Ischemic post-conditioning attenuates the intestinal injury induced by limb ischemia/reperfusion in rats

The purpose of this study was to investigate the protective effects of ischemic post-conditioning on damage to the barrier function of the small intestine caused by limb ischemia-reperfusion injury. Male Wistar rats were randomly divided into 3 groups (N = 36 each): sham operated (group S), lower limb ischemia-reperfusion (group LIR), and post-conditioning (group PC). Each group was divided into subgroups (N = 6) according to reperfusion time: immediate (0 h; T1), 1 h (T2), 3 h (T3), 6 h (T4), 12 h (T5), and 24 h (T6). In the PC group, 3 cycles of reperfusion followed by ischemia (each lasting 30 s) were applied immediately. At all reperfusion times (T1-T6), diamine oxidase (DAO), superoxide dismutase (SOD), and myeloperoxidase (MPO) activity, malondialdehyde (MDA) intestinal tissue concentrations, plasma endotoxin concentrations, and serum DAO, tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) concentrations were measured in sacrificed rats. Chiu’s pathology scores for small intestinal mucosa were determined under a light microscope and showed that damage to the small intestinal mucosa was lower in group PC than in group LIR. In group PC, tissue DAO and SOD concentrations at T2 to T6, and IL-10 concentrations at T2 to T5 were higher than in group LIR (P < 0.05); however, tissue MPO and MDA concentrations, and serum DAO and plasma endotoxin concentrations at T2 to T6, as well as TNF-α at T2 and T4 decreased significantly (P < 0.05). These results show that ischemic post-conditioning attenuated the permeability of the small intestines after limb ischemia-reperfusion injury. The protective mechanism of ischemic post-conditioning may be related to inhibition of oxygen free radicals and inflammatory cytokines that cause organ damage.

Pkd1 Haploinsufficiency Increases Renal Damage and Induces Microcyst Formation following Ischemia/Reperfusion

Mutations in PKD1 cause the majority of cases of autosomal dominant polycystic kidney disease (ADPKD). Because polycystin 1 modulates cell proliferation, cell differentiation, and apoptosis, its lower biologic activity observed in ADPKD might influence the degree of injury after renal ischemia/reperfusion. We induced renal ischemia/reperfusion in 10- to 12-wk-old male noncystic Pkd1(+/-) and wild-type mice. Compared with wild-type mice, heterozygous mice had higher fractional excretions of sodium and potassium and higher serum creatinine after 48 h. In addition, in heterozygous mice, also cortical damage, rates of apoptosis, and inflammatory infiltration into the interstitium at time points out to 14 d after injury all increased, as well as cell proliferation at 48 h and 7 d. The mRNA and protein expression of p21 was lower in heterozygous mice than wild-type mice at 48 h. After 6 wk, we observed dilated tubules, microcysts, and increased renal fibrosis in heterozygotes. The early mortality of heterozygotes was significantly higher than that of wild-type mice when we extended the duration of ischemia from 32 to 35 min. In conclusion, ischemia/reperfusion induces a more severe injury in kidneys of Pkd1-haploin-sufficient mice, a process that apparently depends on a relative deficiency of p2l activity, tubular dilation, and microcyst formation. These data suggest the possibility that humans with ADPKD from PKD1 mutations may be at greater risk for damage from renal ischemia/reperfusion injury.

Effects of Partial Liver Ischemia Followed by Global Liver Reperfusion on the Remote Tissue Expression of Nitric Oxide Synthase: Lungs and Kidneys

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 1 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.

