Sustained Isoprostane E2 Elevation, Inflammation and Fibrosis after Acute Ischaemia-Reperfusion Injury Are Reduced by Pregnane X Receptor Activation.
ABSTRACT: Liver grafts donated after cardiac death are increasingly used to expand the donor pool but are prone to ischaemic-type biliary lesions. The anti-inflammatory effects of the activated pregnane X receptor have previously been shown to be beneficial in a number of inflammatory liver conditions. However, its role in reducing peri-portal inflammation and fibrosis following ischaemia-reperfusion injury has not been investigated. Hepatic injury and its response to pregnane X receptor activation was examined after partial hepatic ischaemia-reperfusion injury induced by surgically clamping the left and middle lobar blood vessels in rats. Molecular and pathological changes in the liver were examined over the following 28 days. Ischaemia-reperfusion injury resulted in transient cholestasis associated with microvillar changes in biliary epithelial cell membranes and hepatocellular injury which resolved within days after reperfusion. However, in contrast to chemically-induced acute liver injuries, this was followed by sustained elevation in isoprostane E2, peri-portal inflammation and fibrosis that remained unresolved in the ischaemic reperfused lobe for at least 28 days after clamping. Administration of pregnenolone-16?-carbonitrile--a rodent-specific pregnane X receptor activator--resulted in significant reductions in cholestasis, hepatic injury, ischaemic lobe isoprostane E2 levels, peri-portal inflammation and fibrosis. Hepatic ischaemia-reperfusion injury therefore results in inflammatory and fibrotic changes that persist well beyond the initial ischaemic insult. Drug-mediated activation of the pregnane X receptor reduced these adverse changes in rats, suggesting that the pregnane X receptor is a viable drug target to reduce ischaemic-type biliary lesions in recipients of liver transplants donated after cardiac death.
Project description:BACKGROUND: Besides its haematopoietic effect, erythropoietin (EPO) has multiple protective effects, i.e. antiapoptotic, antioxidant and angiogenic properties. The neuroprotective effects of EPO against ischaemia have all been demonstrated in cell culture and animal models. The aim of the study was to evaluate the effect of erythropoietin on ischaemia-reperfusion injury (I/R injury) of the liver. METHODS: Forty-eight adult male Sprague-Dawley rats weighing 250-300 g were divided into three groups: group I, hepatic ischaemia-reperfusion (Hepatic I/R); group II, hepatic ischaemia-reperfusion + EPO (Hepatic I/R+ EPO); group III, sham. Hepatic ischaemia was created by placing a microvascular clamp on the hepatic pedicle for 45 minutes. EPO was given to group II at a dose of 1000 U/kg 120 minutes before the onset of the ischaemia. Blood samples and liver tissues were obtained after 45 minutes of reperfusion from half of the rats in each group. The remaining rats were killed after a 24-hour observation period and blood and tissue samples were obtained. Blood alanine aminotransferase, tumour necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2) and liver tissue malondialdehyde (MDA) levels were determined. Liver tissue histopathology was also evaluated by light microscopy. RESULTS: In rats with hepatic ischaemia, serum levels of ALT, TNF-alpha, IL-2 and liver tissue levels of MDA were reduced by the administration of erythropoietin and the histopathological score was also less severe. CONCLUSION: This study demonstrates that pre-ischaemic administration of EPO has protective effects on hepatic I/R injury.
