Review of remote ischemic preconditioning: from laboratory studies to clinical trials

Abstract

In remote ischemic preconditioning (RIPC) short periods of non-lethal ischemia followed by reperfusion of tissue or organ prepare remote tissue or organ to resist a subsequent more severe ischemia-reperfusion injury. The signaling mechanism of RIPC can be humoral communication, neuronal stimulation, systemic modification of circulating immune cells, and activation of hypoxia inducible genes. Despite promising evidence from experimental studies, the clinical effects of RIPC have been controversial. Heterogeneity of inclusion and exclusion criteria and confounding factors such as comedication, anesthesia, comorbidities, and other risk factors may have influenced the efficacy of RIPC. Although the cardioprotective pathways of RIPC are more widely studied, there is also evidence of benefits in CNS, kidney and liver protection. Future research should explore the potential of RIPC, not only in cardiac protection, but also in patients with threatening ischemia of the brain, organ transplantation of the heart, liver and kidney and extensive cardiovascular surgery. RIPC is generally well-tolerated, safe, effective, and easily feasible. It has a great prospect for ischemic protection of the heart and other organs.

Keywords:

• Remote ischemic preconditioning
• myocardial protection
• cerebral protection
• spinal cord protection

Introduction

Ischemic preconditioning (IPC) is a phenomenon in which short periods of non-lethal ischemia protect against a subsequent and more severe ischemia-reperfusion insult. Local ischemic preconditioning was discovered in the mid-1980s in experimental models of myocardial infarction. A significant reduction in the size of an infarction was observed when four 5-minute cycles of coronary occlusion were executed prior to 40 minutes of coronary occlusion in a canine model.[1] A few years later, animal studies showed that ischemia in one coronary artery territory protects myocardium perfused by another coronary artery.[2] The finding that ischemia in a non-cardiac organ protects the heart at a distance led to the concept of remote ischemic preconditioning (RIPC).[3] In 2002 the first clinically relevant large animal (porcine) model showed that RIPC performed by cycles of hind limb tourniquet occlusions reduced the size of myocardial infarction. This finding aroused interest to subsequent clinical trials in humans.[4]

Pathways from remote organ to target protection

The most fascinating part of RIPC is its ability to produce protective factors from distance to the actual target tissue or organ. Several signaling mechanisms of RIPC have been proposed, including humoral pathways, neuronal stimulation, communication via systemic modification of circulating immune cells, and activation of hypoxia inducible genes (Figure 1).

Figure 1. Mechanisms of RIPC.

Humoral pathway

It has been suggested that humoral factors generated in remote preconditioning organs have to be washed out into the bloodstream toward the more susceptible organ (e.g. heart, central nervous system (CNS)). This was shown in a study where cardioprotection was induced in an unpreconditioned rabbit heart using the transfer of coronary perfusate from a RIPC heart. This protective effect was also detected while studying cross-species transfer.[5] The presence of a circulating cardioprotective factor after RIPC was also demonstrated in a porcine model with a denervated donor heart.[6]

Several studies have attempted to identify a specific circulating factor that might be the transmitter of RIPC. The shear-stress-related release of nitric oxide with reactive hyperemia after transient limb ischemia is expected to increase cardioprotective nitrite levels in the blood.[7] Plasma levels of cardioprotective stromal-derived factor-1α were increased after RIPC in rats.[8] A microribonucleic acid (microRNA) was shown to play a role in the pre-conditioning effect of RIPC.[9] Extracellular vesicles (exosomes and microvesicles) are membrane-bound structures secreted by mammalian cells and these vesicles were showed to participate in a carrier mechanism of the cardioprotective effect of RIPC.[10] Although aforementioned factors are clearly humoral transfer signals, they cannot entirely explain RIPC phenomenon.

As ischemia is an unspecific injury that results in disturbances in multiple cellular processes, an obvious explanation is that RIPC activates the release of several humoral factors and involves multiple endogen-protective mechanisms. However, the preconditioning stimulus has to be recognized by the cellular sensor, and the cell must be prepared for the upcoming stress. The elements of RIPC might include the sensor of the stress signal, transducers of the stimulus, or effectors of the tolerance.[11]

