X-ray-Based 3D Histology of Murine Hearts Using Contrast-Enhanced Microfocus Computed Tomography (CECT) and Cryo-CECT

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors of the work hearts-2713659, I make some constructive observations on your work:

Lines 34 to 36  The idea requires bibliographical citation

Lines 38 to 40 The idea requires bibliographical citation

Lines 193-194  The papillary muscles of the LV should be described as: antero-lateral papillary muscle and postero-medial papillary muscle. (Not MP1 and MP2).

Lines 267 to 274     Ideas require bibliographic citation

Lines 388 to 390  The idea should be included as an example of congenital heart disease

As explained in Figure 1 the authors show a decrease in heart volume as an effect of paraffin embedding processing, however, in H&E Figure 2M, it is similar to 2A and 2D.

Please discuss the differential budget costs of the methodological procedures of the objective of the work.

Author Response

Answer to reviewer 1:

We thank the reviewer for his/her constructive comments on our work. Please see below the answer for each point that was mentioned.

Lines 34 to 36  The idea requires bibliographical citation

Lines 38 to 40 The idea requires bibliographical citation

We thank the reviewer for noticing missing bibliographical citations. Two references have been added for the epidemiology of ischemic heart disease (lines 34 to 36) and one for the decrease of deaths due to CVDs (lines 38 to 40).

Lines 193-194  The papillary muscles of the LV should be described as: antero-lateral papillary muscle and postero-medial papillary muscle. (Not MP1 and MP2).

The identification of the two papillary muscles has been clarified in the text and in the reference to video 1.

Lines 267 to 274     Ideas require bibliographic citation

Thank you for notifying missing references. This has been corrected by adding the needed bibliographic citations for each ideas mentioned in the text.

Lines 388 to 390  The idea should be included as an example of congenital heart disease

It has been clarified that Tetralogy of Fallot and wrong positioning of the main vessels were two examples of congenital heart diseases.

As explained in Figure 1 the authors show a decrease in heart volume as an effect of paraffin embedding processing, however, in H&E Figure 2M, it is similar to 2A and 2D.

We thank the reviewer for this interesting remark. It is true that the tissue shrinkage is not as visible on 2D sections (figure 2M) compared to the volumetric comparison (Figure 1E). A sentence has been added in lines 284-287 to suggest an hypothetical explanation. Indeed, we hypothesize that the tissue relaxes on hot water between the 2D sectioning using a microtome and the tissue collection on a glass slide before colorimetric staining and imaging under the microscope.

Please discuss the differential budget costs of the methodological procedures of the objective of the work.

We added a sentence mentioning the costly infrastructures required for microCT, that are not easily accessible in comparison to the well-established classical histology (lines 389-392).

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Pestiaux describes a comparative analyses between classical 2D histological assessment of the cardiac microarchitecture and X-ray-based 3D histology using CECT and cry-CECT. The authors provide a very elaborated comparison between all the techniques and conclude that CECT and cry-CECT are superior maintaining heart volume as compared to classical parafine-embedded methods. Furthermore they demonstrate that coronary vessels as well as valve leaflets can be nicely identified, reconstructed and measured using both CECT and cry-CECT methods. 

Overall the manuscript is interesting and provides nice data on the comparison between distinct modes of heart analyses. While the authors claim the benefits of CECT and cry-CECT methods as non-invasive (o less invasive) as compared to classical 2D histology, as far as I understand, the heart is also required to be fixed and perfused. Thus  I would suggest to the authors to trim down such claims.

 

Author Response

Answer to reviewer 2:

The manuscript by Pestiaux describes a comparative analyses between classical 2D histological assessment of the cardiac microarchitecture and X-ray-based 3D histology using CECT and cry-CECT. The authors provide a very elaborated comparison between all the techniques and conclude that CECT and cry-CECT are superior maintaining heart volume as compared to classical parafine-embedded methods. Furthermore they demonstrate that coronary vessels as well as valve leaflets can be nicely identified, reconstructed and measured using both CECT and cry-CECT methods. 

Overall the manuscript is interesting and provides nice data on the comparison between distinct modes of heart analyses. While the authors claim the benefits of CECT and cry-CECT methods as non-invasive (o less invasive) as compared to classical 2D histology, as far as I understand, the heart is also required to be fixed and perfused. Thus  I would suggest to the authors to trim down such claims.

We thank the reviewer for taking the time to review our manuscript and providing constructive comments on our results and conclusion. With both CECT and cryo-CECT, the heart was indeed fixed and stained by immersion in the CESA. We have highlighted in the description of the limitations of both CECT and cryo-CECT that the hearts needs to be explanted, fixed and stained, as for classical 2D histology. It should also be noted that fixation is not mandatory but, because staining takes still quite some time, and tissue degradation would be a danger in this time frame, fixation is usually done.

 

Author Response File: Author Response.docx