Repeated hyperbaric oxygen induces ischemic tolerance in gerbil hippocampus

Hyperbaric oxygen HBO; 100% oxygen at 2 atmospheres absolute was administered for 1 h to male Mongolian gerbils either for a single session or every other day for five sessions. Two days after HBO pretreatment, the gerbils were subjected to 5 min of forebrain ischemic by occlusion of both common carotid arteries under anesthesia. Seven days after recirculation, neuronal density per 1-mm length of the CAI sector in the hippocampus was significantly better preserved in the five-session HBO pretreatment group ( n = 10: 175.7 (47.8/mm, 54.9% of normal) than in the ischemic control group ( n = 10: 26.2 (11.6/mm, 8.0% of normal) and in the single-session HBO pretreatment group ( n = 7: 37.3 (21.7/mm, 11.4010 of normal). Immunohistochemical staining for the 72-kDa heat-shock protein (HSP-72) in the CAI sector performed 2 days following pretreatment revealed that the five-session HBO pretreatment increased the amount of HSP-72 present compared with that in the ischemic control group and in the single HBO pretreatment group. These results suggest that tolerance against ischemic neuronal damage was induced by repeated HBO pretreatment, which is thought to occur through the induction of HSP-72 synthesis.     

Remote limb ischemic postconditioning protects mouse brain against cerebral ischemia/reperfusion injury via upregulating expression of Nrf2, HO-1 and NQO-1 in mice

Remote ischemic postconditioning (RIPostC) is a promising therapeutic intervention, which has been discovered to reduce ischemia/reperfusion (I/R) injury in heart, kidney, brain and skeletal muscle experimentally. However, its potential protective mechanisms have not been well elucidated. The aim of this study was to investigate the protective effect of RIPostC in cerebral I/R injury and explore the new putative mechanisms of neuroprotection elicited by it. Focal cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) in male CD1 mice. RIPostC was generated by three cycles of 5-min reperfusion/5-min occlusion of the bilateral femoral artery on the bilateral limbs at the onset of middle cerebral artery reperfusion. RIPostC significantly improved neurological outcome, lessened infarct volume and brain edema, upregulated the expression of Nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and quinone oxidoreductase-1 (NQO-1) and activity of superoxide dismutase (SOD), and downregulaed the formation of malondialdehyde (MDA) (p < 0.05). Taken together, these findings demonstrated that RIPostC protected the brain from I/R injury after focal cerebral ischemia by reducing oxidative stress and activating the Nrf2–ARE (antioxidant response element) pathway.     

Effect of hyperbaric oxygen on GABA transport in rat brain synaptosomes

Summary Initial velocities of uptake of GABA have been measured in rat brain synaptosomes from animals which had been exposed to oxygen at high pressure (OHP) and compared to similar measurements in normobaric controls. For hypothalamus, no changes in GABA uptake occurred subsequent to exposure to OHP. For cortical synaptosomes, however, exposure to OHP resulted in a decreased velocity of GABA uptake at all combinations of [Na] and [GABA] used. The OHP data were found to fit the same transport model as found previously for control data. Thus, OHP exposure did not alter the basic mechanism by which sodium and GABA interact with the carrier in the process of transport. However, the constants which quantitate the model were changed by OHP exposure. As a consequence, the several kinetic parameters which are calculated from the model change in the OHP animals. These kinetic parameters are compared to similar calculations for both normobaric control animals and normobaric aged animals. Although the effects of OHP do not precisely parallel the effects of aging, the alterations in kinetic parameters are in several ways similar in the aged and OHP animals.     

