Knowledge regarding the relationship between muscles and the corresponding motor neurons would allow therapeutic genes to transport into specific spinal cord segments. Retrograde tracing technique by targeting the motor endplate (MEP), a highly specialized structure that offers direct access to the spinal motor neurons, has been used to elucidate the connectivity between skeletal muscles and the innervating motor neuron pools. However, current injection strategies mainly based on blind injection or the local MEP region might lead to an underestimation of the motor neuron number due to the uneven distribution of MEP in skeletal muscles. In this work, we proposed a novel intramuscular injection strategy based on the 3D distribution of the MEPs in skeletal muscles, applied the 3D intramuscular injection to the gastrocnemius and tibialis anterior for retrograde tracing of the corresponding motor neurons, and compared this with the existing injection strategy. The intramuscular diffusion of the tracer demonstrated that 3D injection could maximize the retrograde transport by ensuring a greater uptake of the tracer by the MEP region. In combination with optical clearing and imaging, we performed 3D mapping and quantification of the labeled motor neurons and confirmed that 3D injection could label more motor neurons than the current injection method. It is expected that 3D intramuscular injection strategy will help elucidate the connective relationship between muscles and motor neurons faithfully and becomes a promising tool in the development of gene therapy strategies for motor neuron diseases.

中文翻译：

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基于小鼠后肢肌肉运动终板空间分布最大化逆行运输的方法。

关于肌肉和相应运动神经元之间关系的知识将允许治疗基因转运到特定的脊髓节段。通过靶向运动终板 (MEP) 的逆行追踪技术，这是一种高度专业化的结构，可直接访问脊髓运动神经元，已被用于阐明骨骼肌和支配运动神经元池之间的连接。然而，由于 MEP 在骨骼肌中的分布不均匀，目前主要基于盲注或局部 MEP 区域的注射策略可能会导致对运动神经元数量的低估。在这项工作中，我们提出了一种基于 MEP 在骨骼肌中的 3D 分布的新型肌肉注射策略，将 3D 肌内注射应用于腓肠肌和胫骨前肌以逆行追踪相应的运动神经元，并将其与现有的注射策略进行比较。示踪剂的肌内扩散表明 3D 注射可以通过确保 MEP 区域更多地吸收示踪剂来最大化逆行运输。结合光学清除和成像，我们对标记的运动神经元进行了 3D 映射和量化，并确认 3D 注射可以比当前注射方法标记更多的运动神经元。预计3D肌肉注射策略将有助于忠实地阐明肌肉和运动神经元之间的连接关系，并成为开发运动神经元疾病基因治疗策略的有前途的工具。