Oxygen-glucose Deprivation-induced Cell Death Research Articles

Therapeutic Role of a Cysteine Precursor, OTC, in Ischemic Stroke Is Mediated by Improved Proteostasis in Mice.

Oxidative stress aggravates brain injury following ischemia/reperfusion (I/R). We previously showed that ubiquilin-1 (Ubqln1), a ubiquitin-like protein, improves proteostasis and protects brains against oxidative stress and I/R-induced brain injury. Here, we demonstrate that a small molecule compound, L-2-oxothiazolidine-4-carboxylic acid (OTC) that functions as a precursor of cysteine, upregulated Ubqln1 and protected cells against oxygen-glucose deprivation-induced cell death in neuronal cultures. Further, the administration of OTC either at 1h prior to ischemia or 3h after the reperfusion significantly reduced brain infarct injury and improved behavioral outcomes in a stroke model. Administration of OTC also increased glutathione (GSH) level and decreased superoxide production, oxidized protein, and neuroinflammation levels in the penumbral cortex after I/R in the stroke mice. Furthermore, I/R reduced both Ubqln1 and the glutathione S-transferase protein levels, whereas OTC treatment restored both protein levels, which was associated with reduced ubiquitin-conjugated protein level. Interestingly, in the Ubqln1 knockout (KO) mice, OTC treatment showed reduced neuroprotection and increased ubiquitin-conjugated protein level when compared to the similarly treated non-KO mice following I/R, suggesting that OTC-medicated neuroprotection is, at least partially, Ubqln1-dependent. Thus, OTC is a potential therapeutic agent for stroke and possibly for other neurological disorders and its neuroprotection involves enhanced proteostasis.

Melatonin Acts in Synergy with Hypothermia to Reduce Oxygen-Glucose Deprivation-Induced Cell Death in Rat Hippocampus Organotypic Slice Cultures

Background: Hypoxic-ischemic encephalopathy is a major cause of neonatal morbidity. Therapeutic hypothermia, while beneficial, still leaves many treated infants with lifelong disabilities. Thus, adjunctive therapies, such as melatonin, are needed to provide additional neuroprotection. Objectives: The aim of this study was to determine a range of melatonin concentrations that could result in neuroprotective synergy with hypothermia. Methods: Hypoxia-ischemia was simulated by transient oxygen-glucose deprivation (OGD) in organotypic hippocampal slice cultures derived from neonatal rats. Cell damage was quantified by propidium iodide (PI) labeling. Results: Melatonin reduced OGD- induced cell death in a concentration-dependent manner (1–100 μM) with an EC₅₀ of about 25 μM. Hypothermia attenuated cell death in a time-dependent manner, with a nearly full protection upon 24-h exposure (78%) and partial protection (40%) upon 6-h exposure. When submaximal effective concentrations of melatonin (25 or 50 μM, resulting in 54 and 64% protection) were combined with 6 h of hypothermia, nearly full protection (73 and 78%, respectively; p < 0.05 and p < 0.01) was observed. Conclusion: Melatonin acts in synergy with hypothermia in attenuating OGD-induced damage in organotypic hippocampal cultures. This reductionist approach allows the determination of a range of concentrations of melatonin capable of enhancing hypothermic neuroprotection. This information, coupled with pharmacokinetic data, will help to define the therapeutic dosage of melatonin in vivo and, ultimately, in patients.

