Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: uses and limitations.

Pulmonary distribution of particles given by intratracheal instillation or by aerosol inhalation.

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.

Regeneration of hamster tracheal epithelium after mechanical injury. I. Focal lesions: quantitative morphologic study of cell proliferation.

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 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. 78-23 This 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.

Effects of vitamin A-deprivation on hamster tracheal epithelium. 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.

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

Vitamin A, which maintains cellular differentiation of epithelial tissues, also has an effect on metaplastic and neoplastic lesions in the respiratory tract. The surface morphology of hamster tracheal epithelium during vitamin A deficiency and during reversal upon the administration of vitamin A was studied by scanning electron microscopy. Focal squamous metaplasia of the tracheal epithelium, confined upon the upper two thirds of the ventral surface of the trachea, occurs in vitamin A deficiency. Synchronous differentiation of the epithelium with sloughing of the existing surface occurs after reversal of the deficient state. This change is rapid, occurring within 4 days of the administration of vitamin A. The SEM enabled documentation of extent distribution and surface changes in vitamin A deficiency and reversal.

Surface Morphology of Tracheal Epithelium in Vitamin A Deficiency and Reversal

A technique has been developed that permits the intratracheal instillation of volumes as large as 1.5 ml into the respiratory tract of young adult Syrian golden hamsters. 2 factors are of unique importance for the success of this technique: 1. the use of halothane anesthesia that permits total recovery in as little as 2 minutes, and 2. the use of an intratracheal speculum that seals at the larynx and prevents loss of suspension from the lungs. Suspensions consisting of 2% (w/v) ferric oxide, or benzo(a)pyrene-ferric oxide, in 0.5% gelatin-saline have been instilled with a technique mortality rate of less than 5%. Histopathological examination of the lungs at 4 weeks revealed no significant changes related to the technique. Distribution of a suspension containing black ink was uniform when volumes larger than 1 ml were instilled; smaller volumes were less well distributed. Examination of the gastrointestinal tract within 5 hours after instillation showed that no significant loss of ink suspension from the lungs had occurred. Additional studies are in progress to determine the tumorigenic potential of benzo(a)pyrene-ferric oxide when administered in 2 doses, 7 days apart, of 1.5 ml each (total dose = 30 mg BaP) to the same amount of carcinogen administered in 10 or more doses of 0.25 ml at weekly intervals.

Large-Volume Intratracheal Instillation of Particulate Suspensions to Hamsters

Mongolian gerbils fed diets containing lead acetate maintained body weight comparable to gerbils fed the same diet without added lead. Intranuclear lead inclusion bodies in epithelial cells of the proximal convoluted tubules of the kidney were first observed at 4 weeks, and increased in number to about 50 per high power field at 12 weeks. At this time, a corticomedullary area of empty-appearing tubules was prominent. Transmission electron microscopy confirmed the increase in number and size of nuclear lead inclusion over the 12-week period. Cytoplasmic changes observed in proximal tubule cells containing lead inclusions were considered indicative of acute lethal injury. Distinct cytoplasmic fibrillar structures, first apparent at 8 weeks, were present in some proximal tubular lining cells and strongly resembled newly formed intranuclear lead inclusions. After 12 weeks, the total amount of lead present in the gerbil kidney was four to six times greater than that in rat kidney as determined by atomic absorption spectrophotometry. A hypothesis has been formulated that relates the more efficient nephron of the gerbil kidney to the rapid and extensive development of intranuclear inclusion bodies and the greater accumulation of total lead.

The Mongolian gerbil as a model for lead toxicity. I. Studies of acute poisoning.

The Mongolian gerbil as a model for chronic lead toxicity

Hamster-adapted A/PR/8 influenza virus infection followed by a secondary insult consisting of 0.25 ml of 0.5% gelatin-saline instilled directly into the trachea causes epithelial hyperplasia of both the trachea and lung. The extent of the response is related to, and dependent upon, the time interval between virus infection and gelatin-saline administration; the optimal time interval is between 12 and 18 hr. The presence of hyperplasia was measured by 3H-thymidine incorporation. Hyperplasia of the trachea is first apparent 3 days after virus infection, peaks at the fourth day, and is no longer present by the twentieth day. Hyperplasia in the lung begins 4 days after virus infection, peaks at the sixth day, and is undetectable by the twentieth day. Additional studies are in progress to determine the effect of virus-induced hyperplasia on benzo(a)pyrene-ferric oxide initiated respiratory carcinogenesis.

David W. Baxter

Papers

Surface Morphology of Tracheal Epithelium in Vitamin A Deficiency and Reversal

Large-Volume Intratracheal Instillation of Particulate Suspensions to Hamsters

The Mongolian gerbil as a model for lead toxicity. I. Studies of acute poisoning.

The Mongolian gerbil as a model for chronic lead toxicity

Influenza-Virus-Induced Hyperplasia of the Respiratory Tract of the Hamster