The Changes in Expression of NaV1.7 and NaV1.8 and the Effects of the Inhalation of Their Blockers in Healthy and Ovalbumin-Sensitized Guinea Pig Airways
Round 1
Reviewer 1 Report
The manuscript by Kocmalova et al. investigated the role of Nav1.7 and Nav1.8 blockers in airway defense mechanisms in animal asthma model. This reviewer has some major concerns about the manuscript. 1. Fig. 5: The protein expression levels of Nav1.7 and Nav1.8 was detected by ELISA, and OVA induced an increase in Nav1.8 expression, mainly in the bronchus and slightly in the lung. The authors should detect the mRNA expression of Nav1.8 using RT-PCR, in order to identify whether the synthesis of Nav1.8 was increased after chronic allergic inflammation. Western blot analysis is better provided to further demonstrate the protein expression of Nav1.8 in OVA-induced chronic allergic inflammation. 2. What types of cells in the bronchus and lung express Nav1.7 and Nav1.8? Immunohistochemistry should be performed to detect the expression of these channels in these tissues. 3. Why did the authors only investigate the effect of Nav1.8 blockers, but not Nav1.7 blockers, in combination with salbutamol/ipratropium? Nav1.7 blockers also reduced cough reflex and CBF.Author Response
Manuscript MDPI membranes-1250877
Response to Reviewers
Review 1
The manuscript by Kocmalova et al. investigated the role of Nav1.7 and Nav1.8 blockers in airway defense mechanisms in animal asthma model. This reviewer has some major concerns about the manuscript.
Comment 1: Fig. 5: The protein expression levels of Nav1.7 and Nav1.8 was detected by ELISA, and OVA induced an increase in Nav1.8 expression, mainly in the bronchus and slightly in the lung. The authors should detect the mRNA expression of Nav1.8 using RT-PCR, in order to identify whether the synthesis of Nav1.8 was increased after chronic allergic inflammation. Western blot analysis is better provided to further demonstrate the protein expression of Nav1.8 in OVA-induced chronic allergic inflammation.
Author response: Thank you for this suggestion. Detection of mRNA using RT-PCR is commonly used to detect immediately changes. Investigation by RT-PCR will require further experiments as qPCR or Western blot to confirm positive or negative result. The reason why we used ELISA in this step of experiment is that we would like to prove quantitative changes in membrane expression of known protein structure. ELISA method is frequently used for such detection. Western blot and ELISA both can analyse these proteins. The advantages and disadvantages of these methods largely depends on the purpose. ELISA is to choose if you want to quantify a specific (known) protein which is in a mix of various proteins in cell lysates or serum. If we want to see the size of protein we should use western blot. Western blot is to some degree also used to quantify but is not nearly as reliable as ELISA for that purpose, while in ELISA we will not see any size changes to protein as long as the epitope of the protein that is recognized in the ELISA is there. Also, if you are screening many samples for the concentration of a specific protein, ELISA is definitely to prefer.
Comment 2: What types of cells in the bronchus and lung express Nav1.7 and Nav1.8? Immunohistochemistry should be performed to detect the expression of these channels in these tissues.
Author response: Thank you for pointing this out. The reviewer is correct. We agree that this is a potential limitation of the study. One of our goals was to examine what types of cells in the lung and bronchus tissue express Nav1.7 and Nav1.8. We performed immunohistochemical staining from collected tissue. Unfortunately visualization of reaction between both channels protein and antibody by chromogen failed. It would have been interesting to explore this aspect. However, in our study, this would not be possible repeat the reaction because we did not have enough tissue.
Comment 3: Why did the authors only investigate the effect of Nav1.8 blockers, but not Nav1.7 blockers, in combination with salbutamol/ipratropium? Nav1.7 blockers also reduced cough reflex and CBF.
Author response: Nav1.7 reduced cough reflex and ciliary beating frequency but physiological role, expression increase and constant effect on airways reactivity confirmed us in the decision to investigate only effect of Nav1.8 blocker in combination. We think this is an excellent suggestion and we would like to provide these tests in future research.
In addition to the above comments, all spelling and grammatical errors pointed out by the reviewers have been corrected. We look forward to hearing from you in due time regarding our submission and to respond to any further questions and comments you may have.
Sincerely,
Michaela Kocmalova
First author
Author Response File: 
Author Response.docx
Reviewer 2 Report
To Authors:
I highly recommend the authors to proofread the manuscript by a native English speaker and active voice should be used instead of passive voice to facilitate reading For eg Line 92, 144, 276, 361, 372-373, 393, 464-466. The abstract, results, discussion, and conclusion need to be revised since it loses flow. The figures could be simplified to facilitate easy reading.
