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A single endotracheal instillation of elastase initiates a series of changes in animal lungs that results in a condition resembling human panlobular emphysema. An ultrastructural examination of this series of changes was conducted on the lungs of male golden hamsters exposed to 3H-methylated pancreatic elastase and sacrificed at intervals between 4 hour and 24 days after exposure to enzyme. Lung tissue between 4 and 48 hours showed evidence of hemorrhage and progressive degradation of elastic fibers. Very little indication of epithelial cell damage accompanied these changes. Four days after exposure to elastase, synthesis of new elastic fibers began with the appearance of small clumps of microfibrils in close association with interstitial cells, fibroblasts, and smooth muscle cells. There was also evidence of alterations in alveolar type II cells at this time. Small fibrillar elastic fibers continued to be present in the lung through twenty-four days and may represent a slow repair process or may indicate a structural difference in elastic fibers synthesized after exposure to elastase. Evidence of the continued degradation of elastic fiber could be found up to 16 days after exposure to elastase, revealing that repair processes were occurring in some areas of the lung while destructive process still predominated in other areas.

The ultrastructure of fast-twitch-oxidative-glycolytic (FOG), fast-twitch-glycolytic (FG) and slow-twitch-oxidative (SO) fibers in plantaris and soleus muscles of normal and streptozotocin-diabetic rats was studied. In the diabetic animals, the mitochondria of FOG and SO fibers showed a loss of cristae and an increase in electron-dense granules. There was also an increased number of lipid droplets in close proximity to the mitochondria and the nuclei, and a separation of individual muscle nuclei to form satellite cells. Higher incidences of surface projections and sarcoplasmic splittings at the nuclear region were noticed in SO fibers. The FG fibers showed some disorientation of the T-tubular system. It is concluded that streptozotocin-diabetes has differential effects on the fine structure of the three fiber types of rat skeletal muscle.

A single endotracheal instillation of porcine pancreatic elastase into hamster lungs induces morphological changes that can be detected as early as 15 min after the instillation of enzyme. Lung tissue from animals sacrificed at six time points between 15 min and 3 hr after enzyme instillation was examined for ultrastructural alterations. There were few of these alterations and they were highly focal, but they included damaged epithelial cells and partially digested elastic fibers that occurred both in the parenchyma and the pleura. A cytochemical technique employing N-t-Boc-L-alanine-p-nitrothiophenyl ester as a substrate for elastase-like enzymes was also used in an effort to locate pancreatic elastase shortly after instillation into the lungs. Reaction product was observed on the alveolar surface and in pinocytotic vesicles of alveolar type I cells, in connective tissue areas, in fibroblasts, and in pinocytotic vesicles of capillary endothelial cells. The location of reaction product coupled with ultrastructural alterations in the tissue suggests that pancreatic elastase instilled into the trachea may reach the interstitium in two ways: First, transport may take place across intact alveolar type I cells via pinocytotic vesicles, and second, a small amount of enzyme may gain access to the connective tissue after the disruption of epithelium in a few highly focal areas.

Rete mirabile and gas gland epithelium from the swim bladders of six species of marine fishes were assayed for catalase, glutathione peroxidase, and superoxide dismutase activity. Correlation of the results of these assays with measurements of the concentration of oxygen in the lumen of the normal steady state swim bladders revealed that swim bladders in species containing higher levels of oxygen also exhibited higher levels of superoxide dismutase activity in the rete mirabile/gas gland epithelium region. There appeared to be no correlation between oxygen concentration and the level of catalase or glutathione peroxidase activity. Induction of the inflatory reflex in Opsanus tau by a single deflation of the swim bladder resulted in an increase in the percent of oxygen in the swim bladder lumen 18 to 24 hours later, but this was not accompanied by any significant increases in antioxidant enzyme activity. Swim bladders that were deflated three times at 24-hour intervals showed further increases in oxygen concentration at the end of the 72-hour period but no alteration in superoxide dismutase activity.

