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1.  A Randomized Pilot Trial of Remote Ischemic Preconditioning in Heart Failure with Reduced Ejection Fraction 
PLoS ONE  2014;9(9):e105361.
Remote ischemic preconditioning (RIPC) induced by transient limb ischemia confers multi-organ protection and improves exercise performance in the setting of tissue hypoxia. We aimed to evaluate the effect of RIPC on exercise capacity in heart failure patients.
We performed a randomized crossover trial of RIPC (4×5-minutes limb ischemia) compared to sham control in heart failure patients undergoing exercise testing. Patients were randomly allocated to either RIPC or sham prior to exercise, then crossed over and completed the alternate intervention with repeat testing. The primary outcome was peak VO2, RIPC versus sham. A mechanistic substudy was performed using dialysate from study patient blood samples obtained after sham and RIPC. This dialysate was used to test for a protective effect of RIPC in a mouse heart Langendorff model of infarction. Mouse heart infarct size with RIPC or sham dialysate exposure was also compared with historical control data.
Twenty patients completed the study. RIPC was not associated with improvements in peak VO2 (15.6+/−4.2 vs 15.3+/−4.6 mL/kg/min; p = 0.53, sham and RIPC, respectively). In our Langendorff sub-study, infarct size was similar between RIPC and sham dialysate groups from our study patients, but was smaller than expected compared to healthy controls (29.0%, 27.9% [sham, RIPC] vs 51.2% [controls]. We observed less preconditioning among the subgroup of patients with increased exercise performance following RIPC (p<0.04).
In this pilot study of RIPC in heart failure patients, RIPC was not associated with improvements in exercise capacity overall. However, the degree of effect of RIPC may be inversely related to the degree of baseline preconditioning. These data provide the basis for a larger randomized trial to test the potential benefits of RIPC in patients with heart failure.
Trial Registration +++++NCT01128790
PMCID: PMC4152122  PMID: 25181050
2.  The role of remote ischemic preconditioning in the treatment of atherosclerotic diseases 
Brain and Behavior  2013;3(6):606-616.
Remote ischemic preconditioning (RIPC) is the application of a transient and brief ischemic stimulus to a distant site from the organ or tissue that is afterward exposed to injury ischemia, and has been found to reduce ischemia–reperfusion injury (IRI) in various animal models. RIPC appears to offer two distinct phases of endothelial IRI protection, which are presumably mediated through neuronal and humoral pathways.
We conducted a comprehensive literature review on the available published data about the potential effect of RIPC in patients undergoing IRI in one or more vital organs.
Our search highlighted 24 randomized clinical trials about the effect of RIPC on variable clinical settings (abdominal aortic aneurysm repair, open heart surgery, percutaneous coronary intervention, living donor renal transplantation, coronary angiography, elective decompression surgery, carotid endarterectomy, recent stroke, or transient ischemic attack combined with intracranial carotid artery stenosis). Most of the trials focused on postoperative cardiac or renal function after RIPC with conflicting results. Preconditioning protocols, age limits, comorbidities, and concomitant drug use varied significantly across trials, and therefore no firm conclusions can be drawn using the available data. However, no severe local adverse events were observed in any patient undergoing limb or arm preconditioning.
RIPC is a safe and well-tolerated procedure that may constitute a potentially promising innovative treatment in atherosclerotic diseases. Large, multicenter, randomized clinical trials are required to determine an optimal protocol for the RIPC procedure, and to evaluate further the potential benefits of RIPC in human ischemic injury.
PMCID: PMC3868166  PMID: 24363964
Aortic aneurysm; atherosclerosis; coronary artery disease; ischemic stroke; peripheral arterial disease; remote ischemic preconditioning
3.  Activities of cardiac tissue matrix metalloproteinases 2 and 9 are reduced by remote ischemic preconditioning in cardiosurgical patients with cardiopulmonary bypass 
Transient episodes of ischemia in a remote organ or tissue (remote ischemic preconditioning, RIPC) can attenuate myocardial injury. Myocardial damage is associated with tissue remodeling and the matrix metalloproteinases 2 and 9 (MMP-2/9) are crucially involved in these events. Here we investigated the effects of RIPC on the activities of heart tissue MMP-2/9 and their correlation with serum concentrations of cardiac troponin T (cTnT), a marker for myocardial damage.
In cardiosurgical patients with cardiopulmonary bypass (CPB) RIPC was induced by four 5 minute cycles of upper limb ischemia/reperfusion. Cardiac tissue was obtained before as well as after CPB and serum cTnT concentrations were measured. Tissue derived from control patients (N = 17) with high cTnT concentrations (≥0.32 ng/ml) and RIPC patients (N = 18) with low cTnT (≤0.32 ng/ml) was subjected to gelatin zymography to quantify MMP-2/9 activities.
In cardiac biopsies obtained before CPB, activities of MMP-2/9 were attenuated in the RIPC group (MMP-2: Control, 1.13 ± 0.13 a.u.; RIPC, 0.71 ± 0.12 a.u.; P < 0.05. MMP-9: Control, 1.50 ± 0.16 a.u.; RIPC, 0.87 ± 0.14 a.u.; P < 0.01), while activities of the pro-MMPs were not altered (P > 0.05). In cardiac biopsies taken after CPB activities of pro- and active MMP-2/9 were not different between the groups (P > 0.05). Spearman’s rank tests showed that MMP-2/9 activities in cardiac tissue obtained before CPB were positively correlated with postoperative cTnT serum levels (MMP-2, P = 0.016; MMP-9, P = 0.015).
Activities of MMP-2/9 in cardiac tissue obtained before CPB are attenuated by RIPC and are positively correlated with serum concentrations of cTnT. MMPs may represent potential targets for RIPC mediated cardioprotection.
Trial registration identifier NCT00877305.
PMCID: PMC4234318  PMID: 24712447
Cardioprotection; Ischemia/reperfusion injury; Matrix metalloproteinases; Myocardial damage; Remote ischemic preconditioning
4.  Remote ischemic preconditioning in patients with intermittent claudication 
Clinics  2013;68(4):495-499.
Remote ischemic preconditioning (RIPC) is a phenomenon in which a short period of sub-lethal ischemia in one organ protects against subsequent bouts of ischemia in another organ. We hypothesized that RIPC in patients with intermittent claudication would increase muscle tissue resistance to ischemia, thereby resulting in an increased ability to walk.
