This study was examined and compared the immunocytochemical distribution of the two calcium-binding proteins calbindin D-28K and parvalbumin immunreactive neurons in the medulla oblongata and spinal cord after transection of spinal cord in rats. In this experiment, calbindin D-28K immnunoreactive neurons were mainly found in many pyramidal cells distributed medulla oblongata and spina1 cord of rats. Parvalbumin immunoreactive cells were demonstrated in all lamina of the gray matter of the spinal cord. These immunoreactive cells had the most high density in the severa1 nuclei of the ventra1 horn of the all segments of the spina1 cord. Calbindin D-28K neuropil labeling was strongly noted in spina1 all segments of the spinal cord. In contrast parva1bumin immunoreactive, little differences were found in distribution, size and morphology of calbindin D-28K cell body or neuropil staining in the spinal cord. The number of parvalbumin immunoreactive cells were more than twice in the medulla oblongata and spinal cord compared to the calbindin D-28K immunoreactive cells. Calbindin D-28K and parvalbumin-immmoreactive somata were round, ova1, spind1e and polygona1 in shape, and the immunoreactive neurons were unipolar, bipolar, multipolar and horizontal in shape. The diameters of the somata of the two immunoreactive neurons were 40 ~50 micrometer, respectively. Also dendrites of two immunoreactive neurons were densely arrayed in network. These results suggest that CB-IR and PV-IR most high density in the of the VII~X layers in the ventra1 horn of the all segments of the spina1 cord.
Animals ; Calbindins* ; Calcium-Binding Proteins ; Dendrites ; Horns* ; Medulla Oblongata* ; Neurons* ; Neuropil ; Pyramidal Cells ; Rats* ; Spinal Cord Injuries* ; Spinal Cord*

The sclerotium of Poria cocos Wolf, which grows on the roots of pine trees, has long been used as a sedative, diuretic, and anti-inflammatory agent. The accumulating data revealed that certain ingredients of the sclerotium of Poria cocos showed anti-tumor activities. Although the mechanism of anti-tumor activity is not known, the polysaccharides may potentiate the host defense mechanism through the activation of immune system. In the present study we show that PCSC22, a polysaccharide isolated from the sclerotium of Poria cocos with one percent sodium carbonate, significantly induces nitric oxide (NO). Immunohistochemical staining of inducible NO synthase (iNOS) showed that the increase of NO was due to the induction of iNOS production. To further study the mechanism responsible for the induction of iNOS gene expression, we investigated the effect of PCSC22 on the activation of p38 kinase, which is important in the gene expression of inflammatory cytokines including iNOS. Western blot assay showed that PCSC22 produced phosphorylation of p38 kinase. In conclusion, we demonstrate that PCSC stimulates macrophages to express iNOS gene through the activation of p38 kinase.
Blotting, Western ; Carbon ; Cocos* ; Cytokines ; Gene Expression ; Immune System ; Macrophages* ; Nitric Oxide ; Nitric Oxide Synthase ; Phosphorylation ; Phosphotransferases ; Pinus ; Polysaccharides ; Poria* ; Sodium ; Wolves

In this present study, we show that 3HK-induced reactive oxygen species (ROS) accumulation and caspase activation lead to apoptotic cell death. Pretreatment with N-acetylcysteine (NAC), an effective antioxidant, significantly attenuated 3HK-induced apoptosis by way of a reduction of ROS accumulation and caspase activity. SKN-SN cells were protected from 3HK-induced cytotoxicity by heat shock protein (HSP). HSP90 effectively attenuated 3HK-mediated ROS accumulation and apoptosis. In addition, the protective effect of HSP90 was abolished by pretreatment with HSP90 anti-sense oligonucleotide, but not when pretreated with anti-senses for other HSPs. These results suggest that HSP90 protects SKN-SH cells from 3HK-induced cytotoxicity by reducing ROS levels and caspase activity.
Acetylcysteine ; Apoptosis ; Cell Death* ; Heat-Shock Proteins* ; Hot Temperature* ; Reactive Oxygen Species*

