Non-N-methyl- -aspartate glutamate receptors mediate oxygen–glucose deprivation-induced oligodendroglial injury

Cells of oligodendroglial lineage are susceptible to oxygen and glucose deprivation. When oligodendrocyte-like cells differentiated from CG-4-immortalized rat O-2A progenitor cells were exposed to hypoxia alone or glucose deprivation alone for 48 h, release of lactate dehydrogenase (LDH) into the culture medium did not increase. However, when cells were deprived of both oxygen and glucose for 6 or 12 h preceding reoxygenation for 2 h, LDH release increased. Adding glucose to the medium protected against cell death and increased lactate production in a concentration-dependent manner. Cell damage induced by deprivation of oxygen and glucose was prevented by calcium-free medium or by non-N-methyl- -aspartate glutamate receptor (GluR) antagonists, such as 6-cyano-7-nitroquinoxaline-2,3-dione or LY293558, but not by the voltage-dependent calcium channel blocker, nimodipine, or by the N-methyl- -aspartate GluR antagonist, MK-801. The glutamate concentration in the medium from cells exposed to oxygen–glucose deprivation for 12 h was 49.70±3.04 μM/l, which is sufficient to activate GluRs during deprivation of oxygen and glucose. Apoptotic cells detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) or Hoechst 33258 staining did not increase in cells exposed to oxygen–glucose deprivation for 12 h and subsequent reoxygenation for 2 h. No DNA laddering was detected by agarose gel electrophoresis from cells exposed to deprivation of oxygen and glucose. Neither acetyl-YVAD-CHO, an inhibitor of caspase-1-like proteases, nor acetyl-DEVD-CHO, an inhibitor of caspase-3-like proteases, prevented oxygen–glucose deprivation-induced injury. Thus, oxygen and glucose deprivation causes calcium-influx-induced necrotic cell damage in cells of oligodendroglial lineage via non-N-methyl- -aspartate GluR channels.     

Ibudilast prevents oxygen-glucose deprivation-induced oligodendroglial injury]

Previously we have demonstrated that ibudilast, which is used clinically for treating patients with asthma and cerebrovascular diseases, prevents excitotoxicity of oligodendroglial lineage mediated by Ca2+ influx via non-N-methyl-D-aspartate (NMDA) glutamate receptor (GluR) channels. We here present a finding that ibudilast prevents oxygen-glucose deprivation (OGD)-induced oligodendroglial injury. The oligodendrocyte-like cells (OLC), differentiated from the CG-4 cell line established from rat oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, were exposed to hypoxia in the absence of glucose for 12 h and subsequent reoxygenation for 2 h. Cell damage was evaluated by measuring activity of lactate dehydrogenase (LDH) released into the culture medium. OGD for 12 h induced 30 to 50% LDH release into the medium. OLC damage induced by deprivation of oxygen and glucose was prevented by ibudilast at concentrations of > or = 50 microM. The protection given by ibudilast against OGD-induced injury was enhanced by prostacyclin (PGI2). OGD-induced OLC injury was prevented by 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), an inhibitor of non-NMDA GluR or deprivation of Ca2+ from culture medium. While ibudilast increased intracellular cAMP at concentrations of > or = 10 microM, at least 100 microM concentrations were needed to increase intracellular cGMP. Therefore, we concluded that ibudilast prevented OGD-induced oligodendroglial injury possibly by increasing intracellular cAMP which modulates Ca2+ influx via non-NMDA GluR channels.     

Dual inhibition of protein phosphatase-1/2A and calpain rescues nerve growth factor-differentiated PC12 cells from oxygen-glucose deprivation-induced cell death

In the present study, we examined how the cell survival signaling via cyclic AMP-responsive element binding protein (CREB) and Akt, and the cell death signaling via cystein proteases, calpain and caspase-3, are involved in oxygen-glucose deprivation (OGD) followed by reoxygenation (OGD/reoxygenation)-induced cell death in nerve growth factor (NGF)-differentiated PC12 cells. OGD/reoxygenation-induced cell death was evaluated by LDH release into the culture medium. The level of LDH release was low (9.0% +/- 4.1%) immediately after 4 hr of OGD (0 hr of reoxygenation), was significantly increased to 28.6% +/- 6.6% at 3 hr of reoxygenation, and remained at similar levels at 6 and 20 hr of reoxygenation, suggesting that reoxygenation at least for 3 hr resulted in the loss of cell membrane integrity. After 4 hr of OGD followed by 3 hr of reoxygenation, dephosphorylation of phosphorylated CREB (pCREB), but not phosphorylated Akt (pAkt), was induced. Under these conditions, calpain- but not caspase-3-mediated alpha-spectrin breakdown product was increased, indicating that OGD/reoxygenation also induced an increase in calpain activity. The restoration of pCREB by protein phosphatase (PP)-1/2A inhibitors or the inhibition of excessive activation of calpain by calpain inhibitor did not reduce OGD/reoxygenation-induced LDH release. Cotreatment with PP-1/2A and calpain inhibitors reduced OGD/reoxygenation-induced LDH release. The present study suggests that a balance in the phosphorylation and proteolytic signaling is involved in the survival of NGF-differentiated PC12 cells.     

