Analysis Of Thrombus Components After Thrombectomy In Acute Ischemic Stroke And Study Of Hemorrhagic Transformation Model In Mice

Background:Acute ischemic stroke(AIS)is one of the leading causes of death and disability worldwide.Cerebral artery occlusion caused by thrombosis is the most common type.The sudden interruption of cerebral blood flow after cerebral artery occlusion occurs will lead to a series of cascade reactions including local blood cell damage and destruction,platelet and leukocyte adhesion,and activation of the coagulation system.The most effective treatment currently available is reperfusion therapy,consisting mainly of intravenous thrombolysis and endovascular therapy,but all of these treatment modalities are limited by a narrow time window.It is now agreed that the time of ischemia is an important factor affecting prognosis,and early revascularization after cerebral artery occlusion can improve patient prognosis.Then quantification of cerebral thrombus components in different ischemic time states will help clinicians to better understand the pathological changes of thrombus after ischemic stroke attack and provide more evidence to guide individualized treatment.Some progress has been made in the study of thrombus components based on different etiologies,treatments and clinical characteristics of cerebral infarction.However,the pattern of changes of these components with ischemic time during vascular occlusion remains incompletely understood,as well as the relationship with peripheral blood components and prognosis is unclear.Hemorrhagic transformation(HT)after reperfusion therapy affects the clinical prognosis of patients as its important complication,and studies have shown that the incidence and severity of HT correlate with the duration of ischemia.Therefore,in order to improve the salvage rate of AIS,it is of great clinical significance to study the change pattern of ischemic time and cerebral thrombus components and the relationship between ischemic time and hemorrhagic transformation.Part I.Correlation between changes in thrombus composition and the duration of ischemia after thrombectomy in acute ischemic stroke and prognostic analysis.Objectives:Correlation of changes in each thrombotic component with ischemic time and the relationship between changes in thrombotic components and prognosis.Methods:1.206 cerebral thrombi from the First Affiliated Hospital of Army Military Medical University and the Third People’s Hospital of Zigong City who underwent mechanical embolization for ischemic stroke from December 2017 to November 2020 were collected according to the inclusion and exclusion criteria.The clinical data of the patients were collected and divided into three groups according to the ischemia to reperfusion time <4h(T4),4-8h(T4-8)and >8h(T8).2.Cerebral thrombi grouped by T4,T4-8 and T8 were analyzed by Martius Scarlett Blue(MSB)staining method to determine the changes in the content of each component of erythrocytes,fibrin/platelets and leukocytes in thrombi at different ischemic times.3.The levels of thrombus neutrophils and neutrophil extracellular traps NETs(NETs)were quantified by immunohistochemical staining,and the patterns of changes in thrombus neutrophils and NETs at different ischemic times were investigated.4.The thrombus composition was correlated with the peripheral blood composition,collateral circulation and neurological function prognosis of the patients.Results:1.Analysis of clinical baseline data,history of atrial fibrillation,TOAST(atherosclerotic type,cardiogenic type),thrombolytic therapy,surgical procedure(stent embolization),time of surgery,number of surgical operations,National Institutes of Health Stroke Scale NIHSS(National Institutes of Health Stroke Scale)in each ischemic time group score(preoperative),NIHSS score(postoperative),and incidence of hemorrhagic transformation were different(P< 0.05).2.By MSB staining,the leukocyte content in cerebral thrombosis was significantly higher in the T4-8 group(p=0.002)and the T8 group(p=0.001).There was no significant difference in erythrocyte composition(p=0.059)and fibrin/platelet composition(p=0.055)between the ischemic time groups.3.By immunohistochemical staining,the number of leukocytes in cerebral thrombosis was significantly more in the T4-8 group than in the T4(p=0.005)and T8 groups(p=0.002);the number of neutrophils was significantly more in the T4-8 group than in the T4(p=0.003)and T8 groups(p=0.001);the number of NETs gradually increased in T4 and T4-8,but there was no significant difference between the two groups.There was no significant difference between the two groups,and the number was significantly reduced in the T8 group compared with the T4-8 group(p=0.041).4.Correlation analysis: leukocyte content in cerebral thrombus was positively correlated with leukocyte content in peripheral blood(r=0.146,p=0.049),neutrophil count in cerebral thrombus was positively correlated with neutrophil count in peripheral blood(r=0.322,p=0.014);NETs content in thrombus was positively correlated with NIHSS score(preoperative)(r=0.4024,p=0.003)and NETs in thrombus was positively correlated with NIHSS score(postoperative)(r=0.2745,p=0.047);higher postoperative NIHSS score was a closely associated risk factor for clinical prognosis(OR 0.796,95% CI 0.737-0.859,p<0.01);5.NETs and collateral circulation: the longer the ischemic time in the T4,T4-8,and T8 groups,the richer the side branches of the patients(p=0.038).Patients with good collateral circulation had lower levels of NETs in the thrombus compared with patients with poor collateral circulation(p=0.039).Conclusion:1.The content of leukocytes,centrophils and NETs in