Attempt to Develop Rat Disseminated Intravascular Coagulation Model Using Yamakagashi (Rhabdophis tigrinus) Venom Injection

Round 1

Reviewer 1 Report

Thank you for this interesting research study. I would like to see it published and respect the challenge of writing in a secondary language. 

With respect to content: 

1. The title, as written, suggests this is a new model for venom-induced DIC. I think the authors mean to say something like: "A rat model of Yamakagashi venom induced DIC" This is specific to the study and it is reasonable for the authors to assert/discuss the general applicability of their model to others. 
2. DIC is seen in vipers, colubrids and elapids. Within the text, it is often confusing if the authors are making a general statement about venom induced DIC or misidentifying the classification of the snake within the sentence (e.g. Line 179 would lead casual reader to impression that the snake venom in this study is a viper). 
3. The authors observe that the time course to DIC is faster in their model than observed in clinical practice. This observation may be explained by the fact that they are injecting the animals intramuscularly, but that would not likely ever happen in a human patient. It could, however, happen in a small prey item such as a rodent. This is a minor point, but should be in the discussion. Lines 197-2xx attribute this to the quantity of venom administered. This likely accounts for the reproducibility, but is an incomplete explanation for the time course compared to humans.  
4. I agree with the authors that this venom could be an excellent general; model for venom-induced DIC. This is an important point that can be made in the abstract conclusion and in the discussion. The authors, however, do not take the opportunity to suggest what they might do with such a model. For example, the effect of different antidotes could be tested. Varespladib is being tested for snakebite envenoming, as are metalloprotease inhibitors. This could be a great model for testing the effects of small molecules on venom induced DIC (Review of small molecule inhibitors by Bulfone in 2018. Additional recent papers by e.g. Bittenbinder on Factor X 2018 (and others even more recently by the Fry group e.g. Zdenek 2020), Alangode Russell's viper (2020), another colubrid the Banded cat-eye snake (Torres-Bonilla 2020) and a fascinating combination of inhibitors in Nature Communications last month (Albescue 2020 with the Casewell group in Liverpool as well as those by Wang 2018, 2020 and Xie et al also 2020). Given that colubrid bites represent a minority of bites and are unlikely to attract antivenom manufacturing for a specific antidote--understanding how this model can be used to test for paraspecificity of antivenoms or newer approaches (Laustsen) make this model appealing will help the authors gain a wider audience and also attract readers to this interesting paper. 

Really nice work. I think it should be published but requires significant editing and some focus on the potential for broad utility of this particular venom to be a model for venom-induced DIC if that is what the authors think is a reasonable use for their venom and DIC model. Given the small number of venom DIC models in the literature, it is conceivable the authors could make a table summarizing the venoms, method of venom administration and studies. This is a very very low level suggestion (ie if the authors were inspired to do so). 

I hope these are helpful comments. I will be happy to review this paper again. 

Author Response

1. DIC is seen in vipers, colubrids and elapids. Within the text, it is often confusing if the authors are making a general statement about venom induced DIC or misidentifying the classification of the snake within the sentence (e.g. Line 179 would lead casual reader to impression that the snake venom in this study is a viper).

"Snakebite symptoms vary depending on the type of venom possessed by the venom-ous snake, but some venomous snakes, such as vipers, colubrids" because the word "viper" was inappropriately used in the discussions pointed out. we changed it to something like "and elapidsvipers exhibit DIC symptoms".  

2. It was pointed out that there was insufficient consideration of the difference between the elapsed time of DIC in humans and the DIC speaking time in this rat model.

   We added a discussion explanation to this point.

3. Pointed out that there is not enough consideration on how to use the rat model created in this paper.

Using the literature on reports of treatments for snakebites using small molecule antidotes, which was given as a hint, we added that it is possible to select antidotes using this model.

Reviewer 2 Report

The authors present an in vivo investigation wherein they claim to have created a rat model of VICC with Rhabdophis tigrinus venom.  While they have made a good start, this work is far from complete.

Introduction

1. Please indicate explicitly why this model was created. State a hypothesis or justified goal.

Results

1. Figure 1. Indicate the number of rats per group in figure legend.
2. Table 2. Indicate the number of rats per group in legend.
3. Figure 2. Indicate what panel A represents. Indicate the number of rats per condition, present mean and standard deviation.
4. Figure 3. Indicate the number of rats per group. Mean and SD?
5. Figure 4. Indicate the number of rats per group. Mean and SD?
6. Figure 5. Rat number? Why does panel A have mean and SD but the other two do not?
7. Why are there no statistical methods used in this work? Why did the authors only use 1-3 rats per condition, making it impossible to compare conditions statistically? It is typical to have at least n=6 to have any sort of statistical power or significance in such a model.

