nitric oxide ( no ) is a highly reactive molecule with a range of physiological functions [ 1 , 2 ] . this messenger plays an important role in the modulation of vascular tone , neurotransmission [ 4 , 5 ] , and immune system [ 68 ] . in addition to the constitutive forms of the enzyme ( endothelial ( enos or nos3 ) and neuronal ( nnos or nos1 ) ) , there is also an inducible form ( inos or nos2 ) . this last is most commonly associated with inflammatory conditions in which no is produced in large amounts . there is strong evidence to suggest the involvement of inos in the development of neurodegenerative disease . induction of inos , no , and no byproducts has been found in multiple sclerosis ( ms ) patients and animal models and correlates with disease severity and level of inflammatory infiltrate [ 912 ] . interestingly , however , inos - deficient mice developed a more severe ms model [ 13 , 14 ] . it was found that the elimination of inos does not improve , and may actually aggravate , demyelination in the cuprizone - demyelinating model , which suggests that the inos / no system may be neuroprotective . more information is required to understand the role of inos / no in inflammatory response , oligodendrocyte cell death , and myelin damage / loss . the cyclic guanosine 3,5-monophosphate ( cgmp ) signaling pathway is an important no - signaling molecule . no binds to soluble guanylyl cyclase ( sgc ) and increases concentration of cgmp , activating signaling cascades and leading to cgmp - dependent responses [ 16 , 17 ] . the cgmp signal can be terminated by the action of several phosphodiesterases ( pde ) . the cgmp - selective pde5 is expressed in the cardiovascular , neural , and immune systems . studies have shown that selective pde5 inhibitors , widely used in the treatment of erectile dysfunction in humans , such as sildenafil ( viagra ; pfizer ) and vardenafil ( levitra ; bayer ) , raise cgmp levels in the brain and offer protective effects , improve cognition and memory , reduce neuronal cell death following ischemic cerebrovascular injury , decrease white matter damage , and regulate inflammatory responses in ms models [ 21 , 22 ] . recent studies by the authors of an ms model induced in wild - type c57bl6 mice found that sildenafil has an anti - inflammatory action , reducing levels of proinflammatory cytokines and cyclooxygenase-2 ( cox-2 ) and protecting the myelin structure . however , the mechanism of sildenafil neuroprotection remains unknown .   in the present study , inflammatory demyelination was induced in inos mice , and sildenafil was administered for four weeks . the focus of this study was to identify the role of a potent inflammation - associated molecule , inos - derived no , in protective mechanisms related to sildenafil . the present study also aimed to clarify the role of no protective and/or deleterious mechanisms in the demyelinating model . five inos knockout ( b 6.129 p2- nos2 ) mice , aged 7 to 10 weeks , weighing 15 to 20  g , were used per group . the mice were examined for health status , acclimated to the laboratory environment at 25c and 12  h light / dark photoperiod , and housed in metal cages . the control group received standard laboratory diet and pure water . over a four - week period , the experimental groups received either 0.2% cuprizone ( oxalic - bis - cyclohexylidenehydrazide sigma - aldrich inc . , st . louis , mo , usa ) mixed into the chow and pure water or 0.2% cuprizone in the chow and 25  mg / kg of body weight of sildenafil ( viagra ; pfizer inc . , new york , ny , usa ) administered through the drinking water [ 2224 ] . body weight was accessed every week and the drug concentration in the water was adjusted to maintain the dose . all experiments were carried out in compliance with ethical guidelines for animal experimentation ( l-10/2010-ceua ; 05/10-cibio fiocruz ) . after treatment , the animals were anaesthetized ( i.m . ) with ketamine ( 115  mg / kg ) and xylazine ( 10  mg / kg ) ( sespo comrcio e indstria ltda . , the animals were transcardially perfused with physiological saline ( 20  ml ) , followed by 4% paraformaldehyde ( sigma - aldrich ) ( 40  ml ) in 0.1  m phosphate ( sodium phosphate monobasic and dibasic heptahydrate , sigma - aldrich ) buffered saline ( pbs ) , ph 7.2 . cerebella were dissected and immersed in 15% sucrose overnight , followed by 30% sucrose for a second night ( 36 hours total ) . the specimens were then embedded in oct - tissue tek compound ( sakura finetek , torrance , ca , usa ) and frozen in n - hexane ( dinmica , so paulo , sp , brazil ) cooled with liquid nitrogen . cryosections ( 8  m thick ) were permeabilized ( 0.3% triton x-100 ) and incubated for 1  h with blocking solution ( 3% bsa plus 0.2% tween 20 in tris buffered saline ) . subsequently , the sections were incubated with antibodies for glial fibrillar acidic protein ( gfap ) ( dakocytomation , cat . sections were incubated with primary antibodies overnight and then incubated with polyclonal cy3-conjugated secondary antibodies ( jackson , cat . no . 