Bradykinin Postconditioning Protects Pyramidal CA1 Neurons Against Delayed Neuronal Death in Rat Hippocampus

Aims The present study was undertaken to evaluate possible neuroprotective effect of bradykinin against delayed neuronal death in hippocampal CA1 neurons if applied two days after transient forebrain ischemia in the rat. Methods Transient forebrain ischemia was induced in male Wistar rats by four-vessel occlusion for 8 min. To assess efficacy of bradykinin as a new stressor for delayed postconditioning we used two experimental groups of animals: ischemia 8 min and 3 days of survival, and ischemia 8 min and 3 days of survival with i.p. injection of bradykinin (150 μg/kg) applied 48 h after ischemia. Results We found extensive neuronal degeneration in the CA1 region at day 3 after ischemia/reperfusion. The postischemic neurodegeneration was preceded by increased activity of mitochondrial enzyme MnSOD in cytoplasm, indicating release of MnSOD from mitochondria in the process of delayed neuronal death. Increased cytosolic cytochrome c and subsequently caspase-3 activation are additional signs of neuronal death via the mitochondrial pathway. Bradykinin administration significantly attenuated ischemia-induced neuronal death, and also suppressed the release of MnSOD, and cytochrome c, and prevented caspase-3 activation. Conclusions Bradykinin can be used as an effective stressor able to prevent mitochondrial failure leading to apoptosis-like delayed neuronal death in postischemic rat hippocampus.     

Diazepam promotes ATP recovery and prevents cytochrome c release in hippocampal slices after in vitro ischemia

Benzodiazepines protect hippocampal neurons when administered within the first few hours after transient cerebral ischemia. Here, we examined the ability of diazepam to prevent early signals of cell injury (before cell death) after in vitro ischemia. Ischemia in vitro or in vivo causes a rapid depletion of ATP and the generation of cell death signals, such as the release of cytochrome c from mitochondria. Hippocampal slices from adult rats were subjected to 7 min of oxygen-glucose deprivation (OGD) and assessed histologically 3 h after reoxygenation. At this time, area CA1 neurons appeared viable, although slight abnormalities in structure were evident. Immediately following OGD, ATP levels in hippocampus were decreased by 70%, and they recovered partially over the next 3 h of reoxygenation. When diazepam was included in the reoxygenation buffer, ATP levels recovered completely by 3 h after OGD. The effects of diazepam were blocked by picrotoxin, indicating that the protection was mediated by an influx of Cl(-) through the GABA(A) receptor. It is interesting that the benzodiazepine antagonist flumazenil did not prevent the action of diazepam, as has been shown in other studies using the hippocampus. Two hours after OGD, the partial recovery of ATP levels occurred simultaneously with an increase of cytochrome c (approximately 400%) in the cytosol. When diazepam was included in the reoxygenation buffer, it completely prevented the increase in cytosolic cytochrome c. Thus, complete recovery of ATP and prevention of cytochrome c release from mitochondria can be achieved when diazepam is given after the loss of ATP induced by OGD.     

Chlortetracycline and demeclocycline inhibit calpains and protect mouse neurons against glutamate toxicity and cerebral ischemia

Minocycline is a potent neuroprotective tetracycline in animal models of cerebral ischemia. We examined the protective properties of chlortetracycline (CTC) and demeclocycline (DMC) and showed that these two tetracyclines were also potent neuroprotective against glutamate-induced neuronal death in vitro and cerebral ischemia in vivo. However, CTC and DMC appeared to confer neuroprotection through a unique mechanism compared with minocycline. Rather than inhibiting microglial activation and caspase, CTC and DMC suppressed calpain activities. In addition, CTC and DMC only weakly antagonized N-methyl-D-aspartate (NMDA) receptor activities causing 16 and 14%, respectively, inhibition of NMDA-induced whole cell currents and partially blocked NMDA-induced Ca2+ influx, commonly regarded as the major trigger of neuronal death. In vitro and in vivo experiments demonstrated that the two compounds selectively inhibited the activities of calpain I and II activated following glutamate treatment and cerebral ischemia. In contrast, minocycline did not significantly inhibit calpain activity. Taken together, these results suggested that CTC and DMC provide neuroprotection through suppression of a rise in intracellular Ca2+ and inhibition of calpains.     

