Microdissection by ultrasonication: application to early chick embryos.

A technique utilizing microdissection by ultrasonication was applied to scanning electron microscopy of chick embryos during the first three days of incubation. Using a tank cleaner operating at 80 kHz, whole embryos immersed in pure acetone were sonicated until fragmentation became evident. At 12 hr incubation disintegration occurred by one second or less. At 18 hr, three sonic bursts of one second each produced only partial fragmentation. All three germ layers retained their original relationships to each other. During the second day of incubation, large pieces of integument were removed and somites began to microdissect after 10-20 seconds of sonication. Late in the third day of incubation, sonication for 1 min or more was required to produce significant microdissection. Living embryos exposed to 0.1% collagenase for 10 min prior to standard fixation fragmented in a different manner. Lamellipodia and filopodia were most sensitive and were largely destroyed. The three major germ layers (ectoderm, endoderm, mesoderm), however, retained their structural integrity and original relationships to each other. Factors contributing to the results reported here include: 1) extracellular fibrils of varying chemical composition, 2) primitive cell junctions, 3) biomechanical stability in the nonfibrillar portions of the extracellular matrix, and 4) effects of technical procedures performed prior to sonication. Sonicated tissues of early embryos reveal features that are difficult to demonstrate in other ways and may be unrecognized in conventional preparations.

Scanning electron microscopy of developing primary endoderm during first 6 hours of incubation in the chick.

Development of primary endoderm in the domestic fowl (Gallus domesticus) is described in scanning electron microscopy (SEM) supplemented by transmission electron microscopy (TEM). Although complicated by great variability, the ventral surface of the blastoderm reveals this process during the first 6 hours of incubation. Primary endoderm arises (1) from the hypoblast, (2) from the margin of the area pellucida, and (3) from intervening portions of the area pellucida. The early hypoblast becomes several cells thick while individual cells are still spherical. TEM reveals a variety of immature cell junctions. During subsequent flattening of these cells into primary endodermal epithelium, numerous filopodia arise from their surfaces. These are 0.20-0.25 microns in diameter. They become long and branched, attaching to each other and to other cell bodies. Similar filopodial processes are present less conspicuously among cells in the margin of the area pellucida. Here, there is pseudopodial evidence that cells or cell sheets creep along the ventral surface of the epiblast. The filopodia disappear as cell flattening proceeds. The ventral surface of the exposed epiblast delaminates cells that become free after their exploratory filopodia and lamellipodia are put forth. Lateral contacts among cell bodies from the above three sources increase until a continuous epithelium is formed. The primary endoderm of the embryo, a simple squamous epithelium that separates the connective tissue space above from the gastrocoele below, is generated by these developmental events.

Porosity of the basement membrane overlying Peyer's patches in rats and monkeys.

The porosity of the epithelial basement membrane (basal lamina) overlying lymphoid follicles within Peyer's patches was studied in rats and monkeys by scanning electron microscopy. Basement membranes of lymphoid follicles are markedly porous, more conspicuously so than those of adjacent villus cores. The porosity increases centrifugally from the apex of the follicle to its periphery, where the basement membrane continues into the cul-de-sacs of the crypts. Such porosity may facilitate bidirectional passage of lymphocytes during an immune response. The unique structure of the basement membrane overlying lymphoid follicles suggests a biologic adaptation of this tissue boundary to a specific physiologic activity of the organism.

Scanning electron microscopy of Thebesian ostia (microdissection by ultrasonication: enzymatic digestion).

Thebesian vasculature provides for communication between the coronary system and the chambers of the heart. Anatomic, embryologic, physiologic, and therapeutic investigations have involved this component of cardiac anatomy from the early 18th century to the present time. The scanning electron microscope (SEM) now affords an innovative approach to the study of the ostia of these veins as they open into the chambers of the heart. The surface of the intact endocardium is continuous, whether it is treated with boric acid or not, as long as it remains intact. Enzymatic microdissection of tissues with trypsin, hyaluronidase and pronase, followed by similar treatment with boric acid, reveals continuity of successive component layers of the endocardium extending into Thebesian substructure. Thebesian tributaries are easily visualized from the ostia but the deeper capillary network of the Thebesian system is not demonstrable by this approach. Valvular structures such as might prevent retroflow during the cardiac cycle are not present. Our observations with SEM support anatomic relationships indicated by previously published work.

