Machine Perfusion Enables 24-h Preservation of Vascularized Composite Allografts in a Swine Model of Allotransplantation.

The current gold standard for preserving vascularized composite allografts (VCA) is 4°C static cold storage (SCS), albeit muscle vulnerability to ischemia can be described as early as after 2 h of SCS. Alternatively, machine perfusion (MP) is growing in the world of organ preservation. Herein, we investigated the outcomes of oxygenated acellular subnormothermic machine perfusion (SNMP) for 24-h VCA preservation before allotransplantation in a swine model. Six partial hindlimbs were procured on adult pigs and preserved ex vivo for 24 h with either SNMP (n = 3) or SCS (n = 3) before heterotopic allotransplantation. Recipient animals received immunosuppression and were followed up for 14 days. Clinical monitoring was carried out twice daily, and graft biopsies and blood samples were regularly collected. Two blinded pathologists assessed skin and muscle samples. Overall survival was higher in the SNMP group. Early euthanasia of 2 animals in the SCS group was linked to significant graft degeneration. Analyses of the grafts showed massive muscle degeneration in the SCS group and a normal aspect in the SNMP group 2 weeks after allotransplantation. Therefore, this 24-h SNMP protocol using a modified Steen solution generated better clinical and histological outcomes in allotransplantation when compared to time-matched SCS.

VCA supercooling in a swine partial hindlimb model.

Vascularized composite allotransplantations are complex procedures with substantial functional impact on patients. Extended preservation of VCAs is of major importance in advancing this field. It would result in improved donor-recipient matching as well as the potential for ex vivo manipulation with gene and cell therapies. Moreover, it would make logistically feasible immune tolerance induction protocols through mixed chimerism. Supercooling techniques have shown promising results in multi-day liver preservation. It consists of reaching sub-zero temperatures while preventing ice formation within the graft by using various cryoprotective agents. By drastically decreasing the cell metabolism and need for oxygen and nutrients, supercooling allows extended preservation and recovery with lower ischemia-reperfusion injuries. This study is the first to demonstrate the supercooling of a large animal model of VCA. Porcine hindlimbs underwent 48 h of preservation at - 5 °C followed by recovery and normothermic machine perfusion assessment, with no issues in ice formation and favorable levels of injury markers. Our findings provide valuable preliminary results, suggesting a promising future for extended VCA preservation.

Effect of Subnormothermic Machine Perfusion on the Preservation of Vascularized Composite Allografts After Prolonged Warm Ischemia.

BACKGROUND: Warm ischemia time (WIT) and ischemia-reperfusion injury are limiting factors for vascularized composite allograft (VCA) transplantation. Subnormothermic machine perfusion (SNMP) has demonstrated the potential to extend WIT in organ transplantation. This study evaluates the effect of SNMP on VCA viability after prolonged WIT. METHODS: Rat hindlimbs underwent WIT for 30, 45, 60, 120, 150, or 210 min, followed by 3-h SNMP. Monitoring of perfusion parameters and outflow determined the maximum WIT compatible with limb viability after SNMP. Thereafter, 2 groups were assessed: a control group with inbred transplantation (Txp) after 120 min of WIT and an experimental group that underwent WIT + SNMP + Txp. Graft appearance, blood gas, cytokine levels, and histology were assessed for 21 d. RESULTS: Based on potassium levels, the limit of WIT compatible with limb viability after SNMP is 120 min. Before this limit, SNMP reduces potassium and lactate levels of WIT grafts to the same level as fresh grafts. In vivo, the control group presented 80% graft necrosis, whereas the experimental group showed no necrosis, had better healing (P = 0.0004), and reduced histological muscle injury (P = 0.012). Results of blood analysis revealed lower lactate, potassium levels, and calcium levels (P = 0.048) in the experimental group. Both groups presented an increase in interleukin (IL)-10 and IL-1b/IL-1F2 with a return to baseline after 7 to 14 d. CONCLUSIONS: Our study establishes the limit of WIT compatible with VCA viability and demonstrates the effectiveness of SNMP in restoring a graft after WIT ex vivo and in vivo, locally and systemically.

The Superficial Inferior Epigastric Artery Axial Flap to Study Ischemic Preconditioning Effects in a Rat Model.

Fasciocutaneous flaps (FCF) have become the gold standard for complex defect reconstruction in plastic and reconstructive surgery. This muscle-sparing technique allows transferring vascularized tissues to cover any large defect. FCF can be used as pedicled flaps or as free flaps; however, in the literature, failure rates for pedicled FCF and free FCF are above 5%, leaving room for improvement for these techniques and further knowledge expansion in this area. Ischemic preconditioning (I.P.) has been widely studied, but the mechanisms and the optimization of the I.P. regimen are yet to be determined. This phenomenon is indeed poorly explored in plastic and reconstructive surgery. Here, a surgical model is presented to study the I.P. regimen in a rat axial fasciocutaneous flap model, describing how to safely and reliably assess the effects of I.P. on flap survival. This article describes the complete surgical procedure, including suggestions to improve the reliability of this model. The objective is to provide researchers with a reproducible and reliable model to test various ischemic preconditioning regimens and assess their effects on flap survivability.

