Inhibition of phosphodiesterase-5 rescues age-related impairment of synaptic plasticity and memory

Behavioural Brain Research 240 (2013) 11–20

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Inhibition of phosphodiesterase-5 rescues age-related impairment of synaptic plasticity and memory Agostino Palmeri a , Lucia Privitera a , Salvatore Giunta b , Carla Loreto b , Daniela Puzzo a,∗ a b

Department of Bio-Medical Sciences - Section of Physiology, University of Catania, Catania, 95125, Italy Department of Bio-Medical Sciences - Section of Anatomy, University of Catania, Catania, 95125, Italy

h i g h l i g h t s     

We used 26–30 months-old wild type mice as a physiological model of aging. We analyzed hippocampal basal synaptic transmission and long-term potentiation. We analyzed spatial learning, reference and recognition memory. Sildenafil improved synaptic function and memory in aged mice. Sildenafil restored central CREB phosphorylation in old mice.

a r t i c l e

i n f o

Article history: Received 5 September 2012 Received in revised form 26 October 2012 Accepted 30 October 2012 Available online 19 November 2012 Keywords: Aging Sildenafil Synaptic plasticity Memory CREB Hippocampus

a b s t r a c t Aging is characterized by a progressive cognitive decline that leads to memory impairment. Because the cyclic nucleotide cascade is essential for the integrity of synaptic function and memory, and it is down-regulated during aging and in neurodegenerative disorders, we investigated whether an increase in cGMP levels might rescue age-related synaptic and memory deficits in mice. We demonstrated that acute perfusion with the phosphodiesterase-5 inhibitor sildenafil (50 nM) ameliorated long-term potentiation in hippocampal slices from 26–30-month-old mice. Moreover, chronic intraperitoneal injection of sildenafil (3 mg/kg for 3 weeks) improved age-related spatial learning and reference memory as tested by the Morris Water Maze, and recognition memory as tested by the Object Recognition Test. Finally, sildenafil restored central cAMP responsive element-binding protein (CREB) phosphorylation, which is crucial for synaptic plasticity and memory. Our data suggest that inhibition of phosphodiesterase-5 may be beneficial to treat age-related cognitive dysfunction in a physiological mouse model of aging. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Aging is a complex biological process where progressive functional and structural impairment of physiological processes leads to increased susceptibility to disease and, ultimately, to death. Besides the age-related somatic changes (i.e. organ failure, phenotype modifications), senescence is characterized by a “mind” decline, as demonstrated by a greater incidence of dementia and neurodegenerative disorders such as Alzheimer’s disease (AD). The increasing number of older citizens and spiraling healthcare spending have been prompting investigations aimed at discovering new drugs to slow the inexorable senile decline [1], although success has so far been limited. One of the most extensively investigated

∗ Corresponding author at: Department of Bio-Medical Sciences - Section of Physiology, Viale A. Doria 6 (ed. 2), Catania, 95125, Italy. Tel.: +39 095 738 4033; fax: +39 095 738 4217. E-mail address: [email protected] (D. Puzzo). 0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2012.10.060

targets is the cyclic nucleotide cascade, which is down-regulated during aging and in neurodegenerative disorders [2]. Furthermore, cAMP and cGMP are known to cooperate [3] to ensure earlyand late-phase long-term potentiation (LTP), a form of synaptic plasticity that is thought to provide the molecular basis of learning [4] and to underpin memory acquisition and consolidation [5,6]. Over the last few years we have examined the involvement of the cGMP pathway in learning and memory. Considerable attention has been devoted to phosphodiesterase 5 inhibitors (PDE5-I) [7], which raise cGMP levels by inhibiting its degradation. In particular, the PDE5-I sildenafil has recently been proposed to treat a variety of neurodegenerative disorders including AD [8,9]. We have previously demonstrated that enhancement of cGMP signaling by cGMP analogs rescued amyloid-beta (A␤)-induced impairment of LTP and phosphorylation of cAMP Responsive Element Binding protein (CREB) [10], a transcription factor involved in memory. Moreover, treatment with sildenafil rescued synaptic and memory deficits in a transgenic mouse model of amyloid deposition, restoring

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Table 1 Experimental design. From top to bottom, treatment, samples and detailed experimental time-line are showed. IHC = immunohistochemistry; WB = western blot; T1 = first day of object recognition test; T2 = second day of object recognition test; Hidden = hidden platform test; Visible = visible platform test.

Day 0 - Day 20 Sildenafil chronic treatment (3 mg/kg i.p. for 3 weeks)

Sildenafil acute treatment (50 nM)