Scanning electron microscopy of the subarachnoid space in the dog: inflammatory response after injection of defibrinated chicken erythrocytes.

The leptomeningeal reaction and the cerebrospinal fluid reaction of the canine inflammatory response were investigated concurrently. One-half milliliter cerebrospinal fluid (CSF) was withdrawn from the cisterna magna of 17 anesthetized mongrel dogs and analyzed. Using this same spinal tap, control and experimental animals were injected with 0.5 ml sterile saline and 0.5 ml defibrinated chicken erythrocytes, respectively. A second spinal tap was performed 2 to 168 hr later. The CSF from the first spinal tap contained less than 1 WBC/mm3. The cell population was unchanged in the second spinal tap of control animals. In experimental animals, the WBC population increased more than 100-fold by 24 hr. Polymorphonuclear cells (PMNs) appeared in the CSF first, followed by lymphocytes and monocytes. Injected erythrocytes seemed trapped in the subarachnoid space (SAS), especially in the inner sheet of the arachnoid mater. The leptomeninges had a substantial increase in free cells without fibrosis. Pial and leptomeningeal cells of the arachnoid trabeculae appeared swollen. Two hours after injection, chicken erythrocytes were phagocytosed by pial cells, macrophages, and free cells adherent to the leptomeninges. The epiplexus cell populations for saline-control and erythrocyte-experimental animals were similar, suggesting that the choroid plexuses were not a gateway for PMN, lymphocyte, or monocyte infusion into the SAS.

Ultrasonic microdissection of rat cerebellum for scanning electron microscopy.

The cerebelli of rats were initially fixed with aldehydes (modified Karnovsky's fixative; 503 mOsM/L) by cardiac perfusion. Blocks of tissue were razor-cut, usually longitudinal to folia, and immersed in the same fluid for 2-4 hours. Three separate methods of treatment followed: (1) immersion in 1% aqueous boric acid, or (2) in 2% phosphate buffered OsO4 followed by boric acid or (3) in an 8/2 mixture of boric acid and OsO4. After 18-48 hours immersion the blocks were dehydrated in ascending grades of acetone. They were then exposed to ultrasound in 100% acetone at frequencies of 80 kHz or 40 kHz for 10 to 20 minutes. Microdissection of cut surfaces (erosion) occurs after all three treatments. It is least extensive after boric acid, moderate after OsO4 and greatest after the combined mixture. All cerebellar cell types are recognizable as are numerous fibers according to morphology and position. Variable erosion accommodates analysis of different levels of neural organization. In general, structural situations not involving great depth of field are best revealed by H3BO3 or OsO4. Blood vascular relationships to other structures are best demonstrated in deeply eroded specimens.

Microdissection by ultrasonication: porosity of the intestinal epithelial basal lamina.

The porosity of the epithelial basal lamina of normal rat intestine was studied by SEM. Epithelial removal was accomplished by prolonged fixation of tissue samples in OsO4 or immersion in aqueous H3BO3, followed by dehydration in acetone and microdissection by ultrasonic vibration. The underlying basal lamina of intestinal epithelium reveals numerous pores of variable size. These pores are more numerous in small than in large intestine and penetrate the entire thickness of the basal lamina. Within the basal lamina overlying lymph nodules, they are numerically increased. Their occurrence is evident in fixed and unfixed, sonicated and unsonicated tissue samples. Microprojections of epithelial cytoplasm are often observed within these pores. The results of this study suggest that migrating cells or epithelial-cell processes induce pore formation in epithelial basal laminae and that these pores may be eventually repaired.

