Reawakening inflammation in the chronically injured spinal cord using lipopolysaccharide induces diverse microglial states

Rehabilitative training is an effective method to promote recovery following spinal cord injury (SCI), with lower training efficacy observed in the chronic stage. The increased training efficacy during the subacute period is associated with an adaptive state induced by the SCI. A potential link is SCI-induced inflammation, which is elevated in the subacute period, and as injection of lipopolysaccharide (LPS) alongside training improves recovery in chronic SCI, suggesting LPS could reopen a window of plasticity late after injury. Microglia may play a role in LPS-mediated plasticity as they react to LPS and are implicated in facilitating recovery following SCI. However, it is unknown how microglia change in response to LPS following SCI to promote neuroplasticity. Here we used single-cell RNA sequencing to examine microglial responses in subacute and chronic SCI with and without an LPS injection. We show that subacute SCI is characterized by a disease-associated microglial (DAM) signature, while chronic SCI is highly heterogeneous, with both injury-induced and homeostatic states. With LPS injection, microglia shifted away from the homeostatic signature to a primed, translation-associated state and increased DAM in degenerated tracts caudal to the injury. Our results contribute to an understanding of how microglia and LPS-induced neuroinflammation contribute to plasticity following SCI. Female rats received a dorsolateral quadrant hemitransection injury, and then were perfused with HBSS(-/-). A 2 cm spinal cord section around the lesion site was extracted from rats at 8 days following injury (acute), 60 days following injury (chronic with saline), 60 days following injury and 3 days following an LPS injection (chronic with LPS). Spinal cords were manually cold disocciated using a dounce homogenizer into a single cell suspension and then fluorescent-activated cell sorting (FACS) was completed for live, nucleated and Cd11b+ cells.