Ultrastructural stereologic analysis of fetal rabbit type II alveolar epithelial cells following maternal pilocarpine treatment or fasting

We have previously reported that subcutaneous administration of 5 mg/kg pilocarpine HC1, a muscarinic agonist, to pregnant white rabbits on days 24 through 27 of gestation results in an acceleration of fetal lung maturation. In the present study the ultrastructural differences observed within alveolar type II cells of 28-day fetuses from the pilocarpine and control groups were quantified by stereologic analysis. In addition, maturational changes occurring within the lungs of 28-day fetuses obtained from pregnant rabbits which were fasted for 7 days (days 21–28) were similarly analyzed. Stereologic analysis revealed that the fetal type II cells in the pilocarpinetreated group possessed a significantly greater volume density of lamellar inclusion bodies and rough endoplasmic reticulum and a significantly smaller volume density of glycogen than did controls. The volume density values for fetal type II cells of the fasted group were, with the exception of the Golgi apparatus, found to be intermediate between those of the control and the pilocarpine-treated groups. It was also noted that a high percentage of the lamellar inclusion bodies within type II cells of the fasted group appeared morphologically abnormal, being characterized by a highly irregular outline and tightly packed lipid membranes. The results of this study substantiate and quantify our previously reported observation of accelerated maturation in pilocarpine-treated fetuses. In addition, they suggest a similar, though not as pronounced, effect of maternal fasting prior to delivery. However, the abnormal morphology of the lamellar inclusion bodies in the fasted group may reflect a qualitative change in the pulmonary surfactant produced by these fetuses.     

Hypophysectomy and porcine fetal lung development

The effect of hypophysectomy on the development of the lung parenchyma and maturation of pulmonary alveolar type II cells was examined in the fetal pig. In fetuses from four different gilts, hypophysectomy was performed on gestational day 69 or 70. Littermates from the same gilts served as controls. Fetuses were delivered by caesarean section at term (113 +/- 1 days of gestation), and the lungs were fixed by intratracheal instillation. Plasma cortisol and thyroxine concentrations in the umbilical artery were markedly lower for hypophysectomized fetuses compared with control fetuses. Body weight was similar for both groups of fetuses. Total lung volume was 53% smaller in hypophysectomized fetuses compared with control fetuses. However, alveolar septal tissue and capillary luminal volumes were similar in both groups. Total lung alveolar surface area was twice as great in control animals compared with hypophysectomized animals. The volumes of epithelium, interstitium, and endothelium of centriacinar alveolar septa per unit surface area of epithelial basal lamina were 3.7, 4.8, and 2.4 times greater in hypophysectomized fetuses compared with control fetuses. Alveolar type II cell composition also differed significantly between groups. The volume fraction of glycogen in type II cells was 51% for hypophysectomized fetuses and 12% for control fetuses, while lamellar body volume fraction was 8% in hypophysectomized fetuses and 23% in control fetuses. The frequency of alveolar type II cell contact with mesenchymal interstitial cells via foot processes was 5 times greater in the lungs of control animals compared with hypophysectomized animals. These findings demonstrate significant effects of hypophysectomy on the morphogenetic and cytodifferentiation activities of all major tissue compartments of the pulmonary gas exchange area during the final trimester of fetal development.     

Morphologic Development of Fetal Rabbit Lung and Its Acceleration with Cortisol

The influence of cortisol on the maturation of fetal lungs of rabbits has been studied. The normal developmental pattern of the various lobes is also documented and it is shown that there is a definite sequence of maturation among the five lobes of the lung. At a given gestational age, both upper lobes are consistently most mature. The right middle lobe is somewhat less mature while right and left lower lobes are least mature. The difference in the rate of maturation between the most advanced right apex and the least advanced base of lower lobes is as much as 1½ days out of a total gestation of 30 days. Cortisol, injected into the fetuses 2 days prior to delivery, accelerated the overall maturation of the lung by about 1½ days. The maturation of the type II epithelial cells also was accelerated and their inclusion bodies were more numerous than those of the controls. As reported elsewhere, prematurely delivered cortisol-injected rabbits of 26 to 28 days' gestation are more viable, breathe better and their lungs are better aerated when compared with control littermates. The surface activity of the alveolar lining of these animals is greater than that of the controls as indicated by higher ratios of bubble stability. These findings are apparently the results of accelerated maturation of the type II alveolar epithelial cells, which contain surfactant or its precursors as inclusions within the cytoplasm.     

