Methylphenidate exerts neuroprotective effects through the AMPK signaling pathway

Abstract

Purpose:

Cerebral ischemia is the main cause of permanent adult disabilities worldwide. This study investigated the reparative effects and potential mechanisms of methylphenidate (MPH), a medication for the treatment of attention-deficit/hyperactivity disorder.

Methods:

In vitro oxygen-glucose deprivation/reperfusion (OGD/R) and in vivo cerebral ischemia-reperfusion models were established. Sprague-Dawley (SD) rats were randomly divided into four groups (n = 20): Sham, Model, and MPH (0.5 and 1 mg/kg). Rats in MPH groups were treated with 0.5 or 1 mg/kg MPH via intraperitoneal injection for 7 days. Rats in the Sham and Model groups were treated with PBS during the same period. Cell viability was measured using MTT assay. Apoptosis was detected by Annexin V/PI staining. Protein expression was detected by Western blot. The volume of cerebral infarction was detected by triphenyltetrazolium chloride (TTC) staining. The DNA damage in ischemic brain tissues was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.

Results:

MPH treatment significantly reduced OGD/R-induced cell damage, shown by the increased cell viability and decreased apoptotic rate. p-AMPK and p-ACC protein expression increased in the OGD/R model after MPH treatment. The addition of AMPK inhibitor largely abolished the neuroprotective effects of MPH, evidenced by the reduced cell viability, increased apoptotic rate, and decreased protein expression of p-AMPK as well as p-ACC. Moreover, MPH treatment significantly alleviated the cerebral ischemia-reperfusion injury and decreased apoptosis in brain tissues, which may be associated with the AMPK/ACC pathway.

Conclusions:

MPH exerted protective activities against oxidative stress in the OGD/R model and ameliorated brain damage of rats in the middle cerebral artery occlusion model, at least in part, through activating the AMPK pathway. These data demonstrated neuroprotective properties of MPH and highlighted it as a potential therapeutic agent against cerebral ischemia-reperfusion injury.

Keywords

Methylphenidate oxygen-glucose deprivation/reperfusion cerebral ischemia-reperfusion injury AMPK pathway

Introduction

As a common vascular disease in the central nervous system (CNS), ischemic stroke is one of the most common causes of death and long-term disability worldwide.^1,2 Of the 15 million people who have a stroke each year, more than 5 million die and a further 5 million are left permanently disabled. Ischemic stroke caused by sudden blood vessel occlusion with an embolism or thrombus accounts for approximately 80% of all stroke cases.³ There is overwhelming evidence that apoptosis contributes significantly to cell death subsequent to cerebral ischemia-reperfusion (I/R), in addition to necrosis.^4,5 Pathogenic mechanisms following cerebral I/R are associated with elevation of the intracellular Ca²⁺ level, energy failure, release of excitatory neurotransmitters, inflammation, and oxidative stress.⁶ However, the exact mechanism has not been established.

One of the fundamental requirements for all cells is to balance ATP generation and consumption. AMP-activated protein kinase (AMPK), which is a highly conserved sensor of intracellular adenosine nucleotide levels and activated when intracellular ATP levels are low, plays a critical role in regulating growth and reprogramming metabolism.⁷ AMPK has a protective role in myocardial ischemia, angiogenesis, diabetic cardiac injury, and fluid shear stress.^8–11 Acetyl coenzyme A carboxylase (ACC) is an enzyme catalyzing the conversion of acetyl coenzyme A to malonyl coenzyme A, which is involved in the energy metabolism of fatty acids in animals as a promising target for metabolic syndrome intervention. ACC has been demonstrated as a potential immune modulatory target of cerebral ischemic stroke.^12,13 Previous studies showed that energy metabolism dysfunction plays a significant role in ischemic stroke. AMPK/ACC resists cerebral ischemic injury by switching on catabolic pathways and switching off ATP-consuming processes, which may be a potent target for ischemic stroke treatment.^14,15

Cultured cells subjected to hypoxia, fuel deprivation and re-oxygenation replicate the features of I/R. Thereafter, an oxygen and glucose deprivation-reperfusion (OGD/R) cell model is commonly used to simulate the state of neuronal I/R injury that results in neuronal apoptosis.¹⁶ Many animal stroke models have focused on the middle cerebral artery (MCA) because most ischemic strokes occur in the MCA territory. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory. This technique can be used to model transient or permanent occlusion.¹⁷

