| Objective(s):A model of synergistic damage of tree shrew BBB by METH and HIV-Tat protein has been established,to observe the differences of different groups of tree shrews after administration from three different perspectives of animal behavior,histomorphology and molecular biology,and to investigate the role of GLUT]and GLUT3 in the synergistic damage of tree shrew BBB by METH and HIV-Tat protein,and whether gastrodin can interfere with the synergistic damage of tree shrew BBB.Methods:Seventy healthy adult male tree shrews were randomly divided into control groups,experimental groups and gastrodin intervention groups.The control groups included normal saline control group and gastrodin control group;the experimental groups included METH group,HIV-Tat protein group and METH+HIV-Tat protein group;the gastrodin intervention groups were composed of low gastrodin+METH+HIV-Tat protein group and high gastrodin+METH+HIV-Tat protein group,respectively.Ten tree shrews were included in each group to establish the model of synergistic damage of tree shrew BBB by METH and HIV-Tat protein.Tree shrews were put into the experimental box to adapt and allowed to move freely,one hour a day for three consecutive days.On the third day of adaptation,observations were carried out through video surveillance,and the related indicators such as mental status,movement status and behavioral changes of tree shrews in the experimental box were tracked and recorded by using Super Maze series animal behavior analysis software of Shanghai Xinruan Informatlon Technology Co.,Ltd.Starting from the fourth day,the shrews were administered for 10 consecutive days,and relevant indicators such as mental status,movement status and behavioral changes of tree shrews in the experimental box were observed and recorded.Gastrodin control group was intraperitoneally injected with gastrodin at 8 mg/kg day;the normal saline control group was intraperitoneally injected with normal saline according to the standard of the gastrodin control group;the METH group was intraperitoneally injected with METH at 8 mg/kg day;the HIV-Tat protein group was intravenous injected with HIV-Tat protein through tail vein at 100ng/treeshrew·day;the METH+HIV-Tat protein group was intraperitoneally injected with METH at 8 mg/kg day immediately after intravenous injection of HIV-Tat protein at 100 ng/treeshrew·day through tail vein;the low gastrodin+METH+HIV-Tat protein group was intraperitoneally injected with gastrodin at 10 mg/kg·day,and then administered in the same way as in the METH+HIV-Tat protein group after 1h;the high gastrodin+METH+HIV-Tat protein group was intraperitoneally injected with gastrodin at 20 mg/kg·day,and then administered in the same way as in the METH+HIV-Tat protein group after 1h.After 24 hours of the last administration,samples were taken according to the experimental requirements.Then the content of EB and NaF in the brain tissues of tree shrews was detected by microplate reader,the ultrastructural changes of striatum in the brains of tree shrews were observed by transmission electron microscopy,and the expression of GLUT1,GLUT3,JAM-A,Occludin and ZO-1 in the prefrontal cortex of tree shrews was detected by Western Blot.Results:(1)The model of synergistic damage of tree shrew BBB by METH and HIV-Tat protein was successfully established.(2)Single-person operation method of tail vein injection in the awake state of tree shrew was successfully established:a canvas glove was used first to bind the tree shrew and then the glove was tightened with a folder;the tail of the tree shrew was fixed by the left hand,and drugs with a 1ml disposable syringe were injected by the right hand.(3)After injecting EB and NaF into tail vein,the color changes of brain tissues and the contents of EB and NaF in brain tissues of tree shrews in control group,experimental group and gastrodin intervention group were not the same(P<0.05),and the color of brain tissues of tree shrews in experimental group changed significantly with increased EB and NaF,especially in METH+HIV-Tat protein group;the degree of color change of brain tissues and the contents of EB and NaF in the intervention group was between the control group and the experimental group,and the change in low gastrodin+METH+HIV-Tat group was more obvious than that in high gastrodin+METH+HIV-Tat group.