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Marginal liver grafts characterization: 1) Imaging and in situ spectral analysis of Nile Red-induced lipid fluorescence in MCD-induced liver steatosis
No full textReclaiming 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
A Transparency Maturity Model for Government Software Tenders
Get PDFThis work was supported in part by the Project Transparency in Public Electronic Procedures, from the Universidad de La Frontera in conjunction with the Universidad de Los Andes, under Grant Idea2019001. The authors gratefully acknowledge financial support from CONICYT PIA-BASAL AFB180003 and FONDECYT-Chile, grants 1200221 (Sebastian Maldonado) and 11200007 (Carla Vairetti)
Bilirubin: an autofluorescence bile biomarker for liver functionality monitoring.
No full textExcitation at 366-465 nm of bilirubin in aqueous solution with solubilizing agents results in emission spectra composed by two main bands. The variation of their relative contributions as shown by changes in the spectral shape are consistent with the bilirubin bichromophore nature. This latter accounts for an exciton-coupling phenomenon, intramolecular interchromophore energy transfer efficiency being affected by microenvironment. Excitation at 366 nm, despite the poor absorption of bilirubin, gives rise to appreciable emission signals from both pure compounds and bile - collected from functionally altered rat livers - favouring the spectral shape response to environment and molecular conformation changes. As compared to the merely bile flow estimation, real-time detection of fluorescence, revealing composition variations, improves near-UV optical-biopsy diagnostic potential in hepatology
Decreased apoptosis in fatty livers submitted to subnormothermic machine-perfusion respect to cold storage
Get PDFMachine perfusion at subnormothermic temperature (20°C), MP20, was developed by Vairetti et al. and showed to afford a better preservation of fatty livers respect to traditional cold storage (CS) in terms of enzyme release into the perfusate and bile, glycogen stores, energy charge and oxidative stress. Here we investigated whether it also caused decreased cell death by apoptosis. Fatty and lean Zucker rats were submitted to MP20 or CS for 6 h and reperfused normothermically for 2 h. Apoptotic cells were revealed by immunohistochemistry of activated caspase-3 and M30 (new epitope on CK18 degraded by caspase-3) and by the TUNEL assay. Portal pressure was also determined. A statistically significant reduction of hepatocyte apoptosis, but especially of sinusoidal cells was determined for fatty livers submitted to MP20 respect to CS. Portal pressure was significantly lower after MP20 respect to CS. The reduction of sinusoidal cell death by apoptosis without need for anti-apoptotic therapies appears particularly positive since apoptotic sinusoidal cells hinder microcirculation in the sinusoids and are thrombogenic. These results further confirm the potential of MP20 for preserving fatty livers that would be otherwise discarded as grafts, and thus for increasing the donor pool for liver transplantation
Musica, movimenti e mutamenti sociali. Idee e strumenti per la formazione dell’identità sociale
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Autofluorescence properties of rat liver under hypermetabolic conditions.
Get PDFAutofluorescence response to oxygen supply modulation has been investigated in livers of rats under the hypermetabolic state associated to a pathological condition-hyperthyroidism-that is known to enhance hepatocyte metabolic activities involving both NAD, i.e. oxidative pathways engaged in ATP synthesis, and NADP, i.e. reductive bio-synthesis and antioxidant functions. Experiments have been performed on rats in normal condition or submitted to long-term thyroxine (T(4)) administration. Histological inspection did not show any appreciable morphological alteration in liver parenchyma; biochemical analysis indicated an increase in both NADP(+) and NADPH contents. Autofluorescence properties have been monitored in vivo, via a fiber optic probe, on exposed livers both during induction of global ischemia and after restoration of blood circulation. Alteration of oxygen supply modulated liver autofluorescence properties, mainly as to NAD(P)H contribution, in dependence of changes in pyridine coenzymes redox state. With respect to euthyroid, hyperthyroid rat livers exhibited higher autofluorescence signals in all phases of the experiment, and a faster signal decay time upon reoxygenation. The results have been interpreted on the basis of a larger content of NADPH-the coenzyme not directly oxidized in respiratory processes and likely providing an almost constant autofluorescence background contribution-and of uncoupling effects facilitating the respiratory NADH oxidation, associated with the hyperthyroid condition. The results obtained in the liver hypermetabolic model provide interesting perspectives for a further improvement of the diagnostic implications of autofluorescence
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