Reperfusion Phase Research Articles

Tonabersat Prevents Inflammatory Damage in the Central Nervous System by Blocking Connexin43 Hemichannels.

The cis benzopyran compound tonabersat (SB-220453) has previously been reported to inhibit connexin26 expression in the brain by attenuating the p38-mitogen-activated protein kinase pathway. We show here that tonabersat directly inhibits connexin43 hemichannel opening. Connexin43 hemichannels have been called "pathological pores" based upon their role in secondary lesion spread, edema, inflammation, and neuronal loss following central nervous system injuries, as well as in chronic inflammatory disease. Both connexin43 hemichannels and pannexin channels released adenosine triphosphate (ATP) during ischemia in an in vitro ischemia model, but only connexin43 hemichannels contributed to ATP release during reperfusion. Tonabersat inhibited connexin43 hemichannel-mediated ATP release during both ischemia and reperfusion phases, with direct channel block confirmed using electrophysiology. Tonabersat also reduced connexin43 gap junction coupling in vitro, but only at higher concentrations, with junctional plaques internalized and degraded via the lysosomal pathway. Systemic delivery of tonabersat in a rat bright-light retinal damage model (a model for dry age-related macular degeneration) resulted in significantly improved functional outcomes assessed using electroretinography. Tonabersat also prevented thinning of the retina, especially the outer nuclear layer and choroid, assessed using optical coherence tomography. We conclude that tonabersat, already given orally to over 1000 humans in clinical trials (as a potential treatment for, and prophylactic treatment of, migraine because it was thought to inhibit cortical spreading depression), is a connexin hemichannel inhibitor and may have the potential to be a novel treatment of central nervous system injury and chronic neuroinflammatory disease.

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats.

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Ischemic preconditioning with a ketogenic diet improves brain ischemic tolerance through increased extracellular adenosine levels and hypoxia-inducible factors

Ischemic tolerance reduces brain damage and neurological dysfunction after brain ischemia. A ketogenic diet (KD) has disease-modifying effects in several neurodegenerative disorders. In this study, we fed mice with a KD for three weeks and performed reversible middle cerebral artery occlusion (MCAO) in the animals. KD-fed mice had a significantly reduced infarct volume, increased regional cerebral blood flow (rCBF) and extracellular adenosine levels in both the ischemic and the reperfusion phases. In vitro and in vivo experiments revealed that the KD-induced neuroprotection was mediated through the adenosine A1 receptor. The KD increased Akt and ERK1/2 phosphorylation via A1R activation. Besides, the KD also upregulated robustly HIF-1α/HIF-2α and HIF regulated genes, such as VEGF and EPO. A three-week preconditioning period with a KD improved ischemic tolerance in mice with MCAO. The underlying mechanisms might include elevated extracellular adenosine levels, and increased Akt and ERK1/2 phosphorylation via A1 adenosine receptor activation, together with upregulated HIFs and HIF-regulated genes.

Moderate therapeutic hypothermia induces multimodal protective effects in oxygen-glucose deprivation/reperfusion injured cardiomyocytes

