Skip to main content
NCBI home page
PLOS One logoLink to PLOS One
. 2021 Apr 15;16(4):e0249487. doi: 10.1371/journal.pone.0249487

D-(+)-Galactose-induced aging: A novel experimental model of erectile dysfunction

Mathania Silva de Almeida Rezende 1, Arthur José Pontes Oliveira de Almeida 2, Tays Amanda Felisberto Gonçalves 1, Fátima de Lourdes Assunção Araújo de Azevedo 1, Sabine Helena Dantas 3, Sonaly de Lima Silva 2, Evyllen Myllena Cardoso Soares 4, Hayaly Felinto Alves 4, Thais Trajano Lima 4, Javanyr Frederico de Souza Júnior 4, Ricardo Romão Guerra 5, Islania Giselia Albuquerque Araújo 3, Isac Almeida de Medeiros 1,*
Editor: Michael Bader6
PMCID: PMC8049229  PMID: 33857158

Abstract

Erectile dysfunction (ED) is defined as the inability to achieve and/or maintain penile erection sufficient for satisfactory sexual relations, and aging is one of the main risk factors involved. The D-(+)-Galactose aging model is a consolidated methodology for studies of cardiovascular aging; however, its potential for use with ED remain unexplored. The present study proposed to characterize a new experimental model for ED, using the D-(+)-Galactose aging model. For the experiments, the animals were randomly divided into three groups receiving: vehicle (CTL), D-galactose 150 mg/kg (DGAL), and D-(+)-galactose 150 mg/Kg + sildenafil 1.5 mg/Kg (DGAL+SD1.5) being administered daily for a period of eight weeks. All of the experimental protocols were previously approved by the Ethics Committee on the Use of Animals at the Federal University of Paraíba n° 9706070319. During the treatment, we analyzed physical, molecular, and physiological aspects related to the aging process and implicated in the development of ED. Our findings demonstrate for the first time that D-(+)-Galactose-induced aging represents a suitable experimental model for ED assessment. This was evidenced by an observed hyper-contractility in corpora cavernosa, significant endothelial dysfunction, increased ROS levels, an increase in cavernous tissue senescence, and the loss of essential penile erectile components.

Introduction

Erectile dysfunction (ED) is defined as the inability to achieve and/or maintain sufficient erection for satisfactory sexual relations [1]. Its prevalence tends to increase throughout the individual’s life, affecting mainly men over 40 years old [2]. With the global increase in life expectancy, it is estimated that by the year 2025, the worldwide prevalence of ED will reach 322 million men [3].

Aging is a multifactorial process characterized by molecular, cellular, and physiological changes which increase the individual’s susceptibility to the development of disease; it is also considered the main risk factor for ED [3, 4]. Of the changes observed in aging and implicated in the pathophysiology of ED we find: 1) Endothelial dysfunction, 2) Increased contractility and decreased vasodilation of the corpus cavernosum, 3) Oxidative stress, and 4) Increased vascular senescence [5, 6]. Together, these processes lead to tissue remodeling and the development of ED [6, 7].

New pharmacological tools that aim to combat and/or prevent diseases such as ED require the use of experimental animal models [8]. The accelerated D-(+)-galactose-induced aging model is widely accepted; it is based on metabolic theory, and presents many aspects similar to the natural aging process [9]. In the body, D-(+)-galactose is oxidized by galactose oxidase to form hydrogen peroxide (H2O2). In addition, this monosaccharide can react with amine groups in various proteins, forming advanced glycation products, and promoting oxidative stress [10]. Recent studies have demonstrated that chronic administration of D-(+)-galactose also leads to immune system dysregulation, sex hormone deficiencies, increasing inflammatory cytokine levels, cellular apoptosis, and diminished total antioxidant capacity [10, 11]. Taken together, these effects mimic aging and impel the subject toward development of age-related diseases [12].

However, despite D-(+)-galactose-induced accelerated aging being used as a consolidated methodology for studies of cardiovascular aging, its potential for use with ED is still unexplored. [13–15]. Thus, the present study aims to characterize a new model of ED in rats using D-(+)-galactose induced accelerated aging.

Materials and methods

Standards and reagents

In the present study, the following substances were used: D-(+)-galactose, Phenylephrine (Phe), Acetylcholine (ACh), sodium nitroprusside (SNP), dihydroethidium (DHE), 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (x-gal), dimethyl sulfoxide (DMSO), OCT (Optimal Cutting Temperature) Compound (Tissue Plus®), and glutaraldehyde. All were obtained from Sigma-Aldrich (Brazil). Ketamine and xylazine were purchased from Syntec (Brazil). Sildenafil was obtained from Roval Pharmacy (Brazil); heparin (Hepamax-s®) from Blau Farmacêutica S.A. (Brazil); formaldehyde 10% from Medi Química Indústria Farmacêutica Ltda (Brazil); and hematoxylin-eosin from Química Especializada Erich Ltda (Brazil). The carbogen mixture (95% O2 and 5% CO2) was acquired from White Martins (Brazil).

Animals

Forty male Wistar rats (Rattus novergicus), eight weeks old, from the Animal Production Unit of the Institute for Research in Drugs and Medicines (IPeFarM) of the Federal University of Paraíba (UFPB) were used. The animals were kept under appropriate environmental conditions, temperature (22 ± 1°C), a 12-hour light-dark cycle (6–18 hours), with free access to water and food (Nuvilab CR-1, Quimtia®), while recording the physical and mental health of the animals on a daily basis. After confirmation of anesthesia induced by the intraperitoneally administration of xylazine and ketamine (10 and 75 mg/Kg, respectively), the animals were euthanized by exsanguination.

All experimental protocols were carried out according to the guidelines established by the Brazilian National Council for Animal Experiment Control (Conselho Nacional de Controle de Experimentação Animal—CONCEA), obeying law No. 11.794/2008, submitted and previously approved by the Ethics Committee on the Use of Animals (Comissão de Ética no Uso de Animais—CEUA) of the UFPB, n° 9706070319.

Experimental design

The animals were randomly assigned into two three experimental groups: the control group (CTL), which received physiological saline solution (NaCl 0.9%) intraperitoneally (IP), the D-galactose group (DGAL), which received D-(+)-galactose at 150 mg/Kg via IP, and the sildenafil group (DGAL+SD1.5) which received both D-(+)-galactose at 150 mg/Kg via IP and sildenafil 1.5 mg/Kg by oral gavage. All of the animals were subjected to eight weeks of treatment with daily administration. The IP administrations were standardized at a volume less than or equal to 2 mL/Kg [16].

The administered dose of D-(+)-galactose (150 mg/Kg) was chosen based on a review of the literature, citing doses sufficient to induce aging in the animals [17, 18]. Sildenafil was administered at 1.5 mg/Kg, corresponding (approximately) to a dose of 100 mg administered to an adult man with 70 Kg of body weight [19].

Monitoring body weight and blood glucose

Variations in animal body weight were assessed throughout the treatment. The animals were weighed individually three times a week, always before administration of their respective treatments. The values were expressed as average weekly weight in grams (g). Glycemic analysis was performed at the end of treatment on the day of euthanasia. For this, one drop of blood was collected from the end of the caudal vein and introduced to a strip attached to an Accu-chek Guide glucometer (Roche®, Brazil). Glycemic values were expressed in mg/dL.

Erectile function measurements—ICP/MAP ratio

Erectile function was assessed using the ICP/MAP (intra-cavernous pressure/mean arterial) pressure ratio methodology adapted according to that previously described by Kim and colleagues [20]. Briefly, at eight weeks of treatment, the animals were anesthetized with a mixture of xylazine and ketamine (10 and 75 mg/Kg, respectively, via IP). A polyethylene (PE) catheter, filled with heparinized saline (200 IU/mL) was then implanted into the right common carotid artery to the measure the mean arterial pressure (MAP).

