Diffuse reflectance spectroscopy accurately quantifies various degrees of liver steatosis in murine models of fatty liver disease

Background: A real-time objective evaluation for the extent of liver steatosis during liver transplantation is currently not available. Diffuse reflectance spectroscopy (DRS) rapidly and accurately assesses the extent of steatosis in human livers with mild steatosis. However, it is yet unknown whether DRS accurately quantifies moderate/severe steatosis and is able to distinguish between micro-and macrovesicular steatosis. Methods: C57BL/6JolaHsd mice were fed wit a choline-deficient l-amino acid-defined diet (CD-AA) or a choline-sufficient l-amino acid-defined control diet (CS-AA) for 3, 8, and 20 weeks. In addition B6. V-Lepob/OlaHsd (ob/ob) mice and their lean controls were studied. A total of 104 DRS measurements were performed in liver tissue ex vivo. The degree of steatosis was quantified from the DRS data and compared with histopathological analysis. Results: When assessed by histology, livers of mice fed with a CD-AA and CS-AA diet displayed macrovesicular steatosis (range 0-74 %), ob/ob mice revealed only microvesicular steatosis (range 75-80 %), and their lean controls showed no steatosis. The quantification of steatosis by DRS correlated well with pathology (correlation of 0.76 in CD-AA/CS-AA fed mice and a correlation of 0.75 in ob/ob mice). DRS spectra did not distinguish between micro-and macrovesicular steatosis. In samples from CD-AA/CS-AA fed mice, the DRS was able to distinguish between mild and moderate/severe steatosis with a sensitivity and specificity of 86 and 81 %, respectively. Conclusion: DRS can quantify steatosis with good agreement to histopathological analysis. DRS may be useful for real-time objective evaluation of liver steatosis during liver transplantation, especially to differentiate between mild and moderate/severe steatosis

Replacement of Retinyl Esters by Polyunsaturated Triacylglycerol Species in Lipid Droplets of Hepatic Stellate Cells during Activation

Activation of hepatic stellate cells has been recognized as one of the first steps in liver injury and repair. During activation, hepatic stellate cells transform into myofibroblasts with concomitant loss of their lipid droplets (LDs) and production of excessive extracellular matrix. Here we aimed to obtain more insight in the dynamics and mechanism of LD loss. We have investigated the LD degradation processes in rat hepatic stellate cells in vitro with a combined approach of confocal Raman microspectroscopy and mass spectrometric analysis of lipids (lipidomics). Upon activation of the hepatic stellate cells, LDs reduce in size, but increase in number during the first 7 days, but the total volume of neutral lipids did not decrease. The LDs also migrate to cellular extensions in the first 7 days, before they disappear. In individual hepatic stellate cells. all LDs have a similar Raman spectrum, suggesting a similar lipid profile. However, Raman studies also showed that the retinyl esters are degraded more rapidly than the triacylglycerols upon activation. Lipidomic analyses confirmed that after 7 days in culture hepatic stellate cells have lost most of their retinyl esters, but not their triacylglycerols and cholesterol esters. Furthermore, we specifically observed a large increase in triacylglycerol-species containing polyunsaturated fatty acids, partly caused by an enhanced incorporation of exogenous arachidonic acid. These results reveal that lipid droplet degradation in activated hepatic stellate cells is a highly dynamic and regulated process. The rapid replacement of retinyl esters by polyunsaturated fatty acids in LDs suggests a role for both lipids or their derivatives like eicosanoids during hepatic stellate cell activation

Diffuse reflectance spectroscopy: toward real-time quantification of steatosis in liver

Assessment of fatty liver grafts during orthotopic liver transplantation is a challenge due to the lack of real-time analysis options during surgery. Diffuse reflectance spectroscopy (DRS) could be a new diagnostic tool to quickly assess steatosis. Eight hundred and seventy-eight optical measurements were performed in vivo in 17 patients in liver tissue during surgery and ex vivo on liver resection specimens from 41 patients. Liver steatosis was quantified from the collected optical spectra and compared with the histology analysis from the measurement location by three independent pathologists. Twenty two patients were diagnosed with <5% steatosis, 15 patients had mild steatosis, and four had moderate steatosis. Severe steatosis was not identified. Intraclass correlation between the pathologists analysis was 0.949. A correlation of 0.854 was found between the histology and DRS analyses of liver steatosis ex vivo. For the same liver tissue, a correlation of 0.925 was demonstrated between in vivo and ex vivo DRS analysis for steatosis quantification. DRS can quantify steatosis in liver tissue both in vivo and ex vivo with good agreement compared to histopathology analysis. This analysis can be performed real time and may therefore be useful for fast objective assessment of liver steatosis in liver surgery

LD redistribution during HSC activation is microtubule dependent.

<p><b>A.</b> Frames from time lapse life cell analysis revealing redistribution of dynamic LDs towards growing cellular extensions. Tracking shows the typical movements of a dynamic LD from 72 h to 84 h of HSC activation. Arrows indicate other regions of dynamic LDs (trackings not shown). <b>B.</b> To examine microtubule involvement in the LD redistribution process, freshly isolated HSCs were after 24 h in culture either treated with 10 µM nocodazole or vehicle (control) for 72 h at 37°C and after fixation, morphology and LD localization were analyzed by differential interference contrast microscopy (DIC) and fluorescence microscopy after Bodipy staining.</p

HSC activation results in a preferential decrease in retinyl esters.

<p><b>A.</b> Neutral lipid composition of quiescent HSCs (day 0) analyzed by HPLC-APCI-MS. The results represent the means ± SEM of three experiments. <b>B.</b> Quantification of RE, cholesterol esters (CE), total TAG (TAG), and TAG(18:2,18-2,18:2) content in HSC at day 0, 4 and 7. Values are expressed relative to the level of lipid present at day 0. The results represent the means ± SEM of three experiments.</p

HSCs contain a metabolically homogenous population of LDs.

<p><b>A.</b> Freshly isolated HSCs were cultured for 6 days and additionally incubated with 25 µM deuterated arachidonic acid (AA-<i>d</i>8) for another 24 h. After fixation confocal Raman microspectroscopy was performed as described. Raman images in the 1595 cm⁻¹ (RE) and 2180–2280 cm⁻¹ (AA-<i>d</i>8) regions are shown in arbitrary units for LD enriched sites perinuclearly (upper panels) and at the cell extension (from a different cell; lower panels). <b>B.</b> To determine metabolic activity of HSC LDs, freshly isolated HSCs were cultured for 4 days and subsequently incubated for 5 h with 25 µM Bodipy C-12. After fixation cells were analyzed by fluorescence microscopy.</p

HSC activation results in a decrease in retinyl esters in HSC LDs.

<p>Freshly isolated HSCs were cultured and fixed after 2 h (day 0; quiescent state), and day 4 and day 7 (activated state). Confocal Raman microscpectroscopy on LD enriched regions was performed as described in the Method section. Cluster image (20×20 µm²) was constructed from Raman imaging data of the square area in the white light image. Each color represents a different cluster. The cluster averages show the average Raman spectra extracted from the black, pink, green and blue clusters displayed in the cluster image. * indicates (characteristic) RE peaks; # indicates characteristic acyl peak.</p