Effects of N3SA Analogues on Cerebral and Peripheral Arteriolar Vasomotion in Spontaneously Hypertensive Rats

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for your valuable work in this publication, there some question / notes I will need explanatio or edition. 

In the introduction part the aim of this study is missing, i will suggest to add hipotesis in the introduction section. 

Lines 87 - 109. Is this your study results or is it citation? So the question is: Is it cited or this is Authors thoughts? Either way in the introduction part supposed to be only referenced and interpreted literature.

There is no Conclusion part in the manuscript, needs to be included. 

Methods: There is no clear information what total amount of animals were used. 

Is there was adaptation period for animals? What was keeping and handling procedures and protocols in this study. 

Line 304: Animals were anesthetized with α-chloralose (60 mg/kg, intraperitoneally). This is chemical material is rodenticide, is that appropriate to use it on animals? What analgesia protocol were used? 

Lines 306: what induction was used? Who intubation was performed? What material were used? 

Regarding ARRIVE2.0 guidelines, methods must be improved to clear the information of procedures what were done for animals. 

 

Comments on the Quality of English Language

Truly I am happy with authors English language.

Author Response

Comments 1: In the introduction part the aim of this study is missing, i will suggest to add hipotesis in the introduction section. 

Response 1: in agreement with the suggestion, we modified the Introduction section clarifying the aim of our study (page 3, line: 104).

Comments 2: Lines 87 - 109. Is this your study results or is it citation? So the question is: Is it cited or this is Authors thoughts? Either way in the introduction part supposed to be only referenced and interpreted literature.

Response 2: in lines 87 – 95 we reported what Stefanovska et al. described in the paper “Wavelet analysis of oscillations in the peripheral blood circulation measured by laser Doppler technique. IEEE Trans. Biomed. Eng. 1999,46, 120–129”, and in lines 96 – 109 the results we previously described in the 18^th reference.

Comments 3: Here is no Conclusion part in the manuscript, needs to be included. 

Response 3: Following the reviewer’s suggestion, we separated Discussion section from Conclusions section, which in the original version of the manuscript had been joined.

Comments 4: Methods: There is no clear information what total amount of animals were used. 

Response 4: in the section Materials and Methods, sub-paragraph 5.1 “Experimental group” (pag. 8, lines: 287 – 306) the total amount of animals were used had been reported in detail.

Comments 5: Is there was adaptation period for animals? What was keeping and handling procedures and protocols in this study. 

Response 5: The animals we used, arrived to our lab 4 weeks old and we utilized them at least after 20 weeks. During this time, the animals were handled for 10 minutes a day by the same experimenter, so that they became accustomed to being handled and showed no signs of anxiety or stress. We have not added this information in the text because we usually perform the experimental procedures according to the ARRIVE guidelines.

Comment 6: Line 304: Animals were anesthetized with α-chloralose (60 mg/kg, intraperitoneally). This is chemical material is rodenticide, is that appropriate to use it on animals? What analgesia protocol were used? 

Response 6: Alpha-chloralose was selected because it is known to preserve arteriolar vasomotor responses, minimizing interference with vascular reactivity and autonomic regulation. This characteristic is essential for the objectives of the present study, as the use of alternative anesthetic agents could significantly alter the physiological parameters under investigation. Throughout the entire duration of the experiment, the depth of anesthesia was continuously monitored by assessing appropriate reflex responses, ensuring that animals remain under a stable and adequate anesthetic plane.

Our experimental protocol involved non-recovery procedures only. Therefore, no analgesia protocol needs.

In addition, alpha-chloralose has been consistently used in our previous experiments, allowing us for direct comparability of the collected data and ensuring continuity with established and validated experimental protocols.

Comments 7: Lines 306: what induction was used? Who intubation was performed? What material were used? 

Response 7: A 3 Fr polyurethane catheter, approximately 25 mm in length with an inner diameter of 2.5 mm, was used. We added this information in the text (page 8, lines: 311-312). This size ensures stable positioning within the trachea during mechanical ventilation while avoiding excessive length, which could result in overly deep placement or an increase in anatomical dead space.

Regarding ARRIVE2.0 guidelines, methods must be improved to clear the information of procedures what were done for animals. 

Response: Following the reviewer’s suggestion, we modified the “Experimental procedures” section.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript from Lapi et al. tries to elucidate the underlying mechanisms through which thiazides and especially N3SA and two chemical analogs of it (N2SA7 and Suramin) exert their blood pressure-lowering effects. To achieve that the researchers used spontaneous hypertensive rats (SHR) which received the three chemical substances and performed in vivo analysis of cerebral and peripheral microcirculation. By the use of epi-illumination microscopy, they visualized the microvascular networks and identified six distinct frequency components associated with vasomotor activity. They concluded that subtle structural differences between chemical compounds can differentially affect the six different frequency components associated with the arterial tone, as well as their vasodilatory effects. The strength of the manuscript is that it combines in vivo imaging with power spectral analysis enabling the authors to investigate structure-activity relationships and identify how minor structural differences can lead to a substance exhibiting slightly different effects and temporal profiles at central and peripheral microcirculation.

