Calcitonin gene-related peptide levels in tear fluid are elevated in migraine patients compared to healthy controls

Abstract

Background

Calcitonin gene-related peptide (CGRP) released from trigeminal nerve fibres indicates trigeminal activation and has a key role in migraine pathophysiology. The trigeminal nerve directly innervates the eye. Therefore, in this study, we compared Calcitonin gene-related peptide in tear fluid of migraine patients and healthy controls.

Methods

Calcitonin gene-related peptide concentrations in tear fluid and plasma of 48 episodic and 45 chronic migraine patients and 48 controls were assessed using ELISA.

Results

Calcitonin gene-related peptide levels in tear fluid (0.94 ± 1.11 ng/ml) were ∼140 times higher than plasma concentrations (6.81 ± 4.12 pg/ml). Tear fluid CGRP concentrations were elevated in interictal migraine patients (1.10 ± 1.27 ng/ml, n = 49) compared to controls (0.75 ± 0.80 ng/ml, p = 0.022). There was no difference in tear fluid CGRP levels between interictal episodic and chronic migraine patients (episodic: 1.09 ± 1.47 ng/ml, n = 30 and chronic: 1.10 ± 0.89 ng/ml, n = 19) and no correlation of tear fluid CGRP levels with headache frequency in interictal patients (rho = 0.062, p = 0.674). Unmedicated ictal migraine patients had even more elevated tear fluid CGRP levels than interictal migraine patients (1.92 ± 1.84 ng/ml, n = 13, p = 0.102), while medicated ictal migraine patients had lower levels (0.56 ± 0.47 ng/ml, n = 25, p = 0.011 compared to interictal patients), which were undistinguishable from controls (p = 0.609). In contrast to tear fluid, no significant group differences were found in plasma CGRP levels.

Conclusion

To the best of our knowledge, this study shows, for the first time, increased CGRP tear fluid levels in migraine patients compared to healthy subjects. Detection of calcitonin gene-related peptide in tear fluid is non-invasive, and likely allows a more direct access to CGRP released from the trigeminal nerve than plasma sampling.

Keywords

Episodic migraine chronic migraine triptan NSAID CGRP

Introduction

Calcitonin gene-related peptide (CGRP) plays a major role in migraine pathophysiology. CGRP is a 37 amino acid neuropeptide, derived via alternative splicing from the calcitonin gene (1 –3). It is expressed in ∼50% of the trigeminal C fibres, mainly those innervating intracranial vessels (4,5).

Its role in migraine is now firmly established based on increased CGRP levels detected in interictal migraine patients, which are even more elevated during a migraine attack (6 –11), and ictal CGRP levels being reduced by triptans (11,12). In addition, intravenous CGRP induces attacks in migraine patients and selective CGRP receptor antagonists and CGRP-(receptor) antibodies are effective acute and preventive migraine treatments (13 –16). Today, it is thought that during the migraine attack, CGRP is released from the peripheral endings of (especially first division) trigeminal nerve fibres, for example, those terminating at intracranial blood vessels, inducing vasodilation, neurogenic inflammation and consequently sensitization of nociceptive afferents, resulting in migraine pain (17). This makes quantification of trigeminal CGRP release an important tool in migraine research.

Previous approaches relied on blood from the cranial circulation (external jugular vein (10,12)), peripheral blood (6 –9), and saliva (18). Salivary CGRP levels might reflect CGRP release from the third branch of the trigeminal nerve; however, the first branch is more important in migraine. Dilution of CGRP released from trigeminal fibres, and probably also contamination from other CGRP sources, are major drawbacks when using peripheral blood and even external jugular vein blood, of which only ∼20% actually stems from the intracranial circulation (6,7,19,20).

Here, we propose that detection of CGRP from tear fluid might be a more direct and sensitive approach to quantify CGRP release from first division trigeminal nerve fibres in migraine patients. Possible sources of CGRP in tear fluid are sensory, parasympathetic and sympathetic nerves innervating the cornea, conjunctiva, meibomian and lacrimal glands (21,22), but CGRP has been shown to be very scarce in parasympathetic and sympathetic ganglia (23,24) and CGRP-positive fibres in the cornea and conjunctiva almost exclusively originate from the trigeminal ganglion where ∼50% of the neurons express CGRP (4,25 –27). Little contribution is expected from the lacrimal and meibomiam glands (28 –30). Therefore, CGRP in tear fluid can by hypothesized to reflect trigeminal activation. The feasibility of CGRP detection in tear fluid has been shown before in ophthalmology and it is supposed to have several functions in the eye; for example, in inflammation, allergy and wound healing (31,32).

