Search

Ischemic preconditioning (IP) reduces brain damage after subsequent ischemic strokes by activating endogenous protective mechanisms in rodents. Transient ischemic attack (TIA) induces tolerance in the human brain after ischemic strokes; defining mechanisms of IP effects may provide therapeutic targets to improve recovery of patients with ischemic strokes. Iron transported across the blood-brain barrier (BBB) is required for brain functions, including myelination, and its levels should be finely regulated to avoid harmful effects. This study aimed to determine whether IP enhances repair processes by modulating iron metabolism during the post-stroke chronic phase. Male mice were divided into sham and IP groups, and IP was induced 24 h before a transient focal ischemic stroke. Sensorimotor recovery was observed over 8 weeks after the stroke, and brain volumes and levels of proteins related to repair processes and iron metabolism in the ischemic brains were examined 8 weeks after the stroke. There was significantly less ischemic brain atrophy in the IP group than in the sham group, with no differences in sensorimotor recovery between the groups. Levels of tight junction proteins of BBB, neurites outgrowth markers, and myelin sheath proteins and markers for mature oligodendrocytes were significantly increased in the IP group. Iron import proteins, transferrin receptor 1 and DMT1, were also increased in the IP group. These results indicate that IP increases brain repair processes and iron uptake during the chronic phase after an ischemic stroke, and provide new insights to understand the molecular mechanisms of TIA effects on post-stroke recovery., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ischemic stroke is a medical condition that arises because of poor blood supply to the brain. Reperfusion being salvage to the brain further causes, exacerbation of tissue injury, known as reperfusion injury. Ischemic preconditioning (IPC) has been known to provide benefits against ischemia reperfusion (I/R) injury. Dopamine D2/D3 receptor mediated several pathways are also reported as mediators in the IPC mediated neuroprotection. This study investigates the possible involvement of D2/D3 receptor activation in IPC mediated neuroprotection in the I/R brain. Global cerebral ischemia/reperfusion (GCI/R) injury in swiss albino mice was induced by occluding the common carotid arteries for 17 min, followed by 24 h reperfusion. IPC was provided by giving 3 episodes of I/R prior to Ischemia (17 min). Morris water maze (MWM) was used to assess the spatial learning, memory and Rota rod, lateral push test as well as inclined beam test were conducted to assess the motor functions in animals. Cerebral oxidative markers (thiobarbituric acid reactive species-TBARS, reduced glutathione-GSH, superoxide dismutase-SOD, and catalase-CAT), inflammatory markers (IL-6, IL-10, TNF-α, and myeloperoxidase-MPO), acetylcholinesterase activity-AChE, infarct size (% weight and % volume), and histopathological changes were also assessed. Haloperidol (0.05 mg/kg, i.p) was used to antagonize the effects of D2/D3 receptor activation. I/R animals showed reduction in memory, motor function, increase in cerebral oxidative stress, inflammation, AChE activity, infarct size and histopathological changes. Episodes of IPC prior to ischemia, attenuated the deleterious effects of I/R injury. Administration of haloperidol abolished the protective effects of IPC. Hence, it may be concluded that IPC mediated neuroprotection may involves dopamine D2/D3 receptor activation in mice., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Remote ischemic perconditioning (RIPerC) results in collateral enhancement and a reduction in middle cerebral artery occlusion (MCAO) induced ischemia. RIPerC likely activates multiple metabolic protective mechanisms, including effects on matrix metalloproteinases (MMPs) and protein kinases. Here we explore if RIPerC improves neuroprotection and collateral flow by modifying the activities of MMP-9 and AMPK/e-NOS. Age matched adult male Sprague Dawley rats were subjected to MCAO followed one hour later by RIPerC (3 cycles of 15 min ischemia). Animals were euthanized 24 h post-MCAO. Haematoxylin and Eosin (H&E) staining 24 h post-MCAO revealed a significant (p < 0.02) reduction in the infarction volume in RIPerC treated animals (24.9 ± 5.4%) relative to MCAO controls (42.5 ± 4.2, %). TUNEL staining showed a 42.6% reduction in the apoptotic cells with RIPerC treatment (p < 0.01). Immunoblotting in congruence with RT-PCR and Zymography showed that RIPerC significantly reduced MMP-9 expression and activity in RIPerC + MCAO group compared to MCAO group (218.3 ± 19.1% vs. 148.9 ± 12.05% (p < 0.01). Immunoblotting revealed that RIPerC was associated with a significant 2.5-fold increase in activation of p-AMPK compared to the MCAO group (p < 0.01) which was also associated with a significant increase in the e-NOS activity (p < 0.01). RIPerC resulted in reduction of infarction volume, decreased apoptotic cell death and attenuated MMP-9 activity. This together with the increased activity of p-AMPK and increase in p-eNOS may, in part explain the neuroprotection and sustained increase in blood flow observed with RIPerC following acute stroke., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: It has been shown that remote ischemic preconditioning (RIPreC) attenuates ischemic injury after stroke in healthy rats or mice. The present study aims to examine whether RIPreC offers neuroprotection against ischemic stroke in streptozotocin-induced diabetic mice., Methods: Streptozotocin (STZ, 120 mg/kg) was intraperitoneally injected into the mice to induce type 1 diabetic model. The immune and inflammatory changes were analyzed 2 days after reperfusion by flow cytometry and multiplex cytokine assay analysis, respectively., Results: We found that RIPreC reduced infarct sizes and alleviated neurological impairment in diabetic mice. RIPreC decreased CD8 T cells infiltrated into the brain, and attenuated the decreases of CD8 T cells in the blood, CD4 T cells and CD8 T cells in the spleen. Results from multiplex cytokine assay showed that RIPreC treatment decreased IL-6, IL-1 beta and TNF alpha levels in the cortex, while it inhibited IL-6 level in the hippocampus and striatum, and TNF alpha level in the hippocampus. RIPreC treatment also downregulated IL-6 and IFN gamma level in the blood, which increased after cerebral ischemic injury. In addition, RIPreC reduced pro-apoptotic protein BAX expression in the ischemic brain., Conclusions: Our results indicate that RIPreC attenuates cerebral injuries in streptozotocin-induced diabetic mice via anti-inflammatory response and anti-apoptosis in the ischemic brain., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objectives: This study was designed to investigate whether immunomodulation and Microglia polarization is involved in the anti-inflammatory and neuroprotective effect induced by hypoxic preconditioning (HPC) in the middle cerebral artery occlusion (MCAO) brain injury model., Methods: Longa method, (neurological disability status scale) NDSS method and TTC staining were used to evaluate the degree of cerebral infarction injury under different treatments (Sham, HPC, MCAO and co-treatment with HPC and MCAO). Western blot was used to detect expression profiles of apoptosis and related factors of neurological function. Flow cytometry was performed to analyze changes in the ratio of helper T cells, toxic T cells and NK cells in peripheral immune cells. And immunohistochemistry was used to examine the changes in microglial morphology. ELISA was used to evaluate the levels of nerve growth factors and neurogenesis conditions. Finally, RT-PCR was determined to analyze the transformation of microglia phenotype after HPC and MCAO treatment., Results: MCAO dramatically induced local formation of cerebral infarction. HPC relieved MCAO-induced cerebral infarction and increased rat cognition. HPC affected activation of microglia without significantly affecting in peripheral immune cell populations. After HPC co-treatment with MCAO, the M1 phenotype of microglia was changed and there was a transformation to M2., Conclusion: The treatment of HPC remarkably affected the polarization of microglia cells in MCAO rats, and reduced the cerebral nerve injury and played a protective role in MCAO model., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Ischemic preconditioning (IPC) exerts protective effects against ischemic cerebral injury. In the present study, an in vitro model of cerebral ischemia (oxygen and glucose deprivation, OGD) was established to investigate the neuroprotective mechanism of IPC. We found that conditioned medium (C.M.) from astrocytes rather than neurons nor microglia cell line BV2 exerted neuroprotection. Moreover, exosomes derived from OGD preconditioned astrocytes can be taken up by neurons and attenuated OGD-induced neuron death and apoptosis. High-throughput microRNA (miRNA) sequencing revealed that miR-92b-3p levels in exosomes released from preconditioned astrocytes were increased. Overexpression of miR-92b-3p in neurons with miR-92b-3p mimic achieved the same protective effects as C.M. from astrocytes. Thus, we propose that the mechanism of IPC may associate with astrocytes, and that exosome-mediated miR-92b-3p shuttle from preconditioned astrocytes to neurons participate in these process., (Copyright © 2019. Published by Elsevier B.V.)