Role of Nogo-A in neuronal survival in the reperfused ischemic brain

Nogo-A is an oligodendroglial neurite outgrowth inhibitor, the deactivation of which enhances brain plasticity and functional recovery in animal models of stroke. Nogo-A''s role in the reperfused brain tissue was still unknown. By using Nogo-A^−/− mice and mice in which Nogo-A was blocked with a neutralizing antibody (11C7) that was infused into the lateral ventricle or striatum, we show that Nogo-A inhibition goes along with decreased neuronal survival and more protracted neurologic recovery, when deactivation is constitutive or induced 24 h before, but not after focal cerebral ischemia. We show that in the presence of Nogo-A, RhoA is activated and Rac1 and RhoB are deactivated, maintaining stress kinases p38/MAPK, SAPK/JNK1/2 and phosphatase-and-tensin homolog (PTEN) activities low. Nogo-A blockade leads to RhoA deactivation, thus overactivating Rac1 and RhoB, the former of which activates p38/MAPK and SAPK/JNK1/2 via direct interaction. RhoA and its effector Rho-associated coiled-coil protein kinase2 deactivation in turn stimulates PTEN, thus inhibiting Akt and ERK1/2, and initiating p53-dependent cell death. Our data suggest a novel role of Nogo-A in promoting neuronal survival by controlling Rac1/RhoA balance. Clinical trials should be aware of injurious effects of axonal growth-promoting therapies. Thus, Nogo-A antibodies should not be used in the very acute stroke phase.     

七氟醚单次给药预处理缺血再灌注大鼠脑蛋白激酶Cε的转位激活及其延迟性保护作用

BACKGROUND： Brief exposure to the anesthetic sevoflurane results in delayed neuroprotection, However, few studies have addressed delayed neuroprotection after preconditioning with a single administration of sevoflurane. OBJECTIVE： To explore the relationship between a single preconditioning administration of sevoflurane and reactive oxygen species production and protein kinase C-epsilon （PKC-ε ） translocation. DESIGN, TIME, AND SETTING： The randomized, controlled, animal experiment was conducted at the Central Laboratory, Xiangya Hospital, Central South University, China from November 2007 to April 2008. MATERIALS： A total of 120 healthy, male, Sprague Dawley rats were equally and randomly assigned into five groups： sham operation, ischemia/reperfusion, sevoflurane, 2-mercaptopropionylglycine （2-MPG, a selective reactive oxygen species scavenger） ＋ sevoflurane （MPG ＋ sevoflurane）, and MPG. Sevoflurane （Baxter, USA） and MPG （Sigma, USA） were used in this study. METHODS： Intervention consisted of three procedures. （1） MPG injection： a selective reactive oxygen species scavenger, MPG （20 mg/kg）, was infused into the rat caudal vein in the MPG and MPG ＋ sevoflurane groups. （2） Sevoflurane preconditioning： 30 minutes following MPG injection, rats in the sevoflurane and MPG ＋ sevoflurane groups breathed a mixed gas of 2.4% sevoflurane and 97.6% oxygen for 60 minutes. Rats in the sham operation, ischemia/reperfusion, and MPG groups breathed 100% pure oxygen for 60 minutes. （3） IschemiaJreperfusion： 24 hours after sevoflurane or pure oxygen preconditioning, middle cerebral artery occlusion models were established in the ischemia/reperfusion, sevoflurane, MPG ＋ sevoflurane, and MPG groups. Following 2 hours ischemia/6 hours and 24 hours reperfusion, the carotid artery was separated, but the middle cerebral artery was not occluded, in the sham operation group. MAIN OUTCOME MEASURES： In the ischemic hemisphere, PKC-ε translocation in the rat parietal cortex was measured by Western blot analysis. Infarct volume was calculated using the TTC assay. Neurological deficits were evaluated in rats using a scoring system of 8 points. RESULTS： After 6 hours reperfusion, the ratio of PKC-ε in membrane/（cytosol ＋ membrane） was significantly less in the sham operation group than in the ischemia/reperfusion, sevoflurane, MPG ＋ sevoflurane）, and MPG groups （P 〈 0.05）. The ratio of PKC-ε in membrane/（cytosol ＋ membrane） was significantly greater in the sevoflurane group than in the sham operation, ischemia/reperfusion, MPG ＋ sevoflurane, and MPG groups （P 〈 0.05）. No significant differences were observed in the ischemiaJreperfusJon, M PG ＋ sevoflurane, and MPG groups （P 〉 0.05）. Following 24 hours reperfusion, the ratio of PKC-ε in membrane/（cytosol ＋ membrane） was significantly less in the sham operation group than in the ischemia/reperfusion, sevoflurane, MPG ＋ sevoflurane, and MPG groups （P 〈 0.05）. No significant differences were detected in the ischemia/reperfusion, sevoflurane, MPG ＋ sevoflurane, and MPG groups （P 〉 0.05）. Compared with the ischemia/reperfusion, MPG ＋ sevoflurane, and MPG groups, infarct volume was significantly smaller, and neurological deficits were significantly improved, in the sevoflurane group （P 〈 0.05）. No significant differences in infarct volume and neurological deficits were observed among the ischemia/reperfusion, MPG ＋ sevoflurane, and MPG groups （P 〉 0.05）. Infarcts or neurological deficits were not detected in the sham operation group. CONCLUSION： A single preconditioning administration of sevoflurane reduced infarct volumes and improved neurological deficits in ischemic rats. Delayed neuroprotection may be mediated by reactive oxygen species and correlated to PKC- ε activation.     

