The protocol of this review was registered on the “International Prospective Register of Systematic Review” (PROSPERO), with the registration number CRD42020205808. We followed the “Preferred Reporting Items for Systematic Reviews and Meta-Analyses” (PRISMA) guideline (2020) (Page et al., 2021) for conducting and writing the review.

Eligible papers were searched by keywords on PubMed, Web of Science, and EMBASE databases through July 2021, with a combination of the following terms: (“ischemia” OR “ischemic” OR “stroke” OR “cerebral vascular accident”) AND (“Mdivi-1” OR “Mitochondrial division inhibitor-1” OR “fission inhibition”) AND (“mitochondria” OR “mitochondrial” OR “mitochondrion” OR “mitophagy” OR “brain” OR “neuron” OR “neural apoptosis” OR “neural cell death”). In brief, the titles and abstracts of studies were read to exclude duplicates and irrelevant studies that did not provide the required information of ischemic stroke and Mdivi-1 in their abstracts. Then the full texts were read all and the studies were selected based on eligibility criteria. Two independent evaluators carried out the study selection procedure. When disagreement occurred, two evaluators discussed with a third consultant to make the final decision.

The text, graphs, and tables of reviewed studies were read to extract data, including study characteristics and outcomes. If required data were not mentioned in the included papers, the corresponding authors were contacted for requests. The process of data extraction was conducted by two independent reviewers. For study characteristics, we extracted the data of the first author’s name, published year, models (for in vivo models: type, species, sex; for in vitro models: cell type and cell models), and Mdivi-1 treatment (dosage and timing). For the outcome extraction, we extracted and grouped data into two parts: (1) the effects of Mdivi-1 on neural mitochondrial dysfunction, including neural mitochondrial respiratory function and neural mitochondrial quality-control in I/R-induced brain injury after ischemic stroke; and (2) the effects of Mdivi-1 on the neural mitochondria-mediated apoptosis in I/R-induced brain injury after ischemic stroke.

The quality of in vivo studies was evaluated using the “Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies” (CAMARADES) checklist with 10 items (Auboire et al., 2018). The predesigned TOXRTOOL quality checklist was used to evaluate the quality of in vitro studies (Schneider et al., 2009). Two authors independently evaluated and filled in the predesigned datasheets of the CARAMADES checklist and TOXRTOOL checklist. Then, two results were compared, and the differences were discussed between two examiners and a third consultant.

The results of the search procedure were reported by the PRISMA flowchart (2020 version) and the narrative synthesis. We used the table and text to provide the data on study characteristics and outcomes. For presentation of study characteristics, the summaries of study designs, ischemic stroke models, Mvidi-1 treatment, and types of outcomes were provided. For presentation of outcomes, the effects of Mvidi-1 treatment on neural mitochondrial dysfunction and neural mitochondria-mediated apoptosis in I/R injury after ischemic stroke were described. In addition, the methodology quality of studies and the risk of bias were synthesized in two predesigned tables (used for in vivo or in vitro studies).

Using the mentioned term formula, 302 potential articles were found from PubMed (n = 73), Web of Science (n = 106), and EMBASE (n = 123). Then, 99 duplicates were removed. After title and abstract screening, 182 irrelevant-topic studies were removed. After full-text review, 9 studies were excluded, including 06 conference abstracts and 03 studies irrelevant-outcome studies. Finally, 12 studies were included in the current systematic review for analysis (Figure 1).

Regarding in vivo studies, the median CAMARADES score was 6. All studies were conducted with the appropriate models of I/R-induced brain injury and clearly provided anesthetics procedures. Moreover, all studies stated that they complied with available regulatory requirements and declared no conflicts of interest. Six studies (75%) reported data on the control temperature. Six studies (75%) randomized the experimental groups. However, none of the studies carried out allocation concealment, and only one study conducted the blinded outcome assessment. The sample size was also not calculated by all studies (Table 2).

For in vitro studies, according to the TOXRTOOL checklist, two studies reached a sore of 18, 1 study reached a score of 17, and 2 study reached a score of 16. Moreover, all studies met all red items (the important items in the TOXRTOOL checklist). Therefore, the evidence from these studies were considered to be reliable without restrictions (Table 3).

