Diabetes Differentially Alters Glial Cells in Different Brain Regions
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Author's
I would only like to draw attention to the need to make minor corrections, the suggestions of which are presented below:
- clarify in Introduction the hypothesis and research objectives to better position the study within the current research landscape, because, as the Authors themselves state in Introduction, such research has already been carried out, so what is the purpose of this research?
- please address the limitations of the study.
best regards
Author Response
We would like to thank each of the reviewers for their insightful comments, which helped us to improve the manuscript. All the changes are highlighted in red throughout the manuscript. Our responses to the comments are provided in italics.
I would only like to draw attention to the need to make minor corrections, the suggestions of which are presented below: -
clarify in Introduction the hypothesis and research objectives to better position the study within the current research landscape, because, as the Authors themselves state in Introduction, such research has already been carried out, so what is the purpose of this research? - please address the limitations of the study
We have clarified the objective of the study in the Introduction and have added a section on limitations at the end of the manuscript.
Reviewer 2 Report
Comments and Suggestions for AuthorsDiabetes mellitus (DM) significantly increases the susceptibility to central nervous system (CNS) diseases, including stroke, vascular dementia, cognitive impairment, and Alzheimer's disease.
Blood brain barrier (BBB) damage based on significant neuroglial dysfunction is a critical neurovascular complication of diabetes mellitus that negatively impacts CNS health and function. From the title of this article, it is clear that the authors wanted to further investigate the morphological and functional basis of this metabolic problem and its central consequences.
However, although the topic is of considerable importance and timeliness, this article is poorly presented and even lacks a description of the technique used to induce brain damage. What type of ischemia is it? How is it induced? etc.
The morphological data from in situ hybridization lack quantification and relative statistical values. Furthermore, the line that anticipates the results on mRNA levels in Fig. 1 should be removed from the introduction.
The photos in Fig. 2 are inserts only at higher magnification, it is necessary to also insert the photo at lower magnification to understand exactly from which side they were selected. Furthermore, the positive GFAP profiles do not seem to me to be the maximum of the possible acquisitions! I think it is not appropriate to insert in that script paragraph 3.3, the first sentence on the data of other previous studies.
The quality of figure 3 is really very poor and not publishable in that way. The same goes for figure 5 and in any case for all the panels it is necessary to show lower magnifications from which they were "sectioned" and shown at higher magnification.
The discussion is bizarre and without a solid thread supported by the data presented.
Author Response
We would like to thank each of the reviewers for their insightful comments, which helped us to improve the manuscript. All the changes are highlighted in red throughout the manuscript. Our responses to the comments are provided in italics.
However, although the topic is of considerable importance and timeliness, this article is poorly presented and even lacks a description of the technique used to induce brain damage. What type of ischemia is it? How is it induced? etc.
We have added additional information on our ischemic model in the method section.
The morphological data from in situ hybridization lack quantification and relative statistical values. Furthermore, the line that anticipates the results on mRNA levels in Fig. 1 should be removed from the introduction.
Fig.1 is removed from the introduction and is placed in the Results as Fig.2. These data are used for morphological and anatomical expression. We do not have access to the original tissue and are unable to provide quantification.
The photos in Fig. 2 are inserts only at higher magnification, it is necessary to also insert the photo at lower magnification to understand exactly from which side they were selected. Furthermore, the positive GFAP profiles do not seem to me to be the maximum of the possible acquisitions!
We have added the lower resolution images in Fig.3 to indicate the area of higher magnification image. We have increased the quality of all images.
I think it is not appropriate to insert in that script paragraph 3.3, the first sentence on the data of other previous studies.
The sentence is removed.
The quality of figure 3 is really very poor and not publishable in that way. The same goes for figure 5 and in any case for all the panels it is necessary to show lower magnifications from which they were "sectioned" and shown at higher magnification.
We have provided original figures for better quality.
The discussion is bizarre and without a solid thread supported by the data presented
The discussion has been rewritten to provide more continuity and support by the data.
Reviewer 3 Report
Comments and Suggestions for AuthorsRashmi Kumari and colleagues' article details how diabetes differentially alters glial cells in different brain regions. In this study, the authors have investigated the changes in baseline activation of astrocytes and microglia and their impact on vascular permeability in various brain regions, and the results show glial cell activation in the diabetic brain causing impaired inflammatory response and/or degenerating cerebral blood vessels which contribute to neuronal cell death upon CNS injury.
The overall quality of the article should be improved, and the following changes should be made.
Figure 1 shows that the resolution is poor, and a high-resolution image should be provided. Also, it would be good if the authors could provide the scale bar in the image.
The scale bar is missing in all the microscopic images. The authors must add the scale bars to each Figure.
In Figures 2 and 3, the authors should add an inset image showing the GFAP-positive astrocytes and Iba-1 positive microglia/macrophage, respectively. The arrow can be added to highlight a particular cell type.
In Figure 5, the current version does not provide clear information about occludin staining in blood vessels. The authors should provide the image on the same scale.
One of the key experiments missing from this study is the confirmation of GFAP and Iba-1 protein expression. The authors should confirm the study's finding by showing the western blots for these proteins, and their quantitative analysis should be compared with the analysis in Figures 2B and 3B. The authors should include the findings of these experiments in the discussion section.
Author Response
We would like to thank each of the reviewers for their insightful comments, which helped us to improve the manuscript. All the changes are highlighted in red throughout the manuscript. Our responses to the comments are provided in italics.
