Effect And Mechanism Of Tongue Muscle Training On Upper Airway Stability In Obese Rats

Background: Obstructive sleep apnea syndrome(OSAS)is mainly manifested by repeated partial or complete collapse of the upper airway during nighttime sleep,which leads to intermittent hypoxemia and intermittent sleep interruption.It can seriously endanger the public health,and is closely related to cardiovascular and cerebrovascular diseases,insulin resistance,and can also lead to the damage of cognitive function.Obesity is the first high-risk factor of OSAS and can increase the prevalence and severity of sleep apnea.This may be related to the increased collapse of the upper airway due to the abnormal function of the upper airway muscles.The previous test results of our team found that the upper airway critical pressure(Pcrt)in obese rats increased with weight gain,and in addition,obesity can also cause abnormal neuromuscular reactivity in the upper airway dilator muscle.The muscles involved in the maintenance of the upper airway tone are mainly derived from the innervation of the hypoglossal nerves.The mentocogingual muscle is the most representative tongue muscle,and the control of the sublingual motor neurons can maintain the opening and stability of the upper airway.However,during sleep,the motor output of the sublingual motor neurons decreases,leading to decreased tension in the upper airway muscles(such as the mento-lingual muscle),thus causing upper airway collapse,snoring,obstructive sleep apnea,and intermittent hypoxia.If the motor output of sublingual motor neurons can be increased and the tension of upper airway muscle group can be improved,the upper airway collapse can be effectively avoided,maintain the stability of upper airway,and then relieve snoring and reduce the occurrence of obstructive sleep breathing events.Our team’s early clinical trial confirmed that tongue muscle training can effectively alleviate the symptoms of sleep apnea and reduce the incidence of AHI.And its benefit is related to the improvement of central excitability of lingual cortex.The benefits of tongue muscle training,in addition to the structural changes of the muscle itself,can be attributed to the improvement of tongue muscle function and the central and peripheral nerve plasticity.The possible locations of neural plasticity induced by this training include: sublingual motor neuron,sublingual neuromuscular junction or lingual muscle.Synaptogenesis may also occur in the synaptic afferents of sublingual motor neurons(such as glutamatergic synaptic afferents and serotonin synaptic afferents)after tongue muscle training.Previous studies have confirmed that the improvement of central excitability in the genioglossal cortex of rats after tongue muscle training is related to the up-regulation of the expression level of brain-derived neurotrophic factor(BDNF)in the hypoglossal nucleus,and its expression in the hypoglossal nucleus can improve the stability of the airway.In addition,the increase of synaptic afferent(pentahydroxytryptaminergic synaptic afferent)of sublingual motor neurons after lingual muscle training suggests that there may be synaptogenesis,in which neurotrophic factors(such as BDNF)can be used as an important element to start the cascade reaction,which has the function of protecting neurons(improving the survival and function of neurons),thus promoting synaptogenesis.Irisin is a newly discovered secretory myogenic cytokine,which is derived from the polypeptide fragment formed by the hydrolysis of type III fibronectin component containing protein 5(FNDC5)(from the precursor protein FNDC5).Irisin is expressed in skeletal muscle,heart,tongue,rectum and other muscle tissues and organs,as well as brain tissues.FNDC5 m RNA and Irisinare widely expressed in the adipose tissue of rodents and humans,also known as adipocytokines.Iridin is involved in the treatment of metabolic diseases such as obesity and insulin resistance,and its expression has peroxisome proliferator-activated receptor γCo-activator(PGC-1 α).Dependent and exercise-induced,and the level of serum Irisin is positively correlated with its physiological function.Iridin-FNDC5 gene expression regulation has obvious exercise induction and PGC-1 α dependence.Aerobic exercise can increase the serum level of iris in non obese male patients with diabetes to twice,and the level of iris in obese individuals increases even higher.Iris FNDC5 is involved in the process of body heat production,fat conversion and obesity.From