Microdissection by ultrasonication: scanning electron microscopy of the epithelial basal lamina of the alimentary canal in the rat.

The epithelial basal lamina of the various parts of the alimentary canal of the rat was exposed by removal of the overlying epithelium. This was achieved by prolonged fixation in OsO4 or immersion in aqueous boric acid or both, followed by dehydration in acetone and exposure to ultrasonic vibration. The surface of the esophageal basal lamina is undulating with smooth hills and valleys, the smallest of which model the basal surfaces of the germinal cells of the epithelium. The stomach presents a perforated appearance because of ostia formed by evaginations of the basal lamina to enclose glands. In the small intestine, clavate rather than cylindrical villous cores are separated by ostia of intestinal crypts. In the large intestine, ostia are separated by broad areas of basal lamina in the cecum but are close together in the colon. The complex contours of the basal lamina are largely determined by the basal surface of the overlying epithelium but may be affected by structures in the underlying interstitium. Subepithelial lymph nodes, for example, are covered by a conspicuously porous basal lamina. Each nodule may be surrounded by ostia of as many as 20 crypts of Lieberkühn. The basal lamina of the ileocecal valve displays gradual transition from ileum to cecum.

The relationship of proteoglycans to developing transalveolar fibres in the alveolar bone of the mouse.

Proteoglycans are of interest because of their complex role in the development and maintenance of connective tissues. The present study demonstrates changes in the distribution of proteoglycans in the developing alveolar bone of the mouse using techniques of light and high-voltage electron microscopy. In early development proteoglycans are uniformly distributed throughout the interdental septum. By day 25 there is a loss of proteoglycans, probably as a result of mineralization and the maturation of the collagen of transalveolar fibres. By day 45 proteoglycans are distributed only in tissues adjacent to transalveolar fibres. These proteoglycans occupy the channels surrounding transalveolar fibres and the adjacent bone matrix. It is proposed by the authors that these proteoglycans are involved in the maintenance of the collagen of the transalveolar fibres. Because of the absence of fibroblasts adjacent to transalveolar fibres, proteoglycans are thought to be essential carriers of tropocollagen to these large fibres.

Transalveolar fibers of the mouse periodontium: a scanning electron microscopic study.

Transalveolar fibers have been described by many authors using techniques of light microscopy. However, a study of these fibers using techniques of scanning electron microscopy has not been reported. In the present study, scanning electron microscopic techniques revealed that transalveolar fibers were composed of a bundle of smaller fibers. This fiber bundle passed through channels in bone and was attached to the bone by anchoring fibers. Transalveolar fibers branched within bone. Near points of branching, channels were often dilated. Channels were continuous with the connective tissue space of the periodontal ligament at the bone margin. The results presented herein suggested a revised concept for the anchorage of fibers of the periodontal ligament to bone.

Epiphyseal plate development in the zinc-deficient rat.

The effect of zinc deficiency on shearing strength, histological changes and proline utilization of the epiphyseal plate of the tibia of the weanling male rat was studied. A diet was fed based on sprayed egg white and containing less than 2 mg of zinc per kilogram. Over 27 day, the force required to displace the epiphysis of the zinc-deficient (ZD) rats was always less than that required for pair-fed (PF) controls. After 18 days, approximately 15% more force was required to displace the epiphysis of the PF rats than was required in the ZD rats. The thickness of the outside compact bone next to the epiphyseal plate region as determined by scanning electron microscopy was thicker in the ZD rats than in the PF controls of comparable age. The epiphyseal plates narrowed as the rats aged, and were clearly discernible in PF controls but not in ZD rats. The incorporation of L-[U-14C]proline into the epiphysis was significantly less in rats deprived of zinc for 16-22 days than in PF controls.

Development of intraosseous fibers of the mouse periodontium: a high-voltage electron microscopic study.

