| BackgroundSpontaneous intracerebral hemorrhage(ICH)is a common disease with high mortality and morbidity,accounting for 15 to 20%of all stokes and affecting more than 2 million people worldwide each year.The mortality rate of ICH is more than 40%and only 20%of survivors can live independently within six months.But until now,there has been no satisfactory treatment in clinical practice,mainly because the mechanisms of brain damage after ICH are unclear.Increasing researches show that inflammatory response plays important roles in ICH-induced secondary brain damage.Brain inflammation after ICH is characterized by accumulation of activated inflammatory cells,such as blood-derived cells(macrophages,leukocytes)and brain resident cells(astrocytes,microglia and mast cells).These reactive cells can release inflammatory mediators,including chemokines,cytokine,protease,prostaglandins and other immunoactive molecules.The microglia are the first cells to react to brain damage among all the inflammatory cells.Activated microglia have a similar shape to the blood-derived macrophages,so microglia are also called as the brain macrophages.Increasing evidence indicate that microglia/macrophages are activated early following ICH and release a series of toxic factors,including chemokines,cytokines,ROS,cyclooxygenase-2,protease,HO-1 and prostaglandins..Therefore,it is thought that microglia/macrophages play important roles in the secondary brain damage.T cell immunoglobulin and mucin domain 3(Tim-3)is a new immunolo-regulation molecule found in 2002,which is expressed specially in activated Th1 cells.Moreover,it has been proved that Tim-3 is also expressed in the cells of the innate immune system,including macrophages,mast cells and dendritic cells.Further research find that Tim-3 expression in microglia/macrophages upregulate and induce production of proinflammatory cytokines,such as tumor necrosis factor-a(TNF-a)and interleukin-1 ?(IL-1?),which can aggravate inflammation and secondary brain damage.However,until now the effect of Tim-3 on inflammatory response following ICH has been unclear.Considering that the microglia/macrophages are the key cells inducing brain inflammation and secondary brain damage,and the Tim-3 can regulate the function of microglia/macrophages,we hypothesized that Tim-3 possibly took a critical part in ICH-induced inflammation by regulating the function of microglia/macrophages.This experiment was done to prove our hypothesis.Part I:Preclinical research of the effect of T cell immunoglobulin and mucin domain 3 on the inflammatory reaction in peri-hematomal brain tissuePurposes:To detect the expression and cellular resource of Tim-3 in peri-hematomal brain tissue.To study the effect of Tim-3 on the inflammation reaction after ICH.Methods:In this study,we investigated Tim-3 expression,the inflammatory cytokines tumor necrosis factor-a(TNF-a)and interleukin-1?(IL-1?),and brain water content in peri-hematomal brain tissue at 12 hours,and at 1,3,5,and 7 days post-ICH in wild type(WT)ICH and Tim-3-/-ICH mice.The numbers of Tim-3 positive cells,astrocytes,neutrophils and microglia/macrophages were detected using immunofluorescence staining.Cytokines were measured by ELISA.Double immunoflurorescence labeling was performed to identify the cellular source of Tim-3 expression.Mouse neurological deficit scores were assessed through animal behavior.Results:1.The number of Tim-3 positive cells and Tim-3 mRNA in the WT ICH group began to increase at 12 hours,peaked at Day 1,and decreased at Day 3 after ICH.There were a significant difference when compared with those of WT sham mice in all time-tested points(P<0.01),2.Double-immunofluorescence staining revealed that expression of Tim-3 was preponderant in CD11b+ cells(microglia/macrophages),was lower in MPO+ cells(neutrophils),and was lowest in GFAP+ cells(astrocytes).3.IL-1? and TNF-? increased at 12 hours after ICH,and peaked at Day 1,then decreased at Day 3.There was significant difference between the WT ICH group and the WT sham group(P<0.01).Brain water content heightened at 12 hours after ICH,peaked at Day 1 and Day 3,and then gradually declined.There was a significant difference between the WT ICH group and the WT sham group(P<0.01).4.The expression of Tim-3 was positively correlated with IL-1?(r = 0.618,P<0.001),TNF-a(r = 0.610,P<0.001)and brain water content(r = 0.566,P = 0.001).5.The concentration of both IL-1? and TNF-a in the peri-hematomal brain tissues notably decreased in the Tim-3-/-ICH group,and there was significant difference compared to those in the WT ICH group(the Tim-3-/-ICH group versus the WT ICH group,IL-1?:12 h P<0.01;d P<0.05;3dP<0.05;5d P<0.05;7d P<0.01.TNF-a:12 h P<0.05;Id P<0.05;3d P<0.01;5d P<0.01;7d P<0.01).Brain water content also markedly declined in the Tim-3-/-ICH group in all tested time points(the Tim-3-/-ICH group versus the WT ICH group,12 h P<0.01;Id P<0.01;3 d P<0.01;5d P<0.01;7d P<0.05.)At 12 hours and day 1,there was no difference of NDS between the Tim-3-/-ICH group and the WT ICH group.At 3 days,the NDS of the Tim-3-/-ICH group was lower than that of the WT ICH group,but there was no statistical