I have major concerns about the manuscript which are outlined below:
Abstract:
Line 13-15: Abstract needs to be re-written. Its very generic and no specific information about blockers is provided. Further, an abstract needs a revised background. The authors have presented their results in the abstract background.
Line 13: It should be Nav1.7 and Nav1.8
Line 24: NaV1.7 and NaV1.8 should be Nav1.7 and Nav1.8
Materials and Methods:
Line 120: Please include a table or flow chart to indicate the groups.
Line 199: Please indicate that which tissues were used to prepare homogenates. Whole lung tissue and bronchus.
Line 210: The authors stated that SEM was used, however figure legends indicate that SD was used. Please clarify.
Results:
Line 222: Please eliminate “Obtained experimental results”
Line 244-247: Please elaborate the results clearly.
Line 255: Which Nav1.8 blocker was used?
Line 255-261: Please elaborate the results clearly.
Line 266: Please use lung tissue instead of pulmonary tissue.
The authors must justify dose selection, and also clearly state which concentration was used for which inhibitor since inhibitors used in the study are non-specific and can block other sodium channels. Further, concentrations should be discussed in the discussion.
Discussion:
Line 355: Nav1.8.. blockers is missing.
Line 400: Please use reports instead of manuscripts.
Line 409: Please change effecting to activating
Conclusion:
Needs a revised conclusion with active voice.
Figures:
Line 295: Says figures, tables, and schemes, but only figures provided.
Fig 1: Please indicate physiological and pathological for panels.
Fig 4: It looks like Ova – bar is repeated throughout.
Author Response
Response to Reviewers
Review 2
I highly recommend the authors to proofread the manuscript by a native English speaker and active voice should be used instead of passive voice to facilitate reading For e.g. Line 92, 144, 276, 361, 372-373, 393, 464-466. The abstract, results, discussion, and conclusion need to be revised since it loses flow. The figures could be simplified to facilitate easy reading.
Author response: Thank you for pointing this out. The reviewer is correct, and we have provide complex language correction of manuscript by certified editor, expert in this field.
I have major concerns about the manuscript which are outlined below:
Abstract:
Comment 1: Line 13-15: Abstract needs to be re-written. It’s very generic and no specific information about blockers is provided. Further, an abstract needs a revised background. The authors have presented their results in the abstract background.
Author response: We agree with the reviewer’s assessment. Therefore, the formulation of background in abstract was changed as follows: The presented study evaluated the suppositional changes in the airway expression of Nav1.8 and Nav1.7 and their role in the airway defense mechanisms in healthy animals and in an experimental asthma model.
Comment 2: Line 13: It should be Nav1.7 and Nav1.8
Author response: As suggested by the reviewer, we have corrected this typo.
Comment 3: Line 24: NaV1.7 and NaV1.8 should be Nav1.7 and Nav1.8
Author response: As suggested by the reviewer, we have corrected this typo.
Materials and Methods:
Comment 4: Line 120: Please include a table or flow chart to indicate the groups.
Author response: We think this is an excellent suggestion. We agree with this and have incorporated your suggestion into the manuscript (Table 1).
|
Group |
The name in the graphs |
Description of the treatment |
Number of animals in the group |
|
OVA- saline |
OVA- |
5 min inhalation of 0.9% NaCl |
7 |
|
OVA+ saline |
OVA+ |
5 min inhalation of 0.9% NaCl after OVA |
7 |
|
Codeine |
codeine |
per oral administration, dose 10mg/kg before and after OVA |
7 |
|
Salbutamol |
salbutamol |
5 min inhalation of 4.10-3mol/L before and after OVA |
7 |
|
A803467 10-8 |
A8 |
5 min inhalation of 10-8mol/L before and after OVA |
7 |
|
A803467 10-7 |
A7 |
5 min inhalation of 10-7mol/L before and after OVA |
7 |
|
A803467 10-6 |
A6 |
5 min inhalation of 10-6mol/L before and after OVA |
7 |
|
ProTx III 10-8 |
P8 |
5 min inhalation of 10-8mol/L before and after OVA |
7 |
|
ProTx III 10-7 |
P7 |
5 min inhalation of 10-7mol/L before and after OVA |
7 |
|
ProTx III 10-6 |
P6 |
5 min inhalation of 10-6mol/L before and after OVA |
7 |
|
Huwentoxin IV 10-9 |
H9 |
5 min inhalation of 10-9mol/L before and after OVA |
7 |
|
Huwentoxin IV 10-8 |
H8 |
5 min inhalation of 10-8mol/L before and after OVA |
7 |
|
Huwentoxin IV 10-7 |
H7 |
5 min inhalation of 10-7mol/L before and after OVA |
7 |
|
A803467 10-8 + salbutamol |
A8 + SAL |
5 min inhalation of 10-8 A8 and 4.10-3mol/L SAL after OVA |
7 |
|
A803467 10-8 + ipratropium |
A8 + IPR |
5 min inhalation of 10-8 A8 and 10-5mol/L IPR after OVA |
7 |
|
The total number of animals |
105 |
||
Comment 5: Line 199: Please indicate that which tissues were used to prepare homogenates. Whole lung tissue and bronchus.
