Secretory protein synthesis in the stimulated rat parotid gland. Temporal dissociation of the maximal response from secretion.

Administration of the beta-adrenergic drug, isoproterenol (IPR), affects the release of 98% of stored amylase from rat parotid gland acinar cells. A period of 6 h elapses from the onset of secretion to the maximum [(14)C]phenylalanine (Phe) incorporation into total protein and amylase. 10 h after IPR administration the rate of [(14)C]Phe incorporation into total protein was no longer elevated above that of control. Incorporation into amylase, however, remained elevated above the control by 2.3 times. This latent period may reflect: (a) reduced amounts of available ATP which occurs as a result of the process of secretion as well as (b) the time required for reorganization of cellular organelles and membranes after secretion. The latent period after IPR-induced secretion appears similar to the latent period which has recently been reported to occur after physiologic release of amylase from the parotid gland during the diurnal feeding cycle of the rat. These observations support the existence of a positive feedback system operant in the parotid acinar cell linking the release of secretory proteins with their synthesis. The period of greatest protein synthesis is, however, temporally dissociated from the secretory process.

The behavior of subcultivated stratified squamous epithelial cells on reconstituted extracellular matrices: initial interactions.

Stratified squamous epithelial cells derived from the ventral surface of the rat tongue interact with matrices composed of hydrated dermal collagens (types I and III) and with a bovine basement membrane in distinctly different ways. When compared with the behavior of the cells on the basement membrane, the attachment and subsequent migration of the cells on the collagens were inefficient and slow. The resultant epithelial colonies did, however, stratify and differentiate more rapidly than those formed on the membrane. The fibrillar meshwork of the type III collagen gel provided better initial support for the cells than did the gel of type I collagen in which the fibrils were arranged as coarse aggregates. The physical characteristics of the type III gel also allowed the cells to condense the surface resulting in even better epithelial support. The basement membrane encouraged rapid epithelial coverage of large areas by promoting cell attachment and migration. This important property indicates that epithelial discontinuities can be rapidly repaired, providing the cells can migrate along a preexistent basement membrane. The use of such defined extracellular matrices in culture can provide important insight into the function and structural organization of subepithelial connective tissues and basement membranes.

Growth of stratified squamous epithelium on reconstituted extracellular matrices: long-term culture.

Oral keratinocytes grown at an air-liquid interface on stabilized matrices of collagen or a basement membrane exhibit a pattern of tissue organization more similar to the parent tissue than the same cells cultured conventionally. An orderly sequence of cell migration and differentiation is maintained, and the full complement of terminally differentiated cells is retained on the surface of the culture for up to 65 days following subculture. The pattern of histodifferentiation of cultured stratified squamous epithelium differs according to the matrix upon which it is grown. Pliant, fine meshed gels of type III collagen are corrugated by the cultured keratinocytes with adjustments occurring in the various suprabasal cell strata that result in the retention of a flat stratum corneum. Such pliant gels can be stabilized by pouring a supporting underlayer of coarse type I guinea pig collagen. Keratinocytes grown directly on the irregular surface of guinea pig type I collagen migrate into spaces between collagen fibrillar bundles and aberrantly keratinize 20-30 days following subculture. Keratinocytes grown on a basement membrane do not aberrantly keratinize, suggesting that contact with a basement membrane may suppress signals for keratinocyte differentiation. Keratinocytes also form hemidesmosomes opposite a basement membrane but not opposite collagen fibrils. The keratin pattern of oral keratinocytes cultured in different configurations does not change; a finding that indicates that a greater degree of tissue organization does not automatically result in the synthesis of keratins more characteristic of upper cell strata or cornified cells in the native tissue.

The growth and structure of human oral keratinocytes in culture.

Human keratinocytes derived from explants of cheek (buccal) mucosa grow vigorously in culture and can be subcultivated twice. The structure of the oral keratinocytes in vitro is the same in primary cultures and subcultures. The cells stratify, are characterized by well-developed tonofibrillar-desmosomal complexes, and rarely exhibit signs of terminal differentiation. Unique features of the culture system that favor keratinocyte growth are: incubation at 34 degrees C, inclusion of 0.5% dimethyl sulfoxide in the culture medium, and initiating subcultures as 5.0 mm colonies containing 100,000/20 microliter of medium. One primary culture can yield 6 first-passage subcultures, which subsequently achieve confluence in 10-12 days. Such cultures are a useful source of human keratinocytes that stratify but generally do not undergo terminal differentiation.

Changes in aqueous immunoglobulin and albumin levels following penetrating keratoplasty.