ObjectiveTherapeutic hypothermia has been shown to attenuate myocardial cell death due to ischemia/reperfusion injury. However, cellular mechanisms of cooling remain to be elucidated. Especially during reperfusion, mitochondrial dysfunction contributes to cell death by releasing apoptosis inductors. The aim of the present study was to investigate the effects of moderate therapeutic hypothermia (33.5°C) on mitochondrial mediated apoptosis in ischemia/reperfusion-injured cardiomyocytes. MethodsIschemic injury was simulated by oxygen-glucose deprivation for 6h in glucose/serum-free medium at 0.2% O2 in mouse atrial HL-1 cardiomyocytes. Simulation of reperfusion was achieved by restoration of nutrients in complete supplemented medium and incubation at 21% O2. Early application of therapeutic hypothermia, cooling during the oxygen-glucose deprivation phase, was initiated after 3h of oxygen-glucose deprivation and maintained for 24h. Mitochondrial membrane integrity was assessed by cytochrome c and AIF protein releases. Furthermore, mitochondria were stained with MitoTracker Red and intra-cellular cytochrome c localization was visualized by immunofluorescence staining. Moreover, anti-apoptotic Bcl-2 and Hsp70 as well as phagophore promoting LC3-II protein expressions were analyzed by Western-blot analysis. ResultsTherapeutic hypothermia initiated during oxygen-glucose deprivation significantly reduced mitochondrial release of cytochrome c and AIF in cardiomyocytes during reperfusion. Secondly, anti-apoptotic Bcl-2/Bax ratio and Hsp70 protein expressions were significantly upregulated due to hypothermia, indicating an inhibition of both caspase-dependent and -independent apoptosis. Furthermore, cardiomyocytes treated with therapeutic hypothermia showed increased LC3-II protein levels associated with the mitochondria during the first 3h of reperfusion, indicating the initiation of phagophores formation and sequestration of presumably damaged mitochondrion. ConclusionEarly application of therapeutic hypothermia effectively inhibited cardiomyocyte cell death due to oxygen-glucose deprivation/reperfusion-induced injury via multiple pathways. As hypothermia preserved mitochondrial membrane integrity, which resulted in reduced cytochrome c and AIF releases, induction of both caspase-dependent and -independent apoptosis was minimized. Secondly, cooling attenuated intrinsic apoptosis via Hsp70 upregulation and increasing anti-apoptotic Bcl-2/Bax ratio. Moreover, therapeutic hypothermia promoted mitochondrial associated LC3-II during the early phase of reperfusion, possibly leading to the sequestration and degradation of damaged mitochondrion to attenuate the activation of cell death.

Inhibition of Intracellular Type 10 Adenylyl Cyclase Protects Cortical Neurons Against Reperfusion-Induced Mitochondrial Injury and Apoptosis.

Mitochondrial injury significantly contributes to the neuronal death under cerebral ischemia and reperfusion. Within several signaling pathways, cyclic adenosine monophosphate (cAMP) signaling plays a substantial role in mitochondrial injury and cell death. Traditionally, the source of cellular cAMP has been attributed to the membrane-bound adenylyl cyclase, whereas the role of the intracellular localized type 10 soluble adenylyl cyclase (sAC) in neuronal pathology has not been considered. Since neurons express an active form of sAC, we aimed to investigate the role of sAC in reperfusion-induced neuronal apoptosis. For this purpose, the in vitro model of oxygen/glucose deprivation (simulated ischemia, 1h), followed by recovery (simulated reperfusion, 12h) in rat embryonic neurons, was applied. Although ischemia alone had no significant effect on apoptosis, reperfusion led to an activation of the mitochondrial pathway of apoptosis, hallmarked by mitochondrial depolarization, cytochrome c release, and mitochondrial ROS formation. These effects were accompanied by significantly augmented sAC expression and increased cellular cAMP content during reperfusion. Pharmacological suppression of sAC during reperfusion reduced cellular cAMP and ameliorated reperfusion-induced mitochondrial apoptosis and ROS formation. Similarly, sAC knockdown prevented neuronal death. Further analysis revealed a role of protein kinase A (PKA), a major downstream target of sAC, in reperfusion-induced neuronal apoptosis and ROS formation. In conclusion, the results show a causal role of intracellular, sAC-dependent cAMP signaling in reperfusion-induced mitochondrial injury and apoptosis in neurons. The protective effect of sAC inhibition during the reperfusion phase provides a basis for the development of new strategies to prevent the reperfusion-induced neuronal injury.

