The Effect Of Noradrenergic Neurons In A7 Cell Group On The Central Regulation Of Genioglossus In Chronic Intermittent Hypoxic Rats

Objective:Obstructive sleep apnea?OSA?is a common respiratory and sleep related disease characterized by recurrent upper airway collapse and chronic intermittent hypoxia?CIH?,during sleep,but not during wakefulness.Upper airway patency depends on the balance between negative airway pressure and neuromuscular activities,in which the genioglossus plays the most critical role.In normal situations,the genioglossus is modulated by central nervous system through the hypoglossal nerve.The modulation has been proved to be influenced by rapid eye movement?REM?sleep and hypoxia.Under long-term hypoxia,the central regulation of genioglossus is compensatively increased to defend upper airway collapse in OSA patients during wakefulness.However,during REM sleep,the loss of this compensatory effect will lead to a more easily collapse of upper airway,which will furtherly aggravate the disease progression.Both 5-hydroxytryptamine?5-HT?and norepinephrine in the hypoglossal nucleus can affect the activity of hypoglossal nerve during sleep,in which norepinephrine may account for nearly 90%.Our research team has conducted a series of experiments to investigate how 5-HT and norepinephrine participate in the central regulation of genioglossus under normoxia and hypoxia condition in rats.We found that the serotonergic neurons in raphe nucleus may involved in the central regulation of genioglossus in CIH rats,whose effects increased with the prolongation of hypoxia.Microinjection of prazosin??₁-adrenergic receptors antagonists?in hypoglossal nucleus could significantly decrease the transcranial magnetic stimulation?TMS?response of genioglossus.This result suggested that norepinephrine can provide a positive drive to the central regulation of genioglossus through?₁-adrenergic receptors on hypoglossal nucleus.The effect can be significantly enhanced by CIH.All these conclusions indicated that norepinephrine plays an important role in the central regulation of genioglossus.Serotonergic neurons in raphe nucleus activate hypoglossal nerve and genioglossus by stimulating 5-HT_1AA receptors on hypoglossal nucleus.As a result,we suspected that the norepinephrine secreted to hypoglossal nucleus is also derived from noradrenergic neurons from upper central nervous system.And the neurons may also participate in the central regulation of genioglossus in CIH rats.Anatomical evidence shows that the noradrenergic terminals distributed in the hypoglossal nucleus mainly originated from the A5,LC,A7and A1/C1 noradrenergic cell group in brainstem.And the A7 noradrenergic cell group provided the relativity highest density of innervation to the hypoglossal nucleus compared with other noradrenergic cell groups.Receptor located on cell membranes which can bind to norepinephrine are called adrenergic receptors and belong to G protein-coupled receptors.The results showed that?₂-adrenergic receptors were mainly expressed in A7noradrenergic neurons.As a self-receptor,?₂-adrenergic receptors,which can negatively regulate norepinephrine release by pre-synaptic feedback.G protein-gated inwardly rectifying potassium channel?GIRK?is widely distributed in the central nervous system.GIRK channels are usually involved in mediating the inhibitory effect of neurotransmitters on postsynaptic,and can be regulated by various neurotransmitters such as norepinephrine.Western blotting results showed that the expression of GIRK channels were positive in A7region.Therefore,we hypothesize that noradrenergic neurons in A7 region may participate in the central regulation of genioglossus and be regulated by?