Inhibition of PTEN protects PC12 cells against oxygen-glucose deprivation induced cell death through mitoprotection

Mitochondria involve in the determination of ischemic neuronal cell fate through regulation of apoptotic and necrotic cell death. Phosphatase and tensin homolog (PTEN) protein negatively regulates Akt/PKB signaling which is the major cell survival pathway. The current study aimed to examine the impact of SF1670, a potent PTEN inhibitor, on mitochondria-mediated cell survival pathways in an in vitro stroke-like model. PC12 cells were exposed to one hour oxygen and glucose deprivation (OGD) followed by different time points of reperfusion (0, 30, 60, 120 and 180 min) and SF1670 treatments. Our findings showed that OGD/R exposure increased reactive oxygen species (ROS) levels, reduced phosphorylated Akt (p-Akt), ratios of Bcl-2/BAX, intracellular ATP, mitochondrial vital activity and mitochondrial membrane potential (Δψm). OGD/R exposure also increased cleaved caspase 3/pro-caspase 3 and cleaved caspase 9/pro-caspase 9 ratios associated with low cell viability, high lactate dehydrogenase (LDH) release, and greater apoptotic cell death in the TUNEL assay. Conversely, inhibition of PTEN by SF1670 were associated with increased expression of p-Akt and anti-apoptotic proteins (Bcl-2), attenuated pro-apoptotic proteins (BAX) and oxidative stress index (ROS), improved mitochondrial function (restored MMP and ATP), and decreased apoptotic cell death. These results strongly suggest that neuroprotective effect of SF1670 against OGD/R-induced cell death at least is partially mediated through mitoprotective properties of SF1670.

Pretreatment With Argon Protects Human Cardiac Myocyte-Like Progenitor Cells from Oxygen Glucose Deprivation-Induced Cell Death by Activation of AKT and Differential Regulation of Mapkinases.

The noble gas argon induces cardioprotection in a rabbit model of myocardial ischemia and reperfusion. However, no studies in human primary cells or subjects have been performed so far. We used human cardiac myocyte-like progenitor cells (HCMs) to investigate the protective effect on the cellular level. HCMs were pretreated with 30% or 50% argon before oxygen-glucose deprivation (OGD) and reperfusion. We evaluated apoptotic states by flow cytometry and the activation of mitogen-activated protein kinase (MAPKs) members extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), p38 MAPkinase, and protein kinase B (Akt) by Westernblot analysis and by activity assays of downstream transcription factors. Specific inhibitors were used to proof a significant participation of these pathways in the protection by argon. Beneficial effects were further assessed by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay, lactate dehydrogenase (LDH), mitochondrial deoxyribonucleic acid (mtDNA), and cytokine release. Pretreatment with 30% or 50% argon for 90 min before OGD resulted in a significant protection of HCMs against apoptosis. This effect was reversed by the application of MAPK and Akt inhibitors during argon exposure. Argon 30% reduced the release of LDH by 33% and mtDNA by 45%. The release of interleukin 1β was reduced by 44% after OGD and more than 90% during reperfusion. Pretreatment with argon protects HCMs from apoptosis under ischemic conditions via activation of Akt, Erk, and biphasic regulation of JNK. Argon gas is cheap and easily administrable, and might be a novel therapy to reduce myocardial ischemia-reperfusion injury.

P-288 - Nrf2 redox signalling in brain endothelial cells adapted to physiological O2: an in vitro model for ischaemia-reperfusion injury in stroke

Sulforaphane (SFN) has been shown to protect against damage sustained following transient ischaemic stroke via activation of the redox-sensitive transcription factor Nrf2. To further understand the protective effects of SFN on the cerebral endothelium, mouse brain endothelial cell line bEnd.3 was subjected to oxygen-glucose deprivation (OGD), an in vitro model of cerebral stroke. Pre-treatment of bEnd.3 cells with SFN (2.5 µM, 12 h) afforded increased protection against OGD induced cell death. Our lab recently reported that long-term culture under physiological oxygen, 5% O2, results in a phenotype markedly different to cells cultured under ambient air, 18% O2, shown by downregulation of selected Nrf2 target genes by Nrf2 activator. In the present study, the bEnd.3 cell phenotype was determined following long-term (5 days) culture under 5% or 18% O2. Under 5% O2, the intracellular O2 content of bEnd.3 cells was 3.6% O2, and a reduced rate of cell proliferation was observed. Furthermore, bEnd.3 cells cultured under 5% O2 exhibited greater sensitivity to OGD-induced cell death. The present study highlights the importance of conducting cell culture studies under physiological O2 levels.

