Sildenafil restores cognitive function without affecting β-amyloid burden in a mouse model of Alzheimer's disease
2011; Wiley; Volume: 164; Issue: 8 Linguagem: Inglês
10.1111/j.1476-5381.2011.01517.x
ISSN1476-5381
AutoresMar Cuadrado‐Tejedor, Isabel Hervías, Ana Ricobaraza, Elena Puerta, JM Pérez-Roldán, Carolina García, Rafael Franco, Norberto Aguirre, Ana Garcı́a-Osta,
Tópico(s)Cholinesterase and Neurodegenerative Diseases
ResumoBritish Journal of PharmacologyVolume 164, Issue 8 p. 2029-2041 Free Access Sildenafil restores cognitive function without affecting β-amyloid burden in a mouse model of Alzheimer's disease M Cuadrado-Tejedor, M Cuadrado-Tejedor Division of Neurosciences, CIMA, University of Navarra, Pamplona, SpainSearch for more papers by this authorI Hervias, I Hervias Department of Pharmacology, School of Medicine, University of Navarra, Pamplona, Spain These authors contribute equally to this work.Search for more papers by this authorA Ricobaraza, A Ricobaraza Division of Neurosciences, CIMA, University of Navarra, Pamplona, Spain These authors contribute equally to this work.Search for more papers by this authorE Puerta, E Puerta Department of Pharmacology, School of Medicine, University of Navarra, Pamplona, Spain These authors contribute equally to this work.Search for more papers by this authorJM Pérez-Roldán, JM Pérez-Roldán Division of Neurosciences, CIMA, University of Navarra, Pamplona, SpainSearch for more papers by this authorC García-Barroso, C García-Barroso Division of Neurosciences, CIMA, University of Navarra, Pamplona, SpainSearch for more papers by this authorR Franco, R Franco Division of Neurosciences, CIMA, University of Navarra, Pamplona, SpainSearch for more papers by this authorN Aguirre, N Aguirre Department of Pharmacology, School of Medicine, University of Navarra, Pamplona, SpainSearch for more papers by this authorA García-Osta, Corresponding Author A García-Osta Division of Neurosciences, CIMA, University of Navarra, Pamplona, SpainAna García-Osta, Division of Neurosciences, CIMA, University of Navarra, Av. Pio XII 55, 31008 Pamplona, Spain. E-mail: agosta@unav.esSearch for more papers by this author M Cuadrado-Tejedor, M Cuadrado-Tejedor Division of Neurosciences, CIMA, University of Navarra, Pamplona, SpainSearch for more papers by this authorI Hervias, I Hervias Department of Pharmacology, School of Medicine, University of Navarra, Pamplona, Spain These authors contribute equally to this work.Search for more papers by this authorA Ricobaraza, A Ricobaraza Division of Neurosciences, CIMA, University of Navarra, Pamplona, Spain These authors contribute equally to this work.Search for more papers by this authorE Puerta, E Puerta Department of Pharmacology, School of Medicine, University of Navarra, Pamplona, Spain These authors contribute equally to this work.Search for more papers by this authorJM Pérez-Roldán, JM Pérez-Roldán Division of Neurosciences, CIMA, University of Navarra, Pamplona, SpainSearch for more papers by this authorC García-Barroso, C García-Barroso Division of Neurosciences, CIMA, University of Navarra, Pamplona, SpainSearch for more papers by this authorR Franco, R Franco Division of Neurosciences, CIMA, University of Navarra, Pamplona, SpainSearch for more papers by this authorN Aguirre, N Aguirre Department of Pharmacology, School of Medicine, University of Navarra, Pamplona, SpainSearch for more papers by this authorA García-Osta, Corresponding Author A García-Osta Division of Neurosciences, CIMA, University of Navarra, Pamplona, SpainAna García-Osta, Division of Neurosciences, CIMA, University of Navarra, Av. Pio XII 55, 31008 Pamplona, Spain. E-mail: agosta@unav.esSearch for more papers by this author First published: 31 May 2011 https://doi.org/10.1111/j.1476-5381.2011.01517.xCitations: 124 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract BACKGROUND AND PURPOSE Inhibitors of phosphodiesterase 5 (PDE5) affect signalling pathways by elevating cGMP, which is a second messenger involved in processes of neuroplasticity. In the present study, the effects of the PDE5 inhibitor, sildenafil, on the pathological features of Alzheimer's disease and on memory-related behaviour were investigated. EXPERIMENTAL