Oxidative stress and increased formation of vasoconstricting F2-isoprostanes in patients with reversible cerebral vasoconstriction syndrome

Oxidative stress and increased formation of vasoconstricting F2-isoprostanes in patients with reversible cerebral vasoconstriction syndrome

Free Radical Biology and Medicine 61 (2013) 243–248 Contents lists available at SciVerse ScienceDirect Free Radical Biology and Medicine journal hom...

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Free Radical Biology and Medicine 61 (2013) 243–248

Contents lists available at SciVerse ScienceDirect

Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed

Original Contribution

Oxidative stress and increased formation of vasoconstricting F2-isoprostanes in patients with reversible cerebral vasoconstriction syndrome Shih-Pin Chen a,b,c, Yu-Ting Chung d,e, Tsung-Yun Liu d,e,f, Yen-Feng Wang a,b,c, Jong-Ling Fuh a,b,c,g,n, Shuu-Jiun Wang a,b,c,g,n a

Department of Neurology, Neurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan Faculty of Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan Brain Research Center, National Yang-Ming University, Taipei, Taiwan d Institute of Environmental and Occupational Health Sciences, National Yang-Ming University School of Medicine, Taipei, Taiwan e Department of Medical Research & Education, Taipei Veterans General Hospital, Taipei, Taiwan f Department and Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei g Institute of Brain Science, National Yang-Ming University, Taipei, Taiwan b c

art ic l e i nf o

a b s t r a c t

Article history: Received 13 January 2013 Received in revised form 28 March 2013 Accepted 13 April 2013 Available online 19 April 2013

The pathophysiology of reversible cerebral vasoconstriction syndrome (RCVS) is unknown. Oxidative stress is detrimental to endothelial function and vascular reactivity. We hypothesized that the oxidative stress marker 8-iso-prostaglandin F2α, which is also a potent vasoconstrictor, might contribute to the pathogenesis of RCVS. Recruited participants included 103 RCVS patients, 53 patients with primary headache with acute severe attacks, and 54 healthy controls. Subjects recruited prior to 2009 were discovery cohort, whereas those after 2009, replication cohort. Urine samples were obtained from all patients at registration and from 79 patients with RCVS again at remission stage. Urine 8-iso-prostaglandin F2α was analyzed by liquid chromatography-tandem mass spectrometry. Patients with RCVS received magnetic resonance angiography and transcranial color-coded sonography. In RCVS patients, the urine 8-iso-prostaglandin F2α level was higher than that in the other groups in discovery, replication, and combined cohorts (RCVS, 0.2970.18; primary headache with acute severe attacks, 0.2170.19; control, 0.1870.09 ng/mg creatinine; Po0.001), and it was positively correlated with the flow velocities of major intracranial arteries, especially within the first week of disease onset (middle cerebral artery, Spearman's correlation coefficient [rs]¼ 0.580, P¼0.002; anterior cerebral artery, rs ¼0.472, P¼0.042; posterior cerebral artery, rs ¼ 0.457, P¼ 0.022; basilar artery, rs ¼ 0.530, P¼ 0.002). The 8-iso-prostaglandin F2α level decreased from the ictalto remission stage in RCVS patients (0.3170.21 vs 0.1670.10 ng/mg creatinine, Po0.001). 8-Iso-prostaglandin F2α was higher in patients with RCVS and correlated with the severity of vasoconstrictions. Further studies are required to explore its potential pathogenic role. & 2013 Elsevier Inc. All rights reserved.

Keywords: Reversible cerebral vasoconstriction syndrome Thunderclap headaches 8-Iso-prostaglandin F2α Endothelial dysfunction Oxidative stress

Introduction Reversible cerebral vasoconstriction syndrome (RCVS) is characterized by abrupt severe headaches (thunderclap headaches) and reversible cerebral vasoconstrictions [1]. Several large-scale studies have demonstrated that RCVS is not uncommon and should be recognized early because of its substantial risk of devastating complications, such as posterior reversible

