The pineal gland of nocturnal mammals. I. The pinealocytes of the bat (Nyctalus noctula, Schreber)

The ultrastructure of the pinealocytes of noctule bats, mammals which live most of the time in darkness or very low light intensities, was examined and compared with the pinealocytes of other mammals. Two different populations of pinealocytes (I and II) were observed. They differ in general aspect, in location and especially in their content of cell organelles involved in synthetic processes. Mitochondria, ribosomes, lysosomes and lipid inclusions were present in the perikaryon of pinealocytes of both populations. In the pinealocytes of population I some granular vesicles, of presumed Golgi origin, and some other structures were observed. Pinealocytes of population II are characterized by many glycogen granules, more or less associated with a large vacuolar system. Moreover, some small vacuoles originating from cisterns of the granular endoplasmic reticulum and containing flocculent material of a moderate electron density are described. The possibility is discussed that these small vacuoles are involved in one of the secretory processes of the pineal gland while the granular vesicles of the pinealocyte of the population I are the products of another.     

Electron microscopic studies on the aryl-sulphatase activity of the pineal gland

On applying p-Nitrocatechinsulphate (2-Hydroxy-5-nitrophenylsulphate) as substrate, characteristic positivity was demonstrable in the pineal gland. The reaction was localized to the Golgi system and to the smooth endoplasmic reticulum, producing in them a homogenous, in the primary lysosomes a fine granulated strongly electron scattering precipitate. The development of primary lysosomes was well detectable with histochemical reaction at the level of both the Golgi apparate and smooth endoplasmic reticulum. Membrane limited bodies occurring in the cells corresponded to autolysosomes. Negative reaction was obtained with para-nitro-phenylsulphate and acethyl-phenylsulphate. The presence of KCN and pCMB inhibited the reaction.     

Co-localization of serotonin and FMRFamide-like immunoreactivities in the central nervous system of the earthworm, Eisenia fetida

In addition to receptor-type pinealocytes, the mammalian pineal organ contains small and large neurons and ependymal/glial cells as well. Axons of pinealocytes form synaptic ribbon-containing axo-dendritic synapses on large secondary pineal neurons and/or terminate as neurohormonal endings on the basal lamina of the vascular surface of the organ. The small pineal neurons were found to be gamma-aminobutyric acid (GABA)-immunoreactive, while large secondary neurons and pinealocytes contained immunoreactive amino acids (glutamate and aspartate). Glutamate accumulated presynaptically in pinealocytic axon terminals on large secondary neurons and in the axons of these neurons. Glutamate immunoreactive axons of pineal neurons were traced via the pineal tract to the habenular nucleus. Axons containing granular vesicles and coming from extrapineal perikarya are glutamate immunoreactive as well. Aspartate and GABA are also present in some of the myelinated axons, supposedly pinealopetal in the pineal tract.     

Ultrastructural localization of thyrocyte acid phosphatase in the presence of thyrocyte hyperfunction in rats

Acid phosphatase was found by electron microscopy in the lysosomes which appeared in great numbers in the follicular cells of rat hyperplastic thyroid gland. The other types of granules (mature secretory granules and lipids) whose amounts were also greatly increased in cases of functional thyrocyte strain were also nonreactive. The lysosomes were subdivided into three main groups according to distribution of the reaction product: the lysosomes with dense homogeneous deposit and with deposit in the form of densely or loosely packed dark round granules. The lysosome heterogeneity was apparently connected with their different functions also found within the colloid droplets in the form of inclusions of rarely located dark granules. The authors believe such granules to be the result of the merging of the colloid droplets and lysosomes. The acid phosphatase of the latter participated in the hydrolysis of the product of cell secretion with the formation of active substances.     

