If You Have Ms This Might Be Pertinent.

[mrd]

If you have MS you might want to look into this further and talk to your Dr about it. It might be worth a try.

 

Let the jokes about Viagra begin!

 

 

Public release date: 19-May-2011

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Contact: Agustina Garcia

Agustina.garcia@uab.cat

34-935-812-802

Universitat Autonoma de Barcelona

Viagra could reduce multiple sclerosis symptoms

 

 

 

 

 

IMAGE: The research team at UAB was headed by Agustina Garcia.

Click here for more information.

 

 

 

Universitat Autònoma de Barcelona researchers have discovered that Viagra® drastically reduces multiple sclerosis symptoms in animal models with the disease. The research, published in Acta Neuropathologica, demonstrates that a practically complete recovery occurs in 50% of the animals after eight days of treatment. Researchers are confident that clinical trials soon will be carried out in patients given that the drug is well tolerated and has been used to treat sexual dysfunction in some multiple sclerosis patients.

 

Multiple sclerosis is the most common chronic inflammatory disease of the central nervous system and one of the main causes of disability among young adults. The disease is caused by the presence of multiple focuses of demyelination (loss of myelin sheaths around the axons, affecting the ability of neurons to communicate) and neurodegeneration in different areas of the central nervous system. There is currently no cure for the disease, although some drugs have proven effective in fighting symptoms and preventing it from progressing.

 

A research team from the UAB Institute of Biotechnology and Biomedicine directed by Dr Agustina García, in collaboration with the research team directed by Dr Juan Hidalgo from the UAB Institute of Neurosciences, has studied the effects of a treatment using sildenafil, sold as Viagra®, in an animal model of multiple sclerosis known as experimental autoimmune encephalomyelitis (EAE). Researchers demonstrated that a daily treatment with sildenafil after disease onset quickly reduced clinical signs, with a practically complete recovery in 50% of the cases after eight days of treatment. Scientists observed how the drug reduced the infiltration of inflammatory cells into the white matter of the spinal cord, thus reducing damage to the nerve cell's axon and facilitating myelin repair.

 

Sidenafil, together with tadalafil (Cialis®) and vardenafil (Levitra®), form part of a group of vasodilator drugs known as phosphodiesterase type 5 (PDE5) inhibitors, used in the treatment of erectile dysfunction and pulmonary arterial hypertension. Recent studies in animal models of central nervous system pathologies already pointed to the fact that in addition to vasodilation, these drugs could contain other neuroprotective actions and suggest their usefulness as possible treatments of both acute (cerebrovascular stroke) and chronic (Alzheimer's) neuropathologies. In fact, in a research published in 2010 in the Journal of Neurochemistry, the same research group from UAB demonstrated that one of these inhibitors reduced neuroinflammation and neuronal damage in animal models of traumatic brain injury.

 

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[+JuztBudz]

Good find Mrd, thank you. j.b.

[mrd]

Zap,

 

Sorry but the study was done on female rats. I am passing on this info so any one afflicted can decide if taking it for a week is a good idea or not. Since the drug is already used on men who have MS related impotence the question of how it affects people have been looked into.

 

 

 

 

ORIGINAL PAPER

Sildenafil (Viagra) ameliorates clinical symptoms

and neuropathology in a mouse model of multiple sclerosis

Paula Pifarre • Judith Prado • Marı´a Antonia Baltrons •

Merce Giralt • Pere Gabarro • Douglas L. Feinstein •

Juan Hidalgo • Agustina Garcia

Received: 29 October 2010 / Revised: 21 December 2010 / Accepted: 30 December 2010 / Published online: 15 January 2011

Springer-Verlag 2011

Abstract Cyclic GMP (cGMP)-mediated pathways regulate

inflammatory responses in immune and CNS cells.

