Vardenafil Long-Term Administration Improves Episodic Memory in Aging Female Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled "Chronic administration of Vardenafil improves cognition in aging female mice" by Dadomo and colleagues studied the effect of a specific phosphodiesterase inhibitor in aged female mice. I have some technical concerns regarding the manuscript and this study that need to be addressed. 

1. The abstract section needs to be completely rephrased as the current abstract indulges a lot more on the background than the actual study, its results, conclusion, and future direction. The first 2 or 3 lines of any abstract are generally enough for the introduction of the topic. So the abstract needs to be rewritten and must follow the standard practices.

2. In the introduction part (also in the abstract section), the authors are strong wordily trying to prove a notion about gender and cognition, which is actually not the ultimate goal of the current study. This line of argumentation would have worked best if the current study would have been assessing the impact of chronic vardenafil treatment on cognition in both male and female genders and compared the magnitude of the impact on both genders.

3. In the title as well as in the material and methods section, the authors mentioned the 32-day treatment as chronic which is a misleading term. The female animals are 2 years old (700+ days) and 32-day treatment doesn't qualify as a chronic. '"Continuous" 32-day treatment' is a much better description of the treatment regime.

4. The age of the animals used in the current experiment seems to be a mystery according to the manuscript. In the 'abstract' section, the 24 months is mentioned while in the 'experimental procedure' section the age of the animals was mentioned as 48 months.

5. The authors should clarify the reason for not sacrificing the animals at the end of the trial and not opting for the biochemical, histological, and expression analysis of the blood and the brain tissue. The observational results collected from EPM, free exploratory test, and ORT need to be substantiated biochemically.

Author Response

Vardenafil and aging. Peer review commentary and authors’ responses

Dear Editor,

Thank you very much for opportunity to revise out manuscript. We have found the many comments and suggestions provided by the reviewers very constructive and we have tried to change our original work following them. We strongly believe that the current manuscript is improved. In the following we provide our responses to the comments. These responses can be found in bold following each reviewer’s comment.

Best regards,

Paola Palanza, PhD

Responses to the reviewers’ comments

Rev 1. The manuscript entitled "Chronic administration of Vardenafil improves cognition in aging female mice" by Dadomo and colleagues studied the effect of a specific phosphodiesterase inhibitor in aged female mice. I have some technical concerns regarding the manuscript and this study that need to be addressed. 

1. The abstract section needs to be completely rephrased as the current abstract indulges a lot more on the background than the actual study, its results, conclusion, and future direction. The first 2 or 3 lines of any abstract are generally enough for the introduction of the topic. So the abstract needs to be rewritten and must follow the standard practices. We agree with the comment of the reviewer and we modified the abstract.
2. In the introduction part (also in the abstract section), the authors are strong wordily trying to prove a notion about gender and cognition, which is actually not the ultimate goal of the current study. This line of argumentation would have worked best if the current study would have been assessing the impact of chronic vardenafil treatment on cognition in both male and female genders and compared the magnitude of the impact on both genders. We agree with the reviewer. We have eliminated the focus on sex difference and wrote a new introduction that focuses on female aging and the effects that aging has on cognition and memory.
3. In the title as well as in the material and methods section, the authors mentioned the 32-day treatment as chronic which is a misleading term. The female animals are 2 years old (700+ days) and 32-day treatment doesn't qualify as a chronic. '"Continuous" 32-day treatment' is a much better description of the treatment regime. We found a mistake in our original submission and we appreciate that this mistake has been emphasized. Mice were exposed to a 32 days treatment with Vardenafil. In relation to the definition of “chronic” treatment, initially we followed the same terminology that has been used in the scientific literature. Nonetheless, when the term “chronic” was referred to our study we have changed “chronic” to “continuous” or”long-term”. Otherwise, we have maintained the definition of the treatment described in the original studies that we have cited.
4. The age of the animals used in the current experiment seems to be a mystery according to the manuscript. In the 'abstract' section, the 24 months is mentioned while in the 'experimental procedure' section the age of the animals was mentioned as 48 months. We apologize for this mistake. Animals were maintained for and tested at 24 months (2 years) of age. Vardenafil treatment begun 30 days before the ORT (Trial 1 of the ORT occurred on treatment day 31; Trial 2 on day 32).
5. The authors should clarify the reason for not sacrificing the animals at the end of the trial and not opting for the biochemical, histological, and expression analysis of the blood and the brain tissue. The observational results collected from EPM, free exploratory test, and ORT need to be substantiated biochemically. We do not understand the comment. While the neurobiological/biochemical support would have added important information about proximate mechanisms, we, as ethologists, have focused specifically on behavior.

Rev 2.

1. Lack of Advancement: The study does not provide significant advancements in the field. We disagree with this statement. We believe that by focusing on very old females, something that to the best of our knowledge nobody has done yet, we provide new data and evidence on the topic of aging, memory and the NO systems interplay.
2. Absence of Data for Male Mice: The title suggests a comparison of sex differences, but there is no data comparing male mice to female mice, and proper controls are missing. We agree and we changed the focus our original manuscript.
3. Inconsistent Age Information: The authors themselves are unsure about the age of the mice used in the experiment, as they mention 24 months old female mice in the abstract (line 19) and 48 months old female mice in the methods section (line 93). This was a mistake; mice were 24 months old (2 yo)
4. Experimental Design Concerns: The overall experimental design is problematic. The authors need to establish that mice at their designated age (24 or 48 months old) had memory deficits before treatment with Vardenafil. The study lacks appropriate controls for this claim. We are not sure that we understand this comment. Certainly the reviewer would be correct if we were interested in a longitudinal effect of age. However, our goal here was to test object recognition in very old females as a function of vardenafil treatment.
5. Lack of Data on Male Mice: The authors mention sex-specific effects throughout the manuscript but do not provide any data for male mice, which is misleading and false. Despite the reviewer’s strong language (“false”), we agree with h** that our original manuscript was off target. We have changed it and we hope it is improved.
6. Additional Memory Tests: The study should include other memory tests, such as fear-associated memory (active and passive avoidance test), spatial temporal memory (Morris water maze and Y-maze), and reward-associated memory (Y-maze) to provide a more comprehensive analysis. We are not sure why this should be the case; There are many studies that focused exclusively on the ORT so we do not understand why we cannot focus on it.
7. Graphs for Behavior Test Results: Instead of presenting the results in text format, the authors should include graphs for behavior tests to enhance readability and accessibility for non-experts. If the Editor thinks that these graphs will help the readers we will be glad to add them. Otherwise we prefer to keep it simple and straightforward.
8. Consistency in Figures: Ensure consistent formatting and style for representing groups throughout the manuscript. Thank you. We have tried to follow the suggestion.