We hypothesized that CGRP levels in tear fluid (1) are significantly higher compared to those detected in peripheral blood, (2) are elevated in interictal migraine patients compared to healthy controls, (3) are elevated during acute migraine attacks compared to the interictal state, and (4) are reduced after intake of acute headache medication.

To test these hypotheses, we compared CGRP levels in tear fluid and peripheral blood between healthy controls and episodic and chronic migraine patients interictally and during treated and untreated migraine attacks.

Methods

Participants

Participants were recruited from our outpatient headache center and by advertisements at the University Hospital of the Ludwig-Maximilians-University Munich. The study was conducted in accordance with the Declaration of Helsinki and was approved by the ethics committee at the Ludwig-Maximilians-University Munich (526-13). Prior to participation, participants gave written informed consent.

Participants had to meet the following inclusion criteria. All subjects: Age between 18–65 years; migraine patients: Fulfilling the criteria of episodic migraine with or without aura or chronic migraine according to the ICHD-3 (33) based on a thorough interview with a headache specialist, normal clinical examination and normal neuroimaging if indicated; healthy subjects: Fewer than two mild headache days/month without any migraine characteristics (pulsating pain, unilateral pain, exacerbation with physical activity, nausea or vomiting, photophobia and phonophobia).

Exclusion criteria were the presence of other types of primary headache in migraine patients, and (for all subjects) the use of contact lenses on the day of examination, other neurological conditions or ophthalmologic conditions interfering with the ocular surface or lacrimation such as infection, allergic conjunctivitis or known autoimmune disorders of the eye, severe medical or psychiatric disorders, and pregnant or breast-feeding women. Subjects with previously diagnosed arterial hypertension, or blood pressure of > 140/90 mmHg at the time of examination were excluded as previously reported (12) because of possible effects of arterial hypertension on CGRP levels (34,35).

In the present study, migraine patients both within and outside migraine attacks were included. For analysis, we further subdivided this mixed sample into interictal migraine and medicated and unmedicated ictal migraine patients (non-overlapping groups). For the purpose of the present study, interictal migraine was defined as: No headache in the last 48 hours and no intake of acute headache medication within the last 48 hours. Forty-nine migraine patients (30 episodic migraine and 19 chronic migraine) fulfilled these criteria. Ictal migraine patients were defined as patients who had reported headache at the time of tear fluid sampling or within the previous 24 hours, further subdivided into those that had refrained from taking acute headache medication within the last 48 hours (unmedicated ictal migraine patients) and those who had taken acute headache medication (medicated ictal migraine patients). Thirteen patients (three episodic migraine, 10 chronic migraine) were classified as unmedicated ictal migraine patients and 25 patients (12 episodic migraine, 13 chronic migraine) were classified as medicated ictal migraine patients.

Power analysis conducted with G*Power (36) indicated that a sample size of 37 per group is sufficient to detect an effect of f = 0.30 (medium to large size effect) between three groups (controls, episodic migraine, chronic migraine) with a power of 0.8 at p < 0.05 using a one way analysis of variance (ANOVA) and that a two-sided t-test is able to detect a medium size effect (Cohen's d = 0.6), respectively a large size effect (Cohen's d = 0.8) with a sample size of 45, respectively 26 per group with a power of 0.8 at p < 0.05. We therefore aimed at 45 participants per group (controls, episodic migraine, chronic migraine) and of 26 per group for the subgroup analyses.

Experimental procedures

Within 24 hours prior to the experimental session, participants had not consumed alcohol and healthy participants had not taken analgesics within 48 hours. Sampling was conducted between 9:00 a.m. and 5:00 p.m. in a non-fasting condition. Prior to sampling, headache frequency, intensity and duration in the last 3 months, the current use of migraine preventive medication, as well as the presence of headache at the time of sampling, or in the previous 24 and 48 hours and the intake of acute headache medication (only triptans and/or NSAIDs in the present sample) in the previous 48 hours were assessed. Then, participants rested supine for 5 minutes and blood pressure was measured. Next, tear fluid was collected separately from the right and left eye. A plastic capillary was used as previously reported (31,37), which has the advantage of allowing easy detection of the collected volume by measuring the filled part of the capillary. One end of the plastic capillary (plastic capillaries (ref. no. 100012), Sanguis, Nümbrecht, Germany), sampling up to 10 µl, was dipped into the tear fluid at the lateral canthus and removed after complete filling or a maximum sampling time of 1 minute. Much care was taken not to irritate the eye during tear fluid collection and the procedure was immediately stopped if the eye showed signs of irritation with excessive tearing, in order to prevent dilution of samples. The amount of tear fluid obtained was measured (range: 1.4 to 10.0 µl) and the capillary was immediately immersed in a 1.5 ml tube containing 500 µl of tissue protein extractor solution (TPER; Pierce Rockford, IL) and stored at −80℃. The amount of tear volume was not significantly different between interictal and ictal migraine patients and control subjects (interictal episodic migraine: 4.25 ± 1.5 µl, interictal chronic migraine: 3.75 ± 1.05 µl; Z = 1.1, p = 0.286; controls: 4.43 ± 1.57 µl; Z = 1.1, p = 0.285; ictal: 4.5 ± 1.49 µl; ictal vs. controls: Z = 0.07, p = 0.787; ictal vs. interictal: Z = 2.2, p = 0.142). Also, there was no significant difference in the amount of tear volume collected from medicated and unmedicated ictal patients (unmedicated ictal: 4.15 ± 1.64 µl, medicated ictal: 4.68 ± 1.41 µl, Z = 1.6, p = 0.201).

