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

Matt J. Herring, Mark V. Avdalovic, Cheryl L. Quesenberry, Lei F. Putney, Nancy K. Tyler, Frank F. Ventimiglia, Judith A. St. George, Dallas M. Hyde

Abstract

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.

  • stereology
  • alveolar loss
  • alveolar duct enlargement

over the last several decades, the median age of the US population has increased by 20 yr. The number of people age 65 yr and over is projected to increase from 35 million in 2000 to an estimated 71 million in 2030, with the largest increase in individuals age 80 yr and above (US Census 2005; http://www.census.gov/#). Numerous diseases are more frequently diagnosed in the aging lung including chronic obstructive pulmonary disease, pulmonary fibrosis, pneumonia, and lung cancer. Emphysema occurs more frequently in aged individuals and implies greater susceptibility of the aged lung to this disease (2). With the costs associated with maintaining an aging population that is increasing, it has become important to understand the process of aging in the lung and the diseases that can be associated with the aging process. An appropriate research model needs to be identified and characterized, both structurally and functionally, to understand the role of the aging process and how diseases exploit that process.

Landmark work by Fletcher and Peto (4) demonstrated that the forced expiratory flow in 1 s (FEV1) declines with age and that this decline is accelerated in active smokers with associated lung disease. In addition to accelerated decline of FEV1, there is an age-related loss of elastic recoil and decreased dynamic compliance with an increase in air flow resistance (1). The aging lung in humans is associated with numerous changes both functionally and structurally. The structural changes include an increase in alveolar duct volume, mean linear intercept, and the number of interalveolar pores (7, 23). However, a lack of representation of the oldest humans in physiological studies limits our understanding of pulmonary aging (30). Additionally there are very few studies that have compared the difference in the aging lung between men and women despite data that suggest that women may be at higher risk for chronic lung disease compared with men (9). Loss of lung function may be related to multiple factors including loss of elastic recoil, decrease in alveolar number or increase in alveolar size, and possibly a combination of all of these. In dogs, normal aging has been associated with a decline of alveolar surface area without a decrease in lung volume, decreased alveolar surface-to-volume ratio, and loss of alveoli (11, 22). Research in mice has indicated a sexual dimorphism in lung growth and a possible role for female sex hormones in both alveologenesis and alveolar regeneration (16, 18). However, a truly relevant model to study structure function relationships in the aging human lung is currently lacking.

The structure of the nonhuman primate lung is more similar to humans in the number of generations, the type of distal airways, and the number of alveoli than other laboratory animals (21, 25, 26). In addition, the rhesus monkey is a longer lived species than rodents with a year of age in a rhesus macaque being equivalent to 3 yr in a human (6) allowing for longer exposures to ambient air. The population of rhesus macaques at the California National Primate Research Center represents a population of humans that are exposed to the same environmental contaminants for an extended period of time. A sample cohort of otherwise healthy male and female rhesus macaques from about 9 to 33 yr of age was selected to answer our primary aim of whether age and sex influence a morphometric change in alveolar ducts, alveolar number and size, and structural difference in the lung matrix.

MATERIALS AND METHODS

Animals, necropsy, lung volume, and tissue collection.

