A Study On The Alterations Of Electrophysiological Characters And Proliferative Function In Rat Hippocampal Astrocytes Following Oxygen And Glucose Deprivation

Part One The effections of Oxygen and Glucose Deprivation on the Electrophysiological Characters of Astrocytes in Rat HippocampusObjective:To study the effections of Oxygen and Glucose Deprivation on the electrophysiological characters of astrocytes in rat hippocampus.Methods:In this study,acute hippocampal slices were prepared from 23-day-old rats. we applied glucose and oxygen-deficient artificial cerebrospinal fluid(aCSF)(oxygen and glucose deprivation(OGD) solution) to acute rat hippocampus slices and examined astrocyte Vm in the physiological condition and OGD condition by patch-clamp recording.Result:In the physiological condition,mature astrocytes predominantly express linearⅠtoⅤconductance that can be evoked by a series of de-and hyperpolarization voltage steps in CA1 area.Such a linear whole-cellⅠtoⅤrelationship is predominately due to the expression of leak K+ channels,When the recording chamber was continually perfused with OGD solution for 30 mins,the astrocyte Vm showed a multiphasic,rather than linear,depolarization.This was characterized by an initial small-amplitude depolarization phase in the first 11min of OGD process.At the end of this phase,the Vm depolarization amounced to an average o(?)mV; followed by a 6min accelerating depolarizing period,and a second plateau.On OGD withdrawal,this depolarized Vm returned rapidly to the resting Vm level and was followed by a transient further 3.4±1.6mV hyperpolarization below the initial pre-OGD level.The mean astrocyte Vm response over 30 mins OGD under this nonperfusion condition showed similar response and a much stronger depolarization(P＜0.05).Conclusion:Mature astrocytes predominantly express linearⅠtoⅤconductance in CA1 area,and they have a low membrane potential and membrane resistance.The reversal potential of mature astrocytes is much closer to the equilibrium potential of K. This indicated K+ channels were involed in astrocytes conductance;Astrocytes are more resistant to OGD damage than neurons;OGD Induces a multiphase astrocyte membrane potential depolarization, Part Two The Underling mechanisms of the Electrophysiological Changes of Rat Hippocampal Astrocytes following Oxygen and Glucose DeprivationObjective:To investigate the basical mechanisms of the acute electrophysiological response of astrocytes in rat hippocampus following OGD.Methods:In this study,acute hippocampal slices were prepared from 23-day-old rats. we applied glucose and oxygen-deficient artificial cerebrospinal fluid(aCSF)(oxygen and glucose deprivation(OGD) solution) to acute rat hippocampus slices and examined astrocyte Vm in OGD condition and inhibitors interfered condition by patch-clamp recording.We added inhibitors for AMPA receptors(30 mmol/L NBQX), NMDA receptors(50 mmol/L D,L-AP5),glutamate transporters(100 mmol/L TBOA),P2X receptors antagonist(100 mmol/L suramins),GABAA receptor antagonist(10 mmol/L bicuculline) and gap junction blocker(100mmol/L MFA) to the OGD solution, then we observed the effections of astrocytic gap junction,ionotropic receptors and transporter on OGD induced astrocyte membrane potential changes;we added iodoacetate(2 mmol/L),antimycin(25 mmol/L)and rotenone(50 mmol/L) to the OGD solution to investigated the effections of glycolytic and mitochondrial oxidative phosphorylation on OGD induced astrocyte membrane potential changes.Result:These inhibitors did not significantly affect OGD-induced astrocyte Vm depolarization(P＞0.05);When glycolytic and mitochondrial ATP production was inhibited,astrocytes showed a rapid depolarization,We found that the glycolysis inhibitor iodoacetate(2 mmol/L) produced a similar Vm changes as the three inhibitors applied together for an 8-min OGD treatment.Conclusion:OGD induced astrocyte membrane potential changes were not effected by the gap junction,glutamate receptors/transporters and GABA receptors,this indicated extracellular K+ increase was primarily responsible for the astrocytes embrane potential multiphasic.changes following OGD;Glycolysis plays a preferential role in keeping the Vm and Na-K-ATP pump functions in the early period of OGD. Part Three Effects of ischemia and anoxia on cell activation and cell cycle of cultured astrocyte in vitro.Objective:To study the effects of ischemia and anoxia on cell activation and cell cycle of astrocyte in vitro.Methods:We measured the cell cycles and the proliferation of astrocyte in different time after ischemia and anoxia by flow cytometry and Brdu pulse labeling; and detected the expression of GFAP,PCNA and cyclinD1 with the fluorescence immunocytochemistry.Result:After ischemia and anoxia in vitro,S phase of astrocyte significantly increased compared with the normal,and the peak appeared at 6h;and the proliferated ability of astrocyte is highest at 6h by Brdu pulse labeling test,but the S phase and proliferated ability is decreased after 6h.At the early of ischemia and anoxia,the positive staining of GFAP increased;but after ischemia and anoxia 12 hours,the positive staining of GFAP is weak,The levels of PCNA increased after the damage,and reached the peak at 6 h.The expression of cyclinD1 is gradually increased after damaged,the peak is appeared at 24h.Conclusion:Astrocyte cells are activated to proliferate and enter into new cell cycle events by ischemia and anoxia;PCNA and cyclinD1 counsel the adjustment of astrocyte's proliferation and repairing;The cell cycle events are related with proliferation and activation of astrocyte closely.