(G-254) Raman spectroscopy shows comparable functional tissue oxygenation with and without packed RBCs during machine perfused rat livers by measuring mitochondrial redox state in real-time

Introduction::

Oxygen supply is vital to cellular energy production and metabolism. During transplants livers are cut off from sufficient oxygenation after recovery from the donor for several hours. 4ºC storage is used to maintain lower metabolic activity during this period to reduce the most severe form of injury, followed by reperfusion in the recipient¹. Current clinical standard of reattaching the organ immediately after cold storage is being challenged with the novel machine perfusion technology, where an organ is metabolically recovered by maintaining a physiological flow and temperature before re-attachment to the recipient using artificial oxygenation. Since the metabolic activity is restored due to exposure to a higher physiological temperature, the demand for oxygen becomes higher compared to cold storage. However, there is little consensus over the optimum mode of oxygen delivery to the tissue during reperfusion. Several perfusate mediums have been proposed that either use cellular oxygen carriers such as RBCs, acellular hemoglobin-based oxygen carrier, or no carriers but higher partial pressure of oxygen for efficient oxygenation of the liver tissue. There is a significant gap of methodologies to test the effectiveness of oxygenation of the perfused organ using these different types of perfusate in real-time during perfusion.

We propose a resonance Raman spectroscopy-based method for the assessment of oxygenation in real-time during machine perfusion in non-contact benchtop format using a 441 nm laser for excitation. In current study, we test this device in fresh livers recovered from rats to compare the oxygenation using RBC and acellular perfusate during machine perfusion.

Materials and Methods::

We used Lewis rats as a model to test the efficiency of perfusion with and without acellular perfusate as per previously published protocol². Briefly, the livers are resected from the rats under deep anesthesia, and immediately flushed with heparin and saline solutions. During the flush, the rats were euthanized as results of the exsanguination. The liver is then cannulated at the portal vein and the inferior vena cava is transected. It is immediately attached upon recovery to a machine perfusion system that consists of a pump, an oxygenator, and a bubble trap. The perfusate in the control group consisted of Williams Medium E solution (Sigma-Aldrich, St Louis, MO, USA) prepared according to the manufacturer’s instructions, and supplemented with dexamethasone, insulin, heparin, Glutamax, and bovine serum albumin. The experimental group of livers are perfused with the perfusate supplemented with packed RBCs from Lewis rats to maintain a hematocrit of 15-20%. The Raman spectroscopy probe excites the tissue near the point of analysis and carries the resulting spectrum to a spectrometer for recording (described before³). The spectrometer is connected to the computer which is used for analysis via custom software. Briefly, the device stores libraries of completely oxidized and reduced mitochondria among other molecular libraries, which are fitted onto the unknown spectrum by regression. The ratio of reduced mitochondria to total mitochondria is called the resonance Raman reduced mitochondrial ratio (or 3RMR) is calculated and used as a standard value to compare oxygenation between the groups with and without the packed RBCs.

Results, Conclusions, and Discussions::

We were successfully able to measure tissue mitochondrial redox state for both the experimental and control group of livers that were perfused with and without packed RBCs as oxygen carriers. We observed higher 3RMR values at the beginning of the perfusion for acellular perfusate group which goes down significantly towards the end of the perfusion. This may be due to the reason that immediately upon reperfusion, the demand for oxygen is higher due to the short period of ischemia after recovery when there is no supply of oxygen and perfusate. The packed RBC group on the other hand does not show any significant difference in oxygenation as measured by 3RMR at the beginning or at the end of the perfusion indicating high degree of tissue oxygenation right from the beginning. However, the 3RMR was not significantly different between the two groups at the beginning of the perfusion (p=0.12, n=4), which may indicate that a higher partial pressure of oxygen was sufficient for oxygenating the liver tissue during normothermic machine perfusion. This method would provide a strong tool to assess oxygenation during machine perfusion. It can be used to test oxygenation efficiency with different types of perfusates, and with different durations of cold and warm ischemia when the livers are exposed to longer and more severe oxygen deprivation. Such studies may show really interesting trends in oxygenation with and without RBCs in the perfusate, and could be really useful to predict viability of livers after different durations of storage, and eventually help in designing the optimum perfusate for all such different conditions of storage and recovery.

Acknowledgements (Optional): : We gratefully acknowledge funding to SNT from the US National Institute of Health (K99/R00 HL1431149; R01HL157803; R01DK134590), American Heart Association (18CDA34110049), Harvard Medical School Eleanor and Miles Shore Fellowship, and the Claflin Distinguished Scholar Award on behalf of the MGH Executive Committee on Research. Further, we acknowledge the National Science Foundation under Grant No. EEC 1941543 to  SNT. We also thankfully acknowledge support provided by the Fund for Medical Discovery (FMD) Clinical Research Fellowship by the Executive Committee on Research at the Massachusetts General Hospital, and the Afdhal/McHutchison LIFER Award by the American Association for the Study of Liver Diseases (AASLD) to RJ.

References (Optional): : 1. Hepatic microcirculatory perfusion failure is a determinant of liver dysfunction in warm ischemia-reperfusion. - PMC. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1887483/.
2. de Vries RJ, Cronin SEJ, Romfh P, Pendexter CA, Jain R, Wilks BT, et al. (2021) Non-invasive quantification of the mitochondrial redox state in livers during machine perfusion. PLoS ONE 16(10): e0258833. https://doi.org/10.1371/journal.pone.0258833
3. 

Responsive monitoring of mitochondrial redox states in heart muscle predicts impending cardiac arrest.Sci. Transl. Med.9,eaan0117(2017).DOI:10.1126/scitranslmed.aan0117