The purpose of this study, the effects on the cerebral cortex of the rats after brain irradiation was to investigate the change of distribution and morphology of neuropeptide-Y (NPY) neurons. Radiation was produced by the linear accelerator 6MV X-ray. The animals were categorized into control and experimental groups and we use 45 Sprague-Dawley rats weighing about 200 ~250 gm. The head areas of the animals were positioned within the radiation field of 15 cmx20 cm and with the radiation depth of 2 cm. Sodium chloral hydrate-anesthetized rats were exposed to the radiation with the dose rate of 240 cGy/min. The total dose was 1,800 cGy(rad). Animals were sacrificed on 2 hours, 5 hours, 1 day, 2 days, 3 days, 1 week, weeks, 3 weeks after brain irradiation. Using ABC immunohistochemistry, morphology and distribution of neuropeptide-Y immunoreactive neurons (NPY-IR)were studied on the cerebral cortex of the control and brain-irradiated rats. We used light and confocal laser scanning microscopy (CLSM). The following results were obtained : 1. On control group, NPY-IR neurons were found in all layers of the primary motor and sensory cerebral cortex, and the NPY-IR neurons were concentrated within the layer II, III, IV, V and VI. The typical NPY-IR perikarya was bipolar and multipolar shape. 2. On 2 hours, 5 hours after X-irradiation, decreased number of NPY-IR neurons were detected in the primary motor and sensory cerebral cortex of the rats. Also shrunken and transformed NPY-IR neurons were detected in the primary motor and sensory cerebral cortex of the rats. 3. On 1 day, 2 days, 3 days after X-irradiation, morphology and distribution of NPY-IR neurons in the primary motor and sensory cerebral cortex was generally restored. 4. On 1 week, 2 weeks, 3 weeks after X-irradiation, morphology and distribution of NPY-IR neurons in the primary motor and sensory cerebral cortex was almost similar to control group. 5. In optical serial section analysis of NPY-IR neurons, high intensity of immunofluorescence were observed in a part of the 8 ~11 sections of the control and all irradiated groups. In optical single section analysis of NPY-IR neurons, red color (high fluorescence intensity) were observed in a part of 6, 7 sections of the control and all irradiated groups. From the above results, it was concluded that the release of neurotransmitters and transcapillary leakage of blood substance were occurred on 2 hours, 5 hours, 1 day after X-irradiation, but the condition was generally restored on 3 days and 7 days following X-irradiation.
Animals ; Brain ; Cerebral Cortex* ; Fluorescence ; Fluorescent Antibody Technique ; Head* ; Immunohistochemistry ; Microscopy, Confocal ; Neurons ; Neurotransmitter Agents ; Particle Accelerators ; Rats* ; Rats, Sprague-Dawley ; Sodium

In addition to the central and the peripheral nervous system, calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) has been identified throughout the enteric nervous system. Several functions of the CGRP in gastrointestinal (G-I) tract has been identified, but the effect of CGRP on G-I motility is unclear. The distribution of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) in the murine small bowel were studied by using immunohistochemistry, also analyzed functionally by using electrophysiological method. Immunohistochemical studies demonstrated that CGRP-LI is localized in both nerve fibers and myenteric ganglion cells in the whole-mount preparation of murine small intestine. Double labelling with CGRP and c-kit investigated by confocal microscope was shown that CGRP-LI enteric nerve fiber surrounded the c-kit positive interstitial cells of Cajal (ICC). Electrophysiological finding revealed that treatment of CGRP inhibited electrical activity on culture ICC. Our results suggest a CGRP innervation of murine small bowel ICC. The released CGRP from enteric nerve terminals may induce relaxation of small bowel through the inhibition of ICC.
Animals ; Calcitonin Gene-Related Peptide* ; Calcitonin* ; Enteric Nervous System ; Ganglion Cysts ; Immunohistochemistry ; Interstitial Cells of Cajal ; Intestine, Small* ; Mice ; Nerve Fibers ; Peripheral Nervous System ; Relaxation

