mocetinostat and Ischemia

Resuscitation induced ischemia/reperfusion predisposes trauma patients to systemic inflammation and organ dysfunction. We investigated the effect of remote ischemic conditioning (RIC), a treatment shown to prevent ischemia/reperfusion injury in experimental models of hemorrhagic shock/resuscitation, on the systemic immune-inflammatory profile in trauma patients in a randomized trial. We conducted a prospective, single-centre, double-blind, randomized, controlled trial involving trauma patients sustaining blunt or penetrating trauma in hemorrhagic shock admitted to a Level 1 trauma centre. Patients were randomized to receive RIC (four cycles of 5-min pressure cuff inflation at 250 mmHg and deflation on the thigh) or a Sham intervention. The primary outcomes were neutrophil oxidative burst activity, cellular adhesion molecule expression, and plasma levels of myeloperoxidase, cytokines and chemokines in peripheral blood samples, drawn at admission (pre-intervention), 1 h, 3 h, and 24 h post-admission. Secondary outcomes included ventilator, ICU and hospital free days, incidence of nosocomial infections, 24 h and 28 day mortality. 50 eligible patients were randomized; of which 21 in the Sham group and 18 in the RIC group were included in the full analysis. No treatment effect was observed between Sham and RIC groups for neutrophil oxidative burst activity, adhesion molecule expression, and plasma levels of myeloperoxidase and cytokines. RIC prevented significant increases in Th2 chemokines TARC/CCL17 (P < 0.01) and MDC/CCL22 (P < 0.05) at 24 h post-intervention in comparison to the Sham group. Secondary clinical outcomes were not different between groups. No adverse events in relation to the RIC intervention were observed. Administration of RIC was safe and did not adversely affect clinical outcomes. While trauma itself modified several immunoregulatory markers, RIC failed to alter expression of the majority of markers. However, RIC may influence Th2 chemokine expression in the post resuscitation period. Further investigation into the immunomodulatory effects of RIC in traumatic injuries and their impact on clinical outcomes is warranted.ClinicalTrials.gov number: NCT02071290.

Topics: Cytokines; Humans; Ischemia; Ischemic Preconditioning; Peroxidase; Prospective Studies; Shock, Hemorrhagic; Shock, Traumatic; Treatment Outcome

Testicular torsion/detorsion-induced ischemia/reperfusion injury is partly due to the overgeneration of reactive oxygen species. Baicalein, a main bioactive constituent derived from the dried root of

Topics: Animals; Antioxidants; Ischemia; Male; Malondialdehyde; NADPH Oxidases; Peroxidase; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Spermatic Cord Torsion; Testis; Tumor Necrosis Factor-alpha

The most effective way of treating liver cancer is surgical resection, which usually requires blocking the hepatic portal circulation, and may result in hepatic ischemia-reperfusion injury (HIRI). It is of paramount importance to control HIRI for liver cancer surgical resection. In this study, a 70% ischemia-reperfusion (I/R) model of rat liver was established, and the protective effect and mechanism of limb ischemic post-conditioning (LIPOC) on HIRI was investigated. We show that LIPOC has a protective effect on hepatic ischemia-reperfusion injury in rats, which reduces the elimination of superoxide dismutase, thereby increasing oxygen free radical scavenging, decreasing lipid peroxidation, inhibiting neutrophil aggregation, as well as reducing TNFα, IL1β, and other inflammatory cytokines. In addition, LIPOC inhibited the apoptosis of hepatocytes induced by I/R injury, and decreased the Bax/Bcl-2 ratio. Furthermore, LIPOC promoted the phosphorylation of Akt and ERK1/2. The use of PI3K inhibitor LY294002 and ERK1/2 blocker PD98059 inhibited the phosphorylation of Akt and ERK1/2 caused by LIPOC and abolished the injury protection of liver I/R. Moreover, through 16 cases of hepatocellular carcinoma resections, we found that short-term LIPOC treatment significantly suppressed the elevated alanine aminotransferase, aspartic transaminase, and total bilirubin in the early post-operation of liver resection, and reduced reperfusion injury to the ischemic liver. In summary, our study demonstrates that LIPOC could be an effective method for HIRI in the clinical implementation of liver resection and uncovers the potential mechanism of LIPOC in the protective effects of HIRI.

Topics: Adult; Aged; Alanine Transaminase; Animals; Apoptosis; Blotting, Western; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Humans; In Situ Nick-End Labeling; Ischemia; Ischemic Postconditioning; Liver Neoplasms; Male; Malondialdehyde; MAP Kinase Signaling System; Middle Aged; Peroxidase; Phosphatidylinositol 3-Kinases; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase

Colonic ischemia after aortic surgery is associated with increased mortality and morbidity rates. This study was conducted as a single-center side arm to a multicenter, randomized, placebo-controlled study to evaluate the effect of dopexamine hydrochloride on its incidence.. Thirty patients, mean age 65.1 years (range, 46-84), undergoing elective infrarenal aortic surgery were entered. Preoperative hemodynamic and respiratory parameters were optimized. Patients were then randomly assigned to receive a perioperative infusion of dopexamine at 2 microg/kg per minute (n = 12) or 0.9% saline placebo (n = 18). All patients underwent colonoscopy and biopsy preoperatively and 1 week postoperatively. Specimens were assessed for evidence of mucosal ischemia, presence of mast cell tryptase, myeloperoxidase activity, and both the inducible and endothelial isoforms of nitric oxide synthase.. There was no significant difference in perioperative fluid and blood requirements or hemodynamic and respiratory parameters between the two groups. However, there was significantly less evidence of mucosal ischemic changes in dopexamine-treated patients (n = 1) compared with placebo (n = 8) (P =.049). Furthermore, when preoperative biopsies were compared with those performed 1 week postoperatively, nine (50%) patients in the placebo group and two (16.7%) in the dopexamine group scored worse. Although there was no significant difference in inflammatory markers between the two groups, both mast cell tryptase and myeloperoxidase expression were increased in patients with histologic evidence of ischemia (P <.05). Furthermore, inducible nitric oxide synthase staining within the vascular (P =.001) and lamina propria (P <.05) components of the mucosa was also significantly greater.. A perioperative dopexamine infusion affords significant histologic protection to colonic mucosa after aortic surgery.

Topics: Adult; Aged; Aged, 80 and over; Aorta, Abdominal; Biopsy; Colon; Colonoscopy; Dopamine; Dopamine Agonists; Female; Humans; Immunohistochemistry; Inflammation Mediators; Infusions, Intravenous; Intestinal Mucosa; Ischemia; Male; Middle Aged; Neutrophils; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Perioperative Care; Peroxidase; Postoperative Complications; Prospective Studies; Serine Endopeptidases; Tryptases; Vasodilator Agents

The aim of the study was to investigate the time course of neutrophil activation after skeletal muscle ischemia in humans and to assess the effect of xanthine oxidase inhibitor allopurinol or cyclooxygenase inhibitor indomethacin. In patients undergoing tourniquet ischemia of the upper limb, polymorphonuclear neutrophils (PMN) were simultaneously isolated from antecubital vein blood of both the contralateral control arm and the tourniquet arm. PMN-superoxide production (PMN-SOP) was determined by a cytochrome C reduction assay, PMN-myeloperoxidase activity (PMN-MPO) by guaiacol oxidation and serum PMN-elastase concentration by an enzyme immunoassay. At 60 min after release of the tourniquet, significant increases of PMN-SOP, PMN-MPO, and serum elastase concentrations were observed in tourniquet arms as compared with control arms (p < .05). Allopurinol (300 mg orally, 12 and 2 h before ischemia) significantly inhibited the increase of PMN-SOP, PMN-MPO, and serum elastase (p < .05). Indomethacin (50 mg orally, 2 h before ischemia) prevented increased PMN-MPO and serum elastase, but prevented increased PMN-SOP only when neutrophils were incubated in the presence of their autologous plasma. These findings suggest that ischemia/reperfusion of human skeletal muscle involves both xanthine oxidase-dependent oxygen free radicals and cyclooxygenase metabolites. These pathways could activate circulating neutrophils which potentially inflict local and remote endothelial injury.

Topics: Adolescent; Adult; Allopurinol; Cyclooxygenase Inhibitors; Enzyme Inhibitors; Female; Humans; Indomethacin; Ischemia; Leukocyte Count; Leukocyte Elastase; Male; Middle Aged; Muscle, Skeletal; Neutrophil Activation; Peroxidase; Xanthine Oxidase

Histomorphologic/morphometric evaluation, leukocyte scintigraphy, and myeloperoxidase activity were used to determine whether neutrophils accumulate in the large colon of horses during low-flow ischemia and reperfusion. Twenty-four adult horses were assigned to 1 of 3 groups: group 1, sham-operated (n = 6); group 2, 6 hours of ischemia (n = 9); and group 3, 3 hours of ischemia and 3 hours of reperfusion (n = 9). Low-flow ischemia of the large colon was induced in horses of groups 2 and 3 by reducing colonic arterial blood flow to 20% of baseline. Radiolabeled (99mTc) autogenous neutrophils were injected at 175 minutes, which corresponded to 5 minutes prior to reperfusion in group-3 horses. Full-thickness biopsy specimens of the left ventral colon were collected at baseline and at 30-minute intervals for 6 hours; a portion of the biopsy specimen was placed in formalin for histologic examination, and the remainder was used to measure mucosal radioactivity and myeloperoxidase activity. There were no differences in baseline mucosal neutrophil index, mucosal neutrophil numbers, submucosal venular neutrophil numbers, mucosal radioactivity, or mucosal myeloperoxidase activity among groups, or over time in group-1 horses. Neutrophils accumulated in the colonic mucosa during ischemia and further increased at reperfusion, as indicated by neutrophil index (morphology) and mucosal neutrophil numbers (morphometry); mucosal neutrophil index was significantly (P < 0.05) greater in group-3 horses during reperfusion than at the corresponding periods of ischemia in group-2 horses. Neutrophil numbers were significantly (P < 0.05) increased in submucosal venules at 10 minutes of reperfusion in group-3 horses and were significantly (P < 0.05) greater in group-3 than in group-2 horses during the interval from 3 to 6 hours. Mucosal radioactivity significantly (P < 0.05) increased at reperfusion in group-3 horses; there was a trend (P = 0.076) toward greater mucosal radioactivity in group-3, compared with group-2 horses, throughout the 3- to 6-hour interval. There were no differences in mucosal myeloperoxidase activity among or within any of the 3 groups over time. Neutrophils accumulated in the large colon of horses during low-flow ischemia and reperfusion. Neutrophil infiltration was detected by histologic examination and leukocyte scintigraphy, but not by measurement of myeloperoxidase activity. The accumulation of neutrophils during ischemia and the further neutrophil infilt

Topics: Animals; Colitis, Ischemic; Colon; Female; Horse Diseases; Horses; Intestinal Mucosa; Ischemia; Male; Neutrophils; Peroxidase; Radionuclide Imaging; Reperfusion Injury

Sixteen horses were allotted at random to 3 groups: vehicle only; low dosage (vehicle and 3 mg of U-74389G/kg of body weight); high dosage (vehicle and 10 mg of U-74389G/kg). These solutions were given prior to reperfusion. The ascending colon was subjected to 2 hours of ischemia followed by 2 hours of reperfusion. Before, during, and after ischemia, full-thickness colonic tissue biopsy specimens were obtained for measurement of malondealdehyde (MDA) concentration and myeloperoxidase activity and for morphologic evaluation. Although increases were not significant, MDA concentration and myeloperoxidase activity increased during ischemia and reperfusion. Administration of U-74389G did not have significant effects on MDA concentration and myeloperoxidase activity. However, the lower dosage tended (P = 0.08) to reduce myeloperoxidase activity at 30 and 60 minutes of reperfusion. In horses of the vehicle-only group, ischemia induced a decrease in mucosal surface area that was continued into the reperfusion period (P < or = 0.05). Administration of U-74389G at both dosages (3 and 10 mg/kg) prevented the reperfusion-induced reduction in mucosal surface area, which was significant at 60 minutes (high dosage; P = 0.05) and 90 minutes (low and high dosages; P = 0.02). After initial reduction in horses of all groups, mucosal volume increased for the initial 60 minutes of reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Analysis of Variance; Animals; Antioxidants; Biopsy; Colon; Female; Horse Diseases; Horses; Ischemia; Male; Malondialdehyde; Peroxidase; Pregnatrienes; Reperfusion Injury; Time Factors

We previously demonstrated that prehabilitation by running on a treadmill leads to improved survival after gut ischemia reperfusion (I/R) in mice. The purpose of this research was to examine whether prehabilitation attenuates inflammatory responses after gut I/R in mice.. Male C57BL/6J mice (n = 92) were assigned to the sedentary (n = 46) or the exercise (n = 46) group. The exercise group ran on a treadmill for 4 wk, while the sedentary mice did not exercise. After the 4-week pretreatment, all mice underwent gut I/R and the blood, urine, small intestine, lung, liver, and gastrocnemius were harvested prior to ischemia or at 0, 3, 6, or 24 h after reperfusion. Histologically demonstrated organ damage, cytokine levels in the blood, gut and gastrocnemius, myeloperoxidase activity in the gut, 8-hydroxy-2'-deoxyguanosine levels in urine and the gut, and adenosine triphosphate (ATP) and ATP + ADP + adenosine monophosphate levels in the gut and gastrocnemius were evaluated.. The treadmill exercise reduced gut and lung injuries at 3 h and liver injury at 6 h after reperfusion. Running on the treadmill also decreased proinflammatory cytokine levels in the blood at 6 h, gut at 3 h and gastrocnemius at 6 h after reperfusion, myeloperoxidase activity in the gut prior to ischemia, and 6 h after reperfusion and the urinary 8-hydroxy-2'-deoxyguanosine level at 24 h after reperfusion, while ATP levels in exercised mice prior to ischemia and 3 h after reperfusion were increased in the intestine as compared to the levels in sedentary mice.. Prehabilitation with treadmill exercise reduces inflammatory responses after gut I/R and may exert protective actions against gut I/R.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adenosine Triphosphate; Animals; Antioxidants; Cytokines; Ischemia; Male; Mice; Mice, Inbred C57BL; Peroxidase; Physical Conditioning, Animal; Preoperative Exercise; Reperfusion Injury

Remote ischaemic conditioning (RIC) has been shown to reduce ischaemia-reperfusion injury(IRI) in multiple organ systems. IRI is seen in multiple bowel pathologies in the newborn, including NEC. We investigated the potential of RIC as a novel therapy for various intestinal pathologies in the newborn.. We used an established intestinal IRI model in rat pups which results in similar intestinal injury to necrotising enterocolitis (NEC). Animals were randomly allocated to IRI only(n = 14), IRI + RIC(n = 13) or sham laparotomy(n = 10). The macroscopic extent of intestinal injury is reported as a percentage of total small bowel. Injury severity was measured using Chiu-Park scoring. Neutrophil infiltration/activation was assayed by myeloperoxidase activity. Immunohistochemistry was used to assess the expression of hypoxia-inducible factor alpha (HIF-1α). Data are median (interquartile range).. Animals that underwent RIC showed a decreased extent of macroscopic injury from 100%(85-100%) in the IRI only group to 58%(15-84%, p = 0.003) in the IRI + RIC group. Microscopic injury score was significantly lower in animals that underwent RIC compared to IRI alone (3.5[1.25-5] vs 5.5[4-6], p = 0.014). Intestinal myeloperoxidase activity in animals exposed to IRI was 3.4 mU/mg of tissue (2.5-3.7) and 2.1 mU/mg(1.5-2.8) in the IRI + RIC group(p = 0.047). HIF-1α expression showed a non-significant trend towards reduced expression in the IRI + RIC group.. RIC reduces the extent and severity of bowel injury in this animal model, supporting the hypothesis that RIC has therapeutic potential for intestinal diseases in the newborn.

Topics: Animals; Animals, Newborn; Ischemia; Ischemic Preconditioning; Peroxidase; Rats; Reperfusion Injury

Mildronate is a useful anti-ischemic agent and has antiinflammatory, antioxidant, and neuroprotective activities. The aim of this study is to investigate the potential neuroprotective effects of mildronate in the experimental rabbit spinal cord ischemia/reperfusion injury (SCIRI) model.. Rabbits were randomized into 5 groups of 8 animals as groups 1 (control), 2 (ischemia), 3 (vehicle), 4 (30 mg/kg methylprednisolone [MP]), and 5 (100 mg/kg mildronate). The control group underwent only laparotomy. The other groups have the spinal cord ischemia model by a 20-minute aortic occlusion just caudal to the renal artery. The malondialdehyde and catalase levels and caspase-3, myeloperoxidase, and xanthine oxidase activities were investigated. Neurologic, histopathologic, and ultrastructural evaluations were also performed.. The serum and tissue myeloperoxidase, malondialdehyde, and caspase-3 values of the ischemia and vehicle groups were statistically significantly higher than those of the MP and mildronate groups (P < 0.001). Serum and tissue catalase values of the ischemia and vehicle groups were statistically significantly lower than those of the control, MP, and mildronate groups (P < 0.001). The histopathologic evaluation showed a statistically significantly lower score in the mildronate and MP groups than in the ischemia and vehicle groups (P < 0.001). The modified Tarlov scores of the ischemia and vehicle groups were statistically significantly lower than those of the control, MP, and mildronate groups (P < 0.001).. This study presented the antiinflammatory, antioxidant, antiapoptotic, and neuroprotective effects of mildronate on SCIRI. Future studies will elucidate its possible use in clinical settings in SCIRI.

Topics: Animals; Antioxidants; Caspase 3; Catalase; Disease Models, Animal; Ischemia; Malondialdehyde; Methylprednisolone; Neuroprotective Agents; Peroxidase; Rabbits; Reperfusion Injury; Spinal Cord; Spinal Cord Ischemia

Over the past decade, histone deacetylases (HDACs) has been proven to manipulate development and exacerbation of cardiovascular diseases, including myocardial ischemia/reperfusion injury, cardiac hypertrophy, ventricular remodeling, and myocardial fibrosis. Inhibition of HDACs, especially class-I HDACs, is potent to the protection of ischemic myocardium after ischemia/reperfusion (I/R). Herein, we examine whether mocetinostat (MGCD0103, MOCE), a class-I selective HDAC inhibitor in phase-II clinical trial, shows cardioprotection under I/R in vivo and in vitro, if so, reveal its potential pharmacological mechanism to provide an experimental and theoretical basis for mocetinostat usage in a clinical setting. Human cardiac myocytes (HCMs) were exposed to hypoxia and reoxygenation (H/R), with or without mocetinostat treatment. H/R reduced mitochondrial membrane potential and induced HCMs apoptosis. Mocetinostat pretreatment reversed these H/R-induced mitochondrial damage and cellular apoptosis and upregulated CREB, p-CREB, and PGC-1α in HCMs during H/R. Transfection with small interfering RNA against PGC-1α or CREB abolished the protective effects of mocetinostat on cardiomyocytes undergoing H/R. In vivo, mocetinostat was demonstrated to protect myocardial injury posed by myocardial I/R via the activation of CREB and upregulation of PGC-1α. Mocetinostat (MGCD0103) can protect myocardium from I/R injury through mitochondrial protection mediated by CREB/PGC-1α pathway. Therefore, activation of the CREB/PGC-1α signaling pathway via the inhibition of Class-I HDACs may be a promising new therapeutic strategy for alleviating myocardial reperfusion injury.

Topics: Animals; Apoptosis; Benzamides; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Ischemia; Myocardial Reperfusion Injury; Myocytes, Cardiac; Protein Isoforms; Pyrimidines; Rats; Rats, Sprague-Dawley; Signal Transduction

Topics: Animals; Endothelial Cells; Hydroxyeicosatetraenoic Acids; Hypochlorous Acid; Ischemia; Mice; Neovascularization, Pathologic; Neutrophils; Peroxidase

Oxidative damage is critical in the pathogenesis of ovarian ischaemia/reperfusion (I/R) injury, and statins have been reported to exert antioxidant activity. However, the role of VCAM-1 and xanthine oxidase (XO)/uric acid (UA) in ovarian I/R injury is not known. Also, whether or not atorvastatin exerts antioxidant activity like other statins is unclear.. This study investigated the involvement of VCAM-1 and XO/UA in ovarian I/R injury and the likely protective role of atorvastatin.. Forty female Wistar rats were randomized into sham-operated, ischaemia, ischaemia/reperfusion (I/R), ischaemia and atorvastatin, and I/R and atorvastatin.. In comparison with the sham-operated group, atorvastatin blunted ischaemia and I/R-induced distortion of ovarian histoarchitecture and follicular degeneration. Also, atorvastatin alleviated ischaemia and I/R-induced rise in XO, UA, and malondialdehyde, which was accompanied by inhibition of ischaemia and I/R-induced reductions in reduced glutathione level, enzymatic antioxidant activities and increase in myeloperoxidase activity and TNF-α and IL-6 levels by atorvastatin treatment. Additionally, atorvastatin blocked ischaemia and I/R-induced increase in VCAM-1 expression, caspase 3 activity, 8-hydroxydeoxyguanosine level and ovarian DNA fragmentation index.. For the first time, this study revealed that atorvastatin-mediated downregulation of VCAM-1 and XO/UA/caspase 3 signaling averts oxidative injury, inflammation, and apoptosis induced by ovarian ischaemia/reperfusion injury.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antioxidants; Apoptosis; Atorvastatin; Caspase 3; Down-Regulation; Female; Glutathione; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Interleukin-6; Ischemia; Malondialdehyde; Oxidative Stress; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Tumor Necrosis Factor-alpha; Uric Acid; Vascular Cell Adhesion Molecule-1; Xanthine Oxidase

To investigate whether the mechanism underlying the anti-inflammatory effects of electroacupuncture (EA) at ST36 involves dopamine (DA) and its receptor and whether it is mediated by the vagus nerve in a rat model of intestinal ischaemia-reperfusion (I/R) injury.. Rats were subjected to gut ischaemia for 30 min and then received EA for 30 min with or without abdominal vagotomy or intraperitoneal administration of butaclamol (D1 receptor antagonist) or spiperone (D2 receptor antagonist). Plasma levels of DA and tumour necrosis factor (TNF)-α were assessed 1 or 4 h after reperfusion. Myeloperoxidase (MPO) activity and malondialdehyde (MDA) content in intestinal tissues were assessed using enzyme-linked immunosorbent assay (ELISA) kits. Intestinal tissue injury was assessed by observation of the pathological lesions and permeability to 4 kDa fluorescein isothiocyanate (FITC)-dextran.. EA significantly increased levels of DA and lowered levels of TNF-α. EA also inhibited intestinal levels of MPO and MDA and intestinal tissue injury and decreased intestinal permeability to FITC-dextran. Abdominal vagotomy and intraperitoneal administration of butaclamol (but not spiperone) inhibited the effects of EA.. These findings suggest that EA at ST36 could attenuate intestinal I/R-induced inflammatory injury and that the underlying mechanism may involve EA-induced increases in levels of DA, mediated by the vagus nerve and D1 receptors.

Topics: Acupuncture Points; Animals; Disease Models, Animal; Dopamine; Electroacupuncture; Humans; Intestines; Ischemia; Male; Peroxidase; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha

This study explored the role of the depressor arm of renin-angiotensin system (RAS) on ischemic renal injury (IRI)-associated cardio-hepatic sequalae under non-diabetic (ND) and diabetes mellitus (DM) conditions. Firstly, rats were injected with Streptozotocin (55 mg/kg i.p.) to develop DM. ND and DM rats underwent Bilateral IRI followed by 24 h of reperfusion. Further, ND and DM rats were subjected to AT2R agonist-Compound 21 (C21) (0.3 mg/kg/day, i.p.) or ACE2 activator- Diminazene Aceturate (Dize), (5 mg/kg/day, p.o.) per se or its combination therapy. As results, IRI caused cardio-hepatic injuries via altered oxidant/anti-oxidant levels, elevated inflammatory events, and altered protein expressions of ACE, ACE2, Ang II, Ang-(1-7) and urinary AGT. However, concomitant therapy of AT2R agonist and ACE2 activator exerts a protective effect in IRI-associated cardio-hepatic dysfunction as evidenced by inhibited oxidative stress, downregulated inflammation, and enhanced cardio-hepatic depressor arm of RAS under ND and DM conditions.

Topics: Acute Kidney Injury; Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme 2; Animals; Diabetes Mellitus, Experimental; Diminazene; Imidazoles; Ischemia; Kidney; Liver; Male; Myocardium; Oxidative Stress; Peptide Fragments; Peroxidase; Rats, Wistar; Receptor, Angiotensin, Type 2; Sulfonamides; Thiophenes

To investigate the effect of sevoflurane preconditioning on ischemia/reperfusion (I/R)-induced pulmonary/hepatic injury.. Fifty-one Wistar rats were randomly grouped into sham, I/R, and sevoflurane groups. After reperfusion, the structural change of the lung was measured by Smith score, the wet and dry weights (W/D) were determined, malondialdehyde (MDA) myeloperoxidase (MPO) content was determined colorimetrically and by fluorescence, respectively, and matrix metalloprotein-9 (MMP-9) mRNA was quantified by RT-PCR. Biopsy and morphological analyses were performed on liver tissue, activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined, and tumor necrosis factor-alpha (TNF-α) level was determined.. The sham group showed no changes in tissue structure. Structural lesions in the sevoflurane and I/R groups were mild and severe, respectively. Smith score, W/D, MDA, MPO, and MMP mRNA showed the same trend, and were increased in the I/R group and recovered in the sevoflurane group, compared with the sham group (both P<0.05). AST and ALT were significantly increased compared to the sham group (AST: 655±52.06 vs . 29±9.30 U/L; ALT: 693±75.56 vs . 37±6.71 U/L; P<0.05). In the sevoflurane group, AST and ALT levels were significantly decreased (464±47.71 and 516±78.84 U/L; P<0.001). TNF-α presented similar results.. The protection of lung and liver by sevoflurane may be mediated by inhibited leukocyte recruitment and MMP-9 secretion.

Topics: Alanine Transaminase; Anesthetics, Inhalation; Animals; Aspartate Aminotransferases; Disease Models, Animal; Ischemia; Ischemic Preconditioning; Liver; Lung; Male; Malondialdehyde; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Sevoflurane; Tumor Necrosis Factor-alpha

During chronic limb ischemia, oxidative damage and inflammation are described. Besides oxidative damage, the decrease of tissue oxygen levels is followed by several adaptive responses. The purpose of this study was to determine whether supplementation with N-acetylcysteine (NAC) is effective in an animal model of chronic limb ischemia. Chronic limb ischemia was induced and animals were treated once a day for 30 consecutive days with NAC (30mg/kg). After this time clinical scores were recorded and soleus muscle was isolated and lactate levels, oxidative damage and inflammatory parameters were determined. In addition, several mechanisms associated with hypoxia adaptation were measured (vascular endothelial growth factor - VEGF and hypoxia inducible factor - HIF levels, ex vivo oxygen consumption, markers of autophagy/mitophagy, and mitochondrial biogenesis). The adaptation to chronic ischemia in this model included an increase in muscle VEGF and HIF levels, and NAC was able to decrease VEGF, but not HIF levels. In addition, ex vivo oxygen consumption under hypoxia was increased in muscle from ischemic animals, and NAC was able to decrease this parameter. This effect was not mediated by a direct effect of NAC on oxygen consumption. Ischemia was followed by a significant increase in muscle myeloperoxidase activity, as well as interleukin-6 and thiobarbituric acid reactive substances species levels. Supplementation with NAC was able to attenuate inflammatory and oxidative damage parameters, and improve clinical scores. In conclusion, NAC treatment decreases oxidative damage and inflammation, and modulates oxygen consumption under hypoxic conditions in a model of chronic limb ischemia.

Topics: Acetylcysteine; Animals; Disease Models, Animal; Hindlimb; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Interleukin-6; Ischemia; Lactic Acid; Male; Muscle, Skeletal; Nitrates; Nitrites; Oxidative Stress; Oxygen; Oxygen Consumption; Peroxidase; Rats; Rats, Wistar; Thiobarbituric Acid Reactive Substances; Vascular Endothelial Growth Factor A

This study was aimed to investigate the protective effect of methylene blue against lung injury induced by reperfusion of ischemic hindlimb in a rat model.. Twenty-four healthy adult male Sprague-Dawley rats were equally randomized into three groups: sham (SM) group, ischemia reperfusion (IR) group, and methylene blue (MB) group. Rats in both IR and MB groups were subjected to 4 h of ischemia by clamping the left femoral artery and then followed by 4 h of reperfusion. Treatment with 1% methylene blue (50 mg/kg) was administrated intraperitoneally at 10 min prior to reperfusion in the MB group. After 4 h of reperfusion, malondialdehyde (MDA) level, myeloperoxidase (MPO), and superoxide dismutase (SOD) activities in lung tissue were detected; inflammatory cytokines, including IL-1. Hindlimb IR caused remarkable morphological abnormalities and edema in both muscle and lung tissues. SOD activity was decreased, both the MPO activity and MDA level in lung tissue, as well as IL-1. Methylene blue attenuates lung injury induced by hindlimb IR in rats, at least in part, by inhibiting oxidative stress.

Topics: Animals; Hindlimb; Interleukin-1beta; Interleukin-6; Ischemia; Lung Injury; Male; Methylene Blue; Oxidative Stress; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase

During transplant surgeries, the lung experiences an ischaemia-reperfusion (I/R)-induced damage identified as a significant cause of morbidity and mortality. However, the mechanisms by which I/R induces leucocyte accumulation and subsequent tissue damage in lung surgeries remain unknown. Therefore, the present study aims to assess the role of monocyte chemotactic protein 1 (MCP-1) and macrophage inflammatory protein 2 (MIP-2) in leucocyte chemotaxis related to lung injury secondary to I/R.. Six pigs were subjected to an orthotopic left caudal lobe lung transplantation with a subsequent 60-min graft reperfusion (Transplant group). In addition, six animals underwent to sham surgery (Sham Group). Plasma samples and lung biopsies were collected before the beginning of pneumonectomy, before starting the reperfusion, and 30 min and 60 min after the beginning of the reperfusion. Plasma levels of intercellular adhesion molecule 1 (ICAM-1) and lung expressions of MCP-1, MIP-2, myeloperoxidase (MPO), and lung oedema were measured.. Lung I/R caused substantial damage observed as pulmonary oedema. The oedema was evident after the ischemic insult and increased after reperfusion. After reperfusion, increased levels of MPO were observed which suggests an activation and infiltration of neutrophils into the lung tissue. After 30 min of reperfusion, MCP-1, MIP-2, and ICAM-1 levels were significantly increased compared to prepneumonectomy levels (p < 0.05) and a further increase was observed after 60 min of reperfusion (p < 0.05).. The present study demonstrates that activated neutrophils, as well as MCP-1, MIP-2, and ICAM-1, are involved in inflammatory response induced by ischaemia-reperfusion-induced lung injury.

Topics: Acute Lung Injury; Animals; Chemokine CCL2; Chemokine CXCL2; Ischemia; Lung Transplantation; Peroxidase; Pneumonectomy; Pulmonary Edema; Reperfusion; Reperfusion Injury; Swine

Cholecystokinin (CCK), as a gastrointestinal hormone, has an important protective role against sepsis or LPS-induced endotoxic shock. We aim to address the role of CCK in hepatic ischemia followed by reperfusion (I/R) injury.. A murine model of 60min partial hepatic ischemia followed by 6h of reperfusion was used in this study. CCK and CCKAR Levels in blood and liver were detected at 3h, 6h, 12h and 24h after reperfusion. Then the mice were treated with CCK or proglumide, a nonspecific CCK-receptor (CCK-R) antagonist. Mice were randomly divided into four groups as follows: (1) sham group, in which mice underwent sham operation and received saline; (2) I/R group, in which mice were subjected to hepatic I/R and received saline; (3) CCK group, in which mice were subjected to hepatic I/R and treated with CCK (400μg/kg); (4) proglumide group (Pro), in which mice underwent hepatic I/R and treated with proglumide (3mg/kg); CCK and proglumide were administrated via tail vein at the moment of reperfusion. Serum AST (sAST) and serum ALT (sALT) were determined with a biochemical assay and histological analysis were performed with hematoxylin-eosin (H&E). Cytokines (IL-1β, IL-6, IL-10, TNF-α) expressions in blood were determined with enzyme-linked immunosorbent assay (ELISA). The MPO (myeloperoxidase) assay were used to measure neutrophils' infiltration into the liver. The apoptotic index (TUNEL-positive cell number/total liver cell number×100%) was calculated to assess hepatocelluar apoptosis. Finally, activation of NF-κB and phosphor-p38 expression in liver homogenates were analyzed with Western Blot (WB).. Our findings showed that 1) CCK and CCK-AR were upregulated in our experimental model over time; 2) Treatment with CCK decreased sAST/sALT levels, inflammatory hepatic injury, neutrophil influx and hepatocelluar apoptosis, while proglumide aggravated hepatic injury.. These findings support our hypothesis and suggest that CCK played a positive role in the ongoing inflammatory process leading to liver I/R injury.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Cholecystokinin; Cytokines; Ischemia; Liver; Male; Mice, Inbred C57BL; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Peroxidase; Receptor, Cholecystokinin A; Receptor, Cholecystokinin B; Reperfusion Injury

The aim of the current study was to investigate whether intestinal ischemic preconditioning (IP) reduces damage to the liver during hepatic ischemia reperfusion (IR). Sprague Dawley rats were used to model liver IR injury, and were divided into the sham operation group (SO), IR group and IP group. The results indicated that IR significantly increased Bax, caspase 3 and NF‑κBp65 expression levels, with reduced expression of Bcl‑2 compared with the IP group. Compared with the IR group, the levels of AST, ALT, MPO, MDA, TNF‑α and IL‑1 were significantly reduced in the IP group. Immunohistochemistry for Bcl‑2 and Bax indicated that Bcl‑2 expression in the IP group was significantly increased compared with the IR group. In addition, IP reduced Bax expression compared with the IR group. The average liver injury was worsened in the IR group and improved in the IP group, as indicated by the morphological evaluation of liver tissues. The present study suggested that IP may alleviates apoptosis, reduce the release of pro‑inflammatory cytokines, ameloriate reductions in liver function and reduce liver tissue injury. To conclude, IP provided protection against hepatic IR injury.

Topics: Alanine Transaminase; Animals; Apoptosis Regulatory Proteins; Aspartate Aminotransferases; Interleukin-1beta; Intestines; Ischemia; Ischemic Preconditioning; Liver; Male; Malondialdehyde; Peroxidase; Rats, Sprague-Dawley; Reperfusion Injury; Tumor Necrosis Factor-alpha

The aim of this study was to observe the therapeutic effect of insulin-loaded linear poly(ethylene glycol)-brush-like poly(l-lysine) block copolymer poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(l-lysine)) (PEG-b-(PELG-g-PLL) on renal ischemia/reperfusion-induced lung injury through downregulating hypoxia-inducible factor (HIF) as compared to free insulin. Sprague Dawley rats were pretreated with 30 U/kg insulin or insulin/PEG-b-(PELG-g-PLL) complex, and then subjected to 45 minutes of ischemia and 24 hours of reperfusion. The blood and lungs were collected, the level of serum creatinine and blood urea nitrogen were measured, and the dry/wet lung ratios, the activity of superoxide dismutase and myeloperoxidase, the content of methane dicarboxylic aldehyde and tumor necrosis factor-α, and the expression of HIF-1α and vascular endothelial growth factor (VEGF) were measured in pulmonary tissues. Both insulin and insulin/PEG-b-(PELG-g-PLL) preconditioning improved the recovery of renal function, reduced pulmonary oxidative stress injury, restrained inflammatory damage, and downregulated the expression of HIF-1α and VEGF as compared to ischemia/reperfusion group, while insulin/PEG-b-(PELG-g-PLL) significantly improved this effect.

Topics: Animals; Blood Urea Nitrogen; Cell Survival; Creatinine; Hypoxia-Inducible Factor 1, alpha Subunit; Insulin; Ischemia; Kidney; Lung; Lung Injury; Male; Malondialdehyde; Molecular Weight; Organ Size; Particle Size; Peroxidase; Polyethylene Glycols; Polylysine; Proton Magnetic Resonance Spectroscopy; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

This study aimed to investigate the protection of ischemic preconditioning (IPreC), ischemic postconditioning (IPostC) and combined treatment on ischemia reperfusion injury (IRI) of testis. A rabbit testicular ischemia reperfusion (IR) model was established with determining of rabbit serum testosterone, nitric oxide (NO), malondialdehyde (MDA), protein carbonyl (PC), superoxide dismutase (SOD), myeloperoxidase (MPO), glutathione peroxidase (GSH-Px), and tissues pathology. After IR, the NO, MDA, PC, SOD, MPO, and GSH-Px expression significantly increased in torsive testis, and significantly decreased after IPreC, IPostC, and combined treatment in torsive testis when compared to contralateral testis. In torsive testis, testicular tissues was severely damaged with spermatogenic cells disappearing, and were filled with light eosin edema liquid. Cell apoptosis index significantly increased, and the ratio of Bcl-2/Bax significantly decreased. After IPreC, IPostC, and combined treatment, testicular tissues were restored to normal, cell apoptosis index significantly decreased, and the ratio of Bcl-2/Bax significantly increased. It indicates that IPreC, IPostC, and combined treatment has an obvious protective effect on testicular IRI, by decreasing the oxidative stress index and cell apoptosis, provides a significant reference for the treatment of testicular torsion induced infertility, and exhibits a great value in clinical applications.

Topics: Animals; Apoptosis; Enzyme-Linked Immunosorbent Assay; Glutathione Peroxidase; Ischemia; Ischemic Postconditioning; Ischemic Preconditioning; Male; Malondialdehyde; Nitric Oxide; Oxidative Stress; Peroxidase; Protein Carbonylation; Rabbits; Spermatic Cord Torsion; Spermatogenesis; Superoxide Dismutase; Testis; Testosterone

The widespread application of tourniquets has reduced battlefield mortality related to extremity exsanguinations. Tourniquet-induced ischemia-reperfusion injury (I/R) can contribute to muscle loss. Postischemic conditioning (PostC) confers protection against I/R in cardiac muscle and skeletal muscle flaps. The objective of this study was to determine the effect of PostC on extremity muscle viability in an established rat hindlimb tourniquet model.. Rats were randomly assigned to PostC-1, PostC-2, or no conditioning ischemic groups (n = 10 per group). Postischemic conditioning, performed immediately after tourniquet release, consisted of four 15-second cycles (PostC-1) or eight 15-second cycles (PostC-2) of alternating occlusion and perfusion of hindlimbs. Twenty-four hours later, muscles were excised. The primary end points were muscle edema and viability; secondary end points were histologic and markers of oxidative stress.. Ischemia-reperfusion injury decreased viability in all tourniquet limbs, but viability was not improved in either PostC group. Likewise, I/R resulted in substantial muscle edema that was not reduced by PostC. The predominant histologic feature was necrosis, but no significant differences were found among groups. Markers of oxidative stress were increased similarly among groups after I/R, although myeloperoxidase activity was significantly increased only in the no conditioning ischemic group. A protective effect from PostC was not observed in our model suggesting that PostC was not effective in reducing I/R skeletal muscle injury or any benefits of PostC were not sustained for 24 hours when tissues were assessed.. These negative findings are pertinent as the military investigates different strategies to extend the safe time for tourniquet application.

Topics: Animals; Edema; Glutathione; Hindlimb; Ischemia; Ischemic Postconditioning; Lipid Peroxidation; Male; Muscle, Skeletal; Muscular Diseases; Nitric Oxide; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Thiobarbituric Acid Reactive Substances; Tissue Survival; Tourniquets

Liver ischemia reperfusion (IR) injury triggers a systemic inflammatory response and is the main cause of organ dysfunction and adverse postoperative outcomes after liver surgery. Pentoxifylline (PTX) and hypertonic saline solution (HTS) have been identified to have beneficial effects against IR injury. This study aimed to investigate if the addition of PTX to HTS is superior to HTS alone for the prevention of liver IR injury.. Male Wistar rats were allocated into three groups. Control rats underwent 60 minutes of partial liver ischemia, HTS rats were treated with 0.4 mL/kg of intravenous 7.5% NaCl 15 minutes before reperfusion, and HPTX group were treated with 7.5% NaCl plus 25 mg/kg of PTX 15 minutes before reperfusion. Samples were collected after reperfusion for determination of ALT, AST, TNF-alpha, IL-6, IL-10, mitochondrial respiration, lipid peroxidation, pulmonary permeability and myeloperoxidase.. HPTX significantly decreased TNF-alpha 30 minutes after reperfusion. HPTX and HTS significantly decreased ALT, AST, IL-6, mitochondrial dysfunction and pulmonary myeloperoxidase 4 hours after reperfusion. Compared with HTS only, HPTX significantly decreased hepatic oxidative stress 4 hours after reperfusion and pulmonary permeability 4 and 12 hours after reperfusion.. This study showed that PTX added the beneficial effects of HTS on liver IR injury through decreases of hepatic oxidative stress and pulmonary permeability.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Capillary Permeability; Disease Models, Animal; Drug Synergism; Evans Blue; Free Radical Scavengers; Interleukin-1; Interleukin-10; Ischemia; Lipid Peroxidation; Liver Diseases; Lung; Male; Oxidative Stress; Pentoxifylline; Permeability; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Saline Solution, Hypertonic; Tumor Necrosis Factor-alpha

Hepatic injury induced by ischemia-reperfusion (I/R) after transplantation or lobectomy is a major clinical problem. The potential benefit of remifentanil in these hepatic surgeries remains unknown. The current study investigated whether remifentanil protects the liver against I/R injury in a rat model and whether the underlying mechanism involves the modulation of interleukin (IL)-18 signaling.. Male Sprague-Dawley rats were subjected to 45 minutes of partial hepatic ischemia followed by 6 hours of reperfusion. Then, they received an intravenous saline or remifentanil (0.4, 2, or 10 μg/kg per minute) infusion from 30 minutes before ischemia until the end of ischemia with or without previous administration of naloxone, a nonselective opioid receptor antagonist. Serum aminotransferase, hepatic morphology, and hepatic neutrophil infiltration were analyzed. The expression of hepatic IL-18; IL-18-binding protein (BP); and key cytokines downstream of IL-18 signaling were measured.. Remifentanil significantly decreased serum aminotransferase levels and profoundly attenuated the liver histologic damages. Liver I/R injury increased the expression of both hepatic IL-18 and IL-18BP. Although remifentanil pretreatment significantly decreased I/R-induced IL-18 expression, it further upregulated IL-18BP levels in liver tissues. The I/R-induced increases of hepatic interferon-γ, tumor necrosis factor-α and IL-1β expression, and neutrophil infiltration were also significantly reduced by remifentanil. Naloxone inhibited the remifentanil-induced downregulation of IL-18, but not the elevation of IL-18BP, and significantly attenuated its protective effects on liver I/R injury.. Remifentanil protects the liver against I/R injury. Modulating the hepatic IL-18/IL-18BP balance and inhibiting IL-18 signaling mediate, at least in part, the hepatoprotective effects of remifentanil.

Topics: Analgesics, Opioid; Animals; Gene Expression Regulation; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Interferon-gamma; Interleukin-18; Ischemia; Liver; Male; Naloxone; Neutrophils; Peroxidase; Piperidines; Rats; Rats, Sprague-Dawley; Remifentanil; Reperfusion Injury; Signal Transduction; Transaminases

In patients with acute kidney injury (AKI) mortality remains high, despite the fact that the patients are treated with continuous renal replacement therapy. The interaction between the kidney and the immune system might explain the high mortality observed in AKI. In order to elucidate the interaction between the kidney and immune system we developed a two-hit model of AKI and endotoxemia. Our hypothesis was that ischemia/reperfusion (I/R) of the kidney simultaneously with endotoxemia would generate a more extensive inflammatory response compared to I/R of the hind legs. Our expectation was that elevated levels of cytokines would be found in both blood and in organs distant to the kidneys. Forty mice were divided into five groups. The mice were subjected to the following operations: A: Sham only, no lipopolysaccharide (LPS); B: I/R of both kidneys + LPS; C: LPS only; D: Nephrectomy + LPS; E: I/R of both hind legs + LPS. In groups B and E, I/R times were identical. All mice were kept alive for 24 h and then sacrificed. Levels of interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor-α were measured in the blood. The activity of myeloperoxidase (MPO) in lungs, kidneys, and liver was evaluated as an indirect measurement of accumulation of granulocytes. In this study, significantly higher amount of IL-6 and IL-10 in the plasma was observed following renal I/R compared to hind leg I/R. The elevated levels of cytokine in plasma were observed following nephrectomy and endotoxemia. The neutrophil infiltration of distant organs measured by the levels of MPO in the lung and liver also showed a significantly higher level in renal I/R compared to hind leg I/R. Renal I/R is associated with a more pronounced inflammatory response in blood and distant organs. The high cytokine levels measured following nephrectomy might be explained by compromised elimination of cytokines by the kidney in AKI.

Topics: Acute Kidney Injury; Animals; Disease Models, Animal; Endotoxemia; Inflammation; Interleukin-10; Interleukin-1beta; Interleukin-6; Ischemia; Kidney; Lipopolysaccharides; Liver; Lung; Male; Mice; Mice, Inbred C57BL; Nephrectomy; Neutrophil Infiltration; Peroxidase; Reperfusion Injury; Tumor Necrosis Factor-alpha

In human beings or animals, ischemia/reperfusion (I/R) injury of the liver may occur in many clinical conditions, such as circulating shock, liver transplantation and surgery and several other pathological conditions. I/R injury has a complex pathophysiology resulting from a number of contributing factors. Therefore, it is difficult to achieve effective treatment or protection by individually targeting the mediators. This study aimed at studying the effects of local somatothermal stimulation preconditioning on the right Qimen (LR14) on hepatic I/R injury in rats.. Eighteen male Sprague-Dawley rats were randomly divided into three groups. The rats were preconditioned with thermal tolerance study, which included one dose of local somatothermal stimulation (LSTS) on right Qimen (LR14) at an interval of 12 h, followed by hepatic ischemia for 60 min and then reperfusion for 60 min. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) have been used to assess the liver functions, and liver tissues were taken for the measurements such as malondialdehyde (MDA), glutathione (GSH), catalase (CAT), superoxidase dismutase (SOD), and myeloperoxidase (MPO).. The results show that the plasma ALT and AST activities were higher in the I/R group than in the control group. In addition, the plasma ALT and AST activities decreased in the groups that received LSTS. The hepatic SOD levels reduced significantly by I/R injury. Moreover, the hepatic MPO activity significantly increased by I/R injury while it decreased in the groups given LSTS.. Our findings show that LSTS provides a protective effects on the liver from the I/R injury. Therefore, LSTS might offer an easy and inexpensive intervention for patients who have suffered from I/R of the liver especially in the process of hepatotomy and hepatic transplantation.

