The incidence, mortality, and prevalence of neurological disorders are increasing worldwide, primarily because of the growing elderly population (1). Stroke is one of the most common neurovascular conditions with a prevalence of 101.5 million people worldwide (2021 Heart Disease and Stroke Statistical Update) (1). Of these strokes, 76% were classified as ischemic stroke (~77.2 million), ~ 20% as intracerebral hemorrhage (~20.7 million), and about 8% as subarachnoid hemorrhage (8.4 million) (1). Acute ischemic stroke (AIS) occurs when a major artery that supplies oxygen and nutrients to the brain becomes obstructed, leading to the formation of two injury zones: The ischemic core and the “penumbra.” The infarct core is severely hypoperfused, such that neurons undergo rapid and irreversible necrotic cell death (2). In response to ischemia and cell death in the core, inflammatory signals are released into the peripheral circulation, attracting immune cells to the damaged area and exacerbating the inflammatory response. The core of the ischemic region is surrounded by a relatively hypoperfused zone called the penumbra, which defines the tissue at risk for further infarction (3). Because cell death in this penumbral region occurs gradually, it is possible to rescue this peri-infarct area in the hyper-acute phase of AIS prior to cell death and infarct expansion.

One important factor contributing to the viability of penumbral tissue is the existence of strong collateral circulation that reduces ischemia in the penumbra, reducing injury, and improving the clinical outcomes (4). Pial collateral vessels—also called leptomeningeal collaterals–are auxiliary vascular networks on the brain surface that connect the distal part of major branches of the anterior and posterior cerebral artery (ACA, PCA) with the distal branches of the middle cerebral artery (MCA). These vascular anastomoses provide oxygen and essential nutrients via retrograde blood flow to the deprived ischemic tissue when the primary artery is blocked (5). Currently approved treatments for AIS, such as thrombolysis through recombinant tissue plasminogen activator (rtPA) administration or recanalization via mechanical endovascular treatment (EVT, i.e., mechanical thrombectomy), work in a time dependent manner and have a limited therapeutic window (6). While good collateral blood flow can extend this window, rapid restoration of flow to the brain remains the best treatment for acute stroke. However, this is restricted to ~10–20 percent of stroke sufferers who can make it to a primary stroke treatment center in time. Treatments that can improve collateral blood flow may extend the window for recanalization therapy and improve outcome for stroke patients (5).

Even after flow is restored in an occluded cerebral vessel, cellular injury can be exacerbated by reperfusion injury (7). Recanalization of the occluded artery can lead to damage to the integrity of the capillary endothelium, known as ischemia/reperfusion (IR) injury. Restored flow can increase BBB permeability when a high blood volume re-enters the already collapsed vasculature. Following this reperfusion, activated endothelial cells (ECs) produce reactive oxygen species (ROS), which further triggers the influx of inflammatory cells to the ischemic site (8). Increased leukocyte stimulation, trafficking and release of proinflammatory chemoattractant substances amplifies local inflammation. Elevated expression of adhesion molecules on ECs can further potentiate interactions between circulating blood cells and ECs, particularly neutrophil-endothelial interactions that can lead to neutrophil aggregation in the capillary bed (9). Reducing IR injury is key to improving outcome after recanalization therapy. So far, several approaches have been attempted to inhibit leukocytes aggregation and attenuate IR injury, but none have proven effective in clinic (10). Additional therapies are urgently needed to protect brain tissue from the ischemic and post-reperfusion damage. One such approach may be remote ischemic conditioning (RIC) (11, 12). RIC has shown to be a clinically safe and straightforward intervention which helps to attenuate the detrimental effects of ischemia. Multiple molecular signaling pathways contribute to the protective effects of RIC against reperfusion injury, with key signaling pathways converging on transcription factors that regulate cell survival and apoptosis (13). Of these signaling cascades, the reperfusion injury salvage kinase (RISK) and the survivor activating factor enhancement (SAFE) pathways are well-characterized. Below, we will review RIC for stroke treatment, its established mechanisms, and discuss RIC induced modulation of inflammatory immune cells and their gene expression profiles.

Remote Ischemic Conditioning (RIC) is a therapy that involves brief, intermittent episodes of sublethal ischemia and reperfusion that is applied to a peripheral tissue, organ or a vascular territory. This peripheral signal is then transmitted to the distal target organ (e.g., brain or heart) to relay protection against prolonged ischemia and subsequent IR injury (14, 15).

