Intravital visualization of dynamic alteration of hepatic microenvironment in nonalcoholic fatty liver disease

Abstract

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease that affects about 25% of adults globally, yet there are still no effective pharmacological therapies for NAFLD. There have been numerous efforts to understand the underlying pathophysiological factors for NAFLD, but research to elucidate unknown pathological drivers in NAFLD has been chiefly performed by ex vivo methods. Ex vivo methods from sacrificed animals can provide valuable biological information but have difficulty acquiring dynamic information. To observe the constantly varying pathological features in various stages of NAFLD progression in a complex in vivo hepatic microenvironment, spatiotemporal intravital visualization of dynamic alterations in the hepatic micro-environment in the NAFLD progression is a highly desirable approach. Here, we developed intravital liver imaging techniques to visualize the hepatic microenvironment based on custom-design video-rate laser-scanning confocal and two-photon intravital microscopy and longitudinally visualized the various pathological phenotypes in the NAFLD progression.

We successfully visualized the subcellular-level hallmarks of NAFLD in vivo by utilizing a custom-design video-rate laser-scanning confocal microscopy system. We three-dimensionally visualized and quantitatively analyzed the gradual increase of hepatic lipid droplets (LDs) accumulation and their size with the liver vascu-lature simultaneously during the development of hepatic steatosis in the methionine and choline-deficient (MCD) diet-induced NAFLD mouse model. We identified the distinct spatial distribution of different sized hepatic LDs in vivo according to the location in the NAFLD liver. In addition, we could identify the subcellular-level displacement of nuclei in a single hepatocyte and hepatocyte enlargement due to the formation of macrovesicular LDs in a hepatocyte in vivo.

We achieved an in vivo subcellular-level visualization of hepatocellular steatosis and fibrosis in the liver of streptozotocin (STZ)-treated high-fat diet (HFD)-induced NAFLD with hyperglycemia mouse model using the custom-design intravital two-photon microscopy system. Based on two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) imaging, the hepatic steatosis, the deposition of collagen fibers in the perisinusoidal region, and advanced severe fibrosis accompanying the fibrous septa were clearly observed and quantified with the three-dimensional manner and subcellular level in vivo. Our result of significantly increased hepatic LD diameter and perisinusoidal collagen deposition by STZ treatment in HFD-fed mice suggests insulin deficiency can be a strong driving factor aggravating NAFLD.