8 slices from 8 mice per condition MALES ONLY

14 mice per condition SEX-BALANCED

10 min perfusion of hippocampal slices

Morris Water Maze

14 mice per condition SEX-BALANCED

4 mice per condition MALES ONLY

IHC and WB for p-CREB

Object Recognition Test

Tetanus

Day 21: T1 -> sildenafil Basal Synaptic Transmission

Day 22: T2

Day 21: Brain removal

Long-Term Potentiation

Day 21: Hidden 1 -> sildenafil Hidden 2 -> sildenafil

Day 22: Hidden 3 -> sildenafil Hidden 4 -> sildenafil

Day 23: Hidden 5 -> sildenafil Hidden 6 -> sildenafil

phospho-CREB (p-CREB) levels [8]. Other studies have shown that sildenafil may improve memory in rodents [11–15] and macaques [16]; moreover, sildenafil has recently been used to counteract cognitive impairment in mouse models of aging [17]. Here, we aimed to evaluate whether treatment with sildenafil improves age-related impairment of hippocampal synaptic plasticity and memory in wild type (WT) mice aged 26–30 months. 2. Methods 2.1. Animals We used C57Bl/6J WT mice from a colony kept in the animal facility of the Department of Bio-Medical Sciences – Section of Physiology (University of Catania). The animals were maintained on a 12 h light/dark cycle (lights on at 06:00 h) in temperature- and humidity-controlled rooms; food and water were available ad libitum. All experiments were performed in parallel using 4 groups of mice per experiment: 1. young mice treated with vehicle; 2. young mice treated with sildenafil; 3. old mice treated with vehicle; 4. old mice treated with sildenafil. Each group included: 8 males for electrophysiological recordings; 14 sex-balanced mice (6–8 males and 6–8 females) for behavioral tests; 3–4 males for western blot (WB) and immunohistochemistry (IHC). Animals were first treated with intraperitoneal (i.p.) sildenafil or vehicle for 3 weeks, then they were assigned to one of the following experimental protocols: Morris Water Maze (MWM), Object Recognition Test (ORT), WB analysis, or IHC. Acute slice perfusion of vehicle or sildenafil was performed for electrophysiological experiments (please see Table 1 for the detailed experimental plan). For electrophysiology, WB analysis and IHC mice were sacrificed by cervical dislocation followed by decapitation and brain removal. The protocol was approved by the University Institutional Animal Care and Use Committee. 2.2. Drug preparation and administration Sildenafil was synthesized as described [8]. The administration protocol was based on previous findings [8] and on preliminary data indicating that in vitro perfusion with 50 nM sildenafil exerts an acute effect on hippocampal slices before tetanic stimulation, whereas acute administration in vivo before or after training does not influence behavioral performances. The minimum effective concentration was 3 mg/kg/day, the minimum effective period was 3 weeks. We first assessed the acute effects of sildenafil by perfusing hippocampal slices for 10 min before tetanus. For behavioral experiments animals received 3 mg/kg/day by i.p. injection for 3 weeks; control mice received the same volume of saline solution (vehicle). For the behavioral studies sildenafil was administered immediately after training (Table 1). 2.3. Electrophysiological measurements Electrophysiological recordings were performed as described previously [18]. Transverse hippocampal slices (400 (m) were cut and maintained in a recording

Day 24: Probe

Day 25: Visible 1

Day 26: Visible 2

chamber at 29 o C, continuously bubbled with 95% O2 and 5% CO2 and perfused with artificial cerebrospinal fluid (ACSF; composition in mM: 124.0 NaCl, 4.4 KCl, 1.0 Na2 HPO4 , 25.0 NaHCO3 , 2.0 CaCl2 , 2.0 MgSO4 , 10.0 glucose). Field excitatory postsynaptic potentials (fEPSP) were recorded by placing the stimulating electrode at the level of the Schaffer collateral fibers and the recording electrode in the CA1 stratum radiatum. Basal synaptic transmission (BST) was assayed by plotting the stimulus voltages against slopes of fEPSP. After recording BST, a 15 min baseline was recorded every minute at an intensity that evokes a response ∼35% of the maximum evoked response. LTP was induced using a ␪-burst stimulation (4 pulses at 100 Hz, with the bursts repeated at 5 Hz and each tetanus including 3 ten-burst trains separated by 15 s). Responses were recorded for 2 h after tetanus and measured as fEPSP slope expressed as “percentage of baseline” at 120 min after LTP induction.

2.4. Behavioral studies MWM experiments were performed as described [18]. Mice were trained for 3 days in 2 daily sessions each consisting of three 1 min trials; sessions were held 4 h apart, so that mice had to rely on long-term memory of platform location. Time taken to reach the hidden submerged platform (latency), distance swum to reach it, and swimming speed were recorded. The 4th day the platform was removed and spatial memory retention was tested in 4 probe trials. The maze was divided into 4 quadrants, the target quadrant (TQ; i.e. the one previously containing the platform), and 3 non-target quadrants (AL = adjacent left, AR = adjacent right, and OQ = opposite quadrant). The percent time spent in each quadrant was recorded and analyzed with a video tracking system (Netsense srl, Catania, Italy). On the 5th and 6th day visual, motor, and motivation skills were tested in 2 sessions/day (each consisting of three 1 min trials) by measuring the time taken to reach a visible platform (randomly positioned in a different place each time) marked with a green flag, the distance swum to reach it, and swimming speed. For ORT, we used the protocol suggested by van Goethem et al. [19]. Two days before training, mice were handled gently for 5 min and then allowed to familiarize with the apparatus (a plastic box 50 cm long, 35 cm wide, and 15 cm high) for 10 min/day. The ORT consisted of 2 trials, one per day, lasting 10 min instead of 3 min; the longer time of exposure to the stimuli allowed the animal to learn the task. In the first trial (T1), two identical objects were placed in the central part of the box, equally distant from the perimeter. The mouse was placed in the apparatus and allowed to explore the objects. Exploration was defined as the mouse pointing its nose toward the object from a distance not >2 cm (as marked by a reference circle). Then the mice were returned to their cages. The second trial (T2) was performed 24 h later to test memory retention. Mice were presented with two objects, respectively a “familiar” (i.e. the one used for T1) and a “novel” object. The latter object was placed on the left or the right side of the box in a randomly but balanced manner, to minimize potential biases due to a preference for particular locations or objects. To avoid olfactory cues, the objects and the apparatus were cleaned with 70% ethanol after each trial. The following parameters were evaluated: (i) time of exploration of each object and total time of exploration of the two objects expressed as % exploration of the novel and % exploration of the familiar object; (ii) discrimination (D) index calculated as “exploration of novel object minus exploration of familiar object/total

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Fig. 1. Synaptic function is impaired in old mice. (A) BST is impaired in 28-monthold mice, whereas it is normal up to 22 months. The summary graph shows fEPSP slopes vs. stimulation intensities ranging from 5 to 35 V. (B) LTP in the hippocampal CA1 region is already reduced in 22-month-old compared with 3-month-old mice. Arrows indicate tetanus delivery.

exploration time”; (iii) latency to first approach to novel object; (iv) total exploration time. 2.5. Western blot analyses Crude extracts were prepared by homogenizing whole mouse brains in a buffer containing 20 mM Tris (pH 7.4), 2 mM EDTA, 0.5 mM EGTA, 50 mM mercaptoethanol, 0.32 mM sucrose and a protease inhibitor cocktail (Roche Diagnostics, Milano, Italy) in a Teflon–glass homogenizer followed by sonication. Protein concentrations were determined by Bradford’s method [20] using bovine serum albumin as a standard. Sample proteins (60 ␮g) were diluted in 4× sodium dodecyl sulphate (SDS) protein gel loading solution (Invitrogen, Monza, Italy), boiled for 5 min, separated on 4–12% Bis-tris gel (Invitrogen) and electroblotted onto nitrocellulose membranes (Invitrogen). Non-specific binding was blocked for 2 h at 37 ◦ C with 10% non-fat dry milk in Tween–Tris-buffered saline (TTBS). Membranes were incubated overnight at 4 ◦ C with the following antibodies: anti-p-CREB (Ser133) and anti-CREB (both from Millipore, Billerica, MA, USA, 1:1000), anti-␤-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:200). The secondary antibodies (Amersham Biosciences, Milano, Italy) were diluted 1:7500 (p-CREB), 1:5000 (CREB), and 1:10,000 (␤-tubulin). All antibodies were prepared in 10% non-fat dry milk solution in TTBS. Blots were developed using the enhanced chemiluminescence ECL technique (Amersham Biosciences) and relative band densities quantified using ImageQuantTL software. No signal was detected when the primary antibody was omitted (data not shown). 2.6. Immunohistochemistry Brains were removed and fixed in 4% paraformaldehyde solution (0.06 M, pH 7.4) (Immunofix, Bio-Optica, Milano, Italy). After an overnight wash, brains were dehydrated in graded ethanol and embedded in paraffin, preserving their

