Effect of Young Plasma Therapy on Cognition, Oxidative Stress, miRNA-134, BDNF, CREB, and SIRT-1 Expressions and Neuronal Survey in the Hippocampus of Aged Ovariectomized Rats with Alzheimer’s

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript brainsci-3020446 entitled Effect of young plasma therapy on cognition, oxidative stress, miRNA-134, BDNF, CREB, and SIRT-1 expressions and neuronal survey in the hippocampus of aged ovariectomized rats with Alzheimer by Parisa Habibi and coworkers, aimed to use young plasma therapy (YPT) to improve dementia caused by AD in aged ovariectomized rats.

Seventy female Wistar rats were divided into 2 main groups; a) young (CY) (180–220 g, 2-3 months) and b) Old groups (CO) (250–350 g, 22-24 months).

Ovariectomy and Alzheimer caused significant cognitive impairment (p<0.001), upregulated miR-134a (p<0.001), down-regulated SIRT-1, CREB, and BDNF proteins expression (p<0.001), and decreased antioxidant marker levels (p<0.001) compared to the Sham group. YPT significantly restored miR-134a (p<0.001), SIRT-1 (p<0.001), CREB (p<0.001), and BDNF (p<0.001) proteins expression in OVX+AD rats.

YPT as much as or more than ERT significantly improved oxidative stress and down-regulated miR-134a expression and up-regulation SIRT-1, CREB, and BDNF proteins in OVX+AD rats (p<0.001). YPT significantly improved histological abnormalities compared to the OVX+AD group (p<0.001).

The manuscript is well written and interesting.

Hypothesis is logic and the scientific work appropriate.

Figures are informative.

The discussion is consistent with results.

Language requires a moderate revision.

Major points:

1. a quantification of the steroids in brain is missing. It would be interesting to see levels of steroids (as well as oxysterols, sterols) in the brain samples comparing the different groups

2. the method used of r oxidative stress quantification is rather weak

Other comments

Page 2: the quality of the graphical abstract should be improved

Line 192: FRAP is mainly used to asses antioxidant capacity.

MDA, 4-hydroxynonenal, oxysterols are solid markers of oxidative stress as well as 8-OHdG/8-Hydroxyguanosine as marker of DNA oxidative damage.

MDA and TTG are derived calculation, not first hand quantifications.

Line 268, figure 3: the size of the 4 panels could be reduced and composed in one figure

Line 305: figure 4: the four panels of the figure should be reduced in size and composed together in one figure

Line 372: figure 5: the five panels of the figure should be reduced in size and composed together in one figure

Line 459: figure 6: the four panels of the figure should be reduced in size and composed together in one figure

Comments on the Quality of English Language

Language requires a moderate revision.

Author Response

Response to reviewers

Manuscript ID: brainsci-3020446

Editor-in-Chief

Thank you for your kind letter of the decision on the manuscript entitled “Effect of young plasma therapy on cognition, oxidative stress, miRNA-134, BDNF, CREB, and SIRT-1 expressions and neuronal survey in the hippocampus of aged ovariectomized rats with Alzheimer's" We believe that the manuscript has been dramatically improved and hope it has reached your magazine's standard. Once again, we acknowledge your comments very much, which are valuable in improving the quality of our manuscript.

Sincerely yours

Siamak Shahidi

The comments are answered as follows.

1. We sent you an email earlier suggesting you keep only one corresponding

author, please check the email and reply to us as soon as possible.

Response: Your suggestion was made. This revised manuscript has one major corresponding author (Prof. Siamak Shahidi).

2. We notice that the *repetition rate* is higher than our journal

requirements. Some high-repetition parts are highlighted in *the Duplication

report attached*. Please revise your manuscript according to the report and

make sure that there is no large part repetition with other published papers.

Response: The repetition rate was considered and revised the manuscript according to the report.

3. There is a 3rd referee who was interested in reviewing your manuscript. We

will send you a short note if we receive the 3rd report.

Response: With thanks. We received and responded to the 3rd reviewer's comments.

(I) Please check that all references are relevant to the contents of the

manuscript.

Response: Thank you. All references are checked for relevance to the manuscript content.

(II) Any revisions to the manuscript should *be highlighted*, such that any

changes can be easily reviewed by editors and reviewers.

Response: All revisions and corrections throughout the manuscript were highlighted in yellow color.

(III) Please provide a cover letter to explain, *point by point*, the details

of the revisions to the manuscript and your responses to the referees’

comments.

Response: With thanks. All corrections and explanations on the base of reviewers' comments are mentioned in a cover letter and attached to the revised manuscript online via the journal website. 

(IV) If the reviewer(s) recommended references, please critically analyze

them to ensure that their inclusion would enhance your manuscript. If you

believe these references are unnecessary, you should not include them.

Response: Thank you for your guidance.

(V) If you found it impossible to address certain comments in the review

reports, please include an explanation in your appeal.

Response: Thank you for your guidance.

(VI) The revised version will be sent to the editors and reviewers.

With thank.

Reviewer no. 1

The manuscript was edited for writing and grammatically by an expert and a native of the English language. All corrections were highlighted throughout its text.

1. 1. In the introduction section, it is necessary to clearly explain the background and significance of miRNA-134, BDNF, CREB, and SIRT-1 expressions.

Response: With thanks the introduction was revised and added necessary explanations and backgrounds about miRNA-134, BDNF, CREB, and SIRT-1 expressions were added and highlighted. (Page 3- lines 66-91).

2. Were there any specific criteria for selecting the rats used in the study, particularly regarding age and health status? Why are 60 rats in the old group and only 10 rats in the young generation?

