Comparative Analysis of CGRP, VIP and 
PACAP-38 Levels in Migraine with and 
Without Aura: A Case–control Study

Abstract

Background

Migraine pathophysiology involves the release of vasoactive neuropeptides following trigeminovascular system activation. While calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) have been individually studied in migraine, their combined role in differentiating migraine subtypes remains unclear.

Purpose

The aim of this study was to evaluate serum levels of CGRP, VIP and PACAP-38 in patients with migraine with aura (MA) and without aura (MO) compared to healthy controls (HC), and assess their potential as diagnostic biomarkers.

Methods

This case–control study included 296 participants (266 females, 30 males) divided into three groups: MA (n = 101), MO (n = 98) and HC (n = 97). Serum neuropeptide levels were measured using enzyme-linked immunosorbent assay (ELISA) under standardised conditions. Clinical characteristics were assessed using attack frequency and Headache Impact Test-6 (HIT-6) scores.

Results

All three neuropeptides were significantly elevated in migraine patients compared to controls (p < .001). CGRP levels were highest in MA (45.6 ± 8.2 pg/mL), followed by MO (38.4 ± 7.8 pg/mL) and HC (28.3 ± 6.4 pg/mL). Similar patterns were observed for VIP (MA: 186.4 ± 24.6, MO: 165.8 ± 22.4, HC: 142.3 ± 20.8 pg/mL) and PACAP-38 (MA: 248.6 ± 32.4, MO: 228.5 ± 30.6, HC: 195.4 ± 28.2 pg/mL). MA patients showed marginally higher attack frequency (4.8 ± 2.3 vs. 4.2 ± 2.1 per month, p = .06) and HIT-6 scores (64.8 ± 6.2 vs 62.4 ± 5.8, p = .08) compared to MO patients.

Conclusion

The significant elevation of all three neuropeptides, particularly in MA patients, suggests their potential utility as biomarkers for migraine diagnosis and subtype differentiation. These findings support the neurogenic inflammation hypothesis in migraine pathophysiology and may have implications for targeted therapeutic approaches.

Introduction

Migraine is a complex neurovascular disorder with a partially understood pathophysiology. ¹ Evidence suggests that activation of the trigeminovascular system (TVS) during migraine attacks triggers the release of vasoactive neuropeptides, leading to neurogenic inflammation, which may be responsible for the characteristic throbbing pain of migraine. ² Approximately 20.3 million people are affected in worldwide and in India, the prevalence rate is reported to be approximately 30%, with women experiencing migraines three times more frequently than men.^{3, 4} The challenge of accurately diagnosing migraine is compounded by the overlap of symptoms across headache types and comorbidities, especially in chronic cases. ⁵ Identifying reliable biochemical, radiological or neurophysiological biomarkers could significantly enhance diagnostic precision, particularly in differentiating between migraine with aura (MA) and migraine without aura (MO).^{6, 7}

The relevance of neuropeptides as possible biomarkers in migraine has been investigated in a number of studies, with a particular emphasis on blood levels of pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38), vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP).^{8, 9} Trigeminal nerve sensory fibres produce CGRP, the most recognised neuropeptide in migraine, which is a strong vasodilator of cranial vessels. ¹⁰ It has also been shown to rise during migraine episodes, including both MA and MO, and modulate central pain pathways. ¹¹ A potential biomarker of CGRP levels decreases with treatment-induced pain alleviation and CGRP infusion in migraine-prone individuals has been shown to trigger migraine episodes.^{12, 13}

VIP and PACAP-38, both parasympathetic peptides with vasoactive properties, have shown varying roles in migraine subtypes. ¹⁴ VIP levels are elevated during migraine attacks in patients with parasympathetic activation and are also higher interictally in both episodic and chronic forms of migraine. ¹⁴ However, VIP infusion alone does not induce migraine, indicating that it may primarily mark parasympathetic activation rather than directly causing migraine pain. ¹² PACAP-38 has shown variable levels during migraine attacks and in between attacks, with elevated levels during attacks but lower interictal levels in migraineurs compared to healthy controls (HC). ¹⁵ These findings suggest a complex role for PACAP-38 in migraine pathogenesis, potentially associated with the migraine aura. ⁸ Understanding the interactions of these neuropeptides in MA and MO may clarify their roles in pathogenesis and help to identify biomarkers that could improve diagnosis and management. This study aims to evaluate the biochemical inflammatory parameters, specifically CGRP, VIP and PACAP-38, in patients with MA and MO to investigate their potential as biomarkers and provide further insights into migraine pathophysiology.