The feline model of induced rejection of corneal allografts was employed to define the changes in the concentrations of immunoglobulins and albumin in the anterior chamber prior to, and concomitant with the rejection of the transplanted cornea. Fourteen animals received unilateral exchange corneal allografts. Aqueous humor obtained by anterior chamber paracentesis at regular intervals prior to and following the performance of the penetrating keratoplasties was analyzed for IgG, IgM and albumin concentrations using the micro enzyme-linked immunosorbant assay (ELISA). Two patterns of anterior chamber protein modulation were observed. Eight of the animals demonstrated a biphasic pattern in which both immunoglobulin and albumin concentrations were elevated two- to five-fold above presurgical values 14 days postkeratoplasty, returning to preoperative values by day 42. Three to 5 weeks after corneal rejection was induced increases in protein concentrations were observed that correlated with the appearance of clinical signs of rejection. A second, monophasic pattern of anterior chamber protein modulation following keratoplasty was observed in four of the animals. It was distinguishable from the biphasic pattern in that levels did not return to baseline values after the initial rise following keratoplasty until the rejection process was completed. The monophasic response was found to be characteristic of more rapid and vigorous corneal rejection. Examination of albumin to immunoglobulin ratios suggested that all changes in protein levels following keratoplasty were a result of increased influx of serum proteins into the anterior chamber, rather than due to local immunoglobulin synthesis.

Complications associated with bovine corneal endothelial cell-lined homografts in the cat.

Cultured bovine corneal endothelial cells were subcultured onto feline corneas from which the native endothelium had been mechanically removed, and transplanted into cats via penetrating keratoplasty. Although the transplants remained thin and clear in the immediate postoperative period, correlative clinical and morphologic analysis disclosed evidence of a host response directed against the heterologous endothelium by the ninth postoperative day. Eyes with rotational autografts or transplanted homografts did not disclose evidence of a similar host response.

Evidence for corneal endothelial cell hypertrophy during postnatal growth of the cat cornea.

Endothelial cell counts made from specular micrographs of 1-month-old kitten and adult cat corneas demonstrate that a progressive increase in endothelial cell size and a reduced endothelial cell density occurs during the postnatal development of the cat cornea. In addition to confirming the difference in cell size, scanning electron micrographs show that kitten endothelial cells are much more pleomorphic than those of the adult. When the number of corneal endothelial cells/mm2 and the size of the whole cornea are calculated for the kitten and adult, hypertrophy rather than mitosis appears to be the principal mechanism responsible for maintaining a confluent endothelial cell monolayer during the postnatal development of the feline cornea. Hypertrophy also appears to play a role in establishing the adult corneal endothelial cell population of the rabbit when the previously published data of others are treated in a similar manner to those of the kitten and adult cat. Thus, endothelial cell hypertrophy plays a role in establishing an "adult" endothelial cell monolayer in species that have a widely divergent corneal endothelial cell mitotic capacity.

Effect of 1% sodium hyaluronate (Healon) on a nonregenerating (feline) corneal endothelium.

A series of experiments were performed to investigate the effect of 1% sodium hyaluronate (Healon) on the nonregenerating corneal endothelium of the cat. Aqueous humor replacement with 1% sodium hyaluronate resulted in mild, transient elevations of intraocular pressure compared to eyes that were injected with balanced salt solution. Sodium hyaluronate 1% protected the feline endothelium against cell loss incurred by contact with hyaluronate-coated intraocular lenses compared to endothelial contact with lenses that were not coated with sodium hyaluronate. The use of intraoperative 1% sodium hyaluronate, however, did not protect against endothelial cell loss incurred by penetrating keratoplasty or prevent subsequent skin graft-induced corneal homograft rejections. Homograft rejections were milder, however, in some eyes that received grafts coated with 1% sodium hyaluronate. Image analysis of photographs of trypan blue- and alizarin red-stained corneal buttons after trephining, stretching of Descemet's membrane, rubbing against iris-lens preparations, or immediately after penetrating keratoplasty demonstrated that the stretching of the posterior cornea is an important cause of endothelial damage that would not be protected against by a viscoelastic coating.

Postnatal development of corneal endothelium.

Comparison specular micrographs of infant and adult corneas from cats, cows, dogs, rabbits, and humans demonstrate that a large decrease in central endothelial cell density occurs during maturation of the cornea. Central endothelial cell counts of developing cat, dog, and rabbit corneas decrease rapidly during the first months of life. This rapid decline in endothelial cell density correlates with growth of the cornea to the adult size. Central endothelial cell counts of adult cat, cow, deer, dog, pig, rabbit, and human corneas are similar (2500 cells/mm2) despite a wide variation in corneal size. Comparison of observed endothelial cell counts with two hypothetical situations, one of unrestricted endothelial mitosis and the other of only endothelial hypertrophy, indicates that hypertrophy of individual cells is primarily responsible for achieving the adult cell density of 2500 cells/mm2 for these species. This observation is true for species that have a high adult endothelial mitotic capacity (rabbit) as well as those that do not (cat). The human cornea is a special case because the decline in central endothelial cell density indicates that a large apparent corneal endothelial cell loss (approximately 45%) occurs early in postnatal development.

