Pilot: Hypoxia Induces Upregulation of CD39/73 and Immunomodulation in CNS Injury and Repair

Abstract Hundreds of thousands of people in the US alone live with spinal cord injury (SCI), have cervical-level damage and are currently in the chronic stage of injury. Despite the likelihood of lifelong respiratory, motor, and sensory deficits, many studies investigating SCI pathology and outcomes employ acute-stage therapies that focus on a single or select few outcomes. As exercise and hypoxia have individually shown benefits as treatments after CNS injury, we adapted “High-Low” training, a regimen employed by elite athletes, as an experimental treatment combining both interventions in a rodent model of chronic cervical SCI. High-Low (H-L) combines periods of rest at high altitude conditions (i.e. hypoxia) with regular exercise at typical conditions (i.e. normoxia). We implemented voluntary overnight exercise followed by sustained exposure to hypoxia starting 6 weeks after SCI in adult female rats. General anxiety, motor, and respiratory measures were assessed at various time points before and after treatment, and sensory function was assessed at the endpoint. The H-L experiment recently concluded and data analyses of outcomes is underway, with many tissues collected and banked for future evaluation. The H-L group exhibited significant reduction in anxiety like behavior, improved motor coordination, and enhanced respiratory recovery compared to untreated, exercise-only, and hypoxia-only controls. This Pilot Project proposes to use banked H-L rat samples followed by in vivo/ex vivo mouse studies to identify mechanisms supporting multimodal efficacy. The mouse studies will focus on hypoxia-induced alterations to adaptive immune responses and ATP catabolism, while future studies will examine the specific role of exercise. Aim 1 will determine if 8 weeks of H-L induces a significant increase in CD39 and CD73 expression on isolated rat splenic lymphocytes, resulting in improved mitochondrial energetics, using banked tissues. Aim 2 will determine if repeated hypoxia treatment in mice results in anti-inflammatory adaptive immune cells that have reduced ROS generation and improved response to a mitochondrial stress test. We will use flow cytometry, molecular (Adenosine production), and biochemical (Seahorse, Oroboros) assays to identify the novel mechanisms by which combinatorial H-L induces an anti-inflammatory, neuro-reparative phenotype in chronic SCI.