Role of transporters and ion channels in neuronal injury under hypoxia

The aims of the current study were to 1) examine the effects of hypoxia and acidosis on cultured cortical neurons and 2) explore the role of transporters and ion channels in hypoxic injury. Cell injury was measured in cultured neurons or hippocampal slices following hypoxia (1% O(2)) or acidosis (medium pH 6.8) treatment. Inhibitors of transporters and ion channels were employed to investigate their roles in hypoxic injury. Our results showed that 1) neuronal damage was apparent at 5-7 days of hypoxia exposure, i.e., 36-41% of total lactate dehydrogenase was released to medium and 2) acidosis alone did not lead to significant injury compared with nonacidic, normoxic controls. Pharmacological studies revealed 1) no significant difference in neuronal injury between controls (no inhibitor) and inhibition of Na(+)-K(+)-ATP pump, voltage-gated Na(+) channel, ATP-sensitive K(+) channel, or reverse mode of Na(+)/Ca(2+) exchanger under hypoxia; however, 2) inhibition of NBCs with 500 microM DIDS did not cause hypoxic death in either cultured cortical neurons or hippocampal slices; 3) in contrast, inhibition of Na(+)/H(+) exchanger isoform 1 (NHE1) with either 10 microM HOE-642 or 2 microM T-162559 resulted in dramatic hypoxic injury (+95% for HOE-642 and +100% for T-162559 relative to normoxic control, P < 0.001) on treatment day 3, when no death occurred for hypoxic controls (no inhibitor). No further damage was observed by NHE1 inhibition on treatment day 5. We conclude that inhibition of NHE1 accelerates hypoxia-induced neuronal damage. In contrast, DIDS rescues neuronal death under hypoxia. Hence, DIDS-sensitive mechanism may be a potential therapeutic target.     

Progression of Neuronal Damage in an In Vitro Model of the Ischemic Penumbra

Improvement of neuronal recovery in the ischemic penumbra around a brain infarct has a large potential to advance clinical recovery of patients with acute ischemic stroke. However, pathophysiological mechanisms leading to either recovery or secondary damage in the penumbra are not completely understood. We studied neuronal dynamics in a model system of the penumbra consisting of networks of cultured cortical neurons exposed to controlled levels and durations of hypoxia. Short periods of hypoxia (pO₂≈20mmHg) reduced spontaneous activity, due to impeded synaptic function. After ≈6 hours, activity and connectivity partially recovered, even during continuing hypoxia. If the oxygen supply was restored within 12 hours, changes in network connectivity were completely reversible. For longer periods of hypoxia (12–30 h), activity levels initially increased, but eventually decreased and connectivity changes became partially irreversible. After ≈30 hours, all functional connections disappeared and no activity remained. Since this complete silence seemed unrelated to hypoxic depths, but always followed an extended period of low activity, we speculate that irreversible damage (at least partly) results from insufficient neuronal activation. This opens avenues for therapies to improve recovery by neuronal activation.     

Isoform-Specific Effects of Transforming Growth Factors-β on Degeneration of Primary Neuronal Cultures Induced by Cytotoxic Hypoxia or Glutamate

Abstract: The transforming growth factors-β (TGFs-β) are multifunctional peptide-growth factors that have been localized in neuronal and glial cells of the C'NS of mice. rats, and chick embryos. We tested the TGF-β isoforms 1. 2. and 3 for their protective ctlects against neuronal degeneration caused by cytotoxic hypoxia or by the excitatory amino acid i -glutamate. A cytotoxic hypoxia was induced in cultured chick embryo telencephalic neurons by adding I m.l/ sodium cyanide to the culture medium tor a period of 30min. I reatment with TGF-81 (1-30 ng/ml) led to a statistically significant increase in cell viability, neuronal ATP levels. and protein content of the cultures assessed 72 h after the toxic insult. TGF-33 was able to reduce the cyanide-induced neuronal damage at concentrations of 0.3 and 1 ng/ ml. whereas TGF-33, only showed neuroprotective activity at concentrations of 30 and 50 ng/ml. Both pre- and posttreatment with TGF-31, also prevented the degeneration of cultured chick embryo telencephalic neurons that had been exposed to I mM L-glutamate in a buffered salt solution for a period of 60 min. Furthermore, TGF-β1 (0.3-3 ng/ml). and to a lesser extent TGF-β3 (0.1-1 ng/ml). significantly reduced excitotoxic injury of cultured neurons from rat cerebral cortex that had been exposed to serum-free culture medium supplemented with 1 m.M L-glutamate. These results demonstrate that the TGFs-β are able to prevent the degeneration of primary neuronal cultures, which was caused by energy depletion and activation of glutamate receptors, in an isoform-specific manner.     

