Graft Reperfusion Injury Research Articles

Calcineurin Inhibitor Sparing Immunosuppression in a 12-year-old Heart Transplant Recipient with a Single Kidney

Introduction Renal dysfunction requires renal transplant or dialysis in up to 10% of transplant patients at 15 years. Those with chronic renal disease or single kidney are at high risk, but there has been limited study of calcineurin inhibitor (CNI)-sparing heart transplantation (HT) in pediatrics. Case Report A 12 year old female with history of Wilms tumor status post left nephrectomy developed anthracycline-induced dilated cardiomyopathy. She had decompensated heart failure bridged to transplant with milrinone. Given prior recurrent acute kidney injury (AKI) in the setting of a single kidney, she was at increased risk of receiving dialysis post-operatively, but a combined heart-kidney transplant was not felt indicated. She was induced with thymoglobulin and initially managed with concomitant mycophenolate mofetil and methylprednisolone. At approximately 3 weeks post-transplant, sirolimus was added. Within 2 week postoperatively, she developed hypertension, acute tubular necrosis, and oliguric AKI. Prior to starting Sirolimus, she also developed pleural and pericardial effusions requiring a chest tube, pericardiocentesis, and pericardial window. Despite these complications there was no evidence of transplant rejection or graft reperfusion injury on biopsy, but filling pressures were upwards of 25 mmHg bilaterally. Aggressive hypertensive management and judicious diuretic use improved her filling pressures and overall clinical status. She was discharged home 60 days after HT. At her 3 month post-op catheterization she had normal filling pressures and no signs of rejection (ISHLT grade A0, AMR grade 0) on low dose prednisone, mycophenolate, and sirolimus. Her AKI has resolved and she remains dialysis free. Summary AKI after HT is due to a constellation of renal hypoperfusion, low cardiac output, and drug-induced nephrotoxicity requiring dialysis in some patients. Prior studies showed improved renal function in those converted from CNIs to mTOR inhibitors. In our case, the morbidity associated with AKI was deemed too severe to pursue standard use of CNIs. While some pediatric patients have undergone immunosuppression with reduced CNI dosing, few have completed HT without CNI. Her case illustrates the utility of mTOR inhibitors for its nephrotoxic sparing profile while maintaining adequate immunosuppression protection and graft function.

Morphofunctional evaluation of liver grafts obtained from standard donors and expanded criteria donors

Donor organ shortage stimulated an active use of donors with expanded evaluation criteria. The main evaluation method was a histological examination of liver graft biopsy specimens before (time-zero biopsy) and after reperfusion (time-1 biopsy). Severe ischemic and reperfusion injuries among recipients who received a liver graft made 20.4% and 16.6% respectively. The study showed no impact of small droplet, medium droplet, or even large droplet steatosis (less than 50%) on graft reperfusion injury.

Eculizumab for Prevention and Treatment of Kidney Graft Reperfusion Injury, Preliminary Results of RCT.

Eculizumab for Prevention and Treatment of Kidney Graft Reperfusion Injury, Preliminary Results of RCT.

Eculizumab reverses the potentially fatal effects of kidney graft reperfusion injury

Half an hour after reperfusion, the kidney, transplanted to the infant from an adult brain dead standard criteria donor, became flabby and acquired blue color. Hyperacute rejection was suspected as a consequence of false negative cross match, and eculizumab was administered with the purpose to treat antibody-mediated injury, with fast and clear effect. The patient's blood was tested for donor-specific antibodies on the next day, and results were negative. We attribute graft damage to reperfusion injury and explain eculizumab's effectiveness to its ability to prevent progression of reperfusion injury.

The Effect of Perfusion Speed on Graft Reperfusion Injury after Rat Orthotopic Liver Transplantation

ObjectiveThe objective of this study was to investigate the effects of various perfusion speeds on reperfusion injury of grafts after liver transplantation. MethodsLiver transplantation was performed from Sprague-Dawley (SD) to SD rats. Recipients were divided into 4 groups according to perfusion speed: 50 mL/h for group A, 100 mL/h for group B, 150 mL/h for group C, and 200 mL/h for group D. Peripheral blood was collected from the caudal vein. All survivors were humanely killed at 24 hours posttransplantation. The morphological changes in grafts were evaluated using light microscopy, serum tumor necrosis factor-α (TNF-α), and endothelial nitric oxide synthase (eNOS) proteins as well as their messenger RNA (mRNA)-intragraft levels. ResultsThe pathohistological damage of grafts in the 150 mL/h and 200 mL/h groups were compared with the 50 mL/h and 100 mL/h groups. TNF-α in serum was increased at 6 hours, reaching a peak at 12 hours posttransplantation. The TNF-α levels in the 50 mL/h and 100 mL/h groups were significantly lower than the 150 mL/h and 200 mL/h groups; the 50 mL/h group was greater than the 100 mL/h group as well as the 200 mL/h group was greater than the 150 mL/h group. The expressions of eNOS protein and mRNA intragraft in the 150 mL/h and 200 mL/h groups were significantly reduced compared with the 50 mL/h and 100 mL/h groups, and the 50 mL/h group was decreased when compared with the 100 mL/h group. There was no significant difference between the 150 mL/h group and the 200 mL/h group. ConclusionThe optimal perfusion speed for harvesting rat liver graft is 100 mL/h. High-speed perfusion may impair sinusoid endothelial cells and low-speed perfusion may extend the warm ischemia time and increase microthrombus formation.

