ELECTRON MICROSCOPY SIGNS OF NORMAL AND DYSFUNCTIONAL RAT AORTIC ENDOTHELIUM

Highlights

• A fundamental distinguishing feature of normal endothelial cell morphotypes from pathological ones is their preserved orientation along the direction of blood flow in the absence of cytoplasmic or membrane defects.
• The main characteristics of dysfunctional endothelial cells include a spherical shape (indicating a loss of cellular orientation along the direction of blood flow), the presence of large vacuoles within the cell, cytoplasmic vacuolization, impaired plasma membrane integrity, reduced contrast between the nucleus and cytoplasm, and partial detachment of the endothelial cell from the basement membrane.
• The condition of organelles (mitochondria, Golgi complex, and endoplasmic reticulum) and impaired basement membrane integrity are not sensitive or specific markers of dysfunctional endothelium compared to the aforementioned features

Aim. To analyze the electron microscopic features of normal and dysfunctional endothelium using the descending aorta of rats (characterized by laminar blood flow).

Methods. The study was conducted on 5 male Wistar rats (age ≈ 6 months, body weight ≈ 500 g). The extracted aortas were chemically fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide solution with 1.5% potassium ferrocyanide, incubated with 1% thiocarbohydrazide, stained in 2% aqueous osmium tetroxide solution, contrasted in 1% phosphotungstic acid, stained with 2% gadolinium triacetate, dehydrated in ascending concentrations of ethanol, isopropanol, and acetone, embedded in a mixture of acetone and epoxy resin, and then in pure Araldite 502 epoxy resin, followed by its polymerization. After grinding and polishing, the aorta samples were contrasted with lead citrate, coated with carbon, and visualized using backscattered scanning electron microscopy.

Results. Electron microscopic analysis identified three main morphotypes of normal endothelial cells: 1) elongated shape along the direction of blood flow and an elongated nucleus; semicircular shape with an oval, round, kidney-shaped, or polymorphic nucleus with a less pronounced but clearly visible orientation along the direction of blood flow; 3) large nucleus with an even less pronounced but visible orientation along the direction of blood flow. The basement membrane of normal endothelial cells often contained various defects. Dysfunctional endothelium also exhibited several morphotypes, characterized by different combinations of the following features: spherical shape and loss of cell orientation along the direction of blood flow, presence of large vacuoles within the cell, cytoplasmic vacuolization, disruption of plasma membrane integrity, reduced contrast between the nucleus and cytoplasm, and partial detachment of the endothelial cell from the basement membrane. However, pronounced changes in the structure of organelles or the adjacent basement membrane were often not observed in dysfunctional endothelial cells.

Conclusion. The preservation of orientation along the direction of blood flow indicates a normal phenotype of endothelial cells in the absence of other signs of dysfunctional endothelium (vacuoles within the cytoplasm, cytoplasmic vacuolization, disruption of plasma membrane integrity, reduced contrast between the nucleus and cytoplasm, and partial detachment of the endothelial cell from the basement membrane).

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