Ischemia-reperfusion Injury In Pigs Research Articles

Pilot Trial of Extended Hypothermic Lung Preservation to Analyze Ischemia-reperfusion Injury in Pigs

BackgroundIn lung transplantation (LT), the length of ischemia time is controversial as it was arbitrarily stablished. We ought to explore the impact of extended cold-ischemia time (CIT) on ischemia-reperfusion injury in an experimental model. MethodsExperimental, randomized pilot trial of parallel groups and final blind analysis using a swine model of LT. Donor animals (n=8) were submitted to organ procurement. Lungs were subjected to 6h (n=4) or 12h (n=4) aerobic hypothermic preservation. The left lung was transplanted and re-perfused for 4h. Lung biopsies were obtained at (i) the beginning of CIT, (ii) the end of CIT, (iii) 30min after reperfusion, and (iv) 4h after reperfusion. Lung-grafts were histologically assessed by microscopic lung injury score and wet-to-dry ratio. Inflammatory response was measured by determination of inflammatory cytokines. Caspase-3 activity was determined as apoptosis marker. ResultsWe observed no differences on lung injury score or wet-to-dry ratio any given time between lungs subjected to 6h-CIT or 12h-CIT. IL-1β and IL6 showed an upward trend during reperfusion in both groups. TNF-α was peaked within 30min of reperfusion. IFN-γ was hardly detected. Caspase-3 immunoexpression was graded semiquantitatively by the percentage of stained cells. Twenty percent of apoptotic cells were observed 30min after reperfusion. ConclusionsWe observed that 6 and 12h of CIT were equivalent in terms of microscopic lung injury, inflammatory profile and apoptosis in a LT swine model. The extent of lung injury measured by microscopic lung injury score, proinflammatory cytokines and caspase-3 determination was mild.

Effect of Intravenous Lidocaine on Inflammatory and Apoptotic Response of Ischemia-Reperfusion Injury in Pigs Undergoing Lung Resection Surgery.

Background Ischemia-reperfusion injury is one of the most critical phenomena in lung transplantation and causes primary graft failure. Its pathophysiology remains incompletely understood, although the inflammatory response and apoptosis play key roles. Lidocaine has anti-inflammatory properties. The aim of this research is to evaluate the effect of intravenous lidocaine on the inflammatory and apoptotic responses in lung ischemia-reperfusion injury. Methods We studied the histological and immunohistochemical changes in an experimental model of lung transplantation in pigs. Twelve pigs underwent left pneumonectomy, cranial lobectomy, caudal lobe reimplantation, and 60 minutes of graft reperfusion. Six of the pigs made up the control group, while six other pigs received 1.5 mg/kg of intravenous lidocaine after induction and a 1.5 mg/kg/h intravenous lidocaine infusion during surgery. In addition, six more pigs underwent simulated surgery. Lung biopsies were collected from the left caudal lobe 60 minutes after reperfusion. We conducted a double study on these biopsies and assessed the degree of inflammation, predominant cell type (monocyte-macrophage, lymphocytes, or polymorphous), the degree of congestion, and tissue edema by hematoxylin and eosin stain. We also conducted an immunohistochemical analysis with antibodies against CD68 antigens, monocyte chemoattractant protein-1 (MCP-1), Bcl-2, and caspase-9. Results The lungs subjected to ischemia-reperfusion injury exhibited a higher degree of inflammatory infiltration. The predominant cell type was monocyte-macrophage cells. Both findings were mitigated by intravenous lidocaine administration. Immunohistochemical detection of anti-CD68 and anti-MCP-1 showed higher infiltration in the lungs subjected to ischemia-reperfusion injury, while intravenous lidocaine decreased the expression. Ischemia-reperfusion induced apoptotic changes and decreased Bcl-2 expression. The group treated with lidocaine showed an increased number of Bcl-2-positive cells. No differences were observed in caspase-9 expression. Conclusions In our animal model, intravenous lidocaine was associated with an attenuation of the histological markers of lung damage in the early stages of reperfusion.