705 - 165 - 147 ) against rabbit immunoglobulin ( 1  :  200 ) for 1  h. the slides were washed and mounted in fluorescent prolong gold antifade medium ( life technologies , cat . p36930 ) for observation under an inverted fluorescence microscope ( zeiss microimaging gmbh ) equipped with a camera ( zeiss axiocam mrm ) and the release 4.7.2 image analysis software . after perfusion as described for if , cerebella were immediately removed and postfixed in the same fixative overnight . the samples were dehydrated in an ethanol series ( isofar chemical co. , rj , brazil ) , cleared in xylene , and embedded in paraffin ( merck , catalog no . sections ( 5  m thick ) were cut on an rm 2035 microtome ( reichert s , leica ) , rehydrated , washed in 0.05  m pbs , and incubated in this buffer with 1% bovine serum albumin ( bsa , fraction v ) ( miles , naperville , il , usa ) for one hour . endogenous peroxidase was blocked and antigen retrieval was performed , pretreating the sections with 20  mm citrate buffer , ph 6.0 , at 100c , for 30  min . all groups were incubated with the rabbit polyclonal anti - cox-2 ( abcam , canada / us , cat . ab15191 ) ( 1  :  100 , overnight at 4c ) . after washing , the sections were overlaid for 1  h with a biotin - conjugated secondary antibody using an hrp kit ( dakocytomation , ca , usa , biotinylated link universal hrp ; cat . the sections were then weakly counterstained with harris ' hematoxylin and mounted in entellan ( merck , cat . cerebella were quickly dissected , and each group was homogenized in an extraction cocktail ( 10  mm edta , amresco , solon , usa ; 2  mm phenylmethane sulfonyl - fluoride , 100  mm naf , 10  mm sodium pyrophosphate , 10  mm navo4 , 10  g of aprotinin / ml , and 100  mm tris , ph 7.4 ) . cerebella of five animals were mixed and homogenized to form a pool from each group . briefly , the proteins ( 40  g total ) were separated on 6.5% ( ifn- ) , 10% ( tnf- , il-1 ) , or 12% ( gfap , enos ) acrylamide gel . after overnight in 5% nonfat milk , the primary antibodies against gfap ( 1  :  10,000 , dakocytomation , cat . zo 334 ) , cox-2 ( 1  :  1000 , abcam , cat . no . ab15191 ) , tnf- ( 1  :  1000 , peprotech , nj , usa , cat . 500p119 ) , il-1 ( 1  :  2500 , genway biotech , inc . , ca / usa , cat . no . 18 - 732 - 292194 ) , gst3/gstpi ( 1  :  250 , abcam , canada , cat . ab53943 ) , and enos ( 1  :  1000 , bd biosciences , cat . 610299 ) were incubated for four hours followed by horseradish peroxidase - conjugated antibodies anti - rabbit ( 1  :  80,000 , sigma - aldrich , cat . a9169 ) , anti - mouse ( 1  :  1,000 , sigma - aldrich , cat . a0168 ) , or anti - goat secondary antibody ( 1  :  100,000 , sigma - aldrich , cat . , the pixel density of each band was determined using the image j 1.38 software ( http://rsbweb.nih.gov/ij/download.html ; developed by wayne rasband , nih , bethesda , md , usa ) . for each protein investigated , the results were confirmed in three sets of experiments . immunoblotting for -actin ( 1  :  1,000 , sigma - aldrich , cat . after perfusion as described for if , the samples were dehydrated in a graded ethanol series , cleared in xylene , and embedded in paraffin . sections ( 5  m thick ) were cut on an rm 2035 microtome ( reichert s , leica ) . myelin was detected using luxol - fast blue ( lfb ) staining ( solvent blue 38 ; sigma - aldrich ) in accord with nunes et al . . the sections were observed under an inverted microscope ( zeiss microimaging gmbh ) equipped with a camera ( zeiss axiocam mrm ) and release 4.7.2 image analysis software ( zeiss ) . after anesthesia , the animals were sacrificed by transcardial perfusion with physiological saline ( 20  ml ) , followed by 40  ml of fixative2.5% glutaraldehyde and 4% paraformaldehyde in 0.1  m sodium cacodylate acid ( sigma - aldrich ) buffer , ph 7.2 . next , cerebellum fragments were washed twice in the same buffer and postfixed in a solution containing 1% osmium tetroxide ( sigma - aldrich ) , 2  mm calcium chloride , and 0.8% potassium ferricyanide ( sigma - aldrich ) in 0.1  m cacodylate buffer , ph 7.2 , dehydrated in