Procedure for cold exposure

In STC-CPH, participants fasted overnight. Two hours before the PET/CT scan the participants were put in a water-perfused cooling vest covering the upper body with a continuous flow of water at a fixed temperature of 14 °C (fixed cooling protocol⁴⁰). One hour before a 2-bed PET/CT scan ¹⁸F-FDG was injected intravenously (97–100 MBq, mean ± SD: 99.07 ± 0.96 MBq). The 5-min per bed PET scans were performed on a Biograph TruePoint (Siemens, Knoxville, TN) with a low dose CT and 3D-OSEM reconstruction with PSF using 3 iterations 21 subsets and a 2 mm FWHM Gaussian filter (2 × 2x3 mm voxels). Heparin blood samples were collected just before and after 2 h of cold induction. Participants were monitored for shivering (observed by the researcher or/and self-reported by the participant) due to the cold exposure. BAT parameters were analyzed using Mirada RTx 1.0.2 (Mirada Medical Ltd, Oxford, UK) analysis to calculate standardized uptake value (SUV) maximum, mean, and BAT volume on PET.

In STC-LEI, participants underwent a 10 h overnight fast. To activate BAT the participants were imbedded between two water perfused cooling blankets. Once shivering was detected visually by researchers and self-reported by the participants, water temperature increased and a personalized cooling protocol⁴⁰ started for 2 h. After 1 h 2 MBq/kg ¹⁸F-FDG was injected intravenously, and after one more hour of cold exposure, the PET/CT scan was performed for BAT quantification. Blood samples were collected at thermoneutral before cold and at time 110 min after cold induction⁶. BAT parameters were analyzed using the Beth Israel plugin for the FIJI program as described elsewhere^58–60.

BAT was quantified using an SUV mean threshold at > 1.5 g/ml, Hounsfield units from -250 to -10 and anatomic position defined from the vertebrae C3 to T7 in participants from both STC-CPH and STC-LEI. We observed one individual with a low amount of BAT, but all individuals had visually detected BAT, and we did not observe any BAT negative individuals.

In LTC, patients were randomized to a core body temperature of either 33 or 36 °C for 24 h after cardiac arrest. The protocol included a 4 h induction period to achieve the target temperature. Hereafter, the target temperature was maintained for 24 h with subsequent rewarming to 37 °C with a maximum of 0.5 °C/hour. Plasma samples were taken before cold induction and 28 h after (4 h of induction period + 24 h of cold). Cooling was achieved using Thermowrap^21,53.

Wild-type female mice (C57B6) were randomized and housed at 23 ℃ (n = 8) or 4 ℃ (n = 8) for 16 h, at the Panum Institute (University of Copenhagen) in a temperature-controlled facility with a 12-h dark/light cycle and fed a standard chow diet. Blood samples were taken after 16 h of housing at 23 ℃ or 4 ℃ and blinded for plasma analysis. All procedures were approved by the Animal Experiments Inspectorate, Ministry of Justice, Denmark, and reported according to the ARRIVE guidelines.