| Facial nerve injury is a common disease and frequently occurring in clinic,which can be caused by many factors such as trauma,oppression,infection and operation.It is considered to be one of the most important causes of peripheral facial paralysis.Facial expression is a very important non-verbal means of communication.The occurrence of facial paralysis can affect the normal communication of patients,significantly reduce the quality of life,learning and work,and even affect the mental health of patients.In recent years,the mechanism researches of facial nerve injury have shown that there is not only degeneration of nerve fibers after facial nerve injury,but also neuronal apoptosis due to retrograde degeneration of axons.Therefore,how to promote the regeneration of nerve fibers as well as to reduce the programmed death of neurons is an important research direction in the treatment of facial nerve injury.Currently,there are lots of methods commonly used in clinic to promote nerve repair,such as nerve anastomosis,nerve transplantation,nerve conduit and so on.In addition,local nerve growth factor administration and stem cell therapy can be used as an auxiliary method of promoting nerve fiber regeneration.But these neurotrophic factors,which can simultaneously promote axon growth and neuronal survival,are difficult to act directly on the neurons through the blood-brain barrier by exogenous administration in virtue of the fact that they are large molecular polypeptides,and what's more,long-term use of high doses can cause significant side effects.Therefore,developing a small molecule drug that can quickly penetrate the blood-brain barrier and play the role of protecting neurons and promoting the regeneration of nerve fibers has become a focus of attention in recent years.VPA can rapidly penetrate the blood-brain barrier and directly target central neurons because of the characteristic that it is a branched short-chain fatty acid,which means it can be applied to the central and peripheral nervous system injuries and plays an important role in neuroprotection and nerve regeneration.As a clinical drug for more than 40 years,VPA has been proved to be safe and effective,and most importantly,it can be administered systemically.Recent studies have shown some promising results in several animal models of nerve injury and neurodegenerative diseases including optic nerve,sciatic nerve,spinal cord,Alzheimer's disease and Parkinson's disease.However,it remains unclear whether VPA has protective effect on FMNs after facial nerve injury.In the present study,we evaluated the recovery of nerve function after injury by establishing an animal model of facial trunk transection injury and assessed neuronal morphological changes,apoptosis and changes in protein expression level after facial nerve injury.Further study was made to investigate the possible effects of VPA on FMNs in the adult rats and its molecular mechanism so as to pursue potential protective factors of FMNs and the treatment of facial paralysis after facial nerve injury.Methods1.150 healthy adult Wistar male and female rats(weighed 280-310g)were randomly divided into three groups:facial nerve trunk exposure without any injury or treatment(Sham group),facial nerve lesion model plus normal saline treatment(NS group)and facial nerve lesion model plus VPA treatment(VPA group).Rats in VPA group were administrated with a dose of 300 mg/kg VPA daily instead of normal saline in NS group.2.Blink reflex,whisker movement and tip position were observed daily for 28 days postoperatively in order to evaluate the recovery of facial nerve function.3.Rats in three groups were executed to collect the facial nerve nucleus according to the stereotaxic map of the rat brain at 1,7,14,21,28 days after the facial nerve injury,respectively.4.Nissl staining was used to observe the survival of facial neurons,and TUNEL staining was used to observe the apoptosis of facial neurons.5.Immunofluorescence and Westernblot were used to evaluate the variation of the protein expression of Bax,cleaved caspase-3,BDNF and GAP-43 in the facial nerve nucleus in three groups.Results1.The facial nerve function evaluation results showed that the facial nerve axotomy resulted in complete paralysis and facial nerve function recovered with prolonged observation time in NS group.The difference of function recovery between the 28th day and the 7th day after the operation was statistically significant.The function scores of VPA group at each time point were significantly higher than those of the NS group,while no abnormality of facial nerve function was observed in Sham group.2.The results of Nissl staining showed that the survival rate of neurons in the nucleus of facial nerve in NS group decreased with time,which were 92.95±2.99%,75.2116.25%,70.30±2.25%,68.30±3.35%,69.38±3.30%at 1,7,14,21,28 days after operation,respectively.The survival rate of the facial neurons in VPA group was significantly higher than that in NS group,and the difference had statistical significance.3.The results of TUNEL staining showed that the apoptosis of the facial neurons in NS group began to occur on the 7th day after the operation and reached the peak on the 14th day after operation.The number of apoptotic cells on 7,14,21 and 28 days after injury were 10.7±2.96,15.7±1.45,12.3±1.20 and 9.0±0.58,respectively.While,the number of apoptotic cells in VPA group was significantly lower than that in NS group,and the difference was statistically significant.4.Immunofluorescence results showed that the expression of Bax,cleaved caspase-3,BDNF and GAP-43 in Sham group could only be detected by weak fluorescence.However,the fluorescence and color luminance of all factors in NS group were significantly enhanced,especially on the 14th day after injury.The expression of Bax and cleaved caspase-3 in VPA group was significantly weaker than that in NS group,but all of them were stronger than those in Sham group.The luminance of BDNF and GAP-43 was stronger than that of NS group and Sham group,but there was no obvious difference in the fluorescent color of the above factors at all time points.5.Western blot results showed that Bax,cleaved caspase-3,BDNF and GAP-43 protein expression in the facial nucleus after facial nerve injury were markedly increased and reached the peak at the 14th day after injury,then decreased gradually.Compared with the NS group,the expression of Bax and cleaved caspase-3 in the VPA group was reduced,while BDNF and GAP-43 expression were increased,and the difference was statistically significant.ConclusionThe results of this study showed not only the morphologic changes of the cell bodies,but also the apoptosis of the FMNs,and the corresponding changes in the protein expression in the facial nerve nucleus,which may be associated with changes of facial motor function after facial nerve injury.VPA can inhibit the apoptosis of facial neurons induced by nerve injury,increase the number of FMNs survival and promote the normal function of facial neurons and facial motor function recovery.The protective effect of VPA on neurons may be achieved by inhibiting the expression of apoptosis related factors(Bax,cleaved caspase-3)and increasing the expression of survival related factors(BDNF and GAP-43)of FMNs. |
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