Effect of pre-treatment with catecholamines on cold preservation and ischemia/reperfusion-injury in rats

Treatment of organ donors with catecholamines reduces acute rejection episodes and improves long-term graft survival after renal transplantation. The aim of this study was to investigate the effect of catecholamine pre-treatment on ischemia/reperfusion (I/R)- and cold preservation injury in rat kidneys. I/R-injury was induced by clamping the left kidney vessels for 60 min along with a contralateral nephrectomy. Cold preservation injury was induced by storage of the kidneys for 24 h at +4°C in University of Wisconsin solution, followed by syngeneic transplantation. Rats were pre-treated with either dopamine (DA), dobutamine (DB), or norepinephrine (2, 5, and 10 μg/kg/min, each group) intravenously via an osmotic minipump for 24 h before I/R- and cold preservation injury. Pre-treatment with DA (2 or 5 μg/kg/min) and DB (5 μg/kg/min) improved recovery of renal function after I/R-injury and dose dependently reduced mononuclear and major histocompatibility complex class II-positive cells infiltrating the kidney after I/R-injury. One day after I/R-injury, upregulation of transforming growth factor (TGF)-β 1 and 2 and phosphorylation of p42/p44 mitogen-activated protein kinases was observed in kidneys of animals treated with DA or DB. DA (5 μg/kg/min) and DB (5 μg/kg/min) pre-treatment reduced endothelial cell damage after 24 h of cold preservation. Only DA pre-treatment improved renal function and reduced renal inflammation after 24 h of cold preservation and syngeneic transplantation. Our results demonstrate a protective effect of pre-treatment with catecholamines on renal inflammation and function after I/R- or cold preservation injury. This could help to explain the potent organoprotective effects of catecholamine pre-treatment observed in human kidney transplantation

Indocyanine green elimination test in orthotopic liver recipients.

OBJECTIVE: To determine its predictive capability on graft quality and resultant clinical outcome, the indocyanine green (ICG) elimination test was performed by a spectrophotometric method and a noninvasive finger-piece method with 50 orthotopic liver transplantations. BACKGROUND: Early detection of poor-functioning hepatic grafts is one of the most important issues in liver transplantation, but no reliable methods exist. METHODS: The ICG test was performed after 50 orthotopic liver transplantations on postoperative days 1, 3, and 7. Indocyanine green elimination constants (K(ICG)) were measured by both a standard spectrophotometric analysis (K(ICG)-B) and by a finger-piece method (K(ICG)-F). The patients were followed for a minimum of 3 months after transplantation. Results of ICG tests were correlated with various clinical determinations. RESULTS: Twelve of the 50 grafts were lost within three months, of which 7 were related to graft failure. Multivariate analysis using the Cox proportional hazard model revealed that K(ICG) on postoperative day 1 was a better predictor of liver-related graft outcome than any of the conventional liver function tests. Furthermore, K(ICG) values showed significant correlation with the severity of preservation injury, longer intensive care unit (ICU) and hospital stay, prolonged liver dysfunction, and septic complications. Correlation of K(ICG) values by the spectrophotometric method with those by the finger-piece method was highly satisfactory in the grafts that had K(ICG)-B <0.15 min-1 (y = 0.868x -0.011, r = .955). CONCLUSION: The ICG elimination test, conducted spectrophotometrically or optically on the day after liver transplantation, is a reliable indicator of graft quality and subsequent graft outcome early after liver transplantation

Hyaluronate levels in donor organ washout effluents: a simple and predictive parameter of graft viability

