Hypoxia/hypoglycemia preconditioning prevents the loss of functional electrical activity in organotypic slice cultures

In cerebral ischemic preconditioning (IPC), a first sublethal ischemia increases the resistance of neurons to a subsequent severe ischemia. Despite numerous studies, the mechanisms are not yet fully understood. Our goal is to develop an in vitro model of IPC on hippocampal organotypic slice cultures. Instead of anoxia, we chose to apply varying degrees of hypoxia that allows us various levels of insult graded from mild to severe. Cultures are exposed to combined oxygen and glucose deprivation (OGD) of varying intensities, ranging from mild to severe, assessing both the electrical activity and cell death. IPC was accomplished by exposure to the mildest ischemia condition (10% of O2 for 15 min) 24 h before the severe deprivation (5% of O2 for 30 min). Interestingly, IPC not only prevented delayed ischemic cell death 6 days after insult but also the transient loss of evoked potential response. The major interest and advantage of this system over both the acute slice preparation and primary cell cultures is the ability to simultaneously measure the delayed neuronal damage and neuronal function.     

A new model of ischemic preconditioning using young adult hippocampal slice cultures

In ischemic preconditioning (IPC), brief sublethal ischemia protects neurons from a subsequent lethal ischemia. In vivo models faithfully display preconditioning, yet, these models are technically challenging, time-consuming and expensive. In vitro models of preconditioning have also been developed that are technically easier and less expensive. A drawback of pre-existing in vitro models is that since susceptibility to ischemic injury is age-dependent; neuroprotection is being studied in neurons that have intrinsic resistance to oxygen-glucose deprivation (OGD). This study introduces a new in vitro model of ischemic preconditioning in hippocampal slice cultures isolated from 20-30-day-old rats. Slice cultures show a high susceptibility and sharp thresholds toward ischemia that is comparable to that found in vivo. A 5-min OGD treatment was not neurotoxic to young adult slice cultures, while a 10-min OGD treatment was neurotoxic. In addition, the sublethal 5-min OGD treatment protected against a 10-min OGD treatment that was delivered 24 h later. Neuroprotection was seen in preconditioned slice cultures stained with propidium iodide (PI) or with antisera against the neuron-specific antigen NeuN. Energy failure is hypothesized to trigger ischemic preconditioning and a 5-min OGD treatment induced transient energy failure in young adult slice cultures. This model may assist in the search for new therapeutics for the prevention and/or treatment of stroke.     

Glucose/oxygen deprivation induces the alteration of synapsin I and phosphosynapsin

Synapsin I is believed to be involved in regulating neurotransmitter release and in synapse formation. Its interactions with the actin filaments and synaptic vesicles are regulated by phosphorylation. Because exocytosis and synapsin I phosphorylation are a Ca(2+)-dependent process, it is possible that an ischemic insult modifies the presynaptic proteins. However, the neuronal damage and the changes in synapsin I as well as its phosphorylation level as a result of glucose/oxygen deprivation (GOD) and reperfusion in organotypic hippocampal slice cultures have not been established. In this study, the level of synapsin I and phosphosynapsin was measured in organotypic hippocampal slice cultures in order to determine the role of synapsin I in the presynaptic nerve terminals during GOD/reperfusion. Propidium iodide fluorescence was observed in the CA1 area after GOD for 30 min, which could be detected in the whole pyramidal cell layer during reperfusion for 24 h. The immunofluorescence of the neuron specific nuclear protein, NeuN, showed a negative correlation with the PI fluorescence. During GOD/reperfusion, the immunofluorescence of synapsin I increased in the stratum radiatum and the stratum oriens of the CA1 area and the stratum lucidum and the stratum oriens of the CA3 area. The phosphosynapsin level evidently increased in the stratum lucidum of the CA3 area after GOD for 30 min, which was reduced to the control level after reperfusion. These results suggested that the neuronal damage and degenerations were observed as a result of GOD/reperfusion and the increase in synapsin I and its phosphorylation might play a role in modulating the release of neurotransmitters via exocytosis and in the formation of new synapses after brain ischemia.     

