Inhibition of proline-rich tyrosine kinase 2 restores cardioprotection by remote ischaemic preconditioning in type 2 diabetes.

BACKGROUND AND PURPOSE: Remote ischaemic preconditioning (rIPC) for cardioprotection is severely impaired in diabetes, and therapeutic options to restore it are lacking. The vascular endothelium plays a key role in rIPC. Given that the activity of endothelial nitric oxide synthase (eNOS) is inhibited by proline-rich tyrosine kinase 2 (Pyk2), we hypothesized that pharmacological Pyk2 inhibition could restore eNOS activity and thus restore remote cardioprotection in diabetes. EXPERIMENTAL APPROACH: New Zealand obese (NZO) mice that demonstrated key features of diabetes were studied. The consequence of Pyk2 inhibition on endothelial function, rIPC and infarct size after myocardial infarction were evaluated. The impact of plasma from mice and humans with or without diabetes was assessed in isolated buffer perfused murine hearts and aortic rings. KEY RESULTS: Plasma from nondiabetic mice and humans, both subjected to rIPC, caused remote tissue protection. Similar to diabetic humans, NZO mice demonstrated endothelial dysfunction. NZO mice had reduced circulating nitrite levels, elevated arterial blood pressure and a larger infarct size after ischaemia and reperfusion than BL6 mice. Pyk2 increased the phosphorylation of eNOS at its inhibitory site (Tyr656), limiting its activity in diabetes. The cardioprotective effects of rIPC were abolished in diabetic NZO mice. Pharmacological Pyk2 inhibition restored endothelial function and rescued cardioprotective effects of rIPC. CONCLUSION AND IMPLICATIONS: Endothelial function and remote tissue protection are impaired in diabetes. Pyk2 is a novel target for treating endothelial dysfunction and restoring cardioprotection through rIPC in diabetes.

NGAL in the Development of Acute Kidney Injury in a Murine Model of Remote Ischaemic Preconditioning and Liver Ischaemia Reperfusion.

Acute kidney injury (AKI) is common following liver transplantation and is associated with liver ischeamia reperfusion (IR) injury. The purpose of this study was to use a mouse model of liver IR injury and AKI to study the role of Neutrophil Gelatinase Associated Lipocalin (NGAL), a biomarker of AKI, in liver IR injury and AKI. We demonstrate an adapted, reproducible model of liver IR injury and AKI in which remote ischemic preconditioning (RIPC) by repeated episodes of hindleg ischemia prior to liver IR reduced the severity of the IR injury. In this model, serum NGAL at 2 h post reperfusion correlated with AKI development early following IR injury. This early rise in serum NGAL was associated with hepatic but not renal upregulation of NGAL mRNA, suggesting NGAL production in the liver but not the kidney in the early phase post liver IR injury.

Changes in excitatory amino acid transporters in response to remote ischaemic preconditioning and glutamate excitotoxicity.

The successful implementation of remote ischaemic conditioning as a clinical neuroprotective strategy requires a thorough understanding of its basic principles, which can be modified for each patient. The mechanisms of glutamate homeostasis appear to be a key component. In the current study, we focused on the brain-to-blood glutamate shift mediated by glutamate transporters (excitatory amino acid transports [EAATs]) and the effect of remote ischaemic preconditioning (RIPC) as a mediator of ischaemic tolerance. We used model mimicking ischaemia-mediated excitotoxicity (intracerebroventricular administration of glutamate) to avoid the indirect effect of ischaemia-triggered mechanisms. We found quantitative changes in EAAT2 and EAAT3 and altered membrane trafficking of EAAT1 on the cells of the choroid plexus. These changes could underlie the beneficial effects of ischaemic tolerance. There was reduced oxidative stress and increased glutathione level after RIPC treatment. Moreover, we determined the stimulus-specific response on EAATs. While glutamate overdose stimulated EAAT2 and EAAT3 overexpression, RIPC induced membrane trafficking of EAAT1 and EAAT2 rather than a change in their expression. Taken together, mechanisms related to glutamate homeostasis, especially EAAT-mediated transport, represents a powerful tool of ischaemic tolerance and allow a certain amount of flexibility based on the stimulus used.

Remote Ischaemic Preconditioning Accelerates Brain to Blood Glutamate Efflux via EAATs-mediated Transport.

