Selective mGluR1 Antagonist EMQMCM Inhibits the Kainate-Induced Excitotoxicity in Primary Neuronal Cultures and in the Rat Hippocampus

Abundant evidence suggests that indirect inhibitory modulation of glutamatergic transmission, via metabotropic glutamatergic receptors (mGluR), may induce neuroprotection. The present study was designed to determine whether the selective antagonist of mGluR1 (3-ethyl-2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methanesulfonate (EMQMCM), showed neuroprotection against the kainate (KA)-induced excitotoxicity in vitro and in vivo. In in vitro studies on mouse primary cortical and hippocampal neuronal cultures, incubation with KA (150 μM) induced strong degeneration [measured as lactate dehydrogenase (LDH) efflux] and apoptosis (measured as caspase-3 activity). EMQMCM (0.1-100 μM) added 30 min to 6 h after KA, significantly attenuated the KA-induced LDH release and prevented the increase in caspase-3 activity in the cultures. Those effects were dose- and time-dependent. In in vivo studies KA (2.5 nmol/1 μl) was unilaterally injected into the rat dorsal CA1 hippocampal region. Degeneration was calculated by counting surviving neurons in the CA pyramidal layer using stereological methods. It was found that EMQMCM (5-10 nmol/1 μl) injected into the dorsal hippocampus 30 min, 1 h, or 3 h (the higher dose only) after KA significantly prevented the KA-induced neuronal degeneration. In vivo microdialysis studies in rat hippocampus showed that EMQMCM (100 μM) significantly increased γ-aminobutyric acid (GABA) and decreased glutamate release. When perfused simultaneously with KA, EMQMCM substantially increased GABA release and prevented the KA-induced glutamate release. The obtained results indicate that the mGluR1 antagonist, EMQMCM, may exert neuroprotection against excitotoxicity after delayed treatment (30 min to 6 h). The role of enhanced GABAergic transmission in the neuroprotection is postulated.     

姜黄素对AMPA/KA受体介导大鼠海马神经元钙内流的影响

目的 探讨姜黄素对α-氨基-3-羧基-5-甲基异恶唑-4-丙酸(AMPA)/海人酸(KA)受体介导大鼠海马神经元钙内流的影响.方法 选用胚胎17dSD鼠分离海马,离体培养海马神经元,借助活体钙荧光染色和激光共聚焦钙成像技术观察100μmol/LKA刺激海马神经元内钙的变化,不同浓度(5、10、15、30、50 μmol/L)姜黄素预孵育海马神经元30min对100μmol/L KA刺激下细胞内钙变化的影响,15 μmol/L姜黄素对不同浓度(10、30、50、100、200、300 μmol/L)KA刺激海马神经元内钙变化的影响.应用钴染色技术观察(30、100 μmol/L KA)刺激后海马神经元钴阳性染色细胞变化.姜黄素预孵育30min对KA刺激导致钴阳性染色细胞变化的影响.结果 不同浓度姜黄素预孵育30 min均可以明显缓解100 μmol/L或30 μmol/L KA导致的细胞内钙升高程度.差异均有统计学意义(P<0.05),其中15 μmol/L姜黄素作用最为明显.30μmol/L或100 μmol/LKA刺激均可以引起海马神经元钴染色阳性细胞增加,15 μmol/L姜黄素预处理30 min后明显减少钴染色阳性细胞,差异有统计学意义(P<0.05),而其他浓度(5 μmol/L或30 μmol/L)姜黄素未见明显影响.结论 一定浓度的姜黄素可以影响AMPA/KA受体介导大鼠海马神经元钙内流.这可能是姜黄素抗癫痫作用的一个机制.     

