An electron microscope study of the aorta in young and in aging mice.
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Biomedical subjects
Publications and source records attributed to H E KARRER.
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Diluted India ink was instilled into the nasal cavity of mice and the lungs of some animals were fixed with osmium tetroxide at various intervals after one instillation. The lungs of other animals were fixed after 4, 7, 9, 16, or 18 daily instillations. The India ink was found to be phagocytized almost exclusively by the free alveolar macrophages. A few particles are occasionally seen within thin portions of alveolar epithelium, within the "small" alveolar epithelial cells, or within occasional leukocytes in the lumina of alveoli. The particles are ingested by an invagination process of the plasma membrane resulting in the formation of intracellular vesicles and vacuoles. Ultimately large amounts of India ink accumulate in the cell, occupying substantial portions of the cytoplasm. The surfaces of phagocytizing macrophages show signs of intense motility. Their cytoplasm contains numerous particles, resembling Palade particles, and a large amount of rough surfaced endoplasmic reticulum. These structures are interpreted as indicative of protein synthesis. At the level of resolution achieved in this study the membranes of this reticulum appear as single dense "lines." On the other hand, the plasma membrane and the limiting membranes of vesicles and of vacuoles often exhibit the double-line structure typical of unit membranes (Robertson, 37). The inclusion bodies appear to be the product of phagocytosis. It is believed that some of them derive from the vacuoles mentioned above, and that they correspond to similar structures seen in phase contrast cinemicrographs of culture cells. Their matrix represents phagocytized material. Certain structures within this matrix are considered as secondary and some of these structures possess an ordered form probably indicative of the presence of lipid. The possible origin and the fate of alveolar macrophages are briefly discussed.
The interconnections and the surfaces of the striated muscle cells which occur in thoracic and in lung veins of the mouse were studied with the electron microscope. The osmium-fixed tissues were embedded in methacrylate or in araldite and sectioned with a Porter-Blum microtome. Many preparations were stained before embedding with phosphotungstic acid or after sectioning with uranyl acetate. Typical intercalated discs are observed in this muscle. They are similar to the discs found in heart muscle. These intercalated discs represent boundaries between separate muscle cells. Along the discs, cells are joined in planes normal to their myofilaments. The same cells are also joined in planes parallel to the myofilaments by means of lateral interconnections. These lateral cell boundaries are in continuity with the intercalated discs. Three morphologically distinct parts occur within the lateral cell interconnections: One is characterized by small vesicles along the plasma membrane, the second part has the structure of desmosomes, and a third part represents an external compound membrane (formed by the two plasma membranes of the adjoining cells) and is termed "quintuple-layered cell interconnection." Small vesicles and plasma membrane enfoldings along the free surface of muscle cells are interpreted as products of a pinocytosis (phagocytosis) process. Some of them are seen to contain small membrane-bounded bodies or granules. The free cell surface shows a characteristic outer dense layer ("basement membrane") which accompanies the plasma membrane. The topographic relation of this dense layer with the plasma membrane seems to vary in different preparations. The significance of this variation is not well understood. On two occasions a typical arrangement o vesicles and tubules was observed at Z band levels, just beneath the plasma membrane. These structures are believed to represent endoplasmic reticulum. Their possible significance for the conduction of excitation is discussed.
The musculature of small lung veins, of the thoracic portion of the inferior vena cava, and of other thoracic veins of the mouse have been studied in the electron microscope. Tissues were fixed in 1 per cent osmium tetroxide buffered with veronal, to which either sodium chloride or sucrose had been added. Methacrylate or araldite served as embedding matrices. Phosphotungstic acid or uranyl acetate was used to stain some of the preparations. Thin sections were examined in a Siemens and Halske Elmiskop Ib electron microscope. The entire musculature of the veins examined was of the striated type. It represents a variety of cardiac muscle, characterized by centrally located nuclei, typical mitochondria, and narrow I bands. Many I bands cannot be recognized at all. H and M bands are likewise indistinct. There is a double array of primary and secondary myofilaments. Mitochondria are large and numerous and contain many cristae. The endoplasmic reticulum consists of longitudinal tubules which run through the whole sarcomeres and bypass Z bands, and of transverse tubules which accompany Z bands. Some "triads," located at Z levels, consist of flattened vacuoles flanked by such transverse tubules. Small vesicles located at Z bands, close to the nucleus, and beneath the plasma membrane may represent still other portions of the reticulum.
Free alveolar macrophages of normal mouse lung have been studied in the electron microscope. The tissue was obtained from several young adult white mice. One other animal was instilled intranasally with diluted India ink 1(1/2) hours prior to the removal of the lung. Thin sections of the osmium-fixed, methacrylate-embedded tissue were examined either in an RCA EMU 2 electron microscope or in a Siemens and Halske Elmiskop I b. A few thick sections obtained from the same embeddings were stained for iron. The normal alveolar macrophages, which are usually in contact with the alveolar epithelium, were found to contain a variety of inclusion bodies, along with the usual cytoplasmic components like mitochondria, endoplasmic reticulum, and Palade granules. Another typical component of the cytoplasm of these cells which appears as small ( approximately 6 mmicro) very dense granules of composite fine structure is interpreted as ferritin. It is assumed that this ferritin is formed from red blood cells ingested by the alveolar macrophages. The macrophages in the alveoli were found to phagocytize intranasally instilled India ink particles. Such cells, with engulfed India ink particles, were often of more rounded form and the particles were frequently seen lying inside membrane-bound vacuoles or vesicles of the cytoplasm. The membrane of a few vesicles containing India ink particles was seen as the invaginated portion of the cell plasma membrane, and in one instance these same vesicles were seemingly interconnected with a rough surfaced cisterna of the endoplasmic reticulum. The process of phagocytosis is recognized as related to the "normal" process of pinocytosis.
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The fine structure of the alveolar basement membrane of mouse lung was discussed on the basis of three electron micrographs. The basement membrane, i.e., the intercellular layer between endothelium and alveolar epithelium, was found to be of variable width. In its thin parts it appeared rather homogeneous, and did not reveal well defined layers of fibrils. In its thicker portions, some of which may be due to oblique sectioning, cell fragments could be seen lying inside the basement membrane layer. Their exact nature was not determined. In other thickened portions of the membrane bundles of slender (about 23 to 25 mmicro) fibrils were found and were tentatively interpreted as collagen fibrils, in spite of the fact that a periodicity could not be observed.
The general architecture of capillary and alveolar walls of the mouse lung was studied by means of the electron microscope. In order to minimize tissue damage and to improve the cutting properties of embeddings, several modifications in the tissue processing methods were adopted. These modifications were: fixation by infusion, a prolonged time of dehydration, of impregnation, and of polymerization, the use of acetone for dehydration, ammonium sulfide treatment of the fixed and washed tissue, and an elevated (80 degrees C.) polymerization temperature combined with the use of prepolymerized methacrylate. The generally favorable effects of these modified methods upon preservation and cutting properties of embedded tissue are discussed. Both capillary endothelium and alveolar epithelium were found continuous and without pores. The endothelium was seen to be thinnest in those portions that were adjacent to alveolar air spaces. Two morphological "types" of alveolar epithelial cells were found. One protruded into the alveolar lumen with its thick portion containing the nucleus. The other was often located in a niche of the alveolar wall, and contained peculiar dark inclusions amidst numerous mitochondria. Both were attenuated at their periphery to form the thin epithelial layer. The layer between endothelium and epithelium was designated as basement membrane. It was seen to be generally thin and structureless, but was found thickened in some areas where it also contained collagen fibrils.
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