The Role And Mechanism Of NETs In Microthrombosis And Early Brain Injury After Subarachnoid Hemorrhage

Subarachnoid hemorrhage(SAH),which is mainly caused by ruptured intracranial aneurysm,is one of the most common critical diseases in neurosurgery.Because of its sudden onset and dangerous condition,it is harmful to patients,often resulting in disability and even death.The pathophysiological mechanism of brain injury after SAH is complex.Among which,delayed cerebral vasospasm(CVS)and early brain injury(EBI)are the two main complications that usually faced by patients with SAH,and are the important causes of disability and death.In order to alleviate the brain injury after SAH and effectively improve the poor prognosis of patients,researchers have carried out a lot of clinical and basic research work for decades.In the early stage,researchers mainly focused on the delayed cerebral vasospasm,which was thought to be the main reason for poor prognosis of SAH patients.However,the results of a multicenter,randomized,double-blind clinical study of cerebral vasospasm showed that the prognosis of patients with SAH did not improve well even when CVS was well controlled.Based on the results of this study,it is speculated that after SAH,besides CVS,there are other mechanisms of neurological damage that can lead to poor prognosis of patients.In recent years,experts have recognized the importance of early brain injury(EBI),that is,brain injury that occurs from the beginning of SAH to 72 hours.Studies have found that early brain injury may be an important factor leading to secondary neurological dysfunction and poor prognosis after SAH.Previous studies have shown that microthrombosis plays an important role in the poor prognosis after aneurysmal subarachnoid hemorrhage.The number of thrombotic microvessels may be associated with cerebral blood barrier disruption and neuronal damage,but the specific mechanism of microthrombosis is still unknown.Therefore,taking microthrombosis as the target may provide a new strategy for the treatment of subarachnoid hemorrhage.Neutrophil extracellular traps(NETs)are one of the important ways for neutrophils to function.When neutrophils are activated by various external stimulations,they will release the nuclear chromatin into the extracellular environment in different ways to form a high concentration of reticular DNA complex.The reticular complex takes chromatin DNA as the main skeleton,on which a variety of enzymes and antibacterial proteins are attached.When the body is attacked by exogenous infection,NETs can effectively capture pathogenic microorganisms and play an anti-infection role.In addition to infectious diseases,the formation of NETs has also been found to be involved in the pathological processes of a variety of non infectious disease,such as autoimmune diseases,atherosclerosis,kidney injury,Alzheimer’s disease and tumor metastasis.Recently,the formation of NETs has also been reported to be involved in the pathological processes of various central nervous system(CNS)diseases.Studies have shown that the use of DNase Ⅰ to degrade NETs in ischemic stroke and traumatic brain injury improves neural function.NETs also play an important role in the early brain injury after SAH.NETs released by neutrophils after SAH can induce microglia to change into pro-inflammatory subtypes,thus promoting neuroinflammation and leading to adverse outcomes.Promoting coagulation and inducing microthrombosis is one of the important mechanisms of NETs.Studies have shown that microthrombosis after ischemic stroke is closely related to NETs,and the generation of NETs is also an important factor for blood hypercoagulability and pulmonary microthrombosis in patients with novel coronavirus pneumonia.Based on the existing research results,we propose the hypothesis that NETs may also play an important role in the formation of microthrombosis after SAH.This study focuses on the role of NETs in early microthrombosis and early brain injury after SAH,thus exploring new effective targets for clinical treatment of SAH.Part Ⅰ:Expression of NETs in brain tissue of experimental mice after SAH and its relationship with microthrombosisObjective:To observe the expression of NETs in the cerebral cortex of mice at different time points after SAH,and preliminarily study the relationship between NETs and microthrombosis.Methods:There were 2 parts in this experiment.In the first part,the mouse SAH model was established by internal carotid artery endovascular perforation.The study was divided into two groups:sham operation group(Sham group)and subarachnoid hemorrhage group(SAH group).The subarachnoid hemorrhage group was divided into four groups according to the sequence of time:4 hours after SAH group,12 hours after SAH group,24 hours after