Rev 3. In this manuscript, the authors address a gap in the literature regarding the effect of phosphodiesterase 5 inhibitors in aged female rodents on cognitive and anxiety-like outcomes following chronic treatment with Vardenafil. Overall, the study is well presented and the manuscript, well written. I only have minor comments that the authors may wish to address before publication:

1) Given the background presented on the lack of studies addressing sex differences and the failure to include female mice, I was surprised to find that only female mice were used in this study. The authors do address this as a limitation and a line of research for future investigation, nevertheless, I found myself wanting to know how males would have fared under the same conditions. Especially since very old males (i.e. 48 months) are generally not included in studies either. Although the lack of sex as a biological variable in prior research is very important and should be noted, I would suggest reorganizing the introduction so that it isn't the first, and main point, of this section. Instead, I would lead with the third paragraph describing the effect of aging on cognition and the suspected role of cyclic nucleotides. Thank you. We have followed the suggestion and have rewritten the introduction

2) Please note the number of animals ultimately used for the ORT in the legend or text. Based on the description in the methods, I think there were 9 treated mice and 8 controls, but please verify. We have proceeded by adding this info in the figures

3) For the figures, it would be helpful to see the individual values. Figure 1 and the text indicate that time is an important factor in the outcomes for the discrimination index. It's not clear however, if the 24 h control group did not reach significance (p = 0.05) simply due to one mouse with a lower DI. We actually found an outlier. So we thank the reviewer for his suggestion. See the follow response for a quick discussion on the stats.

4) Figure 2 illustrates the significant main effect of treatment, but not all outcomes of the RM ANOVA (i.e. effect of time), which I find myself looking at Figure 1 to understand. Is there a significant difference between controls and treated mice at 1 or 24 h? Or is the effect significant only when the values are pooled? I am also not sure that repeated measures ANOVA should be used here, since a new novel object was introduced at 24 h with the original object (i.e. trial 1 = a and A, 1 h = A and B, 24 h = A and C). If 24 h is the second object (i.e. B) and a new object (i.e. C), please make that clear in the methods. We mistakenly mentioned a repeated ANOVA when in reality  in the original manuscript we used a mixed factorial ANOVA (treatment x trial); we believe that this would be a good approach because the same animal was tested twice making trial a within subject variable; especially when someone hypothesizes that from T1 to T2 the treatment may affect differently memory performance, the use of a mixed factorial ANOVA is preferred. However, given that small sample size and the lack of normality, in the new version we opted for a non-parametric analysis: first we checked that controls were in fact able to discriminate the novel object (Wilcoxon signed rank test); then we compared controls vs Vardenafil treated animals at T1 and T2.  

Even without an interaction, given that there are main effects of time and treatment, comparisons can be made between groups at the different time points (and/or combination of objects).  Was there any difference in the % time exploring the novel objects? We object to this comment by pointing out that mixed ANOVA are used to test the interaction effects and if the interaction effect is not significant then post-hoc analyses on cross effects (i.e. control t1 vs control t2 or control t1 vs treatment t1) should not be tested. Yet, as described above, we decided to change our approach and focused on the two trials, independently. This allows us to explore the reviewer’s suggestions, especially given the small sample, the lack of normal distribution of the dependent variables and by the presence of an outlier in the control group that was eliminated. The results show that despite the surprising effect that the controls were performing above chance also after 24 hours, Vardenafil treated animals outperformed control at both T1 and T2.

 

Comments on the Quality of English Language

In general, the English is very good and only requires minor editing. One exception is the first sentence of the second paragraph of the introduction: "Despite the increased interest toward a gender medicine animal models of aging still focus on male rodents as target experimental subjects or fail to use sex as a moderator variable." I'm not sure what "gender medicine" refers to; a word or two may be missing, and there should be a comma after medicine indicating the end of the clause: "Despite the increased interest..., animal models of aging...". Thank you for your suggestion.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Harold Dadomo et al. attempted to address sex-specific effects of chronic Vardenafil administration. However, the manuscript lacks experimental evidence to support substantial claims made in the title and throughout. The formatting is poor, and the experimental design lacks essential controls.

1. Lack of Advancement: The study does not provide significant advancements in the field.
2. Absence of Data for Male Mice: The title suggests a comparison of sex differences, but there is no data comparing male mice to female mice, and proper controls are missing.
3. Inconsistent Age Information: The authors themselves are unsure about the age of the mice used in the experiment, as they mention 24 months old female mice in the abstract (line 19) and 48 months old female mice in the methods section (line 93).
4. Experimental Design Concerns: The overall experimental design is problematic. The authors need to establish that mice at their designated age (24 or 48 months old) had memory deficits before treatment with Vardenafil. The study lacks appropriate controls for this claim.
5. Lack of Data on Male Mice: The authors mention sex-specific effects throughout the manuscript but do not provide any data for male mice, which is misleading and false.
6. Additional Memory Tests: The study should include other memory tests, such as fear-associated memory (active and passive avoidance test), spatial temporal memory (Morris water maze and Y-maze), and reward-associated memory (Y-maze) to provide a more comprehensive analysis.
7. Graphs for Behavior Test Results: Instead of presenting the results in text format, the authors should include graphs for behavior tests to enhance readability and accessibility for non-experts.
8. Consistency in Figures: Ensure consistent formatting and style for representing groups throughout the manuscript.

