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• W2018161712 abstract "AMP-activated protein kinase (AMPK) signaling is reported to protect neurons under pathologic conditions; however, its effect on oligodendrocytes (OLs) remains to be elucidated. We investigated whether AMPK signaling protects OLs to restore central nervous system (CNS) functions in an experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. Increased inflammation and demyelination in the CNS and peripheral immune responses were consistent with the observed clinical impairments in EAE animals, which were attenuated by treatment with metformin compared with vehicle. In addition, expressions of neurotrophic factors and of signatory genes of OL lineages were increased in the CNS of metformin-treated EAE animals. Likewise, metformin attenuated inflammatory response and enhanced expressions of neurotrophic factors, thereby protecting OLs via AMPK activation in mixed glial cultures stimulated with lipopolysaccharide/interferon γ in vitro, as evidenced by analysis of the expression of signatory genes of O1+/MBP+ OLs and their cellular populations. Metformin also attenuated oxidative stress and malondialdehyde-containing protein levels, with corresponding induction of antioxidative defenses in OLs exposed to cytokines via AMPK activation. These effects of metformin were evident in the CNS of EAE animals. These data provide evidence that AMPK signaling is crucial to protect OLs and, thus, CNS functions in EAE animals. We conclude that AMPK activators, including metformin, have the potential to limit neurologic deficits in multiple sclerosis and related neurodegenerative disorders. AMP-activated protein kinase (AMPK) signaling is reported to protect neurons under pathologic conditions; however, its effect on oligodendrocytes (OLs) remains to be elucidated. We investigated whether AMPK signaling protects OLs to restore central nervous system (CNS) functions in an experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. Increased inflammation and demyelination in the CNS and peripheral immune responses were consistent with the observed clinical impairments in EAE animals, which were attenuated by treatment with metformin compared with vehicle. In addition, expressions of neurotrophic factors and of signatory genes of OL lineages were increased in the CNS of metformin-treated EAE animals. Likewise, metformin attenuated inflammatory response and enhanced expressions of neurotrophic factors, thereby protecting OLs via AMPK activation in mixed glial cultures stimulated with lipopolysaccharide/interferon γ in vitro, as evidenced by analysis of the expression of signatory genes of O1+/MBP+ OLs and their cellular populations. Metformin also attenuated oxidative stress and malondialdehyde-containing protein levels, with corresponding induction of antioxidative defenses in OLs exposed to cytokines via AMPK activation. These effects of metformin were evident in the CNS of EAE animals. These data provide evidence that AMPK signaling is crucial to protect OLs and, thus, CNS functions in EAE animals. We conclude that AMPK activators, including metformin, have the potential to limit neurologic deficits in multiple sclerosis and related neurodegenerative disorders. The critical role of oligodendrocytes (OLs) in producing and maintaining the myelin sheath around axons that supports rapid salutatory conduction in central nervous system (CNS) neurons is well established.1Groves A.K. Barnett S.C. Franklin R.J. Crang A.J. Mayer M. Blakemore W.F. Noble M. Repair of demyelinated lesions by transplantation of purified O-2A progenitor cells.Nature. 