| Spinal cord I/R injury is one of the common secondary spinal cord injuries in the spinesurgery, neurosurgery and other areas, the research into whose injury mechanism has becomea hot spot recently. With the in-depth study of basic spinal cord I/R injury theory andmechanisms, together with the references of the clinical case studies, we found that the rateof the paralyzed incidence and other secondary injuries which are related to spinal cord I/Rinjury presents an upward tendency. Along with the aging process of the world's population,the cases of spinal degenerative have increased every year. Under the circumstance that thespinal cord I/R injury disability rates rises increasely and the origin of spinal cord I/R injuryhas not been found, the research into spinal cord I/R injury endogenous mechanisms andprevention measures is of prime importance. The in-depth research about how the cellfunction executant, protein levels, suspends the beginning and the development of secondaryspinal cord injury is imminent.The research into spinal cord I/R injury focuses on its occurrence mechanism at homeand abroad now. The study of the spinal cord the I/R injury is usually explored and discussedby some combined methods, including ischemia mechanism which can lead to edema,inflammation, the mechanism of local circulation, the mechanism of neuronal apoptosis,protein in the water channel damaged mechanism, the immunological injury mechanismsand various types of cytokines causing the injury mechanism. Although the studies aboutspinal cord I/R injury have made some progress compared to the previous, there is littlesubstantive progress on the diagnosis and treatment of spinal cord I/R injury.We expect tofind a landmark protein performing as an effective therapeutic target for spinal cord I/Rinjury.We have acquired early proteome expression profile changes, and screened significantdifferential expressed proteins during the spinal cord I/R injury through the study of thepre-proteomics. We found that the reported neural-like proteins that have close relationshipwith the nerve cell survival and axonal regeneration process: α-tubulin, NF-M and NF-L.The expressions of the three nerve-repair proteins that have been reported were parallel,indicating that they have participated in the process of spinal cord I/R injury and may have functional relationship.Tubulin proteins and neurofilament proteins are part of the cytoskeleton. Tubulinproteins are kinetically assembled polymers of α-tubulin and β-tubulin that exist in alleukaryotic cells. As part of the cytoskeleton, microtubules are known to be involved indifferent cellular processes, including cell motility, mitosis, intracellular transport, cellshape,secretion and polarization. Microtubules proactively participate in the first steps ofneuronal polarization, the organization of intracellular compartments, the remodeling ofdendritic spines and the trafficking of cargo molecules in neurons.Neurofilament proteins are also abundant in neurons, especially in large-diameter axons.They are made of different three submit proteins, including NF-L(68kDa),NF-M(160kDa)and NF-H(200kDa)in different tissues. NF expression is closely associated with axonalgrowth and maintenance of neuronal homeostasis. Recent studies have shown that NFprotein expression is closely associated with continued growth of normal axons and theregeneration of impaired axons. NF phosphorylation regulates NF axonal transport inneurons, plays a significant role in NF translocation, morphology, and some other functions.And NF significantly stabilizes microtubules within axons, thereby promoting axonalregeneration. Now there are no reports about the fountion of α-tubulin,NF-M and NF-Lproteins in the spinal cord I/R injury.Previous study also found that the step-up of NF-L protein expression lag behindNF-M, so we think that the synthesis of NF-L regulates the expression of NF-M, whichillustrates that these different stages of GOD proteins relate the progress and repair of thespinal cord I/R injury is important, that is to say, these proteins are involved in the importantpathological process of spinal cord I/R injury. Through the validation to such proteins, theeffect that such proteins have on the spinal cord I/R injury pathological change process hasbeen further proved, in the meantime, such protein can be considered as a landmark proteinin the spinal cord I/R injury. It has a great practical significance in clinical spinal cord I/Rinjury pre-diagnosis and cell regulation of molecular identification, etc. There are not relatedreports seen at home and abroad yet.Objective:The changing characteristics and mechanism of action of the active up-regulatednerve-like proteins have been validated through the further studies during the spinal cord I/Rinjury, which will lay a solid foundation to find a specific diagnosis or a new treatment targetof the spinal cord I/R injury. Methods:This subject of research builds acute spinal cord segmental mild I/R injury model usingwaist3,4,5artery occlusion method. The injured segments of spinal cord specimens isanalysed by DIGE technology and proteomics mass spectrometry research, so as to obtainthe amount, types and changes of significant different proteins. And then the researcher willidentify the selected up-regulated expression of proteins by IHC and WB, and preliminaryanalyse the function of α-tubulin, NF-M and NF-L proteins in spinal cord I/R.Results:The acute spinal cord segmental mild I/R model has been successfully established usingwaist3,4,5artery occlusion method. Through the comparison between sham control groupand each0,12,24,48-hour reperfusion group by two-dimensional electrophoresis research,21the differentially double expressed proteins have been obtained ultimately by massspectrometry proteomics technology, containing three up-regulated model protein,respectively α-tubulin, NF-M and NF-L protein. The researcher has identified theup-regulated model proteins by the combination of IHC and WB technology, The resultsshowed that α-tubulin, NF-M and NF-L protein were localized in the cell membrane ofneurons, axons and cytoplasm. Those distribution of gray matter are more significantly thanin white matter in the spinal cord(P＜0.05),but the distribution of the spinal anterior hornand posterior horn is not significant differences(P>0.05).Analyzied the change curve of thethree proteins, we found that the three kinds of protein expression were gradually increasedin the reperfusion injury, reached a peak24hours and then gradually return to back, theresults consistent with the previous proteomic experiments.We have found that NF-M, NF-Lprotein and phosphorylated NF-M, NF-L protein expression owned the same trend.. Westernblot experiments on the normal group and sham-operated control group, together with thequantitative analysis of the I/R spinal cord specimens organization up-regulated expressionproteins between sham-operated group and erery time group shows that there is nosignificant change (P>0.05) between different expression protein in sham-operated controlgroup and nomal group.Conclusion and Innovation:The combination of motor function score and light and electron microscopichistopathological observation proves that the segmental lumbar artery ischemia reperfusionmodel method possess a clear target with high rate of success to the spinal cord mild injury.The identification and functional analysis of differentially expressed proteins in the spinal cord I/R injury process are joint used immunohistochemistry, immunoblotting and DIGEtechnology innovatively. Differentially double expressed proteins in the rabbit spinal cordI/R are obtained by differential proteomics technology (DIGE), draw the power curve ofregulated proteins, and then researchers suggest that the protected critical time point onneurological function in spinal cord I/R injury is reperfusion of24hours. Comprehensiveproteomics, IHC and WB findings prove that a simple surgical procedure and no undergoingischemia reperfusion operation for the model of spinal cord tissue, the target proteinexpression had no effect. At the same time, we have analyzed the result of WB, α-tubulin,NF-M and NF-L protein participating in the sham-operated group and I/R each time points,got the fact that WB, α-tubulin, NF-M and NF-L protein play a part in the protection andrestoration of spinal cord neurons during the process of the spinal cord I/R injury, andsuggested that they may promote the spinal cord recovery by promoting the role of axonprotruding extension, curl and growth cone. According to the findings of phosphorylatedNF-M and NF-L protein WB, the functions of the spinal cord protection and restoration ofNF-M and NF-L protein in the spinal cord I/R injury are achieved by their phosphorylationforms. Alpha-tubulin, NF-M and NF-L protein are involved in the mechanism of spinalcord I/R injury protection with the combination of Western blotting studies describing fromthe protein levels.
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