Acute and repeated restraint stress influences cellular damage in rat hippocampal slices exposed to oxygen and glucose deprivation

Several studies have shown that high corticosteroid hormone levels increase neuronal vulnerability. Here we evaluate the consequences of in vivo acute or repeated restraint stress on cellular viability in rat hippocampal slices suffering an in vitro model of ischemia. Cellular injury was quantified by measuring lactate dehydrogenase (LDH) and neuron-specific enolase released into the medium. Acute stress did not affect cellular death when oxygen and glucose deprivation (OGD) was applied both immediately or 24h after restraint. The exposure to OGD, followed by reoxygenation, resulted in increased LDH in the medium. Repeated stress potentiated the effect of OGD both, on LDH and neuron-specific enolase released to the medium. There was no effect of repeated stress on the release of S100B, an astrocytic protein. Additionally, no effect of repeated stress was observed on glutamate uptake by the tissue. These results suggest that repeated stress increases the vulnerability of hippocampal cells to an in vitro model of ischemia, potentiating cellular damage, and that the cells damaged by the exposure to repeated stress+OGD are mostly neurons. The uptake of glutamate was not observed to participate in the mechanisms responsible for rendering the neurons more susceptible to ischemic damage after repeated stress.     

Extracellular alkalinity exacerbates injury of cultured cortical neurons.

BACKGROUND AND PURPOSE: We have previously shown that extracellular acidity protects cultured fetal murine neocortical neurons from glutamate toxicity and combined oxygen-glucose deprivation injury, an action at least in part mediated by reduction in N-methyl-D-aspartate receptor activation. We now investigate the effect of extracellular alkalinity on both glutamate neurotoxicity and injury due to combined oxygen-glucose deprivation. METHODS: The effects of extracellular alkalinity during injury induced by exposure of murine neocortical cultures to glutamate (0.5 mM for 5 minutes) or oxygen-glucose deprivation are characterized morphologically and quantitated by efflux of lactate dehydrogenase from both neurons and glia to the bathing medium. Calcium accumulation is measured with calcium-45. RESULTS: Moderate extracellular alkalinity is well tolerated by cortical cells but significantly potentiates both glutamate neuronal toxicity and oxygen-glucose deprivation neuronal injury. In contrast, glial viability in the face of combined oxygen-glucose deprivation is little affected by extracellular alkalinity. Increased accumulation of calcium-45 during oxygen-glucose deprivation in alkalotic medium and blockade of this increase by MK-801 is demonstrated. CONCLUSIONS: These observations suggest that alkaline pH can exacerbate excitotoxic neuronal injury, most likely because of increased N-methyl-D-aspartate receptor activation. Metabolic alkalosis of any etiology may sensitize neurons to ischemic injury and potentiate reperfusion injury.     

白藜芦醇预处理对神经干细胞氧糖剥夺损伤的保护作用简

目的探讨白藜芦醇预处理对神经干细胞氧糖剥夺损伤的保护作用。方法用含有白藜芦醇的细胞培养基预培养神经干细胞1h，更换低糖培养基后将细胞培养在缺氧培养盒内6h进行氧糖剥夺实验（OGD）；采用噻唑蓝（MTI＂）比色实验检测细胞活性；用LDH试剂盒测细胞培养基中乳酸脱氢酶（LDH）活性。结果白藜芦醇预处理（PRC）组细胞MTT值高于对照组（n=7，P〈0．01），PRC组细胞培养基上清中LDH低于对照组（n=5，P〈0．01）。结论白藜芦醇预处理对神经干细胞氧糖剥夺损伤有保护作用。     

