The Study On The Role Of Calpains In Ischemic Neuronal Injury And The Neuroprotection Of FNS And Its Mechanism

Objective To explore the role of calpains in neuronal injury during focal cerebral ischemia and ischemia reperfusion and elucidate the neuroprotection of FNS and its mechanism of regulating the activation of calpains, changes in affected cortex were characterized over time following focal ischemia and ischemia reperfusion to the brain.Methods Wistar rats with or without FNS preconditioning were subjected to permanent middle cerebral artery occlusion(MCAo) and reperfusion after MCAo for 3 hours and sacrificed 1,3,6,12,24h later. The affected tissue was processed for immunocytochemistry to detect calpain-induced fodrin breakdown (150kDa FBDP) and expression of calpains and calpastatin, for RT-PCR to detect expression of calpastatin mRNA, for immunological electron microscopic observation to detect the translocation of calpains, for cresyl violet staining (Nissl's staining) and HE staining to observe the pathological injury of neurons, as well as for Western blots to detect calpain-induced fodrin proteolysis. Results1. After permanent focal cerebral ischemia in rats without FNS preconditioning, significant increase in fodrin breakdown occurred within the first hour of occlusion, with further, dramatic increase in the marker continuing over time. While in the rats with FNS preconditioning, the change in fodrin breakdown was significantly inhibited at every time point. But DN electrical stimulation preconditioning showed no effect on the change.2. After permanent focal cerebral ischemia in rats without FNS preconditioning, significant increases in expression of calpains were observerved within the first hour of occlusion, but 3 hours later, they began to decrease and almost disappeared 6 hours later. In rats with FNS preconditioning, the increase in calpains was the similar ,but later ,their decrease was significantly inhibited. They disappeared until 12 hours later. DN electrical stimulation preconditioning showed no effect on the change of calpains.3. After permanent focal cerebral ischemia in rats without FNS preconditioning, significant incease in expression of calpastatin occurred within the first hour of occlusion. 3 hours later,the marker began to decease, with further decrease continuing over time. In the rats with FNS preconditioning, the increse in expression of calpastatin was significantly enhanced at every time point. While DN electrical stimulation preconditioning showed no effect on the change.4. During focal cerebral ischemia reperfusion in rats without FNS preconditioning, dramatic increase in fodrin breakdown occurred within the first hour of reperfusion , with further, marked increase in the marker continuing over time. While in the rats with FNS preconditioning, the change in fodrin breakdown was significantly inhibited.5. During focal cerebral ischemia reperfusion in rats without FNS preconditioning, the immunoreactivity of calpains dereased in the first hour of reperfusion, with further decrease continuing over time. In 3 hours after reperfusion, the immunoreactivity of calpains near to be disappeared. While in the rats with FNS preconditioning, the decrease in calpains were significantly inhibited. They never disappeared and began to increase between 6 to 12 hours , but again decreased after 24 hours of reperfusion.6. During focal cerebral ischemia reperfusion in rats without FNS preconditioning, the decrease of the immunoreactivity of calpastatin was observed in the first hour of reperfusion,with further decrease continuing over time. In 24 hours after reperfusion, the immunoreactivity of calpastatin nearly disappeared. While in the rats with FNS preconditioning, the decrease in calpastatin were significantly inhibited and the expression of calpastatin began to increase after 24 hours of reperfusion.7. After permanent focal cerebral ischemia in rats without FNS preconditioning, the score by neuronal shape, size and number increased in the first hour of occlusion, with further, gradual increase continuing over time. While in the...