Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

Summary

Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment.

Abstract

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 µm < d ≤ 3 µm), intermediate vesicles (3 µm < d ≤ 9 µm), and macrovesicles (d > 9 µm), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.

Introduction

Lipid droplets (LDs), classically viewed as energy depots, are specialized cellular organelles that mediate lipid storage, and they comprise a hydrophobic neutral lipid core, which mainly contains cholesterol esters and triglycerides (TGs), encapsulated by a phospholipid monolayer^1,2,3.

LD biogenesis occurs in the endoplasmic reticulum (ER), starting with the synthesis of triacylglycerol (TAG) and sterol esters. Neutral lipids are diffused between the leaflets of the ER bilayer at low concentrations but coalesce into oil lenses that grow and bud in....

Protocol

All animal procedures performed in this study were approved by the Coimbra Institute for Clinical and Biomedical Research (iCBR) Animal Welfare Body (ORBEA, #9/2018) and complied with the Animal Care National and European Directives and with the ARRIVE guidelines.

1. Experimental design

1. Pair-house 13 week old male Wistar rats in ventilated cages under controlled environmental conditions of temperature (22 °C ± 1 °C), humidity (50%-60%), and light (12 h light-dark cycle) and with ad libitum access to tap water and standard rodent chow.
2. After a 2 week period of acclimatization, ar....

Results

The successful execution of this technique should result in clear lipid droplet staining for the simultaneous characterization of the LD morphology (shape and lipid core density based on the 3D reconstruction) along with their spatial distribution, number per total area, and average size (assessed with the pipeline above described, Figure 1).

Discussion

This BODIPY 493/503 fluorescence-based protocol for LD assessment aimed to develop a new imaging approach for the evaluation of hepatic steatosis. Given the strong correlation between obesity and fatty liver disease, the Western-style high-fat diet was used to establish an animal model of hepatic steatosis²⁶. A robust increase in hepatic TG contents was confirmed by a quantitative triglycerides colorimetric assay kit, which suggested a heightened hepatic lipidosis scenario in the HFD-fed animals. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.6 mm I.D. silicone tubing, I.V mini drip set	Fisher Scientific
4,4-difluoro-1,3,5,7,8-pentametil-4-bora-3a,4a-diaza-s-indaceno (BODIPY 493/503)	Sigma-Aldrich, Lyon, France	D3922
4',6-diamidino-2-phenylindole (DAPI)	Molecular Probes Inc, Invitrogen, Eugene, OR	D1306
70% ethanol	Honeywell	10191455
Adobe Illustrator CC	Adobe Inc.	Used to design the figures
Automatic analyzer Hitachi 717	Roche Diagnostics Inc., Mannheim, Germany	8177-30-0010
Barrier pen (Liquid blocker super pap pen)	Daido Sangyo Co., Ltd, Japon	_
Blade	Leica	221052145	Used in the cryostat
Cell Profiler version 4.2.5	https://cellprofiler.org/releases/	Used to analyse the acquired images
Coverslips	Menzel-Glaser, Germany	_
Cryomolds	Tissue-Tek	_
Cryostat (including specimen disc and heat extractor) CM3050 S	Leica Biosystems	_
Dimethyl Sulfoxide (DMSO)	Sigma-Aldrich, Lyon, France	D-8418	Used to dissolve Bodipy for the 5 mg/mL stock solution. CAUTION: Toxic and flammable. Vapors may cause irritation. Manipulate in a fume hood. Avoid direct contact with skin. Wear rubber gloves, protective eye goggles.
Dry ice container (styrofoam cooler)	Novolab	A26742
Dumont forceps	Fine Science Tools, Germany	11295-10
Glass Petri dish (H 25 mm, ø 150 mm)	Thermo Scientific	150318	Used to weigh the liver after dissection
Glycergel	DAKO Omnis	S303023
GraphPad Prism software, version 9.3.1	GraphPad Software, Inc., La Jolla, CA, USA
High-fat diet	Envigo, Barcelona, Spain	MD.08811
Ketamine (Nimatek  100 mg/mL)	Dechra	791/01/14DFVPT	Used at a final concentration of 75 mg/kg
Laser scanning confocal microscope  (QUASAR detection unit; )	Carl Zeiss, germany	LSM 710 Axio Observer Z1 microscope
Medetomidine (Sedator 1 mg/mL)	Dechra	1838 ESP / 020/01/07RFVPT	Used at a final concentration of 1 mg/kg
Needle	BD microlance	300635
No 15 Sterile carbon steel scalpel Blade	Swann-Morton	205
Objectives 10x (Plan-Neofluar 10x/0.3), 20x (Plan-Apochromat 20x/0.8) and 40x (Plan-Neofluar 40x/1.30 Oil)	Carl Zeiss, Germany
Paint brushes	Van Bleiswijck Amazon B07W7KJQ2X	Used to handle cryosections
Peristaltic pump (Minipuls 3)	Gilson	1004170
Phosphate-buffered saline (PBS, pH ~ 7.4)	Sigma-Aldrich, Lyon, France	P3813
Scalpel handle, 125 mm (5"), No. 3	Swann-Morton	0208
Slide staining system StainTray	Simport Scientific	M920
Standard diet	Mucedola	4RF21
Superfrost Plus microscope slides	Menzel-Glaser, Germany	J1800AMNZ
Tissue-Tek OCT mounting media	VWR CHEMICALS	361603E
Triglycerides colorimetric assay kit	Cayman Chemical	10010303
Ultrasonic bath	Bandelin Sonorex	TK 52
Vannas spring scissors - 3 mm cutting edge	Fine Science Tools, Germany	15000-00
ZEN Black software	Zeiss