The principal cause of primary non-function in orthotopic liver transplantation is thought to be preservation injury to the microvasculature. We, therefore, evaluated if effluent levels of hyaluronate, whose uptake is an endothelial cell marker, could predict early graft function and ultimate graft outcome in orthotopic liver transplantation. A total of 102 cases were studied in two phases. In the first phase, we attempted to determine if a correlation existed between effluent hyaluronate levels, early graft function and ultimate graft outcome. This phase of the study was also used to determine hypothetical cut-off values for hyaluronate which could discriminate between good and bad livers. Thirty-two livers orthotopically transplanted to randomly selected primary recipients were studied. After varying periods of static cold storage (4°C) in University of Wisconsin solution, the livers were reinfused with cold (4°C) lactated Ringer’s solution. The first 50 ml of the reperfusion effluent was collected from the infrahepatic vena cava. Effluent samples were analyzed for hyaluronate. Linear regression analysis demonstrated a significant correlation between effluent hyaluronate levels and post-operative aspartate and alanine aminotransferase levels (p<0.001 for both). Logistic regression demonstrated a highly significant correlation (p = 0.0056) between effluent hyaluronate levels and ultimate graft outcome. Generation of Receiver Characteristics Curves indicated that a level between 400 and 430 μg·l(−1) could possibly discriminate between good livers and those at risk of early graft failure. The authenticity of this hyaluronate cut-off level was further confirmed in the second phase of the study where 70 consecutive primary crossmatch-negative transplants were performed. A highly significant difference was observed in peak aspartate and alanine aminotransferase levels in the first week (p<0.0006 and p<0.0005, respectively) between livers with effluent hyaluronate levels≤400 μg·l(−1) and livers with hyaluronate levels higher than 400 μg·l(−1) Logistic regression revealed a highly significant correlation between effluent hyaluronate levels and graft success (p=0.0001). Since hyaluronate uptake by the microvascular endothelial cell is significantly greater than production, high hyaluronate effluent levels in failed livers would be due to decreased hyaluronate uptake by the injured microvascular endothelial cell. We therefore conclude that effluent hyaluronate levels may prove to be a reliable preoperative test to assess early graft function and outcome in clinical orthotopic liver transplantation

Investigation Of Molecular Mechanisms Of Liver Preservation Injury: A Complication Preceding Organ Transplantation

Of the over 108,000 American awaiting a life-saving organ transplant today, over 12,000 (11%) of those need a new liver (OPTN, 2020). Last year, only 35% of patients on the waiting list for an organ were transplanted. Improving the quality of marginal organs by preventing or reversing preservation injury could vastly increase the number of transplants performed. Washout of circulating blood during liver procurement with cold University of Wisconsin solution flushes out any endogenous pro-survival signaling molecules. We investigated lysophospholipid (LPL) surface receptors (G-protein coupled receptors for lysophosphatidic acid (LPA) and sphingosine- 1-phosphate (S1P)) and their role in protecting hepatocytes against preservation injury. Using an in vitro model of organ preservation, we found that treatment with exogenous LPA during cold storage does not prevent preservation injury. However, the method of lipid delivery may be to blame. The ratio of intracellular S1P:ceramide, an indicator of cell health, is decreased in hepatocytes following cold storage. Inhibition of sphingosine kinase-2 (SK2), the enzyme responsible for much of the intracellularly acting S1P, with ABC294640 (a selective SK2 inhibitor) is devastating in rodent liver transplant, ex vivo perfusion, and in vitro models. Upon further investigation, the mechanism of ABC294640 toxicity is two-fold: this compound directly inhibits complex I of the mitochondrial electron transport chain, independent of its effects on SK2. These data clarify the detrimental phenotype associated with ABC294640 treatment of the liver during cold storage. Modulating LPL signaling pathways, upstream of mitochondrial activity and cytoskeleton conformation may improve liver graft function following preservation

Evaluation of protocol before transplantation and after reperfusion biopsies from human orthotopic liver allografts: Considerations of preservation and early immunological injury

Light microscopic, immunohistochemical and ultrastructural analysis of protocol before transplantation and after reperfusion biopsy specimens from 87 randomly selected patients was performed to assess the contribution of preservation and immunological injury to early graft failure. Most biopsy specimens were essentially normal by light microscopy before transplantatio, and no particular feature could be relied on to predict function after transplantation. Ultrastructural examination of biopsy specimens before transplantation demonstrated preferential degeneration of sinusoidal lining cells, but no strict correlation was seen between ultrastructural sinusoidal integrity before transplantation and function after transplantation. The presence of zonal or severe focal necrosis and a severe neutrophilic exudate in biopsy specimens after reperfusion presaged a poor early postoperative course in most, but not all, patients. The presence of preformed lymphocytotoxic antibodies had no effect on the early clinical course, but was associated with Kupffer cell hypertrophy in needle biopsy specimens taken after transplantation. No definite evidence was seen of hyperacute rejection as a result of preformed lymphocytotoxic antibodies as detected in conventional assays. These findings suggest that preservation injury accounts for only a subset of grafts that fail to function after transplantation. Other perioperative or “recipient” factors may be of equal or greater importance in early graft dysfunction or failure.(HEPATOLOGY 1990;11:932‐941.). Copyright © 1990 American Association for the Study of Liver Disease

Perioperative donor bone marrow infusion augments chimerism in heart and lung transplant recipients

