Title: Development of a novel model of cholecystectomy in subsequently ovariectomized mice
and characterization of metabolic and gastrointestinal phenotypes: A pilot study
Version: 1 Date: 30 Dec 2020
Reviewer:  Birgit Rathkolb
Reviewer's report:
The manuscript "Development  of a novel model of cholecystectomy in subsequently
ovariectomized mice and characterization of metabolic and gastrointestinal phenotypes: A
pilot study" by Alexander et al. introduces a novel mouse model derived from a combination
of previously described models of cholecystectomy with modeling human menopause
by ovariectomy, and provides some preliminary characterization concerning some aspects
previously analyzed in the models combined.
Taking the human situation into account, with cholecystectomy most frequently applied in
women at the onset of menopause, and the fact that in deed previous preclinical research on
effects of cholecystectomy using mouse models was limited mainly to studies in male animals,
the development of such a model appears reasonable.
There are some secondary effects discussed that seem to be associated with cholecystectomy,
such as effects on gut microbiome associated with increased cancer risk or effects on metabolic
phenotype. The authors introduce the reader to some of these studies in the introduction.
However, especially concerning the association of metabolic disease such as NAFLD there are
contradictory results available from different studies, a fact that is not sufficiently adressed by
the authors in the introduction in my eyes.
It implies on one hand, that there are no or rarely very strong obvious effects in humans, and on
the other hand, that the clinical relevance of observations made so far, is still unclear. Therefore
some well-planned studies using such a model might help getting a clearer view.
The method applied for the construction of the model are well described, but description of
phenotyping procedures is incomplete:
The method used for euthanasia is not described and information about the fact whether heart
puncture was performed on the opend body is missing. Further blood collection by heart puncture
frequently results in considerable degrees of artificial hemolysis. This should be mentioned, if
observed, since to my experience severe hemolysis can interfere with cholesterol measurements.
Also a description of sample processing after collection is missing (duration and temperature
of storage before plasma being separated from cells, centrifugation step, tubes used for plasma
samples). These details should be given.
Also a description of the procedures performed to collect and measure fat tissue from different
locations is missing, although data is presented in the methods part
The paragraph of sample processing from the liver, only includes formalin
fixation. for pathohistological investigations. It is also stated that triglycerides
were extracted and analyzed from liver tissue. However, the given reference here
does not provide a detailed description of the procedure but refers to another
publication (Reference lipid extraction and quantification from tissue: Rector et
al. 2008 ; https://journals.physiology.org/doi/full/10.1152/ajpgi.00428.2007?rfr_dat=cr_pub+
+0pubmed&url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org). In this reference it is
stated, that liver samples were snap frozen and then formalin fixed or placed in buffer. Lipid
extraction is stated to be done from powdered liver, but even in this publication it is unclear
whether this was fresh frozen or formalin fixed liver. The procedure used in this study should
at least be shortly described and it should be carefully checked, whether the referenced paper
really describes the respective procedure.
Major weaknesses of the study are the missing age-matched controls after cholecystectomy in
the S1 study, and the small numbers of animals in the different experimental groups of both, the
S1 and the S2 study. However, these facts are sufficientyl adressed by the fact, that the study is
presented as "pilot-study" and that the authors discuss results with caution and state, that hints
obtained from their preliminary data have to be followed up by additional studies.
Considering the small n-numbers I recommend to stick to non-parametric statistics and to use
figures showing single data points instead of or additionally to the box-plots used.
To provide a better overview of the study it would be good to include the age of the animals in
Figure 1 additionally to the study week.
In Figure 4 presenting the resluts of relative gene expresion, data of controls in S1 are presented
as single value, although in the legend n=4 is stated for all groups. Hve the samples been pooled
for analysis? Details have to included in legend and methods descriptions.
Besides these points, there are few minor additional issues:
Either don´t use abbreviations for week, month and year in the running text, or include
abbreviations in the abbreviation list.
Line 106: Incomplete sentence with abbreviation placed in wrong position.
Line 143: "The fat pad was replaced" should be rephrased ("relocated back in the peritoneal
cavity" instead of "replaced"), since I believe that it was not replaced by something else.
In the methods part clinical chemistry results are presented as serum parameters although plasma
was used. I recommend to replace serum by plasma.
Over all, I consider the model introduced of potential value, and the data obtained from this
study of limited scientific value, due to very low n-numbers.
