The Correlation Between Dopamine Transporter And Melatonin In The Different Damaged Midbrain Dopaminergic Neurons Of Rats

Objective:1. To observe dynamic changes in striatal DAT in progression of PD, and to investigate the relationships between DAT and TH.2. To investigate the density and function of DAT in different damaged midbrain dopaminergic neurons in the striatum of rats.3. To investigate the serum MLT levels in different damaged midbrain dopaminergic neurons of rats.4. To study the correlation between the number of TH positive cells in the substantia nigra and the radioactive count of DAT in the striatum of the different damaged midbrain dopaminergic neurons of rats.5. To study the correlation between the number of TH positive cells in the substantia nigra and the serum MLT levels in the different damaged midbrain dopaminergic neurons of rats.6. To investigate the correlation between DAT and MLT in the different damaged midbrain dopaminergic neurons of rats.Methods:1. A hemiparkinsonium rat model was established by a unilateral injection of6-OHDA into the right substantia SNc and the right MFB. The different damaged midbrain dopaminergic neurons of rats model were established by a unilateral injection of 6-OHDA into the right MFB through injecting different dose.2. The rotational behavior was induced by APO after 1, 2, 4, 6 and 8 weeks postlesion. The successful parkinson’s disease model was considered that the number of rotations was more than 210r/30 min.3. The rats were injected 10% chloral hydrate overdose at 9-10 am and room temperature between 20°C-25°C. After depth of anesthesia, fastened the rats’ limbs,opened the chest quickly, peeled the connective tissue around the heart, exposed the heart fully, cut the right atrial appendage, taked blood with centrifuge tube quickly.After centrifugation, the serum was preserved at-20°C. The same batch detected.4. Rats were injected 99 m Tc-TRODAT-1 by tail vein at 2, 4 and 6 weeks post-lesion respectively. Both the left and right striata were removed after 1 h, the densities of DAT were measured usingγradioactive counting meter, then the radioactive counts rate per unit mass and radioactivity of the striatum count ratio were calculated.5. After anesthesia by isoflurane, the rats were injected 99 m Tc-TRODAT-1 in the tail vein to label dopamine transporter(DAT). The changes of density and function of DAT in the striatum were detected by the small animal in vivo imaging system.6. ELISA was used to detect the serum MLT levels, to investigate the relationship between the MLT and the rotation number and DAT in the different damaged midbrain dopaminergic neurons of rats.7. The DA neurons were assayed by imunohistochemical staining to study the relationship among the DAT radioactive count and the MLT levels.Results:1. The radioactive counts in the lesioned side of SN in PD rats was significantly lower than that of unlesioned side, and it was decreased by 16.8%、35.9% and 50.1%respectively when compared with the unlesioned side at 2, 4, and 6 weeks(p<0.05~0.001, n=5). There was a significant positive correlation between radioactive counts and TH immunoreactive positive cells in PD rats at 2, 4, and 6 weeks(p<0.01, r=0.9,n=15)2. The 2mg 6-OHDA group exhibited no marked rotational response to APO,7while the 4mg and 8mg 6-OHDA groups responded with significantly increased number of rotations towards the control and vehicle-treated rats( p<0.001).3. The serum MLT levels of the different damaged midbrain dopamine neurons of rats were increased as compared to control and vehicle at 8 weeks post lesion( p<0.001). The MLT levels of control were(44.67±1.10) pg/ml, vehicle(45.21±1.19)pg/ml, 2mg(49.44±1.54) pg/ml, 4mg(58.35±1.73) pg/ml, 8mg(66.27±1.94) pg/ml.4. The TH positive neurons in lesion side of substantia nigra in 2mg, 4mg and8mg 6-OHDA groups was significantly lower than that in the unlesion side, and the number of TH immunoreactive positive cells gradually decreased( p <0.001). The TH immunoreactive positive cells in the lesion side of 2mg, 4mg and 8mg 6-OHDA groups were decreased 43.62%±15.6%, 59.9%±8.74%, 95.17%±4.64% compared with the unlesion side at 8 weeks post lesion. The TH positive neurons in unlesion side of substantia nigra in 8mg 6-OHDA group were significantly decreased as compared to control and vehicle at 8 weeks post lesion( p<0.05). There were no significant difference in the lesion side and the unlesion side of the control and the vehicle groups.5. The DAT radioactivity in lesion side of striatal in 2mg, 4mg and 8mg 6-OHDA groups were significantly decreased as compared to control at 8 weeks post lesion( p<0.05). The DAT radioactivity in unlesion side of striatal in 2mg, 4mg and 8mg6-OHDA groups were significantly decreased as compared to the control and vehicle groups at 8 weeks post lesion( p<0.05). The DAT radioactivity in lesion side of control was 1569.23±295.38 bq/mm3, 2mg group 1258.97±181.96 bq/mm3, 4mg group1033.45±116.66 bq/mm3, 8mg group 994.15±248.86 bq/mm3. The DAT radioactivity in unlesion of control was 1498.57±275.73 bq/mm3, 2mg group 1174.73±65.94 bq/mm3,4mg group 943.32±47.63 bq/mm3, 8mg group 679.84±131.79 bq/mm3.6. There was a significant negative correlation between the serum MLT level and the lesion side of striatal DAT radioactivity of the different damaged midbrain dopamine neurons of rats at 8 weeks post lesion, |r|=0.873, p<0.01(n=16). There was a significant negative correlation between the serum MLT level and the lesion side of TH positive neurons, |r|=0.94, p<0.01(n=16). There was a significant positive correlation between DAT radioactivity and MLT level, |r|=0.863, p<0.01(n=16).Conclusion:1. The density of DAT decreased gradually by the PD progression, and99mTc-PRODAT-1 distribution of radioactivity may reflect the changes in presynaptic dopaminergic neurons.2.The DAT levels of the differen damaged midbrain dopaminergic neurons of rats gradually decreased by the increasing of damaged degree, which may be associated with the progression of PD3.The MLT levels of the different damaged midbrain dopaminergic neurons of rats gradually increased by the increasing of damaged degree, which may be associated with the progression of PD.4.The MLT levels of serum and the DAT level of brain may become a potential indicator for the severity of clinical judgment of PD.