Roles Of Ca-A/K Channel In Autophagic Death Of Hippocampus In Newborn Rats With Hypoxic-ischemic Brain Damage

Objective: Through the establishment of hypoxic-ischemic brain injury(Hypoxic-ischemic brain damage, HIBD) model, to study autophagy and the expression ofCa-A/K channel subunit in hippocampal neurons. Then, by applying autophagy inhibitor3-methyladenine (3-MA) and autophagy activator Rapamycin to inhibit or activateautophagy, explore the relationship between autophagy and Ca-A/K channel in HIBDhippocampal neurons.Method: The HIBD model was established by using SD rats (7postnatal days) andaccording to the modified Rice-Vannucci method. Rats were randomly divided into fourgroups (Sham group, HIBD group,3-MA group, and RAPA group), then were subdividedinto seven subgroups after0h,1h,3h,6h,12h,24h and48h after HIBD operation. HEstaining was used in every group to understand the cellular level injury of hippocampus;The mRNA expressions of Beclin-1, LC3, GluR1and GluR2were detected by RT-qPCR,while we detected the concentration of Ca-A/K channel subunits (GluR1, GluR2) inprotein level by Western Blot assay. Finally, the position and quantity of LC3, GluR1andGluR2were assessed by immunohistochemistry in cellular level.Results：1. HE staining showed that the left hippocampal neurons in Sham group wereconical or round,3-4cell layers, dense and orderly, centered transparent nucleus.We could see only a small amount of neuronal degeneration or apoptosis. In HIBDgroup, cell structure were incomplete, disorganized, cell swelling and ruptured,vague and ill-defined contours, significant loss, while cell space in hippocampuswere loose and increased. Injuries of hippocampal cell were lighter in3-MA groupcompared with HIBD group, while neuronal apoptosis, necrosis and cell loss inthe big scope were researched in RAPAgroup.2. RT-qPCR showed that GluR2was in low expression while GluR1, Beclin-1andLC3all showed high levels of expression in HIBD group. As for the time point, the expression level of GluR2significantly decreased at6h after HIBD (P<0.05),and reached the lowest level in24h; the expression of GluR1, Beclin-1and LC3were significantly increased at6h, and were still elevated at48h. Compared withHIBD group, the expression of LC3, Beclin-1and GluR1were reduced in3-MAgroup,24h was the lowest, continued until at least48h (P <0.05); while in RAPAgroup, the expression of LC3, Beclin-1, GluR1were peaked at24h, remained at ahigh level in48h (P <0.05).3. Western Blot showed that the expression of GluR2was low while GluR1was inhigh levels in HIBD group. As for the time point, the expression level of GluR2significantly decreased at6h after HIBD (P<0.05), and reached the lowest level in24h; the expression of GluR1expression were significantly increased at6h, andwere still elevated at48h.4. Immunohistochemical staining showed that the left hippocampal neurons in eachgroup expressed LC3, GluR1and GluR2. The expression of LC3was mainlylocated in the cytoplasm, whereas GluR1and GluR2were mainly in cellmembrane. Compared with Sham group, the expression level of LC3, GluR1wereat a high level, while GluR2was in a low level in HIBD group. Compared withHIBD group, the expression of LC3, GluR1in hippocampal neurons were reducedin3-MA group, while GluR2actually increased. RAPA group was in oppositeresult.Conclusions：1. The hippocampal cells were damaged in a short term after HIBD, the damageappeared askaryolysis and karyorrhexis, parts of the cells had blurred boundary.Apoptosis, autophagy and necrosis were involved and the three processes haddynamic changes in this model.2. The expressions of autophagy marker molecular LC3, Beclin-1and Ca-A/Kchannel subunit GluR1, GluR2were changed in HIBD.3. The left hippocampal neurons in each group expressed LC3, GluR1and GluR2,but the location and quantity had differences.4.3-MA, rapamycin resulted in the changes of autophagy and Ca-A/K channel structure, suggested that Ca-A/K channel might involved in autophagy.