Iopamiro

Images were acquired with the use of two different CT scanners: a four‐row Multi‐detector row computed tomography (MDCT) unit (Somatom Volume Zoom, Siemens, Germany) from January 2009 to October 2016 and a 64‐row MDCT unit (Somatom Definition AS Siemens, Erlangen, Germany) from October 2016 to November 2018. The cross‐sectional images were acquired on conscious rabbits, restrained within a device, the VetMouseTrap. Scan settings included a pitch of 1.5, tube potential of 120 kVp, reference tube current of 160 mA, slice thickness of 1.5 mm, matrix 512 × 512, and reconstruction with low and high‐frequency algorithms. A bolus of 740 mg iodine/kg of non‐ionic iodinated contrast medium (Iopamiro 370 mg/ml, Bracco, Manno, Switzerland and Iomeron 350 mg/ml, Bracco) was injected through an angiocatheter placed within the marginal auricular vein. The rabbits examined from 2009 to 2015, had the contrast agent administered manually, followed by 1.5 ml of saline solution flush and post‐contrast images were acquired within 1 min after the contrast administration. From October 2016 onwards, the contrast was administered via a power injector pump (CT Exprès™ Injector Unit, Bracco Injeneering S.A., Lausanne, Switzerland) with a flow velocity of 0.8 ml/s and post‐contrast images were acquired within 18 s of contrast administration.

All patients were examined either with an 8-slice CT scanner (n = 9; GE LightSpeed Qx/I Ultra, General Electric Healthcare, Milwaukee, WI), 64-slice CT scanner (n = 15; GE Healthcare Light-Speed VCT, General Electric Healthcare) or 256-slice CT scanner (n = 18, Brilliance iCT, Philips Healthcare, Cleveland, OH) in supine position. Scanning parameters were as follows: tube voltage of 120 kVp; tube current of 200–400 mA (depending on patient size); beam collimation and rotation time, 1.28 mm and 0.35 seconds for 8-slice CT scanner, 0.625 mm and 0.35 seconds for 64-slice CT scanner, 0.75 mm and 0.27 seconds for 256-slice CT scanner respectively; reconstructed image thickness 3 mm in unenhanced, corticomedullary, and nephrographic phases, and if necessary, image thickness 1.25 mm with a interval of 1.25 mm was reconstructed for analysis. All patients underwent precontrast scan followed by corticomedullary and nephrographic phases scanning at 30 and 90 seconds delay following intravenous injection of 90 to 100 mL nonionic contrast medium (Iopamiro 370 mg I/mL, Bracco; Ultravist 370 mg I/mL, Bayer Healthcare or Omnipaque 350 mg I/mL, GE Healthcare) at a rate of 3.5 mL/s using an automatic injector (MissouriTM, Ulrich Medical, Ulm, Germany). Oblique coronal or sagittal reconstruction was performed for analysis.

All patients had undergone conventional contrast-enhanced upper abdominal CT using the same scanner (LightSpeed VCT 64, GE Medical Systems, Waukesha, WI). The scan range was from the right diaphragmatic surface to the inferior border of the liver in the cranio-caudal direction. After administering a non-ionic contrast medium, iopamidol (370 mg of iodine/mL; Iopamiro; Bracco, Milan, Italy) at a dose of 1.5 mL/kg (maximum dose: 100 mL) with a double-tube high-pressure syringe, at a rate of 3.5 mL/s, hepatic images were acquired at a fixed time point, with the portal venues phase (PVP) at a 70-s delay. Scan parameters were as follows: 120 kV; automatic tube current modulation, 80–500 mA; collimation: 64 × 0.625; noise index, 7; pitch/table speed, 0.984/39.37 mm/rot; rotation time, 0.6 s; field of view, 300–450 mm; matrix, 512 mm. Image reconstruction was performed with a soft tissue kernel and a slice thickness of 1.25 and 5 mm.

For EP applications on liver, 3 electrode prototypes were used: one electrode with zero divergence and two electrode prototypes with different divergence (20° and 10°) and with 5 and 4 needles, respectively (see Table 1). The configuration with 4 electrodes did not have the central needle. The treatment was carried out under ultrasound guidance. The irreversible EP treatment was performed, both in laparoscopic procedure and in open surgery, using the Cliniporator Vitae (Igea SpA, Carpi, Italy) and setting the following electrical parameters to obtain irreversible electroporation: 80–120 pulses, as indicated in the Table 1; 100 µs of pulse duration; 5 kHz of pulse frequency; variable voltage based on electrode prototype and on deployment of needles (see Figure 1 and Table 1).
A Computed Tomography (CT) with iodate contrast medium injection (non-ionic, water solution contrast medium—Iopamiro, Bracco, Milan, Italy) was performed 3 h after the treatment. The animal was then sacrificed and liver specimens removed for vital staining (Tetrazolium) and/or histology and immunohistochemistry evaluation.

Multimodality imaging was performed with a small-animal SPECT scanner (µSPECT, MILabs, Houten, the Netherlands) and a preclinical CT system (Super Argus, SEDECAL, Algete, Spain). SPECT and CT images were acquired 15 min and 24 h after subcutaneous administration of an average 300 µCi dose (no significant differences between groups) of radiolabeled EVs (3 mg of protein, 400 µL of PBS or PEG–cECM solution). Six Balb/C, 15-week-old female mice of 23.3 ± 1.8 g were divided in two groups; half of them (n = 3) received EVs in PBS and the rest EVs in PEG–cECM. To co-register the SPECT and CT images, each animal was placed on an in-house multimodal bed surrounded by three noncoplanar capillaries filled with a mixture of ^99mTc and Iopamiro (Bracco Imaging S.p.A, Milan, Italy), which was visible in both modalities. The SPECT acquisition parameters were an isotropic voxel size of 0.75 mm and an acquisition time of 15 min and 1.25 h. SPECT images were reconstructed using two-dimensional ordered subset expectation maximization (OSEM-2D) with 16 subsets and 1 iteration. The CT was obtained immediately after completion of SPECT imaging. CT study was acquired using an X-ray beam current of 240 mA and a tube voltage of 40 kVp, and reconstructed using an FDK algorithm [42 (link)]. SPECT-CT images were co-registered following the method of García-Vazquez V. [43 ].