_2A-adrenergic receptor on noradrenergic neurons through GIRK channels.CIH has been proved to involve in the central compensation regulation of genioglossus.However,it is still unclear that how norepinephrine participates in the central regulation of genioglossus in CIH rats.The aim of this study is to investigate detailed mechanism of noradrenergic neurons in A7 region participate in the central regulation of genioglossus.In this experiment,CIH model were established to simulate the characteristics of repeated intermittent hypoxia in OSA patients.The temporal changes of electromyogram?EMG?,TMS and related receptors were observed to explore the possible mechanism at different time points of hypoxia.It may helpfully provide a new theoretical basis for clinical diagnosis and follow-up treatment of OSA patients.Methods:1.The effect of noradrenergic neurons,?₂-adrenergic receptors and GIRK channels in A7 cell group on the central regulation of genioglossus under normoxia condition in ratsAll experiments were carried out using SPF adult male Wistar rats?290-310g?in Section One.All animals were randomly divided into normal control or normoxic group?NO group,n=8?,IgG-SAP microinjection group?IgG group,n=8?,Anti-DBH-SAP microinjection group?SAP group,n=8?,normal saline microinjection group?NACL group,n=8?,Clonidine microinjection group?CLO group,n=8?,RS-79948 microinjection group?RS group,n=8?,ML297 microinjection group?ML group,n=8?,Tertiapin-Q microinjection group?TER group?,Tertiapin-Q/Clonidine microinjection group?TC group,n=8?and ML297/RS-79948 microinjection group?MR group,n=8?.According to previous researches,the distribution of A7 norepinephrine neurons were defined by immunohistochemistry and immunofluorescence.0.6%agarose tissue phantoms were prepared to stimulate the drugs diffusion characteristics of rat brain tissue in vivo.And the microinjection sites of A7 norepinephrine neurons were verified by stereotaxic apparatus.Anti-DBH-saporin?a noradrenergic neuron lesioning agent?,RS-79948??₂-adrenoceptor antagonists?,clonidine??₂-adrenoceptor agonist?,tertiapin-Q?GIRK channel blocker?,and ML297?GIRK channel activator?were microinjected into the A7 region of Wistar rats.Equivalent dosage of IgG-SAP or normal saline was microinjected as control group.TMS and EMG was recorded in specific time to investigate the effect of norepinephrine neurons,?₂-adrenergic receptors and GIRK channels on the central regulation of genioglossus after rats recovered from surgery.2.The effect and mechanism of noradrenergic neurons and?₂-adrenergic receptors in A7 cell group on the central regulation of genioglossus under intermittent hypoxia condition in ratsAll experiments were carried out using SPF adult male Wistar rats?290-310g?in Section Two.To establish the chronic intermittent hypoxia model,all rats were placed in oxycycler with 188 s cycle of normoxia?21%O2 for 60 s?and hypoxia?10%O2 for 45 s?,8 h/day?from 8:00 a.m.to 4:00 p.m.?for consecutive four cycles of 28 days.All rats subjected to chronic intermittent hypoxia were divided into simple CIH group?CIH group,n=32?,IgG-SAP microinjection group under CIH condition?CIH+SAP group?,Anti-DBH-SAP microinjection group under CIH condition?CIH+SAP group,n=8?,normal saline microinjection group under CIH condition?CIH+NACL group,n=8?,Clonidine microinjection group under CIH condition?CIH+CLO group,n=8?and RS-79948microinjection group under CIH condition?CIH+RS group,n=8?.According to previous researches,Anti-DBH-saporin?a noradrenergic neuron lesioning agent?was microinjected in A7 region of CIH+SAP group.Equivalent dosage of IgG+SAP was microinjected as control group.RS-79948??₂-adrenoceptor antagonists?and clonidine??₂-adrenoceptor agonist?was respectively microinjected in the A7 region of CIH+RS and CIH+CLO group on the day of the experiment.Equivalent dosage of normal saline was microinjected as control group.After recovering from the surgery,TMS and EMG was recorded on the 7^th,14^th,21^st and 28^th day to investigate the effect of noradrenergic neurons and?₂-adrenergic receptors on the central regulation of genioglossus under intermittent hypoxia condition in rats.At each time point of intermittent hypoxia,the rats in CIH group were deeply anesthetized and intracardially perfused with cold 4%paraformaldehyde.The brains were removed,postfixed,cryoprotected,embedded and sectioned.The expression of dopamine?hydroxylase?DBH?in A7 noradrenergic neurons and noradrenergic terminals,?