In a claudication clinic, 52 ambulatory patients who presented with complaints of intermittent claudication in the lower limbs associated with an absent or reduced arterial pulse in the symptomatic limb and/or an ankle-brachial index <0.90 were recruited for this study. The patients were randomly divided into three groups (A, B and C). All of the patients underwent two tests on a treadmill according to the Gardener protocol. Group A was tested first without RIPC. Group A was subjected to RIPC prior to the second treadmill test. Group B was subjected to RIPC prior to the first treadmill test and then was subjected to a treadmill test without RIPC. In Group C (control group), both treadmill tests were performed without RIPC. The first and second tests were conducted seven days apart. Brazilian Clinical Trials: RBR-7TF6TM.
Group A showed a significant increase in the initial claudication distance in the second test compared to the first test.
RIPC increased the initial claudication distance in patients with intermittent claudication; however, RIPC did not affect the total walking distance of the patients.
PMCID: PMC3634960  PMID: 23778346
Intermittent Claudication; Ischemic Preconditioning; Peripheral Arterial Disease
5.  Profiling of cell stress protein expression in cardiac tissue of cardiosurgical patients undergoing remote ischemic preconditioning: implications for thioredoxin in cardioprotection 
Transient episodes of ischemia in a remote organ (remote ischemic preconditioning, RIPC) can attenuate myocardial ischemia/reperfusion injury but the underlying mechanisms of RIPC in the target organ are still poorly understood. Recent animal studies suggested that the small redox protein thioredoxin may be a potential candidate for preconditioning-induced organprotection. Here we employed a human proteome profiler array to investigate the RIPC regulated expression of cell stress proteins and particularly of thioredoxin in heart tissue of cardiosurgical patients with cardiopulmonary bypass (CPB).
RIPC was induced by four 5 minute cycles of transient upper limb ischemia/reperfusion using a blood pressure cuff. Right atrial tissue was obtained from patients receiving RIPC (N = 19) and control patients (N = 19) before and after CPB. Cell stress proteome profiler arrays as well as Westernblotting and ELISA experiments for thioredoxin (Thio-1) were performed employing the respective tissue samples.
Protein arrays revealed an up-regulation of 26.9% (7/26; CA IX, Cyt C, HSP-60, HSP-70, pJNK, SOD2, Thio-1) of cell stress associated proteins in RIPC tissue obtained before CPB, while 3.8% (1/26; SIRT2) of the proteins were down-regulated. Array results for thioredoxin were verified by semi-quantitative Westernblotting studies which showed a significant up-regulation of thioredoxin protein levels in cardiac tissue samples of RIPC patients taken before CPB (RIPC: 5.36 ± 0.85 a.u.; control: 3.23 ± 0.39 a.u.; P < 0.05). Quantification of thioredoxin levels in tissue of RIPC and control patients by ELISA experiments further confirmed the Westernblotting results (RIPC: 0.30 ± 0.02 ng/mg protein; control: 0.24 ± 0.02 ng/mg protein; P < 0.05).
We provide evidence for thioredoxin as a RIPC-induced factor in heart tissue of cardiosurgical patients and identified several cell stress associated proteins that are regulated by RIPC and may play a role in RIPC-mediated cardioprotection.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-015-0403-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4316390  PMID: 25622749
Remote ischemic preconditioning; Cardioprotection; Cardiac surgery; Protein expression; Thioredoxin
6.  Characterization of acute ischemia‐related physiological responses associated with remote ischemic preconditioning: a randomized controlled, crossover human study 
Physiological Reports  2014;2(11):e12200.
Remote Ischemic Preconditioning (RIPC) is emerging as a new noninvasive intervention that has the potential to protect a number of organs against ischemia–reperfusion (IR) injury. The standard protocols normally used to deliver RIPC involve a number of cycles of inflation of a blood pressure (BP) cuff on the arm and/or leg to an inflation pressure of 200 mmHg followed by cuff deflation for a short period of time. There is little evidence to support what limb (upper or lower) or cuff inflation pressures are most effective to deliver this intervention without causing undue discomfort/pain in nonanesthetized humans. In this preliminary study, a dose–response assessment was performed using a range of cuff inflation pressures (140, 160, and 180 mmHg) to induce limb ischemia in upper and lower limbs. Physiological changes in the occluded limb and any pain/discomfort associated with RIPC with each cuff inflation pressure were determined. Results showed that ischemia can be induced in the upper limb at much lower cuff inflation pressures compared with the standard 200 mmHg pressure generally used for RIPC, provided the cuff inflation pressure is ~30 mmHg higher than the resting systolic BP. In the lower limb, a higher inflation pressure, (~55 mmHg > resting systolic BP), is required to induce ischemia. Cyclical changes in capillary blood O2, CO2, and lactate levels during the RIPC stimulus were observed. RIPC at higher cuff inflation pressures of 160 and 180 mmHg was better tolerated in the upper limb. In summary, limb ischemia for RIPC can be more easily induced at lower pressures and is much better tolerated in the upper limb in young healthy individuals. However, whether benefits of RIPC can also be derived with protocols delivered to the upper limb using lower cuff inflation pressures and with lesser discomfort compared to the lower limb, remains to be investigated.
Remote Ischemic Preconditioning (RIPC) is emerging as a new noninvasive intervention that has the potential to protect a number of organs against ischemia‐reperfusion (IR) injury. Currently, there is little evidence to support what limb (upper or lower) or cuff inflation pressures are most effective to deliver this intervention without causing undue discomfort/pain in nonanesthetized humans. In this study we have demonstrated that limb ischemia for RIPC can be more easily induced at lower pressures and is much better tolerated in the upper limb compared with the lower limb, in young healthy individuals.
PMCID: PMC4255807  PMID: 25413320
Characterization; cuff inflation pressure; remote ischemic preconditioning; tolerability
7.  Impact of remote ischemic preconditioning preceding coronary artery bypass grafting on inducing neuroprotection (RIPCAGE): study protocol for a randomized controlled trial 
Trials  2014;15:414.
Neurological complications after cardiac surgery have a profound impact on postoperative survival and quality of life. The increasing importance of strategies designed to improve neurological outcomes mirrors the growing risk burden of the contemporary cardiac surgical population. Remote ischemic preconditioning (RIPC) reduces adverse sequelae of ischemia in vulnerable organs by subjecting tissues with high ischemic tolerance to brief periods of hypoperfusion. This trial will evaluate the neuroprotective effect of RIPC in the cardiac surgical arena, by employing magnetic resonance imaging (MRI) and neurocognitive testing.