Blood was drawn from the antecubital vein into pre-cooled 5 ml tubes prepared with 15% EDTA and the protease inhibitor aprotinin (Trasylol 10 mg/ml, Sigma-Aldrich, Germany), immediately placed on ice and centrifuged for 10 minutes at 2000 g at 4℃. Plasma aliquots were stored at −80℃. The whole procedure took a maximum of 15 min.

Tear CGRP levels of the right and left eye, respectively, and plasma CGRP levels were determined using a commercial CGRP sandwich Enzyme-linked Immunosorbent Assay (ELISA) kit (CUSABIO®, Wuhan, China; detection range: 1.56–100 pg/ml, minimal detectable dose: 0.39 pg/ml), following manufacturer's instructions. Intra-assay precision and inter-assay precision is declared with a coefficient of variation (CV) of < 8%, respectively < 10%. Duplicate measurements were performed for each sample. Absorption levels were read using a BioRad spectrometer (Bio-Rad Laboratories Inc., USA) and CGRP concentrations were determined from calibration curves using a 4PL fitting as implemented in elisaanalysis.com, resulting in a fit with R² > 0.99 in every case. The final CGRP level of each sample was calculated as the average of the two measurements.

Statistics

Data is presented as mean ± standard deviation. As some variables turned out to be not normally distributed, non-parametric tests were used throughout. For comparison of age and gender between groups, Kruskal-Wallis ANOVA and chi-square tests were used. For comparison of tear fluid CGRP levels between the left and right eye, the Wilcoxon test was used. Tear fluid and plasma CGRP levels were compared between groups using a Kruskal-Wallis ANOVA or a Mann-Whitney U test, as appropriate. For correlations Spearman's rho was used. Statistical analysis was performed with SPSS Statistics 25 (IBM Corp., Armonk, NY, USA). Significance was accepted at p < 0.05 (two-tailed).

Results

In total, 48 episodic migraine patients, 45 chronic migraine patients and 48 healthy control subjects were included (see Table 1 for description of the sample). There were no significant group differences in age (χ²[2] = 3.3, p = 0.191) or gender (χ²[2] = 5.5, p = 0.065). Also, there was no gender difference in headache intensity (χ²[2] = −1.7, p = 0.09) and headache frequency (χ²[2] = −1.8, p = 0.074).

Table 1.

Description of the study population. Values are mean ± standard deviation or numbers of subjects.

	Controls	Migraine patients
Total sample	Episodic migraine	Chronic migraine
n (% female)	48 (69)	93 (85)	48 (88)	45 (82)
Age (years)	33.2 ± 9.6	36.1 ± 12.1	37.7 ± 12.0	34.4 ± 12.1
Aura n (% female)	–	23 (78)	11 (91)	12 (67)
Headache frequency (days/month)	–	14.7 ± 9.8	7.6 ± 3.5	22.2 ± 6.6
Attack duration⁺ (hours)	–	22.8 ± 14.7	22.3 ± 14.9	23.4 ± 14.6
Headache intensity* [0–10]	–	8.1 ± 1.3	7.9 ± 1.5	8.3 ± 1.2
Interictal² n (% female)	–	49 (84)	30 (87)	19 (79)
Ictal³ n (% female)	–	38 (87)	15 (93)	23 (83)
Unmedicated ictal⁴ n (% female)	–	13 (92)	3 (100)	10 (90)
Medicated ictal⁵ n (% female)	–	25 (84)	12 (92)	13 (77)
Preventive medication n (% female)	–	37^§ (92)	16 (100)	21 (86)

Patients were asked to rate the average duration of their migraine attacks in hours.

Patients were asked to rate their mean headache intensity during a migraine attack on a numeric rating scale (NRS) from 0 ( = no pain) to 10 ( = worst pain ever).