Remote limb ischemic post conditioning during early reperfusion alleviates cerebral ischemic reperfusion injury via GSK-3β/CREB/ BDNF pathway

Remote limb ischemic post conditioning (RIPOC) has been reported to attenuate cerebral ischemic reperfusion (I/R) injury, while the molecular mechanisms remain elusive. Various studies have highlighted the involvement of glycogen synthase kinase (GSK-3β) in cerebral I/R injury and cognitive disorders. Hence, the present study was designed to explore the role of GSK-3β and its downstream regulators in RIPOC mediated neuroprotection against cerebral I/R injury and associated cognitive impairment. Male Wistar rats are randomly assigned into four groups: Sham, bilateral common carotid arteries occlusion (BCCAO), RIPOC and BCCAO+RIPOC. BCCAO was achieved by transient occlusion of bilateral common carotid arteries for 20min, followed by reperfusion. Non-invasive RIPOC was induced by 3 cycles each of 10min occlusion and reperfusion of both femoral arteries by using tourniquets, during early reperfusion phase. A battery of behavioral and cognitive tests were performed. Biochemical estimation of oxidative markers, anti-oxidants and pro-inflammatory markers were estimated. Levels of GSK-3β, CREB and BDNF were estimated to confirm the molecular mechanism. Hippocampal structural abnormalities were confirmed by H and E staining. The neurobehavioral analysis revealed that neurological and cognitive deficits caused by BCCAO, were reduced by RIPOC intervention. Meanwhile, the results of biochemical tests suggested that RIPOC attenuates the BCCAO induced oxidative damage, neuroinflammation and hippocampal structural abnormalities. Further, RIPOC prevented the elevation of BCCAO induced GSK-3β. RIPOC exerts neuroprotective effect against I/R injury, putatively by attenuating GSK-3β expression and upregulating the levels of CREB and BDNF.

Effects and mechanisms of compound Chinese medicine and major ingredients on microcirculatory dysfunction and organ injury induced by ischemia/reperfusion.

Microcirculation dysfunction and organ injury after ischemia and reperfusion (I/R) result from a complex pathologic process consisting of multiple links, with metabolism impairment in the ischemia phase and oxidative stress in the reperfusion phase as initiators, and any treatment targeting a single link is insufficient to cope with this. Compound Chinese medicine (CCM) has been applied in clinics in China and some Asian nations for >2000years. Studies over the past decades revealed the protective and therapeutic effect of CCMs and major ingredients on I/R-induced microcirculatory dysfunction and tissue injury in the heart, brain, liver, intestine, and so on. CCM contains diverse bioactive components with potential for energy metabolism regulation; antioxidant effect; inhibiting inflammatory cytokines release; adhesion molecule expression in leukocyte, platelet, and vascular endothelial cells; and the protection of thrombosis, albumin leakage, and mast cell degranulation. This review covers the major works with respect to the effects and underlying mechanisms of CCM and its ingredients on microcirculatory dysfunction and organ injury after I/R, providing novel ideas for dealing with this threat.

Cardioprotective Effects of Methanolic Extract of Scrophularia frigida on Ischemia-Reperfusion-Induced Injuries in Isolated Rat Heart.

Myocardial infarction is a common presentation of coronary artery disease and the leading cause of death worldwide. The present study investigated potential resistance to ischemia-reperfusion (I/R) injuries following administration of methanolic (MeOH) extract of Scrophularia frigida (S. frigida) in isolated rat heart. Male Wistar rat hearts (n= 8-10) were isolated and allowed to equilibrate for 20 min, and then subjected to 30 min regional ischemia followed by 120 min reperfusion. In the control group, Krebs-Henseleit (K/H) solution was perfused. However, in the treatment groups K/H solution containing 1, 5, and 10 µg/cc of extract was infused. In addition, total phenol, total flavonoid content and antioxidant property were evaluated and extract was subjected to phytochemical analysis. Administration of extract improved the flow rate, developed pressure as well as max and min left ventricular dp/dt. Number and duration of VT were significantly reduced by all extract concentrations in ischemic phase. In reperfusion phase, significant decreases in single and total arrhythmias were seen. Furthermore, concentrations of 5 and 10 µg/cc of extract remarkably decreased the infarct size. Generally, the methanolic extract of S. frigida exhibited a protective effect against I/R-induced injures, which might be due to the antioxidant activiies of iridoids and phenolics.