_1A-adrenergic receptors and?_2A-adrenergic receptors in hypoglossal nucleus were detected by freezing section immunohistochemistry.The aim of the experiment was to investigate the possible mechanism which A7 noradrenergic neurons participate in the central regulation of genioglossus under intermittent hypoxia condition in rats.Results:1.The effect of noradrenergic neurons,?₂-adrenergic receptors and GIRK channels in A7 cell group on the central regulation of genioglossus under normoxia condition in rats1.1 Under normoxia condition,A7 noradrenergic neurons participated in and exerted an excitatory effect on the central regulation of genioglossus.Creating lesions in the A7 cell group noradrenergic neurons may induce weaker EMG activities,longer TMS latencies and lower TMS amplitudes?p<0.05?.1.2 Under normoxia condition,?₂-adrenergic receptors on A7 noradrenergic neurons participated in and exerted an inhibitory effect on the central regulation of genioglossus.Activating?₂-adrenergic receptors may induce weaker EMG activities,longer TMS latencies and lower TMS amplitudes?p<0.05?.1.3 Under normoxia condition,GIRK channels on A7 noradrenergic neurons participated in and exerted an inhibitory effect on the central regulation of genioglossus.Activating GIRK channels may induce weaker EMG activities,longer TMS latencies and lower TMS amplitudes?p<0.05?.1.4 Under normoxia condition,the?₂-adrenergic receptors located in A7 cell group were functionally linked to GIRK channels,and this pathway participated in the central regulation of genioglossus.2.The effect and mechanism of noradrenergic neurons and?₂-adrenergic receptors in A7 cell group on the central regulation of genioglossus under intermittent hypoxia condition in rats2.1 Under intermittent hypoxia condition,A7 noradrenergic neurons participated in and functioned as an excitatory drive on the central regulation of genioglossus.The excitatory effect lasted 28 days.Creating lesions in the A7 cell group noradrenergic neurons may induce longer TMS latencies and lower TMS amplitudes?p<0.05?.2.2 Under intermittent hypoxia condition,?₂-adrenergic receptors on A7 noradrenergic neurons participated in and functioned as an inhibitory drive on the central regulation of genioglossus.The inhibitory effect lasted 28 days.Acting?₂-adrenergic receptors may induce longer TMS latencies and lower TMS amplitudes,and lower EMG activities?p<0.05?.2.3 Under intermittent hypoxia condition,the expression of DBH increase in A7noradrenergic neurons?p<0.05?.But no difference in the number of neurons was detected by freezing section immunohistochemistry?p>0.05?.2.4 Under intermittent hypoxia condition,the distribution of noradrenergic terminals increased in hypoglossal nucleus?p<0.05?.But no difference in the mean density was detected by freezing section immunohistochemistry?p>0.05?.2.5 Under intermittent hypoxia condition,the expression of?_1A-adrenergic receptors increased in hypoglossal nucleus?p<0.05?.But no difference in the number of positive neurons was detected by freezing section immunohistochemistry?p>0.05?.2.6 Under intermittent hypoxia condition,the expression of?_2A-adrenergic receptors was decreased in hypoglossal nucleus?p<0.05?.But no difference in the number of positive neurons was detected by freezing section immunohistochemistry?p>0.05?.Conclusion:1.Under normoxia condition,A7 norepinephrine neurons participated in and exerted an excitatory effect on the central regulation of genioglossus.?₂-adrenergic receptors and GIRK channels were functionally coupled and effected as an inhibitory effect on central regulation of genioglossus.2.Under intermittent hypoxia condition,A7 noradrenergic neurons participated in and functioned as an excitatory drive on the central regulation of genioglossus,while?₂-adrenergic receptors exerted the opposite effect.3.Chronic intermittent hypoxia can influence the DBH expression in A7 noradrenergic neurons,the distribution of noradrenergic terminals in hypoglossal nucleus and the expression?_1A-adrenergic and?_2A-adrenergic receptors expression in hypoglossal nucleus.