Canalicular Period Research Articles

Ultrasound-based radiomics for the evaluation of fetal rat lung maturity: A noninvasive assessment method (Ultrasound-based radiomics in fetal rat lung).

To establish a classification model for the evaluation of rat fetal lung maturity (FLM) using radiomics technology. A total of 430 high-throughput features were extracted per fetal lung image from 134 fetal lung ultrasound images (four-cardiac-chamber views) of 67 Sprague-Dawley (SD) fetal rats with a gestational age of 16-21days. The detection of fetal lung tissues included histopathological staining and the expression of surface proteins SP-A, SP-B, and SP-C. A machine learning classification model was established using a support vector machine based on histopathological results to analyze the relationship between fetal lung texture characteristics and FLM. The rat fetal lungs were divided into two groups: terminal sac period (SD1) and canalicular period (SD2). The mRNA transcription and protein expression level of SP-C protein were significantly higher in the SD1 group than in the SD2 group (p<0.05). The diagnostic performance of the rat FLM classification model was measured as follows: area under the receiver operating characteristic curve (AUC), 0.93 (training set) and 0.89 (validation set); sensitivity, 89.26% (training set) and 87.10% (validation set); specificity, 85.87% (training set) and 79.17% (validation set); and accuracy, 87.79% (training set) and 83.64% (validation set). Ultrasound-based radiomics technology can be used to evaluate the FLM of rats, which lays a foundation for further research on this technology in human fetal lungs.

Divergent expression of claudin -1, -3, -4, -5 and -7 in developing human lung

BackgroundClaudins are the main components of tight junctions, structures which are associated with cell polarity and permeability. The aim of this study was to analyze the expression of claudins 1, 3, 4, 5, and 7 in developing human lung tissues from 12 to 40 weeks of gestation.Methods47 cases were analyzed by immunohistochemisty for claudins 1, 3, 4, 5 and 7. 23 cases were also investigated by quantitative RT-PCR for claudin-1, -3 and -4.ResultsClaudin-1 was expressed in epithelium of bronchi and large bronchioles from week 12 onwards but it was not detected in epithelium of developing alveoli. Claudin-3, -4 and -7 were strongly expressed in bronchial epithelium from week 12 to week 40, and they were also expressed in alveoli from week 16 to week 40. Claudin-5 was expressed strongly during all periods in endothelial cells. It was expressed also in epithelium of bronchi from week 12 to week 40, and in alveoli during the canalicular period. RT-PCR analyses revealed detectable amounts of RNAs for claudins 1, 3 and 4 in all cases studied.ConclusionClaudin-1, -3, -4, -5, and -7 are expressed in developing human lung from week 12 to week 40 with distinct locations and in divergent quantities. The expression of claudin-1 was restricted to the bronchial epithelium, whereas claudin-3, -4 and -7 were positive also in alveolar epithelium as well as in the bronchial epithelium. All claudins studied are linked to the development of airways, whereas claudin-3, -4, -5 and -7, but not claudin-1, are involved in the development of acinus and the differentiation of alveolar epithelial cells.

Epithelial N-cadherin and nuclear β-catenin are up-regulated during early development of human lung

BackgroundThe aim of this study was to analyze the cell-specific expression of E- and N-cadherin and β-catenin in developing human lung tissues from 12 to 40 weeks of gestation.MethodsFortyseven cases of developing human lung including pseudoglandular, canalicular, saccular and alveolar periods were analyzed by immunohistochemisty for E- and N-cadherin and β-catenin and twentyone cases were also investigated by RT-PCR for E- and N-cadherin and β-catenin. For identifying the lung cells, the sections were also stained with antibodies against thyroid transcription factor-1 (TTF-1) and caveolin-1. Normal adult lung tissue was used as a control.E-cadherin was strongly expressed in epithelium of bronchi and large bronchioles from week 12 onwards and it was also positive in alveoli in pretype II cells and type II cells. N-cadherin was present in most of the epithelial cells of bronchi and the largest bronchioles during the pseudo-glandular and canalicular periods. N-cadherin was not detected in epithelium of developing alveoli. β-catenin was strongly membrane-bound and positively expressed in bronchial epithelium from week 12 to week 40; it showed nuclear positivity in both developing airway epithelium and in the cells underneath the epithelium during pseudo-glandular period and to a lesser degree also in the canalicular period. β-catenin was positive in pretype II cells as well as in type I and type II pneumocytes within alveoli.RT-PCR analyses revealed detectable amounts of RNAs of E- and N-cadherin and β-catenin in all cases studied. The amounts of RNAs were higher in early stages of gestation.ConclusionsE-cadherin is widely expressed in bronchial and alveolar epithelial cells. N-cadherin exhibit extensive epithelial positivity in bronchial epithelial cells during early lung development. The presence of β-catenin was observed in several cell types with a distinct location in tissue and cells in various gestational stages, indicating that it possesses several roles during lung development. The expressions of protein and mRNAs of E- and N-cadherin and β-catenin were higher in early gestation compared to of the end. Moreover, the expressions of these factors were higher during the lung development than in the adult human lung.