Currently, there are two therapeutic strategies applied to cerebral I/R injury. The best treatment is to restore blood supply by administering thrombolytic agents. However, thrombolytic therapy is limited by a very narrow therapeutic window and can also lead to hemorrhagic complications.¹⁸ Neuroprotection, as the other strategy, has attracted more attention in recent years. Methylphenidate (MPH) is a dopamine-reuptake inhibitor approved for the treatment of attention-deficit/hyperactivity disorder (ADHD). Effects of MPH on the nervous system are controversial due to the dosage. High doses (10 or 20 mg/kg) caused mitochondrial dysfunction, oxidative stress and inflammatory changes in brain cells and induced neurodegeneration in the hippocampus and cerebral cortex of rats.^19–21 However, at a low concentration, MPH exerted neuroprotective activities against oxidative stress in vitro.^22,23 This study was to investigate the neuroprotective effects of MPH in OGD/R and MCAO rat models, and further explore the underlying mechanisms.

Materials and methods

Chemicals and reagents

3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium-bromide (MTT) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Caspase-3 and caspase-9 activity assay kits were purchased from R&D Systems (Minneapolis, MN, USA). The TUNEL assay kit was purchased from Roche (Mannheim, Germany). The Annexin V/PI apoptosis assay kit was purchased from ThermoFisher (Waltham, MA, USA). All solvents and chemicals were of analytical grade and purchased from Sinopharm Co. Ltd. (Shanghai, China). The AMPK inhibitor Dorsomorphin (purity: 99.65%) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Methylphenidate hydrochloride was purchased from the National Institutes for Food and Drug Control (Beijing, China) and dissolved in phosphate-buffer saline (PBS).

Cell culture and oxygen-glucose deprivation/reperfusion (OGD/R)

Primary neurons were isolated from newborn Sprague-Dawley (SD) rats (Vital River Ltd., Beijing, China). The hippocampal tissues were firstly digested with 0.125% trypsin for 10 min. After separation and centrifugation, neurons were inoculated into 6-well plates treated by poly-d-lysine (Sigma–Aldrich, St. Louis, MO, USA) overnight. Cells were maintained with neurobasal medium containing 2% B27, 1% glutamine and cytarabine at 37°C.²⁴ To establish the oxygen-glucose deprivation (OGD) model, the culture medium was removed and cells were washed twice using RPMI1640 medium without glucose. Cells were then transferred to 37°C, N₂/CO₂ (95%/5%) in a hypoxic incubator chamber (Thermo Fisher, Waltham, MA, USA) for 10 min. RPMI1640 medium without glucose was added to cells for 2 h in the hypoxic incubator chamber (OGD phase). Next, cells were cultured in RPMI1640 medium containing 7.5% horse serum and 10% FBS in an air/CO₂ (95%/5%) incubator for 24 h (reperfusion [R] phase). Control cells were cultured under normoxic conditions without OGD treatment. MPH at different concentrations (1, 3, and 10 µM) was added 2 h prior to OGD. The concentrations of MPH were based on our preliminary study and literatures.^22,23 To further validate the MPH mechanism of action, the specific inhibitor of AMPK (10 µM Dorsomorphin) was added 2 h before MPH treatment.

Cell viability assay

The cell viability was assessed using MTT assay. After treatment, cells were incubated with 0.5 mg/mL MTT at 37°C for 1 h. The formazan was dissolved in DMSO and the absorbance was determined at 570 nm with a microplate reader.²⁵

Apoptosis analysis

After treatment, 2 × 10⁶ cells were collected and re-suspended in 500 µl of binding buffer with 5 µl of Annexin V-FITC/PI for 10 min in the dark. Apoptosis was detected with the FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). Data were analyzed using FlowJo V10.

Rat ischemic/reperfusion model and treatment

One hundred and twenty male SD rats, weighing 260–280 g, were purchased from Vital River Ltd. (Beijing, China). Rats were maintained in a temperature-controlled (23 ± 2°C) and humidity-controlled (40–70%) facility under specific pathogen-free condition. Rats had access to food and water ad libitum. The Ethics Committee of Hunan Provincial Brain Hospital has reviewed and approved this study. All experimental procedures were carried out according to the Chinese legislation on the Use and Care of Animals.

