Mannitol

4-week-old WT and KO rats were lightly anaesthetised to allow tail vein catheter placement. Animals received a solution of fluorescently conjugated tracers (fluorescein isothiocyanate (FITC) wheat lectin from Triticum vulgaris (Sigma-Aldrich), tetramethylrhodamine (TMR) dextran (65–80 kDa) (Sigma-Aldrich) and cadaverine-647 (Thermofisher)) into the tail vein. For a positive control a rat received a tail vein injection of mannitol (7.7 ml/kg 20%, Fresenius Kabi Canada) just prior to tracer injection. Negative control animal was given tracer containing only FITC–wheat lectin from Triticum vulgaris. Animals were allowed to recover before euthanasia via intraperitoneal injection of Euthatal (150 mg/kg; Merial) 1 h after tracer injection. Brains were removed and fresh frozen before sections were cut at 10 μm thickness using a cryostat. Slices were collected in series so that each slide had a spread of coronal sections of the deep white matter. Slides were mounted using Vectashield Vibrance (Vector Laboratories).

The main study was a prospective, randomized, single‐center, double‐blind controlled study. The patients were randomized 1:1 to either a dextran‐based priming solution (PrimECC, XVIVO Perfusion AB, Gothenburg, Sweden) or a crystalloid‐based priming solution (Ringer acetate (Fresenius Kabi AB, Uppsala, Sweden) and mannitol (Fresenius Kabi AB)). The primary endpoint was oncotic pressure during CPB. The study’s main results have been published elsewhere.¹⁴For the present secondary analysis, blood samples for analysis of five brain injury markers (GFAP, S100B, NSE, tau, and NfL) and the blood–brain barrier injury marker β‐trace protein were collected from the arterial line at three time points: before surgery (after induction of anesthesia); 2 h after CPB, and 24 h after CPB. At the same time points, the degree of hemolysis was measured as it is known that hemolysis interacts with the analysis of NSE.¹⁵ Patient data, type of surgery, CPB time, aortic cross‐clamp time and total operating time were registered for all patients. Concentrations of brain injury markers 2 h postoperatively were correlated to patient age, operation time, and hemolysis. No neurocognitive tests were performed.

Technetium pertechnetate ^99mTcO₄⁻ was provided by elution of ⁹⁹Mo/^99mTc generator (Mallinckrodt-Tyco, Petten, The Netherlands) with sterile saline solution. Sodium maleate, D-fructose, colchicine, probenecid and L-lysine were purchased from Sigma (Sigma-Aldrich, Saint Luis, MO, USA). Furosemid (Takeda Pharma AB, Stockholm, Sweden), mannitol (Fresenius Kabi AB, Uppsala, Sweden), and Gelofusine (B. Braun Melsungen AG, Melsungen, Germany) were purchased as solutions for injections. Radioactivity was measured by an automated γ-spectrometer with a NaI(Tl) detector (1480 Wizard, Wallac, Turku, Finland).