Comparative Proteomics Reveals Proteins Expression Differences Between The Motor And Sensory Fascicles Of Peripheral Nerve

Comparative proteomics reveals proteins expression differences between the motor and sensory fascicles of peripheral nerveWith high disability rate, the Peripheral Nerve (PN) injury has always troubled surgeons. PN injury results in significant functional and emotional sequel, although microsurgical repair techniques allowing more accurate axon alignment have been advanced. Differentiated nerve fascicle repair can maximize the functional recovery for the patient. Unfortunately, no method currently utilized can rapidly and accurately distinguish between the motor and sensory nerve fascicles during surgery. Surgeons today estimate the properties of both stumps of damaged PN fascicles through more-or-less experience, and perform inter-fascicular repair with limited insight as to outcome. The inability to find the specific molecule in motor or sensory nerve fascicles as a molecular marker in fresh and dated injured PN is partially responsible for failures in identifying nerve fascicles. Identifying specific marker in motor or sensory nerve fascicles will be intriguing and promising, which would allow surgeons to perform fascicular sutures elaborately and accurately in vivo, and result in the best recovery of the PN function. To this objective, we systematically reviewed the methods of identifying the motor or sensory fascicles and related technologies of today's Proteomics research which the predecessors had proposed, and appllied Comparative Proteomics to reveal proteins expression differences between the motor and sensory fascicles of PN. We clarified the volume changes of PN fascicles on different time points after the motor and sensory injured, revealed significant protein for PN regeneration, explored new clues to the identifying between motor and sensory fascicles and the molecular mechanism to damage repair of PN.The principles, shortcomings, and progress of diverse methods of sensory and motor nerve fascicle identification, including nerve morphology, electric stimulation, spectroscopy, enzymatic staining (AchE and CA enzymatic staining methods), radiation biochemical methods and immunochemical methods, were systematically reviewed. Microsurgical techniques literature offered several papers which meticulously describe the nature of peripheral nerve and presumes that differentiating between the sensory and motor fascicles was difficult. The cross sections of the proximal and distal stumps of a nerve, which suffered from a substantial traumatic defect, fail to show mirror images and differ in their appearances. Nerve morphology alone could not far contribute to its identification. Various electric stimulation methods once tried to apply during the course of operation, which stimulated the dissected fascicles of the distal and proximal stumps. However, electric stimulation methods required local anesthesia and were imprecise or unpleasant to the patients. The majority of relevant papers mentioned enzymatic staining methods. However, nerve fascicles differentiation based on measurement of AchE or CA activity is limited since it requires lots of time on incubation, and cannot be used on dated PN injury that the enzyme activities step down in distal nerve stump for Wallerian degeneration. The radiochemical technical needed requirements and high costs, is limited in clinical applications. Methods such as intra-neural topography, electric stimulation, enzymatic staining and radiation biochemical methods used in attempting to solve this problem have either been imprecise, too time consuming, or unsuitable for intra-operative determination, are not suitable for clinical application. It is feasible in theory on identifying the motor or sensory fascicles of PN with immunochemical methods. But the marker proteins which the literature mentioned are challenged by many experts. How to choose a high-throughput screening around motor and sensory fascicles of PN, is a focus on identifying between motor and sensory fascicles of PN.Through systematically reviewing the Proteomics and related technologies system of today's Proteomics, we believe that 2-D DIGE comparative proteomics approach and MALDI-TOF can be applied to choose a high-throughput screening around motor and sensory fascicles of PN. With these comparative proteomics approach, specific or high difference proteins can be found, and these proteins can give clues to the identifying between motor and sensory fascicles and the molecular mechanism to damage repair of PN.To this objective, we made the following four experiments, and obtained corresponding results.Experiment one, animal models:We optimized and established the Wistar rat PN injury animal models. Through deriving nerve sample from normal motor (motor branches of femoral nerve) and sensory (saphenous nerve) fascicles of PN,5mm far ends nerve sample from motor (motor branches of femoral nerve) and sensory (saphenous nerve) fascicles of 8 hours after PN Sunderland V injury,5mm far ends nerve sample from motor (motor branches of femoral nerve) and sensory (saphenous nerve) fascicles of 8 days after PN Sunderland V injury, we achieved the thoroughbred Wistar rats, the same injury model, the same injury time and the same sample preparation model.Experiment two,2D-DIGE:Through optimizing the sample protein extraction method, protein samples purification, protein samples quantification, protein samples grouping and fluorescent labeling of extraction protein, we conducted fluorescence difference gel electrophoresis (DIGE), Typhoon 9400 laser scanner, DeCyder difference software analysis, DeCyder-BVA software matching proteins and got high differences proteins spots. The Results were that we established the fluorescent protein map of the motor branches of femoral nerve and saphenous nerve of PN, high differences proteins were high-throughput chosen between motor and sensory fascicles of PN. With the BVA software, gel analysis showed that an average of 1586 protein spots, protein spots matching rate of about 82% on nine