Decreased survival in rat liver transplantation with extended cold preservation: role of portal vein clamping time

Primary liver graft dysfunction is currently related to cold ischemia-reperfusion injury, although a wide survival range has been reported using 24-hour preservation in cold University of Wisconsin (UW) solution. We hypothesized that the portal vein clamping time (PVCT) played a more important role than cold preservation injury in the postoperative outcome. Rat liver transplantation was performed using different clamping times after 24-hour cold ischemia in the UW solution. Survival rates, plasma tumor necrosis factor (TNF), and nitrate/nitrite levels were examined. Subsequently, the effect of clamping time was evaluated on hepatocyte and sinusoidal endothelial cell (SEC) function using isolated perfused livers. Survival rate was directly related to clamping time length. Marked increases in TNF and nitrate/nitrite levels were found after surgery, particularly after long clamping times. In perfusion studies, the SEC function was already markedly altered after preservation alone and was not further modified by transplantation. By contrast, the hepatocyte function was moderately altered after transplantation, irrespective of clamping times, even when rats operated with long clamping times were in terminal conditions. In rats, 24-hour preservation in cold UW solution is not a severely compromising condition leading to primary liver nonfunction. Long PVCTs are associated with an endotoxemia-like syndrome more related to a warm intestinal ischemia than to cold ischemia injury of the liver.     

Ischemia/reperfusion injury in human kidney transplantation: an immunohistochemical analysis of changes after reperfusion

Organs used for transplantation undergo varying degrees of cold ischemia and reperfusion injury after transplantation. In renal transplantation, prolonged cold ischemia is strongly associated with delayed graft function, an event that contributes to inferior graft survival. At present, the pathophysiological changes associated with ischemia/reperfusion injury in clinical renal transplantation are poorly understood. We have performed an immunohistochemical analysis of pre- and postreperfusion biopsies obtained from cadaver (n = 55) and living/related donor (LRD) (n = 11) renal allografts using antibodies to adhesion molecules and leukocyte markers to investigate the intragraft changes after cold preservation and reperfusion. Neutrophil infiltration and P-selectin expression were detected after reperfusion in 29 of 55 (53%) and 24 of 55 (44%) cadaver renal allografts, respectively. In marked contrast, neutrophil infiltration was not observed in LRD allografts, and only 1 of 11 (9%) had an increased level of P-selectin after reperfusion. Immunofluorescent double-staining demonstrated that P-selectin expression resulted from platelet deposition and not from endothelial activation. No statistically significant association was observed between neutrophil infiltration and P-selectin expression in the glomeruli or intertubular capillaries despite the large number of cadaver renal allografts with postreperfusion changes. Neutrophil infiltration into the glomeruli was significantly associated with long cold ischemia times and delayed graft function. Elevated serum creatinine levels at 3 and 6 months after transplantation were also associated with the presence of neutrophils and platelets after reperfusion. Our results suggest that graft function may be influenced by early inflammatory events after reperfusion, which can be targeted for future therapeutic intervention.     

ICAM-1 upregulation in distant tissues after hepatic ischemia/reperfusion: a clue to the mechanism of multiple organ failure

BACKGROUND/PURPOSE: Endothelial cell adhesion molecules (ECAMs) are felt to play an important role in ischemia/reperfusion (I/R) injury by causing adhesion of leukocytes to endothelial cells. It is possible that ECAMs play a role in multiple organ system failure. ICAM-1 is one of the adhesion molecules that has been shown to be upregulated in response to cytokines. This upregulation leads to leukocyte endothelial cell interaction (adhesion) and to neutrophil infiltration of the affected tissue. The purpose of our study was to measure ICAM-1 expression in the liver and other organs after hepatic ischemia/reperfusion (I/R). METHODS: A laparotomy was performed on 14 Sprague-Dawley rats; 45 minutes of occlusive ischemia to the left lateral lobe was followed by 5 hours of reperfusion. The rat was injected with I125-labeled ICAM-1 MAb and I131-labeled nonbinding MAb (to control for nonspecific accumulation of ICAM-1 MAb). Entire organs were harvested and accumulated activity was measured in each organ. ICAM-1 levels were expressed as percent injected dose per gram of tissue. Control animals underwent sham laparotomy. RESULTS: ICAM-1 was upregulated in the ischemic lobe of the liver, nonischemic lobe of the liver, heart, kidney, intestine, and pancreas. Up-regulation in the lung was not significant. Both the lung and liver had high constitutive levels of ICAM-1. CONCLUSIONS: These data show that (1) significant hepatic upregulation of ICAM-1 after hepatic ischemia/reperfusion and (2) significant ICAM-1 upregulation in other tissues (heart, kidney, and intestine) after hepatic ischemia/reperfusion. The ICAM-1 upregulation in distant organs is likely mediated by cytokines such as tumor necrosis factor (TNF). These data show that leukocyte endothelial cell interactions in distant organs may be mediated by hepatic ischemia/reperfusion. This is a possible explanation for how failure of one organ can lead to failure of others in multiple organ system failure.     

