Up-regulation of PSD-95 in the Rat Hippocampal Formation after Chronic Renal Failure / 대한해부학회지

The mechanism of central nervous system (CNS) dysfunction in uremia are multifactorial and only partially characterized. Studies using hippocampal formation (HF) evaluate the relationship between the uremia and memory impairment. Immunoblots with calcium permeable NMDA (N-methyl-D-aspartate) and AMPA (2-amino-3-hydroxy-5-methylisoxazole-4-propinoic acid) receptors and their associated PSD-95 proteins after chronic renal failure (CRF) provided significant new informations. CRF rats induced by 5/6 nephrectomized had significant effects on up-regulation of PSD-95 protein rather than those of calcium permeable NMDA and AMPA receptor subunits. Up-regulation of PSD-95 after CRF might be associated with the enhanced activity of NMDA and/or AMPA receptors, thereby leads to the intracellular Ca2+ accumulation and functional neuronal cell damage subsequently. Degradation of intermediate filament 200 (NF200) in the axon after CRF may induce an impairment of intracellular transport and eventual cellular dysfunction through destruction of the neuronal cytoarchitecture. These data suggest that up-regulation of PSD-95 in CRF may increase the functional derangement between the nerve cells and ultimately lead to memory impairment.

Bosentan Attenuates Compensatory Left Ventricular Hypertrophy Induced by Aortocaval Fistula in Rats

BACKGROUND AND OBJECTIVES: Endothelin-1 (ET-1) is increased in advanced congestive heart failure and pulmonary hypertension associated with increased pulmonary blood flow. The role of ET-1 and the protective effect of the dual endothelin receptor antagonist, Bosentan, were investigated in overcirculation-induced compensatory left ventricular (LV) hypertrophy using aortocaval fistula (AVF) rats. MATERIALS AND METHODS: Twenty one 8-week-old rats were randomized into sham control, AVF and Bosentan (100 mg/kg/day) treatment groups. Four weeks later, the cardiac hypertrophy, pulmonary artery morphometry, plasma and tissue levels of ET-1 and the immunoreactive signal of ET-1 were evaluated in the heart and lung tissues. RESULTS: Chronic AVF developed LV hypertrophy and markedly increased the plasma and tissue ET-1 levels in the heart and lung compared to those in the controls (p<0.05), and these changes were attenuated by Bosentan treatment (p<0.05). However, the wall thickness of the pulmonary arteriole did not change. In addition, the immunoreactive signal of ET-1 was increased in the heart after AVF compared with the controls, and was also slightly decreased with Bosentan treatment. However, there were no remarkable differences in the lung tissue. CONCLUSION: ET-1 was up-regulated in compensatory LV hypertrophy induced by AVF. Bosentan attenuates cardiac hypertrophy and decreases the ET-1 levels in the plasma, heart and lung. Therefore, it is speculate that chronic treatment of an ET-1 antagonist may provide an additional strategy for AVF-induced compensatory LV hypertrophy.

Nonspecific association of 2',3'-cyclic nucleotide 3'-phosphodiesterase with the rat forebrain postsynaptic density fraction

The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a protein of unknown function in vivo, is abundantly expressed in myelinating glia in two isoforms, CNP1 and CNP2. In this study, immunoblot analysis showed that CNP1 is the major isoform in adult forebrain, and that both isoforms are included in the postsynaptic density (PSD) fraction and tyrosine-phosphorylated at the basal level. However, subcellular distribution and detergent extraction data showed that CNP is nonspecifically associated with the PSD fraction. Immunocytochemistry revealed that CNP is detected, in a weak but punctate pattern, in dissociated rat hippocampal neurons of 3 days to 2 weeks in vitro. The CNP-positive punctae were distributed throughout soma and dendrites, and distinct from PSD95-positive ones. Immunoblot analysis indicated that CNP is also expressed in neuronal stem cell lines, HiB5 and F11. Interestingly, in addition to the known two isoforms, a new CNP isoform of MW 45 kDa was expressed in these cell lines and was the major type of isoform in F11 cells. Taken together, our data suggest that CNP is expressed in the early stage of in vitro development and nonspecifically included in the adult rat PSD fraction.

