Role of Rac1 GTPase in NADPH Oxidase Activation and Cognitive Impairment Following Cerebral Ischemia in the Rat

Background

Recent work by our laboratory and others has implicated NADPH oxidase as having an important role in reactive oxygen species (ROS) generation and neuronal damage following cerebral ischemia, although the mechanisms controlling NADPH oxidase in the brain remain poorly understood. The purpose of the current study was to examine the regulatory and functional role of the Rho GTPase, Rac1 in NADPH oxidase activation, ROS generation and neuronal cell death/cognitive dysfunction following global cerebral ischemia in the male rat.

Methodology/Principal Findings

Our studies revealed that NADPH oxidase activity and superoxide (O₂ ⁻) production in the hippocampal CA1 region increased rapidly after cerebral ischemia to reach a peak at 3 h post-reperfusion, followed by a fall in levels by 24 h post-reperfusion. Administration of a Rac GTPase inhibitor (NSC23766) 15 min before cerebral ischemia significantly attenuated NADPH oxidase activation and O₂ ⁻ production at 3 h after stroke as compared to vehicle-treated controls. NSC23766 also attenuated “in situ” O₂ ⁻ production in the hippocampus after ischemia/reperfusion, as determined by fluorescent oxidized hydroethidine staining. Oxidative stress damage in the hippocampal CA1 after ischemia/reperfusion was also significantly attenuated by NSC23766 treatment, as evidenced by a marked attenuation of immunostaining for the oxidative stress damage markers, 4-HNE, 8-OHdG and H2AX at 24 h in the hippocampal CA1 region following cerebral ischemia. In addition, Morris Water maze testing revealed that Rac GTPase inhibition after ischemic injury significantly improved hippocampal-dependent memory and cognitive spatial abilities at 7–9 d post reperfusion as compared to vehicle-treated animals.

Conclusions/Significance

The results of the study suggest that Rac1 GTPase has a critical role in mediating ischemia/reperfusion injury-induced NADPH oxidase activation, ROS generation and oxidative stress in the hippocampal CA1 region of the rat, and thus contributes significantly to neuronal degeneration and cognitive dysfunction following cerebral ischemia.     

Effect of Antioxidant Treatment in Global Ischemia and Ischemic Postconditioning in the Rat Hippocampus

Ischemic postconditioning is a very effective way how to prevent delayed neuronal death. Effect of Ginkgo biloba extract (EGb 761; 40 mg/kg) posttreatment was studied on the rat model of transient forebrain ischemia and ischemia/postconditioning. Global ischemia was produced by four-vessel occlusion in Wistar male rats. Two experimental protocols were used: (a) 10 min of ischemia/7 days of reperfusion with or without EGb 761 treatment or (b) 10 min of ischemia/2 days of reperfusion/5 min of ischemia (postconditioning), following 5 days of reperfusion. EGb 761 was applied as follows: 30 min before 10 min of ischemia then 5 h, 1 and 2 days after 10 min of ischemia. Fluoro Jade B, marker for neuronal degeneration, was used for quantitative analysis of the most vulnerable hippocampal CA1 neurons. Cognitive and memory functions were tested by Morris water maze, as well. Administration of EGb 761 30 min before 10 min of ischemia or 5 h after ischemia has rather no protective effect on neuronal survival in CA1 region. Ten minutes of ischemia following ischemic postconditioning after 2 days of reperfusion trigger a significant neuroprotection of CA1 neurons, but it is abolished by EGb 761 posttreatment. Ischemia/postconditioning group showed a significant improvement of learning and memory on the seventh day of reperfusion. Protection of the most vulnerable CA1 neurons after ischemia/postconditioning is abolished by exogenous antioxidant treatment used in different time intervals after initial ischemia. Moreover, combination of EGb 761 administration with repeated stress (5 min ischemia used as postconditioning) causes cumulative injury of CA1 neurons.     

