TNF-α Regulate EAAT2Expression On Astrocyte And Its Effect On Oxygen-Glucose Deprivated Neuron Survival

BackgroundAcute ischemic stroke is a serious disease with high incidence and high mortality, which is harmful to human health. Inflammation and excitatoxic effect caused by gluamate were considered as the possible underlying mechanisms. In physiological condition, glutamate is mainly removed by excitatory amino acid transporter (EAAT), which terminate the neuron communication by reuptaking extracellular glutamate into cell. There five different types of EAAT (from EAAT1to EAAT5) were found in brain. EAAT2is mainly expressed on the astrocyte. It was considered that EAAT2removed the most of extracellular glutamate. In ischemic stroke, it was found that EAAT2was significantly changed, causing glutamate accumulation in extracellular interstice and excitatoxity. So, It is impotant to understand what factors affect the EAAT2expression on astrocyte after ischemic stroke. And it is possible benefit to ischemic neuron survival after inhibiting these factors. Astrocyte and microglial will release many cytokines after ischemic stroke. Tumor necrosis factor (TNF-a) is the main one of these factors, which was found significantly increase after stroke. Some literature reported that TNF-a may be involved in the downregulation of EAAT2expression. However, other studys suggest that TNF-a can promote the functions of EAAT2. The reason for such different opinion may be due to the interference of reactive gliosis after stroke and complicated temporal and spatial characteristics of TNF-a. So, we investigate the effect of TNF-a with different concentrations and exposed time on EAAT2expression, and GFAP expression, a specific marker of reactive gliosis.ObjectivesWe aim to investigate the effects of TNF-a concentration and exposed time on EAAT2expression in astrocyte treated by OGD, and identify whether TNF-a has some possible protective effect against excitatoxicity.Methods(1) Pure primary cultures of astrocytes were obtained by cortical tissue of newborn SD rats. Pure primary cultures of neurons were obtained by hippocampus of fetal SD rats. Astrocyte and neuron were co-cultured by a method that putting cover-slip with neuronsupside down on the astrocyte layer.(2) Astrocytes are incubated by exogenous TNF-a with different concentration and exposed time.(3) Mixed cultured cells were treated by oxygen glucose deprivation (OGD) for2hours. After reperfusion for24hours, cells are harvested.(4) Neuron suvival were detected and assessed by MTT assay.(5) The expression of EAAT2and GFAP were detected using Western Blot technique.Results(1) In the role of low concentration of TNF-a (≤10ng/ml), the EAAT2protein in astrocytes gradually increased. At the concentration of10ng/ml, the role of increasing expression was most significant. However, in the role of high concentration of TNF-a(>20ng/ml), EAAT2protein expressin decreased gradually. While GFAP expression in various concentrations of TNF-a have no significant difference. These results suggested low concentration of TNF-a can promote the expression of EAAT2, which is not attributed to the proliferation of glial cells.(2) Incubationing astrocytes of TNF-a within24h, EAAT2protein expression gradually incresaed. Which was significantly at8h,12h,24h of TNF-a administration. But with longer incubation of TNF-a (≥24h), EAAT2expression gradually decreased. At the same time, GFAP expression in various time under TNF-a administration have no significant difference. These results suggested short time incubation of TNF-a can promote the expression of EAAT2, while a long period incubation of TNF-a will inhibit the expression of EAAT2. Which is not attributed to the proliferation of glial cells.(3) OGD severly damage neurons. Short-time incubation of astrocytes by low concentrations of TNF-a and a method that putting cover-slip with neuronsupside down on the astrocyte layer can enhance the resistance of neurons to OGD injury. But the point time of the protective effect didn’t match the time of the enhancement of EAAT2expression.(4) In OGD condition, after short-time incubation of different concentration of TNF-a, EAAT2expression level was mildly elevated. And the degree of increasing was less significant than under the conditions of non-OGD. At the same time, the increasing of EAAT2expression was along with the increasing expression of GFAP. These result suggested that in OGD conditions, TNF-a can not effectively increase the expression of EAAT2. The enhancement of resistance of neurons to OGD injury by TNF-a may not be entirely attributed to its EAAT2regulation..ConclusionTNF-a with low concentration (<10ng/ml)in early phase (<24h) can promote the EAAT2expression on astrocyte. While TNF-a with high concentration in late phase will attenuate the EAAT2expression on astrocyte. This effect was not due to the gliosis response. But the dual effect of TNF-a on EAAT2expression was significantly inhibited by OGD treatment. Therefore, the effect of TNF-a on neuron survival after OGD might be not due to the expression of EAAT2.