This R21 application uses the high risk, high reward technique of mass cytometry (abbreviated as CyTOF) to identify immune cell subsets that mediate neuroinflammation in the central nervous system (CNS) in a well- established orthopedic mouse model of postoperative delirium (POD). Dysregulated immunity is a hallmark of normal aging; it is also recognized as a key feature of many neurological disorders including dementia and perioperative complications like delirium. POD is common, occurring in up to 50% of older patients after a fracture repair. The strongest risk factors for POD are advanced age and dementia. Interestingly, the postoperative emergence of delirium may presage dementia. In fact, delirium superimposed on dementia (DSD) contributes to a faster trajectory of cognitive decline as well as significant mortality. This is significant because of the millions of elderly patients that routinely undergo orthopedic or other surgeries every year. The biologic mechanisms that drive POD and DSD during aging are unknown and without approved drugs to treat or prevent it. We have pioneered a clinically-relevant orthopedic surgery murine model that displays systemic inflammation, alters microglial activation, and causes memory deficits in mice. In our model, surgery mobilizes discrete immune cell populations with pro-inflammatory signaling responses that mediate damage to the blood-brain barrier, damage the neurovascular unit (NVU) and impair normal synaptic transduction. Translationally, similar immune signatures with the same pro-inflammatory markers have now been described in fluid biomarkers of delirious patients. However, current immunophenotyping is limited to selected non-specific cytokines and pro- inflammatory molecules such as TNF, IL-1, IL-6, MCP-1 and S100b in brain tissue (rodents) and cerebrospinal fluid (humans). No study has yet attempted to unbiasedly profile the immune subset specific response to orthopedic surgical trauma in the CNS. We propose two specific aims: (1) to define how age differences between young adult (6mos) and elderly (22mos) male and female mice modulate immune cell subsets in the CNS and blood after orthopedic surgery; and (2) to determine the impact of Alzheimer’s (AD)-like pathologic features using 5xFAD transgenic male and female mice at 6mos of age (when AD and postoperative neuroinflammation become pathologically significant) on immune cell subsets in the CNS and blood after orthopedic surgery. The ability to resolve immune cell subsets and align them with discrete repertoires of pro-inflammatory signaling molecules with CyTOF will be key to understanding whether POD results from neuroinflammation due to peripherally migrating and/or CNS-resident immunocytes. These experiments will provide the foundation for future RO1 studies in which we pursue key findings focused on dysfunction of the NVU associated with neurodegeneration in Alzheimer’s disease as sought by NOT-AG-19-033. We expect our findings to have an...