Role Of Inos-no-cgmp Signaling In Modulation Of Inflammatory And Myelination Processes

Nitric oxide (NO) is the main activator of the soluble guanylate cyclase (sGC)-guanosine 3'5' cyclic monophosphate (cGMP) pathway. The level of cGMP is regulated by phosphodiesterases (PDEs), which break down cGMP. It has been reported that levels of NO in the central nervous system (CNS) can greatly increase during demyelination and/or neuroinflammation. Controversially, in demyelination models, mice without iNOS may develop more severe cases of disease. Furthermore, cGMP accumulation caused by PDE inhibitors has an anti-inflammatory/neuroprotective effect in MS-models. The role of the NO-cGMP pathway in the nervous tissue is, therefore, complex and not fully understood. The aim of the present study was to contribute to existing knowledge of the role of this pathway in the CNS. Wild type (WT - C57BL/6) and iNOS-/- animals were treated with sildenafil (25mg/kg) for 8 weeks. Control animals were not treated. VCAM and ICAM (adhesion proteins), GFAP and Iba-1 (astrocyte and microglia markers, respectively), PKG (cGMP-dependent protein kinase), sGC, eNOS (constitutive endothelial NO sinthase) and GSTpi (a marker of mature oligodendrocytes) were evaluated in the cerebellum using immunohistochemistry or western blotting. Myelin was assessed by luxol fast blue staining and electron transmission microscopy. Treatment with sildenafil reduced ICAM and VCAM levels (anti-inflammatory effect) and increased GFAP and Iba-1 expression (clearance phenotype) in WT animals. The expression of VCAM, ICAM, GFAP, PKG and sGC was lower in iNOS-/- mice than in WT control animals. The treatment of iNOS-/- animals with sildenafil resulted in an increase of all proteins (pro-inflammatory effect). There was overexpression of eNOS in untreated iNOS-/- mice. The myelin structure of iNOS-/- animals was damaged in comparison with WT control. Sildenafil increased GSTpi and resulted in an improved myelin structure in iNOS-/- mice. In conclusion, NO-cGMP signaling plays a role in the regulation of inflammation and myelination processes. The accumulation of cGMP produced opposite effects in WT and iNOS-/- mice. This can be explained by the overexpression of eNOS in iNOS-/- mice, unbalancing cGMP signaling, or cGMP has a dual role in inflammation. Drugs that modulate the NO-sGC-cGMP pathway may be clinically beneficial in the treatment of neuroinflammatory/demyelinating disorders, but further studies of the regulation of this pathway are required. © 2014 Elsevier Inc.1046073Agullo, L., Baltrons, M.A., Garcia, A., Calcium-dependent nitric oxide formation in glial cells (1995) Brain Res., 686, pp. 160-168Arnett, H.A., Hellendall, R.P., Matsushima, G.K., Suzuki, K., Laubach, V.E., Sherman, P., Ting, J.P., The protective role of nitric oxide in a neurotoxicant-induced demyelinating model (2002) J. Immunol., 168, pp. 427-433Baratti, C.M., Boccia, M.M., Effects of sildenafil on long-term retention of an inhibitory avoidance response in mice (1999) Behav. Pharmacol., 10, pp. 731-737Bellamy, T.C., Garthwaite, J., Sub-second kinetics of the nitric oxide receptor, soluble guanylyl cyclase, in intact cerebellar cells (2001) J. Biol. Chem., 276, pp. 4287-4292Benjamins, J.A., Nedelkoska, L., Cyclic GMP-dependent pathways protect differentiated oligodendrocytes from multiple types of injury (2007) Neurochem. Res., 32, pp. 321-329Borán, M.S., García, A., The cyclic GMP-protein kinase