The Effects And Mechanisms Of Repeated Transcranial Magnetic Stimulation On The Upper Airway Stability In Obese Rats

Objective: Obstructive sleep apnea(OSA)seriously harms public health and is closely related to cardiovascular and cerebrovascular diseases and insulin resistance.In addition,it can also cause cognitive impairment such as anxiety and depression disorder.OSA patients are mainly manifested by repetitive partial or complete collapse of the upper airway(UA)during nightly sleep,which leads to intermittent hypoxemia and sleep fragmentation.The activity of the upper airway dilator muscles is an important force to against the collapsibility of upper airway and maintains UA patency.The genioglossus plays the most critical role in UA muscles.The contraction functional shortage of the upper airway dilator muscle during sleep is an important cause of apnea events.Compared with normal people,the genioglossus EMG activity in OSA patients is compensatively increased during wakefulness which plays a major role in maintaining the patency of the upper airway,and the deficiency of such compensation during sleep causes apnea events.Excessive fat accumulation of neck related obesity can cause the upper airway stenosis and anatomical abnormalities which ultimately increase the collapsibility of the upper airway and the risk for sleep apnea.Transcranial magnetic stimulation(TMS)can be used to explore the central regulation of the genioglossus.TMS response of the genioglossus motor cortex is enhanced in OSA patients during wakefulness,but reduced during sleep especially during apnea.It suggests that the waking-related increased central regulation is hypothesized as a compensatory effect to maintain the upper airway patency for OSA patients while awake.Our research team confirmed that this compensatory effect is related to serotonin(5-HT)and norepinephrine(NE)system through a series experiments in rats.The genioglossus is mainly innervated by hypoglossal nerve and hypoglossal nucleus,the motor neurons in the hypoglossal nucleus are further regulated by serotonergic neurons in raphe nucleus and noradrenergic neurons in brainstem A7 and nucleus locus by stimulating 5-HT2 A and ?1-adrenergic receptor on hypoglossal nucleus.Studies suggested that 5-HT and norepinephrine participate in the hypoglossal nerve discharging activity together,and the simultaneous inhibition of 5-HT2 A receptor and ?1-adrenergic receptor of the hypoglossal nucleus can significantly reduce the genioglossus activity in rats.Conversely,the activation of these two receptors can significantly increase the activity of the genioglossus and improve the collapsibility of the upper airway.TWIK-related acid-sensitive potassium channel(TASK-1)is widely distributed in brainstem motor neurons,especially in the hypoglossal nucleus and sensitive to hypoxia and changes of PH value,and TASK-1 channel participates in the regulation of the respiratory system by initiating hypoxia/chemistry sensitive mechanisms.Serotonergic and noradrenergic neurotransmitters have a significant inhibitory effect on TASK-1 channel current.The decrease in the release of such neurotransmitters during sleep will reduce the inhibitory effect to TASK-1 and further decreased the excitability of neurons,suggesting that TASK-1 could participate in the regulation of respiratory center.And the abnormal activity of TASK-1 channel may be the intrinsic reason for the dysfunction of the chemical reflex of respiratory center and genioglossus contraction function.Therefore,we hypothesize that serotonergic and noradrenergic inputs of the hypoglossal nucleus can regulate the central reactivity of the genioglossus through the TASK-1 channel and affect the stability of the upper airway.The occurrence of apnea events during sleep is closely related to the increase in the collapsibility of the upper airway caused by the decompensation of the genioglossus,so whether there are any methods could maintain the stability of the upper airway by enhancing this compensatory effect? Repetitive transcranial magnetic stimulation(r TMS)technology is a painless and harmless physical stimulation technology developed on the basis of TMS.r TMS in the primary motor cortex of brain can cause contraction of target muscles,and this effect on brain function will continue for some time after the finish of stimulation.r TMS has been widely applied to movement disorders caused by many reasons.Researchers applied this physical therapy technique to OSA patients,it suggested that increased output of magnetic stimulation within a certain range could increase the TMS responses of the cortical area and respiratory enhancement effects unconsciously.This indicates that r TMS is expected to become a new physical therapy strategy to enhance the genioglossus muscle activity and maintain the stability of the upper airway.Therefore,this study would explore the effects of repeated transcranial magnetic stimulation on the central responses of the genioglossus motor cortex,EMG activity and upper airway stability in obese rats,and the possible mechanisms of these effects.It may help provide a new physical method and idea for the therapy of OSA patients.Methods: 1.The effects of repeated transcranial magnetic stimulation on the stability of upper airway in obese rats.In the determination of magnetic stimulation parameters,adult male Wistar rats(24)were randomly divided into high-frequency group(10Hz),low-frequency group(1Hz)and sham-stimulation group according to the random number table method.The rats of every group were subjected to corresponding magnetic stimulation parameters.TMS responses of genioglossus in each group rats were recorded on the 0th,1st,3rd,5th,and 6th days of repetitive transcranial magnetic