Meningeal lymphatic CGRP signaling governs pain via cerebrospinal fluid efflux and neuroinflammation in migraine models
Nathan P. Nelson-Maney, László Bálint, Anna L.S. Beeson, D. Stephen Serafin, Bryan M. Kistner, Elizabeth S. Douglas, Aisha H. Siddiqui, Alyssa M. Tauro, Kathleen M. Caron
Nathan P. Nelson-Maney, László Bálint, Anna L.S. Beeson, D. Stephen Serafin, Bryan M. Kistner, Elizabeth S. Douglas, Aisha H. Siddiqui, Alyssa M. Tauro, Kathleen M. Caron
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Research Article Cell biology Vascular biology

Meningeal lymphatic CGRP signaling governs pain via cerebrospinal fluid efflux and neuroinflammation in migraine models

Abstract

Recently developed antimigraine therapeutics targeting calcitonin gene–related peptide (CGRP) signaling are effective, though their sites of activity remain elusive. Notably, the lymphatic vasculature is responsive to CGRP signaling, but whether meningeal lymphatic vessels (MLVs) contribute to migraine pathophysiology is unknown. Mice with lymphatic vasculature deficient in the CGRP receptor (CalcrliLEC mice) treated with nitroglycerin-mediated (NTG-mediated) chronic migraine exhibit reduced pain and light avoidance compared with NTG-treated littermate controls. Gene expression profiles of lymphatic endothelial cells (LECs) isolated from the meninges of Rpl22HA/+;Lyve1Cre RiboTag mice treated with NTG revealed increased MLV-immune interactions compared with cells from untreated mice. Interestingly, the relative abundance of mucosal vascular addressin cell adhesion molecule 1–interacting (MAdCAM1-interacting) CD4+ T cells was increased in the deep cervical lymph nodes of NTG-treated control mice but not in NTG-treated CalcrliLEC mice. Treatment of cultured hLECs with CGRP peptide in vitro induced vascular endothelial–cadherin (VE-cadherin) rearrangement and reduced functional permeability. Likewise, intra cisterna magna injection of CGRP caused rearrangement of VE-cadherin, decreased MLV uptake of cerebrospinal fluid (CSF), and impaired CSF drainage in control mice but not in CalcrliLEC mice. Collectively, these findings reveal a previously unrecognized role for lymphatics in chronic migraine, whereby CGRP signaling primes MLV-immune interactions and reduces CSF efflux.

Authors

Nathan P. Nelson-Maney, László Bálint, Anna L.S. Beeson, D. Stephen Serafin, Bryan M. Kistner, Elizabeth S. Douglas, Aisha H. Siddiqui, Alyssa M. Tauro, Kathleen M. Caron

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Figure 5

RiboTag and in vitro changes are recapitulated in NTG-mediated chronic migraine.

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RiboTag and in vitro changes are recapitulated in NTG-mediated chronic m...
(A) Whole-mount microscopy of decalcified meninges from mice treated with vehicle or NTG. Secondary-only immunofluorescence included as negative control. Top, costaining of LYVE-1 (green) and Pentraxin3 (magenta). Bottom, increased magnification images of the white dashed square in top row. Black, Pentraxin3. Black dashed lines, MLV outlines. Scale bar: 20 μm. (B) Quantification of PTX3 fluorescence relative to background fluorescence in MLV endpoints. Significance calculated using 1-way ANOVA with n = 3–6 animals with at least 2 endpoints assessed per animal. 2 independent cohorts were assessed. (C) Flow cytometry gating strategy. Quantification of flow cytometric analysis of LPAM1+ (α4/β7 integrin+) CD4+ T cells in (D) DCLNs (draining meninges) and (E) inguinal lymph nodes (distal lymph nodes) of NTG-treated chronic migraine CalcrliLEC mice. n = 3 animals per group, pooled left and right DCLN from 2 independent cohorts, performed in duplicate. (F) Schematic indicating proposed relationship between CGRP, MADCAM1, and α4/β7 integrin+ (LPAM1+) CD4+ T cell interaction with LECs. Significance for all graphs calculated using 2-way ANOVA with Tukey’s multiple comparisons test. P value shown if less than 0.05. Graphs show mean ± SD.