Magnetic resonance perfusion
This case is submitted with written consent by next of kin. A 1.5 Tesla Philips Ingenia MRI scanner, software version R 5.4 (Philips Healthcare, Best, Netherlands), was used for perfusion analysis. An anatomical T2-weighted sequence was used to identify lung infiltrates with the following parameters: MultiVane, 2D field-of-view 460×460 mm, voxel size 0.9×0.9×4 mm, echo/repetition times at 100/2458 ms with a compressed sensing-sensitivity encoding factor 2, 1 average and 36 slices. For the dynamic contrast series, we used 4D time-resolved MRI angiography with a keyhole T1-weighted gradient-recalled-echo with field-of-view 500×500 mm, voxel size 1.5×1.5×4 mm, echo/repetition times at 1.8/3.78 ms, a compressed sensing-sensitivity encoding factor 3.6, 1 average with 16 dynamic phases and 60 slices. A Max 3 contrast injector (Ulrich Medical, Ulm, Germany) was used to administer gadolinium-based contrast agent: gadobutrol solution, 1.0 mmol/ml, 2 ml, followed by 20 ml 0.9% saline solution with 5 ml/s. The first phase acquired the entire K-space in 8.3 s and subsequent phases used a keyhole acceleration with 20% scanning, resulting in a temporal resolution of 1.7 s per phase. TTP maps were generated using the T1 MRI perfusion application in Philips Intellispace v10.1.3 (Philips Healthcare, Best, Netherlands).
Computed tomography pulmonary angiography
A consecutive cohort of all patients with RT-PCR-confirmed COVID-19 undergoing CTPA at Karolinska University Hospital in Huddinge, Stockholm, Sweden, between March 2nd (first patient admitted) and May 20th were retrospectively and independently evaluated by two raters (T.G., radiologist; J.A., 4th-year medical student) according to a standardised scheme,19 after consultation of a senior thoracic radiologist (M.K.). The ratings of the radiologist was considered the gold standard and the inter-rater agreement was assessed by intraclass correlation coefficient. All CTPA imaging was performed on Siemens SOMATOM Definition Flash (Siemens Healthineers, Erlangen, Germany), GE Discovery CT750 HD and Revolution CT (GE Healthcare, Milwaukee, USA). A flow chart describing the CTPA cohort can be found in Fig. 4.
Echocardiography
Echocardiography data was retrospectively analysed in a sub-sample (N=50) of patients undergoing CTPA imaging by a senior clinical physiologist (G.A.) to extract the TR-Vmax, SPAP and RVOT-AT, as previously described, with normative values from the litterature.13,14 All echocardiography was performed on GE Vivid S70 (GE Healthcare, Milwaukee, USA). The CTPA and echocardiography sub-study was approved by the Swedish Ethical Review Authority (no. 2020-01895); Informed consent was waived due to the retrospective nature of the study.
Clinical and preclinical measurement of PA pressure
This case is submitted with written consent by next of kin. A Swan-Ganz catheter (Edwards Lifesciences, Irvine, USA) for measurements of PA pressure was placed in the reported patient and in all of the swines. In the patient, it was placed from the jugular vein and in the swines from the femoral vein and connected to a HemoSphere and Vigilance monitor (Edwards Lifesciences, Irvine, USA) respectively.
Swine experimental setup
This study took place at the Karolinska Experimental Research and Imaging Centre, Karolinska University Hospital, Stockholm, between May 11th and May 15th, 2020. All animal studies were conducted according to Karolinska Institutet guidelines for animal experiments. The study was approved by the Regional Ethics Committee for Animal Research in Stockholm, Sweden (no. 6716-2020). Three swines with weights between 36 and 39 kg were used in this study. Each animal fasted for 12 hours with free access to water before the procedure. They arrived sedated after premedication with intramuscular cepetor vet 1 mg/ml-zoletil 100 (Vetmedic/Virbac, Thirsk, UK) 0.8–1 mg/kg. Induction of anaesthesia was conducted with pentobarbital (Sandoz, Holzkirchen, Germany) 1–3 mg/kg and fentanyl (B. Braun, Melsungen, Germany) 2.5 μg/kg as an intravenous bolus dose. Maintenance of anesthesia was achieved with continuous infusion of pentobarbital (0.1–0.2 mg/kg/min) and morphine (Meda, Solna, Sweden) (0.1–0.25 mg/kg/h) titrated to a moderate depth of anesthesia. No muscle relaxants were used during the experiment.
