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Vascularized composite allografts (VCAs) refer to en bloc heterogenous tissue that is transplanted to restore form and function after amputation or tissue loss. Rat limb VCA has emerged as a robust translational model to study the pathophysiology of these transplants. However, these models have predominately focused on hindlimb VCAs which does not translate anatomically to upper extremity transplantation, whereas the majority of clinical VCAs are upper extremity and hand transplants. This work details our optimization of rat forelimb VCA procurement and sub-normothermic machine perfusion (SNMP) protocols, with results in comparison to hindlimb perfusion with the same perfusion modality. Results indicate that compared to hindlimbs, rat forelimbs on machine perfusion mandate lower flow rates and higher acceptable maximum pressures. Additionally, low-flow forelimbs have less cellular damage than high-flow forelimbs based on oxygen uptake, edema, potassium levels, and histology through 2 hours of machine perfusion. These results are expected to inform future upper extremity VCA preservation studies. [ABSTRACT FROM AUTHOR]

The current gold standard of Static Cold Storage (SCS), which is static cold storage on ice (about + 4 °C) in a specialized media such as the University of Wisconsin solution (UW), limits storage to few hours for vascular and metabolically active tissues such as the liver and the heart. The liver is arguably the pinnacle of metabolism in human body and therefore metabolic pathway analysis immediately becomes very relevant. In this article, a Nash Equilibrium (NE) approach, which is a first principles approach, is used to model and simulate the static cold storage and warm ischemia of a proposed model of liver cells. Simulations of energy depletion in the liver in static cold storage measured by ATP content and energy charge are presented along with comparisons to experimental data. In addition, conversion of Nash Equilibrium iterations to time are described along with an uncertainty analysis for the parameters in the model. Results in this work show that the Nash Equilibrium approach provides a good match to experimental data for energy depletion and that the uncertainty in model parameters is very small with percent variances less than 0.1%. [ABSTRACT FROM AUTHOR]

In transplantation, livers are transported to recipients using static cold storage (SCS), whereby livers are exposed to cold ischemic injury that contribute to post-transplant risk factors. We hypothesized that flushing organs during procurement with cold preservation solutions could influence the number of donor blood cells retained in the allograft thereby exacerbating cold ischemic injury. We present the results of rat livers that underwent 24 h SCS after being flushed with a cold University of Wisconsin (UW) solution versus room temperature (RT) lactated ringers (LR) solution. These results were compared to livers that were not flushed prior to SCS and thoroughly flushed livers without SCS. We used viability and injury metrics collected during normothermic machine perfusion (NMP) and the number of retained peripheral cells (RPCs) measured by histology to compare outcomes. Compared to the cold UW flush group, livers flushed with RT LR had lower resistance, lactate, AST, and ALT at 6 h of NMP. The number of RPCs also had significant positive correlations with resistance, lactate, and potassium levels and a negative correlation with energy charge. In conclusion, livers exposed to cold UW flush prior to SCS appear to perform worse during NMP, compared to RT LR flush. [ABSTRACT FROM AUTHOR]

Burn injury mediated hypermetabolic syndrome leads to increased mortality among severe burn victims, due to liver failure and muscle wasting. Metabolic changes may persist up to 2 years following the injury. Thus, understanding the underlying mechanisms of the pathology is crucially important to develop appropriate therapeutic approaches. We present detailed metabolomic and lipidomic analyses of the liver and muscle tissues in a rat model with a 30% body surface area burn injury located at the dorsal skin. Three hundred and thirty-eight of 1587 detected metabolites and lipids in the liver and 119 of 1504 in the muscle tissue exhibited statistically significant alterations. We observed excessive accumulation of triacylglycerols, decreased levels of S-adenosylmethionine, increased levels of glutamine and xenobiotics in the liver tissue. Additionally, the levels of gluconeogenesis, glycolysis, and tricarboxylic acid cycle metabolites are generally decreased in the liver. On the other hand, burn injury muscle tissue exhibits increased levels of acyl-carnitines, alpha-hydroxyisovalerate, ophthalmate, alpha-hydroxybutyrate, and decreased levels of reduced glutathione. The results of this preliminary study provide compelling observations that liver and muscle tissues undergo distinctly different changes during hypermetabolism, possibly reflecting liver-muscle crosstalk. The liver and muscle tissues might be exacerbating each other's metabolic pathologies, via excessive utilization of certain metabolites produced by each other. [ABSTRACT FROM AUTHOR]