Interleukin 15 activates Akt to protect astrocytes from oxygen glucose deprivation-induced cell death

Astrocytes play a pivotal role in neuronal survival under the condition of post-ischemic brain inflammation, but the relevant astrocyte-derived mediators of ischemic brain injury remain to be defined. IL-15 supports survival of multiple lymphocyte lineages in the peripheral immune system, but the role of IL-15 in inflammatory disease of the central nervous system is not well defined. Recent research has shown an increase of IL-15-expressing astrocytes in the ischemic brain. Since astrocytes promote neuron survival under cerebral ischemia by buffering excess extracellular glutamate and producing growth factors, recovery of astrocyte function could be of benefit for stroke therapy. Here, we report that IL-15 is the pro-survival cytokine that prevents astrocyte death from oxygen glucose deprivation (OGD)-induced damage. Astrocytes up-regulate expression of the IL-15/IL-15Rα complex under OGD, whereas OGD down-regulates the levels of pSTAT5 and pAkt in astrocytes. IL-15 treatment ameliorates the decline of pAkt, decreases the percentage of annexin V+ cells, inhibits the activation of caspase-3, and activates the Akt pathway to promote astrocyte survival in response to OGD. We further identified that activation of Akt, but not PKCα/βI, is essential for astrocyte survival under OGD. Taken together, this study reveals the function of IL-15 in astrocyte survival via Akt phosphorylation in response to OGD-induced damage.

Electromagnetic field (10 Hz, 1 mT) protects mesenchymal stem cells from oxygen-glucose deprivation-induced cell death by reducing intracellular Ca2+ and reactive oxygen species

Protective effects of electromagnetic fields (EMFs) against oxygen and glucose deprivation (OGD)-induced human mesenchymal stem cell (MSC) death were studied. Cell survival, intracellular calcium and ROS/RNS levels were measured after culturing MSCs for 3h under OGD with or without EMF exposure. The survival rate of cells cultured under OGD condition was significantly reduced compared to control cells, while cells cultured in OGD with 10Hz/1mT EMF exposure had higher survival ratio than that in equivalent non-exposed cells. This protective effect of EMF was not observed at different frequency/intensity combinations such as 10Hz/0.01mT, 10Hz/0.1mT, 50Hz/1mT and 100Hz/1mT. ROS/RNS levels of cells cultured under OGD conditions significantly increased compared to the control level while 10Hz/1mT EMF alleviated this effect. Intracellular calcium levels in OGD group were higher than control while those in OGD plus 10Hz/1mT EMF group were significantly lower than OGD group. Addition of Ca2+ chelator promoted protective effects of EMF against OGD-induced MSC death. Our results suggest that 10Hz/1mT EMF exposure protects MSCs from OGD-induced cell death and the underlying mechanisms of the protection are reduction of intracellular levels of Ca2+ and ROS/RNS.

High-Content Genome-Wide RNAi Screen Reveals CCR3 as a Key Mediator of Neuronal Cell Death.

Neuronal loss caused by ischemic injury, trauma, or disease can lead to devastating consequences for the individual. With the goal of limiting neuronal loss, a number of cell death pathways have been studied, but there may be additional contributors to neuronal death that are yet unknown. To identify previously unknown cell death mediators, we performed a high-content genome-wide screening of short, interfering RNA (siRNA) with an siRNA library in murine neural stem cells after exposure to N-methyl-N-nitroso-N'-nitroguanidine (MNNG), which leads to DNA damage and cell death. Eighty genes were identified as key mediators for cell death. Among them, 14 are known cell death mediators and 66 have not previously been linked to cell death pathways. Using an integrated approach with functional and bioinformatics analysis, we provide possible molecular networks, interconnected pathways, and/or protein complexes that may participate in cell death. Of the 66 genes, we selected CCR3 for further evaluation and found that CCR3 is a mediator of neuronal injury. CCR3 inhibition or deletion protects murine cortical cultures from oxygen-glucose deprivation-induced cell death, and CCR3 deletion in mice provides protection from ischemia in vivo. Taken together, our findings suggest that CCR3 is a previously unknown mediator of cell death. Future identification of the neural cell death network in which CCR3 participates will enhance our understanding of the molecular mechanisms of neural cell death.