An electrophysiological analysis of the protective effects of felbamate,lamotrigine, and lidocaine on the functional recovery from in vitro ischemia in rat neocortical slices

We used field potential recording techniques to examine whether felbamate (FBM), lamotrigine (LTG), and lidocaine (LID) protect against the irreversible functional damage induced by transient ischemia. Five minutes of ischemia caused a depression of the field potential in rat cortical slices, which did not recover even after more than 1 h of washout. The N-methyl-D-aspartate (NMDA) antagonist ketamine (50 μM) protected against depression of the field caused by ischemia. On the other hand, the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2.3-dione (CNQX) (10 μM) had protective effects only if co-applied with ketamine. We found that either FBM (30–300 μM), which did not modify the amplitude of the field EPSP, or LTG (10–300 μM), which reversibly depressed the excitatory synaptic transmission, had a marked protective effect when superfused before and during the ischemic insult. After FBM (100 μM) and LTG (100 μM), the field EPSP recovered by 84 ± 1% and 73 ± 2.7% of control, respectively. Furthermore, LID (30–300 μM) was less effective than FBM and LTG in inducing a functional recovery from the damage caused by ischemia (58 ± 1.8%). The rank order of potency, based on the maximal protection caused by the three drugs, was FBM > LTG > LID. Our results suggest that a noticeable neuroprotection can be obtained during glucose and O₂ deprivation by preventive therapeutic regimens which use the two recently marketed anticonvulsant drugs, FBM and LTG. Synapse 30:371–379, 1998. © 1998 Wiley-Liss, Inc.     

Autophagy and inflammation in ischemic stroke

Appropriate autophagy has protective effects on ischemic nerve tissue,while excessive autophagy may cause cell death.The inflammatory response plays an important role in the survival of nerve cells and the recovery of neural tissue after ischemia.Many studies have found an interaction between autophagy and inflammation in the pathogenesis of ischemic stroke.This study outlines recent advances regarding the role of autophagy in the post-stroke inflammatory response as follows.(1)Autophagy inhibits inflammatory responses caused by ischemic stimulation through mTOR,the AMPK pathway,and inhibition of inflammasome activation.(2)Activation of inflammation triggers the formation of autophagosomes,and the upregulation of autophagy levels is marked by a significant increase in the autophagy-forming markers LC3-II and Beclin-1.Lipopolysaccharide stimulates microglia and inhibits ULK1 activity by direct phosphorylation of p38 MAPK,reducing the flux and autophagy level,thereby inducing inflammatory activity.(3)By blocking the activation of autophagy,the activation of inflammasomes can alleviate cerebral ischemic injury.Autophagy can also regulate the phenotypic alternation of microglia through the nuclear factor-κB pathway,which is beneficial to the recovery of neural tissue after ischemia.Studies have shown that some drugs such as resveratrol can exert neuroprotective effects by regulating the autophagy-inflammatory pathway.These studies suggest that the autophagy-inflammatory pathway may provide a new direction for the treatment of ischemic stroke.     

