Changes in SLITRK1 Level in the Amygdala Mediate Chronic Neuropathic Pain-Induced Anxio-Depressive Behaviors in Mice.

BACKGROUND: Comorbid chronic neuropathic pain (NPP) and anxio-depressive disorders (ADD) have become a serious global public-health problem. The SLIT and NTRK-like 1 (SLITRK1) protein is important for synaptic remodeling and is highly expressed in the amygdala, an important brain region involved in various emotional behaviors. We examined whether SLITRK1 protein in the amygdala participates in NPP and comorbid ADD. METHODS: A chronic NPP mouse model was constructed by L5 spinal nerve ligation; changes in chronic pain and ADD-like behaviors were measured in behavioral tests. Changes in SLITRK1 protein and excitatory synaptic functional proteins in the amygdala were measured by immunofluorescence and Western blot. Adeno-associated virus was transfected into excitatory synaptic neurons in the amygdala to up-regulate the expression of SLITRK1. RESULTS: Chronic NPP-related ADD-like behavior was successfully produced in mice by L5 ligation. We found that chronic NPP and related ADD decreased amygdalar expression of SLITRK1 and proteins important for excitatory synaptic function, including Homer1, postsynaptic density protein 95 (PSD95), and synaptophysin. Virally-mediated SLITRK1 overexpression in the amygdala produced a significant easing of chronic NPP and ADD, and restored the expression levels of Homer1, PSD95, and synaptophysin. CONCLUSION: Our findings indicated that SLITRK1 in the amygdala plays an important role in chronic pain and related ADD, and may prove to be a potential therapeutic target for chronic NPP-ADD comorbidity.

Detection of apoptotic cells based on in situ hybridization chain reaction using specific hairpins.

There are an increasing number of experiments to study programmed cell death/apoptosis, one of the characteristics of which is DNA fragmentation. The only current method for in situ detection of DNA fragmentation is Terminal deoxynucleotidyl transferase mediated-dUTP Nick End Labeling, TUNEL. In this study, a new method for in situ detection of apoptotic DNA fragments, namely In Situ Hybridization Chain Reaction, isHCR, was established. The principle of the assay is that the sticky end sequence of the apoptotic cell DNA fragment non-specifically initiates a hybridization chain reaction that specifically detects the apoptotic cell. The results of the combined TUNEL and isHCR method demonstrated that the majority of isHCR-positive cells were also labeled by TUNEL. In situ HCR often detect DNA fragments in the cytoplasm that the classical TUNEL method couldnot, and these cells may be in the early stages of apoptosis. It also indicates that DNA fragments are transferred to the cytoplasm during apoptosis. Because the staining process does not require terminal deoxynucleotidyl transferase as TUNEL staining does, isHCR staining cost low and can be performed on a large number of tissue specimens. It is believed that isHCR has the potential to detect DNA fragmentation of apoptotic cells in situ.

Transient expression of thyrotropin releasing hormone peptide and mRNA in the rat hippocampus following global cerebral ischemia/reperfusion injury.

INTRODUCTION: The role of extra-hypothalamic thyrotropin-releasing hormone (TRH) has been investigated by pharmacological studies using TRH or its analogues and found to produce a wide array of effects in the central nervous system. METHODS: Immunofluorescence, In situ labeling of DNA (TUNEL), in situ hybridization chain reaction and quantitative real-time polymerase chain reaction were used in this study. RESULTS: We found that the granular cells of the dentate gyrus expressed transiently a significant amount of TRH-like immunoreactivity and TRH mRNA during the 6-24 h period following global cerebral ischemia/reperfusion injury. TUNEL showed that apoptosis of neurons in the CA1 region occurred from 48 h and almost disappeared at 7 days. TRH administration 30 min before or 24 h after the injury could partially inhibit neuronal loss, and improve the survival of neurons in the CA1 region. CONCLUSION: These data suggest that endogenous TRH expressed transiently in the dentate gyrus of the hippocampus may play an important role in the survival of neurons during the early stage of ischemia/reperfusion injury and that delayed application of TRH still produced neuroprotection. This delayed application of TRH has a promising therapeutic significance for clinical situations.