1993; 362: 453-455Crossref PubMed Scopus (352) Google Scholar In addition, OLs are reported to provide trophic factors and metabolic support for neurons.2Oluich L.J. Stratton J.A. Xing Y.L. Ng S.W. Cate H.S. Sah P. Windels F. Kilpatrick T.J. Merson T.D. Targeted ablation of oligodendrocytes induces axonal pathology independent of overt demyelination.J Neurosci. 2012; 32: 8317-8330Crossref PubMed Scopus (91) Google Scholar, 3Hulshagen L. Krysko O. Bottelbergs A. Huyghe S. Klein R. Van Veldhoven P.P. De Deyn P.P. D'Hooge R. Hartmann D. Baes M. Absence of functional peroxisomes from mouse CNS causes dysmyelination and axon degeneration.J Neurosci. 2008; 28: 4015-4027Crossref PubMed Scopus (83) Google Scholar, 4Kassmann C.M. Lappe-Siefke C. Baes M. Brugger B. Mildner A. Werner H.B. Natt O. Michaelis T. Prinz M. Frahm J. Nave K.A. Axonal loss and neuroinflammation caused by peroxisome-deficient oligodendrocytes.Nat Genet. 2007; 39: 969-976Crossref PubMed Scopus (247) Google Scholar The CNS has the inherent capacity to generate myelin-forming OLs after episodes of demyelination under various pathologic conditions.5Dubois-Dalcq M. Armstrong R. The cellular and molecular events of central nervous system remyelination.Bioessays. 1990; 12: 569-576Crossref PubMed Scopus (50) Google Scholar Persistent demyelination is one of the pathologic hallmarks of multiple sclerosis (MS) and spinal cord (SC) trauma, which is responsible for neurologic deficits in affected individuals.6Blakemore W.F. Smith P.M. Franklin R.J. Remyelinating the demyelinated CNS.Novartis Found Symp. 2000; 231: 289-298Crossref PubMed Google Scholar, 7Murray P.D. McGavern D.B. Sathornsumetee S. Rodriguez M. Spontaneous remyelination following extensive demyelination is associated with improved neurological function in a viral model of multiple sclerosis.Brain. 2001; 124: 1403-1416Crossref PubMed Scopus (60) Google Scholar Therefore, a major question in neurobiology today is how to protect OLs in CNS demyelinating diseases, including MS, to limit neurologic deficits in affected individuals. Pro- and anti-inflammatory cytokines are known to play critical roles in the survival and generation of myelin-forming OLs that are lost after a demyelination attack.8Kornek B. Storch M.K. Weissert R. Wallstroem E. Stefferl A. Olsson T. Linington C. Schmidbauer M. Lassmann H. Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions.Am J Pathol. 2000; 157: 267-276Abstract Full Text Full Text PDF PubMed Scopus (769) Google Scholar, 9Irvine K.A. Blakemore W.F. Remyelination protects axons from demyelination-associated axon degeneration.Brain. 2008; 131: 1464-1477Crossref PubMed Scopus (286) Google Scholar OLs are required for the induction of remyelination to restore the structural integrity of the axon-myelin unit and axonal conduction properties in the MS brain.9Irvine K.A. Blakemore W.F. Remyelination protects axons from demyelination-associated axon degeneration.Brain. 2008; 131: 1464-1477Crossref PubMed Scopus (286) Google Scholar Proinflammatory cytokine signaling is reported to induce OL death and is antagonized by anti-inflammatory cytokine signaling mechanisms.10Lin W. Kemper A. Dupree J.L. Harding H.P. Ron D. Popko B. Interferon-gamma inhibits central nervous system remyelination through a process modulated by endoplasmic reticulum stress.Brain. 2006; 129: 1306-1318Crossref PubMed Scopus (165) Google Scholar, 11Balabanov R. Strand K. Kemper A. Lee J.Y. Popko B. Suppressor of cytokine signaling 1 expression protects oligodendrocytes from the deleterious effects of interferon-gamma.J Neurosci. 2006; 26: 5143-5152Crossref PubMed Scopus (53) Google Scholar, 12Zhang J. Zhang Y. Dutta D.J. Argaw A.T. Bonnamain V. Seto J. Braun D.A. Zameer A. Hayot F. Lopez C.B. Raine C.S. John G.R. Proapoptotic and antiapoptotic actions of Stat1 versus Stat3 underlie neuroprotective and immunoregulatory functions of IL-11.J Immunol. 