Exploration for sorting serum exosome as a measure of spinal cord injury severity

Abstract

This study aimed to extract exosomes from the serum of rats with spinal cord injury and explore their potential as indicators for assessing the severity of spinal cord injury. Control rats were compared with rats in the mild and severe injury groups. Exosomes were isolated from rat serum using an optimized threshold condition with an ultra-high-speed flow cell sorter. Nanoparticle tracking analysis (NTA) was performed to determine the size distribution of exosomes, transmission electron microscopy (TEM) was used to observe the morphology of exosomes, and Western blot analysis was conducted to assess the expression of CD63(cluster of differentiation antigen 63) and CD9(cluster of differentiation antigen 9) proteins on the surface of exosomes. NanoFlow cytometry was utilized to evaluate the expression of CD63 and CD9. The optimal threshold condition for flow cytometry of exosomes was determined to be 0.002. Exosomes exhibited a cup-shaped morphology with a clean background. The percentages of exosomes in the control, mild injury, and severe injury groups were determined to be 6.4 × 1011 particles/mL, 6.2 × 1011 particles/mL, and 2.3 × 1011 particles/mL, respectively. The mean sizes of exosomes were 129.25 nm ± 18.3, 117.93 nm ± 39.9, and 116.38 nm ± 36.5, respectively. The percentages of CD9 were 15.5% ± 0.037, 10.4% ± 0.082, and 7.4% ± 0.051, while the percentages of CD63 were 1.2% ± 0.025, 2.6% ± 0.067, and 1.5% ± 0.038 for the control, mild injury, and severe injury groups, respectively. Comparison analysis revealed that the diameter of exosomes in rat serum ranged from 80 nm to 150 nm. The percentage of exosomes decreased, and CD9 expression significantly decreased as the severity of spinal cord injury increased. Flow cytometry allows for the isolation of exosomes from serum with high purity and uniform particle size. The percentage of serum exosomes and the expression of CD9 may serve as potential diagnostic indicators for spinal cord injury models.