Response: Ok thank you for your interested question. The healthy young and aged females were selected by simple randomization. To determine the sample size a pilot study was done on 5 rats in each group and then using the mean, standard error of mean, variance, and given α-value in a formula the final number of rats in each experimental group was counted. We used only one group of healthy young rats (n=10) and also one group of healthy female old rats (n=10) as normal control group to compare the tested parameters, because we tried to investigate the effects of the aging process on all studied parameters with compare to control young rats. On the other hand, all studied parameters in OVX, OVX-AD, as well as treated groups, were compared statistically with healthy control old rats. A total of 10 rats were used in control young female rats but 60 aged female rats were divided into 6 sub-groups with 10 in each because the rats in each group were divided into 2 sub-groups for biochemical and gene expression (n=6) and histological evaluation (n=4).    

3. Clarify the reasoning behind choosing the specific cognitive behavior tests (NOR, MWM, PAL) used in the study. Were there any alternative tests considered?

Response: Thank you for your question. The AD has a high prevalence among postmenopausal women and is characterized by progressive impairment of memory and other cognitive functions.  The increase in the deposition of Aβ plaques in the brain of postmenopausal women probably indicates the relationship between estrogen deficiency and neurological disorders in middle-aged women. Since during aging and menopause followed by estrogen hormone deficiency in female rats, the synaptic plasticity will disrupt because of gradual neuronal apoptosis in some brain areas involve the cognition so learning and memory impairment will happened. There are different experimental tests for cognitive evaluation in rodents. We selected NOR, MWM and PAL tests for cognitive evaluation because after aging and estrogen deficiency in aged female rats the spatial learning and memory, novel object recognition of new objects and also another type of passive avoidance memory disrupt due to neuronal death and lower electrical signaling in hippocampus and other brain areas.

References:

1. Association, A.s., W. Thies, and L. Bleiler, 2013 Alzheimer's disease facts and figures. Alzheimer's & dementia, 2013. 9(2): p. 208-245.
2. Mielke, M.M., P. Vemuri, and W.A. Rocca, Clinical epidemiology of Alzheimer’s disease: assessing sex and gender differences. Clinical epidemiology, 2014. 6: p. 37.
3. Ross, R.K., et al., Cardiovascular benefits of estrogen replacement therapy. American journal of obstetrics and gynecology, 1989. 160(5): p. 1301-1306.
4. Mosconi, L., et al., Perimenopause and emergence of an Alzheimer’s bioenergetic phenotype in brain and periphery. PloS one, 2017. 12(10): p. e0185926.
5. Mosconi, L., et al., Sex differences in Alzheimer risk: Brain imaging of endocrine vs chronologic aging. Neurology, 2017. 89(13): p. 1382-1390.
6. Li, J. and R.B. Gibbs, Detection of estradiol in rat brain tissues: Contribution of local versus systemic production. Psychoneuroendocrinology, 2019. 102: p. 84-94.
7. 4.  Were any adjustments made for multiple comparisons in the statistical analysis to reduce the risk of type I errors?

 Response: Thank you for your comment. Yes, we have normalized the data and used the one-way and also repeated measures one-way ANOVA followed by Tukey's post hoc test for multiple analyses with application at least 80% power potential of statistic tests.

5. How do this study's findings contribute to our understanding of the underlying mechanisms involved in Alzheimer's disease (AD) and the potential effects of YPT?

 Response: Thank you for your question: The results of the current study showed that in aged and menopausal rats’ neuronal loss (neuronal plaques found in histological tests), cognitive impairment (in behavioral tests), oxidative stress, and alterations in some genes expression have occurred. These are disadvantages for neural normal functions that are followed by dementia type of AD (References). We found that Young plasma therapy could reverse these negative effects significantly.

References:

• Emerging roles of oxidative stress in brain aging and Alzheimer's disease. https://doi.org/10.1016/j.neurobiolaging.2021.07.014

2-     Therapeutic potential of natural molecules against Alzheimer's disease via SIRT1 modulation. https://doi.org/10.1016/j.biopha.2023.114474

3-     Gene- and Gender-Related Decrease in Serum BDNF Levels in Alzheimer’s disease. Int. J. Mol. Sci. 2022, 23(23), 14599; https://doi.org/10.3390/ijms232314599

6. Are there any alternative explanations for the observed effects, and if so, how were they considered in interpreting the results?

Response: With thanks. Yes, we can explain that the ovarian sex hormones especially estrogen have a key role as neuroprotective. Estrogen deficiency after menopause causes down-regulation of estrogen alpha and beta receptors in brain hippocampus and other important areas of the brain for cognition. In the current study, we used the young rat plasma therapy that may contain such hormones and other biochemical molecules and can compensate for those losses. On the other hand in parallel research work, it appeared that the electrical properties of these areas of the brain involved in cognition were disrupted (by long-term potentiation recording and measuring some neurotransmitters such as glutamate and acetylcholine).

7. To what extent can the findings of this study be generalized to other populations, such as humans or different animal models of AD?

 Response: Thank you for your question. Current findings show that aging and estrogen deficiency due to menopause could degenerate the nervous system (sensory, motor areas, and brain integrative functions of these areas in brain) and cause disrupted memory consolidation in humans and rodents. The same impairments could be found in different types of animal models of AD such as AD induced by brain trauma, PD, and induced by injection of the STZ. About YPT, we need to do a clinical trial with our clinical colleagues (physicians) to continue applied research and our findings about YPT to be generalized to humans.

8. What are the next steps in this line of research, and how do you envision this study contributing to future investigations or potential clinical applications?

 Response: with thanks. We can suggest doing the clinical trial with our clinical colleagues (physicians) to continue applied research in such patients. This will be possible by preparing the young plasma from taken blood samples. Because the dignity and preservation of life of the elderly is of great importance.

9. Are there any limitations or areas for improvement identified in this study that could be addressed in future research?

Response: Thank you for a good question. Yes, there are some limitations. Some of these are as follows;

1. We should be careful that the donors of plasma have no diseases.
2. Since the prepared young plasma has different molecules (proteins, enzymes, large molecules ……) it may involve immunity resistance after administration. So needs this status examined before the use of young plasma for treatment.
3. The authors extensively discussed their results with articles published in 2021 and older but failed to discuss articles published in 2022, 2023, and 2024. This concern needs to be addressed.