Author response: As suggested by the reviewer, we have corrected filled this information. The revised text reads as follows on page 5, section 2.6:
“Homogenates from bronchus and whole lung tissue were prepared by sonication (2 min) with a power output of 700 W (homogenizer, Stuart SHM2, ECOMED, Žilina) ac-cording to the manufacturer’s instructions.”
Comment 6: Line 210: The authors stated that SEM was used, however figure legends indicate that SD was used. Please clarify.
Author response: Thank you for pointing this out. The mistake has been corrected. The revised text reads as follows in figure legends:
“sRaw values are expressed as means ± S.E.M.”
Results:
Comment 7: Line 222: Please eliminate “Obtained experimental results”
Author response: As suggested by the reviewer, we have corrected Obtained experimental results to Results.
Comment 8: Line 244-247: Please elaborate the results clearly.
Author response: The authors apologize for unclearness and improved the result description. The revised text reads as follows on page 6, section 3.1.2:
“The pharmacodynamic effect of Nav1.7 as well as Nav1.8 blockers did not change during the development of inflammation (Figure 3). ProTx III (Figure 3, Part A) significantly relieved the bronchoconstriction after histamine or metacholine inhalation but only at the highest concentration. Huwentoxin IV (Figure 3, Part B) did not reduce the sRaw values after histamine or metacholine inhalation. A803467 reduced the sRaw values regardless of the bronchoconstrictor tested (Figure 3, Part C) and, moreover, had an even more significant effect, according to some measurements, than the clinically used bronchodilator salbutamol.”
Comment 9: Line 255: Which Nav1.8 blocker was used?
Author response: As suggested by the reviewer, we have filled this information Nav1.8 blocker A803467.
Comment 10: Line 255-261: Please elaborate the results clearly.
Author response: This part was corrected according to reviewer suggestion. The revised text reads as follows on page 6-7, section 3.1.3:
“Generally, TTX and NaV1.7 and NaV1.8 blockers modulated the CBF but had relatively little effect when tested under physiological conditions: TTX and ProTx III did not significantly influence ciliary beating, Huwentoxin IV significantly decreased the CBF only at the highest concentration used. The IC50 of the NaV1.8 blocker A803467 significantly inhibited the CBF, the main parameter determining mucociliary clearance; however, higher doses did not have a similar impact on cilia movements. Higher doses of A803467 increased the CBF but without statistical significance.
Under the OVA-induced inflammatory conditions, the CBF was significantly and dose-dependently suppressed by the non-selective NaV channel blocker TTX. Furthermore, the frequency of cilia movements was significantly reduced compared to that in the OVA- group. All the tested concentrations of ProTx III and A803467 as well as two higher concentrations of huwentoxin IV significantly decreased the CBF in the control group, which underwent sensitization by OVA, to the level of the CBF values in the healthy control group.”
Comment 11: Line 266: Please use lung tissue instead of pulmonary tissue.
Author response: This part was corrected according to reviewer suggestion.
Comment 12: The authors must justify dose selection, and also clearly state which concentration was used for which inhibitor since inhibitors used in the study are non-specific and can block other sodium channels. Further, concentrations should be discussed in the discussion.
Author response: Thank you for pointing this out. The missing information has been filled into methods. The revised text reads as follows on page 4, section 2:
“For experimental purposes, the IC50s of the tested drugs were used. For the dose-dependence test, 1-fold higher and 1-fold lower concentrations were used.”
Discussion:
Comment 13: Line 355: Nav1.8..blocker is missing.
Author response: As suggested by the reviewer, we have filled this information.
Comment 14: Line 400: Please use reports instead of manuscripts.
Author response: Thank you for pointing this out. The mistake has been corrected.
Comment 15: Line 409: Please change effecting to activating
Author response: Thank you for pointing this out. The mistake has been corrected.
