Nimodipine

The present study was carried out at two different crossed electron-molecular/cluster beam setups. The single-molecule data was collected at the Wippi apparatus in Innsbruck, a detailed description can be found in ref. ^{37 (link)}. An oven serves as inlet for the NIMO sample. A capillary of 1 mm diameter is mounted onto it to guide the evaporated sample towards the interaction region. As ionisation source serves a hemispherical electron monochromator (HEM). It provides electrons with a narrow energy distribution (~100 meV) with Gaussian profile. The attachment processes take place in the region where molecular beam and electrons cross. Measurements at different electron energies are enabled by applying an appropriate acceleration potential in the HEM shortly before the interaction region. The negatively charged parent and fragment ions formed are subsequently extracted into a quadrupole mass analyser by a weak electrostatic field. The quadrupole has a nominal mass range of 2048 u and is utilised for mass selection. Thus, combining the HEM and the mass filter, the formation efficiency of selected fragments at varying energies can be studied. The ions are detected by a channel electron multiplier and counted by a preamplifier with analog-to-digital converter unit. The mass spectrometer is operated under high vacuum (~10⁻⁸ mbar background pressure).
For cluster experiments, the CLUster Beam (CLUB) apparatus in Prague was used, for a detailed review refer to ref. ^{38 (link)}. In the present study, the configuration of the experiment was identical to that one described in ref. ^{25 (link)}. For cluster production, helium or neon gas is humidified by a Pergo gas humidifier. A Nafion tubing gas line passes through a water bath and its membrane selectively permeate water vapour. The humidified gas is introduced into a heated oven filled with NIMO. At the opposite end a 90 μm conical nozzle is mounted. The mixture of humidified buffer gas and NIMO is co-expanded through the nozzle, which leads to the formation of NIMO(H₂O)_n clusters. The cluster beam is skimmed after a distance of ~2.5 cm and crossed by an electron beam in the interaction region ~1.5 m downstream. The electron energy can be varied by an accelerating potential. The created anions are extracted by a 2 μs long high-voltage pulse into a reflectron time-of-flight (RTOF) mass analyser with a mass resolution of ~5 × 10³. A delay of 0.5 μs between electron pulse and ion extraction excludes any effects caused by those. With each extraction pulse, all anions are analysed, detected by a multichannel plate and recorded as mass spectrum.

Following aneurysm occlusion, all patients were closely monitored on a designated neurointensive care unit with clinical examinations and daily transcranial doppler sonography (TCD) (Figure 1). In patients where clinical examination was precluded (i.e., comatose and/or analgosedated), brain tissue oxygen (p_tiO₂) (Neurovent PTO, Raumedic, Helmbrechts, Germany), and cerebral microdialysis catheters (71 High Cut-Off Brain Microdialysis Catheter, μdialysis, Stockholm, Sweden) were inserted unilaterally into the territory carrying the ruptured aneurysm. Microdialysis catheters were perfused at 0.3 μl/min with standardized electrolyte perfusion fluid; dialysates were analyzed in 3-h intervals with shorter sampling intervals available on an as needed basis.
DCI was diagnosed according to the following criteria set by Vergouwen et al. (8 (link)): new focal neurological deficit or decrease in Glasgow Coma Scale ≥2 for a duration ≥1 h or reversible after treatment and not ascribable to other reasons. The aforementioned definition was expanded to include unconscious patients: events with functional deterioration, namely oxygenation crisis (p_tiO₂ < 20 mmHg), metabolic derangement as determined by cerebral microdialysis (lactate/pyruvate ratio ≥ 40) or characteristic hypoperfusion on CT perfusion (CTP), precluding any competing causes (e.g., infection, hydrocephalus) were also counted as DCI.
If DCI was suspected, systolic arterial blood pressure was raised to ≥180 mmHg using noradrenaline infusion. If clinical and functional improvement was not observed, relevant hypoperfusion was verified via CTP, followed by DSA to confirm flow limiting vasoconstriction and to direct ERT. For TBA of proximal intracranial vessel segments, Gateway or Maverick balloons (Stryker Neurovascular, Fremont, USA) were used. A bolus of nimodipine (1–2 mg/h) was applied as needed to optimize endovascular accessibility. For continuous vasodilator administration, microcatheters (Echelon^TM10/14, Covidien/ev3/Medtronic, Irvine, USA; Rebar^TM18, Covidien/ev3, Irvine, USA; Excelsior® SL-10®, Stryker Neurovascular, Fremont, USA) were positioned in up to three locations depending on the angiographic findings (internal carotid arteries, one vertebral artery) and separately perfused with heparinized saline solution (target partial thromboplastin time 50–60 s) and nimodipine (Carinopharm, Elze, Germany; initial dose 1–2 mg/h) at a total rate of 20 ml/h over opaque infusion lines. Analgosedation, induced hypertension, minimal handling protocol, and weight-adapted intravenous application of tirofiban (Aggrastat®, Correvio, Heathrow, United Kingdom) were maintained until termination of treatment. Stepwise de-escalation of ERT was initiated as soon as possible, guided by a combination of neurological examination, multimodal neuromonitoring and perfusion imaging. Routine clinical follow-up included assessment according to the Glasgow Outcome Scale at 3 months after discharge.

Danio renio was maintained under defined conditions [95 (link)] in our zebrafish core facility (Nussallee 10, 53115 Bonn, University of Bonn) according to the German law on the Protection of Animals (§11), the European Union directive 2010/63/EU, and the European Union regulation (EU) 2019/1010, the “Principles of Laboratory Animal Care” (NIH publication no. 86-23, revised 1985), as well as the “Animal Research: Reporting of In Vivo Experiments”(ARRIVE) guidelines [92 (link)]. All experiments were performed before the zebrafish larvae reached an age of 5 days after fertilization.
A 14-h light period and a 10-h dark period were ensured. All animals were fed two times a day with artemia salinae and dry pellets (AquaPro2000, Bückenburg, Germany). Tg(Olig2:eGFP) [96 (link)] and Tg(claudinK:eGFP) [70 (link)] were paired as previously described [95 (link)]. After 30 min, eggs were collected and maintained in Danieau water at 28 °C. Methylene blue was used to avoid the growth of fungi. The pigmentation of fish was prevented using 0.003% phenylthiocarbamide (PTU) in Danieau water after the first 24 h. In addition, fish were decoronated and treated with 1 µM nimodipine, 10 µM nimodipine, or DMSO as a control for 48 h between 24 and 72 h post-fertilization. Fish were anesthetized with ethyl 3-aminobenzoate methanesulfonate (MS222) (Sigma), placed in 96-well glass plates (Cellvis, Mountain View, CA, USA) individually, and imaged with an EnSight Multimode Plate Reader (PerkinElmer, WA, USA). Z-stacks consisting of n = 6 images with z = 50 µm/step size were acquired at a wavelength of 465 nm and 50 ms light exposure. Olig2⁺ cells were counted using PerkinElmer’s Kaleido software (PerkinElmer) [55 (link)]. ClaudinK⁺ cells were counted manually using the ImageJ software.

Nimodipine and BayK8644 were provided (batch: BXR4H3P, research grade) by Bayer AG (Leverkusen, Germany) on the basis of a material transfer agreement. Both substances were dissolved in DMSO (Thermo Fisher Scientific, Waltham, MA, USA) to obtain a 10 mM stock solution. We used a dose of 1 µM or 10 µM for in vitro experiments following recommendations of previously published studies [85 (link),86 (link),87 (link),88 (link),89 (link),90 (link)] and our own experience [20 (link)].