Mechanism Of Ischemia-reperfusion Injury Research Articles

Establishment of a Reperfusion Model in Rabbits with Acute Myocardial Infarction

Clinically effective cardioprotection under acute myocardial infarction (AMI) can only be achieved by establishing the mechanisms of reperfusion-induced cardiac cell death. In spite of the numerous earlier studies on the prevention of ischemia-reperfusion injury of myocardium, the problem of cardiac cell death upon reperfusion is not yet resolved. Even though animal models provide an immense opportunity in the understanding of the mechanisms of ischemia-reperfusion injury, clinically relevant animal models through which translation of this knowledge into clinic are lacking. In this work, we have established a reperfusion model in rabbits with induced AMI by obstructing and releasing the left anterior ventricular branch of left circumflex coronary artery, which is clinically more relevant. This was achieved by cutting the two left ribs of the rabbit followed by obstructing and releasing the artery unlike the traditional approach, which involves incision through sternum and blocking the anterior descending coronary artery. This animal model of ischemia-reperfusion more closely mimics the physiological condition and also the trauma the animal suffers is much smaller with higher survival rate and thus is a potentially better model for studying the pathology related to ischemia-reperfusion injury.

Urine neutrophil gelatinase-associated lipocalin is a marker of graft recovery after kidney transplantation

Delayed graft function (DGF), especially long-lasting DGF, complicates kidney transplant outcome. Neutrophil gelatinase-associated lipocalin (NGAL) is an acute kidney injury marker; therefore, we tested whether urine NGAL could predict DGF, prolonged DGF (lasting over 14 days), or the quality of kidney function in transplant recipients without DGF (non-DGF). We collected urine samples from 176 recipients transplanted with deceased donor kidneys before and various days after transplantation. A total of 70 transplantations had DGF, of which 26 were prolonged. Patients who developed DGF had a significantly slower decrease in urinary NGAL compared with those without DGF, such that day 1 NGAL predicted DGF (area under the curve (AUC) 0.75) and predicted DGF in 15 of 112 cases with day 1 urine output over 1 l (AUC 0.70) and in 19 of 86 cases with a day 1 decrease in creatinine over 50 μmol/l (AUC 0.74). The urinary NGAL level on day 1 predicted prolonged DGF (AUC 0.75), which had significantly worse 1-year graft survival (73%), compared with shorter DGF (100%). In non-DGF, high day 3 NGAL (greater than the mean) was associated with significantly worse kidney function at 3 weeks compared with low NGAL, but not at 3 months and 1 year. NGAL did not correlate with long-term function in DGF. Hence, day 1 urinary NGAL predicted DGF even when it was not clinically expected early on, and importantly, it predicted prolonged DGF that led to worse graft survival.

Mechanisms and Consequences of Cardiac Ischemia-Reperfusion Injury: Insights and Evidence to Improve Outcomes

More than 1.3 million percutaneous interventions (PCIs) and 400,000 coronary artery bypass graft procedures for cardiac revascularization are performed in the United States annually. Although restoring blood flow is the best method for reducing the extent of heart muscle damage after myocardial infarction (MI), reperfusion of ischemic myocardium itself can be injurious. Reperfusion-related complications after PCI for MI and coronary artery bypass grafting include myocardial stunning, the no-reflow phenomenon, reperfusion arrhythmias, and lethal cardiac ischemia-reperfusion (IR) injury, which results in irreversible injury and death of cardiomyocytes. Identified mechanisms that contribute to IR injury include adenosine triphosphate depletion, oxidative stress, inflammation, and intracellular calcium ion overload. At present, although multiple modalities targeting these and other IR injury mechanisms have been tested, effective, proved approaches are lacking, and the need to develop cardioprotective agents to reduce infarct size in patients with MI and prevent perioperative MIs in patients who undergo heart surgery remains unmet. Several pharmacotherapies that have shown promise in preclinical studies, such as MC-1, cariporide, and pexelizumab, have failed to live up to that promise in phase III clinical trials, highlighting the need for clinically relevant, disease-specific models for translating experimental data to efficacy in patients. The development of new pharmacologic agents also will depend on a better understanding of the underlying cellular and subcellular mechanisms of IR injury. This interactive program highlights novel investigational approaches to protection against IR injury. Online Access: http://cmeaccess.com/cme/ajc_cir_program/. This CME Multimedia Activity is also available through the Web site of The American Journal of Cardiology ( http://www.ajconline.org). Click on the Multimedia tab at the top of the Homepage, and select CME Multimedia Activities to access this program through the Journal Homepage.