The Effect of Tumor Necrosis Factor-alpha in Cultured Neonatal Rat Cardiomyocytes

PURPOSE: Tumor necrosis factor-alpha(TNF-alpha) is a pro-inflammatory cytokine that has been implicated in the pathogenesis of cardiovascular disease. Serum levels of TNF-alpha are elevated in many human cardiac related pathogenic conditions, including heart failure. It is well known that TNF-alpha inhibits myocardial contractility and induces apoptosis of adult rat cardiomyocytes via stimulation of TNF receptor 1. But pathophysiologically relevant low levels of TNF-alpha can not induce apoptosis of neonatal cardiomyocytes. So, we evaluated the effects of different concentrations of TNF-alpha in cultured rat neonatal cardiomyocytes : apoptosis or necrosis. METHODS: Neonatal ventricular myocytes were isolated from 3-day-old rats by stepwise collagenase dissociation, and the cells were cultured for 3 days. After that, cardiomyocytes were treated with low(25 ng/mL) and high(250 ng/mL) concentration of TNF-alpha for 48 hours. Apoptosis was determined by terminal deoxynucleotidyl transfer-mediated end labelling(TUNEL) staining, and cell viability was evaluated by lactate dehydrogenase(LDH) measurements using cell culture supernatants. RESULTS: Low dose TNF-alpha did not induce apoptosis compared with controls(10.5 +/- 3.5% : 10.4 +/- 4.3%). And high dose TNF-alpha also did not induce significant apoptosis(10.2 +/- 3.6% : 10.4 +/- 4.3%). There was no detectable morphological changes of cardiomyocytes after low and high concentration of TNF-alpha treatment. LDH levels after TNF-alpha treatment was not significant compared with control(control : low : high, 3.2 +/- 0.1% : 3.1 +/- 0.2% : 3.3 +/- 0.3%). CONCLUSIONS: Our results suggest that high concentration of TNF-alpha alone can not induce apoptosis and significant cytotoxicity in neonatal rat cardiomyocytes.

Expression of the NR2A and 2B Subunits of N-Methyl-D-aspartate Receptors in Hypoxic Neonatal Rat Hippocampus

PURPOSE: The developing brain has been reported to be extremely susceptible to toxicity of ischemia and/or hypoxia during a restricted developmental period. Hippocampal neuronal cell death is a typical type of perinatal hypoxic brain lesion and often coexists with other forms of cerebral hypoxic injuries. In the present study, we examined whether transcriptional changes of NR2A and NR2B subunits of the N-Methyl-D-aspartate(NMDA) receptors related to the neuronal cell death to hypoxic toxicity are involved in developing neurons in the hippocampus. METHODS: We examined the lesion produced by in vivo direct exposure of 92% N2 and 8% O2 for 2 hours at postnatal day 7. Hippocampal sections from the 7th and 14th days after hypoxia were obtained, and the amount of the NR2A and NR2B mRNA subunits were measured by in situ hybridization using the antisense probe to the NR2A and NR2B subunits. To determine the effects of molecular changes of NMDA receptor subunits, morphological changes of neurons and/or accompanying astrocytosis were evaluated by H&E and immunohistochemical stain. RESULTS: Fourteen days after hypoxia, the expression of NR2B significantly increased whereas NR2A showed distinct reduction compared with that of control rat pups. At this time, unexpectedly, neurons in CA3 region showed prominant reduction of the actual numbers and accompanied reactive astrocytosis. CONCLUSION: Alteration of NR2A and NR2B expression to hypoxic insults, suggest the possibility that changes of the NR2 subunits which can alter the function of the NMDA receptor play a crucial role in the occurrence of developmentally specific hippocampal neuronal injury.