Our previous studies have demonstrated that limb ischemic preconditioning (LIP) induced brain ischemic tolerance and up-regulated the expression of p38 MAPK and ERK in the hippocampal CA1 region in rats. The present study was undertaken to investigate the role of adenosine in brain protection and up-regulation of p38 MAPK and ERK induced by LIP. It was found that adenosine A1 receptor antagonist DPCPX dose-dependently inhibited the protective effect of LIP. The up-regulation of p38 MAPK and ERK induced by LIP could be blocked by DPCPX. Furthermore, we observed the effect of adenosine on the brain ischemia. The results showed that pre-administration of adenosine could partly mimic the neuroprotective effect on the brain, up-regulate the expression of p38 MAPK and ERK. Based on the above results, it can be concluded that adenosine participated in brain protection and up-regulation of the expression of p38 MAPK and ERK during the induction of brain ischemic tolerance after LIP., (Copyright © 2018. Published by Elsevier B.V.)

Background: The inflammatory reaction of the spleen is an important component in the pathophysiology of cerebral ischaemia (CI). Regulatory B cells (Bregs) derived from the spleen can inhibit the expansion of inflammation and reduce the damage caused by CI., Aim: The aim of the present study was to explore changes in spleen function and Bregs production due to lipopolysaccharide preconditioning (LPS PC) in ischaemia/reperfusion (I/R) and to uncover potential protective effect of LPS PC on stroke., Methods: Focal cerebral I/R mice were induced by middle cerebral artery occlusion (MCAO). Infarct size and inflammatory cell infiltration in brain tissue, athletic ability, and immune status were analysed by immunostaining, behavioural analyses, and flow cytometry, respectively., Results: The volume of the cerebral infarct was significantly decreased in I/R mice with LPS PC (LPS + I/R) compared to I/R mice, and neuronal apoptosis was ameliorated by LPS PC. After preconditioning with LPS, locomotor activity, forelimb strength, motor endurance, motor coordination, and short-term memory were improved to varying degrees. Moreover, blood-brain barrier (BBB) dysfunction was reversed, and CD11b + , major histocompatibility complex-II positive (MHC-II + ), and Gr-1 + cell infiltration in the brains of LPS + I/R mice was also significantly reduced. B cell-activating factor (BAFF), tumour necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-6 in the brain and spleen in the LPS + I/R group decreased to different degrees, while the levels of transforming growth factor-β (TGF-β) and IL-10 increased. LPS PC alleviated atrophy of the spleen following I/R. In addition, the number of CD8 + T cells, macrophages, TNF + cells, NF-κB + cells, and neutrophils in the spleen was reduced, while the number of proliferating cells and CD19 + -IL10 + Bregs was significantly increased. The number of follicular B (FO B) cells and marginal zone B (MZ B) cells in the spleens of LPS + I/R mice was also increased., Conclusions: I/R mice preconditioned with LPS showed significantly reduced pathological damage, motor dysfunction, cognitive dysfunction, and inflammatory responses. LPS PC may initiate anti-inflammatory protective mechanism in the spleen after stroke, may increase the number of anti-inflammatory cells, such as Bregs, in the spleen, and may play a protective role in stroke., (Copyright © 2018. Published by Elsevier B.V.)