Remote ischaemic conditioning (RIC) becomes an attractive strategy for the endogenous stimulation of mechanisms protecting neurons against ischaemia. Although the processes underlying the RIC are not clearly understood, the homeostasis of glutamate seems to play an important role. The present study is focused on the investigation of the brain to blood efflux of glutamate in a condition mimicking ischaemia-mediated excitotoxicity and remote ischaemic preconditioning (RIPC). The animals were pre-treated with a hind-limb tourniquet one hour before the intraventricular administration of glutamate and its release was monitored as the concentration of glutamate/glutathione in blood and liquor for up to 1 h. The transport mediated by excitatory amino acid transporters (EAATs) was verified by their inhibition with Evans Blue intraventricular co-administration. RIPC mediated the efflux of glutamate exceeding from CSF to blood in the very early stage of intoxication. As a consequence, the blood level of glutamate rose in a moment. EAATs inhibition confirmed the active role of glutamate transporters in this process. In the blood, elevated levels of glutamate served as a relevant source of antioxidant glutathione for circulating cells in RIPC-treated individuals. All of those RIPC-mediated recoveries in processes of glutamate homeostasis reflect the improvement of oxidative stress, suggesting glutamate-accelerated detoxication to be one of the key mechanisms of RIPC-mediated neuroprotection.

Cardioprotective efficacy of limb remote ischaemic preconditioning in rats: discrepancy between a meta-analysis and a three-centre in vivo study.

AIMS: Remote ischaemic preconditioning (RIPC) is a robust cardioprotective intervention in preclinical studies. To establish a working and efficacious RIPC protocol in our laboratories, we performed randomized, blinded in vivo studies in three study centres in rats, with various RIPC protocols. To verify that our experimental settings are in good alignment with in vivo rat studies showing cardioprotection by limb RIPC, we performed a systematic review and meta-analysis. In addition, we investigated the importance of different study parameters. METHODS AND RESULTS: Male Wistar rats were subjected to 20-45 min cardiac ischaemia followed by 120 min reperfusion with or without preceding RIPC by 3 or 4 × 5-5 min occlusion/reperfusion of one or two femoral vessels by clamping, tourniquet, or pressure cuff. RIPC did not reduce infarct size (IS), microvascular obstruction, or arrhythmias at any study centres. Systematic review and meta-analysis focusing on in vivo rat models of myocardial ischaemia/reperfusion injury with limb RIPC showed that RIPC reduces IS by 21.28% on average. In addition, the systematic review showed methodological heterogeneity and insufficient reporting of study parameters in a high proportion of studies. CONCLUSION: We report for the first time the lack of cardioprotection by RIPC in rats, assessed in individually randomized, blinded in vivo studies, involving three study centres, using different RIPC protocols. These results are in discrepancy with the meta-analysis of similar in vivo rat studies; however, no specific methodological reason could be identified by the systematic review, probably due to the overall insufficient reporting of several study parameters that did not improve over the past two decades. These results urge for publication of more well-designed and well-reported studies, irrespective of the outcome, which are required for preclinical reproducibility, and the development of clinically translatable cardioprotective interventions.

Strategies for post-cardiac surgery acute kidney injury prevention: A network meta-analysis of randomized controlled trials.

Objects: Cardiac surgery is associated with acute kidney injury (AKI). However, the effects of various pharmacological and non-pharmacological strategies for AKI prevention have not been thoroughly investigated, and their effectiveness in preventing AKI-related adverse outcomes has not been systematically evaluated. Methods: Studies from PubMed, Embase, and Medline and registered trials from published through December 2021 that evaluated strategies for preventing post-cardiac surgery AKI were identified. The effectiveness of these strategies was assessed through a network meta-analysis (NMA). The secondary outcomes were prevention of dialysis-requiring AKI, mortality, intensive care unit (ICU) length of stay (LOS), and hospital LOS. The interventions were ranked using the P-score method. Confidence in the results of the NMA was assessed using the Confidence in NMA (CINeMA) framework. Results: A total of 161 trials (involving 46,619 participants) and 53 strategies were identified. Eight pharmacological strategies {natriuretic peptides [odds ratio (OR): 0.30, 95% confidence interval (CI): 0.19-0.47], nitroprusside [OR: 0.29, 95% CI: 0.12-0.68], fenoldopam [OR: 0.36, 95% CI: 0.17-0.76], tolvaptan [OR: 0.35, 95% CI: 0.14-0.90], N-acetyl cysteine with carvedilol [OR: 0.37, 95% CI: 0.16-0.85], dexmedetomidine [OR: 0.49, 95% CI: 0.32-0.76;], levosimendan [OR: 0.56, 95% CI: 0.37-0.84], and erythropoietin [OR: 0.62, 95% CI: 0.41-0.94]} and one non-pharmacological intervention (remote ischemic preconditioning, OR: 0.76, 95% CI: 0.63-0.92) were associated with a lower incidence of post-cardiac surgery AKI with moderate to low confidence. Among these nine strategies, five (fenoldopam, erythropoietin, natriuretic peptides, levosimendan, and remote ischemic preconditioning) were associated with a shorter ICU LOS, and two (natriuretic peptides [OR: 0.30, 95% CI: 0.15-0.60] and levosimendan [OR: 0.68, 95% CI: 0.49-0.95]) were associated with a lower incidence of dialysis-requiring AKI. Natriuretic peptides were also associated with a lower risk of mortality (OR: 0.50, 95% CI: 0.29-0.86). The results of a sensitivity analysis support the robustness and effectiveness of natriuretic peptides and dexmedetomidine. Conclusion: Nine potentially effective strategies were identified. Natriuretic peptide therapy was the most effective pharmacological strategy, and remote ischemic preconditioning was the only effective non-pharmacological strategy. Preventive strategies might also help prevent AKI-related adverse outcomes. Additional studies are required to explore the optimal dosages and protocols for potentially effective AKI prevention strategies.

