acid-phosphatase and Reperfusion-Injury

Acute primary open angle glaucoma is an optic neuropathy characterized by the elevation of intraocular pressure, which causes retinal ischemia and neuronal death. Rat ischemia/reperfusion enhances endocytosis of both horseradish peroxidase (HRP) or fluorescent dextran into ganglion cell layer (GCL) neurons 24 h after the insult. We investigated the activation of autophagy in GCL-neurons following ischemia/reperfusion, using acid phosphatase (AP) histochemistry and immunofluorescence against LC3 and LAMP1. Retinal I/R lead to the appearance of AP-positive granules and LAMP1-positive vesicles 12 and 24 h after the insult, and LC3 labelling at 24 h, and induced a consistent retinal neuron death. At 48 h the retina was negative for autophagic markers. In addition, Western Blot analysis revealed an increase of LC3 levels after damage: the increase in the conjugated, LC3-II isoform is suggestive of autophagic activity. Inhibition of autophagy by 3-methyladenine partially prevented death of neurons and reduces apoptotic markers, 24 h post-lesion. The number of neurons in the GCL decreased significantly following I/R (I/R 12.21±1.13 vs controls 19.23±1.12 cells/500 µm); this decrease was partially prevented by 3-methyladenine (17.08±1.42 cells/500 µm), which potently inhibits maturation of autophagosomes. Treatment also prevented the increase in glial fibrillary acid protein immunoreactivity elicited by I/R. Therefore, targeting autophagy could represent a novel and promising treatment for glaucoma and retinal ischemia.

Topics: Acid Phosphatase; Adenine; Animals; Apoptosis; Astrocytes; Autophagy; Caspase 3; Cell Count; Drug Design; Endocytosis; Intraocular Pressure; Lysosomes; Male; Microtubule-Associated Proteins; Neurons; Neuroprotective Agents; Rats; Rats, Wistar; Reperfusion Injury; Retina; Retinal Ganglion Cells; Time Factors

Cold preservation of the liver before transplantation may change uptake and excretory functions of hepatocytes. We hypothesized that an increase in the duration of preservation would result in a progressive decrease in the hepatic uptake and/or biliary excretion of indocyanine green (ICG), which would be attenuated by pharmacologic interventions.. Donor rats (n = 40) were administered saline (control) or single 5 mg/kg doses of methylprednisolone (MP) or its liver-targeted prodrug (DMP) 2 h prior to liver harvest. Following preservation in cold University of Wisconsin solution for 0, 24, 48, or 72 h, livers were reperfused in a single-pass manner for 30 min in the presence of ICG (approximately 4 microg/ml), followed by 60 min of ICG-free perfusion. The inlet, outlet, and bile concentrations of ICG were measured periodically by high performance liquid chromatography (HPLC), and kinetic parameters were estimated.. Effects of duration of preservation: In unpreserved livers, a significant portion of ICG dose (16%) was effluxed from the liver during the washout period. Cold preservation for 24-72 h progressively increased (p < 0.05) the efflux of ICG (>2-fold at 72 h). Similarly, average extraction ratio showed a modest (30-40%) decrease with increasing preservation time (p < 0.05). However, biliary excretion of ICG showed the most sensitivity to the preservation time (14 to >800-fold decline). Effects of pretreatment: DMP caused significant (p < 0.05) increases in biliary ICG levels (>12-fold) and bile flow rates (6-15-fold) of preserved livers. Although MP pretreatment significantly (p < 0.05) increased (6-fold) bile flow rates in 48-h preserved livers, its effects on biliary ICG levels were not significant (p > 0.05).. Biliary excretion of ICG is the most sensitive kinetic parameter to prolonged cold ischemia-reperfusion injury in a rat liver perfusion model. The injury may be significantly attenuated by pharmacologic pretreatment of the liver donors.