The middle cerebral artery occlusion (MCAO) method was used to establish the cerebral I/R injury.^26,27 Rats were anesthetized with intraperitoneal injection of a mixture of 50 mg/kg ketamine and 10 mg/kg xylazine. The common left carotid artery was exposed and isolated, while the middle cerebral artery was occluded by inserting a nylon filament (0.24–0.28 mm diameter) into the internal carotid artery. This was advanced 18–20 mm past the carotid bifurcation until a slight resistance was felt. After 2 h of ischemia, the nylon filament was slowly removed for 24 h of reperfusion. Throughout the procedure, the body temperature was maintained at 36.5 to 37.5°C on a temperature-regulated heating pad. Rats that exhibited abnormal post-operative condition, including heavy neurological deficits such as unconscious and no neurological deficits (neurological score <1), were excluded from the study. In the Sham group, rats were anaesthetized and the carotid artery was exposed, without a filament insert. Rats were randomly divided into four groups (n = 20): Sham, Model, and MPH (0.5 and 1 mg/kg). The MPH doses used were based on the literatures.^28,29 Rats in MPH groups were administered 0.5 or 1 mg/kg MPH via i.p. injection for 7 days. Rats in the Sham and Model groups received PBS (vehicle) during the same period. The rats were treated 30 min after ischemia.

Evaluation of neurological deficits

Neurological deficit testing was assessed by an investigator blinded to the experimental groups using a five-point scale (0–4) Bederson’s test (Table 1).^30,31

Table 1.

Bederson’s five-point test

Neurological function	Score
No obvious neurological deficits	0
Forelimb is flexed and cannot be fully extended	1
As for 1, plus decreased resistance to lateral push	2
Unidirectional circling	3
Longitudinal spinning or seizure activity	4

Triphenyltetrazolium chloride (TTC) staining

Rat brains were quickly removed and frozen at −20°C for 10 min. 2 mm thick coronal brain sections were sliced and incubated with 2% TTC (Sigma–Aldrich, St. Louis, MO, USA) at 37°C in the dark for 20 min, followed by fixation with 4% paraformaldehyde for 24 h.^31,32 Images were photographed using a digital camera. The infarct volume was analyzed by one examiner blinded to the experimental groups using ImageJ 1.43 (National Institutes of Health, Rockville, MD, USA).

Brain water content measurement

Rat brains were quickly removed and samples from the ischemic hemisphere were rapidly weighed to obtain the wet weight (WW), and then dried in an oven at 100°C for 24 h to obtain a dry weight (DW). The brain water content was calculated as: (WW − DW)/WW × 100%.

TUNEL assay

Rat brains were quickly removed and fixed with 4% paraformaldehyde in PBS (pH 7.4) overnight. Brains were then dehydrated using a gradient sucrose solution. Sections (10 µm thick) were made using a freezing microtome. The TUNEL assay was performed according to the manufacturer’s instructions. 3,3′-diaminobenzidine (DAB) was used for the coloration of apoptotic cells. Sections were mounted and the number of TUNEL-positive cells was counted under the BX 60 microscope (Olympus, Tokyo, Japan).^31,32

Caspase activity measurement

Rat brains were quickly removed. Brains were homogenized in PBS (pH 7.4) and centrifuged at 3000 rpm for 15 min to remove cellular debris. Supernatants were kept at −80°C and used for further tests. Caspase-3 (Ac-DEVD-Amc, 390/475 nm) and caspase-9 (Ac-LEDH-Afc, 400/505 nm) activities were measured using assay kits according to the manufacturer’s instructions.

Western blot analysis

After treatment, samples were lysed with a lysis buffer (Sigma–Aldrich) containing protease and phosphatase inhibitors to obtain the protein. The protein was quantified using a BCA assay kit (Biyotime, Shanghai, China). Equal amounts of protein (40 μg/lane) were subjected to 4–12% sodium dodecyl sulfate polyacrylamide gel and then transferred onto polyvinylidene difluoride membranes. Membranes were then blocked with 1% bovine serum albumin for 1 h and incubated with the primary antibodies (anti-Bax rabbit mAb ab182733, 1:3000; anti-Bcl-2 rabbit pAb ab194583, 1:1000; anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) rabbit mAb ab133448, 1:3000; anti-AMPK alpha 1 + AMPK alpha 2 rabbit mAb ab207442, 1:1000; anti-ACC (phospho S79) rabbit mAb ab68191, 1:5000; anti-ACC rabbit mAb ab45174, 1:1500; anti-β-actin rabbit pAb ab8227, 1:3000) at 4°C overnight. After three washes, membranes were incubated with the secondary antibody (goat anti-rabbit IgG H&L HRP, ab205718, 1:5000) for 2 h at room temperature. Membranes were exposed to ECL substrates (Thermo Scientific, MA, USA), followed by the X-ray film development.³³

Statistical analysis

Data were analyzed with SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). The changes between groups were analyzed by one-way ANOVA followed by LSD post hoc analysis. The neurological score was expressed as the median and analyzed using Kruskal–Wallis one-way analysis of variance on ranks test followed by the post hoc Dunn’s multiple comparison test. A level of p < 0.05 was considered statistically significant.