Comments on the Quality of English Language

The language of the manuscript was good.

Author Response

Vardenafil and aging. Peer review commentary and authors’ responses

Dear Editor,

Thank you very much for opportunity to revise out manuscript. We have found the many comments and suggestions provided by the reviewers very constructive and we have tried to change our original work following them. We strongly believe that the current manuscript is improved. In the following we provide our responses to the comments. These responses can be found in bold following each reviewer’s comment.

Best regards,

Paola Palanza, PhD

Responses to the reviewers’ comments

Rev 1. The manuscript entitled "Chronic administration of Vardenafil improves cognition in aging female mice" by Dadomo and colleagues studied the effect of a specific phosphodiesterase inhibitor in aged female mice. I have some technical concerns regarding the manuscript and this study that need to be addressed. 

1. The abstract section needs to be completely rephrased as the current abstract indulges a lot more on the background than the actual study, its results, conclusion, and future direction. The first 2 or 3 lines of any abstract are generally enough for the introduction of the topic. So the abstract needs to be rewritten and must follow the standard practices. We agree with the comment of the reviewer and we modified the abstract.
2. In the introduction part (also in the abstract section), the authors are strong wordily trying to prove a notion about gender and cognition, which is actually not the ultimate goal of the current study. This line of argumentation would have worked best if the current study would have been assessing the impact of chronic vardenafil treatment on cognition in both male and female genders and compared the magnitude of the impact on both genders. We agree with the reviewer. We have eliminated the focus on sex difference and wrote a new introduction that focuses on female aging and the effects that aging has on cognition and memory.
3. In the title as well as in the material and methods section, the authors mentioned the 32-day treatment as chronic which is a misleading term. The female animals are 2 years old (700+ days) and 32-day treatment doesn't qualify as a chronic. '"Continuous" 32-day treatment' is a much better description of the treatment regime. We found a mistake in our original submission and we appreciate that this mistake has been emphasized. Mice were exposed to a 32 days treatment with Vardenafil. In relation to the definition of “chronic” treatment, initially we followed the same terminology that has been used in the scientific literature. Nonetheless, when the term “chronic” was referred to our study we have changed “chronic” to “continuous” or”long-term”. Otherwise, we have maintained the definition of the treatment described in the original studies that we have cited.
4. The age of the animals used in the current experiment seems to be a mystery according to the manuscript. In the 'abstract' section, the 24 months is mentioned while in the 'experimental procedure' section the age of the animals was mentioned as 48 months. We apologize for this mistake. Animals were maintained for and tested at 24 months (2 years) of age. Vardenafil treatment begun 30 days before the ORT (Trial 1 of the ORT occurred on treatment day 31; Trial 2 on day 32).
5. The authors should clarify the reason for not sacrificing the animals at the end of the trial and not opting for the biochemical, histological, and expression analysis of the blood and the brain tissue. The observational results collected from EPM, free exploratory test, and ORT need to be substantiated biochemically. We do not understand the comment. While the neurobiological/biochemical support would have added important information about proximate mechanisms, we, as ethologists, have focused specifically on behavior.

Rev 2.

1. Lack of Advancement: The study does not provide significant advancements in the field. We disagree with this statement. We believe that by focusing on very old females, something that to the best of our knowledge nobody has done yet, we provide new data and evidence on the topic of aging, memory and the NO systems interplay.
2. Absence of Data for Male Mice: The title suggests a comparison of sex differences, but there is no data comparing male mice to female mice, and proper controls are missing. We agree and we changed the focus our original manuscript.
3. Inconsistent Age Information: The authors themselves are unsure about the age of the mice used in the experiment, as they mention 24 months old female mice in the abstract (line 19) and 48 months old female mice in the methods section (line 93). This was a mistake; mice were 24 months old (2 yo)
4. Experimental Design Concerns: The overall experimental design is problematic. The authors need to establish that mice at their designated age (24 or 48 months old) had memory deficits before treatment with Vardenafil. The study lacks appropriate controls for this claim. We are not sure that we understand this comment. Certainly the reviewer would be correct if we were interested in a longitudinal effect of age. However, our goal here was to test object recognition in very old females as a function of vardenafil treatment.
5. Lack of Data on Male Mice: The authors mention sex-specific effects throughout the manuscript but do not provide any data for male mice, which is misleading and false. Despite the reviewer’s strong language (“false”), we agree with h** that our original manuscript was off target. We have changed it and we hope it is improved.
6. Additional Memory Tests: The study should include other memory tests, such as fear-associated memory (active and passive avoidance test), spatial temporal memory (Morris water maze and Y-maze), and reward-associated memory (Y-maze) to provide a more comprehensive analysis. We are not sure why this should be the case; There are many studies that focused exclusively on the ORT so we do not understand why we cannot focus on it.
7. Graphs for Behavior Test Results: Instead of presenting the results in text format, the authors should include graphs for behavior tests to enhance readability and accessibility for non-experts. If the Editor thinks that these graphs will help the readers we will be glad to add them. Otherwise we prefer to keep it simple and straightforward.
8. Consistency in Figures: Ensure consistent formatting and style for representing groups throughout the manuscript. Thank you. We have tried to follow the suggestion.