Recently, cGMP phosphodiesterase inhibitors such as sildenafil,

commonly used to treat sexual dysfunction in

humans including multiple sclerosis (MS) patients, have

been reported to be neuroprotective in animal models of

stroke, Alzheimer’s disease, and focal brain lesion. In this

work, we have examined if sildenafil ameliorates myelin

oligodendrocyte glycoprotein peptide (MOG35–55)-induced

experimental autoimmune encephalomyelitis (EAE), a

mouse model of MS. We show for the first time that

treatment with sildenafil after disease onset markedly

reduces the clinical signs of EAE by preventing axonal loss

and promoting remyelination. Furthermore, sildenafil

decreases CD3? leukocyte infiltration and microglial/

macrophage activation in the spinal cord, while increasing

forkhead box transcription factor 3-expressing T regulatory

cells (Foxp3 Tregs). However, sildenafil treatment did not

significantly affect MOG35–55-stimulated proliferation or

release of Th1/Th2 cytokines in splenocytes but decreased

ICAM-1 in spinal cord infiltrated cells. The presence of

reactive astrocytes forming scar-like structures around

infiltrates was enhanced by sildenafil suggesting a possible

mechanism for restriction of leukocyte spread into healthy

parenchyma. These results highlight novel actions of sildenafil

that may contribute to its beneficial effects in EAE

and suggest that treatment with this widely used and welltolerated

drug may be a useful therapeutic intervention to

ameliorate MS neuropathology.

Introduction

Experimental autoimmune encephalomyelitis (EAE) has

been extensively used as an animal model of multiple

sclerosis (MS) since it shares with the human autoimmune

disease the presence of inflammatory infiltrates in the CNS

parenchyma, demyelination and axonal loss predominantly

in the spinal cord, and paralysis [13]. Like MS, EAE seems

to be initiated by myelin antigen-specific CD4? T-lymphocyte

infiltration into the CNS. CD4? cells together

with infiltrated macrophages, dendritic cells, and resident

microglia constitute the ultimate effector cells of neuroinflammation,

progression of demyelination, and axonal

damage [7, 13]. In contrast, accumulating evidence indicates

that local astroglial activation is neuroprotective.

As in locally triggered innate immune responses caused

by trauma or stroke, reactive astrocytes in EAE have

been reported to form scar-like barriers that restrict leukocyte

infiltration into the CNS parenchyma [33, 35].

Electronic supplementary material The online version of this

article (doi:10.1007/s00401-010-0795-6) contains supplementary

material, which is available to authorized users.

P. Pifarre J. Prado M. A. Baltrons

P. Gabarro A. Garcia (&)

Institute of Biotechnology and Biomedicine,

Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain

e-mail: agustina.garcia@uab.cat

M. A. Baltrons A. Garcia

Department of Biochemistry and Molecular Biology,

Universitat Autonoma de Barcelona, Bellaterra, Spain

M. Giralt J. Hidalgo

Institute of Neurosciences and Department of Cellular Biology,

Physiology and Immunology, Universitat Autonoma de

Barcelona, Bellaterra, Spain

D. L. Feinstein

Department of Anesthesiology,

University of Illinois, Chicago, IL, USA

123

Acta Neuropathol (2011) 121:499–508

DOI 10.1007/s00401-010-0795-6

Additionally, astrocytes are known to release anti-inflammatory

cytokines, ROS scavengers, and growth factors that

could restrict local inflammation and promote nerve

recovery [18]. Interestingly, recent evidence indicates that

demyelination and oligodendrocyte degeneration follows

inflammation-induced astrocyte dysfunction [30]. High

levels of inflammatory mediators (cytokines, chemokines,

and NO) are secreted by infiltrating immune cells and

resident CNS cells and characterize the inflammatory

environment during disease [28]. Up-regulation of NO

synthase type 2 (NOS2) has been reported in MS and EAE,

and NO and peroxynitrite, the highly reactive product of

NO reaction with superoxide, have been implicated in

tissue damage [28]. However, NOS2-deficient mice

develop more severe EAE [38], suggesting that NO may be

neuroprotective. A major target for NO in many cell types

including CNS cells and lymphocytes is NO-sensitive

guanylyl cyclase (GC) [12]. It is well established that NO

via cGMP regulates important neuronal functions such as

synaptic plasticity processes involved in memory formation;

however, very little is known about the function of

this pathway in glial cells or the contribution of cGMP to

NO effects during neuroinflammation. In both neurons and

astrocytes, the NO-cGMP pathway has been reported to

activate antiapoptotic mechanisms [32]. Furthermore, we

have recently shown that in astrocytes this signaling

pathway is involved in the rearrangement of actin and

GFAP filaments that results in astrocyte stellation and

enhanced motility suggesting a role in the regulation of the

reactive phenotype [3]. In agreement with this, other

authors have demonstrated that the same pathway is

involved in up-regulation of GFAP, a marker of astrogliosis

[4]. Immune and glial cells also produce cGMP by

stimulation of natriuretic peptide (NP) receptors [26].

