Paulo N. Martins

Age and immune response in organ transplantation.

The immune system undergoes a complex and continuous remodeling as the result of aging. These changes have a major impact on allorecognition and alloresponse. In addition, immunosuppression in the elderly is challenging as a consequence of an increased incidence of associated comorbidities and altered pharmacokinetics. Both advanced donor and recipient age should be considered independent risk factors for poor patient and graft survival rates, albeit acting in a synergistic manner. Consequently, modifications of the immune system because of aging may request an age-adapted allocation and immunosuppression in parallel with close patient monitoring. Interventions to selectively target changes associated with the senescence process seem to be promising therapeutic strategies to improve transplantation outcome. Here, we are going to review the immunologic changes associated with the aging process relevant for transplantation and their impact on immunosuppressive protocols, organ allocation policies, and transplantation outcome.

Rodent models of partial hepatectomies

Small rodents are the most used experimental models in liver surgical research. Hepatic resections in rodents are commonly performed to study liver regeneration, acute liver failure, hepatic metastasis, hepatic function, ‘small‐for‐size’ transplantation and metabolic response to injury. Most resections require only basic skills, are fast, reliable and highly reproducible. The partial hepatectomy technique in rodents can be improved by microsurgical techniques, which permit individualized dissection and ligature of the vascular and biliary branches with minimal operative morbidity and mortality. This is particularly relevant for murine models of liver resection. However, it requires advanced microsurgical skills. Here, we review the models, surgical techniques, results and limitations of partial liver resections in rodent models. We also reported for the first time segmentectomies of the median lobe in rodent models.

Subnormothermic Machine Perfusion for Ex Vivo Preservation and Recovery of the Human Liver for Transplantation

To reduce widespread shortages, attempts are made to use more marginal livers for transplantation. Many of these grafts are discarded for fear of inferior survival rates or biliary complications. Recent advances in organ preservation have shown that ex vivo subnormothermic machine perfusion has the potential to improve preservation and recover marginal livers pretransplantation. To determine the feasibility in human livers, we assessed the effect of 3 h of oxygenated subnormothermic machine perfusion (21°C) on seven livers discarded for transplantation. Biochemical and microscopic assessment revealed minimal injury sustained during perfusion. Improved oxygen uptake (1.30 [1.11–1.94] to 6.74 [4.15–8.16] mL O2/min kg liver), lactate levels (4.04 [3.70–5.99] to 2.29 [1.20–3.43] mmol/L) and adenosine triphosphate content (45.0 [70.6–87.5] pmol/mg preperfusion to 167.5 [151.5–237.2] pmol/mg after perfusion) were observed. Liver function, reflected by urea, albumin and bile production, was seen during perfusion. Bile production increased and the composition of bile (bile salts/phospholipid ratio, pH and bicarbonate concentration) became more favorable. In conclusion, ex vivo subnormothermic machine perfusion effectively maintains liver function with minimal injury and sustains or improves various hepatobiliary parameters postischemia.

Injury to peribiliary glands and vascular plexus before liver transplantation predicts formation of non-anastomotic biliary strictures.

BACKGROUND & AIMS The peribiliary glands of large bile ducts have been identified as a niche of progenitor cells that contribute to regeneration of biliary epithelium after injury. We aimed to determine whether injury to the peribiliary glands of donor livers is a risk factor for development of non-anastomotic biliary strictures (NAS) after liver transplantation. METHODS In 128 liver transplant procedures, biopsies were taken from the donor bile duct and injury was assessed using an established histological grading system. Histological severity of injury was subsequently compared in liver grafts that later developed biliary structures vs. uncomplicated liver grafts. RESULTS Luminal biliary epithelial loss >50% was observed in 91.8% of the grafts before transplantation, yet NAS occurred in only 16.4%. Periluminal peribiliary glands were more severely injured than deep peribiliary glands located near the fibromuscular layer (>50% loss in 56.9% vs. 17.5%, respectively; p<0.001). Injury of deep peribiliary glands was more prevalent and more severe in livers that later developed NAS, compared to grafts without NAS (>50% loss in 50.0% vs. 9.8%, respectively; p=0.004). In parallel, injury of the peribiliary vascular plexus was more severe in livers that developed NAS, compared to grafts without NAS (>50% vascular changes in 57.1% vs. 20.3%; p=0.006). CONCLUSION Injury of peribiliary glands and vascular plexus before transplantation is strongly associated with the occurrence of biliary strictures after transplantation. This suggests that insufficient regeneration due to loss of peribiliary glands or impaired blood supply may explain the development of biliary strictures.

