Elizabeth M. McDowell

Regeneration of hamster tracheal epithelium after mechanical injury

In companion papers we described in detail the morphologic changes that occur during tracheal regeneration following focal mechanical injury (Keenan et al. 1982a), and the proliferative events that occur following multifocal injury, particularly the dominant role played by the secretory cell population in the regenerative process. In this paper the histogenesis of epidermoid metaplasia and restoration of normal mucociliary epithelium following mechanical injury was studied in hamster tracheal epithelium with single pulse or continuous infusion of tritiated thymidine (3HTdR) combined with colchicine blockade of metaphase mitoses. Both small and large wounds were produced in the same animal, in the dorsal and ventral tracheal semicircles respectively. At 12 h following injury, 14.2% of the dorsal epithelial cells and 34.6% of the ventral epithelial cells were lost. Viable cells migrated into the denuded wound sites, covering the small wounds by 12 h and the larger wounds by 48 h. By 27 h a peak mitotic rate (MR) was reached in the dorsal epithelium (MR = 18.1%), but mitotic activity in the ventral epithelium did not peak until 33 h (MR = 13.3%). In both tracheal semicircles, secretory cells accounted for over 76% of the labeled mitotic cells. Ciliated cells did not label or divide. Epidermoid metaplasia was first observed at 33 h in both semicircles. It was transient in the small wounds, but persisted through 168 h in the large wounds. Almost all the epidermoid cells were labeled by continuous3HTdR infusion. A very few pre-ciliated cells appeared first at 33 h but they peaked in numbers at 72 h in both the dorsal (8.0%) and ventral (5.5%) semicircles. These large, pale staining cells were not labeled by single pulse3HTdR but were labeled by continuous3HTdR infusion. Later, large, pale staining cells morphologically similar to pre-ciliated cells, and with identical labeling patterns, were observed budding cilia. Ciliated cell numbers were restored to control level in the dorsal semicircle by 168 h, but only to 84% of control values in the ventral semicircle, where epidermoid metaplasia persisted. These data suggest that secretory cells have a greater proliferative potential than basal cells in regenerating tracheal epithelium, and that they contribute to the development of both transient and persistent epidermoid metaplasia and the production of new secretory cells. Moreover, these proliferating secretory cells produce ciliated cells via a transient pre-ciliated cell which develops cilia and matures in the regenerating epithelium.SummaryAll stages of epithelial regeneration following a focal denuding mechanical injury have been quantified in hamster trachea. The epithelium was divided into wound and non-wound sites and every cell around the entire tracheal circumference was counted and catergorized according to cell type. The control hamster tracheal epithelium was composed of about 33% ciliated cells, 57% secretory cells and 10% basal cells. Proliferative activity, measured as mitotic rate (MR) following a 6 h colchicine metaphase blockade, was low and confined to the secretory and basal cells. The total cell population had a MR of 0.12% (0.08% secretory cells; 0.04% basal cells).Following a focal denuding wound in the ventral portion of the epithelium, 18% of all the epithelial cells were lost by 6 h. This loss increased to 31% by 12 h. Secretory cells and basal cells from the adjacent non-wounded epithelium flattened into a squamous morphology and during the first 12 h migrated into the wound at about 0.5 μ per min to cover the defect. Cell division at the wound was low at 12 h (MR = 0.4%), but by 24 h an exponential increase in cell proliferation had occured in the wound site (MR = 31.1%). Secretory cells (MR = 19.9%), basal cells (MR = 1.4%) and squamous cells-a mixture of flattened secretory and basal cells (MR = 9.8%)-contributed to this proliferative activity. Mitotic activity in the non-wounded epithelium remained low (MR= 0.6%).Cell proliferation at the wound site produced a multilayered epidermoid metaplastic epithelium by 36 and 48 h. Mitotic activity remained high at these times (36 h MR = 21%, 48 h MR = 12%). Thereafter (60 h–120 h) mitotic activity fell to near control levels, and the wound epithelium was gradually replaced by recognizable basal, secretory and preciliated cells. The latter, first seen in the wound at 48 h, and recognized as very large pale non-ciliated cells, developed cilia through 60, 72, and 96 h so that a nearly normal epithelium was restored by 120 h.