Negative interaction between nitrates and remote ischemic preconditioning in patients undergoing cardiac surgery: the ERIC-GTN and ERICCA studies.

Remote ischaemic preconditioning (RIPC) using transient limb ischaemia failed to improve clinical outcomes following cardiac surgery and the reasons for this remain unclear. In the ERIC-GTN study, we evaluated whether concomitant nitrate therapy abrogated RIPC cardioprotection. We also undertook a post-hoc analysis of the ERICCA study, to investigate a potential negative interaction between RIPC and nitrates on clinical outcomes following cardiac surgery. In ERIC-GTN, 185 patients undergoing cardiac surgery were randomized to: (1) Control (no RIPC or nitrates); (2) RIPC alone; (3); Nitrates alone; and (4) RIPC + Nitrates. An intravenous infusion of nitrates (glyceryl trinitrate 1 mg/mL solution) was commenced on arrival at the operating theatre at a rate of 2-5 mL/h to maintain a mean arterial pressure between 60 and 70 mmHg and was stopped when the patient was taken off cardiopulmonary bypass. The primary endpoint was peri-operative myocardial injury (PMI) quantified by a 48-h area-under-the-curve high-sensitivity Troponin-T (48 h-AUC-hs-cTnT). In ERICCA, we analysed data for 1502 patients undergoing cardiac surgery to investigate for a potential negative interaction between RIPC and nitrates on clinical outcomes at 12-months. In ERIC-GTN, RIPC alone reduced 48 h-AUC-hs-cTnT by 37.1%, when compared to control (ratio of AUC 0.629 [95% CI 0.413-0.957], p = 0.031), and this cardioprotective effect was abrogated in the presence of nitrates. Treatment with nitrates alone did not reduce 48 h-AUC-hs-cTnT, when compared to control. In ERICCA there was a negative interaction between nitrate use and RIPC for all-cause and cardiovascular mortality at 12-months, and for risk of peri-operative myocardial infarction. RIPC alone reduced the risk of peri-operative myocardial infarction, compared to control, but no significant effect of RIPC was demonstrated for the other outcomes. When RIPC and nitrates were used together they had an adverse impact in patients undergoing cardiac surgery with the presence of nitrates abrogating RIPC-induced cardioprotection and increasing the risk of mortality at 12-months post-cardiac surgery in patients receiving RIPC.

Remote ischaemic preconditioning - translating cardiovascular benefits to humans.

Remote ischaemic preconditioning (RIPC), induced by intermittent periods of limb ischaemia and reperfusion, confers cardiac and vascular protection from subsequent ischaemia-reperfusion (IR) injury. Early animal studies reliably demonstrate that RIPC attenuated infarct size and preserved cardiac tissue. However, translating these adaptations to clinical practice in humans has been challenging. Large clinical studies have found inconsistent results with respect to RIPC eliciting IR injury protection or improving clinical outcomes. Follow-up studies have implicated several factors that potentially affect the efficacy of RIPC in humans such as age, fitness, frequency, disease state and interactions with medications. Thus, realizing the clinical potential for RIPC may require a human experimental model where confounding factors are more effectively controlled and underlying mechanisms can be further elucidated. In this review, we highlight recent experimental findings in the peripheral circulation that have added valuable insight on the mechanisms and clinical benefit of RIPC in humans. Central to this discussion is the critical role of timing (i.e. immediate vs. delayed effects following a single bout of RIPC) and the frequency of RIPC. Limited evidence in humans has demonstrated that repeated bouts of RIPC over several days uniquely improves vascular function beyond that observed with a single bout alone. Since changes in resistance vessel and microvascular function often precede symptoms and diagnosis of cardiovascular disease, repeated bouts of RIPC may be promising as a preclinical intervention to prevent or delay cardiovascular disease progression.

Repeated remote ischaemic preconditioning can prevent acute mountain sickness after rapid ascent to a high altitude.