Background.: We and others have demonstrated that a low level of donor cell chimerism was present for years after transplantation in tissues and peripheral blood of heart and lung recipients; it was associated, in the latter, with a lower incidence of chronic rejection. To augment this phenomenon, we initiated a trial combining simultaneous infusion of donor bone marrow with heart or lung allotransplantation. Methods.: Between September 1993 and January 1995, 15 nonconditioned patients received either heart (n = 10) or lung (n = 5) allografts concurrently with an infusion of unmodified donor bone marrow (3.0 × 108 cells/kg), and were maintained on an immunosuppressive regimen consisting of tacrolimus and steroids. Results.: There was no complication associated with the infusion of donor bone marrow. Chimerism was detectable in 73% of bone marrow-augmented patients up to the last sample tested. Of the 5 control recipients who did not receive bone marrow infusion, only 1 had detectable chimerism by flow on postoperative day 15, which dwindled to an undetectable level by postoperative day 36. None of the patients had evidence of donor-specific immune modulation by mixed lymphocyte reaction. Conclusions.: The combined infusion of donor bone marrow and heart or lung transplantation, without preconditioning of the recipient, is safe and is associated with an augmentation of donor cell chimerism. © 1995 The Society of Thoracic Surgeons

Immuno-molecular profiling and modulation of donor livers during normothermic machine perfusion

Normothermic machine perfusion (NMP) is an increasingly adopted preservation strategy in liver transplantation that facilitates organ assessment, safely prolongs preservation and is associated with reduced ischaemia reperfusion injury (IRI) related complications. However, these IRI-related benefits are less apparent in higher risk organs, which maintain a propensity to develop severe preservation injury despite NMP. The underlying mechanisms and contributory factors associated with this persistent injury are unclear, but may be related to molecular processes occurring during NMP or other preservation factors such as the length of cold ischaemia prior to NMP. This thesis aims to characterise the immunological and molecular processes occurring during NMP and devise interventions targeting these processes to reduce preservation injury. Through an evaluation of samples from clinical trials of human liver perfusion, I have demonstrated that ex situ reperfusion injury/inflammation (ERI) is an immunologically and molecularly distinct entity observed consistently across livers preserved with NMP. I have shown that the presence and length of prior static cold storage (pSCS) can impact the molecular signature of donor livers affecting key pathways and processes involved in mitochondrial function and cellular energetics. I have found that circulating damage associated molecular patterns (DAMPs), constitute an important element in ERI and alongside immune cells and inflammatory cytokines within the perfusate, propagate injury and inflammation ex situ, likely impacting the immunogenicity of the graft. I developed a large animal model to investigate these observations and explore possible modulatory interventions. Finally, I demonstrated that by mitigating ERI, ex situ graft function could be improved and preservation injury reduced in a porcine NMP model. This is achieved by the removal of highly pro-inflammatory nuclear DAMPs (neutrophil extracellular traps (NETs)/nucleosomes, free histones and cell free DNA) from the circulating perfusate during NMP with a specialised column

Luminal Preservation Protects the Small Intestine in a Brain-dead Rat Model

Background. Intestinal transplantation depends on donation after brain death (DBD). Luminal preservation (LP) has been beneficial against preservation injury in previous studies in animal models, but none include DBD. This study aims to investigate whether these benefits occur also with DBD. Methods. Wistar rats (male, N = 9) underwent brain death for 2 h. Thereafter, vascular perfusion was done with University of Wisconsin solution (UW). The small intestine was then explanted and randomized into 3 groups: control (empty segment), LP+PEG (with polyethylene glycol 3350 solution), or LP+UW (with UW), treated and tied shut. Ice-cold UW was used for cold storage. Samples were taken at procurement and after 4 (t = 4) and 8 h (t = 8) of preservation. Histopathological scorings were performed for intestinal preservation injury, subepithelial space, absence of epithelial lining, and hemeoxygenase-1 expression. Results. There was low-level mucosal injury (median intestinal preservation injury score 2) at procurement. At t = 4, bowels treated without LP had more damage than LP-treated samples (control score 4, LP+PEG 2 and LP+UW 2, P < 0.001 control versus LP+UW). At t = 8, no benefit of LP was observed (control 2, LP+PEG 3, LP+UW 2). Subepithelial space increased with time and the presence of LP; epithelial lining was better conserved in LP-treated samples. Hemeoxygenase-1 staining showed increased intensity with increased damage, irrespective of treatment. Conclusions. Luminal perfusion of the small intestine with UW or PEG protects the mucosa in brain-dead rats for up to 4 h. Fewer benefits of LP were found than previously described in non-DBD models. To mimic the clinical situation, DBD should be included in future animal studies on intestinal preservation