EXPRESS] Could an endoneurial endothelial crosstalk between Wnt/β-catenin and Sonic Hedgehog pathways underlie the early disruption of the infra-orbital blood-nerve barrier following chronic constriction injury

Background: Blood-nerve barrier (BNB) disruption is pivotal in the development of neuroinflammation, peripheral sensitization and neuropathic pain after peripheral nerve injury. Activation of Toll-Like Receptor 4 (TLR4) and inactivation of Sonic Hedgehog (SHH) signaling pathways within the endoneurial endothelial cells (EEC) are key events, resulting in the infiltration of harmful molecules and immunocytes within the nerve parenchyma. However, we showed in a previous study that preemptive inactivation of TLR4 signaling or sustained activation of SHH signaling did not prevent the local alterations observed following PNI (Moreau et al. 2016), suggesting the implication of another signaling pathway. Methods: Using a classical neuropathic pain model, the infra-orbital nerve chronic constriction injury (IoN-CCI), we investigated the role of the Wnt/beta-catenin pathway in CCI-mediated-BNB disruption and in its interactions with the TLR4 and SHH pathways. In the IoN-CCI model vs control, mRNA expression levels and/or immunochemical detection of major Wnt/SHH pathway (Frizzled-7, VE-cadherin, Patched-1, Gli-1) and/or Tight Junction (TJ) proteins (Claudin 1, Claudin 5, Occludin) readouts were assessed. Vascular permeability (VP) was assessed by sodium fluorescein extravasation. Results: IoN-CCI induced early alterations in the VE-cadherin/beta-catenin/Frizzled-7 complex, shown to participate in local BNB disruption via a beta-catenin-dependent TJ protein downregulation. Wnt pathway also mediated a crosstalk between TLR4 and SHH signaling within EEC. Nevertheless, preemptive inhibition of Wnt/beta-catenin signaling before IoN-CCI could not prevent the downregulation of key SHH pathway readouts or the disruption of the infra-orbital BNB, suggesting that SHH pathway inhibition observed following IoN-CCI is an independent event responsible for BNB disruption. Conclusion: A crosstalk between Wnt/beta-catenin- and SHH-mediated signaling pathways within EEC could mediate the chronic disruption of the BNB following IoN-CCI, resulting in increased irreversible endoneurial VP and neuropathic pain development.