Title: Development of a novel model of cholecystectomy in subsequently ovariectomized mice
and characterization of metabolic and gastrointestinal phenotypes: A pilot study
Version: 1 Date: 30 Dec 2020
Reviewer:  Birgit Rathkolb
Reviewer's report:
The manuscript "Development  of a novel model of cholecystectomy in subsequently
ovariectomized mice and characterization of metabolic and gastrointestinal phenotypes: A
pilot study" by Alexander et al. introduces a novel mouse model derived from a combination
of previously described models of cholecystectomy with modeling human menopause
by ovariectomy, and provides some preliminary characterization concerning some aspects
previously analyzed in the models combined.
Taking the human situation into account, with cholecystectomy most frequently applied in
women at the onset of menopause, and the fact that in deed previous preclinical research on
effects of cholecystectomy using mouse models was limited mainly to studies in male animals,
the development of such a model appears reasonable.
There are some secondary effects discussed that seem to be associated with cholecystectomy,
such as effects on gut microbiome associated with increased cancer risk or effects on metabolic
phenotype. The authors introduce the reader to some of these studies in the introduction.
However, especially concerning the association of metabolic disease such as NAFLD there are
contradictory results available from different studies, a fact that is not sufficiently adressed by
the authors in the introduction in my eyes.
It implies on one hand, that there are no or rarely very strong obvious effects in humans, and on
the other hand, that the clinical relevance of observations made so far, is still unclear. Therefore
some well-planned studies using such a model might help getting a clearer view.
The method applied for the construction of the model are well described, but description of
phenotyping procedures is incomplete:
The method used for euthanasia is not described and information about the fact whether heart
puncture was performed on the opend body is missing. Further blood collection by heart puncture
frequently results in considerable degrees of artificial hemolysis. This should be mentioned, if
observed, since to my experience severe hemolysis can interfere with cholesterol measurements.
Also a description of sample processing after collection is missing (duration and temperature
of storage before plasma being separated from cells, centrifugation step, tubes used for plasma
samples). These details should be given.
Also a description of the procedures performed to collect and measure fat tissue from different
locations is missing, although data is presented in the methods part
The paragraph of sample processing from the liver, only includes formalin
fixation. for pathohistological investigations. It is also stated that triglycerides
were extracted and analyzed from liver tissue. However, the given reference here
does not provide a detailed description of the procedure but refers to another
publication (Reference lipid extraction and quantification from tissue: Rector et
al. 2008 ; https://journals.physiology.org/doi/full/10.1152/ajpgi.00428.2007?rfr_dat=cr_pub+
+0pubmed&url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org). In this reference it is
stated, that liver samples were snap frozen and then formalin fixed or placed in buffer. Lipid
extraction is stated to be done from powdered liver, but even in this publication it is unclear
whether this was fresh frozen or formalin fixed liver. The procedure used in this study should
at least be shortly described and it should be carefully checked, whether the referenced paper
really describes the respective procedure.
Major weaknesses of the study are the missing age-matched controls after cholecystectomy in
the S1 study, and the small numbers of animals in the different experimental groups of both, the
S1 and the S2 study. However, these facts are sufficientyl adressed by the fact, that the study is
presented as "pilot-study" and that the authors discuss results with caution and state, that hints
obtained from their preliminary data have to be followed up by additional studies.
Considering the small n-numbers I recommend to stick to non-parametric statistics and to use
figures showing single data points instead of or additionally to the box-plots used.
To provide a better overview of the study it would be good to include the age of the animals in
Figure 1 additionally to the study week.
In Figure 4 presenting the resluts of relative gene expresion, data of controls in S1 are presented
as single value, although in the legend n=4 is stated for all groups. Hve the samples been pooled
for analysis? Details have to included in legend and methods descriptions.
Besides these points, there are few minor additional issues:
Either don´t use abbreviations for week, month and year in the running text, or include
abbreviations in the abbreviation list.
Line 106: Incomplete sentence with abbreviation placed in wrong position.
Line 143: "The fat pad was replaced" should be rephrased ("relocated back in the peritoneal
cavity" instead of "replaced"), since I believe that it was not replaced by something else.
In the methods part clinical chemistry results are presented as serum parameters although plasma
was used. I recommend to replace serum by plasma.
Over all, I consider the model introduced of potential value, and the data obtained from this
study of limited scientific value, due to very low n-numbers.
