The Effect Of Rotenone On The Dopaminergic Neurons In SNc Of Rats And Its Effect Combined With Iron

Rotenone was widely used as a kind of safety and reliable pesticide. However, it was recently found that systemic exposure of rotenone could cause a syndrome like Parkinson's disease(PD) including pathological and behavior changes. Can direct exposure of rotenone into substantia nigra compact (SNc) cause PD-like motor disorder and cellular toxic effect? And what kind of physiological and biochemical changes happened in dopaminergic (DA-ergic) neurons in SNc after the toxicity of rotenone, which sequently induced progressive impairment of DA-ergic neurons? These were important to illuminate neural mechanism of rotenone-induced PD. However, it is still lack of systemic experimental evidence, especially intra-cellular analytic evidence of in vivo exposure. Otherwise, related clinical analysis indicated that iron level was significantly increased in SNc of PD patients, which hinted that iron element is closely correlative with PD. However, it still lacks of the evidence that whether the higher lever of iron in SNc has synergistic effect of accelerating the impairment of DA-ergic neurons with the neurotoxin—rotenone. In present study, rotenone and ferri ion were directly microinjected into SNc of rats. Using methods of behaviour, immunohistochemistry, fluorescent labeling and biochemistry, mechanisms of impairment of DA-ergic neurons in SNc induced by rotenone and its effect combined with iron was investigated.The principal results are as follows:1. When compared with both the control group and the same group before exposure, latencies of rats' moving on the vertical grids were significantly increased after the exposure of rotenone and its combined exposure with iron. The number of rats' sliding down the inclined plane was also significantly increased after exposure. Compared with the group exposed with the same concentration of rotenone alone, the number of rats' sliding down was increased in the group of combined exposure with rotenone and iron. Compared with both the control group and the same group before exposure, the number of rats' dipping into the holes and time of rearing were significantly decreased, and the time of rats' inactive sitting was significantly increased after exposure of rotenone and its exposure combined with iron in the open-field test. Furthermore, some results had dose-effect relationship in the open-field test.2. The density of Tyrosine Hydroxylase(TH) immunopositive neurons in SNc reduced significantly after the exposure of rotenone. The surviving neurons presented the progressively degenerating characteristics such as shrinking of cell body and disappearing of neurite. Compared with both the control group and the group exposed alone with the same concentration of rotenone, the density of TH-positive neurons in the co-exposure group decreased significantly. Using Hoechst33342 fluorescent staining, apoptosis neurons were specifically labeled in SNc. The result showed that the number of apoptosis neurons in SN was significantly increased after the exposure of rotenone and its exposure combined with iron. Furthermore, compared with the group exposed alone with rotenone, the number of apoptosis neurons in the co-exposure group was larger.3.Mitochondrial membrane potential (ΔΨm) of SNc neurons in rats decreased when compared with the control group. It reflected that rotenone exposure initiated dysfunction of mitochondria in these neurons of SN. After exposure of rotenone, the activity of lactate dehydrogenase (LDH) increased, which indicated that the toxic effect of rotenone caused deficient of energy supply in these cells. Moreover, the significant increase of reactive oxygen species (ROS) lever had also been detected after exposure. Whereas, the activity of Glutathione peroxidase (GSH-PX), a kind of free radical cleaning enzyme, was significantly reduced after exposure. When compared with the group exposed alone with rotenone, the lever of ROS increased significantly in the co-exposure group, while the GSH-PX activity decreased significantly.The results above indicated that:1. Exposure of rotenone directly into SNc in vivo could induce typical PD-like symptoms in rats such as resting tremor, rigidity and bradykinesia. Exposure of proper iron could enhance rotenone-induced behavior disturbance.2. Exposure of rotenone and its co-exposure with iron could cause pathological characteristics of progressively degeneration, as well as obvious absence of DA-ergic neurons in SNc.The neurotoxicity of rotenone mainly causes the apoptosis of DA-ergic neurons in SNc, and the co-exposure with iron could intensify the loss of neurons and its apoptosis.3. The functional disturbance of mitochondria induced byΔΨm drop could be the initial factor of rotenone-induced impairment of DA-ergic neurons in vivo. The accumulation of ROS and energy metabolism disturbance may be the main factors which induced the apoptosis of DA-ergic neurons of SNc. 4. Iron has synergistic neurotoxic effect with rotenone, mainly via the intracellular oxidative and anti-oxidative system of DA-ergic neurons.In current study, it was observed in vivo cytobiology effect of direct exposure of rotenone into SNc neurons in rats. It was investigated the neurotoxicologic mechanism of rotenone induced PD in the aspects of system, cellular and intra-cellular physiology. The results indicated that the drop ofΔΨm of cells induced by rotenone was the important factor initiating the impairment of SNc neurons . The accumulation of ROS and energy metabolism disturbance in SNc neurons may be the two main causes induced the apoptosis of these neurons. It was firstly observed that iron has synergistic effect with neurotoxicity of rotenone mainly via intracellular oxidative and anti-oxidative system of SNc neurons. These results provided important evidences to further investigation of the mechanisms of sporadic PD induced by environmental factors. With the improvement of living conditions, the problem of excessive iron intake increasingly appears. Considering the importance of iron-mediated oxidative stress involved in PD, it is urgent for us to strengthen the research on the safety dose of iron enriched food and tonic food, as well as promulgate health-regulating measures related.