In a previous study changes in the macrodistribution of fibronectin during rat-lung development were examined. Using the peroxidase-antiperoxidase immunocytochemical technique, we have demonstrated the presence of fibronectin in embryonic, neonatal, and adult rat lung at the ultrastructural level. In the embryo, fibronectin is found both in an intra- and extracellular association with isolated pneumoblasts, and in a periodic distribution along the basal lamina. The neonate displays fibronectin in an intracellular association with early type-I cells and on their basal and luminal surfaces, but not in association with type-II cells. Neonatal basal lamina is diffusely labeled by anti-fibronectin antiserum. Fibronectin in adult tissue is found both intracellularly and on the basal and luminal surfaces of type-I cells but not in type-II cells. The basal lamina and interstitial connective tissue are slightly or non-reactive. These observations confirm and extend our initial suggestion that fibronectin is involved in rat-lung development.

In order to further clarify the morphologic changes which occur in the gingiva and the nature of the transendothelial passage of nutrients across the vessel walls, gingival biopsies from the incisor area of Jackson strain mice were examined. Our initial investigations revealed the following changes in the diabetic animals: (1) a marked increase in keratohyalin granules in the stratum granulosum and (2) an apparent thickening of the basement membrane of the gingival blood vessels which ranged from 2000–6000 A. Statistical evaluation of these changes is currently underway.

Electron microscopic observations have been made on the squamous epithelium lining of human oral mucosa with a view to exploring the ultrastructural changes associated with the partial keratinization and the total desquamation processes characteristic of this tissue. The keratohyaline granules appear to possess an inherent electron density demonstrable not only in osmium-fixed material but also in formalin-fixed material. These granules are widely scattered through the cytoplasm of normal epithelial cells from the intermediary and superficial layers of human oral epithelium. This is true even though the epithelium may not be classified as keratinized from a convenitonal optical microscopic point of view, under which the dimensions of these granules tend to be too small to be readily detectable. The keratohyaline granules appear to arise in association with (1) certain foldings of the nuclear membrane, (2) nucleolar fragmentations and formation of nucleolar satellites migrating to the nucleolar surface, and (3) the formation of cytoplasmic complexes in near proximity to the nuclei containing granules of various dimensions and mitochondrial fragments besides the high-density keratohyaline granules. The possible nucleolar contribution to the origin and to the organic nature of the keratohyaline granules has been discussed. The possible additional existence of some electron-dense mineral components in these granules has also been considered. In addition to these changes in the internal machinery of the keratinizing epithelium, certain ultrastructural changes in the peripheral cell membranes have been described. These were characterized by a great increase in total cell surface and by the development of deep foldings of the cell membrane interdigitating with grooves in adjacent cells. Finally, it was noted that, while the deeper cells appeared to be held together by a relatively dense cementing substance, the superficial cells separated more readily and had little or no intercellular cementing substance between them.

Techniques have been described by which thin sections for electron microscopy can be prepared from both undemineralized and artificially demineralized specimens of carious enamel and dentine. Observations on the typical appearance of these tissues have been reported and illustrated with representative electron micrographs. The relation of the findings to present concepts on the histopathology of dental caries has been discussed.

Lymphopoiesis was studied by electron microscopy in the palatine tonsil of the rabbit from 18 days gestation to 5 days after birth. At 18 days tonsils formed as mounds of mesenchyma covered with epithelium. At 19 days the basal epithelial cells started to increase in number, eventually forming ‘buds‘ which projected into the mesenchyme. Simultaneously, lymphocytes appeared nearthe epithelium or buds. There was marked resemblance between the basal epithelial cells and the lymphocytes. Budding slowed down after the 25th day, but individual basal cells continued to migrate into the mesenchyme and lymphocytes increased in number. Ultrastructure wassimilar in both types of cells, and differentfrom mesenchymal cells. At 29 days lymphocytes were found in the basal epithelial layer behind an intact basement membrane. The evidence indicated that lymphocytes were derived from epithelium.