BACKGROUND: This study assessed the effectiveness of 4 different repeated remote ischaemic preconditioning (RIPC) protocols varying in duration and frequency for preventing acute mountain sickness (AMS) after rapid ascent to high altitude. METHODS: In a randomized but not blinded design, participants were assigned to receive either of the four RIPC treatments at low altitude (Group A, once daily for 1 week; Group B, twice daily for 1 week; Group C, once daily for 4 weeks; and Group D, twice daily for 4 weeks) or control (no specific sham treatment). Participants were then flown to a high altitude (3650 m). The primary outcome was the incidence and severity of AMS evaluated by the Lake Louise score (LLS) after arrival; vital signs were collected simultaneously. RESULTS: A total of 250 participants (50 per group; mean age 38.56 ± 0.76 years) were included. The overall AMS incidence was 26.4%. A total of 20 AMS cases (40%) occurred in the control group, 15 cases (30%) both in the RIPC A and RIPC B groups (RR 1.3; 95%CI 0.8-2.3; χ2 = 1.099; p = 0.29), and 8 cases (16%) both in the RIPC C and D groups (RR 2.5; 95%CI 1.2 - 5.2; χ2 = 7.143, p < 0.01), with significantly lower LLSs in the RIPC C and D groups (F = 6.51, p < 0.001). The scores of gastrointestinal symptoms (F = 7.42, p < 0.001) and dizziness (F = 9.82, p < 0.001) but not headache (F = 0.60, p > 0.05) were lower in the RIPC groups compared to control. The blood oxygen saturation level (SpO2) decreased less in the RIPC B, C and D groups compared to control after arrival at a high altitude (F = 11.42, p < 0.001). The number of RIPC treatments received was moderately correlated with SpO2 (R = 0.38, p < 0.001), and SpO2 was moderately inversely correlated with the LLS (R = -0.48, p < 0.001). CONCLUSION: This study demonstrated that a four-week RIPC intervention but not a one-week regimen reduced AMS incidence and severity; however, a placebo effect might have contributed to these results.

HIF-1α mediates the protective effect of plasma extracellular particles induced by remote ischaemic preconditioning on oxidative stress injury in human umbilical vein endothelial cells.

Remote ischaemic preconditioning (RIPC) is considered to alleviate myocardial ischaemia/reperfusion (I/R) injury. The present study explored whether blood plasma particulate matter, which is termed extracellular particles (EPs), and is released from cells during RIPC, could reduce H2O2-induced damage in human umbilical vein endothelial cells (HUVECs). Firstly, EPs were derived from volunteers who did or did not undergo RIPC. To induce RIPC in volunteers, a blood pressure cuff was alternatively inflated for 5 min and deflated for the same duration for four successive cycles. HUVECs were assigned to two groups: i) Group 1 was preincubated for 24 h with EPs from volunteers after sham-RIPC, then treated with H2O2 (1 mM; 6 h) to mimic the in vivo conditions of I/R-induced oxidative stress; and ii) group 2 was preincubated for 24 h with EPs from volunteers after RIPC, then treated with H2O2. Subsequently, EPs were derived from rats received sham-RIPC or RIPC and/or cadmium (Cd) pre-treatment. To induce RIPC in rats, a remote hind limb preconditioning stimulus was delivered using a blood pressure cuff attached at the inguinal level of the rat. The blood pressure cuff was alternatively inflated for 5 min and deflated for the same time period for four successive cycles. HUVECs were assigned to six groups: i) Group 1 was untreated; ii) group 2 received only H2O2 treatment (1 mM; 6 h); iii) group 3 was preincubated for 24 h with EPs from rats exposed to sham-RIPC, then treated with H2O2; iv) group 4 was preincubated for 24 h with EPs from rats that received an intraperitoneal injection of 1 mg/kg Cd [a pharmacological inhibitor of hypoxia-inducible factor 1-α (HIF-1α) in vivo] 180 min before sham-RIPC, then treated with H2O2; v) group 5 was preincubated for 24 h with EPs from rats exposed to RIPC, then treated with H2O2; and vi) group 6 was preincubated for 24 h with EPs from rats that received an intraperitoneal injection of 1 mg/kg Cd 180 min before RIPC, then treated with H2O2. Cell viability and cytotoxicity were monitored using Cell Counting Kit-8 and lactate dehydrogenase assays. Cell apoptosis and necrosis were assessed via flow cytometry and western blot analysis. A notable increase in EP concentration in the plasma of volunteers after RIPC compared with that in the plasma of volunteers after sham-RIPC was observed. RIPC-associated EPs (RIPC-EPs) from volunteers could improve cell viability and reduce cytotoxicity, cell apoptosis and necrosis in HUVECs treated with H2O2 in vitro. Furthermore, RIPC caused a significant increase in HIF-1α expression in the rat limb musculature. The apoptosis-reducing effect of RIPC-EPs was demonstrated to be counteracted by an intraperitoneal injection of Cd before RIPC in rats. A significant decrease in the EP levels precipitated from the plasma of rats that received Cd treatment before RIPC was observed compared with rats that did not receive Cd treatment. The present study suggested that HIF-1α mediated at least partly the protective effect of plasma RIPC-EPs on oxidative stress injury in HUVECs.

