The Effect Of Toll-Like Receptor 4 On Inflammation And Neuron Apoptosis After Cerebral Hemorrhage In Rat

Cerebral hemorrhage is a common disease in neurology,which has both high disability and fatality and greatly threatens health and quality of life.Recently,it has been heatedly forcused on the role of inflammation after cerebral hemorrhage in secondary lesion,which might lead to apoptosis.Until now,the mechanism of inflammation after cerebral hemorrhage is not clear and the beginning of inflammation is not certain,too.Toll-like receptors(TLRs) are a large family of evolutionarily conserved pattern recognition receptors.TLR4,as the gate of membrane receptor,can identify biological and non-biological stimulation and transmits these signals to nuclear factor(NF—κB),which may trigger and amplify inflammation,causes the waterfall-like release of multiple inflammatory factors and results in damage effect.In this research, Horseley-Clarke technique is used to inject 50μL of autogenetic blood from femoral artery into caudate nucleus of mouses,TUNEL staining is chosen to observe apoptosis, immunohistochemistry is used to measure the expression levels of TLR4,NF-κB/p65 and caspase-3,RT-PCR is used to observe the expression of TLR4 mRNA,which aims to find out whether TLR mRNA is expressed and distributed and then to discuss the role of TLR4 pathway in secondary lesion after cerebral hemorrhage.Materials and methodsMaterials1.grouping90 male Wister mouses that weigh from 250 to 300g were randomly grouped into control group and experiment group,which respectively was sub-grouped by 6h,1d, 3dand 7d.(sub-group:n=10;normal control group:n=10).2.major equipmentsstereotaxic apparatus,electronic analytical balance,ime analysis apparatus, spectrophotometry,PCR amplification apparatus,refrigerated centrifuge and gel-image analysis system.3.major reagentTLR4 rabbit anti-mouse polyclonal antibody,NF-κB rabbit anti-mouse polyclonal antibody,Caspase-3 rabbit anti-mouse polyclonal antibody,immunohistochemistry test kit,instant SABC immunohistochemistry test kit,DAB test kit,Trizol,RNA PCR Kit, TLR4,primer,DNA Maker,DEPC,chloroform and avantin.4.models of cerebral hemorrhageModels of slow injection with autoblood.Mouses were drawn 70uL arterial blood, put in prone position on stereotaxic apparatus,needled 0.2mm ahead of anterior fontanel,3.0mm right in median in depth of 6mm(caudate nucleus) and injected with 50uL arterial blood.Mouses in control group were injected with NS,while normal control group received no disposition.5.making samplesmouses were taken from each group after the corresponding time of operation, immediately received heart exposition,infused with 4%paraform and got brains off. The taken sample were fixed in 4%paraform,dehydrated in alchohol,transparent by xylol,dipped in wax and embedded.Successive coronal-slice,about 5 um thick,and then stain.5 mouses were taken from each group after corresponding time of operation and anesthetized,decapitated and got brain off into Trizol at a temperature of -80℃for RT-PCR.Measure1.TUNEL positive cell testAdd the slice with drops of TDT and digoxin-marked dUTP reaction fluid, incubated at 37℃;then add biotinylation anti-digoxin antibody,incubated at 37℃for 30min,washed by 0.01MTBS(pH7.5) 3min for 3 times;stain with DAB,restain with hematoxylin,dehydrate,transparent and mount.Use microgram analysis system to collect images and analyze positive cell spectrodensity.2.measuring expressed TLR4 protein by immunohistochemistryMeasure expressed TLR4 protein in two steps.Add 50 uL rabbit anti mouse polyclonal TLR4 antibody(1:150),stay overnight 4℃,washed by PBS for 3min 3 times; add a drop of goat anti rabbit IgG antibody-HRP polymer,washed by PBS for 3min 3 times,DAB developer,slightly restain by hematoxylin,dehydrate,transparent and mount.Use the microgram analysis system to collect images,and analyze spectrodensity of positive cells.3.measuring NF-κB/p65,Caspase-3 protein by immunohistochemistryUse SABC to measure positive cells which express NF-κB/p65 protein,Caspase-3 protein.Add the slice with 50uL-antifluid(rabbit anti mouse polyclonal NF-κB/p65 antibody(1:400),rabbit anti mouse Caspase-3 multiclonal antibody(1:150)),stay overnight at 4℃;add goat anti rabbit IgG,at 37℃for 20min;add SABC at 37℃fro20min;every step washed by PBS for 3min 3 times;DAB developer,slightly restain by hematoxylin,dehydrate,transparent and mount.Use the microgram analysis system to collect images,and analyze spectrodensity of positive cells.4.measuring TLR4 mRNA by RT-PCRTake 100mg tissue surrounding the hematoma,use Trizol to extract total RNA under no existence of RNA enzyme according