Topics: Acupuncture Points; Alanine Transaminase; Animals; Aspartate Aminotransferases; Catalase; Glutathione; Hyperthermia, Induced; Ischemia; Ischemic Preconditioning; Liver; Liver Diseases; Male; Malondialdehyde; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase; Temperature

Preeclampsia is associated with oxidative stress, which is suspected to play a role in hypertension, placental ischemia, and fetal demise associated with the disease. Various cellular sources of oxidative stress, such as neutrophils, monocytes, and CD4(+) T cells have been suggested as culprits in the pathophysiology of preeclampsia. The objective of this study was to examine a role of circulating and placental CD4(+) T cells in oxidative stress in response to placental ischemia during pregnancy. CD4(+) T cells and oxidative stress were measured in preeclamptic and normal pregnant women, placental ischemic and normal pregnant rats, and normal pregnant recipient rats of placental ischemic CD4(+) T cells. Women with preeclampsia had significantly increased circulating (P=0.02) and placental CD4(+) T cells (P=0.0001); lymphocyte secretion of myeloperoxidase (P=0.004); and placental reactive oxygen species (P=0.0004) when compared with normal pregnant women. CD4(+) T cells from placental ischemic rats cause many facets of preeclampsia when injected into normal pregnant recipient rats on gestational day 13. On gestational day 19, blood pressure increased in normal pregnant recipients of placental ischemic CD4(+) T cells (P=0.002) compared with that in normal pregnant rats. Similar to preeclamptic patients, CD4(+) T cells from placental ischemic rats secreted significantly more myeloperoxidase (P=0.003) and induced oxidative stress in cultured vascular cells (P=0.003) than normal pregnant rat CD4(+)Tcells. Apocynin, a nicotinamide adenine dinucleotide phosphate inhibitor, attenuated hypertension and all oxidative stress markers in placental ischemic and normal pregnant recipient rats of placental ischemic CD4(+)Tcells (P=0.05). These data demonstrate an important role for CD4(+) T cells in mediating another factor, oxidative stress, to cause hypertension during preeclampsia.

Topics: Acetophenones; Adolescent; Adult; Animals; Antioxidants; CD4-Positive T-Lymphocytes; Cells, Cultured; Disease Models, Animal; Female; Humans; Hypertension; Ischemia; Muscle, Smooth, Vascular; Oxidative Stress; Peroxidase; Placenta; Pre-Eclampsia; Pregnancy; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Young Adult

Ischemia-reperfusion (IR) has been reported to be associated with augmented reactive oxygen radicals and cytokines. Currently, we aimed to examine the influence of fluoxetine, which is already used as a preoperative anxiolytic, in the context of IR induced by occlusion of infrarenal abdominal aorta (60 min of ischemia) and its effects on renal oxidative status, inflammation, renal function, and cellular integrity in reperfusion (120 min post-ischemia). Male rats were randomly assigned as control, IR, and pretreated groups. The pretreated group animals received fluoxetine (20 mg/kg, i.p.) once daily for 3 days. Renal tissue oxidative stress, myeloperoxidase activity, proinflammatory cytokines (tumor necrosis factor-α, interleukin-1β, interleukin-6), histology, and function were assessed. As an anti-inflammatory cytokine, interleukin-10 was also assessed. IR led to a significant increase in lipid hydroperoxide, malondialdehyde, and pro-oxidant antioxidant balance and decrease in superoxide dismutase activity and ferric reducing/antioxidant power level (p < 0.05), but fluoxetine was able to restore these parameters. High concentrations of tumor necrosis factor-α, interleukin-1β, interleukin-6, and myeloperoxidase activity caused by IR were significantly decreased in kidney tissue with fluoxetine. In addition, interleukin-10 levels were high in fluoxetine pretreated group. IR resulted in disrupted cellular integrity, infiltration of tissue with leukocytes, and decreased serum creatinine-urea levels (p < 0.05). Fluoxetine significantly restored impaired redox balance and inflammation parameters of rats subjected to IR to baseline values. This beneficial effect of fluoxetine on redox balance might be addressed to an improvement in renal function.

Topics: Acute Kidney Injury; Animals; Anti-Inflammatory Agents; Antioxidants; Cytokines; Drug Evaluation, Preclinical; Fluoxetine; Ischemia; Kidney; Male; Oxidation-Reduction; Oxidative Stress; Peroxidase; Rats, Sprague-Dawley; Reperfusion Injury

The aim of this study was to compare the ischaemia and reperfusion phases of two tourniquet application models (Group 1: expressing the blood by a sterile rubber bandage and Group 2: elevation of the limb for several minutes) using an analysis of ischaemia/reperfusion parameters and blood pH. Sixteen New Zealand rabbits were used. Muscle samples were extracted from the triceps surae; at phase A (baseline: just before tourniquet application), phase B (ischaemia: 3h after tourniquet inflation) and phase C (2h after tourniquet deflation). Nitrite, nitrate, reduced glutathione, myeloperoxidase, malondyaldehyde were measured in the samples. Blood pH was also measured at each phase. Group 2 had significantly decreased nitrite (p=0.007) and nitrate (p=0.01) levels compared to Group 1 while passing from phase A to phase B. The pH decrease through the phases was significant within Group 1 (p=0.006) and was not significant within Group 2 (p=0.052). Lower levels of NO metabolites nitrate and nitrite, result from tourniquet use with incomplete venous blood expression by elevation. Also, with this technique severe acidosis is less likely to occur than when a tourniquet is used with expression of the venous blood by rubber bandage. These findings may help in the decision of which tourniquet technique is to be used for potentially long operations which may exceed 2h.

Topics: Acidosis; Animals; Biomarkers; Ischemia; Malondialdehyde; Muscle, Skeletal; Nitrates; Nitrites; Orthopedic Procedures; Peroxidase; Rabbits; Reperfusion Injury; Tourniquets

Perinatal asphyxia is an important cause of injury to fetal tissues such as the brain, heart, liver and gastrointestinal system. Fetal skin has also been shown to be vulnerable to intrauterine injury after intrauterine ischaemia/reperfusion (I/R) injury.. To examine the effect of dexamethasone on fetal skin in intrauterine I/R injury in rats.. The response of rat fetal skin to I/R injury and maternal dexamethasone treatment were assessed by determining thiobarbituric acid reactive substances (TBARS), and myeloperoxidase (MPO) and nitric oxide (NO) metabolites. We also examined the ultrastructural changes of fetal skin. Bilateral utero-ovarian artery clamping was performed to produce ischaemia for 30 min in rats at day 19 of pregnancy, and reperfusion was achieved by removing the clamps for 60 min before fetal tissue was collected. The treatment group was given dexamethasone intraperitoneally 20 min before I/R was performed.. TBARS, MPO and NO all increased significantly in fetal rat skin after I/R injury. Levels of TBARS, MPO and NO were significantly lower in the dexamethasone-treated group than in the I/R-only group. I/R injury produced ultrastructural damage in the epidermis. Oedema and mitochondrial damage were less severe in the dexamethasone-treated group.. Maternal treatment with dexamethasone may have a protective effect on fetal skin in cases of I/R injury.

Topics: Animals; Anti-Inflammatory Agents; Dexamethasone; Disease Models, Animal; Female; Fetal Diseases; Ischemia; Nitric Oxide; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Skin

To assess the protective effect of exogenous hydrogen sulfide (H₂S) against myocardial injury after skeletal muscle ischemia/reperfusion (IR) in rats and explore the mechanism.. Thirty-one Wistar rats were randomized into normal control (n=8), IR group (n=8, with a 4-h reperfusion following a 4-h ischemia of the bilateral hindlimbs induced using a tourniquet), NaHS group (n=8, with IR and intraperitoneal injection of 14 µmol/kg NaHS), and DL-propargylglycine (PPG) group (n=7, with IR and intraperitoneal injection of 50 mg/kg PPG). The plasma levels of CK-MB and the levels of MPO, TNF-α, MDA, T-SOD, and CuZn-SOD in the plasma and myocardial tissues were measured. The expression of TNF-α in the myocardium was examined using immunohistochemistry. RESULTS Skeletal muscle IR induced significantly increased plasma CK-MB level (P<0.05) and the levels of MPO, TNF-α, and MDA in the plasma and myocardium, and significantly decreased plasma and myocardial levels of T-SOD and CuZn-SOD (P<0.05). NaHS treatment significantly decreased plasma CK-MB level (P<0.05), reduced plasma and myocardial levels of MPO, TNF-α, and MDA, and increased plasma and myocardial T-SOD and CuZn-SOD in rats with IR (P<0.05), whereas PPG treatment did not produce any obvious responses (P>0.05). Immunohistochemistry showed an obviously reduced expression of TNF-α in the myocardium in rats with NaHS treatment compared with those in IR group.. H₂S treatment can alleviate myocardial injury induced by skeletal muscle IR in rats by inhibiting the inflammatory cytokines and oxidative stress.

Topics: Animals; Creatine Kinase, MB Form; Disease Models, Animal; Hydrogen Sulfide; Ischemia; Male; Malondialdehyde; Muscle, Skeletal; Myocardial Reperfusion Injury; Oxidative Stress; Peroxidase; Rats; Rats, Wistar; Superoxide Dismutase; Tumor Necrosis Factor-alpha

To explore the role of myeloperoxidase (MPO) and tumor necrosis factor-α (TNF-α) in myocardial injury induced by hind-limb ischemia-reperfusion (IR) in rats.. Rat models of bilateral hindlimb IR established using a tourniquet were randomized into 9 groups, including a normal control group normal, 2 ischemic groups with hindlimb ischemia for 2 and 4 h, and 6 IR groups with a 4-h ischemia followed by reperfusion for 0.5, 2, 4, 6, 12, and 24 h. The plasma and myocardial levels of MPO and TNF-α in each group were measured, and the myocardial expression of TNF-α was determined with immunohistochemistry.. Compared with the normal control group, the rats with a 2-h ischemia showed significantly increased levels of MPO and TNF-α in the plasma and myocardium. Compared with those in rats with a 4-h ischemia, the plasma and myocardial MPO levels increased significantly at 0.5 and 2 h of reperfusion, respectively; the plasma TNF-α level increased significantly at 4 h of reperfusion and myocardial TNF-α level decreased obviously at 12 h; plasma levels of MPO and TNF-α both significantly decreased at 24 h. The plasma MPO and TNF-α and myocardial TNF-α reached the peak levels at 4 h of reperfusion, and the peak myocardial MPO level occurred at 6 h. Immunohistochemistry showed that TNF-α positivity moderately increased after hindlimb ischemia, and further increased at 4 h of reperfusion but obviously reduced at 24 h.. The activation of systemic and local neutrophils and inflammatory cytokines may play an important role in myocardial injury induced by hindlimb IR in rats.

Topics: Animals; Disease Models, Animal; Hindlimb; Ischemia; Male; Myocardium; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Tumor Necrosis Factor-alpha

Stabilizing mast cells (MCs) can either inhibit or augment inflammation; however, how improved therapeutic benefits against small intestinal ischemia-reperfusion injury (IIRI) can be achieved by stabilizing MCs remains to be elucidated. The present study was designed to evaluate different treatments with cromolyn sodium (CS, an MC stabilizer), which was administrated either prior to ischemia or after reperfusion. Kunming mice were randomized into a sham-operated group (SH), a sole IIR group (M), in which mice were subjected to 30 min superior mesenteric artery occlusion followed by 3 day or 3 h reperfusion, or IIR, treated with CS 15 min prior to ischemia or 15 min after reperfusion in the PreCr and PostCr groups. The survival rate and Chiu's scores were evaluated. The levels of ET-1, histamine, TNF-α and IL-6, and expression of MC protease 7 (MCP7), MC counts and myeloperoxidase (MPO) activity were quantified. IIR resulted in severe injury as demonstrated by significant increases in mortality and injury score. IIR also led to substantial elevations in the levels of ET-1, histamine, TNF-α and IL-6, expression of MCP7, MC counts and MPO activities (P<0.05, M vs. SH groups). All biochemical changes were markedly reduced in the PostCr group (P<0.05, PostCr vs. M groups), whereas pretreatment of IIR mice with CS prior to ischemia exhibited no changes of ET-1 levels, injury score and inflammation (P>0.05, PreCr vs. M groups). In conclusion, administration of CS after reperfusion, but not prior to ischemia, attenuates IIRI by downregulating ET-1 and suppressing sustained MC activation.

Topics: Animals; Anti-Asthmatic Agents; Cromolyn Sodium; Down-Regulation; Endothelin-1; Histamine; Interleukin-6; Intestinal Mucosa; Ischemia; Male; Mast Cells; Mice; Peptide Hydrolases; Peroxidase; Reperfusion Injury; Severity of Illness Index; Survival Rate; Tumor Necrosis Factor-alpha

Polyunsaturated fatty acid (PUFA) metabolites are bioactive autoacoids that play an important role in the pathogenesis of a vast number of pathologies, including gut diseases. The induction and the resolution of inflammation depend on PUFA metabolic pathways that are favored. Therefore, understanding the profile of n-6 (eicosanoids)/n-3 (docosanoids) PUFA-derived metabolites appear to be as important as gene or protein array approaches, to uncover the molecules potentially implicated in inflammatory diseases. Using high sensitivity liquid chromatography tandem mass spectrometry, we characterized the tissue profile of PUFA metabolites in an experimental model of murine intestinal ischemia reperfusion. We identified temporal and quantitative differences in PUFA metabolite production, which correlated with inflammatory damage. Analysis revealed that early ischemia induces both pro-inflammatory and anti-inflammatory eicosanoid production. Primarily, LOX- (5/15/12/8-HETE, LTB4, LxA4) and CYP- (5, 6-EET) metabolites were produced upon ischemia, but also PGE3, and PDx. This suggests that different lipids simultaneously play a role in the induction and counterbalance of ischemic inflammatory response from its onset. COX-derived metabolites were more present from 2 to 5 hours after reperfusion, fitting with the concomitant inflammatory peaks. All metabolites were decreased 48 hours post-reperfusion except for to the pro-resolving RvE precursor 18-HEPE and the PPAR-γαμμα agonist, 15d-PGJ2. Data obtained through the pharmacological blockade of transient receptor potential vanilloid-4, which can be activated by 5, 6-EET, revealed that the endogenous activation of this receptor modulates post-ischemic intestinal inflammation. Altogether, these results demonstrate that different lipid pathways are involved in intestinal ischemia-reperfusion processes. Some metabolites, which expression is severely changed upon intestinal ischemia-reperfusion could provide novel targets and may facilitate the development of new pharmacological treatments.

Topics: Animals; Chromatography, Liquid; Cytokines; Fatty Acids, Unsaturated; Inflammation; Inflammation Mediators; Intestine, Small; Ischemia; Male; Mice; Mice, Inbred C57BL; Peroxidase; Reperfusion Injury; Survival Rate; Tandem Mass Spectrometry

Acute intestinal ischaemia/reperfusion injury (AII/R) is an adaptive physiologic response during critical illness, involving mesenteric vasoconstriction and hypoperfusion. Prevention of AII/R in high risk patient populations would have a significant impact on morbidity and mortality. The purpose of this study was to investigate the protective effects of VSL#3 probiotic treatment in a murine model of AII/R. Adult 129/SvEv mice were subjected to an experimental AII/R model using superior mesenteric artery occlusion. Animals were pre-treated with either three days or two weeks of VSL#3 probiotics. Local tissue injury markers were assessed by levels of myeloperoxidase and activation of nuclear factor kappa B (NFкB). Systemic and local cytokines, including interleukin (IL)-1β, IL- 10, TNFα, and interferon gamma were measured by ELISA and multiplex fluorescent detection. VSL#3 probiotics reduced local tissue inflammation and injury due to AII/R. A two-week course of VSL#3 was more effective than a shorter three-day course. The reduction in local inflammation from the two-week course of VSL#3 is correlated to a significant reduction in levels of active IL-1β, and tissue levels of myeloperoxidase. Levels of active NFкB were significantly elevated in the vehicle-fed AII/R mice, corroborating with tissue inflammation, which were attenuated by VSL#3 administrations. VSL#3 did not cause any systemic inflammation or lung injury. VSL#3 probiotics are effective in reducing local tissue injury from AII/R by down-regulating pro-inflammatory mediators and immune cell recruitment. This study highlights a potential role for VSL#3 in management of patients at high risk for AII/R.

Topics: Animals; Cytokines; Disease Models, Animal; Intestinal Diseases; Ischemia; Mice; NF-kappa B; Peroxidase; Probiotics; Reperfusion Injury; Treatment Outcome

The aim of this study was to analyze the effects of cryotherapy on the biochemical and morphological changes in ischemic and reperfused (I/R) gastrocnemius muscle of rats. Forty male Wistar rats were divided into control and I/R groups, and divided based on whether or not the rats were submitted to cryotherapy. Following the reperfusion period, biochemical and morphological analyses were performed. Following cryotherapy, a reduction in thiobarbituric acid-reactive substances and dichlorofluorescein oxidation levels were observed in I/R muscle. Cryotherapy in I/R muscle also minimized effects such as decreased cellular viability, levels of non-protein thiols and calcium ATPase activity as well as increased catalase activity. Cryotherapy also limited mitochondrial dysfunction and decreased the presence of neutrophils in I/R muscle, an effect that was corroborated by reduced myeloperoxidase activity in I/R muscle treated with cryotherapy. The effects of cryotherapy are associated with a reduction in the intensity of the inflammatory response and also with a decrease in mitochondrial dysfunction.

Topics: Analysis of Variance; Animals; Biomarkers; Cell Survival; Cryotherapy; Disease Models, Animal; Ischemia; Male; Mitochondria, Muscle; Muscle, Skeletal; Oxidative Stress; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury

Proteases and proteinase-activated receptor (PAR) activation are involved in several intestinal inflammatory conditions. We hypothesized that serine proteases and PAR activation could also modulate the intestinal injury induced by ischemia-reperfusion (I-R). C57Bl/6 mice were subjected to 90 minutes of intestinal ischemia followed or not by reperfusion. Sham-operated animals served as controls. After ischemia, plasma and tissue serine protease activity levels were increased compared to the activity measured in plasma and tissues from sham-operated mice. This increase was maintained or further enhanced after 2 and 5 hours of reperfusion, respectively. Trypsin (25 kDa) was detected in tissues both after ischemia and 2 hours of reperfusion. Treatment with FUT-175 (10 mg/kg), a potent serine protease inhibitor, increased survival after I-R, inhibited tissue protease activity, and significantly decreased intestinal myeloperoxidase (MPO) activity and chemokine and adhesion molecule expression. We investigated whether serine proteases modulate granulocyte recruitment by a PAR-dependent mechanism. MPO levels and adhesion molecule expression were significantly reduced in I-R groups pre-treated with the PAR(1) antagonist SCH-79797 (5 mg/kg) and in Par(2)(-/-)mice, compared, respectively, to vehicle-treated group and wild-type littermates. Thus, increased proteolytic activity and PAR activation play a pathogenic role in intestinal I-R injury. Inhibition of PAR-activating serine proteases could be beneficial to reduce post-ischemic intestinal inflammation.

Topics: alpha-Macroglobulins; Animals; Benzamidines; Chemokines; Cysteine Proteinase Inhibitors; Granulocytes; Guanidines; Intestine, Small; Ischemia; Leucine; Male; Mice; Mice, Inbred C57BL; Peroxidase; Protease Inhibitors; Receptor, PAR-1; Receptor, PAR-2; Reperfusion Injury; Serine Proteinase Inhibitors; Trypsin

Studies assessing the effects of partial-hepatic ischemia/reperfusion (I/R) injury focused on the damage to the ischemic-lobe, whereas few data are available on non-ischemic lobe. This study investigated whether acute liver I/R does affect non-ischemic lobe function via modulation of extracellular matrix remodeling. Male Sprague-Dawley rats underwent left lateral- and median-lobe ischemia for 30 min and reperfusion for 60 min or sham operation. After reperfusion, blood samples and hepatic biopsies from both the ischemic (left-lobe, LL) and the non-ischemic lobe (right-lobe, RL) were collected. Serum hepatic enzymes and TNF-alpha, tissue matrix metalloproteinases (MMP-2, MMP-9), liver morphology, malondialdehyde (MDA), and myeloperoxidase (MPO) were evaluated. Liver I/R injury was confirmed by altered increased hepatic enzymes and TNF-alpha. I/R induced an altered morphology and an increase in MMP-2 and MMP-9 activity not only in left-ischemic lobe (LL) but also in the right-non-ischemic (RL) lobe. A lobar difference was detected for MDA formation and MPO activity in both sham and I/R submitted rats, with higher levels in the left lobe for both groups. This study indicates that an increase in MMPs, which may be TNF-alpha-mediated, occurs in both the ischemic- and the non-ischemic lobes; the heterogeneous lobe concentrations of MDA and MPO suggest that the random sampling of liver tissue should be avoided.

Topics: Animals; Extracellular Matrix; Ischemia; Liver; Male; Malondialdehyde; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tumor Necrosis Factor-alpha; Up-Regulation

The aim of the present study was to evaluate the effects of phosphodiesterase type 5 (PDE5) inhibitory drugs, Tadalafil and Sildenafil, on inducible NOS (iNOS), endothelial NOS (eNOS) and p53 genes expressions and apoptosis in ischemia/reperfusion (I/R) induced oxidative injury in rat renal tissue. Eighty Sprague-Dawley rats (300-350 g) were divided into four groups. In ischemia/reperfusion group, rats were subjected to renal ischemia by clamping the left pedicle for 60 min, and then reperfused for 90 min. On the other hand, in other two groups the rats were individually pretreated with Tadalafil and Sildenafil 1 h before the induction of ischemia. Malondialdehyde (MDA) is determined in renal tissue homogenates by high-performance liquid chromatography, the number of apoptotic cell were calculated by TUNEL method and p53 and eNOS expression were detected with immunohistochemistry. On the other hand, myeloperoxidase (MPO) levels were measured by spectrophotometric method and the mRNA level of iNOS in renal tissue was determined by Real-time PCR (RT-PCR). Our results indicate that MDA and MPO levels were increased in the I/R group than those in the control group. Both Tadalafil and Sildenafil treatment decreased the MDA levels in ischemia/reperfusion group, whereas this effect was more potent with Sildenafil. RT-PCR results showed that, iNOS gen expression increased in the I/R group, but decreased in the PDE5 inhibitory drugs treated group. Apoptotic cells, eNOS levels and p53 positive cells were also decreased in PDE5 inhibitory drugs treated group. We suggest that Tadalafil and Sildenafil have beneficial effects against I/R related renal tissue injury and this protective effect is clearer for Sildenafil than Tadalafil.

Topics: Animals; Apoptosis; Carbolines; Gene Expression; Ischemia; Kidney; Male; Malondialdehyde; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidative Stress; Peroxidase; Phosphodiesterase 5 Inhibitors; Piperazines; Purines; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sildenafil Citrate; Sulfones; Tadalafil; Tumor Suppressor Protein p53

Serum IL-6 is increased in patients with acute kidney injury (AKI) and is associated with prolonged mechanical ventilation and increased mortality. Inhibition of IL-6 in mice with AKI reduces lung injury associated with a reduction in the chemokine CXCL1 and lung neutrophils. Whether circulating IL-6 or locally produced lung IL-6 mediates lung injury after AKI is unknown. We hypothesized that circulating IL-6 mediates lung injury after AKI by increasing lung endothelial CXCL1 production and subsequent neutrophil infiltration. To test the role of circulating IL-6 in AKI-mediated lung injury, recombinant murine IL-6 was administered to IL-6-deficient mice. To test the role of CXCL1 in AKI-mediated lung injury, CXCL1 was inhibited by use of CXCR2-deficient mice and anti-CXCL1 antibodies in mice with ischemic AKI or bilateral nephrectomy. Injection of recombinant IL-6 to IL-6-deficient mice with AKI increased lung CXCL1 and lung neutrophils. Lung endothelial CXCL1 was increased after AKI. CXCR2-deficient and CXCL1 antibody-treated mice with ischemic AKI or bilateral nephrectomy had reduced lung neutrophil content. In summary, we demonstrate for the first time that circulating IL-6 is a mediator of lung inflammation and injury after AKI. Since serum IL-6 is increased in patients with either AKI or acute lung injury and predicts prolonged mechanical ventilation and increased mortality in both conditions, our data suggest that serum IL-6 is not simply a biomarker of poor outcomes but a pathogenic mediator of lung injury.

Topics: Acute Kidney Injury; Acute Lung Injury; Animals; Antibodies; Biomarkers; Capillary Leak Syndrome; Cell Line; Chemokine CXCL1; Interleukin-6; Ischemia; Mice; Mice, Inbred C57BL; Nephrectomy; Neutrophil Infiltration; Peroxidase; Pneumonia; Receptors, Interleukin-8B

Temporary ligation, primary anastomosis, and temporary shunt have been reported to deal with superior mesenteric artery (SMA) injuries. We aimed to investigate which brought minimal ischemia reperfusion injury in a hypothermic traumatic shock swine model.. SMA was completely clamped while pigs were hemorrhaged to a mean arterial pressure (MAP) of 40 mm Hg. Animals were then randomized into temporary ligation (A, n=8), primary anastomosis (B, n=8), temporary shunt (C, n=8), and control groups (n=4). Animals in group A remained SMA interrupted for additional 1h while the other groups underwent the corresponding procedures immediately. Intestine injury was assessed by histologic examination and measurement of lipid peroxidations at the end of ischemia and experiment.. Overall mortality rate was 50%, 25%, and 0% in groups A, B, and C, respectively (P<0.05). The total intestine ischemia time was predominantly shorter in group C in the other groups. Remarkable elevations of malonaldehyde (MDA) in small intestine were noted after reperfusion in group A. Animals in other groups, however, did not exacerbate during the 6-h reperfusion (resuscitation period). Group C showed the lowest MDA level at the end of experiment. Myeloperoxidase (MPO) levels showed no significant elevations during the ischemia or early reperfusion period; nevertheless, it reached approximately 3- to 6-fold in groups A and B (compared with baseline, P<0.01), and remained unchanged in group C at the end of experiment.. Our study suggests that temporary shunt insertion might be preferred as it shortens ischemia time, alleviates intestinal ischemia/reperfusion injury, and thus decreases early mortality in this animal model.

Topics: Anastomosis, Surgical; Animals; Blood Pressure; Female; Hemodynamics; Ileum; Ischemia; Ligation; Malondialdehyde; Mesenteric Artery, Superior; Mesenteric Ischemia; Models, Animal; Peroxidase; Reperfusion Injury; Shock, Traumatic; Surgical Procedures, Operative; Swine; Vascular Diseases

Hepatic ischemia/reperfusion (IR) has been extensively studied, but reperfusion after acute hepatic congestion caused by venous occlusion is poorly understood. Congestion/reperfusion (CR) is not uncommon with the development of partial liver transplantation and liver resection. The purpose of this study was to compare the impairments caused by acute hepatic CR or IR using a murine model.. Mice were randomly divided into IR, CR, and a sham operation (SO) group. The portal vein and hepatic artery of the left anterior hepatic lobe (LAHL) were clamped in the IR group, while the hepatic vein of the LAHL was temporarily occluded in the CR Group. This occurred for 75 min followed by reperfusion. The animals were sacrificed at 2, 6, and 24h after reperfusion. Blood and liver samples were collected for hepatic function, histology, myeloperoxidase (MPO), intravital microscopy, and real-time PCR analysis.. Both IR and CR groups showed elevated liver function, histologic damage, cellular apoptosis, and microcirculatory dysfunction compared with the SO group. Compared with the IR group, the CR group revealed higher hepatic enzyme activities (ALT: 838.5 ± 155.6 versus 474.6 ± 123.8 P<0.05, AST: 792.5 ± 93.5 versus 574.8 ± 188.4 P<0.05), increased sinusoidal nonperfusion rate at 2h after reperfusion (27.4% ± 1.97% versus 23.8% ± 1.93%, P<0.05), and raised MPO level at 24h (0.34 ± 0.11 versus 0.15 ± 0.04, P<0.01). The mRNA levels of IL-1β at 6h and MCP-1 at 2 and 6h were markedly higher in the CR group than in the IR group.. Hepatic reperfusion after acute congestion provokes an increased inflammatory response and causes more severe impairments in the liver compared with ischemia/reperfusion in a murine model.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Constriction; Hepatic Veins; Ischemia; Liver; Male; Mice; Mice, Inbred C57BL; Models, Animal; Peroxidase; Portal Vein; Prevalence; Reperfusion Injury; Trauma Severity Indices

The aim of this study was to evaluate urinary uric acid (UA) and lipid peroxidation levels, plasma myeloperoxidase (MPO) and adenosine deaminase (ADA) activities, and serum UA in neonatal rats subjected to hypoxia-ischemia neonatal HI model. The relevance of the findings is the fact that urinary lipid peroxidation and UA levels were significantly higher in 8 days in HI group when compared with the control, returning to baseline levels 60 days after HI. Hence, being an indication of purinic degradation during these first days post-HI. Furthermore, the higher levels of malondialdehyde (MDA) in urine in this period may be related to inadequate scavenging abilities of the immature nervous system and being noninvasive it may suggest the use of urinary MDA measurement as a marker for lipid peroxidation after HI insult. In application terms, these findings can help develop therapeutic interventions as soon as 8 days after HI.

Topics: Albumins; Animals; Animals, Newborn; Disease Models, Animal; Hypoxia; Ischemia; Lipid Peroxidation; Male; Peroxidase; Rats; Rats, Wistar; Statistics, Nonparametric; Time Factors; Uric Acid

The aim of this study is to evaluate the role of Rho-kinase in the pathogenesis of lung injury induced by intestinal ischemia/reperfusion (I/R) and the preconditioning effects of fasudil hydrochloride. The novel therapeutic approach of using Rho-kinase inhibitors in the treatment of intestinal I/R is introduced.. Sprague-Dawley (SD) rats were divided into 4 groups: intestinal I/R group, two fasudil pretreatment groups (7.5 mg/kg and 15 mg/kg), and controls. Intestinal and lung histopathology was evaluated; myeloperoxidase (MPO) and superoxide dismutase (SOD) levels in lung parenchyma were determined. Serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured. eNOS and P-ERM expression were measured by Western Blot.. Lung and intestinal injury were induced by intestinal I/R, characterized by histological damage and a significant increase in BALF protein. Compared to controls, serum TNF-α, IL-6, and lung MPO activity increased significantly in the I/R group, while SOD activity decreased. A strongly positive P-ERM expression was observed, while eNOS expression was weak. After fasudil administration, injury was ameliorated. Serum TNF-α, IL-6, lung MPO and P-ERM expression decreased significantly as compared to the I/R group, while SOD activity and eNOS expression increased significantly.. Rho-kinase plays a key role in the pathogenesis of lung injury induced by intestinal I/R. The inhibition of the Rho-kinase pathway by fasudil hydrochloride may prevent lung injury.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acute Lung Injury; Animals; Interleukin-6; Intestines; Ischemia; Lung; Male; Nitric Oxide Synthase Type III; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; rho-Associated Kinases; Superoxide Dismutase; Tumor Necrosis Factor-alpha

To evaluate the effects of growth hormone (GH) as an antioxidant and tissue-protective agent and analyse the biochemical and histopathological changes in rat ovaries due to experimental ischemia and ischemia/reperfusion injury.. Forty-eight adult female rats were randomly divided into eight groups. In Group 1, a period of bilateral ovarian ischemia was applied. In Groups 2 and 3, 1 and 2 mg/kg of GH was administered, and 30 min later, bilateral ovarian ischemia was applied (after a 3-h period of ischemia, both ovaries were surgically removed). Group 4 received a 3-h period of ischemia followed by 3h of reperfusion. Groups 5 and 6 received 1 and 2 mg/kg of GH, respectively, 2.5 h after the induction of ischemia. At the end of a 3-h period of ischemia, bilateral vascular clips were removed, and 3h of reperfusion continued. Group 7 received a sham operation plus 2mg/kg of GH. Group 8 received a sham operation only. After the experiments, superoxide dismutase and myeloperoxidase activity and levels of glutathione and lipid peroxidation were determined, and histopathological changes were examined in all rat ovarian tissue.. Ischemia and ischemia/reperfusion decreased superoxide dismutase activity and glutathione levels in ovarian tissue, but increased lipid peroxidation levels and myeloperoxidase activity significantly in comparison to the sham group. The 1 and 2 mg/kg doses of GH before ischemia and ischemia/reperfusion decreased lipid peroxidation levels and myeloperoxidase activity in the experimental groups. The administration of GH before ischemia and ischemia/reperfusion treatments also increased superoxide dismutase and glutathione levels. The histopathological findings also suggested a protective role of GH in ischemia/reperfusion injury. That is, ovarian tissues in the ischemia groups showed histopathological changes, such as haemorrhage, cell degeneration, and necrotic and apoptotic cells, but these changes in the GH groups were lesser. Moreover, in the ischemia/reperfusion groups, acute inflammatory processes--such as neutrophil adhesion and migration, apoptotic and degenerative cells, stromal oedema and haemorrhage--were present. However, the ovarian tissues of the IR+GH (1 mg) group had minimal apoptotic cells, and the IR+GH (2 mg) group had no apoptotic cells. In addition, the general ovarian histological structures of these groups were similar to those of the healthy control group.. The administration of GH is protective against ischemia and/or ischemia/reperfusion-induced ovarian damage. This protective effect can be attributed to the antioxidant properties of GH.

Topics: Animals; Antioxidants; Apoptosis; Dose-Response Relationship, Drug; Edema; Female; Glutathione; Hemorrhage; Human Growth Hormone; Ischemia; Lipid Peroxidation; Neutrophil Activation; Ovary; Oxidative Stress; Peroxidase; Random Allocation; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase; Torsion, Mechanical

Tropisetron is widely used to counteract chemotherapy-induced emesis. Evidence obtained from human and animal studies shows that tropisetron possesses anti-inflammatory properties. In this study, we assessed the effect of tropisetron on brain damage in a rat thromboembolic model of stroke. Stroke was rendered in rats by introduction of an autologous clot into the middle cerebral artery (MCA). Tropisetron (1 or 3mg/kg); m-chlorophenylbiguanide (mCPBG), a selective 5-HT(3) receptor agonist (15 mg/kg); tropisetron (3mg/kg) plus mCPBG (15 mg/kg); granisetron (3mg/kg); tacrolimus (1mg/kg); or tacrolimus (1mg/kg) plus tropisetron (3mg/kg) were administered intraperitoneally 1h prior to embolization. Behavioral scores and infarct volume as well as myeloperoxidase (MPO) activity and tumor necrosis factor-alpha (TNF-α) level were determined in the ipsilateral cortex 4h and 48 h following stroke induction. Forty-eight hours after embolization, tropisetron (1 or 3mg/kg), tropisetron (3mg/kg) plus mCPBG (15 mg/kg), tacrolimus (1mg/kg), or tacrolimus (1mg/kg) plus tropisetron (3mg/kg) significantly curtailed brain infarction, improved behavioral scores, diminished elevated tissue MPO activity, and reduced TNF-α levels compared to control group (n=6; P<0.05). mCPBG or granisetron had no effect on the mentioned parameters. Tropisetron attenuates brain damage after a thromboembolic event. Beneficial effects of tropisetron in this setting are receptor independent.

Topics: Analysis of Variance; Animals; Biguanides; Blood Gas Analysis; Brain Edema; Brain Infarction; Brain Injuries; Cerebral Cortex; Disease Models, Animal; Dose-Response Relationship, Drug; Immunosuppressive Agents; Indoles; Ischemia; Male; Nervous System Diseases; Peroxidase; Rats; Rats, Wistar; Seizures; Serotonin Antagonists; Stroke; Tacrolimus; Tropisetron; Tumor Necrosis Factor-alpha

Nonalcoholic fatty liver disease (NAFLD) refers to an increasingly diagnosed condition involving triglyceride accumulation into hepatocytes resulting in a broad spectrum of liver injury. The progression of NAFLD, a relatively benign condition, to nonalcoholic steatohepatitis (NASH) involves the hepatic infiltration of inflammatory cells and subsequent hepatocellular injury. Ischemia/reperfusion (I/R) injury of the liver is a major complication of liver resection, hepatic trauma, and liver transplantation. To date, there have been no studies that have evaluated the effects of hepatic I/R on models of NASH.. Evaluate the effects of hepatic I/R on a mouse model of NASH.. A mouse model of progressive NASH was developed and evaluated using C57BL/6 mice fed a methionine choline deficient diet for 3, 6, 9, and 12 wk. Mice subsequently underwent 90 min of partial hepatic ischemia with reperfusion of 1, 4, and 8 h. Mice were sacrificed after the indicated periods, and blood and liver samples were taken for analysis.. Mice fed the MCD diet showed a rapid induction of hepatic steatosis, inflammation, and fibrosis by 3 wk that persisted over the 12-wk period of diet, as demonstrated by histologic examination, alanine aminotransferase (ALT), and liver content of myeloperoxidase (MPO). The response to I/R in livers with progressive NASH fed MCD diet for 3, 6, 9, and 12 wk showed marked neutrophil recruitment and hepatocyte necrosis.. These data suggest the inflammatory response from I/R is augmented in livers with NASH histopathology compared with normal liver.

Topics: Alanine Transaminase; Animals; Diet; Disease Models, Animal; Disease Progression; Fatty Liver; Ischemia; Liver; Male; Methionine; Mice; Mice, Inbred C57BL; Non-alcoholic Fatty Liver Disease; Peroxidase; Reperfusion Injury

Renal ischemia-reperfusion injury causes acute renal failure, and the hallmarks of renal ischemia-reperfusion injury are inflammation, apoptosis, necrosis, and capillary dysfunction. Milk fat globule-epidermal growth factor-factor VIII (MFG-E8), a membrane-associated secretory glycoprotein, is produced by immune cells and reported to participate in multiple physiologic processes associated with tissue remodeling. We have recently shown that MFG-E8 treatment attenuates organ injury, inflammatory responses, and survival after sepsis through the enhancement of phagocytosis of apoptotic cells. The purpose of this study was to determine whether administration of MFG-E8 attenuates renal ischemia-reperfusion injury.. Prospective, controlled, and randomized animal study.. : A research institute laboratory.. Male C57BL/6J mice (20-25 g).. : Renal ischemia-reperfusion injury with bilateral renal pedicle clamping for 45 mins, followed by reperfusion. A recombinant murine MFG-E8 (0.4 μg/20 g) was given intraperitoneally at the beginning of reperfusion.. MFG-E8 levels, organ injury variables, inflammatory responses, histology, apoptosis, and capillary functions were assessed at 1.5 and 20 hrs after reperfusion. A 60-hr survival study was conducted in MFG-E8 and recombinant murine MFG-E8-treated wild-type mice. After renal ischemia-reperfusion injury, MFG-E8 mRNA and protein expressions were significantly decreased in the kidneys and spleen. Treatment with recombinant murine MFG-E8 recovered renal dysfunction, significantly suppressed inflammatory responses, apoptosis, necrosis, and improved capillary functions in the kidneys. In the survival study, MFG-E8 mice showed a significant deterioration and, in contrast, recombinant murine MFG-E8-treated wild-type mice showed a significant improvement of survival compared with vehicle-treated wild-type mice.. MFG-E8 can be developed as novel treatment for renal ischemia-reperfusion injury. This protective effect appears to be mediated through the enhancement of apoptotic cell clearance and improvement of capillary functions in the kidneys.

Topics: Animals; Antigens, Surface; Apoptosis; Blotting, Western; Disease Models, Animal; In Situ Nick-End Labeling; Inflammation; Ischemia; Kidney; Liver; Male; Mice; Mice, Inbred C57BL; Milk Proteins; Peroxidase; Recombinant Proteins; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Spleen

It is unknown if pro- and anti-inflammatory mediators in acute lung inflammation induced by intestinal ischemia and reperfusion (i-I/R) can be modulated by low-level laser therapy (LLLT).. A controlled ex vivo study was developed in which rats were irradiated (660 nm, 30 mW, 0.08 cm² of spot size) on the skin over the right upper bronchus 1 hour post-mesenteric artery occlusion and euthanized 4 hours later. For pretreatment with anti-tumor necrosis factor (TNF) or IL-10 antibodies, the rats received either one of the agents 15 minutes before the beginning of reperfusion.. Lung edema was measured by the Evans blue extravasation and pulmonary neutrophils influx was determined by myeloperoxidase (MPO) activity. Both TNF and IL-10 expression and protein in lung were evaluated by RT-PCR and ELISA, respectively.. LLLT reduced the edema (80.1 ± 41.8 µg g⁻¹  dry weight), neutrophils influx (0.83 ± 0.02 × 10⁶  cells ml⁻¹), MPO activity (2.91 ± 0.60), and TNF (153.0 ± 21.0 pg mg⁻¹  tissue) in lung when compared with respective control groups. Surprisingly, the LLLT increased the IL-10 (0.65 ± 0.13) in lung from animals subjected to i-I/R. Moreover, LLLT (0.32 ± 0.07 pg ml⁻¹) reduced the TNF-α level in RPAECs when compared with i-I/R group. The presence of anti-TNF or IL-10 antibodies did not alter the LLLT effect on IL-10 (465.1 ± 21.0 pg mg⁻¹  tissue) or TNF (223.5 ± 21.0 pg mg⁻¹ tissue) in lung from animals submitted to i-I/R.. The results indicate that the LLLT attenuates the i-I/R-induced acute lung inflammation which favor the IL-10 production and reduce TNF generation.

Topics: Acute Disease; Animals; Edema; Interleukin-10; Intestines; Ischemia; Lasers, Semiconductor; Low-Level Light Therapy; Lung; Male; Neutrophils; Peroxidase; Pneumonia; Random Allocation; Rats; Rats, Wistar; Reperfusion; Tumor Necrosis Factor-alpha

Hepatic ischemia/reperfusion (I/R) activates Kupffer cells and initiates severe oxidative stress with enhanced production of reactive oxygen species (ROS) and tumor necrosis factor-alpha (TNF-alpha). ROS and TNF-alpha mediate the expression of nuclear factors and kinases, activating the signal transduction pathway, and triggering apoptosis. The aim of our study was to evaluate the potential protective effect of (-)-epigallocatechin-3-gallate (EGCG) administration in inhibition of apoptosis by attenuating the expression of NF-kappaB, c-Jun, and caspase-3 in a model of severe hepatic I/R.. Thirty Wistar rats were allocated into three groups. Sham operation, I/R, and I/R-EGCG 50mg/kg. Hepatic ischemia was induced for 60min by Pringle's maneuver. Malondialdehyde (MDA), myeloperoxidase (MPO), light histology, scanning electron microscopy, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and immunocytochemistry for NF-kappaB, c-Jun, caspase-3, analysis on liver specimens and aspartate (AST), and alanine (ALT) transferases analysis in serum, were performed 120min after reperfusion.. Apoptosis as indicated by TUNEL and caspase-3 was widely expressed in the I/R group but very limited in the EGCG treated group. Liver was stained positive for NF-kappaB and c-Jun in the I/R group but failed to be stained positive in the EGCG treated group. MDA, MPO, AST, and ALT showed marked increase in the I/R group and significant decrease in EGCG treated group. Significant alterations of liver specimens were observed by light histology and transmission electron microscopy whilst pretreatment with EGCG resulted in parenchymal preservation.. Administration of EGCG is likely to inhibit I/R-induced apoptosis and protect liver by down-regulating NF-kappaB and c-Jun signal transduction pathways.

Topics: Alanine Transaminase; Animals; Apoptosis; Aspartate Aminotransferases; Catechin; Down-Regulation; Immunohistochemistry; In Situ Nick-End Labeling; Ischemia; Liver Diseases; Malondialdehyde; NF-kappa B; Peroxidase; Proto-Oncogene Proteins c-jun; Rats; Rats, Wistar; Reperfusion Injury

We examined the role of cysteinyl leukotrienes (CysLTs) in the gastric ulcerogenic response to ischemia/reperfusion (I/R) in mice. Experiments were performed in male C57BL/6J mice after 18-h fasting. Under urethane anesthesia, the celiac artery was clamped for 30 min, and then reperfusion was achieved by removing the clamp. The stomach was examined for lesions 60 min thereafter. The severity of I/R-induced gastric damage was reduced by prior administration of pranlukast [CysLT receptor type 1 (CysLT(1)R) antagonist] as well as 1-[[5'-(3''-methoxy-4''-ethoxycarbonyl-oxyphenyl)-2',4'-pentadienoyl]aminoethyl]-4-diphenylmethoxypiperidine [TMK688; 5-lipoxygenase (5-LOX) inhibitor]. On the contrary, these lesions were markedly worsened by pretreatment with indomethacin, and this response was abrogated by the coadministration of TMK688 or pranlukast. The gene expression of CysLT(1)R but not 5-LOX was up-regulated in the stomach after I/R, but both expressions were increased under I/R in the presence of indomethacin. I/R slightly increased the mucosal CysLT content of the stomach, yet this increase was markedly enhanced when the animals were pretreated with indomethacin. The increased CysLT biosynthetic response to indomethacin during I/R was attenuated by TMK688. Indomethacin alone caused a slight increase of CysLT(1)R expression and markedly up-regulated 5-LOX expression in the stomach. We concluded that I/R up-regulated the expression of CysLT(1)R in the stomach; CysLTs play a role in the pathogenesis of I/R-induced gastric damage through the activation of CysLT(1)R; and the aggravation by indomethacin of these lesions may be brought about by the increase of CysLT production and the up-regulation of 5-LOX expression, in addition to the decreased prostaglandin production.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonate 5-Lipoxygenase; Chromones; Gastric Acid; Gastric Mucosa; Indomethacin; Ischemia; Leukotriene Antagonists; Leukotrienes; Male; Mice; Mice, Inbred C57BL; Peroxidase; Piperidines; Receptors, Leukotriene; Reperfusion Injury; RNA, Messenger; Stomach Ulcer

Acute renal failure (ARF) is a common morbidity factor among patients in the intensive care unit, reaching an incidence from 3% to 30% depending on the definition of ARF and the population. Although the majority of the patients with ARF are treated with continuous renal replacement therapy, the mortality rate still remains above 50%. The causes of death are primarily extra-renal and include infection, shock, septicemia, and respiratory failure. We wanted to evaluate the cell-mediated inflammatory response of renal ischemia-reperfusion (I/R) and non-renal I/R, in blood and in distant organs. In our study, 80 mice were divided into four groups. The following surgeries were performed on the groups compared: bilateral renal I/R by clamping, unilateral renal ischemia, anesthesia only, and unilateral hind leg I/R. Half of the animals were killed after 2 h and the other half after 24 h. To assess the inflammatory response, we measured myeloperoxidase (MPO) in the organs, and CD 11b and major histocompatibility complex (MHC) II-positive cells in the blood. Non-renal I/R elicited the most elevated levels of MPO in extra-renal tissue such as the lungs. There was a trend toward higher MPO levels in the kidney following renal I/R. All kinds of I/R induced an upregulation of the adhesion molecule CD 11b and a downregulation of MHC II. Renal and non-renal I/R induced neutrophil infiltration in distant organs. Renal I/R does not induce a larger cell-mediated inflammatory response in blood and organs than non-renal I/R.