In 1986, ischemic preconditioning was described by Murry et al. in relation to cardiac ischemia (16). A preconditioning (PC) intervention was directly applied to the dog heart via four cycles (each for 5 min) of alternative occlusion/reflow of the left anterior descending (LAD) coronary artery prior to initiation of 40 min cardiac ischemia (16). Their results showed that PC was associated with a considerable reduction in myocardial infarction size. However, in another animal cohort, the same PC protocol preceding 3 h of sustained coronary occlusion failed to salvage the heart tissue injury, suggesting that PC has a protective time window and it may only delay the cellular death up to a few hours and then dissipates (16). Thereafter, additional investigations advanced the theory of “two time windows for protection” based upon these results (17–19). The early phase of protection occurs immediately, within minutes after the PC application, and lasts for ~3 h. It is thought that the early phase is mainly caused by rapid alterations in protein kinase signaling pathways that converge on the mitochondria to stop the apoptotic pathways (20, 21). The late phase starts 18–24 h after PC and lasts for ~4 days. The protection during the late period is probably due to de novo synthesis of proteins that are involved in inflammation, ischemia and vascular dynamics (12, 22, 23), and the suppression of genes involved in IR injury.

In 1993 the conditioning concept was extended to remote ischemic conditioning (RIC), in which ischemia is induced to an organ far from the target organ, often using a blood pressure cuff, offering a safe and feasible approach (24).

The remote application of RIC provides a safe, non-invasive and clinically applicable method, often involving intermittent cycles of inflation and deflation of a blood pressure cuff around the upper arm in humans and upper hind limb in preclinical studies in rodents (25). RIC has been used in three temporal windows during or after cerebral ischemia: (1) remote ischemic preconditioning (RIPreC) is applied prior to the injurious ischemia. While less practical as a therapeutic approach, because the stroke event is not always predictable, RIPreC can be used as a preventive measure for post-operative ischemic complications in known hospital settings. For example, prior administration of RIPreC to patients undergoing endovascular procedure can potentially reduce the high risk of ischemic or haemorrhagic stroke insult after surgical treatments for several clinical settings including intracranial aneurysms and carotid endarterectomy (26–29); (2) remote ischemic per-conditioning (RIPerC), which is applied during the ischemic event (prior to any recanalization); and (3) remote ischemic post-conditioning (RIPostC), which is applied after the ischemic event (i.e., following recanalization) or during reperfusion. The latter two conditioning paradigms have promise for translation, as they are non-invasive and can be administered pre-hospital (i.e., in an ambulance while transferring stroke victims to the emergency center) or following recanalization therapy (14).

Several parameters might affect the overall efficacy of RIC in reducing the infarct size following the focal ischemic stroke, including sex, age, animal species and different models of focal ischemia (30). A recent meta-analysis and systematic review has shown that there is no significant difference in RIC beneficial effects between reperfusion (e.g., intraluminal and embolism models) and permanent (e.g., cauterization, use of a permanent clip, permanent distal MCA ligation, permanent intraluminal suture) models of focal brain ischemia (30). However, RIC was shown to be more efficacious in male rodents relative to their female counterparts (30). As expected, older animals show significantly larger ischemic damage when compared with younger adult group due to several factors, namely, rarefaction of cerebral collaterals, decreased arteriole dimeter, higher tortuosity in cerebral vessel (31–33). All these factors affect the aged animals' ability to compensate for the poor blood flow circulation. On the other hand, the cellular and biochemical alterations associated with aging process, such as higher expression levels of pro-inflammatory cytokines and exacerbated oxidative stress, will reduce the cell survival rate in aged population and increases the neuronal cell injury and death (34, 35). Consequently, aged stroke groups may benefit less from the neuroprotective effects of RIC and may show more limited functional recovery in both pre-clinical and clinical settings (36).

Extensive research has been conducted in the preclinical and clinical settings to investigate the underlying mechanisms of RIC. Still, the primary molecular pathways are somewhat equivocal, possibly due to the contribution of several complex and overlapping signaling pathways. Although much of the research to date focuses on the protective role of RIC on cardiomyocytes in the heart, there is a growing focus on brain ischemia, and the underlying mechanisms of cardio- and neuro protection likely overlap. Multiple hypotheses have been proposed on how the protective signal is transferred from the periphery to the target organ. Generally, three pathways are thought to play a role in RIC protection (Figure 1).