Complete replication of human sperm genome in egg extracts from Xenopus laevis

Significant amounts of D-aspartate (Asp) are found in mammalian tissues and D-Asp is presumed to play some significant, but as yet undefined physiological role. However, it is not known whether D-Asp is synthesized in mammals. In this study, we addressed this issue in cultured rat pinealocytes, parenchymal cells of the pineal gland, which contain significant amounts of D-Asp. Biosynthesis of D-Asp was found to be minimal to non-existent in cultured rat pinealocytes. We then investigated the mechanism of uptake of D-Asp into these cells and its consequent effect on cell function. D-Asp was efficiently taken up into cells, in a time- and dose-dependent manner. Interestingly, the L-Asp levels in the cells and media decreased concomitantly with the uptake of D-Asp. This decrease was not due to D-Asp cytotoxicity, since the cellular levels of othernted. D-Serine and D-alanine were not taken up efficiently into the cells and the cellular levels of L-serine and L-alanine were unchanged. Also, immunocytochemical staining with anti-D-Asp antibody showed that D-Asp, which had been taken up into the cells, was dispersed throughout the cytoplasm. In response to norepinephrine stimulation, pinealocytes, which had been pretreated with D-Asp released D-Asp as well as L-Asp. In these cells, norepinephrine-induced secretion of melatonin, a pineal hormone, was suppressed. The mechanism of this suppression is discussed here.     

A novel pineal night-specific ATPase encoded by the Wilson disease gene

We have identified a pineal night-specific ATPase (PINA), a novel splice variant of the ATP7B gene disrupted in Wilson disease (WD). PINA expression exhibits a dramatic diurnal rhythm in both pineal gland and retina with 100-fold greater expression at night than at day. PINA is expressed in pinealocytes and a subset of photoreceptors in adult rats and is transiently expressed in the retinal pigment epithelium and the ciliary body during retinal development. Nocturnal pineal expression of PINA is under the control of a suprachiasmatic nucleus clock mediated by superior cervical ganglion innervation of the pineal. In vitro, PINA expression in pineal cells can be stimulated by agents activating the cAMP signal transduction pathway. PINA is able to restore copper transport activity in Saccharomyces cerevisiae deficient in the homologous copper-transporting ATPase CCC2, suggesting that this protein may function as a copper transporter in rat pinealocytes. These studies suggest a potential role of rhythmic copper metabolism in pineal and/or retina circadian function.     

Permeability of the pineal organ of the golden hamster (Mesocricetus auratus) to HRP with special reference to different types of blood capillaries

The pineal organ of the golden hamster consists of deep and superficial portions which are connected to each other by a stalk. The permeability of capillaries for horseradish peroxidase (HRP) injected intravenously was examined in sections of entire portions of the gland that cut either along coronal or sagittal planes. Two distinct portions of the parenchyma, i.e., dorsal major and ventral minor ones, were found in the superficial gland. Most of the capillaries in the dorsal portion were of the continuous type of endothelium, whereas those in the ventral portion were fenestrated. In the dorsal portion, HRP readily crossed the endothelium, permeated the basal lamina, flowed into the perivascular connective tissue space and intruded into the intercellular clefts of the parenchyma. In contrast, HRP was not found to penetrate through the endothelium of the capillaries in the ventral portion to reach the perivascular area, thereby leaving the intercellular clefts of the parenchyma free of HRP. In the deep gland the capillaries were exclusively of the nonfenestrated type. Intravenously injected HRP was prevented from crossing the endothelium by the tight junction. In some areas, HRP penetrated through the capillaries in the pia mater, and crossed the outer limiting membrane to reach the intercellular clefts of the parenchyma and the basal lamina of the capillaries in the peripheral region of the deep pineal gland. The junctions between endothelial cells were not penetrated by HRP. The observations indicate that the type of capillary, absence of perivascular spaces, and permeability in the deep pineal are all similar to these factors in the general brain tissue; they differ from these in the superficial pineal gland, in which the dorsal portion shows characteristics found in other endocrine glands, but the ventral zone exhibits a unique situations: the presence of a blood-pineal barrier with a pericapillary connective tissue area.     

