Suppression of the Excitability of Nociceptive Secondary Sensory Neurons Following Systemic Administration of Astaxanthin in Rats
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe study by Chida et al. provides significant insights into the acute effects of AST on both nociceptive and non-nociceptive mechanical stimulation in vivo. The findings, indicating a dose-dependent inhibition of the mean firing frequency of neurons in response to mechanical stimuli, suggest potential therapeutic applications for AST. However, several points require clarification and further consideration:
- Discussion (Line 366, Point i):
- The statement that Ca influx through Ca channels further depolarizes the membrane potential should be reconsidered. Given the extracellular Ca concentration is approximately 1 mM and the Ca influx is in the micromolar range, it is unlikely that this would cause significant further depolarization of the membrane potential. It is recommended to delete this statement to avoid any potential misconceptions.
- Consideration of Mechanical Stimuli:
- Since the study involves mechanical stimuli, it is crucial to consider other relevant ion channels in the final model. In addition to TRPA1 and ASIC3, PIEZO channels, known to be mechanically activated, should also be taken into account. Including these channels will provide a more comprehensive understanding of the mechanistic pathways involved.
- Conclusion and Therapeutic Potential:
- The conclusion discusses the potential therapeutic intervention of AST for nociceptive pain. However, based on the study, the effect of 5 mM AST lasts only 10-20 minutes in rats. This limited duration suggests that the therapeutic potential of AST might be constrained by its short duration of action. It is recommended to temper the conclusion to reflect this limitation and suggest potential avenues for future research to address the duration of AST's effects.
Addressing these points will enhance the clarity and impact of the study.
Author Response
MS# anesthres-3136949-R1
Title: “Suppression of the Excitability of Rat Nociceptive Secondary Sensory Neurons following Systemic Administration of astaxanthin　in Rats
Authors: Risako Chida, Sana Yamaguchi, Syogo Utugi, Yukito Sashide and Mamoru Takeda
***********************************************************************************************************************
Reviewer #1:
The study by Chida et al. provides significant insights into the acute effects of AST on both nociceptive and non-nociceptive mechanical stimulation in vivo. The findings, indicating a dose-dependent inhibition of the mean firing frequency of neurons in response to mechanical stimuli, suggest potential therapeutic applications for AST. However, several points require clarification and further consideration:
Answer: We appreciate Reviewer’s careful reviews on our manuscript. We have carefully checked reviewer’s constructive and helpful comments and made necessary corrections to revised manuscript (marked by red font) We believe that your comments helped to improve our manuscript. According to editor’s suggestion, concerning self citation and similarity rate, we had revised the style of the parts of the main text that overlap with previous papers (marked by blue font) in revised manuscript, as much as possible. Also, to reduce the number of self-citation paper, we have reduced the number of our papers as much as possible and rearranged the reference section.
Comment 1
- Discussion (Line 366, Point i):
The statement that Ca influx through Ca channels further depolarizes the membrane potential should be reconsidered. Given the extracellular Ca concentration is approximately 1 mM and the Ca influx is in the micromolar range, it is unlikely that this would cause significant further depolarization of the membrane potential. It is recommended to delete this statement to avoid any potential misconceptions.
Answer
We greatly appreciate the reviewer’s careful review on our manuscript. According to reviewer’s comment, we have revised manuscript in the discussion section (page7) as follows; This influx has an important consequence: Ca2+ acts as a second messenger, leading to the activation of many cellular processes, such as contraction, secretion and gene transcription
Comment 2
- Consideration of Mechanical Stimuli:
Since the study involves mechanical stimuli, it is crucial to consider other relevant ion channels in the final model. In addition to TRPA1 and ASIC3, PIEZO channels, known to be mechanically activated, should also be taken into account. Including these channels will provide a more comprehensive understanding of the mechanistic pathways involved.
Answer
We greatly appreciate the reviewer’s careful review on our manuscript. According to reviewer’s comment, we have added PIEZO channels in revised Figure 6 and legend (page 9) as follows; When noxious mechanical stimulation is applied to the skin, mechanosensitive ion channels (TRPA1/ASIC3/PIEZO) open,…
Comment 3
- Conclusion and Therapeutic Potential:
The conclusion discusses the potential therapeutic intervention of AST for nociceptive pain. However, based on the study, the effect of 5 mM AST lasts only 10-20 minutes in rats. This limited duration suggests that the therapeutic potential of AST might be constrained by its short duration of action. It is recommended to temper the conclusion to reflect this limitation and suggest potential avenues for future research to address the duration of AST's effects.
