Reviewer ’s report  
Title: A New Method to Induce Nonalcoholic Steatohepatitis (NASH) in Mice  
Version: 4 Date: 26 Jun 2019  
 
Reviewer: Reviewer 2  
Reviewer's report:  
Dear Editor -in-Chief  
BMC Gastroenterology  
Thank you for giving us a chance to revise our manuscript entitled: "A New Method to Indu ce 
Nonalcoholic Steatohepatitis (NASH) in Mice ". We answered and revised our manuscript 
according to editor and reviewer’s comments. For your convenience all changes made in the 
revised manuscript are highlighted (in green) and reviewer comments together with the 
corresponding responses are presented below.  
 
Differently of what stated, the authors did not apply the same amount of digits after the comma 
to all different values in Table 1 and 2.  
Re: corrected  
 
Figure 1 is not mentioned anywhere in the text.  
Re: corrected  
 
In Figure 1 several SD bars are missing (with n=8 is sounds unlikely that SD is equal to 0).  
Re: corrected  
 
It's reviewer's opinion that bars with cigarette smoke exposure should be painted differently and 
maybe grouped together (since sta tistical comparison is related to C+CS control group).  
Re: Done  
 
Whiskey plot is probably a better way to show results. Probably the use of connecting line to 
better representing the comparison with its own relative control group (ctrl and C+CS) is 
probably better. Similar changes should be applied to Figure 3.  
Re: Box plots with whiskers were plotted according to respect reviewer's request.  
  
Figure 2: panel labels are not present. In the legend, authors refer as (A), (B), etc…but such 
labelling has not been applied. It is not clear why Fru+CS condition has an addition al panel (E) 
(is the magnitude the same as the other images?)  
Re: A, B, etc… labels were added to figure 2.  
Re: As described in the text (page 9, paragraph 13), two mice in Fru+Cs subgroup had shown 
amyloidosis in addition to inflammation that marked as un iform pink sediments located in 
hepatic sinusoidal wall. The nature of amyloid sediments was confirmed by Congo red staining 
which became orange. Its image was placed as a separate panel (E) with same magnitude in 
figure 2.  "E: Congo Red" also was added t o Figure 2 legend.  
 
The evaluation of a critical event in pathogenesis such as inflammatory probably deserves 
additional analysis to one cytokine only (TNF alpha) quantification. It looks like smoking 
condition increases TNF alpha hepatic concentration of approx.25%. Is such increase consistent 
with the different diets? Please check comparison between smoking and non -smoking diets 
(ctrl/C+CS; Fru/ Fru+CS is similar to HFD/HFD+CS or WD/WD+Cs)? Similar comparison to 
Figure 1 is largely suggested.  
Re: Done  
I hope that the answers to the comments made are clear and meet your requirements for 
accepting for publication in your high quality and respected journal.  
Best regards Seyyed Ali Mard  
Are the methods appropriate and well described?  
If not, please specify what is requi red in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, plea se explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require furth er 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competi ng interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either now or i n the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the  content of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have  any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
This reviewer has been recruited by a partner organization, Research Square. Reviewers with 
declared or apparent competin g interests are not utilized for these reviews. This reviewer has 
agreed to publication of their comments online under a Creative Commons Attribution License 
attributed to Research Square and was paid a small honorarium for completing the review within 
a specified timeframe. Honoraria for reviews such as this are paid regardless of the reviewer 
recommendation.  
 
I agree to the open peer  review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the author s' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ).  I understand that any comments 
which I do not wish to be included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
Title: A New Method to Induce Nonalcoholic Steatohepatitis (NASH) in Mice  
Version: 4 Date: 26 Jun 2019  
 
Reviewer: Reviewer 2  
Reviewer's report:  
Dear Editor -in-Chief  
BMC Gastroenterology  
Thank you for giving us a chance to revise our manuscript entitled: "A New Method to Indu ce 
Nonalcoholic Steatohepatitis (NASH) in Mice ". We answered and revised our manuscript 
according to editor and reviewer’s comments. For your convenience all changes made in the 
revised manuscript are highlighted (in green) and reviewer comments together with the 
corresponding responses are presented below.  
 
Differently of what stated, the authors did not apply the same amount of digits after the comma 
to all different values in Table 1 and 2.  
Re: corrected  
 
Figure 1 is not mentioned anywhere in the text.  
Re: corrected  
 
In Figure 1 several SD bars are missing (with n=8 is sounds unlikely that SD is equal to 0).  
Re: corrected  
 
It's reviewer's opinion that bars with cigarette smoke exposure should be painted differently and 
maybe grouped together (since sta tistical comparison is related to C+CS control group).  
Re: Done  
 
Whiskey plot is probably a better way to show results. Probably the use of connecting line to 
better representing the comparison with its own relative control group (ctrl and C+CS) is 
probably better. Similar changes should be applied to Figure 3.  
Re: Box plots with whiskers were plotted according to respect reviewer's request.  
  
