Spatial Transcriptomics and Dual Dye Mapping Identify Wnt-Driven BBB Protection in Endothelial EphA4-Deficiency

Area of Science:

• Neuroscience
• Molecular Biology
• Biomedical Engineering

Background:

• Blood-brain barrier (BBB) disruption is a critical consequence of traumatic brain injury (TBI).
• The precise molecular mechanisms and spatial dynamics of BBB permeability post-TBI are not fully understood.
• Understanding these dynamics is crucial for developing effective TBI therapies.

Purpose of the Study:

• To investigate the temporal and spatial characteristics of BBB permeability after controlled cortical impact (CCI) injury.
• To explore the role of EphA4 in regulating BBB integrity following TBI.
• To identify molecular pathways, such as Wnt signaling, involved in BBB protection and repair after TBI.

Main Methods:

• Development of a dual dye-labeling system using Evans Blue Dye (EBD) and sodium fluorescein (NaFl) to assess BBB permeability.
• Utilizing EphA4 conditional knockout (KO) mice and wild-type (WT) littermates subjected to CCI.
• Integration of spatial transcriptomics with dye quantification to analyze gene expression changes in the injured brain.

Main Results:

• Distinct temporal patterns of BBB leakage were observed for NaFl (7 days) and EBD (4 days) post-CCI.
• EphA4 deletion in endothelial cells (ECs) significantly enhanced BBB integrity.
• EphA4 EC ablation upregulated key BBB-related genes (e.g., Tjp2, Cldn1) and neuroprotective genes (e.g., Nr4a1).
• Wnt signaling pathway genes were upregulated in KO mice, and Wnt pathway activation via Frizzled-4 (FZD4) agonist FZM1.8 reduced lesion volume and BBB disruption.

Conclusions:

• Spatial transcriptomics and dual-dye labeling are effective tools for studying TBI-induced BBB disruption.
• EphA4 signaling plays a significant role in regulating BBB integrity post-TBI.
• The Wnt signaling pathway represents a promising therapeutic target for mitigating secondary brain injury and promoting BBB repair following TBI.