Sildenafil induces cGMP accumulation through phosphodiesterase-5 (PDE5) inhibition. cGMP-pathways protect oligodendrocytes and modulate astroglial and microglial reactions. Microglia and astrocytes play an important role in perpetuating multiple sclerosis (MS), a chronic inflammatory disease characterized by demyelination. Therefore, sildenafil can be a potential tool for MS treatment. The present study investigated the effects of sildenafil on the myelin structure and astrocyte/microglia-mediated neuroinflammation in an animal model of MS. Cuprizone-induced demyelination and neuroinflammation in rodents has been widely used as a model for MS. Herein, five male C57BL/6 mice (7-10. weeks old) were used per group. Over a 4-week period, the different groups received the following: (1) cuprizone (0.2%) mixed into the chow; (2) cuprizone in the chow and sildenafil (Viagra®; 3, 25 or 50. mg/kg) in the drinking water; or (3) pure chow and water (control group). Cerebella were analyzed using transmission electron microscopy, western blotting, immunohistochemistry and luxol fast blue staining. Cuprizone induced tissue damage, with an increase in GFAP, Iba-1 and COX-2 and demyelination in comparison to the control group. However, cuprizone did not affect the expression of cytokines (TNF-α, IFN-γ, IL-1β and IL-2). Sildenafil reduced GFAP (25 and 50. mg/kg) and Iba-1 expression (25. mg/kg) in comparison to the cuprizone group, indicating the modulation of astrocytes and microglia, respectively. Sildenafil preserved myelin and axons ultrastructure and strongly reduced IFN-γ, TNF-α, IL-1β, IL-2 and COX-2 expression in comparison to the control and/or cuprizone groups. The results demonstrate a protective effect of sildenafil in the cerebellum. Thus, well-designed clinical trials may demonstrate that the oral administration of sildenafil can be suitable for individuals with MS and other neuroinflammatory/neurodegenerative diseases, providing additional benefits to current treatments. © 2012 Elsevier Ltd.602540551Block, M.L., Zecca, L., Hong, J.S., Microglia-mediated neurotoxicity uncovering the molecular mechanisms (2007) Nat Rev Neurosci, 8, pp. 57-69McGeer, E.G., McGeer, P.L., The role of anti-inflammatory agents in Parkinson's disease (2007) CNS Drugs, 21, pp. 789-797Trapp, B.D., Nave, K.A., Multiple sclerosis: an immune or neurodegenerative disorder? (2008) Annu Rev Neurosci, 31, pp. 247-269Moharregh-Khiabani, D., Blank, A., Skripuletz, T., Miller, E., Kotsiari, A., Gudi, V., Effects of fumaric acids on cuprizone induced central nervous system de- and remyelination in the mouse (2010) PLoS One, 5, pp. 117-169Noseworthy, J.H., Lucchinetti, C., Rodriguez, M., Weinshenker, B.G., Multiple sclerosis (2000) New Engl J Med, 343, pp. 938-952Trapp, B.D., Peterson, J., Ransohoff, R.M., Rudick, R., Mörk, S., Bö, L., Axonal transaction in the lesions of multiple sclerosis (1998) New Engl J Med, 338, pp. 278-285Gao, Z., Tsirka, S.E., Animal Models of MS reveal multiple roles of microglia in disease pathogenesis (2011) Neurol Res Int, 2011, p. 383087Kipp, M., Norkus, A., Krauspe, B., Clarner, T., Berger, K., van der Valk, P., The hippocampal fimbria of cuprizone-treated animals as a structure for studying neuroprotection in multiple sclerosis (2011) Inflamm Res, 60, pp. 723-726Frank-Cannon, T.C., Alto, L.T., McAlpine, F.E., Tansey, M.G., Does neuroinflammation fan the flame in neurodegenerative diseases? (2009) Mol Neurodegener, 4, p. 47Mrak, R.E., Griffin, W.S., Glia and their cytokines in progression of neurodegeneration (2005) Neurobiol Aging, 26, pp. 349-354Johnson, K.P., Brooks, B.R., Ford, C.C., Goodman, A., Guarnaccia, J., Lisak, R.P., Sustained clinical benefits of glatiramer acetate in relapsing multiple sclerosis patients observed for 6years. Copolymer 1 Multiple Sclerosis Study Group (2000) Mult Scler, 6, pp. 