another known perspective,as a preconditioning condition,daily intermittent hypoxia stimulation(d AIH)24 can enhance respiratory plasticity by up-regulating FNDC5 and BNDF in the spinal motor nucleus.So,can the tongue muscle training,which can also increase the expression of FNDC5 and BNDF in the sublingual neuron pool,be similar to d AIH,and can be used as a precondition to strengthen the plasticity of the respiratory motor nervous system,that is,enhance its facilitation response to hypoxia?Or is it possible that this kind of lingual muscle training can only serve as an influencing factor to enhance the long-term facilitation of hypoglossal nerve? In addition,based on the important role of PGC-1α/FNDC5/Irisin system in the recovery of sensorimotor nervous system and neural plasticity,d AIH has also been proved to enhance the function of non-respiratory motor nervous system and promote the recovery of limb function?The above conjectures need to be confirmed by further experiments.Objective: This study aims to investigate the effects of different modes of tongue muscle training on genioglossus muscle function,cortical center excitability and upper airway stability in obese rats,and to determine whether obese rats up regulate peripheral and central Irisin levels through tongue muscle training,thereby improving genioglossoid muscle and its central regulation,and this effect is mediated by PGC-1α/FNDC5/Irisin pathway.Methods:1.The effects of different modes of tongue muscle training on the function of genioglossus muscle and the excitability of cortex center in obese rats,and the stability of upper airway in obese rats,and the mechanism of selecting the best mode and intensity of tongue muscle trainingForty adult male SD rats of clean grade were selected and randomly divided into obesity group and control group by random number table method.The obesity group was fed with 45% high-fat rat food for 8 weeks,and the weight of rats was 20% higher than that of the control group as the standard of obesity rats.The two groups of rats were evaluated for the functional behavior of lingual muscle,and the Pcrit,genioglossal myoelectric EMG and TMS reactivity of genioglossal motor cortex were detected.TMS and EMG changes were continuously recorded at the 5min,10 min,20min,40 min,80min and 120 min of each detection time point.The myosin heavy chain(MHC)typing of genioglossus muscle,genioglossus muscle and sternohyoid muscle were analyzed.The rats with successful modeling and obesity model were randomly divided into four groups,seven in each group,seven in the normal diet group,specifically,the normal feeding control group(Normal control,n=7),the fat control group(Fat control,n=7),the obese function training group 1(Tongue task training 1,n=7),the obese function training group 2(Tongue task training 2,n=7),and the obese strength training group(Tongue strength training,n=7);After 8 weeks of tongue muscle training,the TMS response(amplitude and latency)of the genioglossal cortex at different time points(0,2,4,8weeks)was measured by transcranial magnetic stimulation,and the dynamic characteristics of the upper airway(pcrit,Vimax,etc.)were measured by negative pressure aspiration method.After 60 days,the genioglossal muscle was killed and taken out for MHC examination by Western blot technique,and the structural changes of the genioglossal muscle were examined by projection electron microscopy.And choose the best mode and intensity of tongue muscle training.2.To explore the role of PGC-1α-FNDC5-BDNF system in mediating the excitability of genioglossal motor cortex,airway stability,and long-term facilitation of hypoglossal nerve and genioglossal muscle in obese rats with tongue muscle trainingIn this experiment,28 rats with successful obesity model were selected and randomly divided into obesity control group(Fat control,n=7),obesity training group+cerebrospinal fluid(FST,n=7),obesity training group+inhibitor(FTI,n=7),obesity training group+agonist(FTA,n=7)by random number table method;In addition to FC group,rats in other groups were injected with PGC-1 αAgonists,PGC-1 αBlocker or artificial cerebrospinal fluid micro-penetration,the localization point is located at the ventral side of the caudal nucleus of the hypoglossal nucleus in rats.After 5 days of adaptive water limit,resistance threshold,training time and daily total training threshold were gradually increased according to the preset resistance.After 8 weeks of tongue muscle training,TMS response(amplitude and