The protective effect of vagus nerve stimulation against myocardial ischemia/reperfusion injury: pooled review from preclinical studies

Aims: Myocardial ischemia-reperfusion (I/R) injury markedly undermines the protective benefits of revascularization, contributing to ventricular dysfunction and mortality. Due to complex mechanisms, no efficient ways exist to prevent cardiomyocyte reperfusion damage. Vagus nerve stimulation (VNS) appears as a potential therapeutic intervention to alleviate myocardial I/R injury. Hence, this meta-analysis intends to elucidate the potential cellular and molecular mechanisms underpinning the beneficial impact of VNS, along with its prospective clinical implications. Methods and Results: A literature search of MEDLINE, PubMed, Embase, and Cochrane Database yielded 10 articles that satisfied the inclusion criteria. VNS was significantly correlated with a reduced infarct size following myocardial I/R injury [Weighed mean difference (WMD): 25.24, 95% confidence interval (CI): 32.24 to 18.23, p < 0.001] when compared to the control group. Despite high heterogeneity (I2 = 95.3%, p < 0.001), sensitivity and subgroup analyses corroborated the robust efficacy of VNS in limiting infarct expansion. Moreover, meta-regression failed to identify significant influences of pre-specified covariates (i.e., stimulation type or site, VNS duration, condition, and species) on the primary estimates. Notably, VNS considerably impeded ventricular remodeling and cardiac dysfunction, as evidenced by improved left ventricular ejection fraction (LVEF) (WMD: 10.12, 95% CI: 4.28; 15.97, p = 0.001) and end-diastolic pressure (EDP) (WMD: 5.79, 95% CI: 9.84; −1.74, p = 0.005) during the reperfusion phase. Conclusion: VNS offers a protective role against myocardial I/R injury and emerges as a promising therapeutic strategy for future clinical application.


Introduction
Myocardial infarction (MI) remains a primary global cause of mortality and disability.Prompt and successful reperfusion of the ischemic myocardium through thrombolytic therapy or primary percutaneous coronary intervention is the most efficacious strategy to salvage ischemic myocardium, mitigate myocardial injury, and enhance clinical outcomes (Hausenloy and Yellon, 2013).However, the process of myocardial reperfusion may trigger cardiomyocyte death and exacerbate cardiac dysfunction.This paradoxical occurrence, known as myocardial ischemia/reperfusion (I/R) injury, curtails the beneficial effects of revascularization strategies (González-Montero et al., 2018;Yang, 2018).While the exact molecular mechanisms of reperfusion-related cardiomyocyte death remain not fully clarified, it thus implicates a pressing need for deep exploration and succedent unmarked novel therapeutic targets (Piper et al., 1998).
Vagus nerve stimulation (VNS) was originally employed for treating refractory epilepsy and depression, leveraging its potential advantages in autonomic neuromodulation (George et al., 2007;González et al., 2019).Subsequent research has increasingly suggested that VNS can also confer protection against heart failure progression, due to the restoration of autonomic balance, baroreceptor sensitivity, and electrical stability (Capilupi et al., 2020;Verrier et al., 2022;Elamin et al., 2023).Recent studies have progressively unveiled the role of VNS in mitigating myocardial I/R injury through the activation of the cholinergic antiinflammatory pathway, anti-oxidative stress response, or antiapoptotic response (Chen et al., 2020;Wang et al., 2020;Deng et al., 2022).However, the intricate mechanisms underlying VNSmediated cardioprotection in experimental studies, along with limited clinical evidence, pose obstacles to its broader application in clinical practice.
Hence, a comprehensive systematic review and meta-analysis are warranted to evaluate the effectiveness of VNS during myocardial I/R injury and provide a deeper understanding of the underlying mechanisms of this therapeutic approach.

Search strategy
We conducted a systematic literature search for animal studies assessing the cardioprotection of VNS in myocardial I/R injury in MEDLINE, PubMed, Embase, and Cochrane Database from the inception to July 2023, with no language restriction.The following search terms were used: "myocardial ischemia/reperfusion injury" OR "myocardial I/R injury" OR "myocardial ischemia-reperfusion injury" AND "vagal nerve stimulation".Moreover, we searched the references of comments, meeting abstracts, and review articles for additive studies.