Please review this paper.
user
Title: Development of a novel model of cholecystectomy in subsequently ovariectomized mice
and characterization of metabolic and gastrointestinal phenotypes: A pilot study
Version: 1 Date: 30 Dec 2020
Reviewer:  Birgit Rathkolb
Reviewer's report:
The manuscript "Development  of a novel model of cholecystectomy in subsequently
ovariectomized mice and characterization of metabolic and gastrointestinal phenotypes: A
pilot study" by Alexander et al. introduces a novel mouse model derived from a combination
of previously described models of cholecystectomy with modeling human menopause
by ovariectomy, and provides some preliminary characterization concerning some aspects
previously analyzed in the models combined.
Taking the human situation into account, with cholecystectomy most frequently applied in
women at the onset of menopause, and the fact that in deed previous preclinical research on
effects of cholecystectomy using mouse models was limited mainly to studies in male animals,
the development of such a model appears reasonable.
There are some secondary effects discussed that seem to be associated with cholecystectomy,
such as effects on gut microbiome associated with increased cancer risk or effects on metabolic
phenotype. The authors introduce the reader to some of these studies in the introduction.
However, especially concerning the association of metabolic disease such as NAFLD there are
contradictory results available from different studies, a fact that is not sufficiently adressed by
the authors in the introduction in my eyes.
It implies on one hand, that there are no or rarely very strong obvious effects in humans, and on
the other hand, that the clinical relevance of observations made so far, is still unclear. Therefore
some well-planned studies using such a model might help getting a clearer view.
The method applied for the construction of the model are well described, but description of
phenotyping procedures is incomplete:
The method used for euthanasia is not described and information about the fact whether heart
puncture was performed on the opend body is missing. Further blood collection by heart puncture
frequently results in considerable degrees of artificial hemolysis. This should be mentioned, if
observed, since to my experience severe hemolysis can interfere with cholesterol measurements.
Also a description of sample processing after collection is missing (duration and temperature
of storage before plasma being separated from cells, centrifugation step, tubes used for plasma
samples). These details should be given.
Also a description of the procedures performed to collect and measure fat tissue from different
locations is missing, although data is presented in the methods part
The paragraph of sample processing from the liver, only includes formalin
fixation. for pathohistological investigations. It is also stated that triglycerides
were extracted and analyzed from liver tissue. However, the given reference here
does not provide a detailed description of the procedure but refers to another
publication (Reference lipid extraction and quantification from tissue: Rector et
al. 2008 ; https://journals.physiology.org/doi/full/10.1152/ajpgi.00428.2007?rfr_dat=cr_pub+
+0pubmed&url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org). In this reference it is
stated, that liver samples were snap frozen and then formalin fixed or placed in buffer. Lipid
extraction is stated to be done from powdered liver, but even in this publication it is unclear
whether this was fresh frozen or formalin fixed liver. The procedure used in this study should
at least be shortly described and it should be carefully checked, whether the referenced paper
really describes the respective procedure.
Major weaknesses of the study are the missing age-matched controls after cholecystectomy in
the S1 study, and the small numbers of animals in the different experimental groups of both, the
S1 and the S2 study. However, these facts are sufficientyl adressed by the fact, that the study is
presented as "pilot-study" and that the authors discuss results with caution and state, that hints
obtained from their preliminary data have to be followed up by additional studies.
Considering the small n-numbers I recommend to stick to non-parametric statistics and to use
figures showing single data points instead of or additionally to the box-plots used.
To provide a better overview of the study it would be good to include the age of the animals in
Figure 1 additionally to the study week.
In Figure 4 presenting the resluts of relative gene expresion, data of controls in S1 are presented
as single value, although in the legend n=4 is stated for all groups. Hve the samples been pooled
for analysis? Details have to included in legend and methods descriptions.
Besides these points, there are few minor additional issues:
Either don´t use abbreviations for week, month and year in the running text, or include
abbreviations in the abbreviation list.
Line 106: Incomplete sentence with abbreviation placed in wrong position.
Line 143: "The fat pad was replaced" should be rephrased ("relocated back in the peritoneal
cavity" instead of "replaced"), since I believe that it was not replaced by something else.
In the methods part clinical chemistry results are presented as serum parameters although plasma
was used. I recommend to replace serum by plasma.
Over all, I consider the model introduced of potential value, and the data obtained from this
study of limited scientific value, due to very low n-numbers.
assistant