Effects of Remote Ischaemic Preconditioning on the Internal Thoracic Artery Nitric Oxide Synthase Isoforms in Patients Undergoing Coronary Artery Bypass Grafting.

Remote ischaemic preconditioning (RIPC) is a medical procedure that consists of repeated brief periods of transient ischaemia and reperfusion of distant organs (limbs) with the ability to provide internal organ protection from ischaemia. Even though RIPC has been successfully applied in patients with myocardial infarction during coronary revascularization (surgery/percutaneous angioplasty), the underlying molecular mechanisms are yet to be clarified. Thus, our study aimed to determine the role of nitric oxide synthase (NOS) isoforms in RIPC-induced protection (3 × 5 min of forearm ischaemia with 5 min of reperfusion) of arterial graft in patients undergoing urgent coronary artery bypass grafting (CABG). We examined RIPC effects on specific expression and immunolocalization of three NOS isoforms - endothelial (eNOS), inducible (iNOS) and neuronal (nNOS) in patients' internal thoracic artery (ITA) used as a graft. We found that the application of RIPC protocol leads to an increased protein expression of eNOS, which was further confirmed with strong eNOS immunopositivity, especially in the endothelium and smooth muscle cells of ITA. The same analysis of two other NOS isoforms, iNOS and nNOS, showed no significant differences between patients undergoing CABG with or without RIPC. Our results demonstrate RIPC-induced upregulation of eNOS in human ITA, pointing to its significance in achieving protective phenotype on a systemic level with important implications for graft patency.

Glucagon-like peptide-1 (GLP-1) receptor activation dilates cerebral arterioles, increases cerebral blood flow, and mediates remote (pre)conditioning neuroprotection against ischaemic stroke.

Stroke remains one of the most common causes of death and disability worldwide. Several preclinical studies demonstrated that the brain can be effectively protected against ischaemic stroke by two seemingly distinct treatments: remote ischaemic conditioning (RIC), involving cycles of ischaemia/reperfusion applied to a peripheral organ or tissue, or by systemic administration of glucagon-like-peptide-1 (GLP-1) receptor (GLP-1R) agonists. The mechanisms underlying RIC- and GLP-1-induced neuroprotection are not completely understood. In this study, we tested the hypothesis that GLP-1 mediates neuroprotection induced by RIC and investigated the effect of GLP-1R activation on cerebral blood vessels, as a potential mechanism of GLP-1-induced protection against ischaemic stroke. A rat model of ischaemic stroke (90 min of middle cerebral artery occlusion followed by 24-h reperfusion) was used. RIC was induced by 4 cycles of 5 min left hind limb ischaemia interleaved with 5-min reperfusion periods. RIC markedly (by ~ 80%) reduced the cerebral infarct size and improved the neurological score. The neuroprotection established by RIC was abolished by systemic blockade of GLP-1R with a specific antagonist Exendin(9-39). In the cerebral cortex of GLP-1R reporter mice, ~ 70% of cortical arterioles displayed GLP-1R expression. In acute brain slices of the rat cerebral cortex, activation of GLP-1R with an agonist Exendin-4 had a strong dilatory effect on cortical arterioles and effectively reversed arteriolar constrictions induced by metabolite lactate or oxygen and glucose deprivation, as an ex vivo model of ischaemic stroke. In anaesthetised rats, Exendin-4 induced lasting increases in brain tissue PO2, indicative of increased cerebral blood flow. These results demonstrate that neuroprotection against ischaemic stroke established by remote ischaemic conditioning is mediated by a mechanism involving GLP-1R signalling. Potent dilatory effect of GLP-1R activation on cortical arterioles suggests that the neuroprotection in this model is mediated via modulation of cerebral blood flow and improved brain perfusion.

Cardiovascular events in patients undergoing hip fracture surgery treated with remote ischaemic preconditioning: 1-year follow-up of a randomised clinical trial.