materials and methods closely followed those published previously by Hyde et al. (12) and comply with the guidelines set forth in Hsia et al. (10). Care and housing of animals complied with the provisions of the Institute of Laboratory Animal Resources and conformed to practices established by the Association for Assessment and Accreditation of Laboratory Animal Care. Animal Studies conformed to applicable provisions of the Animal Welfare Act and other federal statutes and regulations relating to animals (Guide for the Care and Use of Laboratory animals; National Institutes of Health, revised 1985). Experimental protocols were reviewed and approved by the University of California, Davis Institutional Animal Care and Use Committee. Thirty-four animals were selected from the outdoor field cages at the California National Primate Research Center in Davis, California. All monkeys were given a physical examination by a staff veterinarian to determine overall state of health and absence of respiratory disease. Monkeys were anesthetized with Diprivan (0.1–0.2 mg·kg−1·min−1 iv) and euthanized by using an overdose of pentobarbital after sedation by Telazol (8 mg/kg im) with the dose adjusted as necessary by the attending veterinarian. Necropsy of monkeys followed exsanguination via the posterior vena cava. Pathology findings at necropsy are reported in Table 1, but there was no evidence of injury or inflammation in the lung either grossly or microscopically. The trachea was cut, and the lungs were removed en bloc. The lungs were inflated and fixed with 1% glutaraldehyde [1% paraformaldehyde in cacodylate buffer (330 mosM, pH 7.4) at 25 cm fluid pressure]. After 8 h, the lungs were tied off at the trachea to maintain fixation pressure and placed in the same fixative to ensure complete fixation. After about a week, the lungs were removed from the fixative, the trachea and extrapulmonary bronchi were removed, and lung volume (VL) was estimated by its buoyant weight in PBS. The tissue was prepared for isotropic uniform random sampling of the lungs. As previously described and illustrated (12), the lungs were then embedded in 4% agar, isotropically oriented using an orientator, sliced into 5-mm slabs, and sampled using smooth fractionators to select 8 to 10 5×5×15-mm blocks for histology. Blocks were embedded in paraffin, four 5-μm serial sections were cut and stained with hematoxylin and eosin (12). A separate section from the paraffin embedded blocks was stained using a pentachrome stain to localize collagen and elastin in the parenchyma (19). Due to tissue shrinkage during fixation, all surface and volume estimates were corrected for global shrinkage using the estimate of global volume change previously reported in Hyde et al. (12).

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Table 1.

Pathology findings at necropsy for 34 adult rhesus macaques

Whole slide scanning and sampling.

Whole image scanning was used to capture images of four serial sections from each block of tissue. Images were captured using Olympus VS110 whole slide scanner running software (VS-ASW-FL, version 2.2, build 7667) and saved as TIF files. Lenses used were Olympus PlanApo N2 ×0.08 NA for the overview and Olympus PlanApo ×10 NA 0.40 for the image. The TIF files of the images were uploaded to a computer with VIS software version 3.4.1.5, using Modules Acquisition, MicroImager, NewCast, Automated Physical Disector, and Scan Imager (Visiopharm, Denmark). All stereological sampling was done using VIS software.

Estimation of alveolar components.

To estimate the number of alveoli, we identified alveolar openings into alveolar ducts, marked by their entrance rings forming the network-like duct wall. Network topology is represented by an invariant number: Euler characteristic χ = 1 − n, where n is the number of alveolar entrances. Counting the number of entrance rings in paired sections by the disector technique allows estimation of total number of alveoli in the lung (12, 14, 20). Two images were used from the serial sections (i.e., sections 1 and 3 with section 2 ignored, all 5-μm thick) that provided a disector height of 10 μm. Bridges and islands in the images are used for counting. Bridges are connections of interalveolar septa that appear in one section but not the other. The absence of a bridge represents an opening of the alveolus. Islands are a new isolated portion of septa. The calculations are based on the Euler characteristic of Δn = (I−B)/2, with I representing islands and B representing bridges counted in a disector both directions, hence divided by 2. The fractionator principle was used in conjunction with the Euler characteristic to determine the total number of alveoli in the lung (Nalv, lung) where n is the number of alveolar openings per sample and SF is total sampling fraction, the product of the fractions of tissue bars, bricks, heights, and areas that are sampled at each level (12): Nalv, lung = ∑Δn/SF

The height sampling fraction was estimated as a ratio of the disector height (10 μm) to the block width that was cut equal to the block height. The area sampling fraction was estimated as the ratio of the sampling field area to the field displacement to the next sampled field in xy using the VIS software.

The number-weighted mean alveolar volume (V̄n alv) was calculated as follows (10): V̄n alv = VL × Vv alv, lung/Nalv, lung

In this equation, VL is the total lung volume and Vv alv, lung is the volume density of alveoli in the lung. In this calculation, each alveolus is given an equal statistical “weight” even though volumes will be different. The number-weighted mean volume means “mean particle size” in the ordinary sense. These particles or alveoli in this case are sampled according to their presence.