Enhanced expression of P2X4 purinoceptors in pyramidal neurons of the rat hippocampal CA1 region may be involved ischemia-reperfusion injury.

Ischemic stroke is the most serious disease that harms human beings. In principle, its treatment is to restore blood flow supply as soon as possible. However, after the blood flow is restored, it will lead to secondary brain injury, that is, ischemia-reperfusion injury. The mechanism of ischemia-reperfusion injury is very complicated. This study showed that P2X4 receptors in the pyramidal neurons of rat hippocampus were significantly upregulated in the early stage of ischemia-reperfusion injury. Neurons with high expression of P2X4 receptors are neurons that are undergoing apoptosis. Intraventricular injection of the P2X4 receptor antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) and PSB-12062 can partially block neuronal apoptosis, to promote the survival of neurons, indicating that ATP through P2X4 receptors is involved in the process of cerebral ischemia-reperfusion injury. Therefore, identifying the mechanism of neuronal degeneration induced by extracellular ATP via P2X4 receptors after ischemia-reperfusion will likely find new targets for the treatment of ischemia-reperfusion injury, and will provide a useful theoretical basis for the treatment of ischemia-reperfusion injury.

Expression of P2X receptors in the rat anterior pituitary.

In this study, the distribution patterns of P2X1 to P2X7 receptors in the anterior pituitary cells of rat were studied with single-, double-, and triple-labeling immunofluorescence, combined method of immunofluorescence and in situ hybridization, and Western blot. The results showed that the expression level of the P2X4 receptor protein was highest, followed by P2X5, P2X3, P2X2, P2X6, and P2X7 receptor proteins, but no P2X1 receptor protein was detected. Strong P2X4 receptor-immunoreactivity was detected in almost all the anterior pituitary cells. Different combinations of P2X receptors were detected in each individual cell type of the rat anterior pituitary. Gonadotrophs express P2X4, P2X5, and P2X6 receptors. Corticotrophs express P2X3 and P2X4 receptors. Folliculo-stellate cells express P2X2 and P2X4 receptors, and somatotrophs, lactotrophs, and thyrotrophs express only P2X4 receptors. The macrophages with Iba-1-ir expressed P2X7 receptors. The possible functions of these P2X receptors in each individual cell type of the rat anterior pituitary are discussed.

LncRNA GAS5 inhibits microglial M2 polarization and exacerbates demyelination.

The regulation of inflammation is pivotal for preventing the development or reoccurrence of multiple sclerosis (MS). A biased ratio of high-M1 versus low-M2 polarized microglia is a major pathological feature of MS Here, using microarray screening, we identify the long noncoding RNA (lncRNA) GAS5 as an epigenetic regulator of microglial polarization. Gain- and loss-of-function studies reveal that GAS5 suppresses microglial M2 polarization. Interference with GAS5 in transplanted microglia attenuates the progression of experimental autoimmune encephalomyelitis (EAE) and promotes remyelination in a lysolecithin-induced demyelination model. In agreement, higher levels of GAS5 are found in amoeboid-shaped microglia in MS patients. Further, functional studies demonstrate that GAS5 suppresses transcription of TRF4, a key factor controlling M2 macrophage polarization, by recruiting the polycomb repressive complex 2 (PRC2), thereby inhibiting M2 polarization. Thus, GAS5 may be a promising target for the treatment of demyelinating diseases.

Expression of P2X1 receptors in somatostatin-containing cells in mouse gastrointestinal tract and pancreatic islets of both mouse and human.

With immunohistochemical and Western blot techniques, P2X1 receptors were detected in the whole mouse gastrointestinal tract and pancreatic islets of mouse and human. (1) δ Cells containing somatostatin (SOM) in the stomach corpus, small intestines, distal colon, pancreatic islets of both mouse and human express P2X1 receptors; (2) strong immunofluorescence of P2X1 receptors was detected in smooth muscle fibers and capillary networks of the villus core of mouse intestine; and (3) P2X1 receptor-immunoreactive neurons were also detected widely in both mouse myenteric and submucosal plexuses, all of which express SOM. The present data implies that ATP via P2X1 receptors is involved in SOM release from pancreatic δ cells, enteric neurons, and capillary networks in villi.