Role of glutathione in hepatic bile formation during reperfusion after cold ischemia of the rat liver

BACKGROUND/AIMS: Liver reperfusion following cold ischemia is frequently associated with diminished bile flow in patients undergoing liver transplantation. Glutathione is a major determinant of bile-acid independent bile flow, and the effects of cold ischemia on biliary glutathione excretion are unknown. METHODS: We examined the effects of cold ischemia (University of Wisconsin solution (4 degrees C), 24 h) with subsequent reperfusion (100 min) on biliary glutathione excretion in a recirculating system. Since glutathione might represent an important antioxidant within the biliary tract and oxidative stress in the biliary tract during reperfusion could contribute to the pathogenesis of bile duct injury after liver transplantation, we also assessed bile duct morphology in reperfused livers of mutant TR- -rats, in whom biliary excretion of glutathione is already impaired. RESULTS: Hepatic bile formation was diminished in reperfused Wistar rat livers after cold ischemia. Biliary glutathione concentrations and output were significantly decreased and correlated with postischemic changes in bile secretion. An increased biliary oxidized glutathione/glutathione ratio, indicating oxidative stress, was detected only immediately after the onset of reperfusion. Basal bile flow rates in TR- -rat livers which were already markedly reduced in control-perfused livers, decreased further during the early but not the later reperfusion period. Reperfusion of both Wistar and TR- -rat livers was not associated with electron microscopic evidence of bile duct damage. CONCLUSIONS: We conclude that impaired biliary excretion of glutathione contributes to decreased bile flow after cold ischemia. The absence of biliary glutathione does not appear to promote ultrastructural evidence of bile duct injury during reperfusion in the isolated perfused rat liver.     

Preservation of pig liver allografts after warm ischemia: normothermic perfusion versus cold storage

Warm ischemia is known to induce substantial damage to the liver parenchyma. With respect to clinical liver transplantation, the tolerance of the liver to warm ischemia and the preservation of these organs have not been studied in detail. In isolated reperfused pig livers we proceeded according to the following concept: Livers were subjected to 1 or 3 h of warm ischemia. Subsequently, these organs were preserved by either normothermic perfusion or cold storage (histidine-tryptophan-alpha-ketoglutarate, HTK) for 3 h each. After storage, liver function was assessed in a reperfusion circuit for another 3 h. Parameters under evaluation were bile flow, perfusion flow, oxygen consumption, enzyme release into the perfusate (creatine kinase, glutamic oxaloacetic transaminase (GOT), lactic dehydrogenase, and glutamic pyruvic transaminase), and histomorphology. Damage to the liver was lowest after warm ischemia of 1 h. The results after cold storage were superior to those after normothermic perfusion (GOT: 3.2 +/- 0.3 and 2.6 +/- 0.2 U/g liver; cumulative bile production: 14.7 +/- 2.1 and 9.4 +/- 1 ml, respectively; P < 0.05). In contrast, we found substantial damage at the end of reperfusion in livers undergoing 3 h of warm ischemia under both preservation techniques with severe hepatocellular pyknoses and essentially altered nonparenchymal cells. The results suggest that pig livers undergoing 1 h of warm ischemia and cold storage for 3 h with HTK solution may lead to functioning after transplantation.     

