Protective Effect and Mechanism of Xbp1s Regulating HBP/O-GlcNAcylation through GFAT1 on Brain Injury after SAH
Round 1
Reviewer 1 Report
The paper addresses a topic of great interest, exploring the possibility of neuroprotective molecules in the course of SAH. There are some problems that authors should address and fix.
Abstract. Here the MCAO technique is mentioned, while SAH is then produced in mice with a totally different methodology. There are some typos and text formatting errors (missing or excessive spaces).
Introduction. In the last sentence on page 2 authors should add “in an animal model of SAH”.
Materials and Methods. 2.1 Animals on page 2 Rats are mentioned instead of mice.
2.2 SAH model. Authors should detail how many mice in the study, how many sham mice, how many SAH mice, how many SAH mice were given Thiamet G.
Discussion. On page 6, when describing SAH outcomes, authors should add cognitive impairment to neeropsychiatric sequelae (the cited reference is still OK). On page 7 it is not clear why the functional anatomy of the hippocampus is explored in this way. moreover, the possibility of treating the entorhinal cortex with improved results of SAH is mentioned, but no study is referred to in this regard. it is also reported that the entorhinal cortex may play a role in both the prevention and treatment of SAH. Again, no studies are referenced to support this claim.
Author Response
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Author Response File: 
Author Response.docx
Reviewer 2 Report
In this manuscript, the authors investigated the roles of the IRE1/XBP1s/O-GlcNAc axis as a target for neuroprotection in subarachnoid hemorrhage (SAH) induced by transient occlusion of the middle cerebral artery (MCAO) in mice. Th authors used Thiamet-G to increase O-GlcNAcylation. They suggested that the IRE1/XBP1 branch of unfolded protein reaction plays a key role in subarachnoid hemorrhage as well as mentioned that IRE1/XBP1 branch is a new idea to regulate protein glycosylation modification, and also provides a promising strategy for clinical perioperative prevention and treatment of SAH. This study is interesting, but the data presentation in this form of the manuscript is poor. However, the authors need to be solved the following issues:
1) Figure 1: No numbering is found in the presented images/data but has been numbered as (a), (b), (c), (d) in the figure legend. Why the brain size in SAH group is smaller than the control group? Please also show the brain image and HE staining for SAH + TG group. The quantitative data for western blot are not consistent with the images.
2) Figure 2: It is very difficult to read the signal of each marker including GFAP as the resolution of images on the right panel is poor. Please replace those images with higher magnification so that the signals can be clearly readable. In figure 2b-f, please provide legends that clearly indicate the control, SAH, and SAH + TG group (shown as white, black or ash bar).
3) Figure 3: Figure numbered as (a), (b), (c) and (d). Why did the authors again number the quantitative data as (c) for O-GlcNAc immunoreactivity, and (d) for apoptosis cell (%)? As it may make confusion to the readers, please correctly and sequentially present the data in this figure and corresponding text in the result section. The quantitative data for western blot are not consistent with the images.
4) There is no figure number and legend for figure shown on page 8. Why did the authors separate this figure from the other figures shown in pages 9-13? After numbering this figure, the authors also should briefly mention it in the text of the manuscript.
5) Please provide dot and bar plot instead of bar plot for all data.
6) The word limit of the abstract should be about 200 words. As the current abstract has more than 300 words and the background is quite long, please properly summarize the abstract. Please correct the following sentence with full form of HBP “About 5% of glucose in human cells goes through (HBP)” (page 1, line 16). Change “udp-glcnac” to “UDP-GlcNAc” (page 1, line 17) as well as please check other abbreviations throughout the manuscript.
7) Authors should measure neurological scores as well as neuronal staining to show protective effects by SAH + Thiamet-G group.
8) Please confirm whether it should be “heteroamine biological pathway (HBP)” or hexosamine biosynthetic pathway (HBP) (page 2, line 51).
9) In figure 3 legend (page 12, line 421; page 13, lines 422-424), why did the authors write “HT22 cell” and “ht22 cell”. Please check all those types of error throughout the manuscript.
10) Provide catalog number for each antibody and material used in this study.
11) Provide full forms of CA1, DG and EC in page 3, line 104 as well as in the legend of figure 2.