Remote ischaemic preconditioning reduces the risk of myocardial injury within 4 days of hip fracture surgery. We aimed to investigate the effect of remote ischaemic preconditioning on the incidence of major adverse cardiovascular events 1 year after hip fracture surgery. We performed a phase-2, multicentre, randomised, observer-blinded, clinical trial between February 2015 and September 2017. We studied patients aged ≥ 45 years with a hip fracture and a minimum of one cardiovascular risk factor. Patients were allocated randomly to remote ischaemic preconditioning applied just before surgery or no treatment (control group). Remote ischaemic preconditioning was performed on the upper arm with a tourniquet in four cycles of 5 min ischaemia and 5 min reperfusion. Primary outcome was the occurrence of major adverse cardiovascular events within 1 year of surgery. A total of 316 patients were allocated randomly to the remote ischaemic preconditioning group and 309 patients to the control group. Major adverse cardiovascular events occurred in 43 patients (13.6%) in the remote ischaemic preconditioning group compared with 51 patients (16.5%) in the control group (adjusted hazard ratio (95%CI) 0.83 (0.55-1.25); p = 0.37). Fewer patients in the remote ischaemic preconditioning group had a myocardial infarction (11 (3.5%) vs. 22 (7.1%); hazard ratio (95%CI) 0.48 (CI 0.23-1.00); p = 0.04). Remote ischaemic preconditioning did not reduce the occurrence of major adverse cardiovascular events within 1 year of hip fracture surgery. The effect of remote ischaemic preconditioning on clinical cardiovascular outcomes in non-cardiac surgery needs confirmation in appropriately powered randomised clinical trials.

Can exercise training enhance the repeated remote ischaemic preconditioning stimulus on peripheral and cerebrovascular function in high-risk individuals?

BACKGROUND: Repeated exposure to remote ischaemic preconditioning (rIPC; short bouts of non-lethal ischaemia) enhances peripheral vascular function within 1 week; whereas, longer periods of rIPC (~ 1 year) may improve cerebral perfusion. Increasing the 'dose' of rIPC may lead to superior effects. Given the similarities between exercise and rIPC, we examined whether adding exercise to the rIPC stimulus leads to greater adaptation in systemic vascular function. METHODS: Nineteen individuals with increased risk for cardiovascular disease (CVD) were randomly allocated to either 8 weeks of rIPC (n = 9) or 8 weeks of rIPC + exercise (rIPC + Ex) (n = 10). rIPC was applied three times per week in both conditions, and exercise consisted of 50 min (70% heart rate max) of cycling 3 times per week. Peripheral endothelial function was assessed using flow-mediated dilation (FMD) before and after ischaemia-reperfusion (IR). Cerebrovascular function was assessed by dynamic cerebral autoregulation (dCA) and cerebrovascular reactivity (CVR), and cardio-respiratory fitness (VO2peak) using a maximal aerobic capacity test. RESULTS: FMD% increased by 1.6% (95% CI, 0.4, 2.8) following rIPC + Ex and by 0.3% (- 1.1, 1.5) in the only rIPC but this did not reach statistical significance (P = 0.65). Neither intervention evoked a change in dCA or in CVR (P > 0.05). VO2peak increased by 2.8 ml/kg/min (1.7, 3.9) following the rIPC + Ex and by 0.1 ml/kg/min (- 1.0, 1.4) following the rIPC only intervention (P = 0.69). CONCLUSION: Combining exercise with rIPC across an 8-week intervention does not lead to superior effects in cerebrovascular and peripheral vascular function compared to a repeated rIPC intervention in individuals at risk of CVD.

Moderate hypothermia with remote ischaemic preconditioning improves cerebral protection compared to deep hypothermia: a study using a surviving porcine model.

OBJECTIVES: The optimal temperature management of hypothermic circulatory arrest is still controversial. Moderate hypothermia preserves cerebral autoregulation and shortens cardiopulmonary bypass (CPB) duration. However, moderate hypothermia alone has inferior organ protection to deep hypothermia, so adjuncts that increase the ischaemic tolerance are needed. Thus, we hypothesized that a combination of remote ischaemic preconditioning (RIPC) and moderate hypothermia would be superior to deep hypothermia alone. METHODS: Sixteen pigs were randomized to either RIPC or control groups (8 + 8). The RIPC group underwent 4 cycles of transient hind limb ischaemia. The RIPC group underwent cooling with CPB to 24°C, and the control group underwent cooling with CPB to 18°C, followed by a 30-min arrest period and subsequent rewarming to 36°C. Measurements of cerebral metabolism were made from sagittal sinus blood samples and common carotid artery blood flow. The permissible periods of hypothermic circulatory arrest were calculated based on these measurements. Neurological recovery was evaluated daily during a 7-day follow-up, and the brain was harvested for histopathological analysis. RESULTS: Six pigs in the RIPC group reached normal neurological function, but none in the control group reached normal neurological function (P = 0.007). The composite neurological score of all postoperative days was higher in the RIPC group than in the control group [55 (52-58) vs 45 (39-51), P = 0.026]. At 24°C, the estimated permissible periods of hypothermic circulatory arrest were 21 (17-25) min in the RIPC group and 11 (9-13) min in the control group (P = 0.007). CONCLUSIONS: RIPC combined with moderate hypothermia provides superior cerebral protection.

The impact of acute remote ischaemic preconditioning on cerebrovascular function.

