Assessing Steatotic Liver Function after Ischemia-Reperfusion Injury by In Vivo Multiphoton Imaging of Fluorescein Disposition

Abstract

Ischemia-reperfusion injury, a common complication during liver surgery where steatotic livers are more prone to the injury, may become more prevalent in the growing obese population. This study characterizes liver morphology toward understanding changes in subcellular function in steatotic livers exposed to ischemiareperfusion injury through quantitative description of fluorescein distribution obtained by minimally invasive in vivo multiphoton microscopy using a physiologic pharmacokinetic model. Rats were fed a high-fat diet for 7 days to induce liver steatosis. Partial ischemia was induced after reperfusion for 4 hours, when fluorescein (10 mg/kg) was injected intravenously. Liver images, bile, and blood were collected up to 180 minutes after injection. Ischemia-reperfusion injury was associated with an increase in alanine transaminase levels and apoptosis. In addition, steatosis featured lipid droplets and an increase in fluorescein-associated fluorescence observed in hepatocytes via multiphoton imaging. Analysis of the hepatic concentrationtime profiles has suggested that the steatosis-induced increase in fluorescein-associated fluorescence mainly arises by inducing hepatic fluorescein metabolism. The combination of ischemiareperfusion with steatosis exacerbates these effects further. This was confirmed by fluorescence lifetime imaging microscopy showing a decreased average fluorescence lifetime of the liver, which is indicative of an increased production of the metabolite. Our results show the potential of noninvasive dye imaging for improving our understanding of liver disease induced by subcellular changes in vivo, providing further quantitative measures of metabolic and biliary liver function, and hence extending the qualitative liver function tests now available.

Diagnostic imaging and therapeutic application of nanoparticles targeting the liver

“…51 CT imaging is usually limited by its poor soft-tissue 20 contrast, while fluorescence imaging is often restricted by the low penetration depth of the excitation and emission light. 77 Thus, a novel nanoparticle-based imaging platform has been proposed recently to combine the merits of each imaging modality.…”

Real-time histology in liver disease using multiphoton microscopy with fluorescence lifetime imaging

“…Body temperature was controlled by placing mice on a heating pad set to 37°C. A midline laparotomy was performed to expose the liver as previously described [22,23]. MPM-FLIM images of unfixed live livers were collected within 30 min after surgical procedures started.…”

Visualizing liver anatomy, physiology and pharmacology using multiphoton microscopy

“…In addition, fluorescein has also been used to reflect the hepatobiliary excretory function. The uptake and clearance of fluorescein has also been found to be delayed in mice with liver steatosis and hepatic I/R injury using TPEF imaging, and confirmed by FLIM . Conventional methods for studying the hepatobiliary excretory function usually involve biochemical analyses of contents in the bile, liver, blood or urine, while MPM enables intravital imaging of fluorescent probes in the liver at the subcellular level.…”

“…Since MPM allows simultaneous visualization of the organ autofluorescence and molecule fluorescence, it is able to determine the levels of fluorescent molecules in the sinusoids, hepatocytes and the bile respectively (Figure a), which enables dividing the liver into subcompartments for pharmacokinetic modelling, as seen in Figure b. A physiologically based pharmacokinetic (PBPK) model has been developed to characterize the kinetics of fluorescein at the single‐cell resolution in health and diseased liver in vivo . Using the same intravital imaging technique, half‐life of Rh123 is calculated by fitting the decay of fluorescence intensity in hepatocytes vs. time profile into the exponential equation .…”