Fig. 2. Sildenafil improves synaptic function in old mice. (A) Ten minute perfusion with 50 nM sildenafil rescues age-related BST impairment. No BST differences are recorded in 3-month-old mice treated with vehicle vs. sildenafil. (B) Sildenafil rescues LTP impairment in aged mice but does not affect synaptic plasticity in young animals. (C) The inhibitor has no effect on basal neurotransmission in experiments where tetanic stimulation is not applied. Bars represent the time of application of sildenafil or vehicle. Arrows indicate tetanus delivery. anatomical orientation. Sections 4–6 ␮m in thickness were obtained according to routine procedures, mounted on sialane-coated slides and air-dried. Endogenous peroxidase activity in deparaffinized and rehydrated sections was quenched with 3% H2 O2 for 10 min. Non-specific antibody binding was blocked with normal horse/goat serum diluted 1:20 in phosphate buffered saline (PBS; Ml07; Bio-Optica). Sections were irradiated (5 min × 3) in capped polypropylene slide-holders with

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Fig. 3. Three-week treatment with i.p. sildenafil (3 mg/kg) in old mice boosts spatial learning and reference memory. (A) Spatial learning in the MWM is enhanced by prior sildenafil treatment. (B) In the probe test sildenafil-treated aged mice spend significantly longer time in the TQ, where the platform was located during training, than vehicletreated old mice or either group of young mice. (C) Sildenafil reduces the distance swum to find the platform and (D) increases swimming speed. ** significant difference (p < 0.0001).

citrate buffer (pH 6.0) using a microwave oven (750 W) to unmask antigen sites. They were then rinsed in PBS and incubated with anti-p-CREB antibody (1:100) before overnight incubation in a moist chamber at 4 ◦ C. Immune complexes were treated with the secondary biotinylated rabbit antimouse linking antibody and peroxidase-labeled streptavidin, both incubated for 20 min at room temperature (Dako LSAB + kit, HRP, Glostrup, Denmark). After rinsing in 3 changes of PBS, immunoreactivity was visualized by development with 0.1% 3,3 -diaminobenzidine and 0.02% hydrogen peroxide (DAB substrate kit, Vector Laboratories, Burlingame, CA, USA) for 2 min. Finally, sections were lightly counterstained with Mayer’s hematoxylin and observed with an Axioplan light microscope (Zeiss, Oberkochen, Germany). Photographs were taken with a digital camera (Canon, Tokyo, Japan). To quantify p-CREB immunohistochemical staining, 10 sections/animal were analyzed in stepwise fashion as a series of consecutive fields with a 20× objective and the stained area/total area was expressed as a percentage. Values from all consecutive images of each biopsy were averaged. Positive controls consisted of tissue specimens with known antigen positivity. Negative control sections were processed like the experimental slides, except that they were incubated with PBS instead of the primary antibody.

2.7. Statistics All experiments were blind with respect to treatment. Data were expressed as mean ± standard error mean (SEM). Statistical analysis was performed by using a dedicated software (Systat, Chicago, IL, USA). For electrophysiological analyses we used two-way ANOVA with repeated measures; for MWM data we applied two-way ANOVA with repeated measures for latency, distance swum, speed, and time spent in TQ vs. other quadrants, and two samples t-test to compare the time spent in TQ by each group; for ORT data we used two-samples t-test to analyze the difference in exploration time of the familiar vs. the novel object, and one sample t-test to compare the D index with zero, to establish whether animals recognized the familiar object in T2; two-way ANOVA or two-samples t-test was applied to compare other factors within 4 or 2 groups, respectively. Finally, we used two samples t-test for WB analysis and IHC. The level of significance was set at p < 0.05.

3. Results 3.1. Treatment with sildenafil improves synaptic function in aged mice We first had to decide what was the most suitable age to study our mice. To do so we conducted preliminary electrophysiological studies and found that BST, an indicator of synaptic strength, begins to be impaired at 26–28 months (F(1,14) = 0.508, p = 0.048; Fig. 1A), whereas it is unchanged in mice up to 22 months of age compared with 3-month-old mice (F(1,14) = 0.01, p = 0.897, Fig. 1A). In contrast, LTP was already impaired in 22-month-old mice (170.96 ± 19.68% of baseline slope 120 min after LTP induction, n = 6, F(1,12) = 6.89, p = 0.022 vs. young mice; Fig. 1B) and even more so in animals aged 26-30 months (163.30 ± 13.80% of baseline slope 120 min after LTP induction, F(1,14) = 11.99, p = 0.004 vs. young mice; Fig. 1B). Mice aged 26–30 months were therefore used, because they were sure to exhibit synaptic impairment. Next we needed to test whether synaptic function impairment in old mice was rescued by acute sildenafil administration. The BST reduction documented in old mice was improved by 10 min perfusion with 50 nM sildenafil (41% increase in the fEPSP slope at 35 V in sildenafil-treated vs. vehicle-treated slices; F(1,14) = 6.04, p = 0.028; Fig. 2A), whereas the drug did not affect BST in young mice (F(1,14) = 0.007, p = 0.936 vs. vehicle; Fig. 2A). Hippocampal slices were then tetanized by -burst stimulation of the Schaffer collateral pathway to induce LTP in the CA1 region. The fEPSP decrease recorded in old animals after tetanus was rescued by

