Early Phase Of Ischemia-reperfusion Injury Research Articles

Clec4e-Receptor Signaling in Myocardial Repair After Ischemia-Reperfusion Injury

The bacterial C-type lectin domain family 4 member E (CLEC4E) has an important role in sterile inflammation, but its role in myocardial repair is unknown. Using complementary approaches in porcine, murine, and human samples, we show that CLEC4E expression levels in the myocardium and in blood correlate with the extent of myocardial injury and left ventricular (LV) functional impairment. CLEC4E expression is markedly increased in the vasculature, cardiac myocytes, and infiltrating leukocytes in the ischemic heart. Loss of Clec4e signaling is associated with reduced acute cardiac injury, neutrophil infiltration, and infarct size. Reduced myocardial injury in Clec4e -/- translates into significantly improved LV structural and functional remodeling at 4weeks' follow-up. The early transcriptome of LV tissue from Clec4e -/- mice versus wild-type mice reveals significant upregulation of transcripts involved in myocardial metabolism, radical scavenging, angiogenesis, and extracellular matrix organization. Therefore, targeting CLEC4E in the early phase of ischemia-reperfusion injury is a promising therapeutic strategy to modulate myocardial inflammation and enhance repair after ischemia-reperfusion injury.

Sevoflurane Attenuates Ischemia-Reperfusion Injury in a Rat Lung Transplantation Model

Sevoflurane is one of the most commonly used volatile anesthetic agents with the fastest onset and offset, replacing isoflurane in modern anesthesiology. Preconditioning and postconditioning using volatile anesthetics can attenuate ischemia-reperfusion injury (IRI). However, no previous studies have evaluated the effect of sevoflurane in lung transplantation after cold ischemic injury. We aimed to study the effects of donor and recipient treatment with sevoflurane in a rat lung transplantation model. Lewis rats were allocated to four groups: control, PreC (preconditioning), PostC (postconditioning), and PreC+ PostC. Donor rats in the PreC and PreC+PostC groups were exposed to 1.5% sevoflurane for 30 minutes before donor operation. Donor lungs were flushed with Perfadex and stored for 12 hours at 4°C before transplantation. Recipients received orthotopic left lung transplantation. In the PostC and PreC+ PostC groups, sevoflurane was initiated 2 minutes before reperfusion and maintained for 30 minutes. Two hours after reperfusion, lung function was evaluated, and samples were collected for histologic, inflammatory, and cell death assessment. Preconditioning and postconditioning using sevoflurane significantly improved the oxygenation of lung grafts (partial arterial gas pressure of oxygen: 198mm Hg in control, 406.5 mm Hg in PreC, 472.4 mm Hg in PostC, and 409.7 mm Hg in PreC+ PostC, p < 0.0001) and reduced pulmonary edema. Sevoflurane treatment reduced levels of interleukin-1β, interleukin-6, and tumor necrosis factor-α. Moreover, sevoflurane significantly inhibited apoptotic cells by a decrease in cytochrome c release into cytosol and caspase-3 cleavage. Preconditioning or postconditioning of lungs using sevoflurane exhibits a significant protective effect against early phase of ischemia-reperfusion injury in a rat lung transplantation model.

The Effect of mTOR-Inhibition on NF-κB Activity in Kidney Ischemia-Reperfusion Injury in Mice