Given the very preliminary nature of this investigation, while it is likely that the authors have created a model to test antivenoms or other interventions with this venom, we have no way of being assured the reliability and vigor of this model statistically.

Author Response

1. Pointed out in the introduction that the purpose of creating this model is inadequate.

Added the purpose of creating this DIC model to the introduction.

2. The following individual points regarding the results

• Figure 1. Indicate the number of rats per group in figure legend.

As you pointed out, we entered the number of rats used in legend.

• Table 2. Indicate the number of rats per group in legend.

As you pointed out, we entered the number of rats used in legend.

• Figure 2. Indicate what panel A represents. Indicate the number of rats per condition, present mean and standard deviation.

As you pointed out, we entered the number of rats used, the average value and SD in the legends.

• Figure 3. Indicate the number of rats per group. Mean and SD?

As you pointed out, we entered the number of rats used, the average value and SD in the legends.

• Figure 4. Indicate the number of rats per group. Mean and SD?

As you pointed out, we entered the number of rats used, the average value and SD in the legends.

• Figure 5. Rat number? Why does panel A have mean and SD but the other two do not?

As you pointed out, we entered the number of rats used, the average value and SD in the legends.

• Why are there no statistical methods used in this work? Why did the authors only use 1-3 rats per condition, making it impossible to compare conditions statistically? It is typical to have at least n=6 to have any sort of statistical power or significance in such a model.

As you pointed out, we could not add data for 6 animals in 1 group, but we have compiled data for 3 animals in all groups. We also performed a statistical analysis of the results.

Reviewer 3 Report

The authors of the review entitled "Establishment of rat disseminated intravascular coagulation model by Yamakagashi (Rhabdophis tigrinus) venom injection " described a work based on IM administration of a fixed amount of R. tigrinus venom and resulted in changes in blood clotting and fibrinolytic factors over time resembling human blood pathology in the snake bite. In general, the study is important because the authors consider that the use of the rat model is very effective in validating the therapeutic effect of the human disseminated intravascular coagulation condition due to snakebite. This model could be used to analyze snake related DIC therapy.

The manuscript is well written, the biological analysis was well carried out, and the results are interesting. There are minor comments as follow:

1. It is important to remember that scientific names must be written in italics, see on page 1, in the title of the manuscript and on line 7, the scientific name Rhabdophis tigrinus does not appear in italics.
2. It is clear that there are limitations in the use of the DIC rat model that the authors have proposed, since the male SD rats used in the experiment are 12 weeks old, although it is logical that the size of the rat should be large. I suggest the authors to do the experiment using other more common rat strains, such as Wistar rats.
3. Is there any report about the chemical profile of the Yamakagashi venom? If yes, the authors must expand the discussion and explain those.

Overall, the manuscript is good and should be accepted with minor corrections.

Author Response

1. It is important to remember that scientific names must be written in italics, see on page 1, in the title of the manuscript and on line 7, the scientific name Rhabdophis tigrinus does not appear in italics.

As you pointed out, the scientific name of the snake is italicized.

2. It is clear that there are limitations in the use of the DIC rat model that the authors have proposed, since the male SD rats used in the experiment are 12 weeks old, although it is logical that the size of the rat should be large. I suggest the authors to do the experiment using other more common rat strains, such as Wistar rats.

The reason for using SD strain rats instead of Wister strain rats was added at the end of the discussion.

3. Is there any report about the chemical profile of the Yamakagashi venom? If yes, the authors must expand the discussion and explain those.

Added the scientific profile of the Yamakagashi toxin you pointed out to the Introduction.

Reviewer 4 Report

The manuscript entitled "Establishment of rat disseminated intravascular coagulation model by Yamakagashi (Rhabdophis tigrinus) venom injection" describes the analysis of Yamakagashi snake venom regarding its lethal dose and the coagulation effects. The aim of the study was to design a DIC rat model. In general, I believe that thisa topic of general interest to Toxin reads, since there are a few studies with colubridae venoms. However, the manuscript need an extensive revision in terms of methods and writing. Firstly, there are many missing words, and some paragraphs are difficult to understand. Secondly, some methods described need to be improved - for example, intravenous injection group has only one animal. Is that correct? Could it be called as a "group"?  Also, there is no statistical analysis.

I listed a few points below that I believe should be worked on the manuscript for the next submission. The manuscript must be improved before being considered for publication, which include more in vivo experiments for some groups with low number of animals, and the statistical analysis of data presented.