Neuroprotective actions of a histidine analogue in models of ischemic stroke

Histidine is a naturally occurring amino acid with antioxidant properties, which is present in low amounts in tissues throughout the body. We recently synthesized and characterized histidine analogues related to the natural dipeptide carnosine, which selectively scavenge the toxic lipid peroxidation product 4-hydroxynonenal (HNE). We now report that the histidine analogue histidyl hydrazide is effective in reducing brain damage and improving functional outcome in a mouse model of focal ischemic stroke when administered intravenously at a dose of 20 mg/kg, either 30 min before or 60 min and 3 h after the onset of middle cerebral artery occlusion. The histidine analogue also protected cultured rat primary neurons against death induced by HNE, chemical hypoxia, glucose deprivation, and combined oxygen and glucose deprivation. The histidine analogue prevented neuronal apoptosis as indicated by decreased production of cleaved caspase-3 protein. These findings suggest a therapeutic potential for HNE-scavenging histidine analogues in the treatment of stroke and related neurodegenerative conditions.     

In vivo neuroprotective role of NMDA receptors against kainate-induced excitotoxicity in murine hippocampal pyramidal neurons

Activation of NMDA receptors has been shown to induce either neuronal cell death or neuroprotection against excitotoxicity in cultured cerebellar granule neurons in vitro. We have investigated the effects of pretreatment with NMDA on kainate-induced neuronal cell death in mouse hippocampus in vivo. The systemic administration of kainate (30 mg/kg), but not NMDA (100 mg/kg), induced severe damage in pyramidal neurons of the hippocampal CA1 and CA3 subfields 3-7 days later, without affecting granule neurons in the dentate gyrus. An immunohistochemical study using an anti-single-stranded DNA antibody and TdT-mediated dUTP nick end labeling analysis both revealed that kainate, but not NMDA, induced DNA fragmentation in the CA1 and CA3 pyramidal neurons 1-3 days after administration. Kainate-induced neuronal loss was completely prevented by the systemic administration of NMDA (100 mg/kg) 1 h to 1 day previously. No pyramidal neuron was seen with fragmented DNA in the hippocampus of animals injected with kainate 1 day after NMDA treatment. The neuroprotection mediated by NMDA was prevented by the non-competitive NMDA receptor antagonist MK-801. Taken together these results indicate that in vivo activation of NMDA receptors is capable of protecting against kainate-induced neuronal damage through blockade of DNA fragmentation in murine hippocampus.     

Neuroprotective role of delta-opioid receptors in cortical neurons

We recently demonstrated that delta-opioid receptor (DOR) activation protects cortical neurons against glutamate-induced injury. Because glutamate is a mediator of hypoxic injury in neurons, we hypothesized that DOR is involved in neuroprotection during O2 deprivation and that its activation/inhibition may alter neuronal susceptibility to hypoxic stress. In this work, we tested the effect of opioid receptor activation and inhibition on cultured cortical neurons in hypoxia (1% O2). Cell injury was assessed by lactate dehydrogenase release, morphology-based quantification, and live/dead staining. Our results show that 1) immature neurons (days 4 and 6) were not significantly injured by hypoxia until 72 h of exposure, whereas day 8 neurons were injured after only 24-h hypoxia; 2) DOR inhibition (naltrindole) caused neuronal injury in both day 4 and day 8 normoxic cultures and further augmented hypoxic injury in these neurons; 3) DOR activation ([D-Ala2,D-Leu5]enkephalin) reduced neuronal injury in day 8 cultures after 24 h of normoxic or hypoxic exposure and attenuated naltrindole-induced injury with prolonged exposure; and 4) mu- or kappa-opioid receptor inhibition (beta-funaltrexamine or nor-binaltorphimine) had little effect on neurons in either normoxic or hypoxic conditions. Collectively, these data suggest that DOR plays a crucial role in neuroprotection in normoxic and hypoxic environments.     