MCPG在神经元氧糖剥夺模型中的抗凋亡作用

目的通过神经元氧糖剥夺模型研究Ⅰ组代谢型谷氨酸受体拮抗剂α-甲基-4-羧苯基甘氨酸（MCPG）对神经元损伤的保护作用,并初步探讨其机制。方法大鼠皮层神经元原代培养2w后,采用氧糖剥夺法建立损伤模型,通过乳酸脱氢酶（LDH）活性测定及碘化丙啶（PI）/Hoechst33342双染鉴定神经元损伤程度;加入Ⅰ组代谢型谷氨酸受体拮抗剂MCPG（1mmol/L）,通过脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TUNEL）检测神经元凋亡情况,并采用蛋白印迹法研究凋亡相关因子caspase-3、细胞外信号调节激酶1/2（ERK1/2）、磷酸化ERK1/2（p-ERK1/2）的表达变化,讨论MCPG抗凋亡作用与ERK1/2信号通路的关系。结果 MCPG能抑制ERK1/2信号通路的活化并降低凋亡相关因子caspase-3的表达,减轻氧糖剥夺造成的神经元凋亡。结论 MCPG能够通过ERK1/2信号通路减轻氧糖剥夺造成的神经元凋亡。     

Effect of varying levels of glucose on oxygen deprivation-induced delayed neuronal cell death in primary cerebrocortical cultures

Calcium accumulation and neuronal injury of rat cortical cell cultures in vitro were examined following oxygen deprivation under conditions of normal and low glucose.⁴⁵Ca²⁺ uptake and lactate dehydrogense (LDH) release, measured at 12 and 24 h after oxygen deprivation, were significantly elevated in cultures exposed to combined oxygen deprivation and low glucose (1.7 or 0.6 mM). Although those cultures deprived of oxygen combined with no glucose displayed delayed increases in⁴⁵Ca²⁺ influx, no significant elevation in LDH release at 24 h was observed.     

Glucose insufficiency alters neuronal viability and increases susceptibility to glutamate toxicity

Complete glucose deprivation has been shown to induce neuronal apoptosis, but the effect of moderate glucose deprivation under normal and pathological conditions is not fully understood. We investigated the effect of a restricted supply of glucose on neuronal vulnerability to glutamate by assaying cellular ATP levels (cellular energy production), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction (mitochondrial function), lactate dehydrogenase (LDH) release (cellular viability) and activation of caspase-3 (apoptosis) in rat hippocampal neurons cultured in media (1.7, 5 and 25 mM glucose) with or without 100 microM glutamate. Cellular ATP levels were significantly reduced in neurons cultured in 1.7 mM glucose, while addition of glutamate markedly lowered cellular ATP levels even at the normal glucose concentration. MTT reduction was also significantly inhibited by 1.7 mM glucose; however, unlike cellular ATP levels, glutamate inhibition of MTT reduction was glucose concentration dependent. The LDH assay suggested that neuronal survival declines with decreasing glucose concentration in media, and glutamate potentiates this effect. Since low glucose media caused a decrease in cellular ATP and cell viability, we investigated apoptosis-related changes in cultured neurons by examining activity of caspase-3. Low glucose media (1.7 and 5 mM glucose) increased caspase-3 activity, and glutamate potentiated this effect. Our results suggest that a low glucose supply in culture media activates an apoptosis mediator and markedly increases susceptibility to glutamate toxicity. Thus, even moderate glucose deprivation could be a serious risk factor that potentiates the pathophysiological consequences of certain neurodegenerative diseases.     

亚低温对体外缺氧缺糖损伤后神经元的保护作用及其对谷氨酸转运体、亲代谢型谷氨酸受体表达的影响

目的通过研究亚低温对体外缺氧缺糖损伤的神经细胞谷氨酸转运体(GLT-1)、亲代谢型谷氨酸受体(mGluR2/3)表达的影响,探讨亚低温的神经保护途径。方法分组培养大鼠大脑皮层细胞,亚低温组在33 C条件、常温组在37 C条件,缺氧缺糖培养2h后,复氧复糖37 C培养,经时(6h、12h、24h、3d)检测LDH释放量、GLT-1和mGluR2/3蛋白表达量。结果在复氧复糖后,两组LDH释放均呈现上升趋势,其中24h和3d亚低温组的LDH上升水平显著降低(P<0.05);两组GLT-1表达均呈现先下降后上升的趋势,其中6h和12h亚低温组的GLT-1下降水平显著降低(P<0.05);两组mGluR2/3表达均呈现上升趋势,其中12h和24h亚低温组的mGluR2/3升高水平显著增加(P<0.05)。结论亚低温能够在体外水平,通过抑制GLT-1蛋白的表达下调,促进mGluR2/3的表达上调,抑制神经元兴奋性损伤。     