A comparative study of PNIPAM nanoparticles of curcumin, demethoxycurcumin, and bisdemethoxycurcumin and their effects on oxidative stress markers in experimental stroke

Oxidative stress and inflammatory damage play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. The development of new strategies for enhancing drug delivery to the brain is of great importance in diagnostics and therapeutics of central nervous diseases. The present study examined the hypothesis that intranasal delivery of nanoformulation of curcuminoids would reduce oxidative stress-associated brain injury after middle cerebral artery occlusion (MCAO). The rats were subjected to 2 h of MCAO followed by 22 h reperfusion, after which the grip strength, locomotor activity was performed. The effects of treatment in the rats were assessed by grip strength, locomotor activity and biochemical studies (glutathione peroxidase, glutathione reductase, lipid peroxidation, superoxide dismutase, and catalase) in the brain. Pretreatment with polymeric N-isopropyl acryl amide (PNIPAM) nanoparticles formulation of all three curcuminoids (curcumin (Cur), demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC)) at doses (100 μg/kg body weight) given intranasally was effective in bringing significant changes on all the parameters. While nanoformulation of curcumin at a dose of 100 μg/kg body weight was most active in the treatment of cerebral ischemia as compared to others nanoformulation of curcuminoids. The potency of antioxidant activity significantly decreased in the order of PNIPAM nanoformulation of Cur > DMC >> BDMC, thus suggesting the critical role of methoxy groups on the phenyl ring.     

Protective Effect of Oren-gedoku-to Against Induction of Neuronal Death by Transient Cerebral Ischemia in the C57BL/6 Mouse

We examined the neuroprotective effects of oren-gedoku-to (TJ15), a herbal medicine, after transient forebrain ischemia. Transient forebrain ischemia was induced by occlusion of both common carotid arteries for 15 min in C57BL/6 mice treated with TJ15. In the control ischemic group without TJ15 treatment, histologic examination of brain tissue collected seven days after reperfusion showed death of pyramidal cells in CA2-3 area of the hippocampus, unilaterally or bilaterally. In mice treated with oral TJ15 (845 mg/kg/day) for five weeks, the frequency of ischemic neuronal death was significantly lower. Immunohistochemistry for Cu/Zn-superoxide dismutase (Cu/Zn-SOD) showed strongly reactive astrocytes in the hippocampus of ischemic mice treated with TJ15. Damage to nerve cells by free radicals plays an important role in the induction of neuronal death by ischemia-reperfusion injury. Our results suggest that TJ15 protects against ischemic neuronal death by increasing the expression of Cu/Zn-SOD and suggest that oren-gedoku-to reduces the exposure of hippocampal neurons to oxidative stress.     

姜黄素后处理通过SIRT1/FOXO1信号通路拮抗小鼠脑缺血再灌注损伤

目的:探究姜黄素后处理是否通过激活SIRT1/FOXO1信号通路抵抗小鼠脑缺血再灌注损伤。方法:小鼠脑缺血30 min,再灌注24 h建立脑缺血再灌注模型。手术前脑室内注射SIRT1特异性抑制剂EX527。再灌注后腹腔注射姜黄素。小鼠随机分为以下6组:假手术组;单纯姜黄素后处理组;缺血再灌注组;缺血再灌注+姜黄素后处理组;EX527预处理+缺血再灌注+姜黄素后处理组;EX527预处理+脑缺血再灌注组。再灌注24 h检测脑梗体积、Complex I活性、ROS含量以及SIRT1、Ac-FOXO1、Bax、Bcl-2、Caspase-3蛋白表达情况。结果:与手术组相比,姜黄素后处理组梗死区脑组织SIRT1的表达量及活性明显增加,脑梗体积降低,ROS含量降低而Complex I活性增高,Bcl-2的表达增高而Bax和Caspase-3的表达量降低(均P0.05)。阻断SIRT1信号通路后上述姜黄素脑保护作用均减弱(P0.05)。结论:我们的研究首次证实姜黄素后处理通过激活SIRT1/FOXO1信号通路,进而降低氧化应激与凋亡,最终减轻脑缺血再灌注损伤。     

A Hydrophilic Peptide Comprising 18 Amino Acid Residues of the Prosaposin Sequence Has Neurotrophic Activity In Vitro and In Vivo

