Experiment Study Of Promoting The Regeneration Of Rat Sciatic Nerve With The RGD Peptide Grafted Poly[LA-(Glc-Lys)] Sustained-release FK506 And NGF Incorporated Diaphragm

I. Background, objective and significanceThe experiment had proved that nerve growth factor(NGF) can accelerate the regeneration and functional recovery in lesioned rat sciatic nerve. Tacrolimus(FK506) is a macrolide produced by a Streptomycete, while a strong immunosuppressive agent. Its immunosuppressive ability is proximately 100 times than cyclosporine and it can inhibits the mixed lymphocyte reaction,cytotoxic T cell generation,the production of T cell-derived soluble mediators such as interleukin 2(IL-2),interleukin 3(IL-3) and gamma-interferon and expression of the IL-2 receptor. However, FK506 can also promote the regeneration and functional recovery in rat sciatic nerve. The relation of promoting the regeneration and neurotrophy of FK506 was explored by many researchers such as Lyons W E,et al, who discovered that the amount of FK506-binding protein of PC12 cell can be up-regulated by FK506 and FK506 markedly augmented the effect of NGF by increasing sensitivity to NGF, Thus FK506 reduced by a factor of 20-50 the NGF concentration needed to elicit maximal outgrowth, Anti-NGF markedly reduced the regeneration effects of FK506 in rat sensory ganglia. When the peripheral nerve is lesioned, the distal stump and the proximal several Ranvier's nodus of the axon become Wallers degeneration; then, Schwann cells generate and become Bungner'sbelt, subsequently the regenerated axon goes through the Bungner' s belt and extend forward. So, the generation of Schwann cells play a role of importance in nerve regeneration. Yang J,et al, observed that FK506 may promote the proliferation and secreting NGF of Schwann cell. Wu ZW,et al,also observed that the time of Schwann cell migrating out of explant from human embryo sciatic nerve,the distance of Schwann cell migrating from explant from human embryo sciatic nerve and the vitality of Schwann cell in groups cultured with FK506 were significantly excellent over that of contrast. Hanawa H,et al, proved in rat experiment autoimmune myocarditis that the extent of inflammation infiltration in the groups gived by muscle injection with different FK506 dosage of 0.1mg/kg/day, 0.32 mg/kg/day and 1.0mg/kg/day respectively was slighter than that of the contrast,and the immunosuppressive effect of FK506 was dose-dependent—more larger dose,less lighter extent of inflammation infiltration. However, Wu ZW,et al,also discovered that there were no significantly difference in the time of Schwann cell migrating out of explant from human embryo sciatic nerve,the distance of Schwann cell migrating from explant from human embryo sciatic nerve and the vitality of Schwann cell among 0.1 ug/ml,1 ug/ml and 10 ug/ml FK506.With regards to dose-dependent effect of FK506 in immunosuppressive ability while no that of the influence to Schwann cell, Gold BG,et al explained that FK506-binding protein 52 (not FK506-binding protein 12) mediated the neurotrophic action of FK506,and FK506's binding to the FK506-binding protein-12 was the base of immunosuppressive effect of FK506, immunosuppressive effect and neurotrophic effect were mediated via two different signal trasmittion path. Sarikcioglu L,et al, in the model of crushed rat sciatic nerve and intrathecal administration ,a daily dose of 0.05 mg/kg/day of FK506 proved effective in accelerating the nerve regeneration. It showed that topographic administration of FK506 ,as well as systematic administration, was benefical to the regeneration of nerve,and could reduce its usage. Based on above facts, we would make the RGD peptide grafted poly[LA-(Glc-Lys )] sustained-release FK506 and NGF incorporated diaphragm,which can better let FK506 promote the regeneration of peripheral nerve through slowly releasing FK506 and make use of the cooperation action of FK506 and NGF, so that it can provide some scientific facts for clinical application that the RGD peptide grafted poly[LA-(Glc-Lys)] sustained-release FK506 and NGF incorporated diaphragm can make better use of the effect of accelerating regeneration of peripheral nerve and neurotrophy of FK506 while reducing its immunosuppressive effect.II. Methods1. 