Combined Oxygen-glucose Deprivation Research Articles

Immunosuppressants attenuate HMGB1 expression and release from primary astrocyte cultures exposed to combined oxygen-glucose deprivation

Immunosuppressants attenuate HMGB1 expression and release from primary astrocyte cultures exposed to combined oxygen-glucose deprivation

Astrocyte responses to injury: VEGF simultaneously modulates cell death and proliferation

Hypoxia is linked to changes in blood-brain barrier (BBB) permeability, and loss of BBB integrity is characteristic of many pathological brain diseases including stroke. In particular, astrocytes play a central role in brain homeostasis and BBB function. We investigated how hypoxia affects astrocyte survival and assessed whether VEGF release through hypoxia-inducible factor-1alpha (HIF-1alpha) induction plays a role in tolerance of these cells to insult. Thus primary astrocytes were subjected to normoxic (21% O(2)), hypoxic (1% O(2)), or near-anoxic (<0.1% O(2)) conditions in the presence or absence of glucose. Cell death was significantly initiated after combined oxygen glucose deprivation, and, surprisingly, astrocyte proliferation increased concomitantly. Near anoxic, but not hypoxic, conditions stabilized HIF-1alpha protein and provoked DNA binding activity, whereas oxygen and glucose deprivation accelerated HIF-1alpha accumulation. Unexpectedly, Hif-1alpha knockdown studies showed that elevated VEGF levels following increased insult was only partially due to HIF-1alpha induction, suggesting alternative mechanisms of VEGF regulation. Notably, endogenous VEGF signaling during insult was essential for cell fate since VEGF inhibition appreciably augmented cell death and reduced proliferation. These data suggest Hif-1 only partially contributes to VEGF-mediated astrocyte responses during chronic injury (as occurs in clinical hypoxic/ischemic insults) that may ultimately be responsible for disrupting BBB integrity.

Glutamate preconditioning prevents neuronal death induced by combined oxygen–glucose deprivation in cultured cortical neurons

The study of ischemic tolerance is critical in the development of strategies for the treatment of ischemic stroke. We used the oxygen and glucose deprivation (OGD) paradigm in cultured cortical neurons as an in vitro approach to elucidate the mechanism of protection conferred by glutamate preconditioning. Pretreatment of neurons with N-methyl-d-aspartate (NMDA) receptor antagonists prevented OGD-induced cell death whereas α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor and voltage-dependent Ca++ channel (VDCC) blockers were without effect. Neurons preconditioned with glutamate exhibited resistant to damage induced by OGD. The ischemic tolerance depended on the duration of preconditioning exposure and the interval between preconditioning exposure and test challenge. Protective efficacy was blocked by the NMDA or AMPA receptor antagonists but not by the VDCC blocker. Furthermore, neuroprotective effect was not seen if extracellular Ca++ was omitted or removed with EGTA. Pretreatment with staurosporin and 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN93) but not 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one (LY294002) or 1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio] butadiene (U0126) significantly reduced ischemic tolerance. Preconditioning increased phosphorylated levels of cAMP responsive element binding protein (CREB) and pretreatment with CRE-decoy oligonucleotide completely blocked preconditioning-induced increase in cell viability. Importantly, glutamate preconditioning increased Bcl-2 expression that was blocked by KN93, staurosporin and CRE-decoy oligonucleotide. These results suggest that preconditioning with glutamate conferred neuroprotection against subsequent OGD by inducing p-CREB-mediated Bcl-2 expression.

The individual and combined neuroprotective effects of propofol and ketamine on rat mixed cortical cultures exposed to oxygen-glucose deprivation-reperfusion injury

