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 .
 we recently demonstrated that sildenafil reduces the expression of cytokines , cox-2 , and gfap in a demyelinating model induced in wild - type ( wt ) mice . 
 herein , the understandings of the neuroprotective effect of sildenafil and the mediation of inos / no system on inflammatory demyelination induced by cuprizone were investigated . 
 the cerebella of inos/ mice were examined after four weeks of treatment with cuprizone alone or combined with sildenafil . 
 cuprizone increased gfap , iba-1 , tnf- , cox-2 , il-1 , and ifn- expression , decreased expression of glutathione s - transferase pi ( gstpi ) , and damaged myelin in inos/ mice . 
 sildenafil reduced iba-1 , ifn- , and il-1 levels but had no effect on the expression of gfap , tnf- , and cox-2 compared to the cuprizone group . 
 sildenafil elevated gstpi levels and improved the myelin structure / ultrastructure . 
 inos/ mice suffered from severe inflammation following treatment with cuprizone , while wt mice had milder inflammation , as found in the previous study . 
 it is possible that inflammatory regulation through inos - feedback is absent in inos/ mice , making them more susceptible to inflammation . 
 sildenafil has at least a partial anti - inflammatory effect through inos inhibition , as its effect on inos/ mice was limited . 
 further studies are required to explain the underlying mechanism of the sildenafil effects . 
1. Background 
2. Materials and Methods
3. Results
4. Discussion