Mild activation of poly(ADP-ribose) polymerase (PARP) is neuroprotective in rat hippocampal slice models of ischemic tolerance

Ischemic tolerance is a phenomenon in which exposure to a mild preconditioning stress results in resistance to a subsequent lethal ischemic insult. Here we investigated the role of poly(ADP-ribose) polymerase (PARP) in the development of ischemic tolerance by using organotypic rat hippocampal slices exposed to 30 min oxygen-glucose deprivation (OGD), which leads to selective injury of the CA1 subregion 24 h later. We developed models of pharmacological preconditioning by exposing slices to subtoxic concentrations of either N-methyl-D-aspartate (NMDA) or (S)-3,5-dihydroxyphenylglycine (DHPG) and then, 24 h later, to 30 min OGD. Under these conditions, we observed a significant reduction in OGD-induced CA1 damage. Exposure of slices to the PARP-1 and -2 inhibitors TIQ-A, PJ-34 and UPF 1069 during preconditioning prevented the development of OGD tolerance in a concentration-dependent manner. NMDA and DHPG preconditioning increased the activity of PARP, as detected by immunoblots using antibodies against the poly(ADP-ribose) polymer product, but was not associated with consumption of cellular NAD(+) or ATP. Neuroprotection induced by preconditioning was also prevented by the caspase inhibitor Z-VAD-FMK. The modest but significant increase in caspase-3/7 induced by preconditioning, however, was not associated with PARP-1 cleavage, as occurred with staurosporine. Finally, TIQ-A prevented the activation of ERK1/2 and Akt induced by NMDA preconditioning, suggesting that the protective mechanism evoked by PARP requires activation of these prosurvival mediators. Our results suggest that preconditioning with appropriate pharmacological stimuli may promote neuroprotective mechanisms triggered by the sublethal activation of two otherwise deleterious executioners such as PARP and caspase-3/7.     

Induction of tolerance against traumatic brain injury by ischemic preconditioning.

We tested the hypothesis that a transient non-lethal ischemic insult lasting 2 min would protect against subsequent moderate traumatic brain injury. Sprague-Dawley rats were randomized into three experimental groups, including sham ischemia procedures and ischemic preconditioning (IPC) followed 48 h later by moderate traumatic brain injury (TBI) provoked by parasagittal fluid percussion injury (1.8-2.1 atm) and IPC followed by 48 h sham TBI. Seven days after the secondary insult, animals were perfusion-fixed for quantitative histopathological analysis. The CA3 necrotic cell count was decreased by 63% in TBI animals that had undergone IPC as compared to TBI animals that underwent sham IPC. TBI animals that had undergone IPC demonstrated significantly smaller contusion volumes than the TBI alone group (6.44 +/- 1.51 vs 1.37 +/- 0.63 mm3, mean +/- s.e.m.) These data indicate that IPC applied 2 days before moderate fluid percussion brain injury increases the brain resistance to traumatic brain damage.     

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.     

Protection by rapid chemical preconditioning of stressed hippocampal slice: a study of cellular swelling using optical scatter imaging

It has been demonstrated that anoxic preconditioning protects against a subsequent 'lethal' injury in the hippocampal slice. The goal of this paper was to test the hypothesis that chemical preconditioning could help reduce the cellular swelling observed in excitotoxically injured hippocampal slices. The control slice was given a 10-min insult of 100 microM N-methyl-D-aspartate (NMDA) to simulate ischemic injury, followed by 30-min perfusion of standard Ringers solution. Cellular swelling was observed with a microscope designed to image light scatter changes resulting from cellular swelling. After the control NMDA injury, the average peak scatter change for CA1, CA3 and DG regions was 31.0 +/- 3.4, 22.4 +/- 4.8 and 27.6 +/- 4.6%, respectively. The peak scatter change of the overall slice was 26.0 +/- 3.6%. The experimental slices were preconditioned by three short 100 microM NMDA insults of 15 s each separated by 10 min of standard Ringers solution perfusion. The slices then received 10 min of 'lethal' injury by 100 microM NMDA. It was observed that the overall scatter signal, as a measure of cellular swelling, was reduced by 8.0% (P<0.05, n=11) after preconditioning. A regional heterogeneity in the responses was also observed. Cellular swelling in CA1, CA3 and DG were reduced by 9.8% (P<0.001, n=11), 9.2% (P<0.005, n=11) and 7.7% (P<0.05, n=11), respectively, when compared to the control. This study presents experimental evidence that short episodes of preconditioning may protect against acute cellular swelling under ischemic conditions.     