Clinical Outcomes and Genetic Expression Profile in Human Liver Graft Dysfunction During Ischemia/Reperfusion Injury

Introduction. This study aims to compare the molecular gene expression during ischemia reperfusion injury. Several surgical times were considered: in the beginning of the harvesting (T0), at the end of the cold ischemia period (T1), and after reperfusion (T2) and compared with graft dysfunction after liver transplant (OLT). Methods. We studied 54 patients undergoing OLT. Clinical, laboratory data, and histologic data (Suzuki classification) as well as the Survival Outcomes Following Liver Transplantation (SOFT) score were used and compared with the molecular gene expression of the following genes: Interleukin (IL)-1b, IL-6, tumor necrosis factor-a, perforin, E-selectin (SELE), Fas-ligand, granzyme B, heme oxygenase-1, and nitric oxide synthetase. Results. Fifteen patients presented with graft dysfunction according to SOFT criteria. No relevant data were obtained by comparing the variables graft dysfunction and histologic variables. We observed a statistically significant relation between SELE at T0 (P ¼ .013) and IL-1b at T0 (P ¼ .028) and early graft dysfunction. Conclusions. We conclude that several genetically determined proinflammatory expressions may play a critical role in the development of graft dysfunction after OLT.

Role of mitochondria in ischemia/reperfusion mediated brain injury : mechanisms and pharmacology interventions

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2004

Mitochondrial reactive oxygen species generation triggers inflammatory response and tissue injury associated with hepatic ischemia-reperfusion: Therapeutic potential of mitochondrially targeted antioxidants.

Mitochondrial reactive oxygen species generation has been implicated in the pathophysiology of ischemia-reperfusion (I/R) injury; however, its exact role and its spatial-temporal relationship with inflammation are elusive. Herein we explore the spatial-temporal relationship of oxidative/nitrative stress and inflammatory response during the course of hepatic I/R and the possible therapeutic potential of mitochondrial-targeted antioxidants, using a mouse model of segmental hepatic ischemia-reperfusion injury. Hepatic I/R was characterized by early (at 2h of reperfusion) mitochondrial injury, decreased complex I activity, increased oxidant generation in the liver or liver mitochondria, and profound hepatocellular injury/dysfunction with acute proinflammatory response (TNF-α, MIP-1α/CCL3, MIP-2/CXCL2) without inflammatory cell infiltration, followed by marked neutrophil infiltration and a more pronounced secondary wave of oxidative/nitrative stress in the liver (starting from 6h of reperfusion and peaking at 24h). Mitochondrially targeted antioxidants, MitoQ or Mito-CP, dose-dependently attenuated I/R-induced liver dysfunction, the early and delayed oxidative and nitrative stress response (HNE/carbonyl adducts, malondialdehyde, 8-OHdG, and 3-nitrotyrosine formation), and mitochondrial and histopathological injury/dysfunction, as well as delayed inflammatory cell infiltration and cell death. Mitochondrially generated oxidants play a central role in triggering the deleterious cascade of events associated with hepatic I/R, which may be targeted by novel antioxidants for therapeutic advantage.

Preconditioning with Triiodothyronine Improves the Clinical Signs and Acute Tubular Necrosis Induced by Ischemia/Reperfusion in Rats.

BACKGROUND Renal ischemia/reperfusion (I/R) injury is manifested by acute renal failure (ARF) and acute tubular necrosis (ATN). The aim of this study was to evaluate the effectiveness of preconditioning with 3, 3, 5 triiodothyronine (T3) to prevent I/R renal injury. METHODOLOGY/PRINCIPAL FINDINGS THE RATS WERE DIVIDED INTO FOUR GROUPS: sham-operated, placebo-treated (SO-P), sham-operated T3- treated (SO- T3), I/R-injured placebo-treated (IR-P), and I/R-injured T3-treated (IR- T3) groups. At 24 h before ischemia, the animals received a single dose of T3 (100 μg/kg). Renal function and plasma, urinary, and tissue variables were studied at 4, 24, and 48 h of reperfusion, including biochemical, oxidative stress, and inflammation variables, PARP-1 immunohistochemical expression, and ATN morphology. In comparison to the SO groups, the IR-P groups had higher plasma urea and creatinine levels and greater proteinuria (at all reperfusion times) and also showed: increased oxidative stress-related plasma, urinary, and tissue variables; higher plasma levels of IL6 (proinflammatory cytokine); increased glomerular and tubular nuclear PARP-1 expression; and a greater degree of ATN. The IR-T3 group showed a marked reduction in all of these variables, especially at 48 h of reperfusion. No significant differences were observed between SO-P and SO-T3 groups. CONCLUSIONS This study demonstrates that preconditioning rats with a single dose of T3 improves the clinical signs and ATN of renal I/R injury. These beneficial effects are accompanied by reductions in oxidative stress, inflammation, and renal PARP-1 expression, indicating that this sequence of factors plays an important role in the ATN induced by I/R injury.

Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death.

Ischemia/reperfusion (I/R) is a pivotal mechanism of liver damage after liver transplantation or hepatic surgery. We have investigated the effects of cannabidiol (CBD), the nonpsychotropic constituent of marijuana, in a mouse model of hepatic I/R injury. I/R triggered time-dependent increases/changes in markers of liver injury (serum transaminases), hepatic oxidative/nitrative stress (4-hydroxy-2-nonenal, nitrotyrosine content/staining, and gp91phox and inducible nitric oxide synthase mRNA), mitochondrial dysfunction (decreased complex I activity), inflammation (tumor necrosis factor α (TNF-α), cyclooxygenase 2, macrophage inflammatory protein-1α/2, intercellular adhesion molecule 1 mRNA levels; tissue neutrophil infiltration; nuclear factor κB (NF-κB) activation), stress signaling (p38MAPK and JNK), and cell death (DNA fragmentation, PARP activity, and TUNEL). CBD significantly reduced the extent of liver inflammation, oxidative/nitrative stress, and cell death and also attenuated the bacterial endotoxin-triggered NF-κB activation and TNF-α production in isolated Kupffer cells, likewise the adhesion molecule expression in primary human liver sinusoidal endothelial cells stimulated with TNF-α and attachment of human neutrophils to the activated endothelium. These protective effects were preserved in CB(2) knockout mice and were not prevented by CB(1/2) antagonists in vitro. Thus, CBD may represent a novel, protective strategy against I/R injury by attenuating key inflammatory pathways and oxidative/nitrative tissue injury, independent of classical CB(1/2) receptors.

Myocardial tolerance to ischemia-reperfusion injury, training intensity and cessation.

Training has been shown to induce cardioprotection. The mechanisms involved remain still poorly understood. Aims of the study were to examine the relevance of training intensity on myocardial protection against ischemia/reperfusion (I/R) injury, and to which extent the beneficial effects persist after training cessation in rats. Sprague-Dawley rats trained at either low (60% [Formula: see text]) or high (80% [Formula: see text]) intensity for 10 weeks. An additional group of highly trained rats was detrained for 4 weeks. Untrained rats served as controls. At the end of treatment, rats of all groups were split into two subgroups. In the former, rats underwent left anterior descending artery (LAD) ligature for 30 min, followed by 90-min reperfusion, with subsequent measurement of the infarct size. In the latter, biopsies were taken to measure heat-shock proteins (HSP) 70/72, vascular endothelial growth factor (VEGF) protein levels, and superoxide dismutase (SOD) activity. Training reduced infarct size proportionally to training intensity. With detraining, infarct size increased compared to highly trained rats, maintaining some cardioprotection with respect to controls. Cardioprotection was proportional to training intensity and related to HSP70/72 upregulation and Mn-SOD activity. The relationship with Mn-SOD was lost with detraining. VEGF protein expression was not affected by either training or detraining. Stress proteins and antioxidant defenses might be involved in the beneficial effects of long-term training as a function of training intensity, while HSP70 may be one of the factors accounting for the partial persistence of myocardial protection against I/R injury in detrained rats.

JNK Inhibition Reduced Retinal Ganglion Cell Death after Ischemia/Reperfusion In Vivo and after Hypoxia In Vitro.