Abstract: Prosaposin, a 517-amino-acid glycoprotein, not only acts as the precursor of saposin A, B, C, and D but also possesses neurotrophic activity to rescue hippocampal CA1 neurons from ischemic damage in vivo and to promote neurite extension of neuroblastoma cells in vitro. Recently, the trophic activity of prosaposin on human neuroblastoma cells has been shown to reside in the NH₂-terminal hydrophilic sequence (LIDNNRTEEILY) of the human saposin C. Here we show that prosaposin, saposin C, and a peptide comprising the 18-amino-acid sequence (18-mer peptide; LSELIINNATEELLIKGL) located in the NH₂-terminal hydrophilic sequence of the rat saposin C-domain promoted survival and neurite outgrowth of cultured rat hippocampal neurons in a dose-dependent manner. Moreover, infusion for 7 days of the 18-mer peptide into the lateral ventricle of gerbils, starting either 2 h before or immediately after 3 min of forebrain ischemia, protected ischemia-induced learning disability and hippocampal CA1 neuronal loss. Thus, we ascribe the in vitro and in vivo trophic actions of prosaposin on hippocampal neurons to the linear 18-mer sequence and raise the possibility that this peptide can be used as an agent for the treatment of forebrain ischemic damage.     

Impact of Ginkgo Biloba Extract EGb 761 on Ischemia/Reperfusion – Induced Oxidative Stress Products Formation in Rat Forebrain

Dysbalance in reactive oxygen/nitrogen species is involved in the pathogenesis of cerebral ischemia/reperfusion injury (IRI). Ginkgo biloba extract (Egb 761) pre-treatment was used to observe potential antioxidant/neuroprotective effect after global ischemia/reperfusion. Egb 761 significantly decreased the level of lipoperoxidation (LPO) in rat forebrain total membrane fraction (homogenate) induced by in vitro oxidative stress (Fe²⁺+H₂O₂). In animals subjected to four-vessel global ischemia for 15 min and 2–24 h reperfusion the EGb pretreatment slightly decreased LPO in forebrain homogenate. However, as detected in EGb treated group, the LPO-induced lysine conjugates are attenuated in comparison to non-treated IRI animals. EGb significantly improved parameters which indicate forebrain protein oxidative damage after IRI. The intensity of tryptophane fluorescence was increased by the 18.2% comparing to non-treated IRI group and bityrosine fluorescence was significantly decreased in ischemic (21%) and 24 h reperfused (15.9%) group in comparison non-treated IRI group. In addition, the level of total free SH- groups in pre-treated animals was significantly higher comparing to non-treated animals. Our results indicate that extract of EGb 761 has potent antioxidant activity and could play a role to attenuate the IRI-induced oxidative protein modification and lipoperoxidation in the neuroprotective process.     

Noradrenaline Metabolism in Neocortex and Hippocampus Following Transient Forebrain Ischemia in Rats: Relation to Development of Selective Neuronal Necrosis

Noradrenaline (NA) metabolism in the neocortex and hippocampus was examined in rats at 1, 24, and 48 h following 15 min of reversible forebrain ischemia. As assessed by the ratio of accumulated 3,4-dihydroxyphenylalanine (DOPA) to the tissue NA level after inhibition of DOPA decarboxylase, the NA turnover rates were markedly increased (120-148% above the control) at 1 h postischemia in both the neocortex and hippocampal formation (CA1 and CA3 plus dentate gyrus). The DOPA:NA ratio went back to control levels after longer postischemic survival times. The ratio between levels of the deaminated NA metabolite, 3,4-dihydroxyphenylethyleneglycol (DOPEG), and NA, which gives another measure of NA turnover rate, showed similar changes. In the neocortex and the CA3 plus dentate gyrus, the DOPEG:NA ratio was markedly increased (89-118%) 1 h after the ischemia, but this change had disappeared at 24 and 48 h. Thus, both the DOPA accumulation experiments and the NA and DOPEG measurements indicate that following transient forebrain ischemia, there is an increased NA turnover in the hippocampus and cortex only in the early recirculation period and not after longer postischemic survival times. The degree of neuronal necrosis in the CA1 region was examined light microscopically on celestine blue-acid fuchsin-stained sections at 24, 48, and 96 h following the ischemic insult. The neuronal damage in CA1 was sparse after 24 h of recovery, had increased markedly after 48 h, and was very pronounced at 96 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of rosiglitazone on global ischemia-induced hippocampal injury and expression of mitochondrial uncoupling protein 2