Acidotoxicity via ASIC1a Mediates Cell Death during Oxygen Glucose Deprivation and Abolishes Excitotoxicity.

Ischemic reperfusion (I/R) injury is associated with a complex and multifactorial cascade of events involving excitotoxicity, acidotoxicity, and ionic imbalance. While it is known that acidosis occurs concomitantly with glutamate-mediated excitotoxicity during brain ischemia, it remains elusive how acidosis-mediated acidotoxicity interacts with glutamate-mediated excitotoxicity. Here, we investigated the effect of acidosis on glutamate-mediated excitotoxicity in acute hippocampal slices. We tested the hypothesis that mild acidosis protects against I/R injury via modulation of NMDAR, but produces injury via activation of acid sensing ion channels (ASIC1a). Using a novel microperfusion approach, we monitored time course of injury in acutely prepared, adult hippocampal slices. We varied the duration of insult to delay the return to preinsult conditions to determine if injury was caused by the primary insult or by the modeled reperfusion phase. We also manipulated pH in presence and absence of oxygen glucose deprivation (OGD). The role of ASIC1a and NMDAR was deciphered by treating the slices with and without an ASIC or NMDAR antagonist. Our results show that injury due to OGD or low pH occurs during the insult rather than the modeled reperfusion phase. Injury mediated by low pH or low pH OGD requires ASIC1a and is independent of NMDAR activation. These findings point to ASIC1a as a mediator of ischemic cell death caused by stroke and cardiac arrest.

Pre-ischaemic mitochondrial substrate constraint by inhibition of malate-aspartate shuttle preserves mitochondrial function after ischaemia-reperfusion.

Pre-ischaemic administration of aminooxiacetate (AOA), an inhibitor of the malate-aspartate shuttle (MAS), provides cardioprotection against ischaemia-reperfusion injury. The underlying mechanism remains unknown. We examined whether transient inhibition of the MAS during ischaemia and early reperfusion by AOA treatment could prevent mitochondrial damage at later reperfusion. The AOA treatment preserved mitochondrial respiratory capacity with reduced mitochondrial oxidative stress during late reperfusion to the same extent as ischaemic preconditioning (IPC). However, AOA treatment, but not IPC, reduced the myocardial interstitial concentration of tricarboxylic acid cycle intermediates at the onset of reperfusion. The results obtained in the present study demonstrate that metabolic regulation by inhibition of the MAS at the onset of reperfusion may be beneficial for the preservation of mitochondrial function during late reperfusion in an IR-injured heart. Mitochondrial dysfunction plays a central role in ischaemia-reperfusion (IR) injury. Pre-ischaemic administration of aminooxyacetate (AOA), an inhibitor of the malate-aspartate shuttle (MAS), provides cardioprotection against IR injury, although the underlying mechanism remains unknown. We hypothesized that a transient inhibition of the MAS during ischaemia and early reperfusion could preserve mitochondrial function at later phase of reperfusion in the IR-injured heart to the same extent as ischaemic preconditioning (IPC), which is a well-validated cardioprotective strategy against IR injury. In the present study, we show that pre-ischaemic administration of AOA preserved mitochondrial complex I-linked state 3 respiration and fatty acid oxidation during late reperfusion in IR-injured isolated rat hearts. AOA treatment also attenuated the excessive emission of mitochondrial reactive oxygen species during state 3 with complex I-linked substrates during late reperfusion, which was consistent with reduced oxidative damage in the IR-injured heart. As a result, AOA treatment reduced infarct size after reperfusion. These protective effects of MAS inhibition on the mitochondria were similar to those of IPC. Intriguingly, the protection of mitochondrial function by AOA treatment appears to be different from that of IPC because AOA treatment, but not IPC, downregulated myocardial tricarboxilic acid (TCA)-cycle intermediates at the onset of reperfusion. MAS inhibition thus preserved mitochondrial respiratory capacity and decreased mitochondrial oxidative stress during late reperfusion in the IR-injured heart, at least in part, via metabolic regulation of TCA cycle intermediates in the mitochondria at the onset of reperfusion.