Propofol and ketamine are have been known to have neuroprotective effects. However, the effect of combined therapy with these 2 drugs is not well known with in vitro model. This study was conducted to determine whether combined administration of propofol and ketamine could have additive effects in protecting cortical neurons from the oxygen-glucose deprivation (ischemia) - reoxygenation (reperfusion) injury. Methods: Thirteen-day-old primary mixed cortical cultures were exposed to a 5-min combined oxygen-glucose deprivation (OGD, in vitro ischemia model), followed by 2 hr of reperfusion. Propofol (1, 10, 25, 50, 100μM) and ketamine (1, 2.5, 5, 10, 50μM) were added as alone or combination from the initiation of the OGD injury to the end of the reperfusion periods. The survived cells were counted using trypan-blue staining. The data were converted to the cell death rate. Statistical analysis was done by oneway-ANOVA tests and Bonferroni's test. P ＜ 0.05 was considered as statistically significant. Results: OGD-reperfusion demonstrated about a 70% cell death rate. 5−50μM of ketamine decreased the cell death rate compared with the no drug treated group (P ＜ 0.05). 10−100μM of propofol decreased the cell death rate compared with the no drug treated group (P ＜ 0.05). Combined administration of ketamine 2.5μM + propofol 50, 100μM, ketamine 10μM + propofol 100μM and propofol 1, 10μM + ketamine 5, 10μM decreased cell death rate compared with the same dosage of propofol or ketamine alone treated group (P ＜ 0.05). Conclusions: Propofol or ketamine demonstrated neuroprotective effects. And, combined administration ofpropofol and ketamine demonstrated additive neuroprotective effects against OGD-reperfusion injury.

Activation of cPLA2 and sPLA2 in astrocytes exposed to simulated ischemia in vitro

Both cytosolic PLA(2) (cPLA(2)) and secretory PLA(2) (sPLA(2)) have been implicated in pathology of cerebral ischemia. However, which of PLA(2) isoforms in astrocytes is responsible for arachidonic acid (AA) release contributing to their ischemic injury remains to be determined. The aim of the present study was to investigate the time-dependent activation of cPLA(2) and sPLA(2) in astrocytes exposed to combined oxygen glucose deprivation (OGD) as well as to evaluate the effectiveness of their pharmacological blockage as a method of preventing ischemic damage of the glial cells. It was shown that exposure of cultured astrocytes to OGD (0.5-24h) causes an increase in cPLA(2) and sPLA(2) expression and activity. The role of AA liberated mainly by cPLA(2) in the process of apoptosis was also demonstrated. To confirm the specific role of cPLA(2) and sPLA(2) in the mechanism of cells injury by OGD exposure, the effect of AACOCF(3) as cPLA(2) inhibitor and 12-epi-scalaradial as sPLA(2) inhibitor on AA release was examined. It was proved that simultaneous pharmacological blockade of enzymatic activity of cPLA(2) and sPLA(2) during OGD by AACOCF(3) and 12-epi-scalaradial substantially improves survival of ischemic injured glial cells.

Purinergic modulation of glutamate release under ischemic-like conditions in the hippocampus

The aim of the present study was to explore whether endogenous activation of different purine receptors by ATP and adenosine contributes to or inhibits excess glutamate release evoked by ischemic-like conditions in rat hippocampal slices. Combined oxygen–glucose deprivation (OGD) elicited a substantial, [Ca 2+] o-independent release of [ 3H]glutamate, which was tetrodotoxin (1 μM)-sensitive and temperature-dependent. The P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS, 0.1–10 μM), and the selective P2X 7 receptor antagonist Brilliant Blue G (1–100 nM), decreased OGD-evoked [ 3H]glutamate efflux indicating that endogenous ATP facilitates ischemia-evoked glutamate release. The selective A 1-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1–250 nM) and the selective A 2A receptor antagonists 4-(2-[7-amino-2-)2-furyl(triazolo-[1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385, 0.1–20 nM) and 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261, 2–100 nM) decreased OGD-evoked [ 3H]glutamate efflux, indicating that endogenous adenosine also facilitates glutamate release under these conditions. The effect of DPCPX and ZM241385 was reversed, whereas the action of P2 receptor antagonists was potentiated by the selective ecto-ATPase inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL67156, 50 μM). The binding characteristic of the A 2A ligand [ 3H]CGS21680 to hippocampal membranes did not change significantly in response to OGD. Taken together these data suggest that while A 1 receptors might became desensitized, A 2A and P2X receptor-mediated facilitation of glutamate release by endogenous ATP and its breakdown product adenosine remains operational under long-term OGD. Therefore the inhibition of P2X/A 2A receptors rather than the stimulation of A 1 adenosine receptors could be an effective approach to attenuate glutamatergic excitotoxicity and thereby counteract ischemia-induced neurodegeneration.

