The Role Of Mitochondrial Dynamics In Central Nervous System Oxygen Toxicity

In underwater operations,underwater warfare and clinical hyperbaric oxygen therapy,oxygen with partial pressure higher than normal pressure(21kPa)will be used,which is called high pressure oxygen.However,high pressure oxygen can be toxicity to the body if the oxygen is breathed for too long and the oxygen partial pressure is too high(over 50kPa),which is referred to as oxygen toxicity.Oxygen toxicity is one of the main factors restricting oxygen used in diving and clinical treatment.How to prevent and cure oxygen toxicity is the key and difficult point in the field of diving medicine.Oxygen toxicity is mainly affected in lung and central nervous system,and central nervous system oxygen toxicity(CNS-OT)is mainly concerned in this study.The main manifestations of central nervous system oxygen toxicity include local muscle fibrillation,paroxysmal spasm and systemic muscle stiffness,which may eventually lead to severe nerve injury and death.At present,the pathogenesis of central nervous system oxygen toxicity has not been fully understood,and there are no good prevention and treatment methods except strict monitoring,intermittent oxygen inhalation.However,in the field of competitive and military diving,the above prevention and control measures are difficult to fully implement,so the incidence of CNS-OT is still high.To study the new pathogenesis of CNS-OT and develop corresponding preventive measures is expected to open up a new situation for basic research and clinical application in related fields.More and more evidence show that the imbalance of mitochondrial dynamics is involved in the occurrence and development of various nervous system diseases.The nervous system with high metabolic demand is strongly dependent on mitochondrial function,so the neuronal activity is greatly affected by mitochondrial dynamics.In this study,we conducted a preliminary experimental analysis and speculated that mitochondrial dynamics changes were involved in the occurrence and development of CNS-OT.Studies have shown that α7 nicotinic acetylcholine receptor mediated cholinergic antiinflammatory pathway plays a role in the occurrence and development of a variety of nervous system diseases,and activation of α7 nicotinic acetylcholine receptor can also have extensive effects on neuronal mitochondrial dynamics.In this study,we demonstrated experimentally that activation of α7 nicotinic acetylcholine receptor delayed the occurrence of CNS-OT by improving mitochondrial dynamics.Polyethylene glycol(PEG)is a kind of low immunogenicity polymer,which is widely used in medicine field.PEG has a good application effect in neurodegenerative diseases,spinal cord injury,epilepsy and other diseases,but whether it is effective in CNS-OT has not been reported.We investigated the prevention and treatment of CNS-OT with polyethylene glycol and its safety.This study is divided into four parts:1.The mechanism of mitochondrial dynamics in central nervous system oxygen toxicity;2.Mechanism of activation of α7 nicotine acetylcholine receptor on CNS-OT in mice;3.Mechanism of α7 nicotine acetylcholine receptor activation on hyperbaric oxygen injury of cultured neurons;4.Effects of polyethylene glycol on CNS-OT in mice.It is expected to provide new ideas for studying the pathogenesis of CNS-OT and lay a foundation for the prevention and treatment of CNS-OT by targeting mitochondrial dynamics.Part Ⅰ:Mechanism of mitochondrial dynamics in central nervous system oxygen toxicityC57 mice were randomly divided into Control group,HBO group and HBO+Mdivi-1 group.No special treatment under normal pressure air in Control group;HBO group was put into hyperbaric oxygen chamber and treated with 6 ATA pure oxygen.The HBO+Mdivi-1 group was pretreated with the mitochondrial division factor Drpl inhibitor Mdivi-1 and then subjected to the same hyperbaric oxygen exposure as the HBO group.After 30 min exposure,the animals were taken out and sacrificed,and the brain hippocampus were taken for follow-up experiments:1.The tissues were fixed and sliced,and the morphology,size and distribution of mitochondria in hippocampal neurons were observed by transmission electron microscopy.2.Tissue RNA was extracted,and the mitochondrial dynamics related factors Drpl,Fis1,Opal and α7nAchR were detected by RT-PCR.3.Mitochondrial proteins were extracted,and the mitochondrial dynamics related factors