In this study, we investigated the effects of remote ischemic preconditioning on post resuscitation cerebral function in a rat model of cardiac arrest and resuscitation. The animals were randomized into six groups: 1) sham operation, 2) lateral ventricle injection and sham operation, 3) cardiac arrest induced by ventricular fibrillation, 4) lateral ventricle injection and cardiac arrest, 5) remote ischemic preconditioning initiated 90min before induction of ventricular fibrillation, and 6) lateral ventricle injection and remote ischemic preconditioning before cardiac arrest. Reagent of Lateral ventricle injection is neuroglobin antisense oligodeoxynucleotides which initiated 24h before sham operation, cardiac arrest or remote ischemic preconditioning. Remote ischemic preconditioning was induced by four cycles of 5min of limb ischemia, followed by 5min of reperfusion. Ventricular fibrillation was induced by current and lasted for 6min. Defibrillation was attempted after 6min of cardiopulmonary resuscitation. The animals were then monitored for 2h and observed for an additionally maximum 70h. Post resuscitation cerebral function was evaluated by neurologic deficit score at 72h after return of spontaneous circulation. Results showed that remote ischemic preconditioning increased neurologic deficit scores. To investigate the neuroprotective effects of remote ischemic preconditioning, we observed neuronal injury at 48 and 72h after return of spontaneous circulation and found that remote ischemic preconditioning significantly decreased the occurrence of neuronal apoptosis and necrosis. To further comprehend mechanism of neuroprotection induced by remote ischemic preconditioning, we found expression of neuroglobin at 24h after return of spontaneous circulation was enhanced. Furthermore, administration of neuroglobin antisense oligodeoxynucleotides before induction of remote ischemic preconditioning showed that the level of neuroglobin was decreased then partly abrogated neuroprotection of remote ischemic preconditioning. These date suggested that neuroglobin involved in neuroprotective effect of remote ischemic preconditioning. In conclusion, remote ischemic preconditioning attenuated post resuscitation cerebral dysfunction and the neuroprotection was mediated partly by high level of neuroglobin in a rat model of cardiac arrest and resuscitation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Ischemic preconditioning with sublethal stress triggers defensive mechanisms against ischemic brain damage; however, such manipulations are potentially dangerous and, therefore, safe stimuli have been sought. Hyperoxia preconditioning by administration of hyperbaric (HBO) or normobaric oxygen (NBO) may have neuroprotective potential. The aim of this study was to determine whether preconditioning with HBO and air (HBA) applied at 2.5 absolute pressure (ATA) or NBO preconditioning induces ischemic tolerance in the brain of gerbils subjected to 3min transient cerebral ischemia. Neuronal cell survival, changes in brain temperature, the generation of factors involved in neurodegeneration and basic behavior in nest building were all tested. Hyperoxic preconditioning prevented ischemia-induced neuronal cell loss, reduced the number of TUNEL positive cells in the CA1 region of the hippocampus and improved the nest building process compared to untreated ischemic animals. Preconditioning also suppressed the production of reactive oxygen species and increased Bax expression normally observed after an ischemic episode. Only HBO preconditioning inhibited ischemia-evoked increases in brain temperature. Our results show that hyperoxic preconditioning results in induction of ischemic tolerance and prevents ischemia-induced neuronal damage in the gerbil brain. Pressurized air preconditioning was as effective as HBO or NBO preconditioning in providing neuroprotection. The observed neuroprotection probably results from mild oxidative stress evoked by increased brain tissue oxidation and activation of antioxidant and antiapoptotic defenses., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The purpose of the present study was to determine whether a unilateral photothrombotic brain lesion induces bilateral ischemic tolerance towards a subsequent severe ischemia performed 5 days later. Severe ischemia was induced by transient (1h; t) or permanent (p) occlusion of the middle cerebral artery (MCAO). Rats were sacrificed 24h later. Preconditioning reduced the size of subsequent infarcts in both hemispheres. This effect was most prominent with tMCAO, and ipsilateral preconditioning was more effective than contralateral preconditioning (% of hemispheric volume, mean ± SD: 31.9 ± 3.7 to 19.0 ± 10.3 with ipsilateral tMCAO; 31.9 ± 3.7 to 22.9 ± 4.9 with contralateral tMCAO; 64.7 ± 4.3% to 47.2 ± 12.5% with ipsilateral pMCAO; 64.7 ± 4.3% to 53.1 ± 8.9% with contralateral pMCAO). Ischemic preconditioning was associated with a successive bilateral up-regulation of superoxide dismutases which may be involved in the development of ischemic tolerance. Our data suggest that a focal ischemic brain lesion induces neuroprotective mechanisms in extensive brain areas and thus cause bilateral ischemic tolerance within a certain time window., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Hypoxic preconditioning (HPC) initiates intracellular signaling pathway to provide protection, but the role of p38 mitogen-activated protein kinase (p38 MAPK) in HPC-induced neuroprotection against cerebral ischemic injuries is a matter of debate. In this study, we found that HPC could reduce 6h middle cerebral artery occlusion (MCAO)-induced infarct volume, edema ratio and cell apoptosis, as well as enhancing the up-regulated p38 MAPK phosphorylation (P-p38 MAPK) levels in the peri-infarct region of mice after 6h MCAO. However, intracerebroventricular injection of p38 MAPK inhibitor SB203580 abolished this HPC-induced neuroprotection. HPC significantly increased the translocation of anti-apoptotic Bcl-2-related protein Bcl-xL from the cytosol to the mitochondria in the peri-infarct region of MCAO mice. Interestingly, the results of reciprocal immunoprecipitation showed that Bcl-xL and P-p38 MAPK were coimmunoprecipitated reciprocally only in the peri-infarct region of HPC and MCAO treated mice, while Bcl-xL and total p38 (T-p38 MAPK), not P-p38 MAPK, could be coimmunoprecipited by each other in the brain of normal control mice. In addition, we found SB203580 significantly decreased P-p38 MAPK levels, and inhibited HPC-induced mitochondria translocation of Bcl-xL in the brain of HPC and MCAO treated mice. Taken together, our findings suggested that P-p38 MAPK mediates HPC-induced neuroprotection against cerebral ischemic injury via mitochondria translocation of Bcl-xL, which might be a key anti-cell apoptotic mechanism of HPC., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Purposes: To investigate whether activation of adenosine A1 receptor (A1R) through limb remote ischemic preconditioning (RIPC) by a noninvasive tourniquet contribute neuroprotective effects against rat focal cerebral ischemic injury induced by transient middle cerebral artery occlusion (MCAO)., Methods: One hundred twenty-eight Sprague-Dawley (SD) rats were randomly assigned into eight groups (n=16 each): MCAO, Control, 8-cyclopentyl-1,3-dipropulxanthine (DPCPX, Adenosine A1 receptor antagonist), RIPC, DPCPX+RIPC, Vehicle+RIPC, 2-chloro-N(6)-cyclopentyladenosine (CCPA, Adenosine A1 receptor agonist) and CCPA+DPCPX groups. All animals underwent right middle cerebral artery occlusion (MCAO) for 2 h. Limb RIPC consisted of three cycles of 5-minute ischemia followed by 5-minute reperfusion in right hind-limb by tourniquet application. Neurological deficit scores were evaluated 24 h after reperfusion, and then the infarct volume was assessed with diffusion weighted imaging (DWI) and 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Inflammation was assessed by serum tumor necrosis factor α (TNF(α)) and NO; oxidative stress was estimated by malondialdehyde (MDA) and 4-hydroxyalkenals (4-HAD), superoxide dismutase (SOD) activity and GSH., Results: Animals in the RIPC, Vehicle+RIPC and CCPA groups developed lower neurological deficit scores and smaller brain infarct volumes than the Control group (P<0.01). Animals in the DPCPX, DPCPX+RIPC and CCPA+DPCPX groups developed higher neurological deficit scores and larger brain infarct volumes than the RIPC, Vehicle+RIPC and CCPA groups (P<0.01). DPCPX abolished the protective effects of RIPC and CCPA. RIPC or CCPA induced a significant increase in brain MnSOD (manganese SOD) activity and NO generation, and this activity was abolished by DPCPX pretreatment. RIPC or CCPA induced a significant reduction (P<0.05) in the GSH and MDA+4HDA concentration and an accumulation in the GSSG concentration in both compartments (serum and tissue) as compared with the MCAO group., Conclusions: The present study demonstrates that limb RIPC induced by noninvasive tourniquet reduces cerebral ischemic injury in rats, and the effect of neuroprotection may depend on the activation of adenosine A1 receptors. CCPA pretreatment can induce delayed ischemic tolerance against cerebral ischemia/reperfusion injury. These protective effects are associated with a reduction in oxidative stress, inflammation and endogenous antioxidant preservation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Ischemic stroke is one of the leading causes of mortality and disability worldwide. Our previous studies have shown that hyperbaric oxygen (HBO) preconditioning can afford significant neuroprotection against cerebral ischemia-reperfusion (I/R) injury in rats. However, it is still unknown whether HBO preconditioning can directly protect primary cultured cortical neurons against oxygen-glucose deprivation (OGD). Peroxisome proliferator-activated receptor-gamma (PPAR γ) plays a central role in the regulation of apoptosis, oxidative stress and inflammation as well as affords significant neuroprotection against cerebral I/R injury. 15-deoxy-∆(12,14)-prostaglandin J(2) (15d-PGJ(2)) is an endogenous ligand with a high affinity for PPAR γ. Recently, some studies demonstrate that activation of PPAR γ mediates lipopolysaccharide and anesthetic preconditioning. In the present study, we firstly found that OGD exposure caused the significant damage of cultured cortical neurons evaluated by cell viability, lactate dehydrogenase (LDH) release and caspase-3 activity, which were significantly ameliorated by HBO preconditioning. Furthermore, HBO preconditioning significantly increased the levels of PPAR γ mRNA and protein, PPAR γ DNA binding activity, 15d-PGJ(2) and antioxidant enzymatic activities in primary cultured cortical neurons with OGD exposure. Moreover, PPAR γ antagonist GW9662 dose-dependently abolished the protection of HBO preconditioning in OGD-exposed neurons. GW9662 blocked the increase of PPAR γ DNA binding activity and antioxidant enzymatic activities, but did not influence the 15d-PGJ(2) level in OGD-exposed neurons with HBO preconditioning. However, the cyclooxygenase (COX)-2 inhibitor NS-398 blocked the production of 15d-PGJ(2) in OGD-exposed neurons with HBO preconditioning. In addition, 15d-PGJ(2) preconditioning could also protect cultured neurons against OGD injury. These results demonstrate that HBO preconditioning has directly beneficial effects on ODG-exposed cortical neurons by the activation of PPAR γ subsequent to the production of 15d-PGJ(2), which in turn increases the downstream antioxidant enzymatic activities., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

[d-Ala2, d-Leu5] enkephalin (DADLE) is a synthetic δ-opioid agonist that induces hibernation and promotes survival of neurons and glial cells in the central nervous system. Several mechanisms for the attenuation of hypoxic injury have been suggested, including control of intracellular signaling pathways via the δ-opioid receptor (DOR). However, the cellular and molecular mechanisms of DADLE in hypoxic injury are largely unknown. To investigate neuronal injury after oxygen-deprivation (OD) and DOR stimulation by DADLE, we used a lactate dehydrogenase assay, MTT assay, and immunofluorescence live/dead staining. And we used cDNA microarrays to investigate the influence of DADLE exposure on transcription after OD in rat cortical glial and neuronal co-culture. DADLE reduced neuronal injury after 24 h of OD. Preconditioning with DADLE before 24 h hypoxia exposure also altered gene expression in comparison with 24h OD without pretreatment. After DADLE exposure and hypoxia, 1917 of 39,511 genes (4.9%) were significantly induced or repressed at least 2.5-fold. Assigning differentially expressed ESTs (expressed sequence tags) to molecular functional groups revealed that DADLE affected many pathways including apoptosis, intracellular ion homeostasis, molecular chaperones, and glucose metabolism. We observed a coordinated change in expression of many genes (increased expression of potentially protective genes and decreased expression of potentially harmful genes) after DADLE exposure. A comprehensive list of regulated genes should prove valuable in advancing our understanding of the neuroprotective mechanisms of DADLE under OD., (Copyright © 2012 Elsevier B.V. All rights reserved.)