12) Please provide references for each methodology in Materials and Methods. Also provide HE staining protocol in Materials and Methods.
13) Provide the number of animals and/or experiments in each figure legend.
14) Please change Thiamet G to Thiamet G (TG) in page 2, line 83. Also change “tg/Tg” to “TG” (page 5, lines 235, 242, 248).
15) Please change “Ab” to “antibody” as well as full form of “pAb” and “mAb” in Materials and Methods.
16) As authors shown mentioned that statistical data have been shown as mean ± SEM, mean ± SD or the median (page 4, lines 198-199) please mention it in each figure legend accordingly.
Comments for author File: 
Comments.pdf
Author Response
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Author Response File: Author Response.docx
Reviewer 3 Report
Before further review, one issue has to be clarified, the SAH model. What happened to the animals after the external carotid was cut? The authors note that SAH was created with a 10s duration
Author Response
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Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
authors revised the manuscript accrding to reviewers' comments
Author Response
Thank you for your valuable feedback on our manuscript. We are grateful for the time and effort you have put into reviewing our work. We have carefully considered your comments and revised our manuscript accordingly. We hope that our revised manuscript meets your expectations and we look forward to your further input.
Reviewer 2 Report
The authors revised the manuscript; however still the writing of manuscript is not so scientific, found some flaws in data consistency and text of the manuscript, and lot of errors in English grammar throughout the manuscript.
1) In the result section for Figure 1 (page 5, lines 27-30), the authors mentioned that “Brain tissue received SAH operation could be seen obvious blood in the subarachnoid space. HE staining of brain tissue showed that there were red blood cells accumulated in the subarachnoid space of SAH group, indicated that the SAH model had achieved success (Fig. 1a, b)”. In the result section, the authors should mention what is the effect of Thiamet-G on brain hemorrhage. The authors also mentioned that “In western blot assay, the expression of P-Perk, Bip, Chop, Xbp1s and ATF6 in Control group and SAH group were different (p<0.01), while the expression of p-Perk, Bip and Chop in SAH group and SAH+ Thiamet-G group were different (p<0.01). It was noteworthy that the difference between Xbp1s and ATF6 was not significant (p>0.01) (Fig. 2a). What does “different” mean? Is it increased or decreased? Why do they put western blot data in figure 2? The arrangement of figures is very poor, including the numbering of figures in the result sections, for example, in the sentence “It was noteworthy that the difference between Xbp1s and ATF6 was not significant (p>0.01)”, the figure number should be “Fig. 2a and 2b)”. The quantitative data of P-perk and ATF6 shown in figure 2a are not consistent with images.
2) The authors did not put animal numbers in the figure legends as I requested in the previous revision.
3) GFAP expression is increased only in CA region by SAH+ Thiamet-G compared to SAH, but not other regions. The text in the result section is not clear regarding this issue. I am not clear what is the relation between SAH + Thiamet-G and astrocyte activation in this study?
Comments for author File: Comments.pdf
Author Response
Dear reviewer,
We appreciate your comments and have carefully considered them in our revisions. We are sorry to hear that you still find the writing of the manuscript lacking scientific rigor and that you have found some flaws in data consistency and the text of the manuscript. We have taken your comments seriously and have made additional revisions to address these issues.
We have carefully considered your comments and suggestions, and have made the necessary revisions to improve the quality and clarity of our paper. Specifically, we have addressed the following issues that you raised in your review:
Regarding your first question, we apologize for any confusion our use of the term "different" may have caused. In the context of our study, "different" refers to a statistically significant change in the parameter being measured. We will make sure to clarify this in the revised manuscript.
In response to your second question .We appreciate your attention to detail and understand the importance of minimizing errors in research. We want to acknowledge that in regards to the protein images, there may be some sensory discrepancies due to the large volume of proteins processed and the need for normalization to an internal reference. We apologize for any confusion this may have caused and recognize that we have limitations in our ability to handle data. We are eager to learn from you and actively work towards correcting any errors. Thank you again for your understanding and for your valuable feedback.
We apologize for the confusion caused by our misunderstanding of your previous request regarding the animal numbers. We thought you only wanted us to indicate the animal numbers in the statistical figures by dots, and we are sorry that our revision did not meet your expectations. We have now added the animal numbers for each group in the figures, as you have requested. We appreciate your attention to detail and thank you for bringing this to our attention.
