Preliminary Study On The Mechanism Of PTEN In The Process Of Recurrent Laryngeal Nerve Injury And Repair In Rats

Objective:1.Establish a recurrent laryngeal nerve(RLN)crush injury model in SD rats.2.To investigate the role of PTEN via PI3K/AKT pathway in RLN injury repairment.Methods:1.Male SD rat right RLN crush injury model,which is validated by micro vessel clamp,was built and undergone larynx movement observation by laryngoscope,neurological function evaluation with EMG,and the nerve fibers microstructure observation with TEM.2.Construction of PTENshRNA and PTEN overexpressed vector via lentivirus vector.3.To regulate PTEN expression bi-directionally,the vectors were transfected into RLN crush injury rat model by local injection.Expression level of PTEN、PI3K、Akt、GSK-3β、BDNF、GDNF mRNA were quantified by realtime qPCR on day3 and 1,3,6,10 and 16 week after injury,as well as larynx movement observation,neurological function evaluation and TEM observation.Results:1.Male SD rat right RLN crush injury model was successfully established.Comparing with the sham-operated group,EMG,voice,laryngeal movement and and TEM structure recovered 16 week after injury.2.The pLenti X1 Puro-PTENshRNA-eGFP vectors and pLV.ExBi.P/Puro-CMV-PTEN-IRES-eGFP vector were successfully constructed,and verified by sequencing.3.Within 16 weeks after injury,PTEN mRNA expression level of PTENshRNA(KD)group was significantly lower than shRNA-N(NC)group,HBSS(CON)group,nerve crush injury(crush)group and sham-operated(sham)group;its expression level of PI3K,Akt and GSK-3β mRNA were significantly higher than NC,CON,crush and sham group.Within 10 weeks after injury,PTEN mRNA expression level of PTEN overexpress(OE)group was significantly higher than NC,CON,crush and sham group:while its expression level of PI3K,Akt and GSK-3β mRNA were significantly lower than NC,CON,crush and sham group.Within 10 weeks after injury,PTEN mRNA expression level of NC,CON and crush group were significantly lower than sham group;while their expression level of PI3K,Akt and GSK-3β mRNA were significantly higher than sham group.The voice of KD group recovered on 10th week after the operation,NC,CON and crush group recovered on 16th week,but OE group kept hoarseness on 16th week.The larynx movement and EMG of KD group recovered on 10th week after the operation,NC,CON and crush group recovered on 16th week,but OE group not totally recovered on 16th week.The TEM structure of KD group regenerated firmly on 10th week after the operation,NC,CON and crush group regenerated firmly on 16th week,but OE group was still regenerating on 16th week.Conclusion:1.It is feasible to investigate molecular mechanism of peripheral nerve injury repair with male SD rat right RLN crush injury model.2.By bi-directional transfection of PTEN on rat RLN crush models,we illustrated that PTEN is a negative regulator in the repair of recurrent laryngeal nerve.After nerve injury,PTEN is downregulated to promote the repair of RLN,which activates PI3K/Akt/GSK-3β pathway.