2011; 187: 1129-1141Crossref PubMed Scopus (30) Google Scholar, 13Butzkueven H. Zhang J.G. Soilu-Hanninen M. Hochrein H. Chionh F. Shipham K.A. Emery B. Turnley A.M. Petratos S. Ernst M. Bartlett P.F. Kilpatrick T.J. LIF receptor signaling limits immune-mediated demyelination by enhancing oligodendrocyte survival.Nat Med. 2002; 8: 613-619Crossref PubMed Scopus (236) Google Scholar In MS brain lesions, spontaneous remyelination follows the demyelination attack in acute lesions.7Murray P.D. McGavern D.B. Sathornsumetee S. Rodriguez M. Spontaneous remyelination following extensive demyelination is associated with improved neurological function in a viral model of multiple sclerosis.Brain. 2001; 124: 1403-1416Crossref PubMed Scopus (60) Google Scholar In contrast, remyelination fails in chronic lesions, irrespective of the presence of OL progenitor cells (OPCs).14Wolswijk G. Oligodendrocyte survival, loss and birth in lesions of chronic-stage multiple sclerosis.Brain J Neurol. 2000; 123: 105-115Crossref PubMed Scopus (185) Google Scholar OPCs transplanted into demyelinated SC lesions, however, are reported to enhance remyelination in adult rats.1Groves A.K. Barnett S.C. Franklin R.J. Crang A.J. Mayer M. Blakemore W.F. Noble M. Repair of demyelinated lesions by transplantation of purified O-2A progenitor cells.Nature. 1993; 362: 453-455Crossref PubMed Scopus (352) Google Scholar Consistently, we earlier documented that lovastatin enhances the differentiation of OPCs into myelin-forming OLs that eventually enhance remyelination in experimental autoimmune encephalomyelitis (EAE), a murine model of MS.15Paintlia A.S. Paintlia M.K. Khan M. Vollmer T. Singh A.K. Singh I. HMG-CoA reductase inhibitor augments survival and differentiation of oligodendrocyte progenitors in animal model of multiple sclerosis.FASEB J. 2005; 19: 1407-1421Crossref PubMed Scopus (86) Google Scholar, 16Paintlia A.S. Paintlia M.K. Singh I. Skoff R.B. Singh A.K. Combination therapy of lovastatin and rolipram provides neuroprotection and promotes neurorepair in inflammatory demyelination model of multiple sclerosis.Glia. 2009; 57: 182-193Crossref PubMed Scopus (48) Google Scholar In light of this information, we assessed whether a pharmacologic agent that can influence OL survival holds promise to restore CNS integrity and functions in MS. Accumulating evidence suggests that AMP-activated protein kinase (AMPK)–mediated restoration of the CNS energy balance is critical to protect the brain under pathologic conditions.17Lopez M. Varela L. Vazquez M.J. Rodriguez-Cuenca S. Gonzalez C.R. Velagapudi V.R. Morgan D.A. Schoenmakers E. Agassandian K. Lage R. Martinez de Morentin P.B. Tovar S. Nogueiras R. Carling D. Lelliott C. Gallego R. Oresic M. Chatterjee K. Saha A.K. Rahmouni K. Dieguez C. Vidal-Puig A. Hypothalamic AMPK and fatty acid metabolism mediate thyroid regulation of energy balance.Nat Med. 2010; 16: 1001-1008Crossref PubMed Scopus (504) Google Scholar, 18Ronnett G.V. Ramamurthy S. Kleman A.M. Landree L.E. Aja S. AMPK in the brain: its roles in energy balance and neuroprotection.J Neurochem. 2009; 109: 17-23Crossref PubMed Scopus (219) Google Scholar We earlier documented that AMPK activators, ie, 5-amino-4-imidazole carboxamide riboside (AICAR) and metformin, attenuate clinical impairments in EAE animals.19Nath N. Khan M. Paintlia M.K. Singh I. Hoda M.N. Giri S. Metformin attenuated the autoimmune disease of the central nervous system in animal models of multiple sclerosis.J Immunol. 2009; 182: 8005-8014Crossref PubMed Scopus (253) Google Scholar, 20Nath N. Giri S. Prasad R. Salem M.L. Singh A.K. Singh I. 5-Aminoimidazole-4-carboxamide ribonucleoside: a novel immunomodulator with therapeutic efficacy in experimental autoimmune encephalomyelitis.J Immunol. 