In addition to direct effects on target organs, RIC has direct effects on improving blood flow in vulnerable tissue. Preclinical stroke studies in mice suggest that RIPerC is effective alone and in combination with i.v. r-tPA in enhancing penumbral flow in young male mice, ovariectomized female mice, and 12-month old male mice (14, 47–49). Remote ischemia has also been associated with increased cerebral blood flow in humans (50–54).

The collateral circulation is a key determinant of infarct progression in AIS. Good collateral flow is associated with reduced infarct expansion and better stroke outcome (55–64). However, a progressive constriction of collateral arterioles over time after ischemic onset may contribute to infarct growth (36, 65, 66). RIC may improve collateral flow by preventing narrowing of key collateral vessels, and is associated with improved collateral flow and reduced infarct in preclinical studies (4, 65, 67–69). Thus, preventing collateral failure is thus critical to improve outcome in stroke patients, and RIC may improve collateral flow. However, the exact mechanisms of enhanced collateral flow due to RIC are not defined.

RIC may increase cerebral blood flow (CBF) either through formation of new vascular branches (angiogenesis and/or arteriogenesis) or strengthening of the existing vasculature. Some of the major signaling mediators of CBF enhancement are discussed below.

RIC increases tolerance and viability of brain tissue during cerebral ischemia by activating signaling that supports survival and inhibits apoptosis, and by reducing inflammation. Several protective signaling pathways and pro-survival kinases and mediators have been shown to be involved in RIC-induced protection (Figure 2). The two most widely studied pathways are (1) the reperfusion injury salvage kinase (RISK) pathway, with its major signaling via Akt and Erk1/2, and (2) the survivor activating factor enhancement (SAFE) pathway, with its major signaling via Janus Kinase (JAK) and signal transducer and activator of transcription 3 (STAT-3) (15, 80–83). These signaling pathways can be triggered by a variety of factors, including SDF-1α, MIF, HIF-1α, heat shock proteins (HSPs), nitric oxide (NO), mammalian target of rapamycin, MMPs, adenosine, bradykinin, erythropoietin (EPO), endocannabinoids, and tumor necrosis factor-α (TNF-α) (15, 80, 84), and are discussed in greater detail below. Furthermore, AMPK signaling pathway is increasingly recognized as a potential mediator of cell survival following RIC, and is discussed below (85–88).

The alternative hypothesis to the “signal transfer through the activation of neural pathway” is that the protective signal generated locally in the remote site (like a limb) may have humoral (blood-borne) nature. Several blood-borne mediators have been identified as important for protection via RIC. These factors can travel via the circulation toward target tissue, wherein they can modulate inflammation and cell death.

Preclinical and clinical evidence suggests that RIC confers neuroprotection in the setting of AIS (144). A growing number of studies are suggesting part of this effect might be due to differential responsiveness of peripheral circulating immune cells following the conditioning stimulus (140, 145). The immune response to ischemic conditioning is itself composed of molecular, cellular, and systemic mediators that may play a role in conditioning tolerance (145). The conditioning stimulus can prime the brain in advance by mobilizing both innate and adaptive immune responses so that by the time severe IR injury happens, the brain enters the “resolution of inflammation” or “recovery phase.” Thus, immunomodulation may contribute to conditioning-induced protection in brain and heart (140, 145–147) (Figure 3).

Several studies have demonstrated that molecular and cellular profile of inflammation change following the conditioning stimulus (140). In a rat model of focal ischemia, Liu et al. reported that RIPreC-mediated neuroprotection was associated with altered immune cell populations and cytokine profiles (140). In this study, a shift in the phenotype of splenic monocytes toward less or non-inflammatory (non-classical) monocytes (CD43⁺/CD172a⁺) was observed in the RIPreC-treated rats (140). Monocytes with inflammatory phenotype (classical monocytes) can infiltrate the brain and lead to highly inflammatory type of cell death via their potent ability to secrete inflammatory mediators and free radicals and to differentiate into macrophages and dendritic cells (148). Thus, RIPreC-mediated change in favor of non-inflammatory monocytes prior to focal stroke has been beneficial against ischemic attack (140). In addition, the expression levels of proinflammatory cytokines such as TNF-α and IL-6 were significantly elevated by RIPreC, suggesting that conditioning induced manipulation of the immune response may be a key mechanism of protection (140) (Figure 3).

