Min Of Oxygen-glucose Deprivation Research Articles

Oxygen/Glucose Deprivation Induces a Reduction in Synaptic AMPA Receptors on Hippocampal CA3 Neurons Mediated by mGluR1 and Adenosine A3Receptors

Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighboring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca(2+), resulting in delayed cell death. However, it is unclear whether the same mechanisms exist in CA3 neurons and whether this underlies the differential sensitivity to ischemia. Here, we investigated the consequences of OGD for AMPAR function in CA3 neurons using electrophysiological recordings in rat hippocampal slices. Following a 15 min OGD protocol, a substantial depression of AMPAR-mediated synaptic transmission was observed at CA3 associational/commissural and mossy fiber synapses but not CA1 Schaffer collateral synapses. The depression of synaptic transmission following OGD was prevented by metabotropic glutamate receptor 1 (mGluR1) or A(3) receptor antagonists, indicating a role for both glutamate and adenosine release. Inhibition of PLC, PKC, or chelation of intracellular Ca(2+) also prevented the depression of synaptic transmission. Inclusion of peptides to interrupt the interaction between GluA2 and PICK1 or dynamin and amphiphysin prevented the depression of transmission, suggesting a dynamin and PICK1-dependent internalization of AMPARs after OGD. We also show that a reduction in surface and total AMPAR protein levels after OGD was prevented by mGluR1 or A(3) receptor antagonists, indicating that AMPARs are degraded following internalization. Thus, we describe a novel mechanism for the removal of AMPARs in CA3 pyramidal neurons following OGD that has the potential to reduce excitotoxicity and promote neuroprotection.

Transient oxygen–glucose deprivation causes immediate changes in redox activity in mouse brain tissue

Redox activity is an important property of living cells, and decreases in redox activity are likely to be an upstream event in ischemic brain injuries. In this study, immediate changes in redox activity caused by ischemic injury were investigated in oxygen–glucose deprivation (OGD) treated mouse brain tissue. Adult mouse brain slices were subjected to 10 min or 15 min OGD treatments and were immediately stained with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) staining procedure. After 10 min OGD, the redox activity decreased in the lateral globus pallidus (LGP), medial globus pallidus (MGP), pyramidal cell layer of hippocampus CA1 (CA1 PL) and the granular layer of the cerebellum (cereb GL). After 15 min OGD, decreases also occurred in the substantia nigra (SN) and several other areas of the brain stem. Hoechst 33342 was used to confirm that changes in redox activity occurred before morphological alterations in the cellular nuclei — morphological changes were not observed even after a 60 min OGD. The results presented here indicate that functional ischemic vulnerability exists in several brain regions, and will be helpful for systematic research on mammalian brain injury caused by transient metabolic stress.

CB1 receptors and post-ischemic brain damage: Studies on the toxic and neuroprotective effects of cannabinoids in rat organotypic hippocampal slices

Cannabinoids (CBs) are implicated in a number of physiological and pathological mechanisms in the central nervous system, but their exact role in post-ischemic brain injury is unclear. The toxic and neuroprotective effects of synthetic and endogenous CBs were evaluated in rat organotypic hippocampal slices exposed to 20 min oxygen–glucose deprivation (OGD) and in gerbils subjected to bilateral carotid occlusion for 5 min. When present in the incubation medium, the synthetic CB agonists WIN 55212-2 and CP 55940 (1–30 μM) and the CB1 agonist ACEA exacerbated CA1 injury induced by OGD, whereas the CB1 receptor antagonists AM 251 and LY 320135 were neuroprotective with maximal activity at 1 μM. AM 251 (at 3 mg/kg, i.p.) also attenuated CA1 pyramidal cell death in gerbils in vivo. The endocannabinoid 2-arachidonoylglycerol (2-AG) reduced OGD injury in hippocampal slices at 0.1–1 μM, whereas anandamide (AEA) was neurotoxic at the same concentrations. The effects of WIN 55212-2, AEA and 2-AG in slices were all dependent on the activation of CB1 but not CB2 receptors, except for the toxic effects of AEA that were also dependent on vanilloid TRPV1 receptors. Our results suggest that exogenous administration of CB1 agonists and the production of endocannabinoids “on demand” may produce different, if not opposite, effects on the fate of neurons following cerebral ischemia.

