Differential Effects of Ischemia and Inflammation on Plasma-Derived Extracellular Vesicle Characteristics and Function in a Mouse Model
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript investigates EV characteristics after stroke vs systemic inflammation in a well characterised animal model. The results show differences in the characteristics of the EV that are generated in these two pathologies but that they have similar functional effects on recipient cells.
Comments
"EV" have become "Ev" in many places in the text
10^10 have become 1010 in many places in the text
ICAM should be ICAM-1; VCAM should be VCAM-1; IL-1 should be IL-1ß
More detail is required for the EM methodology - instrument used
Author Response
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Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe study of Prof. Yvonne Couch focuses on determining the quantity and antigenic composition of extracellular vesicles collected from the blood plasma of mice after arterial clamping or induced inflammation. It also examines the effects of injecting a vesicle suspension into the brain on the animal's condition. Although the results of this study are more of a negative/confirmatory nature (no significant effects were observed), such research is still valuable as it contributes to the body of scientific knowledge. I have a few minor comments regarding the article.
- The work uses the most up-to-date methods for working with vesicles, but extracellular vesicles themselves are so artifact-ridden that it is impossible to avoid misinterpretation. The authors provide a detailed discussion of all possible sources of artifacts, which I believe confuses the reader. Therefore, I recommend that the discussion be significantly shortened, focusing only on the extent to which each result can be trusted.
- It's clear that in many cases, the use of animal models is the only option for studying a system. However, these are experimental models, not full-scale human studies, and should therefore be clearly identified in the title. It's also worth indicating in the abstract which animal models were used. Since the main obstacle to studying extracellular vesicles is the methodological approach (cytometry sees only large vesicles, microscopy sees too few vesicles for statistics), it is imperative to mention it in the Abstract.
- It is impossible to make out what exactly is shown in Figure 2. Is it the mean fluorescence value minus the background? How correct is it to compare the fluorescence values of different dyes? The colors of different points cannot be distinguished. Please consider rearranging the Figure to make it more informative
Figure 4: what do “Contra” and “Ipsi” labels mean? Please correct
Line 91 “cell culture supernatant”
In some cases “EVs”, and in some cases “Evs” is used to denote extracellular vesicles, please, chose one.
Author Response
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Reviewer 3 Report
Comments and Suggestions for AuthorsThis article focuses on the characteristics and functional differences of extracellular vesicles (EVS) under different injury situations, providing basic data for the study of stroke related EVs, which has certain academic value. However, there are some problems in the article, which should be revised.
- "EVs" and "EVs", "MCAO" and "MCAO" are mixed in the text. It is suggested to unify the abbreviation format.
- The separation purity of EVs in this paper directly affects the quantitative accuracy of NTA and the interpretation of the results of subsequent functional experiments. It is suggested to supplement the experiments related to the purity identification of EVs.
- For some experiments in this paper, the sample size of each group is not uniform, which may have an impact on the interpretation of the experimental results. It is recommended to unify the experimental sample size.
- Figure 4 is marked with "IL-1 β", but the experimental results and annotations are written with "IL-1". At the same time, the subheadings and annotations of the histogram in Figure 5 are only partially marked, and the information is incomplete, which affects understanding. It is recommended to check and confirm the modification of the expression and supplement the figure notes.
- The experimental grouping information is missing in the text, and it is suggested to supplement it at the beginning of "materials and methods" or "results".
- In stereotactic surgery, there is a lack of basis for determining the dose of EVs, which is recommended to be supplemented.
- In the method, the PCR primer sequence "(5 '3-') is incorrectly expressed, which is recommended to be modified.
- More references in the past five years should be cited in the references, and the citation format of some literatures is not unified. It is recommended to supplement the literatures in the past five years and unify the document format.
Author Response
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Reviewer 4 Report
Comments and Suggestions for Authorsijms-4054742
The submitted manuscript, “Differential effects of ischemia and inflammation on plasma-derived extracellular vesicle characteristics and function,” addresses a relevant topic and reports extensive experimental work. However, the manuscript has weaknesses in methodology and data presentation. Before I can recommend acceptance of this manuscript to the publication, I will ask the author for revision of findings, mainly in immunohistochemistry (image quality) and EV analysis.
