Coordinated clearance of periciliary liquid and mucus from airway surfaces.

Airway surface liquid is comprised of mucus and an underlying, watery periciliary liquid (PCL). In contrast to the well-described axial transport of mucus along airway surfaces via ciliary action, theoretical analyses predict that the PCL is nearly stationary. Conventional and confocal microscopy of fluorescent microspheres and photoactivated fluorescent dyes were used with well-differentiated human tracheobronchial epithelial cell cultures exhibiting spontaneous, radial mucociliary transport to study the movements of mucus and PCL. These studies showed that the entire PCL is transported at approximately the same rate as mucus, 39.2+/-4.7 and 39.8+/-4.2 micrometer/sec, respectively. Removing the mucus layer reduced PCL transport by > 80%, to 4.8+/-0.6 micrometer/sec, a value close to that predicted from theoretical analyses of the ciliary beat cycle. Hence, the rapid movement of PCL is dependent upon the transport of mucus. Mucus-dependent PCL transport was spatially uniform and exceeded the rate expected for pure frictional coupling with the overlying mucus layer; hence, ciliary mixing most likely accelerates the diffusion of momentum from mucus into the PCL. The cephalad movement of PCL along airway epithelial surfaces makes this mucus-driven transport an important component of salt and water physiology in the lung in health and disease.

Beware Cold Agglutinins in Organ Donors! Ex Vivo Lung Perfusion From an Uncontrolled Donation After Circulatory-Determination-of-Death Donor With a Cold Agglutinin: A Case Report.

BACKGROUND: We began to recover lungs from uncontrolled donation after circulatory determination of death to assess for transplant suitability by means of ex vivo lung perfusion (EVLP) and computerized tomographic (CT) scan. Our first case had a cold agglutinin with an interesting outcome. CASE REPORT: A 60-year-old man collapsed at home and was pronounced dead by Emergency Medical Services personnel. Next-of-kin consented to lung retrieval, and the decedent was ventilated and transported. Lungs were flushed with cold Perfadex, removed, and stored cold. The lungs did not flush well. Medical history revealed a recent hemolytic anemia and a known cold agglutinin. Warm nonventilated ischemia time was 51 minutes. O2-ventilated ischemia time was 141 minutes. Total cold ischemia time was 6.5 hours. At cannulation for EVLP, established clots were retrieved from both pulmonary arteries. At initiation of EVLP with Steen solution, tiny red aggregates were observed initially. With warming, the aggregates disappeared and the perfusate became red. After 1 hour, EVLP was stopped because of florid pulmonary edema. The lungs were cooled to 20°C; tiny red aggregates formed again in the perfusate. Ex vivo CT scan showed areas of pulmonary edema and a pyramidal right middle lobe opacity. Dissection showed multiple pulmonary emboli-the likely cause of death. However, histology showed agglutinated red blood cells in the microvasculature in pre- and post-EVLP biopsies, which may have contributed to inadequate parenchymal preservation. CONCLUSIONS: Organ donors with cold agglutinins may not be suitable owing to the impact of hypothermic preservation.

Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus.

The evolutionary origins of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) are unknown. Current evidence suggests that insectivorous bats are likely to be the original source, as several 2c CoVs have been described from various species in the family Vespertilionidae Here, we describe a MERS-like CoV identified from a Pipistrellus cf. hesperidus bat sampled in Uganda (strain PREDICT/PDF-2180), further supporting the hypothesis that bats are the evolutionary source of MERS-CoV. Phylogenetic analysis showed that PREDICT/PDF-2180 is closely related to MERS-CoV across much of its genome, consistent with a common ancestry; however, the spike protein was highly divergent (46% amino acid identity), suggesting that the two viruses may have different receptor binding properties. Indeed, several amino acid substitutions were identified in key binding residues that were predicted to block PREDICT/PDF-2180 from attaching to the MERS-CoV DPP4 receptor. To experimentally test this hypothesis, an infectious MERS-CoV clone expressing the PREDICT/PDF-2180 spike protein was generated. Recombinant viruses derived from the clone were replication competent but unable to spread and establish new infections in Vero cells or primary human airway epithelial cells. Our findings suggest that PREDICT/PDF-2180 is unlikely to pose a zoonotic threat. Recombination in the S1 subunit of the spike gene was identified as the primary mechanism driving variation in the spike phenotype and was likely one of the critical steps in the evolution and emergence of MERS-CoV in humans.IMPORTANCE Global surveillance efforts for undiscovered viruses are an important component of pandemic prevention initiatives. These surveys can be useful for finding novel viruses and for gaining insights into the ecological and evolutionary factors driving viral diversity; however, finding a viral sequence is not sufficient to determine whether it can infect people (i.e., poses a zoonotic threat). Here, we investigated the specific zoonotic risk of a MERS-like coronavirus (PREDICT/PDF-2180) identified in a bat from Uganda and showed that, despite being closely related to MERS-CoV, it is unlikely to pose a threat to humans. We suggest that this approach constitutes an appropriate strategy for beginning to determine the zoonotic potential of wildlife viruses. By showing that PREDICT/PDF-2180 does not infect cells that express the functional receptor for MERS-CoV, we further show that recombination was likely to be the critical step that allowed MERS to emerge in humans.