Systemic application of carbon monoxide-releasing molecule 3 protects skeletal muscle from ischemia-reperfusion injury

ObjectiveIschemia-reperfusion (IR) is a limb- and life-threatening complication of acute limb ischemia and musculoskeletal trauma. Carbon monoxide-releasing molecules (CORMs) have recently been shown to protect microvascular perfusion and to reduce inflammation and injury in various ischemic animal models. The purpose of this study was to examine the effects of water-soluble CORM-3 on the extent of IR-induced muscle injury. MethodsWistar rats were randomized into three groups: sham (no ischemia), IR + CORM-3 (10 mg/kg intraperitoneally), and IR + inactive CORM-3 (iCORM-3; 10 mg/kg intraperitoneally). No-flow ischemia was induced by the application of a tourniquet to the hind limb for 2 hours; tourniquet release commenced the reperfusion phase. Both CORM-3 and iCORM-3 were injected immediately after tourniquet release. Temporal changes in microvascular perfusion, cellular tissue injury (ethidium bromide and bisbenzimide staining), and inflammatory response (leukocyte recruitment) within the extensor digitorum longus muscle were assessed using intravital video microscopy every 15 minutes for a total of 90 minutes after initiation of reperfusion. Systemic levels of tumor necrosis factor-α were also measured. ResultsHind limb IR resulted in (1) a significant no-reflow phenomenon followed by progressive increase in microvascular perfusion deficit (21% ± 2% continuously perfused capillaries in IR vs 76% ± 4% in sham [P < .001]; 52% ± 8% nonperfused capillaries in IR vs 13% ± 2% in sham at 90 minutes of reperfusion [P < .001]), (2) tissue injury (ethidium bromide and bisbenzimide staining of 0.52 ± 0.07 in IR vs 0.05 ± 0.03 in sham at 90 minutes of reperfusion [P < .001]), (3) leukocyte recruitment (13.7 ± 0.9 adherent leukocytes/30 seconds/1000 μm2 in IR vs 1.8 ± 0.5 adherent leukocytes/30 seconds/1000 μm2 in sham at 90 minutes of reperfusion [P < .001]), and (4) an increase in circulating tumor necrosis factor-α levels. Systemic administration of CORM-3 (but not of iCORM-3) effectively reduced the IR-associated skeletal muscle perfusion deficits, tissue injury, and inflammatory activation. ConclusionsCORM-3 displays potent protective and anti-inflammatory effects in an experimental model of hind limb IR, suggesting a potential therapeutic application of CORMs in treatment of ischemic conditions.

Abstract 146: Transient Receptor Potential Vanilloid 1 (TRPV1) Channel Contributes to Cerebral Reperfusion after Stroke

Introduction: The TRPV1 channel is a non-selective ion channel that is activated by capsaicinoids, temperature (&gt;43 °C), and multiple factors produced during inflammation or injury. At this time, there is very little reported regarding a role of this channel in regulation of cerebral blood flow. Here we provide new evidence for TRPV1 in promoting microvascular reperfusion following stroke. Hypothesis: Endothelial TRPV1 channels contribute to cerebral reperfusion following ischemic stroke. Methods: Experiments were performed in male WT and TRPV1 knockout mice of C57BL/6 background. Immunofluorescence was performed in brain sections with TRPV1 and Pecam-1 antibodies. Pressurized artery preparations were performed with isolated superior cerebellar arteries. Relative cerebral blood flow measurements were performed by laser Doppler and laser speckle flowmetry in mice before, during, and after MCAO (20 or 30 min). Reperfusion was monitored continuously for 2-3 hours and again at 24 hours. Results: TRPV1 expression was positively detected by immunofluorescence in the cerebral microvasculature, as assessed by Pecam-1 co-localization. TRPV1 KO mice were used as a negative control. Pressurized cerebral arteries demonstrated modest vasodilation to TRPV1 agonist (capsaicin) that was significantly potentiated with H 2 O 2 pre-treatment. The potentiated component of vasodilation was lost with removal of the endothelium, demonstrating the endothelial source of the response. Following brief MCA occlusion (20 or 30 min), WT mice demonstrated an initial return of cerebral perfusion followed by a transient drop in perfusion (~25% drop). This drop in perfusion started at 20-30 minutes into the reperfusion phase and lasted approximately one hour, at which point perfusion was restored to pre-stroke baseline value. In TRPV1 KO mice, the drop in perfusion was more profound (~65% drop) and persisted through 3 hours. By 24 hours, flow was restored to baseline in both groups. Conclusions: These data demonstrate a functional role for TRPV1 channels during reperfusion. We speculate that endothelial TRPV1 channels are functionally potentiated during injury and contribute to the vasodilatory capacity of the cerebral microvasculature following stroke.

