The Role Of P2X7Receptor In Secondary Injury After Subarachnoid Hemorrhage Of Rats And The Relevant Mechanism

BackgroundSubarachnoid hemorrhage (SAH) is a devastating cerebrovascular disease, accounting for only5%of all strokes. Approximately85%of cases of spontaneous SAH are due to the rupture of an intracranial aneurysm. Despite recent improvements our knowledge of SAH pathophysiology and the management of ruptured aneurysms, which can include diagnosis method, surgical clipping, endovascular treatment and peri operation therapy, the rebleeding rate among SAH patients has decreased. However, SAH remains a serious and significant health problem with high mortality and morbidity. During the past four decades, considerable efforts have been made to investigate vasospasm as the primary mechanism of delayed cerebral ischemia and poor outcomes underlying SAH injury. However, that theory targeting vasospasm is being questioned increasingly. Vasospasm is now considered as one pathophysiology after SAH, even only a late manifestation. In recent years, an emerging body of evidence at present suggests that SAH is likely to have a multifactorial etiology beyond vasospasm, such as early brain injury (EBI), cortical spreading depolarization and impaired microcirculatory function, may closely dictate fatal prognosis in patients with SAH. EBI has been considered a primary target to combat SAH.EBI refers to immediate injury and lasts72h until the development of vasospasm. EBI has emerged as a new frontier, which includes mechanical impact, raised intracranial pressure, decreases in cerebral blood flow and cerebral perfusion pressure, cortical spreading depolarization, apoptosis, necrosis, autophagy, ions disturbance, inflammation, blood brain barrier disruption, and cerebral edema. However, EBI requires more investigation to understand specific mechanisms and signal pathways.Inflammation and neuronal apoptosis play a key role in EBI.Inflammatory response starts early after SAH. The level of cytokines in cerebrospinal fluid and serum of the patients with SAH is correlated with the patients’prognosis. Among a variety of inflammatory cytokines, interleukin-1β(IL-1β) is well accepted as a pivotal cytokine in neuroinflammation following SAH. However, it remains unclear how IL-1β is produced and secreted after SAH. Cryopyrin inflammasome can be activated by some stimuli, including pathogen-associated molecular patterns and damage-associated molecular pattern molecules. Upon activation, cryopyrin inflammasome results in the autoactivation of pro-caspase-1, which in turn activates pro-IL-1β/IL-18. Low intracellular K+induced by P2X7R, which can active cryopyrin inflammasome. It is reported that active P2X7R is the upstream of inflammasome activation. However, the specific role of P2X7R/cryopyrin inflammasome in SAH has not yet been established.Apoptosis of neuronal cells is believed to contribute to brain injury after SAH. A variety of pro-apoptotic and anti-apoptotic molecules and signaling pathways are involved in SAH-induced brain injury. In experimental studies, inhibition of apoptosis is beneficial for SAH animals. Caspase-3is a frequently activated death protease, which is crucial mediator of apoptosis. The therapeutic strategy targeting on caspase-3is very important for the outcome of SAH.The patients with SAH often present with multiple organ dysfunction and systemic inflammatory response, which include heart arrest, pulmonary and gastrointestinal injury. SAH patients that develop neurogenic pulmonary edema would have a higher mortality rate. Two different mechanisms of neurogenic pulmonary edema likely coexist. One is an ’inflammatory’ mechanism and the other is a hemodynamic mechanism. P2X7R is expressed on several lung cell types, which indicated that P2X7R may play a crucial role in lung inflammation. However, it remains unclear whether the P2X7R is involved in neurogenic pulmonary edema after SAH.Rapid expanding literature suggested that P2X7R antagonists