An immunohistochemical study of the expression of surfactant apoprotein in the hypoplastic lung of rabbit fetuses induced by oligohydramnios.

We shunted amniotic fluid from alternate gestational sacs into the maternal peritoneal cavity between 23 and 30 days gestation in fetal rabbits (full term, 31 days) to investigate the effect of oligohydramnios on surfactant apoprotein A (SP-A) expression by an immunohistochemical morphometric analysis. The amniotic shunt produced a significant decrease in the amniotic fluid volume (P < 0.02), as well as a reduction in the lung weight (P < 0.005) and lung/body weight ratio (P < 0.001), which indicated lung hypoplasia. These fetuses also showed a statistically significant reduction of SP-A expression, ie, SP-A-positive type II cells per unit area (P < 0.05), SP-A-positive type II cells/total cells ratio (P < 0.001), the percentage of SP-A-positive area per unit area (P < 0.005), and the SP-A-positive area/alveolar epithelium area plus the lung interstitium area ratio (P < 0.005). These results suggest that oligohydramnios significantly retards and modifies the structural growth and functional development of alveolar type II cells in SP-A expression. This animal model of hypoplastic lung in fetuses is thus considered to be useful in helping to further develop the treatment for hypoplastic lung.     

Surfactant-associated glycoproteins accumulate in alveolar cells and secretions during reparative stage of hyaline membrane disease

Surfactant-associated (SA) glycoproteins are lung-specific proteins produced in the human lung by alveolar type II cells and Clara cells. The distribution of these proteins was studied immunohistochemically in lung tissue obtained postmortem from 12 stillborn fetuses and 49 infants with hyaline membrane disease (HMD). By 21 weeks of gestation, SA glycoproteins were detected in the fetal alveolar epithelium and within Clara cells. The staining increased in intensity and extent with advancing gestational age. Infants with HMD who survived less than 48 hours did not generally exhibit stainable material either within type II cells or secretions, but staining was often noted in Clara cells as well as focally beneath hyaline membranes. In infants surviving more than 48 hours, intense staining of hyaline membranes, alveolar secretions, proliferating alveolar type II cells, and Clara cells was evident. Immunoreactivity was intense in hypertrophic type II cells that formed a continuous alveolar epithelial lining in lungs with bronchopulmonary dysplasia. Included in the population of infants with HMD were 15 infants with pulmonary hypoplasia. The lungs of these infants showed minimal staining for SA glycoproteins regardless of postnatal survival time. The results provide an immunomorphologic basis for defining normal and abnormal lung maturation. They also indicate that enhanced SA glycoprotein production is a sustained response of regenerating and hypertrophic type II cells in premature infants.     

小鼠肺泡发育与肺泡上皮细胞分化的电镜观察

目的　观察小鼠胚胎及生后肺泡发育及肺泡上皮分化。方法　小鼠胚胎 14天至生后 14天肺组织 ,隔天取材 ,HE染色光镜观察及透射电镜观察。结果　胚胎 14～ 18天 ,小鼠肺的发育以支气管树分支和管壁结构的逐渐完善为主 ,终蕾上皮为柱状或立方状的未分化细胞。胚胎 19天 ,支气管远端形成许多内壁光滑的原始肺泡 ,其上皮分化出Ⅱ型肺泡细胞。生后 1～ 4天 ,肺泡上皮出现少量扁平的Ⅰ型肺泡细胞 ,但仍以Ⅱ型肺泡细胞为主。生后 5～ 14天 ,成熟肺泡形成 ,肺泡上皮以Ⅰ型肺泡细胞为主。结论　出生时 ,小鼠的肺发育只完成了其大体形态的发生 ,肺泡上皮以Ⅱ型肺泡细胞为主。成熟肺泡的形成、数量的增加及Ⅰ型肺泡细胞的大量出现持续到出生后。     

An ultrastructural, morphometric analysis of rabbit fetal lung type II cell differentiation in vivo

