Corticosterone Excess-Mediated Mitochondrial Damage Induces Hippocampal Neuronal Autophagy in Mice Following Cold Exposure

Round 1

Reviewer 1 Report

This is a well-executed, well presented and comprehensive study documenting the phenomenon of autophagy in the hippocampus of mice following cold exposure. The aim of the study is clearly significant and the description of findings is accurate.

Minor corrections:

1) In the discussion should be explained why the phenomenon of autophagy is more severe in males than in females.

2) Figure 10: Authors should indicate mitochondria with arrows.

3) Figure 12: Authors should use a mouse brain (in box).

4) Keywords: replace mitochondria damage with Mitochondria damage

Author Response

Dear Editor,

Thank you for your work for our manuscript. Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied comments carefully and modified the manuscript accordingly. The changes have been highlighted in yellow throughout the manuscript. We hope the modifications have addressed all the shortcomings outlined.

 

Reviewer #1:

 

This is a well-executed, well presented and comprehensive study documenting the phenomenon of autophagy in the hippocampus of mice following cold exposure. The aim of the study is clearly significant and the description of findings is accurate.

 

Minor corrections:

 

1) In the discussion should be explained why the phenomenon of autophagy is more severe in males than in females.

Reply：Thank you for your professional advice. The contents have added in manuscript.

 

2) Figure 10: Authors should indicate mitochondria with arrows.

Reply：Thank you for your professional advice. Figure 10 has revised.

3) Figure 12: Authors should use a mouse brain (in box).

Reply：Thank you for your professional advice. Figure 12 has revised.

 

4) Keywords: replace mitochondria damage with Mitochondria damage

Reply：Thank you for your professional advice. It has revised.

We tried our best to improve the manuscript and made some changes in the manuscript.These changes will not influence the content and framework of the paper. We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the correction will meet with approval.

Once again, thank you very much for your comments and suggestions.

We look forward to hearing from you soon.

With kind regards,

Yours sincerely,

Huanmin Yang

College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, 163319, P. R. China.

Email: [email protected]; Tel: +86-0459-6819200

 

Reviewer 2 Report

In their manuscript, the Authors have investigated the neurodegenerative role of cold stress by in vitro and in vivo tests. For in vitro experiments the Authors used CORT treatment whit or without RU486 incubation in HT22 cells to investigate the correlation between CORT exposition and autophagy. The in vivo experiments were performed to demonstrate that increases of CORT level, after cold exposure, induced the autophagy machinery resulting in an increased risk of neurodegenerative disorders. The topic is really interesting and the experiments are well designed. For this reason, I suggest only some corrections.
1. During the in vitro tests, the Authors used a CORT stimulation to demonstrate the mitochondrial damage in HT22 cells. Could the Authors explain why they didn't use MTS/MTT assay to study cell viability as a function of time and concentration dependence after CORT administration?
2. In figure 7, JC-1 was used to measure the mitochondrial stress (ΔѰm assay), but this assay needs a fluorescence quantification, in fact, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio.
3. It’s not completely clear the reason why the Authors use only the hippocampus area in the experiments.
4. At the end, the neurodegenerative effect of cold stress requires a stronger evidence. I suggest to make a TUNEL assay to evaluate the neurodegeneration.

Author Response

Dear Editor,

Thank you for your work for our manuscript. Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied comments carefully and modified the manuscript accordingly. The changes have been highlighted in yellow throughout the manuscript. We hope the modifications have addressed all the shortcomings outlined.

 

Reviewer #2:

 

In their manuscript, the Authors have investigated the neurodegenerative role of cold stress by in vitro and in vivo tests. For in vitro experiments the Authors used CORT treatment whit or without RU486 incubation in HT22 cells to investigate the correlation between CORT exposition and autophagy. The in vivo experiments were performed to demonstrate that increases of CORT level, after cold exposure, induced the autophagy machinery resulting in an increased risk of neurodegenerative disorders. The topic is really interesting and the experiments are well designed. For this reason, I suggest only some corrections.

During the in vitro tests, the Authors used a CORT stimulation to demonstrate the mitochondrial damage in HT22 cells. Could the Authors explain why they didn't use MTS/MTT assay to study cell viability as a function of time and concentration dependence after CORT administration?

Reply：Thank you for your valuable advice. In order to investigate the effect on cell states after CORT treated, the cell viability was measured by Annexin V-FITC/propidium iodide (PI) staining, the results indicated the apoptosis cells were significant after CORT treated.

 

In figure 7, JC-1 was used to measure the mitochondrial stress (ΔѰm assay), but this assay needs a fluorescence quantification, in fact, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio.

Reply：Thank you for your professional advice. It has added.

 

It’s not completely clear the reason why the Authors use only the hippocampus area in the experiments.

Reply：Thank you for your professional advice. It has added in the introduction.

 

At the end, the neurodegenerative effect of cold stress requires a stronger evidence. I suggest to make a TUNEL assay to evaluate the neurodegeneration.

Reply：Thank you for your professional advice. This study focus on the response of autophagy in hippocampus of cold stress mice, we would measure more impact on hippocampus from cold stress in future. More than this, TUNAL staining was already published in our previous study[1].

We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper. We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the correction will meet with approval.

Once again, thank you very much for your comments and suggestions.

We look forward to hearing from you soon.

With kind regards,

Yours sincerely,

Huanmin Yang

College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, 163319, P. R. China.

Email: [email protected]; Tel: +86-0459-6819200

 

 

Xu, B., et al., HMGB1-mediated differential response on hippocampal neurotransmitter disorder and neuroinflammation in adolescent male and female mice following cold exposure. Brain, behavior, and immunity, 2019. 76: p. 223-235.