cerebral thrombosis showed a pattern of increase followed by decrease with the prolongation of ischemia time;2.The amount of leukocytes in cerebral thrombosis was positively correlated with the amount of leukocytes in preoperative peripheral blood;the neutrophil count in cerebral thrombosis was positively correlated with the neutrophil count in preoperative peripheral blood;3.The level of NETs in cerebral thrombus was positively correlated with NIHSS(postoperative),and patients with high levels of NETs in thrombus had poorer collateral circulation,and higher NIHSS(postoperative)was a closely related risk factor for clinical prognosis;4.The analysis of clinical data showed a high incidence of HT complications after thrombectomy and a time-dependent relationship between the incidence and the time of ischemia,and the relationship between the time of ischemia and the pattern of HT occurrence will be further explored in a mouse animal model in a follow-up study.Part II Establishment of a novel mouse model of hemorrhagic transformation after recanalization of bilateral common carotid artery occlusion and correlation of the occurrence of hemorrhagic transformation and prognosis with the duration of ischemiaObjective: The first part of the study found a high incidence of HT in ischemic stroke patients after mechanical thrombolysis,and the pattern of occurrence was time-dependent with the ischemic time.Considering the complex confounding interference of clinical studies,we will further explore HT in an animal model.therefore,this part of the study aims to establish a novel model of spontaneous HT after ischemia-reperfusion and to investigate the pattern of ischemia time and HT occurrence,as well as the relationship with prognosis.Methods: 1.Model establishment and validation: A reversible vascular blocking device was designed to block-reperfuse the bilateral common carotid arteries in mice using the spring pressure principle to establish an ischemia-reperfusion model.The blocking efficacy of the homemade device was also verified by in vivo fluorescence(fluorescein-dextran isothiocyanate)imaging and digital subtraction angiography(DSA).2.Whether HT could be induced in mice after 2h/4h recanalization of blocked bilateral common carotid arteries for 72 h was examined using 2,3,5-triphenyltetrazolium chloride(TTC)staining and 7T magnetic resonance imaging(MRI)in small animals.3.The incidence of HT,the amount of bleeding and the site of bleeding after recanalization at different ischemic times were investigated by HE staining according to the ischemic time of 1/2/3/4/5/6h divided into six groups after recanalization for 72 h,and the correlation between the incidence of HT,the amount of bleeding and the site of bleeding and the ischemic time was investigated.4.The mice were divided into six groups according to the ischemic time of 1/2/3/4/5/6h,and the NSS-R score was performed before and 72 h after recanalization to assess the neurological function,and the mice were observed for survival for 1 week,and the pattern of different ischemic time and prognosis was investigated.Results: 1.A mouse model of spontaneous HT with bilateral common carotid artery block recanalization was successfully established using a homemade reversible vascular block device.2.The pattern of HT incidence: The incidence of HT in each ischemic time group of 1/2/3/4/5/6h gradually increased in 1-3h,while gradually decreased in 3-6h(HT incidence: 1h 0%,2h 33.3%,3h 100%,4h 83.3%,5h 60%,6h 16.7%,p=0.002).3.Pattern of HT severity: HT severity was positively correlated with ischemia time at 1-4h(r=0.677,p<0.001);and negatively correlated with ischemia time at 4-6h(r=-0.539,p=0.025).Another index of severity,HT volume percentage,also showed a consistent trend,with HT volume percentage positively correlated with ischemic time at 1-4 h of ischemia(r=0.585,p=0.00214)and negatively correlated at 4-6 h of ischemia(r=-0.503,p=0.0379).4.Site of HT occurrence: there was a tendency for hemorrhage conversion susceptibility areas to be concentrated in the thalamus and midbrain,but there was no statistically significant difference(p=0.153).5.Neurological function and survival assessment: with the prolongation of ischemia time,the neurological function of animals after recanalization gradually deteriorated and the survival rate decreased.The ischemic 1-3h group showed significant benefit in neurological function after opening(P < 0.05)and long-term survival rate ≥ 50%;no significant improvement in neurological function after 4h ischemic recanalization(P = 0.566)and long-term survival rate 16.7%;aggravated deterioration in neurological function after 5-6h ischemic recanalization(P < 0.05)and poor prognosis for long-term survival(survival rate 5h,16.7%;6h,0%).Conclusion: 1.In this study,a novel ischemia-reperfusion spontaneous HT model was successfully established by blocking and recanalizing the bilateral common carotid arteries of mice using a homemade reversible vascular blocking device.2.The incidence and severity of HT increased and then decreased with the prolongation of ischemia time,and the incidence of HT peaked at 3 h and the severity of HT peaked at 4 h.This is the first time that the pattern of increasing and then decreasing of HT with the prolongation of ischemia time is presented in an animal model.3.The pattern of ischemia time affecting neurological function and survival prognosis after ischemia recanalization was revealed.Neurological function and long-term survival rate benefited after ischemia 1-3h recanalization,and there was no significant improvement of neurological function after 4-6h recanalization,and long-term survival rate was significantly reduced.