Experimental Study On The Rapid Identification Of Peripheral Nerve Fascicles

It is well known that the peripheral nerves is composed of motor andsensory fibers, and in one fascicle the numbers of motor fiber or sensor fiber ofis different. We decide the method of operation depend on the distributing ofthe functional fascicles and the identification of them. As for the nerve injures,it is a hard work to differentiate these two kinds of entirely different nervefibers, then carry out accurate and rapid operation. It need too much work to dofor the scholar and microsurgery doctor. Moreover we need to identify thediffernt functional fascicles in the nerve graft and shift equally.The nervetransplant and shift operation have already obtained well clinical result inrecent years. If we have a well known about the identification and operateaccording to it, we can carry out the surgical operation successfully, with thebest healing and the least loss of function in supply area. At the same time wecan economize the nerve fibers with maximum degree.As far as it goes the existed method of identifing different nerve fribers ishard to make a progress in clinic work, because they have inherent blemishesand limitations. For example the fixed position diagram, because the nervebisection often have distortion, so the original and normal position change. Wecan't accurately anastomose them according to it. The electricity incitementmethod need the patient to keep awake, and it have no reaction as for the datedinjure. This method can't accurate differentiate the functional bundle also. Withthe enzyme histochemistry only can we differentiate the different bundleswhich is injured freshly, but it need too much time. From the mid 70's peoplehave discovered various nerve protein, and have a further understand about thegenerate and growth process of neuron by histochemistry. The method ofhistochemistry makes use of the principle that particular antibody only reactwith a fixed antigen. In the organize and cell it can make sure of the chemistrycomposition and quality of the cell's structure and organize. It can connect thestructure with the function and the metabolism. In the amplitude of the scope,the histochemisry can make sure the chemistry composition of the organizationand cell structure in home position in microcosmic world, attaining a standardthat is to determine the nature credibility, to have orientation accurately and toconfirm the quality possiblly.Although the histochemistry method has above advantage, it is the keyproblem about how to make sure the motor and sensory nerve fascicles withthe specially marking thing. After reading relatively reference, this experimentfocus on the Agrin and the Annexin V. We hope to indicate the speciality ofthem in different nerve fribers, so as to attain the purpose to discriminatedifferent functional fascicles. The Ping analyze the biochemistry character ofmotor and sensor neuron in spinal nerves, separate and distill a distinct protein35KD. It is proved to be a fixed antigen in sensor neuron by westblotexerperiment. She analyzed and identified this protein, and by combined usedof liquid chromatography and mass spectrometric sequencing, the 35KDprotein was identified as Annexin V. Annexin V is a calcium-binding proteinwith a mol. wt of 35 KD, and is a member of the annexin family. Annexin V isknown to have various functions such as anticoagulant activity,anti-inflammatory actions, and calcium/Ca2+ion channel activity. Annexin Vis widely present in various organs and tissues. Western blot analysis showsthat annexin V level differs among organs: level in the myocardium, lung andkidney is high, compared with that in the brain. It is also found have thefunction of promote axone growth. The Annexin V mainly distribute in theaxon of the sensory nerve in the surroundings nerve, and we didn't find a reportthat discovers it in the motor nerve.In 1998 there was a report that a special 190KD protein was found bySDS gel electrophoresis, and the histochemistry experiment laid a foundationfor the special protein. The histochemistry experiment indicate that the 190KDprotein distribute in cell bodies and axons in the motor neuron of spinal cord,and is the unique protein in motor neuron of spinal cord. After consulting someliterature and analyzing some protein's mol. wt about 190KD in nerve organise,we found that it is Agrin very possible. Agrin is an extracellular matrix proteininvolved in the aggregation of nicotinic acetylcholine receptors in the neuralorganize in the formation of the postsynaptic apparatus of the neuromuscularjunction. Agrin is about a mol. wt of 200KD-600KD protein extracted byelectric ray. Most scholars are consistent with the fact that Agrin is asynapse-organizing protein synthesized and externalized by motor neurons inthe spinal cord, and is transported to axon terminals. Except for a few thescholar confirms glial cell also expresses the agrin, most scholars are consistenton the opnion that agrin mainly distribute in the nerve motor nerve in thesurrounding