MAJOR COMMENTS
1. Missing Controls
Claims of elevated ICAM reactivity lack adequate comparative evidence. The author should include visualisation against other endothelial markers (e.g., CD31, CD34) or negative controls to rule out technical artefacts or inherent staining variation between brain regions and specimens.
2. Image Quality and Interpretation
The six GFAP images presented in Figure 6 do not demonstrate clear staining or meaningful differences between groups. Similarly, microglial reactivity (Iba-1) images lack resolution to assess specific cellular identity. If the author intends to demonstrate no significant difference, a single representative image with an explicit statement would be more efficient.
It would be beneficial if the author employed immunofluorescence with confocal microscopy for quantification and clear cellular identification (e.g. colocalisation). Furthermore, high-magnification detail images (40× or 60×) demonstrating cellular and subcellular antigen localisation should be included.
3. DAB visualisation
The author uses immunohistochemistry with DAB visualisation and attempts densitometric quantification on this inherently qualitative method. This approach risks producing misleading results, as pixel intensity measurements capture non-specific staining and background noise rather than the true biological signal.
The author should use quantification methods appropriate for immunohistochemistry (e.g., cell density, integrated density per cell) with proper normalisation. If quantitative analysis is retained, please clarify the sampling strategy: Were regions selected blindly? Were identical settings used for all compared images? Provide statistical analysis with appropriate controls.
4. EV analysis by NTA vs. DLS
The author employs only simple scatter‑mode NTA to characterise EVs after SEC. Although acceptable under the current MISEV 2023 guidelines, scatter‑mode NTA alone is not fully adequate as a stand‑alone method for an EV‑focused manuscript. It is highly sensitive to PBS quality and background particles, and SEC does not achieve absolute EV purity. Complementary fluorescence‑mode NTA (even with simple membrane staining) would enhance specificity and reduce ambiguity in quantification. Please acknowledge the limitations of scatter‑mode NTA in the discussion section..
Nota bene, in Figure 1 (L187), EVs were characterised by DLS. DLS is not mentioned in the M&M section.
Figure A1. The legend explaining the red and blue bars is missing. I assume that particle concentration was measured by NTA in scatter mode. Am I right? Or DLS was used? How were 200 µL fractions collected? Did you use a fraction collector? Information is missing in the M&M section.
MINOR COMMENTS
5. Abstract
The abstract contains a conceptual error: "lack of distinct glial and astrocyte reactivity" (L29) is imprecise, as glia is an umbrella term encompassing astrocytes and microglia. The correct phrasing should specify "microglial and astrocyte reactivity."
Additionally, the abstract states proinflammatory cytokines (L28) were induced but fails to specify which cytokines.
L26-28: The term “expression” appears twice in succession (“induced expression of pro-inflammatory cytokine gene expression”). Please revise.
6. Abbreviations
Abbreviation sections must be completely revised to include abbreviations used in the manuscript, and irrelevant abbreviations, such as MDPI, should be omitted.
7. References
MISEV 2018 vs 2023. Why does the Author refer to the outdated guidelines (MISEV 2018 Ref 23) if recent guidelines (MISEV 2023) are already included as reference 12?
8. Clarity Notes
Figure 1 A & B: Is "Vehicles" a typographical error in the legend?
L253: The sentence appears incomplete as the scale bar length is not specified.
Please provide complete information about the LPS used (manufacturer, bacterial origin, etc.) in the M&M section.
9. Formal Notes
Add spaces between numbers and units throughout (e.g., L91: "50-200 nm" not "50-200nm")
Correct all indexes and exponents to proper superscripts (e.g., 10¹⁰ particles/mL, not 1010).
L650: Correct "200um" to "200 µm".
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsThe paper has been revised according to the requirements and is ready for publication.
Author Response
Comments: The paper has been revised according to the requirements and is ready for publication.
Response: Thanks very much.
Reviewer 4 Report
Comments and Suggestions for Authorsijms-4054742 – V2
The revised version of the manuscript, “Differential effects of ischemia and inflammation on plasma-derived extracellular vesicle characteristics and function in a mouse model,” corrected minor and formal errors in the original version. However, the manuscript still has serious weaknesses in histological methodology and the presentation of immunohistochemical results.