Topics: Acid Phosphatase; Alanine Transaminase; Animals; Aspartate Aminotransferases; Bile; Cryopreservation; Dextrans; Drug Combinations; Indocyanine Green; Ischemia; L-Lactate Dehydrogenase; Liver; Liver Failure; Liver Transplantation; Methylprednisolone Hemisuccinate; Organ Preservation; Organ Preservation Solutions; Perfusion; Prodrugs; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors; Tissue Donors

We studied the state of lysosomal apparatus and pro- and antioxidant activity in the liver of rats with different resistance to hypoxia during postischemic recovery. Under normal conditions the lysosomal apparatus did not differ in highly and low resistant animals. During ischemia and reperfusion the damage to hepatic lysosomal membranes in rats highly resistant to hypoxia was less pronounced than in low resistant animals. These differences also concerned labilization of lysosomes during exposure to damaging factors (hypotonia and Triton X-100). The rats highly resistant to hypoxia differed from low resistant animals by higher stability of lysosomal membranes, lower prooxidant activity (malonic dialdehyde content), and higher tissue concentration of alpha-tocopherol during reperfusion.

Topics: Acid Phosphatase; alpha-Tocopherol; Animals; beta-Galactosidase; Hypoxia; Intracellular Membranes; Ischemia; Liver; Lysosomes; Male; Malondialdehyde; Octoxynol; Oxidative Stress; Rats; Rats, Wistar; Reperfusion Injury; Ribonucleases; Time Factors

The incidence of steatosis in livers retrieved for organ transplantation is up to 30%. Due to the shortage of donor organs, many of these livers are accepted for clinical transplantation, although a high rate of graft dysfunction is associated with ischemic preservation of steatotic livers. The present study was intended to reduce the ischemia/reperfusion injury of steatotic grafts by the use of venous systemic oxygen persufflation during cold storage.. A histologically-documented mild to moderate steatosis was induced in livers of Wistar rats by fasting for 2 days and subsequent feeding of a fat-free diet enriched in carbohydrates. Fatty livers were retrieved and flushed via the portal vein with 60 ml of HTK. In group A, livers were then stored ischemically at 4 degrees C for 24 h. Livers of group B were additionally connected to a gaseous oxygen supply and persufflated with O2 via the venous vascular system during the cold storage period. Viability of the livers was then assessed upon isolated perfusion in vitro with oxygenated Krebs-Henseleit buffer.. Venous systemic oxygen sufflation resulted in a relevant and significant reduction of parenchymal (ALT: 132+/-90 vs 434+/-172 U/l; p<0.01) and mitochondrial (GLDH: 116+/-57 vs 633+/-241 U/l; p<0.001) enzyme release during reperfusion. Moreover, Kupffer cell activation, as evaluated from acid phosphatase activity in the perfusate, was reduced to about 1/3 (4.0+/-1.3 vs 11.9+/-5.3 U/l; p<0.01). Electron microscopic analysis revealed that the liver mitochondria and sinusoidal endothelial lining were better preserved after oxygen persufflation, which was in line with the data on enzyme release and the increased portal perfusion pressure in the untreated group, while normal values were found after venous systemic oxygen sufflation.. Venous oxygen persufflation may thus represent a useful tool for the safe and improved preservation of ischemia-sensitive steatotic livers.

Topics: Acid Phosphatase; Animals; Cryopreservation; Fatty Liver; Kupffer Cells; Liver; Liver Transplantation; Male; Mitochondria, Liver; Organ Preservation; Oxygen; Perfusion; Portal Vein; Rats; Rats, Wistar; Reperfusion Injury; Venous Pressure

The purpose of this study was to evaluate the effect of prostaglandin E1 (PGE1) on protecting against hepatic endothelial cell damage and increasing graft viability after cold preservation and reperfusion, using an isolated perfused rat liver (IPRL) model. The grafts were divided into three groups, according to the cold preservation time and PGE1 administration, namely: 4h preservation (group 1, n = 9), 6h preservation (group 2, n = 9), and 6h preservation followed by PGE1 infusion (group 3, n = 9). After cold storage, the grafts were put on the recirculating IPRL system, then reperfused for 120 min at 37 degrees C with oxygenated Krebs-Henseleit buffer containing hyaluronic acid (HA). To examine the function of the sinusoidal endothelial cells and hepatocytes, serial measurements of HA, tumor necrosis factor-alpha (TNFalpha), thromboxane B2 (TXB2), acid phosphatase, and conventional parameters in the perfusate were made. After perfusion, the trypan blue exclusion test was performed to assess the presence of any microscopic sinusoidal lining cell damage. In group 3, the bile output and HA clearance were significantly greater, while glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, TNFalpha, TXB2, and acid phosphatase in the perfusate were significantly lower than in group 2. Histologically, less endothelial cell damage and hepatocyte damage than in group 2 was also confirmed. These results therefore suggest that the improvement of hepatic graft viability by PGE1 administration is mainly due to sinusoidal endothelial cell protection.