Neuroprotective effects of the β-carboline abecarnil studied in cultured cortical neurons and organotypic retinal cultures

Presently there is no neuroprotective pharmacological treatment of proven clinical safety and efficacy available. The purpose of this study was to investigate whether the β-carboline, abecarnil (Abe), which has already passed clinical phase III trials in patients with anxiety disorders, is neuroprotective in in vitro models of cerebral ischemia or excitotoxicity. Abe (100 nM) protected cultured cortical neurons when applied 20 min before or 20 min after combined oxygen glucose deprivation (OGD). Furthermore, cultured cortical neurons were protected from NMDA excitotoxicity when Abe (100 nM) was administered 20 min before or concurrent with 100 μM NMDA. In contrast, in adult rat organotypic retinal cultures, Abe failed to protect retinal ganglion cells (RGCs) against glutamate (Glu) excitotoxicity. Thus, although our data demonstrate that Abe is a potential neuroprotectant in cultured neurons, the lack of effect in an organotypical model of Glu toxicity indicates that further study is required before Abe might be considered for human neuroprotection trials.

Influence of hypoxia and ischemia preconditioning on bilirubin damage to astrocytes

Hypoxia–ischemia in the perinatal period is a common cause of neurologic disability in children and is often associated with neonatal morbidity and mortality. Another frequent condition of the newborn is hyperbilirubinemia and it is well known that deposition of unconjugated bilirubin (UCB) in the central nervous system can damage nerve cells and cause encephalopathy. Interestingly, some studies report the onset of cerebral hypoxia–ischemia as a risk factor for UCB encephalopathy, since that condition often precedes neonatal hyperbilirubinemia. However, the cellular mechanisms triggered by hypoxia–ischemia that may enforce UCB deleterious effects are not well elucidated. Therefore, we designed this study to investigate whether hypoxia (HP) or combined oxygen–glucose deprivation (OGD) followed by reoxygenation, modifies glial cell susceptibility to UCB injury. Thus, cultured astrocytes were exposed to HP or OGD for 4 h and returned to normoxic conditions for another 12 h prior to incubation with UCB for 4 h. HP and OGD effects in UCB toxicity were compared to normoxic conditions. Our results demonstrate that HP and OGD preconditioning increase the vulnerability of glial cells to UCB damage by enhancing some of the deleterious effects of UCB, namely cell death by both apoptosis and necrosis. This preconditioning also augments the UCB-induced stimulation of an inflammatory response by an effect that involves the activation of the nuclear factor κB activation. These findings provide a novel basis for the increased risk of brain damage in jaundiced newborns that were previously exposed to hypoxia or ischemia during the perinatal period, namely during delivery.

Glutamate-Independent Calcium Toxicity

It is widely accepted that a critical factor in determining neuronal death during cerebral ischemia is the progressive accumulation of intracellular Na+ ([Na+]i) and Ca2+ ([Ca2+]i) ions, which can precipitate necrosis and apoptosis of vulnerable neurons. Whereas the detrimental action of [Na+]i increase is attributable to both cell swelling and microtubular disorganization—2 phenomena that lead to cell necrosis1—a change in [Ca2+]i has been shown to be a key factor in ischemic brain damage, for it modulates several death pathways, including oxidative and nitrosative stress, mitochondrial dysfunction, and protease activation. Since Olney’s seminal work firstly suggested that excitatory aminoacids could elicit neurotoxicity,2 a large amount of work has been accumulated showing that glutamate extracellular concentrations briskly rise during acute brain injury, thus triggering an influx of Ca2+ and Na+ ions into neurons through ionotropic glutamate receptor subtypes. This evidence has led to the elaboration of the paradigm of glutamate excitotoxicity that explained ischemic neuronal cell death as a mere consequence of Na+ and Ca2+ influx through glutamate receptors.3 Although this theory has been guiding basic research in the field of neurodegeneration for almost 3 decades, more recently it has become the object of serious criticism and reassessment. What has aroused such skepticism among researchers has been the fact that although first, second, and third generation glutamate receptor antagonists have long yielded promising results in animal models of brain ischemia, they have failed to elicit a neuroprotective action in stroke and traumatic brain injury in humans.4 Therefore, the theory of excitotoxicity, though a fascinating paradigm, can only explain some of the events occurring in the acute phase of anoxic insult but cannot be seen as a major target for …

