Protected HT22 Cells Research Articles

Neuroprotective Effect of Steamed and Fermented Codonopsis lanceolata.

Codonopsis lanceolata has been used as an herbal medicine for several lung inflammatory diseases, such as asthma, tonsillitis, and pharyngitis. Previously, we showed the neuroprotective effect of steamed and fermented C. lanceolata (SFC) in vitro and in vivo. In the current study, the treatment of HT22 cells with SFC decreased glutamate-induced cell death, suggesting that SFC protected HT22 cells from glutamate-induced cytotoxicity. Based on these, we sought to elucidate the mechanisms of the neuro-protective effect of SFC by measuring the oxidative stress parameters and the expression of Bax and caspase-3 in HT22 cells. SFC reduced contents of ROS, Ca2+ and NO. Moreover, SFC restored contents of glutathione and glutathione reductase as well as inhibited Bax and caspase-3 activity in HT22 cells. These results indicate that steamed and fermented C. lanceolata (SFC) extract protected HT22 cells by anti-oxidative effect and inhibition of the expression of Bax and caspase-3.

Functionalized acridin-9-yl phenylamines protected neuronal HT22 cells from glutamate-induced cell death by reducing intracellular levels of free radical species

The in vitro neuronal cell death model based on the HT22 mouse hippocampal cell model is a convenient means of identifying compounds that protect against oxidative glutamate toxicity which plays a role in the development of certain neurodegenerative diseases. Functionalized acridin-9-yl-phenylamines were found to protect HT22 cells from glutamate challenge at submicromolar concentrations. The Aryl1-NH-Aryl2 scaffold that is embedded in these compounds was the minimal pharmacophore for activity. Mechanistically, protection against the endogenous oxidative stress generated by glutamate did not involve up-regulation of glutathione levels but attenuation of the late stage increases in mitochondrial ROS and intracellular calcium levels. The NH residue in the pharmacophore played a crucial role in this regard as seen from the loss of neuroprotection when it was structurally modified or replaced. That the same NH was essential for radical scavenging in cell-free and cell-based systems pointed to an antioxidant basis for the neuroprotective activities of these compounds.

Lithium Prevents Acrolein-Induced Neurotoxicity in HT22 Mouse Hippocampal Cells

Acrolein is a highly electrophilic alpha, beta-unsaturated aldehyde to which humans are exposed in many situations and has been implicated in neurodegenerative diseases, such as Alzheimer's disease. Lithium is demonstrated to have neuroprotective and neurotrophic effects in brain ischemia, trauma, neurodegenerative disorders, and psychiatric disorders. Previously we have found that acrolein induced neuronal death in HT22 mouse hippocampal cells. In this study, the effects of lithium on the acrolein-induced neurotoxicity in HT22 cells as well as its mechanism(s) were investigated. We found that lithium protected HT22 cells against acrolein-induced damage by the attenuation of reactive oxygen species and the enhancement of the glutathione level. Lithium also attenuated the mitochondrial dysfunction caused by acrolein. Furthermore, lithium significantly increased the level of phospho-glycogen synthase kinase-3 beta (GSK-3β), the non-activated GSK-3β. Taken together, our findings suggest that lithium is a protective agent for acrolein-related neurotoxicity.

Neuroprotective Action of Deer Bone Extract Against Glutamate or Aβ1–42-Induced Oxidative Stress in Mouse Hippocampal Cells