Reactive Astrocytosis in Post-ischemic Rat Hippocampal Formation / 대한해부학회지

In the present studies, changes of the glial fibrillary acidic protein (GFAP) expression in the astrocytes of the rat hippocampal formation were examined in response to the bilateral carotid artery occlusion for 10 minutes along with a decrease of mean arterial blood pressure (MABP) to 50 mmHg. Their relations to neuronal viability were also studied by H&E staining. In early postischemic period, mild increase of the GFAP expression was observed and this was not only confined to the mild-necrotic (CA3 and dentate gyrus) regions but also in the non-necrotic regions (CA1 and subiculum) at postischemic 8 h. This suggest that astrocytosis during early postischemic period may be resulted from nonspecific reaction associated with changes in brain environment. In contrast, in late phase of the postischemia, a marked increase of the GFAP expression was observed at day 4. Moreover, cell bodies were significantly larger and many prominent and numerous processes were observed, suggesting that this may also contribute to the significant increase in the GFAP expression. Importantly, these cellular changes were only confined to the regions of massive necrosis such as subiculum and inner granular cell layer of dentate gyrus and were not observed in the non-necrotic regions (except CA1). In contrast, the GFAP expression in astrocytes were returned to control levels in mildly damaged CA3 region by 4 days. Thus reactive astrocytosis with upregulation of the GFAP in the late postischemic period with structural transformation in the regions of massive necrosis may contribute to the damages in the neighboring neurons.

p53 mRNA Expression after Hypoxia and Reoxygenation in Hippocampal CA1 and CA3 Regions / 대한해부학회지

In the present study, we examined the p53 mRNA expression in neuronal cell injury using a hypoxia and reoxygenation model of neuronal toxicity in hippocampal CA1 and CA3 regions. Reoxygenation for 6 hours to 3 days, after an exposure to hypoxic condition for 2 hours, produced a significant increase in p53 mRNA expression both in CA1 and CA3 regions compared to those in the control. In order to determine whether these changes in p53 mRNA expression in CA1 and CA3 have an effect on hypoxia-induced apoptotic or necrotic changes, TUNEL and H & E staining were applied to the hippocampal neurons. Interestingly, the CA1 region only showed most of strong TUNEL positive reaction whereas TUNEL positive reaction was weak in the CA3 region at reoxygenation time points. In addition, one of the particular morphological changes in CA1 neurons is the shrinkage of some neurons although most of neurons showed normal. However, the prominent neuronal changes in the CA3 region was that there were extensive red neurons containing eosinophilic cytoplasm and several dark neurons showing pyknotic nuclei, expanded perineuronal spaces, and cork-screw processes which are considered to typical necrotic degeneration. These results suggest that increased p53 expression might play important roles in hippocampal cell injury and their molecular mechanisms underlying cell injury may strongly depends upon the different properties of each hippocampal regions.

Changes of NR2B and c-fos mRNA Expressionin Rat Hippocampal CA1 Neuronal ChangeFollowing Hypoxia and Reoxygenation / 대한해부학회지

Vulnerability of hippocampal CA1 neurons after brain hypoxia is coupled with high concentration of NMDA recep-tor subunits especially NR2 subunits. These subunits can be regarded as modulators, since NMDA receptors display different properties depending on which of the NR2 subunits assembles with NR1. Stimulation of NMDA receptors opens a receptor-gated Ca2+ channel; and the Ca2+ influx is believed to play a critical role in c-fos expression that require alterations in gene expression. We examined the effect of the NMDA subunits (NR1, NR2A, NR2B) mRNA level on the c-fos activation and CA1 neuronal change by in situ hybridization and TUNEL method following hypoxia and reoxygenation. Present studies show that high expression and variability of NR2B mRNA in CA1 region, compared with those of NR1 and NR2A, at our experimental time window suggest that NR2B is main functional subunit in the determining of properties of NMDA receptor in hypoxic hippocampal CA1 cells. From 0 hour to 6 hours, TUNEL positive reaction and c-fos mRNA expression were steadily increased. Thereafter, TUNEL positive reaction was decreased compared with that of 6 hours and c-fos mRNA expression was decreased and then steadily re-increased from 3 days to 6 days. Therefore, high expression of NR2B and subsequent Ca2+ influx may play important roles in the hippocampal CA1 neuronal apoptosis and c-fos expression. In addition, c-fos expression may play different roles in neuronal apoptosis in a time-dependent manner.

