| ObjectiveIntracerebral hemorrhage (ICH) is defined as non-traumatic cerebral parenchymal hemorrhage, which is a kind of disease commonly and frequently occurred in the nervous system. ICH has both high disability and fatality and greatly threatens the health of the patient and the quality of life. Post hemorrhage cerebral edema plays an important role in the deterioration of clinical condition. The cerebral brain hernia induced by it can also lead to death of patients. Treatment options were limited to osmotic agents such as hypertonic dehydration agents and diuretics, surgical decompression, non of which correct the molecular-level mechanisms responsible for brain edema. Endothelial Barrier Antigen (EBA) is a protein triplet of molecular weights 30,25, and 23.5 kDa located in the endothelial plasma membrane of cerebral microvessels, which is selectively expressed by endothelial cells (ECs) of rat blood-brain barrier (BBB). EBA is the key to the cerebral edema that is caused by cerebral ischemia, subarachnoid hemorrhage, brain trauma and etc. In this research, Horseley-Clarke technique is used to inject 50μL of autogenetic blood from femoral artery into caudate nucleus of mouses, immunohistochemistry is used to measure the expression levels of EBA and Fibrinogen, western blot is used to observe the expression of Fibrinogen, permeability of blood-brain barrier(BBB) and brain water content in rats, which aims to find out whether EBA is expressed and distributed in the brain tissue around hematoma, and then to discuss the role of EBA pathway in secondary lesion after cerebral hemorrhage.Materials and methodsMaterials1. grouping150 male Sprague Dawley mouses that weigh from 250 to 300g were randomly grouped into normal control group, sham-operated control group, saline control group, experimental group. The last two groups were respectively sub-grouped by 6h,1d,3d and 7d, n=15.2. major equipments Experimental cranial sterotaxic apparatus, electronic analytical balance, micro-image analysis apparatus, ultraviolet spectrometer, microsyringe, pyro-drying cabinet, tissue homogenater, electronic trophoresis apparatus, and gel-image analysis system.3. major reagentEvens blue, formamide, EBA rabbit anti-mouse polyclonal antibody, Fibrinogen sheep anti-mouse polyclonal antibody, instant SABC immunohistochemistry test kit, DAB test kit, Tris, Acr, and methanol.4. models of cerebral hemorrhageModels of slow injection with autoblood. Mouses 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. Mouses in saline control group were injected with NS, Sham-operated control group without injection, while normal control group received no disposition.5. making samples(1)5 mouses were taken from each group after the corresponding time of operation, and anesthetized, decapitated and got brain off for brain water content and permeability of BBB.(2) 5 mouses 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 for 4 hours, dehydrated in alchohol, transparent by xylol, dipped in wax and embedded. Successive coronal-slice, about 5 um thick, and then stain.(3)5 mouses were taken from each group after the corresponding time of operation, and anesthetized, decapitated and got brain off for Fibrinogen Western Blot detection。Measure1. brain water contentAnterior brain tissue of needle puncture was resectioned and determined by [(wet-dry weight)/wet weight]%. With brain water content as degree of brain edema.2. permeability of BBBPosterior lateral brain tissue of needle puncture was recetioned and assessed by Evens blue stain, as Belayev's method. Permeability of the BBB is assessed by EB content (OD/mg).3. measuring EB A, Fibrinogen protein by immunohistochemistryUse SABC to measure positive cells which express EBA, Fibrinogen protein. Add the slice with 50uL-antifluid (rabbit anti mouse polyclonal EBA antibody (1:200), sheep anti mouse polyclonal Fibrinogen antibody (1:50)), stay overnight at 4℃; add goat anti rabbit IgG, rabbit anti goat IgG at 37℃for 30min; add SABC at 37℃for 30min; 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.4. measuring Fibrinogen protein by.Western BlotRemove the specimen 100mg, low-temperature extract proteins, SDS-PAGE separation of samples. Transfer film, closed, plus a goat anti-mouse polyclonal Fibrinogen(1:100), incubation bath, washing, add secondary antibody, then incubation bath, washing, color, photo film.Statistical analysisAll datas were demonstrated by mean±standard deviation(x±SD), using SPSS 16.0 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.Result1. brain water contentExperimental ICH in rats were significantly higher brain water content,6h began to increase,3d reached the