To record intra-cavernous pressure (ICP), a 30G gauge needle, connected to a PE tube (10 mm) filled with heparinized saline (200 IU/mL), was inserted in the crural region of the left corpus cavernosum. Subsequently, the cavernous nerve was identified and a bipolar bronze stimulator (Animal Nerve Stimulating Electrode, MLA0320, ADinstruments, United States of America) was placed and electrically stimulated with 1 millisecond (ms) pulses, at 6 volts (V), and 16 Hz lasting 60 seconds (s). Two cycles of electrical stimulation were performed, the interval between each stimulation was at least 5 minutes. MAP and ICP variations were measured using pressure transducers (Disposable BP Transducer, MLT0699, ADinstruments) coupled to the PowerLab® data acquisition system (LabChart® software, version 8.1; ADInstruments, USA).

Vascular reactivity

After euthanasia, the animal’s penises were carefully isolated and immediately placed in a Krebs-Ringer nutrient solution (NaCl 118.0; KCl 4.7; CaCl2 2.50; KH2PO4 1.20; MgSO4 1.17; NaHCO3 25.00; and glucose 5.60 (mM)) for dissection and removal of the corpus cavernosum [21]. Corpora cavernosa were suspended vertically in isolated organ baths (Panlab Multi Chamber Organ Baths, ADIntruments, Australia) by two stainless steel metallic rods and immediately submerged in 10 mL of 37°C Krebs-Ringer solution, with a carbogenic mixture (95% O2, and 5% CO2), maintained at pH 7.4, and under a stabilizing tension of 0.5 g, for 60 minutes. Voltage changes were measured using isometric transducers (MLT020, ADInstruments, Australia) and recorded in a PowerLab® data acquisition system (ML870/P, LabChart version 7.0, ADInstruments, Australia).

The contractility of the corpus cavernosum was assessed against an increasing and cumulative addition of Phe (10 nM– 300 μM), via electrical field stimulation (EFS) using different frequencies (1, 2, 4, 8, and 16 Hz) with 50 V electrical pulses of 1 ms duration. The treated groups’ corpus cavernosum relaxing responses were evaluated by increasing and cumulative addition of ACh (1 nM—10 μM), and SNP (100 pM—100 μM).

ROS measurements

Redox-sensitive fluorescent dye (DHE) was used to evaluate ROS (reactive oxygen species) formation. The corpus cavernosum was isolated and embedded in OCT compound, and then immediately frozen using liquid nitrogen for 5 minutes, before transferred and stored in a freezer at -80°C until the next step experimentation. Microtomy of the tissue in cryostat was performed at -20°C, in which cuts with 8 μM thickness were obtained. The tissue was fixed on slides, washed with phosphate-saline buffer (PBS) (161.0 mM NaCl; 1.8 mM NaH2PO4.H2O, and 15.8 mM Na2HPO4), and incubated with DHE (5 μM) for 30 minutes, at 37°C, in a humid chamber protected from light [22]. Subsequently, the sections were washed (twice) before being mounted in Fluorescence Mounting Medium (DAKO©) with coverslips. Images were obtained with a Fluorescence Eclipse Ti-U Nikon® microscope (Japan). Quantification (of levels of staining) was performed using NIS-element® software. The data were normalized using the CTL group, and expressed as percentage fluorescence.

Morphometric analysis

To perform histological sections, tissue sections of the mid-transversal part of the penis were fixed in buffered formaldehyde (10%) and incorporated into paraffin blocks with 5 μm thickness. Hematoxylin-eosin staining was used for morphometric measurement. The images were obtained using an Olympus BX-60 microscope and an Olympus camera coupled with the Olympus CellSens Dimension digital image capture program (USA). The morphometric areas were acquired using the “polygon area” function of the Olympus CellSens Dimension Program according to the given methodology, as modified by Correa et al. [23].

Histochemical analysis of SA-β-galactosidase

Analysis of Senescence Associated β-galactosidase (SA-β-galactosidase) was adapted as previously described by Chang and colleagues [14]. The animal penile segments were embedded in OCT compound and immediately frozen in liquid nitrogen (3 min). After freezing, microtomes (5 μm) of the tissue in cryostat were performed at -20°C. Subsequently, the tissue was washed with PBS and then fixed with a solution of formaldehyde (2%) and glutaraldehyde (0.2%), for a period of 5 minutes. In sequence, the tissues were washed with PBS and incubated with the x-gal staining solution; (150 mM NaCl, 2 mM NaCl2, 5 mM C6N6FeK4, 5 mM C6N6FeK4, 5 mM C6N6FeK3), 1 mg/mL of x-gal buffer, and citrate-phosphate buffer (pH 6.0 40 mM), for a maximum period of 18 h, at 37°C, in a humid chamber protected from light [24]. Subsequently, the sections were washed with PBS solution to remove the excess x-gal staining solution and taken immediately to analysis under a microscope (Nikon Eclipse Ti-E, Nikon, Japan).

Statistical analysis

The data were expressed as mean ± standard error of the mean (SEM). For statistical analysis of the concentration-response curves, the maximum effect (Emax) values were used as calculated from non-linear regression of the responses obtained. The student’s t-test and two-way analysis of variance (ANOVA), with the Bonferroni post-test were used. The data were considered significant when p < 0.05. All analyses performed were calculated using the Graph Pad Prism® version 7.0 statistical program.

Results

Evaluation of physical characteristics, body weights, and blood glucose levels

The animals studied presented differences in their appearance at the end of each treatment (Fig 1). The rats in the CTL group had smooth, healthy-looking, and shiny hair with uniform colors, however, the animals in the DGAL group presented curly, coarse, and opaque hair, with darker regions, and severe hair loss (Fig 1A and 1B). The animals both in the CTL and DGAL groups presented similar graduated increases in their body weights without statistical differences (n = 5; p > 0.05) (Fig 1C). At the end of the eight-week treatment, glycemic levels in both the CTL and DGAL animal groups (121.2 ± 4.09 mg/dL and 118.8 ± 5.73 mg/dL, respectively), were similar and without statistical differences (n = 5; p > 0.05).

Fig 1. Physical appearance at eight-weeks of treatment for (A) CTL, and (B) DGAL animals, (C) Average body weight (g) in function of time (8 weeks total) CTL (●) and DGAL (■).

Fig 1

Open in a new tab

The data are expressed as mean ± SEM (n = 5). The data were analyzed using the two-way ANOVA statistical test, followed by the Bonferroni post-test. Groups: CTL = vehicle; DGAL = D-(+)-galactose 150 mg/Kg.

D-(+)-galactose accelerated aging model induced ED in rats

The erectile function of both groups was assessed at eight weeks of treatment. The animals in the DGAL group exhibited a significant decrease (0.470 ± 0.007; n = 5; p < 0.05) in ICP/MAP when compared to the CTL group (0.733 ± 0.040; n = 5). The animals in the DGAL+SD1.5 group showed a significant increase (0.855 ± 0.01; n = 5) in ICP/MAP when compared to the DGAL group (0.470 ± 0.007; n = 5; p < 0.05) (Fig 2).

Fig 2. Original record (A) and statistical graph (B) of the ICP/MAP in response to electrical stimulation (16 Hz, 6V, 1 ms for 60 s) of the cavernous nerve in CTL and DGAL animals at eight weeks.

Fig 2

Open in a new tab

Groups: CTL (vehicle); DGAL (D-(+)-galactose 150 mg/Kg); and DGAL+SD1.5 (D-(+)-galactose 150 mg/Kg + Sildenafil 1.5 mg/Kg). The results are expressed as mean ± SEM (n = 5). The data were analyzed using the Student’s t-test. * p < 0.05 vs CTL. ES: Electrical stimulation.

D-(+)-galactose accelerated aging model induced hyper-contractility and endothelial dysfunction in isolated corpus cavernosum in rats

The increasing and cumulative addition of Phe (10 nM—300 μM) for the DGAL group promoted a significant increase in contractile response (Emax = 171.95 ± 19.24%; n = 5; p < 0.05) as compared to the CTL group (Emax = 100.00 ± 9.44%; n = 5), without statistical differences in potency, according to the pD2 values of the CTL groups (pD2 = 4.853 ± 0.08) and DGAL (pD2 = 4.93 ± 0.09) (Fig 3A and 3B).