Conclusion:
Comment 16: Needs a revised conclusion with active voice.
Author response: We agree with the reviewer and text was revised by the language site.
Figures:
Comment 17: Line 295: Says figures, tables, and schemes, but only figures provided.
Author response: Thank you for pointing this out. The mistake has been corrected. The revised text reads as Figures.
Comment 18: Fig 1: Please indicate physiological and pathological for panels.
Author response: Thank you for pointing this out. The mistake has been corrected in the figure legend.
Comment 19: Fig 4: It looks like Ova – bar is repeated throughout.
Author response: Thank you for pointing this out. The reviewer is correct, and we have used the means of all used controls for more exact statistics.
In addition to the above comments, all spelling and grammatical errors pointed out by the reviewers have been corrected. We look forward to hearing from you in due time regarding our submission and to respond to any further questions and comments you may have.
Sincerely,
Michaela Kocmalova
First author
Author Response File: Author Response.docx
Reviewer 3 Report
Comments and Suggestions for Authors
In the manuscript by Kocmalova et al. a set of experiments is described which are aimed at characterizing the role of voltage gated sodium channels (Nav1.7 and Nav1.8) blockers in relieving asthma. The goal of the study described and examined an array of classical physiological methods and ELISA assays of Nav1.7 and 1.8 in healthy and unhealthy guinea pig. Overall, the work conveys the role a Nav1.8 blocker, along with Nav1.7 blocker, on the airways defense mechanisms in healthy animals and asthma animal model. Further the authors show that Nav1.7 and 1.8 are present in bronchus and lung with an increased level of Nav1.8 in unhealthy animals compared to healthy animals, suggesting a connection in Nav1.7/8 channels to the animal asthma model.
However, the text needs clarity to provide a better description of the results. There appear to be errors throughout the results section and figures and figure legends. The methods are also unclear in describing protocols and the number of animals used per treatment. It may be helpful to add n values in figure legends. Further grammatical and sentence structure errors make the manuscript very difficult to read. Clarification is needed for the following:
Title is unclear, it may be typos
Lines 227-228 is not supported by figure1, panel D
Asterisks appear to be missing in figure 1, panel F
Lines 293-294 is not supported by figure 8
What does M/L mean in figure legend 1, 6,7,8? Assumption is that it was a typo and stood for mols/L
Define AC, line 149
What does # stand for in figure 7?
Define sRaw sooner.
Lines 250-251, sentence is unclear
Describe the results in the same order that the figures are displayed to contribute to a better flow of the text.
Text does not mention Figure 3 panels A and B.
Regarding homogenate preparations, why were different conditions used for ELISA assays? The different procedures may contribute to the levels of expression. This should be addressed for clarity and to strengthen results.
The major issue which may arise from a lack of clarity in the manuscript or my lack of understanding the experiments is the authors do not clearly support differences between the OVA- and OVA+ animals in the number of coughs, sRaw and CBF values. Since higher levels of Nav1.8 protein is detected in the OVA+ than OVA- animals, the values would be expected to be higher prior to drug administration and likely after drug administration. Is it possible for the authors to address this issue by addressing this in the text of the results, statistically, and/or added panels to the figures?
Other questions that arise from the study include: Are there other Nav channel subunits expressed in the airways? Is the activity of Nav channels altered? Could authors clarify why they did not study Nav1.7 blockers in experiments used for Figures 6,7,8?
Discussion is lengthy and unclear due to sentence structure and grammatical errors.
Author Response
Manuscript MDPI membranes-1250877
Response to Reviewers
Review 3
In the manuscript by Kocmalova et al. a set of experiments is described which are aimed at characterizing the role of voltage gated sodium channels (Nav1.7 and Nav1.8) blockers in relieving asthma. The goal of the study described and examined an array of classical physiological methods and ELISA assays of Nav1.7 and 1.8 in healthy and unhealthy guinea pig. Overall, the work conveys the role a Nav1.8 blocker, along with Nav1.7 blocker, on the airways defense mechanisms in healthy animals and asthma animal model. Further the authors show that Nav1.7 and 1.8 are present in bronchus and lung with an increased level of Nav1.8 in unhealthy animals compared to healthy animals, suggesting a connection in Nav1.7/8 channels to the animal asthma model.
Comment 1: However, the text needs clarity to provide a better description of the results. There appear to be errors throughout the results section and figures and figure legends. The methods are also unclear in describing protocols and the number of animals used per treatment. It may be helpful to add n values in figure legends. Further grammatical and sentence structure errors make the manuscript very difficult to read. Clarification is needed for the following:
Author response: Thank you for pointing this out. The authors apologize for unclearness and improved the result description. All mistakes in results part were corrected. Number of animals were added into figure legends.
