research article sildenafil (viagra) protective effects on

Hindawi Publishing CorporationMediators of InflammationVolume 2013 Article ID 321460 11 pageshttpdxdoiorg1011552013321460

Research ArticleSildenafil (Viagra) Protective Effects onNeuroinflammation The Role of iNOSNO System in anInflammatory Demyelination Model

Catarina Raposo1 Ana Karolina de Santana Nunes12

Rayana Leal de Almeida Luna12 Shyrlene Meiry da Rocha Arauacutejo2

Maria Alice da Cruz-Houmlfling1 and Christina Alves Peixoto2

1 Departamento de Histologia e Embriologia Instituto de Biologia Universidade Estadual de Campinas (UNICAMP)Rua Monteiro Lobato 255 13083-862 Campinas SP Brazil

2 Laboratorio de Ultraestrutura Centro de Pesquisas Aggeu Magalhaes Fundacao Oswaldo Cruz Av Professor Moraes Regosn Cidade Universitaria 50670-420 Recife PE Brazil

Correspondence should be addressed to Catarina Raposo cataraposagmailcom

Received 27 March 2013 Revised 12 June 2013 Accepted 13 June 2013

Academic Editor Geeta Ramesh

Copyright copy 2013 Catarina Raposo et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

We recently demonstrated that sildenafil reduces the expression of cytokines COX-2 and GFAP in a demyelinating model inducedin wild-type (WT) mice Herein the understandings of the neuroprotective effect of sildenafil and the mediation of iNOSNOsystem on inflammatory demyelination induced by cuprizone were investigated The cerebella of iNOSminusminus mice were examinedafter four weeks of treatment with cuprizone alone or combined with sildenafil Cuprizone increased GFAP Iba-1 TNF-120572 COX-2IL-1120573 and IFN-120574 expression decreased expression of glutathione S-transferase pi (GSTpi) and damaged myelin in iNOSminusminus miceSildenafil reduced Iba-1 IFN-120574 and IL-1120573 levels but had no effect on the expression of GFAP TNF-120572 and COX-2 compared to thecuprizone group Sildenafil elevated GSTpi levels and improved the myelin structureultrastructure iNOSminusminus mice suffered fromsevere inflammation following treatment with cuprizone whileWTmice had milder inflammation as found in the previous studyIt is possible that inflammatory regulation through iNOS-feedback is absent in iNOSminusminus mice making them more susceptible toinflammation Sildenafil has at least a partial anti-inflammatory effect through iNOS inhibition as its effect on iNOSminusminus mice waslimited Further studies are required to explain the underlying mechanism of the sildenafil effects

1 Background

Nitric oxide (NO) is a highly reactive molecule with arange of physiological functions [1 2] This messenger playsan important role in the modulation of vascular tone [3]neurotransmission [4 5] and immune system [6ndash8] NOis produced from L-arginine by NO synthases (NOSs) Inaddition to the constitutive forms of the enzyme (endothelial(eNOS or NOS3) and neuronal (nNOS or NOS1)) there isalso an inducible form (iNOS or NOS2) This last is mostcommonly associatedwith inflammatory conditions inwhichNO is produced in large amounts

There is strong evidence to suggest the involvementof iNOS in the development of neurodegenerative diseaseInduction of iNOS NO and NO byproducts has been foundin multiple sclerosis (MS) patients and animal models andcorrelates with disease severity and level of inflammatoryinfiltrate [9ndash12] Interestingly however iNOS-deficient micedeveloped a more severe MS model [13 14] It was found thatthe elimination of iNOS does not improve and may actuallyaggravate demyelination in the cuprizone-demyelinatingmodel [15] which suggests that the iNOSNO systemmay beneuroprotective More information is required to understand

2 Mediators of Inflammation

the role of iNOSNO in inflammatory response oligodendro-cyte cell death and myelin damageloss

The cyclic guanosine 3101584051015840-monophosphate (cGMP) sig-naling pathway is an important NO-signaling molecule NObinds to soluble guanylyl cyclase (sGC) and increases concen-tration of cGMP activating signaling cascades and leading tocGMP-dependent responses [16 17]The cGMP signal can beterminated by the action of several phosphodiesterases (PDE)[18]The cGMP-selective PDE5 is expressed in the cardiovas-cular neural and immune systems [18] Studies have shownthat selective PDE5 inhibitors widely used in the treatmentof erectile dysfunction in humans such as sildenafil (ViagraPfizer) and vardenafil (Levitra Bayer) raise cGMP levels inthe brain and offer protective effects improve cognition andmemory [19] reduce neuronal cell death following ischemiccerebrovascular injury [20] decrease white matter damageand regulate inflammatory responses in MS models [21 22]Recent studies by the authors of an MS model induced inwild-type C57BL6 mice found that sildenafil has an anti-inflammatory action reducing levels of proinflammatorycytokines and cyclooxygenase-2 (COX-2) and protecting themyelin structure [22] However the mechanism of sildenafilneuroprotection remains unknown

In the present study inflammatory demyelination wasinduced in iNOSminusminusmice and sildenafil was administered forfour weeks The focus of this study was to identify the roleof a potent inflammation-associatedmolecule iNOS-derivedNO in protective mechanisms related to sildenafil Thepresent study also aimed to clarify the role of NO protectiveandor deleterious mechanisms in the demyelinating model

2 Materials and Methods

21 Experimental Design Five iNOS knockout (B 6129 P2-Nos2) mice aged 7 to 10 weeks weighing 15 to 20 g wereused per group The mice were examined for health statusacclimated to the laboratory environment at 25∘C and 12 hlightdark photoperiod and housed in metal cages The con-trol group received standard laboratory diet and pure waterOver a four-week period the experimental groups receivedeither 02 cuprizone (oxalic-bis-cyclohexylidenehydrazideSigma-Aldrich Inc St Louis MO USA) mixed into thechow and pure water or 02 cuprizone in the chow and25mgkg of bodyweight of sildenafil (Viagra Pfizer Inc NewYork NY USA) administered through the drinking water[22ndash24] Body weight was accessed every week and the drugconcentration in the water was adjusted tomaintain the doseAll experiments were carried out in compliance with ethicalguidelines for animal experimentation (L-102010-CEUA0510-CIBIO FIOCRUZ) After treatment the animals wereanaesthetized (im) with ketamine (115mgkg) and xylazine(10mgkg) (Sespo Comercio e Industria Ltda Sao Paulo SPBrazil)

22 Immunofluorescence (IF) After anesthesia the ani-mals were transcardially perfused with physiological saline(20mL) followed by 4 paraformaldehyde (Sigma-Aldrich)(40mL) in 01M phosphate (sodium phosphate monobasic

and dibasic heptahydrate Sigma-Aldrich) buffered saline(PBS) pH 72 Cerebella were dissected and immersed in15 sucrose overnight followed by 30 sucrose for a secondnight (36 hours total) The specimens were then embeddedin OCT-Tissue Tek compound (Sakura Finetek TorranceCA USA) and frozen in n-hexane (Dinamica Sao PauloSP Brazil) cooled with liquid nitrogen Cryosections (8120583mthick) were permeabilized (03Triton X-100) and incubatedfor 1 h with blocking solution (3 BSA plus 02 Tween20 in Tris buffered saline) Subsequently the sections wereincubated with antibodies for Glial Fibrillar Acidic Pro-tein (GFAP) (DakoCytomation cat no ZO 334) and Iba-1(Wako Osaka Japan cat no 019-19741) (both 1 100) Sec-tions were incubated with primary antibodies overnight andthen incubated with polyclonal Cy3-conjugated secondaryantibodies (Jackson cat no 705-165-147) against rabbitimmunoglobulin (1 200) for 1 h The slides were washedand mounted in fluorescent Prolong Gold Antifade medium(Life Technologies cat no P36930) for observation underan inverted fluorescence microscope (Zeiss MicroImagingGmbH) equipped with a camera (Zeiss AxioCamMRM) andthe Release 472 image analysis software