Fig 3. Representative traces (A) and concentration-response curves (B) for Phe; representative traces (C) and frequency-response curves (D) for the electrical field stimulation EFS; representative traces (E) and concentration-response curves (F) for ACh; representative traces (G) and concentration-response curves (H) for SNP; in the corpus cavernosum isolated from rats at eight weeks of treatment, for both the CTL (●) and DGAL (■) groups.

Fig 3

Open in a new tab

Groups: CTL (vehicle); DGAL (D-(+)-galactose 150 mg/Kg). The results are expressed as mean ± SEM. The data were analyzed using the two-way ANOVA statistical test, followed by the Bonferroni post-test. * p < 0.05 vs CTL.

The EFS (1, 2, 4, 8, and 16 Hz) in the DGAL group promoted greater contractility at all frequencies tested (1 Hz: 44.34 ± 12.56; 2 Hz: 74.58 ± 16.49; 4 Hz: 104.56 ± 22.63; 8 Hz: 163.13 ± 22.23; 16 Hz: 228.36 ± 17.79%, n = 5), as compared to the CTL group (1 Hz: 13.60 ± 6.15; 2Hz: 21.20 ± 9.37; 4 Hz: 39.30 ± 15.85; 8 Hz: 65.50 ± 20.56; 16 Hz: 93.42 ± 15.68%, n = 5; p < 0.05) (Fig 3C and 3D).

The relaxation response induced by the increasing and cumulative addition of ACh (1 nM—10 μM) was significantly lower in the DGAL group (Emax = 51.75 ± 5.09%; n = 5; p < 0.05) when compared to the CTL group (Emax = 75.424 ± 1.74%; n = 5) (Fig 3E and 3F).

The relaxation response induced by the increasing and cumulative addition of SNP (100 pM– 100 μM) did not result in a significant difference in maximum effect (p > 0.05). However, there was a significant reduction in the potency (p < 0.05) for the DGAL group (Emax = 94.72 ± 11.04%; pD2 = 6.62 ± 0.19; n = 5) as compared to the CTL group (Emax = 113.24 ± 5.59%; pD2 = 7.72 ± 0.16; n = 4) (Fig 3G and 3H).

D-(+)-galactose accelerated aging model induced increased levels of superoxide anions in the corpus cavernosum isolated from rats

Superoxide anions measurements were performed in the corpus cavernosum isolated from Wistar rats. Redox-sensitive DHE fluorescent dye was used in both the CTL and DGAL groups. The animals in the DGAL group presented a significant increase in fluorescent intensity (233.58 ± 13.69%, n = 4) when compared to the CTL group (100.00 ± 13.16, n = 4; p < 0.05) (Fig 4).

Fig 4. Representative image (A) and quantitative analysis (B) of superoxide anion production as measured by fluorescent intensity emitted by the DHE probe.

Fig 4

Open in a new tab

Corpus cavernosum (8 μM) isolated from both CTL and DGAL rat groups, treated for eight weeks (20x objective). Groups: CTL (vehicle); DGAL (D-(+)-galactose 150 mg/Kg). Scale bars, 100 μm. Data are expressed as mean values of the percentage of fluorescence relative to the control ± SEM (n = 4). The data were analyzed using the Student’s t-test. * p < 0.05 vs CTL.

D-(+)-galactose accelerated aging model induced a decrease in the total corpus cavernosum area isolated from rats

The histo-morphometry analysis of animals in the DGAL group revealed a significant decrease in the corpus cavernosum by total area (4.35x106 ± 1.83x105 μm2, n = 3) when compared to the CTL group (4.99x106 ± 2.93x105 μm2, n = 3; p < 0.05) (Fig 5).

Fig 5. Photomicrographs (A) and histomorphometric analysis (B) of the total corpus cavernosum area (8μM) isolated from rats, at eight weeks of treatment.

Fig 5

Open in a new tab

Groups: CTL (vehicle); DGAL (D-(+)-galactose 150 mg/Kg). Scale bars, 200 μm. The data are expressed as mean values of total corpus cavernosum area ± SEM (n = 3). The data were analyzed using the Student’s t-test. * p < 0.05 vs CTL.

D-(+)-galactose accelerated aging model induced an increase in senescence-associated β-galactosidase activity in the corpus cavernosum isolated from rats

The SA-β-galactosidase activity of animals in the DGAL group revealed a significant increase (205.189 ± 6.572%, n = 4) when compared to the CTL group (100.00 ± 13.85, n = 4; p < 0.05) (Fig 6).

Fig 6. Representative image (A) and quantitative analysis (B) of SA-β-galactosidase activity (%) relative to the CTL group in the corpus cavernosum (5 μM) isolated from rats, at eight weeks of treatment (20x objective).

Fig 6

Open in a new tab

Groups: CTL (vehicle); DGAL (D-(+)-galactose 150 mg/Kg). Scale bars, 100 μm. The data are expressed in mean percentage values (activity) in relation to the control ± SEM (n = 4). The data were analyzed using the Student’s t-test. * p < 0.05 vs CTL.

Discussion

In the present study, a novel ED model associated with mimetic aging induced by D-(+)-galactose in Wistar rats was characterized. The daily administration of 150 mg/Kg D-(+)-galactose, via IP (eight weeks), reduced erectile function in vivo, promoting hyper-contractility and endothelial dysfunction in isolated corpus cavernosum, as well as promoting oxidative stress, reducing the proportion of erectile components, and increasing senescence markers in penile tissue.

Chronic administration of D-(+)-galactose for a period of six to ten weeks is well described as a model to accelerate the natural aging process [25, 26]. Physiologically, the monosaccharide, is converted to glucose by galactose-1-phosphate-uridyltransferase and galactokinase [27]. Yet if in excess, deleterious metabolic disturbances are generated, with several effects such as immune system cell dysfunction, sexual hormone deficiencies, increases in inflammatory cytokines, increases in cellular apoptosis, and decreases in both total antioxidant capacity and oxidative stress (via oxidative metabolism) [26, 28, 29]. Taken together, these effects, mainly mediated by persistent oxidative stress, favor the development of disease by affecting both structure and function in pertinent tissues and organs [26, 30].

Despite D-(+)-galactose being widely used for aging research, the potential for association with ED remains unexplored. To test the hypothesis that the aging model induced by D-(+)-galactose can trigger ED, we treated Wistar rats with a chronic daily administration of D-(+)-galactose (150 mg/Kg) for eight weeks. Initially, we observed the rat’s physical appearance, and at the end of the treatment period, the animals in the DGAL group presented physical characteristics such as severe hair loss, and curly or opaque hair with darker regions. This was in contrast to animals in the CTL group which presented smooth hair with a healthy look, and a bright and uniform color. Such aging characteristics were also observed in a study developed by Zhao and colleagues [29] in rats treated with D-(+)-galactose for eight weeks.

The animals’ body weights were also monitored during the eight weeks of treatment. During this period it was observed that the animals of the experimental groups all similarly presented a gradual increase in their body weights, demonstrating that administration of D-(+)-galactose did not interfere in the animals’ body weights. This was also observed in studies developed by Cardoso and colleagues [31]. There was also no significant change in glycemic levels among animals in the treated groups, demonstrating that D-(+)-galactose does not interfere in glucose metabolism.

After the treatment period, the most used method for in vivo evaluation of erectile function in rats, the ICP/MAP ratio was assessed [32]. Electrical stimulation of the cavernous nerve promotes nitrergic discharge inducing relaxation of the corpus cavernosum with consequent elevation of ICP [33]. The ICP/MAP ratio in the DGAL group was reduced significantly as compared to the CTL group, demonstrating, for the first time in the literature, that the D-(+)-galactose induced aging model was effective in promoting ED. Similar results which demonstrated ED were observed in a study demonstrating that elderly rats (physiological aging) presented an ICP/MAP ratio decrease [34]. Treatment with sildenafil, in animals in the DGAL+SD1.5 group, promoted a significant increase in the ICP/MAP ratio as compared to animals in the DGAL group, demonstrating that the treatment prevented ED. This result can be explained by the increase in cGMP via PDE-5 inhibition in the corpus cavernosum, as well as decreases in oxidative stress, and restoration of pro-oxidant/antioxidant equilibrium, which reduces endothelial damage and increases nitric oxide (NO) bioavailability [10, 35, 36]. These mechanisms favor relaxation of trabecular smooth muscle, and result in penile erection.

