Microscopic evaluation of vesicles shed by rat erythrocytes at elevated temperatures

• Number of vesicles shed by erythrocytes increases at elevated temperatures.
• Eighty percent of erythrocyte released vesicles are smaller than 0.45 μm.
• Freshly drawn rat blood contains more than 1 million vesicles per one microliter.
• Erythrocyte vesicles can serve as diagnostic tool of heat stress.

It is hypothesized that the elevation of the temperature of the blood during heat stress may cause an increase of the shedding of erythrocyte membrane vesicles. Therefore, the increase of vesicle numbers following heat stress may be indicative of and proportional to the level heat stress. In order to test this hypothesis, erythrocytes and the vesicles shed by erythrocytes were collected from rat blood and analyzed after the elevation of body temperature by exposure to external heat. The images of erythrocytes and vesicles were analyzed by a custom light microscopy system with spatial resolution of better than 90 nm. The samples were observed in an aqueous environment and required no freezing, dehydration, staining, shadowing, marking or any other manipulation. The elevation of temperature from 36.7±0.3 to 40.3±0.4 °C resulted in significant increase of the concentration of vesicles in blood. At a temperature of 37 °C, mean vesicle concentrations and diameters found in rat blood were (1.4±0.2)×106 vesicles/μL and 0.436±0.03 μm, respectively. The concentration of free vesicles increased after exposure to heat to (3.8±0.3)×106 vesicles/μL. It was estimated that 80% of all vesicles found in rat blood are smaller than 0.45 μm. The increase in the number of vesicle associated with elevated temperatures may be indicative of the heat stress level and serve as diagnostic test of erythrocyte stability and heat resistance.