Beside the importance of peripheral immune cells in conditioning effect, the brain's resident immune cells, microglia and astrocytes, are also considered to be cellular mediators of conditioning stimulus, as they contribute to resolution of neuroinflammation by promoting immunosuppression (145). These cells can release anti-inflammatory cytokines (e.g., TGFβ and IL-10) to inhibit the inflammatory response. In addition, astrocytes and neuronal cells arrange the “repair and regeneration” phase by producing growth factors such as insulin-like growth factor (IGF) and vascular endothelial growth factor (VEGF) that promote neuronal sprouting and angiogenesis (145).

Microglia exhibit a high level of TLR4 expression on their cell surface which helps them to initiate the innate immune response. While hyperactivation of these receptors are known to aggravate the inflammatory status, partial activation during a brief ischemic conditioning may confer neuroprotection by priming the brain against severe and prolonged ischemia (149). Pradillo et al. reported that prior exposure to ischemic preconditioning (IPC, 6-min occlusion of bilateral common carotid arteries) can induce immunological tolerance and therefore protect against the permanent MCAO in wildtype (WT) mice with normal TLR4 expression (149), as shown by a better neurologic outcome and reduced infarct size. However, genetic deletion of TLR4 receptors abolished the protective properties of IPC, indicating the importance of TLR4 for activation of innate immunity and induction of ischemic tolerance by IPC (149). They further observed IPC upregulated the protein levels of TNF-α, iNOS, and COX-2, p65 subunit of NF-κβ transcription factor and downregulated inhibitory kappa B alpha (IκBα). These molecular proteins have been suggested to mediate the ischemic tolerance by IPC (42, 149). Likewise, all the results were reversed in TLR4-deficient mice. Taken together, TLR4 signaling pathway mediates the IPC-induced neuroprotection via activation of transcription factor NF-κβ and therefore upregulation of TNF-α, iNOS, and COX-2 (149, 150).

Considering the fact that peripheral and resident immune cell activation can dramatically change the inflammatory status during and after the cerebral ischemia, RIC's ability to modify the immune response and thereby improve the stroke outcome poses this novel treatment as a potential therapeutic adjunct to already approved stroke therapies.

Gene expression in circulating peripheral immune cells rapidly changes after AIS, and several studies have utilized gene expression profiling to investigate transcriptomic alterations in these cells (151–153). Genes differentially expressed after stroke in humans including neutrophils and monocytes (151, 152), suggesting neutrophils and monocytes play key roles in the genomic responses of circulating blood cells to AIS (151, 152, 154). Alterations in genomic patterns happens at an early stage (<3 h) after stroke onset, and these rapid changes can be used to make an early diagnosis of AIS in humans (151). Notably, neutrophils are the first immune cells to arrive at ischemic brain tissue and are key contributors to BBB permeability, cerebral edema and brain injury (154). Therefore, therapeutic approaches that target the deleterious aspects of neutrophil activation, including neutrophil-mediated BBB disruption, neutrophil transmigration and infiltration, and their interaction with the neurovascular unit (NVU), may be helpful to reduce brain edema and therefore improve the stroke outcome (155).

The significance of transcriptional gene screening of blood cells lies in its potential to identify and validate specific genes as molecular biomarkers in ischemic stroke diagnosis and prognosis (152, 156). A refined gene expression signature would allow a readily available clinical evaluation by blood test, especially when brain imaging facilities are limited (153). Unraveling the differential expression of transcriptomic profile in the whole blood as well as the isolated immune cell populations would not only help to understand the underlying mechanisms during the stroke pathology, but also aid in the development of novel treatments for stroke. Due to RIC's early potential in reducing brain ischemic infarct caused by severe AIS, evaluation of transcriptome in peripheral blood cells following RIC and its comparison with stroke-related changes in gene expression may provide key mechanistic insight into neuroprotection.

