The Role And Mechanism Of TLR4in Intracerebral Hemorrhage Injury In Mice Brain

Intracerebral hemorrhage (ICH) is a common and serious acute cerebrovasculardisease, account for10%-30%cerebrovascular disease, which dominate one of the leadingplace in the rate of mortality and disability. The one-mortality rate reaches30%to50%and70%to80%survivor lose the labor. Therefore ICH is one of the most threaten disease inChina. With the coming aging of population and change of living style, more and morepatients suffered with diabete, hypertension, obesity will possibly contribute to rapid ICHmorbidity rising. The pathogenesis of ICH is not completely elucidated till now, even withseveral years investigation. Thus it is urgent and significant to explore the pathogenesisdeeply. More and more clinical reseach results indicated that the early stage of ICH is notthe serious duration, however2-3day post ICH collateral neural injury comes out whichaggravate brain injury and neural deficit. However, colateral injuries followed ICH, such aslysis of red blood cell induce inflammation, regional cerebral blood flow decrease, severebrain edema formation and cell degeneration in brain parenchymal, are the most criticalfactor that affect ICH prognosis. Collateral injury followed ICH will cause neural functiondefect which leading to diability and delayed death. But it is a pity that the mechanism ofcollateral injury is still uncovered and sufficient of effective method to prevent ithappenning. Current results indicated that microglial and other inflammatory cell activationwith increase release of inflammatory factors, like TNF-α and IL-1, the increase ofintercellular adhesion molecules, periphery infiltrated inflammtory cells migration willresults in cascade inflammation aggravate brain injury and neural function deficit.It is important and urgent to explore the mechanism of collateral injury, the interventionmeasures to extenuate the injury of ICH followed ICH. And it is of clinical significant toimprove patients’ tolerance and prognosis.TLRs (Toll-like receptors) are ancient receptors that induce innate immunity. There are 11type of TLRs were found in human being, named TLR1to TLR11, are the members ofIL-1superfamily. TLR4is the receptor of LPS also take roles in infection by Gram-negativebacteria, septicemia shock. The following evidence indicated that TLR4also play importantrole in cerebral ischemia, Alzheimer disease and other CNS inflammtory injury. The recentevidence indicated that heme, one of the lysis component of RBC, are able to activate TLR4on macrophage leading to inflammatory factors release, results in inflammatory injury,which is the possible mechanism that affact tissue injury. With the autologous bloodinjection ICH model and in vitro microglial stimulation model, we explore the qualitativeand quantitative change of TLR4, the microglial inflammtion by hematoma when TLR4knock out. We try to clarify the heme/TLR4signaling pathway as one of the leading initiaterole in ICH injury.Main results:1. The achievement ratio of ICH was87%. It was found that the mice exhibit serveralnon-symmetry movement, such as contralateral limb paralysis, lack of motor coordination,delayed actin and spontanoeous left turn. The neurological deficit scores (NDS) resultsindicated that ICH have great influence on mice movement.2. To explore the role of TLR4in inflammation after ICH, we first analyzed TLR4mRNA expression on day1,2,3, and5post-ICH using a real-time RT-PCR. The resultshowed that TLR4mRNA was significantly upregulated in perihematoma brain tissuesfollowing ICH at all tested time-points when compared to sham-operated control mice.TLR4mRNA expression peaked on day3(P <0.01) and started decreasing on day5(P <0.01). The NDS was statistical correlated with TLR4mRNA expression.3. It was found that TLR4expressed in all these cell types and the protein expressionwas significantly upregulated when compared to that of the sham group (P <0.01).Although both neuron and reactive astrocyte exhibited TLR4expression,double-fluorescent staining showed that TLR4predominantly expressed in CD11b-positivecells.4. Compared to WT mice, tlr4-/-mice showed a significantly lower brain watercontent and NDS. These suggested that tlr4-/-mice had less extent of neurologicalimpairments after ICH. ELISA showed that tlr4-/-mice exhibited significantly lowerexpression of IL-6, TNF-α, and IL-1β in perihematoma tissues3days after ICH. 5. Using primary cultured microglial cells, we found that heme treatment significantlyincreased TLR4expression in cultured microglia while bilirubin and FeSO4showed noeffect, the expression of TNF-α by activated microglia was significantly increased.Moreover, TLR4neutralizing antibody and TLR4knock out were able to block theactivation of microglia.Main conclusion:1. The TLR4mRNA exrpression was up-regulated in peri-hematoma area andcorrelated with NDS. ICH significantly upregulates TLR4expression in microglial cells,which correlated with the NDS.2. The ICH inflammation, the number of infiltrating cells and NDS decrease whenTLR4was knocked out, which indicated that TLR4induce mice ICH injury.3. Administration of heme significantly induced the expression of TNF-α, IL-1β, andIL-6in microglia from WT mice but not from tlr4-/-mice, suggesting that TLR4depletionsuppressed heme-stimulated microglial activation and TLR4is a putative target to the ICHinjury.In sum, after ICH heme triggers TLR4-mediated inflammatory injury. NeutralizingTLR4with antibody at the time of ICH provides beneficial neuroprotection. To ourknowledge, the present study demonstrates, for the first time, that heme induces TLR4activation which might be an upstream event in the inflammatory process in response toICH, suggesting that TLR4is a promising target for prevention and therapeutic treatment ofICH. The results provided fundamental evidences for clinical ICH treatment.