Project Summary Aging is a major risk factor for the development of chronic pain with 50% of adults over the age of 65 suffering from at least one chronic pain condition. Unfortunately, there is a major knowledge gap regarding the interaction between age and pain. There is an urgent need for basic research using aged animal models to validate targets relevant to chronic pain treatment in this specific population. As humans and animals age, senescent cells accumulate in tissues throughout the body, and if not effectively cleared by the immune system, can disrupt homeostasis. Certain senescent cells secrete factors that induce inflammation, known collectively as the senescence-associated secretory phenotype (SASP), and include cytokines, chemokines, and proteases. Interestingly, several of these SASP factors are known pain-inducing cytokines released in the dorsal root ganglion (DRG) where primary sensory neuron cell bodies reside and drive hyperexcitability. Senescent cells therefore warrant investigation within the pain circuit in aged mice, and even in young mice after peripheral nerve injury. Our central hypothesis is that age-and injury-induced senescent neurons promote DRG hyperexcitability through production of SASP factors, causing persistent pain following peripheral injury. This hypothesis is founded on our robust preliminary evidence confirming: 1) Induction of senescent neurons within the DRG after peripheral nerve injury, 2) Increased senescent cell burden in the DRG of aged compared to young mice with and without injury as indicated by increased expression of senescent markers, p21 and p16, and 3) Localization of the SASP factor and pain mediator, IL6, to senescent neurons. Additionally, we have preliminary data demonstrating that treatment with a senolytic (anti-senescence) drug improves spared nerve injury (SNI)-induced mechanical allodynia while maintaining overall sensory function in young adult and aged mice. Therefore, a potential mechanism underlying enhanced pain susceptibility following injury in aged mice may be the combination of age-related and injury-induced senescent cells. The overall goal of this proposal is to rigorously validate senescent cells as a target for future therapeutic development. We will pursue validation by genetically, functionally, and phenotypically characterizing deleterious SASP-producing senescent DRG neurons using cross-disciplinary approaches across multiple laboratories. Our approach will leverage three clinically-relevant mouse models (SNI, paw incision and orthopaedic trauma) as well as human post-mortem DRG tissue to strengthen the evidence that our target is likely to be robust in translation. This research will be the first of its kind to investigate and validate cellular senescence in pre-clinical mouse models and has the potential to open a new therapeutic avenue, using senolytic agents, to alleviate pain.