Patients scheduled for elective coronary artery bypass grafting with the use of cardiopulmonary bypass will be screened for the study. Eligible patients will be randomized to undergo either a validated RIPC protocol or a sham procedure. The RIPC will be induced by inflation of a blood pressure cuff to 200 mmHg for 5 minutes, followed by a 5-minute reperfusion period. Three sequences of interchanging cuff inflations and deflations will be employed. Neurocognitive testing and MRI imaging will be performed preoperatively and on postoperative day 7. Paired pre- and postoperative neurocognitive and neuroimaging data will then be compared. The primary composite outcome measure will consist of new ischemic lesions on brain MRI, postprocedural impairment in brain connectivity on resting-state functional MRI (rs-fMRI), and significant new declines in neurocognitive performance. The secondary endpoint measures will be the individual components of the primary endpoint measures, expressed as continuous variables, troponin T release on postoperative day 1 and the incidence of major adverse cardiovascular events at 3 months postoperatively. Major adverse cardiovascular events, including accumulating cardiovascular mortality, stroke, nonfatal myocardial infarction, and rehospitalization for ischemia, will form a composite endpoint measure.
This trial will aim to assess whether RIPC in patients subjected to surgical myocardial revascularization employing cardiopulmonary bypass initiates a neuroprotective response. Should the results of this trial indicate that RIPC is effective in reducing the incidence of adverse neurological events in patients undergoing coronary artery bypass grafting, it could impact on the current standard of care.
Trial registration NCT02177981.
PMCID: PMC4223850  PMID: 25348157
coronary artery bypass grafting; functional; magnetic resonance imaging; neurocognitive outcome; remote ischemic preconditioning
8.  Novel perspectives on the PHD-HIF oxygen sensing pathway in cardioprotection mediated by IPC and RIPC 
Reperfusion of ischemic cardiac tissue is the standard treatment for improving clinical outcome following myocardial infarction but is inevitably associated with ischemia-reperfusion injury (IRI). Ischemic myocardial injury can be alleviated by exposing the heart to brief episodes of sublethal ischemia-reperfusion prior to the ischemic insult, a phenomenon that has been termed ischemic preconditioning (IPC). Similarly, remote IPC (RIPC) is defined as transient episodes of ischemia at a distant site before a subsequent prolonged injury of the target organ. In this setting, adaptive responses to hypoxia/ischemia in peripheral tissues include the release of soluble factors that have the potential to protect cardiomyocytes remotely. Oxygen fluctuations is a hallmark of insufficient tissue perfusion and ischemic episodes. Emerging evidence indicates that prolyl hydroxylase oxygen sensors (PHDs) and hypoxia-inducible transcription factors (HIFs) are critical regulators of IPC and RIPC. In this review, we discuss recent findings concerning the role of the PHD-HIF axis in IPC and RIPC-mediated cardioprotection and examine molecular pathways and cell types that might be involved. We also appraise the therapeutic value of targeting the PHD-HIF axis to enhance cardiac tolerance against IRI.
PMCID: PMC4438228  PMID: 26042040
ischemic preconditioning; heart; PHD oxygen sensors; hypoxia-inducible factors; remote ischemic preconditioning
9.  Apolipoprotein A-I Is a Potential Mediator of Remote Ischemic Preconditioning 
PLoS ONE  2013;8(10):e77211.
Remote ischemic preconditioning (RIPC) has emerged as an attractive strategy in clinical settings. Despite convincing evidence of the critical role played by circulating humoral mediators, their actual identities remain unknown. In this study, we aimed to identify RIPC-induced humoral mediators using a proteomic approach.
and Results Rats were exposed to 10-min limb ischemia followed by 5- (RIPC 5′) or 10-min (RIPC 10′) reperfusion prior to blood sampling. The control group only underwent blood sampling. Plasma samples were analyzed using surface-enhanced laser desorption and ionization - time of flight - mass spectrometry (SELDI-TOF-MS). Three protein peaks were selected for their significant increase in RIPC 10′. They were identified and confirmed as apolipoprotein A-I (ApoA-I). Additional rats were exposed to myocardial ischemia-reperfusion (I/R) and assigned to one of the following groups RIPC+myocardial infarction (MI) (10-min limb ischemia followed by 10-min reperfusion initiated 20 minutes prior to myocardial I/R), ApoA-I+MI (10 mg/kg ApoA-I injection 10 minutes before myocardial I/R), and MI (no further intervention). In comparison with untreated MI rats, RIPC reduced infarct size (52.2±3.7% in RIPC+MI vs. 64.9±2.6% in MI; p<0.05). Similarly, ApoA-I injection decreased infarct size (50.9±3.8%; p<0.05 vs. MI).
RIPC was associated with a plasmatic increase in ApoA-I. Furthermore, ApoA-I injection before myocardial I/R recapitulated the cardioprotection offered by RIPC in rats. This data suggests that ApoA-I may be a protective blood-borne factor involved in the RIPC mechanism.
PMCID: PMC3796499  PMID: 24155931
10.  Remote Ischemic Preconditioning in Children Undergoing Cardiac Surgery With Cardiopulmonary Bypass: A Single‐Center Double‐Blinded Randomized Trial 
Remote ischemic preconditioning (RIPC) harnesses an innate defensive mechanism that protects against inflammatory activation and ischemia‐reperfusion injury, known sequelae of cardiac surgery with cardiopulmonary bypass. We sought to determine the impact of RIPC on clinical outcomes and physiological markers related to ischemia‐reperfusion injury and inflammatory activation after cardiac surgery in children.
