Response of the macaque nasal epithelium to ambient levels of ozone. A morphologic and morphometric study of the transitional and respiratory epithelium.

Although ozone (O3)-induced bronchiolitis has been morphologically characterized, effects of O3 on the upper respiratory tract have not been thoroughly investigated. The purpose of this study was to determine whether exposures to ambient levels of O3 induce lesions in the nasal mucosa. Bonnet monkeys were exposed to 0.00, 0.15, or 0.30 ppm O3 for 6 or 90 days, 8 hours/day. After exposure, nasal mucosa was processed for light and electron microscopy. Quantitative changes were evident in the nasal transitional and respiratory epithelium. At 6 or 90 days of exposure to 0.15 or 0.30 ppm O3 lesions consisted of ciliated cell necrosis, shortened cilia, and secretory cell hyperplasia. Inflammatory cell influx was only present at 6 days of exposure. Ultrastructural changes in goblet cells were evident at 90 days. Ambient levels of O3 can induce significant nasal epithelial lesions, which may compromise upper respiratory defense mechanisms.     

Differentiated bronchiolar epithelium in alveolar ducts of rats exposed to ozone for 20 months.

The effects of exposure to 1.0 ppm of ozone for twenty months were studied in male Fischer 344 rats. Light microscopic, morphometric, and immunohistological approaches were used to determine the distribution and degree of differentiation of ciliated and nonciliated bronchiolar epithelial (Clara) cells lining alveolar ducts of the central acinus, a primary target of ozone-induced lung injury. Alveolar duct pathways extending beyond the level of the most proximal alveolar outpocketing of terminal bronchioles were isolated in longitudinal profile. The distance that ciliated and nonciliated bronchiolar epithelial (Clara) cells projected down each alveolar duct pathway was determined by placing concentric arcs radiating outward from a single reference point at the level of the first alveolar outpocketing. A high degree of heterogeneity in the magnitude of bronchiolar epithelial cell extension into alveolar ducts was noted for each isolation and animal. Age-matched control animals also demonstrated variation in the degree of bronchiolar epithelial cell extension down alveolar ducts. In animals exposed to ozone, a striking similarity was noted by scanning electron microscopy in the surface characteristics of cells lining both terminal bronchioles and alveolar ducts. The presence of Clara cell secretory protein in cells of bronchioles and alveolar ducts was also detected immunohistochemically and visualized using confocal laser scanning microscopy in the reflectance mode. Well-differentiated ciliated and nonciliated bronchiolar epithelial cells were found lining alveolar septal tips and alveoli up to a depth of 1,000 mu into the pulmonary acinus after 20 months of exposure to ozone. No evidence of inflammation was present in alveolar ducts, suggesting that epithelial cell transformations in alveolar ducts is a natural consequence of lifetime exposures to oxidant gases.     

The response of the macaque tracheobronchial epithelium to acute ozone injury. A quantitative ultrastructural and autoradiographic study.

The tracheal epithelium of a variety of laboratory species is widely used as a model system in studies of epithelial biology and respiratory carcinogenesis. The purpose of this study was to evaluate the response of the tracheal epithelium to cytotoxic injury in a primate species that may have an epithelium more representative of that in man than smaller laboratory species. This study evaluated changes in the light-microscopic, surface, and ultrastructural appearance of the tracheobronchial epithelium of bonnet monkeys exposed for 3 or 7 days to 0.64 ppm ozone. Population densities, epithelial volumetric densities, and thymidine labeling indexes were determined for cells from posterior membranous and anterior cartilaginous trachea and mainstem bronchus. Ozone-induced epithelial changes were characterized by decreased numbers of ciliated cells, loss of cilia, and necrosis of ciliated cells. There were alterations in mucous (goblet) cell granules. There was an increase in extracellular space and focal epithelial stratification that was associated with increased numbers of small mucous granule cells and the presence of an epithelial cell type not seen in control animals (intermediate cells). There was an increase in cytoplasmic filaments and desmosomal attachments in basal cells, small mucous granule cells, and intermediate cells. Regional differences in lesion distribution were demonstrated by scanning electron microscopy. Longitudinal streaks of ciliary loss were evident in posterior membranous trachea, but ciliary loss in the ventral trachea was most prominent over the posterior border of the cartilaginous rings. The thymidine labeling index and numbers of necrotic ciliated cells were greater after 3 days than after 7 days of continuous exposure. Foci of stratification were often associated with increased numbers of labeled nuclei in the suprabasal region of the epithelium. The results of this study suggest that small mucous granule cells and intermediate cells are important participants in the repair of chemically injured airway epithelium; stratification and increased amounts of cytoplasmic filament bundles and desmosomal attachments, rather than being evidence of squamous metaplasia or dysplastic change, might be stereotypic responses of airway epithelium to injury; and the ciliated cell population becomes less susceptible to ozone-induced necrosis with continuing exposure.     

Reflex-mediated desquamation of bronchiolar epithelium in guinea pigs exposed acutely to sulfuric acid aerosol.

Terminal conducting airways are known to be vulnerable to direct injury by a variety of noxious aerosols. Sulfuric acid aerosol, a by-product of fossil fuel combustion, produces desquamation of terminal bronchiolar epithelium in guinea pigs that is believed to result from direct deep lung irritation, an effect separable from reflex airway constriction induced by sulfuric acid. To characterize desquamation of bronchiolar epithelium, 20 guinea pigs were exposed to 32.6 mg/cu m sulfuric acid aerosol with a mass median aerodynamic diameter of 1.0 micron for 4 hours. The guinea pigs were killed upon termination of the exposure, or 24 hours later, and airway alterations were evaluated by light and transmission electron microscopy. To test whether the development of bronchiolar epithelial desquamation is independent of reflex airway constriction, 24 guinea pigs were exposed to an identical aerosol for 4 hours after pretreating half with 5 mg/kg atropine sulfate intraperitoneally to inhibit airway constriction. Sulfuric acid produced diffuse pulmonary hyperinflation with areas of consolidation and atelectasis. Epithelial desquamation occurred in airways supplying regions of developing atelectasis and was most extensive in terminal bronchioles. Parasympathetic effector blockade with atropine eliminated epithelial desquamation. These results indicate that sulfuric acid-produced desquamation of terminal bronchiolar epithelium is not separable from reflex airway constriction and that terminal conducting airways are vulnerable not only to direct injury by noxious aerosols but also to indirect, reflex-mediated injury.     

Atypical cilia in the bronchiolar epithelium of pigs experimentally infected with hog cholera virus.

To study the effect of hog cholera virus on the epithelial cells of the bronchiolar mucosa, 12 pigs were inoculated with a highly virulent strain. Immunohistochemical and ultrastructural examination of the ciliated epithelial cells demonstrated an increase in the number of atypical cilia. The latter showed alterations in the microtubular pattern, possibly resulting from viral interference with the normal metabolism of the epithelial cells.     

Effects of ischaemia and preservation on the ultrastructure of the bronchiolar epithelium

In ten cases of clinical human single-lung transplantation, the nontransplanted Euro-Collins-preserved contralateral lungs were examined using electron microscopy to determine the effects of ischaemia on the bronchiolar epithelium. Existing structural damage at the time of transplantation was characterized using this approach, and nine nonpreserved canine single lungs were also investigated to identify the impact of ischaemia. The study revealed a significant correlation between the duration of ischaemia and the mitochondrial surface-to-volume ratio, which can serve as a morphometric criterion for mitochondrial damage, in canine lungs. However, this correlation was not found in the human donor lungs. Further examination of human donor lungs showed slight to moderate damage to the endoplasmic reticulum and nuclear chromatin. In addition, various degrees of damage to mitochondrial structure, ranging from inconspicuous to severe, were found. The mitochondrial surface-to-volume ratio can be considered to be a suitable criterion for the quantification of ischaemic damage of the bronchiolar epithelium under experimental conditions. Ultrastructural analysis of human donor lungs revealed intact bronchiolar epithelial cell structures at the time of transplantation, reflecting adequate organ preservation with Euro-Collins solution.     

Fluid-overload pulmonary oedema in mice: the intercellular junctions of bronchiolar epithelium and arterial endothelium.

