Immunohistochemical study of the lymphatic vessels in major salivary glands of the rat.

This study was designed to examine whether lymphatic vessels are present in the lobules of major salivary glands in the rat. Immunostaining with an antibody against podoplanin, a lymphatic endothelial cell marker, was performed on sections of the submandibular, sublingual and parotid glands. Light microscopy demonstrated podoplanin-positive lymphatic vessels around the interlobular ducts and the interlobular arteries and veins in the interlobular connective tissue in all of the major salivary glands. No podoplanin-positive lymphatic vessels were found in the lobules. Electron microscopy also demonstrated lymphatic endothelial cells showing podoplanin expression only in the interlobular connective tissue. These findings suggest that the lymphatic system of the rat major salivary glands originates in the interlobular connective tissue, and not in the lobules.

Changes in the properties of secretory granules in the palatine gland acinar cells of the postnatally developing rat.

This study was designed to examine whether or not phospholipid is contained in the secretory granules of the rat palatine gland acinar cells, and if present, to examine the movements of phospholipid in the secretory granules during postnatal development. The palatine glands of male Wistar rats aged 0 to 56 days were used. Acid-hematin staining showed a few positive acinar cells with a faint reaction in the acini on day 0, numerous positive cells with an intense reaction on day 7, a weakening reaction in the cells on day 14, and almost no reactivity on day 35 and after. In contrast, alcian blue staining showed acinar cells with a weak reaction on day 7, a gradual increase in the reaction from day 14, and the presence of many cells with an intense reaction on day 28 and after. Electron probe microanalysis (EPMA) revealed a higher density of phosphorus in samples on day 7 than on day 56. These findings suggest that developing rat palatine gland acinar cells contain phospholipid in the secretory granules, being particularly more conspicuous around postnatal day 7, but that the amount of phospholipid decreases as the cells change to mature mucous cells.

Effects of exogenous thyroid hormone on the postnatal morphogenesis of the rat parotid gland.

Administration of thyroid hormone has been shown to accelerate the early postnatal development of the rat parotid gland, but these studies have dwelt almost entirely on biochemical changes. The objective of this study was to describe the effects of exogenous thyroid hormone on morphologic aspects of the developing parotid gland, in particular the transient appearance of scattered mucous cells in this otherwise serous gland. Pups were given a daily subcutaneous injection of thyroxine (T(4)) of 0.1, 0.5, or 5.0 microg/g body weight, vehicle only (injection control), or no injection (normal control) beginning at 4 days, and killed for the collection of blood and parotid glands at intervals through 15 days. The serum was analyzed for T(4) and the glands were examined by light and electron microscopy. The results indicated that both serum T(4) and the pace of gland development were proportional to the dose of T(4). In particular, T(4) accelerated decreases in acinar size and gland area occupied by stroma and translocation of a subset of cells with small secretory granules, deeply stained with periodic acid-Schiff, from acini to intercalated ducts. However, the chronology of mucous cell disappearance was indifferent to treatment. In addition, signs of toxicity, including slower gain in body weight and greatly increased apoptosis and vacuoles in the glands, occurred with the higher doses of T(4).

Strain-specific and endocrine control of granular convoluted tubule cells and epidermal growth factor expression in the mouse submandibular gland.