Nitric Oxide-Mediated Cytotoxicity of Manganese in Basal Ganglia Neuronal Cells / 예방의학회지

OBJECTIVES: We have investigated to manifest whether manganese-induced neurotoxicity is mediated by nitric oxide(NO) in the rat primary neuronal cultures and assess the effect of Mn2+ on the N-methyl-D aspartate(NMDA) receptors. METHODS: We have used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay to examine the effect of cytotoxicity of MnCl2 in neuronal cells . NO production was determined by measuring nirites, a stable oxidation product of NO. The neurons in the rat that contains neuronal nitric oxide synthase(nNOS) were examined by immunofluorescence and confocal microscopy. The effects of Mn2+ on the NMDA receptors was assesed by the whole cell voltage clamp technique. RESULTS: We showed that the NO release and NOS expression were increased with 500uM MnCl2 treatment and an NOS inhibitors, NG-nitro-L-arginine , prevented neurotoxicity elicited by manganese. In the electrophysiological study, Mn2+ does not block or activate the NMDA receptors and not pass through the NMDA receptors in a neurons of basal ganglia. CONCLUSIONS: It is concluded that manganese neurotoxicity in basal ganglia was partially mediated by nitric oxide in the cell culture model.

Effects of Transient Cerebral Ischemia on the Expression of N-methyl-D-aspartate receptor subunit 2A, 2B and Neurofilament 200 in the Rat Cerebral Cortex / 대한해부학회지

Transient cerebral ischemia was induced by bilateral common carotid artery ligation with reperfusion to understand its effect on the expression of NMDA receptor subunits 2A (NR2A), 2B (NR2B), and NF200 The changes of the expressions of NR2A, NR2B, and NF200 in cerebral postsynaptic density (PSD) were evaluated through immunoblot analyses. The expressions of NR2A and NF200 were markedly decreased until 18 hours after reperfusion, while that of NR2B was increased. The immunohistochemistry with NFIGO antibody showed that NF200 protein, which is a marker for neuronal damage, was also significantly decreased at this time point indicating neuronal damages, and the morphological damages of neuronal cells were evident by hyperchromatic condensation of nucleus, irregular cell membrane, displacement of nucleus, and chromatolysis of Nissl substances in toluidine blue stain. However, from 18 hours to 3 day after reperfusion, immunoblot analyses showed that NF200 was increased significantly, while the expression of NR2A were recovered to the control level and that of NR2B was returned to somewhat higher level than control. The NR1/NR2B-type receptor is known to have a longer offset decay time than NR1/NR2A-type ones, and to be more potent in Ca2 influxing. Therefore, our results suggest that, until 18 hours, neurons are damaged by overinflux of Ca2 through NR1/NR2B receptors which helps to degrade NF200 by Ca2 sensitive professes resulting in damages to intracellular transport. The fact that the expression of NF200 was increased even though the NR2A and NR2B are control level during 18 hours to 3 days after damage suggests that NMDA receptor subunits expressed at this time may not form functional receptors. The worsening of some neuronal damages after 3 days may indicate that an abnormal reorganization of elevated NF200 between 18 hours to 3 days further disturb intracellular transport and functions of cell membrane which cause cell death.

NR1/NR2B receptor Activation induces Cytoskeletal Proteolysis in Partial Ischemic Rat Hippocampal Formation / 대한해부학회지

In the rat brain, partial ischemia causes a delayed neuronal degeneration that occurs hours to days after reoxygenation. It is generally thought that the ischemic damage is initiated by neurotoxicity mediated through glutamate receptors, particulaly NMDA subtypes. Calcium entry through the NMDA receptor is responsible for the synaptic plasiticity and neuronal pathology. Degradation of MAP-2 and NF200, a major components of neuronal cytoskeleton, by Ca2+-dependent protease after NMDA receptor activation has been postulated in delayed neuronal damage. Calcium-activated protease calpain, excessive degradation of MAP-2, together with the calpain-sensitive microtubule and neurofilaments, would be expected to disrupt intracellular transport- and membrane-related functions that is vital to neurons. Changed of NR subunit 2A, 2B, MAP2 and NF200 in rat hippncampal postsynaptic density[PSD] after partial ischemic injury were investigated though immunoblot analyses. To understand the effect of Ca2+, influx through NMDA receptors on neuronal damage which is manifested by cytoskeletal disruption, morphological change was examined through immunohistochemistry and routine staining method. We found that immunoreactivity to NR2B receptor subuit in the hippocampal formation PSD was upregulated while MAP2 and NF200 was down-regulted at 18 hours after initial partial ischemic insult. On the other hand, morphological changes of neuronal cell in partial ischemic conditions were manifested as eosinophilic inclusion bodies in the cytoplasm which is progression of neuronal damage after 6 days. Calcium influx through NR1/NR2B receptor channel may activate intracellular proteases which would degrade cytoskeleton. Proteolysis of cytoskeleton leads to its reorganization and eventually damages normal function of cell membrane which cause neuronal cell death.