Functional validation of adult hippocampal organotypic cultures as an in vitro model of brain injury

To determine whether hippocampal pyramidal neurons retain authentic functional properties in mature organotypic culture, hippocampal slice cultures were established from young adult rats (P20-21). Cultures maintained 7 days in vitro retained tight organization of neuronal layers, as opposed to the widening restructure of pyramidal neurons often observed in perinatal slices. CA3 and CA1 pyramidal neurons fired action potentials in response to current injection and exhibited spontaneous and evoked synaptic currents, indicating intact neuronal function and normal hippocampal neural circuitry. We also tested neuronal sensitivity of slice cultures to ischemic injury. Acute ischemic paradigm resulted in selective death of pyramidal neurons in the CA1 region, which was prevented by treatment with an NMDA-antagonist, MK-801. Robust efflux of excitatory and inhibitory amino acid neurotransmitters was detected during ischemia, consistent with changes shown in acute slices. In summary, hippocampal organotypic cultures prepared from young adult rats maintained neuronal architecture and synaptic activity in vitro and can be used in parallel with an acute slice system to model mature brain tissue to examine ischemic pathophysiology and neuroprotective treatment.     

Sevoflurane-induced preconditioning protects against cerebral ischemic neuronal damage in rats

In the present study, we tested the ability of sevoflurane to induce early and late preconditioning against ischemic neuronal injury using an in vivo model of global cerebral ischemia in the rat. Seven-minute global ischemia was induced by cardiac arrest, followed by resuscitation and recovery for 7 days. Hippocampal slices were then prepared and the amplitude of extracellularly recorded, orthodromically evoked, CA1 population spikes (neuronal function) was quantified. Rats were preconditioned for 30 min with 1.0 minimum alveolar concentration (MAC) of sevoflurane once or on 4 consecutive days, 15 min (single exposure, early) or 24 h (four exposures, late preconditoning) prior to cardiac arrest. After early or late preconditioning, sevoflurane reduced ischemic neuronal damage from 43 +/- 3% [sham rats, (mean +/- SEM)] to 30 +/- 3% and 35 +/- 4%, respectively. Histopathology demonstrated a preserved morphology of the CA1 region of preconditioned rats, whereas pyknosis was present in control and sham-treated rats. Sevoflurane-induced preconditioning confers neuroprotection during an early as well as late time window.     

Extracellular signal-regulated kinase and c-Jun N-terminal protein kinase in ischemic tolerance.

The alterations and involvement of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK) activation were examined in the hippocampal CA1 region in a rat model of global brain ischemic tolerance. Western blotting study showed that ERK activation (diphosphorylation) level was decreased (3.75-, 0.56-, and 0.23-fold vs sham control) and JNK activation level was increased (3.82-, 4.63-, and 5.30-fold vs sham control) 3 days after more severe ischemic insults (6 min, 8 min, and 10 min of ischemia, respectively). These alterations were significantly prevented by pretreatment with preconditioning ischemia, which also provided neuronal protection against ischemic injury. Inhibition of ERK activation after preconditioning ischemia by PD98059, a specific ERK kinase inhibitor, significantly prevented the inhibitory effects of preconditioning ischemia on both JNK activation and ischemic injury. The results suggest that ERK activation after preconditioning ischemia may result in the prevention of JNK activation and thus be involved in the protective responses in ischemic tolerance in hippocampal CA1 region.     