255-266Reder, A.T., Ebers, G.C., Traboulsee, A., Li, D., Langdon, D., Goodin, D.S., Cross-sectional study assessing long-term safety of interferon-beta-1b for relapsing-remitting MS (2010) Neurology, 74, pp. 1877-1885Yoshioka, A., Yamaya, Y., Saiki, S., Kanemoto, M., Hirose, G., Pleasure, D., Cyclic GMP/cyclic GMP-dependent protein kinase system prevents excitotoxicity in an immortalized oligodendroglial cell line (2000) J Neurochem, 74, pp. 633-640Huizinga, R., van der Star, B.J., Kipp, M., Jong, R., Gerritsen, W., Clarner, T., Phagocytosis of neuronal debris by microglia is associated with neuronal damage in multiple sclerosis (2012) Glia, 60, pp. 422-431Meda, L., Cassatella, M.A., Szendrei, G.I., Otvos, L., Baron, P., Villalba, M., Activation of microglial cells by ß-amyloid protein and interferon-gamma (1995) Nature, 374, pp. 647-650Pifarré, P., Prado, J., Baltrons, M.A., Giralt, M., Gabarro, P., Feinstein, D.L., Sildenafil (Viagra) ameliorates clinical symptoms and neuropathology in a mouse model of multiple sclerosis (2011) Acta Neuropathol, 121, pp. 499-508Rosenberg, G.A., Neurological diseases in relation to the blood-brain barrier (2012) J Cereb Blood Flow MetabPuzzo, D., Sapienza, S., Arancio, O., Palmeri, A., Role of phosphodiesterase 5 in synaptic plasticity and memory (2008) Neuropsychiatr Dis Treat, 4, pp. 371-387Prickaerts, J., van Staveren, W.C., Sik, A., Markerink-van Ittersum, M., Niewöhner, U., van der Staay, F.J., Effects of two selective phosphodiesterase type 5 inhibitors, sildenafil and vardenafil, on object recognition memory and hippocampal cyclic GMP levels in the rat (2002) Neuroscience, 113, pp. 351-361Boccia, M.M., CM. Effects of oxytocin and an oxytocin receptor antagonist on retention of a nose-poke habituation response in mice (1999) Acta Physiol Pharmacol Ther Latinoam, 49, pp. 155-160Wakita, H., Tomimoto, H., Akiguchi, I., Lin, J.X., Ihara, M., Ohtani, R., Ibudilast protect against white matter damage under chronic cerebral hypoperfusion in the rat (2003) Brain Res, 992, pp. 53-59Manson, S.C., Burke, G., Voets, N., Palace, J., Matthews, P.M., Effect of sildenafil citrate on CBF and BOLD activation in multiple sclerosis patients (2005), 21st congress of the European Committee for the Treatment and Research in Multiple SclerosisPoster No. 237 Thessaloniki, GreeceZhang, L., Yu, W., Schroedter, I., Kong, J., Vrontakis, M., Galanin transgenic mice with elevated circulating galanin levels alleviate demyelination in a cuprizone-induced MS mouse model (2012) PLoS One, 7, pp. e33901Silvestroff, L., Bartucci, S., Pasquini, J., Franco, P., Cuprizone-induced demyelination in the rat cerebral cortex and thyroid hormone effects on cortical remyelination (2012) Exp NeurolZhao, L., Mason, N.A., Strange, J.W., Walker, H., Wilkins, M.R., Beneficial effects of phosphodiesterase 5 inhibition in pulmonary hypertension are influenced by natriuretic peptide activity (2003) Circulation, 107, pp. 234-237Saraiva, K.L., Silva, A.K., Wanderley, M.I., De Araújo, A.A., De Souza, J.R., Peixoto, C.A., Chronic treatment with sildenafil stimulates Leydig cell and testosterone secretion (2009) Int J Exp Pathol, 90, pp. 454-462Saraiva, K.L., Silva, V.A., Dias, E.S., Peixoto, C.A., Morphological changes in the testis induced by diethylcarbamazine (2006) Reprod Toxicol, 22, pp. 754-759Riccitelli, G., Rocca, M.A., Pagani, E., Martinelli, V., Radaelli, M., Falini, A., Mapping regional grey and white matter atrophy in relapsing-remitting multiple sclerosis (2012) Mult SclerShields, S.D., Cheng, X., Gasser, A., Saab, C.Y., Tyrrell, L., Eastman, E.M., A channelopathy contributes to cerebellar dysfunction in a model of multiple sclerosis (2012) Ann Neurol, 71, pp. 186-194Skripuletz, T., Bussmann, J.H., Gudi, V., Koutsoudaki, P.N., Pul, R., Moharregh-Khiabani, D., Cerebellar cortical demyelination in the murine cuprizone model (2010) Brain Pathol, 20, pp. 