Project description:Experimental studies have suggested that end-ischaemic dual hypothermic oxygenated machine perfusion (DHOPE) may restore hepatocellular energy status and reduce reperfusion injury in donation after circulatory death (DCD) liver grafts. The aim of this prospective case-control study was to assess the safety and feasibility of DHOPE in DCD liver transplantation.In consecutive DCD liver transplantations, liver grafts were treated with end-ischaemic DHOPE. Outcome was compared with that in a control group of DCD liver transplantations without DHOPE, matched for donor age, donor warm ischaemia time, and recipient Model for End-stage Liver Disease (MELD) score. All patients were followed for 1 year.Ten transplantations involving liver grafts treated with DHOPE were compared with 20 control procedures. There were no technical problems. All 6-month and 1-year graft and patient survival rates were 100 per cent in the DHOPE group. Six-month graft survival and 1-year graft and patient survival rates in the control group were 80, 67 and 85 per cent respectively. During DHOPE, median (i.q.r.) hepatic adenosine 5'-triphosphate (ATP) content increased 11-fold, from 6 (3-10) to 66 (42-87) µmol per g protein (P = 0·005). All DHOPE-preserved livers showed excellent early function. At 1 week after transplantation peak serum alanine aminotransferase (ALT) and bilirubin levels were twofold lower in the DHOPE group than in the control group (ALT: median 966 versus 1858 units/l respectively, P = 0·006; bilirubin: median 1·0 (i.q.r. 0·7-1·4) versus 2·6 (0·9-5·1) mg/dl, P = 0·044). None of the ten DHOPE-preserved livers required retransplantation for non-anastomotic biliary stricture, compared with five of 20 in the control group (P = 0·140).This clinical study of end-ischaemic DHOPE in DCD liver transplantation suggests that the technique restores hepatic ATP, reduces reperfusion injury, and is safe and feasible. RCTs with larger numbers of patients are warranted to assess the efficacy in reducing post-transplant biliary complications.
Project description:Ischaemia-reperfusion injury (IRI) in the liver, a major complication of haemorrhagic shock, resection and transplantation, is a dynamic process that involves the two interrelated phases of local ischaemic insult and inflammation-mediated reperfusion injury. This Review highlights the latest mechanistic insights into innate-adaptive immune crosstalk and cell activation cascades that lead to inflammation-mediated injury in livers stressed by ischaemia-reperfusion, discusses progress in large animal experiments and examines efforts to minimize liver IRI in patients who have received a liver transplant. The interlinked signalling pathways in multiple hepatic cell types, the IRI kinetics and positive versus negative regulatory loops at the innate-adaptive immune interface are discussed. The current gaps in our knowledge and the pathophysiology aspects of IRI in which basic and translational research is still required are stressed. An improved appreciation of cellular immune events that trigger and sustain local inflammatory responses, which are ultimately responsible for organ injury, is fundamental to developing innovative strategies for treating patients who have received a liver transplant and developed ischaemia-reperfusion inflammation and organ dysfunction.
Project description:Effects of ischaemia-reperfusion injury (I/R) of liver on expression of rat glutathione S-transferase (rGST) isoenzymes that metabolize products of oxidative stress were examined. Rats underwent lobar liver ischaemia for 30 min followed by reperfusion. In ischaemic lobes, rGSTA1/A2 transcript levels increased significantly 12 h after I/R (2.94-fold) and protein levels increased significantly at 24 h (1.45-fold); increased transcript levels were also observed in nonischaemic lobes (1.78-fold). Superoxide dismutase prevented I/R and the increases in transcript and protein levels in ischaemic and non-ischaemic lobes. By in-situ hybridization, increases in transcript levels at 6 h were present in zones 2 and 3 of the ischaemic lobes and peaked at 12 h (2.5-fold zone 2, 4.5-fold zone 3). Significant increases in transcript levels also were observed at 24 h in zones 2 (2.0-fold) and 3 (2.9-fold) of non-ischaemic lobes. Nuclear run-off assays showed a 1.8-fold increase in rGSTA1/A2 transcription rates in ischaemic lobes at 3 h. We conclude that I/R causes increased rGSTA1/A2 expression in the zone of the hepatic lobule most susceptible to oxidative injury and that this expression may be an important defence against injury.