Lung stem cell update: promise and controversy.

Currently, there is great enthusiasm about potential stem cell therapies for intractable diseases. We previously reviewed the topic of stem cells in lung injury and repair, including the role of endogenous, tissue (somatic) stem cells and the contribution of circulating cells to the lung parenchyma. Our purpose here is to provide a concise update in this fast-moving field. New information and ongoing debate focus attention on basic issues in lung stem cell biology and highlight the need for additional studies to establish the feasibility of cell therapies to prevent or treat lung diseases.

Cytochemical localization and biochemical evaluation of a lysosomal serine protease in lung: dipeptidyl peptidase II in the normal rat.

Dipeptidyl peptidase II (DPP II) in normal rat lung was evaluated by the enzymes' ability to hydrolyze Lys-Ala or Lys-Pro derivatives of 4-methoxy-2-naphthylamine (MNA). For visualization of this activity, the liberated MNA was coupled with fast blue B for light microscopy (LM) or hexazotized pararosaniline with osmication for electron microscopy (EM). Granular to diffuse reaction product was noted in many lung cells in frozen sections for LM, including alveolar and tissue macrophages, fibroblasts, chondrocytes, bronchial and bronchiolar epithelial cells and mast cells. Reaction product at the EM level was seen in the lysosomal structures of the above cells, although lysosomal heterogeneity with regard to reactivity was noted. Cellular content of reaction product by EM correlated with LM staining intensity. Additional structures, not obviously reactive by LM, such as the lamellar bodies of type II cells and lysosomes in other cell types, were seen to contain reaction product ultrastructurally. A modified biochemical assay for the quantitation of DPP II in tissue homogenates was used to determine the activity of the enzyme in rat lung. Enzyme activity in polyacrylamide isoelectric focusing gels indicate that Lys-Ala-MNA was the more specific substrate but, by virtue of its rapid hydrolysis, Lys-Pro-MNA was more sensitive.

Cell type-specific lectin staining of the tracheobronchial epithelium of the rat: quantitative studies with Griffonia simplicifolia I isolectin B4.

We compared lectin staining patterns to cell population densities, as determined by morphological criteria in rat airways. Eight lectins were studied: Griffonia simplicifolia I isolectin B4 (GSI-B4), Arachis hypogaea (PNA), Wisteria floribunda (WFA), Glycine maximus (SBA), Dolichos biflorus (DBA), Helix pomatia (HPA), Ulex europaeus (UEA-1), and Maclura pomifera (MPA). Two of the lectins strongly stained morphologically distinct cell subpopulations. GSI-B4 stained basal cells, and MPA stained non-ciliated bronchiolar (Clara) cells. The specificity and sensitivity of GSI-B4 as a marker for basal cells was examined. In the trachea, 35% of all cells were GSI-B4 positive; 84% of these were basal cells, 7% were unidentified cells, 5% were serous/mucous cells, and 4% were ciliated, brush, or inflammatory cells. Comparison to cell population density data strongly suggested that all basal cells were GSI-B4 positive. The segmental bronchus was a transitional area; GSI-B4 positive basal cells were present in the region closest to the lobar bronchus but were absent in the distal region; instead, MPA-positive Clara cells appeared. When dissociated tracheal cells were obtained by pronase digestion, 43% were GSI-B4 positive. These results show that GSI-B4 is a sensitive and relatively specific marker for basal cells in the rat trachea which can be used to study dissociated epithelial cells.

Biochemical quantitation and histochemical localization of cathepsin B, dipeptidyl peptidases I and II, and acid phosphatase in pulmonary granulomatosis and fibrosis in rats.

