Background. In the pig-to-baboon model, acute vascular rejection remains the main hurdle for successful long-term xenograft survival. The production of galactosyl knockout pigs could solve concomitantly the problem of hyperacute and acute vascular rejection. This work studies in vitro the cell-mediated cytotoxicity of natural killer (NK) and T cells after priming of baboon peripheral blood lymphocytes (PBLs) with pig antigens to evaluate whether cytotoxicity is galactosyl-dependent. Material and Methods. PBLs from naive and primed baboons were used as effectors on primary porcine aortic endothelial cells (PAECs) to assess cytotoxicity. Untreated or galactosidase-digested PAECs were used to evidence the role of galactosyl residues on cell-mediated cytotoxicity. Two rat-anti baboon monoclonal antibodies were tested to inhibit either T+NK cells (LO-CD2b) or NK cells alone (LO-CD94). Results. When using PBLs from naive animals, spontaneous lysis occurred and was inhibited by both LOCD-2b and LO-CD94. In comparison, lysis of PAECs was significantly higher when baboon PBLs were first primed in vivo with pig xenoantigens. In this case, cytotoxicity was completely inhibited by LO-CD2b but only partially by LO-CD94. Reduction of galactosyl residues by galactosidase digestion showed that PAEC lysis almost completely disappeared with naive baboon PBLs but not with primed baboon PBLs, thereby indicating that anti-pig T-cell response is not dependent on galactosyl residues. Conclusion. Galactosyl knockout pigs could solve hyperacute rejection and also prevent the activation of NK cells even after xenogeneic priming. T cells will then be the next hurdle for the success of xenografting.
Introduction: Brief bubble, and subsequent surface oxygenation is an alternative oxygenation technique for membrane-oxygenated kidneys during HMP. The aim of this study was to evaluate the metabolic effect of interruption of surface oxygenation (mimicking organ transport) during HMP as compared to continuous surface and membrane oxygenation in a pig kidney ex vivo preservation model. Methods: A kidney of a ±40 kg pig was exposed to 30 minutes of warm ischemia and preserved according to one of the following study groups: 1) 22h HMP+intermittent surface oxygenation (30 min at start, 4h interruption followed by 17h30 surface oxygenation) during 22h HMP (n=12), 2) 22h HMP+continuous membrane oxygenation (n=6), and 3) 22h HMP+continuous surface oxygenation (n=7). Brief O2 uploading of the perfusion fluid before kidney perfusion was obtained either by a hollow fiber membrane oxygenator (study group 2) or by direct bubble oxygenation (study group 1 and 3). Results: O2 uploading of the perfusion fluid by minimum 15 minutes of direct bubble oxygenation was as efficient as membrane oxygenation to achieve pO2 levels above 450-500 mmHg (at 4°C) before connecting the kidney to the perfusion device (Figure 1). Metabolic analysis (i.e. lactate, succinate, glutamate, ATP, ADP, AMP, NADH, NAD+ and Flavin Mononucleotide (FMN)) on end-preservation cortical and medullar tissue biopsies demonstrated a similar mitochondrial protection/preservation in all study groups (Figure 2). FMN measurement by fluorescence demonstrated no difference between all study groups during the first 270 minutes of preservation, however perfusate FMN levels were significantly higher at the end of the preservation period in the membrane-oxygenated groups as compared by both surface-oxygenated HMP groups. Conclusion: Brief bubble and intermittent surface oxygenation of the perfusate during standard HMP at procurement site might be an effective, user-friendly, and less expensive preservation strategy to protect mitochondria when compared with membrane-oxygenated kidneys eliminating the need for a membrane oxygenator and oxygen source during transport. Organ Recovery System.