Answer
We greatly appreciate the reviewer’s careful review on our manuscript. According to reviewer’s comment, we have revised manuscript in the discussion section (page 9) as follows; The present study provides the first evidence that, in the absence of inflammatory or neuropathic pain, acute intravenous administration of AST produces a shot-term inhibition of trigeminal sensory transmission, including nociception, possibly by inhibiting Cav channels and excitatory glutamate neuronal transmission (Figure 6). Although further studies are needed to elucidate this mechanism, AST may be considered a potential therapeutic agent for the treatment of trigeminal nociceptive pain without side effects.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for AuthorsCurrent Ms is devoted to in vitro effect of astaxanthin on rat Cav channels. The data obtained by the authors show suppression of SpVc nociceptive transmission. The authors suggest that AST can be considered as an alternative therapeutic agent for the treatment of trigeminal nociceptive pain. Although this suggestion should be verified in future, in my opinion this manuscript is suitable for publication in Anesthesia Research journal. See my concerns below.
In the Introduction section, authors often go off from main idea of MS. I have provided some recommendations below, but Authors should still significantly reorganize the Introduction section according to the journal's guidelines.
Line 44-46. This sentence is not related to the following idea of paragraph. Please, remove this sentence.
Line 45-46: “especially after the failure of conventional Western medicine”. It is nor politically correct phrase. Please, could you rephrase this view?
Line 46-49. There is no logical connection between this sentence and the following ones. Please remove this sentence from the MS.
Line 51-53: Merge to one sentence
Line 62-67. It is unclear why the authors cite data from their previous study. After all, the current study is devoted to Astaxanthin and not "theanine, resveratrol and α-lipoic acid".
Line 68: “these findings”. What data are Authors talking about? Please clarify.
Line 68-74. This paragraph should be completely rewritten. The last paragraph in the Introduction section, as a rule, briefly explains what the study is about and (also very briefly) what data was obtained in general. For more details, see the MDPI tutorial for Authors.
In the Material and Discussion section, the authors discuss the data obtained in previous studies with the current one. Such discussion should be moved to the Discussion section. Or these discussions should be reduced to a minimum.
Line 76: “reported herein” such phrases have to be removed all over the MS. It is already clear to the reader what kind of research is being discussed.
Line 100: “(1986)” replace to numeric reference.
Line 113: “We have previously identified”. If Authors are talking about a preliminary experiment within the current work, then it is necessary to describe in the supplementary materials? Else - Authors have to provide a link to the work.
In the Result section, the Authors duplicate the description of the figures, which is already given in the figure captions themselves. The same phrase Authors have to avoid in Result section of MS. The Authors also partially discuss and compare obtained results with previous data. In the Result section, the Authors should pay more attention to the results they obtained, and all reasoning should be done in the Discussion section.
Line 151, Line 202: “as described previously” such phrases are a discussion element. I suggest either deleting this pharses or moving it to the Discussion section. Authors should do this all over the MS.
Line 159-161: This sentence is a caption to the figure Fig1D. From the caption of the figure itself it is obvious that the relationship between mechanical stimulation intensity and mean discharge frequency is shown. Please remove this sentence. Please, check all over the MS and remove similar sentences (phrases) from Result section.
Line 196-198: The same – see above.
Line 209-211: The same – see above.
Line 215-219: These suggestions belong to the Discussion section. Please move this phares to Discussion section.
Discussion section
Line 428-445. These arguments are not connected in any way with the data obtained by the authors. In these paragraphs, the authors show data obtained on other substances. I recommend deleting or reducing these paragraphs.
Line 446-449. The authors already present these data in the Introduction section. This is duplication.
Line 456-458. The data obtained by the authors do not confirm this statement. Please correct or remove this phrase.
Line 458-460. It is not clear which namely studies or data obtained by the authors confirm this effect? ​​Please could Authors clarify?