Atrial natriuretic peptide (ANP) has been shown to affect

innate and adaptive immune responses and to reduce production

of proinflammatory mediators by macrophages and

microglia [21, 34]. Neuroprotective actions of NPs have

been reported in animal models of cerebral ischemia [37]

and traumatic brain injury [16].

The cGMP signal can be terminated by the action of

several members of the large family of cyclic nucleotide

phosphodiesterase (PDE) [8]. cGMP-selective PDE5, well

known for its important actions in the cardiovascular system,

is also expressed in neural and immune cells [8].

PDE5 inhibitors, such as sildenafil, which are widely used

for treatment of erectile dysfunction in humans, have been

shown to improve cognition in unimpaired and impaired

rodents [27, 29], to increase neurogenesis and enhance

functional recovery after stroke [40] and to regulate glial

inflammatory responses and decrease neuronal cell death

after cortical cryoinjury [25]. In this work, we show that

sildenafil administration after disease onset rapidly

ameliorates MOG35–55-induced EAE in mice by preventing

axonal loss and enhancing remyelination. Sildenafil regulation

of inflammatory cell infiltration and glial reactivity

may underlay these beneficial effects. Results suggest that

this drug may be beneficial in MS.

Materials and methods

Animals and treatments

Two-month-old female C57BL/6 mice (Charles River) were

housed in the animal care facility of the Universitat Autonoma

de Barcelona (UAB) under constant temperature and

provided food and water ad libitum. EAE was induced by

immunization with MOG35–55 peptide (Scientific Technical

Service, Universitat Pompeu Fabra, Barcelona, Spain). On

day 0, mice (n = 34) were injected subcutaneously (s.c.) into

the hind flanks with an emulsion of 100 ll MOG35–55 (3 mg/

ml) and 100 ll CFA (Sigma) supplemented with 4 mg/ml

Mycobacterium tuberculosis H37RA (Difco). Additionally,

animals received an intraperitoneal injection of 500 ng pertussis

toxin (Sigma), which was repeated 2 days after

immunization. As controls, a group of mice were immunized

with BSA (n = 6). Animals were treated with sildenafil

(10 mg/kg, s.c.; extracted from Pfizer Viagra tablets according

to [11]) or vehicle (water) once a day starting at 18 days

post-immunization (dpi). Mice were clinically scored for

EAE daily according to the following criteria: 0 no signs of

disease; 0.5 partial loss of tail tonus; 1 loss of tail tonus; 2

moderate hind limb paraparesis; 2.5 severe hind limb paraparesis;

3 partial hind limb paralysis; 3.5 hind limb paralysis;

4 tetraplegy, and 5 death. Mice were sacrificed under isofluorane

anesthesia at 21 of 26 dpi (3 or 8 days post-initiation of

treatment (dpt) respectively), and spinal cords and spleens

were removed. Experiments were approved by the UAB

Animal and Human Experimentation Ethics Committee.

Histological methods

The lumbar-thoracic region of the spinal cords was fixed in

paraformaldehyde and embedded in paraffin for histological

analysis. Longitudinal sections (8 lm) were stained with

hematoxylin and eosin (H&E) or Luxol Fast Blue (LFB) for

evidence of infiltrates, and demyelination and histological

scores were evaluated blindly according to the following

criteria. For cell infiltration (H&E): 0 no inflammation; 1

cellular infiltrates only around blood vessels and meninges; 2

moderate cellular infiltrates in parenchyma, less than 50% of

the white matter (WM); 3 severe infiltrates in parenchyma,

deep and/or more than 50% of the WM. For demyelination

(LFB): 0 no demyelination; 0.5 little demyelination only

around infiltrates; 1 superficial demyelination,which involves

500 Acta Neuropathol (2011) 121:499–508

123

less than 25%of theWM; 1.5 superficial demyelinationwhich

involvesmore than 25%but less than 50%of theWM; 2 deep

demyelination and/or demyelination that involves more than

50% of the WM; 3 diffuse and widespread demyelination.