Results

MPH reduced OGD/R-induced cell damage

Cell viability was examined using the MTT assay and apoptosis was detected by Annexin V/PI staining. Our data showed that MPH treatment alone had no obvious effects on cell viability (Figure 1(a)), which was consistent with findings of the previous study.²³ OGD/R significantly decreased the cell viability and increased the apoptotic rate. However, MPH treatment increased cell viability (Figure 1(b)) and decreased the apoptotic rate (Figure 1(c) and (d)) in a concentration-dependent manner (p < 0.05), thereby reducing OGD/R-induced cell damage.

Figure 1.

MPH protected neurons against OGD/R-induced toxicity. Primary neurons were isolated from newborn SD rats. The cell viability was assessed using the MTT assay and cell apoptosis was detected by Annexin V/PI staining. MPH treatment alone had no apparent effects on cell viability (a). MPH at different concentrations (1, 3, and 10 µM) was added 2 h prior to OGD/R. (b) MPH pre-treatment increased cell viability and (c) decreased the apoptotic rate. Representative flow cytometry graphics showed the gating scheme and apoptosis was defined as PI⁺Annexin V⁺ (d). Data were expressed as mean ± SD (n = 3). ^##p < 0.01 vs. control; *p < 0.05, **p < 0.01 vs. OGD/R group. Experiments were independently performed three times.

MPH exerted protective effects via the AMPK pathway

To explore the MPH mechanism of action on neuroprotective effects in the OGD/R model, the AMPK signaling pathway was investigated. AMPK requires phosphorylation of threonine 172 in the activation loop of the kinase domain for maximal activity. The AMPK activity is represented as the ratio of p-AMPK over total AMPK.³⁴ Our data showed that MPH treatment increased the protein expression of p-AMPK and p-ACC, while not changing the protein expression of total AMPK and total ACC (Figure 2(a)). To confirm whether AMPK-dependent signaling pathway was the main contributor, the AMPK specific inhibitor was applied prior to MPH exposure. Our results demonstrated that AMPK inhibitor largely abolished MPH-induced neuroprotective effects, evidenced by the decreased cell viability (Figure 2(b)), increased apoptotic rate (Figure 2(c)) and decreased protein expression of p-AMPK as well as p-ACC (Figure 2(d)) (p < 0.05).

Figure 2.

MPH exerted protective effects through the AMPK pathway. Primary neurons were isolated from newborn SD rats. The cell viability was assessed using the MTT assay and cell apoptosis was detected by Annexin V/PI staining. MPH was added 2 h prior to OGD/R. To validate the MPH mechanism of action, the specific inhibitor of AMPK Dorsomorphin (10 µM) was added 2 h before MPH treatment. MPH treatment increased p-AMPK and p-ACC protein expression, while not changing total AMPK and total ACC protein expression (a). Pre-treatment with Dorsomorphin (10 µM) largely abolished the neuroprotective effects of MPH, shown by the (b) decreased cell viability, (c) increased apoptotic rate, and (d) decreased protein expression of p-AMPK as well as p-ACC. Data were expressed as mean ± SD (n = 3). ^##p < 0.01 vs. control; **p < 0.01 vs. OGD/R group; ^&p < 0.05 vs. MPH group. Experiments were independently performed three times.

MPH treatment ameliorated brain damage caused by I/R

To further confirm the neuroprotective effects of MPH, the MCAO model was used. Rats in the Sham group did not show any neurological deficits or brain infarct. I/R caused significant neurological damage, evidenced by higher neurological scores, more water content and a larger cerebral infarct size (p < 0.01). However, MPH treatment significantly lowered the neurological scores (Figure 3(a)), water content (Figure 3(b)), and the cerebral infarct size (Figure 3(c)) (p < 0.05), indicating that the brain damage was ameliorated.

Figure 3.

