RESUMO
The ontogeny of protein gene product 9.5 (PGP 9.5), serotonin (5-HT), calcitonin gene-related peptide (CGRP), and calcitonin (CT) immunoreactivity was evaluated in small-granule endocrine cells of hamster laryngotracheal epithelium from fetal day 11 to adulthood. Two centrifugal (proximal-to-distal) patterns of differentiation occur. The first pattern begins during fetal life. Endocrine cells, single and clustered in groups (presumptive- or protoneuroepithelial bodies, pNEBs), initially colocalize immunostaining for PGP 9.5, 5-HT, and CGRP in the larynx and proximal 2/3 of the trachea on day 12 and spread to the caudal trachea on day 13. 5-HT disappears fleetingly during the 24 h preceding birth; otherwise immunoreactivity for all three substances persists into adulthood. The clusters of endocrine cells survive beyond birth but are so diluted by expansion of the nonendocrine epithelium as to become inconspicuous. Since innervation was not actually observed, these clusters may persist as pNEBs, without developing connections to afferent or efferent nerve fibers. The second pattern concerns single small-granule cells stainable for CGRP but not for 5-HT. These cells first appear in the larynx and cartilaginous part of the cranial trachea on postnatal day 3, and in the middle and caudal trachea, on day 5. The cells increase in number on day 7. In adults, they predominate among endocrine cells of the cartilaginous region. A subset of these cells begins to co-express CT proximally on postnatal day 10, reaching the caudal end of the trachea by 3 weeks. A few elements of the older 5-HT-positive population may also become immunoreactive for CT in juvenile hamsters.
Assuntos
Laringe/citologia , Mesocricetus/anatomia & histologia , Sistemas Neurossecretores/citologia , Traqueia/citologia , Animais , Biomarcadores/análise , Calcitonina/análise , Peptídeo Relacionado com Gene de Calcitonina/análise , Diferenciação Celular , Divisão Celular , Cricetinae , Células Epiteliais , Feminino , Idade Gestacional , Laringe/embriologia , Laringe/crescimento & desenvolvimento , Masculino , Mesocricetus/embriologia , Mesocricetus/crescimento & desenvolvimento , Sistemas Neurossecretores/química , Sistemas Neurossecretores/embriologia , Sistemas Neurossecretores/crescimento & desenvolvimento , Serotonina/análise , Tioléster Hidrolases/análise , Traqueia/embriologia , Traqueia/crescimento & desenvolvimento , Ubiquitina TiolesteraseRESUMO
Results of this and the preceding study reveal 3 patterns of endocrine cell development in hamster airway. The first, a prenatal wave, begins in the larynx and sweeps down the extra- and intrapulmonary conducting airway to the bronchioloalveolar portals. Cells differentiate singly and in groups (presumptive neuroepithelial bodies, pNEBs), colocalize immunoreactivity for serotonin (5-HT) and calcitonin gene-related peptide (CGRP), and persist throughout adulthood. Postnatally a few cells also express calcitonin (CT). Appearance of 5-HT and CGRP staining correlates with the onset of local, NEB-associated mitogenesis in fetal hamster airway epithelium. The second pattern begins after birth and is unique to the larynx and cartilaginous trachea. It involves differentiation of single cells which stain for CGRP but not 5-HT. Later, a proportion also stain for CT. This pattern seemingly accounts for the predominance of single cells in laryngotracheal epithelium of adult animals. In the third pattern, cells immunoreactive for peptide YY (PYY) differentiate, singly at first and later among cells of tiny pNEBs. This begins postnatally in alveoli, spreading centripetally with retrograde differentiation of alveolar epithelium back into the bronchiolar terminations. Restricted distribution and lack of immunoreactivity for 5-HT, CGRP, or CT suggest that the PYY-positive endocrine cells form a regional subset performing special roles in pulmonary homeostasis.