 Response: Thank you for your attention and notice. With the revision of the manuscript, we added new texts with new references (2021-2024).

Reviewer no. 2:

Major points:

1. A quantification of the steroids in the brain is missing. It would be interesting to see levels of steroids (as well as oxysterols, and sterols) in the brain samples comparing the different groups.

Response: With thanks. The measure of the levels of steroids in brain tissue samples is a limitation of current research. Unfortunately measuring and comparing the levels of steroids, oxysterols, and strolls in the brain samples of different experimental groups were not done because this was not foreseen in the special objectives of our research proposal. It is a good and just idea, we will try to measure and compare the levels of steroids in brain samples in our future projects. Of course in the text of the introduction, based on the references, new explanations have been added about the normal levels of steroid hormones and their changes in the conditions of aging or menopause in laboratory mice, which can be seen as highlights.

2. The method used for oxidative stress quantification is rather weak.

Response: With thanks. To measure oxidative stress factors in rats' brain tissue samples, we used the methods introduced in the valid published papers in this field (References). We used the same method in our previous works, whose articles were published. However, other methods are different in terms of accuracy, but we could not do them due to their high cost.

References;

1. Benzie, I.F. and J. Strain, [2] Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration, in Methods in enzymology. 1999, Elsevier. p. 15-27.
2. Erel, O., A new automated colorimetric method for measuring total oxidant status. Clinical biochemistry, 2005. 38(12): p. 1103-1111.

 Other comments

Page 2: the quality of the graphical abstract should be improved.

Response: Ok, thank you for your attention. The graphic abstract was improved in quality and was replaced.

Line 192: FRAP is mainly used to assess antioxidant capacity.

Response: With thanks. Yes, you are right we also used to assess the antioxidant capacity.

MDA, 4-hydroxynonenal, and oxysterols are solid markers of oxidative stress as well as 8-OHdG/8-Hydroxyguanosine as a marker of DNA oxidative damage.

Response: Yes, you are right. Thank you for your reminder.

MDA and TTG are derived calculations, not first-hand quantifications.

Response:  The MDA and TTG were assayed with using the valid ELISA kits introduced in other published articles.

Line 268, figure 3: the size of the 4 panels could be reduced and composed in one figure.

Response: Thank you for your suggestion. All mentioned figures with you were changed on the base of your guide.

Line 305: figure 4: the four panels of the figure should be reduced in size and composed together in one figure.

Response: Was done.

Line 372: figure 5: the five panels of the figure should be reduced in size and composed together in one figure.

Response: Was done.

Line 459: figure 6: the four panels of the figure should be reduced in size and composed together in one figure.

Response: Was done.

Comments on the Quality of English Language

Language requires moderate revision.

Response: With thanks. The manuscript was edited by an expert and native English language and corrected throughout its text. The corrections were highlighted. 

Reviewer no. 3

The manuscript entitled "Effect of young plasma therapy on cognition, oxidative stress, 2 miRNA-134, BDNF, CREB, and SIRT-1 expressions and neu-3 ronal survey in the hippocampus of aged ovariectomized rats 4 with Alzheimer" aimed to use young plasma therapy (YPT) to improve dementia caused by AD in aged ovariectomized rats. Several concerns arise from reading this manuscript in its current form and the AD model it uses.

Response: With thanks. In the current study, we induced AD in rats with the administration of Aβ_1-42 (5 μg/5 μL/rat, dissolved in distilled water (Sigma Aldrich, St. Louis, MO, USA)) was infused into the lateral ventricles (References)

References;

1. Mosconi, L., et al., Perimenopause and emergence of an Alzheimer’s bioenergetic phenotype in brain and periphery. PloS one, 2017. 12(10): p. e0185926.

The introduction is insufficient because it lacks an adequate context to explain the relationship between the markers searched for. For example, it refers in a very general way to microRNAs but fails to delve into the evidence surrounding mir143, the reason for its choice in particular. 

Response: With thanks the introduction was revised and added necessary explanations and backgrounds about miRNA-134, BDNF, CREB, and SIRT-1 expressions were added and highlighted. (Page 3- lines 66-91).

In general, the rationale of the present manuscript is not adequately described either in the introduction or in the description of results.

Response: In the introduction according to the new references inserted in the text, more explanations have been added which are marked with yellow color. It was revised according to the reviewers' comments. The evidence surrounding mir-143, BDNF, and SIRT-1 with related references were added.  (Page 3- lines 66-91).

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

1.       In the introduction section, it is necessary to clearly explain the background and significance of miRNA-134, BDNF, CREB, and SIRT-1 expressions.

2.       Were there any specific criteria for selecting the rats used in the study, particularly regarding age and health status? Why are 60 rats in the old group and only 10 rats in the young generation?

3.       Clarify the reasoning behind choosing the specific cognitive behavior tests (NOR, MWM, PAL) used in the study. Were there any alternative tests considered?

4.       Were any adjustments made for multiple comparisons in the statistical analysis to reduce the risk of type I errors?

5.       How do this study's findings contribute to our understanding of the underlying mechanisms involved in Alzheimer's disease (AD) and the potential effects of YPT?

6.       Are there any alternative explanations for the observed effects, and if so, how were they considered in interpreting the results?

7.       To what extent can the findings of this study be generalized to other populations, such as humans or different animal models of AD?

8.       What are the next steps in this line of research, and how do you envision this study contributing to future investigations or potential clinical applications?

9.       Are there any limitations or areas for improvement identified in this study that could be addressed in future research?

10.   The authors extensively discussed their results with articles published in 2021 and older but failed to discuss articles published in 2022, 2023, and 2024. This concern needs to be addressed.

The manuscript is recommended for major revision before acceptance.