Surgical anatomy of the liver, hepatic vasculature and bile ducts in the rat

Background: The rat is the most used experimental model in surgical research. Virtually all procedures in clinical liver surgery can be performed in the rat. However, the use of the rat model in liver surgery is limited by its small size and limited knowledge of the liver anatomy. As in humans, the rat liver vasculature and biliary system have many anatomical variations. The development of surgical techniques, and the study of liver function and diseases require detailed knowledge of the regional anatomy.

Injury to Peribiliary Glands and Vascular Plexus Before Liver Transplantation Predicts Formation of Non-Anastomotic Biliary Strictures

C1650 The Knockout of Rap1 Accelerates Liver Regeneration After Transplantation. C. Li, C. Lo, K. Ng, X. Qi, W. Geng, Y. Ma, X. Liu, H. Liu, K. Man. Surgery, The University of Hong Kong, Hong Kong, China. Introduction and aim: Impaired of liver regeneration severe affects the tissue repair following liver surgery. Cytokine, growth factor and metabolic networks play important roles in theprogression of liver regeneration. Rap1 is a part of the shelterin complex at mammalian telomeres, involved in protecting chromosome ends and promoting gene silencing. Recent research revealed its novel roles in the regulation of metabolism andinfl ammatory response through binding to extra-telomeric sites. We recently also showed that the knockout of Rap1 attenuated liver graft injury after transplantation. Here, we aimed to investigate the role of Rap1 in liver regeneration and to explore underlying mechanism. Methods:Clinically, the intragraft Rap1 expression and its correlation among liver function, infl ammatory cytokines/chemokines, and macrophage infi ltration were analyzed in human liver graft biopsies after transplantation. To investigate the direct role of Rap1 in hepatocyte proliferation and liver regeneration, Rap1 knockoutand wild type mice were subjected to major hepatectomy plus partial hepatic ischemia/ reperfusion injury (IRI). Mouse hepatocyte proliferation, histological damage, liver function and gene expressions were compared between Rap1 knockoutand wild type group. Results: Intrahepatic Rap1 expression was increased in small-for-size graft in comparison to normal graft (2.9 vs 1.9 folds of normal liver, p<0.05), and associated with higher expressions of infl ammatory cytokines/chemokines and more infi ltrations of macrophage and neutrophil at 2 hours after transplantation. Furthermore, overexpression of Rap1 was signifi cantly correlated with impaired liver function after transplantation (ALT: day 1, 3, 4 p<0.05; AST: day 0, 3, 4, 5 p<0.05). In mouse model, the knockout of Rap1 signifi cantly accelerated liver regeneration at day 2 after major hepatectomy and hepatic IRI compared to wild type group (Ki67: 56 vs 11/ HPF, p<0.05; PCNA: 46 vs 29/HPF, p<0.05). The knockout of Rap1 also attenuated histological damage and liver function. Furthermore, reduced expression of PPAR-α and increased expressions of G0S2 and IL1β were detected in Rap1 knockout mice. Conclusion: The knockout of Rap1 accelerates liver regeneration and associated with the regulation of cell cycle control, PPAR-α and cytokine signaling. Abstract# C1651 Injury of Peribiliary Glands and Vascular Plexus Before Liver Transplantation Predicts Formation of Non-Anastomotic Biliary Strictures. N. Karimian,1 S. op den Dries,1,3 A. Westerkamp,1 A. Gouw,2 J. Markmann,3 T. Lisman,1 H. Yeh,3 K. Uygun,3 P. Martins,3 R. Porte.1 1Department of Surgery, Section of Hepatopancreatobiliary Surgery and Liver Transplantation, University Medical Center Groningen (UMCG), Groningen, Netherlands; 2Department of Pathology, University Medical Center Groningen (UMCG), Groningen, Netherlands; 3Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston. C1651 Injury of Peribiliary Glands and Vascular Plexus Before Liver Transplantation Predicts Formation of Non-Anastomotic Biliary Strictures. N. Karimian,1 S. op den Dries,1,3 A. Westerkamp,1 A. Gouw,2 J. Markmann,3 T. Lisman,1 H. Yeh,3 K. Uygun,3 P. Martins,3 R. Porte.1 1Department of Surgery, Section of Hepatopancreatobiliary Surgery and Liver Transplantation, University Medical Center Groningen (UMCG), Groningen, Netherlands; 2Department of Pathology, University Medical Center Groningen (UMCG), Groningen, Netherlands; 3Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston. Peribiliary glands of large bile ducts have been identifi ed as a niche of progenitor cells that contribute to regeneration of biliary epithelium after injury. It is unknown whether injury of the peribiliary glands is a risk factor for the development of non-anastomotic biliary strictures (NAS) after liver transplantation. Moreover, it is unknown whether pretransplant biliary injury is different in livers donated after brain death (DBD) or cardiac death (DCD). In 128 liver transplant procedures, biopsies were taken from the extrahepatic bile duct and injury was assessed using a systematic histological grading system. Histological injury was correlated with the occurrence of posttransplant biliary strictures and a comparison wasmade between DBD (n=97) and DCD livers (n=29). Biliary epithelial loss >50% was observed in 91.8% of the grafts before transplantation, yet