Shear-Wave Elastography Using Commercially Available Ultrasound in a Mouse Model of Chronic Liver Disease

Round 1

Reviewer 1 Report

Shear wave elastography is a well-established non-invasive method to assess liver fibrosis in cirrhotic patients. Therefore, it could not be argued that the emphatic advantages can be obtained either clinically or non-clinically in the conclusion for which the authors intention in this study. Further, studies investigating the benefits of applying such a device to the fibrotic mouse are well established (2014 IEEE International Ultrasonics Symposium, 2014, pp. 1140-1143). In the results, the key data showing comparison with other groups is missing in Figure 3.

Author Response

Response to the comments of reviewers

We thank the editor and reviewers for the positive assessment of our manuscript and for identifying areas that required corrections and/or modification. The red-colored text in the revised manuscript is the corrected/modified text. All line numbers mentioned in each response to each comment refer to the small-size numbers that appear on the right margin of the text of the revised manuscript.

 

Reviewer 1

Shear wave elastography is a well-established non-invasive method to assess liver fibrosis in cirrhotic patients. Therefore, it could not be argued that the emphatic advantages can be obtained either clinically or non-clinically in the conclusion for which the authors intention in this study. Further, studies investigating the benefits of applying such a device to the fibrotic mouse are well established (2014 IEEE International Ultrasonics Symposium, 2014, pp. 1140-1143). In the results, the key data showing comparison with other groups is missing in Figure 3.

Thank you for your important comments. Reference 6 (Yeh CL et al., IEEE Trans Ultrason Ferroelectr Freq Control 2015) was very helpful in this study. We believe the strength of this study is that we were able to examine the use of commercially available equipment rather than using home-made equipment. In Figure 3, according to your comments, we added some descriptions which would be helpful to readers’ understanding in our revised manuscript (line 204, 215-6). 

Reviewer 2 Report

The authors present a feasiblity/methods paper regarding SWE in mice to detect hepatic fibrosis in three groups (naive/untreated, ccl4, and hfd).  The author's present appropriate data for the focus of the paper and also highlight limitations in their conclusion.  Although the data is likely not available based on the limitations cited, it would be helpful in the conclusion to perhaps speculate regarding long term follow up using swe in longer term studies. Further it would be on benefit to the readers to have the author's speculate on other models of liver injury in the context of SWE (as they noted the potential limitations in a hfd model, perhaps also commenting on other models such as ETOH or ConA induced injury and the potential expected deliniation of acute versus chronic findings via SWE)

Author Response

Response to the comments of reviewers

We thank the editor and reviewers for the positive assessment of our manuscript and for identifying areas that required corrections and/or modification. The red-colored text in the revised manuscript is the corrected/modified text. All line numbers mentioned in each response to each comment refer to the small-size numbers that appear on the right margin of the text of the revised manuscript.

 

Reviewer 2

The authors present a feasiblity/methods paper regarding SWE in mice to detect hepatic fibrosis in three groups (naive/untreated, ccl4, and hfd).  The author's present appropriate data for the focus of the paper and also highlight limitations in their conclusion.  Although the data is likely not available based on the limitations cited, it would be helpful in the conclusion to perhaps speculate regarding long term follow up using swe in longer term studies. Further it would be on benefit to the readers to have the author's speculate on other models of liver injury in the context of SWE (as they noted the potential limitations in a hfd model, perhaps also commenting on other models such as ETOH or ConA induced injury and the potential expected deliniation of acute versus chronic findings via SWE).

Thank you for your wonderful comments. We sincerely appreciated to your comments.

Round 2

Reviewer 1 Report

Maybe it seems that the authors did not understand the pointing out. The concern was the novelty issue. Referring to the literature (2014 IEEE International Ultrasonics Symposium, 2014, pp. 1140-1143), unique findings (what differs from the previously known study) should be highlighted in an appropriate section.

Author Response

Response to the comments of reviewers

We thank the editor and reviewers for the positive assessment of our manuscript and for identifying areas that required corrections and/or modification. The red-colored text in the revised manuscript is the corrected/modified text. All line numbers mentioned in each response to each comment refer to the small-size numbers that appear on the right margin of the text of the revised manuscript.

