The objective of this proposal is to decipher the mechanistic role of the pro-inflammatory cytokine IL-12 and its downstream effector molecules in mediating cytotoxicity and inflammation after spinal cord injury (SCI), a severe condition with devastating consequences for the neurological function and overall health of affected persons. Despite some beneficial inflammatory effects on tissue preservation after SCI, excessive and prolonged inflammation in the injured spinal cord has emerged as a crucial mediator of secondary tissue damage, significantly expanding the tissue loss beyond that created by the initial trauma. Paradoxically, SCI also causes systemic immunosuppression, with resultant high risk of life-threatening infections like pneumonia, making it a crucial task to target the detrimental intraparenchymal inflammation within the spinal cord, without interfering with beneficial immune reactions both in the spinal cord parenchyma and systemically. We have previously shown that IL-12 leads to significantly impaired locomotor recovery and tissue damage after SCI. IL-12 and its receptors are predominantly expressed on astrocytes after SCI. However, the mechanism of IL-12-mediated tissue damage, if directly via cytotoxic effector molecules or indirectly by regulating the inflammatory response, is currently unknown. This information is of high importance for the development of future therapeutic approaches. We hypothesize that IL-12 signaling originating in astrocytes promotes secondary tissue damage resulting in functional impairment. We designed the following three aims to address this hypothesis: (i) We will determine if cell-specific deletion of IL-12 signaling in astrocytes or microglia improves functional locomotor recovery and reduced autonomic impairment. This will provide information about the cell types initiating IL-12 mediated tissue damage. (ii) We will then determine the effect of specific, local inhibition of STAT1 and STAT4, known signaling factors of downstream IL-12 effector functions. We will determine the effect of blocking these specific signals, using well-established techniques of intraspinal injection of AAV-driven shRNA, followed by SCI, and determination of tissue preservation and the inflammatory response, using cell-specific transcriptomic and spatial profiling approaches. Finally, we will (iii) assess the functional impact of STAT1 and STAT4 on locomotor and autonomic recovery. In summary, this proposal will provide valuable insights into the mechanism of pro-inflammatory cytokine function after SCI. It can contribute to developing novel therapeutic approaches in the future with the goal to reduce tissue damage and improve function. This project has high significance for the active military population, veterans, and civilians, given the incidence of spine trauma and SCI, and the immense potential in targeting inflammation for mitigating secondary damage after SCI to improve functional outcomes, health, and quality...