Changes Of Hippocampal Neural Lipids After Status Epilepticus In Mice

Seizures cause increased release of excitatory glutamic acid,and excessive release of excitatory neurotransmitter glutamic acid will lead to lipid homeostasis imbalance,which is excitatory toxicity to nerve cells,and will further aggravate neuronal damage.Seizures activate phospholipases,and the consequent degradation of phospholipids(Phospholipid,PL)leads to the massive release of free fatty acids(Free fatty acid,FAA).After epileptic seizure,the self-defense system of neurons was damaged,and the endosomal-autophagosomal-lysosomal pathway was activated,with the increase of membranous components.A large number of substances could not be degraded in time in lysosomes,which eventually led to lysosome rupture,leading to neuronal death.At present,research on lipid injury after status epilepticus is relatively rare,especially the spatial and temporal changes of lipid changes during the development of neuronal death are not completely clear.The purpose of this experiment is to investigate the relationship between delayed neuronal death and lipid changes in the hippocampus of mice at different stages of epilepsy by detecting neuronal death and changes in phospholipids and free fatty acids.Methods:In this experiment,the seizure model was induced by kainic acid(Kainic acid,KA)injection to the right lateral cerebral ventricle in mice which were sacrificed at 2 h,8 h,16 h,1 d,3 d and 5 d.The level of the PLs was detected by a phospholipid quantitation kit;the level of FAAs was detected with a free fatty acid quantitation kit;neuron death was detected by Fluoro-Jade B staining;the changes of neuronal phospholipids in hippocampal CA3 region were detected by Luxol Fast Blue and Nile red staining;the changes of free cholesterol in hippocampal CA3 neurons were detected by filipin III staining.Results:1.Phospholipid quantitation showed that compared with the control group,the PLs of hippocampus in the experimental group began to decrease at 2h after KA injection,and the PLs continuously decreased and were significantly lower than those in the control at8 h and 16 h.PLs in the hippocampus were increased at days 1 and 3 and returned to the control level.At day 5,the PLs of hippocampus decreased again and was significantly lower than the control value.2.Free fatty acid quantitation showed that the FFAs in KA-injured hippocampus was significantly increased compared with the control group at 2 h,slightly increased at8 h,and reached the maximum level at 16 h.It then decreased at day 1,when the FFAs remained above the control level.By 3d,the FFAs in the hippocampus had decreased and returned to normal.At day 5,the FAAs again increased significantly.3.Fluoro-Jade B staining results showed that no Fluoro-Jade B positive cells were found in the hippocampus of the control group.A small number of positive cells appeared in CA3 region of mice in the experimental group 16 h after KA injection.On day 1 after KA injection,the number of Fluoro-Jade B positive cells in CA3 region increased further,and the neuronal degeneration was more significant.The positive cells could be continuously observed in hippocampal CA3 region on days 3 and 5 after KA injection.4.Luxol Fast Blue staining results showed that: in the control group,the cells in hippocampus were arranged regularly and no obvious abnormality was found.Hippocampal CA3 region was positively stained 16 h after KA injection compared to the control group.The number of positive cells in hippocampal CA3 was gradually increased on day 1 after KA injection.Luxol Fast Blue positive signals were persistently detected in hippocampal CA3 on days 3 and 5 after KA injection.5.Nile red staining showed that the neurons in hippocampal CA3 region of the control group were weakly stained.Nile red positive signals began to appear in the neuronal cytoplasm in hippocampal CA3 region at 16 h after KA injection,and Nile red positive signals in the neurons in hippocampal CA3 region could be continuously detected on days 1d,3d and 5d after KA injection.6.Filipin III staining results showed that the hippocampal neurons in the control group were lightly stained or not stained.Filipin III staining of neurons in hippocampal CA3 began to appear 16 h after KA injection and Filipin III-positive cells were continuously detected in the hippocampus on days 1d,3d and 5d after KA injection.Conclusions:1.After status epilepticus,the levels of phospholipids and free fatty acids in hippocampus of mice changed continuously,suggesting that the structure and function of neuronal cell membrane were seriously damaged.2.The death of hippocampal neurons is accompanied by a large number of intracellular membrane structures,suggesting that cell membrane damage may be one of the important mechanisms of neuron death.