G pathway regulates cytoskeleton dynamics and motility in astrocytes (2007) J. Neurochem., 102, pp. 216-230Borán, M.S., Baltrons, M.A., García, A., The ANP-cGMP-protein kinase G pathway a phagocytic phenotype but decreases inflammatory gene expression in microglial cells (2008) Glia, 56, pp. 394-411Danilov, A.I., Andersson, M., Bavand, N., Wiklund, N.P., Olsson, T., Brundin, L., Nitric oxide metabolite determinations reveal continuous inflammation in multiple sclerosis (2003) J. Neuroimmunol., 136, pp. 112-118De Luca, G., Di Giorgio, R.M., Macaione, S., Calpona, P.R., Di Paola, E.D., Costa, N., Cuzzocrea, S., De Sarro, G., Amino acid levels in some brain areas of inducible nitric oxide synthase knockout mouse (iNOS-/-) before and after pentylenetetrazole kindling (2006) Pharmacol. Biochem. Behav., 85, pp. 804-812Derbyshire, E.R., Marletta, M.A., Biochemistry of soluble guanylate cyclase (2009) Handb. Exp. Pharmacol., 191, pp. 17-31Fenyk-Melody, J.E., Garrison, A.E., Brunnert, S.R., Weidner, J.R., Shen, F., Shelton, B.A., Mudgett, J.S., Experimental autoimmune encephalomyelitis is exacerbated in mice lacking the NOS2 gene (1998) J. Immunol., 160, pp. 2940-2946Forshammar, J., Block, L., Lundborg, C., Biber, B., Hansson, E., Naloxone and ouabain in ultralow concentrations restore Na+/K+-ATPase and cytoskeleton in lipopolysaccharide-treated astrocytes (2011) J. Biol. Chem., 286, pp. 31586-31597Forshammar, J., Jörneberg, P., Biörklund, U., Westerlund, A., Lundborg, C., Biber, B., Hansson, E., Anti-inflammatory substances can influence some glial cell types but not others: cytokine release from microglia (2013) Brain Res., , (in press)Frank-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. 47Gay, F.W., Drye, T.J., Dick, G.W., Esiri, M.M., The application of multifactorial cluster analysis in the staging of plaques in early multiple sclerosis. Identification and characterization of the primary demyelinating lesion (1997) Brain, 120, pp. 1461-1483Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids (1982) Anal. Biochem., 126, pp. 131-138Greenwood, J., Etienne-Manneville, S., Adamson, P., Couraud, P.O., Lymphocyte migration into the central nervous system: implication of ICAM-1 signalling at the blood-brain barrier (2002) Vascul. Pharmacol., 38, pp. 315-322Huang, P.L., Mouse models of nitric oxide synthase deficiency (2000) J Am Soc Nephrol, 11, pp. S120-S123Huang, P.L., Dawson, T.M., Bredt, D.S., Snyder, S.H., Fishman, M.C., Targeted disruption of the neuronal nitric oxide synthase gene (1993) Cell, 75, pp. 1273-1286Huang, P.L., Huang, Z., Mashimo, H., Bloch, K.D., Moskowitz, M.A., Bevan, J.A., Fishman, M.C., Hypertension in mice lacking the gene for endothelial nitric oxide synthase (1995) Nature (Lond), 377, pp. 239-242Kahl, K.G., Zielased, J., Uttenthal, L.O., Rodrigo, J., Toyka, K.V., Schmidt, H.H., Protective role of the cytokine-inducible isoform of nitric oxide synthase induction and nitrosative stress in experimental autoimmune encephalomyelitis of the DA rat (2003) J. Neurosci. Res., 73, pp. 198-205Ke, X., Terashima, M., Nariai, Y., Nakashima, Y., Nabika, T., Tanigawa, Y., Nitric oxide regulates actin reorganization through cGMP and Ca(2+)/calmodulin in RAW 264.7 cells (2001) Biochim. Biophys. Acta, 1539, pp. 101-113Klotz, L., Diehl, L., Dani, I., Neumann, H., von Oppen, N., Dolf, A., Endl, E., Knolle, P., Brain endothelial PPARgamma controls inflammation-induced CD4+ T cell adhesion and transmigration in