The Role Of Mitochondrial Permeability Transition Pore (mPTP) On The Ischemic Postconditioning Against Focal Cerebral Ischemia In Rats

Background: It has been reported that several brief episodes of reperfusion/ischemia before reperfusion, termed as ischemic postconditioning (IPost), could induce neuroprotection from ischemia/reperfusion (I/R) injury. IPost is a new intervention measure, which can alleviate cerebral I/R injury. IPost with a series of mechanical interruptions of reperfusion reduces I/R injury. However, the effective time of IPost agaist focal cerebral I/R injury is still unclear.Mitochondrial permeability transition pore (mPTP), as a nonspecific channel, remains closed during the ischemic period and only opens in the first few minutes of reperfusion. The open of mPTP can lead to mitochondrial swelling and efflux of cytochrome c, which induces apoptosis in the setting of I/R injury. IPost is taking reperfusion as point of cut-in to against I/R injury and relationship between IPost and mPTP in neuroprective as not been reported previously. Thus, the object of this study is to explore the effective time of IPost against focal cerebral I/R injury and the role of mPTP in the neuroprotection induced by IPost. Part 1 Neuroprotective effect of ischemic postconditioning against focal cerebral ischemia and compare the neuroprotective effects between ischemic postconditioning with ischemic preconditioningMethods1. The optimal protocol of ischemic postconditioning against focal cerebral I/R injury in rats60 male SD rats weighing 290-310g were randomized into six groups: Control group and IPost groups with different time intervals (Post-15s/30s/1min/2min/5min). Control group underwent occlusion of the middle cerebral artery occlusion (MCAO) for 90min. IPost was performed by three cycles of reperfusion/ischemia of different time intervals (15s/30s/1min/2min/5min respectively) after MCAO for 90min. The neurological defict scores (NDS) were evaluated 24h after reperfusion. Infarct volume, as a percentage of volume at normal cerebral hemisphere, was determined by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining.2. Compare the protective effect of ischemic postconditioning with that of ischemic preconditioning30 male SD rats were divided into three groups: Control group, IPost group and ischemic preconditioning (IPC). The rats in IPC group were subjected to 20min MCAO and reperfusion for 24h before 90min MCAO. The NDS was evaluated at 24h after reperfusion. The infarct volume was then assessed with TTC staining after the NDS. Results1. The NDS in Post-15s/30s/1min/2min groups at 24h after reperfusion were significantly lower than those in the Control. And there was no significant difference between the Control and the IPost-5min group. The infarct volume at 24h after reperfusion in Post-15s/30s/1min/2min groups were significant smaller than that in the Control. But there was no significant difference between the Control and IPost-5min. These results indicate the effective time of IPost is three cycles of 15s reperfusion/ischemia. It was determined to be used in the subsequent studies.2. The NDS in IPost-15s group and IPC group were better than that in the Control. The infarct volume at 24h after reperfusion in IPost-15s and IPC were significant smaller than that in the Control. But, the infarct volume in IP-15s group was larger than that in IPC.Part 2 The neuroprotective effect of ischemic postconditioning is mediated by inhibiting the mitochondrial permeability transition pore in ratsMethods1. The dose-response of Atractyloside in middle cerebral artery occlusion 30 male SD rats were divided into three groups: Control group, rats in Atractyloside-1 (Atr-1) and Atr-2 groups received intracerebroventricular injection 15μl, 4mmol/L and 2mmol/L respectively 15min before reperfusion. The NDS was evaluated at 24h after reperfusion. The infarct volume was then assessed with TTC staining after the NDS. Thus the optimal dose for subsequent experiments can be determined.2. Neuroprotective effect of ischemic postconditioning is mediated by inhibition of mPTP opening --- in vivo study70 male SD rats were divided into seven groups: Control group, IPost group and the additional groups of rats separately intracerebroventricular injection 15μl, 2μmol/L of Cyclosporin A (CsA,which inhibits mPTP opening) and 15μl, 2mmol/L Atractyloside(Atr, a mPTP opener) respectively 10min and 15min before reperfusion during Control group and IPost group. And the last group received 15μl Alcohol (the solvent of CsA) given at reperfusion. The NDS were assessed at 24, 48 and 72h after reperfusion. Then at the 72h after reperfusion, the animals were decapitated and brain infarct volumes were evaluated with TTC staining.3. Neuroprotective effect of ischemic postconditioning --- the ultrastructure of mitochondria changes detected by transmission electron microscopeExperimental groups as described in experiment 2. After performing MCAO,the ultrastructure of mitochondria of the hippocampus of injured hemisphere was detected by transmission electron microscope at 72h after reperfusion.4. Neuroprotective effect of ischemic postconditioning is mediated by inhibition of mPTP opening --- in vitro study70 male SD rats were divided into seven groups: Ischemic group: 90 min of ischemia and no reperfusion. The additional groups as described in experiment 2. All animals underwent 90 min of ischemia and 15min of reperfusion except the ischemic group. Then at the 15min after reperfusion, the animals were decapitated and brain mitochondria were isolated. The absorbance at 520nm (A520) of isolated mitochondria was measured by spectrophotometer. Opening of the mPTP was determined by Ca2+-induced mitochondrial swelling. The decrease in light scattering closely parallels the percentage of the mitochondrial population undergoing permeability transition.Results1. At 24h after reperfusion,the NDS in Atr-1 group was significantly lower than that in the Control. The NDS in Atr-2 group was significantly highly than that in the Atr-1 group.The infarct volume at 24h after reperfusion, Atr-1 group was significant larger than that in the Control. Atr-2 group was significant small than that in the Atr-1 group. These results indicate the optimal dose of Atr is 2mmol/L, 15μl. It was determined to be used in the subsequent studies.2. All the rats survived until 72h after reperfusion. At 24, 48 and 72h after reperfusion, the NDS in Control+CsA and IPost group were significantly higher than that in the Control. The NDS in IPost+Atr group was significantly lower than that in IPost. But, there was no significant difference between in IPost and IPost+CsA.The infarct volume at 72h after reperfusion in Control+CsA and IPost were significantly smaller than that in the Control. The infarct volume of IPost+Atr was significantly larger than that in the IPost. But, there was no significant difference between in IPost and IPost+CsA. And there was no significant difference between in Alcohol and Control. These results indicate mPTP activation aggravates cerebral tissue injury.3. The electron micrographs indicated that the shapes of mitocbondria were round and had numerous transversae cristae which exhibited parallel alignment. The outer and inner mitochondrial membranes were clearly distinguishable. This was a normal mitochondrial structure. Mitochondria swelling, loss of mitochondria crests and vesicle mitochondria were observed in the Control and administration of Atr. In the IPost and Administration of CsA, the structure of mitochondrial was nearly normal. But mitochondria crest were slightly solution.4. The charge of A520 of ischemic group decreased compared with rest six groups, which confirms that mPTP remains closed during the ischemia period and only open in the first few minutes of reperfusion. The charge of A520 of administration of CsA and IPost were significantly lower than that in the Control, and the absorbance of administration of Atr was significantly higher than that in the IPost. However, there was no significant difference between in IPost and IPost+CsA.Conclusions1. The optimal protocol of IPost against focal cerebral ischemia was three cycles of 15s reperfusion/ischemia, and the neuroptrotection of IPC is superior to that of IPost.2. IPost produced neuroprotection against focal cerebral ischemia and improves function of mitochondrial.3. The improvement of mitochondrial function was probably mediated by inhibiting mPTP opening during reperfusion.