2005; 175: 566-574PubMed Google Scholar The protective effects of these AMPK activators were ascribed to their immunomodulatory activities and the restoration of blood-brain barrier integrity in EAE animals.19Nath N. Khan M. Paintlia M.K. Singh I. Hoda M.N. Giri S. Metformin attenuated the autoimmune disease of the central nervous system in animal models of multiple sclerosis.J Immunol. 2009; 182: 8005-8014Crossref PubMed Scopus (253) Google Scholar, 20Nath N. Giri S. Prasad R. Salem M.L. Singh A.K. Singh I. 5-Aminoimidazole-4-carboxamide ribonucleoside: a novel immunomodulator with therapeutic efficacy in experimental autoimmune encephalomyelitis.J Immunol. 2005; 175: 566-574PubMed Google Scholar, 21Prasad R. Giri S. Nath N. Singh I. Singh A.K. 5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside attenuates experimental autoimmune encephalomyelitis via modulation of endothelial-monocyte interaction.J Neurosci Res. 2006; 84: 614-625Crossref PubMed Scopus (54) Google Scholar AMPK activity was reduced in the brain at the onset and peak of EAE disease.22Meares G.P. Qin H. Liu Y. Holdbrooks A.T. Benveniste E.N. AMP-activated protein kinase restricts IFN-gamma signaling.J Immunol. 2013; 190: 372-380Crossref PubMed Scopus (64) Google Scholar Consistently, we earlier documented that the genetic ablation of AMPK causes severe clinical impairments in EAE animals.23Nath N. Khan M. Rattan R. Mangalam A. Makkar R.S. de Meester C. Bertrand L. Singh I. Chen Y. Viollet B. Giri S. Loss of AMPK exacerbates experimental autoimmune encephalomyelitis disease severity.Biochem Biophys Res Commun. 2009; 386: 16-20Crossref PubMed Scopus (55) Google Scholar In addition, we documented that AICAR inhibits the expressions of inflammatory mediators in astrocytes, microglia, and peritoneal macrophages that are known to play critical roles in EAE/MS pathogenesis.24Giri S. Nath N. Smith B. Viollet B. Singh A.K. Singh I. 5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside inhibits proinflammatory response in glial cells: a possible role of AMP-activated protein kinase.J Neurosci. 2004; 24: 479-487Crossref PubMed Scopus (237) Google Scholar Recently, AMPK signaling was reported to interfere with interferon γ (IFN-γ)–mediated activation of astrocytes and microglia22Meares G.P. Qin H. Liu Y. Holdbrooks A.T. Benveniste E.N. AMP-activated protein kinase restricts IFN-gamma signaling.J Immunol. 2013; 190: 372-380Crossref PubMed Scopus (64) Google Scholar as well as induction of the inflammatory response in liver cells.25Myerburg M.M. King Jr., J.D. Oyster N.M. Fitch A.C. Magill A. Baty C.J. Watkins S.C. Kolls J.K. Pilewski J.M. Hallows K.R. AMPK agonists ameliorate sodium and fluid transport and inflammation in cystic fibrosis airway epithelial cells.Am J Respir Cell Mol Biol. 2010; 42: 676-684Crossref PubMed Scopus (86) Google Scholar, 26Buler M. Aatsinki S.M. Skoumal R. Komka Z. Toth M. Kerkela R. Georgiadi A. Kersten S. Hakkola J. Energy-sensing factors coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1alpha) and AMP-activated protein kinase control expression of inflammatory mediators in liver: induction of interleukin 1 receptor antagonist.J Biol Chem. 2012; 287: 1847-1860Crossref PubMed Scopus (42) Google Scholar These studies provide evidence that CNS bioenergetics and inflammation are linked, thus understanding of their regulatory mechanisms has the scope to identify new therapeutic interventions for MS. Recently, AMPK signaling was reported to protect neurons under pathologic conditions.27Wang P. Xu T.Y. Guan Y.F. Tian W.W. Viollet B. Rui Y.C. Zhai Q.W. Su D.F. Miao C.Y. Nicotinamide phosphoribosyltransferase protects against ischemic stroke through SIRT1-dependent adenosine monophosphate-activated kinase pathway.Ann Neurol. 