Submandibular glands (SMGs) of 11-week-old mice from four strains, ICR, C57BL/6J, BALB/c, and C3H/HeN were examined by immunohistochemistry for epidermal growth factor (EGF). In addition to sex-related differences in granular convoluted tubules (GCTs), the GCT cells were significantly larger in ICR mice than in other three strains. In males from each of the strains, almost all the GCT cells were strongly positive for EGF. The EGF-positive cells in the females, however, were markedly fewer in number, and were stained weaker in C57BL/6J, BALB/c, and C3H/HeN mice than in ICR mice. The GCT cells and their EGF expression in the F1 progeny from ICR and C3H/HeN strains were approximately intermediate between those of the parent strains of the same sex. T(3) and/or dihydrotestosterone (DHT) enhanced the GCT phenotype in the C3H/HeN mice, and remarkably increased the EGF-positive cells in females. Electron microscopy revealed that gold-labeling of EGF was confined to the secretory granules, and that the GCT cells in females, given T(3) + DHT, had a well-developed Golgi apparatus and net-like RER but few basal infoldings, whereas the equivalent cells in the untreated females had poor RER and prominent basal infoldings. These results suggest that the EGF concentration in SMGs is genetically high in ICR mice and low in other strain mice and that, considering the same response of GCT cells to T(3) and/or DHT between the high and low EGF strains, the low EGF concentrations might be partly caused by a lesser sensitivity of the GCT cells to thyroid hormones.

Effect of glucocorticoid on the differentiation and development of terminal tubules in the fetal rat submandibular gland.

Glucocorticoids (CORT) are known to promote branching of the epithelial cords during the development of the rat submandibular gland. The aim of this study was to examine the effect of CORT (triamcinolone) on the differentiation of cells forming the terminal tubules in the developing fetal rat submandibular gland and the properties of the secretory granules. Light and electron microscopy showed that the terminal tubules of the glands in the experimental group contained more type III cells, which have been identified as proacinar cells, than those in the control group, whereas the relative number of type I cells, which have been identified as terminal tubule cells, was reduced. Immunoelectron microscopy using an antibody against neonatal submandibular gland secretory protein B (SMGB) revealed the presence of more gold particles over type III cell granules in the experimental group than in the control group. Lectin histochemistry demonstrated more wheat germ agglutinin (WGA)-labeled gold particles over type III cell granules in the experimental group than in the control group. These findings suggest that CORT promote the differentiation of type III cells, and moreover stimulate the production of secretory granules reactive for SMGB and WGA by acting on the terminal tubules of the developing rat submandibular gland.

Histological analysis of the sublingual gland in rats with streptozotocin-induced diabetes.

This study was designed to examine whether the sublingual gland parenchyma is influenced by the development of insulin-dependent diabetes mellitus. The sublingual glands of rats with streptozotocin-induced diabetes were examined by light and electron microscopy. In order to define the limiting membrane of mucous granules in more detail, samples processed by rapid freezing following by freeze-substitution in addition to chemical fixation were also prepared for electron microscopy. Light and electron microscopy showed vacuole-like structures considered to be lipid droplets in the cytoplasm of serous demilune cells, the largest reaching 4 microm in diameter. Electron microscopy of the chemically fixed samples revealed granule-like structures in addition to the mucous granules proper in the mucous cell cytoplasm. However, electron microscopy of the freeze-substitution fixed samples demonstrated no limiting membrane on the surface of the granule-like structures, although this was clearly observed on the surface of the mucous granules. Accordingly, the granule-like structures present in the mucous cell cytoplasm appeared to be lipid droplets. These findings suggest that the sublingual gland mucous cells become dysfunctional during the development of insulin-dependent diabetes mellitus, although to a slighter degree than the serous demilune cells.

Morphological changes in the rat sublingual gland parenchyma with aging.

BACKGROUND: The characteristics of mucous cells in the aging rat sublingual gland were investigated in this study. Particular attention was paid to accumulated amyloid protein and changes of the properties of the secretory granules at the histochemical and ultrastructural level. OBJECTIVE: This study was designed to examine age-related morphological changes in the sublingual gland of male Wistar rats from 12 to 27 months. METHODS: For light microscopy, the sublingual glands were fixed with 10% neutral-buffered formalin, embedded in paraffin, and processed for Alcian blue, Congo red, and TUNEL staining. For transmission electron microscopy, some of the samples were fixed with Karnovsky solution, postfixed with 2% osmium tetroxide, and embedded in epoxy resin for pronase treatment. RESULTS: The sublingual gland showed slight shrinkage after 21 months. After 24 months, Congo red staining showed positive reaction to the intralobular connective tissue surrounding the terminal portions and to the interlobular connective tissue around the blood vessels and the excretory ducts. At 27 months, some of the granules in the serous demilunes had difficulty in digesting with pronase treatment. The appearance rate of TUNEL-positive cells was low in both mucous and serous portions during the observation period, though the positive cell number was higher in the serous than in the mucous portion. CONCLUSIONS: These findings indicate that the rat sublingual gland accumulates amyloid protein in the parenchyma and changes the properties of secretory granules of the acinar cells in the serous demilune with aging, though apoptosis of the parenchymal cells and the decrease of the gland weight are slight.

