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  1. Author Response:

    Reviewer #1:

    In this work, the authors present a model for double nerve transfers in the forelimb of the rat. The authors provide a detailed description of how the model is developed and they characterize neuromuscular regeneration through nerve crush, neurotomy, behavioral analysis, and retrograde labeling. The peripheral innervation of muscle with a double nerve transfer is compared to that with a single nerve transfer.

    Major strengths:

    • Strong motivation for necessity of this model given
    • Experimental design and surgical techniques are clearly described. The authors include methodologies, materials used, figures, and supplementary videos to support the discussion of how the experimental model is developed.
    • Large number of animals are used for both the double nerve transfer and single nerve transfer, and results appear to be consistent within these populations.

    Thank you for your comments.

    Weaknesses:

    • The work assumes specialized knowledge of peripheral nerve anatomy and some surgical techniques. The article may be less accessible to someone without a background in these areas who seeks to learn more about nerve transfer models.

    Thank you for your feedback, which helped us to improve our manuscript! We agree that the work assumes some knowledge of peripheral nerve anatomy and some surgical techniques. For better understandability, we included more information on nerve transfers in the manuscript to make it more accessible to a wider readership. Please see p11 line 342-343.

    The authors do a rigorous job of describing the techniques used to develop the double nerve transfer model. The experimental design and surgical methods provide detailed accounts of how the model is realized, including descriptions of the techniques as well as highlighting materials that are necessary for the procedures. This is particularly valuable for a reader who desires to replicate this model. The efficacy of the nerve transfer is examined in multiple ways and compared to a single nerve transfer model. These results, which are statistically verified, demonstrate that the double nerve transfer more effectively reinnervates muscles and, in some measures, that there is no difference between animals with double nerve transfers and healthy comparisons. This provides confidence and excitement for how this model may be used in the future for studies involving therapeutics.

    We thank reviewer #1 for the thorough summary and for pointing out major strengths and room for improvement in our work. Indeed, our study proposes a model for single nerve and double nerve transfer to a single target muscle and aims to provide the detailed model for optimal reproducibility .

    Reviewer #2:

    The paper is well-written with sufficient sample sizes. The figures are generally clear and easy to understand.

    The clinical utility of multiple nerve transfers should be better delineated in the introduction. Current limitations, difficulties, and strategies to mitigate such limitations should also be overviewed to provide better context to the reader as to the importance of this work.

    Thank you for this helpful comment. We agree that the reader benefits from more information on the clinical utility of multiple nerve transfers and their limitations. Therefore, we revised the discussion (p8 line 280-284) and the introduction (p3 line 85-89) to better inform the reader and included current limitations.

    What were the relative proportions of innervation between the two nerves in the dual-innervation model based on the retrograde labeling?

    We agree that this is a very interesting aspect of double reinnervation nerve transfers. In this project, the experimental setup did not allow such an investigation yet. However, we are already planning to investigate this aspect via sequential double retrograde labeling (Katada et al., 2006). In addition, we are currently developing an EMG system to reliably quantify relative proportions of innervation. This project is currently in development and will be started following ethical approval and funding.

    Section 3.4.1: Please present the raw data and mentioned scatterplot? What was the correlation coefficient for the linear regression?

    We are pleased to fulfill this request. The raw dataset will be made available on Dryad. Please see Figure 2 – figure supplement 1 for the scatterplot and the correlation coefficients are as follows; SNT: R2 linear=0.390, DNT: R2 linear=0.516.

    Section 3.2. The distribution of scores from each group should be presented in a graphical or tabular format. What was the course of recovery? Were the evaluations performed anytime before 12 weeks?

    We included the data in table 1, as to your suggestion. The evaluation was conducted in all animals once at 12 weeks to see if any restrictions in motion persisted, which was not the case. For testing, evaluations were also performed in some randomly selected animals within the first weeks after surgery (which all achieved the maximum score). We only see a brief reduction in function during the initial healing phase, but not that can be attributed to the denervation of the target and donor nerve.

    Section 3.3: SNT vs DNT should be evaluated in a comparative fashion.

    Thank you for suggestions. We included this information in essential revision 1).

    Section 3.4 How is 'adequate' muscle fibrillation determined?

    Thank you for this important query. We defined an ‘adequate’ response as a macroscopically and clearly recognizable response similar to the control side. An inadequate fibrillation was defined as a non-observable or almost not existing contraction upon crushing. Judged by the two staff members grading the response independently, we observed reproducible and unmistakable responses in all cases. Please see Video 2 for an adequate muscle fibrillation in control animals.

