Biochemical and morphometric analyses in hypoplastic lungs

We report the results of biochemical and morphometric studies on lungs of infants with bilateral lung hypoplasia either with or without oligohydramnios (OH or NOH) in comparison with findings in normally grown lungs. The OH and NOH lungs were equally hypoplastic in terms of DNA content but OH lungs had a significantly lower disaturated phosphatidylcholine (DSPC) concentration than NOH or normal lungs, apart from a subgroup with gastrointestinal or airway obstruction. Hydroxyproline concentration in OH lungs was higher than that in NOH or normal lungs. Desmosine concentrations did not differ significantly between groups despite the obvious lack of elastin in the septal crests of the OH group on histology. Morphometry revealed low lung volume, low radial alveolar counts, low alveolar numbers, and low alveolar surface area in both OH and NOH groups. Alveoli and alveolar ducts constituted a higher proportion of lung volume in NOH than in OH lungs. The similarity of most morphometric indices in the two groups implies that maturation does not depend on quantitative elaboration of airways and alveoli. The finding of impaired epithelial maturation despite the high hydroxyproline concentration in the OH lungs suggests an abnormality in epithelial-mesenchymal interaction that is not present in the equally small lungs of the NOH group.     

Biochemical and Morphometric Analyses in Hypoplastic Lungs

We report the results of biochemical and morphometric studies on lungs of infants with bilateral lung hypoplasia either with or without oligohydramnios (OH or NOH) in comparison with findings in normally grown lungs. The OH and NOH lungs were equally hypoplastic in terms of DNA content but OH lungs had a significantly lower disaturated phosphatidylcholine (DSPC) concentration than NOH or normal lungs, apart from a subgroup with gastrointestinal or airway obstruction. Hydroxyproline concentration in OH lungs was higher than that in NOH or normal lungs. Desmosine concentrations did not differ significantly between groups despite the obvious lack of elastin in the septal crests of the OH group on histology. Morphometry revealed low lung volume, low radial alveolar counts, low alveolar numbers, and low alveolar surface area in both OH and NOH groups. Alveoli and alveolar ducts constituted a higher proportion of lung volume in NOH than in OH lungs. The similarity of most morphometric indices in the two groups implies that maturation does not depend on quantitative elaboration of airways and alveoli. The finding of impaired epithelial maturation despite the high hydroxyproline concentration in the OH lungs suggests an abnormality in epithelial-mesenchymal interaction that is not present in the equally small lungs of the NOH group.     

Effect of metopirone on the synthesis of lung surfactant in does and fetal rabbits.

The possible role of endogenous corticosteroids in the induction of synthesis of lung surfactant in both maternal and fetal rabbits was investigated. Metopirone (SU 4885), which specifically inhibits B hydroxylation in the adrenal gland, thus preventing endogenous cortisol production was used in this study. Pregnant rabbits received one of four treatment regimens: saline, metopirone, cortisone or metopirone plus steroid for 6 days before delivery. Studies of the lungs of the adult rabbits revealed that lung weights and percent dry weight were not influenced by drug injection. However, lung DNA content (mg/g) in the metopirone-treated group was higher than the control group, indicating an increase in cell number. Compared to the saline-treated group there was a significant decrease in lung phospholipid content in the other three groups (p smaller than 0.005). A decrease in lung lecithin and phosphatidylethanolamine was also noted in these three groups indicating that the inhibition was in the early steps of biosynthesis of lung lecithin. The specific activity of 14C-labelled palmitate in lung lecithin was not altered by drug treatment indicating that saturated fatty acid incorporation into lung lecithin is not influenced by metopirone, steroid or a combination of both drugs. Quasi-static deflation pressure-volume characteristics of the lung were also similar in all groups. However, lungs of animals receiving both metopirone and steroid had a small but statistically significant decrease in minimum surface tension (p smaller than 0.05). The change in surface tension did not correlate with a change in lung retractive forces or with lung lipid content and was, therefore, of dubious biological significance. Metopirone administration to pregnant rabbits did not alter surface active properties in the fetus from the 26th to the 29th day of gestation when compared to control fetuses. At day 30-31 gestation the offsprings of metopirone-treated rabbits had lung total phospholipid and lecithin contents, and deflation pressure-volume curves similar to control group, but minimum surface tension of minced lung extracts were high in about 60% of the fetal lungs studied (p smaller than 0.05). These results suggest that endogenous steroid production may play a significant role in the maturation of the fetal lung.     