Lung protection in cardio-pulmonary bypass.

Since the invention of the heart-lung machine paediatric cardiac surgery developed rapidly. For correction of complex cardiac malformations the application of a cardio-pulmonary bypass (CPB) has become indispensable but possible negative effects of this technique should not be neglected. Especially, both bypassed organs i.e. heart and lung are not perfused during the procedure and therefore are threatened by ischemia and reperfusion injury. Additionally, CPB was developed with a non-pulsatile flow but there are clinical observations that pulsatile flow might be superior with improved patient outcomes. Thus, the aim of our study was to evaluate the effect of CPB on lung structure and to assess whether different flow modalities (pulsatile vs. non-pulsatile flow) or application of the antibiotic minocycline might be advantageous. Thirty five piglets of four weeks age were examined and divided into five experimental groups: control (no CPB) without or with minocycline, CPB (non-pulsatile flow) without or with minocycline and CPB with pulsatile flow. CPB was performed for 90 min followed by a 120 min reperfusion and recovery phase. Thereafter, adenosine triphosphate-content of lung biopsies and histology was carried out. We found that CPB was associated with a significant thickening of alveolar wall accompanied by an infiltration of neutrophil leucocytes. Moreover, markers for hypoxia, apoptosis, nitrosative stress, inflammation and DNA damage were significantly elevated after CPB. These cellular damages could be partially inhibited by minocycline or pulsatile flow. Both, minocycline and pulsatile flow attenuate lung damage after CPB.

Intracellular Activation of Complement 3 Is Responsible for Intestinal Tissue Damage during Mesenteric Ischemia.

Intestinal ischemia followed by reperfusion leads to local and remote organ injury attributed to inflammatory response during the reperfusion phase. The extent to which ischemia contributes to ischemia/reperfusion injury has not been thoroughly studied. After careful evaluation of intestinal tissue following 30 min of ischemia, we noticed significant local mucosal injury in wild-type mice. This injury was drastically reduced in C3-deficient mice, suggesting C3 involvement. Depletion of circulating complement with cobra venom factor eliminated, as expected, injury recorded at the end of the reperfusion phase but failed to eliminate injury that occurred during the ischemic phase. Immunohistochemical studies showed that tissue damage during ischemia was associated with increased expression of C3/C3 fragments primarily in the intestinal epithelial cells, suggesting local involvement of complement. In vitro studies using Caco2 intestinal epithelial cells showed that in the presence of LPS or exposure to hypoxic conditions the cells produce higher C3 mRNA as well as C3a fragment. Caco2 cells were also noted to produce cathepsins B and L, and inhibition of cathepsins suppressed the release of C3a. Finally, we found that mice treated with a cathepsin inhibitor and cathepsin B-deficient mice suffer limited intestinal injury during the ischemic phase. To our knowledge, our findings demonstrate for the first time that significant intestinal injury occurs during ischemia prior to reperfusion and that this is due to activation of C3 within the intestinal epithelial cells in a cathepsin-dependent manner. Modulation of cathepsin activity may prevent injury of organs exposed to ischemia.

