MECHANISMS OF DOPAMINERGIC CELL DEATH IN THE SUBSTANTIA NIGRA OF ANIMAL MODELS OF PARKINSON'S DISEASE

Abstract

Parkinson's disease is a common neurodegenerative disease with the pathological feature of dopaminergic cell death in the substantia nigra (SN). Although a gene mutation has been recently found in the rare familial form of Parkinson's disease, the mechanisms under lying the dopaminergic cell death are still obscure. There are increasing evidence suggesting that oxidative stress may be involved in the cell death. Iron, a transitional metal, is a very important factor in the oxidative stress because of its ability to stimulate the formation of toxic hydroxyl radicals. It was found to be significantly elevated in the SN of human Parkinsonian subjects, compared with the age matched control. Nevertheless, it remains controversial whether the iron increase is a causative factor or an epiphenomenon of Parkinson's disease. Using iron histochemistry (Perls' stain) with computer-aided image analysis and nuclear microscopy, a powerful tool for the detection of trace metals, the present study has first established that iron increase occurs in the SN of 6-hydroxydopamine ( 6-0HDA) induced Parkinsonian rats (26%) and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced Parkinsonian monkeys (61%). Subsequent time course study shows that dopaminergic cell death precedes iron increase. In rats, significant loss of tyrosine hydroxylase (TH) positive dopaminergic cells in the SN occurs at 1 week after 6-0HDA injection, while iron increase, compared with the contralateral side, is significant only one month after injection. In monkeys, dopaminergic cell loss in the SN is significant at one week, and iron increase significant at 4. 5 months after unilateral MPTP lesioning. Moreover, iron increase is significantly correlated to the dopaminergic cell death in both Parkinsonian models. These findings imply that increased iron in the SN is highly unlikely to be the primary cause of dopaminergic cell death in the Parkinsonian rats and monkeys. Rather, it is most likely the result of cell death. However, iron may still be involved in the cascade of events leading to oxidative stress, and be responsible for the progression of chronic nigral cell death in the Parkinson's disease. Attempts were also made to study the mechanisms of iron increase in the Parkinsonian SN. Studies focused on the expression of transferrin receptor (TfR), transferrin and ferritin in the SN of Parkinsonian rats, as these proteins had been reported to be responsible for iron homeostasis. The time course study shows a 15-22% decrease (p<0.05) in the number of TfR positive cells in the rat SN after 6-OHDA injection, but no significant changes (p>0.05) in the density of TfR staining in microvessels in the SN. There is decreased (p<0.05) expression of transferrin immunoreactivity. These results suggest that TfR and transferrin may not be responsible for iron accumulation in the SN of 6-OHDA induced Parkinsonian rats. Using serial thin sections for immunohistochemistry, a colocalisation of TfR and TH was demonstrated in the rat SN. Electron microscope study has localised TfR immunoreactive products in some ribosome and cell membrane in the nigral neurons. In agreement with previous studies, ferritin immunoreactivity was observed in microglia and oligodendrocytes in the rat SN. The study shows a 31-63% increase (p<0.05) in the number of ferritin positive microglia cells from 1 week to 1 month after unilateral 6-0HDA lesioning, then a gradual return to the normal level. The results are further confirmed by OX 42 and OX6 immunohistchemistry which especially stain microglial cells. The numbers of OX42 and OX6 positive microglia are increased at 1 week to 1 month after 6-0HDA injection. This returns to normal 3 months later. The temporary increase in the number of ferritin positive microglia is most likely a glial reaction to cell death. It is possible that the increased expression of ferritin in microglia may partly contribute to the iron accumulation at the early stage. Nitric oxide (NO) and glutamate have been implicated in neurodegeneration, and are possibly related to iron metabolism. Hence, investigations on NO and glutamate receptor subunits (GluR1, GluR2/3 and GluR4) were also carried out in the Parkinsonian SN of rats. The results from the study on nitriergic reactivity are in agreement with those of previous studies. Only blood vessels and nerve fibres are NADPH-d reactive. In the rat some neurons in the SN are also nNOS immunoreactive. Using NOS radiometric assay, an increased activity of NOS has been demonstrated in the rat SN after 6-0HDA lesioning, suggesting an increased NO production. The increased NO may be involved in the mechanism of dopaminergic cell death and iron accumulation in the SN after 6-0HDA lesioning. GluR1, GluR2/3 and GluR4 positive cells have different distributions in various parts of the rat SN. Their immunoreactive products are located in some ribosomes in the cytoplasm and in post-synaptic membrane of the nigral neurons. This is in agreement with previous study in other areas of the rat brain. The numbers of GluR1 and GluR2/3 immunopositive cells are significantly decreased (14-41% of GluR1 positive cells, p<0.05; 13-19% of GluR2/3 positive cells, p<0.05) in the rat SN, while there are no significant changes in the number of GluR4 positive cells. These results suggest different susceptibility of different subtypes of glutamate receptors to the neurotoxin 6-0HDA. Lastly, the study has found evidence of apoptotic cell death in the Parkinsonian SN of rats and monkeys, using the TUNEL method for morphological assessment.