Protective Effect Of Ischemic Preconditioning Research Articles

Molecular mechanism underlying the protective effects of ischemic preconditioning in total knee arthroplasty

ProposeTo investigate the molecular mechanisms underlying the protective effects of ischemic preconditioning (IPC) in patients undergoing total knee arthroplasty. MethodsGSE21164 was extracted from an online database, followed by an investigation of differentially expressed genes (DEGs) between IPC treatment samples at 2 time points (T0T and T1T). Function and pathway enrichment analyses were performed on the DEGs. A protein-protein interaction network was constructed to identify hub genes according to 5 different algorithms, followed by enrichment analysis. In addition, long noncoding RNAs (lncRNAs) were identified between the T0T and T1T samples. Furthermore, a competing endogenous RNA network was predicted based on the identified lncRNA-messenger RNA (mRNA), lncRNA-microRNA (miRNA), and mRNA-miRNA relationships revealed in this study. Finally, a drug-gene network was investigated. Statistical analyses were performed using GraphPad Prism 8.0. Differences between groups were determined using an unpaired t-test. p < 0.05 was considered significant. ResultsA total of 343 DEGs at T0 and 10 DEGs at T1 were identified and compared with their respective control groups, followed by 100 DEGs between T0T and T1T. Based on these 100 DEGs, protein-protein interaction network analysis revealed 9 hub genes, mainly with mitochondria-related functions and the carbon metabolism pathway. Six differentially expressed lncRNAs were investigated between T0T and T1T. A competing endogenous RNA network was constructed using 259 lncRNA–miRNA–mRNA interactions, including alpha-2-macroglobulin antisense RNA 1-miR-7161-5p-iron-sulfur cluster scaffold. Finally, 13 chemical drugs associated with the hub genes were explored. ConclusionIron-sulfur cluster scaffold may promote IPC-induced ischemic tolerance mediated by alpha-2-macroglobulin antisense RNA 1-miR-7161-5p axis. Moreover, IPC may induce a protective response after total knee arthroplasty via mitochondria-related functions and the carbon metabolism pathway, which should be further validated in the near future.

Inhibition of the protective effects of preconditioning in ischemia–reperfusion injury by chronic methadone: the role of pAkt and pSTAT3

Cardiac ischemic preconditioning (Pre) reduces cardiac ischemia–reperfusion injury (IRI) by stimulating opioid receptors. Chronic use of opioids can alter the signaling pathways. We investigated the effects of chronic methadone use on IRI and Pre. The experiments were performed on isolated hearts of male Wistar rats in four groups: IRI, Methadone + IRI (M-IRI), Pre + IRI (Pre-IRI), Methadone + Pre + IRI (M-Pre-IRI). The infarct size (IS) in the Pre-IRI group was smaller than the IRI group (26.8% vs. 47.8%, P < 0.05). In the M-IRI and M-Pre-IRI groups, the infarct size was similar to the IRI group. Akt (Ak strain transforming) phosphorylation in the Pre-IRI, M-IRI, and M-Pre-IRI groups was significantly higher than in the IRI group (0.56 ± 0.15, 0.63 ± 0.20, and 0.93 ± 0.18 vs 0.28 ± 0.17 respectively). STAT3 (signal transducer and activator of transcription 3) phosphorylation in the Pre-IRI and M-Pre-IRI groups (1.38 ± 0.14 and 1.46 ± 0.33) was significantly higher than the IRI and M-IRI groups (0.99 ± 0.1 and 0.98 ± 0.2). Thus, chronic use of methadone not only has no protective effect against IRI but also destroys the protective effects of ischemic preconditioning. This may be due to the hyperactivation of Akt and changes in signaling pathways.

Hippocampal metabolic recovery as a manifestation of the protective effect of ischemic preconditioning in rats