The gingival sulcus wall of Dutch Belted rabbits was used to study vascular transudation and exudation with Pelikan ink as a tracer for light microscopy and saccharated iron oxide as an electron microscopic tracer. The marker was apparently transported by numerous plasmalemmal vesicles, and also passed through the interendothelial junctions. The sulcular fluid was an inflammatory exudate rather than a transudate. Ultrastructural alterations of the gingiva following inflammation were studied. The interepithelial space increased more on the sulcular than the attached gingival side. The wider interepithelial space in the sulcular area and the non-keratinizing nature of this tissue may have contributed to the ease of passage of the gingival fluid in that region.

A cell which has not been previously described was observed in the gills of Helostoma temmincki . This cell contains long mitochondria, over 3 μ , in parallel array, surrounded by an extensive system of coated channels. There is a 200 A space between the membranes of the mitochondria and the channels. The particles coating the channels are seen in the center of this space. These particles are about 80 A in diameter with 60 A spaces between them. A connection is sometimes seen between the membranes of the channels and the particles. The close association between mitochondria and the coated channels is a characteristic of cells thought to be functioning in the active transport of ions and water. This morphological similarity to other transport cells, and additional experiments in which the specimens were subjected to gradients of hypertonic and hypotonic water, suggest that this cell also functions in the active transport of a substance.

An intercellular junction known as the nexus has been identified in developing granulosa tissue of rat ovaries. Nexuses are common in Graafian follicles of mature cycling rats and in similar follicles of immature animals stimulated by exogenous gonadotrophins. The angle at which the plane of section intersects a nexus significantly governs its internal appearance. Within obliquely cut nexuses there is often a cross-striated pattern of uniform lines in specimens prepared by conventional fixation and staining procedures. These lines could be significant because they may represent points of continuity between communicating cells. Two forms of nexuses are observed. The abutment form is found at cell surfaces, is continuous with plasma membranes, and is frequently associated with cell processes. The other form of nexus is ring-shaped and appears free within the cytoplasm. These circular profiles probably represent transverse views through nexus-girdled cell processes as well as nexus-bound spheres which may have pinched off the processes. Since the profiles from the two sources are ordinarily indistinguishable, both are designated “annular nexuses” even though some may be cell inclusions. Lanthanum tracer can percolate into the central region of abutment nexuses but is absent from the large majority of annular nexuses, suggesting that many of them are isolated from the extracellular space. Reconstruction of serial sections reveals that some annuli are parts of cell inclusions. Sphere formation could be a means of removing nexuses from cell surfaces.

The fine structure of the epidermis of the external gills of the larval frog, Rana pipiens, was observed during the period of gill degeneration. Many of the epidermal cells underwent morbid alterations. In typical degenerating epidermal cells the nucleus became irregular in outline, chromatin formed electron-dense clumps and the nuclear envelope increased in width. Lysosomes became prevalent in the cytoplasm of these cells and progressively larger autophagic vacuoles appeared. As degeneration continued, apical epidermal cells were found embedded deeper within the epidermis. In some instances cell processes from two neighboring epidermal cells extended partially around the relocated degenerating apical epidermal cells probably contributing to their translocation; phagocytosis was completed by a single cell in other instances. The degree of participation of the phagocytic epidermal cells in the degenerative process was uncertain. The distribution of acid phosphatase activity was followed in specimens treated by the Gomori technique. Profiles of cisterna-like elements, positive for the enzyme, suggested that perhaps entire Golgi cisternae contributed to the formation of autophagic vacuoles in combination with small vesicles. Heterophils and macrophages observed in the degenerating gills were believed to participate in the removal of the dying cells.

Epidermoid carcinomas, preceded by dysplastic leukoplakic lesions, were produced on the right lateral borders of the tongues of Syrian hamsters by the application, three times per week, of DMBA in acetone after the area had been scratched with a root canal broach. Animals were killed at 12 to 13 weeks and 15 to 16 weeks. Electron microscopic studies of both the dysplastic leukoplakic lesions and the well-differentiated epidermoid carcinomas revealed clumping of tonofilaments and widening of intercellular spaces, in addition to the cellular pleomorphism, altered nuclear-cytoplasmic ratio, and prominent nucleoli observed with the light microscope. In the carcinomas, there were variations in the morphology of mitochondria, with tubular forms in evidence. The basal lamina in the carcinomas was discontinuous. Pseudopodia arising from basal cells were seen in areas of basal lamina discontinuity.