Apoptotic characteristics of cell death and the neuroprotective effect of homocarnosine on pheochromocytoma PC12 cells exposed to ischemia

We recently improved an in vitro ischemic model, using PC12 neuronal cultures exposed to oxygen-glucose deprivation (OGD) for 3 hr in a special device, followed by 18 hr of reoxygenation. The cell death induced in this ischemic model was evaluated by a series of markers: lactate dehydrogenase (LDH) release, caspase-3 activation, presence of cyclin D1, cytochrome c leakage from the mitochondria, BAX cellular redistribution, cleavage of poly (ADP-ribose) polymerase (PARP) to an 85-kDa apoptotic fragment, and DNA fragmentation. The OGD insult, in the absence of reoxygenation, caused a strong activation of the mitogen-activated protein kinase (MAPK) isoforms extracellular regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and stress-activated protein kinase (SAPK), also known as p-38. The detection of apoptotic markers and activation of MAPKs during the ischemic insult strongly suggest that apoptosis plays an important role in the PC12 cell death. Homocarnosine, a neuroprotective histidine dipeptide, present in high concentrations in the brain, was found to provide neuroprotection, as expressed by a 40% reduction in LDH release and caspase-3 activity at 1 mM. Homocarnosine reduced OGD activation of ERK 1, ERK 2, JNK 1, and JNK 2 by 40%, 46%, 55%, and 30%, respectively. These results suggest that apoptosis is an important characteristic of OGD-induced neuronal death and that antioxidants, such as homocarnosine, may prevent OGD-induced neuronal death by inhibiting the apoptotic process and/or in relation to the differential attenuation of activity of MAPKs.     

Concurrent assessment of calpain and caspase-3 activation after oxygen-glucose deprivation in primary septo-hippocampal cultures.

The contributions of calpain and caspase-3 to apoptosis and necrosis after central nervous system (CNS) trauma are relatively unexplored. No study has examined concurrent activation of calpain and caspase-3 in necrotic or apoptotic cell death after any CNS insult. Experiments used a model of oxygen-glucose deprivation (OGD) in primary septo-hippocampal cultures and assessed cell viability, occurrence of apoptotic and necrotic cell death phenotypes, and protease activation. Immunoblots using an antibody detecting calpain and caspase-3 proteolysis of alpha-spectrin showed greater accumulation of calpain-mediated breakdown products (BDPs) compared with caspase-3-mediated BDPs. Administration of calpain and caspase-3 inhibitors confirmed that activation of these proteases contributed to cell death, as inferred by lactate dehydrogenase release. Oxygen-glucose deprivation resulted in expression of apoptotic and necrotic cell death phenotypes, especially in neurons. Immunocytochemical studies of calpain and caspase-3 activation in apoptotic cells indicated that these proteases are almost always concurrently activated during apoptosis. These data demonstrate that calpain and caspase-3 activation is associated with expression of apoptotic cell death phenotypes after OGD, and that calpain activation, in combination with caspase-3 activation, could contribute to the expression of apoptotic cell death by assisting in the degradation of important cellular proteins.     

Inhibition of caspase-3-like activity reduces glutamate induced cell death in adult rat retina

Retinal cell death induced by over-stimulation of glutamate receptors is related to the programmed cell death or apoptosis. However, little is known about the intracellular events that lead to this cell death process in the retina. In this study, we asked if caspase-3 family cysteine proteases regulate cell death in an explant culture of adult rat retina after exposure to excessive glutamate. Cells with DNA fragmentation were first detected in the ganglion cell layer 3 h after a brief exposure to 20 mM glutamate; whilst those in the inner nuclear layer were first observed 6 h after the glutamate lesion. Caspase-3-like activity, as indicated by immunostaining of the fractin antibody that recognizes actin fragments generated by caspase-3 family proteases, was seen 40 min after glutamate treatment. Staining was first detected in the ganglion cell layer and then in the inner nuclear layer, preceding the appearance of cells with DNA fragmentation in these layers. Colocalization study showed that all cells with DNA breaks were fractin positive, indicating that caspase-3 family activity was involved in the glutamate-induced cell death in the adult rat retina. Furthermore, DEVD-CHO, a tetrapeptide inhibitor for caspase-3 family members, reduced dramatically the fractin staining and significantly alleviated glutamate-induced cell death and DNA fragmentation in the ganglion cell layer and inner nuclear layer. Inhibitor for caspase-1-like activity, YVAD-CHO, neither reduced the fractin staining nor showed comparable neuroprotective effects to the retina. We conclude that glutamate-induced apoptotic cell death in adult rat retina is mediated by a specific activation of cysteine proteases related to the caspase-3 family, and an intervention to the caspase-3 proteases provides effective protection to retinal neurons against glutamate excitotoxicity.     