Rabbit lung type II cell differentiation was evaluated by use of ultrastructural, morphometric techniques. Fetal lung epithelial cells decreased in size dramatically from day 19 to day 21 of gestation. Thereafter, the cell and cytoplasmic cross-sectional area declined gradually until the neonatal time point. The tall columnar cell shape characteristic of fetal lung epithelial cells at early stages of development became cuboidal by day 24 of gestation. The number of mitochondria per micron2 cytoplasmic area in presumptive alveolar epithelial cells and the mitochondrial volume density increased toward the end of gestation. The volume density of glycogen pools within fetal lung epithelial cells reached a plateau on day 21 of gestation and then declined sharply on day 26 of gestation in lamellar body-containing, type II epithelial cells. Lamellar bodies increased in number and volume density in epithelial cells starting on day 26 of gestation and peaked with respect to these parameters in the neonatal lung tissue. Multivesicular bodies, which are thought to be a precursor to the lamellar body, became more prominent in differentiated type II cells on day 26 of gestation and increased in volume density from day 28 of gestation to the adult time point. The distance between mesenchymal and epithelial cells in fetal lung tissue declined sharply between days 24 and 26 of gestation but remained relatively constant thereafter. Foot processes extending from connective tissue cells contiguous to the epithelium were generally more numerous than those extending from the basal plasma membrane of epithelial cells at every stage of development examined. These data quantitate for the first time key ultrastructural events that occur during the differentiation of fetal lung epithelial cells in vivo.     

Glucocorticoid effects on rabbit fetal lung maturation in vivo: An ultrastructural morphometric study

Maternal administration of glucocorticoids is known to stimulate fetal lung maturation. In the present study, we used microscopy and stereology to evaluate the morphological effects of maternal glucocorticoid treatment on rabbit fetal lung tissue. Betamethasone was administered to pregnant rabbits on days 25 and 26 of gestation at a dose of 0.2 mg/kg body weight. The animal were sacrificed on day 27 of gestation. Glucocorticoid treatment significantly increased the presumptive airspace in the fetal lung tissue but did not alter the relative proportion of epithelium, connective tissue, or vasculature in the tissue. In addition, glucocorticoid treatment significantly increased the proportion of type II cells in the prealveolar epithelium, increased the rate of phosphatidylcholine synthesis, and increased the content of the major surfactant-associated protein, SP-A, in the fetal lung tissue. We could detect no effect of betamethasone on lamellar body crosssectional area, numerical density, or volume density within fetal lung type II cells. Glucocorticoid treatment of the pregnant doe caused a decrease in the volume density of intracellular glycogen and an increase in the volume density of mitochondria in fetal lung type II cells. Betamethasone treatment did not alter the distance between fetal lung epithelial cells and subadjacent connective tissué cells. However, glucocorticoid treatment increased the number of connective tissue foot processes that pierced the epithelial basal lamina. Thus, glucocorticoid treatment of the pregnant doe results in structural changes in the fetal lung tissue, an acceleration of some aspects of type II cell defferentiation, and a concomitant increase in epithelial-mesenchymal interactions.     

Glucocorticoid effects on rabbit fetal lung maturation in vivo: an ultrastructural morphometric study.

Maternal administration of glucocorticoids is known to stimulate fetal lung maturation. In the present study, we used microscopy and stereology to evaluate the morphological effects of maternal glucocorticoid treatment on rabbit fetal lung tissue. Betamethasone was administered to pregnant rabbits on days 25 and 26 of gestation at a dose of 0.2 mg/kg body weight. The animals were sacrificed on day 27 of gestation. Glucocorticoid treatment significantly increased the presumptive airspace in the fetal lung tissue but did not alter the relative proportion of epithelium, connective tissue, or vasculature in the tissue. In addition, glucocorticoid treatment significantly increased the proportion of type II cells in the prealveolar epithelium, increased the rate of phosphatidylcholine synthesis, and increased the content of the major surfactant-associated protein, SP-A, in the fetal lung tissue. We could detect no effect of betamethasone on lamellar body cross-sectional area, numerical density, or volume density within fetal lung type II cells. Glucocorticoid treatment of the pregnant doe caused a decrease in the volume density of intracellular glycogen and an increase in the volume density of mitochondria in fetal lung type II cells. Betamethasone treatment did not alter the distance between fetal lung epithelial cells and subadjacent connective tissue cells. However, glucocorticoid treatment increased the number of connective tissue foot processes that pierced the epithelial basal lamina. Thus, glucocorticoid treatment of the pregnant doe results in structural changes in the fetal lung tissue, an acceleration of some aspects of type II cell differentiation, and a concomitant increase in epithelial-mesenchymal interactions.     