N-3 Polyunsaturated Fatty Acids Reduce Neonatal Hypoxic/Ischemic Brain Injury by Promoting Phosphatidylserine Formation and Akt Signaling.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) attenuate neonatal hypoxic/ischemic (H/I) brain damage, but the underlying mechanisms are not fully understood. This study tested the hypothesis that n-3 PUFAs enhance Akt-dependent prosurvival signaling by promoting the biosynthesis of phosphatidylserine in neuronal cell membranes. Dietary n-3 PUFA supplementation was initiated on the second day of pregnancy in dams. H/I was induced in 7-day-old rat pups by ipsilateral common carotid artery occlusion followed by hypoxia (8% oxygen for 2.5 hours). Neurological outcomes, brain tissue loss, cell death, and the activation of signaling events were assessed after H/I. The effects of n-3 PUFAs (docosahexaenoic acid and eicosapentaenoic acid) on oxygen-glucose deprivation-induced cell death and the underlying mechanism of protection were also examined in primary cortical neuron cultures. n-3 PUFAs reduced brain tissue loss at 7 days after H/I and improved neurological outcomes, whereas inhibition of PI3K/Akt signaling by LY294002 partially abrogated this neuroprotective effect. Docosahexaenoic acid/eicosapentaenoic acid also prevented ischemic neuronal death through the Akt prosurvival pathway in vitro. Furthermore, docosahexaenoic acid/eicosapentaenoic acid increased the production of phosphatidylserine, the major membrane-bound phospholipids, after ischemia both in vitro and in vivo. A reduction in membrane phosphatidylserine by shRNA-mediated knockdown of phosphatidylserine synthetase-1 attenuated Akt activation and neuronal survival after docosahexaenoic acid/eicosapentaenoic acid treatment in the oxygen-glucose deprivation model. n-3 PUFAs robustly protect against H/I-induced brain damage in neonates by activating Akt prosurvival pathway in compromised neurons. In addition, n-3 PUFAs promote the formation of membrane phosphatidylserine, thereby promoting Akt activity and improving cellular survival.

Vasculogenesis in Experimental Stroke After Human Cerebral Endothelial Cell Transplantation

Despite the reported functional recovery in transplanted stroke models and patients, the mechanism of action underlying stem cell therapy remains not well understood. Here, we examined the role of stem cell-mediated vascular repair in stroke. Adult rats were exposed to transient occlusion of the middle cerebral artery and 3 hours later randomly stereotaxically transplantated with 100K, 200K, or 400K human cerebral endothelial cell 6 viable cells or vehicle. Animals underwent neurological examination and motor test up to day 7 after transplantation then euthanized for immunostaining against neuronal, vascular, and specific human antigens. A parallel in vitro study cocultured rat primary neuronal cells with human cerebral endothelial cell 6 under oxygen-glucose deprivation and treated with vascular endothelial growth factor (VEGF) and anti-VEGF. Stroke animals that received vehicle infusion displayed typical occlusion of the middle cerebral artery-induced behavioral impairments that were dose-dependently reduced in transplanted stroke animals at days 3 and 7 after transplantation and accompanied by increased expression of host neuronal and vascular markers adjacent to the transplanted cells. Some transplanted cells showed a microvascular phenotype and juxtaposed to the host vasculature. Infarct volume in transplanted stroke animals was significantly smaller than vehicle-infused stroke animals. Moreover, rat neurons cocultured with human cerebral endothelial cell 6 or treated with VEGF exhibited significantly less oxygen-glucose deprivation-induced cell death that was blocked by anti-VEGF treatment. We found attenuation of behavioral and histological deficits coupled with robust vasculogenesis and neurogenesis in endothelial cell-transplanted stroke animals, suggesting that targeting vascular repair sets in motion a regenerative process in experimental stroke possibly via the VEGF pathway.

