Experimental Study Of Ultrasonic Stimulation Of Dopamine Release From Parkinson’s Disease Model

Parkinson’s disease(PD)is a common neurodegenerative disorder characterized by the reduce of the striatal DA level,which is related to the degeneration,death and deletion of dopaminergic neurons in the substantia nigra pars compacta(SNc),as well as the decrease of dopamine biosynthesis ability of the surviving neurons.A series of clinical symptoms including resting tremor,rigidity,bradykinesia and postural instability will appear when DA content is lower than 70%.The gold standard for the treatment of PD is Levodopa therapy,which is aimed at increasing the availability of striatal dopamine and typically result in a brisk symptomatic improvement in most PD patients.Unfortunately,this response becomes less reliable and less predictable over time,medication related complications such as dyskinesia and "on-off" fluctuations develop in a majority of patients.Because of the side effect of Levodopa therapy,researchers started to explore new therapies for PD.In this research,we established a dopaminergic cell model and MPTP mouse model of PD,then ultrasound was used to stimulate the two models to explore the effects of ultrasound on dopamine release.First of all,we detected the DA release from dopaminergic cell model after ultrasonic stimulation to investigate the effects of ultrasound at the cellular level.Secondly,we detected the content of dopamine in the striatum of the animal models after ultrasonic stimulation to investigate the effects of ultrasound at animal level.The experimental study of the effect of ultrasonic stimulation in vivo and in vitro will provide the data base for further research for PD therapy.Chapter 1 Establish the Dopaminergic Cell Model and PD Animal ModelPC 12 cells were induced by 50 ng/mL NGF to proliferate and differentiate for several days,during the process,the cells will examined by counting the number of neurite-positive cells,which was defined as cells exhibiting one or more neurites with lengths equal to or more than twice the diameter of the cell body.When the cell model induced to 72h,the length of neurites increased and woven into a net,which showed typical morphology of neurons.At this time,the length of the neurites reaches 2.669 times of the diameter of the cell body,which is consistent with the standard of dopaminergic cell models induced by the literature.The animal model of PD was performed on male adult mice(C57/BL6).The mice received i.p.injection of 30mg/kg/d MPTP and continued for 5 days,then evaluated by means of mouse behavioral detection,measurement of dopamine content in striatum,immunohistochemistry of TH positive neurons in SNc.HE staining in the SNc and striatum.After treated the mice with MPTP for 5 days,the animals showed a series of symptoms which was similar to PD,such as tremor,rigidity,bradykinesia.There were significant differences between the PD model group and control group in the pole climbing test,the open field test,the DA content in the striatum and the TH positive neurons in SNc.Within the PD model group,there were some vacuoles in SNc which were related to the degeneration,death and deletion of dopaminergic neurons.Moreover,in the section of striatum appeared neurons and fibers degeneration and necrosis.These results indicated that the clinical symptoms,pathological changes and neurobiological changes of PD were well simulated by the C57/BL6 mice treated with MPTP,which accorded with the standard of PD model.Chapter 2 Experimental Study of Ultrasonic Stimulation of Dopamine Release from Dopaminergic Cell ModelIn this chapter,the dopaminergic cell model was constructed according to the method described in the Section 1 of Chapter 1,then using the ultrasound with different parameters(f=1MHz,P=0.1W/cm2～0.3W/cm2,t=10s～60s,T=0h～24h)stimulate the cell model.DA concentration was detected by ELISA.Moreover,MTT proliferation test and apoptosis assay were used to detect the effects of ultrasonic stimulation on dopamine release from the cell model.In the experiments we observed that,with the increase of ultrasonic power,the prolong of ultrasonic stimulation time and the delay of observation time,DA concentration was increased firstly and then decreased,the amount of DA content eventually reached the top value in the treatment of f=1MHz,P=0.2W/cm2,t=40s,T=12h.Under such condition,there was no significant difference compare to the control group in MTT proliferation test and apoptosis assay.Analysis of experimental datas could be drawn,the optimal effect of dopamine release on the basis of the survival rate of cells after treatment with certain ultrasound(f=1MHz,P=0.2W/cm2,t=40s,T=12h).Chapter 3 Experimental Study of Ultrasonic Stimulation of Dopamine Release from MPTP Model of PDIn this chapter,the MPTP model of PD were established in the same way as described in Section 2 of Chapter 1,then using the ultrasound with a frequency of 1 MHz at an intensity of 0.3W/cm2 stimulate the head of mouse which was shaved the hair and coated with ultrasound coupling gel.The differences between the ultrasonic stimulation group,the PD observation group and the control group were evaluated by the animal behavior tests,biochemical index detection and pathological section.At the same time,the tissues of the ultrasonic stimulation pathway(head skin,skull,Brain tissue)were dyed by HE to show the effects of ultrasound on the cells.In this experiment we observed that,with the prolonge of ultrasonic stimulation time,the delay of observation time,the amount of DA in the striatum of the ultrasonic treatment group was increasing.Using ultrasound with a frequency of 1 MHz at an intensity of 0.3W/cm2 over 15min for Id,5d or 10d respectively,in each group,there was a significant difference in the content of DA in the striatum compare to PD observation group at the same observation time(P<0.05).Especially,the DA content in the striatum of the mice treated with the ultrasound for 10d was not statistically significant compared with the control group(P>0.05).Combined with immunohistochemical results,under this parameter condition,the number of TH positive neurons in SNc,the results of climbing test and open field test and general behavioral changes,had no significant difference between control group(P>0.05).After ultrasonic stimulation,the head skin of the mouse in every group was normal,without blood and swollen.HE stain results showed the cell morphology of head skin,skull,brain tissue were normal and no obvious pathological changes compare to the control group.In summary,ultrasound can increase the release of DA from the dopaminergic cell model and PD animal model.At the cellular level,ultrasound can stimulate the release of DA under the premise of the normal survival of cells.At the animal level,ultrasonic stimulation can increase the release of DA,restore the number of TH-positive neurons and improve the motor capacity of PD model,simultaneously the tissues in the radiation pathway without abnormal pathological changes.It suggests that ultrasound may be a safe and effective new method of PD treatment.