2011; 69: 360-374Crossref PubMed Scopus (252) Google Scholar, 28Fu J. Jin J. Cichewicz R.H. Hageman S.A. Ellis T.K. Xiang L. Peng Q. Jiang M. Arbez N. Hotaling K. Ross C.A. Duan W. Trans-(-)-epsilon-Viniferin increases mitochondrial sirtuin 3 (SIRT3), activates AMP-activated protein kinase (AMPK), and protects cells in models of Huntington Disease.J Biol Chem. 2012; 287: 24460-24472Crossref PubMed Scopus (152) Google Scholar In addition, we earlier documented that AMPK activity was reduced in OLs exposed to psychosine, a galactosylsphingosine lipid that accumulates in the Krabbe brain.29Giri S. Khan M. Nath N. Singh I. Singh A.K. The role of AMPK in psychosine mediated effects on oligodendrocytes and astrocytes: implication for Krabbe disease.J Neurochem. 2008; 105: 1820-1833Crossref PubMed Scopus (61) Google Scholar Based on this knowledge, we investigated whether AMPK signaling can protect OLs in the CNS of EAE animals. By using in vitro and in vivo approaches, we demonstrated that AMPK signaling influences OL survival and, thereby, restores CNS integrity and functions in EAE animals treated with metformin (first-line drug of choice for type 2 diabetes mellitus). Unless otherwise stated, all the chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco's modified Eagle's medium (containing 4.5 g/L of glucose), fetal bovine serum, poly-d-lysine, rat IgG, and rabbit polyclonal IgG (control primary antibodies) and secondary antibodies, ie, Alexa Fluor conjugated with anti-rabbit IgG or anti-mouse IgG, were purchased from Life Technologies (Grand Island, NY). Recombinant platelet-derived growth factor (PDGF)-aa, basic fibroblast growth factor-2, ciliary neurotrophic factor (CNTF), tumor necrosis factor α (TNF-α) and IL-1β proteins were purchased from R&D Systems (Minneapolis, MN). Anti–myelin basic protein (MBP) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). Anti-A2B5 MicroBeads and MS columns were purchased from MACS, Miltenyi Biotec Inc. (Auburn, CA). Anti-O1, anti-A2B5, anti–TNF-α, anti–nitric oxide synthase-II (NOS-II), anti–heme oxygenase-1, anti–manganese superoxide dismutase 2 (SOD2), anti–phospho-AMPKα, and anti-AMPKα1 antibodies were purchased from Millipore (Billerica, MA). Rabbit anti-malondialdehyde (MDA) polyclonal antibodies were purchased from Cell Biolabs (San Diego, CA). Electrochemiluminescence-detecting reagents and nitrocellulose membranes were purchased from GE Healthcare Biosciences (Pittsburg, PA). Adult female Lewis rats weighing 250 to 300 g or pregnant Sprague Dawley rats at embryonic gestation day 18 were purchased from Charles River Laboratories (Wilmington, MA) and were housed in the animal care facility at the Medical University of South Carolina (Charleston, SC) throughout the experiment and were provided with food and water ad libitum. All the animal experiments were conducted in accordance with accepted standards of humane care, as outlined in the ethical guidelines and approved by the Medical University of South Carolina's Animal Ethics Committee. The procedures used for induction of EAE are as described in previous publications with slight modifications.15Paintlia A.S. Paintlia M.K. Khan M. Vollmer T. Singh A.K. Singh I. HMG-CoA reductase inhibitor augments survival and differentiation of oligodendrocyte progenitors in animal model of multiple sclerosis.FASEB J. 2005; 19: 1407-1421Crossref PubMed Scopus (86) Google Scholar, 30Paintlia A.S. Paintlia M.K. Singh A.K. Stanislaus R. Gilg A.G. Barbosa E. Singh I. Regulation of gene expression associated with acute experimental autoimmune encephalomyelitis by Lovastatin.J Neurosci Res. 2004; 77: 63-81Crossref PubMed Scopus (69) Google Scholar In brief, the rats received an