Neuroprotective Effect Of Low-intensity Pulsed Ultrasound On MPP~+/MPTP-induced Dopaminergic Neuron Injury

ObjectiveParkinson’s disease(PD)is a chronic progressive neurodegenerative disease.Most of the traditional treatments of PD are means to relieve symptoms and improve the quality of life,and there are many application limitations.With the deepening of disease research,many new neuromodulations and treatment methods have been developed continuously.Among them,non-invasive brain stimulation methods have shown good application prospects in the treatment of PD.With the advantages of non-invasiveness and strong penetration,ultrasound,as a new type of neuromodulation,has received extensive attention in the field of neuroscience recently.Low-intensity pulsed ultrasound(LIPUS)is a safe therapy with the low power of ultrasound,which is commonly used in the clinical rehabilitation of fractures.It has been found that LIPUS can modulate and protect the central nervous system disease or injury.Therefore,in this study,PD models induced by1-methyl-4-phenylpyridinium(MPP~+)and 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine(MPTP)were studied to investigate whether LIPUS could inhibit the neurotoxicity of MPP~+/MPTP and play a protective role on dopaminergic neurons.Methods1.A PD cell model was constructed with MPP~+(concentration:0.75mmol/L)inducing the mouse neuroblastoma cell lines N2a injury.2.Cell viability of each group was determined by CCK8 assay.The content of reactive oxygen species(ROS)and the potential of mitochondrial membrane were investigated with the help of two kinds of fluorescent probes,DCFH-DA and JC-1,respectively.Annexin V-FITC/PI double staining method was used to detect the apoptosis of N2a cells.And mRNA expression level of transient receptor potential melastatin 7(TRPM7)was determined by real-time fluorescence quantitative PCR(qPCR)after LIPUS treatment.3.Intraperitoneal injection of MPTP(30 mg/Kg)for 5 consecutive days to establish a subacute PD model of adult male C57BL/6 mice.4.The motor function of mice was evaluated by the rotarod test and pole test,and the changes of dopaminergic neurons in the substantia nigra(SN)region compacta were observed by tyrosine hydroxylase(TH)staining and TUNEL staining.5.Hematoxylin-eosin(HE)staining and Nissl staining were used to evaluate the safety of LIPUS in vivo.Results1.After 24 h,compared with the control group,the viability of N2a cells incubated with MPP~+was significantly decreased,the level of intracellular ROS and cell apoptosis rate were significantly increased(all P<0.01).2.After irradiation with LIPUS,cell viability and mitochondrial membrane potential of MPP~+-induced cells were increased significantly(all P<0.01).ROS production and cell apoptosis rate were decreased significantly(all P<0.01).The result of qPCR showed that the expression of TRPM7 in MPP~+group decreased,and the expression of TRPM7 in MPP~++LIPUS group increased significantly(all P<0.01).LIPUS irradiation alone did not cause any cytotoxicity(all P>0.05).3.The motor ability of mice in MPTP group was decreased obviously,which was manifested in the shortening of staying time on the rotator(latency to fall)and the prolongation of climbing pole time(locomotor activity time,LAT)(all P<0.01).The number of TH positive neurons and apoptosis of cells in the SN region of PD mice decreased significantly(all P<0.01).4.The movement and balance dysfunction in PD mice were improved with LIPUS treatment.TH staining and TUNEL staining showed that LIPUS could inhibit dopaminergic neuron loss and apoptosis in the SN region of mice induced by MPTP(all P<0.01).5.Compared with the control group,no histological and neuronal abnormalities were observed with LIPUS irradiation alone.Conclusions1.The PD models induced by MPP~+/MPTP were successfully established.2.LIPUS attenuates the neurotoxicity of MPP~+/MPTP and protects dopaminergic neurons.3.LIPUS irradiation alone did not cause any damage to cells or tissues in vivo or in vitro.