Sergei P. Sorokin

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

Abstract In animal studies concerned with the deposition of particulate matter in the lung, two methods for delivery of particles are commonly used, aerosol inhalation and intratracheal instillation of particle suspensions. We have attempted to evaluate the distribution patterns of each of these methods. Particles labeled with 99m Tc were administered to both rats and hamsters. The animals were subsequently killed. The lungs were excised, weighed, inflated, dried, and divided into 54 pieces which were counted individually in a Nuclear-Chicago Model 4230 Automatic Gamma Scintillation System. Groups receiving intratracheal instillations demonstrated nonuniform distribution patterns with preferential deposition in the dependent portions of the lung. The aerosol groups evidence more even distribution with preferential deposition in the apical lobes.

A study of development in organ cultures of mammalian lungs

Abstract Explants of lungs from 16 litters of fetal rats (13–19 days) and from three litters of fetal guinea pigs (27, 30, and 60 days) were grown as organs on solid media containing 40% serum, glucose, and 1.5% agar. Most components of the lungs developed, though growth of the vascular tree was restricted. The columnar epithelium of the early lungs progressively became lower as branching proceeded and in some cultures even attenuated sharply at points where alveolus-like portions differentiated. Mitotic rates in cultures from slowly developing guinea pigs were comparable to similarly aged lungs in vivo but in rats were considerably diminished. Glycogen concentrated in the budding terminal epithelium, where mitoses were most frequent, and disappeared after formation of the flattened “alveolar” lining. Other histochemical patterns seen in vivo were also closely reproduced in vitro. Comparable results in morphology and histochemistry were obtained in explants grown with added cyanide or malonate, but all cultures failed when given fluoride. Thus, nearly full potential for the formation of lungs is contained within the explants, differentiations occur independent of growth, and anaerobic metabolism chiefly supplies the developing organ. These conclusions are weighed in the discussion, along with some emergent speculation on developmental mechanisms operating in the lungs.

Development of Neuroepithelial Bodies in Fetal Rabbit Lungs.: I. Appearance and Functional Maturation as Demonstrated by High-Resolution Light Microscopy and Formaldehyde-Induced Fluorescence

Developing lungs of fetal rabbits aged 13 days through early postnatal stages were examined for periodic acid-Schiff (PAS)-lead hematoxylin staining, serotonin fluorescence, and argyrophilia, methods selective for small-granule (neuro) endocrine cells. Later stages were also studied by electron microscopy. These cells arise from precursors that first appear around days 18-19 in the endodermal epithelium; the latter stand out as clear cells from their undifferentiated, glycogen laden neighbors. The cells first form in the main and lobar bronchi, then continue to arise in the lining of newly laid down branches of the extending bronchial tree, tending to concentrate near points of branching. Some cells have been seen to divide, and clusters of two, four, or more soon appear. Morphologically mature neuroepithelial bodies begin to appear in larger bronchi around 23 days, although the largest examples occur in 25-day and older fetuses. After 23 days, additional bodies mature from clear-cell precursors in more recently laid down distal lung, so that by the terminal sac period (around days 28-29) all conducting airways contain them, and the clear cells and clear-cell clusters have virtually disappeared. A pinkish staining by PAS-lead hematoxylin and dense-core secretory granules, which begin to appear in the clear cells, are present in mature small-granule cells whether solitary or aggregated. Argyrophilia is less consistently demonstrable. Fluorescence for serotonin is first evident in mature neuroepithelial bodies at 23 days, increases considerably by 26 days, and reaches a prenatal peak around days 28-29, when neuroepithelial bodies and solitary small-granule cells along the whole conducting bronchial tree may participate. Fluorescence appears to decrease during days 30-31 but strengthens again after birth. Composed of these solitary small-granule cells and neuroepithelial bodies, the pulmonary APUD system of rabbits thus appears substantially functional during the final quarter of gestation.

