Yinghua Tian

Novel Short-term Hypothermic Oxygenated Perfusion (HOPE) System Prevents Injury in Rat Liver Graft From Non-Heart Beating Donor

Objective:To assess a machine perfusion system in rescuing liver grafts from non-heart-beating donors (NHBD). Summary Background Data:The introduction of extracorporeal liver perfusion systems in the clinical routine depends on feasibility. Conceivably, perfusion could be performed during recipient preparation. We investigated whether a novel rat liver machine perfusion applied after in situ ischemia and cold storage can rescue NHBD liver grafts. Methods:We induced cardiac arrest in male Brown Norway rats by phrenotomy and ligation of the subcardial aorta. We studied 2 experimental groups: 45 minutes of warm in situ ischemia + 5 hours cold storage versus 45 minutes of warm in situ ischemia + 5 hours cold storage followed by 1 hour hypothermic oxygenated extracorporeal perfusion (HOPE). In both groups, livers were reperfused in a closed sanguineous isolated liver perfusion device for 3 hours at 37°C. To test the benefit of HOPE on survival, we performed orthotopic liver transplantation in both experimental groups. Results:After cold storage and reperfusion, NHBD livers showed necrosis of hepatocytes, increased release of AST, and decreased bile flow. HOPE improved NHBD livers significantly with a reduction of necrosis, less AST release, and increased bile flow. ATP was severely depleted in cold-stored NHBD livers but restored in livers treated by HOPE. After orthotopic liver transplantation, grafts treated by HOPE demonstrated a significant extension on animal survival. Conclusions:We demonstrate a beneficial effect of HOPE by preventing reperfusion injury in a clinically relevant NHBD model.

Prevention of reperfusion injury and microcirculatory failure in macrosteatotic mouse liver by omega‐3 fatty acids

Macrovesicular hepatic steatosis has a lower tolerance to reperfusion injury than microvesicular steatosis with an abnormally high ratio of omega‐6 (n‐6): omega‐3 (n‐3) polyunsaturated fatty acids (PUFAs). We investigated the influence of PUFAs on microcirculation in steatotic livers and the potential to minimize reperfusion injury in the macrosteatotic liver by normalization of PUFAs. Ob/ob mice were used as a model of macrovesicular hepatic steatosis and C57/Bl6 mice fed a choline‐deficient diet for microvesicular steatosis. Steatotic and lean livers were subjected to 45 minutes of ischemia and 3 hours of reperfusion. Hepatic content of omega‐3 and omega‐6 PUFAs was determined. Microcirculation was investigated using intravital fluorescence microscopy. A second group of ob/ob mice was supplemented with dietary omega‐3 PUFAs and compared with the control diet–fed group. Microcirculation, AST, and Kupffer cell activity were assessed. Macrosteatotic livers had significant microcirculatory dysfunction correlating with high omega‐6: omega‐3 PUFA ratio. Dietary omega‐3 PUFA resulted in normalization of this ratio, reduction of intrahepatic lipids, and decrease in the extent of macrosteatosis. Defective microcirculation was dramatically ameliorated with significant reduction in Kupffer cell activity and protection against hepatocellular injury both before ischemia and after reperfusion. Conclusion: Macrosteatotic livers disclosed an abnormal omega‐6: omega‐3 PUFA ratio that correlates with a microcirculatory defect that enhanced reperfusion injury. Thus, protective strategies applied during or after ischemia are unlikely to be useful. Preoperative dietary omega‐3 PUFAs protect macrosteatotic livers against reperfusion injury and might represent a valuable method to expand the live liver donor pool. (HEPATOLOGY 2007;45:855–863.)