Pulmonary Maturation in Canine Foetuses From Early Pregnancy to Parturition

Development of the foetal respiratory system includes both pulmonary growth and maturation. In human medicine, a higher incidence of respiratory distress is reported in newborn males. This study aimed to identify different phases of canine foetal lung maturation throughout pregnancy, to determine the stage of pregnancy in which surfactant production begins and to compare pulmonary development of male and female foetuses. Pregnant bitches (34) were subjected to elective ovariohysterectomy and allocated into four groups, according to the stage of pregnancy: 30-40 days of pregnancy (n = 10), 41-50 days (n = 10), 51-60 days (n = 10) and bitches in the first stage of parturition (n = 4). Foetal lungs were histologically processed and evaluated by optical microscopy. The pseudoglandular phase was identified between the 35th day and 46th day of gestation; the onset of canalicular and saccular periods was observed, respectively, from the 48th day and 60th day of pregnancy. Lungs from foetuses at term were in the saccular phase; thus, the development into the alveolar period occurs in the neonatal period. The histological analyses revealed that respiratory tract development is centrifugal, from upper to lower airways. Therefore, it is possible to identify distinct development periods in different portions of the same organ. In conclusion, the saccular phase of lung development begins around 57 and 60 days of pregnancy, the period in which surfactant production is believed to occur. Male and female foetuses present similar pulmonary development from early pregnancy until parturition.

Expression of Hoxb‐5 during human lung development and in congenital lung malformations

We have previously shown that the Hox gene Hoxb-5 is necessary for normal mouse lung branching morphogenesis. Abnormal Hoxb-5 regulation causes specific alterations in airway branching. We hypothesized that Hoxb-5 is similarly involved in human lung branching morphogenesis, and is abnormally expressed in bronchopulmonary sequestration (BPS) and congenital cystic adenomatoid malformation (CCAM), both of which are congenital lung malformations with abnormal airway development. The temporal, spatial, and cellular expression of the Hoxb-5 protein was evaluated in normal human lung and BPS and CCAM tissue using Western blot analysis and immunocytochemistry. The expression of Hoxb-5 during human lung development showed strong similarities to that during mouse lung development. Western blots showed high Hoxb-5 protein levels in the pseudoglandular period (PSG), decreased but sustained levels in the canalicular period (CAN), and negligible levels during the alveolar period (ALV). Immunocytochemistry showed Hoxb-5 protein expression in mesenchymal cells around branching airways in the pseuodglandular period, subepithelial fibroblast localization (especially at airway branch points) in the CAN and minimal expression in the ALV. In BPS and CCAM tissue, Hoxb-5 protein levels were increased compared to age- and developmentally-matched lung tissue, and were more similar to the PSG and CAN with Hoxb-5-positive cells in mesenchyme surrounding abnormally branched airways. Hoxb-5 expression during human lung branching morphogenesis, which is similar to that observed in mouse lung development, indicates that it plays a role in controlling airway patterning. This notion is supported by results from BPS and CCAM tissue, in which Hoxb-5 is maintained in a manner typical of an earlier developmental stage and is associated with development of abnormal lung tissue.

Distribution and mRNA expression of tenascin-C in developing human lung.

