Experimental Study Of Asynchronously Growing Axons During Facial Nerve Regeneration

Objective:To date,it is believed that functional improvements appear to be consistently associated with reduced polyinnervated motor end-plates after facial nerve repair.However,the reason for the formation of polyinnervated motor end-plates is unclear.We hypothesize that this is because early reaching terminal axons become sprouting along the surface of muscle fibers and establishing polyinnervated motor end-plates,induced by denervated muscle fibers.To test this we plan to evaluate the effects of asynchronously growing axons after facial nerve crushed or transection and immediate end to end suture in the rat,combined structural and functional analyses.Methods:The vibrissal motor performance is monitored periodically using a video-based motion analysis in order to determine the time of motor recovery.A critical feature of our videographic method is to measure tagged vibrissae movements while leaving all others intact in body restrained rats without head fixation.At 4 selective time point,neuron counts of target reinnervation are studied using postoperative retrograde labeling in the bilateral vibrissal pad muscles.1.Thirty two adult female Sprague-Dawley rats were handled daily for 2 weeks.When they grew accustomed to being tightly held by the handler for 10 min,animals were inserted into a custom made rodent restrainer daily for a week.Animals were randomized to four groups(n = 8 per group).A group: right main trunk experimental crush injury;B group: right main trunk transection and end to end suture;C group: right main trunk sham surgery control;D group: right main trunk transection denervation control.Rats were mildly food deprived prior to testing.To increase visibility of observed vibrissae with neighboring vibrissae present,we used eyelash pomade to tag bilateral three large vibrissae in row B.Whisking behavior was elicited in response to the same stimulus—intermittent presentation of peanut butter as a reinforcer.All rats underwent baseline preoperative whisking testing to verify normal whisking behavior.Thereafter,whisking recovery was measured on a scheduled basis for 2,4,8 and 12 weeks.Rats underwent a 5-min videographic recording session during which tagged vibrissae were continuously monitored.The video sequences were slowly reviewed and 5-s sequence fragments containing the most vigorous whisking were analyzed offline using Proanalyst motion analysis software.The selection criteria used were stable position of the head,frequency of whisking,and amplitude of vibrissal protraction.2.Fifty two adult female Sprague-Dawley rats were randomized to four groups.A group(n=24): right main trunk experimental crush injury;B group(n=24): right main trunk transection and end to end suture;C group(n=2): right main trunk sham surgery control;D group(n=2): right main trunk transection denervation control.Rats in A group and B group were randomized to 4 time groups of postoperative 2,4,8,12 weeks,respectively(n = 6 per time group).1,3,7,11 weeks after surgery,The fluorescent tracer Fluoro-Gold(FG)was injected bilaterally into the whisker pad of the rats in A and B groups.One week after surgery,FG was injected bilaterally into the whisker pad of the rats in C group.11 weeks after surgery,FG was injected bilaterally into the whisker pad of the rats in D group.7 days after labeling,the brains were removed and 30 ?m-thick axial sections were cut through the brainstems on a freezing microtome.All retrogradely labeled motoneurons with distinguishable cell nucleus in the 30 ?m-thick sections were counted under fluorescence microscopy.Results:1.Evaluation of preoperative whisking parameters for the top three most vigorous whisks demonstrated bilaterally symmetric and synchronous motor pattern.There were no statistical differences in any preoperative whisking parameters between the manipulated side and intact side.A group: By postoperative week 2,the mean whisking amplitude was approximately 77% of its value on the intact sides,and animals undergoing crush injuries recovered approximately 96% whisking amplitude within 12 weeks.B group: At 10–14 days,the vibrissae “rose” again to the level of the mouth and acquired a posterior orientation with only slight tremor-like movements;by the fourth week,animals showed evidence of rhythmical whisking,at which point rats had approximately 40% of recovery of whisking amplitude compared to the intact sides;by postoperative weeks 12,the mean whisking amplitude was approximately 77% of its value on the intact sides.Since the fourth week,A and B group showed that the whisking frequency is similar in both sides.At postoperative days 14,all animals in C group exhibited normal whisking function compared with the preoperative vigorous whisking,and were not tested further.D group animals underwent complete loss of whisking on the manipulated sides,did not recover any significant whisking function for the duration of the study,and were therefore not assessed for whisking function.As expected,the recovery of whisking function is better and earlier in A group.2.Postoperative 2,4,8,12 weeks,there were no statistical significance differences between A group and B group in numbers of FG-labeled neurons on the intact side.A group: postoperative 2 to 12 weeks,the numbers of FG-labeled neurons on the operated side increased gradually;by postoperative week 2,the labeled neurons were 19.27% of its value on the intact side,postoperative 4 weeks,the labeled neurons were 86.15% of its value on the intact side;postoperative 8,12 weeks,the labeled neurons on the operated side increased significantly and were 126.5% and 128.49% of its value on the intact side,respectively.B group: postoperative 2 weeks,there was no detectable labeled neuron on the operated side;postoperative 4 to 12 weeks,the numbers of FG-labeled neurons on the operated side increased gradually;postoperative 4 weeks,the labeled neurons were 58.46% of its value on the intact side;postoperative 8,12 weeks,the labeled neurons on the operated side increased significantly and were 116.27% and 131.91% of its value on the intact side,respectively.C group: postoperative 2 weeks,the numbers of FG-labeled neurons were similar in two rats.D group: postoperative 12 weeks,there was no detectable labeled neuron on the operated side.Conclusions:1.Observations on body restrained rats show that majority of free whisking on the both sides is synchronous and symmetric,which allows us to compare vibrissal motor data between intact and manipulated side after facial nerve injury.The videographic technique,when used in conjunction with body restrained procedures and motion analysis soft ware,may be an objective tool to evaluate motor recovery from facial nerve manipulation in the rat model.2.The present study provides preliminary evidences that there are asynchronously growing axons during facial nerve regeneration.Our results may be significant in further determining the relationship between asynchronously growing axons and formation of polyinnervated motor end-plates in the target muscle.