It has been reported previously that fluid leaks through the intercellular junctions of arterial endothelium and bronchiolar epithelium in fluid-overload pulmonary oedema in mice. In this study, freeze fracture technique was used to see morphological changes in the intercellular junctions at these sites. In arterial endothelium, the intercellular junction consists of a combination of gap junctions and tight junctions. In this experiment, only the tight junctions were disrupted. In bronchiolar epithelium, the tight junction was disrupted. The other sites in the vascular system and respiratory surface did not show any changes in the morphology of intercellular junctions. Thus, this study confirms the previous study and provides some structural correlation for the changes of permeability in fluid-overload pulmonary oedema.     

Phenobarbital pretreatment protects against morphologic changes in rat bronchiolar epithelium caused by an impurity of malathion.

Oral administration (20 mg/kg) of O,O,S-trimethyl phosphorothioate (OOS) causes delayed toxicity in rats; ie, death occurs as late as 28 days after treatment. OOS-treated rats show morphologic changes in the bronchiolar epithelium of the lung; nonciliated (Clara) cells are fewer but larger 3 days after treatment. We have now found that pretreatment with the P-450-dependent mixed-function oxidase inducer, phenobarbital, protects against the morphologic changes caused by OOS. These results support the view that the lung is a target organ of the delayed toxicity caused by OOS and that OOS detoxification is mediated by P-450-dependent metabolism.     

The response of the rat tracheal epithelium to ozone exposure. Injury, adaptation, and repair.

Although acute ozone (O3) exposure injures tracheal epithelium, the response of the tracheal epithelium to prolonged O3 exposure, and the degree of repair following cessation of exposure have not been previously reported. The purpose of this experiment was to characterize the morphologic response of rat tracheal epithelium to acute (3 days) and prolonged (60 days) exposure to 0.96 ppm O3 as well as to evaluate repair in a 7- and 42-day post-60-day exposure period. Quantitative light- and electron-microscopic evaluation and thymidine labeling indices showed that after 3 days of O3 exposure there was ciliary damage, cell necrosis, an increased density of intermediate cells, and an elevated thymidine labeling index. Following 60 days of exposure, the only major change from controls was the presence of ciliated cells with uniformly short cilia. Tracheal superoxide dismutase levels did not differ between control and 60-day exposure groups. Our findings suggest that the tracheal epithelium adapts to prolonged ozone exposure with the exception of cilia formation in ciliated cells. Complete epithelial recovery occurred by 42 days after exposure.     

Response of bonnet macaque dyads to an acute foraging task under different motivational conditions.

To explore behavioral patterns in bonnet macaque mother-infant dyads under environmental challenge, the motivation of infants to contact their mothers during a 1-hr foraging period was manipulated in two ways. First, sessions were given in which the infants were separated from their mothers for 3 hr prior to the foraging period. Second, sessions were given in which the infants were allowed access to food prior to the foraging period, thereby presumably reducing their motivation to separate from mother to seek food for themselves during the foraging period. Neither manipulation resulted in heightened conflict within the dyads. Separation of the infants prior to the foraging period did result, in general, in mothers forfeiting foraging task engagement to attend to their infants. The extent to which a mother exhibited this pattern correlated with her level of foraging effectiveness.     

Early postnatal exposure to allergen and ozone leads to hyperinnervation of the pulmonary epithelium

Airway injury in infant monkeys exposed to ozone and/or house dust mite allergen (HDMA) is associated with a loss of epithelial innervation. In this study, we evaluated for persistence/recovery of the altered epithelial innervation. Thirty-day-old rhesus monkeys were exposed to repeated episodes of HDMA and/or ozone from 1 to 6 months of age and subsequently allowed to recover for 6 months in the absence of further ozone exposure and/or minimal HDMA challenge (sufficient to maintain allergen sensitization). At 1 year of age, nerve density in intrapulmonary airways was immunohistochemically evaluated using antibodies directed against protein gene product 9.5. Hyperinnervation and irregular epithelial nerve distribution was observed in both HDMA- and ozone-exposed groups; most prominent alterations were observed in animals exposed to HDMA plus ozone. Therefore, while adaptive mechanisms exist that re-establish epithelial innervation following cessation or diminution of exposure to HDMA and/or ozone, the recovery is associated with persistent proliferative mechanisms that result in hyperinnervation of the airways.     

Effects of an ambient level of ozone on primate nasal epithelial mucosubstances. Quantitative histochemistry.

