Oxidative phosphorylated neurofilament protein M protects spinal cord against ischemia/reperfusion injury

Abstract

Previous studies have shown that neurofilament protein M expression is upregulated in the early stage of spinal cord ischemia/reperfusion injury, indicating that this protein may play a role in the injury process. In the present study, we compared protein expression in spinal cord tissue of rabbits after 25 minutes of ischemia followed by 0, 12, 24, or 48 hours of reperfusion with that of sham operated rabbits, using proteomic two-dimensional gel electrophoresis and mass spectrometry. In addition, the nerve repair-related neurofilament protein M with the unregulated expression was detected with immunohistochemistry and western blot analysis. Two-dimensional gel electrophoresis and mass spectrometry showed that, compared with the sham group, upregulation of protein expression was most significant in the spinal cords of rabbits that had undergone ischemia and 24 hours of reperfusion. Immunohistochemical analysis revealed that neurofilament protein M was located in the membrane and cytoplasm of neuronal soma and axons at each time point after injury. Western blot analysis showed that neurofilament protein M expression increased with reperfusion time until it peaked at 24 hours and returned to baseline level after 48 hours. Furthermore, neurofilament protein M is phosphorylated under oxidative stress, and expression changes were parallel for the phosphorylated and non-phosphorylated forms. Neurofilament protein M plays an important role in spinal cord ischemia/reperfusion injury, and its functions are achieved through oxidative phosphorylation.

Keywords: NSFC grant; ischemia/reperfusion; nerve regeneration; neural regeneration; neurofilament protein M; neuroprotection; phosphorylation; proteomics; spinal cord injury.

Figure 1

Two-dimensional fluorescence electrophoresis analysis comparing protein expression in rabbits with and without spinal cord ischemia/reperfusion (I/R) injury. (A) Two-dimensional fluorescence electrophoresis analysis of 10 μL sample (pH 8.0–9.0, final concentration 5 mg/mL) of I25minR24h group. (B) I25minR24h group, showing protein spots with ≥ twofold difference from sham. A total of 29 differentially expressed proteins were found.

Figure 2

Protein fingerprinting. (A) The most obvious different spots are between I0minR0h and I25minR24h groups by paired experiments, we finally selected I25minR24h for electrophoresis (Cy3 and Cy5 fluorescence scanning). Cy3 and Cy5 are different fluorescent probes (B, C). Different protein spots were detected in the polyacrylamide gel by DeCyder software.

Figure 3

Neurofilament protein M expression in rabbit spinal cord after ischemia/reperfusion (I/R) injury (immunohistochemistry). (A) Sham group; (B) I25minR0 group; (C) I25minR12h group; (D) I25minR24h group; (E) I25minR48h group; (F) higher magnification image from the I25minR24h group showing neurofilament protein M expression in the neuronal membrane and axon. Arrows showed neurofilament protein M immunopositive cells. Original magnification: A–E, × 200; F, × 400.

Figure 4

Neurofilament protein M (NF-M) phosphorylation in rabbit spinal cord tissue after ischemia/reperfusion (I/R) injury. Protein expression of NF-M and phosphorylated NF-M increased during the spinal cord I/R injury process. Peak expression occurred at 24 hours of reperfusion. Data are expressed as the optical density ratio of NF-M to GAPDH (mean ± SEM; n = 3). The difference between groups was com-pared using one-way analysis of variance. *P < 0.05, vs. other groups.