• Molecules and Cells
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• 제37권4호
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• pp.345-355
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• 2014

Mitigating secondary delayed neuronal injury has been a therapeutic strategy for minimizing neurological symptoms after several types of brain injury. Interestingly, secondary neuronal loss appeared to be closely related to functional loss and/or death of astrocytes. In the brain damage induced by agonists of two glutamate receptors, N-ethyl-D-aspartic acid (NMDA) and kainic acid (KA), NMDA induced neuronal death within 3 h, but did not increase further thereafter. However, in the KA-injected brain, neuronal death was not obviously detectable even at injection sites at 3 h, but extensively increased to encompass the entire hemisphere at 7 days. Brain inflammation, a possible cause of secondary neuronal damage, showed little differences between the two models. Importantly, however, astrocyte behavior was completely different. In the NMDA-injected cortex, the loss of glial fibrillary acidic protein-expressing ($GFAP^+$) astrocytes was confined to the injection site until 7 days after the injection, and astrocytes around the damage sites showed extensive gliosis and appeared to isolate the damage sites. In contrast, in the KA-injected brain, $GFAP^+$ astrocytes, like neurons, slowly, but progressively, disappeared across the entire hemisphere. Other markers of astrocytes, including $S100{\beta}$, glutamate transporter EAAT2, the potassium channel Kir4.1 and glutamine synthase, showed patterns similar to that of GFAP in both NMDA- and KA-injected cortexes. More importantly, astrocyte disappearance and/or functional loss preceded neuronal death in the KA-injected brain. Taken together, these results suggest that loss of astrocyte support to neurons may be a critical cause of delayed neuronal death in the injured brain.

• 대한한방내과학회지
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• 제24권2호
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• pp.348-357
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• 2003

Objectives : The purpose of this investigation is to evaluate the effects of Aucklandiae Radix Moschus(木香 麝香)and to study the mechanism for neuronal death protection in hypoxia with Embryonic day 20 (E20) cortical cells of a rat (Sprague Dawley). Methods : E20 cortical cells used in this investigation were dissociated in Neurobasal media and grown for 14 days in vitro (DIV). On 14 DIV, Aucklandiae Radix Moschus(木香 麝香) was added to the culture media for 72 hrs. On 17 DIV, cells were given a hypoxic shock and further incubated in normoxia for another three days. On 20 DIV, Moschus(麝香)'s effects for neuronal death protection were evaluated by LDH assay and the mechanisms were studied by Bcl-2, Bak, Bax, caspase family. Results : This study indicate that Aucklandiae Radix(木香)'s effects for neuronal death protection in normoxia and Scutellariae Radix(麝香)'s effects for neuronal death protection in hypoxia were confirmed by LDH assay in culture method of Embryonic day 20(E20) cortical neuroblast. Moschus(麝香)'s mechanism for neuronal death protection in hypoxia is to increase the anti-apoptosis protein Bcl-2. Conclusions : It may be reasonable to propose that Moschus(麝香) protects delayed neuronal death in hypoxia by increasing Bcl-2, thereby reducing mitochondrial permeability transition(PT) pores, the cytochrome c channels.

• The Korean Journal of Physiology and Pharmacology
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• 제2권3호
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• pp.279-286
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• 1998

It has been well documented that transient forebrain global ischemia causes selective neuronal degeneration in hippocampal CA1 pyramidal neurons with a delay of a few days. The mechanism of this delayed hippocampal CA1 pyramidal neuronal death (DND) is still controversial. To delineate the mechanisms of the DND, the effects of treatment with MK-801, an NMDA receptor antagonist, kynurenic acid, a NMDA/non-NMDA receptor antagonist, and/or cycloheximide, a protein synthesis inhibitor, on the DND were investigated in male Wistar rats. To examine the participation of apoptotic neuronal death in the DND, TUNEL staining was performed in ischemic brain section. Global ischemia was induced by 4-vessel occlusion for 20 min. All animals in this study showed the DND 3 and 7 days after the ischemic insult. The DND that occured 3 days and 7 days after the ischemia were not affected by pretreatment with MK-801 (1 mg/kg), but markedly attenuated by the pretreatment with kynurenic acid (500 mg/kg). Treatment with cycloheximide (1 mg/kg) also markedly inhibited the DND. The magnitudes of attenuation by the two drugs were similar. The magnitude of attenuation by co-treatments with kynurenic acid and cycloheximide was not greater than that with any single treatment. TUNEL staining was negative in the sections obtained 1 or 2 days after the ischemic insults, but it was positive at hippocampal CA1 pyramidal cells in sections collected 3 days after the ischemia. These results suggested that the DND should be mediated by the activation of non-NMDA receptor, not by the activation of NMDA receptor and that the activation of AMPA receptor should induce the apoptotic process in the DND.