Axonal density was evaluated by Bielschowsky’s silver

impregnation.

Immunostaining of longitudinal lumbar-thoracic sections

was performed as previously described [25]. Antigen retrieval

was done by the appropriate standard protocol (citrate, EDTA

or protease type XIV; Sigma). Sections were incubated with

the primary antibodies: polyclonal rabbit purified anti-glial

fibrillary acidic protein (GFAP; Dako, Cat. no. Z0334; 1:900)

for astrocytes; polyclonal rabbit purified anti-Iba1 (WAKO,

Cat. no. 19-19741; 1:250) for macrophages/microglia; polyclonal

rabbit purified anti-CD3 (Dako,Cat. no. A0452; 1:100)

for T lymphocytes; monoclonal anti-Foxp3 (a gift from Dr.

G. Roncador, Monoclonal Antibodies Core Unit, Spanish

National Cancer Research Center (CNIO), Madrid [1])

for Tregs; monoclonal purified anti-ICAM-1 (Pharmingen,

Cat. no. 554967; 1:100); monoclonal purified anti-SMI-32

(Covance, Cat. no. SMI-32R; 1:300) for axonal damage.

Appropriate secondary antibodies conjugated to biotin (Vector,

1:200) were used and detected using streptavidin/

horseradish peroxidase (Vector, 1:300) and the peroxidase

substrate DAB Kit (Vector). Control sections were incubated

in the absence of primary antibodies. Sections were counterstainedwith

0.25%cresylEcht Violet (Sigma) or hematoxylin

before mounting. Images were acquired using Nikon Digital

camera DXM 1200F and Nikon Act-1 software.

Staining quantifications

Four to eight areas (1 mm2) randomly chosen along the

length of each of 3–4 different longitudinal spinal cord

sections separated at least 300 lm were analyzed by two

independent investigators blinded to treatment and clinical

score. The analysis software Scion Image (NIH) was used

to quantify LFB, Bielschowsky, SMI-32, ICAM-1, Iba1

and GFAP staining. Quantification of CD3- and Foxp3-

positive cells was performed manually in infiltrates (4–8

infiltrates per section).

Splenocyte responses

Splenocytes were isolated from the spleens of animals killed

at 21 dpi. Cells were flushed out from the spleens gently

using a syringe plunger with PBS and centrifuged (2209g,

4C). The pellet was resuspended and left 5 min at 37C in

5 ml of RBC lysis buffer (Sigma, 17 mM Tris, 140 mM

NH4Cl) to remove erythrocytes. After further centrifugation,

cells were washed once again with PBS, centrifuged and

resuspended in 10 ml of complete medium (RPMI ?

L-glutamine supplemented with 15% FCS (Ingelheim

diagnostic, Barcelona, Spain), pen/strep antibiotics, 1 mM

sodium piruvate and 50 lMb-mercaptoethanol). Cells were

seeded at a density of 2–4 9 106 cells/ml in 96- or 24-well

plates and exposed to MOG35–55 (10 lg/ml, 72 h). Proliferation

was measured in 96-well plates using EZ4U kit

(Biomedica Gruppe, Vienne, France). Cytokine release was

determined in the media of 24-well plates with BD CBA

mouse Th1/Th2 cytokine kit.

Fig. 1 Sildenafil ameliorates clinical symptoms associated with

EAE. C57BL/6 mice immunized with MOG35–55 developed clinical

symptoms of EAE after 1 week. Sildenafil administration (10 mg/kg,

s.c.) daily starting 18 dpi significantly decreased disease severity after

4 days when compared to vehicle-treated animals. Values are

mean ± SEM (n = 12–13). Statistical significance: *p\0.05;

***p\0.001

Table 1 Clinical scores of EAE mice treated or not with sildenafil

Treatment EAE clinical score before treatment

(18 dpi, n = 17 per group)

EAE clinical score after treatment

(26 dpi, n = 12–13 per group)

Mortality Grade of remission

Incidence Average score Cumulative score Incidence Average score Cumulative score

Vehicle 90.9 1.75 ± 0.27 9.28 ± 1.55 100 2.41 ± 0.36 29.45 ± 2.9 1 0.66 ± 0.24

Sildenafil 90.3 2.09 ± 0.22 10.97 ± 1.53 84.61 0.92 ± 0.27 18.81 ± 3.16* 0 -1.17 ± 0.23***