Despite the absorption of inhaled oxidant gases by the nasal cavity, little effort has been made to characterize the effects of these oxidants on the nasal mucosa. This study defines the effects of ambient concentrations of ozone on the character and amount of mucosubstances in epithelium of nasal mucosa. Bonnet monkeys were exposed to 0.00 or 0.15 ppm O3 (8 hr/day) for 6 or 90 days, anesthetized, and exsanguinated. Nasal cavities were fixed with Karnovsky's fixative, decalcified, and processed for light microscopy, and sections were stained with alcian blue (pH 2.5)/periodic acid-Schiff or high iron diamine. Volume densities of secretory material in nasal epithelium were determined with the use of a Quantimet 900 image analyzer. After 6 days' exposure there were significant increases in both acidic and neutral glycoconjugates stored in transitional and respiratory epithelium. After 90 days there was significantly less mucosubstance than at 6 days. Only in the transitional epithelium did the total and sulfated mucosubstance remain greater than that of controls. Nasopharyngeal epithelium was minimally affected after 6 days of O3 and unchanged after 90 days. It is concluded that exposures to ambient levels of O3 induce significant changes in the stored secretory product of nasal epithelium.     

Laser capture microdissection and real-time reverse transcriptase/ polymerase chain reaction of bronchiolar epithelium after bleomycin

Terminal airways are affected in many lung diseases and toxic inhalations. To elucidate the changes in terminal airways in these diverse situations it will be helpful to profile and quantify gene expression in terminal bronchiolar epithelium. We used laser capture microdissection (LCM) to collect terminal bronchiolar epithelial cells from frozen sections of lungs of mice subjected to intratracheal bleomycin. The RNA from these cells was used for analysis of select messenger RNAs (mRNAs) by quantitative real-time polymerase chain reaction (PCR). In parallel, we used real-time PCR to analyze mRNAs in whole-lung homogenates prepared from other mice given intratracheal bleomycin. We found reductions of Clara cell-specific protein and keratinocyte growth factor receptor mRNAs in both terminal bronchiolar epithelium and whole-lung homogenates 7 d after bleomycin. In contrast, terminal bronchiolar epithelial transforming growth factor (TGF)-alpha mRNA was reduced but whole-lung TGF-alpha mRNA was not changed, whereas terminal bronchiolar epithelial epidermal growth factor (EGF) receptor mRNA was not changed but whole-lung EGF receptor was reduced. We conclude that LCM can isolate terminal bronchiolar epithelial cells for studies of cell-specific gene expression by quantitative real-time PCR, and that cell-specific gene expression in terminal bronchiolar epithelium is not necessarily reflected in analysis of whole-lung gene expression.     

Morphology of a naphthalene-induced bronchiolar lesion.

Nonciliated bronchiolar epithelial (Clara) cells are selectively damaged by intraperitoneal administration of naphthalene. We examined these changes using light microscopy, transmission electron microscopy, and scanning electron microscopy. Naphthalene administration causes the Clara cells to expand and exfoliated shortly thereafter. Following exfoliation the remaining ciliated cells show morphologic abnormalities, including cilia loss and ballooning of remaining cilia. Upon regeneration of the Clara cells the ciliated cells gradually return to their normal appearance. One possible explanation for these findings is that the Clara cell secretions directly affect the physiologic state of the surrounding ciliated cells.     

Response latency of macaque area MT/V5 neurons and its relationship to stimulus parameters.