SAH group,and 48 hours after SAH group.After the establishment of the SAH model,the mice were sacrificed at the corresponding time point,and the protein was extracted from the temporal lobe cortex of the mouse brain,and the expression of NETs was detected by Western blot.In the second part,the mouse SAH model was established by internal carotid artery endovascular perforation.The study was divided into two groups:sham operation group(Sham group)and subarachnoid hemorrhage group(SAH group).The mice were sacrificed 24 hours after SAH,and the brain tissue was taken to observe the formation of NETs and microthrombosis and the position relationship between them by immunofluorescence staining.Results:1.Western blotting analyses demonstrated that the level of NETs in the brain was significantly increased at 4h after SAH,and peaked at 24h,after which the expression of NETs gradually decreased.2.The results of immunofluorescence staining showed that a large number of microthrombi were formed in the microvessels,and some were located in the paravascular space.Furthermore,immunofluorescence staining showed that the microthrombi and NETs were co-labeled.Conclusion:After SAH,the production of NETs in the cerebral cortex of mice increased significantly,and there were also a large number of microthrombosis.Immunofluorescence staining showed that NETs and microthrombos were co-located in the cerebral cortex,suggesting that NETs might be involved in the formation of microthrombos in the cerebral cortex of mice after SAH.Part Ⅱ The role and mechanism of NETs in microthrombosis and microcirculatory disturbance after SAH in miceObjective:To observe the effects of inhibition of NETs production after SAH on cerebral cortex microthrombosis,nerve function damage,brain water content,blood-brain barrier damage,neuron apoptosis and microcirculation disturbance in mice.To further clarify the role and mechanism of NETs in microthrombosis and early brain injury after SAH.MethodsThere were 3 parts in this experiment.In the first part,the mouse SAH model was established by internal carotid artery endovascular perforation.The study was divided into three groups:Sham group,SAH+Vehicle group,and SAH+anti-Ly6G group.For neutrophil depletion,anti-Ly6G antibody was intravenously injected into mice at a dose of 5μg/g mouse at 24h before SAH,and mice in the SAH+Vehicle group was injected with the corresponding volume of normal saline.At 24h after SAH induction,the mice were scored for neurological function(beam balance and modified Garcia tests).Then the mice were sacrificed,and the arterial blood was drawn from the left ventricle for routine blood test.The brain tissue was measured for SAH score and brain water content.The permeability of the blood-brain barrier in different groups was evaluated through the determination of Evans blue dye extravasation.The cerebral cortex tissue proteins were extracted and the expression of NETs and blood-brain barrier related proteins were examined by Western blotting.Immunofluorescence staining was performed to detect the formation of NETs and microthrombosis,FJC and tunel staining were performed to detect the neuronal injury and apoptosis.The damage and apoptosis of neurons after SAH in mice were observed by FJC and TUNEL staining.In the second part,the mouse SAH model was established by internal carotid artery endovascular perforation.The study was divided into three groups:Sham group,SAH+Vehicle group,and SAH+DNase Ⅰ group.The SAH+DNase Ⅰ group was injected with DNase I intraperitoneally and intravenously 1h after the SAH induction,and the SAH+Vehicle group was injected with corresponding volume of normal saline.At 24 h after SAH induction,SAH severity、neurological scores(beam balance and modified Garcia tests),Evans blue dye extravasation and brain water content were measured.The cerebral cortex tissue proteins were extracted and the expression of NETs and blood-brain barrier related proteins were examined by Western blotting.Immunofluorescence staining was performed to detect the formation of NETs and microthrombosis.The injury and apoptosis of neurons were observed by FJC and Tunel staining respectively.In the third part,the mouse SAH model was established by internal carotid artery endovascular perforation.The study was divided into three groups:Sham group,SAH+Vehicle group,and SAH+DNase Ⅰ group.The SAH+DNase Ⅰ group was injected with DNase I intraperitoneally and intravenously 1h after the SAH induction,and the SAH+Vehicle group was injected with corresponding volume of normal saline.Six hours after the induction of SAH,the cerebral cortex blood flow of mice was monitored by laser speckle blood monitor.Results:1.Anti-Ly6G antibody plays a protective role in early brain injury after SAH by clearing neutrophils and