Kidney ischemia-reperfusion injury (IRI) is associated with a robust inflammatory response, which is regulated by nuclear factor-kappaB (NF-κB), mainly its heterodimeric form p65/p50. Considering immunomodulatory properties of mammalian target of rapamycin (mTOR) inhibitors, the effect of everolimus on NF-κB activation in kidney IRI was determined in this study. IRI was induced in C57/BL6 mice by clamping both renal pedicles for 45 minutes. Application of everolimus (0.25 mg/kg bw subcutaneously daily) was started one day before IRI induction. Both everolimus-treated and nontreated mice were sacrificed at several times starting at 30 minutes and finishing on day 7 after IRI induction. The NF-κB activity, proinflammatory cytokines IL-1β, TNF-α, and anti-inflammatory cytokine IL-10 production were determined in kidneys. Compared with nontreated animals, everolimus-treated animals showed significantly increased TNF-α (2741.6 ± 201.72 pg/mg; 1925 ± 185.81 pg/mg, P < .05) and IL-1β (11.47 ± 1.2 pg/mg; 4.3 ± 0.13 pg/mg, P < .01) production on day 2 after IRI induction accompanied by significantly greater NF-κB/DNA binding activity and p65 nuclear expression (P < .01). Two hours after IRI induction, everolimus-treated animals showed significantly increased IL-1β mRNA expression (P < .05) followed by increased IL-1β protein concentrations when compared with nontreated animals measured 6 hours after IRI induction (11.71 ± 1.5 pg/mg; 7.5 ± 1.11 pg/mg, P < .01). Both experimental groups showed increased NF-κB/DNA binding activity at 7 days after IRI induction. Significantly increased nuclear p65 expression was measured in nontreated animals (P < .01), whereas everolimus-treated hosts showed significantly increased nuclear RelB expression (P < .01). These data suggested that everolimus potentiated innate immunity in the early phase of IRI, stimulating the production of NF-κB–driven proinflammatory cytokines such as TNF-α and IL-1β. The NF-κB activity was potentiated under m-TOR inhibition during kidney IRI, implicating a possible beneficial role of alternative NF-κB activation during the repair phase.

L-arginine in the ischemic phase protects against liver ischemia-reperfusion injury

To investigate the effects of intravenous L-arginine (LG) infusion on liver morphology, function and proinflammatory response of cytokines during the early phase of ischemia-reperfusion injury (IRI). Thirty rabbits were subjected to 60 minutes of hepatic ischemia and 120 minutes of reperfusion. An intravenous injection of saline or L-arginine was administered five minutes before the ischemia and five minutes before initiating the reperfusion and at the 55th and 115th minutes after the ischemia. Samples were collected for histological analysis of the liver and measurements of the serum AST, ALT and LDH and the cytokines IL-6 and TNF-alpha. It was observed a significant reduction of sinusoidal congestion, cytoplasmic vacuolization, infiltration of polymorphonuclear leukocyte, nuclear pyknosis, necrosis and steatosis in liver tissue, as well as AST, ALT and LDH after injection of LG in the ischemia (p <0.001). Lower levels of IL-6 and TNF-alpha were associated with LG infusion during ischemia. Higher levels these proteins were observed in animals receiving LG during reperfusion. L-arginine protects the liver against ischemia/reperfusion injury, mainly when is administered during the ischemic phase.

N-3 PUFA Supplementation Triggers PPAR-α Activation and PPAR-α/NF-κB Interaction: Anti-Inflammatory Implications in Liver Ischemia-Reperfusion Injury

Dietary supplementation with the n-3 polyunsaturated fatty acids (n-3 PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to rats preconditions the liver against ischemia-reperfusion (IR) injury, with reduction of the enhanced nuclear factor-κB (NF-κB) functionality occurring in the early phase of IR injury, and recovery of IR-induced pro-inflammatory cytokine response. The aim of the present study was to test the hypothesis that liver preconditioning by n-3 PUFA is exerted through peroxisone proliferator-activated receptor α (PPAR-α) activation and interference with NF-κB activation. For this purpose we evaluated the formation of PPAR-α/NF-κBp65 complexes in relation to changes in PPAR-α activation, IκB-α phosphorylation and serum levels and expression of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in a model of hepatic IR-injury (1 h of ischemia and 20 h of reperfusion) or sham laparotomy (controls) in male Sprague Dawley rats. Animals were previously supplemented for 7 days with encapsulated fish oil (General Nutrition Corp., Pittsburg, PA) or isovolumetric amounts of saline (controls). Normalization of IR-altered parameters of liver injury (serum transaminases and liver morphology) was achieved by dietary n-3 PUFA supplementation. EPA and DHA suppression of the early IR-induced NF-κB activation was paralleled by generation of PPAR-α/NF-κBp65 complexes, in concomitance with normalization of the IR-induced IκB-α phosphorylation. PPAR-α activation by n-3 PUFA was evidenced by enhancement in the expression of the PPAR-α-regulated Acyl-CoA oxidase (Acox) and Carnitine-Palmitoyl-CoA transferase I (CPT-I) genes. Consistent with these findings, normalization of IR-induced expression and serum levels of NF-κB-controlled cytokines IL-lβ and TNF-α was observed at 20 h of reperfusion. Taken together, these findings point to an antagonistic effect of PPAR-α on NF-κB-controlled transcription of pro-inflammatory mediators. This effect is associated with the formation of PPAR-α/NF-κBp65 complexes and enhanced cytosolic IκB-α stability, as major preconditioning mechanisms induced by n-3 PUFA supplementation against IR liver injury.