    Was any electrophysiology performed to assess the quality of reinnervation and nerve conduction velocity? Compound muscle action potentials or motor unit counts would help identify the proportion of the muscle that was reinnervated.

    In the current study, we did perform first preliminary EMG analyses in selected animals to test a novel electrophysiology setup. Here, we did see proper EMG responses to stimulation. Due to a limited N and further improvements needed for thorough analyses, we did not include this data here. We are currently working on a next trial to properly assess EMG response, CMAP to stimulation of either donor nerve or both simultaneously. We look forward to these results, which we hope to publish in a follow-up study in the near future.

    Can the authors please comment on the way in which the DNT was adopted by the animals as opposed to the SNT? Was there any noticeable difference in the functional recovery of the muscle or retraining process? The neuroplastic adaptation would be an interesting characterization.

    Thank you for the interesting inquiries. We did not observe any behavioral differences nor in the functional recovery between the SNT and DNT group. Two motor nerves innervating the same muscle did not result in noticeable differences. However, neuroplastic adaptation has not been investigated in this work but future research focusing on that is necessary to characterize potential neuronal changes. In previous models with single nerve transfers to the lateral head of the biceps, even within the denervation phase, little burden to the animal was noted. We believe that the animal can easily compensate the loss of function and therefore, the double innervation may therefore not be evident in functional analyses.

    In the discussion, the authors suggest that "hindlimb models do not adequately represent the physiology of upper extremity nerve transfers and targeted muscle reinnervation procedures." based on outcomes for lower vs upper transfer. A number of additional factors, including usage of the limbs, weight bearing, sensorimotor circuitry etc. play a role and should be accounted for.

    We gratefully accept these proposals and included these additional factors in the discussion (p6 line 188-189)

    In the DNT model, was the distance between entry points of the coaptation held constant between animals or optimized? The increased muscle mass observed in the DNT group is likely a result of a better axon : myocyte ratio and spatial distribution. This could be studied and optimized to improve the outcomes and utility of DNT.

    We tried to keep the coaptation sites of the two nerves constant with approximately 2-3mm of distance to each other with the UN placed proximal and the AIN placed distal. We also believe that the increase in muscle mass observed in the DNT group is probably due to a better ratio between axons and muscle fibers. We look forward to incorporating these considerations into our next projects.

    The discussion should more thoroughly explore the limitations of this model and experimental constraints.

    We agree that a thorough discussion on the limitations of this model and the experimental constraints will improve the manuscript and have included and discussed relevant aspects in more detail. Please see p8 line 280-284.

    What remains to be optimized prior to clinical translation? What types of scientific questions can this help answer? In which types of clinical cases would DNT not be appropriate?

    We agree, many things about nerve transfer physiology are unknown but at the same time utterly fascinating. Before clinical translation, a clear understanding of the quality and quantity of innervation must be acquired. Furthermore, the question of which portions of the muscle are innervated by either of the two nerves and if influencing factors which modify this reinnervation process can be identified. It must also be investigated whether a patient can actually control the muscle voluntarily with two nerves after double reinnervation which is crucial for prosthetic interfacing. We believe that a thorough EMG analysis and the assessment of neuro-muscular-junctions by imaging may help answering these questions. In addition, double innervation may be important for prosthetic interfacing to acquire EMG signals rather than functional reconstruction of a muscle. This is another fascinating topic to investigate in the future.

    Reviewer #3:

    The authors aimed to establish a rodent upper limb model to test double vs. single nerve transfers, and provided base results for histological retrograde labeling, topographic findings, functional (behavioural) analysis and outcomes for reinnervated vs. control muscle mass. The manuscript is well balanced, and contains a detailed description to reproduce the experimental model.

    The authors demonstrated equal functional outcomes for both types of transfers and have in this reviewers opinion succeeded in establishing a novel model for future (experimental) tests before (clinical) application.

    Thank you for acknowledging the good balance and detailed description in our study, which we hope will help other researchers to use this model for their investigations. For this purpose, we provided diligent photo documentation in addition to an extensive description of the surgical nerve transfer procedures.

