Plastic, Aesthetic and Reconstructive Head and Neck Surgery and the Comprehensive Hearing Center, Department of Oto-Rhino-Laryngology, University of Wuerzburg Josef-Schneider-Strasse 11, D-97080 Wuerzburg, Germany.
Cells. 2021 Oct 19;10(10):2802. doi: 10.3390/cells10102802.
Causal therapies for the auditory-pathway and inner-ear diseases are still not yet available for clinical application. Regenerative medicine approaches are discussed and examined as possible therapy options. Neural stem cells could play a role in the regeneration of the auditory pathway. In recent years, neural stem and progenitor cells have been identified in the cochlear nucleus, the second nucleus of the auditory pathway. The current investigation aimed to analyze cell maturation concerning cellular calcium activity. Cochlear nuclei from PND9 CD rats were microscopically dissected and propagated as neurospheres in free-floating cultures in stem-cell medium (Neurobasal, B27, GlutaMAX, EGF, bFGF). After 30 days, the dissociation and plating of these cells took place under withdrawal of the growth factors and the addition of retinoic acid, which induces neural cell differentiation. Calcium imaging analysis with BAPTA-1/Oregon Green was carried out at different times during the differentiation phase. In addition, the influence of different voltage-dependent calcium channels was analyzed through the targeted application of inhibitors of the L-, N-, R- and T-type calcium channels. For this purpose, comparative examinations were performed on CN NSCs, and primary CN neurons. As the cells differentiated, a significant increase in spontaneous neuronal calcium activity was demonstrated. In the differentiation stage, specific frequencies of the spontaneous calcium oscillations were measured in different regions of the individual cells. Initially, the highest frequency of spontaneous calcium oscillations was ascertainable in the maturing somata. Over time, these were overtaken by calcium oscillations in the axons and dendrites. Additionally, in the area of the growth cones, an increasing activity was determined. By inhibiting voltage-dependent calcium channels, their expression and function in the differentiation process were confirmed. A comparable pattern of maturation of these channels was found in CN NSCs and primary CN neurons. The present results show that neural stem cells of the rat cochlear nucleus differentiated not only morphologically but also functionally. Spontaneous calcium activities are of great relevance in terms of neurogenesis and integration into existing neuronal structures. These functional aspects of neurogenesis within the auditory pathway could serve as future targets for the exogenous control of neuronal regeneration.
目前，用于听觉通路和内耳疾病的因果治疗方法尚未应用于临床。再生医学方法被认为是可能的治疗选择。神经干细胞可能在听觉通路的再生中发挥作用。近年来，人们已经在听觉通路上的第二个核——耳蜗核中鉴定出了神经干细胞和祖细胞。本研究旨在分析细胞成熟与细胞钙活性的关系。从 PND9 CD 大鼠的耳蜗核中分离出组织，在无血清悬浮培养中作为神经球在干细胞培养基（Neurobasal、B27、GlutaMAX、EGF、bFGF）中进行培养。30 天后，撤去生长因子并加入视黄酸，以诱导神经细胞分化，对这些细胞进行解离和培养。在分化阶段的不同时间点，通过 BAPTA-1/Oregon Green 进行钙成像分析。此外，通过靶向应用 L-、N-、R-和 T-型钙通道抑制剂，分析不同电压依赖性钙通道的影响。为此，对 CN NSCs 和原代 CN 神经元进行了比较研究。随着细胞分化，自发神经元钙活性显著增加。在分化阶段，在单个细胞的不同区域测量了自发钙振荡的特定频率。最初，在成熟的胞体中可检测到最高频率的自发钙振荡。随着时间的推移，这些被轴突和树突中的钙振荡所取代。此外，在生长锥区域，确定了活性增加。通过抑制电压依赖性钙通道，证实了它们在分化过程中的表达和功能。在 CN NSCs 和原代 CN 神经元中发现了这些通道的类似成熟模式。本研究结果表明，大鼠耳蜗核的神经干细胞不仅在形态上而且在功能上都发生了分化。自发钙活性在神经发生和整合到现有神经元结构中具有重要意义。听觉通路上神经发生的这些功能方面可以作为神经元再生的外源控制的未来目标。
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