Role of P2X7 receptors in release of glutamate and γ-aminobutyric acid during oxygen-glucose deprivation in rat hippocampus and neuronal synaptosome

Objective To evaluate the role of P2X7 receptors in release of glutamate （Glu） and γ-aminobutyric acid （GABA） during oxygen-glucose deprivation （OGD） in rat hippocampus and neuronal synaptosome.Methods Healthy male SD rats weighing 150-200 g were decapitated. Their hippocampi were isolated and cut into slices 400 μm thick or made into neuronal synaptosomes. The hippocampal slices and neuronal synaptosomes were incubated in artificial cerebro-spinal fluid （aCSF） at 35℃ for 30 min and divided into 3 groups （ n = 32 or 24 each）： control group （group C）; group OGD and group OGD ＋ BBG （brilliant blue G, a specific P2X7 receptor antagonist）. OGD was induced by incubating the slices and synaptosomes in glucose-free aCSF aerated with 95% N2-5% CO2. In group OGD ＋ BBG the slices and synaptosomes were incubated in O2-glucose deprived aCSF containing BBG 1 μmol/L 2 ml. Release of Glu and GABA from hippocampal slices and synaptosomes was determined by HPLC at 0, 20, 40, 60 min of OGD （T1-4）. Hippocampal slices were examined with microscope.Results （ 1 ） The release of Glu and GABA from hippocampal slices and synaptosomes were significantly increased after OGD （ P 〈 0.05）. （2） Glu released from hippocampal slices was significantly decreased at T3-4 and Glu released from synaptosomes increased at T2-4 in group OGD ＋ BBG as compared with group OGD （ P 〈 0.05）. （3）GABA released from hippocampal slices was significantly decreased at T4 in group OGD ＋ BBG as compared with group OGD （ P 〈 0.05）. There was no significant difference in GABA released from synaptosomes between group OGD and OGD ＋ BBG （P 〉 0.05）. （4） Microscopic examination showed that OGD induced significant histopathological damage to hippocampal slices which was attenuated by BBG treatment. Conclusion P2X7 receptors mediates the release of Glu and GABA during OGD in rat hippocampus and the P2X7 receptors in glial cells plays a leading role. Key words: Receptors, purinergic P2; Glutamic acid; gamma-Aminobutyric acid; Brain ischemia; Hippocamous; Neurons; Synaptosomes

Role of P2X receptors in synthesis and release of IL-1β during oxygen-glucose deprivation in rat hippocampus

Objective To evaluate the role of P2X receptors in the synthesis and release of IL-1β during oxygen-glucose deprivation (OGD) in rat hippocampus. Methods Male SD rats weighing 150-200 g were anesthetized with ether and decapitated. The hippocampi were removed and sagittally sliced (400 μm thick) and placed in artificial cerebrospinal fluid (aCSF) aerated with 95% O2-5% CO2. One hundred and sixty hippocampal slices were randomly divided into 4 groups ( n = 40 each): Ⅰ control group (group C); Ⅱ OGD group; Ⅲ OGD +BBG group; Ⅳ OGD + anti-P2X4 group (group OP). In group C, the hippocampal slices were continously incubated with aCSF aerated with 95% O2-5% CO2 . In group OGD, the hippocampal slices were incubated with glucose-free aCSF and aerated with 95% N2-5%CO2 . In group OGD + BBG, the hippocampal slices were incubated with aCSF containning P2X7 receptor-specific antagonist G (BBG, final concentration 1 μmol/L) and aerated with 95% O2-5% CO2 for 20 min, then exposed to OGD, and BBG (final concentration 1 μ mol/L) was added in glucose-free aCSF. In group OP, the hippocampal slices were incubated with aCSF containning P2X4 receptor antibody (final concentration 1.5 μg/ml) and aerated with 95% O2-5% CO2 for 60 min, then exposed to OGD, and P2X4 receptor antibody (final concentration 1.5 μg/ml) was added in glucose-free aCSF. LDH and IL-1β release was detected before OGD and at 20, 40 and 60 min of OGD. Histological changes were observed using HE staining.Intracellular pro-IL-1β precursor protein expression was detected by Western blot at 60 min of OGD. Results LDH and IL-1β release and expression of intracellular pro-IL-1β precursor protein were signifcantly higher in the other groups than in group C ( P ＜ 0.05 ). Compared with group OGD, LDH and IL- 1 β release was signifcantly decreased, while the expression of intracellular pro-IL-1β precursor protein un-regulated in group OGD + BBG ( P ＜0.05), but no signifcant difference was found in the prarameters mentioned above in group OP ( P ＞ 0.05). Conclusion P2X7 receptor mediates the synthesis and release of IL-1β during OGD in rat hippocampus, but P2X4 receptor does not. Key words: Receptors, purinergic P2; Interleukin-1 beta; Hypoxia-ischemia,brain; Hippocampus

