Transcriptomic profiling of oligodendrocytes, astrocytes, and microglia in an experimental autoimmune encephalomyelitis (EAE) model of demyelination

The experimental autoimmune encephalomyelitis (EAE) mouse model is widely used to study the pathophysiology of multiple sclerosis (MS), including brain inflammation and demyelination. This study investigates cell-type-specific transcriptional changes in oligodendrocytes, astrocytes, and microglia at day 30 post-induction of EAE using MOG peptide (amino acids 35–55) emulsified in complete Freund's adjuvant (CFA) containing Mycobacterium tuberculosis, followed by pertussis toxin injections. CFA control mice received only CFA and pertussis toxin, without MOG peptide. Oligodendrocytes, astrocytes, and microglia were isolated using magnetic-activated cell sorting (MACS) from brain and spinal cord of CFA-treated and EAE-induced mice. Bulk RNA sequencing was conducted to uncover transcriptional changes associated with EAE-induced demyelination. These findings will enhance our understanding of glial cell contributions to MS pathogenesis and help identify potential therapeutic targets. Overall design: Three-month-old C57BL/6 mice were received CFA and pertussis toxin (CFA control mice), or MOG peptide (35–55) emulsified in CFA and pertussis toxin to induce EAE. At day 30 post-induction,brain cell suspensions were prepared, and oligodendrocytes, astrocytes, and microglia were isolated using magnetic-activated cell sorting (MACS) with cell type-specific markers. Experimental groups for RNA sequencing: (1) Oligodendrocytes from CFA control mice (CFA_Ctrl_OL); (2) Oligodendrocytes from EAE mice (MOG_Ctrl_OL); (3) Astrocytes from CFA control mice (CFA_Ctrl_Astro); (4) Astrocytes from EAE mice (MOG_Ctrl_Astro); (5) Microglia from CFA control mice (CFA_Ctrl_MG); (6) Microglia from EAE mice (MOG_Ctrl_MG). Bulk RNA sequencing was performed for all cell types and conditions. Each group has 3 or 4 repeats.

实验性自身免疫性脑脊髓炎 (experimental autoimmune encephalomyelitis, EAE) 小鼠模型是研究多发性硬化症 (multiple sclerosis, MS) 病理生理学（涵盖脑部炎症与脱髓鞘过程）的广泛应用模型。本研究针对经髓鞘少突胶质细胞糖蛋白（MOG）肽段（氨基酸35–55）免疫诱导的EAE模型，在诱导后第30天，探究少突胶质细胞 (oligodendrocytes)、星形胶质细胞 (astrocytes) 与小胶质细胞 (microglia) 的细胞类型特异性转录组变化：造模方案为使用含结核分枝杆菌 (Mycobacterium tuberculosis) 的完全弗氏佐剂 (complete Freund's adjuvant, CFA) 乳化MOG肽段，辅以百日咳毒素 (pertussis toxin) 注射；CFA对照组小鼠仅接受CFA与百日咳毒素注射，不给予MOG肽段。研究人员从经CFA处理的对照组小鼠及EAE造模小鼠的脑与脊髓中，通过磁激活细胞分选 (magnetic-activated cell sorting, MACS) 分离上述三类胶质细胞，随后开展批量RNA测序，以揭示EAE诱导脱髓鞘相关的转录组改变。本研究结果将深化我们对胶质细胞在MS发病机制中贡献的认知，并助力潜在治疗靶点的发掘。实验整体设计：选取3月龄C57BL/6品系小鼠，分为两大组：CFA对照组小鼠仅注射CFA与百日咳毒素，EAE造模组小鼠则使用CFA乳化的MOG肽段（35–55）联合百日咳毒素进行造模。造模后第30天，制备脑组织单细胞悬液，通过结合细胞类型特异性标志物的磁激活细胞分选（MACS）分离少突胶质细胞、星形胶质细胞与小胶质细胞。用于批量RNA测序的实验分组如下：1. CFA对照组小鼠的少突胶质细胞（CFA_Ctrl_OL）；2. EAE模型小鼠的少突胶质细胞（MOG_Ctrl_OL）；3. CFA对照组小鼠的星形胶质细胞（CFA_Ctrl_Astro）；4. EAE模型小鼠的星形胶质细胞（MOG_Ctrl_Astro）；5. CFA对照组小鼠的小胶质细胞（CFA_Ctrl_MG）；6. EAE模型小鼠的小胶质细胞（MOG_Ctrl_MG）。所有细胞类型与分组均开展批量RNA测序，每组设置3~4次生物学重复。