Dantrolene Prevents Glutamate Cytotoxicity and Ca2+ Release from Intracellular Stores in Cultured Cerebral Cortical Neurons

Using primary cultures of cerebral cortical neurons, it has been demonstrated that the antihyperthermia drug dantrolene completely protects against glutamate-induced neurotoxicity. Furthermore, in the presence of extracellular calcium, dantrolene reduced the glutamate-induced increase in the intracellular calcium concentration by 70%. In the absence of extracellular calcium, this glutamate response was completely blocked by dantrolene. Dantrolene did not affect the kinetics of [3H]glutamate binding to membranes prepared from similar cultures. These results indicate that release of calcium from intracellular stores is essential for the propagation of glutamate-induced neuronal damage. Because it is likely that glutamate is involved in neuronal degeneration associated with ischemia and hypoxia, the present findings might suggest that dantrolene and possibly other drugs affecting intracellular calcium pools might be of therapeutic interest.     

新生SD大鼠皮质神经元的体外培养和鉴定

目的：建立高纯度的新生SD大鼠皮质神经元原代培养方法。方法：取24h内的新生SD大鼠皮质,用木瓜酶和DNaseⅠ共同消化,5％胎牛血清终止消化,吹打分离组织获得单细胞悬液,进行细胞计数,用无血清DMEM／F12种植培养,4h后换成用无血清Neurobasal配制的维持培养液继续培养,尼氏小体染色和免疫荧光法鉴定神经元的纯度。结果：培养第10d,神经元胞体饱满,结构清晰完整,光晕明显,折光性强,可见粗长的树突和轴突,相邻细胞形成紧密网状联系,神经元纯度达到96％以上。结论：经改良和优化,无须添加阿糖胞苷抑制胶质细胞的生长即能够获得生长状态良好、高纯度的神经元。     

Sustained up-regulation of semaphorin 3A, Neuropilin1, and doublecortin expression in ischemic mouse brain during long-term recovery

Strategies to provide neuroprotection and to promote regenerative axonal outgrowth in the injured brain are thwarted by the plethora of axon growth inhibitors and the ligand promiscuity of some of their receptors. Especially, new neurons derived from ischemia-stimulated neurogenesis must integrate this multitude of inhibitory molecular cues, generated as a result of cortical damage, into a functional response. More often than not the response is one of growth cone collapse, axonal retraction and neuronal death. Therefore, characterization of the expression of inhibitory molecules in long-term surviving ischemic brains following stroke is important for designing selective therapeutics. Here, we describe a long-term recovery mouse model for cerebral ischemia in which a brief transient occlusion of the middle cerebral artery (30 min) was followed by up to 30 days of long-term reperfusion. Significantly decreased grip strength motor function and increased expression of one of the major repulsive guidance cues, Semaphorin 3A (Sema3A) and its receptor Neuropilin1 (NRP1) occurred in brains of these mice. Interestingly, increased Doublecortin (DCX) expression occurred only in the lateral ventricular wall zone, but not in the dentate gyrus granule cell layer on the ischemic side of the brain. Importantly, no DCX positive cells were detected in the infarct core region after 30 d ischemic recovery. Collectively, these studies demonstrated the sustained elevation of Sema3A/NRP1 expression in the ischemic territory, which may contribute to the inhibitory microenvironment responsible for preventing new neurons from entering the infarct area. This model will be of use as a platform for testing anti-inhibitory therapies to stroke.     

Role of the phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways in the neuroprotective effects of cilnidipine against hypoxia in a primary culture of cortical neurons

Cilnidipine, a calcium channel blocker, has been reported to have neuroprotective effects. We investigated whether cilnidipine could protect neurons from hypoxia and explored the role of the phosphatidylinositol 3-kinase (PI3K) and extracellular signal-related kinase (ERK) pathways in the neuroprotective effect of cilnidipine. The viability of a primary culture of cortical neurons injured by hypoxia, measured by trypan blue staining and lactate dehydrogenase (LDH) assay, was dramatically restored by cilnidipine treatment. TUNEL and DAPI staining showed that cilnidipine significantly reduced apoptotic cell death induced by hypoxia. Free radical stress and calcium influx induced by hypoxia were markedly decreased by treatment with cilnidipine. Survival signaling proteins associated with the PI3K and ERK pathways were significantly increased while death signaling proteins were markedly decreased in the primary culture of cortical neurons simultaneously exposed to cilnidipine and hypoxia when compared with the neurons exposed only to hypoxia. These neuroprotective effects of cilnidipine were blocked by treatment with a PI3K inhibitor or an ERK inhibitor. These results show that cilnidipine protects primary cultured cortical neurons from hypoxia by reducing free radical stress, calcium influx, and death-related signaling proteins and by increasing survival-related proteins associated with the PI3K and ERK pathways, and that activation of those pathways plays an important role in the neuroprotective effects of cilnidipine against hypoxia. These findings suggest that cilnidipine has neuroprotective effects against hypoxia through various mechanisms, as well as a blood pressure-lowering effect, which might help to prevent ischemic stroke and reduce neuronal injury caused by ischemic stroke.     