Baicalin attenuates focal cerebral ischemic reperfusion injury through inhibition of nuclear factor κB p65 activation

Baicalin is a flavonoid compound purified from plant Scutellaria baicalensis Georgi. We aimed to evaluate the neuroprotective effects of baicalin against cerebral ischemic reperfusion injury. Male Wistar rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion for 24 h. Baicalin at doses of 50, 100 and 200 mg/kg was intravenously injected after ischemia onset. Twenty-four hours after reperfusion, the neurological deficit was scored and infarct volume was measured. Hematoxylin and eosin (HE) staining was performed to analyze the histopathological changes of cortex and hippocampus neurons. We examined the levels of NF-κB p65 in ischemic cortexes by Western blot analysis and RT-PCR assay. The results showed that the neurological deficit scores were significantly decreased from 2.0 ± 0.7 to 1.2 ± 0.4 and the volume of infarction was reduced by 25% after baicalin injection. Histopathological examination showed that the increase of neurons with pycnotic shape and condensed nuclear in cortex and hippocampus were not observed in baicalin treated animals. Further examination showed that NF-κB p65 in cortex was increased after ischemia reperfusion injury, indicating the molecular mechanism of ischemia reperfusion injury. The level of NF-κB p65 was decreased by 73% after baicalin treatment. These results suggest that baicalin might be useful as a potential neuroprotective agent in stroke therapy. The neuroprotective effects of baicalin may relate to inhibition of NF-κB p65.

Improving outcome in patients undergoing liver surgery

Liver surgery is associated with many factors, which may affect outcome. Preoperative assessment of patient's general condition, resectability, and liver reserve are paramount for success. The Child-Pugh score and other scoring systems only partially enables to assess the risk associated with liver surgery. The presence of portal hypertension per se is a major risk factor for hepatectomy. Intraoperatively, any attempts should be made to minimize blood loss. Low central venous pressure and inflow occlusion best prevent bleeding. Ischemic preconditioning and intermittent clamping are routinely applied in many centers to protect against long periods of ischemia, although the mechanisms of protection remain unclear. In this review we describe recent advances in activated pathways associated with protection against ischemia. Postoperatively, the best factor impacting on outcome probably resides in experienced medical care particularly in the intensive care setting. Currently, no drug or gene therapy approaches has reached the clinic. The future relies on new insight into mechanisms of ischemia-reperfusion injury.

Mild episodes of tourniquet-induced forearm ischaemia-reperfusion injury results in leukocyte activation and changes in inflammatory and coagulation markers