EAE incidence (% diseased animals) and clinical scores of MOG-immunized mice before (18 dpi) and after 8 days of sildenafil treatment (26

dpi). Cumulative score (sum of scores from disease onset) and grade of remission (difference between score at 18 and 26 dpi) were significantly

different in sildenafil-treated animals. Results are mean ± SEM of the indicated number of animals at each time point. Statistical significance:

* p\0.05, *** p\0.001

Acta Neuropathol (2011) 121:499–508 501

123

Statistical analysis

Differences in clinical scores, LFB, Bielschowsky, histological

scores, CD3, and cytokines were analyzed by twoway

ANOVA followed by Bonferroni’s post-hoc test.

Student’s t test was used for two group comparisons

(ICAM, Foxp3, Iba-1, GFAP, and SMI32). Results shown

are mean ± SEM of the indicated number of animals.

Results and discussion

Sildenafil administration after disease onset ameliorates

clinical symptoms and neuropathology in a chronic

model of EAE

C57BL/6 mice immunized with MOG35–55 developed EAE

clinical symptoms after 7 days and at 18 dpi, when the

incidence of clinical EAE was 90–91% and the average

score around 2; animals (16–17 per group) were injected

s.c. with sildenafil (10 mg/kg) or vehicle (water) daily and

were killed 3 (21 dpi) or 8 (26 dpi) days later for central

and peripheral immune response analysis. As shown in

Fig. 1 and Table 1, disability in vehicle-treated mice continued

to increase, whereas mice treated with sildenafil

showed a lower cumulative score and a rapid recovery that

was already significant after 4 days of treatment. During

5–8 days of treatment, the clinical score stabilized around

1, and interestingly more than half of the animals presented

virtually full recovery (score 0, n = 2; score 0.5, n = 7). In

a different experiment, animals given 10 doses of 5 mg/kg

sildenafil every other day starting at 20 dpi showed a

smaller but significant improvement in clinical score by

43 dpi (controls: 2.2 ± 0.2, n = 10; sildenafil-treated:

1.6 ± 0.2, n = 7), indicating that the effect depends on the

dose. A group of mice (n = 6) immunized in parallel with

BSA did not present clinical symptoms of EAE (not

shown).

Fig. 2 Sildenafil ameliorates

neuropathology associated with

EAE. Longitudinal lumbarthoracic

spinal cord sections

from MOG-immunized mice

treated or not with sildenafil for

3 (n = 4) or 8 days

(n = 12–13) starting at 18 dpi

were stained to evaluate

a demyelination (LFB),

b axonal loss (Bielschowsky),

and c axonal damage (SMI-32)

(bars 50 lm in a, b; 20 lm in

c). Demyelination scores and

staining intensities [arbitrary

units (a.u.)/mm2] were

quantified as described in

‘‘Materials and methods’’. Note

that immunization with MOG

causes severe demyelination

and axonal damage at 26 dpi

that is significantly reduced by

8 day treatment with sildenafil.

Values are mean ± SEM.

Statistically significant

difference versus vehicletreated

animals: *p\0.05;

***p\0.001; or versus 3 dpt

(21 dpi): ###p\0.001

502 Acta Neuropathol (2011) 121:499–508

123

To investigate if clinical improvement was accompanied

by decreased neuropathology, we examined demyelination

and axonal loss in longitudinal sections of the lumbarthoracic

region of spinal cords by LFB and Bielschowsky’s

silver staining in comparison to BSA-immunized mice that

presented no pathology (Supplemental Fig. 1). As shown in

Fig. 2a, the decrease in LFB staining intensity and the

corresponding demyelination score were similar in vehicletreated

MOG-immunized mice at both 21 and 26 dpi.

