Walter S. Tyler

Comparison of daily and seasonal exposures of young monkeys to ozone

Oxidant air pollution tends to occur in both seasonal and daily cycles of polluted and clean air. To compare the effects of these 2 cycles, we exposed 2 groups of 7-month-old male monkeys to 0.25 ppm (0.49 mg/m3) of ozone (UV photometric standard) 8 h/day either daily or, in the seasonal model, days of alternate months during a total exposure period of 18 months. This is a longer ozone exposure period than any previously reported. A control group breathed only filtered air. Young monkeys were studied as their lungs are similar to those of man and their lungs, like those of man, grow over a period of several years. Monkeys from the seasonal exposure model, but not those exposed daily, had significantly increased total lung collagen content, chest wall compliance, and inspiratory capacity. All monkeys exposed to ozone had respiratory bronchiolitis with significant increases in related morphometric parameters. The only significant difference between seasonal and daily groups was in the volume fraction of macrophages. Even though the seasonally exposed monkeys were exposed to the same concentration of ozone for only half as many days, they had larger biochemical and physiological alterations and equivalent morphometric changes as those exposed daily. Lung growth was not completely normal in either exposed group. Long-term effects of oxidant air pollutants which have a seasonal occurrence may be more dependent upon the sequence of polluted and clean air than on the total number of days of pollution. Estimations of the risks of human exposure to seasonal air pollutants from effects observed in animals exposed daily may underestimate long-term pulmonary damage.

Respiratory bronchiolitis following long-term ozone exposure in bonnet monkeys: a morphometric study.

To quantitate the response of respiratory bronchiolar (RB) epithelium and peribronchiolar connective tissue (PCT) to chronic exposure to high ambient levels of ozone, two groups of 8 adult male bonnet monkeys each were subjected 8 h daily for one year to 0.64 ppm (UV standard) ozone or filtered air, respectively. Blocks of tissue selected throughout the lung and from first generation RBs following airway microdissection had the following significant exposure-related changes: 57% greater volume of RB in the lung, 27% smaller diameter of RB lumen, 179% thicker media and intima of peribronchiolar arterioles, 61% thicker RB epithelium, and 77% thicker PCT. The increase in thickness of the RB wall resulted primarily from an 84% increase in PCT, with the remainder from the epithelium. Estimates of cellular numerical density showed an 81% increase in cuboidal bronchiolar cells and an 87% decrease in type 1 pneumocytes in the exposed group. Cell volumes from serial section reconstruction showed significantly larger cuboidal bronchiolar (79%), ciliated (117%), and type 2 (66%) cells over controls. Significant PCT changes included more amorphous extracellular matrix (288%), neutrophils (1523%), and lymphocytes/plasma cells (307%). The number of fibroblasts and the volume of extracellular fibers were larger than control values by 44% and 31% in the exposed group, but these changes were not statistically significant. Centriacinar changes due to exposure to long-term, high ambient ozone in bonnet monkeys results in narrowing of respiratory bronchioles primarily by peribronchiolar inflammation (inflammatory cells, fibers, amorphous matrix) and secondarily through hyperplasia of cuboidal bronchiolar cells.

Comparative aspects of the strength of pulmonary capillaries in rabbit, dog, and horse.

In previous studies of rabbit and dog lung, we demonstrated stress failure of pulmonary capillaries at high transmural pressures (Ptm). The Ptm necessary to elicit stress failure was 40 cmH2O higher in dog than rabbit, and the total blood-gas barrier (BGB) thickness was greater in dog than rabbit. This suggests that stress failure may be related to BGB thickness, and is consistent with the Laplace relationship which states that wall stress is proportional to capillary radius but inversely proportional to wall thickness. In the present studies, we compared BGB thickness and an index of capillary radius in lungs from 3 rabbits, 3 dogs, and 2 horses perfusion fixed at a Ptm of approximately 30 cmH2O. Thicknesses of the BGB were measured at right angles to the barrier at random points on the capillary wall determined by test line intersections. Capillary radius was determined from the mean of major and minor axes measured on electron micrographs. Capillary pressure for failure in the horse was taken to be the mean of pulmonary arterial and left atrial pressures observed in galloping thoroughbreds known to develop exercise-induced pulmonary hemorrhage, although the actual pressure required for failure may be less than this. Average capillary radii were 3.6, 3.4, and 3.2 microns for rabbits, dogs, and horses, respectively. We found that the BGB was thinnest in the rabbit, intermediate in the dog, and thickest in the horse. Calculated capillary wall stress values for the median total BGB thickness at a nominal Ptm of 30 cmH2O were 2.5 x 10(4), 1.7 x 10(4), and 1.5 x 10(4) N.m-2 for rabbits, dogs, and horses, respectively. This species ranking fits with the pressures required to cause stress failure which are approximately 50, 90, and 130 cmH2O in rabbit, dog, and horse, respectively. We conclude that the differences in capillary radius of curvature and BGB thickness account for some of the observed differences in Ptm necessary to cause stress failure. However, other factors may also be important in determining the strength of the BGB.