The first study of genome expression in human leukocytes following RIPreC was reported by Konstantinov et al. who found that conditioning stimulus achieved by transient forearm ischemia (3 cycles of 5 min I/R) in healthy individuals significantly downregulated the expression of proinflammatory genes in leukocytes (46). These suppressed genes are known to be responsible for the inflammatory responses, including genes involved in TLR4-signaling, proinflammatory cytokine release (TNF-α), leukocyte chemotaxis and extravasation (PI3KCA), leukocyte adhesion (e.g., integrins, ADAM 8,10, PECAM), and exocytosis and secretory granule release (SNAP-23) (46). In another study by the same research group in 2010, these alterations in human leukocyte gene expression showed strong correlation with functional responses of neutrophils, in particular a significant reduction in neutrophil adhesion and phagocytosis ability (40) (Figure 3).

RIPreC was also associated with a 3-fold reduction in synaptosome-associated protein (SNAP-23) in leukocytes, a protein known to mediate exocytosis in mast cells and neutrophils. Lack of SNAP-23 prevents the formation of ternary complex with other SNARE proteins and therefore inhibits the fusion of granules in these cells (46). It is well-established that neutrophils mainly contribute to the inflammatory responses through secretion of specific cytoplasmic granules containing cytotoxic species and proteolytic enzymes; therefore, RIPreC-induced downregulation of SNAP-23 gene may partially explain the mechanisms that underlie the protection (46, 157). In addition, decreased levels of platelet endothelial cell adhesion molecule (PECAM1 or CD31) gene expression after RIPreC may be responsible for the observed reduction in the chemotactic ability of neutrophils (46, 157). PECAM1 is known to stabilize and preserve the integrity of BBB, and it is normally expressed on endothelial cells, platelets, neutrophils, monocytes and specific members of leukocytes. However, in neuroinflammation, PECAM1 mediates paracellular diapedesis across the vascular wall and its blockage abolishes the leukocyte migration (158). Therefore, lower expression of PECAM1 mRNA in RIPreC group compared to the controls may reduce the neutrophil transmigration in the brain (46).

RIPreC also suppresses the expression of the CCR2 gene that encodes for C-C chemokine receptor type 2, an essential protein needed for monocyte migration, infiltration and macrophage trafficking. This result is aligned with a significant reduction in the number of tightly adherent leukocytes and reduced leukocyte accumulation at the inflammatory sites in CCR2-deficient mice, suggesting that CCR2 modulation by RIC may reduce leukocyte adhesion (159). Taken together, RIC modulatory effects on immune responsive cells results in attenuation of inflammatory responses. This modulation includes reduction of excessive release of proinflammatory mediators during AIS, and enhancing the release of anti-inflammatory cytokines such as IL-6 and IL-10 (46).

Extracellular vesicles (EVs) are submicron-sized membrane-derived particles that are generated from different cell types under physiological and pathological conditions (160). Their contents include lipids, proteins, and genetic materials (i.e., microRNAs and circRNAs). EVs function via transferring their cargo, especially miRNAs, to neighboring target cells, or can act over long distances as an intercellular messenger (160). Fundamental biological processes in the target cells (e.g., proliferation, apoptosis, survival, and differentiation) can be modulated by EVs (160). EVs are classified into three main groups based on size distribution, chemical composition, and route of biogenesis: exosomes (30–150 nm), microvesicles or microparticles (MVs or MPs, 150–1,000 nm), and apoptotic bodies (500–5,000 nm) (160). However, since there is no strict size distribution for these sub types, and because different physiological or pathophysiological situations may affect their size and surface protein expression, it is recommended by the International Society for Extracellular Vesicles (ISEV) to use the general term “Extracellular Vesicles” while referring to the three subsets (161). EVs inherit their composition and physicochemical properties from their parent cells (162). Other than general EV markers, like tetraspanins CD9, CD63, and CD81, EVs carry signature markers of their cells of origin (e.g., common surface markers in humans are CD146+ for endothelial-derived EVs, CD41+ for platelet-EVs, CD45+ for leukocyte-EVs, and CD235+ for erythrocyte-EVs) (163). The ubiquitous nature and abundant presence of EVs in most body fluids and their ability to reflect cellular and molecular alterations under pathological states qualify them as promising and powerful tool in biomarker studies (163). For instance, platelet-derived microvesicles (PMVs) are known to play a key role in the pathogenesis of acute atherothrombotic events, such as thrombosis, recurrent ischemia, stroke, and vascular inflammation (164). Notably, there is a correlation between an increase in microvesicles released from platelets and endothelial barrier dysfunction (164, 165). Under normal physiological conditions, the majority of the circulating EV population is derived from circulating platelets and platelet precursors in the bone marrow (163–165). However, EV number, origin, and composition can change in pathology (164).