The main findings from evidence of included studies are synthesized as follow: (1) Mdivi-1 treatment could attenuate neural mitochondrial respiratory deficiency in the I/R-induced brain injury, as evidenced by the recovery of neural mitochondrial complex I-IV activities, the mitochondrial membrane potential, and the ATP production; (2) Mdivi-1 treatment could attenuate neural mitochondrial quality-control dysregulation in the I/R-induced brain injury, as evidenced by increases in the neural mitochondrial biogenesis regulators (PGC-1, TFAM, and NRF-1) and the level of mitochondrial DNA, as well as reductions in neural mitochondrial fission (Drp-1) and the neural mitophagy; (3) Mdivi-1 treatment could reduce neural mitochondria-mediated apoptosis in I/R-induced brain injury, as evidenced by reductions in mitochondria-mediated caspase-independent apoptotic factor (AIF), mitochondria-mediated caspase-dependent apoptotic upstream factor (Bax), and mitochondrial-mediated caspase-dependent apoptotic downstream factors (cytochrome c, caspase-9, and caspase-3) as well as increases in anti-apoptotic factor (Bcl-2). Taken together, Mdivi-1 treatment can improve neural mitochondrial respiratory function and neural mitochondrial quality-control, thereby reducing neural apoptosis through suppressing the mitochondria-mediated apoptotic pathways in I/R-induced brain injury (Figure 2). Therefore, we hypothesize that Mdivi-1 might be a promising therapeutic compound to attenuate I/R-induced brain injury after ischemic stroke.

The included study suggested that Mdivi-1 can normalize the activities of electron transport system, including complexes I–IV, attenuating neural mitochondrial respiratory deficiency in I/R-induced brain injury (Li et al., 2016). In support of this, a previous study showed that an intravenous injection of Mdivi-1 (1.2 mg/kg) enhanced the protein expressions of neural mitochondrial complex I in the brains of Sprague Dawley rats after subarachnoid hemorrhage (Fan et al., 2017). However, another study showed that, when neural mitochondrial respiration was impaired in neurons, Mdivi-1 (25–100 μM) inhibited the excessive activity of complex I to reduce neural mitochondria complex I-dependent oxidative stress (Bordt et al., 2017). It should be noted that neural oxidative stress is one of the main mechanisms in I/R-induced brain injury, which is partially caused by mitochondrial dysfunction and in turn promotes further neural mitochondrial damage (Stepien et al., 2017). We hypothesize that Mdivi-1 might act as a regulator in the activities of electron transport system to prevent the excessive release of reactive oxidative species in neurons and thereby reduce oxidative stress-induced damage to neural mitochondria in I/R-induced brain injury. Consistent with this hypothesis, the included study suggested that Mdivi-1 could reduce the oxidative stress induced by dysfunctional mitochondria in I/R neurons (Wang et al., 2014). Additionally, a previous study also showed that Mdivi-1 treatment attenuated hydrogen peroxide-induced mitochondrial dysfunction in PC12 cells (Song et al., 2019). Mdivi-1 appears to have dual effects that could interrupt the interdependence between neural mitochondrial respiratory deficiency and neural oxidative stress in I/R-induced brain injury after ischemic stroke.

Under physiological condition, mitochondrial membrane potential (MMP) is involved in Krebs cycle, which is the energy storage form used to synthesis ATP (Zorova et al., 2018). In addition, the normal MMP was proven to be a key factor in the removal of dysfunctional mitochondria in cells (Zorova et al., 2018). In ischemic stroke, the depolarization of MMP is promoted, leading to ATP depletion and neural cell death (Hu et al., 2018; Zorova et al., 2018). The review provided that Mdivi-1 can normalize MMP and thereby enhance ATP production both in vivo and in vitro (Zhao et al., 2014; Li et al., 2016; Zhou et al., 2017; Wang et al., 2018; Huang et al., 2021), suggesting that Mdivi-1 treatment can attenuate neural mitochondrial respiratory deficiency as well as maintain the number of healthy neural mitochondria in I/R-induced brain injury. Of note, MPP is produced by the activities of neural mitochondrial complexes I, III, and IV (Zorova et al., 2018). Thus, the reviewed finding suggested that Mdivi-1 might restore MMP by normalizing the activities of neural mitochondrial complexes as mentioned above.

