Delayed Ischemic Postconditioning Protects Against Global Cerebral Ischemia Brain Injury By Preserving Mitochondrial Integrity Via Akt/GSK3β Signaling

Objective:Making four-vessel occlusion (4-VO) global cerebal ischemia (GCI) animal model, to study the mechanism of delayed ischemia postconditioning blocking mitochondria damage via upregulating Akt/GSK3βsignaling pathway in hippocampal CA1 region of the rats.Methods:1. Adult male Sprague-Dawley (SD) rats, weighing 250～300 g, were subjected to global cerebal ischemia (8 min) by 4-VO. Experimental animals were randomly divided into five groups: sham group, ischemia/reperfusion group (I/R), delayed ischemia postconditioning group (PostC), delayed ischemia postconditioning + vehicle group (PostC+Vehicle group), delayed ischemia postconditioning + LY294002 group (PostC + LY294002 group). As I/R group, four-vessel occlusion was induced by occluding the common arteries with aneurysm clips for 8 min; sham controls were obtained by using the same surgical procedures except that the arteries were not occluded; PostC group, the 3 min ischemia was induced at 48h after 8 min ischemia; LY294002 and vehicle were injected intracerebroventricularly (icv) into the left lateral ventricle 20 minutes before 3 min ischemia, the protocol of the study is shown in Figure.1.2. Immunofluorescence of NeuN was used to observe the neurons survival of hippocampal CA1 region of rats.3. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling assay (TUNEL) staining was used to observe the apoptosis of neurons in the happocampal CA1region of rats. 4. Transmission electron microscope (TEM) was used to observe the ultrastructural of neurons in hippocampal CA1 neurons of rats.5. Immunogold electron microscopy for the localization of Cytochrome C in hippocampal CA1 neurons.6. The methods of immunofluorescence and Western blot were used to observe the location and the level of change of Akt, GSK3β, Cyt c and Bax.7. JC-1 staining was used to detect the change of mitochondria membrane potential (MMP) in hippocampal CA1 neurons of rats.Results:1. PostC protects aginst delayed neuronal cell apoptosis in the hippocampal CA1 region 5 days after GCI (Fig. 2). I/R (Vehicle) induced a significant loss of hippocampal CA1 region neurons, as indicated by a marked decrease of NeuN-positive cells compared with sham group, whereas the number of NeuN-positive cells was significantly increased in the PostC group. Furthermore, the neuroprotective effect of PostC appeared to be abolished by LY294002, an inhibitor of PI3K (upstream kinase of Akt), indicated by the decrease of NeuN-positive cells. Staining for TUNEL, an apoptotic marker, revealed that I/R + vehicle increased the number of TUNEL-positive cells in hippocampal CA1 region compared with sham group, with PostC markedly prevented from the injury caused by I/R. The neuroprotective effect was abolished by LY294002 with increase of TUNEL-positive cells.2. PostC preserved the mitochondrial integrity following by GCI, but the effects were prevented by icv injection LY292004. In addition, PostC also attenuated the damage of blood-brain barrier and myelin sheath induced by inschemic reperfusion injury. As can be seen in Fig. 3A, the structure of the mitochondria in hippocampal CA1 region was intact in sham group; In I/R group, neuronal ultrastructure appeared severe impair such as the nuclear membrane distorted and hyperchromasia, mitochondria swollen or vacuolized with some partially broken cristae, even disaggregation; In contrast, we found that same as sham control, the sections from PostC groups clearly showed that nearly normal ultrastructure of neurons, in which nuclear membrane appeared unruffled and the nucleus was round and regular, and majority mitochondrial structure was intact with round and very few broken cristae; Furthermore, LY294002 abolished the protective effect of PostC, displayed as obvious mitochondrial damage compared with PostC groups. Fig. 3B-C showed that PostC prevented the damage of gap junction and myelin sheath induced by inschemic reperfusion injury. All results strongly suggest that delayed Ischemic PostC may protect against GCI by preserving mitochndrial integrity via PI3K-Akt signaling pathway. 3. PostC induced elevation of p-Akt and p-GSK3βin mitochondrial fraction of hippocampal CA1 region (Fig. 4). p-Akt levels in mitochondrial fraction significantly increased in PostC groups at 30 min, 6 h and 1d of reperfusion compared with the same time points of I/R without undergoing PostC, which in line with the changes of p-GSK3β. Akt and GSK3βlevels were not significantly changed at any time point with or without undergoing PostC. In agreement with these results, confocal analysis showed PostC significantly enhanced the levels of p-Akt, and p-GSK3βat 6 h reperfusion in mitochondria collocated with COXⅣ(mitochondrial marker) compared with I/R groups, which mirrored the results of Western blot.4. PostC prevents mPTP opening through upregulating Akt/GSK3βactivation in hippocampal CA1 region (Fig. 5). In I/R group, green fluorescence was stronger than red fluorescence, mitochondrial membrane potential decreased significantly, and neurons were apoptosis. In PostC group, the red fluorescence was more obvious, mitochondrial membrane potential was normal, and neurons were survival. In LY294002 group, the results are the same as I/R group.5. PostC attenuates ischemia-induced the release of Cyt c from mitochondrial to cytoplasm (Fig. 6). Cyt c immunoreactivity of hippocampus CA1 region was examined by Western blot at several time points (sham, 30 min, 3 h, 6 h, 1 d) after 8 min of ischemia with or without PostC. The level of Cyt c in cytosol were significant increased at 30 min and 6 h with PostC compared with sham; but it reduced obviously at the same time point (30 min and 6 h) in ischemia without PostC compared with PostC. To further investigate the effect of PostC on distribution of Cyt c in hippocampus CA1 region neuron at 6 h time-point after Post C, immunogold staining was performed, allowing direct visual location of Cyt c in mitochodria. Numerous gold particles were located over the mitochondrial crista in PostC group, and very few particles were seen on the outer of mitochondria. By contrast, in I/R group gold particles of Cyt c showed much more distribution in cytoplasm than that in the mitochondria paralleled by cristae ruptured and dissolved.6. PostC attenuates Bax translocation from cytoplasm to mitochondria following GCI in hippocampal CA1 region (Fig. 7). Bax levels of total protein were no significant change at any time points with or without PostC, whereas in cytosolic fraction Bax levels markedly increased at reperfusion 30 min, 6 h and 1 d undergoing PostC compared with the same time point of I/R without PostC, which was accompanied with an opposite change of Bax level in the mitochondrial fraction.7. LY294002 significantly abolished the effects of PostC on levels of p-Akt and p-GSK3β, Cyt c releasing, as well as Bax translocation following GCI in hippocampal CA1 region. LY294002 significantly attenuated p-Akt and p-GSK3β, and enhanced the levels of Bax in mitochondrial fraction compared with PostC + vehicle groups, whereas the levels of Akt, GSK3βand internal mitochondrial control (COXⅣ) had no significant changes. Additionally, LY294002 increased the level of Cyt c in responding to PostC at 6 h reperfusion (Fig. 8).Conclusion:The apoptotic neuronal death in the vulnerable hippocampal CA1 region following GCI was considerably attenuated by PostC, the effect associated with a critical role for Akt/GSK3βsignaling in preservation of mitochondrial integrity and attenuation of Bax translocation and Cyt c release by PostC in the mitochondrial (intrinsic) apoptotic pathway. Therefore, we have provided new mechanistic insights into the anti-apoptotic effect induced by delayed ischemic PostC and the application may have clinical efficacy as a potential neuroprotective treatment in ischemic stroke.