Magnetic resonance imaging of the normal craniovertebral junction.

Sagittal magnetic resonance images of the normal craniovertebral junction in 25 patients were examined for visualization of bony, synovial, and ligamentous structures. The excellent delineation of soft tissue by magnetic resonance imaging enabled recognition of the joint space between the dens and anterior arch of C1 in 14 out of 25 patients. High-signal-intensity tissue was noted immediately superior to the dens in all patients; an anatomic specimen confirmed the fibrofatty nature of this tissue. The medullary space of the dens had lower signal intensity than did the marrow in the body of C2 in more than one half of the cases. Additional thin-section images suggested that this was a partial-volume artifact. Understanding of the normal appearances of structures in this region is necessary to assess correctly the presence or absence of disease.

Ultrastructure of cultured canine oral keratinocytes.

Keratinocytes from explants of the oral mucosa of dogs were grown in culture for five passages. The ultrastructure of primary cultures and fully developed subcultures passaged 1, 3, and 5 times was examined. At every stage, the cells had the morphologic characteristics of epithelial cells and formed a multilayered squamous epithelium. The basal cells had the characteristics of metabolically active cells, whereas the suprabasal cells and the cells at the media interface expressed many, but not all, of the organelles and cell surface characteristics associated with keratinocyte differentiation. Keratohyalin granules were located in the suprabasal and superficial cells. Cell size and shape and the relationship between cells in the layers also reflected the morphologic characteristics of the parent tissue. Cells maintained this typical structure through all passages and the cultures changed minimally for up to a week after development.

Evidence for autoregulation of cell division and cell transit in keratinocytes grown on collagen at an air-liquid interface.

Oral and epidermal rat keratinocytes when cultured on a matrix of type I collagen fibrils at the interface between the gaseous and liquid phases of a culture form a highly ordered stratified squamous epithelium. Autoradiographic studies of cells labeled by tritiated thymidine indicate that the keratinocytes are capable of autoregulating cell division. Early confluent cultures exhibit 51% of basal cells labeled, a percentage that decreases to 18% when a full differentiated stratified squamous epithelium is formed. Such a decrease in labeling occurs in cultures where the mitotically active basal cells have unimpeded access to culture medium supplied from below and when no cell type other than the keratinocyte is present in the culture. Additionally, the transit of keratinocytes from the basal cell layer through other viable cell strata to the layer of terminally differentiated cells can be followed by tracking cells labeled with tritiated thymidine. In cultures of oral keratinocytes, cells move from the basal cell layer to the cornified layer at a maximum rate of 7 days, while virtually all labeled cells (91%) are localized in the terminally differentiated cell layer 14 days following labeling. Keratinocyte cultures grown in culture at an air-liquid interface exhibit tissue organization that closely resembles the native, parent tissue. Such cultures can be useful in studying the effects of pharmacologic mediators of cell division and cell transit.

Gradient-echo MR imaging of the temporomandibular joint: diagnostic pitfall caused by the superficial temporal artery.

OBJECTIVE: In routine MR imaging of the temporomandibular joints, a low-signal structure posterosuperior to the mandibular condyle is occasionally seen on the two-dimensional gradient-echo sequence. The structure is ovoid and may have a higher-signal core, simulating a loose body within the joint. We undertook a clinical and cadaveric study to determine the cause of this finding. MATERIALS AND METHODS: In a clinical study, we reviewed the MR images of 100 temporomandibular joints. We scored each joint for the presence and appearance of a low-signal structure posterosuperior to the mandibular condyle: type 1 was ovoid with a low-signal rim and a higher-signal core, type 2 was ovoid with uniformly low signal, type 3 was a low-signal structure inseparable from the posterior wall of the glenoid fossa, and type 4 was normal. Using this scoring system, we determined the appearance and frequency of the finding on two-dimensional gradient-echo, T1-, T2-, and proton density-weighted images. To determine the cause of the finding, we correlated the imaging and anatomic findings in a cadaveric specimen. RESULTS: Of the 100 MR images of joints reviewed, 22 showed a type 1 structure, 24 a type 2 structure, 11 a type 3 structure, and 43 were normal. The finding was seen only on the two-dimensional gradient-echo sequences, never on the spin-echo sequences. Correlation between the imaging and anatomic findings in the cadaveric specimen showed that the finding was caused by the superficial temporal artery. The variability in its appearance is thought to result from the complex manifestations of flowing blood within this artery. CONCLUSION: The finding posterosuperior to the mandibular condyle seen on two-dimensional gradient-echo MR images is a flow phenomenon within the superficial temporal artery. Recognition of the nature of this finding will avoid mistaking it for disease, such as an intraarticular loose body.

