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:1Date:22 August 2014
Reviewer: Joel C Bornstein
Reviewer's report:
Major Compulsory Revisions
This should be an interesting piece of work describing some key effects of a high
fat diet on the mouse small intestine. Unfortunately, the methods are poorly
described, the analytical approaches do not provide useful information (notably in
relation to neuron numbers and neurochemistry), the statistical analysis may be
flawed (insufficient detail is provided to assess this) and there is no attempt to
identify potential mechanisms. These factors severely limit the utility of the data
as currently presented. The good news is that nearly all of these problems can
be addressed without further experimentation, they require reanalysis of data and
better description of what was actually done.
As an example of the problems with the methods (see below for detailed
queries), the specific measures are repeatedly stated to be for a particular
number per animal, but samples of duodenum, jejunum and ileum are actually
being compared. Should a reader assume that the particular number needs to be
divided by 3 to give the number per region or was it really that number per region
per animal? A similar issue arises in the statistical approaches as it is not stated
anywhere whether comparisons are made between animals, sections, individual
neurons or villi, etc. As the degrees of freedom used for the comparison depends
on what is being compared, the absence of information about this severely
impacts on the reliability of the conclusions. Note, the proper basis for
comparison is between animals with the within animal variance being accounted
for by using analysis of variance, rather than a t test.
The problems with the neuronal analysis are two fold. First, density measures in
whole mounts are only really meaningful if the degree of stretch is standardised
across preparations. As this is impossible with the methods used and the
changes in structure identified in the morphometric analysis, the proper measure
is numbers of neurons per ganglion. This is still problematic, but at least there is
a clear structural standardisation. Per ganglion data also allows conclusions to
be drawn as to whether there is a change in the neurochemical profile of the
neurons, by allowing the number of nNOS neurons per ganglion to be compared
with the total number of neurons per ganglion.
The second problem is more of a missed opportunity. The immunohistochemistry
has all been done with primary antisera raised in rabbits, thereby precluding
double labelling studies (in the absence of special methods) that would have
allowed direct measurement of changes in proportions of nNOS neurons and
alterations of VIP colocalization with nNOS. As there are excellent antisera
raised in goat/sheep, this is unfortunate. It is also unfortunate that other labels
known to not colocalise with nNOS in mouse small intestine (see Sang & Young
1997, Qu et al 2008) were not examined, especially markers of cholinergic
neurons (e.g. ChAT, calbindin, calretinin). These data would greatly enhance the
impact of the work, but what has been measured is valuable if properly analysed.
While the experimental design does not include any mechanistic elements, so
the data are purely descriptive, information about the nature of the lipids within
the lard used to create the HFD and how these differ from other HFDs giving
somewhat different results would be useful. For example, Baudry et al found that
a HFD (with high protein) did not change the number of neurons per ganglion in
the jejunum (there may have been a slight increase in nNOS neurons), how did
the lipid composition differ from the present study? The authors point to lipid
composition as an important factor in the Introduction, but then provide no
analysis of their own HFD.
Minor compulsory revisions
There are many small issues that need to be dealt with (note that in the absence
of adequate analysis or explanation of methods, I did not feel it was appropriate
to provide detailed feedback on the Discussion).
1 line 37 some comment about total energy content per gram of the diet would be
useful as would an indication of the changes in total (and type of) protein
2 line 41 the Student’s t test is not appropriate for this type of data, ANOVA (or
the equivalent non-parametric test, Kruskal-Wallis) is much more relevant
3 line 46 “area of vasoactive intestinal varicosities” is ambiguous. Do you mean
the area covered by individual varicosities, something that is problematic to
measure given the small size of varicosities and the limits of resolution of the
microscope? Or do you mean the area within ganglia covered by VIP
varicosities?
What is the significance of either measure when the degree of stretch of the
preparation cannot be standardised and where within the plexus were
varicosities measured?
Why not use nNOS varicosities, as nNOS is cytoplasmic and hence should fill the
varicosity, while VIP is vesicular and thus does not provide a true indication of
varicosity size?
4 line 97 nNOS and VIP are colocalized in many myenteric neurons in mouse
small intestine, although the overlap is not 100%. This needs to be mentioned
here to provide context. It would also be useful to acknowledge that most nNOS
negative neurons in the overall myenteric plexus population are cholinergic and
hence measuring nNOS numbers in comparison to the total population gives a
rough estimate of cholinergic neuron numbers.
5 lines 101-102 information about lipid content in human diets would be useful
here as would information about the types of lipids in the HFD studied
6 line 105 “shown” rather than “demonstrated” to avoid repetition
7 line 107 need to explain the significance of lard as compared to butter fat,
duration of feeding is drastically different in these two studies so is this the key?
8 line 109 8.4 weeks? This is a very strange number, what is meant by 0.4
weeks? Is this a typo or is there some significance in a duration of feeding that
corresponds to a fraction of a day?