Regional differences in the pineal gland of the cotton rat, Sigmodon hispidus: light microscopic, electron microscopic, and immunohistochemical observations

Light microscopic, electron microscopic and immunohistochemical observations of the various portions of the pineal gland of the cotton rat (Sigmodon hispidus) were made. The volume of the proximal half occupied about 30% of the whole organ, and pinealocytes were slightly smaller in size in the proximal portion than elsewhere. The distal and intermediate portions contained few interstitial cells and numerous astrocytes, but the proximal portion lacked interstitial cells and had more abundant astrocytes than elsewhere. Astrocytes, which were immunoreactive for glial fibrillary acidic protein, mainly lined the pericapillary spaces in the distal and intermediate portions, but in the proximal portion these cells often surrounded isolated or groups of pinealocytes. In the distal and intermediate portions, abundant sympathetic fibers and less numerous non-sympathetic, peptidergic fibers were mainly localized in the pericapillary spaces; these fibers were sparsely distributed in the parenchyma close to interstitial cells or astrocytes. In the proximal portion, non-sympathetic fibers were scarce and sympathetic fibers were distributed abundantly and almost exclusively in the parenchyma. Most of the sympathetic fibers were adjacent to astrocytes and, occasionally, made specialized contact with them. Fenestrae in the capillary endothelium were numerous in the distal portion but absent in the proximal portion. Thus, marked differences in structure existed between the distal and proximal portions of the pineal gland of the cotton rat suggesting that both portions are functionally dissimilar. In addition, the present study indicates that the proximal portion of the cotton rat was well developed and showed morphological features similar to the deeply situated pineal glands of other mammals.     

Survival and pulmonary hemodynamics in patients with sequelae of pulmonary tuberculosis who received home oxygen therapy

The pineal organ possesses highly fenestrated capillaries, and is devoid of the so-called blood-brain barrier. The present study indicated that the pineal epithelium of the teleost fish, ayu Plecoglossus altivelis, possesses an unusually thick and convoluted basement membrane (2.2-2.4 microm in width) which is visible even under the light microscope. This pineal basement membrane was observed by scanning and transmission electron microscopy and its detailed composition and relationships with the fenestrated capillaries and the perivascular space were investigated. As the basement membrane was composed of three to eight layers of basal laminae interspersed with laminae lucidae, we termed it the "multilayered basement membrane". In consideration of our previous demonstration that macromolecules such as HRP are trapped by the basement membrane, it is suggested that this multilayered basement membrane may prevent foreign substances from reaching the pineal epithelium.     

Nasopharyngeal carriage of community-acquired, antibiotic-resistant Streptococcus pneumoniae in a Zambian paediatric population

The present study was conducted to examine the response of amoeboid microglial cells in the postnatal rat brain to colchicine administration. One-day-old postnatal rats were given intraperitoneal injections of colchicine and sacrificed at 7, 14 and 21 days of age. In rats killed at 7 days age, the number of OX-42, OX-18 and ED1 positive amoeboid microglial cells was considerably reduced when compared with the control rats. At 14 and 21 days, the number of cells immunoreactive with the above antibodies was comparable to that of the control rats. The intensity of the immunoreaction with the various antibodies was also comparable in colchicine injected and control rats. When rhodamine isothiocyanate (RhIC) was administered, amoeboid microglial cells emitted a bright fluorescence in control rats as well as in colchicine-injected rats, although in the latter, the number of RhIC labelled cells was considerably reduced. With the antibody bromodeoxyuridine a large number of stained cells were observed in the control rats. On the other hand, occasional labelled cells were recognized in colchicine-injected rats. Apoptotic amoeboid microglial cells were observed in 4-day-old colchicine-injected rats. At the electron microscopic level, amoeboid microglial cells in colchicine-injected rats killed at 7 days of age showed a large number of phagosomes in their cytoplasm compared with the corresponding control rats. At 14 and 21 days, in colchicine-injected and control rats, amoeboid microglial cells did not display any noticeable differences. It is concluded from the present study that colchicine suppresses the number of amoeboid microglial cells, and that this may be attributed to the antimitotic effect of the drug as well as apoptosis induced by it; the phagocytic activity, however, was not affected. The cells returned to their normal population and morphological features once the drug was discontinued, indicating the reversible nature of the drug effect.     

