Scanning electron microscopy of the subarachnoid space in the dog. III. Cranial levels.

Young dogs were anesthetized by intrathoracic injection of sodium pentobarbital and perfused with buffered aldehydes. Lining tissue samples from the cranial subarachnoid space were prepared for scanning electron microscopy (SEM) by postfixing in buffered OSO4. Samples were then dehydrated, dried in a Critical Point Drying System, and coated with carbon and palladium-gold. Specimens were viewed in a Cambridge S4 scanning electron microscope. After thorough scanning, selected samples were routinely prepared for transmission electron microscopy (TEM) and viewed in a Philips EM-200 transmission electron microscope. This study depicts the surface morphology of the meningeal linings of the cranial subarachnoid space. The cranial pia mater possesses natural gaps or fenestrations between cells. SEM reveals a more complex morphology of arachnoid trabeculae than previously interpreted from light and transmission electron microscopy. Many free cells are observed on the meningeal linings of the subarachnoid space. The present study establishes that these free cells are macrophages by means of definitive TEM correlates. Microvillous-like processes extending between macrophages and the pial surface are present. The frequency and the nature of these thin processes suggest the possibility of a plasmalemma-mediated system of communication.

Scanning electron microscopy of the subarachnoid space in the dog. IV. Subarachnoid macrophages.

Young dogs of both sexes were used in this study. Transmission and scanning electron microscopy were utilized for the examination of the spinal cord and choroid plexus with emphasis on the study of free cells. These procedures were modified so that, in certain cases, the same cells observed in scanning electron microscopy could be analyzed internally by transmission electron microscopy. One half of the animals were injected under anesthesia with horseradish peroxidase for observation of phagocytosis. This study confirms that the free cells observed in the subarachnoid space with the scanning and transmission electron microscopes are identical. The internal morphology of these cells corresponds to that of macrophages. This is further substantiated by the ability of these cells to localize horseradish peroxidase in discrete vacuoles within their cytoplasms. Both pial macrophages and epiplexus cells localize peroxidase in an identical manner in the same animal after one injection. In addition macrophages on the surface of the pia mater respond to extravasated red blood cells in a characteristic manner including phagocytosis. The plentiful population of macrophages on the surface of the pia mater supports the concept that these cells are of major importance in maintaining asepsis in the subarachnoid space.

Scanning electron microscopy of the subarachnoid space in the dog. V. Macrophages challenged by bacillus Calmette-Guerin.

Mongrel dogs were anesthetized intraperitoneally with pentobarbitol. One cc of cerebrospinal fluid was drawn through a needle inserted into the cisterna magna and mixed with 1 cc (4-9 million viable BCG organisms) of freeze-dried bacillus Calmette-Guerin. One minute later this mixture was injected by the same needle into the cisterna magna. At 1 and 12 days postinjection, experimental animals were perfused with buffered aldehydes. Samples of the leptomeninges were post-fixed in OsO4 and routinely prepared for scanning and transmission electron microscopy. Leptomeningeal samples of untreated, control animals were similarly prepared. Scanning and transmission microscopy confirm that free cells resting on the subarachnoid linings and within the subpial connective tissue space of control animals possess the morphology of macrophages (Malloy and Low, '76). Viable BCG in the subarachnoid space produces a 3-fold increase in the free cell population of the leptomeninges in 24 hours and a 10-fold increase in 12 days. These cells tend to form associations varying from loose aggregates to tight clusters. Approximately 80% of these free cells express macrophage morphology, with abundant plasma-lemmal microappendages and cytoplasmic vacuoles. Transmission electron microscopy of the free cell population of BCG-stimulated animals reveals at least two other members of the leukocyte series on the leptomeningeal linings.

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.

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.

Morphological and histochemical evidence of mitoribosomes in Manduca sexta.

The mitochondria found in the neurons of the frontal ganglion of Manduca sexta contained numerous mitoribosomes. The mitochondria of the glial and perineural cells did not contain mitoribosomes. The mitoribosomes were digested in RNase whereas phospholipase C digested the cellular membranes but had no effect on the mitoribosomes.

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.

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.

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.

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: 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.

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.

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.

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 cardiac endothelium of the dog.

Tissue obtained from young dogs was fixed in buffered aldehydes by vascular perfusion or direct immersion. Selected hearts were maintained in modified mammalian Ringer's solution for three to five minutes prior to fixation. The chambers of the heart and related valves were exposed by dissection and prepared by routine techniques for observation by scanning electron microscopy (SEM). Subsequent to SEM studies, selected specimens were embedded in Epon 812 and sectioned for transmission electron microscopy (TEM). The cardiac endothelium, when fixed immediately in buffered aldehydes, presents an essentially invariable surface throughout the interior of the heart. The predominant nuclear bulges and attenuated peripheral plasmatemma are consistently smooth, with occasional marginal ruffles, scattered microvilli and small blebs. Apart from the higher population of nuclear bulges on valvular surfaces, local variations in SEM of endocardium occur in response to the various stages of systole and diastole encountered. These physiological changes do not produce microappendages. Immersion for three to five minutes in (Chenoweth's) Ringer's solution, prior to fixation, produces a substantial population of microappendages. The cellular surface acquires a swirled appearance erupting in microvilli, blebs and ruffles. These exhibit considerable pleomorphism. There is great lability of the endocardial surface in response to a classic "holding solution" widely used in preparatory techniques. In preparing soft tissues for SEM caution must be used if physiological "holding solutions" are used prior to fixation.

Mitotic cells and their microappendages in the primitive streak of the chick embryo.

Fresh pullet eggs (White Leghorn Strain) were incubated to the primitive streak stage of development. Blastoderms were fixed in situ with isotonic aldehyde fixatives and prepared for scanning electron miscropy by means of post-osmication, critical point drying and gold-palladium coating. Cells judged to be in various stages of mitosis by their surface contours were numerous on the ventral surface of the chick blastoderm. Cells which were in the late preparatory stages for mitosis had rounded up from their surroundings. Microvilli dominated the surface. The degree of separation and number of microvilli increased until late metaphase or anaphase. Mitotic cells did not completely separate themselves from adjacent cells. Ruffles and blebs were not prominent during mitotis and long filopodia were absent. A definite localization of microappendages (microvilli, blebs, ruffles) to the area of cytokinesis was evident in early telophase and persisted through daughter cell formation.