Regeneration of hamster tracheal epithelium after mechanical injury. IV. Histochemical, immunocytochemical and ultrastructural studies

All stages of regeneration in hamster tracheal epithelium were studied following a denuding mechanical injury. At 1 h all the cells had sloughed from the wound site leaving a bare and sometimes disrupted basal lamina. Viable cells at the wound margins rapidly changed shape, flattened and migrated to cover the denuded lesion by 12 h. In addition, epithelial cells that remained viable demonstrated sublethal changes that included the rapid discharge of mucous granules from secretory cells, internalization of cilia by ciliated cells and evidence of heterophagy in both cell types. By 24 h a wave of epithelial cell divisions occurred, primarily by secretory cells. This produced a multilayered epidermoid metaplasia that was best developed at 48 h. The metaplastic epithelium was largely composed of cells with both secretory (mucous granules) and epidermoid (tonofilament bundles and numerous desmosomes) characteristics. The peroxidase-antiperoxidase (PAP) method demonstrated a few keratin-positive cells in the wound as early as 12 h post-wounding and keratin was demonstrated in more cells by 24 h. All cells in the metaplastic wound epithelium were keratin-positive by 48 h. Following 48 h some of the most superficial keratinized cells sloughed from the epithelium and the keratin content of the remaining cells began to decline. At 72 h pre-ciliated and pre-secretory cells were seen in the wound. Pre-ciliated cells were characterized by an abundant electron-lucent cytoplasm, large pale nucleus, filiform apical microvilli and evidence of ciliogenesis, similar to that seen during fetal development. Pre-ciliated cells often contained apical mucous granules, apparently carried over from the parent secretory cells. With the appearance of these columnar cells the normal mucociliary morphology was restored in small wounds by 120 h, but some persistent epidermoid metaplasia remained in the large wounds through 168 h post-wounding. These data provide further evidence for the important role of secretory cells in the histogenesis of epidermoid metaplasia and the regeneration of normal morphology following injury. The implications of these findings in understanding the histogenesis of other lesions in the tracheo-bronchial epithelium are discussed.     

The effect of vitamin A on cellular differentiation and mucous glycoprotein synthesis in long-term rat tracheal organ cultures.

Rat tracheal explants maintained as organ cultures exhibited a normal mucocillary epithelium for at least 46 days in the presence of retinyl acetate. In the absence of vitamin A the explant epithelium became quiescent or underwent a metaplastic change to a keratinizing squamous epithelium. This process was accelerated if explants were derived from vitamin A-deficient animals. Autoradiographic examination showed that [3H]glucosamine label accumulated in various cell types in the explant, but especially in the epithelium. It was found that the explants secreted mucous glycoproteins into the medium and that the production and biochemical characteristics of a specific mucin fraction were dependent upon the vitamin A status of the explant.     

Effects of vitamin A-deprivation on hamster tracheal epithelium. A quantitative morphologic study

The effects of vitamin A-deprivation on the tracheal epithelium were studied in 35-day old hamsters that had been raised since birth on a vitamin A-deficient diet. Colchicine and 3HTdR were given 6 hours before death and the proliferative activities of basal cells and mucous cells were quantified separately by 3HTdR labeling indices and mitotic rates. Vitamin A-deprivation decreased replication of basal cells and mucous cells in tracheal epithelium which showed minimal morphologic change. The mitotic rates and labeling indices were reduced 3 to 4-fold in basal cells and 14-fold in mucous cells (analyzed as percent of total number of each cell type) compared with controls. Thus, replication of mucous cells was more inhibited by lack of vitamin A, than replication of basal cells. The disparate hypoplasia of basal cells and mucous cells in epithelium showing minimal change, resulted in a relative increase in the proportion of basal cells and a relative decrease in the proportion of mucous cells, which could be erroneously interpreted as "basal cell hyperplasia". Proportions of preciliated and ciliated cells were also decreased compared to controls. At foci of stratification and epidermoid metaplasia, cell replication rates were increased over controls and more than 70% of all mitotic activity was associated with "non-basal" cells. Genesis of these lesions was coincident with cell death and cell loss. The histogenesis of stratification and epidermoid metaplasia was characterized. Morphological evidence indicated that these lesions were closely related histogenetically and were composed, for the most part, of altered mucous cells which expressed dual phenotypes i.e. keratinization and mucus synthesis.     