Author Response

Response to reviewers

Manuscript ID: brainsci-3020446

Editor-in-Chief

Thank you for your kind letter of the decision on the manuscript entitled “Effect of young plasma therapy on cognition, oxidative stress, miRNA-134, BDNF, CREB, and SIRT-1 expressions and neuronal survey in the hippocampus of aged ovariectomized rats with Alzheimer's" We believe that the manuscript has been dramatically improved and hope it has reached your magazine's standard. Once again, we acknowledge your comments very much, which are valuable in improving the quality of our manuscript.

Sincerely yours

Siamak Shahidi

The comments are answered as follows.

1. We sent you an email earlier suggesting you keep only one corresponding

author, please check the email and reply to us as soon as possible.

Response: Your suggestion was made. This revised manuscript has one major corresponding author (Prof. Siamak Shahidi).

2. We notice that the *repetition rate* is higher than our journal

requirements. Some high-repetition parts are highlighted in *the Duplication

report attached*. Please revise your manuscript according to the report and

make sure that there is no large part repetition with other published papers.

Response: The repetition rate was considered and revised the manuscript according to the report.

3. There is a 3rd referee who was interested in reviewing your manuscript. We

will send you a short note if we receive the 3rd report.

Response: With thanks. We received and responded to the 3rd reviewer's comments.

(I) Please check that all references are relevant to the contents of the

manuscript.

Response: Thank you. All references are checked for relevance to the manuscript content.

(II) Any revisions to the manuscript should *be highlighted*, such that any

changes can be easily reviewed by editors and reviewers.

Response: All revisions and corrections throughout the manuscript were highlighted in yellow color.

(III) Please provide a cover letter to explain, *point by point*, the details

of the revisions to the manuscript and your responses to the referees’

comments.

Response: With thanks. All corrections and explanations on the base of reviewers' comments are mentioned in a cover letter and attached to the revised manuscript online via the journal website. 

(IV) If the reviewer(s) recommended references, please critically analyze

them to ensure that their inclusion would enhance your manuscript. If you

believe these references are unnecessary, you should not include them.

Response: Thank you for your guidance.

(V) If you found it impossible to address certain comments in the review

reports, please include an explanation in your appeal.

Response: Thank you for your guidance.

(VI) The revised version will be sent to the editors and reviewers.

With thank.

Reviewer no. 1

The manuscript was edited for writing and grammatically by an expert and a native of the English language. All corrections were highlighted throughout its text.

1. 1. In the introduction section, it is necessary to clearly explain the background and significance of miRNA-134, BDNF, CREB, and SIRT-1 expressions.

Response: With thanks the introduction was revised and added necessary explanations and backgrounds about miRNA-134, BDNF, CREB, and SIRT-1 expressions were added and highlighted. (Page 3- lines 66-91).

2. Were there any specific criteria for selecting the rats used in the study, particularly regarding age and health status? Why are 60 rats in the old group and only 10 rats in the young generation?

Response: Ok thank you for your interested question. The healthy young and aged females were selected by simple randomization. To determine the sample size a pilot study was done on 5 rats in each group and then using the mean, standard error of mean, variance, and given α-value in a formula the final number of rats in each experimental group was counted. We used only one group of healthy young rats (n=10) and also one group of healthy female old rats (n=10) as normal control group to compare the tested parameters, because we tried to investigate the effects of the aging process on all studied parameters with compare to control young rats. On the other hand, all studied parameters in OVX, OVX-AD, as well as treated groups, were compared statistically with healthy control old rats. A total of 10 rats were used in control young female rats but 60 aged female rats were divided into 6 sub-groups with 10 in each because the rats in each group were divided into 2 sub-groups for biochemical and gene expression (n=6) and histological evaluation (n=4).    

3. Clarify the reasoning behind choosing the specific cognitive behavior tests (NOR, MWM, PAL) used in the study. Were there any alternative tests considered?

Response: Thank you for your question. The AD has a high prevalence among postmenopausal women and is characterized by progressive impairment of memory and other cognitive functions.  The increase in the deposition of Aβ plaques in the brain of postmenopausal women probably indicates the relationship between estrogen deficiency and neurological disorders in middle-aged women. Since during aging and menopause followed by estrogen hormone deficiency in female rats, the synaptic plasticity will disrupt because of gradual neuronal apoptosis in some brain areas involve the cognition so learning and memory impairment will happened. There are different experimental tests for cognitive evaluation in rodents. We selected NOR, MWM and PAL tests for cognitive evaluation because after aging and estrogen deficiency in aged female rats the spatial learning and memory, novel object recognition of new objects and also another type of passive avoidance memory disrupt due to neuronal death and lower electrical signaling in hippocampus and other brain areas.

References:

1. Association, A.s., W. Thies, and L. Bleiler, 2013 Alzheimer's disease facts and figures. Alzheimer's & dementia, 2013. 9(2): p. 208-245.
2. Mielke, M.M., P. Vemuri, and W.A. Rocca, Clinical epidemiology of Alzheimer’s disease: assessing sex and gender differences. Clinical epidemiology, 2014. 6: p. 37.
3. Ross, R.K., et al., Cardiovascular benefits of estrogen replacement therapy. American journal of obstetrics and gynecology, 1989. 160(5): p. 1301-1306.
4. Mosconi, L., et al., Perimenopause and emergence of an Alzheimer’s bioenergetic phenotype in brain and periphery. PloS one, 2017. 12(10): p. e0185926.
5. Mosconi, L., et al., Sex differences in Alzheimer risk: Brain imaging of endocrine vs chronologic aging. Neurology, 2017. 89(13): p. 1382-1390.
6. Li, J. and R.B. Gibbs, Detection of estradiol in rat brain tissues: Contribution of local versus systemic production. Psychoneuroendocrinology, 2019. 102: p. 84-94.
7. 4.  Were any adjustments made for multiple comparisons in the statistical analysis to reduce the risk of type I errors?

 Response: Thank you for your comment. Yes, we have normalized the data and used the one-way and also repeated measures one-way ANOVA followed by Tukey's post hoc test for multiple analyses with application at least 80% power potential of statistic tests.