Oxygen and Glucose Deprivation in an Organotypic Hippocampal Slice Model of the Developing Rat Brain: The Effects on N-Methyl-d-Aspartate Subunit Composition

Organotypic hippocampal slices (OHS) are commonly used to screen for neuroprotective effects of pharmacological agents relevant to pediatric brain injury. The importance of donor rat pup age and N-methyl-D-aspartate (NMDA) receptor subunit composition have not been addressed. In this study, we evaluated the age-dependent effect of oxygen-glucose deprivation (OGD) in the developing rat brain and determined whether OGD modulates the NMDA receptor subunit composition. OHS were prepared from rat pups on postnatal days (PND) 4, 7, 14, and 21 and cultured 7 days in vitro. The slices were exposed to OGD for durations of 5-60 min. After 24 and 72 h, OHS survival and NMDA subunit composition were assessed. Cell death was evident in OHS prepared from PND 14 and 21 rat pups (P < 0.001) with OGD durations of 5 and 10 min, respectively. In OHS prepared from PND7 rat pups, neurodegeneration was not evident until 20 min OGD (P < 0.001). Exposure to OGD in OHS prepared from PND4 and PND7 rat pups was associated with a transition in the NMDA receptor subunit composition from NR2B predominant to NR2A predominant subunit composition. This in vitro neonatal rat pup investigation using OHS supports both an age and an NMDA receptor subunit composition-dependent relationship between OGD and neuronal cell death.

The adenosine A2A receptor antagonist ZM241385 enhances neuronal survival after oxygen-glucose deprivation in rat CA1 hippocampal slices.

Activation of adenosine A(2A) receptors in the CA1 region of rat hippocampal slices during oxygen-glucose deprivation (OGD), a model of cerebral ischaemia, was investigated. We made extracellular recordings of CA1 field excitatory postsynaptic potentials (fepsps) followed by histochemical and immunohistochemical techniques coupled to Western blots. OGD (7 or 30 min duration) elicited an irreversible loss of fepsps invariably followed by the appearance of anoxic depolarization (AD), an unambiguous sign of neuronal damage. The application of the selective adenosine A(2A) receptor antagonist, ZM241385 (4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-ylamino]ethyl)phenol; 100-500 nmolxL(-1)) prevented or delayed AD appearance induced by 7 or 30 min OGD and protected from the irreversible fepsp depression elicited by 7 min OGD. Two different selective adenosine A(2A) receptor antagonists, SCH58261 and SCH442416, were less effective than ZM241385 during 7 min OGD. The extent of CA1 cell injury was assessed 3 h after the end of 7 min OGD by propidium iodide. Substantial CA1 pyramidal neuronal damage occurred in untreated slices, exposed to OGD, whereas injury was significantly prevented by 100 nmolxL(-1) ZM241385. Glial fibrillary acid protein (GFAP) immunostaining showed that 3 h after 7 min OGD, astrogliosis was appreciable. Western blot analysis indicated an increase in GFAP 30 kDa fragment which was significantly reduced by treatment with 100 nmolxL(-1) ZM241385. In the CA1 hippocampus, antagonism of A(2A) adenosine receptors by ZM241385 was protective during OGD (a model of cerebral ischaemia) by delaying AD appearance, decreasing astrocyte activation and improving neuronal survival.

Selective PARP‐2 inhibitors increase apoptosis in hippocampal slices but protect cortical cells in models of post‐ischaemic brain damage