The author did not address my major comments (No. 1-3) regarding immunohistochemistry correctly. In the current state, the entire histology section (Figure 6) does not support any of the conclusions made in this work. Without a clear demonstration of stained cells using images at appropriate magnification, histology cannot be used as the author did, i.e., simply by counting coloured pixels. Furthermore, the author did not prove that the images used for this quantification were taken from comparable microscope fields of view, although within a similar brain location (Figure A4). The striatum is a complex structure, and every fraction of a millimetre can make a difference in the ultrastructure of brain tissue. For pure quantification of ICAM, GFAP and Iba-1 in the striatum, other quantitative methods, e.g. WB, would be more appropriate. Due to serious weaknesses in histology, I cannot recommend this work for publication.
Author Response
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Author Response File: Author Response.pdf
Round 3
Reviewer 4 Report
Comments and Suggestions for Authorsijms-4054742 – V3
In the 3rd revised version of the manuscript, “Differential effects of ischemia and inflammation on plasma-derived extracellular vesicle characteristics and function in a mouse model,” the author partially corrected the problematic Figure 6 (by simply zooming up). I agree with the reply cover letter, and the selected examples of published works are correct. However, even in this version of the manuscript, the micrographs in Figure 6 are hardly readable, and I cannot recommend this manuscript for publication at this stage.
In order to substantiate the core statement, i.e. that there are no differences between the experimental groups, it is essential to show the histological images that were actually used for quantification in a comparable resolution and quality to those presented, for example, in Arenas et al. 2024 [1]. I therefore kindly ask you to provide high-resolution histology micrographs with clearly readable DAB (brown) staining and visible scale bars (for example, 50 µm, as in Figures 1,2,3,5 etc in Arenas et al., 2024 [1] or Figures 6 and 9 in [2] ), so that staining specificity and tissue morphology can be independently assessed. These images could be incorporated either by enlarging (and/or splitting) Figure 6 in the main manuscript or, if space is limited, by providing the analysed fields as supplementary material.
In the current version of the manuscript, I can only speculate about the micrographs:
(1) Figure 6A (ICAM): Poor-quality images showing blurred low to moderate reactivity in the vascular wall. The apparent brown signal depends on the number of vascular structures captured in each image, which compromises the image's interpretability.
(2) Figure 6A (GFAP): I can not see a specific staining in any of those images.
(3) Figure 6A (Iba-1): shows perhaps some brown staining in the LPS group, but the image quality is too low to be sure. However, the apparent contrast in the LPS image does not align with the data in Figure 6F, further complicating interpretation.
(4) Figure 4A predominantly depicts the striatum with partial inclusion of the corpus callosum in the upper region. In several images, a tangentially sectioned vessel with a constitutively higher astrocyte density forming the blood-brain-barrier is visible. If those images were used for quantitative comparison of the DAB signal between, there is a bias potential.
(5) Figure A5 is the only panel where specific staining of ICAM1, GFAP and Iba-1 appears evident; however, according to the description, these images were not included in the quantification… Why? From which brain region and which animal group were they taken?
Since the Author concludes that there is no difference between the groups, it is particularly important to demonstrate that the used method is sensitive, the quantified staining is specific, background is subtracted, and the images used for analysis are of sufficient quality and morphologically comparable to support this conclusion. Without clear, high-quality images at an appropriate resolution, I cannot recommend this manuscript for publication.
[1] Arenas YM, López-Gramaje A, Montoliu C, Llansola M, Felipo V. Increased levels and activation of the IL-17 receptor in microglia contribute to enhanced neuroinflammation in cerebellum of hyperammonemic rats. Biol Res. 2024 Apr 27;57(1):18. doi: 10.1186/s40659-024-00504-2. PMID: 38671534; PMCID: PMC11055256.
[2] Arenas YM, Montoliu C, Llansola M, Felipo V. A transient blood IL-17 increase triggers neuroinflammation in cerebellum and motor incoordination in hyperammonemic rats. J Neuroinflammation. 2024 Nov 30;21(1):314. doi: 10.1186/s12974-024-03310-5. PMID: 39616376; PMCID: PMC11608494.
