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.
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.
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 plates1-3 (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 physiological states, drugs, and disease. For example, fenestrations are larger in the fasted than in the fed state4; 2-di-iodoamphetamine increases fenestration number;5,6 and a reduction in size and number of fenestrations per cell occurs in ageing and many disease states7-13. Accurate measurement of the size and number of fenestrations is important for understanding how LSEC morphology is influenced by various disease, toxic, and physiological states; their impact on liver function; and for developing fenestration-modulating therapeutic interventions1.
The study of fenestrations is difficult. The diameter of fenestrations lies below the resolution of conventional light microscopy, so previously only observation using electron microscopy both in intact liver tissue or cultured LSECs has been possible. The scanning electron microscope (SEM) has most frequently been used to study fenestration size, frequency and porosity (the percentage of LSEC membrane that is perforated by fenestrations) because SEM allows for the observation of large areas of the endothelial surface and measurement of thousands, if not tens of thousands of fenestrations. Despite its utility, the results which are reported from SEM-based studies for LSEC parameters such as fenestration size, number, frequency and porosity vary widely in the literature (Table 1).
Fenestrations and sieve plates are fragile structures that contract, break, dilate or coalesce during specimen preparation, thus careful processing is needed to preserve their integrity. Elevated perfusion pressure14; incorrect osmolarity of the fixative and buffers15; inadequate fixation or fixation time; and speed of post-fixation dehydration and drying are all areas of processing for SEM that may produce artefacts that interfere with preservation of ultrastructure (Figure 2). Loss of fenestrations (‘defenestration’) and fenestration shrinkage can occur as a result of poor fixation, resulting in reduced fenestration diameter and cell porosity. Methods to improve the preservation of specimens for SEM analysis have been described previously 15-17 and will be discussed here with additional tips on how to improve specimen preservation. The main goals of the specimen preservation are to remove blood from the sinusoids so that the surface of the LSEC can be visualized and to avoid LSEC damage from either high pressure or delayed fixation. Whole liver perfusion of fixative via the portal vein is the preferred method for liver fixation. As described in detail elsewhere16,18 perfusion must be undertaken at low pressure (eg 10 cm of H20) to avoid pressure-related perfusion artefacts and damage to the LSEC, typically manifested as large gaps within in the cell membrane. However, reasonable fixation can often be obtained using needle perfusion of liver biopsies from humans and animals, as described in detail elsewhere19. This technique involves directly injecting fixative into the tissue until blood is flushed out of the sample and the tissue is firm and fixed. Fixation of samples for electron microscopy needs to be performed as quickly as possible following cessation of blood flow to prevent ultrastructural changes occurring as a result of the livers extremely rapid autolytic processes.
We also present a method of image analysis that minimizes the inclusion of artefacts, and standardizes the measurement of fenestrations. Variation in the selection of sinusoids for micrographs, image analysis of artefacts, and measurement of cell area for porosity and fenestration frequency have led to major discrepancies in published results. A standardized approach for evaluation and measurement of fenestrations and the minimum requirements for data presentation have not been clearly addressed in the literature previously4,10,20-31.
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
2. Perfusion Fixation of Liver
3. Needle Perfusion
NOTE: Needle fixation is a variant of perfusion fixation that involves injection of fixative directly into biopsied liver tissue. The aim is for fixative to be flushed through the blood vessels in order to exsanguinate them and expose all of the tissue block to fixative. Care must be taken to keep the injecting pressure low to avoid pressure injury18,19.
4. Preparation for Scanning Electron Microscopy
5. Mounting
NOTE: Correct specimen mounting will maximize the number of clearly delineated sinusoids available for analysis under SEM.
6. Coating
NOTE: Coating the specimen with a fine film of conductive metal (gold or platinum) in a sputter coater grounds the specimen and protects it from damage from the electron beam. If the coating is too thick structures of interest may be obscured.