vitro (2007) J. Neuroimmunol., 190, pp. 34-43Kyritsis, N., Kizil, C., Brand, M., Neuroinflammation and central nervous system regeneration in vertebrates (2014) Trends Cell Biol., 24, pp. 128-135Lartey, F.M., Ahn, G.O., Shen, B., Cord, K.T., Smith, T., Chua, J.Y., Rosenblum, S., Loo, B.W., PET imaging of stroke-induced neuroinflammation in mice using [18F]PBR06 (2014) Mol. Imaging Biol., 16, pp. 109-117Laubach, V.E., Shesely, E.G., Smithies, O., Sherman, P.A., Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death (1995) Proc. Natl. Acad. Sci. U S A, 92, pp. 10688-10692Lee, S.J., Benveniste, E.N., Adhesion molecule expression and regulation on cells of the central nervous system (1999) J. Neuroimmunol., 98, pp. 77-88Lieb, K., Engels, S., Fiebich, B.L., Inhibition of LPS-induced iNOS and NO synthesis in primary rat microglial cells (2003) Neurochem. Int., 42, pp. 131-137MacMicking, J.D., Nathan, C., Hom, G., Chartrain, N., Fletcher, D.S., Trumbauer, M., Stevens, K., Mudgett, T.S., Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase (1995) Cell, 81, pp. 641-650Manson, 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, GreeceMcCarthy, K.D., de Vellis, J., Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue (1980) J. Cell Biol., 85, pp. 890-902Mc Guire, C., Prinz, M., Beyaert, R., van Loo, G., Nuclear factor kappa B (NF-κB) in multiple sclerosis pathology (2013) Trends Mol. Med., 19, pp. 604-613Mrak, R.E., Griffin, W.S., Glia and their cytokines in progression of neurodegeneration (2005) Neurobiol. Aging, 26, pp. 349-354Murthy, K.S., Activation of phosphodiesterase 5 and inhibition of guanylate cyclase by cGMP-dependent protein kinase in smooth muscle (2001) Biochem. J., 360, pp. 199-208Nossaman, B.D., Kadowitz, P.J., Stimulators of soluble guanylyl cyclase: future clinical indications (2013) Ochsner J., 13, pp. 147-156Nunes, A.K., Raposo, C., Luna, R.L., Cruz-Höfling, M.A., Peixoto, C.A., Sildenafil (Viagra®) down regulates cytokines and prevents demyelination in a cuprizone-induced MS mouse model (2012) Cytokine, 60, pp. 540-551Papapetropoulos, A., Abou-Mohamed, G., Marczin, N., Murad, F., Caldwell, R.W., Catravas, J.D., Downregulation of nitrovasodilator-induced cyclic GMP accumulation in cells exposed to endotoxin or interleukin-1 beta (1996) Br. J. Pharmacol., 118, pp. 1359-1366Pifarre, P., Prado, J., Baltrons, M.A., Giralt, M., Gabarro, P., Feinstein, D.L., Hidalgo, J., Garcia, A., Sildenafil (Viagra) ameliorates clinical symptoms and neuropathology in a mouse model of multiple sclerosis (2011) Acta Neuropathol., 121, pp. 499-508Prickaerts, J., van Staveren, W.C., Sik, A., Markerink-van Ittersum, M., Niewöhner, U., van der Staay, F.J., Blokland, A., de Vente, 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. 351-361Puzzo, D., Privitera, L., Leznik, E., Fà, M., Staniszewski, A., Palmeri, A., Arancio, O., Picomolar amyloid-beta positively modulates synaptic plasticity and memory in hippocampus (2008) J. Neurosci., 28, pp. 14537-14545Pyriochou, A., Papapetropoulos, A., Soluble guanylyl cyclase: more secrets revealed (2005) Cell. Signal., 17, pp. 407-413Ramesh, G., Maclean, A.G., Philipp, M.T., Cytokines and chemokines at the crossroads of neuroinflammation, neurodegeneration, and neuropathic pain (2013) Mediators Inflamm., 2013, p. 480739Raposo, C., Nunes, A.K., Luna, R.L., Araújo, S.M., Cruz-Höfling, M.A., Peixoto, C.A., Sildenafil (Viagra) protective effects on neuroinflammation: the role of iNOS/NO