How to protect liver graft with nitric oxide

Organ preservation and ischemia reperfusion injury associated with liver transplantation play an important role in the induction of graft injury. One of the earliest events associated with the reperfusion injury is endothelial cell dysfunction. It is generally accepted that endothelial nitric oxide synthase (e-NOS) is cell-protective by mediating vasodilatation, whereas inducible nitric oxide synthase mediates liver graft injury after transplantation. We conducted a critical review of the literature evaluating the potential applications of regulating and promoting e-NOS activity in liver preservation and transplantation, showing the most current evidence to support the concept that enhanced bioavailability of NO derived from e-NOS is detrimental to ameliorate graft liver preservation, as well as preventing subsequent graft reperfusion injury. This review deals mainly with the beneficial effects of promoting "endogenous" pathways for NO generation, via e-NOS inducer drugs in cold preservation solution, surgical strategies such as ischemic preconditioning, and alternative "exogenous" pathways that focus on the enrichment of cold storage liquid with NO donors. Finally, we also provide a basic bench-to-bed side summary of the liver physiology and cell signalling mechanisms that account for explaining the e-NOS protective effects in liver preservation and transplantation.

Recipient intramuscular administration of naked plasmid TGF-β1 attenuates lung graft reperfusion injury

Background Gene therapy may be an effective strategy for modulating lung graft ischemia–reperfusion injury. We investigated whether recipient intramuscular (IM) naked plasmid gene transfer of transforming growth factor β1–active (TGF-β1–active) ameliorates lung graft ischemia–reperfusion injury. Methods Preliminary studies in F344 rats demonstrated that gastrocnemius muscle transfection of TGF-β1–active produced muscle and plasma protein expression at 24 and 48 hours after transfection. Recipients ( n = 8) received IM injection of naked plasmid-encoding chloramphenicol acetyl transferase (CAT), TGF-β1–latent or TGF-β1–active, respectively, at 24 or at 48 hours before left lung transplantation. We did not treat the control group before transplantation (18-hour cold ischemia). Donor lungs were flushed with low-potassium dextran–1% glucose and stored for 18 hours at 4°C. All groups were killed at 24 hours after transplantation. Immediately before killing the animals, we clamped the contralateral right hilum and assessed graft function. We measured wet-to-dry ratio (W/D), myeloperoxidase, pro-inflammatory cytokines (interleukin 1 [IL-1], tumor necrosis factor α [TNF-α], interferon-γ [INF-γ], and IL-2) and performed immunohistochemistry. Results Arterial oxygenation was greatest in the recipient group transfected with TGF-β1–active at 24 hours before transplantation compared with CAT, TGF-β1–latent, and 18-hour cold ischemia groups ( p < 0.01). The W/D ratio and myeloperoxidase decreased in both 24- and 48-hour groups, with TGF-β1–active compared with CAT, and 18-hour cold ischemia groups (W/D, p < 0.02 and p < 0.004, respectively; myeloperoxidase, p < 0.05 and p < 0.01, respectively). All pro-inflammatory cytokines decreased in the 24-hour TGF-β1–active group compared with CAT, TGF-β1–latent, 18-hour and 1-hour cold ischemia, and non-treated lung groups (IL-1β, p < 0.03; TNF-α, p < 0.02; IFN-γ, p < 0.001; IL-2, p < 0.0001). In 24- and 48-hour groups with TGF-β1–active, immunohistochemistry showed marked staining of Type I and Type II alveolar cells and of macrophages from the apical to the caudal sections of the lung grafts. Conclusions Recipient IM administration of naked plasmid encoding TGF-β1–active before transplantation ameliorates lung isograft reperfusion injury after prolonged ischemia.