Neuronal pathway

Evidence from several experimental models proposes that transient ischemia in itself is not a requirement for remote protection. Multiple alternative triggers capable to result in ischemia protection in remote organ have been proposed. Peripheral nociception, initiated by surgical skin incision, “remote pre-conditioning of trauma”, have been demonstrated to induce remote cardioprotection.[12] Local nerve stimulation appears to have an effect similar to RIPC, whereas a nerve blocker of the peripheral nerve abolishes the protection.[13] Cardioprotection by RIPC seems to be dependent on afferent innervation of the remote organ and intact parasympathetic activity, while delayed remote postconditioning relies on different signaling pathways.[14] The recent study in rat model have shown that the circulating factors of RIPC are produced and released into the systemic circulation by the visceral organs innervated by the posterior gastric branch of the vagus nerve.[15] In brain protection, neuronal involvement in RIPC has been reported in several studies, mainly via a blockade of sensory inputs that reduced the beneficial effects of RIPC.[16,17] In contrast, evidence for spinal cord protection and neuronal pathway involvement is not as strong, as the effects of RIPC on the spine were not eliminated by a ganglionic blocker, but by antioxidants, indicating the importance of humoral factor.[18]

The neuronal pathway includes the somatosensory and autonomous nervous systems and the spinal cord. The causal involvement of peripheral nociceptive sensory nerves is obvious, but the nature and transfer of the released transmitter molecule to the target organ through humoral or neuronal pathways remains unclear. It has been proposed that endogenous substances (adenosine, bradykinin, calcitonin gene-related peptide (CGRP)) are released in a remote preconditioned organ and further stimulate afferent nerve fibers, which transmit the effects to the target organ, promoting protective cellular processes.[19] However, ischemic protection was able to transfer to a recipient heart by blood-derived dialysate after local peripheral adenosine or capsaicin administration or RIPC, suggesting humoral transfer of a neuronally released signal molecule.[13] Human study showed that the diabetic neuropathy was associated with impaired response to the dialysate mediated remote ischemic conditioning indicating that the release mechanism involves neuronal pathways.[20]

When knowledge of mediating factors accumulates, it is evident that the protective cascade is a complex process including both humoral and neuronal pathways. More information is needed on the effects on different organs because it has not been fully confirmed that the same factors that affect the CNS also affect (for example) the heart.

Systemic response

Evidence of suppression of the inflammatory response is detected after RIPC. It has been shown that specific adhesion molecules (intercellular adhesion molecule-1 (ICAM-1), P-selectin) decrease in cell membranes after RIPC.[21] It seems that leukocyte–endothelium interactions are regulated by RIPC, and a decreased number of activated leukocytes is detected in the ischemic area.[22] Moreover, RIPC seems to reduce pro-inflammatory gene expression in circulating leukocytes. These genes are associated with leukocyte activation, cell adhesion, and intracellular signaling.[23] These studies suggest that RIPC has a direct effect on the inflammatory system, and thus prevents exacerbation of ischemic injuries.

Molecular mechanisms of RIPC

Mitochondrial dysfunction is thought to be the major contributor to ischemia–reperfusion-mediated injury. Thus, it seems logical that several reviews of the mechanisms underlying RIPC and IPC point to evidence that several protein kinases are activated during ischemic preconditioning and are targeted toward maintenance of mitochondrial function, especially inhibition of the opening of the mitochondrial permeability transition pores (mPTP).[11,24]

Initially, sublethal ischemic insults lead to transient decreases in cellular ATP levels. It has been demonstrated that the ATP/ADP ratio decreases after minutes of mitochondrial ATP synthase inhibition.[25] The consequential increase in ATP metabolites, such as ADP, AMP, and adenosine, may act to induce ischemia tolerance pathways. For example, adenosine pretreatment has been found to attenuate ischemic insults in in vivo and in vitro studies.[26,27] The decreased ATP/ADP ratio may also directly or indirectly activate mitochondrial K⁺_ATP channels. These channels seem to play a crucial role in ischemic tolerance.[26]

The studies to identify the signaling molecules or mechanisms of RIPC have mostly focused on signals identified in local ischemic precondition studies.[28] However, most signaling mechanisms have thus far only been determined in rodent hearts, and further studies are warranted to translate findings to larger mammals or humans. With these limitations, there is evidence for a causal involvement of the adenosine,[29] bradykinin,[30] interleukin-10 (in delayed RIPC),[31] and stromal-derived factor-1α.[8]

One of the key elements of ischemic tolerance is the adenosine triphosphate-sensitive potassium channel (K_ATP channel), located in the sarcolemma and mitochondria. The opening of K_ATP channels is considered to be one of the main mechanisms of RIPC. In animal studies, the adenosine A1 receptor antagonist weakened the protective effects of RIPC.[32] The K_ATP channel activator diazoxide mimicked the protective effect of preconditioning, also implying that the K_ATP channel is the mediator of the protective effect.[33]

Bradykinin B₂ receptor activation results in protein kinase PKCɛ activation.[30] The PKCɛ translocation to mitochondria accelerates the activity of mitochondrial K_ATP channels.[34] The action of interleukin 10 results in increased phosphorylation of protein kinase B and endothelial nitric oxide synthase.[31]