Interictal is defined as no headache and no intake of acute medication in the last 48 hours.

³Ictal is defined as headache in the last 24 hours.

This can be further divided into unmedicated ictal meaning headache in the last 24 hours, but no intake of acute medication in the last 48 hours or ⁵medicated ictal meaning headache in the last 24 hours and intake of acute medication – either triptans or NSAR – in the last 48 hours.

Details of preventive medication in migraine patients: Onabotulinumtoxin A n = 9, antiepileptic medication n = 9, antidepressive medication n = 11, antihypertensive medication n = 8.

Averaged over all subjects (n = 141), CGRP levels in tear fluid were 1.01 ± 1.31 ng/ml (right eye) and 0.87 ± 1.19 ng/ml (left eye) without a significant difference between eyes (Z = −1.5, p = 0.126). Plasma CGRP levels were 6.81 ± 4.12 pg/ml. On average, CGRP levels in tear fluid were 138 times those found in plasma. There was a significant correlation between CGRP levels from the right and left eye (rho = 0.525; p < 0.001). There was a small but also significant correlation between tear fluid (left and right eye averaged) and plasma CGRP levels (rho = 0.243; p = 0.004). For the remainder of this study, tear fluid CGRP levels from the left and right eye are averaged.

Interictal migraine patients vs. controls

CGRP levels in interictal migraine patients compared to controls are illustrated in Figure 1. There was no significant difference in tear fluid or plasma CGRP levels between interictal episodic and chronic migraine patients (tear fluid episodic: 1.09 ± 1.47 ng/ml, n = 30, chronic: 1.10 ± 0.89 ng/ml, n = 19, U = 248, Z = −0.7, p = 0.448; plasma episodic: 6.38 ± 2.78 pg/ml, chronic: 6.24 ± 3.59 pg/ml; U = 253, Z = −0.7, p = 0.511). There was also no significant correlation between tear fluid or plasma CGRP levels and headache frequency (tear fluid: rho = 0.062, p = 0.674; plasma: rho = −0.141, p = 0.335) in interictal migraine patients. Therefore, data from episodic and chronic migraine patients were combined for further analysis. Tear fluid CGRP levels in interictal migraine patients (chronic and episodic migraine combined, 1.10 ± 1.27 ng/ml, n = 49) were significantly higher than those of the control group (0.75 ± 0.80 ng/ml, n = 48, U = 858, Z = −2.3, p = 0.022).

Figure 1.

CGRP levels in tear fluid (a) and plasma (b) in interictal episodic and chronic migraine patients and healthy controls. Interictal migraine was defined as no headache in the last 48 hours and no intake of acute headache medication within the last 48 hours. (a) There was a significant difference in tear fluid CGRP levels when episodic and chronic migraine patients combined were compared to controls (p = 0.022) but there was no significant difference between episodic and chronic migraine patients (p = 0.448). (b) There were no group differences in plasma CGRP levels. Values are mean ± standard error.

In contrast, no group difference was found in plasma samples (migraine patients: 6.32 ± 3.08 pg/ml; controls: 6.57 ± 4.25 pg/ml; U = 1089, Z = −0.6, p = 0.528).

Ictal vs. interictal migraine patients and controls

When CGRP tear fluid levels of unmedicated (n = 13) and medicated (n = 25) ictal migraine patients were compared with those of interictal migraine patients (n = 49) and controls (n = 48), there was a significant main effect of group (Kruskal Wallis ANOVA, χ²[3] = 14.1, p = 0.003; Figure 2). Pairwise comparisons were used to further test for group differences.

Figure 2.

CGRP levels in tear fluid (a) and plasma (b) in interictal migraine patients, unmedicated and medicated ictal migraine patients, and healthy controls. (a) Unmedicated ictal migraine patients showed a trend for significantly elevated tear fluid CGRP levels compared to interictal migraine patients (p = 0.102). In medicated ictal migraine patients, significantly lower CGRP concentrations in tear fluid were detected compared to interictal migraine patients (p = 0.011) and unmedicated ictal migraine patients (p = 0.004). No significant difference was seen in medicated ictal migraine patients compared to controls (p = 0.609). The significant difference between controls and interictal migraine patients is reported in detail in Figure 1. (b) There were no group differences in plasma CGRP levels. Values are mean ± standard error.

Unmedicated ictal patients showed a trend for significantly higher tear fluid CGRP levels compared to interictal migraine patients (unmedicated ictal: 1.92 ± 1.84 ng/ml, n = 13; interictal: 1.10 ± 1.27 ng/ml, n = 49; U = 224, Z = −1.6, p = 0.102) and significantly higher tear fluid CGRP levels compared to controls (controls: 0.75 ± 0.80 ng/ml, n = 48, U = 164, Z = −2.6, p = 0.009). It must be noted that this comparison can only be regarded as exploratory because of the low number of unmedicated ictal migraine patients.