Previous studies reported that neural mitochondrial biogenesis regulators (e.g., PGC-1α, TFAM, NRF-1, and NRF-2) were upregulated as a compensatory response to energy demand and reductions in neural mitochondrial content in I/R-induced brain injury (Ma et al., 2016; Carinci et al., 2021). Therefore, neural mitochondrial biogenesis was considered the target for therapeutic agents in I/R-induced brain injury after stroke (Carinci et al., 2021). In addition, the upregulation of PGC-1α has been shown to protect neurons against oxidative stress (Yang et al., 2018; Carinci et al., 2021). The included studies showed that the protein levels of mitochondrial biogenesis regulators (i.e., PGC-1α, TFAM, and NRF-1) as well as the level of mitochondrial DNA were further increased by Mdivi-1 treatment (Ma et al., 2016; Huang et al., 2021). Supportively, a previous study showed that treatment with Mdivi-1 (25 and 75 μM) could increase the protein levels of PGC-1α, NRF-1, and NRF-2 in N2a cells with Drp1 RNA silenced (Manczak et al., 2019). Collectively, the current evidence indicates that the enhancement of neural mitochondrial biogenesis might be one of the therapeutic effects of Mdivi-1 treatment on I/R-induced brain injury after ischemic stroke. We also hypothesize that PGC-1α activation is the neural mitochondria-related mechanism by which Mdivi-1 attenuates oxidative stress in I/R-induced brain injury after ischemic stroke.

Drp-1 inhibition is the typical characteristic of Mdivi-1, which was repeatedly confirmed in I/R-induced brain injury in the included studies (Zhang et al., 2013; Wang et al., 2014; Li et al., 2015, 2016; Chuang et al., 2016; Ma et al., 2016; Zhou et al., 2017). Likewise, a previous study showed that treatment with Mdivi-1 (1.2 mg/kg, intravenous injection) significantly reduced neural mitochondrial fission in rats with subarachnoid hemorrhage (Wu et al., 2017). However, another previous study showed that Mdivi-1 could inhibit neural mitochondrial fission in NMDA-treated neurons with Drp-1 knockdown, suggesting that the fission-inhibitory effects of Mdivi-1 might not completely depend on Drp-1 (Ruiz et al., 2018). Therefore, the explanations for therapeutic effects of Mdivi-1 in I/R-induced brain injury are not only based on the Drp-1 inhibition.

Regarding the effects of Mdivi-1 on neural mitochondrial fusion, the included studies showed that Mdivi-1 only increased neural outer membrane mitochondrial fusion (e.g., Mfn1 factor) but did not affect the neural inner membrane mitochondrial fusion (e.g., Opa1 factor) in I/R-induced brain injury (Ma et al., 2016; Huang et al., 2021). The effects of Mdivi-1 on neural mitochondrial fusion were also controversial among previous studies. A previous study showed that Mdivi-1 (20 mg/kg, i.p.) did not change the protein levels of two typical neural mitochondrial fusion factors (Opa1 and Mfn1) in kainic acid-damaged hippocampal cells of 4-week old mice (Kim et al., 2016). However, one study reported that Mdivi-1 treatment (1.2 mg/kg, i.p.) increased the protein level of Opa1 against lipopolysaccharide-induced brain damage (Deng et al., 2018). Another study showed that Mdivi-1 treatment (25 and 75 μM) also enhanced the levels of fusion proteins, including Mfn1, Mfn2, and Opa1 in N2a cells with Drp-1 RNA silenced (Manczak et al., 2019). This discrepancy might partially be explained by the differences in the animal models and Mdivi-1 administrations among studies. Due to lack of data, the current systematic review cannot indicate the effects of Mdivi-1 on neural mitochondrial fusion in I/R injury after ischemic stroke. It should be noted that fusion and fission of neural mitochondria were suggested to be important in determining the extent of ischemic injury after stroke (Carinci et al., 2021). Because Mdivi-1 could attenuate neural mitochondrial fission and might not induce negative impacts on neural mitochondrial fusion as observed in the included studies, Mdivi-1 could reduce the extent of I/R-induced brain injury after ischemic stroke.

In the included studies, Mdivi-1 treatment reduced the level of dysfunctional mitochondrial detectors (i.e., PINK1 and Parkin) and maintained the number of healthy mitochondria, suggesting that Mdivi-1 might attenuate the impairment of neural mitochondrial functions to reduce the neural mitophagy in I/R-induced brain injury (Zhang et al., 2014; Huang et al., 2021). However, it is unclear whether the mitophagy-inhibitory effects of Mdivi-1 is the therapeutic effect or not because the roles of neural mitophagy varies in ischemic stroke (Shao et al., 2020). Accumulated evidence has shown that neural mitophagy is both beneficial and harmful, partially depending on the severity and stage of ischemic injury, which interact with neural apoptosis (Shao et al., 2020). Therefore, further studies should compare the effects of Mdivi-1 on mitophagy and neural apoptosis in any stages of ischemic stroke to comprehensively clarify the therapeutic effects of Mdivi-1.