14,15-EET Suppresses Neuronal Apoptosis in Ischemia–Reperfusion Through the Mitochondrial Pathway

Neuronal apoptosis mediated by the mitochondrial apoptosis pathway is an important pathological process in cerebral ischemia–reperfusion injury. 14,15-EET, an intermediate metabolite of arachidonic acid, can promote cell survival during ischemia/reperfusion. However, whether the mitochondrial apoptotic pathway is involved this survival mechanism is not fully understood. In this study, we observed that infarct size in ischemia–reperfusion injury was reduced in sEH gene knockout mice. In addition, Caspase 3 activation, cytochrome C release and AIF nuclear translocation were also inhibited. In this study, 14,15-EET pretreatment reduced neuronal apoptosis in the oxygen–glucose deprivation and re-oxygenation group in vitro. The mitochondrial apoptosis pathway was also inhibited, as evidenced by AIF translocation from the mitochondria to nucleus and the reduction in the expressions of cleaved-caspase 3 and cytochrome C in the cytoplasm. 14,15-EET could reduce neuronal apoptosis through upregulation of the ratio of Bcl-2 (anti-apoptotic protein) to Bax (apoptosis protein) and inhibition of Bax aggregation onto mitochondria. PI3K/AKT pathway is also probably involved in the reduction of neuronal apoptosis by EET. Our study suggests that 14,15-EET could suppress neuronal apoptosis and reduce infarct volume through the mitochondrial apoptotic pathway. Furthermore, the PI3K/AKT pathway also appears to be involved in the neuroprotection against ischemia–reperfusion by 14,15-EET.     

Molecular mechanisms of neuroprotective action of immunosuppressants--facts and hypotheses

Cyclosporin A (CsA) and FK506 (Tacrolimus) are short polypeptides which block the activation of lymphocytes and other immune system cells. Immunosuppressants exert neuroprotective and neurotrophic action in traumatic brain injury, sciatic nerve injury, focal and global ischemia in animals. Their neuroprotective actions are not understood and many hypotheses have been formed to explain such effects. We discuss a role of drug target - calcineurin in neuroprotective action of immunosuppressants. Protein dephosphorylation by calcineurin plays an important role in neuronal signal transduction due to its ability to regulate the activity of ion channels, glutamate release, and synaptic plasticity. In vitro FK506 protects cortex neurons from NMDA-induced death, augments NOS phosphorylation inhibiting its activity and NO synthesis. However, in vivo experiments demonstrated that FK506 in neuroprotective doses did not block excitotoxic cell death nor did it alter NO production during ischemia/reperfusion. Tissue damage in ischemia is the result of a complex pathophysiological cascade, which comprises a variety of distinct pathological events. Resident non-neuronal brain cells respond rapidly to neuronal cell death and may have both deleterious and useful role in neuronal damage. There is increasing evidence that reactive gliosis and post-ischemic inflammation involving microglia contribute to ischemic damage. We have demonstrated that FK506 modulates hypertrophic/proliferative responses and proinflammatory cytokine expression in astrocytes and microglia in vitro and in focal transient brain ischemia. Our findings suggest that astrocytes and microglia are direct targets of FK506 and modulation of glial response and inflammation is a possible mechanism of FK506-mediated neuroprotection in ischemia.     

Down-regulation Cdc42 attenuates neuronal apoptosis through inhibiting MLK3/JNK3 cascade during ischemic reperfusion in rat hippocampus

JNK signaling pathway is activated and involved in the selective neuronal death in the hippocampal CA1 subfield following cerebral ischemia. However, little is known about upstream partner controlling the pathway. Here we reported that ischemia/reperfusion significantly elevated Cdc42 activity, enhanced assembly of the Cdc42-MLK3 complex and activation of JNK pathway. Most importantly, knock-down endogenous Cdc42 selectively suppressed the MLK3/MKK7/JNK3 cascade, and subsequently blocked the phosphorylation of c-Jun and FasL expression. Meanwhile, Bcl-2 was inactivated and the release of cytochrome c was diminished. These alterations eventually perturbed the caspase-3 activation as well as post-ischemic neuronal cell death. Taken together, our findings strongly suggest that Cdc42 serves as an upstream activator and modulates JNK-mediated apoptosis machinery in vivo, which ultimately results in neuronal apoptosis via nuclear and non-nuclear pathways. Thus, Cdc42 may be a potential therapeutic target in ischemic brain injury.     