Medicated ictal patients showed significantly lower tear fluid CGRP levels compared to unmedicated ictal patients (medicated ictal: 0.56 ± 0.47 ng/ml, n = 25, unmedicated ictal: 1.92 ± 1.84 ng/ml, n = 13, U = 70, Z = −2.8, p = 0.004), compared to interictal patients (interictal: 1.10 ± 1.27 ng/ml, n = 49; U = 389, Z = −2.6, p = 0.011) and no significant difference compared to controls (controls: 0.75 ± 0.80 ng/ml, n = 48, U = 556, Z = −0.5, p = 0.609).

When plasma CGRP levels were compared between the four groups (medicated and unmedicated ictal migraine, interictal migraine, controls), there was no significant main effect of group (χ²[3] = 2.4, p = 0.492).

Regarding the type of acute headache medication used, medicated ictal patients (n = 25) had taken a triptan only (n = 14), a non-steroidal anti-inflammatory drug (NSAID) only (n = 6) or a combination of triptan and NSAID (n = 5). No significant difference in tear fluid CGRP levels emerged between the three categories (NSAID: 0.49 ± 0.41 ng/ml, triptan: 0.62 ± 0.45 ng/ml, combination: 0.49 ± 0.63 ng/ml; χ²[2] = 0.7, p = 0.702).

We also tested if tear fluid CGRP levels in medicated ictal patients were different between those patients who were headache-free at the time of tear fluid collection (n = 18) and those who still (or again) reported headache (n = 7). There were no significant differences (medicated ictal: Headache-free: 0.58 ± 0.49 ng/ml, n = 18; headache: 0.52 ± 0.43 ng/ml, n = 7, p = 0.762). A similar analysis in unmedicated ictal patients (three were free of headache, 10 reported headache at the time of tear fluid collection) also did not reveal significant differences (unmedicated ictal headache-free: 2.23 ± 1.56 ng/ml, n = 3; headache: 1.82 ± 1.99 ng/ml, n = 10; p = 0.735). However, it must be emphasized that these comparisons are only exploratory because of the low numbers of subjects per group.

Discussion

Main findings of this study are 1) that CGRP could be detected in tear fluid at a ∼140 × higher concentration compared to plasma, 2) that tear fluid (but not plasma) CGRP was significantly increased in interictal migraine patients compared to controls, 3) that tear fluid (but not plasma) CGRP was further increased (trend for significance) in ictal patients who had not used acute headache medication (unmedicated ictal patients) compared to interictal migraine patients and 4) that tear fluid (but not plasma) CGRP was significantly decreased in medicated ictal patients compared to unmedicated ictal and interictal patients.

The high CGRP concentrations found in tear fluid are consistent with previous reports, obtained, for example, in patients before and after keratectomy (38) and in patients with allergic conjunctivitis before and after allergen exposure (32). However, the present study is – to the best of our knowledge – the first to study CGRP levels in migraine.

Our hypothesis was that trigeminal activation leads to a detectable CGRP release from trigeminal fibres innervating the eye, making tear fluid CGRP levels a direct and sensitive marker of trigeminal activation. Consistently, we found elevated tear fluid CGRP levels in migraine patients compared to controls. Interestingly, we found elevated CGRP levels even in interictal migraine patients, compared to healthy controls, independent of headache frequency. Elevated CGRP levels in interictal migraine patients compared to controls have been reported in peripheral blood (6 –8) and in saliva (18). Some studies detected elevated interictal CGRP only in chronic migraine (7). In contrast, our and previous data point towards an increased baseline release of CGRP without a relation to headache frequency (6,18). However, our sample of episodic migraine patients showed a relatively high headache frequency (7.6 ± 3.5 days/month), and CGRP levels might be lower in patients with more seldom migraine attacks. Elevated interictal CGRP levels could be part of the migraine disorder, maybe reflecting an increased vulnerability of the trigeminovascular system towards additional CGRP release during the attack. Alternatively, increased interictal CGRP could be reactive to repeated high levels of CGRP release; for example, due to downregulation of CGRP receptors (39,40) with consequent upregulation of baseline CGRP release. In any case, the elevated interictal CGRP tear fluid levels suggest an ongoing activation of the trigeminal system.