31P nuclear magnetic resonance study of phospholipids in ischemia/reperfusion injury in a rat fatty liver model

Obese Zucker rats are susceptible to increased hepatic ischemia/reperfusion (I/RP) injury. Increased lipid peroxidation occurs in this model with warm ischemia. We hypothesized that a severe depletion of phospholipids (PL) occurs with warm I/RP in fatty livers. Obese (Ob) and lean (Ln) Zucker rats were subjected to 90 min of in vivo partial hepatic warm I followed by RP. Total lipids extracted from one gm of liver (median lobe) taken at the end of 1, 2 and 6 hr of RP and sham (Sh) surgery (n=5 Ln & Ob) were analyzed by 202.3 MHz 31P NMR, which provided good resolution of individual PL. Obese (Sh) rats contained 22% more PL than Ln (P= < 0.01). Ischemia caused similar decreases in PL in both Ob (to 67% Sh) and Ln rats (62%). Following 2 hr RP, PL in Ob rats decreased further (46% Sh) and recovered only marginally at 6 hr (53%), in marked contrast to the rapid recovery in Ln to preischemic levels (110% Sh at both 2 and 6 hr; P=<0.001). Mole percents of individual PL did not change significantly except for lysophosphatidylcholine, which increased from 0.43 to 1.3% (Sh vs. 6 hr RP) in the Ob, but decreased from 0.98 to 0.52% in Ln animals (P = <0.001). Fatty livers thus are more vulnerable to phospholipid depletion in response to warm ischemia/reperfusion than normal livers.     

Blocking of classical complement pathway inhibits endothelial adhesion molecule expression and preserves ischemic myocardium from reperfusion injury

Myocardial injury after ischemia (I) and reperfusion (R) is related to leukocyte activation with subsequent release of cytokines and oxygen-derived free radicals as well as complement activation. In our study, the cardioprotective effects of exogenous C1 esterase inhibitor (C1 INH) were examined in a rat model of myocardial I + R (i.e., 20 min + 24 hr or 48 hr). The C1 INH (10, 50 and 100 U/kg) administered 2 min before reperfusion significantly attenuated myocardial injury after 24 hr of R compared to vehicle treated rats (P < .001). Further, cardiac myeloperoxidase activity (i.e., a marker of PMN [polymorphonuclear leukocyte] accumulation) in the ischemic area was significantly reduced after C1 INH treatment compared to vehicle treated animals (0.81 +/- 0.1, 0.34 +/- 0.13, 0.13 +/- 0.1 vs. 1.44 +/- 0.3 U/100 mg tissue, P < .001). In addition, C1 INH (100 U/kg) significantly attenuated myocardial injury and neutrophil infiltration even after 48 hr of reperfusion compared to vehicle treatment. Immunohistochemical analysis of ischemic-reperfused myocardial tissue demonstrated activation of classical complement pathway by deposition of C1q on cardiac myocytes and cardiac vessels. In addition, expression of the endothelial adhesion molecules P-selectin and intercellular adhesion molecule 1 (ICAM-1) was observed after reperfusion of the ischemic myocardium. In this regard, C1 INH administration abolished expression of P-selectin and ICAM-1 on the cardiac vasculature after myocardial ischemia and reperfusion. Blocking the classical complement pathway by exogenous C1 INH appears to be an effective means to preserve ischemic myocardium from injury after 24 and 48 hr of reperfusion. The mechanisms of this cardioprotective effect appears to be due to blocking of complement activation and reduced endothelial adhesion molecule expression with subsequent reduced PMN-endothelium interaction, resulting in diminished cardiac necrosis.     

Effects of a metalloproteinase that truncates P-selectin glycoprotein ligand on neutrophil-induced cardiac dysfunction in ischemia/reperfusion

