Abstract
Brain edema is a common and serious complication of ischemic stroke with limited effective treatment. We previously reported that methylene blue (MB) attenuated ischemic brain edema in rats, but the underlying mechanisms remained unknown. Aquaporin 4 (AQP4) in astrocytes plays a key role in brain edema. We also found that extracellular signal-regulated kinase 1/2 (ERK1/2) activation was involved in the regulation of AQP4 expression in astrocytes. In the present study, we investigated whether AQP4 and ERK1/2 were involved in the protective effect of MB against cerebral edema. Rats were subjected to transient middle cerebral artery occlusion (tMCAO), MB (3 mg/kg, for 30 min) was infused intravenously through the tail vein started immediately after reperfusion and again at 3 h after ischemia (1.5 mg/kg, for 15 min). Brain edema was determined by MRI at 0.5, 2.5, and 48 h after tMCAO. The decreases of apparent diffusion coefficient (ADC) values on diffusion-weighted MRI indicated cytotoxic brain edema, whereas the increase of T2 MRI values reflected vasogenic brain edema. We found that MB infusion significantly ameliorated cytotoxic brain edema at 2.5 and 48 h after tMCAO and decreased vasogenic brain edema at 48 h after tMCAO. In addition, MB infusion blocked the AQP4 increases and ERK1/2 activation in the cerebral cortex in ischemic penumbra at 48 h after tMCAO. In a cell swelling model established in cultured rat astrocyte exposed to glutamate (1 mM), we consistently found that MB (10 μM) attenuated cell swelling, AQP4 increases and ERK1/2 activation. Moreover, the ERK1/2 inhibitor U0126 (10 μM) had the similar effects as MB. These results demonstrate that MB improves brain edema and astrocyte swelling, which may be mediated by the inhibition of AQP4 expression via ERK1/2 pathway, suggesting that MB may be a potential choice for the treatment of brain edema.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Stokum JA, Gerzanich V, Simard JM. Molecular pathophysiology of cerebral edema. J Cereb Blood Flow Metab. 2016;36:513–38.
Wang W, Jiang B, Sun H, Ru X, Sun D, Wang L, et al. Prevalence, incidence, and mortality of stroke in China: results from a nationwide population-based survey of 480 687 adults. Circulation. 2017;135:759–71.
Posada-Duque RA, Barreto GE, Cardona-Gomez GP. Protection after stroke: cellular effectors of neurovascular unit integrity. Front Cell Neurosci. 2014;8:231.
Seyedsaadat SM, Kallmes DF. Memantine for the treatment of ischemic stroke: experimental benefits and clinical lack of studies. Rev Neurosci. 2019;30:203–20.
Simard JM, Kent TA, Chen M, Tarasov KV, Gerzanich V. Brain oedema in focal ischaemia: molecular pathophysiology and theoretical implications. Lancet Neurol. 2007;6:258–68.
Walberer M, Ritschel N, Nedelmann M, Volk K, Mueller C, Tschernatsch M, et al. Aggravation of infarct formation by brain swelling in a large territorial stroke: a target for neuroprotection? J Neurosurg. 2008;109:287–93.
Wang WW, Xie CL, Zhou LL, Wang GS. The function of aquaporin4 in ischemic brain edema. Clin Neurol Neurosurg. 2014;127:5–9.
Wen Y, Li W, Poteet EC, Xie L, Tan C, Yan LJ, et al. Alternative mitochondrial electron transfer as a novel strategy for neuroprotection. J Biol Chem. 2011;286:16504–15.
Wiklund L, Sharma A, Sharma HS. Neuroprotection by methylene blue in cerebral global ischemic injury induced blood-brain barrier disruption and brain pathology: a review. CNS Neurol Disord Drug Targets. 2016;15:1181–7.
Tucker D, Lu Y, Zhang Q. From mitochondrial function to neuroprotection-an emerging role for methylene blue. Mol Neurobiol. 2018;55:5137–53.
Yang SH, Li W, Sumien N, Forster M, Simpkins JW, Liu R. Alternative mitochondrial electron transfer for the treatment of neurodegenerative diseases and cancers: methylene blue connects the dots. Prog Neurobiol. 2017;157:273–91.
Delport A, Harvey BH, Petzer A, Petzer JP, Azure B. and a synthetic structural analogue of methylene blue, ethylthioninium chloride, present with antidepressant-like properties. Life Sci. 2014;117:56–66.
Talley Watts L, Long JA, Chemello J, Van Koughnet S, Fernandez A, Huang S, et al. Methylene blue is neuroprotective against mild traumatic brain injury. J Neurotrauma. 2014;31:1063–71.
