Xu Jianyi, Xuan Ang, Liu Zhang, Li Yusha, Zhu Jingtan, Yao Yingtao, Yu Tingting, Zhu Dan
Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, China.
MoE Key Laboratory for Biomedical Photonics, School of Engineering Sciences, Huazhong University of Science and Technology, Wuhan, China.
Front Cell Neurosci. 2021 Aug 11;15:707982. doi: 10.3389/fncel.2021.707982. eCollection 2021.
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.
了解肌肉与相应运动神经元之间的关系，将有助于治疗性基因转运至特定脊髓节段。通过靶向运动终板（MEP）进行逆行示踪技术，MEP是一种高度特化的结构，可直接通向脊髓运动神经元，已被用于阐明骨骼肌与支配运动神经元池之间的连接。然而，目前主要基于盲目注射或局部MEP区域的注射策略，可能会因MEP在骨骼肌中分布不均而导致对运动神经元数量的低估。在这项研究中，我们基于骨骼肌中MEP的三维分布提出了一种新型肌肉内注射策略，将三维肌肉内注射应用于腓肠肌和胫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。示踪剂在肌肉内的扩散表明，三维注射可通过确保MEP区域对示踪剂的更大摄取来最大化逆行转运。结合光学清除和成像，我们对标记的运动神经元进行了三维映射和定量分析，并证实三维注射比当前注射方法能标记更多的运动神经元。预计三维肌肉内注射策略将有助于忠实地阐明肌肉与运动神经元之间的连接关系，并成为运动神经元疾病基因治疗策略开发中有前景的工具。