Poly(ADP-ribose) polymerases (PARP)-1 and PARP-2 play complementary tasks in the maintenance of genomic integrity, but their role in cell death or survival processes is rather different. A recently described series of selective PARP-2 inhibitors (UPF-1035, UPF-1069) were used to study the role of PARP-1 and PARP-2 in post-ischaemic brain damage. We evaluated post-ischaemic brain damage in two different in vitro models: rat organotypic hippocampal slices exposed to oxygen-glucose deprivation (OGD) for 20-30 min, a model characterized by apoptosis-like cell death and mouse mixed cortical cell cultures exposed to 60 min OGD, a model in which cells die with mostly necrosis-like features. In organotypic hippocampal slices, PARP-2 inhibition with UPF-1069 (0.01-1 micromolxL(-1)) caused a concentration-dependent exacerbation (up to 155%) of OGD-induced CA1 pyramidal cell death. Higher concentrations, acting on both PARP-1 and PARP-2, had no effect on OGD injury. In mouse mixed cortical cells exposed to OGD, on the contrary, UPF-1069 (1-10 micromolxL(-1)) significantly reduced post-ischaemic damage. Selective PARP-2 inhibitors increased post-OGD cell death in a model characterized by loss of neurons through a caspase-dependent, apoptosis-like process (hippocampal slice cultures), but they reduced post-OGD damage and increased cell survival in a model characterized by a necrosis-like process (cortical neurons). UPF-1069 may be a valuable tool to explore the function of PARP-2 in biological systems and to examine the different roles of PARP isoenzymes in the mechanisms of cell death and survival.

Neuroprotective effects of mebudipine and dibudipine on cerebral oxygen–glucose deprivation/reperfusion injury

In the present study, we investigated the effects of mebudipine and dibudipine, two new Ca 2+ channel blockers, on primary murine cortical neurons exposed to oxygen–glucose deprivation/reperfusion. The experiments were performed on cells after 11–16 days of culture. To initiate oxygen–glucose deprivation /reperfusion, the culture medium was replaced by glucose-free medium, and the cells were transferred to a humidified incubation chamber in a mixture of 95% N 2 and 5% CO 2 at 37 °C for 30 min. The cultures were pretreated with mebudipine and dibudipine 3 h prior to oxygen–glucose deprivation/reperfusion, in order to explore their effects on neurons under oxygen–glucose deprivation conditions. Cell viability and nitric oxide (NO) production were assessed by MTT assay and the modified Griess method, respectively. Exposure of murine cortical neuronal cells to 30 min oxygen–glucose deprivation significantly decreased cell viability and increased NO production. Pretreatment of the cultures with mebudipine and dibudipine significantly increased cell viability and decreased NO generation in a dose-dependent manner. However, the drugs had no protective effect in cells subjected to oxygen–glucose deprivation for 60 min. Pretreatment of cultures with MK-801 (10 µM), a non-competitive NMDA antagonist, decreased neuronal death after 30-min oxygen–glucose deprivation, while application of NBQX (30 µM), a selective AMPA-kainate receptor antagonist, partially attenuated the cell injury. oxygen–glucose deprivation -induced cytotoxicity and NO production were also inhibited by N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor and MK-801. We conclude that mebudipine and dibudipine could protect cortical neurons against oxygen–glucose deprivation /reperfusion-induced cell injury in a dose-dependent manner, and that this could be mediated partially by decreased NO production.

Elevated Synaptic Activity Preconditions Neurons against an in Vitro Model of Ischemia

Tolerance to otherwise lethal cerebral ischemia in vivo or to oxygen-glucose deprivation (OGD) in vitro can be induced by prior transient exposure to N-methyl-D-aspartic acid (NMDA): preconditioning in this manner activates extrasynaptic and synaptic NMDA receptors and can require bringing neurons to the "brink of death." We considered if this stressful requirement could be minimized by the stimulation of primarily synaptic NMDA receptors. Subjecting cultured cortical neurons to prolonged elevations in electrical activity induced tolerance to OGD. Specifically, exposing cultures to a K(+)-channel blocker, 4-aminopyridine (20-2500 microm), and a GABA(A) receptor antagonist, bicuculline (50 microm) (4-AP/bic), for 1-2 days resulted in potent tolerance to normally lethal OGD applied up to 3 days later. Preconditioning induced phosphorylation of ERK1/2 and CREB which, along with Ca(2+) spiking and OGD tolerance, was eliminated by tetrodotoxin. Antagonists of NMDA receptors or L-type voltage-gated Ca(2+) channels (L-VGCCs) applied during preconditioning decreased Ca(2+) spiking, phosphorylation of ERK1/2 and CREB, and OGD tolerance more effectively when combined, particularly at the lowest 4-AP concentration. Inhibiting ERK1/2 or Ca(2+)/calmodulin-dependent protein kinases (CaMKs) also reduced Ca(2+) spiking and OGD tolerance. Preconditioning resulted in altered neuronal excitability for up to 3 days following 4-AP/bic washout, based on field potential recordings obtained from neurons cultured on 64-channel multielectrode arrays. Taken together, the data are consistent with action potential-driven co-activation of primarily synaptic NMDA receptors and L-VGCCs, resulting in parallel phosphorylation of ERK1/2 and CREB and involvement of CaMKs, culminating in a potent, prolonged but reversible, OGD-tolerant phenotype.