Comment 2: Title is unclear, it may be typos.
Author response: While we appreciate the reviewer’s feedback, we respectfully disagree. We think that the title describes main goals of study. All typos and grammar errors were corrected. The revised text reads:
“The changes in expression of Nav1.7 and Nav1.8 and the effects of the inhalation of their blockers in healthy and ovalbumin-sensitized guinea pig airways.”
Comment 3: Lines 227-228 is not supported by figure1, panel D
Author response: Thank you. The authors agree that understanding the text may be difficult due to language problems. In figure 1, panel D is shown data supported decreased the number of coughs with statistical significance only in case, when measurement executed 60 minutes after inhalation of ProTx III in concentration 10-6 mols/L in comparison to Figure 1 , panel A.
Comment 4: Asterisks appear to be missing in figure 1, panel F
Author response: The authors thank reviewer for this comment. We checked several times our data and statistics and there is no missing asterisks. Significance was below the line.
Comment 5: Lines 293-294 is not supported by figure 8
Author response: Thank you for pointing this out. We agree with the reviewer and the mistake has been corrected. The revised text reads as:
“On the other hand, the combination of A803467 and ipratropium decreased the CBF less than A803467 alone, and this decline was not statistically significant.”
Comment 6: What does M/L mean in figure legend 1, 6, 7, 8? Assumption is that it was a typo and stood for mols/L
Author response: The reviewer is correct, and we have corrected this typo in the whole text for mol/L.
Comment 7: Define AC, line 149
Author response: As suggested by the reviewer, we have filled this description.
Comment 8: What does # stand for in figure 7?
Author response: Thank you for pointing on this error. It was corrected in the legend for Figure 7. The revised text reads as:
“+p<0.05, ++p <0.01, +++p <0.001 vs OVA+. #p<0.05 vs salbutamol”
Comment 9: Define sRaw sooner
Author response: Definition of sRaw is explained in the Introduction of the manuscript in the line 99 when it was mentioned at first.
Comment 10: Lines 250-251, sentence is unclear
Author response: We are apologize for unclearness. Sentence was modified. The revised text reads as:
“As expected (Figure 4), the CBF values were significantly increased in the group of animals that underwent ovalbumin sensitization.“
Comment 11: Describe the results in the same order that the figures are displayed to contribute to a better flow of the text.
Author response: The required change was provided – results description was modified as reviewer suggests.
Comment 12: Text does not mention Figure 3 panels A and B.
Author response: Thank you for pointing this out. The missing information has been corrected. The revised text reads as:
“ProTx III (Figure 3, Part A) significantly relieved the bronchoconstriction after histamine or metacholine inhalation but only at the highest concentration. Huwentoxin IV (Figure 3, Part B) did not reduce the sRaw values after histamine or metacholine inhalation. A803467 reduced the sRaw values regardless of the bronchoconstrictor tested (Figure 3, Part C) and, moreover, had an even more significant effect, according to some measurements, than the clinically used bronchodilator salbutamol.”
Comment 13: Regarding homogenate preparations, why were different conditions used for ELISA assays? The different procedures may contribute to the levels of expression. This should be addressed for clarity and to strengthen results.
Author response: Thank you for pointing this out. Although we agree that this is an important consideration. However, in our study, this would not be possible because tissue homogenates were prepared according to manual of used ELISA kits. If we used different approach, than manufacturer suggests, we can have difficulties with samples dilution as well as their interpretation.
Comment 14: The major issue which may arise from a lack of clarity in the manuscript or my lack of understanding the experiments is the authors do not clearly support differences between the OVA- and OVA+ animals in the number of coughs, sRaw and CBF values. Since higher levels of Nav1.8 protein is detected in the OVA+ than OVA- animals, the values would be expected to be higher prior to drug administration and likely after drug administration. Is it possible for the authors to address this issue by addressing this in the text of the results, statistically, and/or added panels to the figures?
Author response: Thank you for pointing this out. Although we agree that this is an important consideration. We will fill this information into results. The revised text reads as follows on page 6, section Results:
“The changes of all tested parameter between OVA- and OVA+ group is are obvious. We recorded increase in sRaw values between OVA- and OVA+ group is almost 3 times higher and the number of coughs efforts grows almost by 50%.”
Comment 15: Other questions that arise from the study include: Are there other Nav channel subunits expressed in the airways?