The hypoxia-inducible transcription factor (HIF), consisting of a labile α subunit and a stable β subunit, accumulates during hypoxia. Hypoxia response is triggered by the HIF through the induction of over 100 genes whose products promote cellular survival under ischemic conditions, such as those for erythropoietin (EPO), transferrin, vascular endothelial growth factor (VEGF), and many glycolytic enzymes. The amount of HIF accumulation depends on the alpha subunit. The HIF α subunit is regulated through prolyl hydroxylation by α-ketoglutarate (αKG)-dependent dioxygenases known as EGLNs. Of the three EGLN paralogs, EGLN1 is the primary regulator of HIFα. EGLNs act as “O₂ sensors” in metazoans and coordinate cellular responses that promote adaptation to hypoxia and ischemia.[35]

RIPC is known to increase HIF-1-α protein levels in cardiac atrial tissue in humans.[36] Both local and remote preconditioning is attenuated in HIF1α± mice.[37] However, RIPC was demonstrated to induce cardioprotection in partially HIF-1-α-deficient and pharmacologically HIF-1-α-inhibited mice and rats, indicating the complexity of mechanisms behind the protective effects of RIPC.[38] These results support that HIF protects the heart during acute myocardial infarct. However, long-term changes in HIF levels might cause undesirable effects such as decreased mitochondrial function, which would be an unfortunate side effect in any therapies attempting to protect the myocardium via HIF modulation.[39]

Timing of remote ischemic preconditioning

In most studies the RIPC is executed 30–40 minutes prior to subsequent ischemic insult. But pre-treatment is not essential for RIPC-induced protection. Reduction of the infarction size has also been shown with simultaneous application of the RIPC during coronary occlusion (remote ischemic per-conditioning) or at the time of reperfusion (remote ischemic post-conditioning).[40] However, there are two temporally distinct types of RIPC: early and delayed protection. Both types of ischemic tolerance have been detected in the brain and the heart, although the brain usually appears to follow the delayed pattern. It is widely accepted that the early type of protection is independent of protein synthesis, primarily targeting posttranslational modifications, and the protective effect is brief and transient (lasting minutes), whereas the delayed pattern involves new protein synthesis and lasts hours to days.[41]

RIPC and myocardial protection

The effects of RIPC on cardiac protection have been widely studied. Despite promising results in animal models, the clinical effects of RIPC are controversial. The first small clinical study of RIPC in coronary artery bypass graft surgery (CABG) patients could not demonstrate differences in creatine kinase myocardial band.[42] However, a study of RIPC in pediatric patients who underwent surgical repair of congenital heart defects showed reduced cardiac enzyme release.[43] The most of earlier and often small clinical studies showed attenuated biomarker release demonstrating cardioprotective potential of RIPC. This was first associated with beneficial clinical outcome in a single-center study evaluating the effect of RIPC on patients undergoing CABG. RIPC provided perioperative myocardial protection and significantly improved the prognosis of patients.[44] After percutaneous coronary intervention (PCI), major adverse cardiac and cerebral events were significantly lower in RIPC-treated patients.[45] RIPC before hospital admission in conjunction with PCI increased myocardial salvage in a randomized trial [46] and remote ischemic post-conditioning with PCI reduced infarct size.[47] The combined RIPC and postconditiong along with PCI in ST-segment elevation myocardial infarction (STEMI) improved myocardial salvage.[48] RIPC prior to primary PCI in STEMI reduced myocardial infarction size in randomized trial.[49] However, a large systematic meta-analysis of 2200 patients undergoing cardiovascular surgery did not reveal significant benefits regarding perioperative adverse events.[50] Heterogeneity of inclusion and exclusion criteria and the type of preconditioning stimulus might limit the evaluation of RIPC effects. Confounding factors such as age, comedication, anesthesia, comorbidities, and other risk factors may have influenced the efficacy of RIPC.[51]

A recently published clinical multicenter, double-blinded, randomized, prospective, controlled human trial called the RIPHeart study, with patients undergoing CABG with or without valve surgery using cardiopulmonary bypass, could not demonstrate significant differences between the treatment groups.[52] Similar results were published in the ERICCA study with patients undergoing CABG with or without valve surgery.[53] Though the ERICCA and the RIPHeart studies are to be commended for excellent study protocol and execution, there are some important points that one should consider before rejecting the notion of RIPC altogether. A propofol anesthesia was used on all the patients, which has the effect of dampening RIPC-induced cardioprotection.[54] Concomitant therapy with beta-blockers [55] and statins [56] is cardioprotective and may interfere with the cardioprotective effect of RIPC. The limitation was also that the patients were quite heavily laden with co-morbidities to show any benefits of RIPC. The ERICCA and the RIPHeart studies have shown us that RIPC is not a cure-all protection method that benefits all patient groups that undergo cardiac surgery with a cardiopulmonary bypass. These results are a significant milestone in assessing the efficacy and effects of RIPC in intermediate risk populations, but there is still justification to study the possible effects of RIPC in smaller subgroups to ascertain whether it could be beneficial to a specific patient group. Remote ischemic preconditioning might prove beneficial, for example, in healthier patients undergoing a specific type of operation.