Line 464: “as previously described [26, 27].”. From the sentence (Line 462-464), it is not obvious why the authors provide references to these works? Please correct this sentence (Line 462-464).
Author Response
MS# anesthres-3136949-R1
Title: “Suppression of the Excitability of Rat Nociceptive Secondary Sensory Neurons following Systemic Administration of astaxanthin　in Rats
Authors: Risako Chida, Sana Yamaguchi, Syogo Utugi, Yukito Sashide and Mamoru Takeda
***********************************************************************************************************Reviewer #2:
Current Ms is devoted to in vitro effect of astaxanthin on rat Cav channels. The data obtained by the authors show suppression of SpVc nociceptive transmission. The authors suggest that AST can be considered as an alternative therapeutic agent for the treatment of trigeminal nociceptive pain. Although this suggestion should be verified in future, in my opinion this manuscript is suitable for publication in Anesthesia Research journal. See my concerns below.
Answer
We appreciate Reviewer’s careful reviews on our manuscript. We have carefully checked reviewer’s constructive and helpful comments and made necessary corrections to revised manuscript (marked by red font) We believe that your comments helped to improve our manuscript. According to editor’s suggestion, concerning self citation and similarity rate, we had revised the style of the parts of the main text that overlap with previous papers (marked by blue font) in revised manuscript, as much as possible. Also, to reduce the number of self-citation paper, we have rearranged the references.
- In the Introduction section, authors often go off from main idea of MS. I have provided some recommendations below, but Authors should still significantly reorganize the Introduction section according to the journal's guidelines.
Answer
Thank you for reviewer’s thoughtful comments
Comment 1
Line 44-46. This sentence is not related to the following idea of paragraph. Please, remove this sentence.
Comment 2
Line 45-46: “especially after the failure of conventional Western medicine”. It is nor politically correct phrase. Please, could you rephrase this view?
Answer (comments 1 and 2)
According to reviewer’s comment, we have deleted following sentence and references (page 1);
Complementary alternative medicine (CAM) therapies, such as herbal medicines and acupuncture, are often used in pain management, especially after the failure of conventional Western medicine or when adverse side effects are a concern [8-12].
Comment 3
Line 46-49. There is no logical connection between this sentence and the following ones. Please remove this sentence from the MS.
Answer
According to reviewer’s comment, we have deleted following sentence and references (page 2); Previous studies have reported that various dietary constituents can potentially affect protective biological mechanisms, such as those in the cardiovascular, neural, and anticancer systems [13,14]
Comment 4
Line 51-53: Merge to one sentence
Answer
According to reviewer’s comment, we have merged as follows (pages1-2); AST is a more powerful antioxidant than other carotenoids, including lutein and zeaxanthin [7-9] and has many biological activities, including anti-inflammatory, anti-tumor, anti-diabetic and immunomodulatory effects [10-13].
Comment 5
Line 62-67. It is unclear why the authors cite data from their previous study. After all, the current study is devoted to Astaxanthin and not "theanine, resveratrol and α-lipoic acid".
Answer
According to reviewer’s comment, we have deleted following sentence and references (page 2); In a previous study, we showed that acute intravenous administration of natural products, such as dietary constituents, theanine, resveratrol and α-lipoic acid, suppressed nociceptive SpVc WDR neuronal activity in a concentration–dependent manner by inhibiting Cav and glutaminergic noxious synaptic transmission in the SpVc, implicating natural products as a potential complementary and alternative therapeutic agent for the treatment of trigeminal nociceptive pain [26-28].
Comment 6
Line 68: “these findings”. What data are Authors talking about? Please clarify.
Answer
According to reviewer’s comment,to ,to make clear the contents of paragraph, we have deleted following sentence (Page 2); Taken together, these findings strongly suggest that systemic AST administration may suppress sensory transmission, including nociception, by blocking Cav channels and glutamate neuronal transmission.
Comment 7
Line 68-74. This paragraph should be completely rewritten. The last paragraph in the Introduction section, as a rule, briefly explains what the study is about and (also very briefly) what data was obtained in general. For more details, see the MDPI tutorial for Authors.
