The Study Of Tight Junctions And Vasculogenesis In BSCB Dysfunction After Spinal Cord Injury In Rats

Background and ObjectiveSpinal cord injury is a central nerve trauma severely endangering survival and quality of human life. Along with the rising popularity of universal movement and increasing transportation, the incidence of SCI has significantly increased in recent years. Studies showed that the spinal cord suffered ischemia-edema-free radical-edema-ischemia. The vicious cycle caused by microcirculation dysfunction after SCI is one of the main processes that resulting in irreversible degeneration, necrosis and apoptosis of spinal cord. Blood spinal cord barrier plays an important role in this process. This study focuses on the ultrastructure of intercellular tight junctions and the expression of tight junctions proteins (Claudin-5and ZO-1) as well as the neovascularization, in order to investigate the probable mechanism between tight junctions and vasculogenesis in BSCB function after SCI.Method1. A total of80SD rats were used in this experiment. Quarter was as normal group, others were as SCI group by using modified Allen’s method,1day,3day,7day group after SCI were divided randomly, each group of20samples. T10section of spinal cord tissue was used for the following test.2. Using transmission electron microscope to observe ultrastructure of tight junctions between spinal cord capillary at different point of time, i.e. normal,1day,3day and7day after SCI, each point of4samples, a total of16samples.3. Using immunofluorescence and Western Blotting techniques to test the spatial distribution and expression of TJ proteins (Claudin-5and ZO-1) at different point of time (i.e. normal,1day,3day and7day after SCI), each point of4samples, a total of32samples. All the results of immunofluorescence were analyzed by ImagePro Plus software.4. Using immunofluorescence techniques to make a two-dimensional evaluation on spatial distribution and expression of SD rats’CD34+cell. The samples were extracted on normal and1day,3day,7day after SCI, each point of4samples, a total of16samples. CD34was common with Claudin-5and ZO-1respectively. All data was analyzed by ImagePro Plus software.5. Injecting Evans blue in vivo, the extravasation of Evans blue was observed after SCI by fluorescence microscope, and using spectrophotometer to detect the content of Evans blue at different point of time, i.e. normal,1day,3day and7day after SCI, each point of4samples, a total of32samples.6. All statistical analysis was used SPSS13.0statistics software, The T test was applied in the group comparison. P<0.05was statistically significant.Results1. Tight junctionsTransmission electron microscopy (TEM) observation confirms that the spinal cord vascular endothelial cells connect closely in normal group. The tight junctions between endothelial cells open after acute SCI, the most significant of which occur in the third day after SCI, then gradually recover as time continues, but still have not reached normal levels in the7day after SCI.2. Tight junctions proteins (Claudin-5and ZO-1)Immunofluorescence assay observation confirms that the tight junctions proteins (Claduin-5and ZO-1) are consistent with spinal cord vascular distribution. The distribution and expression are abnormal at1day,3day,7day after SCI, significantly occurs in the first day after injury. Though the expression increases gradually after lday post SCI, have not reached to normal levels in7day after injury. The same results are found in Western Blotting test.3. CD34+cellImmunofluorescence assay observation confirms that the CD34+cells are consistent with blood vessel endothelium. After SCI, there are lacks of CD34+cells at the epicenter zone as well as the expression decreases, significantly occurs in the first day after injury. Though the expression increases gradually after1day post SCI, have not reached to normal levels in7day after injury. At the3day after SCI, there is a few of CD34+cells are observed at the epicenter zone. Until7day after SCI, the CD34+cells are obviously observed in the same region.4. BSCB permeabilityThe extravasation of EB is hardly observed by fluorescence microscope in normal group, however, there is an overt extravasation of EB at1,3,7day after SCI, especially at3day post-SCI. The same outcome is obtained in quantitative analyze.Conclusion1. The distribution and expression of TJ proteins (Claudin-5and ZO-1) decline after SCI, especially at1day post-SCI and increase gradually.2. The spinal cord vascular network is damaged after SCI, especially at1day post-SCI; Vasculogenesis can be observed at3day after SCI at epicenter zone.3. The tight junctions, the decrease expression of tight junctions proteins (Claudin-5and ZO-1) and vasculogenesis may result in dysfunction of BSCB after SCI.