The purpose of this study was to quantitate biochemically and to localize histochemically the proteases cathepsin B (Cath B), dipeptidyl peptidase I (DPP I), and dipeptidyl peptidase II (DPP II) in experimental pulmonary granulomatosis and fibrosis. These were compared to the prototypical lysosomal hydrolase acid phosphatase (AP). Granulomatosis was induced by the intravenous injection of complete Freund's adjuvant (CFA, 0.2 ml) and fibrosis was induced by the intratracheal instillation of bleomycin sulfate (1 unit) in rats (Wistar, 200 g). Total Cath B, DPP I, and AP activities were markedly elevated over control values five days following both treatments when expressed as activity per lung or as specific activity per milligram protein or milligram DNA. By 14 and 28 days, total activity was elevated for all three enzymes, and activity per milligram DNA remained elevated for Cath B following both treatments and for DPP I 28 days following CFA treatment. Total lung activity of DPP II was significantly elevated at 28 days for both treatments. Histochemical staining indicated that these changes are due, in part, to the influx of inflammatory monocytes and their maturation to macrophages. This study provides a basis for examining the role of these proteases in connective tissue matrix injury during inflammatory processes in the lungs.

Rat tracheal epithelial cell differentiation in vitro.

In vitro culture conditions enabling rat tracheal epithelial (RTE) cells to differentiate to mucociliary, mucous, or squamous phenotypes are described. Medium composition for rapid cell growth to confluence in membrane insert cultures was determined, and the effects of major modifiers of differentiation were tested. Retinoic acid (RA), collagen gel substratum, and an air-liquid interface at the level of the cell layer were required for expression of a mucociliary phenotype which most closely approximated the morphology of the tracheal epithelium in vivo. Large quantities of high molecular weight, hyaluronidase-resistant glycoconjugates, most likely mucin glycoproteins, were produced in the presence of RA when the cells were grown with or without a collagen gel and in submerged as well as in interface cultures. However, extensive ciliagenesis was dependent on the simultaneous presence of RA, collagen gel, and an air-liquid interface. When RA was omitted from the media, the cells became stratified squamous and developed a cornified apical layer in air-liquid interface cultures. This phenotype was accompanied by loss of transglutaminase (TGase) type II and keratin 18 and expression of the squamous markers TGase type I and keratin 13. The ability to modulate RTE cell phenotypes in culture will facilitate future studies investigating molecular regulation of tracheal cell proliferation, differentiation, and function.

Rat tracheal epithelial cell differentiation in vitro.

In vitro culture conditions enabling rat tracheal epithelial (RTE) cells to differentiate to mucociliary, mucous, or squamous phenotypes are described. Medium composition for rapid cell growth to confluence in membrane insert cultures was determined, and the effects of major modifiers of differentiation were tested. Retinoic acid (RA), collagen gel substratum, and an air-liquid interface at the level of the cell layer were required for expression of a mucociliary phenotype which most closely approximated the morphology of the tracheal epithelium in vivo. Large quantities of high molecular weight, hyaluronidase-resistant glycoconjugates, most likely mucin glycoproteins, were produced in the presence of RA when the cells were grown with or without a collagen gel and in submerged as well as in interface cultures. However, extensive ciliagenesis was dependent on the simultaneous presence of RA, collagen gel, and an air-liquid interface. When RA was omitted from the media, the cells became stratified squamous and developed a cornified apical layer in air-liquid interface cultures. This phenotype was accompanied by loss of transglutaminase (TGase) type II and keratin 18 and expression of the squamous markers TGase type I and keratin 13. The ability to modulate RTE cell phenotypes in culture will facilitate future studies investigating molecular regulation of tracheal cell proliferation, differentiation, and function.

Isolation and culture of airway epithelial cells from chronically infected human lungs.

We describe procedures for isolating and culturing airway epithelial cells from chronically infected human lungs. Experience in our laboratory demonstrated the need to balance pathogen eradication against antibiotic toxicity to epithelial cells. To provide a logical basis for antibiotic selection and dose, we systematically analyzed the cytotoxicity of antibiotics useful against typical pathogens. Alone, colistin, ciprofloxacin, doxycycline, and tobramycin were moderately toxic at concentrations close to those used in cell culture, whereas amphotericin, ceftazidime, chloramphenicol, imipenem, meropenem, piperacillin, sulfamethoxazole/trimethoprim, and vancomycin were nontoxic even at concentrations many times the antimicrobial level. Epithelial cytotoxicity of combined antibiotics was additive, with no evidence of competition or synergism. Antibiotics had little effect on initial cell attachment and did not acutely lyse cells, but inhibited subsequent growth. Interestingly, cytotoxicity decreased markedly with increasing epithelial cell density. Cystic fibrosis (CF) and non-CF epithelial cells showed no differences in sensitivity to the antibiotics tested and initial exposure to antibiotics did not affect the electrophysiologic properties of resistance or short circuit current in well-differentiated cells. Tailored combinations of antibiotics at appropriate doses killed even multidrug-resistant bacteria. Thus, epithelial cells can usually be cultured from chronically infected CF airways.