23 Immunohistochemistry (IH) After perfusion as de-scribed for IF cerebella were immediately removed andpostfixed in the same fixative overnight The samples weredehydrated in an ethanol series (Isofar Chemical Co RJBrazil) cleared in xylene and embedded in paraffin (Merckcatalog no 1071642504) Sections (5120583m thick) were cut on anRM 2035 microtome (Reichert S Leica) rehydrated washedin 005M PBS and incubated in this buffer with 1 bovineserum albumin (BSA fraction V) (Miles Naperville ILUSA) for one hour Endogenous peroxidase was blockedand antigen retrieval was performed pretreating the sec-tions with 20mM citrate buffer pH 60 at 100∘C for30min All groups were incubated with the rabbit polyclonalanti-COX-2 (Abcam CanadaUS cat no ab15191) (1 100overnight at 4∘C) After washing the sections were overlaidfor 1 h with a biotin-conjugated secondary antibody usingan HRP kit (DakoCytomation CA USA Biotinylated LinkUniversal HRP cat no K0690) and visualized with 31015840-3-diaminobenzidine (DAB) as the chromogen The sectionswere then weakly counterstained with Harrisrsquo hematoxylinand mounted in entellan (Merck cat no 1079610100)

24Western Blotting (WB) Cerebella were quickly dissectedand each group was homogenized in an extraction cocktail(10mM EDTA Amresco Solon USA 2mM phenylmethanesulfonyl-fluoride 100mM NaF 10mM sodium pyrophos-phate 10mM NaVO

4

10 120583g of aprotininmL and 100mMTris pH 74) Cerebella of five animals were mixed andhomogenized to form a pool from each group The WB wasdone in accord with Nunes et al [22] Briefly the pro-teins (40 120583g total) were separated on 65 (IFN-120574) 10(TNF-120572 IL-1120573) or 12 (GFAP eNOS) acrylamide gelAfter overnight in 5 nonfat milk the primary antibodiesagainst GFAP (1 10000 DakoCytomation cat no ZO 334)COX-2 (1 1000 Abcam cat no ab15191) TNF-120572 (1 1000

Mediators of Inflammation 3

Peprotech NJ USA cat no 500-P64) IFN-120574 (1 2500Peprotech cat no 500P119) IL-1120573 (1 2500 GenWay BiotechInc CAUSA cat no 18-732-292194) GST3GSTpi (1 250Abcam Canada cat no ab53943) and eNOS (1 1000BD Biosciences cat no 610299) were incubated for fourhours followed by horseradish peroxidase-conjugated anti-bodies anti-rabbit (1 80000 Sigma-Aldrich cat no A9169)anti-mouse (1 1000 Sigma-Aldrich cat no A0168) oranti-goat secondary antibody (1 100000 Sigma-Aldrichcat no A5420) For quantification the pixel density ofeach band was determined using the Image J 138 soft-ware (httprsbwebnihgovijdownloadhtml developed byWayne Rasband NIH BethesdaMDUSA) For each proteininvestigated the results were confirmed in three sets ofexperiments Immunoblotting for 120573-actin (1 1000 Sigma-Aldrich cat no A2228) was performed as a control

25 Luxol Fast-Blue (LFB) After perfusion as described forIF the samples were dehydrated in a graded ethanol seriescleared in xylene and embedded in paraffin Sections (5 120583mthick) were cut on an RM 2035 microtome (Reichert SLeica) Myelin was detected using Luxol-Fast Blue (LFB)staining (Solvent Blue 38 Sigma-Aldrich) in accord withNunes et al [22] The sections were observed under aninverted microscope (Zeiss MicroImaging GmbH) equippedwith a camera (ZeissAxioCamMRM) andRelease 472 imageanalysis software (Zeiss)

26 Transmission Electron Microscopy (TEM) After anes-thesia the animals were sacrificed by transcardial perfusionwith physiological saline (20mL) followed by 40mL offixativemdash25 glutaraldehyde and 4 paraformaldehyde in01M sodium cacodylate acid (Sigma-Aldrich) buffer pH72 Cerebella were quickly dissected and postfixed in thesame fixative overnight Next cerebellum fragments werewashed twice in the same buffer and postfixed in a solutioncontaining 1 osmium tetroxide (Sigma-Aldrich) 2mMcalcium chloride and 08 potassium ferricyanide (Sigma-Aldrich) in 01M cacodylate buffer pH 72 dehydrated inacetone and embedded in SPIN-PON resin (Embed 812-Electron Microscopy Science Washington PA USA) Resinpolymerization was performed at 60∘C for 3 days Semithinsections (05 120583m in thickness) were placed on glass slidesstained with toluidine blue Ultrathin sections (70 nm inthickness) were placed on 300-mesh nickel grids counter-stainedwith 5uranyl acetate (ElectronMicroscopy Science)and lead citrate (Sigma-Aldrich) and examined using a FEIMorgagni 268D transmission electron microscope

27 Statistical Analysis The densitometric values of theimmunoreactive bands (immunoblotting) were analyzedusing the GraphPad Prism software package (San DiegoCA USA) One-way analysis of variance (ANOVA) followedby Dunnettrsquos andor Tukeyrsquos posttest was used to comparegroups The results were expressed as means plusmn SE whenappropriate A119875 valuelt 005 indicated statistical significance

3 Results

Clinical analysis revealed that cuprizone-treated animalssuffered frommotor limitations such as tremors and abnor-mal walking and posture The group treated with silde-nafil (25mgkg) exhibited normal walking and posture andtremors were either mild or nonexistent

The clinical signs were observed and recorded by threeobservers The iNOSminusminus control animals exhibited normalmotor function and posture and explored their environmentnormally This group was classified as score 0 (no sign) Themice treated with cuprizone exhibited arched (shortened)posture and tremors and had difficulty in exploring theenvironment This group was classified as score 2 Micetreated with 25mgkg of sildenafil both walked and wereable to explore the environment normally with no or mildtremors and were classified as score 1

Cuprizone increased GFAP TNF-120572 and COX-2 expres-sion in cerebellum indicating astrocyte activation (reactivegliosis) and neuroinflammation Sildenafil treatment did notreduce the levels of these proteins in mice without iNOS

Western Blotting (WB) analysis showed that GFAP amarker of astrocyte activation (reactive gliosis) was presentin the cerebellum of untreated iNOSminusminus animals (control)(Figure 1(a)) Treatment with 02 cuprizone for four weekssignificantly increased expression of this protein (Figures 1(a)and 1(c) 119875 lt 0001) Animals that concomitantly receivedsildenafil (25mgKg) and cuprizone also exhibited a highlevel of GFAP in comparison to control (Figures 1(a) and 1(c)119875 lt 0001)

Immunofluorescence (IF) analysis of GFAP in the cere-bellum revealed the expression and location of this cytoskele-tal intermediate filament protein in the animals In themolecular layer of the cerebellum GFAP labeling revealedlong astrocytic processes with a typical arrangement that wasperpendicular to the pia mater membrane (Bergmann glia)(Figure 2(e)) Astrocytic processes were also seen aroundother cells and vessels (arrowheads in Figures 2(b) 2(d) and2(e)) The control group showed basal expression of GFAPlocalized normally (Figures 2(a) and 2(d)) Treatment withCPZ induced astrogliosis increasing the intensity of labelingin the astrocytes (Figures 2(b) and 2(e)) In the sildenafilgroup GFAP labeling was more intense than for control(Figures 2(c) and 2(f))