Antiphospholipid antibodies enhance rat neonatal cardiomyocyte apoptosis in an in vitro hypoxia/reoxygenation injury model via p38 MAPK

A significant amount of myocardial damage during a myocardial infarction (MI) occurs during the reperfusion stage, termed ischaemia/reperfusion (I/R) injury, and accounts for up to 50% of total infarcted tissue post-MI. During the reperfusion phase, a complex interplay of multiple pathways and mechanisms is activated, which ultimately leads to cell death, primarily through apoptosis. There is some evidence from a lupus mouse model that lupus IgG, specifically the antiphospholipid (aPL) antibody subset, is pathogenic in mesenteric I/R injury. Furthermore, it has previously been shown that the immunodominant epitope for the majority of circulating pathogenic aPLs resides in the N-terminal domain I (DI) of beta-2 glycoprotein I (β2GPI). This study describes the enhanced pathogenic effect of purified IgG derived from patients with lupus and/or the antiphospholipid syndrome in a cardiomyocyte H/R in vitro model. Furthermore, we have demonstrated a pathogenic role for aPL containing samples, mediated via aPL–β2GPI interactions, resulting in activation of the pro-apoptotic p38 MAPK pathway. This was shown to be inhibited using a recombinant human peptide of domain I of β2GPI in the fluid phase, suggesting that the pathogenic anti-β2GPI antibodies in this in vitro model target this domain.

The protection effects of (1E,6E)-1,7-diphenylhepta-1,6-diene-3,5-dione, a curcumin analogue, against operative liver injury in rats

The relationship between the chemistry characteristic and the hepatoprotective effects of (1E,6E)-1,7-diphenylhepta-1,6-diene-3,5-dione (DDD), a curcumin analogue, in operative liver injury rats was investigated to reveal the mechanism of hepatic protection effects of DDD. DDD (1.2–4.8mmol/kg) was administrated 10min before reperfusion phase in hepatic ischemia-reperfusion injury (IRI) rats. DDD (4.8mmol/kg) administrated 10min before ischemia and N-acetylcysteine (NAC) (4.8mmol/kg) administrated 10min before reperfusion were included for comparative studies. The plasma liver enzyme activities, histopathological indices and markers of lipid peroxide were determined to evaluate the hepatic protection effects. Effects of DDD on succinate dehydrogenase (SDH) activity were also investigated. DDD showed dose-dependent hepatocyte protections when administrated 10min before reperfusion stages in hepatic IRI rats. DDD showed almost equivalent hepatoprotective effects when administrated 10min before ischemia phase demonstrating that DDD acted on the reperfusion stages selectively against the hepatic IRI, instead of ischemia phase. NAC was not effective against hepatic IRI when treated 10min before reperfusion because of the higher pKa of NAC. In additional, DDD had no effect on the SDH both in hepatic IRI rats and in mitochondria. In conclusion, DDD had dose-dependent hepatocyte protections in the reperfusion stages in hepatic IRI rats, while the observed hepatocyte protections of DDD did not involve SDH activities. β-Diketone structures of DDD were crucial for the hepatocyte protections. The abilities of DDD to clear up the unsaturated aldehydes related with the enolate nucleophilicity and the pKa. DDD might be a promising candidate to treat hepatic IRI.

Inorganic nitrite modulates miRNA signatures in acute myocardial in vivo ischemia/reperfusion

Acute myocardial infarction is the leading cause of mortality in the industrialized world. While it is essential to attempt an early reperfusion of ischemic myocardial territories, reperfusion itself adds damage to the heart, the ischemia–reperfusion (I/R) injury. Particularly the injury resulting from the very first minutes of reperfusion remains incompletely understood. MicroRNAs (miRNAs) are dynamic regulators in I/R injury. Nitric oxide (•NO) signaling, in turn, interacts with miRNA signaling. Our previous investigations showed that •NO signaling in I/R could be modulated by nitrite. We therefore sought to investigate the role of miRNAs in nitrite cardioprotection with focus on the first few minutes of reperfusion. The study was conducted in mice in vivo with 30 min of ischemia and 5 min of reperfusion. Mice received a single-dose of nitrite or saline intracardially 5 min prior to reperfusion. We identified nine miRNAs to be up-regulated after 5 min of reperfusion. The up-regulation of almost half of those miRNAs (miR-125a-5p, miR-146b, miR-339-3p, miR-433) was inhibited by nitrite treatment, perpetuating baseline values. In silico analysis revealed the Irak-M gene to be a target of miR-146b and miR-339-3p. Correspondingly, a rise in Irak-M transcript and protein levels occurred by nitrite treatment within the early phase of reperfusion. The results demonstrate that already a very short phase of reperfusion is sufficient for significant dysregulation in cardiac miRNAs expression and that nitrite preserves baseline values of miRNAs in the scale of only a few minutes. These findings hint at a potential novel cardioprotective mechanism of nitrite signaling.