PURPOSE: Remote ischaemic preconditioning (RIPC) refers to the protection conferred to tissues and organs via brief periods of ischaemia in a remote vascular territory, including the brain. Recent studies in humans report that RIPC provides neuroprotection against recurrent (ischaemic) stroke. To better understand the ability of RIPC to improve brain health, the present study explored the potential for RIPC to acutely improve cerebrovascular function. METHODS: Eleven young healthy (females n = 6, age; 28.1 ± 3.7 years) and 9 older individuals (females n = 4, age 52.5 ± 6.7 years) at increased risk for stroke (cardiovascular disease risk factors) underwent assessments of cerebrovascular function, assessed by carbon dioxide (CO2) reactivity and cerebral autoregulation during normo- and hypercapnia (5% CO2) following 40 mins of bilateral arm RIPC or a sham condition. Squat-to-stand manoeuvres were performed to induce changes in blood pressure to assess cerebral autoregulation (0.10 Hz) and analysed via transfer function. RESULTS: We found no change in middle cerebral artery velocity or blood pressure across 40 mins of RIPC. Application of RIPC resulted in no change in CO2 reactivity slopes (sham vs RIPC, 1.97 ± 0.88 vs 2.06 ± 0.69 cm/s/mmHg P = 0.61) or parameters of cerebral autoregulation during normocapnia (sham vs RIPC, normalised gain%, 1.27 ± 0.25 vs 1.22 ± 0.35, P = 0.46). CONCLUSION: This study demonstrates that a single bout of RIPC does not influence cerebrovascular function acutely in healthy individuals, or those at increased cardiovascular risk. Given the previously reported protective role of RIPC on stroke recurrence in humans, it is possible that repeated bouts of RIPC may be necessary to impart beneficial effects on cerebrovascular function.

Neuro-autonomic changes induced by remote ischemic preconditioning (RIPC) in healthy young adults: Implications for stress.

The mechanisms underlying the protective effects of remote ischemic preconditioning (RIPC) are not presently clear. Recent studies in experimental models suggest the involvement of the autonomic nervous system (ANS) in cardioprotection. The aim of this study was to investigate the changes in ANS in healthy young volunteers divided into RIPC (n = 22) or SHAM (n = 18) groups. RIPC was induced by 1 cycle of 4 min inflation/5 min deflation followed by 2 cycles of 5 min inflation/5 min deflation of a cuff placed on the upper left limb. The study included analysis of heart rate (HR), blood pressure (BP), heart rate variability (HRV), measurements of microcirculation and porphyrin fluorescence in the limb before and after the RIPC. RIPC caused reactive hyperemia in the limb and reduced blood porphyrin level. A mental load (serial sevens test) and mild motor stress (hyperventilation) were performed on all subjects before and after RIPC or corresponding rest in the SHAM group. Reduction of HR occurred during the experiments in both RIPC and SHAM groups reflecting RIPC-independent adaptation of the subjects to the experimental procedure. However, in contrast to the SHAM group, RIPC altered several of the spectral indices of HRV during the serial sevens test and hyperventilation. This was expressed predominantly as an increase in power of the very low-frequency band of the spectrum, increased values of detrended fluctuation analysis and weakening of correlation between the HRV parameters and HR. In conclusion, RIPC induces changes in the activity of ANS that are linked to stress resistance.

Remote ischaemic preconditioning modifies serum apolipoprotein D, met-enkephalin, adenosine, and nitric oxide in healthy young adults.

Remote ischaemic preconditioning (RIPC) has been employed as a non-invasive protective intervention against myocardial ischaemia-reperfusion injury in animal studies. However, the underlying mechanisms are incompletely defined in humans and its clinical efficacy has been inconclusive. As advanced age, disease, and drugs may confound RIPC mechanisms in patients, our aim is to measure whether RIPC evokes release of adenosine, bradykinin, met-enkephalin, nitric oxide, and apolipoproteins in healthy young adults. Healthy subjects (n = 18, 9 males, 23 ± 1.5 years old; 9 females, 23 ± 1.8 years old) participated after informed consent. RIPC was applied using a blood pressure cuff to the dominant arms for four cycles of 5-minute cuff inflation (ischaemia) and 5-minute cuff deflation (reperfusion). Blood was sampled at baseline and immediately after the final cuff deflation (Post-RIPC). Baseline and Post-RIPC plasma levels of adenosine, bradykinin, met-enkephalin, apolipoprotein A-1 (ApoA-1), apolipoprotein D (ApoD), and nitric oxide (as nitrite) were measured via ELISA and high-performance liquid chromatography. Mean (±SD) baseline levels of adenosine, bradykinin, met-enkephalin, ApoA-1, ApoD, and nitrite in healthy young adults were 13.8 ± 6.5 ng/mL, 2.6 ± 1.9 µg/mL, 594.1 ± 197.4 pg/mL, 3.0 ± 0.7 mg/mL, 22.2 ± 4.0 µg/mL, and 49.8 ± 13.4 nmol/L, respectively. Post-RIPC adenosine and nitrite levels increased (59.5 ± 37.9%, P < .0001; 32.2 ± 19.5%, P < .0001), whereas met-enkephalin and ApoD levels marginally decreased (5.3 ± 14.0%, P = .04; 10.8 ± 20.5%, P = .04). Post-RIPC levels were not influenced by sex, age, blood pressure, waist circumference, or BMI. RIPC produces increased levels of adenosine and nitrites, and decreased met-enkephalin and ApoD in the plasma of young healthy adults.