Na(+)/Ca(2+) exchanger 2 is neuroprotective by exporting Ca(2+) during a transient focal cerebral ischemia in the mouse

Na(+)/Ca(2+) exchanger (NCX), by mediating Na(+) and Ca(2+) fluxes bi-directionally, assumes a role in controlling the Ca(2+) homeostasis in the ischemic brain. It has been suggested that the three isoforms of NCX (NCX1, 2 and 3) may be differentially involved in permanent cerebral ischemia. However, the role of NCX2 has not been defined in ischemic reperfusion injury after a transient focal cerebral ischemia. Furthermore, it is not known whether NCX2 imports or exports intracellular Ca(2+) ([Ca(2+)](i)) following ischemia and reperfusion. To define the role of NCX2 in ischemia and reperfusion, we examined mice lacking NCX2, in vivo and in vitro. After an in vitro ischemia, a significantly slower recovery in population spike amplitudes, a sustained elevation of [Ca(2+)](i) and an increased membrane depolarization were developed in the NCX2-deficient hippocampus. Moreover, a transient focal cerebral ischemia in vivo produced a larger infarction and more cell death in the NCX2-deficient mouse brain. In particular, in the wild type brain, NCX2-expressing neurons were largely spared from cell death after ischemia. Our results suggest that NCX2 exports Ca(2+) in ischemia and thus protects neuronal cells from death by reducing [Ca(2+)](i) in the adult mouse brain.     

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.     

Insulin blocks cytochrome c release in the reperfused brain through PI3-K signaling and by promoting Bax/Bcl-XL binding

The critical event of the intrinsic pathway of apoptosis following transient global brain ischemia is the release of cytochrome c from the mitochondria. In vitro studies have shown that insulin can signal specifically via phosphatidylinositol-3-OH-kinase (PI3-K) and Akt to prevent cytochrome c release. Therefore, insulin may exert its neuroprotective effects during brain reperfusion by blocking cytochrome c release. We hypothesized that insulin acts through PI3-K, Akt, and Bcl-2 family proteins to inhibit cytochrome c release following transient global brain ischemia. We found that a single bolus of insulin given immediately upon reperfusion inhibited cytochrome c release for at least 24 h, and produced a fivefold improvement in neuronal survival at 14 days. Moreover, insulin's ability to inhibit cytochrome c release was completely dependent on PI3-K signaling and insulin induces phosphorylation of Akt through PI3-K. In untreated animals, there was an increase in mitochondrial Bax at 6 h of reperfusion, and Bax binding to Bcl-X_L was disrupted at the mitochondria. Insulin prevented both these events in a PI3-K-dependent manner. In summary, insulin regulates cytochrome c release through PI3-K likely by activating Akt, promoting the binding between Bax and Bcl-X_L, and by preventing Bax translocation to the mitochondria.     

Trehalose Inhibits Protein Aggregation Caused by Transient Ischemic Insults Through Preservation of Proteasome Activity,Not via Induction of Autophagy

