In this study, we hypothesized that an increase in integrin αvβ3 and its co-activator vascular endothelial growth factor play important neuroprotective roles in ischemic injury. We performed ischemic preconditioning with bilateral common carotid artery occlusion for 5 minutes in C57BL/6J mice. This was followed by ischemic injury with bilateral common carotid artery occlusion for 30 minutes. The time interval between ischemic preconditioning and lethal ischemia was 48 hours. Histopathological analysis showed that ischemic preconditioning substantially diminished damage to neurons in the hippocampus 7 days after ischemia. Evans Blue dye assay showed that ischemic preconditioning reduced damage to the blood-brain barrier 24 hours after ischemia. This demonstrates the neuroprotective effect of ischemic preconditioning. Western blot assay revealed a significant reduction in protein levels of integrin αvβ3, vascular endothelial growth factor and its receptor in mice given ischemic preconditioning compared with mice not given ischemic preconditioning 24 hours after ischemia. These findings suggest that the neuroprotective effect of ischemic preconditioning is associated with lower integrin αvβ3 and vascular endothelial growth factor levels in the brain following ischemia.

Although urgently needed in clinical practice, a cardioprotective therapeutic approach against myocardial ischemia/ reperfusion injury remains to be established. Remote ischemic preconditioning (rIPC) and ischemic preconditioning (IPC) represent promising tools comprising three entities: the generation of a protective signal, the transfer of the signal to the target organ, and the response to the transferred signal resulting in cardioprotection. However, in light of recent scientific advances, many controversies arise regarding the efficacy of the underlying signaling. We here show methods for the generation of the signaling cascade by rIPC as well as IPC in a mouse model for in vivo myocardial ischemia/ reperfusion injury using highly reproducible approaches. This is accomplished by taking advantage of easily applicable preconditioning strategies compatible with the clinical setting. We describe methods for using laser Doppler perfusion imaging to monitor the cessation and recovery of perfusion in real time. The effects of preconditioning on cardiac function can also be assessed using ultrasound or magnetic resonance imaging approaches. On a cellular level, we confirm how tissue injury can be monitored using histological assessment of infarct size in conjunction with immunohistochemistry to assess both aspects in a single specimen. Finally, we outline, how the rIPC-associated signaling can be transferred to the target cell via conservation of the signal in the humoral (blood) compartment. This compilation of experimental protocols including a conditioning regimen comparable to the clinical setting should proof useful to both beginners and experts in the field of myocardial infarction, supplying information for the detailed procedures as well as troubleshooting guides.

We determined the impact of sex on the magnitude of cardioprotection by local and remote ischemic preconditioning (IPC and RIPC) and of ischemic/reperfused peripheral tissue mass on protection by RIPC. Hearts of female and male Lewis rats were excised, perfused with buffer, and underwent either IPC by 3 × 5/5 min global zero-flow ischemia/reperfusion (GI/R) or time-matched perfusion (TP) before 30/120 min GI/R. In a second approach, anesthetized female and male Lewis rats underwent RIPC, 3 × 5/5 min ischemia/reperfusion of one or both hindlimbs (1-RIPC or 2-RIPC), or placebo. Thirty minutes after the RIPC/placebo protocol, hearts were excised and subjected to GI/R. In female and male hearts, infarct size was less with IPC than with TP before GI/R (IPC+GI/Rfemale : 12 ± 5%; IPC+GI/Rmale : 12 ± 7% vs. TP+GI/Rfemale : 33 ± 5%; TP+GI/Rmale : 37 ± 7%, P < 0.001). With 2-RIPC, infarct size was less than with 1-RIPC in female and male rat hearts, respectively (2-RIPC+GI/Rfemale : 15 ± 5% vs. 1-RIPC+GI/Rfemale : 22 ± 7%, P = 0.026 and 2-RIPC+GI/Rmale : 16 ± 5% vs. 1-RIPC+GI/Rmale : 22 ± 8%, P = 0.016). Infarct size after the placebo protocol and GI/R was not different between female and male hearts (36 ± 8% vs. 34 ± 5%). Sex is no determinant of IPC- and RIPC-induced cardioprotection in isolated Lewis rat hearts. RIPC-induced cardioprotection is greater with greater mass of ischemic/reperfused peripheral tissue.

This study examined the hypothesis that acute hyperglycemia (HG) blocks ischemic preconditioning (IPC) by inhibiting Akt phosphorylation. Brief HG of approximately 400 mg/dL was induced in C57BL/6 mice via intraperitoneal injection of 20% dextrose (2 g/kg). All mice underwent 40 min LAD occlusion and 60 min reperfusion. The IPC protocol was 2 cycles of 5 min ischemia and 5 min reperfusion prior to index ischemia.

