| ObjectiveIn the study, we explore the dynamic changes of endothelial barrier antigen(EBA) and vascular endothelial growth factor(VEGF) positive expression in cerebral cortex under the condition of blood-brain barrier(BBB) damage in rats after CT X-ray irradiation, then we analyze the correlation between the BBB permeability changes and EBA, VEGF expression changes, which provides an important reference for the clinical treatment of early vascular lesions following radiation-induced brain injury. Methods1. Preparation for the experimental animals: Adult clean grade male SD rats(n=72,body weight 210-230g)were kept on the environment of constant humidity and 22±2?. All the animals were given a standard laboratory diet, natural circadian rhythm lighting and distilled water ad libitum for one week.2. Radiation-induced brain injury model making: 24 rats were divided into the sham-operated group(n=6) and the injury group(n=18) by using stochastic indicator method. The rats in injury group were randomly divided into three subgroups for analysis on day 7, 14 and 28 after brain irradiation(each n=6). After rats were anesthetized by using 20% urethane(5 mL/kg) through intraperitoneal injection, the injury model was established by using 16 detector row helical CT to irradiate the specified area of brain, the irradiation area was between the lambdoid suture and coronal suture(using a dedicated self-shielded lead mold to cover other area). The rats in sham-operated group were not irradiated after anesthesia. The success of the injury model was determined by detecting the radioactive sensitive protein.3. The detection of indicators: 24 rats were divided into the sham-operated group(n=6) and 7d, 14 d, 28 d after brain irradiation group(each n=6). The injury model was established by the same way. Rats were injected into 3% Evans Blue by tail vein injection according to the dose of 3 mL/kg, then the blood vessels of cerebral cortex were exposed after having a craniotomy. We observed the capillary permeability of cerebral cortex by microcirculation microscope and recorded the result by microscope vascular camera device( × 400). The permeability of BBB was evaluated by measuring the integrated optical density(IOD) of Evans Blue outside the blood vessels with the help of Image-proplus 6.0 medical image analysis software. After 24 rats were grouped and the injury model was established by the same way, we measured the expression levels of EBA and VEGF in the cerebral cortex by immunohistochemistry staining in each group. Five non-overlapping high-power(×400) images of each section were captured and the tan cells were positive. The expression levels of EBA and VEGF were counted by measuring the IOD of each image with the help of Image-proplus 6.0.4. Correlation analysis: We did correlation analysis between the Evans Blue extravasation and EBA, VEGF expression levels by using Pearson correlation analysis. Result1. The expression levels of radioactive sensitive protein such as glial fibrillary acidic protein(GFAP) and S100 B protein changed regularly on day 7, 14 and 28 after brain irradiation(it has been reported by senior fellow apprentice and senior sister apprentice), which proved that using CT X-ray can make the radiation-induced brain injury model of rats successfully.2. Evans Blue was limited into the blood vessels in the sham-operated group. Evans Blue extravasation was obvious at day 7 after CT X-ray irradiation, which dyed a blue area outside the blood vessels. Evans Blue extravasation was subsequently decreased from day 14 to 28, but both were higher than the sham-operated group.3. The expression of EBA and VEGF in the cerebral cortex in each group was measured by immunohistochemistry staining. The expression levels of EBA were decreased on day 7 after brain irradiation compared with the sham-operated group and subsequently recovered from day 14 to 28, but both were still reduced compared with the sham-operated group. On the contrary, the expression levels of VEGF were increased on day 7 and then recovered from day 14 to 28, but both were still higher than the sham-operated group.4. Evans Blue extravasation levels were negatively correlated with EBA expression levels and positively correlated with VEGF expression levels. Conclusions1. We made radiation-induced brain injury model of rats by using the CT X-ray irradiation in this experiment, which was a new approach of making model with high repeatability, this approach was simple and had a good clinical simulation.2. The changes of BBB permeability were assessed by using Evans Blue combined with microscope vascular camera device, we made an important progress on the analysis of BBB permeability in the body by using Evans Blue as tracer.3. In this study we measured the positive expression of EBA and VEGF by IHC staining, which proved that the CT X-ray irradiation can be used to make radiation-induced brain injury model successfully on the molecular level.4. The increases of Evans Blue extravasation had relation to the decreases of EBA expression and increases of VEGF expression, which proved that the two proteins can be considered as an important index of the BBB damage and repair. This can provide an important reference for the clinical treatment of early vascular lesions following radiation-induced brain injury. |
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