Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A longitudinal rat forelimb model for assessing in vivo neuromuscular function following extremity reperfusion injury

Lab Animal volume 54, pages 259–269 (2025)Cite this article

Subjects

Abstract

Rhabdomyolysis following revascularization of the ischemic upper extremity can lead to life- and limb-threatening sequelae. In the context of replantations and vascularized composite allografting, a reconstructive procedure usually reserved for upper limb amputees, prolonged tissue ischemia is detrimental to extremity functional recovery. Currently, validated survival small animal models of extremity reperfusion injury that permit longitudinal assessment of limb function are lacking. So far, studies that evaluated reperfusion injury-induced neuromuscular impairment have relied on terminal ex vivo procedures and did not provide clinically translatable measurements. Here we present a reliable rat model of extremity post-reperfusion syndrome (PRS) that comprehensively recapitulates the biochemical hallmarks of rhabdomyolysis secondary to upper-extremity reperfusion injury and allows the monitoring of in vivo upper limb function using clinically relevant electrodiagnostic and kinematic metrics. In addition to inducing severe metabolic derangements, our forelimb PRS model provided insights on gross motor and electrophysiological alterations following upper-extremity reperfusion injury. We identify gait coordination parameters—such as stride frequency and the forelimb–hindlimb coordination index—and electrophysiological metrics, including compound muscle action potential amplitude, as objective and noninvasive outcome measures for assessing limb function in small animal models of extremity PRS. This comprehensive, validated functional model can serve as an invaluable tool to evaluate therapeutics or preconditioning regimens to attenuate PRS and mitigate resulting neuromuscular dysfunction.

This is a preview of subscription content, access via your institution

Access options

Fig. 1: An exsanguinating digital tourniquet device effectively induces acute forelimb ischemia and clinically relevant PRS in a rat model.
Fig. 2: Forelimb PRS results in extensive biochemical and metabolic derangements in a rat model.
Fig. 3: Forelimb IRI triggers systemic inflammatory response syndrome and immune dysfunction.
Fig. 4: Motor coordination is impaired following forelimb reperfusion injury.
Fig. 5: Forelimb IRI model induces persistent conduction block.
Fig. 6: Loss of motor unit action potentials and NMJ denervation following forelimb PRS.

Similar content being viewed by others

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Apichartpiyakul, P., Shinlapawittayatorn, K., Rerkasem, K., Chattipakorn, S. C. & Chattipakorn, N. Mechanisms and interventions on acute lower limb ischemia/reperfusion injury: a review and insights from cell to clinical investigations. Ann. Vasc. Surg. 86, 452–481 (2022).

    PubMed  Google Scholar 

  2. Carroll, W. R. & Esclamado, R. M. Ischemia/reperfusion injury in microvascular surgery. Head Neck 22, 700–713 (2000).

    CAS  PubMed  Google Scholar 

  3. He, J., Khan, U. Z., Qing, L., Wu, P. & Tang, J. Improving the ischemia–reperfusion injury in vascularized composite allotransplantation: clinical experience and experimental implications. Front. Immunol. 13, 998952 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Walker, P. M. Ischemia/reperfusion injury in skeletal muscle. Ann. Vasc. Surg. 5, 399–402 (1991).

    CAS  PubMed  Google Scholar 

  5. Ton, C. et al. Methods of ex vivo analysis of tissue status in vascularized composite allografts. J. Transl. Med. 21, 609 (2023).

    PubMed  PubMed Central  Google Scholar 

  6. Žargi, T., Drobnič, M., Stražar, K. & Kacin, A. Short-term preconditioning with blood flow restricted exercise preserves quadriceps muscle endurance in patients after anterior cruciate ligament reconstruction. Front. Physiol. 9, 1150 (2018).

    PubMed  PubMed Central  Google Scholar 

  7. Kubo, Y. et al. Association between serum n-3 polyunsaturated fatty acids and quadriceps weakness immediately after total knee arthroplasty. PLoS ONE 15, e0228460 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Longchamp, A. & Deglise, S. The strength of reperfusion: the dark side of ischaemia. Eur. J. Vasc. Endovasc. Surg. 58, 257 (2019).

