Effects Of Hypoglossal Nucleus 5-HT Receptor On The Central Control Of Genioglossus In Chronic Intermittent Hypoxic Rats

Objective:Obstructive Sleep Apnea,OSA)is mainly caused by recurrent upper airway collapse during sleep and intermittent hypoxia resulting therefrom,but does not occur when the patient awakens.Foreign data show that the prevalence rate of OSA among adults is 2%-4%,and the epidemiological survey results of many provinces and cities in China show that the prevalence rate of OSA is about 4%.OSA is a systemic disease,which can cause a variety of target organ damage:hypertension,coronary heart disease,arrhythmia,heart failure,type 2 diabetes。Mechanism of OSA:When a person inhales,the gas passes through the nose,pharynx,and trachea and eventually enters the lungs.The important mechanism of keeping the pharyngeal cavity open in OSA patients lies in the increased contractile activity and tension of the inspiratory phase of the upper airway dilator muscles,while in the sleep state the whole body muscle activity and neuromuscular reflexes are weakened,and as the force of keeping the upper airway open decreases or disappears,collapse and closure are more likely to occur under the action of negative thoracic pressure,which leads to airflow restriction.Prolonged recurrent apnea hypoventilation can lead to intermittent hypoxemia,resulting in interrupted sleep and thus awakening.Therefore,it is important to keep the upper airway open in patients with OSA.Factors that maintain upper airway opening include upper airway osseous structures,upper airway neuromuscular regulation,and negative airway pressure.The bony structure is difficult to intervene,and the negative airway pressure is difficult to regulate,so our main focus is on the neuromuscular regulation of the upper airway.As the main dilator of the upper airway,the genioglossuse is innervated by the hypoglossal nerve from the nucleus accumbens,and its related transmitters include 5-HT and norepinephrine.Current studies suggest that dysfunction of the genioglossuse may play an important role in the pathogenesis of OSA.The function of genioglossus is closely related to the development of OSA.A large number of experiments have confirmed that the intermittent hypoxia caused by apnea and hypoventilation in OSA can damage the function of the upper airway dilator muscle,and at the same time,the damage of muscle function can lead to further increase of upper airway collapsibility,forming a vicious circle,which eventually makes the condition of OSA patients gradually aggravated and leads to adverse consequences.Chronic intermittent hypoxia is the most important and representative pathophysiological feature of obstructive sleep apnea.It is currently believed that abnormal function of upper airway dilator muscles can lead to OSA,while chronic intermittent hypoxia caused by OSA may impair the function of upper airway dilator muscles,and chronic intermittent hypoxia may also cause compensatory effects of upper airway dilator muscles.The effect of this pathophysiological process of CIH on thegenioglossus and its possible mechanisms are discussed.Anatomically,the genioglossus is mainly innervated by the hypoglossal nerve（HN）,which is the efferent fiber of the hypoglossal nerve,and excitation of the HN causes contraction of the genioglossuse,thus opening the pharyngeal cavity.The serotonergic neurons located in the hypoglossal nerve are involved in the composition of the motor neurons of the upper airway dilator muscle,and 5-HT excites the hypoglossal nerve to increase the muscle tone of the genioglossus.5-HT responses are dependent on the presence of multiple 5-HT receptors in the presynaptic and postsynaptic membranes of the neurons.Intermittent hypoxia induced an increase in the density of 5-HT and noradrenergic terminals and an increase in the expression of 5-HT₂ Receptor（5-hydroxytryptamine 2 Receptor,5-HT₂R）in the hypoglossal nucleus of rats.5-HT₂R is mainly divided into three subtypes,5-HT_2A,5-HT_2B and 5-HT_2C,which have similar molecular structures and pharmacological effects.Among them,the gene polymorphism of 5-HT_2AR(5-hydroxytryptamine 2A Receptor,5-HT_2AR)is associated with OSA.So,is the CIH-induced increase in response in the motor cortex of the chin tongue muscle mediated by 5-HT_2AR in the hypoglossal nucleus?What is the specific pathway through which 5-HT_2AR in the hypoglossal nucleus regulates the central response of the genioglossuse under CIH conditions?PKC is an intracellular factor that can be activated by 5-HT_2AR and is highly expressed in neural tissue.Experiments have shown that in vivo experiments 5-HT induces long-lasting vulnerability in the hypoglossal nucleus via the 5-HT_2AR-mediated PKC pathway.However,experiments have also shown that 5-HT_2AR antagonists only partially eliminate5-HT from causing apnea.Nerves and skeletal muscle interact through two modes:electrical activity and neurotrophic regulation.Nerve