have potential to treat several central nervous system diseases. This study aimed to explore whether P2X7R inhibition can provide neuroprotective effect for SAH and investigate whether those neuroprotection is associated with anti-inflammatory response, anti-apoptosis and attenuation of neurogenic pulmonary edema.Part Ⅰ. The role of P2X7R/cryopyrin inflammasome axis in neuroinflammation after subarachnoid hemorrhageObjectiveThe study is to investigate the change of brain expression of cryopyrin inflammasome components, including cryopyrin, ASC, and pro-caspase-1, and downstream molecules, IL-1βand IL-18after SAH. The study further aims to test the role of P2X7R/cryopyrin inflammasome in the pathophysiology of SAH, in order to extend the knowledge of inflammation after SAH. The study may provide us a novel therapeutic strategy of the inhibition of inflammation after SAH.MethodsFour separate experiments were conducted (Experiments1to4). Experiment1:SAH was induced using the endovascular perforation model. The rats were randomly divided into6groups (sham, and SAH after2,6,24,48, and72h). The temporal expression of cryopyrin, P2X7R and mature IL-1β were detected by Western Blot. The co-location of cryopyrin or P2X7R and the microglia marker, ionized calcium bindingadaptor molecule-1(Iba-1), were examined by immunofluorescence.Experiment2:The animals were randomly divided into5groups:sham, SAH+vehicle (SAH+saline, intracerebroventricular injection), SAH+scramble siRNA, SAH+cryopyrin siRNA and SAH+P2X7R siRNA. Cryopyrin siRNA and P2X7R siRNA were administered by intracerebroventricular infusion into the left lateral ventricle. Neurobehavioral deficits were blindly evaluated with the modified Garcia test plus the beam balance test. Brain edema was evaluated by the wet weight/dry weight method. The expression of cryopyrin, P2X7R, cleaved caspase-1and mature IL-1β/IL-18were detected by Western Blot.Experiment3:The rats were randomly divided into5groups:naive, naive+LPS, naive+LPS+BzATP, naive+LPS+BzATP+Scramble siRNA, and naive+LPS+BzATP+Cryopyrin siRNA. Naive rats received an intracerebroventricular priming dose of LPS to induce neuroinflammation. BzATP, a P2X7R agonist, was administrated intracerebroventricularly3h after LPS injection. Cryopyrin siRNA or scramble siRNA was injected21h prior to LPS injection. The expression of cryopyrin, cleaved caspase-1and mature IL-1β were detected by Western Blot. This experiment is executed to test the relationship between P2X7R and cryopyrin inflammasome.Experiment4:The rats were divided into5groups:sham, SAH+vehicle, SAH+Brilliant Blue G (BBG)(5mg/kg), SAH+BBG (30mg/kg) and SAH+BBG (100mg/kg). Neurobehavioral deficits were blindly evaluated with the modified Garcia test plus the beam balance test at24h and72h after SAH. Brain edema was evaluated by the wet weight/dry weight method. The expression of P2X7R, cleaved caspase-1, mature IL-1β/IL-18and myeloperoxidase (MPO) were detected by Western Blot. The neutrophil infiltration was evaluated by staining of MPO.Results(1) Western Blot showed that cryopyrin and mature IL-1β levels reached its peak around24h after SAH, respectively. However, P2X7R level did not increase during72h following SAH. Immunofluorescence double-staining demonstrated that both cryopyrin and P2X7R were co-located with Iba-1, indicated cryopyrin and P2X7R were expressed in microglia.(2) Either cryopyrin siRNA or P2X7R siRNA treatment significantly abolished caspase-1activation and subsequent mature IL-1β/IL-18secretion at24h after SAH. Furthermore, cryopyrin siRNA and P2X7R siRNA attenuated SAH-induced brain edema and ameliorated neurological impairment.(3)In LPS-priming naive rat model, BzATP, a P2X7R agonist, enabled the increase of cleaved caspase-1level as well as the level of mature IL-1β. Cryopyrin siRNA prevented the proinflammatory effect of BzATP.