Tenascin-C is an extracellular matrix glycoprotein that is spatially expressed during organogenesis, in inflammatory and fibrotic disorders, and in neoplasms. The aim of this study was to analyze its expression in developing human lung tissues during pseudoglandular, canalicular, saccular, and alveolar periods corresponding to Weeks 12 to 40. Lung tissues were obtained at autopsy from 34 nonmalformed cases. An immunohistochemical analysis and a messenger RNA (mRNA) in situ hybridization method combined with light microscopy were used. The extent of tenascin-C immunoreactivity was scored as absent, low, moderate, or strong in and around different types of pulmonary cells. The immunohistochemical expression for tenascin-C was strong beneath the airway epithelium, especially at the sites of airway subdivision during Weeks 12 to 23, whereas its expression was moderate or weak underneath alveolar and bronchiolar epithelia between Weeks 24 and 40. The expression for tenascin-C was strong in the intima of veins, especially in the canalicular period, i.e., Weeks 17 to 28. A moderate or strong immunoreactivity for tenascin-C was also observed around chondrocytes in every case studied during all periods. The increased expression of tenascin-C mRNA was most often seen in the cells below the airway epithelium. Taken together, tenascin-C is expressed in human lung during all developmental periods, and its expression is especially strong below the airway epithelium at the sites of airway subdivision.

Expression of heterogeneous nuclear ribonucleoprotein A2/B1 changes with critical stages of mammalian lung development.

Recent reports have demostrated a link between expression of members of the family of heterogeneous nuclear ribonucleoproteins (hnRNPs) and cancer. Overexpression of hnRNP A2/B1 correlated with the eventual development of lung cancer in three different clinical cohorts. We have studied the expression of hnRNP A2/B1 messenger RNA (mRNA) and protein during mammalian development. The expression of hnRNP A2/B1 mRNA and protein are parallel but change dynamically during critical periods in mouse pulmonary development. hnRNP A2/B1 is first detected in the lung in the early pseudoglandular period, peaks at the beginning of the canalicular period, and remains high during the saccular (alveolar) period. In mouse and rat, hnRNP A2/B1 expression is first evident in the earliest lung buds. As lung development progresses, the cuboidal epithelial cells of the distal primitive alveoli show high levels of the ribonucleoprotein, which is almost undetectable in the proximal conducting airways. The expression of hnRNP A2/ B1 is restricted in mature lung. Similar dynamic pattern of expression through lung development was also found in rat and human lung. Upregulated expression of hnRNP A2/B1 at critical periods of lung development was comparable to the level of expression found in lung cancers and preneoplastic lesions and is consistent with hnRNP A2/B1 overexpression playing an oncodevelopmental role.

Calcitonin gene-related peptide expression is altered in pulmonary neuroendocrine cells in developing lungs of rats with congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is associated with high neonatal mortality from lung hypoplasia and persistent pulmonary hypertension. Pulmonary neuroendocrine cells (PNEC) produce calcitonin gene-related peptide (CGRP), a potent vasodilator. We previously reported altered distribution of CGRP-positive PNEC in full-term rats with CDH, that may lead to an imbalance in vasoactive mediators. In the present study we examined the expression of CGRP-positive PNEC during lung development in rats with CDH induced by 2,4-dichlorophenyl-p-nitrophenylether (Nitrofen). Cesarean sections were performed on Days 16, 18, 20, or 22, and the lungs were immunostained for CGRP and immunoreactive cells were quantitated through image analysis. On Day 16, CGRP-immunoreactive staining was negative; on Day 18, CGRP-immunoreactive cells were found in all controls (not exposed to Nitrofen), whereas in CDH pups, CGRP-positive cells were present in only four of six cases. On Day 20, CGRP immunoreactivity was similar in CDH pups, Nitrofen-exposed pups without CDH, and controls. On Day 22 (term), significantly more CGRP-positive cells (i.e., number of positive cells per surface area [mm2] or lung volume [mm3]) were found in ipsilateral lungs of CDH pups than in controls (P < 0.05). The difference was even more striking in contralateral lungs of CDH pups (P < 0.001), ruling out nonspecific effects of Nitrofen. In CDH lungs, the proportion of immunostained epithelium and the size of the neuroendocrine cell clusters (neuroepithelial bodies [NEB]) were not significantly different from those of controls. On Day 22, supraoptimal dilution immunocytochemistry yielded similar results in CDH pups and controls. We conclude that in CDH, CGRP expression in PNEC and NEB is delayed during early stages of lung development. Because CGRP also exhibits growth factor-like properties for endothelium and epithelial cells, the lack of this factor during a crucial developmental stage (canalicular period) may be causally related to lung hypoplasia.

