Ischemic stroke (IS), one of the devastating disorders, is the intended second leading cause of mortality and disability followed by vascular occlusion and irreversible damage of the brain tissue [1, 2]. Despite the rising aged population, the incidence of stroke is expected to grow thereby a demand to accede a novel and more effective therapeutic approach is increasing, particularly for patients suffering from acute cerebral ischemia [3, 4]. It has been proved that long-lasting autophagy besides a variety of other neurologic conditions plays a crucial role in cerebral ischemic injury. However, growing pieces of evidence demonstrated that autophagy has the potential to exert controversial effects (either detrimental or beneficial) in cerebral IS [5]. In better words, regulated and moderate autophagy may provide a neuroprotection effect while an excessive or inappropriate activation of autophagy could trigger deleterious effects to develop cell death [6, 7]. Autophagy, a catabolic-conserved process through the breakdown and subsequent recycling of cellular constituents, is an essential physiological intracellular process for maintaining cellular homeostasis and simultaneously participates in bio-energetic procedures under various stress conditions [8]. This phenomenon is highly regulated by numerous molecules such as microtubule-associated protein 1A light chain 3 (LC3), Beclin-1, and P62 (a scaffold protein) that have a necessary role in the regulation of the autophagy signaling pathway [9]. Of note, the excessive activation of autophagy and related effectors in neural cells have been firmly established in a variety of focal ischemic stroke models such as experimental middle cerebral artery occlusion (MCAO). Moreover, recent evidence demonstrated that the over-activity of neuronal autophagy through persistent stress, such as cerebral ischemia, results in cell damage, especially in the border area of lesion sites [10, 11]. Therefore, autophagy regulation could be considered a potential target for IS treatment [12]. In contrast, it has also been reported that pre-activation of autophagy in the brain tissue could enhance brain ischemic tolerance, facilitate cellular energy production, and prevent neuronal apoptosis during subsequent exposure to the ischemic conditions [13]. For instance, rapamycin, as a well-known autophagy inducer has a palliative effect on pre-clinical IS damage through the activation of mitophagy, suggesting that autophagy has a beneficial effect on ischemia/reperfusion injury.Although there is no debate regarding autophagy participation in cerebral ischemia, the accurate function of autophagy in IS remains controversial. In hence, the main purpose of this systematic review refers to uncover a total pattern of infarct volume evolution after autophagy modulation quantitatively via meta-analysis in the experimental models of stroke.

To the best of our knowledge, IS, as a more common type of stroke and a devastating disease, is mainly characterized by the major lack of regional cerebral blood supply in a distinct area of the cerebral tissue [42]. IS could be defined as one of the major leading causes of a corresponding loss of neurologic function, particularly in the aging population [43, 44]. Besides the dysregulated autophagy, it has been also documented that other pathological conditions such as mitochondrial dysfunction, oxidative stress, acidosis, calcium overload, and inflammatory response are associated with the pathogenesis of cerebral ischemia-reperfusion injury (IRI) [45]. The current systematic review and meta-analysis aimed to clarify autophagy modulation (either inhibition or induction) and its possible effects on the histological and infracted volume restoration in animal models of ischemic stroke. As mentioned earlier, the basal level of autophagy is considered as an obligatory factor for neuronal normal activity while autophagy dysregulation promotes neurodegeneration, as well as misfolded protein aggregation [46]. Moreover, mounting evidence highlighted the causal role of autophagy activity during IS [47, 48, 49]. In detail, recent publications indicated that following the acute and severe IS, autophagic subtypes including mitophagy, pexophagy, lipophagy, and endoplasmic reticulophagy are predominantly involved in IS progression [50]. To further establish this finding, using transmission electron microscopy represented the increased amount of autophagosomes, named bilayer-membrane autophagic vacuoles, in the damaged ischemic neurons, which further highlighted the autophagy involvement in cerebral pathology induced by IS [51]. In this line, our results pointed out that autophagy efficacy predominately emerges in a time course between 6–72 h in terms of both cell protection and cell death status. Meanwhile, other variables such as gender, different anesthetic drugs used, route of administration, and different procedures for stroke induction had no significant bearing on autophagy consequences. Even so, the clinical application of autophagy modulators in diagnosed stroke patients is still restricted due to the plenty of contradictory studies. Given the limited studies conducted in this era, it could be assumed that there is a high risk of bias and suggesting further pre-clinical studies to confirm the exact role of autophagy in terms of the stroke volume modification with considering time-dependent effectiveness; However, to further establish of these findings, a comprehensive estimation of 29 studies, in which determined the infarct size using either percentage (%) or ischemic area measurement (mm${}^3$) revealed that 17 and 12 articles through the autophagy cessation and stimulation gained the parallel results and markedly decreased infarct size by 15.41% and 35.65 mm${}^3$, respectively. According to the recent systematic review, rapamycin (Sirolimus), an immunosuppressive drug that induces moderate autophagy by inhibition of the mammalian target of rapamycin (mTOR), exhibited a great beneficial effect for infarct volume reduction and ongoing neuroprotection effect, particularly in lower doses (8 ng) [52, 53]. Similarly, our analysis also demonstrated that the neuroprotective effects of different agents or conditions mostly mediated by autophagy inhibition resulted in infracted volume reduction. This outcome strongly implicated that prolonged stimulation, as well as the overexpression of autophagy, plays a major role in infarct size progression in stroke subjects, which negatively could exhibit in the high level of rapamycin (20 mg/kg), as well. In a study conducted by Chi et al. [26], it has been shown that mTOR, as a main target of rapamycin, exerts an imperative role not only in the maintenance of the cellular survival also governs the oxygen balance following the cerebral IRI likely through AKT and S6K1 phosphorylation in the cerebral cortex. Therefore, the high dose of rapamycin can increase infarct volume via mTOR inhibition as well as limitation of O${}_2$ consumption during reperfusion [26]. Notably, the protective effects in neural cells induced by autophagy, are predominately mediated using mTOR1 inhibitors such as rapamycin and metformin preconditioning as well as mTOR2 activation [54]. While utilizing rapamycin in low doses may also have enhanced autophagy activity enough in a non-mTORC2 manner to maintain neuronal survival following ischemia. Another protective activity of autophagy intercedes by scavenging accumulated misfolded proteins and cytoplasmic worn-out components in response to acute IS [55, 56]. To interrogate the exact role of the multi-phase autophagy process, a primary clinical trial to clarify the autophagy inhibitory/induction effect on mTOR2 is highly recommended in the context of IS. Another critical issue refers to the MCAO-induced IS leading to neuronal death by autophagosome accumulation and blocks autophagy flux in which increases the intracellular LC3, Beclin-1 (well-known autophagic biomarkers), and P62 (an adaptor protein) that conversely shows autophagy flux [57, 19]. The effect size of both autophagy modulations for infarct volume reduction was approximately equal. Together, there is no significant publication bias regarding the mean infarct volume percentage while publication bias was observed in mean differences of infarction volume amount (mm${}^3$) between studied groups.