Intubation using an endotracheal tube (7.0) was performed after induction of anesthesia and during spontaneous breathing. The animals were normoventilated by pressure-controlled ventilation with Siemens Servoventilator 900C (Siemens Healthineers, Erlangen, Germany) with a total tidal volume of 10 mL/kg and with an inspiratory oxygen fraction (FiO2) of 0.21. End-tidal CO2 was monitored with a capnography incorporated in the surveillance system (Datex, GE Healthcare, Milwaukee, USA). Ventilation and respiratory frequency were adjusted to maintain end-tidal CO2 between 4.7–5.3 kPa. A 5–lead electrocardiogram was recorded continuously as well as arterial pulse oximetry (SpO2) for peripheral oxygen saturation (Datex, GE Healthcare, Milwaukee, USA). The Pulse oximetry was placed on the tail. All animals received a urine catheter and hourly diuresis was recorded. Prior to skin incision, local anesthesia (lidocaine 10 mg/ml, Aspen Nordic, Ballerup, Denmark) <0.4 mg/kg) were given. A 7 F central venous catheter (Therumo, Tokyo, Japan) was inserted by open technique via the left internal jugular vein for administration of drugs. ANGII infusion started at 20 ng/kg/min in Swine #1 and were stepwise elevated to 80 ng/kg/min in 60 minutes, followed by a stepwise increase to a maximum dose of 240 ng/kg/min after 515 minutes except for Swine #2 who suffered acute right ventricular heart failure at a dose of 100 ng/kg/min and died. This schedule was then used in subsequent swines. Bleeding time was assessed by cutting the skin of the ear for 10-15 mm and removing any blood every 15 seconds. When no further bleeding could be identified, this was determined to be the bleeding time.
Preclinical imaging
All conventional angiography was performed with a Philips XD20 angiographic system and 3DRA workstation (Philips Healthcare, Best, Netherlands). Visipaque 270 contrast agent (GE Healthcare, Milwaukee, USA) was used for all contrast-enhanced applications. Xper-CT images obtained were reviewed using the XD20 systems XperCT high dose program and soft tissue algorithms.
For endovascular access, we punctured the vessels using a micropuncture set (Merit Medical AB, Stockholm, Sweden) guided by ultrasonography with Siemens Acuson Sequoia 512 (Siemens Healthineers, Erlangen, Germany). Access was established with an introducer in the right femoral artery, the right femoral vein (Swine #1 and #3) and the left femoral artery (Swine #2). We used a 7 French introducer except for the PA catheter where we used an 8 F introducer (Terumo, Tokyo, Japan). We placed the 7.5 F PA catheter (Edwards Lifesciences, Irvine, USA) by fluoroscopy guidance and the location was confirmed by invasive pressure. A distal access guide (Envoy 6 F, Cordis, Santa Clara, USA) was then placed in the proximal part of the descending aorta for continuous monitoring of aortic pressure and a 7 F pigtail catheter was placed in the right atrium of Swine #2 for pulmonary angiography. Ultrasonography was performed every second hour during the experiment to exclude deep venous thrombosis in the hind legs.
Pre-clinical chemistry analysis
Blood samples were acquired at baseline and at 120, 240, 360, 435, 545, 665, 760 minutes or directly prior to death when systolic blood pressure dropped below 70 mmHg. Arterial blood gases were obtained at baseline, 75, 270, 345, 435, 545, 665, 750 min or when close to circulatory collapse. Citrated platelet-poor plasma and serum samples were analysed using proprietary assays at the Karolinska University Laboratory, accredited according to ISO 15189 by the Swedish Board for Accreditation and Conformity Assessment. Coagulation parameters were analysed on the Sysmex CS-5100 System (Siemens Healthineers, Erlangen, Germany), chemistry analyses were analysed on the Cobas 6000 Analyzer (Roche Diagnostics, Basel, Switzerland) and osmolality in serum was analysed with the Osmometer Advanced 2020 Multi-Sample (Advanced Instruments, Norwood, USA).
Pre-clinical histological analysis
Lungs samples were placed in 4% formaldehyde at +8°C for 24 hours. The formaldehyde was then exchanged. Tissue samples were then snap-frozen in isopentane containing dry ice and cut using a Leica CM 3050 S cryostat (Leica, Wetzlar, Germany). Samples were collected on Superfrost plus glas (Thermo Fisher Scientific, Waltham, USA) and Mayer's Hematoxylin and Eosin 0.2% (Histolab AB, Stockholm, Sweden). Glasses were mounted with glycerol:PBS with cover glasses (VWR International, Radnor, USA), put in a Zeiss Axio Scope.A1 (Carl Zeiss AG, Oberkochen, Germany) and pictures were acquired by a Samsung Galaxy S10 Plus (Samsung, Seoul, South Korea) through the microscope oculars.
Statistical analysis
Statistical analyses were performed using MATLAB (version R2019b, The Mathworks Inc., Natick, USA) and IBM SPSS Statistics version 25 for Mac (IBM, Armonk, USA). A threshold of P<0.05 was considered to be statistically significant. Statistical analysis of manually drawn regions of interest in normal-appearing lung tissue on MRI perfusion were analysed using MATLAB. Extrapulmonary and extramediastinal tissues were manually masked. Other descriptive statistics on MRI perfusion were also generated in MATLAB. Inter-rater agreement of CTPA measurements were calculated using intraclass correlation coefficient of average measurements in SPSS.
Data availability
The authors will freely make available any materials and information associated with their publication that are reasonably requested by others for the purpose of academic, non-commercial research.