1. Introduction
Senescence is an unavoidable process that affects every living organism. As a consequence of advancing age, biological systems lose their function, giving rise to chronic diseases, morbidity, and eventually death. By 2050, the estimated number of people that will be affected by aging-related dementia is expected to be three times higher than currently known [1]. Aging appears to differentially affect the two sexes, especially in terms of cognitive function. For example, although sex differences in the incidence of Alzheimer’s disease are small, sex appears to moderate the relationship between aging and cognitive decline, with women who present with particular vulnerabilities showing a steeper cognitive decline [2]. In mammalian females, memory and cognition are influenced by the effects that fluctuating gonadal steroids have on different brain areas. Consequently, for women, the menopause transition, when the levels of estrogen and progesterone are reduced, is a very challenging period of life characterized by mood disorders and cognitive declines [3].
Gonadal steroids, and especially estrogen, have neuroprotective and neuroplastic effects in the hippocampus [4], while ovariectomy has detrimental effects on memory tasks that depend on the hippocampus [5]. For example, estrogen is known to contribute to dendritic and synaptic plasticity and to influence long-term potentiation (LTP) in the hippocampus [6]. In several regions of the brain, such as the hippocampus and the hypothalamus, estrogens interact with the nitric oxide (NO) system [4]. The interplay between gonadal steroids and the NO system is exemplified by the progesterone-induced lordosis required for copulation [7], by the estrogen-dependent increased immunostaining for neuronal NOS in several brain areas of mice [4], and by estrogen modulation of NOS in the hippocampus of rats [8].
Among the different biological mechanisms that are important players in the regulation of age-dependent cognitive decline, the downregulation of the cyclic nucleotide cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) cascades appears to play a major role [9]. cAMP and cGMP have been implicated in the processes of long-term potentiation (LTP) and memory, and their intracellular production is modulated by NO [10,11]. Substances that block the synthesis of NO through the inhibition of nitric oxide synthase (NOS) have been shown to impair cognitive performance in animal models of learning and memory, specifically in the novel object recognition task (ORT) [12,13,14], an animal model of episodic memory. On the contrary, selective inhibitors of the phosphodiesterase enzymes (PDEs) have been found to counteract these cognitive impairments. PDEs are a large family of enzymes that, as part of homeostatic negative feedback, degrade either one or both of the cyclic nucleotides [15]. Thus, the use of drugs that inhibit PDEs has been investigated, producing some interesting results, especially in light of possible treatments for aging-related cognitive declines in Alzheimer [16].
Good examples of PDE inhibitor drugs with positive effects on cognition are Sildenafil and Vardenafil, two drugs that specifically inhibit PDE5, an enzyme that degrades cGMP and regulates the NO-cGMP/PKG pathway. This pathway induces the phosphorylation of cAMP response element-binding protein (CREB) [17], and it is implicated in the CREB-dependent LTP [18,19]. Despite their role as vasodilators and their successful use in the treatment of erectile dysfunction, both Sildenafil and Vardenafil were able to rescue cognitive performance in the ORT in rodents [20,21,22] and in animal models of Alzheimer’s disease [23,24]. For example, it has been shown that a three-week continuous treatment with Sildenafil increased CREB phosphorylation and improved memory performance in transgenic mice expressing the human amyloid precursor protein APP [25,26]. Similar results were reported for Vardenafil, although concomitantly with roflumilast, a PED4 inhibitor [24]. These studies strongly suggest that PDE5 inhibitors can enhance neuroplasticity and memory formation through their enhancement of the NO-cGMP pathway.
In the present study, we tested the effects of Vardenafil on the episodic memory of very old female mice as they performed the ORT. The performance in the ORT of female rodents depends on the levels of gonadal steroids [27,28] and, ultimately, on the cellular cascade of molecular messengers that includes the cyclic nucleotide monophosphates cGMP [20] and cAMP, both implicated in long-term potentiation (reviewed in Winters et al. [29]). Several cortical and subcortical regions, such as the hippocampus and the perirhinal cortex, are necessary for memory formation in the ORT [29,30], and these areas are key targets of gonadal steroids.