MPH treatment significantly ameliorated brain damage caused by I/R. The MCAO method was used to establish I/R injury. Rats were randomly divided into four groups (n = 20): Sham, Model, and MPH (0.5 and 1 mg/kg). In each group, rats were randomly divided into four sub-groups (n = 5) for different sample process. Rats in MPH groups were administered 0.5 or 1 mg/kg MPH via i.p. injection for 7 days. Rats in the Sham and Model groups received PBS during the same period. Rats were treated at 30 min after ischemia. Compared with the model group, MPH treatment significantly decreased (a) the neurological deficit score; (b) brain water content; and (c) cerebral infarct size shown by TTC staining. Data were expressed as median (a, n = 20) or mean ± SD ((b) and (c), n = 5). ^##p < 0.01 vs. Sham group; *p < 0.05, **p < 0.01 vs. model group.

MPH treatment decreased apoptosis in brain tissues

TUNEL assay was used to detect the apoptosis in I/R-injured brain tissues. A significant number of TUNEL-positive cells were observed in the Model group, whereas brain sections from the Sham group showed few TUNEL-positive cells. MPH treatment significantly decreased DNA fragmentation (Figure 4(a)) and caspase activities (Figure 4(b)). Moreover, expression of the anti-apoptotic protein Bcl-2 increased, but pro-apoptotic protein Bax expression decreased after MPH treatment (Figure 4(c)) (p < 0.05).

Figure 4.

MPH treatment decreased apoptosis in brain tissues induced by I/R. The MCAO method was used to establish I/R injury. Rats were randomly divided into four groups (n = 20): Sham, Model, and MPH (0.5 and 1 mg/kg). In each group, rats were randomly divided into four sub-groups (n = 5) for different sample process. Rats in MPH groups were administered 0.5 or 1 mg/kg MPH via i.p. injection for 7 days. Rats in the Sham and Model groups received PBS during the same period. Rats were treated at 30 min after ischemia. Apoptosis was determined based on: DNA damage shown by the (a) TUNEL assay; (b) caspase activities; and (c) apoptosis-related proteins. Data were expressed as mean ± SD (n = 5). ^##p < 0.01 vs. Sham group; *p < 0.05, **p < 0.01 vs. Model group.

MPH treatment increased the protein expression of p-AMPK and p-ACC in brain tissues

To further confirm the MPH mechanism of action, the protein expression of AMPK and ACC was examined. MPH treatment markedly increased the relative protein expression level of p-AMPK and p-ACC (p < 0.05), compared with the Model group, while not changing total AMPK and total ACC protein expression (Figure 5).

Figure 5.

MPH treatment increased p-AMPK and p-ACC protein expression in rat brain tissues. The MCAO method was used to establish I/R injury. Rats were randomly divided into four groups (n = 20): Sham, Model, and MPH (0.5 and 1 mg/kg). In each group, rats were randomly divided into 4 sub-groups (n = 5) for different sample process. Rats in MPH groups were administered 0.5 or 1 mg/kg MPH via i.p. injection for 7 days. Rats in the Sham and Model groups received PBS during the same period. Rats were treated 30 min after ischemia. MPH treatment increased the protein expression of p-AMPK and p-ACC, while not changing the protein expression of total AMPK and total ACC. Data were expressed as mean ± SD (n = 5). ^##p < 0.01 vs. Sham group; *p < 0.05, **p < 0.01 vs. Model group.

Discussion

Stroke is a leading cause of disability and accounts for 9% of deaths worldwide. Reactive oxygen species (ROS) have been implicated in brain injury after an ischemic stroke. Studies have shown that a rapid increase in ROS production after an acute ischemic stroke overwhelms antioxidant defenses, thus causing further tissue damage.³⁵ Current treatments to protect the brain against severe stroke damage lack efficacy. A number of agents have been tested and significantly ameliorated the neurological damage associated with focal cerebral I/R injury through inhibiting inflammatory responses and oxidative stress.^31,32,36 Unlike these leading compounds, MPH is a first-line stimulant used to treat ADHD. Studies have demonstrated that MPH could ameliorate hypoxia-induced mitochondrial damage in human neuroblastoma SH-SY5Y cells through inhibition of oxidative stress and improve the glutathione status in brains of Wistar rats,^22,37 implying its neuroprotective effects. This study investigated protective effects of MPH in OGD/R and MCAO models. Our data indicated that MPH treatment significantly increased cell viability and ameliorated brain damage, which may be associated with the AMPK signaling pathway. Other pathways such as anti-oxidative stress or anti-inflammatory responses may also be involved in the neuroprotective effects of MPH, which will be further investigated in future corollary studies.

