Serum MicroRNAs as Biomarkers of Sepsis and Resuscitation

Round 1

Reviewer 1 Report

Overview: the authors have assessed the response of microRNAs to sepsis and resuscitation in a porcine model.  They found that two microRNAs are elevated in sepsis and respond (by decreasing) to a higher dose of resuscitation.  They also evaluated the relationship between these microRNAs and protein expression and found a relationship to Toll-like receptor signaling.  This is a straightforward large-animal model and the findings point towards the potential utility of these microRNAs as biomarkers of sepsis and/or of the response to resuscitation.  The paper is very well written. 

The authors have identified and discussed the main limitations of the study.  For example, the model likely does not involve a large amount of tissue trauma, so any effect of sterile systemic inflammatory response syndrome caused by trauma cannot be assessed.  The live E coli infusion is certainly preferable to infusion of endotoxin in reproducing human infection, although other models of sepsis in the porcine model could be used to further evaluate the findings in future studies.  It is a relatively small study with a limited 5 hours of follow up. 

In addition, it is noteworthy that the study design does not replicate all aspects of clinical care in that, for example, there is no use of vasoactive medications, no antibiotics, and fluid resuscitation is not given in response to clinical endpoints.  This point is worth mentioning.

Would the authors speculate on the potential mechanisms that lead to increased expression of microRNAs in this model?

Minor comments:

Define ‘miRNAs’ as meaning ‘microRNAs’ in the Introduction

Methods

“We studied available biological samples from control (non septic) and septic pigs.” -- Am not sure what this means.

Recommend stating that the protocol was approved by, or conducted in accordance with the guidelines established by, the relevant body with respect to animal care and use.

Recommend giving the weight, age, gender, breed, and source of pigs.

“See supplementary material for details on miRNA isolation from serum, miRNA cDNA Preparation and RT-qPCR are detailed.”--> rephrase

Results

“IL-1β, TNFα and IL-6 serum concentrations, as well as IL-1β in the renal medulla and (not reaching statistical significance) in the renal cortex concentrations increased in the septic group”-- rephrase.  In other words, this should say something like, “Serum concentrations of IL-1β, TNFα and IL-6, and IL-1β levels in the renal medulla, were higher in the septic group. IL-1β concentrations in the renal cortex trended higher in the septic group (not significant)”

Discussion

“In addition, we provide for the first time evidence that the expression of miR-146a-5p is sensitive to the quality fluid resuscitation.”--> rephrase to something like “…sensitive to the dose of fluid…”

“However, these findings have not been previously reported in a clinically relevant large animal model of sepsis, nor has the association with resuscitation being described.”--> rephrase (being --> been)

Comments for author File: Comments.docx

Author Response

We appreciate the reviewers criticisms in general, that helped improve our manuscript. We answer below point by point the issues raised by the reviewer. Modifications are in blue font in the revised version of he manuscript. We hope our answers are found to be satisfactory.

Author Response File: Author Response.docx

Reviewer 2 Report

In “Serum microRNAs as biomarkers of sepsis and resuscitation”, the authors studied the differences in miRNAs expression in a big animal model of sepsis. The objective was to identified new biomarkes of sepsis and evaluate the variation of the expression of this miRNAs biomarkers during different resuscitation protocols. They found new biomarkers for diagnosis and for evaluation of the therapeutic approach (responding to resuscitation protocol). The subject is relevant and could have important implications. The animal model used (pigs) is appropriate to reproduce the human condition and easier to translate the results to clinical practice, however the evolution of the condition (5 hours) seems to be limited. This limitation should be included in the limitation section (4.2). Other limitations of the study has been explained and discussed in the text. The statistic is correct, correction methods for multiple testing have been used. The authors did not speculate on mechanisms based of the biomarkers identified, and I appreciate it. The understanding of the mechanisms would require other complementary omic techniques, and I understand it is out of the scope of this paper. Overview, the study provide relevant new results. The conclusions are correct based on the limitations of the study.

Minor:

• Abstract-

Line 2 :  definition of miRNA

line 4 : Number of treated animals “ … or an intravenous live E. coli (N= ?)”

 

• Introduction:

 

Page 2 line 11: definition of MiRNAs. Sometimes is defined as MiRNAs, sometimes miRNAs (line 14).

 

• Material and Methods:

2.1 Animal preparation: It’s not clear the number of pigs in control and septic groups

2.2 Protocol and measurements: It’s not clear again the number of animals per group

 

• Figure 2: The resolution is not enough to understand the conections.

Comments for author File: Comments.docx

Author Response

We appreciate the reviewers criticisms in general, that helped improve our manuscript. We answer below point by point the issues raised by the reviewer. Modifications are in blue font in the revised version of he manuscript. We hope our answers are found to be satisfactory.

Author Response File: Author Response.docx