General points

• In case of hemorrhagic viperidae venom, it is common to observe dermonecrotic activity on the local which the venom was injected. In the introduction section, it was said that this venom has hemorrahagic activity and I am curious to know if the authors observed this effect during their experiments.
• You can used "envenomation / snake envenomation" instead of snakebite. When the physiopathological effects of venom are described, this is a more suitable term, since not all snakebites lead to envenomation (check Naik, 2007 work).
• I consider that statistical analysis is very important in case of in vivo experiments, since it is known the biological variation among animals. This helps to validate the observed data, and avoid wrong conclusions. Also, a similar number of animals in all group is important ( there are groups with only 1 or 2 animals that makes the group sample too small).
• To validate the DIC rat model for this snake venom, would be interest to perform a parallel experiment using a validate model (such as endotoxin, for example). 

Reference:

Naik, B. S. (2017). “Dry bite” in venomous snakes: A review. Toxicon, 133, 63-67.

Materials and Methods

Methods section should be better elaborate, and the text needs to be revised also.

1. In “5.2. Snake venoms”, how many snakes had their gland extracted (or an estimation)? Were snakes both males and females?
2. Line 243 – “R, tigrinus”

There is a typo – the scientific name should be R. tigrinus

3. In “5.3. Anti-snake venom equine antibody” What is the ED50 dose? Please, include this information.
4. Line 247 "The intravenous LD50  value of the venom was 5.1 μg/20 g mouse [26]"  Please,  revise the LD50 reference. I could not found this information in the [26] article. Is it correct?
5. In “5.4 - 5.4. Experimental protocols”

I don`t undestand why the experimental groups have different number of animals. Why intravenous injection group (a) has only one animal? Is that correct? The results of a single animal cannot be considered since there are many biological variations.  In the results section, it was said that 100% of the animals of this group have died, but this information is biased since only one animal was used. Moreover, the antivenom group (d) also has very few animals (n=2).

5. In “5.6. Principle of test in the measurement of PT” and “5.8. Principle of test in the measurement of D-dimer”, was the samples analyzed individually to ever biological replicates, or as group pool? Please, add this information in the text.
6. No statistical analysis was cited in the methods section. Please, include.

Introduction

1. I miss more details about the studied snake. What is the region(s) that Yamakagashi can be found? How many accidents are caused by this snake per year?

2. Line 35- “Venoms from snakebites have”

I suggest modify the text to “snakes” instead of “snakebites”.

3. Line 36-37 – “ (…) is the most common and most important”

Most important what? I think authors miss the word “symptom”, or else.

Results

1. Line 63 “Rats were administered 150, 200, 250, and 300 μg Yamakagashi venom”

This phrase needs to a grammar revision. My suggestion “Rats were administered with Yamakagashi venom with different amounts (150 , 200, 250 and 300 μg)

2. Line 74-75 “Moreover, when a specific antibody was administered to a rat to which 300 μg venom was administered”

Please, include in the results section that the antivenom was administred after 2 h of the envenomation.

3. Please include standard deviation in all graphs. Some of them have it , but other don't.

4. All figures should include statistical analysis. Are the results statiscially significant? 

Discussion

Discussion should be revised, since it is a little bit repetitive (there are copy of same paragraphs, such as line 154 and 185). This section should include the discussion of statistic results.

Author Response

1. General points

Of the items pointed out as general points, we corrected it according to the comment that "envenomation" is used instead of "snakebite". In addition, statistical calculations were carried out with the number of rats in one group being the same as three. However, we could not respond to the parallel experiment you pointed out in a short period of time. However, since many DIC models using rats have been published, it may be possible to cite them in the discussion of those documents.

2. Individual points

Materials and Methods

Methods section should be better elaborate, and the text needs to be revised also.

• In “5.2. Snake venoms”, how many snakes had their gland extracted (or an estimation)? Were snakes both males and females?

We have entered the information you indicated in the location of “5.2. Snake venoms”.

• Line 243 – “R, tigrinus”

There is a typo – the scientific name should be R. tigrinus

Corrected the comma you pointed out to a period.

• In “5.3. Anti-snake venom equine antibody” What is the ED50 dose? Please, include this information.

There is no description in 5.3. Regarding the ED50 you pointed out.

• Line 247 "The intravenous LD50 value of the venom was 5.1 μg/20 g mouse [26]" Please, revise the LD50 reference. I could not found this information in the [26] article. Is it correct?

The description of LD50 and the literature were incorrect, so we corrected them.