Hydrogen-Enriched Preservation Protects the Isogeneic Intestinal Graft and Amends Recipient Gastric Function During Transplantation

Inhaled hydrogen gas exerts antioxidant and anti-inflammatory effects in rat intestinal transplantation. Here, we investigated whether ex vivo donor organ treatment with dissolved hydrogen would prevent intestinal graft injury. Isogeneic intestinal transplantation was performed in Lewis rats with vascular flush, luminal preservation, and cold graft storage in nitrogen-bubbled (SITxN2) or hydrogen-bubbled (SITxH2) preservation solution. Lactated Ringer's solution and 3-hr cold ischemia time were used for mechanistic investigations, whereas survival experiments were performed with University of Wisconsin solution and 6-hr cold ischemia time. During the early phase of ischemia-reperfusion injury, hydrogen-enriched solution significantly preserved mucosal graft morphology, diminished graft malondialdehyde levels demonstrating substantial reduction potential and blunted proinflammatory molecular responses (early growth response gene [EGR-1], interleukin [IL]-6, IL-1ß, and inducible nitric oxide synthase) within the reperfused intestinal graft muscularis. During the late phase of ischemia-reperfusion injury, circulating IL-6 protein and lactate dehydrogenase levels were significantly ameliorated in SITxH2 animals, which were associated with a favorable functional outcome in in vivo liquid gastrointestinal transit and recipient solid gastric emptying of chrome steel balls, and marked prevention of the posttransplant associated suppression of in vitro muscarinic jejunal contractility. Reflecting improved graft preservation, hydrogen preloading of grafts increased recipient survival rates from 41% to 80%. Anti-inflammatory and antiapoptotic heme oxygenase-1 was significantly upregulated in the hydrogen-treated graft muscularis but not mucosa before reperfusion. Graft preloading with hydrogen demonstrated superior morphologic and functional graft protection in rodent intestinal transplantation, ultimately facilitating recipient survival. Antioxidant capacity and muscularis heme oxygenase-1 upregulation are possible protective mechanisms.

Blocking Fas Ligand on Leukocytes Attenuates Kidney Ischemia-Reperfusion Injury

Inflammation contributes to the pathogenesis of ischemic acute kidney injury (AKI), and T cells mediate the early phase of ischemia-reperfusion injury (IRI). The Fas/Fas ligand (FasL) pathway modulates the balance of T cell subsets in the peripheral circulation as well as multiple inflammatory responses, suggesting that FasL may mediate ischemic AKI. Here, we induced bilateral renal IRI in mice bearing a loss-of-function mutation of FasL (the gld mutation) and in wild-type mice. Compared with wild-type mice, serum creatinine was lower in gld mice (1.4 ± 0.9 mg/dl versus 2.6 ± 0.4) at 24 hours after IRI (P<0.05). In addition, gld mice had fewer TNF-α-producing T lymphocytes in the kidneys and renal lymph nodes. Furthermore, pharmacologic blockade of FasL protected the kidneys of wild-type mice from IRI. Analysis of bone marrow chimeric mice suggested that the pathogenic effect of FasL involves leukocytes; reconstitution of wild-type mice with gld splenocytes attenuated IRI. In contrast, reconstitution of gld mice with wild-type splenocytes enhanced IRI. These data demonstrate that FasL, particularly on leukocytes, mediates ischemic AKI.