Heparin and in-vitro experimental lung hypoplasia

 Pulmonary hypoplasia (PH) is a leading contributor to the lethality of congenital diaphragmatic hernia (CDH). Studies now suggest that PH arises prior to visceral herniation. Growth factors (GF) are pivotal to this embryonic lung growth. With striking in-vitro effects on lung morphogenesis, GF are under investigation as therapies for PH. Heparin modulates the kinetics of heparan-sulphate binding ligands that drive lung development. We hypothesised that heparin may rescue PH by favourable alteration of endogenous pulmonary GF activity. Normal and hypoplastic lung primordia were microdissected on day 13.5 of gestation and cultured for up to 78 h in plain media with and without heparin. In-vitro morphological development was studied by serial measurements of terminal bud count, lung area, and lung perimeter. Nitrofen-exposed lungs cultured with heparin showed no significant improvements in terminal bud count, lung area, and lung perimeter at 30, 54, and 78 h compared to untreated hypoplastic lungs maintained in vitro. In normal lungs heparin demonstrated no sustained significant morphological effects compared to untreated control lungs. In this study, heparin did not stimulate branching morphogenesis of normal or hypoplastic lungs in our organ culture system. Known at higher concentrations to inhibit smooth-muscle proliferation, heparin may ameliorate pulmonary vascular hypermuscularisation with the prospect of benefiting CDH infants on extracorporeal membrane oxygenation. Future studies will address the impact of exogenous GF on hypoplastic lung development in organ culture.     

Is the intake of palm oil (palmitic acid) in meals associated with the low incidence of respiratory distress syndrome in Nigeria? (author's transl)

Comparative gaschromatographic assays of different domestic oils used in West Germany and the domestic oil (palm oil) used in Nigeria and other West African countries were performed. It was discovered that the palm oil contains extremely high amounts of palmitic acid and linoleic acid compared to the other oils used in Germany. In view of the fact that previous studies have shown that palmitic acid is required for the biosynthesis of lung lecithin, which is closely related to fetal maturation, we performed animal experiments with rabbits to determine the incorporation rate of 3H-marked palmitic acid into various fetal organs: (fetal lung, placenta, fetal liver, fetal intestine etc.) after a previous intravenous injection of the 3H-marked palmitic acid to the mother rabbit. The radiochromatogram showed high incorporation rates of palmitate into the lecithin by fetal lung and placenta. These results seem to document that the high content of palm oil in Nigerian meals can partly be related to the low incidence of respiratory distress syndrom in Nigerian premature infants.     

Transcutaneous absorption of vitamin A in newborn rats

Very low birth weight neonates have low tissue concentrations of vitamin A, which may contribute to the development of lung disease. These infants, however, may not receive vitamin A supplementation for several days after birth. We determined if the relatively permeable skin of a newborn could be used to administer vitamin A. 25 control rat pups were killed and lungs and livers were collected. 20 microl (1,000 IU) of retinyl palmitate were applied to the skin surface of an additional 50 two-day-old pups. At 2.5 and 5 h after application, 25 pups were killed, and lungs and livers were collected. Concentrations of retinyl palmitate and retinol were significantly higher in the lungs of pups 5 h after administration of vitamin A compared with controls. There were no differences in concentrations of retinyl palmitate or retinol in livers. We conclude that transcutaneous administration may be an effective means of delivering vitamin A to the lungs of newborn rats.     

A study of cartilage development in pulmonary hypoplasia

To discriminate between different forms of pulmonary hypoplasia (PH), 24 hypoplastic lungs were studied for their development of bronchial cartilage plates and peripheral air spaces. In 6 lungs from premature infants with oligohydramnios, normal amounts of immature and irregularly shaped cartilages were distributed with mitoses concentrated toward the periphery of the bronchi. Pulmonary acini appeared markedly immature. In 5 lungs from infants with diaphragmatic hernia, large numbers of cartilage bars were clustered around the proximal bronchi, whose branching was much reduced. Peripheral air spaces were small but structurally mature. In Potter syndrome, small amounts of tiny, mature cartilages were observed irregularly around the proximal bronchi and poorly distributed into the peripheral bronchi. The acinar structure was very immature. In 4 anencephalic infants, a marked decrease in the volume of mature cartilage was present, with cartilage seen only around proximal bronchi; the acini were atelectatic and less well developed. It is suggested that the earlier the action of a teratogen, the greater the abnormality of bronchial branching, cartilage distribution, and later lung development.     