Given this in vivo observation of changes in erectile function, the next step would be to assess whether changes in the contractile and relaxing reactivity of corpus cavernosum isolated from the rats is involved in this process. These results are important, since erectile function is a hemodynamic process, and any imbalance is closely related to ED [37].

Therefore knowing that noradrenergic discharge and stimulation of α-adrenergic receptors favors increases in corpus cavernosum smooth muscle tone, and consequently impairs the state of erection [38], the response of the corpus cavernosum in contractile reactivity was evaluated using cumulative Phe and EFS curves. After the treatment period, in response to Phe and EFS, rats of the DGAL group presented increased hyper-contractility of the corpus cavernosum as compared to the CTL group. This effect may have been related to over-regulation of the contractile pathways in the corpus cavernosum; autonomic neuropathy (caused by exacerbation of sympathetic activity), and/or greater noradrenergic receptor sensitivity [39].

NO is another important factor and plays a key role in corpus cavernosum tonus regulation. Changes in NO synthesis or bioavailability can favor corpus cavernosum contraction, and consequently the development of ED [40]. We therefore evaluated whether NO release was affected by the treatments due to the action of ACh in the endothelial cells. ACh, an endothelial muscarinic agonist, was evaluated for its role in endothelium-dependent relaxation impairment. We observed that endothelium-dependent relaxation mediated by ACh was significantly impaired in the DGAL corpus cavernosum strips as compared to the CTL strips.

Age-related changes result in altered endothelial cell function, and cause reductions in cellular nitric oxide levels with subsequent impairment in penile smooth muscle relaxation. ACh, to induce its vasorelaxant effect releases NO to target muscarinic (M3) receptors in endothelial cells. In our experimental conditions, animals of the DGAL group presented a significantly impaired relaxation response to ACh, as compared to the CTL group. This effect reveals an endothelial dysfunction that may be associated with decreased NO bioavailability, yielding impaired corpus cavernosum relaxation [10, 36, 39]. Lafuente-Sanchis and colleagues [41] have demonstrated that reductions in endothelium-dependent vasodilation, in response to ACh in elderly animals, is likely related to endothelial dysfunction in the cavernous trabeculae.

In addition to assessing endothelium-dependent relaxation, we also investigated impairment in pathways directly involved in relaxation of corpus cavernosum smooth muscle tissue. For this, the SNP was used, whose induced relaxation did not present statistical differences between the groups in the maximum response, did promote a reduction in the potency of the relaxation response of the DGAL group, compared to the CTL group, suggesting that the functionality of the smooth muscle cells of the corpus cavernosum may thus be altered.

Recent studies suggest that endothelial dysfunction in age-induced ED is likely related to oxidative stress [42]. Similarly, the D-(+)-galactose accelerated aging model revealed an increase in ROS levels which lead to oxidative damage [26]. Further, increased oxidative stress has also been linked to lower NO concentrations. In age-related ED, ROS has been postulated as a principal cause of impaired cavernous function. We thus evaluated whether ROS would also increase in corpus cavernosum isolated from rats using histological sections from both the CTL and DGAL groups, measuring fluorescent intensity as emitted by a DHE probe. In these experiments, the animals in the DGAL group presented a significant difference in fluorescent intensity as compared to the CTL group. This suggested an increase in superoxide anions levels, contributing to cavernous tissue remodeling, a key event in the pathophysiology of ED. Corroborating our findings, Gur and his group [43] have demonstrated an increase in ROS levels in the smooth muscle and the endothelium of the corpus cavernosum in elderly rats as compared to young animals.

In addition to functional abnormalities, age-related ED is associated with structural changes resulting in the loss of essential penile erectile components [44]. Morphologically, a significant reduction in the muscle cell layer was observed in the DGAL group, as compared to the CTL group, suggesting a loss in erectile components essential for the penile erection. This reduction functionally alters the smooth muscle of the corpus cavernosum, and revealed a significant reduction in SNP potency in the DGAL group as compared to the CTL group. Yet it is likely that such morphological changes do not sufficiently modulate functionality so as to alter the SNP response maximum. These data are in agreement with several previous studies, which reveal that both in aged men and aged animals, a decline in erectile capability is associated with a diminishing number of smooth muscle tissue cells [45–48]. Similar data were also observed in an ED model induced by diabetes [49]. Reduction of erectile function with aging has been extensively reported and related to multiple functional, morphometric, molecular, and cellular changes that lead to significant loss of erectile capability.

Accumulation of senescent cells is a biological marker of aging, and is associated with increased lysosomal SA-β-galactosidase activity. We found that in cavernous tissues, the DGAL group presented an increase in SA-β-galactosidase activity when compared to the CTL group, suggesting an accumulation of senescent cells. Similar results have been demonstrated in the cardiac tissue of animals receiving the same treatment with D-(+)-galactose [14].

D-gal is a known normal substance in the body, however, at high levels, accumulating free D-gal is converted into secondary metabolites such as galactitol, hydrogen peroxide, and Schiff’s base, which in turn, induce inflammation, cellular apoptosis, and degenerative changes, this resulting in aging and age-related disorders. Further, this model was characterized by increased inflammatory cytokines, and up-regulated P16, P53, and P21 gene expression [13, 26, 50]. One of the main limitations of the present study is that the model poorly relates real physiological and biochemical changes. In addition, in the present study, inflammatory mediators, P53-P21, PI3K/Akt, and AMPK/ULK1 pathways were not measured. Nevertheless, due to its ability to mimic the senescent characteristics of natural aging, D-galactose-induced aging is potentially an ideal model for anti-aging therapeutic intervention studies.

In summary, our results demonstrate for the first time that the D-(+)-galactose aging model was able to promote ED in Wistar rats, through hyper-contractility and endothelial dysfunction in the rat corpus cavernosum. These effects may be related to oxidative stress, decreased erectile components, and accumulation of senescence cells in the corpora cavernosa of these animals.

Conclusion

The present study reports on a novel ED rat model, successfully induced by D-(+)-galactose (daily, during 8 weeks), and validated based on functional, cellular, molecular, and morphometric analysis. The D-(+)-galactose-induced aging model was able to mimic ED in Wistar rats. The present study found in isolated rat corpus cavernosum that ED is associated with hyper-contractility and endothelial dysfunction. The effects appear to be associated with the β-galactosidase activity through an increase in oxidative stress, loss of erectile components, and increased cell senescence.

Supporting information

S1 File

(XLSX)

Click here for additional data file. (24.6KB, xlsx)

Acknowledgments

The authors are grateful to Instituto UFPB de Desenvolvimento da Paraíba (IDEP) and Instituto de Pesquisa em Fármacos e Medicamentos (IPeFarM) for their technical help, respectively, for ROS measurements and Histochemical analysis of SA-β-galactosidase.

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

- Mathania Silva de Almeida Rezende received a scholarship from CAPES (process number 88887507825/202000) Isac Almeida de Medeiros received grant from CNPQ (process number 427783/2016-0).