Sampling of alveoli according to their volume results in estimates of volume-weighted mean alveolar volume (V̄v alv). Estimation of V̄v alv was determined by using a point-sampled intercept method using a CAST-grid system in VIS software. This estimates the volume of alveoli in the tissue that was sampled using isotropic uniformed random sampling. The estimation was determined by V̄v alv = (π/3) × IAS3 × (1/Fsgv), where IAS3 is the mean of the cubed point-sampled intercepts of alveoli. Fsgv is the factor of shrunken global volume, the units are in micrometers to the third power from Hyde et al. (12). In this calculation, each alveolus is weighted by its volume. Using the volume-weighted mean alveolar volume and number-weighted alveolar volumes, we calculated the coefficient of variation of the distribution of number-weighted mean alveolar volumes. This calculation was previously described (14) as CVnalv=(V¯valv/V¯nalv)1

This number is used to describe the variation in sizes of alveoli. The higher the number equates to greater variation of small and large alveoli, i.e., a more heterogeneous size distribution of alveoli.

Estimation of mean linear intercept.

Mean linear intercept (Lm) was measured using the equation Lm = 2·d·P(airspace)/I(a). This unbiased method uses a point and intersection method by using points that hit airspaces in parenchymal volume, P(airspaces), and counting intersections of a test line system with interalveolar septal surface, I(a) (10).

Estimation of alveolar surface.

Similar to Lm, surface area of interalveolar septal tissue in the lung was estimated using a point and intersection method (10). The surface of interalveolar septa/volume of lung (Sv IAS) was estimated as Sv IAS = 2 * IIAS/LLung, where IIAS is the number of intersections with interalveolar septa by a linear probe and LLung is the total probe length in sections of lung tissue (mm2/mm3) or millimeters to the first power.

The total surface of interalveolar septa within the lung (SIAS) was estimated as SIAS = Sv IAS *[1/(Fsgv)2/3] * VL, where (Fsgv)2/3 is the factor for shrunken global volume raised to the two-thirds power, and the units are converted to meter squared from Hyde et al. (12).

Estimation of the volume of parenchymal components.

One section from the serial sections of each block was sampled into a montage of images at 1,650 × 1,320 μm to ensure no overlapping of images. Each image was evaluated in Stereology Toolbox using a double lattice test system of 25 course/100 fine points (13). The lungs were evaluated for parenchymal and nonparenchymal components using point counting. Fine points were used to count rare features such as arteries, veins, and respiratory bronchioles, while course points were used to count common features such as alveoli, alveolar ducts, and interalveolar septa. The volumes of parenchyma (Vpar), alveoli (Valv), interalveolar septa (VIAS), alveolar duct core air (Vad), and nonparenchyma (Vnp) were determined by multiplying their volume densities (Vv) by lung volume (VL) in units of centimeters to the third power (12).The same method was used to estimate the volume of collagen and elastin in the parenchyma. Collagen was stained yellow with the pentachrome stain, while elastin was stained black (19). Movat Pentachrome stain has been used in other studies and verified to be comparable to immunoblotting and reverse transcriptase polymerase chain reaction for determination of elastin and collagen content (3). Fine points within the double lattice test system were used to count the points of elastin or collagen as well as interalveolar septal tissue. Collagen and elastin were expressed as a volume ratio to total interalveolar septal tissue in the lung.

Statistical analyses.

Statistical analyses were based on linear and nonlinear regression (SPSS; IMB, Armonk, NY). Age, VL, and body weight were used as predictors against the measured variables, and a series of models were fitted to each. Three functions were considered for each outcome: linear, quadratic, and a two-parameter exponential function. The models fitted to each outcome were linear: y =B0+B1Age+e; quadratic: y = β012Age2+e; and two-parameter exponential: y = B0exp(−B1Age)+e

Adjusted R2 value (Adj R2; coefficient of determination) was used to determine the best model fit. The test for R2 is whether it is statistically significant from zero. If the model is linear, it will decrease/increase in a uniform fashion. If it is either quadratic or exponential, it will change at a given moment indicating specific inflection points along the curve. For the linear and two-parameter exponential functions, the minimum R2 value is 0.19 for females and 0.22 for males for significance. For the quadratic function, the R2 value is 0.21. Sex was treated as a moderator of model parameters to allow for sex differences in all regression coefficients. Values for B0, B1, and B2 are listed in Tables for all measured parameters. These values represent the positive or negative interactions even if the regression coefficients may not be significant (see Tables 4 and 5). For all lung volume and body weight as predictors, the equations and regression analysis data can be found in Table 5. Along with regression coefficients, an ANOVA was run for each regression line with significance defined as P < 0.05. A one-way ANOVA was performed to compare young versus old rhesus macaques in number of alveoli. In models where both male and female had a significant regression coefficient, an F test was calculated using residual sum of squares for each gender regression (Rss1 and Rss2) plus a combined gender regression (Rss3), the degrees of freedom for each gender regression (dfe1+dfe2) and the change in degrees of freedom (Δp) to determine a significant difference between lines: F=[Rss3(Rss1+Rss2)]/Δp[Rss1+Rss2]/(dfe1+dfe2)

