The Profile of Retinal Ganglion Cell Death and Cellular Senescence in Mice with Aging

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study by Wang et al., investigates the profile of RGC survival in mice along aging (from 2 to 18 months). On the basis of the results obtained, the authors conclude that the gradual increase in senescent cells as well as pyroptosis and ferroptosis is the cause of a progressive RGC reduction in mice along aging. The study is of potential interest and results presented in the manuscript provide a support to the conclusion offered by the authors. The study tried to investigate some mechanisms underlying these observations. However, the study remains descriptive and has not addressed a causal link. It is worth considering adding the following content to make the structure of the manuscript more complete.

Major Concerns:

- The authors used C57BL/6J and B6.Cg-Tg(Thy1-YFP)HJrs/J (Thy1-YFP-H) mice (2 – 18-month-old). Why they used two different strains of mice? Furthermore, the authors have to specify the sex of mice used. It would be interesting to evaluate is there is a sex-difference in RGC survival along aging.

- The resolution of several result figures requires improvement. Specifically, figures 3,4,5,6 need enhancement in image clarity. High-resolution images should be provided for publication-quality reproduction.

- Related to possible age-related mechanisms of RGC survival, the following references may be helpful to you:

 - PMID: 22178719

I suggest to improve the Introduction and Discussion section. The rationale for performing such experiments, should be also clearly explained in the text and considered in the discussion, to highlight the specific contribution to this study.

- The RNA sequencing analysis results in Figure 7 and Table 1require improvement in two aspects:

• Enhancement of image resolution for better visualization of plots
• Comprehensive annotation of all significantly differentially regulated pathways.

Author Response

Dear the Reviewer,

We greatly appreciate the comments and suggestions from the reviewers. We have revised the manuscript accordingly with yellow highlights. 

 

Reviewer 1

1. The authors used C57BL/6J and B6.Cg-Tg(Thy1-YFP)HJrs/J (Thy1-YFP-H) mice (2 – 18-month-old). Why they used two different strains of mice? Furthermore, the authors have to specify the sex of mice used. It would be interesting to evaluate is there is a sex-difference in RGC survival along aging.

Response: Thank you for the question. Since C57BL/6J mice cannot show the dendritic morphology of individual RGC, Thy1-YFP-H mice instead were used in the RGC dendrite analyses. Clarification has been supplemented in the revised manuscript (Page 16, line 319). Also, we apologize for the missing information on the sex of the mice, which has been added to the Methods section of the revised manuscript (Page 16, line 317).

 

2. The resolution of several result figures requires improvement. Specifically, figures 3,4,5,6 need enhancement in image clarity. High-resolution images should be provided for publication-quality reproduction.

Response: We apologize for the unclear images. High-resolution images have been separately uploaded to the manuscript submission system.

 

3. Related to possible age-related mechanisms of RGC survival, the following references may be helpful to you: PMID: 22178719

Response: Thank you for the suggestion. PMID 22178719 has been added to the revised manuscript (Reference 17; Page 13, line 256-258).

 

4. I suggest to improve the Introduction and Discussion section. The rationale for performing such experiments, should be also clearly explained in the text and considered in the discussion, to highlight the specific contribution to this study.

Response: Thank you for the suggestion. The Introduction and Discussion sections have been refined accordingly in the revised manuscript (Page 3, line 54 – Page 4, line 62; Page 15, line 299-303).

 

5. The RNA sequencing analysis results in Figure 7 and Table 1require improvement in two aspects: • Enhancement of image resolution for better visualization of plots; • Comprehensive annotation of all significantly differentially regulated pathways.

Response: Thank you for the suggestions. Figure 7 has been refined accordingly in the revised manuscript. For the Table 1, the functional clusters with enrichment score > 1.3 are the statistically significant pathways, which have been all listed in the table.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

I appreciate the opportunity to review your interesting manuscript on profiling retinal ganglion cell (RGC) survival in mice during aging. The study presents valuable observations and data on cellular senescence and neurodegeneration-related pathways. However, there are several areas where the manuscript would benefit from substantial improvements, particularly in presentation, grammar, and overall clarity. Some of the results and interpretations are also not fully convincing in their current form.

Below are my specific comments and suggestions:

1. Use of "along aging":
   Throughout the manuscript—including the title—the phrase "along aging" is repeatedly used. This is not standard in English or scientific writing. I recommend replacing it with more appropriate alternatives such as "during aging," "with aging," or "as aging progresses."

