The Regulated Role Of KCC2-GABA_A Receptor Pathway In Propofol Post-conditioning Against Oxygen And Glucose Deprivation Injury In The Hippocampal Brain Slices

Objective:Cerebral ischemia-reperfudion injury in clinic can severely affect the quality of patients’life induced by neurosurgy, cardiac arrest and so on. Choosing the appropriate anesthetics which could preserve the compromised brain function for the patients with high risk of perioperative stroke is a major research endeavor for anesthetists. Propofol is commonly used in clinical intravenous anesthetic. It can reduce cerebral metabolic rate, intracranial pressure, and has anti-oxidation and inhibition of apoptosis, which can reduce cerebral ischemia-reperfusion injury. Our previous studies found that propofol-postconditioning induced neuroprotection through excitatory glutamate receptor AMPA receptor. In recent years, more and more scholars agree with ischemia-reperfusion injury is a excitotoxic injury, caused by the disturbance of balance between excitation and inhibition in CNS. GABAA receptor is the major inhibitory receptor in mature mammalian CNS and KCC2is the guarantee of inhibitory postsynaptic transmission mediated by GABAA receptor. Wether they are involved in the neuroprotection induced by propofol postconditioning is unclear. This study aimed to investigate in vitro hippocampal slices, whether propofol could protected brain slices from OGD injury, and to further explore KCC2-GABAA receptor pathway is involved, so as to provide a theoretical basis for the reasonable choice of anesthetic in clinical.Method:This study chose hippcampal brain slices to make OGD-reperfusion model in vitro to simulate the in vivo model of ischemia-reperfusion injury. The experiment included three parts. The first part, investigated whether the propofol postconditioning could protect the slices from OGD injury. Firstly, selected a suitable OGD time. Selected7min,10min,12min,15min as OGD duration, respectively denoted01-04group. All slices were incubated1h after OGD. Evoked action potentials (AP) was recorded with whole-cell patch-clampto evalued the effect of OGD on the excitability of pyramidal neurons in CA1region. Secondly, divided54slices into3groups (n=18)randomly:control group (group C) cultured in normal ACSF7min+lh; group OGD, transfered into normal ACSF1h after OGD7min; propofol-postconditioning group (group P), transfered into normal ACSF containing propofol (1.2μg/ml) for1h. The amplitude and frequencys of sEPSC and sIPSC was recorded with whole-cell patch clamp technique to explore the effect of propofol on CA1synaptic transmission after OGD. Compared the degree of injury between groups with propidium iodide staining. The second part, to investigate the role of GABAa receptor in the propofol postconditioning,72slices was randomly divivded into4groups(n=18):groupC, group OGD, group P, group P+Bic. Mininature inhibitory postsynaptic current (mIPSC), mediated by GABAa receptor was recorded; evaluated the mortality of CA1with PI staining; measured the changes of chloride ion concentration with MQAE used laser scanning confocal microscopy. The third part, to study the regulated role of KCC2in propofol postconditioning,144slices was randomly divivded into6groups(n=24):the amplitude and frequencys of mIPSC and chloride ion concentration was measured, the expression of KCC2was detected with western blots and RT-PCR.Result:Part Ⅰ. during exploring the duration of OGD, compared with group C, only the frequency of evoked action potentials in group O1reduced(P<0.05), group02and O3showed no significant difference (P>0.05); while compared with the group01, frequency of AP in group02and O3was higher (P<0.05), probably due to the changes of membrane potential caused by OGD very closed to the equilibrium potential of the sodium ion, which suggested more severely damage. Therefore we selected7min as OGD time. Compared with the group Con, the amplitude and frequency of sEPSC increased in group OGD and group Pro+OGD, while the amplitude and frequency of sIPSC decreases, the PI fluorescence intensity and the mortality increased (P<0.05); compared with group OGD, the amplitude and frequency of sEPSC reduced in group Pro+OGD, while the changes of sIPSC was just opposite and cell death relative decrease (P<0.05). Part II:Compared with group C, the amplitude and frequency of mIPSC of the remaining three groups reduced, cell death and intracellular chloride concentration increased (P<0.05); compared with the group OGD, the amplitude and frequency of mIPSC of group P increased, cell death and intracellular chloride ion concentration decreased (P<0.05); group P+Bic showed no statistically significant compared with group OGD (P>0.05).Part III:compared with group C, the amplitude and frequency of mIPSC decreased in group OGD and group P, while the intracellular chloride concentration was higher and KCC2expression was lower (P<0.05); compared with group OGD, the amplitude and frequency of mIPSC in group P increases, the intracellular chloride concentration decreased and KCC2expression levels up-regulated (P<0.05). Given NEM to each group, the trend remained.Conclusion:Propofol postconditioning could protect the hippocampal brain slices from oxygen-glucose deprivation injury. The mechanism may be the up-regulated expression of KCC2and maintaining the inbibiyory nature of the GABAA receptor...