Reviewer ’s report  
 
Title: The Ussing chamber system for measuring intestinal permeability in health and disease  
 
Version: 1 Date: 12 Feb 2019  
 
Reviewer: Dominic Webb  
 
Reviewer's report:  
 
These authors have an impressive and relevant publication history. The more senior authors are 
heavily cited by o ther research groups and are well placed to publish an authoritative paper on 
the setup and use of an Ussing chamber. The most junior authors have a bright future.  
 
The Ussing chamber system, including modern instruments, was recently reviewed (Westerhout 
J, In: The Impact of Food Bioactives on Health: in vitro and ex vivo models [Internet]. Cham 
(CH): Springer; 2015. Chapter 24.). Another recent review covered issues like historical 
perspectives on what has been achieved a nd the future (Herrmann JR & Turner JR., Am J 
Physiol Cell Physiol. 2016 Mar 15;310(6):C423 -31). The present manuscript goes further by 
detailing a modern instrument and software in actual use, reporting experimental data in rodent 
and human tissue. Th is manuscript has value to those learning the method and I would like to see 
it published. However, the writing throughout really needs to be cleaned up.  
 
Comments  
 
Introduction section  line 78 -79: This is not entirely true. The sugar tests are rather g ood at 
discerning small from large intestine permeability in vivo. There is a lack of standardization.  
 
 
Methods section  
Line 229. There are different forms of lucifer yellow. I suspect the carbohydrazide (CH) form is 
the most commonly used for permeabi lity. Please state along with counter ion (lithium or 
potassium?). Also, since lucifer yellow and FITC -dextran are critical reagents, please specify 
catolog numbers.  
 
Results section  
Line 272 "and humans": this particular section, along with legend (and figure) 4, only describes 
FOXP3 -DTR mice. Nothing about humans. I suggest deleting "and humans".  
 
Line 307: remove "direct", unless you can concretely 1) state a definition for "direct" and 2) how 
you ruled out "indirect" as a competing mode l. 
 
Line 555 - I believe (C) should read (D).  
 
Fig 4A: Since recordings were made for 3 different colon segments in each animal, the data for 
each animal could be normalized against one of the segments to illustrate whether those animals 
with highest pr oximal resistance also had highest mid and distal resistance, etc.  
This affects whether or not the claim that mid colon resistance "is naturally more permeable", as 
stated in lines 276 -279, is supported by the data for all the mice or on average (mean ing not all 
mice). In its present form, figure 4A only shows that mid colon resistance is on average lower, 
with sufficient overlap that it could be the same or higher in many cases.  
 
 
Figures/Legends  
 
Fig 5B Spell out Colon Length (cm) as units in Y ax is. 
 
Fig 5E: For completeness, in including /h in the units, this can be viewed as a rate. If you have 
any data, it would be informative to indicate somewhere if FITC -Dextran (or Lucifer Yellow) 
leakage is linear over time. In other words, is the signal twice as big if one waits 2 hours as 
compared to 1 hour and does the system reach equilibrium in 1 hour or some other amount of 
time? Consider a new person learning this method. Resistance is recorded over time (5D) while 
FITC -dextran is not (although it could have been).  
 
Fig 7A versus C. On average, the two panels yield same conclusion. Fine so far. However, in 7A, 
there is more spread in right colon. In fig 7C, there is more spread in left colon. Please do a 
correlation analy sis to give a sense of the extent to which resistance and lucifer yellow correlate.  
 
Fig 7C. Should units not be µg/ml/h/Cm^2 as stated in Methods section and shown in fig 6 for 
FITC -dextran? Maybe h got lost because time was 1 hour? Please fix or expl ain what is different 
from FITC -dextran.  
 
 
Minor  
Grammar and wording throughout could be improved. Given that the authors are in the UK, they 
can easily identify mispellings, lack or incorrect use of commas, and reword a bit for readability. 
Examples:  first two sentences in background section (use of wording on barrier between the two 
sentences), line 21 (comma after (23)) and line 135 -136 (housed), line 145 (ARRIVE is perhaps 
an acronym? Caps?). Line 148 (Setup, one word?), line 218 ("10 mM cold glucose" odd 
structure"), line 269 (sentence structure), Line 304 (tissue's). Some of this might be explained by 
regional grammar, but others are definitely errors. Please go through.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comment s to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical revie w? 
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in t he manuscript:  
 
Needs some lang uage corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this  manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied f or patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the  authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Common s 
CC-BY license (http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: The Ussing chamber system for measuring intestinal permeability in health and disease  
 
Version: 1 Date: 12 Feb 2019  
 
Reviewer: Dominic Webb  
 
Reviewer's report:  
 
These authors have an impressive and relevant publication history. The more senior authors are 
heavily cited by o ther research groups and are well placed to publish an authoritative paper on 
the setup and use of an Ussing chamber. The most junior authors have a bright future.  
 