Topics: Animals; CD11b Antigen; Immunity, Cellular; Ischemia; Kidney; Male; Mice; Mice, Inbred C57BL; Neutrophil Infiltration; Peroxidase; Reperfusion; Tumor Necrosis Factor-alpha

The aim of this study was to evaluate the effects of atorvastatin as an antioxidant and tissue protective agent and study the biochemical and histopathological changes in experimental ischemia and ischemia/reperfusion (I/R) injury in rat ovaries. The experiment used 48 adult female rats, and the experimental groups can be summarized as: group I, a sham operation; group II, a sham operation +10 mg/kg atorvastatin; group III, bilateral ovarian ischemia; and groups IV and V, bilateral ovarian ischemia +5 and 10 mg/kg atorvastatin before 30 min of ischemia, respectively (after a 3-h period of ischemia, the bilateral ovaries were surgically removed); group VI, 3-h period of ischemia followed by 3-h reperfusion; groups VII and VIII received 5 and 10 mg/kg atorvastatin, respectively, 2.5 h after the induction of ischemia, and at the end of a 3-h period of ischemia, bilateral vascular clips were removed and 3-h reperfusion continued. After the experiments, superoxide dismutase (SOD) and myeloperoxidase (MPO) activity and levels of glutathione (GSH) and lipid peroxidation (LPO) were determined, and histopathological changes were examined in all rat ovarian tissue. Ischemia and I/R increased the LPO level and MPO activity while decreasing the SOD activity and GSH level significantly in comparison to the sham group. The 5- and 10-mg/kg doses of atorvastatin before ischemia and I/R reversed the trend in LPO level and MPO activity. The levels of SOD and GSH were decreased by ischemia and I/R. The administration of atorvastatin before ischemia and I/R treatments also reversed the trend in the SOD and GSH levels. In the I/R plus atorvastatin groups, although minimal vascular dilation in the ovary stoma and some degenerative cell clusters were seen, most of the cellular structures showed no pathological changes. Administration of atorvastatin is effective in reversing tissue damage induced by ischemia and/or I/R in ovaries.

Topics: Animals; Antioxidants; Atorvastatin; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Glutathione; Heptanoic Acids; Ischemia; Lipid Peroxidation; Ovary; Peroxidase; Pyrroles; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase

Hepatic ischemia followed by reperfusion (IR) results in mild to severe remote organ injury. Oxidative stress and nitric oxide (NO) seem to be involved in the IR injury. Our aim was to investigate the effects of liver I/R on hepatic function and lipid peroxidation, leukocyte infiltration and NO synthase (NOS) immunostaining in the lung and the kidney. We randomized 24 male Wistar rats into 3 groups: 1) control; 2) 60 minutes of partial (70%) liver I and 2 hours of global liver R; and 3) 60 minutes of partial (70%) liver I and 6 hours of global liver R. Groups 2 and 3 showed significant increases in plasma alanine and aspartate aminotransferase levels and in tissue malondialdehyde and myeloperoxidase contents. In the kidney, positive endothelial NOS (eNOS) staining was significantly decreased in group 3 compared with group 1. However, staining for inducible NOS (iNOS) and neuronal NOS (nNOS) did not differ among the groups. In the lung, the staining for eNOS and iNOS did not show significant differences among the groups; no positive nNOS staining was observed in any group. These results suggested that partial liver I followed by global liver R induced liver, kidney, and lung injuries characterized by neutrophil sequestration and increased oxidative stress. In addition, we supposed that the reduced NO formation via eNOS may be implicated in the moderate impairment of renal function, observed by others at 24 hours after liver I/R.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Immunohistochemistry; Ischemia; Kidney; Lung; Male; Malondialdehyde; Neutrophils; Nitric Oxide Synthase; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury

Leucocytes play crucial roles in ischemia reperfusion injury that is inevitable in kidney transplantation. Leucocyte-depleted hemoreperfusion improved post-ischemic renal function was demonstrated in our previous study and its underlying mechanisms were further investigated in this study.. Porcine kidneys were subjected to 7 min warm ischemia and 2 h cold storage, and preserved by hemoreperfusion with or without leucocyte depletion for 6 h on an isolated organ perfusion system.. Tubulointerstitial damage was improved by leucocyte depletion, which was accompanied by reduced myeloperoxidase+ cell infiltration up to 91%. Apoptotic cells in tubular and interstitial areas were increased by hemoreperfusion, but tubular apoptosis was decreased by leucocyte depletion. The raised caspase-3 activity by hemoreperfusion was almost completely abolished by leucocyte depletion. In addition, the expression of IL-1β active subunit was enhanced by hemoreperfusion, but partially reduced by leucocyte depletion, although IL-1β precursor and HSP70 were increased by hemoreperfusion regardless of leucocyte depletion. Furthermore, myeloperoxidase+ cells were associated with caspase-3 activity, both of which were positively correlated with tubular apoptosis, IL-1β active subunit, tubulointerstitial damage, and serum creatinine, while HSP70 was linked to renal blood flow.. Leucocyte depletion improved post-ischemic renal function and structure was mainly due to reduced infiltration of myeloperoxidase+ cells, which was associated with decreased apoptosis, caspase-3 activity and IL-1β activation.

Topics: Animals; Apoptosis; Cardiopulmonary Bypass; Caspase 3; DNA Nucleotidylexotransferase; Hemoperfusion; HSP70 Heat-Shock Proteins; Interleukin-1beta; Ischemia; Kidney; Kidney Function Tests; Kidney Tubules; Leukocyte Count; Leukocyte Reduction Procedures; Organ Preservation; Peroxidase; Reperfusion Injury; Swine

Neutrophil activation and tissue sequestration are crucial events in intestinal ischemia-reperfusion injury, but their role in the gut wall after clinical cardiopulmonary bypass (CPB) remains unclear. We tested whether local post-CPB inflammatory response in the gut wall would be associated with intestinal mucosal perfusion.. Twenty pigs underwent 60 min of aortic clamping and 75 min of normothermic perfusion. Intestinal biopsies were taken after 120 min of reperfusion. Based on ileal myeloperoxidase activity (MPO), the animals were divided into 2 groups, CPB-induced increase in MPO (MPO+) versus no such increase (MPO-), for comparison of the parameters that measure gut mucosal perfusion. Ileal p(CO)((2)) and intramucosal pH were determined, and arterial gases were analyzed. Additionally, several hemodynamic parameters and blood thrombin-antithrombin complexes (TAT) were measured.. Myocyte degeneration, endothelial activation and vasculitis were more pronounced in the MPO+ group (p < 0.05), while the MPO- group showed significantly increased pi(CO)((2)) and lower mucosal pH values during reperfusion. Hemodynamics and TAT levels did not differ between the groups.. Tissue sequestration of neutrophils was poorly associated with perturbed mucosal perfusion after CPB. Mechanisms of gut wall injury after a low-flow/reperfusion setting can differ from those in reperfusion injury after total ischemia.

Topics: Animals; Cardiopulmonary Bypass; Female; Hemodynamics; Hydrogen-Ion Concentration; Intestines; Ischemia; Male; Neutrophil Activation; Peroxidase; Reperfusion Injury; Sus scrofa

Moderate hypothermic circulatory arrest with selective cerebral perfusion has been developed for cerebral protection during thoracic aortic surgery. However, visceral organs, particularly the kidneys, suffer greater tissue damage under moderate hypothermic circulatory arrest, and acute renal failure after hypothermic circulatory arrest is an independent risk factor for early and late mortality. This study investigated whether atrial natriuretic peptide could prevent the reduction in renal perfusion and protect renal function after moderate hypothermic circulatory arrest. Twelve pigs cooled to 30 degrees C during cardiopulmonary bypass were randomly assigned to a peptide-treated group of 6 and a control group of 6. Moderate hypothermic circulatory arrest was induced for 60 min. Systemic arterial mean pressure and renal artery flow did not differ between groups during the study. However, renal medullary blood flow increased significantly in the peptide-treated group after hypothermic circulatory arrest. Myeloperoxidase activity was significantly reduced in the medulla of the peptide-treated group. Renal medullary ischemia after hypothermic circulatory arrest was ameliorated by atrial natriuretic peptide which increased medullary blood flow and reduced sodium reabsorption in the medulla. Atrial natriuretic peptide also reduced the release of an inflammatory marker after ischemia in renal tissue.

Topics: Animals; Atrial Natriuretic Factor; Blood Flow Velocity; Blood Pressure; Cardiopulmonary Bypass; Circulatory Arrest, Deep Hypothermia Induced; Creatinine; Disease Models, Animal; Female; Inflammation Mediators; Ischemia; Kidney; Laser-Doppler Flowmetry; Natriuresis; Peroxidase; Recombinant Proteins; Renal Circulation; Swine; Time Factors; Ultrasonography; Urination

NADPH oxidase is an enzyme that converts molecular oxygen into reactive oxygen species, which cause severe damage in several organs. Cyclooxygenase (COX)-2 is an inducible enzyme that is important in gastric mucosal defense and repair processes. It is unclear whether NADPH oxidase is related to COX expression in the gastric mucosa, so we investigated the correlation. Under urethane anesthesia, a male Sprague Dawley rat stomach was mounted in an ex-vivo chamber, and ischemia/reperfusion (I/R) was performed through a cannula in the femoral vein. I/R significantly increased NADPH oxidase activity, H(2)O(2) production, and myeloperoxidase (MPO) activity. In contrast, ischemia alone clearly enhanced both NADPH oxidase activity and H(2)O(2) production but not MPO activity. Pretreatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI) suppressed I/R-induced mucosal damage. On the other hand, the selective COX-2 inhibitor rofecoxib exhibited a tendency to enhance the severity of gastric damage induced by I/R, although the selective COX-1 inhibitor SC-560 and the nonselective COX inhibitor indomethacin had no effect. I/R also increased the expression of COX-2, and this increase was suppressed by pretreatment with DPI. These findings suggest that the increase in NADPH oxidase activity is involved in the occurrence of gastric mucosal damage induced by I/R and that this enzyme activity may be causally related to the upregulation of COX-2 during I/R.

Topics: Animals; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Enzyme Inhibitors; Gastric Mucosa; Hydrogen Peroxide; Indomethacin; Ischemia; Lactones; Male; NADPH Oxidases; Onium Compounds; Peroxidase; Pyrazoles; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sulfones; Up-Regulation

Patients with acute kidney injury frequently have pulmonary complications. Similarly ischemic acute kidney injury or bilateral nephrectomy in rodents causes lung injury characterized by pulmonary edema, increased pulmonary capillary leak and interstitial leukocyte infiltration. Interleukin-6 is a pro-inflammatory cytokine that is increased in the serum of patients with acute kidney injury and predicts mortality. Here we found that lung neutrophil infiltration, myeloperoxidase activity, the neutrophil chemokines KC and MIP-2 and capillary leak all increased within 4 h following acute kidney injury in wild-type mice. These pathologic factors were reduced in interleukin-6-deficient mice following acute kidney injury or bilateral nephrectomy. The lungs of mutant mice had reduced KC but MIP-2 was similar to that of wild type mice. Wild-type mice, treated with an interleukin-6 inactivating antibody, had decreased lung myeloperoxidase activity and KC levels following acute kidney injury. Our study shows that interleukin-6 contributes to lung injury following acute kidney injury.

Topics: Acute Kidney Injury; Animals; Capillary Permeability; Chemokines; Interleukin-6; Ischemia; Lung Diseases; Mice; Mice, Knockout; Nephrectomy; Neutrophil Infiltration; Peroxidase

To study the protective mechanism of ischemic preconditioning (IPC) and diazoxide preconditioning (DPC) against myocardium ischemia-reperfusion (I/R) injury.. The hearts were taken out from 30 male Wistar rats and were divided randomly into 3 equal groups: I/R group undergoing 30-min equilibration perfusion and 30-min ischemia and then 60-min reperfusion, IPC group undergoing 10-min equilibration perfusion and then two cycles of 5 min ischemia interspersed with 5 min reperfusion prior to 30 min ischemia and a 60-min reperfusion, and DPC group undergoing 10-min equilibration perfusion and 2 cycles of 5 min of 100 microM diazoxide perfusion followed by 5-min drug-free period before the 30 min ischemia and 60-min reperfusion. Frozen sections of myocardium at the cardiac apex were made and immunohistochemical staining was used to detect the expression of peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha). Ultrathin sections 70 nm thick were made and transmission electron microscopy was used to detect the structure of the mitochondria with the Flameng scoring system.. The PGC-1alpha expression of the IPC and DPC groups were significantly higher than that of the I/R group (P<0.01 and P<0.05), however, there was no significant difference in PGC-1alpha is expression between the IPC and DPC groups (P >0.05). The Flameng scores of the IPC and DPC groups were 0.44 +/- 0.13 and 0.47 +/- 0.10 respectively, both significantly higher than that of the I/R group (1.78 +/- 0.14, both P <0.01), however, there was no significant difference between IPC and DPC groups (P>0.05).. IPC and DPC can protect myocyte mitochondria from the injury of ischemia/ reperfusion. The cardioprotective effects of IPC and DPC may be concerned with the activation and high expression of PGC-1alpha.

Topics: Animals; Diazoxide; Immunohistochemistry; Ischemia; Ischemic Preconditioning, Myocardial; Male; Microscopy, Electron, Transmission; Mitochondria, Heart; Myocardial Reperfusion Injury; Myocardium; Peroxidase; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Rats; Rats, Wistar; RNA-Binding Proteins; Transcription Factors; Vasodilator Agents

Intestinal ischemia-reperfusion (I/R) is an important event in the pathogenesis of multiple organ dysfunction syndrome (MODS). The aim of this study is to determine the effects of ginsenoside Rb1 on liver injury induced by intestinal I/R in rats. Adult male Wistar rats were randomly divided into four groups: (1) a control, sham-operated group (sham group); (2) an intestinal I/R group subjected to 1 h intestinal ischemia and 2 h reperfusion (I/R group); (3) a group treated with 20 mg/kg ginsenoside Rb1 before reperfusion (Rb1-20 group); and (4) a group treated with 40 mg/kg ginsenoside Rb1 before reperfusion (Rb1-40 group). Liver and intestinal histology was observed. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) level in serum and malondialdehyde (MDA) level in intestinal tissues were measured. Myeloperoxidase (MPO), TNF-alpha, MDA level and immunohistochemical expression of NF-kgr;B and intracellular adhesion molecule-1 (ICAM-1) in liver tissues was assayed. In addition, a western blot analysis of liver NF-kappaB expression was performed. Results indicated intestinal I/R induced intestinal and liver injury, which was characterized by increase of AST and ALT in serum, MDA level in intestine, MPO, TNF-alpha and MDA level and ICAM-1 and NF-kappaB expression in the liver tissues. Ginsenoside Rb1 (20, 40 mg/kg) ameliorated liver injury, decreased MPO, TNF-alpha and MDA level, NF-kappaB and ICAM-1 expression in liver tissues. In conclusion, ginsenoside Rb1 ablated liver injury induced by intestinal I/R by inhibiting NF-kappaB activation.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Biomarkers; Ginsenosides; Intercellular Adhesion Molecule-1; Intestinal Mucosa; Intestines; Ischemia; Liver; Liver Diseases; Male; Malondialdehyde; NF-kappa B; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Tumor Necrosis Factor-alpha

To investigate potential associations between-463 G/A myeloperoxidase (MPO) promoter polymorphism and susceptibility to, and clinical features of giant cell arteritis (GCA).. A total of 156 patients with biopsy-proven GCA who were residents of Reggio Emilia, Italy, and 235 population-based controls from the same geographic area were genotyped for-463 G/A promoter polymorphism of the MPO gene by molecular methods. The patients were subgrouped according to the presence or absence of polymyalgia rheumatica and severe ischaemic complications (visual loss and/or cerebrovascular accidents).. The distribution of the MPO-G/A genotype differed significantly between patients with GCA and the controls (p(corr) = 0.003). Allele G was significantly more frequent in patients with GCA than in the controls (p(corr) = 0.0002, OR 2.0, 95% CI 1.4 to 2.9). Homozygosity for the G allele was significantly more frequent in patients with GCA than in controls (p(corr) = 0.0002, OR 2.2, 95% CI 1.4 to 3.4). No significant associations were found when patients with GCA with and without polymyalgia rheumatica or with and without severe ischaemic complications were compared.. Our findings show that the-463 G/A promoter polymorphism of the MPO gene is associated with GCA susceptibility and support a role for MPO in the pathophysiology of GCA.

Topics: Aged; Aged, 80 and over; Female; Gene Frequency; Genetic Predisposition to Disease; Giant Cell Arteritis; Humans; Ischemia; Male; Middle Aged; Peroxidase; Polymorphism, Genetic; Polymyalgia Rheumatica; Promoter Regions, Genetic; Registries

We have previously shown oxidative stress and oedema, caused by both xanthine oxidase-derived oxidants and infiltrating neutrophils, within skeletal muscle after contractile-induced claudication. The purpose of this study was to determine whether supplementation with antioxidant vitamins attenuates the oxidative stress, neutrophil infiltration and oedema associated with an acute bout of contractile-induced claudication. Rats received vehicle, vitamin C, vitamin E or vitamin C + E for 5 days prior to contractile-induced claudication. Force production was significantly reduced in the claudicant limbs of all groups compared with the control (sham) limb of control animals. Contractile-induced claudication caused a significant increase in protein oxidation, lipid peroxidation, neutrophil infiltration and oedema compared with sham muscles. Supplementation with vitamin C, E or C + E prevented the increases in each of these, and there were no differences between groups. These findings suggest that, in an animal model of exercise-induced claudication, neutrophil chemotaxis is caused by oxidizing species and that antioxidant supplementation can prevent oxidative damage, neutrophil infiltration and oedema following an acute bout of contractile-induced claudication.

Topics: Animals; Antioxidants; Ascorbic Acid; Disease Models, Animal; Edema; Electric Stimulation; Intermittent Claudication; Ischemia; Lipid Peroxidation; Male; Muscle Contraction; Muscle, Skeletal; Neutrophil Infiltration; Neutrophils; Oxidative Stress; Peroxidase; Protein Carbonylation; Rats; Rats, Sprague-Dawley; Severity of Illness Index; Vitamin E

Intestinal ischemia/reperfusion (I/R) produces reactive oxygen species (ROS) activating signal transduction and apoptosis. The aim of this study was to evaluate the effect of (-)-epigallocatechin-3-gallate (EGCG) administration in inhibition of apoptosis by attenuating the expression of NF-kB, c-Jun and caspace-3 in intestinal I/R. Thirty male wistar rats were used. Group A sham operation, B I/R, C I/R-EGCG 50 mg/kg ip. Intestinal ischemia was induced for 60 min by clamping the superior mesenteric artery. Malondialdehyde (MDA), myeloperoxidase (MPO), light histology, Fragment End Labelling of DNA (TUNEL), immunocytochemistry for NF-kB, c-Jun and caspace-3 analysis in intestinal specimens were performed 120 min after reperfusion. Apoptosis as indicated by TUNEL and Caspace-3, NF-kB and c-Jun was widely expressed in I/R group but only slightly expressed in EGCG treated groups. MDA and MPO showed a marked increase in the I/R group and a significant decrease in the EGCG treated group. Light histology showed preservation of architecture in the EGCG treated group. In conclusion, EGCG pre-treatment is likely to inhibit intestinal I/R-induced apoptosis by down-regulating the expression of NF-kB, c-Jun and caspase-3.

Topics: Animals; Apoptosis; Caspase 3; Catechin; Down-Regulation; In Situ Nick-End Labeling; Intestinal Mucosa; Intestines; Ischemia; Male; Malondialdehyde; Microscopy; Necrosis; NF-kappa B; Peroxidase; Proto-Oncogene Proteins c-jun; Rats; Rats, Wistar; Reperfusion Injury

To investigate the mechanism of the injury of intestinal mucosal induced by intestinal ischemia-reperfusion and the protect effects of Urinary Trypsin Inhibitor (UTI).. Thirty male Wistar rats were randomly divided into three groups, the sham operation group (SO), ischemia 45 minutes and reperfusion 6 hours group (I/R), UTI-treated group (UTI). Using clamping and then releasing the superior mesenteric artery the model of intestinal ischemia-reperfusion in rats was made. UTI group was given UTI 2 x 10(4) U/KG by administering intravenously before 30 minutes of operation, while the group SO and I/R were intravenously injected with saline. Blood, intestinal tissue and lymph node were obtained after 6 hours of reperfusion. The level of intestinal fatty acid binding protein (IFABP), Tumor Necrosis Factor-alpha (TNF-alpha), Nitric Oxide (NO), malondialdehyde (MDA), superoxide dismutase (SOD), myeloperoxidase (MPO) and the rate of bacterial translocation (BT) in each group were examined. Intestinal tissue samples were also taken for histological analysis by light microscopy and electron microscopy.. The content of IFABP, TNF-alpha, NO, MDA and MPO were significantly lower in group UTI than in group I/R [IFABP (520.87 +/- 75.41) pg/ml vs (493.57 +/- 136.35) pg/ml, NO (58.97 +/- 7.06) micromol/L vs (95.15 +/- 9.13) micromol/L, TNF-alpha (15.38 +/- 1.70) pg/ml to (23.55 +/- 4.34) pg/ml, MDA (4.5 +/- 1.1) nmol/mg vs (9.2 +/- 2.6) nmol/mg, MPO (1.98 +/- 0.22) U/g vs (3.02 +/- 0.55) U/g, SOD (77.08 +/- 7.14) U/mg vs (60.61 +/- 6.83) U/mg, P < 0.01]. There was significant difference in the rate of lymph node BT between the group UTI and I/R (P < 0.05). Histological changes showed that milder damage of intestinal mucosal in group UTI as to group I/R.. Intestinal ischemia-reperfusion may result in intestinal mucosal damage, the mechanism may be involved in the release of abnormal TNF-alpha, NO, reactive oxygen and activated PMN; UTI can protect intestinal mucosal against intestinal ischemia-reperfusion injury, which may be associated with inhibiting the release of NO and TNF-a, ameliorating reactive oxygen damage, decreasing the aggregation and activation of PMN.

Topics: Animals; Bacterial Translocation; Glycoproteins; Intestinal Mucosa; Intestines; Ischemia; Ischemic Preconditioning; Male; Malondialdehyde; Nitric Oxide; Peroxidase; Random Allocation; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase; Trypsin Inhibitors

Testicular torsion/detorsion (T/D) results in enhanced formation of free radical metabolites, which contributes to the pathophysiology of tissue damage. We investigated the protective effects of ethyl pyruvate (EP) against testis tissue damage in an experimental model of testicular torsion.. Sprague-Dawley rats were divided into 5 groups. In those animals that underwent T/D, right testes were rotated 720 degrees for 1 hour. Group 1 control rats underwent sham operation. In group 2, the rats underwent T/D. The EP was prepared and injected in the form of Ringer's ethyl pyruvate solution. The rats in group 3, 4, and 5 received 2 doses of 20, 50, and 100 mg/kg EP (30 minutes before and after detorsion), respectively. The right testes of 6 animals from each group were excised 4 hours after detorsion for the measurement of lipid peroxidation, myeloperoxidase (MPO), and antioxidant enzymes activities. Germ cell apoptosis was determined in right testes of 8 animals per group 24 hours after detorsion. The epididymal sperm concentration and motility were evaluated 1 month after treatments.. Germ cell apoptosis indices were significantly higher in group 2 compared with control group. The level of lipid peroxidation and MPO activity increased, whereas antioxidant enzymes activities decreased after T/D. Sperm count and motility were also reduced 1 month after T/D in group 2 rats. However, EP treatment at doses of 50 and 100 mg/kg significantly decreased the early apoptotic damage and improved long-term sperm count and motility. In the same dosing groups, we observed normalization of oxidant/antioxidant balance and decrement of MPO activity. However, administration of 20 mg/kg of EP conferred no protective effect.. Administration of Ringer's ethyl pyruvate solution (in appropriate doses) is protective against apoptotic tissue damage following testicular torsion and improves long-term testicular function. The antioxidant and anti-inflammatory properties of EP seem responsible for the protective effects. Our findings suggest this resuscitation solution as a possible substitute for fluid and electrolyte maintenance during surgical detorsion.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Dose-Response Relationship, Drug; Germ Cells; Ischemia; Lipid Peroxidation; Male; Oxidative Stress; Peroxidase; Postoperative Care; Pyruvates; Rats; Rats, Sprague-Dawley; Sperm Count; Sperm Motility; Spermatic Cord Torsion; Testis

Activated protein C (APC) is a serine protease with anticoagulant and antiinflammatory activities. The delaying effects of remote reperfusion injury on the wound-healing process in colonic anastomoses have been previously shown. In this study, we aimed to investigate whether APC protects against deleterious systemic effects of intestinal ischemia/reperfusion (I/R) injury on colonic anastomotic wound healing process.. Male Wistar-albino rats were randomly allocated into 4 groups, and a left colonic anastomosis was performed in all animals: (1) sham-operated group, simultaneously with left colonic anastomosis, the superior mesenteric artery and collateral branches were divided from the celiac axis, and the inferior mesenteric artery were isolated but not occluded (group 1, n = 12), (2) sham + APC group, identical to group 1 except for APC treatment (100 microg/kg, intravenously, 15 minutes before construction of the colonic anastomosis), (group 2, n = 12), (3) intestinal I/R group, 60 minutes of superior mesenteric ischemia followed by reperfusion (group 3, n = 12), and (4) APC-treated group, (100 microg/kg, intravenously, 15 minutes before reperfusion) (group 4, n = 12). All animals were sacrificed, and colonic anastomotic bursting pressures were measured in vivo on day 7. Tissue samples were obtained for analysis of hydroxyproline contents, nitrate/nitrite levels, and activities of oxidative and antioxidative enzymes. The plasma levels of proinflammatory cytokines and D-dimer were also measured.. Intestinal I/R led to significant decreases in colonic anastomotic bursting pressures, tissue hydroxyproline contents, and activities of antioxidative enzymes, along with increases in tissue nitrate/nitrite levels, activities of oxidative enzymes, and plasma levels of proinflammatory cytokines and D-dimer (P < .05). However, APC treatment led to significant increases in colonic anastomotic bursting pressures, tissue hydroxyproline contents, and activities of antioxidative enzymes, along with decreases in tissue nitrate/nitrite levels, activities of oxidative enzymes, and plasma levels of proinflammatory cytokines and D-dimer (P < .05).. This study clearly showed that APC treatment prevented the delaying effects of remote I/R injury on colonic anastomotic wound healing process. Further clinical studies are required to determine whether APC has a useful role in the enhancement of colonic anastomotic wound healing after particular operations in which I/R injury occurs.

Topics: Analysis of Variance; Anastomosis, Surgical; Animals; Chi-Square Distribution; Glutathione; Glutathione Reductase; Interleukin-6; Intestines; Ischemia; Male; Malondialdehyde; Mesenteric Arteries; Mice; Nitrates; Nitrites; Peroxidase; Protein C; Random Allocation; Reperfusion Injury; Tumor Necrosis Factor-alpha; Wound Healing; Xanthine Oxidase

The purpose of this study was to examine the effects of simvastatin pretreatment in the setting of acute limb ischemia-reperfusion injury in an experimental diabetes model that is associated with a high risk for limb loss.. Adult male Sprague-Dawley rats were randomized into two groups. Diabetes was induced in the first group by intravenous streptozotocin injection. The second group served as the nondiabetic group. Eight weeks after the streptozotocin injection, half of the rats in the diabetic and the nondiabetic groups were further randomized to receive either intraperitoneal simvastatin (1 mg/kg per day) or saline treatment for 6 weeks. Bilateral hind-limb ischemia was induced for 4 hours by the tourniquet method. After 24 hours of reperfusion, tissue samples were collected from the gastrocnemius and anterior tibial muscles bilaterally for measurement of muscle edema, percentage of necrosis, and malondialdehyde (MDA), glutathione, and myeloperoxidase (MPO) levels.. Ischemic injury was more prominent in diabetic animals. The diabetic animals with limb ischemia exhibited a 7% increase in tissue edema, a 47% increase in muscle necrosis and MPO level, and a 15% reduction in glutathione levels compared with the nondiabetic animals (P < .05). Simvastatin treatment with 1 mg/kg for 6 weeks reduced the ischemic injury. Simvastatin pretreatment led to a 71% reduction in muscle necrosis in diabetic animals (P < .001). The protective effects of simvastatin pretreatment also correlated with a 23% improvement in tissue edema, a 75% reduction in tissue myeloperoxidase content, and a 71% increase in glutathione levels in diabetic animals (P < .01). Furthermore, skeletal muscle injury, characterized by tissue edema and leucosequestration, was significantly less severe with simvastatin pretreatment compared with the nondiabetic animals (P < .01).. Simvastatin pretreatment reduced limb ischemia-reperfusion injury in diabetic and nondiabetic animals. We conclude that simvastatin pretreatment may be a potential therapeutic intervention for skeletal muscle ischemia-reperfusion injury in the clinical setting.

Topics: Animals; Diabetes Mellitus, Experimental; Edema; Glutathione; Hindlimb; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Ischemia; Male; Malondialdehyde; Muscle, Skeletal; Necrosis; Peroxidase; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Severity of Illness Index; Simvastatin; Time Factors; Tourniquets

We have reported that chronically administered pravastatin prevented coronary artery reperfusion-induced lethal ventricular fibrillation (VF) in anesthetized rats without lowering the serum cholesterol level. The present study was undertaken to evaluate whether pravastatin prevents ischemia-induced lethal VF, simultaneously examining myeloperoxidase (MPO) activity in ischemic myocardial tissues. Anesthetized rats were subjected to 30-min ischemia and 60-min reperfusion after chronic administration of pravastatin (0.02, 0.2, and 2 mg/kg), fluvastatin (2 and 4 mg/kg), or vehicle for 22 days, orally, once daily. ECG and blood pressure were continually recorded, and MPO was measured by a spectrophotometer. Pravastatin and fluvastatin significantly (P<0.05) decreased MPO activities, but only pravastatin decreased the incidence of ischemia-induced lethal VF. Both statins had no significant effects on body weight, blood pressure, heart rate, and QT interval as we reported earlier. Our results prove further that pravastatin has benefits to decrease cardiovascular mortality beyond its cholesterol-lowering effect. Pravastatin is more potent than fluvastatin in prevention of arrhythmias. A decrease in the neutrophil infiltration may be partly involved in the inhibitory effect of pravastatin on the ischemia-induced VF.

Topics: Administration, Oral; Anesthesia; Animals; Arrhythmias, Cardiac; Coronary Vessels; Dose-Response Relationship, Drug; Drug Administration Schedule; Fatty Acids, Monounsaturated; Fluvastatin; Hydroxymethylglutaryl CoA Reductases; Indoles; Ischemia; Male; Peroxidase; Pravastatin; Rats; Rats, Sprague-Dawley

Free radicals are important in development of intestinal ischemia-reperfusion (I/R) injury, leading to intestinal and pulmonary damage. We evaluated the effects of peroxynitrite decomposition catalyst FeTMPyP in infant intestinal I/R. Suckling rats underwent 40 min intestinal ischemia + 90 min reperfusion. At reperfusion, animals received saline or FeTMPyP. Groups were (n = 11 per group): 1) control+saline; 2) I/R+saline; 3) I/R+FeTMPyP. Increased histologic injury and ICAM-1 expression were observed in ileum of both I/R+saline and I/R+FeTMPyP rats, but P-selectin expression was increased in I/R+saline animals only versus controls. Myeloperoxidase (neutrophil infiltration marker) was increased in ileum and lungs of I/R+saline rats, but FeTMPyP prevented this in the ileum. I/R+saline animals showed higher malondialdehyde (lipid peroxidation marker) in ileum and lungs versus both control+saline and I/R+FeTMPyP rats. Glutathione was decreased in all I/R animals, but oxidized and total glutathione were higher in I/R+FeTMPyP than the I/R+saline group. Nitrate+nitrite concentration (systemic nitric oxide production) was elevated in I/R+saline but not in I/R+FeTMPyP animals. FeTMPyP provides limited protection against intestinal I/R in neonatal rats by reducing ileal P-selectin expression, systemic nitric oxide production, neutrophil infiltration in ileum and lipid peroxidation in both lungs and ileum; and preserving intestinal antioxidant capacity.

Topics: Animals; Animals, Suckling; Endothelial Cells; Glutathione; Intercellular Adhesion Molecule-1; Intestinal Mucosa; Intestines; Ischemia; Malondialdehyde; Metalloporphyrins; Nitrates; Nitrites; P-Selectin; Peroxidase; Peroxynitrous Acid; Random Allocation; Rats; Reperfusion Injury; Vascular Cell Adhesion Molecule-1

In this study, we have investigated the role of the cannabinoid CB(2) (CB(2)) receptor in an in vivo mouse model of hepatic ischemia/reperfusion (I/R) injury. In addition, we have assessed the role of the CB(2) receptor in TNF-alpha-induced ICAM-1 and VCAM-1 expression in human liver sinusoidal endothelial cells (HLSECs) and in the adhesion of human neutrophils to HLSECs in vitro. The potent CB(2) receptor agonist HU-308, given prior to the induction of I/R, significantly attenuated the extent of liver damage (measured by serum alanine aminotransferase and lactate dehydrogenase) and decreased serum and tissue TNF-alpha, MIP-1alpha, and MIP-2 levels, tissue lipid peroxidation, neutrophil infiltration, DNA fragmentation, and caspase 3 activity. The protective effect of HU-308 against liver damage was also preserved when given right after the ischemic episode. HU-308 also attenuated the TNF-alpha-induced ICAM-1 and VCAM-1 expression in HLSECs, which expressed CB(2) receptors, and the adhesion of human neutrophils to HLSECs in vitro. These findings suggest that selective CB(2) receptor agonists may represent a novel, protective strategy against I/R injury by attenuating oxidative stress, inflammatory response, and apoptosis.

Topics: Alanine Transaminase; Animals; Apoptosis; Cannabinoid Receptor Agonists; Cannabinoids; Cell Adhesion; Chemokines; Endothelial Cells; Humans; Inflammation; Inflammation Mediators; Intercellular Adhesion Molecule-1; Ischemia; L-Lactate Dehydrogenase; Leukocytes; Lipid Peroxidation; Liver; Mice; Oxidative Stress; Peroxidase; Reperfusion Injury; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Ischemia and reperfusion (I/R) damage involves adhesion and transmigration of lymphocytes and neutrophils. FTY720 is an immunosuppressive agent that reduces the number of neutrophils and monocytes in peripheral blood as well as tissue lymphocyte infiltration. This study investigated the effect of FTY720 during hind limb I/R.. Male C57/BL6 mice underwent temporary ligation of the infrarenal aorta for 4 hours. After 48 hours of reperfusion, animals were killed by exsanguination. Tissue myeloperoxidase content reflecting neutrophil infiltration and reverse transcription polymerase chain reaction analysis of local cytokine transcription in lung, liver, and kidney were performed.. After I/R, treatment with FTY720 improved survival and prevented upregulation of pro- and anti-inflammatory cytokines in evaluated organs, whereas no changes were detected in myeloperoxidase content after treatment with FTY720.. Whereas neutrophil infiltration was not affected by treatment with FTY720, other immunocompetent or intrinsic cells appear to be involved in changes of cytokine production in different organs.

Topics: Analysis of Variance; Animals; Biomarkers; Cytokines; Disease Models, Animal; Fingolimod Hydrochloride; Hindlimb; Immunosuppressive Agents; Interleukin-1; Ischemia; Male; Mice; Mice, Inbred C57BL; Peroxidase; Probability; Propylene Glycols; Random Allocation; Reference Values; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Sphingosine; Survival Rate

Remote preconditioning (RPC) can protect from ischemia/reperfusion injury (IRI). We investigated the influence of RPC in pancreatic IRI.. Wistar rats were randomized to 2 hours of ischemia and 2 hours of reperfusion of a pancreatic tail segment with or without 15 minutes of infrarenal ischemia 60 minutes before IRI. Microcirculatory measurements before ischemia and 1 and 2 hours after reperfusion included functional capillary density and leukocyte adherence in postcapillary venules, quantified by intravital fluorescence microscopy. Histology and tissue myeloperoxidase activity were further parameters of pancreatic injury.. Remote preconditioning caused an improvement of microcirculation (functional capillary density: 1 hour after reperfusion, 460 +/- 13 vs 350 +/- 9 cm/cm2; 2 hours after reperfusion, 437 +/- 13 vs 295 +/- 13 cm/cm2; P < 0.01) and reduced inflammatory tissue response (leukocyte adherence in postcapillary venules: 2 hours after reperfusion, 155 +/- 55 vs 748 +/- 187 cells/mm2; P < 0.01). Histology was significantly better in preconditioned animals (IR, 8.1+/- 1.3 score points; RPC, 6.2 +/- 1.3 score points; P < 0.05). The difference in myeloperoxidase activity was not significant (ischemia/reperfusion [IR], 105 +/- 72; RPC, 245 +/- 209 mU x min(-1) x mg(ti)(-1); P = 0.13).. With our dynamic functional microcirculatory measurements, we could demonstrate that RPC is a feasible method to reduce experimental pancreatic IRI. This was seen in an attenuation of nutritive tissue perfusion and a reduction of inflammatory tissue response and a lower histological damage. Because it is easy to perform before organ harvest, RPC could be a step to improve organ procurement in pancreas transplantation. Clinical studies are the next step to evaluate RPC in pancreas transplantation.

Topics: Animals; Aorta; Capillaries; Cell Adhesion; Disease Models, Animal; Feasibility Studies; Hemodynamics; Ischemia; Ischemic Preconditioning; Leukocytes; Ligation; Male; Microscopy, Video; Pancreas; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Time Factors; Venules

Ischemia and reperfusion of the skeletal muscle tissue may cause remote lung injury. We aimed to evaluate the protective effect of ischemic preconditioning (IP) on the lung during unilateral lower limb ischemia reperfusion (IR).. Four groups of rats were used in this study: (i) the sham group (sham, n = 6) served as time controls, they remained anesthetized for the whole duration of the study; (ii) the ischemia and reperfusion group (IR, n = 10) underwent 4 h of left lower limb ischemia followed by 2 h of reperfusion; (iii) the ischemic preconditioning group (IP, n = 10), the left lower limbs of rats were exposed to three cycles of IP (10 min of ischemia followed by 10 min of reperfusion); and (iv) the ischemic preconditioning plus ischemia reperfusion group (IP/IR, n = 10) underwent IP followed by IR as in the IP and IR groups. Plasma and tissue samples were taken at the end of the study period for determination of lung tissue myeloperoxidase activity (MPO) and polymorphonuclear leukocyte count (PMNL), histological lung injury score and plasma thiobarbituric acid reactive substances (TBARS) level.. PMNL count and MPO activity in the lung tissue, and plasma TBARS level were higher in the IR group compared with other groups while there were no differences between the sham and the IP and between the sham and the IP/IR groups. Histological lung injury score was higher in the IR group than in the IP/IR and sham groups. The plasma TBARS level in the IP group was significantly lower than in the IP/IR group.. IP pretreatment reduces lipid peroxidation and lung injury caused by lower limb IR.

Topics: Animals; Disease Models, Animal; Hindlimb; Ischemia; Ischemic Preconditioning; Lipid Peroxidation; Lung; Lung Diseases; Male; Muscle, Skeletal; Neutrophils; Peroxidase; Rats; Rats, Wistar; Thiobarbituric Acid Reactive Substances; Time Factors; Tourniquets

Monocyte chemotactic protein-1 (MCP-1) is important in macrophage recruitment and activation. However, the magnitude and temporal sequence of MCP-1 expression in relation to tissue injury and regeneration following ischemic injury remains unknown.. Hind limb ischemia was induced by femoral artery excision (FAE) in C57Bl/6J mice; a sham surgery was performed on the contralateral leg. Muscle lysates were used to measure MCP-1 and activities of creatine kinase, lactate dehydrogenase, and myeloperoxidase. Histology and immunohistochemistry were used to localize inflammation and MCP-1.. FAE resulted in a prolonged period of ischemia and the administration of MCP-1 did not alter the restoration of perfusion. One day after femoral artery excision, extensive muscle necrosis and neutrophils were prevalent throughout the musculature of the lower leg. By 3 days, a mononuclear cell infiltrate predominated in association with robust muscle regeneration as indicated by myoD expression. Concomitantly, myeloperoxidase was maximally increased. Muscle enzymes (creatine kinase and lactate dehydrogenase) were maximally decreased within 3 days and returned to baseline levels by day 14, a time course consistent with injury and regeneration observed by histology. In parallel with these inflammatory and regenerative events, MCP-1 in muscle was maximally increased at day 3. By immunohistochemistry, MCP-1 was within vascular endothelial cells and infiltrating macrophages in areas of ischemic injury.. The transient increases and selective tissue distribution of MCP-1 during early inflammation and muscle regeneration support the hypothesis that this cytokine participates in the early reparative events preceding the restoration of vascular perfusion following ischemic injury.

Topics: Animals; Chemokine CCL2; Creatine Kinase; Inflammation; Ischemia; L-Lactate Dehydrogenase; Leukocyte Count; Male; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Organ Size; Peroxidase; Receptors, CCR2; Receptors, Chemokine; Regeneration; RNA, Messenger

Activation of extravascular coagulation has been reported in acute lung injury models of sepsis and acute respiratory distress syndrome. Thrombin, the main effector protease of extravascular coagulation, activates proinflammatory cell types, including macrophages, endothelial cells, and neutrophils, each of which participates in lung ischemia-reperfusion injury. We used hirudin, a potent, specific direct thrombin inhibitor, to define the role of thrombin in lung ischemia-reperfusion injury.. Rats were pretreated with hirudin 30 minutes before warm, in situ left lung ischemia and reperfusion. Multiple in vivo assessments of lung injury were determined, and mechanistic studies assessed transcriptional regulation early in reperfusion and proinflammatory protein secretion late in reperfusion. Immunohistochemistry localized thrombin activation.. Thrombin localized to macrophages and endothelial and epithelial cells early in reperfusion. Hirudin significantly limited lung ischemia-reperfusion injury-induced derangements in vascular permeability and intraalveolar inflammatory cell sequestration, resulting in improved arterial oxygenation after ischemia and 4 hours of reperfusion. The protection was transcriptionally mediated by attenuated activator protein-1 and early growth response-1 transactivation, but not nuclear factor kappa B transactivation. This was associated with reduced chemokine, but not tumor necrosis factor alpha, secretion late in reperfusion.. Thrombin promotes lung ischemia-reperfusion injury, as hirudin protected against experimental acute lung injury. Hirudin conferred protection through a mechanism independent of nuclear factor kappa B and tumor necrosis factor alpha, suggesting that its effects may be mediated by a parallel, synergistic inflammatory pathway through activator protein-1 and early growth response-1.

Topics: Animals; Capillary Permeability; Disease Models, Animal; Humans; Inflammation; Ischemia; Peroxidase; Pulmonary Circulation; Rats; Rats, Long-Evans; Reperfusion; Reperfusion Injury; Respiratory Function Tests; Thrombosis

To investigate the effects of propofol and caffeic acid phenethyl ester (CAPE) on prevention of lung injury as a remote organ after performing hindlimb ischaemia-reperfusion (IR) in a rat model.. The animals were divided randomly into one of four groups: sham, no IR (n = 8), control, IR, (n = 8), CAPE group, IR with CAPE, (n = 8), propofol group, IR with P, (n = 8). After the rats were anaesthetised, the animals in the CAPE group received CAPE of 10 micromol, in the propofol group received propofol 50 mg/kg, in the control group received a similar volume of saline solution by means of intraperitoneal injection 1 h before reperfusion. After 4 h of ischaemia the tourniquet was removed and the animals were released for reperfusion for 4 h thereafter. At the end of the reperfusion period, a median sternotomy was performed. A blood sample was obtained for plasma malondialdehyde (MDA). The lung tissues were also removed for MDA assays, myeloperoxidase (MPO) activity, and histopathological examination.. Plasma and lung MDA levels, and lung MPO activity were significantly higher in the control group compared to the other groups (p < 0.0005). In the CAPE group, these were significantly lower compared to the control group (p < 0.0005). Also, propofol caused a marked reduction in the MDA levels and MPO activity compared with control group (p < 0.0005), with no significant difference compared to that of the sham group. Histopathologically, the scores resulted in a grade zero (8/8) in the sham group, 3 (3/8) or 4 (5/8) in the control group, 1 (2/8) or 2 (6/8) in the CAPE group, and 1 (3/8) or 2 (5/8) in the propofol group.. Propofol and CAPE seem to be effective in protecting against lung injury caused by increased oxidative stress and neutrophil accumulation after hindlimb IR in a rat model.

Topics: Animals; Caffeic Acids; Free Radical Scavengers; Hindlimb; Ischemia; Lung; Lung Diseases; Male; Malondialdehyde; Oxidative Stress; Peroxidase; Phenylethyl Alcohol; Positive-Pressure Respiration; Propofol; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Treatment Outcome

To investigate the preventive effect of ischemic preconditioning (IPC) on ischemia/reperfusion (I/R)-induced apoptosis and injury in the rat intestine.. We divided 30 male Wistar rats, weighing 300-350 g, randomly into three groups. The control group rats (n = 10) were subjected to laparotomy only; the I/R group (n = 10) rats were subjected to occlusion of the superior mesenteric artery for 45 min, followed by reperfusion for 60 min; and the IPC group (n = 10) rats were subjected to IPC, achieved with two cycles of 5 min ischemia and 5 min reperfusion immediately before the I/R, as in the I/R group. Blood samples were collected by cardiac puncture, to measure nitrate and myeloperoxidase (MPO) levels. Histopathological and immunohistochemical studies were done to evaluate the I/R-induced apoptosis and injury.. The blood MPO and nitrate levels were increased in the I/R group, but IPC prevented their increase. There were significantly fewer apoptotic cells in the IPC group than in the I/R group, and this finding was supported by the caspase-3 expression in the ileum. The intestinal histopathology was also protected by IPC against I/R-induced injury.. Ischemic preconditioning clearly prevented I/R-induced injury and apoptosis by a mechanism related to the caspase-3-dependent pathway. We also showed that IPC inhibited leukocyte activation, with the suppression of myeloperoxidase levels in I/R and nitric oxide-related oxidoinflammatory pathway upregulation.

Topics: Analysis of Variance; Animals; Apoptosis; Biomarkers; Caspase 3; Caspases; Disease Models, Animal; Immunohistochemistry; Intestine, Small; Ischemia; Ischemic Preconditioning; Laparotomy; Male; Peroxidase; Probability; Random Allocation; Rats; Rats, Wistar; Reference Values; Reperfusion Injury; Risk Factors; Sensitivity and Specificity

In recent years it has become increasingly clear that a cross-talk between the inflammatory response and blood coagulation exists, although many of the underlying mechanisms remain unclear. In the present study we investigated the potential anti-inflammatory properties of two different anticoagulant compounds, i.e. active-site inactivated FVIIa (FVIIai) and fondaparinux sodium, a selective FXa inhibitor, administered as pretreatment in a model of intestinal I/R in rats.. Endothelial barrier permeability was assessed using the vascular leakage of radiolabelled human serum albumin, tissue neutrophil sequestration was quantitated by myeloperoxidase (MPO) activity, and plasma levels of macrophage inflammatory protein (MIP)-2 were examined using an enzyme-linked-immuno-sorbent assay after 40 min of intestinal ischaemia and 6 h of reperfusion in the rat (n = 34). Pretreatment with FVIIai or fondaparinux sodium was administered 90 min before initiation of ischaemia.. Endothelial-barrier permeability in all examined organs, myeloperoxidase activity in the lungs, and ileum and MIP-2 levels in plasma increased after intestinal I/R. Pretreatment with FVIIai decreased the endothelial barrier permeability and MPO activity in the ileum, and a tendency towards decreased permeability was also observed in the lungs. Fondaparinux did not affect the endothelial barrier permeability or MPO activity. Both FVIIai and fondaparinux decreased the MIP-2 levels in plasma after intestinal I/R.. Inhibition of the TF-FVIIa complex by FVIIai can attenuate inflammatory responses in connection with intestinal I/R-injury and could represent a potentially important therapeutic strategy for the prevention of organ dysfunction. Potential anti-inflammatory properties of fondaparinux and other inhibitors of FXa are not excluded and need further investigation.

