Neurol Sci DOI 10.1007/s10072-014-1679-7

ORIGINAL ARTICLE

Is serum S100B protein an useful biomarker in migraine? Asuman Celikbilek • Seda Sabah • Nermin Tanik Hakan Ak • Tugay Atalay • Neziha Yilmaz

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Received: 2 January 2014 / Accepted: 6 February 2014 Ó Springer-Verlag Italia 2014

Abstract Experimental data have demonstrated a role for S100B protein through the release of proinflammatory cytokines, following trigeminal nerve activation, implicated in the pathology of migraine. We investigated serum levels of S100B protein, as a peripheral glial biomarker, in patients with migraine. In total, 49 migraineurs and 35 ageand gender-matched controls were enrolled in this prospective clinical study. The migraine diagnosis was made according to the International Classification of Headache Disorders II diagnostic criteria. Serum samples were obtained for the measurement of S100B levels from all participants and were analyzed using commercial enzymelinked immunosorbent assay kits. Serum S100B levels were significantly lower in migraineurs than controls (p \ 0.001). S100B levels did not significantly differ in migraineurs with or without aura (p [ 0.05). In addition, there was no correlation between serum S100B levels and headache characteristics, including attack severity, frequency and duration, and disease duration (p [ 0.05). These findings suggest that serum S100B levels were

A. Celikbilek (&)
N. Tanik Department of Neurology, Bozok University, Medical School, 66200 Yozgat, Turkey e-mail: [email protected] S. Sabah Department of Medical Biology, Bozok University, Medical School, Yozgat, Turkey H. Ak
T. Atalay Department of Neurosurgery, Bozok University, Medical School, Yozgat, Turkey N. Yilmaz Department of Infectious Diseases and Microbiology, Bozok University, Medical School, Yozgat, Turkey

significantly decreased in migraine patients, but further research is needed to ascertain the contribution of S100B in the clinical evaluation of migraine. Keywords Glial cells
Migraine
Neurogenic inflammation
S100B protein
Trigeminal nerve

Introduction Migraine that affects an estimated 15 % of the population worldwide producing a considerable disability, is a complex, chronic, painful, neurovascular disorder characterized by episodic activation of the trigeminovascular system [1, 2]. Adult trigeminal ganglia comprise neuronal cells and glial cells [3]. Glial cells, which were thought to play only a supportive role, are now known to directly modulate neuronal function, survival, and activity [4]. There are two types of glial cells in the ganglia: satellite glial cells that surround neuronal cell bodies, and Schwann cells, responsible for myelin production [3, 4]. Satellite glial cells have been reported to modulate the excitability state of ganglion neurons and play an important role in peripheral sensitization [4]. It has been demonstrated experimentally that following trigeminal nerve activation, release of calcitonin gene-related peptide (CGRP) from neuronal cell bodies activates satellite glial cells that then release nitric oxide (NO) and other proinflammatory cytokines, and initiate inflammatory events in the ganglia that contribute to peripheral sensitization in migraineurs [1, 3, 5– 7]. CGRP and NO are regarded as key mediators in the pathophysiology of migraine [6, 7]. Recent experimental data have demonstrated the increased spinal trigeminal activity, accompanying the increased CGRP as well as neuronal NO synthase (nNOS) after pretreatment of rats

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with nitrovasodilators, which indicates a role for these proteins in the generation of migraine pain [6, 8]. Accordingly, CGRP receptor inhibition has been shown to block neuronal activity induced by activation of intracellular NO receptors in the rat spinal trigeminal nucleus [7]. Taken together, CGRP receptor antagonists are predominantly thought to be effective in preventing experimental headaches [9, 10]. S100B is a calcium-binding protein, produced mostly and released by glial cells in the central nervous system [11]. In addition to intracellular and extracellular functions, S100B can be secreted from cells in response to inflammatory stimuli [3]. Recently, an in vivo model of trigeminal nerve activation showed that stimulation of trigeminal nerves caused a rapid increase in the expression of the inflammatory protein, S100B, in both neurons and satellite glial cells. It was proposed that increased S100B activity would contribute to peripheral sensitization of trigeminal primary sensory neurons and have an impact on the generation and maintenance of inflammatory pain [3]. There are limited data examining serum levels of S100B protein in migraine, indicating involvement in neurovascular inflammation in the pathogenesis of migraine. In one study examining the levels of S100B in children with headaches, serum S100B levels were significantly higher in patients with migraine than tension-type headache [12]. In another, Teepker et al. [13] reported increased serum S100B levels in migraine patients. More recently, a clinical study by Yilmaz et al. [14] showed increased serum levels of S100B, ictally or interictally, in a group of patients with migraine without aura, which were suggested to be associated with glial and/or neuronal damage in migraineurs. In the present study, we investigated serum levels of S100B protein and any relationship with headache characteristics in migraine patients with and without aura.