Drp1 and Fis1 were detected by automated Western blotting(simple western).The mitochondria of Control group were normal oval shape and clear internal structure under electron microscope.Mitochondria in HBO group lost their normal morphology,mitochondria in the process of splitting appeared,mitochondrial crista disappeared,and the proportion of abnormal mitochondria was higher than that in Control group.The mitochondrial morphology of HBO+Mdivi-1 group was recovered,and the proportion of abnormal mitochondria was lower than that of HBO group.At the mRNA level,the expression of mitochondrial fission factors Drpl and Fis1 in HBO group was higher than that in Control group,the expression of mitochondrial fusion factor Opal was lower than that in Control group,and the expression of α7nAchR was higher than that in Control group.At the level of mitochondrial protein,the expressions of mitochondrial fission factors Drp1 and Fis1 in HBO group were higher than those in Control group.The NAD+/NADH ratio and mitochondrial membrane potential in HBO group were lower than those in Control group.In the process of hyperbaric oxygen-induced central nervous system oxygen toxicity in mice,hippocampal mitochondrial fission is hyperactive,mitochondrial morphology disorders,mitochondrial function declines,α7nAchR expression increases.Mitochondrial dynamics and α7nAchR may be involved in the occurrence and development of CNS-OT.Part Ⅱ:The mechanism of α7 nicotinic acetylcholine receptor activation on central nervous system oxygen toxicity of miceC57 mice were randomly divided into Control group,HBO group,HBO+PNU group and HBO+Mdivi-1 group.No special treatment under normal pressure air in Control group;HBO group was put into hyperbaric oxygen chamber and treated with 6ATA pure oxygen.HBO+PNU group was pretreated with α7nAchR agonist PNU-282987,and then exposed to hyperbaric oxygen as HBO group.The HBO+Mdivi-1 group was pretreated with the mitochondrial fission factor Drpl inhibitor Mdivi-1 and exposed to hyperbaric oxygen as HBO group.Animals exposed for 30 minutes were taken out and killed.Followup experiments were carried out:1.Oxygen convulsive latency was recorded,and neurological severity scores of mice were recorded when exiting the cabin;2.The brain hippocampus was fixed and sectioned,and the mitochondrial morphology,size and distribution of hippocampal neurons were observed by transmission electron microscopy.3.Tissue RNA was extracted,and the mitochondrial dynamics related factors Drp1 and Fis1 were detected by RT-PCR.4.Tissue protein was extracted,and the mitochondrial dynamics related factors Drpl and Fis1 were detected by western-blot.5.Tissue GSH,MDA,Mn-SOD and other oxidative stress indexes were determined.The latency of oxygen convulsion in cabin and the score of neurological severity scores after leaving cabin in HBO+PNU group were higher than those in HBO group.Mitochondria were observed under electron microscope.The mitochondria in HBO group were small,some mitochondrial cristae disappeared,and the proportion of abnormal mitochondria was higher than that in Control group.The morphology and structure of mitochondria in HBO+PNU group were recovered,and the proportion of abnormal mitochondria in HBO+PNU group was lower than that in HBO group.In mRNA levels,mitochondrial fission factors Drpl and Fis1 in HBO+PNU group were lower than those in HBO group.The protein levels of mitochondrial fission factors Drpl and Fis1 in HBO group were higher than those in Control group.The expressions of Drpl and Fis1 in HBO+PNU and HBO+Mdivi-1 groups were lower than those in HBO group.Compared with the Control group,GSH,MDA and Mn-SOD in HBO group were changed,and the above oxidative stress indexes in HBO+PNU group were recovered compared with HBO group.Activation of α7nAchR can improve mitochondrial dynamics,reduce excessive mitochondrial fission,relieve oxidative stress,delay the occurrence of oxygen convulsion,and improve neurological function after CNS-OT.Part Ⅲ:The mechanism of activating α7 nicotine acetylcholine receptor on hyperbaric oxygen injury of neuron cellsHT22 mice hippocampal neurons were selected and divided into Control group,HBO group,HBO+PNU group and HBO+Mdivi-1 group.The HBO+PNU group was givenα7nAchR agonist Pnu-282987(5 μM,10μM)1h before exposure,and the HBO+Mdivi-1 group was given Drp1 inhibitor Mdivi-1(25 μM,50μM)1h before exposure.The Control group was incubated in a constant temperature