The role of glutamate transporter-1a in the induction of brain ischemic tolerance in rats

This study was undertaken to determine the role of glutamate transporter-1a (GLT-1a), one of the splice variants of glutamate transporter-1, in the induction of brain ischemic tolerance by cerebral ischemic preconditioning (CIP). We used a rat global cerebral ischemic model and assessed changes by neuropathological evaluation, Western blot analysis, immunohistochemistry, real-time PCR, in vivo brain microdialysis, and high performance liquid chromatography. We found that CIP induced a significant upregulation of GLT-1a expression in the CA1 hippocampus in a time course corresponding to that of neuroprotection of CIP against brain ischemia. Severe brain ischemia for 8 min induced delayed downregulation of GLT-1a, an obvious increase in glutamate concentration and delayed neuronal death of the pyramidal neurons in the CA1 hippocampus. When the animals were pretreated with CIP before the severe ischemia, the above changes normally induced by the severe ischemia were effectively prevented. Importantly, such a preventive effect of CIP on these changes was significantly inhibited by intracerebroventricular administration of GLT-1a antisense oligodeoxynucleotides, which have been proven to specifically inhibit the expression of GLT-1a protein and mRNA, and had no effect on the expression of GLT-1b. In addition, the concentration of aspartate was also elevated after severe brain ischemic insult. However, CIP had no effect on the elevated aspartate concentrations. These results indicate that GLT-1a participated in the brain ischemic tolerance induced by CIP in rats.     

Persimmon leaf flavonoid promotes brain ischemic tolerance

Persimmon leaf flavonoid has been shown to enhance brain ischemic tolerance in mice, but its mechanism of action remains unclear. The bilateral common carotid arteries were occluded using a micro clip ...     

Reversed operation of glutamate transporter GLT-1 is crucial to the development of preconditioning-induced ischemic tolerance of neurons in neuron/astrocyte co-cultures

Sublethal ischemia leads to increased tolerance against subsequent prolonged cerebral ischemia in vivo. In the present study, we investigated the roles of the astrocytic glutamate (Glu) transporter GLT-1 in preconditioning (PC)-induced neuronal ischemic tolerance in cortical neuron/astrocyte co-cultures. Ischemia in vitro was simulated by subjecting cultures to both oxygen and glucose deprivation (OGD). A sublethal OGD (PC) increased the survival rate of neurons significantly when cultures were exposed to a lethal OGD 24 h later. The extracellular concentration of Glu increased significantly during PC, and treatment with an inhibitor of N-methyl-D-actetate (NMDA) receptors significantly reversed the PC-induced ischemic tolerance of neurons, suggesting that the increase in extracellular concentration of Glu during PC was critical to the development of PC-induced neuronal ischemic tolerance via the activation of NMDA receptors. Treatment with a GLT-1 blocker during PC suppressed this increase in Glu significantly, and antagonized the PC-induced neuronal ischemic tolerance. This study suggested that the reversed operation of GLT-1 was crucial to the development of neuronal ischemic tolerance.     