A Study of Cartilage Development in Pulmonary Hypoplasia

To discriminate between different forms of pulmonary hypoplasia (PH), 24 hypoplastic lungs were studied for their development of bronchial cartilage plates and peripheral air spaces. In 6 lungs from premature infants with oligohydramnios, normal amounts of immature and irregularly shaped cartilages were distributed with mitoses concentrated toward the periphery of the bronchi. Pulmonary acini appeared markedly immature. In 5 lungs from infants with diaphragmatic hernia, large numbers of cartilage bars were clustered around the proximal bronchi, whose branching was much reduced. Peripheral air spaces were small but structurally mature. In Potter syndrome, small amounts of tiny, mature cartilages were observed irregularly around the proximal bronchi and poorly distributed into the peripheral bronchi. The acinar structure was very immature. In 4 anencephalic infants, a marked decrease in the volume of mature cartilage was present, with cartilage seen only around proximal bronchi; the acini were atelectatic and less well developed. It is suggested that the earlier the action of a teratogen, the greater the abnormality of bronchial branching, cartilage distribution, and later lung development.     

Pulmonary Hypoplasia in Neonatal Hypophosphatasia

Morphological, biophysical, and biochemical parameters of lung growth were studied at autopsy on a male infant with hypophosphatasia who died with asphyxia immediately after birth. The lungs were hypoplastic because of a marked decrease in airspace formation but lung maturation was normal for gestational age by all the parameters used. Diaphragmatic development, assessed by weight and fiber measurement, was in keeping with the decreased chest size. The proposed mechanism for this late onset type of pulmonary hypoplasia, attributed to decreased thoracic volume, is correlated with antenatal ultrasonographic observations of normal fetal breathing movements in the affected infant.     

Pulmonary hypoplasia in neonatal hypophosphatasia

Morphological, biophysical, and biochemical parameters of lung growth were studied at autopsy on a male infant with hypophosphatasia who died with asphyxia immediately after birth. The lungs were hypoplastic because of a marked decrease in airspace formation but lung maturation was normal for gestational age by all the parameters used. Diaphragmatic development, assessed by weight and fiber measurement, was in keeping with the decreased chest size. The proposed mechanism for this late onset type of pulmonary hypoplasia, attributed to decreased thoracic volume, is correlated with antenatal ultrasonographic observations of normal fetal breathing movements in the affected infant.     

Hypoplastic lungs and abnormal phospholipids in asphyxiating thoracic dystrophy

The respiratory impairment of asphyxiating thoracic dystrophy previously has been attributed to slower growth of the ribs which reduces chest size and limits chest expansion during breathing. Two siblings with this condition are described. One was found to have an abnormally low crying vital capacity; in the other the peak flow rate was reduced markedly. Chest X-rays and ventilation-perfusion nuclear scans were suggestive of hypoplastic lungs. Nasopharyngeal aspirates of airway secretions were found to contain significantly less total phospholipids and differences in phospholipid composition in comparison with a normal control group. These findings raise the possibility that the lungs are hypoplastic and have an abnormal phospholipid content in asphyxiating thoracic dystrophy.     

Hypoplastic lungs and abnormal phospholipids in asphyxiating thoracic dystrophy

Abstract The respiratory impairment of asphyxiating thoracic dystrophy previously has been attributed to slower growth of the ribs which reduces chest size and limits chest expansion during breathing. Two siblings with this condition are described. One was found to have an abnormally low crying vital capacity; in the other the peak flow rate was reduced markedly. Chest X-rays and ventilation-perfusion nuclear scans were suggestive of hypoplastic lungs. Nasopharyngeal aspirates of airway secretions were found to contain significantly less total phospholipids and differences in phospholipid composition in comparison with a normal control group. These findings raise the possibility that the lungs are hypoplastic and have an abnormal phospholipid content in asphyxiating thoracic dystrophy.     