 

Maybe it seems that the authors did not understand the pointing out. The concern was the novelty issue. Referring to the literature (2014 IEEE International Ultrasonics Symposium, 2014, pp. 1140-1143), unique findings (what differs from the previously known study) should be highlighted in an appropriate section.

Thank you for your kind and appropriate comment. I agree to your comment, and added some descriptions in our revised manuscript in discussion section (line 278-80, 311-8). Your comment has helped us to demonstrate our novelty of this study. Thank you very much.

Round 3

Reviewer 1 Report

The issue has been well addressed. There is no concern to raise.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Authors aimed to develop a shear-wave elastography (SWE) measurement method using ultrasound in mice and to compare its results with those of other noninvasive tests for liver fibrosis.
There are major comments to be addressed.
1. Authors should address whether histological data were availalble in this study. If not, it shoudl be discussed as a limitation.
2. MRI protocols were validated in other studies ? 
3. Authors can suggest the optimal cutoffs ? 

Author Response

Reviewer 1

Authors aimed to develop a shear-wave elastography (SWE) measurement method using ultrasound in mice and to compare its results with those of other noninvasive tests for liver fibrosis.
There are major comments to be addressed.

1. 
   Authors should address whether histological data were availalble in this study. If not, it shoudl be discussed as a limitation.

Thank you for your valuable comment. In our study, we took imaging data over time on the same mouse. Therefore, we could not obtain histological data at 0 and 2 week during our study period. According to your comment, we added some descriptions in our manuscript as a limitation. (line 428-30).

2. 
   MRI protocols were validated in other studies? 

Thank you for your important comment. The protocol for this experiment has already been reported (PMID: 35091025, ref 12). In our previous report, we measured the T1rho and T2 values by 7T-MRI in the acute liver inflammation model mice administered carbon tetrachloride (CCl4), and we compared and examined whether each relaxation time can be used for detecting acute liver inflammation. In the report, the T1rho value changed significantly compared to the T2 value, and a continuous change was observed even after 6 days. Therefore, T1rho mapping can diagnose acute liver inflammation. We believe that this protocol can be used in chronic hepatitis models. We added some descriptions in our manuscript (line 62-3).

3. 
   Authors can suggest the optimal cutoffs? 

According to your comment, we investigated the cutoff value of SWE in mouse liver fibrosis model (Figure 3C). Thank you for your great comment, we could add very significant data to our revised manuscript  (line 179-82, line 218-20, Figure 3C, line 274).

Reviewer 2 Report

comments are attached.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

 The authors investigated the use of SWE to monitor the progression of liver fibrosis in a mouse study. They have three groups: normal, liver fibrosis, and fatty liver. They compare the SWE with MRI and gene expression. The manuscript can not be accepted in its current form. The manuscript can be improved by considering the following comments:

 

1. The authors need to clarify the contributions in the introduction. As SWE assessments of fibrosis in the rat models were investigated previously, why is there a need of developing SWE assessments of fibrosis in the mouse model?

Thank you for your critical comments. According to your comments we added some descriptions in introduction of our revised manuscript (line 69-70). To establish the utility of SWE measurements by commercially available US in mouse liver disease models should be important to both verify the effectiveness of therapeutic drugs and for experiments using genetically modified mice.

 

2. The claim of the development of SWE measurement to assess liver diseases in a mouse model is completely wrong. The authors did not develop the SWE measurement. Rather, they use SWE measurement using a commercially available ultrasound system.

Thank you for your impotant comment. Your comment is well taken. According to your comment, we modified some descriptions in our revised manuscript (line 32-3, line 393-4, line 438-9).

 

3. SWE is used in clinics to diagnose liver fibrosis. What is the innovation of this study using a small animal model?

Thank you for your critical comments. According to your comments we added some descriptions in introduction of our revised manuscript (line 69-70). As described above, we believe to establish the utility of SWE measurements by commercially available US in mouse liver disease models should be important to both verify the effectiveness of therapeutic drugs and for experiments using genetically modified mice.

 

4. The authors need to improve the writing of the manuscripts. Due to word choice and sentence structure, it was difficult to follow some texts.

Thank you for your comments. According to your comments, we corrected our manuscript carefully.

 

5. Was the fibrosis model diffusive or localized? How did the authors match the plane in SWE measurements for the longitudinal study?

We used CCl₄-induced liver fibrosis model in this study. This model induces diffuse liver fibrosis. In our study, we measured mouse liver SWE in same position by same operator as is reported in other study [1].