Recipient intramuscular gene transfer of naked plasmid TGFβ1 ameliorates lung graft reperfusion injury

Patients in the control group consisted of: 1)the ‘twin‘ recipient of an index single lung transplant (contralateral lung from the same donor) and 2) where no ‘twin’ existed (in single and double lung transplants), the recipients of transplants performed immediately before and after an index case in patients with the same indication for transplantation. Twenty-five single and 14 double lung recipients formed the index group; thirty-six single and 29 double lung recipients formed the control group. No significant differences were found between groups in A-a gradient or pO2/FiO2 ratio at 1 or 6 hours postoperatively; pulmonary artery pressures at 1, 6, or 24 hours postoperatively; or time to extubation. Index patients demonstrated trends toward shorter ICU stay (p5.09) and length of hospitalization (p5.13). Cold ischemic time were significantly longer (p5.008) for index cases. Index patients showed a strong trend (p5.057) toward greater survival at 1 year (81.6% vs. 68.3%), 3 years (81.6% vs. 60.4%) and 5 years (65.9% vs. 53.7%) posttransplant. Analysis of radiographic data, rejection episodes and BOS scores in the two groups will be discussed. In conclusion, controlled reperfusion in human lung transplantation may impact favorably on outcomes and warrant further study.

Transhepatic neutrophil and monocyte activation during clinical liver transplantation.

During experimental liver transplantation, neutrophil sequestration results in increased oxygen free radical production and correlates inversely with graft viability. Neutrophil activation in clinical liver transplantation is poorly understood. We assessed leukocyte sequestration and transhepatic differences of neutrophil and monocyte CD11b expression, neutrophil free radical production, and plasma concentrations of interleukin 6 and interleukin 8 in nine patients during liver transplantation. Significant hepatic neutrophil sequestration occurred during initial graft rewarming with portal blood, after inferior vena cava declamping, and after hepatic artery declamping (all P<0.05). A positive transhepatic difference (i.e., outcoming - ingoing) in CD11b expression of neutrophils was observed after portal vein declamping (51+/-32 relative fluorescence unit [RFU]) and in CD11b expression of monocytes during initial graft rewarming (67+/-86 RFU, both P<0.05). A transcoronary increase in both unstimulated (74+/-80 RFU) and N-formyl-methionyl-leucylphenylalanine-stimulated (112+/-168 RFU) neutrophil free radical production took place after hepatic artery declamping (both P<0.05). A negative transcoronary difference of interleukin 6 occurred during initial graft rewarming (-192+/-176 pg/ml) and a positive difference of interleukin 8 occurred after hepatic artery declamping (17+/-23 pg/ml, both P<0.05). Hepatic sequestration and transhepatic activation of neutrophils, and hepatic production of interleukin 8 occur during clinical liver transplantation. A splanchnic influx of interleukin 6 occurs to the graft, possibly modulating neutrophil-mediated graft reperfusion injury.

Alterations of Na + homeostasis in hepatocyte reoxygenation injury

Reperfusion injury represents an important cause of primary graft non-function during liver transplantation. However, the mechanism responsible for cellular damage during reoxygenation has not yet been completely understood. We have investigated whether changes in intracellular Na + distribution might contribute to cause hepatocyte damage during reoxygenation buffer after 24 h of cold storage. Hepatocyte reoxygenation resulted in a rapid increase in cellular Na + content that was associated with cytotoxicity. Na + accumulation and hepatocyte death were prevented by the omission of Na + from the incubation medium, but not by the addition of antioxidants. Blocking Na +/H + exchanger and Na +/HCO 3 − co-transporter by, respectively, 5-( N,N-dimethyl)-amiloride or omitting HCO 3 − from the reoxygenation medium significantly decreased Na + overload and cytotoxicity. Stimulation of ATP re-synthesis by the addition of fructose also lowered Na + accumulation and cell death during reoxygenation. A significant protection against Na +-mediated reoxygenation injury was evident in hepatocytes maintained in an acidic buffer (pH 6.5) or in the presence of glycine. The cytoprotective action of glycine or of the acidic buffer was reverted by promoting Na + influx with the Na +/H + ionophore monensin. Altogether, these results suggest that Na + accumulation during the early phases of reoxygenation might contribute to liver graft reperfusion injury.

Ex vivo gene therapy prevents chronic graft vascular disease in cardiac allografts