The Ussing chamber system, including modern instruments, was recently reviewed (Westerhout 
J, In: The Impact of Food Bioactives on Health: in vitro and ex vivo models [Internet]. Cham 
(CH): Springer; 2015. Chapter 24.). Another recent review covered issues like historical 
perspectives on what has been achieved a nd the future (Herrmann JR & Turner JR., Am J 
Physiol Cell Physiol. 2016 Mar 15;310(6):C423 -31). The present manuscript goes further by 
detailing a modern instrument and software in actual use, reporting experimental data in rodent 
and human tissue. Th is manuscript has value to those learning the method and I would like to see 
it published. However, the writing throughout really needs to be cleaned up.  
 
Comments  
 
Introduction section  line 78 -79: This is not entirely true. The sugar tests are rather g ood at 
discerning small from large intestine permeability in vivo. There is a lack of standardization.  
 
 
Methods section  
Line 229. There are different forms of lucifer yellow. I suspect the carbohydrazide (CH) form is 
the most commonly used for permeabi lity. Please state along with counter ion (lithium or 
potassium?). Also, since lucifer yellow and FITC -dextran are critical reagents, please specify 
catolog numbers.  
 
Results section  
Line 272 "and humans": this particular section, along with legend (and figure) 4, only describes 
FOXP3 -DTR mice. Nothing about humans. I suggest deleting "and humans".  
 
Line 307: remove "direct", unless you can concretely 1) state a definition for "direct" and 2) how 
you ruled out "indirect" as a competing mode l. 
 
Line 555 - I believe (C) should read (D).  
 
Fig 4A: Since recordings were made for 3 different colon segments in each animal, the data for 
each animal could be normalized against one of the segments to illustrate whether those animals 
with highest pr oximal resistance also had highest mid and distal resistance, etc.  
This affects whether or not the claim that mid colon resistance "is naturally more permeable", as 
stated in lines 276 -279, is supported by the data for all the mice or on average (mean ing not all 
mice). In its present form, figure 4A only shows that mid colon resistance is on average lower, 
with sufficient overlap that it could be the same or higher in many cases.  
 
 
Figures/Legends  
 
Fig 5B Spell out Colon Length (cm) as units in Y ax is. 
 
Fig 5E: For completeness, in including /h in the units, this can be viewed as a rate. If you have 
any data, it would be informative to indicate somewhere if FITC -Dextran (or Lucifer Yellow) 
leakage is linear over time. In other words, is the signal twice as big if one waits 2 hours as 
compared to 1 hour and does the system reach equilibrium in 1 hour or some other amount of 
time? Consider a new person learning this method. Resistance is recorded over time (5D) while 
FITC -dextran is not (although it could have been).  
 
Fig 7A versus C. On average, the two panels yield same conclusion. Fine so far. However, in 7A, 
there is more spread in right colon. In fig 7C, there is more spread in left colon. Please do a 
correlation analy sis to give a sense of the extent to which resistance and lucifer yellow correlate.  
 
Fig 7C. Should units not be µg/ml/h/Cm^2 as stated in Methods section and shown in fig 6 for 
FITC -dextran? Maybe h got lost because time was 1 hour? Please fix or expl ain what is different 
from FITC -dextran.  
 
 
Minor  
Grammar and wording throughout could be improved. Given that the authors are in the UK, they 
can easily identify mispellings, lack or incorrect use of commas, and reword a bit for readability. 
Examples:  first two sentences in background section (use of wording on barrier between the two 
sentences), line 21 (comma after (23)) and line 135 -136 (housed), line 145 (ARRIVE is perhaps 
an acronym? Caps?). Line 148 (Setup, one word?), line 218 ("10 mM cold glucose" odd 
structure"), line 269 (sentence structure), Line 304 (tissue's). Some of this might be explained by 
regional grammar, but others are definitely errors. Please go through.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comment s to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical revie w? 
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in t he manuscript:  
 
Needs some lang uage corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this  manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied f or patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the  authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Common s 
CC-BY license (http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: The Ussing chamber system for measuring intestinal permeability in health and disease  
 
Version: 1 Date: 12 Feb 2019  
 
Reviewer: Dominic Webb  
 
Reviewer's report:  
 
These authors have an impressive and relevant publication history. The more senior authors are 
heavily cited by o ther research groups and are well placed to publish an authoritative paper on 
the setup and use of an Ussing chamber. The most junior authors have a bright future.  
 