Reduction of the prenatal hypoxic-ischemic brain edema with noscapine

Cytotoxic free radicals and release of several neurotransmitters such as bradykinin contribute to the pathogenesis of hypoxic-ischemic brain damage. We have studied the efficacy of noscapine, an opium alkaloid and a bradykinin antagonist, in reducing post-hypoxic-ischemic damage in developing brain of 7-d-old rat pups. Hypoxic-ischemic injury to the right cerebral hemisphere was produced by legation of the right common carotid artery followed by 3 h of hypoxia with 8% oxygen. Thirty to 45 min before hypoxia the rat pups received noscapine (dose = 0.5-2 mg/kg) or saline. Pups were scarified at 24 h post recovery for the assessment of cerebral damage by histological methods. Our results showed that noscapine was an effective agent in reducing the extent of brain injury after hypoxic-ischemic insult to neonatal rats. Therefore, it is concluded that noscapine may be a useful drug in the managements of patients after stroke.     

Study of neuroprotection of donepezil, a therapy for Alzheimer's disease

Donepezil is a potent acetylcholinesterase inhibitor used for the treatment of Alzheimer's disease. Although acetylcholinesterase inhibitors are thought to be symptomatic treatment of Alzheimer's disease, it is not clear whether they are effective against progressive degeneration of neuronal cells. In this study, we investigated the neuroprotective effects of donepezil against ischemic damage, N-methyl-d-aspartate (NMDA) excitotoxicity, and amyloid-beta (Abeta) toxicity using rat brain primary cultured neurons. Lactate dehydrogenase (LDH) released into the culture medium was measured as a marker of neuronal cell damage. As an ischemic damage model, we used oxygen-glucose deprivation in rat cerebral cortex primary cultured neurons. Pretreatment with donepezil (0.1, 1 and 10muM) significantly decreased LDH release in a concentration-dependent manner. However, other acetylcholinesterase inhibitors (galantamine, tacrine and rivastigmine) did not significantly decrease LDH release. In a NMDA excitotoxicity model, pretreatment with donepezil (0.1, 1 and 10muM) decreased the LDH release in a concentration-dependent manner. In binding assay for glutamate receptors, donepezil at 100muM only slightly inhibited binding to the glycine and polyamine sites on NMDA receptor complex. We further examined the effect of donepezil on Abeta (1-40)- and Abeta (1-42)-induced toxicity in primary cultures of rat septal neurons. Pretreatment with donepezil (0.1, 1 and 10muM) significantly decreased LDH release induced by Abetas in a concentration-dependent manner. However, other acetylcholinesterase inhibitors (galantamine and tacrine) and NMDA receptor antagonists (memantine and dizocilpine (MK801)) did not significantly decrease LDH release. These results demonstrate that donepezil has protective effects against ischemic damage, glutamate excitotoxicity and Abeta toxicity to rat primary cultured neurons and these effects are not dependent on acetylcholinesterase inhibition and antagonism of NMDA receptors. Thus, donepezil is expected to have a protective effect against progressive degeneration of brain neuronal cells in ischemic cerebrovascular disease and Alzheimer's disease.     

Cortical cell elution by sedimentation field-flow fractionation.

As a cell sorter, Sedimentation field-flow fractionation (SdFFF) can be defined as an effective tool for cell separation and purification, respecting integrity and viability as well as providing enhanced recovery and purified sterile fraction collection. The complex cell suspension containing both neurons and glial cells of all types, obtained from cerebral cortices of 17-day-old rat fetuses, is routinely used as a model of primary neuronal culture. Using SdFFF, this complex cell mixture was eluted in sterile fractions which were collected and cultured. SdFFF cell elution was conducted under strictly defined conditions: rapid cell elution, high recovery (negligible cell trapping), short- and long-term cell viability, sterile collection. After immunological cellular type characterization (neurons and glial cells) of cultured cells, our results demonstrated the effectiveness of SdFFF to provide, in less than 6 min, viable and enriched neurons which can be cultured for further investigations.     