NAS occurred in 16.4%. Periluminal peribiliary glands were more severely injured than the deep peribiliary glands located near the fi bromuscular layer (>50% loss in 56.9% versus 17.5%, respectively; p<0.001). Injury of deep peribiliary glands was more prevalent and more severe in livers that later developed NAS, compared to uncomplicated grafts (>50% loss in 50.0% versus 9.8%, respectively; p=0.004). In parallel, injury of the peribiliary vascular plexus was more severe in livers that developed NAS, compared to uncomplicated grafts (>50% vascular changes in 57.1% versus 20.3%; p=0.006). Comparison of DBD and DCD livers revealed signifi cantly more vascular injury in the latter (p=0.005). Conclusion: Injury of peribiliary glands and vascular plexus before transplantation is strongly associated with the occurrence of biliary strictures after transplantation. This suggests that insuffi cient regeneration due to loss of peribiliary glands and blood supply may explain the development of biliary strictures. Abstract# C1652 N-Acetylcysteine Treatment Attenuates ROS Mediated Endoplasmic Reticulum Stress and Apoptosis During Liver Ischemia Reperfusion Injury. H. Lu, F. Zhang, J. Rao, Y. Sun, X. Qian, L. Lu, X. Wang. Liver Transplantation Center, First Affiliated Hospital of Nanjing Medical University(NJMU); Key Laboratory of Living Donor Liver Transplantation of Ministry of Public Health, Nanjing, China. Background: The protective effects of N-acetylcysteine (NAC) treatment have been demonstrated during ischemia reperfusion injury (IRI) in several organs, but its underlying mechanism has not been suffi ciently elucidated. Our previous data has shown that ER stress is critical for the development of liver IRI. This study investigated effects of NAC on ER stress and tissue injury during liver IRI. Methods: Mice were injected with NAC (300mg/kg, ip) 2 hours before ischemia. The level of reactive oxygen species (ROS) was analyzed by GSH and MDA. Hepatic injury was evaluated based on sALT and histopathology. Apoptosis was analyzed by TUNEL staining and Caspase-3 activities. ER stress molecules (GRP78, ATF4 and CHOP) were determined by real-time PCR and western blotting in vivo and in vitro. Antiapoptotic molecules related to ER stress (Bcl-2 and Bcl-xl) were assessed after reperfusion. To analyze the roles of NAC on ROS-mediated ER stress and apoptosis, LDH was examined in cultured hepatocytes treated by H2O2 or Thapsigargin (TG). Results: NAC treatment signifi cantly attenuated ROS-mediated liver injury after IRI. Importantly, ROS-mediated ER stress was signifi cantly inhibited in NAC treated mice after IRI. As demonstrated by experiments in vitro, NAC treatment signifi cantly reduced the upregulation of ER stress molecules after H2O2 treatment. Furthermore, NAC treatment signifi cantly reduced caspase-3 activity after reperfusion, which was in line with the protein expression of Bcl-2 and Bcl-xl. Similarly, NAC treatment signifi cantly inhibited LDH release from hepatocytes treated by H2O2 or TG. Conclusions: This work provides a new evidence for the protective effects of NAC treatment on hepatocytes from IRI. Through the inhibition of ROS-mediated ER stress, NAC may be critical for inhibiting the ER stress related apoptosis pathway. Abstract# C1653 Inhibition of 12/15-Lipoxygenase Prevents Cell Death After Hepatic Ischemia and Reperfusion. M. Drefs, M. Thomas, A. Khandoga, F. Haak, J. Andrassy, M. Guba, J. Werner, M. Rentsch. Department of General, Visceral, Transplantation, Vascular and Thoracic Surgery, Hospital of the University of Munich, Munich, Germany. Introduction Graft failure secondary to ischemia-reperfusion-injury (IRI) still represents a major complication in liver transplantion. 12/15-Lipoxygenase (LOX), a protein of the glutathione-peroxidase-4 (GPx-4) signaling cascade, has been proven to substantially mediate cerebral postischemic apoptosis. Aim of this study was to investigate the impact of 12/15-LOX inhibition on hepatic IRI and thus gain insights into GPx-4 dependent signaling in the liver. Methods Livers of C57BL/6 mice were exposed to 60 minutes of warm ischemia by clamping the common pedicle of the median and left lateral liver lobe and subsequent reperfusion for 90 minutes. Baicalein, an inhibitor of 12/15-LOX, was administered intraperitoneally 30 minutes before operation (group 1). Controls were treated with vehicle dimethylsulfoxide(DMSO) (group 2) or untreated (group 3).Tissue samples were analyzed by TUNEL assay and Western Blot for pro-apoptotic proteins p44/42 MAP kinase (ERK1/2), Jun-amino-terminal kinase (JNK), Poly-ADP-ribose polymerase (PARP) and Caspase-3. Results Analysis of hepatic cell death by fl uorescence TUNEL labeling showed a signifi cant reduction of apoptotic cells in liver samples pretreated with baicalein (group 1: -64.8%; p<0,001) and with DMSO (group 2: -23.2%) compared to untreated samples (group 1). Western Blot analysis revealed a considerable downregulation of ERK1/2 (-36,7%) and PARP (-73,8%), as well as a slight reduction of JNK and Caspase-3 after Baicalein administration. The pretreatment with DMSO also showed a slight down-regulation regarding the investigated pro-apoptotic protein cascade, however in a discrete fashion. Conclusion Inhibition of 12/15-LOX leads to a signifi cant decr