BackgroundMonocytes and neutrophils are examples of phagocytic leukocytes, with neutrophils being considered as the 'chief' phagocytic leukocyte. Both monocytes and neutrophils have been implicated to play a key role in the development of ischaemia-reperfusion injury, where they are intrinsically involved in leukocyte-endothelial cell interactions. In this pilot study we hypothesised that mild episodes of tourniquet induced forearm ischaemia-reperfusion injury results in leukocyte activation and changes in inflammatory and coagulation markers.MethodsTen healthy human volunteers were recruited after informed consent. None had any history of cardiovascular disease with each subject volunteer participating in the study for a 24 hour period. Six venous blood samples were collected from each subject volunteer at baseline, 10 minutes ischaemia, 5, 15, 30, 60 minutes and 24 hours reperfusion, by means of a cannula from the ante-cubital fossa. Monocyte and neutrophil leukocyte sub-populations were isolated by density gradient centrifugation techniques. Leukocyte trapping was investigated by measuring the concentration of leukocytes in venous blood leaving the arm. The cell surface expression of CD62L (L-selectin), CD11b and the intracellular production of hydrogen peroxide (H2O2) were measured via flow cytometry. C-reactive protein (CRP) was measured using a clinical chemistry analyser. Plasma concentrations of D-dimer and von Willebrand factor (vWF) were measured using enzyme-linked fluorescent assays (ELFA).ResultsDuring ischaemia-reperfusion injury, there was a decrease in CD62L and an increase in CD11b cell surface expression for both monocytes and neutrophils, with changes in the measured parameters reaching statistical significance (p =< 0.05). A significant decrease in peripheral blood leukocyte concentration was observed during this process, which was measured to assess the degree of leukocyte trapping in the micro-circulation (p =< 0.001). There was an increase in the intracellular production of H2O2 production by leukocyte sub-populations, which was measured as a marker of leukocyte activation. Intracellular production of H2O2 in monocytes during ischaemia-reperfusion injury reached statistical significance (p = 0.014), although similar trends were observed with neutrophils these did not reach statistical significance. CRP was measured to assess the inflammatory response following mild episodes of ischaemia-reperfusion injury and resulted in a significant increase in the CRP concentration (p =< 0.001). There were also increased plasma concentrations of D-dimer and a trend towards elevated vWF levels, which were measured as markers of coagulation activation and endothelial damage respectively. Although significant changes in D-dimer concentrations were observed during ischaemia-reperfusion injury (p = 0.007), measurement of the vWF did not reach statistical significance.ConclusionTourniquet induced forearm ischaemia-reperfusion injury results in increased adhesiveness, trapping and activation of leukocytes. We report that, even following a mild ischaemic insult, this leukocyte response is immediately followed by evidence of increased inflammatory response, coagulation activity and endothelial damage. These results may have important implications and this pilot study may lead to a series of trials that shed light on the mechanisms of ischaemia-reperfusion injury, including potential points of therapeutic intervention for pathophysiological conditions.

The effect of anaesthetics on the myocardium - new insights into myocardial protection

A variety of laboratory and clinical studies clearly indicate that exposure to anaesthetic agents can lead to a pronounced protection of the myocardium against ischaemia-reperfusion injury. Several changes in the protein structure of the myocardium that may mediate this cardioprotection have been identified. Ischaemia-reperfusion of the heart occurs in a variety of clinical situations including transplantations, coronary artery bypass grafting or vascular surgery. Ischaemia may also occur during a stressful anaesthetic induction. Early restoration of arterial blood flow and measures to improve the ischaemic tolerance of the tissue are the main therapeutic options (i.e. cardioplegia and betablockers). There exists increasing evidence that anaesthetic agents interact with the mechanisms of ischaemia-reperfusion injury and protect the myocardium by a 'preconditioning' and a 'postconditioning' mechanism. Hence, the anaesthesiologist may substantially influence the critical situation of ischaemia-reperfusion during surgery by choosing the appropriate anaesthetic agent. This review summarizes the current understanding of the mechanisms of anaesthetic-induced myocardial protection. In this context, three time windows of anaesthetic-induced cardioprotection are discussed: administration (1) during ischaemia, (2) after ischaemia-during reperfusion (postconditioning) and (3) before ischaemia (preconditioning). Possible clinical implications of these interventions will be reviewed.

Increased susceptibility to oxidant injury in hepatocytes from rats with intra-abdominal hypertension.