Chronic treatment of gonadectomized female rats with estradiol induces neuronal nitric oxide synthase [31]. In normally cycling female mice, the number of NO-expressing neurons in different brain areas, such as the hippocampus, varies as a function of the levels of estrogen [31]. These data suggest that the performance of female rodents in the ORT depends on a close interplay between the estrogenic milieu and the NO-cGMP pathway. Given that the low levels of ovarian steroids that characterize female aging have negative effects on rodents’ performance in the ORT [32], it is possible that these deficits may be caused by a reduced activity of the nitronergic system. Consequently, inhibition of the PED5 enzyme is expected to rescue the performance in the ORT of aged females.
Previous studies that looked at the role of Vardenafil on ORT performance have demonstrated that both acute and chronic exposure improved memory in rats [20,33,34]. Similar results were reported in young and older mice and in a transgenic mouse model of AD for Sildenafil, a similar but weaker PDE5 inhibitor [22,23,35]. However, these studies have mainly focused on males or used mixed-sex groups. To the best of our knowledge, no studies have directly investigated the effects of PDE5 inhibitors and specifically of Vardenafil on object recognition memory in very old female mice. The present study aims at filling this gap.
2. Materials and Methods
2.1. Animals and Husbandry
Experimental animals were CD1 mice derived from Charles River Italia (Calco, Italy), but born and reared in our colony room at the Laboratory of Behavioral Biology at the University of Parma. Mice were kept in rooms at 22 ± 2 °C in a 12 h light-dark cycle (lights on 07:00–19:00). Throughout the study, food (4rf21 standard diet, Mucedola, Italy) and water were available ad libitum. After weaning (25–28 days), mice were housed in same-sex groups of siblings (5 ± 1 per cage) in Plexiglas cages (45 cm × 25 cm × 20 cm) with wood shaving bedding. The cages were changed weekly. Housing and experiments were conducted in accordance with the animal experimentation directive of the European Communities Council Directive of 22 September 2010 (2010/63/EU) and approved by the Italian Institute of Health.
2.2. Experimental Procedure
Twenty-five females were assigned to the study, and at the beginning of the test session, the females were 24 months old. Of these 25 mice, three died before the beginning of the study. Of the remaining twenty-two mice, eleven were randomly assigned to the continuous Vardenafil treatment, and 5 animals (3 controls) died during the period of the experiment. On day 1 of the procedure, females were individually housed in plexiglass cages (27 cm × 21 cm × 15 cm) with food and water ad libitum. Beginning on day 9, animals in the treatment group were intraperitoneally injected with 2 mg/kg of Vardenafil, while controls were exposed to intraperitoneal injections of saline solution. On day 29, mice were exposed to a battery of tests and were sacrificed at the end of the study after the last treatment. Treatment was carried out at the same time every day and after the end of the tests. Body weight was measured throughout the experimental period.
2.3. Elevated plus Maze (EPM) Test
Mice were tested in the EPM on day 29 of the experimental procedure. The time spent and frequency of entry in the closed and open arms of the maze were measured and used to derive the levels of anxiety-related behaviors. The testing procedure used in this study was based on a method previously described and used in our laboratory [36]. The EPM consisted of a black wooden platform covered with transparent plastic, with two open arms (30 cm × 5 cm) and two closed arms (30 cm × 5 cm × 40 cm) arranged in the shape of a plus sign. The arms extended from a central platform (5 cm × 5 cm). The maze was elevated 50 cm above the floor. Mice were individually placed on the central platform facing an open arm and allowed to explore the maze freely for 5 min. The frequency of entries into the open arms and closed arms of the maze and time spent in the respective arms were recorded with a video camera situated 2 m above the apparatus. To reduce any lingering olfactory cues, between successive trials, the maze was cleaned with a clean, damp cloth and a solution of 2% ethylic alcohol. Latency to enter, duration within, and frequency of transitions between different arms were recorded on a SD memory card and later analyzed and scored by a trained research assistant using specific software (Observer XT 9.0, Noldus).
