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A mouse model of volumetric muscle loss and therapeutic scaffold implantation

Nature Protocols (2024)Cite this article

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Abstract

Skeletal myofibers naturally regenerate after damage; however, impaired muscle function can result in cases when a prominent portion of skeletal muscle mass is lost, for example, following traumatic muscle injury. Volumetric muscle loss can be modeled in mice using a surgical model of muscle ablation to study the pathology of volumetric muscle loss and to test experimental treatments, such as the implantation of acellular scaffolds, which promote de novo myogenesis and angiogenesis. Here we provide step-by-step instructions to perform full-thickness surgical ablation, using biopsy punches, and to remove a large volume of the tibialis anterior muscle of the lower limb in mice. This procedure results in a reduction in muscle mass and limited regeneration capacity; the approach is easy to reproduce and can also be applied to larger animal models. For therapeutic applications, we further explain how to implant bioscaffolds into the ablated muscle site. With adequate training and practice, the surgical procedure can be performed within 30 min.

Key points

  • A surgical procedure for the full-thickness surgical ablation of ~20–60% of the mouse tibialis anterior using a commercial 2–3-mm biopsy punch allows the ablation size to be customized. The model is representative of skeletal muscle loss.

  • The surgically ablated muscles’ uniform geometry does not fully reproduce the complexity of traumatic muscle injury, which includes other injuries associated with trauma to the bone, nerves or tendons.

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Fig. 1: Schematic overview of creating VML injury to the TA muscle using a biopsy punch.
Fig. 2: Intraoperative images of the VML surgical procedure followed by scaffold implantation.
Fig. 3: Histological staining and force measurements of TA muscle at 21 d after muscle ablation (2-mm and 3-mm models).
Fig. 4: Histological staining of TA muscle at 3 weeks after muscle ablation with collagen scaffold implantation.

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Data availability

All data generated or analyzed during this study are derived from our original research study or are included in this paper. Source data are provided with this paper.

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Acknowledgements

This research received funding from the Alliance for Regenerative Rehabilitation Research and Training (AR3T), which is supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institute of Neurological Disorders and Stroke (NINDS) and National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the National Institutes of Health under Award Number P2CHD086843. This study was supported by grants to N.F.H. from the US National Institutes of Health (R01 HL142718, R41HL170875 and R21 HL172096-01), the National Science Foundation (1829534 and 2227614) and the Department of Veterans Affairs (RX001222 and 1I01BX004259). N.F.H is a recipient of a Research Career Scientist award (IK6BX006309) from the Department of Veterans Affairs. K.H.N. was supported by grants from the National Institutes of Health (R01AR080150, R00HL136701) and AR3T (CNVA00048860).

Author information

Authors and Affiliations

  1. Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA

    Caroline Hu, Gladys Chiang, Marco Quarta, Thomas A. Rando & Ngan F. Huang

  2. The Stanford Cardiovascular Institute, Stanford University, Palo Alto, CA, USA

    Alex H.-P. Chan & Ngan F. Huang

  3. Department of Cardiothoracic Surgery, Stanford University, Palo Alto, CA, USA

    Alex H.-P. Chan & Ngan F. Huang

  4. Department of Biomedical Engineering, Oregon Health and Science University, Portland, OR, USA

    Cynthia Alcazar & Karina H. Nakayama

  5. Department of Neurology, University of California Los Angeles, Los Angeles, CA, USA

    Thomas A. Rando

  6. Department of Chemical Engineering, Stanford University, Palo Alto, CA, USA

    Ngan F. Huang

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Contributions

C.A., C.H., M.Q. and K.H.N. optimized the muscle ablation model and scaffold implantation procedure. G.C. and A.H.-P.C provided technical assistance with tissue histology. N.F.H. and T.A.R. interpreted the data. C.A., C.H. and N.F.H. planned and wrote the manuscript, with input from all authors. All authors gave approval for the final version to be published.

Corresponding author

Correspondence to Ngan F. Huang.

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The authors declare no competing interests.

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Nature Protocols thanks Kimberley Huey, Pablo Fernandez-Marcos and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Hu, C. et al. Bioengineering 9, 37 (2022): https://doi.org/10.3390/bioengineering9010037

Alcazar, C. A. et al. Biomat. Sci. 8, 5376–5389 (2020): https://doi.org/10.1039/d0bm00990c

Quarta, M. et al. NPJ Regen. Med. 3, 18 (2018): https://doi.org/10.1038/s41536-018-0057-0

Quarta, M. et al. Nat. Commun. 8, 15613 (2017): https://doi.org/10.1038/ncomms15613

Supplementary information

Reporting Summary

Supplementary Video 1

Video showing the surgical procedure of collecting the TA muscle at 3 weeks after induction of VML.

Source data

Source Data Fig. 3

Statistical source data.

Source Data Table 1

Statistical source data.

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Hu, C., Chiang, G., Chan, A.HP. et al. A mouse model of volumetric muscle loss and therapeutic scaffold implantation. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-01059-y

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