Effect Of BDNF On The Central Regulation Of Genioglossus And Upper Airway Stability In Tongue Strength Training Rats

Objective: Obstructive sleep apnea(OSA),charactered by the upper airway repeatedly collapses during sleep,is a sleep-disordered breathing disorder with an incidence rate of9%-38% in the population and as high as 49% in the elderly.The genioglossus muscle is one of the most typical upper airway dilators,and it is very important for the maintenance of upper airway opening.However,studies have shown that the EMG activity of the genioglossus muscle of OSA patients is significantly reduced during sleep when compared with the normal control group.About one-third of OSA patients have poor nocturnal genioglossus muscle responsiveness which defined as the maximum increase of genioglossus EMG activity is less than 0.1% for every increase of one centimeter of water column of epiglottic negative pressure.These results indicated that the functional imbalance of the upper airway dilator muscles is one of the important mechanisms of OSA.So,as oropharyngeal training focused on improving the contractility of upper airway dilator muscles,could it be a promising therapy for OSA patients? Previous studies have shown that oropharyngeal muscle training could reduce the AHI of patients with moderate to severe OSA,and improve their daytime sleepiness,sleep disturbance and subjective snoring symptoms.Our previous study in which 10 moderate OSA patients were performed one week tongue training found that the overall AHI of the OSA patients in the tongue training group decreased at night when compared with the control group,but the efficacy of each OSA patient in the treatment group was different.63.6% of OSA patients have good efficacy,while the remaining patients have poor efficacy.Researchers currently speculate that the reason why OSA patients can benefit from oropharyngeal muscle training is that,in addition to the contractile function of the muscle itself and the improvement of anatomical structure,it may be derived from the adjustment of the central and peripheral nerve plasticity of the tongue.However,whether the tongue training first induces the plasticity of the cortical motor center or increases the muscle EMG activity is still inconclusive,and there is no research to explore the dynamic influence of the oropharyngeal muscle training on the stability of the upper airway.As one of the most typical neurotrophic factors,brain-derived neurotrophic factor(BDNF)plays an important role in axon growth,neuron survival and differentiation,cognition,learning,and memory.The preliminary experiment of our research group also found that 8 weeks of tongue strength training(TST)could uprate the expression of BDNF and its high-affinity receptor-tyrosine kinase receptor B(Trk B)in the hypoglossal nucleus of rats.So,is the benefit of tongue training based on neuroplasticity dependent on BDNF synthesis?Extracellular signal-regulated kinase(ERK)is a serine/threonine protein kinase,which can be activated by a variety of signals,such as neurotrophic factors and neurotransmitters.It plays an important role in neuroplasticity,cell proliferation and differentiation,learning,and cognition.After BDNF binds to its receptor Trk B,it promotes the translocation of ERK from the nucleus to the cytoplasm by increasing the phosphorylation at position 515 of the Trk B receptor.The activation of ERK further leads to the phosphorylation of c AMP-response element binding protein(CREB)at position133.Activated CREB can then up-regulate immediate early gene expression,promote cytoskeletal protein synthesis,and promote dendritic growth and differentiation.Some scholars conducted resistance training on epilepsy model mice for 4 weeks and found that the number of epileptic seizures in the training group was significantly less than that in the control group.At the same time,training could reverse the reduction in the expression of ERK and CREB caused by epilepsy in the hippocampus of rats.Previous studies have also found that 4 weeks of strength training could rescue the spatial memory impairment of mice caused by streptozotocin.Fundamentally,such improvement may be due to the increase of ERK and CREB activation levels in the hippocampus.The above findings suggested that exercise training may enhance central plasticity by activating ERK and CREB through the combination of