We also apologize for any confusion caused by the text in the results section regarding the expression of GFAP in different regions. We have revised the text to make it clearer that the increase in GFAP expression was observed only in the CA region in the SAH+Thiamet-G group compared to the SAH group. Regarding the relation between SAH+Thiamet-G and astrocyte activation in our study, we hypothesize that Thiamet-G, an inhibitor of O-GlcNAcase, may have a protective effect on astrocytes in the CA region after SAH, leading to a decrease in astrocyte activation as evidenced by the lower GFAP expression. However, the mechanism behind this protective effect and the specificity of the effect on the CA region require further investigation. GFAP staining is a method that can be used to evaluate the impact of SAH on neural injury. GFAP is a specific cytoskeletal protein highly expressed in astrocytes, and its expression level is considered as an indicator of astrocyte activation and damage. In SAH studies, GFAP staining can be used to evaluate the extent of astrocyte damage, thereby providing insight into the effects of SAH on neural injury.
We hope that these revisions have addressed your concerns and have improved the quality of the manuscript. Please let us know if you have any further comments or suggestions.
Thank you again for your valuable feedback.
Sincerely,
Kefan,Wu
Reviewer 3 Report
The manuscript is of interest because it highlights a new research direction for SAH. Comments:
General: why study the hippocampus, a structure located far away from the lesion site ?
DISCUSSION shall start with the aim of the study the main conclusion of the study
Figure 2, GFAP immunohistochemistry is not clear, doe snot look like GFAP; please provide an enlarged inset for clarification. Similarly, western blot is overexposed, guess how the vaues on the graphs have been calculated because of overexposure
Author Response
Dear Reviewer
Thank you for taking the time to review our manuscript entitled [Protective effect and mechanism of Xbp1s regulating HBPO-GlcNAcylation through GFAT1 on brain injury after SAH]. We appreciate the insightful comments and constructive feedback you provided. Your suggestions have helped us to improve the manuscript.
The hippocampus is a critical brain region that is known to play a crucial role in a variety of cognitive processes, including learning and memory. Although it is located far away from the lesion site, the hippocampus can still be affected by damage to other brain regions or by changes in neural activity patterns that result from the injury. Furthermore, many brain regions are interconnected and form complex networks that enable the brain to perform its various functions. Thus, damage to one brain region can have far-reaching effects on other regions, and understanding the role of the hippocampus in these networks is important for gaining a comprehensive understanding of brain function and dysfunction. Overall, studying the hippocampus can provide valuable insights into the mechanisms underlying cognitive processes and the effects of brain injury on brain function.
Thank you for reminding us to review the requirements for the discussion section. We have carefully revised the manuscript and placed the study's aims at the beginning of the discussion, as you suggested. We appreciate your valuable feedback and are grateful for your time and effort in reviewing our work.
Thank you for your feedback on Figure 2. We apologize for the unclear GFAP immunohistochemistry image and agree that it does not appear to show GFAP staining. We have taken your suggestion and provided an enlarged inset for clarification. We hope that this revision will address your concern and we appreciate your attention to detail in reviewing our work. Thank you for bringing up the possibility that the image quality issue might be due to the PDF compression. We confirm that all the images we submitted were saved in the high-resolution 300dpi format in the original Word document. We will take measures to ensure that the images are properly formatted and uncompressed in the final version of the manuscript. Once again, thank you for your feedback and for your careful review of our work.
Thank you for your feedback on our Western blot results. We acknowledge that overexposure might have affected the values on the graphs and made it challenging to obtain accurate measurements. To mitigate the effects of overexposure, we used image analysis software that is designed to correct for saturation and manually adjusted the background levels to estimate the values. We recognize that these methods can introduce some level of error and might not provide entirely accurate measurements. In future studies, we will optimize the exposure time and signal intensity to obtain more accurate results. We appreciate your attention to detail in reviewing our work and your valuable feedback.
We hope that our revised manuscript meets your expectations and is now suitable for publication in [Biomedicines]. Thank you once again for your valuable input, which has greatly enhanced the quality of our manuscript.
Sincerely,
Kefan, Wu