The ever-present risk of brain ischemic events in humans and its full prevention make the detailed studies of an organism's response to ischemia at different levels essential to understanding the mechanism of the injury as well as protection. We used the four-vessel occlusion as an animal model of forebrain ischemia to investigate its impact on the metabolic alterations in both the hippocampus and the blood plasma to see changes on the systemic level. By inducing sublethal ischemic stimuli, we focused on the endogenous phenomena known as ischemic tolerance. NMR spectroscopy was used to analyze relative metabolite levels in tissue extracts from rats' hippocampus and blood plasma in three various ischemic/reperfusion times: 3 h, 24 h, and 72 h. Hippocampal tissues were characterized by postischemically decreased glutamate and GABA (4-aminobutyrate) tissue content balanced with increased glutamine level, with most pronounced changes at 3 h reperfusion time. Glutamate (as well as glutamine) levels recovered towards the control levels on the third day, as if the glutamate re-synthesis would be firstly preferred before GABA. These results are indicating the higher feasibility of re-establishing of glutamatergic transmission three days after an ischemic event, in contrast to GABA-ergic. Tissue levels of N-acetylaspartate (NAA), as well as choline, were decreased without the tendency to recover three days after the ischemic event. Metabolomic analysis of blood plasma revealed that ischemically preconditioned rats, contrary to the non-preconditioned animals, did not show hyperglycemic conditions. Ischemically induced semi-ketotic state, manifested in increased plasma ketone bodies 3-hydroxybutyrate and acetoacetate, seems to be programmed to support the brain tissue revitalization after the ischemic event. These and other metabolites changes found in blood plasma as well as in the hippocampus were observed to a lower extent or recovered faster in preconditioned animals. Some metabolomic changes in hippocampal tissue extract were so strong that even single metabolites were able to differentiate between ischemic, ischemically preconditioned, and control brain tissues.

Integrative analysis of the roles of lncRNAs and mRNAs in ischaemic preconditioning to alleviate liver ischaemia–reperfusion injury in mice

The objective of our study was to elucidate the possible underlying mechanism for the protective effect of ischaemic preconditioning (IPC) against ischaemia-reperfusion (I/R) injury and to provide new research perspectives of long non-coding RNAs (lncRNAs). In this study, serum and liver tissue samples were collected to measure indexes of liver injury from a mouse liver model in sham, I/R injury and I/R + IPC groups. Furthermore, liver samples from 5 randomly selected mice per group were extracted and subjected to the microarray and subsequent bioinformatics analysis. IPC ameliorated liver damage by lowered liver transaminase levels and pro-inflammatory cytokines. A total of 167 lncRNAs and 108 messenger RNAs (mRNAs) were significantly differentially expressed genes (DEGs) between the I/R + IPC and I/R groups. Gene Ontology (GO) analysis revealed that these genes were mainly related to unfolded proteins, responses to topologically incorrect proteins, responses to temperature stimuli, protein folding and protein refolding. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis indicated that the DEGs were enriched in the following pathways: protein processing in the endoplasmic reticulum; antigen processing and presentation; and fructose and mannose metabolism. Additionally, the 7 selected DEGs (Hspa1ab, Chka, Clec2h, Mvd, Adra1a, AK085737 and AK088966) were validated in modules of the lncRNA-mRNA weighted coexpression network, which agreed with the qRT–PCR and chip data. And the identified differentially expressed lncRNAs and mRNAs may provide new clues to understand the pivotal pathophysiological mechanism by which IPC alleviates I/R-caused liver damage.

Glutathione S-Transferase and Clusterin, New Players in the Ischemic Preconditioning Renal Protection in a Murine Model of Ischemia and Reperfusion.

Renal ischemia and reperfusion injury (IRI) involves oxidative stress, disruption of microvasculature due to endothelial cell damage, loss of epithelial cell polarity secondary to cytoskeletal alterations, inflammation, and the subsequent transition into a mesenchymal phenotype. Ischemic preconditioning (IPC) has been proposed as a therapeutic strategy to avoid/ameliorate the IRI. Since previous results showed that IPC could have differential effects in kidney cortex vs. kidney medulla, in the present study we analyzed the effectiveness and molecular mechanisms implicated in IPC in both kidney regions. We evaluated 3 experimental groups of BALB/c male mice: control (sham surgery); renal ischemia (30 min) by bilateral occlusion of the renal pedicle and reperfusion (48 hours) (I/R); and renal IPC (two cycles of 5 min of ischemia and 5 min of reperfusion) applied just before I/R. Acute kidney injury was evaluated by glomerular filtration rate (GFR), Neutrophil Gelatinase-Associated Lipocalin (NGAL) blood level, and histologic analysis. Oxidative stress was studied measurement the Glutathione S-Transferase (GST) activity, GSH/GSSG ratio, and lipoperoxidation levels. Inflammatory mediators (IL-1β, IL-6, Foxp3, and IL-10) were quantified by qRT-PCR. The endothelial (PECAM-1), epithelial (AQP-1), mesenchymal (Vimentin, Fascin, and Hsp47), iNOS, clusterin, and Hsp27 expression were evaluated (qRT-PCR and/or Western blot). The IPC protocol prevented the decrease of GFR, reduced the plasma NGAL, and ameliorated morphological damage in the kidney cortex after I/R. The IPC also prevented the downregulation of GST activity, lipoperoxidation and ameliorated the oxidized glutathione. In addition, IPC prevented the upregulation of vimentin, fascin, and Hsp47, which was associated with the prevention of the downregulation of AQP1 after I/R. The protective effect of IPC was associated with the upregulation of Hsp27, Foxp3, and IL-10 expression in the renal cortex. However, the upregulation of iNOS, IL-1β, IL-6, and clusterin by I/R were not modified by IPC. IPC conferred better protection in the kidney cortex as compared to the kidney medulla. The protective effect of IPC was associated with amelioration of oxidative stress, tubular damage, and the induction of markers of Treg lymphocytes activity in the cortical region. Further studies are needed to evaluate if lower tubular cell stress/damage after I/R may explain the preferential induction of Treg response in the kidney cortex induced by IPC.