The effect of remote ischemic preconditioning (RIPC) has been proposed that mediates the protective response in ischemia reperfusion injury (IRI) of various organs. In this study, we investigated the effect of RIPC in hepatic IRI, by assessing biomarker of oxidative stress and inflammatory cytokines. Moreover, we intended to demonstrate any such protective effect through nitric oxide (NO). Twenty-five rats were divided into the 5 groups: (1) Sham; (2) RIPC; (3) hepatic IRI; (4) RIPC + hepatic IRI; (5) C-PTIO, 2-(4-carboxyphenyl)-4,5dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3oxide, + RIPC + hepatic IRI. RIPC downregulated the level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), histologic damage, and activity of Malondialdehyde (MDA). However, there was no significant reduction in the level of tumor necrosis factor-alpha (TNF-α) and nuclear factor kappa B (NF-κB). AST and ALT levels, and hepatic tissue morphology in the C-PTIO group showed a significant improvement compared to those of the RIPC + hepatic IRI group. The application of RIPC before hepatic ischemia downregulated the oxidative stress, not the inflammatory cytokines. Moreover, these protective effect of RIPC would be mediated through the activation of NO as well as anti-oxidant effect.

This study reports a novel strategy for investigating the key factors responsible for the protective effect of remote ischemic preconditioning (RIPC) against renal ischemia-reperfusion (IR) injury, which remains the leading cause of the acute kidney injury that increase the morbidity and mortality in patients with renal impairment.

Neurodegeneration can benefit from ischemic preconditioning, a natural adaptive reaction to sublethal noxious stimuli. Although there is growing interest in advancing preconditioning to preserve brain function, preconditioning is not yet considered readily achievable in clinical settings. One of the most challenging issues is that there is no fine line between preconditioning stimuli and lethal stimuli. Here, we show deleterious effect of preconditioning on oligodendrocyte precursor cells (OPCs). We identified Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3), a mitochondrial BH3-only protein specifically involved in OPCs loss after preconditioning. Repeated ischemia stabilized BNIP3 and increased the vulnerability of OPCs to subsequent ischemic events. BNIP3 became mitochondrial-bound and was concurrent with the dysfunction of monocarboxylate transporter 1 (MCT1). Inhibition of BNIP3 by RNAi or necrostatin-1 (Nec-1) and knocking out of BNIP3 almost completely prevented OPCs loss and preserved white matter integrity. Together, our results suggest that the unfavorable effect of BNIP3 on OPCs should be noted for safe development of ischemic tolerance. BNIP3 inhibition appears to be a complementary approach to improve the efficacy of preconditioning for ischemic stroke.

Remote ischemic preconditioning (RIP) and pharmacological preconditioning are the effective methods that can be used to prevent ischemia reperfusion (IR) injury. The aim of this study was to evaluate the effects of RIP and N-Acetylcysteine (NAC) with RIP in the rat hepatic IR injury model.

Ischemic preconditioning (IP) is one of the most important endogenous mechanisms that protect the cells against ischemia-reperfusion (I/R) injury. However, the exact molecular mechanisms remain unclear. In this study, we showed that changes in the level of agmatine were correlated with ischemic tolerance. Changes in brain edema, infarct volume, level of agmatine, and expression of arginine decarboxylase (ADC) and nitric oxide synthases (NOS; inducible NOS [iNOS] and neural NOS [nNOS]) were analyzed during I/R injury with or without IP in the rat brain. After cerebral ischemia, brain edema and infarct volume were significantly reduced in the IP group. The level of agmatine was increased before and during ischemic injury and remained elevated in the early reperfusion phase in the IP group compared to the experimental control (EC) group. During IP, the level of plasma agmatine was increased in the early phase of IP, but that of liver agmatine was abruptly decreased. However, the level of agmatine was definitely increased in the ipsilateral and contralateral hemisphere of brain during the IP. IP also increased the expression of ADC-the enzyme responsible for the synthesis of endogenous agmatine-before, during, and after ischemic injury. In addition, ischemic injury increased endogenous ADC expression in the EC group. The expression of nNOS was reduced in the I/R injured brain in the IP group. These results suggest that endogenous increased agmatine may be a component of the ischemic tolerance response that is induced by IP. Agmatine may have a pivotal role in endogenous ischemic tolerance.

