Background & Aims

Migraine is a highly prevalent, debilitating neurological disorder characterized by painful headaches. These headaches are thought to reflect the sensitization and activation of trigeminal primary afferent neurons innervating the cortical meninges. Several lines of evidence suggest that meningeal immune cells are a primary source of mediators responsible for the sensitization of meningeal primary afferent neurons. Conversely, primary afferent neurons release neuropeptides that can modulate immune cell activity. Of particular importance in the context of migraine and migraine pain is the neuropeptide calcitonin-gene related peptide (CGRP), as suggested by the efficacy of recent anti-CGRP migraine therapeutics. However, the mechanism of action of these drugs is incompletely understood. We hypothesize that the efficacy of anti-CGRP migraine therapeutics is mediated by their ability to inhibit pro-inflammatory macrophages in the meninges.

Methods

Optogenetic spreading depression (OSD), a minimally invasive, optogenetic approach to induce cortical spreading depolarization, was used as a model of migraine. Identification of and changes to immune cell populations in the cortical meninges were assessed using flow cytometry. Fluorescence-activated cell sorting (FACS) paired with RT-qPCR as well as RNA fluorescent in situ hybridization (FISH) were used to assess the effects of CGRP and OSD on meningeal macrophage gene expression. Finally, two-photon microscopy was used to assess changes to macrophage morphology in response to CGRP and OSD in vivo.

Results

We have confirmed the ability to generate cortical spreading depolarization with blue light stimulation in mice expressing channelrhodopsin-2 under the Thy1 promoter (Thy1-ChR2-YFP mice). We have optimized our gating strategy for flow cytometry, replicating previous results of sex differences in meningeal immune cell subpopulations. FACS followed by RT-qPCR support the expression of CGRP receptor subunit mRNA (Calcrl and Ramp1) in meningeal macrophages, including in the CX3CR1-positive subset of meningeal macrophages. This is further supported by transcript visualization by RNA FISH. We have additionally established a method to visualize changes in morphology, and thereby activation state, of CX3CR1-positive macrophages in the meninges through an intact thinned skull.

Conclusions

Results from gene expression studies support a mechanism for the direct influence of CGRP on meningeal macrophages. Our in vivo two-photon microscopy imaging paradigm provides a longitudinal, complementary approach to assess changes in macrophage activity. Future studies will aim to assess the effects of CRGP and OSD on putative transcriptional changes in meningeal macrophages and correlate changes in macrophage phenotype with changes in macrophage morphology in vivo.

References

Avona, A, Burgos-Vega, C, Burton, MD, Akopian, AN, Price, TJ, and Dussor, G. Dural calcitonin gene-related peptide produces female-specific responses in rodent migraine models. J Neurosci, 2019. 39(22): 4323-4331.

Harriott, AM, Chung, DY, Uner, A, Bozdayi, RO, Morais, A, Takizawa, T, Qin, T, and Ayata, C. Optogenetic spreading depression elicits trigeminal pain and anxiety behavior. Ann Neurol, 2021. 89(1): 99-110.

Houben, T, Loonen, IC, Baca, SM, Schenke, M, Meijer, JH, Ferrari, MD, Terwindt, GM, Voskuyl, RA, Charles, A, van den Maagdenberg, AM, and Tolner, EA. Optogenetic induction of cortical spreading depression in anesthetized and freely behaving mice. J Cereb Blood Flow Metab, 2017. 37(5): 1641-1655.

McIlvried, LA, Cruz, JA, Borghesi, LA, and Gold, MS. Sex-, stress-, and sympathetic post-ganglionic-dependent changes in identity and proportions of immune cells in the dura. Cephalalgia, 2017. 37(1): 36-48.

McWhorter, FY, Wang, T, Nguyen, P, Chung, T, and Liu, WF. Modulation of macrophage phenotype by cell shape. Proc Natl Acad Sci U S A, 2013. 110(43): 17253-17258.

Schain, AJ, Melo-Carrillo, A, Borsook, D, Grutzendler, J, Strassman, AM, and Burstein, R. Activation of pial and dural macrophages and dendritic cells by cortical spreading depression. Ann Neurol, 2018.

Presenting Author

Talia Adi

Poster Authors

Talia Adi

BA

University of Pittsburgh School of Medicine

Lead Author

Keith J. Kaufman

MA

Lead Author

Ross S. Williamson

PhD

Lead Author

Michael S. Gold

PhD

University of Pittsburgh

Lead Author

Topics

  • Specific Pain Conditions/Pain in Specific Populations: Migraine