Jiang Z, Watts LT, Huang S, Shen Q, Rodriguez P, Chen C, et al. The effects of methylene blue on autophagy and apoptosis in MRI-defined normal tissue, ischemic penumbra and ischemic core. PLoS One. 2015;10:e0131929.
Fang Q, Yan X, Li S, Sun Y, Xu L, Shi Z, et al. Methylene blue ameliorates ischemia/reperfusion-induced cerebral edema: an MRI and transmission electron microscope study. Acta Neurochir Suppl. 2016;121:227–36.
Schlaug G, Benfield A, Baird AE, Siewert B, Lovblad KO, Parker RA, et al. The ischemic penumbra: operationally defined by diffusion and perfusion MRI. Neurology. 1999;53:1528–37.
Moseley ME, Kucharczyk J, Mintorovitch J, Cohen Y, Kurhanewicz J, Derugin N, et al. Diffusion-weighted MR imaging of acute stroke: correlation with T2-weighted and magnetic susceptibility-enhanced MR imaging in cats. AJNR Am J Neuroradiol. 1990;11:423–9.
Shen Q, Meng X, Fisher M, Sotak CH, Duong TQ. Pixel-by-pixel spatiotemporal progression of focal ischemia derived using quantitative perfusion and diffusion imaging. J Cereb Blood Flow Metab. 2003;23:1479–88.
Shen Q, Fisher M, Sotak CH, Duong TQ. Effects of reperfusion on ADC and CBF pixel-by-pixel dynamics in stroke: characterizing tissue fates using quantitative diffusion and perfusion imaging. J Cereb Blood Flow Metab. 2004;24:280–90.
Shen Q, Ren H, Cheng H, Fisher M, Duong TQ. Functional, perfusion and diffusion MRI of acute focal ischemic brain injury. J Cereb Blood Flow Metab. 2005;25:1265–79.
Schitine C, Nogaroli L, Costa MR, Hedin-Pereira C. Astrocyte heterogeneity in the brain: from development to disease. Front Cell Neurosci. 2015;9:76.
Vella J, Zammit C, Di Giovanni G, Muscat R, Valentino M. The central role of aquaporins in the pathophysiology of ischemic stroke. Front Cell Neurosci. 2015;9:108.
Manley GT, Fujimura M, Ma T, Noshita N, Filiz F, Bollen AW, et al. Aquaporin-4 deletion in mice reduces brain edema after acute water intoxication and ischemic stroke. Nat Med. 2000;6:159–63.
Papadopoulos MC, Manley GT, Krishna S, Verkman AS. Aquaporin-4 facilitates reabsorption of excess fluid in vasogenic brain edema. FASEB J. 2004;18:1291–3.
Yao X, Derugin N, Manley GT, Verkman AS. Reduced brain edema and infarct volume in aquaporin-4 deficient mice after transient focal cerebral ischemia. Neurosci Lett. 2015;584:368–72.
He ZP, Lu H. Aquaporin-4 gene silencing protects injured neurons after early cerebral infarction. Neural Regen Res. 2015;10:1082–7.
Yuan F, Wang T. Glutamate-induced swelling of cultured astrocytes is mediated by metabotropic glutamate receptor. Sci China C Life Sci. 1996;39:517–22.
Shi Z, Zhang W, Lu Y, Lu Y, Xu L, Fang Q, et al. Aquaporin 4-mediated glutamate-induced astrocyte swelling is partially mediated through metabotropic glutamate receptor 5 activation. Front Cell Neurosci. 2017;11:116.
Lai TW, Zhang S, Wang YT. Excitotoxicity and stroke: identifying novel targets for neuroprotection. Prog Neurobiol. 2014;115:157–88.
Hu Y, Duan M, Liang S, Wang Y, Feng Y. Senkyunolide I protects rat brain against focal cerebral ischemia-reperfusion injury by up-regulating p-Erk1/2, Nrf2/HO-1 and inhibiting caspase 3. Brain Res. 2015;1605:39–48.
Lopez-Morales MA, Castello-Ruiz M, Burguete MC, Jover-Mengual T, Aliena-Valero A, Centeno JM, et al. Molecular mechanisms underlying the neuroprotective role of atrial natriuretic peptide in experimental acute ischemic stroke. Mol Cell Endocrinol. 2018;472:1–9.
Liu R, Yuan H, Yuan F, Yang SH. Neuroprotection targeting ischemic penumbra and beyond for the treatment of ischemic stroke. Neurological Res. 2012;34:331–7.