The Parkinson's disease‐associated gene PINK1 protects neurons from ischemic damage by decreasing mitochondrial translocation of the fission promoter Drp1

Our previous study has shown that PTEN-induced novel kinase 1 (PINK1) knocking down significantly induced mitochondrial fragmentation. Although PINK1 is proved to be associated with autosomal recessive parkinsonism and its function in this chronic pathological process is widely studied, its role in acute energy crisis such as ischemic stroke is poorly known. In this study by employing an oxygen-glucose deprivation (OGD) neuronal model, we explored the function of PINK1 in cerebral ischemia. Human PINK1, two PINK1 mutants W437X and K219M, or Pink1 shRNA were transduced before OGD using lentiviral delivery. Our results showed that over-expression of wild-type PINK1 significantly ameliorated OGD induced cell death and energy disturbance including reduced ATP generation and collapse of mitochondrial membrane potential. PINK1 over-expression also reversed OGD increased mitochondrial fragmentation, and suppressed the translocation of the mitochondrial fission protein dynamin-related protein 1 (Drp1) from the cytosol to the mitochondria. Transduction of the mutant PINK1 failed to provide any protective effect, while knockdown of Pink1 significantly increased the severity of OGD-induced neuronal damage. Importantly, inhibition of Drp1 reversed the effects of knocking down Pink1 on neuronal death and ATP production in response to OGD. This study demonstrates that PINK1 prevents ischemic damage in neurons by attenuating mitochondrial translocation of Drp1, which maintains mitochondrial function and inhibits ischemia-induced mitochondrial fission. These novel findings implicate a pivotal role of PINK1 regulated mitochondrial dynamics in the pathology of ischemic stroke. In this study by employing an oxygen-glucose deprivation (OGD) neuronal model, we explored the function of PINK1 in cerebral ischemia. We indicated that PINK1 significantly ameliorated OGD induced cell death and energy disturbance including reduced ATP generation and collapse of mitochondrial membrane potential by attenuating mitochondrial translocation of Drp1, which maintains mitochondrial function and inhibits ischemia-induced mitochondrial fission.

Effects of roscovitine, a cell cycling-dependent kinase inhibitor, on intraocular pressure of rabbit and retinal ganglion cell damage

Glaucoma is characterized by increased intraocular pressure (IOP) and the death of retinal ganglion cells. Previously, we reported that roscovitine, a cell cyclin-dependent kinase (CDK) inhibitor, strongly induced relaxation of porcine trabecular meshwork cells, implicating an interaction with lowered IOP. In addition, the activity of CDKs is known to increase in response to high IOP, which is linked to retinal ganglion cell damage. However, the effects of roscovitine on IOP and retinal damage have not been investigated. Roscovitine has racemic isomers that differ in their inhibition of CDKs. Therefore, we investigated the effects of both the R-isomer and the S-isomer on the IOP of rabbits and on the death of cultured retinal ganglion cells. In the in vivo rabbit experiment, instillation of both isomers significantly lowered the IOP. In the in vitro cell experiment, the R-isomer amplified the effects of tunicamycin, an endoplasmic reticulum stress inducer, and increased oxygen–glucose deprivation-induced cell death, whereas S-isomer significantly inhibited this cell death. Therefore, both isomers of roscovitine can lower the IOP, but from the perspective of neuroprotective effects, the S-isomer was superior to the R-isomer. The S-isomer of roscovitine may be useful as an agent for lowering IOP and its neuroprotective effects.