Next, we found that tear fluid CGRP levels were further elevated (although only at the level of a trend) in patients with headache within the last 24 hours who had not taken acute migraine medication (unmedicated ictal patients). This is consistent with the concept of CGRP representing a marker of nociceptive trigeminal activation (10) and with previous results showing increased blood CGRP levels during spontaneous (10,41) and NO-induced migraine attacks (11). The fact that the additional CGRP elevation in unmedicated ictal patients was relatively small and significant only at the trend level might be due to different factors, including that a) the group of unmedicated ictal patients was small (n = 13), b) the sample might have included rather mild headaches as patients decided not to take acute headache medication, and c) we performed only group comparisons, not within-subject comparisons (i.e. assessing patients during the interictal and the ictal state).

Finally, ictal patients who had taken acute headache medication (NSAIDs, triptans, or both), showed decreased CGRP concentrations in tear fluid compared to ictal unmedicated and interictal patients. In fact, their tear fluid CGRP levels were undistinguishable from those found in controls. This is in line with previous findings showing that ictal CGRP levels from external jugular blood decreased after intake of sumatriptan (12) and that ictal CGRP levels from saliva decreased after intake of sumatriptan (18) or rizatriptan (42). It is also consistent with the mechanism of action of triptans, which prevent CGRP release from peripheral nerve fibres via the activation of 5-HT-1D receptors (43). The effect of NSAIDs on ictal CGRP levels has not been tested before in humans. In vitro studies showed that NSAIDs inhibit CGRP release from cultured trigeminal ganglion neurons (44,45). The present study did not reveal significant differences between patients that had taken triptans or NSAIDs, nor between patients who were headache-free versus those who still reported headache at the time of tear fluid collection. However, numbers of patients in these subgroup analyses are quite low so results have to be interpreted with caution. Clearly, larger studies specifically investigating ictal patients will be needed to address these questions.

Peripheral blood CGRP concentrations in the present study were rather low compared to previous reports (7,12). However, peptide concentrations measured in blood are dependent on the test and kit used and vary highly in the existing literature (13 ± 4 pg/ml to 254 ± 195 pg/ml (7,8,10,41,46,47)). No significant group differences in blood CGRP levels were detected in the present study, either between migraine patients and controls or between ictal and interictal migraine patients. This might be due to peripheral blood CGRP being a rather diluted indicator of trigeminal CGRP release that may also be contaminated by other CGRP sources. Although significant ictal and interictal elevations of CGRP levels in peripheral blood have been reported (6,7,11), others have found effects in blood stemming from the cranial circulation but not in peripheral blood (10,41) and one study found no difference between ictal and interictal CGRP levels, either in external jugular or in peripheral blood, despite using two different assays (47). Similar to our results, a recent study detected no differences in peripheral blood CGRP levels between interictal episodic or chronic migraine patients and controls, and also no differences between ictal and interictal samples (19).

In any case, our results corroborate our hypothesis that tear fluid CGRP is a more sensitive indicator of trigeminal CGRP release in migraine than peripheral blood CGRP.

Limitations and perspectives

Several limitations should be considered. First, CGRP is a peptide with a half-life of approximately 7 minutes, at least in blood. We closely monitored storage of tubes on ice, fast sample processing and freezing to minimize degradation. Second, variations in tear fluid production might affect CGRP levels. There were no group differences in tear fluid volume sampled. However, to better control this possible confounder, we plan to assess tear fluid flow during collection in future studies. An alternative approach would be to assess total tear fluid protein and normalize tear fluid CGRP to this value. Third, for organizing reasons we did not contact interictal patients 48 hours after tear fluid collection to ensure that no migraine attack occurred in this time period. Therefore, we cannot exclude that some of our interictal patients might in fact have been preictal. Fourth, although the CGRP assay was the same, pre-processing necessarily differed between plasma and tear fluid samples due to different volumes and composition (see methods) and this might have affected absolute CGRP levels. Therefore, although concentrations in tear fluid were clearly much higher than in plasma, the exact figure might differ from the 138 times that were determined in the present study. Fifth, although we chose the most sensitive ELISA available on the market, some of the data points were located in the lower part of the standard curve, indicating that accuracy of these values may be less than optimal. This could not be avoided due to the need for dilution of the tear fluid samples before measurement.

In the present study, CGRP measurements from the left and right eye were averaged. Whether tear fluid CGRP levels are higher on the side of current or usual headache is an interesting question. In the present sample clearly unilateral headaches were rare, so this aspect could not be investigated. However, this constitutes a topic for further investigation, especially in patients with (strictly unilateral) cluster headache. Interpretation of the present results is also limited by the cross-sectional approach. Changes of tear fluid CGRP throughout the migraine attack would be another interesting application of the method.

Footnotes

Key findings

The present data suggest that CGRP measurement in tear fluid is a non-invasive and sensitive method to detect elevated interictal and ictal CGRP release from the trigeminal nerve.

