Comparison of Retinal Metabolic Activity and Structural Development between rd10 Mice and Normal Mice Using Multiphoton Fluorescence Lifetime Imaging Microscopy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Line 45: "....and specifically study the region of interest." please can you make this clearer this is too vague.

Line 59: you state that there are only two examples of the technique used in eye/ retinal disease- this is not correct there are other eye pathologies some in the retina and some in other ocular tissues where FLIM has been used in vivo, invitro and mounted......

Figure 1: the description of the figure is not sufficient for the reader to comprehend all elements; can you please revise to make it clear what each element is (it is there but doesn't link with the heatmap which needs to be stated how this relates/ localizes with the sections).

 

Results: you state that mice were images at different timepoints in the first 3 months of their life- I presume this was invivo imaging? please clarify.

Results/ discussions: you state that structurally initially the retinas were comparable and later differed significantly: can you please elaborate more on this observation: i.e. how did the thickness change, any particular layer thinning and if so how much or total absence or remodelling?

Author Response

Dear Editor,

We would like to thank you and the reviewers, for reviewing our manuscript. We are grateful for the meticulous and insightful remarks. We have addressed all the comments, and hope that the manuscript meets your expectations for publication.

 

Reviewer 1 comments:

Line 45: "....and specifically study the region of interest." please can you make this clearer this is too vague.

• We have clarified this statement and edited it to say “...generate an optical section of the sample at the desired plane of focus” Please see line 47 in the document that has tracked changes on.

Line 59: you state that there are only two examples of the technique used in eye/ retinal disease- this is not correct there are other eye pathologies some in the retina and some in other ocular tissues where FLIM has been used in vivo, invitro and mounted......

• We performed another literature search, which has yielded further FLIM studies investigating RPE and sub-RPE deposits in AMD eyes. To the best of our knowledge, there have not been other peer-reviewed, published articles studying inherited retinal degeneration using FLIM, though there have been studies published on these topics and other retinal pathologies like hydroxychloroquine retinopathy and AMD using FLIO, a similar imaging modality. We have now edited the statement above and added appropriate sources (please see lines 61-68, which has now been edited to “To date, FLIM eye studies have mostly been performed on healthy, functional eyes. Of the ocular pathologies investigated so far, FLIM has been performed on eyes with hydroxychloroquine retinopathy and age-related macular degeneration. To the best of our knowledge, there have not been other FLIM articles studying inherited retinal degeneration, though this topic has been explored using fluorescence lifetime imaging ophthalmoscopy (FLIO), a similar imaging modality.”

Figure 1: the description of the figure is not sufficient for the reader to comprehend all elements; can you please revise to make it clear what each element is (it is there but doesn't link with the heatmap which needs to be stated how this relates/ localizes with the sections).

• We have rectified this in the revised figure caption.

Results: you state that mice were images at different timepoints in the first 3 months of their life- I presume this was in vivo imaging? please clarify.

• This was in vitro imaging, as the mice were sacrificed, their eyes enucleated, sectioned, then imaged using FLIM, as described in the methodology section. We hope this clarifies Reviewer 1’s query.

Results/ discussions: you state that structurally initially the retinas were comparable and later differed significantly: can you please elaborate more on this observation: i.e. how did the thickness change, any particular layer thinning and if so how much or total absence or remodeling?

• These differences can be appreciated in Figure 2. We have revised the figure legend to further clarify. As mentioned in the figure legend, retinal layers were fully differentiable by P10 in both strains and remained morphologically similar up to P15. After P15, rd10 retinas showed progressive thinning, mainly of the photoreceptors including the inner and outer segments of photoreceptors and the outer nuclear layer. At P56 and later, the rd10 retinas showed almost total loss of photoreceptors, and the boundaries between the different inner retinal layers were harder to distinguish compared to the WT retinas.

Reviewer 2 Report

Comments and Suggestions for Authors

Major Comments:

1. Title and Abstract:
• The title is somewhat lengthy and could be more concise. Consider simplifying it while still conveying the main focus of the study.
• The abstract should provide a clear and concise summary of the study, including key findings and implications. Ensure that it reflects the structure of the paper.
2. Introduction:
• The introduction is comprehensive but could benefit from more explicit statements about the objectives and hypotheses of the study.
• Clearly state the research questions or hypotheses that the study aims to address.
• Consider providing more background on the importance of studying retinal metabolism and its relevance to retinal degeneration.
3. Methods:
• The methods section is well-detailed, but it may be helpful to provide a brief overview at the beginning of the section to guide readers.
• Clarify the rationale for choosing specific time points for sacrifice and enucleation. Why were these time points selected?
4. Results:
• Figures and tables are essential for understanding the results. Ensure that figure captions are comprehensive and explain the key findings.
• In Figure 2, it would be beneficial to include labels or annotations indicating the specific retinal layers being referred to.
5. Discussion:
• Connect the results more explicitly to the existing literature. How do your findings align with or differ from previous studies?
• Discuss potential limitations and how they might impact the interpretation of results.
• Clearly state the clinical or biological relevance of your findings. What implications do your results have for understanding retinal degeneration?
6. Conclusion:
• The conclusion should succinctly summarize the main findings and their significance.