Classification of Headache Disorders II diagnostic criteria [15]. Of the patients, 29 had migraine with aura, while the remainder had migraine without aura. The control subjects were healthy individuals with no headache of any kind. Migraine patients were evaluated according to headache characteristics, including severity, frequency and duration of the migraine attack, and duration of the disease. Based on a visual analog scale, the headache was defined as mild (score 1–3), moderate (4–6), severe (score 7–8), or very severe (score 9–10) [16]. Migraine headache attack frequency was noted in terms of the number of attacks per month [17]. Duration of the headache attack was defined in hours, whereas disease duration was in years. All patients were studied during a headache-free period and they were not on any medication. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters [18]. Fasting venous blood samples were taken from all subjects, and routine laboratory analyses were performed using standard methods in our laboratory. The study protocol was approved by the Bozok University Local Research Ethics Committee. Written informed consent was obtained from all participants. S100B assay Blood samples were collected in Vacutainer tubes without anticoagulant supplements. All blood samples were centrifuged (3,000 rpm, 10 min) and the supernatant was immediately removed and stored at -80 °C until assayed by an investigator blinded to patient’s status. Serum samples were analyzed for human S100B using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (human S100B, BioVendor Research and Diagnostic Products; Heidelberg; Germany). Serum S100B concentrations are expressed in pg/mL. Statistical analysis

Methods Study population In total, 49 migraineurs and 35 age- and gender-matched controls, 18–50-year-olds, were enrolled in this cross-sectional prospective study, conducted in the Yozgat region of Turkey. Patients with malignancies, chronic renal, hepatic, or cardiovascular disease, diabetes, thyroid disease, inflammatory or autoimmune disease, psychiatric illness, and a history of local trauma or surgery were excluded. In addition, those who were pregnant, morbidly obese, current smokers, or current consumers of alcohol were excluded. Patient medical histories, physical, and neurological examinations were performed by the same neurologist. The migraine diagnosis was made according to the International

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A Shapiro–Wilk’s test, histograms, and q–q plots were used to test the normality of the data, and Levene’s test was used to assess variance homogeneity. Independent-sample t tests and Mann–Whitney U tests were used to compare differences between continuous variables, and Chi squared (v2) analyses were used to assess differences between categorical variables. Values are expressed as frequencies and percentages, means and standard deviations, or medians and interquartile ranges. Pearson correlations were used to examine relationships between S100B and headache characteristics, including attack severity, frequency and duration, and disease duration. Analyses were conducted using the SPSS software (ver. 15.0, SPSS Inc., Chicago, IL, USA). Statistical significance was set at p \ 0.05.

Neurol Sci Table 1 Demographic and laboratory data of control and migraine patients Variables

Control (n = 35)

Migraine (n = 49)

p

Age (years)

30 (26–38)

34 (29.5–43.5)

0.062

Gender (female/ male)

29 (82.9)/6 (17.1)

45 (91.8)/4 (8.2)

0.210

BMI (kg/m )

25.8 (23.8–26.7)

25.7 (23.1–27.2)

0.543

Fasting glucose (mg/dL)

84.7 ± 5.9

86.2 ± 6.7

0.287

2

Creatinine (mg/dL)

0.7 (0.6–0.8)

0.7 (0.6–0.8)

0.197

WBC (103/mm3)

7.6 (6.5–9)

6.9 (6.3–8.7)

0.175

Hemoglobin (mg/dL)

13.4 (12.7–14.6)

13.3 (12.1–14)

0.341

Platelet (103/mm3)

262.7 ± 65.6

285.5 ± 48.8

0.070

AST (IU/L)

17.1 ± 6.4

16.5 ± 5.2

0.657

ALT (IU/L)

19 (15–27)

18 (14.5–24)

0.403

TSH (uIU/mL)

1.6 (1–2.5)

1.9 (1.4–2.7)

0.169

S100B (pg/mL)

17.4 (3.9–52.6)

2.3 (0.01–10.3)

\0.001

Values are expressed as n (%), mean ± SD or median (25th–75th percentiles) BMI body mass index, WBC white blood cells, AST aspartate aminotransferase, ALT alanine aminotransferase, TSH thyroid stimulating hormone

Table 2 Serum S100B levels in migraine patients Variable

Without aura (n = 20)

With aura (n = 29)

p

S100B (pg/mL)

5.82 (0.01–17.29)

0.89 (0.01–6.27)

0.123

Values are expressed as median (25th–75th percentiles) Table 3 Correlation coefficients between serum S100B levels and headache characteristics in migraine patients (n = 49) Variables

S100B

Attack severity

r = -0.009 p = 0.950

Attack frequency

r = 0.213 p = 0.142

Attack duration

r = 0.30 p = 0.840

Disease duration

r = 0.123 p = 0.399

Results Demographic and laboratory data of the migraine patients and controls are summarized in Table 1. No significant difference was found between the groups with respect to age or gender (p [ 0.05). Routine laboratory results were similar between the groups (p [ 0.05). Serum S100B levels were significantly lower in migraine patients than in controls (p \ 0.001, Table 1). Serum S100B levels did not significantly differ in migraineurs with or without aura (p [ 0.05, Table 2). There were no correlations between serum S100B levels and headache characteristics,

including attack severity, frequency and duration, and disease duration in migraineurs (p [ 0.05, Table 3).