MicroRNAs (miR) are single-stranded short RNA molecules that regulate gene expression by degradation or translational repression of mRNA. It has been reported that the downregulation of miR-199a plays an important role in cardiac ischemic tolerance. We examined the expression of miR-199a after 3-nitropropionic acid (3-NPA) preconditioning in rat brain. 3-NPA (20mg/kg), an irreversible inhibitor of succinate dehydrogenase, was injected intraperitoneally to induce ischemic tolerance in rats. For comparison, the control group received intraperitoneal injections of vehicle (0.9% sodium chloride). Quantitative RT-PCR assay was applied to detect the expression of miR-199a. Luciferase reporter assays and Western blotting were used to verify the target genes of miR-199a. In cortex and striatum, miR-199a was downregulated at two separate time intervals (the 2nd and 4th day), while in the hippocampus, it was downregulated on the 2nd day after 3-NPA preconditioning. The maximum reduction of miR-199a was 66.3% in striatum (4th day), 54.9% in hippocampus (2nd day), and 27.6% in cortex (2nd day). The level of sirt1 protein, a putative target of miR-199a and a known mediator of neuroprotective effect in brain ischemic tolerance, decreased significantly in hippocampal neurons by overexpression of miR-199a, while it increased with knockdown of miR-199a. Taking these results together, we hypothesize miR-199a may have a role in the formation of cerebral ischemic tolerance., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The signal transducers and activators of transcription 3 (STAT3) has been suggested to have neuroprotective roles. However, its role in ischemic preconditioning (PC) is still obscure. In this study, we examined the phosphorylation status of ser727-STAT3, which is necessary for activation of STAT3, and its roles in a rat global ischemia model with or without PC. PC was induced by 3 min of nonlethal ischemia 48 h before 5 min of lethal ischemia. Western blot analysis showed that phospho-ser727-STAT3 significantly increased from 8 to 48 h after nonlethal ischemia, while it increased only for 1h after lethal ischemia and returned to the baseline within 24h. In the preconditioned brains, phospho-ser727-STAT3 was induced at 1 to 4h after lethal ischemia, and decrease of its levels delayed compared to the nonconditioned brains. Immunohistochemistry revealed that phospho-ser727-STAT3 was expressed mainly in CA1 neurons after nonlethal ischemia. Additionally, STAT3 inhibitor peptide treatment prevented PC induced-neuroprotection. These results indicate that phosphorylation of ser727-STAT3 plays an important role in PC induced- neuroptotection., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Our previous studies have demonstrated that hyperbaric oxygen (HBO) preconditioning induces tolerance to focal cerebral ischemia. The present study aimed to investigate whether autophagy is involved in the neuroprotection elicited by HBO preconditioning in a rat model of transient focal cerebral ischemia. Twenty-four hours after the completion of HBO preconditioning (2.5 atm absolute in 100% oxygen for 60 min per day for 5 consecutive days), male Sprague-Dawley rats were subjected to focal cerebral ischemia by middle cerebral artery occlusion (MCAO) for 120 min. The neurobehavioral score and infarct volume were used to evaluate cerebral ischemic injury. An intracerebroventricular injection of the autophagy inhibitor 3-methyladenine (3-MA) or the autophagy inducer rapamycin was administered before HBO preconditioning or MCAO. We found that after reperfusion the protein expression of LC3-II and Beclin 1 and the formation of autophagosomes were increased by HBO preconditioning or ischemia, but the increase following HBO preconditioning was higher than the increase following ischemia. 3-MA suppressed the increases in LC3-II and Beclin 1 induced by HBO preconditioning and attenuated the neuroprotection of HBO preconditioning against cerebral ischemia. Furthermore, 3-MA treatment before MCAO aggravated subsequent cerebral ischemic injury. In contrast, pretreatment with rapamycin up-regulated LC3-II and Beclin 1 after reperfusion and mimicked the neuroprotective effect of HBO preconditioning. These results indicate that HBO preconditioning elevates autophagic activity, which elicits a neuroprotective effect against ischemic injury in the brain, and suggest a novel mechanism of HBO preconditioning-induced tolerance against transient focal cerebral ischemia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Attenuation of ischemic injury can be achieved by priming the brain with a sub-lethal ischemic insult, a phenomenon known as ischemic preconditioning (IP). We sought to determine if subjecting a distant organ, such as the lower limb, to a similar priming ischemic insult would result in protection of the brain from a subsequent severe ischemic injury, as induced by middle cerebral artery occlusion (MCAo) and if this protection is mediated via a neurogenic pathway. Adult Wistar male rats were subjected to either remote preconditioning (RPC) or sham surgery and then subsequently underwent 2 h MCAo 24, 48 or 72 h after the RPC/sham RPC stimulus. Of the animals undergoing RPC, only those that sustained MCAo 24 h later showed significantly smaller cerebral infarct volumes (150.34±30.91 mm(3)) and better clinical neurological outcomes (1.15±0.69) as compared to the sham RPC group (infarct volume 250.25±26.98mm(3); neurological score 1.80±0.87) (P<0.05). RPC animals sustaining MCAo at 48 and 72 h later did not show significant differences in cerebral infarct volumes or clinical neurological outcomes as compared to the sham RPC group. Furthermore, attenuation of the neuroprotective effect by the ganglion blocker hexamethonium suggested a neurogenically mediated pathway responsible for this phenomenon. Remote sub-lethal ischemic injury to both lower limbs results in cerebral protection from subsequent ischemia within 24 h of initiation of the RPC stimulus and this protection in part may be mediated via a neurogenic pathway., (Copyright © 2011 Elsevier B.V. All rights reserved.)

It is well known that a brief anoxia or hypoxia episodes can render brain resistant to a subsequent ischemia. Recent investigations indicate that mechanisms of such stimulated endogenous neuroprotection are related to the family of hypoxia-inducible factors (HIF), however there are still little data available on the role of HIF family members in hippocampus-a brain structure, highly sensitive to oxygen deficiency. We have used the model of cultured hippocampal slices and single-cell quantitative RT-PCR to study HIF-1α and HIF-3α mRNA expression following triple 5-min mild anoxia, 30-min oxygen-glucose deprivation and their combination. We also tested the effects of HIF prolyl-hydroxylase inhibition with 2,4-pyridinedicarboxylic acid diethyl ester pre-treatment followed by a 30-min oxygen-glucose deprivation. It was found that neuronal damage induced by oxygen-glucose deprivation was accompanied by a significant decrease in both HIF-1α and HIF-3α mRNA levels in CA1 but not CA3 neurons. Anoxia preconditioning did not affect cell viability and HIF mRNA levels but applied before oxygen-glucose deprivation prevented neuronal damage and suppression of HIF-1α and HIF-3α mRNA expression. It was also found that effects of the prolyl-hydroxylase inhibitor were similar to anoxia preconditioning. These results suggest that anoxia preconditioning increases anti-ischemic neuronal resistance which to a certain extent correlates with the changes of HIF-1α and HIF-3α expression., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Recent studies show helium may be one kind of neuroprotective gas. This study aimed to examine the short and long-term neuroprotective effects of helium preconditioning in an established neonatal cerebral hypoxia-ischemia (HI) model. Seven-day-old rat pups were subjected to left common carotid artery ligation and then 90 min of hypoxia (8% oxygen at 37°C). The preconditioning group inhaled 70% helium-30% oxygen for 5 min three times with an interval of 5 min 24h before HI insult. Pups were decapitated 24h after HI and brain morphological injury was assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining, Nissl and TUNEL staining. Caspase-3 activity in the brain was measured. Five weeks after HI, postural reflex testing and Morris water maze testing were conducted. Our results showed that helium preconditioning reduced the infarct ratio, increased the number of survival neurons, and inhibited apoptosis at the early stage of HI insult. Furthermore, the sensorimotor function and the cognitive function were improved significantly in rats with helium preconditioning. The results indicate that helium preconditioning attenuates HI induced brain injury., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Neurons preconditioned with non-injurious hypoxia or the anesthetic isoflurane express different genes but are equally protected against severe hypoxia/ischemia. We hypothesized that neuroprotection would be augmented when preconditioning with isoflurane and hypoxic preconditioning are combined. We also tested if preconditioning requires intracellular Ca(2+) and the inositol triphosphate receptor, and if gene expression is similar in single agent and combined preconditioning. Hippocampal slice cultures prepared from 9 day old rats were preconditioned with hypoxia (95% N(2), 5% CO(2) for 15 min, HPC), 1% isoflurane for 15 min (APC) or their combination (CPC) for 15 min. A day later cultures were deprived of O(2) and glucose (OGD) to produce neuronal injury. Cell death was assessed 48 h after OGD. mRNA encoding 119 signal transduction genes was quantified with cDNA micro arrays. Intracellular Ca(2+) in CA1 region was measured with fura-2 during preconditioning. The cell-permeable Ca(2+) buffer BAPTA-AM, the IP(3) receptor antagonist Xestospongin C and RNA silencing were used to investigate preconditioning mechanisms. CPC decreased CA1, CA3 and dentate region death by 64-86% following OGD, more than HPC or APC alone (P<0.01). Gene expression following CPC was an amalgam of gene expression in HPC and APC, with simultaneous increases in growth/development and survival/apoptosis regulation genes. Intracellular Ca(2+) chelation and RNA silencing of IP(3) receptors prevented preconditioning neuroprotection and gene responses. We conclude that combined isoflurane-hypoxia preconditioning augments neuroprotection compared to single agents in immature rat hippocampal slice cultures. The mechanism involves genes for growth, development, apoptosis regulation and cell survival as well as IP(3) receptors and intracellular Ca(2+)., ((c) 2010 Elsevier B.V. All rights reserved.)