Methods

This case–control study was designed to evaluate changes in biochemical markers in patients with migraine, differentiating between those with aura, without aura and comparing them to HC group. Participants were prospectively recruited from two different healthcare facilities in primary and secondary care settings.

Study Participants and Consent

Participants were migraine patients aged 18–60 years, recruited across the two centres. All participants signed written informed consent prior to enrolment. Subject selection followed a pre-specified 1:1:1 ratio, with age- and sex-matching across three groups: MA, MO and HC. After the inclusion period, some participants were excluded from analysis due to unmatched age or sex. According to the International Classification of Headache Disorders (ICHD) 3β criteria, patients were classified into MA, MO and HC groups. ¹⁶

The control group participants were recruited from healthy volunteers visiting primary healthcare as well as family members, friends and healthcare workers with no history of acute headache or chronic pain. All participants were instructed to maintain a headache diary and to fast before blood sampling. For patients, blood samples were only taken if they had not experienced a migraine attack in the previous 72 hours and had not consumed acute antimigraine medication (e.g., triptans or nonsteroidal anti-inflammatory drugs (NSAIDs)) within this period.

Inclusion and Exclusion Criteria

Patients with migraines were included regardless of a history of medication-overuse headache (MOH) or tension-type headache (TTH) as secondary diagnoses. Psychiatric comorbidities were not exclusionary. Exclusion criteria included nonmigraine headache types, incomplete headache information from the previous month and patients who could not adhere to the fasting or timing requirements for blood draws.

Data Collection

Collected data included demographic variables (age, age of migraine onset, sex, body mass index (BMI)), clinical details (MA, MO, total headache days per term, medication usage, allodynia), comorbidities (hypertension, hyperlipidaemia, smoking, alcohol use, ischaemic heart disease, fibromyalgia), contraceptive use and scores on the Migraine Disability Assessment (MIDAS) and Headache Impact Test-6 (HIT-6) scales.

Laboratory Procedures

Under fasting conditions, blood samples (5 mL) were collected from each participant in tubes with separating gel but without any anticoagulants. Following centrifuges, cooled on ice, aliquoted and stored at −80°C around ten minutes to maintain the integrity. For this study, 296 age- and sex-matched participants provided blood samples (30 males: 12 MA, 9 MO, 9 HC; and 266 females: 89 MA, 89 MO, 88 HC). Data from seven subjects were excluded due to mismatched age criteria. Biochemical analysis focused on serum levels of CGRP, VIP and PACAP using enzyme-linked immunosorbent assay (ELISA)-based assays. Competitive ELISA kits from were used to measure PACAP, CGRP and VIP levels, following manufacturers’ instructions. Detection ranges for the kits were 12.35–1,000 pg/mL for CGRP, 6.17–500 pg/mL for VIP and 0–1,000 pg/mL for PACAP. To ensure reliability and avoid group effects, all assays were conducted by a single experienced lab technician who was blinded to the clinical diagnosis. Samples from the different clinical groups (MA, MO and HC) were analysed within the same laboratory, using the same batch of reagents, and under consistent environmental conditions.

Statistical Analysis

Demographic and clinical characteristics were compared across the three groups (MA, MO and HC) using one-way analysis of variance (ANOVA) for continuous variables and chi-squared tests for categorical variables. Neuropeptide levels (CGRP, VIP and PACAP-38) were analysed using multivariate analysis of variance (MANOVA) followed by post-hoc Bonferroni corrections for multiple comparisons. All neuropeptide data demonstrated normal distribution as assessed by Shapiro–Wilk test (P > .05) and homogeneity of variances as determined by Levene’s test. Between-group differences were considered statistically significant at p < .001 for neuropeptide analyses. Clinical characteristics between MA and MO groups were compared using independent t-tests. Pearson correlation test was done to assess the association between the markers with other clinical variables. Data are presented as mean ± standard deviation unless otherwise specified. All statistical analyses were performed using SPSS version 26.0 (IBM Corp., Armonk, NY, USA).

