• 동의생리병리학회지
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• 제22권4호
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• pp.771-777
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• 2008

Chungpaesagan-tang which is used for treating patients of brain in cerebrovascular disease frequently from clinical doctor has not reported about the effect of neuronal aptosis caused of brain ischemia. The aim of this study is to investigate effect of Chungpaesagan-tang protecting neuronal cells from being damaged by brain ischemia through using organotypic hippocampal slice cultures. We caused ischemic damage to organotypic hippocampal slice cultures by oxygen and glucose deprivation. And added Chungpaesagan-tang extract to cultures. thereafter we measured area percentage of propidium iodide (PI)-stained neuronal cell, lactate dehydrogenase (LDH) levels in culture media and Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells. Area percentage of PI-stained neuronal cells and count of TUNEL-positive cells in CA1 and DG area of organotypic hippocampal slice culture were significantly decreased in pertinent density level of Chungpaesagan-tang extract. LDH levels in culture media of organotypic hippocampal slice culture were significantly decreased in pertinent density level of Chungpaesagan-tang extract. Within pertinent density level, Chungpaesagan-tang has cell protection effect that prevents brain ischemia damaging neuronal cells and apoptosis increasing.

• 대한한방내과학회지
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• 제29권1호
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• pp.231-242
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• 2008

Objectives : We can find out the experimental reports of Yanggyuksanhwa-tang, which has the function of regulating blood pressure related with cerebral disease, and increasing local cerebral blood stream volume, also has the recoveries for the damage of vessel endothelium, and endothelium hypertrophy caused by angiospasm after subarachnoid hemorrhage, and reduces the contraction of smooth muscle, so simultaneously improves necrosis. The aim of this study is to investigate effect of Yanggyuksanhwa-tang protecting neuronal cells from being damaged by brain ischemia through using organotypic hippocampal slice cultures. Methods : We caused ischemic damage to organotypic hippocampal slice cultures by oxygen and glucose deprivation, and Yanggyuksanhwa-tang extract was added to cultures. Thereafter we measured area percentage of propidium iodide (PI)-stained neuronal cell, lactate dehydrogenase (LDH) levels in culture media and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells. Results : Area percentage of PI-stained neuronal cells and count of TUNEL-positive cells in CA1 and DG area of organotypic hippocampal slice culture were significantly decreased in pertinent density level of Yanggyuksanhwa-tang extract. LDH levels in culture media of organotypic hippocampal slice culture were significantly decreased in pertinent density level of Yanggyuksanhwa-tang extract. Conclusions : Within pertinent density level, Yanggyuksanhwa-tang has cell protection effect that prevents brain ischemia damaging neuronal cells and apoptosis increasing.

• 동의신경정신과학회지
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• 제21권1호
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• pp.43-57
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• 2010

Objectives: This study was designed to assess neuroprotective effects of herb medicine against Alzhheimer's disease related brain damage in organotypic hippocampal slice culture. Methods: We induced dementia related brain damage in organotypic hippocampal slices by $\beta$-amyloid. Those slices were treated with herb medicines - Hwangryeonhaedoktang, Sopungsoongiwon. Using by PI staining, the extents of cell death were assessed. After that, we selected the best effective one among those herb medicines and the major components of that medicine were studied to reveal neuroprotective effects and related proteins by using PI stating. Results: In PI staining, Sopungsoongiwon is the best effective herb medicine between Hwangryeonhaedoktang and Sopungsoongiwon. Notopterygii Rhizoma, Corni Fructus, Areca Catechu, Aurantii Fructus Immaturus, Plantaginis Semen is the best effective one among the components of Sopungsoongiwon. Conclusions: We suggested that purgative effect would be the best effetive medicine on dementia related brain damage between clearing heat and toxic materials.

• Natural Product Sciences
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• 제11권3호
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• pp.179-182
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• 2005

We investigated that water extract of Acanthopanax sessiliflorus roots rescued the N-methyl-D-aspartate (NMDA), agonist of glutamate receptor, -induced toxicity in rat organotypic hippocampal slice culture. When the cell death in NMDA only-treated hippocampal slices was set 100%, A. sessiliflorus decreased the cell death to 75.4, 51.6, 48.9, and 40.6% at 1, 10, 50, and $100\;{\mu}g/ml$ treatment, respectively. On the basis of these results, the water extract of A. sessiliflorus roots may be a preventive agent against NMDA-induced cytotoxicity.