神经肽Y对海马神经元“癫痫样”动作电位的影响

目的探讨神经肽Y(neuropeptide Y,NPY)对海马神经元"癫痫样"动作电位的影响。方法用无镁细胞外液处理原代培养12 d的海马神经元3 h,诱导海马神经元癫痫样放电,建立海马神经元癫痫样放电模型;用全细胞膜片钳电流钳模式检测神经元动作电位,分别给予0.1μmol/L和1μmol/L NPY各1μL,给药时间10 s,观察其对神经元动作电位频率及波幅的影响。结果无镁细胞外液处理神经元3 h,可以形成稳定的海马神经元癫痫样放电模型,频率16～23 Hz,波幅75～96 mV。模型组神经元动作电位频率为(18.00±2.32)Hz,而0.1μmol/L和1μmol/L NPY组分别为(4.75±1.04)Hz和(1.50±0.75)Hz。与模型组相比较,两种浓度NPY组均降低了动作电位发放的频率(P<0.05)。模型组神经元动作电位波幅为(82.25±5.17)mV,而0.1μmol/L和1μmol/L NPY组分别为(49.75±2.49)mV和(40.00±2.20)mV。与模型组相比较,两种浓度NPY组均降低了动作电位发放的波幅(P<0.05)。两种浓度NPY之间相比较,也有统计学差异(P<0.05)。1μmol/LNPY明显抑制了动作电位发放的频率和波幅。结论 NPY能够抑制无镁细胞外液诱发的神经元癫痫样电活动,为应用NPY抑制癫痫发作提供了细胞电生理学证据。     

Coenzyme Q10 Ameliorates Neurodegeneration, Mossy Fiber Sprouting, and Oxidative Stress in Intrahippocampal Kainate Model of Temporal Lobe Epilepsy in Rat

Temporal lobe epilepsy (TLE) is the most common form of epilepsy in adults and the most resistant type to treatment. Novel treatment approaches are strongly required to prevent or even reverse the cellular and molecular mechanisms of epileptogenesis. In this study, we investigated the possible neuroprotective effect of coenzyme Q10 (CoQ10) in an intrahippocampal kainate model of TLE in rat. Kainate injection caused a higher seizure severity during status epilepticus and spontaneous seizure phases, and CoQ10 pretreatment significantly attenuated its severity and incidence rate. Intrahippocampal kainate also led to elevation of malondialdehyde (MDA) and nitrite, and CoQ10 significantly attenuated the increased MDA and nitrite content. In addition, intrahippocampal kainate induced a significant degeneration of neurons in CA1, CA3, and hilar regions of the hippocampus, and CoQ10 significantly attenuated these changes in CA1 and CA3 regions. Timm’s staining data showed a robust mossy fiber sprouting (MFS) in dentate gyrus of kainate-lesioned rats and CoQ10 significantly lowered MFS intensity. These data suggest that CoQ10 pretreatment could attenuate spontaneous recurrent seizures and inhibit hippocampal neuronal loss and aberrant MFS in kainate-induced model of TLE in rat, and part of its beneficial effect is due to its potential to mitigate oxidative stress.     

Development of kainic acid and N-methyl-d-aspartic acid toxicity in organotypic hippocampal cultures

The excitotoxic effects of N-methyl-d-aspartic acid (NMDA) and kainic acid (KA) were studied in organotypic hippocampal slices maintained in vitro for various periods of time. Cultures aged to equivalent Postnatal Day (EPD) 10–12,15–17, and 23–26 were exposed to 50 μM KA or 50 μM NMDA and were analyzed at 0, 3, 6, 9, 12, 24, 48 h, or 5 days after the initiation of the excitotoxin exposure. Neuronal injury was determined by: (1) propidium iodide (PI) uptake; (2) lactate dehydrogenase (LDH) release; (3) morphological damage in hematoxylin and eosin (H/E) stained sections; (4) loss of Nissl stain. Changes in PI uptake and LDH release after KA or NMDA treatment indicated that there was a developmental shift towards increasing sensitivity to KA toxicity during in vitro development, whereas cultures of all ages were equally sensitive to NMDA toxicity. The profile of damage in H/E-stained sections after treatment with KA or NMDA indicated a transient phase of damaged morphology at 12 and 24 h that was not evident after 5 days. To determine whether the disappearance of morphological manifestations of neuronal damage 5 days after treatment was due to recovery of morphology or to neuronal death, neuronal loss in Nissl-stained sections was also quantified. KA treatment did not cause significant neuronal loss in any hippocampal region in EPD 10–12 cultures, indicating that the neurons were able to successfully recover from the damage demonstrated in H/E sections at 12 and 24 h in these cultures. KA treatment in mature cultures (EPD 23–26) and NMDA treatment in all cultures produced a marked loss of identifiable Nissl-stained neurons at 5 days, indicating neuronal death and disintegration. The results provide further support for the similarities between the organotypic hippocampal culture model and in vivo excitotoxic models and also confirm that excitotoxic neuronal injury can be reversible under some conditions.     

