Influence Of 3-Nitropropionic On The Expression Of Caspase-3 After Ischemic Preconditioning

Background :Cerebral ischemic preconditioning and 3-NPA preconditioning can induce ischemic tolerance that can protect brain issue against the more severe ischemia followed. This phenomenon has been confirmed by some studies reported these years. The mechanisms are still not clear, maybe relevant to the expression of apoptosis related gene. But there is no report about the further influence of 3-nitropropionic on the expression of caspase-3 after ischemic preconditioning.Objective :To investigate effects of 3-NPA intervention after cerebral ischemic preconditioning on neurological function, infarct volume, and the expression of caspase-3 in brain issue after brain ischemia again, and to investigate the brain protection mechanism produced by 3-NPA intervention after cerebral ischemia preconditioning.Methods :72 healthy male Sprague-Dawley (SD) rats (weighted 200~250g) were randomly divided into 3 groups: ischemia group (n=24), ischemia preconditioning group (namely preconditioning group, n=24), and ischemia preconditioning plus 3-NPA preconditioning group (namely intervention group, n=24). Each group was divided into 4 subgroups according to 6h, 1d, 2d, 4d after ischemia again. Ischemia group: A thread was inserted to anterior cerebral artery (ACA) in the right side directly through common carotid artery (CCA). Ischemia preconditioning group: a focal-focal cerebral ischemic model was used according to Koizumi, adopting the thread embolism method. After exposing right CCA, external carotid artery (ECA), and internal carotid artery (ICA) of the anaesthetized rat, distal ECA was ligated and cut. A small hole on the proximal ECA was cut through which a thread (0.205mm diameter carbon fishing line) wasinserted through ICA to ACA to block middle carotid artery (MCA) blood flow by 30 minutes. Then the thread was pull out, the ECA nub was ligated and the skin was sewed up. Half hour after preconditioning 20ml.kg-1 sodium chloride was given through intraperitoneal injection. After 24 hours, a thread was inserted to right ACA again through CCA for ever. Intervention group: The method of operation was the same as above. Half hour after preconditioning 20ml.kg-1 3-NPA (1mg/ml) was given through intraperitoneal injection. The following indexes in 3 groups were observed respectively: neurological function according to Bederson method; the infarction volume by stained with 2% 2, 3, 5-tripheny tetrazolium chloride (TTC); cerebral tissue pathomorphology through hematoxylin-eosin (HE) staining; and the expression of caspase-3 in the hippocampus with immunohistochemistry method.Results:1. The score of neurological deficits: At the same point of time after ischemia, neurological deficit in preconditioning group was much less than that in ischemia group, the difference was significant (p<0.05). The score was lower in intervention group than those in preconditioning group and ischemia group. The difference was significant (p<0.05).2. The infarction volume: At the same point of time after ischemia,brain infarct volume of the rats in preconditioning group was smaller than that of ischemia group, the difference was significant (p<0.05). The infarct volume in intervention group was smaller than that of preconditioning group and ischemia group. The difference was significant (p<0.05).3. Immunohistochemistry: At the same point of time, the expression of caspase-3 positive-cells was lower in the hippocampus tissue in preconditioning group than that in ischemia group (p<0.05). This expression was also lower in intervention group than those in preconditioning group and ischemia group (p<0.05). Expression of caspase-3 positive-cells in the hippocampus tissue began at 6 hours after ischemia and increased progressively, reaching the peak 1- 2 days after ischemia and then gradually decreased. The difference between any two points of time in each group was significant (p<0.05).Conclusion:1. Cerebral ischemia preconditioning could protect the brain against ischemia again, decrease neurological deficits and infarct volume, inhibit the expression of caspase-3, and increase the tolerance of brain tissue to ischemia.2. 3-NPA intervention can give the brain more protection. The expression of caspase-3 could be further lowered, indicating that ischemia preconditioning and the intervention of 3-NPA may have combined effect on the protection of neuron.3. The expression of caspase-3 of different group at the same point has significant difference, so the inhibition of apoptosis maybe one of the mechanisms that ischemia preconditioning and the intervention of 3-NPA induce ischemic tolerance.