Discussion Two main findings emerged from the present study. First, markedly decreased serum levels of S100B, with no difference between subgroups, with and without aura, were observed in migraine patients. Second, no correlation between serum S100B levels and headache characteristics was found in migraineurs. In recent years, S100B has been suggested to be involved in neurodegeneration and/or neuroinflammation. There are many clinical studies investigating the relationship between S100B and disorders, including systemic lupus erythematosus [19], multiple sclerosis [20], Parkinson’s disease [21], and Alzheimer’s disease [22]. S100B has intracellular functions, such as modulation of cytoskeleton proteins and regulation of cellular cycles, and extracellular functions, which are concentration-dependent. At nanomolar concentrations, S100B has neurotrophic and gliotrophic actions, and promotes neuronal survival, and astrocytic proliferation [11]. At micromolar concentrations, it may be neurotoxic, and may act as a neurodegenerative molecule [11]. S100B protein exerts both intracellular and extracellular regulatory activities, mainly via activation of the receptor for advanced glycation end-products (RAGE) [23]. S100B has also been shown to stimulate the production of pro-inflammatory cytokines, and up-regulate inducible NO synthase, thereby inducing NO release in the glia [14, 24, 25]. Both in vivo and in vitro studies have provided evidence of NO and other proinflammatory cytokines secretion following trigeminal nerve activation through the release of CGRP in migraine pathogenesis [1, 3, 5–7]. CGRP is expressed in a major part of primary afferent neurons [6, 9]. After pretreatment of rats with nitrovasodilators, the proportion of trigeminal ganglion neurons immunoreactive for CGRP as well as for nNOS was increased [8]. More recently, Seiler et al. experimentally showed that systemic pretreatment with nitroglycerin modulates immunofluorescence of CGRP receptor components and intracellular NO receptors, at the level of the trigeminal ganglion [6]. Furthermore, Feistel et al. demonstrated that the increased nitroglycerin-induced spinal trigeminal activity might be prevented by the CGRP receptor inhibition in a rat model [7]. Considered together, CGRP is closely involved in the cascade of molecular events leading to migraine painful crisis [6, 9]. Based on this data, we propose that increased NO and cytokine release from glial cells could be via S100B secretion, and that this may contribute to the local inflammatory responses in the pathology of migraine. Accordingly, elevated

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S100B levels as a result of trigeminovascular system activation was expected. However, we found markedly decreased serum levels (pg/mL) of S100B in migraineurs in contrast to the previous data [12–14]. It remains unknown whether this indicates neurotrophic and/or gliotrophic effects of this protein following inflammatory damage related to the migraine. Our results differ from some previous reports [12, 13]. One explanation for the difference may be that our measurements were made only during headache-free periods. If the analysis was performed ictally, we might find an elevated concentration of serum S100B, because a higher level of cytokines would be expected to associate with higher activation of trigeminal nerves, resulting in a higher degree of inflammation during the headache attack [26]. Another explanation may be sampling time; here, it was between 8:30 and 10:00 am. If samples had been taken in the afternoon, the results might change due to the suggestion that chronic activation of glia in response to daily stress leads to overproduction of inflammatory molecules, such as S100B, resulting in excessive neuroinflammation in the glia [14, 25]. Furthermore, it is often impossible to detect a real change in the levels of cytokines and proteins. For example, S100B has a short half-life of 30 min in serum [27], and decreased concentrations may be the result of rapid serum clearance, because of the fluctuation in circulating cytokine levels in migraine patients [28]. To our knowledge, this is the first report to include migraineurs with and without aura. The median values were about five times higher in migraineurs without aura than that of those with aura, but, such a quite difference did not reach statistical significance, probably due to the larger distribution width of the samples. In other words, the decreased serum levels of S100B did not differ in these subgroups. Because more prominent neuroinflammation [29], and consequently higher S100B levels, would be expected in migraine patients with aura, it is difficult to explain a mechanistic pathway for this condition. We also did not find any correlation between serum S100B levels and headache characteristics in migraineurs. This may be related to the headache-free period of the sampling time in our study group. As the circulating cytokine and S100B levels may change during the migraine attack [27, 28], S100B may be expected to correlate with, at least, attack severity or duration if the samples were taken during an attack. The present study had several limitations. First, it is necessary to validate these findings with a larger cohort to reach a more definitive conclusion. Second, we used a single S100B measurement at a single time point, which may not reflect the actual patient status, whereas repeated measurements, especially ictally and interictally, and/or multiple measurements over time and changes in those

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measurements, may provide a more accurate picture of the underlying mechanism of migraine. Third, we lacked cerebrospinal fluid analysis, which might offer more reliable results than a serum analysis on this issue, due to the ethical reasons in asymptomatic population.

Conclusions Based on the present findings, serum S100B levels were significantly decreased, as well as were not correlated with the subgroups, with and without aura, or the headache characteristics in patients with migraine. Further research is needed to ascertain the contribution of S100B in the clinical evaluation of migraine. Conflict of interest

None.

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