Microvesicles shed from microglia activated by the P2X7-p38 pathway are involved in neuropathic pain induced by spinal nerve ligation in rats.

Microglia are critical in the pathogenesis of neuropathic pain. In this study, we investigated the role of microvesicles (MVs) in neuropathic pain induced by spinal nerve ligation (SNL) in rats. First, we found that MVs shed from microglia were increased in the cerebrospinal fluid and dorsal horn of the spinal cord after SNL. Next, MVs significantly reduced paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). In addition, the P2X7-p38 pathway was related to the bleb of MVs after SNL. Interleukin (IL)-1ß was found to be significantly upregulated in the package of MVs, and PWT and PWL increased following inhibition with shRNA-IL-1ß. Finally, the amplitude and frequency of spontaneous excitatory postsynaptic currents increased following stimulation with MVs. Our results indicate that the P2X7-p38 pathway is closely correlated with the shedding of MVs from microglia in neuropathic pain, and MVs had a significant effect on neuropathic pain by participating in the interaction between microglia and neurons.

Inhibition of P2X7 receptors improves outcomes after traumatic brain injury in rats.

Traumatic brain injury (TBI) is the leading cause of death and disability for people under the age of 45 years worldwide. Neuropathology after TBI is the result of both the immediate impact injury and secondary injury mechanisms. Secondary injury is the result of cascade events, including glutamate excitotoxicity, calcium overloading, free radical generation, and neuroinflammation, ultimately leading to brain cell death. In this study, the P2X7 receptor (P2X7R) was detected predominately in microglia of the cerebral cortex and was up-regulated on microglial cells after TBI. The microglia transformed into amoeba-like and discharged many microvesicle (MV)-like particles in the injured and adjacent regions. A P2X7R antagonist (A804598) and an immune inhibitor (FTY720) reduced significantly the number of MV-like particles in the injured/adjacent regions and in cerebrospinal fluid, reduced the number of neurons undergoing apoptotic cell death, and increased the survival of neurons in the cerebral cortex injured and adjacent regions. Blockade of the P2X7R and FTY720 reduced interleukin-1ßexpression, P38 phosphorylation, and glial activation in the cerebral cortex and improved neurobehavioral outcomes after TBI. These data indicate that MV-like particles discharged by microglia after TBI may be involved in the development of local inflammation and secondary nerve cell injury.

Neuron-Derived ADAM10 Production Stimulates Peripheral Nerve Injury-Induced Neuropathic Pain by Cleavage of E-Cadherin in Satellite Glial Cells.

BACKGROUND: Increasing evidence suggests the potential involvement of metalloproteinase family proteins in the pathogenesis of neuropathic pain, although the underlying mechanisms remain elusive. METHODS: Using the spinal nerve ligation model, we investigated whether ADAM10 proteins participate in pain regulation. By implementing invitro methods, we produced a purified culture of satellite glial cells to study the underlying mechanisms of ADAM10 in regulating neuropathic pain. RESULTS: Results showed that the ADAM10 protein was expressed in calcitonin gene-related peptide (CGRP)-containing neurons of the dorsal root ganglia, and expression was upregulated following spinal nerve ligation surgery invivo. Intrathecal administration of GI254023X, an ADAM10 selective inhibitor, to the rats one to three days after spinal nerve ligation surgery attenuated the spinal nerve ligation-induced mechanical allodynia and thermal hyperalgesia. Intrathecal injection of ADAM10 recombinant protein simulated pain behavior in normal rats to a similar extent as those treated by spinal nerve ligation surgery. These results raised a question about the relative contribution of ADAM10 in pain regulation. Further results showed that ADAM10 might act by cleaving E-cadherin, which is mainly expressed in satellite glial cells. GI254023X reversed spinal nerve ligation-induced downregulation of E-cadherin and activation of cyclooxygenase 2 after spinal nerve ligation. ß-catenin, which creates a complex with E-cadherin in the membranes of satellite glial cells, was also downregulated by spinal nerve ligation surgery in satellite glial cells. Finally, knockdown expression of ß-catenin by lentiviral infection in purified satellite glial cells increased expression of inducible nitric oxide synthase and cyclooxygenase 2. CONCLUSION: Our findings indicate that neuron-derived ADAM10 production stimulates peripheral nerve injury-induced neuropathic pain by cleaving E-cadherin in satellite glial cells.

