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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.
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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 evaluate the role of μ-δ heterodimer in down-regulation of the expression of excitatory amino acid transporter 3 (EAAT3) in hippocampi caused by reinstatement of morphine-induced conditioned place preference (CPP) in rats.Methods Thirty-two healthy clean-grade male Sprague-Dawley rats,weighing 200-240 g,were assigned into 4 groups (n =8 each) using a random number table method:control group (group C),extinction group (group E),reinstatement group (group R) and reinstatement plus interference plasmid group (group RI).The model of morphine-induced CPP was established,and extinction of CPP was gradually induced by stopping administration.A small dose of morphine 5 mg/kg was intraperitoneally injected again to induce CPP reinstatement,and dwell time around the medicine box was recorded.μ-δ heterodimer interference plasmid 5 μl was injected into the lateral cerebral ventricle after successful establishment of CPP model in group RI.The content of glutamate (Glu) in hippocampi was measured using high-performance liquid chromatography.The EAAT3 expression in hippocampal CA1 and CA3 regions was detected using Western blot.Results Compared with group C,no significant change was found in the dwell time around the medicine box or content of Glu in hippocampi (P＞0.05),and the expression of EAAT3 in hippocampal CA1 and CA3 regions was significantly up-regulated in group E,and the dwell time around the medicine box was significantly prolonged,the content of Glu in hippocampi was increased (P＜0.05),and no significant change was found in the expression of EAAT3 in hippocampal CA1 and CA3 regions in group R (P＞0.05).Compared with group E,the dwell time around the medicine box was significantly prolonged,the content of Glu in hippocampi was increased,and the expression of EAAT3 in hippocampal CA1 and CA3 regions was down-regulated in group R (P＜0.05).Compared with group R,the dwell time around the medicine box was significantly shortened,the content of Glu in hippocampi was decreased,and the expression of EAAT3 in hippocampal CA 1 and CA3 regions was upregulated in group RI (P＜0.05).Conclusion μ-δ heterodimer is involved in down-regulation of EAAT3 expression in the hippocampus caused by reinstatement of morphine-induced CPP in rats.
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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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Objective To investigate the effect of phenolic alkaloids from Menispermum dauricum( PAMd)on the mRNA expression of the glutamate transporter EAAC1 in hippocampal neurons of rats subjected to focal cerebral ischemia and to elucidate its neuroprotective mechanisms. Methods A total of 42 Sprague Dawley(SD)rats were randomly divided into three groups:sham group,model control group,and PAMd(10 mg·kg-1 ,i.g)group(n = 14 each).The focal cerebral ischemia model of rat was induced by the middle cerebral artery occlusion( MCAO). The 2,3,5-triphenyltetrazolium chloride( TTC) staining was applied to measure the cerebral infarct size and the reverse transcriptase-polymerase chain reaction(RT-PCR)assay to detect EAAC1 mRNA expression in hippocampal neurons. Results After 24 h ischemia,the cerebral infarct volumes were (0.0±0.0)%,(35.3±2.9)% and(21.3±3.8)% in sham group,model control group and PAMd group,respectively(P<0.05). The relative expression levels of EAAC1 mRNA were 0.97±0.04,2.46±0.13,and 1.91±0.15 in the three groups,respectively (P<0. 05). Conclusion PAMd may protect against cerebral ischemia by up-regulating EAAC1 mRNA expression and alleviating the excitotoxicity of glutamic acid.
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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.