We investigate the effect of rosiglitazone, a ligand for peroxisome proliferator-activated receptor-gamma (PPARgamma) with anti-inflammatory and anti-oxidative actions, on hippocampal injury and its roles in mitochondrial uncoupling protein 2 (UCP2) expression caused by transient global ischemia (TGI) in rats. Increased UCP2 expression was observed in mitochondria of hippocampal CA1 2-24h after TGI/reperfusion, with maximal expression levels at 6-18h. Administration of rosiglitazone to hippocampus 30min prior to the onset of TGI further enhanced mitochondrial UCP2 expression 2-6h following TGI/reperfusion. Rats subjected to TGI/reperfusion displayed a significant increase in lipid peroxidation, based on increased malondialdehyde (MDA) levels, in hippocampal CA1 mitochondria 2-6 h after reperfusion. Rosiglitazone significantly attenuated TGI/reperfusion-induced lipid peroxidation and suppressed hippocampal CA1 neuronal death based on the surviving neuronal counts. In conclusion, our results provide correlative evidence for the "PPARgamma-->UCP2-->neuroprotection" cascade in ischemic brain injury.     

Phosphorylation of JNK Increases in the Cortex of Rat Subjected to Diabetic Cerebral Ischemia

Previous studies have demonstrated that the c-Jun N-terminal kinase (JNK) pathway plays an important role in inducing neuronal apoptosis following cerebral ischemic injury. JNK signaling pathway in activated during cerebral ischemic injury. It participates in ischemia-induced neuronal apoptosis. However, whether JNK signaling is involved in the process of neuronal apoptosis of diabetes-induced cerebral ischemia is largely unknown. This study was undertaken to evaluate the influence of cerebral ischemia–reperfusion injury on phosphorylation of JNK in diabetic rats. Twenty-four adult streptozotocin induced diabetic and 24 adult non-diabetic rats were randomly subjected to 15 min of forebrain ischemia followed by reperfusion for 0, 1, 3, and 6 h. Sixteen sham-operated diabetic and non-diabetic rats were used as controls. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). Protein expression of phospho-JNK was examined by immunohistochemistry and Western blot. The numbers of TUNEL-positive cells and phospho-JNK protein expression in the cerebral cortices after 1, 3 and 6 h reperfusion was significantly higher in diabetic rats compared to non-diabetic animals subjected to ischemia and reperfusion (p < 0.05). Western blot analysis showed significantly higher phospho-JNK protein expression in the cerebral cortices of the diabetic rats after 1 and 3 h reperfusion than that was presented in non-diabetic animals subjected to ischemia and reperfusion (p < 0.05). These findings suggest that increased phosphorylation of JNK may be associated with diabetes-enhanced ischemic brain damage.     

Tat-indoleamine 2,3-dioxygenase 1 elicits neuroprotective effects on ischemic injury

It is well known that oxidative stress participates in neuronal cell death caused production of reactive oxygen species (ROS). The increased ROS is a major contributor to the development of ischemic injury. Indoleamine 2,3-dioxygenase 1 (IDO-1) is involved in the kynurenine pathway in tryptophan metabolism and plays a role as an anti-oxidant. However, whether IDO-1 would inhibit hippocampal cell death is poorly known. Therefore, we explored the effects of cell permeable Tat-IDO-1 protein against oxidative stress-induced HT-22 cells and in a cerebral ischemia/reperfusion injury model. Transduced Tat-IDO-1 reduced cell death, ROS production, and DNA fragmentation and inhibited mitogen-activated protein kinases (MAPKs) activation in H₂O₂ exposed HT-22 cells. In the cerebral ischemia/reperfusion injury model, Tat-IDO-1 transduced into the brain and passing by means of the blood-brain barrier (BBB) significantly prevented hippocampal neuronal cell death. These results suggest that Tat-IDO-1 may present an alternative strategy to improve from the ischemic injury.     