During I/R injury, apoptosis plays a pivotal role in brain damage associated with stroke.³⁸ The Bcl-2 family mainly exerts its pro- or anti-apoptotic activities through the mitochondrial apoptotic pathway. Bax and bid are positive regulators that promote cell death. In contrast, bcl-2 and bcl-xl are negative regulators that prevent cells from undergoing apoptosis. The bcl-2 proteins may facilitate the passage of cytochrome-c or other apoptosis-related factors that trigger the activation of a caspase cascade, resulting in apoptosis.^39,40 The caspase family promotes and implements apoptosis in mammalian cells. As an executor, caspase-3 is a key downstream apoptosis protease in the caspase cascade and activates factors of DNA fragmentation, which in turn activate endonucleases to cleave nuclear DNA and cause cell death.⁴¹ In this study, pre-treatment with MPH significantly reduced the loss of cell viability and apoptosis in cultured neurons. Moreover, MPH treatment significantly decreased the activities of caspase-3/9 and DNA fragmentation in brains of MCAO rats. Pro-apoptotic protein Bax expression decreased, but anti-apoptotic protein Bcl-2 expression increased after MPH treatment.

AMPK is activated during events, such as nutrient starvation, which activates a set of energy-producing pathways and inhibits energy-consuming pathways. Thereafter, AMPK acts to balance the levels of ATP consumption and generation via anabolic and catabolic pathways. This role for AMPK is particularly relevant in tissues with a high energy demand and robust ATP consumption.^42,43 AMPK is widely but distinctly distributed in different organs, which might explain the susceptibility of different organs to ischemic stimuli.⁴⁴ In the brain, ischemia is one of the most important forms of energy deprivation. AMPK is activated by increases in AMP/ATP or ADP/ATP ratios under energy stress. Therefore, AMPK acts as an early sensor of energy deprivation for maintaining metabolic homeostasis.^45,46 Neuron apoptosis is usually secondary to ischemic injury and serves as an index of ischemic extent, whereas this process may be ameliorated by AMPK. Cerebral ischemic pre-conditioning can activate AMPK and ameliorate cell apoptosis in the peri-infarct region, evidenced by the reduced percentage of TUNEL-positive cells, suggesting the ability of AMPK to reduce apoptosis in ischemic stroke.⁴⁷ Na⁺/K⁺-ATPase is a membrane-bound enzyme necessary to maintain neuronal excitability. MPH treatment could increase Na⁺/K⁺-ATPase activity in the cerebrum of young and adult rats.⁴⁸ Therefore, we hypothesized that MPH exerted its neuroprotective effects in association with AMPK. Our results showed that MPH reduced cell damage through activating the phosphorylation of AMPK, which was confirmed by the addition of an AMPK inhibitor.

Some study limitations should be mentioned. First, it is uncertain whether the data from in vitro and in vivo models accurately reflect molecular changes in human. Second, although clear effects were observed after MPH treatment, its activities need to be confirmed by other animal models. Finally, pharmacokinetic assays are needed to determine the concentration of MPH in different organs and tissues.

In conclusions, this study demonstrated that MPH relieved cerebral I/R injury, at least in part, through activating AMPK. Our data might supply the new insight into the potential of MPH in the treatment of cerebral ischemia.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

X Huang

References

Feigin

Krishnamurthi

Parmar

, et al. Update on the global burden of ischemic and hemorrhagic stroke in 1990–2013: the GBD 2013 study. Neuroepidemiology 2015; 45: 161–176.

Benjamin

Blaha

Chiuve

, et al. Heart disease and stroke statistics-2017 update: a report from the American Heart Association. Circulation 2017; 135: 146–603.

Banerjee

Williamson

Habib

, et al. Human stem cell therapy in ischaemic stroke: a review. Age Ageing 2011; 40: 7–13.

Hong

Xin

, et al. Neuroprotective effects of leonurine on ischemia/reperfusion-induced mitochondrial dysfunctions in rat cerebral cortex. Biol Pharm Bull 2010; 33: 1958–1964.

Zhang

Cui

, et al. The neuroprotection of oxymatrine in cerebral ischemia/reperfusion is related to nuclear factor erythroid 2-related factor 2 (nrf2)-mediated antioxidant response: role of nrf2 and hemeoxygenase-1 expression. Biol Pharm Bull 2011; 34: 595–601.

