Inhibition of P2X7 receptor ameliorates transient global cerebral ischemia/reperfusion injury via modulating inflammatory responses in the rat hippocampus

Ketan Chu 0 2 4 Bo Yin 0 2 4 Jingye Wang 4 6 Guoping Peng 0 2 4 Hui Liang 0 2 4 Ziqi Xu 0 2 4 Yue Du 0 2 4 Marong Fang 4 5 Qiang Xia 4 Benyan Luo 0 2 4 7 0 Brain Medical Center, First Affiliated Hospital, Zhejiang University School of Medicine , Hangzhou , China 1 Qingchun Road , Hangzhou 310003 , China 2 Department of Neurology, First Affiliated Hospital, Zhejiang University School of Medicine , 76 3 Yuhangtang Road , Hangzhou 310058 , China 4 School of Medicine , 388 5 Institute of Anatomy and Cell Biology, Medical College, Zhejiang University , Hangzhou , China 6 Department of Neurology, First Affiliated Hospital, Anhui Medical University , Hefei , China 7 Key Laboratory of Medical Neurobiology, Ministry of Health , Hangzhou , China Background: Neuroinflammation plays an important role in cerebral ischemia/reperfusion (I/R) injury. The P2X7 receptor (P2X7R) has been reported to be involved in the inflammatory response of many central nervous system diseases. However, the role of P2X7Rs in transient global cerebral I/R injury remains unclear. The purpose of this study is to determine the effects of inhibiting the P2X7R in a rat model of transient global cerebral I/R injury, and then to explore the association between the P2X7R and neuroinflammation after transient global cerebral I/R injury. Methods: Immediately after infusion with the P2X7R antagonists Brilliant blue G (BBG), adenosine 5-triphosphate-2,3-dialdehyde (OxATP) or A-438079, 20 minutes of transient global cerebral I/R was induced using the four-vessel occlusion (4-VO) method in rats. Survival rate was calculated, neuronal death in the hippocampal CA1 region was observed using H & E 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-1, TNF- 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 (10 g) and A-0438079 (3 g), and a low dosage of OxATP (1 g) significantly increased survival rates, reduced I/R-induced learning memory deficit, and reduced I/R-induced neuronal death, DNA cleavage, and glial activation and inflammatory cytokine overexpression in the hippocampus. Conclusions: Our study indicates that inhibiting P2X7Rs 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. - Background Transient 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 may paradoxically exacerbate brain injury, which is called cerebral ischemia/reperfusion(I/R) injury [1]. Therefore, efforts need be made that not only preserve cerebral blood flow, but also prevent the actual mechanisms that trigger brain damage after I/R injury [2]. 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 bloodbrain barrier disruption and delayed neuronal death [3]. 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 [3,4]. The P2X7 receptor(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 [5]. 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-1, TNF- and IL-6 [6,7]. Recently, the P2X7R has been reported to be involved in neuroinflammation in many CNS diseases including Alzheimers disease (AD), epilepsy, spinal cord injury and multiple sclerosis, and treatment with P2X7R antagonists reduces experimentally induced neuroinflammation in animal models of such diseases [6,8-12]. The P2X7R has also been reported to participate in cerebral ischemic injury. In vitro and in vivo studies have shown that inhibition of P2X7Rs reduced oxygen and glucose deprivation-induced oligodendrocyte death [13] as well as infarct volume after transient middle cerebral artery occlusion(MCAO) injury [14,15]. However, ischemic injury exacerbation by P2X7R antagonists has also been reported [16,17]. To date, the contribution of the P2X7R to cerebral ischemic injury remains an issue, and whether inhibition of P2X7R has beneficial or harmful effects in global cerebral I/R injury has not been studied. We, therefore, designed experiments using two widely used P2X7R antagonists, Brilliant blue G (BBG) and adenosine 5-triphosphate-2,3-dialdehyde (OxATP) [6], and the selective P2X7R antagonist A-438079 [18] to investigate the role of P2X7R in a rat model of transient global cerebral I/R injury. We also explored the association between the P2X7R and neuroinflammation after transient global cerebral I/R injury. Methods Animals and surgical procedures Male SpragueDawley rats weighing 260320 g were provided by the Animal Center of Zhejiang University. All procedures used in this study were carried out according to the guidelines of the NIH Guide for the Care and Use of Laboratory Animals and have been approved by the Ethics Committee for the Use of Experimental Animals in Zhejiang University. Twenty minutes of global cerebral ischemia was induced by the four-vessel occlusion (4-VO) method with slight modification, as established by Pulsinelli [19], and routinely used in our laboratory [20,21]. Briefly, anesthesia was induced with 4% (w/v) choral hydrate (400 mg/kg, intraperitoneally (i.p.)), then the bilateral common carotid arteries (CCAs) were freed and both vertebral arteries were permanently electrocauterized. Rats were allowed to recover for 24 hours after closing the surgical incisions. On the following day (+0D), anesthesia was applied, the surgical incision in the neck was opened and both CCAs were occluded with aneurysm clips to induce global cerebral ischemia. The clips were removed for reperfusion. Rectal temperature was maintained at 36.5 to 37.5C throughout the (...truncated)