Effect of carotenoids on the respiratory burst of rat peritoneal macrophages

Numerous factors are involved in the spread of secondary damage in spinal cord after traumatic injury, including ischemia, edema, increased excitatory amino acids, and oxidative damage to the tissue from reactive oxygen species. Neutrophils and macrophages can produce reactive oxygen species when activated and thus may contribute to the lipid peroxidation that is known to occur after spinal cord injury. This study examined the rostral-caudal distribution of neutrophils and macrophages/microglia at 4, 6, 24, and 48 h after contusion injury to the T10 spinal cord of rat (10 g weight, 50 mm drop). Neutrophils were located predominantly in necrotic regions, with a time course that peaked at 24 h as measured with assays of myeloperoxidase activity (MPO). The sharpest peak of MPO activity was localized between 4 mm rostral and caudal to the injury. Macrophages/microglia were visualized with antibodies against ED1 and OX-42. Numerous cells with a phagocytic morphology were present by 24 h, with a higher number by 48 h. These cells were predominantly located within the gray matter and dorsal funiculus white matter. The number of cells gradually declined through 6 mm rostral and caudal to the lesion. OX-42 staining also revealed reactive microglia with blunt processes, particularly at levels distant to the lesion. The number of macrophages/microglia was significantly correlated with the amount of tissue damage at each level. Treatments to decrease the inflammatory response are likely to be beneficial to recovery of function after traumatic spinal cord injury.     

Intracellular behaviour of samarium and europium in lactating mammary gland

The subcellular localization of samarium and europium, two rare-earths, increasingly used in both medical and industrial fields, has been studied in several organs such as liver and kidney but never in the mammary gland despite of its importance in the biology of lactation and nutrition domains. The intracellular behaviour of samarium and europium after their intra-peritoneal administration in the lactating mammary gland cells was investigated. The results showed the presence of very electron dense deposits in the glandular epithelial cell lysosomes. These particular lysosomes were never observed in the mammary cell lysosomes of control rats. These intralysosomal deposits were probably composed of insoluble samarium or europium phosphates by analogy with previous studies, the transmission electron microscopy, the ion mass microscopy and the electron probe microanalysis, and other techniques allowing the identification of the chemical structure of the intralysosomal deposits.     

A fluid-phase endocytotic capacity and intracellular degradation of a foreign protein (horseradish peroxidase) by lysosomal cysteine proteinases in the rat junctional epithelium

We investigated the co-localization of lysosomal cathepsins B, H and L, and horseradish peroxidase (HRP) in junctional epithelial (JE) cells both as a fluid-phase endocytotic marker to demonstrate the fluid-phase endocytotic capacity of JE cells, and to understand the morphological relationships of the endocytosed foreign substances to lysosomal cathepsins in these cells. The diaminobenzidine (DAB) histochemical and cytochemical methods and immunohistochemical avidin-biotin-peroxidase complex and immunocytochemical post-embedding colloidal gold methods were used. Under light microscopy, DAB reaction products based on HRP were found in JE but were rare or absent in the oral sulcular epithelium and oral epithelium. Immunolabeling for cathepsins B and H was found in the granular structures of the cells, but no cathepsin L was identified. With electron microscopy, DAB reaction products, which indicated both HRP and the azurophil granules of neutrophils, were endocytosed into JE cells. Using a post-embedding technique, gold particles indicating HRP were present on the plasma membrane of JE cells, at the periphery of electronlucent vacuoles, and in the electrondense granules. Gold particles indicating cathepsin B or H were found in the electrondense granules. With different sizes of colloidal golds, the co-localization of cathepsin B or H with HRP was indicated only in the electrondense portion of the larger vacuoles consisting of electronlucent and -dense parts. This study provided the first morphological data which indicate that JE has a fluid phase endocytotic capacity, and which suggest that the lysosomal cathepsins B and H are involved in the intracellular degradation of foreign substances invading through the gingival sulcus in JE cells.     

