Methylene Blue Reduces Acute Cerebral Ischemic Injury via the Induction of Mitophagy
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The treatment of stroke is limited by a short therapeutic window and a lack of effective clinical drugs. Methylene blue (MB) has been used in laboratories and clinics since the 1890s. Few studies have reported the neuroprotective role of MB in cerebral ischemia-reperfusion injury. However, whether and how MB protects against acute cerebral ischemia (ACI) injury was unclear. In this study, we investigated the effect of MB on this injury and revealed that MB protected against ACI injury by augmenting mitophagy. Using a rat middle cerebral artery occlusion (MCAO) model, we demonstrated that MB improved neurological function and reduced the infarct volume and necrosis after ACI injury. These improvements depended on the effect of MB on mitochondrial structure and function. ACI caused the disorder and disintegration of mitochondrial structure, while MB ameliorated the destruction of mitochondria. In addition, mitophagy was inhibited at 24 h after stroke and MB augmented mitophagy. In an oxygen-glucose deprivation (OGD) model in vitro, we further revealed that the elevation of mitochondrial membrane potential (MMP) by MB under OGD conditions mediated the augmented mitophagy. In contrast, exacerbating the decline of MMP during OGD abolished the MB-induced activation of mitophagy. Taken together, MB promotes mitophagy by maintaining the MMP at a relatively high level, which contributes to a decrease in necrosis and an improvement in neurological function, thereby protecting against ACI injury.
Stroke is a primary cause of disability and death worldwide. The treatment of stroke is constrained by a therapeutic window consisting of a few hours, and effective drugs for the treatment of stroke are extremely scarce. Methylene blue (MB) has been demonstrated to perform diverse biological functions and has been used for various medical applications (1). MB readily crosses the blood-brain barrier and accumulates in the mitochondrial matrix (2,3); therefore, its biological functions primarily involve improving mitochondrial functions, such as enhancing cytochrome c oxidase activity (4), oxygen consumption (5,6) and ATP production (7) and reducing reactive oxygen species (ROS) generation (8). Clinically, MB has been approved by the FDA for methemoglobinemia and as an antidote to cyanide poisoning (9,10). A few studies have shown that MB reduces cerebral ischemia-reperfusion injury (11, 12, 13). In fact, fewer than 5% of patients with ischemic stroke can obtain thrombolytic treatment in the therapeutic window (14). That is, more than 95% of patients suffer permanent cerebral ischemia due to not receiving reperfusion in time. Acute cerebral ischemia (ACI) is one of the most common cerebral vascular diseases and here refers to the process whereby artery blockage causes brain tissue ischemia without reperfusion, resulting in brain dysfunction. The importance and necessity of ACI treatment lies in early intervention, without considering reperfusion or not, to reduce the mortality and disability. Therefore, the research and development of neuroprotective drugs for the treatment of acute ischemic stroke is clinically significant. Here, we demonstrate for the first time that MB attenuates ACI injury and that its protective effect is related to the induction of mitophagy.
Mitochondria are the principal target of ischemic injury (15). Ischemia generally leads to mitochondrial dysfunction (16,17). Dysfunctional mitochondria can be selectively eliminated via mitophagy (18, 19, 20). In this process, damaged mitochondria are enveloped by double-membrane structures known as autophagosomes, which are then delivered to lysosomes for degradation (21, 22, 23). Thus, mitophagy plays a crucial role in controlling the quality of mitochondria by preventing the generation of ROS by dysfunctional mitochondria (24,25). Deficiency in mitophagy is associated with neurodegenerative diseases such as Parkinson’s disease (26,27), Alzheimer’s disease (28,29) and Huntington’s disease (30). Stroke is another type of neurodegenerative disease. However, whether mitophagy is involved in the pathogenesis of stroke and whether MB interrupts this process remain unclear.
