Machenisms Of Mitochondrial Oxidative Stress And Protection Effect Of Edaravone In Vascular Dementia Rats Induced By Chronic Cerebral Ischemia

Vascular dementia (VD),caused by cerebrovascular disease ,is a group of syndromes characterized by cognitive impairment. As the third most common cause of dementia after Alzheimer's disease, Vascular dementia which affects the quality of life severely accounts for approximately 10-50% of cases in China. Oxidative stress has been thought to play a important role in VD through causing the oxidative lesion of biomacromolecule and cell apoptosis. Recently, researchers have focused on the mitochondria oxidative stress and free radical toxicity. Edaravone is a new free radical cleaner which can clean hydroxy radical specificly and inhibit oxidative stress and cell apoptosis. It's unknown of the researches about Edaravone in VD, although its cerebral protection has been supported in the acute cerebral infarction. Based on this background, we study the mechanisms of mitochondria oxidative stress in VD and the protective mechanisms of edaravon in the rat model of VD established through permanent bilateral occlusion of both common carotid arteries. We hope it can provide precise theory base for the treatment of VD in the use of Edaravone.I. The establishment and evaluation of the vascular dementia rat modelAfter the Morris Water Maze pre-test, we selected 100 female rats, which were 3-4 months and 300-350g in weight, and divided them into the control group and the model group in random. After establishing the VD rat model through permanent bilateral occlusion of both common carotid arteries, we have operations to cut down the heads of rats for brain in the 1st month and 2nd month respectively.Morris Water Maze was used to detect the changes of spatial learning -memory capacity of the rats. The result showed: the escape latency of model group was significantly longer than that of control group in the 1st month and 2nd month. It was proved that learning -memory capacity of rats after operation declined statistically.The morphologic changes.The pyramidal cells inhippocampus CA1 of the rats in control group distributed evenly and their appearance were nearly normal; the hippocampal pyramidal cell layers in model group were disturbed and interrupted. Ultrastructure of hippocampus CA1 region. We can see the neuron of hippocampus in the control group normal nucleus, perikaryon with abundant organelles, normal polyribosomes, mitochondria and Golgi apparatus,roughendoplasmic reticulum (RER). The neuropil with crowded dendrites, many mitochondria, axon containing neurotubules and many synapses with adequate vesicles, dense postsynaptic constituents and clear cleft . We can see the hippocampus neuron in the model group severe edematous neuropils, the dark neurons and gliocyte increasing, dilated dendrites ,and axon in which vacuoles confused to form irregular cysts ,axons with obscure neurotubules, mitochondria loss crista and marked decrease in number of synapses.The results suggest that chronic cerebral ischemia can lead to the decline of learning -memory capacity in VD rat model. It's reliable of the establishment of the VD rat model.II. The etiopathogenisis research about the effect of the mitochondria oxidative stress in the vascular dementia rat1 . The research about the activity of SOD, the content of MDA, and the change of mitochondria membrame'fluridity in vascular dementia rat hippocampus.Superoxide dismutase(SOD) is a free radical cleaner which can clean superoxide anion and protect cell against reactive oxygen species (ROS) overload and subsequent damage. It can maintain the balance between the oxidation and antioxidation, and it is one of the markers to reflect the cleaning ability of free radical. Malonialdehyde (MDA) is the metabolite of unsaturated fatty acid in oxidation response in biomembrane resulted from free radical. So it can indirectly reflect the severity degree of the injury of cell. Mitochondrial membrane mainly consist of phospholipid molecule. It is very important for mitochondrion membrane to execute normal function that keeping its fluidity and permeability. Increased production of free radicals can destruct mitochondrion membrane's fluidity and permeability, thus metabolites of mitochondrion are released into intercellular substance, which can lead to apoptosis and necrosis of cells.We measure the activity of SOD, the content of MDA by UV-spectrophotometer and measure the fluidity of mitochondrial membrane by fluorescence spectrophotometer. The result showed: the activity of SOD in the model group decreased significantly than that of the control group (p<0.05) in the 1st month and 2nd month after operation, the activity of lactoperoxidase (LPO)( representing for the content of MDA)and the viscous coefficient (η) of mitochondrial membrane (representing for the fluidity of mitochondrial membrane) of model group was significantly higher than that of the control group (p<0.05).We can conclude that chronic cerebral ischemia in the nuron cell of rats decreases the cleaning ability of free radical, which can increase the content of MDA and reduce the fluidity of mitochondrial membrane. Above all, It is a mechanism of mitochondrial oxidative lesion in VD.2. The activity of hippocampus mitochondrial COX and the change of genetic expression in VD ratsCytochrome C oxidase (COX) is the end enzyme of mitochondrion electron transport chain, and also is the rate-limiting enzyme of mitochondrion electron transport chain which have important effect on energy metabolism of cell. Thus activity of COX is closely correlated with the function of neuron and is the marker of neuron metabolism. We measure the activity of COX and the change of genetic expression by UV-spectrophotometer and fluorescence spectrophotometer. The results showed: the activity of COX in the control group decreased significantly than that of the model group (p<0.05) in the 1st month and 2nd month after operation. The expression of mitochondrial COXⅡin the model group decreased than that of the control group through RT-PCR. Different from the control group, the COXⅡin the model group occurs 4 base substitution,1 base deletion ,1 other base substitution in the same place, and losses 4 genetic mutable points. The COXⅢoccurs 3 genetic mutable points(base substitution),and losses 1 mutable