Ozone-induced Adaptive and Reactive Cellular Changes in Respiratory Bronchioles of Bonnet Monkeys

To characterize the response of respiratory bronchioles (RBs) to chronic high ambient levels of ozone, bonnet monkeys were exposed for 90 days to 0, 0.4, or 0.64 ppm ozone (UV photometric standard; 3 monkeys/exposure). Morphologic changes in respiratory bronchiolar epithelium and interstitium were evaluated quantitatively at both the light and transmission electron microscopic levels. Significant changes in respiratory bronchioles following exposure included: a thicker wall and a narrower lumen, a thicker epithelial compartment and a much thicker interstitial compartment, shifts in epithelial cell populations with many more nonciliated bronchiolar epithelial cells and fewer squamous type I epithelial cells, larger nonciliated bronchiolar epithelial cells with a larger complement of cellular organelles associated with protein synthesis, greater amounts of both interstitial fibers and amorphous ground substance, greater numbers of interstitial smooth muscle cells per epithelial basal lamina surface area, and greater volumes of interstitial smooth muscle, macrophages, mast cells, and neutrophils per epithelial basal lamina surface area. These observations imply that chronic ozone exposure causes a concentration-dependent reactive peribronchiolar inflammatory response and an adaptive response consisting of hypertrophy and hyperplasia of the nonciliated bronchiolar cell.

Long-term consequences of exposure to ozone. I. Lung collagen content

Lung collagen content of rats and monkeys (Macaca fascicularis) exposed to ozone for 1 to 13 weeks and for 1 year, respectively, was quantified by measurement of 4-hydroxyproline in hydrolysates of whole lungs. In addition, ratios of type I to type III collagen in the lungs of the same monkeys were also evaluated by cyanogen bromide peptide mapping techniques. We observed elevated levels of collagen in lungs of both species of animals exposed to ozone. We conclude that elevations in collagen synthesis rates in lungs of rats and monkeys acutely exposed to high levels of ozone are reflected by corresponding increases in lung collagen content over subchronic and chronic time frames. Preliminary results on young rats also suggest that removal of rats from atmospheres containing ozone does not cause reversal of such increases in lung collagen content. To the contrary, recovery periods of up to 6 weeks seem to exacerbate the observed increases in lung collagen content.

Age-related morphometric differences in responses of rat lungs to ozone

The influence of age on morphologic changes in lungs of rats exposed to ozone was studied in female Sprague-Dawley rats, aged 60 and 444 days. Rats of both age groups were exposed continuously for 72 hr to either 0.35 or 0.80 ppm ozone, or to filtered air. Tissues were evaluated using light microscopic morphometry and scanning electron microscopy. The lungs from ozone-exposed 60-day-old rats had larger volume fractions of centriacinar lesions than lungs from exposed 444-day-old rats. Within each age group there was an observed dose response, with rats exposed to 0.80 ppm ozone having larger volume fractions of lesions than those exposed to 0.35 ppm. Only the 444-day-old rats lost body weight during the exposure period. They also had smaller fixed lung volumes than same-aged controls. All 60-day-old rats gained weight during the exposure period, although rats exposed to 0.80 ppm ozone gained less than filtered air controls. Lesions observed in both age groups of female rats were qualitatively similar to those previously described in young adult male rats. We conclude that there are age-related differences in the morphometric responses of rats to ozone exposure. Younger rats had larger proportions of centriacinar lesions and macrophages while older rats had greater body weight and lung volume changes.