 

6. How did the author match the SWE versus the MRI imaging plane?

We set up the MRI slice at the lower edge of the diaphragm. In US analysis, we measured SWE almost same imaging plane.

 

7. Did the authors perform SWE after placing mice in the stereotaxic frame? The motion due to breathing or hand-held will impact the SWE measurements. Did the authors correct the motion artifacts?

We did not use stereotaxic frame in this study as described in previous report [1]. The variation of our measured SWE values was very low, with S.D. less than 0.1 m/s. We also did not correct the motion artifacts in our study.

 

8. The authors should use Proton density fat fraction from MRI to quantify the fatty liver.

Thank you for your valuable comment. Our facility has not yet installed a sequence for PDFF, so we could not measure PDFF in this study. We would like to consider this in the future when PDFF is installed.

 

9. Histology analysis: The authors need to quantify the presence of fibrosis and inflammatory cell in the histology slides. Showing one or two slides is not enough.

In this study, we demonstrated mouse histology. The figures showed representative histological pictures. The location of the tissue is approximately the same as the location of the SWE measurement. Quantification of liver fibrosis, such as hydroxyproline quantification, may be a good idea as you suggest. In our study, we did not perform quantitative measurement of liver fibrosis. Instead, we measured serum liver fibrosis marker and hepatic gene expressions.

 

10. Some discussion texts are wrong like performing correlation analysis using groups 1 and 2 but mentioning only 2; there was no correlation analysis between SWE and M2BP but claiming the correlation.

We apologize for the missing table (Table 1) in our submitted manuscript. We added Table 1 to our revised manuscript. In this Table 1, we performed correlation analysis using group 1, 2, and 3. Thank you for your critical comment.

 

11. 2: provide dimensions of the slide

Thank you for your important comment. We added scale bar to each figure in Figure 2.

 

12. 3: provide axis label

Thank you for your comment. We added axis label to Figure 3.

 

13. 4: provide a y-axis label

 

Thank you for your comment. We added y-axis label to Figure 4.

 

References

1. Morin, J., et al., Application of Ultrasound and Shear Wave Elastography Imaging in a Rat Model of NAFLD/NASH. J Vis Exp, 2021(170).

Reviewer 3 Report

The authors describe the mouse model of assessing liver stiffness by SWE and correlate it with non-invasive markers of liver fibrosis (M2BP), MRI liver stiffness assessment and they also describe changes of expression of some selected genes. 

However, the article does not bring new information. The correlation of SWE and MRI together with non-invasive markers has been described. Also the role of M2BP. 

The studied cohorts are very small to draw robust data. 

The selection of the genes looks random, the terminology is to exact (line 155, there are not primers), the role of the genes and the results is neither described nor discussed.  The gene names must be in capital letters and in italics. 

Author Response

Reviewer 3

The authors describe the mouse model of assessing liver stiffness by SWE and correlate it with non-invasive markers of liver fibrosis (M2BP), MRI liver stiffness assessment and they also describe changes of expression of some selected genes. However, the article does not bring new information. The correlation of SWE and MRI together with non-invasive markers has been described. Also the role of M2BP. 

Thank you for your important comments. We include a response to each of your comments as followings. SWE measurements are already used in humans and are certainly not new. The significance of this paper is that SWE can be measured over time in the same individual mouse using commercially available ultrasound equipment. We believe this will simplify the evaluation of experiments using genetically engineered mice and drug administration experiments.

 

The studied cohorts are very small to draw robust data. 

As for the sample size, it is indeed small, but the SWE measurement shows a significant difference with relatively small S.D. We consider it to be the minimum number necessary. The Ethics Review Committee for Animal Experimentation of of our institute has requested that the number be reduced from an animal welfare perspective.

 

The selection of the genes looks random, the terminology is to exact (line 155, there are not primers), the role of the genes and the results is neither described nor discussed.  The gene names must be in capital letters and in italics. 

Primer sequences and the role of each gene were added (line 161-9). The gene names were corrected to italic. In the case of humans, all the names of genes are written in capital letters, but in this study of mice, the names of genes are written with the first letter capitalized and the rest in lowercase letters.