point. The results told us: genetic mutable rate of COXⅡ, COXⅢin the hippocampus mtDNA in the VD model group rats is higher than that of the control group, and mutable modus of COXⅡis multiplicitily and complicated.The activity of COX can be determined by the genes of COXⅡand COXⅢwhich leads to the change of amino acid sequence through translation in the way of genetic mutation. The mechanism of VD rats this research confirmed is correlated to the activity of hippocampus mitochondrial COX or mitochondrial oxidative stress.III. The protective effect of Edaravone in the vascular dementia rats1. The establishment of the vascular dementia rat model The 100 healthy male Wistar rats (300-500g in weight) were divided into 4 groups randomly as the control group, the model group (VD+ 0.9%Sodium Chloride), the Edaravone group (VD+ Edaravone), the Duxil group (VD+ Duxil) after place navigation. From 7th day, the rats in the Edaravone group were injected with Edaravone 3mg/Kg every day, for 20 days. The rats in the Duxil group received gastric perfusion with Duxil 20 mg/Kg every day, for 20 days. In the 1st month and 2nd month the heads of rats were cut down for brain.2. The effect of Edaravone to VD rats on praxiologic and histologic changesAfter one month's treatment, the escape latencies in the Edaravone group and the Duxil group rats were more significantly shorten than the model group(p<0.05).That means the learning–memory capacity in the two groups were improved. It's nearly same. After two months's treatment, the escape latencies in the Edaravone group were more shorten than the Duxil group(p<0.05).It indicates that vascular cognitive impairment may be associated with the hippocampus neuronal lesion induced by chronic cerebral ischemia. And Edaravone can lessen injures of hippocampal neurons and improve the spatial learning and memory abilities.The ultrastructure of hippocampus neural cell in the Edaravone group and the Duxil group were similar to the control group in the light microscope. In the electron microscope we can see the severe edematous neuropils the dark neurons and gliocyte increasing, dilated dendrites, and axon in which vacuoles confused to form irregular cysts ,axons with obscure neurotubules, mitochondria loss crista and marked decrease in number of synapses in the Edaravone group, which is similar to the Duxil group.Vascular cognitive impairment is correlated to hippocampus neuron lesion causing by chronic cerebral ischemia. The results showed: the pathologic changes observed by electron microscope in nerve cell of the Edaravone group is fairly light and metabolic activity is activive. So we realized that Edaravone can avoid hippocampal neurons from injures of chronic cerebral ischemia.3. The effect of the activity of SOD, the content of MDA, the activity of COX and the change of mitochondria membrame'fluridity in VD rats after administrated for Edaravone.the activity of SOD in the Edaravone group and the Duxil group was higher significantly than that of the model group (p<0.05) in the 1st month and 2nd month after operations, the content of MDA and the viscous coefficient (η) of mitochondrial membrane (representing for the fluidity of mitochondrial membrane) of model group was significantly higher than that of the Edaravone group and the Duxil group (p<0.05).And the effect of indexs was better in the 2nd month than those in the 1st month. The expression of mitochondrial COXⅡthrough RT-PCR in the model group decreased than that of the Edaravone group(p<0.05) which was similar to the Duxil group. Compared with the control group, the COXⅡin the model group occurs 4 base substitution, 1 base deletion, 1 other base substitution in the same place, and losses 4 genetic mutable points. In the Edaravone group the COXⅡoccurs 7 base substitution, 1 base deletion which is similar to the control group, and losses 5 genetic mutable points. The COXⅢin the model group occurs 3 genetic mutable points (base substitution), and losses 1 mutable point. In the Edaravone group the COXⅢoccurs 2 genetic mutable points, and genetic mutable rate decreases. Compared with the model group, the COXⅡin the Edaravone group occurs 6 base substitution and losses 3 genetic mutable points. In the Edaravone group the COXⅢoccurs 2 genetic mutable points.It can decrease the production of free radical or increase the cleaning of that, which reduce the level of reactive oxygen species and metabolite of fatty acid in oxidative response and lessen mitochondria oxidative lesion. It can protect central nerve system through delaying the retrogradation or death of the nerve cells.Conclusion:1. To establish the vascular dementia rat model through permanent bilateral occlusion of both common carotid arteries, and evaluate praxiologic and histologic changes of rats after Morris Water Maze and place navigation, it demonstrates further that the model is reliable through the changes of hippocampal microanatomy and ultrastructure in the rats and declining of learning -memory capacity.2. The mechanisms in vascular dementia rats which this research confirmed is associated to mitochondria oxidative stress, such as the activity of SOD decrease and the content of MDA increases, the mitochondria membrame'fluridity decreases.3. It demonstrates further that the activity of COX and the expression of COX take part in the mechanisms in the vascular dementia rats,such as the activity of COX and the expression of COXⅡdecrease, and COXⅡand COXⅢgenetic mutable rate increases.4. Edaravone has the protective and therapeutic effects in the vascular dementia rats through increasing the learning -memory capacity and lessening the hippocampal ischemia.5. Edaravone can clean free acids and enhance the ability of antioxidation through increasing the activity of SOD and that of COX, the mitochondria membrame'fluridity, and decreasing the content of MDA, COXⅡand COXⅢgenetic mutable rates.New points in this research:1. So far it's the first time to report the mechanisms of mitochondria oxidative stress in the vascular dementia rats which this research confirmed in China.2. At present it's the first time to report the protective effect of Edaravone in the rat model of vascular dementia.3. This research which enlarges the indications of Edaravone provides precise theory bases for the mitochondria oxidative stress and the treatment of vascular dementia.