Topics: Animals; Anticoagulants; Blood Cell Count; Cell Membrane Permeability; Chemokine CXCL2; Chemokines, CXC; Endothelium; Factor VIIa; Factor Xa Inhibitors; Fondaparinux; Hemostasis; Intercellular Signaling Peptides and Proteins; Interleukin-8; Intestines; Ischemia; Male; Neutrophil Infiltration; Peroxidase; Polysaccharides; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Reperfusion Injury

The aim of this study was to determine whether nuclear factor-kappaB (NF-kappaB) inhibitors are efficient against hepatic ischemia/reperfusion (I/R) injury. We previously demonstrated that xanthine oxidase-derived reactive oxygen species activate NF-kappaB during ischemia. However, the role of NF-kappaB activation during ischemia in post-reperfusion injury remains unclear. Therefore, while we examined the effects of NF-kappaB inhibitors, sulfasalazine and pyrrolidinedithiocarbamate on hepatic I/R injury using a rat lobar hepatic I/R model, we estimated the relationship between NF-kappaB activation during ischemia and following hepatic damage caused by reperfusion. The portal vein and the hepatic artery were clamped for 1 hr followed by reperfusion for up to 24 hr. NF-kappaB activation was determined by Western blot analysis. NF-kappaB activation was observed in the ischemic lobe of the liver, and the activation was prevented by pre-administration with NF-kappaB inhibitors. Although the serum ALT level, hepatic MPO activity and BSP clearance, as an index of hepatic injury, were increased after reperfusion, the increase was attenuated by pre-administration with NF-kappaB inhibitors. These findings suggest that NF-kappaB activation during ischemia is relevant to hepatic I/R injury. Moreover, we first showed that pre-administration with NF-kappaB inhibitors is effective against hepatic I/R injury.

Topics: Alanine Transaminase; Animals; Ischemia; Liver; Male; NF-kappa B; Peroxidase; Proline; Rats; Rats, Wistar; Reperfusion Injury; Sulfobromophthalein; Thiocarbamates

Ischemic acute renal failure is characterized by damages to the proximal straight tubule in the outer medulla. Lesions include loss of polarity, shedding into the tubule lumen, and eventually necrotic or apoptotic death of epithelial cells. It was recently shown that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) increases keratinocyte survival after an inflammatory reaction. Therefore, whether PPARbeta/delta could contribute also to the control of tubular epithelium death after renal ischemia/reperfusion was tested. It was found that PPARbeta/delta+/- and PPARbeta/delta-/- mutant mice exhibited much greater kidney dysfunction and injury than wild-type counterparts after a 30-min renal ischemia followed by a 36-h reperfusion. Conversely, wild-type mice that were given the specific PPARbeta/delta ligand L-165041 before renal ischemia were completely protected against renal dysfunction, as indicated by the lack of rise in serum creatinine and fractional excretion of Na+. This protective effect was accompanied by a significant reduction in medullary necrosis, apoptosis, and inflammation. On the basis of in vitro studies, PPARbeta/delta ligands seem to exert their role by activating the antiapoptotic Akt signaling pathway and, unexpectedly, by increasing the spreading of tubular epithelial cells, thus limiting potentially their shedding and anoikis. These results point to PPARbeta/delta as a remarkable new target for preconditioning strategies.

Topics: Acetates; Animals; Apoptosis; Blotting, Western; Cells, Cultured; Creatinine; Dose-Response Relationship, Drug; Epithelial Cells; Humans; In Situ Nick-End Labeling; Inflammation; Ischemia; Keratinocytes; Kidney; Kidney Tubules; Ligands; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Fluorescence; Necrosis; Neutrophils; Peroxidase; Phenols; Phenotype; Phenoxyacetates; PPAR delta; PPAR-beta; Proto-Oncogene Proteins c-akt; Renal Insufficiency; Signal Transduction; Sodium; Time Factors

Anti-inflammatory action of Antithrombin III (AT III) is still not well understood in ischemia/reperfusion (I/R) injury. In the present study, we aimed to investigate the anti-inflammatory action of AT III on remote lung and local skeletal muscle tissue injury in a rat model of bilateral lower limb I/R model.. Bilateral lower limb ischemia and reperfusion were produced by means of tourniquets occlusions and releases, respectively. Three groups of rats were used in this controlled study: sham group (sham, n=3) underwent 5 h of anesthesia only; control group (I/R, n=7) underwent 3 h of bilateral lower limb ischemia followed by 2 h of reperfusion; and AT III pretreated group (I/R-AT III, n=6) underwent the same procedure as the control group, but also received i.v. 250 U kg-1 AT III 30 min before ischemia induction under midazolam and fentanyl anesthesia.. Lung and muscle tissue accumulation of polymorphonuclear leukocytes (PMN) were assessed by measuring tissue myeloperoxidase (MPO) activity. Histopathological changes in tissues were assessed by PMN counts in the lung, and muscle tissues and by histological lung injury score. Plasma 6-keto prostaglandin F(1alpha) and tumor necrosis factor alpha levels were measured by an enzyme immunoassay technique. Myeloperoxidase activity could not be detected in the muscle tissues of all groups. The lung and muscle tissue PMN counts in the I/R group were significantly higher compared with the I/R-AT III group (P<0.05).. Data from the present study provides some evidence that AT III pretreatment attenuates remote lung and local skeletal muscle tissue injury caused by lower limb I/R.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Anticoagulants; Antithrombin III; Disease Models, Animal; Ischemia; Ischemic Preconditioning; Lower Extremity; Lung; Muscle, Skeletal; Neutrophil Infiltration; Neutrophils; Peroxidase; Pilot Projects; Rats; Rats, Wistar; Reperfusion; Reperfusion Injury; Tumor Necrosis Factor-alpha

This study examined the effects of estrogen supplementation on markers of neutrophil infiltration and damage in skeletal muscle of rats following ischemia. Male and female gonad-intact rats, with or without 14 days of estrogen supplementation were subjected to two hours of hind-limb ischemia and sacrificed at 24, 48 or 72 hours post-ischemia. Control animals were sacrificed without ischemia. Plantaris and red and white gastrocneimus muscles were removed and assayed for myeloperoxidase (MPO), a marker of neutrophil infiltration, and glucose-6-phosphate dehydrogenase (G6PD) and beta-glucuronidase (betaGLU), as markers of muscle damage. Significant elevations of MPO, G6PD and betaGLU activities were observed at various time points post-ischemia. No systematic differences between genders were noted in any of the measures. Estrogen supplementation in both male and female animals failed to significantly attenuate post-ischemia increases in MPO, G6PD and betaGLU activities in any of the muscles studied and in some cases accentuated activities of some of these measures. Unlike previous findings following exercise in skeletal muscle, this study failed to demonstrate estrogen-induced attenuation of indices of neutrophil infiltration or damage in skeletal muscles of rats up to 72 hours following ischemia. This demonstrates that estrogen may not consistently attenuate neutrophil infiltration and that a number of variables including damage modality, tissue or estrogen level may influence this.

Topics: Animals; Biomarkers; Estrogens; Female; Glucosephosphate Dehydrogenase; Glucuronidase; Ischemia; Male; Muscle, Skeletal; Neutrophil Infiltration; Neutrophils; Peroxidase; Rats; Rats, Sprague-Dawley; Time Factors

Cyclooxygenase (COX)-2 has been identified as an important mediator elaborated during ischemia/reperfusion, with pro- and anti-inflammatory properties having been reported. As the role of COX-2 in the small intestine remains unclear, we hypothesized that COX-2 expression would mediate mesenteric ischemia/reperfusion-induced gut injury, inflammation, and impaired transit and that these deleterious effects could be reversed by the selective COX-2 inhibitor, N-[2-(cyclohexyloxy)-4-nitrophenyl] methanesulphanamide (NS-398). Additionally, we sought to determine the role of peroxisome proliferator-activated receptor gamma (PPARgamma) in mediating protection by NS-398 in this model. Rats underwent sham surgery or were pretreated with NS-398 (3, 10, or 30 mg/kg) intraperitoneally 1 h before 60 min of superior mesenteric artery occlusion and 30 min to 6 h of reperfusion. In some experiments, NS-398 (30 mg/kg) was administered postischemia. Ileum was harvested for COX-2 mRNA and protein, PGE2, myeloperoxidase (inflammation), histology (injury), intestinal transit and PPARgamma protein expression, and DNA-binding activity. COX-2 expression and PGE2 production increased after mesenteric ischemia/reperfusion and were associated with gut inflammation, injury, and impaired transit. Inhibition of COX-2 by NS-398 (30 mg/kg, but not 3 or 10 mg/kg) not only reversed the deleterious effects of COX-2, but additionally induced expression and nuclear translocation of PPARgamma. NS-398 given postischemia was equally protective. In conclusion, COX-2 may function as a proinflammatory mediator in a rodent model of mesenteric ischemia/reperfusion. Reversal of gut inflammation, injury, and impaired transit by high-dose NS-398 is associated with PPAR activation, suggesting a potential role for PPAR-gamma in shock-induced gut protection.

Topics: Active Transport, Cell Nucleus; Animals; Cell Nucleus; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Cytoplasm; Cytosol; Dinoprostone; DNA; DNA Primers; Dose-Response Relationship, Drug; Gastrointestinal Tract; Inflammation; Intestine, Small; Ischemia; Male; Mesentery; Nitrobenzenes; Peroxidase; PPAR gamma; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfonamides; Time Factors

Increased microvascular permeability and extravasation of inflammatory cells are key events of lung ischemia-reperfusion (IR) injury. The purpose of this study was to investigate the role of matrix metalloproteinases (MMP) in IR-induced alveolar capillary membrane disruption after experimental lung transplantation. We used a rat model of lung orthotopic transplantation (n = 86) with a prolonged cold ischemic phase. MMP2 and MMP9 were elevated 4 h after the onset of ischemia and further increased during reperfusion. Compared to sham values, the alveolar-capillary membrane permeability increased by 105% and 82.6% after 4 h of ischemia and 2 h or 24 h of reperfusion, respectively. A 4- and 5-fold increase of the infiltration of ischemic tissue by neutrophils was also observed after 2 h and 24 h of reperfusion. The PO2/FIO2 ratio dropped significantly from 244 to 76.6 after 2 h of reperfusion and from 296.4 to 127.6 after 24 h of reperfusion. A nonselective inhibitor of MMP, administered to the rats and added to the preservation solution, reduced significantly the alveolar-capillary leakage, the transmigration of neutrophils and improved gas exchanges in animals submitted to 4 h of ischemia combined with 2 h or 24 h of reperfusion. We conclude that inhibition of MMP attenuates IR injury after experimental lung transplantation.

Topics: Animals; Capillaries; Coloring Agents; Enzyme Inhibitors; Evans Blue; Hypoxia; Ischemia; Lung; Lung Transplantation; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Microscopy, Fluorescence; Neutrophils; Peroxidase; Placebos; Rats; Rats, Inbred F344; Reperfusion Injury; Time Factors

Antithrombin (AT) reveals its antiinflammatory activity by promoting endothelial release of prostacyclin (PGI(2)) in vivo. Since neuroinflammation is critically involved in the development of ischemia/reperfusion (I/R)-induced spinal cord injury (SCI), it is possible that AT reduces the I/R-induced SCI by attenuating the inflammatory responses. We examined this possibility using rat model of I/R-induced SCI in the present study. AT significantly reduced the mortality and motor disturbances by inhibiting reduction of the number of motor neurons in animals subjected to SCI. Microinfarctions of the spinal cord seen after reperfusion were markedly reduced by AT. AT significantly enhanced the I/R-induced increases in spinal cord tissue levels of 6-keto-PGFIalpha, a stable metabolite of PGI2. AT significantly inhibited the I/R-induced increases in spinal cord tissue levels of TNF-alpha, rat interleukin-8 and myeloperoxidase. In contrast,Trp(49) -modified AT did not show any protective effects. Pretreatment with indomethacin significantly reversed the protective effects of AT. An inactive derivative of factor Xa, which selectively inhibits thrombin generation, has been shown to fail to reduce SCI. Taken together, these observations strongly suggested that AT might reduce I/R-induced SCI mainly by the antiinflammatory effect through promotion of endothelial production of PGI(2). These findings also suggested that AT might be a potential neuroprotective agent.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Antithrombins; Coloring Agents; Disease Models, Animal; Epoprostenol; Factor Xa; Humans; Inflammation; Interleukin-8; Ischemia; Male; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Spinal Cord; Spinal Cord Injuries; Tetrazolium Salts; Time Factors; Tryptophan; Tumor Necrosis Factor-alpha

CD39 (ecto-nucleoside triphosphate diphosphohydrolase-1; E-NTPDase-1), is highly expressed on quiescent vascular endothelial cells and efficiently hydrolyzes extracellular ATP and ADP to AMP and ultimately adenosine. This action blocks extracellular nucleotide-dependent platelet aggregation and abrogates endothelial cell activation. However, CD39 enzymatic activity is rapidly lost following exposure to oxidant stress. Modulation of extracellular nucleotide levels may therefore play an important role in the pathogenesis of vascular injury. Acute ischemic injury of the bowel is a serious medical condition characterized by high mortality rates with limited therapeutic options. Here we evaluate the effects of cd39-deletion in mutant mice and the use of supplemental NTPDase or adenosine in influencing the outcomes of intestinal ischemia-reperfusion. Wild-type, cd39-null, or hemizygous cd39-deficient mice were subjected to intestinal ischemia. In selected animals, 0.2 U/g apyrase (soluble NTPDase) was administered prior to re-establishment of blood-flow. In parallel experiments adenosine/amrinone was infused over 60 min during reperfusion periods. Survival rates were determined, serum and tissue samples were taken. Intravital videomicroscopy and studies of vascular permeability were used to study platelet-endothelial cell interactions and determine capillary leakage. In wild-type animals, ischemia reperfusion injury resulted in 60% mortality within 48 hours. In mutant mice null or deficient for cd39, ischemia reperfusion-related death occurred in 80% of animals. Apyrase supplementation protected all wild-type animals from death due to intestinal ischemia but did not fully protect cd39-null and cd39-hemizygote mice. Adenosine/amrinone treatment failed to improve survival figures. In wild type mice, platelet adherence to postcapillary venules was significantly decreased and vascular integrity was well preserved following apyrase administration. In cd39-null mice, ischemia-reperfusion induced marked albumin leakage indicative of heightened vascular permaeability when compared to wild-type animals (p=0.04). Treatment with NTPDase or adenosine supplementation abrogated the increased vascular permeability in ischemic jejunal specimens of both wild-type mice and cd39-null. CD39 activity modulates platelet activation and vascular leak during intestinal ischemia reperfusion injury in vivo. The potential of NTPDases to maintain vascular integrity suggests potential pharm

Topics: Adenosine; Adenosine Diphosphate; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Blood Platelets; Capillaries; Cyclic AMP; Cytokines; Endothelium, Vascular; Gene Deletion; Intestine, Small; Ischemia; Male; Mice; Mice, Inbred C57BL; Microscopy, Video; Oxidative Stress; Permeability; Peroxidase; Platelet Activation; Reperfusion Injury; Time Factors; Vascular Endothelial Growth Factor A

Endogenous nitric oxide (NO) is known to modulate post-ischemic inflammatory response in various organs. However, the role of nitric oxide synthase isoforms (NOS) in mediating pulmonary post-ischemic inflammatory response is poorly understood. We therefore studied post-ischemic endothelial adhesion molecule expression and leukocyte migration in endothelial NOS knockout (eNOS-KO) mice subjected to pulmonary ischemia and reperfusion in vivo. Under anesthesia and mechanical ventilation, the left pulmonary hilum in wild-type (WT) and eNOS-KO mice was clamped for 1 hour, followed by reperfusion for up to 24 hours. In WT mice, we observed a selective up-regulation of both eNOS mRNA and protein in lung tissue, while inducible NOS (iNOS) and neuronal NOS (nNOS) remained unchanged. Survival in eNOS-KO mice was reduced due to severe pulmonary edema, underlining an increased susceptibility to ischemia-reperfusion (I/R) injury. Interstitial tissue infiltration by CD18- and CD11a-positive white blood cells as well as lung tissue water content peaked at 5 hours of reperfusion and were found significantly higher than in WT mice. Enhanced leukocyte-endothelial interaction was associated with pronounced up-regulation of vascular cell adhesion molecule (VCAM) in eNOS-KO mice during post-ischemic reperfusion. We conclude that eNOS attenuates post-ischemic inflammatory injury to the lung most probably via inhibition of endothelial adhesion molecule expression.

Topics: Animals; Body Water; Chemotaxis, Leukocyte; Disease Models, Animal; Female; Gene Expression Regulation, Enzymologic; Immunohistochemistry; Inflammation; Intercellular Adhesion Molecule-1; Ischemia; Isoenzymes; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Peroxidase; Reperfusion; Reverse Transcriptase Polymerase Chain Reaction; Vascular Cell Adhesion Molecule-1

Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA, plays an important role in the tissue injury associated with ischemia-reperfusion and inflammation. Splanchnic artery occlusion and reperfusion causes an enhanced formation of reactive oxygen species which contribute to the pathophysiology of shock. The aim of the present study was to investigate the effects of 5-aminoisoquinolinone (5-AIQ), a potent water-soluble inhibitor of poly(ADP-ribose) polymerase (PARP), in the pathogenesis of splanchnic artery occlusion shock. Splanchnic artery occlusion shock was induced in rats by clamping both the superior mesenteric artery and the celiac artery for 45 min, followed thereafter by release of the clamp (reperfusion). At 60 min after reperfusion, all animals were sacrificed for histological examination and biochemical studies. Treatment of rats with 5-AIQ (3 mg/kg i.v.), attenuated the fall of mean arterial blood pressure caused by splanchnic artery occlusion shock. 5-AIQ also attenuated the ileum injury as well as the increase in the tissue levels of myeloperoxidase and malondialdehyde caused by splanchnic artery occlusion shock in the ileum. The immunohistochemical examination also demonstrated a marked increase in the immunoreactivity to PAR, nitrotyrosine, and intercellular adhesion molecule (ICAM-1) in the necrotic ileum from splanchnic artery occlusion-shocked rats. 5-AIQ treatment significantly reduced the increase of positive staining for PAR, nitrotyrosine and ICAM-I. In conclusion, these results show that 5-AIQ, a new water-soluble potent inhibitor of poly(ADP-ribose) polymerase, exerts multiple protective effects in splanchnic artery occlusion/reperfusion shock.

Topics: Animals; Celiac Artery; Disease Models, Animal; Intercellular Adhesion Molecule-1; Intestine, Small; Ischemia; Isoquinolines; Lipid Peroxidation; Male; Malondialdehyde; Mesenteric Artery, Superior; Mesenteric Vascular Occlusion; Peroxidase; Poly(ADP-ribose) Polymerase Inhibitors; Rats; Rats, Sprague-Dawley; Reperfusion; Reperfusion Injury; Solubility; Tyrosine; Water

The role of 5-lipoxygenase (5-LOX) in the pathophysiology of renal ischemia/reperfusion (I/R) injury is not known. Here we investigate the effects of 1) the 5-LOX inhibitor zileuton and 2) 5-LOX gene knockout (5-LOX(-/-)) mice on renal dysfunction and injury caused by I/R of the kidney in mice. Wild-type mice treated with zileuton (3 mg/kg i.v.) or 5-LOX(-/-) mice were subjected to bilateral renal artery occlusion (30 min) followed by reperfusion (24 h). Plasma urea, creatinine, and aspartate aminotransferase (AST) were measured as markers of renal dysfunction and reperfusion injury. Kidneys were used for histological evaluation of renal injury. Renal myeloperoxidase activity was measured and used as an indicator of polymorphonuclear leukocyte (PMN) infiltration and renal expression of intercellular adhesion molecule-1 (ICAM-1) was determined using immunohistochemistry. Administration of zileuton before I/R significantly reduced the degree of renal dysfunction (urea, creatinine) and injury (AST, histology). In addition, zileuton reduced the expression of ICAM-1 and the associated PMN infiltration caused by I/R of the mouse kidney. Compared with wild-type mice, the degree of renal dysfunction, injury, and inflammation caused by I/R in 5-LOX(-/-) mice was also significantly reduced, confirming the pathophysiological role of 5-LOX in the development of renal I/R injury. We propose that 1) endogenous 5-LOX metabolites enhance the degree of renal injury, dysfunction, and inflammation caused by I/R of the kidney by promoting the expression of adhesion molecules, and 2) inhibitors of 5-LOX may be useful in the treatment of conditions associated with I/R of the kidney.

Topics: Animals; Arachidonate 5-Lipoxygenase; Disease Models, Animal; Hydroxyurea; Inflammation; Intercellular Adhesion Molecule-1; Ischemia; Kidney Diseases; Leukotriene B4; Lipoxygenase Inhibitors; Mice; Mice, Knockout; Neutrophils; Peroxidase; Reperfusion Injury

Aortic ischemia-reperfusion (AIR) induced lung injury has already been documented. Kupffer cell blockage (KCB) with gadolinium chloride (GdCl3) has also been shown to attenuate remote organ damage caused by ischemia reperfusion. The present study was designed to examine the effect of GdCl3 in lung ischemia-reperfusion injury induced by aortic occlusion. Thirty-two rats were randomly allocated to four groups as follows: SHAM (Sham Laparotomy), SHAM+KCB, AIR, and AIR+KCB. An atraumatic microvascular clamp was placed across the infrarenal abdominal aorta just after its origin from the aorta for 30 minutes. The microvascular clamp on the infrarenal abdominal aorta was removed and reperfused for 60 minutes. GdCl3 was given 24 hours prior to the experiment. Malondialdehyde (MDA) level and myeloperoxidase (MPO) activity were assayed in lung tissues. MDA level and MPO activity in the AIR group were significantly higher than those in the other groups. When compared to AIR group, KCB with GdCl3 significantly decreased MDA level and MPO activity in the AIR+KCB group. These results suggest that GdCl3 attenuates the lung injury caused by AIR. The effects of GdCl3 on reduced lung damage may be mediated through significant decreases in both MDA level and MPO activity.

Topics: Animals; Anti-Inflammatory Agents; Aorta; Arterial Occlusive Diseases; Female; Gadolinium; Ischemia; Lung Diseases; Male; Malondialdehyde; Peroxidase; Rats; Rats, Wistar; Reperfusion; Reperfusion Injury

Female mice are much more resistant to ischemia/reperfusion (I/R)-induced kidney injury when compared with males. Although estrogen administration can partially reduce kidney injury associated with I/R, we demonstrated that the presence of testosterone, more than the absence of estrogen, plays a critical role in gender differences in susceptibility of the kidney to ischemic injury. Testosterone administration to females increases kidney susceptibility to ischemia. Dihydrotestosterone, which can not be aromatized to estrogen, has effects equal to those of testosterone. Castration reduces the I/R-induced kidney injury. In contrast, ovariectomy does not affect kidney injury induced by ischemia in females. Testosterone reduces ischemia-induced activation of nitric oxide synthases (NOSs) and Akt and the ratio of extracellular signal related kinase (ERK) to c-jun N-terminal kinase (JNK) phosphorylation. Pharmacological (Nomega-nitro-L-arginine) or genetic (endothelial NOS or inducible NOS) inhibition of NOSs in females enhances kidney susceptibility to ischemia. Nitric oxide increases Akt phosphorylation and protects Madin-Darby canine kidney epithelial cells from oxidant stress. Antagonists of androgen or estrogen receptors do not affect the gender differences. In conclusion, testosterone inhibits the post-ischemic activation of NOSs and Akt and the ratio of ERK to JNK phosphorylation through non-androgen receptor-medicated mechanisms, leading to increased inflammation and increased functional injury to the kidney. These findings provide a new paradigm for the design of therapies for ischemia/reperfusion injury and may be important to our understanding of the pathophysiology of acute renal failure in pregnancy where plasma androgen levels are elevated.

Topics: Animals; Extracellular Signal-Regulated MAP Kinases; Female; Intercellular Adhesion Molecule-1; Ischemia; JNK Mitogen-Activated Protein Kinases; Kidney; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Peroxidase; Pregnancy; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Reperfusion Injury; Sex Characteristics; Testosterone

Volatile anesthetics protect against cardiac ischemia-reperfusion injury via adenosine triphosphate-dependent potassium channel activation. The authors questioned whether volatile anesthetics can also protect against renal ischemia-reperfusion injury and, if so, whether cellular adenosine triphosphate-dependent potassium channels, antiinflammatory effects of volatile anesthetics, or both are involved.. Rats were anesthetized with equipotent doses of volatile anesthetics (desflurane, halothane, isoflurane, or sevoflurane) or injectable anesthetics (pentobarbital or ketamine) and subjected to 45 min of renal ischemia and 3 h of reperfusion during anesthesia.. Rats treated with volatile anesthetics had lower plasma creatinine and reduced renal necrosis 24-72 h after injury compared with rats anesthetized with pentobarbital or ketamine. Twenty-four hours after injury, sevoflurane-, isoflurane-, or halothane-treated rats had creatinine (+/- SD) of 2.3 +/- 0.7 mg/dl (n = 12), 1.8 +/- 0.5 mg/dl (n = 6), and 2.4 +/- 1.2 mg/dl (n = 6), respectively, compared with rats treated with pentobarbital (5.8 +/- 1.2 mg/dl, n = 9) or ketamine (4.6 +/- 1.2 mg/dl, n = 8). Among the volatile anesthetics, desflurane demonstrated the least reduction in plasma creatinine after 24 h (4.1 +/- 0.8 mg/dl, n = 12). Renal cortices from volatile anesthetic-treated rats demonstrated reduced expression of intercellular adhesion molecule 1 protein and messenger RNA as well as messenger RNAs encoding proinflammatory cytokines and chemokines. Volatile anesthetic treatment reduced renal cortex myeloperoxidase activity and reduced nuclear translocation of proinflammatory nuclear factor kappaB. Adenosine triphosphate-dependent potassium channels are not involved in sevoflurane-mediated renal protection because glibenclamide did not block renal protection (creatinine: 2.4 +/- 0.4 mg/dl, n = 3).. Some volatile anesthetics confer profound protection against renal ischemia-reperfusion injury compared with pentobarbital or ketamine anesthesia by attenuating inflammation. These findings may have significant clinical implications for anesthesiologists regarding the choice of volatile anesthetic agents in patients subjected to perioperative renal ischemia.

Topics: Anesthetics, Inhalation; Animals; ATP-Binding Cassette Transporters; Creatinine; Cytokines; Electrophoretic Mobility Shift Assay; Immunoblotting; Inflammation; Inflammation Mediators; Intercellular Adhesion Molecule-1; Ischemia; KATP Channels; Kidney; Kidney Cortex; Kidney Diseases; Kidney Function Tests; Male; Necrosis; NF-kappa B; Peroxidase; Potassium Channels; Potassium Channels, Inwardly Rectifying; Proteins; Rats; Rats, Sprague-Dawley; Renal Circulation; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Tumor Necrosis Factor-alpha

The objective of this study was to define the relationship among Kupffer cells, O(2)(-) production, and TNF-alpha expression in the pathophysiology of postischemic liver injury following short and long periods of ischemia. Using different forms of superoxide dismutase with varying circulating half-lives, a monoclonal antibody directed against mouse TNF-alpha, and NADPH oxidase-deficient mice, we found that 45 or 90 min of partial (70%) liver ischemia and 6 h of reperfusion (I/R) produced time-dependent increases in liver injury and TNF-alpha expression in the absence of neutrophil infiltration. Furthermore, we observed that hepatocellular injury induced by short periods of ischemia were not dependent on formation of TNF-alpha but were dependent on Kupffer cells and NADPH oxidase-independent production of O(2)(-). However, liver injury induced by extended periods of ischemia appeared to require the presence of Kupffer cells, NADPH oxidase-derived O(2)(-), and TNF-alpha expression. We conclude that the sources for O(2)(-) formation and the relative importance of TNF-alpha in the pathophysiology of I/R-induced hepatocellular injury differ depending on the duration of ischemia.

Topics: Alanine Transaminase; Animals; Cytokines; Hepatectomy; Humans; Ischemia; Kupffer Cells; Liver; Liver Circulation; Mice; Mice, Inbred C57BL; Mutation; NADPH Oxidases; Oxygen Consumption; Peroxidase; Reactive Oxygen Species; Superoxide Dismutase; Time Factors; Tumor Necrosis Factor-alpha

Antithrombin (AT) supplementation in patients with severe sepsis has been shown to improve organ failures in which activated leukocytes are critically involved. However, the precise mechanism(s) for the therapeutic effects of AT is not well understood. We examined in rats whether AT reduces ischemia/reperfusion (I/R)-induced renal injury by inhibiting leukocyte activation. AT markedly reduced the I/R-induced renal dysfunction and histologic changes, whereas neither dansyl glutamylglycylarginyl chloromethyl ketone-treated factor Xa (DEGR-F.Xa), a selective inhibitor of thrombin generation, nor Trp49-modified AT, which lacks affinity for heparin, had any effect. Renal tissue levels of 6-keto-PGF(1 alpha), a stable metabolite of prostacyclin (PGI(2)), increased after renal I/R. AT enhanced the I/R-induced increases in renal tissue levels of 6-keto-PGF(1 alpha), whereas neither DEGR-F.Xa nor Trp49-modified AT had any effect. AT significantly inhibited I/R-induced decrease in renal tissue blood flow and the increase in the vascular permeability. Ischemia/reperfusion-induced increases in renal tissue levels of tumor necrosis factor-alpha, cytokine-induced neutrophil chemoattractant, and myeloperoxidase were significantly inhibited in animals given AT. Pretreatment of animals with indomethacin reversed the effects induced by AT. Iloprost, an analog of PGI(2), produced effects similar to those induced by AT. These observations strongly suggest that AT reduces the I/R-induced renal injury by inhibiting leukocyte activation. The therapeutic effects of AT might be mainly mediated by PGI(2) released from endothelial cells through interaction of AT with cell surface glycosaminoglycans.

Topics: 6-Ketoprostaglandin F1 alpha; Amino Acid Chloromethyl Ketones; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antithrombin III; Capillary Permeability; Drug Evaluation, Preclinical; Endothelium, Vascular; Epoprostenol; Factor Xa; Iloprost; Indomethacin; Ischemia; Kidney; Lymphocyte Activation; Male; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Specific Pathogen-Free Organisms; Tumor Necrosis Factor-alpha

We have shown previously that ischemia results in reactive oxygen species production by lung endothelium that occurs within 3-5 s after flow cessation and is followed by lipid peroxidation at 15-30 min as determined by assay of thiobarbituric acid-reactive substances, conjugated dienes, and protein carbonyls in lung homogenate. The present study evaluated membrane lipid peroxidation in isolated, ventilated rat lungs using a fluorescence imaging method that permits continuous observation of pulmonary subpleural microvascular endothelial cells in situ. Diphenyl-1-pyrenylphosphine (DPPP), a fluorescent probe which localizes in the plasma membrane and shows increased fluorescence emission after its oxidation by lipid hydroperoxides, was used for detection of membrane lipid peroxidation. Compared to continuously perfused control lungs, endothelial cell DPPP fluorescence increased significantly within 1 min of ischemia (i.e., flow cessation); these changes were prevented by pretreatment with 0.5 mM alpha-tocopherol succinate (vitamin E) added to the perfusate. Increased DPPP fluorescence was confirmed by spectrofluorometry of lipid extracts of lung homogenates. These data indicate that DPPP can be used for the real-time detection of lipid peroxidation in an intact organ. Ischemia results in peroxidation of the pulmonary microvascular endothelial cell membrane and this insult can be detected as early as 1 min after the onset of ischemia compatible with a radical-mediated process.

Topics: Animals; Antioxidants; Cell Membrane; Endothelium, Vascular; Fluorescence; Fluorescent Antibody Technique; Fluorescent Dyes; Hypoxia; Immunoenzyme Techniques; Intracellular Membranes; Ischemia; Lipid Peroxidation; Lipid Peroxides; Lung; Male; Organophosphorus Compounds; Oxidants; Peroxidase; Pyrenes; Rats; Rats, Sprague-Dawley; Vitamin E

This study was designed to study the effects of Melatonin (Mel) and N-Acetylcystein (NAC) on hepatic ischemia/reperfusion (I/R) injury in rats. For this purpose Wistar albino rats were subjected to 45 minutes of hepatic ischemia followed by 60 minutes of reperfusion period. Melatonin (10 mg/kg) or NAC (150 mg/kg) were administered alone or in combination, intraperitoneally, 15 minutes prior to ischemia and just before reperfusion. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were determined to assess liver functions. Liver tissues were taken for determination of malondialdehyde (MDA) levels, an end product of lipid peroxidation; glutathione (GSH) levels, a key antioxidant; protein carbonyl concentration (protein oxidation) (PO), a specific marker of oxidative damage of proteins; and myeloperoxidase (MPO) activity, as an indirect index of neutrophil infiltration. Plasma ALT and AST activities were higher in ischemia/reperfusion group than in control. They were decreased in the groups given Mel, NAC or the combination. Hepatic GSH levels, significantly depressed by I/R, were elevated to control levels in the combination group, whereas treatment with Mel or NAC alone provided only a limited protection. Hepatic MDA and PO levels, and MPO activity were significantly increased by I/R. The increase in these parameters were partially decreased by Mel or NAC alone, whereas treatment with the combination reduced these values back to control levels. In conclusion, considering the dosages used, Mel appeared to be significantly more potent than NAC in reversing the oxidative damage induced by I/R. Our findings show that Mel and NAC have beneficial effects against the I/R injury and due to their synergistic effects, when administered in combination, may have a more pronounced protective effects on the liver.

Topics: Acetylcysteine; Animals; Antioxidants; Biomarkers; Female; Free Radical Scavengers; Glutathione; Ischemia; Lipid Peroxidation; Liver; Liver Circulation; Liver Function Tests; Male; Malondialdehyde; Melatonin; Neutrophil Infiltration; Oxidation-Reduction; Peroxidase; Proteins; Rats; Rats, Wistar; Reperfusion Injury

We investigated kidney and lung alterations caused by intercellular adhesion molecule type 1 (ICAM-1) blockade after ischemia and reperfusion of hind limb skeletal muscles. Rats were submitted to ligature of the infrarenal aorta for 6 h. The animals were randomized into three groups of 6 rats each: group I, sacrificed after ischemia; group II, reperfusion for 24 h, and group III, reperfusion for 24 h after receiving monoclonal anti-ICAM-1 antibodies. At the end of the experiment, blood samples were collected for creatinine, lactate dehydrogenase, creatine phosphokinase, potassium, pH and leukocyte counts. Samples were taken from the muscles of the hind limbs and from the kidneys and lungs for histological analysis and measurement of the neutrophil infiltrate by myeloperoxidase staining. The groups did not differ significantly with regard to the laboratory tests. There were no major histological alterations in the kidneys. An intense neutrophil infiltrate in the lungs, similar in all groups, was detected. Myeloperoxidase determination showed that after reperfusion there was significantly less retention of polymorphonuclear neutrophils in the muscles (352 +/- 70 vs 1451 +/- 235 x 10(2) neutrophils/mg; P<0.01) and in the kidneys (526 +/- 89 vs 852 +/- 73 10(2) neutrophils/mg; P<0.01) of the animals that received anti-ICAM-1 before perfusion compared to the group that did not. The use of anti-ICAM-1 antibodies in this experimental model minimized neutrophil influx, thus reducing the inflammatory process, in the muscles and kidneys after ischemia and reperfusion of the hind limbs.

Topics: Animals; Antibodies, Monoclonal; Cell Adhesion; Hindlimb; Intercellular Adhesion Molecule-1; Ischemia; Kidney; Lung; Muscle, Skeletal; Neutrophils; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury

Statins are lipid-lowering drugs with anti-inflammatory and antioxidant properties. This study explores the potential of these commonly prescribed agents to ameliorate lung ischemia-reperfusion injury.. Left lungs of Long-Evans rats were rendered ischemic for 90 minutes and reperfused for up to 4 hours. Treated animals received simvastatin orally (0.5 mg/kg) for 5 days before the experiment. Injury was quantitated in terms of tissue myeloperoxidase content, vascular permeability ((125)I bovine serum albumin extravasation), and bronchoalveolar lavage leukocyte and cytokine content. Changes in nuclear translocation of transcription factors were evaluated by electromobility shift assay. Additional animals received N(G)-nitro-L-arginine methyl ester before ischemia-reperfusion to assess whether inhibition of nitric oxide synthase could reverse simvastatin's protective effects. The presence of nicotinamide adenine dinucleotide phosphate oxidase was also evaluated using enzyme staining both histologically and in native electrophoresis.. Lung vascular permeability was reduced in treated animals by 71% compared with positive controls (P <.001). Administration of N(G)-nitro-L-arginine methyl ester reversed this protection. The protective effects of statin pretreatment correlated with a 68% reduction in tissue myeloperoxidase content (P <.01), marked reductions in bronchoalveolar lavage leukocyte accumulation, and decreased expression of proinflammatory cytokines. Nicotinamide adenine dinucleotide phosphate oxidase expression also decreased with statin treatment.. In addition to its antioxidant properties, the protective effects of simvastatin are likely mediated by modulation of endothelial nitric oxide synthase. The potential to pretreat recipients of lung transplantation with statins to ameliorate reperfusion injury is promising.

Topics: Animals; Bronchoalveolar Lavage Fluid; Capillary Permeability; Cell Count; Cytokines; Disease Models, Animal; Enzyme Inhibitors; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypolipidemic Agents; Ischemia; Lung; Lung Diseases; Male; Models, Cardiovascular; NADPH Oxidases; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Peroxidase; Rats; Rats, Long-Evans; Reperfusion Injury; Simvastatin; Statistics as Topic; Time Factors; Transcription Factors; Treatment Outcome

Acutely increased intra-abdominal pressure (IAP) can lead to multiple organ failure. As blood flow to intra-abdominal organs is reduced by high venous resistance, ischemia-reperfusion (I/R) injury plays an important role in the pathogenesis of abdominal compartment syndrome (ACS) following IAP. Melatonin, a secretory product of the pineal gland, is known to have free radical scavenging and antioxidative properties in several oxidative processes. The objective of this study was to examine the potential protective properties of melatonin on the oxidative organ damage in a rat model of ACS. Under ketamine anesthesia, an arterial catheter was inserted intraperioneally (i.p.) and using an aneroid manometer connected to the catheter, IAP was kept at 20 mmHg (ischemia group; I) for 1 hr. In the ischemia/reperfusion (I/R) group, pressure applied for an hour was decompressed and a 1-hr reperfusion period was allowed. In another IR group, melatonin was administered (10 mg/kg, i.p.) immediately before the decompression of IAP. The results demonstrate that tissue levels of malondialdehyde (MDA) and myeloperoxidase activity (MPO; index of tissue neutrophil infiltration) were elevated, while glutathione (GSH; a key to antioxidant) levels were reduced in both I and I/R groups (P < 0.05-0.001). Melatonin treatment in I/R rats reversed these changes (P < 0.01-0.001). Moreover, melatonin given to the I/R group reduced the elevations in serum aspartate aminotransferase, alanine aminotransferase and blood urea nitrogen levels and abolished the increase in serum creatinine levels. Our results indicate that melatonin, because of antioxidant and free radical scavenging properties, ameliorates reperfusion-induced oxidative organ damage. In conclusion, the results of the present study suggest that the therapeutic value of melatonin as a 'reperfusion injury-limiting' agent must be considered in ACS.

Topics: Abdomen; Animals; Compartment Syndromes; Free Radical Scavengers; Glutathione; Ischemia; Lipid Peroxidation; Malondialdehyde; Melatonin; Oxidative Stress; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury

To assess the physiologic and biochemical changes resulting from ischemia and reperfusion. Effective therapy for ischemic priapism reestablishes corporal venous outflow and arterial inflow and results in increased corporal partial pressure of oxygen. Data are limited concerning reperfusion injury of ischemic erectile tissue associated with reactive oxygen species (ROS) and the potential role of ROS scavengers in the clinical therapy of ischemic priapism.. Anesthetized adult New Zealand white male rabbits (n = 7) were exposed to a low oxygen tension breathing gas to achieve hypoxia within the corpora cavernosa. This resulted in a mean systemic oxygen saturation of 60%. The pelvic nerve was electrically stimulated to induce penile erection, and the base of the erect penis was clamped. After varying durations of ischemia, the clamp was removed to allow reperfusion. We determined the intracavernosal oxygen tension, histologic changes, myeloperoxidase activity, and lipid peroxidation.. Corporal partial pressure of oxygen progressively decreased as the duration of priapism increased. A statistically significant increase was noted in myeloperoxidase activity and lipid peroxidation with corporal reperfusion. Polymorphonuclear leukocyte infiltration was documented in the ischemic reperfused tissue.. In the management of ischemic priapism, reperfusion causes erectile tissue injury owing to the presence of ROS. There is a need to investigate the utility of ROS scavengers and antioxidants in the management of ischemic priapism.

Topics: Animals; Chemotaxis, Leukocyte; Hypoxia; Ischemia; Lipid Peroxidation; Male; Models, Animal; Neutrophils; Oxidative Stress; Penis; Peroxidase; Priapism; Rabbits; Reactive Oxygen Species; Reperfusion Injury

The aim of this study was to evaluate the effect of allopurinol, methylene blue, and a monoclonal antibody to the adhesion molecule ICAM-1 in intestinal ischemia and reperfusion injury.. The rats were divided into 5 groups. CG (n = 8) was untreated controls, SISG (n = 11) received sterile isotonic saline solution, ICAMG (n = 12) received a monoclonal antibody to rat ICAM-1, ALLOG (n = 12) received allopurinol, and MBG (n = 14) received methylene blue. Intestinal ischemia was performed for 60 minutes followed by 60 minutes of reperfusion. The agents were injected 10 minutes before the reperfusion to animals. After 60 minutes of reperfusion, the plasma samples for myeloperoxidase (MPO) activity, tumor necrosis factor alpha (TNF-alpha) and uric acid levels, and the intestinal biopsies of ileum and jejunum for histopathologic examination were taken.. The mucosal damage was attenuated, and TNF-alpha level significantly decreased in ALLOG and ICAMG compared with SISG. The MPO activity was the lowest in ICAMG, and uric acid level was significantly decreased in ALLOG compared with the other groups. Methylene blue decreased TNF-alpha response to reperfusion injury but significantly increased the grade of the mucosal damage and the MPO activity.. This study shows that prereperfusion application of allopurinol and monoclonal antibody to the adhesion molecule ICAM-1 may attenuate the damage caused by intestinal ischemia and reperfusion, but the different time-points for application, the effects observed in the different ischemia and reperfusion durations, and the long-term results also should be investigated in the same experimental model before the final conclusion. Methylene blue was not effective to prevent or attenuate the intestinal tissue injury, but because this was the first study examining the effect of methylene blue on intestinal reperfusion injury, further studies with the different doses, ischemic duration, and application times will be needed.

Topics: Allopurinol; Animals; Antibodies, Monoclonal; Drug Evaluation, Preclinical; Ileum; Intercellular Adhesion Molecule-1; Intestines; Ischemia; Jejunum; Male; Methylene Blue; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tumor Necrosis Factor-alpha; Uric Acid

To investigate the effect of liposome-carried metallothionein (lipo-MT) on secondary ischemia-reperfusion injury in the rat island flap.. An abdominal island flap was created in the Wistar rat. The animals were divided into four groups: the sham group, the secondary ischemia-reperfusion group, the group treated with blank liposome and the group treated with lipo-MT. The malondialdehyde (MDA) content, the myeloperoxidase (MPO) activity was assayed immediately, at 30 minutes and 7 days after the secondary venous ischemia-reperfusion. The level of endothelin (ET) and lactic dehydrogenase (LDH) of the rat plasma was measured at 30 minutes after secondary venous ischemia-reperfusion. The content of MT of the flap was assayed by Cd-hemoglobin saturation method at 7 days after the operation.. The treatment of lipo-MT significantly decreased the content of MDA, MPO of the flap, decreased the activity of ET, LDH of the rat palsma, increased the content of MT of the flap and improved the flap viability.. Lipo-MT can improve flap survival by reducing ischemia-reperfusion injury.

Topics: Animals; Endothelins; Graft Survival; Ischemia; Lactate Dehydrogenases; Liposomes; Malondialdehyde; Metallothionein; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Surgical Flaps; Time Factors

To investigate the effects of carbachol on local inflammation in gut tissue during ischemia/reperfusion (I/R).. A jejunal sac was formed in Wistar rats. The superior mesenteric artery was occluded (SMAO) for 60 minutes followed by reperfusion for another 60 minutes. Animals were divided into three groups, pretreatment group (carbachol was injected into the jejunal sac 30 minutes after SMAO, 0.1 mg/kg), treatment group (carbachol was injected in same dosage into the jejunal sac 30 minutes after reperfusion), and controls (saline injection). The contents of tumor necrosis factor-alpha(TNF-alpha) and activity of myeloperoxidase (MPO) in gut tissue were determined at 1 hours, 2.5 hours and 6 hours after SMAO.. The contents of TNF-alpha and activity of MPO were significantly decreased in pretreatment and treatment groups compared with control group at 2.5 hours after SMAO (both P<0.05). There were no differences in both contents between pretreatment group and treatment group at any specified time. It was also found that there were less inflammatory pathological changes in the gut tissues in the two treated groups than that of control.. The RESULTS suggest that carbachol could alleviate gut inflammatory response during gut ischemia/reperfusion injury by inhibiting proinflammatory cytokine release.

Topics: Animals; Carbachol; Gastroenteritis; Intestines; Ischemia; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Tumor Necrosis Factor-alpha

Renal ischemia/reperfusion injury could arise as a consequence of clinical conditions such as renal transplantation, shock, cardiac arrest, hemorrhage and renal artery surgery. In this experimental study, we aimed to determine the preventive effects of amrinone on bilateral renal ischemia/reperfusion injury in rats. A total of 60 Wistar-albino rats were divided into six groups ( n=10). Midline laparotomies were made under ketamine anesthesia. In the sham, amrinone1 and amrinone2 without ischemia (AWI1 and AWI2) groups saline, 5 and 10 mg/kg of amrinone was infused, respectively. In the ischemia, ischemia plus amrinone1 (IPA1) and ischemia plus amrinone2 (IPA2) groups, saline and 5 and 10 mg/kg of amrinone was infused, respectively, at the beginning of reperfusion, subsequent to 45 min of bilateral renal artery occlusion. Following 6 h of reperfusion, blood was drawn to study serum BUN and creatinine and a bilateral nephrectomy was done to determine tissue malonyldialdehyde ( MDA) and myeloperoxidase (MPO) levels. The results were analysed by Mann-Whitney U-test. The parameters studied were statistically higher in the ischemia group compared with the other groups ( P<0.05 for each comparison), indicating renal I/R injury. These parameters were lower in the amrinone without ischemia groups (AWI1 and AWI2) than in the sham group, however there were no significant differences between the groups ( P>0.05, for each comparison). The treatment groups IPA1 and IPA2 had statistically similar results compared with the sham group, showing the preventive effect of amrinone on renal I/R injury at the given doses. We conclude that amrinone prevented experimental renal ischemia/reperfusion injury in rats, independently of the administered doses. This preventive effect of the agent could depend on its effect of regulating the microcirculation, in decreasing intracellular calcium and in preventing neutrophil activation. We propose that this preventive effect of amrinone - which has gained clinical application especially in cases of cardiac insufficiency - could also be exploited in clinical conditions related with renal ischemia/reperfusion.

