Effect of lipid accumulation after spinal cord injury in autofluorescence intensity of injury site

Abstract

To investigate the relationship between lipid accumulation and autofluorescence intensity of injury site after spinal cord injury (SCI), and explore whether CuSO⁴ can eliminate autofluorescence in the injury site after SCI. Thirty six Wild Type mice at age of 8 to 12 weeks (weight 18 to 24 g) were randomly divided into normal control group (n=4) and SCI group (n=32). Respectively, 8 mice of SCI group were sacrificed randomly at 1 week, 2 weeks, 4 weeks and 8 weeks after injury. Frozen sections of spinal cord tissue with injury site at the center were made to observe autofluorescence under green channel of fluorescence microscope (Specimens of normal control group were taken from the same segment of the spinal cord, and fixed with 4% paraformaldehyde solution). Oil Red O staining was applied to visualize the lipid accumulation in the injury site, and correlation between lipid accumulation and autofluorescence intensity was analyzed. Furthermore, sections were incubated with CuSO⁴ buffer to eliminate autofluorescence, and CuSO⁴ concentration and incubation time was optimized. No obvious autofluorescence or lipid staining was found in normal spinal cord tissue sections. By contrast, autofluorescence appeared in the injury site of spinal cord sections, and the intensity increased with passing time after injury. Oil Red O staining showed that lipid accumulated in the injury site with passing time after injury as well, and the correlation between lipid accumulation and autofluorescence intensity was positive. After incubation with CuSO⁴ buffer, the autofluorescence in the injury site was significantly reduced, especially after optimizing CuSO⁴ concentration and incubation time. Lipid accumulation may play an important role to determine the autofluorescence intensity of injury site after SCI, and the autofluorescence intensity can be used as a simple index for evaluating lipid peroxidation damage. Optimized method of using CuSO⁴ can significantly reduce the autofluorescence in the injury site after SCI, which will be beneficial to the application of immunofluorescence staining technique in the study of SCI.