Background Remote ischemic preconditioning (RIPC) attenuates myocardial damage during elective and primary percutaneous coronary intervention. Recent studies suggest that coronary microcirculatory function is an important determinant of clinical outcome. The aim of this study was to assess the effect of RIPC on markers of microcirculatory function. Methods and Results Patients referred for cardiac catheterization and fractional flow reserve measurement were randomized to RIPC or sham. Operators and patients were blinded to treatment allocation. Comprehensive physiological assessments were performed before and after RIPC/sham including the index of microcirculatory resistance and coronary flow reserve after intracoronary glyceryl trinitrate and during the infusion of intravenous adenosine. Thirty patients were included (87% male; mean age: 63.1±10.0 years). RIPC and sham groups were similar with respect to baseline characteristics. RIPC decreased the calculated index of microcirculatory resistance (median, before RIPC: 22.6 [interquartile range [IQR]: 17.9-25.6]; after RIPC: 17.5 [IQR: 14.5-21.3]; P=0.007) and increased coronary flow reserve (2.6±0.9 versus 3.8±1.7, P=0.001). These RIPC-mediated changes were associated with a reduction in hyperemic transit time (median: 0.33 [IQR: 0.26-0.40] versus 0.25 [IQR: 0.20-0.30]; P=0.010). RIPC resulted in a significant decrease in the calculated index of microcirculatory resistance compared with sham (relative change with treatment [mean±SD] was -18.1±24.8% versus +6.1±37.5; P=0.047) and a significant increase in coronary flow reserve (+41.2% [IQR: 20.0-61.7] versus -7.8% [IQR: -19.1 to 10.3]; P<0.001). Conclusions The index of microcirculatory resistance and coronary flow reserve are acutely improved by remote ischemic preconditioning. This raises the possibility that RIPC confers cardioprotection during percutaneous coronary intervention as a result of an improvement in coronary microcirculatory function. Clinical Trial Registration URL: www.anzctr.org.au/ . Unique identifier: CTRN12616000486426.

Increased histone deacetylase (HDAC) activity and the resulting dysregulation of protein acetylation is an integral event in retinal degenerations associated with ischemia and ocular hypertension. This study investigates the role of preconditioning on the process of acetylation in ischemic retinal injury. Rat eyes were unilaterally subjected to retinal injury by 45 min of acute ischemia, and retinal neuroprotection induced by 5 min of an ischemic preconditioning (IPC) event. HDAC activity was evaluated by a fluorometric enzymatic assay with selective isoform inhibitors. Retinal localization of acetylated histone-H3 was determined by immunohistochemistry on retina cross sections. Cleaved caspase-3 level was evaluated by Western blots. Electroretinogram (ERG) analyses were used to assess differences in retinal function seven days following ischemic injury. In control eyes, analysis of HDAC isoforms demonstrated that HDAC1/2 accounted for 28.4 ± 1.6%, HDAC3 for 42.4 ± 1.5% and HDAC6 activity 27.3 ± 3.5% of total activity. Following ischemia, total Class-I HDAC activity increased by 21.2 ± 6.2%, and this increase resulted solely from a rise in HDAC1/2 activity. No change in HDAC3 activity was measured. Activity of Class-II HDACs and HDAC8 was negligible. IPC stimulus prior to ischemic injury also suppressed the rise in Class-I HDAC activity, cleaved caspase-3 levels, and increased acetylated histone-H3 in the retina. In control animals 7 days post ischemia, ERG a- and b-wave amplitudes were significantly reduced by 34.9 ± 3.1% and 42.4 ± 6.3%, respectively. In rats receiving an IPC stimulus, the ischemia-induced decline in ERG a- and b-wave amplitudes was blocked. Although multiple HDACs were detected in the retina, these studies provide evidence that hypoacetylation associated with ischemic injury results from the selective rise in HDAC1/2 activity and that neuroprotection induced by IPC is mediated in part by suppressing HDAC activity.

Purpose The purpose of this study was to examine whether an individual's IPC-mediated change in cold pain sensitivity is associated with the same individual's IPC-mediated change in exercise performance. Methods Thirteen individuals (8 males; 5 females, 27 ± 7 years, 55 ± 5 ml.kgs-1.min-1) underwent two separate cold-water immersion tests: with preceding IPC treatment and without. In addition, each participant undertook two separate 5-km cycling time trials: with preceding IPC treatment and without. Pearson correlation coefficients were used to assess the relationship between an individual's change in cold-water pain sensitivity following IPC with their change in 5-km time trial performance following IPC. Results During the cold-water immersion test, pain intensity increased over time (p < 0.001) but did not change with IPC (p = 0.96). However, IPC significantly reduced the total time spent under pain (-9 ± 7 s; p = 0.001) during the cold-water immersion test. No relationship was found between an individual's change in time under pain (r = -0.2, p = 0.6) or pain intensity (r = -0.3, p = 0.3) following IPC and their change in performance following IPC. Conclusion These findings suggest that IPC can modulate sensitivity to a painful stimulus, but this altered sensitivity does not explain the ergogenic efficacy of IPC on 5-km cycling performance.