Using a rat middle cerebral artery occlusion (MCAO) model of stroke, we have demonstrated that mitophagy is induced in the early stage of stroke but is inhibited shortly thereafter (data not shown). In this study, we showed that 24 h of ischemia suppressed mitophagic activity but that MB treatment rescued it. We confirmed this effect of MB using an oxygen-glucose deprivation (OGD) model in vitro and identified that the protection conferred by MB during OGD was abolished due to the inhibition of mitophagy. As mitophagy is directly induced by the loss of mitochondrial membrane potential (MMP) (27,31, 32, 33), we further explored whether the effect of MB on mitophagy was mediated by the regulation of MMP. We found that the MMP dramatically declined after cells were exposed to OGD, whereas MB attenuated this decline. Moreover, we verified that the relatively high MMP maintained by MB during OGD contributed to the activation of mitophagy.
Materials and Methods
Middle Cerebral Artery Occlusion and Methylene Blue Treatment
Adult male Sprague Dawley rats were purchased from Vital River Laboratories (Beijing, China). Focal ischemia was induced via right MCAO as described previously (34). Briefly, focal cerebral ischemia was induced by inserting a 4-0 nylon monofilament through the right internal carotid artery. Different doses (1),5 or 10 mg/kg) of MB or vehicle (normal saline) were intraperitoneally (IP) injected immediately after occlusion. The animal protocol was approved by the Institutional Animal Care and Use Committee of the Academy of Military Medical Science, and all measures were taken to reduce animal numbers and suffering.
Neurological Deficit Score Assessment
Total neurological deficit score. This test, which consisted of two sections, was conducted at 24 h after MCAO with or without MB treatment: (1) the postural reflex test was performed to evaluate upper body posture while the rat was suspended by the tail, and (2) the forelimb placing test was performed to examine sensor motor integration in forelimb placing responses to visual, tactile and proprioceptive stimuli. The total neurological deficit score was graded on a scale from 0 (normal score) to 12 (maximal score) (35).
Grid-walking test. Each animal was placed individually on a grid with an area of 110 × 10 × 100 cm (length × width × height) containing 9-cm2 wire mesh and were allowed to freely walk for 5 min. During this 5-min period, the total number of foot faults by the ipsilateral forelimb was counted. If the rat was resting with the grid at the level of the wrist, this positioning was also considered as a fault. If the rat was not able to step for 5 min, the maximal score of 15 was assigned (36).
Infarct Volume Analysis
The rats were anesthetized and euthanized 3, 6, 9, 12 or 24 h after MCAO. The brains were rapidly removed and were placed in a rat mold to generate 2-mm coronal sections. The sections were stained with 1% (w/v) 2,3,5-triphenyltetrazolium chloride (TTC) solution at room temperature for 30 min and then fixed in 4% paraformaldehyde. The infarct size was measured and analyzed using ImageJ software. To compensate for the effect of brain edema, the corrected infarct volume was calculated as described previously: corrected infarct area = left hemisphere area — (right hemisphere area — infarct area) (37).
Hematoxylin and Eosin Staining
The brain tissues from the rats were embedded in paraffin and 6-µm-thick serial coronal sections were generated and mounted on slides. The sections were stained using hematoxylin and eosin (H&E) according to a previously described standard protocol (38).
Transmission Electron Microscopic Examination
0: Normal mitochondrion.
1: Normal mitochondrion but lacking granular deposits.
2: Loss of matrix granules and clarification of the matrix without disruption of cristae.
3: Loss of matrix granules, uniform clarification of the matrix and disruption of cristae.
4: Loss of matrix granules, uniform disruption of cristae, and loss of mitochondrial membrane integrity.
The samples were visualized and photographed using a transmission electron microscope (TEM) at 80 kV.
FJB and TUNEL Labeling
Cell necrosis was determined via Fluoro-Jade B (FJB; Millipore, Billerica, MA, USA) labeling and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling (TUNEL). At 24 h after MCAO, the rats were anesthetized and transcardially perfused with saline followed by cold 4% paraformaldehyde (PFA) solution. The brains were rapidly removed and cryoprotected in 30% sucrose. FJB and TUNEL labeling were performed using in situ death detection kits as described previously (41). Fluorescence signals were visualized via fluorescence microscopy.