Abbreviations: RCVS, reversible cerebral vasoconstriction syndrome. n Corresponding authors at: Department of Neurology, Neurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan, 112. Fax: 886 2 28765215. E-mail addresses: [email protected] (S.-P. Chen), [email protected] com (Y.-T. Chung), [email protected] (T.-Y. Liu), [email protected] (Y.-F. Wang), [email protected] (J.-L. Fuh), [email protected] (S.-J. Wang). 0891-5849/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2013.04.022

encephalopathy syndrome, ischemic stroke, intracerebral hemorrhage, and cortical subarachnoid hemorrhage [2–7]. Severe vasoconstriction, especially of the middle and posterior cerebral arteries, is associated with posterior reversible encephalopathy syndrome or ischemic stroke [3,4]. Despite a gradual delineation of clinical features, the pathophysiology of RCVS remains elusive. Endothelial dysfunction and sympathetic overactivity might be involved in the pathogenesis of RCVS; however, direct evidence for these connections is lacking. Oxidative stress has complex interactions with endothelial dysfunction or sympathetic overactivity, is detrimental to vascular reactivity, and is associated with vasoconstriction [8–12].Therefore, it is likely to be associated with RCVS, and an objective measurement for oxidative stress may be useful for assessing disease severity.

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F2-isoprostanes are produced in vivo by the nonenzymatic free radical peroxidation of arachidonic acid [10,13]. Among the isoprostanes, 8-iso-prostaglandin F2α (also known as Iso-PGF2α Type-III or 15-F2t-isoprostane) is the most stable and reliable marker for oxidative stress [10,13]. In addition, 8-iso-prostaglandin F2α has been found to be a potent vasoconstrictor in most species and vascular beds, both in vitro and in vivo [14]. In patients with aneurismal subarachnoid hemorrhage, 8-iso-prostaglandin F2α has been identified in the cerebrospinal fluid and its level is particularly high in those with delayed vasospasm [15,16]. RCVS shares commonalities with subarachnoid hemorrhage including the presence of thunderclap headaches and cerebral vasoconstrictions, although they occur in different patterns. These similarities suggest that RCVS and subarachnoid hemorrhage might share some common pathophysiological pathways. These evidences point to a plausible hypothesis that 8-iso-prostaglandin F2α plays a role in the pathogenesis of RCVS and might correlate with disease severity. The purpose of this study was to address this hypothesis.

Materials and methods Participants and clinical settings Consecutive patients with RCVS were recruited from the headache clinic, neurologic wards, and emergency department of Taipei Veterans General Hospital. Taipei Veterans General Hospital is a 2909-bed national medical center located in Taipei City (Capital of Taiwan) that serves both veterans and nonveteran citizens. The headache clinic, which has been in operation since 1997, has a headache patient pool of410,000 patients, and sees an average of 30–35 RCVS patients per year (based on data from the last 5 years). The diagnosis of RCVS required fulfillment of the following criteria: (1) at least two acute-onset severe headaches (thunderclap headache), with or without focal neurological deficits; (2) vasoconstrictions demonstrated on magnetic resonance angiography; (3) reversibility of vasoconstrictions, as demonstrated by at least one follow-up magnetic resonance angiography within 3 months; and (4) subarachnoid hemorrhage or other intracranial disorders ruled out by appropriate investigations, but cortical subarachnoid hemorrhage in RCVS was allowed. The diagnostic criteria were based on the definition of “benign (or reversible) angiopathy of the central nervous system” proposed by the International Classification of Headache Disorders, second edition (ICHD-2) (Code 6.7.3) [17] with the exception of the duration criterion D. They also fulfilled the essential diagnostic elements of RCVS proposed by Calabrese et al. [1]. To enhance homogeneity, only patients with RCVS within 30 days of headache onset were eligible. The second recruited group included patients who presented with primary headache with acute severe attacks. These patients visited the headache clinic because of acute occurrence or worsening of headaches, and were eventually diagnosed as migraines or cluster headache. To be eligible, the headache intensity had to be at least 7 on an 11-point (0–10) numerical rating scale. Volunteer control subjects who had neither headache history nor severe medical illness were recruited as normal controls. Subjects were eligible for participation in the study if they were aged 20–65 years, could fully understand the objectives of the study, and were willing to join the study. Subjects who smoked cigarettes or had uncontrolled hypertension (systolic blood pressure 4160 mm Hg, diastolic blood pressure 4100 mm Hg), known cardiovascular or cerebrovascular disease, or any neovascularization-associated condition, such as cancer, Moyamoya disease, or diabetic retinopathy, were excluded, because these conditions might influence the urine level of 8-iso-PGF2α. A matched proportion of control subjects with grade 1 hypertension (systolic blood pressure 140–159 mm Hg, diastolic