9 line 110 what is meant by “medium-chain triglycerides”? Please be more
precise and also identify the key differences between lard and corn oil. These
sorts of issues raise the question of why lard was used in the present study and
why comparisons with another fat source were not made to help identify general
vs specific lipid effects.
10 line 112-113 how does diet vary in route of administration? This is illogical and
should be removed.
11 line 115-116 despite the claim that effects of HFD on myenteric neurons have
not been studied, lines 95-98 identify such a study and there are many other
examples in the literature
12 line 129 did mice have to be both 42 days old and 34 g in weight? What
leeway was allowed in these measures, eg if an animal was 36 g when 42 days
of age was it excluded?
13 line 132-134 The HFD and the control dies differed markedly in all major
ingredients. This should be stated here. Why was lard used, what is its lipid
composition and how does this compare with the lipids in the control diet? It is
not sufficient to say that someone else has used a diet, specific justification is
needed, especially when virtually every variable differs between control and
treatment.
14 line 137 what was the concentration of the vincristine solution? Giving the
volume without specifying concentration gives no indication of dose.
15 line 155-158 why only single staining? Much information is lost this way, see
above.
16 line 174 a 40X objective does not seem to have the resolution to adequately
measure varicosity size, indeed the theoretical limits of resolution for
conventional fluorescence microscopy are such as to make these measurements
problematic. Can the authors provide a justification for these experiments?
17 line 178 this is the first of many examples of ambiguity in the number of
preparations analysed. Was it 30 images per animal or 30 images per region per
animal (150 images for each dietary regime)? How were the images chosen?
18 line 179 the area measures are given to an unrealistic and unhelpful degree of
precision, this is especially true given that no effort was made to standardise the
degree of stretch within the whole-mount preparations prior to fixation. The
number of neurons per unit area is not helpful, see general comments above.
19 line 181 again the issue of neurons per animal vs neurons per region per
animal needs to be clarified. How were the neurons chosen, this is critical given
that the data suggest altered phenotype and the systematic variation in neuronal
size with neurochemical phenotype that has been reported by many
researchers?
20 line 182 the same issues arise for VIP varicosities, but also need to indicate
how varicosities were identified and defined, how overlapping varicosities were
discriminated from each other and so on. The varicosities illustrated in Fig 5 were
clearly not within the myenteric ganglia, so were they in the circular muscle or the
tertiary plexus? One might expect varicosities of motor neurons to have different
properties to those of interneurons.
21 line 194 How was “randomly” achieved and was the observer blind to the
treatment?
40 measurements were made per animal? This seems remarkably few and again
does not take into account the different regions, the number of sections, etc. This
needs to be clarified.
22 line 203 What is the HE method? Please provide references and indicate what
parameters allow the analysis of proliferation to be undertaken.
23 line 210 Again the numbers are ambiguous, also line 212 and line 215 (how
are crypt cells defined?).
24 line 220 The statistical analyses used are inappropriate for this type of data.
Where parametric tests can be used the appropriate analysis would be 2 way
ANOVA testing for differences in both diet and region with a post hoc test for
individual differences. Comparisons should be for region per animal, not
individual measurements. If parametric tests are not appropriate, then a
non-parametric ANOVA is needed. Both types of test are available in PRISM.
Furthermore, when presenting these comparisons in the text the parameters of
the analysis (degrees of freedom and type of test should also be provided).
25 line 230 9.9% seems too precise (how does this differ meaningfully from
10%?) and the actual lengths should be provided in the text here.
26 line 231 similarly, how does 29.2% differ meaningfully from 30% and what
were the actual measurements for duodenum, jejunum and ileum?
27 line 236 The distances between ganglia and the number of ganglia per field
can both be quantified in whole mounts and this quantification should be
provided for all three regions, along with the neurons per ganglion data
requested above.
28 line 245 the increases in density of neurons are only meaningful if counts are
done on preparations under the same amount of tension and this was not
controlled in the present study, see general comments.
29 line 255-258 If this material is to remain in the manuscript, then far more detail
about the measurements and statistical analysis is required, see above
30 line 270 insert “proliferation of” after “rate of”
31 line 272-274 consider eliminating digits after decimal point in the percentages
as these make no material difference to the conclusions and are implausibly
precise
32 Figure descriptions are inadequate as they do not properly describe the
material presented. This is especially the case for Figs 3-5. E.g. in fig 5 no
indication of what is in the insets is provided, nor is the location within the plexus
of the images. In my version of the pdf, there is no lettering on Figs 3 and 4 so it
is impossible to assess them
33 A figure showing micrographs identifying the parameters measured and
compared in the sections of intestinal wall including EE cells, proliferation, etc,
would greatly increase the comprehensibility of the manuscript
34 data in Table 3 is too precise, for example it is not credible that body weight
can be meaningfully estimated to 10 µg accuracy. Similarly, measuring intestinal
length to an accuracy of 100 µm is not credible
35 similarly the data in Table 4 are given with vastly greater precision than is
credible. For example, villus height is given to an accuracy 2 orders of magnitude
(in the SEM) greater than the resolution of a light microscope
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