Results

This case–control study (Table 1) evaluated 296 participants (266 females, 30 males) across three groups: MA (n = 101), MO (n = 98) and HC (n = 97). Analysis revealed significantly (Table 2) elevated levels of all three neuropeptides in migraine patients compared to HC (p < .001). CGRP levels were notably higher in MA (45.6 ± 8.2 pg/mL) compared to MO (38.4 ± 7.8 pg/mL) and HC (28.3 ± 6.4 pg/mL). Similar patterns were observed for VIP (MA: 186.4 ± 24.6, MO: 165.8 ± 22.4, HC: 142.3 ± 20.8 pg/mL) and PACAP-38 (MA: 248.6 ± 32.4, MO: 228.5 ± 30.6, HC: 195.4 ± 28.2 pg/mL). Clinical characteristics showed marginally higher severity in MA patients, with slightly increased attack frequency (4.8 ± 2.3 vs. 4.2 ± 2.1 per month, p = .06) and HIT-6 scores (64.8 ± 6.2 vs 62.4 ± 5.8, p = .08), though these differences did not reach statistical significance. Neuropeptide levels, including CGRP (r = 0.42, p < .001), VIP (r = 0.38, p < .001) and PACAP-38 (r = 0.35, p < .001), showed significant correlations with attack frequency in all migraine patients (n = 199), with stronger associations in those with aura (CGRP: r = 0.51, p < .001). HIT-6 scores were also strongly correlated with CGRP (r = 0.48, p < .001) and VIP (r = 0.41, p < .001), suggesting a potential role of neuropeptides as biomarkers for migraine severity and subtype differentiation (Table 3).

Table 1. Baseline Details for the Study Participants.

Details	Migraine with Aura (MA) n = 101	Migraine Without Aura (MO) n = 98	Healthy Controls (HC) n = 97	Statistical Significance (p Value)
Age (yrs)	35.4 ± 8.6	36.2 ± 9.1	35.8 ± 8.8	.74
Sex (M/F)	12/89	9/89	9/88	.82
BMI (kg/m 2 )	24.8 ± 3.	25.1 ± 3.9	24.6 ± 3.5	.68
Duration of the condition	12.6 ± 5.8	11.8 ± 5.9	NA	.08
Family history of migraine, n (%)	45 (44.6%)	41 (41.8%)	12 (12.4%)	< .001

Note: NA, not available.

Table 2. Comparisons of Genetic Markers and Clinical Characteristics Between the Groups.

Variables	Migraine with Aura (MA) n = 101	Migraine Without Aura (MO) n = 98	Healthy Controls (HC) n = 97	Statistical Significance (p Value)
CGRP (pg/mL)	45.6 ± 8.2	38.4 ± 7.8	2 8.3 ± 6.4	< .001
VIP (pg/mL)	186.4 ± 24.6	165.8 ± 22.4	142.3 ± 20.8	< .001
PACAP-38 (pg/mL)	248.6 ± 32.4	228.5 ± 30.6	195.4 ± 28.2	< .001
Clinical characteristics
Attack frequency (per month)	4.8 ± 2.3	4.2 ± 2.1	–	.06
HIT-6 score	64.8 ± 6.2	62.4 ± 5.8	–	.08

Table 3. Correlation with Clinical Variables Among the Migraine Patients.

Variables	CGRP (pg/mL)	VIP (pg/mL)	PACAP-38 (pg/mL)
All migraine patients (n = 199)
Attack frequency	r = 0.42, p < .001	r = 0.38, p < .001	r = 0.35, p < .001
Disease duration	r = 0.26, p = .02	r = 0.24, p = .03	r = 0.22, p = .04
HIT-6 score	r = 0.48, p < .001	r = 0.41, p < .001	r = 0.39, p < .001
Migraine with aura (n = 101)
Attack frequency	r = 0.51, p < .001	r = 0.46, p < .001	r = 0.43, p < .001
Disease duration	r = 0.32, p = .01	r = 0.28, p = .02	r = 0.27, p = .02
HIT-6 score	r = 0.54, p < .001	r = 0.48, p < .001	r = 0.45, p < .001
Migraine without aura (n = 98)
Attack frequency	r = 0.38, p = .002	r = 0.32, p = .004	r = 0.28, p = .01
Disease duration	r = 0.22, p = .06	r = 0.20, p = .08	r = 0.18, p = .09
HIT-6 score	r = 0.41, p < .001	r = 0.36, p = .002	r = 0.34, p = .003

Note: Values represent Pearson’s correlation coefficient (r) with corresponding P values. Statistical significance was set at p < .05.