Water extracts of deer bone, called nokgol in Korean, and deer antlers have been traditionally used as anti-aging medicines. Deer antler extract is known to possess various activities, including anti-aging or anti-amnesic activity. However, there are no reports about the neuroprotective effect of deer bone extract (DBE). The objective of this study was to examine the neuroprotective effect of DBE on glutamate-induced cell death of mouse hippocampal cells (HT-22 cells) and to elucidate the mode of neuroprotective action of DBE. In this study, HT-22 cells was pretreated with DBE before stimulation with glutamate, and then, the effects of DBE on cell viability, oxidative stress markers, and MAP kinases were determined. Separately, the effect of DBE on H₂O₂ or amyloid beta peptide (1-42) (Aβ₁₋₄₂)-induced cytotoxicity of HT-22 cells was evaluated. DBE protected HT-22 cells from glutamate-induced cell death and prevented the increase in lactate dehydrogenase leakage in HT-22 cells. DBE also prevented glutamate-induced oxidative stress, as indicated by increased reactive oxygen species and lipid peroxidation as well as by decreases in glutathione (GSH) levels and GSH peroxidase activity. In addition, DBE inhibited glutamate-induced activation of c-Jun N-terminal kinases (JNK), p38, and extracellular signal-regulated kinase, indicators of oxidative stress-induced cell death. Furthermore, DBE also protected against H₂O₂ and Aβ₁₋₄₂-induced cytotoxicity. These results suggest that DBE may be a useful functional agent for the prevention against neurodegenerative disorders involving oxidative stress.

Anandamide protects HT22 cells exposed to hydrogen peroxide by inhibiting CB1 receptor-mediated type 2 NADPH oxidase.

Background. Endogenous cannabinoid anandamide (AEA) protects neurons from oxidative injury in rodent models; however the mechanism of AEA-induced neuroprotection remains to be determined. Activation of neuronal NADPH oxidase 2 (Nox2) contributes to oxidative damage of the brain, and inhibition of Nox2 can attenuate cerebral oxidative stress. We aimed to determine whether the neuronal Nox2 was involved in protection mediated by AEA. Methods. The mouse hippocampal neuron cell line HT22 was exposed to hydrogen peroxide (H2O2) to mimic oxidative injury of neurons. The protective effect of AEA was assessed by measuring cell metabolic activity, apoptosis, lactate dehydrogenase (LDH) release, cellular morphology, intracellular reactive oxygen species (ROS), and antioxidant and oxidant levels and Nox2 expression. Results. HT22 cells exposed to H2O2 demonstrated morphological changes, decreased LDH release, reduced metabolic activity, increased levels of intracellular ROS and oxidized glutathione (GSSG), reduced levels of superoxide dismutase (SOD), and reduced glutathione (GSH) and increased expression of Nox2. AEA prevented these effects, a property abolished by simultaneous administration of CB1 antagonist AM251 or CB1-siRNA. Conclusion. Nox2 inhibition is involved in AEA-induced cytoprotection against oxidative stress through CB1 activation in HT22 cells.

Downregualtion of dynamin-related protein 1 attenuates glutamate-induced excitotoxicity via regulating mitochondrial function in a calcium dependent manner in HT22 cells

Glutamate-mediated excitotoxicity is involved in many acute and chronic brain diseases. Dynamin related protein 1 (Drp-1), one of the GTPase family of proteins that regulate mitochondrial fission and fusion balance, is associated with apoptotic cell death in cancer and neurodegenerative diseases. Here we investigated the effect of downregulating Drp-1 on glutamate excitotoxicity-induced neuronal injury in HT22 cells. We found that downregulation of Drp-1 with specific small interfering RNA (siRNA) increased cell viability and inhibited lactate dehydrogenase (LDH) release after glutamate treatment. Downregulation of Drp-1 also inhibited an increase in the Bax/Bcl-2 ratio and cleavage of caspase-9 and caspase-3. Drp-1 siRNA transfection preserved the mitochondrial membrane potential (MMP), reduced cytochrome c release, enhanced ATP production, and partly prevented mitochondrial swelling. In addition, Drp-1 knockdown attenuated glutamate-induced increases of cytoplasmic and mitochondrial Ca2+, and preserved the mitochondrial Ca2+ buffering capacity after excitotoxicity. Taken together, these results suggest that downregulation of Drp-1 protects HT22 cells against glutamate-induced excitatory damage, and this neuroprotection may be dependent at least in part on the preservation of mitochondrial function through regulating intracellular calcium homeostasis.