Pilot Trial of Extended Hypothermic Lung Preservation to Analyze Ischemia-reperfusion Injury in Pigs

BackgroundIn lung transplantation (LT), the length of ischemia time is controversial as it was arbitrarily stablished. We ought to explore the impact of extended cold-ischemia time (CIT) on ischemia-reperfusion injury in an experimental model. MethodsExperimental, randomized pilot trial of parallel groups and final blind analysis using a swine model of LT. Donor animals (n=8) were submitted to organ procurement. Lungs were subjected to 6h (n=4) or 12h (n=4) aerobic hypothermic preservation. The left lung was transplanted and re-perfused for 4h. Lung biopsies were obtained at (i) the beginning of CIT, (ii) the end of CIT, (iii) 30min after reperfusion, and (iv) 4h after reperfusion. Lung-grafts were histologically assessed by microscopic lung injury score and wet-to-dry ratio. Inflammatory response was measured by determination of inflammatory cytokines. Caspase-3 activity was determined as apoptosis marker. ResultsWe observed no differences on lung injury score or wet-to-dry ratio any given time between lungs subjected to 6h-CIT or 12h-CIT. IL-1β and IL6 showed an upward trend during reperfusion in both groups. TNF-α was peaked within 30min of reperfusion. IFN-γ was hardly detected. Caspase-3 immunoexpression was graded semiquantitatively by the percentage of stained cells. Twenty percent of apoptotic cells were observed 30min after reperfusion. ConclusionsWe observed that 6 and 12h of CIT were equivalent in terms of microscopic lung injury, inflammatory profile and apoptosis in a LT swine model. The extent of lung injury measured by microscopic lung injury score, proinflammatory cytokines and caspase-3 determination was mild.

Reduction of myocardial ischemia reperfusion injury in pigs by (over) expression of human membrane co-factor protein

Edible films and barriers derived from polysaccharides, proteins and lipids provide several functions which prevent mass transfer, for example, water vapor, gas, and volatile compounds, between foods and environment. The preparation of materials can be either by solution casting commonly used in the laboratory or a more commercially extrusion process. Plasticizers are used to increase deformability reducing the glass transition temperature (Tg) of the materials which can be determined using differential scanning calorimetry (DSC) and dynamic mechanical thermal analysis (DMTA). This chapter also demonstrates the effects of plasticizers, other components, and treatments on the Tg values of various edible films. Moreover, the non-equilibrium state of the edible film matrices causes several time-dependent changes during storage or “aging,” for example, plasticizer loss, lipid destabilization, and crystallization which subsequently impacts functional and mechanical properties of the films.

Systemic Dosing of Thymosin Beta 4 before and after Ischemia Does Not Attenuate Global Myocardial Ischemia-Reperfusion Injury in Pigs.

The use of cardiopulmonary bypass (CPB) and aortic cross-clamping causes myocardial ischemia-reperfusion injury (I-RI) and can lead to reduced postoperative cardiac function. We investigated whether this injury could be attenuated by thymosin beta 4 (TB4), a peptide which has showed cardioprotective effects. Pigs received either TB4 or vehicle and underwent CPB and aortic cross-clamping for 60 min with cold intermittent blood-cardioplegia and were then followed for 30 h. Myocardial function and blood flow was studied by cardiac magnetic resonance and PET imaging. Tissue and plasma samples were analyzed to determine the amount of cardiomyocyte necrosis and apoptosis as well as pharmacokinetics of the peptide. In vitro studies were performed to assess its influence on blood coagulation and vasomotor tone. Serum levels of the peptide were increased after administration compared to control samples. TB4 did not decrease the amount of cell death. Cardiac function and global myocardial blood flow was similar between the study groups. At high doses a vasoconstrictor effect on mesentery arteries and a vasodilator effect on coronary arteries was observed and blood clot firmness was reduced when tested in the presence of an antiplatelet agent. Despite promising results in previous trials the cardioprotective effect of TB4 was not demonstrated in this model for global myocardial I-RI.

Effect of dexmedetomidine preconditioning on myocardial ischemia-reperfusion injury in pigs

