Experimental and clinical studies have demonstrated that RIC protection is dependent on an intact neural pathway to the remote organ or tissue with local resection of the neural pathway abolishing RIC protection [27, 63]. However, the actual nature of the neural pathway in terms of its afferent, central, and efferent components remains unclear. The current paradigm has proposed that in response to the RIC stimulus, autacoids such as adenosine [23, 62, 86] and bradykinin [95] are produced in the remote organ or tissue resulting in the nitric oxide-dependant stimulation of local afferent sensory nerves. At the HCI Workshop, Kharbanda (Oxford, UK) presented unpublished human data investigating whether adenosine provides the ‘trigger’ for the limb RIC stimulus in IHD patients undergoing coronary angiography. Utilizing the human forearm model, they found that local arterial infusion of caffeine (a non-specific adenosine receptor antagonist) into the trigger arm blocked the beneficial effects of RIC on preventing ischemia-induced endothelial dysfunction, and inhibited the production of a cardioprotective plasma dialysate. Furthermore, the administration of an arterial infusion of adenosine into the femoral artery resulted in the production of a cardioprotective plasma dialysate in patients undergoing coronary angiography, confirming the findings in experimental animal studies that adenosine acted as a ‘trigger’ for limb RIC [86]. Most recent experimental data have suggested that the sensory arm of the neural pathway leading from the remote organ or tissue may be recruited by the activation of transient receptor potential vanilloid (TRPV) receptors, which are prevalent in unmyelinated small diameter (Aδ & C) sensory fibers [6, 47, 81]. Experimental studies have demonstrated that the activation of these fibers by topical capsaicin or nociceptive stimuli can recapitulate limb RIC cardioprotection [6, 47, 81].

However, the neural components of the pathway downstream of this sensory afferent neural pathway in the remote organ or tissue remain unclear. Jones et al. [47] found that cardioprotection elicited by peripheral nociception was blocked by spinal transection at T7 but not C7, suggesting that direct stimulation of cardiac nerves may be responsible for conveying the cardioprotective signal to the heart. In contrast to this study, and using an elegant experimental optogenetic approach, Gourine (London, UK) [64] has recently shown that the activity of the brainstem vagal preganglionic neuronsis required to mediate the protective effect of limb RIC on the heart, with their activation inducing powerful cardioprotection and their inhibition abrogating the beneficial effects of RIC [64]. To study the role of the efferent vagal pathway to limb RIC cardioprotection, Donato et al. [22] showed that resection of the vagal nerve and atropine abolished the MI-limiting effects of limb RIC in the rabbit heart and stimulation of the vagal nerve recapitulated limb RIC cardioprotection. However, dependency of limb RIC cardioprotection on the parasympathetic nervous system appears to preclude a role for a blood-borne cardioprotective factor.

Whether an efferent neural pathway is actually required to convey the cardioprotective signal to the heart or whether this is simply mediated by a blood-borne cardioprotective factor to the heart is not fully resolved. Kingma et al. [52] reported that neither the ganglionic blocker (hexamethonium) nor cardiac denervation abolished renal RIC protection of the canine heart. Similarly, Rassaf et al. [79] found that MI size reduction by limb RIC in the murine heart persisted despite femoral nerve resection (although the sciatic nerve was not resected in this model). Clearly, further studies are required to elucidate the details of the neural pathway underlying limb RIC cardioprotection.

The earliest experimental evidence for a blood-borne cardioprotective factor released by RIC was provided in 1999 by Dickson et al. [21], who demonstrated that the cardioprotective effect elicited by ischemic preconditioning of the heart and kidney in one rabbit could be transferred via whole blood transfusion to a non-preconditioned rabbit. Since then, a number of experimental studies have attempted to identify the blood-borne cardioprotective factor(s), resulting in a number of candidate factors being proposed including calcitonin gene-related peptide [87], opioids [73], endogenous cannabinoids [30], and hypoxia-inducible factor-1α (HIF-1α) [50].