    PubMed  Google Scholar 

  9. Tu, H. et al. A comparison of acute mouse hindlimb injuries between tourniquet- and femoral artery ligation-induced ischemia-reperfusion. Injury 52, 3217–3226 (2021).

    PubMed  Google Scholar 

  10. Gok, E. et al. Single muscle fibre contractility testing in rats to quantify ischaemic muscle damage during reperfusion injury. Eur. J. Vasc. Endovasc. Surg. 58, 249–256 (2019).

    PubMed  Google Scholar 

  11. Kern, B. et al. A novel rodent orthotopic forelimb transplantation model that allows for reliable assessment of functional recovery resulting from nerve regeneration. Am. J. Transplant. 17, 622–634 (2017).

    CAS  PubMed  Google Scholar 

  12. Pendexter, C. A. et al. Development of a rat forelimb vascularized composite allograft (VCA) perfusion protocol. PLoS ONE 18, e0266207 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Yi, H., Kim, M. A., Back, S. K., Eun, J. S. & Na, H. S. A novel rat forelimb model of neuropathic pain produced by partial injury of the median and ulnar nerves. Eur. J. Pain 15, 459–466 (2011).

    PubMed  Google Scholar 

  14. Chen, X. K., Rathbone, C. R. & Walters, T. J. Treatment of tourniquet-induced ischemia reperfusion injury with muscle progenitor cells. J. Surg. Res. 170, e65–e73 (2011).

    PubMed  Google Scholar 

  15. Corrick, R. M. et al. Dexamethasone protects against tourniquet-induced acute ischemia–reperfusion injury in mouse hindlimb. Front. Physiol. 9, 244 (2018).

    PubMed  PubMed Central  Google Scholar 

  16. de Carvalho, E. G., Corsini, W. & Hermes, T. A. Severe muscle damage after a short period of ischemia and reperfusion in an animal model. Surgery 174, 363–368 (2023).

    PubMed  Google Scholar 

  17. Cearra, I., Herrero De La Parte, B., Moreno-Franco, D. I. & García-Alonso, I. A reproducible method for biochemical, histological and functional assessment of the effects of ischaemia–reperfusion syndrome in the lower limbs. Sci. Rep. 11, 19325 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Petrasek, P. F., Homer-Vanniasinkam, S. & Walker, P. M. Determinants of ischemic injury to skeletal muscle. J. Vasc. Surg. 19, 623–631 (1994).

    CAS  PubMed  Google Scholar 

  19. Charles, A.-L. et al. Muscles susceptibility to ischemia–reperfusion injuries depends on fiber type specific antioxidant level. Front. Physiol. 8, 52 (2017).

    PubMed  PubMed Central  Google Scholar 

  20. Utagi, B., Kumar, R. & Bhagavan, K. R. Endovascular management of two uncommon cases of acute upper limb ischemia in young. J. Health Allied Sci. NU 13, 431–435 (2023).

    Google Scholar 

  21. Weibrecht, K., Dayno, M., Darling, C. & Bird, S. B. Liver aminotransferases are elevated with rhabdomyolysis in the absence of significant liver injury. J. Med. Toxicol. 6, 294–300 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Kodadek, L. et al. Rhabdomyolysis: an American Association for the Surgery of Trauma Critical Care Committee Clinical Consensus Document. Trauma Surg. Acute Care Open 7, e000836 (2022).

    PubMed  PubMed Central  Google Scholar 

  23. Herrero de la Parte, B. et al. The prevention of ischemia–reperfusion injury in elderly rats after lower limb tourniquet use. Antioxidants 11, 1936 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Kuroda, Y. et al. Oxidative stress evaluation of skeletal muscle in ischemia–reperfusion injury using enhanced magnetic resonance imaging. Sci. Rep. 10, 10863 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Torres, P. A., Helmstetter, J. A., Kaye, A. M. & Kaye, A. D. Rhabdomyolysis: pathogenesis, diagnosis, and treatment. Ochsner J. 15, 58–69 (2015).

