Response to Reviewers

PONE-D-22-08060

Effect of preconditioning on propofol-induced neurotoxicity during the developmental period

PLOS ONE

Dear Dr. Shibuta

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Response

We submitted a copy of the ARRIVE guidelines with this letter. Thank you.

3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

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Response

We submitted data set as files “PPF 100n.xlsx”, “PPF-PCーSR-PO.xlsx” and “PC-PPF(imaging)-PO.xlsx”

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Response

In accordance with your suggestion, we deleted “data not shown” and revised.

[Note: HTML markup is below. Please do not edit.] Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: Partly Reviewer #2: Yes Reviewer #3: No

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes

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Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Response to Reviewer #1:

Thank you very much for your valuable comments. Your comments have helped us improve our manuscript. We have stated the changes made to the manuscript and have answered your questions below. I hope these revisions meet your approval.

All abbreviations should be used full name in first using in abstract, introduction, and ... .

Response

We confirmed that all abbreviations were spelled out at their first occurrence, as per your comments.

Why was the PPF (PPF-PC) at 100 nM or 1 μM concentration diluted DMSO+PBS regimen chosen?

Response

PPF at 1 μM did not kill neurons significantly in our preliminary experiment. This concentration was the maximum concentration that did not affect the survival rate of the primary cultured neurons. However, 10 μM PPF killed neurons significantly. We also used PPF at 100 nM in case PPF 1μM affected neuronal survival (but it did not) or PPF-PC improved survival rate (but it did not).

Why were the 17-day chosen to remove the fetuses?

Response

Cultures of the embryonic and fetal mammalian central nervous system have been used in many experiments due to its reliability, while the in vitro maintenance of adult mammal neurons has hitherto been largely unsuccessful. We identified that E16–E17 was the best survival rate until 28DIV. The brains of fetuses on E<15 were too small to remove, and therefore, sometimes, we could not collect as many neurons as we anticipated.

What was the criteria for identifying a neuron?

Response

As we previously described, in order to confirm the purity of the neuronal culture, cells were immunostained with anti-MAP2 or anti-GFAP antibody, before and after the experiment. More than 97% expressed MAP2 and less than 2% of the cells expressed GFAP, regardless of the duration of the experiments. This demonstrated that most of the cells in our cultures were neurons. (J Neurosci Res. 72(5):613-21, 2003; Br. J. Anaesth. 104: 52-58, 2010)

The other techniques such as western blotting or RT-PCR should be used to confirm that cell death pathways (apoptosis, ..) is not involved in the administration of PPF.

Response

Moderate to high concentrations of PPF administration during the developmental period (till DIV8) are involved in neuronal death, as shown in our previous reports (Neurotoxicology 69: 320-9, 2018). In the present study, our main purpose was to investigate whether PC alleviates PPF-induced neurotoxicity but not to elucidate cell death pathways. To minimize the number of experimental animals we used, we did not investigate death pathways in the present study.

The magnification and quality of fig. 4 and Fig. 5 low. The scale bar or magnification should be added to the figures.

Response

We added scale bars in the Figures following your valuable comments.

What is the main difference between the present study and reference number 9?

Response

Reference 9 (our previous report, 2018) demonstrated that exposure to moderate – high doses of PPF during the early developmental period leads to neural death. Based on this finding, we tried preconditioning treatments to alleviate this PPF-induced neurotoxicity. PC with low-dose NMDA was effective against subsequent glutamate insults, as Sragovich et al (2012) demonstrated. However, our result showed that PPF-PC did not improve survival rate of the neurons from later moderate – high doses of PPF exposure during the early developmental period.

Thank you very much for your valuable comments, again.

Reviewer #2: Dear editor,

The study entitled “Effect of preconditioning on propofol-induced neurotoxicity during developmental period” presented a set of results showing that low concentrations of propofol using a preconditioning treatment did not alter the cytotoxic effects of propofol in moderate and high concentrations in fetal rat brain cells. The methodology used is satisfactory and in accordance with the conclusion presented. The manuscript is informative about the neurotoxicity shown by propofol during the embryogenesis of rats. After a minor review, the manuscript should be considered in PLOS ONE Journal.

Response to reviewer #2

Thank you for your valuable comments. Your comments encouraged us to improve our manuscript.

We have stated the changes made to the manuscript and have answered your questions below. I hope these revisions meet your approval.

Minor points:

In the introduction, please provide more robust data from literature where preconditioning treatments were positively functioning against a certain drug cytotoxicity.

Response

According to your helpful suggestion, we have added two references (Sragovich, S. et al 2012 and Navon et al 2012). The addition of these two references helped improve our manuscript significantly.

Please provide the chemical structure of propofol.

Response

According to your comment, we have provided the chemical structure of PPF as Figure 1.

Page 9, line 139: How many rat fetuses were used to obtain the 46 culture dishes of neurons?

Page 10, line 155: How many rat fetuses were used to obtain the 69 culture dishes of neurons?

Response

I am unable to provide the exact number of rat fetuses. However, we usually obtain approximately 20–30 cultured dishes from one pregnant rat. Each pregnant rat has 4–12 fetuses. In the present study, we confirmed that we purchased and used 12 pregnant rats in total. We mentioned this in the materials and method section of the revised manuscript.

Reviewer #3: The present study describes some interesting phrmacology of propofol in its neurotoxic capacity in the early development in experimental animals.

The works are interesting for further considerstion. However, befor the animal study, some basic and bridge translation between in vivo and in vitro are required. The [Ca2+] internalization via its specific chanels are not defined, although the event is extremely important. In addinio, its coupling with Na+ and K+ trsansporters are not explained through the experimental design. Other basic approaches they assessed are encouraged.

In conclusion, the study is interesting in our readers but the present form is too premature to justify the claims.

Response

Thank you very much for your valuable comments.

The involvement of L-type calcium channels in PPF-induced neurotoxicity has already been reported by Kahraman et al (2008). Our preliminary studies also showed the same result, therefore, we did not perform further investigations on this. We understand that a major limitation of this experiment is that the degree of effect on cortical neurons in vitro may not necessarily correlate with the neurotoxic effect in vivo. In addition, we comprehend the importance of the involvement of Na+ and K+ transporters in PPF-induced neurotoxicity to clarify the neurotoxic mechanism as you mentioned. We are greatly inspired by your valuable comments for our future study. Still, we believe the alleviation of neurotoxicity is one of the main interests of not only anesthesiologists, pediatricians, and obstetricians, but all medical doctors. Since preconditioning is considered to be one of the useful therapies, in addition to hypothermia and medication as a neuroprotection tool, we believe PlosOne will provide the appropriate audience for our research.

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Reviewer #2: Yes: Leonardo Pereira Franchi Reviewer #3: Yes: Cheorl-Ho Kim

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