Mitogen-activated protein kinases (MAPKs) are key regulators that have been linked to cell survival and death. Among the main classes of MAPKs, c-jun N-terminal kinase (JNK) has been shown to mediate cell stress responses associated with apoptosis. In Vitro, hypoxia induced a significant increase in 661W cell death that paralleled increased activity of JNK and c-jun. 661W cells cultured in presence of the inhibitor of JNK (D-JNKi) were less sensitive to hypoxia-induced cell death. In vivo, elevation in intraocular pressure (IOP) in the rat promoted cell death that correlated with modulation of JNK activation. In vivo inhibition of JNK activation with D-JNKi resulted in a significant and sustained decrease in apoptosis in the ganglion cell layer, the inner nuclear layer and the photoreceptor layer. These results highlight the protective effect of D-JNKi in ischemia/reperfusion induced cell death of the retina.

Mitochondrial K+ transport and cardiac protection during ischemia/reperfusion

Mitochondrial ion transport, oxidative phosphorylation, redox balance, and physical integrity are key factors in tissue survival following potentially damaging conditions such as ischemia/reperfusion. Recent research has demonstrated that pharmacologically activated inner mitochondrial membrane ATP-sensitive K+ channels (mitoK ATP) are strongly cardioprotective under these conditions. Furthermore, mitoK ATP are physiologically activated during ischemic preconditioning, a procedure which protects against ischemic damage. In this review, we discuss mechanisms by which mitoK ATP may be activated during preconditioning and the mitochondrial and cellular consequences of this activation, focusing on end-effects which may promote ischemic protection. These effects include decreased loss of tissue ATP through reverse activity of ATP synthase due to increased mitochondrial matrix volumes and lower transport of adenine nucleotides into the matrix. MitoK ATP also decreases the release of mitochondrial reactive oxygen species by promoting mild uncoupling in concert with K+/H+ exchange. Finally, mitoK ATP activity may inhibit mitochondrial Ca2+ uptake during ischemia, which, together with decreased reactive oxygen release, can prevent mitochondrial permeability transition, loss of organelle function, and loss of physical integrity. We discuss how mitochondrial redox status, K+ transport, Ca2+ transport, and permeability transitions are interrelated during ischemia/reperfusion and are determinant factors regarding the extent of tissue damage.

CPU0213, a novel endothelin type A and type B receptor antagonist, protects against myocardial ischemia/reperfusion injury in rats

The efficacy of endothelin receptor antagonists in protecting against myocardial ischemia/reperfusion (I/R) injury is controversial, and the mechanisms remain unclear. The aim of this study was to investigate the effects of CPU0123, a novel endothelin type A and type B receptor antagonist, on myocardial I/R injury and to explore the mechanisms involved. Male Sprague-Dawley rats weighing 200-250 g were randomized to three groups (6-7 per group): group 1, Sham; group 2, I/R + vehicle. Rats were subjected to in vivo myocardial I/R injury by ligation of the left anterior descending coronary artery and 0.5% sodium carboxymethyl cellulose (1 mL/kg) was injected intraperitoneally immediately prior to coronary occlusion. Group 3, I/R + CPU0213. Rats were subjected to identical surgical procedures and CPU0213 (30 mg/kg) was injected intraperitoneally immediately prior to coronary occlusion. Infarct size, cardiac function and biochemical changes were measured. CPU0213 pretreatment reduced infarct size as a percentage of the ischemic area by 44.5% (I/R + vehicle: 61.3 ± 3.2 vs I/R + CPU0213: 34.0 ± 5.5%, P < 0.05) and improved ejection fraction by 17.2% (I/R + vehicle: 58.4 ± 2.8 vs I/R + CPU0213: 68.5 ± 2.2%, P < 0.05) compared to vehicle-treated animals. This protection was associated with inhibition of myocardial inflammation and oxidative stress. Moreover, reduction in Akt (protein kinase B) and endothelial nitric oxide synthase (eNOS) phosphorylation induced by myocardial I/R injury was limited by CPU0213 (P < 0.05). These data suggest that CPU0123, a non-selective antagonist, has protective effects against myocardial I/R injury in rats, which may be related to the Akt/eNOS pathway.