inhibiting the production of NETs1.1 Anti-Ly6g antibody significantly reduced the number of neutrophils in peripheral blood after subarachnoid hemorrhage.1.2.Anti-Ly6G antibody significantly reduced the brain water content and attenuated the neurological deficits at 24h after SAH.1.3.Anti-Ly6G antibody reduced the extravasation of EB dye and significantly increased the expression of blood-brain barrier related proteins ZO-1 and Occludin after SAH.1.4.Anti-Ly6G antibody significantly reduced neuronal cell damage and apoptosis at 24h after SAH.1.5.Anti-Ly6G antibody significantly reduced the production of NETs in the cerebral cortex of mice after subarachnoid hemorrhage,and thereby reduced microthrombosis in the cerebral cortex.2.DNase Ⅰ plays a protective role in early brain injury after SAH by clearing NETs and inhibiting microthrombosis2.1.DNase Ⅰ significantly reduced the brain water content and attenuated the neurological deficits at 24h after SAH.2.2.DNase Ⅰ reduced the extravasation of EB dye and significantly increased the expression of blood-brain barrier related proteins ZO-1 and Occludin after SAH.2.3.DNase Ⅰ significantly reduced neuronal cell injury apoptosis at 24h after SAH.2.4.DNase Ⅰ significantly reduced the production of NETs in the cerebral cortex of mice after subarachnoid hemorrhage,and thereby reduced microthrombosis in the cerebral cortex.2.5.DNase Ⅰ significantly reduce the early cerebral cortex hypoperfusion after SAH in mice.Conclusion:The formation of NETs in the cerebral cortex of mice after SAH is closely related to microthrombosis.Intervention with anti Ly6G antibody and DNase Ⅰ can reduce NETs formation can inhibit microthrombosis formation,thereby reducing cerebral cortex hypoperfusion,brain water content,the blood brain barrier damage and reducing the number of neuronal cell damage and apoptosis in the early stage of SAH in mice,improving neural dysfunction in mice,and thus playing a neuroprotective role in the early brain injury after SAH.Part Ⅲ The role of NETs in the disturbance of cerebrospinal fluid circulation and secondary hydrocephalus after SAH in miceObjective:To observe the protective effect of DNase Ⅰ on CSF flow dysfunction and hydrocephalus after SAH,and further study the role and mechanism of NETs in brain injury after subarachnoid hemorrhage.Methods:There were 2 parts in this experiment.In the first part,the mouse SAH model was established by internal carotid artery endovascular perforation.The study was divided into three groups:Sham group,SAH+Vehicle group,and SAH+DNase Ⅰ group.DNase Ⅰ was dissolved in saline.DNase Ⅰ(50μg in 250μL of saline intraperitoneally and a second dose of 10μg intravenously)was injected 1h after SAH induction,and mice in the SAH+Vehicle group was injected with the corresponding volume of normal saline.24h after the SAH model was established,Evans blue solution was injected into the cisterna magna,and the mice were sacrificed after 1 hour of circulation.The distribution of Evans blue at the skull base was observed,and the concentration of Evans blue in the forebrain and deep cervical lymph nodes was measured.In the second part,the mouse SAH model was established(the method and grouping were the same as above).On the first day and the third day after the establishment of the SAH model,the brain of the mice was scanned with a 7.0T small animal magnetic resonance scanner,and the volume of the ventricles was measured.Results:1.At 24h after SAH,by observing the distribution of EB dye in the skull base and detecting the concentration of EB dye in the forebrain and deep cervical lymph nodes,it was found that the circulation of brain fluid in SAH+Vehicle group mice was significantly blocked;DNase Ⅰ intervention can promote the distribution of EB dye in the skull base of S AH+DNase I group mice,and increase the concentration of EB dye in the forebrain and deep cervical lymph node tissues,suggesting that DNase Ⅰ intervention can reduce the disturbance of the circulation of brain effusion and deep cervical lymph drainage in the subarachnoid space of mice.2.By analyzing the MRI images of brain tissue,it was found that the ventricles of mice were significantly dilated on the first and third days after SAH,while DNase Ⅰ intervention could significantly reduce the degree of ventricular dilation at the corresponding time point,suggesting that DNase Ⅰ intervention could reduce the hydrocephalus after SAH in mice.Conclusion:DNase Ⅰ treatment effectively attenuated CSF flow dysfunction and lymphatic drainage disorder,so as to reduce the degree of acute hydrocephalus in mice after subarachnoid hemorrhage.This suggests that NETs may play an important role in the dysfunction of CSF flow,impaired lymphatic drainage and acute hydrocephalus in mice after subarachnoid hemorrhage.