2. Abstract section – Grammar and clarity:
   The abstract contains several grammatical errors and would benefit from careful proofreading. For example, the sentence:
   "The numbers of senescent cells in ganglion cell layer (GCL) were significantly and progressively increased since 8-months"
   should be revised to:
   "The number of senescent cells in the ganglion cell layer (GCL) significantly and progressively increased starting at 8 months of age."

3. Figure 1:
   The current line graph may not effectively represent the data. Converting it to a bar graph could improve clarity and interpretation.

4. Figure 2:
   The OCT image is too small to discern any meaningful differences. Additionally, there is a lack of proper labeling within the figure panels. Were histological data (e.g., H&E staining) included to support the OCT findings? If not, I recommend adding such complementary data.

5. Figure 3:
   The immunofluorescence images indicate a reduction starting at 12 months; however, from visual inspection, there appears to be a decrease as early as 4 or 6 months. Please verify the image interpretations to ensure accuracy and avoid misleading readers. It would also be helpful to include nuclear counterstaining or co-staining with a marker such as Brn3a to improve image interpretation.

6. Figure 5:
   The cleaved caspase-1 signal appears higher at 10 months than at 12 or 14 months, which contradicts the progression trend. Similarly, the 4-HNE staining appears quite similar between 6, 8, and 10 months, though the 8-month sample may show slightly stronger signal. Please re-express this data using bar graphs, which would help clarify these comparisons visually.

In addition, I have suggested several in-line edits and comments within the manuscript, highlighted in yellow. Please refer to the attached annotated version for detailed corrections.

Overall, I believe the manuscript would greatly benefit from improved clarity, conciseness, and grammatical correctness—especially in the results, figure legends, and discussion sections. Enhancing the overall presentation and refining some of the language would significantly improve readability and the impact of your findings.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The manuscript is clear and well-organized for a scientific context, but it contains several grammatical, and clarity issues that can be improved for better readability and professionalism. Proper proofreading of the English language needs to be done before consideration for publication. 

Author Response

Dear the Reviewer,

We greatly appreciate the comments and suggestions from the reviewers. We have revised the manuscript accordingly with yellow highlights. 

 

Reviewer 2

1. Use of "along aging": Throughout the manuscript—including the title—the phrase "along aging" is repeatedly used. This is not standard in English or scientific writing. I recommend replacing it with more appropriate alternatives such as "during aging," "with aging," or "as aging progresses."

Response: Thank you for the comment and suggestion. The phrase “along aging” has been changed throughout the manuscript.

 

2. Abstract section – Grammar and clarity: The abstract contains several grammatical errors and would benefit from careful proofreading. For example, the sentence: "The numbers of senescent cells in ganglion cell layer (GCL) were significantly and progressively increased since 8-months" should be revised to: "The number of senescent cells in the ganglion cell layer (GCL) significantly and progressively increased starting at 8 months of age."

Response: Thank you for the comment. The grammatical errors have been corrected throughout the revised manuscript.

 

3. Figure 1: The current line graph may not effectively represent the data. Converting it to a bar graph could improve clarity and interpretation.

Response: Thank you for the comment. The line graph in Figure 1 has been changed to the bar chart format in the revised manuscript.

 

4. Figure 2: The OCT image is too small to discern any meaningful differences. Additionally, there is a lack of proper labeling within the figure panels. Were histological data (e.g., H&E staining) included to support the OCT findings? If not, I recommend adding such complementary data.

Response: Thank you for the comment. The label for whole retina has been added in the revised Figure 2. The changes in the thickness of ganglion cell complex are small but significant and functionally meaningful (Yao Y et al., Cell Prolif 2024). Our previous study (Yang Y et al., Invest Ophthalmol Vis Sci. 2014) has confirmed the close correlation of the retinal layer thickness measurement in the hematoxylin and eosin staining with that by optical coherence tomography.

 

5. Figure 3: The immunofluorescence images indicate a reduction starting at 12 months; however, from visual inspection, there appears to be a decrease as early as 4 or 6 months. Please verify the image interpretations to ensure accuracy and avoid misleading readers. It would also be helpful to include nuclear counterstaining or co-staining with a marker such as Brn3a to improve image interpretation.

