Ischemic heart disease remains the single leading cause of death in the United States, with acute myocardial infarction (MI) accounting for the considerable morbidity and mortality from this condition (1). Final infarct size, which is the main predictor of outcomes following MI, is directly related to the duration of time from symptom onset to definitive treatment restoring coronary flow and myocardial tissue perfusion (2–5). Thus, the key to maximal salvage is summarized by the phrase “Time is Muscle”—shortening patients' symptom-to-door and door-to-balloon time in order to minimize the final volume of infarcted myocardium (4, 6, 7). Strategies to reduce the final infarct size include implementation of mild to moderate systemic hypothermia, which has been shown to protect against MI and slow the rate of infarction, allowing for increased ischemic time prior to reperfusion without increasing infarct size (8, 9). Multiple clinical trials have since evaluated the effect of endovascular cooling during primary percutaneous coronary intervention (PCI). It has been shown that patients who are systemically cooled to <35°C early during MI have a significant reduction in infarct size at the time of reperfusion (10). However, the induction of systemic hypothermia is resource intensive and must be performed with supervision by medical professionals, making the challenge of initiating these therapies rapidly after initial symptom onset likely unsurmountable (7).

We recently identified an alternative therapeutic hypothermia approach, topical neck cooling (NC), which could potentially exert similar infarct-sparing effects to systemic hypothermia via stimulation of the vagus nerve by temperature reduction. The link between the vagus nerve and cardioprotection has been reported by our lab (11) and others (12–16), and the vagus nerve has been demonstrated to be directly activated by temperature changes (17). In the current study, we investigated the cardioprotective effect of topical neck cooling, hypothesizing that locally decreased temperatures in the soft tissue and encompassed structures of the neck, without inducing systemic hypothermia, would attenuate myocardial ischemic and reperfusion injury (IRI). Furthermore, we evaluated the role of the timing of NC application and the depth of NC in achieving a protective effect against myocardial IRI. This topical approach to hypothermic therapy, which is non-invasive and easily implemented after MI, may prolong the therapeutic window to reach definitive reperfusion interventions and thereby could lead to improved patient outcomes.

This study complied with the Guide for the Care and Use of Laboratory Animals as recommended by the U.S. National Institutes of Health ensuring that all animals received humane care. The University of Virginia Animal Care and Use Committee reviewed and approved the study protocol.

Using a mouse model of myocardial IRI, we found that NC produced an infarct-sparing effect comparable to that seen with moderate systemic hypothermia. We distinguished that the cardioprotective effect of NC is timing- and dose-dependent; this therapy optimally should be applied early after initial symptom onset and prior to reperfusion. Furthermore, we identified that NC exerted a cardioprotective effect against both ischemic injury and post-ischemic reperfusion injury. The mechanism of this intervention involves activation of the vagal acetylcholine anti-inflammatory pathway to increase the myocardium's resistance to ischemia and reduce inflammatory responses during post-ischemic reperfusion.

Following the onset of an MI, the key to salvaging ischemic myocardium is shortening the time between coronary artery occlusion and reperfusion of the ischemic myocardium (2–5). Early recognition of MI and timely transportation to the hospital remain the only opportunities to shorten ischemic duration (4, 22), but faster transportation to interventional centers is improbable in the current healthcare landscape, and thus far, clinical treatments to protect myocardium during the ischemic period are lacking (7, 23). At this time, most potential therapies target early reperfusion as an adjunct to definitive revascularization (23). During the ischemic period, coronary occlusion precludes delivery of medication to the at-risk myocardium, rendering pharmaceutical interventions ineffectual during the ischemia phase of acute MI. Moreover, several promising preclinical drug therapies have not shown consistent benefit in clinical trials (7, 24). Pre-conditioning, by either ischemic or pharmacologic methods, prior to MI may increase myocardial tolerance to ischemic insult and attenuate ischemic injury (23, 25–28). However, its clinical application in acute MI is not feasible and often can only be delivered in the peri-revascularization period. Furthermore, randomized controlled trials of remote ischemic preconditioning under controlled elective situations have failed to show consistent clinically relevant beneficial effects (27, 29). When used during the MI itself, smaller clinical trials have suggested benefit with remote ischemic conditioning but a large phase III trial showed no difference in heart failure and death after a year (30–32). Mild to moderate systemic hypothermia has been demonstrated to be protective against acute MI and allows for prolonged time to reperfusion without a concomitant increase in infarct size (8–10, 31). This therapeutic approach also allows for its cardioprotective effect to be implemented during both the ischemic and reperfusion phases of acute MI. However, induction of systemic hypothermia is resource intensive and cannot be performed without the presence of medical personnel and thus has been difficult to translate clinically (7, 31). Thus, this practice has not been implemented as part of pre-hospital management of acute coronary syndrome, and a large gap in patient care persists.

