Activating Systemic Inflammation with LPS Drives Microglia-Mediated Degradation of Perineuronal Nets after Cervical Spinal Cord Injury in Female Lewis Rats

STUDY PURPOSE: Previously, we demonstrated that combining lipopolysaccharide (LPS)-induced inflammation with motor task–specific training enhances performance in rats after cervical spinal cord injury (SCI), even after performance had plateaued. However, the biological mechanisms underlying our observation remain unclear. In this study, we first evaluated whether a subacute inflammatory response induced by LPS promotes the breakdown of perineuronal nets (PNNs), thereby removing a barrier to neuroplasticity in adult female Lewis rats with incomplete cervical SCI. Subsequently, we determined whether PNNs degradation is mediated by microglial activation by LPS, providing a mechanistic explanation for the improved performance observed in our prior study. DATA COLLECTED: This study comprised two complementary experiments using a total of 64 age-matched adult female Lewis rats (10–12 weeks old). In the first experiment (n=24), animals received a right-sided C4 dorsolateral quadrant transection. Injury efficacy was confirmed by impaired right forepaw positioning (i.e., “close paw”) 24 h post-SCI. After a 10-day recovery period, rats were randomly assigned to receive a single intraperitoneal injection of LPS (1.5mg/kg) or saline under isoflurane anesthesia. Sickness behavior was longitudinally assessed at 4, 8, 12, 24, 36, and 48h post-injection using a composite score (0–3) evaluating social isolation, piloerection, ptosis, body temperature, and weight loss. Liver weight was recorded at euthanasia. Five days after LPS/saline administration, animals were perfused for histological analysis. Perineuronal nets (PNNs) surrounding ventral motor neurons were detected by co-labeling choline acetyltransferase (ChAT) with Wisteria floribunda agglutinin (WFA) and anti-aggrecan antibody. High-resolution confocal images (63×) were acquired from cervical (rostral to lesion), thoracic, and lumbar segments; only motor neurons with cross-sectional area 350µm² were analyzed. PNN integrity was quantified as fluorescence intensity using Fiji/ImageJ. Microglial density was assessed via Iba1/DAPI co-labeling rostral to the lesion core. Microglial involvement in PNN degradation was evaluated by quantifying Iba1 cell density, phagocytic activity (CD68), and internalization of PNN components (WFA/aggrecan) via 3D Imaris reconstructions. Lesion extent was determined by GFAP immunostaining, calculating the percentage of damaged versus spared tissue at the epicenter. Microglial activation at the lesion core was evaluated by Iba1 fluorescence intensity after background subtraction from an adjacent Iba1-negative region; both raw and background-corrected values are reported.
A second experiment (n=40) investigated whether microglial inhibition prevents LPS-induced PNN degradation. Following identical SCI induction and confirmation, forelimb locomotor function was assessed using the Forelimb Locomotor Scale (FLS) at 5 and 15 days post-injury. After 10 days of recovery, rats were assigned to four groups (n=10/group): Control (daily saline), LPS (single LPS dose followed by saline), Minocycline (daily minocycline 35mg/kg), and LPS+Minocycline (single LPS followed by daily minocycline). Treatments were administered intraperitoneally for five consecutive days. Sickness behavior and liver weight were assessed as in Experiment 1. Histological analyses for PNN integrity, microglial parameters, and lesion extent followed identical protocols to the first experiment. All quantitative image analyses were performed blinded using Fiji/ImageJ. DATA USAGE NOTES: