This study was designed to observe the apoptosis and expression of p53 in the osteoarthritic synovial membrane compared with normal synovial membrane of human. The collected normal and osteoarthritic synovia were dissected and fixed for two hours (in 4% paraformaldehyde and 0.1% glutaraldehyde solution). In this study, TUNEL staining and immunocytochemical gold labeling techniques were used. In the immunocytochemical gold labeling techniques, primary antibodies which was to be monoclonal mouse anti-p53 were used. Donkey anti-mouse IgG tagged with 6 nm colloidal gold particles was used as the secondary antibody. The tissues were observed under JEOL 1200 EX-II transmission electron microscope. The results were as follows. 1. On TUNEL staining, normal synovium were not seen TUNEL positive signal cells. But, in the osteoarthritic synovium, few TUNEL positive cells were seen in synovial membrane and subsynovial layers. 2. On the transmission electron microscopic observation, normal synovium had 1~3 synovial cell layers, which had phagocytic synovial cells and secretory synovial cells. The osteoarthritic synovium had 2~5 synovial cell layers, which consisted with abnormally proliferated secretory synovial cells. These cells had heterochromatin in nucleus and well developed endoplasmic reticulum in the cytoplasm. 3. On the normal synovium of the human knee joint, p53 positive cells were not identified. But, in the osteoarthritic synovium of the human knee joint, p53 positive cells were identified. These cells were recognized secretory synovial cells and apoptotic cells. In the secretory synovial cells, the distributions of p53 were mitochondria and rough endoplasmic reticulum. In the apoptotic cells, p53 were marked on rough endoplasmic reticulum, which showed secretory synovial cells. On the basis of above findings, it is obvious that osteoarthritic synovial membrane has identified the apoptotic cells compared with normal synovium. These apoptotic cells might be identified as mainly secretory synovial cells and a few phagocytic synovial cells. The immunogold of p53 was marked at rough endoplasmic reticulum and in nucleus of apoptotic cells. Apoptosis in the osteoarthritic synovium seemed to be developed through p53 negative dependent pathway.
Animals ; Antibodies ; Apoptosis* ; Cytoplasm ; Endoplasmic Reticulum ; Endoplasmic Reticulum, Rough ; Equidae ; Glutaral ; Gold Colloid ; Heterochromatin ; Humans* ; Immunoglobulin G ; In Situ Nick-End Labeling ; Knee Joint ; Knee* ; Mice ; Microscopy, Immunoelectron ; Mitochondria ; Osteoarthritis ; Synovial Fluid ; Synovial Membrane*

To observe the cellular expression of extracellular matrix components during mouse skin regeneration, the wounded skin samples were processed by immunoelectronmicroscopic methods, using primary antibodies for fibronectin, collagen type IV and laminin. The tissues were observed under transmission electron-microscope. The results were summarized as follows. 1. The granulation tissues and x-cells were observed in the wound margin at 18 hr post injury. The number of fibroblasts was increased in the granulation tissues at 1 day post injury. 2. The expression of fibronectin was observed in x-cells at 18 hr post injury, and in fibroblasts at 1 day post injury. In x-cells, after 1 day post injury, the expression of fibronectin was decreased. 3. At 1 day post injury, the expression of collagen type IV was increased in fibroblasts whereas not in x-cells. 4. The expression of laminin was increased by 18 hr post injury, but decreased after 1 day post injury. On the basis of above findings, in mouse, the regenerations of wounded skin were faster than other animals. The first step, infiltration response, was processed till 18 hr or 1 day post injury. The second step, fibroblast proliferation phase, began at 1 day post injury. In the regenerations of wounded skin, x-cells migrated to the wound region and activated, earlier than fibroblast. Thereafter, x-cells which appeared to be transformed into fibroblasts, played an important role in the synthesis of fibronectin and collagen type IV, and the formation of granulation tissue, with migrated fibroblasts to the wound region.
Animals ; Antibodies ; Collagen Type IV ; Extracellular Matrix* ; Fibroblasts ; Fibronectins ; Granulation Tissue ; Laminin ; Mice* ; Regeneration* ; Skin* ; Wounds and Injuries