Distribution and Number of Clara Cells in the Normal and Disturbed Development of the Human Fetal Lung

In this study the numerical growth and topological distribution of Clara cells were investigated in normal and hypoplastic lungs of fetuses ranging in age from the 10th to the 24th gestational week. In addition, the lungs of premature infants suffering from hyaline membrane syndrome (HMS) and bronchopulmonary dysplasia (BPD) were used as a model of disturbed lung growth in the early postnatal phase. Clara cells were observed to appear in the airway epithelium of fetuses of the 15th gestational week. After the 15th week of gestation the Clara cell number increased monotonously with increasing gestational age, reaching 5.4% Clara cells in the bronchial epithelium and 11.2% in the bronchiolar epithelium at the 24th gestational week. In the investigated period of gestation the Clara cell number was significantly higher in the bronchiolar epithelium compared to the bronchial epithelium. Hypoplastic lungs showed no difference in number and distribution of Clara cells compared to normal age-matched controls. This finding suggests the growth of Clara cells to be relatively accelerated compared to the decreased maturation of the lung parenchyma. The HMS/BPD cases showed normal Clara cell counts in the bronchial epithelium, whereas in the bronchiolar epithelium this value was decreased. This finding is caused by the extreme turnover of the airway epithelium in HMS/BPD; the local distribution of the epithelial damage is speculated to be caused by the physicochemical properties of inhaled oxygen.     

Surfactant abnormality and the sudden infant death syndrome--a primary or secondary phenomenon?

A prospective study of 46 infant deaths occurring between 3 and 100 weeks of age was performed and comprised a structured necropsy followed by collection of lung washings for surfactant phospholipid analysis and samples for microbiological examination. Of the 46 infants studied, 23 died from sudden infant death syndrome (SIDS) alone; SIDS was the cause of death in a further 12 but there were additional clinical or pathological findings insufficient in themselves to account for the death (''SIDS-plus''). In 11 there were other causes of death (''non-SIDS''). The lung washings from infants dying from SIDS had significantly lower concentrations of phosphatidylcholine and a significantly lower palmitate content in the phosphatidylcholine. There was no association between surfactant phospholipid abnormality and the presence of recognised pathogens, histological evidence of pulmonary inflammation, aspiration of stomach contents, age at death, sex, and death-postmortem interval. There were, however, lower concentrations of phosphatidylcholine and phosphatidylcholine palmitate content in infants colonised by organisms with reported phospholipase A2 activity.     

Effect of epidermal growth factor on lung growth in experimental fetal pulmonary hypoplasia

The purpose of this study was (1) to compare the expression of epithelial growth factor receptor (EGFR) in the lung tissues of human fetuses with or without pulmonary hypoplasia, and (2) to investigate the effects of EGF on lung growth in experimental pulmonary hypoplasia in rabbits. Firstly, we investigated the expression of EGFR in lung tissues of human fetuses with or without pulmonary hypoplasia by immunohistochemistry. Secondly, the amniotic fluid was shunted into the maternal abdominal cavity in a group of 12 fetal rabbits, another group (n = 12) received EGF injection (5 microg, i.p.) at day 25 of gestation. The third group (n = 12) was only treated with EGF while littermates not operated on served as the control group (n = 12). On day 29 of gestation, fetuses were delivered by Cesarean section and the lungs removed. The body weight and wet lung and liver weights were measured. As a measure of fetal lung growth, we determined the size of lung acini, the number of terminal airspaces, and diameter of alveoli (n = 6, each groups). We also measured the concentration of phosphatidylcholine (PC) and the lecithin/sphingomyelin (L/S) ratio in lung lavage fluid at birth in some fetuses (n = 6, each groups). In human fetuses with pulmonary hypoplasia, there was a significant decrease in radial alveolar count and expression of EGFR compared with fetuses without pulmonary hypoplasia. Amniotic shunt significantly decreased fetal lung/body weight ratio compared with control. Injection of EGF in the shunted group significantly increased lung/body weight ratio to the control level. The concentration of PC and L/S ratio in lung fluid lavage from rabbit fetuses with hypoplastic lungs was significantly higher than the other three groups. Histopathological examination of fetuses with hypoplastic lungs treated with EGF showed no significant change in the size of acini, number of terminal airspaces or the diameter of alveoli compared with the control group. Our results suggested that EGF was associated with lung growth and maturation of human lung and that treatment of rabbit fetuses with hypoplastic lungs with EGF facilitated lung growth and development.     