The protective effect of nordihydroguaiaretic acid on cerebral ischemia/reperfusion injury is mediated by the JNK pathway

Nordihydroguaiaretic acid (NDGA) is a powerful antioxidant and/or lipoxygenase (LOX) inhibitor which is isolated from Larrea tridentate. NDGA has been shown to have neuroprotective effects both in vitro and in vivo experiments. However, little is known regarding NDGA's protective mechanism in ischemia/reperfusion (I/R) injury. We therefore investigated the potential protective effects of NDGA and explored the underlying mechanisms. Oxygen-glucose deprivation (OGD) was performed in cultured rat cortical neurons for 60min. The effect of NDGA on OGD induced cell death and apoptosis was examined at 24h after reperfusion. Western blot was used to analyze the expression of p-c-jun and p-JNK. Exogenous 5-, 12-, 15-hydroxyeicosatetraenoic acid (HETE) was added respectively to the cells to investigate the contribution of the products of LOX to the c-Jun N-terminal protein kinase (JNK) pathway. Rats were injected intraperitoneally with NDGA before being subjected to middle cerebral artery occlusion (MCAO). At 24h after reperfusion, neurological deficit, brain infarct volume and the expression of p-c-jun and p-JNK were measured. The results showed that NDGA increased cell viability and inhibited apoptosis after OGD in neurons. NDGA suppressed the expression of p-c-jun and p-JNK in cortical neurons, whereas exogenous 12-, 15-HETE attenuated this effect. NDGA improved neurological deficit, reduced infarct volumes, and downregulated the overexpression of p-c-jun and p-JNK after MCAO and reperfusion. In conclusion, these results suggest that NDGA's protective effect against I/R injury is mediated by the suppression of JNK pathway. This effect is probably due to its 12/15-LOX inhibitor property.

Quantitative analysis of phenolic components and glycoalkaloids from 20 potato clones and in vitro evaluation of antioxidant, cholesterol uptake, and neuroprotective activities

Potato (Solanum tuberosum L.) is one of the most important food crops in the world and provides essential nutrients. With an aim to develop potato varieties for functional food or nutraceutial applications, we have conducted metabolomic profiling, total phenolics, chlorogenic acid, anthocyanins, and glycoalkaloids analyses on 20 selected potato clones within the Canadian potato breeding program of Agriculture and Agri-Food Canada. Pigmented potatoes in general contain higher levels of phenolic components, including chlorogenic acid and anthocyanins. Levels of phenolics were retained with granulation processing of pigmented potato tubers, but glycoalkaloids were significantly reduced with granulation. The pigmented potatoes also have higher antioxidant activity reaching up to 35% of that for berries, measured as their potency in scavenging DPPH radicals. Extracts of the 20 potato clones (peel, tuber, and granule) were also evaluated for in vitro effects on liver LDL cholesterol uptake and protection of cortical neurons from cell death caused by oxygen glucose deprivation (OGD). These potato extracts in general showed mild activity in enhancing LDL cholesterol uptake in liver HepG2 cells, and also protected cortical neurons against OGD induced cell death, with extracts from granules of six of the potato clones showing significant neuroprotective effects. The bioactive components are not dependent on pigmentation of potato clones. These novel bioactivities identified in potatoes warrant in-depth investigations in the future. Taken together, our results provide further evidence for the enhanced health beneficial components in potato.