The ameliorating effects of 2,3-dihydroxy-4-methoxyacetophenone on scopolamine-induced memory impairment in mice and its neuroprotective activity

We isolated 2,3-dihydroxy-4-methoxyacetophenone, a neuroprotective compound from Cynenchum paniculatum in our previous study.The present study was conducted to investigate the possible neuroprotective effect of 2,3-dihydroxy-4-methoxyacetophenone that has been previously isolated from Cynenchum paniculatum on hippocampal neuronal cell line, HT22 cells and its possible cognitive-enhancing effect on scopolamine-induced amnesia in mice.Neuroprotective effect against glutamate-induced neurotoxicity in HT22 cells was evaluated by MTT assay. Also, cognitive enhancing effect against scopolamine (1mg/kg, ip) induced learning and memory deficit was measured by Morris water maze test. Oral administered of 2,3-dihydroxy-4-methoxyacetophenone (1, 10, 20, 40 and 50mg/kg) to amnesic mice induced by scopolamine. In Morris water maze test, 2,3-dihydroxy-4-methoxyacetophenone (50mg/kg) improved the impairment of spatial memory induced by scopolamine. 2,3-Dihydroxy-4-methoxyacetophenone protect HT22 cells on glutamate induced cell-death in a dose-dependent manner (EC50 value: 10.94μM). Furthermore, 2,3-dihydroxy-4-methoxyacetophenone was found to inhibit [Ca2+] accumulation in HT22 cells and had antioxidantive activity. The results showed that 2,3-dihydroxy-4-methoxyacetophenone exert neuroprotective and cognitive-enhancing activities through its antioxidant activity. We suggest that 2,3-dihydroxy-4-methoxyacetophenone improves cognitive function and may be helpful for the treatment of Alzheimer’s disease.

Blockade of SOCE protects HT22 cells from hydrogen peroxide-induced apoptosis

Oxidative stress is an established event in the pathology of neurobiological diseases. Previous studies indicated that store-operated Ca2+ entry (SOCE) has been involved in oxidative stress. The present study was carried out to investigate the effects of SOCE inhibition on neuronal oxidative stress injury induced by hydrogen peroxide (H2O2) in HT22 cells, a murine hippocampal neuronal model. H2O2 insult induced significant intracellular Ca2+ overload, mitochondrial dysfunction and cell viability decrease. Inhibition of SOCE by pharmacological inhibitor and STIM1 RNAi significantly alleviated intracellular Ca2+ overload, restored the mitochondrial membrane potential (MMP), decreased cytochrome C release and eventually inhibited H2O2-induced cell apoptosis. These findings suggest that SOCE inhibition exhibited neuroprotection against oxidative stress induced by H2O2 and SOCE might be a useful therapeutic target in neurobiological disorders.

The antioxidant idebenone fails to prevent or attenuate chronic experimental autoimmune encephalomyelitis in the mouse

Oxidative stress and mitochondrial dysfunction appear to contribute to neurodegenerative processes during multiple sclerosis (MS). Thus, antioxidants may represent a therapeutic option for MS. The antioxidant idebenone was proven to be beneficial in Friedreich's ataxia and Leber's hereditary optic neuropathy, two disorders caused by mitochondrial alterations. Here we showed that idebenone protected neuronal HT22 cells from glutamate-induced death in vitro. However, in experimental autoimmune encephalomyelitis, idebenone failed to affect disease incidence or onset when applied preventively, or to reduce disease severity when applied therapeutically. Histopathological examination of CNS from idebenone treated mice showed no improvement in inflammation, demyelination, or axonal damage. Thus, we hypothesize that idebenone treatment will likely not benefit patients with MS.