Impact of Ginkgo Biloba Extract EGb 761 on ischemia/reperfusion - induced oxidative stress products formation in rat forebrain

Dysbalance in reactive oxygen/nitrogen species is involved in the pathogenesis of cerebral ischemia/reperfusion injury (IRI). Ginkgo biloba extract (Egb 761) pre-treatment was used to observe potential antioxidant/neuroprotective effect after global ischemia/reperfusion. Egb 761 significantly decreased the level of lipoperoxidation (LPO) in rat forebrain total membrane fraction (homogenate) induced by in vitro oxidative stress (Fe(2+)+H(2)O(2)). In animals subjected to four-vessel global ischemia for 15 min and 2-24 h reperfusion the EGb pretreatment slightly decreased LPO in forebrain homogenate. However, as detected in EGb treated group, the LPO-induced lysine conjugates are attenuated in comparison to non-treated IRI animals. EGb significantly improved parameters which indicate forebrain protein oxidative damage after IRI. The intensity of tryptophane fluorescence was increased by the 18.2% comparing to non-treated IRI group and bityrosine fluorescence was significantly decreased in ischemic (21%) and 24 h reperfused (15.9%) group in comparison non-treated IRI group. In addition, the level of total free SH- groups in pre-treated animals was significantly higher comparing to non-treated animals. Our results indicate that extract of EGb 761 has potent antioxidant activity and could play a role to attenuate the IRI-induced oxidative protein modification and lipoperoxidation in the neuroprotective process.     

Inductin of neuron-derived orphan receptor-1 in the dentate gyrus of the hippocampal formation following transient global ischemia in the rat

Neuron-derived orphan receptor (NOR-1) is a member of the thyroid/steroid receptor superfamily that was originally identified in forebrain neuronal cells undergoing apoptosis. In addition to apoptotic stimuli, activation of several signal transduction pathways including direct neuronal depolarization regulates the expression of NOR-1. In this study we tested whether the expression of NOR-1 is changed following transient ischemic injury in the adult rat brain. NOR-1 mRNA increased rapidly in the dentate gyrus of the hippocampal formation and piriform cortex 3 h after transient global ischemia and returned to basal level at 6 h. On the other hand, oxygen-glucose deprivation of cultured cerebral cortical neurons did not alter the expression of NOR-1. These results suggest that expression of NOR-1 is differentially regulated in different brain regions in response to globally applied brain ischemia, but that hypoxia is not sufficient to induce its expression.     

Bilobalide, a component of the Ginkgo biloba extract (EGb 761), protects against neuronal death in global brain ischemia and in glutamate-induced excitotoxicity.

In this study, the effect of bilobalide, a purified terpene lactone component of the Ginkgo biloba extract (EGb 761), and EGb 761 against ischemic injury and against glutamate-induced excitotoxic neuronal death was compared. In the case of ischemic injury, neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in the hippocampal regions of gerbils was measured. A significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons at 7-days of reperfusion after 5 min of transient global forebrain ischemia. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected hippocampal CA1 neurons against ischemia-induced neuronal death and reductions in COX III mRNA. In rat cerebellar neuronal cultures, addition of bilobalide or EGb 761 protected in a dose-dependent manner against glutamate-induced excitotoxic neuronal death [effective concentration (EC50) = 5 microg/ml (12 microM) forbilobalide and 100 microg/ml for EGb 761]. These results suggest thatboth EGb 761 and bilobalide protect against ischemia-induced neuronal death in vivo and glutamate-induced neuronal death in vitro by synergistic mechanisms involving anti-excitotoxicity, inhibition of free radical generation, scavenging of reactive oxygen species, and regulation of mitochondrial gene expression.     