Intra-abdominal hypertension leads to visceral organ hypoperfusion, and subsequent decompression may cause ischemia-reperfusion, releasing toxic metabolites. This study focuses on the effect of intra-abdominal hypertension on hepatic antioxidant store and the susceptibility of hepatocytes to oxidant injury. Sprague-Dawley rats (150-180 g) were acclimatized to an environment for 3 days and then divided into two groups according to challenge based on intra-abdominal pressure (0 and 30 cm H2O for control and experimental groups, respectively). After a 90-minute challenge, the rats underwent immediate laparotomy for decompression; after a further 30 minutes, one fragment of liver from the lingual lobe (>0.1 g) was excised to measure glutathione (GSH) in vivo before portal vein perfusion. After hepatocyte isolation (viability rate > 85%), the cell density was set at 1 x 10/mL for each well. The samples were cultured in an incubator for 12 hours, after which varying concentrations of t-butyl hydroperoxide (TBHP) (0.0, 0.5, 1.0, and 2.0 mmol/L) were added into the wells. After another 5-hour incubation, the total store of intracellular GSH in vitro (GSHVT) and the hepatocyte survival rates were measured for different groups of TBHP challenge using GSH assay and MTT kits. The control and experimental groups consisted of 10 and 8 rats, respectively, that successfully completed the entire experimental procedure. Compared with the control group, the in vivo GSH store was significantly reduced after the intra-abdominal pressure challenge (mean +/- SE, 968.1 +/- 63.5 vs. 1,581.0 +/- 115.3 nmol/g of protein; p = 0.001). After the hepatocyte isolation, the GSHVT stores at various TBHP concentrations in the experimental rats were also similarly and significantly decreased relative to the control animals (894.4 +/- 56.4, 804.2 +/- 118.4, 586.9 +/- 86.6, and 410.2 +/- 87.4 nmol/g of protein vs. 1,282.2 +/- 112.0, 1,156.6 +/- 91.0, 995.2 +/- 92.7, and 866.8 +/- 62.4 nmol/g of protein for TBHPs of 0.0, 0.5, 1.0, and 2.0 mmol/L, respectively; all p < 0.05). Moreover, from photocytometry, the hepatocyte survival rates were significantly reduced for the experimental rats compared with the control animals after challenge with various TBHP concentrations (survival was 100%, 91.1%, 81.3%, and 72.8% vs. 100%, 99.2%, 95.0%, and 88.2%, respectively, for TBHPs of 0.0, 0.5, 1.0, and 2.0 mmol/L; p < 0.05 for the last two). This animal study demonstrated that intra-abdominal hypertension and subsequent decompression deplete the total in vivo GSH store in rat livers, probably via the mechanism of ischemia-reperfusion injury, and the GSHVT after hepatocyte isolation, which makes the isolated hepatocytes of rats more susceptible to oxidant challenge.