Effect of ischemic preconditioning on radiation damage to the submandibular gland in rats.

To investigate the effects of radiation on rat submandibular glands and the possible protective effects of ischemic preconditioning, the submandibular glands of Wistar rats were subjected to in situ radiation after ischemic preconditioning. The glands were exposed to X-radiation at a single dose of 20Gy. Ischemic preconditioning was achieved by three min of ischemia and three min of reperfusion, repeated three times before irradiation. Salivary secretion, histological changes, alterations in tight junctions, and the levels of oxidative stress, pro-inflammatory cytokines, and water secretion proteins mediated by the muscarinic acetylcholine M3 subtype receptor were determined at 1 and 12weeks post-irradiation. In glands subjected to irradiation only, the secretion, superoxide dismutase activity, tight junction width, acinar cell number, and M3 receptor and aquaporin-5 levels were lower at 1 and 12weeks than seen in the ischemically preconditioned irradiated glands. In contrast, tumor necrosis factor-α, malondialdehyde, myeloperoxidase activity, and the expression of the tight junction protein claudin-4 were significantly higher in the irradiated only glands. Our study revealed that radiation caused a series of injury-stress responses, especially damage to the water secretion pathway mediated by the M3 receptor that ultimately led to hyposecretion, which might play an important role in the dysfunction of the irradiated only glands. Ischemic preconditioning reduced the radiation-induced injury to submandibular glands and ameliorated salivary hyposecretion.

Ischaemic preconditioning-induced serum exosomes protect against myocardial ischaemia/reperfusion injury in rats by activating the PI3K/AKT signalling pathway.

Ischaemia/reperfusion (I/R) injury can lead to severe arrhythmia and aggravate myocardial damage. Exosomes are small-membrane vesicles that play a protective role in myocardial I/R injury. This study aimed to explore the protective effects of ischaemic preconditioning (IPC)-induced serum exosomes (IPC-Exo) on myocardial I/R injury in rats and its underlying mechanism. Serum exosomes were extracted from IPC rats and quantified using a bicinchoninic acid assay kit. IPC-Exo (50 μg) was injected into the infarcted myocardium immediately after ligation. Rats were randomly divided into Sham, I/R, IPC-Exo + I/R, I/R + LY294002, and I/R + IPC-Exo + LY294002 groups. Haemodynamic parameters were measured by physiological recording. Transthoracic echocardiography was used to detect cardiac function. The serum levels of creatine kinase isomer-MB, lactate dehydrogenase, aspartate transaminase, tumour necrosis factor-alpha, interleukin (IL)-1β, and IL-10 were detected by enzyme-linked immunosorbent assay. Triphenyl tetrazolium chloride staining was used to measure the myocardial infarct size. Apoptosis in myocardial tissues was detected by TUNEL staining. Western blotting was used to detect the levels of PI3K/AKT and apoptosis-related proteins. Our results showed that treatment with IPC-Exo ameliorated cardiac function and reduced inflammatory factor production, cardiomyocyte apoptosis, and myocardial infarct size. Moreover, IPC-Exo treatment promoted the protein expression of Bcl-2, p-PI3K, and p-AKT but inhibited that of caspase-3 and Bax. However, treatment with LY294002 significantly reversed that IPC-Exo-induced increase in p-PI3K and p-AKT levels, improvement of haemodynamics, and decrease of inflammatory factor production and apoptosis in the I/R + IPC-Exo group. Taken together, our results suggest that IPC-Exo may alleviate I/R injury via activating the PI3K/AKT signalling pathway.

Time-related metabolomics study in the rat plasma after global cerebral ischemia and reperfusion: Effect of ischemic preconditioning.

