CSD-Induced Arterial Dilatation and Plasma Protein Extravasation Are Unaffected by Fremanezumab: Implications for CGRP's Role in Migraine with Aura

Abstract

Cortical spreading depression (CSD) is a wave of neuronal depolarization thought to underlie migraine aura. Calcitonin gene-related peptide (CGRP) is a potent vasodilator involved in migraine pathophysiology. Evidence for functional connectivity between CSD and CGRP has triggered scientific interest in the possibility that CGRP antagonism may disrupt vascular responses to CSD and the ensuing plasma protein extravasation (PPE). Using imaging tools that allow us to generate continuous, live, high-resolution views of spatial and temporal changes that affect arteries and veins in the dura and pia, we determined the extent to which CGRP contributes to the induction of arterial dilatation or PPE by CSD in female rats, and how these events are affected by the anti-CGRP monoclonal antibody (anti-CGRP-mAb) fremanezumab. We found that the CSD-induced brief dilatation and prolonged constriction of pial arteries, prolonged dilatation of dural arteries and PPE are all unaffected by fremanezumab, whereas the brief constriction and prolonged dilatation of pial veins are affected. In comparison, although CGRP infusion gave rise to the expected dilatation of dural arteries, which was effectively blocked by fremanezumab, it did not induce dilatation in pial arteries, pial veins, or dural veins. It also failed to induce PPE. Regardless of whether the nociceptors become active before or after the induction of arterial dilatation or PPE by CSD, the inability of fremanezumab to prevent them suggests that these events are not mediated by CGRP, a conclusion with important implications for our understanding of the mechanism of action of anti-CGRP-mAbs in migraine prevention.SIGNIFICANCE STATEMENT The current study identifies fundamental differences between two commonly used models of migraine, CSD induction and systemic CGRP infusion. It raises the possibility that conclusions drawn from one model may not be true or relevant to the other. It sharpens the need to accept the view that there is more than one truth to migraine pathophysiology and that it is unlikely that one theory will explain all types of migraine headache or the mechanisms of action of drugs that prevent it. Regarding the latter, it is concluded that not all vascular responses in the meninges are born alike and, consequently, that drugs that prevent vascular dilatation through different molecular pathways may have different therapeutic outcomes in different types of migraine.

Keywords: CGRP; CSD; PPE; aura; migraine; vasodilation.

Figure 1.

Experiment timeline. Diagram of the timing of injections and imaging for experimental rats.

Figure 2.

In vivo imaging of meningeal vasculature in the rat. A, Example images from an image stack including the skull, dura, and pia of the rat. Intravenous FITC-dextran is green, the second harmonic generation from skull and dura is blue, and intrinsic fluorescence (from lipofuscin or other molecules) is red. B, Orthogonal reconstruction of image stack in A, showing the locations of blood vessels in relationship to skull, dura, pia, and brain. C, Top images were taken before FITC injection and show only intrinsic fluorescence using an excitation wavelength of 800 nm, in the dura (left images) and pia (right images). Bottom images are taken at 890 nm after FITC injection. Note the green border (arrows) at 800 nm around arteries but not veins, as well as a darkened paravascular space surrounding pial arteries (arrowheads in top right image). D, Representative images of a dural artery (top), pial artery (middle), and pial vein (bottom) during CSD. E, Quantification of the fold change in diameter of pial and dural arteries and veins during CSD in a representative rat. Scale bars: A–D, 50 μm.

Figure 3.

Effects of fremanezumab on CSD-induced vasculature changes. A–D, Plots of the average fold change in pial arteries (A), dural arteries (B), pial veins (C), and dural veins (D) during CSD in rats injected with drug (n = 7) and vehicle (n = 9). The p value in C indicates the significant difference between fremanezumab and saline for the normalized vein diameters averaged per rat over the time indicated in blue and red (500–1500 s).

Figure 4.

Effects of fremanezumab on CSD-induced PPEEs. A, Example images of a PPEE occurring on the border of a dural blood vessel (red arrow). B, Representative image showing location of PPEEs (red circles) during CSD in one rat. C, Quantification of average frequency of PPEEs per 250 s bins (red) overlaid with a plot of average dural artery diameter (green) post-CSD. D, Quantification of the average number of PPEEs during CSD in rats injected with drug (red) and vehicle (blue).

Figure 5.

Effects of fremanezumab on CGRP infusion-induced vasculature changes. CGRP was infused into the blood after recovery from CSD in most rats. In two vehicle-treated rats, CGRP was infused before CSD was induced. A–D, Plots of the average fold change in pial arteries (A) pial veins (B), dural arteries (C), and dural veins (D) during CGRP infusion in rats treated with (n = 5) and vehicle (n = 8). The p value in B indicates significant differences between fremanezumab and saline for the normalized dural artery diameter averaged per rat across the time frame indicated in blue and red (20–300 s).

Figure 6.

Effects of fremanezumab on CGRP infusion-induced PPEEs. A, Quantification of the average frequency of PPEEs in 250 s bins during CGRP infusion in fremanezumab- and saline-injected rats. B, Representative image showing locations of PPEEs (red circle) during CGRP infusion in one rat (the same rat as in Fig. 2G).

Figure 7.

Presence of fremanezumab in the dura. A, In vivo images of the dura (left) and pia (right) of a rat 4 h after infusion with Alexa Fluor 594-conjugated fremanezumab (Fr594; representative of n = 2 rats). Labeled fremanezumab is indicated as red fluorescent areas (arrows) in the dura outside of blood vessels (bv). Note the lack of labeling outside of blood vessels in the pia (arrowhead). B, Fluorescent images of fixed dura removed from rats that did not undergo skull thinning and were injected with control nonfluorescent fremanezumab (left) or Fr594 (right; representative of n = 2 rats). C, In vivo images of a mouse expressing EYFP in the brain (thy1-EYFP), after injection with Fr594. The mouse scalp was cut, but the skull was not thinned. Images are shown at 5 m post injection (left and middle), with the EYFP channel displayed only in the left images, and at 180 m post injection (right) (representative of n = 2 mice). Note the presence of fremanezumab in the dura after 180 m (arrow) but not the pia or cortex. Scale bars: A–C, 50 μm.

Figure 8.

Summary diagram. Diagram of the CGRP-dependent and CGRP-independent mechanisms in two scenarios involving CSD-induced headache.