Objective: We hypothesized that ex vivo hyperbaric transfection of antisense oligodeoxynucleotides for blockade of intercellular adhesion molecule–1, an important mediator of cell adhesion and T-cell co-stimulation, would reduce chronic graft vascular disease in cardiac allografts. Methods: PVG hearts underwent ex vivo transfection with antisense, reverse antisense intercellular adhesion molecule–1 oligodeoxynucleotide (80 μmol/L), or saline solution at 3 atm pressure for 45 minutes at 4° C and were transplanted heterotopically into ACI recipients with or without treatment with intercellular adhesion molecule–1 (1A29) or leukocyte function associated antigen–1 (WT.1) monoclonal antibodies. Transfection efficiency was confirmed with fluorescein isothiocyanate–labeled oligodeoxynucleotides and fluorescent microscopy. Efficacy of intracellular adhesion molecule–1 blockade was assessed with the use of immunohistochemistry. Graft reperfusion injury was evaluated at 6 to 24 hours by neutrophil infiltration (myeloperoxidase [MPO]), cardiac edema (%wt/wt), and histologic injury (percent contraction band necrosis). Grafts from recipients treated with cyclosporine A (5 mg/kg per day, days 0 to 9) were scored for chronic graft vascular disease on postoperative day 90 ranging from 0 (no involvement) to 4 (>50% vascular occlusion). Results: Transfection was highly efficient (fluorescein isothiocyanate–labeled oligodeoxynucleotides in 48% ± 5% of total myocardial nuclei) and effective at blocking intracellular adhesion molecule–1 expression (positive area in allografts taken on postoperative day 3 was reduced from 100% ± 0% to 52% ± 14%, n = 4). Blockade with antisense oligodeoxynucleotides versus monoclonal antibodies was less effective at preventing reperfusion injury while more effective at reducing chronic graft vascular disease (score 0.98 ± 0.48, P < 0.05). Reverse antisense oligodeoxynucleotides and vector control (antisense oligodeoxynucleotide infusion without pressure) groups failed to demonstrate this beneficial effect. Conclusion: Hyperbaric transfection of antisense oligodeoxynucleotides proved highly efficient, effective at blockade of intracellular adhesion molecule–1, and demonstrated a sequence-specific reduction in chronic graft vascular disease. This highly targeted alteration of donor organ immunogenicity may have an important future role in clinical immunosuppressive strategies. (J Thorac Cardiovasc Surg 1998;116:386-96)

Attenuation of lung graft reperfusion injury by a nitric oxide donor

Objective: One of the primary features of ischemia-reperfusion injury is reduced production of protective autocoids, such as nitric oxide, by dysfunctional endothelium. Administration of a nitric oxide donor during reperfusion of lung grafts may therefore be beneficial through modulation of vascular tone and leukocyte and platelet function. Methods: Rat lung grafts were flushed with University of Wisconsin solution and reperfused for 1 hour in an ex vivo model incorporating a support animal. Group I grafts ( n = 6) were reperfused immediately after explantation, group II ( n = 6) and III ( n = 5) grafts after 24 hours of storage at 4° C. In group III, glyceryl trinitrate, a nitric oxide donor, was administered during the first 10 minutes of reperfusion at a rate of 200 μg/min. In an additional group ( n = 5), 200 μg/min hydralazine was administered instead, to assess the effect of vasodilation alone. Results: Graft function in group II deteriorated compared with that in group I, with significant reduction of graft effluent oxygen tension and blood flow and elevation of pulmonary artery pressure, peak airway pressure, and wet/dry weight ratio. In contrast, in group III, glyceryl trinitrate treatment improved graft function to baseline levels in all these parameters. Administration of hydralazine, meanwhile, produced mixed results with only two out of five grafts functioning at control levels. Conclusions: In this model, administration of glyceryl trinitrate to supplement the nitric oxide pathway in the early phase of reperfusion has a sustained beneficial effect on lung graft function after 24-hour hypothermic storage, probably through mechanisms beyond vasodilation alone. (J Thorac Cardiovasc Surg 1997;113:327-34)

Backgrounds of early intragraft immune activation and rejection in liver transplant recipients. Impact of graft reperfusion quality.

In solid organ transplantation, acute rejections are most frequent during the first weeks. The aim of this study was to investigate the relationship between graft reperfusion injury and later immune responses against the graft. Intragraft immune activation was routinely monitored by transplant aspiration cytology in 47 recipients of hepatic allografts. As a parameter of reperfusion quality, oxygen saturation of hemoglobin (SO2) in hepatic tissue was determined intraoperatively by a near-infrared spectroscopy. Grafts that presented aspiration cytology scores of 2 or more (i.e., more than 10% of lymphocytes activated) at 1 week after operation (group I, n = 14) were associated with a higher heterogeneity of hepatic tissue SO2 at the end of operation (coefficient of variation in 12 points 18.3 +/- 18.3%, mean +/- SD) than grafts with no or very mild intragraft immune activation (group II, n = 33, 9.2 +/- 4.2%; P < 0.01). Group I was also accompanied by higher postoperative peak glutamic oxalacetic transaminase level (corrected by graft size, P < 0.05) and higher donor age (43.9 +/- 12.9 vs. 32.6 +/- 13.9 years, P < 0.02). Heterogenous reperfusion (P < 0.01), higher peak glutamic oxalacetic transaminase level (P < 0.01), and higher donor age (P < 0.05) were also associated with clinical rejection at 1 week (n = 10), but not with later-onset rejection (n = 11). These data suggest that intragraft immune activation and clinical rejection in the early phase after hepatic engraftment are strongly influenced by graft injury, which can be recognized early after reperfusion.