Our review suggested that Mdivi-1 can inactivate mitochondria-mediated apoptotic pathways, which is supported by the reduced levels of proapoptotic factors, including AIF, Bax, cytochrome c, caspase-9, and caspase-3 and increased levels of antiapoptotic factor (Bcl-2) (Zhang et al., 2013; Wang et al., 2014, 2018; Zhao et al., 2014; Li et al., 2015, 2016; Chuang et al., 2016; Ma et al., 2016; Zhou et al., 2017). Consistently, one study showed that pretreatment with 50 μM Mdivi-1 reduced the proportion of apoptotic cells as well as the levels of Bax, cytosolic cytochrome c, and caspase-3 in glutamate-damaged cortical neurons in vitro, implying that Mdivi-1 might reduce neural apoptosis in the brain against the overexpression of glutamate, a primary mechanism of I/R-induced brain injury after ischemic stroke (Zhou et al., 2018). Likewise, previous studies showed that the intravenous injection of Mdivi-1 (1.2 mg/kg) could reduce the TUNEL-apoptotic positive cells as well as the protein levels of Bax, cytochrome c, and cleaved caspase-3 in the brains of Sprague Dawley rats after subarachnoid hemorrhage (Fan et al., 2017; Wu et al., 2017). One possible explanation for antiapoptotic effects of Mdivi-1 is that Mdivi-1 could attenuate the neural mitochondrial permeability impairment in stroke, preventing the translocation of Bax into neural mitochondria and thus inactivating neural mitochondria-related apoptotic pathways (Zhao et al., 2014). Mdivi-1 also reduced mPTP opening in the inner mitochondrial membrane, thereby inactivate the release of AIF and cytochrome c from neural mitochondria to neural cytosol (Zhou et al., 2017). Of note, mitochondria-mediated apoptotic pathways are typical activated after ischemic stroke, occurring when neural mitochondria functions are impaired and cannot manage the damage of I/R-induced brain injury (Guan et al., 2018; Shao et al., 2020). Thus, the current evidence indicates that Mdivi-1 might suppress mitochondria-related apoptotic pathways by improving the neural mitochondrial functions, thereby protecting the brain against I/R-induced brain injury after ischemic stroke.

In our systematic review, the quality of the included in vivo studies was evaluated by the CAMARADES checklist, which is commonly used in animal studies (Auboire et al., 2018). As a commonplace characteristic of preclinical research, the included in vivo studies herein did not calculate simple size (n = 12, 100%), conduct the allocation concealment (n = 12, 100%) or assess outcome in a blinded manner (n = 11, 92%). However, selection bias was reduced because all studies followed the regulatory requirements and clearly described the animal models and surgery procedures. Moreover, major studies (n = 6, 75%) randomized animals into experimental groups. In addition, the studies were published in peer-reviewed journals and declared no conflicts of interest, thereby minimizing the reporting bias and publication bias for our review findings. Regarding in vitro studies, the TOXRTOOL checklist has been used in many studies to strictly evaluate the quality of in vitro studies (Schneider et al., 2009). The included in vitro studies met most requirements of the checklist, meaning that the results from those studies were considered reliable. The findings from both the in vivo and in vitro included studies were consistent, suggesting that our reviewed findings might be reasonable but not ultimately conclusive due to a lack of data; thus, they need to be supported by further studies.

Several limitations need to be considered in this systematic review. First, we included evidence from full-text English articles but not from papers in other languages, conference abstracts or locally published articles, which might have increased publication bias for our reviewed findings. Second, the included studies used various models of ischemic stroke that increased biases to summarize the benefits of Mdivi-1 on neural mitochondrial functions and neural mitochondria-mediated apoptosis. Third, the dosage and timing of Mdivi-1 treatment were diverse, and only two studies compared several doses of Mdivi-1 in ischemic stroke, restricting the comprehensive provision of the effects of Mdivi-1. Fourth, most studies used male animals, and no study compared the effects of Mdivi-1 on neural mitochondrial functions and neural apoptosis in the two sexes. Therefore, the review cannot provide evidence of sex-specific mitochondria-related mechanisms underlying the effects of Mdivi-1. Finally, this review only provided the effects of Mdivi-1 on neural mitochondrial functions as well as neural mitochondria-related apoptosis, but it did not provide cause-effects for why Mdivi-1 induces those benefits.