Induction of carbon monoxide in donor animals prior to organ procurement reduces graft immunogenicity and inhibits chronic allograft dysfunction.

Background. Nonspecific inflammatory damages occurring prior to organ transplantation reduce long-term graft survival. Here, we tested the beneficial effects of carbon monoxide (CO) induction by methylene chloride (MC). Methods. Fischer-344 (F-344 Rat) or Dark Agouti (DA Rat) donor animals were either treated with MC four hours prior to organ removal or remained untreated. Kidneys were transplanted into Lewis (LEW) recipients. The low responder strain combination (F-344→LEW) was studied for long-term graft changes. Dendritic cells (DCs) migration and early changes were followed in additional groups of a high responding donor/recipient strain combination (DA→LEW). Native kidneys of uninephrectomized, age-matched normal animals served as controls. Results. Following MC application COHb peaked within two hours in donor animals. Renal function and morphology improved significantly in renal allografts of CO induced donor animals and were comparable to native controls long-term (24 wks). Early after transplantation (24 hr) donor-derived DCs, CD4+ T-cells and alloreactive T-cells were significantly reduced following the engraftment of organs from treated donors. In addition, a trend towards a Th1/Th2 shift and a significant intragraft reduction of CD3 mRNA expression was observed. Conclusion. Donor treatment for the induction of CO reduced graft immunogenicity and inhibited chronic allograft nephropathy.

