Objective To investigate the relationship between the timing of diazoxide injected and its effect against brain damage after middle cerebral artery occlusion(MCAO) in male rats,and to establish a simple,sensitive,rapid method of quantitative analysis.Methods 8 mmol/L diazoxide(6 μl) was injected into the lateral cerebral ventricle at the time of 30 min before ischemia,10 min,30 min,and 60 min during ischemia,and 10min during reperfusion,respectively.After reperfusion 22 hours,the tissue of hemisphere was incubated in 2,3,5-triphenyltetrazolium chloride(TTC) for 30 minutes,and then red formazan was extracted by solvent extraction and measured by Microplate Reader.Results Neurological score was improved 22 h later in the animals treated with diazoxide before ischemia and 10 min during ischemia compared with sham treatment(P<0.05).Spectrophotometric measurements of the extract showed diminished formazan coloration in all samples that was experienced ischemia-reperfusion injury compared to sham-operated controls.This apparent hemisphere injury was attenuated in the group of ischemia rats that received diazoxide at the time of 30 min before ischemia(0.28±0.06,P<0.01),10 min during ischemia(0.35±0.06,P<0.01),30 min during ischemia(0.41±0.09,P<0.01) compared to ischemia group without diazoxide administrated(0.52 ±0.06).Conclusion These finding suggest that opening of mitoKATP channels before ischemia and during early ischemia,but not that upon reperfusion,is important for enhancement of brain tolerance against infarction,and solvent extraction and microplate Reader quantitation of formazan has potential utility as an simple,objective way to index experimental brain injury.
Objective To investigate the ability of once and twice isoflurane preconditioning against oxygen and glucose deprivation（OGD） injury in rats brain in vitro.Methods Rat hippocampal slices were exposed to 1vol%,2vol% and 3vol% isoflurane for 30 min under normoxic condition（95% O2/5% CO2） once and twice（n=12 for each group） respectively before OGD.At the end of each exposure,the slices were set with a 15-min washout period interspersed,then slices were exposed to 13-min OGD period（95% N2/5%CO2,glucose-free） followed by 30min reoxygenation.The amplitude CA1 population spikes（PS,neuronal function） was measured and used to quantify the degree of recovery of neuronal function at post OGD period.To assess the role of the mitogen-activated protein kinases（MAPKs） in preconditioning,U0126,an inhibitor of extracellular signal-regulated protein kinase（MEK-ERK1/2）,and SB203580,an inhibitor of p38 MAPK,were used during isoflurane exposure.Results Isoflurane-preconditioning with 1vol%,2vol% and 3vol% once increased the degree of recovery from 4.8%±1.4%（control） to 41.9%±9.2%,55.1%±11.0% and 63.2%±10.8%,respectively,and twice to 53.8%±12.0%,63.5%±11.1% and 76.3%±12.3%, respectively.The effect of twice exposure to 3vol% isoflurane was blocked by U0126（6.1%±1.5%）.Conclusion It is concluded that twice isoflurane preconditioning could achieve better neuroprotection than once via activation of extracellular signal-regulated protein kinase（MEK-ERK1/2）.