Previous studies have shown that neural mitochondrial dysfunction could activate neuroinflammation and further promote neural cell deaths in I/R-induced brain injury (Carinci et al., 2021). Ischemic injury might activate the mPTP to open, releasing mitochondrial proinflammatory factors, including ATP, AIF, and HSP60, from neural mitochondria to extracellular spaces and then interacting with receptors in inflammatory cells, such as astrocytes and microglia, to increase proinflammatory factors (e.g., tumor necrosis factor-alpha, interleukin-1, and interleukin-18) (Galluzzi et al., 2012; Jung and Seong, 2021). These proinflammatory factors in turn activate caspases to induce inflammatory programmed cell death (Jung and Seong, 2021). Our review provided that Mdivi-1 can enhance neural mitochondrial functions, thereby posing a hypothesis that Mdivi-1 attenuates neuroinflammation in I/R-induced brain injury after ischemic stroke. Supportively, one study showed that Mdivi-1 (1.2 mg/kg) injection after subarachnoid hemorrhage inactivated nuclear translocation of NF-κB and thereby reduced the levels of TNF-α, IL-6, and IL-1ß in ischemic-induced brain damage in MCAO rat models (Fan et al., 2017). Another study showed that Mdivi-1 treatment (25 mg/kg, i.p.) reduced neuroinflammatory T-cells (Th1 and Th17) in the brains of an autoimmune encephalomyelitis mouse model (Li et al., 2019). To comprehensively evaluate the therapeutic characteristics of Mdivi-1 in ischemic stroke, further studies are required to investigate the effects of Mdivi-1 on the relationship between mitochondrial dysfunction and neuroinflammation in ischemic brain injury.

Previous studies have shown that ischemic injury also induces neural apoptosis in the brain by promoting the extrinsic-mediated apoptotic pathway, which is accompanied by the augmentation of cellular calcium influx (Carinci et al., 2021). One study showed that Mdivi-1 could control the neural mitochondrial calcium concentration, thereby attenuating neural mitochondrial dysfunction and neural apoptosis in NMDA-treated neurons (Ruiz et al., 2018). However, to the best of our knowledge, there is no evidence regarding the effects of Midi-1 on extrinsic apoptotic pathways in the brain after ischemic stroke. This issue should be addressed by further studies to provide additional evidence on the potential effects of Mdivi-1 on neural apoptosis in ischemic stroke.

The findings from previous studies suggested that Mdivi-1 showed mitochondria-related therapeutic effects on ischemic stroke as well as other disorders in the central nervous system (Fan et al., 2017; Li et al., 2019; Song et al., 2019). Regarding neurodegenerative disorders that might be promoted after ischemic stroke such as Alzheimer’s disease (Vijayan and Reddy, 2016) and Parkinsonism (Harnod et al., 2020), Mdivi-1 has been proven to increase mitochondrial length and ATP level, as well as reduce the mitochondrial depolarization of Aβ-treated hippocampal cells in Alzheimer’s disease (Baek et al., 2017). Likewise, treatment with Mdivi-1 could also enhance mitochondrial respiration and reduce neural mitochondrial fragmentation in the Parkinsonian’s rat model (Bido et al., 2017). Moreover, Mdivi-1 has been shown to have a high capacity to cross the brain-blood barrier, supporting its potential therapeutic effects on the brain (Cui et al., 2016). However, alongside therapeutic benefits mentioned above, the off-target effects of Mdivi-1 has been reported. A previous study showed that treatment with 50 μM Mdivi-1 caused mitochondrial depolarization, oxidative stress, and apoptosis in oligodendrocytes with excitotoxicity (Ruiz et al., 2020), suggesting the complicated effects of Mdivi-1 on mitochondrial functions which might be influenced by many factors, including dosage and targeted cells. Currently, the off-target effects of Mdivi-1 on neural cells under I/R injury have not been explored. In addition, there are no preclinical data on side effects and the optimal administration of Mdivi-1 (dose, time window, injection method) for ischemic stroke, making it impossible to apply Mdivi-1 treatment in humans and thus requiring additional data from further studies.