Rev 3. In this manuscript, the authors address a gap in the literature regarding the effect of phosphodiesterase 5 inhibitors in aged female rodents on cognitive and anxiety-like outcomes following chronic treatment with Vardenafil. Overall, the study is well presented and the manuscript, well written. I only have minor comments that the authors may wish to address before publication:

1) Given the background presented on the lack of studies addressing sex differences and the failure to include female mice, I was surprised to find that only female mice were used in this study. The authors do address this as a limitation and a line of research for future investigation, nevertheless, I found myself wanting to know how males would have fared under the same conditions. Especially since very old males (i.e. 48 months) are generally not included in studies either. Although the lack of sex as a biological variable in prior research is very important and should be noted, I would suggest reorganizing the introduction so that it isn't the first, and main point, of this section. Instead, I would lead with the third paragraph describing the effect of aging on cognition and the suspected role of cyclic nucleotides. Thank you. We have followed the suggestion and have rewritten the introduction

2) Please note the number of animals ultimately used for the ORT in the legend or text. Based on the description in the methods, I think there were 9 treated mice and 8 controls, but please verify. We have proceeded by adding this info in the figures

3) For the figures, it would be helpful to see the individual values. Figure 1 and the text indicate that time is an important factor in the outcomes for the discrimination index. It's not clear however, if the 24 h control group did not reach significance (p = 0.05) simply due to one mouse with a lower DI. We actually found an outlier. So we thank the reviewer for his suggestion. See the follow response for a quick discussion on the stats.

4) Figure 2 illustrates the significant main effect of treatment, but not all outcomes of the RM ANOVA (i.e. effect of time), which I find myself looking at Figure 1 to understand. Is there a significant difference between controls and treated mice at 1 or 24 h? Or is the effect significant only when the values are pooled? I am also not sure that repeated measures ANOVA should be used here, since a new novel object was introduced at 24 h with the original object (i.e. trial 1 = a and A, 1 h = A and B, 24 h = A and C). If 24 h is the second object (i.e. B) and a new object (i.e. C), please make that clear in the methods. We mistakenly mentioned a repeated ANOVA when in reality  in the original manuscript we used a mixed factorial ANOVA (treatment x trial); we believe that this would be a good approach because the same animal was tested twice making trial a within subject variable; especially when someone hypothesizes that from T1 to T2 the treatment may affect differently memory performance, the use of a mixed factorial ANOVA is preferred. However, given that small sample size and the lack of normality, in the new version we opted for a non-parametric analysis: first we checked that controls were in fact able to discriminate the novel object (Wilcoxon signed rank test); then we compared controls vs Vardenafil treated animals at T1 and T2.  

Even without an interaction, given that there are main effects of time and treatment, comparisons can be made between groups at the different time points (and/or combination of objects).  Was there any difference in the % time exploring the novel objects? We object to this comment by pointing out that mixed ANOVA are used to test the interaction effects and if the interaction effect is not significant then post-hoc analyses on cross effects (i.e. control t1 vs control t2 or control t1 vs treatment t1) should not be tested. Yet, as described above, we decided to change our approach and focused on the two trials, independently. This allows us to explore the reviewer’s suggestions, especially given the small sample, the lack of normal distribution of the dependent variables and by the presence of an outlier in the control group that was eliminated. The results show that despite the surprising effect that the controls were performing above chance also after 24 hours, Vardenafil treated animals outperformed control at both T1 and T2.

 

Comments on the Quality of English Language

In general, the English is very good and only requires minor editing. One exception is the first sentence of the second paragraph of the introduction: "Despite the increased interest toward a gender medicine animal models of aging still focus on male rodents as target experimental subjects or fail to use sex as a moderator variable." I'm not sure what "gender medicine" refers to; a word or two may be missing, and there should be a comma after medicine indicating the end of the clause: "Despite the increased interest..., animal models of aging...". Thank you for your suggestion.

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

In this manuscript, the authors address a gap in the literature regarding the effect of phosphodiesterase 5 inhibitors in aged female rodents on cognitive and anxiety-like outcomes following chronic treatment with Vardenafil. Overall, the study is well presented and the manuscript, well written. I only have minor comments that the authors may wish to address before publication:

1) Given the background presented on the lack of studies addressing sex differences and the failure to include female mice, I was surprised to find that only female mice were used in this study. The authors do address this as a limitation and a line of research for future investigation, nevertheless, I found myself wanting to know how males would have fared under the same conditions. Especially since very old males (i.e. 48 months) are generally not included in studies either. Although the lack of sex as a biological variable in prior research is very important and should be noted, I would suggest reorganizing the introduction so that it isn't the first, and main point, of this section. Instead, I would lead with the third paragraph describing the effect of aging on cognition and the suspected role of cyclic nucleotides. 

2) Please note the number of animals ultimately used for the ORT in the legend or text. Based on the description in the methods, I think there were 9 treated mice and 8 controls, but please verify. 

3) For the figures, it would be helpful to see the individual values. Figure 1 and the text indicate that time is an important factor in the outcomes for the discrimination index. It's not clear however, if the 24 h control group did not reach significance (p = 0.05) simply due to one mouse with a lower DI. 

4) Figure 2 illustrates the significant main effect of treatment, but not all outcomes of the RM ANOVA (i.e. effect of time), which I find myself looking at Figure 1 to understand. Is there a significant difference between controls and treated mice at 1 or 24 h? Or is the effect significant only when the values are pooled? I am also not sure that repeated measures ANOVA should be used here, since a new novel object was introduced at 24 h with the original object (i.e. trial 1 = a and A, 1 h = A and B, 24 h = A and C). If 24 h is the second object (i.e. B) and a new object (i.e. C), please make that clear in the methods. 

Even without an interaction, given that there are main effects of time and treatment, comparisons can be made between groups at the different time points (and/or combination of objects).  Was there any difference in the % time exploring the novel objects?

Comments on the Quality of English Language

In general, the English is very good and only requires minor editing. One exception is the first sentence of the second paragraph of the introduction: "Despite the increased interest toward a gender medicine animal models of aging still focus on male rodents as target experimental subjects or fail to use sex as a moderator variable." I'm not sure what "gender medicine" refers to; a word or two may be missing, and there should be a comma after medicine indicating the end of the clause: "Despite the increased interest..., animal models of aging...".