incubator under atmospheric pressure without special treatment.The other groups were put into the hyperbaric oxygen chamber(6ATA,99%oxygen and 0.8%carbon dioxide)for 2 hours.After leaving the chamber,the test was as follows:1.The morphological changes of cells were observed under microscope.2.CCK-8 was used to detect cell viability.3.Oxidative stress indexes such as MDA and GSH were detected;4.RNA was extracted and RT-PCR was used to analyze the mRNA expression changes of Drpl and Fis1.5.Proteins were extracted,and the changes in whole-cell protein and mitochondrial protein expression of Drpl and Fis1 were analyzed using automated Western blotting(simple western).There was no significant difference in cell morphology among all groups.The cell vitality of HBO group was decreased compared with Control group,while the cell vitality of HBO+PNU(10μM)and HBO+Mdivi-1(25 μM,50 μM)groups was increased compared with HBO group.Compared with the Control group,GSH decreased and MDA increased in the HBO group,and the above oxidative stress indexes were recovered in the HBO+PNU group compared with the HBO group.The expression of Drp1 mRNA in HBO group was higher than that in Control group,and the expression in HBO+PNU group was lower than that in HBO group.Fis1 expression in HBO group was higher than that in Control group,and the expression in HBO+PNU(10 μM)group was lower than that in HBO group.No significant difference was found in the levels of Drpl and Fis1 in wholecell protein levels among all groups.The mitochondrial protein levels of Drpl and Fis1 in HBO group were higher than those in Control group.Drpl in HBO+PNU group was lower than those in HBO group,and Fis1 in HBO+PNU(10 μM)group was lower than those in HBO group.Activation of α7 nicotine acetylcholinergic receptor can inhibit the expression of fission factor,improve the mitochondrial dynamics of damaged neurons by hyperbaric oxygen,reduce oxidative stress,and restore mitochondrial function and cell vitality.Part Ⅳ:Effects of polyethylene glycol on central nervous system oxygen toxicity in miceC57 mice were randomly divided into Control group,PEG group,HBO group and HBO+PEG group.No special treatment in Control group;PEG group was pretreated with polyethylene glycol(intraperitoneal injection)without HBO exposure.HBO group was put into hyperbaric oxygen chamber and treated with 6 ATA pure oxygen.HBO+PEG group was given polyethylene glycol(intraperitoneal injection)in advance,then put into hyperbaric oxygen chamber and treated with 6 ATA pure oxygen.Animals exposed for 30 minutes were taken out and killed,and subsequent experiments were carried out:1.The latency of oxygen convulsion was recorded;2.The hippocampus of mice brain was fixed and sectioned,and the morphology,size and distribution of mitochondria of hippocampal neurons were observed by transmission electron microscopy.3.Tissue protein was extracted and the expression of mitochondrial dynamics related factor Drpl was detected by western-blot.4.Morris Water maze test was used to detect the cognitive and memory functions of mice.5.Motor ability of mice was measured by rotarod system.The results showed that the latency of oxygen convulsion of HBO+PEG group was significantly longer than that of HBO group.Transmission electron microscopy(TEM)showed that hippocampal neuron mitochondria in HBO group were round and scattered,with abnormal internal mitochondria structure and endometrial cristae disappearing.The morphology and structure of hippocampal neuron mitochondria in HBO+PEG group were recovered,and the proportion of abnormal mitochondria per unit area was significantly lower than that in HBO group.The expression of Drp1 protein in hippocampus of HBO+PEG group was not significantly different from that of HBO group.In Morris water maze experiment,there was no significant difference in percentage of swimming distance in the target quadrant and residence time in the target quadrant between the Control group and the PEG group.There was no significant difference in the walking time on the rotarod system between the Control group and the PEG group.These results indicate that polyethylene glycol can delay the occurrence of oxygen convulsion of CNS-OT mice,recover the mitochondrial structure of brain hippocampus,and has no obvious side effect on the cognition,memory and motor ability of mice.PEG is relatively safe to mice.But its specific mechanisms of effects on CNS-OT need to be further explored.