The Changes Of Microenvironment After Mg²⁺-free-induced Seizures-like Discharge In Neurons In Vitro

Objective To study the changes of brain-derived neurotrophic factor（BDNF），nerve growth facto（rNGF），glutamate（GLU），interleukin-1beta （IL-1β） and lactic dehydrogenase （LDH） levels in themicroenvironment after Mg²⁺-free-induced seizures and analyze thesources of BDNF.Methods Fetal rat hippocampal neurons （N）and neonatal ratastrocytes （AST） were purified, then divided into four groups：control Ngroup，Mg²⁺-free treated N group，control N+AST co-culture group andMg²⁺-free treated N+AST co-culture group.The Mg²⁺-free treated groupswere exposed to Mg²⁺-free media for3h to induce the repeatedspontaneous seizures-like discharge, and control groups were culturednormally. The levels of BDNF，NGF and IL-1β in each group at differenttime points were analyzed by enzyme linked immunosorbent assay（ELISA），and the levels of IL-1β in each group at different time points were analyzed by high performance liquid chromatography（HPLC），thenthe concentration of LDH at72h were detected by autobiochemistryinstrument.Result （1）Under the microscope, the size and cellular processes ofAST in Mg²⁺-free treated N+AST co-culture group increased sharply after48h.（2）After treated with Mg²⁺-free media for3h, the neuronalepileptiform activity was observed, which continued to exist until theculture fluid returned to be normal for72h （.3）The BDNF levels increasedat24h and48h in control N+AST co-culture group contrast to control Ngroup （p﹤0.05）.Compared with control N+AST co-culture group, BDNFlevels increased at12h,24h and48h in Mg²⁺-free treated N+ASTco-culture group, especially higher at12h and24h （p﹤0.01）. There wereno significant differences in BDNF levels between control N group andMg²⁺-free treated N group.Compared with Mg²⁺-free treated N group,BDNF levels increased at24h in Mg²⁺-free treated N+AST co-culturegroup （p﹤0.05）（.4）No significant differences on NGF levels of each timepoints were found between Mg²⁺-free treated N+AST co-culture group andcontrol N+AST co-culture group.（5）The GLU levels increased begin6hin Mg²⁺-free treated N+AST co-culture group after treated with Mg²⁺-freemedia， and there were significant differences in GLU concentrationbetween control N+AST group and Mg²⁺-free treated N+AST group at48h and72h（p﹤0.05）（.6）Compared with control N+AST co-culture group, the IL-1β levels increased significant at12h、24h and48h in Mg²⁺-freetreated N+AST group（p﹤0.05，p﹤0.01）.（7）The concentration of LDHincreased significantly at72h in Mg²⁺-free treated N+AST co-culturegroup contrasts to control N+AST co-culture group （p﹤0.01）.Conclusion （1）The experimental evidence suggested that theincreasing BDNF in control N+AST co-culture group might come fromboth N and AST，but chiefly from N. And the activated AST may be themain source of significantly increasing BDNF in Mg²⁺-free treated N+ASTco-culture group.（2） It is no significant effect on NGF in themicroenvironment of the N+AST co-culture system afterMg²⁺-free-induced seizures-like discharge.（3）These results explained thatthe GLU and IL-1β levels increased significant in N+AST co-culture groupafter treated with Mg²⁺-free-induced seizures, but there were nodependence between concentration of GLU and IL-1β.（4）The data ofLDH showed that neurons were damaged in the microenvironment afterMg²⁺-free-induced seizures.