Author Response

Vardenafil and aging. Peer review commentary and authors’ responses

Dear Editor,

Thank you very much for opportunity to revise out manuscript. We have found the many comments and suggestions provided by the reviewers very constructive and we have tried to change our original work following them. We strongly believe that the current manuscript is improved. In the following we provide our responses to the comments. These responses can be found in bold following each reviewer’s comment.

Best regards,

Paola Palanza, PhD

Responses to the reviewers’ comments

Rev 1. The manuscript entitled "Chronic administration of Vardenafil improves cognition in aging female mice" by Dadomo and colleagues studied the effect of a specific phosphodiesterase inhibitor in aged female mice. I have some technical concerns regarding the manuscript and this study that need to be addressed. 

1. The abstract section needs to be completely rephrased as the current abstract indulges a lot more on the background than the actual study, its results, conclusion, and future direction. The first 2 or 3 lines of any abstract are generally enough for the introduction of the topic. So the abstract needs to be rewritten and must follow the standard practices. We agree with the comment of the reviewer and we modified the abstract.
2. In the introduction part (also in the abstract section), the authors are strong wordily trying to prove a notion about gender and cognition, which is actually not the ultimate goal of the current study. This line of argumentation would have worked best if the current study would have been assessing the impact of chronic vardenafil treatment on cognition in both male and female genders and compared the magnitude of the impact on both genders. We agree with the reviewer. We have eliminated the focus on sex difference and wrote a new introduction that focuses on female aging and the effects that aging has on cognition and memory.
3. In the title as well as in the material and methods section, the authors mentioned the 32-day treatment as chronic which is a misleading term. The female animals are 2 years old (700+ days) and 32-day treatment doesn't qualify as a chronic. '"Continuous" 32-day treatment' is a much better description of the treatment regime. We found a mistake in our original submission and we appreciate that this mistake has been emphasized. Mice were exposed to a 32 days treatment with Vardenafil. In relation to the definition of “chronic” treatment, initially we followed the same terminology that has been used in the scientific literature. Nonetheless, when the term “chronic” was referred to our study we have changed “chronic” to “continuous” or”long-term”. Otherwise, we have maintained the definition of the treatment described in the original studies that we have cited.
4. The age of the animals used in the current experiment seems to be a mystery according to the manuscript. In the 'abstract' section, the 24 months is mentioned while in the 'experimental procedure' section the age of the animals was mentioned as 48 months. We apologize for this mistake. Animals were maintained for and tested at 24 months (2 years) of age. Vardenafil treatment begun 30 days before the ORT (Trial 1 of the ORT occurred on treatment day 31; Trial 2 on day 32).
5. The authors should clarify the reason for not sacrificing the animals at the end of the trial and not opting for the biochemical, histological, and expression analysis of the blood and the brain tissue. The observational results collected from EPM, free exploratory test, and ORT need to be substantiated biochemically. We do not understand the comment. While the neurobiological/biochemical support would have added important information about proximate mechanisms, we, as ethologists, have focused specifically on behavior.

Rev 2.

1. Lack of Advancement: The study does not provide significant advancements in the field. We disagree with this statement. We believe that by focusing on very old females, something that to the best of our knowledge nobody has done yet, we provide new data and evidence on the topic of aging, memory and the NO systems interplay.
2. Absence of Data for Male Mice: The title suggests a comparison of sex differences, but there is no data comparing male mice to female mice, and proper controls are missing. We agree and we changed the focus our original manuscript.
3. Inconsistent Age Information: The authors themselves are unsure about the age of the mice used in the experiment, as they mention 24 months old female mice in the abstract (line 19) and 48 months old female mice in the methods section (line 93). This was a mistake; mice were 24 months old (2 yo)
4. Experimental Design Concerns: The overall experimental design is problematic. The authors need to establish that mice at their designated age (24 or 48 months old) had memory deficits before treatment with Vardenafil. The study lacks appropriate controls for this claim. We are not sure that we understand this comment. Certainly the reviewer would be correct if we were interested in a longitudinal effect of age. However, our goal here was to test object recognition in very old females as a function of vardenafil treatment.
5. Lack of Data on Male Mice: The authors mention sex-specific effects throughout the manuscript but do not provide any data for male mice, which is misleading and false. Despite the reviewer’s strong language (“false”), we agree with h** that our original manuscript was off target. We have changed it and we hope it is improved.
6. Additional Memory Tests: The study should include other memory tests, such as fear-associated memory (active and passive avoidance test), spatial temporal memory (Morris water maze and Y-maze), and reward-associated memory (Y-maze) to provide a more comprehensive analysis. We are not sure why this should be the case; There are many studies that focused exclusively on the ORT so we do not understand why we cannot focus on it.
7. Graphs for Behavior Test Results: Instead of presenting the results in text format, the authors should include graphs for behavior tests to enhance readability and accessibility for non-experts. If the Editor thinks that these graphs will help the readers we will be glad to add them. Otherwise we prefer to keep it simple and straightforward.
8. Consistency in Figures: Ensure consistent formatting and style for representing groups throughout the manuscript. Thank you. We have tried to follow the suggestion.