blood pressure 90–99 mm Hg) [18] were permitted to enroll since some RCVS patients may have hypertension. Subjects with a history of using vasoconstrictors, illicit drugs, or taking antioxidants regularly were also excluded. For replication purposes, we recruited two independent cohorts by the time of subject recruitment. The discovery cohort consisted of patients who entered the study prior to 2009, and patients who were recruited after 2009 were the replication cohort. The discovery and replication cohorts were pooled together as the combined cohort for final analyses. Clinical evaluations In addition to providing basic demographic information and body mass index, all enrolled subjects were asked to complete a detailed headache intake form, provide medical, headache, and drug histories, and receive comprehensive clinical and neurological examinations upon entering the study. We treated RCVS as an urgency condition so that the diagnostic investigations, including the first magnetic resonance angiography, transcranial color-coded sonographyand/or spinal taps, were performed within the first 2 days that the patient was seen. Protocols for these interventions have been reported elsewhere [3,4]. Sequential magnetic resonance angiographies were performed in all subjects with RCVS until the vasoconstrictions normalized or until 3 months after disease onset [4]. The mean flow velocities of the major cerebral arteries, including the anterior cerebral artery, middle cerebral artery, posterior cerebral artery, and basilar artery, were detected by transcranial color-coded sonography and recorded. Except for the basilar artery, both sides of the cerebral arteries were analyzed, and the worse side was chosen to represent the selected cerebral artery for calculations. Analysis of urine 8-iso-prostaglandin F2α Each participant provided a 15 ml sample of middle-stream urine upon registration. In patients with RCVS or other acute severe headaches, samples were collected prior to administration of any treatment. A remission urine sample was also collected in some RCVS patients at least 3 months after the ictal stage and had discontinued nimodipine for at least one month. Urine samples were centrifuged (1500 g for 10 min at 4 1C), aliquoted, and stored in −80 1C freezer until analysis. The urinary level of 8-iso-prostaglandin F2α was determined by liquid chromatography–tandem mass spectrometry, which was performed with a modified protocol [19]. In brief, freshly thawed urine was centrifuged at 12,000 rpm for 10 min to collect supernatant (1 ml). The supernatant was fortified with 20 ml of deuterated internal standard solution, 8-iso-prostaglandin F2α-d4 (Cayman Chemical Company, Ann Arbor, MI), to produce a final concentration of 20 ng/ml. The samples were acidified with 10 ml of formic acid and 20 ml of methanol was added to the mixture. After gentle vortex-mixing and centrifugation (12,000 rpm for 10 min), the supernatant was subjected to solid-phase extraction with an Oasis HLB 96-well extraction plate (30 μm, 10 mg) (Waters Corporation, Milford, MA). The plate was preconditioned with 1 ml of methanol followed by 1 ml of 5% methanol with 2% formic acid. After washing, the analytes were eluted with 200 μl of methanol containing 0.5% ammonium hydroxide (v/v). Extracts were evaporated to dryness under a stream of air at 40 1C using an Evaporator (Thermo Fisher Scientific, Waltham, MA). Extracted residues were reconstituted in 100 ml of 20% methanol. A 10 ml aliquot of the extracted sample was injected onto a Gemini C18 column (100 mm  2.0 mm, 3 mm, Phenomenex), which was run with a mobile phase of 0.1% ammonium hydroxide in water (A) and 0.1% ammonium hydroxide in acetonitrile (B) at a constant flow rate of 0.3 ml /min. The following linear gradient program for elution was