Inclusion and exclusion criteria
Studies were included based on the following criteria: (a) reported the infarct size measured by triphenyl tetrazolium chloride (TTC) and Evan's blue double staining method, (b) analyzed intervention received VNS treatment merely; comparator intervention received or no treatment, (c) with no cardiovascular-related comorbidity.We excluded studies that did not express infarct size as the percentage of infarct area over the area at risk (AAR) or did not quantify the ischemic area by Evans blue/ TTC staining.

Data extraction
The data were extracted independently by two authors (Yu-Peng Xu and Xin-Yu Lu) from included studies, with discrepancies resolved by consensus.The following details were recorded in Table1: (1) studies' information, including first author's name, country, year of publication number of included animals, and duration of I/R injury; (2) animals' characteristics, including species, gender and anesthetics; (3) the vagal nerve stimulation protocol, including stimulation site, duration, parameters and heart rate reduction; (4) methods for determining the infarct size.The results were expressed in terms of mean and standard deviation to minimize publication bias.The digital ruler software was used to measure the value when some data were only represented by graphs.

Quality assessment
Two reviewers independently evaluated and graded the quality of included studies based on published criteria for animal experiments.One point for each of the following: a peer-reviewed publication, random allocation to groups, blinded assessment of outcome, sample size calculation, compliance with animal welfare regulations, and a statement of a potential conflict of interest.Any discrepancies were arbitrated by a third reviewer.

Statistical analysis
All outcome data were treated as continuous variables in this meta-analysis, presented as the mean and standard deviation.DerSimonian and Laird random effects metaanalysis was used to measure the WMD and the related 95%CIs.Heterogeneity between studies results was evaluated by Cochran's Q test and quantified by I 2 statistics test.Begger's and Egger's test was used to assess the potential publication bias.

Results
A total of 61 studies were initially screened and 10 studies comprising 238 animals matched the inclusion criteria for further quantitative analysis (Figure 1).Of these, 123 animals were treated with VNS and 115 animals were treated with control therapy.Cohort characteristics were presented in Table 1.Half of the studies used rodents with the remaining used rabbits, dogs and

Infarct size
Data on infarct size were available in 10 studies.VNS was associated with a dramatic reduction of infarct size assessed by Evans blue/TTC staining post myocardial I/R injury (WMD: 25.24, 95% CI: 32.24 to −18.23, p < 0.001, Figure 2), accompanied by high heterogeneity (I 2 = 95.3%, p < 0.001).There was no evidence of publication bias according to Begg's and Egger's test.Subsequent sensitivity analysis utilizing the one-study-omit method showed similar findings (Table 3).In addition, stratified analysis according to vagal stimulation site, duration, animal species and  state region and myocardial I/R regimen did not influence the efficacy results of infarct size after I/R assaults (Table 4).Further meta-regression also did not reveal any interaction between the prespecified covaries and VNS-mediated reduction in myocardial I/R damage (Table 5).