Thrombin in ischemic neuronal death

Thrombin plays a role in cerebral ischemia as rats subjected to focal cerebral ischemia were protected by the intracerebral injection of hirudin, a selective thrombin inhibitor. To separate the roles of thrombin in cell death and in coagulation, we have used an in vitro approach to test the effect of hirudin and of protease nexin-1 (PN-1), a cerebral thrombin inhibitor, on neuronal ischemia. Rat organotypic hippocampal slice cultures were subjected to oxygen (5%) and glucose (1 mmol/L) deprivation (OGD) during 30 min. Hirudin or PN-1 administered after OGD significantly prevented neuronal death in the CA1 region. After 24 h, there was a marked increase in thrombin immunoreactivity on Western blots. Thrombin therefore contributes to ischemic damage in neural tissue in vitro.     

Effect of induced tolerance on biochemical disturbances in hippocampal slices from the gerbil during and after oxygen/glucose deprivation.

To evaluate whether the state of tolerance is stable enough to be studied under in vitro conditions after induction by ischemic preconditioning in vivo, metabolic disturbances of hippocampal slices prepared from control and preconditioned gerbils were evaluated during and after oxygen/glucose deprivation (OGD). Slices were subjected to 5, 10 or 15 min OGD with or without 2h recovery. During the state of metabolic stress, changes in energy metabolism were identical in slices taken from control and preconditioned gerbils. Following OGD, however, recovery of protein synthesis was significantly improved in hippocampal slices of preconditioned animals, indicating that the effect of preconditioning on metabolic disturbances induced by transient OGD in vitro or transient ischemia in vivo is similar. It is suggested that the hippocampal slice preparation is an in vitro model suitable for the study of basic mechanisms underlying the induction of tolerance in vivo.     

A synthetic NCAM-derived peptide, FGL, protects hippocampal neurons from ischemic insult both in vitro and in vivo

There is a major unmet need for development of innovative strategies for neuroprotection against ischemic brain injury. Here we show that FGL, a neural cell adhesion molecule (NCAM)-derived peptide binding to and inducing phosphorylation of the fibroblast growth factor receptor (FGFR), acts neuroprotectively after an ischemic insult both in vitro and in vivo. The neuroprotective activity of FGL was tested in vitro on dissociated rat hippocampal neurons and hippocampal slice cultures, using a protocol of oxygen-glucose deprivation (OGD). FGL protected hippocampal neurons from damage and maintained or restored their metabolic and presynaptic activity, both if employed as a pretreatment alone to OGD, and if only applied after the insult. In vivo 24 h pretreatment with a single suboccipital injection of FGL significantly protected hippocampal CA1 neurons from death in a transient global ischemia model in the gerbil. We conclude that FGL promotes neuronal survival after ischemic brain injury.     

Induction of NADPH-diaphorase activity in the hippocampus in a rat model of cerebral ischemia and ischemic tolerance

Preconditioning of the brain with sublethal ischemia induces tolerance to subsequent lethal periods of ischemia (ischemic tolerance). In this study, we used NADPH-diaphorase histochemistry to investigate the postischemic changes of nitric oxide synthase (NOS) in the hippocampus in a rat model of cerebral ischemia and ischemic tolerance. Forebrain ischemia was induced by 4-vessel occlusion for 3 min as an ischemic preconditioning. Three days after the preconditioning or sham operation, second ischemia was induced for 6 min. A transient increase in NADPH-diaphorase activity, beginning after 2 h and maximal after 1 day, was observed in CA1 pyramidal neurons of rats subjected to 3 min of preconditioning ischemia as well as 6 min of subsequent ischemia both with and without preconditioning. In addition, expression of NADPH-diaphorase activity was seen in reactive glial cells in the damaged CA1 region of animals subjected to 6 min of ischemia without preconditioning. Thus, direct involvement of increased NADPH-diaphorase activity in ischemic tolerance was not suggested because the increased NADPH-diaphorase activity preceded the induction of ischemic tolerance which takes place 1–7 days after preconditioning. However, the present findings suggest that the induction of neuronal NADPH-diaphorase activity occurs in response to cerebral ischemia.     