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10 min sildenafil perfusion before tetanus (227.18 ± 15.61% of baseline slope, F(1,14) = 10.08, p = 0.007; Fig. 2B). Sildenafil did not affect LTP in young mice (220.59 ± 5.87% of baseline slope, F(1,14) = 0.42, p = 0.527 Fig. 2B). BST was unchanged in non-tetanized slices (vehicle-treated old mice: 99.13 ± 3.63%; sildenafil-treated old mice: 101.62 ± 5.60%; vehicle-treated young mice: 101.56 ± 7.95%; sildenafil-treated young mice: 97.10 ± 3.69% of baseline slope without tetanic stimulation, n = 4 mice per condition; F(3,12) = 0.30, p = 0.823; Fig. 2C). 3.2. Sildenafil improves cognition in aged mice During training mice were required to find a hidden platform beneath the surface of the water. The older mice took longer to find it, thus confirming the learning impairment due to senescence (overall latency: 43.2 ± 1.68 vs. 34.7 ± 1.78 sec in old and young mice, respectively; F(1,26) = 11.83, p = 0.002; Fig. 3A). Aged mice previously treated with sildenafil (3 mg/kg for 3 weeks) showed a marked latency improvement (overall latency: 26.5 ± 2.10 s) that was even greater than the latency recorded in young animals (F(1,26) = 38.12, p < 0.0001 vs. vehicle-treated aged mice; F(1,26) = 8.78, p = 0.006 vs. vehicle-treated young mice; Fig. 3A). Sildenafil did not affect performance in young mice (33.8 ± 1.34 s; F(1,26) = 0.16 p = 0.692; Fig. 3A). Vehicle-treated old mice failed to remember where the platform was located in the training sessions, as shown by the fact that they spent almost the same amount of time in TQ as in the other quadrants (TQ: 27.2 ± 1.40%; AR: 24.5 ± 0.90%; OQ: 23.4 ± 0.80%; AL: 24.9 ± 0.60%; F(3,52) = 2.76, p = 0.051). Moreover, they spent in TQ less time than did either group of young mice (t(26) = 2.63, p = 0.014). Sildenafil-treated old mice spent longer in TQ (TQ: 43.7 ± 1.30%; AR: 19.6 ± 0.70%; OQ: 16.1 ± 1.30%; AL: 20.6 ± 1.30%; F(3,52) = 115.88, p < 0.0001; t(26) = 8.81, p < 0.0001 vs. vehicle-treated aged mice; Fig. 3B), whereas the time spent in TQ by young animals treated with vehicle or sildenafil was not significantly different (37.0 ± 1.40%, t(26) = 0.75, p = 0.456; Fig. 3B). As in the learning curve, sildenafil-treated aged mice showed an excellent performance that was even better than that of young animals (t(26) = 4.61, p < 0.0001). To rule out any possible motor effects on spatial memory performance the distance swum to reach the platform and swimming speed during the spatial learning test were measured and analyzed. We found that the distance swum by the 4 groups of mice to find the hidden platform was different (F(3,52) = 3.28, p = 0.028) and was greatest in the older mice (734.2 ± 35.51 cm compared to 622.5 ± 33.41 cm in young mice; F(1,26) = 4.43, p = 0.045; Fig. 3C). Sildenafil treatment reduced the distance swum by older mice (571.7 ± 46.01 cm; F(1,26) = 6.68, p = 0.016; Fig. 3C) and also increased their swimming speed (vehicle-treated old = 17 ± 0.38 cm/s vs. vehicle-treated young mice = vs. 18.5 ± 0.34 cm/s; F(1,26) = 4.04, p = 0.05; vehicle-treated old vs. sildenafil-treated old mice = 22 ± 0.77 cm/s; F(1,26) = 34.53, p < 0.0001; Fig. 3C). In the young animals sildenafil affected neither the distance swum (659.3 ± 46.67 cm; F(1,26) = 2.72, p = 0.111) nor swimming speed (19.9 ± 0.77 cm/s; F(1,26) = 0.02, p = 0.373). We also performed a trial with a visible platform, to study motor performances without the influence of memory cues. Data analysis showed that older mice treated with sildenafil took significantly less time to reach the platform than those treated with vehicle (F(1,26) = 4.36, p = 0.047; Fig. 4A) due to higher swimming speed (F(1,26) = 18.54, p < 0.0001; Fig. 4B), whereas the differences among the 4 groups in the distance swum to reach the platform were not significant (F(3,52) = 0.33, p = 0.799; Fig. 4C). For ORT, we first measured the time taken by mice to explore the familiar and the novel object. Vehicle-treated old animals spent almost the same time exploring the familiar as the novel object

Fig. 4. Sildenafil enhances swimming speed in the visible platform trial. (A) Old mice treated with sildenafil need less time to reach the visible platform and (B) swim faster. (C) The distance swum to reach the platform is not changed by the treatment.

(47.78 ± 1.88% vs. 52.21 ± 1.88%; t(26) = 1.65, p = 0.109; Fig. 5A), which indicates a memory impairment; those treated with sildenafil spent less time exploring the familiar than the novel object (39.73 ± 2.15% vs. 60.26 ± 2.15%; t(26) = 6.72, p < 0.0001; Fig. 5A): these data, which reflect a memory enhancement, were similar to those found in young animals (vehicle: 42.57 ± 2.14% familiar vs. 57.42 ± 2.14% novel object; t(26) = 4.90, p < 0.0001; sildenafil: 37.12 ± 1.58% familiar vs. 62.87 ± 1.58% novel object; t(26) = 11.51, p < 0.0001; Fig. 5A). The D index, calculated as “novel object exploration minus familiar object exploration/total exploration time” confirmed that the older animals were unable to learn, because D was not significantly different from zero (t(13) = 1.17, p = 0.262; Fig. 5B). The difference among groups (F(3,52) = 5.44, p = 0.002) reflects the recognition memory impairment exhibited by the old mice, which was rescued by sildenafil (0.04 ± 0.03 vs. 0.20 ± 0.04 in older mice treated with vehicle vs. sildenafil; t(26) = 2.80, p = 0.009; Fig. 5B). A significant difference was also detected between young mice

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Fig. 5. Object recognition memory is improved by sildenafil in aged mice. (A) T2 exploration times (after 24 h retention interval) show that sildenafil-treated old mice spend significantly longer exploring the novel compared with the familiar object, like young mice; this indicates that they are able to learn. (B) Sildenafil-treated aged mice have a higher D index than vehicle-treated littermates. Differences from zero, reflecting the ability to learn, are depicted as hashes (# p < 0.005; ## p < 0.0001). * significant difference (p < 0.05); ** highly significant difference (p < 0.0001). (C) Latency to first approach to the novel object is comparable in the four groups. (D) The shorter total exploration time shown by aged mice is increased by sildenafil treatment.

treated with vehicle vs. sildenafil (0.14 ± 0.04 vs. 0.25 ± 0.03; t(26) = 2.31, p = 0.030; Fig. 5B). In contrast the latency to first approach to the novel object by the 4 groups of mice was not significantly different (F(3,52) = 0.055, p = 0.983; Fig. 5C). Finally, total exploration time was significantly shorter in both groups of older animals (t(26) = 2.06, p = 0.049; Fig. 5D).

in p-CREB in young animals treated with sildenafil (+28% vs. vehicle-treated young mice; t(6) = 2.98, p = 0.025; Fig. 7A,B). Thus, the p-CREB modifications paralleled the changes in synaptic plasticity and memory demonstrated by the other tests.

