| Objectives:This topic is to test the expression of Divalent metal transporter1 (DMT1) and Ferroportin 1(FP1) in substantia nigra (SN) in manganese-induced parkinsonism rats model. To study the relation between dopaminergic neuron injury, accumulation of manganese and iron,and the expression of iron transport protein in manganese-induced parkinsonism rats.To probe the effects of DMT1 and FP1 about accumulation of manganese in manganese-induced parkinsonism rats.Method:80 healthy Sprague Dawley rats (180-200g, male), were assigned at random to control group (n=20), Mn exposed group with 5mg/kg group (n=20), Mn exposed group with 15 mg/kg group (n=20), Mn exposed group with 20mg/kg group (n=20).The rats in first group were injected (i.p) with isotonic sodium chloride solution (NaC1),the rats in other three of groups were injected (i.p) with MnCl2 solution (5,15,20mg/kg,respectively) for 6 day for 16 weeks. We’ll perform three behavioral tests at the 16th week.First,It is Single-Pellet Reaching Task to evaluate to the analysis of the behavior performance; Second, It is the beam test to asscess the rat’s balance after poisoning.Three,It is Morris water maze test on learning and memory ability of rats.Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES) was applied to measure the concentration of Mn in substantia nigra. Meanwhile,the tyrosine hydroxylase was tested by immunohistochemical staining, thus determining whether the manganese-induced parkinsonism rats model was successfully operated. Then, the expression of DMT 1 and FP1 in SN were tested by immunohistochemical staining, and the expressions of their mRNA were detected by real-time RT-PCR.Results:1.The overall weight of rats showed a growing trend, But the weight of Mn exposed groups have slowly growing,especially Mn exposed group with 15,20mg/kg groups. Average weight between four groups have obvious difference, the difference is statistically (F=527.387, P<0.01)2. Behavior test:(1) Single-Pellet Reaching Task:The number of food were found to be significantly different (F=4.253, P=0.007) between the control group and Mn exposed group with 5,15,20mg/kg groups. (2) the beam test:Each rat have scored for three times, and take the average score as the results.The score were found to be significantly different (F=209.15, P<0.01) between the control group and Mn exposed group with 5,15,20mg/kg groups. (3)Morris water maze test:The is prolonging, compared with control group, Mn exposed group with 15,20mg/kg had an significantly increasing escape latency, the rise of which accompanied the rise of manganese dosage (P< 0.05). On the sixth day,compared with control group, Mn exposed group with 5,15,20mg/kg had an significantly decreasing time of crossing platform after removing the platform,, the rise of which accompanied the rise of manganese dosage (P<0.05)3. Compared with the control group (0.56 ± 0.20), the concentration of Mn in substantia nigra in Mn exposed group with 5,15,20mg/kg [(1.72 ± 0.33,2.92±0.77,5.65±1.60) ug/g] were significantly increased(P<O.01).4. Compared with the control group(0.109±0.019), The mean OD of TH positive cells in substantia nigra in Mn exposed group with 5,15,20mg/kg (0.054 ± 0.008,0.016 ± 0.004,0.003 ± 0.001) were significantly decreased(P<0.01). Compared with the control group (0.015±0.007), The mean OD of DMT 1 positive cells in substantia nigra in Mn exposed group with 15,20mg/kg (0.062 ± 0.004,0.116±0.064) were significantly increased(P<0.01). Compared with the control group (0.306 ± 0.081), The mean OD of FP1 positive cells in substantia nigra in Mn exposed group with 5, 15,20mg/kg (0.092±0.011,0.048±0.008,0.008±0.002) were significantly decreased(P<0.01).5. Compared with control group (0.001±0.0004), Mn exposed group with 15,20mg/kg had an significantly increasing expression of nigra DMT1 mRNA (0.052±0.0126,0.124±0.0299) (P<0.01); but the expression of FP1 mRNA was significantly reduced with the increase of manganese dosage(P<0.05). the expression of FP1 mRNA in Mn exposed group with 5,15,20mg/kg (0.059± 0.0076,0.033±0.0094,0.002±0.0007) was significantly reduced as compared with that of the control group (0.162±0.0463) (P<0.01)Conclusion:1.After injecting with MnCl2 solution for 16 weeks, this experimental method could successfully serve in the preparation for manganese-induced parkinsonism rats model.2.The TH positive cells in substantia nigra in Mn exposed group with 5,15,20mg/kg were significantly decreased,which is associated with the loss of dopaminergic neuron.3.These observations suggest that the increasing expression of DMT1 and decreasing expression of FP1 may be involved in the process of Mn accumulation in the substantia nigra and the process of dopaminergic neuron loss in manganese-induced parkinsonism rats model. |
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