A major gap in the field of ischemic preconditioning (IPC) is whether or not long-lasting neuroprotection can be achieved. Moreover, the specific mechanisms underlying IPC and how they can be translated into the clinic remain uncertain. To fill these gaps, we tested the hypothesis that IPC exerts long-lasting structural and functional neuroprotection against ischemic stroke through the master gatekeeper of antioxidant defenses, nuclear factor erythroid 2-related factor 2 (Nrf2). We also tested whether the brain could be pharmaceutically preconditioned with a potent and blood-brain barrier-permeable Nrf2 activator, 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-trifluoethyl amide (CDDO-TFEA).

It has been demonstrated that brief episodes of sublethal ischemia-reperfusion, so-called ischemic preconditioning, provide powerful tissue protection in different tissues such as heart, brain, skeletal muscle, lung, liver, intestine, kidney, retina, and endothelial cells. Although a recent study has claimed that there are no protective effects of ischemic preconditioning in rat testis, the protective effects of ischemic preconditioning on testicular tissue have not been investigated adequately. The present study was thus planned to investigate whether ischemic preconditioning has a protective effect on testicular tissue.

Brain ischemic tolerance is an endogenous protective mechanism activated by a preconditioning stimulus that is closely related to N-methyl-d-aspartate receptor (NMDAR). Glycine transporter type 1 (GlyT-1) inhibitors potentiate NMDAR and suggest an alternative strategy for brain preconditioning. The aim of this work was to evaluate the effects of brain preconditioning induced by sarcosine, a GlyT-1 inhibitor, against global cerebral ischemia and its relation to NMDAR. Sarcosine was administered over 7 days (300 or 500 mg/kg/day, ip) before the induction of a global cerebral ischemia model in Wistar rats (male, 8-week-old). It was observed that sarcosine preconditioning reduced cell death in rat hippocampi submitted to cerebral ischemia. Hippocampal levels of glycine were decreased in sarcosine-treated animals, which was associated with a reduction of [(3)H] glycine uptake and a decrease in glycine transporter expression (GlyT-1 and GlyT-2). The expression of glycine receptors and the NR1 and NR2A subunits of NMDAR were not affected by sarcosine preconditioning. However, sarcosine preconditioning reduced the expression of the NR2B subunits of NMDAR. In conclusion, these data demonstrate that sarcosine preconditioning induces ischemic tolerance against global cerebral ischemia and this neuroprotective state is associated with changes in glycine transport and reduction of NR2B-containing NMDAR expression.

The use of cardiopulmonary bypass (CPB) in cardiac surgeries is known to induce pathological changes in vital organs such as lungs. Remote ischemic preconditioning (RIPC) is a protective strategy that has shown to be able to reduce tissue damage related to ischemia-reperfusion injury (IRI). The current study seeks to evaluate the beneficial effects of limb RIPC on lung tissues and function in a rat CPB model. RIPC, which consisted of three cycles of 5-min ischemia and subsequently 5-min reperfusion, was induced in the hind limbs of the animals via a tourniquet. Bronchoalveolar lavage (BAL) fluid analysis and hematoxylin and eosin staining revealed that limb RIPC could significantly attenuate CPB-induced pulmonary injury, as evidenced by a combination of lower total BAL protein content, less severe alveolar wall thickening and reduced intra-alveolar neutrophil infiltration. Consistently, RIPC was also found to improve the proliferation capacity of the bronchioalveolar stem cells isolated from the lung tissues in rats subjected to surgical procedure with CPB. These beneficial effects translated into significantly improved lung function. Further investigation suggested that RIPC could up-regulate the serum levels of several anti-inflammatory cytokines such as interleukin (IL)-4 and 10, which might play a role in its pulmonoprotective effects. Taken together, the current study provided convincing evidence that limb RIPC could be a useful strategy for minimizing CPB-induced organ injuries in patients undergoing CPB surgery.