Answer
According to reviewer’s comment, we have rewritten as follows (page 2); Thus, the aim of the present study was to investigate whether acute intravenous administration of AST in rats could attenuate the excitability of nociceptive SpVc WDR neuronal activity in vivo in response to mechanical stimulation. In the present study, we found that the mean firing frequency of SpVc WDR neurons in response to mechanical stimuli was inhibited by AST in a dose-dependent and reversible manner, implicating AST as a potential therapeutic agent for the treatment of trigeminal nociceptive pain without side effects.
- In the Material and Discussion section, the authors discuss the data obtained in previous studies with the current one. Such discussion should be moved to the Discussion section. Or these discussions should be reduced to a minimum.
Answer
Thank you for reviewer’s thoughtful comments
Comment 8
Line 76: “reported herein” such phrases have to be removed all over the MS. It is already clear to the reader what kind of research is being discussed.
Answer
According to reviewer’s comment, we have removed following phrase (page 2) “reported herein”all over the MS.
Comment 9
Line 100: “(1986)” replace to numeric reference.
Answer
According to reviewer’s comment, we have revised as follows (page 2); Paxinos and Watson [18].
Comment 10
Line 113: “We have previously identified”. If Authors are talking about a preliminary experiment within the current work, then it is necessary to describe in the supplementary materials? Else - Authors have to provide a link to the work.
Answer
Since this section describes the criteria for identifying WDR neurons, we have rewritten it as follows (page 3): In this study, we identified the criteria for WDR neurons as follows: graded non-noxious and noxious mechanical stimulation applied to the receptive field produces increased firing frequency in proportion to stimulus intensity.
- In the Result section, the Authors duplicate the description of the figures, which is already given in the figure captions themselves. The same phrase Authors have to avoid in Result section of MS. The Authors also partially discuss and compare obtained results with previous data. In the Result section, the Authors should pay more attention to the results they obtained, and all reasoning should be done in the Discussion section.
Answer
Thank you for reviewer’s thoughtful comments
Comment 11
Line 151, Line 202: “as described previously” such phrases are a discussion element. I suggest either deleting this phrases or moving it to the Discussion section. Authors should do this all over the MS.
Answer
According to reviewer’s comment, we have removed following phrase “as described previously（pages 3）
Comment 12
Line 159-161: This sentence is a caption to the figure Fig1D. From the caption of the figure itself it is obvious that the relationship between mechanical stimulation intensity and mean discharge frequency is shown. Please remove this sentence. Please, check all over the MS and remove similar sentences (phrases) from Result section.
Answer
According to reviewer’s comment, we have removed following sentence (page 3); Fig. 1D shows the relationship between mechanical stimulation intensity and mean discharge frequency (stimulus-responsive curve) for WDR neurons.　
Comment 13
Line 196-198: The same – see above.
Answer
According to reviewer’s comment, we have removed following sentence (page 4); Fig. 2 shows a typical example of the effect of intravenous administration of AST (5 mM) on the excitability of SpVc WDR neurons in response to non-noxious mechanical stimulation.
Comment 14
Line 209-211: The same – see above.
Answer
According to reviewer’s comment, we have removed following sentence (page4); Fig. 2 also shows typical examples of the effects of intravenous injection of 5 mM AST on the excitability of SpVc WDR neurons in response to noxious mechanical stimulation (15-60g).
Comment 15
Line 215-219: These suggestions belong to the Discussion section. Please move this phares to Discussion section.
Answer
Concerning the sentence “There were no significant changes in the size of the receptive field before and after AST administration, as previously described [26-28]”, We believe that content was belong to results section. So we have removed the phrase “as previously described [26-28]”. We have revised as follows (page 4); “There were no significant changes in the size of the receptive field before and after AST administration
Concerning the sentence “Local injection of vehicle (DMSO) had no significant effect on spontaneous or non-noxious, noxious mechanical stimulation-evoked SpVc WDR neuron activity (n = 3), as described previously [26-28], since we have already described for vehicle effect, we have revised as follows; “Local injection of vehicle (DMSO) had no significant effect on spontaneous or non-noxious, noxious mechanical stimulation-evoked SpVc WDR neuron activity (n = 3).
- Discussion section
Line 428-445. These arguments are not connected in any way with the data obtained by the authors. In these paragraphs, the authors show data obtained on other substances. I recommend deleting or reducing these paragraphs.