301-312Zhang, R.L., Zhang, Z., Zhang, L., Wang, Y., Zhang, C., Chopp, M., Delayed treatment with Sildenafil enhances neurogenesis and improves functional recovery in aged rats after focal cerebral ischemia (2006) J Neurosci Res, 83, pp. 1213-1219Zhang, Y.Q., Rodesch, C.K., Broadie, K., Living synaptic vesicle marker: synaptotagmin-GFP (2002) Genesis, 34, pp. 142-145Kulkarni, S.K., Patil, C.S., Phosphodiesterase 5 enzyme and its inhibitors: update on pharmacological and therapeutical aspects (2004) Methods Find Exp Clin Pharmacol, 26, pp. 789-799Shimizu-Albergine, M., Rybalkin, S.D., Rybalkina, I.G., Feil, R., Wolfsgruber, W., Hofmann, F., Individual cerebellar Purkinje cells express different cGMP phosphodiesterases (PDEs): in vivo phosphorylation of cGMP-specific PDE (PDE5) as an indicator of cGMP-dependent protein kinase (PKG) activation (2003) J Neurosci, 23, pp. 6452-6459Rosengren, L.E., Lycke, J., Andersen, O., Glial fibrillary acidic protein in CSF of multiple sclerosis patients: relation to neurological deficit (1995) J Neurol Sci, 133, pp. 61-65Petzold, A., Eikelenboom, M.J., Gyeric, D., Markers for differential glial cell responses in multiple sclerosis: clinical and pathological correlations (2002) Brain, 125, pp. 1462-1473Tani, M., Glabinski, A.R., Tuohy, V.K., Stoler, M.H., Estes, M.L., Ransohoff, R.M., In situ hybridization analysis of glial fibrillary acidic protein mRNA reveals evidence of biphasic astrocyte activation during acute experimental autoimmune encephalomyelitis (1996) Am J Pathol, 148, pp. 889-896Voskuhi, R.R., Peterson, R.S., Song, B., Ao, Y., Morales, L.B., Tiwari-Woodruff, S., Reactive astrocytes form scar-like perivascular barriers to leukocutes during adaptative immune inflammation of the CNS (2009) J Neurosci, 29, pp. 11511-11522Glabinski, A.R., Tani, M., Strieter, R.M., Tuohy, V.K., Ransohoff, R.M., Synchronous synthesis of alpha- and beta-chemokines by cells of diverse lineage in the central nervous system of mice with relapses of chronic experimental autoimmune encephalomyelitis (1997) Am J Pathol, 150, pp. 617-630Ambrosini, E., Remoli, M.E., Giacomini, E., Rosicarelli, B., Serafini, B., Lande, R., Astrocytes produce dendritic cell-attracting chemokines in vitro and in multiple sclerosis lesions (2005) J Neuropathol Exp Neurol, 64, pp. 706-715Hardin-Pouzet, H., Krakowski, M., Bourbonnière, L., Didier-Bazes, M., Tran, E., Owens, T., Glutamate metabolism is down-regulated in astrocytes during experimental allergic encephalomyelitis (1997) Glia, 20, pp. 79-85Korn, T., Magnus, T., Jung, S., Autoantigen specific T cells inhibit glutamate uptake in astrocytes by decreasing expression of astrocytic glutamate transporter GlAST: a mechanism mediated by tumor necrosis factor-alpha (2005) FASEB J, 19, pp. 1878-1880Pitt, D., Werner, P., Raine, C.S., Glutamate excitotoxicity in a model of multiple sclerosis (2000) Nat Med, 6, pp. 67-70Micu, J., Jiang, Q., Coderre, E., Ridsdale, A., Zhang, L., Woulfe, J., NMDA receptors mediate calcium accumulation in myelin during chemical ischaemia (2006) Nature, 439, pp. 988-992Back, S.A., Tuohy, T.M., Chen, H., Wallingford, N., Craig, A., Struve, J., Hyaluronan accumulates in demyelinated lesions and inhibits oligodendrocyte progenitor maturation (2005) Nat Med, 11 (9), pp. 966-972. , Epub 2005 August 7Toft-Hansen, H., Füchtbauer, L., Owens, T., Inhibition of reactive astrocytosis in established experimental autoimmune encephalomyelitis favors infiltration by myeloid cells over T cells and enhances severity of disease (2011) Glia, 59, pp. 166-176Bannerman, P., Hahn, A., Soulika, A., Gallo, V., Pleasure, D., Astrogliosis in EAE spinal cord: derivation from radial glia, and relationships to oligodendroglia (2007) Glia, 55, pp. 57-64Pifarré, P., Prado, J., Giralt, M., Molinero, A., Hidalgo, J., Garcia, A., Cyclic GMP phosphodiesterase inhibition alters the glial inflammatory response, reduces