NMOSD IgG Impact Retinal Cells in Murine Retinal Explants

Round 1

Reviewer 1 Report

 

In the present study, the authors investigate the capacity that IgG-anti AQP4, NMOSD-IgG, has to damage retinal cells and produce retinopathy. They used as an experimental model, retinal explants from mice that were kept in culture for 7 days and then exposed to NMOSD-IgG or IgG from controls (health individuals) for 1 or 3 days.

Binding of IgG to AQP4 protein was detected when NMOSD patient samples were used, but was not detected in healthy control patients. An increase in GFAP signal was observed in NMOSD IgG samples, indicating a certain degree of retinopathy for the authors. In that case, but not in the control, levels of certain chemokines were detected, while no changes in complement protein levels were detected. From these experiments, the authors conclude that in deep, IgG-NMOSD produce damage of the retinal cell that is produced in part by an increase in chemokines, but independent of the activation of complement proteins.

 

The study is interesting, experiments are well addressed, although results shown in figure 3 (panels b and c) and figure 5, are imposible to evaluate. Pannels look completely black, making it impossible to evaluate in detail the information shown on them. I would like to see these figures in an appropriate form.

The only parameter indicative of 'retinopathy' damage in retinal cells is the increase in GFAP expression levels, and that would not necessarily be the case. So, more caution is needed to indicate these results.

In general the study is very straight forward, and conclusions can be sustained on results shown.

 

After revising these minor considerations, I think the manuscript could be accepted.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Wolf et al. realized a very interesting article describing the “NMOSD IgG Impact Retinal Cells in Murine Retinal Explants”.  The topic is relevant, and the results add incremental knowledge to the field. However, there are some limitations:

• The topic of NMOSD IgG's impact on retinal cells seems interesting and relevant, studying disease mechanisms in retinal explants could provide useful insights.
• The authors use appropriate methods like immunohistochemistry, PCR, and ELISA to characterize retinal explants and measure markers of interest.
• The results suggest NMOSD IgG binding alters gliosis, chemokine levels, but not complement mRNA in mouse retinal explants. This hints at interesting effects on retinal physiology.
• Sample sizes seem small (5 NMOSD patient samples, 10-11 explants per group) so the results may be considered preliminary. Larger studies would lend more confidence.
• Some concerns about relying just on marker co-expression to definitively identify "transitional" cell populations - this is suggestive but not conclusive evidence.
• The writing seems clear overall but could be tightened in places to avoid redundancy between figures, results text, and discussion.
• The relevance of the in vitro chemokine experiments to the human disease context needs clarifying.

 

Here are some suggestions for revisions that could help strengthen the manuscript:

Introduction

• Provide more background on NMOSD pathogenesis and the rationale for studying IgG effects on retinal cells specifically. Why focus on the retina?
• Expand on prior evidence of NMOSD IgG binding and damaging rodent retinal cells to build rationale.

Methods

• Provide more details on the patient IgG samples used - demographics, disease status, treatments, etc.
• Explain the relevance of the in vitro concentrations/conditions used for the chemokine experiments with regards to the human disease state.

Results

• Include negative control stainings and quantitation for the immunohistochemistry to document specificity.
• Streamline the descriptions of staining results to avoid repetitiveness.
• For PCR data, show individual data points rather than just bar graphs.

Discussion

• Discuss limitations of relying solely on marker co-expression as evidence for transitional phenotypes. Suggest future functional studies.
• Relate the in vitro chemokine results back to the human disease context. Are the conditions reflective of NMOSD pathogenesis?
• Discuss caveats of the mouse retinal model for studying human IgG effects.
• Shorten repetitive portions summarizing the results. Focus more on interpretation, implications, and future directions.

·       Furthermore, I suggest adding data related to recent bulk transcriptomics studies which could represent a strong substrate to enforce the role of described molecular mechanisms, such as the recent PMID: 36290689, PMID: 36490268 and PMID: 32184807.

 

In summary, providing more context for methods/results, tightened writing, and expanded interpretation could help strengthen the conclusions and impact of this study.

The English language requires several important revisions.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Re: NMOSD IgG impact retinal cells in murine retinal explants
Special Issue: Molecular Mechanism and Regulation in Neuroinflammation

Manuscript ID: cimb-2539071

 

 

Dear authors

The current study reported the potential NMOSD IgG mediated primary retinopathy, and it was found that IgG from NMOSD patients specifically interacts with proteins in the mouse retina. This interaction leads to increased GFAP expression in Müller cells. The study is important in its field and well-constructed, however I would like to offer a few points for your consideration, which I hope will be helpful in further enhancing its quality. Please address point by point.

 

1.    The results of the study are based on a small sample (n-values un the current study is very small), please address these limitations in detail.

2.    In figure number 1 b, please make sure that the part of the statistical significance of the asterisk along with the line is like all the other figures in the article

3.    Figure 3b, Figure 3c and Figure 5 completely dark – impossible to distinguish the an error bar graph (standard deviation bar) and the statistical significant results )asterisk(.

4.    I deeply value including the clinical implications of the research. Clinical implications of the research results should be added in detail in the discussion chapter.

 

Minor editing of English language 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Thanks for revising the manuscript. I find it now well for publishing.

Reviewer 2 Report

The authors addressed all suggested points.

The English is now really improved.