Cardiac arrest is one of the major causes of death and disability. The aim of the study was to identify dynamic time-dependent metabolomic changes reflected in rat plasma induced by cerebral ischemia and reperfusion with the focus on the protective effect of ischemic preconditionig. Global cerebral ischemia in rats was induced by the four-vessel occlusion. Blood plasma was collected in three reperfusion times: an early post-acute 3 hr, then 24 hr, as an incipient time for delayed neuronal death induction and 72 hr as prolonged reperfusion period. The metabolomic measurements were conducted via untargeted nuclear magnetic resonance spectroscopy. Plasma of ischemized rats manifested dynamic metabolomic changes over the reperfusion time, such as increased levels of ketone bodies, decreased levels of pyruvate, alanine, and citrate. All three branched chain amino acids showed common pattern during reperfusion time: a decrease in 3 hr compared to sham, then a highest level in 24 hr and decrease in 72 hr reperfusion time, similar to their corresponding ketoacids. The protective effect of ischemic preconditioning was demonstrated by a faster tendency of plasma metabolites to normalize. Results also proved the remarkable metabolomic differences between the control (naïve) and sham-operated anesthetized animals, what warrants for critical evaluation of surgery/anaesthesy in the algorithm of metabolomic animal studies.

Protective effects of ischemic preconditioning against neuronal apoptosis and dendritic injury in the hippocampus are age-dependent.

Ischemic preconditioning with non-lethal ischemia can be protective against lethal forebrain ischemia. We hypothesized that aging may aggravate ischemic susceptibility and reduce brain plasticity against preconditioning. Magnetic resonance diffusion tensor imaging (DTI) is a sensitive tool to detect brain integrity and white matter architecture. This study used DTI and histopathology to investigate the effect of aging on ischemic preconditioning. In this study, adult and middle-aged male Mongolian gerbils were subjected to non-lethal 5-min forebrain ischemia (ischemic preconditioning) or sham-operation, followed by 3days of reperfusion, and then lethal 15-min forebrain ischemia. A 9.4-Tesla MR imaging system was used to study DTI indices, namely fractional anisotropy (FA), mean diffusivity (MD), and intervoxel coherence (IC) in the hippocampal CA1 and dentate gyrus (DG) areas. In situ expressions of microtubule-associated protein 2 (MAP2, dendritic marker protein) and apoptosis were also examined. The 5-min ischemia did not cause dendritic and neuronal injury and any significant change in DTI indices and MAP2 in adult and middle-aged gerbils. The 15-min ischemia-induced significant delayed neuronal apoptosis and early dendritic injury evidenced by DTI and MAP2 studies in both CA1 and DG areas with more severe injury in middle-aged gerbils than adult gerbils. Ischemic preconditioning could improve neuronal apoptosis in CA1 area and dendritic integrity in both CA1 and DG areas with better improvement in adult gerbils than middle-aged gerbils. This study thus suggests an age-dependent protective effect of ischemic preconditioning against both neuronal apoptosis and dendritic injury in hippocampus after forebrain ischemia.

Ischemic Preconditioning Preventing Downregulation of miR-182 Protects Intestine Against Ischemia/Reperfusion Injury by Inhibiting Apoptosis

Intestinal ischemia/reperfusion (I/R) injury is a severe condition associated with high morbidity and mortality. Ischemic preconditioning (IPC) had been found to be the most promising strategies against I/R injury. However, the potential molecular mechanisms underlying the protective effect of IPC have not been fully disclosed. MicroRNA182 (miR-182) is closely related to apoptosis and plays an important role in I/R injury. Our recent study demonstrated that miR-182 was down-regulated in the intestinal mucosa after I/R injury. However, whether miR-182 is involved in the protective effects of IPC in the setting of intestinal I/R injury is unknown. To investigate the role of miR-182 in the protective effect of IPC in intestine after I/R injury and potential mechanisms. AntagomiR-182 was pretreated before IPC in mice with intestinal I/R injury. MiR-182 mimic was administered before oxygen and glucose deprivation and reperfusion (OGD/R) in mice intestinal mucosa epithelial (MIME) cells. IPC partially prevented the downregulation of miR-182 in mice, which was blocked by pretreatment with antagomiR-182. Compared with the IPC group, pretreatment with antagomiR-182 further increased Chiu's scores and diamine oxidase activities. Meanwhile, apoptotic cells and cleaved caspase-3 expression were increased. Compared with the OGD/R group, pretreatment with miR-182 mimic prevented the downregulation of miR-182, improved cell survival, reduced apoptosis and cleaved caspase-3 expression in MIME cells. The downregulation of miR-182 was partially prevented by IPC, which was involved in IPC induced intestinal protection, and the mechanisms may be associated with inhibition of apoptosis.