Assuntos
Brônquios/citologia , Pulmão/citologia , Mesocricetus/anatomia & histologia , Sistemas Neurossecretores/citologia , Animais , Biomarcadores/análise , Brônquios/embriologia , Brônquios/crescimento & desenvolvimento , Calcitonina/análise , Peptídeo Relacionado com Gene de Calcitonina/análise , Cricetinae , Células Epiteliais , Idade Gestacional , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Mesocricetus/embriologia , Mesocricetus/crescimento & desenvolvimento , Sistemas Neurossecretores/química , Sistemas Neurossecretores/embriologia , Sistemas Neurossecretores/crescimento & desenvolvimento , Peptídeo YY , Peptídeos/análise , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/embriologia , Alvéolos Pulmonares/crescimento & desenvolvimento , Serotonina/análise , Tioléster Hidrolases/análise , Ubiquitina TiolesteraseRESUMO
Lungs were removed from hamsters at gestational day 12 and cultured in BGJb medium under the following conditions: (1) alone, (2) with 5% fetal bovine serum (FBS), (3) with defined additives (with and without vitamin A), and (4) with defined additives but lacking both epidermal growth factor (EGF) and vitamin A. Patterns of cell proliferation were determined by immunochemical labeling for bromodeoxyuridine (BrdU) and airway branching was evaluated in each explant. After 4 days in BGJb alone, an orderly but limited branching pattern occurred, and labeling was greater in the epithelium than in the connective tissue. With FBS a relatively normal branching pattern occurred, and labeling was nearly equivalent in connective tissue and epithelium. With defined additives the connective tissue was heavily labeled in the compact region supporting the distorted bronchioles and in the loose flange at the periphery; removing only vitamin A did not alter these patterns. Removing both vitamin A and EGF caused an extensive but distorted epithelial branching pattern that extended to the periphery of the explant (a flange of loose connective tissue did not form); BrdU labeling was sparse. These results indicate that EGF played a major role in contributing to alterations in the growth and development of fetal hamster lung.
Assuntos
Brônquios/embriologia , Pulmão/embriologia , Animais , Brônquios/química , Brônquios/citologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Cricetinae , Meios de Cultura/farmacologia , Meios de Cultivo Condicionados , Fator de Crescimento Epidérmico/farmacologia , Feminino , Feto , Idade Gestacional , Pulmão/química , Pulmão/citologia , Mesocricetus , Técnicas de Cultura de Órgãos , Gravidez , Albumina Sérica/farmacologia , Vitamina A/farmacologiaAssuntos
Brônquios/embriologia , Desenvolvimento Embrionário e Fetal , Alvéolos Pulmonares/embriologia , Animais , Brônquios/citologia , Brônquios/inervação , Brônquios/metabolismo , Diferenciação Celular , Divisão Celular , Cílios , Cricetinae , Células Epiteliais , Epitélio/embriologia , Epitélio/inervação , Epitélio/metabolismo , Glicogênio/metabolismo , Mesocricetus , Sistemas Neurossecretores/citologia , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/metabolismoRESUMO
Clusters of small-granule endocrine cells, neuroepithelial bodies (NEBs), appear in the airway lining of pseudoglandular lungs, but their prenatal function has remained obscure. Transplacental labeling of S-phase cells in Syrian golden hamsters has allowed us to relate NEBs to patterns of replication in the surrounding endoderm. Two methods were used: 1) continuous exposure to 3H-thymidine for the last 25% (4 days) of gestation, and 2) 2-hr exposure to 5-bromo-2'-deoxyuridine (BrdU) on fetal day 15. 3H-thymidine incorporation was assessed in autoradiographs of neonatal lung by grain counting from 923 nonendocrine and 251 endocrine cells in 28 airway epithelial terrains, each centered on a NEB: 12 in the perihilar, 8 in the middle, and 8 in the distal third of the left axial bronchus. Grain densities for 10-25 nonendocrine cells on either side of the NEB were plotted vs. position relative to the endocrine cell cluster and analyzed by rank-order correlation and linear regression. Label was highest in cells closest to NEBs in all 12 terrains (P < 0.05-0.001) in the perihilar airway, in 3 of 8 terrains (P < 0.025-0.001) in the middle third of the bronchus, and in respective, pooled populations (P < 0.001). The effect was not demonstrable in the distal third of the airway. In the 15-day fetus 243 mm of airway perimeter were measured and 3,218 BrdU-labeled epithelial cells counted from sections through the entire length of the left axial airway and the lobar bronchus, intermediate, and terminal bronchioles of the infracardiac (IC) lobe.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Brônquios/embriologia , Sistemas Neurossecretores/embriologia , Animais , Animais Recém-Nascidos , Brônquios/citologia , Divisão Celular , Cricetinae , Células Epiteliais , Epitélio/embriologia , Pulmão/citologia , Pulmão/embriologia , Mesocricetus , Sistemas Neurossecretores/citologia , Fase SAssuntos
