Differential Proteomic Analysis On The Acute Spinal Cord Ischemia-reperfusion Injury In Rabbits

Spinal cord ischemia-reperfusion is a severe spinal cord injury, which has a significant disabling and lifelong effect on many people. Though SCI has been studied extensively using different biochemical assays such as Immuno- histochemistry, Western blot,flow cytometry and immunofluorescence techniques, many evolving molecules remain to be discovered and their interrelationships need to be understood.Proteomics is a term that describes the science and methodology of the investigation of the proteome, which quantitatively acquires the composition of proteins in a cell, a tissue, or an organism. After genomics, proteomics is often considered the next logical step to study biological systems, with the added capacity to describe the spatiotemporal differences in protein expression and protein–protein interactions and protein stability and degradation. both in normal and pathological tissue. Proteomics on spinal cord ischemia-reperfusion injury is not reported before. Proteomic analysis may open the possibility to understand the mechanisms of SCI.【Objective】To establish and optimize the two-dimensional gel electrophoresis technology and to obtain normal and injured spinal cord proteome; To compare protein expression profiles in each group using two-dimensional gel electrophoresis; To select the specific protein and assure the mechanisms of spinal cord injury and its recovery on molecular level.【Methods】An acute focal spinal cord ischemia model was established using traditional Zivin method. Then, the proteins were extracted and separed by polyacrylamide gel electrophoresis. DeCyder v.5.02 DIGE image analysis software was used to analyze the images and select differentially expressed protein. These proteins were identified by mass spectrometry.【Results】Optimized two-dimensional gel electrophoresis maps of the spinal cord were obtained. Protein expression in control group and 24h reperfusion group were significantly different. 49 differentially expressed protein spots were selected and 21 different proteins were finally obtained. According to the changes of, we gained two curves for over expressed protein and less expressed protein after injury. The changing of protein quantity were occurred at time of 24h post reprefusion. Over expressed protein comprised 20 proteins, including some metabolic enzymes, nerve fiber proteins and stress proteins. Less expressed protein included four proteins,among which M andα-actin were involved. So, 24h post-injury is the key point for changing of proteins expression. The proteins we identified involve following processes: metabolism, signal transduction, transport and stress function and so on. These changes are consistent with the characteristics of stress response. We also found that some proteins shifted horizontally on 2-D gel. It suggest that these proteins not only changed in quantity but also phosphorylized during injury.We also found many carbohydrate metabolism and energy metabolism enzymes decreased in 24h post reperfusion, such as succinate dehydrogenase and glycogen phosphorylase, which were not reported before in study of nerve injury.【Conclusion and Innovation point】We successfully established an acute spinal cord ischemia-reperfusion injury model on rabbit. We obtained electrophoresis map at different time points and analyzed the changes of the proteins expression. 24h post reperfusion is the key time point for protein expression.Proteomic analysis of spinal cord ischemia had not been reported.We found a protein, SPNA2, which may be a marker protein for spinal cord ischemia. We also found the expression change of membrane protein-FSCN1, which has not been reported in nerve injury. In addition, we also speculated the reason of expression change of many enzymes in sugar metabolism during the course.