A total of 310 MT/V5 single cells were tested in anesthetized, paralyzed macaque monkeys with moving random-dot stimuli. At optimum stimulus parameters, latencies ranged from 35 to 325 ms with a mean of 87+/-45 (SD) ms. By examining the relationship between latency and response levels, stimulus parameters, and stimulus selectivities, we attempted to isolate the contributions of these factors to latency and to identify delays representing intervening synapses (circuitry) and signal processing (flow of information through that circuitry). First, the relationship between stimulus parameters and latency was investigated by varying stimulus speed and direction for individual cells. Resulting changes in latencies were explainable in terms of response levels corresponding to how closely the actual stimulus matched the preferred stimulus of the cell. Second, the relationship between stimulus selectivity and latency across the population of cells was examined using the optimum speed and direction of each neuron. A weak tendency for cells tuned for slow speeds to have longer latencies was explainable by lower response rates among slower-tuned neurons. In contrast, sharper direction tuning was significantly associated with short latencies even after taking response rate into account, (P = 0.002, ANCOVA). Accordingly, even the first 10 ms of the population response fully demonstrates direction tuning. A third study, which examined the relationship between antagonistic surrounds and latency, revealed a significant association between the strength of the surround and the latency that was independent of response levels (P < 0.002, ANCOVA). Neurons having strong surrounds exhibited latencies averaging 20 ms longer than those with little or no surround influence, suggesting that neurons with surrounds represent a later stage in processing with one or more intervening synapses. The laminar distribution of latencies closely followed the average surround antagonism in each layer, increasing with distance from input layer IV but precisely mirroring response levels, which were highest near the input layer and gradually decreased with distance from input layer IV. Layer II proved the exception with unexpectedly shorter latencies (P< 0.02, ANOVA) yet showing only modest response levels. The short latency and lack of strong direction tuning in layer II is consistent with input from the superior colliculus. Finally, experiments with static stimuli showed that latency does not vary with response rate for such stimuli, suggesting a fundamentally different mode of processing than that for a moving stimulus.     

Exposure of humans to ambient levels of ozone for 6.6 hours causes cellular and biochemical changes in the lung.

An acute (2 h) exposure of humans to 0.4 ppm ozone initiates biochemical changes in the lung that result in the production of components mediating inflammation and acute lung damage as well as components having the potential to lead to long-term effects such as fibrosis. However, many people are exposed to lower levels of ozone than this, but for periods of several hours. Therefore, it is important to determine if a prolonged exposure to low levels of ozone is also capable of causing cellular and biochemical changes in the lung. Nonsmoking males were randomly exposed to filtered air and either 0.10 ppm ozone or 0.08 ppm ozone for 6.6 h with moderate exercise (40 liters/min). Bronchoalveolar lavage (BAL) was performed 18 h after each exposure, and cells and fluid were analyzed. The BAL fluid of volunteers exposed to 0.10 ppm ozone had significant increases in neutrophils (PMNs), protein, prostaglandin E2 (PGE2), fibronectin, interleukin-6 (IL-6), and lactate dehydrogenase (LDH) compared with BAL fluid from the same volunteers exposed to filtered air. In addition, there was a decrease in the ability of alveolar macrophages to phagocytize yeast via the complement receptor. Exposure to 0.08 ppm ozone resulted in significant increases in PMNs, PGE2, LDH, IL-6, alpha 1-antitrypsin, and decreased phagocytosis via the complement receptor. However, BAL fluid protein and fibronectin were no longer significantly elevated. We conclude that exposure of humans to as low a level as 0.08 ppm for 6.6 h is sufficient to initiate an inflammatory reaction in the lung.     

Short term morphologic effects of high ambient levels of ozone on lungs of rhesus monkeys.

Groups of laboratory-reared, young adult rhesus monkeys were exposed to 0.8 p.p.m. or 0.5 p.p.m. of ozone for 8 hours a day on 7 consecutive days. Lesions were studied using correlated techniques which permitted examination of specified levels of airways and adjacent lung parenchyma by light microscopy, scanning electron microscopy, and transmission electron microscopy. Lesions were observed in the trachea and lungs of all exposed animals. The extent and severity of damage, but not its nature, varied with exposure concentration. Damage was most severe in respiratory bronchioles and more distal parenchymal regions were unaffected. Major features of the response within respiratory bronchioles were hyperplasia and hypertrophy of nonciliated bronchiolar epithelial cells and intraluminal accumulations of macrophages. Replacement of type 1 epithelium in alveoli by type 2 cells and forms intermediate between types 1 and 2 were also observed. In large conducting airways, damage to ciliated cells was observed but mlcus-producing cells were morphologically unaltered. Two gradients in severity of ozone-induced lesions were appreciable in the trachea and lungs. The most obvious gradient was in respiratory bronchioles where the degree of damage was most severe in proximal locations. A second gradient in severity was noted in conducting airways in which more severe and extensive lesions occurred in the trachea and major bronchi than in small bronchi and terminal bronchioles.