Figure 2: panel labels are not present. In the legend, authors refer as (A), (B), etc…but such 
labelling has not been applied. It is not clear why Fru+CS condition has an addition al panel (E) 
(is the magnitude the same as the other images?)  
Re: A, B, etc… labels were added to figure 2.  
Re: As described in the text (page 9, paragraph 13), two mice in Fru+Cs subgroup had shown 
amyloidosis in addition to inflammation that marked as un iform pink sediments located in 
hepatic sinusoidal wall. The nature of amyloid sediments was confirmed by Congo red staining 
which became orange. Its image was placed as a separate panel (E) with same magnitude in 
figure 2.  "E: Congo Red" also was added t o Figure 2 legend.  
 
The evaluation of a critical event in pathogenesis such as inflammatory probably deserves 
additional analysis to one cytokine only (TNF alpha) quantification. It looks like smoking 
condition increases TNF alpha hepatic concentration of approx.25%. Is such increase consistent 
with the different diets? Please check comparison between smoking and non -smoking diets 
(ctrl/C+CS; Fru/ Fru+CS is similar to HFD/HFD+CS or WD/WD+Cs)? Similar comparison to 
Figure 1 is largely suggested.  
Re: Done  
I hope that the answers to the comments made are clear and meet your requirements for 
accepting for publication in your high quality and respected journal.  
Best regards Seyyed Ali Mard  
Are the methods appropriate and well described?  
If not, please specify what is requi red in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, plea se explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require furth er 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competi ng interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either now or i n the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the  content of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have  any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
This reviewer has been recruited by a partner organization, Research Square. Reviewers with 
declared or apparent competin g interests are not utilized for these reviews. This reviewer has 
agreed to publication of their comments online under a Creative Commons Attribution License 
attributed to Research Square and was paid a small honorarium for completing the review within 
a specified timeframe. Honoraria for reviews such as this are paid regardless of the reviewer 
recommendation.  
 
I agree to the open peer  review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the author s' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ).  I understand that any comments 
which I do not wish to be included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
Title: A New Method to Induce Nonalcoholic Steatohepatitis (NASH) in Mice  
Version: 4 Date: 26 Jun 2019  
 
Reviewer: Reviewer 2  
Reviewer's report:  
Dear Editor -in-Chief  
BMC Gastroenterology  
Thank you for giving us a chance to revise our manuscript entitled: "A New Method to Indu ce 
Nonalcoholic Steatohepatitis (NASH) in Mice ". We answered and revised our manuscript 
according to editor and reviewer’s comments. For your convenience all changes made in the 
revised manuscript are highlighted (in green) and reviewer comments together with the 
corresponding responses are presented below.  
 
Differently of what stated, the authors did not apply the same amount of digits after the comma 
to all different values in Table 1 and 2.  
Re: corrected  
 
Figure 1 is not mentioned anywhere in the text.  
Re: corrected  
 
In Figure 1 several SD bars are missing (with n=8 is sounds unlikely that SD is equal to 0).  
Re: corrected  
 
It's reviewer's opinion that bars with cigarette smoke exposure should be painted differently and 
maybe grouped together (since sta tistical comparison is related to C+CS control group).  
Re: Done  
 
Whiskey plot is probably a better way to show results. Probably the use of connecting line to 
better representing the comparison with its own relative control group (ctrl and C+CS) is 
probably better. Similar changes should be applied to Figure 3.  
Re: Box plots with whiskers were plotted according to respect reviewer's request.  
  
Figure 2: panel labels are not present. In the legend, authors refer as (A), (B), etc…but such 
labelling has not been applied. It is not clear why Fru+CS condition has an addition al panel (E) 
(is the magnitude the same as the other images?)  
Re: A, B, etc… labels were added to figure 2.  
Re: As described in the text (page 9, paragraph 13), two mice in Fru+Cs subgroup had shown 
amyloidosis in addition to inflammation that marked as un iform pink sediments located in 
hepatic sinusoidal wall. The nature of amyloid sediments was confirmed by Congo red staining 
which became orange. Its image was placed as a separate panel (E) with same magnitude in 
figure 2.  "E: Congo Red" also was added t o Figure 2 legend.  
 
The evaluation of a critical event in pathogenesis such as inflammatory probably deserves 
additional analysis to one cytokine only (TNF alpha) quantification. It looks like smoking 
condition increases TNF alpha hepatic concentration of approx.25%. Is such increase consistent 
with the different diets? Please check comparison between smoking and non -smoking diets 
(ctrl/C+CS; Fru/ Fru+CS is similar to HFD/HFD+CS or WD/WD+Cs)? Similar comparison to 
Figure 1 is largely suggested.  
Re: Done  
I hope that the answers to the comments made are clear and meet your requirements for 
accepting for publication in your high quality and respected journal.  
Best regards Seyyed Ali Mard  
Are the methods appropriate and well described?  
If not, please specify what is requi red in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, plea se explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require furth er 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competi ng interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manuscript, either now or i n the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the  content of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have  any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
This reviewer has been recruited by a partner organization, Research Square. Reviewers with 
declared or apparent competin g interests are not utilized for these reviews. This reviewer has 
agreed to publication of their comments online under a Creative Commons Attribution License 
attributed to Research Square and was paid a small honorarium for completing the review within 
a specified timeframe. Honoraria for reviews such as this are paid regardless of the reviewer 
recommendation.  
 
I agree to the open peer  review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for publication, my named report 
including any attachments I upload will be posted on the website along with the author s' 
responses. I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ).  I understand that any comments 
which I do not wish to be included in my named report can be included as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant