2003 Fiscal Year Final Research Report Summary
Immunohistochemical study of cytokine induction and endotherial cell damage on hypoxic brain injury
| Project/Area Number |
13671141
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Embryonic/Neonatal medicine
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| Research Institution | Kyoto Prefectural University of Medicine |
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Principal Investigator |
HASEGAWA Koh Kyoto Prefectural University of Medicine, Department of Medical Research, Associate Professor, 医学研究科, 講師 (80228444)
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| Project Period (FY) |
2001 – 2003
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| Keywords | hypoxic brain injury / cytokine / nitric oxide / nitrotyrosine / free radical scavenger |
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| Research Abstract |
It is reported that Cytokines play important roles on hypoxic ischemic brain injury. In this study, the relationship between cytokines and nitric oxide (NO) in brain injury was investigated.
[Material and Method]
In each experiment forty cerebrocortical slices, each 350μm thick, were obtained from ten 7-day old Sprague-Dawley rats. Perfused slices had only one injury layer, were allowed 3 hrs to metabolically recover from decapitation, and were studied in a tissue culture well. In each study there was a 30 mm treatment period, such as hypoxia or NMDA, followed by 4 hours of perfusion with oxygenated ACSF. Frozen sections 20 μm thick were cut in the vertical plane of slices, and processed for Immunohistochemistry of IL1-beta. Adjacent frozen sections were used for HE staining and TUNEL staining. Expression of nitrotyrosine (NT), a marker of peroxynitrite was also investigated with/without 7-nitroindazole (7NI), a novel nNOS inhibitor. Apoptosis in slices was detected by TUNEL staining after etarabon (MCI-186) treatment before NMDA perfusion, a kind of free radical scavenger.
[Results]
Hypoxia or NMDA treatment caused IL1-beta expression at 15 to 30 min after the each insult, compared to control slices. 7NI could not decrease neuronal damage and NT positive cells, showing that NO did not cause neuronal death at early phase after hypoxia. Etarabon significantly decreased TUNEL positive cells on brain slices after NMDA treatment.
[Conclusion]
It is suggested that cytokine, which appears in neuronal tissue just after hypoxia, might cause NO induction by activation of the NMDA receptor, resulting in apoptosis of neuronal cells appeared at late phase.
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