Localization of surfactant-associated protein C (SP-C) mRNA in fetal rabbit lung tissue by in situ hybridization.

Surfactant is a lipoprotein substance that is synthesized and secreted by alveolar type II epithelial cells and acts to reduce surface tension at the air-alveolar interface. SP-C is a 5,000-D molecular weight, hydrophobic, surfactant-associated protein. In the present study, we used a ribonuclease protection assay to show that SP-C mRNA is induced in rabbit fetal lung tissue early in development, increases in relative concentration as development proceeds, and is present in maximal concentration at term (31 days of gestation). We also used the technique of in situ hybridization to localize SP-C mRNA in fetal, neonatal, and adult rabbit lung tissue. SP-C mRNA was present in all of the epithelial cells of the prealveolar region of day 19 gestational age rabbit fetal lung tissue, i.e., about 7 days before the appearance of differentiated alveolar type II cells in the fetal lung tissue. By day 27 of gestation, SP-C mRNA was restricted to epithelial cells with the morphologic characteristics of alveolar type II cells. SP-C mRNA was not detected in bronchiolar epithelium at any stage of lung development. The intensity of SP-C mRNA hybridization in the prealveolar and alveolar type II epithelial cells increased as a function of gestational age and was maximal at term. The pattern of SP-C mRNA localization in neonatal and adult rabbit lung tissue was consistent with the restriction of SP-C gene expression to differentiated alveolar type II cells. Our data are suggestive that SP-C may serve some as yet unknown function early in lung development because it is present in fetal lung prealveolar epithelial cells much earlier in gestation than are differentiated, surfactant-producing alveolar type II cells.     

Glucocorticoid regulation of surfactant-associated proteins in rabbit fetal lung in vivo

The effects of a maternally administered synthetic glucocorticoid, betamethasone, on the levels of mRNA for the surfactant proteins SP-A, SP-B, and SP-C and on the levels of SP-A protein were investigated in day 27 gestational age rabbit fetal lung tissue. Betamethasone administration to the pregnant rabbit caused approximately a twofold increase in the fetal lung level of SP-A protein and a threefold increase in fetal lung SP-A mRNA levels when compared to levels in fetuses obtained from saline-treated or uninjected animals. SP-B mRNA was increased fourfold in fetal lung tissue obtained from glucocorticoid-treated pregnant does when compared to levels in fetuses of uninjected pregnant does. However, SP-B mRNA levels in fetal lung tissue from saline-injected controls were also significantly elevated, ～twofold, when compared to fetal lung SP-B mRNA levels in the uninjected control condition. SP-C mRNA levels in lung tissue of fetuses from both saline-injected and betamethasone-injected pregnant does were increased similarly, ～twofold, over SP-C mRNA levels in fetal lung tissue obtained from uninjected control does. These data are suggestive that betamethasone treatment increases fetal lung SP-A and SP-B mRNA levels and that maternal stress alone can increase the expression of SP-B and SP-C mRNA in rabbit fetal lung tissue. Using in situ hybridization, SP-A mRNA was shown to be present primarily in alveolar type II cells in fetuses of control and saline-injected does. However, SP-A mRNA was easily detected in both alveolar type II cells and bronchiolar epithelial cells of rabbit fetal lung tissue following maternal betamethasone treatment. In contrast, SP-B and SP-C mRNA were present only in alveolar type II cells of lung tissue obtained from fetuses of control, saline, or betamethasone-treated does. Thus maternal administration of glucocorticoids increased SP-A protein as well as SP-A and SP-B mRNA levels in rabbit fetal lung tissue. SP-A mRNA was localized to both alveolar type II cells and in smaller amounts in bronchiolar epithelial cells of rabbit fetal lung tissue. However, SP-B and SP-C mRNA were detected only in alveolar type II cells. © 1993 Wiley-Liss, Inc.     

MHC class II antigens on lung epithelial of human fetuses and neonates. Ontogeny and expression in lungs with histologic evidence of infection.