The c‐Jun N‐terminal kinase (JNK) inhibitor XG‐102 enhances the neuroprotection of hyperbaric oxygen after cerebral ischaemia in adult rats

J‐R. Liu, Y. Zhao, A. Patzer, N. Staak, R. Boehm, G. Deuschl, J. Culman, C. Bonny, T. Herdegen and C. Eschenfelder (2010) Neuropathology and Applied Neurobiology 36, 211–224
The c‐Jun N‐terminal kinase (JNK) inhibitor XG‐102 enhances the neuroprotection of hyperbaric oxygen after cerebral ischaemia in adult rats Aim: Both hyperbaric oxygenation (HBO) and inhibition of the c‐Jun N‐terminal kinases (JNKs) by the peptide inhibitor XG‐102 (D‐JNKI‐1) are efficient protective strategies against ischaemia‐induced neurodegeneration. The present study investigated whether the combination of HBO and JNK inhibitor, XG‐102, provides additive neuroprotection against cerebral ischaemia. Methods: Rat middle cerebral artery was occluded (MCAO) for 90 min. XG‐102 [2 mg/kg, intraperitoneally] or HBO (3 ATA, 60 min) was applied 3 h after the onset of MCAO. For the combination treatment, HBO was started 10 min after the injection of XG‐102. Twenty‐four hours after MCAO, the infarct area, the neurological score and the immunohistochemistry staining in brain slices for cleaved‐PARP, transferase‐mediated biotinylated UTP nick end labelling, c‐Jun and phosphorylated (activated) c‐Jun were observed. Results: XG‐102 or HBO alone reduced the total infarct area by 43% and 63%, respectively. The combination diminished total infarct area by 78%, improved the neurological function and reduced brain oedema. Co‐application of HBO and XG‐102 also significantly reduced the cleavage of PARP, by 96% and 91% in cortical penumbra and ischaemic core, respectively. Moreover, cotreatment significantly attenuated the number of cells labelled with transferase‐mediated biotinylated UTP nick end labelling and phosphorylated c‐Jun. Conclusion: Our study demonstrates that HBO reinforces the efficiency of neuroprotective drugs such as XG‐102 and vice versa. Both treatments, physical HBO and pharmacological XG‐102, are already in phase I/II studies and promising strategies for clinical use.     

Enhanced SUMOylation and SENP-1 protein levels following oxygen and glucose deprivation in neurones

Here, we show that oxygen and glucose deprivation (OGD) causes increased small ubiquitin-like modifier (SUMO)-1 and SUMO-2/3 conjugation to substrate proteins in cultured hippocampal neurones. Surprisingly, the SUMO protease SENP-1, which removes SUMO from conjugated proteins, was also increased by OGD, suggesting that the neuronal response to OGD involves a complex interplay between SUMOylation and deSUMOylation. Importantly, decreasing global SUMOylation in cultured hippocampal neurones by overexpression of the catalytic domain of SENP-1 increased neuronal vulnerability to OGD-induced cell death. Taken together, these results suggest a neuroprotective role for neuronal SUMOylation after OGD.     

Detrimental effects of post-treatment with fatty acids on brain injury in ischemic rats

Studies have illustrated that fatty acids, especially polyunsaturated fatty acids (PUFA), have a role in regulating oxidative stress via the enhancement of antioxidative defense capacity or the augmentation of oxidative burden. Elevated oxidative stress has been implicated in the pathogenesis of brain injury associated with cerebral ischemia/reperfusion (I/R). The objective of this study was to assess whether treatment with fatty acids after focal cerebral I/R induced by occlusion of the common carotid arteries and the middle cerebral artery has effects on brain injury in a rat model. PUFA, including arachidonic acid (AA) and docosahexaenoic acid (DHA), and the saturated fatty acid, stearic acid (SA), were administrated 60 min after reperfusion via intraperitoneal injection. AA and DHA aggravated cerebral ischemic injury, which manifested as enlargement of areas of cerebral infarction and increased impairment of motor activity, in a concentration-dependent manner. However, there were no remarkable differences in post-ischemic alterations between the SA and saline groups. The post-ischemic augmentation of injury in AA and DHA treatment groups was accompanied by increases in the permeability of the blood-brain barrier (BBB), brain edema, metalloproteinase (MMP) activity, inflammatory cell infiltration, cyclooxygenase 2 (COX-2) expression, caspase 3 activity, and malondialdehyde (MDA) production, and by a decrease in the brain glutathione (GSH) content. Furthermore, we found that either AA or DHA alone had little effect on free radical generation in neuroglia, but they greatly increased the hydrogen peroxide-induced oxidative burden. Taken together, these findings demonstrate the detrimental effect of PUFA such as AA and DHA in post-ischemic progression and brain injury after cerebral I/R is associated with augmentation of cerebral I/R-induced alterations, including oxidative changes.     