APPROACH Sildenafil was administered to the Tg2576 transgenic mouse model of Alzheimer's disease and to age-matched negative littermates (controls). Memory function was analysed using the Morris water maze test and fear conditioning tasks. Biochemical analyses were performed in brain lysates from animals treated with saline or with sildenafil. KEY RESULTS Treatment of aged Tg2576 animals with sildenafil completely reversed their cognitive impairment. Such changes were accompanied in the hippocampus by a reduction of tau hyperphosphorylation and a decrease in the activity of glycogen synthase kinase 3β (GSK3β) and of cyclin-dependent kinase 5 (CDK5) (p25/p35 ratio). Moreover, sildenafil also increased levels of brain-derived neurotrophic factor (BDNF) and the activity-regulated cytoskeletal-associated protein (Arc) in the hippocampus without any detectable modification of brain amyloid burden. CONCLUSIONS AND IMPLICATIONS Sildenafil improved cognitive functions in Tg2576 mice and the effect was not related to changes in the amyloid burden. These data further strengthen the potential of sildenafil as a therapeutic agent for Alzheimer's disease. Abbreviations Aβ β-amyloid Arc activity-regulated cytoskeletal-associated protein BDNF brain-derived neurotrophic factor CDK5 cyclin-dependent kinase 5 CREB cAMP response element binding CS conditioned stimulus GSK3β glycogen synthase kinase 3β IEG immediate early gene MWM Morris water maze US unconditioned stimulus Introduction Phosphodiesterases (PDEs) are enzymes that hydrolyse the cyclic nucleotides cAMP or cGMP, which act as second messengers in intracellular signalling and in processes of neuroplasticity, such as long-term potentiation (Frey et al., 1993; Son et al., 1998). PDE inhibitors affect signalling pathways by elevating cAMP and/or cGMP levels, which may ultimately lead to gene transcription through activation of cAMP response element binding (CREB) (Impey et al., 1996; Lu et al., 1999). CREB-dependent gene expression has been shown to underlie long-term memory formation in several vertebrate and invertebrate species, probably through the formation of new synaptic connections (Tully et al., 2003). The pathological signs of Alzheimer's disease include (i) the presence of plaques (composed of deposits of amyloid filaments) and neurofibrillary tangles (composed of deposits of hyperphosphorylated tau) surrounded by altered neurite processes and glia; (ii) the loss of synapses; and (iii) a degeneration of the neurons (Selkoe, 2002). One of the earliest manifestations of Alzheimer's disease is the inability of affected individuals to form new memories. Memory impairment appears to significantly predate the death of nerve cells, implying that neuronal dysfunction is responsible for the pathophysiology of early stage Alzheimer's disease. Administration of sildenafil, a selective PDE5 inhibitor, activates the NO/cGMP pathway and significantly increases brain cGMP levels (Hartell, 1996; Prickaerts et al., 2002a,b; Zhang et al., 2002; Puerta et al., 2009). PDE5 inhibitors constitute an effective treatment for erectile dysfunction; however, the presence of PDEs in various regions of the CNS (Loughney et al., 1998; Reyes-Irisarri et al., 2005) and the fact that cGMP has been recognized as a second messenger of key neural phenomena such as synaptic plasticity (Haghikia et al., 2007; Paul et al., 2008) substantiate the potential use of PDE inhibitors for neurological disorders. Moreover, animal studies have shown that sildenafil enhances memory in several models (Prickaerts et al., 2002b; Rutten et al., 2005) and attenuates memory impairment induced by NO synthase (NOS) inhibition (Devan et al., 2006; 2007). Another study shows that sildenafil, dose-dependently, improves performance in the object retrieval task in cynomolgus macaques (Rutten et al., 2008). Cognitive dysfunction by blockade of muscarinic cholinergic receptor (Devan et al., 2004), diabetes or electroconvulsive shock (Patil et al., 2006) is also reversed by sildenafil treatment. Glycogen synthase kinase 3β (GSK3β) and cyclin-dependent kinase 5 (CDK5) are the most relevant kinases involved in the pathogenic mechanisms of Alzheimer's disease through the phosphorylation at