An immunohistochemical study with anti-HLA-DP, anti-HLA-DQ and anti-HLA-DR monoclonal antibodies was carried out on lung tissue from 29 fetuses and 19 infants that died within the 1st year of life. The aim of the study was to verify the influence of lung maturation and lung inflammation, caused by intrauterine infection, on the expression of MHC class II antigens on lung epithelia. No positive reaction of lung epithelia was noticed in 6 fetuses of less than 21 weeks' gestation. In fetuses of more than 21 weeks' gestation, lung inflammation strongly correlated with the expression of MHC class II subregion products on lung epithelia. All 4 fetuses with histopathologic evidence of lung infection but only 4 out of 23 fetuses without lung inflammation bore HLA-DR antigens on their immature cuboidal alveolar epithelium. Within the bronchial epithelial layers only scattered MHC class II positive epithelia were found. After birth, MHC class II antigens were almost constantly detected on cuboidal type II pneumocytes of inflamed and noninflamed lung tissue as well. Only 3 infants that died within the 1st week of life did not show any MHC class II antigen expression on their lung epithelia.     

Reduced cell proliferation in fetal lung after maternal administration of pilocarpine. A scintillation autoradiographic study

Fetuses were obtained on the 28th gestational day from pregnant New Zealand white rabbits treated daily, on the 24th through the 27th gestational day, with pilocarpine HCl, 5 mg/kg in saline, or saline alone. Lung fragments from these fetuses were incubated for two hours in medium containing ³H-thymidine. Scintillation autoradiography of 1-μm-thick sections of these fetal lungs revealed that the lung tissue from pilocarpine-treated fetuses had significantly lower labelled cell indices for both alveolar epithelial cells and interstitial cells. These results indicate that pilocarpine treatment promotes differentiation of immature cells in the fetal lung at the expense of cell proliferation.     

Acceleration of canalicular development in lungs of fetal mice exposed transplacentally to dexamethasone.

Morphometric techniques were used to compare the volume density of air space (Vva) and the degree of maturation of pulmonary epithelium in normal fetal mouse lung and in lungs of fetuses exposed transplacentally to dexamethasone. Pregnant Bagg-Webster Swiss mice of 16 days' gestation were given injections of either saline or dexamethasone in doses ranging from 0.40 to 12.0 microng. per gm. of body weight, and killed at intervals thereafter. Fetuses were removed and weighed and their lungs prepared for morphometry using osmium-fixed, Epon-embedded tissue. In control lungs, Vva increased 10-fold between days 17 and 19, an increase from 1.5 to 15%. A 25-fold increase occurred during the same period in test fetal lungs exposed to 0.40 microng. per gm. or more of dexamethasone. When the degree of air space development was compared 24 hours after exposure, within a single weight group and, according to dose, a linear increase in air space was found; 0.1-microng. per gm. increment in dexamethasone produced a 0.66% increment in Vva. Body weight was an important determinant, in that fetuses in the lower weight range had much less response. The latter showed an increment of approximately 0.25% in Vva for each 0.1-microng. per gm. increment of dexamethasone. It can be emphasized from the present experiments that a maximal development of Vva could be achieved by amounts of dexamethasone too low to depress fetal or lung weight. The proportion of pulmonary epithelial cells containing osmiophilic granules increased in control lungs from 18% on day 17 to 42% on day 18. Test fetuses (17 days old) examined 24 hours after receiving either 0.40 or 0.80 microng. per mg. of dexamethasone showed no significant increase in this proportion; however, a significant increase in the proportion of cells containing osmiophilic granules was found in fetal lungs exposed to 2.0 microng. per mg. Whereas a significant increase in Vva was found within 14 hours of exposure, no increase in the proportion of cells containig osmiophilic granules was detectable at this time. It was concluded that air space development is a sensitive method for evaluating the effect of dexamethasone as it gives a clear dose-response curve in fetuses exposed to it 24 hours prior to sacrifice. Accelerated maturation of the presumptive type II cell could only be demonstrated within 24 hours by using higher doses than those required to initiate air space development. These observations suggest that the steps invovled in canal formation, which are assumed to reflect alterations in mesenchyme, may have a different sensitivity to dexamethasone than do those initiating the maturation of alveolar epithelial cells.     