Germinal center (GC) is a critical site where the humoral immune responses take place. Especially memory cells are known to be generated from the GC. In this experiment, T cells observed in the GC were studied in the aspect of memory T cells. T cells responding to myelin basic protein (MBP) antigen usually have their own specific T cell receptor complex (TCR) consisting of V 2 and V 8. Therefore, MBP antigen enables to trace specific T cells reacting to MBP antigen. This experiment, in which balb/c mice were injected with MBP into footpad and the popliteal lymph nodes were removed, showed that most of V 2 +/- cells were L3T4 +/- cells, and that initially they were located in the deep cortex near the B cell follicles and later they were observed in the GC. In case of the primary injection of MBP, IL -4 +/- cells were observed for the first time, followed by appearance of CD69 and CD2R. In case of the secondary injection, all of IL -4, CD25, CD69, CD2R and CTLA -4 were observed from the 1st day after injection. However IL -4, CD25 and CD69 among them were not observed any more since 2 wks after the secondary injection. These results strongly suggest that T cells observed in the GC during the immune responses for MBP might be memory T helper cells.
Animals ; Germinal Center* ; Immunity, Humoral ; Lymph Nodes ; Memory* ; Mice ; Myelin Basic Protein ; Receptors, Antigen, T-Cell ; T-Lymphocytes* ; T-Lymphocytes, Helper-Inducer

This study was designed to observe the expression of perlecan in the normal and degenerative arthritic synovial membrane. By using the immunohistochemical staining and immuno -electron microscopical gold labeling techniques, we observed five materials of normal and degenerative arthritic synovia each. The results were as follows. 1. By the immunohistochemical methods, perlecan -positive staining was seen on the 1 ~2 cell layers of the normal synovial membrane. But, a weaker staining compared to that seen in the normal synovial membrane was found in the degenerative arthritic synovial membrane. 2. Under the electron microscopic observation, perlecan was largely distributed in the rough endoplasmic reticulum of the secretory synovial cell, and in the vacuoles of the phagocytic synovial cell on the normal synovium of the human knee joint. It was also found in the extracellular matrix of the synovial membrane. 3. Perlecan -positive cells were also identified on the degenerative arthritic synovium of the human knee joint. However, fewer perlecan was observed here than that found in the normal synovium. In conclusion, perlecan is synthesized by the secretory synovial cells and degraded by the phagocytic synovial cells. And it, known as a major component of the basement membrane, also proven to exist in the extracellular matrix of the synovial membrane having no basement membrane. From the fact that less perlecan was observed in the degenerative arthritis, perlecan is might to play a major role in the degenerative process.
Basement Membrane ; Endoplasmic Reticulum, Rough ; Extracellular Matrix ; Humans ; Knee Joint ; Knee* ; Osteoarthritis* ; Synovial Fluid ; Synovial Membrane* ; Vacuoles

Using in situ hybridization technique with digoxigenin-labelled riboprobe, study on the expression of hsp 70 mRNA in the developing mouse brain was performed. The results obtained are as follows; 1. In embryonic day 16 group, cells with strong reactivity to hsp70 mRNA were found in spinal cord. In neuroepithelial layer lining fourth ventricle and external granular layer of cerebellum, moderate reactivity was observed. But the reactivity was weak in the forebrain including cerebral cortex, diencephalon and olfactory bulb. 2. In embryonic day 18 group, the regional pattern of hsp70 mRNA expression was similar to that of embryonic day 16 group. In medulla oblongata, however, stronger reactivity was found in the embryonic day 18 group. 3. In postnatal day 0 mice group, cells with moderate or strong reactivity to hsp70 mRNA were found in the overall area of central nervous system, Especially, cells with moderate reactivity were found in the dentate gyrus of hippocampus, and the supragranular cortical plate and subplate neocortex. 4. In postnatal day 2 mice group, cells with moderate or strong reactivity to hsp70 mRNA were found in the same pattern as in postnatal day 0 group. Further differentiation of cerebral cortex and cerebellum was found. 5. Strong expression of hsp70 mRNA was found in the areas with high rate of cell division. In general, the area of expression moved to more rostral area in central nervous system as development proceeds. Above results suggest that hsp70 play an important role in the development and differentiation of central nervous system.
Animals ; Brain* ; Cell Division ; Central Nervous System ; Cerebellum ; Cerebral Cortex ; Dentate Gyrus ; Diencephalon ; Fourth Ventricle ; Heat-Shock Proteins* ; Hippocampus ; Hot Temperature* ; HSP70 Heat-Shock Proteins* ; In Situ Hybridization ; Medulla Oblongata ; Mice* ; Neocortex ; Olfactory Bulb ; Prosencephalon ; RNA, Messenger ; Spinal Cord