This study was designed to test the effects of polymorphonuclear leukocytes (PMNs) in the presence and absence of a P-selectin blocker, mocarhagin, in provoking cardiac dysfunction in isolated perfused rat hearts following ischemia and reperfusion. Control rat hearts not subjected to ischemia were perfused without blood cells for 80 min. Additional control rat hearts were perfused with 100 x 10(6) PMNs in the presence and absence of 0.2 microgram/ml mocarhagin over a 5-min perfusion followed by a 45-min observation period. No significant reduction in coronary flow (CF), left ventricular developed pressure (LVDP), or the first derivative of LVDP (dP/dt max) was observed at the end of the observation period in any non-ischemic group. Similarly, global ischemia (I) for 20 min followed by 45 min of reperfusion (R) produced no sustained effects on the final recovery of any of these parameters in any group of hearts perfused in the absence of PMNs. I/R hearts perfused with PMNs exhibited decreases of 50-60% in all measurements of cardiac function (P < 0.001). These PMN perfused I/R hearts also exhibited marked increases in cardiac myeloperoxidase (MPO) activity indicating a significant PMN infiltration, and enhanced P-selection expression on the coronary microvascular endothelium. All cardiodynamic effects as well as MPO accumulation and PMN infiltration were attenuated markedly by the metalloproteinase, mocarhagin, which inhibits P-selectin-mediated cell adhesion by cleaving its high-affinity receptor, PSGL-1, present on neutrophils. These results provide evidence that neutrophils provoke post-reperfusion cardiac dysfunction, and that this may be largely due to P-selectin-induced adherence of neutrophils to the endothelium.     

Enhancement of endogenous cyclic AMP signal: a new approach to allow for cold preservation of rat livers from non-heart-beating donors?

BACKGROUND: The organ donor shortage has led to a reconsideration of the use of non-heart-beating donors (NHBDs). However, graft injury due to warm ischemia in NHBD livers strongly affects posttransplant outcome. The present study was aimed at investigating the role of the cellular cyclic (c)AMP second messenger signal with regard to hepatic viability after cold preservation of NHBD livers. METHODS: Cardiac arrest was induced in Wistar rats by frenotomy of the anesthetized nonheparinized animal. After 30 min, the livers were excised and flushed with 20 ml of heparinized saline solution, rinsed with 10 ml of University of Wisconsin (UW) solution, and stored submerged in UW solution at 4 degrees C for 24 hr. In half of the experiments, UW solution was supplemented with glucagon (0.5 microg/ml) to increase the cAMP signal in the liver. Reperfusion was carried out in vitro after all livers were incubated at 25 degrees C in saline solution to replicate the period of slow rewarming during surgical implantation in vivo. RESULTS: Hepatic levels of cAMP (nmol/g dry weight) declined from 1.21+/-0.05 to 0.53+/-0.03 (P<0.01) at 30 min after cardiac arrest. Subsequent storage in UW solution resulted in a further decline to 0.35+/-0.04 after 24 hr in group A, whereas glucagon treatment enhanced cellular cAMP signal to 0.64+/-0.06 (P<0.01). Upon reperfusion, liver integrity was significantly improved after glucagon administration, with 66% reduction in alanine aminotransferase release and a threefold increase in hepatic bile production as compared with untreated livers. Moreover, liver ATP tissue levels were restored to only 2.19+/-0.51 micromol/g in the untreated group but reached 4.97+/-0.41 micromol/g (P<0.05) after treatment with glucagon. CONCLUSIONS: Posthoc conditioning of predamaged livers by glucagon enhances cAMP tissue levels during ischemic preservation and improves hepatic integrity upon reperfusion. This may represent a promising approach for the use of livers from non-heart-beating donors in clinical transplantation.     

The P-selectin glycoprotein ligand-1 is important for recruitment of neutrophils into inflamed mouse peritoneum

The P-selectin glycoprotein ligand-1 (PSGL-1) is a high-affinity ligand of P-selectin on myeloid cells and certain subsets of lymphoid cells. We generated the rat monoclonal antibody (MoAb) 2PH1 that recognizes an epitope within the first 19 amino acids at the N-terminus of the processed form of mouse PSGL-1. This antibody blocks attachment of mouse myeloid cells to P-selectin under both static and flow conditions. Intravenous administration of saturating amounts of 2PH1 reduced the number of rolling leukocytes in venules of the acutely exposed mouse cremaster muscle by 79% (+/-5.7%), whereas an anti-P-selectin MoAb reduced it completely. Examining the effect of the MoAb 2PH1 on the recruitment of neutrophils into chemically inflamed mouse peritoneum showed that blocking PSGL-1 inhibited neutrophil accumulation in the peritoneum by 82% (+/-7%) at 2 hours and by 59% (+/-7.9%) at 4 hours after stimulation. A similar effect was seen with the MoAb against P-selectin. Simultaneous administration of both antibodies at the 4-hour time point blocked neutrophil accumulation by 86% (+/-4.2%), arguing for an additional partner molecule for PSGL-1 besides P-selectin. This is the first demonstration of the importance of PSGL-1 in the recruitment of mouse neutrophils into inflamed tissue.     