Methods and Results
Overall, 299 children (aged neonate to 17 years) were randomized to receive an RIPC stimulus (inflation of a blood pressure cuff on the left thigh to 15 mm Hg above systolic for four 5‐minute intervals) versus a blinded sham stimulus during induction with a standardized anesthesia protocol. Primary outcome was duration of postoperative hospital stay, with serial clinical and laboratory measurements for the first 48 postoperative hours and clinical follow‐up to discharge. There were no significant baseline differences between RIPC (n=148) and sham (n=151). There were no in‐hospital deaths. No significant difference in length of postoperative hospital stay was noted (sham 5.4 versus RIPC 5.6 days; difference +0.2; adjusted P=0.91), with the 95% confidence interval (−0.7 to +0.9) excluding a prespecified minimal clinically significant differences of 1 or 1.5 days. There were few significant differences in other clinical outcomes or values at time points or trends in physiological markers. Benefit was not observed in specific subgroups when explored through interactions with categories of age, sex, surgery type, Aristotle score, or first versus second half of recruitment. Adverse events were similar (sham 5%, RIPC 6%; P=0.68).
RIPC is not associated with important improvements in clinical outcomes and physiological markers after cardiac surgery in children.
Clinical Trial Registration
URL: Unique identifier: NCT00650507.
PMCID: PMC4310383  PMID: 25074698
congenital; heart defects; pediatrics; remote ischemic preconditioning; surgery
11.  ggstThe role of tendon microcirculation in Achilles and patellar tendinopathy 
Tendinopathy is of distinct interest as it describes a painful tendon disease with local tenderness, swelling and pain associated with sonographic features such as hypoechogenic texture and diameter enlargement. Recent research elucidated microcirculatory changes in tendinopathy using laser Doppler flowmetry and spectrophotometry such as at the Achilles tendon, the patellar tendon as well as at the elbow and the wrist level. Tendon capillary blood flow is increased at the point of pain. Tendon oxygen saturation as well as tendon postcapillary venous filling pressures, determined non-invasively using combined Laser Doppler flowmetry and spectrophotometry, can quantify, in real-time, how tendon microcirculation changes over with pathology or in response to a given therapy. Tendon oxygen saturation can be increased by repetitive, intermittent short-term ice applications in Achilles tendons; this corresponds to 'ischemic preconditioning', a method used to train tissue to sustain ischemic damage. On the other hand, decreasing tendon oxygenation may reflect local acidosis and deteriorating tendon metabolism. Painful eccentric training, a common therapy for Achilles, patellar, supraspinatus and wrist tendinopathy decreases abnormal capillary tendon flow without compromising local tendon oxygenation. Combining an Achilles pneumatic wrap with eccentric training changes tendon microcirculation in a different way than does eccentric training alone; both approaches reduce pain in Achilles tendinopathy. The microcirculatory effects of measures such as extracorporeal shock wave therapy as well as topical nitroglycerine application are to be studied in tendinopathy as well as the critical question of dosage and maintenance. Interestingly it seems that injection therapy using color Doppler for targeting the area of neovascularisation yields to good clinical results with polidocanol sclerosing therapy, but also with a combination of epinephrine and lidocaine.
PMCID: PMC2397381  PMID: 18447938
12.  Effect of Remote Ischemic Preconditioning on Acute Kidney Injury in Nondiabetic Patients Undergoing Coronary Artery Bypass Graft Surgery: A Secondary Analysis of 2 Small Randomized Trials 
American Journal of Kidney Diseases  2010;56(6-2):1043-1049.
Novel treatment strategies are required to reduce the development of acute kidney injury (AKI) in patients undergoing cardiac surgery. In this respect, remote ischemic preconditioning (RIPC), a phenomenon in which transient nonlethal ischemia applied to an organ or tissue protects another organ or tissue from subsequent lethal ischemic injury, is a potential renoprotective strategy.
Study Design
Secondary analysis of 2 randomized trials.
Setting & Participants
78 consenting selected nondiabetic patients in a university teaching hospital undergoing elective coronary artery bypass graft (CABG) surgery recruited to 2 previously reported randomized studies.
RIPC consisted of three 5-minute cycles of right forearm ischemia, induced by inflating a blood pressure cuff on the upper arm to 200 mm Hg, with an intervening 5 minutes of reperfusion, during which time the cuff was deflated. The control consisted of placing an uninflated cuff on the arm for 30 minutes.
AKI measured using Acute Kidney Injury Network (AKIN) criteria, duration of hospital stay, in-hospital and 30-day mortality.
Numbers of participants with AKI stages 1, 2, and 3 were 1 (3%), 3 (8%), and 0 in the intervention group compared with 10 (25%), 0, and 0 in the control group, respectively (P = 0.005). The decrease in AKI was independent of the effect of concomitant aortic valve replacement and cross-clamp times, which were distributed unevenly between the 2 groups.
Retrospective analysis of data. More patients in the RIPC group underwent concomitant aortic valve replacement with CABG; although we have corrected statistically for this imbalance, it remains an important confounding variable.
RIPC induced using transient forearm ischemia decreased the incidence of AKI in nondiabetic patients undergoing elective CABG surgery in this retrospective analysis. A large prospective clinical trial is required to study this effect and clinical outcomes in patients undergoing cardiac surgery.
PMCID: PMC2991586  PMID: 20974511
Remote ischemic preconditioning; transient limb ischemia; coronary artery bypass graft surgery; acute kidney injury
13.  Effect of remote ischemic post-conditioning on systemic inflammatory response and survival rate in lipopolysaccharide-induced systemic inflammation model 
Remote ischemic preconditioning (RIPC) and postconditioning (RpostC) have protective effects on ischemia and reperfusion injury. The effects have been reported to activate heme oxygenase-1 (HO-1) and attenuate nuclear factor kappa B (NF-κB) and subsequently reduce systemic inflammation. Ischemic preconditioning prevented inflammatory responses by modulating HO-1 expression in endotoxic shock model. Therefore, we investigated whether RpostC could have protective effects on lipopolysaccharide (LPS)-induced systemic inflammation.
The LPS-induced sepsis mice received LPS (20 mg/kg) intraperitoneally. Remote ischemic conditioning was induced with three 10-min ischemia/10-min reperfusion cycles of the right hind limbs using tourniquet before LPS injection (RIPC) or after LPS injection (RpostC). The effects of RIPC and RpostC were examined for the survival rate, serum cytokines, NF-κB, HO-1 and liver pathology in the LPS injected mice.
Survival rate within 120 hours significantly increased in the LPS injected and remote ischemic conditioned mice than in LPS only injected mice (60-65% vs 5%, respectively, p < 0.01). Tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) increased markedly in the LPS only injected mice, however, remote ischemic conditioning suppressed the changes (p < 0.05). Interleukin-10 (IL-10) level was significantly higher in the LPS injected and RpostC treated mice than in the LPS only injected mice (p = 0.014). NF-κB activation was significantly attenuated (p < 0.05) and HO-1 levels were substantially higher in the LPS injected and remote ischemic conditioned mice than in the LPS only injected mice. Neutrophil infiltration was significantly attenuated in the LPS injected and remote ischemic conditioned mice than in the only LPS injected mice (p < 0.05).