The nerves innervating the sublingual gland of the rat was investigated using PRV (pseudorabies virus) as a neural tracer. The neural tracer was injected into left sublingual gland of the rat. In the central nervous system, PRV immunoreactive neurons were labeled bilaterally and tended to be more densely labeled in the left side. PRV immunoreactive neuronal cell bodies and fibers were observed in insular cortex, paraventricular nucleus, deep mesencephalic nucleus, spinal trigeminal tract, lateral paragigantocellular nucleus, parvicellular reticular nucleus, raphe obscurus, gigantocellular reticular nucleus and gigantocellular reticular nucleus, alpha. The more densely labeled PRV immunoreactive neurons were found in the deep mesencephalic nucleus, spinal trigeminal tract and lateral paragigantocellular nucleus. These results may provide a neuroanatomical data on the nerves innervating the sublingual gland in the rat brain.
Animals ; Brain* ; Central Nervous System ; Herpesvirus 1, Suid* ; Neurons ; Paraventricular Hypothalamic Nucleus ; Pseudorabies* ; Raphe Nuclei ; Rats* ; Sublingual Gland* ; Trigeminal Nucleus, Spinal

Bile acids and their synthetic derivatives induced apoptosis in various kinds of cancer cells and anticancer effects. It has been reported that the synthetic chenodeoxycholic acid (CDCA) derivatives showed apoptosis-inducing activity on various cancer cells in vitro. It wasn't discovered those materials have apoptosis-inducing effects on G361 human melanoma cells. The present study was done to examine the synthetic bile acid derivatives, HS-1199 and HS-1200, induced apoptosis on G361 cells and such these apoptosis events. The viability of G361 cells was assessed by the MTT assay. Induction of apoptosis was confirmed by DNA electrophoresis and Hoechst staining. Westen blot analysis and immunofluorescent staining were performed to study the alterations in expression level and translocation of apoptosis-related proteins. Proteasome activity and mitochondrial membrane potential (MMP) change were also assayed. Tested G361 cells showed several lines of apoptotic manifestation such as activation of caspase-3, DFF and PARP, DNA degradation (HS-1200 only), nuclear condensation, inhibition of proteasome activity, reduction of mitochondrial membrane potential, and the release of cytochrome c and AIF to cytosol. Between two synthetic derivatives, HS-1200 showed stronger apoptosis-inducing effect than HS-1199 did. Taken collectively, we here demonstrated for the first time that synthetic CDCA dedrivatives induce apoptosis of human melanoma cells through the proteasome, mitochondria and caspase pathway. Therefore our data provide the possibility that HS-1200 could be considered as a novel therapeutic strategy for human melanoma cells from its powerful apoptosis-inducing activity.
Apoptosis* ; Bile ; Bile Acids and Salts ; Caspase 3 ; Chenodeoxycholic Acid* ; Cytochromes c ; Cytosol ; DNA ; Electrophoresis ; Humans* ; Melanoma* ; Membrane Potential, Mitochondrial ; Mitochondria ; Proteasome Endopeptidase Complex

In the present study, we investigated influences of glycogen synthase kinase (GSK) 3beta on the development and/or progression of amyotrophic lateral sclerosis (ALS). We used transgenic mice expressing a human Cu/Zn superoxide dismutase mutant (SOD1G93A) as an in vivo model of ALS and examined expressional changes of GSK3beta immunohistochemically in the spinal cord, brain stem and cerebellum. With these experiments we demonstrate that the neurons in these regions of symptomatic SOD1G93A transgenic mice showed increased GSK3beta immunoreactivities compared with wild-type SOD1 transgenic mice. In contrast to symptomatic SOD1G93A transgenic mice, few GSK3beta immunoreactivity changes were detected in 8w- and 13w-old presymptomatic SOD1G93A transgenic mice. These data suggest the possibility that GSK3 functions as a modulating factor of apoptosis-related alterations in ALS and that GSK3beta exert differential functions in the development and/or progression of ALS. But the exact functional significances of these changes require further elucidation.
Amyotrophic Lateral Sclerosis ; Animals ; Brain Stem ; Central Nervous System* ; Cerebellum ; Glycogen Synthase Kinases* ; Glycogen Synthase* ; Glycogen* ; Humans ; Mice ; Mice, Transgenic* ; Neurons ; Spinal Cord ; Superoxide Dismutase