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applied: from 0–7 min, linear increase from 7 to 20% B; from 7–9 min, linear increase from 20 to 90% B; from 9-10 min, hold at 90% B, and then return to the initial conditions. The optimized mass spectrometry parameters were as follows: capillary voltage of 4000 V, drying gas temperature of 325 1C, drying gas flow rate of 10 liters/min, nitrogen nebulizer pressure of 30 psi, and dwell time of 100 ms. Detection was carried out by selected reaction monitoring acquisition, which was operated in electrospray negative mode. The fragmentor voltage (V), collision energy (V), and selected reaction monitoring transitions monitored were as follows: 144, 22, m/z 353-193 for 8-iso-prostaglandin F2α; 144, 22, m/z 357-251 for 8-iso-prostaglandin F2α-d4 (m/z 357251 was adopted instead of m/z 197 because interfering peaks were noted when the latter was used, see Supplemental Fig. 1). The monitored precursor and product ions were acquired with the Agilent MassHunter Workstation Data Acquisition software and quantified with the Agilent MassHunter Quantitative software. Ethics The study protocol was approved by the Institutional Review Board of Taipei Veterans General Hospital. All participants provided written informed consent before entering the study. All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. The corresponding authors had full access to all of the data in the study and had final responsibility for the decision to submit for publication. Statistics Descriptive statistics are presented as the mean 7 standard deviation or as the number (percentage). Derived urinary 8-isoprostaglandin F2α(corrected for creatinine excretion to adjust for dilutional effects) was analyzed and correlated with clinical parameters. The discovery, replication, and combined cohorts were compared. For comparisons among multiple groups, one-way

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analysis of variance with post hoc least significant difference test was used for continuous variables, and the chi-square test was used for categorical variables. Between-group differences in variables were analyzed with a general linear model to adjust for confounders. Paired t test was used to compare the difference of urinary 8-iso-prostaglandin F2α levels between ictal and remission stages in patients with RCVS. The Kolmogorov–Smirnov test was used to test for normality. Bivariate correlations were performed to examine relationships between the level of 8-iso-prostaglandin F2α and clinical parameters, and the Pearson correlation coefficient r or Spearman correlation coefficient rs was applied as appropriate. The time trend curve of 8-iso-prostaglandin F2α was derived by the distance-weighted least squares method, as follows: a polynomial regression was calculated for each value on the X-variable scale (duration after headache onset) to determine the corresponding Y value (8-iso-prostaglandin F2α) such that the influence of individual data points on the regression decreased with their distance from the particular X value. All calculated P values were two-tailed. Statistical significance was defined as a P o 0.05. All analyses were performed with the IBM SPSS Statistics software package, version 18.0. The time trend curve was derived with STATISTICA 10.

Results Participants and their characteristics The discovery cohort consisted of 51 patients with RCVS, 27 patients with primary headache with acute severe attacks, and 27 normal controls. The replication cohort included 52 patients with RCVS, 26 patients with primary headache with acute severe attacks, and 27 normal controls. Pooled together, there were 103 patients with RCVS, 53 patients with primary headache with acute severe attacks (migraine (n ¼41) or cluster headaches (n ¼12)), and 54 normal controls eligible for final analysis. Comparisons of

Table 1 Demographics and associated medical conditions among patients with reversible cerebral vasoconstriction syndromes, patients with primary headache with acute severe attacks, and subjects without any headaches. Discovery cohort

Age, years (mean7 SD) Gender (M/F) Hypertension, n (%) Diabetics, n (%) BMI, kg/m2 (mean7 SD)

RCVS (n ¼51)

Primary headaches (n¼ 27)

No headaches (n¼ 27)

P value

48.07 10.8 6/45 6 (11.8) 3 (5.9) 23.6 7 3.0

47.2 77.8 6/21 3 (11.1) 2 (7.4) 23.3 73.9

45.0 7 8.5 5/22 4(14.8) 0 (0) 22.7 7 2.9

0.466 0.457 0.903 0.385 0.667

RCVS (n ¼52)

Primary headaches (n¼ 26)

No headaches (n¼ 27)

50.17 9.0 6/46 8 (15.4) 2 (3.8) 23.3 7 3.5

45.3 7 12.6 6/20 4 (15.4) 1 (3.8) 23.0 7 2.6

48.37 10.1 7/20 4 (14.8) 0 (0) 24.0 73.3

RCVS (n ¼103)

Primary headaches (n¼ 53)

No headaches (n¼ 54)