Sevoflurane Protects Rat Mixed Cerebrocortical Neuronal–Glial Cell Cultures against Transient Oxygen–Glucose Deprivation

The purpose of this study was to clarify the role of glutamate and reactive oxygen species in sevoflurane-mediated neuroprotection on an in vitro model of ischemia-reoxygenation. Mature mixed cerebrocortical neuronal-glial cell cultures, treated or not with increasing concentrations of sevoflurane, were exposed to 90 min combined oxygen-glucose deprivation (OGD) in an anaerobic chamber followed by reoxygenation. Cell death was quantified by lactate dehydrogenase release into the media and cell viability by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium by mitochondrial succinate dehydrogenase. Extracellular concentrations of glutamate and glutamate uptake were assessed at the end of the ischemic injury by high-performance liquid chromatography and incorporation of L-[H]glutamate into cells, respectively. Free radical generation in cells was assessed 6 h after OGD during the reoxygenation period using 2',7'-dichlorofluorescin diacetate, which reacts with intracellular radicals to be converted to its fluorescent product, 2',7'-dichlorofluorescin, in cell cytosol. Twenty-four hours after OGD, sevoflurane, in a concentration-dependent manner, significantly reduced lactate dehydrogenase release and increased cell viability. At the end of OGD, sevoflurane was able to reduce the OGD-induced decrease in glutamate uptake. This effect was impaired in the presence of threo-3-methyl glutamate, a specific inhibitor of the glial transporter GLT1. Sevoflurane counteracted the increase in extracellular level of glutamate during OGD and the generation of reactive oxygen species during reoxygenation. Sevoflurane had a neuroprotective effect in this in vitro model of ischemia-reoxygenation. This beneficial effect may be explained, at least in part, by sevoflurane-induced antiexcitotoxic properties during OGD, probably depending on GLT1, and by sevoflurane-induced decrease of reactive oxygen species generation during reoxygenation.

Calcineurin and Erk1/2-signaling pathways are involved in the antiapoptotic effect of cyclosporin A on astrocytes exposed to simulated ischemia in vitro

The present study focused on mechanisms involved in the anti-apoptotic effect of cyclosporin A (CsA) towards ischemic injured astrocytes in vitro [under combined oxygen glucose deprivation (OGD)]. We investigated whether this action might be mediated through activation of extracellular signal regulated kinases 1 and 2 (Erk1/2) or attenuation of calcineurin (CaN) by immunosuppressant in ischemic astrocytes. Additionally, the influence of CsA on phosphorylation of Akt kinase was determined. After 21 days of in vitro culture, astrocytes were subjected to OGD (for 8 h) and CsA (0.25-10 microM); 0.25 microM CsA distinctly stimulated the Erk1/2 pathway in astrocytes exposed to OGD. This protective effect of CsA was strongly associated with CaN inhibition, increased expression of anti-apoptotic factors such as Bcl-X(L) and NF-kappaB, as well as suppression of caspase-3 activity. Maximum p-Akt kinase expression was observed following treatment with 1 microM CsA. Finally, we also demonstrated that the beneficial effect of CsA at a concentration of 10 microM is related mainly to strong CaN inhibition. The results obtained suggest that, depending on the concentration used, CsA might act as a protective agent towards ischemia-injured astroglial cells through alternative intracellular pathways associated with increased p-Erk1/2 and p-Akt expression or CaN inactivation.

Inhibition of Arachidonic Acid Release by Cytosolic Phospholipase A2 Is Involved in the Antiapoptotic Effect of FK506 and Cyclosporin A on Astrocytes Exposed to Simulated Ischemia In Vitro

In the present study, we investigated whether the protective effect of FK506 and cyclosporin A (CsA) against in vitro ischemic injury of astrocytes might be mediated through attenuation of cytosolic isoform of phospholipase A2 (cPLA2) expression and activity as well as inhibition of arachidonic acid (AA) release. On the 21st day in vitro, cultures of rat astrocytes were subjected to ischemia-simulating conditions (combined oxygen glucose deprivation) for 8 h and exposed to FK506 (10 – 1000 nM) and CsA (0.25 – 10 µM). Obtained data suggest the crosstalk between the action of 0.25 – 10 µM CsA as well as 1 µM FK506 on calcineurin (CaN) and cPLA2 in anti-apoptotic signal transduction pathways. Moreover, we have shown that immunosuppressants at these concentrations protected glial cells against ischemia-induced apoptosis through the increase of cell viability, mitochondrial function restoration, and attenuation of oxidative stress. Finally, in our study, low concentrations of FK506 (10 and 100 nM) exerted limited effects on the assessed parameters. Our findings document a key role either for CaN or cPLA2 expression attenuation and AA release inhibition in the antiapoptotic effect of FK506 and CsA in ischemic astrocytes.

