Damage To The Plasma Membrane And Changes In The Endosomal-autophagosomal-lysosomal System In Hippocampal Neurons After Status Epilepticus

Seizures lead to delayed neuronal death through complex mechanisms,including oxidative stress and mitochondrial damage.As a self-defense system in neurons,the endosomal-autophagosomal-lysosomal system becomes activated in early SE to resist injury,and the membrane organelles increase.When the injury is aggravated,endosomes,autophagosomes and lysosomes rupture,which further promotes neuronal death.With the rupture of membranous organelles,phospholipids and cholesterol are released from the membrane,leading to changes in the membranous composition of the cytoplasm.There have been fewer studies on changes in the endosomalautophagosomal-lysosomal system of injured neurons after SE.In addition,the change of phospholipids and cholesterol in injured neurons also requires future studies.In this experiment,mice under anesthesia were injected with kainic acid to induce neuronal death in the hippocampus CA3 region.This study aimed to elucidate the relationship between delayed neuronal death and plasma membrane changes,providing new clues to understand epilepsy-induced delayed neuronal death.Methods:1.Dead neurons in the hippocampal CA3 region after SE was detected by FluoroJade B and propidium iodide(PI)tissue staining.2.Nuclei containing fragmented DNA was shown by TUNEL staining in the hippocampal CA3 of the post-SE.3.The changes of neurons,microglia and astrocytes in the hippocampal CA3 after SE were detected by Neu N,Iba1 and GFAP immunofluorescence.4.Temporal and spatial changes of early endosomes,autophagosomes and lysosomes of the hippocampal CA3 neurons after SE were detected by Rab5,LC3A/B and LAMP1 immunofluorescence.5.Phospholipid and cholesterol in the hippocampal CA3 neurons after SE were detected by Nile red and filipin III staining,respectively.Results:1.Fluoro-Jade B positive cells began to appear in the hippocampal CA3 region on Day 1 after SE compared with the control group.The number of Fluoro-Jade B positive cells reached a maximum on Day 3 after SE.Positive neurons could be observed until Day 7 after SE.Compared with the control group,PI staining of neuronal cytoplasm in the hippocampal CA3 region was weakened on Day 1 after SE,and then gradually decreased from Days 2 to 7 d after SE.2.Compared with the control group,Neu N immunopositivity in the hippocampal CA3 region gradually decreased from Days 1 to 7 after SE.On Day 7 after SE,the Neu N immunopositivity of neurons in the hippocampal CA3 region almost disappeared.Compared with the control group,the number of Iba 1-positive and GFAP-positive cells gradually increased from Days 1 to 7 after SE.3.TUNEL-positive signals appeared in the hippocampal CA3 region on Day 1after SE compared with the control group.On Day 2 after SE,some of TUNEL-positive signals in the hippocampal CA3 region began to diminish.On Day 7 after SE,the TUNEL-positive signals further reduced in the hippocampal CA3 region.4.A small number of Rab5-positive granules were present in the cytoplasm of hippocampal CA3 neurons in the control group.On Day 1 after SE,the number of Rab5-positive granules in the hippocampal CA3 neurons increased compared with the control group.On Days 2 and 3 after SE,Rab5-positive structures showed diffuse distribution in the hippocampal CA3 region.On Days 5 and 7 after SE,the positivity of Rab5 was reduced in the hippocampal CA3 region.5.A small amount of LC3A/B-positive particles were present in the cytoplasm of neurons in the control group.On Day 1 after SE,the number of LC3A/B-positive particles in the hippocampal CA3 region increased.On Day 2 after SE,the LC3A/B positivity in neurons of the hippocampal CA3 region was diffusely distributed.On Day3 after SE,the LC3A/B positivity showed patchy distribution in the hippocampal CA3 neurons.6.LAMP1-positive particles were uniformly distributed in the cytoplasm of hippocampal CA3 neurons in the control group.On Day 1 after SE,the LAMP1-positivity began to be diffusely distributed in the hippocampal CA3 neurons.On Day 2after SE,the distribution of LAMP1-positivity in the hippocampal CA3 neurons was more diffuse.On Day 3 after SE,the LAMP1-positivity in the hippocampal CA3 neurons decreased.7.Nile red and filipin III staining showed light staining in the cytoplasm of hippocampal CA3 neurons in the control group.Compared with the control group,Nile red and filipin III staining of neurons in the hippocampal CA3 region were increased on Day 1 after SE.The positive neurons were observed up to Day 7 after SE.Conclusions:1.Rab5,LC3A/B,and LAMP1 increased and then decreased during SE,suggesting that the endosomal-autophagosomal-lysosome system was activated during early SE.2.Delayed neuronal death was closely associated with lipid changes,and there was an increase in phospholipid and cholesterol components in dead neurons.3.PI tissue staining can label the hippocampal CA3 dead neurons in the early postSE.In summary,this study find that SE not only activated the endosomalautophagosomal-lysosomal system during the early post-SE but increased phospholipids and cholesterol levels in the cytoplasm of dead neurons in the hippocampal CA3 region.Exploring these phenomena may be helpful to clarify the mechanism of SE-induced delayed neuronal death,which will provide new clues for the treatment of epilepsy.