| Objective:OSA is a common disorder characterized by repetitive partial or complete upper airway collapse causes sleep fragmentation and intermittent hypoxemia and is often associated with increased risk of motor vehicle accidents,excessive daytime sleepiness,impaired cognitive performance,and reduced quality of life.Besides,OSA is proved to be a strong and independent risk factor for cardiovascular and cerebrovascular disease,together with the associated all-cause mortality,the need for effective long-term treatment of OSA is urgent.In recent years,both animal and human studies have shown the capacity of tongue exercise to benefit tongue function.Notably,tongue exercise enhances GG corticomotor excitability,facilitate upper airway patency and alleviate sleep apnea syndrome.Progressive resistance exercise of tongue has been shown to have beneficial effects on tongue muscle strength and a trend toward an increase in muscle fiber size.there are also evidence revealing that exercise-induced augmentation in 5HT input to the hypoglossal nucleus.A characteristic finding is that exercise increased 5-HT and BDNF in hypoglossal nucleus,an area involved in controlling upper airway muscle dilator and stiffness.Given that BDNF is a molecular important for synaptic plasticity,we proposed that these exercise-induced increases in hypoglossal nucleus might underlie the ability of exercise to enhance respiratory neural plasticity.As a member of the neurotrophin family,BDNF is known to play a prominent role in the survival,growth,and maintenance of neurons during development and also the ability to modulate synaptic-plasticity in the adult brain,which can be seen in diverse tasks it manages.Respiratory long-term facilitation(LTF)is a long-lasting enhancement of respiratory activity following repeated exposure to respiratory stimulation.As a typical ideal model of respiratory plasticity,LTF is proposed to be an important protective mechanism of respiratory homeostasis during sleep,particularly in patients who suffer from obstructive sleep apnea.Previously,studies have found that LTF in phrenic/XII motor output requires new synthesis of BDNF,and activation of its high affinity receptor tyrosine,TrkB.Respiratory LTF exhibits metaplasticity,since preconditioning with chronic intermittent hypoxia(CIH)(7 days)or daily exposure to acute intermittent hypoxia(dAIH)enhanced phrenic and XII LTF.We proposed that these exercise-induced increases in hypoglossal nucleus might underlie the ability of exercise to enhance respiratory neural plasticity,namely XII LTF,or respiratory metaplasticity.It is of significant clinical significant to determine if tongue exercise utilizes BDNF action to exert its beneficial effects in respiratory neuronal plasticity,as exercise may be potentially combined with other interventions which might impact BDNF-mediated mechanism.Furthermore,most UA muscle training model in previous research were focused on the strength gaining.The progressive resistance exercise of tongue was deemed as an efficiency way to increase the maximal voluntary contraction and neuroplasticity.Although this traditional method gained some therapeutic effect,it inevitably increased the physical cross-sectional area of the muscle,which further narrowed the upper airway.Thus,this method has limitations.Considering that the activity level of the neuralmuscular system plays an important role in the increment of muscle strength and intramuscular coordination.Then,in order to maximize the muscle training-induced neuroplasticity,new elements should be added into the training program,so as to increase the recruitment of the hypoglossal motor activity and the corticomotor control of the tongue.This study aimed at investigate the effect of two different tongue training model on the tongue corticomotor control and upper airway dynamics and its associated underlying mechanism,using techniques such as transcranial magnitic stimulation,upper aiway critical accessment,genioglossus EMG activity,etc.Based on the animal experimental results,tongue training might be evolved into a novel therapeutic program,the latter of which needs criteria to target on the OSA patients with appropriate indications,such as those with the upper airway muscle dysfunction.In this view,it is important to establish a set of assessment tools to evaluate the tongue mechanical properties and screening those with tongue dysfuntion.The group of patients with tongue dysfuntion is more likely to benefit more from the tongue training therapy.Thus,the final part of the experiment will investigate the mechanical performance(ie.strength,endurance,fatigability and strength stability)of tongue muscle using elevation and protrusion tasks in patients with a wide range of severities of obstructive sleep apnea events.Methods:1.The Different Effect of Tongue Motor Task Training(TTT)and Strength Training(ST)on the genioglossus(GG)ultrastructure and function,the modulation of GG Corticomotor Excitability and EMG activity in response to tongue traning and AIH,as well as upper airway dynamics changes in Rats18 water-restricted adult male Sprague-Dawley rats were trained to lick an isotonic force-sensing disc at targeting forces for water reinforcement with sound feedback.In TTT group,30-50%of maximal protrusion force(MPF)was used as target window,while the ST paradigm used a progressive resistance training program with targeting force set above50%,60%and 70%of MPF,progressively.The rats in both groups were trained daily for two months,with the same training intensity(1000 g·s per day)quantified by total tongue muscle work.GG EMG activity and Corticomotor excitability evaluated by motor evoked potentials(MEPs)in GG muscle evoked by transcortical magnetic stimulation(TMS)was assessed at four sessions:at baseline before training,every 10 minutes within 2 hours after the 4,6 and 8 weeks training.The GG Corticomotor Excitability and EMG activity in response to AIH was also measured dynamicly for an hour.After 8 weeks,the upper airway dynamic changes were assessed among different groups.At the end of experiment,all