Résumé
Objective To evaluate the role of excitatory amino acid transporter 2 ( EAAT2) in can-nabinoid receptor 2 ( CB2 receptor) activation-induced attenuation of microglial injury caused by glutamate. Methods N9 microglial cells were divided into 4 groups ( n=26 each) using a random number table meth-od: control group ( group Con) , glutamate group ( group Glu) , CB2 receptor agonist AM1241 plus gluta-mate group (group AM1241+Glu) and AM1241 plus EAAT inhibitor TBOA plus glutamate group (group AM1241+TBOA+Glu) . The cells were routinely cultured for 30 h in group Con. In group Glu, the cells were routinely cultured for 6 h, and then were incubated for 24 h in the culture medium containing gluta-mate 10 mmol∕L. In group AM1241+Glu, the cells were incubated for 4 h in the culture medium containing AM12412 μmol∕L, and then were routinely cultured for 2 h, and then were incubated for 24 h in the cul-ture medium containing glutamate 10 mmol∕L. In group AM1241+TBOA+Glu, the cells were incubated for 4 h in the culture medium containing AM12412 μmol∕L and TBOA 100 μmol∕L, and then were routinely cultured for 2 h, and then were incubated for 24 h in the culture medium containing glutamate 10 mmol∕L. The cell viability was measured by MTT assay, the activity of lactic dehydrogenase ( LDH) in supernatant was determined using colorimetric method, and the expression of EAAT2 was determined by Western blot. Results Compared with group Con, the cell viability was significantly decreased and LDH activity was in-creased in Glu, AM1241+Glu and AM1241+TBOA+Glu groups, and the expression of EAAT2 was signifi-cantly up-regulated in Glu and AM1241+Glu groups ( P<0. 05) . Compared with group Glu, the cell viabil-ity was significantly increased, LDH activity was decreased, and the expression of EAAT2 was up-regulated in group AM1241+Glu ( P<0. 05) , and no significant change was found in the parameters mentioned above in group AM1241+TBOA+Glu ( P>0. 05) . Compared with group AM1241+Glu, the cell viability was sig-nificantly decreased, LDH activity was increased, and the expression of EAAT2 was down-regulated in group AM1241+TBOA+Glu ( P<0. 05) . Conclusion The mechanism by which the activation of CB2 re-ceptor attenuates microglial injury caused by glutamate is related to up-regulating the expression of EAAT2.
Résumé
In the central nervous system, gap junctions exist between neurons and glial cells. Among them, connexin 43 (Cx43) is one of the most abundant connexin proteins in the central nervous system,involved in the metabolic coupling of intercellular substance exchange and electrical coupling of electrical signaling. It plays an important role in regulating cell metabolism, homeostasis, and cell differentiation. After cerebral ischemia, the uncoupling of gap junctions and abnormal hemichannel activity cause a steady-state imbalance of the internal and external environment of the cells, eventually leading to brain tissue damage.Therefore, maintaining the normal function of Cx43 is essential for protecting brain tissue from neuronal damage induced by cerebral ischemia-reperfusion.
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Objective To investigate the effect of rhynchophylline on mRNA expression of excitatory amino acid transporter 2 (EAAT2 ) and N-methyl-D-aspartic acid receptor 2B (NR2B) after astrocyte oxygen-glucose deprivation. Methods The subcultured third generation astrocytes from the hippocampus were inoculated into 6-well plates, and they were divided into blank control group, hypoxia-ischemia group,low-dose rhynchophylline group (0. 02 mg/ml) and high-dose rhynchophylline group (0. 2 mg/ml) after the cells were attached to the wall and grew out protrusion. The total RNAs in each group were extracted.Real-time fluorescence quantitative polymerase chain reaction was used to detect the expression levels of EAAT2 and NR2B mRNA in astrocytes of each group. Results Compared with the blank control group, the expression levels of NR2B and EAAT2 mRNA in astrocytes of the ischemia-hypoxia group were significantly higher (all P < 0. 05 ). The expression levels of NR2B and EAAT2 mRNA in the low-dose rhynchophylline group were lower than those in ischemia-hypoxia group, but there was no significant difference. The expression levels of NR2B and EAAT2 mRNA in the high-dose rhynchophylline group were significantly lower than the ischemia-hypoxia group and the low-dose rhynchophylline group (all P < 0. 05).Conclusion The expression of EAAT2 and NR2B mRNA in astrocytes of hippocampus cultured in vitro was significantly increased after ischemia and hypoxia, and rhynchophylline intervention could significantly reduce its expression in a concentration dependent manner.
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OBJECTIVE: To investigate the effects of prolonged exposure to isoflurane on the expression of glial fibrillary acidic protein (GFAP) and excitatory amino acid transporter-2 (EAAT2) in cortex of frontal lobe and hippocampus of neonatal rats. METHODS: Forty Wistar rats at postnatal day 7 were randomly divided into isoflurane group and control group according to the random number table method (n=20). Isoflurane group were exposed to 1.1% isoflurane (equivalent to 0.5 MAC for neonatal rats) for 6 h, the others were exposed to the gas mixture of 30% of the oxygen and nitrogen for 6 h in the control group. Five neonatal rats were sacrificed 12, 24 h, 3 and 7 d after exposure in each group. The brain were frozen and sliced, brain sections were double-stained with GFAP and EAAT2 markers. The fluorescence intensity of GFAP and EAAT2 double-labeled immunofluorescence was quantified in cortex of frontal lobe and hippocampus at 12, 24 h, 3 and 7 d after exposure by the Image J programme. RESULTS: Compared with the control group, the immunofluorescence intensity of GFAP in cortex of frontal lobe after exposure 12, 24 h in isoflurane group was significantly decreased(P < 0.01), whereas there was no significant difference after exposure 3, 7 d. The immunofluorescence intensity of GFAP in hippocampus after exposure 12, 24 h and 3 d in isoflurane group decreased compared with control group(P < 0.01), except 7 d after exposure. Double-labeled immunofluorescence showed lowered expression of GFAP and EAAT2 co-stained region in cortex of frontal lobe and hippocampus at 12, 24 h, 3 and 7 d after exposure in isoflurane group when compared with control group(P < 0.01). CONCLUSION: The 1.1% isoflurane prolonged exposure transiently reduces the expression of GFAP in the cortex and hippocampus, and delays the development of cytoskeleton. Whereas inhibiting the expression EAAT2 of astrocytes is prolonged, that may be one of the mechanisms for isoflurane-induced neurotoxicity.
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Glutamate is an essential excitatory neurotransmitter which regulates brain functions.An increase in extracellular glutamate could excessively activate ionotropic glutamate receptors,initiate calcium overload,and lead to cell death after cerebral ischemia.Glutamate transporter-1 (GLT-1) is one of the major glutamate transporters expressed predominantly in astrocytes.Astrocytes also express the enzyme glutamine synthetase (GS) which converts the glutamate to glutamine; the latter is then 'recycled' into neurons.Pretreatment with ceftriaxone (CEF),ischemia and intermittent hypobaric hypoxia could lead to neuroprotection by increasing the expression of GLT-1 and regulating the activity of glutamate transporter in brain.