Minor Comments:

1. Check for typographical errors and grammatical issues.
2. Be consistent in the usage of abbreviations and acronyms. Spell out terms upon first use.
3. Consider reorganizing or rephrasing certain sentences for clarity and flow.
4. Provide more context for the figures in the text to aid understanding.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Dear Editor,

We would like to thank you and the reviewers, for reviewing our manuscript. We are grateful for the meticulous and insightful remarks. We have addressed all the comments, and hope that the manuscript meets your expectations for publication.

 

Reviewer 2 comments:

The title is somewhat lengthy and could be more concise. Consider simplifying it while still conveying the main focus of the study. The abstract should provide a clear and concise summary of the study, including key findings and implications. Ensure that it reflects the structure of the paper.

• We agree that the title is long, however, most of it is because of “Multiphoton Fluorescence Lifetime Imaging Microscopy”. We feel the current title is the shortest that can convey the focus of the study. . The abstract is in the format requested by the journal and states the findings, and has been edited to include the study’s implications (please see lines 24-26).

The introduction is comprehensive but could benefit from more explicit statements about the objectives and hypotheses of the study. Clearly state the research questions or hypotheses that the study aims to address. Consider providing more background on the importance of studying retinal metabolism and its relevance to retinal degeneration.

• Please see lines 69-76, which more explicitly state the objectives and research aims of the study. Please also see lines 41-42 which provide additional context for studying retinal metabolic changes in retinal degeneration models.

The methods section is well-detailed, but it may be helpful to provide a brief overview at the beginning of the section to guide readers.

Clarify the rationale for choosing specific time points for sacrifice and enucleation. Why were these time points selected?

• The rationale for choosing these timepoints has been indicated in the Materials and Methods section (please see lines 83-89).

Figures and tables are essential for understanding the results. Ensure that figure captions are comprehensive and explain the key findings.

• We have edited the legends for Figure 1 (see lines 108-121), Figure 2 (see lines 154-162), and Figure 4 (see lines 212-217) for clarification.

In Figure 2, it would be beneficial to include labels or annotations indicating the specific retinal layers being referred to.

• In Figure 2, please see the retinal layers labeled in the P84 WT image, as these layers were the best defined and most obvious. The abbreviations for the layers have been added, please see lines 159-162.

Connect the results more explicitly to the existing literature. How do your findings align with or differ from previous studies?

• Please see lines 251-253: “As consistent with Kooragayala et al.’s findings in P30 rd1 and rd10 mice, OXPHOS (and by proxy respiration) was shown to be elevated in the setting of significant retinal degeneration in almost all time points compared to WT mice.”
• Additionally see lines 254-256: “In prior studies, young retinas were observed to use glycolysis for biosynthesis rather than OXPHOS during proliferation; which is also seen in both normal and cancerous cells” in connection to lines 254-267: “. It is likely that such a phenomenon is reflected in our data as well, with the WT and rd10 mice retinas showing initially decreasing OXPHOS from P0 to P30 and from P0 to P10, respectively (likely in direct exchange for glycolysis), then OXPHOS later increases then plateaus.”
• Please also see lines 268-273: “In our data, WT and rd10 retinas were confirmed to remain structurally the same throughout the first 15 days of development. As per Samardzija et al., and as confirmed in our results, after P15, WT photoreceptor outer segments continue growing till P28, where then their size stays relatively stable. In our rd10 mice, and those in the literature, after P15, photoreceptor outer segments visibly degenerated, and almost completely disappeared by P28.”
• And lastly see lines 284-288: “In terms of comparing the retinal layers (outer versus inner), we noted freer NAD(P)H, or more glycolysis, in the outer retina relative to the inner retina. Our findings were consistent with our previous study and Browne and colleagues’ work in human organoids, which showed more glycolysis in the outer layers and more OXPHOS in the inner layers of the organoids.”

Discuss potential limitations and how they might impact the interpretation of results.

• Please see lines 291-294 for potential limitations and implications.

Clearly state the clinical or biological relevance of your findings. What implications do your results have for understanding retinal degeneration?

• We have added lines 301-303 as a conclusion sentence and to tie our findings back to the bigger picture.

The conclusion should succinctly summarize the main findings and their significance.

• Based on the journal template, there is no separate heading for conclusion. We expanded on the last paragraph of the discussion as suggested.

Check for typographical errors and grammatical issues. Be consistent in the usage of abbreviations and acronyms. Spell out terms upon first use. Consider reorganizing or rephrasing certain sentences for clarity and flow.

• We have looked over the manuscript again for clarity and grammatical errors.

Provide more context for the figures in the text to aid understanding.