Increased Acinar Complexity with Polyhydramnios

We report 4 cases that presented a contradiction in terms of intrauterine lung growth. In all cases there was increased acinar complexity despite an array of influences known to be associated with pulmonary hypoplasia. In 2 of the 4 cases the lungs were severely hypoplastic by weight. Following an analysis of the factors operative in each case regarding lung growth, we conclude that diffuse increased acinar complexity occurs in selected cases with massive polyhydramnios. The observation is of importance because it is consonant with an intimate and dynamic relationship between amniotic fluid volume, lung fluid production, and fetal breathing movements in normal intrauterine lung growth and maturation. Although lung hypoplasia is known to occur in association with polyhydramnios, the reported alterations in lung structure were not previously observed.     

Increased acinar complexity with polyhydramnios

We report 4 cases that presented a contradiction in terms of intrauterine lung growth. In all cases there was increased acinar complexity despite an array of influences known to be associated with pulmonary hypoplasia. In 2 of the 4 cases the lungs were severely hypoplastic by weight. Following an analysis of the factors operative in each case regarding lung growth, we conclude that diffuse increased acinar complexity occurs in selected cases with massive polyhydramnios. The observation is of importance because it is consonant with an intimate and dynamic relationship between amniotic fluid volume, lung fluid production, and fetal breathing movements in normal intrauterine lung growth and maturation. Although lung hypoplasia is known to occur in association with polyhydramnios, the reported alterations in lung structure were not previously observed.     

Type IV collagen in developing human lung: a comparison between normal and hypoplastic fetal lungs

Human lung tissue obtained from 24 aborted fetuses of varying gestational ages (beyond 24 weeks) was examined for immunolocalisation of type IV collagen, using monoclonal antibody. Twelve cases of lung hypoplasia associated with oligohydramnios were compared with 12 normally developed lungs over the same gestational age range acting as control. Type IV collagen was located in alveolar septa and subepithelial basement membranes in all the lungs examined but showed less prominently in hypoplastic lungs than in control lungs of a similar gestational age. The findings suggest a potential role of type IV collagen in maturation of the developing human lung.     

The effects of preterm delivery and mechanical ventilation on human lung growth

The effects of preterm birth and mechanical ventilation on growth of the alveolar region of the lung were assessed by morphometric and/or quantitative biochemical methods in the lungs from 104 perinatal and infant autopsies. The lungs of 4 preterm infants who died at 4-16 weeks age without having received mechanical ventilation were large relative to body weight but showed normal alveolar number and alveolar surface area. Infants treated by mechanical ventilation for hyaline membrane disease (HMD) and who died at ages from 1 week up to 14 months showed impairment in alveolar development evidenced by low alveolar number and a low alveolar surface area. Lung volume and total lung DNA values were relatively normal. Dilated alveolar ducts were a feature at all ages with emphysematous changes apparent in the longest surviving infants. Biochemical features included a high concentration of hydroxyproline, reflecting collagen, and a high desmosine concentration, reflecting elastin, in infants dying at less than 60 weeks postconceptional age. Changes in the lungs of infants ventilated at low pressures for conditions other than HMD were of a similar nature but less severe than those seen in the HMD group. These findings indicate that preterm birth alone may have little adverse influence on lung development but that conditions necessitating mechanical ventilation may lead to permanent impairment in alveolar development. We postulate that the standard technique of applying positive pressure ventilation may itself lead to impaired alveolar growth, although the effect is enhanced by concomitant HMD and BPD.     

Quantitative aspects of perinatal lung growth

Weight, DNA, protein, hydroxyproline and disaturated phosphatidylcholine (DSPC) content were investigated in lungs of 97 normally formed infants over an age range from 22 to 75 postconceptional weeks, including 25 cases of hyaline membrane disease (HMD) and 13 small-for-dates infants (SFD). Lung weight and lung DNA relative to body weight were markedly lower in infants who died at 37-41 weeks than in those who died at shorter gestations or in early infancy. Total lung DSPC and DSPC concentration had a narrow peak at 36-41 weeks. The DSPC concentration per milligram of lung DNA in the first few months of infant life was similar to that in infants at 24 weeks gestation. Lung protein concentrations increased steadily but were variable at all ages. SFD infants had significantly higher concentrations of hydroxyproline and showed a peak DSPC concentration at an earlier gestation than the normals. Lungs of HMD infants showed some increase in hydroxyproline concentration but little other quantitative evidence of difference from the normals. We suggest that the relatively small lung size in many infants who die near term may result from recurrent intrauterine stress. Lung changes in small for dates infants are compatible with an advance in lung maturation, while the increased hydroxyproline concentration in the lungs of cases of HMD implies an early proliferative response to lung injury.