米诺环素对新生鼠缺氧后脑室周围区域谷氨酸清除的影响
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摘要
目的:探讨米诺环素对新生大鼠缺氧后脑室周围区域谷氨酸的作用及其可能的机制。方法:对出生后1 d的大鼠给予系统性缺氧构建缺血缺氧性脑损伤模型(hypoxic-ischemic brain damage,HIBD)。采用液相色谱-串联质谱联用技术检测缺氧后4 h和1 d脑室周围区域谷氨酸水平;Western blot观察该区域EAAT1和EAAT2发育期变化以及EAAT1、EAAT2、Iba-1、IL-1β、TNF-α和TGF-β1蛋白在缺氧后4 h和1 d的动态变化,并同时观察给予米诺环素后对谷氨酸水平及上述蛋白的影响。结果:谷氨酸水平检测发现,缺氧后4 h和1 d,脑室周围区域谷氨酸水平明显上升;给予米诺环素(45 mg/kg)后,谷氨酸水平显著下降。Western blot结果显示,出生后第1周EAAT1和EAAT2低表达,但在出生后第2周表达显著上升;缺氧后1 d,EAAT1、EAAT2、Iba-1、IL-1β和TGF-β1表达上调;给予米诺环素后能促进EAAT1和TNF-α的表达,但却抑制EAAT2的产生。结论:缺氧后早期,米诺环素能降低脑室周围区域谷氨酸水平,其机制可能与它选择性调控谷氨酸转运体,而非抑制该区域炎症反应有关。
AIM: To investigate the role of minocycline on glutamate uptake in the periventricular zone and its putative mechanism after hypoxic exposure in neonatal rats. METHODS: A model of hypoxic-ischemic brain damage( HIBD) was developed by putting postnatal 1 d rat pups in 5% O2 for 3. 5 h. The glutamate level in periventricular zone was measured by liquid chromatography coupled with tandem mass spectrometry assay( LC-MS / MS) after hypoxic exposure for 4 h and 1 d. The dynamic changes of glutamate transporters EAAT1,and EAAT2 during developmental period in periventricular zone were determined by Western blot. Moreover,the expression of EAAT1,EAAT2,Iba-1,IL-1β,TNF-α and TGF-β1 was also detected by Western blot after hypoxic exposure for 4 h and 1 d in that region. The effects of minocycline on all parameters mentioned above were tested after minocycline treatment at the same time points and in the same region. RESULTS: After hypoxic exposure,glutamate level was increased,but it was decreased after minocycline treatment. EAAT1 and EAAT2 kept a low expression level at the first postnatal week,but a predominant elevation was found at the end of the second postnatal week. The expression of EAAT1,EAAT2,Iba-1,IL-1β and TGF-β1 was increased at 1 d after hypoxic exposure. EAAT1 and TNF-α expression was significantly up-regulated,while EAAT2 was down-regulated after minocycline treatment. CONCLUSION: Minocycline inhibits the increase in the glutamate level after hypoxia in periventricular region of the neonatal rats. The mechanism may relate to the selective regulation of glutamate transporters,rather than the inhibition of neuroinflammation in periventricular zone.
AIM: To investigate the role of minocycline on glutamate uptake in the periventricular zone and its putative mechanism after hypoxic exposure in neonatal rats. METHODS: A model of hypoxic-ischemic brain damage( HIBD) was developed by putting postnatal 1 d rat pups in 5% O2 for 3. 5 h. The glutamate level in periventricular zone was measured by liquid chromatography coupled with tandem mass spectrometry assay( LC-MS / MS) after hypoxic exposure for 4 h and 1 d. The dynamic changes of glutamate transporters EAAT1,and EAAT2 during developmental period in periventricular zone were determined by Western blot. Moreover,the expression of EAAT1,EAAT2,Iba-1,IL-1β,TNF-α and TGF-β1 was also detected by Western blot after hypoxic exposure for 4 h and 1 d in that region. The effects of minocycline on all parameters mentioned above were tested after minocycline treatment at the same time points and in the same region. RESULTS: After hypoxic exposure,glutamate level was increased,but it was decreased after minocycline treatment. EAAT1 and EAAT2 kept a low expression level at the first postnatal week,but a predominant elevation was found at the end of the second postnatal week. The expression of EAAT1,EAAT2,Iba-1,IL-1β and TGF-β1 was increased at 1 d after hypoxic exposure. EAAT1 and TNF-α expression was significantly up-regulated,while EAAT2 was down-regulated after minocycline treatment. CONCLUSION: Minocycline inhibits the increase in the glutamate level after hypoxia in periventricular region of the neonatal rats. The mechanism may relate to the selective regulation of glutamate transporters,rather than the inhibition of neuroinflammation in periventricular zone.
