Data_Sheet_1_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.

外周神经损伤会引发大量免疫介导的症状，对疼痛、抑郁及整体生活质量造成负面影响。多种疼痛状态下的性别二态性，其底层机制正是神经免疫差异。先天免疫系统正是这类性别差异的来源，它通过与神经系统的双向信号传导，促进炎症发生与伤害感受敏化。白细胞与感觉神经元之间的时空相互作用，或是解释性别间此类差异的关键所在。迄今为止，相关研究始终难以直观呈现这类相互作用。我们有望解答一系列关键科学问题，即神经损伤后，外周白细胞向疼痛系统关键组织——背根神经节（dorsal root ganglia, DRG）与坐骨神经——的募集机制。我们采用全组织透明化技术处理完整背根神经节与坐骨神经，并联合使用多光子显微镜（multi-photon microscopy）与转基因报告株系（transgenic reporter lines），以可视化观测神经损伤后参与神经病理性疼痛发生发展的白细胞动态变化。我们观察到，神经损伤后，腰段背根神经节的白细胞募集存在显著的性别二态性。此外，我们还对免疫细胞的大小与形态进行了评估，以解析神经组织炎症环境下的免疫细胞激活状态。不过，雌雄个体免疫系统对神经损伤的应答机制仍有待进一步研究；而结合先进全组织图像分析的下一代成像技术的持续应用，仍是解析神经元与非神经元细胞间双向相互作用的重要研究手段。