However, in 21 dpi animals treated with sildenafil for the

last 3 days, demyelination was already less evident and by

26 dpi, after 8 days of sildenafil treatment, there was a

significant increase in LFB staining and a decrease in

demyelination score suggesting that sildenafil promotes

remyelination. In contrast, quantification of Bielschowsky’s

staining showed that axonal loss increased from 21 to

26 dpi in vehicle-treated animals but not in sildenafiltreated

animals (Fig. 2b), indicating that sildenafil prevents

further axon degeneration. In agreement with this, staining

of non-phosphorylated neurofilaments with anti-SMI-32, a

Fig. 3 Sildenafil decreases

inflammatory cell infiltration

and ICAM-1 expression and

increases Foxp3? cells in spinal

cords of EAE mice. Spinal cord

sections from MOG-immunized

mice treated or not with

sildenafil for 3 (n = 4) or

8 days (n = 12–13) starting at

18 dpi were stained to evaluate

a general cell infiltration

(H&E), b T lymphocytes (anti-

CD3), c Tregs (anti-Foxp3), and

d ICAM-1. Arrowheads points

to infiltrated cells and arrow

points to blood vessel (bars

50 lm in a; 20 lm in b–d). Cell

infiltration score, percentage of

CD3? and Foxp3? cells in

infiltrates and ICAM-1 staining

were quantified as indicated in

‘‘Materials and methods’’.

Sildenafil significantly

decreased inflammatory cell

infiltration and ICAM-1 in

infiltrates and increased

Foxp3? cells after 3 days of

treatment. Inflammatory cell

infiltration decreased

significantly from 21 to 26 dpi

in vehicle-treated animals, and

8-day sildenafil treatment did

not reduce it further. Values are

mean ± SEM. Statistically

significant difference versus

vehicle-treated animals:

*p\0.05; **p\0.01;

***p\0.001; or versus 3 dpt

(21 dpi): ###p\0.001

Acta Neuropathol (2011) 121:499–508 503

123

marker of axonal damage, was significantly lower in 8-day

sildenafil-treated animals compared to vehicle-treated

controls (Fig. 2c). Taken together, these results indicate

that the functional recovery produced by sildenafil treatment

in MOG-immunized animals results from axonal

protection and remyelination.

Sildenafil treatment decreases inflammatory cell

infiltration and ICAM-1 expression while increasing

the proportion of Foxp3? Tregs in spinal cord of EAE

mice

Autoreactive T cells infiltrating the CNS are the initiator and

early effector cells in EAE development, but infiltrated macrophages,

dendritic cells, and residentmicroglia constitute the

ultimate effector cells that amplify neuroinflammation and

tissue damage. Thus, we next examined the effect of sildenafil

on cellular infiltration in the spinal cord.As estimated byH&E

staining at 21 dpi, administration of sildenafil for the

previous 3 days dramatically decreased the severity of

cell infiltration, being the infiltrates smaller and largely

confined to the submeningeal area (Fig. 3a). Quantification

of CD3? T cells in infiltrates showed that sildenafil

significantly reduced the proportion of this cell population

at 3 dpt (Fig. 3b). At 26 dpi, vehicle-treated animals

presented significantly lower infiltration score (H&E)

and number of CD3? T cells per infiltrate respect to 21

dpi, and levels were no different to those determined in

animals treated with sildenafil for 3 or 8 days (Fig. 3a,

b). However, activated macrophages/microglia in WM

(strongly Iba1? globoid cells) were significantly reduced

by sildenafil at both 3 and 8 dpt (Fig. 4a, b). These

results agree with a recent report showing that inflammatory

cell infiltration in MOG-immunized mice

decreases after peak disease despite generalized axonal

loss and demyelination and persistent clinical disability

[15] and suggest that decreased macrophage/microglial

activation may bear a more direct relation to the axon

protective effect of sildenafil than decreased T cell

infiltration. Additionally, a contribution of a direct effect

of sildenafil in axons and oligodendrocytes potentiating

cGMP-mediated neuroprotective pathways cannot be

ruled out [2, 14, 22].

To investigate if regulation of the peripheral immune

response might be involved in sildenafil reduction of

inflammation in the spinal cord, we examined the immune

response of splenocytes upon ex vivo stimulation with

MOG35–55. No significant differences were found in the

proliferative response or the release of INF-c, TNF-a, IL-2

or IL-6 in splenocytes from vehicle- and sildenafil-treated

animals 3 dpt (Fig. 5), thus ruling out that sildenafil compromises

the ability of T cells to be activated.

ICAM-1, a type-1 membrane-bound glycoprotein

expressed in most leukocyte subtypes, endothelial cells,

and CNS glial cells, is involved in leukocyte entry, lymphocyte

activation, and other immune responses and plays

a central role in the development of MS and EAE [6, 17].