Studies on the pathophysiology of acute renal failure

SummaryAcute renal failure was induced in male rats by the subcutaneous injection of 4 mg HgCl2 per kg body weight. Changes in the proximal tubule were studied by light and electron microscopy at six time intervals from 15 min to 24 h. Renal function was monitored at 6 and 24 h.Between 15 min and 3 h changes were similar in all regions of the proximal tubule (pars convoluta and pars recta). Dispersion of cytoplasmic polysome groups was widespread and mitochondrial matrices were condensed in some cells. No changes were noted in the brush border but increased endocytotic activity occurred in some convoluted tubules at 1 and 3 h. At 6 h severe changes had occurred in the pars recta in the medullary rays. Microvilli of the brush border were focally absent, the mitochondria were swollen and the endoplasmic reticulum was dilated. At this time only subtle changes occurred in the pars recta in the outer stripe of the outer medulla. However by 24 h necrosis was widespread throughout the pars recta, yet changes in the proximal convoluted portion were minimal.A significant azotemia, decreased GFR and increased FENa+ and FEK+ occurred at 6 and 24 h after HgCl2 injection. Thus HgCl2 at 4 mg per kg body weight produced reproducible renal failure and necrosis involving the pars recta of every nephron but necrosis did not begin in the pars recta until after 6 h while acute renal failure was probably initiated much earlier.The following hypothesis is presented. HgCl2 initially interacts with the entire proximal tubule. Although injury is sublethal in the pars convoluta it is responsible for greatly diminished sodium reabsorption and is related to the pathogenesis of the renal failure through feedback mechanisms involving the macula densa and release of renin. This results in renal hemodynamic alterations, decreased GFR and other functional disturbances associated with renal failure. The development of necrosis in the pars recta appears to be a relatively late event, possibly due to further accumulation of Hg++ in this region. In any case, the necrosis appears pathogenetically dissociable from the mechanism of acute renal failure.

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

SummaryThe 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 and3HTdR were given 6 hours before death and the proliferative activities of basal cells and mucous cells were quantified separately by3HTdR 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 disparatehypoplasia 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 The opinions and assertions contained herein are the private views of the authors and are not to be construed official or as reflecting the views of the Department of Defense The experiments reported herein were conducted according to the principles set forth in the “Guide for the Care and Use of Laboratory Animals” Institute of Laboratory Animal Resources, National Research Council, DHSW Publ. No. 78-23This is contribution No. 1554 from the Cellular Pathobiology Laboratory of the Department of Pathology, University of Maryland School of Medicine 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.

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

SummaryIn 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 and3HTdR 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 morphologic 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 of preciliatedplus 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 cellsand 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.

Regeneration of Hamster Tracheal Epithelium After Mechanical Injury II. Multifocal Lesions: Stathmokinetic and Autoradiographic Studies of Cell Proliferation*