The Carboxyl-Terminal Domain of Inducible Hsp70 Protects from Ischemic Injury in vivo and in vitro

Heat shock protein (Hsp)70 can suppress both necrosis and apoptosis induced by various injuries in vivo and in vitro. However, the relative importance of different functions and binding partners of Hsp70 in ischemic protection is unknown. To explore this question, we tested the ability of Hsp70-K71E, an adenosine triphosphate (ATP)ase-deficient point mutant, and Hsp70-381-640, a deletion mutant lacking the ATPase domain and encoding the carboxyl-terminal portion, to protect against ischemia-like injury in vivo and in vitro. Heat shock protein 70-wild type (-WT), -K71E, -381-640, and control vector plasmid LXSN were expressed in primary murine astrocyte cultures. Astrocytes overexpressing Hsp70-WT, -K71E, or -381-640 were all significantly protected from 4 h combined oxygen-glucose deprivation and 24 h reperfusion when assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay or propidium iodide staining and cell counting (P < 0.05). Brains of rats were transfected with plasmids encoding Hsp70-WT, -K71E, -381-640, or LXSN 24 h before 2 h middle cerebral artery occlusion followed by 24 h reperfusion. Animals that overexpressed either of the mutant proteins or Hsp70-WT had significantly better neurological scores and smaller infarcts than control animals. Protection by both mutants was associated with reduced protein aggregation, as assessed by ubiquitin immunohistochemistry and reduced nuclear translocation of apoptosis-inducing factor. The results show that the carboxyl-terminal portion of Hsp70 is sufficient for neuroprotection. This indicates that neither the ability to fold denatured proteins nor interactions with cochaperones or other proteins that bind the amino-terminal half of Hsp70 are essential to ischemic protection.

Roles of the C-terminal peptide binding domain of human inducible HSP70 in focal ischemic brain injury

The inducible heat shock protein 70 (Hsp70) is a molecular chaperone that is expressed primarily in response to stress, including heat shock, ischemia and glucose deprivation. Hsp70 consists of a 44 kDa amino terminal ATPase domain, an 18 kDa peptide or substrate binding domain, and a 10 kDa carboxy terminal domain. While a large number of proteins interact with Hsp70, only in some cases has the domain with which they interact been identified. Although it is accepted that Hsp70 can protect cells from ischemic injury, the mechanism is not known. Possible protective effects include prevention of protein aggregation, refolding denatured proteins and reduction of apoptosis. The relative importance of the ATP-binding domain compared to the peptide binding domain of HSP70 in ischemic protection is unknown. To explore this question we tested whether two Hsp70 mutant proteins could protect against ischemia-like injury of primary cultured murine astrocytes and reduce focal ischemic injury induced by transient (2 h) middle cerebral artery occlusion (MCAO). The two mutants studied were 1) Hsp70 K71E, a point mutation that abrogates ATP binding and renders the protein deficient in folding ability and 2) Hsp70 381-640 a deletion mutant lacking the ATP binding domain. Hsp70 wild type (WT), -K71E, -381-640 and vector plasmid LXSN were expressed in primary murine astrocyte cultures using retroviral mediated transfection. After G418 selection, more than 95% of cells expressed these proteins by immunostaining. Cultures were then subjected to combined oxygen-glucose deprivation (OGD) and cell survival was assessed by MTT assay or PI staining and cell counting. Astrocytes overexpressing Hsp70 -WT, -K71E or -381-640 were all significantly protected from 4 h OGD (cell viability was increased about 50%, p < 0.05). We then tested the ability of these mutant proteins to protect rats from transient focal ischemic injury. Plasmids encoding Hsp70 -K71E, -381-640, or the control backbone plasmid LXSN were stereotactically injected into the left lateral ventricle 24 h prior to MCAO. These mutant proteins were expressed in both astrocytes and neurons as determined by double immunostaining. Animals that overexpressed either of these two mutant proteins had significantly better neurological scores and smaller infarct volumes (30-35%) assessed at 24 hr (infarction volume (mm3): 233.538.6 in control group; 152.2543.95 in Hsp70 K71E group; and 162.7132.5 in Hsp70 381-640 group, P<0.05 compared to control, n=8 in each group). Protection by both mutants was associated with reduced protein aggregation and apoptosis, as assessed by ubiquitin immunohistochemistry and Klenow staining. Both the point mutant and the deletion mutant were found to reduce ischemic injury. Since the carboxy terminal half of the protein is sufficient for protection, interaction with protein partners that bind the aminoterminal half of the protein are not essential to ischemic protection. Also, since neither mutant can facilitate folding, their efficacy against ischemia suggests that inhibiting protein aggregation is sufficient to reduce ischemic injury and apoptotic cell death.