Early Immunological Effects of Ischemia-Reperfusion Injury: No Modulation by Ischemic Preconditioning in a Randomised Crossover Trial in Healthy Humans.

Ischemic preconditioning (IPC) has been protective against ischemia-reperfusion injury (IRI), but the underlying mechanism is poorly understood. We examined whether IPC modulates the early inflammatory response after IRI. Nineteen healthy males participated in a randomised crossover trial with and without IPC before IRI. IPC and IRI were performed by cuff inflation on the forearm. IPC consisted of four cycles of five minutes followed by five minutes of reperfusion. IRI consisted of twenty minutes followed by 15 min of reperfusion. Blood was collected at baseline, 0 min, 85 min and 24 h after IRI. Circulating monocytes, T-cells subsets and dendritic cells together with intracellular activation markers were quantified by flow cytometry. Luminex measured a panel of inflammation-related cytokines in plasma. IRI resulted in dynamic regulations of the measured immune cells and their intracellular activation markers, however IPC did not significantly alter these patterns. Neither IRI nor the IPC protocol significantly affected the levels of inflammatory-related cytokines. In healthy volunteers, it was not possible to detect an effect of the investigated IPC-protocol on early IRI-induced inflammatory responses. This study indicates that protective effects of IPC on IRI is not explained by direct modulation of early inflammatory events.

Clearance of damaged mitochondria via mitophagy is important to the protective effect of ischemic preconditioning in kidneys

ABSTRACTIschemic preconditioning (IPC) affords tissue protection in organs including kidneys; however, the underlying mechanism remains unclear. Here we demonstrate an important role of macroautophagy/autophagy (especially mitophagy) in the protective effect of IPC in kidneys. IPC induced autophagy in renal tubular cells in mice and suppressed subsequent renal ischemia-reperfusion injury (IRI). The protective effect of IPC was abolished by pharmacological inhibitors of autophagy and by the ablation of Atg7 from kidney proximal tubules. Pretreatment with BECN1/Beclin1 peptide induced autophagy and protected against IRI. These results suggest the dependence of IPC protection on renal autophagy. During IPC, the mitophagy regulator PINK1 (PTEN induced putative kinase 1) was activated. Both IPC and BECN1 peptide enhanced mitolysosome formation during renal IRI in mitophagy reporter mice, suggesting that IPC may protect kidneys by activating mitophagy. We further established an in vitro model of IPC by inducing ‘chemical ischemia’ in kidney proximal tubular cells with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Brief treatment with CCCP protected against subsequent injury in these cells and the protective effect was abrogated by autophagy inhibition. In vitro IPC increased mitophagosome formation, enhanced the delivery of mitophagosomes to lysosomes, and promoted the clearance of damaged mitochondria during subsequent CCCP treatment. IPC also suppressed mitochondrial depolarization, improved ATP production, and inhibited the generation of reactive oxygen species. Knockdown of Pink1 suppressed mitophagy and reduced the cytoprotective effect of IPC. Together, these results suggest that autophagy, especially mitophagy, plays an important role in the protective effect of IPC.Abbreviations: ACTB: actin, beta; ATG: autophagy related; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; BUN: blood urea nitrogen; CASP3: caspase 3; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; COX4I1: cytochrome c oxidase subunit 4I1; COX8: cytochrome c oxidase subunit 8; DAPI: 4ʹ,6-diamidino-2-phenylindole; DNM1L: dynamin 1 like; EGFP: enhanced green fluorescent protein; EM: electron microscopy; ER: endoplasmic reticulum; FC: floxed control; FIS1: fission, mitochondrial 1; FUNDC1: FUN14 domain containing 1; H-E: hematoxylin-eosin; HIF1A: hypoxia inducible factor 1 subunit alpha; HSPD1: heat shock protein family D (Hsp60) member 1; IMMT/MIC60: inner membrane mitochondrial protein; IPC: ischemic preconditioning; I-R: ischemia-reperfusion; IRI: ischemia-reperfusion injury; JC-1: 5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethylbenzimidazolylcarbocyanine iodide; KO: knockout; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; mito-QC: mito-quality control; mRFP: monomeric red fluorescent protein; NAC: N-acetylcysteine; PINK1: PTEN induced putative kinase 1; PPIB: peptidylprolyl isomerase B; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; RPTC: rat proximal tubular cells; SD: standard deviation; sIPC: simulated IPC; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling

Effect of reperfusion injury from distant ischemia to small intestine

BACKGROUND The ileum is the most vulnerable part of the small intestine that plays an important role as the motor of multisystem organ failure. Villous damage is demonstrated after ligation of supply artery in mice; however, there is no study on the ileum after distant ischemic organs. Thus, this study was aimed to find out ileal villous changes following reperfusion injury, the protective effects of ischemic hypothermia and ischemic preconditioning.METHODS An experimental study conducted enrolled 21 subjects of Oryctolagus cuniculus. Ischemia is induced by ligation of the femoral artery for 4 hours. Eight hours after ligation was released, ileum and duodenal specimens were taken through laparotomy. H&amp;E stained specimens were examined for histomorphological changes. Villi change scores, tissue level of hypoxia-inducible factor-1α (HIF-1α), malondialdehyde (MDA), and occludin were statistically analyzed in four treatment groups, namely ischemia, ischemic hypothermia, ischemic preconditioning, and control.RESULTS Intestinal villi changes were found following ischemic-induced arterial ligation. Ileal villi changes showed differences with the duodenum and controls as indicated by the villi damage scores, increased tissue HIF-1α and MDA, and decreased occludin levels. Ileal villi changes in the ischemic and ischemic hypothermia groups showed significant changes with controls; whereas the ischemic preconditioning group showed no significant differences.CONCLUSIONS Ischemia at a distance leads to both histomorphological and biochemical damage of the ileal villi and disrupts the integrity of the intestinal mucosal barrier. In addition, the study showed a protective effect of ischemic preconditioning.

Astrocytic gap junction inhibition by carbenoxolone enhances the protective effects of ischemic preconditioning following cerebral ischemia

BackgroundStroke is the second leading cause of death worldwide and the most common cause of adult-acquired disability in many nations. Thus, attenuating the damage after ischemic injury and improving patient prognosis are of great importance. We have indicated that ischemic preconditioning (IP) can effectively reduce the damage of ischemia reperfusion and that inhibition of gap junctions may further reduce this damage. Although we confirmed that the function of gap junctions is closely associated with glutamate, we did not investigate the mechanism. In the present study, we aimed to clarify whether the blockade of cellular communication at gap junctions leads to significant reductions in the levels of glutamate released by astrocytes following cerebral ischemia.MethodsTo explore this hypothesis, we utilized the specific blocking agent carbenoxolone (CBX) to inhibit the opening and internalization of connexin 43 channels in an in vitro model of oxygen-glucose deprivation/re-oxygenation (OGD/R), following IP.ResultsOGD/R resulted in extensive astrocytic glutamate release following upregulation of hemichannel activity, thus increasing reactive oxygen species (ROS) generation and subsequent cell death. However, we observed significant increases in neuronal survival in neuron-astrocyte co-cultures that were subjected to IP prior to OGD/R. Moreover, the addition of CBX enhanced the protective effects of IP during the re-oxygenation period following OGD, by means of blocking the release of glutamate, increasing the level of the excitatory amino acid transporter 1, and downregulating glutamine expression.ConclusionsOur results suggest that combined use of IP and CBX represents a novel therapeutic strategy to attenuate damage from cerebral ischemia with minimal adverse side effects.

The Protective Effect of Ischemic Preconditioning and HMGB1 in Steatotic Liver Grafts from Brain-Dead Donors Submitted to Transplant

Background and Aims Currently, 80% of grafts are taken from brain-dead (BD) donors. However, BD markedly reduces the tolerance of liver grafts to preservation/reperfusion injury and reduces graft survival. In addition, steatosis is currently estimated to be present in up to 50% of deceased donor livers and is recognized as a key donor variable predicting post-transplant outcome. HMGB1 has been described in different inflammatory disorders, and the deleterious effects of brain death may counteract the protection conferred by ischemic preconditioning. Therefore, our study examined how HMGB1 may affect preconditioned and un-preconditioned steatotic liver grafts from brain death donors undergoing transplantation. Methods Steatotic grafts from non-BD and BD donors were cold stored for 6 hours and then transplanted. HMGB1 was pharmacologically modulated in liver grafts from brain death donors alone or in combination with ischemic preconditioning. Before the implantation of liver grafts in the recipient and after transplantation, hepatic damage and inflammatory response was analyzed and HMGB1-underlying mechanisms was characterized. Results and Discussion We report that BD reduces HMGB1 in preconditioned and un-preconditioned livers, which is associated with inflammation and damage. Exogenous HMGB1 in BD donors activates PI3k/Akt and reduces hepatic inflammation (neutrophil accumulation, oxidative stress) and hepatic damage, altogether increasing the survival of recipients. Combination of exogenous HMGB1 and preconditioning shows additional benefits and stronger protection against damage, oxidative stress and neutrophil accumulation than HMGB1 treatment alone. This superior beneficial effect may derive from the anti-oxidant and anti-inflammatory properties of different mediators generated by preconditioning, which may act dependently of HMGB1. Conclusions We show the injurious effects of BD in steatotic liver transplantation, which was associated with reduced HMGB1 expression. Pharmacological interventions to activate the HMGB1 signaling pathway in combination with ischemic preconditioning could be useful to reduce the incidence of postoperative complications in steatotic liver transplantation from deceased donors.