Although the actual identity of the factor remains unclear, biochemical studies have suggested that the factor may be a peptide less than 30 kDa in size [58, 84]. Using proteomic analysis of plasma following RIC to identify the blood-borne cardioprotective factor(s) has been challenging. At the HCI Workshop, a number of novel candidates for the blood-borne cardioprotective factor(s) of RIC were proposed, each with varying degrees of experimental evidence: including (1) stromal-derived factor-1α or SDF-1α (S Davidson, London, UK) [19]; (2) exosomes (Giricz and Ferdinandy, Budapest, Hungary) [28]; nitrite (Heusch, Essen, Germany) [78, 79]; (3) microRNA-144 (Redington, Toronto, Canada) [60]; (4) HIF-1α (Prunier, Anger, France) [48]; and (5) Apolipoprotein a-I (Prunier) [41]. Of these, the most promising candidates for the blood-borne cardioprotective factor of RIC in terms of the available experimental evidence are probably SDF-1α, nitrite, and microRNA-144, as in these three cases limb RIC was demonstrated to elevate levels of the putative factor in the plasma, and blocking the factor also abolished the cardioprotective effect of RIC. However, these studies have failed to provide direct evidence that the factor secreted into the blood was actually responsible for the cardioprotective effect. Furthermore, it is important to note that none of these studies actually provided evidence that the production of the putative factor in response to RIC was dependent on an intact neural pathway to the limb, an important omission given that the blood-borne cardioprotective factor has been shown to be released downstream of the neural pathway (see next section).

The neural and humoral pathways underlying limb RIC have been known to interact to mediate the protective effect, but the actual nature of this relationship has not been clear until very recently (see Fig. 1 for a hypothetical scheme). Emerging studies from Redington’s and Botker’s research groups have begun to unravel the interplay between these two pathways in the setting of limb RIC. The major advance in this regard, has been facilitated by their use of an experimental model in which cardioprotective plasma dialysate harvested from animals or humans treated with limb RIC is demonstrated to reduce MI size in naïve animal hearts. Using this experimental model, they have been able to provide evidence showing that the blood-borne cardioprotective factor is produced downstream of the neural pathway. Redington’s group has shown that the cardioprotective plasma dialysate can be produced in animals and human volunteers in response to sensory neural stimulation of the limb using a number of different approaches including direct nerve stimulation [81], transcutaneous electrical nerve stimulation [68], electro-acupuncture [80] and even topical capsaicin [6, 81]. Botker’s group has demonstrated that diabetic patients with a peripheral sensory neuropathy in their upper limbs do not produce the cardioprotective plasma dialysate in response to limb RIC, when compared to diabetic patients with no sensory neuropathy [46]. Therefore, the combined evidence suggests that the blood-borne cardioprotective factor is most likely produced downstream of the neural pathway. But of course questions remain as to where along the neural pathway is the cardioprotective factor released into the blood stream, and which cell is actually responsible for its release.

The current paradigm suggests that the cardioprotective signal initiated by limb RIC recruits signal transduction pathways (such as PI3K-Akt) in the target organ or tissue, which are known to be mediators of direct ischemic preconditioning and postconditioning [36, 37, 61]. In the HCI Workshop, data were presented implicating two novel mediators of limb RIC cardioprotection including aldehyde dehydrogenase-2 (ALDH-2) and phospho-myozenin-2. Kharbanda presented recent data showing in an animal MI model and human volunteers that the protective effect of limb RIC was abolished in the presence of an ALDH-2 inhibitor [13]. Interestingly, in support of a role for ALDH-2, human volunteers with a Glu504Lys polymorphism in ALDH-2 were found to be resistant to RIC protection against ischemia-induced endothelial dysfunction [13]. Further study is required to determine where in the mechanistic pathway ALDH-2 plays its mediatory role and to identify its downstream effectors. Suleiman (Bristol, UK) presented recent data investigating cardiac phosphoproteomics in the setting of limb RIC, demonstrating the phosphorylation of the cardiac sarcomeric protein, phospho-myozenin-2. These findings suggest that RIC may have functional effects on myocardial contractile function [1]. The importance of this to the cardioprotective effect induced by RIC remains to be investigated.

It has been well established in the neuroprotection experimental literature that RIC can limit cerebral infarct size following an acute ischemic stroke [29]. At the HCI Workshop, Botker presented a recent clinical study investigating the effect of limb RIC in patients thrombolysed for an acute ischemic stroke—no clear benefit was found in terms of cerebral infarct size and functional recovery [44]. However, a small clinical study by Meng et al. [67] comprising 63 patients with prior stroke or transient ischemic accident demonstrated that RIC repeated twice daily for 300 days was able to reduce the recurrence of stroke and to improve functional recovery.