L’hydroxyéthylamidon 130/0,4 atténue les dommages hépatiques précoces causés par les lésions d’ischémie/reperfusion

Ischemia/reperfusion injury (IRI) remains a clinical challenge. We tested the hypothesis that fluid therapy using hydroxyethyl starch (HES) 130/0.4 during the early phase of IRI in rat liver decreases markers of hepatic injury. We induced liver IRI in three groups of rats anesthetized with ketamine and chlorpromazine by means of 60 min of segmental hepatic ischemia followed by 120 min of reperfusion. At the onset of reperfusion, Group 1 (IRI + HES; n = 12) was given 13 mL.kg(-1) of HES; Group 2 (IRI + HS; n = 12) received the same volume of 7.5% saline (HS), and Group 3 (IRI-only; n = 12) received no fluid. Three other groups of 12 animals each were sham-operated and received the same fluid as the test groups. We euthanized the animals after three hours, drew blood for alanine aminotransferase (ALT) quantification, and took ischemic liver samples for histomorphological study. Serum ALT activity was greater in all of the IRI groups than in the sham-operated animals. The ALT activity was 1,081 +/- 575 IU.L(-1) in IRI + HES Group 1; 2,363 +/- 1,839 IU.L(-1) in IRI + HS Group 2; and 2,866 +/- 2,491 IU.L(-1) in IRI-only Group 3. There was a statistically significant difference between the IRI + HES and the IRI-only groups (P = 0.001), but not between the IRI + HS and the IRI-only groups (P > 0.05). Likewise, histological scores were greater in all IRI groups compared with the sham-operated animals. Scores were higher in the IRI-only group (median 3.5) than in the groups receiving fluid (IRI + HES median 2; IRI + HS median 3). The difference between IRI + HES and IRI-only was statistically significant (P = 0.008) but not so between IRI + HS and IRI-only (P > 0.05). Giving HES 130/0.4 attenuates rat liver IRI compared with no fluid, while giving HS does not. This suggests a role for HES in hepatoprotection associated with liver IRI.

N-acetylcysteine Improves Early Cardiac Isograft Function in a Rat Heterotopic Transplantation Model

Reactive oxygen species play an important role in the early phase of ischemia-reperfusion injury. N-acetylcysteine (NAC), an antioxidant, has shown protective effects in ischemia-reperfusion injuries in some organ transplantations; however, its roles in cardiac transplantation have not been thoroughly evaluated. Lewis rats were divided into three groups ( n = 8 in each group): sham, control, and NAC group. They were subjected to abdominal heterotopic cardiac transplantation and followed for 24 hours postoperation. For the NAC group, a bolus of NAC (150 mg/kg) was infused at 30 minutes before reperfusion, followed by 20 mg/kg/h for 1 hour; another 150 mg/kg NAC intraperitoneally administered 12 hours after reperfusion. Cardiac function and blood inflammation markers were measured at both 2 and 24 hours after reperfusion. Oxidative stress markers and neutrophil infiltration were evaluated in heart tissue at 24 hours postoperatively. Echocardiography showed that NAC significantly improved the postoperative fractional shortening of isografts ( P < .01), although NAC had no effect on the heart rate. Serum lactate dehydrogenase also indicated a significant decrease in the NAC group at 24 hours posttransplantation ( P = .02) Serum tumor necrosis factor-α and interleukin-1 concentrations which rapidly increased postoperatively were reduced by administration of NAC. In transplanted hearts, the increased malondialdehyde level and myeloperoxidase activity were partially reversed by NAC (both P < .01); NAC also replenished endogenous glutathione. Finally, neutrophil infiltration in isografts was reduced in the NAC group. Recipient treatment with continuous intravenous NAC protected cardiac isografts against posttransplantation ischemia-reperfusion injury, probably through its antioxidant and anti-inflammatory properties.

