Anti-fibrinolytic Effect Of Neutrophil Extracellular Traps After Intraventricular Hemorrhage

Objective: To identify the existence of neutrophil extracellular traps in the rat IVH model and evaluate the influence of neutrophil extracellular traps on the fibrinolysis of tissue-type plasminogen activatorMethods: First,the IVH model was induced by Injection of autologous arterial blood into rat lateral cerebral ventricle,and then the distribution characteristics of NETs were detected by citrullinated histone(Cit H3)and DAPI immunofluorescence staining.Secondly,the rats were randomly divided into 4 groups: sham group,DNA group,Blood group and Blood+DNA group.The sham group only required a needle injection into the right ventricle.The DNA group,Blood group and Blood+DNA group were induced by injection of 200μl of DNA(2μg)solution,whole blood,or whole blood+DNA(2μg)mixture into the ventricle.At 1,3,and 7 days after operation,MRI was used to measure the volume of the ventricle,and the m Nss score and forelimb placement test were used to evaluate the behavioral injury of the animal.On the 7th day,immunofluorescence staining was used to detect the proliferation of astrocytes around the affected side of the ventricle.The effect of exogenous DNA on IVH rats.Thirdly,the rats were randomly divided into 5groups: sham group,Vehicle group,tPA group,tPA+DNAse1 group and tPA+DNA group.The Vehicle group,tPA group and tPA+DNAse1 group were given 2μl of saline,tPA(10μg/μl),and tPA+DNAse1(tPA,10 μg/μl;DNAse1,1000 IU/μl),respectively,1 hour after intraventricular injection of whole blood.In the tPA+DNA group,the blood +DNA mixtures were injected into the right ventricle,followed by tPA fibrinolysis 1hour later.The volume of the ventricle was measured after the operation,and the behavioral changes of the animals and the proliferation of astrocytes around the affected ventricle were observed.Finally,the whole blood or whole blood+DNA(2μg)mixture was injected into the ventricle.On the third day,the fresh blood clot in the ventricle was taken out and put into 1ml normal saline,tPA(1 μg/ml)and tPA+DNAse1(tPA,1 μg/ml,DNAse1,100IU/ml)solution for in vitro fibrinolysis determination.Results: The results of immunofluorescence staining showed that NETs and their network-like structures were detected in the blood clot at 1 hour after IVH.Secondly,we found that the injection of exogenous DNA solution into the ventricle did not affect the ventricular volumn and behavior test result,but injection with the mixture of exogenous DNA and whole blood into the rat cerebral ventricle could aggravate the dilation of the ventricle,further deteriorated the functional defects in rats and promoting periventricular astrocyte proliferation.And tPA can effectively reduce the dilation of the ventricle caused by IVH,improve the functional outcome and reduce periventricular astrocyte proliferation.Compared with tPA group,the fibrinolytic efficacy of tPA was enhanced in the tPA+DNAse1 group On the contrary,the administration of exogenous DNA delayed the degradation of blood clots,and attenuated the fibrinolytic efficacy of tPA.Conclusion: Neutrophil extracellular traps were present in the blood clot and impaired tPA fibrinolysis after intraventricular hemorrhage,and degradation of NETs by the DNAse1 may be a new target to improve this situation.