Protein aggregation has been proved to be a pathological basis accounting for neuronal death caused by either transient global ischemia or oxygen glucose deprivation (OGD), and inhibition of protein aggregation is emerging as a potential strategy of preventing brain damage. Trehalose was found to inhibit protein aggregation caused by neurodegenerative diseases via induction of autophagy, whereas its effect is still elusive on ischemia-induced protein aggregation. In this study, we investigated this issue by using rat model of transient global ischemia and SH-SY5Y model of OGD. We found that pretreatment with trehalose inhibited transient global ischemia-induced neuronal death in the hippocampus CA1 neurons and OGD-induced death in SH-SY5Y cells, which was associated with inhibition of the formation of ubiquitin-labeled protein aggregates and preservation of proteasome activity. In vitro study showed that the protection of trehalose against OGD-induced cell death and protein aggregation in SH-SY5Y cells was reversed when proteasome activity was inhibited by MG-132. Further studies revealed that trehalose prevented OGD-induced reduction of proteasome activity via suppression of both oxidative stress and endoplasmic reticulum stress. Particularly, our results showed that trehalose inhibited OGD-induced autophagy. Therefore, we demonstrated that proteasome dysfunction contributed to protein aggregation caused by ischemic insults and trehalose prevented protein aggregation via preservation of proteasome activity, not via induction of autophagy.     

Distinct Ca2+ thresholds determine cytochrome c release or permeability transition pore opening in brain mitochondria.

In diseases associated with neuronal degeneration, such as Alzheimer's or cerebral ischemia, the cytosolic Ca2+ concentration ([Ca2+]cyt) is pathologically elevated. It is still unclear, however, under which conditions Ca2+ induces either apoptotic or necrotic neuronal cell death. Studying respiration and morphology of rat brain mitochondria, we found that extramitochondrial [Ca2+] above 1 M causes reversible release of cytochrome c, a key trigger of apoptosis. This event was NO-independent but required Ca2+ influx into the mitochondrial matrix. The mitochondrial permeability transition pore (PTP), widely thought to underlie cytochrome c release, was not involved. In contrast to noncerebral tissue, only relatively high [Ca2+] (is approximately equal to 200 M) opened PTP and ruptured mitochondria. Our findings might reflect a fundamental mechanism to protect postmitotic neuronal tissue against necrotic devastation and inflammation.     

Kynurenic acid attenuates MPP(+)-induced dopaminergic neuronal cell death via a Bax-mediated mitochondrial pathway

Kynurenic acid (KYNA), a tryptophan metabolite in the kynurenine pathway, is protective against various insults. However, the molecular mechanism of this protective effect has not been identified. In this study, we examined the protective effects of KYNA against 1-methyl-4-phenylpyridinium (MPP(+)), the best-characterized toxin inducing pathological changes resembling Parkinson's disease (PD), using SH-SY5Y and SK-N-SH human neuroblastoma cells. Pre-treatment of KYNA attenuated MPP(+)-induced neuronal cell death in SH-SY5Y and SK-N-SH cells. MPP(+)-induced cell death was preceded by increases in Bax expression and mitochondrial dysfunction, such as collapse of mitochondrial membrane potential (DeltaPsi(m)), release of cytochrome c from mitochondria into the cytoplasm, and increases in caspase-9/-3 activities. KYNA effectively inhibited all of these mitochondrial apoptotic processes. Our results indicate that KYNA plays a protective role by down-regulating Bax expression and maintaining mitochondrial function in MPP(+)-induced neuronal cell death, and suggest that KYNA may have therapeutic potential in PD.     

SM-20 is a novel mitochondrial protein that causes caspase-dependent cell death in nerve growth factor-dependent neurons

Sympathetic neurons undergo protein synthesis-dependent apoptosis when deprived of nerve growth factor (NGF). Expression of SM-20 is up-regulated in NGF-deprived sympathetic neurons, and ectopic SM-20 is sufficient to promote neuronal death in the presence of NGF. We now report that SM-20 is a mitochondrial protein that promotes cell death through a caspase-dependent mechanism. SM-20 immunofluorescence was present in the cytoplasm in a punctate pattern that colocalized with cytochrome oxidase I and with mitochondria-selective dyes. Analysis of SM-20/dihydrofolate reductase fusion proteins revealed that the first 25 amino acids of SM-20 contain a functional mitochondrial targeting sequence. An amino-terminal truncated form of SM-20 was not restricted to mitochondria but instead localized throughout the cytosol and nucleus. Nevertheless, the truncated SM-20 retained the ability to induce neuronal death, similar to the wild type protein. SM-20-induced death was accompanied by caspase-3 activation and was blocked by a general caspase inhibitor. Additionally, overexpression of SM-20, under conditions where cell death is blocked by a general caspase inhibitor, did not result in widespread release of cytochrome c from mitochondria. These results indicate that SM-20 is a novel mitochondrial protein that may be an important mediator of neurotrophin-withdrawal-mediated cell death.     

