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Objective To investigate the effect of hemoglobin on structure and function of blood-brain barrier (BBB) in rat models after intracerebral hemorrhage.Methods One hundred and eight male SD rats were randomized into normal control group (n=12),intracerebral hemoglobin injection group (Hb group,n=48) and sham-operated group (n=48); according to the different observation time points,rats in the Hb group and sham-operated group were divided into 4 subgroups (6 and 24 h,3 and 7 d after the injection,n=1 2).Histological changes and iron deposition of the brain tissues were examined with HE staining and iron staining,respectively.BBB permeability was evaluated by the permeation of Evens blue.The expressions of claudin-5 and ZO-1 in perihematomal brain tissues were detected by immunofluorescence staining and real-time fluorescence quantitative PCR.Results Evident edema and necrosis were observed in the perihematomal zone of Hb group,and iron deposition was found 3 and 7 days after the injection.The permeation of Evens blue in Hb group was significantly increased as compared with that in sham-operated group (P＜0.05).Immunofluorescence staining indicated that expressions ofclaudin-5 and ZO-1 were low and discontinuous in Hb group; the mRNA expression levels of claudin-5 and ZO-1 in Hb group were significantly lower than those in the sham-operated group (P＜0.05).Conclusion After intracerebral hemorrhage,Hb may induce the damage of BBB and participate in the course of brain edema.
RÉSUMÉ
Objective To explore the effects of hemoglobin level on Rho kinase Ⅱ of endothelial cells and tight junction protein clandin-5 in blood-brain barrier (BBB) of rats after intracerebral hemorrhage (ICH).Methods Male SD rats were randomized into normal control group (n=18) and hemoglobin inducement group (n=72); hemoglobin inducement group was divided into 4 subgroups (24 and 48 h,and 3 and 7 d after surgery,n=18).Rat ICH models in the hemoglobin inducement group were established by hemoglobin injection into the brain tissues.The expression ofRho kinase Ⅱ of endothelial cells and phosphorylated myosin light chain protein in BBB were observed by immunohistochemical staining,and BBB permeability was evaluated by Evans Blue dye; The protein and mRNA expressions of claudin-5 in the perihematomal brain tissues were detected by immunofluorescence staining and real-time quantitative PCR.Results Immunohistochemical staining indicated that expressions of Rho kinase Ⅱ of endothelial cells and phosphorylated myosin light chain in BBB of normal control group were rare,and those in the hemoglobin inducement group were high; as compared with that in the normal control group,mean optical density of Rho kinase Ⅱ and phosphorylated myosin light chain proteins in the endothelial cells of BBB in hemoglobin inducement group increased significantly at 48 h and 3 d after inducement (P＜0.05).The permeation of Evans Blue in hemoglobin inducement group significantly increased (P＜0.05).Immunofluorescence staining showed that the expression of clandin-5 in hemoglobin inducement group was uncontinuous and low as compared with that in the normal control group at 24 and 48 h and 3 d after inducement.PCR showed that the mRNA expression of claudin-5 in hemoglobin inducement group was significantly lower than that in the normal control group at 24 and 48 h and 3 and 7 d after the inducement (P＜0.05).Conclusion After intracerebral hemorrhage,hemoglobin can lead to increased expression of phosphorylated myosin light chain,and reduce the expression of tight junction protein claudin-5 of BBB by increasing the expression ofRho kinase Ⅱ in endothelial cells of BBB,which may be an important mechanism in the disruption of BBB and the occurrence of brain edema after intracerebral hemorrhage.
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Objective To investigate the changes of tight junction (TJ) protein occludin expression between the mierovascular endothelial cells of the blood-brain barrier (BBB) of rats after intracerebral hemorrhage (ICH). Methods Ninety-three male SD rats were randomly divided into normal control group and experimental group; rats of the experimental group were divided into 6 subgroups (6, 24, 48 and 72 h, and 7 and 14 d after intraeerebral hemorrhage). Rat models of intracerebral hemorrhage in the experimental group were established by autologous blood injection.Morphology of the brain tissues was detected by hematoxylin-eosin (HE) staining.The tight junctions between the microvascular endothelial cells of the BBB were sampled for ultrastructural observation using electron microcopy.Immunofluorescence and quantitative real time-PCR were used to analyze the protein and mRNA expressions of occludin,respectively. Results The brain edema was found in brain tissues around the hematoma. Necrosis and inflammatory infiltration could be found in perihematomal brain tissues and it was most obvious at 48 h after intracerebral hemorrhage. Obvious paracellular clefts were found between the adjacent endothelial cells in experimental groups. Strong expression of occludin was noted in the normal brain tissue; expression of occludin was positive 6 h after the injection and weak positive at 24, 48 and 72 h after the injection in the experimental groups.Quantitative real time-PCR showed that the mRNA expression of occludin in the experimental group was significantly reduced as compared with the normal brain tissue at 6,24,48 and 72 h after the injection with statistically significant difference (P＜0.05). Conclusion After intra-cerebral hemorrhage,as a major component of TJ of the BBB,occludin expression is down-regulated,which may be one of the most impotant molecular basis of disruption of BBB.
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[Objective]To explore the distribution and expression changes of tight junctional protein JAM-1 in rat models after intracerebral hemorrhage (ICH) and their significance.[Methods]One hundred and twenty-eighty healthy male SD rats were randomly divided into normal control group (n=16) and ICH group (n=112),and the ICH models were induced by stereotactically injecting 75 uL autologous blood into the right caudate nucleus.Seven time points after ICH (6,12,24 and 48 h,and 3,7 and 14 d after ICH,16 rats for each time point) were chosen.BBB permeability was evaluated by Evans blue dye extravasation.The distribution and expression of JAM-1 were detected by immunofluorescence and real-time quantitative PCR.[Results] As compared with that in the normal control group,BBB permeability in the ICH group significantly increased at 24 and 48 h,and 3 and 7 d after ICH (P＜0.05).JAM-1 expression decreased at blood vessels at 12,24 and 48 h after ICH,and JAM-1 expressed at the circulatingleukocytes3 dafterlCH,and abundant JAM-1 positive cells around hematoma were noted in the ED-l-positve macrophages 7 d after ICH.JAM-I mRNA significantly decreased at 12,24 and 48 h after ICH,and significantly increased 7 d after ICH as compared with that in the normal control group (P＜0.05).[Conclusion] JAM-1 experssion changes not only participate in regulation of BBB permeability but also play roles in inflammatory insult after ICH.