However, there are some points that can be improved to enhance the quality of the manuscript.

 

Major comments

1. The most significant drawback of the research design, according to my opinion, is the number of animals used. In page 8, lines 299-302, is stated that four animals were used per group of chemical substance. Three animals were used for the evaluation of cerebral microcirculation and one animal per group was used to assess peripheral microcirculatory dynamics. Although the procedures of in vivo imaging were complicated and it would be for sure difficult to implement them on more animals, it would be ideal to have more animals per group as this would ensure replicability of the results. Even if four animals had to be used, then it would be better to allocate two animals in cerebral microcirculation study and two in peripheral microcirculation study. The existence of only one animal in the second group (peripheral microcirculation) does not allow SE determination and significance comparisons.
2. Page 3, lines 81-86: this paragraph would be more suitable for the “materials and methods” section. Probably only the six frequency components could be mentioned in the last paragraph of the introduction, line 100.
3. Page 4, lines 121-133: numbering of figure 2 graphs does not coincide with figure legend and figure 2 numbering. For example, line 127 after “with baseline conditions” “(Figure 2B I, II)” should be added. Line 128 “(Figure 2B II)” should be added after “baseline conditions”. Line 129 “(Figure 2B II)” should be replaced by “(Figure 2B III)”. Line 131 “(Figure 2B III)” should be replaced by “(Figure 2B IV)”.
4. Figure 2 legend and especially numbering should be corrected. Line 143, “(I)” should be added after “basal conditions”. Line 144, “(II)” should be added after “N3SA administration”, and “(III)” should be added after “N2SA7 administration”. Line 145, “(III)” should be replaced by “(IV)”. Line 148, "compared to/with" would be better used instead of "respect to".
5. Figure 3 legend and especially numbering should be corrected. Line 169, “(I)” should be added after “basal conditions”. Line 170, “(II)” should be added after “N3SA administration”, and “(III)” should be added after “N2SA7 administration”. Line 171, “(III)” should be replaced by “(IV)”.
6. Page 5, line 172, I would suggest “us” to be added between “allowed” and “to identify”.
7. Pages 5-6, lines 178-185, numbering of figure 3 in the text does not coincide with figure 3 graphs. Page 5, line 179, “(Figure 3B I, II)” should be used instead of “(Figure 3B I)”. Page 6, line 182, “(Figure 3B III)” should be used instead of “(Figure 3B II)”. Line 185, “(Figure 3B IV)” should be used instead of “(Figure 3B III)”.
8. Pages 4 and 5, it would be better to state in the legends of figures 2 and 3 (and on the diagrams maybe) how many animals are included in each group, if the values are presented as mean±SE and the meaning of significance stars.

Comments on the Quality of English Language

Minor comments

1. Page 3, line 79: a verb is missing from this sentence. "is" or "we tested" could be suitable.
2. Page 3, line 109: the word “of” should be added between “profile” and “an antihypertensive agent”.
3. Page 5, lines 153 and 157, “respect to baseline” could be replaced with “compared to baseline values” or “with respect to baseline values”.
4. Page 5, line 158, “(Figure 3A)” should be added after “vasoconstriction”.
5. Page 5, line 154, the word "nadir" is maybe not suitable here since it means the lowest point of events and when used in the text it refers to the most pronounced difference. Maybe "zenith" could be used or paraphrased as "with the most pronounced difference of the effect observed at 40 minutes".
6. Page 6, line 205, maybe “difference” should be replaced by “different”.
7. Page 8, line 285 maybe “with” should be added just before “a basal systolic”.
8. Page 9, line 364, a verb is missing in the sentence. "Are depicted as" may be suitable.

Author Response

Comments 1: The most significant drawback of the research design, according to my opinion, is the number of animals used. In page 8, lines 299-302, is stated that four animals were used per group of chemical substance. Three animals were used for the evaluation of cerebral microcirculation and one animal per group was used to assess peripheral microcirculatory dynamics. Although the procedures of in vivo imaging were complicated and it would be for sure difficult to implement them on more animals, it would be ideal to have more animals per group as this would ensure replicability of the results. Even if four animals had to be used, then it would be better to allocate two animals in cerebral microcirculation study and two in peripheral microcirculation study. The existence of only one animal in the second group (peripheral microcirculation) does not allow SE determination and significance comparisons.

Response 1: We thank the reviewer for the comment. Initially our aim was to study the effects on the cerebral microcirculation and we used three animals. Subsequently, given the results obtained, we decided to use one animal from each group we had available to evaluate the effects on a peripheral microcirculation. We are aware that we cannot obtain statistical data using just one animal, but we thought it was interesting to show the recorded effects. For this reason, we cannot use two animals for the microcirculation of the femoral muscle.

Comments 2 : Page 3, lines 81-86: this paragraph would be more suitable for the “materials and methods” section. Probably only the six frequency components could be mentioned in the last paragraph of the introduction, line 100.