SummaryRegeneration of hamster tracheal epithelium following multifocal mechanical injury was studied with the combined use colchicine metaphase blockade and single pulse tritiated thymidine (3HTdR) labeling. Control hamster tracheal epithelium had a low labeling index (LI = 0.63%) and a low mitotic rate (MR = 0.19%), but of the cells labeled and in mitosis, more were secretory cells (LI = 0.36%, MR = 0.14%) than basal cells (LI = 0.26%, MR = 0.05%).Following injury, 38% of the epithelial cells was lost by 6 h, and this loss increased to 43% by 12 h. During these times LI and MR remained near control values. However, at 24 h there was a dramatic rise in LI (14.1%) and MR (10.6%) as a broad peak of proliferation with rapid movement through the cell cycle. Secretory cells, which comprised 71 % of the epithelial cells, accounted for about 77% of the mitoses observed and 65% of the3HTdR labeling. In contrast, basal cells comprised 13% of the total cells present, 12% of the mitoses, and 21% of the3HTdR labeling. Non-ciliated squamous cells (a mixed population of flattened secretory and basal cells) comprised 5.4% of the epithelium, 11% of the mitoses and 14% of the3HTdR labeling. When only the labeled cells were considered as discrete populations at 24 h post-injury labeled secretory cells had 88% of their number in mitosis and 12% in interphase, compared to labeled basal cells with 45% in mitosis and 55% in interphase. These data indicate that secretory cells passed through the DNA synthesis phase into mitosis at twice the rate of basal cells. Thus secretory cells played the dominant role in tracheal regeneration due to the number of cells involved and their proliferative rate.Between 24 and 60 h, proliferative activity gradually declined and epidermoid metaplasia developed in the epithelium. By 72 h the LI and MR had declined to almost control values. Preciliated cells, first seen at 48 h, were most evident at 72 h when LI (0.7%) and MR (0.4%) were low. Pre-ciliated cells were not labeled by3HTdR, indicating that these post-mitotic cells did not synthesize DNA. Thereafter, LI and MR remained low and the majority of the epithelial cells in the wound assumed normal morphology by 120 h. Nevertheless, some epidermoid metaplastic cells did remain.The wound sites also contained considerable submucosal mesenchymal proliferation during the period of epithelial regeneration. At 24 h capillary endothelial cells were labeled and dividing, and by 36 h large numbers of fibroblasts were labeled and in mitosis. This mesenchymal proliferation remained sustained for a longer period than the epithelial proliferation, similar to the situation observed in developing fetal and neonatal airways.

Studies on the pathogenesis of ischemic cell injury

SummaryFlocculent densities in the matrix of mitochondria have become quite important in cell pathology since, when prominent, they indicate irreversible cell injury. The morphology and chemical nature of these flocculent densities have been studied in kidney after various periods of autolysis in vitro in whole tissue samples and in isolated mitochondria. After 30 to 60 min of ischemia, flocculent densities were seen only occasionally and they were most prominent in samples subjected to mechanical damage during isolation. However, in 2- and 4-h samples numerous densities were seen. The size of the densities increased with time, being about 1,400 Å in diameter at 4 h. Densities were also seen in mitochondria isolated in medium containing EDTA. They were seen only in the mitochondrial matrix, and could occasionally be found in condensed mitochondria. Small densities were generally round but larger ones varied in shape and often appeared as aggregates of smaller densities. Digestion of the densities from water-soluble glycol methacrylate embedded samples was successful with pronase, but neither acid nor lipid solvents were effective. Calcium or inorganic phosphate content of isolated mitochondria did not show an increase parallel to the occurrence of flocculent densities. The results suggest that the densities consist predominantly of protein and are probably formed through denaturation of proteins of the mitochondrial matrix and/or of the inner membrane.

Studies on the pathophysiology of acute renal failure. II. A histochemical study of the proximal tubule of the rat following administration of mercuric chloride.

SummaryAcute renal failure was induced in male rats by the subcutaneous injection of 4 mg HgCl2 per kg body weight. Enzyme activities of the proximal tubule were studied histochemically at six time intervals from 15 min to 24 h. The enzymes studied were alkaline phosphatase, 5′-nucleotidase, acid phosphatase, α-glycerophosphate dehydrogenase (NAD-independent), malic dehydrogenase, succinic dehydrogenase, latic dehydrogenase, glucose-6-phosphate dehydrogenase and glucose-6-phosphatase. Decreases in activity were observed for alkaline phosphatase and 5′-nucleotidase after 15 min. Acid phosphatase was decreased after 30 min. These three enzymes returned to control levels after 3 h, but malic dehydrogenase and α-glycerophosphate dehydrogenase were decreased at this time interval. Succinic dehydrogenase was first decreased after 6 h. The earliest morphological changes detectable by light microscopy were observed in pars recta tubules in the medullary rays after 6 h, a time when all enzymes studied showed widespread decreased activity throughout the proximal tubule. After 24 h, the pars convoluta appeared morphologically normal but the pars recta was necrotic and exhibited calcification, whereas enzyme activity was decreased (absent in some cases) in both pars convoluta and pars recta.These results support the hypothesis that Hg+ +, when given in a sublethal dose, is associated with early histochemical changes in the brush border of the proximal tubule, which may be related to early changes in sodium reabsorption and to the subsequent development of acute renal failure. The observation that changes in plasma membrane-associated enzymes occur early and prior to alterations in enzymes of mitochondria and the endoplasmic reticulum suggests that Hg++ interacts initially with the plasma membrane.

