Dear Editor :
Thank you very much for your letter and the comments from the referees
about our paper. Overall the comments have been fair, encouraging and
constructive, and we have learned much from it. After carefully studying the
reviewers’ comments and advice, we have made corresponding changes to the
paper. We also responded point by point to the reviewer’s comments as listed
below, along with a clear indication of the location of the revision. We hope you
will fins this version of the manuscript acceptable for publication in BMC
Psychiatry.
The authors of this manuscript thank the referees for their helpful comments
on how to improve our manuscript. We believe that we have now produced a
better account of our work and the revised manuscript was more suitable for
publication in BMC Psychiatry.
Thank the reviewers and the editor again for your hard work in effort to
improve our manuscript.
Sincerely yours
Dongjuan Liu, Bing Xiao, Fang Han, Feifei Luo, Enhua Wang, Yuxiu Shi
My responses on the comments in my paper
List of Major Changes:
1) Revised English writing throughout the manuscript
2) Added comprehensive changes and additions the content of the article.
3) Added a quantitative content analysis of all experimental.
4) Rearranged experimental order.
5) Added the full name and affiliation of the ethics committee that approved the
animal studies in full to a revised manuscript.
Comment of reviewer #1:
Majors
(1) The stress paradigm used includes exposure to ether until loss of
consciousness, which might per se be harmful to cells and promote apoptosis. It
is extremely relevant to consider the effects of ether per se on apoptosis and its
related mechanisms. It is possible that all the molecular and cellular results here
presented could be caused by ether and not stress (or “PTSD-like” symptoms).
This concern is of particular relevance if one considers the numerous previous
studies by the same research group on consequences of the SPS protocol on
apoptosis in the hippocampus and amygdala, which reveal essentially the same
findings (peak of apoptosis-like events 7 days after SPS; are there any brain
structures where there is no increase in apoptosis after SPS?). The authors
have to mention those similarities and to discuss their possible implementations.
Ether anesthesia has been wildely used in animal studies. Although ether
could affect apoptosis, the rats in the normal control group in this study
also received ether treatment. Our data clearly demonstrated that
although the rats in control group has some apoptotic cells, this could not
account for the significant increase of apoptotic cells found in the SPS
treated rats.
Our previous studies observed apoptosis in the hippocampus and
amygdale, with the peak at day 7 after SPS. This suggests that it may
occur in other parts of the brain. In this study, we focused on dorsal raphe
nucleus.
In particular, it remains to be shown whether (i) apoptosis, indeed, affects
neurons (as repeatedly stated by the authors) and (ii) the apoptosis-related
events have any consequences on the affected brain structure. In case of the
dorsal raphe this could be achieved e.g. by measurements of 5-HT release.
(i) We demonstrate that apopotosis does occur, which affects neurons by
reducing the number of the neuron cells of dorsal raphe nucleus.
(ii) the apoptosis-related events have any consequences on the affected
brain structure. In case of the dorsal raphe this could be achieved e.g. by
measurements of 5-HT release.
We previously have shown increase of 5-HT 1A release by SPS stimulate
. (Luo FF, Han F, Shi YX ：Change in 5-HT1A receptor in the dorsal raphe
nucleus in a rat model of post-traumatic stress disorder. Molecular
Medicine Reports. 2011, 4:843-847)
(2) A general remark concerning the Results section is the lack of F values and
degree of freedom notation when ANOVA tests were performed. The report of
such data is essential for the consistency of the presented data and because
test results could be verified by any interested reader.
We forgot to provide F value and degree of freedom notation when ANOVA
tests we now have added it to the resluts and figure legends.
(3) Have the authors ever checked for markers of inflammation at different time
points after SPS?
No, we did not check markers for inflammation.
(4) Language is a main issue in this paper and needs considerable review.
We have made a big effort to revise English writing throughout the
manuscript. We believe that it has significantly improved the quality of the
writing in this revised manuscript.