oxidative stress and cell death and increases angiogenesis following focal brain injury (2010) J Neurochem, 112, pp. 807-817Rott, O., Fleischer, B., Cash, E., Interleukin-10 prevents experimental allergic encephalomyelitis in rats (1994) Eur J Immunol, 24, pp. 1434-1440Stevens, D.B., Gould, K.E., Swanborg, R.H., Transforming growth factor-beta 1 inhibits tumor necrosis factor-alpha/lymphotoxin production and adoptive transfer of disease by effector cells of autoimmune encephalomyelitis (1994) J Neuroimmunol, 51, pp. 77-83Kreutzberg, G.W., Microglia: a sensor for pathological events in the CNS (1996) Trends Neurosci, 19, pp. 312-318Khoshakhlagh, P., Bahrololoumi-Shapourabadi, M., Mohammadirad, A., Ashtaral-Nakhai, L., Minaie, B., Abdollahi, M., Beneficial effect of phosphodiesterase-5 inhibitor in experimental inflammatory bowel diseasemolecular evidence for involvement of oxidative stress (2007) Toxicol Mech Methods, 17 (5), pp. 281-288Suzumura, A., Ito, A., Yoshikawa, M., Sawada, M., Ibudilast suppressesTNF alpha production by glial cells functioning mainly as type III phosphodiesterase inhibitor in the CNS (1999) Brain Res, 837, pp. 203-212da Cruz-Höfling, M.A., Rapôso, C., Verinaud, L., Zago, G.M., Neuroinflammation and astrocytic reaction in the course of Phoneutria nigriventer (armed-spider) blood-brain barrier (BBB) opening (2009) Neurotoxicology, 30, pp. 636-646Palumbo, M.L., Trinchero, M.F., Zorrilla-Zubilete, M.A., Schinder, A.F., Genaro, A.M., Glatiramer acetate reverts stress-induced alterations on adult neurogenesis and behavior. Involvement of Th1/Th2 balance (2011) Behav Brain Res, 218, pp. 121-128Zhao, S., Zhang, L., Lian, G., Wang, X., Zhang, H., Yao, X., Sildenafil attenuates LPS-induced pro-inflammatory responses through down-regulation of intracellular ROS-related MAPK/NF-κB signaling pathways in N9 microglia (2011) Int Immunopharmacol, 11, pp. 468-474Wang, L., Chopp, M., Szalad, A., Liu, Z., Bolz, M., Alvarez, F.M., Phosphodiesterase-5 is a therapeutic target for peripheral neuropathy in diabetic mice (2011) Neuroscience, 193, pp. 399-410Benjamins, J.A., Nedelkoska, L., Cyclic GMP-dependent pathways protect differentiated oligodendrocytes from multiple types of injury (2007) Neurochem Res, 32, pp. 321-329Wang, X., Robinson, P.J., Cyclic GMP-dependent protein kinase and cellular signaling in the nervous system (1997) J Neurochem, 68, pp. 443-456Abramovitz, M., Adam, M., Boie, Y., Theutilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs (2000) Biochim Biophys Acta, 1483, pp. 285-293Bilak, M., Wu, L., Wang, Q., Haughey, N., Conant, K., St Hillaire, C., PGE2 receptors rescue motor neurons in a model of amyotrophic lateral sclerosis (2004) Ann Neurol, 56, pp. 240-248Carlson, N.G., Rojas, M.A., Black, J.D., Redd, J.W., Hille, J., Hill, K.E., Microglial inhibition of neuroprotection by antagonists of the EP1 prostaglandin E2 receptor (2009) J Neuroinflammation, 6, p. 5Farooqui, A.A., Horrocks, L.A., Farooqui, T., Modulation of inflammation in brain: a matter of fat (2007) J Neurochem, 101, pp. 577-599Dore-Duffy, P., Donaldson, J.O., Koff, T., Longo, M., Perry, W., Prostaglandin release in multiple sclerosis: correlation with disease activity (1986) Neurology, 36, pp. 1587-1590Neu, I.S., Metzger, G., Zschocke, J., Zelezny, R., Mayatepek, E., Leukotrienes in patients with clinically active multiple sclerosis (2002) Acta Neurol Scand, 105, pp. 63-66Dore-Duffy, P., Ho, S.Y., Donovan, C., Cerebrospinal fluid eicosanoid levels: endogenous PGD2 and LTC4 synthesis by antigen-presenting cells that migrate to the central nervous system (1991) Neurology, 41, pp. 322-324Muthian, G., Raikwar, H.P., Johnson, C., Rajasingh, J., Kalgutkar, A., Marnett, L.J., COX-2 inhibitors modulate IL-12 signaling through JAK-STAT pathway leading to Th1 response in experimental allergic encephalomyelitis (2006) J Clin Immunol, 26, pp. 73-85