latency)of genioglossal cortex at different time points(0,15,30,60 days)was measured by transcranial magnetic stimulation,and the dynamic characteristics of upper airway(pcrit,Vimax,etc.)were measured by negative pressure suction method,After 8 weeks,the genioglossus muscle was removed from the rats and the MHC was examined by Western blot,and the structural changes of the genioglossus muscle were examined by projection electron microscopy.The expression level of PGC-1α,FNDC5/Irisin,BDNF and the protein expression of AMPK,PGC-1α,FNDC5 and Irisin in the hypoglossal nucleus of rats in each group were detected;Brain stem samples and brain stem specimens were taken from all rats.The expression of PGC-1α,FNDC5,BDNF and Irisin in the hypoglossal nucleus after lingual muscle movement and the relationship between them were observed morphologically by immunohistochemistry Result:1.The effects of different modes of tongue muscle training on the mental and lingual muscle function and cortical central excitability in obese rats,and on the stability of the upper airway in obese rats,and the optimal mode and intensity of tongue muscle training were selected for mechanism discussion.(1)Obese rats increased significantly from the baseline after 8 weeks of training,and the maximal tongue extension strength increased with time.(2)Compared with obese rats,tms reactivity was reduced by prolonged latency of tms and decreased amplitude(4.47±0.24 vs.4.75 ± 0.43,p <0.05);EMG activity decreased in the mentinguentalis muscle(p <0.05).After 8 weeks of tongue muscle training,all three training modes could cause the persistent increase in TMS response(including the decrease of motor evoked potential latency and increased amplitude)in the motor cortical area of the mental and tongue muscle,and the increase in excitability could last for 120 minutes.(3)Compared with normal rats,the TMS reactivity in the motor cortical area of the motor cortex was increased and increased)after upper airway negative pressure stimulation,and the increase in excitability lasted for 120 min.(4)Compared with normal recombinant rats,in terms of skeletal muscle fiber structure,the high-fat fed obese rats had disorder,blurred sarcomeres,some mitochondria swelling,disordered mitochondrial cristae,mitochondrial membrane rupture,and lipid droplets were visible.After 8 weeks of tongue muscle training,electron microscopy of rats in the strength group showed the ultrastructure of mento-lingual muscle(mitochondrial vacuoles,etc.);(5)The proportion of MHC type 1 muscle fibers was decreased in the obese group(p<0.05),and the proportion of MHC type I muscle fibers was increased in the power and functional group 2 compared to the obese group(p <0.05).(6)Compared with normal recombinant rats,the upper airway Pcrt value in obese rats is increased,indicating that they are more likely to collapse.Compared with the obese group,both the tongue muscle strength group and the functional group 2 can cause the tendency of increasing the negative value of the upper airway closure pressure in rats,which can be reduced.2.To Explore the mediating role of PGC1a-FNDC5-BDNF system in the excitability,airway stability and the changes of the protein expression level of AMPK-PGC1a-FNDC5-Irisin pathway in the mental and tongue muscle.(1)Compared with the obese rats,the electro EMG of the agonist group peaked in the first 20 minutes,with the difference in the heart rate and respiratory rate of the rats.(2)AMPK-PGC1a-FNDC5/Irisin-BDNF pathway protein expression levels in mental-lingual muscle changed,and the level of changes was associated with lingual muscle strength training and associated with injected agonist or inhibitor.(3)After 8 weeks of lingual muscle training,the expression levels of each protein in AMPK-PGC1a-FNDC5/Irisin-BDNF pathway changed,and the level of change correlated with lingual muscle strength training and with injected agonist or inhibitor.Conclusion:1.Our research shows that the stability of the airway in obese rats is decreased,and the central regulatory ability of the genioglossus muscle is decreased2.Through 8 weeks of tongue muscle training,tongue muscle function training and tongue muscle strength training can improve the stability of upper airway and the central regulation of genioglossal muscle in obese rats;Tongue muscle strength training improved the most.3.AMPK-PGC1a-FNDC5/Irisin pathway mediated the improvement of central excitability and upper airway stability of the menti-lingual muscle in obese rats.