The development of the rat lung is a process of continuing morphological change. Indications from work in other mammalian systems suggest that fibronectin may be important in the control of this process. The present study has examined embryonic, neonatal, and adult lung tissue of the rat by means of the peroxidase-antiperoxidase (PAP) technique to demonstrate fibronectin at the light-microscopic level. Positive reaction was observed with anti-fibronectin serum in all stages examined. Control sections treated with pre-immune serum or no primary serum gave negative results in each case. Fibronectin in adult tissue was localized to the alveolar surface and alveolar basal lamina. Neonatal tissue showed fibronectin on pulmonary tubule walls and in basal lamina while embryonic tissue revealed localization of the protein in the basal lamina and in association with small groups of cells at the base of septal buds. These findings suggest a role for fibronectin in the control of rat lung development. The results are discussed in terms of the known functions of fibronectin as a preliminary matrix for the subsequent deposition of collagenous connective tissue, as a cellular adhesion protein, and as surface-bound material for cellular migration.

An emphysemalike condition can be induced in animal lungs by the instillation of a single dose of elastase. Autoradiography was used to determine the location of 3H-methylated porcine pancreatic elastase in hamster lungs at four time points. Six hours after instillation of radiolabeled enzyme the distribution of silver grains was very patchy, but in heavily labeled areas grains were concentrated over macrophages, connective tissue areas and over some fibroblasts. By 24 hr the labeling of connective tissue areas was no longer evident and almost all silver grains were associated with macrophages or with the edema fluid that filled many alveoli at this time. By 4 days only macrophages exhibited concentrations of silver grains. The labeling of macrophages was still evident at 7 days. Elastase inactivated by N-acetyl-(L-alanyl)3-L-alanine chloromethyl ketone showed a different distribution 6 hr after instillation. Silver grains were concentrated over macrophages and alveolar type II cells but showed no affinity for connective tissue areas or fibroblasts. By 24 hr almost all grains were located over heavily labeled macrophages.

The anterior chamber of the swimbladder of the toadfish Opsarmustau L. is lined by a single layer of columnar gas gland cells, cuboidal cells that resemble gas gland cells but are located outside of the gas gland region, and squamous cells. Multilamellar bodies are numerous in the gas gland cells and the cuboidal cells and are present in smaller numbers in the squamous cells. Capillaries lie in the lamina propria directly below the epithelial lining. A thick continuous muscularis mucosae and a submucosa consisting of tightly packed cells, cell processes, and connective tissue may contribute to the impermeability to gases of the wall of the anterior chamber. The posterior chamber of the swimbladder is lined by a single type of squamous epithelial cell. Multilamellar bodies were occasionally observed in these cells also. Other types of cells frequently form a partial second layer between the epithelial lining and the basement lamina. A thin muscularis mucosae lies directly below the basement lamina and the capillaries of the posterior chamber are located in the submucosa. The tunica externa is a layer of dense connective tissue that surrounds both the anterior and posterior chambers. Collagen fibrils in the form of tactoids are present in this layer.

An ultrastructural study of skeletal muscle fiber biopsies from the tongue and soleus muscle of diabetic Chinese hamsters revealed the presence of lipid droplets within the muscle fibers. These droplets often were found in the cyptoplasm near mitochondira, sometimes inside or surrounded by one or more mitochondria. Glycogen granules were found in great quantities in the cytoplasm of the tongue and soleus muscle. Granules which appeared to be glycogen were also found within the mitochondria of the tongue muscle.

The antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase were measured in the rete mirabile and gas gland epithelium area of the swim bladder of the toadfish Opsanus tau. When the concentration of enzyme in the swim bladder was compared with the concentration in other organs (kidney, heart, gills) of the same fish, the swim bladder was found to have the highest concentration of superoxide dismutase but relatively low levels of glutathione peroxidase and catalase. Cytochemical assay for the peroxidatic activity of catalase confirmed that virtually no catalase is present in epithelial cells of the gas gland. A similar assay for peroxidase revealed a cyanide-sensitive peroxidase in the multilamellar bodies of these cells. Most of the catalase and peroxidase in the rete mirabile appears to be confined to the granules of neutrophils and the cytoplasm of erythrocytes. Enzyme activity in the neutrophils is not inhibited by 10(-1) M KCN. Cyanide does not appear to inhibit the peroxidase activity in erythrocytes but has little effect on catalase in these cells.