impulses generated in the central nervous system trigger muscle contraction through electromechanical coupling.On the other hand,neurotrophic control acts through the release of neurotrophic factors（including neurotrophic factors）and regulates the development,differentiation,survival and function of nerve endings.The most widely studied neurotrophic factor is the brain-derived neurotrophic factor（BDNF）.BDNF is initially synthesized as the precursor pro BDNF,which is cleaved by intracellular or extracellular proteases into the mature isoform m BDNF.Both isoforms induce different or even opposite functions by preferentially binding to the low-affinity nerve growth factor receptor（p75）or the tropomyosin-related kinase B receptor（TrkB）induces different or even opposite functions.The high-affinity receptor of BDNF,TrkB receptor,plays an important role in the cascade of responses that cause long-lasting chemotaxis,and TrkB receptor activation induces a wide range of downstream signaling,including ERK/MAP kinases.Experiments have shown that in chronic intermittent hypoxia,TrkB receptor blockers can inhibit the long-range vulnerability of thegenioglossuss,and that CIH induces an increase in 5-HT_2AR-dependent BDNF synthesis in the cervical medullary diaphragm nucleus,while activation of PKC induces an increase in BDNF,with PKC playing an important role in 5-HT_2A→BDNF.In the cascade of responses that cause long-lasting diaphragmatic vulnerability,the high-affinity receptor of BDNF,TrkB receptor,plays an important role and TrkB receptor activation induces a wide range of downstream signaling,including ERK/MAP kinases,causing long-lasting diaphragmatic vulnerability.Therefore,we hypothesize that the central regulation of the genioglossus by serotonergic neurons in the hypoglossal nucleus acts through the 5-HT-5-HT_2AR-PKC pathway-BDNF/TrkB pathway.Methods:1.Effects of chronic intermittent hypoxia on the structure,function and central control of rat genioglossusSixteen male Wistar rats（SPF grade）were divided into two groups according to the random number table:air control group（NO）and chronic intermittent hypoxia group（CIH）.The CIH group was given hypoxic treatment and was placed in a hypoxic chamber（8 hours/day）from 8:00 to 16:00 every day.The hypoxic mode was:10%oxygen concentration for 45 seconds,21%oxygen concentration for 60 seconds,188Seconds/loop.On the 1st,7th,14th and 21st day of the experiment,transcranial magnetic stimulation was used to measure the reactivity（amplitude and latency）of the genioglossus motor cortex at different time points in the CIH group and the NO group,and the genioglossus muscle was recorded..At the end of the experiment,the genioglossus tissue of each group was taken for the preparation and observation of electron microscopy,the measurement of mitochondrial membrane potential and the analysis of genioglossus muscle fiber typing..2.Effects of 5-HT_2A receptor in the hypoglossal nucleus on the structure,function and central control of rat genioglossus under chronic intermittent hypoxiaEstablishment of chronic intermittent hypoxic model and hypoxic conditions As described above,24 male Wistar rats（SPF grade）were divided into three groups according to random number table:simple chronic intermittent hypoxia group（CIH）,chronic intermittent low Oxygen was administered to the solvent PBS group（CIH+PBS,CPBS group）and chronic intermittent hypoxia to the 5-HT_2Areceptor antagonist（MDL100907）group（CIH+MDL,CMDL group）.One week before the start of chronic intermittent hypoxia,the hypoglossal nucleus was located according to the rat brain stereotactic map（The Rat Brain in Stereotaxic Coordinates,6th Ed）.The position was:13.6 mm after the anterior iliac crest,depth 9.0 mm（AP:-13.6 mm.DV:9.0 mm）.Hypoxia was started on the 7th day after surgery,and the concentration of 5-HT_2Areceptor antagonist was 1 m M daily at a dose of 60 nl.The latency and amplitude of the EMG of the rats in each group and the TMS response of the genioglossus cortex were measured on days 1,7,14,and 21 of the experiment,respectively.The recording method is the same as before.3、Expression levels of various proteins in the hypoglossal nucleus 5-HT_2Areceptor-PKC-BDNF/Trk pathway under chronic intermittent hypoxiaThe brain stem of each group of rats was prepared and immunohistochemically prepared.The expression of 5HT_2AR,PKC,BDNF and TrkB proteins in the hypoglossal nucleus was observed.Results:1.The effect of chronic intermittent hypoxia on the structure,function and central control of rat genioglossus muscle.1)Compared with the air group,the electron microscopy of the genioglossus muscle in chronic intermittent hypoxic rats showed that the ultrastructure of the genioglossus muscle was destroyed（the light and dark bands disappeared,the mitochondrial arrangement was disordered,the mitochondrial vacuoles changed,the intima disappeared,and the ridge Phenomenon such as increase in number,etc.）