Marginal liver grafts characterization: 1) Imaging and in situ spectral analysis of Nile Red-induced lipid fluorescence in MCD-induced liver steatosis

Reclaiming fatty liver grafts for transplantation appears mandatory due to the shortage of donors. We demonstrated that obese Zucker rat livers were damaged by conventional cold-storage, but were better preserved by subnormothermic machine perfusion (Vairetti et al., 2009). Steatosis induced by a Methionine-Choline deficient (MCD) diet in the liver of Wistar rats is being now investigated respect to an isocaloric diet. We characterized the lipid content using the fluorescent probe Nile Red (NR): under excitation in the 400–500nm range, NR bound to triglycerides or cholesteryl esters emits a bright yellow fluorescence and a dimmer red emission when bound to phospholipids. 1–4 and 9 weeks of MCD or control diet were considered. Unfixed cryostat liver sections were stained with a 1.6 M solution of NR in glycerol. Areas occupied by yellow-emitting neutral lipid droplets were quantified by image analysis using a fluorescence Zeiss Axioscope 2 Plus microscope, a Canon EOS 1100D digital camera and Image Pro Plus 4.5 software. Spectral analysis was performed by microspectrofluorometry, using a Leitz microspectrograph with an EG&G Optical Multichannel Analyzer (excitation: 436 nm). Spectra were normalized for a direct comparison of the emission profile, and hence of the lipid nature (triglycerides: 530–620 nm; phospholipids: 630–700 nm). Control livers contained yellow-fluorescent micro lipid droplets whose area increased steadily from the 1st to the 4th week and stabilized henceforth. Response to MCD was highly variable in terms of yellow-fluorescent areas. Severe macrosteatotic changes occurred from the2wtreatment onwards as demonstrated by the several-fold increase in yellow emission-positive areas. In MCD-treated animals, spectral analyses revealed a strong relative increase of the yellow but also of the red signals respect to the controls. The latter suggest abundance of phospholipid-bound vesicles, consistent with autophagocytosis that will be investigated at the EM level. Supported by Fondazione CARIPLO, grant n◦ 2011-0439.