Department of Neurology, Ruhr-University Bochum, St. Josef Hospital;
Department of Neurology, Ruhr-University Bochum, St. Josef Hospital.
J Vis Exp. 2023 Feb 10(192). doi: 10.3791/64768.
The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.
髓鞘形成过程对于在神经系统中实现快速和充分的信号转导至关重要。在外周神经系统中，神经元和施万细胞之间存在复杂的相互作用，以控制轴突的髓鞘形成。这种相互作用的干扰和髓鞘的破坏是炎症性神经病的标志，并在外周神经退行性疾病中继发发生。在这里，我们提出了一个背根神经节外植体和施万细胞的共培养模型，该模型可在外周轴突上形成稳健的髓鞘化，以研究外周神经系统中的髓鞘形成过程、研究轴突-施万细胞相互作用，并评估治疗剂对每种细胞类型的潜在影响。从方法学上讲，从胚胎大鼠（E13.5）中收获背根神经节，将其与周围组织分离，并作为整个外植体培养 3 天。从 3 周龄成年大鼠中分离出施万细胞，并对坐骨神经进行酶消化。通过磁性激活细胞分选纯化得到的施万细胞，并在神经调节素和 forskolin 丰富的条件下培养。在背根神经节外植体培养 3 天后，将 30,000 个施万细胞添加到含有抗坏血酸的一个背根神经节外植体中。在共培养的第 10 天，通过免疫细胞化学染色中髓鞘碱性蛋白的散在信号，可以检测到髓鞘形成的最初迹象。从第 14 天开始，髓鞘形成并沿轴突传播。髓鞘化可以通过髓鞘碱性蛋白染色进行定量，作为髓鞘化区域与轴突区域的比率，以说明轴突密度的差异。该模型提供了研究体外外周髓鞘化各个方面的实验机会，这对于理解炎症性和神经退行性疾病中周围神经系统脱髓鞘和神经退行性变的病理学以及可能的治疗机会至关重要。
