Hasseldam Henrik, Rasmussen Rune Skovgaard, Johansen Flemming Fryd
Department of Biomedical Sciences, Faculty of Health, University of Copenhagen, Ole Maaloes vej 5, DK-2200, Copenhagen, Denmark.
J Neuroinflammation. 2016 Sep 15;13(1):246. doi: 10.1186/s12974-016-0707-3.
Multiple sclerosis is widely accepted as an inflammatory disease. However, studies indicate that degenerative processes in the CNS occur prior to inflammation. In the widely used animal model experimental autoimmune encephalomyelitis (EAE), we investigated the significance of degenerative processes from mitochondrial membrane potentials, reactive oxidative species, cell death markers, chemokines, and inflammatory cell types in brain, spinal cord, and optic nerve tissue during the effector phase of the disease, before clinical disease was evident.
Sixty-two rats were placed in eight groups, n = 6 to 10. Four groups were immunized with spinal cord homogenate emulsified in complete Freund's adjuvant (one served as EAE group), three groups were immunized with complete Freund's adjuvant only, and a control group was injected with phosphate buffered saline only. Groups were sacrificed 3, 5, 7, or 12-13 days after the intervention and analyzed for early signs of CNS degeneration.
Loss of mitochondrial membrane potential and oxidative changes was observed days before clinical disease debut at day 9.75 ± 0.89. The early mitochondrial changes were not associated with cytochrome C release, cleavage of caspases 9 (38/40 kDa) and 3 (17/19 kDa), and cleavage of PARP (89 kDa) or spectrin (120/150 kDa), and apoptosis was not initiated. Axonal degeneration was only present at disease onset. Increases in a range of cytokines and chemokines were observed systemically as a consequence of immunization with complete Freund's adjuvant, whereas the encephalitogenic emulsion induced an upregulation of the chemokines Ccl2, Ccl20, and Cxcl1, specifically in brain tissue, 7 days after immunization.
Five to seven days after immunization, subtle decreases in the mitochondrial membrane potential and an increased reactive oxygen species burden in brain tissue were observed. No cell death was detected at these time-points, but a specific expression pattern of chemokines indicates activity in the CNS, several days before clinical disease debut.
多发性硬化被广泛认为是一种炎症性疾病。然而，研究表明中枢神经系统（CNS）的退行性变过程在炎症之前就已发生。在广泛使用的动物模型实验性自身免疫性脑脊髓炎（EAE）中，我们在疾病效应期、临床疾病出现之前，研究了脑、脊髓和视神经组织中线粒体膜电位、活性氧化物质、细胞死亡标志物、趋化因子和炎症细胞类型等退行性变过程的意义。
62只大鼠分为8组，每组n = 6至10只。4组用完全弗氏佐剂乳化的脊髓匀浆免疫（一组作为EAE组），3组仅用完全弗氏佐剂免疫，对照组仅注射磷酸盐缓冲盐水。干预后3、5、7或12 - 13天处死各组大鼠，分析CNS早期退变迹象。
在临床疾病于9.75±0.89天首次出现前数天，观察到线粒体膜电位丧失和氧化变化。早期线粒体变化与细胞色素C释放、半胱天冬酶9（38/40 kDa）和3（17/19 kDa）的裂解、聚（ADP - 核糖）聚合酶（89 kDa）或血影蛋白（120/150 kDa）的裂解无关，且未启动凋亡。轴突变性仅在疾病发作时出现。由于用完全弗氏佐剂免疫，全身观察到一系列细胞因子和趋化因子增加，而致脑炎乳剂在免疫后7天诱导趋化因子Ccl2、Ccl20和Cxcl1上调，特别是在脑组织中。
免疫后5至7天，观察到脑组织中线粒体膜电位轻微下降和活性氧负担增加。在这些时间点未检测到细胞死亡，但趋化因子的特定表达模式表明在临床疾病首次出现前数天CNS就有活动。