A t-test was also used on the same parameters to determine differences between young and old animals in the same sex. Animals were divided into young animals (17 yr and younger) and old animals (18 yr and older) and the mean value of the parameter was used in the t-test to determine whether there was a significant difference between young and old. All values for the t-test are listed in Table 2. Significance was determined if P < 0.05. We used SPSS Statistics 19 (IBM) for all statistical analysis.

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Table 2.

Comparison of means of estimated parameters for both sexes using a t-test

RESULTS

General characteristics.

All rhesus macaques (Macaca mulata) for this study were colony-born from the California National Primate Research Center and maintained in outdoor enclosures for their lifetime. Ages in females ranged from 9 to 33 yr and males ranged from 9 to 29 yr old (Table 3). Lung volume (VL) in females ranged from 409 to 675 cm3 with a mean of 476.61 cm3. VL in males ranged from 387 to 787 cm3 with a mean of 592.31 cm3 (see Table 3). Body weight in females ranged from 4.13 to 10.99 kg with a mean of 7.87 kg. Body weight in males ranged from 7.03 to 12.37 kg with a mean of 9.30 kg (see Table 3).

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Table 3.

Descriptive statistics of the morphological parameters in rhesus monkeys

General age-related changes.

This study focuses on parenchymal sections of the lung which included interalveolar septa, airspace, and small arteries and veins. The anatomical features of the aging lung that were examined included: alveoli, alveolar ducts, respiratory bronchioles, veins, and arteries. Elderly female lungs appeared different from males due to larger alveoli as well as fewer alveoli and increased size of alveolar ducts. Appearance of alveolar ducts and alveoli changed significantly between ages 9 and 33 in both males and females. In Fig. 1, alveolar ducts appeared as longer, winding passages which branched down to alveoli that appear like small sacs. The younger animals appear to have a more uniform alveolar size and distribution while the older females have far larger alveoli and an increase in the volume of alveolar duct. The lungs of males still retain some smaller alveoli, while the female does not appear to do so. The older animals appear to have larger pores of Kohn in both males and females. As seen in Fig. 1C, the alveoli in an older female have several openings that can also be seen in Fig. 1C, insert. These may be the result of alveolar destruction or an increase in pore size. Respiratory bronchioles appeared not to change during the aging process. Larger airways were not included in this study. Small arteries were ascertained by their association with bronchioles as well as containing smooth muscle in the walls. Veins were typically smaller and rectangular in shape and not associated with airways. The older animals showed an increase in vascular wall thickness; most notably in the smooth muscle of arteries.

Fig. 1.

Representative lung histological images from hematoxylin and eosin-stained samples that illustrate the increase in alveolar size and corresponding decrease in alveolar number with age especially in the female. A: sample from 10-yr-old female. B: sample from 10-yr-old male. C: sample from 33-yr-old female. Note the large increases in alveolar size evident in this elderly female. D: sample from 29-yr-old male. Bar = 1 mm; inset bar = 200 μm.

Alveolar components.

The number of alveoli decreased with age and in females declined from 544 million to 168 million while in males, the decrease was from 492 million to 299 million. The loss of alveoli relative to age was best fit by a quadratic function (Adj R2 = 0.786 for females and 0.216 for males), indicating a gradual decline at first that became slightly steeper with age. This quadratic function was observed in both males and females with a significantly greater slope in females (Fig. 2). Using an F test, we determined that the regression line for females was significantly different than males, indicating a greater rate of decline in females. The lower Adj R2 values indicated that there was no correlation between the number of alveoli and body weight or with lung volume. We divided the adult age groups at 18 yr to designate young versus elderly. The mean of the number of alveoli were compared in both males and females by using a t-test. Females were found to have a significant difference between young and old with P < 0.001 (Table 2). There was no significant difference in males between young and old animals (P = 0.103).

Fig. 2.

Number of alveoli (represented in log format) has a quadratic function in decreasing during the aging process with a significant decrease in females compared with males. The log of number of alveoli in the lung (Nalv, lung) vs. age (yr) for females (● with solid line) and males (◇ and dotted line) are plotted according to a quadratic function (Table 4).