Regarding the latent underlying mechanisms of action involved in autophagy regulation, Zhang et al. [58] indicated that chloride channel-3, as a signal molecule, exerted a neuroprotective role, which can directly activate autophagy machinery through the interaction between Beclin1 and Vps34 in a self-protective manner to impede infarct volume progression following acute IS (AIS), in vivo. In contrast, it has been reported that FK506 binding protein 5 (FKBP5), as a novel prognostic and diagnostic value, is upregulated in subjects with AIS and participates in disease severity. FKBP5 by autophagy induction through the downstream AKT/FOXO3 blocking could promote AIS exacerbation [59]. Another target signaling pathway to suppress dysregulated autophagy refers to the AKT/mTOR axis stimuli as well as autophagy-related gene 7 (Atg 7) downregulation emerging by dichloromethane therapy against IS in rats [60]. Notably, the results of a recent study conducted by Cai et al. [61] also showed that one of the substantial mechanisms involved in the neuroprotective role of tissue-type plasminogen activator (tPA), a well-known thrombolytic medication in the clinical treatment of cerebral IRI, e.g., IS, is mainly related to the activation of FUN14 domain-containing 1 (FUNDC1)-mediated mitophagy to retrieve mitochondrial dysfunction following the AMPK phosphorylation and subsequent apoptotic cell reduction. Previously, it has been reported that the elevated level of inflammatory mediators, such as annexin A1 and monomeric C-reactive protein, can worsen the prognosis of the post-ischemic aged brain, in vivo [62, 63]. Interestingly, a cross-talk between autophagy and inflammation has also been delineated, which corroborated the benefits of moderate autophagy in facing post-stroke inflammatory response through the mTOR/AMPK pathway and subsequent inflammasome inhibitions [64]. Collectively, beyond the existing conventional therapies, novel therapeutic approaches such as hypothermia-induced infarct size reduction and autophagy modulation are of great significance, recently [50, 62]. Even so, as a limitation of the current study, the possible effect of some critical risk factors including aging, co-morbidities, and raised inflammatory mediators should be considered in upcoming studies, as well.

Given the conflict effects of autophagy regarding the infarct volume reduction, the studies included in this meta-analysis mostly reported a negative relation between autophagy induction and stroke volume development due to excessive autophagy activity following severe IS; in hence, it seems that further studies are also required to explore the underlying mechanisms to clarify the exact intervention role of autophagy modulation during cerebral ischemia for translating the potential therapeutic target in stroke patients.

AIS, Acute Ischemic stroke; CI, confidence intervals; CMA, Comprehensive Meta-analysis; FKBP5, FK506 binding protein 5; IS, Ischemic stroke; IRI, ischemia-reperfusion injury; LC3, microtubule-associated protein 1A light chain 3; MCAO, middle cerebral artery occlusion; mTOR, mammalian target of rapamycin; SD, standard deviation.

AR—Designed the study; NV and FS—Performed search strategy; HH—Performed the methodological analysis; RR—Revised the final draft; YS—Contributed to writing the manuscript; SS—Interpreted the analyzed Data.

The ethic number approved by Ethics Committee of Tabriz University of Medical Sciences for this study is IR.TBZMED.REC.1398.294.

Not applicable.

This review was supported by the Aging Research Institute of the Tabriz University of Medical Science with code number: 62827.

The authors declared no conflict of interest.

See Table 3, Fig. 5.

Fig. 5.

Search and selection process of systematic review.