Effects of retinoic acid on the growth and morphology of hamster tracheal epithelial cells in primary culture

SummaryHamster tracheal epithelial cells were grown in primary culture on a collagen gel substrate in hormone-supplemented serum-free Ham’s F12 medium with 10-8 M retinoic acid (RA +), or without retinoic acid (RA -). On days 1 and 2, the colonies were composed of large (secretory) cells and lesser numbers of small (basal) cells; ciliated cells were rare. At these times, cell number, thymidine incorporation, and total labelling indices (small and large cells, combined) were similar in RA+ and RA-cultures, but the large cells became flat in RA-medium on day 2. On days 3–5, thymidine incorporation and total labelling indices were less in RA-than RA+ cultures, and on days 4–6, cell numbers were decreased in RA-cultures. On day 3, the large cells of the RA-colonies had flattened further and clusters of small basal cells had formed. On day 4, the RA+ colonies were composed of densely-packed cuboidal secretory cells, small basal cells were inconspicuous; the total labelling index was about 27%. The RA-colonies were composed of large flat secretory cells and numerous small basal cells which were clustered in groups; the total labelling index was about 7%. Since large and small cells could be discriminated by size in RA-colonies, a labelling index was generated based on cell size. On days 2, 3 and 4, the labelling index of the small basal cells in the RA-colonies was 44%, 43% and 24% respectively, whereas the labelling index of the large secretory cells fell rapidly over the same period (56%, 14% and 2%). On days 5 and 6, the cuboidal secretory cells in the RA + cultures had differentiated further and the cells were stratified focally. Some new ciliated cells had formed on day 6. In RA cultures, mucous granules were not observed in the large flat cells and ciliated cells were not seen. The total labelling indices were 11% and 0.35% in RA+ cultures, and 0.5% and 0.25% in RA-cultures on days 5 and 6, respectively.The study shows that the target cell for vitamin A in the hamster tracheal epithelium is the secretory (mucous) cell. When retinoic acid was deficient, the secretory cells flattened and their capacity to divide was greatly diminished. Since the basal cells continued to replicate when the secretory cells did not, the population density of the basal cells increased disproportionally, which could be interpreted erroneously as a “basal cell hyperplasia.” Real hyperplasia and epidermoid metaplasia were late secondary events which occurred in this study following focal disintegration of the epithelial cell sheet. Then the large secretory cells became keratinized and expressed an epidermoid phenotype.

Changes in Secretory Cells of Hamster Tracheal Epithelium in Response to Acute Sublethal Injury: A Quantitative Study