Response: Thank you for the comment. The RGC density was calculated as the average data of 8 fields for each retina and the mean of the results from 5 mice. Also, the βIII-tubulin-stained cells on the upper surface of retina has been confirmed as RGCs by co-staining with Brn3a in our previous study (Cen LP et al., Stem Cells 2018).

 

6. Figure 5: The cleaved caspase-1 signal appears higher at 10 months than at 12 or 14 months, which contradicts the progression trend. Similarly, the 4-HNE staining appears quite similar between 6, 8, and 10 months, though the 8-month sample may show slightly stronger signal. Please re-express this data using bar graphs, which would help clarify these comparisons visually.

Response: We apologize for the confusion. The retinal sections were analyzed by the number of positively stained cells instead of the signal intensities. Detailed information has been supplemented in the Methods section of the revised manuscript (Page 20, line 407-408).

 

7. In addition, I have suggested several in-line edits and comments within the manuscript, highlighted in yellow. Please refer to the attached annotated version for detailed corrections.

Response: Thank you for the comments and corrections. Changes have been made accordingly throughout the revised manuscript.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The dynamics of retinal ganglion cell (RGC) death and cellular senescence is monitored in mice along aging. Although the current data show RGC death and cellular senescence, it is difficult to find the new findings.

Figure 1's each labeling should be well-characterized. Enlarged images to detect RGC are needed. However, this finding is already known.

Figure 2's ganglion cell complex thickness change is not rigorous. It may need more clear imaging analysis with representative images.

Figure 3's RGC density should be provided depending on the regions in the retina. It is not clear how density is measured and where it was calculated. These data are also known factors based on previous literature.

In Figure 5, retinal sections seem not convincing. There is no consistency in each section. Some have lots of cells in the retina; then this aspect is gone in some image but after that time point, cell number is increased. This should be removed and revised to show the clear comparisons. It is better to show one or two main timepoints.

Figure 6 is the same case with Figure 5.

To get a high impact and originality in this topic, factors such as sex, mouse or rat types, or left and right eyes, and functional analyses with brain-eye might be required.

 

 

 

Author Response

Dear the Reviewer,

We greatly appreciate the comments and suggestions from the reviewers. We have revised the manuscript accordingly with yellow highlights. 

 

Reviewer 3

1. Figure 1's each labeling should be well-characterized. Enlarged images to detect RGC are needed. However, this finding is already known.

Response: Thank you for the comment and suggestion. The labels in Figure 1 were refined, and the enlarged image is presented in Supplementary figure 1.

 

2. Figure 2's ganglion cell complex thickness change is not rigorous. It may need more clear imaging analysis with representative images.

Response: Thank you for the comment. The changes in the thickness of ganglion cell complex are small but significant and functionally meaningful (Yao Y et al., Cell Prolif 2024). High-resolution images have been separately uploaded to the manuscript submission system.

 

3. Figure 3's RGC density should be provided depending on the regions in the retina. It is not clear how density is measured and where it was calculated. These data are also known factors based on previous literature.

Response: We apologize for the missing information. The locations of the images taken on the retina, and the calculation of RGC density have been added to the Methods section of the revised manuscript (Page 18, line 368 – Page 19, line 374 and Supplementary figure 4).

 

4. In Figure 5, retinal sections seem not convincing. There is no consistency in each section. Some have lots of cells in the retina; then this aspect is gone in some image but after that time point, cell number is increased. This should be removed and revised to show the clear comparisons. It is better to show one or two main timepoints.

Response: We apologize for the confusion in the images. The images of the main time points have been selected, and Figure 5 has been refined accordingly in the revised manuscript.

 

5. Figure 6 is the same case with Figure 5.

Response: We apologize for the confusion in the images. The images of the main time points have been selected, and Figure 6 has been refined accordingly in the revised manuscript.

 

6. To get a high impact and originality in this topic, factors such as sex, mouse or rat types, or left and right eyes, and functional analyses with brain-eye might be required.

Response: We apologize for the missing information on the sex of the mice, which has been added to the Methods section of the revised manuscript (Page 16, line 317).

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The Authors answered all my queries.

Reviewer 3 Report

Comments and Suggestions for Authors

The comments are partially addressed and still it is difficult to find the new aspect in this model with the current dataset. Those data in the current study have been already known in previous literature.