Durukan

Tatlisumak

. Acute ischemic stroke: overview of major experimental rodent models, pathophysiology, and therapy of focal cerebral ischemia. Pharmacol Biochem Behav 2007; 87: 179–197.

Kahn

Alquier

Carling

, et al. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 2005; 1: 15–25.

Sun

Yuan

, et al. C1q/tumor necrosis factor-related protein-3, a newly identified adipokine, is a novel antiapoptotic, proangiogenic, and cardioprotective molecule in the ischemic mouse heart. Circulation 2012; 125: 3159–3169.

Yuan

Lau

, et al. C1q-TNF-related protein-9, a novel cardioprotetcive cardiokine, requires proteolytic cleavage to generate a biologically active globular domain isoform. Am J Physiol Endocrinol Metab 2015; 308: 891–898.

10.

Zhao

Fan

Zhang

, et al. Adiponectin enhances bone marrow mesenchymal stem cell resistance to flow shear stress through AMP-activated protein kinase signaling. Sci Rep 2016; 6: 28752.

11.

Zheng

Yuan

, et al. C1q/TNF-related proteins, a family of novel adipokines, induce vascular relaxation through the adiponectin receptor-1/AMPK/eNOS/nitric oxide signaling pathway. Arterioscler Thromb Vasc Biol 2011; 31: 2616–2623.

12.

Wakil

Abu-Elheiga

. Fatty acid metabolism: target for metabolic syndrome. J Lipid Res 2009; 50: S138–S143.

13.

Wang

Zhou

Tang

, et al. ACC1 (acetyl coenzyme A carboxylase 1) is a potential immune modulatory target of cerebral ischemic stroke. Stroke 2019; 50: 1869–1878.

14.

Gadalla

Pearson

Currie

, et al. AICA riboside both activates AMP-activated protein kinase and competes with adenosine for the nucleoside transporter in the CA1 region of the rat hippocampus. J Neurochem 2004; 88: 1272–1282.

15.

Turnley

Stapleton

Mann

, et al. Cellular distribution and developmental expression of AMP-activated protein kinase isoforms in mouse central nervous system. J Neurochem 1999; 72: 1707–1716.

16.

Ryou

Mallet

. An in vitro oxygen-glucose deprivation model for studying ischemia-reperfusion injury of neuronal cells. Methods Mol Biol 2018; 1717: 229–235.

17.

Chiang

Messing

Chou

. Mouse model of middle cerebral artery occlusion. J Vis Exp 2011; 48: 2761–2763.

18.

Powers

Rabinstein

Ackerson

, et al. 2018 guidelines for the early management of patients with acute ischemic stroke. Stroke 2018; 49: 46–97.

19.

Martins

Reinke

Petronilho

, et al. Methylphenidate treatment induces oxidative stress in young rat brain. Brain Res 2006; 1078: 189–197.

20.

Motaghinejad

Motevalian

Shabab

, et al. Effects of acute doses of methylphenidate on inflammation and oxidative stress in isolated hippocampus and cerebral cortex of adult rats. J Neural Transm 2017; 124: 121–131.

21.

Gomes

Inacio

Valvassori

, et al. Superoxide production after acute and chronic treatment with methylphenidate in young and adult rats. Neurosci Lett 2009; 465: 95–98.

22.

Zhu

Tian

Zhang

, et al. Methylphenidate ameliorates hypoxia-induced mitochondrial damage in human neuroblastoma SH-SY5Y cells through inhibition of oxidative stress. Life Sci 2018; 197: 40–45.

23.

Ludolph

Schaz

Storch

, et al. Methylphenidate exerts no neurotoxic, but neuroprotective effects in vitro. J Neural Transm 2006; 113: 1927–1934.

24.

Wang

Liu

, et al. Inhibition of miR-19a protects neurons against ischemic stroke through modulating glucose metabolism and neuronal apoptosis. Cell Mol Bio Lett 2019; 24: 37.

25.

Kumar

Nagarajan

Uchil

. Analysis of cell viability by the MTT assay. Cold Spring Harb Protoc 2018; 2018: DOI:10.1101/pdb.prot095505

26.

Guo

Xia

, et al. Sevoflurane postconditioning involves an up-regulation of HIF-1α and HO-1 expression via PI3K/Akt pathway in a rat model of focal cerebral ischemia. Brain Res 2012; 1463: 63–74.