Cell Culture and Oxygen-Glucose Deprivation
A hypoxia-sensitive neuron-like PC12 cell line was used to simulate cerebral ischemia in an OGD model. The cells were cultured at 37°C in RPMI 1640 medium supplemented with 10% fet al bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin in 5% CO2. For OGD, the cultures were placed in a hypoxic incubator containing 0.3% O2 and a gas mixture of 95% N2/5% CO2, and the medium was replaced with glucose-free RPMI 1640 medium for 2 h. For treatment, MB (0.5 µmol/L) dissolved in PBS (phosphate buffered saline) or an inhibitor (3-MA, 2 mmol/L; carbonyl cyanide m-chlorophenylhydrazone (CCCP), 1 µmol/L) was added to the culture medium immediately when the cells were exposed to OGD.
Brain sections that included the ischemic area and corresponding regions from the contralateral hemisphere or cells were collected, and protein extracts were prepared as described previously (34). The primary antibodies used for Western blot-targeted receptor-interacting protein 1 or 3 (RIP1/RIP3), microtubule-associated protein light chain 3 (LC3), translocase of the outer membrane of mitochondrion (Tom20), Beclin1, P62 or β-actin. The signals were detected with the use of an ECL Western blot substrate (Thermo Fisher Scientific Inc., Waltham, MA, USA). Quantitative analyses were performed using the ImageJ software (NIH, Bethesda, MD, USA).
The intracellular ROS levels were measured using the oxidation-sensitive fluorescent probe CM-H2DCFDA after OGD. The cells were incubated in 10 µmol/L CM-H2DCFDA in the dark at 37°C for 30 min and then washed with PBS. The suspended cells were analyzed via flow cytometry.
Mitochondrial Membrane Potential Assessment
The MMP was determined by using the fluorescent probe JC-1 two hours after OGD in PC12 cells according to the manufacturer’s directions. The ratio of red:green fluorescence of JC-1 is dependent on the MMP. After treatment, the cells were incubated in 2.5 µmol/L JC-1 for 20 min at 37°C and then rinsed twice with PBS. Afterward, the cells were collected and analyzed via flow cytometry (42).
The data are presented as the means ± SE. Differences between the control and experimental groups were examined via one-way analysis of variance (ANOVA). Values of P <0.05 were considered to be significant. At least three independent experiments were performed for each experiment.
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MB Attenuates the Infarct Volume and Improves Neurological Functions
MB Alleviates the Destruction of Brain Tissue Morphology
MB Decreases Necrosis Rather than Apoptosis
RIP1 and RIP3 are the key signaling molecules in programmed necrosis (44,45). To verify the effects of MB on necrosis, we measured the protein levels of RIP1 and RIP3 via Western blot. Compared to the contralateral (uninjured) side, ischemia (in the affected side) caused an elevation in the expression of both RIP1 and RIP3; however, MB suppressed this elevation of RIP1 and RIP3 expression (Figures 3D–F), suggesting a protective effect of MB against necrosis.
MB Improves the Structure of Mitochondria
MB Increases the Induction of Mitophagy
Mitophagy Mediates the Protection of MB against Acute Ischemic Injury
The MB-Induced Elevation of MMP Contributes to the Activation of Mitophagy
Stroke is the fourth leading disease that threatens human life and health (48). Acute cerebral ischemia is the primary cause of stroke. At present, there is a lack of effective drugs for the treatment of ACI injury. Another issue is that fewer than 5% of stroke patients can obtain thrombolytic therapy within the therapeutic window. Therefore, seeking effective neuroprotective drugs is an immediate focus. MB is a lipophilic compound that has been used for the treatment of multiple disorders, including methemoglobinemia, malaria and cyanide poisoning (9,10,49). Recently, MB has been shown to play neuroprotective roles in a variety of mitochondria-associated cytotoxicity paradigms, such as stroke, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease and optic neuropathy (30,47). In this study, we demonstrated for the first time that MB attenuates ACI injury by increasing the induction of mitophagy.
We have identified that MB reduces the infarct volume and the neurological dysfunction caused by ACI injury. To investigate the impact of MB on brain tissue structure after stroke, we observed the changes in cell morphology. Cell swelling and nuclear pyknosis were reduced due to MB treatment. Based on further analysis of apoptosis and necrosis, we determined the role of MB in ameliorating necrosis. We were also surprised to find that MB did not decrease but rather increased apoptosis. The reason for this increase in apoptosis appears to be related to its reduction of necrosis, representing a compromise mechanism that avoids additional necrosis in stroke. The expression of key signaling molecules in programmed necrosis, RIP1 and RIP3 was decreased significantly by MB, suggesting the suppression of necrotic activity. Collectively, these results supported that hindering the necrotic process contributes to the protective effect of MB against ACI injury.
