Author's response to reviews
Title: Protective Effect of Edaravone for Tourniquet-Induced
Ischemia-Reperfusion Injury on Skeletal Muscle in Murine Hindlimb
Authors:
Kazuichiro Hori (khori@hsph.harvard.edu)
Masaya Tsujii (m-t727@clin.medic.mie-u.ac.jp)
Takahiro Iino (tiino@clin.medic.mie-u.ac.jp)
Haruhiko Satonaka (stnkhcyk@clin.medic.mie-u.ac.jp)
Takeshi Uemura (jpywf380@yahoo.co.jp)
Koji Akeda (k_akeda@clin.medic.mie-u.ac.jp)
Masahiro Hasegawa (masahase@clin.medic.mie-u.ac.jp)
Atsumasa Uchida (atsumasa@mie-u.ac.jp)
Akihiro Sudo (a-sudou@clin.medic.mie-u.ac.jp)
Version: 4 Date: 22 February 2013
Author's response to reviews: see over

Dear Dr. Homa Tajsharghi,
Attached is our revised manuscript of ‘Manuscript ID: 1649706976869862. We revised the manuscript according to the suggestions by reviewers and replied to all the questions raised by them. We totally agree with your comments and attached point by point responses. We would appreciate your consideration of our manuscript for publication in
The Journal of BMC Musculoskeletal Disorders.
Sincerely,
Corresponding author: Masaya Tsujii
Department of Orthopaedic Surgery,
Mie University Graduate School of Medicine
2-174 Edobashi, Tsu city, Mie prefecture 514-8507, Japan
Tel: +81-59-231-5022
Fax: +81-59-231-5211 e-mail: m-t727@clin.medic.mie-u.ac.jp

Response to Reviewer 1
Materials and Methods
Comment 1: Was there any animal lost during the experiment?
Response 1: None of the Animals were lost in the 1.5h model for this study. We have added the sentence as suggested by reviewer. (page12, line163) In hindlimb muscle IRI research, multi-system organ failure called crush syndrome is the most important complication as you say. We have been performing in the models with 6h ischemia and found some lost animals. Thank you for your comments.
Comment 2: Could they explain the interest of closing the artery and vein? The vein is a fragile vessel, and the authors have a risk of important lesions.
Response 2: As the reviewer says tourniquet use during long periods is an important risk of thrombosis, especially at deep vein. Nevertheless we could not find the thrombosis in the leg analyzed in this research. We added the sentence about the thrombosis. (page12, line172) Furthermore, we believe that the model in this study had fewer risk of thrombosis because the orthodontic rubber band had less tension than the classical band as reported by Crawford et al. Please refer to reference 5.
Comment 3: How did they control the reperfusion? About animals: the authors said that they anesthetized animals with pentobarbital.
Response 3: We added the sentence about how we conducted reperfusion. (page7, line95) We duplicated crawford et al method.
Comment 4: When they reperfused, were the animals maintained anesthetized? Or have they injected some antalgics?
Response 4: Mice remained anesthetized throughout the duration of ischemia. (page7, line94) We did not apply any antalgics.
Comment 5: How are the drugs prepared and dissolved ? which solvent is used to dissolve and inject the drugs into the mice. Were the control mices treated with solvent
(solvent control) ?
Response 5: We have added a paragraph of drug administration methods. How we prepared the drugs as pointed by the reviewer. (Page8, line100)
Comment 6: This sentence is not clear “ The other 3 animals not subject to I/R injury

served as normal control.”
Response 6: Normal control was used for pathological analysis of the hindlimb muscle in our preliminary experiment for the determinant in the proper size of TA and GC number of field (GC15, TA10). Therefore, the sentence has been deleted. We apologize.
Results and Discussion
Comment: It is unclear why these investigators chose to study the effect of Edaravoneon iNOS protein expression (Figures 6) but not on endothelial nitric oxide synthase
(eNOS) protein expression. The eNOS is known to induce synthesis/release of NO in endothelial cells and NO is known to cause vasodilation and angiogenesis. Therefore, it is more meaningful to study the effect. Edaravone on eNOS than on iNOS protein expression.
There is no real discussion.
Response: We totally agree with your comments. Nevertheless, this time we concentrated on inflammation, so we focused on iNOS. In a separate study we hope to examine the effect of eNOS protein expression. Therefore, we added the sentence about the importance of iNOS in I/R injury of skeletal muscle using the reference regarding
NO in skeletal muscles of iNOS knockout mice. (Page18, line274)
References
Comment: References should be checked, because they aren’t up-to-date.
Please check Ref 25. Effect of a novel free radical scavenger, edaravone
(MCI-186), on acute brain infarction. Randomized, placebo-controlled, double-blind study at multicenters.
Edaravone Acute Infarction Study Group.
Response: We have updated our references by including the most recent publication we could find. Including the author Reference25 has been changed to Reference23.
Response to Reviewer 2
Comment(1): In the Materials & Methods section, the description of pre-treatment needs to be re-written and clarified.
Response(1): We have rewritten in the animal models including the application of the drug, and added the sentence about methods of drug administration as suggested by reviewer. (Page8, line100) Furthermore, we have clarified the clinical pretreatment in methods. (Page7, Line86)
Comment(2): Labelling of figures is incorrect throughout the article. Figures are

labelled 1-8 in the article but are labelled 1-5 by the authors. This needs to be corrected as references to figures throughout the article are inappropriate.
Response(2): We apologize for the confusion that resulted while uploading images to the journal’s website. We have re-labelled the figures and updated the manuscript accordingly.
Comment(3): In Fig 4 (as labelled in the article), error bars and asterisk need to be
Corrected
Response(3): We apologize for out of alignment, we have corrected error bars and asterisk in Fig 4. Because of format application, Figgue4 has been changed to Figure5.
Comment(4): A key to the final two figures is needed to demonstrate Ed and Saline groups
Response(4): We have added demonstration of Ed and Saline groups in Figure 3A B.
Discretionary Revisions:
(1) Perhaps a more detailed discussion of Edaravone would be useful. What is its mechanism of action, usage in Japan, usage internationally, side-effects, half-life, etc?
Response(1) We have added effect of edaravone in I/R injury to discussion.(Page17, line243) Edaravone exerts neuroprotective effects by inhibiting endothelial injury and by ameliorating neuronal damage in brain ischemia. Widely used for stroke in Japan.
(2) p-values for results of significance could be included
Response(2) Thank you for your comments. We have added p-values.
(3) Discussion could be expanded regarding research to date on how to attenuate effects of I/R injury and the results other investigators have achieved
Response(3) We added further clinical possibility of edaravone to I/R injury in discussion. (Page17, line255)
(4) Perhaps a comment from the authors on where they see their research going in the future - ? human trial
Response(4) Now we are conducting the timing of conditioning, and in the future we hope to design a human trial to prevent compartment syndrome and crush syndrome.