Minors
(a)Figure 2 lacks a stronger explanation of what is depicted. The electron-dense
black granule could be marked by an arrow. For this result section on
enzymohistochemistry analysis of Cyt-C by TEM, no information is provided on
how measurement and analysis was performed to quantify mitochondrial cristae
fracture, mitochondrial vacuolization and rupture of mitochondria. There is a p
value for a change (translocation of Cyt-C from mitochondria to cytoplasm),
however, the data is not shown and information on how this analysis was
performed is not presented.
1) we have added more detailed figure legends.
2) we have added arrows to indicate black granules.
3) As requested by this reviewer, we now have added the percentage of
mitochondria with morphological change and positive expression of Cyt-C
( Figure. 1b).
(b) In figure 5, the authors depicted the distribution of TMP in lysosomes by
electron microscopy and describe intracellular lysosomes in control group as
having “normal structure”. However, there is no explanation on how they define
“normal” neither which features they considered in SPS groups to define them
as not normal. Additionally, there is no description on the quantification method
employed, no quantitative data is shown, although the text suggests quantitative
analyses, once a p value is presented for the result concerning the enzymatic
cytochemistry of TMP using transmission electronic microscopy.
Normal lysosomeis round shape with evenly, high density of TMP. SPS
stimulate rats has irregular shape of lysosome with release of TMP due to
rupture of lysosome. We now have quantified the perentage of lysosome
with increased TMP expression (Figure. 6b)
(c) For the morphological change of the dorsal raphe nucleus by TEM, there is
no specific information on what kind of quantitative analysis or statistical test
was performed in order to obtain the significant difference described.
As requested by the reviewer, we now have added the percentage of the
apoptosis cells (Figure.4b).
(d) There is no paper reference or empirical evidence on this paper to support
the claim: “Apoptosis and overexpression of TPM were pathophysiological
foundation of behavior disorder induced by PTSD”.
We now changed to “apoptosis and overexpression of TMP may be one of
the pathophysiological foundation for the behavior disorder in PTSD
patients.”
(e) The statement: “SPS induced apoptosis in dorsal raphe nucleus neuron of
rats and smaller volume of the dorsal raphe nucleus” can not be confirmed by
the data presented since no volume measurement was performed on this study.
We agree that we did not directly measure the volume of dorsal raphe
nucleus. We now have modified this sentence “SPS induced apoptosis in
dorsal raphe nucleus neuron of rats, which may lead to smaller volume of
the dorsal raphe nucleus.”
(f) Reference number 7 is listed together with information about the employment
SPS method for PTSD research; however, it is actually a review on virus and
apoptosis.
Thanks for catching the error and we have deleted the reference.
(g) Reference number 9 is referred as a meta-analysis when it is actually an
original paper in which an evaluation of brain volume is compared among
children victims of traumatic events, with and without PTSD. It is important to
re-state this information, first because it is not a meta-analysis study and second
because the information that the study was conducted in children might have
biological relevance, since the figure could be completely different in the adult
and the animal experiments herein performed were conducted in adult subjects.
Single-prolonged stress is a accepted animal model of PTSD and has been
used in the PTSD research field. It induces time-dependent dysregulation
of the HPA axis. , We think children and adult have no major difference in
this regard.
(h) Figure 1A needs more detailed information on what is specifically shown,
what does the X and Y axes represent and what the green dots mean. It lacks a
deeper explanation on how apoptosis rate was calculated.
Green dot in Flow cytometer cell point suggested that cells were clustered
distribution, the X-axis and Y-axis represents the relative intensity values
of the fluorescence signal (fluorescein isothiocyanate and iodinated
Fang dinyl) ;
We have added detailed description and provided quantitation for the
apopotosis rate.The apoptosis rate =（number of apoptosis /total cells)
×100%. ）
(i) It is not clear how enhanced expression of cytochrome c, as revealed by
Western blotting analyses, can suggest its release from mitochondria to
cytosol,once the observed signal could also come from internal mitochondrial
cytochrome c.