This study was performed to investigate the effect of Matrigel, a reconstituted basement membrane, on the expression of the anterior pituitary hormones in culture. Rat pituitary cells cultured for 6 days on Matrigel showed 3-dimensional, lobular structures with connecting cells while those on plastic showed flat, polygonal cells forming a monolayer. Western blot analysis showed that prolactin (PRL) content in the anterior pituitary cells was higher compared to those cultured on plastic. In comparison, TSH expression was not increased in cultures on Matrigel. The total cell number and the proportion of fibroblasts was decreased. These results suggested that Matrigel is a useful culture substrate for the enhanced expression of PRL but not for TSH. Further studies are needed in order to find a useful culture substrate for TSH cells.
Animals ; Basement Membrane* ; Blotting, Western ; Cell Count ; Fibroblasts ; Pituitary Hormones, Anterior* ; Plastics ; Prolactin ; Rats* ; Thyrotrophs

These study was designed to observe the appearance and the characteristics of apoptotic cells during the development of knee joint in rat. The fetus were collected on the 16th, 17th, 18th, 19th, and 20th day of pregnancy. In this study, TUNEL staining, electron microscopic investigation and immunocytochemical gold labeling techniques were used. In the immuno-cytochemical gold labeling techniques, primary antibodies were used, which were to be polyclonal rabbit anti-mouse/ rat Bax, polyclonal rabbit anti-tissue transglutaminase C, and polyclonal goat anti-cpp32p20. The samples were observed under JEOL 1200 EX-II transmission electron microscope. The results were as follows. 1. In a 16-day-old fetus, between femur and tibia cartilages, mesenchymal cells were observed. Mesenchymal cells had marginated heterochromatin and dilated rough endoplasmic reticulum. 2. In a 17-day-old fetus, the knee joint clefts were first formed. In the primordial cruciate ligaments between the cartilages, capillaries were scattered. The apoptotic cells, which had fragmented and condensed nucleus, showed in the synovium. And necrotic cells, which had nuclear chromatin margination, perinuclear cisternae, and dilated rough endoplasmic reticulum, also were observed in the joint cleft surface. 3. From the 18-day-old fetus, phagocytic synovial cells and secretory synovial cells could be confirmed. The apoptotic cells were not seen. 4. In a 17-day-old fetus, a few cells were positive for TUNEL reaction in the joint cleft region. 5. In a 17-day-old fetus, Bax were marked on the mitochondria, endoplasmic reticulum of apoptotic cells. Also, it was marked at the phagocytosed apoptotic bodies in the neighboring cells. 6. In a 17-day-old fetus, the tissue Transglutaminase C were marked in the perinuclear region, vacuoles, cell membrane and extracellular matrix of the apoptotic cells. Also, it was marked at the phagocytosed apoptotic bodies in the neighboring cells. 7. In a 17-days-old fetus, CPP32 labeling were marked in the cytoplasm of the apoptotic cells. Practically, it was distributed between the phagocytosed apoptotic bodies and the neighboring cells. On the basis of above findings, it is obvious that the joint cleft are first formed in a 17-day-old fetus, a few cells are to be TUNEL positive signals, and the apoptotic cells contain Bax, tissue Transglutaminase C, and CPP32. Therefore the apoptotic cells and the necrotic cells are appeared in the 17-day-old fetus, and these cells are concerned with joint cleft formation.
Animals ; Antibodies ; Apoptosis ; Capillaries ; Cartilage ; Cell Membrane ; Chromatin ; Cytoplasm ; Endoplasmic Reticulum ; Endoplasmic Reticulum, Rough ; Extracellular Matrix ; Femur ; Fetus ; Goats ; Heterochromatin ; In Situ Nick-End Labeling ; Joints ; Knee Joint* ; Knee* ; Ligaments ; Mitochondria ; Pregnancy ; Rats* ; Synovial Membrane ; Tibia ; Vacuoles

Retina, a part of CNS, has served valuable and accessible tissue for elucidating the cellular properties of neurons and glia due to its similarity to brain. Unlike mammalian counterpart, avian retina is devoid of vessels and astrocytes. However little is known about glial reaction to neuronal injuries in this species. Therefore, this study was performed to investigate the microglial responses in the quail retina following neuronal injuries. The retinae from normal and optic nerve transected adult quails were studied immunohistochemically with anti-QH1, a marker known to be specific for microglia. In the normal retina, QH1-labeled microglial cells displayed typical feature of ramified (resting) form and were localized mainly in the inner plexiform layer. After optic nerve transection (ONT) morphology of microglial cells changed from the ramified to the amoeboid form. This feature of microglial cells maintained throughout the post operational periods until 28 days after ONT. Particularly, at 14 and 21 days after ONT amoeboid microglia displayed cell bodies with stout and bushy processes, suggesting active phagocytosis. The distribution pattern of microglia also changed in accord to ganglion cell degeneration: they gradually moved to layers of ganglion cells and optic nerve fibers where ganglion cell bodies and axons were under degeneration. This change of microglial distribution was most prominent at 14 days of ONT. The result of this study is generally consistent with that reported in mammalian counterpart and this similarity between the avascular avian retina and the vascularized mammalian counterpart suggests that processes of microglial activation, such as migration and phagocytosis, can occur in the vessel-free CNS tissue.
Adult ; Astrocytes ; Axons ; Brain ; Ganglion Cysts ; Humans ; Microglia* ; Neuroglia ; Neurons ; Optic Nerve Injuries* ; Optic Nerve* ; Phagocytosis ; Quail ; Retina ; Retinaldehyde*