Endogenous insulin signaling protects cultured neurons from oxygen–glucose deprivation-induced cell death

Curiosity surrounding the physiological relevance of neural insulin signaling has gradually developed since the discovery that nervous tissue contains both the hormone and its receptor. Similar to other receptor tyrosine kinases, ligand interaction with the insulin receptor (IR) activates a variety of intracellular signaling pathways, particularly those relevant to cellular survival. Consequently, one explanation for the presence of the insulin pathway in the brain may involve participation in the response to neuronal injury. To investigate this possibility, the present study began by examining the effect of oxygen–glucose deprivation (OGD), a well-characterized in vitro model of ischemia, on ligand-binding, surface expression, and function of the IR in cultured rat neurons that were prepared under serum-free conditions. Reduced insulin-binding was observed following OGD, although surface expression of the receptor was not altered. However, OGD did significantly decrease the ability of insulin to stimulate phosphorylation of the transmembrane IR β-subunit, without affecting protein expression of this subunit. Subsequent experiments focused on the manner in which pharmacologically manipulating IR function affected neuronal viability after OGD. Application of the IR sensitizer metformin moderately improved neuronal viability, while the specific IR tyrosine kinase inhibitor tyrphostin A47 was able to dramatically decrease viability; both compounds acted without affecting IR surface expression. Our study suggests that not only does the IR appear to play an important role in neuronal survival, but also that neurons may actively maintain IRs on the cell surface to compensate for the OGD-induced decrease in the ability of insulin to phosphorylate its receptor.

Ionotropic glutamate receptors and glutamate transporters are involved in necrotic neuronal cell death induced by oxygen–glucose deprivation of hippocampal slice cultures

Organotypic hippocampal slice cultures represent a feasible model for studies of cerebral ischemia and the role of ionotropic glutamate receptors in oxygen–glucose deprivation-induced neurodegeneration. New results and a review of existing data are presented in the first part of this paper. The role of glutamate transporters, with special reference to recent results on inhibition of glutamate transporters under normal and energy-failure (ischemia-like) conditions is reviewed in the last part of the paper. The experimental work is based on hippocampal slice cultures derived from 7 day old rats and grown for about 3 weeks. In such cultures we investigated the subfield neuronal susceptibility to oxygen–glucose deprivation, the type of induced cell death and the involvement of ionotropic glutamate receptors. Hippocampal slice cultures were also used in our studies on glutamate transporters reviewed in the last part of this paper. Neurodegeneration was monitored and/or shown by cellular uptake of propidium iodide, loss of immunocytochemical staining for microtubule-associated protein 2 and staining with Fluoro-Jade B. To distinguish between necrotic vs. apoptotic neuronal cell death we used immunocytochemical staining for active caspase-3 (apoptosis indicator) and Hoechst 33342 staining of nuclear chromatin. Our experimental studies on oxygen–glucose deprivation confirmed that CA1 pyramidal cells were the most susceptible to this ischemia-like condition. Judged by propidium iodide uptake, a selective CA1 lesion, with only minor affection on CA3, occurred in cultures exposed to oxygen–glucose deprivation for 30 min. Nuclear chromatin staining by Hoechst 33342 and staining for active caspase-3 showed that oxygen–glucose deprivation induced necrotic cell death only. Addition of 10μM of the N-methyl- d-aspartate glutamate receptor antagonist MK-801, and 20μM of the non- N-methyl- d-aspartate glutamate receptor antagonist 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo( F)quinoxaline to the culture medium confirmed that both N-methyl- d-aspartate and non- N-methyl- d-aspartate ionotropic glutamate receptors were involved in the oxygen–glucose deprivation-induced cell death. Glutamate is normally quickly removed, from the extracellular space by sodium-dependent glutamate transporters. Effects of blocking the transporters by addition of the dl-threo-beta-benzyloxyaspartate are reviewed in the last part of the paper. Under normal conditions addition of dl-threo-beta-benzyloxyaspartate in concentrations of 25μM or more to otherwise untreated hippocampal slice cultures induced neuronal cell death, which was prevented by addition of 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo( F)quinoxaline and MK-801. In energy failure situations, like cerebral ischemia and oxygen–glucose deprivation, the transporters are believed to reverse and release glutamate to the extracellular space. Blockade of the transporters by a subtoxic (10μM) dose of dl-threo-beta-benzyloxyaspartate during oxygen–glucose deprivation (but not during the next 48 h after oxygen–glucose deprivation) significantly reduced the oxygen–glucose deprivation-induced propidium iodide uptake, suggesting a neuroprotective inhibition of reverse transporter activity by dl-threo-beta-benzyloxyaspartate during oxygen–glucose deprivation under these conditions. Adding to this, other results from our laboratory have demonstrated that pre-treatment of the slice cultures with glial cell-line derived neurotrophic factor upregulates glutamate transporters. As a logical, but in some glial cell-line derived neurotrophic factor therapy-related conditions clearly unwanted consequence the susceptibility for oxygen–glucose deprivation-induced glutamate receptor-mediated cell death is increased after glial cell-line derived neurotrophic factor treatment. In summary, we conclude that both ionotropic glutamate receptors and glutamate transporters are involved in oxygen–glucose deprivation-induced necrotic cell death in hippocampal slice cultures, which have proven to be a feasible tool in experimental studies on this topic.