Inhibition of Drp1 provides neuroprotection in vitro and in vivo

Impaired regulation of mitochondrial dynamics, which shifts the balance towards fission, is associated with neuronal death in age-related neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. A role for mitochondrial dynamics in acute brain injury, however, has not been elucidated to date. Here, we investigated the role of dynamin-related protein 1 (Drp1), one of the key regulators of mitochondrial fission, in neuronal cell death induced by glutamate toxicity or oxygen-glucose deprivation (OGD) in vitro, and after ischemic brain damage in vivo. Drp1 siRNA and small molecule inhibitors of Drp1 prevented mitochondrial fission, loss of mitochondrial membrane potential (MMP), and cell death induced by glutamate or tBid overexpression in immortalized hippocampal HT-22 neuronal cells. Further, Drp1 inhibitors protected primary neurons against glutamate excitotoxicity and OGD, and reduced the infarct volume in a mouse model of transient focal ischemia. Our data indicate that Drp1 translocation and associated mitochondrial fission are key features preceding the loss of MMP and neuronal cell death. Thus, inhibition of Drp1 is proposed as an efficient strategy of neuroprotection against glutamate toxicity and OGD in vitro and ischemic brain damage in vivo.     

Cerebral ischemia-reperfusion-induced autophagy protects against neuronal injury by mitochondrial clearance

Cerebral ischemia-reperfusion (I-R) is a complex pathological process. Although autophagy can be evoked by ischemia, its involvement in the reperfusion phase after ischemia and its contribution to the fate of neurons remains largely unknown. In the present investigation, we found that autophagy was activated in the reperfusion phase, as revealed in both mice with middle cerebral artery occlusion and oxygen-glucose deprived cortical neurons in culture. Interestingly, in contrast to that in permanent ischemia, inhibition of autophagy (by 3-methyladenine, bafilomycin A₁, Atg7 knockdown or in atg5^?/? MEF cells) in the reperfusion phase reinforced, rather than reduced, the brain and cell injury induced by I-R. Inhibition of autophagy either with 3-methyladenine or Atg7 knockdown enhanced the I-R-induced release of cytochrome c and the downstream activation of apoptosis. Moreover, MitoTracker Red-labeled neuronal mitochondria increasingly overlapped with GFP-LC3-labeled autophagosomes during reperfusion, suggesting the presence of mitophagy. The mitochondrial clearance in I-R was reversed by 3-methyladenine and Atg7 silencing, further suggesting that mitophagy underlies the neuroprotection by autophagy. In support, administration of the mitophagy inhibitor mdivi-1 in the reperfusion phase aggravated the ischemia-induced neuronal injury both in vivo and in vitro. PARK2 translocated to mitochondria during reperfusion and Park2 knockdown aggravated ischemia-induced neuronal cell death. In conclusion, the results indicated that autophagy plays different roles in cerebral ischemia and subsequent reperfusion. The protective role of autophagy during reperfusion may be attributable to mitophagy-related mitochondrial clearance and inhibition of downstream apoptosis. PARK2 may be involved in the mitophagy process.     

Imbalance of neurotrophin receptor isoforms TrkB-FL/TrkB-T1 induces neuronal death in excitotoxicity