27.

Dong

Yang

Zhang

, et al. Hemopexin alleviates cognitive dysfunction after focal cerebral ischemia-reperfusion injury in rats. BMC Anesthesiol 2019; 19: 13.

28.

Berridge

Devilbiss

Andrzejewski

, et al. Methylphenidate preferentially increases catecholamine neurotransmission within the prefrontal cortex at low doses that enhance cognitive function. Biol Psychiatry 2006; 60: 1111–1120.

29.

Navarra

Clark

Gargiulo

, et al. Methylphenidate enhances early-stage sensory processing and rodent performance of a visual signal detection task. Neuropsychopharmacology 2017; 42: 1326–1337.

30.

Bederson

Pitts

Tsuji

, et al. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 1986; 17: 472–476.

31.

Kaundal

Sharma

. Ameliorative effects of GW1929, a nonthiazolidinedione PPARγ agonist, on inflammation and apoptosis in focal cerebral ischemic-reperfusion injury. Curr Neurovasc Res 2011; 8: 236–245.

32.

Kaundal

Shah

Sharma

. Neuroprotective effects of NU1025, a PARP inhibitor in cerebral ischemia are mediated through reduction in NAD depletion and DNA fragmentation. Life Sci 2006; 79: 2293–2302.

33.

Taylor

Posch

The design of a quantitative western blot experiment. Biomed Res Int 2014; 2014: 361590.

34.

Stein

Woods

Jones

, et al. The regulation of AMP-activated protein kinase by phosphorylation. Biochem J 2000; 345: 437–443.

35.

Rodrigo

Fernandez-Gajardo

Gutierrez

, et al. Oxidative stress and pathophysiology of ischemic stroke: novel therapeutic opportunities. CNS Neurol Disord Drug Targets 2013; 12: 698–714.

36.

Kabra

Thiyagarajan

Kaul

, et al. Neuroprotective effect of 4-amino-1,8-napthalimide, a poly(ADP ribose) polymerase inhibitor in middle cerebral artery occlusion-induced focal cerebral ischemia in rat. Brain Res Bull 2004; 62: 425–433.

37.

Loureiro-Vieira

Costa

Duarte

, et al. Methylphenidate clinically oral doses improved brain and heart glutathione redox status and evoked renal and cardiac tissue injury in rats. Biomed Pharmacother 2018; 100: 551–563.

38.

Jung

Park

, et al. Postischemic administration of pravastatin reduces neuronal injury by inhibiting Bax protein expression after transient forebrain ischemia in rats. Neurosci Lett 2015; 594: 87–92.

39.

Miller

Moulder

Knudson

, et al. Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death. J Cell Biol 1997; 139: 205–217.

40.

Cory

Adams

. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2002; 2: 647–656.

41.

Prabhakar

Vona-Davis

Murray

, et al. Phosphocreatine restores high-energy phosphates in ischemic myocardium: implication for off-pump cardiac revascularization. J Am Coll Surg 2003; 197: 786–791.

42.

Carling

. The AMP-activated protein kinase cascade-a unifying system for energy control. Trends Biochem Sci 2004; 29: 18–24.

43.

Hardie

DG.

Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology 2003; 144: 5179–5183.

44.

Culmsee

Monnig

Kemp

, et al. AMP-activated protein kinase is highly expressed in neurons in the developing rat brain and promotes neuronal survival following glucose deprivation. J Mol Neurosci. 2001; 17: 45–58.

45.

Lewinska

Adamczyk-Grochala

Deregowska

, et al. Sulforaphane-induced cell cycle arrest and senescence are accompanied by DNA hypomethylation and changes in microRNA profile in breast cancer cells. Theranostics 2017; 7: 3461–3477.

46.

Oakhill

Steel

Chen

, et al. AMPK is a direct adenylate charge-regulated protein kinase. Science 2011; 332: 1433–1435.

47.

Jiang

Zhu

, et al. Ischemic preconditioning provides neuroprotection by induction of AMP-activated protein kinase-dependent autophagy in a rat model of ischemic stroke. Mol Neurobiol 2015; 51: 220–229.

48.

Scherer

Matte

Ferreira

AGK

, et al. Methylphenidate treatment increases Na(+), K (+)-ATPase activity in the cerebrum of young and adult rats. J Neural Transm 2009; 116: 1681–1687.