nerve.It would be viable to discriminating nerve fascicles makinguse of agrin.Moreover because the spectrum of raman can analyze and indicate thestructure characteristic of the material at the molecule level, in the past yeares,people have been try to make use of the spectrum of raman to indicate thestructure feature of biology molecule, and expect to attain a goal to rapidlydiagnose disease without wound and pain, or with a little pain and wound. Thespectrum of raman not only can reflect the structure characteristic of thebiology molecule in organization, which is relate with the biology behavior,and but also can indicate the feacture of every organizatio at the molecule levelby reflecting the quality and ratio of protein, nucleic acid, glucide and all kindsof lipid. With the development of raman spectrum technique particularly microraman spectra, fiber-optic technique and minimizing of instrument, the ramanspectrum technique in the apply of the medical clinic particularly in theanalyze of nerve identification is possible. Micro raman techique is painless,unwound, rapid, specially, it can analyze and diagnose small district ororganize. It can also detect the quantity or be certain the quality.In this researchwe apply spectrum technique to analyze the structure of the surroundings nervefascicles, and hope to acquire the particularity of the motor nerve and sensorynerve structure, so as to discriminate nerve bundles quickly.We expect to approve that the unique feature of Annexin v and Agrin ineither of the peripheral nerve through westblot and immunohistochemical react.We hope to get the specific raman spectrum through this new method.According to these methods we hope to differentiate different nerve fascicles.ResultWestern blot analysis shows that sensor neurons express Annexin Vlargely, and have little express in motor neurons. As for the Agrin the situationis reverse. Annexin v express mostly in the axon and dorsal boot of spinalnerve. This experiment also approved that Agrin is the special subject that onlyexist in cell bodies and axons of motor neuron in ventral corner of spinal, anddidn't find it in dosal ganglion and sensor nerves.The experiment proves the same kind specimen shows same details andthe feature is similar, but the different specimen shows distinct features, suchas the intensity and band of the raman peak. These indicate the differentcontents of special subject and the different surrounding of the subject in.Therefore, the difference of these particularities can acted as the basis for theidentify, thus we resolve the hard problem of discriming the two kinds ofnerves . There are raman peaks at 1088cm-1,1276cm-1 ,1439cm-1,1579cm-1,1659cm-1 ,2720cm-1 ,2881cm-1both in motor nerve and sensor nerve.Significant differences were found in the spectum at1088cm-1,1276cm-1 ,1439cm-1,1579cm-1,1659cm-1. We compare 16 motor nerves with sensornerves at the ratio of 1276cm-1 /1439cm-1, and found that the ratio ofI1276cm-1 /1439cm-1 of sensor is higher than that of motor nerves. The ratio ofsensor is 1.17±0.08, motor is 0.95±0.06. Throug T test(T=7.89), the differencebetween the two mean value is very significant, p<0.05. We can get aconclusion from the Experiment that the ratio of 1.06 can acted as a standard,the nerve whose ration is higher than 1.06 is sensor nerve, contrarily it is motornerve. Appling the standard for another 16 rabbit in identifing motor nerve, thedelicacy, diversiy, positive predicting value and negative predicting value is87.5%,93.8%,93.3%,88.2% respectively. The same date for the sensor is93.8%,87.5%,88.2%,93.3% respectively. At the range of 2700cm-1-3500cm-1,we can find the peak of motor nerve is edge and narrow, and that of sensor isbounty.Conclusionthe Annexin V is special substance in sensor nerve and Agrin is thespecial substance in mortor nerve. We get through the obstacle that there is nota sign that can differentiate the perpherial nerve with immunohistochemicalmethods, and get a new method for the identify.The feature of moror and sensor nerve raman spectum is different, and wecan make use of it for the differencing. The vary between the two specimen areinvariablenes and this research laid a foundation for clinic work.InnovationIt is the first time that we apply Annexin V and Agrin as marker todiffienciate the two kinds of nerve fribers, so that we resolve the difficultproblem that there is not a molecule marker in this area. This reasearch laid afoundation for neuron growth, regeneration, enginery and recessive diseases,and will do further work for reasearch of structure and function of neuron.It is the first time that raman spectrum was used as a mean to identifingnerve bundles, and we discover that there are connection between specialraman spectrum and different structure of organization. In the future, weshould apply the Micro Raman spectrum as a rapid mean to provide evidencefor the operation, so as to get the best healing state. We believe that the MicroRaman spectrum will be a new characteristic differentiating method beforelong .