Trichloroethylene induces dopaminergic neurodegeneration in Fisher 344 rats

Abstract

Trichloroethylene, a chlorinated solvent widely used as a degreasing agent, is a common environmental contaminant. Emerging evidence suggests that chronic exposure to trichloroethylene may contribute to the development of Parkinson's disease. The purpose of this study was to determine if selective loss of nigrostriatal dopaminergic neurons could be reproduced by systemic exposure of adult Fisher 344 rats to trichloroethylene. In our experiments, oral administration of trichloroethylene induced a significant loss of dopaminergic neurons in the substantia nigra pars compacta in a dose-dependent manner, whereas the number of both cholinergic and GABAergic neurons were not decreased in the striatum. There was a robust decline in striatal levels of 3, 4-dihydroxyphenylacetic acid without a significant depletion of striatal dopamine. Rats treated with trichloroethylene showed defects in rotarod behavior test. We also found a significantly reduced mitochondrial complex I activity with elevated oxidative stress markers and activated microglia in the nigral area. In addition, we observed intracellular alpha-synuclein accumulation in the dorsal motor nucleus of the vagus nerve, with some in nigral neurons, but little in neurons of cerebral cortex. Overall, our animal model exhibits some important features of Parkinsonism, and further supports that trichloroethylene may be an environmental risk factors for Parkinson's disease.

Fig. 1

TCE-induced dopaminergic neuron degeneration in a dose-dependent manner. (a) Representative TH immunostaining in the substantia nigra of 6-week TCE (200, 500 and 1000 mg/kg) and vehicle-treated rats. Scale bar = 500 μm. (b) Unbiased stereological cell counting showed a significant reduction in the total number of TH-positive neurons in the substantia nigra of 500 and 1000 mg/kg TCE-treated rats as compared with vehicle treatment. *p < 0.05, **p < 0.01 vs. vehicle, n = 6 per group. (c) Silver staining revealed degenerating neurons with metallic silver deposits (arrow) and the surrounding of glia-like cells (arrowheads) in the substantia nigra after 6-week 1000 mg/kg TCE treatment. Scale bar = 50 μm.

Fig. 2

There was no obvious damage for non-dopaminergic neurons in central nervous system after 6-week TCE (1000 mg/kg) treatment. (a) ChAT and DARPP-32 immunostaining were respectively visualized for cholinergic and GABAergic neurons in the striatum; and no significant cell loss in TCE compared with vehicle-treated rats. Scale bar = 500 μm. (b) Giemsa staining showed intact Purkinje cell monolayer (arrow) in the cerebellum of both TCE and vehicle-treated group. Scale bar = 1 mm. (c) Luxol fast blue staining revealed no myelin disruption in the brain of both TCE and vehicle-treated animals. Scale bar = 1 mm.

Fig. 3

TCE (1000 mg/kg) treatment affected dopamine’s metabolites in the striatum and led to movement dysfunction. (a) The levels of DOPAC and HVA were significantly decreased in TCE compared with vehicle-treated rats. (b) Rotarod testing analysis showed the duration on the rotating drum was significantly decreased on the 5th and 6th week of TCE treatment when compared with vehicle-treated animal. *p < 0.05, **p < 0.01 vs. vehicle, n = 8 per group.

Fig. 4

Mitochondrial dysfunction, oxidative stress and inflammation were found in the substantia nigra following 1000 mg/kg TCE treatment. (a) Mitochondrial complex I enzyme activity as measured by NADH substrate oxidation was significantly reduced in the TCE-treated rats as compared with vehicle treatment. (b) Cleaved caspase 3 immunostaining showed more positive signals (arrows) in TCE-treated rats (scale bar = 100 μm). (c) The level of protein carbonyls was significantly increased at 2 weeks, 4-HNE was increased at 6 weeks and 3-NT was increased at both time-points after TCE treatment in comparison with vehicle treatment (left panel). 3-NT immunostaining and counter-staining with hematoxylin indicated that tyrosine nitration was mostly localized in the neuron-like cells (arrows) in TCE-treated rats (right panel, scale bar = 100 μm). (d) Representative OX-42 staining revealed activated microglia with large cell bodies and stout processes at 2 weeks after TCE (the inset depicts a high-magnification image, scale bar = 200 μm). *p < 0.05, **p < 0.01 vs. vehicle, n = 9 per group.

Fig. 5

Intracellular α-synuclein accumulation was observed in brainstem and substantia nigra pars compacta at 6 weeks after 1000 mg/kg TCE treatment. Immunohistochemistry for α-synuclein and counter-staining with hematoxylin showed marked intracellular α-synuclein protein accumulation in the dorsal motor nucleus of the vagus nerve (a, outlined area, scale bar = 200 μm), some in nigral cells (b, arrows, scale bar = 10 μm) and little in neurons of cerebral cortex (c, scale bar = 200 μm) of TCE-treated animals in comparison of vehicle treatment.