Objective To investigate the effects of dexmedetomidine preconditioning on myocardial ischemia-reperfusion injury in pigs.Methods Twenty healthy pigs of both sexes,weighing 25-35 kg,were randomly divided into two groups (n =10 each):control group (group C),and dexmedetomidine group (Dex group).Ketamine 10 mg/kg combined with midazolam 0.1 mg/kg were injected intramuscularly,and the trachea was intubated after the incision of trachea followed by mechanical ventilation.Anesthesia was maintained with sevoflurane (1.5％-2.0％).A loading dose of dexmedetomidine 0.5 μg/kg was injected intravenously over 10 min after intubation followed by infusion at 0.5 μg/(kg·h) until the occlusion of descending coronary artery.After the infusion of study drugs,the hearts were subject to 30 min of regional ischemia followed by 60 min of reperfusion.Mean arterial pressure (MAP),heart rate (HR),and central venous pressure (CVP) were recorded at the time point of pre-ischemia,10 and 30 min after ischemia,and 10,30 and 60 min after reperfusion.The blood samples from the internal jugular vein were collected,cTnI and CK-MB were determined by enzyme linked immunosorbent assay (ELISA).The arrhythmia was scored 20 min after the reperfusion.The descending coronary artery was reoccluded after the end of reperfusion,and the left ventricles which were not blocked were stained with Evans blue.The left ventricles were sliced from base to the tip into 10-mm slices.The infarct size (IS％) was calculated.Results CK-MB and cTnI were higher in group C than in Dex group at re-perfusion 30 min and 60 min (P ＜ 0.05).The scores of the arrhythmia were (3.300 ±0.675) in group C,higer than in Dex group (2.000 ±0.677) (P ＜ 0.05).IS％ in group C was 42.4％,higher than in Dex group (23.5％,P ＜0.05).Conclusion Dexmedetomidine preconditioning can reduce myocardial ischemia-reperfusion injury in pigs. Key words: Dexmedetomidine ; Myocardial ischemia; Reperfusion injury ; Preconditioning

The “Pavia Model” of Experimental Small Bowel Transplantation in Pigs: Technical Variations for Ischemia Reperfusion Injury Studies

Ischemia reperfusion injury (IRI) is a major field of study in small bowel transplantation because of its implications regarding intestinal immunity. In this study, we have introduced some variations to the described models of IRI in pigs to make possible a complete isolation of the small bowel for IRI studies. In swine, two anatomical barriers make impossible a complete isolation of the small bowel at the origin of superior mesenteric artery (SMA) and vein (SMV): the main colic vessels, which originate distally to form SMA and SMV, and the blood supply of the distal portion of the duodenum and the cephalic part of the pancreas. In a group of Large White pigs (n = 5), we have performed a complete isolation of the small bowel, including sub-total colectomy and pancreaticoduodenectomy. Both SMA and SMV were isolated at the origin from the aorta and at the junction of the splenic vein, respectively. Intestinal continuity was restored with duodenojejunal anastomosis and with ileotransverse colon anastomosis. One pig died on postoperative day 5 from intestinal occlusion due to adhesions. The remaining four pigs were killed on postoperative day 7 after an uneventful postoperative course. No complications were found at autopsy. In swine, resection of part of the pancreas and duodenum and removal of the large bowel does not affect short-term survival, allowing a full isolation of the entire small bowel mimicking the transplantation procedure. Thus, this model appears to be attractive for IRI studies in the field of intestinal transplantation.

Effects of a Preconditioning Oral Nutritional Supplement on Pig Livers after Warm Ischemia

Background. Several approaches have been proposed to pharmacologically ameliorate hepatic ischemia/reperfusion injury (IRI). This study was designed to evaluate the effects of a preconditioning oral nutritional supplement (pONS) containing glutamine, antioxidants, and green tea extract on hepatic warm IRI in pigs. Methods. pONS (70 g per serving, Fresenius Kabi, Germany) was dissolved in 250 mL tap water and given to pigs 24, 12, and 2 hrs before warm ischemia of the liver. A fourth dose was given 3 hrs after reperfusion. Controls were given the same amount of cellulose with the same volume of water. Two hours after the third dose of pONS, both the portal vein and the hepatic artery were clamped for 40 min. 0.5, 3, 6, and 8 hrs after reperfusion, heart rate (HR), mean arterial pressure (MAP), central venous pressure (CVP), portal venous flow (PVF), hepatic arterial flow (HAF), bile flow, and transaminases were measured. Liver tissue was taken 8 hrs after reperfusion for histology and immunohistochemistry. Results. HR, MAP, CVP, HAF, and PVF were comparable between the two groups. pONS significantly increased bile flow 8 hrs after reperfusion. ALT and AST were significantly lower after pONS. Histology showed significantly more severe necrosis and neutrophil infiltration in controls. pONS significantly decreased the index of immunohistochemical expression for TNF-α, MPO, and cleaved caspase-3 (P < 0.001). Conclusion. Administration of pONS before and after tissue damage protects the liver from warm IRI via mechanisms including decreasing oxidative stress, lipid peroxidation, apoptosis, and necrosis.