Protective Effect of Gastric Distension Preconditioning on Renal Ischemia/Reperfusion Injury in Rats.

The stomach mechanoreceptors can be stimulated by gastric distension (GD) and through afferent vagal nerve, increased activity of the renal sympathetic pathways. Because renal sympathectomy can abolish the protective effect of ischemic preconditioning, it seems that GD preconditioning can be effective in renal ischemia/reperfusion (I/R) injury. Gastric inflate (8 ml of 37°C water for 20 min) by a latex balloon inserted into the stomach through the fundus; I/R group was subjected to 45 min of bilateral ischemia and 24 h of reperfusion. GD preconditioning decreases blood urea nitrogen, creatinine, kidney damage score, and alkaline phosphatase levels compared to the sham GD group (P < 0.05). GD preconditioning may protect renal I/R injury through anti-inflammatory activity, but this efficacy requires extensive studies on the methods and mechanisms.

The role of pharmacological preconditioning in renal ischemic and reperfusion injury

Renal ischemic and reperfusion injury resulting in acute renal failure is a multidisciplinary problem at the junction of pathophysiology, transplantology, urology, nephrology, cardiac surgery and pharmacology. One of renal protection strategies is using the phenomenon of preconditioning. Preconditioning is one of the ways to adopt a tissue to repeated short-term effects of damaging factors to induce an enhanced tolerance to the long period of hypoxia and/or ischemia. There are multiple cellular and molecular mechanisms of the renal protective effects of preconditioning stimuli, but the key effectors and signaling molecules are ATP-dependent potassium channels, nitric oxide synthase, nitric oxide, and mitochondrial pore. Contradictory data on the protective effect of ischemic preconditioning allow searching for approaches to pharmacological correction of ischemic and reperfusion injuries. The article provides data on possible ways of using erythropoietin, darbepoetin and phosphodiesterase 5 inhibitors.

Role of miR‑21 on vascular endothelial cells in the protective effect of renal delayed ischemic preconditioning.

Vascular endothelial cells may serve crucial roles in the development of acute kidney injury (AKI). microRNA (miR)-21, which possesses a renal protective function has been found on vascular endothelial cells. The present study aimed to test the hypothesis that miR-21 may protect vascular endothelial cells against injury, which may contribute to the protective effects of renal delayed ischemic preconditioning (IPC). Preconditioned (15 min ischemia) or Sham mice (not clamped) were subjected to 35 min occlusion of bilateral renal pedicles 4 days following preconditioning or Sham treatment. Human umbilical vein endothelial cells (HUVECs) were treated with cobalt(II) chloride (CoCl2) to establish an in vitro hypoxia model. Locked nucleic acid-modified anti-miR-21 or scrambled control oligonucleotides were transfected into cells or delivered into mice via tail vein injection <1 h prior to IPC. Following 24 h of reperfusion or hypoxia, morphological and functional parameters, apoptosis and miR-21 and programmed cell death 4 (PDCD4) expression were assessed in vivo and in vitro. Treatment of HUVECs with CoCl2 led to an upregulation of miR-21 expression, a downregulation of PDCD4 protein expression and attenuation of apoptosis. Inhibition of miR-21 expression led to increased expression levels of PDCD4 protein and apoptosis in HUVECs. IPC attenuated renal IR injury in mice. The protective effect of IPC appeared to be dependent on upregulated miR-21 expression. IPC-induced upregulation of miR-21 expression also occurred in HUVECs, and IPC also led to reduced PDCD4 expression and vascular permeability in mouse kidneys. The effects of IPC were attenuated by the inhibition of miR-21; miR-21 expression attenuated damage in vascular endothelial cells, which may contribute to the protective effects of delayed IPC on renal IR injury. The present study suggested a novel target for the prevention and repair of AKI in the future.