3.3. Sildenafil restored CREB phosphorylation in aged mice

In this study we demonstrated that the PDE5-I sildenafil improved synaptic plasticity and memory in aged mice. It also restored CREB phosphorylation, which is crucial for learning and memory processes [21–23], as well as synaptic plasticity [24–26]. Although genetically- or drug-induced animal models of aging were available [27–30], we felt that a physiological model of aging would provide more realistic information on the natural development of the aging process. In a preliminary electrophysiological study we established the age at which synaptic impairment begins. LTP, which reflects the functional process underlying synaptic plasticity, was already impaired at 22 months, whereas BST, a stimulus–response parameter that allows assessment of the synaptic structure, was unaffected up to 26 months. These data prompted the study of very old animals, whose synapse function and structure is already compromised. In addition to synaptic alterations these

WB analysis showed a 72% decrease in p-CREB in old vs. young mice (t(10) = 9.966; p < 0.0001). Sildenafil partially restored p-CREB levels in the older animals (+32% vs. vehicle-treated aged mice; t(10) = 4.098, p = 0.002; Fig. 6A,B) and also raised p-CREB in young mice (+27% vs. vehicle-treated young mice; t(10) = 4.95; p = 0.001). In contrast total CREB levels were not significantly changed in the 4 groups of mice (F(3,20) = 0.54, p = 0.659; Fig. 6A,C). To confirm these data we explored p-CREB expression in the hippocampus, particularly in the CA1 region. p-CREB expression was significantly reduced in old vs. young animals (−71%; t(6) = 15.565, p < 0.0001); this reduction was largely rescued by treatment with sildenafil (+61% vs. vehicle-treated aged mice; t(6) = 15.27; p < 0.0001; Fig. 7A,B). The IHC data confirmed the moderate increase

4. Discussion

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Fig. 6. Sildenafil rescues CREB phosphorylation in aged mice. (A) WB analysis comparing p-CREB and CREB expression in young and aged mice. ␤-tubulin expression is shown as an internal control. (B) Bar graphs showing the results of WB analysis reported in A indicate that sildenafil raised p-CREB expression in both old and young animals. (C) Bar graphs showing that CREB levels are not modified by age and treatment. * significant difference (p < 0.05).

mice were then demonstrated to have severely impaired spatial learning and reference memory as tested by the MWM, as expected [31,32]. Recognition memory was investigated by ORT, a task based on the natural tendency of rodents to explore unfamiliar objects. As recently reported by several studies, this ability depends on the integrity of the hippocampus, the perirhinal cortex, and the medial temporal lobe [33–35]. Like other models of aging our mice showed a markedly impaired recognition memory [36,37]. Since activation of the nitric oxide (NO)/cGMP cascade is involved in hippocampal plasticity and memory [38], we decided to explore whether a stronger cGMP signal would rescue the age-related cognitive deficit. Increased cGMP levels can be obtained with different strategies based on the physiological cyclic nucleotide pathway. cGMP production is catalyzed by the enzyme guanylate cyclase, which in turn is activated by the gaseous neurotransmitter NO; in contrast cGMP is degraded by PDE enzymes.

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The cGMP signal can therefore be boosted by NO-mimetics, cGMP analogs, or PDE-I. Recently, a number of studies have focused on PDE5-I, downstream NO generation, because of their ubiquitous expression in several brain regions associated with cognitive function and of their long-standing use in clinical practice [7]. Among PDE5-I, sildenafil (Viagra® ) [39] was originally developed to treat arterial hypertension and angina pectoris; it then became the first drug to be approved for erectile dysfunction (ED), where its favorable risk–benefit balance has made it the chief treatment. Sildenafil has now been proposed for the treatment of pulmonary artery hypertension (Revatio® ) [40]. Recently, several studies have shown its potential in enhancing memory performance in animals without cognitive impairment [11–16] and in countering cognitive modifications in several impairment models; for instance, it partially offset the learning deficits induced by blockage of cholinergic muscarinic receptors [41], inhibition of NO synthase [42], hyperammonemia [43], lipopolysaccharides [44], diabetes, and electroconvulsive shock [45,46]. Therefore, sildenafil and other PDE5-I have been proposed as therapeutic weapons against neurodegenerative diseases characterized by cognitive impairment, such as AD [7,9]. We have previously demonstrated that sildenafil rescued synaptic plasticity and memory impairment in animal models of AD (APP/PS1) [8]; in our conditions it also reduced the A␤ load, whereas in studies of Tg2576 animals it enhanced cognitive function without changing the amyloid burden [47]. In a recent study SAMP8 mice, used to investigate age-related dementia, displayed better spatial learning and memory after treatment with 7.5 mg/kg sildenafil for 4 weeks [17]. Here we report that 3 week i.p. sildenafil rescued age-related synaptic and memory dysfunction. These data are consistent with previous works showing that: (i) an increase in the NO/cGMP signal positively modulates hippocampal LTP [48]; (ii) NO donors, cGMP analogs, and PDE5-I rescued the LTP reduction induced by exogenous A␤ [10] or found in Tg models of AD [8]; and (iii) NO-mimetics may reverse cognitive impairment in AD [49]. Other studies have demonstrated that PDE5 inhibition improves ORT performance by young but not old mice [50]; the difference could however be due to the different experimental protocol or type of compound used by the authors. In fact, they used zaprinast, which has an IC50 = 0.6 ␮M compared with the more potent inhibition exerted by sildenafil (IC50 = 3 nM); moreover ORT was performed in a single day instead of 2 days as in our protocol. In line with other studies [8,12–15,44–46], we found that the PDE5-I exerted its effects if administered immediately after training (after T1 in ORT, and after each acquisition trial in the hidden spatial learning task), indicating that PDE5 may be involved in memory consolidation. Another finding reported in this study is that sildenafil rescued the CREB phosphorylation impairment in aged animals, paralleling the improvement in synaptic plasticity and memory whereas, as previously demonstrated, CREB levels were unaffected [51,52]. LTP and memory are known to involve an early protein-independent phase and a late phase [53] that requires transcription genes, such as CREB, to turn new information into stable synaptic modifications [54]. CREB phosphorylation could be influenced by the modulation of cyclic nucleotide signals [55–57]. In this regard, we have previously shown that A␤ inhibited CREB phosphorylation by reducing cGMP levels [10] and that the reduction in p-CREB in APP/PS1 animals was rescued by sildenafil [8]. The beneficial effect of sildenafil is reminiscent of the effect of rolipram, a PDE4-I that elevates cAMP levels and therefore activates CREB through protein kinase A [56,58]. Indeed, PDE4 and PDE5 share many similarities since, though acting on different targets, their action converges onto the same molecule, CREB. However, since current PDE4-I have several undesirable side effects whereas PDE5-I have long been used in clinical practice, inhibiting PDE5