To circumvent the logistical difficulties of inducing systemic hypothermia, we developed a therapeutic hypothermia approach of topical neck cooling and demonstrated that it attenuates myocardial IRI to a similar degree to systemic hypothermia (Figure 2). It has been reported that mild (35°C) and, to an even more significant degree, moderate (32°C) systemic hypothermia result in an infarct-sparing effect (33). Multiple clinical trials have since evaluated the effect of endovascular cooling during primary PCI, and it has been shown that patients with anterior STEMI who were cooled to <35°C early during MI have a significant reduction in infarct size at the time of reperfusion (10). Using an in vivo mouse model undergoing 40 min of LCA occlusion and 60 min of reperfusion, we found that moderate systemic hypothermia attenuates myocardial infarct size by 60% in comparison to normothermic mice. By cooling the neck to 15°C while maintaining normal core temperature, we found that simple topical neck cooling produced a similar infarct-sparing effect to that seen with moderate systemic hypothermia (Figure 2A). The infarct-sparing effect of NC is dose-dependent and effective only when it is applied before reperfusion (Figure 3). Given that the neck subcutaneous tissue temperature is similar between mild NC, which did not result in an infarct-sparing effect, and systemic hypothermia (data not shown), the cardioprotective mechanisms underlying NC may differ from that of systemic hypothermia.

Therapeutic hypothermia produces multifactorial beneficial effects resulting in an overall protective anti-inflammatory state, inhibition of apoptosis, and activation of cell survival pathways (34, 35). One of the mechanisms underlying hypothermic cardiac protection is vagal activation to modulate the IRI-associated inflammatory response (17, 36), possibly via a postsynaptic process (36). Preclinical and small clinical studies have demonstrated electrical vagal nerve stimulation itself has been clinically effective in reducing infarct size (14–16, 31). Moreover, neck cooling (via the application of a neck wrap containing frozen ice packs) (37) and electrical stimulation of the vagus nerve at the neck have been shown to relieve migraine headaches (38), raising the possibility that activation of the vagus nerve could be a shared mechanism underlying the beneficial effects of neck cooling In support of this, a recent clinical trial demonstrated that topical neck cooling at the lateral neck stimulates the vagus nerve and produces a parasympathetic nervous system response in healthy participants (39). In the current study, the infarct-sparing effect of NC was abolished by vagotomy at the GEJ (Figures 3B,C), further suggesting that the cardioprotective effect of NC against IRI is mediated by vagus nerve stimulation in the neck region. There was also evidence of parasympathetic activation by NC, with a significant decrease in heart rate observed during NC (Figure 8). In addition to being a major component of the parasympathetic nervous system, the vagus nerve has several other cardioprotective mechanisms (40), and has a recognized anti-inflammatory role with its efferent fibers mediating the cholinergic anti-inflammatory pathway (CAP) (41). The CAP is mediated through the binding of acetylcholine released from distal vagus nerve terminals to α-7-nicotinic cholinergic receptors (α7nAChR) to ultimately inhibit the release of pro-inflammatory cytokines, such as TNFα, by peripheral macrophages (42) and from the spleen (Figure 9) (43). In this study, NC significantly decreased splenic tissue acetylcholine levels while concomitantly increasing plasma acetylcholine (Figure 7), indicating that NC does modulate acetylcholine release, with the ultimate result of increased splenic clearance of this neurotransmitter, likely after its binding to downstream targets, and entry into peripheral circulation. Our previous studies have demonstrated that the spleen plays a central role in amplifying inflammatory responses and mediating myocardial IRI (2, 18), and that therapies to modulate the spleen to an anti-inflammatory phenotype attenuate myocardial IRI (11). Moreover, a cardioprotective role of the vago-splenic axis has been implicated in a preclinical study demonstrating infarct reduction via remote ischemic preconditioning in myocardial IRI, further establishing the role of both the spleen and vagus nerve in mediating the progression of infarction (13, 44). Taken together, these results suggest that the cardioprotective effect of NC may be attributable to activation of the vagal nerve, possibly via the CAP, to modulate splenic-derived inflammatory responses involved in post-ischemic myocardial reperfusion injury.