Topics: Amrinone; Animals; Blood Urea Nitrogen; Creatinine; Dose-Response Relationship, Drug; Ischemia; Isomerism; Malondialdehyde; Peroxidase; Phosphodiesterase Inhibitors; Rats; Rats, Wistar; Renal Circulation; Reperfusion Injury

The aim of this study was to investigate the role of inducible nitric oxide (NO) synthase (iNOS) and NO on the modulation of the inflammatory response caused by splanchnic ischemia and reperfusion. A severe model of mesenteric ischemia and reperfusion was produced by subjecting mice to 45 min occlusion followed by reperfusion of the superior mesenteric artery and celiac trunk. In this experimental protocol, wild-type mice treated with GW274150 (5 mg/kg i.p.), a novel, potent, and selective inhibitor of iNOS activity, and mice lacking of the gene for iNOS (iNOS 'knock-out', iNOS-KO) exhibited no difference in the rate of mortality in comparison with wild-type control mice. In a second study, using a less severe model of mesenteric injury obtained by occlusion of the superior mesenteric artery only for 45 min, we evaluated the survival rate. Under these conditions, wild-type mice treated with GW274150 and iNOS-KO mice showed a significant difference in the rate of mortality in comparison with wild-type. Therefore, wild-type mice treated with GW274150 and iNOS-KO mice when compared with wild-type littermates showed a significant reduction of the mesenteric injury, upregulation of P-selectin and intercellular adhesion molecule-1, and neutrophil infiltration, as well as a significant inhibition of the degree of oxidative and nitrosative damage, indicated by malondialdehyde levels, formation of nitrotyrosine and poly(ADP-ribose)polymerase (PARP), respectively. Plasma levels of the proinflammatory cytokines tumour necrosis factor-alpha, interleukin (IL) 6, and IL-1beta were also significantly reduced in iNOS-KO mice in comparison with control wild-type mice. Wild-type mice treated with GW274150 and iNOS-KO mice were also found to have reduced activation of the transcriptional factor nuclear factor-kappaB in the ileum. These results suggest that the induction of iNOS and NO production are essential for the upregulation of the inflammatory response in splanchnic ischemia/reperfusion and participate in end organ damage under these conditions.

Topics: Animals; Cytokines; Disease Models, Animal; Immunohistochemistry; Inflammation Mediators; Ischemia; Lipid Peroxidation; Male; Malondialdehyde; Mesenteric Arteries; Mesenteric Vascular Occlusion; Mice; Mice, Knockout; Neutrophil Infiltration; Nitric Oxide; Nitric Oxide Synthase; P-Selectin; Peroxidase; Reperfusion Injury; Sensitivity and Specificity; Survival Rate

Ischemia/reperfusion (I/R) injury induces an inflammatory response and production of reactive oxygen species (ROS), which affects the organs remote to the sites of I/R. The aim was to assess the hepatic changes after renal I/R injury.. Twenty mice were subjected to either sham operation or varying degrees of renal I/R injury. Hepatic TNF-alpha levels, myeloperoxidase (MPO), superoxide dismutase (SOD), and catalase (CAT) activities and reduced glutathione (GSH) levels, thiobarbituric acid-reactive substances (TBARS), and protein carbonyl levels were evaluated to show hepatic response to renal I/R injury.. Hepatic tumor necrosis factor-alpha levels were found to be increased significantly after 30 min ischemia-1 h reperfusion and remained elevated through 60 min ischemia-1 h reperfusion. Supporting the neutrophil recruitment, about 10-fold increase in MPO activity was detected after 30 min ischemia-1 h reperfusion. Antioxidant enzymes were detected to be decreased after 30 min ischemia-1 h reperfusion and reached to the minimum levels after 60 min ischemia-1 h reperfusion. Decreased levels of GSH and increased levels of TBARS and protein carbonyls after 60 min ischemia-1 h reperfusion supported the ROS-mediated biomolecular alterations.. A minumum of 30 min ischemia-1 h reperfusion is enough to elicit remote effects of renal I/R injury. Care should be taken to protect other organs remote from I/R sites especially during renal surgery.

Topics: Animals; Catalase; Ischemia; Kidney; Lipid Peroxidation; Liver; Male; Mice; Oxidation-Reduction; Peroxidase; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Tumor Necrosis Factor-alpha

Prior ischemia leads to resistance against subsequent ischemic insults. The mechanisms that underlie this adaptive response remain unidentified. Thus, we studied whether the reduced susceptibility of mice previously subjected to the ischemia to ischemia/ reperfusion injury is related with altered inflammatory responses. Thirty minutes of bilateral kidney ischemia results in significantly increased plasma creatinine and blood urea nitrogen levels in BALB/c male mice. There is severe disruption of actin cytoskeleton of proximal tubular cells in the outer stripe of the outer medulla 24 hours post-ischemia. When mice are subjected to 30 minutes of bilateral ischemia 8 days later, there is no increase in plasma creatinine and blood urea nitrogen levels and the post-ischemic disruption of actin cytoskeleton of proximal tubular cells is much less. Inflammatory responses have highly implicated with ischemia/reperfusion injury. Ischemia results in the increased tissue myeloperoxidase (MPO) activity that is a marker of leukocyte infiltration. There is, however, no the post-ischemic increase of MPO activity in kidneys previously subjected to ischemia. Post-ischemic expression of tissue intercellular adhesion molecule-1 (ICAM-1) is greater in the kidney previously sham-operated than in the kidneys previously subjected to ischemia. In conclusion, prior ischemia protects kidney function and morphology against subsequent ischemia 8 days later. The resistance is associated with the reduced post-ischemic leukocyte infiltration due to the reduced post-ischemic ICAM-1 expression.

Topics: Adaptation, Physiological; Animals; Blood Urea Nitrogen; Creatinine; Gene Expression Regulation; Inflammation; Intercellular Adhesion Molecule-1; Ischemia; Kidney; Kidney Tubules, Proximal; Male; Mice; Mice, Inbred BALB C; Peroxidase; Renal Circulation; Reperfusion Injury

In order to ascertain whether erythropoietin plays a role in early and late repair processes following ischaemic skin flap injury, a dorsal, caudally based skin flap was created in rats. The rats were successively divided into four groups. Group 1 was not treated. The other groups were treated with a subcutaneous administration of 0.9% NaCl saline solution (group 2), a subcutaneous administration of vehicle (group 3) or a subcutaneous administration of 300 IU/kg/day of recombinant human erythropoietin (group 4). We evaluated the possible relationships between neutrophil accumulation, myeloperoxidase activity and content in flap tissue, flap survival, flap temperature (using telethermography) and flap revascularization (using videocapillaroscopy). Necrosis in the flap was significantly less extensive in group 4 than in groups 1, 2 and 3. A significant increase in neutrophil infiltration occurred between the 1st and 24th hour in these groups, but this was not observed in group 4. These findings were confirmed by biochemical data of myeloperoxidase activity and malonyldialdehyde content. Between the 1st and 7th days, we recorded an increase of about 20% in flap temperature in groups 1, 2 and 3, whereas no significant variation was observed in group 4. On the 7th day, videocapillaroscopic findings showed an increase in the mean vascularization index in group 4. Our findings suggest that recombinant human erythropoietin administration can improve the wound healing process, in both early and late stages of injury, by reducing inflammatory response, increasing the density of capillaries in ischaemic flaps and allowing earlier repair of a damaged area.

Topics: Animals; Erythropoietin; Ischemia; Malondialdehyde; Microscopic Angioscopy; Neovascularization, Physiologic; Peroxidase; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Skin; Surgical Flaps; Thermography; Wound Healing

Multiple organ dysfunction syndrome (MODS) is mediated by complex mechanisms in which interactions between activated leukocytes and endothelial cells play a central role. ICAM-1 (intercellular adhesion molecule-1) mediates firm adhesion and transendothelial migration of activated leukocytes from postcapillary venules into the tissue. The present study evaluated the ICAM-1 expression in various organs after 40 min of intestinal ischemia and 1, 3, 6, 12 h of reperfusion (I/R) in the rat, using a dual monoclonal antibody technique (n = 36). Endothelial barrier permeability, using the vascular leakage of radiolabeled human serum albumin was also assessed (n = 12). Neutrophil sequestration in the lungs was quantitated by myeloperoxidase activity and plasma protease inhibitor levels were measured with electroimmunoassay. Significant regional differences were found in ICAM-1 expression between organs, both constitutively and after I/R-injury. The highest constitutive levels were observed in the liver and lungs, followed by the kidneys. The constitutive ICAM-1 expression in the intestines and in the heart was about 1/20 compared with that found in the liver and lungs. The brain and muscle had levels of about 1/150 of that in the liver and lungs. After intestinal I/R, significant increases (17-45%) were found in the lungs, intestines, brain, heart, and muscle. Albumin leakage index (ALI) in all examined organs and myeloperoxidase activity in the lungs increased after I/R-injury. Serum levels of albumin and most protease inhibitors decreased significantly after I/R challenge. Intestinal I/R results in an increase of systemic ICAM-1 expression with marked organ variability. The upregulation of ICAM-1 could represent a crucial step in the adherence- and migration process of activated leukocytes and potentially in the development of tissue injury.

Topics: Albumins; Animals; Antibodies, Monoclonal; Endothelium, Vascular; Inflammation; Intercellular Adhesion Molecule-1; Intestinal Mucosa; Intestines; Ischemia; Male; Molecular Biology; Neutrophils; Permeability; Peroxidase; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Reperfusion Injury

We tested the hypothesis that occlusion of the superior mesenteric artery induces late preconditioning against myocardial infarction and examined the effects of pharmacological modifiers of inducible nitric oxide synthase activity on the late preconditioning in anesthetized rats.. Rats underwent an intestinal ischemia preconditioning protocol (30 min occlusion of the superior mesenteric artery) or were sham-operated. They were subjected to a sustained 30 min of coronary occlusion and 180 min of reperfusion 24 h later.. In rats receiving no pharmacological intervention, the percentage of myocardial infarct within the area at risk and left ventricle was 72+/-4% and 31+/-2%, respectively, in sham-operated rats, and these were significantly reduced to 44+/-4% and 23+/-2% (P<0.01) 24 h after intestinal ischemia preconditioning. Myeloperoxidase activity was significantly reduced by intestinal ischemia preconditioning. Administration of aminoguanidine (300 mg/kg, s.c.) or S-methylisothiourea sulfate (3 mg/kg, i.v.), both relative inducible NO synthase inhibitors, 60 or 30 min before sustained myocardial ischemia not only abolished the late preconditioning afforded by intestinal ischemia, but also inhibited the ability of intestinal ischemia preconditioning to significantly reduce neutrophil infiltration. A change in inducible NO synthase activity was not observed in normal myocardium 24 h after intestinal ischemia, but 30 min of coronary occlusion significantly increased the inducible NO synthase activity in the preconditioned group, which was abolished by aminoguanidine or S-methylisothiourea sulfate.. These data provide pharmacological evidence that induction of inducible nitric oxide synthase, following intestinal ischemia, is associated with increased myocardial tolerance to infarction 24 h later.

Topics: Analysis of Variance; Animals; Enzyme Inhibitors; Guanidines; Intestinal Mucosa; Intestines; Ischemia; Ischemic Preconditioning, Myocardial; Isothiuronium; Male; Mesenteric Artery, Superior; Myocardial Ischemia; Myocardium; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Peroxidase; Rats; Rats, Sprague-Dawley

To determine effect of leukocyte depletion on hematologic, morphologic, and metabolic variables of equine jejunum after induction of arterial low-flow ischemia and reperfusion by use of an extracorporeal circuit.. 14 healthy adult horses.. A segment of jejunum was surgically removed and maintained in an isolated circuit for 3 hours (control group), arterial flow was reduced to 20% of baseline for 40 minutes followed by 1 hour of reperfusion (low-flow group), or leukocyte depletion was filter-induced, and low-flow ischemia and reperfusion were conducted as in the low-flow control group (filter-treated group). Various metabolic, hemodynamic, and histomorphologic variables were evaluated, including effects of electrical field stimulation and L-N-nitro-arginine-methyl-ester (L-NAME) on contractile activity.. The extracorporeal circuit appeared to maintain the jejunum within physiologic limits for an extended period. Low-flow ischemia with reperfusion induced significant differences in various measurements, compared with control specimens. Significant differences were not detected between the low-flow and filter-treated groups. Myeloperoxidase activity was greater in the low-flow group than the control group, whereas a difference was not detected between control and filter-treated groups.. The extracorporeal circuit maintained intestine for 3 hours in a physiologic state and may be used for simulation of tissue injury. Leukocyte depletion generally did not attenuate the effects of low-flow ischemia and reperfusion on equine small intestine.

Topics: Animals; Blood Flow Velocity; Blood Pressure; Colon; Electric Stimulation; Extracorporeal Circulation; Horses; In Vitro Techniques; Intestinal Mucosa; Ischemia; Jejunum; Leukocytes; Models, Biological; Muscle Contraction; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Peroxidase; Regional Blood Flow; Reperfusion

We sought to determine whether mice deficient in the proinflammatory caspase-1, which cleaves precursors of IL-1 beta and IL-18, were protected against ischemic acute renal failure (ARF). Caspase-1(-/-) mice developed less ischemic ARF as judged by renal function and renal histology. These animals had significantly reduced blood urea nitrogen and serum creatinine levels and a lower morphological tubular necrosis score than did wild-type mice with ischemic ARF. Since caspase-1 activates IL-18, lack of mature IL-18 might protect these caspase-1(-/-) mice from ARF. In wild-type animals, we found that ARF causes kidney IL-18 levels to more than double and induces the conversion of the IL-18 precursor to the mature form. This conversion is not observed in caspase-1(-/-) ARF mice or sham-operated controls. We then injected wild-type mice with IL-18-neutralizing antiserum before the ischemic insult and found a similar degree of protection from ARF as seen in caspase-1(-/-) mice. In addition, we observed a fivefold increase in myeloperoxidase activity in control mice with ARF, but no such increase in caspase-1(-/-) or IL-18 antiserum-treated mice. Finally, we confirmed histologically that caspase-1(-/-) mice show decreased neutrophil infiltration, indicating that the deleterious role of IL-18 in ischemic ARF may be due to increased neutrophil infiltration.

Topics: Acute Kidney Injury; Animals; Apoptosis; Caspase 1; Cell Movement; Interleukin-18; Ischemia; Kidney Tubules; Mice; Mice, Mutant Strains; Neutralization Tests; Neutrophils; Peroxidase; Protein Processing, Post-Translational

The objective of this study was to assess the role of inducible nitric oxide synthase (iNOS) in ischemia- and reperfusion (I/R)-induced liver injury. We found that partial hepatic ischemia involving 70% of the liver resulted in a time-dependent increase in serum alanine aminotransferase (ALT) levels at 1-6 h following reperfusion. Liver injury at 1, 3, and 6 h post-ischemia was not due to the infiltration of neutrophils as assessed by tissue myeloperoxidase (MPO) activity and histopathology. iNOS-deficient mice subjected to the same duration of ischemia and reperfusion showed dramatic and significant increases in liver injury at 3 but not 6 h following reperfusion compared to their wild type controls. Paradoxically, iNOS mRNA expression was not detected in the livers of wild type mice at any point during the reperfusion period and pharmacological inhibition of iNOS using L-N(6)(iminoethyl)-lysine (L-NIL) did not exacerbate post-ischemic liver injury at any time post-reperfusion. These data suggest that iNOS deficiency produces unanticipated genetic alterations that renders these mice more sensitive to liver I/R-induced injury.

Topics: Alanine Transaminase; Animals; Crosses, Genetic; Ischemia; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Knockout; Neutrophils; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Peroxidase; Reperfusion Injury

Hepatic ischemia/reperfusion (I/R) results in tumor necrosis factor (TNF) release. Kupffer cells (KC) are one source of this TNF. This study investigates the effects of hepatic I/R combined with lipopolysaccharide (LPS) on the lung and liver injury that follow hepatic I/R and on hepatic release of TNF, epithelial neutrophil activating protein (ENA-78), and macrophage inflammatory protein-2 (MIP-2). The effects of these experimental conditions on TNF production by primary rat KC in vitro were also investigated. Rats were subjected to hepatic I/R alone, hepatic I/R + LPS, sham laparotomy alone, or sham laparotomy + LPS and pulmonary MPO, pulmonary microvascular permeability, hepatic neutrophil influx, hepatic injury, and hepatic TNF, ENA-78, and MIP-2 production were measured. These experiments demonstrated that hepatic I/R in conjunction with LPS results in a more severe lung and liver injury and increased hepatic TNF, ENA-78, and MIP-2 release. The effects of these experimental conditions on rat KC TNF production demonstrated that hepatic I/R + LPS results in a more significant release of TNF as compared to LPS alone or I/R alone. Hepatic I/R plus LPS results in a more severe lung and liver injury and is likely secondary to a more significant and prolonged release of TNF by KC. This may provide a mechanism for development of multiple organ system failure in some patients undergoing hepatic resection, hepatic transplantation, complex vascular operations, or in the setting of hypovolemic shock. Portal endotoxemia related to mesenteric venous congestion or other systemic insults may have a significant impact on post-operative complications and recovery in the setting of a local or global hepatic I/R injury.

Topics: Animals; Cells, Cultured; Chemokine CXCL2; Chemokine CXCL5; Chemokines; Chemokines, CXC; Interleukin-8; Ischemia; Kupffer Cells; Lipopolysaccharides; Liver; Lung; Lung Injury; Male; Neutrophils; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tumor Necrosis Factor-alpha

The pathophysiologic and potential therapeutic role of selectins in renal ischemia-reperfusion injury (IRI) is not fully understood, due in part to redundancy in the roles of individual selectins. We hypothesized that blockade of ligands for all three selectins using a novel small molecule (TBC-1269) would improve the course of renal IRI by overcoming redundancy issues. This was investigated in a rat model of renal IRI.. Rats were treated with TBC-1269 either during or post-IRI. The effects of TBC-1269 were investigated in two models of renal IRI: moderate IRI (30 minutes bilateral renal artery clamping) and severe IRI (45 minutes clamping). The combination of anti-E- and anti-P-selectin antibodies also was investigated in rats subjected to moderate IRI. Renal function, histological injury and mortality were assessed.. Rats treated with TBC-1269 during moderate IRI showed significantly reduced serum creatinine (SCr) and tubular necrosis post-ischemia compared to control animals. By contrast, delayed treatment (post-IRI) did not show a reduction in SCr. In rats with severe IRI, TBC-1269 treatment during IRI significantly reduced mortality at 48 hours post-ischemia. Rats with moderate IRI and treated with the combination of anti-E- and anti-P-selectin antibodies showed significantly reduced SCr compared to control rats at 24 hours post-ischemia.. Small molecule selectin ligand inhibition provides a novel and effective approach to attenuate ischemic acute renal failure. Timing of treatment is crucial to success.

Topics: Acute Kidney Injury; Animals; Antibodies; Biphenyl Compounds; CD4 Lymphocyte Count; E-Selectin; Immunohistochemistry; Ischemia; Kidney; Kidney Tubules; Male; Mannose; Mannosides; P-Selectin; Peroxidase; Rats; Rats, Sprague-Dawley; Renal Circulation; Selectins

To investigate the effects of intestinal ischemia and reperfusion (I/R) on the pulmonary vascular endothelium and smooth muscle.. Respiratory failure is an important cause of death and complications after intestinal I/R. Although the mechanism of respiratory failure in this setting is complex and poorly understood, recent studies of lung injury suggest that endothelial dysfunction may play a significant role.. A rat model of acute lung injury was studied after 60 minutes of superior mesenteric arterial occlusion followed by either 120 or 240 minutes of reperfusion. The pulmonary vasomotor function was examined in isolated lungs perfused at a constant flow rate.. Sixty minutes of intestinal ischemia followed by 120 or 240 minutes of reperfusion led to a significant reduction in the ability of the pulmonary vasculature to respond to angiotensin II, acetylcholine, and calcium ionophore but not to nitroglycerin. The vasoconstriction response to N(G)-nitro-L-arginine methyl ester, which is a measure of basal nitric oxide release, was diminished in the 240-minute reperfusion group. Intestinal I/R was also associated with pulmonary leukosequestration and increased pulmonary microvascular leakage.. Basal and agonist-stimulated release of nitric oxide from the pulmonary vascular endothelium and the ability of pulmonary smooth muscle to contract in response to angiotensin II were impaired by intestinal I/R. Such functional impairment in both pulmonary vascular endothelium and smooth muscle may contribute to the alveolocapillary dysfunction and pulmonary hypertension found in acute lung injury after intestinal I/R.

Topics: Animals; Capillary Permeability; Endothelium, Vascular; Intestines; Ischemia; Lung; Male; Microcirculation; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Respiratory Distress Syndrome; Vascular Resistance; Vasomotor System

Lower torso ischemia and reperfusion leads to remote organ leukosequestration and injury. We now examine the intermediary role of selectins and complement in mediating lung and liver injury after hindlimb ischemia.. Mice underwent a 2-hour bilateral tourniquet hind-limb ischemia followed by 3 hours of reperfusion.. Neutrophil depletion significantly decreased lung vascular permeability index (PI), measured by the extravasation of 125I-albumin, and liver injury as assessed by serum alanine aminotransferse levels. Lung PI and serum alanine aminotransferse levels were also reduced in mice treated with recombinant soluble P-selectin glycoprotein ligand-immunoglobulin fusion protein. Complement inhibition with soluble complement receptor type 1 decreased lung PI and serum alanine aminotransferse levels. C5-deficient mice exhibited a similar decrease in lung PI and liver injury. Lung and liver injury were restored in C5-deficient mice reconstituted with wild-type serum.. Remote organ injury after lower torso reperfusion is selectin and complement dependent.

Topics: Alanine Transaminase; Animals; Capillary Permeability; Complement C5; Complement System Proteins; Disease Models, Animal; Hindlimb; Ischemia; Liver Diseases; Lung Diseases; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Multiple Organ Failure; Neutrophils; Peroxidase; Receptors, Complement; Reperfusion Injury; Selectins

Controlled reperfusion with a modified solution limits pulmonary injury following ischemia. Our initial studies infused this modified reperfusate at a pressure of 40 to 50 mm Hg to insure distribution. However, perhaps a lower pressure, which is closer to the normal physiologic pressure in the lung, would improve results by decreasing sheer stress.. Fifteen adult pigs underwent 2 hours of lung ischemia by clamping the left bronchus and pulmonary artery. Five (group 1) then underwent uncontrolled reperfusion by removing the vascular clamps and allowing unmodified blood to reperfuse the lung at a pulmonary artery pressure of 20 to 30 mm Hg. The other 10 pigs underwent controlled reperfusion by mixing blood from the femoral artery with a crystalloid solution, and infusing this modified reperfusate into the ischemic lung through the pulmonary artery for 10 minutes before removing the arterial clamp. In 5 (group 2), the modified solution was infused at a pressure of 40 to 50 mm Hg, and in 5 (group 3) 20 to 30 mm Hg. Lung function was assessed 60 minutes after reperfusion and expressed as percentage of control.. Compared to uncontrolled reperfusion (group 1), controlled reperfusion at a pressure of 40 to 50 mm Hg (group 2) significantly improved postreperfusion pulmonary compliance (77% versus 86%; p<0.001 versus group 1), and arterial/alveolar ratio (a/A) ratio (27% versus 52%; p<0.001 versus group 1); as well as decreased pulmonary vascular resistance (PVR) (198% versus 154%; p<0.001 versus group 1), lung water (84.3% versus 83.5%; p<0.001 versus group 1), and myeloperoxidase (0.35 versus 0.23 optical density/min/mg protein). Reducing the pressure of the modified reperfusate to 20 to 30 mm Hg further improved postreperfusion compliance (92%+/-1%; p<0.001 versus groups 1 and 2) and a/A ratio (76%+/-1%; p<0.001 versus groups 1 and 2); and lowered PVR (133%+/-2%; p<0.001 versus groups 1 and 2), lung water (82.7%+/-0.1%; p<0.001 versus groups 1 and 2), and myeloperoxidase (0.16%+/-0.01%; p<0.001 versus groups 1 and 2).. After 2 hours of pulmonary ischemia, a severe lung injury occurs following uncontrolled reperfusion, controlled reperfusion with a modified solution reduces this reperfusion injury, and lowering the pressure of the modified reperfusate to more physiologic levels (20 to 30 mm Hg) further reduces the reperfusion injury improving pulmonary function.

Topics: Animals; Blood; Blood Pressure; Body Water; Bronchi; Constriction; Ischemia; Lung; Lung Compliance; Organ Preservation Solutions; Peroxidase; Pressure; Pulmonary Artery; Pulmonary Edema; Pulmonary Gas Exchange; Reperfusion; Reperfusion Injury; Rheology; Swine; Vascular Resistance

The relative contribution of xanthine oxidase (XO) and leukocytes to tissue injury after short-term ischemia is unknown. In this study, we subjected three groups of rat spinotrapezius muscles to 30-min ischemia and 1-h reperfusion: 1) ischemia-reperfusion (I/R) + 0.9% saline, 2) I/R + superoxide dismutase, and 3) I/R + oxypurinol. A fourth group served as nonischemic control. We quantified the increase in resistance (%DeltaR) caused by leukocyte-capillary plugging concurrently with myocyte uptake of propidium iodide (PI) [expressed as no. of PI spots per total volume of perfused tissue (N(PI)/V)] and performed assays to quantify XO activity, thiobarbituric acid-reactive substances (TBARS), and myeloperoxidase (MPO). Groups 2 and 3 exhibited significant decreases in N(PI)/V relative to group 1. MPO levels and TBARS were similar among all groups, and mean %DeltaR was significantly reduced in groups 2 and 3 relative to group 1. However, elevated XO was observed in groups 1 and 2 relative to group 3 and nonischemic controls. These data are consistent with the hypothesis that XO, rather than toxic species produced by plugging or venule-adherent leukocytes, is responsible for postischemic damage in this model.

Topics: Animals; Capillaries; Cell Adhesion; Cell Movement; Female; Ischemia; Leukocytes; Lipid Peroxides; Muscle, Skeletal; Oxidants; Oxypurinol; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sodium Chloride; Superoxide Dismutase; Time Factors; Vascular Resistance; Xanthine Oxidase

One of the early events in physiological shock is the generation of activators for leukocytes, endothelial cells, and other cells in the cardiovascular system. The mechanism by which these activators are produced has remained unresolved. We examine here the hypothesis that pancreatic digestive enzymes in the ischemic intestine may be involved in the generation of activators during intestinal ischemia. The lumen of the small intestine of rats was continuously perfused with saline containing a broadly acting pancreatic enzyme inhibitor (6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfate, 0.37 mM) before and during ischemia of the small intestine by splanchnic artery occlusion. This procedure inhibited activation of circulating leukocytes during occlusion and reperfusion. It also prevented the appearance of activators in portal venous and systemic artery plasma and attenuated initiating symptoms of multiple organ injury in shock. Intestinal tissue produces only low levels of activators in the absence of pancreatic enzymes, whereas in the presence of enzymes, activators are produced in a concentration- and time-dependent fashion. The results indicate that pancreatic digestive enzymes in the ischemic intestine serve as an important source for cell activation and inflammation, as well as multiple organ failure.

Topics: Animals; Benzamidines; Bile; Blood Pressure; Guanidines; Intestinal Mucosa; Intestines; Ischemia; Leukocytes; Liver; Lung; Male; Pancreas; Peroxidase; Protease Inhibitors; Rats; Rats, Wistar; Serine Endopeptidases; Shock; Trypsin

Ruptured abdominal aortic aneurysm (RAAA) repair, a combination of hemorrhagic shock and lower-torso ischemia, is associated with a 50-70% mortality. Myocardial dysfunction may contribute to the high rate of mortality after aneurysm repair. We attempted to determine whether RAAA repair results in cardiac dysfunction mediated by tumor necrosis factor-alpha (TNF-alpha). We modeled aortic rupture and repair in the rat by inducing hemorrhagic shock to a mean blood pressure of 50 mmHg for 1 h, followed by supramesenteric clamping of the aorta for 45 min. After 90 min of reperfusion, cardiac contractile function was assessed with a Langendorff preparation. Myocardial TNF-alpha, ATP and creatine phosphate (CP) levels, and markers of oxidant stress (F(2)-isoprostanes) were measured. Cardiac function in the combined shock and clamp rats was significantly depressed compared with sham-operated control rats but was similar to that noted in animals subjected to shock alone. Myocardial TNF-alpha concentrations increased 10-fold in the combined shock and clamp rats compared with sham rats, although there was no difference in myocardial ATP, CP, or F(2)-isoprostanes. TNF-alpha neutralization improved cardiac function by 50% in the combined shock and clamp rats. Hemorrhagic shock is the primary insult inducing cardiac dysfunction in this model of RAAA repair. An improvement in cardiac contractile function after immunoneutralization of TNF-alpha indicates that TNF-alpha mediates a significant portion of the myocardial dysfunction in this model.

Topics: Adenosine Triphosphate; Aneurysm, Ruptured; Animals; Antibodies; Aorta; Aortic Aneurysm, Abdominal; Cardiomyopathies; Constriction; Ischemia; Male; Myocardium; Peroxidase; Phosphocreatine; Rats; Rats, Sprague-Dawley; Shock, Hemorrhagic; Tumor Necrosis Factor-alpha; Ventricular Function, Left

We assessed a kallikrein-like amidase activity probably related to the kallikrein-kinin system, as well as the participation of leukocyte infiltration in renal ischemia and reperfusion. Male C57BL/KSJmdb mice were subjected to 20 or 60 min of ischemia and to different periods of reperfusion. A control group consisted of sham-operated mice, under similar conditions, except for ischemia induction. Kallikrein-like amidase activity, Evans blue extravasation and myeloperoxidase activity were measured in kidney homogenates, previously perfused with 0.9% NaCl. Plasma creatinine concentration increased only in the 60-min ischemic group. After 20 min of ischemia and 1 or 24 h of reperfusion, no change in kallikrein-like amidase activity or Evans blue extravasation was observed. In the mice subjected to 20 min of ischemia, edema was evident at 1 h of reperfusion, but kidney water content returned to basal levels after 24 h of reperfusion. In the 60-min ischemic group, kallikrein-like amidase activity and Evans blue extravasation showed a similar significant increase along reperfusion time. Kallikrein-like amidase activity increased from 4 nmol PNA mg protein-1 min-1 in the basal condition to 15 nmol PNA mg protein-1 min-1 at 10 h of reperfusion. For dye extravasation the concentration measured was near 200 microg of Evans blue/g dry tissue in the basal condition and 1750 microg of Evans blue/g dry tissue at 10 h of reperfusion. No variation could be detected in the control group. A significant increase from 5 to 40 units of DeltaAbs 655 nm g wet tissue-1 min-1 in the activity of the enzyme myeloperoxidase was observed in the 60-min ischemic group, when it was evaluated after 24 h of reperfusion. Histological analysis of the kidneys showed migration of polymorphonuclear leukocytes from the vascular bed to the interstitial tissue in the 60-min ischemic group after 24 h of reperfusion. We conclude that the duration of ischemia is critical for the development of damage during reperfusion and that the increase in renal cortex kallikrein-like amidase activity probably released from both the kidney and leukocytes may be responsible, at least in part, for the observed effects, probably through direct induction of increased vascular permeability.

Topics: Amidohydrolases; Animals; Capillary Permeability; Creatinine; Ischemia; Kallikreins; Kidney; Male; Mice; Mice, Inbred C57BL; Microcirculation; Peroxidase; Reperfusion Injury

To examine whether adenosine reduces ischemia/reperfusion (I/R)-induced liver injury by inhibiting leukocyte activation via A(2) receptor (A(2)R) stimulation, we investigated the effects of adenosine and selective A(2A) receptor (A(2A)R) agonists (YT-146 and CGS21680C) on I/R-induced liver injury in rats. Adenosine, YT-146, and CGS21680C, in the concentration of 10(-7) to 10(-5) M, significantly inhibited neutrophil elastase release by about 30 to 40% and increased intracellular Ca(2+) concentrations in isolated neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine (fMLP) in vitro. Adenosine, YT-146, and CGS21680C, in the concentration of 10(-7) to 10(-5) M, significantly inhibited tumor necrosis factor (TNF)-alpha production by monocytes stimulated with endotoxin by about 50%. Although ZM241385, a selective A(2A)R antagonist, significantly enhanced the increase in neutrophil elastase release and intracellular Ca(2+) concentrations in neutrophils stimulated with fMLP, this agent did not affect the endotoxin-induced TNF-alpha production by monocytes. Rats were subjected to liver ischemia for 60 min. Serum levels of transaminases increased after hepatic I/R, peaking at 12 h after reperfusion. The i.v. infusion of adenosine (1 and 10 mg/kg/h), YT-146 (0.1 and 1 mg/kg/h), and CGS21680C (0.1 and 1 mg/kg/h) significantly inhibited the I/R-induced increase in serum transaminase levels 12 h after reperfusion. The I/R-induced decrease in hepatic tissue blood flow was significantly prevented by adenosine and YT-146. Hepatic levels of TNF-alpha, cytokine-induced neutrophil chemoattractant (equivalent to human interleukin-8), and myeloperoxidase were significantly increased after I/R. These increases were significantly inhibited by the administration of adenosine, YT-146, and CGS21680C. Although the histological neutrophil accumulation in the liver was significantly increased after I/R as evaluated by the naphthol AS-D chloroacetate technique, the administration of adenosine, YT-146, and CGS21680C significantly inhibited this increase. These findings suggest that adenosine reduces I/R-induced liver injury both by inhibiting the synthesis of inflammatory mediators and by inhibiting neutrophil degranulation directly, probably through A(2A)R stimulation.

Topics: Adenosine; Alkynes; Animals; Calcium; Cells, Cultured; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Ischemia; Leukocyte Elastase; Lipopolysaccharides; Liver; Male; Monocytes; Neutrophil Activation; Peroxidase; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptor, Adenosine A2A; Reperfusion Injury; Triazines; Triazoles; Tumor Necrosis Factor-alpha

L-selectin plays an important role in the early phase of PMNs recruitment in the hepatic microvasculature following liver ischemia and reperfusion (I/R). Leukocyte cytokine chemoattractants (chemokines) cause polymorphonuclear neutrophil (PMN) activation in I/R injury. In this study, we examined the role of L-selectin in the production of chemokines in the liver and lung inflammatory response following 90 min of warm ischemia.. Thirty-six C57BL/6 mice were subjected to partial liver ischemia for a period of 90 min. Three groups of animals were included (n = 12 per group)-sham group, ischemic control, and the ischemic group receiving monoclonal antibody against L-selectin. We evaluated at 3 h: liver injury measurements, serum chemokines (MIP-2 and MIP-1alpha), liver and lung tissue myeloperoxidase (MPO), and liver and lung histology. Statistical analysis included ANOVA, Student-Newman-Keuls', and Kruskal-Wallis multiple comparison Z-value tests.. The ischemic group treated with anti-L-selectin showed significant decreases in liver enzyme levels and a marked decrease in serum MIP-2 (P < 0.05) when compared to ischemic controls. No reduction in serum MIP-1alpha was noted; however, neutrophil infiltration was significantly ameliorated in the liver and in the lung, as reflected by decreased MPO levels (P < 0.05). Improved histopathological features were observed in the anti-L-selectin-treated group compared to ischemic controls in the liver and the lung.. Our study suggests an important role for L-selectin in the pathogenesis of liver I/R and the production of chemokines. Anti-L-selectin treatment resulted in improved liver function, decreased neutrophil infiltration, and decreased MIP-2 chemokine response.

Topics: Animals; Chemokine CCL3; Chemokine CCL4; Chemokine CXCL2; Ischemia; L-Selectin; Liver; Macrophage Inflammatory Proteins; Mice; Mice, Inbred C57BL; Monokines; Peroxidase; Reperfusion Injury

Peroxynitrite-mediated DNA strand breaks trigger poly (ADP-ribose) synthetase (PARS) activation, resulting in intracellular energetic failure and organ dysfunction. We investigated the role of PARS activation on the inflammatory and functional response of the intestine to mesenteric ischemia-reperfusion injury. Anesthetized rats exposed to 15 min occlusion of the superior mesenteric artery showed an increased mucosal PARS activity (ex vivo incorporation of radiolabelled NAD+ in gut mucosal scrapings) as soon as 10 min after reperfusion. During the first 30 min of reperfusion, significant mucosal damage developed, as well as mucosal hyperpermeability to a 4000 MW fluorescent dextran (FD4). These alterations were significantly reduced by treatment with the NO synthase inhibitor L-NMA, which blocks the production of peroxynitrite, as well as with the PARS inhibitors 3-aminobenzamide and nicotinamide, whereas they were markedly enhanced by the glutathione depletor L-buthionine-(S,R)-sulfoximine. Also, PARS inhibition significantly reduced ileal neutrophil infiltration (myeloperoxidase activity) at 3 h reperfusion. In a second set of experiments, the effects of 15 or 30 min ischemia followed by 3 h reperfusion were evaluated in PARS knockout and wild-type mice. Significant protection against histological damage, neutrophil infiltration, and mucosal barrier failure (evaluated by the mucosal-to-serosal FD4 clearance of everted ileal sacs incubated ex vivo) was noted in PARS knockout mice, who also showed reduced alterations in remote organs, as shown by lesser lipid peroxidation (malondialdehyde formation) and neutrophil infiltration in the lung and liver. In conclusion, PARS plays a crucial role in mediating intestinal injury and dysfunction in the early and late phases of mesenteric reperfusion. Pharmacological inhibition of PARS may be a novel approach to protect tissues from reperfusion-related damage.

Topics: Animals; Arterial Occlusive Diseases; Chemotaxis, Leukocyte; DNA Damage; Energy Metabolism; Enzyme Induction; Enzyme Inhibitors; Ileum; Intestinal Mucosa; Ischemia; Lipid Peroxidation; Liver; Lung; Male; Mesenteric Artery, Superior; Mesentery; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophil Infiltration; Neutrophils; Nitrates; Nitric Oxide Synthase; omega-N-Methylarginine; Oxidative Stress; Peroxidase; Poly(ADP-ribose) Polymerases; Rats; Rats, Wistar; Reperfusion Injury

Leukocyte adhesion/infiltration in response to renal ischaemia/reperfusion (I/R) injury is a well-known but poorly understood phenomenon. The identification, kinetics, and exact role of these inflammatory cells in I/R injury and regeneration are still matters of debate.. Uninephrectomized rats were submitted to 60 min renal ischaemia by clamping of renal vessels.. Severe acute renal failure was observed, with maximum functional impairment on day 2. By 12 h after the ischaemic event, up to 80% of proximal tubular cells in the outer stripe of outer medulla (OSOM) were already severely damaged. Proliferation (proliferating cell nuclear antigen (PCNA) staining) started after 24 h, reaching maximum activity on day 3. Regeneration of tubular morphology started on the 3rd day, and after 10 days 50% of tubules had regenerated completely. Interstitial leukocytes (OX-1 immunohistochemical staining) were already prominent at day 1, thereafter gradually increasing with time. The so-called neutrophil-specific identification methods (myeloperoxidase (MPO), chloroacetate esterase, mAb HIS-48) proved to be non-specific, since they also stained for macrophages, as demonstrated by flow cytometry and the combination of these stainings with the macrophage-specific ED-1 staining. MPO activity was already significantly increased at 1 h post-I/R (439+/-34%, P<0.005), reaching its maximum activity after 12 h of I/R (1159+/-138%, P<0.0005), declining thereafter. On the other hand, neutrophil presence investigated by H&E staining revealed only a few neutrophils in glomeruli, medullary rays, and OSOM at 24 h after the ischaemic event (4.7+/-4.2 cells/mm(2) vs controls=2.3+/-2.0 cells/mm(2) (n.s.)), and remained unchanged over the next 10 days. In contrast, significant monocyte/macrophage adhesion/infiltration (ED-1 staining) occurred at the OSOM at 24 h post-ischaemia (at 24 h, 120+/-46 cells/mm(2) vs. sham=18+/-4 cells/mm(2) (P<0.05)), became prominent at day 5 (1034+/-161 cells/mm(2) vs sham=18+/-18 cells/mm(2) (P<0.05)), and almost disappeared after 10 days. CD4(+) cells (W3/25) gradually increased from day 5, reaching a maximum at day 10. A few CD8(+) cells (OX-8) were apparent from days 3 until 10, but no B-cells (OX-33) were observed.. After severe warm I/R renal injury, a pronounced acute tubular necrosis occurs during the first 12-24 h in the absence of a marked cellular infiltrate, but with an important renal MPO activity, reflecting the activation of the adhering inflammatory cells (polymorphonuclear cells (PMNs) and mainly monocytes/macrophages). Only later at the time and site (OSOM) of regeneration a sequential accumulation of monocytes/macrophages and T cells becomes prominent, in contrast with the low number of neutrophils found in the kidney during the 10-day post-ischaemic period. The non-specificity of the so-called neutrophil-specific identification methods (MPO activity, naphthol AS-D chloroacetate esterase, or mAb HIS-48 staining), cross-reacting with monocytes/macrophages, explains the controversy in literature concerning the number of PMNs in post-ischaemic injury.

Topics: Animals; Blood Cells; Extracellular Space; Ischemia; Kidney; Kidney Medulla; Kinetics; Leukocytes; Macrophages; Male; Monocytes; Peroxidase; Proliferating Cell Nuclear Antigen; Rats; Rats, Inbred Lew; Regeneration; Renal Circulation; Reperfusion Injury; Staining and Labeling

Endothelial cell injury after hemorrhage and resuscitation (HEM/RES) might contribute to intestinal hypoperfusion and mucosal ischemia. Our recent work suggests that the injury might be the result of complement activation. We hypothesized that HEM/RES causes complement-mediated endothelial cell dysfunction in the small intestine.. Male Sprague-Dawley rats (195-230 g) were anesthetized and HEM to 50% of baseline mean arterial pressure for 60 minutes. Just before RES, animals received either soluble complement receptor-1 (sCR1, 15 mg/kg) to inhibit complement activation or saline vehicle. Resuscitation was with shed blood and an equal volume of saline. Two hours after RES, the small bowel was harvested to evaluate intestinal nitric oxide synthase activity (NOS), neutrophil influx, histology, and oxidant injury.. HEM/RES induced tissue injury, increased neutrophil influx, and reduced NOS activity by 50% (vs. SHAM), all of which were completely prevented by sCR1 administration. There were no observed differences in oxidant injury between the groups.. Histologic tissue injury, increased neutrophil influx, and impaired NOS activity after HEM/RES were all prevented by complement inhibition. Direct oxidant injury did not seem to be a major contributor to these alterations. Complement inhibition after HEM might ameliorate reperfusion injury in the small intestine by protecting the endothelial cell, reducing neutrophil influx and preserving NOS function.

Topics: Animals; Complement Activation; Dinoprost; Disease Models, Animal; Intestinal Mucosa; Ischemia; Linear Models; Male; Neutrophil Activation; Nitric Oxide Synthase; Peroxidase; Rats; Rats, Sprague-Dawley; Resuscitation; Shock, Hemorrhagic

The pathogenesis of generalized microvascular injury following hemorrhagic shock and total ischemia appears to be dependent on leukocytes interacting with the venular endothelium. The purpose of this study was to compare leukocyte adherence and sequestration following hemorrhagic shock with that of total ischemia in the small bowel mesentery of rats. Leukocyte adherence and sequestration was measured by direct visualization in vivo using intravital microscopy. In addition, sequestration was also quantitated by measuring tissue levels of myeloperoxidase, a marker of leukocyte infiltration. Mean arterial blood pressure was decreased to 40 mm Hg for 30 min (hemorrhagic shock group). In the total ischemia group, both the superior and inferior mesenteric arteries were clamped for 30 min followed by reperfusion. Hemorrhagic shock (9.4+/-1.5 cell/100 microm) and total ischemia (8.3+/-3 cell/100 microm) caused a statistically significant increases in leukocyte adherence 60 min postinsult as compared with controls (.9+/-1.5 cell/100 microm). However, the increase in leukocyte adherence appeared earlier and to a greater degree initially following total ischemia. Leukocyte sequestration as measured by intravital microscopy was significant only after total ischemia [(24.6+/-1.7 cell/(100 microm)2; p<.01] and not hemorrhagic shock [3.4+/-.6 cell/(100 microm)2] versus controls [2.2+/-.2 cell/(100 microm)2]. This difference in sequestration was also confirmed by tissue levels of myeloperoxidase. The results of this study suggest that the microvascular response following hemorrhagic shock is different than that of total ischemia, and caution is warranted when extrapolating the experimental results of one to the other.

Topics: Animals; Cell Adhesion; Endothelium, Vascular; Ischemia; Leukocytes; Male; Mesentery; Microscopy; Neutrophils; Peroxidase; Rats; Rats, Sprague-Dawley; Shock, Hemorrhagic

Ischemia/reperfusion injury is a commonly occurring event with severe pathologic consequences. Reperfusion initiates both the local and systematic damage in part through rapid oxygen generation. The glutathione system is a major mechanism of reducing this oxidative stress. If this system can be maintained or augmented during this stress then less damage may occur. Glutamine provides the source of glutamate to this system and has been shown to preserve total glutathione levels after injury/ischemia to both hepatic and gut models. To test this effect, we looked at glutamine and its role in ischemia/reperfusion injury in a rat hind limb model.. Fifty male HSD/Holtzman rats weighing 350-400 g were randomized to receive glutamine (3% sol) or normal saline via intraperitoneal injections. The groups were then subjected to 2 hours of ischemia to their hind limbs using the Tourni-Cot method. Animals were then randomized to reperfusion groups of 30 minutes, 2 hours, and 4 hours. Muscle tissue assays were performed for lipid peroxidation (LPO), total glutathione (GSH), and myeloperoxidase (MPO). Peripheral blood was analyzed for creatinephosphokinase levels (CPK).. Animals that received glutamine showed a general trend of less lipid peroxidation products than the normal saline groups. In animals that received glutamine and underwent 2 hours of ischemia and reperfusion times of 0 minutes, 30 minutes, and 2 hours, there were significantly less percent changes in lipid peroxidation products from controls (4.6% vs 48.2%, P <0.05), (18.9% vs 123%, P <0.05), (12.6% vs 115%, P <0.05). A general trend upward was noted in CPK levels in both groups. In animals receiving 2 hours of ischemia and 30 minutes of reperfusion, there was a significantly greater level of creatinephosphokinase (CPK) calculated as percent change from control in the normal saline group as compared with the glutamine group (209.2% vs 92.7%). Myeloperoxidase assay of muscle tissue revealed a progressive increase as the reperfusion times grew. In animals receiving 2 hours of ischemia and 30 minutes of reperfusion, the normal saline group had a significantly larger percent increase from controls than the group that received glutamine (1126.4% vs 108%, P <0.05). Also, in those animals receiving 4 hours of reperfusion, the normal saline group had a significantly higher percent increase in MPO content than the glutamine group (6245% vs 108%, P <0.05). Total glutathione levels decreased rapidly as reperfusion occurred in both the normal saline and glutamine groups. No significant difference between the groups was noted.. Total glutathione levels during reperfusion were not significantly different in the groups receiving glutamine versus normal saline. Glutamine may provide an initial protective effect on reperfusion injury after moderate reperfusion times in the hind limb model as defined by CPK and LPO levels. Glutamine may blunt neutrophil recruitment after longer reperfusion times (4 hours) in the ischemic hind limb. Total glutathione levels decreased significantly after moderate levels of ischemia (2 hours) and reperfusion (30 minutes, 2 hours).