24 male Wistar rats were randomly divided into three groups,eight per group. Group A: the RGD peptide grafted poly[LA-(Glc-Lys)] sustained-release diaphragm incorporated FK506 and NGF ,group B: the RGD peptide grafted poly[LA-(Glc-Lys)] sustained-release diaphragm incorporated FK506 ,group C: the RGD peptide grafted poly[LA-(Glc-Lys)] sustained-release diaphragm.2. The rats were given 0.4% pentobarbital sodium by intrperitoneal injection with a dosage of 1 ml/100g. After that the left rat sciatic nerve was exposed,cut and bridged with correspondent diaphragm. First fixed the two ends of the sciatic nerve to the either end of diaphragm, 5mm defect of sciatic nerve left; then rolled the diaphragm up to pipe and sutured both edges of the diaphragm . Gruoup A with FK506 and NGF amount of 0.48mg and 2 ug; group B with 0.72mg of FK506; group C for control.3. Three months postoperatively, exposed the bilateral gastrocnemius muscles and the regenerated sciatic nerve ,observed the size of regenerated sciatic nerve and quanlity, absorbtion and adhesion to the surrounding tissue of the diaphragm; then placed the incentive electrical pole to the proximal sciatic nerve and the recording electrical pole to the gastrocnemius muscle, examined the motor nerve conduct velocity(MNCV), compound muscle active potential (CMAP) and refractory period of the sciatic nerve with the 4000-type evoked potential/electromyography instrument made in Denmark.4. After the electrophysiologial examination, removed the regenerated sciatic nerve quickly and immersed it into the 2.5% Glutaraldehyde; subsequently, cut thoroughly the bilateral gastrocnemius muscles , the experimental gastrocnemius muscles weight/the other ratio was calculated as the recovery rate of the experimental gastrocnemius muscle.5. Three months postoperatively,choiced the distal sciatic nerve of the diaphragm and firstly fixed it in the 2.5% Glutaraldehyde for two hours, then did it into 1% Osmium Tetroxide for one hour, orientally embedded it in Epon 812,made the semi-section of thickness in 1.0um and stained in methylene blue. Finishing that, observed the regenerated nerve in light microscope and recorded the number of the regenerated mylinated fiber, myelin sheath thickness and diameter of the axon with HPIAS—1000 color pathological image-analysis system.6. Three months postoperatively,choiced the distal sciatic nerve of the diaphragm and firstly fixed it in the 2.5% Glutaraldehyde for two hours, then did it into 1% Osmium Tetroxide for one hour, orientally embedded it in Epon 812,made the ultra-section of thickness in 60 run and stained in Uranyl Acetate-Lead Citrate, finished that, observed the regenerated nerve in electrical microscope. 7. The experiment data was evaluated by the statistical software of SPSS 11.5,the statistical results were expressed in (x|-)±s.The statistical method was One-way ANOVA,and the difference between groups was either LSD(Least-Significant Diffeence) method or S-N-K (Student-Neuman-keuls) method. (α=0.05)III. Results1. General observation14 days postoperation, the foot of two rats occured ulcer in Group A, as did the foot of one rat in Group B and C. But the ulcer of four rats all recovered one week later. After three months of surgery,the gastrocnemius muscles in all animals became atrophied compared with the other, but the Group A was less serious than Group B and C; the diaphragm remains intact in appearance, but became crisp and soft ,had no apparently adhesion to surrounding tissue; the regenerated sciatic nerve had gone through the defect and bridged the two ends of the lesioned nerve,and the regenerated sciatic nerve of Group A was larger in size than that of Group B and C,and it had no apparently difference in size with the proximal nerve.2. Electrophysiologial examinationThree months postsurgery, the results of the motor nerve conduct velocity (MNCV),compound muscle active potential (CMAP) and refractory period of the sciatic nerve were list on Table 1. the motor nerve conduct velocity(MNCV) in Group A was faster than that in Group B, the difference was statistically significant (P=0.016), the motor nerve conduct velocity (MNCV) in Group A was faster than that in Group C , the difference was statistically significant(P=0.000), the motor nerve conduct velocity (MNCV) in Group B was faster than that in Group C , the difference was statistically significant (P=0.030); the compound muscle active potential (CMAP) in Group A was higher than that in Group B , the difference was statistically significant (P=0.049), the compound muscle active potential (CMAP) in