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HepatologyVolume 33, Issue 6 p. 1556-1557 CorrespondenceFree Access Reply: Hartmut Jaeschke, Hartmut Jaeschke Ph.D. Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, ARSearch for more papers by this authorJaspreet S. Gujral, Jaspreet S. Gujral M.D. Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, ARSearch for more papers by this authorThomas J. Bucci, Thomas J. Bucci D.V.M., Ph.D. Pathology Associates, Inc. and National Center for Toxicological Research, Jefferson, ARSearch for more papers by this authorAnwar Farhood, Anwar Farhood M.D. Department of Pathology, University of Texas Health Science Center, Houston, TXSearch for more papers by this author Hartmut Jaeschke, Hartmut Jaeschke Ph.D. Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, ARSearch for more papers by this authorJaspreet S. Gujral, Jaspreet S. Gujral M.D. Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, ARSearch for more papers by this authorThomas J. Bucci, Thomas J. Bucci D.V.M., Ph.D. Pathology Associates, Inc. and National Center for Toxicological Research, Jefferson, ARSearch for more papers by this authorAnwar Farhood, Anwar Farhood M.D. Department of Pathology, University of Texas Health Science Center, Houston, TXSearch for more papers by this author First published: 30 December 2003 https://doi.org/10.1053/jhep.2001.0103306le03AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 Gujral JS, Bucci TJ, Farhood A, Jaeschke H. Mechanism of cell death during warm hepatic ischemia-reperfusion in rats: apoptosis or necrosis? Hepatology 2001; 33: 397– 405. MEDLINE 2 Kohli V, Selzner M, Madden JF, Bentley RC, Clavien PA. Endothelial cell and hepatocyte deaths occur by apoptosis after ischemia-reperfusion injury in the rat liver. Transplantation 1999; 67: 1099– 1105. MEDLINE 3 Cursio R, Gugenheim J, Ricci JE, Crenesse D, Rostagno P, Maulon L, Saint-Paul MC, et al. A caspase inhibitor fully protects rats against lethal normothermic liver ischemia by inhibition of liver apoptosis. FASEB J 1999; 13: 253– 261. MEDLINE 4 Grasl-Kraupp B, Ruttkay-Nedecky B, Koudelka H, Bukowska K, Bursch W, Schulte-Hermann R. In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis and autolytic cell death: a cautionary note. Hepatology 1995; 21: 1465– 1468. MEDLINE 5 Lawson JA, Fisher MA, Simmons CA, Farhood A, Jaeschke H. Inhibition of Fas receptor (CD95)-induced hepatic caspase activation and apoptosis by acetaminophen in mice. Toxicol Appl Pharmacol 1999; 156: 179– 186. MEDLINE 6 Sasaki H, Matsuno T, Tanaka N, Orita K. Activation of apoptosis during the reperfusion phase after rat liver ischemia. Transplant Proc 1996; 28: 1908– 1909. MEDLINE 7 Wyllie AH, Kerr JF, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 1980; 68: 251– 306. MEDLINE 8 Bajt ML, Lawson JA, Vonderfecht SL, Gujral JS, Jaeschke H. Protection against Fas receptor-mediated apoptosis in hepatocytes and nonparenchymal cells by a caspase-8 inhibitor in vivo: evidence for postmitochondrial processing of caspase-8. Toxicol Sci 2000; 58: 109– 117. MEDLINE 9 Jaeschke H, Fisher MA, Lawson JA, Simmons CA, Farhood A, Jones DA. Activation of caspase-3 (CPP32)-like proteases is essential for TNF—induced hepatic parenchymal cell apoptosis and neutrophil-mediated necrosis in a murine endotoxin shock model. J Immunol 1998; 160: 3480– 3486. MEDLINE 10 Jaeschke H. Mechanisms of reperfusion injury after warm ischemia of the liver. J Hepatobiliary Pancreat Surg 1998; 5: 402– 408. MEDLINE 11 Kane DJ, Ord T, Anton R, Bredesen DE. Expression of bcl-2 inhibits necrotic neural cell death. J Neurosci Res 1995; 269– 275. 12 Yamabe K, Shimizu S, Kamiike W, Waguri S, Eguchi Y, Hasegawa J, Okuno S, et al. Prevention of hypoxic liver cell necrosis by in vivo human bcl-2 gene transfection. Biochem Biophys Res Commun 1998; 243: 217– 223. MEDLINE 13 Jaeschke H. Mechanisms of ischemia-reperfusion injury. In: AL Gerbes, U Beuers, D Jungst, GR Pape, M Sackmann, eds. Hepatology 2000, Falk Symposium 117. Dordrecht: Kluwer Academic Publishers, 2000: 259– 269. Volume33, Issue6June 2001Pages 1556-1557 ReferencesRelatedInformation

Effects of Physiologic Concentration of Estrogen on Ischemia/Reperfusion-induced Apoptosis in Rat Myocardium