Hoxb-5 expression in the developing mouse lung suggests a role in branching morphogenesis and epithelial cell fate.

Hoxb-5 is one of the few homeobox genes strongly expressed in the developing mouse lung. To explore the hypothesis that Hoxb-5 acts to regulate epithelial cell fate and branching morphogenesis in the developing lung, we studied the temporal, spatial, and cell-specific expression of Hoxb-5 from gestational day (d) 13.5 to postnatal day (P) 2. Immunocytochemistry demonstrated regional localization of Hoxb-5 protein to developing conducting airways and surrounding mesenchyme. The cellular expression pattern changed from diffusely positive nuclei of mesenchymal cells on d13.5 to become more localized to nuclei of subepithelial fibroblasts and some adjacent columnar and cuboidal epithelial cells on d14.5. After d14.5, Hoxb-5 protein expression continued to decrease in mesenchymal cells distal from developing airways, but persisted in fibroblasts underlying conducting airways. Hoxb-5 protein expression persisted in nuclei of columnar and cuboidal epithelial cells on d16.5 and d17.5, with expression in low cuboidal epithelial cells as well from d17.5 to P2. Western blot analysis showed temporal and quantitative changes in Hoxb-5 protein expression with peak expression on d14.5-15.5. We conclude that Hoxb-5 protein is developmentally regulated in a temporal, spatial, and cell-specific manner throughout the pseudoglandular, canalicular, and terminal saccular periods of lung development in the mouse. This localization and expression pattern suggests that Hoxb-5 may influence branching morphogenesis, cell-cell communication, cell fate, and differentiation of conducting airway epithelia.

Fetal congenital cystic adenomatoid malformations of the lung: a clinicopathologic study of eleven cases.

We describe the pathological examination of 11 cases of fetal congenital cystic adenomatoid malformation of the lung. All patients were treated in utero between 21 and 27 weeks of gestation with either lobectomy or placement of a thoracoamniotic shunt. Ten cases involved a single lobe, and one case involved two lobes. The lesions contained both solid and cystic areas. On the basis of microscopic appearances, we separated the malformations into two distinct types. The first type consisted of seven cases showing scattered bronchiole-like dilated spaces lined by pseudostratified ciliated epithelium with intervening tightly packed small tubules lined by columnar cells with subnuclear vacuoles. This pattern superficially resembled the pseudoglandular period of lung development. The remaining four cases were of the other histologic type. They contained scattered bronchiole-like structures with intervening irregularly branching glands lined by cuboidal epithelium within loose mesenchymal stroma. This pattern superficially resembled the canalicular period of lung development. We observed that the malformations contained cysts of various sizes and that cyst size varied widely within a single lesion. Moreover, predominantly cystic and predominantly solid lesions could not be separated histologically. Thus, we identify two patterns of fetal congenital cystic adenomatoid malformations, pseudoglandular and canalicular, the clinical significance of which is yet to be determined.

Lectin binding beneath the epithelium and in smooth muscle cells in the developing bronchial tree

Components of the subepithelial stratum in developing rat lung reacted transiently with Maclura pomifera agglutinin (MPA) and Aleuria aurantia agglutinin (OFA) conjugated to horseradish peroxidase. These lectins possess selective affinity for and serve to localize glycoconjugates (GCs) with terminal Gal / GalNAc and Fuc, respectively. Staining was strongest with both lectins in the proximal bronchial tree and decreased peripherally to growing buds where it was absent. MPA staining of subepithelial structures decreased from the pseudoglandular through the canalicular period and disappeared by the terminal sac stage. Disappearance of this subepithelial reactivity coincided with appearance of apical MPA-positive glycoconjugate in the canalicular period. OFA stained selectively a layer of flattened cells and a thin extracellular stratum under the epithelium of proximal bronchi in the canalicular period. This lectin affinity extended farther peripherally in the pseudoglandular interval and diminished thereafter. The layer of OFA-positive cells underlying the epithelium was identified immunohistochemically as immature smooth muscle. These muscle cells gained contractile protein while losing surface lectin reactivity during fetal development. The high iron diamine method localized sulfated GC in basement membrane of proximal respiratory passages in the fetal lung. The results attest to the involvement of specific GCs in mediating epithelial-mesenchymal cell interaction during critical stages of bronchial morphogenesis.