Author response: Thank you for this question. Navs are expressed in a number of “non-excitable” cells, including fibroblasts, glia, immune cells, and cancer cells. Existence of Navs has also been reported in cultured human smooth muscle cells such as bronchial smooth muscle cells (Nav1.1, Nav1.2, Nav1.4 and Nav1.8), coronary arterial smooth muscle cells (Nav1.3), pulmonary arterial smooth muscle cells (Nav1.5) and aortic smooth muscle cells. (Jo et. Al, 2004)
Comment 16: Is the activity of Nav channels altered?
Author response: We think this is an excellent question. The changes in Nav channels are possible. It would have been interesting to explore if there is changes in irritation threshold or there is changed duration for opening the channel. However, we did not performed electrophysiological evaluation and from our experiments it is not possible exactly respond to this question.
Comment 17: Could authors clarify why they did not study Nav1.7 blockers in experiments used for Figures 6, 7, 8?
Author response: Nav1.7 reduced cough reflex and ciliary beating frequency but physiological role, expression increase and constant effect on airways reactivity confirmed us in the decision to investigate only effect of Nav1.8 blocker in combination. We think this is an excellent suggestion and we would like to provide these tests in future research.
Comment 18: Discussion is lengthy and unclear due to sentence structure and grammatical errors.
Author response: We agree with the reviewer and text was revised by the language site.
In addition to the above comments, all spelling and grammatical errors pointed out by the reviewers have been corrected. We look forward to hearing from you in due time regarding our submission and to respond to any further questions and comments you may have.
Sincerely,
Michaela Kocmalova
First author
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Although the authors responded to my previous comments carefully, they did not perform additional experiments to support their idea.
This reviewer strongly suggested that the authors should perform additional experiments such as RT-PCR and Western blot (previous Ccomment 1 ), since RT-PCR and Western blot can detect whether the protein changes were due to mRNA changes. These methods can determine whether the protein expression levels of Nav1.7 and Nav1.8 are due to changes in protein synthesis (Increased protein levels may also due to decreased protein degradation).
This reviewer also suggested that the author should perform immunohistochemical staining for Nav1.7 and Nav1.8 (previous comment 2), since the location of these channels is important for their functions. This reviewer did not understand why they failed to detect the expression of Nav1.7 and Nav1.8 in the tissue. The authors should modify the experimental conditions. In addition, the author should use additional animals to have enough tissues for these experiments.
For comment 3, if the authors can not investigate the effect of Nav1.7 blockers in combination with salbutamol/ipratropium, they should discuss it in the Discussion section.
Author Response
Manuscript MDPI membranes-1250877
Response to Reviewers
Dear Dr. Editor,
On behalf of all authors, let us thank to all reviewers for their careful assessment of the revised manuscript “The changes in expression of NaV1.7 and NaV1.8 and the effects of the inhalation of their blockers in healthy and ovalbumin-sensitized guinea pig airways” for publication in the MDPI Journal special issue Membrane Channels. We would like to thank you for the positive reactions on the corrected changes we made. Allow us to reply the remaining comments:
Review 1
Although the authors responded to my previous comments carefully, they did not perform additional experiments to support their idea.
This reviewer strongly suggested that the authors should perform additional experiments such as RT-PCR and Western blot (previous Comment 1), since RT-PCR and Western blot can detect whether the protein changes were due to mRNA changes. These methods can determine whether the protein expression levels of Nav1.7 and Nav1.8 are due to changes in protein synthesis (Increased protein levels may also due to decreased protein degradation).
This reviewer also suggested that the author should perform immunohistochemical staining for Nav1.7 and Nav1.8 (previous comment 2), since the location of these channels is important for their functions. This reviewer did not understand why they failed to detect the expression of Nav1.7 and Nav1.8 in the tissue. The authors should modify the experimental conditions. In addition, the author should use additional animals to have enough tissues for these experiments.
For comment 3, if the authors cannot investigate the effect of Nav1.7 blockers in combination with salbutamol/ipratropium, they should discuss it in the Discussion section.
Review 2
The revised manuscript is significantly improved.
Review 3
All the points raised have been addressed.
Author response:
Nav activity is associated with the presence of a functional alpha subunit. The presence of functional subunit and it`s increase were demonstrated by ELISA methodology. The increase / expression also corresponds to the increase in the activity of the previously mentioned ion channels. The possibility that degraded ion channel protein was detected by ELISA is not supported by these facts. mRNA Expression may not be result of the functional protein translation. Several studies have been published where the combination of either evidence of mRNA or protein synthesis and verification of its function is considered sufficient.
However, we agree that the use of the combination of different methods demonstrating the synthesis of the corresponding mRNA and protein would increase the validity of the results. The authors are willing to add this fact to the discussion as one of the limits of the work.