Protective effect of preservation of canine pancreas by the two-layer (University of Wisconsin solution/perfluorochemical) method against rewarming ischemic injury during implantation

Rewarming ischemia during implantation severely compromises posttransplant pancreas graft survival because the graft has already been subjected to warm and cold ischemia before implantation. The purpose of this study was to examine whether preservation of the pancreas graft by the two-layer method ameliorates rewarming ischemic injury of the graft during implantation using a canine model. After flushing with cold University of Wisconsin solution (UW), the pancreas grafts were preserved by the two-layer (UW/perfluorochemical [PFC]) method (group 1) or simple cold storage in UW (group 2) for 24 hr and then autotransplanted. In control, the pancreas grafts were flushed out with cold UW and immediately autotransplanted without preservation (group 3). After completion of vascular anastomosis, vascular clamp was not released until 90, 120, or 150 min of rewarming ischemia, including anastomosis time, had elapsed. After 90 min of rewarming ischemia, graft survival rates were 5/5, 100%, 5/5, 100%, and 5/5, 100%, in groups 1, 2, and 3, respectively. After 120 min, all the grafts in groups 2 and 3 failed (0/5, 0%, and 0/5, 0%, respectively); however, all the grafts in group 1 survived (5/5, 100%). Even after 150 min, 1 of 3 grafts in group 1 survived (1/3, 33%). After 24 hr preservation, tissue ATP levels of the grafts in group 1 were about 2-fold the reference values before harvesting (8.23 +/- 0.72 vs. 4.44 +/- 0.49 mumol/g dry weight, P < 0.05) and significantly higher compared with group 2 (8.23 +/- 0.72 vs. 1.76 +/- 0.52 mumol/g dry weight, P < 0.01). After 120 min of rewarming ischemia, tissue ATP levels in group 1 were 84% of the reference values and significantly higher compared with group 2 (3.75 +/- 0.25 vs. 1.57 +/- 0.48 mumol/g dry weight, P < 0.05). Two hours after reperfusion, ATP levels in group 1 were 42% of reference values but significantly higher compared with group 2 (1.86 +/- 0.36 vs. 1.03 +/- 0.18 mumol/g dry weight, P < 0.05). We conclude that the two-layer (UW/PFC) method ameliorates rewarming ischemic injury of the pancreas graft during implantation by increasing tissue ATP contents during preservation and consequently maintaining tissue ATP levels during implantation.     

Chemokine expression during hepatic ischemia/reperfusion-induced lung injury in the rat. The role of epithelial neutrophil activating protein

The liver is highly susceptible to a number of pathological insults, including ischemia/reperfusion injury. One of the striking consequences of liver injury is the associated pulmonary dysfunction that may be related to the release of hepatic-derived cytokines. We have previously employed an animal model of hepatic ischemia/reperfusion injury, and demonstrated that this injury causes the production and release of hepatic-derived TNF, which mediates a neutrophil-dependent pulmonary microvascular injury. In this study, we have extended these previous observations to assess whether an interrelationship between TNF and the neutrophil chemoattractant/activating factor, epithelial neutrophil activating protein-78 (ENA-78), exists that may be accountable for the pathology of lung injury found in this model. In the context of hepatic ischemia/reperfusion injury, we demonstrated the following alterations in lung pathophysiology: (a) an increase in pulmonary microvascular permeability, lung neutrophil sequestration, and production of pulmonary-derived ENA-78; (b) passive immunization with neutralizing TNF antiserum resulted in a significant suppression of pulmonary-derived ENA-78; and (c) passive immunization with neutralizing ENA-78 antiserum resulted in a significant attenuation of pulmonary neutrophil sequestration and microvascular permeability similar to our previous studies with anti-TNF. These findings support the notion that pulmonary ENA-78 produced in response to hepatic-derived TNF is an important mediator of lung injury.     