peak,7d gradually reduced, as compared with the control group, but there are significant differences (P<0.05).2. measure the EB content in brain tissueAfter experimental ICH in rats brain the EB content increased,6h is significantly higher,3d reached the highest,7d group was gradually reduced, as compared with the control group, but there are significant differences(P<0.05).3. expression of EBA after cerebral hemorrhageNormal control rats cortex capillary endothelial cells showed strong positive expression of EBA, sham-operated control group and the saline control group compared with normal group there was no significant difference. After intracerebral hemorrhage, in 6h group the EBA expression in the ipsilateral cortex around the hematoma and endothelial cell membrane of micro vascular is decreased.1d EBA expression was significantly reduced; 3d the EBA expression was significantly reduced; the degree of EBA expression in 7d was increased than 3d, but still lower than the 6h and 1d.4. expression of Fibrinogen after cerebral hemorrhageNormal control rats have no Fibrinogen expression in the surrounding cortex microvessels. The expression of Fibrinogen in Sham-operated control group and the saline control group increased, but compared with the normal group, there was no significant difference. In 6h group, the cerebral cortex around the hematoma had weakly positive expression of Fibrinogen, and the positive sites were mainly around the capillaries.1d Fibrinogen expression levels increased significantly;3d Fibrinogen expression level reached the peak, coloring the most; 7d Fibrinogen expression level gradually decreased, but compared with the control group there are significant differences(P<0.05).5. western blot detection of protein expression FibrinogenFibrinogen almost has no expression in normal control group, Sham-operated control group and the saline control group Fibrinogen expression compared with normal group there was no significant difference. After intracerebral hemorrhage Fibrinogen protein 34KD band can be found in each group.6h Fibrinogen weakly positive expression;ld and 3d group Fibrinogen expression gradually increased,3d reached the peak;7d the expression of Fibrinogen has gradually reduced, but compared with the control group there are significant differences(P<0.05).DiscussionWe injected autoblood from femoral artery into right caudate nucleus to make models of cerebral hemorrhage, used immunohistochemistry to measure EBA and found out that there was change of expression of EBA protein after cerebral hemorrhage. In addition, the experiment after the BBB permeability of ICH was measured in two ways, exogenous tracers (EB) and endogenous tracer (Fibrinogen). And further confirmed the accuracy and reliability of endogenous tracer. In recent years, many experimental studies have confirmed that, EBA participated in the occurrence and development of brain edema caused by a variety of reasons. The test results showed cerebral hemorrhage group and the control group, EBA expression began to decrease after 6h, 1d and 3d group gradually reduced EBA expression, when 3d the EBA expression of the least gradually restored expression later,7d group recovered than before. The changes of BBB permeability and brain water content are in the opposite. The BBB permeability and brain water content result can be seen:6h after ICH slightly elevated, ld-3d gradually increased,3d reached its peak, and then gradually decreased, and 7d was still higher than normal levels. BBB permeability and brain edema was positively correlated. The expression of EBA was negatively correlated with BBB permeability and the extent of brain edema. After ICH, the decrease of EBA expression in brain tissue and the openness of BBB permeability all participate in the pahtophysilosical mechanisms in cerebral edema post hemorrhage. At the same time, the BBB permeability and the expression of EBA are negatively associated, suggesting that the decrease of EBA expression may indirectly influence the BBB permeability involved in the formation of brain edema. On the one hand, the study on the pathophysiological mechanism about the expression of EBA in post ICH cerebral edema may improve the possible mechanisms of brain edema after ICH, on the other hand, it may provide a sufficient theoretical basis for the effective treatment of the ICH.Conclusion1. These results indicate that in the EBA expression in brain tissue of rates reduced after intracerebral hemorrhage.2. EBA may influence the BBB permeability and participate in the pahtophysilosical mechanisms in cerebral edema post hemorrhage. |
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