Optimization of In vivo Imaging Provides a First Look at Mouse Model of Non-Alcoholic Fatty Liver Disease (NAFLD) Using Intravital Microscopy - PubMed (original) (raw)
Optimization of In vivo Imaging Provides a First Look at Mouse Model of Non-Alcoholic Fatty Liver Disease (NAFLD) Using Intravital Microscopy
Rachelle P Davis et al. Front Immunol. 2020.
Abstract
Non-alcoholic fatty liver disease is a spectrum of liver pathology ranging from simple steatosis to steatohepatitis and can progress to diseases associated with poor outcomes including cirrhosis and hepatocellular carcinoma (HCC). NAFLD research has typically focused on the pathophysiology associated with lipid metabolism, using traditional measures such as histology and serum transaminase assessment; these methods have provided key information regarding NAFLD progression. Although valuable, these techniques are limited in providing further insight into the mechanistic details of inflammation associated with NAFLD. Intravital microscopy (IVM) is an advanced tool that allows for real-time visualization of cellular behavior and interaction in a living animal. Extensive IVM imaging has been conducted in liver, but, in the context of NAFLD, this technique has been regularly avoided due to significant tissue autofluorescence, a phenomenon that is exacerbated with steatosis. Here, we demonstrate that, using multiple imaging platforms and optimization techniques to minimize autofluorescence, IVM in fatty liver is possible. Successful fatty liver intravital imaging provides details on cell trafficking, recruitment, function, and behavior in addition to information about blood flow and vessel dynamics, information which was previously difficult to obtain. As more than 30% of the global population is overweight/obese, there is a significant proportion of the population at risk for NAFLD and complications due to NAFLD (liver decompensation, cirrhosis, HCC). IVM has the potential to elucidate the poorly understood mechanisms surrounding liver inflammation and NAFLD progression and possesses the potential to identify key processes that may be targeted for future therapeutic interventions in NAFLD patients.
Keywords: NAFLD; fatty liver; intravital imaging; mice; technique.
Copyright © 2020 Davis, Surewaard, Turk, Carestia, Lee, Petri, Urbanski, Coffin and Jenne.
Figures
Figure 1
Establishment of a mouse model of NAFLD. Animals were fed either SD or HFD for 20 w and animal weight (A) and plasma ALT levels (B) were measured. Histology of the mouse liver reveals normal tissue architecture in animals fed SD (Ci), whereas, animals fed HFD (Cii) display pronounced steatosis and hepatocyte ballooning. IVM imaging of unstained liver (i.e., no exogenous fluorophores or dyes added) shows an overall autofluorescence dotted by focal intense spots of fluorescence (white spots in the overlay image) (scale bar = 100 μm) (D). ***p < 0.001.
Figure 2
Optimization of IVM of murine NAFLD. (A) Schematic representation of the imaging strategy utilizing narrower emission filters (red and blue boxes) that have been shifted away from peak fluorophore emission to limit the collection of non-specific spectral overlap (yellow shading). IVM of liver using resonant scanning confocal microscopy with conventional filter settings in animals fed SD (Bi) or HFD (Bii). RSC imaging of livers from mice fed SD (Biii) or HFD (Biv) using optimized (narrowed filters, off-peak fluorescence collection, sequential excitation) imaging parameters yields substantially less background autofluorescence and allows for clear visualization of multiple labeled immune cell populations. Cell populations were labeled with i.v. fluorophore-conjugated antibodies 10 min prior to imaging (scale bar = 50 μm). Additionally, lipid deposits are visible in the liver of mice fed HFD (Bv, white outlines; same FOV as Biv). IVM of liver using spinning disk microscopy in animals fed SD (Ci) or HFD (Cii). Animals were i.v. injected with fluorophore-conjugated antibodies 10 min prior to imaging (scale bar = 50 μm).
Figure 3
Assessment of fatty liver physiology using IVM. Mapping of liver vasculature in mice fed SD (Ai) or HFD (Aii) using an i.v. injection of FITC-conjugated albumin (red). Overlay of hepatocyte fluorescence with vascular contrast agent (SD, Aiii; HFD, Aiv) (scale bar = 50 μm). (B) Mean liver sinusoidal diameter as measured by IVM (values represent vascular diameter in μm, mean of >10 sinusoids/FOV; 5 FOV/animal, n = 3 animals per group). Mean sinusoidal blood flow rate as determined by IVM (C). Rate was calculated by measuring the sinusoidal cross-sectional area and blood velocity (as determined by velocity of i.v. injected fluorescent beads) and values are reported as μm3/s. ***p < 0.001; **p < 0.01.
Figure 4
Tracking of immune cell behavior and function in fatty liver by IVM. Fluorescent antibody labeling of multiple immune cell populations enables tracking of cell behavior and function by IVM (A). Cells were labeled by i.v. injection fluorophore-conjugated antibodies 10 min prior to imaging. Scale bar = 50 μm. (B) Quantification of the number of neutrophils (Ly6G+) and cytotoxic T cells (CD8+) by IVM in the livers of mice fed HFD (values represent the number of cells/FOV; minimum of 5 FOV/animal; n = 3 animals). (C) Quantification of the number of macrophage (F4/80+) by IVM in the livers of mice fed SD or HFD (values represent the number of cells/FOV; minimum of 5 FOV/animal; n = 3 animals per group). (D) IVM assessment of neutrophil behavior in the livers of mice HFD; 5 FOV/animal; n = 3 animals. (E) Determination of neutrophil (Ly6G+), cytotoxic T cell (CD8+) or intravascular bead velocity in the livers of mice fed HFD (values represent mean object velocity in μm/s from 5 FOV/animal; n = 3 animals). (F) Representative image obtained by IVM of sterile bead capture (bright green) by liver macrophage (F4/80+; red). Scale bar = 50 μm. (G) Quantification of bead capture in the liver (values represent the number of beads/FOV; minimum of 5 FOV/animal; n = 3 animals). (H) Determination of the fraction of macrophage that have captured a minimum of 1 bead (values represent the % of macrophages binding beads/FOV; minimum of 5 FOV/animal; n = 3 animals). (I) Determination of fraction of macrophage that have bound multiple beads (values represent the % of macrophages binding multiple beads/FOV; minimum of 5 FOV/animal; n = 3 animals). ***p < 0.001; **p < 0.01; *p < 0.05.
References
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