stimulation(r TMS)to select the possible efficient frequency of magnetic stimulation;In the modeling part of obese rats,5 week old male SPF Wstar rats(130-150g)were randomly divided into obese group(n = 8)and control group(n = 8).Rat model of nutritional obesity were made by feeding high-fat diets with 45% fat content.Weighing more than 20% of the control group rats was used as the criterion for successful modeling of obese rats.The TMS responses,genioglossus myoelectric activity(EMG)and upper airway critical pressure(Pcrit)values of two groups were recorded;high-fat-induced obese rat model was made according to the above method,obese rats were randomly divided into r TMS group(n = 8)and sham-stimulation group(n = 8).TMS responses,EMG and Pcrit values of two groups were recorded on the 0th,7th and 14 th days of magnetic stimulation to verify the influence of r TMS on the upper airway obese rats.2.Effects of 5-HT/5-HT2 A receptor,NE/?1 adrenergic receptor and TASK-1 on the stability of upper airway in r TMS-treated rats.Obese rats were randomly divided into r TMS group(n = 8),artificial cerebrospinal fluid group(r TMS + ACSF group,n = 8),5-HT2 A receptor antagonist group(r TMS + MDL group,n = 8)),?1 adrenergic receptor antagonist group(r TMS + Prazosin group,n = 8),r TMS + MDL + Prazosin group(n = 8),r TMS + MDL + Prazosin + TASK-1 antagonist group(r TMS + MDL + Prazosin + ML365,n = 8).Localization and verification of the hypoglossal nucleus region in obese rat were operated according to the stereotactic atlas of the rat brain and related references.After the catheter was successfully implanted in the hypoglossal nucleus region,rats in the experimental group were given specific antagonists and the control group was given the same dose of ACSF.TMS responses,EMG and Pcrit values of every group were recorded on day 0,7,and 14 of r TMS to verify whether serotonergic/ noradrenergic inputs and TASK-1 participate in the regulation of r TMS on the upper airway of obese rats.Obese rats(n = 12)were randomly divided into r TMS group(n = 6)and sham stimulation group(n = 6.The rats were sacrificed at the end of the experiment.The hypoglossal nucleus region of the brainstem was used for immunohistochemistry of 5-HT/5-HT2 A receptors,DBH/?1A receptors and TASK-1 channel proteins.The differences in immunohistochemistry staining results between two groups at different time-points were detected.Results: 1.The effects of repeated transcranial magnetic stimulation on the stability of upper airway in the obese rats.1.1 High-frequency repetitive transcranial magnetic stimulation can increase the TMS responses of the genioglossus motor cortex in rats(P <0.05).1.2 Compared with normal weight group,the TMS responses of the genioglossus in obese rats were reduced,manifested by the extended latency and decreased amplitude(P <0.05);the EMG activity of the genioglossus were decreased P <0.05);Nutritional obesity increased the Pcrit value of upper airway in rats,and made the upper airway more likely to collapse(P <0.05).1.3 High-frequency repetitive transcranial magnetic stimulation increased the TMS responses of the genioglossus in obese rats(P <0.05),enhanced the EMG activity of the genioglossus(P <0.05),and improved the stability of the upper airway in obese rats(P <0.05).2.Effects of 5-HT/5-HT2 A receptor,NE/?1 adrenergic receptor and TASK-1 on the stability of upper airway in r TMS-treated rats.2.1 High-frequency repetitive transcranial magnetic stimulation increased central reactivity and EMG activity in obese rats.The microinjection of 5-HT2 A receptor antagonist(MDL)in the hypoglossal nucleus inhibited this positive regulatory effect(P < 0.05).2.2 The microinjection of ?1 adrenergic receptor antagonist(Prazosin)in the hypoglossal nucleus inhibited the positive effect of high frequency repetitive transcranial magnetic stimulation on the central regulation of the genioglossus in obese rats and reduced the TMS responses,EMG activity and the collapsibility of upper airway(P <0.05).2.3 Simultaneous administration of 5-HT2 A receptor antagonist and ?1 adrenergic receptor antagonist in the hypoglossal nucleus significantly reduced the positive effects of high-frequency r TMS on central regulation of genioglossus and upper airway stability in obese rats,increased the collapsibility of upper airway in obese rats(P <0.05).2.4 The microinjection of TASK-1 blocker after 20 minutes of administeration with dual antagonist(MDL + Prazosin)in the hypoglossal nucleus improved the inhibitory effects of 5-HT2 A receptor antagonist and ?1 adrenergic receptor antagonist on the central regulation of genioglossus in high-frequency r TMS rats(P <0.05),but there were no synergistic effects in two drugs.2.5 High-frequency r TMS increased the number of serotonergic and noradrenergic terminals of the hypoglossal nucleus caudal and rostral region in obese rats(P <0.05).2.6 High frequency r TMS increased the expression of 5-HT2 A receptors,noradrenergic ?1 receptors and TASK-1 channel protein in the rostral/caudal region of hypoglossal nucleus in obese rats(P <0.05).Conclusions: 1.High frequency repetitive transcranial magnetic stimulation can increase the central regulation of the genioglossus,reduce the collapsibility of the upper airway and improve the stability of the upper airway in obese rats.2.The positive effect of high frequency repetitive transcranial magnetic stimulation on the upper airway stability in obese rats is related to the 5-HT2 A receptor and ?1 adrenergic receptor in the hypoglossal nucleus;high frequency repetitive transcranial magnetic stimulation could influence the expression levels of 5-HT/5-HT2 A receptor, noradrenergic terminals/?1A receptor and TASK-1 channel protein in the hypoglossal nucleus in obese rats.