PMVs are important mediators of vascular homeostasis, inflammation, and angiogenesis (165). Accordingly, PMVs can contribute to the vascular homeostasis by maintaining the balance between their procoagulant and anticoagulant properties, depending on the composition of their surface markers or molecular contents. While the expression of phosphatidylserine (PS) and tissue factor (TF) on these vesicles can trigger the activation of coagulation cascades (166), the presence of glycoprotein 1b and annexin V is necessary for activation of protein C and its co-factor protein S, which are best characterized for their roles in anticoagulation pathways (167). PMVs can also play immunomodulatory role in modulating inflammation (165). PMVs exert pro-inflammatory actions mainly via provoking monocytes and neutrophils, thereby inducing them to release inflammatory mediators, including IL-1β, TNF-α, MCP-1, and MMP-9 (168). Notably, PMVs can boost the immune response by promoting leukocyte-endothelial interactions (169), via PMV uptake by activated neutrophils (polymorphonuclear cells, PMNs) and endothelial cells (ECs). Activation of neutrophils and ECs by PMV uptake was confirmed by an increased surface expression of CD11b and adhesion molecules (ICAM-1 and P-selectin), respectively (169).

Endothelial-derived microvesicles (EMVs) are also important players during various aspects of inflammation (170). They are believed to be secreted from activated ECs as an early response to any alterations in vascular homeostasis. In particular, EMVs contribute to fundamental processes affecting vascular endothelial cell fate, such as apoptosis, cell survival and proliferation, and homeostasis (170). A recent study investigated the content and vascular effects of endothelial-derived microvesicles (EMVs) during inflammation (171). It was shown that the protein levels of c-Src kinase inside the isolated EMVs from mouse was elevated (171) and led to dissociation of endothelial adherens junctions and increased vascular permeability (171). Additionally, src kinase hyperactivity caused higher adhesion and interaction between neutrophils and ECs, as was shown by elevated expression of adhesion molecules (ICAM-1 and VCAM-1) and integrins (CD11b) on the endothelium and neutrophils, respectively (171). In a rat model of focal brain injury and CNS inflammation (induced by IL-1β microinjection into the striatal region), Couch and colleagues demonstrated that the number of circulating CD31-positive EVs (i.e., endothelial cell origin) significantly increased in the acute phase of brain injury compared to age-matched controls (172). Proteomic analysis revealed that circulating EVs in the bloodstream after stroke contain upregulated proinflammatory proteins and can activate peripheral immune cells to induce an inflammatory response (172).

Numerous studies have been conducted to identify the conveyor of RIC protective signals from the remote site to the target organ. EVs may be a potential carrier of this signal (40, 173–177). EVs can transmit cargo (e.g., lipids, proteins, and nucleic acids) from the donor cells to nearby or far-away target cells to modify biological processes in them (178). In this regard, Shan et al. investigated whether transfusion of isolated PMVs from RIPreC-treated rats (donor) to rats who underwent transient MCAO (recipient) can confer protection (179). Their findings revealed a significant increase in PMV (CD41+ and annexin V+) levels in the PMV-treated recipient mice compared to control mice, with a resultant reduced infarct size and better neurologic outcome, indicating that PMVs may be a carrier of the RIPreC protective signal (179). Likewise, Li et al. reported neuroprotection conferred by RIPreC (three cycles of 10-min occlusion/reopening of mouse hindlimb) against permanent MCAO in mice was associated with increased levels of exosomes (<100 nm in diameter) in plasma (180). Interestingly, the transfer of purified exosomes from RIPreC-treated mice (donor) to non-treated stroked mice (recipient) reduced infarct volume and improved neurologic outcome in the recipient mice, indicating RIPreC protective signal may be conveyed through exosomes. Furthermore, RIPreC treatment upregulated the HIF-1α in the purified exosomes compared to the control group (180). Notably, sublethal hypoxic or ischemic conditioning also upregulates transcription factor HIF-1α, which in turn translocates into the nucleus and dimerizes with HIF-1β (181). After dimerization, HIF-1α binds to the hypoxia response elements on specific target genes, such as VEGF and erythropoietin (EPO), thereby counteracts the cell apoptosis. The neuroprotective properties of VEGF and EPO in ischemic brain have been linked to their ability to induce angiogenesis and neurogenesis, respectively (182, 183).