5. How do this study's findings contribute to our understanding of the underlying mechanisms involved in Alzheimer's disease (AD) and the potential effects of YPT?

 Response: Thank you for your question: The results of the current study showed that in aged and menopausal rats’ neuronal loss (neuronal plaques found in histological tests), cognitive impairment (in behavioral tests), oxidative stress, and alterations in some genes expression have occurred. These are disadvantages for neural normal functions that are followed by dementia type of AD (References). We found that Young plasma therapy could reverse these negative effects significantly.

References:

• Emerging roles of oxidative stress in brain aging and Alzheimer's disease. https://doi.org/10.1016/j.neurobiolaging.2021.07.014

2-     Therapeutic potential of natural molecules against Alzheimer's disease via SIRT1 modulation. https://doi.org/10.1016/j.biopha.2023.114474

3-     Gene- and Gender-Related Decrease in Serum BDNF Levels in Alzheimer’s disease. Int. J. Mol. Sci. 2022, 23(23), 14599; https://doi.org/10.3390/ijms232314599

6. Are there any alternative explanations for the observed effects, and if so, how were they considered in interpreting the results?

Response: With thanks. Yes, we can explain that the ovarian sex hormones especially estrogen have a key role as neuroprotective. Estrogen deficiency after menopause causes down-regulation of estrogen alpha and beta receptors in brain hippocampus and other important areas of the brain for cognition. In the current study, we used the young rat plasma therapy that may contain such hormones and other biochemical molecules and can compensate for those losses. On the other hand in parallel research work, it appeared that the electrical properties of these areas of the brain involved in cognition were disrupted (by long-term potentiation recording and measuring some neurotransmitters such as glutamate and acetylcholine).

7. To what extent can the findings of this study be generalized to other populations, such as humans or different animal models of AD?

 Response: Thank you for your question. Current findings show that aging and estrogen deficiency due to menopause could degenerate the nervous system (sensory, motor areas, and brain integrative functions of these areas in brain) and cause disrupted memory consolidation in humans and rodents. The same impairments could be found in different types of animal models of AD such as AD induced by brain trauma, PD, and induced by injection of the STZ. About YPT, we need to do a clinical trial with our clinical colleagues (physicians) to continue applied research and our findings about YPT to be generalized to humans.

8. What are the next steps in this line of research, and how do you envision this study contributing to future investigations or potential clinical applications?

 Response: with thanks. We can suggest doing the clinical trial with our clinical colleagues (physicians) to continue applied research in such patients. This will be possible by preparing the young plasma from taken blood samples. Because the dignity and preservation of life of the elderly is of great importance.

9. Are there any limitations or areas for improvement identified in this study that could be addressed in future research?

Response: Thank you for a good question. Yes, there are some limitations. Some of these are as follows;

1. We should be careful that the donors of plasma have no diseases.
2. Since the prepared young plasma has different molecules (proteins, enzymes, large molecules ……) it may involve immunity resistance after administration. So needs this status examined before the use of young plasma for treatment.
3. The authors extensively discussed their results with articles published in 2021 and older but failed to discuss articles published in 2022, 2023, and 2024. This concern needs to be addressed.

 Response: Thank you for your attention and notice. With the revision of the manuscript, we added new texts with new references (2021-2024).

Reviewer no. 2:

Major points:

1. A quantification of the steroids in the brain is missing. It would be interesting to see levels of steroids (as well as oxysterols, and sterols) in the brain samples comparing the different groups.

Response: With thanks. The measure of the levels of steroids in brain tissue samples is a limitation of current research. Unfortunately measuring and comparing the levels of steroids, oxysterols, and strolls in the brain samples of different experimental groups were not done because this was not foreseen in the special objectives of our research proposal. It is a good and just idea, we will try to measure and compare the levels of steroids in brain samples in our future projects. Of course in the text of the introduction, based on the references, new explanations have been added about the normal levels of steroid hormones and their changes in the conditions of aging or menopause in laboratory mice, which can be seen as highlights.

2. The method used for oxidative stress quantification is rather weak.

Response: With thanks. To measure oxidative stress factors in rats' brain tissue samples, we used the methods introduced in the valid published papers in this field (References). We used the same method in our previous works, whose articles were published. However, other methods are different in terms of accuracy, but we could not do them due to their high cost.

References;

1. Benzie, I.F. and J. Strain, [2] Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration, in Methods in enzymology. 1999, Elsevier. p. 15-27.
2. Erel, O., A new automated colorimetric method for measuring total oxidant status. Clinical biochemistry, 2005. 38(12): p. 1103-1111.

 Other comments

Page 2: the quality of the graphical abstract should be improved.

Response: Ok, thank you for your attention. The graphic abstract was improved in quality and was replaced.

Line 192: FRAP is mainly used to assess antioxidant capacity.

Response: With thanks. Yes, you are right we also used to assess the antioxidant capacity.

MDA, 4-hydroxynonenal, and oxysterols are solid markers of oxidative stress as well as 8-OHdG/8-Hydroxyguanosine as a marker of DNA oxidative damage.

Response: Yes, you are right. Thank you for your reminder.

MDA and TTG are derived calculations, not first-hand quantifications.

Response:  The MDA and TTG were assayed with using the valid ELISA kits introduced in other published articles.

Line 268, figure 3: the size of the 4 panels could be reduced and composed in one figure.

Response: Thank you for your suggestion. All mentioned figures with you were changed on the base of your guide.

Line 305: figure 4: the four panels of the figure should be reduced in size and composed together in one figure.

Response: Was done.

Line 372: figure 5: the five panels of the figure should be reduced in size and composed together in one figure.

Response: Was done.

Line 459: figure 6: the four panels of the figure should be reduced in size and composed together in one figure.

Response: Was done.

Comments on the Quality of English Language

Language requires moderate revision.

Response: With thanks. The manuscript was edited by an expert and native English language and corrected throughout its text. The corrections were highlighted. 