    PubMed  PubMed Central  Google Scholar 

  26. Nance, J. R. & Mammen, A. L. Diagnostic evaluation of rhabdomyolysis. Muscle Nerve 51, 793–810 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Brow, T. D., Kakkar, V. V. & Das, S. K. The significance of creatine kinase in cardiac patients with acute limb ischaemia. J. Cardiovasc. Surg. 40, 637–644 (1999).

    CAS  Google Scholar 

  28. Clemens, M. S. et al. Extracorporeal filtration of potassium in a swine model of bilateral hindlimb ischemia–reperfusion injury with severe acute hyperkalemia. Mil. Med. 183, e335–e340 (2018).

    PubMed  Google Scholar 

  29. Tricarico, D., Capriulo, R. & Camerino, D. C. Involvement of K(Ca2+) channels in the local abnormalities and hyperkalemia following the ischemia–reperfusion injury of rat skeletal muscle. Neuromuscul. Disord. 12, 258–265 (2002).

    PubMed  Google Scholar 

  30. Cearra, I. et al. Effects of folinic acid administration on lower limb ischemia/reperfusion injury in rats. Antioxidants 10, 1887 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Korei, C. et al. Hematological, micro-rheological, and metabolic changes modulated by local ischemic pre- and post-conditioning in rat limb ischemia–reperfusion. Metabolites 11, 776 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Deune, E. G. et al. Prevention of ischemia–reperfusion injury with a synthetic metalloprotein superoxide dismutase mimic, SC52608. Plast. Reconstr. Surg. 98, 711–718 (1996).

    CAS  PubMed  Google Scholar 

  33. Yassin, M. M. I., Harkin, D. W., Barros D’Sa, A. A. B., Halliday, M. I. & Rowlands, B. J. Lower limb ischemia–reperfusion injury triggers a systemic inflammatory response and multiple organ dysfunction. World J. Surg. 26, 115–121 (2002).

    PubMed  Google Scholar 

  34. Ege, T., Us, M. H., Sungun, M. & Duran, E. Cytokine response in lower extremity ischaemia/reperfusion. J. Int. Med. Res. 32, 124–131 (2004).

    CAS  PubMed  Google Scholar 

  35. Ferreira, J. et al. Higher levels of cytokines in patients with chronic limb-threatening ischemia. Ann. Vasc. Surg. 106, 255–263 (2024).

    PubMed  Google Scholar 

  36. Orfany, A. et al. Mitochondrial transplantation ameliorates acute limb ischemia. J. Vasc. Surg. 71, 1014–1026 (2020).

    PubMed  Google Scholar 

  37. Islam, M. N., Bradley, B. A. & Ceredig, R. Sterile post-traumatic immunosuppression. Clin. Transl. Immunol. 5, e77 (2016).

    Google Scholar 

  38. Horner, E., Lord, J. M. & Hazeldine, J. The immune suppressive properties of damage associated molecular patterns in the setting of sterile traumatic injury. Front. Immunol. 14, 1239683 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Lai, Y.-R. et al. Clinical disease severity mediates the relationship between stride length and speed and the risk of falling in Parkinson’s disease. J. Pers. Med. 12, 192 (2022).

    PubMed  PubMed Central  Google Scholar 

  40. Krizsan-Agbas, D. et al. Gait analysis at multiple speeds reveals differential functional and structural outcomes in response to graded spinal cord injury. J. Neurotrauma 31, 846–856 (2014).

    PubMed  PubMed Central  Google Scholar 

  41. Lakes, E. H. & Allen, K. D. Gait analysis methods for rodent models of arthritic disorders: reviews and recommendations. Osteoarthritis Cartilage 24, 1837–1849 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Schoen, M. et al. Ischemic preconditioning prevents skeletal muscle tissue injury, but not nerve lesion upon tourniquet-induced ischemia. J. Trauma 63, 788–797 (2007).

    PubMed  Google Scholar 

  43. Juel, V. C. in Handbook of Clinical Neurology Vol. 160 (eds Levin, K. H. & Chauvel, P.) 303–310 (Elsevier, 2019).

  44. Barkhaus, P. E., Nandedkar, S. D., de Carvalho, M., Swash, M. & Stålberg, E. V. Revisiting the compound muscle action potential (CMAP). Clin. Neurophysiol. Pract. 9, 176–200 (2024).