Topics: Animals; Creatine Kinase; Disease Models, Animal; Glutamic Acid; Glutamine; Glutathione; Hindlimb; Injections, Intraperitoneal; Ischemia; Lipid Peroxidation; Male; Muscle, Skeletal; Neutrophils; Oxidative Stress; Peroxidase; Protective Agents; Random Allocation; Rats; Rats, Inbred Strains; Reperfusion Injury; Single-Blind Method; Time Factors

Ischemia followed by reperfusion leads to severe organ injury and dysfunction. Inflammation is considered to be the most important cause of tissue injury in organs subjected to ischemia. The mechanism that triggers inflammation and organ injury after ischemia remains to be elucidated, although different causes have been postulated. We investigated the role of apoptosis in the induction of inflammation and organ damage after renal ischemia. Using a murine model, we demonstrate a relationship between apoptosis and subsequent inflammation. At the time of reperfusion, administration of the antiapoptotic agents IGF-1 and ZVAD-fmk (a caspase inactivator) prevented the early onset of not only renal apoptosis, but also inflammation and tissue injury. Conversely, when the antiapoptotic agents were administered after onset of apoptosis, these protective effects were completely abrogated. The presence of apoptosis was directly correlated with posttranslational processing of the endothelial monocyte-activating polypeptide II (EMAP-II), which may explain apoptosis-induced influx and sequestration of leukocytes in the reperfused kidney. These results strongly suggest that apoptosis is a crucial event that can initiate reperfusion-induced inflammation and subsequent tissue injury. The newly described pathophysiological insights provide important opportunities to effectively prevent clinical manifestations of reperfusion injury in the kidney, and potentially in other organs.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blood Urea Nitrogen; Caspases; Chemotaxis, Leukocyte; Cysteine Proteinase Inhibitors; Cytokines; Depression, Chemical; Drug Administration Schedule; Epidermal Growth Factor; Humans; In Situ Nick-End Labeling; Insulin-Like Growth Factor I; Ischemia; Kidney; Male; Mice; Neoplasm Proteins; Nephritis; Peroxidase; Protein Processing, Post-Translational; Recombinant Proteins; Reperfusion Injury; RNA-Binding Proteins

We used IL-6 knock-out (KO) mice to evaluate a possible role for IL-6 in the pathogenesis of splanchnic artery occlusion shock (SAO). SAO shock was induced by clamping both the superior mesenteric artery and the celiac trunk, followed by release of the clamp. There was a marked increase in the peroxynitrite formation in the plasma of the SAO-shocked IL-6 wild-type (WT) mice after reperfusion. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine in the necrotic ileum in shocked IL-6 WT mice. SAO-shocked WT mice developed a significant increase of tissue myeloperoxidase (MPO) and malondialdehyde (MDA) activity and marked histological injury to the distal ileum. SAO shock was also associated with a significant mortality (0% survival). Reperfused ileum tissue sections from SAO-shocked WT mice showed positive staining for P-selectin. Little specific staining was observed in sham-WT mice. Staining of ileum tissue obtained from sham-operated WT mice with anti-ICAM-1 antibody showed weak but diffuse staining, demonstrating that ICAM-1 is constitutively expressed. However, after SAO shock the staining intensity increased substantially in the ileum section from WT mice. Intensity and degree of P-selectin and ICAM-1 were markedly reduced in tissue section from SAO-shocked IL-6 KO mice. SAO-shocked IL-6 KO mice also show significant reduction of neutrophil infiltration into the reperfused intestine, as evidenced by reduced MPO activity, improved histological status of the reperfused tissues, reduced peroxynitrite formation, reduced MDA levels, and improved survival. In vivo treatment with anti-IL-6 significantly prevents the inflammatory process. Our results clearly demonstrate that IL-6 plays an important role in ischemia and reperfusion injury and allows the hypothesis that inhibition of IL-6 may represent a novel and possible strategy. Part of this effect may be due to inhibition of the expression of adhesion molecules and subsequent reduction of neutrophil-mediated cellular injury.

Topics: Animals; Antibodies, Monoclonal; Arterial Occlusive Diseases; Celiac Artery; Constriction; Cytokines; Ileum; Immunity, Innate; Intercellular Adhesion Molecule-1; Interleukin-6; Ischemia; Leukocyte Count; Lipid Peroxidation; Malondialdehyde; Mesenteric Artery, Superior; Mice; Mice, Knockout; Neutrophils; Nitrates; Nitric Oxide; P-Selectin; Peroxidase; Reperfusion Injury; Shock; Splanchnic Circulation; Tyrosine

Topics: Acetylcysteine; Adenosine Triphosphate; Allopurinol; Animals; Glutathione; Heart; Intestine, Small; Ischemia; Liver; Lung; Malondialdehyde; Mesenteric Arteries; Multiple Organ Failure; Myocardium; Oxidative Stress; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Somatostatin

Topics: Adenosine Triphosphate; Animals; Glutathione; Intestine, Small; Ischemia; Malondialdehyde; Mesenteric Artery, Superior; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Somatostatin

Platelet-activating factor (PAF), cytokines, proteases, and other factors are probably involved in the development of gut barrier dysfunction following intestinal ischemia and reperfusion (I/R), although the act underlying pathophysiological mechanisms has not yet been fully clarified. The aim of the present study was to clarify the relationship of intestinal barrier integrity to systemic levels of interleukin-1beta, interleukin-6, and protease inhibitor levels and local leukocyte accumulation in a rat model of intestinal ischemia for 40 min followed by 3 or 12 h reperfusion, with or without treatment with a PAF inhibitor.. Myeloperoxidase (MPO) content in the small intestinal mucosa, serum levels of interleukin-1beta and -6, and plasma protease inhibitors, and intestinal endothelial and epithelial permeability were assessed, with or without treatment with the PAF antagonist lexipafant.. Intestinal I/R resulted in intestinal barrier dysfunction with pronounced plasma leakage to the intestinal lumen, the leakage being aggravated following a longer reperfusion period. Proteolytic plasma activity was evident by low levels of the plasma protease inhibitors measured. MPO content increased significantly after I/R, as did serum levels of interleukin-1beta and -6, without difference between the two periods of reperfusion. Treatment with the PAF inhibitor lexipafant partly, though not fully, restored the changes caused by I/R.. PAF seems to be involved in the release of cytokines, such as interleukin-1 and -6, consumption of protease inhibitors, and impaired intestinal barrier integrity seen following intestinal I/R. Treatment with a PAF antagonist was effective in restoring the changes caused by intestinal I/R, though not reaching complete normal levels.

Topics: Animals; Interleukin-1; Interleukin-6; Intestinal Mucosa; Intestines; Ischemia; Male; Mesenteric Arteries; Permeability; Peroxidase; Platelet Activating Factor; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Ischemia-reperfusion injury, a common source of renal dysfunction in adults, is associated with tubular epithelial cell damage. Since fibroblast growth factors (FGF) attenuated tissue injury after transient myocardial ischemia, we hypothesized that acidic fibroblast growth factor (aFGF; FGF-1) would attenuate renal ischemia-reperfusion injury. We studied the effects of FGF-1 in a rat model of acute renal failure induced by bilateral renal ischemia (60 min) and 1, 2 or 7 days reperfusion. After FGF-1 administration at the onset of renal reperfusion, there was less functional impairment of the kidneys. The histological changes were not as severe as in controls. Increases in serum creatinine and blood urea nitrogen 24 h after reperfusion were attenuated by 35% (p< 0.01) and by 53% (p< 0.001), respectively, in FGF-1-treated animals compared to vehicle-treated rats. The ischemia/reperfusion-induced increase in tissue myeloperoxidase, a marker of neutrophil infiltration, was mitigated (67% reduction, p< 0.05) with FGF-1 treatment. As shown by histology, neutrophil infiltration and tubular cell necrosis in medulla were less pronounced (p< 0.0001 and p< 0.05, respectively) in animals receiving FGF-1. Furthermore, ischemia-induced apoptosis, prevalent in tubular cells of the cortex, was also attenuated by FGF-1-treatment (83% reduction, p< 0.0001). Pretreatment of animals with Nw-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide synthase, abolished the attenuating effects of FGF-1 on neutrophil infiltration, suggesting that nitric oxide might participate in the anti-inflammatory effects of FGF-1 in this experimental design. Our data support a role for FGF-1 in attenuation of renal damage or failure after ischemia-reperfusion injury of the kidney, in part at least by inhibition of neutrophil infiltration.

Topics: Animals; Apoptosis; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Ischemia; Kidney; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury

The objective of this study was to investigate the effects of the micronized purified flavonoid fraction Daflon 500 mg (90% diosmin and 10% hesperidin) on I/R-induced microvascular leukocyte-endothelium interaction and leakage of the high molecular weight plasma tracer FITC-dextran (relative molecular mass, 150 kd) as assessed in the striated skin muscle of the dorsal skin fold chamber model in the hamster. Intravital fluorescence microscopy was used for analysis of microvascular perfusion, leukocyte-endothelium interaction, and macromolecular leakage of FITC-dextran 150 kd in the striated skin muscle of the hamster. A tourniquet ischemia of 4 hours' duration was induced followed by reperfusion. Animals were treated with an oral administration of Daflon 500 mg (n = six) or its vehicle (5% Arabic gum solution, n = six) for 8 days at a daily dose of 30 mg/kg body weight. Measurements in the microcirculation were made before the 8-day feeding protocol before induction of ischemia and at 0.5, 2, and 24 hours of reperfusion. In the absence of I/R, no differences in microvascular perfusion, leukocyte-endothelium interaction, and macromolecular leakage were found in Daflon 500 mg and vehicle-treated control animals before and after administration of the drugs. Induction of ischemia and reperfusion, however, elicited a significant increase in venular leukocyte rolling and sticking in vehicle-treated animals, which was accompanied by enhancement of leakage of FITC-dextran 150 kd into the perivascular tissue. Treatment with Daflon 500 mg had no effect on postischemic leukocyte rolling and sticking, and macromolecular leakage of FITC-dextran 150 kd from arterioles and postcapillary venules was significantly reduced. These data indicate that Daflon 500 mg preserves the endothelial barrier function of striated skin muscle arterioles and venules after I/R, which appears to be independent of an action on postischemic intravascular leukocyte rolling and sticking.

Topics: Animals; Cell Adhesion; Cricetinae; Diosmin; Disease Models, Animal; Endothelium, Vascular; Ischemia; Leukocytes; Male; Mesocricetus; Microcirculation; Muscle, Skeletal; Peroxidase; Reperfusion Injury; Skin

To assess the effects of gabexate mesilate (FOY), a protease inhibitor, on a canine model of pulmonary ischemia-reperfusion injury. FOY has been applied clinically to treat acute pancreatitis and disseminated intravascular coagulation (DIC) and has been found to suppress some leukocyte-mediated tissue injuries in both in vitro and in vivo studies.. Comparison of four experimental groups: group 1 (untreated control, n = 8), unilateral (left) pulmonary ischemia due to perfusion and ventilation obstruction followed by reperfusion, without receiving any specific treatment; group 2 (negative control, sham operation, n = 8), left pulmonary hilar dissection without ischemia; group 3 (FOY posttreatment, n = 8), FOY treatment during the reperfusion stage only; and group 4 (FOY pretreatment, n = 8), FOY treatment before ischemia and then continued during reperfusion.. University animal laboratory.. Heart-worm-free mongrel dogs (12 to 15 kg body wt) were anesthetized with pentobarbital and mechanically ventilated.. Lung ischemia was made by snaring the left pulmonary artery and veins and clamping the bronchus with peribronchial tissue for 90 min followed by reperfusion for 18 h. Animals of the two treatment groups received a 1 mg/kg bolus of FOY at the beginning of reperfusion, with infusion of 2 mg/kg/h of FOY continuously starting 30 min before ischemia (group 4) or after reperfusion (group 3). During this study the following were measured: hemodynamics and aerodynamics, blood gas, bronchoalveolar lavage (BAL) fluid neutrophil percentage and protein concentration, lung wet to dry weight ratio (W/D ratio), myeloperoxidase (MPO) activity of the lung tissue, alveolar neutrophil infiltration, and degree of injury.. This model of lung ischemia-reperfusion induced significant pulmonary hypertension, increased pulmonary vascular resistance, decreased pulmonary dynamic compliance and arterial hypoxemia, increased BAL fluid total protein amount and neutrophil percentage, and increased alveolar neutrophil infiltration, histological injury score, and lung tissue MPO assay (group 1). Animals of the sham operation (negative control, group 2) showed only minimal changes in the above parameters. Treatment with FOY significantly attenuated the injury by decreasing the lung W/D ratio, alveolar neutrophil infiltration, histological injury score, lung tissue MPO assay, BAL fluid neutrophil percentage, and protein amount. Pretreatment with FOY (group 4) attenuated the injury to a significantly greater degree than it did when administered at the reperfusion stage only (group 3), which was reflected by the above-mentioned parameters, and as well significantly improved gas exchange function. FOY treatment was found to have little effect in altering hemodynamics and aerodynamics at most time points in this model of lung injury.. FOY can attenuate the ischemia-reperfusion-induced acute lung injury in dogs by ameliorating the degree of alveolar membrane permeability change, neutrophil aggregation, and activation. FOY treatment starting before ischemia attenuated this injury to a significantly higher degree than its use after ischemia. However, the effect of FOY may be partial because it cannot alter the hemodynamics or aerodynamics as prominently as other parameters in this type of lung injury. Concomitant use of FOY with other agents will have additive or synergic effects in preventing lung ischemia-reperfusion injury.

Topics: Animals; Bronchoalveolar Lavage Fluid; Dogs; Gabexate; Hemodynamics; Ischemia; Lung; Neutrophils; Peroxidase; Reperfusion Injury; Respiratory Distress Syndrome; Serine Proteinase Inhibitors

1 Reactive oxygen species have been demonstrated to play a critical role in post-ischaemic tissue injury. The present experiment was designed to evaluate the effects of SB 211475, a hydroxylated metabolite of the new beta-adrenoceptor antagonist, carvedilol, on rat splanchnic ischaemia (SI, 60 min) and reperfusion(R)-induced shock and tissue injury. 2 Administration of SB 211475 two min before R attenuated SI/R injury in a dose-dependent manner. At doses of 0.5 mg kg(-1) and 1.0 mg kg(-1), SB 211475 exerted significant anti-shock and endothelial protective effects, characterized by prolonged survival times, increased survival rates, attenuated increases in tissue myeloperoxidase activity and haematocrits, and preserved endothelium-dependent vasorelaxation. 3 Administration of 1 mg kg(-1) carvedilol attenuated shock-induced tissue injury and endothelial dysfunction. However, administration of 0.5 mg kg(-1) carvedilol had no protective effects on post-ischaemic tissue injury. 4 Previous studies have shown that SB 211475 has virtually no beta-blocking activity but possesses more potent antioxidant activity than carvedilol. In the present study, SB 211475 exerted more potent protective effects than the parent compound, suggesting that this metabolite of carvedilol is superior to carvedilol with regard to its protection against post-ischaemia tissue injury.

Topics: Adrenergic beta-Antagonists; Animals; Antioxidants; Blood Pressure; Carbazoles; Carvedilol; Endothelium, Vascular; In Vitro Techniques; Ischemia; Male; Mesenteric Artery, Superior; Muscle Relaxation; Muscle, Smooth, Vascular; Peroxidase; Propanolamines; Rats; Rats, Sprague-Dawley; Reperfusion Injury

The purpose of this study was to determine whether depletion of circulating neutrophils, using an antineutrophil monoclonal antibody (RP3), would attenuate ischemia/reperfusion injury in rat skeletal muscle. A 3- and 5-hr period of ischemia was induced unilaterally into the hindlimbs of rats; the isolated limbs were then reperfused for 24 hr after ischemia. The gastrocnemius muscle was then removed, and blood was taken simultaneously. The hematologic parameters were measured, muscle neutrophil sequestration was assessed by myeloperoxidase (MPO) activity, free radical production was evaluated by the tissue lipid peroxides (LPO) levels, muscle viability was assessed by tissue levels of adenosine triphosphate (ATP) and creatine phosphate (PCr) levels, and muscle wet/dry weights were determined. Treatment with RP3 selectively and sufficiently depleted the circulating neutrophil population, markedly reduced MPO, and significantly attenuated LPO and the tissue water content after both 3- and 5-hr of ischemia. After 3 hr of ischemia, ATP and PCr levels were significantly increased by neutrophil depletion; however, after 5 hr of ischemia, the same effect was not demonstrated. These results suggest that neutrophil depletion after 3 hr of ischemia restrains free radical production and edema formation, and also attenuates skeletal muscle ischemia reperfusion injury; however, after 5 hr of ischemia, ischemic damage was so severe, that neutrophil depletion did not reduce ischemia reperfusion injury.

Topics: Adenine Nucleotides; Animals; Antibodies, Monoclonal; Hindlimb; Ischemia; Lipid Peroxidation; Male; Muscle, Skeletal; Neutrophils; Peroxidase; Rats; Rats, Inbred Strains; Reperfusion Injury

Increased production of oxygen free radicals and infiltration of neutrophils into tissue subjected to ischemia-reperfusion have emphasized that neutrophils play a direct role in the development of injury. The present study was designed to elucidate the effect of FK506, a new immunosuppressive drug, on 11 hours of complete ischemia and reperfusion of the inguinal island skin flaps in rats. Group 1 (n = 10) control animals underwent ischemia and reperfusion and no treatment. Group 2 (n = 10) animals received FK 506 0.3 mg/kg/day, and group 3 (n = 9) animals received 0.5 mg/kg/day intramuscularly for 3 days before the ischemia. The effect of the drug was evaluated by measuring flap survival and tissue malondialdehyde content and myeloperoxidase activity and also by histopathologic examination of the skin specimens taken at the 1st and 24th hour after reperfusion. The survival of flaps controlled for 7 days was found to be significantly improved in group 2 (65.0 +/- 10.93 percent) and group 3 (93 +/- 6.25 percent) when compared with the control group (14 +/- 10.12 percent) (p < 0.04 and p < 0.0001). The tissue contents of malondialdehyde and activities of myeloperoxidase were significantly lower in groups 2 and 3 than in the control group. Three days of pretreatment with FK506 significantly reduced neutrophil infiltration in groups treated with either of the doses. These results showed that neutrophils play an important role in island flap survival associated with ischemia-reperfusion injury. Increased neutrophil infiltration was found related with increased levels of malondialdehyde and myeloperoxidase. Flap necrosis and the increase in malondialdehyde, myeloperoxidase, and neutrophil infiltration were improved by FK506 pretreatment, a neutrophil modulating agent.

Topics: Animals; Free Radicals; Graft Survival; Groin; Immunosuppressive Agents; Injections, Intramuscular; Ischemia; Leukocyte Count; Male; Malondialdehyde; Neutrophil Activation; Neutrophils; Peroxidase; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Skin; Skin Transplantation; Surgical Flaps; Tacrolimus

Intestinal ischemia-reperfusion (I-R) is associated with lung injury and the acute respiratory distress syndrome. The hypothesis of this study was that intestinal I-R activates circulating neutrophils to promote elastase-mediated lung injury. Isolated rat lungs were perfused with blood or plasma obtained after intestinal I-R, and lung neutrophil retention and injury and bronchoalveolar lavage (BAL) elastase were measured. Perfusion with I-R blood caused lung neutrophil accumulation and injury and increased BAL elastase. These effects were attenuated by the elastase inhibitor L-658758. Interference with neutrophil adherence before gut reperfusion blocked BAL elastase accumulation. The role of endothelial junction proteins (cadherins) in I-R-elicited lung damage was also evaluated. Activated human neutrophils proteolyzed cadherins in human umbilical vein endothelial cells. Furthermore, plasma of patients with acute respiratory distress syndrome contained soluble cadherin fragments. The results of this study suggest that the elastase released by systemically activated neutrophils contributes to lung neutrophil accumulation and pulmonary microvascular injury. Elastase-mediated proteolysis of endothelial cell cadherins may represent the mechanism through which lung microvascular integrity is disrupted after intestinal I-R.

Topics: Animals; Blood Pressure; Bronchoalveolar Lavage Fluid; Cadherins; Capillaries; Capillary Permeability; Cells, Cultured; Cephalosporins; Endothelium, Vascular; Humans; In Vitro Techniques; Intestines; Ischemia; Leukocyte Elastase; Lung; Male; Neutrophils; Peroxidase; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Respiratory Distress Syndrome; Serine Proteinase Inhibitors; Umbilical Veins

The objective of this study was to directly study a role for mast cells in ischemia-reperfusion (I/R)-induced mucosal and microvascular dysfunction. I/R was induced in the intestine and skeletal muscle (gastrocnemius and cremaster muscle) of wild-type mice and mast cell-deficient mice (W/Wv). Changes in mucosal permeability (blood-to-lumen clearance of 51Cr-EDTA), leukocyte infiltration (myeloperoxidase activity in the intestine and intravital microscopy in the cremaster muscle), and vascular permeability (tissue wet-to-dry weight ratio and FITC-albumin leakage) were measured as indexes of tissue dysfunction. In wild-type animals, intestinal I/R induced a significant increase in mucosal permeability, leukocyte infiltration, and vascular permeability. Mast cell-deficient animals were completely protected from I/R-induced mucosal dysfunction. However, skeletal muscle I/R induced a significant increase in leukocyte infiltration, FITC-albumin leakage, and edema formation to the same degree in both wild-type and mast cell-deficient animals. These data suggest that mast cells may be important mediators of I/R-induced mucosal and microvascular dysfunction in the intestine but not of microvascular dysfunction in skeletal muscle.

Topics: Animals; Capillary Permeability; Chromium Radioisotopes; Edetic Acid; Inflammation; Intestinal Mucosa; Ischemia; Jejunum; Leukocytes; Mast Cells; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Mutant Strains; Muscle, Skeletal; Peroxidase; Reperfusion

The function of cytokine-induced neutrophil chemoattractant (CINC), which is the rat counterpart to human growth-related gene product belonging to the CXC chemokine subgroup, is based principally on neutrophil-specific chemotactic activity. In addition, we previously reported that plasma CINC was elevated during the period of small intestinal ischemia-reperfusion injury, and that there was a correlation between the degree of mucosal damage and the peak level of CINC after reperfusion, suggesting that CINC may play a major role in neutrophil infiltration into the rat small intestinal ischemia-reperfusion injury site. Thus, we investigated whether administration of anti-CINC monoclonal antibodies (mAbs) reduces small intestinal ischemia-reperfusion injury. Small intestine was subjected to ischemia for 3 h by occlusion of the anterior mesenteric artery with an atraumatic vascular clump. After infusion of anti-CINC mAbs or isotype-matched mAbs, the intestine was subjected to reperfusion. The pretreatment with anti-CINC mAbs attenuated ischemia-reperfusion injury in the small intestine, in association with the reduction of tumor necrosis factor-alpha and myeloperoxidase production, and resulted in the prolongation of survival. It is concluded that CINC plays an important role in the onset of rat small intestinal ischemia-reperfusion injury. In addition, blocking the action of CINC, namely, the neutrophil chemotactic activity, may be useful in preventing ischemia-reperfusion injury in the small intestine.

Topics: Animals; Antibodies, Monoclonal; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Growth Inhibitors; Growth Substances; Intercellular Signaling Peptides and Proteins; Intestinal Mucosa; Intestine, Small; Ischemia; Ligation; Male; Mesenteric Arteries; Peroxidase; Rats; Rats, Inbred Lew; Reperfusion Injury; Survival Analysis; Tumor Necrosis Factor-alpha

Topics: Acetylcysteine; Amylases; Animals; Biomarkers; Glutathione; Intestine, Small; Ischemia; Malondialdehyde; Myocardium; Necrosis; Peroxidase; Rats; Rats, Wistar; Reperfusion; Somatostatin

Tissue subjected to a period of ischemia undergoes morphological and functional damage that increases during the reperfusion phase. The aim of the present work was to assess the possible improvement induced by exogenous administration of nitric oxide (NO) on renal injury and inflammatory reaction in an experimental animal model of renal ischemia-reperfusion (I-R).. Ischemia was achieved by ligation of the left arteria and vein for 60 min, followed first by contralateral nephrectomy and then reestablishment of blood flow. Molsidomine, used as an NO donor, was administered by systemic injection 30 min before reperfusion. The effect of molsidomine was compared with the effect of hydralazine, a non-NO donor hypotensive agent.. Treatment with molsidomine improved the renal dysfunction (increase in plasma creatinine and urea levels) caused by I-R. Moreover, molsidomine blunted the enhanced production of proinflammatory cytokines (tumor necrosis factor [TNF]-alpha and interleukin [IL] 1alpha), the increase in tissular levels of superoxide anions and oxygen free radical scavengers, and the neutrophilic infiltration observed in the ischemic kidney. One hundred percent survival was achieved in the group of animals treated with the NO donor, whereas the groups of animals undergoing I-R that did not receive molsidomine showed a 40% mortality from the second day after reperfusion.. The present work demonstrated that systemic treatment with an NO donor before reperfusion improved renal function and diminished inflammatory responses in a kidney subjected to an I-R process.

Topics: Animals; Blood Pressure; Cytokines; Free Radical Scavengers; Ischemia; Kidney; Kidney Function Tests; Male; Nephritis; Nitric Oxide; Peroxidase; Rats; Rats, Wistar; Reactive Oxygen Species; Renal Circulation; Reperfusion Injury; Superoxides; Survival Analysis

We investigated the effects of the xanthine oxidase inhibitor, BOF-4272, on the production of cytokine-induced neutrophil chemoattractant (CINC) following reperfusion injury in rat liver. Ischemia was induced for 30 minutes by portal vein occlusion. Animals were pretreated with intravenous injection of BOF-4272 (1 mg/kg) or heparin (50 U/kg) 5 minutes before vascular clamp. Both BOF-4272 and heparin limited increases in the chemoattractant compared with nonpretreated rats. Pretreatment with BOF-4272 plus heparin resulted in an additive effect. Most cells immunostained for chemoattractant were macrophages in sinusoids. In vitro chemoattractant production by Kupffer cells isolated from animals pretreated with heparin or BOF-4272 was significantly lower than by Kupffer cells from nonpretreated animals. Expression of transcripts in liver for chemoattractant peaked 3 hours after reperfusion in nonpretreated animals, while pretreatment with heparin or BOF-4272 significantly decreased chemoattractant mRNA levels. In vitro chemoattractant transcription and production could be induced in naive Kupffer cells by hypoxanthine and xanthine oxidase, but BOF-4272 prevented these increases. We conclude that Kupffer cells release chemoattractant in response to oxygen radicals reducible by xanthine oxidase inhibition.

Topics: Animals; Chemokines, CXC; Chemotactic Factors; Enzyme Inhibitors; Growth Substances; Heparin; Intercellular Signaling Peptides and Proteins; Ischemia; Kupffer Cells; Liver; Liver Circulation; Male; Neutrophils; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger; Triazines; Xanthine Oxidase

Splanchnic artery occlusion and reperfusion (SAO/R) results in a severe form of circulatory shock that has a high mortality rate. To examine the time course of the early events involved in SAO/R, occlusion of the superior mesenteric artery (SMA) and the celiac artery (120 min) were followed by reperfusion periods of 0, 2.5, 5, 20, 30, 60, or 120 min. Relaxation of isolated SMA vascular rings to the endothelium-dependent vasodilator ACh was unimpaired following 120 min of ischemia (86 +/- 5%); however, significant (P < 0.01) reductions in endothelium-dependent vasorelaxation were observed following 2.5 min (53 +/- 6%) of reperfusion with severe dysfunction (P < 0.001) observed at 20 min (29 +/- 4%). Neutrophil adherence to the endothelium increased as a function of reperfusion time with a 2.3-fold increase observed at 20 min (P < 0.01) and a 3.4-fold increase observed at 120 min (P < 0.001). Intestinal myeloperoxidase activity was significantly increased 30 min after reperfusion (P < 0.05), whereas surface expression of P-selectin progressively increased at 5 (P < 0.05) and 30 min (P < 0.001) postreperfusion. These findings demonstrate that endothelial dysfunction is a very early event in the pathophysiology of SAO/R, subsequently resulting in increased surface expression of P-selectin and the adherence of neutrophils to the endothelium that leads to neutrophil accumulation in the splanchnic viscera.

Topics: Animals; Arteries; Blood Pressure; Cell Adhesion; Endothelium, Vascular; Intestinal Mucosa; Ischemia; Male; Mesenteric Arteries; Neutrophils; P-Selectin; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Splanchnic Circulation; Time Factors

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.. 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.. 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.. Cytokine-induced neutrophil chemoattractant production after ischemia-reperfusion in the liver is mediated by neutrophil elastase and activation of coagulation within the hepatic microcirculation.

Topics: Animals; Antithrombin III; Aspartate Aminotransferases; Chemokines, CXC; Chemotactic Factors; Drug Synergism; Glycine; Growth Substances; Heparin; Intercellular Signaling Peptides and Proteins; Ischemia; Leukocyte Count; Leukocyte Elastase; Liver; Liver Circulation; Male; Neutrophils; Peroxidase; Rats; Rats, Wistar; Reperfusion; RNA, Messenger; Serine Proteinase Inhibitors; Sulfonamides

The aim of this study was to determine whether the length of ischemia in skeletal muscle influences the return of normal microvascular permeability during reperfusion in addition to influencing the size of the initial changes. In anesthetized rabbits, the transvascular clearance of labeled albumin was measured in the gastrocnemius and soleus muscles during the first, second, third, or fourth hour of reperfusion after 1, 2, 3, or 4 h of ischemia. The size of the increases in albumin clearance, tissue water, and myeloperoxidase activity during the first hour of reperfusion was dependent on the length of ischemia. The return of the albumin clearance to control values during the fourth hour of reperfusion was independent of the length of ischemia. Tissue water, extravascular mass of native albumin, and myeloperoxidase activity remained elevated during the 4 h of reperfusion. After 4 h of ischemia, the solvent-drag reflection coefficient for albumin was significantly less than control during the first hour of reperfusion. The value during the fourth hour of reperfusion was not significantly different from control. These results suggest that the inflammatory mediators producing a change in permeability are washed out of the microvasculature during the first few hours of reperfusion.

Topics: Animals; Body Water; Female; Ischemia; Male; Microcirculation; Muscle, Skeletal; Peroxidase; Rabbits; Reference Values; Reperfusion; Serum Albumin; Time Factors

An ischemia-reperfusion injury on the pancreas is involved in the pathophysiology of acute pancreatitis or tissue injuries after pancreas transplantation. On the other hand, recent studies have demonstrated that ischemia-reperfusion induces apoptosis in several organs such as kidney, heart, and brain. In the present study, we sought to characterize a pattern of injury during ischemia-reperfusion on the pancreas and determined whether ischemia-reperfusion on the pancreas causes the apoptotic process. Ischemia-reperfusion was induced by blocking the inferior splenic artery and removing the clamp in pentobarbital-anesthetized rats. Rats were sacrificed at 0-72 hr following a 60-min ischemia. Evans blue extravasation showed 3.5-fold increase at 2 hr after reperfusion, indicating a rapid increase of vascular permeability. Tissue myeloperoxidase activity, an index of neutrophil accumulation, significantly increased in a time-dependent manner until 48 hr after reperfusion. Histological analysis revealed the existences of interstitial cell infiltration and edema. DNA breaks of acinar cells were detected by gel electrophoresis and in situ nick end-labeling, and the numbers strikingly increased at 48 hr after reperfusion. Furthermore, Bax protein, an effector of apoptotic cell death, was expressed in acinar cells. The results indicate that an ischemia-reperfusion injury on the pancreas in rats resembles many features of acute pancreatitis. Apoptosis in acinar cells may be one of the specific features of the ischemia-reperfusion injury on the pancreas.

Topics: Amylases; Animals; Apoptosis; DNA; Interleukin-1; Ischemia; Male; Pancreas; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tumor Necrosis Factor-alpha

We used adenosine A1 receptor agonist N6-1(phenyl-2R-isopropyl)-adenosine (PIA), A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and ATP-sensitive K+ (KATP) channel blockers sodium 5-hydroxydecanoate (5-HD) and glibenclamide (Glib), as probes to investigate the role and mechanism of adenosine in ischemic preconditioning (IPC) of noncontractile skeletal muscle against infarction, using the pig latissimus dorsi muscle flap model. Except for Glib, all drugs were delivered to each muscle flap by 10-min local intra-arterial infusion to avoid systemic effects. Muscle flaps that were subjected to 4 h of global ischemia and 48 h of reperfusion sustained 40 +/- 2% infarction. IPC with three cycles of 10 min ischemia and reperfusion, preischemic adenosine, or PIA treatment reduced (P < 0.05) muscle infarction to 24 +/- 2, 18 +/- 2, and 24 +/- 2%, respectively. The anti-infarction effect of IPC and adenosine was blocked by DPCPX, 5-HD, and Glib (P < 0.05). Preischemic adenosine treatment also maintained higher muscle contents of phosphocreatine, ATP, and energy charge potential and lower muscle contents of dephosphorylated metabolites and lactate during ischemia and a lower muscle myeloperoxidase (MPO) activity during reperfusion compared with the control (P < 0.05). Preischemic adenosine treatment did not increase muscle content of adenosine during ischemia or reperfusion. Furthermore, adenosine given at the onset of reperfusion was not effective in attenuating muscle MPO activity or infarction. Taken together, these observations indicate that adenosine, through A1 receptors, initiates the mechanism of IPC with postreceptor involvement of KATP channels in skeletal muscle. However, adenosine is unlikely to play a key role in the effector mechanism. Presently, the cause and role of energy sparing and neutrophil inhibitory effects associated with the anti-infarction effect of preischemic adenosine treatment are unknown.

Topics: Adenosine; Animals; Decanoic Acids; Glyburide; Hydroxy Acids; Infarction; Ischemia; Ischemic Preconditioning; Muscle, Skeletal; Neutrophils; Peroxidase; Phenylisopropyladenosine; Potassium Channel Blockers; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Reperfusion; Swine; Time Factors; Xanthines

The purpose of this study was to investigate the role of neutrophils in ischemic tissue injury and the possible inhibition by pretreatment with FK506, a neutrophilic modulating agent. A dorsal caudally based skin flap (3 x 9 cm) was used as an ischemic injury model in experimental groups. Prior to flap elevation, FK506 at doses of 0.3 mg per kilogram (group 2), 0.5 mg per kilogram (group 3), and 1.0 mg per kilogram (group 4) was given for 3 days intramuscularly. The relationship among neutrophil accumulation (histopathologically), myeloperoxidase (MPO) activity, malondialdehyde (MDA) content (biochemically) of the flap tissue, and flap survival were studied. Skin flaps showed reduced necrosis in the FK506-treated groups (p < 0.08, p < 0.0001, and p < 0.0001 respectively). The increase in accumulation of neutrophils, and MDA and MPO levels (which were induced by ischemia) observed 1 and 24 hours after flap elevation was diminished by FK506 pretreatment. The increased neutrophilic infiltration, and raised tissue MDA content and MPO activity revealed involvement of both free radical production and neutrophils in ischemia. This injury was decreased by FK506, probably by inhibition of neutrophilic chemotaxis, infiltration, and releasing factors.

Topics: Animals; Graft Survival; Immunosuppressive Agents; Ischemia; Male; Malondialdehyde; Neutrophils; Peroxidase; Rats; Rats, Wistar; Skin; Surgical Flaps; Tacrolimus

The aim of this study was to look at the role of alpha 1-acid glycoprotein as a natural anti-inflammatory agent with particular respect to its antineutrophil and anticomplement activity. A recombinantly engineered form of sialyl Lewisx (sLe(x))-bearing alpha 1-acid glycoprotein (sAGP) was administered intravenously to pentobarbital-anesthetized rats after 50 min of intestinal ischemia just before 4 h of reperfusion. A non-sLe(x)-bearing form of AGP (nsAGP) was used as control. sAGP-treated animals had a 62% reduction (P < 0.05) in remote lung injury, assessed by 125I-albumin permeability, compared with those treated with nsAGP (permeability index of 3.61 +/- 0.15 x 10(-3) and 5.18 +/- 0.67 x 10(-3), respectively). There was a reduction in pulmonary myeloperoxidase levels in sAGP-treated rats compared with nsAGP-treated rats. Complement-dependent intestinal injury, assessed by 125I-albumin permeability was reduced by 28% (P < 0.05) in animals treated with sAGP (7.58 +/- 0.63) compared with those treated with nsAGP (10.4 +/- 0.54). We conclude that sAGP ameliorates both complement- and neutrophil-mediated injuries.

Topics: Animals; Cell Membrane Permeability; Complement Pathway, Alternative; Humans; Intestines; Iodine Radioisotopes; Ischemia; Lung; Male; Mesenteric Vascular Occlusion; Oligosaccharides; Orosomucoid; Peroxidase; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reperfusion; Sialyl Lewis X Antigen

Studies in the rat have pointed to a role for intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of acute tubular necrosis. These studies used antibodies, which may have nonspecific effects. We report that renal ICAM-1 mRNA levels and systemic levels of the cytokines IL-1 and TNF-alpha increase 1 h after ischemia/ reperfusion in the mouse. We sought direct proof for a critical role for ICAM-1 in the pathophysiology of ischemic renal failure using mutant mice genetically deficient in ICAM-1. ICAM-1 is undetectable in mutant mice in contrast with normal mice, in which ICAM-1 is prominent in the endothelium of the vasa recta. Mutant mice are protected from acute renal ischemic injury as judged by serum creatinine, renal histology, and animal survival . Renal leukocyte infiltration, quantitated morphologically and by measuring tissue myeloperoxidase, was markedly less in ICAM-1-deficient than control mice. To evaluate whether prevention of neutrophil infiltration could be responsible for the protection observed in the mutant mice, we treated normal mice with antineutrophil serum to reduce absolute neutrophil counts to < 100 cells/mm3. These neutrophil-depleted animals were protected against ischemic renal failure. Anti-1CAm-1 antibody protected normal mice against renal ischemic injury but did not provide additional protection to neutrophil-depleted animals. Thus, ICAM-1 is a key mediator of ischemic acute renal failure likely acting via potentiation of neutrophilendothelial interactions.

Topics: Animals; Intercellular Adhesion Molecule-1; Ischemia; Kidney; Kidney Diseases; Mice; Mice, Inbred Strains; Mice, Knockout; Neutrophils; Peroxidase

The purpose of this study was to investigate whether treatment with TCV-309, a PAF antagonist, improves life-sustaining function of renal grafts that have suffered warm ischemia (WI) prior to cold storage (CS) and whether TCV-309 influences leukocyte sequestration in tissues. Syngeneic kidneys with 20 min of WI and 24 hr of CS were transplanted into bilateral nephrectomized rats. In the treated group, TCV-309 was administered (i.v. 1 mg/kg) 5 min before reperfusion. Rats in the control group received saline. On day 14, 80% rats survived in the treated group, which was higher than the controls (0%). At 24 hr of reperfusion, myeloperoxidase (MPO) activity, a marker enzyme for PMNs, in the treated kidney was significantly lower than the controls, but did not differ from the normal values. The MPO activity in the controls was higher than the normal values. In conclusion, the PAF antagonist improves posttransplant function of rat kidneys subjected to a period of WI and CS. PMNs are involved in postischemic renal injury, which is, at least partially, mediated by PAF. The effectiveness of PAF antagonist in treatment of recipients may lead to its clinical application in transplantation of ischemically injured kidneys.

Topics: Animals; Hot Temperature; Immunity, Cellular; Ischemia; Isoquinolines; Kidney; Kidney Cortex; Kidney Transplantation; Lung; Macrophages; Monocytes; Neutrophils; Organ Preservation; Peroxidase; Platelet Activating Factor; Platelet Aggregation Inhibitors; Pyridinium Compounds; Rats; Rats, Inbred Lew; Reperfusion Injury; Tetrahydroisoquinolines; Time Factors

Ischemia and reperfusion injury of rodent intestine is complement mediated. P-selectin antagonism reduces local injury, yet neutrophil depletion does not. This study tests the thesis that the protective mechanism of P-selectin antagonists involves complement inhibition.. We subjected rats (n = 86) to 50 minutes of complete mesenteric ischemia and 4 hours of reperfusion. Treatment with a monoclonal antibody (PB1.3) against P-selectin reduced intestinal injury as judged by 125I-albumin permeability index (7.33 +/- 0.40) compared with saline solution treatment (11.4 +/- 0.49) (p < 0.05).. However, intestinal neutrophil sequestration assessed by myeloperoxidase assay was unchanged. Immunohistochemistry revealed that mucosal C5b-9 was deposited in animals treated with saline solution and was absent in the sham group. PB1.3 treatment reduced C5b-9 deposition in the intestinal mucosa compared with that in animals treated with saline solution (p < 0.05). Neutrophil-dependent remote lung injury assessed by 125I-albumin permeability and pulmonary myeloperoxidase assay were not significantly reduced by PB1.3. Treatment with a soluble form of P-selectin ligand, sialyl Lewisx (sLex), reduced intestinal myeloperoxidase (0.065 +/- 0.006) compared with saline solution treatment (0.136 +/- 0.02) (p < 0.05), but it did not reduce permeability. Remote lung permeability was reduced (4.52 +/- 0.65 x 10(-3)) by sLex compared with saline solution treatment (6.11 +/- 0.41 x 10(-3)) (p < 0.05).. Antagonizing the lectin domain of P-selectin and thereby neutrophil adhesion was without local benefit in this model. In contrast, PB1.3 exerted a novel antagonism of P-selectin and reduced complement deposition.

Topics: Animals; Antibodies, Monoclonal; Complement Membrane Attack Complex; Intestines; Ischemia; Male; Neutrophils; P-Selectin; Permeability; Peroxidase; Rats; Rats, Sprague-Dawley

During postischaemic revascularization neutrophil-endothelial adhesion in the skeletal muscle microcirculation, promoted by the neutrophil adhesion molecule Mac-1, may impair muscle blood flow and release oxygen free radicals and proteolytic enzymes which causes further tissue injury. This study has assessed the effect of an anti-Mac-1 monoclonal antibody on the severity of skeletal muscle injury in a rat model of 6-h hindlimb ischaemia and 4-h reperfusion. In control animals a sustained impairment of muscle perfusion was associated with neutrophil sequestration, muscle infarction and muscle oedema (P < 0.001 versus normal rats). In contrast, intravenous administration of anti-Mac-1 monoclonal antibody before revascularization prevented neutrophil recruitment, reduced muscle necrosis and improved postischaemic muscle perfusion at 120 and 240 min (not significantly different from normal), thus confirming that neutrophils are important cellular mediators of skeletal muscle reperfusion injury. Monoclonal antibodies targeting neutrophil adhesion molecules may, therefore, have a role in the prevention of this complication of limb revascularization.

Topics: Animals; Antibodies, Monoclonal; Edema; Hindlimb; Ischemia; Macrophage-1 Antigen; Male; Microcirculation; Muscle, Skeletal; Neutrophils; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury

The effects of 48 hr of hypothermic (4 degrees C ischemia) and short-term reperfusion. (I-R) on intestinal function and metabolism were studied in dogs utilizing Collins flush alone or with the putative cytoprotectant amino acid, glycine. Intestinal blood flow after hypothermic ischemia in Collins-flushed segments briefly rose at reperfusion, rapidly declined after 5 min, and plateaued over the 60-minute reperfusion period. Paired intestinal segments flushed with 5 mM glycine demonstrated parallel changes in blood flow over the reperfusion period, but the blood flow values were significantly higher (100-300%), relative to the Collins segments. Intestinal oxygen consumption (VO2) was about 50% of normal nonischemic intestinal segments at all times after reperfusion. The glycine-flushed intestinal segments significantly consumed about 100% more oxygen, relative to the paired control intestines. Intestinal fluid and protein flux into the lumen significantly increased after I-R in both glycine- and Collins-flushed segments. Mucosal tissue myeloperoxidase (MPO) activity, a biochemical marker of neutrophils, significantly increased after 48 hr of cold ischemia with Collins flush and 1 hr of reperfusion, relative to tissue obtained before ischemia. The reperfusion-induced increase in MPO activity was abolished in intestinal segments flushed with glycine. Mucosal synthesis of the chemoattractant leukotriene B4 (LTB4) significantly increased after I-R and glycine flush abolished these increases. Nitric oxide synthesis by mucosal tissue in Collins-flushed segments subjected to 48 hr of hypothermic ischemia and 1 hr of reperfusion was significantly higher, compared with nonischemic tissue or mucosal tissue subjected to cold ischemia without reperfusion. Glycine flush did not alter this pattern of NO synthesis. Light microscopic analysis in both Collins- and glycine-flushed segments revealed that intestinal hypothermic ischemia and reperfusion caused significant morphologic changes characterized by loss of villus epithelium, decreased villus height, and venous congestion. These data indicate that glycine significantly improve oxygenation after hypothermic ischemia and reperfusion and prevented the I-R-induced increase in tissue neutrophil infiltration and leukotriene synthesis.

Topics: Animals; Dogs; Glycine; Hypertonic Solutions; Hypothermia, Induced; Intestinal Mucosa; Intestine, Small; Ischemia; Leukotriene B4; Neutrophils; Nitric Oxide; Organ Preservation; Peroxidase; Reperfusion Injury

The combination of ischemia and reperfusion after lung transplantation is characterized by endothelial damage, neutrophil sequestration, and decreased release of endothelial nitric oxide. Because nitric oxide has been shown to selectively dilate the pulmonary vasculature, abrogate neutrophil adherence, and restore endothelial dysfunction, we hypothesized that inhaled nitric oxide given for 4 hours during initial reperfusion might attenuate reperfusion injury in a porcine model of left single-lung transplantation. We tested hemodynamic and gas exchange data, lung neutrophil sequestration, and pulmonary artery endothelial dysfunction after 4 and 24 hours of reperfusion in 12 pigs randomly assigned to nitric oxide and control groups. Harvested lungs were preserved in normal saline solution for 24 hours at 4 degrees C. During transplantation, inflatable cuffs were placed around each pulmonary artery to allow separate evaluation of each lung by occluding flow. Compared with the transplanted lungs in the control group, transplanted lungs in pigs treated with inhaled nitric oxide significantly improved gas exchange, pulmonary vascular resistance, shunt fraction, and oxygen delivery at 4 and 24 hours after reperfusion. Neutrophil sequestration, as measured by the neutrophil-specific enzyme myeloperoxidase and the alveolar leukocyte count per light microscopic field, was significantly lower at 24 hours after reperfusion in the transplanted lungs of the nitric oxide group. The nitric oxide-treated native right lungs exhibited significantly reduced increase in neutrophil accumulation compared with that in control native right lungs. After 24 hours of reperfusion, endothelium-dependent relaxation to acetylcholine was similarly and severely altered in both groups. We conclude that short-term inhaled nitric oxide given during the first 4 hours of reperfusion after lung transplantation significantly attenuates reperfusion injury, improving graft function as long as 24 hours after operation. This effect is probably mediated by a decrease in neutrophil sequestration. A protective effect on the contralateral lung was also observed. Inhaled nitric oxide may be a suitable agent when an acute reperfusion phenomenon is anticipated.