RIPC and cerebral protection

Although the cardioprotective pathways of RIPC are more widely studied, there is also evidence of benefits in CNS protection. Neuronal ischemic preconditioning was demonstrated in vivo 1990.[57] Remote ischemic protection of the brain was first tested in a rat model of global cerebral ischemia, and the results were promising.[58] Experimental and clinical studies about the neuroprotective effect of RIPC on the CNS have been rather limited so far. In a large experimental animal model with 1 hour of HCA, significantly better EEG recovery rates, superior behavioral scores, and better histopathological scores were recorded in the RIPC arm of the study 7 days post-operation.[59] In subsequent studies RIPC reduced the amount of circulating cerebrocortical leukocytes and provided a degree of cell and organ preservation.[22]

Adults undergoing carotid endarterectomy benefitted from RIPC.[60] More interestingly, RIPC has been associated with significantly better motor learning in healthy adults.[61] Additionally, RIPC has shown promising results in focal brain ischemia as an adjunct to thrombolysis in acute ischemic strokes, and was able to reduce the risk of infarction in brain tissue.[62] However, the main brain structure mediating this protection has not been identified yet, indicating that the studies on this subject are justified.[63]

RIPC and spinal cord protection

Despite the favorable trend in the outcome of extensive thoracoabdominal aneurysm repair, the incidence of temporary or permanent spinal cord injury still varies between 3% and 20% depending on the extent of the aortic pathology and operative urgency.[64] Nevertheless, paraplegia and paraparesis still exist postoperatively, leaving room for new protective innovations. The beneficial effect of RIPC has been documented in the experimental studies of spinal cord injury.[65] A recent study documented a significant increase in MEP amplitude and a decrease in onset latency after hind limb RIPC in a porcine model.[66] Spinal cord protection by RIPC was confirmed in a study of 40 patients who underwent cervical decompression surgery. A significantly better recovery rate was observed in RIPC-treated patients 3 months after surgery.[67] Additionally, the cardioprotective effect of RIPC was lost if the spinal cord was chemically blocked.[68] In contrast, the effect of RIPC on the spinal cord was not abolished by ganglionic blockers but by using antioxidants.[18] Thus, the spinal cord might play a role in transmitting the effect of ischemic bouts from the periphery to the target organ, but the protective mechanisms might be different in various organs. Remote ischemic preconditioning causes initial oxidative stress, which seems to irritate the spinal cord either via neural, humoral, or systemic (immunological) pathways.

Although no clinical trials have been conducted to date, RIPC has the potential to become a novel therapeutic modality for the amelioration of ischemic insults to the spinal cord in the setting of thoracoabdominal aortic aneurysm repair.

Safety of remote ischemic preconditioning

Remote ischemic preconditioning is beneficial in many ways, and it is smoothly applicable to cardiac operations as well as to aortic arch and descending aortic aneurysm operations, using both endovascular and open procedures. It is not surprising that, in clinical studies of RIPC, arm or leg ischemia is most often used because of the ease of application. RIPC is generally well-tolerated and safe. The microdialysis study measuring possible injurious effects of RIPC on the limbs did not find any indications of permanent cell damage.[69] The clinical trial in phase I tested the safety and feasibility of RIPC and did not find any indications of neurovascular injury. The only disadvantage was the temporary pain created by inflation–deflation cycles.[70]

Conclusions

Numerous positive findings from experimental animal works and human studies support the protective effect of RIPC for ischemia-reperfusion injury of the heart and other organs.[71] Details of local release of the protective signal at the remote site and participation of neuronal and humoral pathways are not clear yet. Also, signal transfer to the target organ is partly indefinite. Translation of RIPC strategies from successful animal experiments to the clinic has been somewhat disappointing. Findings from early experiments were neither unequivocal nor confirmed in larger mammalian models and planning of clinical trials was incomplete.[51] Upcoming studies should concentrate on better understanding of the signal transduction to solve the confounding influence of comorbidities and comedication. RIPC distinctly has a feasible prospect for ischemic protection of the heart and other organs. Future research should explore the potential benefit of RIPC, not only in cardiac protection, but also in patients with threatening ischemia of the brain, organ transplantation of the heart, liver and kidney, and extensive cardiovascular surgery.[72]

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.