Oxidative stress and proinflammatory cytokines contribute to demyelination and axonal damage in a cerebellar culture model of neuroinflammation.
Oxidative stress and proinflammatory cytokines contribute to demyelination and axonal damage in a cerebellar culture model of neuroinflammation.
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DOI：
10.1371/journal.pone.0054722
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Villoslada P
中科院分区：
文献类型：
--
作者：
di Penta A;Moreno B;Reix S;Fernandez-Diez B;Villanueva M;Errea O;Escala N;Vandenbroeck K;Comella JX;Villoslada P
脱髓鞘和轴突损伤是多发性硬化症（MS）发病机制中的关键过程。炎症引起的氧化应激和促炎细胞因子介导组织损伤。为了监测与小胶质细胞激活相关的脱髓鞘和轴突损伤，我们采用了脂多糖（LPS）刺激的小脑器官型培养物模型。 LPS 激活的小胶质细胞释放促炎细胞因子（IL-1β、IL-6 和 TNFα），并增加诱导型一氧化氮合酶 (iNOS) 的表达和活性氧 (ROS) 的产生。这种激活与小脑培养物中的脱髓鞘和轴突损伤有关。非磷酸化神经丝的存在、轴突球体中线粒体的积累以及轴突横断所揭示的轴突损伤与更强的 iNOS 表达和随之而来的 ROS 增加有关。此外，我们使用 iNOS 抑制剂丙酮酸乙酯、游离清除剂和黄嘌呤氧化酶抑制剂别嘌呤醇，以及使用 Fc-TNFR1 构建体阻断促炎细胞因子，分析了促炎细胞因子和氧化应激在脱髓鞘和轴突变性中的作用。我们发现用丙酮酸乙酯或别嘌呤醇阻断小胶质细胞活化可显着减少轴突损伤，并在较小程度上减少脱髓鞘。阻断 TNFα 可显着减少脱髓鞘，但不能防止轴突损伤。此外，多发性硬化症最常见的治疗方法——干扰素-β，被用作可在该模型中进行测试的免疫调节剂化合物的示例。在体外，干扰素-β 治疗可减少 LPS 刺激后的氧化应激（iNOS 和 ROS 水平）和促炎细胞因子的释放，从而减少轴突损伤。使用内毒素刺激的小脑培养物的神经炎症模型模拟了氧化应激和促炎细胞因子组合介导的髓磷脂和轴突损伤。该模型既可以促进对神经炎症相关事件的理解，又有助于开发治疗多发性硬化症和其他神经退行性疾病的神经保护疗法。
Demyelination and axonal damage are critical processes in the pathogenesis of multiple sclerosis (MS). Oxidative stress and pro-inflammatory cytokines elicited by inflammation mediates tissue damage. To monitor the demyelination and axonal injury associated with microglia activation we employed a model using cerebellar organotypic cultures stimulated with lipopolysaccharide (LPS). Microglia activated by LPS released pro-inflammatory cytokines (IL-1β, IL-6 and TNFα), and increased the expression of inducible nitric oxide synthase (iNOS) and production of reactive oxygen species (ROS). This activation was associated with demyelination and axonal damage in cerebellar cultures. Axonal damage, as revealed by the presence of non-phosphorylated neurofilaments, mitochondrial accumulation in axonal spheroids, and axonal transection, was associated with stronger iNOS expression and concomitant increases in ROS. Moreover, we analyzed the contribution of pro-inflammatory cytokines and oxidative stress in demyelination and axonal degeneration using the iNOS inhibitor ethyl pyruvate, a free-scavenger and xanthine oxidase inhibitor allopurinol, as well as via blockage of pro-inflammatory cytokines using a Fc-TNFR1 construct. We found that blocking microglia activation with ethyl pyruvate or allopurinol significantly decreased axonal damage, and to a lesser extent, demyelination. Blocking TNFα significantly decreased demyelination but did not prevented axonal damage. Moreover, the most common therapy for MS, interferon-beta, was used as an example of an immunomodulator compound that can be tested in this model. In vitro, interferon-beta treatment decreased oxidative stress (iNOS and ROS levels) and the release of pro-inflammatory cytokines after LPS stimulation, reducing axonal damage. The model of neuroinflammation using cerebellar culture stimulated with endotoxin mimicked myelin and axonal damage mediated by the combination of oxidative stress and pro-inflammatory cytokines. This model may both facilitate understanding of the events involved in neuroinflammation and aid in the development of neuroprotective therapies for the treatment of MS and other neurodegenerative diseases.