Response 2: Following the reviewer’s suggestion, we eliminated lines 85-86 from the Introduction section, since the six components are listed in the “Materials and Methods” section.

Comments 3: Page 4, lines 121-133: numbering of figure 2 graphs does not coincide with figure legend and figure 2 numbering. For example, line 127 after “with baseline conditions” “(Figure 2B I, II)” should be added. Line 128 “(Figure 2B II)” should be added after “baseline conditions”. Line 129 “(Figure 2B II)” should be replaced by “(Figure 2B III)”. Line 131 “(Figure 2B III)” should be replaced by “(Figure 2B IV)”.

Response 3: We are grateful to the reviewer for having highlighted some errors in the text which we have corrected (page 4, lines 125 - 131).

Comments 4: Figure 2 legend and especially numbering should be corrected. Line 143, “(I)” should be added after “basal conditions”. Line 144, “(II)” should be added after “N3SA administration”, and “(III)” should be added after “N2SA7 administration”. Line 145, “(III)” should be replaced by “(IV)”. Line 148, "compared to/with" would be better used instead of "respect to".

Response 4: We are grateful to the reviewer for having highlighted some errors in the text which we have corrected (page 4, lines 143 - 145).

Comments 5: Figure 3 legend and especially numbering should be corrected. Line 169, “(I)” should be added after “basal conditions”. Line 170, “(II)” should be added after “N3SA administration”, and “(III)” should be added after “N2SA7 administration”. Line 171, “(III)” should be replaced by “(IV)”.

Response 5: We are grateful to the reviewer for having highlighted some errors in the text which we have corrected (page 5, lines 171 - 173).

Comments 6: Page 5, line 172, I would suggest “us” to be added between “allowed” and “to identify”.

Response 6: Following the reviewer’s suggestion, we added “us” between “allowed” and “to identify”.

Comments 7: Pages 5-6, lines 178-185, numbering of figure 3 in the text does not coincide with figure 3 graphs. Page 5, line 179, “(Figure 3B I, II)” should be used instead of “(Figure 3B I)”. Page 6, line 182, “(Figure 3B III)” should be used instead of “(Figure 3B II)”. Line 185, “(Figure 3B IV)” should be used instead of “(Figure 3B III)”.

Response 7: We are grateful to the reviewer for having highlighted some errors in the text which we have corrected (page 6, lines 181 - 186).

Comments 8: Pages 4 and 5, it would be better to state in the legends of figures 2 and 3 (and on the diagrams maybe) how many animals are included in each group, if the values are presented as mean±SE and the meaning of significance stars.

Response 8: Following the reviewer’s suggestion we added the information requested in the legend of figure 2 (page 4 lines 148 - 149). In our original text, this information was inserted in the Materials and Methods section, sub-paragraph Statistical analysis and now it is removed (page 9, line 370).

Minor comments

Comments 1: Page 3, line 79: a verb is missing from this sentence. "is" or "we tested" could be suitable.

Response 1: Following the reviewer’s suggestion, we added “we tested” (page 3, line 79).

Comments 2: Page 3, line 109: the word “of” should be added between “profile” and “an antihypertensive agent”.

Response 2: We are grateful to the reviewer for having highlighted the error in the text which we have corrected (page 3, line 109).

Comments 3: Page 5, lines 153 and 157, “respect to baseline” could be replaced with “compared to baseline values” or “with respect to baseline values”.

Response 3: Following the reviewer’s suggestion, we replaced “respect to baseline” with “compared to baseline values” (page 5, lines: 154 – 159).

Comments 4: Page 5, line 158, “(Figure 3A)” should be added after “vasoconstriction”.

Response 4: Page 5, line 160, we added “(Figure 3A)” after “vasoconstriction”.

Comments 5: Page 5, line 154, the word "nadir" is maybe not suitable here since it means the lowest point of events and when used in the text it refers to the most pronounced difference. Maybe "zenith" could be used or paraphrased as "with the most pronounced difference of the effect observed at 40 minutes".

Response 5: Following the reviewer’s suggestion, we replaced “nadir” with “the most pronounced difference” and we added “observed” (page 5, lines 155-156). 

Comments 6: Page 6, line 205, maybe “difference” should be replaced by “different”.

Response 6: We changed “difference” with “different” (page 6, line 207).

Comments 7: Page 8, line 285 maybe “with” should be added just before “a basal systolic”.

Response 7: We added “with” (page 8, line 288).

Comments 8: Page 9, line 364, a verb is missing in the sentence. "Are depicted as" may be suitable.

Response 8: we do not understand what is requested.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors made a reasonable changes into the manuscript. 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors replied adequately to all of my comments and corrected the errors in the manuscript, which is now more clear for the readers. Unfortunately, they have not been able to provide more animals to evaluate the effects on peripheral microcirculation as suggested in major point 1, which would provide statistical data. However, they explained adequately why this is not feasible according to the initial design of the experiment and it is better to show the results even from one animal than not presenting them at all. In any case, the manuscript is improved and I would recommend acceptance.