A better understanding of the mechanisms underlying neuronal death in cerebral ischemia is required for the development of stroke therapies. Here we analyze the contribution of the tropomyosin-related kinase B (TrkB) neurotrophin receptor to excitotoxicity, a primary pathological mechanism in ischemia, which is induced by overstimulation of glutamate receptors of the N-methyl-D-aspartate type. We demonstrate a significant modification of TrkB expression that is strongly associated with neurodegeneration in models of ischemia and in vitro excitotoxicity. Two mechanisms cooperate for TrkB dysregulation: (1) calpain-processing of full-length TrkB (TrkB-FL), high-affinity receptor for brain-derived neurotrophic factor, which produces a truncated protein lacking the tyrosine-kinase domain and strikingly similar to the inactive TrkB-T1 isoform and (2) reverse regulation of the mRNA of these isoforms. Collectively, excitotoxicity results in a decrease of TrkB-FL, the production of truncated TrkB-FL and the upregulation of TrkB-T1. A similar neuro-specific increase of the TrkB-T1 isoform is also observed in stroke patients. A lentivirus designed for both neuro-specific TrkB-T1 interference and increased TrkB-FL expression allows recovery of the TrkB-FL/TrkB-T1 balance and protects neurons from excitotoxic death. These data implicate a combination of TrkB-FL downregulation and TrkB-T1 upregulation as significant causes of neuronal death in excitotoxicity, and reveal novel targets for the design of stroke therapies.     

Endoplasmic reticulum stress-induced apoptosis requires bax for commitment and Apaf-1 for execution in primary neurons

Apoptosis triggered by endoplasmic reticulum (ER) stress is associated with various pathophysiological conditions including neurodegenerative diseases and ischemia. However, the mechanism by which ER stress induces neuronal apoptosis remains controversial. Here we identify the pathway of apoptosis carried out in sympathetic neurons triggered to die by ER stress-inducing agent tunicamycin. We find that ER stress induces a neuronal apoptotic pathway which upregulates BH3-only genes DP5 and Puma. Importantly, we show that ER stress commits neurons to die before cytochrome c release and this commitment requires Bax activation and c-jun N-terminal kinase signaling. Furthermore, ER stress engages the mitochondrial pathway of death as neurons release cytochrome c and Apaf-1 deficiency is sufficient to block apoptosis. Our findings identify a critical function of Bax in committing neurons to ER stress-induced apoptosis and clarify the importance of the apoptosome as the non-redundant caspase activation pathway to execute neuronal apoptosis in response to ER stress.     

The regulation and role of neuronal gap junctions during neuronal injury

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI) and epilepsy. The coupling of neurons by gap junctions (electrical synapses) increases during neuronal injury. In a recent study with the use of in vivo and in vitro models of cortical ischemia in mice, we have demonstrated that the ischemic increase in neuronal gap junction coupling is regulated by glutamate via group II metabotropic glutamate receptors (mGluR). Specifically, we found that activation of group II mGluRs increases background levels of neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein), whereas inactivation of group II mGluRs prevents the ischemia-mediated increases in the coupling and Cx36 expression. Using the analysis of neuronal death, we also established that inactivation of group II mGluRs or genetic elimination of Cx36 both dramatically reduce ischemic neuronal death in vitro and in vivo. Similar results were obtained using in vitro models of TBI and epilepsy. Our study demonstrated that mechanisms for the injury-mediated increase in neuronal gap junction coupling are part of the mechanisms for glutamate-dependent neuronal death.     

Block of P2X7 receptors could partly reverse the delayed neuronal death in area CA1 of the hippocampus after transient global cerebral ischemia

Transient global ischemia (which closely resembles clinical situations such as cardiac arrest, near drowning or severe systemic hypotension during surgical procedures), often induces delayed neuronal death in the brain, especially in the hippocampal CA1 region. The mechanism of ischemia/reperfusion (I/R) injury is not fully understood. In this study, we have shown that the P2X7 receptor antagonist, BBG, reduced delayed neuronal death in the hippocampal CA1 region after I/R injury; P2X7 receptor expression levels increased before delayed neuronal death after I/R injury; inhibition of the P2X7 receptor reduced I/R-induced microglial microvesicle-like components, IL-1β expression, P38 phosphorylation, and glial activation in hippocampal CA1 region after I/R injury. These results indicate that antagonism of the P2X7 receptor and signaling pathways of microglial MV shedding, such as src-protein tyrosine kinase, P38 MAP kinase and A-SMase, might be a promising therapeutic strategy for clinical treatment of transient global cerebral I/R injury.