Effects of Letrozole Treatment and Vitamin C Supplementation on Morphology, Endoplasmic Reticulum Stress, Programmed Cell Death, and Oxidative Stress in the Small Intestine of Adult Male Rats

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

thank you for providing this manuscript. Please find my comments per line and some questions below:

Line 13: please delete an

Line 16: please delete – in endoplasmic

Line 19: please delete – in morphometrical

Line 20: please delete – in perimeter

Line 22: please delete – in measurements

Line 23: please delete – in treatment

Line 25: please delete – in supplementation and check the whole document for this

Could you please use capital letters for all abbreviations so they are easier to follow?

Could you please define the middle segment of the small intestinum and name it? Is it Duodenum, Jejunum or Ileum you investigated?

Why did you change the administration of your substances in group IV? How did you make sure that this animals reached the same Vit. C dose then the animals given the foodpellet with the Vit. C? Please describe the Water intake measurement?

As crypts consist of at least 4 different cell types: How did you take this into account in your calculation system of enterocytes?

Why didn´t you use rat specific antibodies?

Line 131: please set )

Table 2: Are there no significances at XBP-1?

Statistics: Why did you include 4 groups and do not present the comparisons of all 4 groups? Please do a correct statistical analysis of the 4 groups presented. Use ANOVA and a multiple comparison post test corrected for the number of groups. A Mann-Whitney- U Test is used for only 2 groups, you have 4 groups to test.

Line 200: The headline (Discussion) is missing, please add

Line 218: As you used male rats how can you fix this part of your discussion. Do you have some results on female rats? Please show this results or revise the discussion to the results presented

Discussion: please discuss your findings! Did you check microtubules function? Did you use several doses of Vit. C? Did you measure estrogens? Did you check pathways? Did you work with (breast) cancer cells? Please show the corresponding results or revise

Line 272: the sentence starting with However seems not to be finished. Please check

As Caspase 3 is not alone responsible for apoptosis how much effect do you think you miss by only examining caspase 3? Do you have data on effectorcaspase 6 and 7 also? Please show. For the proof of apoptosis the tunnel assay would be state of the art. Did you do this assay? Please show the data.

Did you proof your histological findings with a factual technique for example qPCR?

kind regards

Comments on the Quality of English Language

Deare authors,

the manuscript is good to read but there are lots of words separated by - although they are not separated by line. Please check!

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study was conducted to find out if blocking the conversion of androgens into estrogens using letrozole has an effect on the histology of the small intestine of Wistar rats, and if vitamin C donation rescues the intestinal cells.

The hypothesis was that estrogens can moderate ER stress, so lack of estrogens increases this stress, and vitamin C can counterbalance this effect.

The rats' diet was either not supplemented, or supplemented with either vitamin C or letrozole, or both vitamin C and letrozole. Tissue was analysed histologically and using immunocytochemistry of markers such as PERK, an ER stress marker, caspase 3, an apoptosis marker, catalase and XBP-1.

While the hypothesis is timely and interesting and the choice of markers seems correct, the results are very confusing. Moreover, the results are displayed in a way that makes them extremely difficult to understand, and the discussion omits some of them and some of them are interpreted in a way that does not help the reader. So I am afraid I can not recommend publishing this manuscript at this stage.

It is almost impossible to understand the results because most of them are only given in a table, with a few exceptions (Fig. 2) there are no graphs. 

From what I can see in the table 2, the effects of letrozol and vitamin C differ depending on which aspect was looked at: the crypt areas and perimeters went up in letrozol treatment, and further up in letrozol plus vitamin C treatment, so vitamin C exacerbated the effect of letrozol in this case. The cell size also went up with letrozol treatment and further up with letrozol and vitamin C treatment, and so did the PERK level, indicating that vitamin C exacerbated ER stress. 

XBP-1 –showed huge changes between the different groups, but the changes were not significant, so their interpretation seems difficult. 

Catalase levels went down in letrozol group, but up much further than the controls when letrozol and vitamin C were given. 

 

Only in the caspase 3 levels did vitamin C seem to show a rescue effect: these levels went up in the letrozol group but down in the group treated with letrozol and vitamin C.  

The discussion is confusing and neither the results of catalase, nor of XBP-1 are discussed yet these stainings are included in the results so they warrant mention in the discussion. 

 

 

The few graphs that were made show the percentage of nuclear area, cytoplasm area, and goblet cell area in the four groups in pie charts (Fig. 2). Unfortunately it is almost impossible to compare these since it is not possible to determine the source of the changes: For example, if the cytomplasm percentage goes up (Control plus vitamin C), this could be because the cells swell up, or their size could remain the same but  the nuclei and goblet cells shrink. So absolute numbers for the areas should have been used. 

In the discussion the authors attempt to interpret these pie charts, implying a protective effect of vitamin C against the changes induced by letrozole donation. However, the authors omit the fact that vitamin c on its own seems to have a larger effect than letrozol and letrozol plus vitamin c. So unfortunately I can not agree with the statemen in lines 236 – 243 unless the effect becomes visible in absolute numbers.

If the authors were to resubmit this manuscript I would like to ask them to

- produce graphs of the data shown in Table 2

- describe all the results in the results section (right now some of them are not described in the text of this section)

- show absolute numbers, in graphs, rather than percentages in Fig. 2 

- Make sure all the results shown find a place in the discussion and are correctly interpreted.

 

Minor points:

Throughout: remove extra hyphens, there are especially many in the abstract (for example, line 18, en-do-plasmic has two hyphens that should not be there; line 36 testo-sterone).

ERet: please use the usual abbreviation “ER”

I suggest sticking to “ER stress” (as in line 203) instead of using a complicated abbreviation such as "ERetS".

Line 272 truncated sentence: “However, the lack of observed changes in the level of unspliced XBP”.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

thank you for taking my comments into account! The manuscript has improved and I think it is ready for publication now. Congratulations! 

kind regards

Author Response

Thank you for your advice and constructive comments concerning our manuscript entitled, “Effects of letrozole treatment and vitamin C supplementation on morphology, endoplasmic reticulum stress, programmed cell death, and oxidative stress in the small intestine of adult male rats”. Thank you for allowing us to publish it. 

Reviewer 2 Report

Comments and Suggestions for Authors

Though many issues were adressed I still have two major points:

(i) Even if software default settings caclulate percentages, they are not meaningful in this case. I disagree with the percentages and would like to see absolute values to detect if they can be interpreted in a meaningful way. This concerns quite a large part of the paper: figure two, its description in the results and its interpretation.

(ii) though some confusing results were deleted, the results and their interpretation are still confusing. I do not see what your main message is, what you have learned from your experimental data, what we as readers can learn from the data. Please elaborate. 

Author Response

Please see the attachment

Author Response File: Author Response.pdf