48.87 10.0 12/91 14 (13.6) 5 (4.9) 23.4 7 3.3

46.0 7 10.5 12/41 7 (13.2) 3 (5.7) 23.1 73.3

46.7 7 9.4 12/42 8 (14.8) 0 (0) 23.4 7 3.2

Replication cohort

Age, years (mean7 SD) Gender (M/F) Hypertension, n (%) Diabetics, n (%) BMI, kg/m2 (mean7 SD)

0.113 0.216 0.998 0.586 0.627

Combined cohort

Age, years (mean7 SD) Gender (M/F) Hypertension, n (%) Diabetics, n (%) BMI, kg/m2 (mean7 SD)

BMI, body mass index; RCVS, reversible cerebral vasoconstriction syndrome.

0.164 0.117 0.967 0.230 0.894

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demographics and medical illness among these 3 groups are summarized in Table 1. There was no difference between these 3 groups. Patients with RCVS had an average of 5.074.3 (range 2– 16) thunderclap headache attacks in a mean period of 10.476.7 days (range 1–30 days) prior to entering the study. Triggers were identified in 80 (77.7%) patients. Five patients (4.9%) developed posterior reversible encephalopathy syndromes and 6 (5.8%) developed ischemic stroke. Four of these patients had both posterior reversible encephalopathy syndromes and ischemic stroke. One patient had intracerebral hemorrhage in addition to posterior reversible encephalopathy syndromes and ischemic stroke. In addition, one patient was found to have cortical subarachnoid hemorrhage. Studies of the cerebrospinal fluid were undertaken in 26 (25.2%) patients. All 26 patients had clear and colorless cerebrospinal fluid, and the pressure, cell counts, and metabolic and immunological analyses were normal.

A total of 79 (76.7%) patients with RCVS had a follow-up urine sample taken during the remission stage (214.6785.3 days from headache onset), including 32 patients in the discovery stage and 47 patients in the replication stage. The average urine 8-iso-prostaglandin F2α level during remission differed from that of the ictal stage in either the discovery (0.157 0.10 vs 0.3170.19 ng/mg creatinine, P o0.001; paired t test) or replication cohort (0.1670.11 vs 0.3170.21 ng/mg creatinine, P o0.001; paired t test). Combined together, the urine 8-iso-prostaglandin F2α level during remission was lower than that of the ictal stage (0.1670.10 vs 0.3170.21 ng/mg creatinine, P o 0.001; paired t test), but was not different from that of the normal control group (P ¼ 0.953). The demographics, medical condition, and timing of initial urine sample collection did not differ between patients with or without remission samples (data not shown).

Comparison of urine 8-iso-prostaglandin F2α between the groups

In all participants (n ¼ 210), the level of urine 8-iso-PGF2α did not correlate with age (r ¼0.043, P ¼0.548), body mass index (r ¼−0.003, P ¼0.972), or gender (male, 0.2470.21; female, 0.2570.16 ng/mg creatinine, P ¼0.753). It did not differ between subjects with or without hypertension (0.2470.14 vs 0.2570.18 ng/mg creatinine, P ¼0.744) or diabetes mellitus (0.2270.08 vs 0.2570.17 ng/mg creatinine, P ¼0.423). In patients with RCVS (n¼ 103),the level of 8-iso-prostaglandin F2α differed between different time points, with the peak level occurring at around the second to third week of headache onset (Fig. 2). The urine 8-iso-prostaglandin F2α level was positively correlated with mean flow velocities of basilar artery (rs ¼0.23, P¼0.007), right anterior cerebral artery (rs ¼0.256, P ¼0.013), and right posterior cerebral artery (rs ¼ 0.256, P ¼ 0.009). Because the mean flow velocities of the major cerebral arteries were not normally distributed in this study (according to the Kolmogorov– Smirnov test), Spearman's correlation was used to evaluate their relationship with urine 8-iso-prostaglandin F2α. When analyzing the data according to the different time periods, we found that the correlations between urine 8-iso-prostaglandin F2α level and the mean flow velocities of cerebral arteries was primarily attributable to the significant correlation within the first week of disease onset (rs ¼0.580, P ¼ 0.002 for middle cerebral artery; rs ¼ 0.472, P¼0.042

