|Budget Amount *help
¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 1998 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1997 : ¥2,400,000 (Direct Cost : ¥2,400,000)
The present project aims to elucidate the role of sphingosine-1-phosphate (Sph-1-P) in the pathway of delayed neuronal death in CA1 neuronal cells. While Sph-1-P has recently been reported to have an important role in intracellular calcium release as an intracellular messenger, the role of Sph-1-P is unknown in ischemic delayed neuronal death, in which increase of intracellular calcium concentraion is a pivotal step toward cell death.
First, we have established the assay system for Sph-1-P and examined Sph-1-P contents in various regions of the brains of normal control gerbils. We extracted Sph-1-P by ice cold chloroform/methanol (1 : 2) and measured it by HPLC and radioimmunoassay. The Sph-1-P contents measured by radioimmunoassay in the hippocampus, cerebral cortex and cerebellum were 5.42*1.90, 0.84*0.21, 4.29*1.67 nmol/mg (mean*SEM, n=5), respectively. Measurement by HPLC could not give sufficient sensitivity and Sph-1-P was not detected in the hippocampus and cerebral cortex.
we examined changes in Sph-1-P contents in the brains after transient cerebral ischemia. Male mongolian gerbils (60-8Og) were subjected to 5 minute forebrain ischemia. Bilateral carotidarteries were occluded with aneurysmal clips for 5 min under halothane anesthesia. The rectal and temporal muscle temperatures were controlled at 37.5ﾟC using heating lamp and blanket. Brains were removed from gerbils before ischemia, 6 hours, 12 hours, 24 hours, and 48 hours after ischemia and Sph-1-P contents in the hippocampus, cerebral cortex and cerebellum were determined by above-mentioned method The Sph-1-P contents in the regions were as follows. Values are presented in the order of ; before ischemia, 6 hours, 12 hours, 24 hours, and 48 hours after ischemia.
Hippocapmus : 5.42*1.90, 4 63*1.24, 4.62*1.55, 4.89*1.73 (nmol/mg, mean*SEM, n=5)
Cerebral cortex : 0.84*0.21, 0.67*0.19, 0.72*0.12, 0.63*0.15 (nmol/mg, mean*SEM, n=5)
Cerebellum : 4.29*1.67, 4.56*1.71, 6.04*1.37, 6.34*2.26 (nmol/mg, mean*SEM, n=5)
Thus, no chronological changes were found in the Sph-1-P contents after ischemia.
These results suggested that Sph-1-P is not involved in the pathological pathway of neuronal death in CA1 regions. However, the present assay system could not allow cell-specific, or intracellular site-specific measurement of Sph-1-P and whether intracellular local changes of Sph-1-P are associated with cell death pathway should await further studies. Less