2.4. Free Exploratory Test
On day 31 of the experimental procedure, mice were exposed to the free exploratory open field test. The unfamiliar area was a rectangular arena of 73 cm × 110 cm bordered along by a 50 cm high polypropylene wall and divided into 20 squares in which a bright and a dark area were created. Tests were conducted between 14:00 and 18:30 h in a darkened room illuminated with a white light, which was positioned behind and above the wall of the open field (OF) so as to cast a shadow along the length of the OF. In particular, eight zones can be observed: a dark area near the home cage; a bright area near the home cage; far dark, far bright, wall dark, wall bright, center dark, and center bright areas (see Bartolomucci et al. [37]). During observation, the experimenter sat 2.5 m away from the apparatus. Five minutes before testing, the home cage was placed at one end of the rectangular OF. Once the barrier was removed, mice were given a total of 10 min to emerge from the home cage and enter the OF. Risk assessment, behaviors, and time spent exploring each zone were recorded from the first approach to the entrance. If the mice were to enter the OF with the four paws, a maximum time of 5 min of exploration was given. To reduce any lingering olfactory cues, the apparatus was wiped with a clean, damp cloth and a solution of 2% ethylic alcohol between each test. Sessions were recorded on videotape by a video camera situated 2 m above the apparatus.
2.5. Object Recognition Test (ORT)
The test was carried out from day 39 to day 40 of the experiment, at which point mice were exposed to a daily injection of Vardenafil or vehicle for 32 days. The test was designed following the guidelines described by Ennaceur and Delacour [12]. The apparatus consisted of a square arena (54 cm × 54 cm × 40 cm) made of polyethylene with a gray floor and constant illumination. The day before the test, each mouse was allowed to freely explore the arena for ten minutes. On the first day of the test, two objects were placed symmetrically, about 5 cm from the wall, and each object was available in duplicate. The objects were a cone (polyethylene), a sealed 0.5 mL glass bottle filled with an opaque solution, and a cube made of plexiglass and filled with sawdust. Animals were brought into the testing room at least 30 min before the beginning of the session in order to become accustomed to the experimental room. A testing session comprised three trials, each lasting five minutes. In the first trial of familiarization (T1), two identical objects were placed on opposite sides of the arena, and a mouse was placed in the corner. The starting corners were alternated between animals receiving the same treatment. The second (T2) and third (T3) trials occurred 60 min and 24 h after the end of T1, respectively. In T2 and T3, mice were placed back in the arena, but now with dissimilar objects: a familiar one (present in T1) and a new one. In these two trials, the familiar object was the same. The time spent exploring each object during each trial was recorded by means of a video camera and analyzed with the program The Observer (Noldus). Exploration was defined as the mouse’s nose placed at a distance of no more than 2 cm and/or touching the object.
2.6. Drug Preparation and Treatment
The Vardenafil (Bayer, Italy) solution was prepared according to Dadomo et al. [38]. Briefly, Vardenafil (20 mg) tablets were ground into a fine powder using a mortar and pestle. As the good solubility in water of Vardenafil is 1.1 mg/mL, the drug was dissolved in 100 mL of saline solution (0.9% NaCl) and passed through a 40-micron filter (PTFE Filters, Cole-Parmer, General control, Milan, Italy) to eliminate residues of excipients. The final concentration, 0.2 mg/mL, was checked by high-performance liquid chromatography (HPLC; see later drug analysis). The resulting solution was kept at 4 °C. A drug solution was prepared every 4 days, and the stability of the solution was spectrometrically determined. The drug solution was brought to room temperature for 2 h prior to injections. The dosages for mice were calculated using allometric dose translation of BSA by the formula: Mice dosage (mg/kg) = Human dosage (mg/kg)/(mice K.M./human K.M.). The final dosage of the drug is 2 mg/kg. Dosages of 2 mg/kg, injection volumes, and time of injection are based on extensive previous experience in the lab with the current drugs [38]. Moreover, for reasons of generalizability of the results, 2 mg/kg is a dose that is functionally equivalent to the dosage used in humans. Vardenafil solution was injected intraperitoneally (i.p.) in a volume of 1 mL per 100 g of mouse body weight in the evening, after the behavioral tests.
