A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Summary

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

Abstract

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

Introduction

The liver sinusoidal endothelial cells (LSECs) are highly differentiated endothelial cells that line the wall of the hepatic sinusoid. LSECs are perforated with fenestrations that are non-diaphragmed, transcellular pores 50-250 nm in diameter. Up to 20% of the surface of LSECs is covered by fenestrations, which are usually in groups of tens to hundreds called sieve plates^1-3 (Figure 1). Fenestrations allow transfer of plasma and nanosubstrates between blood and hepatocytes, creating a highly efficient ultrafiltration system. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiologi....

Protocol

NOTE: All procedures involving the use of animals are carried out according to the local legislation. Our work is approved by the Sydney Local Health District Animal Welfare Committee. The allowed procedures are described in the project license documentation and follow guidelines that ensure the welfare of the animal at all times. Ensure adherence to the legislation on animal experimentation of the country where the work is performed.

1. Protocol for Preparing EM Fixative

1. For 100 ml of fixative, prepare paraformaldehyde by adding 2 g of paraformaldehyde powder to 25 ml distilled water in a conical flask, heat to 65
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Results

Initial visualization at low magnification by scanning electron microscopy reveals a flat surface of the liver specimen with an exposed area large enough to observe many large liver vessels and sinusoids (Figure 1A). Ensuring correct liver block placement on the mounting stub is essential for obtaining clear images of the sinusoids and the Glisson’s capsule of the liver should be avoided for this reason (Figure 1B). Increasing the magnification allows closer inspection of the dense vascu.......

Discussion

The ability to accurately and reproducibly measure the status of the liver sinusoidal endothelium is an important step in understanding the biology of these highly specialized cells. Newer techniques such as structured illumination microscopy³², atomic force microscopy³³ and d-STORM (direct Stochastic Optical Reconstrucion Microscopy) ³⁴ will impart important information regarding the morphology of these cells in vitro but SEM remains the primary methodology to visualize and.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
25% EM grade Glutaraldehyde	ProSciTech	C001	Store stock at -20 C until needed, avoid refreeze
Paraformaldehyde powder	Sigma Aldrich	158127	Always prepare Paraformaldehyde fresh
Sodium Cacodylate powder	Sigma Aldrich	C0250	Prepare 0.2 M stock, pH 7.4 by dissolving powder in dH2O, used mostly at 0.1 M by preparing 1:2 dilution
Calcium Chloride	Sigma Aldrich	C1016	Prepare 1 M CaCl2by dissolving powder in dH2O
Osmium tretroxide	ProSciTech	C011	Wash ampoules in weak acid prior to use to avoid contamination. Prepare 2 % stock in glass bottle
Ethanol- Absolute	Sigma Aldrich	459836	100 % Ethanol must be high grade and stored with Molecular Sieve
Other grades of Ethanol	Labtech	EL5	Prepare graded Ethanols with dH2O
Hexamethyldisilazane	Sigma Aldrich	52619	Allow to reach room temperature before use
Cannulas	Terumo	TSROX1832C, TSROX2225C, TSROX2419C	18 G is suitable for most rats, 22 G is suitable for most mice, but it is good to have a few 24 G on hand in case of very small mice
Conductive Carbon tape	ProSciTech	IA0201
Carbon Paint	ProSciTech	I003
Ketamine			Must be obtained under license
Xylazine			Must be obtained under license
Molecular Sieve	Sigma Aldrich	208647	Removes water from the 100 % Ethanol