| Parkinson's disease (PD), a profound movement disorder resulting from nigrostriatal dopaminergic (DA) system degeneration, has been linked to living in a rural environment, farming, and occupational exposure to agricultural chemicals, suggesting an environmental exposure basis for the disease. Exposure to paraquat (PQ) as a risk factor for sporadic PD has been associated with Parkinsonism. Other agricultural chemicals, including dithiocarbamate fungicides such as maneb (MB), are widely used in the same geographical regions and also impact dopamine systems, suggesting that mixtures may be more relevant to sporadic PD.The chemical constitution of PQ is extremely similar to MPP+, the active metabolite of MPTP which is known as a neurotoxin and their pathogenesis mechanism is also similar. Additionally, it was shown that MB is able to alter the biodisposition of DA and PQ, resulting in a prolonged exposure to reactive oxygen species (ROS) and reactive nitrogen species generating compounds. MB is capable of converting a non-toxic dose of PQ and other xenobiotics into a toxic dose through alterations in toxicokinetics.Epidemiological studies demonstrate long-term co-exposure to PQ and MB obviously increases the risk of PD onset. Experiments indicate that PQ selectively kills dopaminergic neurons in the substantia nigra and produces some of the syndrom of PD in laboratory animals, while, it is clear that MB enhances the PQ toxicity.Dietary restriction has been shown to have several health benefits including increased insulin sensitivity, stress resistance, reduced morbidity in some disease, and increased life span, while the mechanism remains unclear. Studies reveal it plays significantly therapeutical effects on Huntington disease (HD). Whether DR has the protective effects on the damage of dopaminergic neurons induced by systemic exposure to combination of PQ and MB? There is few related report yet. This study plans to use the combination of PQ and MB to set up the PD model mimicing the environmental toxin related PD, to demonstrate whether DR has the protective effects on nerve injury induced by PQ and MB, and to reveal the possible mechanism of its protective effects. Experimental contents as follows:Experiment 1Objective: To investigate the insult of PQ and MB on DA neurons in SNpc and the expression of glial fibrillary acidic protein (GFAP) in substantia nigra in C57BL/6J mice. Methods: Animals were divided into saline and PQMB group randomly. All were injected with either Vehicle (0.9%) or a combination of PQ (8 mg/kg) and MB (24 mg/kg) twice a week (Tuesday and Thursday, 9:00 am) for 6 weeks intraperitoneally. Before the first injection and after the last injection, the mice were perfused and the brains were removed and cut with the common practice. Expression of tyrosine hydroxylase (TH) and GFAP in substantial nigra were investigated utilizing the immunohistochemical methods. Results: The survival number of TH positive cells in SNpc in PQMB group was 116.50±7.44, which was less than that of the saline group (178.83±5.68) (P < 0.001). And the DA neurons did not appear as healthy as those in saline-treated animals, with fewer dendrites and less intense TH immunoreactivity within cells. The reactive level of Asts decreased in PQMB group and the△GV of GFAP was 45.98±1.46, which is lower than that of saline group (P < 0.001). Conclusion: The results suggested that combination of PQ and MB can induce loss of DA neurons in SNpc in mice to some extent, which is the characteristic change of pathology in PD patient. PQ and MB can also insult Ast in area of substantia nigra.Experiment 2Objective: To explore the persistent neurotoxic effects of combinded use of PQ and MB on mice, and to observe the protective function of DR to this damage. Methods: The PQ and MB combination injected mice were divided into two groups randomly; one group had access to food ad libitum (PQMB/AL), while another fed on alternate days only (PQMB/DR). Animals were tested on three different time-points, the follow0, 3, 5, 8 weeks, the mice were perfused and the brains were removed and cut with the common practice. Expression of TH in SNpc was investigated utilizing the immunohistochemical methods. Expression of GFAP was localized on frozen sections by immunohistochemical methods and BX51 microscopy, and GFAP content (△Gray Value,△GV) was evaluated by Leica Q570c True colour image analysis system. Results: immunohistochemical results showed that the number of TH positive neurons in SNpc reduced continuously and irreversably at the end of 8 weeks, the survival TH positive cells in SNpc in PQMB/AL group decreased sharply to 82.64±5.10 and was lower than that of 0 week in the samel group (P < 0.05). While, the dietary restriction seemed to be able to slow down this process, the statistical significance for the number of the survival neurons could be found between the PQMB/DR and PQMB/AL group from the 5th week (P < 0.05). The glial reactivity was different among the PQMB groups and Saline groups. The expression level of GFAP in PQMB/Al group reduced first, upregulated to the same level to the control group in 3 weeks, then△GV reached 69.02±1.25 and 63.3±1.67 in 5 and 8 weeks, which was much higher than that of control group (51.66±0.92 and 53.04±1.12), respectively (P < 0.01). While, DR reduced the expression level significantly and the△GV was 48.92±1.28 and 47.5±2.68 in 5 and 8 weeks respectively, which was lower than it of the PQMB/AL group simultaneously (P < 0.01). Conclusion: the nigrostriatal dopaminergic neurotoxicity of the PQ and MB combination was progressive and irreversible. DR can limit the TH neurons necrosis or apoptosis and relieve the reaction of Asts induced by PQ and MB in mice brain, which may be one of the conceivable mechanisms for the neuroprotection of DR.Experiment 3Objective: To explore the effects of DR on behavioral alterations in mice induced by combination of PQ and MB. Methods: Mice were divided into saline and PQMB group randomly. All were injected with either saline (0.9%) or a combination of PQ (8 mg/kg) and MB (24 mg/kg) twice a week (Tuesday and Thursday, 9:00 am) for 6 weeks intraperitoneally. On the last injection, the PQMB treated mice were divided into two groups randomly; one group had access to food ad libitum (PQMB/AL), while another fed on alternate days only (PQMB/DR). The saline treated mice were divided into two groups randomly too, Saline/AL and Saline/DR. The pole test and the open-field test were used to analyze the behavioral abnormalities. Animals were tested on five different time-points, time just before the first injection (-6 weeks), time after the last-injection (0 week) and the following 3, 5, 8 weeks. Results: Injection of PQMB caused behavioral syndromes including restlessness, straub tail, hindlimb abduction, tremor and instability of gait in C57BL/6J mice. Symptoms of Bradykinesia, akinesia and instability of gait appeared after several weeks of PQMB administration. In locomotor activity test, the horizontal movement distance in PQMB/AL group was 935.01±146.27 cm in 0 week, which was lower than that of saline group (1903.38±203.14 cm) (P < 0.01). In the pole test, Tturn (defined as the time taken until mice turned completely downward) and TLA (defined as the time until mice had climbed down to the floor) were 2.59±0.21 s and 13.85±0.69 s respectively in 0 week after the last injection of PQMB, which showed significantly prolongation compared with the saline group (0.90±0.12 s; 6.34±0.37 s) (P < 0.01). And motor deficits were persistent in PQMB group over time, while the horizontal movement distance in locomotor activity test increased (P < 0.05) and TLA in the pole test decreased after 8 weeks'dietary restriction (P < 0.01) and Tturn decreased significantly in PQMB/DR group from the 5th week after the last injection (P < 0.01). Conclusion: PQMB can induce behavioral alteration in C57BL/6J mice and motor deficits were persistent over time. Mid or long term of Dietary restriction may have protection effects on those motor deficits.
|
PDF Full Text Request