Evidence of remodeling in dura mater cardiac valves.

Subcellular changes in 12 dura mater cardiac valves (in the mitral or aortic position) surgically removed after 23 to 108 months of implantation owing to calcification, rupture, or endocarditis show signs of a remodeling process. Significant morphologic changes in the connective tissue fiber matrices and cell populations were noted in the recovered valvular leaflets. Macrophages were found within electronlucent (cleared-out) areas, and they seemed to play an essential role in the remodeling process by ingesting and digesting selected connective tissue components. Fibroblasts found within these "rebuilding" areas in the dura mater tissue possessed small cytoplasmic vesicles (65 nm in diameter) being extruded from the cell. Evidence of early collagen formation was also found in association with both peripheral filaments and peripheral condensations, as well as within the connective tissue matrices surrounding the cellular elements, where electron dense amorphous material was observed. In conclusion, the long-term durability of dura mater bioprosthetic cardiac valves may be directly related to (1) glycerin stabilization and preservation of the collagen fibers, (2) the viability of the fibroblasts and macrophages within the implanted valves, and (3) the unique morphology and fine structure of the double-layered dura mater encephali. We hypothesize that the fibroblasts or myofibroblast-like cells found within the implanted leaflets, no matter what their origin, are capable of giving form and organization to the early developing connective tissue.

Migration of intravascular trophoblast cells in uterine arteries of the golden hamster: a scanning electron microscopic study.

Intravascular trophoblast (IVT) cells, derived from the trophoblast of the developing hamster embryo, are known to migrate in retrograde fashion into the uterine arteries. There they migrate to a certain point, destroy and replace the endothelial lining, and modify the smooth muscle of the arteries. The dilated vessels that result presumably enhance the flow of blood to the placental exchange area. The morphology of IVT cells in the hamster placenta was investigated by scanning and transmission electron microscopy. Although occasional single migrating cells were observed, the IVT generally appear as sheets of large, contiguous, sometimes overlapping cells that spread over the endothelial surfaces of the uterine central terminal arteries and vascular knot arteries. This process seems to be aided by the appearance of filopodia, which make contact either with other intravascular trophoblast cells or the endothelium. After consolidation, the IVT cells act as a functional part of the vessel lining and are readily distinguished from the surrounding endothelium by their numerous microvilli. The final distribution of the IVT cells is patchy rather than uniform.

A new technique for exposure of osteocytes and transalveolar fibres of the interdental septum in the mouse for scanning electron microscopy.

The technique selectively removes the fibrous component of the interdental septum. Mandibles were fixed, demineralized and incubated at 37 degrees C in 0.1 per cent bacterial collagenase. Samples were post-fixed in 2 per cent buffered osmium tetroxide for 24 h prior to dehydration in ascending grades of acetones. While in pure acetone, tissue was ultrasonicated at 80 kHz for 5 min and conventionally processed for scanning electron microscopy. Ultrasonication of tissue without prior collagenase digestion did not expose transalveolar fibres in their entirety. After 6 h exposure to collagenase followed by ultrasonication, bone matrix and osteocytes were removed exposing transalveolar fibres which passed through the interdental septum without interruption, becoming continuous with the periodontium of the adjacent tooth.

Ultrasonic microdissection of the mouse mandible: exposure of the vasculature of alveolar bone and myelinated axons of the pulp.

The present scanning electron microscopic study describes a new method for the exposure of blood vessels of mouse alveolar bone and myelinated nerves of the dental pulp. This technique differs from others because it does not remove the periodontal ligament, allowing study of the vascular continuity between alveolar bone and periodontal ligament. Fixed and demineralized mandibles are digested with bacterial collagenase (1 mg/ml) at 37 degrees C for 12 hours, exposed to buffered osmium tetroxide for 24 hours, and ultrasonicated at 80 kHz for 5 minutes. The technique demonstrates that the vascular distribution of the interdental and interradicular septa is different. Vessels pass horizontally through the interdental septum and are continuous with vessels of the adjacent periodontal ligament. Vessels of the interradicular septum branch from a central vessel, pass toward the adjacent periodontal ligament, and become continuous with its vessels. Thus, the pattern of vessel distribution of the interdental septum of the mouse has little similarity to that of man or of research animals. The present study provides an improved method for demonstration of bone vasculature and pulpal axons while retaining valuable anatomical landmarks.