Changes in the number and distribution of myoepithelial cells in the rat parotid gland during postnatal development.

The mature rat parotid gland shows hardly any cell bodies of myoepithelial cells around the acini, only a few cell processes being visible. However, in the early postnatal period, the rat parotid gland shows many myoepithelial cell bodies around the acini, including the intercalated ducts. In order to clarify the reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development, changes in the number and distribution of myoepithelial cells in the rat parotid gland were examined histochemically and chronologically, with particular reference to cell proliferation and cell death. From day 7 to day 14, many myoepithelial cells showing a positive reaction with anti-actin antiserum were found around the acini and intercalated ducts, but thereafter the number of such cells decreased gradually, particularly around the acini, and had almost disappeared after day 35. BrdU/PCNA-positive myoepithelial cells surrounding the acini were easily detected on day 14, but disappeared by day 21, whereas BrdU/PCNA-positive acinar cells remained numerous even after day 21. TUNEL/ISEL staining showed no positive myoepithelial cells throughout the observation period. Transmission electron microscopy also demonstrated no myoepithelial cells with chromatin condensation characteristic of apoptosis through the observation period. These findings suggest that the main reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development is the large difference between the number of myoepithelial cells and that of acinar cells, because the acinar cells retain their proliferative activity even after myoepithelial cells have become quiescent.

Three-dimensional observation of blood vessels supplying the gland proper and capsule of the mouse submandibular gland.

Previous studies have shown that submandibular glands suffering interruption of the blood circulation from the main supplying artery have more surviving parenchymal cells in their peripheral portion than in their central portion. Although the reason for this difference between the peripheral and central portions has not yet been clarified, the existence of collateral circulation has been suspected. The present study was designed to examine whether or not the vessels binding the gland proper to the capsule provide such collateral circulation. Silicone rubber or methacrylate was injected into the main artery supplying the mouse submandibular gland, in which the gland proper is wrapped by a capsule similar to that of the human submandibular gland, and then the gland was observed by both stereoscopic and scanning electron microscopy. Three-dimensional observations showed no communicating vessels between the gland proper and the capsule. Therefore, it is suggested that the parenchymal cells surviving in the ischemic peripheral portion of the submandibular gland are nourished by permeation of tissue fluid contained in the capsule.

Changes in the distribution and fine structure of the intralobular blood vessels of the submandibular gland in the postnatally developing mouse.

Previous studies have shown that the blood vessels supplying the endocrine organs and the mucosa of the intestinal canals change in terms of not only their distribution but also their structure with the development and growth of each organ. We examined changes in the distribution and structure of intralobular blood vessels, including capillaries, throughout the postnatal development of the submandibular gland, an exocrine organ. The mouse submandibular gland from days 0 (birth) to 49 was investigated chronologically and ultrastructurally. The capillaries changed from continuous to fenestrated on day 10, coincident with an increase in the number of acini to more than the number of terminal tubules. The number of sections of intralobular blood vessels per unit area gradually decreased with increasing acinar size and was lowest on day 21 when pups were weaned; the same number was maintained from then on. In contrast with the reduction in the number of intralobular blood vessels, the number of capillary pores appeared to increase gradually. Acinar size increased further till day 28. Capillary pore number also increased further, till day 35, apparently in relation to the increasing acinar size. These findings suggest that the changes in distribution and structure of the intralobular blood vessels in the submandibular gland of the postnatally developing mouse are closely related to the development of the parenchymal cells in preparation for weaning and sexual maturity.