Selective portal vein embolization and ligation trigger different regenerative responses in the rat liver

Two strategies are clinically available to induce selective hypertrophy of the liver: portal vein embolization (PVE) and portal vein ligation (PVL). The aim of this study was to compare the impact of PVE and PVL on liver regeneration. Rats were subjected to 70% PVL, 70% PVE, 70% partial hepatectomy (PH) (positive control), or sham operation (negative control). PVL and PVE of liver segments were validated by portography and histology, demonstrating obstruction of the involved portal branches. Liver weight and markers of regeneration were assessed at 24, 48, and 72 hours, and 7 days after surgery (n = 5). Sinusoidal perfusion was examined by intravital microscopy. The weight of the regenerating liver segments increased continuously in all groups, with the highest weight gain after PH, which also disclosed the strongest proliferative activity. In Ki‐67 and PCNA stainings, hepatocyte proliferation after PVL was more pronounced than after PVE (P = 0.01). Volumetric blood flow and functional sinusoidal density were lower after PVE than after PVL (P = 0.006, P = 0.02, respectively). The accumulation of Kupffer cells 24 hours after the intervention was highest after PH. Transcript levels of cytokines (interleukin‐1β, tumor necrosis factor‐α, interleukin‐6) peaked at 24 hours and were highest after PH. The embolized part of the liver after PVE showed prominent foreign body reaction in the portal triad with accumulation of macrophages. Conclusion: PVL is superior to PVE in inducing a regenerative response of the remnant liver. The impairment of liver regeneration after PVE may be a consequence of macrophage trapping in the occluded segment due to a foreign body reaction. Lower blood flow and lower accumulation of macrophages, particularly Kupffer cells, in the regenerating part of the liver likewise causes impaired liver regeneration after PVE. (HEPATOLOGY 2008.)

Ischemic preconditioning and intermittent clamping improve murine hepatic microcirculation and Kupffer cell function after ischemic injury

The aim of this study was to evaluate whether the protective effect of intermittent clamping and ischemic preconditioning is related to an improved hepatic microcirculation after ischemia/reperfusion injury. Male C57BL/6 mice were subjected to 75 or 120 min of hepatic ischemia and 1 or 3 hours of reperfusion. The effects of continuous ischemia, intermittent clamping, and ischemic preconditioning before prolonged ischemia on sinusoidal perfusion, leukocyte‐endothelial interactions, and Kupffer cell phagocytic activity were analyzed by intravital fluorescence microscopy. Kupffer cell activation was measured by tissue levels of tumor necrosis factor (TNF)‐α, and the integrity of sinusoidal endothelial cells and Kupffer cells were evaluated by electron microscopy. Continuous ischemia resulted in decreased sinusoidal perfusion rate and phagocytic activity of Kupffer cell, increased leukocyte‐endothelial interactions and TNF‐α levels. Both protective strategies improved sinusoidal perfusion, leukocyte‐endothelial interactions and phagocytic activity of Kupffer cells after 75‐minutes of ischemia, and intermittent clamping also after 120 minutes ischemia. TNF‐α release was significantly reduced and sinusoidal wall integrity was preserved by both protective procedures. In conclusion, both strategies are protective against ischemia/reperfusion injury by maintaining hepatic microcirculation and decreasing Kupffer cell activation for clinically relevant ischemic periods, and intermittent clamping appears superior for prolonged ischemia. (Liver Transpl 2004;10:520–528.)

MRI: the new reference standard in quantifying hepatic steatosis?

Objective The purpose of this study was to assess non-invasive imaging modalities including MRI and CT and compare the quantitative amount of fat with data provided by the pathologist and a chemical lipid assay in leptin-deficient mouse livers. Methods A liver/fat phantom was first used to assess the accuracy of small-animal MRI and human MRI and CT, followed by correlation analysis with ob/ob mouse liver fat quantified by an accurate chemical lipid assay. Similarly, the authors compared the pathologists quantification and the automated software quantification of fat with the lipid assay. The authors then investigated whether hepatic steatosis assessed by MRI correlates with the degree of liver injury in a model of ischaemia/reperfusion in leptin-deficient mice as well as with serious postoperative complications in patients undergoing major liver resection (NCT01234714). Results The authors designed lipid/liver mixtures at various ratios to mimic a wide range of fat liver contents. Small-animal and human MRI detected this fat with a high correlation to the actual fat contents. Mouse livers assessed by human MRI correlated best with total intrahepatic fat by chemical lipid analysis (r=0.975). Human CT, the pathologists assessment and the automated software were less reliable (r=−0.873, 0.512 and 0.873, respectively). There was a significant correlation of the MRI fat quantification with several parameters of liver injury, and MRI data could predict mouse survival after ischaemia/reperfusion injury. In patients undergoing major liver resection, higher liver fat content was associated with more serious postoperative complications, such as liver or multiorgan failure and sepsis, necessitating admission to the intensive care unit. Conclusions With the use of a well-defined set of biological standards, MRI can predict intrahepatic fat with high accuracy. In contrast to biopsies, this method is non-invasive, giving a representative assessment of the whole liver.

