1997 Fiscal Year Final Research Report Summary
Signal transduction of brain capillary endothelial cells in relation to regulation of blood-brain barrier
| Project/Area Number |
07671516
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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 |
Cerebral neurosurgery
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| Research Institution | Kobe University |
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Principal Investigator |
NAGASHIMA Tatsuya Kobe University, 医学部・附属病院, 講師 (80201680)
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| Co-Investigator(Kenkyū-buntansha) |
TAMAKI Norihiko Kobe University, 医学部, 教授 (10030941)
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| Project Period (FY) |
1995 – 1997
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| Keywords | Blood-brain barrier / Endothelial cells / Brain / Capilary / Signal transduction / Free radical / Calcium ion / Confocal Laser Microscopy |
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| Research Abstract |
#1 : The mechanism of reversible osmotic opening of the blood-brain barrier : role of intracellular calcium ion in capillary endothelial cells
Despite clinical and experimental interest in the osmotic opening of the blood-brain barrier (BBB), the mechanism underlying the phenomenon remain undermined. The aim of this study is to investigate the mechanism of intracellular calcium ion change in brain microvascular endothelial cells subjected to hyperosmotic stress.
MATERIALS AND METHODS : Cultured rat brain capillary endothelial cells were obtained by two-step enzymatic purification. Intracellular calcium ion was measured by a confocal laser scanning microscope. After exposing the endothelial cells to 1.4 M mannitol, the change of intracellular calcium ion concentration was monitored.
RESULTS : Intracellular calcium ion concentration increased rapidly and reached its peak value within 10 seconds after the application of mannitol. The calcium ion concentration returned to the basal level with
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in 200 seconds. A specific blocker (KB-R7943) of sodium/calcium exchange did not affect the rapid elevation of intracellular calcium ion. However, it blocked the return phase almost completely. The results indicated that the sodium/calcium exchanger pumped out the increased intracellular sodium/calcium during the return phase.
CONCLUSION : Reversible osmotic disruption and reconstruction of the BBB is not due to simple mechanical shrinkage of the endothelial cells but is due to the intracellular calcium ion-activated complex mechanism.
#2 : Mechanism of BBB Disruption in Reoxygenation Injury of Rat Brain Capillary Endothelial Cells
MATERIALS AND METHODS : Rat and human BCEC were subjected to anoxia/reoxygenation. Enzyme inhibitors including oxypurinol, indomethacin, or L-NAME,or specific free-radical scavengers such as superoxide dismutase (SOD), catalase, or the ferric iron chelator deferoxamine were added before A/R injury. The BCEC were incubated in a range of calcium ion concentrations from 1 mM to 0.01 mM during A/R injury. Cytotoxicity was assayd by release of intracellular lactate dehydrogenase (LDH).
RESULTS : With A/R injury, LDH release from the control group (no protective agents) significantly increased compared with a small increase in a normoxic group. BCEC treated with oxypurinol, indomethacin, or L-NAME showed suppression of LDH release. LDH release was almost totally suppressed by SOD,and partially by catalase or deferoxamine. The LDH release was partly dependent on calcium concentration.
CONCLUSION : BCEC subjected to A/R become potent generators of free radicals, especially superoxide anion. Free radical production depends on both xanthine oxidase and cyclooxygenase pathways. Peroxynitrite and extracellular calcium ion both contribute importantly to reoxygenation injury of BCEC. Less
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