Role of thyroid hormone in the initiation of EGF (epidermal growth factor) expression in the sublingual gland of the postnatal mouse.

The effect of triiodo-L-thyronine (T3) and propylthiouracil (PTU) on the initiation of epidermal growth factor (EGF) expression in the sublingual glands (SLGs) of postnatal mice was investigated by indirect enzyme-labeled and immunogold antibody methods for light and electron microscopy, respectively. In normal males, EGF immunoreactivity first appeared in a few scattered granular cells of striated ducts (SDs) at 5 weeks of age, and the immunoreactive cells had increased in number at 6 weeks of age. No EGF expression was observed in the glands of females at any ages examined. When T3 (1 mg/kg body weight) was given to males every other day for 2 weeks before examination, EGF expression began earlier; the immunoreactive granular cells were first detected at 4 weeks of age, and at later ages they were markedly increased in number compared to those of normal males. Moreover, T3 was capable of inducing EGF in the female glands. After T3 was administered to females in the same manner as in males, a few immunoreactive cells were first detected at 5 weeks of age, and increased numbers were detected at later ages. By contrast, when PTU (1 mg/kg body weight) was given to male mice every other day for 2 weeks before examination, the EGF-immunoreactive cells were markedly decreased in number compared to those of normal males of the same age. Electron microscopy revealed that many SD cells contained secretory granules, and that these cells constituted the granular striated tubule (GST) in a portion of SDs, but they were undetectable by light microscopy, because their secretory granules were minimal in size and few in number. Gold-labeling of EGF was confined to the secretory granules of scattered granular cells, whose secretory granules were far larger in size and more abundant than those of the GST cells. These results suggest that thyroid hormone is essential to differentiation of the cellular phenotype of GST precursor cells into typical granular cells (detectable by light microscopy) that express EGF in the mouse SLG, showing a close resemblance to the submandibular granular convoluted tubule cells.

Lateral expansion of the rat lower dental arch by applying an active plate model to the mandibular first molar region: observation of histological changes in the lower alveolar bone over a 2-week period.

Lateral expansion of the lower dental arch has rarely been conducted clinically because the structure of the lower jaw is considered to be unsuitable for this type of treatment. However, successful lateral expansion of the lower dental arch using SCHWALZ, an orthopedic appliance has been reported in recent years. Therefore, an experimental study was performed to examine the histological changes in the lower alveolar bone when lateral expansion is applied to the lower dental arch for periods of up to 2 weeks. An active plate model based on the Schwarz appliance was attached to the first molar region in rats. The distance between the right and left first molars was measured, and the mandible and first molar tooth were processed for light microscopy at different times after the fitting. The lateral expansion caused by lateral movement of the right and left first molars following the application of the active plate amounted to an average of 1.06 mm over a period of 14 days. Alkaline phosphatase/tartrate-resistant acid phosphatase staining revealed bone deposition on the periosteal surface of the buccal alveolar bone and the periodontal surface of the lingual alveolar bone, whereas bone absorption was observed on the periodontal surface of the buccal alveolar bone and the periosteal surface of the lingual alveolar bone. These findings demonstrate that lateral expansion of the lower dental arch through the application of an active plate model was achieved by bony deposition and absorption of alveolar bone, similar to the process that occurs in association with lateral expansion of the upper dental arch.

Granule types and their morphological changes in terminal cluster and acinar cells in the late pre- and early postnatal rat sublingual gland.