Synergism between platelets and neutrophils in provoking cardiac dysfunction after ischemia and reperfusion: role of selectins

BACKGROUND: Neutrophils (PMNs) are known to contribute to both cardiac dysfunction and myocardial necrosis after reperfusion of an ischemic heart. Moreover, platelets are also important blood cells that can aggravate myocardial ischemic injury. This study was designed to test the effects of PMNs and platelets separately and together in provoking cardiac dysfunction in isolated perfused rat hearts after ischemia and reperfusion. METHODS AND RESULTS: Control rat hearts not subjected to ischemia were perfused without blood cells for 80 minutes. Additional control rat hearts were perfused with 75x106 PMNs, with 100x106 platelets, or with 75x106 PMNs+100x106 platelets over a 5-minute perfusion followed by a 75-minute observation period. No significant reduction in coronary flow, left ventricular developed pressure (LVDP), or the first derivative of LVDP (dP/dtmax) was observed at the end of the observation period in any nonischemic group. Similarly, global ischemia (I) for 20 minutes followed by 45 minutes of reperfusion (R) produced no sustained effects on the final recovery of any of these parameters in any group of hearts perfused in the absence of blood cells. However, I/R hearts perfused with either PMNs or platelets alone exhibited decreases in these variables of 10% to 12% (P<0.05 from control). Furthermore, I/R hearts perfused with both PMNs and platelets exhibited decreases of 50% to 60% in all measurements of cardiac function (P<0.001). These dual-cell-perfused I/R hearts also exhibited marked increases in cardiac myeloperoxidase (MPO) activity, indicating a significant PMN infiltration, and enhanced P-selectin expression on the coronary microvascular endothelium. All cardiodynamic effects as well as MPO accumulation and PMN infiltration were markedly attenuated by a sialyl LewisX-oligosaccharide or a recombinant soluble P-selectin ligand, which inhibits selectin-mediated cell adhesion. CONCLUSIONS: These results provide evidence that platelets and neutrophils act synergistically in provoking postreperfusion cardiac dysfunction and that this may be largely due to cell-to-cell interactions mediated by P-selectin. These findings may help explain the reperfusion injury phenomenon.     

Ischaemia-reperfusion injury of the liver: treatment in theory and in practice

Cold ischaemia-reperfusion injuries are an unavoidable feature of current liver transplantation procedures. Damage to liver grafts accures mainly from hypothermic storage under hypoxic conditions (cold ischaemia), from sustained ischaemia during implantation into the recipient (rewarming ischaemia) and from restoration of blood and oxygen to the graft (reperfusion injury). These three stages are characterized by progressive deteriorations in hepatic function, with sinusoidal endothelial cells most affected during cold ischaemia. Activation of Kupffer cells (hepatic macrophages) at reperfusion augments damage to both endothelial and parenchymal cells by the release of numerous compounds which initiate and perpetrate injury and impair the hepatic microcirculation. The key events in the expression of ischaemia-reperfusion injury are detailed and therapeutic interventions are described which target these steps. The treatments discussed include University of Wisconsin (UW) preservation solution, calcium channel blockade, inhibitors of Kupffer cell activation, promoters of microvascular vasodilation, hepatoprotectants and the use of anti-oxidants.     

The guanylate cyclase-coupled natriuretic peptide receptor: a new target for prevention of cold ischemia-reperfusion damage of the rat liver