MSX3 Switches Microglia Polarization and Protects from Inflammation-Induced Demyelination.

The major challenge for progressive multiple sclerosis therapy is the promotion of remyelination from inflammation-induced demyelination. A switch from an M1- to an M2-dominant polarization of microglia is critical in these repair processes. In this study, we identified the homeobox gene msh-like homeobox-3 (Msx3) as a new pivotal regulator for microglial polarization. MSX3 was induced during microglia M2 polarization and repressed in M1 cells. The expression of MSX3 in microglia was dynamically regulated during experimental autoimmune encephalomyelitis (EAE), which is an animal model of multiple sclerosis. The overexpression of MSX3 in microglia promoted M2 but impeded M1 polarization. Interrupting MSX3 expression in microglia accelerated inflammation-induced demyelination and neurodegeneration. The conditioned medium from MSX3-transduced microglia promoted oligodendrocyte progenitor survival, differentiation, and neurite outgrowth. The adoptive transfer of MSX3-transduced microglia suppressed EAE and facilitated remyelination within the murine CNS in EAE and the LPC model. Mechanically, chromatin immunoprecipitation assays also indicated that MSX3 directly regulated three key genes associated with microglia M2 polarization, including Pparg, Stat6, and Jak3. Importantly, we found that overexpression of MSX3 in human-derived microglia represents the M2 phenotype and ameliorated EAE after intraventricular injection. Our findings suggest a new homeobox protein-dependent mechanism for driving microglia M2 polarization and identify MSX3 as an attractive therapeutic approach for preventing inflammation-induced demyelination and promoting remyelination.

Co-localization of Pirt protein and P2X2 receptors in the mouse enteric nervous system.

P2X2 receptors, with other P2X receptor subtypes, have an important role mediating synaptic transmission in regulating the functions of the gastrointestinal tract. Our recent work has found a new regulator of P2X receptor function, called phosphoinositide-interacting regulator of transient receptor potential channels (Pirt). In the present work, we have shown that Pirt immunoreactivity was localized in nerve cell bodies and nerve fibers in the myenteric plexus of the stomach, ileum, proximal, and distal colon and in the submucosal plexus of the jejunum, ileum, proximal, and distal colon. Almost all the Pirt-immunoreactive (ir) neurons were also P2X2-ir, and co-immunoprecipitation experiments have shown that Pirt co-precipitated with the anti-P2X2 antibody. This work provides detailed information about the expression of Pirt in the gut and its co-localization with P2X2, indicating its potential role in influencing P2X2 receptor function.

Enhanced RAGE Expression in the Dorsal Root Ganglion May Contribute to Neuropathic Pain Induced by Spinal Nerve Ligation in Rats.

OBJECTIVE: There is some evidence implicating receptor for advanced glycation end products (RAGE) signaling in the pathogenesis of neuropathic pain (NP). The objective was to investigate whether RAGE signaling in the dorsal root ganglion (DRG) might contribute to NP following peripheral nerve injury. DESIGN: Experimental study before and after spinal nerve ligation (SNL) surgery. SETTING: Caged in a controlled environment. SUBJECTS: Male Sprague-Dawley rats. METHODS: A SNL rat model of NP was used. Mechanical hyperalgesia was measured by the paw withdrawal threshold (PWT) to mechanical stimuli (1.4-15 g). Protein expressions of RAGE (immunofluorescence and western blotting), glial fibrillary acidic protein (GFAP; satellite glial cell [SGC] activation marker), IL-1ß (ELISA), TNF-α (ELISA), and NF-κB (western blotting) in the DRG were determined. RAGE signaling was inhibited by intrathecal injection of anti-RAGE antibody. RESULTS: After 7 days, SNL surgery reduced the PWT and upregulated the protein expression of RAGE, GFAP, NF-κB, TNF-α, and IL-1ß. Intrathecal injection of RAGE-neutralizing antibody attenuated the SNL-induced mechanical hyperalgesia, activation of SGCs, and upregulation of NF-κB, TNF-α, and IL-1ß in the DRG. CONCLUSION: RAGE signaling may contribute to the pain hypersensitivity observed in the rat SNL model of NP. Although the precise mechanism remains to be established, NF-κB, TNF-α, and IL-1ß likely play a role, together with the activation of SGCs.

