction product content within the respiratory tract. Thirty-four anesthetized, male Wistar rats were exposed to oxygen-18 ((18)O)-labeled ozone at 1.0 ppm for 2 h using a dual-chamber, negative-pressure ventilation system. Frequency was set at 80 (n = 9), 120 (n = 7), 160 (n = 8), or 200 (n = 10) breaths per minute (bpm), while tidal volume (V(t)) was set to provide a constant minute ventilation of 72.8 ml/min/100 g body weight. Airways sampled were from the midlevel trachea and the mainstem bronchi and parenchyma of the cranial and caudal right lobes. (18)O content in each airway sample was quantified and normalized to surface area. Across frequencies, there was significantly greater (p <.05) (18)O content in the trachea and bronchi (conducting airway epithelium) compared to the parenchyma sampling sites. Tracheal (18)O content decreased between 80 and 160 bpm, but then underwent an increase at 200 bpm. In comparison, (18)O content gradually increased between 80 and 200 bpm at the right cranial and caudal bronchi sites. Right cranial parenchymal (18)O content decreased at 200 bpm compared to 80, 120, and 160 bpm. Right caudal parenchymal (18)O content was relatively constant over all breathing frequencies. We concluded that the development of rapid shallow breathing from 80 to 160 bpm results in a reduced deposition of O(3) in the trachea, while only mildly affecting to ozone deposition in parenchyma supplied by short and long airway paths.

Pathogenesis of mucous cell metaplasia in a murine asthma model.

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.

Repeated episodes of ozone inhalation attenuates airway injury/repair and release of substance P, but not adaptation.

To determine the impact of repeated episodes of ozone exposure on physiologic adaptation, epithelial injury/repair, and tracheal substance P levels, adult rats were subjected to episodes of ozone (5 days, 1 ppm, 8 h/day) followed by 9 days of filtered air for four cycles. Rats were sampled on days 1 and 5 of each episode and 9 days after day 5 of episodes 1, 2, and 4. One hour before being euthanized each rat was injected with 5-bromo-2'-deoxyuridine to label proliferating cells. Each 5-day episode showed a characteristic pattern of rapid shallow breathing (days 1 and 2), epithelial injury, and interstitial and intraluminal inflammation. In contrast, the neutrophil component of inflammation, tracheal substance P release, and cell proliferation became attenuated with each consecutive episode of exposure. Concurrent with this cyclic and attenuated response there was progressive hypercellularity and hyperplasia in all airways studied and a progressive remodeling present in the terminal bronchioles. Our findings are consistent with the notion that the cumulative distal airway lesion is at least in part the result of a depressed cell proliferative response to injury in these airways. This depressed cell proliferative response may be in part the result of diminished neutrophil inflammation and/or release of mitogenic neuropeptides in response to ozone-induced injury.