The Study About The Expression Change Of TLR2,NF-κB/p65 And Caspase-3 In Rat Brain After Intracerebral Hemorrhage

ObjectiveIntracerebral hemorrhage (ICH) 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 focused on the role of inflammation after ICH in secondary lesion, which might lead to apoptosis. Until now, the mechanism of inflammation after ICH 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. TLR2, as the gate of membrane receptor, can identify biological and non-biological stimulation and transmits these signals to nuclear factor kappa B (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 rats, TUNEL staining is choosen to observe apoptosis, immunohistochemistry is used to measure the expression levels of TLR2, NF-KB/p65 and caspase-3. And then, investigate the relationship between the three proteins and apoptosis and discuss the role of TLR2 pathway in secondary lesion after ICH.Materials and methodsMaterials1. grouping45 male SD rats 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=5; normal control group:n=5).2. major equipmentsstereotaxic apparatus, electronic analytical balance, image analysis apparatus, spectrophotometry, 3.major reagentsTLR2 rabbit anti-rat polyclonal antibody, NF-KB/p65 rabbit anti-rat polyclonal antibody, Caspase-3 rabbit anti-rat polyclonal antibody, instant SABC immuno-histochemistry test kit, DAB test kit, apoptosis test kit.4.making models of ICHRats 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. Rats in control group were injected with NS, while normal control group received no disposition.5. making samplesRats 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 slices, about 5 um thick.Measure1. TUNEL positive cell testAdd the slices 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 0.01 MTBS(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. SABC immunohistochemistryUse SABC method to measure positive cells which express TLR2 protein, NF-KB/p65 protein and Caspase-3 protein. Add the slices with 50uL-antifluid(rabbit anti rat polyclonal TLR2 antibody(1:200), rabbit anti rat polyclonal NF-κB/p65 antibody(1:300), rabbit anti rat Caspase-3 polyclonal antibody(1:150)), stay overnight at 4℃; add goat anti rabbit IgG, at room temperature for 20min; add SABC at room temperature for 20min; 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. Statistical analysisAll datas were demonstrated by mean±standard deviation(x±s), using SPSS16.0 and Excel software for data processing and analysis of variance(ANOVA), comparing each two groups when statistically significant and using spearman correlation analysis, p<0.05 when difference significant.Results1. the changes of apoptosis after ICHTUNEL positive cells emerged 6h after ICH, 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 ICH, there emerged some typical apoptosis cells, such as crescentiform, horseshoe shape and nuclear debris aggregated apoptotic body. Apoptosis cells are mainly neurons.2. the expression of caspase-3 after ICH6h after ICH 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 1d, a great quantity of positive neurons began to emerge and reached peak after 3d 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. the expression of NF-κB/p65 after ICHNF-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-KB/p65 was also expressed on the opposite side of hematoma. In control group, we found extremely few positive cells.4. the expression of TLR2 after ICHExpression of TLR2 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). DiscussionWe injected autoblood from femoral artery into right caudate nucleus to make models of ICH, used immunohistochemistry to measure TLR2 and found out that there was a change of expression of TLR2 protein after ICH. Recently, many researches confirmed that apoptosis was involved in secondary lesion after ICH. We used Caspase-3 immunohistochemistry and TUNEL staining at the slices, which were coherent in scope and time, which suggested that Caspase-3 played an important role in apoptosis. NF-κB pathway is the most important downstream pathway of TLR2 pathways, and involved in inflammation and apoptosis. We found that expression of TLR2 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 TLR2 is always accompanied by the activation of NF-κB. But this cannot conclude that TLR2 essentially activates NF-κB, while TLR2 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 ICH. Time of inflammatory cell infiltration is positive correlate with time of apoptosis. We can come to a conclusion that NF-κB pathway mediated by TLR2 may be involved in inflammation and aggravates damage and apoptosis of neurons.Conclusion1.increase in expression of TLR2, NF-KB/p65 and caspase-3 in brain tissue surrounding hematoma after ICH and the expression of them changed as the apoptosis.2.NF-κB pathway mediated by TLR2 may be involved in inflammation after ICH and may mediate caspase-3 to cause damage and apoptosis of neurons.