Resonance Raman spectroscopy detects leaky mitochondrion in rat livers with ischemia-reperfusion injury

Ischemia-Reperfusion Injury (IRI) is a critical problem in organ transplantation that hinders clinical translation of novel technologies aiming to extend organ preservation time without continuous oxygenation. Resonance Raman (RR) spectroscopy is an innovative approach to quantitatively assess mitochondrial redox state via rapid organ surface measurements. Since mitochondrial damage contributes greatly to the development of IRI, RR reduced mitochondrial ratio (3RMR), a readout of RR spectroscopy on percentage of mitochondria in reduced state, can be a powerful parameter in organ assessment predictive of transplant success/outcome. In this study, 3RMR surface readings were performed on rat livers during three hours of subnormothermic machine perfusion (SNMP) after fresh procurement, as well as one hour and three hours of warm ischemic time (storage in 37oC saline solution) to evaluate mitochondria health following ischemia-reperfusion stress. Preliminary data confirmed that rat livers with 3 hours of ischemia exhibited classic injury parameters such as absent bile production, elevated lactate level, excessive tissue edema, and significantly lower oxygen consumption during SNMP in comparison to healthy fresh livers (n=4 per group, p<0.05). Interestingly, RR results showed that injured livers had lower percentage of reduced mitochondria (3RMR < 10%) than healthy livers (3RMR ∼ 15-20%), despite complex IV being fully oxidized at maximum capacity of direct oxygen consumption in all study groups. Further RR spectroscopy analysis suggested that complex III of injured livers significantly leaked electrons/protons during reperfusion with less than 8% in a reduced state after 180 minutes of SNMP. Meanwhile, healthy livers had 30% reduced complex III which remained relatively unchanged through SNMP. These observations agreed with published literature that organs which developed IRI post-transplant commonly had leaky mitochondria resulting in ROS production, oxidation stress, and, finally, cell death. In other words, RR spectroscopy enables quantitative mitochondrial assessment beneficial for detection of organ injury following warm ischemic stress.