Comparative histochemical patterns in developing mammalian lungs

Abstract Prenatal and postnatal lungs from two rodents with different gestations, albino rat (23 days) and guinea pig (68 days), were compared to determine the course of histochemical differentiation and its relation to birth. Common patterns exist, but differentiation attained prenatally in guinea pigs is completed postnatally in rats. A partitioning of activity between epithelium and stroma is notable. Species differences in localization of chemical constituents in several mammals and some metabolic implications are discussed. In both animals glycogen is stored in the epithelium during its formation and disappears with its maturation, so that terminal buds are always richest. Mesenchymal glycogen fluctuates in developing rat lungs but is absent from those of guinea pigs. Triglycerides occur only prenatally in guinea pigs but in rats remain for 3 weeks after birth. Both species store fats in alveolar regions. Succinic dehydrogenase activity is uniform in early prenatal lungs but gradually increases in epithelium and decreases in stroma. The strongest reaction appears in the cardiac muscle of pulmonary veins near parturition and persists thereafter. Prenatal acid phosphatase occurs in bronchial and bronchiolar epithelia. It is also found in alveolar septa just prenatally in both animals. Alkaline phosphatase of prenatal rat lungs is present only in terminal buds. It is located elsewhere in guinea pigs. The adult pattern of distribution is reached just before parturition in guinea pigs and early postnatally in rats.

Small-granule (neuro)endocrine cells in the infracardiac lobe of a hamster lung. Number, subtypes, and distribution.

Small-granule APUD (amine precursor uptake and decarboxylation) endocrine cells were surveyed in 600 3 microns glycol methacrylate-embedded, periodic acid-Schiff (PAS)-lead hematoxylin-stained serial sections comprising 95% of the infracardiac lobe of a hamster lung. Results were confirmed by less systematic study of other hamster lungs. Positions of endocrine cells were marked on cardboard profiles of bronchi and bronchioles for assembly into a 70 X enlarged three-dimensional model from which size and branching of the airway were determined. Records were made for computer analysis of the number and staining patterns of endocrine cells, the nature of contiguous epithelial cells, and the presence of underlying smooth muscle and blood and lymph vessels. APUD cells occurred in 95% of all airways, at a mean density of six solitary cells and 10 cell clusters (neuroepithelial bodies) per millimeter of airway length, measured along the bronchial-bronchiolar long axis. Nineteen percent of endocrine cell loci (29% of all cells) were found at bronchioloalveolar portals in all regions of peripheral lung. Twenty percent of loci (28% of all cells) occurred about the origins of lateral airway branches; these included 4% of loci on carinal points of bifurcation. Two groups of APUD cells had distinctive anatomic relationships: 1) 13% of loci (20% of cells) were related to pulmonary capillaries and venules, mainly at bronchioloalveolar portals; and 2) 39% of loci (53% of cells) overlaid peribronchial muscle, mainly in larger airways where changes in diameter might affect ventilation. In this lobe, APUD cells were not related to goblet or mast cells; 74% of loci abutted Clara and/or ciliated cells, 17% great alveolar cells. Few loci were associated with pulmonary arteries and veins. Five APUD cell types were identified by PAS-lead hematoxylin staining. Types I, II, and V, with granules approximately 0.2 micron in diameter, made up 38%, 45%, and 2% of cells, respectively. Types III and IV, 10% and 5% of cells, respectively, had larger granules. Types I, II, III, and V occurred as solitary cells as well as in neuroepithelial bodies. One-third of the neuroepithelial bodies contained a single cell type; the rest were mixed. Type IV cells, with coarse lead hematoxylin-positive granules, usually were found in large neuroepithelial bodies containing two to four cell types and were never seen occurring alone. We conclude that 1) hamster lungs contain different kinds of APUD cells; 2) those likely have a variety of functions; 3) distinctions merely between solitary and clustered cells may not be significant; 4) the histophysiology of many neuroepithelial bodies probably is more complex than previously suspected; and 5) PAS-lead hematoxylin is superior to argyrophilia and amine fluorescence for light microscopic counting and analysis of pulmonary APUD cells in this species.

Ontogeny of endocrine cells in the respiratory system of Syrian golden hamsters

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.

An unusual bronchial carcinoid tumor: Light and electron microscopy

An unusual bronchial carcinoid tumor was studied by light and electron microscopy. The tumor cells, which appeared to be monotonously uniform in hematoxylin and eosin stained sections, were found to be morphologically heterogeneous at the ultrastructural level with regard to the size, number, and morphology of the endocrine granules. Presumptive endocrine granules were seen in all tumor cells, but some cells contained only small round granules (2000 A largest diameter), other cells contained large round granules (some with as large a diameter as 1.0 mu), and some cells contained large polymorphic granules. Many of the cells stained positively at the light microscopic level when selective stains for endocrine cells were applied. All types of granules showed argyrophilia at the ultrastructural level. Numerous clusters of endocrine cells were observed in the otherwise normal bronchial and bronchial glandular epithelium. The spectrum of granule morphologies, as seen in the tumor cells, was displayed in cells of the intraepithelial clusters. Some mucous cells and sparsely ciliated cells within these clusters contained argyrophilic granules. Multiple continuities existed between the epithelial endocrine cell clusters and the underlying tumor mass. The intraepithelial clusters represent foci of carcinoma in situ, the genesis of which is discussed.