Reviewer no. 3

The manuscript entitled "Effect of young plasma therapy on cognition, oxidative stress, 2 miRNA-134, BDNF, CREB, and SIRT-1 expressions and neu-3 ronal survey in the hippocampus of aged ovariectomized rats 4 with Alzheimer" aimed to use young plasma therapy (YPT) to improve dementia caused by AD in aged ovariectomized rats. Several concerns arise from reading this manuscript in its current form and the AD model it uses.

Response: With thanks. In the current study, we induced AD in rats with the administration of Aβ_1-42 (5 μg/5 μL/rat, dissolved in distilled water (Sigma Aldrich, St. Louis, MO, USA)) was infused into the lateral ventricles (References)

References;

1. Mosconi, L., et al., Perimenopause and emergence of an Alzheimer’s bioenergetic phenotype in brain and periphery. PloS one, 2017. 12(10): p. e0185926.

The introduction is insufficient because it lacks an adequate context to explain the relationship between the markers searched for. For example, it refers in a very general way to microRNAs but fails to delve into the evidence surrounding mir143, the reason for its choice in particular. 

Response: With thanks the introduction was revised and added necessary explanations and backgrounds about miRNA-134, BDNF, CREB, and SIRT-1 expressions were added and highlighted. (Page 3- lines 66-91).

In general, the rationale of the present manuscript is not adequately described either in the introduction or in the description of results.

Response: In the introduction according to the new references inserted in the text, more explanations have been added which are marked with yellow color. It was revised according to the reviewers' comments. The evidence surrounding mir-143, BDNF, and SIRT-1 with related references were added.  (Page 3- lines 66-91).

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled "Effect of young plasma therapy on cognition, oxidative stress, 2 miRNA-134, BDNF, CREB, and SIRT-1 expressions and neu-3 ronal survey in the hippocampus of aged ovariectomized rats 4 with Alzheimer" aimed to use young plasma therapy (YPT) to improve dementia caused by AD in aged ovariectomized rats. Several concerns arise from reading this manuscript in its current form and the AD model they use. 

The introduction is insufficient because it lacks an adequate context to explain the relationship between the markers searched for. For example, it refers in a very general way to microRNAs but fails to delve into the evidence surrounding mir143, the reason for its choice in particular. 

In general, the rationale of the present manuscript is not adequately described either in the introduction or in the description of results.

Author Response

Response to reviewers

Manuscript ID: brainsci-3020446

Editor-in-Chief

Thank you for your kind letter of the decision on the manuscript entitled “Effect of young plasma therapy on cognition, oxidative stress, miRNA-134, BDNF, CREB, and SIRT-1 expressions and neuronal survey in the hippocampus of aged ovariectomized rats with Alzheimer's" We believe that the manuscript has been dramatically improved and hope it has reached your magazine's standard. Once again, we acknowledge your comments very much, which are valuable in improving the quality of our manuscript.

Sincerely yours

Siamak Shahidi

The comments are answered as follows.

1. We sent you an email earlier suggesting you keep only one corresponding

author, please check the email and reply to us as soon as possible.

Response: Your suggestion was made. This revised manuscript has one major corresponding author (Prof. Siamak Shahidi).

2. We notice that the *repetition rate* is higher than our journal

requirements. Some high-repetition parts are highlighted in *the Duplication

report attached*. Please revise your manuscript according to the report and

make sure that there is no large part repetition with other published papers.

Response: The repetition rate was considered and revised the manuscript according to the report.

3. There is a 3rd referee who was interested in reviewing your manuscript. We

will send you a short note if we receive the 3rd report.

Response: With thanks. We received and responded to the 3rd reviewer's comments.

(I) Please check that all references are relevant to the contents of the

manuscript.

Response: Thank you. All references are checked for relevance to the manuscript content.

(II) Any revisions to the manuscript should *be highlighted*, such that any

changes can be easily reviewed by editors and reviewers.

Response: All revisions and corrections throughout the manuscript were highlighted in yellow color.

(III) Please provide a cover letter to explain, *point by point*, the details

of the revisions to the manuscript and your responses to the referees’

comments.

Response: With thanks. All corrections and explanations on the base of reviewers' comments are mentioned in a cover letter and attached to the revised manuscript online via the journal website. 

(IV) If the reviewer(s) recommended references, please critically analyze

them to ensure that their inclusion would enhance your manuscript. If you

believe these references are unnecessary, you should not include them.

Response: Thank you for your guidance.

(V) If you found it impossible to address certain comments in the review

reports, please include an explanation in your appeal.

Response: Thank you for your guidance.

(VI) The revised version will be sent to the editors and reviewers.

With thank.

Reviewer no. 1

The manuscript was edited for writing and grammatically by an expert and a native of the English language. All corrections were highlighted throughout its text.

1. 1. In the introduction section, it is necessary to clearly explain the background and significance of miRNA-134, BDNF, CREB, and SIRT-1 expressions.

Response: With thanks the introduction was revised and added necessary explanations and backgrounds about miRNA-134, BDNF, CREB, and SIRT-1 expressions were added and highlighted. (Page 3- lines 66-91).

2. Were there any specific criteria for selecting the rats used in the study, particularly regarding age and health status? Why are 60 rats in the old group and only 10 rats in the young generation?

Response: Ok thank you for your interested question. The healthy young and aged females were selected by simple randomization. To determine the sample size a pilot study was done on 5 rats in each group and then using the mean, standard error of mean, variance, and given α-value in a formula the final number of rats in each experimental group was counted. We used only one group of healthy young rats (n=10) and also one group of healthy female old rats (n=10) as normal control group to compare the tested parameters, because we tried to investigate the effects of the aging process on all studied parameters with compare to control young rats. On the other hand, all studied parameters in OVX, OVX-AD, as well as treated groups, were compared statistically with healthy control old rats. A total of 10 rats were used in control young female rats but 60 aged female rats were divided into 6 sub-groups with 10 in each because the rats in each group were divided into 2 sub-groups for biochemical and gene expression (n=6) and histological evaluation (n=4).    

