Mishra Pooja-Shree, Dhull Dinesh K, Nalini A, Vijayalakshmi K, Sathyaprabha T N, Alladi Phalguni Anand, Raju Trichur R
Department of Neurophysiology, National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore, 560029, India.
Present address: Centre de Recherche de l'Institut Universitaire en Santé Mentale de Québec (CRIUSMQ), Québec, QC, G1J 2G3, Canada.
J Neuroinflammation. 2016 Aug 30;13(1):212. doi: 10.1186/s12974-016-0698-0.
Non-cell autonomous toxicity is one of the potential mechanisms implicated in the etiopathogenesis of amyotrophic lateral sclerosis (ALS). However, the exact role of glial cells in ALS pathology is yet to be fully understood. In a cellular model recapitulating the pathology of sporadic ALS, we have studied the inflammatory response of astroglia following exposure to the cerebrospinal fluid from ALS patients (ALS-CSF).
Various inflammatory markers including pro-inflammatory and anti-inflammatory cytokines, COX-2, PGE-2, trophic factors, glutamate, nitric oxide (NO), and reactive oxygen species (ROS) were analyzed in the rat astroglial cultures exposed to ALS-CSF and compared with the disease control or normal controls. We used immunofluorescence, ELISA, and immunoblotting techniques to investigate the protein expression and real-time PCR to study the messenger RNA (mRNA) expression. Glutamate, NO, and ROS were estimated using appropriate biochemical assays. Further, the effect of conditioned medium from the astroglial cultures exposed to ALS-CSF on NSC-34 motor neuronal cell line was detected using the MTT assay. Statistical analysis was carried out using one-way ANOVA followed by Tukey's post hoc test, or Student's t test, as applicable.
Here, we report that the ALS-CSF enhanced the production and release of inflammatory cytokines IL-6 and TNF-α, as well as COX-2 and PGE-2. Concomitantly, anti-inflammatory cytokine IL-10 and the beneficial trophic factors, namely VEGF and GDNF, were down-regulated. We also found impaired regulation of glutamate, NO, and ROS in the astroglial cultures treated with ALS-CSF. The conditioned medium from the ALS-CSF exposed astroglial cultures induced degeneration in NSC-34 cells.
Our study demonstrates that the astroglial cells contribute to the neuroinflammation-mediated neurodegeneration in the in vitro model of sporadic ALS.
非细胞自主性毒性是肌萎缩侧索硬化症（ALS）发病机制中涉及的潜在机制之一。然而，神经胶质细胞在ALS病理过程中的确切作用尚未完全明确。在一个重现散发性ALS病理的细胞模型中，我们研究了星形胶质细胞暴露于ALS患者脑脊液（ALS-CSF）后的炎症反应。
在暴露于ALS-CSF的大鼠星形胶质细胞培养物中分析各种炎症标志物，包括促炎和抗炎细胞因子、COX-2、PGE-2、营养因子、谷氨酸、一氧化氮（NO）和活性氧（ROS），并与疾病对照或正常对照进行比较。我们使用免疫荧光、酶联免疫吸附测定（ELISA）和免疫印迹技术研究蛋白质表达，并使用实时聚合酶链反应（PCR）研究信使核糖核酸（mRNA）表达。使用适当的生化测定法估计谷氨酸、NO和ROS。此外，使用MTT测定法检测暴露于ALS-CSF的星形胶质细胞培养物的条件培养基对NSC-34运动神经元细胞系的影响。根据适用情况，使用单因素方差分析（ANOVA），随后进行Tukey事后检验或学生t检验进行统计分析。
在此，我们报告ALS-CSF增强了炎症细胞因子IL-6和TNF-α以及COX-2和PGE-2的产生和释放。同时，抗炎细胞因子IL-10以及有益的营养因子，即血管内皮生长因子（VEGF）和胶质细胞源性神经营养因子（GDNF）下调。我们还发现用ALS-CSF处理的星形胶质细胞培养物中谷氨酸、NO和ROS的调节受损。暴露于ALS-CSF的星形胶质细胞培养物的条件培养基诱导NSC-34细胞变性。
我们的研究表明，在散发性ALS的体外模型中，星形胶质细胞促成了神经炎症介导的神经退行性变。
Zotero 插件
