Patients and definitions

For this exploratory study, we prospectively included patients with newly diagnosed IIH according to Friedman criteria from an ongoing prospectively followed observational cohort study (Vienna Idiopathic Intracranial Hypertension Biomarker study [VIIH-BIO]), jointly conducted by the Department of Neurology and Department of Ophthalmology at the Medical University of Vienna starting in January 2021.

Data are collected at the following time points: at diagnosis (D0) and after one day (D1), one (W1) and two weeks (W2), and one (M1), two (M2), three (M3) and six months (M6). Afterwards, patients are followed in a three-month interval. Briefly, standardized VIIH-BIO case reports include demographic data, disease specific parameters as well as documentation of diagnostic and therapeutic procedures. Specialized neurologists and neuro-ophthalmologists perform all examinations. Headache diagnosis is assessed by a headache specialist also using a specific headache diary. Headache phenotype is classified according to ICHD-3 [11]. All patients are treated according to best practice based on recommendation of weight loss, pharmacological treatment with acetazolamide, topiramate and/or furosemide, and invasive treatment options such as serial lumbar punctures and subsequent ventriculoperitoneal (VP) shunt or optic nerve sheath fenestration (ONSF) in case of visual-threatening treatment-refractory papilledema [12].

For the present study, the following data were obtained at each time point: body weight, monthly headache days (MHD) with headache phenotype recorded in a specific headache diary (including changes to those phenotypes during follow-up), and ophthalmological assessment including visual acuity, perimetry, fundoscopy, optical coherence tomography (OCT) and ocular ultrasonography. Patients with IIH with migraine history did not have a migraine attack for at least six months prior to the inclusion in the study or had < 1 migraine headache day on average in the last three months.

Patients with episodic migraine (EM) in a headache-free interval [17] and healthy controls (HC) from the Department of Neurology, Medical University of Innsbruck, Austria, served as control groups in the cross-sectional part of the study. In the EM group, only patients with ≥ 3 consecutive migraine- and headache-free days prior to blood sampling were included. Diagnosis of any primary headache syndrome except for infrequent episodic tension-type headache served as an exclusion criterion for HC. Importantly, none of the patients were on active preventatives or any CGRP targeting therapies.

Ophthalmological assessment

Best-corrected visual acuity was assessed using Sloan charts at distance after subjective refraction. Results were given in logarithm of the minimum angle of resolution (logMAR). Meaningful change was defined as ≥ 0.2 logMAR [13].

For perimetry, we performed automated visual field testing (Humphrey Field Analyzer, Carl Zeiss Meditec, Jena, Germany) using 30 − 2 Swedish Interactive Threshold Algorithm (SITA) standard protocols, quantifying the mean deviation in decibels (dB) of all test locations compared to age-matched controls and defining abnormal perimetry as a mean deviation lower than − 2 dB.

Fundoscopy included assessment of absence or presence of papilledema and secondary optic atrophy. We used the Frisén staging scale to rate papilledema severity, categorizing the swelling of optic discs from stage 0 (no papilledema) to stage 5 (severe papilledema) [14].

For OCT imaging, we used the same spectral-domain OCT (Spectralis OCT, Heidelberg Engineering, Heidelberg, Germany; software Heidelberg eye explorer software version 6.9a) adhering to the OSCAR-IB quality control criteria and describing findings in accordance with the APOSTEL criteria [15, 16]. For peripapillary retinal nerve fiber layer (pRNFL) measurement, a 12° (3.4 mm) ring scan centered on the optic nerve head was used (1536 A-scans, automatic real-time tracking [ART]: 100 averaged frames) [17]. Ganglion cell layer (GCL) volume was measured without pupil dilatation in both eyes of each patient by means of a 20°×20° macular volume scan (centered on the macula with 512 A-scans and 25 B-scans aligned vertically with 16 averaged frames). Volume values characterize the mean volume of the circular area centered around the foveola, corresponding to the 6 mm ring of the circular grid defined by the Early Treatment Diabetic Retinopathy Study [18]. Image processing was semiautomated using the built-in proprietary software for automated layer segmentation and manual correction of obvious errors. Measurements of worse eyes were used for statistical analysis, i.e., higher pRNFL thickness as a marker of oedema and lower GCL volume as a marker of neuroaxonal loss.