Topics: Acid Phosphatase; Alanine Transaminase; Alprostadil; Animals; Aspartate Aminotransferases; Cryopreservation; Endothelium; Graft Survival; Hyaluronic Acid; L-Lactate Dehydrogenase; Liver Transplantation; Male; Rats; Rats, Inbred Lew; Reperfusion Injury; Thromboxane B2; Tumor Necrosis Factor-alpha

The mechanisms by which heparin protects the liver during induced episodes of liver ischemia-reperfusion are poorly understood. Previous work in a swine model demonstrated that serum levels of glycohydrolases and lipid peroxide peaked within 3 h after 45 minutes of hepatic ischemia followed by reperfusion. Serum levels of lactate dehydrogenase and aspartate aminotransferase peaked 20-24 h later. The aim of this study was to evaluate the effect of heparin on these two-phases of enzyme release, using a pig model of hepatic ischemia-reperfusion injury. Twenty male swine were divided into control (n = 8) and heparin (n = 12) groups. In the heparin group, heparin was administered prior to and concurrent with ischemia-reperfusion. Following 45 min of hepatic ischemia, the levels of beta-galactosidase, beta-glucosidase, acid phosphatase, purine nucleoside phosphorylase, lipid peroxides, lactate dehydrogenase, and aspartate aminotransferase in serum were monitored for up to 166 h and compared to pre-ischemic and control levels. With heparin infusion, the peak levels of beta-galactosidase, beta-glucosidase, and the lipid peroxide were reduced to 50-60% of the control levels. Acid phosphatase and purine nucleoside phosphorylase activities in serum were reduced to 25% and 60%, respectively. The peak concentrations of lactate dehydrogenase and aspartate aminotransferase were reduced to about 25% of the control level. In addition, the serum enzymes of control pigs did not return to pre-ischemic levels until 2 weeks after hepatic ischemia, while they normalized in less than 1 week in the heparin-treated animals. Systemic heparinization had different protective effects on the first and secondary phases of liver injury. These differences may reflect heparin protection of different types of liver cells. The protection of the parenchymal cells may be the combined result of reduced sinusoidal cell injury and the anticoagulant properties of heparin.

Topics: Acid Phosphatase; Animals; Aspartate Aminotransferases; beta-Galactosidase; beta-Glucosidase; Heparin; Ischemia; L-Lactate Dehydrogenase; Lipid Peroxides; Liver; Male; Purine-Nucleoside Phosphorylase; Reperfusion Injury; Swine

Abundant fat in the liver has been implicated in poor outcome after liver transplantation or liver surgery, but the reasons for this association are still unclear. The aim of the present study was to examine mechanisms that may be involved in hepatic dysfunction after ischemia-reperfusion (I/R) of the steatotic rat liver. Steatosis was produced by a choline-methionine-deficient (CMDD) diet. In the first experiment, isolated perfused rat livers, subjected to 24-hour cold storage followed by 120-minute reperfusion, were used to investigate hypothermic I/R injury of the steatotic rat liver. In the second experiment, livers were subjected to 60-minute partial left lobar vascular clamping to allow study of normothermic I/R injury. In the first experiment, compared with normal nonsteatotic liver, steatotic livers showed significantly greater injury, as assessed by amounts of hepatic enzymes released into the perfusate, bile production, the concentrations of reduced glutathione (GSH) in the perfusate, as well as in the livers themselves, and electron microscopic findings of sinusoidal microcirculatory injury. The addition of N-acetylcysteine (NAC), a precursor of glutathione, to the liver before cold storage significantly improved these parameters in steatotic livers. The second experiment showed that, compared with nonsteatotic livers, steatotic livers had lower concentrations of GSH and impaired rates of bile production. There was also evidence of increased oxidative stress in polymorphonuclear leukocytes (PMNLs) in liver or peripheral blood of rats with fatty livers. An anti-rat intercellular adhesion molecule-1 (ICAM-1) monoclonal antibody inhibited neutrophil infiltration into pericentral sinusoids and improved these parameters in the steatotic rats. We conclude that sinusoidal microcirculatory injury is involved in hypothermic I/R injury, that oxidative stress produced by PMNLs is involved in normothermic I/R injury, and that NAC and anti-rat ICAM-1 monoclonal antibody restore liver integrity in I/R injury.