Moskowitz MA, Lo EH, Iadecola C. The science of stroke: mechanisms in search of treatments. Neuron. 2010;67:181–98.
Ashwal S, Tone B, Tian HR, Cole DJ, Pearce WJ. Core and penumbral nitric oxide synthase activity during cerebral ischemia and reperfusion. Stroke. 1998;29:1037–46.
Huang YC, Tzeng WS, Wang CC, Cheng BC, Chang YK, Chen HH, et al. Neuroprotective effect of agmatine in rats with transient cerebral ischemia using MR imaging and histopathologic evaluation. Magn Reson Imaging. 2013;31:1174–81.
Qian Q, Huang HT, Xu L, Jin P, Lin M. Prediction of infarct lesion volumes by processing magnetic resonance apparent diffusion coefficient maps in patients with acute ischemic stroke. J Stroke Cerebrovasc Dis. 2016;25:2821–7.
Krueger M, Mages B, Hobusch C, Michalski D. Endothelial edema precedes blood-brain barrier breakdown in early time points after experimental focal cerebral ischemia. Acta Neuropathol Commun. 2019;7:17.
Shi ZF, Zhao WJ, Xu LX, Dong LP, Yang SH, Yuan F. Downregulation of aquaporin 4 expression through extracellular signal-regulated kinases1/2 activation in cultured astrocytes following scratch-injury. Biomed Environ Sci. 2015;28:199–205.
Loubinoux I, Volk A, Borredon J, Guirimand S, Tiffon B, Seylaz J, et al. Spreading of vasogenic edema and cytotoxic edema assessed by quantitative diffusion and T2 magnetic resonance imaging. Stroke. 1997;28:419–26.
Stokum JA, Kurland DB, Gerzanich V, Simard JM. Mechanisms of astrocyte-mediated cerebral edema. Neurochem Res. 2015;40:317–28.
Previch LE, Ma L, Wright JC, Singh S, Geng X, Ding Y. Progress in AQP research and new developments in therapeutic approaches to ischemic and hemorrhagic stroke. Int J Mol Sci. 2016;17:1146.
He Z, Wang X, Wu Y, Jia J, Hu Y, Yang X, et al. Treadmill pre-training ameliorates brain edema in ischemic stroke via down-regulation of aquaporin-4: an MRI study in rats. PLoS One. 2014;9:e84602.
Verkman AS, Smith AJ, Phuan PW, Tradtrantip L, Anderson MO. The aquaporin-4 water channel as a potential drug target in neurological disorders. Expert Opin Ther Targets. 2017;21:1161–70.
Hubbard JA, Szu JI, Binder DK. The role of aquaporin-4 in synaptic plasticity, memory and disease. Brain Res Bull. 2018;136:118–29.
Tan Y, Zhang H, Wang XC, Qin JB, Wang L. The value of multi ultra high-b-value DWI in grading cerebral astrocytomas and its association with aquaporin-4. Br J Radiol. 2018;91:20170696.
Lu H, Hu H, He ZP. Reperfusion of the rat brain tissues following acute ischemia: the correlation among diffusion-weighted imaging, histopathology, and aquaporin-4 expression. Chin Med J. 2011;124:3148–53.
Gunnarson E, Zelenina M, Axehult G, Song Y, Bondar A, Krieger P, et al. Identification of a molecular target for glutamate regulation of astrocyte water permeability. Glia. 2008;56:587–96.
Farr GW, Hall CH, Farr SM, Wade R, Detzel JM, Adams AG, et al. Functionalized phenylbenzamides inhibit aquaporin-4 reducing cerebral edema and improving outcome in two models of CNS injury. Neuroscience. 2019;404:484–98.
Chu H, Huang C, Ding H, Dong J, Gao Z, Yang X, et al. Aquaporin-4 and cerebrovascular diseases. Int J Mol Sci. 2016;17:1249.
Ceccariglia S, D’Altocolle A, Del Fa A, Silvestrini A, Barba M, Pizzolante F, et al. Increased expression of Aquaporin 4 in the rat hippocampus and cortex during trimethyltin-induced neurodegeneration. Neuroscience. 2014;274:273–88.
Miclescu A, Sharma HS, Martijn C, Wiklund L. Methylene blue protects the cortical blood-brain barrier against ischemia/reperfusion-induced disruptions. Crit Care Med. 2010;38:2199–206.
Qi LL, Fang SH, Shi WZ, Huang XQ, Zhang XY, Lu YB, et al. CysLT2 receptor-mediated AQP4 up-regulation is involved in ischemic-like injury through activation of ERK and p38 MAPK in rat astrocytes. Life Sci. 2011;88:50–6.
