Data_Sheet_8_Use of Integrated Optical Clearing and 2-Photon Imaging to Investigate Sex Differences in Neuroimmune Interactions After Peripheral Nerve Injury.zip

Peripheral nerve injury induces a myriad of immune-derived symptoms that negatively impacts pain, depression, and overall quality of life. Neuroimmune differences underlie sexual dimorphisms in various pain states. The innate immune system is a source of these sex differences, which promotes inflammation and pro-nociception through bidirectional signaling with the nervous system. Spatiotemporal interactions between leukocytes and sensory neurons could hold the key to explain ascribed differences between sexes. To date, studies have found it difficult to display these interactions. We are poised to answer important questions regarding the recruitment of peripheral leukocytes to key tissues of the pain system, the dorsal root ganglia (DRG) and sciatic nerve after nerve injury. We optically clear whole DRGs and sciatic nerves and concomitantly use multi-photon microscopy and transgenic reporter lines, to visualize leukocyte dynamics involved in neuropathic pain development following nerve injury. We observed robust sexual dimorphisms in leukocyte recruitment to the lumbar DRGs after nerve injury. We also assessed immune cell size and morphology to understand activation states in the context of nervous tissue inflammation. The altered mechanisms by which the male and female immune systems respond to nerve injury are still topics of further research, however; the continued use of next-generation imaging with advanced whole tissue image analysis remains an important tool in understanding the reciprocal interactions between neuronal and non-neuronal cells.

周围神经损伤（peripheral nerve injury）可引发大量免疫源性症状，对疼痛、抑郁及整体生活质量产生负面影响。多种疼痛状态下的性别二态性（sexual dimorphisms）背后存在神经免疫差异。先天免疫系统（innate immune system）是这类性别差异的来源，其通过与神经系统的双向信号传导促进炎症反应与促伤害感受（pro-nociception）过程。白细胞（leukocyte）与感觉神经元（sensory neurons）之间的时空相互作用，或是解释性别间差异的关键所在。迄今为止，相关研究均难以直观呈现这类相互作用。本研究旨在解答神经损伤后，疼痛系统关键组织——背根神经节（dorsal root ganglia, DRG）与坐骨神经（sciatic nerve）中外周白细胞募集的诸多重要科学问题。我们通过对完整背根神经节与坐骨神经进行光学透明化（optical clearing）处理，并联合使用多光子显微镜（multi-photon microscopy）与转基因报告品系（transgenic reporter lines），实现了对神经损伤后参与神经病理性疼痛（neuropathic pain）发生发展的白细胞动态变化的可视化观测。我们观察到，神经损伤后腰椎背根神经节中的白细胞募集存在显著的性别二态性差异。此外，我们还通过评估免疫细胞的大小与形态，解析了神经组织炎症环境下免疫细胞的激活状态。不过，雌雄免疫系统应对神经损伤的调控机制仍有待进一步研究；而结合先进全组织图像分析（whole tissue image analysis）的下一代成像技术（next-generation imaging），仍是解析神经元与非神经元细胞（neuronal and non-neuronal cells）间双向相互作用的重要研究工具。