The Nerve Conduit With GDNF Microcapsule Bridging Rat Facial Nerve To Induce Facial Nerve Regeneration

As one of the most important peripheral nerves, the facial nerve dominate facialexpressions in human beings, and can be easily injured in many circumstances such astrauma, surgery, imflammation or tumor. Although facial nerve can be repaired throughsurgical reinnervation procedures after injury, the facial motor function can hardly return tonormal, and usually with facial paralysis sequelae such as synkinesis, hemi-facial spasm,which have a significant impact on patients' quality of life.Many in vitro studies have indicated that various exogenous neurotrophic factors caninduce directional outgrowth of regenerating axons, and also comfirmed that the directionof axons growth tends to the direction of higher concentration gradient of neurotrophicfactors. Glial cell line-derived neurotrophic factor (GDNF) is the neurothrophic factorwhich has the strongest effect on motor nerve so far. Studies found that the degree ofabberant reinnervation of the facial nerve axons are related to the concentration of GDNFin a concentration dependent manner, a certain concentration of GDNF can significantlyreduce the abberant reinnervation of peripheral nerve.During the repair process after facial nerve injury, artificial conduit attracts more andmore attention because of its wide range of donor sources, lack of immune response,decrease of surrounding tissue scar invasion, good growth-oriented, casual shaping andeasily added with a variety of active substances. It can repair injured nerves with longdefects, and the reparing effect is similar to autogenous nerve transplant. How to importexogenous neurotrophic factors into the nerve conduit in order to release active moleculesin a more efficient time and space specific manner, has been the direction toward which thebiologists and engineering experts are working hard.In the present study we packed neurotrophic factor GDNF into microcapsules so as toestablish a evaluation system which can assess the inducing regeneration effect in differentconcentration of neurotrophic factor, and delivery GDNF in nerve conduit by microcapsulein a controlled release manner, try to find a more efficient release manner in time andspace of neurotrophic factors an assess its reinnervation effect on abberant reinnervation offacial nerve.The present study is divided into three parts.Part1The preparation of GDNF microcapsules and determination of theircharacteristics Objective: Prepare GDNF microcapsules and determine their characteristics.Methods: GDNF microcapsules were prepared with double emulsion solvent evaporationmethod, and then irradiated for sterilization purpose with radioactive source60Co after theywere freeze-dried. Bacteriological examination was used to evaluate sterilization effect ofthe microcapsules, light microscope and electron microscope were used to assess themorphology and distribution of the microcapsules and calculate the physical properties ofthe microcapsules, GDNF-ELISA were used to assess their in vitro release, and cell culturewere used to evaluate their biological activity. Results: Microcapsules were cultured for7days after preparation and radiation sterilization and found no micro-organism growth,capsules after sterilization could meet the aseptic standards. Microcapsules were seen to bemorphologically intact with smooth surface under scanning electron microscope. Themeasure value of the microcapsule diameter was19.15±0.23um. The production rate ofGDNF microcapsules was79.10%, the drug loading of the microcapsules was0.175ug／mg, and the covered rate was69.6%. In vitro release experiments showed that in the first3days, microcapsules released1/3of the total drug amount, and the release rate graduallystabilized after3days. The majority of drug were released from the microcapsules in arelatively stable rate in the first40days. Determination of the GDNF biological activity inthe microcapsules showed that the number and length of motor neurite in the positivecontrol group had no significant difference compared with those in GDNF microcapsulegroup (P>0.05), however, both were better than those in the blank control group, whichproved the biological activity of GDNF in the GDNF microcapsules we have prepared.Conclusion: GDNF microcapsules were successfully prepared by the double emulsionsolvent evaporation method, and were sterilized by irradiation with radioactive source60Coafter freeze-dried. Tests on the characteristics, sterility and GDNF activity of themicrocapsules showed that the microcapsules which contain the target concentration andactivity of GDNF, were in good morphology, and exhibit a satisfactory controlled-releaseresult.Part2The induced regeneration role of in vitro release of GDNF microcapsules onmotor neuronsObjective: To establish the nerve cells induced regeneration model, and screen thepre-prepared GDNF microcapsules by concentration, in order to identify the the optimalGDNF concentration in microcapsule which can best induce the regeneration of motornerves. Methods: Establish a nerve cell induced regeneration model according to Cao et al protocol, Assess the activity of nerve cells in the chamber by cell culture. Detect theconcentration of GDNF in different partition of agarose by GDNF-ELISA in order tocomfirm the formation of a concentration gradient in the chamber. Then we put themicrocapsules with different concentration into the chamber and detect the inducingregeneration effect on nerve cells under different concentration. Results: A concentrationgradient was formed in the chamber, and a successful nerve cell induced regenerationmodel was established. The agarose in the chamber had no cytotoxicity, so the model isfeasible for the nerve cells. In the aspect of inducing regeneration effect on the motorneurons at different GDNF concentrations, there was no significant difference betweenGDNF200ng/ml group and GDNF100ng/ml group (P＞0.05), and GDNF200ng/mlgroup and GDNF100ng/ml group were better than both blank control group and GDNF50ng/ml group, with a statistical significance (P<0.05), and the difference between blankcontrol group and GDNF50ng/ml group were not significant (P>0.05). Conclusion: Weestablished a motor neuron induced growth model, and use this model to screen theprepared GDNF microcapsules by concentration, and initially determined that the optimalconcentration for GDNF to induce the growth of motor neurons in vitro is100ng/ml.Part3The nerve conduit with GDNF microcapsule bridging Rat facial nerve toinduce facial nerve regenerationObjective: To explore a more effective GDNF release method in the inducedregeneration of the facial nerve by reparing facial nerve with a PLGA/chitosan compositenerve conduit graft containing pre-prepared and screened GDNF microcapsules. Methods:Facial nerve trunk and branches were located through anatomical procedures, Frozensections, fluorescent retrograde tracing method were used to locate the facial nucleus andsubnucleus region in the brain. Animals were divided into three groups according to theirnerve reinnervation procedures in in vitro experiment, the blank control group, the GDNFgel injection group and the GDNF microcapsules injection group. After nine weeks, theabberant reinnervation of facial nerves and functional recovery of facial muscles wereevaluated by facial muscle contractile function score, histology, Abberant reinnervationrate calculated by Fluorescent Retrograde Tracking method and electromyography. Results:Facial nerve trunk and its branches were identified to trace zygomatic branch and buccalbranch so as to evaluate the abberant reinnervation rate of facial nerve. GDNFmicrocapsules injection group were better than both GDNF gel injection group and blank control group in many aspects such as contractile function score, abberant reinnervationrate calculated by fluorescent retrograde tracing method and electromyography, with astatistical significance (P<0.05). Conclusion: Reinnervation of transected facial nerve trunkusing degradable biological conduit graft containing GDNF microcapsules in adult SD ratscan significantly reduce the proportion of abberant reinnervation of facial nerve, which isbetter than reinnervation with blank conduit graft and with conduit in which injected withGDNF gel.Final ConclusionReinnervation of transected facial nerve trunk using degradable biological conduitgraft containing GDNF microcapsules in adult SD rats can significantly reduce theproportion of abberant reinnervation of facial nerve, which can shed a new insight inreducing the abberant reinnervation after facial nerve injury and promote functionalrecovery of facial nerve, and also provide an experimental basis for clinical applications.