CD44 expression in renal ischemia-reperfusion injury in rats

Renal ischemia-reperfusion injury (IRI) is an invariable consequence of transplantation. The tubuloepithelial expression of CD44 is markedly enhanced in autoimmune renal injuries. The aim of this experimental study was to evaluate the effect of IRI on the expression of CD44 in rat kidney. Thirty male Sprague-Dawley rats were used. The rats were divided into three groups. The rats in group 1 (n = 10) underwent laparotomy and left nephrectomy (Sham surgery). The rats in group 2 (n = 10) underwent laparotomy, 1 h renal ischemia, followed by 1 h of reperfusion and then left nephrectomy was performed. The rats in group 3 (n = 10) underwent laparotomy, 1 h renal ischemia, followed by 24 h of reperfusion and then left nephrectomy was performed. Histopathological findings and the immunohistochemical expression of CD44 in ischemic and reperfused rat kidneys were investigated. In histopathologic evaluation, non-specific changes were observed in group 2 and early phase of IRI were present in group 3. CD44 was expressed in both group 2 and 3 but not in group 1. The mean immunohistochemical staining percentages of rat kidneys in group 1, 2, and 3 were 0.00 +/- 0.00, 39.90 +/- 5.53, and 26.20 +/- 8.38, respectively. The immunohistochemical staining pattern was more dense in group 2 than in group 3 (P < 0.001). In conclusion, the expression of CD44 in renal tubuloepithelial cells was significantly increased after IRI. The increase in CD44 expression was more prominent during the early phase of IRI and started to decline after 24 h of reperfusion.

Pharmacological Preconditioning of Rat Liver by Up-Regulation of Heme Oxygenase 1

Purpose We investigated whether pharmacologically induced up-regulation of heme oxygenase 1 by pyrrolidine dithiocarbamate (PDTC) conferred protection against subsequent ischemia-reperfusion injury (IRI) to the rat liver after temporary vascular occlusion of 70% of the organ. Methods Female Wistar rats (200 to 250 g body weight) anesthetized with pentobarbitone were cannulated in the carotid artery and jugular vein. After laparotomy, a rubber band was applied around the entire vascular supply to the median and left lateral lobes, enabling vascular occlusion of 70% of the liver. A laser Doppler miniprobe was placed on the left lateral lobe to monitor peripheral liver blood flow (PLBF). Immediately upon completion of the surgery, the rats were administered either PDTC (50 mg/kg intravenously; n = 8) or its solvent (isotonic NaCl; n = 8). After 60 minutes, regional ischemia was induced for 30 minutes. The animals were then monitored for 2 hours of reperfusion. Blood samples for alanine transferase (ALT) estimation (as a measure of parenchymal injury) were drawn immediately prior to ischemia and reperfusion, as well as 60 and 120 minutes after reperfusion; PLBF was calculated at these times. Results ALT increased in the course of the experiments but there was no difference between the groups. The reduction in PBLF due to ischemia-reperfusion was significantly lower in the PDTC group: about 16% versus 40%, after 2 hours of reperfusion. Conclusion Pretreatment with PDTC attenuated the disturbance of hepatic microcirculation, but not parenchymal injury, in the early phase of IRI.

Improved lung preservation relates to an increase in tubular myelin-associated surfactant protein A

BackgroundDeclining levels of surfactant protein A (SP-A) after lung transplantation are suggested to indicate progression of ischemia/reperfusion (IR) injury. We hypothesized that the previously described preservation-dependent improvement of alveolar surfactant integrity after IR was associated with alterations in intraalveolar SP-A levels.MethodsUsing immuno electron microscopy and design-based stereology, amount and distribution of SP-A, and of intracellular surfactant phospholipids (lamellar bodies) as well as infiltration by polymorphonuclear leukocytes (PMNs) and alveolar macrophages were evaluated in rat lungs after IR and preservation with EuroCollins or Celsior.ResultsAfter IR, labelling of tubular myelin for intraalveolar SP-A was significantly increased. In lungs preserved with EuroCollins, the total amount of intracellular surfactant phospholipid was reduced, and infiltration by PMNs and alveolar macrophages was significantly increased. With Celsior no changes in infiltration or intracellular surfactant phospholipid amount occurred. Here, an increase in the number of lamellar bodies per cell was associated with a shift towards smaller lamellar bodies. This accounts for preservation-dependent changes in the balance between surfactant phospholipid secretion and synthesis as well as in inflammatory cell infiltration.ConclusionWe suggest that enhanced release of surfactant phospholipids and SP-A represents an early protective response that compensates in part for the inactivation of intraalveolar surfactant in the early phase of IR injury. This beneficial effect can be supported by adequate lung preservation, as e.g. with Celsior, maintaining surfactant integrity and reducing inflammation, either directly (via antioxidants) or indirectly (via improved surfactant integrity).