7. Using the Scanning Electron Microscope
8. Analysis
NOTE: ImageJ software which can be downloaded free from the NIH is utilized to quantify fenestration diameter and frequency (www.imagej.nih.gov/ij).
9. Calculations
10. Presentation of Fenestration Data
NOTE: Whenever possible, publications including quantification of fenestration data should include the following information
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 vasculature of the liver and reveals the single cell plates of hepatocytes that divide the vessels (Figure 1C). Poor fixation of the liver results in poor image quality, blood cells and debris obscure the view of the liver (Figure 1D).
Further increasing the magnification allows observation of LSEC fenestrations (Figure 2A). It is essential that the sinusoids are correctly identified – the plates of hepatocytes can, under some circumstances appear like blood vessels but features such as the bile canaliculi assist with orientation (Figure 2B). High perfusion pressure can cause artefacts such as large gaps in the endothelium, thought to be caused by coalescence of the LSEC sieve plates. These are easily identified under SEM (Figure 2C). Avoiding cell membrane directly above the nuclei will assist with accuracy of fenestration density and porosity calculations (Figure 2D).
Analysis of the LSEC with ImageJ allows quantification of LSEC fenestration diameter, density and frequency (Figure 3A). These dimensions provide an empirical measurement of fenestrations and allow measurements of changes induced by aging, disease, toxins or therapies (Figure 3B).
Figure 1. Visualizing the liver with SEM. (A) Initial inspection under the SEM reveals the sinusoids of the liver and larger vessels including those of the portal tract and central venules; (B) The Glisson’s capsule covers all details of the sinusoids; (C) The dense vasculature of the liver and single cell plates of hepatocytes that lie between the vessels; (D) Poor fixation results in collapsed sinusoids and the development of ultrastructural artefacts. Please click here to view a larger version of this figure.
Figure 2. The ultrastructure of the liver. (A) LSEC fenestrations are easily seen in well-fixed tissue; (B) The surface of a hepatocyte with well-preserved bile canaliculi; (C) Large gaps in the endothelium caused by high perfusion pressure; (D) LSEC nuclei indicated by the arrows bulge under the surface of the thin LSEC and should not be included in the area for porosity analysis. Please click here to view a larger version of this figure.
Figure 3. Analysis of the LSEC with ImageJ. (A) The polygonal tool allows measurement of the overall area for measurement and the line tool is used to measure fenestration diameter; (B) A frequency histogram generated by the data gained from Image J analysis. Please click here to view a larger version of this figure.
Diameter (nm) | Frequency (per um2) | Porosity (%) | Reference |
n.a. | n.a | 60 ± 12 | 20 |
127 ± 4 nm | 6.5 ± 0.5 | 4.6 ± 0.4 | 21 |
n.a | n.a | 40.5 | 22 |
n.a. | 0.6 ± 1.4 | 0.01 ± 0.03 | |
n.a. | 2.5 ±0.5 | 0.047 ± 0.012 | 24 |
87.25 ± 1.4 | n.a | 9.22 ± 0.7 | 25 |
100-200 | 4.49 ± 0.69 | 2.55 ± 0.54 | 26 |
n.a. | n.a. | 23 ± 2 | 27 |
85 ± 17 | 11 ±0.4 | 6.6 ± 0.2 | 28 |
n.a | 2.7 ± 1.1 | 4.1 ± 2.3 | 10 |
68 ± 1 | 8 ± 0.6 | 3.4 ± 0.2 | 29 |
n.a. | n.a | 3.9 ± 0.2 | 30 |
73.3 ± 0.4 | n.a | 2.2 ± 0.2 | 31 |
90.7 ± 11.7 | 8.45 ± 2.43 | 5.93 ± 2.05 | 4 |
110.7 ± 0.25 | 9 | 6 | 3 |
104.8 ± 0.22 | 13 | 8 | 3 |
Table 1. Published data on fenestrations from a selection of studies in rats showing a wide range of values for fenestration diameter, frequency and porosity when measured by SEM in intact and preserved liver tissue (Mean ± sem).