Remote ischaemic preconditioning ameliorates sinus rhythm restoration rate through Cox maze radiofrequency procedure associated with inflammation reaction reduction.

Remote ischaemic preconditioning (RIPC) as adjuvant to selective heart surgery attenuates cardiac injury and atrial fibrillation (AF) occurrence. We investigated its effect on sinus rhythm (SR) restoration rate in permanent AF patients undergoing Cox maze (CM) radiofrequency ablation with concomitant mitral valve surgery. From May 2013 to May 2017, 206 patients with rheumatic valve disease concomitant with permanent AF were randomized to receive prosthesis valve replacement and CM radiofrequency ablation procedure with (n = 104) or without (n = 102) RIPC (intermittent arm ischaemia through three cycles of 5-min inflation, followed by 5-min deflation of a blood pressure cuff). The primary end point of the study was freedom from cumulative AF without using antiarrhythmic drugs 1 year after operation; the secondary end points included inflammation reaction index over 48 h postoperatively and clinical outcomes. Baseline characteristics and preoperative data did not differ between groups. The SR restoration rates were significantly higher in the RIPC group, 85.6%, 83.7%, and 82.7%, than those in the control group, 72.5%, 70.6%, and 69.6%, at discharge, 6 months and 12 months, respectively, after the radiofrequency ablation procedure (P < 0.05). The serum concentration of high sensitivity C-reactive protein and neutrophil-lymphocyte ratio were significantly decreased at 12 h, 24 h, and 48 h postoperatively in the RIPC group compared to those in the control group (P < 0.05). RIPC induced by brief ischaemia and reperfusion of the arm ameliorated SR restoration rate in patients with permanent AF through CM radiofrequency ablation procedure and was associated with reduction of postoperative systemic inflammation reaction index.

Can human myocardium be remotely preconditioned? The results of a randomized controlled trial.

OBJECTIVES: No experimental study has shown that the myocardium of a remotely preconditioned patient is more resistant to a standardized ischaemic/hypoxic insult. METHODS: This was a single-centre randomized (1:1), double-blinded, sham-controlled, parallel-group study. Patients referred for elective coronary bypass surgery were allocated to either remote ischaemic preconditioning (3 cycles of 5-min ischaemia/5-min reperfusion of the right arm using a blood pressure cuff inflated to 200 mmHg) or sham intervention. One hundred and thirty-four patients were recruited, of whom 10 dropped out, and 4 were excluded from the per-protocol analysis. The right atrial trabecula harvested on cannulation for cardiopulmonary bypass was subjected to 60 min of simulated ischaemia and 120 min of reoxygenation in an isolated organ experiment. Postoperative troponin T release and haemodynamics were assessed in an in vivo study. RESULTS: The atrial trabeculae obtained from remotely preconditioned patients recovered 41.9% (36.3-48.3) of the initial contraction force, whereas those from non-preconditioned patients recovered 45.9% (39.1-53.7) (P = 0.399). Overall, the content of cleaved poly (ADP ribose) polymerase in the right atrial muscle increased from 9.4% (6.0-13.5) to 19.1% (13.2-23.8) (P < 0.001) after 1 h of ischaemia and 2 h of reperfusion in vitro. The amount of activated Caspase 3 and the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells also significantly increased. No difference was observed between the remotely preconditioned and sham-treated myocardium. In the in vivo trial, the area under the curve for postoperative concentration of troponin T over 72 h was 16.4 ng⋅h/ml (95% confidence interval 14.2-18.9) for the remote ischaemic preconditioning and 15.5 ng⋅h/ml (13.4-17.9) for the control group in the intention-to-treat analysis. This translated into an area under the curve ratio of 1.06 (0.86-1.30; P = 0.586). CONCLUSIONS: Remote ischaemic preconditioning with 3 cycles of 5-min ischaemia/reperfusion of the upper limb before cardiac surgery does not make human myocardium more resistant to ischaemia/reperfusion injury. CLINICAL TRIAL REGISTRATION NUMBER: NCT01994707.