Nito C, Kamada H, Endo H, Narasimhan P, Lee YS, Chan PH. Involvement of mitogen-activated protein kinase pathways in expression of the water channel protein aquaporin-4 after ischemia in rat cortical astrocytes. J Neurotrauma. 2012;29:2404–12.
Ahnstedt H, Mostajeran M, Blixt FW, Warfvinge K, Ansar S, Krause DN, et al. U0126 attenuates cerebral vasoconstriction and improves long-term neurologic outcome after stroke in female rats. J Cereb Blood Flow Metab. 2015;35:454–60.
Rodrigo R, Fernandez-Gajardo R, Gutierrez R, Matamala JM, Carrasco R, Miranda-Merchak A, et al. Oxidative stress and pathophysiology of ischemic stroke: novel therapeutic opportunities. CNS Neurol Disord Drug Targets. 2013;12:698–714.
Shenoda B. The role of Na+/Ca2+ exchanger subtypes in neuronal ischemic injury. Transl Stroke Res. 2015;6:181–90.
Siniscalchi A, Gallelli L, Malferrari G, Pirritano D, Serra R, Santangelo E, et al. Cerebral stroke injury: the role of cytokines and brain inflammation. J Basic Clin Physiol Pharmacol. 2014;25:131–7.
Huang L, Lu J, Cerqueira B, Liu Y, Jiang Z, Duong TQ. Chronic oral methylene blue treatment in a rat model of focal cerebral ischemia/reperfusion. Brain Res. 2018;1678:322–9.
Ryou MG, Choudhury GR, Li W, Winters A, Yuan F, Liu R, et al. Methylene blue-induced neuronal protective mechanism against hypoxia-reoxygenation stress. Neuroscience. 2015;301:193–203.
Roy Choudhury G, Winters A, Rich RM, Ryou MG, Gryczynski Z, Yuan F, et al. Methylene blue protects astrocytes against glucose oxygen deprivation by improving cellular respiration. PLoS One. 2015;10:e0123096.
Dai H, Song D, Xu J, Li B, Hertz L, Peng L. Ammonia-induced Na, K-ATPase/ouabain-mediated EGF receptor transactivation, MAPK/ERK and PI3K/AKT signaling and ROS formation cause astrocyte swelling. Neurochem Int. 2013;63:610–25.
Acknowledgements
We sincerely appreciate the help of Prof. Shao-hua Yang from the Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, for critical comments on the paper. We also thank Prof. Shao-wu Li from the Neurological Imaging Center, Beijing Neurosurgical Institute, for help with MRI data acquisition and Dr Ying Jiang from the Department of Functional Neurosurgery, Beijing Neurosurgical Institute, for guidance related to the MRI data analysis. This project is supported by the National Natural Science Foundation of China (81801159, 8127128, and 51707012).
Author information
Authors and Affiliations
Contributions
ZFS carried out the experiments and contributed to the acquisition, analysis, and interpretation of data and drafting of the paper; QF, YC, LXX, MW, MJ, YL, XXW, YJW, XY, and LPD conducted the experiments, acquired the data, and analyzed the data; FY conceived and designed the experiments for the work and contributed to acquisition, analysis, and interpretation of data and drafting of the paper. All authors read and approved the paper.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Rights and permissions
About this article
Cite this article
Shi, Zf., Fang, Q., Chen, Y. et al. Methylene blue ameliorates brain edema in rats with experimental ischemic stroke via inhibiting aquaporin 4 expression. Acta Pharmacol Sin 42, 382–392 (2021). https://doi.org/10.1038/s41401-020-0468-5
Received:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/s41401-020-0468-5
Keywords
This article is cited by
-
Non-coding RNAs and Aquaporin 4: Their Role in the Pathogenesis of Neurological Disorders
Neurochemical Research (2024)
-
Brain-Targeting Emodin Mitigates Ischemic Stroke via Inhibiting AQP4-Mediated Swelling and Neuroinflammation
Translational Stroke Research (2024)
-
Crosstalk Among Glial Cells in the Blood–Brain Barrier Injury After Ischemic Stroke
Molecular Neurobiology (2024)
-
Macrophage migration inhibitory factor exacerbates asthmatic airway remodeling via dynamin-related protein 1-mediated autophagy activation
Respiratory Research (2023)
-
Tanshinone IIA reduces AQP4 expression and astrocyte swelling after OGD/R by inhibiting the HMGB1/RAGE/NF-κB/IL-6 pro-inflammatory axis
Scientific Reports (2022)