The volume of alveolar air showed a linear decrease for females in relation to age that was significant (Adj R2 = 0.296), while males did not show any correlation between volume of alveolar air and age (Fig. 3), lung volume, or body weight (Tables 4 and 5). The number-weighted volume of alveoli had a quadratic function in females with a significant positive correlation with age (Adj R2 = 0.797), but no significant correlation was found in males (Fig. 4 and Table 4). Females were found to have a significant difference between young and old in the number-weighted volume of alveoli with P < 0.001; however, there was no significant difference in males between young and old animals (P = 0.162) (Table 2). The changes in the coefficient of variation of the number-weighted volume of alveoli over time were best fitted to a quadratic curve (Adj R2 =0.317) in that at age 9 for females, the variance is small, increases until about the age of 20, and then decreases again. Males did not have a significant correlation between age and coefficient of variation of the number-weighted volume of alveoli (Fig. 5 and Table 4). However, neither females nor males showed a significant difference between young and old animals (Table 2).

Fig. 3.

Both males and females indicate a linear decrease in volume of alveolar air during the aging process with no significant difference between the two. The cumulative volume of alveolar air in the lung (Valv) (cm3) vs. age (yr) for females (●, solid line) and males (◇, dotted line) are plotted according to a linear function (Table 4).

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Table 4.

Maximum likelihood estimates for models based on age

Fig. 4.

Females have a significant increase in the number-weighted mean volume of alveoli compared with males during the aging process. The number-weighted mean volume of alveoli (V̄n alv)(cm3) vs. age (yr) for females (●, solid line) and males (◇, dotted line) are plotted according to a quadratic function (Table 4).

Fig. 5.

Coefficient of variation (CV) shows a significant change over time for females vs. males. During early adulthood, females have uniformly small alveoli and slowly change to uniformly large alveoli in adulthood. The CV of the distribution of the number-weighted mean alveolar volume (CVn alv) vs. age (yr) for females (●, solid line) and males (◇, dotted line) are plotted according to a quadratic function (Table 4 ).

The volume of alveolar duct in females had a significant positive correlation between age (Adj R2 = 0.821), while males did not (Fig. 6 and Table 4). Females were found to have a significant difference between young and old in the volume of alveolar duct with P < 0.001; however, there was no significant difference in males between young and old animals (P = 0.085) (Table 2). Interalveolar septal tissue was assessed by measuring both volume and surface area. Surface-to-volume ratio of interalveolar septal tissue (Sv IAS) followed a linear decline in both males and females. Males had a significant correlation with age (P < 0.05), while females showed near significance of correlation at P = 0.073 (Fig. 7 and Table 4). Neither females nor males showed a significant difference between young and old animals for Sv IAS (Table 2). Mean linear intercept (distance between interalveolar septa) had a linear function with relation to age in both males and females (Fig. 8 and Table 4). There was a significant positive correlation in both males (Adj R2 = 0.251) and females (Adj R2 = 0.308) regarding mean linear intercept and age, but no difference between sexes. Neither females nor males showed a significant difference between young and old animals for Lm (Table 2). Total surface area of interalveolar septal tissue in the lung (SIAS) was not significantly changed in males or females between young and old (Table 2), but there was a positive correlation to age with a significance level of P = 0.08 in females (Table 4). This indicates some compensatory growth with increased lung volume (the reference volume) even though there was a marked decline with age in Sv IAS.

Fig. 6.

Females have a significant exponential increase in the volume of alveolar duct as they age compared with males. The cumulative volume of alveolar duct core air in the lung (Vad) (cm3) vs. age (yr) for females (●, solid line) and males (◇, dotted line) are plotted according to a quadratic function (Table 4).

Fig. 7.

Both males and females lose surface area of interalveolar septa per volume of lung at a similar linear rate during the aging process. The surface of interalveolar septa per lung volume vs. age (Sv IAS, Lung) (yr) for females (●, solid line) and males (◇, dotted line) are plotted according to a quadratic function (Table 4).

Fig. 8.

Mean linear intercept (Lm) of alveoli in the lung increases linearly for both males and females at the same rate during the aging process. Lm of alveoli vs. age (yr) for females (●, solid line) and males (◇, dotted line) are plotted according to a quadratic function (Table 4).

