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

The overall goal of this procedure is to analyze the liver sinusoidal endothelium. This is accomplished by first isolating a liver and perfusing it with fixative. Next, the liver is cut into blocks and dehydrated in preparation for electron microscopy.

Then the liver samples are sputter coated and examined under an electron microscope. Finally, the electron micrographs are analyzed and fenestration diameter and frequency and percent porosity are calculated. Ultimately, electron microscopy is used to show changes in the liver sinusoidal endothelium under various conditions.

This method provides us with insight into the ultra structure and morphology of the liver blood vessels in their endothelium. It can also be used to examine the kidney and brain in the same way All procedures involving the use of animals are carried out according to the local legislation. This 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. This is a non survival surgery. After preparing fixative, according to the text protocol, warm the perfusion buffer and fixative at 35 to 37 degrees Celsius.

Once the animal is anesthetized with a single intraperitoneal injection of ketamine and xylazine, assess the level of sedation by hind limb withdrawal and a tail pinch. Next, with blunt ended scissors, make a Y incision in the animal's abdomen to expose the liver and portal vein. Then tie two very loose sutures around the portal vein one proximal to the liver and the other more distally to the liver.

Cannulate the portal vein with an appropriately sized IV cannula. Then tighten the sutures to secure cannula. Now using five to 20 milliliters of PBS warm to 37 degrees Celsius, begin perfusion at a pressure of 10 centimeters of H2O.

Avoid air bubbles entering the liver, which would produce artifacts and air pressure that is too high, which will damage the liver, sever the abdominal and thoracic vena CVA to allow the buffer to exit freely from the liver. This prevents high back pressure, damage to liver, sinusoidal and endothelial cells or LCCs. Once the liver is free of blood, replace the PBS with electron microscopy or em, fixative and perfuse until the liver is hardened and very pale.

Approximately five minutes. Then with the scalpel, cut the liver into one to two cubic millimeter blocks. Use em fixative to post.

Fix the tissue at four degrees Celsius for 24 to 72 hours. Then transfer the tissue into 0.1 molar sodium Cate buffer, and store at four degrees Celsius for up to 12 months to mount the specimens for scanning electron microscopy or SEM. After dehydrating the samples according to the text protocol, label the base of the metal SEM stubs and apply double-sided carbon tape to the top of the stubs under a dissecting microscope, visualize the samples to identify the surface with the best sinusoids for SEM.

Lace, a chosen surface facing up onto the carbon tape surface of the stub and stick it firmly on the stub. After performing sputter coating and SEM according to the text protocol, open an image in image J and use a scale bar embedded in the picture to set the scale using the polygon tool in image J trace around the entire flat area of the LSEC, including fenestrated and fenestrated areas, exclude any large pieces of debris that are on the cell surface that might be obscuring fenestrations. To measure the fenestration diameter, place a line through the longest diameter of each fenestration by selecting the line tool.

Press M to measure the line and D to permanently draw the line on the picture. This line is defined as the fenestration diameter and will now be available in the results box of image J.Measure all gaps which are larger holes in the LSEC cytoplasm over 250 nanometers, as well as fenestrations. Calculate the area of gaps that are not circular by tracing around them and using Image J to calculate their area past the data from the results box in image J into an Excel spreadsheet.

For further analysis, use the following formulas for carrying out fenestration calculations. The average fenestration diameter equals the average of all fenestration diameters. The fenestration area equals pi r squared where R is calculated from individual fenestration diameters.

The porosity percent equals sigma pi R squared over the total area analyzed and microns times 100 exclude the sum area of the gaps from this calculation. To present the data in publications include fenestration diameter with a statement confirming that boundary diameters were used to define fenestrations fenestration frequency and porosity also include a statement confirming whether gaps were included in the analysis and a frequency distribution graph of fenestration diameter and the number of livers blocks as well as images. Low magnification by SEM reveals a flat surface of the liver specimen with an exposed area large enough to observe many large liver vessels and sinusoids, including the portal tract, and central ES as shown here.

Ensuring correct liver block placement on the mounting stub is essential for obtaining clear images of the sinusoids and the glistens capsule, which covers all details of the sinusoids of the liver, should be avoided. For this reason, as shown here, higher magnification allows observation of LSEC fenestrations. The plates of hepatocytes can appear like blood vessels, but features such as the BLI assist with orientation.

This figure demonstrates that high profusion pressure can cause artifacts such as large gaps in the endothelium thought to be caused by coalescence of the LSEC SIV plates that are easily identified under SEM, avoiding cell membrane directly above the nuclei, that can be seen as a bulge under the surface of the LSEC will assist with accuracy of fenestration density and porosity calculations. Finally, quantification of LSEC fenestration diameter density and frequency provide an empirical measurement of fenestrations and allow measurements of changes induced by aging disease, toxins or therapies. Following the perfusion procedure, the tissue can be processed for other techniques like transmission electron microscopy to look at cellular ultra structure, mitochondrial structure.

It can also be used for histology.