Brain and Serum Membrane Vesicle (Exosome) Profiles in Experimental Alcohol-Related Brain Degeneration: Forging the Path to Non-Invasive Liquid Biopsy Diagnostics
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript "Temporal Lobe and Serum Exosome Profiles of Alcohol-Related Brain Degeneration: Forging the Path to Non-Invasive Liquid
Biopsy Diagnostics" by Yang et al focused on understanding the exosome profile in response to chronic ethanol exposure in rat model. The topic of the research which is focused on exosomes which are emerging as hotspot for diagnostic and therapeutic purpose. Overall, the presentation and design of the experiments conducted looks fine but there are certain limitations which needs to be addressed to further improve the quality of the manuscript and attract more readers in the field. Please find below my comments,
1. Authors mentioned in Material and Methods that they use both male and female rats for ethanol exposure however none of the results and figure discuss about sex specific differences in exosome profile for oligodendrocyte pathology markers. Please incorporate if any sex differences found in results.
2. Authors isolated the total exosome from Tissue and Serum which contain diverse population of the exosomes from different tissues and cell types. I believe that isolation of brain-derived exosomes (using oligodendrocyte marker) from serum would provide scientifically more strong basis for the authors hypothesis and interest for readers.
3. Another major limitations are the use of ELISA only for exosome characterization and data for all the OGs related markers proteins. How reliable is to depend on ELISA only for all protein marker quantification?
At least western blot for OGs markers that found to significantly altered in ELISA for TL and serum exosomes need to be included.
Comments on the Quality of English LanguageMinor changes may be required.
Author Response
Please see the attached file containing point-by-point responses to comments.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors try to study the chronic alcohol induced Temporal Lobe and serum exosome profile, which may be related to pathological changes in human alcohol induced neurodegeneration. Overall, findings from the current study have not contributed any novel information to enhance our understanding of alcohol related neurodegeneration.
There are some concerns on this study:
1, It will be questionable whether 8 weeks alcohol treatment of rats can be a good model for chronic human alcohol related neurodegeneration. The authors should provide supporting information to validate that their model is excellent and reasonable. Is there any degenerative phenotype in their rat models under alcohol treatment? The authors also need to explain why they select these proteins as the marker in their study.
2, The protein marker changes in TL, TL and serum exosome are mainly detected by Elisa protocol. However, western blot analysis and quantitative real time RT-PCR are suggested to monitor the changes of protein markers in their samples.
3, why only Temporal Lobe brain tissue is studied? The authors should explain on it.
4, The TL exosome is from TL brain tissue. However, these brain tissue derived exosome may be the intercellular vesicles with similar size of exosome. They may not be called exosome. They should be called endosome. One way it to study the exosome in CSF, which will be better than the endosome inside brain tissue.
Author Response
Please see the attachment for the point-by-point responses.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe study examined serum exosome, temporal lobe tissue, and temporal lobe exosomes from rats given 8 week access to a liquid diet containing 0% or 37% ethanol derived calories, specifically assessing oligodendrocyte glycoprotein, astrocyte, and oxidative stress markers. Many markers were decreased in TL, some were increased and some decreased in TL exosomes, and some were decreased in serum exosomes with relatively little concordance across tissues. The findings are novel but there are concerns about experimental design (specifically, combining data from males and females) and statistics; there are 2 sexes, 2 groups, 3 tissues examined, and multiple readouts per tissue –repeated measures t-test does not seem to be the correct choice.
The introduction lacks rationale for specifically looking at temporal lobe in rats. This region is a target in humans but is it also in rats?
No results are reported for food consumption. Do rats change food intake when given a liquid diet? Or when alcohol is added to the diet?
Do the authors know if HSP70 expression is the same in males and females and if its expression is altered by alcohol?
It looks like all data from males and females were combined – is it known that none of the outcomes are different between sexes? A lot of the data looks bimodal, it would be good to consider the source of this variability.
Author Response
Please see the attachment for the point-by-point responses to the critique.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript quality and presentation improved after incorporating the reviewers comments and suggestions.
Author Response
No further corrections were suggested.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe revised manuscript has been improved significantly. so can be accepted in the current form.
Author Response
No further changes were suggested. The revised manuscript was deemed acceptable.
Reviewer 3 Report
Comments and Suggestions for AuthorsAs noted in the initial review, there are multiple readouts from each sample - at the least, the statistical outcomes should be adjusted for these multiple comparisons.
Line 273 – were the animals “leaner” or did they weigh less?
Table 3 needs some explanation of what the numbers represent (as is done for Table 4)
Author Response
Results were analyzed using repeated-measures T-tests to assess individual effects of ethanol on each analyte and sample source, and by repeated measures ANOVA to compare ethanol’s effects across the spectrum of analytes within the same sample sets. The False Discovery Rate for ANOVA was set at 5%. [added from Line 256]. We have also eliminated statistical trendwise results previously shown in the graphs.
Line 273: We have corrected the sentence to indicate that the rats fed liquid diets weighed less, omitting any comment about leanness. Also, in the legend to Figure S4, we included the diet compositions and noted that the rats fed chow were larger, avoiding comments on adiposity.
The missing headings for Table 3 have been added to explain the tabulated data.