Discussion
As far as we are aware, this is the first meta-analysis ever conducted to demonstrate that VNS is beneficial in protecting the myocardium from ischemia-reperfusion (I/R) injury.By incorporating data from 10 distinct studies, our research evaluated the efficacy of VNS in preclinical studies.These findings indicated that VNS could significantly reduce infarct size during myocardial I/R injury and also improve heart function by reducing LVEDP and increasing LVEF.Intriguingly, these benefits  were observed to be independent of the type and site of VNS or the animal size.Myocardial I/R injury remains a significant clinical challenge despite advancements in reperfusion therapies such as thrombolysis and PCI (Férez Santander et al., 2004).This is primarily because of the intricated pathophysiologic process underlying reperfusion injury, including oxidative stress, calcium overload, inflammation, mitochondrial dysfunction, and cell apoptosis (Davidson et al., 2019).During the reperfusion, excessive reactive oxygen species (ROS) production due to the abrupt increase in oxygen supply and corresponding antioxidant enzyme insufficiency are the critical factor of cardiomyocyte death (Férez Santander et al., 2004;Hausenloy and Yellon, 2013).Meanwhile, previous studies reported that mitochondria are the main source of ROS and mitochondrial damage affects post-injury cardiac function by dysregulated ROS modulation.In a vicious cycle, ROS can also impair the mitochondrial respiratory chain and promote mitochondrial membrane depolarization, leading to impaired ATP production and further exacerbating cell death (Murphy and Steenbergen, 2008;Ong and Hausenloy, 2010;Ong and Gustafsson, 2012).Moreover, non-coding RNA, including mi-RNA and Lnc-RNA have increasingly emerged as key regulators  in various cellular processes such as apoptosis, inflammation, fibrosis, and angiogenesis and have implications for myocardial ischemia-reperfusion (I/R) injury (Ong et al., 2018).Unfortunately, there are currently limited therapeutic options available to prevent heart damage from reperfusion injury.Several pharmacological interventions have been tried to attenuate myocardial I/R injury by targeting the abovementioned cellular and molecular mechanisms.Vitamins C and E, being well-established antioxidant, have been shown to reduce cardiomyocyte death by inhibiting ROS release during the reperfusion injury (Rodrigo et al., 2014).In addition to the anti-inflammatory drugs, calcium channel blockers or cyclosporine also showed similar cardioprotective effects in retarding infarct area extension and subsequent deterioration of systolic function in a preclinical setting (Boden et al., 2000;Piot et al., 2008;Trelle et al., 2011).However, none of them showed the theoretical potential in clinical translation due to the huge gap between compelling experimental evidence and scant clinical data.
The vagus nerves, originating from the medulla oblongata, are the longest cranial nerve and is involved in the regulation of various physiological systems (Berthoud and Neuhuber, 2000).VNS is first identified as a therapeutic approach for Inflammatory disease by activating the cholinergic anti-inflammatory pathway (Bonaz et al., 2016).On the contrary, vagal denervation consistently released the lymphocyte from thymus to spleen and lymph nodes, which indicated the role of vagus nerves in controlling inflammatory status (Antonica et al., 1994;Antonica et al., 1996).Recently, clinical trials and preclinical trials have demonstrated the beneficial effect of VNS in reducing arrhythmias and hospitalizations, improving cardiac contractility and quality of life for patients with heart failure or AF, which suggests a crucial role of VNS in the treatment of heart disease (Li et al., 2004;Zhang et al., 2009;Zannad et al., 2015;Gold et al., 2016).In terms of the physiological properties of VNS, it was also utilized as a promising method for alleviating myocardial reperfusion injury.As expected, VNS modulates inflammatory cytokines and simultaneously inhibits ROS by activation of AMPK cascades (Kong et al., 2012).Additionally, experimental research indicated that VNS preserved the integrity and function of mitochondria by regulating mitochondrial dynamics, biogenesis, and mitophagy, which turns into cardioprotection against myocardial I/R injury (Nuntaphum et al., 2018).Meanwhile, VNS suppresses the sympathetic nerve sprouting and blocks the inflammatory process, which attenuating ventricular remodeling and decreases the incidence of ventricular arrhythmias after reperfusion injury on mechanism, Jak2/STAT3,  NF-κB, Akt/GSK-3β signaling pathway, which are responsible for VNS induced preventive effects on myocardium during reperfusion injury (Buchholz et al., 2015;Zhao et al., 2021) (Yu et al., 2017).It also indicates that suppressed inflammatory response, evidenced by lower IL-6, IL-1β, high-mobility group-box 1 protein 1, and TNF-α, contributes to the favorable effects of tragus stimulation.However, there remains a great challenge to translate the cardioprotective effects of VNS into myocardial infarction patients, and it therefore is still a pressing need for well-designed randomized control trials to further confirm the role of VNS in the setting of myocardial I/R injury, and contemporaneously deeply elucidate the underlying mechanisms.