Rev 3. In this manuscript, the authors address a gap in the literature regarding the effect of phosphodiesterase 5 inhibitors in aged female rodents on cognitive and anxiety-like outcomes following chronic treatment with Vardenafil. Overall, the study is well presented and the manuscript, well written. I only have minor comments that the authors may wish to address before publication:

1) Given the background presented on the lack of studies addressing sex differences and the failure to include female mice, I was surprised to find that only female mice were used in this study. The authors do address this as a limitation and a line of research for future investigation, nevertheless, I found myself wanting to know how males would have fared under the same conditions. Especially since very old males (i.e. 48 months) are generally not included in studies either. Although the lack of sex as a biological variable in prior research is very important and should be noted, I would suggest reorganizing the introduction so that it isn't the first, and main point, of this section. Instead, I would lead with the third paragraph describing the effect of aging on cognition and the suspected role of cyclic nucleotides. Thank you. We have followed the suggestion and have rewritten the introduction

2) Please note the number of animals ultimately used for the ORT in the legend or text. Based on the description in the methods, I think there were 9 treated mice and 8 controls, but please verify. We have proceeded by adding this info in the figures

3) For the figures, it would be helpful to see the individual values. Figure 1 and the text indicate that time is an important factor in the outcomes for the discrimination index. It's not clear however, if the 24 h control group did not reach significance (p = 0.05) simply due to one mouse with a lower DI. We actually found an outlier. So we thank the reviewer for his suggestion. See the follow response for a quick discussion on the stats.

4) Figure 2 illustrates the significant main effect of treatment, but not all outcomes of the RM ANOVA (i.e. effect of time), which I find myself looking at Figure 1 to understand. Is there a significant difference between controls and treated mice at 1 or 24 h? Or is the effect significant only when the values are pooled? I am also not sure that repeated measures ANOVA should be used here, since a new novel object was introduced at 24 h with the original object (i.e. trial 1 = a and A, 1 h = A and B, 24 h = A and C). If 24 h is the second object (i.e. B) and a new object (i.e. C), please make that clear in the methods. We mistakenly mentioned a repeated ANOVA when in reality  in the original manuscript we used a mixed factorial ANOVA (treatment x trial); we believe that this would be a good approach because the same animal was tested twice making trial a within subject variable; especially when someone hypothesizes that from T1 to T2 the treatment may affect differently memory performance, the use of a mixed factorial ANOVA is preferred. However, given that small sample size and the lack of normality, in the new version we opted for a non-parametric analysis: first we checked that controls were in fact able to discriminate the novel object (Wilcoxon signed rank test); then we compared controls vs Vardenafil treated animals at T1 and T2.  

Even without an interaction, given that there are main effects of time and treatment, comparisons can be made between groups at the different time points (and/or combination of objects).  Was there any difference in the % time exploring the novel objects? We object to this comment by pointing out that mixed ANOVA are used to test the interaction effects and if the interaction effect is not significant then post-hoc analyses on cross effects (i.e. control t1 vs control t2 or control t1 vs treatment t1) should not be tested. Yet, as described above, we decided to change our approach and focused on the two trials, independently. This allows us to explore the reviewer’s suggestions, especially given the small sample, the lack of normal distribution of the dependent variables and by the presence of an outlier in the control group that was eliminated. The results show that despite the surprising effect that the controls were performing above chance also after 24 hours, Vardenafil treated animals outperformed control at both T1 and T2.

 

Comments on the Quality of English Language

In general, the English is very good and only requires minor editing. One exception is the first sentence of the second paragraph of the introduction: "Despite the increased interest toward a gender medicine animal models of aging still focus on male rodents as target experimental subjects or fail to use sex as a moderator variable." I'm not sure what "gender medicine" refers to; a word or two may be missing, and there should be a comma after medicine indicating the end of the clause: "Despite the increased interest..., animal models of aging...". Thank you for your suggestion.

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The study examined the impact of Vardenafil on cognitive function in older female mice, using various behavioral parameters to assess cognitive impairment. The study focused on aged female mice to test the hypothesis. However, the study's design is presented in a confusing manner, making it difficult to draw clear conclusions. Additionally, there are a few issues that need to be addressed:

 1.      It is a commendable effort to investigate the impact of the PDE5 inhibitor vardenafil on female cognitive impairment. The study indicates that hormonal dysfunction or imbalance is the root cause of cognitive impairment in females. I am curious to know if the drug treatment with vardenafil can help alleviate hormonal dysfunction in females later in life. Could this be the reason behind the PDE5 inhibitor's ability to improve cognition in female mice? I suggest that the discussion section should explore this possibility.

2.      During the experimental period, did the authors observe any changes in body weight in mice treated with vardenafil?

3.      I am wondering why the authors did not employ a histochemistry study of the mouse brain's hippocampus area?  Additionally, a number of biochemical assays may be useful to justify the hypothesis of this study. Providing these assays in the manuscript would enhance its quality. Just assessing the behavior of mice is not a strong indicator of disease condition. Biochemical and histological studies are useful approaches to clarify the study's methodology.

4.      In section 2.6, drug preparation and treatment, spectrometrically should spelled correctly instead of spettrometrically in line 8 (line # 188 in the manuscript). In line # 202, object should spelled correctly instead of oobject.

5.      What is the rationale behind injecting Vardenafil solution intraperitoneally (i.p.) in the evening, after the behavioral tests instead of prior to the behavior test? Describe briefly.

6.      The manuscript lacks clear and consistent information regarding the duration of treatment.

7.      The manuscript should include an explanation for using CD1 mice. Please ensure that the text is free from any spelling, grammar, or punctuation errors.

 

8.      Although the information provided in this study is good, the hypothesis needs more parameters to be justified. 

Author Response

Dear Editor,

thank you very much for the opportunity to improve our manuscript. First and foremost we would like to underscore that we are aware of the many limitations of our work. To begin with, as correctly pointed out by rev. #2, we have not provided data on younger females nor we have neurochemical data. Neurochemical data would have definitely added strength to our behavioral observation but we did not collect brains and we cannot improve this aspect of the study. Younger females would have added information on actual changes in performance throughout the life span of these animals, thus really pinpointing any effect due to aging. We add to this limitations the lack of data on the ovarian cycle (rev #4). Specifically for this last critical comment we know that female mice cycle throughout their life. However in our case it resulted very difficult to perform vaginal smears as the mice were very fragile and we wanted to avoid the risk of disturbing and hurting them. This limitation and the fact that females treated with the vehicle did not show a reduction in the ORT performance does not allow us to discuss in detail the possible role that vardenafil has in improving ORT performance in females that lack of ovarian steroids (rev #4). However we have briefly discussed this possibility in the discussion. In relation to body weight (rev #4) there were no significant differences: at the beginning of the treatment (experimental day 1) vehicle average BW was equal to 39.92 g (1.51)  while Vardenafil average BW was 39.61 (1.39). At the end of the experiment vehicle BW was 36.32 (0.62) and Vardenafil average BW was 39.05 (1.75). We added this information in the current version of the manuscript.

