Changes Of NCAM And BDNF Expressive Level During Facial Nerve Regeneration In Rat Facial Muscles

ObjectiveCollateral sprouting of regenerating axons and terminal axon sprouting(TAS)are universal phenomenon after peripheral nerve injury.It is well known that major reasons for the poor recovery of motor function after severe peripheral nerve injury are the TAS and polyinnervated end-plates(PEDs).Synaptic connections and in particular neuromuscular junctions(NMJs)are dynamic structures that undergo some structural changes throughout life.In pathological cases,such as the blocking of neurotransmitter release with different toxins or after partial denervation,synaptic plasticity is manifested as extensive terminal axonal sprouting,leading to new NMJs on denervated or paralyzed muscle fibers.The growing environment of the terminal axons may be composed of extracellular matrix,terminal Schwann cells and cell adhesion molecules.Two conceivable candidates for functional involvement are the neural cell adhesion molecule(NCAM)and brain-derived neurotrophic factor(BDNF).Facial muscles undergo a process from complete denervation to imprecise reinnervation following transection and repair of facial nerve.However,the changes of NCAM and BDNF expressive level during facial nerve regeneration are unclear.The relationship between NCAM,BDNF expressive level and PEDs also need to be identified.Based on our previous works,we observed,compared,and analyzed the changes of NCAM and BDNF expression in different time point after facial nerve repair in order to lay the foundation for the future PEDs study.Methods Seventy two adult female Sprague-Dawley rats were randomized to experimental group(n=64)and control group(n=8).Experimental groups included FFA group,FFA+PS group,FFA+P-NCAM-Ab group and FFA+BDNF-Ab group(n=16 per group).FFA group: right main trunk transection and facial–facial anastomosis(FFA).FFA+PS: 4 days after FFA,100μl physiological saline was injected daily in the surface of the right levator labii superioris for a period of 6 days.FFA+P-NCAM-Ab group: 4 days after FFA,100μl polyclonal anti-NCAM antibody(6 μg)was injected daily in the surface of the right levator labii superioris for a period of 6 days.FFA+BDNF-Ab group: 4 days after FFA,100μl anti-BDNF antibody(6 μg)was injected daily in the surface of the right levator labii superioris for a period of 6 days.Control group: animals received no treatment.Rats inexperimental group and control group were randomized to 4 time groups of 2,4,8,12 weeks,respectively(n = 4 per time point in experimental group;n=2 per time point in control group).At 2,4,8,12 weeks,right levator labii superioris preparations in experimental groups and bilateral levator labii superioris preparations in control group were dissected out.The protein and m RNA expressions of NCAM and BDNF in levator labii superioris were determined by western blotting and quantitativereal-time polymerase chain reactions(q RT-PCR).Results 1.Postoperative 2 weeks,the protein and m RNA expressions of NCAM and BDNF of levator labii superioris in all experimental groups were higher than that in the control group,and the NCAM and BDNF expression in FFA and FFA+PS groups were higher than the FFA+P-NCAM-Ab group and FFA+BDNF-Ab groups.2.The protein and m RNA expressions of NCAM and BDNF of levator labii superioris in all experimental groups attained a peak at 4th week,and the NCAM and BDNF expression in FFA and FFA+PS groups were also higher than the FFA+P-NCAM-Ab and FFA+BDNF-Ab groups,m RNA expression of NCAM in FFA+P-NCAM-Ab was significantly lower than other three experimental groups.3.Postoperative 8 weeks,the protein and m RNA expressions of NCAM and BDNF of levator labii superioris in all experimental groups begun to decline,the expressions of NCAM and BDNF were higher than or approximation to the levels at the 2nd week.4.At the 12 th week,the protein and m RNA expressions of NCAM and BDNF of levator labii superioris in all experimental groups continued to decline,FFA group and FFA+PS group deceased more than FFA+P-NCAM-Ab group and FFA+BDNF-Ab group,the protein and m RNA expressions of BDNF corresponded to normal control,however,the protein expressions of NCAM in all experimental groups were higher than that in the control group,the m RNA expression of NCAM in the FFA+P-NCAM-Ab group was similar to control group.Conclusions 1.Changes of NCAM and BDNF expression seem to parallel denervation and reinnervation process of muscle fibers.2.Anti-NCAM antibody and anti-BDNF antibody can depress expression of NCAM andBDNF during denervation and reinnervation of muscle fibers.3.Interfere in NCAM and BDNF expression during denervation and reinnervation process of muscle fibers possibly prevents TAS resulting in reduced PEDs.