Cerebral ischemia-reperfusion injury (IRI) is a complex process resulting in cellular damage and death. Many studies have reported that an ischemic preconditioning could induce protection against ischemic insult. However, the safety concerns and practical feasibility have limited the application of ischemia preconditioning in practice. Subsequently, a number of substances including endotoxin and cytokines etc. have proven effective in inducing ischemic tolerance in the neurocyte. Unfortunately, the application of these substances to the clinical practice of neurosurgery still remains questionable for their toxicity or side effects. Therefore, a novel therapy to protect against cerebral IRI requires further study. Several recent studies confirmed that repeated hyperbaric oxygen preconditioning (HBO-PC) prior to cerebral ischemia or spinal cord ischemia can provide neuroprotection. HBO as a therapeutic measure has been widely accepted for its convenience and safety. However, information about the mechanism of how this neuroprotection works is still very limited. We hypothesize that autophagy induction is involved in HBO-PC induced neuroprotection on IRI in neurocyte. The hypothesis reveals that autophagy may be a new therapeutic target for cerebral IRI., (Copyright 2010 Elsevier B.V. All rights reserved.)

The present study has been designed to expound the significance of cyclic adenosine diphosphoribose receptor activation in ischemic preconditioning induced reversal of ischemia and reperfusion induced cerebral injury in mice. Bilateral carotid artery occlusion of 17 min followed by reperfusion for 24 h was employed in present study to produce ischemia and reperfusion induced cerebral injury in mice. Cerebral infarct size was measured using triphenyltetrazolium chloride staining. Memory was evaluated using Morris water-maze test. Rota-rod test was employed to assess motor incoordination. Bilateral carotid artery occlusion followed by reperfusion produced cerebral infarction and impaired memory and motor co-ordination. Three preceding episodes of bilateral carotid artery occlusion for 1 min and reperfusion of 1 min (ischemic preconditioning) prevented markedly ischemia-reperfusion-induced cerebral injury measured in terms of infarct size, loss of memory and motor coordination. 8-Bromo-cyclic adenosine diphosphate ribose (2 mg/kg, ip), an antagonist of cyclic ADP-ribose receptor, attenuated the neuroprotective effect of ischemic preconditioning. It is concluded that neuroprotective effect of ischemic preconditioning may be due to the adenosine diphosphoribose receptor activation.

Several recent studies suggest that sumo-2/3 modification of proteins occurs following harmful ischemia, however, sumo-2/3-ylation may also be associated with hibernation-mediated neuroprotection. Here we investigate the sumoylation of proteins following ischemia and ischemic tolerance using our established in vitro model of ischemia (oxygen and glucose deprivation; OGD). Following harmful ischemia (120 min OGD), we observed a significant increase in the sumo-2/3-ylation of high molecular weight proteins (>85 kDa), but not sumo-1-ylation of proteins. Sumo-2/3-ylation following 120 min OGD was reduced when cultures were preconditioned with non-harmful 30 min OGD 24 h earlier (delayed ischemic tolerance). However, we observed no change in sumo-2/3-ylation in a model of rapid ischemic tolerance. The effects of preconditioning on sumo-2/3-ylation following harmful ischemia were blocked by the protein synthesis inhibitor cycloheximide (1.0 muM), a known inhibitor of delayed ischemic tolerance. In addition, we observed a reduction in sumo-2/3-ylation using hypothermia (4 degrees C 30 min) as the preconditioning stimuli to induce delayed ischemic tolerance. Further studies show that sumo-2/3-ylation occurs during the ischemic insult and that preconditioning does not change expression of the sumo E1- and E2-ligases (UBA2 and Ubc9) or the sumo specific isopeptidases (SenP1-3). While sumo-2/3-ylation is enhanced under conditions of cell stress, it is not yet clear whether this is a cause or consequence of harmful ischemia-induced cell damage.

Brain ischemia can usually disrupt the blood-brain barrier (BBB), increasing the permeability of BBB. Ischemic preconditioning could not only reduce neurons damage, but also protect the functions of BBB during brain ischemia. In this study, we used BBB model in vitro to examine the effects of preconditioning on cell viability, BBB permeability, tight junction and cell adhesion of rat brain microvascular endothelial cells (BMECs). The rat BMECs were exposed to 2.5 h of oxygen glucose deprivation (OGD) following 3 h of reoxygenation to simulate ischemia/reperfusion in vivo, using 0.5 h OGD as preconditioning. Results showed that OGD/reoxygenation induced cell death, increased BBB permeability, changed the distribution of endothelial cells tight junction protein ZO-1 and cellular framework protein F-actin, and increased the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). OGD preconditioning could partially protect cell viability and BBB permeability, maintain the membrane location of ZO-1 and F-actin, and decrease the expression of ICAM-1 and VCAM-1. Our study suggests that OGD preconditioning could stabilize the tight junction protein and alleviate cell adhesion to protect BBB function during ischemic stress.

The purpose of the present study was to investigate the role of myocyte enhancer binding factor 2C (MEF2C), a common substrate of p38 kinase and extracellular signal-regulated kinase 5 (ERK5) in the hippocampal CA1 region following cerebral ischemia preconditioning (CIP) and without CIP. In animals that did not undergo preconditioning, MEF2C was significantly activated with an early peak at 30 min of reperfusion, which was followed by a pronounced decrease of MEF2C protein levels in the late phase of reperfusion (3-5 d). Co-immunoprecipitation studies failed to show an interaction between ERK5 and MEF2C, and ERK5-antisense oligonucleotide (ERK5-AS) had no effect on MEF2C activation, suggesting that the MEF2C activation is mediated by a kinase other than ERK5. Following preconditioning (3 min ischemia), MEF2C was strongly activated during the late stage of reperfusion (6 h-5 d). Co-immunoprecipitation studies showed that the interaction of ERK5 and MEF2C significantly increased at 3 d of reperfusion, and this increase was markedly inhibited by ERK5-AS. Inhibition of the ERK5-MEF2C pathway resulted in a significant increase in the number of TUNEL-positive apoptotic cells compared with CIP groups in the hippocampal CA1 region, and abolished the neuroprotective effect induced by CIP. Taken together, these results demonstrate that ERK5-MEF2C signaling is significantly enhanced in the hippocampus CA1 following CIP, and that ERK5-MEF2C signaling plays a critical role in the mediation of the anti-apoptotic and neuroprotective actions of ischemic preconditioning.

Mild NMDA receptor activation is correlated with neuroprotection in models of cerebral ischemia. Neuroprotection with NMDA manifests as a form of ischemic tolerance and involves the induction of cellular stress systems sensitive to disturbances in cellular calcium homeostasis. Unilateral micro-injection of 10, 160 and 320 microM NMDA into the prefrontal cortex of a rat 30 min prior to permanent occlusion of the middle cerebral artery (MCAO) significantly reduced the area of infarct observed after 4 h of ischemia. The highest dose of NMDA (320 microM) prevented the propagation of ischemic damage through a direct toxicity on neuronal tissue adjacent to the injection site as demonstrated in thionin-stained sections. As a result, the degree of ischemia-induced damage was similar to that measured in rats pretreated with the low dose of NMDA (10 microM). Expression of heat shock protein (HSP) 70 and glucose-regulated protein (GRP) 94 in cortical samples taken from the region of infarct following MCAO was significantly reduced in rats pretreated with 10 microM NMDA compared to saline-injected control rats and rats pretreated with higher doses of NMDA. Furthermore, 10 microM NMDA did not appear to influence expression of m-calpain or GRP78, however, higher doses of NMDA did significantly induce expression of both proteins as assessed by Western blotting. In summary, our data demonstrate an in vivo rodent model of ischemic tolerance in which 30 min of neuronal preconditioning with 10 microM NMDA confers protection against a 4 h period of MCAO-induced ischemia. This effect may involve modulation of cellular stress signals, in particular HSP70 and GRP94.

Reactive oxygen species (ROS) and the mitochondrial ATP-dependent potassium channel (mitoK(+)(-)(ATP)) play a major role in myocardial preconditioning. The same pathways seem to be involved in cerebral preconditioning. The aim of this study was to evaluate ROS involvement during the initial phase of delayed preconditioning and its relationship with mitoK(+)(-ATP) opening in a rat model of cerebral ischemia-reperfusion. Ischemia was induced by a 1-h occlusion of middle cerebral artery followed by a 24-h reperfusion period. A delayed preconditioning was induced by a 3-min ischemia (IPC), an in situ infusion of hydrogen peroxide (H(2)O(2)), or an administration of mitoK(+)(-ATP) agonist diazoxide, 72 h before the ischemia-reperfusion (I/R). IPC was performed in the presence or not of N-acetyl-cysteine (NAC) or 5-hydroxydecanoate (5-HD). A neuroprotection was induced by IPC and administration of H(2)O(2) or diazoxide. The decrease in infarct size was respectively 24.5%, 45.7% and 24.6%. IPC was abolished by 5-HD and NAC, indicating that mitoK(+)(-ATP) and ROS are involved. The protection induced by H(2)O(2) was blocked by 5-HD and diazoxide triggering was abolished by NAC. This strong relationship between ROS and mitoK(+)(-ATP) needs to be clarified as ROS might be involved both upstream and downstream of mitoK(+)(-ATP) opening.