multiple sites of the microtubule-binding protein, tau (Hanger et al., 1992; Mandelkow et al., 1992; Ishiguro et al., 1993; Tomidokoro et al., 2001; Elyaman et al., 2002; Liu et al., 2002; Otth et al., 2002; Tsai et al., 2004). These kinases are associated with neuronal death, the formation of paired helical filaments and neurite retraction (Plattner et al., 2006; Twomey and Mccarthy, 2006; Lopes et al., 2007). Therefore, inhibition of GSK3β and CDK5 activity has been proposed as a plausible therapeutic target for the treatment of Alzheimer's disease (Lau et al., 2002; Koh et al., 2007). Puzzo et al. (2009) have recently demonstrated that sildenafil produces an immediate and long-lasting improvement of synaptic function, CREB phosphorylation and memory in a mouse model of amyloid deposition. This effect is also associated with a long-lasting reduction of β-amyloid (Aβ) levels. In the present study, we investigated whether sildenafil could reverse the memory impairment in an aged mouse model of Alzheimer's disease with a pathology showing both Aβ deposits and hyperphosphorylated tau. Our results demonstrated that sildenafil restored cognitive deficits in this model of Alzheimer's disease, without affecting the Aβ-burden. Methods Mouse model and treatment All animal care and experimental procedures were in accordance with European and Spanish regulations (86/609/CEE; RD1201/2005) and were approved by the Ethical Committee of the University of Navarra (no. 018/05). Behavioural studies were carried out during light time (from 9 am to 2 pm). In this study, Tg2576 Alzheimer's disease transgenic mice, that express the human 695-aa isoform of the amyloid precursor protein (APP) containing the Swedish double mutation (APPswe) [(APP695)Lys670→Asn, Met671→Leu] driven by a hamster prion promoter, were used. The mice were on an inbred C57BL/6/SJL genetic background. In the Tg2576 Alzheimer's disease mouse model, Aβ peptide content in the brain accumulates exponentially between 7 and 12 months of age and mice show impaired memory in the water maze test at the age of 12–15 months (Hsiao et al., 1996; Reed et al., 2010). Based on effectiveness and toleration, the dose of sildenafil citrate (Viagra;Pfizer, New York, NY, USA) used in patients with erectile dysfunction is 25–100 mg·day−1. The dosage of sildenafil we used in the transgenic Alzheimer's disease mouse model is 15 mg·kg−1·day−1, which is equivalent to 85 mg·day−1 in humans, using the BSA-based dose calculation (Reagan-Shaw et al., 2008). First of all, the effect of sildenafil on the modulation of memory-associated immediate early genes (IEGs), whose expression may be altered in APP transgenic mice (Dickey et al., 2004), was assessed. For this, 14- to 16-month-old female Tg2576 mice were treated once daily with sildenafil (15 mg·kg−1, i.p.) or saline for 5 days. The last injection was given 30 min before being trained in a hippocampal-dependent memory task and mice were killed 2 h after the training session. A second group of animals was used to test the long-term effect of sildenafil in cognitive function. In this set of experiments, 14- to 16-month-old female Tg2576 mice and age-matched negative littermates (controls) were treated once daily with sildenafil (15 mg·kg−1, i.p.) or saline for 5 weeks. Morris water maze test The Morris water maze (MWM) test was used to evaluate spatial memory function in response to treatment with sildenafil, as previously described (Hsiao et al., 1996; Ricobaraza et al., 2009; Reed et al., 2010). After treatments, groups of animals underwent spatial reference learning and memory testing in the MWM. The water maze was a circular pool (diameter 1.2 m) filled with water maintained at 20°C and made opaque by the addition of non-toxic white paint. Mice were trained for three consecutive days (8 trials per day) swimming to a raised platform (visible-platform). No distal visible cues were present during this phase. The same platform location was used for all visible-platform sessions and was changed for the hidden-platform training (submerged 1 cm beneath the surface) conducted over 8 consecutive days (4 trials per day) with all visible distal cues present in this phase. In both visible- and hidden-platform versions, mice were placed pseudo-randomly in selected locations, facing towards the wall of the pool to eliminate the potentially confounding contribution of extramaze spatial cues. Each trial was terminated when the mouse reached the platform or after 60 s, which ever came first. To test the retention, three probe trials were performed at the beginning of 4th, 7th and the last day of the test (day 9). In the probe trials the platform was removed from the pool, and the percentage of time spent in the quadrant where the platform was previously set was recorded. All trials were monitored by a camera above the centre of the pool connected to a SMART-LD program (Panlab S.L., Barcelona, Spain) for subsequent analysis of escape latencies, swimming speed, path length and per cent time spent in each quadrant of the pool during probe trials. All experimental procedures were performed without knowledge of the treatments of the groups. Fear conditioning test After running the MWM, all the animals were trained in the fear conditioning test. The conditioning procedure was carried out in a StartFear system (Panlab S.L.) that allows recording and analysis of the signal generated by the animal's movement through a high sensitivity Weight Transducer system. The analogue signal is transmitted to the FREEZING and STARTLE software modules through the load cell unit for recording purposes and later analysis, in terms of activity/immobility. The conditioning box is housed inside a soundproof chamber, which minimized external stimulation during the conditioning and retention tests. The box was provided with a house light that supplied dim illumination and with a floor grid through which foot shocks could be administered. On a training day, the mice were placed in the conditioning chamber for 2 min before the onset of a tone at 2800 Hz, 85 dB (conditioned stimulus, CS), which lasted for 30 s. The last 2 s of the CS was paired with a 0.3 mA foot shock (unconditioned stimulus, US). After the shock and 10 s of resting the same CS-US was delivered three consecutive times. Finally, 30 s after the last pair of CS-US, mice were returned to their home cages. To test the effect of sildenafil in non-transgenic mice, a lighter paradigm of training (1 CS-US pairing, Ricobaraza et al., 2010) was used to avoid an overtraining that may prevent the detection of a possible memory enhancement. Twenty-four hours after the training, the mice were placed again in the conditioning chamber; after 2 min of exposure, the tone starts for a period of 2 min and freezing time was assessed during the 4 min (no differences were found in the freezing behaviour with or without the tone). Freezing behaviour was defined as the lack of movement except for breathing for at least 2 s, and was analysed to give the percentage time freezing during exposure to the chamber. The conditioning apparatus was controlled by the experimenter with specific software (Packwin, Panlab S.L.) running on a PC computer. Twenty-four hours after the fear conditioning test, the animals were killed and the brains removed for biochemical studies. One hemi brain was post-fixed in 4% paraformaldehyde, (PFA) followed by immersion in 2% PFA (24 h) and cytoprotected in 30% sucrose solution in phosphate buffer overnight at 4°C. Microtome sections (30µm thick) were cut coronally, collected free floating and stored in 30% ethylene glycol, 30% glycerol and 0.1 M phosphate buffer at −20°C until processed. The cortex and hippocampus from the other hemi brain were dissected, homogenized and processed as described below for subsequent Western blot. Determination of Aβ levels For analysis of total (soluble and insoluble) Aβ42 burden, the frontal cortex was homogenized in a buffer containing 5 M guanidine HCl and 50 mM Tris-HCl, pH 8, protease inhibitors (Complete Protease Inhibitor Cocktail, Roche, Barcelona, Spain) and phosphatase inhibitors (0.1 mM Na3VO4, 1 mM NaF). Aβ42 levels detected with 3D6 antibody, specific for amino acids 1–5 of Aβ and shows no cross-reactivity to the endogenous murine Aβ protein at concentrations up to 1 ng·mL−1 (Johnson-Wood et al., 1997), were measured using a sensitive sandwich elisa kit from Biosource (Camarillo, CA, USA) following the