The Effects Of Thrombin Inhibitor Argatroban To The Perihematomal Brain Tissue In Experimental Intracerebral Hemorrhage Rats

Objectives:To investigate the effect of Argatroban intravenously to the perihematomal brain tissue in experimental ICH model of rats in order to elucidate the precise mechanisms of thrombin-induced brain damage.Methods:The ICH in rat model was established by Utilizing stereotactic apparatus and infusing antologous blood into the fight caudate nucleus.The rats were randomly divided into three groups:sham-operated group,control group and argatroban treatment group.In argatroban treatment group:rats were received an intracerebral injection of 60μl autologous blood followed by argatroban(1mg/kg i.v.)injection through tail vein every 24h.In control group:rats received an intracerebral injection of 60μl of autologous blood followed by sterile saline(1mg/kg i.v.)injection through tail vein every 24h.In sham-operated group:rats had only a needle insertion and rats were given the same amount of sterile saline(1 mg/kg i.v.) through tail vein every 24h.The perihematomal brain tissue sampies were extracted on the ICH 1d,3d,5d respectively to determine the differential expression of AQP4,MMP-9 and NF-κB mRNA and protein by immunohistochemical,RT-PCR and Western Blot methods. The rat brain water content,BBB permeability and neurologic defecit scores were also observed.Results:(1)Argatroban group got higher Cacia scores than that in contral group at every time points respectively,especially on the 12 hour,third day and 5^thday after ICH,whereas there was no difference between the argatroban group and the contral group on 6 hour and 7^thday.Argatroban group got nearly normal scores on 7^thday.(2) The brain water content were much higher than that in sham-operated group(p＜0.01), it began to rise at 6h,peaked on days 3 and then declined gradually,still higher on 7^th day.Argatroban group got much brain water content than that in contral group on every time points(p＜0.05).(3)The level of EB began to rise at 6h,peaked at first to third day and declined on 5^thto 7^thday,the content of EB was much lower than that in contral group at each time points respectively(p＜0.05).(4)The AQP4 positive cells in Argatroban group reduced markedly than that in contral group since 12h(p＜0.05),the expression of AQP4 mRNA and protein increased at three time points after ICH and reached the highest level at days 3 after ICH(p＜0.05);the down-regulation of AQP4 mRNA expression by Argatroban was observed at day 1 after ICH,and AQP4 protein level was not changed at day 1 but was markedly decreased at day 3.The changes still lasted at day 5 post-ICH.(5)The MMP-9 positive cells expressed generally around the hematoma and the numbers of positive cell in Argatroban group reduced than those in control group respectively(p＜0.05);the expression of MMP-9 mRNA and protein increased at three time points after ICH(p＜0.05);the down-regulation of MMP-9 mRNA expression by Argatroban was observed at day 1 after ICH,and MMP-9 protein level was not changed at day 1 but was markedly decreased at day 3.The changes still lasted at day 5 post-ICH(p＜0.05).(6)The NF-κB positive cells expressed generally around the hematoma at 6h and peaked at 1 day;the numbers of NF-κB positive cell in Argatroban group reduced than those in control group respectively(p＜0.05);the expression of NF-κB mRNA and protein increased at three time points in control group(p＜0.05);the down-regulation of NF-κB mRNA expression by Argatroban was observed at day 1 after ICH,and NF-κB protein level was not changed at day 1 but was markedly decreased at day 3,the changes still lasted at day 5 post-ICH.(p＜0.05).Conclusions:(1)Argatroban reduced the brain water content,BBB permeability and improved the neurologic defecit score,which may be an effective therapy after ICH in rats.(2)The results demonstrated that the upregulation expression of MMP-9, NF-κB and AQP4 mRNA and protein after ICH was time-dependent,and the brain edema formation were induced through MMP-9,NF-κB and AQP4 expression in rats, and MMP-9,NF-κB and AQP4 may be involved in the pathological mechanisms of ICH.(3)Argatroban-induced cerebral edema clearance was correlated with the down-regulation of MMP-9,NF-κB and AQP4 expression of mRNA and protein in perihematomal area.That may be one of the mechanisms of Argatroban to brain edema clearance.(4)The effect of Argatroban in ICH reflected that thrombin may be involved in the regulation of AQP4,MMP-9 and NF-κB.