Intravital Measurements of Reactive Oxygen/Nitrogen Species in the Injured Liver in Vivo by Electrochemical Microscopy

Reactive oxygen/nitrogen species (ROS/RNS) are formed during normal cellular metabolism and contribute to its regulation, while many pathological processes are associated with ROS/RNS imbalances. Modern methods for measuring ROS/RNS are mainly based on the use of inducible fluorescent dyes and protein sensors, which have several disadvantages for in vivo use. Intravital electrochemical microscopy can be used to quantify ROS/RNS with high sensitivity without exogenous tracers and allows dynamic ROS measurements in vivo . Here, we developed a method for quantifying total ROS/RNS levels in the liver and evaluated our setup in live mice using three common models of liver disease associated with ROS activation: acute liver injury with CCl4 , partial hepatectomy (HE), and induced hepatocellular carcinoma (HCC). We have demonstrated using intravital electrochemical microscopy that any exposure to the peritoneum in vivo leads to an increase in ROS/RNS levels, from a slight increase to an explosion, depending on the procedure. Analysis of the ROS/RNS level in a partial hepatectomy model revealed oxidative stress, both in mice after HE 24 hours after surgery, and in sham-operated ones. We quantified dose-dependent ROS/RNS production in CCl4 -induced injury with underlying neutrophil infiltration and cell death. We expect that in vivo electrochemical measurements of reactive oxygen/nitrogen species in the liver may become a routine approach that provides valuable data in research and preclinical studies.