Study On The Protective Effects Of Ischemic Preconditioning On Ischemia Reperfusion Rat Brain

ObjectiveIschemia, hypoxia and ischemia reperfusion injury of brain are themain factor to death and mutilation in central nervous system diseases.Recent research indicates ischemic preconditioning may improve thetolerance to ischemia. Ischemic preconditioning has another name ofischemic tolerance, it means the phenomenon of once or more timestransient ischemia may be an injury stressor which can activateself-protection mechanism to tolerant long-time ischemia. 1990, Kitagawaproposed firstly that this phenomenon happend in brain tissue,subsequently, a lot of experiments confirmed brain ischemicpreconditioning. However, the mechanism of brain ischemicpreconditioning are not known completely. And related research confirmischemia reperfusion injury of brain is tightly related to intracellularcalcium overload, and intracellular calcium overload predict destructionand death of cells.To confirm the protective effects of ischemic preconditioning on ratbrain and clarify the significance of calcium transport under ischemicpreconditioning, we dividied sixteen Wistar rats into two groupsrandomly, the control group(n=8) and the experimental group(n=8). 30 minglobal brain ischemia is given to the rats of control group, and then24 hours reperfusion; to the experimental group, we give a 3 min ischemicpreconditioning, followed by 30 min global brain ischemia and 24 hoursreperfusion. Then we observed the brain infarction volume of the controlgroup and the experimental group to evaluate the protective effects ofbrain ischemic preconditioning. Brain microsomes of the two groups wereprepared simultaneously, then we observed the effects of brain ischemic preconditioning on Ca²⁺uptake, Ca²⁺release of microsomes and the activityof Na⁺, K⁺-ATPase and Ca²⁺-ATPase to explain the possible mechanism of brainischemic preconditioning.MethodsThe global brain ischemia was induced with four-blood vesselocclusion. The brain infarction volume could be observed by the TTCstaining. Brain microsomes of two groups were prepared with two-stepcentrifugalization, protein concentration was determined by Lowry'smethod. The Ca²⁺uptake and release were observed by UV spectrophotometer.The activity of Na⁺, K⁺-ATPase and Ca²⁺-ATPase were determined with anenzyme-coupled assay.Results1. Effects of ischemic preconditioning on the brain infarction volumeAfter the reperfusion, the rat brain of the control group and theexperimental group were stained by TTC. Brain infarction appeared in bothgroups. The infarction volume in the control group is larger than theexperimental group(14.83±4.37% vs. 5.82±2.98%, P＜0.01),which hasstatistical significance.2. Effects of ischemic preconditioning on the calcium uptake of brainmicrosomesThe maximum Ca²⁺ uptake(nmol/mg protein) of control and experimentalgroups are 17.05±0.22 and 25.47±0.98 respectively (P＜0.01), which hasstatistical significance. The maximum Ca²⁺ uptake of experimental groupis higher than that of control group.The initial velocity of Ca²⁺ uptake (nmol/mg protein/s) of controland experimental groups are 2.25±0.92 and 3.33±0.87 respectively (P＜0.05). The initial velocity of experimental group is higher than thatof control group. 3. Effects of ischemic preconditioning on the calcium release of brainmicrosomesThe maximum Ca²⁺ release(nmol/mg protein) of control and experimentalgroups are 31.12±8.45 and 8.46±2.76 respectively(P＜0.01). The maximumCa²⁺ release of experimental group is lower than that of control group.The initial velocity of Ca²⁺ release(nmol/mg protein/0.01s) of controland experimental groups are 1.86±0.51 and 0.74⁰.31 respectively (P＜0.01), The initial velocity of experimental group is lower than thatof control group.4. Effects of ischemic preconditioning on the activity of Na⁺,K⁺-ATPase and Ca²⁺-ATPase.The activity of Na⁺, K⁺-ATPase(mU/mg protein) of control andexperimental groups are 18.94±1.93 and 32.66±1.69 respectively (P＜0.05),and the activity of Ca²⁺-ATPase (mU/mg protein) of control andexperimental groups are 14.70±1.92 and 29.19±1.71 respectively(P＜0.05).The activity of Na⁺, K⁺-ATPase and Ca²⁺-ATPase of experimental groupincreased 42% and 50% compared with control group.Conclusion1. Ischemic preconditioning can reduce infarction volume after ischemiareperfusion, which can decrease the brain injury degree and protectthe reperfusion rat brain.2. Ischemic preconditioning can increase the ability of Ca²⁺ uptake anddecrease intracellular calcium overload after brain ischemiareperfusion, which can lessen the calcium overload in reperfusioninjury and protect the rat brain.3. Ischemic preconditioning can reduce the ability of Ca²⁺ release, anddecrease intracellular calcium overload after brain ischemiareperfusion, which can lessen the calcium overload in reperfusioninjury and protect the rat brain. 4. Ischemic preconditioning can maitain the activiy of Na⁺, K⁺-ATPase andCa²⁺-ATPase after brain ischemia reperfusion, which can contributesto intracellular ionic balance and protect the brain.5. The results of this study had approved the protective effects ofischemic preconditioning on rat brain, and we had more knowledge aboutthe possible mechanism of ischemic preconditioning, which may helpto apply new ideas about prevention and cure the brain ischemiareperfusion injury.