The design of study fully respects the principles of 3R and we tried to eliminate exogenous factors that distort the results of animal experiments as much as possible. We consider this part to be its strength. Previously mentioned aforementioned lack of material to repeat immunohistochemical analysis and mRNA determination is related to 3R. There is no enough material from 1 animal to perform many different methods and we had to carefully decide which will be performed (2 bronchus, 500-600 mg of lung tissue).
The physiological functions of animals, including the airways defense mechanisms, can be affected not only by age and sex of the animals, but also by the season during which they were born and during which the experiment is performed. Therefore, we think that performing an mRNA analysis and a morphological study on an additional group of animals may not be of sufficient value. In addition, the process of preparing the experiment, adapting the animals, sensitizing of animals and carrying out the experiments will take several months and may affect the publication of a special issue of the journal. The authors will fully respect any decision of the editor.
Comment 3 was filled this into revised manuscript. The revised text reads as follows on page 7 section Results 3.1.5:
Nav1.7 reduced cough reflex and ciliary beating frequency. Physiological role, expression increase and constant effect on airways reactivity confirmed us in the decision to investigate only effect of Nav1.8 blocker in combination. Because of previously mentioned data and from the clinical practice point of view, we focused attention on the NaV1.8 blocker. We tested combinations of A803467 with salbutamol and ipratropium, clinically used relievers of asthma. We observed that such combi-nations affected the number of cough efforts, sRaw values and CBF.
Sincerely,
Michaela Kocmalova
First author
Reviewer 2 Report
The revised manuscript is significantly improved.
Author Response
Manuscript MDPI membranes-1250877
Response to Reviewers
Dear Dr. Editor,
On behalf of all authors, let us thank to all reviewers for their careful assessment of the revised manuscript “The changes in expression of NaV1.7 and NaV1.8 and the effects of the inhalation of their blockers in healthy and ovalbumin-sensitized guinea pig airways” for publication in the MDPI Journal special issue Membrane Channels. We would like to thank you for the positive reactions on the corrected changes we made. Allow us to reply the remaining comments:
Review 1
Although the authors responded to my previous comments carefully, they did not perform additional experiments to support their idea.
This reviewer strongly suggested that the authors should perform additional experiments such as RT-PCR and Western blot (previous Comment 1), since RT-PCR and Western blot can detect whether the protein changes were due to mRNA changes. These methods can determine whether the protein expression levels of Nav1.7 and Nav1.8 are due to changes in protein synthesis (Increased protein levels may also due to decreased protein degradation).
This reviewer also suggested that the author should perform immunohistochemical staining for Nav1.7 and Nav1.8 (previous comment 2), since the location of these channels is important for their functions. This reviewer did not understand why they failed to detect the expression of Nav1.7 and Nav1.8 in the tissue. The authors should modify the experimental conditions. In addition, the author should use additional animals to have enough tissues for these experiments.
For comment 3, if the authors cannot investigate the effect of Nav1.7 blockers in combination with salbutamol/ipratropium, they should discuss it in the Discussion section.
Review 2
The revised manuscript is significantly improved.
Review 3
All the points raised have been addressed.
Author response:
Nav activity is associated with the presence of a functional alpha subunit. The presence of functional subunit and it`s increase were demonstrated by ELISA methodology. The increase / expression also corresponds to the increase in the activity of the previously mentioned ion channels. The possibility that degraded ion channel protein was detected by ELISA is not supported by these facts. mRNA Expression may not be result of the functional protein translation. Several studies have been published where the combination of either evidence of mRNA or protein synthesis and verification of its function is considered sufficient.
However, we agree that the use of the combination of different methods demonstrating the synthesis of the corresponding mRNA and protein would increase the validity of the results. The authors are willing to add this fact to the discussion as one of the limits of the work.
The design of study fully respects the principles of 3R and we tried to eliminate exogenous factors that distort the results of animal experiments as much as possible. We consider this part to be its strength. Previously mentioned aforementioned lack of material to repeat immunohistochemical analysis and mRNA determination is related to 3R. There is no enough material from 1 animal to perform many different methods and we had to carefully decide which will be performed (2 bronchus, 500-600 mg of lung tissue).
The physiological functions of animals, including the airways defense mechanisms, can be affected not only by age and sex of the animals, but also by the season during which they were born and during which the experiment is performed. Therefore, we think that performing an mRNA analysis and a morphological study on an additional group of animals may not be of sufficient value. In addition, the process of preparing the experiment, adapting the animals, sensitizing of animals and carrying out the experiments will take several months and may affect the publication of a special issue of the journal. The authors will fully respect any decision of the editor.