Amelioration of oxygen and glucose deprivation-induced neuronal death by chloroform fraction of bay leaves (Laurus nobilis)

The purpose of this study was to determine whether the Laurus nobilis chloroform fraction (LNCF) protects against cerebral ischemia neuronal damage. Human neuroblastoma SH-SY5Y cells and brain slices from rats were subjected to oxygen and glucose deprivation (OGD), followed by reoxgenation with and without LNCF. The viabilities of SH-SY5Y cells and brain slices from the rats were 58.5±4.9% and 79.7±5.9% in the group subjected to OGD, and 80.4±0.4% and 97.2±1.9% at 4 μg/ml of LNCF, respectively. LNCF also significantly inhibited death-associated protein kinase (DAPK) dephosphorylation. Pretreatment with LNCF at 4 mg/kg significantly decreased infarct size by 79% of vehicle control in the middle cerebral artery occlusion (MCAO) in vivo model. LNCF is a neuroprotective drug candidate against cerebral ischemia neuronal damage.     

Neuroprotective Effects of Alpha Lipoic Acid on Haloperidol-Induced Oxidative Stress in the Rat Brain

Haloperidol is an antipsychotic drug that exerts its' antipsychotic effects by inhibiting dopaminergic neurons. Although the exact pathophysiology of haloperidol extrapyramidal symptoms are not known, the role of reactive oxygen species in inducing oxidative stress has been proposed as one of the mechanisms of prolonged haloperidol-induced neurotoxicity. In the present study, we evaluate the protective effect of alpha lipoic acid against haloperidol-induced oxidative stress in the rat brain. Sprague Dawley rats were divided into control, alpha lipoic acid alone (100 mg/kg p.o for 21 days), haloperidol alone (2 mg/kg i.p for 21 days), and haloperidol with alpha lipoic acid groups (for 21 days). Haloperidol treatment significantly decreased levels of the brain antioxidant enzymes super oxide dismutase and glutathione peroxidase and concurrent treatment with alpha lipoic acid significantly reversed the oxidative effects of haloperidol. Histopathological changes revealed significant haloperidol-induced damage in the cerebral cortex, internal capsule, and substantia nigra. Alpha lipoic acid significantly reduced this damage and there were very little neuronal atrophy. Areas of angiogenesis were also seen in the alpha lipoic acid-treated group. In conclusion, the study proves that alpha lipoic acid treatment significantly reduces haloperidol-induced neuronal damage.     

Lactate and glucose as energy substrates during, and after, oxygen deprivation in rat hippocampal acute and cultured slices

The effects of raised brain lactate levels on neuronal survival following hypoxia or ischemia is still a source of controversy among basic and clinical scientists. We have sought to address this controversy by studying the effects of glucose and lactate on neuronal survival in acute and cultured hippocampal slices. Following a 1-h hypoxic episode, neuronal survival in cultured hippocampal slices was significantly higher if glucose was present in the medium compared with lactate. However, when the energy substrate during the hypoxic period was glucose and then switched to lactate during the normoxic recovery period, the level of cell damage in the CA1 region of organotypic cultures was significantly improved from 64.3 +/- 2.1 to 74.6 +/- 2.1% compared with cultures receiving glucose during and after hypoxia. Extracellular field potentials recorded from the CA1 region of acute slices were abolished during oxygen deprivation for 20 min, but recovered almost fully to baseline levels with either glucose (82.6 +/- 10.0%) or lactate present in the reperfusion medium (108.1 +/- 8.3%). These results suggest that lactate alone cannot support neuronal survival during oxygen deprivation, but a combination of glucose followed by lactate provides for better neuroprotection than either substrate alone.     

In vivo hypoxia-induced neuronal damage in dentate gyrus of rat hippocampus: changes in NMDA receptors and the effect of MK–801