Western blot analysis measures Cyt-C in the cytoplasm that is released
from mitochondria. Therefore, it can meaure the enhancement of Cyt-C
released from mitochondria.
(j) In the Results section, there are discrepancies in the thickness of the sections
(e.g. 12 micro_m vs. 20 micro_m)
We corrected the error (12 μm)
(k) The figure numbers included in the graphs do not correspond to those
mentioned in the text.
We now have checked each figures to make sure they are correctly
mentioned in the text.
(l) Please explain the abbreviations in the text (e.g. TBS, TBST, PVDF etc.). If
an abbreviation was introduced, it should be exclusively used thereafter (e.g.
cyt-c).
We now have described the following in the method: TBS（tris buffered
saline）, TBST (tris buffer with saline and tween 20), PVDF （polyvinylidene
fluoride ）
(m) In the Results section, the authors explain that the results of the Western
blots were expressed relative to the mean of the control group. This implies that
the mean of the controls is 100%, which, however, is not the case in the figure.
SPS group showed weaker expression that normal group, with statistical
significance.
(n) It is important to know whether data were analyzed blind to the experimental
group.
Yes, and we have added this to the manuscript.
(o) The authors have to specify how apoptosis in the dorsal raphe nucleus may
lead to novel treatments for PTSD (last sentence of the Clonclusion).
We have modified the sentence “Further elucidation of the apoptotic
mechanisms of dorsal raphe nucleus cells will improve our understanding
of the mechanisms underlying PTSD”.
(p) Legend to Figure 1: Please explain “**” shown in Fig. 1B.
We now have change to “■P < 0.05 SPS 1d vs. control group. ●P < 0.05
SPS 7d vs. SPS 1d.▲P < 0.05 SPS 14d vs. SPS 7d.”
Major language issues:
(i) Title: Single-prolonged stress is a singular noun and the verb “to induce”
should be conjugated in the singular form, thus, instead of induce, it should read
“induces”.
We have made corresponding corrections in the revised paper.
(ii) Abstract: The sentence “however, the mechanisms that midsagittal area of
the pons was significantly reduced are not well understood” is not
understandable and should be revised. In the methods section of the abstract,
when it is stated “the models of post-traumatic stress disorder (PTSD) were
created”, it seems that the authors meant that they applied the single-prolonged
stress paradigm, since the model was not created by these authors in this article.
In the results section of the abstract,apoptosis rate were significantly increased”
s not grammatically correct, they author should write either apoptosis rates were
significantly increased or apoptosis rate was significantly increased.
We have thoroughly revised the manuscript accordingly.
(iii) Background Third paragraph of the background section: the phrase “the
mechanisms is not yet entirely clear” should be read either the mechanisms are
not entirely clear or the mechanism is not yet entirely clear. Forth paragraph of
the background section: “take part in regulation of apoptosis”, the word take
should be singular, “takes”.
We have made corresponding corrections in the revised paper.
(iv) Methods: The entire Methods section should be revised, since there are
many sentences with major issues and need grammatical correction.
We have done perfection of this content.
(v) Results: “Cristae fractuer” should be read “cristae fracture”
We have made corresponding corrections in the revised paper.
(vi) Discussion: The sentence “Cytochrome c, a key protein in electron transport,
located on mitochondrial inner and outer membrane” has no main verb and, thus,
makes no sense.
We have made corresponding corrections in the revised paper. We very
much appreciate your careful reading of our manuscript and valuable
suggestions.
Comment of reviewer #2:
1. Data and analyses are not sufficiently presented. For each ANOVA, F, df, and
p values should be reported.
We have added to the manuscript. We are sorry forgot the F values, df,
and p values, it is made by our careless. F-values and degrees have been
added in revised paper.
2. Figure 1b shows asterisks on all experimental groups, however the text states
that the 7d group displayed a peak in apoptosis rate. This needs to be presented
in the graph, in addition to statistical information for this peak change.
We have done the perfection of this content.
