Study On The Repair Of Facial Nerve Defect And The Prevention Of Perineural Scar

Part Ⅰ：Study on the prevention of perineural scar using chitosanconduitsExperiment1、 Reduction of extraneural scaring by application of chitosanConduits after injury of rabbit facial nervesObject: After anatomy injury of facial nerve, the extraneural scar formation mayoppress the nerve and hinder axon regeneration, affect the recovery of facial nervefunction. The aim of the study was to observe the effects on facial nerve scarring andblood supply inside the nerve by application of biodegradable chitosan conduits as aphysical barrier.Method: Thirty-six New Zealand white rabbits were performed facial nerve anatomyinjury modeling, the parotids were subtotal resected. The animals were randomlydivided into three groups: superficial coverage group（n=12）,circumferentiallywrapping group（n=12）, autologous control group（n=12）. Four and twelve weeks aftersurgery， using Petersen grading to assess the adhesions between facial nerve andsurrounding tissues，using histological staining to detect the facial nerve scarring andvascularity density, using vibrissae motion，TEM and neurophysiological to assessfacial nerve function.Result：Petersen score was no significant difference between groups four weeks aftersurgery(P>0.05), coverage and wrapped groups were better than autologous grouptwelve weeks after surgery (P <0.01); myelinated nerve fiber diameter, myelin sheaththickness, number of nerve fibers in the coverage group were better than wrapped groupand control group four weeks after surgery (p <0.05); collagen thickness in the controlgroup were greater than in the coverage group and wrapped group four and twelveweeks after surgery, the difference was statistically significance (P <0.01); myelinated nerve fiber diameter, myelin sheath thickness, number of nerve fibers in the coveragegroup were closer to the control group (P>0.05), but better than in the wrapped group(p <0.05).Conclusions：Chitosan conduits as a physical barrier covering the surface of the facialnerve can effectively reduce the formation of perineural scarring, improve the recoveryof facial nerve.Experiment2、Experimental study of hyaluronic acid combined with chitosanconduits on prevention of sciatic nerve scarring in ratsObject: To observe the effect on chitosan conduits combined with hyaluronic acid in ratsciatic nerve function after1cm clamp injury.Method: Sixty SD rats which under clamp injury were randomly divided into fourgroups covering: superficial coverage group（n=15）, coverage/hyaluronic acid group,simple clamping group, clamp/hyaluronic acid groups. The adhesion between sciaticnerve and surrounding tissue, perineural scar, sciatic nerve function and nervefunction index, histological and electrophysiological testing were analyzed four、eightand twelve weeks after surgery.Result： The sciatic nerve function index, petersen score, the nerve fibers, myelinsheath thickness and the diameter of myelinated nerve fibers were no significantdifference between groups four weeks after surgery; but above indexes in the clamp/hyaluronic acid group were better than in the control group eight and twelve weeksafter surgery. The epineurium collagen thickness measurement in other groups weresignificantly lower than the control group, the difference was statistically significant(p <0.01);the epineurium collagen thickness in the clamp/hyaluronic were thickerthan coverage/hyaluronic acid group twelve weeks after surgery, the differencewas statistically significant (p <0.05).Conclusions：Hyaluronic acid combined with chitosan conduits can promote therecovery of rat sciatic nerve injury and reducing its perineural scar generation. Experiment3、The clinical effects of facial nerve function after the implantionof Chitosan conduits in parotid surgeryObject: To compare the effects on facial nerve function between the application ofchitosan conduits and conventional parotid gland surgery in patients.Method:75cases of parotid gland tumar patients receiving superficial lobe of theparotid gland excision or total resection surgery were divided into two groups, oneapplication of chitosan conduits coverage total and every branch, another group forconventional surgery as control. Seven days，one month，three months，six months，nine months after surgery, ultrasound and blood tests were carried out，facial nervefunction were observed according to HB grade system.Result：Follow-up time from9to50months, average21.7months.50patients wereremoval of the superficial lobe of the parotid gland,25patients were under total parotidresection. Postoperative blood and ultrasound examination display no abnormalities,indicating that chitosan does not cause adverse reactions after the conduits implanted inthe body. The incidence of immediate paralysis after seven days in the chitosan group(29/32,90.6%) and in the control group (39/43,90.7%); incidence of permanent facialparalysis incidence of facial paralysis in the chitosan group (0/32,0%) and in thecontrol group (2/43,4.7%) were not statistically different (P>0.05). There were nosignificant differences in facial paralysis severity between the two groups seven daysafter surgery (P>0.05).The facial nerve recovery rate in the chitosan group were fasterthan the control group, the difference was statistically significant (P <0.01).Conclusions：The chitosan conduits have good biocompatibility, implanted in vivowithout adverse reactions.The application of chitosan conduit on the surface of facialnerve after parotid surgery play good result in promoting facial nerve functionalrecovery.Part Ⅱ： Construction of lentiviral vectors containing GDNFgene, viral packaging and the transfection of MSCs cellsObjective: To construct lentiviral vector containing EGFP and GDNF,using thelentiviral vector to transfect mesenchymal stem cells and make the MSCsoverexpression GDNF. Method: By PCR amplification, BamHI and AscI restriction endonuelease digestion,T4DNA ligase, the gene of interest inserted into the lentivirus vector GDNFpLenti6.3_MCS_IRES2-EGFP, construct recombinant lentivirus. In lipofectamine2000will build successfully mediated lentivirus transfected human