The iNOSminusminus control group had a basal level of TNF-120572(Figure 3(a)) WB analysis showed that cuprizone induced asignificant increase of this cytokine indicating neuroinflam-mation (Figures 3(a) and 3(b) 119875 lt 005 compared to controlgroup) Animals that received cuprizone plus sildenafil alsohad a significant increase of TNF-120572 compared to control(119875 lt 001)

COX-2 was analyzed by WB and immunohistochemistry(IH) tests This enzyme was expressed in a minimal amountin the cerebellum of the iNOSminusminus control group (Figures 3(c)and 4(a)) CPZ administration induced a significant increasein COX-2 expression compared to control (Figures 3(c) and3(d) 119875 lt 001) Sildenafil treatment did not decrease COX-2which remained high in comparison with the control group


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Figure 1 Western blotting for GFAP (a) GFAP immunoblot of iNOSminusminus control (Cont) cuprizone (CPZ) and cuprizone plus sildenafil(CPZ + Sild) groups (b) 120573-actin immunoblot (c) Graph represents quantification and statistical analysis The control group showed basalexpression of GFAP CPZ treatment induced a significant increase of this protein and sildenafil plus CPZ did not reduce GFAP expressionwhich remained higher in relation to the control group The experiment was performed in triplicate (119899 = 5 animalsgroup) The results wereexpressed as mean plusmn SE lowast119875 lt 005 lowastlowast119875 lt 001 compared to control

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Figure 2 Immunofluorescence forGFAP (a) and (d) show expression and physiological locations of GFAP inmicewithout iNOS andwithouttreatment CPZ administration ((b) (e)) induced reactive gliosis with thicker andmore numerous astrocytic processes compared to controlGFAP remained high in relation to control after application of sildenafil plus CPZ ((c) (f)) Arrows show astrocytic processes and arrowheadspoint to processes around vessels and other cells W white matter M molecular layer P purkinje layer G granular layer and v vessel Bars20 120583m


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Figure 3 Western blotting for TNF-120572 and COX-2 ((a) (c)) Immunoblots of iNOSminusminus control (Cont) cuprizone (CPZ) and cuprizone plussildenafil (CPZ + Sild) groups ((b) (d)) Graphs represent quantification and statistical analysisThe control group showed basal expression ofTNF-120572 CPZ treatment caused a significant increase of this cytokine and sildenafil plus CPZ did not decrease its expression which remainedhigher in relation to the control group Only minimum amounts of COX-2 were present in the control group CPZ and CPZ + Sild caused asignificant increase of this enzyme compared to controlThe experiment was performed in triplicate (119899 = 5 animalsgroup)The results wereexpressed as mean plusmn SE lowast119875 lt 005 lowastlowast119875 lt 001 compared to control

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Figure 4 Immunohistochemistry for COX-2 ((a)ndash(c)) and immunofluorescence for Iba-1 ((d)ndash(f)) iNOSminusminus control (a) showed very lowCOX-2 expression (arrow) After CPZ treatment (b) COX-2 labeling significantly increased mainly in white matter in relation to controlAnimals treated with CPZ + Sild (c) also increased COX-2 comparing to the control group A basal expression of Iba-1 was seen in controlanimals without iNOS (d) CPZ increased Iba-1 and induced an activated phenotype (arrowheads) of microglia (e) Sildenafil plus CPZdecreased Iba-1 and induced latent phenotype (arrows) of microglia (f)


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Figure 5 Western blotting for IL-1120573 and IFN-120574 ((a) (c)) Immunoblots of iNOSminusminus control (Cont) cuprizone (CPZ) and cuprizone plussildenafil (CPZ + Sild) groups ((b) (d)) Graphs represent quantification and statistical analysis The control group showed basal expressionof IL-1120573 and IFN-120574 CPZ treatment induced a significant increase of these cytokines compared to control Sildenafil plus CPZ significantlydecreased IL-1120573 and IFN-120574 expression in relation to the CPZ group The experiment was performed in triplicate The results were expressedas mean plusmn SE lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared to control 119875 lt 001 119875 lt 0001 compared with CPZ

(Figures 3(c) and 3(d) 119875 lt 001) IH labeling revealed theexpression and location of COX-2 (Figures 4(a)ndash4(c)) whichwas mainly found in the white matter of the cerebellumThe control group had a very low expression of this enzyme(Figure 4(a)) while treated animals had a high expression ofCOX-2 in white matter (Figures 4(b) and 4(c))

Immunoblot control with 120573-actin is shown in Figure 1(b)Cuprizone increased Iba-1 IL-1120573 and IFN-120574 expression

in the cerebellumof iNOSminusminusmiceWhile sildenafil decreasedthe expression of these proteins the level of expressionremained above that of control animals

The microglial marker Iba-1 was analyzed by IF Therewas physiological expression of Iba-1 in the cerebellum ofiNOSminusminus control animals with cells with processes thatwere typically branched thin and weakly labeled (arrowFigure 4(d)) Cuprizone treatment induced a stronger expres-sion of Iba-1 (Figure 4(e)) compared to the control groupwith thicker and more intensely labeled microglial processesThese processes lost their typical thin branched appearanceindicating that the cell phenotype had acquired activatedcharacteristics Sildenafil together with CPZ decreased Iba-1expression and themicroglia exhibited thin highly branchedprocesses typical of a latent state (Figure 4(f))

Immunoblotting for IL-1120573 and IFN-120574 revealed basalexpression of these cytokines in the iNOSminusminus control group(Figures 5(a) and 5(c)) Cuprizone strongly increased theexpression of these cytokines (both 119875 lt 0001) comparedto the control group (Figures 5(a)ndash5(d)) which indicates

neuroinflammation Sildenafil treatment resulted in a signifi-cant decrease of IL-1120573 and IFN-120574 compared to the cuprizonegroup (119875 lt 001) However the levels of IL-1120573 and IFN-120574 remained higher in the sildenafil-treated group whencompared with baseline levels of the control group (119875 lt 001and 119875 lt 0001 resp)

Mice without iNOS underwent a significant increase ineNOS expression compared to wild-type animals

Untreated mice iNOSminusminus (control group 119875 lt 001) andiNOSminusminus animals treated with CPZ or CPZ + Sild (both119875 lt 0001) showed a significant increase in eNOS levelscompared with wild-type mice that did not undergo treat-ment The expression of eNOS in iNOSminusminus mice treated withCPZ and CPZ plus sildenafil was not significantly higherwhen compared to iNOSminusminus control animals but showed apropensity to increase (Figures 6(a) and 6(b))

Cuprizone decreased GSTpi indicating the depletion ofmature oligodendrocytes Sildenafil increased GSTpi expres-sion in the cerebellum of iNOSminusminus mice

GSTpi a marker of myelinating oligodendrocyteswas expressed in the cerebellum of the control group(Figure 7(a)) After treatment with CPZ this markerdecreased significantly (119875 lt 0001) compared to the controlgroup suggesting an impairment ofmature oligodendrocytesand consequentmyelin damage Sildenafil treatment togetherwith CPZ induced partial recovery of the GSTpi markerindicating that sildenafil had a protective effect on mature