The aims of this study were to determine the most optimal timing to start machine perfusion during kidney preservation to improve early graft function and to evaluate the impact of temperature and oxygen supply during machine perfusion in a porcine ischemia–reperfusion autotransplant model. The left kidney of an approximately 40-kg female Belgian Landrace pig was exposed to 30 minutes of warm ischemia via vascular clamping and randomized to 1 of 6 study groups: (1) 22-hour static cold storage (SCS) (n = 6), (2) 22-hour hypothermic machine perfusion (HMP) (n = 6), (3) 22-hour oxygenated HMP (n = 7), (4) 20-hour HMP plus 2-hour normothermic perfusion (NP) (n = 6), (5) 20-hour SCS plus 2-hour oxygenated HMP (n = 7), and (6) 20-hour SCS plus 2-hour NP (n = 6). Graft recovery measured by serum creatinine level was significantly faster for continuous HMP preservation strategies compared with SCS alone and for all end-ischemic strategies. The active oxygenated 22-hour HMP group demonstrated a significantly faster recovery from early graft function compared with the 22-hour nonactive oxygenated HMP group. Active oxygenation was also found to be an important modulator of a faster increase in renal flow during HMP preservation. Continuous oxygenated HMP applied from the time of kidney procurement until transplant might be the best preservation strategy to improve early graft function. The aims of this study were to determine the most optimal timing to start machine perfusion during kidney preservation to improve early graft function and to evaluate the impact of temperature and oxygen supply during machine perfusion in a porcine ischemia–reperfusion autotransplant model. The left kidney of an approximately 40-kg female Belgian Landrace pig was exposed to 30 minutes of warm ischemia via vascular clamping and randomized to 1 of 6 study groups: (1) 22-hour static cold storage (SCS) (n = 6), (2) 22-hour hypothermic machine perfusion (HMP) (n = 6), (3) 22-hour oxygenated HMP (n = 7), (4) 20-hour HMP plus 2-hour normothermic perfusion (NP) (n = 6), (5) 20-hour SCS plus 2-hour oxygenated HMP (n = 7), and (6) 20-hour SCS plus 2-hour NP (n = 6). Graft recovery measured by serum creatinine level was significantly faster for continuous HMP preservation strategies compared with SCS alone and for all end-ischemic strategies. The active oxygenated 22-hour HMP group demonstrated a significantly faster recovery from early graft function compared with the 22-hour nonactive oxygenated HMP group. Active oxygenation was also found to be an important modulator of a faster increase in renal flow during HMP preservation. Continuous oxygenated HMP applied from the time of kidney procurement until transplant might be the best preservation strategy to improve early graft function.
The aim of this feasibility study was to determine an alternative oxygenation technique (easy, cheap, and compatible with air transport) for membrane oxygenation during hypothermic machine perfusion (HMP) to improve early graft function in a porcine ischemia-reperfusion autotransplant model.The left kidney of a ±40- kg pig was exposed to 30 minutes of warm ischemia before 22 hours of preservation and autotransplantation. In the experimental group, oxygenation of the perfusate during HMP was obtained by direct bubble and 30-minute surface oxygenation at start and 1-hour end ischemic (n = 4) and outcome measures compared with historical HMP without active oxygenation (n = 6), 22-hour continuous oxygenated HMP (HMPO2) (n = 8), and 2-hour HMPO2 + 20-hour HMP (n = 6) using membrane oxygenation in both historical oxygenated control groups.Brief bubble and 30-minute surface oxygenation of the perfusate effectively maintained supraphysiological Po2 levels during the first 2 hours of HMP with improved flow dynamics. Although the metabolic profile of the perfusate (ie, flavin mononucleotide) and tissue (ie, glutamate, ATP) after brief O2 uploading at the start of HMP seemed to be slightly better with the use of a membrane oxygenator compared with bubble and interrupted surface oxygenation, both techniques yielded similar, superior early graft function when compared with HMP without active oxygenation.The data presented in this feasibility study support the conclusion that brief bubble and intermittent surface oxygenation could be an alternative oxygenation technique during HMP to achieve an improved kidney graft function compared with HMP without active oxygenation and similar functional outcome when compared with membrane HMPO2.
Background: In the field of vascularized composite tissue allotransplantation, the surgical design of facial subunit grafts is an evolving concept. The purpose of the present article is to study the possibility of dividing the historical nose and lip face transplant into several morphologic and functional subunit grafts, depending on their respective supply. Methods: This study was conducted in 20 adult cadavers. The facial artery and its branches were dissected bilaterally in 16 fresh and four embalmed heads. Nasolabial perfusion was assessed by selective injection of methylene blue and eosin ( n = 2) or India ink ( n = 2) in the superior labial and distal facial arteries. Dynamic perfusion through the distal facial artery was illustrated by fluoroscopy ( n = 3). Three nose–upper lip grafts were harvested and injected with barium sulfate for microangiography computed tomographic analysis. Finally, three isolated nasal and bilabial grafts were procured and their vascular patency assessed by fluoroscopy. Results: The distal facial artery can perfuse the entire nose, septum, and upper lip, without any contribution of the superior labial artery. A dense anastomotic network indeed exists between the respective distal rami of both vessels. Furthermore, the exclusion of the superior labial artery from the harvested nasal subunit allowed safe bilabial subunit procurement, from the same specimen. Conclusions: The authors’ results demonstrate the feasibility of harvesting nasal and labial subunits, in an isolated or a combined manner. These results can find applications in subunit autologous replantation, allotransplantation, allogenic face partial retransplantation, and the emerging field of vascularized composite tissue engineering.