In the discovery cohort, the urine 8-iso-prostaglandin F2α level in RCVS patients was higher than in patients with primary headache with acute severe attacks and controls (0.2970.18 vs 0.2270.19 vs 0.1870.09 ng/mg creatinine, P ¼0.010; post hoc least significant difference test, RCVS vs primary headache with acute severe attacks, P¼ 0.049; RCVS vs control, P¼0.004).The same finding was replicated in the replication cohort (RCVS vs primary headache with acute severe attacks vs controls: 0.2970.18 vs 0.2170.20 vs 0.1970.09 ng/mg creatinine, P ¼0.019; post hoc least significant difference test: RCVS vs primary headache with acute severe attacks, P¼0.049; RCVS vs control, P¼ 0.011). After the two cohorts were pooled together, the difference between patients with RCVS and the other two control groups was even more significant (0.2970.18 vs 0.2170.19 vs 0.1870.09 ng/mg creatinine, P o0.001; post hoc least significant difference test, RCVS vs primary headache with acute severe attacks, P¼ 0.005; RCVS vs control, Po0.001) (Fig. 1). The significant difference remained after adjusting for age, sex, hypertension, and diabetes with the general linear model analysis. The level of urine 8-isoprostaglandin F2α did not differ between patients with primary headache with acute severe attacks and normal controls (P ¼ 0.346).

Fig. 1. Comparison of urine 8-iso-prostaglandin F2α level among patients with reversible cerebral vasoconstriction syndrome, subjects with primary headache with acute severe attacks, and controls with no headache history. The urine 8-isoprostaglandin F2α level was higher in patients with reversible cerebral vasoconstriction syndrome (RCVS) in comparison with patients with primary headache with acute severe attacks (P¼ 0.005) and controls (Po 0.001). There was no difference between patients with other acute severe headaches and control subjects without headache history (P ¼0.346) (Analysis of variance test with post hoc less significant difference test).

Correlation between urine 8-iso-prostaglandin F2α and clinical parameters

Fig. 2. Distribution of urine 8-iso-prostaglandin F2α level and the mean flow velocity of middle cerebral artery at different time points of the disease course.

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for anterior cerebral artery; rs ¼0.457, P¼0.022 for posterior cerebral artery; and rs ¼0.530, P ¼0.002 for basilar artery) (n¼29). All other clinical parameters, including other triggers, blood pressure surge, numbers and durations of thunderclap headaches prior to sample collection, and the presence of posterior reversible encephalopathy syndromes or ischemic stroke, did not associate with the level of urine 8-iso-prostaglandin F2α (Supplemental Table 1).

Discussion Our study demonstrated that the urine level of 8-iso-prostaglandin F2α was significantly elevated in patients with RCVS during the ictal stage compared to those with primary headaches with acute severe attacks or normal controls, and was correlated with the severity of vasoconstriction. In addition, the urine level of 8-iso-prostaglandin F2α was decreased in the patients with RCVS as the disease was remitted. These findings suggested that oxidative stress may play a role in the pathogenesis of RCVS. The overall temporal trend of the urine 8-iso-prostaglandin F2α level was similar to the trend of the vasoconstrictions, which we previously determined by transcranial color-coded sonography and magnetic resonance angiography [3,4]. The level of 8-isoprostaglandin F2α peaked during the second and third weeks of the disease course, when the vasoconstrictions are at their worst point (see Fig. 2) [3,4]. This parallel trend indicates that the level of urine 8-iso-prostaglandin F2α could partially reflect disease activity of RCVS. This association is especially true when urine 8-iso-PGF2α is measured in the early stage of the disease, considering its strong correlations with the flow velocities of major cerebral arteries during the first week of disease. Increased oxidative stress has been identified in patients with primary headache disorders [20,21]. However, in our study, the urine 8-iso-prostaglandin F2α level was higher in patients with RCVS than in patients with migraine or cluster headaches with similar headache intensity. It is plausible that the elevated 8-isoprostaglandin F2α level in RCVS does not come from or account for headaches per se. A recent study found that the serum 8-isoprostaglandin F2α is increased in patients with preeclampsia [22], which is a possible cause of RCVS. Because 8-iso-prostaglandin F2α in the circulation is quickly metabolized and eliminated, the persistently high urine level during the disease course suggests that there is a constantly increased production during the active stage of the disease. In subarachnoid hemorrhage, blood clots are considered to be the main source of lipid peroxidation and reactive oxygen species [15,16]. In RCVS, however, the source of oxidative stress is uncertain. An underlying endotheliopathy and/or sympathetic overactivity could be the culprit. Hypertension or a blood pressure surge might lead to the accumulation of reactive oxygen species and be a confounder of the 8-iso-prostaglandin F2α measurement. However, our study did not find an association between 8-isoprostaglandin F2α production and blood pressure surge or hypertension. It is possible that 8-iso-prostaglandin F2α is not only the oxidative product generated during the disease process but also exerts its direct vasoconstrictive effect on the cerebral vasculature. However, this speculation is yet to be proven. The known vasoconstrictive effects of 8-iso-prostaglandin F2α are mediated through its direct action on thromboxane A2 receptors [23], inducing thromboxane A2 and endothelin-1 released from endothelial cells [24,25], and its counteraction of the effects of nitric oxide [26,27]. In addition to its detrimental effects on vascular tone regulation, 8-iso-PGF2α can stimulate mitogenesis in vascular smooth muscle cells [28], induce monocyte adhesion to endothelial cells [29], activate rapid neutrophil

