PACAP and Other Neuropeptide Targets Link Chronic Migraine and Opioid-induced Hyperalgesia in Mouse Models

Abstract

Chronic use of opioids can produce opioid-induced hyperalgesia (OIH), and when used to treat migraine, these drugs can result in increased pain and headache chronicity. We hypothesized that overlapping mechanisms between OIH and chronic migraine occur through neuropeptide dysregulation. Using label-free, non-biased liquid chromatography-mass spectrometry to identify and measure changes in more than 1500 neuropeptides under these two conditions, we observed only 16 neuropeptides that were altered between the two conditions. The known pro-migraine molecule, calcitonin-gene related peptide, was among seven peptides associated with chronic migraine, with several pain-processing neuropeptides among the nine other peptides affected in OIH. Further, composite peptide complements Pituitary adenylate cyclase-activating polypeptide (PACAP), Vasoactive intestinal peptide (VIP) and Secretogranin (SCG) showed significant changes in both chronic migraine and OIH. In a follow-up pharmacological study, we confirmed the role of PACAP in models of these two disorders, validating the effectiveness of our peptidomic approach, and identifying PACAP as a mechanistic link between chronic migraine and OIH. Data are available via ProteomeXchange with identifier PXD013362.

Keywords: Peptides; animal models; headache; hyperalgesia; mass spectrometry; mouse models; neurobiology*; pain; peptidomics.

Graphical abstract

Fig. 1.

A, Nervous system regions collected for the peptide quantitation analysis. DH, dorsal horn; H, hypothalamus; NAc, nucleus accumbens; PAG, periaqueductal gray; RVM, rostroventral medulla; TG, trigeminal ganglia; TN, trigeminal nucleus caudalis. Treatment paradigm for inducing (B) migraine-associated pain and (C) OIH. Chronic NTG and chronic morphine both produce hyperalgesia. Responses to mechanical von Frey hair stimulation was determined before drug/vehicle injection. D, Mice treated every other day with NTG (10 mg/kg, IP). E, Mice treated with morphine twice daily (days 1–3, 20 mg/kg, day 4, 40 mg/kg, SC). Both data sets were analyzed by two-way repeated measures ANOVA, p < 0.001 time, drug, and interaction, ***p < 0.001 as compared with vehicle day 1 responses. n = 15/group.

Fig. 2.

Experimental design and workflow for tissue collection and peptide extraction followed by label-free MS-based quantitation. Regions of the nervous system were processed and analyzed individually.

Fig. 3.

Select peptide level changes as determined by quantitative mass spectrometry for (A) migraine and (B) OIH models. *p ≤ 0.05, **p ≤ 0.01. The histogram values represent the Log2 transformed value of each peptide signal. Error bars represent standard deviation of mass spectrometric signals corresponding to the respective peptides. Full list of altered peptides given in supplemental Table S3. C, Chord diagram of neuropeptide overlap detected among regions of the nervous system: H: hypothalamus; PAG: periaqueductal gray; TG: trigeminal ganglia; TN: trigeminal nucleus; DH: dorsal horn; RVM: rostroventral medulla; NAc: nucleus accumbens. In this figure, the thickness of the lines between two regions is directly proportional to the extent of overlap between two regions. D, Functional classification of select proteins affected by migraine and OIH conditions. Protein selection was based on detection of significant level differences among protein-derived peptides detected in samples of the nervous system regions collected from migraine and OIH animal models.

Fig. 4.

Composite prohormone profiling.A, An example of proSAAS prohormone with the detected, quantified peptides originated from conventional mono/dibasic cleavage sites (i); quantification of the prohormone from additive signal of detected constituent peptides of proSAAS for the OIH model in the PAG (ii). B, Overlap of the prohormones with the composite prohormone signature significantly different (p ≤ 0.1) between the treatment and control groups for both migraine and OIH. VIP, SCG and PACAP are common to both migraine and OIH conditions. proTRH and PENK are exclusive to the migraine group whereas proSAAS is exclusive to the OIH group. C, Studied regions labeled with the prohormones that are significantly different between the control and treatment groups.

Fig. 5.

PAC1 inhibition by M65 blocked pain induced by chronic NTG and morphine. To induce chronic migraine-associated pain, mice were treated every other day, but periorbital allodynia was determined only on days 1, 5, and 9. Allodynia was assessed before treatment (A, basal responses) and 2 h after drug administration (B, post-treatment responses). M65 (0.1 mg/kg IP) or vehicle (VEH) was injected 1 h, 30 min post-NTG/VEH. (A) M65 blocked the development of basal hypersensitivity, 2-way repeated measures ANOVA, p < 0.01 treatment and interaction, ***p < 0.001 as compared with VEH-VEH on day 1; and (B) acute NTG-induced allodynia, 2-way repeated measures ANOVA, p < 0.001 treatment only. C, To evoke OIH, mice were administered morphine or vehicle twice daily for 4 days (days 1–3, 20 mg/kg; day 4, 40 mg/kg, SC), but periorbital allodynia was only assessed on days 1 and 3 of treatment, and 15–18 h after the final treatment (day 5). Morphine treatment caused significant periorbital allodynia, 2-way repeated measures ANOVA, p < 0.01 drug, time and interaction, **p < 0.01, ***p < 0.001 as compared with VEH-VEH on day 1. D, Administration of M65 24 h after the last injection of morphine/VEH (day 5) reversed OIH, 2-way ANOVA, p < 0.01 pretreatment (VEH/morphine), drug (M65/VEH), and interaction, ***p < 0.001. n = 6/group. PACAP is an overlapping mechanism involved in the development of both chronic migraine-associated pain and opioid-induced hyperalgesia.