system in an inflammatory demyelination model (2013) Mediators Inflamm., 2013, p. 321460RapÔso, C., Odorissi, P.A.M., Oliveira, A.L.R., Aoyama, H., Ferreira, C.V., Verinaud, L., Fontana, K., Cruz-Höfling, M.A., Effect of Phoneutria nigriventer venom on the expression of junctional protein and P-gp efflux pump function in the blood-brain barrier (2012) Neurochem. Res., 37, pp. 1967-1981Riccitelli, G., Rocca, M.A., Pagani, E., Martinelli, V., Radaelli, M., Fajini, A., Comi, G., Filippi, M., Mapping regional grey and white matter atrophy in relapsing-remitting multiple sclerosis (2012) MultScler, 18, pp. 1027-1037Ruuls, S.R., Van der Linder, S., Sontrop, K., Huitinga, I., Dijkstra, C.D., Aggravation of experimental allergic encephalomyelitis (EAE) by administration of nitric oxide (NO) synthase inhibitors (1996) Clin. Exp. Immunol., 103, pp. 467-474Saraiva, 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. 454-462Shields, 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. 186-194Shimouchi, A., Janssens, S.P., Bloch, D.B., Zapol, W.M., Bloch, K.D., CAMP regulates soluble guanylate cyclase beta 1-subunit gene expression in RFL-6 rat fetal lung fibroblasts (1993) Am. J. Physiol., 265, pp. L456-L461Tsutsui, M., Shimokawa, H., Morishita, T., Nakata, S., Sabanai, K., Nakashima, Y., Yanagihara, N., Development of genetically engineered mice lacking all three nitric oxide synthase isoforms (2007) Yakugaku Zasshi, 127, pp. 1347-1355Vollmar, A.M., Förster, R., Schulz, R., Effects of atrial natriuretic peptide on phagocytosis and respiratory burst in murine macrophages (1997) Eur. J. Pharmacol., 319, pp. 279-285Vollmar, A.M., The role of atrial natriuretic peptide in the immune system (2005) Peptides, 26, pp. 1086-1094Wakita, H., Tomimoto, H., Akiguchi, I., Lin, J.X., Ihara, M., Ohtani, R., Shibata, M., Ibudilast protect against white matter damage under chronic cerebral hypoperfusion in the rat (2003) Brain Res., 992, pp. 53-59Wang, L., Chopp, M., Szalad, A., Liu, Z., Bolz, M., Alvarez, F.M., Lu, M., Zhang, Z.G., Phosphodiesterase-5 is a therapeutic target for peripheral neuropathy in diabetic mice (2011) Neuroscience, 193, pp. 399-410Wang, X., Robinson, P.J., Cyclic GMP-dependent protein kinase and cellular signaling in the nervous system (1997) J. Neurochem., 68, pp. 443-456Wei, X.Q., Charles, I.G., Smith, A., Ure, J., Feng, G.J., Huang, F.P., Xu, D., Liew, F.Y., Altered immune responses in mice lacking inducible nitric oxide synthase (1995) Nature (Lond), 375, pp. 408-411Williams, K.C., Hickey, W.F., Traffic of hematogenous cells through the central nervous system (1995) Curr. Top. Microbiol. Immunol., 202, pp. 221-245Woodcock, T., Morganti-Kossmann, M.C., The role of markers of inflammation in traumatic brain injury (2013) Front. Neurol., 4, p. 18Xia, X., Xu, Z., Hu, X., Wu, C., Dai, Y., Yang, L., Impaired iNOS-sGC-cGMP signaling contributes to chronic hypoxic and hypercapnic pulmonary hypertension in rat (2012) Cell Biochem. Funct., 30, pp. 279-285Yoshioka, 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. 633-640Yoshioka, Y., Takeda, N., Yamamuro, A., Atsushi, K., Maeda, S., Nitric oxide inhibits lipopolysaccharide-induced inducible nitric oxide synthase expression and its own production through the cGMP signaling pathway in murine microglia BV-2 cells (2010) J. Pharmacol. Sci., 113, pp. 153-160Zhao, L., Mason, N.A., Strange, J.W., Walker, H., Wilkins, M.R., Beneficial effects of phosphodiesterase 5 inhibitor in pulmonary hypertension are influenced by natriuretic peptide activity (2003) Circulation, 107, pp. 234-23