Coenzyme Q10 decreases amyloid pathology and improves behavior in a transgenic mouse model of Alzheimer's disease

Increased oxidative stress is implicated in the pathogenesis of Alzheimer's disease (AD). A large body of evidence suggests that mitochondrial dysfunction and increased reactive oxygen species occur prior to amyloid-β (Aβ) deposition. Coenzyme Q10 (CoQ10), a component of the mitochondrial electron transport chain, is well characterized as a neuroprotective antioxidant in animal models and human trials of Huntington's disease and Parkinson's disease, and reduces plaque burden in AβPP/PS1 mice. We now show that CoQ10 reduces oxidative stress and amyloid pathology and improves behavioral performance in the Tg19959 mouse model of AD. CoQ10 treatment decreased brain levels of protein carbonyls, a marker of oxidative stress. CoQ10 treatment resulted in decreased plaque area and number in hippocampus and in overlying cortex immunostained with an Aβ42-specific antibody. Brain Aβ42 levels were also decreased by CoQ10 supplementation. Levels of amyloid-β protein precursor (AβPP) β-carboxyterminal fragments were decreased. Importantly, CoQ10-treated mice showed improved cognitive performance during Morris water maze testing. Our results show decreased pathology and improved behavior in transgenic AD mice treated with the naturally occurring antioxidant compound CoQ10. CoQ10 is well tolerated in humans and may be promising for therapeutic trials in AD.     

1MeTIQ Provides Protection Against Aβ-Induced Reduction of Surface Expression of Synaptic Proteins and Inhibits H2O2-Induced Oxidative Stress in Primary Hippocampal Neurons

Alzheimer’s disease (AD) is associated with increased brain levels of β-amyloid (Aβ) peptides, which readily self-aggregate into fibrils and oligomers that have particularly deleterious properties toward synapses of excitatory glutamatergic neurons. Here, we examined the neuroprotective effects of 1-methyl-1,2,3,4,-tetrahydroisoquinoline (1MeTIQ) against Aβ-induced loss of synaptic proteins in cultured primary hippocampal neurons. Exposure of mature primary hippocampal neurons to 10 μM synthetic Aβ1-40 over 72 h resulted in ~60 % reduction in the surface expression of NR1 subunit of the NMDA receptor (NMDAR), PSD-95, and synaptophysin, without causing neuronal death. Concomitant treatment with 500 μM of 1MeTIQ, a low-affinity NMDAR antagonist significantly ameliorated the loss of synaptic protein markers. The neuroprotective properties of 1MeTIQ were compared with those of MK-801, which at 0.5 μM concentration also prevented Aβ1-40-induced loss of synaptic proteins in primary neuronal cultures. Furthermore, we provide novel evidence demonstrating effectiveness of 1MeTIQ in reducing the level of reactive oxygen species (ROS) in primary neuronal culture system. As oxidative stress contributes importantly to neurodegeneration in AD, 1MeTIQ may provide a dual neuroproctective effect in AD both as a NMDARs antagonist and ROS formation inhibitor. 1MeTIQ occurs endogenously at low concentrations in the brain and its synthetic form readily penetrates the blood–brain barrier after the systemic administration. Our results highlight a possibility of the application of 1MeTIQ as a neuroprotective agent in AD-related neurodegeneration.     

