| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2004: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2003: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
We investigated involvement of ER stress response in ischemic tolerance phenomenon in the brain. We hypothesized that brain ischemia causes ER stress response. We also hypothesized that inhibition of ER stress is one of the potential mechanisms for induction of ischemic tolerance. To test the hypothesis, expression of ER stress-related proteins were analyzed following in both global and focal cerebral ischemia.
[Materials and Methods]
Adult male Wistar rats and Spontaneously hypertensive rats were used in production of global cerebral ischemia and focal cerebral ischemia, respectively. In global cerebral ischemia, 2-min bilateral common carotid artery occlusion (CCAO) after bilateral vertebral artery occlusion that causes no damage CA1 sector of the hippocampus was adopted as effective ischemic preconditioning stimuli. On the other hand, 6-min CCAO was used for production of selective neuronal death of CA1 sector. Double ischemia (2 + 6 min CCAO, 72 hour-interval) was used for production
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of ischemic tolerance. In focal cerebral ischemia, bacterial lipopolysaccharide (LPS ; 0.9 mg/kg) was administered intravenously 72 hours prior to the middle artery occlusion (MCAO) to induce cross-ischemic tolerance. Expression of various ER stress-related proteins such as Ire1as a stress sensor protein, Grp78 and Grp94 as chaperon proteins, PERK as another sensor of ER stress sensor, and phosphorylated eIF-2α as a translation initiation factor were analyzed by Western blotting according to various time course after ischemia.
[Results]
In global cerebral ischemia, there were no differences among 2-min, 6-mm, 2 + 6 min-ischemia group in expression of Grp78. Nor phosphorylated PERK or phosphorylated Ire1 expression was observed in all three groups. However, phosphorylated eIF-2α expression was upregulated in 6-min ischemia compared with 2-min and 2 + 6 min-ischemia. In focal cerebral ischemia, expression of Grp78 and Grp94 were gradually upregulated following LPS-preconditioning alone over 72 hours. In LPS-preconditioning group, Grp94 expression was temporary downregulated 6 hours following MCAO. It turned to be upregulated 24 hours after MCAO. On the other hand, there was no change in expression of Grp94 in saline-control group. Grp78 was gradually upregulated up to 4 days following MCAO in LPS-preconditioning group. However, there was no change in expression of Grp78 in saline-control group.
[Discussion]
In this study, we could not verify the upregulated expression of Ire-land PERK which play important roles in initiation of ER stress response. However, we verified inhibition of eIF-2α expression in preconditioning and tolerance groups in global cerebral ischemia. Moreover, Grp78 and Grp 94 were both upregulated in expression in LPS-induced tolerance to focal cerebral ischemia. Further study will be needed to apply the strategy of ER stress-targeted therapy in neuronal rescue for following cerebral ischemia. Less
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