RpostC attenuated inflammatory responses and improved survival outcomes of mice with LPS-induced systemic inflammation. The mechanism may be caused by modifying NF-κB mediated expression of cytokines.
PMCID: PMC4046032  PMID: 24904237
Remote ischemic preconditioning; Remote ischemic postconditioning; Systemic inflammation
14.  Preconditioning Shields Against Vascular Events in Surgery (SAVES), a multicentre feasibility trial of preconditioning against adverse events in major vascular surgery: study protocol for a randomised control trial 
Trials  2015;16:185.
Patients undergoing vascular surgery procedures constitute a ‘high-risk’ group. Fatal and disabling perioperative complications are common. Complications arise via multiple aetiological pathways. This mechanistic redundancy limits techniques to reduce complications that target individual mechanisms, for example, anti-platelet agents. Remote ischaemic preconditioning (RIPC) induces a protective phenotype in at-risk tissue, conferring protection against ischaemia-reperfusion injury regardless of the trigger. RIPC is induced by repeated periods of upper limb ischaemia-reperfusion produced using a blood pressure cuff. RIPC confers some protection against cardiac and renal injury during major vascular surgery in proof-of-concept trials. Similar trials suggest benefit during cardiac surgery. Several uncertainties remain in advance of a full-scale trial to evaluate clinical efficacy. We propose a feasibility trial to fully evaluate arm-induced RIPC’s ability to confer protection in major vascular surgery, assess the incidence of a proposed composite primary efficacy endpoint and evaluate the intervention’s acceptability to patients and staff.
Four hundred major vascular surgery patients in five Irish vascular centres will be randomised (stratified for centre and procedure) to undergo RIPC or not immediately before surgery. RIPC will be induced using a blood pressure cuff with four cycles of 5 minutes of ischaemia followed by 5 minutes of reperfusion immediately before the start of operations. There is no sham intervention. Participants will undergo serum troponin measurements pre-operatively and 1, 2, and 3 days post-operatively. Participants will undergo 12-lead electrocardiograms pre-operatively and on the second post-operative day. Predefined complications within one year of surgery will be recorded. Patient and staff experiences will be explored using qualitative techniques. The primary outcome measure is the proportion of patients who develop elevated serum troponin levels in the first 3 days post-operatively. Secondary outcome measures include length of hospital and critical care stay, unplanned critical care admissions, death, myocardial infarction, stroke, mesenteric ischaemia and need for renal replacement therapy (within 30 days of surgery).
RIPC is novel intervention with the potential to significantly improve perioperative outcomes. This trial will provide the first evaluation of RIPC’s ability to reduce adverse clinical events following major vascular surgery.
Trial Registration NCT02097186 Date Registered: 24 March 2014
PMCID: PMC4414457  PMID: 25903752
remote preconditioning; vascular surgery; perioperative complications
15.  Remote ischemic preconditioning preserves Connexin 43 phosphorylation in the rat heart in vivo 
Remote ischemic preconditioning (RIPC) protects the heart from ischemia and reperfusion (I/R) injury. The underlying molecular mechanisms are unclear. It has been demonstrated that Connexin 43 (Cx43) is critically involved in cardioprotective interventions including classical ischemic preconditioning. In the present study we investigated the influence of RIPC on the expression patterns of Cx43 after I/R in the rat heart in vivo.
Male Wistar rats were subjected to 35 min regional myocardial ischemia followed by 2 h reperfusion with or without 4 cycles of 5 minutes bilateral hind limb ischemia and reperfusion (RIPC), to RIPC without ischemia or underwent no intervention (Sham). Infarct size was measured by TTC staining. The myocardium was divided into area at risk (AAR) and area not at risk (non AAR). Expression of Cx43-mRNA and protein was analyzed by qPCR and Western Blot analysis, respectively. Localization of Cx43 was visualized by confocal immunofluorescence staining.
RIPC reduced the infarct size (I/R: 73 ± 5% vs. RIPC I/R: 34 ± 14%, p < 0.05). Expression of Cx43 mRNA did not differ between groups. I/R caused a strong decrease of relative Cx43 protein expression in the AAR that was partly abolished by RIPC. Furthermore, RIPC decreased the level of ischemia-induced dephosphorylation of Cx43. Confocal immunofluorescence staining showed that I/R caused a loss of the Cx43 signal at the intercalated discs, while the Cx43 signal at the intercalated discs was partly sustained after RIPC.
Preservation of Cx43 protein expression and phosphorylation after RIPC might protect the rat heart in vivo.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-014-0228-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4256705  PMID: 25159820
Cardioprotection; Connexin 43 (Cx43); Remote ischemic preconditioning (RIPC)
16.  Remote ischemic preconditioning delays the onset of acute mountain sickness in normobaric hypoxia 
Physiological Reports  2015;3(3):e12325.
Acute mountain sickness (AMS) is a neurological disorder occurring when ascending too fast, too high. Remote ischemic preconditioning (RIPC) is a noninvasive intervention protecting remote organs from subsequent hypoxic damage. We hypothesized that RIPC protects against AMS and that this effect is related to reduced oxidative stress. Fourteen subjects were exposed to 18 hours of normoxia (21% oxygen) and 18 h of normobaric hypoxia (12% oxygen, equivalent to 4500 m) on different days in a blinded, randomized order. RIPC consisted of four cycles of lower limb ischemia (5 min) and 5 min of reperfusion, and was performed immediately before the study room was entered. A control group was exposed to hypoxia (12% oxygen, n = 14) without RIPC. AMS was evaluated by the Lake Louise score (LLS) and the AMS-C score of the Environmental Symptom Questionnaire. Plasma concentrations of ascorbate radicals, oxidized sulfhydryl (SH) groups, and electron paramagnetic resonance (EPR) signal intensity were measured as biomarkers of oxidative stress. RIPC reduced AMS scores (LLS: 1.9 ± 0.4 vs. 3.2 ± 0.5; AMS-C score: 0.4 ± 0.1 vs. 0.8 ± 0.2), ascorbate radicals (27 ± 7 vs. 65 ± 18 nmol/L), oxidized SH groups (3.9 ± 1.4 vs. 14.3 ± 4.6 μmol/L), and EPR signal intensity (0.6 ± 0.2 vs. 1.5 ± 0.4 × 106) after 5 h in hypoxia (all P < 0.05). After 18 hours in hypoxia there was no difference in AMS and oxidative stress between RIPC and control. AMS and plasma markers of oxidative stress did not correlate. This study demonstrates that RIPC transiently reduces symptoms of AMS and that this effect is not associated with reduced plasma levels of reactive oxygen species.