间充质干细胞外泌体对海马神经元氧化应激的调控作用

目的 探讨间充质干细胞外泌体(Mesenchymal stem cell derived-exosomes,MSC-Exo)对海马神经元氧化应激的作用。方法 首先体外培养原代小鼠海马神经元,用H₂O₂刺激建立氧化应激模型,细胞计数试剂盒(Cell counting kit-8,CCK8)筛选最佳H₂O₂水平并检测不同水平MSC-Exo对细胞活力的作用,试剂盒检测超氧化物歧化酶(Superoxide dismutase,SOD)的活性,酶联免疫吸附测定法(Enzyme linked immunosorbent assay,ELISA)检测DNA氧化损伤分子8-羟基脱氧鸟苷(8-hydroxy-2 deoxyguanosine,8-OHdG)的表达水平,然后免疫荧光和免疫印迹检测应激和损伤分子一氧化氮合成酶(Inducible nitric oxide synthase,iNOS)、高迁移率族蛋白B1(High mobility group box 1,HMGB1)的表达水平。结果 CCK8细胞活力实验显示,10 μg/mL及以上水平的MSC-Exo对海马神经元氧化损伤具有明显的保护作用; 与对照组比较,H₂O₂组SOD的活性显著下降,而在MSC-Exo+H₂O₂组有所提高,趋于正常; 与对照组比较,H₂O₂作用后iNOS,HMGB1,8-OHdG等分子的表达水平显著上调(P<0.01),MSC-Exo作用于模型后与H₂O₂组比较,这些分子的表达水平显著下调(P<0.01)。结论 MSC-Exo作用于H₂O₂诱导的海马神经元氧化应激模型后能够增加SOD的活性,抑制海马神经元iNOS,HMGB1,8-OHdG等分子的表达,表明MSC-Exo对海马神经元氧化应激有一定的调控作用。     

Methylmercury-induced alterations in astrocyte functions are attenuated by ebselen

Methylmercury (MeHg) preferentially accumulates in glia of the central nervous system (CNS), but its toxic mechanisms have yet to be fully recognized. In the present study, we tested the hypothesis that MeHg induces neurotoxicity via oxidative stress mechanisms, and that these effects are attenuated by the antioxidant, ebselen. Rat neonatal primary cortical astrocytes were pretreated with or without 10 μM ebselen for 2h followed by MeHg (0, 1, 5, and 10 μM) treatments. MeHg-induced changes in astrocytic [(3)H]-glutamine uptake were assessed along with changes in mitochondrial membrane potential (ΔΨ(m)), using the potentiometric dye tetramethylrhodamine ethyl ester (TMRE). Western blot analysis was used to detect MeHg-induced ERK (extracellular-signal related kinase) phosphorylation and caspase-3 activation. MeHg treatment significantly decreased (p<0.05) astrocytic [(3)H]-glutamine uptake at all time points and concentrations. Ebselen fully reversed MeHg's (1 μM) effect on [(3)H]-glutamine uptake at 1 min. At higher MeHg concentrations, ebselen partially reversed the MeHg-induced astrocytic inhibition of [(3)H]-glutamine uptake [at 1 min (5 and 10 μM) (p<0.05); 5 min (1, 5 and 10 μM) (p<0.05)]. MeHg treatment (1h) significantly (p<0.05) dissipated the ΔΨ(m) in astrocytes as evidenced by a decrease in mitochondrial TMRE fluorescence. Ebselen fully reversed the effect of 1 μM MeHg treatment for 1h on astrocytic ΔΨ(m) and partially reversed the effect of 5 and 10 μM MeHg treatments for 1h on ΔΨ(m). In addition, ebselen inhibited MeHg-induced phosphorylation of ERK (p<0.05) and blocked MeHg-induced activation of caspase-3 (p<0.05-0.01). These results are consistent with the hypothesis that MeHg exerts its toxic effects via oxidative stress and that the phosphorylation of ERK and the dissipation of the astrocytic mitochondrial membrane potential are involved in MeHg toxicity. In addition, the protective effects elicited by ebselen reinforce the idea that organic selenocompounds represent promising strategies to counteract MeHg-induced neurotoxicity.     