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Figure 6 Western blotting for eNOS (a) Immunoblot of wild-type mice control (WT cont) iNOSminusminus mice control (iNOSminusminus

cont) iNOSminusminus animals treated with cuprizone (iNOSminusminus CPZ) andiNOSminusminus animals treated with cuprizone plus sildenafil (iNOSminusminusCPZ + Sild) (b) Graph represents quantification and statisticalanalysis eNOS was physiologically expressed in WTmice Animalswithout iNOS without treatment showed a significant increase ofthis enzyme compared to WT control After CPZ and CPZ plussildenafil iNOSminusminus animals also showed a significant increase ofeNOS compared to WT animals but no significant differencewas identified between iNOSminusminus control and treated animals Theexperiment was performed in triplicate The results were expressedas mean plusmn SE lowast119875 lt 005 lowastlowastlowast119875 lt 0001 compared to control

oligodendrocytes In the sildenafil group GSTpi decreasedin comparison with control (119875 lt 0001) but increased incomparison to the cuprizone group (119875 lt 0001 Figures 7(a)and 7(b))

Myelin sheath structure was disorganized in animalswithout iNOS which did not undergo treatment (control)Cuprizone induced more severe myelin disruption Sildenafilimproved myelin structure and ultrastructure

Interestingly iNOSminusminus animals without treatment (con-trol) had moderate disorganization of the myelin structureand ultrastructure (Figures 8(a) 8(d) and 8(g)) StandardLFB staining showed that the control group iNOSminusminus hadvacuoles in white matter (arrow in Figure 8(a)) resulting ininhomogeneous and disorganized tissue Qualitative analysisof ultrathin cerebellum sections by TEM revealed that themyelin sheath was often shredded with blanks and withoutthe characteristic lamellar pattern (arrows in Figures 8(d) and8(g))

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Figure 7 Western blotting for GSTpi (a) Immunoblot of iNOSminusminuscontrol (Cont) cuprizone (CPZ) and cuprizone plus sildenafil (CPZ+ Sild) groups (b) Graph represents quantification and statisticalanalysis iNOSminusminus control showed basal expression of GSTpi AfterCPZGSTpi decreased significantly compared to iNOSminusminus cont CPZ+ Sild treatment increased GSTpi expression in relation to the CPZgroup but the levels of this protein remained significantly decreasedcompared to control The experiment was performed in triplicatelowastlowastlowast

119875 lt 0001 compared to control 119875 lt 0001 compared to CPZ

Cuprizone-treated animals exhibitedmore severe damagetomyelin structure and ultrastructure (Figures 8(b) 8(e) and8(h)) LFB staining (Figure 8(b)) showed vacuoles (arrows)and spaces (arrowhead) as slits in white matter characteristicof highly disorganized tissue TEM revealed that CPZ causedserious damage to the myelin sheath ultrastructure whichhad numerous spaces between the shreds in practically allfibers (represented by arrows in Figures 8(e) and 8(h)) Thetypical lamellar pattern was entirely absent

Simultaneous treatment with sildenafil and CPZ resultedin a noticeable improvement of myelin organization (Figures8(c) 8(f) and 8(i)) White matter was more homogeneouspresenting fewer and in general smaller vacuoles and slit-like spaces (Figure 8(c)) Ultrathin section analysis revealeda more preserved myelin sheath which rarely showed signs ofshredding

4 Discussion

The cuprizone model is characterized by primary andreversible demyelination due to peripheral immune system-independent myelin injury [25] In this model demyelina-tion is accompanied by a well-characterized sequence ofevents involving the depletion of mature oligodendrocytesmicroglia activation and astrocyte proliferation [26] There-fore demyelination and resident neuroinflammation induced


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Figure 8 Luxol Fast Blue (LFB) staining ((a)ndash(c)) and electron micrographs ((d)ndash(i)) (a) (d) and (g) represent iNOSminusminus control group (b)(e) and (h) represent CPZ-treated animals (c) (f) and (i) represent CPZ + Sild-treated animals Arrows in (a) (c) vacuoles in the whitematter arrowhead in (b) spaces between fibers arrows in (d) (e) (g) and (h) damaged myelin sheath arrowheads in (f) (i) preservedmyelin Bars = 20120583m ((a)ndash(c)) 2120583m ((d)ndash(f)) 05 120583m ((g)ndash(i))

by cuprizone in rodents have been widely used as a modelfor MS [20 27] In the present study the usefulness ofthe cuprizone model is considerable as it allows evaluationof the role of the iNOSNO-sGC-cGMP pathway in theinflammation and demyelination mediated by resident CNScells

The cerebellum was chosen for analysis as it is an impor-tant CNS affected region in MS patients revealing severewhitematter atrophy [28 29] Furthermore the authors of thepresent study had recently investigated this region [22] Thepresent data relating to iNOSminusminus animals will be discussedwith reference to this previous study Nunes et al (2012)[22] showed that in C57BL6 wild-type (WT)mice cuprizoneinduced tissue damage increased GFAP Iba-1 and COX-2 expression and caused demyelination in comparison tothe control group However cuprizone did not affect theexpression of cytokines (TNF-120572 IFN-120574 IL-1120573 and IL-2) inWT mice In the previous study by the authors sildenafil

reduced GFAP and Iba-1 expression in comparison to thecuprizone group preserved myelin and axon ultrastructureand significantly downregulated IFN-120574 TNF-120572 IL-1120573 IL-2and COX-2 expression in comparison to the control andorcuprizone groups The previous results demonstrated theprotective effect of sildenafil on the cerebellum Sildenafilpromotes the accumulation of cGMP which is the mainNO signaling molecule It was considered important toinvestigate the role of NO in the MS-model and the effectsof sildenafil more profoundlyTherefore in the present studythe relationship between iNOSnull mice the MS-model andthe effects of sildenafil in the cerebellum was investigated

In the absence of iNOSNO cuprizone significantlyincreased expression of GFAP TNF-120572 COX-2 Iba-1 IL-1120573and IFN-120574 In addition cuprizone intoxication decreasedGSTpi a marker for myelinating oligodendrocytes damagedmyelin and induced tremors abnormal walking and pos-tureThis data indicates that cuprizone intoxication occurred


even without the iNOS-NO system and was stronger than inWTmice where cuprizone did not increase cytokine expres-sion [22] Interestingly iNOSminusminus control animals showed analtered myelin structure

It was hypothesized that in the absence of iNOSeNOS may be overexpressed as a compensatory mecha-nism Bernardini et al [30] showed that treatment withendotoxin influenced NOS expression upregulating iNOSand simultaneously downregulating eNOS There appearedto be a regulatory relationship between the expression ofiNOS and eNOS In fact it was found here that eNOSwas strongly expressed in iNOSminusminus animals in comparisonwith WT mice The eNOS levels remained high after theadministration of cuprizone and cuprizone plus sildenafilIn normal conditions low levels of NO produced by botheNOS and nNOS participate in cell signaling and regulatephysiologic processes [31] However eNOS overexpression(and consequently constant high concentration of NO) canbe responsible for myelin changes and proinflammatorysusceptibility in iNOSminusminus animals

On the other hand iNOS possesses an important feed-back mechanism in inflammatory conditions when theincrease of this enzyme is self-regulated and induces a reduc-tion of some proinflammatory proteins [32ndash35] The inhibi-tion of iNOS activity induces enhancement of IL-1120573 IL-6andTNF-120572 levels [36] and subsequently a persistent increaseof iNOS expression downregulating the TNF receptor [35]In the present study absence of iNOS may explain increasedTNF-120572 IFN-120574 and IL-1 after cuprizone treatment in iNOSminusminusmice while cuprizone did not increase these cytokines inWTmice [22] This feedback mechanism is coregulated by a highconcentration of cGMP [32] Therefore another hypothesisfor explaining the more severe inflammation induced bycuprizone in mice without iNOS is the absence of iNOSfeedback mechanism