.2)Compared with the air group,the mitochondrial membrane potential of the genioglossus in chronic intermittent hypoxic rats decreased.Compared with the air group,the MHC type II of the genioglossus increased after 2 and 3 weeks of chronic intermittent hypoxia（P<0.05）,and MHC type I muscle fibers were significantly reduced on days 14and 21（P<0.05）.3)Compared with the air group,with the progress of chronic intermittent hypoxia,the myoglossal muscle myoelectricity gradually increased（P<0.05）.After reaching two weeks,no further increase and maintained at a high potential.The TMS response in the motor cortex of the genioglossus muscle of chronic intermittent hypoxic rats was enhanced on days 1,7,14,and 21（P<0.05）.2.The effect of 5-HT_2A receptor in the hypoglossal nucleus on the regulation of the genioglossus of rats under chronic intermittent hypoxia.1)Compared with simple chronic intermittent hypoxic rats,administration of5-HT2AR antagonist MDL100907 in the hypoglossal nucleus can cause a decrease in the motor cortical area response of the genioglossus muscle,which is manifested as a prolonged latency of motor evoked potentials（P<0.05）.2)Compared with simple chronic intermittent hypoxic rats,there was no significant change in the genioglossus myoelectric activity and genioglossus central reactivity under the condition of intermittent hypoxia given to the solvent PBS group in the hypoglossal nucleus area（P>0.05）.3.Expression levels of various proteins in the hypoglossal nucleus 5-HT_2Areceptor-BDNF-TrkB pathway under chronic intermittent hypoxia1)Compared with the normal group,the number of 5HT_2AR immunoreactive cells after 21 days of chronic intermittent hypoxia was increased in the tail of the hypoglossal nucleus（ventral side）（P<0.05）;while in the tail of the hypoglossal nucleus（dorsal）Side)（P>0.05）;there is no significant change in the expression of the hypoglossal nucleus in the head（dorsal/ventral side）（P>0.05）.In the chronic intermittent hypoxic group,the mean optical density value of 5HT_2AR protein positive expression in the ventral part of the hypoglossal nucleus of the rat increased,and there was no significant change on the1st and 7th days of CIH（P>0.05）,on the 14th and 21st days Days gradually increased and reached the highest on the 21st day（P<0.05）.Under the condition of chronic intermittent hypoxia,after antagonizing 5HT_2AR of hypoglossal nucleus,the average optical density value of 5HT_2AR protein positive expression on the ventral side of the tail of hypoglossal nucleus of rats was compared with that in air group on the 1st and 7th days of CIH There was no significant change（P>0.05）,which was significantly reduced on days 14 and 21 of CIH（P<0.05）,but there was no significant change compared with simple chronic intermittent hypoxia（P>0.05）.2)Compared with the air group and chronically intermittent hypoxic group,there was no significant difference in the expression of PKC on the ventral side of the hypoglossal nucleus compared with the control group（P>0.05）.Compared with the air group,the mean optical density value of the positive expression of BDNF protein in the ventral part of the tail of the hypoglossal nucleus in the chronic intermittent hypoxic group was clearly unchanged at CIH 1,7 but increased on days 14 and 21（P<0.05）.Under chronic intermittent hypoxic conditions,compared with the simple hypoxic group,after antagonizing 5-HT_2AR of the hypoglossal nucleus,BDNF protein expression water decreased on days 14 and 21 of CIH（P<0.05）.Compared with the air group,the mean optical density value of TrkB protein positive expression in the ventral portion of the tail of the hypoglossal nucleus of the chronic intermittent hypoxic group was clearly reduced on days 1,7,14,and 21 of the CIH（P<0.05）.Under chronic intermittent hypoxic conditions,after antagonizing 5-HT_2AR of the hypoglossal nucleus,BDNF protein expression water increased on days 1,7,14,and 21 of CIH（P<0.05）.Conclusions:1.Chronic intermittent hypoxia can directly damage the structure and function of the genioglossus muscle,which is manifested as structural disorder,reduced fatigue resistance,and reduced energy metabolism efficiency.Intermittent hypoxia can occur within 2 weeks of injury,and persists with chronic intermittent hypoxia.Chronic intermittent hypoxia can cause increased excitability of the motor cortical center of the genioglossus muscles and increased electrical activity of the genioglossus muscles.The hypoglossal nucleus 5-HT_2AR participates in the changes of the motor cortex of the genioglossus muscle caused by chronic intermittent hypoxia and plays an exciting role.2.The hypoglossal nucleus 5-HT_2AR participates in the central regulation of the genioglossus muscle in chronic intermittent hypoxic rats and excites the motor cortex area of the genioglossus muscle.3.5-HT_2AR of hypoglossal nucleus of rats participates in the central regulation of genioglossus muscle of chronic intermittent hypoxic rats through the BDNF-TrkB pathway.