Coenzyme Q10 enhances the anticonvulsant effect of phenytoin in pilocarpine-induced seizures in rats and ameliorates phenytoin-induced cognitive impairment and oxidative stress

Conventional antiepileptic drugs fail to adequately control seizures and predispose to cognitive impairment and oxidative stress with chronic usage in a significant proportion of patients with epilepsy. Coenzyme Q10 (CoQ10), an antioxidant compound, exhibits a wide range of therapeutic effects that are attributed to its potent antioxidant capacity. To evaluate the neuroprotective effects of CoQ10 in rats against the observed oxidative stress during seizures induced by pilocarpine, and to study its interactions with the conventional antiepileptic drug phenytoin, two experiments were performed. Experiment 1 was conducted to test the effect of phenytoin, CoQ10, or both on seizure severity and oxidative markers in the pilocarpine model of epilepsy. Experiment 2 was conducted to test the effect of 2 weeks of chronic treatment with phenytoin, CoQ10, or both on oxidative markers and behavioral tests in rats. Overall, CoQ10 reduced the severity of pilocarpine-induced seizures and the severity of oxidative stress. Moreover, it potentiated the antiepileptic effects afforded by phenytoin treatment, with the potential safety and efficacy in ameliorating oxidative stress and cognitive impairment caused by chronic phenytoin therapy. Our findings strongly suggest that CoQ10 can be considered a safe and effective adjuvant to phenytoin therapy in epilepsy both to ameliorate seizure severity and to protect against seizure-induced oxidative damage by reducing the cognitive impairment and oxidative stress associated with chronic use of phenytoin.     

阿托伐他汀对淀粉样β蛋白诱导大鼠原代海马神经元损伤保护作用的研究

目的探讨阿托伐他汀(atorvastatin,Ato)对β淀粉样蛋白(Aβ)诱导的体外培养原代海马神经元损伤保护作用。方法选用出生0~24hSprague-Dawley大鼠乳鼠,解剖显微镜下分离海马,进行海马神经元体外培养,培养10d后用于实验。将培养的海马神经元分为3组:(1)正常对照组:加入含有1%(质量浓度)DMSO培养基;(2)Aβ1-42组:加入1.25μmol/L Aβ;(3)Aβ(1.25μmol/L)+不同浓度阿托伐他汀组(0.1、0.5、1、2.5μmol/L)。应用CellTiter-GloTM荧光细胞活性试剂盒检测培养海马神经元ATP含量,用以反映神经元活力;通过CytoTox-ONETM试剂盒测定培养细胞上清液中乳酸脱氢酶(LDH)含量,用以测定神经元细胞膜的损伤程度;应用免疫荧光染色观察神经元突触素(SYP)、突触后致密蛋白95(PSD-95)、bassoon蛋白表达。Western印迹法半定量检测海马神经元突触蛋白SYP、PSD-95、bassoon的改变。结果与正常对照组比较,培养10d海马神经元经1.25μmol/L Aβ1-42作用48h后,其ATP和LDH水平均明显降低(均P0.01),而0.5、1.0、2.5μmol/L Ato组能够明显抑制Aβ1-42引起的ATP和LDH水平降低(P0.01)。免疫荧光及Western blot结果显示,1.25μmol/L Aβ1-42可使海马神经元SYP、PSD-95、bassoon蛋白表达明显降低,而Ato能够明显抑制Aβ1-42引起的SYP、PSD-95、bassoon蛋白表达降低。结论 Ato对Aβ诱导的体外培养海马神经元毒性有保护作用,这种保护作用可能与Ato对抗Aβ1-42引起的海马神经元SYP、PSD-95、bassoon表达降低有关。     

Coenzyme Q10 provides neuroprotection in iron-induced apoptosis in dopaminergic neurons

The exact molecular mechanism of progressive loss of neuromelanin containing nigrostriatal dopaminergic neurons in Parkinson's disease (PD) remains unknown, yet evidence suggests that iron might play an important role in PD pathology. In this study we have determined the neuroprotective role of coenzyme Q(10) (CoQ(10)) in ironinduced apoptosis in cultured human dopaminergic (SK-N-SH) neurons, in metallothionein gene- manipulated mice, and in alpha-synuclein knockout (alpha-synko) mice with a primary objective to assess a possible therapeutic and anti-inflammatory potential for CoQ(10) in PD. Iron-induced mitochondrial damage and apoptosis were characterized by reactive oxygen species production, increased metallothionein and glutathione synthesis, caspase- 3 activation, NF-kappaB induction, and decreased Bcl-2 expression, without any significant change in Bax expression. Lower concentrations of FeSO4 (1-10 microM) induced perinuclear aggregation of mitochondria, whereas higher concentrations (100-250 microM) induced CoQ(10) depletion, plasma membrane perforations, mitochondrial damage, and nuclear DNA condensation and fragmentation. FeSO(4)-induced deleterious changes were attenuated by pretreatment with CoQ(10) and by deferoxamine, a potent iron chelator, in SK-N-SH cells. 1-Methyl, 4-phenyl, 1,2,3,6- tetrahydropyridine (MPTP)-induced striatal release of free iron, and NF-kappaB expression were significantly increased; whereas ferritin and melanin synthesis were significantly reduced in the substantia nigra pars compacta (SNpc) of MT(dko) mice as compared with control(wt) mice, MT(trans) mice, and alpha-synko mice. CoQ(10) treatment inhibited MPTP-induced NF-kappaB induction in all of the genotypes. These data suggest that glutathione and metallothionein synthesis might be induced as an attempt to combat iron-induced oxidative stress, whereas exogenous administration of CoQ(10) or of metallothionein induction might provide CoQ(10)-mediated neuroprotection in PD.     