Serotonin reverts age-related capillarization and failure of regeneration in the liver through a VEGF-dependent pathway

The function of the liver is well-preserved during the aging process, although some evidence suggests that liver regeneration might be impaired with advanced age. We observed a decreased ability of the liver to restore normal volume after partial hepatectomy in elderly mice, and we identified a pathway that rescued regeneration and was triggered by serotonin. 2,5-dimethoxy-4-iodoamphetamine (DOI), a serotonin receptor agonist, reversed the age-related pseudocapillarization of old liver and improved hepatosinusoidal blood flow. After hepatectomy, the open fenestrae were associated with a restored attachment of platelets to endothelium and the initiation of a normal regenerative response, including the up-regulation of essential growth mediators and serotonin receptors. In turn, hepatocyte proliferation recovered along with regain of liver volume and animal survival. DOI operates through the release of VEGF, and its effects could be blocked with anti-VEGF antibodies both in vitro and in vivo. These results suggest that pseudocapillarization in the aged acts as a barrier to liver regeneration. DOI breaks this restraint through an endothelium-dependent mechanism driven by VEGF. This pathway highlights a target for reversing the age-associated decline in the capacity of the liver to regenerate.

Bile salt toxicity aggravates cold ischemic injury of bile ducts after liver transplantation in Mdr2+/− mice

Intrahepatic bile duct strictures are a serious complication after orthotopic liver transplantation (OLT). We examined the role of endogenous bile salt toxicity in the pathogenesis of bile duct injury after OLT. Livers from wild‐type mice and mice heterozygous for disruption of the multidrug resistance 2 Mdr2 gene (Mdr2+/−) were transplanted into wild‐type recipient mice. Mdr2+/− mice secrete only 50% of the normal amount of phospholipids into their bile, leading to an abnormally high bile salt/phospholipid ratio. In contrast to homozygous Mdr2−/− mice, the Mdr2+/− mice have normal liver histology and function under normal conditions. Two weeks after OLT, bile duct injury and cholestasis were assessed by light and electron microscopy, as well as through molecular and biochemical markers. There were no signs of bile duct injury or intrahepatic cholestasis in liver grafts from wild‐type donors. Liver grafts from Mdr2+/− donors, however, had enlarged portal tracts with cellular damage, ductular proliferation, biliostasis, and a dense inflammatory infiltrate after OLT. Parallel to this observation, recipients of Mdr2+/− livers had significantly higher serum transaminases, alkaline phosphatase, total bilirubin, and bile salt levels, as compared with recipients of wild‐type livers. In addition, hepatic bile transporter expression was compatible with the biochemical and histological cholestatic profile found in Mdr2+/− grafts after OLT. In conclusion, toxic bile composition, due to a high biliary bile salt/phospholipid ratio, acted synergistically with cold ischemia in the pathogenesis of bile duct injury after transplantation. (HEPATOLOGY 2006;43:1022–1031.)

Activation of serotonin receptor-2B rescues small-for-size liver graft failure in mice.

The implantation of grafts below 30% of the normal liver volume is associated with a high risk of failure known as small‐for‐size (SFS) syndrome. Strategies to rescue small grafts may have a dramatic impact on organ shortage. Serotonin is a potent growth factor for the liver. The goal of this study was to determine whether enhanced serotonin signaling could prevent the deleterious effects of SFS syndrome. We performed 30% normal liver volume transplantations in wild‐type C57/BL6 and interleukin‐6 (IL‐6)−/− mice. Some animals received α‐methyl‐5‐HT (DOI), an agonist of serotonin receptor‐2 (5‐HT2B). Endpoints included long‐term survival, serum and hepatic markers of liver injury and regeneration, assessment of hepatic microcirculation by intravital fluorescence microscopy and scanning electron microscopy, and transcript levels of a variety of serotonin receptors, tumor necrosis factor α, and IL‐6. All recipients of small grafts (controls) died within 2‐4 days of transplantation, whereas half of those receiving DOI survived permanently. Control animals disclosed major liver injury, including diffuse microvesicular steatosis in hepatocytes, impairment of microcirculation, and a failure of regeneration, whereas these parameters were dramatically improved in animals subjected to DOI. Blockage of 5‐HT2B blunted the protective effects of DOI. Whereas IL‐6 levels were higher in DOI‐treated animals, IL‐6−/− mice were still protected by DOI, suggesting a protective pathway independent of IL‐6. Conclusion: Serotonin through its action on receptor‐2B protects SFS liver grafts from injury and prevents microcirculation and regeneration. The mechanism of hepato‐protection is independent of IL‐6. (Hepatology 2011;)