Ultrasonic microdissection of immature intermediate human placental villi as studied by scanning electron microscopy.

The human placenta during the first 20 weeks of gestation undergoes rapid and extensive morphological changes. Near the end of this period, the most predominant type of villus present is the immature intermediate placental villus. In order to visualize this complex structure with scanning electron microscopy (SEM), we have developed a microdissection technique to expose tissue components of the placental villus while retaining its normal histological architecture. Placental villi were initially fixed in Karnovsky's fixative, buffered formalin, or 2% osmium tetroxide solution prior to exposure to connective tissue enzymes or detergents alone or in combination. Samples were dehydrated through 100% acetone and ultrasonicated at 80 kHz for 15 minutes prior to critical point drying and SEM examination. The most satisfactory microdissections were obtained by using a combined detergent/ultrasonication technique. By means of this procedure it was possible to remove the syncytiotrophoblast to expose the underlying cytotrophoblast, basal lamina and the stromal core components of the villi. The selective removal of these structures revealed the 3-dimensional relationships of the stromal channels, reticulum cells and Hofbauer cells. Of interest was the pattern of fetal capillaries coursing parallel to the long axis of each villus and terminating in a vascular knot at the tip.

Microdissection by ultrasonication after prolonged OsO4 fixation: a technique for scanning electron microscopy.

A new method of tissue preparation for electron microscopy is described for the selective dissection of mammalian tissues. This technique, based upon brittlization by prolonged osmication, dehydration to pure acetone and subsequent ultrasonication, has been especially useful for the SEM visualization of microcirculation and basal laminas. Epithelial cells of treated tissues were largely fragmented and removed, whereas collagen containing materials, notably epithelial and vascular basal laminas, withstood the vibratory insult. The extent of epithelium and basal lamina removal depended upon tissue type and duration of ultrasonic treatment. Alone or in conjunction with specific enzymatic pretreatment, this new technique offers a method to better visualize known and unrecognized histological relationships.

The effects of insulin-glucose on microappendages of the plasmalemma in the early chick embryo.

Chick embryos incubated for 18-24 hours (stages 4-6) were immersed for 3-5 minutes in Hanks' Balanced Salt Solution containing either insulin (10(-9) M) or D-glucose (10(-1) M) or a mixture of both. The ventral and dorsal surfaces of the entoderm and the ventral aspect of the mesoblast were examined in a scanning electron microscope. D-glucose alone or insulin alone produced negligible change. When combined these ingredients generated highly pleomorphic microappendages of the plasmalemma. The ventral surface of the entoderm was characterized by a large number of microvilli, both long and short. Concentrations of microvilli and blebs appeared in recesses at cell margins. Some of these represented mesoblastic protrusions through intercellular apertures in the entoderm. The dorsal entodermal surface developed stellate clusters of microvilli and blebs. The ventral aspect of the mesoblast became the most pleomorphic area of all. It possessed a variety of microappendages including large populations of microvilli and blebs that resembled the protrusions observed from the subblastodermic cavity. There were also stellate clusters similar to those on the dorsal entodermal surface. The same mesoblastic area in controls was virtually free of microappendages. The mesoblastic protrusions, occurring as they do prior to an established circulation, are interpreted as a "feeding" phenomenon.

A re-examination of the osteocytic network of interdental bone.

A new method for removal of bone minerals and matrix was used in this study to expose osteocytes and osteocytic processes for examination by techniques of scanning electron microscopy. Osteocytes demonstrated more numerous Type II junctions with osteocytic processes than had previously been recognized. The network of osteocytic processes was extensive and exhibited several structural modifications for more efficient transport: rings and branching. Adjacent osteocytes were connected by osteocytic processes suggesting a functional syncytium. The study suggests that the network of osteocytes and osteocytic processes is more extensive and complex than previously recognized.