Elevated CGRP levels in tear fluid can be found in interictal migraine patients compared to healthy controls.

CGRP levels in tear fluid are significantly reduced after intake of acute medication compared to interictal migraine patients.

Acknowledgements

We wish to thank the patients who participated in the present study and Sigrid Langer for technical support.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

References

Recober

Russo

. Calcitonin gene-related peptide: An update on the biology. Curr Opin Neurol 2009; 22: 241–246.

Rosenfeld

Mermod

J-J

Amara

, et al. Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 1983; 304: 129–135.

Russo

. Calcitonin gene-related peptide (CGRP): A new target for migraine. Ann Rev Pharmacol Toxicol 2015; 55: 533–552.

Eftekhari

Salvatore

Calamari

, et al. Differential distribution of calcitonin gene-related peptide and its receptor components in the human trigeminal ganglion. Neuroscience 2010; 169: 683–696.

Hendrikse

Bower

Hay

, et al. Molecular studies of CGRP and the CGRP family of peptides in the central nervous system. Cephalalgia 2019; 39: 403–419.

Ashina

Bendtsen

Jensen

, et al. Evidence for increased plasma levels of calcitonin gene-related peptide in migraine outside of attacks. Pain 2000; 86: 133–138.

Cernuda-Morollón

Larrosa

Ramón

, et al. Interictal increase of CGRP levels in peripheral blood as a biomarker for chronic migraine. Neurology 2013; 81: 1191–1196.

Fusayasu

Kowa

Takeshima

, et al. Increased plasma substance P and CGRP levels, and high ACE activity in migraineurs during headache-free periods. Pain 2007; 128: 209–214.

Gallai

Sarchielli

Floridi

, et al. Vasoactive peptide levels in the plasma of young migraine patients with and without aura assessed both interictally and ictally. Cephalalgia 1995; 15: 384–390.

10.

Goadsby

Edvinsson

Ekman

. Vasoactive peptide release in the extracerebral circulation of humans during migraine headache. Ann Neurol 1990; 28: 183–187.

11.

Juhasz

Zsombok

Modos

, et al. NO-induced migraine attack: Strong increase in plasma calcitonin gene-related peptide (CGRP) concentration and negative correlation with platelet serotonin release. Pain 2003; 106: 461–470.

12.

Goadsby

Edvinsson

. The trigeminovascular system and migraine: Studies characterizing cerebrovascular and neuropeptide changes seen in humans and cats. Ann Neurol 1993; 33: 48–56.

13.

Asghar

Hansen

Amin

, et al. Evidence for a vascular factor in migraine. Ann Neurol 2011; 69: 635–645.

14.

Doods

Arndt

Rudolf

, et al. CGRP antagonists: Unravelling the role of CGRP in migraine. Trends Pharmacol Sci 2007; 28: 580–587.

15.

Hansen

Hauge

Olesen

, et al. Calcitonin gene-related peptide triggers migraine-like attacks in patients with migraine with aura. Cephalalgia 2010; 30: 1179–1186.

16.

Israel

Neeb

Reuter

. CGRP monoclonal antibodies for the preventative treatment of migraine. Curr Pain Headache Rep 2018; 22: 38.

17.

Messlinger

Fischer

Lennerz

. Neuropeptide effects in the trigeminal system: Pathophysiology and clinical relevance in migraine. Keio J Med 2011; 60: 82–89.

18.

Bellamy

Cady

Durham

. Salivary levels of CGRP and VIP in rhinosinusitis and migraine patients. Headache 2006; 46: 24–33.

19.

Lee

S-Y

Cho

, et al. Feasibility of serum CGRP measurement as a biomarker of chronic migraine: A critical reappraisal. J Headache Pain 2018; 19: 53.

20.

Tfelt-Hansen

. Calcitonin gene-related peptide in blood: Is it increased in the external jugular vein during migraine and cluster headache? A review. J Headache Pain 2009; 10: 137–143.

21.

Noseda

Burstein

. Migraine pathophysiology: Anatomy of the trigeminovascular pathway and associated neurological symptoms, cortical spreading depression, sensitization, and modulation of pain. Pain 2013; 154: S44–S53.

22.

Stern

Gao

Siemasko

, et al. The role of the lacrimal functional unit in the pathophysiology of dry eye. Exp Eye Res 2004; 78: 409–416.

23.

Tajti

Möller

Uddman

, et al. The human superior cervical ganglion: Neuropeptides and peptide receptors. Neurosci Lett 1999; 263: 121–124.

24.