(4) The middle dosage of BBG (30mg/kg) improved neurobehavioral functions and attenuated brain edema, which resulted from decreased cleaved caspase-1and the subsequent production of mature IL-1β/IL-18following SAH. Furthermore, BBG suppressed neutrophil infiltration in the cortex at24h after SAH.ConclusionP2X7R/cryopyrin inflammasome axis may be a new target that could be effective for modulating the caspase-1/IL-1β/IL-18after SAH. Hence, blockade of P2X7R/cryopyrin inflammasome axis can exert potentially anti-inflammatory effects for SAH. Experiment1:SAH was induced using the endovascular perforation model. The rats were randomly divided into6groups (sham, and SAH after2,6,24,48, and72h). The temporal expression of cryopyrin, P2X7R and mature IL-1β were detected by Western Blot. The co-location of cryopyrin or P2X7R and the microglia marker, ionized calcium bindingadaptor molecule-1(Iba-1), were examined by immunofluorescence.Experiment2:The animals were randomly divided into5groups:sham, SAH+vehicle (SAH+saline, intracerebroventricular injection), SAH+scramble siRNA, SAH+cryopyrin siRNA and SAH+P2X7R siRNA. Cryopyrin siRNA and P2X7R siRNA were administered by intracerebroventricular infusion into the left lateral ventricle. Neurobehavioral deficits were blindly evaluated with the modified Garcia test plus the beam balance test. Brain edema was evaluated by the wet weight/dry weight method. The expression of cryopyrin, P2X7R, cleaved caspase-1and mature IL-1β/IL-18were detected by Western Blot.Experiment3:The rats were randomly divided into5groups:naive, naive+LPS, naive+LPS+BzATP, naive+LPS+BzATP+Scramble siRNA, and naive+LPS+BzATP+Cryopyrin siRNA. Naive rats received an intracerebroventricular priming dose of LPS to induce neuroinflammation. BzATP, a P2X7R agonist, was administrated intracerebroventricularly3h after LPS injection. Cryopyrin siRNA or scramble siRNA was injected21h prior to LPS injection. The expression of cryopyrin, cleaved caspase-1and mature IL-1β were detected by Western Blot. This experiment is executed to test the relationship between P2X7R and cryopyrin inflammasome.Experiment4:The rats were divided into5groups:sham, SAH+vehicle, SAH+Brilliant Blue G (BBG)(5mg/kg), SAH+BBG (30mg/kg) and SAH+BBG (1OOmg/kg). Neurobehavioral deficits were blindly evaluated with the modified Garcia test plus the beam balance test at24h and72h after SAH. Brain edema was evaluated by the wet weight/dry weight method. The expression of P2X7R, cleaved caspase-1, mature IL-1β/IL-18and myeloperoxidase (MPO) were detected by Western Blot. The neutrophil infiltration was evaluated by staining of MPO.Results(1) Western Blot showed that cryopyrin and mature IL-1β levels reached its peak around24h after SAH, respectively. However, P2X7R level did not increase during72h following SAH. Immunofluorescence double-staining demonstrated that both cryopyrin and P2X7R were co-located with Iba-1, indicated cryopyrin and P2X7R were expressed in microglia.(2) Either cryopyrin siRNA or P2X7R siRNA treatment significantly abolished caspase-1activation and subsequent mature IL-1(3/IL-18secretion at24h after SAH. Furthermore, cryopyrin siRNA and P2X7R siRNA attenuated SAH-induced brain edema and ameliorated neurological impairment.(3) In LPS-priming naive rat model, BzATP, a P2X7R agonist, enabled the increase of cleaved caspase-1level as well as the level of mature IL-1β. Cryopyrin siRNA prevented the proinflammatory effect of BzATP.(4) The middle dosage of BBG (30mg/kg) improved neurobehavioral functions and attenuated brain edema, which resulted from decreased cleaved caspase-1and the subsequent production of mature IL-1β/IL-18following SAH. Furthermore, BBG suppressed neutrophil infiltration in the cortex at24h after SAH.ConclusionP2X7R/cryopyrin inflammasome axis may be a new target that could be effective for modulating the caspase-1/IL-1β/IL-18after SAH. Hence, blockade of P2X7R/cryopyrin inflammasome axis can exert potentially anti-inflammatory effects for SAH. Part Ⅱ. P2X7Receptor antagonism ameliorates neuronal apoptosis after subarachnoid hemorrhageObjectiveThe study is to investigate the