manufacturer's instructions. Immunohistochemistry Floating tissue sections comprising hippocampal formation were processed for 6E10 immunostaining following the protocol previously described by Ricobaraza et al. (2009). Briefly, brain sections were incubated in blocking solution (PBS containing 0.5% Triton X-100, 0.1% BSA and 2% normal goat serum) for 2 h at room temperature. After washing, sections were incubated in 70% formic acid for 7 min to expose the epitope. Sections were incubated with the 6E10 antibody (against amino acids 1–17 of Aβ peptide, 1:200, Chemicon) for 24 h at 4°C, washed with PBS and incubated with the secondary antibody (Alexa Fluor 488 goat anti-mouse highly cross-absorbed, Molecular Probes, Eugene, OR, USA, 1:400) for 2 h at room temperature, protected from light. Fluorescence signals were detected with confocal microscope LSM 510 Meta (Carl Zeiss, Germany); objective Plan-neofluar 40×/1.3 oil DIC. Sections were evaluated in Z-series (0.4 µm steps) using LSM 510 Meta software. Production of protein extracts Mice were killed by cervical dislocation and hippocampi quickly dissected from the brains. Total tissue homogenates were obtained by homogenizing the hippocampus in a cold lysis buffer with protease inhibitors (0.2 M NaCl, 0.1 M HEPES, 10% glycerol, 200 mM NaF, 2 mM Na4P2O7, 5 mM EDTA, 1 mM EGTA, 2 mM DTT, 0.5 mM PMSF, 1 mM Na3VO4, 1 mM benzamidine, 10 µg·mL−1 leupeptin, 400 U·mL−1 aprotinin), centrifuged at 14 000×g 4°C for 20 min and the supernatant was aliquoted and stored at −80°C. Total protein concentrations were determined using the Bio-Rad Bradford protein assay (Bio-Rad Laboratories). For APP carboxy-terminal fragments determination, the prefrontal cortex was homogenized in a buffer containing SDS 2%, Tris-HCl (10 mM, pH 7.4), protease inhibitors (Complete Protease Inhibitor Cocktail, Roche) and phosphatase inhibitors (0.1 mM Na3VO4, 1 mM NaF). The homogenates were sonicated for 2 min and centrifuged at 100 000×g for 1 h. Aliquots of the supernatant were frozen at −80°C and protein concentration was determined by the Bradford method using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Immunoblotting Protein samples were mixed with an equal volume of 2 × Laemmli sample buffer, resolved onto SDS-polyacrylamide gels and transferred to nitrocellulose membrane. The membranes were blocked with 5% milk, 0.05% Tween-20 in PBS or TBS followed by overnight incubation with the following primary antibodies: mouse monoclonal anti-p-tau AT8 (1:1000, Pierce Biotechnology, Inc. Rockford, USA), mouse monoclonal anti-tau (1:5000, clone Tau46, Sigma-Aldrich, St. Luis, MO, USA), rabbit polyclonal anti-pGSK3-Ser-9 (1:1000, Cell Signalling Technology, Beverly, MA), rabbit polyclonal anti-pAkt-Ser-473 (1:1000, Cell Signalling Technology), rabbit monoclonal anti-Akt (1:1000, Cell Signalling Technology), mouse polyclonal anti-pGSK3β-Tyr-216 (1:1000, BD Transduction Laboratories, Lexington KT) rabbit polyclonal anti-pCREB (1:500, Upstate-Millipore, Temecula, CA, USA), rabbit polyclonal anti-CREB (1:1000, Cell Signalling Technology), rabbit polyclonal anti-GSK3 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-c-fos (1:1000, Santa Cruz Biotechnology), rabbit polyclonal anti-Arc (1:1000, Santa Cruz Biotechnology), rabbit polyclonal anti-brain-derived neurotrophic factor (BDNF) (1:1000, Osenses Pty Ltd, Flagstaff Hill, SA, Australia) rabbit polyclonal anti-p35/p25 (1:1000, Cell Signalling Technology), mouse monoclonal anti-actin (1:2000, Sigma-Aldrich, St. Louis, MO, USA) and mouse monoclonal anti-tubulin (1:10000, Sigma-Aldrich) in the corresponding buffer. Following two washes in PBS/Tween-20 or TBS/Tween-20 and one PBS or TBS alone, immunolabelled protein bands were detected by using HRP-conjugated anti-rabbit or anti-mouse antibody (Santa Cruz; dilution 1:5000) following an enhanced chemiluminescence system (ECL, GE Healthcare Bioscience, Buckinghamshire, UK), and autoradiographic exposure to Hyperfilm ECL (GE Healthcare Bioscience). Quantity One software v.4.6.3 (Bio-Rad) was used for quantification. For Western blot analysis of APP-derived fragments, aliquots of the protein extracts were mixed with XT sample buffer plus XT reducing agent or Tricine sample