Mucin production in the middle ear in response to lipopolysaccharides.

OBJECTIVE: This study examined the response of middle ear tissue to establish the lowest dose of lipopolysaccharide to induce mucin production in a rat otitis media model. METHODS: Twenty-six male Sprague-Dawley rats' eustachian tubes were obstructed before transtympanic inoculation of the bulla tympanica with 35 microL of Krebs Ringer or 1, 10, 100, or 1000 microgram/mL lipopolysaccharide. After 7 days the effusion and a lavage were collected for mucin ELISA measurement, and tissue was collected for histologic evaluation. RESULTS: Mucin secretion was significantly increased in the 100 microgram/mL 51.20 +/- 13.6 microgram/mL (SE) and 1000 microgram/mL 69.42 +/- 8.57 microgram/mL groups when compared with the Krebs Ringer control group 1.84 +/- 0.28 microgram/mL (P < 0.05). Histologic evaluation shows goblet cell metaplasia and hyperplasia in the middle ear epithelium in the 1000 and 100 microgram/mL groups. CONCLUSIONS: The histology and ELISA results suggest that a middle ear effusion is generated with a dose of lipopolysaccharide as low as 100 microgram/mL.

Nitric oxide mediates mucin secretion in endotoxin-induced otitis media with effusion.

The mechanisms that regulate mucin release in chronic otitis media with effusion, a leading cause of hearing loss in children, remain largely unknown. We developed an animal model using Sprague-Dawley rats to determine the factors responsible for mucin production in chronic otitis media with effusion. N-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of nitric oxide synthase, was used to investigate the role of nitric oxide in mucin secretion by the middle ear epithelium. All rats underwent eustachian tube obstruction. In the first set of rats, the middle ear was then injected transtympanically with 35 microl of either 300 mOsm Krebs-Ringer bicarbonate buffer (control group) or 1 mg/ml lipopolysaccharide in Krebs-Ringer (experimental group 1). In a second set of rats, the middle ear space was injected with lipopolysaccharide and then infused at a continuous rate for 7 days with either Krebs-Ringer (experimental group 2) or 1 mmol/L L-NAME in Krebs-Ringer (experimental group 3) through an osmotic infusion pump. After 7 days the volume of effusion and the quantity of mucin collected were significantly greater in lipopolysaccharide-exposed ears than in controls. In addition, antimucin immunostaining demonstrated mucous cell hyperplasia in response to lipopolysaccharide. The lipopolysaccharide-induced production of mucin and mucous cell hyperplasia was inhibited in ears treated with lipopolysaccharide and L-NAME. These results suggest that nitric oxide is a mediator in the pathway of mucin secretion in chronic otitis media with effusion.

Mucus clearance, MyD88-dependent and MyD88-independent immunity modulate lung susceptibility to spontaneous bacterial infection and inflammation.

It has been postulated that mucus stasis is central to the pathogenesis of obstructive lung diseases. In Scnn1b-transgenic (Scnn1b-Tg⁺ mice, airway-targeted overexpression of the epithelial Na⁺ channel ß subunit causes airway surface dehydration, which results in mucus stasis and inflammation. Bronchoalveolar lavage from neonatal Scnn1b-Tg⁺ mice, but not wild-type littermates, contained increased mucus, bacteria, and neutrophils, which declined with age. Scnn1b-Tg⁺ mice lung bacterial flora included environmental and oropharyngeal species, suggesting inhalation and/or aspiration as routes of entry. Genetic deletion of the Toll-interleukin-1 receptor adapter molecule MyD88 in Scnn1b-Tg⁺ mice did not modify airway mucus obstruction, but caused defective neutrophil recruitment and increased bacterial infection, which persisted into adulthood. Scnn1b-Tg⁺ mice derived into germ-free conditions exhibited mucus obstruction similar to conventional Scnn1b-Tg⁺ mice and sterile inflammation. Collectively, these data suggest that dehydration-induced mucus stasis promotes infection, compounds defects in other immune mechanisms, and alone is sufficient to trigger airway inflammation.