• In “5.4 - 5.4. Experimental protocols”

I don`t undestand why the experimental groups have different number of animals. Why intravenous injection group (a) has only one animal? Is that correct? The results of a single animal cannot be considered since there are many biological variations.  In the results section, it was said that 100% of the animals of this group have died, but this information is biased since only one animal was used. Moreover, the antivenom group (d) also has very few animals (n=2).

As you pointed out, we have compiled data for 3 animals in all groups.

• In “5.6. Principle of test in the measurement of PT” and “5.8. Principle of test in the measurement of D-dimer”, was the samples analyzed individually to ever biological replicates, or as group pool? Please, add this information in the text.

    No statistical analysis was cited in the methods section. Please, include.

The measurement of PT and D-dimer is performed here using plasma collected from individual rats instead of pooled plasma. we also added statistical calculations to the method part.

Introduction

• I miss more details about the studied snake. What is the region(s) that Yamakagashi can be found? How many accidents are caused by this snake per year?

We added to the introduction about the habitat of Yamakagashi and the number of bites per year.

• Line 35- “Venoms from snakebites have”

I suggest modify the text to “snakes” instead of “snakebites”.

"Snakebites" was a mistype. Fixed to "snakes".

• Line 36-37 – “ (…) is the most common and most important”

Most important what? I think authors miss the word “symptom”, or else.

As you pointed out, "symptom" was missing, so we added it.

Results

• Line 63 “Rats were administered 150, 200, 250, and 300 μg Yamakagashi venom”

This phrase needs to a grammar revision. My suggestion “Rats were administered with Yamakagashi venom with different amounts (150 , 200, 250 and 300 μg)

Fixed as you pointed out.

• Line 74-75 “Moreover, when a specific antibody was administered to a rat to which 300 μg venom was administered”

Please, include in the results section that the antivenom was administred after 2 h of the envenomation.

Fixed as you pointed out.

• Please include standard deviation in all graphs. Some of them have it , but other don't.

As you pointed out, the standard deviation is shown on all graphs.

• All figures should include statistical analysis. Are the results statiscially significant?

As you pointed out, we performed statistical analysis on the data written in all figures. As a result, it became clear that there was a statistically significant difference between the group receiving 300 ug of toxin and the other groups except for the D-dimer value shown in Figure 5.

• Discussion

Discussion should be revised, since it is a little bit repetitive (there are copy of same paragraphs, such as line 154 and 185). This section should include the discussion of statistic results.

Improved the overlap in the discussion part.

Round 2

Reviewer 2 Report

The authors likely need n=6 per group, they need to use repeated one-way and two-way analysis of variance (ANOVA) for their timed series, and an appropriate post hoc test (e.g., Tukey, SNK) is needed to detect differences within group over time and between group over time.  The authors need to indicate what statistical power they can calculate with this preliminary data (pick a couple of variables) and prospectively increase the number of animals per group accordingly.

Author Response

Thank you for your comment on the draft of Toxins-1064978.

Below is a response to each of the two reviewers who received many useful comments.

Reviewer 2.

• The authors likely need n=6 per group, they need to use repeated one-way and two-way analysis of variance (ANOVA) for their timed series, and an appropriate post hoc test (e.g., Tukey, SNK) is needed to detect differences within group over time and between group over time. The authors need to indicate what statistical power they can calculate with this preliminary data (pick a couple of variables) and prospectively increase the number of animals per group accordingly.

As you pointed out, the homogeneity of the variance of each data was tested by the Bartlett test. As a result, when comparing the data groups whose homogeneity was affirmed, the groups were compared by the two-way arrangement method. We also used the Mann-Whitney U test to compare groups for data for which homogeneity was denied. The result was reflected in the result of the treatise.

The data obtained in each experiment was analyzed one by one by following statistical procedures. As a result, in the revised version submitted last time, it was simply detected as a significant difference in examining the homogeneity of variance, but in this analysis, it was judged that there was no significant difference. Thank you for teaching us how to understand the significance of the result data.

Reviewer 4 Report

The authors made the corrections suggested by all reviewers. Introduction was significantly improved.

However I believe it is still necessary to explain a little bit more about your statistical analyses of the following experiments.

-Effects of platelet counts (item 2.2)

-Effects of prothrombin time (item 2.3)

-Effects of FIB concertation (item2.4) 

I see authors inserted the p value , which is below 0.05, however I was a bit confusing of what are you  were comparing. For example, line  150, there is the following insert  "((a) vs. (b)(c): p<0.05).)", however in all cases there is the following data:  (a) represents time course of 300 ug of venom injected, (b) shows time course of different concentrations (150 ug, 200 ug and 250 ug), and (c) time course of 300 ug + antivenom.