Aminas , Biomarcadores , Pulmão/química , Sistemas Neurossecretores/química , Peptídeos , Animais , Humanos , Pulmão/citologia , Especificidade da EspécieRESUMO
We showed previously that the proliferation of hamster airway secretory cells decreases during vitamin A deficiency (VAD) but later increases when submucosal inflammation develops (Virchows Arch [B] 59:231-242, 1990). This observation has important biological implications since two morphological extremes (atrophy and quiescence versus hyperplasia and hyperproliferation) are reported in the literature for VAD tracheal epithelium in vivo. In the present study, histological slides of tracheal rings from 35-day-old control and VAD hamsters (Virchows Arch [B] 45:197-219, 1984) were reviewed again. Rings from VAD hamsters were selected based on the absence or presence of a florid submucosal inflammation. Quantitative analyses were made on the cartilaginous part of rings from the anterior third of the trachea. When inflammation was absent, a mucociliary pseudostratified epithelium was, for the most part, maintained. The mitotic rate (MR, 6 h colchicine blockade) of secretory cells was markedly reduced (29-fold) but that of basal cells was not changed significantly. Moreover, cell density was not changed by VAD but ciliated cells and secretory cells were decreased and basal cells were increased, proportionally. We call this "minimal morphological change." Thinning (atrophy) of the minimally changed epithelium was associated with focal cell sloughing. Small scattered foci of epidermoid metaplasia (multiple layers of highly keratinized cells which were extremely flat, so that the epithelium was thin and attenuated) were also seen. We call this "atrophic epidermoid metaplasia." When inflammation was present, hyperplastic changes (stratification and epidermoid metaplasia) predominated and cells were in mitosis at all epithelial levels (low, middle, superficial) except in the most superficial (terminally differentiated) squames. The tracheal epithelium was thickened and hypercellular. The cells were piled up at the stratified lesions, and epithelial height, cell density and epithelial MR were significantly increased compared with the non-inflamed VAD epithelium. The effects of VAD and inflammation on cell proliferation were analyzed further by studying 7 h bromodeoxyuridine (BrdU) labelling patterns of cells in VAD tracheal epithelium, with and without submucosal inflammation. In addition, inflammation was induced in "minimally changed epithelium" by mild mechanical injury. The BrdU labelling patterns confirmed that DNA synthesis by secretory cells is reduced markedly by VAD. However, this suppression is overidden by the influx of inflammatory cells (the nature of the stimulus is unknown). The results indicate that the morphological contrasts (atrophy and hyperplasia) seen in the trachea during VAD in vivo are related to extremes in proliferation rates of tracheal secretory cells, regulated by VAD alone (minimal replication) and by inflammation (maximal replication).
Assuntos
Traqueíte/patologia , Deficiência de Vitamina A/patologia , Animais , Atrofia/patologia , Bromodesoxiuridina , Cricetinae , Epitélio/patologia , Hiperplasia/patologia , Metaplasia/patologia , Modelos BiológicosRESUMO
Intact fetal hamster lungs were taken for culture on gestational day 12, when only lobar bronchi and primary bronchioles are established and the epithelial cells are undifferentiated. Explants were maintained on Transwell collagen membranes for 2 and 4 days in BGJb medium alone, with 5% FBS, or with the following additives: insulin, transferrin, hydrocortisone, cholera toxin, EGF, and vitamin A. Development of the respiratory tree was affected differently by each medium formulation. BGJb medium with 5% FBS permitted near normal branching of airways and presumptive alveoli. In contrast, BGJb medium alone permitted only limited branching of these structures. BGJb medium with additives permitted branching but markedly altered normal development. The differentiation of endocrine and secretory cells was monitored by immunolabeling for serotonin and calcitonin gene-related peptide, and Clara cell protein, respectively. Ciliated cells were identified by morphology. All medium formulations supported the timely differentiation of endocrine, secretory, and ciliated cells. The ultimate goal of our studies is to characterize factors that influence airway branching and cytodifferentiation during fetal lung development. This study showed that near normal airway branching and cytodifferentiation were supported in vitro by BGJb medium with 5% FBS. Although cytodifferentiation occurred with the two other formulations, airway development was impaired.
