| Objective:To investigate the effect of rolipram on histopathology,memory function and PDE activity following global cerebral injury induced by ischemia-reperfusion (I-R) in rats.Base on this model,we also explore the mechanisms of the effect.Moreover,we investigate the effect of rolipram on proliferation and differentiation of the rat embryonic neural stem cells(NSCs).Methods:The global cerebral ischemia-reperfusion injury model was made by four-vessel occlusion in rats.the rats were randomly divided into sham-operated group,I-R group,rolipram 0.3mg·kg-1 group and rolipram 1mg·kg-1 group.Rolipram was administered once a day with ip from 6 h after the onset of the operation for 2 weeks.Then the learning and memory abilities were tested after Morris water maze and step-though training.After behavioral testing was completed,the brain were immediately removed for nissl(toluidine blue) staining and evaluated the PDE activity of the hippocampus by HPLC.NSCs were isolated from hippocampal,Nestin staining by immunocytochemistry was used to conform the NSCs.MTT and flow cytometry were used to investigate the effect of rolipram on proliferation of the rat embryonic neural stem cells.The capacity of NSCs differentiation toward neurons was determined by immunocytochemistry staining.RESULTS:Histological examination of brains from sham operated animals revealed no cell damage at all.Application of 0.3 mg·kg-1 or 1 mg·kg-1 rolipram resulted in a reduction of neuronal cell loss in the hippocampal CA1 sector,From 2th to 5th day of Morris water maze test,compared with sham-operated group,the escape lantency of the I-R group were significantly longer(63±16 s,38±16 s,26±12 s,18±8 s);Compared with the model group,the escape lantency of the 0.3 mg·kg-1 or 1 mg·kg-1 rolipram group were significantly shorter(36±23 s,16±13 s,17±12 s,7±2 s;41±13 s,22±10 s,17±12 s,11±2 s).In the probe trial test,compared with sham-operated group,the I-R group decreased the exploration time in the target quadrant(19±5 s);these were reversed by rolipram 0.3 and 1 mg·kg-1(4.3±1.8,4.0±1.4).In addition,rolipram also reversed the distracted platform searches induced by ischemia(4.3±1.8,4.0±1.4);In the step-through test,compared with sham-operated group,the lantency of the I-R group were significantly shorter (64±35 s) and the error times were increased statistically significant(1.4±1.0); compared with the model group,the lantency of the 0.3 mg·kg-1 or 1 mg·kg-1 rolipram group were significantly longer(141±54 s,138±71 s),and the error times were decreased statistically significant(0.6±0.5,0.7±0.8);The assay of the HPLC demonstrated that the activities of PDE in hippocampus of model group(16.1±0.9μmol.g-1.min-1) were higher than those in the sham-operated group and rolipram group(13.8±15μmol.g-1.min-1,14.1±0.7μmol.g-1.min-1).The microphotographs of hippocampusal neurons and Nestin staining conformed the neural stem cells. Compared with the control group(0.481±0.077),the MTT value of rolipram(10μM,20μM,40μM ) group increased obviously:0.675±0.048,0.677±0.111,0.684±0.059.Moreover,the results of flow cytometry indicated that the proliferation index of rolipram(10μM,20μM,,0μM ) group increased from 52.04%to 60.46%,64.11%,68.58%.These indicated that rolipram could promote the proliferation of the neural progenitor cells.Furthermore,the neural progenitor cells cultured in 10%FBS could differentiate into NF and GFAP positive cells.The number of NF positive cells detected in rolipram(10μM,20μM,40μM) groups were significantly higher than the control group(from 11 to 22,23,26,respectivly).These indicated that rolipram could promote the neural progenitor cells differentiated into neurons. CONCLUSION:Global cerebral ischemia induced the injury of the hippocampal,memory deficits and increased the PDE activity.The loss of the neurons may be the pathogenesis of the memory deficits.Rolipram reversed ischemia-induced memory deficits,this was associated with the attenuation of ischemia-induced neuronal damage by rolipram via inhibition of PDE4 activity in the hippocampus.Moreover,rolipram could promote the proliferation and differentiation of the neural stem cells,which may be one of the possible action mechanisms.
|
PDF Full Text Request