Baicalein Preventive Treatment Confers Optimal Cardioprotection by PTEN/Akt/NO Activation.

Baicalein is a flavonoid with excellent oxidant scavenging capability. It has been reported to protect against a variety of oxidative injuries including ischemia/reperfusion (I/R). However, the optimal treatment strategy for I/R injury and the protective mechanisms are not fully understood. In this study we employed an established chick cardiomyocyte model of I/R and investigated the effects of three baicalein treatment strategies on reactive oxygen species (ROS) scavenging, nitric oxide (NO) production and cell viability. The molecular signaling pathways were also explored. Compared to the I/R control (cell death 52.2[Formula: see text][Formula: see text][Formula: see text]2.0%), baicalein preventive treatment (25[Formula: see text][Formula: see text]M, pretreated for 72[Formula: see text]h and continued through I/R) conferred the best protection (19.5[Formula: see text][Formula: see text][Formula: see text]3.9%, [Formula: see text]), followed by I/R treatment (treated during I/R) and reperfusion treatment (treated at reperfusion only). Preventive and I/R treatments almost completely abolished ROS generation during both ischemic and reperfusion phases, and increased NO production and Akt phosphorylation. Reperfusion treatment reduced the ROS burst in the early reperfusion phase only, and had no effect on NO production and Akt activation. Further, the phosphorylation of phosphatase and tensin homolog (PTEN), a phosphatase negatively regulating Akt activation, was significantly increased by baicalein preventive treatment and slightly by the I/R treatment. PTEN protein expression was reduced in the same trend accordingly. Baicalein reperfusion treatment had no effects on PTEN phosphorylation and expression. Our results indicate that baicalein preventive treatment confers optimal cardioprotection against I/R injury, and this protection involves effective oxidant scavenging and the activation of PTEN/Akt/NO pathway.

Dexrazoxane Shows No Protective Effect in the Acute Phase of Reperfusion during Myocardial Infarction in Pigs.

Calcium and iron overload participate in the mechanisms of ischemia/reperfusion (I/R) injury during myocardial infarction (MI). Calcium overload induces cardiomyocyte death by hypercontraction, while iron catalyses generation of reactive oxygen species (ROS). We therefore hypothesized that dexrazoxane, an intracellular metal chelator, would attenuate I/R injury. MI was induced in pigs by occlusion of the left anterior descending artery for 1 hour followed by 2 hours reperfusion. Thirty minutes before reperfusion either 5 mg/ml dexrazoxane (n = 5) or saline (n = 5) was infused intravenously. Myocardial necrosis as percentage of the area at ischemic risk was found to be similar in both groups (77.2 ± 18% for dexrazoxane and 76.4 ± 14% for saline group) as determined by triphenyl tetrazolium chloride staining of the ischemic myocardium. Also, serum levels of troponin-I were similar in both groups. A conductance catheter was used to measure left ventricular pressure and volume at all times. Markers for tissue damage due to ROS (HNE), endothelial cell activation (CD31) and inflammation (IgG, C3b/c, C5b9, MCP-1) were assessed on tissue and/or in serum. No significant differences were observed between the groups for the parameters analyzed. To conclude, in this clinically relevant model of early reperfusion after acute myocardial ischemia, dexrazoxane lacked attenuating effects on I/R injury as shown by the measured parameters.