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Spatial transcriptomics and dual tracer mapping reveal Wnt-driven blood-brain barrier permeability Induced by endothelial EphA4 deficiency
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DOI：10.1186/s40478-026-02394-9.png)
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Abstract
En 中文
Blood-brain barrier (BBB) disruption is a key pathological event following traumatic brain injury (TBI), yet its molecular and spatial characteristics remain incompletely understood. Here, we developed a dual-tracer mapping system to assess the temporal and spatial dynamics of BBB permeability following controlled cortical impact (CCI) injury in EphA4f/f VE-Cadherin-CreERT2 (KO) and EphA4f/f (WT) mice. By tracking Evans Blue Dye (EBD), sodium fluorescein (NaFl), and endogenous IgG deposition, we identified distinct patterns of vascular tracer extravasation in the injured cortex and hippocampus. NaFl, a small-molecule tracer, continued to extravasate for 7 days post-injury, whereas EBD leakage diminished after 4 days. Notably, endothelial EphA4 deletion significantly reduced size-dependent vascular tracer extravasation. To further investigate molecular pathways associated with altered BBB permeability, we integrated spatial transcriptomics with tracer quantification, revealing that endothelial EphA4 ablation upregulates BBB-associated genes (Tjp2, Tjp3, Cldn1, and Ocln) and injury-responsive genes linked to injury-responsive genes (Nr4a1 and Npas4). Wnt signaling genes were similarly upregulated in the KO cortex, and pharmacological inhibition of Frizzled-4 (FZD4)/Wnt signaling attenuated the reduction in vascular tracer extravasation observed in KO mice. Conversely, pharmacological activation of Wnt signaling with the FZD4 agonist FZM1.8 in WT mice reduced lesion volume and vascular tracer extravasation. Overall, these findings demonstrate the utility of combining spatial transcriptomics with dual-tracer mapping to identify region-specific transcriptional programs associated with BBB permeability following CCI injury and to define the contribution of EphA4/Wnt signaling to vascular responses after TBI. Together, these findings identify Wnt signaling as a promising therapeutic pathway for reducing vascular permeability and limiting secondary brain injury following TBI.
Keywords:
Traumatic brain injury (TBI)
Eph signaling
Spatial transcriptomics
Brain microvasculature
Blood-brain barrier (BBB)
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