    PubMed  PubMed Central  Google Scholar 

  45. Parry, G. J., Cornblath, D. R. & Brown, M. J. Transient conduction block following acute peripheral nerve ischemia. Muscle Nerve 8, 409–412 (1985).

    CAS  PubMed  Google Scholar 

  46. Stecker, M. M., Baylor, K. & Chan, Y. M. Acute nerve compression and the compound muscle action potential. J. Brachial Plex Peripher. Nerve Inj. 3, 1 (2008).

    PubMed  PubMed Central  Google Scholar 

  47. Iida, H., Schmelzer, J. D., Schmeichel, A. M., Wang, Y. & Low, P. A. Peripheral nerve ischemia: reperfusion injury and fiber regeneration. Exp. Neurol. 184, 997–1002 (2003).

    PubMed  Google Scholar 

  48. Wilson, R. J. et al. Voluntary running protects against neuromuscular dysfunction following hindlimb ischemia-reperfusion in mice. J. Appl. Physiol. 126, 193–201 (2019).

    CAS  PubMed  Google Scholar 

  49. Tömböl, T., Pataki, G., Németh, A. & Hamar, J. Ultrastructural changes of the neuromuscular junction in reperfusion injury. Cells Tissues Organs 170, 139–150 (2002).

    PubMed  Google Scholar 

  50. Harrigan, M. E. et al. Assessing rat forelimb and hindlimb motor unit connectivity as objective and robust biomarkers of spinal motor neuron function. Sci. Rep. 9, 16699 (2019).

    PubMed  PubMed Central  Google Scholar 

  51. Sanders, D. B. et al. Guidelines for single fiber EMG. Clin. Neurophysiol. 130, 1417–1439 (2019).

    PubMed  Google Scholar 

Download references

Acknowledgements

O.A.S. gratefully acknowledges support as a trainee on the NIAMS NIH T32 AR56950 program (PI: Jennifer J. Westendorf, Ph. D.). This work was also supported in part by the Obaid Reconstructive Transplant Award.

Author information

Authors and Affiliations

  1. Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA

    Omar A. Selim, Chunfeng Zhao & Steven L. Moran

  2. Musculoskeletal Research Training (T32) Program, Mayo Clinic, Rochester, MN, USA

    Omar A. Selim

  3. Division of Plastic Surgery, Mayo Clinic, Rochester, MN, USA

    Aida Sarcon, Mehmet Tunaboylu & Steven L. Moran

Authors
  1. Omar A. Selim
    View author publications

    Search author on:PubMed Google Scholar

  2. Aida Sarcon
    View author publications

    Search author on:PubMed Google Scholar

  3. Mehmet Tunaboylu
    View author publications

    Search author on:PubMed Google Scholar

  4. Chunfeng Zhao
    View author publications

    Search author on:PubMed Google Scholar

  5. Steven L. Moran
    View author publications

    Search author on:PubMed Google Scholar

Contributions

O.A.S. and S.L.M. conceptualized and designed the study. O.A.S. developed the methodology and visualized data presentation. O.A.S. and A.S. performed the in vivo experiments. O.A.S. performed in vivo electrophysiology. M.T. assisted in vivo work. O.A.S. wrote the manuscript. O.A.S., C.Z. and S.L.M. analyzed and interpreted the data. O.A.S. and S.L.M supervised the research. C.Z. and S.L.M provided resources. All the authors approved the final version of the manuscript.

Corresponding author

Correspondence to Omar A. Selim.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Lab Animal thanks David Hercher and Huiyin Tu for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Table 1.

Reporting Summary

Supplementary Video 1

Representative pre-injury rat gait.

Supplementary Video 2

Gait analysis 3 h after PRS.

Supplementary Video 3

Gait analysis 14 days after PRS.

Supplementary Data 1

ARRIVE guidelines checklist.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Selim, O.A., Sarcon, A., Tunaboylu, M. et al. A longitudinal rat forelimb model for assessing in vivo neuromuscular function following extremity reperfusion injury. Lab Anim 54, 259–269 (2025). https://doi.org/10.1038/s41684-025-01601-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41684-025-01601-9

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing