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

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. Video LinkThe video component of this article can be found at

Targeting Vascular Endothelial-Cadherin in Tumor-Associated Blood Vessels Promotes T-cell–Mediated Immunotherapy

“…Care was taken to keep the injecting pressure low to avoid destroying the blood vessels. The tumors were cut into smaller pieces over SEM fixative and incubated in SEM fixative for 72 h at 4 C. Specimens were further prepared for scanning electron microscopy as previously described (27) and tumor vasculature was examined on a JEOL 6380 JSM at up to Â3,000 magnification. Four control and four CD5-2 vessel samples were examined.…”

Tracking Fenestrae Dynamics in Live Murine Liver Sinusoidal Endothelial Cells

“…Moreover, we noticed that even fenestrae as large as 200‐400 nm may close very rapidly within 1 minute or may shrink and slowly fade. Following Cogger, we previously omitted fenestrae >300 nm in calculations of porosity . By tracing individual fenestrae with time, however, we noticed that some of them can be as large as 400 nm in diameter, still being functional structures.…”

“…Secondly, we noticed a correlation between gaps and the number of fenestrae. Those large cytoplasmic openings were indicated in the literature as markers of a disease and/or an effect of a toxic environment, which results from fusion of a few fenestrae in the sieve plate . Nonetheless, until now it was not certain if those structures were formed due to improper fixation and even if they were formed in live LSECs at all .…”

“…Following Cogger, we previously omitted fenestrae >300 nm in calculations of porosity. (19,26) By tracing individual fenestrae with time, however, we noticed that some of them can be as large as 400 nm in diameter, still being functional structures. However, FACRs forming >400-nm fenestrae sometimes break, which leads to formation of gaps in the cytoplasm.…”

Dietary Protein to Carbohydrate Ratio and Caloric Restriction: Comparing Metabolic Outcomes in Mice

“…Following post-fixation with osmium tetroxide, graded dehydration in ethanol and hexamethyldisilazane, 1mm 3 blocks of liver were sputter coated with platinum and examined using a JEOL 6380 scanning electron microscope (JEOL, Japan) at 20,000× magnification. Ten random images were taken per sample and fenestration diameter analysed using ImageJ software (Cogger et al, 2015). …”