Development of Neonatal Murine Microglia In Vitro: Changes in Response to Lipopolysaccharide and Ischemia-Like Injury

Hypoxic/ischemic brain injury in the neonate can activate an inflammatory cascade, which potentiates cellular injury. The role of microglia in this inflammatory response has not been studied extensively. We used an in vitro model of murine microglia to investigate changes in microglial cytokine release and injury during early development. Isolated microglia were subjected to lipopolysaccharide (LPS) activation or injury by glucose deprivation (GD), serum deprivation (SD), or combined oxygen-glucose deprivation (OGD) for varying durations. The extent and the type of cell death were determined by trypan blue, terminal deoxynucleotidyl end-nick labeling, and annexin staining. Early-culture microglia (2-3 d in purified culture) showed significantly more apoptotic cell death after SD, GD, and OGD compared with microglia maintained in culture for 14-17 d. Measurements of tumor necrosis factor-alpha (TNF-alpha) and IL-1beta in culture media demonstrated that OGD induced greater release of both TNF-alpha and IL-1beta than LPS activation, with early-culture microglia producing more TNF-alpha compared with late-culture microglia. Microglia that are cultured for a short time are more sensitive to ischemia-like injury in vitro than those that are cultured for longer durations and may contribute to worsening brain injury by increased release of inflammatory cytokines. Inhibition of microglial activation and decreasing proinflammatory cytokine release may be targets for reduction of neonatal hypoxic/ischemic brain injury.

Overexpression of Bcl-xl protects septal neurons from prolonged hypoglycemia and from acute ischemia-like stress

Overexpression of Bcl-xl, a member of the Bcl-2 protein family, is reported to protect from a variety of stresses involving delayed cell death. We tested the ability of Bcl-xl overexpression to protect primary cultures of embryonic rat septal neurons subjected to one of four different stresses: 6 h of combined oxygen–glucose deprivation, which produces rapid cell death, or a 24 h exposure to hypoglycemia, hyperglycemia, or 1mM 3-nitropropionic acid (an inhibitor of mitochondrial respiration), which results in a more slowly-developing death. Prior to the stress neurons were transiently transfected to overexpress either green fluorescent protein only or green fluorescent protein along with wild-type Bcl-xl. Immediately after oxygen–glucose deprivation, many neurons expressing green fluorescent protein only showed process blebbing and disintegration, with only 49% of the initial cells remaining intact with processes. Neurons expressing both green fluorescent protein and Bcl-xl showed less damage (68% intact post-stress, P<0.05). This result indicates that Bcl-xl’s saving effects are not due solely to blocking delayed (apoptotic) death, because death following oxygen–glucose deprivation was rapid and was not accompanied by increased activation of caspase-3. Bcl-xl expression also significantly protected against the hypoglycemic stress (23% intact 24 h post-stress with green fluorescent protein only, compared with 70% with Bcl-xl and green fluorescent protein), but did not protect from hyperglycemia or 3-nitropropionic acid. Thus Bcl-xl does not protect against all forms of delayed death. Bcl-xl’s protective effects may include blocking early damaging events, perhaps by increasing mitochondrial function in the face of low levels of energy substrates. Bcl-xl’s protective effects may require an intact electron transport chain.