The basement membrane, as a specialized form of extracellular matrix, is known to influence the development and differentiation of several kinds of organs and tissues. The development of palatal rugae involves morphological changes of oral epithelium, basement membrane (BM), and mesenchymal tissue and, thus, is thought to be one of the model systems of embryonic development. The BM is thought to influence also the rugae development and defferentiation. If so, the components of the BM would change during rugae development. This study was performed to observe the changes of laminin, fibronectin, and collagen type IV in the oral BM during the fetal ages 16~21 by immunohisto-chemistry. The stainability to laminin began to disappear at the anterior portion of the palate on day 17, and at the end of the gestation showed hardly the immunoreactivity to laminin. Along with the decrease of the stainability through the oral apithlium, the BM of rugae area revealed more decreased reactivity to laminin compared to inter-rugal area and such a phenomina could be observed with anterior rugae first and with the middle and posterior rugae thereafter. The stainin pattern of fibronectin was also similar to that of laminin. In case of collagen IV, it maintained the immunoreac-tivity tro collagen IV with a little decrease at the anterior rugae on day 21 of gestation. As was observed, the components of BM, especially the laminin and fibronectin, changed in its immunoreactivity in parallel to the development of the rugae, which enables us to suggest their relationship to the development and differentiation of the rugae.
Basement Membrane ; Collagen ; Collagen Type IV* ; Embryonic Development ; Epithelium* ; Extracellular Matrix ; Female ; Fibronectins* ; Gestational Age ; Laminin* ; Palate* ; Pregnancy

Nitric oxide (NO) involvement has been demonstrated in mechanisms of synaptic plasticity, particularly in hippocampal long-term potentiation, a mechanism that underlies certain forms of learning and memory. Further, NO has been shown to regulate various neurotransmitters which play an important role in learning and memory. Several findings suggest that NO production may be decreased in the aged rat. Changes in the nNOS-containing neurons with aging were demonstrated by immunocytochemistry and in situ hybridization. NOS-immunoreactive cells in aged rats were present in all cortical areas and the hippocampus, and the pattern of distribution was similar to that of the control group. The number of NOS-immunoreactive cells in the cerebral cortex was significantly decreased in the aged rats, but the extent of changes was variable in each area, and ranged from mild decrease (<30%) to severe decrease (>50%). Severely decreased areas were the cingulate cortex, parietal cortex area 1, temporal cortex area 1, 2, 3, medial part of occipital cortex area 2, monocular and binocular part of occipital cortex area 1, entorhinal cortex, hippocampus proper, dentate gyrus and subiculum. Moderately decreased areas (30~50%) were frontal cortex area 1, 2, 3, parietal cortex area 2, forelimb, hindlimb, lateral part of occipital cortex area 2. Slightly decreased area was insular cortex. Morphologically, the number of dendritic branches seemed to be decreased in aged group and the length of dendrites of NOS-IR neurons showed a tendency to shorten. These results indicate the involvement of neuronal system containing NOS in the aging brain, and provide the first morphological evidence for the loss of NOS neurons in the cerebral cortex of the aged rats by immunocytochemistry. Further multidisciplinary investigations involving normal aging and neurodegenerative disease such as Alzheimer's disease are needed to clarify the importance of nitric oxide changes in the cerebral cortex with aging.
Aging ; Alzheimer Disease ; Animals ; Brain ; Cerebral Cortex* ; Dendrites ; Dentate Gyrus ; Entorhinal Cortex ; Forelimb ; Gyrus Cinguli ; Hindlimb ; Hippocampus ; Immunohistochemistry ; In Situ Hybridization ; Learning ; Long-Term Potentiation ; Memory ; Neurodegenerative Diseases ; Neurons* ; Neurotransmitter Agents ; Nitric Oxide ; Plastics ; Rabeprazole ; Rats* ; Telescopes