The developmental characteristics of serous cells appearing in the rat sublingual gland from the late prenatal to the early postnatal period were investigated in this study. Particular attention was paid to the morphological changes observed in the secretory granules at the histochemical and ultrastructural level. On prenatal day 18, granules with homogeneous high electron density (Type I granules), and mottled granules (Type II granules) with heterogeneous electron density appeared in the narrow luminar cytoplasm of cells constituting the terminal clusters. On prenatal day 19, these granules decreased in number and were replaced by bipartite granules (Type III granules) composed of a highly electron-dense core and a more electron-lucent rim. Pronase treatment almost completely digested the Type I and II granules and the electron-dense core of the Type III granules, although some of the Type I and II granules in serous demilunes at a later stage were insufficiently digested. On prenatal day 19.5, homogeneous granules of low electron density (Type IV granules) appeared in the terminal clusters and acini, and increased in number daily, making up 92.8% of the total granules on postnatal day 28. The granule morphology on electron microscopy, Alcian blue, and periodic acid-Schiff staining strongly suggested that Type I and II granules were serous granules, Type IV granules were mucous granules, and Type III granules were transforming-type granules. None of the secretory cells showed chromatin condensation, which is a characteristic of apoptosis. These findings suggest that the developing rat sublingual gland from the late prenatal to early postnatal period has numerous serous granules in the terminal clusters and acini, and that the majority of granules are replaced by mucous granules through transforming-type granules. In addition, because apoptotic figures of secretory cells could not be detected, it appears that most of the serous cells in the developing rat sublingual gland might have changed to mucous cells.

Effects of gadolinium and tetrodotoxin on the response of slowly adapting type I mechanoreceptors to mechanical stimulation in frog dorsal skin.

To elucidate the excitatory mechanism of mechanoreceptors innervating the frog skin, we examined the effects of gadolinium (Gd3+) and tetrodotoxin (TTX) on the response of single-unit activity of slowly adapting type I mechanoreceptors to mechanical stimulation topically applied to the receptive field (RF). Recordings were made from 46 fibers responding to mechanical stimulation with von Frey hairs, which caused an irregular firing pattern with slow adaptation. Application of a mechanically gated channel blocker, Gd3+ (30 microM), and a Na+ channel blocker, TTX (3 microM), caused the suppression of discharge rates, which was characterized by the conversion of a slowly adapting to a rapidly adapting discharge pattern. The administration of a high-voltage-activated (HVA) Ca2+ channel blocker, Cd2+ (100 microm), inhibited the unit discharge and caused the conversion of a slowly adapting to a rapidly adapting discharge pattern. Tonic discharges evoked by anodal electrical stimulation were inhibited by the application of Gd3+ or TTX. Electron microscopic examination showed that the cytoplasm of Merkel cells seen in the RF contained numerous Merkel granules. These results suggest that the excitatory mechanism of frog cutaneous mechanoreceptors may be mediated by the activation of Gd(3+)-sensitive stretch-activated channels in the Merkel cell-neurite complex, which are related to the Na+ influx via voltage-gated Na+ channels and/or the Ca2+ influx through HVA Ca2+ channels.

Morphological changes in mouse sublingual gland parenchyma subjected to chorda tympani resection.

Morphological changes in the mouse sublingual gland parenchyma subjected to parasympathetic nerve block were investigated. Mice were subjected to unilateral resection of the chorda tympani, near its point of joining with the lingual nerve. After 1, 2, 3, 5, 10 or 20 weeks, the mice were killed and their sublingual glands were removed and processed for light and electron microscopy. Two weeks after resection, the space between the adjoining lobules of the glands on the treated side began to be expanded, and by 10 weeks were 10 times the size of the spaces in the glands of the untreated mice. Three weeks after resection, the lobule area decreased to about 72% of the area of glands in the untreated mice and the acinus area to about 52%. However, no significant difference was seen between the numbers of acini in each group. Electron microscopy showed that the glands on the treated side contained fewer secretory granules than the glands in the untreated mice, though there was no difference in size. Neither the lobules of the glands on the treated side nor those of the glands of the untreated mice contained many TUNEL-positive cells. These findings suggest that following parasympathetic nerve resection, mouse sublingual gland acinar cells undergo atrophy with a reduction size rather than cell death.