Differences in the UV-crosslinking patterns of the poliovirus 5''-untranslated region with cell proteins from poliovirus-susceptible and -resistant tissues

Specific polyclonal antibody was raised against D-aspartate (D-Asp) which had been conjugated to glutaraldehyde and was purified by affinity chromatography. Immunohistochemical staining of rat pineal gland with the antibody demonstrated the presence of D-Asp in the cytoplasm of pinealocytes, the predominant cell type in this gland. D-Asp immunoreactivity was more evident in the distal region than in the proximal region of the gland. Pinealocytes in the distal region are presumably involved in the synthesis and secretion of the pineal hormone, melatonin, and the results of staining may indicate some yet unknown role of D-Asp in the regulation of melatonin secretion.     

Arrangement of subunits in 20 S particles consisting of NSF, SNAPs, and SNARE complexes

The present study attempts to identify the antigen-presenting cells in the retina, utilizing bone marrow-transplanted chimeric rats. Two types of chimeras were used: one produced by transplanting bone marrow cells from F1 hybrids of Lewis and Brown Norway (BN) into sublethally irradiated Brown Norway rats (LBN/F1-->BN), followed by adoptive transfer of S-antigen-specific T cells obtained from Lewis rats; the second produced by transplanting bone marrow cells from BN rats into sublethally irradiated F1 hybrids (BN-->LBN/F1), followed by adoptive transfer of S-antigen-specific T cells obtained from F1 hybrids. As controls, Lewis, F1 hybrids and BN rats also received adoptive transfer of syngeneic uveitogenic T cell lines. All animals were killed on the seventh day after adoptive transfer and their eyes and pineal glands were analysed immunohistochemically, utilizing antibody directed against Lewis specific MHC class II molecules(OX-3). The analyses revealed the development of uveoretinitis and pinealitis in both types of chimeras and in the Lewis and F1 hybrid rats. BN rats did not develop uveoretinitis. OX-3-positive cells were found in the retina and the pineal glands of both types of chimeras, and in the Lewis and F1 hybrid rats but not in the BN rats. These cells in the retina expressed dendritic morphology and perivascular distribution. Retinal pigment epithelia, Müller cells and the vascular endothelia of both chimeras, the two strains, and the F1 hybrid rats did not demonstrate OX-3-positive staining. These results suggest that the bone marrow-derived cells in the retina and pineal gland may present S-antigen to T cells, initiating the cascade of uveoretinitis and pinealitis.     

Women''s perceptions of their general health, with special reference to their risk of coronary artery disease: results of a national telephone survey

We measured the time-dependent morphological changes of microglial cells reacting to ischemic cell damage after transient (2 h) middle cerebral artery occlusion in the rat by means of lectin histochemistry with the B4-isolectin from Griffonia simplicifolia as well as immunohistochemistry with monoclonal antibodies directed against monocyte/microphage (ED1) and major histocompatibility complex (MHC) class II (OX-6) antigens. As early as 1 h after onset of reperfusion, microglia were absent in the severely neuronal damaged preoptic area. However, ameboid-like microglia were evident in an adjacent area containing scattered shrunken neurons. Rod, round and ameboid-like microglia were present in the ischemic lesion between 2 to 10 h of reperfusion. Round and ameboid cells became predominant in the ischemic core lesion and were mingled with highly ramified microglia to the boundary at 22 h of reperfusion. Highly ramified microglia were found in an adjacent area containing morphologically intact neurons. Round and ameboid cells were localized to the inner boundary of the ischemic lesion surrounding the infarct zone at 46 of reperfusion. Round and ameboid cells were present throughout the entire ischemic lesion in the infarct zone from 70-166 h of reperfusion. A marked increase in number and in intensity of highly ramified microglial cells were present in the outer boundary of the lesion during this period. In addition, a significant increase in both ED1- and OX-6-immunoreactive cells in the ischemic region was detected after 10 h of reperfusion and persisted up to 166 h of reperfusion. These data demonstrate that microglia exhibit a time dependent change in morphology after reperfusion and that the severity of injury may be reflected in the state of microglial activation.     