Extracellular matrix.

Alveoli have been stained using a pentachrome procedure previously described in materials and methods. Elastin is stained black and is represented by thin, black fibers. Collagen is stained yellow and is observed mostly in the older animals and around the opening rings of the alveoli. The volume of collagen followed a two-parameter exponential increase with significant P values in relation to age in both males (Adj R2 = 0.404) and females (Adj R2 = 0.553) (Fig. 9 and Table 4). The volume of elastin decreased in a quadratic function for both males and females (Fig. 10 and Table 4). Only females had a significant correlation relative to age (Adj R2 = 0.338). There was no significant sex difference between males and females regarding age for both collagen and elastin (Table 4). Both females and males showed a significant difference between young and old animals for the volume of collagen, while only females showed a significant difference between young and old animals for the volume of elastin (Table 2). Lung volume was significantly correlated with the volume of collagen in both females and males, while there was only a significant correlation between lung volume and the volume of elastin in females (Table 5). Body weight only showed a negative correlation with the volume of elastin in females (Table 5).

Fig. 9.

Volume of collagen in both males and females increase exponentially in both males and females as they are in a similar rate. The cumulative volume of collagen in interalveolar septal tissue in the lung (Vc) (cm3) vs. age (yr) for females (●, solid line) and males (◇, dotted line) are plotted according to a quadratic function (Table 4).

Fig. 10.

Volume of elastin in lung parenchyma decreases in a similar linear rate in both males and females during the aging process. The cumulative volume of elastin in interalveolar septal tissue of the lung (Ve) (cm3) vs. age (yr) for females (●, solid line) and males (◇, dotted line) are plotted according to a quadratic function (Table 4). The amount of elastin in the parenchyma decreases with age in both males and females. There is no difference between males and females.

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Table 5.

Maximum likelihood estimates for models based on lung volume and body weight

It is not surprising that lung volume and body weight were not different between young and old females and males (Table 2), because full somatic growth is completed at ∼7 yr of age in rhesus macaques (6). During postnatal development of rhesus macaques, lung volume is the best predictor of alveolar number; alveoli are added as basic units without a change in the mean number-weighted alveolar volume with increasing lung volume (12). Surprisingly, in the aging lung, the number-weighted mean alveolar volume showed a significant positive correlation with lung volume, but not body weight in both males and females (Table 5).

DISCUSSION

This study shows that female rhesus macaques have significant morphometric changes in the lung associated with aging compared with males. The number of alveoli decreased in both males and females but with a significantly enhanced response to aging in the females. With the significant loss of alveoli, we also detected a significant linear decrease in the volume of alveolar air for females in relation to age, but not for males. The loss in volume of alveoli and the gain in volume of alveolar ducts showed a significant positive correlation with age in females, but not in males. The decreased surface area of interalveolar septal tissue and the increase in the mean linear intercept showed that the loss of alveoli, the increase in alveolar size, and the subsequent increase in the volume of alveolar duct is due to a loss of septal tissue. These findings together with the changes in collagen and elastin indicate significant structural decrements are associated with the aging lung and most of these changes are more pronounced in females.

The number of alveoli in an adult human lung is estimated to be between 200 and 600 million (24). In a more recent study using design-based stereological methods, the authors showed a mean alveolar number of 480 (ranged between 274 and 790 million) with a clear sexual dimorphism and a number-weighted mean alveolar volume of 4.2 × 106 μm3 (19). In this study of the adult rhesus macaque, the alveolar number ranged between 350 and 544 million, which is within the human's range. With smaller lung volumes than humans, rhesus macaques have smaller alveoli than humans (Table 2). In this study, the data indicate that as rhesus macaques age, they lose a significant number of alveoli, with females declining faster than males (Fig. 2). Based on the predicted values obtained from using the equation in Table 4, females started at age 9 with 20% more alveoli but had 34% less alveoli by age 33. Females lost roughly half their alveoli, starting at 480 million in the younger animals and ending with as few as 168 million by age 30.

