A Preliminary Mechanism Study On The Recovery Of Motor Function After Manganese Removal In Manganese-exposured Rats

Objective:To study the recovery of motor function,memory ability,the changes of PARK2/Parkinanddopaminergicneuronsaftermanganeseremovalin manganese-exposured rats for five month.Methods:Sixty healthy adult SPF SD rats（6 weeks old,160-180 g）were randomly divided into control group（normal saline）,1mg/kg Mn²⁺group and 5mg/kg Mn²⁺group,twenty each group.In the morning,rats were intraperitoneally injected with manganese solution.The rats were poisoned once every other day.After continuous intraperitoneal injection for 1 month,ten rats in each group were randomly selected for rod and Y maze experiments to detect motor and memory functions relatively.Secondly,seven rats in ten were randomly selected to collect whole blood after anesthesia with pentobarbital sodium.Graphite furnace atomic absorption spectrometer was used to detect the concentration of manganese in whole blood,real-time reverse transcription polymerase chain reaction was used to detect the expression of PARK2 mRNA in whole blood.Then the brain was separated into midbrain and striatum.The manganese concentration in each brain region was measured by graphite furnace atomic absorption spectrometer,and the expression of PARK2 mRNA in each brain region was measured by real-time reverse transcription polymerase chain reaction.Western blot was used to detect the expression of Parkin in each brain region.Thirdly,three rats in ten were randomly selected.After anesthesia with pentobarbital sodium,they were perfused with paraformaldehyde.The number of dopaminergic neurons was detected by immunohistochemistry.The remaining rats in each group were stopped being dyed manganese,and continued to be routinely reared for 5months.Dynamic monitoring of the expression of PARK2 mRNA in blood taken from venous of eyelid by real-time fluorescence quantitative polymerase chain reaction after ether anesthesia every month.Results:Compared with manganese exposured 1 month,the concentration of striatum and blood manganese in the 5 mg/kg manganese group decreased after 5 months of manganese removal(t_striatum=4.54,t_{whole blood}=13.27,P<0.05),but the concentration of manganese in the midbrain and striatum was still higher than that in the control group(F_midbrain=112.60,F_striatum=9.75,P<0.05),rod distance of rats increased（t=2.12,P<0.05）.the number of dopaminergic neurons in brain increased too,the cell dendrites increased significantly,the cell morphology was fuller,but the cytoplasmic staining was poor.After1 month of demineralization,the expression of PARK2 mRNA of blood in manganese-exposured groups increased（P>0.05）.Compared with manganese exposured 1month,the expression of PARK2 mRNA in the striatum of the 5 mg/kg manganese group increased after 5 months of manganese removal（t=-4.22,P<0.05）,and the expression of Parkin in the midbrain and striatum of rats increased too(F_midbrain=0.52、F_striatum=1.66,P>0.05).Conclusion:1.After manganese removal in manganese-exposured rats for five month,the rats’impaired motor function recovered.2.The decrease of PARK2 mRNA/Parkin level may be one of the mechanisms of manganese-induced central nervous system poisoning.