3. Clarify the reasoning behind choosing the specific cognitive behavior tests (NOR, MWM, PAL) used in the study. Were there any alternative tests considered?

Response: Thank you for your question. The AD has a high prevalence among postmenopausal women and is characterized by progressive impairment of memory and other cognitive functions.  The increase in the deposition of Aβ plaques in the brain of postmenopausal women probably indicates the relationship between estrogen deficiency and neurological disorders in middle-aged women. Since during aging and menopause followed by estrogen hormone deficiency in female rats, the synaptic plasticity will disrupt because of gradual neuronal apoptosis in some brain areas involve the cognition so learning and memory impairment will happened. There are different experimental tests for cognitive evaluation in rodents. We selected NOR, MWM and PAL tests for cognitive evaluation because after aging and estrogen deficiency in aged female rats the spatial learning and memory, novel object recognition of new objects and also another type of passive avoidance memory disrupt due to neuronal death and lower electrical signaling in hippocampus and other brain areas.

References:

1. Association, A.s., W. Thies, and L. Bleiler, 2013 Alzheimer's disease facts and figures. Alzheimer's & dementia, 2013. 9(2): p. 208-245.
2. Mielke, M.M., P. Vemuri, and W.A. Rocca, Clinical epidemiology of Alzheimer’s disease: assessing sex and gender differences. Clinical epidemiology, 2014. 6: p. 37.
3. Ross, R.K., et al., Cardiovascular benefits of estrogen replacement therapy. American journal of obstetrics and gynecology, 1989. 160(5): p. 1301-1306.
4. Mosconi, L., et al., Perimenopause and emergence of an Alzheimer’s bioenergetic phenotype in brain and periphery. PloS one, 2017. 12(10): p. e0185926.
5. Mosconi, L., et al., Sex differences in Alzheimer risk: Brain imaging of endocrine vs chronologic aging. Neurology, 2017. 89(13): p. 1382-1390.
6. Li, J. and R.B. Gibbs, Detection of estradiol in rat brain tissues: Contribution of local versus systemic production. Psychoneuroendocrinology, 2019. 102: p. 84-94.
7. 4.  Were any adjustments made for multiple comparisons in the statistical analysis to reduce the risk of type I errors?

 Response: Thank you for your comment. Yes, we have normalized the data and used the one-way and also repeated measures one-way ANOVA followed by Tukey's post hoc test for multiple analyses with application at least 80% power potential of statistic tests.

5. How do this study's findings contribute to our understanding of the underlying mechanisms involved in Alzheimer's disease (AD) and the potential effects of YPT?

 Response: Thank you for your question: The results of the current study showed that in aged and menopausal rats’ neuronal loss (neuronal plaques found in histological tests), cognitive impairment (in behavioral tests), oxidative stress, and alterations in some genes expression have occurred. These are disadvantages for neural normal functions that are followed by dementia type of AD (References). We found that Young plasma therapy could reverse these negative effects significantly.

References:

• Emerging roles of oxidative stress in brain aging and Alzheimer's disease. https://doi.org/10.1016/j.neurobiolaging.2021.07.014

2-     Therapeutic potential of natural molecules against Alzheimer's disease via SIRT1 modulation. https://doi.org/10.1016/j.biopha.2023.114474

3-     Gene- and Gender-Related Decrease in Serum BDNF Levels in Alzheimer’s disease. Int. J. Mol. Sci. 2022, 23(23), 14599; https://doi.org/10.3390/ijms232314599

6. Are there any alternative explanations for the observed effects, and if so, how were they considered in interpreting the results?

Response: With thanks. Yes, we can explain that the ovarian sex hormones especially estrogen have a key role as neuroprotective. Estrogen deficiency after menopause causes down-regulation of estrogen alpha and beta receptors in brain hippocampus and other important areas of the brain for cognition. In the current study, we used the young rat plasma therapy that may contain such hormones and other biochemical molecules and can compensate for those losses. On the other hand in parallel research work, it appeared that the electrical properties of these areas of the brain involved in cognition were disrupted (by long-term potentiation recording and measuring some neurotransmitters such as glutamate and acetylcholine).

7. To what extent can the findings of this study be generalized to other populations, such as humans or different animal models of AD?

 Response: Thank you for your question. Current findings show that aging and estrogen deficiency due to menopause could degenerate the nervous system (sensory, motor areas, and brain integrative functions of these areas in brain) and cause disrupted memory consolidation in humans and rodents. The same impairments could be found in different types of animal models of AD such as AD induced by brain trauma, PD, and induced by injection of the STZ. About YPT, we need to do a clinical trial with our clinical colleagues (physicians) to continue applied research and our findings about YPT to be generalized to humans.

8. What are the next steps in this line of research, and how do you envision this study contributing to future investigations or potential clinical applications?

 Response: with thanks. We can suggest doing the clinical trial with our clinical colleagues (physicians) to continue applied research in such patients. This will be possible by preparing the young plasma from taken blood samples. Because the dignity and preservation of life of the elderly is of great importance.

9. Are there any limitations or areas for improvement identified in this study that could be addressed in future research?

Response: Thank you for a good question. Yes, there are some limitations. Some of these are as follows;

1. We should be careful that the donors of plasma have no diseases.
2. Since the prepared young plasma has different molecules (proteins, enzymes, large molecules ……) it may involve immunity resistance after administration. So needs this status examined before the use of young plasma for treatment.
3. The authors extensively discussed their results with articles published in 2021 and older but failed to discuss articles published in 2022, 2023, and 2024. This concern needs to be addressed.

 Response: Thank you for your attention and notice. With the revision of the manuscript, we added new texts with new references (2021-2024).

Reviewer no. 2:

Major points:

1. A quantification of the steroids in the brain is missing. It would be interesting to see levels of steroids (as well as oxysterols, and sterols) in the brain samples comparing the different groups.