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Fig. 7. Hippocampal expression of p-CREB. (A) Representative examples of hippocampal slices stained with p-CREB antibody. 40× magnification of the CA1 region. Scale bar: 150 ␮m. (B) Bar graphs showing the intensity of stained area/total area indicate that sildenafil completely restores p-CREB expression in aged mice, while the increase is smaller in young animals. * significant difference (p < 0.05); ** highly significant difference (p < 0.0001).

may be the better strategy to treat nervous system diseases, such as memory dysfunction in the elderly and in AD, or other conditions such as multiple sclerosis [59], chronic neuropathic pain [60], and muscular dystrophy [61]. Another effect of PDE5-I on aged mice was an improvement in motor performance. Analysis of swimming speed and distance swum to reach the platform in the spatial learning task showed that sildenafil-treated aged mice took a shorter route to the platform and swam faster than their untreated counterparts. In the MWM visible platform test latency and swimming speed were boosted by sildenafil, whereas there were no differences in the distance swum to reach the platform. These findings, together with the longer total exploration time measured in vehicle-treated aged mice in ORT and the fact that four of these animals failed to complete the test because they almost drowned in the first trial, can be the starting point for further studies of the role of PDE5 on physical performances based on the involvement of the NO/cGMP pathway in muscle contraction, metabolism and vascularization [62–64] and given the improved exercise capacity of patients treated with sildenafil for ED [65,66]. In conclusion, our findings document the benefits of sildenafil treatment on age-related cognitive dysfunction in mice. However, use of sildenafil and of the other commercially available PDE5-I, vardenafil, both of which are able to cross the blood-brain barrier,

requires further research in humans before it can be considered for the treatment of chronic CNS diseases such as aging and AD. Acknowledgements This work was supported by University of Catania “Progetto di Ricerca d’Ateneo” to A.P. We thank Dr. Ottavio Arancio (Columbia University, New York, USA) for his kind gift of sildenafil and Dario Furnari for his help with animal treatments. References [1] Le Couteur DG, McLachlan AJ, Quinn RJ, Simpson SJ, de Cabo R. Aging biology and novel targets for drug discovery. Journals of Gerontology Series A, Biological Sciences and Medical Sciences 2012;67:168–74. ´ KU, Strosznajder JB. Cyclic GMP and nitric oxide synthase [2] Domek-Łopacinska in aging and alzheimer’s disease. Molecular Neurobiology 2010;41:129–37. [3] Lu YF, Kandel ER, Hawkins RD. Nitric oxide signaling contributes to late-phase LTP and CREB phosphorylation in the hippocampus. Journal of Neuroscience 1999;19:10250–61. [4] Bliss TVP, Collingridge GL. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 1993;361:31–9. [5] Matsumoto Y, Unoki S, Aonuma H, Mizunami M. Critical role of nitric oxidecGMP cascade in the formation of cAMP-dependent long-term memory. Learning and Memory 2006;13:35–44. [6] Rutten K, Prickaerts J, Hendrix M, van der Staay FJ, Sik A, Blokland A. Timedependent involvement of cAMP and cGMP in consolidation of object memory:

A. Palmeri et al. / Behavioural Brain Research 240 (2013) 11–20

[7]

[8]

[9]

[10]

[11]

[12]

[13] [14]

[15]

[16]

[17]

[18] [19]

[20]

[21]

[22]

[23] [24] [25]

[26] [27]

[28]

[29] [30] [31]

[32]

[33]