s.c. injection of 25 μg of guinea pig MBP in 0.1 mL of PBS emulsified with an equal volume of complete Freund's adjuvent supplemented with 2 mg/mL of mycobacterium tuberculosis H37Ra (Difco, Detroit, MI) in the hind limb foot pads on days 0 and 7. Immediately and again 24 hours later, the rats received 200 ng of pertussis toxin i.p. in 0.1 mL of PBS. Pertussis toxin was used as per the standardized protocol reported by us and other investigators for the induction of EAE. Similarly, healthy control group rats received an s.c. injection of PBS and complete Freund's adjuvant emulsion in the hind limb foot pads on days 0 and 7. The rats were examined for clinical scores by an experimentally blinded investigator (A.K.S.) daily. Clinical scores were assessed using a scale from 0 to 5: 0, no clinical disease; 1.0, piloerection; 2.0, loss in tail tonicity; 3.0, hind leg paralysis; 4.0, paraplegia, and 5.0, moribund or dead. At several times during the study, rats were weighed. The clinical data from rats with a clinical score >4.0 were not included in the statistical analysis. At the conclusion of the study, the rats were euthanized, perfusion was performed, and the lumbar SC was removed, snap frozen with liquid nitrogen, and stored at −70°C until use. Alternatively, the SC of each rat was cut into four small pieces and fixed in 4% paraformaldehyde for histopathologic analysis. Metformin, 150 mg/mL, was suspended in PBS and administered by gavages every day in a 200-μL volume. Treatment was started in rats with established EAE (day 11 or 12 after immunization) and continued until the lessening of paralytic symptoms (day 22 after immunization; recovery). Control EAE rats without drug treatment received PBS once daily. Likewise, healthy control rats received vehicle or metformin once daily. Mixed glial cell cultures were generated from the brains of P1–P2 pups obtained from Sprague Dawley mothers as described earlier.31Paintlia A.S. Paintlia M.K. Singh A.K. Orak J.K. Singh I. Activation of PPAR-gamma and PTEN cascade participates in lovastatin-mediated accelerated differentiation of oligodendrocyte progenitor cells.Glia. 2010; 58: 1669-1685Crossref PubMed Scopus (37) Google Scholar Briefly, dissociated cortices in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 4 mmol/L l-glutamine were cultured on flasks coated with 50 μg/mL of poly-d-lysine before use. After 10 days, the cells were used directly for studies or the flasks were shaken (250 rpm) for 2 hours to remove microglia. The flasks were shaken for another 8 hours, and OPCs were dislodged from the astrocyte layer. Pure OPC cultures >99.9% were generated by using anti-A2B5 MicroBeads and passing through MS columns (MACS, Miltenyi Biotec). Purified OPCs (2000 cells/cm2) were plated on poly-d-lysine–coated culture dishes or glass coverslips in serum-free modified Bottenstein-Sato–based medium as described earlier.32Paintlia M.K. Paintlia A.S. Singh A.K. Singh I. S-Nitrosoglutathione induces ciliary neurotrophic factor expression in astrocytes, which has implications to protect the central nervous system under pathological conditions.J Biol Chem. 2013; 288: 3831-3843Crossref PubMed Scopus (25) Google Scholar After 24 hours, Bottenstein-Sato–based medium supplemented with 10 ng/mL of CNTF was replaced, which showed the transformation of OPCs into OLs (O1+) at 48 hours of incubation. These OLs were grown in 24-well cell culture plates or 100 cm2 culture dishes and were exposed to 10 ng/mL of TNF-α and 20 ng/mL of IL-1β, simultaneously, in the presence or absence of metformin. OPC-enriched (20%) mixed glial cultures were plated in 6-well cell culture plates (2000 cells/cm2) in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum and 25 μg/mL of gentamicin (Nunc, Roskilde, Denmark) and