De Alok, Krueger James M, Simasko Steven M
Program in Neuroscience, Department of VCAPP, College of Veterinary Medicine, Washington State University, 205 Wegner Hall, Pullman, WA 99164-6520, USA.
Brain Res. 2005 Aug 16;1053(1-2):54-61. doi: 10.1016/j.brainres.2005.06.044.
Tumor necrosis factor-alpha (TNFalpha) affects several CNS functions such as regulation of sleep, body temperature, and feeding during pathology. There is also evidence for TNFalpha involvement in physiological sleep regulation, e.g., TNFalpha induces sleep and brain levels of TNFalpha increase during prolonged wakefulness. The immediate cause of enhanced TNFalpha production in brain is unknown. We investigated whether glutamate could signal TNFalpha production because glutamate is a neurotransmitter associated with cell activation and wakefulness. We used primary cultures of fetal rat hypothalamic cells to examine the expression and release of TNFalpha. Immunostaining for neuron specific enolase revealed that the cultures were 50-60% neuronal and 40-50% non-neuronal cells. TNFalpha was detected in both the media and cells under basal conditions. Stimulation of the cells with 1 mM glutamate for 2 h produced an increase in media content of TNFalpha, whereas cell content was elevated at earlier time points. Using trypan blue exclusion and MTT assays, there was no evidence of cell toxicity with this stimulation protocol. Immunocytochemical staining revealed that TNFalpha was expressed by approximately 25% of the neurons and approximately 75% of the glial cell in the culture. Stimulation of the cultures with glutamate did not increase the percentage of cells expressing TNFalpha. We conclude that TNFalpha is constitutively expressed and released by healthy cultures of hypothalamic cells and that activation of the cells with a non-toxic challenge of glutamate increases TNFalpha production. These findings support the hypothesis that TNFalpha can participate in normal physiological regulation of sleep and feeding.
肿瘤坏死因子-α（TNFα）在病理状态下会影响多种中枢神经系统功能，如睡眠、体温调节和进食。也有证据表明TNFα参与生理睡眠调节，例如，TNFα可诱导睡眠，且在长时间清醒期间大脑中TNFα水平会升高。大脑中TNFα产生增加的直接原因尚不清楚。我们研究了谷氨酸是否可作为TNFα产生的信号，因为谷氨酸是一种与细胞激活和清醒相关的神经递质。我们使用原代培养的胎鼠下丘脑细胞来检测TNFα的表达和释放。对神经元特异性烯醇化酶进行免疫染色显示，培养物中50%-60%为神经元细胞，40%-50%为非神经元细胞。在基础条件下，培养基和细胞中均检测到TNFα。用1 mM谷氨酸刺激细胞2小时，培养基中TNFα含量增加，而细胞内TNFα含量在较早时间点升高。通过台盼蓝排斥试验和MTT试验，未发现该刺激方案存在细胞毒性的证据。免疫细胞化学染色显示，培养物中约25%的神经元和约75%的胶质细胞表达TNFα。用谷氨酸刺激培养物并未增加表达TNFα的细胞百分比。我们得出结论，健康的下丘脑细胞培养物可组成性表达和释放TNFα，用无毒性的谷氨酸刺激细胞可增加TNFα的产生。这些发现支持了TNFα可参与睡眠和进食正常生理调节的假说。