Assuntos
Brônquios/embriologia , Pulmão/embriologia , Uteroglobina , Animais , Bromodesoxiuridina/análise , Peptídeo Relacionado com Gene de Calcitonina/análise , Diferenciação Celular/fisiologia , Cricetinae , Técnicas de Cultura , Desenvolvimento Embrionário e Fetal/fisiologia , Idade Gestacional , Técnicas Imunoenzimáticas , Laminina/análise , Pulmão/citologia , Mesocricetus , Proteínas/análiseRESUMO
Autoradiographs were prepared from lungs of a newborn Syrian golden hamster exposed continuously to [3H]thymidine throughout the final 4.5 days of gestation. Silver grains were counted over nuclei of 1,145 nonendocrine airway epithelial cells adjacent to 28 mature neuroepithelial bodies (NEBs). Generally, accumulated label was greater in cells nearer a NEB than in those further away. Diminution of label with increasing distance from the closet NEB was confirmed statistically. In 24 of 28 instances, both rank-order correlation and linear regression were significant (P less than 0.001-0.05); in two others, only one test was significant; in another two, neither test was significant. The pattern was consistent and widespread. Rank-order correlations and linear regressions were significant (P less than 0.001) in populations pooled separately from left lung, right upper, and right lower lobes, and the three regression lines were superimposable. Confirmation was obtained in another animal by labeling S-phase cells with a 2-h transplacental pulse of bromodeoxyuridine (BrdU) on fetal day 15. Of 322 BrdU-positive cells counted in 270,204 microns 2 of bronchial epithelium, 174 (54%) lay within 20 microns of a neuroepithelial body. This concentration of dividing cells was significant by chi-square test: chi 2[1] = 101.62; P less than 0.001. We conclude that established NEBs promote growth of the developing airway by stimulating proliferation of local endoderm. A few daughter cells may enter NEBs; most move away to join the expanding nonendocrine airway lining.
Assuntos
Ectoderma/fisiologia , Pulmão/citologia , Animais , Autorradiografia , Bromodesoxiuridina , Divisão Celular , Cricetinae , Replicação do DNA , Ectoderma/citologia , Células Epiteliais , Epitélio/fisiologia , Feto , Pulmão/embriologia , Mesocricetus , Análise de Regressão , Timidina/metabolismo , TrítioRESUMO
Lectin binding was studied in the developing submandibular glands of fetal Syrian golden hamsters (Mesocricetus auratus) from gestational day 12 to 16 (the day of birth). The fetuses were fixed, embedded in paraffin, sectioned and stained with nine lectin-horseradish peroxidase conjugates: concanavalin A (Con A), wheat germ agglutinin (WGA), Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), Maclura pomifera agglutinin (MPA). Griffonia simplicifolia agglutinin I-B4 (GSA I-B4), peanut agglutinin (PNA). Ulex europeus agglutinin I (UEA I) and Limulus polyphemus agglutinin (LPA). The developing glands showed dramatic morphological alterations on a daily basis, accompanied by progressive changes in lectin staining. On day 12 the primitive gland showed only trace lectin staining with WGA, HPA, MPA, PNA and UEA I, but by day 13, strong staining with these lectins, as well as with DBA, was seen at the ductal lumenal surface, after the formation of the ductal lumens. Secretory granules first appeared in cells of the primitive acini on day 14: the secretion products were stained strongly with WGA. DBA, HPA, MPA, PNA and UEA I. On day 15, the secretion products were also stained moderately with GSA I-B4. Secretory differentiation was further developed on day 16, but the staining intensity of the mucins with the different lectins varied among the secretory cells. LPA failed to stain any part of the gland throughout the observation period, and Con A stained only glycogen.
Assuntos
Feto/metabolismo , Lectinas/metabolismo , Glândula Submandibular/metabolismo , Animais , Cricetinae , Idade Gestacional , Histocitoquímica , Mesocricetus , Glândula Submandibular/embriologiaRESUMO
Lectin binding was studied in the developing airways of Syrian golden hamsters on gestational days 11-16 (day 16 is the day of birth). The trachea and lungs were fixed in 4% formaldehyde-1% glutaraldehyde, 6% mercuric chloride-1% sodium acetate-0.1% glutaraldehyde, and 95% ethanol; embedded in paraffin; and stained with eight lectin-horseradish peroxidase conjugates: Triticum vulgare (WGA), Dolichos biflorus (DBA), Helix pomatia (HPA), Maclura pomifera (MPA), Griffonia simplicifolia I-B4 (GSA I-B4), Arachis hypogaea (PNA), Ulex europeus I (UEA I), and Limulus polyphemus (LPA). Each lectin yielded a characteristic staining pattern, which modulated throughout development. In general, changes in staining characteristics of the tracheal epithelium preceded similar changes in the lobar bronchus, bronchiole, and alveolus. In the case of UEA I, MPA, WGA, and HPA, staining increased with time uniformly over the luminal surface of all epithelial cells. However, in the case of PNA, GSA I-B4, and LPA, after the differentiation of ciliated and secretory cells, the apical surfaces of the ciliated cells stained more intensely than the apical surfaces of the secretory cells. Neuraminidase pretreatment enhanced PNA and GSA I-B4 staining in both cell types. In the case of PNA, these light microscopic observations were confirmed by ultrastructural study. Unlike the other lectins, the pattern of staining with DBA was unusual. Staining was moderate at first, then decreased (days 13 and 14), then increased at all airway levels. This study shows that different glycoconjugates modulate in airway epithelial cells throughout fetal development.
Assuntos
Glicoconjugados/metabolismo , Lectinas , Traqueia/metabolismo , Animais , Cricetinae , Endotélio Vascular/metabolismo , Epitélio/metabolismo , Mesocricetus , Alvéolos Pulmonares/metabolismo , Coloração e Rotulagem , Traqueia/irrigação sanguínea , Traqueia/crescimento & desenvolvimentoRESUMO
Antibodies against rabbit cytochrome P-450 reductase (reductase), cytochrome P-450 isozyme 2 (P-450 IIB), and cytochrome P-450 isozyme 5 (P-450 IVB) were used to detect homologous enzymes in the developing lung of the Syrian golden hamster. No immunocytochemical labeling was observed on gestational days 11, 12, and 13. On gestational day 14, light immunoperoxidase labeling for reductase and P-450 IIB was observed over cells lining the trachea and cranial portions of lobar bronchi. On gestational day 15, these enzymes were detected in conducting airways at all anatomic levels, and in the media of the pulmonary vein and its branches. Light labeling for P-450 IVB was first observed over cells lining the trachea and lobar bronchi on gestational day 15, but the smallest bronchioles and the media and endothelium of the pulmonary vein did not label for this enzyme until gestational day 16 (neonatal day 1). Type II pneumocytes and the pleural mesothelium first labeled for each of the three enzymes on neonatal day 1. Although the mesothelium no longer labeled for reductase or P-450 IIB in hamsters 3.5 wk old, the other labeling sites persisted in adult hamsters. Because cytochrome P-450 enzymes are associated with the endoplasmic reticulum, an ultrastructural examination of differentiating secretory cells was made to detect its appearance. At each conducting airway level, smooth endoplasmic reticulum was present in the cells 2 d before cytochrome P-450 enzymes could be detected immunocytochemically. The appearance of these enzymes paralleled the development of the hamster lung; they were first present in the trachea and lobar bronchi, then in the bronchioles, and finally in the alveoli.
Assuntos
Sistema Enzimático do Citocromo P-450/análise , Pulmão/enzimologia , Oxigenases/análise , Animais , Animais Recém-Nascidos , Especificidade de Anticorpos , Cricetinae , Sistema Enzimático do Citocromo P-450/imunologia , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/ultraestrutura , Feminino , Feto , Técnicas Imunoenzimáticas , Isoenzimas/análise , Isoenzimas/imunologia , Pulmão/imunologia , Pulmão/ultraestrutura , Mesocricetus , NADPH-Ferri-Hemoproteína Redutase/análise , NADPH-Ferri-Hemoproteína Redutase/imunologia , Oxigenases/imunologia , Gravidez , Alvéolos Pulmonares/enzimologia , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/ultraestruturaRESUMO
The conducting airway epithelium of fetal Syrian golden hamsters was studied from gestational day 12 to day 15, during normal and uncontrolled diabetic pregnancies. Diabetes was induced in the pregnant hamsters by injecting streptozotocin at 60 mg/kg body weight, subcutaneously, early on gestational day 10. Cells in S-phase were labelled immunochemically with bromodeoxyuridine (BrdU), and the day on which endocrine cells and ciliated cells first appeared was determined. In control fetuses, the BrdU-labelling indices (LI's) of different anatomical airway levels were significantly different from one gestational day to the next. For example, the LI of the lobar bronchus was significantly different on each gestational day (P less than .0001), and the same was true of the bronchioles. Moreover, the difference between LI's of the lobar bronchus and bronchioles-terminal buds was highly significant on day 12 (P less than .0001), and on day 13 the differences between lobar bronchus and bronchioles, lobar bronchus and terminal buds, and bronchioles and terminal buds were also highly significant (P less than .0001). However, on gestational days 14 and 15, the LI's were reduced and were comparable at different airway levels. The BrdU-labelling indices were very consistent among fetuses of the same age, and the differences between the average LI's for pups of different litters was numerically very small. Hyperglycemia (mild, moderate, severe) did not alter LI's in the fetal airway epithelial cells. Furthermore, although glycogen was not depleted from the airway epithelium of the hyperglycemic fetuses as it was in the controls, the endocrine cells first appeared on gestational days 12, 13, and 14, respectively, in the trachea, lobar bronchus and bronchioles, followed 1 day later by the ciliated cells, in the fetuses of control and diabetic mothers. In our experimental model, induction of diabetes in the pregnant hamsters on gestational day 10 did not appear to alter development or differentiation of the fetal conducting airway epithelium.