To identify the potential mechanism by which MB inhibited necrosis, we focused on the effect of MB on mitochondrial structure and function after stroke, considering that the mitochondrion is an important target of MB (50). The observation and scoring of the mitochondrial ultrastructure indicated that the mitochondrial structure was seriously disrupted after stroke, but that MB dramatically ameliorated the disintegration of the mitochondrial structure. This result inspired us to explore whether mitochondrial function was also improved by MB in stroke. Mitophagy functions as selective clearance of damaged mitochondria to reduce necrosis or apoptosis and to sustain cellular activity (31,51). Therefore, mitophagy is considered as a protective response to a variety of stresses. At 24 h after ACI, mitophagy was suppressed to a large extent. However, to our excitement, mitophagy was clearly augmented by MB treatment compared with the VC treatment. Thus, the augmentation of mitophagy may at least partially explain the maintenance of the mitochondrial structure by MB in stroke. To further verify whether mitophagy mediated the protective effect of MB against ACI injury, we examined the ROS levels in an OGD model in vitro. We found that OGD caused a dramatic elevation of ROS, MB greatly suppressed this elevation, and the suppression of MB was abolished after mitophagy was inhibited, demonstrating that mitophagy mediates the protective effect of MB against ACI injury.
MMP is an electrostatic potential generated by the proton gradient across the mitochondrial inner membrane that is driven by the enzymes of electron transport chain (ETC). The MMP is an indicator of the mitochondrial functional status, and deregulation of MMP leads to a large spectrum of major human diseases, including diabetes, neurodegeneration, cancer and others (52). It has been reported that MB maintains the MMP when mitochondria are dysfunctional (2), and the MMP is a regulator of mitophagy (27). Accordingly, we investigated the effect of MB on the MMP and the relationship between the MMP and the induction of mitophagy. We discovered that the dramatic decline in the MMP under OGD conditions was greatly hindered by MB, and unexpectedly, MB in the absence of OGD induced a decrease in the MMP. Based on MB’s redox properties that low MB concentrations favor dimerization and reduction, whereas high concentrations promote oxidation (7), we propose the following hypothesis for this phenomenon: in functional mitochondria, MB in its reduced form donates H+ (47), which tends to cause an elevation of MMP; MB in its oxidized form accepts H+ (53), which tends to cause a decline of MMP. However, in dysfunctional mitochondria, these effects may be just opposite. When the activities of ETC enzymes are decreased, MMP drops rapidly and excessive H+ in the mitochondrial matrix provided by MB in its reduced form cannot be pumped out into the mitochondrial intermembrane space, which aggravates the drop of MMP; in contrast, MB in its oxidized form can accept the H+ that cannot be pumped out due to the decreased activities of ETC enzymes, which hinders the further drop of MMP. It thus seems that 0.5 µmol/L MB we used in this study displayed its oxidation properties. That is, it decreased the MMP in the normal conditions and attenuated the drop of MMP in the OGD conditions. Furthermore, we demonstrated that the augment of mitophagy by MB was completely abolished due to the addition of CCCP. These results suggested that the maintenance of MMP by MB in stroke promoted the induction of mitophagy.
In summary, our study demonstrated that the suppression of mitophagy is involved in the pathogenesis of stroke. More importantly, we confirmed that the augmentation of mitophagy by MB mediates the protection against ACI injury. Thus, we propose that MB is a promising neuroprotective agent against acute ischemic stroke.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
We are grateful to Chang-Hong Ren and Zhi-Feng Gao, Department of Hypoxia/Ischemia, Xuanwu Hospital, Capital Medical University, for their excellent technical assistance in preparing the rat MCAO model and analyzing the scores of neurological dysfunction. This work was supported by the National Basic Research Programs of China (2012CB518200, 2011CB910800), the National Natural Science Foundation of China (81071066),81000856 and 31271211), and the Integrated Drug Discovery Technology Platform of National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (2012ZX09J12201-005).
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