Response: With thanks. The measure of the levels of steroids in brain tissue samples is a limitation of current research. Unfortunately measuring and comparing the levels of steroids, oxysterols, and strolls in the brain samples of different experimental groups were not done because this was not foreseen in the special objectives of our research proposal. It is a good and just idea, we will try to measure and compare the levels of steroids in brain samples in our future projects. Of course in the text of the introduction, based on the references, new explanations have been added about the normal levels of steroid hormones and their changes in the conditions of aging or menopause in laboratory mice, which can be seen as highlights.

2. The method used for oxidative stress quantification is rather weak.

Response: With thanks. To measure oxidative stress factors in rats' brain tissue samples, we used the methods introduced in the valid published papers in this field (References). We used the same method in our previous works, whose articles were published. However, other methods are different in terms of accuracy, but we could not do them due to their high cost.

References;

1. Benzie, I.F. and J. Strain, [2] Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration, in Methods in enzymology. 1999, Elsevier. p. 15-27.
2. Erel, O., A new automated colorimetric method for measuring total oxidant status. Clinical biochemistry, 2005. 38(12): p. 1103-1111.

 Other comments

Page 2: the quality of the graphical abstract should be improved.

Response: Ok, thank you for your attention. The graphic abstract was improved in quality and was replaced.

Line 192: FRAP is mainly used to assess antioxidant capacity.

Response: With thanks. Yes, you are right we also used to assess the antioxidant capacity.

MDA, 4-hydroxynonenal, and oxysterols are solid markers of oxidative stress as well as 8-OHdG/8-Hydroxyguanosine as a marker of DNA oxidative damage.

Response: Yes, you are right. Thank you for your reminder.

MDA and TTG are derived calculations, not first-hand quantifications.

Response:  The MDA and TTG were assayed with using the valid ELISA kits introduced in other published articles.

Line 268, figure 3: the size of the 4 panels could be reduced and composed in one figure.

Response: Thank you for your suggestion. All mentioned figures with you were changed on the base of your guide.

Line 305: figure 4: the four panels of the figure should be reduced in size and composed together in one figure.

Response: Was done.

Line 372: figure 5: the five panels of the figure should be reduced in size and composed together in one figure.

Response: Was done.

Line 459: figure 6: the four panels of the figure should be reduced in size and composed together in one figure.

Response: Was done.

Comments on the Quality of English Language

Language requires moderate revision.

Response: With thanks. The manuscript was edited by an expert and native English language and corrected throughout its text. The corrections were highlighted. 

Reviewer no. 3

The manuscript entitled "Effect of young plasma therapy on cognition, oxidative stress, 2 miRNA-134, BDNF, CREB, and SIRT-1 expressions and neu-3 ronal survey in the hippocampus of aged ovariectomized rats 4 with Alzheimer" aimed to use young plasma therapy (YPT) to improve dementia caused by AD in aged ovariectomized rats. Several concerns arise from reading this manuscript in its current form and the AD model it uses.

Response: With thanks. In the current study, we induced AD in rats with the administration of Aβ_1-42 (5 μg/5 μL/rat, dissolved in distilled water (Sigma Aldrich, St. Louis, MO, USA)) was infused into the lateral ventricles (References)

References;

1. Mosconi, L., et al., Perimenopause and emergence of an Alzheimer’s bioenergetic phenotype in brain and periphery. PloS one, 2017. 12(10): p. e0185926.

The introduction is insufficient because it lacks an adequate context to explain the relationship between the markers searched for. For example, it refers in a very general way to microRNAs but fails to delve into the evidence surrounding mir143, the reason for its choice in particular. 

Response: With thanks the introduction was revised and added necessary explanations and backgrounds about miRNA-134, BDNF, CREB, and SIRT-1 expressions were added and highlighted. (Page 3- lines 66-91).

In general, the rationale of the present manuscript is not adequately described either in the introduction or in the description of results.

Response: In the introduction according to the new references inserted in the text, more explanations have been added which are marked with yellow color. It was revised according to the reviewers' comments. The evidence surrounding mir-143, BDNF, and SIRT-1 with related references were added.  (Page 3- lines 66-91).

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The figures in the revised manuscript are unclear, and some of the markings (for example, A, B, etc.) are not visible. 

Revise the figures, and then the manuscript can be considered for publication.

Author Response

Thank you for your comments.

We revised the figures.

Reviewer 3 Report

Comments and Suggestions for Authors

The background information in the introduction is much improved over the first version. However, there is still a lack of information to support this research and to understand its rationale. Particularly, in the part on microRNAs very little information is given as to why only mir134a was chosen. They also do not mention whether this model of continuous injections of young plasma is comparable to parabiosis models in which two living organisms are surgically joined to share a circulatory system. There is also no mention of the prior history of the dose and isoform of the estradiol used for this study. 

How the results are explained is very unclear, for example in line 267 the authors begin the explanation of their results by saying "As revealed in Figure 2, the CY group had a substantial increase in the value of the discrimination index (DI) in the NOR test compared to other groups". However, this was not the most important point of this first test, the comparison that had to be clear was how treatment with either estradiol or ovariectomy had affected an animal model of "Alzheimer's disease".

The description of the results is confusing and overly technical as it is unclear to the reader what they wanted to obtain from each test and how this related to their hypothesis testing. 

The discussion does not express the limitations of the model and is not presented in an integrated manner but as separate ideas. With the results obtained it is challenging to conclude that "Therefore, plasma therapy may be recommended as a clinical trial for older women with Alzheimer's", this study provides robust information to support this assertion. 

Comments on the Quality of English Language

How western blot gels are presented seems very unreliable. Different treatments or groups were run on different gels and are shown as cutouts in each column. They do not provide supplementary material to clarify how these samples were run. Moreover, some of them appear to be identical only with less contrast. For these reasons, I find these experiments very unreliable. 

Author Response

Please refer to the attachment.

Author Response File: Author Response.pdf