were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cell cultures with 70% to 80% confluence were used to treat with 100 ng/mL of lipopolysaccharide (LPS) and 50 ng/mL of IFN-γ in the presence or absence of metformin or pharmacologic agents. Concentrations of the pharmacologic agents used in the study showed no cell toxicity as revealed by trypan blue exclusion and lactate dehydrogenase release assays. The reactive oxygen species (ROS) was determined using a microplate reader. Cells were plated in 24-well culture plates and were treated with cytokines in the presence or absence of metformin (Cultures and Treatment of OLs). For positive control, before performing the assay, cells were treated with 0.3% H2O2 suspended in culture medium and incubated for 30 minutes at 37°C. After 24 hours of incubation, the cells were washed with PBS (3×) and were incubated with 5 μmol/L cell-permeable fluorescent dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-DCFDA; Life Technologies) in serum-free medium as described previously.33Paintlia M.K. Paintlia A.S. Singh A.K. Singh I. Synergistic activity of interleukin-17 and tumor necrosis factor-alpha enhances oxidative stress-mediated oligodendrocyte apoptosis.J Neurochem. 2011; 116: 508-521Crossref PubMed Scopus (69) Google Scholar After incubating for 30 minutes, the cells were washed with PBS and lyzed in cell lysis buffer (0.1 N of NaOH in 50% methanol). From each well, 200 μL of solution was transferred to a 96-well black microplate. Changes in fluorescence were determined at excitation/emission = 485/530 nm using a SoftMax Pro spectrofluorometer (Molecular Devices, Sunnyvale, CA). Changes in mitochondrial membrane potential were measured in cells using JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolylcarbocyanine iodide) dye. Cells were incubated with 1 μmol/L JC-1 dye for 15 minutes at 37°C. Then cells were rinsed in PBS (2×) and examined by fluorescence microscopy (Olympus BX60) and photographed using the attached Olympus digital camera (Optronics, Goleta, CA). Oxidative stress usually causes the disruption of mitochondrial membrane potential. JC-1 dye indicates mitochondria depolarization by a change from red to green fluorescence. For single labeling, a standard method was used. Briefly, slides were blocked by using an Image-iT fixation and permeabilization kit (Life Technologies) and were incubated with appropriately diluted primary antibody (dilution 1:100) at 4°C overnight followed by washing and further incubation with secondary antibodies, Alexa Fluor 488–conjugated goat anti-rabbit IgG antibodies (dilution 1:500) for 1 hour. For double labeling, slides were incubated simultaneously or separately with both types of primary antibodies after blocking with a serum-PBS solution at 4°C overnight as described for single labeling. Secondary antibodies Alexa Fluor 488–conjugated goat anti-rabbit IgG (CNTF) or Alexa Fluor 594–conjugated rabbit anti-mouse IgG [glial fibrillary acidic protein (GFAP)] antibodies were used. Slides were also incubated with Alexa Fluor–conjugated IgG antibodies without primary antibody as negative controls and an appropriate mouse IgG or rabbit polyclonal IgG as isotype controls. After thorough washings, slides were mounted using Fluoromount-G (Electron Microscopy Sciences, Hatfield, PA) containing Hoechst. Slides were examined using a fluorescence microscope (Olympus BX60), and images were captured at magnifications ×400 using an Olympus digital camera (Optronics) using a dual-bandpass filter. The contrast and brightness of images were processed using Adobe Photoshop CS5 software (Adobe Systems Inc., San Jose, CA). Fixed SC sections of 5 μm were stained with Luxol fast blue and H&E (Sigma-Aldrich) and were examined