PMCID: PMC4393159  PMID: 25742960
AMS; high altitude; oxidative stress; prevention; reactive oxygen species
17.  Remote ischemic preconditioning to reduce contrast-induced nephropathy: study protocol for a randomized controlled trial 
Trials  2014;15:119.
Despite the increasing use of pre- and posthydration protocols and low-osmolar instead of high-osmolar iodine-containing contrast media, the incidence of contrast-induced nephropathy (CIN) is still significant. There is evidence that contrast media cause ischemia-reperfusion injury of the medulla. Remote ischemic preconditioning (RIPC) is a non-invasive, safe, and low-cost method to reduce ischemia-reperfusion injury.
The RIPCIN study is a multicenter, single-blinded, randomized controlled trial in which 76 patients at risk of CIN will receive standard hydration combined with RIPC or hydration with sham preconditioning. RIPC will be applied by four cycles of 5 min ischemia and 5 min reperfusion of the forearm by inflating a blood pressure cuff at 50 mmHg above the actual systolic pressure. The primary outcome measure will be the change in serum creatinine from baseline to 48 to 72 h after contrast administration.
A recent pilot study reported that RIPC reduced the incidence of CIN after coronary angioplasty. The unusual high incidence of CIN in this study is of concern and limits its generalizability. Therefore, we propose a randomized controlled trial to study whether RIPC reduces contrast-induced kidney injury in patients at risk for CIN according to the Dutch guidelines.
Trial registration
Current Controlled Trials ISRCTN76496973
PMCID: PMC3990011  PMID: 24721127
Contrast-induced nephropathy; Remote ischemic preconditioning; Acute kidney injury; Pre- and posthydration; Randomized controlled trial
18.  Remote ischemic preconditioning confers late protection against myocardial ischemia-reperfusion injury in mice by upregulating interleukin-10 
Basic research in cardiology  2012;107(4):277.
Remote ischemic preconditioning (RIPC) induces a prolonged late phase of multi-organ protection against ischemia-reperfusion (IR) injury. In the present study, we tested the hypothesis that RIPC confers late protection against myocardial IR injury by upregulating expression of interleukin (IL)-10. Mice were exposed to lower limb RIPC or sham ischemia. After 24 h, mice with RIPC demonstrated decreased myocardial infarct size and improved cardiac contractility following 30-min ischemia and 120-min reperfusion (I-30/R-120). These effects of RIPC were completely blocked by anti-IL-10 receptor antibodies. In IL-10 knockout mice, RIPC cardioprotection was lost, but it was mimicked by exogenous IL-10. Administration of IL-10 to isolated perfused hearts increased phosphory-lation of the protein kinase Akt and limited infarct size after I-30/R-120. In wild-type mice, RIPC increased plasma and cardiac IL-10 protein levels and caused activation of Akt and endothelial nitric oxide synthase in the heart at 24 h, which was also blocked by anti-IL-10 receptor antibodies. In the gastrocnemius muscle, RIPC resulted in immediate inactivation of the phosphatase PTEN and activation of Stat3, with increased IL-10 expression 24 h later. Myocyte-specific PTEN inactivation led to increased Stat3 phosphorylation and IL-10 protein expression in the gastrocnemius muscle. Taken together, these results suggest that RIPC induces late protection against myocardial IR injury by increasing expression of IL-10 in the remote muscle, followed by release of IL-10 into the circulation, and activation of protective signaling pathways in the heart. This study provides a scientific basis for the use of RIPC to confer systemic protection against IR injury.
PMCID: PMC3596418  PMID: 22752341
Remote ischemic preconditioning; Interleukin-10; Reperfusion injury; Phosphatase and tensin homologue deleted on chromosome ten; Stat3
19.  Effect of ischemic and pharmacological preconditioning of lower limb muscle tissue on tissue oxygenation measured by near-infrared spectroscopy – a pilot study 
BMC Anesthesiology  2014;14:54.
Ischemic or volatile anesthetic preconditioning is defined as tissue protection from impending ischemic cell damage by repetitive short periods of tissue exposure to ischemia or volatile anesthetics. Objective of this study was to elucidate, if ischemic preconditioning and pharmacological preconditioning with sevoflurane have effects on muscle tissue oxygen saturation in patients undergoing surgical revascularization of the lower limb.
In this prospective randomized pilot study ischemic and pharmacological (sevoflurane) preconditioning was performed in 40 patients with lower limb arterial occlusive disease undergoing surgical revascularization. Sevoflurane preconditioning was performed in one group (N = 20) by repetitive application of sevoflurane for six minutes interspersed by six minutes of washout. Thereafter, ischemic preconditioning was performed in all patients (N = 40) by repetitive clamping of the femoral artery for six minutes interspersed by six minutes of reperfusion. The effect of both procedures on leg muscle tissue oxygen saturation (rSO2) was measured by near-infrared spectroscopy during both procedures and during surgery and reperfusion (INVOS® 5100C Oxymeter with Small Adult SomaSensor® SAFB-SM, Somanetics, Troy, Michigan, USA).
Repetitive clamping and reperfusion of the femoral artery resulted in significant cyclic decrease and increase of muscle rSO2 (p < 0.0001). Pharmacological preconditioning with sevoflurane resulted in a faster and higher increase of rSO2 during postoperative reperfusion (Maximal 111% baseline ± 20 versus 103% baseline ± 14, p = 0.008) consistent with an additional effect of pharmacological preconditioning on leg perfusion.
Ischemic preconditioning of lower limb muscle tissue and pharmacological preconditioning with sevoflurane have an effect on tissue oxygenation in patients with lower limb occlusive arterial disease.
Trial registration
The trial has been registrated at, Trial Number: NCT02038062 at 14 January 2014.
