• Natural Product Sciences
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• v.15 no.3
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• pp.125-129
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• 2009

Neurodegenerative diseases such as Alzheimer's disease, stroke, and Parkinson's disease, are caused by neuronal cell death. Apoptosis, oxidative stress, inflammation, excitotoxicity or ischemia are discussed to play a role of neuronal cell death. In order to find the candidate of neuroprotective agent, neuroprotective activity of some medicinal plants was investigated with in vitro assay system using glutamate-induced neurotoxicity in primary cultures of rat cortical cells. The aqueous methanolic extracts of twenty-seven medicinal plants were evaluated the protective effects against glutamate-injured excitotoxicity in rat cortical cells at the concentration of 50 $\mu$g/ml and 100 $\mu$g/ml, respectively. Among them, extracts of Lonicera japonica, Taraxacum platycarpum, Polygonum aviculare, Gardenia jasminoides, Forsythia viridissima, Lygodium japonicum, Panax notoginseng, Akebia quinata, Anemarrhena asphodeloides and Phellodendron amurense showed significantly neuroprotective activities against glutamate-induced neurotoxicity in primary rat cortical cells.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.6
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• pp.353-361
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• 2011

In this study, cyanidin-3-glucoside (C3G) fraction extracted from the mulberry fruit (Morus alba L.) was investigated for its neuroprotective effects against oxygen-glucose deprivation (OGD) and glutamate-induced cell death in rat primary cortical neurons. Cell membrane damage and mitochondrial function were assessed by LDH release and MTT reduction assays, respectively. A time-course study of OGD-induced cell death of primary cortical neurons at 7 days in vitro (DIV) indicated that neuronal death was OGD duration-dependent. It was also demonstrated that OGD for 3.5 h resulted in approximately 50% cell death, as determined by the LDH release assay. Treatments with mulberry C3G fraction prevented membrane damage and preserved the mitochondrial function of the primary cortical neurons exposed to OGD for 3.5 h in a concentration-dependent manner. Glutamate-induced cell death was more pronounced in DIV-9 and DIV-11 cells than that in DIV-7 neurons, and an application of $50{\mu}M$ glutamate was shown to induce approximately 40% cell death in DIV-9 neurons. Interestingly, treatment with mulberry C3G fraction did not provide a protective effect against glutamate-induced cell death in primary cortical neurons. On the other hand, treatment with mulberry C3G fraction maintained the mitochondrial membrane potential (MMP) in primary cortical neurons exposed to OGD as assessed by the intensity of rhodamine-123 fluorescence. These results therefore suggest that the neuroprotective effects of mulberry C3G fraction are mediated by the maintenance of the MMP and mitochondrial function but not by attenuating glutamate-induced excitotoxicity in rat primary cortical neurons.

• YAKHAK HOEJI
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• v.39 no.4
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• pp.444-449
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• 1995

Primary cultures of rat cortical and chicken embryonic brain cells were employed to establish a reliable screening method for natural products blocldng or enhancing glutamate-induced neurotoxicity. Exposure of primary cultured rat cortical cells or chicken embryonic brain cells to high dose of glutamate resulted in the fragmentation of neutites and consequent neuronal death. The level of cytoplasmic lactate dehydrogenase(LDH), indicator for cell survival in cultures, was significantly reduced at exposure to glutamate. For the practical application of the methods, series of concentrations of plants extracts and positive control were applied prior to the glutamate insult on primary cultures of rat cortical and chicken embryonic, brain cells. Relative LDH level in cells was measured for the estimation of the effect of the test materials on the glutamatergic neurons. The validity of the present screening method for natural products acting on glutamatergic neurons was examined with dextromethorphan, a known glutamatergic antagonist. The treatment of 100 $\mu{M}$ dextromethorphan prevented the reduction of LDH in rat cortical and chicken embryonic brain cells caused by glutamate insult keeping 60% and 90% of LDH level in normal control, respectively. Above results indicate that primary cultures of rat cortical and chicken embryonic brain cells could be proper systems for the screening of potential natural agents acting on glutamatergic, neurons. Between the two types of cultures, primary culture of chicken embryonic brain cells seemed to be a better system for the primary screening, since it is technically easier and economical compared to that of rat cortical cells.

• Proceedings of the Ginseng society Conference
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• 2007.12a
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• pp.62-62
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• 2007

• YAKHAK HOEJI
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• v.45 no.4
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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.

• YAKHAK HOEJI
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• v.43 no.4
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• pp.535-540
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• 1999

Glutamate (Glu) receptor-mediated excitoxicity has been implicated in many acute and chronic types of neurological disorders. Exposure of mature rat cortical neurons (15-18 days in culture) to the various concentrations of Glu resulted in a marked neuronal death, whereas immature rat cortical neurons (4∼5 days in culture) were resistant to the Glu-induced toxicity. Glu receptor subtype-specific agonists showed differential extent of toxicity in the immature neurons. The neurons treated with NMDA or kainate (KA) did not exhibit damage. However, quisqualate (QA) treatment induced a considerable cell death (36.1%) in immature enurons. The non-NMDA antagonist DNQX did not reduce this response. Interestingly, the QA-induced toxicity was potentiated by spermine in a concentration-dependent manner. Again, the spermine-enhanced damage was not altered by the polyamine antagonist ifenprodil. Taken together, unlike NMDA or KA, QA can induce neurotoxicity in immature rat cortical neurons and the QA-induced toxicity was potentiated by spermine. The lack of antagonizing effects of DNQX and ifenprodil on QA-induced toxicity and the potentiated toxicity by spermine, respectively, implies that both QA receptor and the polyamine site of NMDA receptor may not mediate the neurotoxicity observed in this study, and that a distinct mechanism(s) may be involved in excitotoxicity in immature neurons.