The effect of peripheral nerve on limb growth and maturation has received little attention after the limb differentiation stage. This study investigated the effects of paralysis (loss of function) on growth of bones in the hind limb of Hymenochirus boettgeri. Complete denervation of the right sciatic nerve was performed at stages 63 and 66, animals were sacrificed after 6 and 7 (Groups I and II) and 12 and 13 (Groups III and IV) weeks. Denervation was assessed by degree of paralysis. Specimens were cleared and double stained with alcian blue and alizarin red for cartilage and bone. Length and area of right and left femurs and length of right and left tibia were measured by using an image analyzer program after photographing, and the ratio of right to left femur length and area was calculated. There were no gross morphological differences between the control and sham groups. In the experimental groups, the ratio of femur length was 93.71% and 95.70% in Group I and II, and 96.12% and 96.06% in Group III and IV. The ratio of tibia length was 94.05% and 96.15% in Group I and II, and 98.12% and 98.22% in Group III and IV. The ratio of femur area was 90.43% and 95.61% in Group I and II, and 96.08% and 95.73% in Group III and IV. Comparison between control and experimental groups showed statistically significant (p<0.05). There was a histologically maturational delay in proximal end of denervated hind limb, comparing with opposite side. These results suggested that denervation of sciatic nerve affect directly the growth and maturation of hind limb bones in Hymenochirus boettgeri and loss of critical period of bony maturation after denervation.
Alcian Blue ; Animals ; Bone Development ; Cartilage ; Critical Period (Psychology) ; Denervation* ; Extremities* ; Femur ; Paralysis ; Peripheral Nerves ; Sciatic Nerve ; Tibia

Retinoids play an important role in growth, reproduction and differentiation. Recently, retinoids have been used to both protect and treat from various animal models of carcinogenesis. In this study the effect of N-(4-hydroxyphenyl) retinamide (fenretinide) on viability of human neuroblastoma cell lines were evaluated. For the evaluation of apoptosis of human neuroblastoma cell lines by fenretinide. MTT assay, cytoplasmic DNA fragmentation, TUNEL stain, and Western blot analysis were performed. In MTT assay, fenretinide inhibited the proliferation of CHP134, IMR32 and SH-SY5Y but not in PC12 cells. Cytoplasmic DNA fragmentation was induced by treament of fenretinide (10 micrometer) for 48 h in IMR32 cells. PARP cleavage was detected by Western blot analysis after 16 h of treatment of fenretinide in CHP134, IMR32 and SH-SY5Y. These fenretinide effects on growth inhibition and increased apoptosis followed to the time dependent manner. The fenretinide treatment did not affect the phosphorylation of MAP kinases (ERK, JNK, p38). There was no change of Bcl-x and Bad expression after treatment of fenretinide (1 micrometer) in neroblastoma cell lines. Pretreatement of PD98059, SB203580, LY294002, or genistein also did not affect fenretinide-induced PARP cleavage in neuroblastoma cell lines. From these results, the fenretinide-induced apoptosis is due to the PARP cleavage which occured MAP kinase signal cascades independently.
Animals ; Apoptosis* ; Blotting, Western ; Carcinogenesis ; Cell Line* ; Cytoplasm ; DNA Fragmentation ; Fenretinide* ; Genistein ; Humans* ; In Situ Nick-End Labeling ; Models, Animal ; Neuroblastoma* ; PC12 Cells ; Phosphorylation ; Phosphotransferases ; Reproduction ; Retinoids