Neuroprotective effects of PEP-1-carbonyl reductase 1 against oxidative-stress-induced ischemic neuronal cell damage

Human carbonyl reductase 1 (CBR1) is a member of the NADPH-dependent short-chain dehydrogenase/reductase superfamily that is known to play an important role in neuronal cell survival via its antioxidant function. Oxidative stress is one of the major causes of degenerative disorders including ischemia. However, the role CBR1 plays with regard to ischemic injury is as yet poorly understood. Protein transduction domains such as PEP-1 are well known and now commonly used to deliver therapeutic proteins into cells. In this study, we prepared PEP-1–CBR1 protein and examined whether it protects against oxidative-stress-induced neuronal cell damage. PEP-1–CBR1 protein was efficiently transduced into hippocampal neuronal HT-22 cells and protected against hydrogen peroxide (H₂O₂)-induced neuronal cell death. Transduced PEP-1–CBR1 protein drastically inhibited H₂O₂-induced reactive oxygen species production, the oxidation of intracellular macromolecules, and the activation of mitogen-activated protein kinases, as well as cellular apoptosis. Furthermore, we demonstrated that transduced PEP-1–CBR1 protein markedly protected against neuronal cell death in the CA1 region of the hippocampus resulting from ischemic injury in an animal model. In addition, PEP-1–CBR1 protein drastically reduced activation of glial cells and lipid peroxidation in an animal model. These results indicate that PEP-1–CBR1 protein significantly protects against oxidative-stress-induced neuronal cell death in vitro and in vivo. Therefore, we suggest that PEP-1–CBR1 protein may be a therapeutic agent for the treatment of ischemic injuries as well as oxidative-stress-induced cell damage and death.     

Autophagy regulates endoplasmic reticulum stress in ischemic preconditioning

Recent studies have suggested that autophagy plays a prosurvival role in ischemic preconditioning (IPC). This study was taken to assess the linkage between autophagy and endoplasmic reticulum (ER) stress during the process of IPC. The effects of IPC on ER stress and neuronal injury were determined by exposure of primary cultured murine cortical neurons to 30 min of OGD 24 h prior to a subsequent lethal OGD. The effects of IPC on ER stress and ischemic brain damage were evaluated in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The results showed that both IPC and lethal OGD increased the LC3-II expression and decreased p62 protein levels, but the extent of autophagy activation was varied. IPC treatment ameliorated OGD-induced cell damage in cultured cortical neurons, whereas 3-MA (5–20 mM) and bafilomycin A₁ (75–150 nM) suppressed the neuroprotection induced by IPC. 3-MA, at the dose blocking autophagy, significantly inhibited IPC-induced HSP70, HSP60 and GRP78 upregulation; meanwhile, it also aggregated the ER stress and increased activated caspase-12, caspase-3 and CHOP protein levels both in vitro and in vivo models. The ER stress inhibitor Sal (75 pmol) recovered IPC-induced neuroprotection in the presence of 3-MA. Rapamycin 50–200 nM in vitro and 35 pmol in vivo 24 h before the onset of lethal ischemia reduced ER stress and ischemia-induced neuronal damage. These results demonstrated that pre-activation of autophagy by ischemic preconditioning can boost endogenous defense mechanisms to upregulate molecular chaperones, and hence reduce excessive ER stress during fatal ischemia.     

Protective effects of L-pGlu-(2-propyl)-L-His-L-ProNH2, a newer thyrotropin releasing hormone analog in in vitro and in vivo models of cerebral ischemia