There is a significant organ shortage in the field of liver transplantation, partly due to a high discard rate of steatotic livers from donors. These organs are known to function poorly if transplanted but make up a significant portion of the available pool of donated livers. This study demonstrates the ability to improve the function of steatotic rat livers using a combination of ex situ machine perfusion and a "defatting" drug cocktail. After 6 hours of perfusion, defatted livers demonstrated lower perfusate lactate levels and improved bile quality as demonstrated by higher bile bicarbonate and lower bile lactate. Furthermore, defatting was associated with decreased gene expression of pro-inflammatory cytokines and increased expression of enzymes involved in mitochondrial fatty acid oxidation. Rehabilitation of marginal or discarded steatotic livers using machine perfusion and tailored drug therapy can significantly increase the supply of donor livers for transplantation. [ABSTRACT FROM AUTHOR]

Ex-vivo liver perfusion (EVLP) is an ideal platform to study liver disease, therapeutic interventions, and pharmacokinetic properties of drugs without any patient risk. Rat livers are an ideal model for EVLP due to less organ quality variability, ease of hepatectomy, well-defined molecular pathways, and relatively low costs compared to large animal or human perfusions. However, the major limitation with rat liver normothermic machine perfusion (NMP) is maintaining physiologic liver function on an ex-vivo machine perfusion system. To address this need, our research demonstrates 24-hour EVLP in rats under normothermic conditions. Early (6 hour) perfusate transaminase levels and oxygen consumption of the liver graft are shown to be good markers of perfusion success and correlate with viable 24-hour post-perfusion histology. Finally, we address overcoming challenges in long-term rat liver perfusions such as rising intrahepatic pressures and contamination, and offer future directions necessary to build upon our work. [ABSTRACT FROM AUTHOR]

Gradual rewarming from hypothermic to normothermic is a novel perfusion modality with superior outcome to sudden rewarming to normothermic. However, the identification of an oxygen carrier that could function at a temperature range from 4 to 7°C or whether it is necessary to use oxygen carrier during kidney rewarming, remains unresolved. This study was designed to test the use of a hemoglobin‐based oxygen carrier (HBOC) during gradual kidney rewarming as an alternative to simple dissolved oxygen. In this study, 10 rat kidneys were randomly divided into the control and the HBOC group. In the control group, no oxygen carrier was used during rewarming perfusion and the perfusion solution was oxygenated only by applying diffused carbogen flow. The protocol mimicked a donor after circulatory death (DCD) kidney transplantation, where after 30 minutes warm ischemia and 120 minutes cold storage in University of Wisconsin solution, the DCD kidneys underwent gradual rewarming from 10 to 37°C during 90 minutes with or without HBOC. This was followed by 30 minutes of warm ischemia in room temperature to mimic the anastomosis time and 120 minutes of reperfusion at 37°C to mimic the early post‐transplant state of the graft. The HBOC group demonstrated superior kidney function which was highlighted by higher ultrafiltrate production, better glomerular filtration rate and improved sodium reabsorption. There was no significant difference between the 2 groups regarding the hemodynamics, tissue injury, and adenosine triphosphate levels. In conclusion, this study suggests better renal function recovery in DCD kidneys after rewarming with HBOC compared to rewarming without an oxygen carrier. [ABSTRACT FROM AUTHOR]

• We bioengineered rat fibroblasts to secrete a biomarker: Gaussia Luciferase. • Machine perfusion was used to engraft these genetically modified cells in rat limbs. • After transplantation we could track the biomarker secretion for 4 days. • It is the first step toward engraftment of biosensor cells before transplantation. • Vascularized Composite Allotransplantation would benefit from in situ cell therapy. Transplantation of the hand or face, known as vascularized composite allotransplantation (VCA), has revolutionized reconstructive surgery. Notwithstanding, there are still several areas of improvement to mitigate immune rejection while sparing systemic adverse effects. The goal of this study was to evaluate the engraftment and viability of a genetically modified cell population pre-engrafted into a VCA transplant, to potentially act as a local biosensor to report and modify the graft in vivo. A rat fibroblast cell line genetically modified to secrete Gaussia-Luciferase (gLuc), which served as a constitutive biomarker of cells, was incorporated into a VCA to study the viability of biosensor cells in a syngeneic rat heterotopic partial hindlimb transplantation model. Five perfusions were first performed as engineering runs to have a stable limb perfusion protocol, followed by 3 perfusions to analyze the cell engraftment during machine perfusion, and finally 4 perfusions to study in vivo persistence of the cell biosensors. Blood samples were collected to monitor gLuc secretion during perfusion and postoperatively. A time-dependent increase in gLuc secretion in the limb perfusion outflow during machine perfusion indirectly verified the presence of biosensors within the graft. After the ex vivo perfusion, VCA hindlimbs were analyzed for near infrared fluorescence emission that showed a presence of dyed engineered cells in all areas of the limbs. Postoperatively, gLuc was detectable 4 to 5 days after transplantation (W = 16, P =.02857). This study demonstrated that engineered cells could be successfully preimplanted into VCAs—an important step toward development of an in vivo biosensor platform to use in modulating acute VCA outcomes. [Display omitted] [ABSTRACT FROM AUTHOR]