Ultrastructural responses of ovarian granulosa cells to exogenous estrogen have been investigated in immature rats. Animals, 21-daysold, were hypophysectomized and a regimen of 6 daily injections was initiated 30 hr after the operation. Some rats received sesame oil only and others were given totals of 3 or 6 mg diethylstilbestrol (DES). Ovarian tissues were prepared for microscopy 24 hr after the last injection. In addition, normal rats were sacrificed at 21 days to monitor ovarian morphology at the time of hypophysectomy. Rapid simultaneous changes, occurring in cells of the pre-ovulatory rat follicle, suggest a necessity for cell communication, particularly in the multilayered avascular granulosa. Therefore, emphasis is placed on an intercellular junction (the nexus) thought to play an important role in communication. A correlation is made between the extent of thecal development and the incidence of granulosa cell nexuses in follicles of normal as well as hypophysectomized rats. If the “thec...

Corneal collagen is shown to contain several of the well known lysine-derived crosslinks. NaBH4 reduction of acid-soluble corneal collagen revealed the reduced aldol-condensation product of two residues of allysine and an unknown component referred to as the pre-hydroxylysine component. The reconstituted fibrils contain hydroxylysinonorleucine and histidinohydroxymerodesmosine. Pepsin treatment of insoluble corneal tissue at 4 °C in 0.5 M acetic acid results in 60–70% solubilization of collagen; however, data obtained suggest the solubilized collagen has been altered in the non-helical terminal regions. Abnormal ribbon-like segment long spacing monomers are formed rather than normal segment long spacing fibrils and chemical reduction reveals complete loss of the pre-hydroxylysine component and hydroxylysinonorleucine. Significant losses of aldol-condensation product and histidinohydroxymerodesmosine occur with increasing duration of pepsin treatment. As demonstrated by electron microscopy the insoluble corneal collagen remaining after acetic acid extraction has no fibrous structure. In addition lysine-derived crosslinks are not detected by NaBH4 reduction. After dialysis of the insoluble residue against 0.15 M NaCl (pH 7.5) normal collagen fibers together with intermolecular crosslinks appear.

A continued study of gingival biopsies from the Jackson strain diabetic mouse revealed not only an amorphous thickening of the basement membranes of the blood vessels, but also a lamellar thickening. The normal control blood vessels had basement membrane widths in the 2000-A range, whereas the diabetic animals exhibited an average width of 8000 A. These changes were significant at the 0.1% level. Other changes seen in our studies were: (1) an alteration in the endothelial lining of the blood vessels which appeared as an increase in pseudopod or flap formation on the luminal side, and (2) the appearance of large dense bodies within the epithelial cell mitochondria in the mouse which to our knowledge has not been previously described.

Plastid development in albescent (al/al) and wild-type (+/al) strains of Zea mays has been studied in the electron microscope. Etiolated seedlings of the mutant are severely deficient in colored carotenoid pigments and accumulate carotenoid precursors tentatively identified as phytoene and phytofluene. The fine structure of proplastids in etiolated wild-type and mutant leaves is similar with 1 notable exception. Osmiophilic bodies found in the wild-type were lacking in all sections of albescent proplastids examined suggesting that these structures may be storage centers for carotenoid pigments. Plastid pigments are destroyed, chlorophyll synthesizing potential is lost, and the ultrastructure of plastids is irreversibly altered when mutant seedlings are placed directly in high intensity light. However, synthesis of plastid pigments and development of the photosynthetic apparatus as seen in the electron microscope is normal, and indistinguishable from that in the wild-type, in seedlings of the albescent mutant preilluminated with low intensity light prior to high intensity illumination. During treatment in low intensity light carotenogenesis is initiated in the mutant and proceeds normally thereafter.