• We have updated our Figure 1, 2, and 4 accordingly.

Reviewer 3 Report

Comments and Suggestions for Authors

I carefully and with great interest read the manuscript of Erin Su et al. entitled "Comparison of Retinal Metabolic Activity and Structural Development between rd10 and Normal Mice using Multiphoton Fluorescence Lifetime Imaging Microscopy", in which the work was aimed at investigating the possibility of using the FLIM method to analyze retinal metabolic activity in mice.

In my opinion, the work is performed at a high scientific and technical level, written in an accessible and understandable language. The topic of the work is of high relevance, and understanding the mechanisms of changes in retinal metabolic activity is an important task.

Nevertheless, there are a number of shortcomings in the work, which should be corrected, before publication in the CIMB journal.

 

Remarks:

1) In the Introduction, there is no clearly stated aim of the study.

2) In Materials and Methods on line 68, the dimensionality of the time points is not clear. Later on, of course, it became clear that they are days, but it is better to specify earlier.

3) In Figure 1 on the phasor diagram, there is no scale or divisions. It is not clear what the oval highlighted area and the color scale inside it mean. The scale bar on the microscopic images is very fine. The direction of the glycolysis-OXPHOS shift is not clear. I liked how it was done in [Ryabova, A.; Romanishkin, I.; Skobeltsin, A.; Markova, I.; Pominova, D.; Linkov, K.; Loschenov, V. Detection of Changes in Macrophage Polarization as a Result of 5-Aminolevulinic Acid Photodynamic Therapy Using Fluorescence-Lifetime Imaging Microscopy. In Proceedings of the Photonics; MDPI, 2022; Vol. 9, p. 961].

4) In Figure 2, indicate where the outer and inner retinal regions are.

5) Figure 3 should indicate the standard deviation to make it clear that there is a decreasing/increasing trend rather than a random variation.

6) In my opinion Figures 3, 4, 5, and 6 have a lot of overlap. Perhaps Figures 5 and 6 should be moved to the supplementary materials.

7) Finally, is it possible to digitize the results obtained and not just indicate the trend - decrease in OXPHOS and increase in glycolysis?

8) Line 241 "OXPHS"

 

I believe that once the deficiencies are corrected, the paper can be published in the CIMB journal.

Author Response

Dear Editor,

We would like to thank you and the reviewers, for reviewing our manuscript. We are grateful for the meticulous and insightful remarks. We have addressed all the comments, and hope that the manuscript meets your expectations for publication.

 

Reviewer 3 comments:

In the Introduction, there is no clearly stated aim of the study.

• We have added more explicit research aims to the end of our introduction (please see lines 69-76).

In Materials and Methods on line 68, the dimensionality of the time points is not clear. Later on, of course, it became clear that they are days, but it is better to specify earlier.

• We have now clarified this in line 83.

In Figure 1 on the phasor diagram, there is no scale or divisions. It is not clear what the oval highlighted area and the color scale inside it mean. The scale bar on the microscopic images is very fine. The direction of the glycolysis-OXPHOS shift is not clear. I liked how it was done in [Ryabova, A.; Romanishkin, I.; Skobeltsin, A.; Markova, I.; Pominova, D.; Linkov, K.; Loschenov, V. Detection of Changes in Macrophage Polarization as a Result of 5-Aminolevulinic Acid Photodynamic Therapy Using Fluorescence-Lifetime Imaging Microscopy. In Proceedings of the Photonics; MDPI, 2022; Vol. 9, p. 961].

• Please find our edited figure legend, which we hope provides enough clarification.

4) In Figure 2, indicate where the outer and inner retinal regions are.

• We have added the retinal layers’ abbreviations to the Figure 2 legend (please see lines 155-159). From these layers, the outer retinal region is from the photoreceptor outer segment to the outer nuclear layer, and the inner retinal region includes from the outer plexiform layer to the retinal nerve fiber layer, as mentioned in lines 125-127. We have reiterated this in the Figure 2 legend as well for clarification (please see lines 158-159).

5) Figure 3 should indicate the standard deviation to make it clear that there is a decreasing/increasing trend rather than a random variation.

• We have included the requested standard deviations in Figure 3 as requested here. We have also added additional tables below for your review (please see attachment below) to show that there is a significant change between certain timepoints.

6) In my opinion Figures 3, 4, 5, and 6 have a lot of overlap. Perhaps Figures 5 and 6 should be moved to the supplementary materials.

• We have moved Figures 5 and 6 to Supplementary Materials.

7) Finally, is it possible to digitize the results obtained and not just indicate the trend - decrease in OXPHOS and increase in glycolysis?

• We agree with Reviewer 3 that numerically indicating the bound NAD(P)H values is valuable and have therefore included the numeric values in our supplemental tables.

8) Line 241 "OXPHS"

• We thank Reviewer 3 for catching this mistake! It has now been corrected.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

All comments have been corrected