References

  • 1.NIH. Impotence: NIH Consensus Development Panel on Impotence. Journal of the American Medical Association. 1993;270(1):83–90. [PubMed] [Google Scholar]
  • 2.Yafi FA, Jenkins L, Albersen M, Corona G, Isidori AM, Goldfarb S, et al. Erectile dysfunction. Nature reviews 2016;2:16003-. 10.1038/nrdp.2016.3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Kessler A, Sollie S, Challacombe B, Briggs K, Van Hemelrijck M. The global prevalence of erectile dysfunction: a review. BJU International. 2019. 10.1111/bju.14813 [DOI] [PubMed] [Google Scholar]
  • 4.Almeida AJPO, Ribeiro TP, Medeiros IA. Aging: Molecular Pathways and Implications on the Cardiovascular System. Oxidative Medicine and Cellular Longevity. 2017;2017:7941563-. 10.1155/2017/7941563 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Aversa A, Bruzziches R, Francomano D, Natali M, Gareri P, Spera G. Endothelial dysfunction and erectile dysfunction in the aging man. International Journal of Urology. 2010;17(1):38–47. 10.1111/j.1442-2042.2009.02426.x [DOI] [PubMed] [Google Scholar]
  • 6.Echeverri Tirado LC, Ferrer JE, Herrera AM. Aging and Erectile Dysfunction. Sexual Medicine Reviews. 2016;4(1):63–73. 10.1016/j.sxmr.2015.10.011 [DOI] [PubMed] [Google Scholar]
  • 7.Gökçe Mİ, Yaman Ö. Erectile dysfunction in the elderly male. Turkish journal of urology. 2017;43(3):247–51. 10.5152/tud.2017.70482 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Wu C, Kovac JR. Models for erectile dysfunction and their importance to novel drug discovery. Expert Opinion on Drug Discovery. 2016;11(2):185–96. 10.1517/17460441.2016.1126243 [DOI] [PubMed] [Google Scholar]
  • 9.Kong SZ, Li JC, Li SD, Liao MN, Li CP, Zheng PJ, et al. Anti-Aging Effect of Chitosan Oligosaccharide on d-Galactose-Induced Subacute Aging in Mice. Marine Drugs. 2018;16(6). 10.3390/md16060181 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Azman KF, Zakaria R. D-Galactose-induced accelerated aging model: an overview. Biogerontology. 2019;20(6):763–82. 10.1007/s10522-019-09837-y [DOI] [PubMed] [Google Scholar]
  • 11.Zheng S. Protective effect of Polygonatum sibiricum Polysaccharide on D-galactose-induced aging rats model. Scientific reports. 2020;10(1):2246-. 10.1038/s41598-020-59055-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Li X, Zhang Y, Yuan Y, Sun Y, Qin Y, Deng Z, et al. Protective Effects of Selenium, Vitamin E, and Purple Carrot Anthocyanins on D-Galactose-Induced Oxidative Damage in Blood, Liver, Heart and Kidney Rats. Biological Trace Element Research. 2016;173(2):433–42. 10.1007/s12011-016-0681-8 [DOI] [PubMed] [Google Scholar]
  • 13.Bo-Htay C, Palee S, Apaijai N, Chattipakorn SC, Chattipakorn N. Effects of d-galactose-induced ageing on the heart and its potential interventions. Journal of Cellular and Molecular Medicine. 2018;22(3):1392–410. 10.1111/jcmm.13472 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Chang YM, Chang HH, Lin HJ, Tsai CC, Tsai CT, Chang HN, et al. Inhibition of Cardiac Hypertrophy Effects in D-Galactose-Induced Senescent Hearts by Alpinate Oxyphyllae Fructus Treatment. Evidence-Based Complementary and Alternative Medicine. 2017;2017:2624384. 10.1155/2017/2624384 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Wu W, Hou CL, Mu XP, Sun C, Zhu YC, Wang MJ, et al. H2S Donor NaHS Changes the Production of Endogenous H2S and NO in D-Galactose-Induced Accelerated Ageing. Oxidative Medicine and Cellular Longevity. 2017;2017:5707830. 10.1155/2017/5707830 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Concea. RESOLUÇÃO NORMATIVA N° 33, DE 18 DE NOVEMBRO DE 2016. 2016:5.
  • 17.Chen WK, Tsai YL, Shibu MA, Shen CY, Chang-Lee SN, Chen RJ, et al. Exercise training augments Sirt1-signaling and attenuates cardiac inflammation in D-galactose induced-aging rats. Aging. 2018;10(12):4166–74. 10.18632/aging.101714 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Dehghani A, Hafizibarjin Z, Najjari R, Kaseb F, Safari F. Resveratrol and 1,25-dihydroxyvitamin D co-administration protects the heart against D-galactose-induced aging in rats: evaluation of serum and cardiac levels of klotho. Aging clinical and experimental research. 2019;31(9):1195–205. 10.1007/s40520-018-1075-x [DOI] [PubMed] [Google Scholar]
  • 19.Sheweita S, Salama B, Hassan M. Erectile dysfunction drugs and oxidative stress in the liver of male rats. Toxicology reports. 2015;2:933–8. 10.1016/j.toxrep.2015.06.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Kim SW, Lee J, Park J, Chai JS, Oh S, Paick JS, et al. Combination of LIM-kinase 2 and Jun Amino-terminal Kinase Inhibitors Improves Erectile Function in a Rat Model of Cavernous Nerve Injury. Urology. 2019;131:136–43. 10.1016/j.urology.2019.06.005 [DOI] [PubMed] [Google Scholar]
  • 21.Claudino MA, Franco-Penteado CF, Priviero FB, Camargo EA, Teixeira SA, Muscara MN, et al. Upregulation of gp91phox subunit of NAD(P)H oxidase contributes to erectile dysfunction caused by long-term nitric oxide inhibition in rats: reversion by regular physical training. Urology. 2010;75(4):961–7. 10.1016/j.urology.2009.05.098 [DOI] [PubMed] [Google Scholar]
  • 22.Foldvari M, Oguejiofor C, Afridi S, Kudel T, Wilson T. Liposome encapsulated prostaglandin E1 in erectile dysfunction: correlation between in vitro delivery through foreskin and efficacy in patients. Urology. 1998;52(5):838–43. 10.1016/s0090-4295(98)00299-4 [DOI] [PubMed] [Google Scholar]
  • 23.Corrêa LNS, Contreiras E, Daleprane J, Boaventura G, Abidu-Figueiredo M, Chagas M. Effects of Transgenic Soybean and Organic Soybean on Rat Penis Morphology during the Aging Process. International Journal of Morphology. 2017;35:1043–9. [Google Scholar]
  • 24.Silva GC, Abbas M, Khemais-Benkhiat S, Burban M, Ribeiro TP, Toti F, et al. Replicative senescence promotes prothrombotic responses in endothelial cells: Role of NADPH oxidase- and cyclooxygenase-derived oxidative stress. Experimental Gerontology. 2017;93:7–15. 10.1016/j.exger.2017.04.006 [DOI] [PubMed] [Google Scholar]
  • 25.Sadigh-Eteghad S, Majdi A, McCann SK, Mahmoudi J, Vafaee MS, Macleod MR. D-galactose-induced brain ageing model: A systematic review and meta-analysis on cognitive outcomes and oxidative stress indices. PloS one. 2017;12(8):e0184122. 10.1371/journal.pone.0184122 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Jing L, Jiang J-R, Liu D-M, Sheng J-W, Zhang W-F, Li Z-J, et al. Structural Characterization and Antioxidant Activity of Polysaccharides from Athyrium multidentatum (Doll.) Ching in d-Galactose-Induced Aging Mice via PI3K/AKT Pathway. Molecules (Basel, Switzerland). 2019;24(18):3364. 10.3390/molecules24183364 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Ma Y, Ma B, Shang Y, Yin Q, Wang D, Xu S, et al. Flavonoid-Rich Ethanol Extract from the Leaves of Diospyros kaki Attenuates D-Galactose-Induced Oxidative Stress and Neuroinflammation-Mediated Brain Aging in Mice. Oxidative Medicine and Cellular Longevity. 2018;2018:8938207-. 10.1155/2018/8938207 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Ni Y, Wu T, Yang L, Xu Y, Ota T, Fu Z. Protective effects of astaxanthin on a combination of D-galactose and jet lag-induced aging model in mice. Endocrine Journal. 2018;65(5):569–78. 10.1507/endocrj.EJ17-0500 [DOI] [PubMed] [Google Scholar]
  • 29.Zhao H, Li J, Zhao J, Chen Y, Ren C, Chen Y. Antioxidant effects of compound walnut oil capsule in mice aging model induced by D-galactose. Food & Nutrition Research. 2018;62. 10.29219/fnr.v62.1371 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Omidi M, Ahangarpour A, Ali Mard S, Khorsandi L. The effects of myricitrin and vitamin E against reproductive changes induced by D-galactose as an aging model in female mice: An experimental study. International Journal of Reproductive BioMedicine (Yazd). 2019;17(11):789–98. 10.18502/ijrm.v17i10.5486 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Cardoso A, Magano S, Marrana F, Andrade JP. D-Galactose High-Dose Administration Failed to Induce Accelerated Aging Changes in Neurogenesis, Anxiety, and Spatial Memory on Young Male Wistar Rats. Rejuvenation Research. 2015;18(6):497–507. 10.1089/rej.2015.1684 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.McMurray G, Casey JH, Naylor AM. Animal models in urological disease and sexual dysfunction. British Journal of Pharmacology. 2006;147 Suppl 2:S62–79. 10.1038/sj.bjp.0706630 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Li WJ, Park K, Paick JS, Kim SW. Chronic treatment with an oral rho-kinase inhibitor restores erectile function by suppressing corporal apoptosis in diabetic rats. The Journal of Sexual Medicine. 2011;8(2):400–10. 10.1111/j.1743-6109.2010.01724.x [DOI] [PubMed] [Google Scholar]
  • 34.Helmy MM, Senbel AM. Evaluation of vitamin E in the treatment of erectile dysfunction in aged rats. Life Sciences. 2012;90(13–14):489–94. 10.1016/j.lfs.2011.12.019 [DOI] [PubMed] [Google Scholar]
  • 35.Akhvlediani ND, Matyukhov IP. [Current role of sildenafil in the management of erectile dysfunction]. Urologiia (Moscow, Russia: 1999). 2018(2):142–6. [PubMed] [Google Scholar]
  • 36.Leal MAS, Aires R, Pandolfi T, Marques VB, Campagnaro BP, Pereira TMC, et al. Sildenafil reduces aortic endothelial dysfunction and structural damage in spontaneously hypertensive rats: Role of NO, NADPH and COX-1 pathways. Vascular pharmacology. 2020;124:106601. 10.1016/j.vph.2019.106601 [DOI] [PubMed] [Google Scholar]
  • 37.Díaz-Díaz E, León M, Arzuaga N, Timossi C, Díaz R, Salinas C, et al. Erectile Dysfunction: A Chronic Complication of the Diabetes Mellitus. In: Nunes K, editor. Erectile Dysfunction—Disease-Associated Mechanisms and Novel Insights into Therapy2012. p. 69–96.
  • 38.Aydin K, Gokcen K, Yildirim S, Bagcivan I, Parlak M, Gokce G. In vitro evaluation of nebivolol effects on nonadrenergic noncholinergic responses in rabbit corpus cavernosum. Andrology. 2018;50(8):e13062. 10.1111/and.13062 [DOI] [PubMed] [Google Scholar]
  • 39.Andersson KE. Mechanisms of penile erection and basis for pharmacological treatment of erectile dysfunction. Pharmacological Reviews. 2011;63(4):811–59. 10.1124/pr.111.004515 [DOI] [PubMed] [Google Scholar]
  • 40.Silva FH, Claudino MA, Calmasini FB, Alexandre EC, Franco-Penteado C, Burnett AL, et al. Sympathetic Hyperactivity, Increased Tyrosine Hydroxylase and Exaggerated Corpus Cavernosum Relaxations Associated with Oxidative Stress Plays a Major Role in the Penis Dysfunction in Townes Sickle Cell Mouse. PloS one. 2016;11(12):e0166291. 10.1371/journal.pone.0166291 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Lafuente-Sanchis A, Triguero D, Garcia-Pascual A. Changes in nerve- and endothelium-mediated contractile tone of the corpus cavernosum in a mouse model of pre-mature ageing. Andrology. 2014;2(4):537–49. 10.1111/j.2047-2927.2014.00213.x [DOI] [PubMed] [Google Scholar]
  • 42.Angulo J, El Assar M, Sevilleja-Ortiz A, Fernandez A, Sanchez-Ferrer A, Romero-Otero J, et al. Short-term pharmacological activation of Nrf2 ameliorates vascular dysfunction in aged rats and in pathological human vasculature. A potential target for therapeutic intervention. Redox Biology. 2019;26:101271. 10.1016/j.redox.2019.101271 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Gur S, Sikka SC, Hellstrom WJG. Novel phosphodiesterase-5 (PDE5) inhibitors in the alleviation of erectile dysfunction due to diabetes and ageing-induced oxidative stress. Expert opinion on investigational drugs. 2008;17(6):855–64. 10.1517/13543784.17.6.855 [DOI] [PubMed] [Google Scholar]
  • 44.Cho MC, Song WH, Paick JS. Suppression of Cavernosal Fibrosis in a Rat Model. Sexual Medicine Reviews. 2018;6(4):572–82. 10.1016/j.sxmr.2018.02.007 [DOI] [PubMed] [Google Scholar]
  • 45.Bakircioglu ME, Sievert KD, Nunes L, Lau A, Lin CS, Lue TF. Decreased trabecular smooth muscle and caveolin-1 expression in the penile tissue of aged rats. The Journal of Urology. 2001;166(2):734–8. [PubMed] [Google Scholar]
  • 46.Calabrò A, Italiano G, Pescatori ES, Marin A, Gaetano O, Abatangelo G, et al. Physiological aging and penile erectile function: a study in the rat. European Urology. 1996;29(2):240–4. [PubMed] [Google Scholar]
  • 47.Wespes E, Goes PM, Schiffmann S, Depierreux M, Vanderhaeghen JJ, Schulman CC. Computerized analysis of smooth muscle fibers in potent and impotent patients. The Journal of Urology. 1991;146(4):1015–7. 10.1016/s0022-5347(17)37990-9 [DOI] [PubMed] [Google Scholar]
  • 48.Wespes E, Sattar AA, Golzarian J, Wery D, Daoud N, Schulman CC. Corporeal Veno-Occlusive Dysfunction: Predominantly Intracavernous Muscular Pathology. The Journal of Urology. 1997;157(5):1678–80. 10.1016/s0022-5347(01)64833-x [DOI] [PubMed] [Google Scholar]
  • 49.Tao M, Tasdemir C, Tasdemir S, Shahabi A, Liu G. Penile alterations at early stage of type 1 diabetes in rats. International Brazilian Journal of Urology. 2017;43:753–61. 10.1590/S1677-5538.IBJU.2016.0454 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Guo B, Guo Q, Wang Z, Shao JB, Liu K, Du ZD, et al. D-Galactose-induced oxidative stress and mitochondrial dysfunction in the cochlear basilar membrane: an in vitro aging model. Biogerontology. 2020;21(3):311–23. 10.1007/s10522-020-09859-x [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Michael Bader

