Department of Biomedical Sciences, College of Osteopathic Medicine, University of New England, Biddeford, Maine.
Center for Excellence in the Neurosciences, University of New England, Biddeford, Maine.
Curr Protoc. 2023 Apr;3(4):e743. doi: 10.1002/cpz1.743.
Central nervous system glial cells are known to mediate many neurocognitive/neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. Similar glial responses have been recognized as critical factors contributing to the development of diseases in the peripheral nervous system, including various types of peripheral neuropathies, such as peripheral nerve injury-induced neuropathic pain, diabetic neuropathy, and HIV-associated sensory neuropathy. Investigation of the central mechanisms of these peripherally-manifested diseases often requires the examination of spinal cord glial cells at cellular/molecular levels in vitro. When using rodent models to study these diseases, many investigators have chosen to use neonatal cerebral cortices to prepare glial cultures or immortalized cell lines in order to obtain sufficient numbers of cells for assessment. However, differences in responses between cell lines versus primary cultures, neonatal vs. adult cells, and brain vs. spinal cord cells may result in misleading data. Here, we describe a protocol for preparing mixed glial cells from adult mouse spinal cord that can be used for direct in vitro evaluations or further preparation of microglia-enriched and microglia-depleted cells. In this protocol, spinal cord tissue is enzymatically dissociated and adult mixed glial cells are ready to be used between 12 and 14 days after the establishment of the culture. This protocol may be further refined to prepare spinal cord glial cells from spinal cord tissues of adult rats and potentially other species. Mixed glial cultures can be prepared from animals of different strains or post-in vivo manipulations and therefore are suitable for studying a variety of diseases/disorders that involve spinal cord pathological changes, such as amyotrophic lateral sclerosis and multiple sclerosis, as well as toxin-induced changes. © 2023 Wiley Periodicals LLC. Basic Protocol: Preparation of primary mixed glial cell cultures from adult mouse spinal cord tissue.
中枢神经系统胶质细胞被认为介导许多神经认知/神经退行性疾病，包括阿尔茨海默病和帕金森病。类似的神经胶质反应已被认为是导致周围神经系统疾病发展的关键因素，包括各种类型的周围神经病变，如周围神经损伤引起的神经性疼痛、糖尿病性神经病和 HIV 相关感觉神经病。研究这些表现为周围的中枢机制疾病通常需要在体外细胞/分子水平上检查脊髓胶质细胞。当使用啮齿动物模型研究这些疾病时，许多研究人员选择使用新生大脑皮质来制备胶质培养物或永生化细胞系，以获得足够数量的细胞进行评估。然而，细胞系与原代培养物、新生细胞与成年细胞、大脑与脊髓细胞之间的反应差异可能导致数据误导。在这里，我们描述了一种从成年小鼠脊髓中制备混合胶质细胞的方案，可用于直接体外评估或进一步制备富含小胶质细胞和耗尽小胶质细胞的细胞。在该方案中，脊髓组织经酶解分离，在培养建立后 12-14 天即可获得成熟的成年混合胶质细胞。该方案可进一步改进，以从小鼠和其他潜在物种的脊髓组织中制备脊髓胶质细胞。混合胶质细胞培养物可从不同品系的动物或体内操作后制备，因此适用于研究涉及脊髓病理变化的各种疾病/障碍，如肌萎缩侧索硬化症和多发性硬化症，以及毒素诱导的变化。© 2023 威立出版社。基本方案：从成年小鼠脊髓组织中制备原代混合胶质细胞培养物。
