Wang Di, Zhao Man, Tang Xiao, Gao Man, Liu Wenjing, Xiang Minghui, Ruan Jian, Chen Jie, Long Bin, Li Jun
College of Life Sciences, Anhui Normal University, Wuhu, Anhui Province, China.
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu Province, China.
Neural Regen Res. 2023 Dec;18(12):2743-2750. doi: 10.4103/1673-5374.373717.
Cynops orientalis (C. orientalis) has a pronounced ability to regenerate its spinal cord after injury. Thus, exploring the molecular mechanism of this process could provide new approaches for promoting mammalian spinal cord regeneration. In this study, we established a model of spinal cord thoracic transection injury in C. orientalis, which is an endemic species in China. We performed RNA sequencing of the contused axolotl spinal cord at two early time points after spinal cord injury - during the very acute stage (4 days) and the subacute stage (7 days) - and identified differentially expressed genes; additionally, we performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses, at each time point. Transcriptome sequencing showed that 13,059 genes were differentially expressed during C. orientalis spinal cord regeneration compared with uninjured animals, among which 4273 were continuously down-regulated and 1564 were continuously up-regulated. Down-regulated genes were most enriched in the Gene Ontology term "multicellular organismal process" and in the ribosome pathway at 10 days following spinal cord injury. We found that multiple genes associated with energy metabolism were down-regulated and multiple genes associated with the lysosome were up-regulated after spinal cord injury, indicating the importance of low metabolic activity during wound healing. Immune response-associated pathways were activated during the early acute phase (4 days), while the expression of extracellular matrix proteins such as glycosaminoglycan and collagen, as well as tight junction proteins, was lower at 10 days post-spinal cord injury than 4 days post-spinal cord injury. However, compared with 4 days post-injury, at 10 days post-injury neuroactive ligand-receptor interactions were no longer down-regulated, up-regulated differentially expressed genes were enriched in pathways associated with cancer and the cell cycle, and SHH, VIM, and Sox2 were prominently up-regulated. Immunofluorescence staining showed that glial fibrillary acidic protein was up-regulated in axolotl ependymoglial cells after injury, similar to what is observed in mammalian astrocytes after spinal cord injury, even though axolotls do not form a glial scar during regeneration. We suggest that low intracellular energy production could slow the rapid amplification of ependymoglial cells, thereby inhibiting reactive gliosis, at early stages after spinal cord injury. Extracellular matrix degradation slows cellular responses, represses the expression of neurogenic genes, and reactivates a transcriptional program similar to that of embryonic neuroepithelial cells. These ependymoglial cells act as neural stem cells: they migrate and proliferate to repair the lesion and then differentiate to replace lost glial cells and neurons. This provides the regenerative microenvironment that allows axon growth after injury.
东方蝾螈在脊髓损伤后具有显著的脊髓再生能力。因此，探索这一过程的分子机制可为促进哺乳动物脊髓再生提供新方法。在本研究中，我们建立了东方蝾螈脊髓胸段横断损伤模型，东方蝾螈是中国的特有物种。我们在脊髓损伤后的两个早期时间点，即极急性期（4天）和亚急性期（7天），对钝口螈脊髓损伤部位进行了RNA测序，鉴定出差异表达基因；此外，我们在每个时间点进行了基因本体论和京都基因与基因组百科全书通路分析。转录组测序表明，与未受伤动物相比，东方蝾螈脊髓再生过程中有13059个基因差异表达，其中4273个基因持续下调，1564个基因持续上调。脊髓损伤后10天，下调基因在基因本体论术语“多细胞生物过程”和核糖体通路中富集程度最高。我们发现，脊髓损伤后多个与能量代谢相关的基因下调，多个与溶酶体相关的基因上调，这表明伤口愈合过程中低代谢活性的重要性。免疫反应相关通路在急性早期（4天）被激活，而脊髓损伤后10天，糖胺聚糖和胶原蛋白等细胞外基质蛋白以及紧密连接蛋白的表达低于脊髓损伤后4天。然而，与损伤后4天相比，损伤后10天神经活性配体-受体相互作用不再下调，上调的差异表达基因在与癌症和细胞周期相关的通路中富集，并且SHH、VIM和Sox2显著上调。免疫荧光染色显示，钝口螈室管膜神经胶质细胞损伤后胶质纤维酸性蛋白上调，这与哺乳动物脊髓损伤后星形胶质细胞的情况相似，尽管钝口螈在再生过程中不形成胶质瘢痕。我们认为，脊髓损伤早期细胞内低能量产生可能会减缓室管膜神经胶质细胞的快速扩增，从而抑制反应性胶质增生。细胞外基质降解减缓细胞反应，抑制神经源性基因的表达，并重新激活类似于胚胎神经上皮细胞的转录程序。这些室管膜神经胶质细胞充当神经干细胞：它们迁移并增殖以修复损伤，然后分化以替代丢失的胶质细胞和神经元。这提供了损伤后允许轴突生长的再生微环境。