The extent of ischemic myocardial injury, defined by the duration from symptom onset to definitive treatment, determines post-ischemic reperfusion injury and therefore also final infarct size, which is the major predictor of clinical outcomes (2–5). Systemic therapeutic hypothermia attenuates ischemic myocardial injury and allows for longer ischemic time prior to reperfusion without consequent increase in infarct size (8–10). In this study we found that NC protects the heart not only from post-ischemic reperfusion injury but also from initial ischemic injury alone. During 40 min of LCA occlusion without reperfusion, NC attenuated ischemic myocardial infarct size by 50% compared to normothermic control as defined by TTC-blue staining (Figure 5) and significantly decreased CP levels of cTnT, cfDNA and HMGB1 (Figure 6). We have previously demonstrated that these two important DAMPs are released from ischemic myocardium during reperfusion and activate inflammatory pathways to induce reperfusion injury; both cfDNA and HMGB1 are vital to induce myocardial IRI (2, 18). The diminished release of cfDNA and HMGB1 from ischemic myocardium following NC mitigates the de novo inflammatory response within ischemic myocardium, as additionally reflected by the reduced CP levels of pro-inflammatory cytokines IFNα and IFNβ (Figure 6), and consequently is conducive to a reduced post-ischemic reperfusion injury as we have reported previously (18). Plasmacytoid dendritic cells are a potential target activated by cfDNA to produce IFNα and IFNβ and effect an injurious pro-inflammatory response (45–47). We plan to further evaluate the role of this innate immune cell subset in mediating myocardial IRI in future experiments.

Another mechanism through which NC-induced vagal activation may prove cardioprotective is through resulting bradycardia, given that NC demonstrated a significant decrease in HR. Bradycardia has long been thought to reduce cardiomyocyte oxygen consumption, and heart rate reduction therapies, including ivabradine or beta blockers such as metoprolol, given during the ischemic period of MI have been investigated with potential, but often equivocal, benefit (48–52). Such drugs may have significant benefit through alternate mechanisms independent of their induced bradycardia, such as with ivabradine (52). Within our study, late application of NC demonstrating decreased infarct size, albeit with less effect than when applied early, implicates a larger impact on the modulation of reperfusion injury. While our study underscores the impact of NC through inflammatory modulation, the effect of bradycardia cannot be excluded. The extent through which bradycardia plays an additional role warrants further investigation for neck cooling.

A limitation of this study is that the possibility of direct cooling of the heart contributing to NC's observed protective effect against ischemic injury and reperfusion injury cannot be excluded. We did find that NC resulted in moderate hypothermia in the pleural cavity (Figures 3A, 4B). Theoretically, NC may have directly lowered myocardial temperature either through local transfer from the neck subcutaneous tissues or return of cooled venous blood to the heart, and resultant reduced metabolic demands on the myocardium could, in part, account for the cardioprotective effect against ischemic and/or reperfusion injury. Studies with either large animals or more targeted cooling devices may help to differentiate these effects. Additionally, the role of the vagal cholinergic anti-inflammatory pathway in facilitating the cardioprotective effect of NC against ischemic myocardial injury and post-ischemic reperfusion injury remains to be defined.

In conclusion, NC without lowering core body temperature attenuated myocardial IRI to a similar degree to moderate systemic hypothermia. We demonstrated that NC reduces both ischemic and post-ischemic reperfusion injury via activation of the vagal cholinergic anti-inflammatory pathway. Clinically, NC may prolong the treatment window to achieve reperfusion without enlarging subsequent infarct size and would avoid the myriad negative side effects associated with systemic hypothermia. NC is a novel treatment that is straightforward to implement, readily accessible, and could be easily applied in the pre-hospital setting by non-medical personnel immediately after onset of acute MI to improve patient outcomes.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by University of Virginia Animal Care and Use Committee.

AZ and ZY contributed to conception and design of the study. AZ, BY, DW, and ZY participated in acquisition of data via experiments. KM and ZY performed the statistical analysis. IK and ZY participated in funding acquisition. AZ wrote the first draft of the manuscript. AZ, KM, ZY, and RR wrote sections of the manuscript and performed critical revisions. ZY supervised all aspects of the study. All authors contributed to manuscript revision, read, and approved the submitted version.

This work was supported in part by NIH R01HL130082, Commonwealth Health Research Board (CHRB) Grant Award #207-12-21, and University of Virginia George A. Beller, M.D. Research Award T32HL007849 (ZY) and T32HL007849-21A1 (IK).

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.

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