Inhibition of NF-κB Activation by β-Glucan Is Not Associated with Protection from Global Ischemia-Reperfusion Injury in Pigs

Pretreatment with β-glucan has been shown to protect against regional ischemia-reperfusion injury, through inhibition of myocardial NF-κB activation. The aim was to examine whether β-glucan pretreatment could protect against the global ischemia-reperfusion injury, which is encountered in the clinical setting during open heart surgery. Twenty-one pigs were randomized to pretreatment with oral β-glucan (SBGo, n = 7), pretreatment with i.p. β-glucan (SBGip, n = 7), and untreated controls (n = 7). The pigs were subjected to cardiopulmonary bypass (CPB) with 1 h of global cardioplegic ischemia followed by wean from CPB and reperfusion for 4 h. Cardiac function was determined by a conductance catheter, and troponin T was sampled from the coronary sinus. Atrial biopsies obtained at baseline, following 30 min, and 3 h of reperfusion were analyzed for phosphorylated NF-κB by Western blot. Following reperfusion, phosphorylated NF-κB increased by 210% in the control group, 197% in the SBGo group, but was reduced by 5% in the SBGip group (P < 0.01 versus control). After 4 h of reperfusion, preload recruitable stroke work dropped by 19% in the control group and 25% in the SBGo group compared with 60% in the SBGip group (P < 0.01 versus control). The area under the curve for troponin T was larger in the SBGip group compared with the control group (P< 0.05) and the SBGo group (P < 0.01). Inhibition of NF-κB activation by i.p. β-glucan does not protect against ischemia-reperfusion injury in pigs subjected to global ischemia and reperfusion, and may be associated with aggravation of ischemia-reperfusion injury.

Effects of Perfusion Solutions on Kidney Ischemia-Reperfusion Injury in Pigs

During ischemia-reperfusion, free oxygen radicals which directly affect renal cells may cause delayed graft function. We investigated whether there was a difference regarding antioxidant enzyme content between use of Ringer’s lactate (RL) versus University of Wisconsin (UW) perfusion solutions in kidney transplantation. Ischemia was achieved by clamping the renal pedicle for 20 minutes followed by perfusion with either solution for 20 minutes and reperfusion for another 20 minutes. A parenchymal biopsy was taken before and after the ischemia, perfusion, and reperfusion (IPR) process. The levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA) were investigated in the biopsy specimens. We used paired t tests within groups and t tests for comparisons between groups. The results were expressed as mean values ± SEM with P < .05 accepted as statistically significant. After IPR, SOD, GPx, and MDA were decreased in all groups: only GPx ( P = .001) and MDA ( P = .04) for the RL group and SOD ( P = .001) and MDA ( P = .05) for the UW group were statistically significant. In the control group, we did not observe any difference ( P > .05). Comparisons between groups did not reveal differences ( P > .05). In our study, no difference was observed between RL and UW regarding their effects on antioxidant enzymes following renal I/R injury in pigs. More investigations are needed to evaluate graft function in this setting.

Ischemic postconditioning during reperfusion activates Akt and ERK without protecting against lethal myocardial ischemia-reperfusion injury in pigs

Transient episodes of ischemic preconditioning (PC) render myocardium protected against subsequent lethal injury after ischemia and reperfusion. Recent studies indicate that application of short, repetitive ischemia only during the onset of reperfusion after the lethal ischemic event may obtain equivalent protection. We assessed whether such ischemic postconditioning (Postcon) is cardioprotective in pigs by limiting lethal injury. Pentobarbital sodium-anesthetized, open-chest pigs underwent 30 min of complete occlusion of the left anterior descending coronary artery and 3-h reflow. PC was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. Postcon was elicited by three cycles of 30-s reperfusion, followed by 30-s reocclusion, after the 30-min occlusion period and before the 3-h reflow. Infarct size (%area-at-risk using triphenyltetrazolium chloride macrochemistry; means +/- SE) after 30 min of ischemia was 26.5 +/- 5.2% (n = 7 hearts/treatment group). PC markedly limited myocardial infarct size (2.8 +/- 1.2%, n = 7 hearts/treatment group, P < 0.05 vs. controls). However, Postcon had no effect on infarct size (37.8 +/- 5.1%, n = 7 hearts/treatment group). Within the subendocardium, Postcon increased phosphorylation of Akt (74 +/- 12%) and ERK1/2 (56 +/- 10%) compared with control hearts subjected only to 30-min occlusion and 15-min reperfusion (P < or = 0.05), and these changes were not different from the response triggered by PC (n = 5 hearts/treatment group). Phosphorylation of downstream p70S6K was also equivalent in PC and Postcon groups. These data do not support the hypothesis that application of 30-s cycles of repetitive ischemia during reperfusion exerts a protective effect on pig hearts subjected to lethal ischemia, but this is not due to a failure to phosphorylate ERK and Akt during early reperfusion.