Neuroprotective effects of ischemic preconditioning on hippocampal CA1 pyramidal neurons through maintaining calbindin D28k immunoreactivity following subsequent transient cerebral ischemia

Ischemic preconditioning elicited by a non-fatal brief occlusion of blood flow has been applied for an experimental therapeutic strategy against a subsequent fatal ischemic insult. In this study, we investi-gated the neuroprotective effects of ischemic preconditioning (2-minute transient cerebral ischemia) on calbindin D28k immunoreactivity in the gerbil hippocampal CA1 area following a subsequent fatal tran-sient ischemic insult (5-minute transient cerebral ischemia). A large number of pyramidal neurons in the hippocampal CA1 area died 4 days after 5-minute transient cerebral ischemia. Ischemic preconditioning reduced the death of pyramidal neurons in the hippocampal CA1 area. Calbindin D28k immunoreactivity was greatly attenuated at 2 days after 5-minute transient cerebral ischemia and it was hardly detected at 5 days post-ischemia. Ischemic preconditioning maintained calbindin D28k immunoreactivity after transient cerebral ischemia. These findings suggest that ischemic preconditioning can attenuate transient cerebral ischemia-caused damage to the pyramidal neurons in the hippocampal CA1 area through maintaining cal-bindin D28k immunoreactivity.     

沙鼠脑缺血耐受的组织学变化及HSP在其中的作用

目的 :观察脑缺血耐受时的组织学变化及 HSP在其中的作用。方法 :通过 HE染色观察脑缺血耐受时的组织学变化 ,并通过免疫组化染色 ,了解 HSP70及 HSP2 7在其中的作用。结果 :一次性 5分钟缺血后 7天海马 CA1区神经元大多坏死 ,若在缺血前给予 2分钟的缺血预处理 ,该区神经元大多保留 ,表现出明显的保护作用。只给一次性 5分钟缺血 ,海马 CA1区神经元无 HSP70染色。若在缺血前给予预处理 ,海马 CA1区神经元可见明显 HSP70染色。而HSP2 7主要在胶质细胞表达 ,海马区的神经元未见其表达。结论 :缺血前给予预处理对以后的缺血有保护作用 ;在缺血耐受过程中 ,HSP70表达出一定的保护作用。     

Ischemic preconditioning: a long term survival study using behavioural and histological endpoints

In this study we sought to determine if ischemic preconditioning provided long term behavioral and histological protection. A second goal was to see if ischemic preconditioning conveys its protective effect on CA1 neurons by altering post-ischemic brain temperature. While preconditioning episodes of short duration ischemia (i.e. 1.5 min) provided significant histological protection of CA1 pyramidal cells against a subsequent severe ischemic insult (i.e. 5 min), this did not result in complete behavioural protection. Preconditioned ischemic animals initially displayed habituation deficits in an open field test that were comparable to untreated ischemic gerbils. A significant decline in CA1 preservation in preconditioned animals was observed when survival time was extended from 10 (81% protection) to 30 (53% protection) days. In addition, protection was not observed in the subiculum and CA2 sector of the hippocampus where consistent damage was observed in 21/22 gerbils. Ischemic preconditioning did not markedly affect post-ischemic brain temperature suggesting that the observed protection was not due to a reduction in temperature during or after the severe ischemic insult. The lack of functional protection within the first 10 days after ischemia, along with the decline of cellular preservation over time, suggests that this paradigm may not provide permanent protection.     

Ischemic preconditioning in 18- to 20-month-old gerbils: long-term survival with functional outcome measures.