Pergolide protects dopaminergic neurons in primary culture under stress conditions

Summary. Dopamine agonists are an important therapeutic strategy in the treatment of Parkinson's disease. They postpone the necessity for and reduce the required dose of L-3,4-dihydroxyphenylalanine (L-DOPA) medication thus protecting against the development of motor complications and potential oxidative stress due to L-DOPA metabolism. In primary cultures from mouse mesencephalon we show that pergolide, a preferential D₂ agonist enhanced the survival of healthy dopaminergic neurons at low concentrations of 0.001 μM. About 100 fold higher concentrations (0.1 μM) were necessary to partially reverse the toxic effects of 10 μM 1-methyl-4-phenylpyridinium (MPP⁺). Pergolide was equally effective in preventing the reduction of dopamine uptake induced by 200 μM L-DOPA. Furthermore, between 0.001–0.1 μM it also reduced lactate production thus promoting aerobic metabolism. The present findings suggest that pergolide protects dopaminergic neurons under conditions of elevated oxidative stress. Received February 4, 2002; accepted February 21, 2002     

Increased susceptibility of glutathione peroxidase-1 transgenic mice to kainic acid-related seizure activity and hippocampal neuronal cell death

Glutathione peroxidase (GSHPx) has been demonstrated in several in vivo studies to reduce both the risk and severity of oxidatively-induced tissue damage. The seizure-inducing neurotoxin kainic acid (KA) has been suggested to elicit its toxic effects in part via generation of oxidative stress. In this study, we report that expression of elevated levels of murine GSHPx-1 in transgenic mice surprisingly results in increased rather than decreased KA susceptibility including increased seizure activity and neuronal hippocampal damage. Isolated transgenic primary hippocampal culture neurons also display increased susceptibility to KA treatment compared with those from wildtype animals. This could be due to alterations in the redox state of the glutathione system resulting in elevated glutathione disulfide (GSSG) levels which, in turn, may directly activate NMDA receptors or enhanced response of the NMDA receptor.     

Protective Efficacy of Coenzyme Q10 Against DDVP-Induced Cognitive Impairments and Neurodegeneration in Rats

The present study was carried out to elucidate the effects of coenzyme Q(10) (CoQ(10)) against cognitive impairments induced by dichlorvos (DDVP). We have previously shown organophosphate, DDVP-induced impairments in neurobehavioral indices viz. rota rod, passive avoidance, and water maze tests. In addition to this, we have also reported that chronic DDVP exposure leads to decreased mitochondrial electron transfer activities of cytochrome oxidase along with altered mitochondrial complexes I-III activity. Administration of CoQ(10) (4.5 mg/kg, i.p. for 12 weeks prior to DDVP administration daily) to DDVP-treated rats improved cognitive performance in passive avoidance task and Morris water maze test. Furthermore, CoQ(10) treatment also reduced oxidative stress (as evident by reduced malondialdehyde, decreased ROS and increased Mn-SOD activity) in DDVP-treated rats' hippocampus region, along with enhanced activity of complexes I-III and complex IV. Electron microscope studies of rat hippocampus mitochondria revealed that CoQ(10) administration leads to near normal physiology of mitochondria with well-defined cristae compared with DDVP-treated animals where enlarged mitochondria with distorted cristae are observed. CoQ(10) administration also attenuated neuronal damage in hippocampus as evident from histopathological studies. These results demonstrate the beneficial effects of CoQ(10) against organophosphate-induced cognitive impairments and hippocampal neuronal degeneration.     

