The Effects And Mechanisms Of TPA And UPA For Fibrinolytic Therapy Following Intracerebral Hemorrhage In Rats

BackgroundIntracerebral hemorrhage(ICH),associated with a high rate of mortality and disability,is the most devastating type of stroke.According to a Nationwide Population-Based Survey published in 2017,the age-standardized incidence rate of stroke was 246.8 per 100,000 person-years in China,and ICH constituted 23.8%,which was much higher than that in most western countries.However,the underlying mechanisms of ICH related brain injury is not clearly understood.The physical mass effects of hematoma as well as perihematomal edema(PHE)can deteriorate patient outcomes after ICH.Thus,removal of the hematoma to relieve the primary and secondary injury was pivotal for the treatment of ICH.However,limited by the location of hematoma or the surgical trauma,traditional craniotomy for hematoma removal is not always feasible.In recent decades,minimally invasive aspiration of hematoma and subsequent fibrinolysis has been developed and,given the limited harm to peripheral normal brain tissues,has been considered a less traumatic and more effective treatment modality for spontaneous ICH.Tissue-type plasminogen activator(t PA)is the most widely used drug for ICH fibrinolysis so far.Although beneficial effects such as resolving hematoma have been reported,many other side effects,including the proedema,proneurotoxic,and proinflammatory effects,have been observed in preclinical trials,which lead the application of t PA for fibrinolytic therapy following ICH to be questioned.Recently,an older thrombolytic drug urokinase-type plasminogen activator(u PA),which is not available in the United States since 1999 for commercial reasons,has gradually resurfaced as a possible fibrinolytic agent.For some patients with supratentorial ICH,u PA is recommended to use in the fibrinolytic treatment.Nevertheless,since no preclinical or clinical studies have evaluated the effects of these 2 agents for fibrinolytic therapy after ICH,the use of which fibrinolytics remains empirical.Therefore,a rat model was used to explore the effects and mechanisms of u PA and t PA for fibrinolytic therapy following ICH from two parts in our study:(1)part 1: evaluate the efficacies on hematoma reduction and the PHE side effects of these 2 fibrinolytics in a rat ICH model;(2)part 2: detect the blood-brain barrier(BBB)related indicators after the fibrinolytics administration to study the related mechanisms.Part ? The effects of t PA and u PA for ICH fibrinolysis in a rat model.ObjectiveHematoma and perihematomal edema,brain water content,evans blue fluorescence and neurological scores were measured to investigate the effects of t PA and u PA for fibrinolytic therapy following ICH in rats.MethodsSeventy rats were randomly divided into 5 groups(14 rats/group): sham,ICH,ICH+saline,ICH+ t PA,and ICH+ u PA.Thirty minutes after hematoma placement,the 2 ?l amount of saline,t PA(10 ?g/?l),or u PA(100 IU/?l)was injected into the core of the hematoma respectively.The sham group required only a needle injection into the right caudate nucleus.Animals were killed on Day 3 after ICH for brain water content(8 rats/group)and Evans blue(EB)fluorescence(6 rats/group)after performing MRI and behavioral tests.Results1.As the MRI indicated,both t PA and u PA treatment reduced the volumes of hematoma and PHE.Besides,significantly decreased brain water content in the ipsilateral hemisphere was observed in the u PA group rather than the t PA group.2.During the behavior tests on Day 3 post-ICH thrombolytic therapy,the u PA group had a lower corner turn score and higher forelimb placing score than did the vehicle group,the t PA group had a trend toward a higher neurological score than did the vehicle group,but there was no significant difference.3.Quantification of perivascular EB fluorescence revealed that both t PA and u PA treatment decreased EB extravasation from the vasculature.ConclusionBoth u PA and t PA displayed similar efficacy in reducing the volumes of hematoma and PHE,and showed a protective effect on BBB after ICH in rats.Part II The effects and mechanisms of u PA and t PA on BBB following ICH in rats.ObjectiveThe BBB tight junction proteins(including claudin-5 and ZO-1),matrix metalloproteinases(MMPs),and nuclear factor–?B(NF-?B)activation were measured to evaluate the efficacies on BBB protection and the related mechanisms of t PA and u PA for fibrinolytic therapy following ICH.MethodsFifty-four rats were randomly divided into 3 groups(18 rats/group): ICH+saline,ICH+u PA,and ICH+t PA.After fibrinolysis,as described above,animals were killed on Day 3 after ICH for WB(9 rats/group: 4 for MMPs and ZO-1,another 5 for NF-?B),RT-PCR(4 rats/group for MMPs),and immunofluorescence(5 rats/group for claudin-5 and ZO-1).Results1.Both u PA and t PA up-regulated the expression of ZO-1 and claudin-5 in the perihematomal area or(and)the ipsilateral cortex.2.Both u PA and t PA effectively decreased the MMP-12 m RNA expression on Day 3 after ICH.However,only u PA significantly reduced the MMP-2 m RNA expression level.In addition,more remarkable MMP-9 m RNA expression was observed following treatment with t PA in comparison with u PA.Moreover,the corresponding WB assays revealed trends in MMP expression levels similar to those in the m RNA assays.3.Quantification of NF-?B pathway activation revealed that t PA,not u PA,resulted in a significant exacerbation of NF-?B pathway activation for ICH fibrinolysis.ConclusionDespite the different effects on the regulation of MMPs expression via NF-?B pathway,both u PA and t PA played a protective role in maintaining the integrity of BBB...