Effects Of Whole Brain Irradiation On Expression Of Occludin And Claudin-5, Tight Junction Associated Proteins, In Blood-brain Barrier Of Rats

Objectives:To investigate the effect and the mechanism of irradiation on the permeability ofblood-brain barrier (BBB), through detecting the concentrations of Carmustine (BCNU) inbrain tissue homogenate, and the expression changes of tight junction associatedprotein,Occludin and Claudin-5,in cerebral blood vessels, afer different doses X-rayirradiation, for the purpose of offering experimental and theoretical basis to clinicalradiochemotherapy in the treatment of brain tumors.Methods:After one-fraction whole-brain irradiation on SD rats, the concentrations ofCarmustine (BCNU) in serum and brain tissue homogenate were measured byhigh-performance liquid chromatography (HPLC)20min after tail vein injection; and themRNA expressions of occludin and claudin-5in rats’ cerebral microvessels were detectedby RT-PCR,on different doses of irradiation at different time points; andimmunohistochemical assay was used for the detection of the expression of the proteinsOccludin and Claudin-5in rat brain vascula among groups of14day after irradiation andgroups of15Gy irradiation at each time point.Result:1.Concentration of BCNU: The concentrations of BCNU in the serum in each grouprats didn’t have statistical significance (F=1.44, P>0.05); however, in the brain tissuehomogenate they gradually increased along with the increase of irradiation doses, exceptfor the differences between20-Gy and15-Gy group showed no significant (P>0.05), thedifferences among the other groups were statistically significant (F=53.32, P <0.05).2.Results of occludin and claudin-52mRNA expression by PCR:1) Effects of occludin mRNA expression along with different doses of X-ray: Compared with the control group, expressions in each group of occludin mRNA afterirradiation were lower, with the difference was statistically significant (P <0.05). But in1to14day(s) after irradiation, occludin mRNA expression results decreased along withirradiation doses increasing, and within the same time points among each group thedifferences were statistically significant (P <0.05), except for the difference between20Gy group and15Gy group, which was not statistically significant (P>0.05). In21daysafter irradiation, the expression quantity of occludin mRNA wasn’t related with thechange of irradiation doses, with no significant statistical difference among each group(P>0.05).2) Effects of occludin mRNA expression along with time:The expression of occludin mRNA in7-21days after irradiation increased along withtime, in the same dose group there were statistically significant differencs among eachtime groups (P <0.05).While there was not statistically significant differencs between7-day groups and1-day groups whith the same dose group（P>0.05）.3) Effects of claudin-5mRNA expression along with different doses of X-ray:Compared with the control group, expressions in each group of claudin-5mRNA afterirradiation were lower, with the difference was statistically significant (P <0.05). But in7days and14days after irradiation, claudin-5mRNA expression results decreased alongwith irradiation doses increasing, and within the same time points among each group thedifferences were statistically significant (P <0.05), except for the difference between20Gy group and15Gy group, which was not statistically significant (P>0.05).In1day and21days after irradiation, between5-Gy groups and10-Gy groups, or20-Gy groups and15-Gy groups,the differences showed all no statistically significance (P>0.05), while in15-Gy groups, compared with10-Gy groups at the same time point, the expression werereduced, and differences were statistically significant (P <0.05).4) Effects of claudin-5mRNA expression along with time:In1to21day(s) after irradiation, the expression of claudin-5mRNA in each irradiatedgroups increased along with time, and the diffence had statistical significance among eachtime-group in the same dose group (P <0.05).3.The results of Occludin and Claudin-5protein in cerebral vascular detected byimmunohistochemitry:Occludin and Claudin-5, tight junction associated proteins, in brain tissue of rats in each group, were positively expressed along the blood vessels; and compared with thecontrol group, the two are all weakened in the irradiated groups, with the difference werestatistically significant (P <0.05). In14days after irradiation, they decreased along withincreasing of irradiation doses, and except for20-Gy group compared with15-Gy group,the differences among the other groups were statistically significant (P <0.05).And theexpression increased along with time with7to21days after irradiation, with statisticallysignificant differences among every groups(P <0.05). But there was no statisticallysignificant differences between7-day group and1-day group after irradiation（P>0.05）.Conclusions:The whole-brain irradiation can increase the permeability of blood-brain barrier.When the irradiation dose is less than15Gy by one-fraction whole-brain irradiation, thepermeability of blood-brain barrier increases along with increasing of doses of X-ray,while the expression of Occludin and Claudin-5, tight junction protein, decreases. Butthere more than15Gy increased dose shows no significant change. In about1or2week(s),after one-fraction whole-brain irradiation, expression of proteins Occludin andClaudin-5gradually increase, and the blood-brain barrier function gradually recovers. Sothe molecular mechanisms of radiation induced blood-brain barrier opening may beinvolved in loss of Occludin and Claudin-5, tight junction associated proteins in vascularendothelial cell.