Efeito da temperatura de descongelação na integridade de espermatozoides criopreservados de cães

Avaliou-se a influência da temperatura de descongelação na integridade de espermatozoides criopreservados de cães. Foram utilizados reprodutores das raças Basset Hound (n=3) e Rottweiler (n=3), submetidos a colheitas de sêmen por manipulação peniana. As amostras de sêmen foram descongeladas a 37ºC/1min (G1) ou 70ºC/6s (G2) e avaliadas quanto à motilidade progressiva, vigor e integridade do acrossoma após 0, 30 e 60 minutos de incubação (37ºC), e ultraestrutura espermática imediatamente após a descongelação. Em todos os tempos de incubação, a motilidade progressiva dos espermatozoides descongelados a 70ºC por 6s (74,6%) foi mais alta (P0,05) entre os grupos, e o porcentual de gametas com acrossomas íntegros foi maior (P<0,05) nos espermatozoides do G1 do que no G2. Lesões ultraestruturais foram identificadas nos espermatozoides descongelados de ambos os grupos, em maior quantidade nos gametas do G2. Conclui-se que amostras congeladas de sêmen de cães devam ser descongeladas a 37ºC por 1min

Tipo de conhecimento sobre inclusão produzido pelas pesquisas Type of knowledge about inclusion produced by research

As pesquisas brasileiras sobre produção de conhecimento no campo da Educação Especial iniciaram-se há mais de 15 anos. No presente objetivou-se analisar o tipo de conhecimento produzido nessa área frente à metodologia empregada nas dissertações e teses financiadas pelo Programa de Apoio a Educação Especial. Os trabalhos de conclusão de curso analisados referiam-se àqueles projetos aprovados pelo Proesp no âmbito do Estado de São Paulo. O período de análise ficou compreendido entre 2004 e 2008. Vinte e sete estudos foram localizados. A análise dos estudos foi realizada por meio de cinco categorias: 1) pesquisas que apresentaram generalização e aplicação imediata dos resultados; 2) pesquisas que apresentaram resultados imediatos para um grupo específico de participantes; 3) pesquisas descritivas com achados inovadores; 4) pesquisas de intervenção com achados inovadores; 5) pesquisas descritivas que corroboraram outras pesquisas. Foi possível concluir que os estudos avançaram nos conhecimentos sobre a inclusão e que o uso de metodologias pouco utilizadas no campo da educação serviu de subsídio para obter os resultados.<br>The Brazilian research on knowledge production in the field of Special Education began more than 15 years ago. The objective was to analyze the type of knowledge produced in the area before the methodology used in dissertations funded by the "Programa de Apoio a Educação Especial - PROESP". The final paper analyzed referred to those projects approved by PROESP within the State of São Paulo. The analysis period was between 2004 and 2008. Twenty-seven studies were found. The review was conducted through five categories: 1) studies that showed generalization and immediate application of the results, 2) research that showed immediate results for a specific group of participants, 3) descriptive research with innovative findings, 4) intervention research with innovative findings and 5) descriptive research that corroborated other studies. It was concluded that the studies advance in knowledge on inclusion and the use of some infrequent methodologies in education served as a subsidy to get the results