Topics: Acetylcysteine; Acid Phosphatase; Alanine Transaminase; Animals; Antibodies, Monoclonal; Aspartate Aminotransferases; Bile; Choline Deficiency; Fatty Liver; In Vitro Techniques; Intercellular Adhesion Molecule-1; Ischemia; L-Lactate Dehydrogenase; Liver; Male; Malondialdehyde; Methionine; Microscopy, Electron; Neutrophils; Perfusion; Rats; Rats, Wistar; Reperfusion Injury

The study was performed on rats divided into 9 groups. Groups 1-3 served as controls. In groups 4 and 5 rat livers were subjected to 90-min ischemia followed by 12- or 24-hour reperfusion. In groups 6 and 7 rats were injected with intraperitoneal chlorfenvinphos (2 mg/kg b.w.) and sacrificed after 12 or 24 hours. In groups 8 and 9 rat livers were subjected to 90-min ischemia, 12- or 24-hour reperfusion and then rats were injected with chlorfenvinphos (2 mg/kg b.w.). Liver sections were evaluated morphologically, histochemically (SDH, LDH, G6Pase, glycogen, Mg2+ ATPase and AcP). The microsomal fraction of the liver was evaluated for cytochrome P450 content and NADPH-cytochrome P-450 reductase activity. It has been found that liver ischemia and reperfusion result in extensive necrosis, enzymatic disturbances, particularly in acinar zone 3. Ischemia as well as reperfusion decrease the cytochrome P450 content of hepatocytic microsomes and the activity of NADPH-cytochrome P-450 reductase. Intraperitoneal injection of chlorfenvinphos during ischemia and reperfusion dramatically intensifies damage to the liver, although chlorfenvinphos alone produces only mild nonspecific effects on the morphological and enzymatic structure of the liver.

Topics: Acid Phosphatase; Adenosine Triphosphatases; Animals; Biomarkers; Chlorfenvinphos; Cytochrome P-450 Enzyme System; Glucose-6-Phosphatase; Ischemia; L-Lactate Dehydrogenase; Liver; Male; NADP; NADPH-Ferrihemoprotein Reductase; Necrosis; Periodic Acid-Schiff Reaction; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Succinate Dehydrogenase

Advances in liver surgery and transplantation have lead to a steady increase in the number of these interventions. Prompt quantitative assessment of hepatic of hepatic function and a patient's subsequent morbidity and mortality following surgery remain difficult despite the currently utilized historic markers of hepatic parenchymal injury (e.g., aspartate transaminase [AST], lactate dehydrogenase [LDH] gamma-glutamyl transpeptidase [GGT]). Increases in serum glycohydrolase activities appear to provide sensitive and quantitative markers of hepatic ischemia/reperfusion injury. In 10 male swine (25 to 35 kg body weight) following 30, 45, and 90 minutes of acute hepatic ischemia, the systemic release of eight different glycohydrolases and lipid peroxides into serum were determined and compared with pre- and postischemic serum levels of LDH, GGT, and AST. The rapid release of glycohydrolases into serum was directly proportional to the length of the ischemic period from 30 to 90 minutes; e.g., beta-glucosidase, mean 1.9-fold increase at 30 minutes; 8.3-fold at 45 minutes; and 22.8-fold at 90 minutes; P < .002) and the activities peaked within the first 3 hours postischemia. In constrast, AST, LDH, and GGT were released slowly and peaked 20 to 30 hours after hepatic blood flow was restored. In swine with fatal outcomes (90 minutes of ischemia), all enzyme levels increased continuously during the final hours of life. However, in swine that survived hepatic ischemia/reperfusion injury (45 minutes of ischemia) the glycohydrolases, but not AST, LDH, and GGT, declined after 2 to 3 hours' postischemia and the serum lipid peroxide levels followed the same pattern. Serum beta-galactosidase and beta-glucosidase levels are sensitive markers that rise as quickly as traditional enzyme markers (AST, LDH, GGT) following hepatic ischemic injury; moreover, the glycohydrolases have the added value of serving as predictors of survival.