studies using selective phosphodiesterase type 2, 4 and 5 inhibitors. European Journal of Pharmacology 2007;558:107–12. Puzzo D, Sapienza S, Arancio O, Palmeri A. Role of phosphodiesterase 5 in synaptic plasticity and memory. Journal of Neuropsychiatric Disease and Treatment 2008;4:371–87. Puzzo D, Staniszewski A, Deng SX, Privitera L, Leznik E, Liu S, Zhang H, Feng Y, Palmeri A, Landry DW, Arancio O. Phosphodiesterase 5 inhibition improves synaptic function, memory, and amyloid-beta load in an Alzheimer’s disease mouse model. Journal of Neuroscience 2009;29:8075–86. Sabayan B, Zamiri N, Farshchizarabi S, Sabayan B. Phosphodiesterase-5 inhibitors: novel weapons against Alzheimer’s disease? International Journal of Neuroscience 2010;120:746–51. Puzzo D, Vitolo O, Trinchese F, Jacob JP, Palmeri A, Arancio O. Amyloid-beta peptide inhibits activation of the nitric oxide/cGMP/cAMP-responsive elementbinding protein pathway during hippocampal synaptic plasticity. Journal of Neuroscience 2005;25:6887–97. Baratti CM, Boccia MM. Effects of sildenafil on long-term retention of an inhibitory avoidance response in mice. Behavioural Pharmacology 1999;10:731–7. Prickaerts J, van Staveren WC, Sik A, Markerink-van Ittersum M, Niewohner U, van der Staay FJ, Blokland A, de Vente J. Effects of two selective phosphodiesterase type 5 inhibitors, sildenafil and vardenafil, on object recognition memory and hippocampal cyclic GMP levels in the rat. Neuroscience 2002;113: 351–61. Singh N, Parle M. Sildenafil improves acquisition and retention of memory in mice. Indian Journal of Physiology and Pharmacology 2003;47:318–24. Prickaerts J, Sik A, van Staveren WC, Koopmans G, Steinbusch HW, van der Staay FJ, de Vente J, Blokland A. Phosphodiesterase type 5 inhibition improves early memory consolidation of object information. Neurochemistry International 2004;45:915–28. Rutten K, Vente JD, Sik A, Ittersum MM, Prickaerts J, Blokland A. The selective PDE5 inhibitor, sildenafil, improves object memory in Swiss mice and increases cGMP levels in hippocampal slices. Behavioural Brain Research 2005;164: 11–6. Rutten K, Basile JL, Prickaerts J, Blokland A, Vivian JA. Selective PDE inhibitors rolipram and sildenafil improve object retrieval performance in adult cynomolgus macaques. Psychopharmacology 2008;196:643–8. ˜ Orejana L, Barros-Minones L, Jordán J, Puerta E, Aguirre N. Sildenafil ameliorates cognitive deficits and tau pathology in a senescence-accelerated mouse model. Neurobiology of Aging 2012;33:625.e11–20. Puzzo D, Privitera L, Palmeri A. Hormetic effect of amyloid-beta peptide in synaptic plasticity and memory. Neurobiology of Aging 2012;33:1484.e15–24. van Goethem NP, Rutten K, van der Staay FJ, Jans LA, Akkerman S, Steinbusch HW, Blokland A, Van’t Klooster J, Prickaerts J. Object recognition testing: Rodent species, strains, housing conditions, and estrous cycle. Behavioural Brain Research 2012;232:323–34. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 1976;72:248–54. Bourtchuladze R, Frenguelli B, Blendy J, Cioffi D, Schutz G, Silva AJ. Deficient long-term memory in mice with a targeted mutation of the cAMP-responsive element-binding protein. Cell 1994;79:59–68. Yin JC, Wallach JS, Del Vecchio M, Wilder EL, Zhou H, Quinn WG, Tully T. Induction of a dominant negative CREB transgene specifically blocks long-term memory in Drosophila. Cell 1994;79:49–58. Lonze BE, Ginty DD. Function and regulation of CREB family transcription factors in the nervous system. Neuron 2002;35:605–23. Montminy M. Transcriptional regulation by cyclic AMP. Annual Review of Biochemistry 1997;66:807–22. Tao X, Finkbeiner S, Arnold DB, Shaywitz AJ, Greenberg ME. Ca2+ influx regulates BDNF transcription by a CREB family transcription factor-dependent mechanism. Neuron 1998;20:709–26. Hu SC, Chrivia J, Ghosh A. Regulation of CBP-mediated transcription by neuronal calcium signaling. Neuron 1999;22:799–808. Takeda T, Hosokawa M, Takeshita S, Irino M, Higuchi K, Matsushita T, Tomita Y, Yasuhira K, Hamamoto H, Shimizu K, Ishii M, Yamamuro T. A new murine model of accelerated senescence. Mechanisms of Ageing and Development 1981;17:183–94. Treuting PM, Hopkins HC, Ware CA, Rabinovitch PR, Ladiges WC. Generation of genetically altered mouse models for aging studies. Experimental and Molecular Pathology 2002;72:49–55. Sprott RL. Animal models of aging: something old, something new. ILAR Journal 2011;52:1–3. Yeoman M, Scutt G, Faragher R. Insights into CNS ageing from animal models of senescence. Nature Reviews Neuroscience 2012;13:435–45. Li Q, Zhao HF, Zhang ZF, Liu ZG, Pei XR, Wang JB, Cai MY, Li Y. Long-term administration of green tea catechins prevents age-related spatial learning and memory decline in C57BL/6J mice by regulating hippocampal cyclic amp-response element binding protein signaling cascade. Neuroscience 2009;159:1208–15. Bergado JA, Almaguer W, Rojas Y, Capdevila V, Frey JU. Spatial and emotional memory in aged rats: a behavioral-statistical analysis. Neuroscience 2011;172:256–69. Barker GR, Bird F, Alexander V, Warburton EC. Recognition memory for objects, place, and temporal order: a disconnection analysis of the role of the medial prefrontal cortex and perirhinal cortex. Journal of Neuroscience 2007;27:2948–57.