Glucose/oxygen deprivation and reperfusion upregulate SNAREs and complexin in organotypic hippocampal slice cultures

Brain ischemia activates Ca²⁺‐dependent synaptic vesicle exocytosis. The synaptosomal‐associated protein 25 (SNAP‐25) and syntaxin proteins, located on presynaptic terminals, are components of the SNARE (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor) complex and play a key role in regulating exocytosis. Changes in the expression of SNAREs could affect SNARE complex formation, fusion of vesicles with the presynaptic membrane, and release of neurotransmitters through exocytosis. To investigate the relationship of glucose/oxygen deprivation (GOD)/reperfusion‐induced neuronal damage and alteration of presynaptic function, we examined the expression of SNAREs and complexin during GOD and reperfusion using organotypic hippocampal slice cultures. Microtubule‐associated protein 2 (MAP‐2) staining and transmission electron microscopy showed that neuronal damage increased in a time‐dependent manner and both types of neuronal death can occur at different times during GOD and reperfusion. The immunoreactivity of SNAREs such as SNAP‐25, vesicle‐associated membrane protein and syntaxin and complexin increased in pyramidal cell bodies in the CA1 and CA3 areas in a time‐dependent manner following reperfusion. Our data suggest that alteration of presynaptic function may play a partial role in delayed neuronal death during GOD and reperfusion in organotypic hippocampal slice cultures.     

阿斯匹林干预对沙鼠脑缺血再灌注后NF-κB、MCP-1表达的影响

目的 通过研究阿斯匹林干预对沙鼠脑缺血再灌注后NF-κB、MCP-1表达的影响，旨在进一步探讨阿斯匹林在脑缺血再灌注损伤中的作用。方法 将50只健康蒙古沙鼠随机分为正常对照组、假手术组、脑缺血再灌注组（I／R组）、阿斯匹林干预组；夹闭双侧颈总动脉10min后松夹，建立沙鼠全脑缺血再灌注模型；用免疫组化法检测缺血脑组织NF-κBp65、MCP-1的表达。结果缺血再灌注后6h～7d NF-κBp65的表达量显著高于正常对照组及假手术组（P〈0．01），且出现MCP-1阳性表达；同I／R组比较，阿斯匹林干预组在缺血再灌注后6h、1d、3d、7d NF-κBp65与MCP-1表达量均下降（P〈0．05）。结论 阿斯匹林能够下调NF-κB、MCP-1的表达而减轻脑缺血再灌注损伤。     

Persimmon leaf flavonoid induces brain ischemic tolerance in mice

The persimmon leaf has been shown to improve cerebral ischemic outcomes; however, its mechanism of action remains unclear. In this study, mice were subjected to 10 minutes of ischemic preconditioning, and persimmon leaf flavonoid was orally administered for 5 days. Results showed that the persimmon leaf flavonoid significantly improved the content of tissue type plasminogen activator and 6-keto prostaglandin-F1 α in the cerebral cortex, decreased the content of thromboxane B2, and reduced the content of plasminogen activator inhibitor-1 in mice. Following optical microscopy, persimmon leaf flavonoid was also shown to reduce cell swelling and nuclear hyperchromatism in the cerebral cortex and hippocampus of mice. These results suggested that persimmon leaf flavonoid can effectively inhibit brain thrombosis, improve blood supply to the brain, and relieve ischemia-induced pathological damage, resulting in brain ischemic tolerance.     

