Heparin

Ibogaine hydrochloride was acquired from Toronto Research Chemicals (North York, Canada), and noribogaine hydrochloride, noribogaine glucuronide lithium salt, and ibogaine-¹³C-d3 from TLC Pharmaceutical Standards (Newmarket, Canada). Distilled water was obtained from B. Braun Medical Supplies (Melsungen, Germany). Isoflurane was purchased from Virbac Nederland (Barneveld, The Netherlands), heparin (5000 IU ml⁻¹) from Leo Pharma (Breda, The Netherlands), and bovine serum albumin (BSA, fraction V) from Roche Diagnostics GmbH (Mannheim, Germany). The chemicals used in the bioanalytical ibogaine assay were of ULC/MS grade and were obtained from Biosolve (Valkenswaard, The Netherlands), except for DMSO, which was purchased from Merck (Darmstadt, Germany). All other chemicals and reagents were obtained from Sigma-Aldrich (Steinheim, Germany).

Murine periosteum‐derived cells (mPDCs) were obtained as described.22 Briefly, tibias and femurs were dissected and cleaned, their epiphyses were embedded in 5% low melting point agarose (SeaPlaque; Lonza, Verviers, Belgium) and periosteal cells were released from the periosteal surface by two collagenase‐dispase digests. The first digest (10 min) was discarded, the second digest (50 min) was washed, pelleted, and seeded. Cells were cultured in αMEM with 2mM GlutaMAX‐I, supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 50 μg/mL streptomycin (all from Gibco, Life Technologies, Ghent, Belgium). For experiments with fibroblast growth factor 2‐pretreated mPDCs (mPDC^FGF2), the culture medium was supplemented with 5 U/mL heparin (LEO Pharma, Ballerup, Denmark) and 5 ng/mL human recombinant FGF2 (R&D Systems, Minneapolis, MN, USA) from isolation until the cells were used for experiments, mostly after 3 weeks.

Head kidney derived macrophages were isolated from cod based on previously described protocols. In short, head kidneys were aseptically removed and transferred to L-15++ (L-15 (Gibco, Invitrogen or PAA Laboratories) supplemented with 25 mM HEPES, 2 mM l-glutamine, 13.7 mM NaCl, 1.8 mM glucose, 4.2 mM NaHCO₃, 20 U/ml penicillin, 20 μg/ml streptomycin and 10 U/ml heparin (LEO Pharma AS)). Head kidney were homogenised (GentleMACS^TM dissociator, Miltenyi Biotec) and minced through a 100 μm nylon Falcon cell strainer (BD Bioscience) and diluted in 90 ml L-15++. Cell suspensions were loaded on discontinuous 28%/45% Percol (GE Healthcare) gradients and separated by centrifugation at 400 × g for 40 min at 4 °C. The interphase containing purified macrophages was washed twice in 50 ml L-15+ (L-15++ without heparin) followed by centrifugation at 300 × g for 10 min at 4 °C. In the last washing step, cells were diluted in L-15+ and seeded at a density of 5–10 × 10⁶cells per well in 24-well culture plates (Nunc or BD Biosciences). Covered by Microplate sealing Tape (Nunc), the culture plates were incubated at 12 °C to the following day when cells were washed twice in L-15+ for further studies.

Doxorubicin was purchased from Meda AS (Slemmestad, Norway), diazoxide, 5-hydroxydecanoate and pentobarbital from Haukeland Hospital Pharmacy (Bergen, Norway), heparin from Leo Pharma A/S (Oslo, Norway), and ingredients for the Krebs-Henseleit bicarbonate buffer from Merck KGaA (Darmstadt, Germany). This study conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and was approved by the Animal Care and User Committee in Norway with document number: ID 1766 FOTS, and certified institution number 066 Vivarium, University of Bergen.

On the seventh day post-injection, the anaesthetized rats were perfused with 0.01 M pH 7.4 phosphate-buffered saline (PBS) containing 0.6 % (v/v) heparin (LEO Pharma A/S, Ballerup, Denmark) via a cardiac approach followed by 4 % paraformaldehyde (Merck, Espoo, Finland) to fix the head. The bullae were isolated after decapitation and decalcified using 10 % EDTA (Sigma-Aldrich, Steinheim, Germany) in the following 4 weeks with weekly solution changes. A standard procedure for paraffin embedding and tissue block was conducted in accordance with the protocol in a previous study [3 (link)].

Total plasma cholesterol (very low-density lipoprotein; VLDL-C, low-density lipoprotein; LDL-C and high-density lipoprotein; HDL-C) and plasma triglycerides were measured after 5 h fasting in week 12 of age using commercially available enzymatic assays (CHOD-PAP and GPO-PAP, respectively; Roche Diagnostics, Almere, The Netherlands). Plasma HDL-cholesterol was measured after precipitation of the apoB-containing lipoproteins (VLDL and LDL) from 20 μL EDTA plasma by adding 10 μL 0.2 M MnCl2 and 10 μL heparin (LEO Pharma, Amsterdam, The Netherlands; 500 U/mL). Mixtures were incubated for 20 min at room temperature and centrifuged for 15 min at 13,000 rpm at 4 °C to precipitate VLDL and LDL particles. The remaining cholesterol in the supernatant (HDL-C) was quantified with the aforementioned assay (CHOD-PAP). Furthermore, for ZDF rats, lipoprotein profiles were obtained by using the AKTA-fast protein liquid chromatography system (Pharmacia, Roosendaal, The Netherlands), as described previously [31 (link)]. The plasma from ZDF or lean rats for both sexes was pooled and analyzed, and the averaged profile of 24 fractions resulting from 2 analyses was presented.

Tumor-bearing mice received IV bolus injections of lysine-fixable TRITC-conjugated 2 mDa dextran solution (50 µg/g, Thermo Fisher Scientific, #D7139). The IV injection flow rate was kept <1 ml/min. After 3 h of TRITC-dextran circulation, mice were anaesthetized with sevoflurane and transcardially perfused. In order to remove the major fraction of erythrocytes, the mouse was first perfused with 5 ml of PBS, followed by the perfusion of 5 ml AF647-conjugated WGA (100 µg/ml, Thermo Fisher Scientific, #W32466) in PBS aimed at marking the intravascular path of the perfusate. The next step of the perfusion procedure varied depending on the perfusion protocol used: in protocol #1—perfusion of 10 ml PBS, in protocol #2—perfusion of 45 ml PBS, and in protocol #3—perfusion of 45 ml PBS with heparin (10 i.u./ml, LEO Pharma). In order to standardize perfusion flow rate, the flow of the perfusate was driven by a syringe pump (Harvard Apparatus, #0-2001) running at a flow rate of 8 ml/min. The flow rate was selected to match the cardiac output of the mouse^{64 (link)} and was proven effective for perfusion of the normal brain vessels (Figs. 2e–j, 3m–p, and Supplementary Fig. 1). The only interruption of the perfusion was a short break for manually injecting the lectin solution to the perfusion system.