引文
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[12]Wixey JA,Reinebrant HE,Buller KM.Inhibition of neuroinflammation prevents injury to the serotonergic network after hypoxia-ischemia in the immature rat brain[J].J Neuropathol Exp Neurol,2011,70(1):23-35.
[13]Hou G,Yang X,Yuan TF.Hippocampal asymmetry:differences in structures and functions[J].Neurochem Res,2013,38(3):453-460.
[14]Lai AY,Dhami KS,Dibal CD,et al.Neonatal rat microglia derived from different brain regions have distinct activation responses[J].Neuron Glia Biol,2011,7(1):5-16.
[15]Butovsky O,Jedrychowski MP,Moore CS,et al.Identification of a unique TGF-beta-dependent molecular and functional signature in microglia[J].Nature Neurosci,2014,17(1):131-143.
[2]Loeliger M,Watson CS,Reynolds JD,et al.Extracellular glutamate levels and neuropathology in cerebral white matter following repeated umbilical cord occlusion in the near term fetal sheep[J].Neuroscience,2003,116(3):705-714.
[3]Olmos G,LladóJ.Tumor necrosis factor alpha:a link between neuroinflammation and excitotoxicity[J].Mediators Inflamm,2014,2014:861231.
[4]Boycott HE,Wilkinson JA,Boyle JP,et al.Differential involvement of TNF alpha in hypoxic suppression of astrocyte glutamate transporters[J].Glia,2008,56(9):998-1004.
[5]Kaur C,Sivakumar V,Zou Z,et al.Microglia-derived proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1beta induce Purkinje neuronal apoptosis via their receptors in hypoxic neonatal rat brain[J].Brain Struct Funct,2014,219(1):151-170.
[6]Wixey JA,Reinebrant HE,Spencer SJ,et al.Efficacy of post-insult minocycline administration to alter long-term hypoxia-ischemia-induced damage to the serotonergic system in the immature rat brain[J].Neuroscience,2011,182:184-192.
[7]Regan MR,Huang YH,Kim YS,et al.Variations in promoter activity reveal a differential expression and physiology of glutamate transporters by glia in the developing and mature CNS[J].J Neurosci,2007,27(25):6607-6619.
[8]Nijboer CH,Heijnen CJ,Degos V,et al.Astrocyte GRK2 as a novel regulator of glutamate transport and brain damage[J].Neurobiol Dis,2013,54:206-215.
[9]Back SA,Rosenberg PA.Pathophysiology of glia in perinatal white matter injury[J].Glia,2014,62(11):1790-1815.
[10]Deng W,Rosenberg PA,Volpe JJ,et al.Calcium-permeable AMPA/kainate receptors mediate toxicity and preconditioning by oxygen-glucose deprivation in oligodendrocyte precursors[J].Proc Natl Acad Sci U S A,2003,100(11):6801-6806.
[11]Colovic M,Caccia S.Liquid chromatographic determination of minocycline in brain-to-plasma distribution studies in the rat[J].J Chromatogr B Analyt Technol Biomed Life Sci,2003,791(1-2):337-343.
[12]Wixey JA,Reinebrant HE,Buller KM.Inhibition of neuroinflammation prevents injury to the serotonergic network after hypoxia-ischemia in the immature rat brain[J].J Neuropathol Exp Neurol,2011,70(1):23-35.
[13]Hou G,Yang X,Yuan TF.Hippocampal asymmetry:differences in structures and functions[J].Neurochem Res,2013,38(3):453-460.
[14]Lai AY,Dhami KS,Dibal CD,et al.Neonatal rat microglia derived from different brain regions have distinct activation responses[J].Neuron Glia Biol,2011,7(1):5-16.
[15]Butovsky O,Jedrychowski MP,Moore CS,et al.Identification of a unique TGF-beta-dependent molecular and functional signature in microglia[J].Nature Neurosci,2014,17(1):131-143.