Mild hypothermia protects liver against ischemia and reperfusion injury

To determine whether mild hypothermia could protect liver against ischemia and reperfusion injury in pigs. Twenty-four healthy pigs were randomly divided into normothermia, mild hypothermia and normal control groups. The experimental procedure consisted of temporary interruption of blood flow to total hepatic lobe for different lengths of time and subsequent reperfusion. Hepatic tissue oxygen pressure (PtiO2) and aspartate aminotransferase (AST) values were evaluated, and ultrastructural analysis was carried out for all samples. Serum AST was significantly lower, and hepatic PtiO2 values were significantly higher in the mild hypothermia group than in the normothermia group during liver ischemia-reperfusion periods (P=0.032, P=0.028). Meanwhile, the histopathologic injury of liver induced by ischemia-reperfusion was significantly improved in the mild hypothermia group, compared with that in the normothermia group. Mild hypothermia can protect the liver from ischemia-reperfusion injury in pigs.

Expression of E-selectin and its transcripts during intestinal ischemia-reperfusion injury in pigs

Background Ischemia-reperfusion injury (IRI) can result in severe organ dys- or nonfunction. Interaction of leukocytes and endothelial cells mediated by E-selectin appears to be a key step for disturbed microcirculation. Therefore we studied gene and protein expression as well as localization of E-selectin during intestinal IRI. Methods Intestinal tissue samples were obtained from extracorporeal perfused intestines (cold ischemia time [CIT] 2 or 20 hours, each n = 5) and additionally in intestinal transplanted pigs (CIT 2 or 20 hours, each n = 1). Mucosal damage was graded according to the Chiu classification. E-selectin mRNA was determined by PCR and quantitative RT-PCR. Localization of E-selectin mRNA was performed by in situ hybridization and of the protein by immunohistochemistry. Results Histologically, mucosal damage occurred during reperfusion and was earlier and more severe after 20 hours of CIT. E-selectin mRNA expression was detected by PCR already after laparotomy and was elevated after reperfusion. Interestingly, mRNA expression was already increased after 20 hours of CIT. E-selectin mRNA was localized to the luminal surface of muscular, submucosal, and mucosal endothelial cells and the protein was detected on submucosal arterial endothelium as early as 2 hours after reperfusion. Conclusion Prolongation of CIT results in more severe mucosal damage during reperfusion, which is associated with protein expression of E-selection that might be used as a marker for activated endothelial cells. Increased E-selectin mRNA at end of 20 hours of CIT might indicate a preactivated state of endothelial cells potentially triggered by bacterial translocation or products.

Cardioselective nitric oxide synthase 3 gene transfer protects against myocardial ischemia-reperfusion injury in pigs

Cardioselective nitric oxide synthase 3 gene transfer protects against myocardial ischemia-reperfusion injury in pigs

Destruction of Kupffer’s cells increases total liver blood flow and decreases ischemia reperfusion injury in pigs

Destruction of Kupffer’s cells increases total liver blood flow and decreases ischemia reperfusion injury in pigs

Evaluation of liver function and morphology following ischemia-reperfusion injury in pigs

Evaluation of liver function and morphology following ischemia-reperfusion injury in pigs

Hypothermia, hepatic oxygen supply-demand, and ischemia-reperfusion injury in pigs

We examined the effects of two degrees of hypothermia on hepatic oxygen delivery and uptake, hepatic lactate uptake as a marker of hepatic function, and the effect of hypothermia on ischemia-reperfusion injury in the liver in miniature pigs (n = 18, 21-30 kg body wt). Hepatic arterial and portal venous blood flows were measured while hepatic oxygen delivery was progressively decreased without venous congestion in the preportal area. With decreases in hepatic blood and oxygen supply, oxygen extraction gradually increased from 50 to 90% in the normothermic group and from 25 to 70 and 84% in the hypothermic (30. and 34 degrees C, respectively) groups. The values of critical hepatic oxygen delivery were between 7.3 and 11.9 ml O2.min-1.100 g-1 without significant differences among the groups. During reperfusion after ischemic insult, hepatic oxygen uptake returned to base-line values in both hypothermic groups but remained substantially below base-line values in normothermic groups of animals. Hepatic enzyme concentrations (lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, and alcohol dehydrogenase) were substantially increased (up to 30-fold) in normothermic animals, but the concentrations did not increase in either of the hypothermic groups. These results demonstrated that hypothermia per se does not affect hepatic oxygen delivery but decreases hepatic oxygen demand and uptake, provides an effective protection from hepatic oxygen deprivation, and lessens reperfusion injury.