BDNF and Tr KB,and ultimately improve cognition,memory,and related nervous system functions.Then,does BDNF also mediate the improvement of the excitability of the genioglossus center and the stability of the upper airway by TST through the signal pathway of Trk B-ERK-CREB? There is no relevant report on it.Therefore,this study aims to explore the effects of BDNF on genioglossus function,central control,and upper airway dynamics of TST rats,using transcranial magnetic stimulation,genioglossus EMG recording,upper airway critical closure pressure measurement,brain stereotaxic,microinjection of hypoglossal nucleus,and adeno-associated virus transfection.The m RNA and protein expression levels of Tr KB,ERK,CREB in the hypoglossal nucleus of each group of rats before and after the tongue training were analyzed by RT-PCT,immunofluorescence and immunohistochemical techniques,and further microinjection of ERK or CREB inhibitor into the hypoglossal nucleus,to detect its effects on the genioglossus TMS responsiveness and upper airway stability of trained rats to explore the related mechanism of BDNF affecting the genioglossus central excitability and upper airway stability after the tongue training.Methods: 1.To explore the effect of BDNF on the central regulation and upper airway of the genioglossus muscle of the tongue training rats84 male SD rats(SPF grade)were randomly divided into normal control group(Normal Control,NC,n=12),tongue strength training group(TST,n= 12),TST+PBS group(n=12),TST+Tr KB-Fc group(n=12),TST+AAV-EGFP group(n=18)and TST+AAV-BDNF group(n=18).After 5-day adaptive water restriction,all the TST group began to perform resistance-fetching training for 8 weeks according to the preset gradually increased resistance threshold,training time,and total daily training threshold.Specifically,the rats protruded the tongue to press the 18 mm diameter disc which connected with the pressure sensor.The rats would get about 0.02 ml of distilled water award when the pressing force reaches the preset pressure threshold.The daily training duration was 10-30 minutes,and the threshold of muscle total work was 1000-3000 gs per day.For the TST+PBS and TST+Tr KB-Fc groups,a microinjection device was inserted into the ventral region of bilateral hypoglossal nucleus(13.4–14.5mm posterior to bregma,±0.2–0.3 mm lateral to the midline,and at a depth of 8.2–8.6 mm)before the tongue training.After the rats recovered 4-5 days after the operation,they began to the water restriction period.During the training period,Tr KB-Fc(a specific inhibitor of BDNF,0.5 ?g/?l,60nl/side)or PBS(0.01 mol,60nl/side)was injected into the hypoglossal nucleus before daily training.Before TST program,AAV8-h Syn-EGFP or AAV8-h Syn-BDNF(0.5?l/side,47425-1,Genechem,Shanghai)were injected into the hypoglossal nucleus of the rats in the TST+AAV-EGFP or TST+AAV-BDNF groups.Three weeks later,6 rats in each group were randomly selected to detect the m RNA and protein expression levels of BDNF in the hypoglossal nucleus.The remaining 12 rats performed the water-restricted period and then started the TST program.Maximum voluntary tongue force,Pcrit,genioglossus EMG and TMS reactivity of the genioglossus motor cortex of rats were evaluated on the 0th day,2nd week,4th week and 8th week of the training in all groups.At each detection time point,TMS and EMG changes were recorded continuously at 5 min,20 min,40 min,80 min,and 120 min.After 8 weeks of training,all the rats were directly decapitated and the brains were removed to obtain the hypoglossal nucleus for RT-PCR analysis of BDNF m RNA expression levels,or brain tissue perfusion and specimen sectioning were performed,and the comparisons level of BDNF protein in hypoglossal nucleus were performed by immunohistochemistry or immunofluorescence techniques.2.To explore the related mechanism of BDNF affecting the central excitability of the genioglossus muscle and the stability of the upper airway after the tongue training In this part of the experiment,first detected the m RNA expression level of Tr KB,ERK1/2 and CREB,and protein expression level of Tr KB,ERK,p-ERK,CREB and p-CREB in the hypoglossal nucleus in NC group,TST group,TST+PBS group,TST+Tr KB-Fc group,TST+AAV-EGFP group and TST+AAV-BDNF group.Then,40SPF-grade male SD rats were divided into NC(n=8),TST group(n=8),TST+PBS(n=8),and TST+ PD98059 group(n=8)and TST+ KG-501(T