Modified Tail Vein and Penile Vein Puncture for Blood Sampling in the Rat Model.

Blood samples are required in most experimental animal designs to assess various hematological parameters. This paper presents two procedures for blood collection in rats: the lateral tail vein puncture and the dorsal penile vein puncture, which offer significant advantages over other previously described techniques. This study shows that these two procedures allow for fast sampling (under 10 min) and yield sufficient blood volumes for most assays (202 µL ± 67.7 µL). The dorsal penile vein puncture must be done under anesthesia, whereas the lateral tail vein puncture can be done on a conscious, restrained animal. Alternating these two techniques, therefore, enables blood draw in any situation. While it is always recommended for an operator to be assisted during a procedure to ensure animal welfare, these techniques require only a single operator, unlike most blood sampling methods that require two. Moreover, whereas these previously described methods (e.g., jugular stick, subclavian vein blood draw) require extensive prior training to avoid harm to or death of the animal, tail vein and dorsal penile vein puncture are rarely fatal. For all these reasons, and according to the context (e.g., for studies including male rats, during the perioperative or immediate postoperative period, for animals with thin tail veins), both techniques can be used alternately to enable repeated blood draws.

Towards Optimizing Sub-Normothermic Machine Perfusion in Fasciocutaneous Flaps: A Large Animal Study.

Machine perfusion has developed rapidly since its first use in solid organ transplantation. Likewise, reconstructive surgery has kept pace, and ex vivo perfusion appears as a new trend in vascularized composite allotransplants preservation. In autologous reconstruction, fasciocutaneous flaps are now the gold standard due to their low morbidity (muscle sparing) and favorable functional and cosmetic results. However, failures still occasionally arise due to difficulties encountered with the vessels during free flap transfer. The development of machine perfusion procedures would make it possible to temporarily substitute or even avoid microsurgical anastomoses in certain complex cases. We performed oxygenated acellular sub-normothermic perfusions of fasciocutaneous flaps for 24 and 48 h in a porcine model and compared continuous and intermittent perfusion regimens. The monitored metrics included vascular resistance, edema, arteriovenous oxygen gas differentials, and metabolic parameters. A final histological assessment was performed. Porcine flaps which underwent successful oxygenated perfusion showed minimal or no signs of cell necrosis at the end of the perfusion. Intermittent perfusion allowed overall better results to be obtained at 24 h and extended perfusion duration. This work provides a strong foundation for further research and could lead to new and reliable reconstructive techniques.

Supercooling: A Promising Technique for Prolonged Organ Preservation in Solid Organ Transplantation, and Early Perspectives in Vascularized Composite Allografts.

Ex-vivo preservation of transplanted organs is undergoing spectacular advances. Machine perfusion is now used in common practice for abdominal and thoracic organ transportation and preservation, and early results are in favor of substantially improved outcomes. It is based on decreasing ischemia-reperfusion phenomena by providing physiological or sub-physiological conditions until transplantation. Alternatively, supercooling techniques involving static preservation at negative temperatures while avoiding ice formation have shown encouraging results in solid organs. Here, the rationale is to decrease the organ's metabolism and need for oxygen and nutrients, allowing for extended preservation durations. The aim of this work is to review all advances of supercooling in transplantation, browsing the literature for each organ. A specific objective was also to study the initial evidence, the prospects, and potential applications of supercooling preservation in Vascularized Composite Allotransplantation (VCA). This complex entity needs a substantial effort to improve long-term outcomes, marked by chronic rejection. Improving preservation techniques is critical to ensure the favorable evolution of VCAs, and supercooling techniques could greatly participate in these advances.

Microvascular assessment of fascio-cutaneous flaps by ultrasound: A large animal study.

Objectives: Blood perfusion quality of a flap is the main prognostic factor for success. Microvascular evaluation remains mostly inaccessible. We aimed to evaluate the microflow imaging mode, MV-Flow, in assessing flap microvascularization in a pig model of the fascio-cutaneous flap. Methods: On five pigs, bilateral saphenous fascio-cutaneous flaps were procured on the superficial femoral vessels. A conventional ultrasound evaluation in pulsed Doppler and color Doppler was conducted on the ten flaps allowing for the calculation of the saphenous artery flow rate. The MV-Flow mode was then applied: for qualitative analysis, with identification of saphenous artery collaterals; then quantitative, with repeated measurements of the Vascularity Index (VI), percentage of pixels where flow is detected relative to the total ultrasound view area. The measurements were then repeated after increasing arterial flow by clamping the distal femoral artery. Results: The MV-Flow mode allowed a better follow-up of the saphenous artery's collaterals and detected microflows not seen with the color Doppler. The VI was correlated to the saphenous artery flow rate (Spearman rho of 0.64; p = 0.002) and allowed to monitor the flap perfusion variations. Conclusion: Ultrasound imaging of microvascularization by MV-Flow mode and its quantification by VI provides valuable information in evaluating the microvascularization of flaps.