• The Korean Journal of Physiology and Pharmacology
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• 제8권1호
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• pp.1-5
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• 2004

Transient forebrain ischemia results in the delayed neuronal death in the CA1 area of the hippo-campus. The present study was performed to determine effects of aminoguanidine, a selective iNOS inhibitor, on the generation of peroxynitrite and delayed neuronal death occurring in the hippocampus following transient forebrain ischemia. Transient forebrain ischemia was produced in the conscious rats by four-vessel occlusion for 10 min. Treatment with aminoguanidine (100 mg/kg or 200 mg/kg, i.p.) or saline (0.4 ml/100 g, i.p.) was started 30 min following ischemia-reperfusion and the animals were then injected twice daily until 12 h before sacrifice. Immunohistochemical method was used to detect 3-nitrotyrosine, a marker of peroxynitrite production. Posttreatment of aminoguanidine (200 mg/kg) significantly attenuated the neuronal death in the hippocampal CA1 area 3 days, but not 7 days, after ischemia-reperfusion. 3-Nitrotyrosine immunoreactivity was enhanced in the hippocampal CA1 area 3 days after reperfusion, which was prevented by the treatment of aminoguanidine (100 mg/kg and 200 mg/kg). Our findings showed that (1) the generation of peroxynitrite in the hippocampal CA1 area 3 days after ischemia-reperfusion was dependent on the iNOS activity; (2) the postischemic delayed neuronal death was attenuated in the early phase through the prevention of peroxynitrite generation by an iNOS inhibitor.

• 대한한의학회지
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• 제23권3호
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• pp.145-163
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• 2002

Objectives: The purpose of this investigation was to evaluate the effects of Woohwangcheongsim-won and to study the mechanism for neuronal death protection in hypoxia with Embryonic day 20 (E20) cortical cells of a rat (Sprague Dawley). Methods: E20 cortical cells were dissociated in neurobasal media and grown for 14 days in vitro (DIV). On 14 DIV, Woohwangcheongsim-won was added to the culture media for 24 hrs or 72 hrs. On 17 DIV, cells were given a hypoxic shock and further incubated in normoxia for another three days. On 20 DIV, Woohwangcheongsim-won's effects for neuronal death protection were evaluated by LDH assay, propidium iodide stain and phospho-H2AX immunostain and the mechanisms were studied by Bcl-2, Bak, Bax, caspase family, PKCα, ca1pain I. Results & Conclusions : 1. This study indicated that Woohwangcheongsim-won's effects for neuronal death protection in hypoxia were confirmed by LDH assay, propidium iodide stain and phospho-H2AX immunostain in culture method of Embryonic day 20(E20) cortical neuroblasts. 2. Woohwangcheongsim-won's mechanisms for neuronal death protection in hypoxia are to reduce the membrane damage fraction, to restrain DNA truncate, to restrain inflow of cytochrome c into cellularity caused by Bak diminution, to reduce the caspase cascade intiator caspase-8 and the effector caspase-3, to reduce the calpain I activity and to increase PKCand its activity in the membrane fraction. (J Korean Oriental Moo 2002;23(3):145～163)

• 대한한의학회지
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• 제23권4호
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• pp.125-139
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• 2002

Objectives: The purpose of this investigation is to evaluate the effects of Woohwangcheongsim-won and to study the mechanism for neuronal death protection in OGD (oxygen-glucose deprivation) model with embryonic day 20 (E20) cortical cells of a rat (Sprague Dawley). Methods: E20 cortical cells were dissociated in neurobasal media and grown for 14 days in vitro (DIV). On 14 DIV, Woohwangcheongsim-won was added to the culture media for 72 hrs. On 17 DIV, cells were given an oxygen-glucose deprivation shock (2hrs and 4hrs) and further incubated in normoxia for another three days. On 20 DIV, Woohwangcheongsim-won's effects for neuronal death protection were evaluated by LDH assay and the mechanisms were studied by Bcl-2, Bak, Bax, caspase family. Results & Conclusions: 1. This study indicates that Woohwangcheongsim-won's effects for neuronal death protection in OGD model is confirmed by LDH assay in culture method of embryonic day 20(E20) cortical neuroblasts. 2. Woohwangcheongsim-won's mechanisms for neuronal death protection in OGD model are to restrain inflow of cytochrome c into cellularity caused by Bcl-2 increase (2hrs and 4hrs), to reduce the caspase cascade initiator caspase-8 (4hrs).