For assessment of the optic nerve sheath diameter (ONSD), we performed transbulbar sonography (ABSolu, Quantel Medical, Cournon d’Auvergne, France) after topical anesthesia with oxybuprocaine eye drops. We performed quantitative measurement of ONSD using standardized amplitude modulation (A-scan) echography with tissue sensitivity settings, placing the 8 MHz A-scan probe on the temporal eye equator in primary gaze position [19, 20]. At least two measurements were taken within 3 mm of the posterior bulb wall, and the highest was documented as the diameter.

Blood collection and separation of plasma

Blood was taken from a cubital vein using EDTA-K tubes (S-Monovetten, Sarstedt, Nümbrecht, Germany) and centrifuged at 4 °C for 5 min with 3,000 rpm/5,000 g. The plasma fraction was taken off with an Eppendorf pipette, transferred to cryovials (Nunc CryoTubes, Merck, Darmstadt, Germany), frozen in liquid nitrogen and stored at 80 °C within 10–12 min after blood sampling.

CGRP sample processing

For detailed information on the rigid (pre-)processing and analysis of the samples, please refer to our previous article [21]. In short, samples were processed with a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA; CGRP Enzyme Immunoassay #A05481, shortly named CGRP EIA, Bertin Bioreagent, Montigny-le-Bretonneux, France) for α- and β-CGRP, with a cross-reactivity with amylin, calcitonin and substance P of < 0.01%. For this purpose, a synthetic interstitial solution was prepared, and protease inhibitors were added to create a standard buffer, which was fitted with human CGRP and diluted to create serial dilutions of CGRP. Furthermore, human blood plasma was used as an alternative to the standard buffer. With these preparations a reference curve was fitted to later determine the individual CGRP concentrations of each sample. Detailed information on the processing and analysis of the sample is available elsewhere [21].

Standard protocol approvals, registrations, patient consents, and reporting

The study was approved by the ethics committee of the Medical University Vienna (ethical approval number: 2216/2020). Written informed consent was obtained from all patients. This study adheres to the reporting guidelines outlined within the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement.

Data availability statement

Data supporting the findings of this study are available from the corresponding author upon reasonable request by a qualified researcher and upon approval by the data-clearing committee of the Medical University Vienna.

Statistics

Statistical analysis was performed using SPSS 26.0 (SPSS Inc, Chicago, IL, USA). Categorical variables were expressed in absolute frequencies and percentages, continuous parametric variables as mean and standard deviation (SD) and continuous non-parametric variables as median with inter-quartile range (IQR) or absolute range (AR) as appropriate. Continuous variables were tested for normal distribution using Kolmogorov–Smirnov test with Lilliefors correction. Logarithmic transformation was used to reduce the skewness of the data. Univariate comparisons were performed by the chi-square test, one-way analysis of variance (ANOVA), and Kruskal-Wallis test with pairwise comparisons as appropriate. Univariate correlation analyses were performed using the Pearson or Spearman test as appropriate. Repeated measures ANOVA was used to determine the change in pCGRP levels over time, adjusted for age, sex, body mass index [BMI], headache duration and migraine history.

To test the intraindividual variability of CGRP levels, the intraclass correlation coefficient (ICC) was calculated based on a mean-rating, absolute-agreement, 2-way mixed-effects model.

Prespecified sensitivity analyses to determine the potential confounding influence were performed with the same statistical analysis setup excluding patients with IIH without papilledema (IIH-WOP).

Missing values were handled by multiple (20 times) imputation using the missing not at random (MNAR) approach with pooling of estimates according to Rubin’s rules [22].

The significance level was set at a two-sided p-value < 0.05.