Neuroprotective and neurotoxic effects of monoamine oxidase-B inhibitors and derived metabolites under ischemia in PC12 cells

Selegiline and rasagiline are selective and irreversible monoamine oxidase-B inhibitors that exert neuroprotective effects in various preclinical models. The aim of the present study was to examine the effect of selegiline and its major metabolite, l-methamphetamine in comparison to rasagiline and its major metabolite, 1- R-aminoindan on oxygen–glucose deprivation induced cell death in nerve growth factor (NGF)-differentiated pheochromocytoma (PC12) cells. Our results show that selegiline reduces oxygen–glucose deprivation induced cell death by 30%. When the cultures were treated with rasagiline at similar concentrations, cell death induced by oxygen–glucose deprivation was reduced by 45–55%. l-methamphetamine, a major selegiline metabolite, but not 1- R-aminoindan, the major rasagiline metabolite, enhanced oxygen–glucose deprivation-induced cell death by 70%. Under normoxic conditions, both metabolites lack neurotoxicity. Concomitant exposure of the cultures under oxygen–glucose deprivation, to a combination of either selegiline and l-methamphetamine or rasagiline and 1- R-aminoindan, indicated that l-methamphetamine, but not 1- R-aminoindan, blocked the neuroprotective effect of the parental drug. These results suggest there may be a neuroprotective advantage of rasagiline over selegiline.

Calcitonin Potentiates Oxygen–Glucose Deprivation-Induced Neuronal Death

Calcitonin is a hormone that decreases plasma calcium through inhibition of osteolysis. It is used in the treatment of osteoporosis and other bone disorders. Salmon calcitonin is typically utilized in individuals for whom use of estrogen is contraindicated, for example, women at high risk for breast cancer. In addition to actions on bone, calcitonin may have effects on the central nervous system. Receptors for calcitonin are present on central neurons, and salmon calcitonin has been shown to alter neuronal activity. Since salmon calcitonin is used clinically, and it can have actions on neurons, the present studies were designed to determine whether salmon calcitonin could alter death of cortical neurons. The effects of salmon calcitonin on neuronal death induced by exposure of murine cortical cultures to serum deprivation, staurosporine, oxygen–glucose deprivation, kainate, and NMDA were tested. Salmon calcitonin had no effect on apoptotic cell death in cortical cultures. However, acute treatment with salmon calcitonin (1–1000 nM) caused significant potentiation of neuronal death induced by oxygen–glucose deprivation. Similarly, salmon calcitonin potentiated cell death induced by exposure to kainate. In contrast, it did not potentiate cell death induced by exposure to NMDA. In fact, addition of a high concentration (1000 nM) of salmon calcitonin attenuated NMDA toxicity. These results indicate that calcitonin is not a survival factor for cortical neurons; however, it can alter excitotoxic cell death. The most interesting, and disturbing, effect is the ability of low concentrations of salmon calcitonin to potentiate oxygen–glucose deprivation-induced cell death.