role of P2X7R antagonism in neuronal apoptosis after SAH. In addition, this study explores whether P2X7R/P38Mitogen-activated protein kinase (MAPK) take part in the neuronal apoptosis after subarachnoid hemorrhage. The study further aims to test the role of apoptosis in the pathophysiology of SAH. The study may provide us a novel therapeutic strategy of the inhibition of apoptosis after SAH.MethodsTwo experiments were conducted (Experiments1and2).Experiment1:The rats were divided into4groups:sham, SAH+vehicle, SAH+BBG, SAH+BBG+BzATP.The animals were intraperitoneally treated with BBG or saline at30minutes after SAH. BzATP, a P2X7receptor agonist, was intracerebroventricularly administered3h before SAH. Neurobehavioral deficits were blindly evaluated with the modified Garcia test plus the beam balance test. Brain edema was evaluated by the wet weight/dry weight method. The expression of P2X7R, phosphorylated p38MAPK, Bcl-2and cleaved caspase-3were detected by Western Blot. Double immunofluorescence staining using P2X7R and NeuN showed the cell type of P2X7R. Neuronal apoptosis was examined by double immunofluorescence staining using TUNEL and neuronal nuclei.Experiment2:The animals were randomly divided into4groups:sham, SAH+vehicle, SAH+scramble siRNA and SAH+P2X7R siRNA. P2X7R siRNA was administered by intracerebroventricular infusion into the left lateral ventricle24h before SAH. Neurobehavioral deficits were blindly evaluated with the modified Garcia test and the beam balance test. The expression of P2X7R, cleaved caspase-3and phosphorylated p38MAPK were detected by Western Blot.Results(1)Immunofluorescence staining showed that P2X7R was expressed in neuron at24h after SAH.(2) BBG attenuated neuronal apoptosis in the subcortex, which was associated with decreased expression of phosphorylated p38mitogen-activated protein kinase and cleaved caspase-3and an increased expression of Bcl-2.(3) The beneficial effects of P2X7R siRNA were also mediated by inhibition of p38mitogen-activated protein kinase pathway and cleaved caspase-3.ConclusionP2X7R is involved in neuronal apoptosis after SAH. Inhibition of P2X7R can prevent p38MAPK and cleaved caspase-3and eventually reduce early brain injury after SAH. It provided a new mechanism for apoptosis after SAH. Part III. Role of P2X7receptor in Neurogenic Pulmonary Edema after Subarachnoid HemorrhageObjectiveThe present study aimed to test the therapeutic potential of BBG, a selective P2X7R antagonist, on neurogenic pulmonary edema (NPE)in a rat SAH model. We wanted to explore the role of P2X7R in the pathophysiology of NPE.Furthermore, the study is to show the effect of inflammation in NPE and to find a novel therapeutic strategy for NPE.MethodsThe rats were randomly divided into sham, vehicle-, or BBG-treatment groups. NPE clinical symptoms, body weight and wet lung weight were measured at24h following SAH. Western blot was performed to test the change of mature IL-1β, myeloperoxidase (MPO) and tight junction proteins ZO-1and occluding. Gelatin zymography is to examine the activity of matrix metallopeptidase-9(MMP-9). Immunofluorescence staining is to show the co-location of P2X7R and Tl-a. Hemotoxylin and eosin staining is to compare the severity of lung damage in all groups.Results(1) P2X7R and the marker of alveolar type I epithelial cells (the mucin-type glycoprotein, T1-α) immunoreactivities were generally co-localized.(2) The incidence of clinical symptoms was correlated with pulmonary index after SAH.(3) BBG administration decreased IL-1β,MPO, and MMP-9activation, but increased tight junction proteins, such as ZO-1and occluding. HE staining showed BBG significantly ameliorated pulmonary injury after SAH.ConclusionWe presented the first experimental study describing clinical symptoms for useful NPE diagnosis after SAH. Furthermore, P2X7R antagonism ameliorates NPE via its anti-inflammatory effects after SAH. This study is helpful to discover a new treatment for NPE and warrants further research.