Novel human bronchial epithelial cell lines for cystic fibrosis research.

Immortalization of human bronchial epithelial (hBE) cells often entails loss of differentiation. Bmi-1 is a protooncogene that maintains stem cells, and its expression creates cell lines that recapitulate normal cell structure and function. We introduced Bmi-1 and the catalytic subunit of telomerase (hTERT) into three non-cystic fibrosis (CF) and three DeltaF508 homozygous CF primary bronchial cell preparations. This treatment extended cell life span, although not as profoundly as viral oncogenes, and at passages 14 and 15, the new cell lines had a diploid karyotype. Ussing chamber analysis revealed variable transepithelial resistances, ranging from 200 to 1,200 Omega.cm(2). In the non-CF cell lines, short-circuit currents were stimulated by forskolin and inhibited by CFTR(inh)-172 at levels mostly comparable to early passage primary cells. CF cell lines exhibited no forskolin-stimulated current and minimal CFTR(inh)-172 response. Amiloride-inhibitable and UTP-stimulated currents were present, but at lower and higher amplitudes than in primary cells, respectively. The cells exhibited a pseudostratified morphology, with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells, and occasional ciliated cells. CF and non-CF cell lines produced similar levels of IL-8 at baseline and equally increased IL-8 secretion in response to IL-1beta, TNF-alpha, and the Toll-like receptor 2 agonist Pam3Cys. Although they have lower growth potential and more fastidious growth requirements than viral oncogene transformed cells, Bmi-1/hTERT airway epithelial cell lines will be useful for several avenues of investigation and will help fill gaps currently hindering CF research and therapeutic development.

The use of 2-thionaphthyl acetate as a substrate for the localization and characterization of nonspecific esterase activity in rat alveolar and peritoneal macrophages.

A 2-thionaphthyl acetate substrate was utilized to assess the subcellular distribution of nonspecific esterases in rat pulmonary alveolar and peritoneal macrophages. The enzymatically liberated 2-thionaphthol was visualized at pH 7.1 by utilizing gold as a capture agent. Glutaraldehyde-fixed macrophages derived from healthy animals using standard lavage techniques exhibited a high affinity for the substrate and reaction times were thus relatively short (30-60 min). Alveolar macrophages had heavy reaction product on the external surface of the plasma membrane and membranes limiting cisternae of rough endoplasmic reticulum, Golgi complex and mitochondria. Only a thin layer of reaction density was observed associated with the limiting membranes of lysosomes and phagosomes. Peritoneal macrophages were similarly but much less intensely reactive, although they generally lacked or had very little plasma membrane-associated staining. The 2-thionaphthyl acetate esterase activities in both alveolar and peritoneal macrophages were sensitive to diisopropylfluorophosphate (DFP), while only the latter was inhibited by sodium fluoride. Polyacrylamide gel isoelectric focusing of whole cell homogenates indicated that the 2-thionaphthyl acetate esterase activity was the same as that for alpha-naphthyl acetate in these cells. The data indicate that a significantly different distribution of nonspecific esterase activity results with use of a 2-thionaphthyl acetate substrate in the presence of gold ions than that previously reported with other methods. The rapid penetrability and sensitivity of this substrate make it a potentially useful tool for evaluating subcellular localization of esterase activity and probing characteristics of cellular organelles.

Ontogeny of rat lung type II cells correlated with surfactant lipid and surfactant apoprotein expression.

During the last stages of intrauterine growth, remarkable changes occur in the alveolar epithelium that include cellular differentiation and increased production of surfactant lipid and apoprotein. We made morphometric measurements of type II cell characteristics from rats aged gestational day 20 to 14 days postnatal. We also measured the amounts of disaturated phosphatidylcholine (DSPC) and surfactant apoprotein (SP-A) in lung tissue, bronchoalveolar lavage, and a lamellar body-rich fraction, and we estimated the lung content of mRNAs for SP-A, SP-B, and SP-C. Lavage and lamellar body surfactant lipid and apoprotein content per lung showed a pattern of a sharp rise in the early postnatal period, then a substantial decline, and a second increase by day 14. When normalized for dry lung weight, the highest DSPC values were found on postnatal day 1 in all compartments. The fraction of whole lung DSPC found in lamellar body or lavage was greatest in the 48-h period surrounding birth. Lamellar body SP-A was greater than lavage SP-A on gestational day 22, but a day later the lavage SP-A was 16 times greater than the lamellar body SP-A. The lung tissue content of all three apoprotein mRNAs increased sharply before birth, fell during the 1st postnatal wk, and then rose again to adult levels. Type II cell number and lamellar body number per milligram of dry lung tissue was highest on post-natal day 1 and fell by one-half during the 1st postnatal wk. The amount of DSPC per unit of lamellar body volume rose to its greatest value on postnatal day 1 and then decreased more than threefold. These findings indicate a pattern of expansion of surfactant cellular and biochemical pools at the time of birth in the rat.