Localization of surfactant-associated proteins SP-A and SP-B mRNA in rabbit fetal lung tissue by in situ hybridization.

Pulmonary surfactant is a lipoprotein substance, comprised of approximately 80% phospholipid and approximately 10% protein, that lowers surface tension at the air-alveolar aqueous interface. Surfactant is synthesized and secreted by alveolar type II epithelial cells where it is stored intracellularly in lamellar bodies. In the present study, we used the technique of in situ hybridization to localize the mRNA for two surfactant-associated proteins, SP-A and SP-B, in developing rabbit fetal lung tissue. We found that SP-A mRNA was first localized in rabbit fetal lung alveolar type II cells on day 26 of gestation, the time at which lamellar bodies are first observed within fetal lung type II cells. On day 28 of gestation, a very small amount of SP-A mRNA was also detectable in the epithelial cells of some bronchioles. In neonatal and adult rabbit lung tissue, SP-A mRNA was primarily restricted to alveolar type II cells; however, the epithelial cells of some bronchioles contained small amounts of SP-A mRNA. SP-B mRNA was first detected in cuboidal epithelial cells in the prealveolar region of the rabbit fetal lung tissue on day 24 of gestation, i.e., at least 2 days before the appearance of SP-A mRNA and lamellar bodies within differentiated alveolar type II cells. SP-B mRNA was detected in most bronchiolar epithelial cells of the rabbit fetal lung tissue at day 28 of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-azetidine-2-carboxylic acid retards lung growth and surfactant synthesis in fetal rats

Maturation of the pulmonary epithelium during late fetal development is controlled at least in part by the underlying fibroblasts. To further investigate this cellular interdependence and the role of collagen in type 2 cell differentiation, we studied the effects of inhibiting fibroblast function in vivo by injecting the proline analog L-azetidine-2-carboxylic acid (LACA) to timed pregnant rats, and examining changes in cell proliferation and surfactant synthesis in fetal lungs. LACA (200 mg/kg) was injected twice daily for 2 days and rats were killed 2 days later at days 19, 20, 21, and 22 of gestation. Fetal lung weight and DNA content were about 50% of controls, hydroxyproline per dry weight was reduced and by electron microscopy, there appeared to be less fibrillar collagen in the lung. Autoradiography after [3H]thymidine pulse-labeling showed reduced cell proliferation on days 19 and 20 mainly due to lower fibroblast growth with a smaller reduction in epithelial labeling. Lung development in LACA-treated rats was retarded; air sacs were slow to open, epithelial cells retained glycogen longer and fewer cells developed lamellar bodies compared with age-matched controls. There was a reduction in the incidence of epithelial-interstitial cell contacts at day 20 only. Measurements of disaturated phosphatidylcholine showed a 50% reduction per dry weight and a lower disaturated phosphatidylcholine/lipid ratio after LACA. The results indicate that LACA administration in vivo slows fibroblast growth and greatly reduces fibrillar collagen deposition with an accompanying reduction in pulmonary surfactant. This suggests that secreted matrix influences growth and differentiation of the alveolar epithelium.     

人胎肺Ⅱ型肺泡细胞中表面蛋白-B的表达及意义

目的检测肺表面蛋白-B(SP-B)及其调控因子甲状腺转录因子-1(TTF-1)在肺泡Ⅱ型细胞发育过程的表达特征,探讨两者对于人胎肺肺泡Ⅱ型细胞发育、分化和成熟的调控作用。方法取16-35周人胎肺组织,常规石蜡包埋切片,用免疫组化技术检测SP-B和TTF-1的表达特征。结果,TTF-1在胎肺16周开始表达,定位于上皮细胞核内,随支气管树的发育分化,远端位置的反应总是较近端者强。SP-B蛋白于18周开始表达,定位于肺泡Ⅱ型细胞胞浆内,阳性反应细胞于早、中期表达丰富;由呼吸道近端逐渐往远端迁移,且强度不断增强。发育末期至成肺中,TTF-1和SP-B阳性反应均在肺泡Ⅱ型细胞中稳定表达。结论TTF-1参与支气管树形态发生和肺泡的发育成熟,并调控肺泡Ⅱ型细胞中SP-B蛋白的分泌,起稳定肺泡直径的作用。因此,SP-B蛋白的分泌反映肺泡Ⅱ型细胞功能的成熟。     

