Endothelial ephrin-B2 knockdown increases post-traumatic disruption of the blood-spinal cord barrier following spinal cord injury

Traumatic Spinal Cord Injury (SCI) disrupts the Blood-Spinal Cord Barrier (BSCB), amplifying injury processes. Targeted therapies require deeper insight into underlying mechanisms. Endothelial Ephrin-B2, a guidance molecule regulating cell adhesion and repulsion, might influence the BSCB, but its role after SCI remains unknown. This study investigates Ephrin-B2 in endothelial cells (ECs) and its influence on BSCB integrity in a mouse SCI model up to 28 days. Transgenic mice with Tamoxifen-inducible EC-specific Ephrin-B2 knockdown (efnb2iΔEC, n = 79) and Cre-negative littermates (efnb2lox/lox, n = 71) received serial Tamoxifen-injections. Animals underwent thoracic clip-compression injury (Th6/7, 60s, 5 g, n = 128) or Sham injury (laminectomy, n = 22). At postoperative days 1, 3, 7, 14 and 28 in vivo analysis included neurological assessment (Basso Mouse Scale, Tally Subscore, CatWalkTM automated gait analysis) and 7 T MRI (T2 Turbo Rare). Ex vivo analysis included Evans Blue assay ( n = 5 per group/timepoint), vessel perfusion, and immunohistochemistry ( n = 4 per group/timepoint) examining vessel density (CD31, FITC-Lectin), pericyte coverage (CD31, Desmin, PDGFR-beta), and junction proteins (Claudin-5, VE-Cadherin). efnb2iΔEC mice showed increased BSCB disruption during the acute phase (1d: p = 0.0117; 3d: p = 0.0153) accompanied by a trend for increased edema on day 1 after SCI ( p = 0.0712). Vascular and junctional markers revealed posttraumatic impairment with regeneration after day 7, without significant inter-group differences ( p > 0.05). Neurological recovery however was not influenced by EC-specific Ephrin-B2 knockdown (p > 0.05). Endothelial Ephrin-B2 is crucial for BSCB resilience after SCI. Its EC- specific knockdown aggravates barrier disruption and influences edema volume, suggesting Ephrin-B2 as a potential therapeutic target for acute SCI. • Ephrin-B2 is upregulated after SCI, but its role remains largely unexplored. • Endothelial Ephrin-B2 knockdown aggravates BSCB breakdown and increases edema. • Endothelial Ephrin-B2 may be a therapeutic target to stabilize the BSCB after SCI.