Assuntos
Gravidez em Diabéticas/metabolismo , Prenhez/fisiologia , Sistema Respiratório/embriologia , Animais , Bromodesoxiuridina/metabolismo , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Cricetinae , Células Epiteliais , Epitélio/embriologia , Epitélio/metabolismo , Epitélio/fisiologia , Feminino , Glicogênio/metabolismo , Imuno-Histoquímica , Mesocricetus , Gravidez , Prenhez/metabolismo , Fenômenos Fisiológicos Respiratórios , Sistema Respiratório/citologia , Sistema Respiratório/metabolismoRESUMO
An antibody to a protein produced by Clara cells in adult Syrian golden hamsters has been used to monitor the development and functional differentiation of secretory cells in the conducting airway epithelium of this species. Lungs from fetal and neonatal hamsters at gestational day 11 and at intervals up to and including 3.5 weeks of age (as well as adults) were studied. The earliest time this Clara cell protein could be identified by immunoperoxidase labeling in the fetal conducting airways was at gestational day 15. On this day, labeling was observed in a few secretory cells lining the trachea, in many lining the lobar bronchi, and in virtually all secretory cells lining the bronchioles. Ciliated cells and endocrine cells were not labeled. Granules first appeared within the apical cytoplasm of the secretory cells on gestational day 15 at all airway levels. To identify the exact subcellular location of this protein, an ultrastructural labeling procedure using protein A gold was employed. The gold particles labeled only electron-dense granules within the secretory cells, indicating that they represent the specific site of this protein. Since secretory cells in the most distal conducting airways began to produce this protein on the same day in development as cells in the larger airways, including the trachea, this expression of functional maturation occurs simultaneously throughout the conducting respiratory tree rather than proceeding sequentially in a cranial to caudal direction. Consequently, secretory cells lining the smaller conducting airways mature more rapidly than those lining the larger airways.
Assuntos
Pulmão/citologia , Proteínas/metabolismo , Uteroglobina , Animais , Animais Recém-Nascidos/metabolismo , Cricetinae , Feminino , Feto/citologia , Feto/metabolismo , Imuno-Histoquímica/métodos , Pulmão/metabolismo , Pulmão/ultraestrutura , Mesocricetus , Microscopia Eletrônica/métodos , GravidezRESUMO
We studied glycogen storage in the developing airway epithelium of Syrian golden hamsters from gestational Day 11 to neonatal Day 2 using concanavalin A (ConA) staining as an adjunct approach to the periodic acid-Schiff (PAS) reaction. One hundred and fourteen fetuses and neonates were fixed in 4% formaldehyde-1% glutaraldehyde, 6% mercuric chloride-1% sodium acetate-0.1% glutaraldehyde, and 95% ethanol, embedded in paraffin, and stained with ConA-horseradish peroxidase conjugate as well as with PAS. ConA staining was abolished by alpha-glucosidase digestion or by pre-treatment with periodic acid, demonstrating that ConA bound to glycogen. In tissues fixed with mercury and/or aldehydes, ConA staining was greatly enhanced by pepsin digestion. Airway glycogen stores, revealed by ConA and PAS, fluctuated during development. At first all the undifferentiated epithelial cells contained abundant glycogen. Then, coincident with the appearance of the first endocrine cells, the glycogen stores were depleted. Thereafter, glycogen accumulated in pre-secretory and basal cells until birth, but by 2 days after birth the glycogen stores were again depleted. The initial depletion of glycogen followed by repletion was observed at all levels of the conducting airways; changes in the trachea preceded those in the bronchi and bronchioles by 1 and 2 days, respectively.
