Vascular Patency

Upon arrival at the organ perfusion laboratory, the livers were placed in an organ bath lying over an ultrasound reflective layer and perfused with 1 L of preservation solution (Soltran, Baxter, Newbury, UK), and the core liver temperature was allowed to return to room temperature (ranging between 24 and 30 °C) before being subjected to histotripsy insonation¹. Perfusion of the organ to maintain viability was administered via the portal vein (Figure 2) with the perfusate draining via the vena cava into an organ bath. The perfusate was not recycled. The perfusion solution was delivered using a perfusion pump (Baxter™, Newbury, UK) to achieve a constant flow rate of 350 mL·h⁻¹, confirming vessel patency before lesions were created by US histotripsy. The HIFU probe was positioned in multiple sequential locations chosen at random over the surface of the perfused liver with 50 pulses applied over approximately one minute at each site. The focal histotripsy lesions could be identified by the puckering of the liver capsule, a pinpoint dimple. Once the lesions were created, these were incised with a surgical blade and the central liquefied core was aspirated using a 20 µL single-channel gauge pipette. The aspirate was subject to pre-culture microscopy and then immediately transferred into culture medium for subsequent assessment of cell number and morphology.

Demographic, clinical and perioperative data were collected. In addition, perioperative ankle-brachial index (ABI) was collected to assess the effects of endovascular approach. Significant restenosis was defined by a focal increase in peak systolic velocity (PSV) greater than 300 cm/s, a PSV ratio greater than 3.0, and uniform PSV less than 50 cm/s throughout the stent [13 (link)]. Follow-up included ultrasound and/or computer tomography angiography (CTA) examinations at 3, 6, and 12 months in the first year and annually thereafter to assess the patency of the treated vessels. The follow-up time was 3–60 months, with an average of 26.8 ± 19.3 months.

The Dartmouth Health Institutional Review Board approved this protocol after full review; informed consent was obtained from participants. Candidates for participation were identified through review of institutional surgical rosters. The inclusion criterion was patients receiving a lower-limb amputation for nononcological indications. Most patients’ amputations were indicated for vascular insufficiency or chronic infection, including nonhealing wounds associated with diabetes mellitus. The exclusion criteria included (1) patients undergoing limb amputation for oncologic indications and (2) patients with peripheral arterial disease that precluded arterial cannulation.
Figure 1 illustrates the experimental setup used in this study. Following standard-of-care amputation, limbs were transported immediately to our surgical laboratory, where a dominant artery (superficial femoral, anterior tibial, or posterior tibial), based upon the level of amputation, vessel diameter, and vessel patency, was dissected and cannulated using a sized vascular cannula (Medtronic, Biomedicus, Minneapolis, Minnesota, United States). Each cannula was secured using a #0 silk suture and was connected to a perfusion circuit (Stockert, 3S, Breisgau, Germany). Limbs were then perfused with sterile saline at 90 to $170\mathrm{mL}/\min$ to maintain physiologic pressures below 120 mm Hg. After confirming successful perfusion, a standard dose ( $1\mathrm{mg}/\mathrm{mL}$ concentration and 10 mL total) of a nerve-specific fluorophore (LGW16-03) was administered to the limb through injection into the circuit via a Luer lock. Perfusion continued for 10 min, whereupon the perfusate was changed to sterile saline for model washout. Instead of venous cannulation for return, limbs were drained by gravity, and the perfusate was collected in a reservoir and then recycled into the circuit. Cardiac pump tubing (Sorin Group USA, SMART RX ¼” × 1/16,” Arvada, Colorado, United States) was used for the return and cannula lines, and an in-line pressure monitor was used to maintain appropriate physiological pressures. Following the conclusion of the perfusion period, the compartment supplied by the dominant cannulated artery was dissected, and the dominant nerve (common peroneal or tibial) was exposed for imaging; for this representative patient, the common peroneal nerve was imaged. In situ fluorescence imaging occurred via an open-field imager (Solaris, Perkin-Elmer, Waltham, Massachusetts, United States), which was set to excite and image at 660 nm. Samples of the target nerve and neighboring tissues (e.g., muscle, adipose, and fascia) were harvested for high-resolution fluorescence scanning using the Odyssey CLx (LI-COR Biosciences, Lincoln, Nebraska, United States), which was set to the 700-nm channel. Histopathology staining was performed on all nerve samples for examination of the tissue structure and nerve health. Following imaging and tissue sampling, the amputated limbs were taken to the Department of Pathology for standard clinical processing.