Remote Ischemic Preconditioning (RIPC) induced by brief episodes of ischemia of the limb protects against multi-organ damage by ischemia-reperfusion (IR). Although it has been demonstrated that RIPC affects gene expression, the proteomic response to RIPC has not been determined. This study aimed to examine RIPC induced changes in the plasma proteome. Five healthy adult volunteers had 4 cycles of 5 min ischemia alternating with 5 min reperfusion of the forearm. Blood samples were taken from the ipsilateral arm prior to first ischaemia, immediately after each episode of ischemia as well as, at 15 min and 24 h after the last episode of ischemia. Plasma samples from five individuals were analysed using two complementary techniques. Individual samples were analysed using 2Dimensional Difference in gel electrophoresis (2D DIGE) and mass spectrometry (MS). Pooled samples for each of the time-points underwent trypsin digestion and peptides generated were analysed in triplicate using Liquid Chromatography and MS (LC-MS). Six proteins changed in response to RIPC using 2D DIGE analysis, while 48 proteins were found to be differentially regulated using LC-MS. The proteins of interest were involved in acute phase response signalling, and physiological molecular and cellular functions. The RIPC stimulus modifies the plasma protein content in blood taken from the ischemic arm in a cumulative fashion and evokes a proteomic response in peripheral blood.

Based on growing evidence linking autophagy to preconditioning, we tested the hypothesis that autophagy is necessary for cardioprotection conferred by ischemic preconditioning (IPC). We induced IPC with three cycles of 5 min regional ischemia alternating with 5 min reperfusion and assessed the induction of autophagy in mCherry-LC3 transgenic mice by imaging of fluorescent autophagosomes in cryosections. We found a rapid and significant increase in the number of autophagosomes in the risk zone of the preconditioned hearts. In Langendorff-perfused hearts subjected to an IPC protocol of 3 x 5 min ischemia, we also observed an increase in autophagy within 10 min, as assessed by Western blotting for p62 and cadaverine dye binding. To establish the role of autophagy in IPC cardioprotection, we inhibited autophagy with Tat-ATG5(K130R), a dominant negative mutation of the autophagy protein Atg5. Cardioprotection by IPC was reduced in rat hearts perfused with recombinant Tat-ATG5(K130R). To extend the potential significance of autophagy in cardioprotection, we also assessed three structurally unrelated cardioprotective agents--UTP, diazoxide, and ranolazine--for their ability to induce autophagy in HL-1 cells. We found that all three agents induced autophagy; inhibition of autophagy abolished their protective effect. Taken together, these findings establish autophagy as an end-effector in ischemic and pharmacologic preconditioning.

Ischemic preconditioning (IPC) by ischemia/reperfusion (I/R) renders resistance to the kidney. Strong IPC triggers kidney fibrosis, which is involved in angiotensin II (AngII) and its type 1 receptor (AT1R) signaling. Here, we investigated the role of AngII/AT1R signal pathway in the resistance of IPC kidneys to subsequent I/R injury. IPC of kidneys was generated by 30 minutes of bilateral renal ischemia and 8 days of reperfusion. Sham-operation was performed to generate control (non-IPC) mice. To examine the roles of AngII and AT1R in IPC kidneys to subsequent I/R, IPC kidneys were subjected to either 30 minutes of bilateral kidney ischemia or sham-operation following treatment with AngII, losartan (AT1R blocker), or AngII plus losartan. IPC kidneys showed fibrotic changes, decreased AngII, and increased AT1R expression. I/R dramatically increased plasma creatinine concentrations in non-IPC mice, but not in IPC mice. AngII treatment in IPC mice resulted in enhanced morphological damage, oxidative stress, and inflammatory responses, with functional impairment, whereas losartan treatment reversed these effects. However, AngII treatment in non-IPC mice did not change I/R-induced injury. AngII abolished the resistance of IPC kidneys to subsequent I/R via the enhancement of oxidative stress and inflammatory responses, suggesting that the AngII/AT1R signaling pathway is associated with outcome in injury-experienced kidney.

Nitric oxide (NO) is cardioprotective and a mediator of ischemic preconditioning (IP). Endothelial nitric oxide synthase (eNOS) is protective against myocardial ischemic injury and a component of IP but the role and location of neuronal nitric oxide synthase (nNOS) remains unclear. Therefore, the aims of these studies were to: (i) investigate the role of nNOS in ischemia/reoxygenation-induced injury and IP, (ii) determine whether its effect is species-dependent, and (iii) elucidate the relationship of nNOS with mitoKATP channels and p38MAPK, two key components of IP transduction pathway.