Effect And Mechanism Of Low Intensity Focused Ultrasound On The Depressive-like Behavior Induced By Lipopolysaccharide In Mice

Objective:Depression is a common and serious mental illness worldwide and it is the main cause of global disability.Despite the use of various treatments,including common antidepressants and psychotherapy,there are still 40%-50%of patients whose symptoms cannot be relieved.Therefore,the development of new strategies is essential for patients with depression.Low intensity focused ultrasound(LIFUS),as a non-invasive physical therapy method,has been proven to inhibit or activate neurons,regulate brain activity,and show positive effects in various neurological diseases.This also provides a new perspective for the treatment of depression.In this study,firstly,the LIFUS stimulation system was established and the safety of the ultrasonic stimulation system was evaluated.And then depressive-like behavior mice model was induced by lipopolysaccharide(LPS)via intraperitioneal injection,and LIFUS was applied to the prefrontal cortex(PFC)of model mice to study the intervention effect of LIFUS stimulation on depression-like behavior,which will provide a theoretical basis for promoting the clinical transformation of depression with ultrasound treatment.Methods:1.Building a LIFUS stimulation system with signal generators,power amplifiers and ultrasonic transducers.2.The experimental animals used in this study are C57BL/6(C57)male mice raised in the SPF environment,aged 8 weeks and weighed 22-25 g.Experimental animals were randomly grouped according to different experimental purpose.3.Research of target location and safety of LIFUS stimulation:normal C57 mice were randomly divided into sham stimulation group(Sham)and ultrasound stimulation group(LIFUS).After LIFUS stimulation on the PFC of mice,the C-fos protein was marked with immunofluorescence to locate the target of LIFUS stimulation,and the safety of LIFUS stimulation was evaluated by Hematoxylin-eosin staining(HE).4.Establishment of the mice model with depression-like behavior:C57 mice were randomly divided into saline group(Nacl),LPS concentration with 0.5mg/kg group(0.5 mg/kg),LPS concentration with 0.83 mg/kg group(0.83 mg/kg),LPS concentration with 1 mg/kg group(1 mg/kg),intraperitoneal injection of LPS or 0.9%sodium chloride injection.The open field test(OFT)was performed after 24 hours of establishment of animal model to evaluate the depression-like behavior of mice.5.Intervention effect and mechanism of LIFUS on depression-like behavior in mice induced by LPS:C57 mice were divided into saline group(Nacl+sham),ultrasound sham stimulation group(LPS+sham)and ultrasound stimulation group(LPS+LIFUS)by random number method.LPS(1 mg/kg)or the same amount of 0.9%sodium chloride injection was injected intraperitoneally according to the body weight of experimental mice.The motion ability of mice was evaluated with Open filed test(OFT)6 hours later,and the mice that failed to be modeled were excluded,and then the LIFUS was applied in the(PFC)of mice with succeeded model.After 24 hours,the depression-like behavior of mice was assessed by Tail suspension test(TST)and Forced swimming test(FTS),and the anxiety-like behavior was evaluated with Elevated plus maze(EPM).In addition,Western-blot(WB)technology was used to detect the expression of tumor necrosis factor alpha(TNF-α),interleukin-6(IL-6)and interleukin-1bate(IL-1β)on the PFC of mice.Results:1.Research of target location and safety of LIFUS stimulation:compared with the Sham group,LIFUS stimulation significantly activated the C-fos protein of PFC of mice[Sham:(73.50±9.4)vs.LIFUS:(204.00±25.57),P<0.01],and the HE staining showed that the PFC stimulated by LIFUS had no obvious tissue damage and bleeding.2.Establishment of the mice model with depression-like behavior:the number entering into the central zone in the OFT of mice in the Nacl,0.5mg/kg,0.83mg/kg and lmg/kg 4 groups were(32.00±4.29),(15.89±3.49),(9.89±1.46),(6.67±1.05)respectively.Compared with mice in the Nacl group,the number of entering into central zone in the OFT of mice in the 0.83 mg/kg and 1 mg/kg group was significantly reduced(P<0.0001),but the number of entering into central zone of mice in the 1 mg/kg group was the least.3.Intervention effect of LIFUS on depression-like behavior in mice induced by LPS:intraperitoneal injection of LPS caused obvious depression-like behavior in mice,which was manifested as the immobility time of FST in the LPS+sham group[(152.40±9.31)s]was significantly higher than the Nacl+sham group[(103.20±10.65)s,P<0.01],and the immobility time of TST in the LPS+sham group[(165.80±7.24)s]was significantly higher than the Nacl+sham group[(124.40±5.81)s,P<0.001].However,the stimulation of LIFUS applied on the PFC significantly improved the depression-like behavior of mice in the LPS+LIFUS group[FST:(111.00±11.70)s,P<0.05;TST:(141.50±6.39)s,P<0.05].LIFUS stimulation also significantly reduced the up-regulation of inflammatory factors in the PFC area caused by LPS,which manifested that compared with the LPS+sham group,the expression of TNF-α、IL-6 and IL-1β three inflammatory factors in LPS+LIFUS group was significantly decreased.Conclusion:1.The LIFUS stimulation system and its parameters used in this study can safely stimulate the PFC of mice.2.In this study,the concentration of LPS induced depression-like behavior mouse model induced was 1mg/kg,which satisfied the needs of experiments.3.The LIFUS stimulation system and ultrasonic parameters used in this study can significantly improve the depression-like behavior of mice induced by LPS,and the mechanism of its anti-depressant effect may be related to the reduction of inflammatory factors of PFC of mice.