In ischemic preconditioning, a sublethal ischemic insult protects neurons from subsequent ischemia. In organotypic hippocampal slice cultures a sublethal 5-minute hypoxia-hypoglycemia treatment prevented neuronal loss after a 10-minute experimental ischemic (EI) treatment of hypoxia-hypoglycemia. Whereas preconditioning protected against EI given 24 h later, it did not protect when EI was given 2 h later, suggesting a slow development of neuroprotection. This model identified two regulators of ischemic preconditioning: the atypical protein kinase M zeta (PKMzeta), and the Na/K ATPase. Two hours following preconditioning, when there was no neuroprotection, Na/K ATPase activity was unchanged. In contrast, Na/K ATPase activity significantly increased 24 h after the preconditioning treatment. Elevated Na/K ATPase activity was accompanied by increased surface expression of the alpha1 and alpha2 isoforms of the Na/K ATPase. Similarly, active PKMzeta levels were increased at 24 h, but not 2 h, after preconditioning. PKMzeta overexpression by sindbis virus vectors also increased Na/K ATPase activity. To examine PKMzeta regulation of Na/K ATPase, occlusion experiments were performed using marinobufagenin to inhibit alpha1, dihydroouabain to inhibit alpha2/3 and a zeta-pseudosubstrate peptide to inhibit PKMzeta. These experiments showed that PKMzeta regulated both the activity and surface expression of the alpha1 isoform of the Na/K ATPase. Marinobufagenin, dihydroouabain, and zeta-pseudosubstrate peptide were used to determine if PKMzeta or the alpha1 and alpha2 Na/K ATPase isoforms protected neurons. All three compounds blocked neuroprotection following ischemic preconditioning. PKMzeta levels were elevated 3 days after ischemic preconditioning. These data indicate key roles of PKMzeta and Na/K ATPase in ischemic preconditioning.

Hypoxic ischemic (HI) injury in neonates damages brain tissues. We examined the mechanism of hyperbaric oxygen preconditioning (HBO-PC) in neonatal HI rat model. Seven-day-old rat pups were subjected to left common carotid artery ligation and hypoxia (8% oxygen at 37 degrees C) for 90 min. HBO (100% O(2), 2.5 atmospheres absolute for 2.5 h) were administered by placing pups in a chamber 24 h before HI insult. Brain injury was assessed by the survival rate, 2,3,5-triphenyltetrazolium chloride (TTC), Nissl, TUNEL straining and caspase-3,caspase-9 activities after HI. In HBO preconditioned animals, survival rate was increased, infarct ratio was decreased, and the positive stained TUNEL cells were reduced, accompanied by the suppression of caspase-3 and -9 activities. These results indicate that a single HBO-PC appears to provide brain protection against HI insult via inhibition of neuronal apoptosis pathways.

Neuroprotection against cerebral ischemia conferred by ischemic preconditioning (IPC) requires translocation of epsilon protein kinase C (epsilonPKC). A major goal in our laboratory is to define the cellular targets by which epsilonPKC confers protection. We tested the hypothesis that epsilonPKC targets the mitochondrial K(+)(ATP) channel (mtK(+)(ATP)) after IPC. Our results demonstrated a rapid translocation of epsilonPKC to rat hippocampal mitochondria after IPC. Because in other tissues epsilonPKC targets mtK(+)(ATP) channels, but its presence in brain mitochondria is controversial, we determined the presence of the K(+)(ATP) channel-specific subunits (Kir6.1 and Kir6.2) in mitochondria isolated from rat hippocampus. Next, we determined whether mtK(+)(ATP) channels play a role in the IPC induction. In hippocampal organotypic slice cultures, IPC and lethal ischemia were induced by oxygen-glucose deprivation. Subsequent cell death in the CA1 region was quantified using propidium iodide staining. Treatment with the K(+)(ATP) channel openers diazoxide or pinacidil 48 h prior to lethal ischemia protected hippocampal CA1 neurons, mimicking the induction of neuroprotection conferred by either IPC or epsilonPKC agonist-induced preconditioning. Blockade of mtK(+)(ATP) channels using 5-hydroxydecanoic acid abolished the neuroprotection due to either IPC or epsilonPKC preconditioning. Both ischemic and epsilonPKC agonist-mediated preconditioning resulted in phosphorylation of the mtK(+)(ATP) channel subunit Kir6.2. After IPC, selective inhibition of epsilonPKC activation prevented Kir6.2 phosphorylation, consistent with Kir6.2 as a phosphorylation target of epsilonPKC or its downstream effectors. Our results support the hypothesis that the brain mtK(+)(ATP) channel is an important target of IPC and the signal transduction pathways initiated by epsilonPKC.

We have studied ischemic tolerance induced by the serine protease thrombin in two different models of experimental ischemia. In organotypic hippocampal slice cultures, we demonstrate that incubation with low doses of thrombin protects neurons against a subsequent severe oxygen and glucose deprivation. L-JNKI1, a highly specific c-jun N-terminal kinase (JNK) inhibitor, and a second specific JNK inhibitor, SP600125, prevented thrombin preconditioning (TPC). We also show that the exposure to thrombin increases the level of phosphorylated c-jun, the major substrate of JNK. TPC, in vivo, leads to significantly smaller lesion sizes after a 30-min middle cerebral artery occlusion (MCAo), and the preconditioned mice were better off in the three tests used to evaluate functional recovery. In accordance with in vitro results, TPC in vivo was prevented by administration of L-JNKI1, supporting a role for JNK in TPC. These results, from two different TPC models and with two distinct JNK inhibitors, show that JNK is likely to be involved in TPC.

Introduction: Ischemic preconditioning (IPC) induces protection to cerebral ischemia. However, it was previously unclear whether this protection resulted from altered susceptibility to ischemia. The current study examines the effects of late phase ischemic preconditioning in a mouse model of middle cerebral artery occlusion (MCAO). Specific examination of the regional cerebral blood flow (rCBF) was conducted., Experimental Procedure: Intra-abdominal radiofrequency probes were implanted in animals and core temperature was regulated. Mice were subjected to MCAO: (1) brief 15 min duration (preconditioning ischemia) and (2) 45 min MCAO (injurious ischemia). Naive (i.e. not preconditioned) animals were compared with preconditioned animals (preconditioning ischemia plus injurious ischemia at 72 h reperfusion). rCBF was measured using laser Doppler flowmetry (LDF) and magnetic resonance cerebral perfusion (MRP) arterial spin labeling. Percentage of brain infarcted was compared between groups., Results: rCBF was significantly improved in the preconditioned cohorts of mice. Naive animals showed flow reductions to 16+/-3.59% (MCAO&#95;45; injurious, unpreconditioned) and 17.1+/-8.6% (MCAO&#95;15; preconditioning ischemia alone) of baseline, while preconditioned animals had flows 33.9+/-13.2% (IPC&#95;45; preconditioned animals with injurious ischemia at 72 h reperfusion) of baseline (p=0.001). Percentage of brain infarcted was 17.2+/-6.2% in naive animals, while it was 5.1+/-4.6% in the preconditioned animals (p=0.003). MRP of the perfusion to the ischemic hemisphere, in a striatal coronal slice of the brain was 26.7+/-5.8% of the contralateral hemisphere in naive animals while preconditioned mice had flows of 38.7+/-6.8% of contralateral (p=0.04)., Conclusions: Improved rCBF is an important factor in the protection of IPC, during injurious MCAO in the mouse. Stringent monitoring of rCBF is required in future studies of IPC.

Recently, cerebral blood flow changes were re-discussed to contribute to tissue protection after preconditioning. In the present study, we demonstrate increased vessel diameters of the leptomeningeal anastomoses after hypoxic preconditioning by visualization of the brain angioarchitecture using the latex perfusion technique. This finding may display the anatomical correlate to previously described perfusion changes after preconditioning.