The overall quality of the article should be improved, and the following changes should be made. Figure 1 shows that the resolution is poor, and a high-resolution image should be provided.
The image is replaced now as Fig.2
Also, it would be good if the authors could provide the scale bar in the image. The scale bar is missing in all the microscopic images. The authors must add the scale bars to each Figure.
Scale bars have been added to all Figures.
In Figures 2 and 3, the authors should add an inset image showing the GFAP-positive astrocytes and Iba-1 positive microglia/macrophage, respectively. The arrow can be added to highlight a particular cell type.
Arrows have been added to highlight cell types.
In Figure 5, the current version does not provide clear information about occludin staining in blood vessels. The authors should provide the image on the same scale.
The image on the same scale has been included. More information regarding occludin has been added to the Discussion.
One of the key experiments missing from this study is the confirmation of GFAP and Iba-1 protein expression. The authors should confirm the study's finding by showing the western blots for these proteins, and their quantitative analysis should be compared with the analysis in Figures 2B and 3B. The authors should include the findings of these experiments in the discussion section.
We appreciate that the protein quantification is important for the validation of changes observed in different brain region. However, at this time, we do not have material from different brain regions for the Western blotting, and are unable to include western blots and has been added in limitation section
Reviewer 4 Report
Comments and Suggestions for AuthorsThe manuscript by Kumari et al. with a title “Diabetes Differentially Alter Glial Cells in Different Brain Regions” analyzes astrocyte and microglia activation levels and blood-brain barrier changes using diabetic mice as a model for obesity-induced type 2 diabetes and metabolic syndrome. The manuscript is well written but needs some corrections (see comments below). The methods chosen are appropriate to answer the scientific questions and are well described. The results are mostly clearly presented but still need some corrections. This manuscript provides additional evidence of changes in the diabetic brain, indicates the need of further research, and has translational potential.
Major:
What kind of mutation do the db/db mice have? This should be explained in the introduction and further literature on this mouse strain should be cited.
All Figures: the figure title should be included only in the legend and deleted from the figure body. The description of Figures 4 and 5 should be restructured similarly to Figure 2 and 3 (the text in the figure). A scale bar should be added to all figures.
Results: the glucose level of db/+ and db/db measured as described in methods should be presented in one of the figures as evidence of obesity-induced type 2 diabetes and metabolic syndrome.
Figure 1: the brain images should be enlarged to be easier to read. If Figure 1 is part of the results, it should be transferred to the “Results”
Figure 4 and 5: is it possible to quantify these results?
Please correct all abbreviation and their consistent use throughout the manuscript.
Line 212: For better understanding without reference to previous publications, “ob/ob mice” should be briefly explained
Minor
Abstract, line 17 “the number of activated astrocytes (GFAP) and microglia/macrophage (Iba-1)” should be changed to “the number of activated astrocytes (GFAP-positive) and microglia/macrophage (Iba-1-positive)”
Lines 19-21 should be rephrased as it is somewhat confusing
Abstract: unnecessary abbreviations could be deleted, as they are introduced in the introduction (HI, BBB, CA3, TJs)
Line 37: “PNS” should be explained
Author Response
We would like to thank each of the reviewers for their insightful comments, which helped us to improve the manuscript. All the changes are highlighted in red throughout the manuscript. Our responses to the comments are provided in italics.
Major: What kind of mutation do the db/db mice have? This should be explained in the introduction and further literature on this mouse strain should be cited.
Mutation details are provided and references are cited in the introduction.
All Figures: the figure title should be included only in the legend and deleted from the figure body.
Figure headings are removed and added to legend.
The description of Figures 4 and 5 should be restructured similarly to Figure 2 and 3 (the text in the figure). A scale bar should be added to all figures.
Figs are changed and scale bar is added.
Results: the glucose level of db/+ and db/db measured as described in methods should be presented in one of the figures as evidence of obesity-induced type 2 diabetes and metabolic syndrome.
New Fig.1 is added for the blood glucose and body weight for db/db and db/+ mouse.
Figure 1: the brain images should be enlarged to be easier to read. If Figure 1 is part of the results, it should be transferred to the “Results”
Fig.1 is now fig.2 and enlarged and moved to the result section.
Figure 4 and 5: is it possible to quantify these results?
I appreciate the suggestion for quantifying the result. However, we cannot produce the quantification at this point and has been added in the limitation.
Please correct all abbreviation and their consistent use throughout the manuscript. Line 212: For better understanding without reference to previous publications, “ob/ob mice” should be briefly explained
Ob/ob explanation is added. Consistent abbreviations have been used throughout the manuscript
Minor:
Abstract, line 17 “the number of activated astrocytes (GFAP) and microglia/macrophage (Iba-1)” should be changed to “the number of activated astrocytes (GFAP-positive) and microglia/macrophage (Iba-1-positive)”.
Changes have been made in abstract.
Lines 19-21 should be rephrased as it is somewhat confusing Abstract: unnecessary abbreviations could be deleted, as they are introduced in the introduction (HI, BBB, CA3, TJs) Line 37: “PNS” should be explained
Changes are made in the abstract, abbreviations are deleted from abstract, and “PNS” is defined.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsNow it has been improved
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors have addressed my comments.
Reviewer 4 Report
Comments and Suggestions for AuthorsThe authors have significantly improved the manuscript in accordance with the reviewers’ comments. I have no more concerns.