acetone and embedded in spin - pon resin ( embed 812-electron microscopy science , washington , pa , usa ) . resin polymerization was performed at 60c for 3 days . semithin sections ( 0.5  m in thickness ) were placed on glass slides , stained with toluidine blue . ultrathin sections ( 70  nm in thickness ) were placed on 300-mesh nickel grids , counterstained with 5% uranyl acetate ( electron microscopy science ) and lead citrate ( sigma - aldrich ) , and examined using a fei morgagni 268d transmission electron microscope . the densitometric values of the immunoreactive bands ( immunoblotting ) were analyzed using the graphpad prism software package ( san diego , ca , usa ) . one - way analysis of variance ( anova ) , followed by dunnett 's and/or tukey 's posttest , was used to compare groups . clinical analysis revealed that cuprizone - treated animals suffered from motor limitations , such as tremors , and abnormal walking and posture . the group treated with sildenafil ( 25  mg / kg ) exhibited normal walking and posture , and tremors were either mild or nonexistent . the inos control animals exhibited normal motor function and posture and explored their environment normally . the mice treated with cuprizone exhibited arched ( shortened ) posture and tremors and had difficulty in exploring the environment . mice treated with 25  mg / kg of sildenafil both walked and were able to explore the environment normally , with no or mild tremors , and were classified as score 1 . cuprizone increased gfap , tnf- , and cox-2 expression in cerebellum , indicating astrocyte activation ( reactive gliosis ) and neuroinflammation . sildenafil treatment did not reduce the levels of these proteins in mice without inos . western blotting ( wb ) analysis showed that gfap , a marker of astrocyte activation ( reactive gliosis ) , was present in the cerebellum of untreated inos animals ( control ) ( figure 1(a ) ) . treatment with 0.2% cuprizone for four weeks significantly increased expression of this protein ( figures 1(a ) and 1(c ) ; p < 0.001 ) . animals that concomitantly received sildenafil ( 25  mg / kg ) and cuprizone also exhibited a high level of gfap in comparison to control ( figures 1(a ) and 1(c ) ; p < 0.001 ) . immunofluorescence ( if ) analysis of gfap in the cerebellum revealed the expression and location of this cytoskeletal intermediate filament protein in the animals . in the molecular layer of the cerebellum , gfap labeling revealed long astrocytic processes with a typical arrangement that was perpendicular to the pia mater membrane ( bergmann glia ) ( figure 2(e ) ) . astrocytic processes were also seen around other cells and vessels ( arrowheads in figures 2(b ) , 2(d ) , and 2(e ) ) . the control group showed basal expression of gfap localized normally ( figures 2(a ) and 2(d ) ) . treatment with cpz induced astrogliosis , increasing the intensity of labeling in the astrocytes ( figures 2(b ) and 2(e ) ) . in the sildenafil group , gfap labeling was more intense than for control ( figures 2(c ) and 2(f ) ) . the inos control group had a basal level of tnf- ( figure 3(a ) ) . wb analysis showed that cuprizone induced a significant increase of this cytokine , indicating neuroinflammation ( figures 3(a ) and 3(b ) ; p < 0.05 , compared to control group ) . animals that received cuprizone plus sildenafil also had a significant increase of tnf- , compared to control ( p < 0.01 ) . this enzyme was expressed in a minimal amount in the cerebellum of the inos control group ( figures 3(c ) and 4(a ) ) . cpz administration induced a significant increase in cox-2 expression , compared to control ( figures 3(c ) and 3(d ) ; p < 0.01 ) . sildenafil treatment did not decrease cox-2 , which remained high in comparison with the control group ( figures 3(c ) and 3(d ) ; p < 0.01 ) . ih labeling revealed the expression and location of cox-2 ( figures 4(a)4(c ) ) , which was mainly found in the white matter of the cerebellum . the control group had a very low expression of this enzyme ( figure 4(a ) ) , while treated animals had a high expression of cox-2 in white matter ( figures 4(b ) and 4(c ) ) . cuprizone increased iba-1 , il-1 , and ifn- expression in the cerebellum of inos mice . while sildenafil decreased the expression of these proteins , the level of expression remained above that of control animals . there was physiological expression of iba-1 in the cerebellum of inos control animals , with cells with processes that were typically branched , thin and weakly labeled ( arrow , figure 4(d ) ) . cuprizone treatment induced a stronger expression