Interestingly although sildenafil had a low anti-inflam-matory effect on iNOSminusminus mice it considerably improvedthe myelin structure of mice without iNOS Cuprizone isa copper chelator which leads to direct oligodendrocytedeath with subsequent demyelination [37] In this modeloligodendrocyte death and demyelination are independent ofimmune and inflammatory response It was found that cGMPanalog (8-Br-cGMP) protects differentiated oligodendrocytesfrom death initiated by staurosporine thapsigargin orkainate [38] It is possible that sildenafil through theaccumulation of cGMP has a direct beneficial effect onoligodendrocytes protecting these cells and improvingmyelination independent of its anti-inflammatory effects

In conclusion the findings of the present study show thatiNOSminusminus mice are more susceptible to cuprizone intoxicationdue to the potential involvement of two mechanisms (1)iNOS-negative feedback mechanism in inflammatory condi-tions is absent and consequently proinflammatory proteinssuch as cytokines and COX-2 are excessively increased (2)eNOS is overexpressed by a compensatory mechanism andgenerates chronically high levels of NO damaging the tissueAlso the results of the present study suggest that sildenafilmay exert its anti-inflammatory effects mainly through iNOS

inhibition by cGMP-iNOS feedback In addition sildenafilmay have a direct protective effect on oligodendrocytes Fur-ther studies are required to explain themolecularmechanismof sildenafil protection in the central nervous system

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Catarina Raposo and Ana Karolina Santana Nunes wereinvolved in the treatment of animals development of WBIF and MET testing procedures analysis and interpretationof results and the drafting of the paper Catarina Raposoand Ana Karolina Santana Nunes contributed equally to thedevelopment of this study Rayana Leal de Almeida Lunaand Shyrlene Meiry da Rocha Araujo contributed to thetreatment of animals development of IH and LFB testingprocedures and analysis and interpretation of results MariaAlice da Cruz-Hofling contributed to the analysis and inter-pretation of data and paper revision Christina Alves Peixotocontributed to the supervision of technical procedures theanalysis and interpretation of data and paper revision andgave final approval of the version to be published

Acknowledgment

The authors are grateful to Ms Josineide Correia AnneGabrielle Vasconcelos Maria da Conceicao Carvalho andStephanie S M Federighi for their excellent technical assis-tance to CPqAM for the animal lodging facilities andCentrode Tecnologias Estrategicas do Nordeste (CETENE) forlaboratory facilities The study was supported by Fundacaode Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Ciencia e Tecnologia do Estadode Pernambuco (FACEPE) Conselho Nacional de Desen-volvimento Cientıfico e Tecnologico (CNPq) Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior (CAPES)and Instituto Nacional de Ciencia e Tecnologia de BiologiaEstrutural e Bioimagem (INBEB) Catarina Raposo is sup-ported by research fellowship from FAPESP Maria Aliceda Cruz-Hofling and Christina Alves Peixoto are supportedby research fellowships from CNPq They acknowledge theEnglish editing by Mr James Young

References

[1] C Nathan and Q-W Xie ldquoNitric oxide synthases roles tollsand controlsrdquo Cell vol 78 no 6 pp 915ndash918 1994

[2] C Nathan and Q-W Xie ldquoRegulation of biosynthesis of nitricoxiderdquo Journal of Biological Chemistry vol 269 no 19 pp13725ndash13728 1994

[3] A C Palmer I M Calder and J T Hughes ldquoSpinal corddegeneration in diversrdquo The Lancet vol 2 no 8572 pp 1365ndash1366 1987

[4] G Garthwaite and J Garthwaite ldquoCyclic GMP and cell deathin rat cerebellar slicesrdquo Neuroscience vol 26 no 1 pp 321ndash3261988


[5] R G Knowles M Palacios R M Palmer and S MoncadaldquoFormation of nitric oxide from L-arginine in the centralnervous system a transduction mechanism for stimulationof the soluble guanylate cyclaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 86 no13 pp 5159ndash5162 1989

[6] J B Hibbs Jr R R Taintor Z Vavrin and EM Rachlin ldquoNitricoxide a cytotoxic activated macrophage effector moleculerdquoBiochemical and Biophysical Research Communications vol 157no 1 pp 87ndash94 1988

[7] D J Stuehr and C F Nathan ldquoNitric oxide a macrophageproduct responsible for cytostasis and respiratory inhibition intumor target cellsrdquo Journal of Experimental Medicine vol 169no 5 pp 1543ndash1555 1989

[8] J V Esplugues ldquoNO as a signalling molecule in the nervoussystemrdquo British Journal of Pharmacology vol 135 no 5 pp1079ndash1095 2002

[9] L Meda M A Cassatella G I Szendrei et al ldquoActivation ofmicroglial cells by 120573-amyloid protein and interferon-120574rdquoNaturevol 374 no 6523 pp 647ndash650 1995

[10] J Prickaerts W C G Van Staveren A Sik et al ldquoEffects oftwo selective phosphodiesterase type 5 inhibitors sildenafil andvardenafil on object recognition memory and hippocampalcyclic GMP levels in the ratrdquo Neuroscience vol 113 no 2 pp351ndash361 2002

[11] E G McGeer and P L McGeer ldquoThe role of anti-inflammatoryagents in Parkinsonrsquos diseaserdquo CNS Drugs vol 21 no 10 pp789ndash797 2007

[12] G A Rosenberg ldquoNeurological diseases in relation to theblood-brain barrierrdquo Journal of Cerebral Blood Flow ampMetabolism vol 32 pp 1139ndash1151 2012

[13] D O Willenborg M Staykova S Fordham N OrsquoBrien and DLinares ldquoThe contribution of nitric oxide and interferon gammato the regulation of the neuro-inflammation in experimentalautoimmune encephalomyelitisrdquo Journal of Neuroimmunologyvol 191 no 1-2 pp 16ndash25 2007

[14] UC Sahrbacher F LechnerH P Eugster K FreiH Lassmannand A Fontana ldquoMice with an inactivation of the induciblenitric oxide synthase gene are susceptible to experimentalautoimmune encephalomyelitisrdquo European Journal of Immunol-ogy vol 28 no 4 pp 1332ndash1338 1998

[15] H A Arnett R P Hellendall G K Matsushima et al ldquoTheprotective role of nitric oxide in a neurotoxicant-induceddemyelinating modelrdquo Journal of Immunology vol 168 no 1pp 427ndash433 2002

[16] A T Bender and J A Beavo ldquoSpecific localized expression ofcGMP PDEs in Purkinje neurons and macrophagesrdquo Neuro-chemistry International vol 45 no 6 pp 853ndash857 2004

[17] J Schlossmann and F Hofmann ldquocGMP-dependent proteinkinases in drug discoveryrdquo Drug Discovery Today vol 10 no9 pp 627ndash634 2005

[18] MKipp TClarner SGingele et al ldquoBrain lipid binding protein(FABP7) as modulator of astrocyte functionrdquo PhysiologicalResearch vol 60 supplement 1 pp S49ndashS60 2011

[19] A Palmeri L Privitera S Giunta C Loreto and D PuzzoldquoInhibition of phosphodiesterase-5 rescues age-related impair-ment of synaptic plasticity and memoryrdquo Behavioural BrainResearch vol 240 pp 11ndash20 2013

[20] L Zhang W Yu I Schroedter J Kong and M VrontakisldquoGalanin transgenic mice with elevated circulating galaninlevels alleviate demyelination in a cuprizone-inducedmsmousemodelrdquo PLoS ONE vol 7 no 3 article e33901 2012

[21] P Pifarre J Prado M A Baltrons et al ldquoSildenafil (Viagra)ameliorates clinical symptoms and neuropathology in a mousemodel of multiple sclerosisrdquoActa Neuropathologica vol 121 no4 pp 499ndash508 2011

[22] A K S Nunes C Raposo R L Luna M A Cruz-Hoflingand C A Peixoto ldquoSildenafil (Viagra) down regulates cytokinesand prevents demyelination in a cuprizone-induced MS mousemodelrdquo Cytokine vol 60 pp 540ndash551 2012

[23] L Zhao N A Mason J W Strange H Walker and M RWilkins ldquoBeneficial effects of phosphodiesterase 5 inhibition inpulmonary hypertension are influenced by natriuretic peptideactivityrdquo Circulation vol 107 no 2 pp 234ndash237 2003

[24] K L A Saraiva A K Silva M I Wanderley A A De AraujoJ R De Souza and C A Peixoto ldquoChronic treatment withsildenafil stimulates Leydig cell and testosterone secretionrdquoInternational Journal of Experimental Pathology vol 90 no 4pp 454ndash462 2009

[25] D Moharregh-Khiabani A Blank T Skripuletz et al ldquoEffectsof fumaric acids on cuprizone induced central nervous systemde- and remyelination in the mouserdquo PLoS ONE vol 5 no 7article e11769 2010

[26] Z Gao and S E Tsirka ldquoAnimal Models of MS reveal multipleroles of microglia in disease pathogenesisrdquo Neurology ResearchInternational vol 2011 Article ID 383087 9 pages 2011

[27] L Silvestroff S Bartucci J Pasquini and P Franco ldquoCuprizone-induced demyelination in the rat cerebral cortex and thyroidhormone effects on cortical remyelinationrdquo Experimental Neu-rology vol 235 no 1 pp 357ndash367 2012

[28] G Riccitelli M A Rocca E Pagani et al ldquoMapping regionalgrey and white matter atrophy in relapsing-remitting multiplesclerosisrdquoMultiple Sclerosis vol 18 no 7 pp 1027ndash1037 2012

[29] S D Shields X Cheng A Gasser et al ldquoA channelopathycontributes to cerebellar dysfunction in a model of multiplesclerosisrdquo Annals of Neurology vol 71 no 2 pp 186ndash194 2012

[30] C Bernardini F Greco A Zannoni M L Bacci E Seren andM Forni ldquoDifferential expression of nitric oxide synthases inporcine aortic endothelial cells during LPS-induced apoptosisrdquoThe Journal of Inflammation vol 9 p 47 2012

[31] W-L Chang C-H Chung Y-C Wu and M-J Su ldquoThevascular and cardioprotective effects of liriodenine in ischemia-reperfusion injury via NO-dependent pathwayrdquo Nitric Oxidevol 11 no 4 pp 307ndash315 2004

[32] D Perez-Sala E Cernuda-Morollon M Dıaz-Cazorla FRodrıguez-Pascual and S Lamas ldquoPosttranscriptional regula-tion of human iNOS by the NOcGMP pathwayrdquo AmericanJournal of Physiology vol 280 pp 466ndash473 2001

[33] P K Datta S Dhupar and E A Lianos ldquoRegulatory effects ofinducible nitric oxide synthase on cyclooxygenase-2 and hemeoxygenase-1 expression in experimental glomerulonephritisrdquoNephrology Dialysis Transplantation vol 21 no 1 pp 51ndash572006

[34] A A Beg and D Baltimore ldquoAn essential role for NF-120581B inpreventing TNF-120572-induced cell deathrdquo Science vol 274 no5288 pp 782ndash784 1996

[35] R S Chanthaphavong P A Loughran T Y Lee M J Scottand T R Billiar ldquoA role for cGMP in inducible nitric-oxidesynthase (iNOS)-induced tumor necrosis factor (TNF) 120572-converting enzyme (TACEADAM17) activation translocationand TNF receptor 1 (TNFR1) shedding in hepatocytesrdquo Journalof Biological Chemistry vol 287 pp 35887ndash35898 2012



[37] K Benardais A Kotsiari J Skuljec et al ldquoCuprizone[bis(cyclohexylidenehydrazide)] is selectively toxic for matureoligodendrocytesrdquo Neurotoxicity Research vol 24 no 2 pp244ndash250 2013

[38] J A Benjamins and L Nedelkoska ldquoCyclic GMP-dependentpathways protect differentiated oligodendrocytes frommultipletypes of injuryrdquo Neurochemical Research vol 32 no 2 pp 321ndash329 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


the role of iNOSNO in inflammatory response oligodendro-cyte cell death and myelin damageloss

The cyclic guanosine 3101584051015840-monophosphate (cGMP) sig-naling pathway is an important NO-signaling molecule NObinds to soluble guanylyl cyclase (sGC) and increases concen-tration of cGMP activating signaling cascades and leading tocGMP-dependent responses [16 17]The cGMP signal can beterminated by the action of several phosphodiesterases (PDE)[18]The cGMP-selective PDE5 is expressed in the cardiovas-cular neural and immune systems [18] Studies have shownthat selective PDE5 inhibitors widely used in the treatmentof erectile dysfunction in humans such as sildenafil (ViagraPfizer) and vardenafil (Levitra Bayer) raise cGMP levels inthe brain and offer protective effects improve cognition andmemory [19] reduce neuronal cell death following ischemiccerebrovascular injury [20] decrease white matter damageand regulate inflammatory responses in MS models [21 22]Recent studies by the authors of an MS model induced inwild-type C57BL6 mice found that sildenafil has an anti-inflammatory action reducing levels of proinflammatorycytokines and cyclooxygenase-2 (COX-2) and protecting themyelin structure [22] However the mechanism of sildenafilneuroprotection remains unknown

In the present study inflammatory demyelination wasinduced in iNOSminusminusmice and sildenafil was administered forfour weeks The focus of this study was to identify the roleof a potent inflammation-associatedmolecule iNOS-derivedNO in protective mechanisms related to sildenafil Thepresent study also aimed to clarify the role of NO protectiveandor deleterious mechanisms in the demyelinating model

2 Materials and Methods

21 Experimental Design Five iNOS knockout (B 6129 P2-Nos2) mice aged 7 to 10 weeks weighing 15 to 20 g wereused per group The mice were examined for health statusacclimated to the laboratory environment at 25∘C and 12 hlightdark photoperiod and housed in metal cages The con-trol group received standard laboratory diet and pure waterOver a four-week period the experimental groups receivedeither 02 cuprizone (oxalic-bis-cyclohexylidenehydrazideSigma-Aldrich Inc St Louis MO USA) mixed into thechow and pure water or 02 cuprizone in the chow and25mgkg of bodyweight of sildenafil (Viagra Pfizer Inc NewYork NY USA) administered through the drinking water[22ndash24] Body weight was accessed every week and the drugconcentration in the water was adjusted tomaintain the doseAll experiments were carried out in compliance with ethicalguidelines for animal experimentation (L-102010-CEUA0510-CIBIO FIOCRUZ) After treatment the animals wereanaesthetized (im) with ketamine (115mgkg) and xylazine(10mgkg) (Sespo Comercio e Industria Ltda Sao Paulo SPBrazil)