• Food Science and Biotechnology
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• 제16권4호
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• pp.632-635
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• 2007

This study examined the neuroprotective effect of scopoletin from Angelica dahurica against oxygen and glucose deprivation-induced neurotoxicity in a rat organotypic hippocampal slice culture. Scopoletin reduced the propidium iodide (PI) uptake, which is an indication of impaired cell membrane integrity. In addition, it inhibited the loss of NeuN, which represents the viability of neuronal cells. The results suggests that scopoletin from A. dahurica protects neuronal cells from the damage caused by oxygen and glucose deprivation.

• 대한한의학회지
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• 제29권5호
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• pp.29-40
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• 2008

Objectives : It has been reported that Sophorae Subprostratae Radix (SSR) has a neuroprotective effect on cerebral ischemia in animals. In the present study, the authors investigated the neuroprotective effect of SSR on glutamate excitotoxicity. Glutamate excitotoxicity was induced by using NMDA, AMPA, and KA in PC12 cells and in organotypic hippocampal slice cultures. Methods :Methanolic extract of SSR was added at 0.5, 5, and 50 ${\mu}$g/ml to culture media for 24 hours. The effects of SSR were evaluated by measuring of cell viability, PI-stained neuronal cell death, TUNEL-positive cells, and MAP-2 immunoreactivity. Results : SSR increased PC12 cell viabilities significantly against AMPA-induced excitotoxicity, but not against NMDA-induced or KA-induced excitotoxicity. In organotypic hippocampal slice cultures damaged by NMDA-induced excitotoxicity, SSR attenuated neuronal cell death significantly in the CA1, CA3, and DG hippocampal regions and reduced TUNEL-positive cells significantly in CA1 and DG regions. In organotypic hippocampal slice cultures damaged by AMPA-induced excitotoxicity, SSR attenuated neuronal cell death and reduced TUNEL-positive cell numbers significantly in the CA1 and DG regions. In organotypic hippocampal slice cultures damaged by KA-induced excitotoxicity, SSR attenuated neuronal cell death significantly in CA3, but did not reduce TUNEL-positive cell numbers in CA1, CA3 or DG. In organotypic hippocampal slice cultures damaged by NMDA-induced excitotoxicity, SSR attenuated pyramidal neuron neurite retraction and degeneration in CA1. Conclusions : These results suggest that the neuroprotective effects of SSR are related to antagonistic effects on the NMDA and AMPA receptors of neuronal cells damaged by excitotoxicity and ischemia.

• 동의생리병리학회지
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• 제21권6호
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• pp.1401-1406
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• 2007

The present study investigated neuroprotective effects Rehmanniae Radix on PC12 cells and hippocampal neural cells. PC12 cells were damage by $H_2O_2$ and nitric oxide and organotypic hippocampal slice cultures were damaged by oxygen-glucose deprivation. Then methanol extract of Rehmanniae Radix was treated with 0.5, 5, and $50\;{\mu}g/ml$ in culture media. Effects of Rehmanniae Radix were evaluated with cell viability assay, PI-staining, and TUNEL-labeling. Treatment of Rehmanniae Radix ($with\;5\;and\;50\;{\mu}g/ml$) produced significant increase of cell viability of PC12 cells damaged by $H_2O_2$ and by SNP-induced nitric oxide. Treatment of Rehmanniae Radix produced significant decrease of PI-uptake % in CA1 ($with\;5\;and\;50\;{\mu}g/ml$) and DG ($with\;50\;{\mu}g/ml$) regions of organotypic hippocampal slice cultures damaged by oxygen-glucose deprivation. Moreover, treatment of Rehmanniae Radix produced significant decrease of TUNEL- positive cells in CA1 ($with\;5\;and\;50\;{\mu}g/ml$) and DG ($with\;50\;{\mu}g/ml$) regions of organotypic hippocampal slice cultures damaged by oxygen-glucose deprivation. These results suggest that methanol extract of Rehmanniae Radix has neuroprotective effects on PC12 cells damaged by oxidative stress and on organotypic hippocampal slice cultures damaged by oxygen-glucose deprivation.

