Abstract
Background
Mitochondrial dysfunction is a critical factor in several diseases, but current in situ assessment methods are severely limited. Non-invasive monitoring of mitochondrial redox state using resonance Raman Spectroscopy (RRS) offers a promising solution. This study aims to demonstrate RRS utility with liver models of warm ischemia-reperfusion injury in organ transplantation.
Methods
Lewis rat (female) and Yorkshire pig (both sexes) livers were evaluated during reperfusion by subnormothermic machine perfusion, with 3-6 replicates per study group, and statistical comparisons using unpaired two-tailed Student’s t-tests with Welch’s correction for potentially unequal variance. RRS provides in situ quantification of the overall mitochondrial redox state, and herein further refined to resolve the redox state of individual complex III and IV.
Results
Here we show that RRS can differentiate non-viable rat livers (3 h warm ischemia, WI) from viable 1 h WI and fresh controls as early as 30 mins into reperfusion. RRS also identifies dysfunction at complex III characterized by hyperoxidation during reperfusion. This guides us to test methylene blue, which acts as an alternate electron donor to bypass complex III, as treatment rescuing mitochondria from WI-induced reperfusion injury. When tested on pig marginal livers with extended WI (30-45 mins), our RRS-guided treatment enables recovery of hemodynamics and oxygen/lactate values that approached controls without WI.
Conclusions
RRS assessment and guided treatment with methylene blue provide two lines of evidence indicating that mitochondrial hyperoxidation, specifically at complex III, is a critical mechanism underlying warm ischemia-reperfusion injury. This study demonstrates the potential of RRS for transplantation and broader applications.
Plain language summary
Healthy mitochondria are crucial for organ function, and their functional failure contributes to poor organ transplantation outcomes. In this study, we used a light-based method called resonance Raman spectroscopy (RRS) to assess mitochondrial health directly on the liver surface, without removing tissue. We find that long periods of warm ischemia (i.e., no blood flow at body temperature) caused significant mitochondrial stress, especially at complex III, which is important for energy production. We then tested methylene blue, an FDA-approved drug that helps mitochondria maintain function while bypassing complex III. In pig livers with injury similar to marginal human donor organs, methylene blue improves oxygen use and blood flow profiles. These results highlight the potential of RRS for improving transplant outcomes.
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Data availability
All data relevant to this study are included in this published article and its Supplementary Material file. Additional information can be provided by the corresponding author upon reasonable request. Source data underlying Figs. 1–5 can be accessed from Supplementary Data 1.
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Acknowledgements
This work was supported by generous funding to S.N.T. from the US National Institutes of Health (R01DK134590). The authors also gratefully acknowledge funding to S.N.T. from the US National Institute of Health (K99/R00 HL1431149; R01HL157803; R24OD034189), National Science Foundation (EEC 1941543), Polsky Family Foundation, and Shriners Children’s Boston (Grant #BOS-85115). In addition, the authors acknowledge funding to K.U. from the US National Institute of Health (R01DK114506, R01DK096075), and support for R.J. by Grant #LIFER23-263034 from the American Association for the Study of Liver Diseases Foundation. This work was also supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program under Award Number W81XWH-19-1-0472 to J.N.K. Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the Department of Defense. The authors express deep gratitude for expert support from highly skilled technicians of the MGH Knight Surgery for the pig liver procurements, that of Pathology Core from Harvard Medical School for histology studies, and that of Mass Spectroscopy Core from Shriners Children’s Boston for the liver energy status evaluation. The authors would like to extend their gratitude to Dr. Robert Balaban and Dr. Armel Femnou from the NIH National Heart, Lung, and Blood Institute for their expertise in mitochondrial energetics and their important assistance in developing the RRS library of individual mitochondrial complexes. In addition, the authors would like to thank Ms. Jesslyn James from Xavier University of Louisiana for her support with pig liver machine perfusion and RRS measurements at MGH during a research experience for undergraduate (REU) program funded by the NSF Grant No. EEC 1941543. The authors would like to thank Dr. Alissa Cutrone and Dr. Arnaud Lyon for their surgical support with pig liver procurement. The authors also deeply appreciate Dr. Reinier J de Vries and Ms. Casie A. Pendexter for their valuable contributions to the human liver experiments. Finally, the authors express sincere gratitude to Mr. Zafiris Zafirelis and HbO2 Therapeutics for their provision of hemoglobin-based oxygen carrier (HBOC) used in this study.
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Contributions
K.T.N., O.S.O. and S.N.T. designed the study. K.T.N. and O.S.O. performed the rat liver machine perfusion. C.T. and K.T.N. performed the pig liver machine perfusion with support from O.S.O. and R.J. K.T.N. was responsible for resonance Raman Spectroscopy (RRS) data collection and interpretation. O.S.O. was responsible for rat and pig liver procurements. K.T.N., O.S.O., C.T., and T.P. contributed to organ viability data generation and interpretation. K.T.N., R.J., and P.R contributed to RRS algorithm optimization and data analysis with support from E.A. and D.V. additionally. J.N.K., P.R., and D.V. contributed to developing the RRS library for individual complex III and IV. K.T.N. performed the data curation, and R.J. performed the statistical hypothesis testing with support from K.T.N., O.S.O., and A.M. Overall, S.N.T. is responsible for all aspects of the research project with support for laboratory resources from K.U., surgical techniques and pig liver resources from S.A.R. and A.A.O., as well as engineering support for RRS technology development from D.V. and P.R. All authors reviewed and edited the manuscript.
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Competing interests
The authors declare competing interests. S.N.T., J.N.K., and P.R. have provisional patent applications relevant to this study. D.V. and P.R. are employees and shareholders of Pendar Technologies. S.N.T.’s are managed by MGH and Partners HealthCare in accordance with their conflict-of-interest policies. J.N.K.’s competing interests are managed by BCH’s conflict-of-interest policies. D.V. and P.R.’s competing interests are subject to the Research Integrity Policy of Pendar Technologies. The following patented technologies have been used in this study: US2020/0281474A1 In-vivo monitoring of cellular energetics with Raman spectroscopy (application). Additional patent applications for use in ophthalmology, tissue viability, and burn injury assessment using Resonance Raman Spectroscopy have been submitted, where R.J. is also an inventor. Finally, a patent disclosure was submitted for the use of methylene blue to overcome ischemia-reperfusion injury, where K.T.N., O.S.O., and S.N.T. are co-inventors. This does not alter our adherence to journal policies on sharing data and materials. All other authors do not have competing interests.
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Nguyen, K.T., Ozgur, O.S., Jain, R. et al. Mitochondrial hyperoxidation contributes to warm ischemia-reperfusion injury in rat and pig livers. Commun Med (2026). https://doi.org/10.1038/s43856-026-01551-4
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DOI: https://doi.org/10.1038/s43856-026-01551-4