23 Nov 2020

PONE-D-20-33703

D-[+]-Galactose-induced aging:  a novel experimental model of erectile dysfunction

PLOS ONE

Dear Dr. Medeiros,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jan 07 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Michael Bader

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please note that PLOS does not permit references to “data not shown.” Authors should provide the relevant data within the manuscript, the Supporting Information files, or in a public repository. If the data are not a core part of the research study being presented, we ask that authors remove any references to these data.

3. Please ensure you have discussed any potential limitations of your study in the Discussion.

4.At this time, we request that you  please report additional details in your Methods section regarding animal care, as per our editorial guidelines:

(1) Please state the number of rats used in the study

(2) Please include the method of euthanasia. Please also clarify whether animals were euthanised prior to isolation of the penis.

(7) Please describe the care received by the animals, including the frequency of monitoring and the criteria used to assess animal health and well-being.

Thank you for your attention to these requests.

5. Please ensure you have correctly described the number of experimental groups in line 92.

6. At this time, we ask that you please provide scale bars on the microscopy images presented in Figures 4 and 6, and refer to the scale bar in the corresponding Figure legend.

7.We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The Authors have conducted a new experimental model of DE, using the D-(+)-Galactose aging model. The Authors found that the D-(+)-Galactose-induced aging represents a new experimental model suitable for the assessment of ED, evidenced by the hypercontractility of the corpora cavernosa, plus a significant endothelial dysfunction.

The logic and presentation of the key idea is not bad. I have following concerns about this work at least in its present form.

1. There was a significant difference in body weight between the CTL group and the DGAL group.

2. There are some typing, writing and grammatical errors in this manuscript. This work does require an extensive edit by a native-English speaker.

Reviewer #2: I thank the researchers for their valuable work and meticulously prepared manuscript. The research is aimed at creating a new animal model of erectile dysfunction. I believe that the comments I have stated in the attachment will increase the power of the publication.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Guldem Mercanoglu

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment

Submitted filename: comments.docx

Click here for additional data file. (15.1KB, docx)
PLoS One. 2021 Apr 15;16(4):e0249487. doi: 10.1371/journal.pone.0249487.r002

Author response to Decision Letter 0

6 Feb 2021

Response to the editor

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Answer: The PLOS ONE’s style requirements have been met.

2. Please note that PLOS does not permit references to “data not shown.” Authors should provide the relevant data within the manuscript, the Supporting Information files, or in a public repository. If the data are not a core part of the research study being presented, we ask that authors remove any references to these data.

Answer: References to "data not shown" have been removed. However, we have performed a new set of experiments to follow reviewer’s suggestions, where we have constructed concentration-response curves to SNP in DGAL treated rats. These new data were presented and discussed in the revised the version of manuscript.

3. Please ensure you have discussed any potential limitations of your study in the Discussion.

Answer: As we know, D-gal is a normal substance in the body, however, at high levels, accumulating free D-gal is converted into secondary metabolites such as galactitol, hydrogen peroxide, Schiff’s base, that, in turn, induce inflammation, cellular apoptosis, degenerative changes, resulting in aging and age-related disorders. Furthermore, this model is characterized by increased inflammatory cytokines, up-regulated P16, P53 and P21 genes expressions [13, 26, 50]. So that one of the main limitations of the present study is that this model can be hard to reflect real physiology and biochemistry changes. In addition, we have not measured inflammatory mediators, P53-P21, PI3K/Akt and AMPK/ ULK1 pathways in the present study. Regardless, D-galactose- induced aging carry the potential to be an ideal model for anti-aging therapeutic interventions studies due to its abilities to mimic senescence characteristics of natural aging. This paragraph has been added to the discussion section (see sentences 438-449).

4. At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines:

(1) Please state the number of rats used in the study

Answer: We added the information concerning the number of animals used in the study (see sentence 85).

“Forty male Wistar rats (Rattus novergicus), eight weeks old, from the Animal Production Unit of the Institute for Research in Drugs and Medicines (IPeFarM) of the Federal University of Paraíba (UFPB) were used.”

(2) Please include the method of euthanasia. Please also clarify whether animals were euthanised prior to isolation of the penis.

Answer: The euthanasia method was added to the manuscript (sentence 90-93), as well as if the animals were euthanized before the isolation of the corpus cavernosum (sentence 141).

Sentence 90-93: “The animals were euthanized by exsanguination, after confirmation of anesthesia induced by the intraperitoneally administration of xylazine and ketamine (10 and 75 mg/Kg, respectively).”

Sentence 141: “After euthanasia, the animal's penises was carefully isolated and immediately placed in a Krebs-Ringer nutrient solution with composition (mM): NaCl 118.0; KCl 4.7; CaCl2 2.50; KH2PO4 1.20; MgSO4 1.17; NaHCO3 25.00 and glucose 5.60, for dissection and removal of the corpus cavernosum (21).”

(3) Please describe the care received by the animals, including the frequency of monitoring and the criteria used to assess animal health and well-being.

Answer: Animal care was described, as well as the frequency of monitoring and the criteria used to assess the animal's health and well-being (see sentence 87-90 and 94-98).

Sentence 87-90: “The animals were kept under appropriate environmental conditions, temperature (22 ± 1 ° C), 12-hour light-dark cycle (6-18 hours), with free access to water and food (Nuvilab CR-1, Quimtia®), keeping track of the physical and mental health of the animals on a daily basis.”

Sentence 94-98: “All experimental protocols were carried out according to the guidelines established by the brazilian National Council for Animal Experiment Control (Conselho Nacional de Controle de Experimentação Animal - CONCEA), obeying the law 11.794/2008, submitted and previously approved by the Ethics Committee on the Use of Animals (Comissão de Ética no Uso de Animais - CEUA) of the UFPB, nº 9706070319.”

5. Please ensure you have correctly described the number of experimental groups in line 92.

Answer: We appreciate the suggestions. Typing errors were corrected in the manuscript (see sentence 100).

“The animals were randomly assigned into three experimental groups: control group (CTL), which received physiological saline solution (NaCl 0.9%) intraperitoneally (IP), D-galactose group (DGAL), which received D-(+)-galactose 150 mg/Kg via IP and sildenafil group (DGAL+SD1.5) which received D-(+)-galactose 150 mg/Kg via IP and sildenafil 1.5 mg/Kg by oral gavage.”

6. At this time, we ask that you please provide scale bars on the microscopy images presented in Figures 4 and 6, and refer to the scale bar in the corresponding Figure legend.

Answer: The scale bars of the images were added, as requested, and the reference to it in the caption of the corresponding figure (see figure 4 and 6) (see sentence 278, 289 and 302).

Sentence 278: Scale bars, 100 μm.

Sentence 289: Scale bars, 200 μm.

Sentence 302: Scale bars, 100 μm.

7. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

Answer: We have mentioned (see sentences 94-98) that all experimental protocols were carried out according to the guidelines established by the brazilian National Council for Animal Experiment Control (Conselho Nacional de Controle de Experimentação Animal - CONCEA), obeying the law 11.794/2008, submitted and previously approved by the Ethics Committee on the Use of Animals (Comissão de Ética no Uso de Animais - CEUA) of the Federal University of Paraiba-UFPB, nº 9706070319.

Importantly, none of the funding institutions restricts the availability of data. All data will be fully available. A file containing all results spreadsheets, was submitted along with manuscript.

Response to Reviewers

1. There are minor typing errors:

Line 28- DE replace to ED

Line 348-the activity has been replaced by The activity

Answer: We thank the reviewers for the thoughtful suggestions and for the good insights for improving our manuscript. Here we tried to answer in the best form possible to all the concerns. Certainly, all the criticisms helped us to improve our manuscript and make it clearer.

The typing errors were corrected in the manuscript.

Sentence 28: Thus, the present study proposed to characterize a new experimental model of ED, using the D-(+)-Galactose aging model.

Sentence 376: The activity of ACh, which is an endothelial muscarinic agonist, was evaluated to determine if there is any impairment of endothelium-dependent relaxation mediated by this molecule.

2. In the material-method section, it should be stated how the D-(+)-galactose and sildenafil doses are selected. Because, as it is known, the dose of the research molecule is the main parameter in studies investigating molecular mechanisms.

Answer: We appreciate the pertinent suggestions. The choice of doses of both, D-galactose and sildenafil, was duly justified in the manuscript (see sentence 108-111).

The administered dose of D-(+)-galactose (150 mg/Kg) was chosen based on the literature review, in sufficient doses to induce aging in the animal (17, 18). Sildenafil was administered in a dose of 1.5 mg/Kg, corresponding, approximately, to a dose of 100 mg administered to an adult man with 70 Kg of body weight, according to the literature (19).

3. In the result part, why were DGAL + SD1.5 results not presented in the relaxation study results (between the 232-235th sentences)?

Answer: This manuscript presents a new model of erectile dysfunction induced by administration of d-galactose however, we have not investigated the in vitro effects of sildenafil in this model. Sildenafil was used only as a pharmacological tool just to check (in vivo) the effectiveness of the model.

4. In the discussion section, the reason for the improvement in ICP / MAP in the DGAL + SD1.5 group was discussed as cGMP increase mediated by PDE-5 inhibition by sildenafil (324-329th sentences). However, recent studies have shown that sildenafil increases NO bioavailability by reducing oxidative stress and restoring pro-oxidant / antioxidant balance (Leal MAS et al. Sildenafil reduces aortic endothelial dysfunction and structural damage in spontaneously hypertensive rats: Role of NO, NADPH and COX-1 pathways. Vascul Pharmacol 124:106601; 2020). Considering the possible mechanism of D-galactose in aging "oxidation to form hydrogen peroxide by galactose oxidase, ROS increase resulting in decreased SOD level and impaired redox homeostasis", (Azman KF, Zakaria R. D-galactose-induced accelerated aging model: an overview. Biogerontology 20:763-782, 2019). I believe that discussing the effect of sildenafil mentioned above in this section will strengthen the results of the study.