Assuntos
Concanavalina A/metabolismo , Glicogênio/metabolismo , Histocitoquímica , Reação do Ácido Periódico de Schiff , Sistema Respiratório/metabolismo , Animais , Animais Recém-Nascidos , Cricetinae , Células Epiteliais , Epitélio/metabolismo , Feto/metabolismo , Peroxidase do Rábano Silvestre , Mesocricetus , Concentração Osmolar , Sistema Respiratório/citologia , Sistema Respiratório/crescimento & desenvolvimentoRESUMO
The effects of vitamin A-deficiency and inflammation were studied in the conducting airways of Syrian golden hamsters. An important goal of the study was to characterize epithelial changes that occur early in vitamin A-deficiency, that might precede yet predispose to infection, and precipitate inflammatory changes in the lungs. Age-matched vitamin A-replete control and vitamin A-deprived hamsters were killed at 33 days of age (preweight-plateau); at 41 days of age (weight plateau-early weight loss); and at 48-55 days of age (prolonged weight plateau followed by weight loss). A tablet containing bromodeoxyuridine (BrdU) was implanted subcutaneously into each hamster 7 h before it was killed. No changes were seen in the conducting airway epithelium of vitamin A-deprived hamsters in the preweight plateau. However, labelling of secretory cells for BrdU was reduced 6-7 fold in the epithelium lining the lobar bronchus (p less than 0.0002) and the bronchioles (p less than 0.0001), and the proportions of ciliated cells were decreased (p less than 0.0001) at both airway levels in vitamin A-deficient hamsters in the weight plateau-early weight loss stage. Changes in cellular morphology were minimal in the intrapulmonary airway epithelium at this time but a few small focal patches of epidermoid metaplasia were seen in the tracheal epithelium. Small foci of inflammation were closely associated with the airways in the weight plateau, and the inflammation became more widespread when the deficiency was prolonged. The results suggest that the defense of the lungs to infection was impaired initially in the vitamin A-deficient hamsters by a widespread reduction in the numbers of ciliated cells throughout the epithelium of the conducting airways (trachea, bronchi, bronchioles). At the foci of inflammation, labelling of epithelial secretory cells for BrdU was greatly increased at all airway levels. A highly stratified cornifying epidermoid metaplasia developed in the tracheal epithelium, and goblet cell metaplasia developed in the cranial portion of the lobar bronchus, in association with submucosal inflammation. Goblet cell metaplasia appeared to be the only abnormality that was not reversed when vitamin A was restored to the diet.
Assuntos
Brônquios/patologia , Bronquite/patologia , Traqueia/patologia , Traqueíte/patologia , Deficiência de Vitamina A/patologia , Fatores Etários , Animais , Bromodesoxiuridina/metabolismo , Cricetinae , Epitélio/patologia , Mesocricetus , MetaplasiaRESUMO
The cytochrome P-450 monooxygenase enzymes, NADPH-reductase and form 2, were demonstrated immunohistochemically in hamster tracheal epithelium that was regenerating after mechanical injury. Bromodeoxyuridine (BrdU), a thymidine analogue, was used to map the location and extent of the wound sites between 8 and 144 h post-injury. In the control and non-wounded areas of the epithelium, the secretory cells were labelled for the monooxygenase enzymes. Label was heaviest in the apical cytoplasm of these columnar cells. At 8 h, secretory cells at the wound margins migrated to cover the wound sites, becoming progressively flattened. Reaction product for monooxygenase enzymes was strong in these flat cells but immunolabelling for BrdU was very low. At 24 h many cells at the wound sites were labelled for BrdU (indicative of a high rate of cell division). Some cells were labelled for monooxygenase but many were not stained at this time. At 48 and 72 h post-injury, none of the cells within the wound sites (regenerating epithelium) were stained. Immunochemical labelling for the monooxygenase enzymes was restored to the nascent secretory cells as they differentiated in the wound sites, beginning at 96 h post-injury. Labelling was stronger at 120 and 144 h post-injury, comparable to that in the control epithelium. The observations suggest that the monooxygenase enzymes were retained by the secretory cells in the wound sites before they divided but were lost from their progeny. Then, the temporal sequence of monooxygenase expression was similar to the pattern of differentiation of nascent secretory cells during fetal development of the tracheal epithelium.
Assuntos
Sistema Enzimático do Citocromo P-450/análise , Oxigenases/análise , Regeneração/fisiologia , Traqueia/enzimologia , Traqueia/fisiologia , Animais , Cricetinae , Epitélio/enzimologia , Técnicas Imunoenzimáticas , Masculino , Mesocricetus , Fatores de TempoRESUMO
Immunohistochemical demonstration of the thymidine analogue bromodeoxyuridine (BrdU) in regenerating cells was useful in determining the size and location of the wounded areas (epithelial and submucosal) during regeneration of the hamster tracheal epithelium, at times late in the healing process (72-144 hr postinjury) when the wound sites and their boundaries were not recognized with certainty in conventionally stained paraffin sections. Cells distant from the wound sites remained unlabelled. The success of this method resulted from prolonged exposure to BrdU released over several hours from a 25mg tablet implanted subcutaneously at 24 hr postwounding at the time when DNA synthesis and cell proliferation are maximal. This simple technique promises to be useful in determining the size and location of wound sites with application to a wide variety of organs and tissues in studies of repair and healing.