• The Korean Journal of Physiology and Pharmacology
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• 제10권3호
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• pp.123-129
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• 2006

In several experimental models, estrogens protect neurons against ischemic insults. However, the recent clinical studies of hormone replacement showed negative results to prevent stroke. Therefore, optimal models to study estrogen replacement for neuroprotection are needed before its clinical ap-plication. Organotypic hippocampal slice under oxygen-glucose deprivation (OGD) has been established as a model of cerebral ischemia and has advantages to study drug effects. We investigated whether estrogen protected CAI neurons and affected activation of Akt (pAkt) in CAI region under OGD. Thus, rat hippocampal slices on day 7 of culture were treated with $17-{\beta}$ estradiol (E, 1 nM) for 7 days before 30 min OGD, and cell death of CAI neurons was quantified by propidium iodide (PI) staining and expression of pAkt was studied by Western blot and immunofluorescence. PI intensity in slices treated with E was significantly reduced 72 hour after OGD compared to that of non-treated slices (p < 0.05). E pretreatment also increased the expression of pAkt 72 hour after OGD compared to that of no treatment (p<0.01). These data suggest that estrogen pretreatment may rescue neurons from ischemic insults through the activation of Akt and also indicate that our model would be a useful alternative method to study the mechanisms and effects of estrogen replacement treatment for neuroprotection.

• Journal of Korean Neurosurgical Society
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• 제29권8호
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• pp.1008-1018
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• 2000

Objective : Glutamate induced excitotoxicity is one of the leading causes of cell death under pathologic condition. However, there is controversy whether excitotoxicity may also participate in the neuronal death under low intensity insult such as simple hypoxia or hypoglycemia. To investigate the role of NMDA receptor in low intensity insult, we chose anoxia as the method of injury and used organotypically cultured hippocampal slice as the material of experiment. Materials & Methods : The hippocampal slices cultured for 2-3 weeks were exposed to 60 minutes of complete oxygen deprivation(anoxia). Neuronal death was assessed with Sytox stain. Corrected optical density of fluorescence in gray scale, used as cellular death indicator, was obtained from pictures taken at 24 and 48 hours following the insult. The well-known in vivo phenomenon of regional difference in susceptibility of hippocampal sub-fields to ischemic insult was reproduced in HOSC(hippocampal organotypic slice culture) by complete oxygen deprivation injury. Results : $CA_1$ was the most vulnerable to complete oxygen deprivation in hippocampus while $CA_3$ was resistant. Oxygen deprivation for 10 and 20 minutes with glucose(6.5g/l) present was insufficient to induce neuronal death in the cultured hippocampal slice. However, after 30 minutes exposure under anoxic condition, neuronal death was able to be detected in the center of $CA_1$ area. The intensity and area of fluorescence indicating cell death correlated with the duration of oxygen deprivation. NMDA receptor and non-NMDA receptor blocking with MK-801(30 & $60{\mu}M$) and CNQX($100{\mu}M$) did not provide cellular protection to HOSC against damage induced by oxygen deprivation, but increased intracellular calcium buffering capacity with BAPTA-AM($10{\mu}M$) was effective in preventing neuronal death (p=0.01, Student's t-test). Cycloheximide($1{\mu}g/ml$, $10{\mu}g/ml$) provided no protection to HOSC against insult of complete oxygen deprivation for 60 minutes and combined therapy of MK-801(30 & $60{\mu}M$) and cycloheximide(1 & $10{\mu}g/ml$) was also ineffective in preventing neuronal death. Conclusion : The results of this study show that the another mechanism not associated with glutamate receptor(NMDA & non NMDA) may play major role in cell death mechanisms induced by complete oxygen deprivation and increased intracellular calcium during anoxia may participate in the neuronal death mechanism of oxygen deprivation. Further investigation of the calcium entry channel activated during oxygen deprivation is necessary to understand the neuronal death of anoxia.

• Applied Microscopy
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• 제42권4호
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• pp.200-206
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• 2012

Induction of neurogenesis can occur in the hippocampus in response to various pathological conditions, such as Alzheimer's disease. The aim of this study was to investigate the changes that occur in endogenous neural stem cells in response to amyloid beta $(A{\beta})_{25-35}$-induced neuronal cell damage in organotypic hippocampal slice cultures. Cresyl violet staining and Fluoro-Jade B staining were used to detect neuronal cell damage and changes of mossy fiber terminals were observed by Timm's staining. The immunofl uorescence staining was used to detect the newly generated cells in the subgranular zone (SGZ) of the dentate gyrus with specific marker, 5-bromo-2'-deoxyuridine (BrdU), Ki-67, Nestin, and doublecortin (DCX). In compared to control slices, neuronal cell damage was observed and the mossy fibers were expanded to CA3 area by treatment with $A{\beta}_{25-35}$. Ki-67/Nestin- and BrdU/DCX-positive cells were detected in the SGZ. In conclusion, these results demonstrate that $A{\beta}$-induced neuronal damage results in an increase in endogenous neural stem cells in rat hippocampal slice cultures not only for gliosis but also for neurogenesis.