Ultrastructure of the crayfish antennal gland revealed by scanning and transmission electron microscopy combined with ultrasonic microdissection.

The antennal gland of the crayfish Pacifasticus leniusculus was studied using standard techniques for scanning electron microscopy as well as newer procedures for ultrasonic microdissection. To clarify relationships in the nephron tubule, transmission electron microscopy was employed. The coelomosac contains elongated cells (podocytes) displaying microvilli and extensive apical blebbing. A smooth basal lamina lines the blood space that furnishes hemolymph to the coelomosac. The labyrinth consists of tall columnar cells displaying apical microvilli, numerous blebs that seem to represent an expansion of apical plasma membrane, and lateral interdigitations. The nephron tubule consists of two distinctly different areas: a proximal region of flattened cells with extensive intercellular fusions, and a distal segment of separate, dome-shaped cells. Despite many similarities between the crayfish kidney and the vertebrate nephron, there are striking differences. The amount of surface blebbing that occurs in the coelomosac and labyrinth far exceeds that of the vertebrate nephron and may reflect its importance in the function of the crayfish kidney. The cells of the coelomosac are taller than are the vertebrate podocytes and possess less obvious arms and pedicels. In addition, the proximal segment of the nephron tubule is notable for its intercellular fusions, which are not present in the vertebrate nephron. Although the function of the intercellular fusions is unknown, they may play a role in cellular communication or the redistribution of fluids or electrolytes between cells.

Strain variation in phagocytosis of Cryptococcus neoformans: dissociation of susceptibility to phagocytosis from activation and binding of opsonic fragments of C3.

Phagocytosis of Cryptococcus neoformans is markedly influenced by the presence of a polysaccharide capsule. We examined activation and binding of C3 fragments to eight isolates of C. neoformans. All isolates were shown to have capsules by light and electron microscopy. These strains differed in susceptibility to phagocytosis by neutrophils. Yeast cells were opsonized by incubation in normal human serum. Five strains were resistant to ingestion, two strains showed intermediate levels of resistance to ingestion, and one strain was quite sensitive to phagocytosis. Yeast cells opsonized with heat-inactivated serum (56 degrees C for 30 min) neither attached nor were ingested by neutrophils. A quantitative estimate of the amount of C3 bound to the yeast cells was determined by use of normal human serum containing 125I-labeled C3. The results showed approximately 5 X 10(6) to 10 X 10(6) C3 molecules per yeast cell regardless of whether the yeast cells were sensitive or resistant to phagocytosis. Bound C3 was eluted from the yeast cells by treatment with 0.1 M NH2OH (pH 10), and the eluted fragments were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Results of this analysis showed that little of the C3 was in the form of C3b, and there was substantial decay to iC3b, the inactive decay product of C3b. This pattern of decay was similar with all strains. Immunoelectron microscopy was used to assess the ultrastructural location of the C3 fragments bound to the yeast cells. C3 fragments were bound to the perimeter of the capsule regardless of whether the isolate was sensitive or resistant to phagocytosis. Thus, phagocytosis-sensitive and phagocytosis-resistant isolates were similar with regard to the amount, molecular form, and ultrastructural location of C3 fragments bound to the cryptococcal capsule. These results further indicate that activation of the complement cascade is necessary but not sufficient for phagocytosis of the yeast cell.

Role of the capsule in phagocytosis of Cryptococcus neoformans.

The capsule is closely associated with the virulence of Cryptococcus neoformans. The capsule inhibits phagocytosis by macrophages, monocytes, and neutrophils. Studies in our laboratory have shown that incubation of encapsulated cryptococci in normal human serum leads to deposition of large amounts of C3 fragments at the surface of the yeast and lesser amounts of IgG within the capsule. Thus, the capsule mediates two biologic activities with opposing effects. It is our current view that phagocytosis of the yeast is dependent on a balance between the antiphagocytic action of cryptococcal polysaccharide and the ability of the yeast to focus opsonically active complement fragments and the IgG at the capsular surface.