Role Of Neutrophil Extracellular Traps And Tolvaptan In Experimental Intracerebral Hemorrhage

Backgroud Intracerebral hemorrhage(ICH)remains a high morbidity and mortality and seriously affects patients' outcome.Due to the complicated pathogenesis and the lack of clear evidence-based medical guidance,the treatments for ICH are quite limited,and the efficacy is far from satisfactory.Effective removal of hematoma is the key to the treatment of ICH.At present,the removal of hematoma mainly depends on surgical methods.However,traditional craniotomy often brings secondary injury.Minimally invasive surgery combined with tissue plasminogen activator(t PA)fibrinolysis,given the small damage to the surrounding normal brain tissue,have been considered as a promising treatment for ICH.However,its hematoma clearance efficiency is not satisfied,which needs to be improved.Neutrophil extracellular traps(NETs)was reported to enhance the fibrinolytic resistance of thrombus,and therefore impaired fibrinolysis in acute ischemic stroke.However,the existence and potential effect of NETs in ICH is unclear so far,which needs further study.Besides the primary injury,secondary injury is also an important target of ICH treatment.As an almost inevitable secondary injury of ICH,cerebral edema(CE)can increase the intracranial pressure,and thus aggravate the ischemia and hypoxia,leading to a worse outcome.At present,the treatment for CE after ICH mainly depends on drug dehydration and adjuvant treatment,but the efficacy is uncertain.Surgical decompression can be used for refractory CE which drug treatment is ineffective for,but the operation itself always causes secondary damage.The treatment of CE after ICH is still a difficult problem to be solved.It is found that once the hypothalamus is damaged,the CE will always be persistent and severe,suggesting that the neuroendocrine system is involved in the occurrence and development of CE.The hypothalamus can release a variety of hormones and play a variety of roles,among which arginine vasopressin(AVP)can regulate the homeostasis of water and electrolyte.It has been reported that the AVP level in plasma is positively correlated with the severity of CE in ICH patients,suggesting that AVP participates in the formation of CE after ICH.What's more,tolvaptan,a specific AVP receptor antagonist,was reported to relieve CE and improve the outcome of patients with craniocerebral injury.Tolvaptan is expected to be a new choice in the treatment of CE.Therefore,we designed animal experiments to verify the therapeutic effect of tolvaptan on CE after ICH.Then,clinical trials about tolvaptan may be carried out to provide a new target for the treatment for CE after ICH.Therefore,we focused on the current main methods for ICH treatment ——removal of hematoma and relieving CE,and designed this animal experiment,in hope to explore the role of NETs and tolvaptan in ICH treatment.Part ? The role of neutrophil extracellular traps in fibrinolysis therapy after intracerebral hemorrhageObjective To observe the distribution of neutrophil extracellular traps(NETs)after ICH in rats,and to explore its effect on t PA fibrinolysis.Methods There were 3 parts in this study.First,at 0.5 h,1 h,and 1.5 h after blood intracranial injection,the brains were collected for NETs detection by immune-staining.Second,ICH rats were given intrahematoma fibrinolysis: rats were randomized to receive the equal amount of saline,DNAse 1,tissue-plasminogen activator(t PA),and t PA + DNAse 1 at 1 hour after hematoma placement.On day 3,animals were sacrificed for terminal deoxynucleotidyl transferase-mediated d UTP Nick-end labeling(TUNEL)staining following MRI and behavioral tests.Third,on day 3 after ICH,the hematoma within brain were collected for ex vivo fibrinolysis assay to further evaluate the effect of NETs in t PA fibrinolysis.Results 1.The confocal images of co-staining of DAPI,H3,and MPO confirmed the presence of NETs in the rat ICH.2.Disintegration of NETs by DNAse 1 promoted t PA-induced hematoma resolution and brain swelling relief after ICH.3.Disintegration of NETs by DNAse 1 potentiated the effect of t PA on attenuating neurologic deficits after ICH.4.Disintegration of NETs by DNAse 1 potentiated the effect of t PA on reducing cell death after ICH.5.Disintegration of NETs by DNAse 1 enhanced t PA-induced fibrinolysis ex vivo.Conclusion The presence of NETs impaired the