Project description:BACKGROUND:Understanding the molecular mechanisms in perturbation of the metabolome following ischaemia and reperfusion is critical in developing novel therapeutic strategies to prevent the sequelae of post-injury shock. While the metabolic substrates fueling these alterations have been defined, the relative contribution of specific organs to the systemic metabolic reprogramming secondary to ischaemic or haemorrhagic hypoxia remains unclear. MATERIALS AND METHODS:A porcine model of selected organ ischaemia was employed to investigate the relative contribution of liver, kidney, spleen and small bowel ischaemia/reperfusion to the plasma metabolic phenotype, as gleaned through ultra-high performance liquid chromatography-mass spectrometry-based metabolomics. RESULTS:Liver ischaemia/reperfusion promotes glycaemia, with increases in circulating carboxylic acid anions and purine oxidation metabolites, suggesting that this organ is the dominant contributor to the accumulation of these metabolites in response to ischaemic hypoxia. Succinate, in particular, accumulates selectively in response to the hepatic ischemia, with levels 6.5 times spleen, 8.2 times small bowel, and 6 times renal levels. Similar trends, but lower fold-change increase in comparison to baseline values, were observed upon ischaemia/reperfusion of kidney, spleen and small bowel. DISCUSSION:These observations suggest that the liver may play a critical role in mediating the accumulation of the same metabolites in response to haemorrhagic hypoxia, especially with respect to succinate, a metabolite that has been increasingly implicated in the coagulopathy and pro-inflammatory sequelae of ischaemic and haemorrhagic shock.
Project description:Hepatic ischaemia/reperfusion (I/R) injury is of primary concern during liver surgery. We propose a new approach for preserving low liver blood perfusion during hepatectomy either by occlusion of the portal vein (OPV) while preserving hepatic artery flow or occlusion of the hepatic artery while limiting portal vein (LPV) flow to reduce I/R injury. The effects of this approach on liver I/R injury were investigated. Rats were randomly assigned into 4 groups: sham operation, occlusion of the portal triad (OPT), OPV and LPV. The 7-day survival rate was significantly improved in the OPV and LPV groups compared with the OPT group. Microcirculatory liver blood flow recovered rapidly after reperfusion in the OPV and LPV groups but decreased further in the OPT group. The OPV and LPV groups also showed much lower ALT and AST levels, Suzuki scores, inflammatory gene expression levels, and parenchymal necrosis compared with the OPT group. An imbalance between the expression of vasoconstriction and vasodilation genes was observed in the OPT group but not in the OPV or LPV group. Therefore, preserving low liver blood perfusion by either the OPV or LPV methods during liver surgery is very effective for preventing hepatic microcirculatory dysfunction and hepatocyte injury.
Project description:Aryl hydrocarbon receptor (AhR) antagonism can mitigate cellular damage associated with cerebral ischaemia and reperfusion (I/R) injury. This study investigated the neuroprotective effects of AhR antagonist administration before reperfusion in a rat stroke model and influence of the timing of AhR antagonist administration on its neuroprotective effects. Magnetic resonance imaging (MRI) was performed at baseline, immediately after, and 3, 8, and 24 h after ischaemia in the sham, control (I/R injury), TMF10 (trimethoxyflavone [TMF] administered 10 min post-ischaemia), and TMF50 (TMF administered 50 min post-ischaemia) groups. The TMF treatment groups had significantly fewer infarcts than the control group. At 24 h, the relative apparent diffusion coefficient values of the ischaemic core and peri-infarct region were significantly higher and relative T2 values were significantly lower in the TMF10 groups than in the control group. The TMF treatment groups showed significantly fewer terminal deoxynucleotidyl transferase dUTP nick-end labelling positive (+) cells (%) in the peri-infarct region than the control group. This study demonstrated that TMF treatment 10 or 50 min after ischaemia alleviated brain damage. Furthermore, the timing of AhR antagonist administration affected the inhibition of cellular or vasogenic oedema formation caused by a transient ischaemic stroke.