The aim of our studies was to investigate hormonal prevention of hepatic preservation damage by the atrial natriuretic peptide (ANP) and the mechanisms involved. Isolated perfusion of rat livers was performed in a nonrecirculating fashion. Twenty minutes of preischemic perfusion was performed with or without different concentrations of ANP, followed by 24-hour storage in cold University of Wisconsin (UW) solution. Two hundred nanomoles of ANP prevented hepatocellular damage during a 2-hour reperfusion period as indicated by a marked attenuation of the sinusoidal efflux of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP), and by reduced Trypan blue uptake. Furthermore, postischemic bile flow as an indicator of liver function was significantly improved by about 60% with 200 nmol/L ANP. No protection was conveyed by 20 nmol/L ANP nor by pretreatment with 200 nmol/L ANP for only 10 minutes. The effects of ANP seemed to be mediated by the guanylate cyclase-coupled A (GC-A) receptor and cyclic guanosine monophosphate (cGMP): whereas expression of both GC-A and GC-B receptors as well as of the GC-C receptor was found, cGMP did protect from ischemia-reperfusion damage, but selective ligands of the B and C receptor did not. To begin to determine the mechanisms of ANP-mediated protection, different parameters were investigated: ANP had no effect on portal pressure as an indicator of hepatic circulation, nor on intracellular energy depletion determined by adenosine nucleotide concentration. However, the marked augmentation of nuclear factor kappaB (NF-kappaB) binding activity during reperfusion was prevented in ANP-pretreated livers. In conclusion, pretreatment with ANP protects the rat liver from cold ischemia-reperfusion damage. This effect is mediated via the GC-A receptor and cGMP, and may be linked to an influence of ANP on NF-kappaB activation. Thus, ANP signaling via the GC-A receptor should be considered as a new pharmacological target to prevent preservation injury of the liver.     

The effects of thyroid hormone modulation on rat liver injury associated with ischemia-reperfusion and cold storage

We investigated the effects of thyroid hormone modulation on liver injury associated with ischemia-reperfusion (I-R) and cold storage in rats. First, euthyroid and thyroxine (T4)-pretreated rats were exposed in vivo to 20-min global liver ischemia, then 30-min reperfusion. Liver injury was assessed by measuring serum alanine aminotransferase (ALT) levels. Liver concentrations of adenine nucleotides, reduced glutathione (GSH), and oxidized glutathione were evaluated. Second, rats were given the antithyroid drug propylthiouracil (PTU). Livers stored at 0-1 degrees C in Euro-Collins' solution for 20 h were reperfused at 37 degrees C for 15 min. Lactate dehydrogenase (LDH) in the effluent perfusate and bile flow were evaluated during reperfusion. Serum ALT levels increased after ischemia and I-R. ALT increased significantly more in T4-pretreated than in euthyroid rats after ischemia and I-R. Preischemic levels of adenosine triphosphate (ATP) were significantly lower in livers from T4-pretreated than in euthyroid rats (6.22 +/- 0.7 and 11 +/- 0.9 nmol/mg protein, respectively; P < 0.05). After ischemia, liver ATP was similarly reduced in T4-pretreated and euthyroid rats. After reperfusion, ATP partially recovered in euthyroid rats but remained low in T4-pretreated rats (6.7 +/- 1.0 and 1.91 +/- 0.7 nmol/mg protein, respectively; P < 0.05). Preischemic levels of liver GSH decreased to 44% in T4-pretreated rats. After ischemia, GSH decreased similarly in euthyroid and T4-pretreated rats. GSH recovered promptly after reperfusion in euthyroid rats but remained low in T4-pretreated rats (13.9 +/- 3.3 and 3.9 +/- 0.9 nmol/mg protein, respectively; P < 0.02). During reperfusion after cold storage, LDH in effluent perfusate was significantly lower and bile flow higher in livers from PTU-pretreated rats than from euthyroid rats. The histopathological changes observed after I-R and cold storage confirmed the biochemical findings. Our results suggest that T4 administration exacerbates pretransplant liver damage by increasing liver susceptibility to I-R, whereas PTU administration reduces the liver injury associated with cold storage. Implications: We studied the effects of thyroid hormone modulation on liver injury associated with ischemia-reperfusion and cold storage in rats. Thyroxine administration increased susceptibility to ischemia-reperfusion injury, whereas the antithyroid agent propylthiouracil reduced the deleterious effects associated with cold storage.     

