Investigation Of Liver Functional Changes In Disease Status Using Multiphoton Microscope

Aim:To investigate liver damage and interrelate drug uptake, metabolism and clearance changes in disease status using multiphoton microscopy and lifetime imagingMethods:(1) Using multiphoton fluorescence lifetime imaging to study the distribution and metabolism of fluorescein and its metabolite in vivo in rat liver. Fluorescence lifetime values in vitro were used to interpret in vivo data;(2) Localized ischemia was induced in male Wistar rats for1hour followed by reperfusion for4hours. Fluorescein (10mg/kg) was injected intravenously, blood and bile collected and images recorded for180min. The liver was harvested at the end of the experiment for mRNA and protein expression determination;(3) Localized ischemia was induced for1hour in male Wistar rats. The liver was reperfused for4,24,48hours or1week, where-after Rhodamine123was injected intravenously. Blood was collected for alanine transaminase measurements and bile was collected and analyzed for Rhodamine123elimination. Multiphoton microscopy images were taken for6hours post injection. The liver was harvested at the end of the experiment for histology, as well as for mRNA and protein expression of P-glycoprotein;(4) Localised hepatic ischemia was induced in male Wistar rats for1hour followed by4hours reperfusion after which liver function and oxidative stress were assessed. Multiphoton and fluorescence lifetime microscopy was conducted prior to ischemia and up to4hours of reperfusion to determine liver cell damage by image auto fluorescence. Results were compared to morphological assessment of liver damage;(5) Steatosis was induced in rat livers by feeding them a high fat diet for7days. Partial ischemia (median and left lobes) was induced for1hour following4hours reperfusion, where after sodium fluorescein (10mg/kg) was injected. Its distribution was visualized using multiphoton microscopy in vivo. Bile was collected for excretion measurements and the liver was harvested for histology analysis. A three-compartment model was applied to calculate pharmacokinetic parameters.Results:(1) Our results show that the mean fluorescence lifetime of fluorescein and its metabolite decreases over time after injection of fluorescein in three different regions of the liver. Furthermore, we were able to convert the lifetime values to concentration ratios of fluorescein and its metabolite over time;(2) Results showed a delayed sinusoidal appearance of and increased hepatocyte concentration of fluorescein in hepatic ischemia reperfusion injury;(3) Ischemia-reperfusion injury resulted in extensive liver damage as indicated by significantly elevated levels of alanine transaminase in plasma at4and24hours, inflammatory cell infiltration at24and48hours and acinus dependent apoptosis at4and24hours. The excretion of rhodamine123was impaired and levels of P-glycoprotein were significantly decreased in ischemia reperfusion injury at48hours. Furthermore, the diminished excretion of rhodamine123 was visualized by multiphoton microscopy showing increased fluorescence at4,24and48hours;(4) Liver function was significantly impaired and oxidative stress was increased at2and4hours of reperfusion. Multiphoton microscopy detected liver damage at1hour of reperfusion, which was manifested by vacuolated cells with a heterogeneous spread of damage over the liver. Most of the damage was localised in the midzonal area of the liver acinus. In addition, fluorescence lifetime imaging showed a decrease in cellular metabolic activity;(5) Ischemia-reperfusion injury resulted in extensive liver damage and increased apoptosis. Bile flow rate was significantly lowered in steatotic livers exposed to ischemia. In vivo imaging demonstrated increased fluorescence intensity that took longer to fade all diseased models compared to sham. The pharmacokinetic model showed significantly increased intrinsic metabolism of fluroescein. As a consequence, the biliary excretion rate was significantly reduced.Conclusions:We have demonstrated a novel method to study a fluorescent compound and metabolite in vivo using multiphoton fluorescence lifetime imaging; Multiphoton and fluorescence lifetime imaging microscopy, by detecting evidence of early ischemia-reperfusion injury both structurally and functionally is useful for following progressive liver injury in vivo; I/R injury impair microcirculation and transporter function; long term ischemia reperfusion caused decreased function of P-glycoprotein, which was confirmed by reduced levels of the protein. These findings show the importance of careful drug dosing immediately after liver surgery; The present study demonstrated that the metabolism of fluorescein was significantly increased in normal and steatotic livers exposed to ischemia-reperfusion injury. The subsequent decreased concentration of fluorescein explained the decreased excretion rate. Our results show the importance of imaging in combination with modeling to study drug transport and reflect on careful drug monitoring in surgery of steatotic livers.