Traumatic brain injury (TBI) is a leading cause of death and disability worldwide, and is indiscriminate in who it affects, including children. Although there are currently no Food and Drug Administration-approved therapeutics, promising results from recent induced pluripotent stem cell-derived neural stem cell (iNSC) studies have demonstrated decreased tissue damage and functional deficits in pre-clinical TBI models. Moreover, while the rest has been traditionally identified as the standard of care following TBI, research now suggests that physical activity postinjury may significantly enhance neuroprotective and regenerative signaling in patients. Combining these two therapies may therefore synergistically improve recovery outcomes in TBI patients. In this study, we evaluated the combined therapeutic efficacy of iNSCs and structured treadmill walking on cellular, tissue, and functional recovery in a translational pediatric pig TBI model. One-month-old piglets received a controlled cortical impact-induced TBI and were randomly assigned to either a PBS (n = 4), PBS + treadmill (n = 4), iNSC (n = 4), or iNSC + treadmill (n = 4) treatment group. Piglets received intraparenchymal transplantations of either iNSCs or PBS 5 days post-TBI. At 1-week post-transplantation, piglets assigned to the treadmill treatment groups began a 12-week progressive walking regimen. Motor function and open field behavior assessments were performed pre-TBI and 12 weeks post-transplantation. Magnetic resonance imaging (MRI) and histological evaluation of collected brain tissue were performed 12 weeks post-transplantation. Immunohistochemistry revealed long-term survival, engraftment, and differentiation of transplanted iNSCs into neurons, astrocytes, and oligodendrocytes in treated piglets. Furthermore, iNSC + treadmill treatment showed increased endogenous neuron and oligodendrocyte survival, increased proliferation of neuroblasts, and decreased populations of reactive astrocytes and immune cells in TBI brain tissue. MRI analysis revealed a significant reduction in lesion volume, midline shift, and white matter degradation with preserved cerebral blood flow following both iNSC and iNSC + treadmill interventions. These cellular and tissue-level effects corresponded with significant motor function recovery as seen through increased step and stride length with decreased stance percentage and time. During open field behavioral assessments, iNSC and iNSC + treadmill-treated piglets demonstrated improved exploratory behaviors. These findings suggest that the combination of iNSCs with structured treadmill walking significantly enhanced TBI recovery beyond the therapeutic potential of iNSCs or exercise alone. Therefore, this novel combination therapy needs to be further explored as a potential transformative treatment option for pediatric TBI patients.
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