Enhancement of N-methyl-D-aspartateNMDA Receptor 2B-mediated Neurotoxi-city after Hypoxia in the Rat Hippocampal Formation / 대한해부학회지

Ischemic brain hippocampal formation has been developed to understand the relationship between delayed neuronal damage and the expression of NMDA receptor subunits[NR2A, NR2B], MAP2, and NF200 in ttle conditions of hypoxia. Changes of NR subunits[NR2A, 2B], MAP2 6nd NF200 in rat brain postsynaptic density[PSD] after hypoxic injury were investigated through immunoblot analyses. To understand the effect of Ca2+ influx through NMDA receptors on neuronal damage which is manifested by morphological change, cytoskeletal disruption was examined through H & E, toluidine blue and immunohistochemical studies. The expression of NR2B was increased than normal at 30 hours after hypoxia. At this time, the expression of MAP2 and NF200 was markedly decreased and their morphology was more eosinophilic than normal and then became darker with expanded perineuronal space. Irreversible neuronal cell damage in hypoxic hippocampal formation is most prominent in CA3 region of hippocampus and the process is triggered by Ca2+ influx through NR1/MR2B receptor channel at 30 hour after initial hypoxic insult. Ca2+ influx through NR1/MR2B receptor channel may activate intracellular proteases which would degrade cytoskeleton. Proteolysis of cytoskeleton leads to its reorganization and eventually damages normal function of cell membrane which causes neuronal cell death. And, morphological changes of neuronal cells in hypoxic conditions were manifested as red neurons in the stage of reactive change, and as dark neuron in the stage of late hypoxic cell damage.

Up-regulation of NMDA Receptor Subunit 2B Induces Degradation of Cytoskeletons in Hypoxic Rat Cerebral Cortex / 대한해부학회지

In the rat brain, global hypoxia cause a delayed neuronal degeneration that occurs hours to days after reoxygenation. It is generally thought that the ischemic damage is initiated by neurotoxicity mediated through glutamate receptors, particulary NMDA subtypes. Calcium entry through the NMDA receptor is responsible for the synaptic plasiticity and neuronal pathology. Degradation of MAP-2 and NF200, a major components of neuronal cytoskeleton, by Ca2+-dependent protease after NMDA receptor activation has been postulated in delayed neuronal damage. Changes of NR subunit 2B, MAP2 and NF200 in rat brain postsynaptic density[PSD] after hypoxic injury were investigated through immunoblot analyses. To understand the effect of Ca2+ influx through NMDA receptors on neuronal damage which is manifested by cytoskeletal disruption, morphological change was examined through immunohistochemistry and H & E staining. We found that immunoreactivity to NR2B antibody in the cerebral cortex PSD was up-regulated while MAP2 and NF200 was down-regulated at 30 hours after initial hypoxic insult. At this time, morphological changes of neuronal cells in hypoxic conditions were manifested as down-regulation of MAP2 and NF200 immunoreactivities, hyperchromatic condensation of cytoplasm and nucleus, homogenizing cell change, expansion of perineuronal space and dispersion of chromatin. From 3 days, NR2B, MAP2, NF200 were up-regulated simultaneously. On the other hand, morphological alterations in hypoxic neurons were progress further. Our present results suggests that Calcium influx through NR1/NR2B receptor channel is effective whithin 30 hours but ineffective from 30 hours. Delayed neuronal cell death triggered by Ca2+ influx through NR1/NR2B receptor channel within 30 hours, which may activate intracellular profeases. Proteolysis of cytoskeleton by activated protease leads to its abnormal reorganization and eventually damages normal function of cell membrane which causes neuronal cell death.

Mosaic-trisomy 22 with Inv(22)(p12-q13) / 체질인류학회지

No abstract available.