Modifying graft immunogenicity and immune response prior to transplantation: potential clinical applications of donor and graft treatment

Many studies have shown a strong association between initial graft injury and poor long‐term graft outcome. Events initiated by unspecific immune‐activating processes including brain death and ischemia/reperfusion injury occurring prior to transplantation reduce graft functionality and amplify the host immune response. These events may be particularly relevant for less than optimal grafts with reduced resistance to unspecific injuries. Several approaches to ameliorate immune activation of the graft by treating the donor or the graft have been studied. While various substances have been shown to have protective effects in experimental transplantation, only a few drugs have been tested clinically and have demonstrated beneficial effects. We review the results of experimental and clinical studies on donor or graft immunomodulation prior to transplantation and analyze the evidence to support clinical application of these strategies.

Age-related differences in hepatic ischemia/reperfusion: gene activation, liver injury, and protective effect of melatonin.

BACKGROUND Ischemia/reperfusion (I/R) causes functional and structural damage to liver cells, this being more pronounced with increasing age of the tissue. Melatonin is a pineal indole that has been shown to play an important role as a free radical scavenger and anti-inflammatory molecule. MATERIAL AND METHODS The age-dependent responses to I/R were compared in 2-mo-old and 14-mo-old male Wistar rats. After 35 min of hepatic ischemia followed by 36 h of reperfusion, rats were sacrificed. Sham-operated control rats underwent the same protocol without real vascular occlusion. Animals were intraperitoneally injected with 10 mg/kg melatonin 24 h before the operation, at the time of surgery, and 12 and 24 h after it. The tissues were submitted to histopathologic evaluation. The levels of ALT and AST were analyzed in plasma. The expression of TNF-α, IL-1β, IL-10, MCP-1, IFN-γ, iNOS, eNOS, Bad, Bax, Bcl2, AIF, PCNA, and NFKB1 genes were detected by RT-PCR in hepatic tissue. RESULTS I/R was associated with significant increases in the expression of pro-inflammatory and pro-apoptotic genes in liver. Older rats submitted to I/R were found to respond with increased liver damage as compared with young rats, with serum ALT and AST levels significantly higher than in young animals. Mature rats also showed more evident increases in expression of pro-inflammatory cytokines (IL-1β, MCP-1, and IFN-γ) as well as a decrease in the mRNA expression of IL-10 as compared with young animals. Pro-apoptotic genes (Bax, Bad, and AIF) were significantly enhanced in liver after I/R, without differences between young and mature animals. However, the expression of Bcl2 gene did not show any change. Melatonin treatment was able to lower the expression of pro-inflammatory cytokines and pro-apoptotic genes and to improve liver function, as indicated by normalization of plasma AST and ALT levels and by reduction of necrosis and microsteatosis areas. CONCLUSIONS Melatonin treatment was able to reduce the I/R-stimulated pro-inflammatory and pro-apoptotic genes in the rat liver. Since older animals showed a more marked increase in inflammation and in liver injury, the treatment was more effective in those subjects.

Basic microsurgery training: comments and proposal

Microsurgical techniques have been applied in many surgical specialties and have also a broad application in surgical research. It demands high technical skills and continued training. The microsurgical training is lengthy, very expensive and demands high commitment. The microsurgical skills should be first mastered in the lab and only then applied in the clinic. Here, we propose a model of a training course in microsurgery. We also suggest that surgical societies involved with microsurgery promote training courses on a regular basis.