Involvement of mitoK ATP channel in protective mechanisms of cerebral ischemic tolerance

Little work has been performed to determine roles of mitochondrial ATP-sensitive potassium channels (mitoK ATP) in ischemic preconditioning (IPC) in brain. To investigate the role on cerebral IPC, we examined effect of 5-hydroxydecanoate (5-HD), a selective mitoK ATP blocker, and diazoxide (DZX), a selective mitoK ATP opener on various IPC models. An IPC model with gerbil: 2 min bilateral common carotid arteries occlusion (BLCO) + 24 h recovery + 5 min BLCO. 5-HD, DZX, vehicle was administered 30 min before 5 min BLCO. Seven days later, surviving CA1 neurons were counted. A focal IPC model with rat: 15 min middle cerebral artery occlusion (MCAO) + 48 h recovery + 90 min MCAO. Twenty-four hours before 90 min MCAO, 5-HD, DZX, or vehicle was administered. One day after 90 min MCAO, neurological symptoms and infarct volumes were evaluated. An in vitro IPC model with primary neuronal cultures: 8 min oxygen–glucose deprivation (OGD) + 24 h recovery + 70 min OGD. Thirty minutes before 70 min OGD, 5-HD or DZX were added. One day later, surviving neurons were counted. Mitochondrial membrane potential was also monitored. 5-HD significantly attenuated the protective effect of IPC in gerbil model, rat model, and in vitro OGD model. DZX significantly facilitated the protective effect of IPC in gerbil and rat model. The mitochondrial membranes were depolarized with IPC, and 5-HD treatment significantly reduced this effect. These results strongly suggest that mitoK ATP channel activation plays a key role in development of a protective mechanism of cerebral IPC.

Neuroprotection by group I mGlu receptors in a rat hippocampal slice model of cerebral ischemia is associated with the PI3K–Akt signaling pathway: A novel postconditioning strategy?

Ischemic postconditioning is defined as a repetitive series of brief interruptions of reperfusion applied immediately after ischemia. In this study, postconditioning was investigated by first exposing rat organotypic hippocampal slices to 30 min oxygen–glucose deprivation (OGD), which promotes selective CA1 pyramidal cell death, and 5 min later to either a brief period (3 min) of OGD or to a low dose (10 μM) of 3,5-dihydroxyphenylglycine (DHPG) for 30 min. Both protocols attenuated CA1 neuronal injury, as revealed 24 h later by measuring the intensity of propidium iodide fluorescence in this region. The beneficial effects were observed when DHPG postconditioning was applied up to 15 min after OGD, but not at later time points, and was not additive with the neuroprotective effects of a preconditioning DHPG treatment. The attenuation of the OGD-induced CA1 injury evoked by postconditioning was prevented when mGlu1 and mGlu5 receptor antagonists and inhibitors of phosphatidylinositol 3-kinase and Akt activity were present in the incubation medium during the 5 min recovery period after OGD and the 30 min exposure to DHPG. The PI3K inhibitor was also able to prevent the reduction of NMDA toxicity induced by the DHPG treatment. Finally, DHPG increased the phosphorylation of Akt in a transient and mGlu1/mGlu5-dependent manner. Our results show that activation of the mGlu1/mGlu5-PI3K–Akt signaling pathway plays a crucial role in the mechanisms of postconditioning evoked by DHPG and point to this strategy as a possible novel therapeutic tool for stroke and cerebral ischemia.