of iba-1 ( figure 4(e ) ) , compared to the control group , with thicker and more intensely labeled microglial processes . these processes lost their typical thin branched appearance , indicating that the cell phenotype had acquired activated characteristics . sildenafil together with cpz decreased iba-1 expression , and the microglia exhibited thin , highly branched processes , typical of a latent state ( figure 4(f ) ) . immunoblotting for il-1 and ifn- revealed basal expression of these cytokines in the inos control group ( figures 5(a ) and 5(c ) ) . cuprizone strongly increased the expression of these cytokines ( both p < 0.001 ) , compared to the control group ( figures 5(a)5(d ) ) , which indicates neuroinflammation . sildenafil treatment resulted in a significant decrease of il-1 and ifn- compared to the cuprizone group ( p < 0.01 ) . however , the levels of il-1 and ifn- remained higher in the sildenafil - treated group when compared with baseline levels of the control group ( p < 0.01 and p < 0.001 , resp . ) . mice without inos underwent a significant increase in enos expression , compared to wild - type animals . untreated mice inos ( control group ; p < 0.01 ) and inos animals treated with cpz or cpz + sild ( both p < 0.001 ) showed a significant increase in enos levels , compared with wild - type mice that did not undergo treatment . the expression of enos in inos mice treated with cpz and cpz plus sildenafil was not significantly higher when compared to inos control animals but showed a propensity to increase ( figures 6(a ) and 6(b ) ) . gstpi , a marker of myelinating oligodendrocytes , was expressed in the cerebellum of the control group ( figure 7(a ) ) . after treatment with cpz , this marker decreased significantly ( p < 0.001 ) compared to the control group , suggesting an impairment of mature oligodendrocytes and consequent myelin damage . sildenafil treatment together with cpz induced partial recovery of the gstpi marker , indicating that sildenafil had a protective effect on mature oligodendrocytes . in the sildenafil group , gstpi decreased in comparison with control ( p < 0.001 ) but increased in comparison to the cuprizone group ( p < 0.001 ; figures 7(a ) and 7(b ) ) . myelin sheath structure was disorganized in animals without inos which did not undergo treatment ( control ) . interestingly , inos animals without treatment ( control ) had moderate disorganization of the myelin structure and ultrastructure ( figures 8(a ) , 8(d ) , and 8(g ) ) . standard lfb staining showed that the control group inos had vacuoles in white matter ( arrow in figure 8(a ) ) , resulting in inhomogeneous and disorganized tissue . qualitative analysis of ultrathin cerebellum sections by tem revealed that the myelin sheath was often shredded , with blanks , and without the characteristic lamellar pattern ( arrows in figures 8(d ) and 8(g ) ) . cuprizone - treated animals exhibited more severe damage to myelin structure and ultrastructure ( figures 8(b ) , 8(e ) , and 8(h ) ) . lfb staining ( figure 8(b ) ) showed vacuoles ( arrows ) and spaces ( arrowhead ) as slits in white matter , characteristic of highly disorganized tissue . tem revealed that cpz caused serious damage to the myelin sheath ultrastructure , which had numerous spaces between the shreds in practically all fibers ( represented by arrows in figures 8(e ) and 8(h ) ) . simultaneous treatment with sildenafil and cpz resulted in a noticeable improvement of myelin organization ( figures 8(c ) , 8(f ) , and 8(i ) ) . white matter was more homogeneous , presenting fewer and , in general , smaller vacuoles and slit - like spaces ( figure 8(c ) ) . ultrathin section analysis revealed a more preserved myelin sheath which rarely showed signs of shredding . the cuprizone model is characterized by primary and reversible demyelination , due to peripheral immune system - independent myelin injury . in this model , demyelination is accompanied by a well - characterized sequence of events involving the depletion of mature oligodendrocytes , microglia activation , and astrocyte proliferation . therefore , demyelination and resident neuroinflammation induced by cuprizone in rodents have been widely used as a model for ms [ 20 , 27 ] . in the present study , the usefulness of the cuprizone model is considerable as it allows evaluation of the role of the inos / no - sgc - cgmp pathway in the inflammation and demyelination mediated by resident cns cells . the cerebellum was chosen for analysis , as it is an important cns affected region in ms patients , revealing severe white matter