PMCID: PMC4134469  PMID: 25132803
Ischemic preconditioning; Sevoflurane; Surgical revascularization; Arterial occlusive disease
20.  Is remote ischaemic preconditioning of benefit to patients undergoing cardiac surgery? 
A best evidence topic in cardiac surgery was written according to a structured protocol. The question addressed was whether remote ischaemic preconditioning (RIPC) is of benefit to patients undergoing cardiac surgery. Altogether, more than 264 papers were found using the reported search, 16 of which represented the best evidence to answer the clinical question. The authors, journal, date and country of publication, patient group studied, study type, relevant outcomes and results of these papers are tabulated. We conclude that RIPC is a safe protocol which could potentially be used in cardiac surgery to provide additional cardiac protection against ischaemia reperfusion injury, although it may not be appropriate for patients on K+ ATPase channel blockers (sulphonylureas) as they seem to eliminate the effect of RIPC. In our study, we found two meta-analyses of cardiac surgery with or without RIPC. Both unequivocally showed 0.81 and 0.74 standardized mean reduction in myocardial necrosis markers in patients receiving RIPC and cardiac or vascular surgery. No difference in perioperative myocardial infarction incidence or 30-day mortality were found. In adult cardiac surgery, we found 11 randomized control trials (RCTs) ranging in size from 45 to 162 patients. Two representative studies reported no difference in postoperative cardiac troponin I concentration in RIPC vs. controls. In one of the studies (CABG ± RIPC) no additional benefit could have been observed for RIPC regarding intra-aortic balloon pump usage (controls 8.5 vs. RIPC 7.5%), inotropic support (39 vs. 50%) or vasoconstrictor usage (66 vs. 64%). On the other hand, in the other study [CABG ± AVR (aortic valve replacement)  ± RIPC] significant reduction of troponin I at 8 h postoperatively (controls, 2.90 µg/l vs. RIPC, 2.54 µg/l, P = 0.043) was shown. Marked reduction in cardiac necrosis markers was also found in several smaller RCTs concerning coronary artery bypass grafting (CABG) patients receiving RIPC preoperatively: with cold crystalloid cardioplegia (44.5% reduction), with cross-clamping and fibrillation (43% reduction) and with cold blood cardioplegia (42.4% reduction). The proof of concept trials summarized here give some early evidence that RIPC may potentially provide some reduction in myocardial injury. If confirmed, in future clinical studies this technique may one day lead to a method to reduce reperfusion injury in clinical practice.
PMCID: PMC3329313  PMID: 22286602
Review; Myocardial protection; Remote ischaemic preconditioning
21.  Liver ischemia preconditions the heart against ischemia-reperfusion arrhythmias 
This study aimed to examine the hypothesis that an antiarrhythmic effect might be obtained by ischemic preconditioning of the liver, and also to characterize the potential underlying mechanisms.
Materials and Methods:
Male Wistar rats were anesthetized by thiopental sodium (50 mg/kg, IP) followed by IV injection of heparin (250 IU). Remote ischemic preconditioning (RIPC) was induced by 3 cycles of 5 min liver ischemia followed by 5 min of reperfusion. The hearts were excised within 5 min after the final cycle of preconditioning and perfused using Langendorff’s system. The isolated perfused hearts were subjected to 30 min global ischemia followed by 90 min reperfusion. The myocardial arrhythmias induced by ischemia- reperfusion (I/R) were determined in accordance with the guidelines of Lambeth Conventions. The potential role of KATP channels on RIPC was assessed by injection of glibenclamide (nonselective KATP blocker) or 5-hydroxydecanoate (mitochondrial KATP blocker) on rats 30 and 15 min before induction of RIPC in the liver, respectively.
Hepatic remote preconditioning of the heart significantly (P<0.0001) prevented the incidence of myocardial arrhythmias induced by I/R in the perfused hearts (5.33±1.54 vs. 32.33±6.44,). However, the protective effects of remote preconditioning was significantly (P<0.01) abolished by the KATP blocker, glibenclamide (25.5±4.9 vs. 5.33±1.54,).
Hepatic RIPC may prevent the arrhythmias induced by I/R in the isolated perfused hearts via KATP channels.
PMCID: PMC4366748  PMID: 25810880
Ischemia-reperfusion arrhythmia; Isolated heart; Liver; Remote ischemic; preconditioning
22.  Effect of remote ischemic preconditioning on cognitive function after off-pump coronary artery bypass graft: a pilot study 
Korean Journal of Anesthesiology  2013;65(5):418-424.
Several studies have shown in animal models that remote ischemic preconditioning (rIPC) has a neuroprotective effect. However, a randomized controlled trial in human subjects to investigate the neuroprotective effect of rIPC after cardiac surgery has not yet been reported. Therefore, we performed this pilot study to determine whether rIPC reduced the occurrence of postoperative cognitive dysfunction in patients who underwent off-pump coronary artery bypass graft (OPCAB) surgery.
Seventy patients who underwent OPCAB surgery were assigned to either the control or the rIPC group using a computer-generated randomization table. The application of rIPC consisted of four cycles of 5 min ischemia and 5 min reperfusion on an upper limb using a blood pressure cuff inflating 200 mmHg before coronary artery anastomosis. The cognitive function tests were performed one day before surgery and again on postoperative day 7. We defined postoperative cognitive dysfunction as decreased postoperative test values more than 20% of the baseline values in more than two of the six cognitive function tests that were performed.
In the cognitive function tests, there were no significant differences in the results obtained during the preoperative and postoperative periods for all tests and there were no mean differences observed in the preoperative and postoperative scores. The incidences of postoperative cognitive dysfunction in the control and rIPC groups were 28.6% (10 patients) and 31.4% (11 patients), respectively.
rIPC did not reduce the incidence of postoperative cognitive dysfunction after OPCAB surgery during the immediate postoperative period.
PMCID: PMC3866337  PMID: 24363844
Cognitive disorders; Neuroprotective effect; Off-pump coronary artery bypass
23.  No Evidence for Activated Autophagy in Left Ventricular Myocardium at Early Reperfusion with Protection by Remote Ischemic Preconditioning in Patients Undergoing Coronary Artery Bypass Grafting 
PLoS ONE  2014;9(5):e96567.