An iron oxide aerosol suitable for animal exposures

Abstract An aerosol generator has been constructed which produces submicronic iron oxide particles from the combustion of iron pentacarbonyl, Fe(CO)5. If operating conditions are chosen with care, CO levels will be below 5 ppm when mass concentrations are as high as 340 mg/m3. The aerosol is suitable for animal exposures since it is relatively nontoxic and is easily quantitated by techniques of chemical analysis. The particles reach the pulmonary alveolar surface where they are readily recognized by light and electron microscopy. These aerosols are ideal for use in morphological studies on the pathways of particle clearance in the lungs.

An unusual bronchial carcinoid tumor analyzed by conjunctive staining

Abstract Two staining methods selective for pulmonary APUD endocrine cells-PAS-lead hematoxylin and a more elaborate procedure termed conjunctive staining-were applied to 2 μm. glycol methacrylate sections of a human bronchial carcinoid tumor, in order to display the advantage they hold over routine pathologic methods in revealing the presence as well as cytochemical characteristics of endocrine cell granules in the tumor cells. PAS-lead hematoxylin alone showed the tumor population to be mixed, with a wide range of staining in the granules, extending between the extremes of PAS only and lead hematoxylin only. The conjunctive staining protocol is described in detail. In tissue previously reacted for argyrophilia, it demonstrates, sequentially, fluorescence for serotonin, PAS alone, and PAS-lead hematoxylin, all in a single section. By comparing photomicrographs of a given field recorded during the process, exact point by point correlations of staining behavior can be made, limited only by the resolving power of the microscope. Conjunctive staining of the tumor revealed 10 distinct cytochemical cell signatures, far exceeding the number of types of endocrine cells known to exist in normal human lung. Preliminary results indicate that three types with PAS and lead hematoxylin positive granules may occur in adult human bronchi, although previous studies have reported but a single type of endocrine cell. Numerous tumorlets in the bronchial epithelium overlying the tumor were an unusual feature of the case. These were composed of cells staining similarly to cells in the tumor mass. Some were connected to it by cords of cells, whereas other tumorlets were interconnected in the epithelium, but a number of smaller ones were clearly isolated from other tumor cells, as shown by serial sections. A few tumorlets contained cells with both mucous and lead hematoxylin positive granules. With a population density that exceeded the expected distribution of normal small granule endocrine cells in the bronchial epithelium and including cells with mixed endocrine and nonendocrine characteristics, the tumorlets provided testimony favorable to the view that carcinoid tumors like this one arise from indifferent cells of the bronchial epithelium that have been driven by oncogenic forces toward expressing an APUD cell phenotype.

FLUCTUATIONS IN THE ACID PHOSPHATASE ACTIVITY OF SPHEROSOMES IN GUARD CELLS OF CAMPANULA PERSICIFOLIA

Acid phosphatase activity fluctuates in mature epidermal guard cells of Campanula persicifolia in response to changing light conditions. As demonstrated both by Gomoris method and by Burstones simultaneous coupling azo dye technique in both unfixed and aldehyde-fixed epidermal strips, the enzyme is active in the spherosomes when the guard cells are flaccid and the stomata are either partially or fully closed. It is inactive when the cells are turgid and the stomata are fully open, regardless of whether opening had resulted from a photoactive or scotoactive process. In living preparations, such inactive spherosomes will gradually become active for acid phosphatase if turgid guarde clls are subjected to partial plasmolysis. Marked activity for acid phosphatase becomes manifest in the vacuoles as well as in the spherosomes of the guard cells if the plants are kept in the dark for many days. In senescent leaves the vacuoles of the guard cells are reactive even though the plants are grown in the light. The walls of the epidermal cells exhibit sporadic activity for the enzyme, but the walls of guard cells have not been observed to react. Nuclear staining sometimes is present after tissues are incubated in the Gomori medium, but it is considered to result from nonenzymic binding of lead on the nuclear surface. The spherosomes evidently are among the principal sites for acid phosphatase activity in plant cells. In guard cells the spherosomes are considered to be enzymically active when the cells are in a relatively catabolic phase of metabolism and to become inactive when the cells enter a relatively anabolic phase.