Reviewer 4 Report

Shear-wave elastography has gained a lot of attention over the years and is on a continuous trend at the moment. The setting proposed by the authors is interesting and may be a starting point for future studies.

Major concerns regarding the manuscript:

 

The authors state that  „We developed an SWE measurement method to assess the degree of liver fibrosis in mouse models of liver disease”  whereas in the title they mention that the shear wave elastography is used by commercially available ultrasound. The method is developed by the manufacturer, as they mention they used a specific device that enables SWE (line 99-101). This is also emphasized in the discussion as well as the conclusion.

 

The Discussions are rather thin, and additional information is required. The authors should focus, on how the transition to a clinical setting might be performed, how new cut-off values might be embedded, and also emphasize the differences with other elastographic techniques used on fibrosis assessment on mice.

Author Response

Reviewer 4

Shear-wave elastography has gained a lot of attention over the years and is on a continuous trend at the moment. The setting proposed by the authors is interesting and may be a starting point for future studies.

Major concerns regarding the manuscript:

 The authors state that „We developed an SWE measurement method to assess the degree of liver fibrosis in mouse models of liver disease,  whereas in the title they mention that the shear wave elastography is used by commercially available ultrasound. The method is developed by the manufacturer, as they mention they used a specific device that enables SWE (line 99-101). This is also emphasized in the discussion as well as the conclusion.

Thank you for your critical comment. According to your comment, we modified some descriptions in our revised manuscript (line 32-3, line 393-4, line 438-9).

 

The Discussions are rather thin, and additional information is required. The authors should focus on how the transition to a clinical setting might be performed, how new cut-off values might be embedded, and also emphasize the differences with other elastographic techniques used on fibrosis assessment on mice.

According to your comment, we investigated the cutoff value of SWE in mouse liver fibrosis model (Figure 3C). In addition, we added some descriptions in discussion (line 418-25). Thank you for your great comment, we could add very significant data to our revised manuscript.

Round 2

Reviewer 1 Report

Authors addresssed raised points appropriately.

Reviewer 2 Report

 

The paper improved significantly from the previous version. I have further following concerns:

1.      The authors should proofread the manuscript by a native English writer to improve the writing. They also need to change the title by mentioning longitudinal monitoring.

2.      The authors need to mention that the previous mouse or rat-based study used a research ultrasound system. They also need to discuss how the clinical ultrasound system is different from the previous study using the research ultrasound system.

3.      Line 66: “Use” instead of “develop” and check other parts of the manuscript.

4.      The response to the comments “Was the fibrosis model diffusive or localized? How did the authors match the plane in SWE measurements for the longitudinal study?”  was not sufficient. The histology image shows localized fibrosis. Even if the same operator scans the mice, the operator needs to use some landmarks to match the plane. The authors need to improve the method section by describing how the operator matches the plane.

5.      The authors need to add text responding to the comments 6-9. Some readers may have similar questions but they will not find the answer in the manuscript.

6.      Histology analysis: As the authors did not quantify the histology, they should discuss how the blood biomarkers and gene expression are related to the extent of the fibrosis in a mouse model by citing previous works.

7.      Tables 1 and 2: define the symbols and acronyms in the caption to make the table standalone.

8.      Perform a similar correlation analysis as Table 1 and 2 after combining groups 1, 2, and 3.

9.      Fig. 3 A and 4A: provide axis labels with dimensions and Fig 3B: provide y-axis label

Reviewer 3 Report

The authors addressed the comment of the review, however, does bring no new ideas. 

Reviewer 4 Report

It is still not clear what are your intentions. As the title mentions, you are testing SWE on mice. This raises some major concerns:

1. Why do you test an already validated technique, which is also used on a daily basis on humans? The abstract and introduction mention you want to develop.... perhaps you want to assess.

2. Why do you compare the technique with Tesla MRI, Mac-2-binding protein, and hepatic genes? Why did you chose so specific? Are thos validated and SWE is not?

3. Was M2BP measured at both 2 and 4 weeks? If not, then was the purpose if the other two methods were measured in a dynamic way? Also, I think there is a confusion here because you did not "develop" the biomarker, you "assessed" the biomarker

4. Is histology the reference standard for SWE? If so, what is the purpose of other techniques? 

5. Did you consider to test the accuracy of all techniques in comparison with histology? Perhaps this design would be better for your work.

The ultrasound device is not fully described.