Thus, we examined if sildenafil was affecting ICAM-1

expression in spinal cords of EAE mice at 21 dpi when

significant reduction in cell infiltration was observed

(Fig. 3b). In control mice, ICAM-1-immunoreactivity was

observed in blood vessels and in numerous cells within and

Fig. 4 Sildenafil decreases macrophage/microglia activation in the

spinal cord of EAE mice. a Spinal cord sections from MOGimmunized

mice treated or not with sildenafil for 3 (n = 4) or 8 days

(n = 12–13) were immunostained for Iba1 to evaluate macrophage/

microglia activation (bar 50 lm); b quantification of Iba1 staining

intensity in WM. Treatment with sildenafil significant reduced

macrophage/microglia activation at both treatment times. Inserts

magnification of GM areas showing that sildenafil promotes a resting

morphology in microglia (bar 20 lm). Values are mean ± SEM.

Statistical significance: *p\0.05

504 Acta Neuropathol (2011) 121:499–508

123

around infiltrates (Fig. 3d). In contrast, in sildenafil-treated

animals, it was almost absent in infiltrates but was still

observed in blood vessels. Studies on ICAM-1 null mice

have evidenced the critical role of ICAM-1 expression in T

cells for the modulation of effector T cell responses [6].

Interestingly, NO via cGMP was reported to reduce T cell

adhesion to human brain microvessels in vitro, but NO did

not modulate adhesion molecule expression in the endothelial

cells, suggesting a direct action on the T cells [39].

Thus, down-regulation by sildenafil of ICAM-1 expression

in T cells could be involved in decreasing T cell infiltration

and/or activation in the spinal cord of EAE mice. Additionally,

by increasing cGMP in macrophages sildenafil

could attenuate TNF-a release and TNF-a-induced

expression of chemokines and adhesion molecules in

endothelial cells [34].

The role of Foxp3? Tregs in suppressing autoreactive T

cells is well established. Additionally, Foxp3? Tregs have

been shown to play a critical role in protection and

recovery from EAE [24]. Since sildenafil afforded both

protection and recovery, we investigated if it affected the

population of Foxp3? cells. As shown in Fig. 3c, at 21 dpi,

Foxp3? cells in spinal cord infiltrates of sildenafil-treated

mice were notably increased compared to vehicle-treated

controls. Thus, up-regulation of Tregs may be another

factor contributing to the beneficial effects of sildenafil in

recovery from EAE. This result is in clear contrast with a

recent report on splenocytes showing that NO inhibits

expression of Foxp3 in myelin basic protein-primed T cells

via cGMP [5]. This discrepancy may be reconciled in light

of the observation that development of Foxp3? Tregs in

response to antigen stimulation is antagonized by Th1/Th2

lineage differentiation activities [36]. This may occur in

splenocytes from immunized mice but not in the spinal

cord of animals treated with sildenafil where T cell infiltration

is greatly reduced.

Sildenafil treatment regulates reactive gliosis

In addition to heavily Iba1-stained globoid macrophages/

microglia within and around infiltrates, MOG-immunized

mice presented activated ramified microglia (strongly Iba1-

stained) throughout the spinal cord, more evident at 21 than

at 26 dpi. In contrast, animals treated with sildenafil for 3

or 8 days showed a decreased intensity of Iba1 staining in

activated microglia and increased numbers of cells with

long and thin ramifications typical of resting microglia

(Fig. 4a, inserts), suggesting that decreased microglial

activation may also contribute to the neuroprotective effect

of sildenafil. In vitro studies have shown that cGMPmediated

pathways reduce expression and release of

inflammatory mediators from both macrophages and

microglia [21, 23, 34]. Furthermore, we have recently

shown that treatment with cGMP-PDE inhibitors decreases

recruitment and activation of macrophages/microglia

around a cortical cryoinjury in rodents [25]. Since

microglia does not seem to express NO-sensitive GC [26],

it is tempting to speculate that increases in cGMP occur in

response to NPs. Interestingly, a recent study showed that

Fig. 5 Sildenafil treatment does not affect in vitro responses to

MOG35–55 in splenocytes from EAE mice. Splenocytes isolated from

vehicle- or sildenafil-treated MOG-immunized mice (21 dpi) were

incubated with or without MOG35–55 (10 lg/ml) for 72 h, and the

proliferative response (a) and the release of cytokines (b) were

measured. Values are mean ± SEM (n = 4). Statistically significant

differences versus control splenocytes: #p\0.05; ##p\0.01;

###p\0.001

Acta Neuropathol (2011) 121:499–508 505

123

administration of brain natriuretic peptide downregulates

microglial activation in murine models of traumatic brain

injury and intracerebral hemorrhage [16].