Short bubble and subsequent surface oxygenation is an innovative oxygenation technique and alternative for membrane oxygenation during hypothermic machine perfusion (HMP). The metabolic effect of the interruption of surface oxygenation for 4 h (mimicking organ transport) during HMP was compared to continuous surface and membrane oxygenation in a pig kidney ex situ preservation model. After 30 min of warm ischemia by vascular clamping, a kidney of a ±40 kg pig was procured and subsequently preserved according to one of the following groups: (1) 22-h HMP + intermittent surface oxygenation (
With oxygenation proposed as a resuscitative measure during hypothermic models of preservation, the aim of this study was to evaluate the optimal start time of oxygenation during continuous hypothermic machine perfusion (HMP). In this porcine ischemia-reperfusion autotransplant model, the left kidney of a ±40 kg pig was exposed to 30 minutes of warm ischemia prior to 22 hours of HMP and autotransplantation. Kidneys were randomized to receive 2 hours of oxygenation during HMP either at the start (n = 6), or end of the perfusion (n = 5) and outcomes were compared to standard, nonoxygenated HMP (n = 6) and continuous oxygenated HMP (n = 8). The brief initial and continuous oxygenated HMP groups were associated with superior graft recovery compared to either standard, nonoxygenated HMP or kidneys oxygenated at the end of HMP. This correlated with significant metabolic differences in perfusate (eg, lactate, succinate, flavin mononucleotide) and tissues (eg, succinate, adenosine triphosphate, hypoxia-inducible factor-1α, nuclear factor erythroid 2-related factor 2) suggesting superior mitochondrial preservation with initial oxygenation. Brief initial O2 uploading during HMP at procurement site might be an easy and effective preservation strategy to maintain aerobic metabolism, protect mitochondria, and achieve an improved early renal graft function compared with standard HMP or oxygen supply shortly at the end of HMP preservation.
Transplantation of heart following donation after circulatory death (DCD) was recently introduced into clinical practice. Ex vivo reperfusion following DCD and retrieval is deemed necessary in order to evaluate the recovery of cardiac viability after the period of warm ischemia. We tested the effect of four different temperatures (4 °C-18 °C-25 °C-35 °C) on cardiac metabolism during 3-h ex vivo reperfusion in a porcine model of DCD heart. We observed a steep fall in high-energy phosphate (ATP) concentrations in the myocardial tissue at the end of the warm ischemic time and only limited regeneration during reperfusion. Lactate concentration in the perfusate increased rapidly during the first hour of reperfusion and slowly decreased afterward. However, the temperature of the solution does not seem to have an effect on either ATP or lactate concentration. Furthermore, all cardiac allografts showed a significant weight increase due to cardiac edema, regardless of the temperature.
Background. The optimal perfusate partial pressure of oxygen (P o 2 ) during hypothermic machine perfusion (HMP) is unknown. The aims of the study were to determine the functional, metabolic, structural, and flow dynamic effects of low and high perfusate P o 2 during continuous HMP in a pig kidney ischemia-reperfusion autotransplant model. Methods. The left kidneys of a ±40 kg pigs were exposed to 30 minutes of warm ischemia and randomized to receive 22-hour HMP with either low perfusate P o 2 (30% oxygen, low oxygenated HMP [HMPO 2 ]) (n = 8) or high perfusate P o 2 (90% oxygen, HMPO 2 high) (n = 8), before autotransplantation. Kidneys stored in 22-hour standard HMP (n = 6) and 22-hour static cold storage (n = 6) conditions served as controls. The follow-up after autotransplantation was 13 days. Results. High P o 2 resulted in a 3- and 10-fold increase in perfusate P o 2 compared with low HMPO 2 and standard HMP, respectively. Both HMPO 2 groups were associated with superior graft recovery compared with the control groups. Oxygenation was associated with a more rapid and sustained decrease in renal resistance. While there was no difference in functional outcomes between both HMPO 2 groups, there were clear metabolic differences with an inverse correlation between oxygen provision and the concentration of major central metabolites in the perfusion fluid but no differences were observed by oxidative stress and metabolic evaluation on preimplantation biopsies. Conclusions. While this animal study does not demonstrate any advantages for early graft function for high perfusate P o 2 , compared with low perfusate P o 2 , perfusate metabolic profile analysis suggests that aerobic mechanism is better supported under high perfusate P o 2 conditions.
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