Enhanced release of synaptic glutamate underlies the potentiation of oxygen–glucose deprivation-induced neuronal injury after induction of NOS-2

Reactive nitrogen oxide species (RNOS) may contribute to the progression/enhancement of ischemic injury by augmentation of glutamate release, reduction of glutamate uptake, or a combination of both. Consistent with this, induction of nitric oxide synthase (NOS-2) in murine neocortical cell cultures potentiated neuronal cell death caused by combined oxygen–glucose deprivation in association with a net increase in extracellular glutamate accumulation. However, uptake of glutamate via high affinity, sodium-dependent glutamate transporters was unimpaired by induction of NOS-2 under either aerobic or anaerobic conditions. Further, blocking possible routes of extra-synaptic glutamate release with NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid], a volume-sensitive organic anion channel blocker, or TBOA ( d, l-threo-β-benzyloxyaspartate), an inhibitor of glutamate transport, exacerbated rather than ameliorated injury. Finally, treatment with riluzole or tetanus toxin attenuated the enhancement in both glutamate accumulation and oxygen–glucose deprivation-induced neuronal injury supporting the idea that increased synaptic release of glutamate underlies, at least in part, the potentiation of neuronal injury by RNOS after NOS-2 induction.

Increased postischemic brain injury in mice deficient in uracil-DNA glycosylase.

Uracil-DNA glycosylase (UNG) is involved in base excision repair of aberrant uracil residues in nuclear and mitochondrial DNA. Ung knockout mice generated by gene targeting are viable, fertile, and phenotypically normal and have regular mutation rates. However, when exposed to a nitric oxide donor, Ung(-/-) fibroblasts show an increase in the uracil/cytosine ratio in the genome and augmented cell death. After combined oxygen-glucose deprivation, Ung(-/-) primary cortical neurons have increased vulnerability to cell death, which is associated with early mitochondrial dysfunction. In vivo, UNG expression and activity are low in brains of naive WT mice but increase significantly after reversible middle cerebral artery occlusion and reperfusion. Moreover, major increases in infarct size are observed in Ung(-/-) mice compared with littermate control mice. In conclusion, our results provide compelling evidence that UNG is of major importance for tissue repair after brain ischemia.

Increased postischemic brain injury in mice deficient in uracil-DNA glycosylase

Uracil-DNA glycosylase (UNG) is involved in base excision repair of aberrant uracil residues in nuclear and mitochondrial DNA. Ung knockout mice generated by gene targeting are viable, fertile, and phenotypically normal and have regular mutation rates. However, when exposed to a nitric oxide donor, Ung–/– fibroblasts show an increase in the uracil/cytosine ratio in the genome and augmented cell death. After combined oxygen-glucose deprivation, Ung–/– primary cortical neurons have increased vulnerability to cell death, which is associated with early mitochondrial dysfunction. In vivo, UNG expression and activity are low in brains of naive WT mice but increase significantly after reversible middle cerebral artery occlusion and reperfusion. Moreover, major increases in infarct size are observed in Ung–/– mice compared with littermate control mice. In conclusion, our results provide compelling evidence that UNG is of major importance for tissue repair after brain ischemia.

Geldanamycin reduces necrotic and apoptotic injury due to oxygen-glucose deprivation in astrocytes

Recent data show that geldanamycin can protect the brain against stroke in vivo, and this may be due to induction of heat shock proteins. Our previous results show that heat shock protein 70 expression by retroviral transfection protects astrocytes from necrotic injury by combined oxygen-glucose deprivation, an in vitro model of ischemia. This study tested the ability of geldanamycin to protect astrocytes from either necrotic or apoptotic injury induced by oxygen-glucose deprivation. Astrocytes were pre-treated with 0.1 µg ml-1 geldanamycin in the medium 4, 8, or 16 h before as well as during oxygen-glucose deprivation. Increased protein levels of heat shock protein 70 were observed after 8 h pre-treatment with geldanamycin and increased further at 16 h pre-treatment, as detected by immunoblotting. Geldanamycin pre-treatment protected mature astrocytes from necrotic cell death and young astrocytes from apoptotic death. Geldanamycin protection of astrocytes against in vitro ischemia is likely due to upregulation of heat shock protein 70.