2.7. Statistical Analysis
To test if Vardenafil affected general motor activity and anxiety-like behaviors, we used a one-way ANOVA on several target measures of the OPF and EPM. In the ORT, preference for the novel object was measured by means of a relative discrimination index (DI) calculated as the difference between the time spent investigating the novel object (b) minus the time spent on the familiar object (a) divided by the total time spent exploring both objects (DI = (b – a)/(a + b)). A DI above 0 indicates preference for the novel object, while a DI equal to or close to zero indicates a lack of discrimination. Due to the lack of normal distribution for the DI and the small sample size, in order to establish if vehicle-exposed animals discriminated between novel and familiar objects, we used a one-sample Wilcoxon-signed rank test where we compared the DI score at T1 (1 h delay) or T2 (24 h delay) from acquisition with a median of 0 [39]. Between-group effects T1 and T2 were assessed using Mann-Whitney tests. In the ORT, one female from the vehicle group scored 3.0 SD below the average for the discrimination at T1 and 2.6 SD below the average for T2. This animal’s scores were considered outliers, and the animal was removed from the statistical analysis. The significance level for all analyses was set at p < 0.05. Analyses were carried out using SPSS 28.0.
3. Results
3.1. Treatment with Vardenafil Does Not Affect Anxiety-like Behaviors and Locomotor Activity in Aged Female Mice
Body weight at the beginning (M vehicle = 39.92 g, SE = 1.51; M Vardenafil = 39.61 g, SE = 1.39) and at the end (M vehicle = 36.32, SE = 0.62; M Vardenafil = 39.05 g, SE = 1.75) of the study did not differ between the two groups. Table 1 presents the results for exploration time of the two objects (familiar and novel) and habituation in the three different trials. e1 corresponds to the total exploration of the two familiar objects during the familiarization trial. e2 and e3 represent the time spent exploring the familiar and novel objects. h is a measure of habituation given by e1 − e2 (h1) and e1 − e3 (h24).
Vardenafil did not influence the time spent in the center, F(1,15) = 0.27, p > 0.05; the total number of visits to the center, F(1,15) = 1.55, p > 0.05; the time spent in the open arms, F(1,15) = 1.01, p > 0.05; total visits in the open arms, F(1,15) = 1.71, p > 0.05; time spent in the closed arms, F(1,15) = 0.47, p > 0.05; and total visits of the open arms, F(1,15) = 0.73, p > 0.05 in the elevated plus maze (Figure 1a,b). Moreover, no differences in the number of transitions were found; F(1,15) = 1.61, p > 0.05. No differences were found between the Vardenafil and saline groups for total walk, F(1,15) = 0.30, p > 0.05, and for immobility, F(1,15) = 0.94, p > 0.05, in the open field test (Figure 1c). These results show that continuous treatment with Vardenafil did not influence anxiety-like behaviors or overall motor activity in aging females. This result implies that anxiety and motor activity are not confounding animals’ performance in the ORT.