You cannot simply say it is statistically significant with so many variants (time, concentration...). Is the data statistically significant for all  time points? For all concentrations?  Why are you comparing the data (a) with (b) and (c)? It should be discuss in a paragraph in the manuscript. Since there are many time points and different concentrations, some of them may not be statistically significant, which is normal and expected.

According to the author`s response, they found that results are statistical significance, however it is necessary to write it in the manuscript. The only text I observed was for "Effects of D-dimer concertation", which had no statistical significance ( Line 170). 

In summary,  please clarify your statistical analyses results with a brief paragraph for each item I pointed out, and also discuss your data in discussion section!

Minor points:

-New table 1 has a typo - It is written "Grupe" instead of "group"

Author Response

Thank you for your comment on the draft of Toxins-1064978.

Below is a response to each of the two reviewers who received many useful comments.

Reviewer 4.

• In summary, please clarify your statistical analyses results with a brief paragraph for each item I pointed out, and also discuss your data in discussion section!

As you pointed out, we briefly added the statistical analysis results between each group to the description of the results of the effects of Rhabdophis tigrinus venom administration on platelet counts, prothrombin time and FIB concentration. Furthermore, we discussed the results of these statistical analyzes in the discussion.

2) Minor points:

-New table 1 has a typo - It is written "Grupe" instead of "group"

We have corrected the words in table 1 as you pointed out.

Round 3

Reviewer 2 Report

The authors tried to address my comments, and they provide the results with n=3 which sometimes do and sometimes do not show significant differences.  I have asked twice for the number of animals to be increased, but there seems to be no interest in doing this. 

I cannot imagine that the results have much statistical power, and I have asked for the authors to figure out what their power is so they can decide on the number of animals to use.  Again, they have not interest.

It would be best to have a statistician advise the authors to make this a top tier work.  If they continue to refuse, then then need to heavily condition their statements and declare their work very preliminary requiring more work to confirm their results given the variation between groups.

Author Response

Dear reviewer 2,

                                                                                                      Feb. 12th, 2021

Thank you for your comment on the draft of Toxins-1064978.

Below is a response to you who received many useful comments.

   We are very grateful to you for your very meaningful comments, including statistical comments.

   We would like to explain why we are cautious about increasing the number of animals to 6 in a group, which are repeatedly requested by reviewers. As the reviewer commented, since it is a paper using data obtained by conducting experiments with 3 animals in 1 group, the significance of the results cannot be strongly asserted. Regarding the conclusions, it was stated that the conclusions were not definitive and that there was a limit that clearly stated that further verification was necessary.

   Here, in order to obtain the experimental results of 6 animals in one group, a re-experiment of almost the same scale as the previous experiments will be performed. In order to carry out a re-experiment of the same scale, it takes at least 3 months or more to submit an animal experiment plan, arrange animals, conduct an experiment using received animals and analyze them at the institute. In addition, COVID-19 is currently in epidemic and there is currently no guarantee that the experiment will be carried out as planned. The experimental system we conducted here has 3 animals per group, but some stable results have been obtained. We would like to publish this data as a treatise once.

We would like to ask a judge based on the above points.

Best regards

Reviewer 4 Report

The modifications in the text regarding statistical analysis were made.

However, there still some minor points to improve, which are listed below:

The new paragraph in line 208-201 is not a discussion, is simply the description of results. I recommend removing it since it is expected that higher doses of venom are more lethal.

Also, line 216-237 is a summary of results; however, I recommend adding some insights - For example: what are the implications of the decrease in PLT? what are the possible explanations of no differences of D-dimer between groups?

Line 235-237 "Since the antivenom-administered group was administered at the peak of the appearance of D-dimer, it is considered that the neutralizing action of venom was not given time."

This phrase is a little bit confusing, should be rewritten. I suggest including a more in-depth discussion about clinical implications, which can validate the DIC model.

There are a few typos errors such as "ug" instead of  "µg" (greek letter for micro) (examples: line 209, line 201). Please, revise the text.

Author Response

Dear reviewer 4,

                                                                                                      Feb. 12th, 2021

Thank you for your comment on the draft of Toxins-1064978.

Below is a response to you who received many useful comments.

   We are very grateful to the reviewers for their meaningful comments to bring the paper closer to completion. This comment pointed out that there was insufficient consideration of the results obtained. We rewrote the consideration, including the statistical analysis results of the data obtained in each experiment.

   In addition, the error of ug pointed out as a minor point has been corrected to μg.

Best regards,

Yamamoto

Akihiko Yamamoto, DVD, Ph. D.

National Institute of Infectious Diseases