Topics: Administration, Inhalation; Animals; Cell Adhesion; Disease Models, Animal; Endothelium, Vascular; Graft Survival; Hemodynamics; Ischemia; Leukocyte Count; Lung; Lung Transplantation; Neutrophils; Nitric Oxide; Organ Preservation; Oxygen; Peroxidase; Pulmonary Artery; Pulmonary Circulation; Pulmonary Gas Exchange; Random Allocation; Reperfusion Injury; Swine; Vascular Resistance; Vasodilator Agents

We investigated the role played by monocytes and lymphocytes in the pathogenesis of experimental shock. Splanchnic artery occlusion (SAO) shock was induced in anaesthetized rats by clamping splanchnic arteries for 45 min followed by reperfusion. Sham operated animals were used as controls. SAO shocked rats had a decreased survival time (80 +/- 11 min, while sham shocked rats survived more than 4 h), increased serum (248 +/- 21 U/ml) and macrophage (145 +/- 15 U/ml) levels of TNF-alpha, enhanced myeloperoxidase (MPO) activity in the ileum (3.38 +/- 0.2 U x 10(-3)/g tissue), decreased number of monocytes, lymphocytes and neutrophils and a profound hypotension. In addition we found an increased expression of vascular cell adhesion molecule-1 (VCAM-1) on aortic endothelium and a reduced percentage of VLA-4 positive monocytes and lymphocytes. Inhibition of TNF-alpha synthesis, reversed the increased endothelial expression of VCAM-1, increased the percentage of integrin VLA-4 positive leukocytes and improved monocyte, lymphocyte and neutrophil count. Furthermore a passive immunization with specific antibodies raised against VCAM-1 (2 mg/kg, i.v. 3 h before SAO) increased survival, reduced MPO activity in the ileum (0.034 +/- 0.04 U x 10(-3)/g tissue) and improved mean arterial blood pressure. Our data suggest that monocytes and lymphocytes participate in the pathogenesis of splanchnic ischaemia-reperfusion injury and may amplify the adhesion of neutrophils to peripheral tissues.

Topics: Animals; Blood Pressure; Endothelium, Vascular; Flow Cytometry; Immunohistochemistry; Intestines; Ischemia; Leukocyte Count; Lymphocytes; Male; Monocytes; Peroxidase; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Reperfusion Injury; Shock; Survival Analysis; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

The present study addressed the effect of interventions aimed to increase NO in the setting of acute renal ischemia/reperfusion (I/R) in uninephrectomized rabbits. In the 60-minute post-I/R period, L-arginine+superoxide (O2.-) dismutase (SOD) synergistically improved the renal functional (69.4% versus 10.4% of the pre-I/R glomerular filtration rate with or without L-arginine+SOD, respectively; p < .01) and histological parameters (82.9% decrease of medullary congestion in L-arginine+SOD, P < .01 versus vehicle) and blocked the I/R-dependent neutrophil accumulation (89.3% reduction). In spite of these results over the short term, a second set of experiments disclosed that the protection by L-arginine+SOD was no longer present at 24 and 48 hours (plasma creatinine in vehicle-treated versus L-arginine+SOD-treated animals [mg/100 mL]: 24 hours after I/R, 9.4 +/- 1.9 versus 8.07 +/- 0.65; 48 hours after I/R, 11.6 +/- 3.6 versus 9.7 +/- 0.9; P = NS in all the cases). Additional experiments were conducted using a milder 30-minute ischemic model, which showed no significant functional or histological protection by using L-arginine+SOD. In conclusion, our experiments disclosed the following: (1) the critical importance of the interaction between NO and O2.- in the acute protective effect of L-arginine (this effect not only improved renal function and histology but also reduced neutrophil accumulation) and (2) the discordance existing between the immediate protection afforded by L-arginine+SOD and the lack of protection observed at 24 and 48 hours. This finding suggests that a punctual intervention on the NO system at the time of I/R is not sufficient to reduce renal damage over the long term.

Topics: Animals; Arginine; Endothelium, Vascular; Hemodynamics; Ischemia; Kidney; Male; Neutrophils; Nitric Oxide; Nitrites; Peroxidase; Rabbits; Reference Values; Renal Circulation; Reperfusion; Time Factors; Vasodilation

Neutrophils accumulate in skeletal muscle subjected to ischemia-reperfusion and appear to contribute to reperfusion-induced microvascular dysfunction. The overall objective of this study was to assess the role of the neutrophilic hydrolytic enzyme elastase in ischemia-reperfusion-induced granulocyte accumulation and microvascular dysfunction in skeletal muscle. We examined the effect of three structurally unrelated elastase inhibitors [eglin C, MeOsuc-Ala-Ala-Val-CH2Cl (MAAPV), or L-658758], administered at the onset of reperfusion, on neutrophil content and the increase in microvascular permeability induced by 4 h of ischemia and 0.5 h of reperfusion in the isolated canine gracilis muscle. Changes in vascular permeability (1 - sigma) were assessed by determining the solvent drag reflection coefficient for total plasma proteins (sigma) in the following groups: 1) 4.5 h of continuous perfusion (nonischemic), 2) ischemia-reperfusion alone, 3) ischemia-reperfusion + eglin C, 4) ischemia-reperfusion + MAAPV, and 5) ischemia-reperfusion + L-658758. Muscle neutrophil content was monitored by assessing tissue myeloperoxidase (MPO) activity in biopsies obtained at the end of the experiments. In nonischemic muscles, 1 - sigma and MPO activity averaged 0.13 +/- 0.03 and 0.7 +/- 0.2 units/g wet wt, respectively. Ischemia-reperfusion was associated with marked increases in microvascular permeability (1 - sigma = 0.39 +/- 0.02) and muscle MPO activity (8.9 +/- 1.2 units/g wet wt) that were attenuated by eglin C, MAAPV, and L-658758 (1 - sigma = 0.21 +/- 0.01, 0.22 +/- 0.02, and 0.21 +/- 0.03, respectively; MPO activity = 2.7 +/- 0.4, 2.1 +/- 0.8, and 2.8 +/- 1.8 units/g wet wt, respectively). These results suggest that granulocyte accumulation in postischemic skeletal muscle is dependent on the release of elastase from activated phagocytic cells. Moreover, neutrophilic elastase appears to play a major role in reperfusion-induced increases in microvascular permeability in skeletal muscle.

Topics: Amino Acid Chloromethyl Ketones; Animals; Capillary Permeability; Cephalosporins; Dogs; Female; Ischemia; Leukocyte Elastase; Male; Microcirculation; Muscle, Skeletal; Neutrophils; Pancreatic Elastase; Peroxidase; Protease Inhibitors; Proteins; Reperfusion; Serine Proteinase Inhibitors; Serpins

The role of platelet-activating factor (PAF) in ischemic acute renal failure was evaluated by administering an oral PAF antagonist (Ro-24-4736) to rats prior to or after interruption of blood flow to both kidneys for 30 min. In animals treated with the PAF antagonist prior to ischemia, renal function was less impaired and histological abnormalities was less pronounced when compared with postischemic kidneys from vehicle-treated animals. Serum creatinine (mg/ dl) 24 h following renal ischemia was 1.58 +/- 0.17 in the PAF antagonist-treated rats compared with 2.19 +/- 0.15 in rats given placebo (P < 0.01). There was less necrosis in the outer medulla of kidneys of PAF antagonist-treated animals (P < 0.01). Tissue myeloperoxidase activity at 48 and 72 h postischemia was lower in kidneys of PAF antagonist-treated rats (P < 0.05). The PAF antagonist was also protective when administered 30 min but not 2 h following the ischemic insult. The coincident use of anti-intercellular adhesion molecule-1 monoclonal antibody did not confer additional protection over that observed with the oral PAF antagonist alone. These data suggest that PAF contributes to the pathophysiology of renal ischemic injury, perhaps by its effects on leukocyte-endothelial interactions. An orally active PAF antagonist can protect against the development of ischemic acute renal failure.

Topics: Acute Kidney Injury; Administration, Oral; Animals; Antibodies; Dose-Response Relationship, Drug; Intercellular Adhesion Molecule-1; Ischemia; Kidney; Male; Peroxidase; Phenanthridines; Platelet Activating Factor; Rats; Rats, Sprague-Dawley; Renal Circulation; Reperfusion; Triazines

Topics: Analysis of Variance; Animals; Antioxidants; Dogs; Edema; In Vitro Techniques; Ischemia; Muscle, Skeletal; Necrosis; Peroxidase; Pregnatrienes; Reperfusion; Reperfusion Injury; Steroids, Heterocyclic; Thromboxane B2; Time Factors

The purpose of this study was to determine whether fructose-1,6-diphosphate (FDP) or adenosine (Ado), administered at the onset of reperfusion, would prevent ischemia/reperfusion (I-R)-induced leukocyte adherence and microvascular dysfunction in skeletal muscle. Changes in vascular permeability and tissue neutrophil content were assessed by measurement of the solvent drag reflection coefficient (delta) for total plasma proteins and muscle myeloperoxidase (MPO) activity, respectively, in continuously perfused, isolated canine gracilis muscles and in muscles subjected to I-R alone, I-R + FDP, and I-R + Ado. To determine whether FDP or Ado would attenuate leukocyte-endothelial cell adhesive interactions induced by I-R, leukocyte adherence and emigration were assessed in postischemic mouse cremaster muscles, using intravital microscopy in the presence and absence of FDP or Ado during reperfusion. I-R was associated with a marked increase in microvascular permeability and muscle MPO activity relative to nonischemic controls. These increases were attenuated by FDP and Ado. I-R also increased the number of adherent and emigrated leukocytes relative to control. I-R-induced leukocyte adherence and emigration were significantly attenuated by either FDP or Ado. These results indicate that FDP and Ado attenuate postischemic microvascular barrier dysfunction in skeletal muscle by a mechanism that may be related to their ability to inhibit leukocyte adhesion and emigration.

Topics: Adenosine; Animals; Capillary Permeability; Cell Adhesion; Dogs; Female; Fructosediphosphates; Immunologic Factors; Ischemia; Leukocytes; Male; Mice; Mice, Inbred Strains; Muscles; Neutrophils; Peroxidase; Reperfusion; Superoxides

Hemorrhagic mucosal lesions are produced during intestinal ischemia and after reperfusion due at least in part to the accumulation and activation of polymorphonuclear leukocytes in the tissue. It has been shown in vitro that adenosine is instrumental in attenuating this pathophysiological process. Acadesine [5-amino-4-imidazolecarboxamide (AICA) riboside], a purine nucleoside analogue, increases the availability of adenosine in the tissue. The aim of the study was therefore to assess the influence of acadesine treatment on neutrophil accumulation, purine metabolism, and mucosal damage after intestinal ischemia and reperfusion. Intestinal ischemia was induced in cats by partial occlusion of the superior mesenteric artery for 2 h. Samples of the small intestine were exercised before and at the end of the hypotensive period as well as 10 and 60 min after reperfusion. Conjugated dienes, myeloperoxidase, and reduced and oxidized glutathione, as well as the purine metabolites, were determined in the tissue samples. The tissue was also examined histologically. Six cats received saline, and six cats were treated initially before ischemia with acadesine (2.5 mg/kg body wt i.v.) over 5 min as a bolus. Thereafter, acadesine (0.5 mg.kg-1.min- i.v.) was given continuously during ischemia and 30 min after reperfusion. Acadesine treatment significantly attenuated the mucosal lesions seen during reperfusion. This improvement was due at least in part to the inhibition of neutrophil accumulation, as judged by low myeloperoxidase levels. The prevention of neutrophil activation resulted most likely from increased adenosine concentrations in the intestinal tissue in the early reperfusion period.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aminoimidazole Carboxamide; Animals; Cats; Cell Movement; Female; Glutathione; Intestinal Mucosa; Intestine, Small; Ischemia; Lipid Peroxides; Male; Neutrophils; Peroxidase; Purines; Reperfusion Injury; Ribonucleosides

Ischemia and reperfusion of the liver are associated with changes in the interaction of leukocyte-endothelium cells. The role of an adhesion molecule, P-selectin, is studied in ischemia and reperfusion injury of the liver.. Total hepatic ischemia was produced in the rat for 90 minutes, using a portosystemic shunt. To determine the role of P-selectin in ischemia and reperfusion, a murine IgG1 monoclonal antibody to P-selectin (1 mg/kg) was used at different times (30 minutes before and at reperfusion and five minutes and 24 hours after reperfusion). Rats survived for seven days, and tests showing hepatic injury, myeloperoxidase in hepatic tissue, and histologic studies were analyzed at four hours postreperfusion.. Survival improved from 15 percent for the rats in the ischemia control group to 55 percent for those in the group receiving anti-P-selectin antibody given 30 minutes before reperfusion (p < 0.05). We observed an improved statistically significant difference in tests demonstrating hepatic injury, myeloperoxidase in hepatic tissue, and histologic studies in the treated and ischemia control groups. The other groups did not show consistent significant differences.. P-selectin has a significant role in ischemia and reperfusion injury of the liver. Early modulation of the interaction between P-selectin and its ligand decreased neutrophil adhesion and migration and consequently diminished damage to the liver.

Topics: Analysis of Variance; Animals; Antibodies, Monoclonal; Disease Models, Animal; Drug Evaluation, Preclinical; Immunoglobulin G; Ischemia; Liver; Male; Mice; P-Selectin; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Statistics, Nonparametric; Time Factors

Gut ischemia/reperfusion (I/R) provokes lung injury via a mechanism that involves neutrophils [polymorphonuclear neutrophils (PMNs)]. CD11b/CD18 (alpha mB2) is the integrin receptor on PMNs critical for adhesion-dependent oxidative burst. The purpose of this study was to investigate the mechanistic role of CD11b in the process of gut I/R-induced lung injury. Sprague-Dawley rats underwent 45 minutes of superior mesenteric artery (SMA) occlusion with and without CD11b monoclonal antibody treatment (IB6) (1 mg/kg, i.v.), before SMA clamping. At 2-hour reperfusion, PMN presence in tissue was quantitated by myeloperoxidase activity and circulating PMN priming determined by the difference in superoxide production with and without N-formyl-methionyl-leucyl-phenylalanine, whereas lung leak was assessed by 125I-albumin lung/blood ratio. In sum, CD11b blockade prevented gut I/R-induced lung leak, but did not attenuate gut I/R-induced PMN priming or tissue PMN accumulation. In conclusion, gut I/R promotes PMN priming and PMN adhesion in both local and distant beds via receptors other than CD11b, but this B2 integrin receptor is critical for PMN-mediated endothelial injury.

Topics: Animals; Intestinal Mucosa; Intestines; Ischemia; Lung; Lung Diseases; Macrophage-1 Antigen; Male; Neutrophils; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion; Reperfusion Injury

Warm and cold ischemia-reperfusion injuries to canine small intestine was compared. In the warm ischemic model, the superior mesenteric artery of mongrel dogs was clamped for 2 h and then released (group A). As a cold ischemia model, canine small intestines were harvested with cold lactated Ringer solution, preserved for 24 h in cold LR solution and then autotransplanted (group B). After ischemia and during reperfusion, activities of maltase (MAL), myeloperoxides (MPO), xanthine dehydrogenase (XD) and xanthine oxidase (XO) were measured as well as hypoxanthine (HX) concentration. MAL activities were not changed during warm or cold ischemia, whereas it was remarkably decreased after revascularization in both the groups. Neutrophil infiltration after reperfusion was shown by the increase of MPO activities to 8 and 1.5 U/mg protein in groups A and B respectively from a normal value of 0.35 U/mg protein. During warm ischemia, %XO (XO/XD + XO) was increased from 18.4 to 84.9% for 2 h. In contrast, %XO was not changed for 24 h of cold ischemia. Tissue accumulation of HX was increased 2.8 times from a normal value of 1.06, 2 h after warm ischemia, but there was almost neither accumulation of HX nor the conversion of XD to XO in 24 h cold ischemia. It was observed that warm and cold ischemia caused similar injury after reperfusion in spite of the striking difference in the conversion of XD to XO and accumulation of HX. Thus, it is suggested that the XO system is not always necessary for ischemia-reperfusion injury.

Topics: alpha-Glucosidases; Animals; Cold Temperature; Dogs; Female; Hot Temperature; Hypoxanthine; Hypoxanthines; Intestinal Mucosa; Intestine, Small; Ischemia; Male; Osmolar Concentration; Peroxidase; Reperfusion; Xanthine Dehydrogenase; Xanthine Oxidase

To quantify simultaneously the pulmonary and gastrointestinal (GI) damage that occurs during uncomplicated surgery requiring cardiopulmonary bypass (CPB), and to examine the relationships between markers of such damage.. Prospective, open.. Adult ICU of a national referral hospital.. Twenty patients undergoing elective CPB surgery.. Pulmonary vascular injury was assessed using the protein accumulation index (PAI), a double isotope technique specific for high permeability pulmonary edema. The relationships of the PAI with percent neutrophils in bronchoalveolar lavage (BAL), serum, and BAL myeloperoxidase (MPO), and bypass time were examined. Splanchnic vascular injury was assessed using tonometry to measure intramucosal pH (pHi) and the ratio of absorbed lactulose to L-rhamnose (L/R ratio) to determine gut mucosal permeability. Positive correlations were observed between bypass time and PAI (r = 0.64, p < 0.01), percent neutrophils in the postoperative BAL and PAI (r = 0.51, p < 0.05), and postoperative serum MPO and PAI (r = 0.77, p < 0.001). The L/R ratio rose significantly following CPB from 0.04 +/- 0.01 in controls to 0.48 +/- 0.05 (p < 0.0001). The L/R ratio in patients who developed a low pHi was 0.59 +/- 0.06 compared with 0.32 +/- 0.07 in those whose pHi remained normal (p < 0.05). No significant correlation between bypass time and pHi (r = -0.3, p = 0.33), bypass time and L/R ratio (r = 0.27, p = 0.26), PAI and L/R ratio (r = 0.2, p = 0.42), PAI and pHi (r = -0.34, p = 0.16), postoperative serum MPO and L/R ratio (r = 0.03, p = 0.90), or postoperative serum MPO and pHi (r = -0.10, p = 0.67) could be demonstrated.. Pulmonary and GI injury are detectable following uncomplicated CPB. The absence of any relationship between the respective markers of dysfunction suggests that differing pathologic processes are responsible.

Topics: Aortic Valve; Bronchoalveolar Lavage Fluid; Capillary Permeability; Cardiopulmonary Bypass; Case-Control Studies; Coronary Artery Bypass; Endothelium, Vascular; Female; Gastrointestinal Diseases; Heart Valve Prosthesis; Humans; Hydrogen-Ion Concentration; Intensive Care Units; Intestinal Absorption; Intestines; Ischemia; Male; Middle Aged; Neutrophil Activation; Peroxidase; Prospective Studies; Respiratory Distress Syndrome

Neutrophils have been identified to play a major role in ischemia/reperfusion injury through several mechanisms. Neutrophil migration into reperfused gut may reduce bacterial translocation, but may also enhance the reperfusion injury. Ethanol ingestion impairs cutaneous chemotaxis, but its effects on neutrophil migration to postischemic small bowel are unknown. This study investigates the effects of ethanol on small bowel accumulation of neutrophils after ischemia/reperfusion. Ninety-five rats were divided into five groups; normal control, sham operation, ethanol-sham, ischemia, and ethanol-ischemia groups. Ethanol was given once acutely by gavage (3 g/kg, 20% solution) to the animals in the ethanol-sham and the ethanol-ischemia groups 4 hr before ischemic injury. Ischemia was produced for 1 hr by placing a vessel loop around the superior mesenteric vessels. After 1 hr, 87% of animals had gut ischemia and the loop was removed. Three hours later the small bowel was examined for necrosis and the reperfused viable small bowel was extirpated for measurement of neutrophil infiltration by colorimetric assay for myeloperoxidase (MPO), an enzyme restricted to neutrophils. Both ethanol and ischemia/reperfusion produced significant independent increase in the MPO activity. When ethanol was given prior to ischemia, the MPO activity was further increased by statistically significant margin. The present study demonstrated that ethanol enhanced the effects of gut ischemia/reperfusion injury on PMN accumulation into the intestinal wall. These observations suggest that ethanol may potentiate ischemic injury to the gut and lead to increased problems when gut blood flow is significantly impaired.

Topics: Alcohol Drinking; Animals; Cell Movement; Ethanol; Intestine, Small; Ischemia; Male; Neutrophils; Organ Size; Peroxidase; Rats; Rats, Sprague-Dawley

Topics: Animals; Blood Urea Nitrogen; Creatinine; Ischemia; Kidney; Male; Malondialdehyde; Neutrophils; Nitroprusside; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion

The human premature newborn is susceptible to necrotizing enterocolitis (NEC) in the first 1 to 3 weeks of life, a time when the gastrointestinal tract is structurally and functionally premature. Studies of NEC are hampered by the lack of a standard, reproducible model in newborn animals. The purpose of this study was to produce a model for intestinal ischemic injury in newborn rats. On Days 14, 18, 22, and 26 of life, newborn rats (10/day) were subjected to 1 hr of superior mesenteric artery occlusion with a microaneurysm clip. Platelet activating factor (PAF, 50 micrograms/kg) was injected into the lumen of the proximal small intestine after occlusion was initiated. Control animals (10/day) underwent sham laparotomy on Days 14, 18, 22, and 26. Animals were autopsied upon demise (7.6 +/- 0.7 hr) or at 24 hr. The intestine was inspected for gross ischemic changes and samples were taken for histology and myeloperoxidase (MPO, an index of neutrophil infiltration). Ischemic injury was graded in a blinded fashion, by a pathologist, using a scale from 0 to 4 (0, no injury; 4, full-thickness necrosis). All animals in the experimental groups had evidence of histologic injury (mean +/- SEM) on Days 14 (1.0 +/- 0.0), 18 (2.5 +/- 0.5), 22 (3.6 +/- 0.3), and 26 (3.1 +/- 0.5). The sham-operated control animals had no injury (P < 0.0001). MPO levels (U/g protein) on Days 18 (27.2 +/- 1.7 vs 13.9 +/- 2.3), 22 (40.9 +/- 5.4 vs 7.6 +/- 0.8), and 26 (29.3 +/- 4.4 vs 7.6 +/- 1.0) were significantly higher in experimental groups vs controls (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Animals, Newborn; Enterocolitis, Pseudomembranous; Female; Intestines; Ischemia; Mesenteric Artery, Superior; Mesenteric Vascular Occlusion; Peroxidase; Platelet Activating Factor; Pregnancy; Rats; Rats, Sprague-Dawley

Topics: Alanine Transaminase; Animals; Antibodies, Monoclonal; Ischemia; Liver; Macrophage-1 Antigen; Male; Mice; Mice, Inbred Strains; Necrosis; Organ Preservation; Peroxidase; Reperfusion Injury; Temperature; Tumor Necrosis Factor-alpha

The potential role of intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of reperfusion injury was investigated in male Fischer rats subjected to 45 min of hepatic ischemia and 24 h of reperfusion. ICAM-1 mRNA levels increased during ischemia in the ischemic liver lobes; however, during reperfusion mRNA levels increased in both the ischemic and nonischemic lobes. Immunohistochemical evaluation indicated ICAM-1 expression only on sinusoidal lining cells in controls; ischemia-reperfusion enhanced ICAM-1 expression in the sinusoids and induced some expression on hepatocytes. The monoclonal anti-ICAM-1 antibody 1A29, but not an immunoglobulin G control antibody, administered at 1 h and 8 h of reperfusion (2 mg/kg) significantly attenuated liver injury as indicated by 51% lower plasma alanine aminotransferase activities and 32-36% less hepatic necrosis at 24 h without affecting reactive oxygen formation by Kupffer cells and hepatic neutrophils. Although 1A29 reduced neutrophil extravasation in a glycogen peritonitis by 60%, the antibody had no significant effect on hepatic neutrophil infiltration during reperfusion. These data suggest that ICAM-1 plays a significant role during the neutrophil-dependent injury phase after hepatic ischemia and reperfusion and therefore blocking this adhesion molecule may have therapeutic potential against postischemic acute liver failure.

Topics: Animals; Gene Expression; Intercellular Adhesion Molecule-1; Ischemia; Liver; Male; Necrosis; Neutrophils; Peroxidase; Rats; Rats, Inbred F344; Reperfusion Injury; RNA, Messenger; Time Factors

This study investigated the effect of neutrophil depletion and neutrophil elastase inhibition on the severity of skeletal muscle reperfusion injury. In a rodent model, indices (experimental/normal limb) of gastrocnemius muscle viability (histochemical staining), oedema (wet:dry weight ratio) and myeloperoxidase content (neutrophil recruitment) were assessed in normal (no ischaemia), ischaemic (6-h unilateral hindlimb ischaemia), control (6-h ischaemia and 4-h reperfusion), neutrophil-depleted rats (given antineutrophil serum) and rats receiving the neutrophil elastase inhibitor Elafin. Neutrophil recruitment muscle infarction and oedema did not occur in normal limbs, or in those subjected to ischaemia without reperfusion. In contrast increased muscle myeloperoxidase levels (P < 0.001), muscle infarction (P < 0.01) and oedema (P < 0.001) all occurred in the reperfused limbs of control animals compared with those of normal and ischaemic rats. Antineutrophil serum and Elafin both reduced neutrophil recruitment during reperfusion (P < 0.001 and P < 0.01 respectively) and muscle viability was preserved. Reperfusion oedema still occurred however, suggesting that altered endothelial permeability is mediated by factors other than neutrophils.

Topics: Acute Disease; Animals; Disease Models, Animal; Edema; Hindlimb; Immune Sera; Ischemia; Leukocyte Elastase; Male; Muscle, Skeletal; Neutropenia; Neutrophils; Pancreatic Elastase; Peroxidase; Proteinase Inhibitory Proteins, Secretory; Proteins; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Serine Proteinase Inhibitors; Time Factors

Reperfusion of ischemic skeletal muscle is associated with neutrophil (PMN) adherence to damaged endothelium and PMN-mediated tissue destruction. Neutrophils may attach to endothelium through surface adhesive molecules, such as CD18. The purpose of this study was to determine whether monoclonal antibody blockade of CD18 would reduce skeletal muscle necrosis associated with ischemia and reperfusion. In rabbits, an entire hindlimb was rendered ischemic for 4 hr, followed by 48 hr of in vivo reperfusion. Animals were allocated to one of five treatment groups: ischemia/reperfusion without treatment (I/R controls), I/R plus treatment with the anti-CD18 antibody IB4 (end-ischemic 2 mg/kg dose), I/R plus treatment with an identical dose of isotype-matched control Ig, I/R plus anterior compartment fasciotomy, or I/R plus both IB4 and fasciotomy. After 48 hr of reperfusion anterior tibial muscle necrosis was assessed (by tetrazolium staining and computerized planimetry), wet:dry muscle weights (W:D) were determined, and muscle PMN sequestration was measured by myeloperoxidase (MPO) activity. IB4-treated animals exhibited markedly reduced muscle MPO activity, compared to untreated animals. Although all interventions reduced edema formation (W:D ratios), none did so significantly. IB4 treatment reduced muscle necrosis when used alone (to 28 +/- 7%, vs. 48% +/- 6% in untreated controls), however this was not statistically significant (P = 0.06).2+ Fasciotomy significantly reduced necrosis (to 22 +/- 2%, P < 0.05); however, the addition of IB4 to fasciotomy resulted in necrosis that was significantly lower than that after fasciotomy alone (12 +/- 4%, P < 0.05 vs fasciotomy group) and the least necrosis of any group.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; CD18 Antigens; Female; Ischemia; Muscles; Necrosis; Neutrophils; Organ Size; Peroxidase; Rabbits; Reperfusion Injury

The pathophysiology of ischemic acute renal failure is complex, and the role of leukocyte adhesion in this process is not well defined. A monoclonal antibody (mAb) against intracellular adhesion molecule 1 (anti-ICAM-1), administered at the time of bilateral renal ischemia in the rat, prevented both functional impairment and histologic changes of acute renal failure. Plasma creatinine measured (mg/dl) 24 hr after 30 min of ischemia was 0.61 +/- 0.05 in the anti-ICAM-1-treated animals compared with 2.4 +/- 0.14 (P < 0.0001) in the vehicle-treated ischemic group. Forty-eight hours after ischemia, creatinine values were 0.46 +/- 0.05 and 2.03 +/- 0.22 (P < 0.0001) in anti-ICAM-1 and vehicle-treated groups, respectively. A low dose of anti-ICAM-1 that was itself nonprotective, when given with partially protective doses of a mAb against lymphocyte function-associated antigen-1 (anti-LFA-1), acted synergistically to prevent renal failure. Anti-ICAM-1 mAb also protected the kidney when administered 0.5 or 2 hr but not 8 hr after restoration of blood flow and when the ischemic period was extended to 40 min. Ischemia-induced increases in tissue myeloperoxidase, a marker of neutrophil infiltration, were mitigated with anti-ICAM-1 treatment. Thus, anti-ICAM-1 mAb protected the kidney against ischemic renal failure, even when the antibody was administered after the ischemic period. These results suggest a critical role for leukocytes and adhesion molecules in the pathophysiology of ischemic injury and may have important therapeutic implications.

Topics: Acute Kidney Injury; Animals; Antibodies, Monoclonal; Cell Adhesion Molecules; Creatinine; Intercellular Adhesion Molecule-1; Ischemia; Kidney; Lymphocyte Function-Associated Antigen-1; Male; Peroxidase; Rats; Rats, Sprague-Dawley; Time Factors

Topics: Animals; Biomarkers; Glycoproteins; Intestinal Mucosa; Ischemia; Jejunum; Male; Muscle, Smooth; Neutrophils; Peroxidase; Rats; Rats, Sprague-Dawley; Reference Values; Reperfusion; Reperfusion Injury; Trypsin Inhibitors

In spite of the interest in free radicals as mediators of ischemic damage, most information on these species in biological systems is derived from indirect measurements. Our aim was to obtain more direct information concerning sources of free radical production during ischemia and reperfusion.. We have performed simultaneous measurement of radical generation, purine metabolites, reduced glutathione, neutrophil infiltration and morphological appearance in the cat small intestine in vivo during 60 minutes of ischemia followed by 60 minutes of reperfusion.. Radical formation increased abruptly on reperfusion and remained elevated in untreated animals. Inhibition by a monoclonal antibody (IB4) against the neutrophil and by allopurinol treatment was paralleled by improvement of biochemical and morphological parameters. The radicals detected during reperfusion could be divided into one component arising directly from the neutrophils, one due to the xanthine oxidase reaction, and one unknown source.. Neutrophils are a major source of radical production during reperfusion after ischemia. Radicals formed in the xanthine oxidase reaction seem to function as a primer for the neutrophils. The nonsignificant linear correlation between radical formation and morphological appearance suggests that factors other than free radicals are important for the development of intestinal damage after a period of ischemia.

Topics: Allopurinol; Animals; Antibodies, Monoclonal; Biopsy; Cats; Female; Hypoxanthine; Hypoxanthines; Intestinal Mucosa; Intestine, Small; Ischemia; Male; Neutrophils; Peroxidase; Reactive Oxygen Species; Reperfusion Injury; Xanthine Oxidase

The importance of sequential events which lead to skin necrosis has significant implications in trauma, vascular injury, and wound healing. In this series of experiments, we tested the hypothesis that xanthine oxidase (XO) activity was increased along an ischemic gradient of a skin flap and that the XO enzyme activity correlated with an increase in neutrophils. There were two animal groups in which the skin flaps were raised and assayed at 0, 1, or 6 hr. In the other group, they were created as bipedicle flaps for 7 days, before the distal attachment was divided and the tissue assayed. In the acutely raised flaps, some animals were treated with the XO inhibitor, allopurinol. Xanthine dehydrogenase (XD) and XO activity was measured with a fluorometric pterin assay and neutrophil concentration was measured using a myeloperoxidase marker. In this model, there was consistent skin necrosis in the distal end of the skin flap (48 +/- 8%). The data showed that both XD and XO activity in the distal ends was statistically significantly increased over the sham control or proximal ends of the skin flaps at 1 hr (P < 0.05). XO activity remained elevated in the distal ends at 6 hr. Allopurinol significantly reduced the neutrophil concentrations in the distal ends of the skin flaps when compared to untreated animals (P < 0.05). Moreover, allopurinol reduced skin necrosis to 12 +/- 1%. Preconditioning of the skin flap reduced the XO activity to sham control levels. The observations implicate XO activity as source of free radical injury in skin necrosis seen in random skin flaps.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Allopurinol; Animals; Dose-Response Relationship, Drug; Histological Techniques; Ischemia; Peroxidase; Rats; Rats, Sprague-Dawley; Skin; Surgical Flaps; Time Factors; Xanthine Dehydrogenase; Xanthine Oxidase

The objective of this study was to determine whether pulmonary endothelial expression of the adhesive glycoprotein P-selectin contributes to the lung injury and leukostasis observed after intestinal ischemia-reperfusion (I/R). The pulmonary capillary filtration coefficient and lung myeloperoxidase activity were determined in rat lungs isolated after 120 min of superior mesenteric artery occlusion and 90 min of reperfusion. Intestinal I/R resulted in a marked increase in the pulmonary capillary filtration coefficient compared with control and sham-operated rats. The increase in pulmonary microvascular permeability elicited by intestinal I/R was effectively prevented by pretreatment with a P-selectin monoclonal antibody (MAb; MAb PB1.3) but was unaffected by a control MAb. The intestinal I/R-induced increase in pulmonary microvascular permeability was accompanied by a dramatic sequestration of granulocytes in the lung compared with control and sham-operated rats; however, neither the P-selectin nor the control MAbs affected this event. These results indicate that P-selectin contributes to the pulmonary microvascular dysfunction observed after intestinal I/R. The inhibition of intestinal I/R-induced lung injury by immunoneutralization of P-selectin appears to be unrelated to the accompanying lung leukosequestration.

Topics: Animals; Antibodies, Monoclonal; Blood Pressure; Capillary Permeability; Cell Adhesion Molecules; Intestines; Ischemia; Leukocyte Count; Lung Diseases; Male; Organ Size; P-Selectin; Peroxidase; Platelet Membrane Glycoproteins; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Reperfusion Injury

To examine the role of neutrophils, their presence and the degree of infiltration, as important determinants of ischemia and reperfusion injury of the liver, male Sprague-Dawley rats were subjected to 60 and 90 min of total-liver ischemia. The presence of neutrophils, assessed by the measurement of liver tissue myeloperoxidase (MPO), and the degree of neutrophil liver infiltration, determined by the naphthol AS-D chloroacetate esterase technique, correlated well with animal survival and response to FK506 and cyclosporine administration. Lipid peroxidation, measured by the malondialdehyde (MDA) test in liver tissue, was another factor closely linked with liver function and survival. Pretreatment with FK506 (0.3 mg/kg) and CsA (5 mg/kg) was given at 4 hr and 1 hr before ischemia and at the time of reperfusion. Control ischemic animals showed increased neutrophil liver infiltration, high MPO and MDA liver levels, and diminished overall survival. FK506 and CsA-treated animals had better survival and diminished neutrophil liver infiltration, as well as MPO and MDA levels. The mechanism by which FK506 and CsA protected the animals from severe liver ischemic injury is unknown. Our data indicated that the presence and the degree of infiltration of neutrophils were important components of liver ischemia/reperfusion injury in the rat. So it is possible that one of the fundamental effects of the FK506 and CsA might be through the inhibition of the presence and infiltration of neutrophils in liver tissue.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Cyclosporine; Ischemia; L-Lactate Dehydrogenase; Liver; Male; Malondialdehyde; Microscopy, Electron; Neutrophils; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Survival Analysis; Tacrolimus

Ischemia in rat hind limbs followed by reperfusion results in local as well as remote organ (lung) injury characterized by increased vascular permeability (125I-labeled bovine serum albumin leakage) and hemorrhage (51Cr-labeled rat erythrocytes extravasation) in skeletal muscle and lung, together with an associated increased tissue content of myeloperoxidase, reflecting neutrophil accumulation. Within 60 minutes of reperfusion following ischemia, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6 plasma levels increased significantly, reaching maximum levels after 2 hours of reperfusion. Polyclonal antibodies to TNF-alpha and IL-1 provided significant protection against vascular injury in both muscle and lung. These results were confirmed by the use of soluble TNF-alpha receptor and IL-1 receptor antagonist. In rat lungs following ischemia and reperfusion, there was immunohistochemical evidence of E-selectin expression in the lung vasculature; this expression was blocked by treatment of animals with anti-TNF-alpha. These data indicate that both local (hind limb) and remote (lung) organ injury after ischemia/reperfusion requires participation of TNF-alpha and IL-1. The cytokines may, in part, be involved in the up-regulation of endothelial adhesion molecules.

Topics: Animals; Cell Adhesion Molecules; E-Selectin; Hemorrhage; Hindlimb; Immunotherapy; Interleukin-1; Interleukin-6; Ischemia; Lung Diseases; Male; Muscles; Peroxidase; Rats; Reperfusion Injury; Tumor Necrosis Factor-alpha; Up-Regulation

Antibodies to the neutrophil CD18 integrin have been shown to ameliorate the local effects of intestinal ischemia and reperfusion (I/R). In addition to local mucosal injury, intestinal I/R results in systemic hypotension and injury to the lungs with lung leukosequestration. This study tests the effect of a CD18 monoclonal antibody on the hypotension and lung injury after intestinal I/R. In anesthetized rabbits, the superior mesenteric artery was clamped for 60 min followed by 3 h of reperfusion. Animals were treated with saline, an anti-CD18 monoclonal antibody (R15.7 MAb), or nonspecific immunoglobulin G. Another non-ischemic group were sham controls. Neutrophil sequestration was assessed by measure of lung myeloperoxidase (MPO) and permeability by lung-to-blood concentration ratio of 125I-labeled bovine serum albumin and wet-to-dry weight ratio. Immediately after reperfusion, mean arterial pressure fell to 49 +/- 2.1 mmHg and remained at this level. The hypotension was unaffected by treatment with R15.7 MAb. Thirty minutes after reperfusion, the circulating white blood cell count fell to 2.91 +/- 0.53 x 10(3)/mm3 vs. sham 6.40 +/- 0.66 x 10(3)/mm3 (P < 0.05). Treatment with R15.7 MAb prevented this fall in white blood cell count (5.75 +/- 1.59 x 10(3)/mm3). At 3 h of reperfusion in saline-treated animals there was increased MPO, 74.8 +/- 4.9 U/g vs. 42.0 +/- 4.8 U/g in sham animals (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Blood Pressure; Capillary Permeability; CD18 Antigens; Endothelium, Vascular; Hypotension; Intestines; Ischemia; Lung; Neutrophils; Peroxidase; Rabbits; Reperfusion Injury; Serum Albumin, Radio-Iodinated

Free radical-initiated lipid peroxidation (LP) following intestinal ischemia/reperfusion (I/R) may disrupt mucosal integrity. It is unknown if inhibition of LP prevents this injury. We analyzed rat ileum, subjected to I/R, for evidence of LP inhibition and structural damage following treatment with the 21-aminosteroid, U74389F, a potent LP inhibitor. Four groups of Lewis rats were studied after superior mesenteric artery occlusion with ligation of collateral arcades: (i) no ischemia, (ii) 10 min ischemia, (iii) 10 min ischemia + 1 hr reperfusion, (iv) 10 min ischemia + 1 hr reperfusion + U74389F (6 mg/kg i.v. prior to clamp removal and reperfusion). Ileal mucosa was analyzed for: 9i0 superoxide dismutase (SOD; U/mg protein), a key antioxidant enzyme, (ii) myeloperoxidase (MPO; U/mg protein), an index of PMN stimulation, (iii) malondialdehyde (MDA; pmole/mg), an end product of LP, and (iv) routine histology. MDA rose from 2.09 +/- 0.44 (mean +/- SE) in Group 1 to 15.10 +/- 2.22 in Group 3 following I/R (P < 0.01). In Group 2 and Group 4, MDA remained unchanged at 3.25 +/- 1.38 and 1.73 +/- 0.15, respectively. MPO, likewise, rose during I/R from 0.59 +/- 0.17 in Group 1 to 1.10 +/- 0.13 in Group 3 (P = 0.08) and 1.49 +/- 0.24 in Group 4 (P < 0.05). SOD did not vary significantly in the four groups studied. Despite PMN stimulation indicated by increased MPO with reperfusion, no PMN infiltration was seen histologically. U74389F normalized MDA, indicating effective inhibition of LP; however, similar epithelial sloughing and edema and hemorrhage in the lamina propria were seen in treated and untreated rats. These data implicate MDA-independent or possibly LP-independent pathways in intestinal morphologic damage occurring with I/R.

Topics: Animals; Constriction; Free Radicals; Ileum; Intestinal Mucosa; Intestines; Ischemia; Lipid Peroxidation; Male; Malondialdehyde; Mesenteric Artery, Superior; Neutrophils; Peroxidase; Pregnatrienes; Rats; Rats, Inbred Lew; Reperfusion Injury

Topics: Alanine Transaminase; Analysis of Variance; Animals; Aspartate Aminotransferases; Ischemia; Jugular Veins; L-Lactate Dehydrogenase; Liver; Liver Function Tests; Male; Malondialdehyde; Mesenteric Veins; Peroxidase; Rats; Rats, Wistar; Reperfusion; Splanchnic Circulation; Time Factors

Activated neutrophils appear to be directly involved in potentiating central nervous system ischemic injury. After initial endothelial adherence, neutrophils can potentiate ischemia by causing capillary plugging and infiltrating ischemic tissue to release toxic metabolites. We used an endothelial component adherence assay to characterize neutrophil adhesion following reversible central nervous system ischemia. Rabbit spinal cord ischemia was produced by 40 min of reversible arterial occlusion. Neutrophils were isolated using density gradient centrifugation and adherence to laminin or fibronectin was determined using a myeloperoxidase assay. The baseline (N = 26) percentage of adherent neutrophils was 3.8 +/- 0.3/5.6 +/- 0.6 (laminin/fibronectin), sham-operated controls (2 h post) (N = 6) 3.3 +/- 0.5/3.9 +/- 0.4, 30 min post ischemia (N = 6) 2.5 +/- 1.1/4.1 +/-1.9, 2 h (N = 6) 7.6 +/- 1.4/9.9 +/- 2.6 (p < .05 to baseline and sham), 18 h (N = 8) 4.9 +/- 1.5/7.1 +/- 3.1, and 42 h (N = 6) 3.4 +/- 0.4/6.6 +/- 2.1. Concurrent serum fibrinogen values were baseline 142 +/- 12, sham (18 h) 141 +/- 14, 2 h 166 +/- 21, 18 h 512 +/- 43 (p < .01), and 42 h 580 +/- 86 (p < .01). These results suggest that neutrophil adherence is increased after acute central nervous system ischemia. There appears to be a limited period of increased adherence that occurs earlier than other acute phase reactants.

Topics: Acute Disease; Animals; Biomarkers; Cell Adhesion; Fibronectins; Ischemia; Laminin; Male; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Peroxidase; Rabbits; Spinal Cord

Ischemia followed by reperfusion in rat limb results in evidence of vascular injury in the limb as well as in the lung as measured by leakage of [125I]albumin and extravasation of [51Cr] red blood cells. Vascular injury in lung and limb was proportional to the time of limb reperfusion and was associated with accumulation of myeloperoxidase, as well as evidence of complement consumption. In this model, the rank order of protective interventions was: neutrophil depletion > catalase + superoxide dismutase = allopurinol > dimethylthiourea = dimethylsulfoxide > deferoxamine = complement depletion. These data suggest that toxic oxygen products of neutrophils are related to the development of vascular injury. There was a reasonable correlation between protective effects of interventions and reduced tissue content of myeloperoxidase. Systemic treatment with the L-arginine antagonists, NG-monomethyl-L-arginine or nitro-L-arginine methyl ester, was also protective against vascular injury, suggesting that metabolic products of L-arginine participate in events leading to injury.

Topics: Animals; Arginine; Capillary Permeability; Complement Hemolytic Activity Assay; Hemorrhage; Hindlimb; Ischemia; Lung; Male; Muscles; Neutrophils; Peroxidase; Rats; Rats, Inbred Strains; Reperfusion Injury; Time Factors

Recent evidence has suggested that the immaturity of the neonatal intestine may play a key role in the development of ischemic injury. However, relatively little data exist on the susceptibility of the neonatal intestine to ischemic injury at various ages especially in the fed versus fasted states. In this study, the levels of xanthine oxidase ([XO] an enzyme which is a known, major source of free radicals in postischemic tissue) and myeloperoxidase ([MPO] an index of tissue neutrophil infiltration) were measured in 1-, 5-, 10-, 15-, and 20-day-old Sprague-Dawley rats. Rats were divided into fed (n = 8/day) and fasted (n = 8/day) groups 4 hours prior to sacrifice. The entire small intestine was removed and divided into five segments: the duodenum, proximal jejunum, distal jejunum, proximal ileum, and distal ileum. The specimens were homogenized and assayed for XO and MPO levels. A significant increase in XO was observed in the fasted animals compared to the fed animals on all days. Peak levels in XO were observed in both groups from day 5 to 10. MPO levels were significantly higher in the fasted versus fed animals on day 1. MPO levels decreased as the animals aged. These data demonstrate dramatic differences in the levels of inflammatory enzymes of the newborn rat in the fed versus fasted states. Also, marked variations with age are seen in both XO and MPO. Whether the XO and MPO levels present at the time of ischemic insult affect severity of injury remains to be seen.

Topics: Animals; Animals, Newborn; Enterocolitis, Pseudomembranous; Fasting; Intestine, Small; Ischemia; Peroxidase; Rats; Rats, Sprague-Dawley; Xanthine Oxidase

Neutrophils (PMNs) are believed to play a key role in the pathogenesis of postinjury adult respiratory distress syndrome. We have previously shown that gut ischemia/reperfusion (I/R) produces lung injury by a process that requires PMNs. More recently, we have shown that xanthine oxidase (XO) plays a role. The purpose of this study was to characterize the mechanistic sequencing of XO activity versus the PMN in this model of gut I/R-induced lung injury. Normal and XO-inactivated (tungsten enriched, molybdenum depleted diet) rats underwent 45 min of superior mesenteric artery occlusion. After 6 hr reperfusion, blood was sampled and gut and lungs harvested. Myeloperoxidase (MPO) was used to quantitate PMN presence in the gut and lungs, while circulating PMN priming was measured as the difference in superoxide production with and without the activating stimulus, fMLP. 125I-labeled albumin leak was used as a marker for lung endothelial permeability. We observed that the gut I/R increased gut MPO levels, primed circulating PMNs, increased lung MPO levels, and provoked distant lung leak. XO inactivation abolished gut MPO activity, attenuated circulating PMN priming, and blocked lung leak. In conclusion, XO plays a proximal role in the pathogenesis of remote organ injury following splanchnic hypoperfusion.