Differential mechanisms underlying neuroprotection of hydrogen sulfide donors against oxidative stress

This study investigated whether slow-releasing organic hydrogen sulfide donors act through the same mechanisms as those of inorganic donors to protect neurons from oxidative stress. By inducing oxidative stress in a neuronal cell line HT22 with glutamate, we investigated the protective mechanisms of the organic donors: ADT-OH [5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione], the most widely used moiety for synthesizing slow-releasing hydrogen sulfide donors, and ADT, a methyl derivative of ADT-OH. The organic donors were more potent than the inorganic donor sodium hydrogensulfide (NaHS) in protecting HT22 cells against glutamate toxicity. Consistent with previous publications, NaHS partially restored glutamate-depleted glutathione (GSH) levels, protected HT22 from direct free radical damage induced by hydrogen peroxide (H2O2), and NaHS protection was abolished by a KATP channel blocker glibenclamide. However, neither ADT nor ADT-OH enhanced glutamate-depleted GSH levels or protected HT22 from H2O2-induced oxidative stress. Glibenclamide, which abolished NaHS neuroprotection against oxidative stress, did not block ADT and ADT-OH neuroprotection against glutamate-induced oxidative stress. Unexpectedly, we found that glutamate induced AMPK activation and that compound C, a well-established AMPK inhibitor, remarkably protected HT22 from glutamate-induced oxidative stress, suggesting that AMPK activation contributed to oxidative glutamate toxicity. Interestingly, all hydrogen sulfide donors, including NaHS, remarkably attenuated glutamate-induced AMPK activation. However, under oxidative glutamate toxicity, compound C only increased the viability of HT22 cells treated with NaHS, but did not further increase ADT and ADT-OH neuroprotection. Thus, suppressing AMPK activation likely contributed to ADT and ADT-OH neuroprotection. In conclusion, hydrogen sulfide donors acted through differential mechanisms to confer neuroprotection against oxidative toxicity and suppressing AMPK activation was a possible mechanism underlying neuroprotection of organic hydrogen sulfide donors against oxidative toxicity.

Neuroprotective Mechanisms of Methylene Blue

BackgroundThe phenothiazine dye methylene blue (MB) has antioxidant properties and augments ATP production, and is selectively internalized by neurons after systemic injection into rats.HypothesisMB protects neurons against hypoxia‐reoxygenation (H/R) injury by enhancing ATP production and activating the Akt/mTOR signaling pathway.MethodsHT22 cells were subjected to 3h oxygen‐glucose deprivation (OGD; 0.5% O2) followed by 24 h reoxygenation (H/R protocol). Cell viability (Calcein AM), and contents of hypoxia inducible factor‐1α, Akt/mTOR pathway components, and actin (immunoblot), erythropoietin (EPO; immunocytochemistry) and ATP content (microplate assay) were analyzed. P values <0.05 were considered statistically different.ResultsOGD reduced cell viability by 40% vs. normoxia, but 50 and 100 nM MB prevented H/R induced cell death. MB (10–1000 nM) concentration‐dependently increased cellular ATP content, in parallel with increased HIF‐1α content and EPO expression. MB (500 μM) increased Akt phosphorylation fourfold and mTOR phosphorylation by 45% vs. H/R control.ConclusionMB protects HT22 cells by both enhancing ATP production and activating EPO's anti‐apoptotic signaling.

A novel domain of amino-Nogo-A protects HT22 cells exposed to oxygen glucose deprivation by inhibiting NADPH oxidase activity.

This study aimed to investigate the protective effect of the M9 region (residues 290-562) of amino-Nogo-A fused to the human immunodeficiency virus trans-activator TAT in an in vitro model of ischemia-reperfusion induced by oxygen-glucose deprivation (OGD) in HT22 hippocampal neurons, and to investigate the role of NADPH oxidase in this protection. Transduction of TAT-M9 was analyzed by immunofluorescence staining and western blot. The biologic activity of TAT-M9 was assessed by its effects against OGD-induced HT22 cell damage, compared with a mutant M9 fusion protein or vehicle. Cellular viability and lactate dehydrogenase (LDH) release were assessed. Neuronal apoptosis was evaluated by flow cytometry. The Bax/Bcl-2 ratio was determined by western blotting. Reactive oxygen species (ROS) levels and NADPH oxidase activity were also measured in the presence or absence of an inhibitor or activator of NADPH oxidase. Our results confirmed the delivery of the protein into HT22 cells by immunofluorescence and western blot. Addition of 0.4μmol/L TAT-M9 to the culture medium effectively improved neuronal cell viability and reduced LDH release induced by OGD. The fusion protein also protected HT22 cells from apoptosis, suppressed overexpression of Bax, and inhibited the reduction in Bcl-2 expression. Furthermore, TAT-M9, as well as apocynin, decreased NADPH oxidase activity and ROS content. The protective effects of the TAT-M9 were reversed by TBCA, an agonist of NADPH oxidase. In conclusion, TAT-M9 could be successfully transduced into HT22 cells, and protected HT22 cells against OGD damage by inhibiting NADPH oxidase-mediated oxidative stress. These findings suggest that the TAT-M9 protein may be an efficient therapeutic agent for neuroprotection.