buffer (Bio-Rad) and boiled for 5 min. Proteins were separated in a Criterion precast Bis-Tris 4–12% gradient precast gel (Bio-Rad) and transferred to nitrocellulose membranes. The membranes were blocked with 5% milk, 0.05% Tween-20 in TBS followed by overnight incubation with the following primary antibodies: mouse monoclonal 6E10 (amino acids 1–17 of Aβ peptide, 1:1000, Millipore, Billenica, MA), rabbit polyclonal anti-APP C-terminal (amino acids 676–695) (1:2000, Sigma-Aldrich). Data analysis and statistical procedures The results were processed for statistical analysis using spss package for Windows, version 15.0 (SPSS, Chicago, IL, USA). Unless otherwise indicated, results are presented as mean ± SEM. In the MWM and fear conditioning test, escape latencies during training were analysed using one-way anova followed by Scheffe's post hoc test. In the MWM, Friedman's test was performed to determine the intra-group comparisons over trials. Biochemical data were analysed using Kruskal–Wallis test followed by Mann–Whitney post hoc test. Student's t-test was used in case two groups were compared. Results Sildenafil facilitated the induction of memory-associated genes in Tg2576 mice The activation of several immediate early genes (IEGs) is crucial for long-term memory formation and amyloid deposition in mice leads to impaired induction of the IEGs expressed by exposure to a novel environment (Dickey et al., 2004). To know whether sildenafil affected the induction of IEGs, 14- to 16-month-old female Tg2576 mice were treated with sildenafil (15 mg·kg−1, i.p.) or saline once a day for 5 days. Animals were given fear conditioning training 30 min after the last injection and they were killed 2 h later (Figure 1A). Gene expression in the hippocampus of transgenic mice that received saline or sildenafil were investigated and compared with a group of aged- and strain-matched non-transgenic mice that underwent the same procedure. Figure 1Open in figure viewerPowerPoint The induction of memory-related genes in the hippocampus of aged Tg2576 mice is facilitated with sildenafil treatment. (A) Scheme showing times of injection, training and death. Administration of sildenafil induces c-fos (B), Arc (C) and pCREB (D) following fear conditioning training. Representative Western blot bands from hippocampal tissues of non-transgenic mice (Non-Tg), transgenic mice treated with saline (Tg2576 saline) or with sildenafil (Tg2576 sildenafil) are shown. The histograms represent the quantification of the immunoreactive bands in the Western blot. Data are expressed as mean percentage (±SEM) of the Non-Tg results (100%). n= 5–6 in each group. *P < 0.05, significantly different from Tg2576 saline mice (Kruskal–Wallis followed by Mann–Whitney post hoc test). The genes for the synaptic activity-dependent proteins, Arc and c-fos, are induced specifically in neurons engaged in memory-encoding processes (Guzowski et al., 1999). Tg2576 mice had a marked reduction in c-fos expression (58 ± 4% reduction; P < 0.05), which was partially reversed by sildenafil treatment (Figure 1B). Moreover, a significant increase (more than twofold) in Arc expression was observed in the sildenafil-treated group compared with Tg2576 saline-treated mice; the level of Arc was similar in wild-type and saline-treated Tg2576 mice (Figure 1C). An important mediator of transcriptional changes associated to memory is the transcription factor CREB protein (Dash et al., 1990). In the Tg2576 group receiving sildenafil there was a significant increase in the expression of hippocampal pCREB compared with that of the transgenic group receiving saline (Figure 1D). Collectively these data indicated that, in the hippocampus of transgenic mice, the induction of IEGs, related to plasticity and memory consolidation, was facilitated by sildenafil. Sildenafil restored cognitive function in Tg2576 mice Cognitive impairment in Tg2576 animals starts at the age of 12 months in the MWM, the memory retention measured in the probe trials being more affected (Hsiao et al., 1996; Westerman et al., 2002; Reed et al., 2010). Studies were performed comparing heterozygous transgenic Tg2576 females with age- and strain-matched transgenic negative littermates (controls) that were treated once daily with sildenafil (15 mg·kg−1, i.p.) or saline for 5 weeks. No significant differences were found among the groups during the training phase of the test (visible-platform, Figure 2B), but there were significant differences in the spatial learning during the hidden-platform between groups [F(2, 238)= 25.35; P < 0.001] (Figure 2C). Tg2576 mice treated with saline were impaired in their performance in this test (days 2–8) compared with age-matched non-transgenic mice (sildenafil- and saline-treated groups) or Tg2576 mice treated with sildenafil. Figure 2Open in figure viewerPowerPoint Chronic treatment with sildenafil reverses the learning deficit in aged Tg2576 mice. (A) Scheme showing times of injection, training and death. Escape latency of the visible- (B) and hidden-platform (C) in the MWM test for the non-transgenic mice and transgenic mice treated with saline (Non-Tg saline; Tg2576 saline) or sildenafil (Non-Tg sildenafil; Tg2576 sildenafil). Results are expressed as mean ± SEM (n= 10–12 in each group). Tg2576 saline mice showed significantly longer escape latencies in the hidden-platform training thatn the Non-Tg saline group (**P < 0.01, ***P < 0.001, anova with Scheffe's post hoc test), Non-Tg sildenafil ($P < 0.01, $$$P < 0.001, anova with Scheffe's post hoc test) and to Tg2576 sildenafil-treated group (††P < 0.01, †††P < 0.001, anova with Scheffe's post hoc test). (D) Percentage of time spent searching for the target quadrant of the probe test. Results are expressed as mean ± SEM n= 10–12 in each group. Tg2576 saline mice performed significantly worse than Non-Tg saline (*P < 0.05, **P < 0.01, anova with Scheffe's post hoc test), Non-Tg sildenafil ($P < 0.05, $$P < 0.01, anova with Scheffe's post hoc test) and Tg2576 sildenafil (†P < 0.05, anova with Scheffe's post hoc test) in the probe trial. (E) Tg2576 saline mice exhibited significantly less freezing than the Non-Tg control mice (*P < 0.05, anova with Scheffe's post hoc tests) and Tg2576 sildenafil mice (†P < 0.05, anova with Scheffe's post hoc tests) in the fear conditioning task. Results are expressed as mean ± SEM n= 10–12 in each group. (F) Aged wild-type (Non-Tg) mice receiving sildenafil for 5 weeks showed a significant enhancement of memory compared with Non-Tg saline-treated mice ($P < 0.05, Student's t-test). Results are expressed as mean ± SEM n= 10–12 in each group. Intra-group comparisons were analysed by the latencies in the hidden-platform training over trials using the Friedman's repeated measure non-parametric test. The mean latencies (time spent) to reach the platform decreased over the training sessions for the non-transgenic mice treated with saline (χ2r = 30.50, P < 0.001), sildenafil (χ2r = 28.00, P < 0.001) and for the transgenic sildenafil-treated group (χ2r = 26.40, P < 0.001, Friedman's test). On the contrary, the latencies exhibited by the saline-treated transgenic group did not significantly decrease over trials (χ2r = 6.15, P > 0.05). The results indicate that non-transgenic and sildenafil-treated animals tended to learn correctly the platform location, whereas saline-treated transgenic animals did not. Specifically, intra-group comparisons of escape latencies showed a significant effect of the training for the non-transgenic and the transgenic sildenafil-treated groups. In contrast, transgenic saline-treated mice did not show any significant reduction in their escape latencies from days 2 to 8, compared with the first training day, reflecting their inability to learn the platform location. As a putative measurement of memory retention mice swam in the pool with the platform removed. On day 4, no significant differences were found among the groups. [F(2, 30)= 2.06, P= 0.10]. One-way anova showed significant differences between groups on days 7 and 9 [F(2, 30)= 8.96, P < 0.01 for day 7; and F(2, 30)= 5.94 P < 0.01, for day 9]. On days 7 and 9 the proportion of time spent in the target quadrant was significantly lower for transgenic mice treated with saline, compared with the non-transgenic mice or the transgenic mice that received sildenafil treatment. Transgenic sildenafil-treated animals spent a
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