(4)Compared with the control group,the striatum ultrastructure of the brain tissue of the tree shrews in the experimental group and the gastrodin intervention group changed in varying degrees,and the most significant change was in the METH+HIV-Tat protein group,which showed significantly swollen vascular endothelial cells,broken basement membrane,severely edematous peripheral vessels and separated microvascular basement membrane;the swelling degree of vascular endothelial cells and perivascular edema in gastrodin intervention group were alleviated.(5)After several minutes of administration,the activity of tree shrews in the experimental groups(METH group,HIV-Tat protein group and METH+HIV-Tat protein group)and gastrodin intervention group(low gastrodin+METH+HIV-Tat group and high gastrodin+METH+HIV-Tat group)increased,with different degrees and duration of stereotyped behavior(P<0.05),which were manifested as:activities accompanied by repeated exploration,repeated climbing and jumping,repeated rapid running and circling along the periphery,repeated head twisting,tail raising,repeated convulsions,hiccups,dorsiflexion,repeated head raising,vertical tail shaking,unstable gait,repeated mouth scratching,self-mutilation,rapid tail rotation in situ,etc.The most significant performance was in the METH+HIV-Tat protein group,followed by the METH group,the low gastrodin+METH+HIV-Tat group,and the HIV-Tat protein group.The stereotype behavior of tree shrews in the high gastrodin+METH+HIV-Tat group is lower than that of the low gastrodin+METH+HIV-Tat group.There was no significant change in the behavior of the tree shrews in the control group(normal saline control group and gastrodin control group).(6)The scoring criteria for tree shrews' stereotypical behaviors was successfully established:0 point,motionless or hardly active;1 point,normal activities accompanied by repeated exploration in situ or no fixed direction of repeated exploration;2 points,rapid running and circling along the periphery,repeated climbing and jumping;3 points,repeated head twisting,tail raising,repeated convulsions,hiccups,dorsiflexion;4 points,repeated head raising,vertical tail shaking,unstable gait,repeated mouth scratching,self-mutilation;5 points,rapid tail rotation in situ.(7)The immobility time,motion trajectory and total motion distance of tree shrews in the control groups,experimental groups and gastrodin intervention groups were different(P<0.05).The immobility time of tree shrews in the control groups in the open field was relatively long,the motion trajectory was clearer and concise,and the total moving distance was relatively short;in the experimental group,the immobility time of tree shrews in the open field was relatively short,and their movements were mainly in circles and more complex,with longer total distance of movement,represented by METH+HIV-Tat protein group;the immobility time,motion trajectory and total motion distance of the tree shrews in the intervention group were between the two groups,and the high gastrodin+METH+HIV-Tat group had longer immobility time,clearer and simpler motion trajectory and shorter total motion distance than the lower gastrodin+METH+HIV-Tat group.(8)Compared with the body weight before the first administration,the body weight of tree shrews in each experimental group and gastrodin intervention group decreased in varying degrees(P<0.05),while the body weight of tree shrews in saline control group and gastrodin control group increased normally.(9)Compared with the control group,the expression of JAM-A,Occludin and ZO-1 in each experimental group and gastrodin intervention group decreased in varying degrees(P<0.05),especially in METH+HIV-Tat group.Compared with METH+HIV-Tat group,the expression of three tight junction proteins was improved after gastrodin intervention,and the improvement was more obvious in high gastrodin+METH+HIV-Tat group.(10)Compared with the control group,the expression of GLUT1 and GLUT3 in each experimental groups and gastrodin intervention groups decreased in varying degrees(P<0.05),especially in METH+HIV-Tat protein group.Compared with METH+HIV-Tat protein group,the expression of GLUT1 and GLUT3 in prefrontal cortex of tree shrews in low gastrodin+METH+HIV-Tat group and high gastrodin+METH+HIV-Tat group increased,especially in high gastrodin+METH+HIV-Tat group.Conclusion(s):(1)METH and HIV-Tat proteins can synergistically damage the tree shrew BBB,and lead to behavioral changes of tree shrews(such as increased stereotypes,significantly increased total distance of exercise,significantly reduced immobility time,etc.).(2)GLUT1 and GLUT3 play an important role in the synergistic damage of tree shrew BBB by METH and HIV-Tat protein.(3)Gastrodin has a good interventional effect on the injury of tree shrew BBB caused by METH and HIV-Tat protein. |
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