Background:Ischemia/reperfusion injury is very important issue in the era of thrombolysis and primary coro- nary angioplasty in acute myocardial infarction. However, the mechanism of ischemia-reperfusion injury is not fully clarified. Estrogen is well known to have protective actions against ischemic heart disease. We tested hypothesis that estrogen may protect myocardium by reducing ischemia/reperfusion-induced apoptosis. Methods:Sprague-Dawley rats that underwent ovariectomy, female controls, and male rats were subjected to 45minutes of left coronary artery occlusion followed by 4 or 24hours of reperfusion. Coronary artery occlusion was performed 1 week after ovariectomy or sham operation. And sham operation was also performed in each group to confirm the effects of ischemia/reperfusion. Results:Ischemia/reperfusion induced apoptosis in myo- cardium, especially at border zone, whereas sham operation did not induce apoptosis. After 4 hours of reperfus- ion the percentages of apoptotic myocytes in border and center zone of reperfused area were 35.7±3.7%, 29.0 ±4.2% in ovariectomized rats (n=3, 40.8±3.7%, 29.5±2.5% in female control rats (n=3, and 39.0±1.6% (p =0.10, 32.4±1.6% (p=0.43 in male rats (n=3. After 24hours of reperfusion the percentages in border and center zone of reperfused area were 20.6±3.1%, 12.9±4.8% in ovariectomized rats (n=3), 19.6±4.1%, 14.0±2.0% in female control rat (n=3, and 21.4±6.6% (p=0.93), 15.1±2.4% (p=0.85in male rats (n=

Postischemic hypothermia diminishes skeletal muscle reperfusion edema

The mechanisms of ischemia-reperfusion injury in skeletal muscle remain controversial. We investigated the ability of postischemic hypothermia to diminish reperfusion edema and improve skeletal muscle pH in a bilateral, in vivo isolated canine gracilis muscle model. In five anesthetized animals, both gracilis muscles were subjected to 6 hr of ischemia followed by 1 hr of reperfusion. After 5 hr of warm ischemia, one gracilis muscle was cooled to 21°C (cold reperfusion, CR) while the contralateral gracilis muscle was maintained at ambient temperature (warm reperfusion, WR). Reperfusion muscle edema was quantitated by measurement of gracilis muscle weight gain. Interstitial muscle pH was monitored by glass microelectrodes. Vascular permeability was measured by analysis of albumin ( 125I-Alb) leak. Results are presented as the means ± SEM. Reperfusion conditions Cold (CR) Warm (WR) P value Ischemic temperature (°C) 34 ± 1 34 ± 1 0.68 Reperfusion temperature (°C) 21 ± 3 34 ± 2 <0.05 pH just before hypothermia 5.97 ± 0.08 5.97 ± 0.06 0.89 pH just before reperfusion 6.40 ± 0.13 5.96 ± 0.05 <0.02 pH after 1 hr of reperfusion 6.77 ± 0.27 6.43 ± 0.29 <0.05 Muscle weight gain (g) 25 ± 4 46 ± 9 <0.05 125I-Alb (counts/min/g) 41 ± 8 45 ± 14 0.13 Postischemic hypothermia significantly increased the interstitial muscle pH and significantly reduced postre-perfusion muscle edema, without changing the vascular permeability to albumin. These data suggest that hypothermia may provide a clinical method for salvaging ischemic skeletal muscle from the postreperfusion edema that can lead to compartment syndromes, reperfusion injury, and subsequent limb loss.

Heparin Decreases Ischemia-Reperfusion Injury in Isolated Canine Gracilis Model

The mechanisms of ischemia-reperfusion injury in skeletal muscle remain controversial. Some investigators have demonstrated that heparin can ameliorate ischemic injury to heart, brain, and renal tissue. We investigated the ability of heparin sodium to decrease ischemia-reperfusion injury in an isolated gracilis muscle model in ten anesthetized mongrel dogs. One gracilis muscle was perfused normally while the contralateral muscle was subjected to six hours of ischemia followed by one hour of reperfusion. Five dogs were given a preischemic bolus of heparin sodium (200 U/kg, intravenously followed by a continuous infusion (15 U/kg/h, intravenously), and five control dogs received no heparin. Quantitation of skeletal muscle ischemia-reperfusion injury was determined by histochemical staining with triphenyl tetrazolium-chloride and computerized planimetry of the infarct size. Results from the ischemic muscle demonstrate a significant beneficial effect of heparinization. The nonheparinized dogs had a 72% +/- 5% infarct size, which was significantly reduced to 24% +/- 8% in the heparinized dogs. The mechanism of this protective effect may be due to heparin's anticoagulant, antiplatelet, or anti-inflammatory action.