• Development and Reproduction
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• v.2 no.2
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• pp.157-163
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• 1998

Preimplantation embryos of the rat was examined by the morphological changes in the cortical granule envelope (CGE), blastomere surface, and zona pellucida (ZP) of embryo after cortical reaction. The ultrastructural characteristics and CGE of embryos were observed with the scanning electron microscope and fluorescence microscope. In the ultrastructural characteristic of embryo surface, surface microvilli were shortened and the CGE-like structure existed above microvilli in eight-cell embryo. Rough spongy surface and decreased network numbers were key characters of embryonic ZP compared to unfertilized oocyte. The CGE formed by cortical reaction existed in perivitelline space during embryo development but it was thin and locally distributed ill contrast to fertilized oocyte. The present results indicate that cortical reaction forms cortical granule envelope in perivitelline space and causes not only zona hardening, but also ultrastructural changes in ZP and cell membrane of preimplantation embryos.

• Clinical and Experimental Pediatrics
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• v.55 no.7
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• pp.238-248
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• 2012

Purpose: Hypoxic-ischemic encephalopathy is an important cause of neonatal mortality, as this brain injury disrupts normal mitochondrial respiratory activity. Carnitine plays an essential role in mitochondrial fatty acid transport and modulates excess acyl coenzyme A levels. In this study, we investigated whether treatment of primary cultures of rat cortical neurons with L-carnitine was able to prevent neurotoxicity resulting from oxygen-glucose deprivation (OGD). Methods: Cortical neurons were prepared from Sprague-Dawley rat embryos. L-Carnitine was applied to cultures just prior to OGD and subsequent reoxygenation. The numbers of cells that stained with acridine orange (AO) and propidium iodide (PI) were counted, and lactate dehydrogenase (LDH) activity and reactive oxygen species (ROS) levels were measured. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the terminal uridine deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were performed to evaluate the effect of L-carnitine (1 ${\mu}M$, 10 ${\mu}M$, and 100 ${\mu}M$) on OGD-induced neurotoxicity. Results: Treatment of primary cultures of rat cortical neurons with L-carnitine significantly reduced cell necrosis and prevented apoptosis after OGD. L-Carnitine application significantly reduced the number of cells that died, as assessed by the PI/AO ratio, and also reduced ROS release in the OGD groups treated with 10 ${\mu}M$ and 100 ${\mu}M$ of L-carnitine compared with the untreated OGD group (P<0.05). The application of L-carnitine at 100 ${\mu}M$ significantly decreased cytotoxicity, LDH release, and inhibited apoptosis compared to the untreated OGD group (P<0.05). Conclusion: L-Carnitine has neuroprotective benefits against OGD in rat primary cortical neurons in vitro.

• Archives of Pharmacal Research
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• v.27 no.4
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• pp.454-459
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• 2004

The present study investigated the effects of gossypin, 3,3',4',5,7,8-hexahydroxyflavone 8-glucoside, on the toxicity induced by oxidative stress or $\beta$-amyloid ($A_{\beta}$) in primary cultured rat cortical cells. The antioxidant properties of gossypin were also evaluated by cell-free assays. Gossypin was found to inhibit the oxidative neuronal damage induced by xanthinelxanthine oxidase or by a glutathione depleting agent, D,L-buthionine (S,R)-sulfoximine. In addition, gossypin significantly attenuated the neurotoxicity induced by $A_{{\beta}(25-35)}$. Furthermore, gossypin dramatically inhibited lipid peroxidation initiated by $Fe^{2+}$ and ascorbic acid in rat brain homogenates. It also exhibited potent radical scavenging activity generated from 1 ,1-diphenyl-2-picrylhydrazyl. These results indicate that gossypin exerts neuroprotective effects in the cultured cortical cells by inhibiting oxidative stress- and $A_{\beta}$-induced toxicity, and that the antioxidant properties of gossypin may contribute to its neuroprotective actions.

• Molecules and Cells
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• v.24 no.3
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• pp.416-423
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• 2007

Alterations in the glycan chains of cell surface glycoconjugates are frequently involved biological processes such as cell-cell interaction, cell migration, differentiation and development. Cultured embryonic (E18) rat cortical neurons underwent apoptosis in response to camptothecin, and lectin histochemistry showed that binding to apoptotic neurons of FITC-conjugated Maackia amurensis agglutinin (MAA), which is specific for terminal ${\alpha}2,3$-sialic acid residues, increased progressively with increasing concentrations of camptothecin. Analysis of the total proteins of apoptotic neurons by SDS-PAGE, and lectin blotting using HRP-labeled MAA, revealed that the expression of terminal ${\alpha}2,3$-sialic acid residues on an unknown protein with an apparent molecular mass of 25.6 kDa also increased in apoptotic neurons. NP-HPLC analysis of the total cellular N-glycans of normal and apoptotic neurons demonstrated that the expression of structurally simpler biantennary types of N-glycans fell by 49% during apoptosis whereas the more branched triantennary types of N-glycans with terminal sialic acid residues increased by up to 59%. These results suggest that increased surface expression of ${\alpha}2,3$-sialic acid residues and hyperglycosylation of N-glycans is a common feature of cellular responses to changes in cell physiology such as tumorigenesis and apoptosis.