Protective Effect of Ischemic Preconditioning on Myocardium Against Remote Tissue Injury Following Transient Focal Cerebral Ischemia in Diabetic Rats.

BackgroundRemote ischemic preconditioning (IPreC) could provide tissue-protective effect at a remote site by anti-inflammatory, neuronal, and humoral signaling pathways.ObjectivesThe aim of the study was to investigate the possible protective effects of remote IPreC on myocardium after transient middle cerebral artery occlusion (MCAo) in streptozotocin- induced diabetic (STZ) and non-diabetic rats.Methods48 male Spraque Dawley rats were divided into eight groups: Sham, STZ, IPreC, MCAo, IPreC+MCAo, STZ+IPreC, STZ+MCAo and STZ+IPreC+MCAo groups. We induced transient MCAo seven days after STZ-induced diabetes, and performed IPreC 72 hours before transient MCAo. Remote myocardial injury was investigated histopathologically. Bax, Bcl2 and caspase-3 protein levels were measured by Western blot analysis. Total antioxidant status (TAS), total oxidant status (TOS) of myocardial tissue were measured by colorimetric assay. Oxidative stress index(OSI) was calculated as TOS-to-TAS ratio. For all statistical analysis, p values < 0.05 were considered significant.ResultsWe observed serious damage including necrosis, congestion and mononuclear cell infiltration in myocardial tissue of the diabetic and ischemic groups. In these groups TOS and OSI levels were significantly higher; TAS levels were lower than those of IPreC related groups (p < 0.05). IPreC had markedly improved histopathological alterations and increased TAS levels in IPreC+MCAo and STZ+IPreC+MCAo compared to MCAo and STZ+MCAo groups (p < 0.05). In non-diabetic rats, MCAo activated apoptotic cell death via increasing Bax/Bcl2 ratio and caspase-3 levels. IPreC reduced apoptotic cell death by suppressing pro-apoptotic proteins. Diabetes markedly increased apoptotic protein levels and the effect did not reversed by IPreC.ConclusionsWe could suggest that IPreC attenuates myocardial injury via ameliorating histological findings, activating antioxidant mechanisms, and inducing antiapoptotic activity in diabetic rats.

The Effectiveness Of Ischemic Preconditioning In Older Physically (in)active Males

Reperfusion is critical for the survival of ischemic tissue, but causes significant additional damage to the endothelium (ischemia-reperfusion [IR]). Ischemic preconditioning (IPC) refers to short repetitive episodes of ischemia, which has demonstrated to reduce IR injury. The protective effects of IPC against endothelial IR injury are attenuated with aging. Possibly, regular physical activity is able to 1) attenuate endothelial IR, and 2) restore effectiveness of IPC in older humans. PURPOSE: To compare endothelial IR-injury and the effectiveness of IPC to protect against endothelial IR-injury between older lifelong physically active versus inactive males. METHODS: We included 11 older athletes (63±4 years, >5 exercise hours/week) and 3 inactive controls (69±6 years, <1 exercise hours/week). We measured brachial artery endothelial function before and after IR injury using flow-mediated dilation (FMD). IR-injury was induced by 20-minute ischemia followed by 20 minutes of reperfusion. Prior to IR-injury, subjects underwent SHAM (35-minute rest) or IPC (3 cycles of 5-minute cuff inflation to 220 mmHg). The order of SHAM and IPC was randomly assigned to the participants. RESULTS: FMD% did not differ between athletes and inactive controls at baseline for SHAM (4.5±4.4% vs. 4.1±2.4%, p=0.87) or IPC (5.1±3.1% vs. 3.3±1.1%, p=0.37). IR-injury showed no significant decrease in FMD% in athletes (FMDpre 4.5±4.4% vs. FMDpost 3.0±2.0%, p=0.32), whereas FMD% markedly decreased in inactive controls (FMDpre 4.1±2.3% vs. FMDpost 0.5±2.5%, p=0.036). Applying IPC before IR-injury did not alter the change in %FMD in both athletes (FMDpre 5.1±3.2% vs. FMDpost 2.8±3.3%, p=0.17) and controls (FMDpre 3.3±1.1% vs. FMDpost 1.5±2.2%, p=0.19). No differences were observed in %FMD change between SHAM and IPC in athletes and inactive controls. CONCLUSIONS: Athletes demonstrate smaller endothelial IR-injury compared to their physically inactive peers. The cardio-protective effects of regular exercise training may, in part, relate to attenuating endothelial IR-injury. Finally, lifelong regular exercise training seems unable to alter the age-related attenuated efficacy of IPC against endothelial IR-injury.