Use of Integrated Optical Clearing and 2-Photon Imaging to Investigate Sex Differences in Neuroimmune Interactions after Peripheral Nerve Injury

Peripheral nerve injury induces a myriad of immune symptoms that impacts pain and overall quality of life. Recent studies have revealed neuroimmune interactions in various pain states are important to mediate sex differences in their etiology. A described source of these sexual dimorphisms is the innate immune system, which promotes inflammation and pro-nociception through bidirectional signaling with the nervous system. The spatiotemporal interactions between macrophages and sensory neurons could hold the key to explain ascribed differences between sexes. To date, studies have found it difficult to adequately display these interactions. We are poised to answer important questions regarding the recruitment and morphology of peripheral macrophages in key tissues of the pain system: the dorsal root ganglia (DRG) and sciatic nerve (ScN). Our approach utilized ScaleS1, an optical clearing method, to clear whole DRGs and ScNs after peripheral nerve injury. With the concomitant use of 2-photon microscopy and transgenic reporter lines, we visualized macrophage dynamics involved in neuropathic pain development following injury. Male and female mice were sacrificed at the peak of nerve injury-induced pain development and DRGs and ScNs were harvested, processed, and cleared. Whole tissue images were captured via 2-photon microscopy and were processed and analyzed using Imaris imaging software. Macrophage infiltration was increased in the ipsilateral DRGs after nerve injury in males. We also assessed macrophage size and morphology to understand activation states in the context of nervous tissue inflammation. We found sex and injury dependent clustering of macrophage morphology populations in both the DRG and ScN. 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 nonneuronal cells. This research is supported by the grants: APS Future Leader's Grant (M.D.B), and the Rita Allen Foundation Grant (M.D.B), NINDS K22NS096030 (M.D.B), and the U.T System STARS.