In the present study, the newly synthesized TRH analog (l-pGlu-(2-propyl)-l-His-l-ProNH₂; NP-647) was evaluated for its effects in in vitro (oxygen glucose deprivation (OGD)-, glutamate- and H₂O₂-induced injury in PC-12 cells) and in vivo (transient global ischemia) models of cerebral ischemic injury. PC-12 cells were subjected to oxygen and glucose deprivation for 6 h. Exposure of NP-647 was given before and during OGD. In glutamate and H₂O₂ induced injury, exposure of NP-647 was given 1, 6 and 24 h prior to exposure of glutamate and H₂O₂ exposure. NP-647, per se found to be non-toxic in 1-100 μM concentrations. NP-647 showed protection against OGD at the 1 and 10 μM. The concentration-dependent protection was observed in H₂O₂- and glutamate-induced cellular injury. In in vivo studies, NP-647 treatment showed protection of hippocampal (CA1) neuronal damage in transient global ischemia in mice and subsequent improvement in memory retention was observed using passive avoidance retention test. Moreover, administration of NP-647 resulted in decrease in inflammatory cytokines TNF-α and IL-6 as well as lipid peroxidation. These results suggest potential of NP-647 in the treatment of cerebral ischemia and its neuroprotective effect may be attributed to reduction of excitotoxicity, oxidative stress and inflammation.     

肢体缺血预处理减轻大鼠海马缺血/再灌注损伤

目的:探讨肢体缺血预处理(LIP)对大鼠全脑缺血/再灌注损伤的影响.方法: 36只大鼠椎动脉凝闭后随机分为假手术(Control)组、脑缺血组、肢体缺血组、LIP 0 d组(LIP后即刻行脑缺血)、LIP 1 d组(LIP后1 d行脑缺血)和LIP 2 d组(LIP后2 d行脑缺血).重复夹闭大鼠双侧股动脉3次(每次10 min,间隔10 min)作为LIP,夹闭颈总动脉进行全脑缺血8 min后再灌注.硫堇染色观察海马CA1区组织学分级及锥体神经元密度以判断海马损伤程度.结果:脑缺血组海马CA1区锥体神经元损伤严重,与Control组比较,组织学分级明显升高,神经元密度明显降低(P<0.01).LIP 0 d组海马CA1区神经元损伤较脑缺血组明显减轻,组织学分级明显降低,神经元密度明显升高(P<0.01).而LIP 1 d组和LIP 2 d组大鼠海马CA1区锥体细胞缺失较多,仍有明显的组织损伤.结论:LIP可减轻随后立即发生的脑缺血/再灌注损伤,但对间隔1 d后的脑缺血/再灌注损伤无显著对抗作用.     

Hyperbaric oxygen treatment: the influence on the hippocampal superoxide dismutase and Na+,K+-ATPase activities in global cerebral ischemia-exposed rats

The influence of hyperbaric oxygen (HBO) treatment on the activities of superoxide dismutase (SOD) and Na⁺,K⁺-ATPase was determined during different time periods of reperfusion in rats exposed to global cerebral ischemia. Ischemic animals were either sacrificed or exposed to the first HBO treatment 2, 24, 48 or 168 h after ischemic insult (for SOD activities measurement) or immediately, 0.5, 1, 2, 6, 24, 48, 72 or 168 h after ischemic procedure (for Na⁺,K⁺-ATPase activities measurement). Hyperbaric oxygenation procedure was repeated for seven consecutive days. The results of presented experiments demonstrated the statistically significant increase in the hippocampal SOD activity 24 and 48 h after global cerebral ischemia followed by a decrease in the enzymatic activity 168 h after ischemic insult. In the ischemic rats treated with HBO the level of hippocampal SOD activity was significantly higher after 168 h of reperfusion in comparison to the ischemic, non HBO-treated animals. In addition, it was found that global cerebral ischemia induced a statistically significant decrease of the hippocampal Na⁺,K⁺-ATPase activity starting from 1 to 168 h of reperfusion. Maximal enzymatic inhibition was obtained 24 h after the ischemic damage. Decline in Na⁺,K⁺-ATPase activity was prevented in the animals exposed to HBO treatment within the first 24 h of reperfusion. Our results suggest that global cerebral ischemia induces significant alterations in the hippocampal SOD and Na⁺,K⁺-ATPase activities during different periods of reperfusion. Enhanced SOD activity and preserved Na⁺,K⁺-ATPase activity within particular periods of reperfusion, could be indicators of a possible benefitial role of HBO treatment in severe brain ischemia.