期刊名称:Proceedings of the National Academy of Sciences
印刷版ISSN:0027-8424
电子版ISSN:1091-6490
出版年度:2019
卷号:116
期号:28
页码:14290-14299
DOI:10.1073/pnas.1819343116
出版社:The National Academy of Sciences of the United States of America
摘要:Animal models of central nervous system (CNS) demyelination, including toxin-induced focal demyelination and immune-mediated demyelination through experimental autoimmune encephalomyelitis (EAE), have provided valuable insights into the mechanisms of neuroinflammation and CNS remyelination. However, the ability to track changes in transcripts, proteins, and metabolites, as well as cellular populations during the evolution of a focal lesion, has remained challenging. Here, we developed a method to label CNS demyelinating lesions by the intraperitoneal injection of a vital dye, neutral red (NR), into mice before killing. We demonstrate that NR-labeled lesions can be easily identified on the intact spinal cord in both lysolecithin- and EAE-mediated demyelination models. Using fluorescence microscopy, we detected NR in activated macrophages/microglia and astrocytes, but not in oligodendrocytes present in lesions. Importantly, we successfully performed RT-qPCR, Western blot, flow cytometry, and mass spectrometry analysis of precisely dissected NR-labeled lesions at 5, 10, and 20 d postlesion (dpl) and found differential changes in transcripts, proteins, cell populations, and metabolites in lesions over the course of remyelination. Therefore, NR administration is a simple and powerful method to track and analyze the detailed molecular, cellular, and metabolic changes that occur within the lesion microenvironment over time following CNS injury. Furthermore, this method can be used to identify molecular and metabolic pathways that regulate neuroinflammation and remyelination and facilitate the development of therapies to promote repair in demyelinating disorders such as multiple sclerosis.