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adhesion [30], inhibit endothelial cell migration [31], and induce neuromicrovascular endothelial cell death [32]. These effects of 8iso-prostaglandin F2α might contribute to prolonged vascular changes, and thus lead to the prolonged vasoconstrictions in RCVS. Further in vivo animal studies are required to test this hypothesis. Our study had some limitations. First, we could not completely exclude the possibility that some patients with RCVS or acute severe headaches had taken nonsteriodal anti-inflammatory drugs prior to entering the study. However, because isoprostanes are cyclooxygenase-independent metabolites of arachnoid acid, the influence of these cyclooxygenase inhibitors should be minimal. Second, all patients recruited in this study were Taiwanese; thus, the results may not be generalizable to other ethnic groups. Third, the urine samples were not collected at the same time of day for all patients. Therefore, the influence of diurnal changes of 8-iso-prostaglandin F2α should be considered. Nevertheless, a previous study found that the urine levels do not exhibit diurnal variations [33]. In conclusion, our findings identified that the oxidative stress marker 8-iso-prostaglandin F2α was correlated with disease severity of RCVS. More detailed study is needed to demonstrate the role of lipid peroxidation, and oxidative stress in general, in the pathogenesis of RCVS. If proven, the search for potential therapeutic targets for RCVS could focus on inhibiting the nonenzymatic peroxidation of arachidonic acids or restoring the underlying endotheliopathy.

Disclosure Dr. Shih-Pin Chen received grants from the National Science Council of Taiwan and Taipei-Veterans General Hospital. Dr. Yu-Ting Chung reports no disclosures. Dr. Tsung-Yun Liu reports no disclosures. Dr. Yen-Feng Wang reports no disclosures. Dr. Jong-Ling Fuh is a member of a scientific advisory board of Elli Lilly, and has as well received research support from the Taiwan National Science Council, Taipei-Veterans General Hospital and Elli Lilly. Dr. Shuu-Jiun Wang has served on the advisory boards of Pfizer, Allergan, and Elli Lilly Taiwan. He has received speaking honoraria from local companies (Taiwan branches) of Pfizer, Elli Lilly, Boehringer Ingelheim and GSK. He has received research grants from the Taiwan National Science Council, Taipei-Veterans General Hospital, and Taiwan Headache Society. Acknowledgments This study was supported by grants from the National Science Council of Taiwan (99-2314-B-075-036-MY3, 100-2314-B-010-019MY2, 100-2314-B-010-018-MY3), Taipei-Veterans General Hospital (V100B-007, VGHUST101-G7-1-1, V101C-106, V101E7-003), NSC support for Center for Dynamical Biomarkers and Translational Medicine, National Central University, Taiwan (NSC 100-2911-I-008001), Brain Research Center, National Yang-Ming University and a grant from Ministry of Education, Aim for the Top University Plan. No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.freeradbiomed. 2013.04.022.

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