| Objective:The aim of this study was to explore the optimal irradiation time,mechanical index,reversibility of Blood-Brain Barrier(BBB)opening and dynamic changes of transmittance in mice by low-frequency transcranial ultrasound combined with microbubbles,and to provide a theoretical basis for the later treatment of Alzheimer's Disease(AD)with low-frequency transcranial ultrasound combined with microbubbles.Methods:1.The feeding conditions provided by the Laboratory Animal Center of Military Medical University are C57BL/6(C57)male mice in SPF environment,aged 2 months,with an average body weight of about 25 g.2.C57 mice were randomly divided into groups according to different experimental purposes.To explore the optimal irradiation time,C57 mice were randomly divided into control group,ultrasound group and ultrasound combined with microbubbles group.According to the different action time of low-frequency ultrasound,the group of ultrasound combined with microbubbles was further divided into four subgroups:400 s,500 s,600 s and 700 s.To explore the optimal mechanical index:C57 mice were randomly divided into control group,ultrasound group,ultrasound combined with microbubbles group.According to the different mechanical index of ultrasound,the ultrasound combined with microbubbles group was further divided into 0.8,0.9,1.To explore the reversibility of BBB opening and the dynamic change of transmittance:C57 mice were randomly divided into control group and experimental group.According to the interval of 1VN injection after low frequency ultrasound combined with microbubbles,the experimental groups were divided into 0 h,0.5 h,1 h,2 h,3 h,8 h and 10 h.3.To explore the optimum irradiation time and the optimum mechanical index:after establishing tail vein passage in mice,the temporal area of mice was irradiated by low-frequency ultrasound,and microbubbles were injected at the same time.After irradiation with different irradiation time or mechanical index,Evans blue was injected into the tail vein,while no microbubbles were observed in the control group,and the same amount of saline was injected into the tail vein.To explore the optimal mechanical index:after establishing tail vein passage in mice,low frequency ultrasound was used to irradiate temporal area of mice,and microbubbles were injected at the same time.After different intervals,the tail vein was injected with Evans blue.The control group had no ultrasound,and the tail vein was injected with the same dose of Evans blue.4.Visual evaluation and quantitative analysis of Evans blue extravasation in brain tissue irradiated by ultrasound were carried out.Hematoxylin-eosin Staining(HE)was used to analyze the pathological damage of bilateral brain parenchyma.Results:1.To explore the optimum irradiation time:no blue staining of brain tissue was observed in control group and ultrasound group,and blue staining of different degrees was observed in irradiated areas in ultrasound combined with microbubbles group at 400 s,500 s,600 s and 700 s,and the area and degree of blue staining increased with the increase of irradiation time;the extraction amount of Evans blue at 400 s,500 s,600 s and 700 s was[(45.43±2.89),(108.50±2.27),(124.49±3.32),(152.96±3.06)?g/g]was higher than that of control group(8.41±0.13)?g/g and ultrasound group(8.91±0.15)?g/g,the difference was statistically significant(P<0.05).HE staining showed that there was erythrocyte exudation in brain parenchyma at 600 s and 700 s,while no erythrocyte exudation was observed in other groups.2.To explore the optimum mechanical index:there was no blue staining of brain parenchyma in control group and ultrasound group,and the mechanical index of ultrasound combined with microbubbles group was 0.8,0.9 and 1 in irradiated area,and the area and depth of blue staining increased with the increase of mechanical index.The extraction quantity of Evans blue at mechanical index 0.8,0.9 and 1 was[(61.26±1.29),(92.54±0.78),(108.50±2.27)?g/g],respectively,which was higher than that of the control group.There was significant difference between the control group(8.41±0.13)?g/g and the ultrasound group(8.91±0.15)?g/g,(P<0.05);no erythrocyte exudation was observed by HE staining.3.The reversibility of BBB opening and the dynamic changes of transmittance:No blue staining of brain parenchyma was found in control group and experimental group at 8 h and 10 h,and the blue staining of brain parenchyma at 0 h,0.5 h,1 h,2 h and 3 h in experimental group was different,and the blue staining degree and area were the largest at 1 h;the content of Evans blue in brain tissue of control group and experimental group at 0 h,0.5 h,1 h,2 h,3 h,8 h and 10 h was[(8.32±0.12),(45.43±2.89),(50.94±1.25),(79.79±1.12),(51.55±1.20),(31.60±1.77),(8.34±0.09),(8.31±0.07)?g/g]respectively(p<0.001).There were no significant differences between the control group and the experimental group at 8h and 10h(P>0.05).Conclusion:1.The optimal irradiation time for opening the blood-brain barrier in mice was 500 seconds when the low-frequency ultrasound frequency was 3 MHz and the pulse repetition frequency was 40 Hz.2.When the frequency of low-frequency ultrasound is 3 MHz and the pulserepetition rate is 40 Hz,the best mechanical index for opening the blood-brain barrier in mice is 1.3.When the low-frequency ultrasound frequency is 3MHz,the mechanicalindex is 1,and the pulse repetition frequency is 40Hz,the opening of BBB in mice is reversible when combined with microbubbles irradiation,and the permeability ofBBB is the highest after 1 hour of ultrasound irradiation and returms to normal after 8 hours of ultrasound irradiation. |
PDF Full Text Request