A subpopulation of gonadotropin-releasing hormone neurons in the adult mouse forebrain is γ-Aminobutyric acidergic.

Gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in reproductive function. GnRH is released in distinct pulses that are regulated by neurotransmitters or neuromodulators. With immunohistochemistry and GAD67-GFP knockin mice, this study shows for the first time that a subset of GnRH neurons in the forebrain of adult mouse is γ-aminobutyric acid (GABA)-ergic. There is a gender difference in the percentage of GnRH neurons expressing GAD67-GFP in female vs. male mice. The percentage of GnRH neurons expressing GAD67-GFP decreased after castration of female mice and increased to the normal female level after estradiol treatment. The percentage of GnRH neurons expressing GAD67-GFP did not change significantly in intact, castrated, or castration + testosterone propionate-treated male mice. During the female estrous cycle, the percentage of GnRH neurons expressing GAD67-GFP was higher during the estrous stage than during the diestrous stage. During sexual maturation of postnatal development, GnRH neurons did not express GAD67-GFP until postnatal day (P) 15, and the gender differences were first detected at P30, which corresponds to the maturation stage. In conclusion, our data suggest that 1) a subset of GnRH neurons in mouse forebrain is GABA-ergic, 2) expression of GAD67-GFP in GnRH neurons is at least in part regulated by estrogen, and 3) GnRH neurons secrete GABA to regulate themselves.

P2X7 receptors and Fyn kinase mediate ATP-induced oligodendrocyte progenitor cell migration.

Recruitment of oligodendrocyte precursor cells (OPCs) to the lesions is the most important event for remyelination after central nervous system (CNS) injury or in demyelinating diseases. However, the underlying molecular mechanism is not fully understood. In the present study, we found high concentrations of ATP could increase the number of migrating OPCs in vitro, while after pretreatment with oxidized ATP (a P2X7 receptor antagonist), the promotive effect was attenuated. The promotive effect of 2'(3')-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) (a P2X7 receptor agonist) was more potent than ATP. After incubation with BzATP, the activity of Fyn, one member of the Src family of kinases, was enhanced. Moreover, the interaction between P2X7 and Fyn was identified by co-immunoprecipitation. After blocking the activity of Fyn or down-regulating the expression of Fyn, the migration of OPCs induced by BzATP was inhibited. These data indicate that P2X7 receptors/Fyn may mediate ATP-induced OPC migration under pathological conditions.

Up-regulation of P2X7 receptors mediating proliferation of Schwann cells after sciatic nerve injury.

Peripheral nerve injury (PNI) is a common disease, which results in a partial or total loss of motor, sensory and autonomic functions, leading to a decrease in quality of life. Schwann cells play a vital role in maintaining the peripheral nervous system and in injury and repair. Using immunohistochemistry, Western blot, calcium assay and bromodeoxyuridine (BrdU) proliferation assay, the present study clearly demonstrated that P2X7 receptors (R) were expressed in myelinating and non-myelinating Schwann cells in longitudinal sections of sciatic nerves. After sciatic nerve injury (SNI), P2X7R expression in Schwann cells of injured sciatic nerves was significantly up-regulated during the early days of SNI. Double immunofluorescence of proliferating cell nuclear antigen (PCNA) and P2X7R implied that P2X7R may be involved in proliferation of Schwann cells. Further experiments on primary cultures of Schwann cells showed that P2X7R are functionally expressed in Schwann cells of rat sciatic nerves; ATP via P2X7R can promote Schwann cell proliferation, possibly via the MAPK/ERK intracellular signalling pathway. Other possible roles of P2X7R on Schwann cells are discussed.

LINGO-1 receptor promotes neuronal apoptosis by inhibiting WNK3 kinase activity.

