Neuroprotective Effects Of Hypoxic Preconditioning And Its Mitochondrial Mechanisms

Hypoxia is a common injurious factor in many clinical diseases and some specific environments such as high altitude, aviation and diving. Central nervous system (CNS) is extremely dependent on aerobic oxidation to supply energy, therefore, it is very sensitive to hypoxia. Long-term or severe hypoxia may lead to dysfunction and pathological changes of CNS. It is very important to looking for the approaches to enhance CNS hypoxia tolerance, and looking for new strategies to prevent and treat clinical hypoxic diseases. Recent studies have described a phenomenon termed hypoxic preconditioning (HP) in which a brief periods of hypoxia protect cells from damage caused by severe hypoxia. HP provides a new way to enhance hypoxia tolerance, and has been paid more and more attentions by researchers. By now HP is thought as an endogenous feature shared by many tissues and organs. But its neuroprotective mechanisms are still uncertain.The mechanisms of CNS injuries caused by hypoxia are very complicated, cellular necrosis and apoptosis have been considered as the major causes of CNS hypoxic injuries. Mitochondria are closely related with brain hypoxic injuries, and have been thought as the "common pathway" in the cell death process. Opening of mitochondrial permeability transition pore (MPTP) results in disruption of mitochondrial membrane potential, uncoupling of oxidative phosphorylation, decreasing of ATP, increasing of reactive oxygen species (ROS), overloading of Ca²⁺ in the cells, and induce cell necrosis, meanwhile, it also causes mitochondrial swelling, rupture of mitochondrial outer membrane, release of apoptosis promoters from intermembrane space of mitochondria, and finally, induces cell apoptosis.Many studies manifest that HP attenuates apoptosis and necrosis caused by severe hypoxia. Based on the key roles of mitochondria in cell death, we hypothesize that HP may attenuate apoptosis and necrosis by inhibiting the opening of MPTP, decreasing cellular Ca²⁺, ROS and inhibiting release of pro-apoptosis factors from mitochondria. In order to verify above presumption and explore the mechanisms of HP, we studied theneuroprotective effects of HP and mitochondria's roles on it in vivo and in vitro. The main results and conclusions are as following:1. By using intact rats, we observed protective effects of HP on the damages of rats hippocampus CA1 neurons and deficits of rats learning and memory ability caused by severe hypoxia, changes of mitochondrial respiratory control rates (RCR), content of adenylic acid, mitochondrial swelling and membrane potential.(1) Severe hypoxia (10000m, lh) resulted in injuries of rat's hippocampus CA1 neuron and damages of mitochondria, induced neuronal apoptosis. HP (5000 m, 4 h/d, 7d) diminished the injuries of neuron and mitochondria, and attenuated the apoptosis caused by severe hypoxia.(2) Severe hypoxia (10000m, lh) caused deficits of learning and memory ability, HP relieved the deficits of the learning and memory ability caused by severe hypoxia.(3) HP decreased the amount of mitochondrial AMP, and increased the amount of mitochondrial ADP.(4) HP decreased the mitochondrial swelling, and inhibited the decrease of mitochondrial membrane potential induced by Ca2+. Cyclosporin A (CsA), which is a blocking agent of MPTP, imitated protective effects of HP on mitochondria. Atractyloside, which is an opening agent of MPTP, blocked the effects of HP. These results suggest that protective effects of HP are related to MPTP.2. By using cultured PC 12 cells, the mitochondrial mechanisms of HP protection were further studied, the main results as following:(1) Hypoxia (2.5%O2, 24h) significantly increased LDH release from PC 12 cells, HP (2.5% O2, 2 h, following 12 hours interval) significantly decreased LDH release from PC12 induced by severe hypoxia cells. The results indicate that the HP protects the PC 12 cells from severe hypoxia injury.(2) Hypoxia significantly decreased viability of PC 12 cells, induced cell necrosis and apoptosis. HP inhibited the decrease of PC 12 cells viability and the increases of necrosis and apoptosis. CsA imitated the protective effects of HP, and ATR blocked the protective effects of HP.(3) Hypoxia caused injury of PC 12 cells as well as decrease of mitochondrial membrane potential. HP prevented severe hypoxia-induced PC 12 cell injury and maintainedthe mitochondrial membrane potential. CsA imitated the protective effects of HP on cell injury and mitochondrial membrane potential. These results indicate that inhibition of MPTP opening and maintaining of mitochondrial membrane potential may contribute to the HP protection.(4) The concentration of Ca2+ and ROS in PC 12 cells of HP group were significantly higher than that in control group, but were significantly lower than that in hypoxic group. The results indicate that HP inhibit the increase of Ca2+ and ROS level in PC 12 cells after severe hypoxia(5) Caspase 3 activity in HP group was significantly higher than that in control group, but lower than that in hypoxic group. It suggests that hypoxia increases Caspase3 activity and HP inhibits the increase of Caspase3 activity.(6) Bcl-2 protein expression in hypoxic PC 12 cells was significantly higher than that in control group, but significantly lower than that in HP group. Cytochrome C content in hypoxic group was significantly higher than that either in control group or HP group. It suggests that HP may increase Bcl-2 expression, and decrease release of Cytochrome C to the cellular plasma.3. By using Bcl-2 transfected PC 12 cell model, the relationship between HP and mitochondria, especially the MPTP was further studied. The main results as following:(1) Eukaryotic expression vector pcDNA3.1/Bcl-2 was successfully constructed and Bcl-2 gene was transfect to PC 12 cells by using cationic liposome and PC 12 cells which stably overexpressed Bcl-2 were obtained.(2) Under normoxic condition, there had no differences in cell activity, and numbers of apoptotic and necrotic cell between the PC 12 cells transfected Bcl-2 and control cells, but under hypoxia, Bcl-2 transfected PC 12 cells showed higher cell activity, and fewer apoptosis and necrosis than the cells in control group. ATR could significantly reverse the preotective effects of Bcl-2 overexpression on hypoxic injury. These results indicated that overexpression of Bcl-2 increases hypoxia tolerance of PC 12 cells. It may result from the inhibition of MPTP opening.(3) Under normoxic condition, mitochondrial membrane potential, intracellular ROS and Ca2+ of PC 12 cells overexpression Bcl-2 had no significant difference with control group cells. But in hypoxia, the concentration of cellular ROS and Ca2+ were significantlylower than that in control group cell, and the mitochondrial membrane potential was higher significantly than that in control group cell. ATR significantly decreased the mitochondrial membrane potential, and significantly increased cellular concentration of Ca2+ of Bcl-2 transfected PC 12 cells in hypoxia, but had no significant effects on the ROS production. These results indicate that overexpression of Bcl-2 decreases the production of ROS, concentration of cellular Ca2+ in PC 12 cells and maintains the mitochondrial membrane potential under hypoxia. HP could upregulate the expression of Bcl-2 which inhibits the opening of MPTP and therefore contribute to the protection for PC 12 cells under hypoxic condition...