Development of intraosseous fibers was studied in mandibles of Swiss white mice, age 17 days to 45 days. Light microscopic (LM) and high-voltage electron microscopic (HVEM) techniques were used. In LM, Wilder's reticular stain revealed intraosseous fibers throughout the interdental septum by day 17. fibers were composed of unit collagen fibrils with abundant interfibrillar matrix. As development proceeded, fibers exhibited less interfibrillar matrix. Channels surrounding the intraosseous fibers became evident. Anchoring fibers attached the intraosseous fiber to bone. Fibroblasts appeared to be absent, suggesting that maintenance of the intraosseous fiber might be the function of the osteocyte. The presence of intraosseous fibers suggested a reevaluation of the fibrous attachment of teeth to bone. The concept of intraosseous fibers may simplify concepts of tooth movement and approximal drift.

The central nervous system in scanning electron microscopy.

The first decade of scanning electron microscopy of the central nervous system has emphasized study of the ventricular system, especially the circumventricular organs. These are largely non-ciliated areas located in close vicinity to the third and fourth ventricles. Supraependymal cells (neurons and phagocytes) are common here along with tanycytes. Occasionally small blood vessels are exposed. Neurosecretory function is established for some of these organs and suspected for most of them. The subarachnoid space is clearly lined by flat surfaced connective tissues with fenestrations. Free cells which are known to be macrophages are common on the natural surfaces. These tissues react to antigens in the manner of connective tissues elsewhere in the body. The "non-surface" areas of the central nervous system have been examined by various techniques utilizing fracturing, isolation and ultrasonication but an established methodology has not yet emerged.

Intercellular junctions in the developing arachnoid membrane in the chick.

The arachnoid membrane of chick embryos was prepared for electron microscopic study by means of thin sections and freeze-fracture replicas. Particular attention was given to the relationships among junctional complexes during arachnoid maturation. By 14 days of incubation, the arachnoid had differentiated into morphologically distinct inner and outer zones. Both desmosomes and gap junctions were present among the cells of both layers at this time. Desmosomes were most numerous in the inner arachnoid layer and their structure remained constant. Gap junctions showed a great variation in structure. The large gap junctions contained a particle packing pattern in which rows of intramembranous particles were separated by particle-free zones. Arched gap junctions were also present. Smaller arrays of gap junctions exhibited a variety of configurations on the membrane P-face. The first tight-junctional stands clearly identifiable in freeze-fractured preparations appeared at 15-17 days. These were closely associated with the particles of gap junctions and consisted of single stands on the P-face. By hatching age (21 days) a "mature" pattern of tight-junctional strands was interwoven in several layers. In the interim, more complex arrangements of tight-junctional strands were in intimate relation with gap junctions.

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.

Development of transalveolar fibres in alveolar bone of the mouse.

Transalveolar fibre development in the mandibles of albino mice aged 12-32 days postnatal was studied. Silver impregnation techniques revealed transalveolar fibres in the alveolar crest bone at day 14 and throughout the interdental septum at day 17. Collagen strains revealed transalveolar fibres in alveolar crest bone at day 19 and throughout the interdental bone by day 25. Changes in orientation occurred in transalveolar fibres of the alveolar crest at day 19 and in oblique transalveolar fibres at day 25. These changes were coincident with the eruption of the teeth and the development of functional occlusion.

Scanning electron microscopy of the subarachnoid space in the dog: evidence for a non-hematogenous origin of subarachnoid macrophages.

Injection of viable BCG into the subarachnoid space of immunized and non-immunized dogs produced a 10-fold increase in the populations of pial free cells. In immunized animals injected three days previously with BCG, stereoscopic SEM revealed that many pial cells had rounded up and were protruding into the subarachnoid space. With continued rounding these cells took on amoeboid characteristics, with shapes that suggested a capacity for cell movement. Internally, these pial cells possessed an increased volume of perinuclear cytoplasm and organelles. Reactive pial cells could be distinguished from macrophages of presumed hematogenous origin on the basis of their surface morphology. These findings suggested that pial cells had the ability to alter their normal structural and behavioral characteristics and to become macrophage-like under these conditions of secondary challenge by BCG.