19

[34] Broadbent NJ, Gaskin S, Squire LR, Clark RE. Object recognition memory and the rodent hippocampus. Learning and Memory 2009;17:5–11. [35] Barker GR, Warburton EC. When is the hippocampus involved in recognition memory? Journal of Neuroscience 2011;31:10721–31. [36] Wei W, Song L, Hui-ping D, Shen L, Yi-yuan T. Differential impairment of spatial and nonspatial cognition in a mouse model of brain aging. Life Sciences 2009;85:127–35. [37] Soontornniyomkij V, Risbrough VB, Young JW, Soontornniyomkij B, Jeste DV, Achim CL. Hippocampal calbindin-1 immunoreactivity correlate of recognition memory performance in aged mice. Neuroscience Letters 2012;516:161–5. [38] Puzzo D, Palmeri A, Arancio O. Involvement of the nitric oxide pathway in synaptic dysfunction following amyloid elevation in Alzheimer’s disease. Reviews in the Neurosciences 2006;17:497–523. [39] Boolell M, Allen MJ, Ballard SA, Gepi-Attee S, Muirhead GJ, Naylor AM, Osterloh IH, Gingell C. Sildenafil: an orally active type 5 cyclic GMP-specific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction. International Journal of Impotence Research 1996;8:47–52. [40] Montani D, Chaumais MC, Savale L, Natali D, Price LC, Jaïs X, Humbert M, Simonneau G, Sitbon O. Phosphodiesterase type 5 inhibitors in pulmonary arterial hypertension. Advances in Therapy 2009;26:813–25. [41] Devan BD, Sierra-Mercado Jr D, Jimenez M, Bowker JL, Duffy KB, Spangler EL, Ingram DK. Phosphodiesterase inhibition by sildenafil citrate attenuates the learning impairment induced by blockade of cholinergic muscarinic receptors in rats. Pharmacology Biochemistry and Behavior 2004;79:691–9. [42] Devan BD, Bowker JL, Duffy KB, Bharati IS, Jimenez M, Sierra-Mercado Jr D, Nelson CM, Spangler EL, Ingram DK. Phosphodiesterase inhibition by sildenafil citrate attenuates a maze learning impairment in rats induced by nitric oxide synthase inhibition. Psychopharmacology 2006;183:439–45. [43] Erceg S, Monfort P, Hernandez-Viadel M, Rodrigo R, Montoliu C, Felipo V. Oral administration of sildenafil restores learning ability in rats with hyperammonemia and with portacaval shunts. Hepatology 2005;41:299–306. [44] Patil CS, Jain NK, Singh VP, Kulkarni SK. Differential effect of the PDE5 Inhibitors, sildenafil and zaprinast, in aging- and lippolysaccharide-induced cognitive dysfunction in mice. Drug Development Research 2004;63:66–75. [45] Patil CS, Singh VP, Singh S, Kulkarni SK. Modulatory effect of the PDE-5 inhibitor sildenafil in diabetic neuropathy. Pharmacology 2004;72:192–5. [46] Patil CS, Singh VP, Kulkarni SK. Modulatory effect of sildenafil in diabetes and electroconvulsive shock-induced cognitive dysfunction in rats. Pharmacological Reports 2006;58:373–80. [47] Cuadrado-Tejedor M, Hervias I, Ricobaraza A, Puerta E, Pérez-Roldán JM, GarcíaBarroso C, Franco R, Aguirre N, García-Osta A. Sildenafil restores cognitive function without affecting ␤-amyloid burden in a mouse model of Alzheimer’s disease. British Journal of Pharmacology 2011;164:2029–41. [48] Bon CL, Garthwaite J. On the role of nitric oxide in hippocampal long-term potentiation. Journal of Neuroscience 2003;23:1941–8. [49] Thatcher GR, Bennett BM, Reynolds JN. Nitric oxide mimetic molecules as therapeutic agents in Alzheimer’s disease. Current Alzheimer Research 2005;2:171–82. ´ [50] Domek-Łopacinska K, Strosznajder JB. The effect of selective inhibition of cyclic GMP hydrolyzing phosphodiesterases 2 and 5 on learning and memory processes and nitric oxide synthase activity in brain during aging. Brain Research 2008;1216:68–77. [51] Foster TC, Sharrow KM, Masse JR, Norris CM, Kumar A. Calcineurin links Ca2+ dysregulation with brain aging. Journal of Neuroscience 2001;21(11):4066–73. [52] Tomobe K, Okuma Y, Nomura Y. Impairment of CREB phosphorylation in the hippocampal CA1 region of the senescence-accelerated mouse (SAM) P8. Brain Research 2007;1141:214–7. [53] Frey U, Krug M, Reymann KG, Matthies H. Anisomycin, an inhibitor of protein synthesis, blocks late phase of LTP phenomena in the hippocampal CA1 region in vitro. Brain Research 1988;452:57–65. [54] Silva AJ, Kogan JH, Frankland PW, Kida S. CREB and memory. Annual Review of Neuroscience 1998;21:127–48. [55] Comery TA, Martone RL, Aschmies S, Atchison KP, Diamantidis G, Gong X, Zhou H, Kreft AF, Pangalos MN, Sonnenberg-Reines J, Jacobsen JS, Marquis KL. Acute gamma-secretase inhibition improves contextual fear conditioning in the Tg2576 mouse model of Alzheimer’s disease. Journal of Neuroscience 2005;25:8898–902. [56] Gong B, Vitolo OV, Trinchese F, Liu S, Shelanski M, Arancio O. Persistent improvement in synaptic and cognitive functions in an Alzheimer mouse model after rolipram treatment. Journal of Clinical Investigation 2004;114: 1624–34. [57] Sierksma AS, Rutten K, Sydlik S, Rostamian S, Steinbusch HW, van den Hove DL, Prickaerts J. Chronic phosphodiesterase type 2 inhibition improves memory in the APPswe/PS1dE9 mouse model of Alzheimer’s disease. Neuropharmacology 2012. July 4. [58] Vitolo OV, Sant’Angelo A, Costanzo V, Battaglia F, Arancio O, Shelanski M. Amyloid beta-peptide inhibition of the PKA/CREB pathway and long-term potentiation: reversibility by drugs that enhance cAMP signaling. Proceedings of the National Academy of Sciences of the United States of America 2002;99:13217–21. [59] Pifarre P, Prado J, Baltrons MA, Giralt M, Gabarro P, Feinstein DL, Hidalgo J, Garcia A. Sildenafil (Viagra) ameliorates clinical symptoms and neuropathology in a mouse model of multiple sclerosis. Acta Neuropathologica 2011;121:499–508. [60] Patil CS, Padi SV, Singh VP, Kulkarni SK. Sildenafil induces hyperalgesia via activation of the NO–cGMP pathway in the rat neuropathic pain model. Inflammopharmacology 2006;14:22–7.

20

A. Palmeri et al. / Behavioural Brain Research 240 (2013) 11–20

[61] Adamo CM, Dai DF, Percival JM, Minami E, Willis MS, Patrucco E, Froehner SC, Beavo JA. Sildenafil reverses cardiac dysfunction in the mdx mouse model of Duchenne muscular dystrophy. Proceedings of the National Academy of Sciences of the United States of America 2010;107:19079–83. [62] Reid MB. Role of nitric oxide in skeletal muscle: synthesis, distribution and functional importance. Acta Physiologica Scandinavica 1998;162:401–9. [63] Marechal G, Gailly P. Effects of nitric oxide on the contraction of skeletal muscle. Cellular and Molecular Life Sciences 1999;55:1088–102. [64] Lacerda AC, Marubayashi U, Balthazar CH, Leite LH, Coimbra CC. Central nitric oxide inhibition modifies metabolic adjustments induced by exercise in rats. Neuroscience Letters 2006;410:152–6.

[65] Bocchi EA, Guimarães G, Mocelin A, Bacal F, Bellotti G, Ramires JF. Sildenafil effects on exercise, neurohormonal activation, and erectile dysfunction in congestive heart failure: a double-blind, placebo-controlled, randomized study followed by a prospective treatment for erectile dysfunction. Circulation 2002;106:1097–103. [66] Lewis GD, Shah R, Shahzad K, Camuso JM, Pappagianopoulos PP, Hung J, Tawakol A, Gerszten RE, Systrom DM, Bloch KD, Semigran MJ. Sildenafil improves exercise capacity and quality of life in patients with systolic heart failure and secondary pulmonary hypertension. Circulation 2007;116: 1555–62.