PONE-D-23-43281Contrast enhanced longitudinal changes observed in an experimental bleomycin-induced lung fibrosis rat model by radial DCE-MRI at 9.4TPLOS ONE

Dear Dr. in 't Zandt,

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, René in ’t Zandt et al describe the application of DCE-MRI to a rat model of bleomycin-induced lung injury. The experiments and analyses were performed well and carefully. The work is novel, as far as I’m aware, no publication exists yet concerning DCE-MRI applied to a bleomycin model.

Major comments

-Animals: 15 rats were used in the study (line 99). In figure 2, data from 6 rats are shown as controls. Therefore, I presume that 9 rats received bleomycin. However, in figure 2, data from 6 rats were summarized at day 7 and data from 5 rats were summarized at day 28. I suppose that the same animals were measured at both time points after bleomycin. Thus, why data from only 6 respectively 5 rats were summarized instead of 9? More clarity about the experimental procedure should be provided regarding this point.

-Figure 2: Enhancement factor is by definition a factor and does not have units. Therefore, a.u. (arbitrary units) should be omitted. Also, the quality of the graphs in the pdf document was poor – increasing the font size and the quality of the graphs reproduction would be welcome.

-The colors are confusing. In fig. 2a the rectangles around the images are green, blue and purple for control, bleomycin day 7 and bleomycin day 28, respectively. However, in fig. 2b, the colors are green, purple and blue for control, bleomycin day 7 and bleomycin day 28, respectively. Please adapt the colors. In order to better discriminate control curves, I suggest plotting them with black fonts.

-Lines 272-273: The fact that the tissue remodeling detected by MRI occurs primarily around main airways has been shown earlier and confirmed histologically by comparison observations in the same regions (doi 10.1002/jmri.22476).

-Signal intensity of the tissue remodeling area around main airways, determined from images before the contrast agent injection, should also be provided. Analysis should be compared with the DCE-MRI results. It might be the case that both measures might be able to discriminate between the inflammatory and more fibrotic (tissue remodeling) phases of the bleomycin model.

-Figure 2B,C: It is not clear to me whether the curves represent the mean of an average of signal over the whole lung in every animal, or whether they are the mean of the signal over the lesions in every rat. I rather suppose that it is the first, as the mean profiles look the same in control and bleomycin animals (except obviously for the peak), which is surprising to me. I would expect the washout period in bleomycin rats to be different (longer) than in controls. I would strongly suggest to perform analyses in the areas around main airways as well, where most of the remodeling occurred, and present the results in a separate figure. Please clarify.

-Figure 3: The same comment concerning color coding made above for figure 2 holds true here.

-Figure 3B: If I understood correctly the distribution of the clusters and compare to the images shown in figure 3A, does it mean that the higher cluster volumes for lower enhancement factors at day 28 after bleomycin are related to diffuse fibrosis? This would be extremely interesting – especially when testing a therapy and comparing the results to the stronger remodeling around main airways in the central lung. Please clarify.

-Discussion: In the Introduction section, the authors mention references 8-10 summarizing clinical DCE-MRI work in IPF patients. They should briefly compare their findings with the clinical observations.

-Contrast agent: Given the fact that the use of Gd-based contrast agents in MRI examinations is being scrutinized due to reported side effects, especially in the kidney, the pros/cons of DCE-MRI in the evaluation of pulmonary fibrosis in comparison to MRI techniques not relying on the administration of contrast material should be briefly discussed.

Minor comments

-Line 74: Suggest replacing “In addition” by “Also”.

-Line 86: Suggest replacing “Therefore” by “Thus”.

-Lines 152-153 & lines 167-221: Sentences in past tense would be more appropriate.

-Results should be described in past tense (e.g. line 257).

Reviewer #2: In the manuscript the authors present their work on dynamic contrast enhanced lung MRI in a rodent model in which lung fibrosis was induced using bleomycin.

For this n = 15 rats were used, of those one group and I went a bleomycin challenge at day 0, the second group served as control and did not underwent any treatment. Lung MRI was performed at day 7 and day 28 using using a 9.4 Tesla scanner and a radial 3D UTE sequence.

Contrast agent was applied intravenously after seven minutes and dynamic contrast enhanced imaging was performed for 21 minutes. The relative enhancement was measured across the imaging time. For quantitive analysis, the enhancement intensity were binned using 6 thresholds; enhancement bins were then compared and statistically analyzed.

The main findings of this study were an increased lung volume between experimental and control group at day 28, determined by manual lung segmentation and a significantly increased DCE during inflammation stage at day 7. Interestingly that appeared not to be any different contrast enhancement in experimental group at day 28 compared to the control group.

1. Please specify the number of used rodents in the study. That included number is not given in the abstract, please add the number of overall included animals here as well as how many were used in experimental and control group. Please also add this in the method section.

I’m not sure if there is data inconsistency here: in the method section you state that n = 15 animals were included. I cannot find any information in the method section how many animals were resorted to the control and to the experimental group.

In figure 1A the n=4 control and n=12 bleomycin rats we’re included which sums in a total of 16 animals. In figure 1B it is not understandable why there are only six animals in the experimental group at a seven and five at day 28.

Please revise your numbers throughout the whole manuscript, check how many animals were used and please add this information as I requested above. Whenever there are different numbers at different time points (day 7 and day 28) please explain the missing n here!

2. Regarding the used UTE sequence: please add the radio trajectory information in the abstract as well. The TE of > 0.3 ms appears quite long here, additionally the flip angle is rather high, this is probably why you only get very low signal from the lung tissue. Was this anticipated? Is this a limit from the Bruker sequence or could TE be reduced? Please comment on this. You further state that the flip angle was set to 40° because of sensitivity to Gd contrast agent, was this done empirically or did you perform any estimations?

3. In the method section, you explained that images were acquired in coronal orientation. In the figures you show transverse images, why did you do that? Pixel resolution was anisotropic with 0.6 x 0.6 x 1.2 mm and you even zerofill to 0.3 x 0.3? This seems odd, did I miss something here? Please comment or elaborate.

4. The binning method that you used for quantification of the DCE signal is very hard to understand! This needs more explanation in the methods section and probably a graphical explanation in a figure, so that its easier to follow.

5. Please try and elaborate why there is no difference in DCE in bleomycin day 28 images. I would definitely expect extracellular accumulation of GBCA during washout phase and some kind of late enhancement when there would be fibrosis, but this cannot be seen here. Why?

Please review the manuscript regarding spelling and grammar. If possible, it should be revised by a native speaker. The manuscript contains some very complicated non idiomatic sentence structures and a few spelling errors.

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Reviewer #1: Yes: Nicolau Beckmann

Reviewer #2: No

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Contrast enhanced longitudinal changes observed in an experimental bleomycin-induced lung fibrosis rat model by radial DCE-MRI at 9.4T

PONE-D-23-43281R1

Dear Dr. Zandt,

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Reviewers' comments:

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: All points that I raised have been carefully addressed by the authors in this revised version. Manuscript became clearer.

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Reviewer #1: Yes: Nicolau Beckmann

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