The Neuroprotective Efficacy Of Toll-like Receptor 4 Antagonist On Mice With Intracerebral Hemorrhage

Background and ObjectiveIntracerebral hemorrhage(ICH)refers to non-traumatic parenchymal brain hemorrhage,accounting for 20%-30%of the total stroke patients in China and its mortality rate in acute phrase is up to 40%.Many of the survivors have obvious nerological dysfunction,which brings heavy burden to their families and society and seriously endangers human health.Currently,there is no effective medical treatment for patients with ICH.Then,it’s of great significance to further study the pathogenesis of ICH and explore novel approaches that can improve functional recovery after ICH.Inflammation plays a critical role in the progression of secondary brain injury after ICH.Toll-like receptors on microglia and macrophages infiltrated into hematoma is essential for the regulation of inflammation after ICH,which may be a potential target for the treatment of ICH.Therefore,we used TLR4 antagonist to bind TLR4 in our experiment to investigate the influence of TLR4 antagonist on the inflammation,brain injury,and neurologic function in mice with ICH,which will provide more theoretical support for the clinical treatment of ICH.Materials and Methods1 Animals and experimental groupsOne hundred and twenty eight male mice(8 to 10 months old,weighing 30 to 35 grams,SPF)were divided into fourgroups according to computer-generated random numbers:sham+vehicle group(the same amount of aseptic saline was injected intraperitoneally at the same time,n=32),sham+TAK-242 group(three times a week before the operation,3mg/Kg,n=32),ICH+vehicle group(n=32)and ICH+TAK-242 group(n=32).2 ICH mouse modelWe injected the left caudate nucleus of mice with 0.075U collagenase VII-S(Sigma-Aldrich,St Louis,MO,USA,C2399)in 0.5ul saline to induce hemorrhage at the following stereotactic coordinates:0.6mm anterior and 2.0mm lateral of the bregma and 3.2mm in depth.Postoperative NDS(neurological deficitscores)below 4 was identified as model failure,then excluded from the experiment.3 Treatment ReagentsT AK-242(3mg/Kg,MCE,HY-11109)or sterile saline was injected intraperitoneally every other day in one week before the operation.4 Experimental assessment(1)Histopathology and Morphology at early stage of ICH:①Luxol Fast Blue/Cresyl Violet staining and Fluoro-Jade B(FJB)histochemical staining were used to test the injury volume and degeneration of neurons respectively on day 3 after ICH(n=8/group).(2)Molecular biology:Blood was collected from the mice on day 1,3,7,and 14 after ICH,and the concentrations of IL-10 and IL-23 in serum were detected by ELISA kits(R&D System,eBioscience).(3)The long-term histopathology and behavioral evaluation after ICH:①Luxol Fast Blue/Cresyl Violet staining was used to evaluate white matter injury,residual lesion volume and brain atrophy on day 28 post-ICH.②Neurological Deficit Scores,forelimb placement test and corner turn test were performed to evaluate the neurological function on day 1,3,7,14,and 28 post-ICH,and the rectal temperature of mouse in four groups were tested at the same time.5 Statistical analysisSPSS 21.0 and Graphpad Prism 5.0 were used for statistical analysis.Parametric data were expressed as mean ± standard deviation,student’s t test was used for comparisons of difference between two groups,one-way analysis of variance or nonparametric test followed by Bonferroni correction were used for comparisons among multiple groups.Chi-square test was used for comparisions of difference between two groups in the rectal temperature and mortality rate.A P<0.05 was considered significant.Result1.The lesion volume and FJB positive cells were obvious in the ICH+vehicle group and the ICH+TAK-242 group on day 3 after ICH.When compared with the ICH+vehicle group,TAK-242 significantly reduced injury volume and the number of FJB positive cells on day 3 after ICH(n=8/group,P=0.015).2.TLR4 antagonist improved long-term histopathology and promoted long-term neurological recovery in mice with ICH.We observed obvious brain atrophy,brain white matter injury and residual lesion in ICH mice treated with vehicle or TAK-242 on day 28 after ICH.When compared with the ICH+vehicle group,TAK-242 significantly reduced residual lesion(n=8/group,P=0.012),brain white matter damage(n=8/group,P=0.012)and brain atrophy(n=8/group,P<0.01)on day 28 after ICH.Additionally,we also evaluated the neurological function with NDS score(H=50.18,49.93,50.82,52.89,58.77,P<0.001,n=12-14/group),forelimb placement(H=54.28,22.85,26.16,26.16,41.04,n=12-14/group,P<0.001)and corner turn test H=47.26,44.35,39.66,40.35,n=12-14/group,P<0.001)in mice with ICH on day 1,3,7,14 and 28 after ICH.The results revealed that TAK-242 significantly reducedneurobehavioral deficits after ICH.3.TLR4 antagonist regulatedserum levels of inflammatory factors after ICH.TLR4 antagonist enhanced the expression of IL-10 in serum when compared with ICH+vehicle group on day 1,3 and 7(F=9.229,18.039,5.657,P=0.001,<0.001,0.008,n=5/group),but not on day 14after ICH(F=-4.882,P=0.633,n=5/group).The concentration of IL-23 in serum of ICH+TAK-242 group was slightly higher than that of ICH+vehicle group,but the difference was not statistically significant except on day 7 after ICH(H=1.743,0.141,9.629,2.229,P=0.627,0.987,0.022,0.526,n=5/group).Conclusion1 TLR4 antagonist can significantly acute andimprovelong-term histopathology,and promoted long-term neurological recovery in mice with ICH.2TLR4 antagonist may exert neuroprotective efficacy byregulating the expressio n of inflammatory factors in mice with ICH.