3.2. Vardenafil Improves Object Recognition in Aged Female Mice
The test of discrimination between the novel and familiar object was calculated as the difference in exploration time between the two objects over the total exploration time. If the mouse explores both objects equally, the discrimination index should be equal to 0. The analysis shows that vehicle-exposed mice show a DI significantly different from 0 after 1 h (T = 26.00, z = 2.02, p < 0.05) and after 24 h (T = 27.00, z = 2.19, p < 0.05) from acquisition. Females exposed to Vardenafil showed a DI significantly different from 0 at 1 h, T = 45.00, z = 2.66, p < 0.01, and again after 24 h, T = 45.00, z = 2.66, p < 0.01, from acquisition. After 1 h (Mann–Whitney U = 59.00; n1 = 7; n2 = 9, p < 0.01) and 24 h (Mann-Whitney U = 57.00; n1 = 7; n2 = 9, p < 0.01), Vardenafil-treated females spent more time in proximity to the novel object. These results show that Vardenafil-exposed females preferred the novel object compared to females from the vehicle group, independent of time from exposure (Figure 2d).
4. Discussion
In this study, we report the effects of Vardenafil, a PDE5 inhibitor, on a cognition task that measures episodic memory in aged female mice. We report that a 32-day exposure to a dose of 2 mg/kg of Vardenafil improved object recognition memory in very old female CD1 mice. The effects of Vardenafil on performance in the ORT were not influenced by its possible action on anxiety, locomotor activity, or exploratory behaviors. In a previous study, we reported that the same continuous exposure to Vardenafil used in the present study induced higher levels of locomotor activity in subordinate male mice [38] while not having effects on anxiety-like behaviors. Since the measurement of performance in the ORT could be subject to individual differences in exploratory behaviors and aging-related reductions in ovarian steroids affect mood disorders, it was of paramount importance to control locomotor activity and emotional behaviors in these very old female mice. Our results support a positive enhancing effect of prolonged exposure to Vardenafil on episodic memory in a late-life stage without affecting activity levels. To the best of our knowledge, this is the first study investigating the effects of a long-term dosage of Vardenafil specifically on very old female mice.
Good performance in object recognition has been related to the functioning of the NO system. Specifically, for the enhancement of memory to occur, either increased NO production or reduced cGMP hydrolysis is required [40]. Aging is associated with reduced levels of cGMP that have an impact on processes related to learning and memory, such as LTP [9,41] and oxidative stress [42]. Old rats have reduced levels of cGMP in several brain regions that are key for successful performance in the ORT, such as the hippocampus [43]. The potentially positive role of PED5 inhibitors on cognitive declines associated with aging relies on their ability to increase cyclic nucleotides, such as cAMP and cGMP that, in turn, have memory-enhancing effects [18]. The anti-aging effects that PED5 inhibitors have on memory and cognition have been observed under chronic treatment in normal-aged mice [23], in mouse models of Alzheimer [24,35], and in models of accelerated senescence [44]. Specifically, several studies have shown that the enhancing effects of Vardenafil on cognition are obtained under prolonged exposure in rodent models of aging [23,25].
In female mice, the expression of the neural nitric oxide synthase (nNOS) in the hippocampus is under the influence of ovarian steroids [31], and ovarian steroids influence several structural and functional outcomes, such as cell proliferation, morphology, and LTP in this brain area [45,46] (reviewed in Yagi and Galea [47]). Given the role of the ovarian cycle on memory performance in the ORT [28,48], it is possible that the poor performance in object memory in very old females is a consequence of a reduced functioning of the NO-cGMP pathway, perhaps a consequence of a prolonged lack of estrogen or diminished responsiveness to steroid hormones with aging [28].
These hypothesized mechanisms could explain the observation that object recognition in 20-month-old female mice is impaired in female Swiss mice [5]. Other studies that investigated episodic memory in intact-aged C57BL/6 females using the ORT reported an overall decrease in object recognition with age [49]. However, studies investigating the positive effects that PED5 inhibitors have on memory in animal models of aging have focused either exclusively on males [35,50] or used aggregated scores of males and females in the statistical analysis [23]. Since aging-related cognitive deficits are sex-specific, i.e., the cognitive outcomes of aging males and females may be affected differently [51], our study improves this line of research by focusing on very old, outbred female mice.