Group A was higher than that in Group C, the difference was statistically significant (P=0.000), the compound muscle active potential (CMAP) in Group B was higher than that in Group C, the difference was statistically significant (P=0.046); the refractory period in Group A was shorter than that in Group B , the difference was statistically significant(P=0.046), the refractory period in Group A was shorter than that in Group C, the difference was statistically significant (P=0.000), the refractory period in Group B was shorter than that in Group C, the difference was statistically significant (P=0.049).3. Examination of the recovery rate of experimental gastrocnemius muscleThree months postsurgery, the recovery rate of experimental gastrocnemius muscle was list on Table 2. the recovery rate of experimental gastrocnemius muscle in Group A was superior to that of Group B, the difference was statistically significant (P=0.050) ; the recovery rate of experimental gastrocnemius muscle in Group A was superior to that of Group C, the difference was statistically significant (P=0.000), the recovery rate of experimental gastrocnemius muscle in Group B was superior to that of Group C, the difference was statistically significant (P=0.044).4. Observation by light microscopeThree months postoperatively, in regenerated sciatic nerve of Group A, the regenerated mylinated nerve fiber was dense , its myelin sheath was thick, the big regenerated mylinated nerve fiber was more than that of Group B and C, the regenerated mylinated nerve fiber had no apparent difference in size and the less cellular substance of Schwann cells was seen than that of Group B and C; in regenerated sciatic nerve of Group B, the regenerated mylinated nerve fiber was sparse , its myelin sheath was thin, great regenerated mylinated nerve fiber was more than that of Group C, the regenerated mylinated nerve fiber had some difference in size and less cellular substance of Schwann cells was seen than that of Group C; in regenerated sciatic nerve of Group C, the regenerated mylinated nerve fiber was rather sparser , its myelin sheath was thinner and the great regenerated mylinated nerve fiber was fewer than that of Group B and A, the regenerated mylinated nerve fiber had apparent difference in size and the more cellular substance of Schwann cells was seen than that of Group B and A.5. Image-analysisThree months postoperatively, the number of the regenerated mylinated fiber in Group A was more than that in Group B, the difference between them was statistically significant (P=0.041), the number of the regenerated mylinated fiber in Group A was more than that in Group C, the difference between them was statistically significant (P=0.000), the number of the regenerated mylinated fiber in Group B was more than that in Group C, the difference between them was statistically significant (P=0.001) ; the diameter of the axon in Group A was larger than that in Group B, the difference between them was statistically significant (P=0.034), the diameter of the axon in Group A was larger than that in Group C, the difference between them was statistically significant (P=0.000), the diameter of the axon in Group B was larger than that in Group C, the difference between them was statistically significant (P=0.042) ; the myelin sheath thickness in Group A was thicker than that in Group B, the difference between them was statistically significant (P=0.046), the myelin sheath thickness in Group A was thicker than that in Group C, the difference between them was statistically significant (P=0.000), the myelin sheath thickness in Group B was thicker than that in Group C, the difference between them was statistically significant (P=0.016).6. Observation by electron microscopeThree months postoperatively, the results of transmission electron microscope showed that in Group A, there were more the regenerated mylinated nerve fiber, myelin sheath was intact and its appearance was exellent and thicker than Group B and C; the regenerated mylinated nerve fiber in Group C was poorest among three groups, the regenerated mylinated nerve fiber was few and spaced sparsely.IV. ConclussionThe RGD peptide grafted poly[LA-(Glc-Lys)] sustained-release FK506 and NGF incorporated diaphragm is effective on accelerating the regeneration of rat sciatic nerve; moreover, the combination usage of the FK506 and NGF can reduce the amount of the immunosuppressant agent of FK506.