Anatomical and electromyographic studies of the digastric muscle.

The techniques and sites for EMG recordings from the digastric muscles are controversial. To re-evaluate old techniques for recording from the digastric muscles, especially the posterior bellies, the morphology of the muscles was studied by conventional dissections and by examination of specimens sectioned in the frontal and the horizontal planes. Based on these anatomical findings, recording sites and approaches to them were developed for the anterior and posterior bellies of the digastric muscles. EMG recordings from the two bellies of the muscle were obtained from five healthy subjects. The EMG recordings were ranked according to muscle activity level and the activity within single muscles and between muscles compared using the Wilcoxon signed rank test. The anterior and posterior bellies had synchronized activity in all mandibular movements but were silent or had negligible activity with the mandible in the rest position, when the head was rotated, and while clenching. Both bellies had marked to very marked activity during jaw opening, and moderate to marked activity during protrusion, retrusion and lateral movements. During swallowing the anterior and posterior bellies had patterns characterized by bursts of activity of high amplitude and short duration. The two bellies were not, however, always synchronously active.

Anatomical and electromyographic studies of the lateral pterygoid muscle.

The relationships of the lateral pterygoid muscle within the infratemporal fossa were observed by conventional dissections and by examination of specimens sectioned in the horizontal and frontal planes. The following less well-known features were noted. At the origins of the superior and inferior heads there are regions in which the fibres are interlaced or closely overlapped by fibres of either the temporalis muscle or the medial pterygoid muscle. Fibres of the superior head insert not only into the meniscus of the temporomandibular joint, but also into the pterygoid fovea at the neck of the mandibular condyle. Specimens sectioned through the origin of the inferior head of the muscle show internal tendon lamellae consistent with a pennate structure. Electromyographic (EMG) activity was recorded in five healthy subjects using concentric needle and fine-wire electrodes. Strong to very strong activity was consistently observed in the superior head during clenching and tooth gnashing. The inferior heads were silent or had negligible to slight activity most of the time during ipsilateral movements or clenching, but were co-activated bilaterally, with strong to very strong activity during jaw opening, protrusion, swallowing, tooth gnashing and during passive retrusion. They showed marked activity unilaterally during contralateral movements.

Condylar cartilage: a scanning electron microscope study of anorganic mammalian condyles.

The mandibular condyles of four mammalian species (sheep, cat, monkey, and human) were rendered anorganic in NaOCl and examined by SEM. A mineralized cartilage front was identified in all specimens on the basis of a comparable morphology of chondrocyte lacunae and a calcospheritic pattern of mineralization. Species-specific differences in the cartilage surfaces were found to exist. The role of this cartilage in condylar remodeling and pathology is discussed. Attention is drawn to the fact that mineralized cartilage was identified on the articular aspect of all adult mammalian anorganic condyles examined.

Variation in basement membrane topography in human thick skin.

Samples of human plantar and palmar skin were excised and incubated in 20 mM EDTA after which the epidermis was gently separated from the dermis with the plane of separation occurring in the lamina lucida. Scanning electron microscopic examination of the dermal component revealed the classically described series of regularly spaced grooves and papillae that characterize the epidermal-dermal junction in thick skin. Primary dermal grooves exhibited evenly spaced tunnels that were originally occupied by sweat gland ducts. The basement membrane (basal lamina) in the primary grooves was relatively smooth but did exhibit a flattened, reticulated pattern at high magnifications. The basement membrane of secondary dermal grooves and papillae was in the form of numerous, elevated microridges off of which septae arose at roughly right angles. The surface appearance of the basement membrane in these areas was that of a honeycomb owing to the numerous compartments and recesses formed by the ridges and septae. Degradation of the basement membrane by trypsin demonstrated that the foundation for the highly folded and compartmentalized basement membrane was composed of dermal collagen fibrils, 60-70 nm in diameter, that were arranged in a series of variably sized, interconnected collagen bundles or walls. Epidermal basal cells extended cytoplasmic (foot) processes into two or more compartments, formed by the ridges and septae, which considerably amplified the basement membrane surface available for epidermal attachment. Scanning electron microscopic studies of the epidermal-dermal junction confirm the variable surface character of this interface previously reported by others using sectioned material.(ABSTRACT TRUNCATED AT 250 WORDS)