Differential effects of natural polyphenols on neuronal survival in primary cultured central neurons against glutamate- and glucose deprivation-induced neuronal death

Neuronal injury in the central nervous system following ischemic insult is believed to result from glutamate toxicity and glucose deprivation. In this study, polyphenols isolated from Scutellaria baicalensis Georgi, including baicalin, baicalein, and wogonin, were investigated for their neuroprotective effects against glutamate/NMDA (Glu/NMDA) stimulation and glucose deprivation in primary cultured rat brain neurons. Cell death was accessed by lactate dehydrogenase (LDH) release assay for necrosis, and mitochondrial activity was accessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction activity assay. It was found that both baicalin and baicalein decreased LDH release of the cultured neurons after 24 h treatment, whereas wogonin profoundly increased LDH release after 2 h treatment and resulted in neuronal death after 24 h. Glu/NMDA treatment profoundly increased LDH release and moderately decreased MTT reduction activity in an NMDA receptor-dependent manner. Both baicalin and baicalein significantly reduced Glu/NMDA-increased LDH release, in which baicalein is much more potent than baicalin. Glu/NMDA-increased intracellular calcium was also significantly attenuated by baicalin and baicalein. Baicalin and baicalein did not affect glutamate receptor binding activity, but baicalein did moderately decrease Glu/NMDA-induced nitric oxide (NO) production. In the glucose deprivation (GD) study, baicalein but not baicalin showed significant protective effects on the GD-increased LDH release, without affecting the GD-induced NO production, in cultured rat brain neurons. These results suggest that baicalein is the most effective compound among three polyphenols tested in preventing neurotoxicity induced by both glutamate and GD, whereas baicalin was only effective in preventing glutamate toxicity. Wogonin might have a neurotoxic effect on the brain.     

Induction of thioredoxin-interacting protein is mediated by oxidative stress, calcium, and glucose after brain injury in mice

Oxidative stress and glucose affect the expression of various genes that contribute to both reactive oxygen species generation and antioxidant systems. However, systemic alteration of oxidative stress-related gene expression in normal brains and in brains with a high-glucose status after ischemic-reperfusion has not been explored. Using a polymerase chain reaction array system, we demonstrate that thioredoxin-interacting protein (Txnip) is induced by both oxidative stress and glucose. We found that Txnip mRNA is induced by ischemic-reperfusion injury and that Txnip is located in the cytoplasm of neurons. Moreover, in vitro oxygen-glucose deprivation (OGD) and subsequent reoxygenation without glucose and in vivo administration of 3-nitropropionic acid also promoted an increase in Txnip in a time-dependent manner, indicating that oxidative stress without glucose can induce Txnip expression in the brain. However, calcium channel blockers inhibit induction of Txnip after OGD and reoxygenation. Using the polymerase chain reaction array with ischemic and hyperglycemic-ischemic samples, we confirmed that enhanced expression of Txnip was observed in hyperglycemic-ischemic brains after middle cerebral artery occlusion. Finally, transfection of Txnip small interfering RNA into primary neurons reduced lactate dehydrogenase release after OGD and reoxygenation. This is the first report showing that Txnip expression is induced in neurons after oxidative or glucose stress under either ischemic or hyperglycemic-ischemic conditions, and that Txnip is proapoptotic under these conditions.     

Immature rat brain slices exposed to oxygen-glucose deprivation as an in vitro model of neonatal hypoxic-ischemic encephalopathy