embryonic kidney cellline (293T), lentiviral packaging production and determination of viral titer. Thepackaged lentivirus infection BMSCs, and the cell line transfected viral transfectioncomplex (MOI value) was measured to select the best MOI. Western, RT-PCR werethen detected by GDNF protein and mRNA expression, flow cytometry purifiedtransfection cells.Result: Successfully constructed the lentiviral vector expressed GDNF and EGFP, theMSCs expressed strong fluorescence96hours after transfection.The expression of cellfluorescence were strong and cell activity were better when the MOI was25. Westernand RT-PCR analysis confirmed the successful expression of GDNF in MSCs, flowcytometry discovered the transfection efficiency was94.2%.Conclusions：Successfully constructed the MSCs which overexpressing EGFP andGDNF, and provide a basis for post-experiment.Part Ⅲ：Correlation of acellular nerve graft combined withGDNF transfected MSCs for repairation of facial nerve defectExperiment1、Compare of different acellular peripheral nerve methodObjective: Application of different approachs to decellularize rabbit facial nerve and toobserve the histomorphology and the biocompatibility, provide experimental evidencefor the selection of facial nerve acellular method.Method: Application of freeze-thaw, chemical and freeze-thaw+enzymatic digestionto decellularize rabbit facial nerve, the acellular nerves were under HE staining,scanning electron microscopy and TEM observation. The acellular nerves weresubcutaneously implanted,at2,4weeks after surgery,the rabbits were sacrificed toperform the general observation and histological observation.Result: Histomorphology observation see after chemical and freeze-thaw+enzymatic digestion process, the myelin was more thoroughly cleared, only a very small amount ofthe visible part of the region of residual myelin; after freeze-thaw treatment, the myelinwas visible shrinkaged, but there is more residual deformation. Experimentalobservation see a little more lymphocytic infiltration, mainly in the basement membranesurrounding the nerve bundles in the gap when the nerve treated by simple freeze-thawprocess. There was no significant inflammatory nerve cells when the nerve underchemical and freeze-thaw+enzymatic digestion process.Conclusions：Thaw+enzymatic digestion treatment method is simple method, thenerve scaffold structure was saved intact, the nerve myelin was thoroughly removed, Itis a good method and provide the basis for the later experiments.Experiment2、Acellular nerve combined with GDNF transfected MSCsto repair facial nerve defectsObjective: The facial nerve acellular scaffold combined with GDNF transfected MSCsto repair facial nerve defects，to observation the effect of facial nerve regenerationand neuronal survival opposite.Method:Sixty New Zealand white rabbits were performed facial nerve anatomy injurymodeling.The animals were randomly divided into: acellular nerve allograft group(ANA), acellular nerve+mesenchymal stem cell group (AN-MSCs), acellular nerve+GDNF transfected mesenchymal stem cells (AN-G-MSCs), nerve autograft group(Control). Four and twelve weeks after surgery， using vibrissae motion andneurophysiological examination to assess facial nerve function，using toluidine blueand transmission Electron microscopy to observation the axon regeneration both inproximal and distal of the transformation nerve.Result: Facial nerve axons morphometric analysis found that the nerve axons count,myelin sheath thickness, myelin diameter in ANA group were significantly lower thanthe other three groups in the total dry place four weeks after surgery (P <0.05); Thereas no significant difference between groups twelve and twenty-four weeks after surgery(P>0.05). The nerve axons count, myelin sheath thickness, myelin diameter in ANAgroup and AN-MSCs group were were significantly lower than the other two groups in the distal part of facial nerve branch twelve weeks after surgery (P <0.05). The nerveaxons count, myelin sheath thickness, myelin diameter in the ANA group were lowerthan the other three groups twenty-four weeks after surgery.Conclusions： The acellular nerve combined with GDNF transfected MSCs cansuccessfully repairing multi-branch facial nerve defect, the effect of reparation wasbetter than the simple application of acellular nerve, and can protect the Survival ofneurons to some extent.Experiment3、Long-term follow-up study on nerve allografts to repairfacial nerve defectObjective: Application of acellular nerve graft to repair facial nerve, and to observe theefficacy and safety of reparation following long-term observation.Method: From2004to2013, due to trauma or parotid tumor invasion resected facialnerve leads to defects in44cases, the final total of35cases of neurological defectspatients completed follow-up study,20cases of autologous nerve graft and15cases ofacellular nerve graft. The shortest postoperative follow-up was9months,usingultrasound and blood examination during follow-up to check the security, Using HBgrade system to analysis the facial nerve function.Result: Followed up from nine months to nine years, facial nerve defect length from10mm to40mm. Immediate surgery to repair a defect in26cases, delayed surgicaltransplant in9cases, the interval between injury to surgery was2days to3months.Ultrasound and blood examination postoperative follow-up during the investigationwere found no abnormalities, indicating acellular nerve did not cause significant adversereactions after implantation in vivo. Final nerve autograft group and acellular nervegraft group meaningful functional recovery were85%(17/20) and80%(12/15), nosignificant difference (P>0.05), but the HB grading compare found that the nervefunction recovery in the autologous group was significantly better than the acellularnerve graft, the difference was statistically significant (P <0.05).Conclusions：In this study, acellular nerve repair facial nerve and long-term follow-upobservation in the clinic and found acellular nerve can repair facial nerve, long-term follow-up found no significant adverse reactions, but the repair effect was significantlyweaker than autologous nerve graft.