• 약학회지
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• 제45권4호
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• pp.419-425
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• 2001

The neuronal cell death induced by excess glutamate (Glu) has been implicated in many acute and chronic neurodegenerative diseases including cerebral ischemia. Glu-induced elevation of intra-cellular $Ca^{2+}$ plays a critical role in the excitotoxicity, partly through the activation of a variety of $Ca^{2+}$ dependent enzymes. In the present study, we investigated the Glu-induced modulation of $Ca^{2+}$/calmodulin-dependent protein kinase IV (CaMK IV), a multifunctional enzyme abundantly present in the nuclei of neurons. The exposure of cultured rat cortical neurons to $100{\mu}$M Glu for 3 min dramatically increased CaMK IV activity up to 4.5-fold of the control-treated enzyme activity. The activation was very rapid, reaching peak at 3 min and then declined gradually. Under the same experimental conditions, time-dependent acute and delayed neuronal cell death was observed. Immunoblot analyses using specific antibodies showed that the expressions of CaMK IV and $CaMKK_{\alpha}$ were time-dependently modulated by Glu. Taken together, these results imply that the modulation of CaMK IV activity by Glu may be involved in the cascade of events resulting in neuronal cell death in cortical cultures.

• 대한한방내과학회지
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• 제24권1호
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• pp.104-112
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• 2003

Objectives : The purpose of this study is to evaluate the effects of Sohaphyang-won and is to study the mechanism for neuronal death protection in hypoxia with Embryonic day 20(E20) cortical cells of a guinea pig(Sprague Dawley). Methods : E20 cortical cells, used in this investigation were dissociated in Neurobasal media and grown for 14 days in vitro (DIV). On 14 DIV, Sohaphyang-won was added to the culture media for 72 hours. On 17 DIV, cells were given a hypoxic shock and further incubated in normoxia for another three days. On 20 DIV, Sohaphyang-won's effects for neuronal death protection were evaluated by LDH assay and the mechanism was studied by Bcl-2, Bak, Bax, caspase family. Results : This study indicates that Sohaphyang-won's effects for neuronal death protection in hypoxia is confirmed by LDH assay by the method of Embryonic day 20(E20) cortical neuroblast. Conclusions : Sohaphyang-won's mechanism for neuronal death protection in hypoxia restrains inflow of cytochrome C into cellularity caused by Bcl-2 increase and reduces the caspase cascade initiator caspase-10 and the effector caspase-3.

• 한국감성과학회:학술대회논문집
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• 한국감성과학회 2009년도 추계학술대회
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• pp.190-192
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• 2009

Kainic acid (KA), an agonist for kainate and AMPA receptors, is an excitatory neurotoxic substance. Vitamin E such as alpha-tocopherol and alpha-tocotrienol is a chain-breaking antioxidant, preventing the chain propagation step during lipid peroxidation. In the present study, we have investigated the neuroprotective effects of alphatocopherol and alpha-tocotrienol on KA-induced neuronal death using organotypic hippocampal slice culture (OHSC). After 15h KA treatment, delayed neuronal death was detected in CA3 region. Alpha-tocopherol and alpha-tocotrienol increased cell survival and reduced the number of TUNEL-positive cells in CA3 region. These data suggest that alpha-tocopherol and alpha-tocotrienol treatment have protective effects on KA-induced cell death

• 한국한의학연구원논문집
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• 제8권1호
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• pp.109-126
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• 2002

The herbal extract (YMT_02) is a modified herbal extracts from Yukmijihwangtang (YMJ) to promote memory-enhancing. The YMJ extracts has been widely used as an anti-aging herbal medicine for hundred years in Asian countries. The purpose of this study is to; 1) quantitatively evaluate the memory-enhancing effect of YMT_02 by hehavior task, 2) identify candidate genes responsible for enhancing memory by cDNA microarray and 3) assess the anti-oxidant effect of YMT_02 on PC12 cell. Memory retention abilities are addressed by passive avoidance task with Sprague-Dawley (SD) male rat. Before the training session, the rats are subdivided into four groups and administrated with YMT_02, Ginkgo biloba, Soya lecithin and normal saline for 10 days. The retention test was performed. 24 hours after the training session. The retention time of the YMT_02 group was significantly (p<0.05) delayed $({\sim}100%)$, whereas Ginkgo biloba and Soya lecithin treatment delayed 20% and 10% respectively. The hippocampi of YMT_02 and control group were dissected and mRNA was further purified. After synthesizing cDNA using oligo-dT primer, the cDNA were applied and mRNA was further purified. After synthesizing cDNA using oligo-dT primer, the cDNA were applied to Incyte rat GEMTM 2 cDNA microarray. The microarray results show that prealbumin(transthyretin), phosphotidy lethanolamine N-methyltransferase, and PEP-19 are expressed abundantly in the YMT_02 treated group. Especially, PEP-19 is a neuron-specific protein, which inhibits apoptotic processes in neuronal cell. On the other hand, transcripts of RAB15, glutamate receptor subunit 2 and CDK 108 are abundant in control group. Besides, neuronal genes involved in neuronal death or neurodegeneration such as neuronal-pentraxin and spectrin are abundantly expressed in control group. Additionally, the YMT_02 shows an anti oxidative effect in the PC12 cell. The list of differentially expressed genes may implicate further insight on the action and mechanism behind the memory-enhancing effect of herbal extracts YMT_02, for example, anti-apoptotic, anti-oxidative, and neuroprotective effects.