Review for 10.1186/s12876-015-0228-z (2015)

Reviewer's report
Title:Intestinal and neuronal myenteric adaptations in the small intestine induced
by a high-fat diet in mice
Version:2Date:23 October 2014
Reviewer: Joel C Bornstein
Reviewer's report:
This manuscript is significantly improved, but still has some problems that need
to be addressed. Some of these relate to the authors omitting to include
information incorporated in the response to reviewers in the manuscript, if it
provides information of use to a reviewer then it should also be provided to a
reader (see below). I am disappointed that fading of fluorescence precludes the
per ganglion analysis that I suggested and suggest that a mouse-on-mouse kit
may well address the problem with high background Hu staining (we also use
other fixation protocols for this sort of staining).
Major compulsory revisions
A key issue relates to the statistical treatment of the data. While I accept that
unpaired t-tests and Mann-Whitney U tests are appropriate given the
experimental goals, the authors do not indicate that they have undertaken any
correction for multiple comparisons. This is essential to avoid false positives.
With the large number of comparisons undertake, each using the same treatment
there is a high probability that some of the differences identified as significant at
P < 0.05 are actually not significant. There are a number of tests available to
correct for multiple comparisons, including in the statistical software used by the
authors, and one of these should be applied and then specified in the text. This
may well change the conclusions of some aspects of the study, while others will
clearly be unaffected. This in turn may well affect the Discussion, which should
be revised to deal with any changes revealed by the correction for multiple
comparisons.
Minor essential revisions
I have been through the revised manuscript and have a series of suggestions
that are listed below.
1 page 2 3rd last line “peptide” rather than “polypeptide”
It would be worthwhile being more specific about what is meant by
“morphometric parameters … increased … decreased”
2 page 4 line 2 “responses are distinct” presumably indicates that different
outcomes are produced by different types of fat, however the treatment that
follows this treats all fat as the same. This needs to be clarified. Are the
outcomes described common to all types of fat with other outcomes that are not
mentioned differing between different fats or are the described outcomes
differentially produced? This needs to be made clear, especially in the light of the
discussion of palmitic acid later on.
3 page 6 line 10 specify the conditions under which the intestinal circumference
was measured. This could be relaxed, stretched, in the presence of a smooth
muscle relaxer, in Krebs’ solution or PBS. Each would be expected to give a
different result, which would also depend on the contractile state of the segment.
It is important to a reader to know the conditions for these measurements and
how reliable they might be.
4 page 6 line 15 It is essential for the precautions and fixation protocols identified
in the authors’ response to my original query to be included here. Without this
information, the quantification of the immunohistochemistry cannot be relied on.
5 page 6 last 2 lines The fact that these were varicosities in the circular muscle
should be stated here, along with an explanation of why this was not simply done
with nNOS varicosities (most inhibitory varicosities will be both VIP and nNOS)
either instead of or in parallel. Should also specify what happens with
overlapping varicosities.
6 page 7 line 18 Either specify how selection of images was randomised, subject
to the rule that images did not overlap, or term the selection as “arbitrary” subject
to the same condition. “random” has a very specific meaning and should not be
used unless well defined randomization procedures are followed and outlined in
the test.
7 page 8 line 15 see point 6, note this sentence reads very strangely as it implies
that the measurements by the blinded observer were unsystematic, whereas it
was the image being studied that was chosen “randomly”
8 page 9 3rd last line this is where correction for multiple comparisons needs to
be specified
9 page 10 line 4 The 29% reduction in the “diameter” of the ileum is one of those
results that may be affected by a correction for multiple comparisons.
Furthermore, in the Methods it is stated that the circumference was measured,
rather than the diameter, was the circumference measured divided by Pi to give
diameter?
10 page 10 line 10 The 16% increase in neuronal density can be more than
accounted for by the increased area of plexus implied by a 29% increase in
either diameter or circumference. This makes any conclusions from this result
highly problematic.
11 page 10 line 14 The same applies to the change in nNOS neuron density
seen in the ileum, while the change in the duodenum is one whose significance
might be affected by correcting for multiple comparisons. This is important
because a major conclusion of the study is that the high fat diet alters
neurochemical phenotype and if this cannot be supported by the density data,
the whole thing is somewhat less interesting.
12 page 10 line 18 What was the P value for the change in size in the jejunum
13 page 12 line 12 The data in the 2 sentences preceding the sentence
beginning “These studies demonstrate …” are insufficiently detailed to support
the assertions in this sentence.
14 page 13, line 2 there is ample evidence in both the work of Sang and Young
and Qu et al that nNOS is in both motor neurons and interneurons in mouse
small intestine. Thus, discussion based exclusively on inhibitory motor neurons is
inadequate. At this point the fact that most nNOS motor neurons also express
VIP should be discussed.
15 page 13 5th last line insert “used in this study” after “HFD”
16 page 14 1st 5 lines This is another point where the substantial colocalization
of nNOS and VIP needs to be discussed. The current treatment implies that they
are in separate neurons, but appropriate processing shows major overlaps and
this has significant implications for the present study.
17
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
I declare that I have no competing interests