Ultrastructural changes in fat cells and blood capillaries of the mammary gland in starved mice

The effects of starvation on fat cells and blood capillaries of the first abdomino-inguinal mammary gland in mice were investigated by light and transmission electron microscopy. The body weight of starved mice abruptly decreased to approximately 70% of that of controls at 3 days of starvation and, thereafter, gradually decreased. In adipose tissues of mammary stroma, multilocular fat cells increased in number and clustered during starvation to a glandular appearance at 6 days. Collagen fibers increased in amount around mammary ducts and buds. By electron microscopy, multilocular fat cells possessed numerous mitochondria, small lipid droplets, and plasmalemmal vesicles, while endothelial cells of the blood capillaries showed numerous pinocytotic vesicles plus short marginal folds and microvillous processes. These observations prove that the number of pinocytotic vesicles in blood capillary endothelium is closely related with the increased amount of lipid of fat cells in the mammary gland during starvation.     

Fine structure of the free-living parakeet pineal in relation to the breeding cycle

Seasonal changes in the ultrastructure of the free-living Rose-Ringed Parakeet Psittacula krameri pineal were examined in relation to the sub-tropical environment and seasonal reproduction. Dark and light pinealocytes of the presumptive neuroendocrine cell line predominated, while supporting cells, ependymal cells, myelinated and non-myelinated nerve fibers with nerve endings, and regressed photoreceptor elements were also observed. Unlike in pineals of many animals, particularly mammals, the presence of dense-core vesicles (DCVs) with varying core density, and absence of clear vesicles and vacuoles with flocullent material, indicate the involvement of DCVs in the synthesis and secretion of pineals principle/s. In November (pre-breeding) when the day length registered a drop to LD 10:14, pinealocytes showed significantly decreased and smaller DCVs and mitochondria, nuclei with heterochromatin, and greater distribution of glycogen and lipid droplets, all indicating low pineal metabolic activity. During the shortest day regime from December to March, when the birds peaked breeding, the number and size of DCVs and mitochondria increased, and Golgi body-endoplasmic reticulum-lysosome complex (GERL) was very well defined. Images of DCVs suggested possible secretion of pineal principle/s by dissolution, and exocytosis. Coincidence of these features with peak gonadotrophic (circulating LH) and spermatogenic and testicular endocrine activity described previously suggested an active turnover of pineal products during this short day length regime when parakeets breed. In contrast, during the post-breeding season (April onwards), when the day-length increased to LD 13:11 and hypophyseal-gonadal function was down, nuclei and RER continued to show active profile, the Golgi body and associated complex were moderately seen, and the DCVs and mitochondria were significantly smaller and lesser. It is therefore probable that the pineal is an important relay to translate cues related to less drastic sub-tropical environmental change into DCV-linked neurohormones that in turn may be involved in modulating seasonal breeding in parakeets.     

Analysis of HAM rat disease with HTLV-I infection, an animal model for HAM/TSP in human-immunohistochemical, immuno-electron microscopic and flow cytometrical analyses of microglia/macrophages in the affected spinal cord]

HAM rat disease is a demyelinating disease in the spinal cord and peripheral nerve of WKAH rats with HTLV-I infection and has been described as an animal model for human T lymphocyte virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in humans. The histopathological characteristic in the affected lesions is a vacuolar myelopathy, with the apoptosis of oligodendrocytes and the massive infiltration of microglia/macrophage lineage cells. To investigate the pathogenetic role of the infiltrating microglias/macrophages in the lesion, the author carried out chronological analyses with immunohistochemistry, immuno-electron microscopy and flow cytometry on the spinal cord specimens of HAM rats. Many OX-42 positive cells were identified as microglias either irregular or plump in shape. Both MHC Class II and LFA-1 expressions were increased in these cells, suggesting they were activated in the lesion. Moreover, many microglias/macrophages showed PCNA positivity, indicating they proliferated locally. Some apoptotic microglias/macrophages were evidenced by immuno-electron microscopy. Flow cytometrical analysis revealed that the majority of the infiltrating cells in the affected lesion was originated from resident microglias, and macrophages from the blood stream appeared to be minimum. Collective evidence suggests that the activated and proliferated resident microglias with HTLV-I infection may play a critical role in the disease course through the production of cytotoxic factors such as TNF-alpha.