Topics: Acid Phosphatase; alpha-Galactosidase; alpha-Glucosidases; Animals; Aorta; beta-Galactosidase; beta-Glucosidase; beta-N-Acetylhexosaminidases; Biomarkers; Glucuronidase; Glycoside Hydrolases; Hepatic Veins; Lipid Peroxidation; Liver; Male; Portal Vein; Reperfusion Injury; Swine; Time Factors

We investigated whether intraportal injection of 150 mg/kg N-acetylcysteine (NAC) into rats reduced hepatic ischemia-reperfusion injury after 48 hours of cold storage and 2 hours of reperfusion. The organ was isolated and perfused to evaluate liver function. The control group received an intraportal injection of 5% dextrose. NAC increased L-cysteine concentrations 15 minutes after injection (1.29 +/- 0.11 mumol/g vs. 2.68 +/- 0.4 mumol/g, P < .05). However, neither treatment modified glutathione liver concentrations relative to preinjection values. After 48 hours of cold storage and 2 hours of reperfusion, livers from NAC-treated rats produced larger amounts of bile than those in the control group (5.04 +/- 1.92 vs. 0.72 +/- 0.37 microL/g liver; P < .05), and showed a significant reduction in liver injury, as indicated by reduced release of lactate dehydrogenase (679.4 +/- 174.4 vs. 1891.3 +/- 268.3 IU/L/g; P < .01), aspartate transaminase (AST) (13.94 +/- 3.5 vs. 38.75 IU/L/g; P < .01), alanine transaminase ALT) (14.92 +/- 4.09 vs. 45.91 +/- 10.58 IU/L/g; P < .05), and acid phosphatase, a marker of Kupffer cell injury (344.4 +/- 89.6 vs. 927.3 +/- 150.8 IU/L/g; P < .01) in the perfusate. Reduced glutathione concentrations in the perfusate were similar in the two groups (805 +/- 69 vs. 798 +/- 252 nmol/L/g), whereas oxidized glutathione (GSSG) concentrations were higher in the control group (967 +/- 137 vs. 525 +/- 126 nmol/L/g; P < .05). Reduced glutathione (GSH) concentrations in liver tissue collected at the end of perfusion were significantly higher in the NAC group (7.3 +/- 0.9 vs. 4.1 +/- 1.0 mumol/g; P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcysteine; Acid Phosphatase; Alanine Transaminase; Animals; Aspartate Aminotransferases; Buthionine Sulfoximine; Cold Temperature; Cysteine; Glutathione; L-Lactate Dehydrogenase; Liver; Male; Methionine Sulfoximine; Oxidation-Reduction; Rats; Rats, Wistar; Reperfusion Injury

The aim of the study was to evaluate the enzyme activity of cellular membranes (GGT), cytosol (ALT, AST) and lysosome (AP, AcP) in the cytosol, whole homogenate and blood serum during declamping shock, following release of abdominal aorta cross-clamping. The aorta was clamped for 60 minutes. An increase in GGT, AP and AcP activities in the cytosol and whole homogenate of the renal cortex, renal medulla, liver, lung, heart and the skeletal muscle occurs after declamping. Rise in the enzymatic activity, especially of acid phosphatase is higher when the aorta above renal arteries was clamped. However, its activity in the blood serum remains unchanged. Alterations in the distribution and the activity of the studied enzymes may indicate that aortic clamping damages the endoplasmic reticulum and lysosomal membranes. Yet, cellular membranes preserve their structural and functional integrity.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Cell Membrane; Constriction; Cytosol; Dogs; gamma-Glutamyltransferase; Kidney; Liver; Lung; Lysosomes; Muscle, Skeletal; Myocardium; Reperfusion Injury; Shock, Surgical; Transaminases