Zaidi

Matthews

. Source and origin of nerve fibres immunoreactive for substance P and calcitonin gene-related peptide in the normal and chronically denervated superior cervical sympathetic ganglion of the rat. Autonom Neurosci 2013; 173: 28–38.

25.

Elsås

Edvinsson

Sundler

, et al. Neuronal pathways to the rat conjunctiva revealed by retrograde tracing and immunocytochemistry. Exp Eye Res 1994; 58: 117–126.

26.

Jones

Marfurt

. Peptidergic innervation of the rat cornea. Exp Eye Res 1998; 66: 421–435.

27.

Luhtala

Palkama

Uusitalo

. Calcitonin gene-related peptide immunoreactive nerve fibers in the rat conjunctiva. Invest Ophthalmol Vis Sci 1991; 32: 640–645.

28.

Chung

Tigges

Stone

. Peptidergic innervation of the primate meibomian gland. Invest Ophthalmol Vis Sci 1996; 37: 238–245.

29.

Matsumoto

Tanabe

Ueda

, et al. Immunohistochemical and enzymehistochemical studies of peptidergic, aminergic and cholinergic innervation of the lacrimal gland of the monkey (Macaca fuscata). J Autonom Nerv Sys 1992; 37: 207–214.

30.

Seifert

Stuppi

Spitznas

, et al. Differential distribution of neuronal markers and neuropeptides in the human lacrimal gland. Graefe's Arch Clinical Exp Ophthalmol 1996; 234: 232–240.

31.

Golebiowski

Chao

Stapleton

, et al. Corneal nerve morphology, sensitivity, and tear neuropeptides in contact lens wear. Optom Vis Sci 2017; 94: 534–542.

32.

Sacchetti

Micera

Lambiase

, et al. Tear levels of neuropeptides increase after specific allergen challenge in allergic conjunctivitis. Mol Vis 2011; 17: 47–52.

33.

Headache Classification Committee of the International Headache Society (IHS). The International Classification of Headache Disorders, 3rd edition (ICHD-3). Cephalalgia 2018; 38: 1–211.

34.

Edvinsson

Erlinge

Ekman

, et al. Sensory nerve terminal activity in severe hypertension as reflected by circulating calcitonin gene-related peptide (CGRP) and substance P. Blood Press 1992; 1: 223–229.

35.

Smillie

S-J

Brain

. Calcitonin gene-related peptide (CGRP) and its role in hypertension. Neuropeptides 2011; 45: 93–104.

36.

Faul

Erdfelder

Lang

A-G

, et al. G* Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Meth 2007; 39: 175–191.

37.

Chen

Duan

, et al. Quantification of tear proteins by SDS-PAGE with an internal standard protein: A new method with special reference to small volume tears. Graefe's Arch Clin Exp Ophthalmol 2010; 248: 853–862.

38.

Mertaniemi

Ylätupa

Partanen

, et al. Increased release of immunoreactive calcitonin gene-related peptide (CGRP) in tears after excimer laser keratectomy. Exp Eye Res 1995; 60: 659–665.

39.

Cottrell

Padilla

Pikios

, et al. Post-endocytic sorting of calcitonin receptor-like receptor and receptor activity-modifying protein 1. J Biol Chem 2007; 282: 12260–12271.

40.

Hay

Walker

. CGRP and its receptors. Headache 2017; 57: 625–636.

41.

Sarchielli

Alberti

Vaianella

, et al. Chemokine levels in the jugular venous blood of migraine without aura patients during attacks. Headache 2004; 44: 961–968.

42.

Cady

Vause

, et al. Elevated saliva calcitonin gene-related peptide levels during acute migraine predict therapeutic response to rizatriptan. Headache 2009; 49: 1258–1266.

43.

Edvinsson

Haanes

Warfvinge

, et al. CGRP as the target of new migraine therapies – successful translation from bench to clinic. Nat Rev Neurol 2018; 14: 338–350.

44.

Neeb

Hellen

Boehnke

, et al. IL-1β stimulates COX-2 dependent PGE2 synthesis and CGRP release in rat trigeminal ganglia cells. PloS One 2011; 6: e17360.

45.

Vellani

Moschetti

Franchi

, et al. Effects of NSAIDs on the release of calcitonin gene-related peptide and prostaglandin E2 from rat trigeminal ganglia. Mediators Inflamm 2017; 2017: 1–7.

46.

Jang

Park

Kho

, et al. Plasma and saliva levels of nerve growth factor and neuropeptides in chronic migraine patients. Oral Dis 2011; 17: 187–193.

47.

Tvedskov

Lipka

Ashina

, et al. No increase of calcitonin gene–related peptide in jugular blood during migraine. Ann Neurol 2005; 58: 561–568.