Lung development following diaphragmatic hernia in the fetal rabbit

Diaphragmatic hernia was created in 39 rabbit fetuses on day 23 of gestation. Fifteen fetuses underwent a sham thoracotomy (SHAM). Thirty-nine non-operated littermates served as internal controls (CTR). Fetuses were harvested by Caesarean section on days 25, 27, 29 and 30 of gestation. Pulmonary response was evaluated by lung to body weight ratio (LBWR), morphometry, and density of type II pneumocytes. No difference was found between CTR and SHAM fetuses at term. CDH fetuses had smaller lungs (LBWR 0.014 +/- 0.004 versus 0.030 +/- 0.04 in CTR, P < 0.0001), a less complex acinus [mean terminal bronchial density (MTBD) 1.786 +/- 0.408 versus 0.917 +/- 0. 188, P < 0.0001], thicker alveolar septa [mean wall transection length (LMW) 0.0221 +/- 0.008 versus 0.0142 +/- 0.002, P = 0.0003], and a lower type II cell count (144.5 +/- 19.33 versus 216.2 +/- 27.85 per high power field, P < 0.0001). The differences in MTBD and LMW were significant from gestational day 25 onwards, and the differences in type II cell count from day 27 onwards. Surgical diaphragmatic hernia in rabbit fetuses in the late pseudoglandular phase reproduces many features of the pulmonary hypoplasia associated with human congenital diaphragmatic hernia, including the delayed maturation. The effects are present within 2 days following experimental diaphragmatic hernia and progress over time.     

Electron microscopic observations on the morphogenesis of the albino rat lung,with special reference to pulmonary epithelial cells

Electron microscopic observations of developing albino rat lung provide further evidence for the endodermal origin of the type I and II pulmonary epithelial cells, and for the mesodermal origin of the interstitial pulmonary cells. Cytoplasmic glycogen increased in the fetal endodermal cells from day 16 to the twentieth day of gestation. Further development revealed a decrease of this substance in the differentiating pulmonary epithelial cells, and a concurrent increase in the mesodermal interstitial pulmonary cells, these being cells formerly characterized by their high lipid content. A continuous basement membrane delineated the mesodermal and endodermal components of the developing rat lung from day 16 to the third postnatal day. Tight junctions between adjacent endodermal epithelial cells were present throughout this same gestational period. Lamellar bodies, which are characteristic of the type II pulmonary epithelial cell, may be found simultaneously with large quantities of cytoplasmic glycogen. This feature is considered characteristic of fetal endodermal components. A definite continuum of ultrastructural changes may be traced from the endodermal, columnar epithelial cells to the definitive type I and II pulmonary epithelial cells. Comparable observations for the mesodermal components reveal a progression leading to mature interstitial pulmonary cells.     

Ultrastructural morphometric characterization of alveolar type II cells of perinatal sheep: a comparison with adults

Summary The quantitative morphologic changes in alveolar type II cells during the perinatal period were characterized morphometrically in the lungs of fetal lambs at 132, 138, and 147 days gestational age (DGA) and in newborns at 2 days postnatal age (2 DPN). Ultrastructural features were compared with those of type II cells of ewes 365 days old. Lamellar body profile number per type II cell profile was highest at term (147 DGA) and 2 DPN. In adults, the number of lamellar body profiles and volume density of lamellar bodies were equal to those of the 132 DGA fetus. Multivesicular bodies were most common at 138 DGA and in adults. The volume density of cytoplasmic glycogen fell dramatically during the latter part of gestation. The volume density of many cellular organelles increased to the level observed in adults by term (147 DGA). Subcellular composition of type II cells of adult sheep differs from that reported for adult rats chiefly by the volume density of lamellar material within the cytoplasm. Plate-like or globe-like inclusions were present only in the type II cells of adults. Cytoplasmic extensions of the type II cell crossing the basal lamina were most abundant in the 132 and 138 DGA fetal sheep. Cytoplasmic extensions were rare in adults. We conclude that morphologic changes of the alveolar type II cell associated with gestational age follow a species-specific time course. In the sheep, this occurs during the later part of gestation and extends into the neonatal period. Morphologic and morphometric changes appear to correspond with cellular interactions between alveolar type II cells and mesenchymal cells of the interstitium.