3. Figure 2 and Figure 5 do not appear to show graphical representations of
quantifications of changes. The authors report significant changes, however
only a p<0.05 is reported in the text, without any statistical information or
graphical representation of data for review (i.e., error bars, etc). This is
especially important since sample sizes were quite small (n=5) in these
experiments.
We have added the appear to show graphical representations of
quantifications of changes.We have discussed the statistics in the result
section. We now have added the scale bar to the Figures .
4. In Figure 3, specificity of the Cyt-C antibody should be shown (i.e., a greater
area of the gel in this figure or a supplementary figure). Similarly to above, a
'peak' at 7d is reported in the text, with no statistical evidence shown.
We have added the statistics to the resluts and figure legend.
5. The analysis of TMP using light microscope does not seem to have a sound
sampling technique. Taking 15 random slides from each group does not allow
for a comprehensive representation of all subjects. Similarly, choosing five
random visual fields from each slide is not accepted as a statistically sound
sampling technique (stereology or counting the entire region would be more
acceptable). Also, neuronal volume is reported to change in the Results section,
but there is no description of how volumes were measured, or any presentation
of volume data. This experiment needs to be better described and validated, in
general.
a. Five sections were selected from each group. Five visual fields of the
dorsal raphe nucleus in each sections was selected for examination.
b．we did not perform quantization of the neuronal volume. We have
deleted “smaller neuronal volume ”.
Minor Essential Revisions:
1. Language, grammar, and typos need to be edited throughout the manuscript.
We have made a big effort to revise English writing throughout the
manuscript. We believe that the quality of the writing has significantly
improved in this revised manuscript.
2. Please check the weight of the subjects. 150-180g seems very small for
56-day old rats
We have made corrections (180-200g) in the revised paper.
Discretionary Revisions:
1. An analysis of dorsal raphe volumes from all groups would add to the validity
of the SPS model,.showing that this animal model yields neuronal loss that
parallels neuronal loss in PTSD
Single-prolonged
stress
(SPS),
which
shows
time-dependent
dysregulation in the HPA axis, is one of the widely used animal models for
PTSD research.
References:
（1）Kohda K, Harada K, Kato K, Hoshino A, Motohashi J, Yamaji T,
Morinobu S, Matsuoka N, Kato N (2007) Glucocorticoid receptor activation
is involved in producing abnormal phenotypes of single-prolonged stress
rats: a putative post-traumatic stress disorder model. Neuroscience
148:22-33.
（ 2 ） Yamamoto S, Morinobu S, Fuchikami M, Kurata A, Kozuru T,
Yamawaki S (2008) Effects of single prolonged stress and D-cycloserine
on contextual fear extinction and hippocampal NMDA receptor expression
in a rat model of PTSD. Neuropsychopharmacology 33: 2108-2116.）
We speculated that this animal model yields neuronal loss that maight
parallel neuronal loss in PTSD.
Comment of reviewer #3:
Reviewer's report:
I am not aware of the model used as being a justified model of PTSD.
Regardless the immobilization should be further described and justified.
The control animals should have been handled.
1.Single-prolonged
stress
(SPS),
which
shows
time-dependent
dysregulation in the HPA axis, is one of the widely used animal models for
PTSD research.
References:
（1）Kohda K, Harada K, Kato K, Hoshino A, Motohashi J, Yamaji T,
Morinobu S, Matsuoka N, Kato N (2007) Glucocorticoid receptor activation
is involved in producing abnormal phenotypes of single-prolonged stress
rats: a putative post-traumatic stress disorder model. Neuroscience
148:22-33.
（ 2 ） Yamamoto S, Morinobu S, Fuchikami M, Kurata A, Kozuru T,
Yamawaki S (2008) Effects of single prolonged stress and D-cycloserine
on contextual fear extinction and hippocampal NMDA receptor expression
in a rat model of PTSD. Neuropsychopharmacology 33: 2108-2116.）
2. We added SPS animal model of establishing in Model establishment and
grouping in detail.