Answer: We rewrite this topic to follow the reviewer suggestion. We added, in the discussion section, the mechanisms that may be involved in the response of sildenafil to improve the ICP/MAP parameter (see sentence 353-356).

“This result can be explained by the increase in cGMP by inhibition of PDE-5 in the corpus cavernosum, as well as by the decrease in oxidative stress, restoration of the pro-oxidant/antioxidant balance that reduces endothelial damage and increases the bioavailability of NO (10, 35, 36). These mechanisms favor the relaxation of the smooth muscles of the trabeculae resulting in penile erection.”

5. In the discussion section, it was not understood what was meant by SHR-CTL and WKY-CTL groups between sentences 344-353. In the material method part, no information about these groups is given.

Answer: Typing errors were corrected throughout the manuscript. For instance, we replaced WKY-CTL with CTL and SHR-CTL with DGAL (see sentence 380-381).

“Thus, in the present study, endothelium-dependent relaxation mediated by ACh was significantly impaired in the strips of the corpora cavernosa of the DGAL when compared to the CTL group.”

6. In the discussion section, between the 354-363th sentences Could the decrease in acetylcholine response be due to a decrease in NO bioavailability rather than a decrease in NO release in DGAL group? The results in the DGAL + SD1.5 group specified in Article 3 can be discussed considering. Because as it is known, in the presence of ROS, ONOO- produced by interacting with NO and ROS. This molecule is the main molecule responsible for nitrosative damage and reduced NO bioavailability. Again, as discussed in Article 4; it was shown that, Sildenafil reverses endothelial dysfunction in spontaneous hypertensive rats by improving vascular relaxation to acetylcholine with increased NO bioavailability via reducing oxidative stress (Azman KF, Zakaria R. D-galactose-induced accelerated aging model: an overview. Biogerontology 20:763-782, 2019).

Answer: We appreciate the suggestion and we have introduced some information on the topic, that included a decrease in the bioavailability of NO in response to the ACh of the DGAL group (see sentence 387-389).

“This effect revealed an endothelial dysfunction that may be associated with a decrease in the bioavailability of NO, with consequent impaired relaxation of the corpus cavernosum (10, 36, 39).”

7. In the discussion section, in the paragraph starting with the 385th sentence, morphological changes are mentioned, and a decrease in smooth muscle cells in the DGAL group is stated. Does this not contradict the nitroprusside non-response referred to in sentences 364-369? Researchers should discuss this and, if necessary, state that the morphological changes are not at a level to create functional changes.

Answer: Answer: Good point. Following the reviewer suggestion, we decided to explore this data in depth in the manuscript. Concentration-response curves to SNP in DGAL treated rats were added to the manuscript.

Fig.(see Figure in the file named Response to Reviewers) Concentration-response curves to SNP in the corpus cavernosum isolated from rats after eight weeks treatment, of both CTL (●) and DGAL (■) groups.

We found that the relaxing response induced by increasing and cumulative addition of SPN (100 pM – 100 µM) did not show significative difference in the maximum effect (p > 0.05), however, there was a significant reduction in the potency (p < 0.05) of the DGAL group (Emax = 94.72 ± 11.04 %; pD2 = 6.62 ± 0.19; n = 5) when compared to the CTL group (Emax = 113.24 ± 5.59 %; pD2 = 7.72 ± 0.16; n = 4) (Figs. 3G and 3H). Furthermore, considering this result, we have added a topic (see sentence 419-423) that discusses the relationship between the loss of erectile components and the relaxing response to SPN.

“This reduction was able to alter the functionality of the smooth muscle of the corpus cavernosum since there was a significant reduction in the potency of the SPN in the DGAL group when compared to the CTL. Nevertheless, most likely, the morphological changes are not at a level to create functional changes sufficient to alter the maximum response to SNP.”

Attachment

Submitted filename: Response to the editor final.docx

Click here for additional data file. (23.8KB, docx)

Decision Letter 1

Michael Bader

9 Feb 2021

PONE-D-20-33703R1

D-[+]-Galactose-induced aging:  a novel experimental model of erectile dysfunction

PLOS ONE

Dear Dr. Medeiros,

Thank you for submitting your manuscript to PLOS ONE. You did not answer to the comments of reviewer 1. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised by this reviewer:

The Authors have conducted a new experimental model of DE, using the D-(+)-Galactose aging model. The Authors found that the D-(+)-Galactose-induced aging represents a new experimental model suitable for the assessment of ED, evidenced by the hypercontractility of the corpora cavernosa, plus a significant endothelial dysfunction.

The logic and presentation of the key idea is not bad. I have following concerns about this work at least in its present form.

1. There was a significant difference in body weight between the CTL group and the DGAL group.

2. There are some typing, writing and grammatical errors in this manuscript. This work does require an extensive edit by a native-English speaker.

Please submit your revised manuscript by Mar 26 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Michael Bader

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Apr 15;16(4):e0249487. doi: 10.1371/journal.pone.0249487.r004

Author response to Decision Letter 1

16 Mar 2021

Response to the Reviewer 1

We thank reviewer 1 for the thoughtful suggestions and for the good insights for improving our manuscript. Here we tried to answer in the best form possible to all the concerns.

1. There was a significant difference in body weight between the CTL group and the DGAL group.

Answer: According to the tables below, the animals both in the CTL and DGAL groups presented similar graduated increases in their body weights without statistical differences (n = 5; p > 0.05) (Fig 1C) (see Results Section, Sentence 206-207).

Statistical analyzes were performed using the bidirectional analysis of variance followed by Bonferroni´s test for post hoc comparisons, using the GraphPad Prism 7.0 program. Differences between groups were considered statistically significant at P <0.05.

Time (week) CTL GROUP DGAL GROUP Statistics

Mean (g) SEM N Mean (g) SEM N Significant? P Value

1 272,00 6,89 5 251,00 5,74 5 No >0,9999

2 284,00 9,13 5 272,60 7,81 5 No >0,9999

3 297,60 13,27 5 282,20 12,96 5 No >0,9999

4 317,80 15,10 5 293,00 13,23 5 No >0,9999

5 328,20 16,56 5 295,80 12,59 5 No 0,3342

6 335,60 16,98 5 313,00 14,12 5 No >0,9999

7 344,80 17,34 5 327,80 12,72 5 No >0,9999

8 359,00 14,43 5 341,60 11,16 5 No >0,9999

2. There are some typing, writing and grammatical errors in this manuscript. This work does require an extensive edit by a native-English speaker.

Answer. The manuscript underwent an extensive review by a native speaker. A declaration of manuscript translation-corrections was included as a second supporting information file.

Attachment

Submitted filename: Response to the editor final.docx

Click here for additional data file. (29.1KB, docx)

Decision Letter 2

Michael Bader

19 Mar 2021

D-[+]-Galactose-induced aging:  a novel experimental model of erectile dysfunction

PONE-D-20-33703R2

Dear Dr. Medeiros,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Michael Bader

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Michael Bader

29 Mar 2021

PONE-D-20-33703R2

D-(+)-Galactose-induced aging: a novel experimental model of erectile dysfunction

Dear Dr. de Medeiros:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Michael Bader

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 File

    (XLSX)

    Click here for additional data file. (24.6KB, xlsx)
    Attachment

    Submitted filename: comments.docx

    Click here for additional data file. (15.1KB, docx)
    Attachment

    Submitted filename: Response to the editor final.docx

    Click here for additional data file. (23.8KB, docx)
    Attachment

    Submitted filename: Response to the editor final.docx

    Click here for additional data file. (29.1KB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting information files.

    Articles from PLoS ONE are provided here courtesy of PLOS

    RESOURCES