Hypoxia is a major cause of ischaemia-induced neuronal damage. In the present study, we examined the effects of in vivo hypoxia on N-methyl-D-aspartate receptors (NMDAR) in the rat hippocampus. This model of in vivo hypoxia involved placing rats in a hypoxic chamber containing 5% O₂ and 95% N₂ for 30 min. In the hippocampus, neuronal cells in the CA3, the hilus of the dentate gyrus and the dentate gyrus (DG) were damaged. In the CA1, which is known to be vulnerable to ischaemic damage, neuronal cells did not show hypoxia-induced damage. In vivo hypoxia-induced damage caused morphological changes in neuronal cells, such as shrunken, spindle or triangular shapes accompanied by pyknotic nuclei, but did not induce the loss of neuronal cells. On the other hand, the number of binding sites for [³H]-1-[1-(2-thienyl)cyclohexyl]-3,4-piperidine hydrochloride (TCP) gradually decreased on and after 7 days, and then maximally decreased by 25% at 21 days after hypoxia. The number of NMDAR1-immunopositive cells was decreased by 22% in the DG, but was unchanged in the CA3. Furthermore, we examined the effect of a non-competitive NMDA antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine hydrogen maleate (MK–801), on against in vivo hypoxia. The administration of MK–801 (3 mg/kg, i.p.), 30 min before hypoxia treatment, partly protected against neuronal damage in the DG, but not in the CA3. These results suggest that hypoxia-induced neuronal damage in the DG involves, in part, the activation of NMDAR.     

Long-term electrophysiological activity and pharmacological response of a human induced pluripotent stem cell-derived neuron and astrocyte co-culture

Human induced pluripotent stem cell (hiPSC)-derived neurons may be effectively used for drug discovery and cell-based therapy. However, the immaturity of cultured human iPSC-derived neurons and the lack of established functional evaluation methods are problematic. We here used a multi-electrode array (MEA) system to investigate the effects of the co-culture of rat astrocytes with hiPSC-derived neurons on the long-term culture, spontaneous firing activity, and drug responsiveness effects. The co-culture facilitated the long-term culture of hiPSC-derived neurons for >3 months and long-term spontaneous firing activity was also observed. After >3 months of culture, we observed synchronous burst firing activity due to synapse transmission within neuronal networks. Compared with rat neurons, hiPSC-derived neurons required longer time to mature functionally. Furthermore, addition of the synapse antagonists bicuculline and 6-cyano-7-nitroquinoxaline-2,3-dione induced significant changes in the firing rate. In conclusion, we used a MEA system to demonstrate that the co-culture of hiPSC-derived neurons with rat astrocytes is an effective method for studying the function of human neuronal cells, which could be used for drug screening.     

Extract from Terminalia chebula Seeds Protect Against Experimental Ischemic Neuronal Damage Via Maintaining SODs and BDNF Levels

The fruit of Terminalia chebula Retz has been used as a traditional medicine in Asia and contains tannic acid, chebulagic acid, chebulinic acid and corilagin. Extract from T. chebula seeds (TCE) has various biological functions. We observed the neuroprotective effects of TCE against ischemic damage in the hippocampal C1 region (CA1) of the gerbil that had received oral administrations of TCE (100?mg/kg) once a day for 7?days before the induction of transient cerebral ischemia. In the TCE-treated ischemia group, neuronal neuclei (a marker for neurons)-positive neurons were distinctively abundant (62% of the sham group) in the CA1 4?days after ischemia-reperfusion (I-R) compared to those (12.2% of the sham group) in the vehicle-treated ischemia group. Four days after I-R TCE treatment markedly decreased the activation of astrocytes and microglia in the ischemic CA1 compared with the vehicle-treated ischemia group. In addition, immunoreactivities of Cu, Zn-superoxide dismutase (SOD1), Mn-superoxide dismutase (SOD2) and brain-derived neurotrophic factor (BDNF) in the CA1 of the TCE-treated ischemia group were much higher than those in the vehicle-ischemia group 4?days after I-R. Protein levels of SOD1, SOD2 and BDNF in the TCE-treated ischemia group were also much higher than those in the vehicle-ischemia group 4?days after I-R. These results indicate that the repeated supplement of TCE protected neurons from ischemic damage induced by transient cerebral ischemia by maintaining SODs and BDNF levels as well as decreasing glial activation.     

叠氮钠损伤的神经元内硫氧还蛋白mRNA水平的变化

氧化应激与许多神经退变病有关,而线粒体损伤是氧化应激加剧的重要原因。本文通过细胞活性检测（MTT法）、形态学观察,分析NaN3对原代培养神经元的损伤作用,并通过RT－PCR半定量检测NaN3损伤后神经元内硫氧还蛋白（Thioredoxin,Trx)mRNA水平的改变,以阐明这一重要的氧还调节蛋白在神经元损伤过程中的作用。实验表明NaN3以浓度和时间依赖方式损伤神经元,降低Trx表达水平。提示：神经元内呼吸链受损引起Trx表达减少,从而减弱神经元内氧还调节功能,最终引起神经元损伤、死亡。