efficacy of t PA for ICH fibrinolysis in rats.Disintegration of NETs enhanced the t PA fibrinolysis.This study provides a new target for fibrinolytic therapy for ICH which might have practical implications.Part II The mechanism of neutrophil extracellular trap on increasing t PA fibrinolysis resistanceObjective To test the hypothesis that cell-free DNA(cf DNA),the main framework of NETs,might be the key reason for the t PA fibrinolysis resistance in a rat intraventricular hemorrhage(IVH)model.Methods There were 4 parts in this study.First,cf DNA were detected in IVH clots by immunofluorescence staining in a rat model of IVH.Second,rats were randomly given intraventricular injection of cf DNA solutions,blood,and blood + cf DNA respectively.Then the ventricular dilatation,animal behavior,and the proliferation of periventricular astrocytes were measured to evaluate the effects of cf DNA on IVH.Third,after blood(with or without exogenous cf DNA)intraventricular injection,IVH rats were given intraventricular infusion of 2 ?L saline,t PA,or t PA + DNAse 1 randomly.Then the ventricular volume,animal behavior,and the reactive astrocytes proliferation were measured to evaluate the effect of cf DNA on t PA fibrinolysis for IVH.Fourth,rats were injected intraventricularly with blood or blood + cf DNA to induce IVH,and the clots were collected and weighed for fibrinolysis assay 3 days later to further evaluate the effect of cf DNA on t PA fibrinolysis in vitro.Results 1.The confocal images of co-staining of H3 and DAPI showed that cf DNA began to appear as early as 1 hour after IVH.2.Intraventricular injection of cf DNA(2 ?g)had no obvious effects on the ventricular volume(measured by MRI),behavioral function(measured by m NSS and forelimb placement test)and proliferation of periventricular astrocytes(measured by immunofluorescence).Compared with blood injection,additional of cf DNA(2 ?g)into the blood delayed the hematoma clearance and aggravated the damages of IVH in rats.3.t PA fibrinolysis significantly alleviated ventricular dilation(measured by MRI),improved neurological deficits(measured by m NSS and forelimb placement test),reduced periventricular astrocytosis(measured by immunofluorescence).Additional of DNAse1 or cf DNA resulted in a strengthening or weakening effect on t PA fibrinolysis effectiveness respectively.4.Additional of cf DNA(2 ?g)into the blood delayed the hematoma clearance and increased t PA fibrinolysis resistance in vitro.Conclusion In experimental IVH,cf DNA may be the key reason for t PA fibrinolysis resistance induced by NETs.This study provided further evidence for that degradation of cf DNA by DNAse 1 could enhance the fibrinolytic efficacy of t PA.Part III Effects and mechanism of tolvaptan on cerebral edema in ratsObjective To observe the changes of cerebral edema(CE),behavioral results and blood-brain barrier(BBB)related indicators after oral tolvaptan treatment in a collagenase-induced ICH rat model,and to verify the efficacy of tolvaptan on CE using a traumatic brain injury(TBI)model.Methods There were 3 parts in this study.First,to observe the safety and efficacy of tolvaptan on CE,SD rats were randomly divided into 3 groups: sham,vehicle,and tolvaptan.Tolvaptan,dissolved in water at a concentration of 10 mg/kg,was administrated to the animals through oral gavage at 12 h,36 h,and 60 h after surgery.The vehicle group received an equal volume(1.5 m L)of water at the same time.Brain swelling(%),brain water content(BWC),neurological scores were evaluated.Second,evans blue(EB)fluorescence and BBB tight junction proteins were measured after tolvaptan treatment.Third,the efficacy of tolvaptan on CE after TBI were assessed.Results 1.Tolvaptan had no obvious effects on the body weight and mean arterial pressure in normal and ICH rats.2.Tolvaptan significantly reduced the brain swelling(%)and BWC of the ipsilateral hemisphere and attenuated the neurological deficits of ICH rats.3.Tolvaptan reduced the extravasation of EB dye,increased the expression of ZO-1 and occludin,and decreased the upregulation of MMP-9 after ICH.4.Tolvaptan significantly reduced the brain swelling(%)and BWC of the ipsilateral hemisphere and improved the MNSs score after TBI.Conclusion Tolvaptan effectively reduced CE and attenuated the neurological deficit of ICH and TBI rats.The underlying mechanism may be related to the protective effects of tolvaptan on BBB.