using light microscopy (Olympus BX60). Images were acquired using an Olympus digital camera (Optronics). The region of SC depicting >10 nuclei was considered as lesion. The meanings, gray matter, and white matter of the sections were scored in a blinded manner based on the presence or absence of infiltrating cells in each region of the SC. Histopathology scores were recorded as the number of lesions in the SC of each group of rats that showed a readily identifiable inflammatory cell infiltrate. Similarly, demyelination in the white matter of the SC was examined and scored. The demyelination degree was scored as grade 0, no disease; grade 1, foci of demyelination/axonal loss that is superficial and involves <25% of the lateral columns; grade 2, deep foci that involve >25% of the lateral columns; and grade 3, diffuse and widespread demyelination and axonal loss. Histologic data are from five rats per group, where three to four sections per rat were examined for the presence of lesions. Cells or tissues were carefully processed for RNA isolation using TRIzol reagent (Invitrogen, Carlsbad, CA) followed by cDNA synthesis and real-time PCR analysis using a Bio-Rad CFX96 system (Bio-Rad Laboratories, Hercules, CA). The primer sets used in the study (Table 1) were designed using PrimerQuest and were synthesized by Integrated DNA Technologies (Coralville, IA). iQ SYBR Green supermix was purchased from Bio-Rad Laboratories. Thermal cycling conditions were as follows: activation of iTaq DNA polymerase (Bio-Rad Laboratories) at 95°C for 10 minutes followed by 40 cycles of amplification at 95°C for 30 seconds and 59°C to 60°C for 30 seconds. The specificity and detection methods for data analysis are as described earlier.31Paintlia A.S. Paintlia M.K. Singh A.K. Orak J.K. Singh I. Activation of PPAR-gamma and PTEN cascade participates in lovastatin-mediated accelerated differentiation of oligodendrocyte progenitor cells.Glia. 2010; 58: 1669-1685Crossref PubMed Scopus (37) Google Scholar In brief, real-time PCR specificity for each analysis was determined by melting curve analysis of the amplified product. The level of target gene transcripts was calculated relative to the expression of reference gene, β-actin, in each sample. The detection of threshold was set above the mean baseline fluorescence determined by the first 20 cycles. A standard curve for each template was generated with a serial dilution of the template (cDNA). Likewise, cytokine and chemokine expressions were measured by using cytokine and chemokine PCR arrays (PARN-150ZD-2; Qiagen Inc., Valencia, CA) with cDNA of each sample, using real-time PCR at conditions described in the product manual. Data were analyzed using website-based RT2 Profiler PCR data analysis software version 3.5 (SABiosciences, a QIAGEN company, Valencia, CA).Table 1Primers Used for Quantitative Real-Time PCR AnalysisGene namePrimer sequenceβ-actinForward: 5′-AGCTGTGCTATGTTGCCCTAGACT-3′Reverse: 5′-ACCGCTCATTGCCGATAGTGATGA-3′TNF-αForward: 5′-CTTCTGTCTACTGAACTTCGGGGT-3′Reverse: 5′-TGGAACTGATGAGAGGGAGCC-3′NOS-IIForward: 5′-GGAAGAGGAACAACTACTGCTGGT-3′Reverse: 5′-GAACTGAGGGTACATGCTGGAGC-3′IFN-γForward: 5′-TTTGAGGTCAACAACCCACAGGTC-3′Reverse: 5′-TTTCCGCTTCCTGAGGCTGGATT-3′IL-17AForward: 5′-TCTGAGCCAGCCAAGAAGAAGTGT-3′Reverse: 5′-TTCCAACCCAAACATAGGCAC-3′ROR" @default.
• W2018161712 created "2016-06-24" @default.
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• W2018161712 date "2013-08-01" @default.
• W2018161712 modified "2023-10-17" @default.
• W2018161712 title "AMP-Activated Protein Kinase Signaling Protects Oligodendrocytes that Restore Central Nervous System Functions in an Experimental Autoimmune Encephalomyelitis Model" @default.
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