The interrelation between intracellular cAMP content and activity of lysosomal hydrolases was studied in rat liver and heart during ischemia of varying genesis and after recirculation. The activity of acid phosphatase (AP) and cathepsin D (CD) was determined in the fraction enriched with lysosomes (FEL) and in the supernatant fraction (SF) at 30,000 x g. Ischemia of isolated perfused heart of 20 to 60 min as described by Langendorff was accompanied by an increase in the SF/FEL ratio. Postischemic reperfusion resulted in a further increase in this ratio. In a terminal state induced by cardiac arrest of 10 min and within the first postresuscitation hours the SF/FEL ratio in the rat liver also increased. Processing of the liver FEL with 0.025% concentration of detergent Triton X-100 was also indicative of lability of lysosomal membranes during recirculation. The intracellular cAMP content changed differently. During ischemia of the myocardium, the cAMP level rose by 40 min and remained increased after 20 and 40 min of reperfusion. The cAMP content in the liver decreased after 10 min of circulatory arrest and increased in the postresuscitation period achieving its peak 4 h after resuscitation. Intra-abdominal injection of lyposomes with incapsulated cAMP to rats in the postresuscitation period and the study of the effect of dibutyryl-cAMP, caffeine and isoproterenol on the activity of acid hydrolases of ischemic heart and after postischemic reperfusion showed that an increase in the cAMP content achieved in various ways was conducive to stabilization of lysosomal membranes.

Topics: Acid Phosphatase; Animals; Cathepsin D; Cyclic AMP; Liver; Lysosomes; Male; Myocardial Reperfusion Injury; Myocardium; Rats; Rats, Inbred Strains; Reperfusion Injury

Reperfusion injury characterized by loss of endothelial cell viability occurs after cold ischemic storage of livers for transplantation surgery. Here, ultrastructural changes in stored rat livers were examined by scanning and transmission electron microscopy. With increasing times of storage in Euro-Collins solution (4 to 24 hr) followed by 15 min of reperfusion at 37 degrees C, a sequence of structural alterations was observed involving endothelial and Kupffer cells. Widening of endothelial fenestrations occurred after 4 hr and progressed over 8 to 24 hr to retraction of cellular processes, ball-like rounding, sinusoidal denudation and ultrastructural derangements consistent with loss of cell viability. Kupffer cells exhibited progressive rounding, ruffling of the cell surface, polarization, appearance of wormlike densities, vacuolization and degranulation over a similar time course. By contrast, the structures of parenchymal and fat-storing cells were relatively undisturbed by cold storage and reperfusion. Alterations to endothelial and Kupffer cells were also studied as a function of time of reperfusion. After 24 hr of storage, endothelial cells showed retraction of cytoplasm before reperfusion that progressed quickly to loss of viability and denudation during reperfusion. Kupffer cell activation (ruffling, degranulation) during reperfusion was slower and occurred after deterioration of endothelial cells. Livers stored in Euro-Collins solution were also compared with livers stored in University of Wisconsin cold storage solution, an improved preservation medium for transplantation. University of Wisconsin solution provided better preservation of endothelial structure and markedly reduced parenchymal cell blebbing and swelling before reperfusion. University of Wisconsin solution also reduced Kupffer cell activation and release of lysosomal enzymes. In conclusion, endothelial cell deterioration followed by Kupffer cell activation occurred after increasing times of cold ischemic storage and reperfusion of rat livers. Both changes may contribute to the pathophysiology of graft failure caused by reperfusion-mediated storage injury.

Topics: Acid Phosphatase; Adenosine; Allopurinol; Animals; Endothelium; Glutathione; Hypertonic Solutions; Insulin; Kupffer Cells; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Strains; Reperfusion Injury; Solutions