rats were killed,and the genioglossus(GG)of rats was isolated to assess the Myosin heavy chain(MHC)isoforms using western blot.The rest of the GGs were rapidly removed and sliced into several tissue pieces at the volume of about 1.0 mm3 for each.They were fixed in 2.5%glutaraldehyde phosphate buffer.Samples were examined under transmission electron microscope.2.To examine the levels of 5HT2AR,PKC,BDNF and TrkB in the hypoglossal nucleus of rats among different groups,as well as the effect of tongue task training on serotonergic activation of hypoglossal motoneurons.After 8 weeks,they were then anesthetized,vagotomized and paralyzed.The dorsal medulla was exposed,and 5-HTP(5mM,20nl)or 5-HT2A receptor antagonist ketanserin(1mM,3x40nl)were microinjected into the XII nucleus while genioglossus electromyographic(EMG)activity was recorded.The EMG-root mean square(RMS)values was measured before and at different times after microinjections.At the end of the experiment,all rats were killed,and the expression of 5HT2AR,PKC,BDNF and Trk B in the hypoglossal nucleus were detected by immunohistochemistry.3.Assessment of tongue mechanical properties using different contraction tasksThis study also assessed tongue muscle strength,strength stability,endurance time,fatigue indices and total muscle work,using elevation and protrusion tasks with repetitive isometric fatiguing contractions in 12 normal+mild,17 moderate and 11 severe patients with OSA,and to assess the influence of body mass index(BMI)and age.Results:1.The Different Effect of Tongue Motor Task Training(TTT)and Strength Training(ST)on the genioglossus(GG)ultrastructure,GG mitochodrial membrane potential,as well as the modulation of GG Corticomotor excitability and EMG activity in response to tongue traning and AIH,together with the upper airway dynamics changes in Rats(1)After 8 weeks,the precision of task performance(percent of peak force inside target window)increased in TTT-treated rats(P=0.04).(2)After 4 weeks in both groups,MEPs latencies of genioglossus evoked by TMS decreased immediately after training and reached the valley 20 minutes after training compared to baseline(3.8±0.3 ms vs.4.8±0.2ms;3.9±0.4 ms vs.4.7±0.1 ms for TTT and ST,respectively).This excitatory effect lasted up to 80 minutes in TTT-treated rats,but it rebound above the baseline value after 20minutes and reached the apex at 80 minutes in ST group(5.3±0.4 ms).After 8 weeks in both groups,MEPs latencies consistently decreased within the 2 hours after training.(3)After 8 weeks,the Pcrit tend to decrease in TTT group,when compared with the normal control.(4)Regarding the GG ultrastructure,TST group revealed disordered structure of the myofibril,such as massive lysis,vacuolar degeneration and necrosis of myofilaments,proliferation of connective tissue among myofibril bundles,aggregated and edematous mitochondria with a variety of sizes of myofibrils,disruption,and lysis of mitochondria cristae,while this above change was not obvious in TTT group.(5)The mitochondrial membrane electrical potential(??m)measurements revealed that the relative value of??m was significantly lower in TST group,when compared with control.(6)Western blot revealed a increase of MHC I isoform in TST group.2.To examine the levels of 5HT2AR,PKC,BDNF and TrkB in the hypoglossal nucleus of rats among different groups,as well as the effect of tongue task training on serotonergic activation of hypoglossal motoneurons.(1)The immunoreactivity of 5HT2AR,PKC,BDNF and TrkB in the hypoglossal nucleus of rats was imfluenced by two models of tongue training approaches.(2)The excitatory effect of 5-HTP on the tonic genioglossus EMG activity was similar in sham-and TTT-treated rats within 20 minutes after injection,while this excitability maintained for 40 minutes only in TTT-treated rats.In contrast,the tonic genioglossus EMG activity was more profoundly reduced 30 minutes following ketanserin injections in TTT-than sham-treated rats[36%±7%(SD)vs.78±11%,P<0.05]without significant changes in central respiratory rate or heart rate.3.Assessment of tongue mechanical properties using different contraction tasks(1)Endurance time was longer in protrusion than elevation task(P=0.01).(2)In both tasks,endurance time was negatively correlated with baseline value of strength coefficient of variation(P<0.01).(3)Compared to other groups,patients with moderate OSA had the lowest total muscle work for protrusion(P=0.01),and shortest endurance time(P=0.04)regardless of type of task.(4)Additionally,in patients with moderate-severe OSA,the total muscle work for both tasks was lower in non-obese compared to obese(P<0.05).Total muscle work for protrusion was positively correlated with apnea hypopnea index(AHI)in obese subjects(P<0.01).(5)Endurance time was shorter(P<0.01)and recovery time was longer(P=0.02)in the old compared to young subjects.Conclusions:1.Tongue task training can increase the precision of task performance and is associated with stable increment of corticomotor excitability in a shorter period of time,while the strength training rats might suffer fatigue at early stages of training.2.strength training could induce muscle damage,which might give rise to peripheral fatigue at early stage of training.3.Consistent with increased neuroanatomic measures of serotonergic innervation of XII nucleus following tongue exercise,ketanserin microinjections revealed a stronger endogenous serotonergic excitatory drive to genioglossus activity in TTT-than sham-treated anesthetized rats.4.The tongue is more prone to fatigue during the elevation task and in patients with moderate OSA.Obesity appeared to prevent alteration of tongue mechanical properties in patients with OSA.Baseline strength stability and endurance were related,illustrating the role of central neuromuscular output in tongue resistance to fatigue.5.These animal and human experiment results could have implications for treatment paradigms related to enhancement of central neuromuscular output of tongue for OSA patients with compromised tongue function. |
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