A comparison of the efficacy and tolerability of dorzolamide and acetazolamide as adjunctive therapy to timolol. Oral to Topical CAI Study Group

BACKGROUND: Recent observations provide evidence that complement is involved in the pathophysiology of ischemia/reperfusion injury. In this study, we assessed the impact of complement inhibition on hepatic microcirculation and graft function using a rat model of liver transplantation. METHODS: Arterialized orthotopic liver transplantation was performed in Lewis rats after cold preservation (University of Wisconsin solution, 4 degrees C, 24 h). Eight animals received the physiological complement regulator soluble complement receptor type 1 (sCR1) intravenously 1 min before reperfusion. Controls received Ringer's solution (n=8). Microvascular perfusion, leukocyte adhesion, and Kupffer cell phagocytic activity were studied 30-100 min after reperfusion by in vivo microscopy. RESULTS: Microvascular perfusion in hepatic sinusoids was improved in the sCR1 group (87+/-0.7% vs. 50+/-1%; P < 0.001). The number of adherent leukocytes was reduced in sinusoids (68.3+/-4.7 vs. 334.1+/-15.8 [adherent leukocytes per mm < or = liver surface]; P < 0.001) and in postsinusoidal venules after sCR1 treatment (306.6+/-21.8 vs. 931.6+/-55.9 [adherent leukocytes per mm < or = endothelial surface]; P < 0.001). Kupffer cell phagocytic activity was decreased in the sCR1 group compared to controls. Postischemic bile production reflecting hepatocellular function was increased by almost 200% (P = 0.004) after complement inhibition. Plasmatic liver enzyme activity was decreased significantly upon sCR1 treatment, indicating reduced parenchymal cell injury. CONCLUSIONS: Our results provide further evidence that the complement system plays a decisive role in hepatic ischemia/reperfusion injury. We conclude that complement inhibition by sCR1 represents an effective treatment to prevent reperfusion injury in liver transplantation.     

Rat whole-limb viability after cold immersion using University of Wisconsin and Euro-Collins solutions

The purpose of this study was to compare University of Wisconsin (UW) solution with Euro-Collins (EC) solution in their cold preservation effects on rat limbs. Thirty-six Lewis rat limbs were preserved in EC solution (n=18) or UW solution (n=18) at 4 degrees C for 72 hr, and grafted orthotopically to a syngeneic rat using microsurgical techniques. The surgeon was blinded to the solution used. We evaluated the vascular patency rate and death rate of both groups at day 7 after surgery and performed histological evaluations of bone, muscle, growth plate, and articular cartilage for each specimen of successful grafts in both groups. The vascular patency rates of the EC and UW groups were 27.7% (5/18) and 11% (2/18), respectively, and showed no significant difference. The death rates of the EC and UW groups were 50% (9/18) and 60% (10/18), which were not significantly different. There were no clear differences in histological viability between both groups, in all tissues exclusive of bone marrow and muscle tissue. Our results showed that in comparing EC and UW solutions, one was not significantly superior to the other as a cold immersion storage medium after a 72 hr ischemia-induced reperfusion injury.     

Neutrophil elastase inhibitor reduces neutrophil chemoattractant production after ischemia-reperfusion in rat liver

BACKGROUND & AIMS: Neutrophils are important in the development of tissue injury induced by ischemia-reperfusion. The ability of an inhibitor of neutrophil elastase (ONO-5046) to protect against ischemia-reperfusion injury in rat liver was investigated by measuring serum concentrations of cytokine-induced neutrophil chemoattractant. METHODS: Liver ischemia was induced in rats by occluding the portal vein for 30 minutes, and ONO-5046 or anticoagulants were injected intravenously 5 minutes before vascular clamping. RESULTS: Serum concentration of cytokine-induced neutrophil chemoattractant increased after reperfusion, reached a maximum at 6 hours, and then gradually decreased. However, pretreatment of animals with heparin (50 U/kg), antithrombin III (250 U/kg), or ONO-5046 (10 mg/kg) resulted in significantly smaller increases in the serum concentration of cytokine-induced neutrophil chemoattractant after reperfusion. Pretreatment with both ONO-5046 and heparin, or both ONO-5046 and antithrombin III, produced additive effects. Pretreatment of rats with both ONO-5046 and heparin or both ONO-5046 and antithrombin III also inhibited the increase in cytokine-induced neutrophil chemoattractant mRNA in liver. These combined treatments significantly reduced the increases in both the number of neutrophils accumulated in the liver and the hepatic activity of myeloperoxidase. CONCLUSIONS: Cytokine-induced neutrophil chemoattractant production after ischemia-reperfusion in the liver is mediated by neutrophil elastase and activation of coagulation within the hepatic microcirculation.