Protection against kainate neurotoxicity by ginsenosides: attenuation of convulsive behavior, mitochondrial dysfunction, and oxidative stress

We previously demonstrated that kainic acid (KA)-mediated mitochondrial oxidative stress contributed to hippocampal degeneration and that ginsenosides attenuated KA-induced neurotoxicity and neuronal degeneration. Here, we examined whether ginsenosides affected KA-induced mitochondrial dysfunction and oxidative stress in the rat hippocampus. Treatment with ginsenosides attenuated KA-induced convulsive behavior dose-dependently. KA treatment increased lipid peroxidation and protein oxidation and decreased the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio to a greater degree in the mitochondrial fraction than in the hippocampal homogenate. KA treatment resulted in decreased Mn-superoxide dismutase expression and diminished the mitochondrial membrane potential. Furthermore, KA treatment increased intramitochondrial Ca(2+) and promoted ultrastructural degeneration in hippocampal mitochondria. Treatment with ginsenosides dose-dependently attenuated convulsive behavior and the KA-induced mitochondrial effects. Protection appeared to be more evident in mitochondria than in tissue homogenates. Collectively, the results suggest that ginsenosides prevent KA-induced neurotoxicity by attenuating mitochondrial oxidative stress and mitochondrial dysfunction.     

Cellular and synaptic basis of kainic acid-induced hippocampal epileptiform activity

The effects of kainic acid (KA) were studied using extracellular and intracellular recordings in the hippocampal slice preparation. In sufficient concentrations, KA led to a loss of all evoked responses. However, the amount of drug needed for this varied according to anatomic region. CA3 was more sensitive (1 microM) than CA1 or the dentate gyrus (10 microM). These results can be understood in terms of a profound and long-lasting depolarization of neurons. Lower concentrations of KA (0.05-0.1 microM) did not change the resting membrane potential or input resistance of hippocampal pyramidal cells but produced spontaneous epileptiform activity which originated in CA3 and propagated to CA1. Epileptiform discharges were not present in the dentate gyrus. Coincident with the induction of paroxysms, the following changes were observed: (1) an increase in the excitability of CA3 and CA1 pyramidal cells as measured by a left shift in the input-output curves of evoked responses and a lowered threshold stimulus intensity necessary for activation of action potentials in single neurons; (2) augmentation and synchronization of bursting in pyramidal cells; and (3) prolonged EPSPs without an increase in their amplitude. These findings indicate that multiple changes, involving both the properties of single neurons and synaptic connections, are involved in the development of hippocampal paroxysms and that CA3 and CA1 have different roles in the generation of these discharges.     

Inhibition of hexokinase leads to neuroprotection against excitotoxicity in organotypic hippocampal slice culture

During seizures, glucose concentrations are high in the hippocampus. Mitochondrial hexokinase (HK) catalyzes the first essential step of glucose metabolism and directly couples extramitochondrial glycolysis to intramitochondrial oxidative phosphorylation. The neuroprotective effects of an HK inhibitor, 3-bromopyruvate (3-BrPA), on kainic acid (KA)-induced excitotoxic injury were investigated. Hippocampal slices were prepared from hippocampi of 6-8-day-old rats using a tissue chopper and placed on a membrane insert. After a treatment with KA (5 μM) for 15 hr, neuronal death was quantified by propidium iodide (PI), cresol violet, and TUNEL staining. KA-induced cell death was significantly prevented by 30 μM 3-BrPA treatment. According to Western blots, the expression level of phospho-Akt increased after 3-BrPA treatment. The induction of long-term potentiation (LTP) at 48 hr after 3-BrPA treatment tended to increase in the CA1 area compared with the KA-only group, but the difference was not significant. Blocking the PI3 kinase/Akt pathway using LY294002 reversed the neuroprotective effect of 3-BrPA. These results suggest that inhibition of HK may play a protective role against neuronal death in KA-induced excitotoxic injury.