Rev # 4 asked for the rationale behind injecting the drug in the evening (after the behavioral test) instead of prior to the test. The reason is mainly due to avoiding to disturb the animals. Mice performance in the EPM and OPF is very sensitive to any environmental or physical stressor. In relation to the use of CD1 mice in this study has to do with our preference for outbred strain when we are interested in complex phenotypic traits such as aging.

Following the inputs of rev#3 and rev #5 we added the figure of the EPM and OPF and the figure of the exploration time in the ORT. We are also more than willing to share our data to the reviewers in case they think it is needed/helpful.

Despite the clear limitations of the study, we believe the finding is interesting and we hope it could (should) stimulate more research on this topic.

Reviewer 5 Report

Comments and Suggestions for Authors

This excellent study aims to mend the gap between sex differences in cognition. The introduction is well-written, gives enough background information, and clearly states the need for this study. The method section describes all the tests done clearly and in an easy-to-understand manner. The authors have also described the statistical tests used for all the experiments with explanations. 

The authors have stated the results well with the appropriate statistics. However, it would be beneficial if graphs for locomotor activity and elevated plus maze were included, even though there were no significant differences. Also, please indicate the significant difference in the figure 1. The discussion is good; the authors understand their data and can derive inferences based on previous studies. This paper will add new insight into studying sex differences; however, some minor changes are required. 

 

Minor comments:

 

1. Define cAMP and cGMP the first time it is used in the introduction. 

 

2. The first paragraph lacks enough references. E.g., “Aging appears to differentially affect the two sexes, especially in terms of cognitive function” (Add reference)

“In mammalian females, memory and cognition are influenced by the effects that fluctuating gonadal steroids have on different brain areas.” (Add reference)

 

3. Please include the figures for locomotor activity and elevated plus maze. This will give the readers an idea of how older female mice behave. 

Author Response

Dear Editor,

thank you very much for the opportunity to improve our manuscript. First and foremost we would like to underscore that we are aware of the many limitations of our work. To begin with, as correctly pointed out by rev. #2, we have not provided data on younger females nor we have neurochemical data. Neurochemical data would have definitely added strength to our behavioral observation but we did not collect brains and we cannot improve this aspect of the study. Younger females would have added information on actual changes in performance throughout the life span of these animals, thus really pinpointing any effect due to aging. We add to this limitations the lack of data on the ovarian cycle (rev #4). Specifically for this last critical comment we know that female mice cycle throughout their life. However in our case it resulted very difficult to perform vaginal smears as the mice were very fragile and we wanted to avoid the risk of disturbing and hurting them. This limitation and the fact that females treated with the vehicle did not show a reduction in the ORT performance does not allow us to discuss in detail the possible role that vardenafil has in improving ORT performance in females that lack of ovarian steroids (rev #4). However we have briefly discussed this possibility in the discussion. In relation to body weight (rev #4) there were no significant differences: at the beginning of the treatment (experimental day 1) vehicle average BW was equal to 39.92 g (1.51)  while Vardenafil average BW was 39.61 (1.39). At the end of the experiment vehicle BW was 36.32 (0.62) and Vardenafil average BW was 39.05 (1.75). We added this information in the current version of the manuscript.

Rev # 4 asked for the rationale behind injecting the drug in the evening (after the behavioral test) instead of prior to the test. The reason is mainly due to avoiding to disturb the animals. Mice performance in the EPM and OPF is very sensitive to any environmental or physical stressor. In relation to the use of CD1 mice in this study has to do with our preference for outbred strain when we are interested in complex phenotypic traits such as aging.

Following the inputs of rev#2 and rev #5 we added the figure of the EPM and OPF and the figure of the exploration time in the ORT. We are also more than willing to share our data to the reviewers in case they think it is needed/helpful.

Despite the clear limitations of the study, we believe the finding is interesting and we hope it could (should) stimulate more research on this topic.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have significantly improved their submitted manuscript according to the reviewer's comments. 

Author Response

Dear Editor,

thank you very much for the opportunity to improve our manuscript. First and foremost we would like to underscore that we are aware of the many limitations of our work. To begin with, as correctly pointed out by rev. #2, we have not provided data on younger females nor we have neurochemical data. Neurochemical data would have definitely added strength to our behavioral observation but we did not collect brains and we cannot improve this aspect of the study. Younger females would have added information on actual changes in performance throughout the life span of these animals, thus really pinpointing any effect due to aging. We add to this limitations the lack of data on the ovarian cycle (rev #4). Specifically for this last critical comment we know that female mice cycle throughout their life. However in our case it resulted very difficult to perform vaginal smears as the mice were very fragile and we wanted to avoid the risk of disturbing and hurting them. This limitation and the fact that females treated with the vehicle did not show a reduction in the ORT performance does not allow us to discuss in detail the possible role that vardenafil has in improving ORT performance in females that lack of ovarian steroids (rev #4). However we have briefly discussed this possibility in the discussion. In relation to body weight (rev #4) there were no significant differences: at the beginning of the treatment (experimental day 1) vehicle average BW was equal to 39.92 g (1.51)  while Vardenafil average BW was 39.61 (1.39). At the end of the experiment vehicle BW was 36.32 (0.62) and Vardenafil average BW was 39.05 (1.75). We added this information in the current version of the manuscript.