Similarly, whether RIC cardioprotection is transferable from the RIC-treated subject's plasma to naïve untreated subjects, and if this is mediated by circulating EVs, has been examined in both rodents and humans (184–186). Notably, these studies suggest RIC increased the release of EVs from the heart, RIC-induced protective signal is conveyed in part to the target organ via EVs, and this protection is transferable intra and across species. Using an ex-vivo langendorff-perfused rat heart method, Giricz et al. assigned the isolated hearts to two “donor” and “recipient” groups (173). A group of donor hearts were preconditioned via 3 alternate cycles of 5 min ischemia/5 min reperfusion prior to 30 min of global ischemia. Western blots against the EV marker (HSP60) revealed higher EV levels in coronary perfusates collected from preconditioned donor hearts compared to untreated control hearts, (173) as well as a smaller infarct, suggesting an EV-mediated transmission of RIC protective effects. Infarct size was significantly decreased in naïve hearts that received the coronary perfusate from the preconditioned donors, while no reduction of infarct size was noted in the hearts recipient of EV-depleted coronary perfusate (173). Likewise, RIPreC-induced cardioprotection has been associated with increased EV concentration and differential expression of specific microRNAs in plasma from patients who underwent coronary bypass surgery (187). Isolated EVs from RIC-treated patients added to cultured rat cardiomyoblasts in vitro conferred the same protection against hypoxia, indicating RIC protection is mediated via circulating EVs, and it is transferable across species (187). These studies also confirmed the presence of a well-recognized endothelial surface marker (i.e., CD146) on the isolated EVs, suggesting endothelial cells as a likely cellular source of RIC-induced EV release (187).

MicroRNAs (miRNAs or miRs) are a key regulator of many fundamental cellular and molecular processes, such as cell growth, differentiation and apoptosis (188). MiRNAs are small single-stranded non-coding nucleic acids (~22 nucleotides long), which function through base-pairing with a complementary region in mRNA transcript and repress their translation into functional proteins (188).

The emerging role of miRNAs in stroke pathogenesis has recently been the focus of investigations in this field (189). MiRNA and target mRNA expression levels can change rapidly after the cerebral ischemia (190–193). Besides, numerous studies demonstrated that RIC alters the miRNA profile and thereby the expression and translation of genes and proteins. This again can reprogram the transcriptional response to the ischemic event (176, 194–196). Therefore, identifying the miRNAs and miRNA targets involved in stroke pathophysiology appears to be a promising candidate as either diagnosis or therapeutic tool.

Evidence from animal studies of cerebral ischemia suggests that miRNA (non-coding genes) genes may have a higher sensitivity to preconditioning (PC) stimulus than protein-coding mRNAs (191, 197, 198); since differential expression was observed in more than 20% of miRNAs, while <5% of coding mRNAs changed following PC application (197, 198). MiRNA profiling analysis by Dharap et al., in the cerebral cortex of preconditioned mice after a 10 min MCA occlusion reported a rapid change in the expression of 51 miRNAs following PC. Bioinformatics and pathway analysis suggested MAP Kinase and mTOR signaling are the main downstream signaling pathways of up-regulated miRNAs (26 out of 51), and Wnt and GnRH signaling pathways are the main targets of down-regulated miRNAs (25 out of 51) (197). Of these 51 differentially expressed miRNAs, the most up-regulated and down-regulated miRNAs 24h after PC were miR-21 (13-fold) and miR-466c (27-fold), respectively. Notably, miR-21 is anti-apoptotic factor that attenuates the expression of certain pro-apoptotic genes, including programmed cell death 4 (PDCD4), phosphatase and tensin homolog (PTEN), tropomyosin1 in neurons (199–203).

