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. 2020 Aug 19;11(9):5132-5146.
doi: 10.1364/BOE.395890. eCollection 2020 Sep 1.

Intravital longitudinal imaging of hepatic lipid droplet accumulation in a murine model for nonalcoholic fatty liver disease

Jieun Moon  1   2 , Eunji Kong  2   3 , Jingu Lee  1   2 , Jinjoo Jung  4 , Eunha Kim  5 , Seung Bum Park  4   6 , Pilhan Kim  1   2   3
Affiliations

Intravital longitudinal imaging of hepatic lipid droplet accumulation in a murine model for nonalcoholic fatty liver disease

Jieun Moon et al. Biomed Opt Express. .

Abstract

Nonalcoholic fatty liver disease (NAFLD) is a rapidly increasing chronic liver disorder worldwide accompanied by hepatic steatosis, inflammation, fibrosis, and severe liver failure. Unfortunately, an effective treatment strategy for NAFLD has not yet been established, which has been hampered by the limited understanding of the pathophysiological drivers for NAFLD. To examine the unknown cellular and molecular mechanisms in the pathogenesis of NAFLD, there is an increasing need for the direct in vivo observation of hepatic microenvironments over extended periods of time. In this work, using a custom-built intravital imaging system and a novel fluorescent lipid droplet labeling dye, Seoul-Fluor 44 (SF44), we established an intravital imaging method to visualize individual lipid droplets and microvasculature simultaneously in the liver of live mice in vivo. In addition, in the nonalcoholic steatosis and steatohepatitis mouse model induced by a methionine and choline-deficient diet, we longitudinally visualized and quantitatively analyzed the development of lipid droplets in hepatocytes and sinusoid at a subcellular resolution during the progression of NAFLD up to 21 days in vivo.

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Conflict of interest statement

The authors declare that there are no conflicts of interest related to this article.

Figures

Fig. 1.
Fig. 1.
Intravital liver imaging setup. (a) Photograph of intravital liver imaging setup. Heating pad for body temperature maintenance of the anesthetized mouse and silicone rubber heater for temperature maintenance of exposed liver were used. (b) Schematic of exposed liver positioning on wet gauze to minimize motion and humidity maintenance.
Fig. 2.
Fig. 2.
Intravital imaging of SF44-labeled hepatic LDs in MCD-diet induced NASH mouse model. (a) In vivo images of hepatic LDs (yellow, SF44) and sinusoid (cyan, CD31) in the liver of mice fed the normal diet or MCD diet for 28 days. Scale bars, 50 µm. (b) Diameter of LD in ND or MCD for 28 days shown on (a). (c) Histopathological hematoxylin and eosin (H&E) staining images of the livers of mice fed the normal diet or MCD diet for 28 days. Scale bars, 50 µm. (d) Graph of temporal changes in the fluorescence intensity of SF44 in the liver of mice fed the MCD diet for 28 days after the intravenous injection of SF44. (e) Wide-area (top row) and magnified (solid-, yellow dotted-line box, respectively) sequential images of the hepatic LDs (yellow) after intravenous injection of SF44 obtained with same excitation laser power and detector sensitivity. Scale bars, 50 µm.
Fig. 3.
Fig. 3.
Spatial distribution of hepatic lipid droplet in MCD diet-induced NASH mouse model. (a) Wide-area in vivo image showing spatially distributed different-sized hepatic lipid droplets in the liver of mouse fed MCD diet for 14 days. Scale bar, 100 µm. (b) Magnified images of (a) marked by yellow solid-lined box (portal vein), white dotted-lined box (periportal) and white solid-lined box (sinusoid), respectively. Scale bars, 20 µm. (c,d) LD diameter and LD area, respectively at portal vein, periportal, and sinusoid shown on (b).
Fig. 4.
Fig. 4.
Longitudinal intravital imaging of hepatic lipid droplet accumulation in MCD-diet induced NASH mouse model. (a) Representative maximum intensity projection (MIP) images of hepatic LD (yellow, SF44) and sinusoid (cyan, CD31) in the liver of mice fed normal diet or MCD diet. Scale bars, 20 µm. (b) Representative magnified in vivo images of single hepatocyte showing the position of nuclei (asterisks) and boundary (dotted line). Scale bars, 10 µm.
Fig. 5.
Fig. 5.
Quantitative analysis of longitudinal accumulation of hepatic lipid droplet in MCD-diet induced NASH mouse model. (a) Representative 3D rendered image of hepatic lipid droplets (green, SF44) and sinusoid (red, CD31) in the liver of mice fed normal diet or MCD diet. Scale bars, 20 µm. (b) Number of hepatic lipid droplets. (c) Volumetric ratio of hepatic lipid droplets. (d) Fractional ratio of groups of hepatic lipid droplets with different diameters; d < 1 µm, 1 < d ≤ 3 µm, 3 < d ≤ 9 µm, d > 9 µm. (e) Fractional ratio of hepatic lipid droplet in each group. (d–e) Data are presented as mean ± standard error of mean. 3 mice were analyzed for each group.
Fig. 6.
Fig. 6.
Quantitative analysis of sinusoid in MCD-diet induced NASH mouse model. (a) Representative image of sinusoid area in the liver of mice fed normal diet or MCD diet. Scale bars, 50 µm. (b) Average diameter of sinusoid in normal diet or MCD diet.
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