Hepcidin Expression Does Not Rescue the Iron-Poor Phenotype of Kupffer Cells in Hfe-Null Mice After Liver Transplantation

BACKGROUND & AIMS Hemochromatosis is a common hereditary disease caused by mutations in HFE and characterized by increased absorption of iron in the intestine. However, the intestine does not appear to be the site of mutant HFE activity in the disease; we investigated the role of the liver-the source of the iron regulatory hormone hepcidin-in pathogenesis in mice. METHODS We exchanged livers between Hfe wild-type (+/+) and Hfe null (-/-) mice by orthotopic liver transplantation (OLT) and assessed histopathology, serum and tissue iron parameters, and hepatic hepcidin messenger RNA expression. RESULTS At 6-8 months after OLT, Hfe(-/-) mice that received Hfe(-/-) livers maintained the hemochromatosis phenotype: iron accumulation in hepatocytes but not Kupffer cells (KC), increased transferrin levels, and low levels of iron in the spleen. Hfe(+/+) mice that received Hfe(-/-) livers had increased levels of iron in serum and liver and low levels of iron in spleen. However, they did not develop the iron-poor KCs that characterize hemochromatosis: KCs appeared iron rich, although hepatic hepcidin expression was low. Transplantation of Hfe(+/+) livers into Hfe(-/-) mice prevented hepatic iron accumulation but did not return spleen and plasma levels of iron to normal; KCs still appeared to be iron poor, despite normal hepcidin expression. CONCLUSIONS In Hfe(-/-) mice, transplantation of livers from Hfe(+/+) mice reversed the iron-loading phenotype associated with hemochromatosis (regardless of Hfe expression in intestine). However, KCs still had low levels of iron that were not affected by hepatic hepcidin expression. These findings indicate an independent, iron-modifying effect of HFE in KCs.

Systemic protection through remote ischemic preconditioning is spread by platelet-dependent signaling in mice

Remote ischemic preconditioning (RIPC), the repetitive transient mechanical obstruction of vessels at a limb remote to the operative site, is a novel strategy to mitigate distant organ injury associated with surgery. In the clinic, RIPC has demonstrated efficacy in protecting various organs against ischemia reperfusion (IR), but a common mechanism underlying the systemic protection has not been identified. Here, we reasoned that protection may rely on adaptive physiological reponses toward local stress, as is incurred through RIPC. Standardized mouse models of partial hepatic IR and of RIPC to the femoral vascular bundle were applied. The roles of platelets, peripheral serotonin, and circulating vascular endothelial growth factor (Vegf) were studied in thrombocytopenic mice, Tph1−/− mice, and through neutralizing antibodies, respectively. Models of interleukin‐10 (Il10) and matrix metalloproteinase 8 (Mmp8) deficiency were used to assess downstream effectors of organ protection. The protection against hepatic IR through RIPC was dependent on platelet‐derived serotonin. Downstream of serotonin, systemic protection was spread through up‐regulation of circulating Vegf. Both RIPC and serotonin‐Vegf induced differential gene expression in target organs, with Il10 and Mmp8 displaying consistent up‐regulation across all organs investigated. Concerted inhibition of both molecules abolished the protective effects of RIPC. RIPC was able to mitigate pancreatitis, indicating that it can protect beyond ischemic insults. Conclusions: We have identified a platelet‐serotonin‐Vegf‐Il10/Mmp8 axis that mediates the protective effects of RIPC. The systemic action, the conservation of RIPC effects among mice and humans, and the protection beyond ischemic insults suggest that the platelet‐dependent axis has evolved as a preemptive response to local stress, priming the body against impending harm. (Hepatology 2014;60:1409–1417)