Answer
According to reviewer’s comment, we have deleted following paragraph (page 8); Complementary alternative medicine (CAM) is currently defined by Western medicine as a medical system that has not been scientifically tested or clinically applied. CAM is mainly used for herbal medicines and acupuncture. Recently, CAM has been actively used for patients with symptoms for whom Western medical treatments, such as drug therapy, do not work, and CAM is expected to be useful for the treatment of chronic pain [8-10]. Previous studies have reported that various dietary constituents can potentially affect protective biological mechanisms, such as those in the cardiovascular, neural, and anticancer systems [13,14].
Comment 16
Line 446-449. The authors already present these data in the Introduction section. This is duplication.
Answer
According to reviewer’s comment, we have deleted following sentences (page 8) ;AST is a naturally occurring carotenoid widely distributed in a variety of living organisms, such as plants, microalgae, crustacean shells, and salmon [15]. It has been demonstrated that AST has many biological activities including anti-inflammatory, anti-tumor, anti-diabetic, and immunomodulatory effects [20-22, 19].
Comment 17
Line 456-458. The data obtained by the authors do not confirm this statement. Please correct or remove this phrase.
Comment 18
Line 458-460. It is not clear which namely studies or data obtained by the authors confirm this effect? ​​Please could Authors clarify?
Answer (comments 17 and 18)
According to reviewer’s comment, we have deleted following sentences (page 8); Since it has been demonstrated that glutamate receptor antagonists played an important role in the management of migraine [37] and systemic administration of AST suppressed excitatory synaptic transmission, including NMDA receptors, it can be assumed that AST might alleviate primary headache syndrome, such as in migraine and cluster headache. In other words, AST may have an effect equivalent to that of ketamine, NMDA receptor blockers, or intravenous anesthetic drugs.
Comment 19
Line 464: “as previously described [26, 27].”. From the sentence (Line 462-464), it is not obvious why the authors provide references to these works? Please correct this sentence (Line 462-464).
Answer
In this study, we found that acute intravenous AST administration suppresses the SpVc nociceptive transmission, possibly by inhibiting Cav channels and excitatory glutamate neuronal transmission. In the previous studies, we demonstrated Iontophoretic application of L-glutamate induced the mean firing frequency of SpVc WDR neuron responding to noxious mechanical stimulation was also inhibited by intravenous administration of resveratrol [19]. In future, we are planning that whether Iontophoretic application of L-glutamate induced the mean firing frequency of SpVc WDR neuron responding to noxious mechanical stimulation was also inhibited by intravenous administration of AST.
So, we have deleted some sentenses and revised in this paragraph as follows (page 8); Although in the present study we could not examine whether AST administration attenuated the inflammatory pain, we found that acute intravenous AST administration suppressed the SpVc nociceptive transmission, possibly by inhibiting Cav channels and excitatory glutamate neuronal transmission, implicating AST as a potential therapeutic agent for the treatment of trigeminal nociceptive pain without side effects. AST suppresses the production of cytokines such as prostaglandin E2 and tumor necrosis factor-α, which are produced in tissues during inflammation and enhance the inflammatory response, in a concentration-dependent manner [38]. Recent findings also produced evidence that AST administration alleviated neuropathic pain through inhibition of MAPKs and nuclear factor kappa B signal cascades in animal models [39]. Taken together, these findings suggest that AST alleviates pathological pain, including inflammatory pain, by suppressing the production of inflammatory signal mediators and inflammatory cytokines expressed in neurons and glial cells. However further studies are needed to elucidate the mechanism underlying the effects of AST on trigeminal inflammatory pain.
We have added to the discussion section 4.2. later parts of 3rd paragraph (page 8) as follows; Previously, using multi-barrel electrode, we demonstrated iontophoretic application of L-glutamate induced the mean firing frequency of SpVc WDR neuron responding to noxious mechanical stimulation was inhibited by intravenous administration of resveratrol [19]. Therefore, further confirmational studies are needed on whether AST modulates iontophoretic application of glutamate-induced SpVc neuronal discharge using multi-barrel electrode.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsAgree to publish the current version
Reviewer 2 Report
Comments and Suggestions for AuthorsNow MS is looking good. Authors implemented all of my recommendations.