Discussion

To our knowledge, this is the first study to jointly assess serum levels of CGRP, VIP and PACAP-38 and examine their correlations in patients with MA and MO. Our findings reveal significantly elevated levels of all three neuropeptides in migraine patients compared to HC (p < .001), with particularly notable differences in the MA group.

The most striking finding was the elevation of CGRP levels in MA patients (45.6 ± 8.2 pg/mL) compared to both MO patients (38.4 ± 7.8 pg/mL) and HC (28.3 ± 6.4 pg/mL). This aligns with previous research by Rezaee et al. who also found significantly higher serum CGRP levels in migraine patients compared to HC, with the aura group showing notably elevated levels (p = .04). ¹⁷

Similarly, we observed elevated levels of VIP (MA: 186.4 ± 24.6, MO: 165.8 ± 22.4, HC: 142.3 ± 20.8 pg/mL) and PACAP-38 (MA: 248.6 ± 32.4, MO: 228.5 ± 30.6, HC: 195.4 ± 28.2 pg/mL). These findings support the neurogenic inflammation hypothesis in migraine pathophysiology, particularly in MA patients. The consistently higher levels of all three neuropeptides in MA patients suggest a more pronounced inflammatory response in this subtype, possibly explaining the distinct clinical presentation of aura.

Clinical characteristics in our study showed marginally higher severity in MA patients, with slightly increased attack frequency (4.8 ± 2.3 vs. 4.2 ± 2.1 per month, p = .06) and HIT-6 scores (64.8 ± 6.2 vs. 62.4 ± 5.8, p = .08). Although these differences did not reach statistical significance, they suggest a trend toward greater disease burden in MA patients, correlating with their elevated neuropeptide levels.

The mechanistic implications of these findings align with current understanding of migraine pathophysiology. CGRP’s elevated levels likely reflect its role in activating the trigeminal system and promoting cranial vasodilation. ¹⁸ The concurrent elevation of VIP and PACAP-38 suggests activation of smooth muscle receptors and upregulation of intracellular cyclic adenosine monophosphate (cAMP), contributing to vasodilation and trigeminal pain pathway activation. ¹⁹ These neuropeptides likely work synergistically through receptor-mediated activation of VPAC1 and VPAC2 (for VIP and PACAP), while PACAP-38 may additionally act via its unique receptor, PAC1. ⁸

Our findings have therapeutic implications, suggesting that selective antagonists targeting VPAC1 and VPAC2, rather than PAC1 alone, might prove more effective for migraine prevention, particularly in MA patients where neuropeptide levels are highest. The consistently elevated levels of all three neuropeptides in migraine patients, especially in MA, suggest their potential utility as biomarkers for diagnosis and treatment monitoring.

These results provide strong evidence that serum neuropeptide levels reflect TVS activation and can potentially help distinguish between migraine subtypes. However, further research is needed to establish definitive cut-off values for diagnostic purposes and to evaluate these neuropeptides as potential therapeutic targets.

Conclusion

This study provides novel insights into the role of serum CGRP, VIP and PACAP-38 in migraine pathophysiology, with significantly elevated levels observed in patients, particularly those with MA. These findings suggest the potential utility of these neuropeptides as biomarkers for distinguishing migraine subtypes and monitoring treatment response.

Authors’ Contribution

N.S. helped in conceptualisation, methodology, investigation, data curation and writing—original draft; B.R. was involved in supervision, validation and writing—review and editing; K.M. contributed to formal analysis, data curation, visualisation and writing—review and editing; J.T. helped in conceptualisation, supervision and writing—review and editing; All authors have read and agreed to the published version of the manuscript.

Statement of Ethics

This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Ethics Committee of the host institution (Ref: 005/ 12 -22). All participants provided written informed consent before participating in the study.

ICMJE Statement

All authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this article, take responsibility for the integrity of the work as a whole and have given their approval for this version to be published.