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  2. Evaluation Summary:

    The study uses a novel rodent surgical model for establishing that dual nerve transfer in the upper extremity improves neuromuscular regeneration in comparison to single nerve transfer. The authors provide a detailed description of how the model is developed and they characterize neuromuscular regeneration through nerve crush, neurotomy, behavioral analysis, and retrograde labeling. The nerve transfer method is clearly delineated for researchers to use in future scientific and clinical applications. The evidence clearly support the main study conclusions. Thus, this manuscript is of great interest to readers in the field of peripheral nerve repair and neural interfaces.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    In this work, the authors present a model for double nerve transfers in the forelimb of the rat. The authors provide a detailed description of how the model is developed and they characterize neuromuscular regeneration through nerve crush, neurotomy, behavioral analysis, and retrograde labeling. The peripheral innervation of muscle with a double nerve transfer is compared to that with a single nerve transfer.

    Major strengths:

    - Strong motivation for necessity of this model given
    - Experimental design and surgical techniques are clearly described. The authors include methodologies, materials used, figures, and supplementary videos to support the discussion of how the experimental model is developed.
    - Large number of animals are used for both the double nerve transfer and single nerve transfer, and results appear to be consistent within these populations.

    Weaknesses:

    - The work assumes specialized knowledge of peripheral nerve anatomy and some surgical techniques. The article may be less accessible to someone without a background in these areas who seeks to learn more about nerve transfer models.

    The authors do a rigorous job of describing the techniques used to develop the double nerve transfer model. The experimental design and surgical methods provide detailed accounts of how the model is realized, including descriptions of the techniques as well as highlighting materials that are necessary for the procedures. This is particularly valuable for a reader who desires to replicate this model. The efficacy of the nerve transfer is examined in multiple ways and compared to a single nerve transfer model. These results, which are statistically verified, demonstrate that the double nerve transfer more effectively reinnervates muscles and, in some measures, that there is no difference between animals with double nerve transfers and healthy comparisons. This provides confidence and excitement for how this model may be used in the future for studies involving therapeutics.

    Read the original source
    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    The paper is well-written with sufficient sample sizes. The figures are generally clear and easy to understand.

    The clinical utility of multiple nerve transfers should be better delineated in the introduction. Current limitations, difficulties, and strategies to mitigate such limitations should also be overviewed to provide better context to the reader as to the importance of this work.

    What were the relative proportions of innervation between the two nerves in the dual-innervation model based on the retrograde labeling?

    Section 3.4.1: Please present the raw data and mentioned scatterplot? What was the correlation coefficient for the linear regression?

    Section 3.2. The distribution of scores from each group should be presented in a graphical or tabular format. What was the course of recovery? Were the evaluations performed anytime before 12 weeks?

    Section 3.3: SNT vs DNT should be evaluated in a comparative fashion.

    Section 3.4 How is 'adequate' muscle fibrillation determined?

    Was any electrophysiology performed to assess the quality of reinnervation and nerve conduction velocity? Compound muscle action potentials or motor unit counts would help identify the proportion of the muscle that was reinnervated.

    Can the authors please comment on the way in which the DNT was adopted by the animals as opposed to the SNT? Was there any noticeable difference in the functional recovery of the muscle or retraining process? The neuroplastic adaptation would be an interesting characterization.

    In the discussion, the authors suggest that "hindlimb models do not adequately represent the physiology of upper extremity nerve transfers and targeted muscle reinnervation procedures." based on outcomes for lower vs upper transfer. A number of additional factors, including usage of the limbs, weight bearing, sensorimotor circuitry etc. play a role and should be accounted for.

    In the DNT model, was the distance between entry points of the coaptation held constant between animals or optimized? The increased muscle mass observed in the DNT group is likely a result of a better axon : myocyte ratio and spatial distribution. This could be studied and optimized to improve the outcomes and utility of DNT.

    The discussion should more thoroughly explore the limitations of this model and experimental constraints.

    What remains to be optimized prior to clinical translation? What types of scientific questions can this help answer? In which types of clinical cases would DNT not be appropriate?

    Read the original source
    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    The authors aimed to establish a rodent upper limb model to test double vs. single nerve transfers, and provided base results for histological retrograde labeling, topographic findings, functional (behavioural) analysis and outcomes for reinnervated vs. control muscle mass. The manuscript is well balanced, and contains a detailed description to reproduce the experimental model.

    The authors demonstrated equal functional outcomes for both types of transfers and have in this reviewers opinion succeeded in establishing a novel model for future (experimental) tests before (clinical) application.

    Read the original source
    Was this evaluation helpful?