miRNAs play an important role in apoptotic signaling pathways through regulation of many pro-apoptotic genes (204–207). For a detailed review on apoptotic factors regulated by many different miRNAs, see Jang and Lee (204). For instance, Wu et al. demonstrated that upregulation of miR-21 (measured by rt-PCR Quantitative Kit) in human keloid fibroblasts were associated with a host of cellular events, which all led to the inhibition of cell apoptosis, including lower ROS, increased ratio of Bcl-2/BAX, decreased cytochrome C release into the cytosol, lower activity of caspase-3 and 9. All these events are critical components involved in the mitochondrial-mediated apoptotic pathway (206). In a rat embolic MCA occlusion model, Buller et al. demonstrated that elevated miR-21 level after focal ischemia attenuated the expression of Fas ligand (FasL) via complementary base-pairing with FasL transcript and blocking its translation into FasL protein ligands (208). Thus, miR-21 attenuates the neuronal cell apoptosis in the ischemic brain area by targeting critical cell death-inducing factors (200, 202, 203). FasL belongs to the TNF family, and its binding to one of the apoptosis signaling receptors FasR (apoptosis antigen-1, APO-1) initiates a cascade of events that leads to the activation of caspases and eventually causes neuronal cell death (209).

An important consideration is the potential use of miRNAs in maintaining the ionic balance in the ischemic region of the brain (176). During brain ischemia, there is a disruption in sodium and calcium balance due to downregulation of necessary ion channels and transporters by regulatory effects of some miRNAs (210, 211). To this end, blocking the expression of these miRNAs can be a therapeutic strategy to interfere with their detrimental behaviour (212). Anti-miRNAs evolution represents an efficient approach to inhibit and alter the action of miRNAs. Interestingly, in a rat model of transient cerebral ischemia, miR-103-1 was shown to downregulate the expression of Na/Ca exchanger (NCX1), a plasma membrane transporter which regulates the ionic homeostasis in ischemic brain. Notably, anti-miR-103-1 could significantly upregulate the expression of NCX1 mRNA and proteins levels in the brain cortex and striatum of ischemic rats, inducing a strong neuroprotective effect (~60% reduction in infarct volume) (211).

Aside from the regulatory role of miRNAs in the underlying mechanisms of conditioning-induced neuroprotection, miRNAs have also shown to be involved in the cardioprotective effects of conditioning (184). For example, Lassen et al. demonstrated that beneficial effects of RIC are delivered through EVs and their miRNA content. The transferability of EV-mediated RIC cardioprotection from the RIC-treated patients to in vitro cultured murine myoblasts was also demonstrated (184). In this study, the three miRNAs were most upregulated in association with cardioprotection after the RIC treatment were miR-144-3p, miR-451a, and miR-16-5p. These miRNAs demonstrated a two-fold upregulation, and each was linked to fibroblast growth factor 2 (FGF2) mRNA. Pathway analysis and gene ontology analyses suggested that all three differentially expressed miRNAs are associated with the mTOR signaling pathway and mediate the protein turnover, stress response, and apoptosis (184). Additionally, IR injury in the mouse myocardium reduces miR-144 expression levels, and this was reversed in mice receiving either RIPreC or systemic injection of miR-144 into the tail vein 30 min prior to global ischemia. These manipulations resulted in a marked reduction in infarct size and improved functional recovery of the heart. However, these beneficial effects were abolished after systemic injection of antagomir-144 (specific antisense oligonucleotide against miR-144), indicating the significance of miR-144 in RIPreC -induced cardioprotection. Moreover, miR-144 elevation led to downregulation of mTOR. Of note, mTOR signaling is an inhibitor of autophagy, which is a vital regulator of cell survival and a natural homeostatic mechanism of cell to remove the unnecessary or damaged components. Improved functional recovery after RIPreC may involve suppression of mTOR signaling and improved cardiomyocyte survival through increased autophagy (213).

Characterization and identification of EVs provides a “snapshot” of the environment of their origin cell at any given time. Additionally, they have a ubiquitous nature with high abundance in most body fluids. These features, along with their capacity as a vehicle for intercellular communications, position them as an ideal diagnostic and/or therapeutic target in many pathological states, including AIS. Moreover, these natural lipid mediators can be modulated for the delivery of specific agents or drugs to the target cells or organs, exhibiting superior properties relative to synthetic nanoparticles, including natural targeting ability, biocompatibility and safety. Thus, by identifying key EV-based mechanisms of RIC, new avenues of therapy to improve outcome after AIS can be developed.

SA-H researched, wrote, and designed figures for the manuscript. IW and GJ designed, co-wrote, and edited the manuscript with SA-H. All authors contributed to the article and approved the submitted version.

Funding for this project was provided by the Canadian Institutes for Health Research (IW), Heart and Stroke Foundation of Canada (IW), and the David Lawson Graduate Scholarship (SA-H, Faculty of Medicine and Dentistry, University of Alberta).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.