Iduna protects HT22 cells from hydrogen peroxide-induced oxidative stress through interfering poly(ADP-ribose) polymerase-1-induced cell death (parthanatos)

Oxidative stress-induced cell death is common in many neurological diseases. However, the role of poly(ADP-ribose) polymerase-1-induced cell death (parthanatos) has not been fully elucidated. Here, we found that hydrogen peroxide (H2O2) could lead to PARP-1 activation and apoptosis-inducing factor nuclear translocation in a concentration dependent manner. Iduna, as a novel regulator of parthanatos, was also induced by H2O2. Down-regulation of Iduna by genetic ablation promoted H2O2-induced cell damage. Up-regulation of Iduna reduced the loss of mitochondrial potential and ATP and NAD+ production, but did not affect the mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio, and Caspase-9/Caspase-3 activity. In contrast, overexpression of Iduna inhibited activation of PARP-1 and nuclear translocation of AIF. Further study showed that PARP-1 specific inhibitor, DPQ, blocked the protective effect of Iduna against H2O2-induced oxidative stress. Moreover, in the presence of proteasome inhibitor (MG-132) or ubiquitin E1 inhibitor (PYR-41), protective effect of Iduna was significantly weaken. These results indicate that Iduna acts as a potential antioxidant by improving mitochondrial function and inhibiting oxidative stress-induced parthanatos, and these protective effects are dependent on the involvement of ubiquitin–proteasome system.

Edaravone Protects HT22 Neurons from H2O2‐induced Apoptosis by Inhibiting the MAPK Signaling Pathway

Oxidative stress is frequently implicated in the pathology of neurodegenerative diseases. This study aimed to investigate the effects and their underlying mechanism(s) of edaravone upon hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis in HT22 cells, a murine hippocampal neuronal model. HT22 cells were treated with H2O2 in the presence of various concentrations of edaravone or in its absence. A CCK-8 assay, Hoechst 33342 staining, and flow cytometry were used to detect cytotoxicity and apoptosis. In addition, the levels of reactive oxygen species (ROS) and the expression of Bcl-2, Bax, p-ERK 1/2, p-JNK, and p-P38 proteins in HT22 cells were examined. Exogenous H2O2 decreased cell viability in a concentration-dependent manner and was associated with increased apoptosis and ROS production. Moreover, H2O2 significantly activated and upregulated the expression of p-ERK 1/2, p-JNK, and p-P38, while edaravon protected HT22 cells against H2O2-induced injury by inhibiting the production of ROS and activating the MAPK signaling pathway. Our results provide the first evidence that edaravone can protect H2O2-induced cell injury in HT22 neurons via its antioxidant action. These findings suggest that edaravone may be useful in the treatment of neurodegenerative disorders in which oxidative stress has been principally implicated.

Inhibition of 12/15‐lipoxygenase as therapeutic strategy to treat stroke

Targeting newly identified damage pathways in the ischemic brain can help to circumvent the currently severe limitations of acute stroke therapy. Here we show that the activity of 12/15-lipoxygenase was increased in the ischemic mouse brain, and 12/15-lipoxygenase colocalized with a marker for oxidized lipids, MDA2. This colocalization was also detected in the brain of 2 human stroke patients, where it also coincided with increased apoptosis-inducing factor. A novel inhibitor of 12/15-lipoxygenase, LOXBlock-1, protected neuronal HT22 cells against oxidative stress. In a mouse model of transient focal ischemia, the inhibitor reduced infarct sizes both 24 hours and 14 days poststroke, with improved behavioral parameters. Even when treatment was delayed until at least 4 hours after onset of ischemia, LOXBlock-1 was protective. Furthermore, it reduced tissue plasminogen activator-associated hemorrhage in a clot model of ischemia/reperfusion. This study establishes inhibition of 12/15-lipoxygenase as a viable strategy for first-line stroke treatment.