Estradiol abolishes reduction in cell death by the opioid agonist Met5-enkephalin after oxygen glucose deprivation in isolated cardiomyocytes from both sexes

There is evidence for differences in the response to the treatment of cardiovascular disease in men and women. In addition, there are conflicting results regarding the effectiveness of pharmacologically induced protection or ischemic preconditioning in females. We investigated whether the ability of Met(5)-enkephalin (ME) to reduce cell death after oxygen-glucose deprivation (OGD) is influenced by the presence of 17beta-estradiol (E(2)) in a nitric oxide (NO)- and estrogen receptor-dependent manner. On postnatal day 7 to 8, murine cardiomyocytes from wild-type or inducible NO synthase (iNOS) knockout mice were separated by sex, isolated by collagenase digestion, cultured for 24 h, and subjected to 90 min OGD and 180 min reoxygenation at 37 degrees C (n = 4 to 5 replicates). Cell cultures were incubated in E(2) for 15 min or 24 h before OGD. ME was used to increase cell survival. Cell death was assessed by propidium iodide. More than 300 cells were examined for each treatment. Data are presented as means +/- SE. As a result, in both sexes, ME-induced cell survival was lost in the presence of E(2), and the ability of ME to improve cell survival was restored after treatment with the estrogen receptor antagonist ICI-182780. Furthermore, iNOS was necessary for ME to increase cell survival following OGD in vitro. We conclude that ME-induced reduction in cell death is abolished by E(2) in a sex-independent manner via activation of estrogen receptors, and this interaction is dependent on iNOS.

Resveratrol attenuates early pyramidal neuron excitability impairment and death in acute rat hippocampal slices caused by oxygen-glucose deprivation

Accumulating evidence indicates that the polyphenol resveratrol (trans-3, 5, 4”-trihydroxystibene, RVT) potently protects against cerebral ischemia neuronal damage due to its oxygen free radicals scavenging and antioxidant properties. However, it is unknown whether RVT can attenuate ischemia-induced early impairment of neuronal excitability. To address this question, we simulated ischemic conditions by applying oxygen-glucose deprivation (OGD) to acute rat hippocampal slices and examined the effect of RVT on OGD-induced pyramidal neuron excitability impairment using whole-cell patch clamp recording. 100 µM RVT largely inhibited the 15 min OGD-induced progressive membrane potential (Vm) depolarization and the reduction in evoked action potential frequency and amplitude in pyramidal neurons. In a parallel neuronal viability study using TO-PRO-3 iodide staining, 20 min OGD induced irreversible CA1 pyramidal neuronal death which was significantly reduced by 100 µM RVT. No similar effects were found with PQQ treatment, an antioxidant also showing potent neuroprotection in the rat rMCAO ischemia model. This suggests that antioxidant action per se, is unlikely accounting for the observed early effects of RVT. RVT also markedly reduced the frequency and amplitude of AMPA mediated spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons, which is also an early consequence of OGD. RVT effects on neuronal excitability were inhibited by the large-conductance potassium channel (BK channel) inhibitor paxilline. Together, these studies demonstrate that RVT attenuates OGD-induced neuronal impairment occurring early in the simulated ischemia slice model by enhancing the activation of BK channel and reducing the OGD-enhanced AMPA/NMDA receptor mediated neuronal EPSCs.

The influence of propofol administration time on oxygen-glucose deprivation-reperfusion injury in rat mixed cortical cultures focused on N-Methyl-D-Aspartate (NMDA) receptors

Background: Propofol has been shown to have neuroprotective properties. However, the effect of propofol administration time on neuroprotection is not well understood. This study was conducted to determine if propofol administration time would influence its neuroprotective effects on an in vitro ischemia-reperfusion model, with special attention directed toward NMDA-induced calcium influx. Methods: Primary mixed cortical cultures of thirteen-day-old rats were exposed to 5 min of oxygen-glucose deprivation (OGD), followed by 2 hr of reperfusion. Propofol (1−100μM) was administered before OGD or administered from the time of OGD to the end of the reperfusion period. In the blank and full kill groups, no drug or NMDA 500μM treatment was given. The surviving cells were counted using trypan-blue staining, and cell death rate (CDR) was determined. To measure the maximum Ca2+ influx, 50μM propofol was pre-treated or co-treated with 100μM NMDA. In the control and NMDA 100μM groups, no drug or NMDA 100μM was given. A P ＜ 0.05 was considered statistically significant. Results: Cells pre-treated with propofol (10−100μM) or co-treated (50−100μM) at the time of OGD injury had a decreased CDR compared to the blank group. Cells pre-treated (2,713 nA) or co-treated (3,626 nA) with propofol had a decreased maximum Ca2+ influx compared to the 100μM NMDA group (4,075 nA). Cells pre-treated with propofol had a decreased maximum Ca2+ influx compared to co-treated rats. Conclusions: Propofol demonstrated neuroprotective effects at lower concentrations when administered prior to OGD injury. This may be partially attributable to the reduction of Ca2+ influx against NMDA receptor activation.