Topics: Animals; Intestines; Ischemia; Lung; Lung Diseases; Male; Neutrophils; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxides; Xanthine Oxidase

The involvement of polymorphonuclear leukocytes (PMN) in reperfusion-mediated vascular injury was studied in a model of ischemia and reperfusion in rabbit hindlimb. Ischemia was induced by 4-h occlusion of the left iliac artery followed by 4-h reperfusion. Plasma creatine kinase (CK) and lactate dehydrogenase (LDH) activities, hindlimb vascular resistance (HVR), and myeloperoxidase (MPO) activity in the postischemic extensor digitorum longus (EDL) muscle were measured to evaluate the extent of vascular and skeletal muscle injury. In addition, the ischemia/reperfusion-induced injury of the hindlimb vasculature was evaluated by electron microscopy. Ischemia and reperfusion (n = 10) was associated with an increase in CK (6,380 +/- 1,346 U/L, p < 0.05) and LDH (552 +/- 76 U/L, p < 0.05) activities which were significantly greater than those observed in sham-operated control animals (CK 1,651 +/- 207 U/L, LDH 246 +/- 14 U/L; n = 6). HVR in sham-operated animals decreased by 20 +/- 3%, but increased in the ischaemic group by 56 +/- 16% (p < 0.05). MPO activity of EDL muscle increased from 7.3 +/- 3.9 U per muscle (sham) to 28.0 +/- 5.9 U per muscle (p < 0.05) after ischemia and reperfusion. Morphologic analysis did not show any alteration in the microvascular bed of the hindlimb. Moreover, 1 mg/kg/h intravenous (i.v.) cloricromene, an antithrombotic drug that inhibits superoxide anion production as well as PMN adhesion to endothelium, reduced the increase in plasma CK and LDH and the increase in MPO and HVR observed in animals subjected to hindlimb ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blood Pressure; Chromonar; Creatine Kinase; Heart Rate; Hindlimb; Ibuprofen; Ischemia; L-Lactate Dehydrogenase; Leukocytes; Male; Microscopy, Electron; Microscopy, Electron, Scanning; Peroxidase; Rabbits; Reperfusion Injury; Vascular Resistance

Phospholipase A2 (PLA2) has recently been implicated as a key enzyme of local inflammation after gut ischemia-reperfusion (I/R). The hypothesis of this study is that PLA2 inhibition decouples remote organ injury from gut I/R.. Sprague-Dawley rats were pretreated with a PLA2 inhibitor, quinacrine (10 mg/kg, intravenously), before the induction of gut ischemia (45 minutes of superior mesenteric artery occlusion) followed by 6 hours of reperfusion. 125I-labeled albumin leak was employed as a marker of pulmonary endothelial permeability and myeloperoxidase as a monitor of neutrophil (PMN) traffic in the gut and lung. To further characterize the impact of PLA2 inhibition, PMNs were harvested at 6 hours of reperfusion and superoxide production was measured in the presence or absence of an activating stimulus, N-formyl-methionyl-leucyl-phenylalanine.. Gut I/R increased gut PLA2 activity, elicited gut PMN influx, and produced lung leak; these events were prevented by PLA2 blockade. Gut I/R also markedly enhanced PMN superoxide production with N-formyl-methionyl-leucyl-phenylalanine, and this priming was ablated by PLA2 inhibition.. These data suggest that PLA2 activation is a proximal step in the pathogenesis of distant organ injury after splanchnic hypoperfusion, a process that appears to involve PMN priming in the gut bed.

Topics: Animals; Capillary Permeability; Intestines; Ischemia; Lung; Male; Neutrophils; Peroxidase; Phospholipases A; Phospholipases A2; Rats; Rats, Inbred Strains; Reperfusion Injury; Serum Albumin; Superoxides

Although previous studies have reported that neutrophils play an important role in mediating the microvascular injury observed after reperfusion of ischemic intestine, the contribution of these phagocytic cells to the mucosal dysfunction remains unclear. Three series of experiments consisting of an untreated group, a short-term monoclonal antibody (MAb) IB4 treatment group (MAb IB4 given on the day of the experiment), and a long-term MAb IB4 treatment group (3-day pretreatment with MAb IB4) were performed using autoperfused segments of cat ileum exposed to 3 hours of ischemia followed by 1 hour of reperfusion. Mucosal myeloperoxidase activity, an index of mucosal granulocyte levels, increased from 12 to 25 U/g wet wt in the untreated group. In the short-term MAb IB4 experiments, baseline values were very similar to those of the untreated group but no increase in myeloperoxidase activity was observed after ischemia/reperfusion. Long-term pretreatment with MAb IB4 reduced baseline values of myeloperoxidase activity to approximately 1 U/g wet wt; the values remained at this level throughout the experiment. The permeability of the mucosal barrier was quantitated by measuring blood-to-lumen clearance to 51Cr-ethyl-enediaminetetraacetic acid (EDTA). The water absorptive capacity of the intestine was also measured. In the untreated group, mucosal permeability to 51Cr-EDTA increased sixfold and water absorption was abolished after reperfusion. Both short-term and long-term administration of MAb IB4 prevented the net fluid loss into the lumen, but only long-term administration of MAb IB4 blunted the increased mucosal permeability induced by ischemia/reperfusion. These data suggest that interstitial granulocytes contribute significantly to the mucosal dysfunction associated with reperfusion of the ischemic intestine.

Topics: Animals; Antibodies, Monoclonal; Cats; Cell Membrane Permeability; Chromium Radioisotopes; Edetic Acid; Granulocytes; Intestinal Absorption; Intestinal Mucosa; Ischemia; Peroxidase; Reperfusion Injury; Water

Injury to nonpulmonary organ systems often initiates systemic processes that cause recruitment of neutrophils to the lung. We found that rats subjected to intestinal ischemia-reperfusion (I/R) had increased transvascular leak of 125I-labeled albumin into lungs and decreased lung ATP levels (P less than 0.05). In addition, rats subjected to intestinal I/R had increased plasma xanthine oxidase (XO) activity, plasma leukotactic activity for neutrophils, and lung neutrophil retention (assessed by morphometry and myeloperoxidase activity) compared with sham-treated rats (P less than 0.05). By comparison, after intestinal I/R, rats fed an allopurinol- or tungsten-enriched diet had decreased plasma and intestinal XO activities, decreased plasma leukotacic and lung myeloperoxidase (MPO) activities, decreased lung leak, and increased lung ATP levels compared with rats fed control diets (P less than 0.05). Further studies suggested a more specific role for circulating rather than tissue XO in mediating lung neutrophil accumulation but not lung leak. Plasma XO, plasma leukotactic, and lung MPO activities, but not lung leak, increased in rats administered purified XO intravenously. In addition, plasma XO, plasma leukotactic, and lung MPO activities, but not lung leak, decreased in rats administered antisera against XO and then subjected to intestinal I/R. We conclude that circulating XO increases acutely and may contribute to pulmonary retention of neutrophils after an ischemic intestinal insult.

Topics: Allopurinol; Animals; Cell Movement; Endotoxins; Injections, Intravenous; Intestines; Ischemia; Lung; Male; Neutrophils; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tungsten; Xanthine Oxidase

Reperfusion of ischemic skeletal muscle is associated with white blood cell (WBC) sequestration and hydroperoxy-conjugated diene (HCF) formation, a marker of free radical-mediated phospholipid peroxidation. The purpose of this study was to define the kinetics of phospholipid fatty acyl peroxidation, deacylation, and remodeling in postischemic skeletal muscle during prolonged reperfusion in vivo, and to determine whether reperfusion with WBC and plasma-depleted blood would attenuate postischemic phospholipid peroxidation and myocyte necrosis. The isolated, paired, canine gracilis muscle model was used. After 5 h of ischemia, muscles underwent unaltered reperfusion or initial reperfusion with WBC-deficient blood cells resuspended in hydroxyethyl starch, followed by return to normal circulation (modified reperfusion). The concentration of native fatty acids and HCDs of linoleic acid extracted from muscle phospholipids was quantified by gas chromatography and positively identified by mass spectrometry. Ischemia and reperfusion resulted in phospholipid deacylation and a selective increase in phospholipid stearic acid content, but had no effect on total phospholipid phosphorus. Modified reperfusion decreased 1) early HCD formation (54%) and 2) postischemic skeletal muscle necrosis (49%). These data suggest that reperfusion results in phospholipid deacylation and remodeling, and that the initial oxidant stress during reperfusion may be a significant determinant of ultimate muscle necrosis.

Topics: Acylation; Animals; Dogs; Fatty Acids, Unsaturated; In Vitro Techniques; Ischemia; Lipid Peroxides; Muscles; Necrosis; Peroxidase; Phospholipids; Phosphorus; Reperfusion

The influence of oxygen free radical scavengers and anti-inflammatory drugs on postischemic lipid peroxidation and myeloperoxidase activity was shown. The best results were obtained from vitamin E and the antiinflammatory treatment with CP and SUL, whereas an iron elimination only showed slight effects on myeloperoxidase activity above all. In experiments without therapy a linear increase of lipid peroxides dependent on reperfusion durance was found, whereas myeloperoxidase already showed a remarkable increase during ischemia and early reperfusion. This difference can be interpreted by scavenging mechanisms, which are overcharged after an appointed durance of reperfusion.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antioxidants; Free Radical Scavengers; Intestinal Mucosa; Intestines; Iron; Ischemia; Lipid Peroxides; Male; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Vitamin E

Neutrophils play an important role in ischemia-reperfusion (I/R)-induced vascular injury in the small intestine. Monoclonal antibodies against the leukocyte adhesion glycoprotein CD11/CD18 afford protection against I/R-induced microvascular injury. It has been suggested that the response to I/R differs between the various layers of the bowel wall, with relatively few granulocytes accumulating in the mucosa compared with the serosa or mesentery. The objectives of this study were to determine whether I/R-induced neutrophil accumulation is 1) homogenous in the different layers of intestine (mucosa, submucosa, muscle, and mesentery) and 2) dependent on the expression and/or activation of the leukocyte adhesion glycoprotein CD11/CD18. Neutrophil infiltration was monitored by measuring myeloperoxidase activity in mucosa, submucosa, muscle, and mesentery of cat small intestine subjected to 3 h ischemia (blood flow reduced to 20% of control) and reperfusion. I/R elicited a comparable degree of polymorphonuclear (PMN) infiltration in mucosa, submucosa, and mesentery, with the muscularis exhibiting a greater response. Pretreatment with the CD18-specific monoclonal antibody (IB4) significantly attenuated the I/R-induced PMN accumulation in all layers of the bowel wall and mesentery, indicating that the granulocyte accumulation elicited by I/R is dependent on the expression and/or activation of the leukocyte adhesion molecule CD11/CD18.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Cats; CD18 Antigens; Granulocytes; Intestines; Ischemia; Peroxidase; Receptors, Leukocyte-Adhesion; Reperfusion; Tissue Distribution

Ischemia and reperfusion of the lower torso lead to leukotriene- and neutrophil (PMN)-dependent lung injury characterized by lung PMN sequestration, increased permeability, and noncardiogenic edema. It is thought that PMNs require adhesion to endothelium to alter barrier function. This study tests the role of CD 18, the PMN adherence receptor, in mediating lung permeability after lower torso ischemia and reperfusion. Anesthetized rabbits (n = 9) underwent 3 hours of bilateral hind limb ischemia. Ten minutes after the release of the tourniquets, plasma leukotriene B4 levels increased to 395 +/- 85 pg/ml, higher than 129 +/- 35 pg/ml in controls (n = 9, p less than 0.01). At this time there was a reduction in circulating white blood cells (x 10(3)), 3.56 +/- 0.49/mm3 relative to 6.07 +/- 0.61/mm3 in controls (p less than 0.01). PMNs were sequestered in the hind limbs, indicated by increased myeloperoxidase activity of 1.06 +/- 0.19 units/g compared with 0.56 +/- 0.09 units/g in controls (p less than 0.05). Four hours after tourniquet release, PMNs were sequestered in the lungs, 52 +/- 4 PMNs per 10 high-power fields, a value higher than 31.5 +/- 3 PMNs per 10 high-power fields in controls; bronchoalveolar lavage fluid protein content increased to 554 +/- 90 micrograms/ml relative to 277 +/- 46 micrograms/ml in controls; and there was lung edema, measured by increased wet weight-to-dry weight ratios of 5.19 +/- 0.10, higher than 4.29 +/- 0.21 in controls (all p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Capillary Permeability; CD18 Antigens; Hindlimb; Ischemia; Male; Neutrophils; Peroxidase; Pulmonary Circulation; Rabbits; Receptors, Leukocyte-Adhesion; Reperfusion; Tourniquets

Oxygen-derived free radicals and leukocytes have been implicated in the pathogenesis of ischemia-reperfusion injury. This study aimed at determining, by using biochemical and histochemical techniques, whether an accumulation of neutrophils occurs in the ischemic reperfused rat liver and whether superoxide free radicals play a role in mediating this neutrophil accumulation. Hepatic ischemia was induced by occluding blood supply to the left and median lobes, and reperfusion was reinstituted by releasing the occlusion. Myeloperoxidase activity of the liver was measured with a tetramethylbenzidine-H2O2 assay after removal of glutathione (by dialysis) and in the presence of 3-aminotriazole (catalase inhibitor). A modification of Graham and Karnovsky's method was used to stain neutrophils in liver frozen sections, and the number of neutrophils was counted. Results showed that ischemia-reperfusion of the liver produced a 4.4-fold increase in myeloperoxidase activity (from 0.073 +/- 0.009 to 0.320 +/- 0.017 units/mg liver, means +/- SE), which was proportional to the number of neutrophils (3.1-fold increase from 18 +/- 7 to 57 +/- 4 cells/mm2) in the liver tissue. Pretreatment with long-acting superoxide dismutase significantly attenuated the elevated myeloperoxidase activity and the number of neutrophils. These results indicate that reperfusion after a period of ischemia induces an accumulation of neutrophils in the liver, and superoxide anion free radicals are important mediators in the mechanism of this neutrophil accumulation.

Topics: Animals; Free Radicals; Ischemia; Leukocyte Count; Liver; Liver Circulation; Male; Neutrophils; Peroxidase; Rats; Rats, Inbred Strains; Reperfusion; Staining and Labeling; Superoxide Dismutase; Superoxides

Pentobarbital-anesthetized rats were subjected to occlusion of both the celiac and superior mesenteric arteries for 90 min followed by reperfusion for 2 h. All seven rats given only the vehicle died within 2 h of reperfusion, whereas rats treated with LY-255283 (3 or 10 mg/kg iv), a leukotriene B4 (LTB4) receptor antagonist given 10 min before reperfusion, exhibited significantly higher survival rates of 57% (4 out of 7) and 75% (6 out of 8), respectively, 2 h after reperfusion. Rats given 1 mg/kg of LY-255283 showed no significant improvement in survival. Splanchnic artery occlusion (SAO)-shock rats treated with LY-255283 (3 or 10 mg/kg) exhibited significantly attenuated accumulation of plasma free amino-nitrogenous compounds and of a myocardial depressant factor. Treatment with LY-255283 (10 mg/kg) markedly (P less than 0.01) ameliorated the deficits of endothelium-dependent relaxation of isolated superior mesenteric artery (SMA) rings in untreated SAO-shock rats. LY-255283 at 10 mg/kg significantly attenuated the increased myeloperoxidase activity in the intestinal tissue of SAO-shock rats. Moreover, LY-189444, a closely related compound having no LTB4 antagonist activity, did not protect rats in SAO shock, whereas a lipoxygenase inhibitor confirmed protection in SAO shock. These results suggest that LTB4 plays a pivotal role in endothelial dysfunction occurring in SAO-shock rats by chemoattraction and activation of neutrophils on the surface of vascular endothelial cells. Moreover, LY-255283 but not LY-189444 inhibited the adherence of rat neutrophils to isolated SMA endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arteries; Blood Pressure; Cathepsin D; Constriction; Endothelium, Vascular; Ileum; Ischemia; Leukotriene B4; Male; Neutrophils; Peroxidase; Rats; Rats, Inbred Strains; Reperfusion; Splanchnic Circulation; Tetrazoles

Haemorrhagic mucosal lesions are produced during intestinal ischaemia and after reperfusion probably mediated by oxygen radicals. Oxygen radicals react with cell membrane lipids and induce cell damage by peroxidation and induce accumulation of polymorphonuclear leucocytes in the tissue. The aim of the study was to elucidate the involvement of polymorphonuclear leucocytes in post-ischaemic intestinal damage. Intestinal ischaemia was induced in cats by partial occlusion of the superior mesenteric artery. Samples from the small intestine were excised before and at the end of the two hour hypotensive period as well as 10 minutes and 60 minutes after reperfusion. Conjugated dienes, myeloperoxidase, and the purine metabolites were determined in the samples. The tissue was also examined histologically. Seven cats were treated before reperfusion with a monoclonal antibody (IB4) which inhibits leucocyte adherence to endothelial cells and its subsequent activation. After reperfusion myeloperoxidase activity increased and the ischaemic mucosal lesions worsened significantly. IB4 treatment prevented an increase in post-hypotensive myeloperoxidase activity and attenuated the normally observed severe mucosal lesions. We conclude that the severe post-ischaemic lesions are induced by polymorphonuclear leucocytes. Such mucosal injury may be appreciably reduced by blocking leucocyte adherence with IB4.

Topics: Animals; Cats; Cell Adhesion; Female; Intestine, Small; Ischemia; Lipid Peroxidation; Male; Neutrophils; Peroxidase; Purines; Reperfusion Injury

Ischemia induced oxygen free radical damage was formerly attributed only to xanthine oxidase in intestine, liver, kidney and heart. A reevaluation indicated neutrophils as one of the major sources of postischemic oxidative tissue damage, chiefly in the intestine. Our data, obtained from the same occlusion time period for intestine, liver and kidney, showed a certain oxidative damage in intestine and kidney already during ischemia, expressed by an increase of thiobarbituric acid reactive substances (TBARS), whereas the liver sustained damage of this kind only during reperfusion. Oxidative stress was expressed by a comparison of the increase of TBARS, though this test is not a measure of a specific product of lipid peroxidation, but rather comprises several breakdown products of free radical damage. Myeloperoxidase as measure of neutrophil stimulation increased in the intestine and liver. The kidney sustained damage without an increase of myeloperoxidase activity, but showed a similar pattern of increase of TBARS as in the intestine. Our data suggest a major role of neutrophils in intestinal ischemia induced damage, where neutrophils can effect initiation and propagation. In the liver neutrophils may play a minor role concerning propagation, but they may act as an important initiating mechanism. Hepatic tissue shows a high ischemic tolerance, which is demonstrated by a missing increase of TBARS in spite of a certain increase of myeloperoxidase activity during ischemia. This can be interpreted by the high capacity of antioxidative mechanisms of liver tissue and the ability of a higher oxygen extraction ratio under nearly ischemic conditions. In the kidney there appears a smaller contribution of neutrophils. The similar pattern of increase of TBARS in kidney and intestine demonstrates a comparable low ischemic tolerance of these two tissues, whereas different initiating and propagating systems may occur.

Topics: Animals; Aorta; Arterial Occlusive Diseases; Intestinal Mucosa; Intestines; Ischemia; Kidney; Lipid Peroxidation; Liver; Male; Peroxidase; Rats; Rats, Inbred Strains; Reperfusion; Thiobarbiturates

The aim of this study was to determine whether phalloidin (1 microM) or antamanide (1 microM), cyclic peptides that stabilize dense peripheral band and stress fiber F-actin in endothelium, would attenuate the increase in microvascular permeability induced by 4 h of ischemia and 30 min of reperfusion (I/R) in the isolated canine gracilis muscle. Changes in microvascular permeability (1 - sigma) were assessed by determining the solvent drag reflection coefficient for total plasma proteins (sigma) in muscles subjected to 4.5 h of continuous perfusion (nonischemic controls), I/R alone, I/R + phalloidin, or I/R + antamanide. Muscle neutrophil content was assessed by determination of myeloperoxidase (MPO) activity in tissue samples obtained at the end of the experiments. Fluorescent detection of nitrobenzoxadiazole-phallicidin in endothelial cell monolayers confirmed that phalloidin enters these cells. I/R was associated with marked increases in microvascular permeability and muscle neutrophil content (1 - sigma = 0.45 +/- 0.07; MPO = 8.9 +/- 0.5 units/g) relative to control (4.5 h continuous perfusion) preparations (1 - sigma = 0.12 +/- 0.03; MPO = 0.5 +/- 0.8 unit/g). These I/R-induced changes were largely prevented by administration of phalloidin (1 - sigma = 0.19 +/- 0.02; MPO = 0.8 +/- 0.4 U/g) or antamanide (1 - sigma = 0.07 +/- 0.11; MPO = 0.9 +/- 0.3 unit/g) at reperfusion. Similar results were obtained when phalloidin was administered before ischemia (1 - sigma = 0.24 +/- 0.04; MPO = 1.2 +/- 1.0 units/g). Although antamanide decreased superoxide production (by approximately 60%) and adherence to plastic (by approximately 75%) by activated neutrophils in vitro, phalloidin failed to alter these aspects of granulocyte function.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Actins; Animals; Blood Proteins; Capillary Permeability; Cytochalasin D; Dogs; Endothelium, Vascular; Female; Ischemia; Leukocyte Adherence Inhibition Test; Male; Muscles; Neutrophils; Peptides, Cyclic; Peroxidase; Phalloidine; Reperfusion; Superoxides

Isolated segments of cat small intestine were subjected to 3 hr of ischemia followed by 1 hr reperfusion (I/R). Mucosal biopsies were obtained for measurement of myeloperoxidase (MPO) activity, an index of tissue neutrophil count, and leukotriene B4 (LTB4) production. Animals were pretreated with either cyclosporin A (CsA) or FK506, which are potent immunosuppressants. Both agents significantly attenuated the neutrophil infiltration induced by I/R. FK506, but not CsA, reduced the elevated mucosal LTB4 production normally observed following reperfusion. The results of this study suggest that FK506 and CsA may be important agents in modulating neutrophil infiltration in acute inflammatory conditions.

Topics: Animals; Cats; Cyclosporine; In Vitro Techniques; Intestine, Small; Ischemia; Leukocyte Count; Leukotriene B4; Neutrophils; Peroxidase; Reperfusion Injury; Tacrolimus

The clinical significance and applicability of interventions aimed at reducing reperfusion injury in postischemic skeletal muscle remain unproven, since long-term muscle salvage has not been demonstrated by most treatment protocols that attenuate early reperfusion injury. We have shown that reperfusion of ischemic skeletal muscle results in an early and prolonged sequestration of white blood cells and activation of the alternative complement cascade. The purpose of this study was to determine if 40 minutes of reperfusion with blood depleted of white blood cells and complement proteins, followed by 2 days of normal perfusion, would reduce muscle necrosis after 5 hours of ischemia. The isolated paired canine gracilis muscle model was used. The treatment muscle was initially reperfused with arterial blood that had been spun, washed, passed through a leukocyte removal filter, and resuspended in hydroxyethyl starch (greater than 99.9% removal of white blood cells and the complement proteins factor B and C4). The contralateral control muscle was subjected to unaltered reperfusion. Blood flow (ml/min/100 gm) was measured by timed collection of gracilis venous blood. Myeloperoxidase activity (absorbance at 655 nm/min/mg tissue protein) in muscle biopsies was used to monitor white blood cell sequestration. After 48 hours of reperfusion in vivo, necrosis was quantified by nitroblue tetrazolium staining. Initial reperfusion with white blood cell and complement depleted blood significantly reduced muscle necrosis (53% +/- 3% vs 29% +/- 8%, p less than 0.0025, paired t test). Early blood flow was improved, (p = 0.0025, repeated measure-ANOVA), but subsequent white blood cell sequestration was not altered (p = 0.33, repeated measure-ANOVA). This suggests that a significant amount of white blood cell mediated injury occurs during the first 40 minutes of reperfusion. Preventing early complement activation and white blood cell mediated reperfusion injury is an intervention that is feasible during surgery and may result in clinically significant salvage of postischemic skeletal muscle.

Topics: Animals; Biopsy; Complement System Proteins; Dogs; Female; In Vitro Techniques; Ischemia; Leukocytes; Male; Muscles; Necrosis; Perfusion; Peroxidase; Reperfusion; Reperfusion Injury; Time Factors

Superoxide radicals produced during acute intestinal ischemia are biochemically related with the presence of hydrogen peroxyde. In this study we have investigated the distribution of peroxidase-catalase activity, histochemically determined, in the ischemic ileal wall. In the rat, complete arterial and venous occlusion produced a progressive increase in extra-vascular peroxidase-catalase activity with a maximum corresponding to the ileal wall. Probably the tissue peroxidase-catalase activity is related to massive degranulation of polymorphonucleates.

Topics: Animals; Catalase; Ileum; Intestinal Mucosa; Ischemia; Neutrophils; Peroxidase; Rats; Rats, Inbred Strains

The objective of this study was to determine whether hydrogen peroxide, iron, and/or hydroxyl radicals play a role in ischemia/reperfusion (I/R)-induced granulocyte infiltration in the feline small intestine and whether a chemoattractant is formed when superoxide or hydrogen peroxide reacts with feline extracellular fluid. In vivo determinations of granulocyte infiltration consisted of measurements of tissue myeloperoxidase activity in either the intestinal mucosa (I/R studies) or dermis (chemotaxis studies), whereas in vitro measurements of granulocyte migration were obtained using a Boyden chamber. Treatment with either catalase or the iron chelator deferoxamine significantly attenuated granulocyte infiltration into the mucosa induced by reperfusion of the ischemic intestine. Two hydroxyl radical scavengers, dimethyl sulfoxide (DMSO) and dimethylthiourea (DMTU), were also evaluated for their ability to modulate I/R-induced granulocyte infiltration. DMTU significantly attenuated the I/R-induced granulocyte accumulation, whereas DMSO had no effect. In other experiments, we were unable to stimulate granulocyte migration with feline plasma exposed to superoxide-generating systems using both in vitro and in vivo models of leukocyte chemotaxis. However, hydrogen peroxide in the presence of either ferrous iron or hemoglobin did significantly increase the chemotactic activity of cat plasma. The results obtained from our studies suggest that either hydrogen peroxide or radical species derived from the interaction of superoxide and hydrogen peroxide with iron elicit I/R-induced granulocyte infiltration in the intestine.

Topics: Animals; Catalase; Cats; Chemotaxis, Leukocyte; Deferoxamine; Dimethyl Sulfoxide; Granulocytes; Hydrogen Peroxide; Intestinal Mucosa; Intestine, Small; Ischemia; Leukotriene B4; Muscle, Smooth; Peroxidase; Reperfusion; Superoxide Dismutase; Superoxides; Thiourea; Zymosan

In the present study we have evaluated the activity of myeloperoxidase (MPO) in ischemic reperfused skeletal muscle of rats, as an index of polymorphonuclear leukocyte (PMNL) accumulation and its time course. A tourniquet model for temporary ischemia was used, in which one hindleg was made ischemic for 1.5, 3 or 5 hours. Muscle biopsies were taken after 12 hours reperfusion from both the injured and the uninjured legs. Controls received anesthesia only. No increase in the MPO levels was observed after 1.5 hours of ischemia followed by 12 hours of reperfusion. With the same reperfusion time and 3 or 5 hours of ischemia, there was a five-fold increase in MPO activity. Prolonging the ischemia from 3 to 5 hours did not cause any further significant MPO increase. With 3 hours of ischemia, biopsies were also taken after 0, 1, 5, 24 and 74 hours reperfusion. The results showed a time dependent increase in MPO activity. A significant increase was first seen after 5 hours reperfusion with the peak at 24 hours. After 74 hours of reperfusion the MPO activity had almost returned to control levels. The uninjured leg had MPO levels similar to those in the control. However, there was a small reduction at 5 and 12 hours of reperfusion. During the ischemic period before reperfusion, the ischemic leg showed a significant decrease in MPO activity. Severe ischemia in skeletal muscle results in a time dependent accumulation of PMNLs during reperfusion, as measured by the changes in MPO activity in the tissue.

Topics: Animals; Ischemia; Male; Muscles; Neutrophils; Peroxidase; Rats; Rats, Inbred Strains

Tissue injury at reperfusion has been reported after partial ischemia. However, previous attempts to demonstrate a component of injury caused by reperfusion after total ischemia have failed. This study was performed to evaluate the hypothesis that in such situations the extent of the tissue injury caused by ischemia itself prevented detection of a reperfusion component. Rats were subjected to near-total intestinal ischemia by means of a hydrostatic pressure clamp that produced preferential venous occlusion (strangulation) for periods from 1 to 90 minutes. Tissue injury was evaluated microscopically by a blinded examiner. Ischemic periods of 20 minutes or less did not induce detectable tissue injury. Longer durations of ischemia caused villous injury: the longer the period of ischemia, the more extensive the tissue injury. However, there was no exacerbation of injury seen after reperfusion, regardless of the duration of ischemia. In a separate series of rats, total arterial occlusion was employed without concomitant venous congestion. Such isolation arterial occlusion of 40 to 60 minutes' duration was followed by a statistically significant exacerbation of tissue injury at reperfusion. Thus total intestinal ischemia may be followed by reperfusion injury if there is no concomitant congestion and if ischemic injury is not too extensive.

Topics: Animals; Blood Pressure; Equipment and Supplies; Intestines; Ischemia; Male; Peroxidase; Rats; Rats, Inbred Strains; Regional Blood Flow; Reperfusion Injury

Recent studies implicate a role for granulocytes in the genesis of the microvascular and parenchymal cell dysfunction, which occurs upon reperfusion of ischemic tissues. Although the molecular mechanisms underlying this neutrophil-mediated injury are not completely understood, it is clear that an essential first step in granulocyte migration from the vascular lumen to the interstitial space is adherence to vascular endothelium. The purpose of this study was to determine whether prevention of neutrophil adherence with monoclonal antibody IB4 directed against the neutrophil CD11/CD18 glycoprotein adherence complex or neutrophil depletion with a specific polyclonal antineutrophil serum would attenuate the microvascular dysfunction seen in postischemic skeletal muscle. Changes in vascular permeability were assessed by measurement of the solvent drag reflection coefficient for total plasma proteins (sigma) in isolated canine gracilis muscle subjected to ischemia/reperfusion, ischemia/reperfusion plus antineutrophil serum, or ischemia/reperfusion plus IB4. Estimates of sigma averaged 0.83 +/- 0.04 in nonischemic, control gracilis muscles, while ischemia/reperfusion was associated with a marked increase in vascular permeability (decrease in sigma to 0.54 +/- 0.04) and vascular resistance (increased by 135 +/- 41% over the control value). Prevention of neutrophil adherence or neutrophil depletion prevented this increase in vascular permeability (sigma = 0.80 +/- 0.03 and 1.01 +/- 0.06, respectively) and resistance (decrease of 16.51 +/- 8.0% and increase of 2.4 +/- 4.6% over control values, respectively). The results of this study suggest that neutrophils play a critical role in the genesis of microvascular dysfunction in postischemic skeletal muscle. Furthermore, neutrophil adherence to vascular endothelium appears to be a prerequisite for the production of this injury.

Topics: Animals; Blood Vessels; Capillary Permeability; Cell Adhesion; Dogs; Female; Granulocytes; Ischemia; Male; Microcirculation; Muscles; Neutrophils; Peroxidase; Reperfusion; Vascular Resistance

Cytokines are recognized as critical early mediators of organ injury. We attempted to determine whether or not severe hepatic ischemia/reperfusion injury results in tumor necrosis factor-alpha (TNF-alpha) release with subsequent local and systemic tissue injury. After 90 min of lobar hepatic ischemia, TNF was measurable during the reperfusion period in the plasma of all 14 experimental animals, with levels peaking between 9 and 352 pg/ml. Endotoxin was undetectable in the plasma of these animals. Pulmonary injury, as evidenced by a neutrophilic infiltrate, edema and intra-alveolar hemorrhage developed after hepatic reperfusion. The neutrophilic infiltrate was quantitated using a myeloperoxidase (MPO) assay; this demonstrated a significant increase in MPO after only 1 h of reperfusion. Anti-TNF antiserum pretreatment significantly reduced the pulmonary MPO after hepatic reperfusion. After a 12-h reperfusion period, there was histologic evidence of intra-alveolar hemorrhage and pulmonary edema. Morphometric assessment showed that pretreatment with anti-TNF antiserum was able to completely inhibit the development of pulmonary edema. Liver injury was quantitated by measuring serum glutamic pyruvic transaminase which showed peaks at 3 and 24 h. Anti-TNF antiserum pretreatment was able to significantly reduce both of these peak elevations. These data show that hepatic ischemia/reperfusion results in TNF production, and that this TNF is intimately associated with pulmonary and hepatic injury.

Topics: Alanine Transaminase; Animals; Hemodynamics; Ischemia; Liver; Liver Diseases; Lung; Neutrophils; Peroxidase; Rats; Reperfusion Injury; Time Factors; Tumor Necrosis Factor-alpha

Polymorphonuclear leukocytes are known to accumulate in tissues subjected to ischemia and reperfusion. Studies on endothelial cell monolayers suggest that limited release of elastase plays an important role, via basement membrane degradation, in the leukocyte diapedesis and extravasation elicited by proinflammatory mediators. Thus the objective of this study was to define the role of elastase in the leukocyte infiltration associated with reperfusion of the ischemic bowel. In one series of experiments the cat small intestine was subjected to 3 h of ischemia (blood flow reduced to 20% of base line) and 1 h of reperfusion. Neutrophil accumulation was quantified by measurement of tissue myeloperoxidase activity in mucosal biopsies obtained during base-line, ischemic, and reperfusion periods. Pretreatment with either of the elastase inhibitors Eglin C and L658,758 significantly attenuated the neutrophil infiltration induced by reperfusion but not by ischemia per se. In another series of experiments, leukocyte adherence and extravasation were monitored in cat mesenteric venules subjected to 1 h of ischemia and reperfusion. Pretreatment with L658,758 significantly attenuated the increased rates of leukocyte adherence and extravasation induced by reperfusion, with a proportionately greater reduction in leukocyte extravasation rate. These results indicate that elastase release is an important factor in reperfusion-induced neutrophil infiltration.

Topics: Animals; Cats; Cell Adhesion; Cephalosporins; Intestinal Mucosa; Intestine, Small; Ischemia; Leukocytes; Neutrophils; Pancreatic Elastase; Peroxidase; Proteins; Pyrrolidines; Reperfusion; Serine Proteinase Inhibitors; Serpins; Splanchnic Circulation; Venules

After skeletal muscle ischemia, tissue damage is augmented during reperfusion. White blood cells (WBCs) and complement proteins may participate in the reperfusion injury. The purpose of this study was to define the kinetics of classical and alternative pathway complement activation and WBC sequestration by postischemic skeletal muscle during the first 48 h of reperfusion in vivo. The isolated canine gracilis muscle model was used. Systemic levels of the complement proteins factor B (alternative pathway) and C4 (classical pathway) were quantitated by hemolytic assay. WBC sequestration was measured by gracilis arterial-venous WBC differences and tissue myeloperoxidase activity. Reperfusion was associated with an 18% decrease in systemic factor B levels but no consistent change in systemic C4 levels. WBCs were sequestered during the first 4 h of reperfusion, and tissue myeloperoxidase activity was elevated 97-fold after 48 h of reperfusion. These results suggest that skeletal muscle ischemia-reperfusion stimulates 1) activation of the alternative but not the classical complement pathway and 2) an immediate and prolonged sequestration of WBCs.

Topics: Animals; Complement Activation; Complement C4; Complement Factor B; Dogs; Ischemia; Leukocytes; Muscles; Peroxidase; Reperfusion

Topics: Animals; Antioxidants; Capillary Permeability; Cats; Cell Adhesion; Intestinal Mucosa; Intestine, Small; Ischemia; Kinetics; Leukocytes; Muscle, Smooth; Peroxidase; Reperfusion; Xanthine Oxidase

Myeloperoxidase (MPO) is an enzyme found in granulocytes of neutrophils, but not in mammalian tissues. Previous studies have directly correlated MPO activity with neutrophil accumulation in tissues. This study presents a method for determining MPO activity in liver. Neutrophil accumulation in rat liver was provoked by creating partial ischemia followed by reperfusion. Liver homogenates prepared by a standard procedure showed no MPO activity. The homogenate was applied to Sephadex G100 and DEAE Sepharose CL6B columns which separated MPO activity from inhibitory activity. The inhibitor was identified as catalase based upon its elution from the columns and removal with 3-amino- 1,2,4-triazole (AT), a catalase inhibitor. Based upon these findings, it was determined that full MPO activity can be assayed in unfractionated liver homogenates by first inactivating catalase with AT.

Topics: Amitrole; Animals; Catalase; Chromatography, Gel; Ischemia; Liver; Male; Methods; Neutrophils; Peroxidase; Rats; Rats, Inbred Strains

In this study the role of free radicals, lipid peroxidation, and neutrophil infiltration as mediators of ischemia and reperfusion-induced intestinal mucosal damage were investigated. We used a rat experimental model in which a ligated loop of the distal ileum was subjected to ischemia and reperfusion and the ensuing mucosal damage was assessed by means of lysosomal enzyme release and intestinal permeability measurements. We also determined the mucosal content of malondialdehyde, a lipid peroxidation product, and the mucosal activity of myeloperoxidase, a neutrophil granulocyte marker. Ischemia and revascularization alone caused increased mucosal permeability to sodium fluorescein, increased N-acetyl-beta-glucosaminidase release from the mucosa into the lumen, increased malondialdehyde content in the mucosa, and increased myeloperoxidase activity in the mucosa. Intravenous injection of enzymatic antioxidant, superoxide dismutase, together with xanthine oxidase inhibitor, allopurinol, prevented the malondialdehyde accumulation and caused attenuation of all the other effects of ischemia. Intravenous pretreatment of hydrocortisone sodium succinate (Solu-Cortef), a steroid and also a nonenzymatic antioxidant, prevented not only malondialdehyde accumulation but also neutrophil infiltration and mucosal damage. These data support a concept that neutrophil infiltration is an important element in ischemic mucosal damage. In addition, the blocking of this phenomenon may have clinical significance in attempts to modulate the potential damaging effects of the increased neutrophil infiltration associated with small-intestinal ischemia.

Topics: Acetylglucosaminidase; Allopurinol; Animals; Disease Models, Animal; Fluoresceins; Free Radicals; Hydrocortisone; Intestinal Absorption; Intestinal Mucosa; Ischemia; Lipid Peroxidation; Malondialdehyde; Neutrophils; Oxygen; Peroxidase; Rats; Reperfusion Injury; Superoxide Dismutase

The influence of quinacrine, a phospholipase A2 inhibitor, and enzymatic scavengers of active oxygen metabolites (superoxide dismutase and catalase) on ischemic small intestinal mucosal damage has been investigated. In the absence of an inhibitor, ischemia and reperfusion caused increased mucosal permeability to sodium fluorescein, increased N-acetyl-glucosaminidase release from the mucosa into the lumen, increased malondialdehyde content, and increased myeloperoxidase and phospholipase A2 (PLA2) activities in the mucosa. All these effects of ischemia were efficiently inhibited by the PLA2 inhibitor quinacrine. On the other hand, superoxide dismutase together with catalase, even if it totally prevented the increased formation of malondialdehyde, was only able to reduce 50 percent of the increases of the other parameters. These findings indicate that, in addition to free radicals, other factors are involved in the pathogenesis of small intestinal mucosal injury after ischemia and reperfusion. We suggest that one such factor is the activation of PLA2 and the generation of various PLA2-dependent compounds such as arachidonic acid metabolites, lysophosphatidyl choline, and platelet-activating factor.

Topics: Acetylglucosaminidase; Animals; Catalase; Free Radicals; Ileum; Intestinal Mucosa; Ischemia; Malondialdehyde; Oxygen; Permeability; Peroxidase; Phospholipases; Phospholipases A; Phospholipases A2; Quinacrine; Rats; Reperfusion; Superoxide Dismutase

We have examined how a Ginkgo biloba extract influences the damaging effects of ischaemia in the small-intestinal mucosa. We used a rat experimental model in which a ligated loop of the distal ileum was subjected to ischaemia and revascularization, and the ensuing mucosal damage assessed by lysosomal enzyme release and intestinal permeability measurements. We also determined the mucosal content of malondialdehyde, a lipid peroxidation product, and the mucosal activity of myeloperoxidase, a neutrophil granulocyte marker. Ischaemia and revascularization alone caused increased mucosal permeability to sodium fluorescein, increased N-acetyl-beta-glucosaminidase release from the mucosa into the lumen, increased malondialdehyde content in the mucosa, and increased myeloperoxidase activity in the mucosa. Intravenous injection of G. biloba extract caused a dose-dependent attenuation of all these effects of ischaemia. It is suggested, therefore, that G. biloba extract may protect the intestinal mucosa against ischaemic damage by reducing neutrophil infiltration and lipid peroxidation.

Topics: Acetylglucosaminidase; Animals; Intestinal Mucosa; Intestine, Small; Ischemia; Malondialdehyde; Models, Biological; Permeability; Peroxidase; Plants, Medicinal; Rats; Trees

The influence of quinacrine on malondialdehyde (MDA) as an index of lipid peroxidation, activities of phospholipase A2 (PLA2), and myeloperoxidase (MPO)--a neutrophil granulocyte maker in plasma--was examined in rats following ischemia and reperfusion. In the absence of quinacrine, ischemia and reperfusion caused increased MDA content and increased activities of PLA2 and myeloperoxidase in the plasma. All these effects were efficiently inhibited by the PLA2 inhibitor quinacrine. The finding indicates that the occurrence of an increased level of MDA following intestinal ischemia may be used for diagnostic purposes and points to the possibility that high plasma MDA might indicate a need for PLA2 inhibitor treatment.

Topics: Animals; Intestine, Small; Ischemia; Malonates; Malondialdehyde; Peroxidase; Phospholipases A; Phospholipases A2; Quinacrine; Rats; Rats, Inbred Strains; Regional Blood Flow

The effects of antioxidant therapy with probucol were evaluated in rats subjected to 1 h renal ischemia and to 24 h reperfusion. Probucol exerted significant antioxidant effects in renal cortical tubules in vitro when exposed to a catalase-resistant oxidant. At 24 h probucol treatment (IP) improved single nephron glomerular filtration rate (SNGFR) (28.1 +/- 3.3 nl/min) in comparison to untreated ischemic (I) rats (15.2 +/- 3.0), primarily as a result of improving SNGFR in a population of low SNGFR, low flow and/or obstructed nephrons. However, absolute proximal reabsorption remained abnormally low in IP rats at 24 h (5.9 +/- 0.8 nl/min), and cell necrosis was greater than in I rats. Kidney GFR remained low in IP rats due to extensive tubular backleak of inulin measured by microinjection studies. Evaluations after 2 h of reperfusion revealed a higher SNGFR in IP (36 +/- 3.1 nl/min) than I rats (20.8 +/- 2.7 nl/min). Absolute proximal reabsorption was essentially normal (11.6 +/- 1.3 nl/min) in IP rats, which was higher than IP rats at 24 h and the concurrent I rats. Administration of the lipophilic antioxidant, probucol, increased SNGFR and proximal tubular reabsorption within 2 h after ischemic renal failure. Although SNGFR remained higher than I rats at 24 h, absolute reabsorption fell below normal levels and tubular necrosis was more extensive in IP rats. Early improvement in nephron filtration with antioxidants may increase load dependent metabolic demand upon tubules and increase the extent of damage and transport dysfunction.

Topics: Acute Kidney Injury; Animals; Chemical Phenomena; Chemistry; Glomerular Filtration Rate; Inulin; Ischemia; Kidney; Kidney Glomerulus; Kidney Tubules; Male; Malondialdehyde; Peroxidase; Phenols; Probucol; Rats

The influence of various inflammatory inhibitors on the damaging effects of ischaemia in the small intestinal mucosa has been investigated. A rat experimental model was used, in which a ligated loop of the distal ileum was subjected to ischaemia and revascularisation and the ensuing mucosal damage assessed by lysosomal enzyme release and intestinal permeability measurements. The mucosal content of malondialdehyde - a lipid peroxidation product - and its activity of myeloperoxidase - a neutrophil granulocyte marker was also determined. In the absence of inhibitor, ischaemia and revascularisation caused increased mucosal permeability to sodium fluorescein, increased N-acetyl-beta-glucosaminidase release from the mucosa into the lumen, increased malondialdehyde content in the mucosa and increased myeloperoxidase activity in the mucosa. All these effects were inhibited by the phospholipase A2 inhibitors, quinacrine and nordihydroguaiaretic acid (NDGA), while the lipoxygenase inhibitor, BW755C, had no influence and the cyclooxygenase inhibitor, indomethacin, potentiated the increases in mucosal permeability and N-acetyl-glucosaminidase release. BN 52021, a specific platelet activating factor antagonist, did not influence the myeloperoxidase activity, but it decreased the formation of malondialdehyde and the increases in mucosal permeability and N-acetyl-beta-glucosaminidase release, although not to the same extent as quinacrine and NDGA. These findings indicate that phospholipase A2 inhibition prevents mucosal damage associated with small intestinal ischaemia and suggest that at least part of the ischaemic damage is mediated by products of phospholipase A2 activity that are not arachidonic acid metabolites.

Topics: Acetylglucosaminidase; Animals; Intestinal Absorption; Intestinal Mucosa; Intestine, Small; Ischemia; Malondialdehyde; Peroxidase; Phospholipases; Phospholipases A; Phospholipases A2; Rats

Topics: Animals; Bromides; Chlorides; Free Radicals; Humans; Hydrogen Peroxide; Hydroxides; Hydroxyl Radical; Inflammation; Iodides; Ischemia; Microcirculation; Neutrophils; Oxygen; Peroxidase; Superoxide Dismutase; Xanthine Oxidase

A growing body of experimental data indicates that reactive oxygen metabolites such as superoxide, hydrogen peroxide, and hydroxyl radical may mediate the mucosal injury produced by reperfusion of ischemic intestine. Xanthine oxidase has been proposed as the primary source of these reduced O2 species because pretreatment with xanthine oxidase inhibitors such as allopurinol or pterin aldehyde prevent postischemic mucosal injury. Another potential source of oxygen radicals is the inflammatory neutrophil. To ascertain whether neutrophils could play a role in the pathogenesis of ischemia-reperfusion injury in the small bowel we examined the effect of ischemia and reperfusion on neutrophil infiltration and tissue levels of reduced glutathione, superoxide dismutase, and catalase. Our studies demonstrate that reperfusion of ischemic intestines results in a dramatic increase (1,800%) in neutrophil infiltration and a concurrent loss of reduced glutathione and superoxide dismutase of 60 and 30%, respectively. Catalase activity was unaffected by ischemia-reperfusion. Pretreatment with allopurinol or administration of superoxide dismutase prevented the influx of neutrophils and retarded the drop in reduced glutathione levels. These results suggest a relationship among xanthine oxidase-generated oxy radicals, neutrophil extravasation, and mucosal damage. We propose that ischemia and reperfusion results in xanthine oxidase-generated, superoxide-dependent accumulation of inflammatory neutrophils in the mucosa where neutrophil-derived reactive oxygen metabolites mediate and/or exacerbate intestinal injury.

Topics: Allopurinol; Animals; Cats; Free Radicals; Glutathione; Intestinal Mucosa; Intestines; Ischemia; Neutrophils; Oxygen; Peroxidase; Superoxide Dismutase; Xanthine Oxidase