Involvement of RhoA/ROCK Signaling Pathway in Methamphetamine-Induced Blood-Brain Barrier Disruption

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Major comments

1) It is already known that METH is related to RhoA/ROCK pathway (Ref. 22). The authors should include more information in the main text (introduction and discussion) about the significance of using HBMEC to verify BBB dysfunction and cytoskeletal reorganization.

2) The brightness of the bands in Fig. 3 should be quantified.

3) It seems that METH exposure promotes a drastic change in the morphology of HBMECs. (For example, see Fig. 2D, E and Fig. 4C, D.) If possible, it would be better to compare not only 3D plot images but also quantified data such as cell size distribution.

Minor comments

1) Please write the number of experiments in the legend of figures and make it a scatter plot of all data.

2) Page 6, line 7: Please change Fig. 5C to Fig. 5C, D.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Hwang et al. investigate in this manuscript the effects of the well-known drug of abuse Methamphetamine in a human blood-brain barrier model based on hBMECs, thereby recapitulating mostly, what has been published already e.g. in 2019 by Xue et al. in rat BMECs.

Overall this manuscript suffers a lot from the lack of experimental details which have been omitted from the figure legends and also the materials and methods section, which only give a bare minimum of technical information. This makes it impossible to thoroughly evaluate the scientific quality and reliability of the presented data and the authors' conclusions drawn from them.

Hence I cannot recommend this manuscript for publication in its present form. Reconsideration after thorough and extensive improvements might be possible.

 

Specific comments:

1) Figure legend 1: Details about the applied statistical tests should be included in every figure legend, not only in the materials&methods. Obviously all data were normalized to controls - also this information is missing and must be added to the figure legend. It is also not clearly stated what the "control" was - untreated cells or mock treated. Which statistical parameter do the "bars" in the graphs indicate? How many biological and technical replicates are included in the analyses (N=... n=...)?

1C: The authors only show the normalized values of the measured TEER. The absolute values should be shown - or mentioned in the results section - as well. It is also not clear how the TEER had been calculated, ohm*cm2?

In 1D the "permeability" is shown as fluorescence values, normalized to controls. Usually the permeability coefficient is calculated from such experiments, taking also into account the permeation of the substrate through blank membranes!

 

2) Figure 2: I don't see, which information one could retrieve from the 3D plot in (c) - I suggest to delete 2C and enlarge the other images instead for better visibility.

3) Figure 3: I appreciate, that the authors supplied at least the partially cut blots which have been included in Figure3, but how did the biological replicate samples look like - these should be provided as well. Why is the p-MLC band in the control in 3D much stronger, as compared to the control in 3C?

The authors make several claims in the results section about increases/decrease of phospho-levels based on single Western blots shown in Figure 3. The blots of the three independent experiments must be quantified and normalized to total protein levels + statistics, in order to make valid assumptions about up/down regulations of signal intensities.

4) Figure 4: Same applies as in Figure 2 - the 3D plot does not reveal other information, than what is directly visible in the IF images... can be deleted.

5) Figure 5: The description of the mouse experiment is extremely sloppy. Neither  number of animals is provided, nor mouse strain, age, etc etc. Also the Y-27632 control  animals are missing.

 

Materials and Methods:

4.1.: Reference for anti-Claudin-5 is missing.

4.5.: How exactely were the TEER values calculated? How many inserts per condition, how many independent experiments?

Comments on the Quality of English Language

The language in large parts of the manuscript is comprehensible, but several spelling- and orthographic mistakes have to be corrected.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

 

Study describes effects of methamphetamine on brain endothelial cells using functional assays (permeability, TEER), western blot for quantitation of tight junction proteins, immunocytochemistry for TJ protein and cytoskeleton, detection of Rho and MLCK activities and short in vivo experiment to evaluate permeability of blood brain barrier in animals. Authors used two Rho inhibitors to prove its involvement in effects of methamphetamine. Study has a number of issues, most importantly it repeats prior study using the same methodologies and outcomes.

1.     There is no quantitative assessment of western blots or imaging studies, which makes interpretation of results suboptimal.

2.     Doses of methamphetamine are very high and have no meaningful pathophysiologic effects of it in humans.

3.     There is no analysis of all changes in dynamics which does not allow evaluation of changes over period of time.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I appreciate the authors' efforts to improve the manuscript, in particular the scientifically appropriate description of applied materials and methods.

In the Abstract there's a typing mistake: "zonula occluden-1". The correct name is "zonula occludens-1".

Otherwise I have no further comments and recommend the manuscript for publication in the J Biomolecules.