Age-related Changes Of Autophagy And Rotenone-induced Dopaminergic Neurotoxicity In SD Rats

Part I Age-associated changes of dopaminergic neuron and oxidative stress of SD rats and responses to Rotenone neurotoxinObjective To investigate the age-associated changes of dopaminergic neuron and oxidative stress of SD rats and responses to Rotenone neurotoxin.Methods Rotenone neurotoxin SD rat model was constructed in different aged (3, 12, 20 months old, n=20 in each group) rats. The rotenone (1.0 mg·kg-1·d-1, at 24h intervals) was injected subcutaneously in back. Then, the weight, tyrosine hydroxylase- immunoreactivity in substantia nigra pars compacta (SNc) were all observed. Oxidative stress was assayed with biochemical method.Results 1. Behavior of Rotenone neurotoxin SD rats was abnormal, showing some symptoms such as PD. The weight of normal aging groups increased while that of Rotenone neurotoxin groups decreased firstly and then retained a certain level but still less than the former (P<0.01). 2. In normal aging groups, 12 months old rats had the highest number of SNc TH positive cells. Compare with 12 months old group, the number of 20 months old rats was significant lower (P<0.01), and so did in 3 months old group(P<0.05). There was a significant decrease of SNc TH positive cells in all Rotenone treated rats with the least number of 20 months old rats. 3. In normal aging groups, SOD activity, CAT activity decreased in 20 months old group, which was significant difference compared with 3 months old group(P<0.05) and so did in 12 months old group(P<0.05). Comparing with normal aging groups, SOD activity, CAT activity increased significantly in all Rotenone treated groups(P<0.01)with the highest number of 20 months old rats. Conclusion Rotenone should be neurotoxic to dopamine neurons. There is an age-related decline in SNc dopaminergic neurons. Rotenone induced more severe dopaminergic neurotoxic impairment in 20 months old rats. The hypersensitivity to Rotenone in aged rats may related to the high level of oxidative stress.Part II The effects of autophagy and aging on dopamine neurotoxin of rotenone neurotonxin SD rats.Objective To study the effects of autophagy and aging on dopamine neurotoxin of rotenone- treated SD rats.Methods Autophagosomes were observed with electron microscopy. Protein levels of caspase-3 and LC3 were determined with immunohistochemistry and Western Blot analysis.Results 1.Electron microscopy revealed that the formation of autophagosomes in the rat striatum in Rotenone treated groups. 2. Immunohistochemistry demonstrated the increase in caspase-3 protein levels in Rotenone treated groups comparing with normal aging groups. 3.Western Blot analysis revealed the increase in cleaved-caspase-3 protein activation and LC3 protein levels comparing with normal aging groups(P<0.01), while the number of LC3 protein was least in 20 months old groups but that of cleaved-caspase-3 protein was highest in 20 months old groups.Conclusion The autophagy activity increased, with the apoptotic cell death increasing in Rotenone treated rats, but this compensatory stage was fail in old Rotenone treated rats, which maybe played a role in the hypersensitivity of old rats to rotenone neurotoxity.