脑缺血耐受大鼠脑组织血管内皮生长因子和血管生成素-1表达的改变及其意义

目的探讨血管内皮生长因子(VEGF)和血管生成素-1(Ang-1)在短暂性脑缺血预处理(IP)诱导的脑缺血耐受大鼠脑组织表达的改变及其意义。方法将99只Wistar大鼠随机分成对照组(9只)、非IP(NIP)组(45只)和IP组(45只)。大脑中动脉线栓法建立局灶性IP模型,NIP组以假手术代替预缺血,分别在IP后1、3、7、14、21 d予以缺血2 h、再灌注22 h,对照组2次均为假手术。大鼠处死前给予神经功能缺损评分(NDS),采用TTC染色测定脑梗死体积,免疫组化染色法检测脑组织VEGF、Ang-1蛋白表达。结果对照组大鼠脑组织无梗死灶,NDS为0。IP 1、3、7 d亚组NDS和梗死体积显著小于NIP组1、3、7 d亚组(P<0.01～0.05),其中IP 3 d亚组NDS最低,梗死体积最小。对照组脑组织少量VEGF蛋白表达,IP 1、3、7 d亚组VEGF蛋白表达显著高于NIP 1、3、7 d亚组(均P<0.05),其中IP 3 d亚组VEGF蛋白表达最高。各组脑组织均有一定程度Ang-1蛋白表达,IP 7 d亚组Ang-1蛋白表达高于NIP 7 d亚组(P<0.05)。结论 IP 1～7 d内脑组织VEGF、Ang-1表达增加,对脑缺血耐受的产生具有重要作用。     

Hyperbaric oxygen treatment induces dynamic ATPase activity changes in the rat brain following transient global cerebral ischemia-reperfusion*

BACKGROUND： Energy depletion, induced by ischemia or hypoxia, is one of the first events in neuronal injury. OBJECTIVE： To investigate the dynamic changes of Na^＋-K^＋-ATPase and Ca^2＋-ATPase activity in the rat brain following transient global cerebral ischemia-reperfusion （IR）, as well as the effects of hyperbaric oxygen （HBO） treatment. DESIGN, TIME AND SETTING： A randomized and controlled animal study was performed in the Department of Biochemistry and Molecular Biology, Capital Medical University between February and December 2006. MATERIALS： Clean-grade, female, Sprague Dawley rats were provided by the Animal Research Department of Capital Medical University （License number： SYXK11-00-0047）. Na^＋-K^＋-ATPase and Ca^2＋-ATPase kits were provided by Nanjing Jiancheng Bioengineering Institute （Nanjing, China）. A hyperbaric oxygen chamber （DWC150-300） was supplied by Shanghai 701 Medical Oxygen Chamber Factory （Shanghai, China）. METHODS： Sixty-three rats were randomly divided into nine groups： sham operated group （sham-O） as control, groups of IR, and groups treated with hyperbaric oxygen （HBO） after IR. Animal from the IR and HBO groups were sacrificed after four different survival intervals of 6, 24, 48 and 96 hours, respectively. Each group consisted of seven rats. The rats of HBO groups were placed into the hyperbaric chamber. The HBO chamber was flushed with pure oxygen for 5 minutes, followed by a gradual rise in pressure over 5 minutes and stabilization at 0.2 MPa. Then, pure oxygen was supplied for 45 minutes in stabilized pressure, followed by gradually reduced pressure over 15 minutes. The rats of the 6-h HBO group were placed into the HBO chamber following reperfusion for 3 hours on the first day, which was repeated on three consecutive days, always at the same time. Rats in the sham-O group and IR group remained under normal atmospheric pressure. MAIN OUTCOME MEASURES： The Na^＋-K^＋-ATPase and Ca^2＋-ATPase activity in rat brain homogenate