Lamellar body formation precedes pulmonary surfactant apoprotein expression during embryonic mouse lung development in vivo and in vitro

Abstract The purpose of this investigation was to determine whether lamellar inclusion body (LB) formation and surfactant apoprotein (SP-35) production are directly coordinated by temporal and positional information during development. In the present study we report a comparison between embryonic B10.A mouse lung morphogenesis and cytodifferentiation in vivo with that observed during organ culture in serumless medium. Precursor LB were first detected at embryonic day 12 (E12d), and progressively larger numbers and forms were produced during subsequent differentiation of respiratory alveolar duct epithelium. SP-35 was first detected during the canalicular period (E16.5d). Lung cultures (E12 d) showed pseudoglandular and canalicular periods of morphogenesis, and both ciliated epithelial and type II cell differentiation. Nonciliated cells produced increasing numbers of lamellar inclusion bodies throughout the culture period. SP-35 was detected at 9 days in vitro (d.i.v.). These observations indicate (i) precursor LB formation precedes SP-35 expression and is not dependent on apoprotein synthesis; (ii) E12d lung development in vitro using serumless medium proceeds at a rate equivalent to 0.5 days in vivo through 11 d.i.v.; and (iii) morphogenesis and differentiation occur in the absence of exogenous hormones and growth factors. The cell-cell interactions that play a role in morphogenesis and cell differentiation appear to be intrinsic to the developmental program for embryonic lung development and are likely to be mediated by autocrine and/or paracrine factors.

Elastin and collagen in the fetal sheep lung. I. Ontogenesis.

The ontogenesis of elastin (desmosine), collagen (hydroxyproline), and DNA concentrations and their rates of increase were studied in fetal sheep lungs from day 60 until term. Elastin increased 13-, 17-, 63- and 11- fold in nondissected parenchyma, dissected (free of tubular structures of greater than 0.1 mm diameter) parenchyma, pleura, and trachea, respectively. Collagen increased 2.1-, 1.8-, 5- and 3-fold, respectively, in the four tissues. A sharp rise in elastin occurred after day 100. The rate of increase in elastin was greater in dissected than in nondissected parenchyma while the reverse was true for collagen. The steepest rise of elastin concentration occurred in the pleura after day 125. DNA concentration peaked on day 125 and was lowest at term. These findings are consistent with 1) the onset of a steep rise in elastin accumulation during the canalicular period, 2) the development of a rigid, mainly collagenous structure of the central airways and blood vessels and a distensible peripheral "gas-exchange tissue," rich in elastin, 3) an important role of elastin in the function of the visceral pleura, and 4) a peak of mitotic activity during the early alveolar period.

Development of neuroepithelial bodies in intact and cultured lungs of fetal rats

Intact, 14- to 21-day fetal rat lung pairs, neonatal lungs, and cultured 15- and 16-day lung explants were examined in 2-micron-thick glycol methacrylate sections stained by PAS-lead hematoxylin. Selected stages were also studied in histochemical preparations for aliesterase and formaldehyde-induced monoamine fluorescence, as well as by scanning and transmission electron microscopy. Neuroepithelial bodies (NEBs) first appear in pseudoglandular lungs at 15 days in vivo as pyramidal groups of basal, diffusely lead-hematoxylin-positive cells in glycogen-depleted epithelium of main and lobar bronchi. By day 16, primitive NEBs occur within three to four generations of the terminal buds, and older, proximal bodies are larger and more distinctive than at 15 days. Aliesterase activity is first detected in basally located, developing NEBs on day 16. During the canalicular and alveolar sac periods, NEBs appear and mature on a proximal-to-distal gradient along the airway, as they do in developing rabbit and human lungs. As earlier-formed airways elongate, additional NEBs appear and supplement the population already present. By days 20-21, NEBs occur at all airway levels down to the bronchiolo-alveolar junctions, and many of the cells have discrete PAS- and lead-hematoxylin-positive, infranuclear granules. Near term some NEBs exhibit serotonin fluorescence after incubation in 5-hydroxytryptophan and have abundant, ca. 100-nm, electron-dense granules. These are concentrated toward the cell base like the stained granules visualized by light microscopy. Similar results were obtained from lungs placed in organ culture. From 2 days in culture to a time equivalent to term, NEB formation parallels that in vivo, indicating that developmental requirements are met in in vitro. Taken altogether, morphologic and cytochemical evidence suggests that NEBs of rats are functional in late fetal life and that their development is relatively independent of extrapulmonary influences and of the intraepithelial ingrowth of sensory nerve endings.