Rev # 4 asked for the rationale behind injecting the drug in the evening (after the behavioral test) instead of prior to the test. The reason is mainly due to avoiding to disturb the animals. Mice performance in the EPM and OPF is very sensitive to any environmental or physical stressor. In relation to the use of CD1 mice in this study has to do with our preference for outbred strain when we are interested in complex phenotypic traits such as aging.

Following the inputs of rev#3 and rev #5 we added the figure of the EPM and OPF and the figure of the exploration time in the ORT. We are also more than willing to share our data to the reviewers in case they think it is needed/helpful.

Despite the clear limitations of the study, we believe the finding is interesting and we hope it could (should) stimulate more research on this topic.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have changed some text and context of the manuscript but still, certain flaws are remaining that are listed below:

 

1.    Lack of Advancement: 

The study does not provide any novelty in the field due to the following reasons. The human study conducted on Vardenafil shows no effect on cognitive performance in healthy subjects (Study title: The effects of the phosphodiesterase type 5 inhibitor vardenafil on cognitive performance in healthy adults: a behavioral- electroencephalography study). Further, the mice model claimed by authors to be aged and cognitively disabled, but there is a difference in the ORT test at Control T1 and Control T2 suggesting that the mice can learn and are not cognitively disabled. This put their whole study design into question. Moreover, without providing data or experimental support and writing fancy terms and overstatements like “NO system interplay” does not provide novelty to the study, one must provide evidence for such claims. Authors themselves cite the effect of different PDE inhibitors on aged and mixed-sex groups (in lines 94-99), but just changing the class of the drug and even that class has no effect on memory in humans cannot be novel or advancement to the field.

 

2.    Experimental Design Concerns: 

The overall experimental design remains problematic. The rationale of picking this 24-month time point was to show age-dependent cognitive decline and restore it with Vardenafil treatment. Still, mice do not have cognitive decline as Control T1 and Control T2 are different in the ORT test, suggesting memory potentiation. The other control missing is the effect on young females. This experiment should have been done to compare the differences as old mice are not cognitively impaired and will give an idea about whether Vardenafil treatment has an age-dependent effect or works similarly in young mice.

 

3.    Additional Memory Tests are needed if the manuscript is addressing cognition: 

ORT only gives information about exploratory behavior and recognition memory of the mice, which comes under cognition, but additional tests are needed to be done to assess cognition thoroughly. In my previous comment, I clearly stated why each behavioral memory test is used and what information it implies. This is an overstatement by the authors by not doing enough behavioral tests and suggesting cognition to be improved. Yes, there are many articles with only ORT, but they do have other information like biochemical data to support the findings. With a single behavioral test, it is not possible to claim cognition to be improved without any other supporting evidence.

 

4.    Graphs for Behavior Test Results: 

 

I hope the senior author understands how the review process happens. If the data is not plotted and presented to the reviewers and is in text format it’s not easy to judge if it’s right or wrong. As a reviewer, we cannot investigate distribution, outliers, and many other things. Even making this comment last time, the authors did not understand the criticality of putting information in the paper. Even the ORT has an exploration time graph missing that will help us understand the discrimination index graph much more easily. The authors have provided only a single graph of the discrimination index in the paper. I am unable to understand why the authors are so shy in putting the information that is critical for the review process. Currently, all journal policies are moving towards transparency of complete data submission, and this is a clear mandate for any scientific publication. Authors must understand that it is not a choice. If the paper is going to reviewers, they should have all the data information requested to judge the work.

Comments on the Quality of English Language

Not Required.

Author Response

Dear Editor,

thank you very much for the opportunity to improve our manuscript. First and foremost we would like to underscore that we are aware of the many limitations of our work. To begin with, as correctly pointed out by rev. #2, we have not provided data on younger females nor we have neurochemical data. Neurochemical data would have definitely added strength to our behavioral observation but we did not collect brains and we cannot improve this aspect of the study. Younger females would have added information on actual changes in performance throughout the life span of these animals, thus really pinpointing any effect due to aging. We add to this limitations the lack of data on the ovarian cycle (rev #4). Specifically for this last critical comment we know that female mice cycle throughout their life. However in our case it resulted very difficult to perform vaginal smears as the mice were very fragile and we wanted to avoid the risk of disturbing and hurting them. This limitation and the fact that females treated with the vehicle did not show a reduction in the ORT performance does not allow us to discuss in detail the possible role that vardenafil has in improving ORT performance in females that lack of ovarian steroids (rev #4). However we have briefly discussed this possibility in the discussion. In relation to body weight (rev #4) there were no significant differences: at the beginning of the treatment (experimental day 1) vehicle average BW was equal to 39.92 g (1.51)  while Vardenafil average BW was 39.61 (1.39). At the end of the experiment vehicle BW was 36.32 (0.62) and Vardenafil average BW was 39.05 (1.75). We added this information in the current version of the manuscript.

Rev # 4 asked for the rationale behind injecting the drug in the evening (after the behavioral test) instead of prior to the test. The reason is mainly due to avoiding to disturb the animals. Mice performance in the EPM and OPF is very sensitive to any environmental or physical stressor. In relation to the use of CD1 mice in this study has to do with our preference for outbred strain when we are interested in complex phenotypic traits such as aging.

Following the inputs of rev#3 and rev #5 we added the figure of the EPM and OPF and the figure of the exploration time in the ORT. We are also more than willing to share our data to the reviewers in case they think it is needed/helpful.

Despite the clear limitations of the study, we believe the finding is interesting and we hope it could (should) stimulate more research on this topic.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have revised the manuscript in a thoughtful and meticulous manner. Although the manuscript has several limitations, it can serve as a good starting point for further research on this topic. Therefore, I recommend it for publication in this journal.

Author Response

Dear Editor,

we have read the reviewer's comment and it appears to suggest publication of our manuscript- Therefore we beleive that there is not further need for revision

Best regards,

Davide Ponzi