Role Of P2X7Receptor In Global Cerebral Ischemia/Reperfusion Injury And Its Related Mechanisms

BackgroundTransient global cerebral ischemia is one of the major complications of clinical emergencies such as cardiac arrest, drowning or severe systemic hypotension during a surgical procedure. Currently, the most adequate treatment for these patients is re-establishing perfusion of the brain as soon as possible. However, reperfusion mayparadoxically exacerbate brain injury, which is called cerebral ischemia/reperfusion (I/R) injury. Therefore, efforts need to be made that not only to preserve cerebral blood flow, but also to prevent the actual mechanisms that trigger brain damage after I/R injury.Neuroinflammation, which is characterized by microglial and astroglial activation, as well as the release of cytotoxic agents (cytokines, matrix metalloproteinases, nitric oxide and reactive oxygen species) can be triggered by cerebral I/R injury, which can contribute to blood-brain barrier disruption and delayed neuronal death. Subsequently, these damaged cells release more toxic mediators, which in turn activate more immune cells. Thus, prolonged inflammation caused by this vicious circle exacerbates brain damage. Taken together, anti-inflammation therapy may become a promising therapeutic strategy for the treatment of cerebral I/R injury.The P2X7receptor(P2X7R), a purinergic receptor, was first discovered in macrophages. In the central nervous system (CNS), the P2X7R is predominantly expressed in microglia which are the resident macrophages of the brain. The P2X7R can be activated by high concentrations of ATP. Stimulating the P2X7R leads to microglial activation, reactive oxygen species production and increased secretion of pro-inflammatory cytokines such as IL-1β, TNF-α and IL-6. Recently, the P2X7R has been reported to be involved in neuroinflammation in many CNS diseasesincluding A lzheimer’s disease (AD), epilepsy, spinal cord injury and multiple sclerosis, and treatment with P2X7R antagonists reduces experimentally induced neuroinflammation in animal models of such diseases.The P2X7receptor(P2X7R), a purinergic receptor, was first discovered in macrophages. In the central nervous system (CNS), the P2X7R is predominantly expressed in microglia which are the resident macrophages of the brain. The P2X7R can be activated by high concentrations of ATP. Stimulating the P2X7R leads to microglial activation, reactive oxygen species production and increased secretion of pro-inflammatory cytokines such as Interleukin-1β(IL-1β), Tumor necrosis factor-α (TNF-a) and Interleukin-6(IL-6). Recently, the P2X7R has been reported to be involved in neuroinflammation in many CNS diseases including Alzheimer’s disease (AD), epilepsy, spinal cord injury and multiple sclerosis, and treatment with P2X7R antagonists reduces experimentally induced neuroinflammation in animal models of such diseases. Only a few study have reported the effect of P2X7R in cerebral ischemic injury, and the results are inconsistent. So it is still worth to be discussed.AimTo clarify the role of P2X7R in global cerebral I/R-induced injury and neruoinflammation, in the present study we performed the following investigations.(1) We examined the P2X7R expression of P2X7R after global cerebral I/R-induced injury in rats.(2) Using P2X7R antagonist, we investigated the role of P2X7R in global cerebral I/R injury. We also explored the association between the P2X7R and neuroinflammation after transient global cerebral I/R injury. (3) We also investigated the interaction between P2X7R and NADPH Oxidiase(NOX), and thier effect in the regulation on neuroinflammation.Part Ⅰ Expression properties of P2X7R in the hippocampal CA1region after global cerebral I/R injury.Methods:Twenty mins global cerebral I/R injury was conducted on male Sprague-Dawley rats weighing260-320g using four-vessel occlusion (4-VO) method. Regional cerebral blood flow (rCBF) from10mins before ischemia to90mins after reperfusion was measured using a laser Doppler flowmetry; HE staining was performing to observe the neuronal death6hours,1day,3days,7days and14days after reperfusion; activated microglia and astrocytes were detected by immunohistochemical staining; P2X7R expression was examined by western bolts and immunobistochemical staining.Results:During the ischemic period, the rCBF was lower than20%; more than90%CA1neurons dead after3days-reperfusion. glial activation, gliosis and P2X7R expression were increased in a time-dependent manner after reperfusion. P2X7R mainly expressed on microglia-like cells.Conclusion:P2X7R is involved in global cerebral I/R injury and is associated with I/R-induced neuroinflammation.Part Ⅱ Inhibition of P2X7R ameliorates transient global cerebral I/R injury via modulating inflammatory responses in the rat hippocampusMethods:Immediately after infusion with the P2X7R antagonists Brilliant blue G (BBG), adenosine5’-triphosphate-2’,3’-dialdehyde (OxATP) or A-438079, tweenty minutes of transient global cerebral I/R was induced using the4-VO method in rats. Survival rate was calculated, neuronal death in the hippocampal CA1region was observed using HE staining, and DNA cleavage was observed by deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL). In addition, behavioral deficits were measured using the Morris water maze, and RT-PCR and immunohistochemical staining were performed to measure the expression of IL-1β,TNF-α and IL-6, and to identify activated microglia and astrocytes.Results:The P2X7R antagonists protected against transient global cerebral I/R injury in a dosage-dependent manner. A high dosage of BBG (10μg) and A-438079(3μg). and a low dosage of OxATP (1μg) significantly increased survival rates, reduced I/R-induced learning memory deficit, and reduced I/R-induced neuronal death, DNA cleavage, glial activation and inflammatory cytokine overexpression in the hippocampus.Conclusions:Our study indicates that inhibiting P2X7R.S protects against transient global cerebral I/R injury by reducing the I/R-induced inflammatory response, which suggests inhibition of P2X7Rs may be a promising therapeutic strategy for clinical treatment of transient global cerebral I/R injury.Part Ⅲ P2X7R-NOX participates in transient global cerebral I/R-induced neuroinflammationMethods:Rats were divided into six group:Sham1, Sham2, Saline, Vehicle, A-438079(3μg i.c.v.), Apocynin (10mg/kg i.p. twice). NOX activity was measured with Lucigenin-enhanced chemiluminescence; expression of gp91phox and p47phox, p47phox translocation were detected by western bloting; neuronal death in the hippocampal CA1region was observed using HE staining and immunohistochemical staining were performed to identify activated microglia and astrocytes.Results:NOX activity was elevated at the third day after global cerebral ischemia reflected by elevated relative light units (RLU) values, and increased gp91phox, p47phox expression and less p47phox translocation. The P2X7R antagonists A-438079suppressed the I/R-induced NOX activity elevation. NOX antagonist apocynin protected against transient global cerebral I/R injury and reduced I/R-induced glial activation.Conclusions:P2X7R-NOX participated in the I/R-induced brain injury and neurolinflammation after I/R injury.