Quantitative three-dimensional reconstruction and carbohydrate cytochemistry of rat nonciliated bronchiolar (Clara) cells.

We have employed three-dimensional reconstructions and cytochemistry to analyze nonciliated bronchiolar epithelial (Clara) cells from rat lungs. We identified a previously unreported ovoid granule type, which was characterized by its relative electron-lucency and eccentric electron-dense core. These electron-lucent granules were less common than electron-dense granules and were polarized toward the Golgi. They occupied 0.51% of the cell volume. Quantitative analysis of cell volume fractions showed the more numerous electron-dense granules to be polarized toward the air border; they occupied 2.5% of the cell volume. Periodic acid-thiocarbohydrazide-silver protein staining for hexose sugars indicated no differences between the granule types with regard to their carbohydrate content. Multivesicular bodies occupied 0.42% of total cell volume and were distinctly polarized toward the surface of the Golgi. Rod-shaped granules were present in 4 of the 6 reconstructed cells, ranging in number from 0 to 9 per cell. Nine apparent secretory events were seen in 6 cells and all appeared as exocytosis. We conclude that rat Clara cells contain at least 3 morphologically distinct granule types located in different volumes of the cell, and that exocytosis is the mode of secretion. The functional relationship between the ovoid granule types could include precursor-product lineage or different secretory products. We suggest that multivesicular bodies and light granules are stages in dark granule genesis.

Epidermal growth factor regulates expression of the mucous phenotype of rat tracheal epithelial cells.

The purpose of this study was to determine whether epidermal growth factor (EGF) regulates mucous differentiation of airway epithelial cells in culture. Reduction of the EGF concentration below 25 ng/ml, which is the concentration routinely used in rat tracheal epithelial cell cultures, resulted in a 2 to 3-fold decrease in the percentage of mucous cells as determined by a mucin monoclonal antibody. The amount of secreted mucin decreased more than 10-fold within 5 days after reducing the EGF concentration. MUC5 gene expression, which was previously shown to correlate with mucous differentiation, was also reduced more than 8-fold. Addition of 25 ng/ml EGF to EGF-deprived cultures resulted in rapid induction of MUC5 expression. Following tissue injury and during inflammation the release of EGF and its functional analogue TGF alpha have been observed and may be involved in the mucus hypersecretion characteristic of many airway diseases.

Neonatal hyperoxia alters the pulmonary alveolar and capillary structure of 40-day-old rats.

High inspired oxygen concentrations during the neonatal period profoundly inhibit rat lung development, an effect that is partly reversed during recovery in air. Persistent effects of neonatal hyperoxia on the size and number of alveoli or the structure of pulmonary capillaries have not been well defined. Using light and electron microscopic morphometry plus quantitative three-dimensional reconstructions of alveoli, we examined the lungs of 40-day-old rats that were exposed to more than 95% oxygen for the first 7 days after birth. Neonatal hyperoxia administered to rats resulted in abnormally enlarged air spaces at age 40 days. The fraction of the lung consisting of parenchyma was significantly increased and alveolar surface area was 13% lower than controls. There was an abnormal enlargement of alveolar ducts, which reduced by 24% the relative amount of air in the alveoli, compared to that in the alveolar ducts. The number of alveoli per lung and the mean volume of an alveolus were not different between the groups, but alveolar size class distributions were different, with significantly more very small and very large alveoli in 40-day-old rats after neonatal hyperoxia. By scanning electron microscopy, the alveolar surface of the exposed animals had a corrugated appearance, which was especially evident along alveolar ducts. Transmission electron microscopy revealed a greater density of capillaries, particularly in the alveolar regions close to terminal airways. Based on a random sample of the entire parenchymal region, capillary blood volume per cm2 of alveolar basal lamina was 18% greater. The results demonstrate that neonatal exposure to hyperoxia can cause abnormalities in the pulmonary alveolar and capillary structure of 40-day-old rats, and that these changes are similar to some features of broncho-pulmonary dysplasia.