The difference in alveolar number and size is illustrated in Fig. 1. In both the young male and female macaques, the alveoli appear uniform in size and distribution. In elderly male macaques, some of the uniform smaller alveoli are still present but an increase in the volume of alveolar duct becomes more distinctive. In contrast, the lungs from elderly female macaques display numerous changes, most notably the increase in alveolar size, possibly due to loss of septal tissue. Total volume of alveolar air in the lung decreases with age in both males and females even though the size of individual alveoli increases. Alveoli become wider and shallower in depth potentially due to loss of individual septa. This corresponds with the change in the number-weighted volume of alveoli; the volume of alveoli increases significantly in females.

There is also a corresponding increase in alveolar duct air, indicating a shortening of interalveolar septal tissue (24). The loss of alveoli coupled with overall loss of interalveolar septal tissue can result in a loss of diffusing capacity in the human lung (20, 29). In the rhesus macaque, we see a similar morphological pattern with age. As seen in Fig. 6, the volume of alveolar duct increases in a linear fashion with age in males and females. In females, the volume of alveolar duct is significantly less than males at approximately age 10 yr, but the linear change rate with age is significantly higher than males resulting in a volume of alveolar duct that was larger than males after ∼23 yr of age.

In this study, we focused only on the collagen and elastin content within interalveolar septa (Fig. 10). Our measurements demonstrated an increase in collagen with a corresponding decrease of elastin in the lung parenchyma. Morphologically, elastin fibers appeared to be unraveled, similar to previous research in human senile lungs (28). In general, elastin has been seen in other organs to undergo fragmentation and thinning with age. This fragmentation would cause ectasia of elastin and a transfer of mechanical load to collagen, which is significantly stiffer than elastin (8). This change in the elastin-to-collagen ratio, if confirmed in the human lung, could explain the decrease in static elastic recoil pressure in the senile human lung. While these same morphological observations and measurements were observed in rhesus macaque lungs, there were no significant differences between males and females regarding changes in age of elastin and collagen. Both males and females showed similar changes in matrix components with age.

The number of alveoli within the lung is the primary parameter estimated in this study that accurately reflected changes in lung structure associated with age in the rhesus macaque. Importantly, the methodology used is not influenced by inflation or shrinkage and combined with the robust sampling method is efficient and unbiased. This methodology and lack of inflation or shrinkage variance may explain why mean linear intercept and surface density of interalveolar septa did not show differences between sexes. The loss of alveoli documented here is notable, at least in part, because of similarity to diseases in the aged human lung. In addition, the data show that the alveolar changes in female rhesus monkey lungs were more strongly correlated to aging than the changes in males (Table 4). Studies have shown that women account for 85% of nonsmoking chronic obstructive pulmonary disease cases (9). Women that do smoke have a faster lung decline over 45 yr of age (5). This increased susceptibility may be due to the increased structural decrements with age.

Defining the mechanism(s) leading to the loss of alveoli may provide therapeutic targets to treat these pulmonary disorders. Estrogen has been shown to be a key hormone in lung growth and development as well as in repair processes of interalveolar septa (1, 16). Hormone replacement therapy with estrogen has been used to prevent lung inflammation and enhance regeneration of alveoli in mice (15, 17, 27). Further research should focus on the loss of estrogen after menopause and its impact on the aging lung in females. In conclusion, this study provides further evidence that the rhesus monkey is a good model for the aging lung in humans, and we have used this model to demonstrate that the lungs of females have accelerated loss of alveoli compared with males.

GRANTS

This work was supported by National Institutes of Health Grants P01-ES-00628 and P51-OD-011107.

DISCLOSURES

No conflicts of interest, financial or otherwise are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: M.J.H., M.V.A., and D.M.H. conception and design of research; M.J.H., C.L.Q., and L.F.P. performed experiments; M.J.H., C.L.Q., L.F.P., N.K.T., F.F.V., and D.M.H. analyzed data; M.J.H., M.V.A., F.F.V., J.A.S.G., and D.M.H. interpreted results of experiments; M.J.H. prepared figures; M.J.H. drafted manuscript; M.J.H., M.V.A., C.L.Q., L.F.P., N.K.T., F.F.V., J.A.S.G., and D.M.H. edited and revised manuscript; M.J.H., M.V.A., C.L.Q., L.F.P., N.K.T., F.F.V., J.A.S.G., and D.M.H. approved final version of manuscript.

ACKNOWLEDGMENTS

The support of Primate Services at the California National Primate Research Center for animal handling, care, and necropsy support and especially the efforts of Sona Santos were critical to this study and are gratefully acknowledged.

REFERENCES

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