Limitations
First, there is no standard protocol for myocardial I/R regimen (i.e., different ischemic or reperfusion duration) or VNS treatment (i.e., different parameters, stimulation site, and type), while subgroup analysis shows remarkable consistent outcomes among the studies.Second, the pooled results from this meta-analysis are based on animals without comorbidities which may impede extrapolating these findings to complicated clinical situations.Third, despite significant heterogeneity that may affect the interpretation of the results, sensitivity analysis and subgroup analyses with robust data substantially support the benefits and reliability of VNS in reducing infarct size and improving cardiac function after reperfusion injury.Meanwhile, the prespecified covariates have no impact on pooled results of both infarct size and LVEF by meta-regression.Finally, the majority of outcomes of included studies concentrate on infarct area and LVEF, rather than mortality or other cardiac functional indicators (e.g., 6-min walking or cardiopulmonary exercise testing), which may more precisely reflect the prognosis and symptoms in clinical practice.

Conclusion
In summary, VNS is a promising therapeutic strategy for preventing lethal myocardial reperfusion injury according to the significant advantages in limiting infarct size and cardiac function from basic studies.It thus provides the theoretical feasibility and reliability to extend the utilization of VNS in ST elevation myocardial infarction patients with revascularization, and implicates the future prospects of clinical application.

FIGURE 1
FIGURE 1Flow chart of the literature screening.

FIGURE 2
FIGURE 2Forest plot of infarct size for VNS treatment against myocardial I/R injury.VNS, vagus nerve stimulation.

FIGURE 3
FIGURE 3Forest plot of LVEF for VNS treatment in myocardial I/R injury.LVEF: left ventricular eject fraction; VNS, vagus nerve stimulation.

FIGURE 4
FIGURE 4Forest plot of LVEDP for VNS treatment in myocardial I/R injury.LVEDP, left ventricular end-diastolic pressure; VNS, vagus nerve stimulation.

TABLE 1
Characteristics of included studies, animals and VNS treatment.
VNS, vagus nerve stimulation; I/R, ischemia/reperfusion; SD, Sprague-Dawley rats; NZ, new zealand rabbit; M, male; RVN, right vagus nerve stimulation; LVN, left vagus nerve stimulation, TTC, triphenyl tetrazolium chloride; HR, heart rate; con, continuous; int, intermittent; NA, none available.Frontiers in Pharmacology frontiersin.orgswine.Continuous, right cervical vagal trunk stimulation was conducted in most of enrolled studies for VNS, the remaining studies performed VNS in left vagal nerve with either continuous or intermittent regimen.The parameters of VNS varied substantially among the studies.The majority of studies reported a 10%-20% heart rate reduction during the procedure to guarantee the biological effect of VNS.Additionally, the potential mechanisms of action of VNS in myocardial I/R injury were detailed in Table2, predominantly involving anti-inflammatory, oxidative stress, mitochondrial dysfunction anti-apoptosis.

TABLE 2
The underlying mechanisms involved in the protective effects of VNS against myocardial I/R injury.
Bruno et al 2015 Consistent vagal stimulation: co-activation of the sympathetic nervous system Intermittent vagal stimulation: activation of the Akt/GSK-3β signaling pathway

TABLE 3
Sensitivity analysis for pooled estimates of infarct size by leaving each study out.

TABLE 4
Subgroup analysis for pooled estimates of infarct size according to vagal stimulation site, duration, animal species, state region and myocardial I/R regimen.

TABLE 5
Meta-regression for infarct size.

TABLE 6
Sensitivity analysis for left ventricular ejection fraction (LVEF) and left ventricular end-diastolic pressure (LVDEP).
LVEF, left ventricular eject fraction; LVEDP, left ventricular end-diastolic pressure; VNS, vagus nerve stimulation; CI, confidence interval.Coefficient* indicates the estimates (WMD) of corresponding covariates for LVEF, or LVDEP, in the context of meta-regression.