Comment 3 was filled this into revised manuscript. The revised text reads as follows on page 7 section Results 3.1.5:
Nav1.7 reduced cough reflex and ciliary beating frequency. Physiological role, expression increase and constant effect on airways reactivity confirmed us in the decision to investigate only effect of Nav1.8 blocker in combination. Because of previously mentioned data and from the clinical practice point of view, we focused attention on the NaV1.8 blocker. We tested combinations of A803467 with salbutamol and ipratropium, clinically used relievers of asthma. We observed that such combi-nations affected the number of cough efforts, sRaw values and CBF.
Sincerely,
Michaela Kocmalova
First author
Reviewer 3 Report
All the points raised have been addressed.
Author Response
Manuscript MDPI membranes-1250877
Response to Reviewers
Dear Dr. Editor,
On behalf of all authors, let us thank to all reviewers for their careful assessment of the revised manuscript “The changes in expression of NaV1.7 and NaV1.8 and the effects of the inhalation of their blockers in healthy and ovalbumin-sensitized guinea pig airways” for publication in the MDPI Journal special issue Membrane Channels. We would like to thank you for the positive reactions on the corrected changes we made. Allow us to reply the remaining comments:
Review 1
Although the authors responded to my previous comments carefully, they did not perform additional experiments to support their idea.
This reviewer strongly suggested that the authors should perform additional experiments such as RT-PCR and Western blot (previous Comment 1), since RT-PCR and Western blot can detect whether the protein changes were due to mRNA changes. These methods can determine whether the protein expression levels of Nav1.7 and Nav1.8 are due to changes in protein synthesis (Increased protein levels may also due to decreased protein degradation).
This reviewer also suggested that the author should perform immunohistochemical staining for Nav1.7 and Nav1.8 (previous comment 2), since the location of these channels is important for their functions. This reviewer did not understand why they failed to detect the expression of Nav1.7 and Nav1.8 in the tissue. The authors should modify the experimental conditions. In addition, the author should use additional animals to have enough tissues for these experiments.
For comment 3, if the authors cannot investigate the effect of Nav1.7 blockers in combination with salbutamol/ipratropium, they should discuss it in the Discussion section.
Review 2
The revised manuscript is significantly improved.
Review 3
All the points raised have been addressed.
Author response:
Nav activity is associated with the presence of a functional alpha subunit. The presence of functional subunit and it`s increase were demonstrated by ELISA methodology. The increase / expression also corresponds to the increase in the activity of the previously mentioned ion channels. The possibility that degraded ion channel protein was detected by ELISA is not supported by these facts. mRNA Expression may not be result of the functional protein translation. Several studies have been published where the combination of either evidence of mRNA or protein synthesis and verification of its function is considered sufficient.
However, we agree that the use of the combination of different methods demonstrating the synthesis of the corresponding mRNA and protein would increase the validity of the results. The authors are willing to add this fact to the discussion as one of the limits of the work.
The design of study fully respects the principles of 3R and we tried to eliminate exogenous factors that distort the results of animal experiments as much as possible. We consider this part to be its strength. Previously mentioned aforementioned lack of material to repeat immunohistochemical analysis and mRNA determination is related to 3R. There is no enough material from 1 animal to perform many different methods and we had to carefully decide which will be performed (2 bronchus, 500-600 mg of lung tissue).
The physiological functions of animals, including the airways defense mechanisms, can be affected not only by age and sex of the animals, but also by the season during which they were born and during which the experiment is performed. Therefore, we think that performing an mRNA analysis and a morphological study on an additional group of animals may not be of sufficient value. In addition, the process of preparing the experiment, adapting the animals, sensitizing of animals and carrying out the experiments will take several months and may affect the publication of a special issue of the journal. The authors will fully respect any decision of the editor.
Comment 3 was filled this into revised manuscript. The revised text reads as follows on page 7 section Results 3.1.5:
Nav1.7 reduced cough reflex and ciliary beating frequency. Physiological role, expression increase and constant effect on airways reactivity confirmed us in the decision to investigate only effect of Nav1.8 blocker in combination. Because of previously mentioned data and from the clinical practice point of view, we focused attention on the NaV1.8 blocker. We tested combinations of A803467 with salbutamol and ipratropium, clinically used relievers of asthma. We observed that such combi-nations affected the number of cough efforts, sRaw values and CBF.
Sincerely,
Michaela Kocmalova
First author
Author Response File: Author Response.docx