Differential role of mGlu1 and mGlu5 receptors in rat hippocampal slice models of ischemic tolerance

Activation of glutamate receptors has been proposed as a key factor in the induction of ischemic tolerance. We used organotypic rat hippocampal slices exposed to 30 min oxygen-glucose deprivation (OGD) to evaluate postischemic pyramidal cell death in the CA1 subregion. In this model, 10 min exposure to OGD 24 h before the exposure to toxic OGD was not lethal and reduced the subsequent OGD neurotoxicity by approximately 53% (ischemic preconditioning). Similarly, a 30 min exposure to the group I mGlu receptor agonist DHPG (10 microM) significantly reduced OGD neurotoxicity 24 h later (pharmacological preconditioning). Ischemic tolerance did not develop when either the selective mGlu1 antagonists LY367385 and 3-MATIDA or the AMPA/KA antagonist CNQX were present in the incubation medium during exposure to sublethal OGD. Neither the NMDA antagonist MK801 nor the mGlu5 antagonist MPEP affected the preconditioning process. On the other hand, pharmacological preconditioning was prevented not only by LY367385 or CNQX, but also by MPEP. In preconditioned slices, the toxic responses to AMPA or NMDA were reduced. The neurotoxicty of 100 microM DHPG in slices simultaneously exposed to a mild (20 min) OGD was differentially altered in the two preconditioning paradigms. After ischemic preconditioning, DHPG neurotoxicity was reduced in a manner that was sensitive to LY367385 but not to MPEP, whereas after pharmacological preconditioning it was enhanced in a manner that was sensitive to MPEP but not to LY367385. Our results show that mGlu1 and mGlu5 receptors are differentially involved in the induction and expression of ischemic tolerance following two diverse preconditioning stimuli.

Cannabinoid modulation of neuronal function after oxygen/glucose deprivation in area CA1 of the rat hippocampus

Endocannabinoids released during cerebral ischemia have been implicated as neuroprotective agents. We assessed the role of cannabinoid receptors in modulating the response of neurons to oxygen/glucose deprivation (OGD), a model for in vitro ischemia, in rat hippocampal slices using extracellular recording techniques. Under control conditions, 15 min OGD resulted in only 50% recovery of CA1 field excitatory postsynaptic potentials (fEPSPs) 60 min post-insult. This post-OGD depression of function was primarily NMDA receptor-dependent as the NMDA receptor antagonist MK-801 (50 μM) promoted recovery of synaptic transmission to 76% of the baseline. Treatment with the CB 1 receptor antagonist AM251 (1 μM), which prevented the depression of excitatory synaptic transmission caused by WIN55,212-2 (1 μM), also markedly enhanced recovery of function (71% of control). The enhanced recovery after OGD in the presence of AM251 was independent of both GABA A receptors and NMDA receptors since co-application of AM251 with either bicuculline (10 μM) or MK-801 (50 μM) did not alter recovery, or further improved recovery, respectively. These results suggest endocannabinoids released during OGD may modulate synaptic transmission and post-OGD neuronal outcome via activation of an AM251-sensitive cannabinoid receptor.

Oxygen–glucose deprivation decreases glutathione levels and glutamate uptake in rat hippocampal slices

Ischemia is a transitory or permanent reduction of blood flow that may provoke an excessive release of glutamate. In that condition, increased reactive oxygen species generation and/or decreased cerebral antioxidant capacity may induce cell death. Antioxidant enzymes and thiols play an important role in the cellular defenses against oxidative stress. The purpose of this study was to evaluate cell viability, glutamate uptake and antioxidant status in rat hippocampal slices exposed to oxygen–glucose deprivation (OGD), an in vitro model of ischemia. After 15 min or 1 h of OGD, hippocampal slices showed a significant reduction of cell viability. Reperfusion during 1 or 2 h did not increase cell death. In this condition, the activities of antioxidant enzymes catalase, glutathione reductase, and peroxidase did not change. However, slices exposed to 15 min OGD and reperfused for 1 or 2 h showed higher superoxide dismutase activity. A significant reduction of glutathione levels was observed after 1 or 2 h of reperfusion in slices previously exposed to 1 h of OGD, although the protein-thiol content was unchanged. Slices exposed to 1 h of OGD and reperfused for 2 h showed reduced sodium-dependent l-[ 3H]glutamate uptake. The reduction of glutamate uptake was partially reversed by dl-dithiothreitol (DTT), a thiol-reducing agent, which may reduce thiol groups in glutamate transporters. Therefore, higher glutamate levels in the synaptic cleft could promote transporter reversal and impair glutamate uptake. Increased extracellular glutamate levels associated with decreased glutathione levels might exacerbate cell damage induced by oxygen and glucose deprivation.

The chloride transporter Na(+)-K(+)-Cl- cotransporter isoform-1 contributes to intracellular chloride increases after in vitro ischemia.

Ischemic episodes in the CNS cause significant disturbances in neuronal ionic homeostasis. To directly measure changes in intracellular Cl- concentration ([Cl-]i) during and after ischemia, we used Clomeleon, a novel ratiometric optical indicator for Cl-. Hippocampal slices from adult transgenic mice expressing Clomeleon in hippocampal neurons were subjected to 8 min of oxygen-glucose deprivation (OGD) (an in vitro model for ischemia) and reoxygenated in the presence of glucose. This produced mild neuronal damage 3 h later that was prevented when the extracellular [Cl-] was maintained at 10 mm during reoxygenation. OGD induced a transient decrease in fluorescence resonance energy transfer within Clomeleon, indicating an increase in [Cl-]i. During reoxygenation, there was a partial recovery in [Cl-]i, but [Cl-]i rose again 45 min later. To investigate sources of Cl- accumulation, we examined the effects of Cl- transport inhibitors on the rises in [Cl-]i during and after OGD. Bumetanide and furosemide, which inhibit Cl- influx through the Na(+)-K(+)-Cl- cotransporter isoform-1 (NKCC-1) and efflux through the K(+)-Cl- cotransporter isoform-2, were unable to inhibit the first rise in [Cl-]i, yet entirely prevented the secondary rise in [Cl-]i during reoxygenation. In contrast, picrotoxin, which blocks the GABA-gated Cl- channel, did not inhibit the secondary rise in [Cl-]i after OGD. [Cl-]i increases during reoxygenation were accompanied by an increase in phosphorylation of NKCC-1, an indication of increased NKCC-1 activity after OGD. We conclude that NKCC-1 plays an important role in OGD-induced Cl- accumulation and subsequent neuronal damage.

Neuroprotection with delayed initiation of prolonged hypothermia after in vitro transient global brain ischemia

Prolonged therapeutic hypothermia (32–34 °C for 12–24 h) improves the functional outcome of comatose cardiac arrest survivors. It is generally believed that rapidly achieving target temperature optimizes neuroprotection. However, this hypothesis has not been tested systematically. In this study, we compared the neuroprotective effect of prolonged hypothermia initiated between 0 and 8 h after reoxygenation using an in vitro model of simulated global brain ischemia. Organotypic hippocampal slices were prepared from 5-day-old Wistar rat pups and cultured for 1 week prior to analysis. Ischemia was simulated by normothermic oxygen–glucose deprivation (OGD). Hypothermia (33 °C) was initiated 0–8 h after reoxygenation and maintained until 24 h post-injury. CA1 regional cell death was quantified by propidium iodide (PI) fluorescence. Release of 14-3-3 β protein was evaluated as a potential surrogate maker for neuroprotection. Hypothermia initiated 0, 1, 2, or 4 h after 30 min OGD reduced 24 h CA1 regional PI fluorescence by 47 ± 34%, 85 ±4%, 88 ± 3%, and 88 ± 5% ( P < 0.05 for all versus normothermic reoxygenation). Direct comparison of hypothermia initiated 4 or 8 h after reoxygenation revealed equivalent neuroprotection following 15 and 30 min OGD, but neither was protective after 60 min OGD. Hypothermia initiated 4 or 8 h after 30 min OGD reduced 14-3-3 β release by 73 ± 11% and 92 ± 4%, respectively ( P < 0.01 for both versus normothermic reoxygenation). In this model, the neuroprotective effect of prolonged post-ischemic hypothermia is both optimal and equivalent when initiated between 1 and 8 h after reoxygenation. These results suggest the need for further in vivo studies to define the therapeutic window within which prolonged hypothermia is optimally neuroprotective after cardiac arrest.