Secretory cells are reported to rapidly increase in number when the tracheobronchial epithelium is irritated. We suggest that this acute change results from accumulation of secretion granules within specialized albeit unobstrusive secretory cells rather than from conversion of undifferentiated cells to secretory cells of the production of new cells by mitosis. This hypothesis was tested in hamster tracheal epithelium 6, 12, and 24 hr after intratracheal instillation of elastase in saline or saline alone. Basal, secretory, and ciliated cells and cells of indeterminate character were quantified by counting 1400 cells per hamster in 2 micrometers thick glycol methacrylate sections stained with periodic acid-Schiff (PAS)-lead hematoxylin. Secretory cells were characterized and quantified depending upon distribution patterns of the PAS-positive secretion granules and on the amount of intracellular secretion product. Thirty-two hamsters were studied; half of them received colchicine 6 hr prior to tissue sampling. Over 24 hr the percentage of ciliated cells showed no significant change and no morphological evidence of alteration was observed in this cell population; thus ciliated cells were excluded from the analysis. When the ciliated cell population was excluded, control and experimental values for the different cell categories remained statistically constant, if unavoidable classification errors at the light microscopic level were compensated for on the basis of ultrastructural features. Average control values for the non-ciliated cell population were: mitotic index 0.030, basal cells 27.8%, secretory cells 57.9%, and cells of indeterminate character 14.3%. Secretory product was decreased in secretory cells at 6 hr but thereafter secretion accumulated in these cells. At 24 hr the overall number of secretory cells appeared to be increased. However, the increase was apparent and not real and was accounted for by accumulation of secretion granules in preexisting but previously unobtrusive secretory cells.

Studies on the pathophysiology of acute renal failure. IV. Protective effect of dithiothreitol following administration of mercuric chloride in the rat.

SummaryStudies were undertaken to investigate the effect of dithiothreitol (DTT), an agent capable of chelating heavy metals, on mercuric chloride (HgCl2)-induced acute renal failure (ARF) in rats. The rats were divided into four groups: Control; HgCl2 (4 mg/kg)-alone; DTT (15.4 mg/kg)-alone; and HgCl2 followed 30 min later by DTT (HgCl2 + DTT). Kidneys were studied by light and electron microscopy and histochemical enzyme analysis at 1, 3, 6, 12, 24 and 48 h following treatment. In addition, renal function was monitored at 6, 12, 24 and 48 h.Functionally, HgCl2-alone induced a progressive non-oliguric form of ARF characterized by increased fractional excretion of sodium (FENa+), a reduction in glomerular filtration rate (GFR) and azotemia, apparent by 12 h and continuing throughout the course of study. Administration of HgCl2 + DTT ameliorated the development of ARF whereas DTT, administered by itself, produced no functional abnormality.Administration of HgCl2-alone caused dispersion of cytoplasmic ribosomes at 1 h and 3 h. Also, a reduction in plasma membrane marker enzyme activities (alkaline phosphatase and 5′-nucleotidase), occurred at 1 h, particularly in the pars convoluta. At 6, 12, 24 and 48 h morphological changes in the first portion of the pars convoluta (P1) tended to be reversible. The injury which occurred in the more terminal portions of the pars convoluta (P2) was sublethal in some animals but progressed at the later time intervals to advanced irreversible cell injury followed by necrosis in others. Activity of all enzymes studied returned or remained at control levels at 3 h. However, activities of all enzymes studied, including acid phosphatase and succinate dehydrogenase, were reduced throughout P1 and P2 at 6, 12, 24 and 48 h. By 24 and 48 h, all pars recta (P3) tubules were necrotic and enzyme activities were minimal.Administration of HgCl2 + DTT produced similar but less severe lesions at 1 h as did HgCl2-alone. However, a protective effect (morphological and enzymatic) was evident at 3 h and during the remainder of the study, when virtually no morphological or substantial enzymatic changes occurred in P1 or P2. Nevertheless, by 48 h, extensive necrosis had developed in P3 of HgCl2 + DTT treated rats as in animals treated with HgCl2-alone. DTT when administered alone produced no morphological or enzymatic abnormalities.The results of this study support the hypothesis that a structural-functional correlation exists between the progression of proximal convoluted tubular injury and the development of HgCl2-induced ARF, which is unrelated to necrosis in the pars recta. According to this hypothesis, HgCl2-induced alterations in proximal tubular handling of Na+ and Cl− lead to an augmentation of a tubulo-glomerular feedback mechanism involving the renin-angiotensin system. This results in vasoconstriction of the afferent arteriole and subsequent filtration failure. DTT ameliorates HgCl2-induced ARF by altering the effect of the heavy metal on Na+ and Cl− transport functions and the subsequent initiation of such a feedback mechanism.