to RNA extraction kit instruction,take the amplification products for electrophoresis,use gel image-analysis system to scan and analyze,calculate ratio of spectrodensity of TLR4 mRNA andβactin mRNA.Statistical analysisAll datas were demonstrated by mean±standard deviation(x±s),using SPSS17.0 and Excel software for data processing and analysis of variance(ANOVA),comparing each two groups when statistically significant and using pearson correlation analysis, p＜0.05 when difference significant.1.changes of apoptosis after cerebral hemorrhageTUNEL positive cells emerged 6h after cerebral hemorrhage,apoptosis cells significantly increased after 1d,reached peak at 3d and gradually decreased after 7d compared with control(p＜0.05).Apoptosis cells mainly existed surrounding hematoma and in cerebral cortex of the same side.3d after cerebral hemorrhage emerged some typical apoptosis cells,such as crescentiform,horseshoe shape and nuclear debris aggregated apoptotic body.Apoptosis cells are mainly neurons.2.expression of caspase-3 after cerebral hemorrhage 6h after cerebral hemorrhage there was a weakly positive expression of caspase-3 surrounding the hematoma and in cerebral cortex of the same side,mainly expressed in kytoplasm.Afer 1 d,a great quantity of positive neurons began to emerge and reached peak after3d with the deepest coloration in nucleus,which meant there was nuclear transfer after schizolysis of capases-3.The quantity decreased after 7d(p＜0.05).3.expression of NF-κB/p65 after cerebral hemorrhageNF-κB/p65 positive cells surrounding the hematoma began to increase after 6h, continued to increase after 1d,reached peak at 3d and after this began to decrease, which was higher for continuous 7 d than control(p＜0.05).It was expressed mainly in gliocytes and some neurons,it was positive when nucleus was cinnamomeous and cannot be stained by hematoxylin.NF-κB/p65 was also expressed on the opposite side of hematoma.In control group,we found extremely few positive cells.4.expression of TLR4Expression of TLR4 surrounding hematoma and in cerebral cortex of the same side was higher than in control,began to increase after 6h,reached peak at 3d,slowly decreased thereafter and still had a significant deviation compared with control(p＜0.05).5.expression of TLR4 mRNAResult showed that even nomal brain tissues can express TLR4 mRNA,TLR4 mRNA increased in NS control group,while there was no significant difference between these two groups.In cerebral hemorrhage group,expression of TLR4 mRNA was higher than control,began to increase after 6h,reached peak at 3d and still had a significant difference compared with control(p＜0.05).DiscussionWe injected autoblood from femoral artery into fight caudate nucleus to make models of cerebral hemorrhage,used RT-PCR and immunohistochemistry to measure TLR4 and found out that there was a change of expression of both TLR4 mRNA and protein after cerebral hemorrhage.Recently,many researches confirmed that apoptosis was involved in secondary lesion after cerebral hemorrhage.We used Caspase-3 immunohistochemistry and TUNEL staining at the slices,which were coherent in scope and time,which suggested that Caspase played an important role in apoptosis.NF-κB pathway is the most important downstream pathway of TLR4 pathways,and involved in inflammation and apoptosis.We found that expression of TLR4 was in positive correlation with expression of NF-κB and increase of positive nucleus and also coherent with peak of inflammatory cell infiltration.This suggests that increase in expression of TLR4 is always accompanied by the activation of NF-κB.But this cannot conclude that TLR4 essentially activates NF-κB,while TLR4 is involved in inflammation after cerebral hemorrhage.Many researches confirmed that TLR4 pathway mainly activates NF-κB,which triggers and amplify inflammation,causes waterfall-like release of multiple inflammation factors and leads to damage effect. Many researches have also confirmed that this inflammation exists surrounding hematoma,which is thought to result in apoptosis after cerebral hemorrhage.Time of inflammatory cell infiltration is positive correlant with time of apoptosis.We can come to a conclusion that NF-κB pathway mediated by TLR4 is involved in inflammation and aggravates damage and apoptosis of neurons.Conclusion1.increase in expression of TLR4 and NF-κB surrounding hematoma after cerebral hemorrhage suggests it may play an important role in the development of inflammation damage in cerebral hemorrhage.2.NF-κB pathway mediated by TLR4 is involved in inflammation after cerebral hemorrhage and may mediate caspase-3 to cause damage and apoptosis of neurons.