| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2005: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
Ischemic neuronal death is mediated by mitochondria. We have demonstrated that mitochondrial membrane potential showed a specific behavior depending on the grade of ischemic impact. Mitochondrial hyperpolarization has been observed after short-term oxygen-glucose deprivation (OGD) in cultured rat hippocampal neurons. Since hyperpolarization putatively links to subsequent death mode, the role of mitochondrial membrane potential may relate to cytosolic Ca^<++> [Ca^<2+>]c buffering capacity, and signaling pathway of PKB, which regulate apoptotic cascade.
1 Cytosolic Ca^<++> [Ca^<2+>]c buffering capacity after OGD
Hippocampal neurons cultured in dish were anaerobically incubated for 30 or 120 min with glucose-tree medium (30OGD, 120OGD). After OGD, neurons were loaded fluo-3 or rhod-2 for monitoring cytosolic and mitochondrial Ca^<++> concentration ([Ca^<2+>]c, [Ca^<2+>]m). To evaluate mitochondrial membrane potential (MMP), TMRE (tetramethylrhodamie, ethyl ester, perchloratye (TMRE)) was lo
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aded. Neurons were exposed to 0.5mM glutamate to induce depolarization.
The [Ca^<2+>]c, was steeply increased by glutamate load and increase in [Ca^<2+>]m followed in control and 30OGD. The [Ca^<2+>]c in 30OGD more rapidly decreased than that in control after it reached peak, and [Ca^<2+>]m progressively increased in 30OGD, while it remained constant in control. The area under the curve of normalized fluorescence of fluo-3 (AUCf) in 30OGD was significantly lower than that oin control, while the area under the curve of normalized fluorescence of rhod-2 (AUCr) in 30OGD was significantly larger than control. In 120OGD, increase in [Ca^<2+>]c and [Ca^<2+>]m was blunted.
Mitochondrial Ca buffering capacity after 30OGD was enhanced. This buffering capacity may link to preconditioning after short term ischemic episode.
2 OGD and Akt
We employed ELISA for detection of phosphorylated Akt. Short-term OGD(30min) increased phosphorylation of Akt, while longer OGD (120min) suppressed phosphorylation. We are now confirming this distinct change by using Western blotting. Less
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