Cognition-enhancing and neuroprotective activities of the standardized extract of Betula platyphylla bark and its major diarylheptanoids

Diarylheptanoids have been the center of the intensive research efforts for Alzheimer's disease and other neurodegenerative diseases. The present study aimed to determine the effect of the standardized extract of B. platyphylla bark and its major diarylheptanoids in scopolamine-induced amnesic mice through cyclic AMP response element-binding protein (CREB) activation. Oral administration of the standardized extract of B. platyphylla bark (100mg/kg body weight), aceroside VIII (1mg/kg body weight) and platyphylloside (1 or 2mg/kg body weight) significantly ameliorated scopolamine-induced amnesia in passive avoidance test. CREB phosphorylation and brain-derived neurotrophic factor (BDNF) expression in the cortex and hippocampus of the scopolamine-treated mice were markedly increased by the treatment of the standardized extract of B. platyphylla bark and platyphylloside. The standardized extract of B. platyphylla bark and its major diarylheptanoids also significantly protected HT22 cells against neurotoxicity induced by glutamate insult. The standardized extract of B. platyphylla bark and platyphylloside may ameliorate memory deficits by activating the CREB–BDNF pathway and prevent a neurodegeneration by inhibiting neuronal cell death.

Effects of Resveratrol and trans-3,5,4'-Trimethoxystilbene on Glutamate-Induced Cytotoxicity, Heme Oxygenase-1, and Sirtuin 1 in HT22 Neuronal Cells

Resveratrol (trans-3,5,4’-trihydroxystilbene) has received considerable attention recently for the potential neuroprotective effects in neurodegenerative disorders where heme oxygenase-1 (HO-1) and sirtuin 1 (SIRT1) represent promising therapeutic targets. Resveratrol has been known to increase HO-1 expression and SIRT1 activity. In this study, the effects of resveratrol and trans-3,5,4’-trimethoxystilbene (TMS), a resveratrol derivative, on cytotoxicity caused by glutamate-induced oxidative stress, HO-1 expression, and SIRT1 activation have been investigated by using murine hippocampal HT22 cells, which have been widely used as an in vitro model for investigating glutamate-induced neurotoxicity. Resveratrol protected HT22 neuronal cells from glutamateinduced cytotoxicity and increased HO-1 expression as well as SIRT1 activity in a concentration-dependent manner. Cytoprotec-tion afforded by resveratrol was partially reversed by the specific inhibition of HO-1 expression by HO-1 small interfering RNA and the nonspecific blockage of HO-1 activity by tin protoporphyrin IX, but not by SIRT1 inhibitors. Surprisingly, TMS, a resveratrol derivative with methoxyl groups in lieu of the hydroxyl groups, and trans-stilbene, a non-hydroxylated analog, failed to protect HT22 cells from glutamate-induced cytotoxicity and to increase HO-1 expression and SIRT1 activity. Taken together, our findings suggest that the cytoprotective effect of resveratrol was at least in part associated with HO-1 expression but not with SIRT1 activation and, importantly, that the presence of hydroxyl groups on the benzene rings of resveratrol appears to be necessary for cytoprotection against glutamate-induced oxidative stress, HO-1 expression, and SIRT1 activation in HT22 neuronal cells.