difference.At 5 and 7 days,there was a significant difference between two groups(P<0.05),which meant that blockage of expression of Tim-3 could improve the neurological deficit after ICH.6.The number of astrocytes,neutrophils and microglia/macrophages in the peri-hematomal brain tissues all increased after ICH.But the peak times of the three cells were different.The number of astrocytes elevated at Day 1,peaked at Day5 and Day 7.The number of neutrophils rose at 12 hours,peaked at Day 3,and then gradually declined.But the number of astrocytes and neutrophils were not different between the WT ICH group and the Tim-3-/-ICH group.The number of microglia/macrophages increased at 12 hours and peaked at Day 1.This variation was the same as that of Tim-3,IL-1? and TNF-a.In Tim-3-/-ICH mice,the number of microglia/macrophages was obviously less than that of WT ICH mice and there was a significant difference between the two groups(P<0.01).Conclusions:Our findings demonstrate that Tim-3 plays an important role in brain inflammation after ICH,and may be a potential treatment target.Part II:The effect of T cell immunoglobulin and mucin domain 3 on the systemic inflammatory reaction of patients with spontaneous intracerebral hemorrhagePurposes:To study the expression of Tim-3 on peripheral blood immunocytes and the relationship between Tim-3 and the systemic inflammatory response or brain injury of patients with intracerebral hemorrhage.Methods:According to the volume of hematoma at 12 hours after ICH onset,60 newly diagnosed ICH patients were divided into the small(volume of hematoma<30ml,30 cases)and large(volume of hematoma?30ml,30 cases)ICH group.The expression of Tim-3 on peripheral blood immunocytes was analyzed by flow cytometry.Real time RT-PCR was used to detect Tim-3mRNA on peripheral blood mononuclear cells(PBMCs).Meanwhile,tumor necrosis factor-a(TNF-a),interleukin-1?(1L-1?)and S-100B protein were measured by ELISA.Glasgow outcome scale(GOS)at Day 30 was used to estimate the prognosis of patients with ICH.Results:1.The leukocyte count,neutrophil count and monocyte count increased after onset of ICH,peaked at Day 3,and decreased at Day 7.Both the leukocyte count and the neutrophil count increased more obviously in the large ICH group and there was a significant difference when compared with those in the small ICH group at all tested time points(P<0.01).The monocyte count in the large ICH group increased remarkably at Day 3 and Day 7.The neutrophil percentage also increased at all tested time points after ICH,but there was a difference between the two ICH groups only at Day 3(P<0.01).Regarding monocyte percentage,there was no difference among three groups.The variation of the lymphocytes was directly opposite to that of the neutrophils and monocytes.The count and percentage of the lymphocyte decreased obviously at all tested time points,particularly in the large ICH group,and there was a significant difference when compared with that in the small ICH group(P<0.01).2.Tim-3 expression on the CD14+ cells in the large ICH group was very much higher than that in the control group(t=4.174,P<0.001).On the CD4+T cells,Tim-3 expression slightly increased in the large ICH group,but there was no statistical significance between the patients in the large ICH and healthy controls(t=1.674,P=0.10).Tim-3 expression on the CD8+T cells in the large ICH group was obviously lower than that in the control group(t=3.801,P<0.001).3.The Tim-3 mRNA expression increased in the two ICH groups at Day 1,peaked at Day 3,and decreased at Day 7 after ICH.There was a significant difference when compared with that in the control group at all tested time points(P<0.01).However,the Tim-3 expression in the large ICH group increased more dramatically than that in the small ICH group and there was an obvious difference between the two ICH groups(P<0.01).4.The concentration of IL-1? and TNF-? increased obviously in the two ICH groups at all tested time points.There was a significant difference between the two ICH groups(P<0.01).S-100B protein increased persistently within 7 days after ICH.However,the concentration of S-100B protein in the large ICH group was much higher and there was a significant difference when compared with that in the small ICH group and the control group(P<0.01).5.The expression of Tim-3 is positively correlated with the concentration of IL-1?(small ICH group:r=0.498,P<0.001;large ICH group:r =0.511,P<0.001),TNF-a(small ICH group:r=0.598,P<0.001;large ICH group:r= 0.682,P<0.001)and S-100B protein(small ICH group:r=0.508,P<0.001;large ICH group:r=:0.477,P<0.001).There was negative correlation between GOS and Tim-3 mRNA(small ICH group:r=0.650,P<0.001;large ICH group:r=0.723,P<0.001),IL-1?(small ICH group:r=0.492,P=0.06;large ICH group:r=0.497,P=0.005),TNF-a(small ICH group:r=0.569,P=0.001;large ICH group:r=0.637,P<0.001),or S-100B protein(small ICH group:r=0.508,P=0.004;large ICH group:r=0.538,P=0.002).Conclusions:The expression of Tim-3 on CD 14+ monocytes involves in systemic inflammatory reaction after ICH and may be a novel treatment target. |
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