22 Immunofluorescence (IF) After anesthesia the ani-mals were transcardially perfused with physiological saline(20mL) followed by 4 paraformaldehyde (Sigma-Aldrich)(40mL) in 01M phosphate (sodium phosphate monobasic

and dibasic heptahydrate Sigma-Aldrich) buffered saline(PBS) pH 72 Cerebella were dissected and immersed in15 sucrose overnight followed by 30 sucrose for a secondnight (36 hours total) The specimens were then embeddedin OCT-Tissue Tek compound (Sakura Finetek TorranceCA USA) and frozen in n-hexane (Dinamica Sao PauloSP Brazil) cooled with liquid nitrogen Cryosections (8120583mthick) were permeabilized (03Triton X-100) and incubatedfor 1 h with blocking solution (3 BSA plus 02 Tween20 in Tris buffered saline) Subsequently the sections wereincubated with antibodies for Glial Fibrillar Acidic Pro-tein (GFAP) (DakoCytomation cat no ZO 334) and Iba-1(Wako Osaka Japan cat no 019-19741) (both 1 100) Sec-tions were incubated with primary antibodies overnight andthen incubated with polyclonal Cy3-conjugated secondaryantibodies (Jackson cat no 705-165-147) against rabbitimmunoglobulin (1 200) for 1 h The slides were washedand mounted in fluorescent Prolong Gold Antifade medium(Life Technologies cat no P36930) for observation underan inverted fluorescence microscope (Zeiss MicroImagingGmbH) equipped with a camera (Zeiss AxioCamMRM) andthe Release 472 image analysis software

23 Immunohistochemistry (IH) After perfusion as de-scribed for IF cerebella were immediately removed andpostfixed in the same fixative overnight The samples weredehydrated in an ethanol series (Isofar Chemical Co RJBrazil) cleared in xylene and embedded in paraffin (Merckcatalog no 1071642504) Sections (5120583m thick) were cut on anRM 2035 microtome (Reichert S Leica) rehydrated washedin 005M PBS and incubated in this buffer with 1 bovineserum albumin (BSA fraction V) (Miles Naperville ILUSA) for one hour Endogenous peroxidase was blockedand antigen retrieval was performed pretreating the sec-tions with 20mM citrate buffer pH 60 at 100∘C for30min All groups were incubated with the rabbit polyclonalanti-COX-2 (Abcam CanadaUS cat no ab15191) (1 100overnight at 4∘C) After washing the sections were overlaidfor 1 h with a biotin-conjugated secondary antibody usingan HRP kit (DakoCytomation CA USA Biotinylated LinkUniversal HRP cat no K0690) and visualized with 31015840-3-diaminobenzidine (DAB) as the chromogen The sectionswere then weakly counterstained with Harrisrsquo hematoxylinand mounted in entellan (Merck cat no 1079610100)

24Western Blotting (WB) Cerebella were quickly dissectedand each group was homogenized in an extraction cocktail(10mM EDTA Amresco Solon USA 2mM phenylmethanesulfonyl-fluoride 100mM NaF 10mM sodium pyrophos-phate 10mM NaVO

4

10 120583g of aprotininmL and 100mMTris pH 74) Cerebella of five animals were mixed andhomogenized to form a pool from each group The WB wasdone in accord with Nunes et al [22] Briefly the pro-teins (40 120583g total) were separated on 65 (IFN-120574) 10(TNF-120572 IL-1120573) or 12 (GFAP eNOS) acrylamide gelAfter overnight in 5 nonfat milk the primary antibodiesagainst GFAP (1 10000 DakoCytomation cat no ZO 334)COX-2 (1 1000 Abcam cat no ab15191) TNF-120572 (1 1000






3 Results









GFAP 53 kDa

(a)

42 kDa

Cont CPZ CPZ + Sild

120573-Actin

(b)

0

50

100

150

200Re

lativ

e exp

ress

ion

com

pare

d to

cont

rol

Cont CPZ + SildCPZ

lowastlowastlowast

(c)


GFAP-control

W

20 120583m

(a)

CPZ

W

v

(b)

W

CPZ + Sild

(c)

Cont

MP

G

v

20 120583m

(d)

CPZ

M

PG

vv

(e)

G

P M

CPZ + Sild

(f)



Cont CPZ CPZ + Sild

17 kDaTNF-120572

0

100

200

300

400

500

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ CPZ + Sild

lowastlowastlowast

COX-2

Cont CPZ CPZ + Sild

64 kDa

0

200

400

600

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ CPZ + Sild

lowastlowast

lowastlowast

(a)

(b)

(c)

(d)


COX-2-control

20 120583m

(a)

CPZ

(b)

CPZ + Sild

(c)

Iba-1-control

20 120583m

(d)

CPZ

(e)

CPZ + Sild

(f)



Cont CPZ + SildCPZ

17 kDaIL-1 120573

0

50

100

150

200

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ + SildCPZ

lowastlowast

lowastlowastlowast

Cont CPZ + SildCPZ

17 kDaIFN-120574

0

50

100

150

200

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ + SildCPZ

lowastlowastlowast

lowastlowastlowast

(a)

(b)

(c)

(d)













eNOS 140 kDa

iNOSminusminus

CPZiNOSminusminus

CPZ + SildWTCont

iNOSminusminusCont

(a)

0

100

200

300

400

500

Rela

tive e

xpre

ssio

nco

mpa

red

to W

T co

ntro

l

lowast


iNOSminusminus

CPZiNOSminusminus

CPZ + SildWT


(b)






GSTpi

Cont CPZ CPZ + Sild

29 kDa

(a)

0

50

100

150

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ CPZ + Sild

lowastlowastlowast

lowastlowastlowast

(b)





4 Discussion



LFB-control

20 120583m

(a)

CPZ

(b)

CPZ + Sild

(c)

MET-control

2 120583m

(d)

CPZ

2 120583m

(e)

CPZ + Sild

2 120583m

(f)

Control

05 120583m

(g)

CPZ

05 120583m

(h)

CPZ + Sild

05 120583m

(i)

















Acknowledgment


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















3 Results









GFAP 53 kDa

(a)

42 kDa

Cont CPZ CPZ + Sild

120573-Actin

(b)

0

50

100

150

200Re

lativ

e exp

ress

ion

com

pare

d to

cont

rol

Cont CPZ + SildCPZ

lowastlowastlowast

(c)


GFAP-control

W

20 120583m

(a)

CPZ

W

v

(b)

W

CPZ + Sild

(c)

Cont

MP

G

v

20 120583m

(d)

CPZ

M

PG

vv

(e)

G

P M

CPZ + Sild

(f)



Cont CPZ CPZ + Sild

17 kDaTNF-120572

0

100

200

300

400

500

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ CPZ + Sild

lowastlowastlowast

COX-2

Cont CPZ CPZ + Sild

64 kDa

0

200

400

600

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ CPZ + Sild

lowastlowast

lowastlowast

(a)

(b)

(c)

(d)


COX-2-control

20 120583m

(a)

CPZ

(b)

CPZ + Sild

(c)

Iba-1-control

20 120583m

(d)

CPZ

(e)

CPZ + Sild

(f)



Cont CPZ + SildCPZ

17 kDaIL-1 120573

0

50

100

150

200

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ + SildCPZ

lowastlowast

lowastlowastlowast

Cont CPZ + SildCPZ

17 kDaIFN-120574

0

50

100

150

200

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ + SildCPZ

lowastlowastlowast

lowastlowastlowast

(a)

(b)

(c)

(d)













eNOS 140 kDa

iNOSminusminus

CPZiNOSminusminus

CPZ + SildWTCont

iNOSminusminusCont

(a)

0

100

200

300

400

500

Rela

tive e

xpre

ssio

nco

mpa

red

to W

T co

ntro

l

lowast


iNOSminusminus

CPZiNOSminusminus

CPZ + SildWT


(b)