Abstract 3494: LOXblock-1 Provides Efficient, Specific, And Long-lasting Neuroprotection Against Transient Focal Ischemia In Mice By Inhibiting 12/15-Lipoxygenase

Background and Purpose - Acute phase stroke treatment options are currently very limited. We have previously demonstrated 12/15-lipoxygenase (12/15-LOX) to initiate an apoptotic cell death cascade, contributing to neuronal cell death, but also vascular leakage and edema formation in the ischemic brain. The current study was designed to test if a novel inhibitor LOXBlock-1, can specifically reduce 12/15-LOX activity in vivo, and provide efficient and long-lasting protection against stroke-induced injury. Methods - In vitro protection by LOXBlock-1 was tested in HT22 cells. A cell-based reporter assay determined activation of HIF1a and NRF2. The lipoxygenase metabolite 12-HETE was measured using a commercial EIA kit, and confirmed by HPLC/mass spectrometry. We injected LOXBlock-1 intraperitoneally in mice subjected to middle cerebral artery occlusion (MCAO). Immunohistochemistry was used to detect MDA-modified lysine in the ischemic brain, TTC and H&E stains were used to determine infarct size. Neurological severity score and corner test were used to evaluate behavioral outcome. Intracerebral hemorrhage was induced by collagenase injection. Results - Nanomolar LOXBlock-1 protected HT22 cells against oxidative glutamate toxicity. LOXBlock-1 reduced 12-HETE levels, without activating HIF1a- and NRF2-dependent pathways. One day after MCAO, MDA-modified lysine co-localized with 12/15-LOX in the peri-infarct cortex, and LOXBlock-1 reduced hemispheric swelling, levels of 12-HETE, and infarct size. After 14 days, infarct size remained reduced, and behavioral outcome was improved. LOXBlock-1 did not increase collagenase-induced hemorrhage. Conclusion - 12/15-LOX inhibition by LOXBlock-1 provides effective and long-lasting protection against transient focal ischemia in mice, without worsening hemorrhage. LOXBlock-1 may be a suitable acute phase stroke treatment.

Glutamate-induced cell death in HT22 mouse hippocampal cells is attenuated by paxilline, a BK channel inhibitor

In the present study, we show that the large conductance calcium-activated potassium channel (BKCa channel) inhibitor paxilline protects neuronal cells against glutamate-induced cell death. In our studies, we used HT22 mouse hippocampal cells as an experimental model and observed that the effect of paxilline was dose-dependent. We also found that other inhibitors of BKCa channels, iberiotoxin and charybdotoxin, were not cytoprotective. Paxillinol, which is a structural analog of paxilline but does not inhibit BKCa channel, also protected HT22 cells against glutamate-induced toxicity. These data suggest that the observed cytoprotection was not related to BKCa channel inhibition by paxilline. In addition, paxilline neither restored glutathione levels nor reduced the amount of reactive oxygen species upon glutamate treatment.Our results suggest that paxilline protects neuronal HT22 cells against glutamate-induced cell death independently of BKCa channel activity and oxidative stress induced by glutamate treatment.

Mechanism for the protective effect of resveratrol against oxidative stress-induced neuronal death

Oxidative stress can induce cytotoxicity in neurons, which plays an important role in the etiology of neuronal damage and degeneration. This study sought to determine the cellular and biochemical mechanisms underlying resveratrol's protective effect against oxidative neuronal death. Cultured HT22 cells, an immortalized mouse hippocampal neuronal cell line, were used as an in vitro model, and oxidative stress and neurotoxicity were induced in these neuronal cells by exposure to high concentrations of glutamate. Resveratrol strongly protected HT22 cells from glutamate-induced oxidative cell death. Resveratrol's neuroprotective effect was independent of its direct radical scavenging property, but instead was dependent on its ability to selectively induce the expression of mitochondrial superoxide dismutase (SOD2) and, subsequently, reduce mitochondrial oxidative stress and damage. The induction of mitochondrial SOD2 by resveratrol was mediated through the activation of the PI3K/Akt and GSK-3β/β-catenin signaling pathways. Taken together, the results of this study show that up-regulation of mitochondrial SOD2 by resveratrol represents an important mechanism for its protection of neuronal cells against oxidative cytotoxicity resulting from mitochondrial oxidative stress.