Tracheal epithelium: cell kinetics and differentiation in normal rat tissue

Abstract. The fate of [³H]thymidine ([³H]Tdr) pulse-labelled cells was followed in tracheal epithelium of young male rats. The time course for cell differentiation, and the relation of events to tissue composition were studied. In vivo labelling and light microscope autoradiography of epoxy embedded sections were used. Labelled and total nuclei for each cell type, and combinations of labelled cells which were adjacent to one another, were tallied. Hierarchical analyses of variance were performed on the several data sets. All cell types, except ciliated, were labelled at 1 hr. A few labelled ciliated cells were seen 24 hr post-label. The frequency of labelled intermediate cells peaked at day 2; goblet and ciliated cells at day 3. No significant changes occurred in the labelling index, but at 24 hr the frequency of adjacent labelled cells (ALC) had increased > 5-fold, and changes had occurred in patterns of ALC combinations. The labelled ciliated cells which were seen at 24 hr were adjacent to labelled intermediate cells. No labelled basal-ciliated cell combinations were seen at any time. Data indicated that ciliated cells can develop from S-phase intermediate cells within 24 hr, and neither basal nor superficial goblet cells are progenitors of ciliated cells. It is proposed that both superficial goblet cell and ciliated cell development is preceded by two divisions: a basal cell division followed by an intermediate cell division.     

Restoration of mucociliary tracheal epithelium following deprivation of vitamin A. A quantitative morphologic study

In order to learn more about the respective roles played by basal cells and mucous cells in the maintenance of tracheal mucociliary epithelium, cell kinetics and epithelial cell morphology were characterized over a 7-day period, during which dietary vitamin A was restored to previously deprived hamsters. Hamsters were reared from birth to 35 days of age on vitamin A-replete or deficient diets. Deprived hamsters were made replete by 5 mg vitamin A-acetate orally, plus a vitamin A-replete diet. Colchicine and 3HTdR were given 6 h before death. The numbers of basal cells, mucous cells, preciliated cells and ciliated cells, and mitotic rates (MR) and labeling indices (LI) of basal cells and mucous cells, were quantified in glycol methacrylate sections stained with PAS-lead hematoxylin. Vitamin A-deprivation decreased replication of basal cells and mucous cells in tracheal epithelium which showed minimal morphological change. The proportion of basal cells was increased and proportions of mucous, preciliated and ciliated cells were decreased. Following restoration of vitamin A to the diet, the basal cell MR remained below control level throughout the experimental period, but the mucous cell MR started to rise on day 2-replete, and on day 3-replete and thereafter the mucous cell MR was within the control range. Basal cell and mucous cell LI's showed similar trends. Preciliated cells were reduced or absent in vitamin A-deprived epithelium. Their number had risen by day 3-replete and thereafter they were generated within the control range. These cells matured into ciliated cells. By day 4-replete, the proportion of basal cells had decreased markedly and the proportions of mucous cells, and preciliated plus ciliated cells had increased, so that at this time cellular proportions were within or near control values. This trend continued so that by day 7-replete, a nearly normal mucociliary epithelium was restored. The results show that vitamin A-levels modulate replication rates of basal cells and mucous cells and indicate that mitotic division of mucous cells is a prerequisite for the genesis of preciliated cells and new mucous cells and for restoration of the mucociliary epithelium following deprivation of vitamin A in the diet.     

Hyperkeratinization and hyperplasia of the forestomach epithelium in vitamin A deficient rats

Specific pathogen free F-344 rats were kept on a vitamin A deficient diet for 3-5 months. After 3 months on this diet the forestomach epithelium showed a mild atrophy. Stereological cytology showed a marked increase in the volume density of cytoplasmic filament bundles and decrease in the volume density of non-bundled cytoplasmic filaments. The volume fraction of keratohyalin granules increased whereas the volume fraction of membrane coating granules decreased. Some animals maintained for 4-5 months on the hypovitaminic diet exhibited the same mild atrophic forestomach epithelium accompanied by hyperkeratinization. Others showed a marked epithelial atrophy and 4 out of 12 animals revealed a marked hyperplasia with increased 3H thymidine labeling index and several papillomatous proliferations. Although many of these alterations reflect a decrease in proliferation as well as hyperkeratinization, the latter changes were not unlike those produced by known carcinogens, and support the putative important role of vitamin A in both carcinogenesis and chemoprevention.