atrophy [ 28 , 29 ] . the present data , relating to inos animals , will be discussed with reference to this previous study . ( 2012 )   showed that in c57bl/6 wild - type ( wt ) mice cuprizone induced tissue damage , increased gfap , iba-1 , and cox-2 expression , and caused demyelination , in comparison to the control group . however , cuprizone did not affect the expression of cytokines ( tnf- , ifn- , il-1 , and il-2 ) in wt mice . in the previous study by the authors , sildenafil reduced gfap and iba-1 expression in comparison to the cuprizone group , preserved myelin and axon ultrastructure , and significantly downregulated ifn- , tnf- , il-1 , il-2 , and cox-2 expression in comparison to the control and/or cuprizone groups . the previous results demonstrated the protective effect of sildenafil on the cerebellum . it was considered important to investigate the role of no in the ms - model and the effects of sildenafil more profoundly . therefore , in the present study , the relationship between inosnull mice , the ms - model , and the effects of sildenafil in the cerebellum was investigated .    in the absence of inos iba-1 , il-1 , and ifn-. in addition , cuprizone intoxication decreased gstpi , a marker for myelinating oligodendrocytes , damaged myelin , and induced tremors , abnormal walking , and posture . this data indicates that cuprizone intoxication occurred even without the inos - no system and was stronger than in wt mice , where cuprizone did not increase cytokine expression . it was hypothesized that , in the absence of inos , enos may be overexpressed as a compensatory mechanism . .   showed that treatment with endotoxin influenced nos expression , upregulating inos and , simultaneously , downregulating enos . in fact , it was found here that enos was strongly expressed in inos animals in comparison with wt mice . the enos levels remained high after the administration of cuprizone and cuprizone plus sildenafil . in normal conditions , low levels of no produced by both enos and nnos participate in cell signaling and regulate physiologic processes . however , enos overexpression ( and consequently , constant high concentration of no ) can be responsible for myelin changes and proinflammatory susceptibility in inos animals . on the other hand , inos possesses an important feedback mechanism in inflammatory conditions , when the increase of this enzyme is self - regulated , and induces a reduction of some proinflammatory proteins [ 3235 ] . the inhibition of inos activity induces enhancement of il-1 , il-6 , and tnf- levels   and , subsequently , a persistent increase of inos expression , downregulating the tnf receptor . in the present study , absence of inos may explain increased tnf- , ifn- , and il-1 after cuprizone treatment in inos mice , while cuprizone did not increase these cytokines in wt mice . another hypothesis for explaining the more severe inflammation induced by cuprizone in mice without inos is the absence of inos feedback mechanism . interestingly , although sildenafil had a low anti - inflammatory effect on inos mice , it considerably improved the myelin structure of mice without inos . cuprizone is a copper chelator which leads to direct oligodendrocyte death with subsequent demyelination . in this model , oligodendrocyte death and demyelination are independent of immune and inflammatory response . it was found that cgmp analog ( 8-br - cgmp ) protects differentiated oligodendrocytes from death initiated by staurosporine , thapsigargin , or kainate . it is possible that sildenafil , through the accumulation of cgmp , has a direct beneficial effect on oligodendrocytes , protecting these cells and improving myelination , independent of its anti - inflammatory effects .    in conclusion , the findings of the present study show that inos mice are more susceptible to cuprizone intoxication due to the potential involvement of two mechanisms : ( 1 ) inos - negative feedback mechanism in inflammatory conditions is absent and , consequently , proinflammatory proteins , such as cytokines and cox-2 , are excessively increased ; ( 2 ) enos is overexpressed by a compensatory mechanism and generates chronically high levels of no , damaging the tissue . also , the results of the present study suggest that sildenafil may exert its anti - inflammatory effects mainly through inos inhibition , by cgmp - inos feedback . in addition , sildenafil may have a direct protective effect on oligodendrocytes . further studies are required to explain the molecular mechanism of sildenafil protection in the central nervous system .