BACKGROUND AND PURPOSE: In young animals, ischemic preconditioning protects CA1 hippocampal neurons against global ischemia. However, cerebral ischemia occurs most frequently in individuals aged >/=65 years. This study examined the protection provided by ischemic preconditioning in a population of aged (18- to 20-month-old) gerbils. METHODS: One group of animals was exposed to two 1.5-minute episodes of global ischemia separated by 24 hours and followed 72 hours later by a 5-minute occlusion of both carotid arteries. A second group was given 2 episodes of preconditioning only. Two other groups were exposed to 5 minutes of ischemia or sham surgery. The animals survived 10, 30, or 60 days. Functional and histological assessments were used to determine the extent of protection. RESULTS: Ten days after ischemia there was >80% protection of CA1 neurons in ischemic preconditioned animals compared with 6% in ischemic gerbils. Nevertheless, these preconditioned animals were impaired in open-field tests of habituation. In addition, CA1 dendritic field potentials were smaller in amplitude compared with those in sham animals. While there was a complete loss of staining for CA1 microtubule-associated protein-2 in ischemic animals, staining in ischemic preconditioned animals was normal. This suggests that dendritic abnormalities per se were not responsible for the observed functional deficits. CA1 cell survival declined to approximately 75% of sham values (P<0.05) at 60 days after ischemia. CONCLUSIONS: Ischemic preconditioning provided substantial neuroprotection in aged gerbils. Nonetheless, the striking dissociation between histological and functional protection provided by ischemic preconditioning in aged animals emphasizes the need to use functional end points and long-term survival when assessing neuroprotection. Although functional recovery was evident with increasing survival time, CA1 cell death continued, thereby raising the possibility that the level of neuroprotection attained was not permanent.     

缺血预处理后海马CA1区反应性星形胶质细胞增生与迟发性神经元缺血耐受性的关系

目的 探讨缺血预处理后海马CA1区反应性星形胶质细胞增生与迟发性神经元缺血耐受性的关系。方法 实验动物被随机分为手术组、缺血组、预缺血组、预缺血后再缺血组。阴断沙土鼠双侧颈总动脉造成前脑缺血模型。采用细胞特异性抗原胶质纤维酸性蛋白（GFAP）免疫组化法标记星形胶质细胞。结果 预缺血后1－7天,海马CA1区GFAP阳性的星形胶质细胞数轻度增加,至28天时增生非常显著（P＜0．01）。预缺血后1－7天再缺血,海马CA1区存活正常神经元数逐渐下降,预缺血后28天再缺血又显著增加（P＜0．01）。结论 缺血预处理后,神经元可出现迟发性缺血耐受,反应性星形胶质细胞增生可能起了重要作用。     

Nitric oxide is involved in anoxic preconditioning neuroprotection in rat hippocampal slices

Sublethal anoxia/ischemia protects against subsequent damaging insults in intact brain or hippocampal slices. To help further understand mechanisms underlying anoxic/ischemic preconditioning, we tested three hypotheses which were that: (a) anoxic preconditioning (APC) improves electrical recovery in rat hippocampal slices; (b) anoxic preconditioning requires nitric oxide (NO); and (c) anoxic preconditioning blocks mitochondrial dysfunction that occurs following re-oxygenation after anoxia. Control hippocampal slices underwent a single 'test' anoxic insult. Experimental slices were preconditioned by 3 short anoxic insults prior to the 'test' insult. Evoked potentials (EPs), and NADH redox status were recorded prior to, during and after preconditioning and/or 'test' anoxic insults. To examine the role of NO, studies sought to determine whether APC could be produced by the NO donor, DEA/NO, and whether APC could be inhibited by NO synthase (NOS) inhibitor (7-nitroindazole). EP amplitudes recovered significantly better after reoxygenation in preconditioned slices and after NO-emulated preconditioning (90.0+/-17.7% and 90.0+/-21.3%, respectively, n=9, ** p<0.01, vs. 17.0+/-7.9%, n=9, in control slices). Inhibition of NOS blocked APC protection (6.8+/-6.8%, n=9). The intensity of NADH hyperoxidation was not significantly different among groups following 'test' anoxia. These data confirm that preconditioning by anoxia improves electrical recovery after anoxia in hippocampal slices. Evidence supports that NO from constitutive hippocampal NOS may be involved in the neuroprotection afforded by preconditioning by a mechanism that does not change the apparent mitochondrial hyperoxidation after anoxia.