Hypoxia preconditioning has been shown to produce tolerance against brain injuries. The hypothesis of this study is that chronic hypobaric hypoxia may also induce acute hypoxia tolerance. We used intracellular recording in slices from rats exposed to chronic hypobaric hypoxia (exposed) and control to investigate the effects of chronic hypobaric hypoxia on the physiology of locus coeruleus (LC) including neuronal excitability. The results showed 35.7% reduced spontaneous firing rate and no change for membrane potential and input resistance in exposed neurons. In response to the alpha-2 adrenoceptor (A2R) agonist clonidine, both the hyperpolarizing potency and efficacy were increased indicated by a decreased EC(50) (control: 30.9 nM and exposed: 19.7 nM) and a 50.5% increase in maximum hyperpolarized potential, respectively. A2R binding sites were also increased 21% in exposed neurons measured by radioligand [(3)H]rauwolscine binding assay. When treated with acute N(2)-hypoxia, the cell survival time (ST) was longer in exposed neurons, suggesting that a tolerance was induced. In addition, the ST for both groups of LC neurons was decreased by the A2R antagonist yohimbine and increased by the glutamate receptor antagonist kynurenic acid but not by MK-801; the decreased percentage of ST by yohimbine was larger and the increased percentage by kynurenic acid was smaller in exposed neurons. The results suggested that up-regulation of A2R and altered non-NMDA glutamate receptor function induced by chronic hypobaric hypoxia may underlie, in part, the decreased LC neuronal excitability and acute hypoxia tolerance.

The patterns of expression of the Bcl-2, Bax, and Bcl-xL proteins were examined immunocytochemically in rat hippocampus and neocortex after severe hypobaric hypoxia (180 Torr for 3 h) and severe hypoxia preconditioned by intermittent mild hypoxia (360 Torr for 2 h daily, for 3 consecutive days, 24 h prior to severe hypoxia). As revealed by TUNEL assay, severe hypobaric hypoxia produced extensive apoptotic damage to the neurons of hippocampal CA1-CA4 and the neocortex but not the dentate gyrus granule cells. Remarkable posthypoxic up-regulation of Bax expression maximal at 24 h was detected in the CA1-CA4 areas of hippocampus and neocortex 3-72 h after severe hypoxia. The preconditioning to severe hypoxia protected neurons from the posthypoxic apoptotic transformations, the up-regulation of Bax expression, and resulted in persistent overexpression of Bcl-2 and Bcl-xL. We conclude that the protective action of hypoxic preconditioning is at least in part mediated by shifting of neuronal Bax/Bcl-2-Bcl-xL ratio to a favor of antiapoptotic proteins Bcl-2 and Bcl-xL.

It has been reported that limb ischemic preconditioning (LIP) could induce brain ischemic tolerance. In the present study, we investigated the role of p38 MAPK in the induction of brain ischemic tolerance by observing expression of phosphorylated p38 (p-p38) MAPK in the hippocampus after LIP and the effect of p38 MAPK inhibitor SB 203580 on the protection of LIP against delayed neuronal death (DND) in the CA1 hippocampus induced normally by brain ischemic insult. The results of Flow cytometry and Western blotting showed that expression of p-p38 MAPK initially increased at 6 h after LIP compared with sham group in the CA1 hippocampus. The increases reached peak at 12 h and lasted to 24 h after LIP. Expression of p-p38 MAPK was also increased in the CA3/dentate gyrus (DG) regions after LIP, but the beginning and peaking times were 1 and 3 days after LIP, which were relatively later than those in the CA1. Histological evaluation showed that LIP protected the CA1 hippocampal pyramidal neurons against DND induced by global brain ischemic insult for 8 min, suggesting the occurrence of brain ischemic tolerance. Pretreatment with SB 203580 at 30 min before LIP effectively blocked the ischemic tolerance induced by LIP. Together, it could be concluded that activation of p38 MAPK played an important role in the brain ischemic tolerance induced by LIP, and that components of the p38 MAPK cascade might be targets to modify neuronal survival in ischemic tolerance.

The potency of hyperbaric preconditioning (HBO-PC) is uncertain compared to well-validated ischemic or hypoxic models and no studies have directly compared HBO-PC to hypoxic preconditioning (HPC). We subjected rat pups to unilateral carotid cauterization followed by 90 min (min) of hypoxia using 8% O(2). Three HBO-PC regimes (maximum 2.5 atmospheres for 150 min) were compared to HPC (150 min of 8% O(2)) for changes in mortality and brain weight. Preconditioning-induced oxidative stress was assessed using aconitase activity and manganese superoxide dismutase (MnSOD) transcript levels. Initial brain weight data revealed a large coefficient of variation and compelled an examination of the temperature sensitivity of the model that revealed a narrow optimal range of 35 to 37 degrees C of variability in brain injury and mortality. With rigorous temperature control, high dose HBO-PC and HPC showed comparable anatomic (mean hemispheric weight decrease: control 42%, HPC 25% (P=0.01), HBO-PC 26% (P=0.01) and mortality protection (control 14.7%, HPC 5.9% HBO-PC 5.7%, P=0.001). High dose HBO-PC, but not HPC, suppressed aconitase activity by 65% at 24 h after the preconditioning stimulus (P=0.001). In contrast, MnSOD mRNA increased 2.5-fold at 24 h after HPC (P=0.007) but not after high dose HBO-PC. Thus, when temperature variability is eliminated, HBO-PC and HPC elicit similar preconditioning efficacy in neonatal brain but invoke different defenses against oxidative stress.

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.

Brain edema formation due to blood-brain barrier (BBB) disruption is a major consequence of cerebral ischemia. Previously, we demonstrated that targeting mitochondrial ATP-sensitive potassium channels (mitoK(ATP)) protects neuronal tissues in vivo and in vitro, however, the effects of mitoK(ATP) openers on cerebral endothelial cells and on BBB functions have never been examined. We investigated the effects of mitoK(ATP) channel opener diazoxide on BBB functions during ischemia/reperfusion injury (I/R). Rats were treated with 6, 20 or 40 mg/kg diazoxide ip for 3 days then exposed to global cerebral ischemia for 30 min. BBB permeability was assessed by administering Evan's-blue (EB) and Na-fluorescein (NaF) at the beginning of the 30 min reperfusion. I/R increased BBB permeability for the large molecular weight EB (ng/mg) in the cortex (control: 146 +/- 12, n = 7; I/R: 1049 +/- 152, n = 11) which was significantly attenuated in diazoxide-treated rats (575 +/- 99, n = 9; 582 +/- 104, n = 8; 20 and 40 mg/kg doses). Diazoxide pretreatment also significantly inhibited the extravasation of the low molecular weight NaF. Edema formation in the cortex was also decreased after diazoxide pretreatment. In cultured cerebral endothelial cells, diazoxide depolarized the mitochondrial membrane, suggesting a direct diazoxide effect on the endothelial mitochondria. Our results demonstrate that preconditioning of cerebral endothelium with diazoxide protects the BBB against ischemic stress.

3-Nitropropionic acid (3-NPA) is a suicide inactivator of succinate dehydrogenase (SDH), commonly used as a pharmacological model of Huntington's disease in rodents. Several studies have shown that a single administration of 3-NPA given systemically provides subsequent ischemic tolerance. The present study has tested the hypothesis that 3-NPA is capable of inducing tolerance in a model of permanent focal cerebral ischemia and whether 3-NPA can be truly applicable as a tolerance-inducer to ischemia. Rats given 3-NPA intraperitoneally revealed that the mortality of 3-NPA of 15, 20, and 25 mg/kg groups was 20.5, 38.8, and 83.3%, respectively. All rats survived without behavioral sequelae at smaller doses. Three days after 3-NPA preconditioning, the rats showing no behavioral changes underwent the permanent middle cerebral artery occlusion. The groups treated with 10 and 15 mg/kg of 3-NPA showed significantly reduced neurological deficits and infarction volumes in comparison with the control group, whereas the groups treated with 5 and 20 mg/kg of 3-NPA revealed no tolerance effects. When the regional SDH activity (% of control) was photometrically semi-quantified, it was observed that the activity was reduced to 90.8, 76.1, 67.8, and 64.3% in the outer layers of the cerebral cortex, and to 79.4, 67.5, 63.2, and 62.9% in the striatum 1 h after 3-NPA application (5, 10, 15, 20 mg/kg), respectively. In conclusion, although the preconditioning with 3-NPA is clearly shown in the setting of permanent ischemia, the preconditioning with this mitochondrial toxin demonstrated a rather narrow safety margin (critical threshold).