Component | Artefact |
Osmolarity of fixative and buffer too low (hypotonic) | Cell swelling |
Osmolarity of fixative and buffer too high(hypertonic) | Cell shrinkage |
Specimen size too large | Fixative cannot penetrate far enough during incubation = autolysis/inadequate fixation |
pH of the fixative and buffer prior to osmium less than 7.2 or over 7.4 | Protein denaturation and structural deformities |
Fixation time too short | Autolysis |
Fixation time too long | May cause cell shrinkage |
Temperature high | Increases fixation speed however may denature proteins |
Temperature low | Inadequate fixation |
Perfusion pressure too high | Gaps, space of Disse enlargement, vacuoles in hepatocytes, loss of sieve plates, widening of fenestration diameter |
Mechanical damage when mincing and/or using forceps | Crushing of cellular structures if the tissue is not hardened well enough by the fixative |
Prolonged time to fixation after death, specimen biopsy, or exsanguination | Ischaemia, autolysis, gap formation, defenestration |
Low fixative incubation volume | Less than 20 times the specimen volume = inadequate penetration of the fixative and autolysis in the centre of blocks. |
Fixative concentration too high | Concentration affects the rate of fixation and penetration of fixative. Artefact formation. |
Fixative concentration too low | Exhaustion of the fixative before the fixation process is complete producing cell blebbing and other artefacts. |
Signs of incomplete fixation in liver | Cell blebbing (swollen microvilli or originating from the cytoplasm), defenestration, gaps, loss of microvilli, swollen mitochondria, cell swelling, sinusoid lumens collapsed or compressed, blood in sinusoidal lumen, irregularly shaped hepatocyte nuclei |
Blood contamination of the fixative | Deactivates fixative |
Dirty perfusion and preparation equipment | Specimen contamination with debris and bacteria |
Incomplete dehydration of the specimens or storage without desiccant after coating | Specimen charge-up on SEM. Adding carbon paint to the block may help reduce charge |
Table 2. Troubleshooting tips for poor specimen and image quality.
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 measure their structure in situ.
The most crucial and technically challenging step is the initial fixation: if the liver is preserved well the subsequent steps will produce easily observable and indeed, beautiful, images of the liver sinusoid. Whole liver perfusion is the most effective method to ensure good fixation, but comparable results are possible in needle perfusion samples that have been fixed quickly and under low pressure. Resorption of water by the dehydrated liver samples is another common reason for poor SEM images, but this can be easily avoided by proper storage of the samples in a desiccator with desiccant.
There is a need for standardization of the quantification of fenestrations so that studies from different research groups can be compared and interpreted. In the past there has been wide variation in the values published with very little methodological information about how these values were obtained. Here we have provided a standardized approach for determining and presenting values that describe fenestration ultrastructure.
Whenever possible, publications including quantification of fenestration data should include the following information: Fenestration diameter, with a statement confirming what boundary diameters where used to define fenestrations (typically between 50 – 250 nm); fenestration frequency (number/µm2 ) and porosity (%); a statement confirming whether gaps have been included in the analysis; and a frequency distribution graph of fenestration diameter. In addition, the number of livers, blocks, images and fenestrations should be included in the analysis.
Fenestration diameter, frequency and porosity are important indicators of liver status and standardizing the collection of this data will be beneficial to the field. The methods discussed here provide a framework for ensuring that studies of the ultrastructure of the LSEC and fenestrations are performed and presented in a comparable way across different research groups. The methodology is easily adapted to measuring fenestrations in isolated and cultured LSECs.
The authors have nothing to disclose.
The authors have no acknowledgements.
Name of material | Company | Catalogue Number | Comments |
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 optained under licence | ||
Xylazine | Must be obtained under licence | ||
Molecular Sieve | Sigma Aldrich | 208647 | Removes water from the 100 % Ethanol |