Morphometry of In Situ and Lavaged Pulmonary Alveolar Macrophages from Control and Ozone-Exposed Rats

Effects of ambient levels of ozone on cell size and compartments were determined morphometrically for both in situ and lavaged pulmonary alveolar macrophages from rats exposed to filtered air or to filtered air with 0.60 ppm ozone. The ozone exposure was 8 hr/day for 3 days. Significant exposure-related compartmental volume density changes of in situ centriacinar macrophages were: decreased endoplasm (p less than 0.01); increased lysosome-like structures (p less than 0.01); decreased primary lysosomes (p less than 0.01); increased small and large secondary lysosomes (p less than 0.001); and decreased phagosomes/autophagosomes (p less than 0.05). In lavaged macrophages, the only significant exposure-related change was an increase in the density of large secondary lysosomes (p less than 0.01). Mean profile areas of in situ centriacinar macrophages from control and exposed rats were 86.94 micrometers2 and 112.04 micrometers2, respectively. The average mean cell volume V and mean caliper diameter D of macrophages lavaged from control rats were 1128.45 micrometers3 and 12.92 micrometers, respectively, whereas those from exposed rats were 1583.08 micrometers3 and 14.46 micrometers, respectively. Exposure-related increases in cell size were seen in both in situ and lavaged macrophages, but more significant differences in cell compartments were seen in the in situ centriacinar macrophages. Morphometry of pulmonary alveolar macrophages after ambient levels of ozone indicated increased uptake, storage, or both rather than cell damage. Comparison of in situ centriacinar and lavaged macrophages from both control and exposed rats revealed significant differences in their volume fractions of nucleus, cytoplasm, ectoplasm, mitochondria, lysosome-like structures, lipid droplets, vacuoles, and phagosome/autophagosomes. These differences between centriacinar and lavaged macrophages indicate different cell populations are sampled by these two methods.

Structural Evaluation of the Respiratory System

The theoretical and practical bases for morphological evaluation of the respiratory system useful for inhalation toxicology are reviewed. For most studies we recommend a comprehensive gross examination followed by in vitro tracheal infusion of a fixative containing both glutaraldehyde and formaldehyde in cacodylate buffer. Lungs fixed in this manner are suitable for LM, SEM, and TEM and lung volumes can be determined. The airway orientation of many lesions and the potential for gradients of damage are considered in the lung sampling plan. While LM of paraffin sections continues to be the basic method for evaluation, the SEM and TEM, especially when ancillary methods are used, provide valuable additional information. The use of backscattered electrons and energy-dispersive X-ray analysis in the SEM provides information concerning the localization and elemental analyses of particles. Cytochemical procedures characterize biological activities of specific cell types and are becoming more widely used. Morphometry permits correlation of quantified structure with physiological and biochemical data.

Effects of ozone on lung and somatic growth. Pair fed rats after ozone exposure and recovery periods

Minor differences in lung growth and development during childhood have been considered as a potential cause of rapid decline in pulmonary function in adulthood. Inhalation of ozone commonly causes changes in both body weight and lung volumes, which complicates interpretation of any changes in lung growth. The effects of ozone on lung growth were studied in rats which were pair fed. This technique permitted comparison of ozone-exposed and filtered-air control rats of the same body weight and body size as well as age and sex. Exposure was to filtered air or to 0.64 or 0.96 ppm ozone (UV standard) 8 h/night for 42 nights. A second control group was fed ad libitum and exposed to only filtered air. Half the rats were studied at the end of the 42-night exposures, the rest after a 42-day post-exposure period during which all rats were fed ab libitum and breathed filtered air. Rats examined at the end of the exposure period had larger saline and fixed lung volumes. These larger lungs had greater volumes of parenchyma, alveoli and respiratory bronchioles. Some of these changes persisted throughout a 42-day post-exposure period. Ozone inhalation by young rats alters lung growth and development in ways likely to be detrimental and those changes persist after ozone inhalation stops.

Composite Vascularized Free-Rib and Pleural Transfer for Laryngotracheal Reconstruction

A free rib pleura composite vascularized transfer for repair of a defect in the canine upper airway is discussed and the results are reported. The procedure was carried out on eight dogs. One dog died from the anesthetic. Three surgical failures resulted from technical difficulties: failure of the vascular anastomosis, torsion of the vascular pedicle, and hemorrhage. Four dogs successfully maintained their airways, although one demonstrated bone absorption. Bone scanning, angiography, tetracycline labeling, and light microscopy confirmed active osseous metabolism of the rib, providing further evidence of the adequacy of periosteal circulation in maintaining normal rib metabolism. Scanning electron microscopy and conventional light microscopy demonstrated the development of a healthy respiratory epithelium. Thus a free flap of rib and attached pleura will provide adequate skeletal support and a moist, functioning, epithelial lining in a single-stage operation for reconstruction of a laryngotracheal defect in the normal dog.