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BACKGROUND: Propofol (2, 6-diisopropylphenol) has been known to have neuroprotective effects. Excitatory amino acid transporter 4 (EAAT4) is a glutamate transporter predominantly expressed in the cerebellar Purkinje cells, which is vulnerable to ischemic injury. Thus, we hypothesized that propofol reverses reduced EAAT4 activity which was induced by oxidative stress and investigated the effects of propofol on EAAT4 under oxidative stress induced by tert-butyl hydroperoside (t-BHP). METHODS: EAAT4 was expressed in Xenopus oocytes by injection of its mRNA. By using two-electrode voltage clamping, membrane currents were recorded before, during, and after application of L-aspartate (3 microM) in the presence or absence of t-BHP and propofol. RESULTS: L-aspartate induced an inward current in EAAT4 expressing oocytes. Exposure of these oocytes to t-BHP (1-20 mM) for 10 min dose-dependently decreased EAAT4 activity (1 +/- 0.01 microC for control; 0.88 +/- 0.05 microC for 1 mM; 0.83 +/- 0.03 microC for 2mM; 0.65 +/- 0.04 microC for 3 mM; 0.51 +/- 0.07 microC for 5 mM; 0.45 +/- 0.03 f microC for 10 mM and 0.24 +/- 0.06 microC for 20 mM). IC50 for t-BTH was 6.05 mM and further study was performed with 10 mM t-BTH. Propofol (3-10 microM) dose-dependently reversed this t-BHP-attenuated EAAT4 activity. CONCLUSIONS: Oxidative stress by t-BHP decreased EAAT4 activity and 3-10 microM propofol restored oxidative stress-reduced EAAT4 activity.
Subject(s)
Amino Acid Transport System X-AG , Aspartic Acid , Constriction , Excitatory Amino Acid Transporter 4 , Glutamic Acid , Inhibitory Concentration 50 , Membranes , Neuroprotective Agents , Oocytes , Oxidative Stress , Propofol , Purkinje Cells , RNA, Messenger , XenopusABSTRACT
Objective To investigate the change in the expression of excitatory amino acid transporter 3 (EAAT3) in the spinal cord neurons in a rat model of chronic morphine tolerance. Methods Forty-five male SD rats were randomly divided into 5 groups ( n = 9 each) : group I sham operation (group S); group II normal saline (group NS); group Ⅰ morphine (group M); group Ⅳ ketamine (group K) and groupV M + K. In group II - V a catheter was placed in the subarachnoid space at L_(3-5) interspace. The animals were observed for 3 days. The animals with motor or sensory paralysis of the hindlimbs were excluded. NS 40 μl,morphine 20 μg, ketamine 30μg,morphine 20μg + ketamine 30μg were injected via intrathecal catheter twice a day for 7 consecutive days. 50% paw withdrawal threshold and latency (PWT, PWL) of the hindpaw to radiant heat were measured before (T_0, baseline) , on day 1, 3, 5, 7 of (T_(1-4)) and 1 day after (T_5 ) IT drug administration. The rats were sacrificed after last pain threshold measurement. The expression of EAAT3 protein in the spinal cord was determined by Western blotting and immuno-histochemistry. Results The sensitivity of the hindpaw to noxious heat stimulation was significantly decreased during (T_(1,2)) and increased after IT administration (T_(4,5)) in group M and was significantly decreased during and after FT administration (T_(1-5)) in group M + K as compared with the baseline values at T_0 and group S and was significant lower in group M + K than in group M. The expression of EAAT3 protein in the spinal cord was significantly decreased in group M and M + K as compared with group S and was significantly lower in group M than in group M + K. Conclusion The down-regulation of the expression of EAAT3 in the spinal dorsal horn neurons is involved in the development of chronic morphine tolerance and the expression of EAAT3 is down-regulated by morphine partly through the activation of NMDA receptor.
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Objective To explore the mechanism of early-onset sub-clinical seizures following acute cerebral infarction. Methods Right middle cerebral artery occlusion (MCAO) rats models were made by threads methods. Sub-clinical seizures were detected with recording apparatus at the same time. Glial glutamate transporter-1 (GLT-1) were detected by immunohistochemical method in rats hippocampus. Results Sub-clinical seizures occurred in 27.4% rats following MCAO. The GLT-1 expressions in CA_1 and CA_3 regions were lower in sub-clinical seizure group[(344.5?35.0)?m~2 and (360.4?13.5)?m~2 respectively] than those in the controls[(447.0?22.8)?m~2 in CA_1 region and (402.3?28.5)?m~2 in CA_3 region]. But there were not significant differences of GLT-1 immunoreactivities in dentate gyrus between the two groups above. Conclusion Early-onset sub-clinical seizures occur following the acute MCAO in rats, which should be related to the decreased expression of GLT-1 in CA_1 and CA_3 of hippocampus.