GSTpi

Cont CPZ CPZ + Sild

29 kDa

(a)

0

50

100

150

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ CPZ + Sild

lowastlowastlowast

lowastlowastlowast

(b)





4 Discussion



LFB-control

20 120583m

(a)

CPZ

(b)

CPZ + Sild

(c)

MET-control

2 120583m

(d)

CPZ

2 120583m

(e)

CPZ + Sild

2 120583m

(f)

Control

05 120583m

(g)

CPZ

05 120583m

(h)

CPZ + Sild

05 120583m

(i)

















Acknowledgment


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GFAP 53 kDa

(a)

42 kDa

Cont CPZ CPZ + Sild

120573-Actin

(b)

0

50

100

150

200Re

lativ

e exp

ress

ion

com

pare

d to

cont

rol

Cont CPZ + SildCPZ

lowastlowastlowast

(c)


GFAP-control

W

20 120583m

(a)

CPZ

W

v

(b)

W

CPZ + Sild

(c)

Cont

MP

G

v

20 120583m

(d)

CPZ

M

PG

vv

(e)

G

P M

CPZ + Sild

(f)



Cont CPZ CPZ + Sild

17 kDaTNF-120572

0

100

200

300

400

500

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ CPZ + Sild

lowastlowastlowast

COX-2

Cont CPZ CPZ + Sild

64 kDa

0

200

400

600

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ CPZ + Sild

lowastlowast

lowastlowast

(a)

(b)

(c)

(d)


COX-2-control

20 120583m

(a)

CPZ

(b)

CPZ + Sild

(c)

Iba-1-control

20 120583m

(d)

CPZ

(e)

CPZ + Sild

(f)



Cont CPZ + SildCPZ

17 kDaIL-1 120573

0

50

100

150

200

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ + SildCPZ

lowastlowast

lowastlowastlowast

Cont CPZ + SildCPZ

17 kDaIFN-120574

0

50

100

150

200

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ + SildCPZ

lowastlowastlowast

lowastlowastlowast

(a)

(b)

(c)

(d)













eNOS 140 kDa

iNOSminusminus

CPZiNOSminusminus

CPZ + SildWTCont

iNOSminusminusCont

(a)

0

100

200

300

400

500

Rela

tive e

xpre

ssio

nco

mpa

red

to W

T co

ntro

l

lowast


iNOSminusminus

CPZiNOSminusminus

CPZ + SildWT


(b)






GSTpi

Cont CPZ CPZ + Sild

29 kDa

(a)

0

50

100

150

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ CPZ + Sild

lowastlowastlowast

lowastlowastlowast

(b)





4 Discussion



LFB-control

20 120583m

(a)

CPZ

(b)

CPZ + Sild

(c)

MET-control

2 120583m

(d)

CPZ

2 120583m

(e)

CPZ + Sild

2 120583m

(f)

Control

05 120583m

(g)

CPZ

05 120583m

(h)

CPZ + Sild

05 120583m

(i)

















Acknowledgment


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cont CPZ CPZ + Sild

17 kDaTNF-120572

0

100

200

300

400

500

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ CPZ + Sild

lowastlowastlowast

COX-2

Cont CPZ CPZ + Sild

64 kDa

0

200

400

600

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ CPZ + Sild

lowastlowast

lowastlowast

(a)

(b)

(c)

(d)


COX-2-control

20 120583m

(a)

CPZ

(b)

CPZ + Sild

(c)

Iba-1-control

20 120583m

(d)

CPZ

(e)

CPZ + Sild

(f)



Cont CPZ + SildCPZ

17 kDaIL-1 120573

0

50

100

150

200

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ + SildCPZ

lowastlowast

lowastlowastlowast

Cont CPZ + SildCPZ

17 kDaIFN-120574

0

50

100

150

200

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ + SildCPZ

lowastlowastlowast

lowastlowastlowast

(a)

(b)

(c)

(d)













eNOS 140 kDa

iNOSminusminus

CPZiNOSminusminus

CPZ + SildWTCont

iNOSminusminusCont

(a)

0

100

200

300

400

500

Rela

tive e

xpre

ssio

nco

mpa

red

to W

T co

ntro

l

lowast


iNOSminusminus

CPZiNOSminusminus

CPZ + SildWT


(b)






GSTpi

Cont CPZ CPZ + Sild

29 kDa

(a)

0

50

100

150

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ CPZ + Sild

lowastlowastlowast

lowastlowastlowast

(b)





4 Discussion



LFB-control

20 120583m

(a)

CPZ

(b)

CPZ + Sild

(c)

MET-control

2 120583m

(d)

CPZ

2 120583m

(e)

CPZ + Sild

2 120583m

(f)

Control

05 120583m

(g)

CPZ

05 120583m

(h)

CPZ + Sild

05 120583m

(i)

















Acknowledgment


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cont CPZ + SildCPZ

17 kDaIL-1 120573

0

50

100

150

200

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ + SildCPZ

lowastlowast

lowastlowastlowast

Cont CPZ + SildCPZ

17 kDaIFN-120574

0

50

100

150

200

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ + SildCPZ

lowastlowastlowast

lowastlowastlowast

(a)

(b)

(c)

(d)













eNOS 140 kDa

iNOSminusminus

CPZiNOSminusminus

CPZ + SildWTCont

iNOSminusminusCont

(a)

0

100

200

300

400

500

Rela

tive e

xpre

ssio

nco

mpa

red

to W

T co

ntro

l

lowast


iNOSminusminus

CPZiNOSminusminus

CPZ + SildWT


(b)






GSTpi

Cont CPZ CPZ + Sild

29 kDa

(a)

0

50

100

150

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ CPZ + Sild

lowastlowastlowast

lowastlowastlowast

(b)





4 Discussion



LFB-control

20 120583m

(a)

CPZ

(b)

CPZ + Sild

(c)

MET-control

2 120583m

(d)

CPZ

2 120583m

(e)

CPZ + Sild

2 120583m

(f)

Control

05 120583m

(g)

CPZ

05 120583m

(h)

CPZ + Sild

05 120583m

(i)

















Acknowledgment


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















eNOS 140 kDa

iNOSminusminus

CPZiNOSminusminus

CPZ + SildWTCont

iNOSminusminusCont

(a)

0

100

200

300

400

500

Rela

tive e

xpre

ssio

nco

mpa

red

to W

T co

ntro

l

lowast


iNOSminusminus

CPZiNOSminusminus

CPZ + SildWT


(b)






GSTpi

Cont CPZ CPZ + Sild

29 kDa

(a)

0

50

100

150

Relat

ive e

xpre

ssio

nco

mpa

red

to co

ntro

l

Cont CPZ CPZ + Sild

lowastlowastlowast

lowastlowastlowast

(b)





4 Discussion



LFB-control

20 120583m

(a)

CPZ

(b)

CPZ + Sild

(c)

MET-control

2 120583m

(d)

CPZ

2 120583m

(e)

CPZ + Sild

2 120583m

(f)

Control

05 120583m

(g)

CPZ

05 120583m

(h)

CPZ + Sild

05 120583m

(i)

















Acknowledgment


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















LFB-control

20 120583m

(a)

CPZ

(b)

CPZ + Sild

(c)

MET-control

2 120583m

(d)

CPZ

2 120583m

(e)

CPZ + Sild

2 120583m

(f)

Control

05 120583m

(g)

CPZ

05 120583m

(h)

CPZ + Sild

05 120583m

(i)

















Acknowledgment


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























Acknowledgment


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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