n=8)according to the random number table method.The genioglossus central responsiveness(TMS + genioglossus EMG)and upper airway dynamics(Prit)were detected after Microinjecting the ERK inhibitors or CREB inhibitors into hypoglossal nucleus.The tongue training model,positioning injection method of the injection group,and the genioglossus central reactivity and other detection methods are the same as before.Results: 1.To explore the effect of BDNF on the central regulation and upper airway of the genioglossus muscle of the tongue training rats(1)Strength training could gradually increase the maximum voluntary tongue extension force of rats in the TST group.Compared with the NC group,the TMS latency of the TST group was shortened,and the EMG activity of the genioglossus was increased,and the degree of the shortening of the TMS latency and the amplitude of the increase of EMG activity increased with the extension of training time,and the duration of this excitement gradually extend from 20 minutes to 80 minutes.Secondly,the Pcrit values of the TST group in the 4th and 8th weeks of training were significantly lower than those of the control group(P<0.05).After 8 weeks of TST,the m RNA and protein levels of BDNF in the hypoglossal nucleus in the training group were significantly higher than those in the control group(P<0.05).(2)Compared with the TST group alone,the voluntary tongue extension strength of rats after BDNF block in the hypoglossal nucleus decreased slightly,and the enhancement of TMS responsiveness in the genioglossus center caused by the tongue training was decreased,which was manifested as TMS the incubation period was prolonged(P<0.05),and the elevated EMG activity of the genioglossus muscle and the improved collapsibility of the upper airway caused by the tongue training were weakened(P<0.05).At the same time,after 8 weeks of tongue training,the m RNA and protein levels of BDNF in the hypoglossal nucleus in the TST+Tr KB-Fc group were lower than those in the TST group(P<0.05).(3)Compared with the TST+AAV-EGFP group,the MVTF of rats increased slightly after overexpression of BDNF in the hypoglossal nucleus,and the enhancement of the TMS reactivity of the genioglossus center caused by the tongue training increased,which was manifested as shortening of TMS latency(P<0.05);Increase of genioglossus EMG activity and improvement of upper airway collapsibility after TST were increased.(P<0.05).Correspondingly,after 8 weeks of training,compared with the TST+AAV-EGFP group,the m RNA and protein levels of BDNF in the hypoglossal nucleus of the TST+AAV-BDNF group were significantly increased(P<0.05).2.To explore the related mechanism of BDNF affecting the central excitability of the genioglossus muscle and the stability of the upper airway after the tongue training(1)After 8 weeks of tongue training,the m RNA levels of Trk B,ERK1/2,and CREB in the hypoglossal nucleus of the TST group increased when compared with the NC group.TST also elevated the level of Trk B,ERK,p-ERK,CREB,and p-CREB proteins.(2)Compared with the TST and TST +PBS groups,the up regulation of Trk B,ERK1/2,CREB m RNA and protein levels in the hypoglossal nucleus of the rats caused by the tongue training were weakened by blocking the function of BDNF in the hypoglossal nucleus(P <0.05).(3)Compared with the TST+AAV-EGFP group,the up-regulation of Trk B,ERK1/2,CREB m RNA and protein levels in the hypoglossal nucleus caused by the tongue training were enhanced by overexpressing of BDNF(P<0.05).(4)Compared with the TST and TST +PBS groups,the MVTF of rats decreased slightly after inhibiting ERK or CREB in the hypoglossal nucleus,and the enhancement of the TMS responsiveness of the genioglossus center caused by tongue training decreased significantly(P<0.05).At the same time,the improvement of genioglossus EMG activity and the upper airway collapsibility after TST were also weakened(P<0.05).conclusion:1.Our research showed that 8 weeks TST could gradually improve the cortical excitability of genioglossus and the genioglossus EMG activity,and ultimately enhance the stability of the upper airway in rats.2.BDNF mediated the central regulation of the genioglossus and the changes of upper airway dynamics in the TST rats.3.BDNF regulated the improvement of genioglossus central excitability and upper airway stability caused by tongue training through Tr KB-ERK-CREB pathway.