To analyze whether exposure to oxygen-glucose deprivation (OGD) of immature rat brain slices might reproduce the main pathophysiologic events leading to neuronal death in neonatal hypoxic-ischemic encephalopathy (NHIE), 500 microm-thick brain slices were obtained from 7-day-old Wistar rats, and incubated in oxygenated physiological solution. In OGD group, oxygen and glucose were removed from the medium for 10-30 min (n = 25); then, slices were re-incubated in normal medium. In control group the medium composition remained unchanged (CG, n = 30). Medium samples were obtained every 30 min for 3 h. To analyze neuronal damage, slices were stained with Nissl and CA1 area of hippocampus and cortex were observed under microscopy. In addition, neuronal death was quantified as LDH released to the medium determined by spectrophotometry. Additionally, medium glutamate (Glu) levels were determined by HPLC and those of TNFalpha by ELISA, whereas inducible nitric oxide synthase expression was determined by Western blot performed on slices homogenate. Optimal OGD time was established in 20 min. After OGD, a significant decrease in the number of neurones in hippocampus and cortex was observed. LDH release was maximal at 30 min, when it was five-fold greater than in CG. Furthermore, medium Glu concentrations were 200 times greater than CG levels at the end of OGD period. A linear relationship between Glu and LDH release was demonstrated. Finally, 3 h after OGD a significant induction of iNOS as well as an increase in TNFalpha release were observed. In conclusion, OGD appears as a feasible and reproducible in vitro model, leading to a neuronal damage, which is physiopathologically similar to that found in NHIE.     

曲札茋苷对二氯化钴诱导PC12细胞氧化应激的保护作用

目的 探讨曲札茋苷注射液对二氯化钴诱导PC12细胞氧化应激的保护作用及其机制.方法 取对数生长期PC12细胞随机分为5组:对照组、模型组、曲札茋苷低水平组、曲札茋苷高水平组、依达拉奉组;甲基三氯硅烷(Methyltrichlorosilane,MTS)法检测细胞活力:分光光度法检测超氧化物歧化酶(Superox-ide...     

Immediate and delayed effects of in vitro ischemia on glutamate efflux from guinea-pig cerebral cortex slices

Immediate and delayed effects of glucose deprivation, oxygen deprivation (hypoxia) and both oxygen and glucose deprivation (in vitro ischemia) on glutamate efflux from guinea pig cerebral cortex slices were studied. Immediate effects were evaluated by measuring changes of glutamate efflux during the metabolic insults. Delayed effects were evaluated by measuring the response of the tissue to a 50 mM KCl pulse applied 60 minafter the metabolic insults. Deprivation of glucose in the medium did not induce either immediate or delayed effects, while hypoxic condition produced an immediate slight stimulation of glutamate efflux without any delayed effect. Conversely, in vitro ischemia produced both immediate and delayed effects on glutamate efflux. During in vitro ischemia glutamate efflux dramatically increased in a calcium-independent and tetrodotoxin-sensitive manner; this effect was potentiated by a low sodium containing medium. The blockade of the sodium/potassium ATP_ase exchanger by ouabain caused a glutamate outflow similar to that induced by in vitro ischemia. On the whole, these data demonstrate the central role played by the sodium electrochemical gradient and by the membrane glutamate uptake system in the glutamate overflow induced by in vitro ischemia. Moreover, in slices previously exposed to both oxygen and glucose deprivation the effect of KCl on glutamate efflux was potentiated. This in vitro ischemia-induced delayed potentiation of neurotransmitter efflux, until now unreported in the literature, was found to be selectively restricted to glutamatergic structures and to be mainly due to an enhancement of the exocytotic component of glutamate release. © 1997 Elsevier Science B.V. All rights reserved.     

Hyperoxia promotes astrocyte cell death after oxygen and glucose deprivation

Astrocyte dysfunction and death accompany cerebral ischemia/reperfusion and possibly compromise neuronal survival. Animal studies indicate that neuronal death, neurologic injury, and oxidative molecular modifications are worse in animals exposed to hyperoxic compared to normoxic ventilation during reperfusion after global cerebral ischemia. It is unknown, however, whether ambient O2 affects brain cell survival using in vitro ischemia paradigms where mechanisms of injury to specific cell types can be more thoroughly investigated. This study tested the hypothesis that compared with the supraphysiological level of 20% O2 normally used in cell culture, lower, more physiological O2 levels protect astrocytes from death following oxygen and glucose deprivation. Primary rat cortical astrocytes were cultured under either 7 or 20% O2, exposed to O2, and glucose deprivation for 4 h, and then exposed to normal medium under either 7 or 20% O2. Cell death and 3-nitrotyrosine and 8-hydroxy-2-deoxyguanosine immunoreactivities were assessed at different periods of reoxygenation. Astrocytes exposed to low levels of O2 during reoxygenation undergo less death and exhibit lower levels of protein nitration and nucleic acid oxidation when compared with those under high levels of O2 during reoxygenation. These results support the hypothesis that the 20% O2 normally used in cell culture exacerbates astrocyte death and oxidative stress in an in vitro ischemia/reperfusion model compared to levels that more closely approximate those that exist in vivo.