The development of the lung in mammals: an analysis of concepts and findings.

To evaluate one model of mammalian-lung development, i.e., division into periods, pre- and postnatal lung development in the CPB-S mouse strain was divided into the currently distinguished periods: the pseudoglandular period, covering establishment of the air-conducting portion; and the canalicular, terminal-sac, and alveolar or postnatal periods, in which the respiratory portion develops. The last three periods would each cover the formation of a different component of the respiratory unit or pulmonary acinus (acinus pulmonaris) (nonalveolated respiratory bronchiole, nonalveolated duct and sac, and alveolar pouch). However, determination of the nature of the relevant structures on the basis of recent findings concerning the epithelia showed that these hypotheses are not tenable. Since the tubule with cuboidal epithelium (appearing in the pseudoglandular and following periods) is the basic structure in the genesis of the pulmonary acinus, the development of the respiratory portion must start in the pseudoglandular period. Likewise, since the definitive components of the acinus are derived from this acinar tubule, their establishment may not be restricted to one of the other periods. Because other postulated divisions of mammalian-lung development were based on similar histological interpretations, they cannot reflect the course of mouse-lung development either. Therefore, a developmental scheme based on the recent findings concerning the epithelia is given as well as a tentative scheme for the human lung. The respiratory portion proved to develop by budding of acinar tubules, the mode of budding being not restricted to any particular pattern.

The morphological development of the pulmonary epithelium of human foetuses studied by light- and electron microscopy.

The morphological development of pulmonary epithelium of 24 human foetal lungs was studied by light- and electron microscopy in embryos with a crown-rump length between 20 and 160 mm. The glandular and the canalicular period in the development of lungs were identified. In the beginning of the glandular period the epithelium was uniformly of tall columnar type. In the later glandular period the epithelium in most peripheral bronchial tubules diminished in thickness to cuboidal type. In larger bronchial tubules ciliated and non-ciliated cells could be distinguished. The cytoplasmic glycogen was abundant, and increasing in amount from 20 to 70 mm crown-rump length, decreasing during the later glandular period. Junctional complexes, desmosomes and a continuous basement membrane were present at 20 mm crown-rump length. In the canalicular stage the epithelial lining of developing alveoli consisted of two cell types identified as alveolar cells of type I and II. Lamellar bodies characteristics of type II cells were only occasionally observed.

The pattern of the terminal air spaces in a premature infant of 30-32 weeks that lived nineteen and a quarter hours.

AbstractThe availability of a perfectly preserved lung from a premature infant of 30–32 weeks that lived nineteen and a quarter hours has made possible the first wax reconstructions of the terminal air spaces in the canalicular period of fetal growth. Here, as in the newborn, the most distal respiratory bronchioles give rise to two pairs of clusters of saccules, but with this difference: each saccule (lined by flat epithelium) is separated from others by a spongy, vascular, connective tissue matrix. By reconstructing just the cavities of the saccules one can study the bizarre shapes presented by the saccules. In pattern the latter closely resemble peripheral alveolar saccules of the child lung modelled by the writer. Seemingly, therefore, this fetal period is the one in which the terminal generations of branches are being elongated and their shapes determined by invading capillaries and connective tissues. Clinically, one may conclude that peripheral diffusion of gases (in the infant or adult lung) does not follow any such simple dichotomous pattern of flow as has been hitherto assumed.