gels. The pixel volume of each spot was calculated, normalized. Every gel was matched with the internal standard image in itself so as to gain the minimal gel-gel variance. To judge differences in protein expression, a cut-off value of a 1.5-fold increase or decrease was used. Among the protein spots detected, totally,160 protein spots were detected as changed upward or downward by>1.5 fold, P<0.05(using t test statistical method).Experiment three, MALDI-TOF:Different protein spots were screened through artificial, then removed the less folds protein spots which might not be identified by mass spectrometry, finally,16 different protein spots were selected to identify. Throughî›–reparing Coomassie staining gel, automatic spots picking, automatic restriction enzyme cutting, automatic spots targeting, then used MALDI-TOF Pro in reflector mode for peptide mass field fingerprinting (PMF map) to analyze and identify. The reliability of search results was evaluated by the expectations and coverage ratio. To search NCBI database using Pro Found software, resulted in 14 protein spots (ApoE,NF-L,TEC,Serpina3n,Peroxiredoxin-2,TPM1,DPYSL2,CaBP1,HSP70. Gng7,LDHB,Enolase2,Serpina 10. PDI A3) were identified exactly.Experiment four:Realtime-PCR. We used Real-time method to validate the accuracy of Proteomics. Extracted total RNA from protein samples, RT-PCR, established PCR reaction system and conditions, and applied statistical analysis [two groups comparison with t test statistical method, multiple mean comparison with single factor variance One-Way ANOVA test and q test statistical method), through cross-groups comparing to one group as control, the other relative expression of the gene changes with multiple relationships, the data were normalized to detect mRNA changes of high difference proteins between the motor and sensory fascicles of PN injury at three time points. The results were that the trends of mRNA changes protein were close to the Proteomics results, thus validated the accuracy of Proteomics results.Through bioinformatics query on high difference proteins, we got lots of the protein structures and functions of information about ApoE. NF-L,TEC,Serpina3n,Peroxiredoxin-2, TPM1,DPYSL2,CaBP1,HSP70,Gng 7,LDHB,Enolase 2,Serpina 10,PDI A3, including: (1) There are indeed existing high difference proteins between the motor and sensory fascicles of normal PN. ApoE,NF-L,TEC,Serpina3n,Peroxiredoxin-2,TPM1 play important roles in the difference of the motor and sensory fascicles of normal PN. (2) High difference proteins TPM1,Serpina 10,DPYSL2,NF-L,CaBP1 are related with the sensory fascicles regeneration. (3) High difference proteins Serpina3n,Peroxiredoxin-2,HSP70,LDHB,Enolase 2,CaBP1,Serpina 10,Gng 7,PDI A3 are related with the motor fascicles regeneration. (4) ApoE in Y/G ratio is-1.94 times, good match, protein stability in all the gel, T-test statistic values are 0.00068, statistically significant;in 8HY/8HG ratio is-2.07 times, good match, protein stability in all the gel, T-test statistic values are 0.0053, statistically significant; in 8DY/8DG ratio is-2.20 times, good match, protein stability in all the gel, T-test statistic values are 0.0069, statistically significant. In the nerve sample from normal motor (motor branches of femoral nerve) and sensory (saphenous nerve) fascicles of PN,5mm far ends nerve sample from motor (motor branches of femoral nerve) and sensory (saphenous nerve) fascicles of 8 hours after PN Sunderland V injury,5mm far ends nerve sample from motor (motor branches of femoral nerve) and sensory (saphenous nerve) fascicles of 8 days after PN Sunderland V injury, ApoE in sensory fascicles are about 2 times upper and lower levels to ApoE in motor fascicles at three time points. ApoE might be served as the marker protein between sensory and motor fascicles, was worthy of further research.The innovations of our research are:(1) It is the first time to apply Comparative Proteomics to screen the high difference proteins between the motor and sensory fascicles of PN. By far, there are no related researches on screening the high difference proteins between the motor and sensory fascicles of PN at different time points. (2) Our research break through the traditional study mode (protein changes in a single time point) which has ever been applied with. After PN injury, proteins amount changes with time (8h or 8d), subsequently, the application of the kind of protein should be changed when we wanted to use it to identify the motor or sensory fascicles of PN. It is a concept breakthrough to traditional concept of using single protein to identify the motor or sensory fascicles of PN at all time points. (3) Our research specifically divided the research mode of the protein changes after PN injury. We made a positive work on the study of proteins amount changes in sensory fascicles and proteins amount changes in motor fascicles at different time points after PN injury. So far, there is no report on the application of Comparative Proteomics to research the related protein amount changes of simple sensory or simple motor fascicles at different time point after PN injury. (4) ApoE might be used in identifying the motor or sensory fascicles of the fresh or dated PN injuries, and this experimental work provides the foundation for the clinical applications to identifying the motor or sensory fascicles of PN.With high incidence of PN injuries and difficulty to cure, it is urgent for clinical work to study in-depth on high difference proteins between sensory and motor fascicles of PN, which can enrich the clinician's understanding of the PN injury and promote the progress on identifying the sensory or motor fascicles of PN. Therefore, our researches have important scientific significance and practical clinical value. And this research provided the follow-up object of study for the role of a single protein, directed the research area and accumulated raw data, carried out the preliminary experiments on identifying the sensory and motor fascicles of PN. At the same time, our research provided a new way of thinking to the repair of PN injury.