Feasibility Studies On Non-Invasive Evaluation Of Parkinson’ Disease Using Dopamine Receptor Imaging Method

Part I Synthesis of 18 F-fallypride and establishment of quality controlObjective TLC and HPLC methods were established for the quality control of 18 F-fallypride and its precursor and purities were also determined.Methods HPLC was performed using a Sep-Pak C18 column (5μm,4.6 x 250 mm, Waters, USA) with mobile phase (acetonitirle:water= 60:40) containing 0.1% triethanolamine; Detection wavelength was 295 nm and low rate was 0.8 mL/min with injection volumn of 10 μL.-fallypride was determined by Mini-scan TLC with a developing solvent (dichloromethane:methanol= 9:1).Results The retention time of 18 F-fallypride precousor was 22.66 min with more than 98% of purity while the Rfof 18F-fallypride was 0.5 with more than 96% of purity.Conclusions The synthesis method of 18 F-fallypride is simple with high purity, which meets the need for clinical use.Part II Biodistrubition of 18 F-fallypride in miceObjective To investigate the biodistribution of 18 F-fallypride in mice and its specific binding ability with DA receptor in brain.Methods The mice were sacrified at 5,15,30,60,90,120 min after i.v. administration of18 F-fallypride. Then, tissues including heart, liver, spleen, lung, kidney, intestines, bone, and brain of mice were removed and weighed. The brain was divided into cerebellum, striatum, thalamus, hippocampus, frontal lobe, cortex, and complement parts. Radioactive counts were caculated by y counter as% ID/g. Meantime, Micro-PET combined with 2D ROI was performed to calculate the radioactive counts of 18 F-fallypride in striatum and cerebellum. Moreover, the specific binding ability of 18 F-fallypride was evaluated by calculate the ratio of radioactive counts in striatum and cerebellum.Results 18F-fallypride was immediately distributed over most of organs after i.v. administration of 18 F-fallypride in mice and high rate of clearance of 18 F-fallypride was observed in liver, spleen, and kidney. In addition,18 F-fallypride was intensively distributed in striatum other than cerebellum, which suggests the high specificity binding DA. Furthermore,19F-fallypride was chosen as the competitive inhibition in order to investigate the specificity binding of 18 F-fallypride to DA. Conclusions 18 F-fallypride is a potential DA receptor imaging agent used for the visualization and evaluation of PD animal model.Part Ⅲ Evaluation of PD mice with traditional methods and non-invasive Micro-PET methodObjective PD mice model was established and evaluated wih both traditional methods and non-invasive Micro-PET method.Methods Intraperitoneal injection of MPTP (25 mg/kg) was performed to establish PD model in mice, which was evaluated by both traditional behavioral tests and Micro-PET method. Traditional behavioral tests consist of normal behavior, swim test, independent performance, immunohistochemistry, and western blotting while calculating uptake values of 18 F-fallypride by selecting regions of interest (ROI) of coronal plane after Micro-PET scan.Rsults The normal behavior of model group mice was similar to symptoms of PD patients, besides, swim time, ambulation, and rearing results were significantly less than those of control groups (P< 0.001) after Intraperitoneal injection of MPTP (25 mg/kg). Immunohistochemistry results showed a significant decreas of positive TH neurons numbers, TH protein expression, DAT numbers, and DAT protein expression in MPTP model mice compared with control groups. The MDA contents of striatum of PD model mice were significantly increased compared to those of blank groups while the GSH-PX and SOD contents were decreased. The uptake values of 18 F-fallypride of model group mice were significantly lower than those of control groups.Conclusions PD mice model was successfully established according to the results observed from both traditional and Micro-PET method. However, the change of DA receptor could be observed more directly using Micro-PET compared with raditional.Part IV Intervention effect of L-dopa on PD model miceObjective To investigate the intervention effect of L-dopa on PD model mice.Methods Intraperitoneal injection of MPTP (25 mg/kg) was performed to establish PD model in mice, which was evaluated by both traditional behavioral tests and Micro-PET method.Results No significant differences of swim time, ambulation, and rearing results were observed between the L-dopa group mice and control group mice after intraperitoneal injection of MPTP (25 mg/kg). The results of TEM showed that the intervention of L-dopa could improve the morphology change of nucleus and cytoplasm of substantia nigra induced by MPTP injection. In addition, the intervention of L-dopa significantly increasd the TH positive numbers and decrease the metabolic rate of DA. Moreover, the uptake values of 18 F-fallypride in striatum of L-dopa group mice were significantly increased compared with those of model groups.Conclusions The intervention of L-dopa could due to the improvement of injury of substantia nigra induced by MPTP injection, the increase of TH positive cell numbers and the DA receptors contents, and the decreas of metabolic rate of DA, all of which, to a certain extent, would alleviate the symptoms of PD.