Remote ischemic preconditioning (RIPC) by repeated brief limb ischemia/reperfusion reduces myocardial injury in patients undergoing coronary artery bypass grafting (CABG). Activation of signal transducer and activator of transcription 5 (STAT5) in left ventricular (LV) myocardium at early reperfusion is associated with such protection. Autophagy, i.e., removal of dysfunctional cellular components through lysosomes, has been proposed as one mechanism of cardioprotection. Therefore, we analyzed whether or not the protection by RIPC is associated with activated autophagy.
CABG patients were randomized to undergo RIPC (3×5 min blood pressure cuff inflation/5 min deflation) or placebo (cuff deflated) before skin incision (n = 10/10). Transmural myocardial biopsies were taken from the LV before cardioplegia (baseline) and at early (5–10 min) reperfusion. RIPC-induced protection was reflected by decreased serum troponin I concentration area under the curve (194±17 versus 709±129 ng/ml × 72 h, p = 0.002). Western blotting for beclin-1-phosphorylation and protein expression of autophagy-related gene 5–12 (ATG5-12) complex, light chain 3 (LC3), parkin, and p62 was performed. STAT3-, STAT5- and extracellular signal-regulated protein kinase 1/2 (ERK1/2)-phosphorylation was used as positive control to confirm signal activation by ischemia/reperfusion.
Signals of all analyzed autophagy proteins did not differ between baseline and early reperfusion and not between RIPC and placebo. STAT5-phosphorylation was greater at early reperfusion only with RIPC (2.2-fold, p = 0.02). STAT3- and ERK1/2-phosphorylation were greater at early reperfusion with placebo and RIPC (≥2.7-fold versus baseline, p≤0.05).
Protection through RIPC in patients undergoing CABG surgery does not appear to be associated with enhanced autophagy in LV myocardium at early reperfusion.
PMCID: PMC4010496  PMID: 24797938
24.  Cardioprotective effect of remote preconditioning of trauma and remote ischemia preconditioning in a rat model of myocardial ischemia/reperfusion injury 
Remote ischemia preconditioning (RIPC) and remote preconditioning of trauma (RPCT) are two methods used to induce a cardioprotective function against ischemia/reperfusion injury (IRI). However, the underlying mechanisms of these two methods differ. The aim of the present study was to investigate the cardioprotective function of the two methods, and also observe whether combining RIPC with RPCT enhanced the protective effect. In total, 70 male Sprague Dawley rats were randomly divided into five groups, which included the sham, control, RIPC + RPCT, RPCT and RIPC groups. With the exception of the sham group, all the rats were subjected to myocardial IRI through the application of 30 min occlusion of the left coronary artery and 180 min reperfusion. Serum cardiac troponin I (cTnI) levels, myocardial infarct size (IS) and the cardiomyocyte apoptotic index (AI) were assessed. The levels of serum cTnI were lower in the experimental groups when compared with the control group (control, 58.59±12.50 pg/ml; RIPC + RPCT, 46.05±8.62 pg/ml; RPCT, 45.98±11.24 pg/ml; RIPC, 43.46±5.05 pg/ml; P<0.05, vs. control), and similar results were observed for the myocardial IS (control, 48.34±6.79%; RIPC + RPCT, 29.64±4.51%; RPCT, 29.05±8.51%; RIPC, 27.72±6.27%; P<0.05, vs. control) and the AI (control, 31.75±10.65%; RIPC + RPCT, 18.32±9.30%; RPCT, 18.51±9.26%; RIPC, 20.41±3.86%; P<0.05, vs. control). However, no statistically significant differences were observed among the three experimental groups (P>0.05). Therefore, RIPC and RPCT exhibit cardioprotective effects when used alone or in combination. However, a combination of RIPC and RPCT does not enhance the cardioprotective effect observed with the application of either single method. Therefore, for patients undergoing major abdominal surgery, RIPC was considered to be unnecessary, while for patients undergoing other types of non-cardiac major surgery and minimally invasive interventional surgery, RIPC may be useful. In addition, patients with embolism diseases are also liable to IRI when reperfusion treatment such as thrombolysis is conducted. Thus RIPC may also be beneficial for these patients.
PMCID: PMC4471708  PMID: 26136887
ischemia/reperfusion injury; ischemia preconditioning; remote ischemia preconditioning; remote preconditioning of trauma; myocardioprotection
25.  Remote preconditioning, perconditioning, and postconditioning: a comparative study of their cardioprotective properties in rat models 
Clinics  2013;68(2):263-268.
Ischemia reperfusion injury is partly responsible for the high mortality associated with induced myocardial injury and the reduction in the full benefit of myocardial reperfusion. Remote ischemic preconditioning, perconditioning, and postconditioning have all been shown to be cardioprotective. However, it is still unknown which one is the most beneficial. To examine this issue, we used adult male Wistar rat ischemia reperfusion models to compare the cardioprotective effect of these three approaches applied on double-sided hind limbs.
The rats were randomly distributed to the following five groups: sham, ischemia reperfusion, remote preconditioning, remote perconditioning, and remote post-conditioning. The ischemia/reperfusion model was established by sternotomy followed by a 30-min ligation of the left coronary artery and a subsequent 3-h reperfusion. Remote conditioning was induced with three 5-min ischemia/5-min reperfusion cycles of the double-sided hind limbs using a tourniquet.
A lower early reperfusion arrhythmia score (1.50±0.97) was found in the rats treated with remote perconditioning compared to those in the ischemia reperfusion group (2.33±0.71). Meanwhile, reduced infarct size was also observed (15.27±5.19% in remote perconditioning, 14.53±3.45% in remote preconditioning, and 19.84±5.85% in remote post-conditioning vs. 34.47±7.13% in ischemia reperfusion, p<0.05), as well as higher expression levels of the apoptosis-relevant protein Bcl-2/Bax following global (ischemia/reperfusion) injury in in vivo rat heart models (1.255±0.053 in remote perconditioning, 1.463±0.290 in remote preconditioning, and 1.461±0.541 in remote post-conditioning vs. 1.003±0.159 in ischemia reperfusion, p<0.05).
Three remote conditioning strategies implemented with episodes of double-sided hind limb ischemia/reperfusion have similar therapeutic potential for cardiac ischemia/reperfusion injury, and remote perconditioning has a greater ability to prevent reperfusion arrhythmia.
PMCID: PMC3584272  PMID: 23525325
Cardioprotective Property; Ischemia/Reperfusion Injury; Models

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