The Ussing chamber system, including modern instruments, was recently reviewed (Westerhout 
J, In: The Impact of Food Bioactives on Health: in vitro and ex vivo models [Internet]. Cham 
(CH): Springer; 2015. Chapter 24.). Another recent review covered issues like historical 
perspectives on what has been achieved a nd the future (Herrmann JR & Turner JR., Am J 
Physiol Cell Physiol. 2016 Mar 15;310(6):C423 -31). The present manuscript goes further by 
detailing a modern instrument and software in actual use, reporting experimental data in rodent 
and human tissue. Th is manuscript has value to those learning the method and I would like to see 
it published. However, the writing throughout really needs to be cleaned up.  
 
Comments  
 
Introduction section  line 78 -79: This is not entirely true. The sugar tests are rather g ood at 
discerning small from large intestine permeability in vivo. There is a lack of standardization.  
 
 
Methods section  
Line 229. There are different forms of lucifer yellow. I suspect the carbohydrazide (CH) form is 
the most commonly used for permeabi lity. Please state along with counter ion (lithium or 
potassium?). Also, since lucifer yellow and FITC -dextran are critical reagents, please specify 
catolog numbers.  
 
Results section  
Line 272 "and humans": this particular section, along with legend (and figure) 4, only describes 
FOXP3 -DTR mice. Nothing about humans. I suggest deleting "and humans".  
 
Line 307: remove "direct", unless you can concretely 1) state a definition for "direct" and 2) how 
you ruled out "indirect" as a competing mode l. 
 
Line 555 - I believe (C) should read (D).  
 
Fig 4A: Since recordings were made for 3 different colon segments in each animal, the data for 
each animal could be normalized against one of the segments to illustrate whether those animals 
with highest pr oximal resistance also had highest mid and distal resistance, etc.  
This affects whether or not the claim that mid colon resistance "is naturally more permeable", as 
stated in lines 276 -279, is supported by the data for all the mice or on average (mean ing not all 
mice). In its present form, figure 4A only shows that mid colon resistance is on average lower, 
with sufficient overlap that it could be the same or higher in many cases.  
 
 
Figures/Legends  
 
Fig 5B Spell out Colon Length (cm) as units in Y ax is. 
 
Fig 5E: For completeness, in including /h in the units, this can be viewed as a rate. If you have 
any data, it would be informative to indicate somewhere if FITC -Dextran (or Lucifer Yellow) 
leakage is linear over time. In other words, is the signal twice as big if one waits 2 hours as 
compared to 1 hour and does the system reach equilibrium in 1 hour or some other amount of 
time? Consider a new person learning this method. Resistance is recorded over time (5D) while 
FITC -dextran is not (although it could have been).  
 
Fig 7A versus C. On average, the two panels yield same conclusion. Fine so far. However, in 7A, 
there is more spread in right colon. In fig 7C, there is more spread in left colon. Please do a 
correlation analy sis to give a sense of the extent to which resistance and lucifer yellow correlate.  
 
Fig 7C. Should units not be µg/ml/h/Cm^2 as stated in Methods section and shown in fig 6 for 
FITC -dextran? Maybe h got lost because time was 1 hour? Please fix or expl ain what is different 
from FITC -dextran.  
 
 
Minor  
Grammar and wording throughout could be improved. Given that the authors are in the UK, they 
can easily identify mispellings, lack or incorrect use of commas, and reword a bit for readability. 
Examples:  first two sentences in background section (use of wording on barrier between the two 
sentences), line 21 (comma after (23)) and line 135 -136 (housed), line 145 (ARRIVE is perhaps 
an acronym? Caps?). Line 148 (Setup, one word?), line 218 ("10 mM cold glucose" odd 
structure"), line 269 (sentence structure), Line 304 (tissue's). Some of this might be explained by 
regional grammar, but others are definitely errors. Please go through.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comment s to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical revie w? 
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in t he manuscript:  
 
Needs some lang uage corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this  manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied f or patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the  authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Common s 
CC-BY license (http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant