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Protective effect of mild hypothermia and barbiturate for cerebral ischemia-Evaluation by using neuronal cells-

Research Project

Project/Area Number 11671474
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Anesthesiology/Resuscitation studies
Research InstitutionHOKKAIDO UNIVERSITY

Principal Investigator

MORIMOTO Yuji  Hokkaido Univ., Grad.School of Med., Assistant Prof., 医学部・附属病院, 講師 (00250457)

Co-Investigator(Kenkyū-buntansha) WATANABE Satoshi  Hokkaido Univ., Grad.School of Med., Staff, 医学研究科, 助手 (20292005)
Project Period (FY) 1999 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥1,800,000 (Direct Cost: ¥1,800,000)
KeywordsCell culture ; PC 12 cells / Cell death ; apoptosis / Hypothermia ; mild hypothermia / Barbiturate ; pentobarbital / Caspase / Reactive oxygen species / Cerebral ischemia
Research Abstract

It has been reported that mild hypothermia and barbiturate are neuroprotecitve. However, the mechanism of this protective effect has not been well clarified. One possible mechanism is the inhibition of neuronal apoptosis. We evaluated the effect of hypothermia (29-35℃) and pentobarbital on apoptosis in neuronal cells using PC 12 cells, which is derived from rat pheochromocytoma. We further evaluated the mechanism of protective effect from the viewpoints of production of reactive oxygen species (ROS) and caspase activation
Apoptosis was induced by depriving serum from the medium, which is one of the most representative methods to induce apoptosis in PC 12 cells. First, cytotoxicity (evaluated by a leakage assay of lactic dehydogenase) and the percentage of apoptotic cells (calculated by flow cytometry with propidium iodide) were evaluated 4 days after induction of apoptosis. Second, production of ROS was measured by flow cytometry with a ROS-specific fluorogen, C-DCDHF-DA 3 or 6 hours after induction. Third, caspase-2 and -3 activation was measured by VDVAD p-nitroaniline and DEVD-p-nitroaniline cleavage assays 15 hours after induction.
Hypothermia (29 - 35℃) and clinical dose of pentobarbital significantly decreased cytotoxicity and the percentage of apoptotic cells. At 37℃ the production of ROS has already increased almost twice compared to control 3 hours after induction of apoptosis. Clinical dose of pentobarbital significantly decreased ROS production whereas hypothermia did not inhibit it. Caspase-2 and -3 activity was not inhibited by hypothermia and pentobarbital, although the activity increased by the induction of apoptosis.
Our data indicated that hypothermia (29 - 35℃) and clinical dose of pentobarbital inhibited induced apoptosis in neuronal cells. Inhibition of ROS production may be partly attributable to this protection by barbiturate. However, production of ROS and caspase activation may not be related to the mechanism of protective effect of hypothermia.

Report

(3 results)
  • 2000 Annual Research Report   Final Research Report Summary
  • 1999 Annual Research Report
  • Research Products

    (10 results)

All Other

All Publications (10 results)

  • [Publications] Morimoto,Y., et al.: "Pentobarbital intubits apopotosis in neuronal cells."Critinal Care Med. 28icine. 1899-1904 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] 森本裕二 他: "脳虚血に対するペントバルビタール,ミダゾラムの保護効果の機序-アポトーシス抑制の可能性-"循環制御. 20:2. 255-258 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Shibano,T.,Morimoto,Y., et al.: "Inhibition of production of reactive oxygen species and caspase activation may not be the mechanisms protective effect of hypothermia on neuronal apoptexis"European J.Anavethacology. 17:2. 91 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Morimoto, Y., Morimoto, Y., Nishihira, J., Kemmotsu, O., Shibano, T., Shikama, H.: "Pentobarbital inhibits apoptosis in neuronal cells"Critical Care Medicine. 28. 1899-1904 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Shikama, H., Morimoto, Y., Shibano, T., Kemmotsu, O.: "Effect of acidosis on the ratio of apoptotic and necrotic cell death due to hypoxia in neuronal cells"European journal of Anaesthesiology. 17. 91 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Shibano, T., Morimoto, Y., Shikama, H., Kemmotsu, O.: "Inhibition of production of reactive oxygen species and caspase activation may not be the mechanisms of protective effect of hypothermia on neuronal apoptosis"European journal of Anaesthesiology. 17. 91 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Shikama, H., Morimoto, Y., Shibano, T., Kemmotsu, O.: "Effect of acidosis and antioxidants on neuronal cell death due to hypoxia."Anesthesiology. 93. A709 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Morimoto, Y: "Pentobarbital inhibits apoptosis in neuranal cells."Crtieal Care Med. 28 icine. 1899-1904 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] 森本裕二: "脳虚血に対するパントバルビタール,ミダゾラムの保護効果の機序-アポトーシス抑制の可能性-"循環制御. 20 : 2. 255-258 (1999)

    • Related Report
      2000 Annual Research Report
  • [Publications] Morinoto, Y: "Inhibition of production of reactive oxrgen species and caspuse activation may not be the mechanisms protective eftect of hypothermia on neuronal apoptosis"European J.Anaesthesiology. 17 : 2. 91 (2000)

    • Related Report
      2000 Annual Research Report

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Published: 1999-04-01   Modified: 2016-04-21  

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