铅暴露对大鼠血脑脊液屏障通透性、分泌及转运功能的影响

Effect of lead exposure on permeability,secretion and transportation function of blood-cerebro-spinal fluid barrier of rats

目的探讨铅暴露对血脑脊液屏障通透、分泌和转运功能的影响,为铅神经毒性机制研究提供依据。方法 SD大鼠分别饮用含有0.05%,0.1%和0.2%醋酸铅饮水8周(相当于醋酸铅80,160和320 mg·kg-1)。应用激光共聚焦免疫荧光法检测血清、脉络丛和脑脊液中铅含量;Morris迷宫测试潜伏期延长和穿台次数。股动脉灌注伊文氏蓝(EB)和荧光索钠(NaFI),检测脑脊液中EB和NaFI含量;应用激光共聚焦法进行脉络丛中紧密连接黏附分子(JAM)、闭合蛋白的表达分布;ELISA方法检测血清和脑脊液中甲状腺素运载蛋白和瘦素含量。结果染铅大鼠血清、脉络丛和脑脊液中的铅含量明显上升,尤其以CSF中铅含量的增加尤为显著。水迷宫数据显示,醋酸铅160和320 mg·kg-1染毒组大鼠的潜伏期分别为52±12和(89±19)s,显著高于正常对照组的(28±7)s(P<0.05);穿台次数显著低于正常对照组(P<0.05)。3个醋酸铅染毒组大鼠脑脊液中NaFI含量分别为0.94±0.09,1.02±0.03和(1.08±0.18)mg·L-1,均显著高于正常对照组(P<0.05)。与正常对照组比较,醋酸铅160和320 mg·kg-1染毒组大鼠脑脊液中EB含量明显增加,差异有统计学意义(P<0.05)。激光共聚焦免疫荧光检测结果显示,醋酸铅160和320 mg·kg-1染毒组大鼠脉络丛紧密连接蛋白JAM表达分别为正常对照组的44.9%和42.9%;且闭合蛋白表达也呈下降趋势。醋酸铅320 mg·kg-1染毒组大鼠脑脊液中转甲状腺素蛋白含量为(32.3±11.7)ng·g-1蛋白,显著低于正常对照组(P<0.05)。醋酸铅160和320 mg·kg-1组大鼠脑脊液瘦素含量显著下降(P<0.05)。结论铅染毒可导致血脑脊液屏障通透性增加、分泌和转运功能下降,这可能是铅致中枢神经系统损伤的机制之一。

OBJECTIVE To investigate the effects of lead exposure on the permeability,secretion and transportation function of blood-cerebro-spinal fluid barrier （BCB）of rats in order to provide the theo-rical basis for elucidating the mechanis m of lead induced neurotoxicity.MEHTODS 60 SPF SD rats were rando mly divided into 4 groups,including a control group and three doses lead exposed groups. Rat in the lead exposure groups were given drinking water containning 0.05%,0.1 % and 0.2% lead acetate （at dose of 80,160,320 mg·kg -1 ）for 8 weeks.Laser scanning confocal microscopy was uti-lized to determine the lead content in seru m,cerebrospinal fluid （CSF）and choroid plexus sa mples. Morris maze was used to test learning and me mory.Fe moral artery perfusion of Evans blue （EB）and fluorescein sodiu m （NaFI）was performed to measure BCB permeability function.Confocal laser scan-ning was applied to detect junction adhesion molecule （JAM）and occludin protein expression in choroid＆amp;nbsp;plexus.ELISA was used to measure the concentration of transthyretin （TTR）and leptin in seru m and CSF.RESULTS The lead content in seru m,choroid plexus and CSF significantly increased,especially the lead level in CSF.Morris water maze data showed that escape latency of rat in lead acetate 160 and 320 mg·kg -1 group were 52 ±12,（89 ±19）s,respectively,longer than that of control group 〔（28 ±7）s, P〈0.05〕.The ti mes across platform of rats in lead acetate 160 and 320 mg·kg -1 group were lower than that of control group（P 〈0.05）.The NaFI content in CSF of rats in all lead acetate exposure groups were 0.94 ±0.09,1 .02 ±0.03 and （1 .08 ±0.18）mg·L -1 ,respectively,and were higher than those of control group〔（0.74 ±0.04）mg·L -1 〕;While the EB content in CSF of rat in lead acetate 160 and 320 mg·kg -1 group were higher than the control group（P 〈0.05）,which indicated that lead acetate exposure at low dose can lead to the increase of permeability of BCB.Laser scanning confocal micro-scope i mages showed that the JAM protein expression of choroid plexus in lead acetate 160 and 320 mg·kg -1 group were 44.9% and 42.9% of the control group.Sa me decline was seen in terms of occludin expression.The TTR content of CSF of rats in lead acetate 80 mg·kg -1 group was （32.3 ± 1 1 .7）ng·g -1 protein,lower than that of the control group,and the difference was significant.This decline was also noted in lead acetate 160 and 320 mg·kg -1 group.The data of TTR in CSF suggested that the low dose lead acetate exposure can disrupt the BCB secretion function.The leptin levels in CSF of lead acetate 160 and 320 mg·kg -1 group were lower than that in the control group （P 〈0.05 ）. CONCLUSION Lead exposure did disrupt the permeability,transportation and secretion function of BCB.Our data suggest that BCB dysfunction might be involved in the mechanis m of lead induced neurotoxicity.