Cerebral IRI arising from perinatal hypoxic-ischemia, results in significant neonatal morbidity and long-term neurological impairment [59], despite the adoption of hypothermic neuroprotection in the developed world [5]. In this regard, N Robertson (London, UK) presented a recent study investigating the effect of limb RIC applied at the onset of reperfusion using a porcine model of neonatal cerebral hypoxia–ischemia. Limb RIC preserved cerebral white matter metabolism on magnetic resonance spectroscopy and reduced white matter cell death following transient global cerebral hypoxia–ischemia, suggesting that RIC may have therapeutic potential as a neuroprotective strategy for mitigating brain injury and improving outcomes in babies with birth asphyxia. This may have important implications in low resource countries where limb RIC could be used as a simple and low-cost neuroprotective intervention.

Limb RIC has been investigated as a renoprotective strategy in several different clinical settings in which there is a risk of acute renal IRI [26]. In patients undergoing either cardiac bypass or major vascular surgery, acute renal IRI is a major determinant of acute kidney injury (AKI), a complication which occurs in 20–30 % of patients and which is associated with worse clinical outcomes. Several clinical studies have investigated a potential protective role of RIC on AKI in these surgical settings, but the results have been inconclusive [11, 77, 91]. The results of the large multicentre ERICCA [33] and RIPHeart [69] trials which are also investigating the effect of limb RIC on AKI should hopefully provide a definitive answer as to whether limb RIC is renoprotective in the setting of cardiac surgery.

Contrast-induced AKI (CI-AKI) is a significant cause of renal impairment in IHD patients undergoing coronary angiography and interventions, and one component of the injury is due to acute renal ischemic injury, and therefore a potential target for limb RIC [88]. Er et al. [24] have investigated in the Renal Protection Trial the effect of limb RIC on the incidence of CI-AKI in 100 high-risk patients undergoing coronary angiography and interventions who were pre-treated with intravenous normal saline and oral N-acetylcysteine—limb RIC reduced the incidence of CI-AKI from 40 to 12 %. The ERIC-CIN study in the UK is currently investigating whether the renoprotective effect of limb RIC is still present in 362 patients pre-treated with sodium bicarbonate prior to coronary angiography and procedures [7]. At the HCI Workshop, Crimi (Pavia, Italy) presented data investigating the effect of limb RIC on CI-AKI in STEMI patients treated by primary percutaneous coronary intervention (PPCI). In the original study, his team had already demonstrated a cardioprotective effect of limb RIC in this patient group with reduced enzymatic myocardial infarct size, and in this post hoc sub-group analysis they found that compared to control, limb RIC appeared to reduce the incidence of AKI in those STEMI patients with impaired renal function prior to PPCI [14, 15]. Finally, The EUROpean and Chinese cardiac and renal Remote Ischemic Preconditioning Study (EURO-CRIPS) trial will investigate both the renal and myocardial protective effects of limb RIC against CI-AKI and peri-procedural myocardial injury in 1,110 patients undergoing elective PCI, respectively [70].

Acute renal IRI sustained during pediatric renal transplantation is a critical determinant of graft function and clinical outcomes. MacAllister (London, UK) presented unpublished data from the REnal Protection Against Ischaemia–Reperfusion in transplantation (REPAIRISRCTN30083294) trial, a randomized double-blinded placebo-controlled trial of 400 living-donor renal transplant patients investigating the effect of limb RIC on renal graft function. He found that in those patients in whom limb RIC was administered to the donor and recipient, the estimated GFR at 6 months post-transplantation was increased compared to control, suggesting limb RIC to be a potential therapeutic strategy for preserving renal graft function post-transplantation.

B Davidson (London, UK) has been investigating in pre-clinical studies the protective effect and mechanisms underlying hepatic protection against acute IRI induced by limb RIC [2, 3, 49]. In the HCI Workshop, data were presented translating this therapeutic approach into the clinical setting, with a small study of 16 patients showing that limb RIC reduced the release of liver enzymes following liver resection surgery (ClinicalTrials.gov Identifier: NCT007965880). The ongoing Remote Ischaemic PreCOnditioning in Liver Transplant (RIPCOLT) study is currently investigating the efficacy of limb RIC in 40 liver transplant patients on liver protection and graft and patient survival.