We have recently demonstrated marked neuroprotective efficacy of a combination therapy with magnesium (calcium- and glutamate-antagonist), tirilazad (antioxidant) and mild hypothermia (MTH) in a rat model of transient focal cerebral ischemia. In the present study, we investigated MTH under conditions of permanent focal cerebral ischemia. In part I, 20 Sprague-Dawley rats were subjected to 6 h of permanent, laser-Doppler flowmetry (LDF) controlled middle cerebral artery occlusion (MCAO). Drugs were administered 30 min before and 1 h after MCAO. Hypothermia (33 degrees C) was maintained for 2 h. Infarct size was planimetrically determined after 6 h. In part II, 29 rats were assigned to the same treatment arms and subjected to 7 days of permanent MCAO. Neurological deficits and body weight were assessed daily. Infarct size was determined on day 7. In part I, MTH significantly reduced infarct formation by 52% after 6 h. In part II, high mortality within the first 3 days was observed in both groups. Treated animals showed a significantly better postoperative weight gain on day 7 and neurological recovery on days 6 and 7 compared to controls without significant differences in infarct volume. MTH seems to exert its neuroprotective properties even in a setting of permanent cerebral ischemia. High mortality and absence of infarct reduction after 7 days might be due to model limitations. Neurological recovery, the most important clinical outcome parameter, is significantly improved in 7-day survivors. Significant neuroprotection under conditions of permanent ischemia and former promising results in transient ischemia justify further investigations of MTH.

Preconditioning describes a variety of treatments that induce neurons to become more resistant to a subsequent ischemic insult. How preconditioned neurons adapt to subsequent ischemic stress is not fully understood, but is likely to involve multiple protective mechanisms. We hypothesized hypoxic preconditioning induces protection by a coordinated up-regulation of antioxidant enzyme activity. To test this hypothesis, we developed two in vitro models of ischemia/reperfusion, involving oxygen-glucose deprivation (OGD) where neuronal cell death was predominantly by necrosis (necrotic model) or programmed cell death (PCD model). Hypoxic preconditioning 24 h prior to OGD significantly reduced cell death from 83% to 22% in the necrotic model and 68% to 11% in the PCD model. Consistent with the hypothesis, the activity of the antioxidant enzymes glutathione peroxidase, glutathione reductase, and Mn superoxide dismutase were significantly increased by 54%, 73% and 32%, respectively, in neuronal cultures subjected to hypoxic preconditioning. Furthermore, superoxide and hydrogen peroxide concentrations following OGD were significantly lower in the PCD model that had been subjected to hypoxic preconditioning.

The main goals of the current study were to assess: (a) whether a sublethal ischemic insult could protect the CA1 subregion of the hippocampus in organotypic slices against a lethal ischemic insult; and (b) whether this protection is long lasting as determined with an accurate immunohistochemical neuronal marker, NeuN. Hippocampal slice cultures were grown for 12-14 days in vitro. Slices were exposed either to oxygen/glucose deprivation (OGD) for 45 min (ischemia), or OGD for 15 min (ischemic preconditioning), 48 h prior to 45 min OGD, or were untreated (sham). Cell death was estimated by propidium iodide fluorescence 1 day after OGD and by NeuN immunohistochemistry 7 days after OGD. Image analysis was employed to measure the relative optical density of the NeuN-signal in all groups. After ischemia, damaged neurons were shrunken or lost and NeuN immunoreactivity was reduced. Relative optical density of NeuN (ROD [NeuN]) was 0.193+/-0.015 in control (sham) (n=9). In slices that underwent ischemia, ROD [NeuN] declined to 0.108+/-0.018 (n=5) in CA1 (*P<0.05 ROD [NeuN] in preconditioned slice cultures was 0.190+/-0.037 (76% higher than the ischemia group). Similar results were found after measuring PI fluorescence. In the CA1 sub-region, PI fluorescence was about 13, 47 and 17% in the sham, ischemic and IPC groups, respectively. We suggest that the immunohistochemical approach validates the dye uptake method used in slice cultures and yields quantitative data specific for neurons. We also conclude that the organotypic hippocampal slice model is useful for studying delayed ischemic preconditioning that is maintained for hours or days after the preconditioning event.

We evaluated the efficacy of cycloheximide, heat stress, NMDA receptor blockade (MK801/AP-5), oxygen--glucose deprivation, hypoxia, hypothermia and TNFalpha preconditioning to protect cortical neurons from in vitro ischemic insults that result in acute necrotic and delayed apoptotic neuronal death. Preconditioning treatments were performed 22--24 h before in vitro ischemia. In vitro ischemia was carried out in 96-well microtitre strip-plates by washing neuronal cultures with a balanced salt solution containing 25 mM 2-deoxy-D-glucose and incubating in an anaerobic chamber. Glutamate receptor blockers were present during in vitro ischemia to induce delayed neuronal death. Cycloheximide, heat stress, MK801 and oxygen--glucose deprivation preconditioning were neuroprotective in both acute and delayed in vitro ischemic neuronal death models. AP-5 preconditioning and a 12 h post-MK801 preconditioning interval protected neurons from acute ischemic neuronal death only. Hypoxia, TNFalpha and hypothermic preconditioning provided no neuronal protection in the in vitro ischemia models. This study has confirmed for the first time that several preconditioning treatments can protect neurons from in vitro ischemia induced acute necrotic and delayed apoptotic neuronal death. In addition, a unique feature of this study is the finding that preconditioning could be induced in near-pure primary cortical neuronal cultures, thus confirming that ischemic tolerance is an intrinsic property of neurons and provides a simplified culture system for identifying neuroprotective proteins.

Pretreatment of the brain with sublethal ischemia has been reported to induce neuronal resistance to otherwise lethal ischemia, a phenomenon designated as ischemic tolerance. The protective mechanisms of the phenomenon are not known yet, however, recent experimental data suggest the involvement of adenosine receptor activation in the acquisition of tolerance. In this study, the effect of theophylline, a non-selective adenosine receptor antagonist, and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an adenosine A1 receptor antagonist, were investigated to ascertain if these drugs could cancel the protective effect of ischemic tolerance in the gerbil. DPCPX or theophylline was administered at 3 h after a short preconditioning ischemia, and 21 h later animals were subjected to lethal ischemia of 5 min duration. DPCPX at a dose of 1.0 mg/kg (i.p) and theophylline at a dose of 20 mg/kg (i.p) significantly reduced the protective effect of preconditioning in the CA1 hippocampal neurons. These findings suggest the involvement of adenosine receptor activation for the development of ischemic tolerance phenomenon.

Background: Our recent studies have shown that prior intracerebral injection of a low dose of thrombin attenuates the brain edema formation that results from either an intracerebral hematoma, an intracerebral injection of a large dose of thrombin or cerebral ischemia. The aim of the current study is to investigate whether thrombin-induced tolerance (thrombin preconditioning; TPC) is associated with activation of p44/42 mitogen activated protein (MAP) kinases., Methods: This study contained three parts. In the first, rats received an intracerebral infusion of either saline or one unit thrombin (the TPC dose) into the right caudate nucleus. After 1, 3 and 7 days, the rats will be killed and brains used to detect p44/42 MAP kinases activation using Western blot analysis and immunohistochemistry. In the second and third parts, rats received intracerebral infusions of either vehicle, one unit thrombin (TPC) or one unit thrombin and 5 nmol PD 098059. These rats were either killed to detect kinases activation after 24 h or received a second intracerebral infusion of five-unit thrombin 7 days later with brain edema being assessed after a further 24 h., Results: Western blot analysis demonstrated that p44/42 MAP kinases were activated in the ipsilateral basal ganglia after the intracerebral infusion of thrombin one unit. Cells immunoreactive for activated p44/42 MAP kinases were found in the ipsilateral basal ganglia and ipsilateral cortex. PD 098059, a MAP kinase kinase inhibitor, abolished thrombin-induced activation of p44/42 MAP kinases. TPC suppressed thrombin-induced brain edema while PD 098059 blocked this protective effect. The water contents in the ipsilateral basal ganglia 24 h after infusion of thrombin five units were 82.6+/-0.8%, 79.2+/-0.4% and 81.8+/-1.9% in the control, TPC alone and TPC plus PD 098059 groups, respectively., Conclusion: Thrombin can activate p44/42 MAP kinases within the brain and the protective effects of thrombin preconditioning on brain edema formation are related to this activation.

SV129 or C57BL/6 mice were exposed to hyperbaric oxygenation (HBO, 5 days, 1 h every day, 100% O(2) at 3 atm absolute). One day after the 5th HBO session focal cerebral ischemia was induced. In SV129 mice, HBO induced tolerance against permanent focal cerebral ischemia (n=42, mean infarct volume reduction 27%, P=0.001), but not against transient (30 or 60 min) focal cerebral ischemia. In the C57BL/6 strain of mice, HBO did not induce tolerance against focal cerebral ischemia, even when the duration of ischemia or the HBO protocol were modified. For the first time we demonstrate that HBO can induce tolerance to focal cerebral ischemia, but this effect is strain dependent.