Project description:Ischaemic preconditioning is a method of protecting tissue against ischaemia-reperfusion injury. It is an innate protective mechanism that increases a tissue's tolerance to prolonged ischaemia when it is first subjected to short burst of ischaemia and reperfusion. It is thought to provide this protection by increasing the tissue's tolerance to ischaemia, therby reducing oxidative stress, inflammation and apoptosis in the preconditioned tissue. We used microarrays to investigate the genomic response induced by ischaemic preconditioning in muscle biopsies taken from the operative leg of total knee arthroplasty patients in order to gain insight into the ischaemic preconditioning mechanism. Overall design: Patients undergoing primary knee arthroplasty were randomised to control and treatment (ischaemic preconditioning) groups. Patients in the treatment group received a preconditioning stimulus immediately prior to surgery. The ischaemic preconditioning stimulus consisted of three five-minute periods of tourniquet insufflation on the lower operative limb, interrupted by five minute periods of reperfusion. All patients had a tourniquet applied to the lower limb after the administration of spinal anaesthesia, as per normal protocol for knee arthroplasty surgery. Muscle biopsies were taken from the quadriceps muscle of the operative knee at the immediate onset of surgery (T0) and at 1 hour into surgery (T1). Total RNA was extracted from biospies of four control and four treatment patients and hybridised to the Affymetrix Human U133 2.0 chip.
Project description:Ischaemic preconditioning is a method of protecting tissue against ischaemia-reperfusion injury. It is an innate protective mechanism that increases a tissue's tolerance to prolonged ischaemia when it is first subjected to short burst of ischaemia and reperfusion. It is thought to provide this protection by increasing the tissue's tolerance to ischaemia, therby reducing oxidative stress, inflammation and apoptosis in the preconditioned tissue. We used microarrays to investigate the genomic response induced by ischaemic preconditioning in muscle biopsies taken from the operative leg of total knee arthroplasty patients in order to gain insight into the ischaemic preconditioning mechanism. Patients undergoing primary knee arthroplasty were randomised to control and treatment (ischaemic preconditioning) groups. Patients in the treatment group received a preconditioning stimulus immediately prior to surgery. The ischaemic preconditioning stimulus consisted of three five-minute periods of tourniquet insufflation on the lower operative limb, interrupted by five minute periods of reperfusion. All patients had a tourniquet applied to the lower limb after the administration of spinal anaesthesia, as per normal protocol for knee arthroplasty surgery. Muscle biopsies were taken from the quadriceps muscle of the operative knee at the immediate onset of surgery (T0) and at 1 hour into surgery (T1). Total RNA was extracted from biospies of four control and four treatment patients and hybridised to the Affymetrix Human U133 2.0 chip.
Project description:BACKGROUND AND PURPOSE: Na+/Ca2+ exchanger (NCX) inhibitors are known to attenuate myocardial reperfusion injury. However, the exact mechanisms for the cardioprotection remain unclear. The present study was undertaken to examine the mechanism underlying the cardioprotection by NCX inhibitors against ischaemia/reperfusion injury. EXPERIMENTAL APPROACH: Isolated rat hearts were subjected to 35-min ischaemia/60-min reperfusion or 20-min ischaemia/60-min reperfusion. NCX inhibitors (3-30 microM KB-R7943 (KBR) or 0.3-1 microM SEA0400 (SEA)) were given for 5 min prior to ischaemia (pre-ischaemic treatment) or for 10 min after the onset of reperfusion (post-ischaemic treatment). KEY RESULTS: With 35-min ischaemia/60-min reperfusion, pre- or post-ischaemic treatment with KBR or SEA neither enhanced post-ischaemic contractile recovery nor attenuated ischaemia- or reperfusion-induced Na+ accumulation and damage to mitochondrial respiratory function. With the milder model (20-min ischaemia/reperfusion), pre- or post-ischaemic treatment with 10 microM KBR or 1 microM SEA significantly enhanced the post-ischaemic contractile recovery, associated with reductions in reperfusion-induced Ca2+ accumulation, damage to mitochondrial function, and decrease in myocardial high-energy phosphates. Furthermore, Na+ influx to mitochondria in vitro was enhanced by increased concentrations of NaCl. KBR (10 microM) and 1 microM SEA partially decreased the Na+ influx. CONCLUSIONS AND IMPLICATIONS: The NCX inhibitors exerted cardioprotective effects during relatively mild ischaemia. The mechanism may be attributable to prevention of mitochondrial damage, possibly mediated by attenuation of Na+ overload in cardiac mitochondria during ischaemia and/or Ca2+ overload via the reverse mode of NCX during reperfusion.