Assuntos
Bromodesoxiuridina , Regeneração , Traqueia/lesões , Cicatrização , Animais , Divisão Celular , Cricetinae , Imuno-Histoquímica , Masculino , Mesocricetus , Traqueia/patologiaRESUMO
Activation of protooncogenes and inactivation of putative tumor suppressor genes are genetic lesions considered to be important in lung carcinogenesis. Fifty-four cases of non-small-cell lung cancer (23 adenocarcinomas, 23 squamous-cell carcinomas, and 8 large-cell carcinomas) were examined for loss of DNA sequences at 13 polymorphic genetic loci. Loss of heterozygosity was seen more frequently in squamous-cell carcinoma than in adenocarcinoma. The loss of DNA sequences from the short arm of chromosome 17 (D17S1 locus) was detected in 8 of 9 heterozygous cases of squamous-cell carcinoma and in only 2 of 11 heterozygous cases of adenocarcinomas. Furthermore, in 7 of these 8 squamous-cell carcinomas, loss of heterozygosity from chromosome 17 was accompanied by loss of DNA sequences from chromosome 11. The spectrum of allelic sequences lost from chromosome 11 was, however, similar in every type of carcinoma studied, and the data show two regions commonly deleted from chromosome 11 (11pter-p15.5 and 11p13-q13) that may have a role in the pathogenesis of all these types of non-small-cell bronchogenic carcinoma. Loss of DNA sequences from chromosome 3 was seen in 16 of 31 cases where the constitutive DNA was heterozygous-i.e., informative. These data included only 6 of 16 cases where loss of heterozygosity involved a chromosomal locus previously shown to be lost consistently in small-cell lung cancer (DNF15S2). Loss of heterozygosity at the chromosome 13q locus, D13S3, was seen in 9 of 21 informative cases, and in 2 cases, both adenocarcinomas, duplication of the intact DNA sequences suggested the possibility that mitotic recombination had occurred. Frequent DNA sequence deletions, including those from chromosome 17, in squamous-cell carcinomas may reflect the extensive mutagenic and clastogenic effects of tobacco smoke that may lead to inactivation of putative tumor-suppressor genes.
Assuntos
Adenocarcinoma/genética , Carcinoma de Células Pequenas/genética , Carcinoma de Células Escamosas/genética , Deleção Cromossômica , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 13 , Cromossomos Humanos Par 17 , Cromossomos Humanos Par 3 , Neoplasias Pulmonares/genética , Triagem de Portadores Genéticos , Humanos , Polimorfismo de Fragmento de Restrição , Proto-OncogenesRESUMO
The morphological and proliferative effects of intratracheal cannulation (ITC) or intralaryngeal cannulation (ILC), with or without the instillation of saline or Fe2O3 particles in saline, were studied in Syrian golden hamsters. Instillation of Fe2O3 in saline at either airway level resulted in a similar distribution of Fe2O3 particles in all lung lobes. ILC produced laryngeal mucosal wounds. ITC produced laryngeal and tracheal mucosal wounds. The cannula-induced wounds were associated with proliferative epithelial lesions. ITC, but not ILC, resulted in significant increases in the mitotic rates (MR, 6-h colchicine blockade) of tracheal epithelial cells at 24 and 32 h postcannulation. Instillation of saline by ITC produced slight increases in intrapulmonary bronchial and bronchiolar MR, but saline given by ILC did not increase MR at any airway level. Instillation of Fe2O3 particles in saline by ITC produced increases in tracheal, intrapulmonary bronchial, and bronchiolar MR. Instillation of Fe2O3 particles in saline by ILC had little effect on tracheal MR, but increased intrapulmonary bronchial and bronchiolar MR. Foci of Fe2O3 particle-laden macrophages were associated with mild bronchiolar-alveolar hyperplasia at the junctions of the terminal bronchioles and the alveolar ducts. The cytokinetic and morphological changes in the intrapulmonary airways were associated with the influx of inflammatory cells in response to Fe2O3 particle deposition. The marked increases in tracheal MR and the localized hyperplastic tracheal epithelial lesions were clearly associated with mechanical wounding from the cannula during ITC. Comparative studies using ILC or ITC instillation techniques allowed further investigations of the important role of tracheal mucosal wounding in the induction of respiratory carcinogenesis, as described in a companion paper (Keenan et al., Cancer Res., 49: 1528-1540, 1989).