Increasing evidence indicates that astrogliosis can play a

crucial role in the pathogenesis and resolution of demyelinating

disease. Astrocytes can promote and perpetuate

immune-mediated demyelination by priming autoreactive T

cells and expressing cytokines, chemokines, and costimulatory

and adhesion molecules [9]. However, astrocytes

also promote anti-inflammatory responses and form perivascular

barriers that restrict the influx of leukocytes into

CNS parenchyma [18, 31]. We have previously shown that

increasing cGMP in astrocytes regulates cytoskeleton

dynamics and accelerates migration in a scratch wound

assay in vitro [3]. In addition, we have shown that treatment

with PDE5 inhibitors enhances astrogliosis around a cortical

cryolesion suggesting that cGMP-mediated pathways

can accelerate glial scar formation [25]. Thus, we examined

the effect of sildenafil on GFAP immunoreactivity in the

spinal cord of EAE mice. Different effects were observed in

WM and gray matter (GM). Severely reactive astrocytes

with long overlapping processes (anisomorphic gliosis)

were observed throughout WM but particularly in areas of

heavy inflammatory cell infiltration (Fig. 6a). Although no

significant differences in GFAP overall staining intensity

were observed between vehicle- and sildenafil-treated animals,

a stronger tendency to form scar-like structures

around confined infiltrates could be generally observed in

the latter (Fig. 6a), suggesting a role in controlling the

spreading of infiltration. In the GM moderate astrogliosis

was widespread in vehicle-treated mice, and in contrast

to WM, sildenafil had a biphasic effect, significantly

decreasing GFAP immunoreactivity after 3 days but

increasing it after 8 days of treatment (Fig. 6b). At this

treatment time, activated astrocytes were evenly distributed

in GM and presented a more stellate shape, features typical

of isomorphic gliosis. Numerous evidences indicate that

these activated astrocytes exert suppressive effects on

Fig. 6 Sildenafil modifies

astrogliosis in the spinal cord of

EAE mice. Spinal cord sections

from MOG-immunized mice

treated or not with sildenafil for

3 (n = 4) or 8 days

(n = 12–13) were

immunostained for GFAP to

evaluate astroglial activation

(bar 20 lm). Quantification of

GFAP staining in the WM

(a) showed no significant

differences between treated and

untreated animals, but scar-like

structures around infiltrates

were more prominent in

sildenafil-treated animals. In the

GM (b), sildenafil had a

biphasic effect on astroglial

activation, decreasing GFAP

staining intensity at 3 dpt but

increasing it at 8 dpt. Values are

mean ± SEM. Statistical

significance: *p\0.05;

***p\0.001

506 Acta Neuropathol (2011) 121:499–508

123

inflammatory cells and release antioxidants and growth

factors that protect neurons and oligodendrocytes [18].

Thus, it will be of interest to investigate how the different

effects of sildenafil on astroglial reactivity in WM and GM

observed at the different treatment times relate to the

reduced inflammation, axon protection, and remyelination

the drug produces in EAE mice.

To our knowledge, this is the first report demonstrating

efficacy of a PDE5 inhibitor in a mouse model of MS.

Sexual dysfunction is a common symptom in MS patients,

and treatment with PDE5 inhibitors including sildenafil

[10] and tadalafil [19] are commonly used to treat these

symptoms. However, to our knowledge, there have not

been any published studies to determine if these treatments

modify MS pathology although a presentation at the 2005

ECTRIMS meeting suggested radiological benefit in a

small cohort of MS patients [20].

Acknowledgments This work was supported by grants SAF2007-

64164 and SGR2005-939 to AG and SAF2008-00435 and RETICS

(REEM, RD07/0060/0002) to JH. We thank Mar Castillo and David

Llige´ for technical support and Dr. Dolores Jaraquemada for helpful

discussion.

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