In agreement with studies that showed improved memory performance in the ORT after exposure to PDE5 inhibitors, the improved memory in very old females treated with Vardenafil could be a consequence of increased levels of cGMP. However, the precise mechanism through which the treatment used in the present study works cannot be inferred from our experiment. The lack of male or younger females in our study design prevents us from shedding light on sex differences in the aging process underlying performance in the ORT and on the possible age-reversal effect of Vardenafil on performance in episodic memory. Moreover, the lack of information on the current ovarian status of the very old female tested in the current study does not allow us to infer the exact role of ovarian steroids on the NO-cGMP pathway. In this regard, several questions remain open and will need further investigation. For example, while there is evidence that acute injections of Vardenafil act specifically in the consolidation phase of memory, how long-term treatment works to improve molecular aspects of synaptic function is not known. Moreover, how the NO-cGMP pathway works in the two sexes to produce behavioral responses in specific cognitive domains and how this pathway is affected by aging in a sex-specific manner or by age-dependent sensitivity to estrogens is a line of research that needs further investigation, especially in light of the reported gender-dependent vulnerabilities in neurodegenerative disorders.
Conceptualization, P.P., H.D. and S.P. (Stefano Parmigiani); methodology, H.D.; validation, P.P., H.D. and S.P. (Stefano Parmigiani); formal analysis, D.P.; investigation, H.D.; resources, P.P.; data curation, S.P. (Silvia Paterlini); writing—original draft preparation, D.P.; writing—review and editing, D.P., P.P. and S.P. (Silvia Paterlini); visualization, D.P.; supervision, P.P.; project administration, P.P.; funding acquisition, P.P. All authors have read and agreed to the published version of the manuscript.
The animal study protocol was approved by the University of Parma Committee of Animal Experimentation prot. N. 130/05. Housing and experiments were conducted in accordance with the animal experimentation directive of the European Communities Council Directive of 22 September 2010 (2010/63/EU).
Not applicable.
The data presented in this study are available in the
The authors declare no conflict of interest.
Footnotes
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Enlarge this image.Figure 1. No effects of Vardenafil on behavioral responses in the elevated plus maze showing the total time spent in (a) and the total number of visits to the different areas of the maze (b); (c) total time spent walking, rearing and not moving in the open field test. Vehicle n = 8; Vardenafil n = 9.
Enlarge this image.Figure 2. Object recognition test. (a) Exploration time between vehicle and Vardenafil in the first session (T1; familiarization session). (b) Exploration time between vehicle and Vardenafil in the second session, T2. (c) Exploration time between vehicle and Vardenafil in the second session, T3. (d) Median and interquartile range (IQR) for the Discrimination Index (DI) at 1 h and 24 h from exposure to the familiar object. * p < 0.05 from a hypothetical median of 0 at 1 h (T2) and 24 h (T3) delay, respectively. + p < 0.10. Vehicle n = 7; Vardenafil n = 9.
Object recognition test.
| Vehicle a | Vardenafil b | |
|---|---|---|
| e 1 | 22.57 (4.44) | 21.11 (2.56) |
| e 2 | 20.14 (3.68) | 24.43 (3.15) |
| e 3 | 30.25 (7.10) | 40.86 (2.74) |
| h 1 | 2.43 (3.76) | −3.32 (3.35) |
| h 24 | −7.68 (5.08) | −19.75 (4.28) |
a n = 7; b n = 9.
Supplementary Materials
The following supporting information can be downloaded at:
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; Ponzi, Davide 1
; Paterlini, Silvia 1
; Parmigiani, Stefano 2 ; Palanza, Paola 1
1 Department of Medicine and Surgery, University of Parma, 43121 Parma, Italy
2 Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, 43121 Parma, Italy;