LINGO-1 is a functional component of the Nogo receptor 1 · p75(NTR) · LINGO-1 and Nogo receptor 1 · TAJ (TNFRSF19/TROY)·LINGO-1 signaling complexes. It has recently been shown that LINGO-1 antagonists significantly improve neuronal survival after neural injury. However, the mechanism by which LINGO-1 signaling influences susceptibility to apoptosis remains unknown. In an effort to better understand how LINGO-1 regulates these signaling pathways, we used an established model of serum deprivation (SD) to induce neuronal apoptosis. We demonstrate that treatment either with a construct containing the intracellular domain of LINGO-1 or with Nogo66, a LINGO-1 receptor complex agonist, resulted in an enhanced rate of apoptosis in primary cultured cortical neurons under SD. Reducing the expression levels of the serine/threonine kinase WNK3 using shRNA or inhibiting its kinase activity had similar effects on the survival of serum-deprived neurons. Consistent with these observations, we found that LINGO-1 and WNK3 co-localized and co-precipitated in cultured cortical neurons and brain tissue. Significantly, this co-association was enhanced by Nogo66 treatment. Binding of WNK3 to the intracellular domain of LINGO-1 led to a reduction in WNK3 kinase activity, as did Nogo66 stimulation. Moreover, in vitro and in vivo evidence indicates that endogenous WNK3 suppresses SD-induced neuronal apoptosis in a kinase-dependent manner, as the expression of either a WNK3 RNAi construct or a kinase-dead N-terminal fragment of WNK3 led to increased apoptosis. Taken together, our results show that LINGO-1 potentiates neuronal apoptosis, likely by inhibiting WNK3 kinase activity.

LPAR6 Participates in Neuropathic Pain by Mediating Astrocyte Cells via ROCK2/NF-κB Signal Pathway.

Neuropathic pain (NPP) is a common type of chronic pain. Glial cells, including astrocytes (AS), are believed to play an important role in the progression of NPP. AS cells can be divided into various types based on their expression profiles, among which A1 and A2 types have clear functions. A1-type AS cells are neurotoxic, while A2-type AS cells exert neuroprotective functions. Some types of lysophosphatidic acid receptors (LPAR) have been shown to play a role in NPP. However, it remains unclear how AS cells and LPAR6 affect the occurrence and progression of NPP. In this study, we established a mouse model of chronic constriction injury (CCI) to simulate NPP. It was found that the expression of LPAR6 in AS cells of the spinal dorsal horn was increased in the CCI model, and the thresholds of mechanical and thermal pain were elevated after knocking out LPAR6, indicating that LPAR6 and AS cells participated in the occurrence of NPP. The experiment involved culturing primary AS cells and knocking down LPAR6 by Lentivirus. The results showed that the NF-κB signal pathway was activated and the number of A1-type AS cells increased in the CCI model. However, LPAR6 knockdown inhibited the NF-κB signal pathway and A1-type AS cells. The results of the mRNA sequencing and immunoprecipitation test indicate an interaction between LPAR6 and ROCK2. Inhibiting ROCK2 by Y-27632 increased mechanical and thermal pain thresholds and alleviated NPP at the molecular level. The study presents evidence that LPAR6 activates the NF-κB pathway through ROCK2 and contributes to the progression of NPP by increasing A1-type AS and decreasing A2-type AS. This suggests that LPAR6 could be a potential therapeutic target for alleviating NPP. Clinical applications that are successful can offer new therapeutic options, enhance the quality of life for patients, and potentially uncover new mechanisms for pain modulation.

Developmental expression of P2X5 receptors in the mouse prenatal central and peripheral nervous systems.

The functions of P2X purinoceptors (P2X1-7) in the nervous system of adults have been widely studied. However, little is known about their roles during embryonic development. Our previous work has reported an extensive expression of P2X5 receptors in the adult mouse central nervous system. In the present study, we have examined the expression pattern of P2X5 receptor mRNA and protein during prenatal development of the mouse nervous system (from embryonic day E8 to E17). P2X5 receptors appeared in the neural tube as early as E8 and were gradually confined to new-born neurons in the cortical plate and ventral horn of the spinal cord. Heavy signals for P2X5 receptors were also found in dorsal root ganglia (DRG), retina, olfactory epithelium, and nerve fibers in skeletal muscles. In conclusion, P2X5 receptors were strongly represented in the developing mouse nervous system. The transient high expression pattern of P2X5 receptors in epithelium-like structures suggests a role during early neurogenesis.

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-1ß 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.