| Background and ObjectiveObstructive sleep apnea (OSA) is a clinic disease with a higher incidence characterized by repetitive upper airway obstruction during sleep, obvious hypopnea or apnea and hypoxia development, which results in some serious consequences and increases the risk of other diseases, such as hypertension, myocardial infarction, stroke, sudden death, and so on. The genioglossus (GG) is the largest upper airway dilator muscle, innervated by hypoglossal nerve from hypoglossal motoneurons. Contraction of the genioglossus results in anterior movement of the tongue and widening of the oropharyngeal airway. Thus it ensures effective lung ventilation and normal respiration. A number of evidence highlighted that pathogenesis of OSA was linked to suppression of genioglossus muscle tone in sleep, especially during REM sleep. To OSA patients with the anatomical deficits present, this reduction in activity leads to a complete obstruction of the upper airway, resulting in repetitive hypoxemia in sleep. There have been reported that plasma orexin A concentrations were significantly lower in patients with OSA. Sleep apneas were frequently observed during REM sleep in orexin knockout mice. These studies suggest that abnormal regulation of orexin is associated with OSA.Orexins (orexin A and orexin B) are neuropeptides secreted by orexin neurons in hypothalamus, which involve in many important physiological process, such as feeding behavior and energy homeostasis, sleep/wake cycle, stress, regulation of cardiorespiratory system, etc. Orexin receptors (OXR) belong to a large family of guanine nucleotides regulatory (G) protein-coupled receptors. They have been categorized into two major subtypes, orexin type 1 receptor (OX1R) and orexin type 2 receptor (OX2R). Orexin A binds to these two types of receptors while orexin B mainly binds to OX2R. Hypoglossal motoneurons not only receive projection from orexin neuron but also express two subtypes of orexin recptors.To date, it is not clear how orexins control upper airway patency. There have been only few studies about the effect of orexin on genioglossus muscle and hypoglossal nerve activity. Some studies demonstrated microinjection with orexin A at hypoglossal nucleus of decerebrated cat increased GG muscle activity. Others showed orexin B microinjected in rat pontine Kolliker-Fuse nucleus enhanced preinspiratory activity of the hypoglossal nerve. However, these limited studies have demonstrated neither effect of endogenous orexins on genioglossus and the mechanisms nor the effect of orexins on genioglossus muscle in sleep/wake state. Therefore, the present study aimed to investigate how endogenous orexin regulates genioglossus muscle and hypoglossal nerve activities and the underlying mechanisms, to clarify how orexins control genioglossus activity in sleep/wake state in freely moving animals, and to observe the role of orexin neurons in hypothalamus and OXRs in medullary hypoglossal nucleus in pathogenesis of OSA by establishing chronic intermittent hypoxia (CIH) rats model to mimic OSA. We hope to provide new theories to pathogenesis and treatment of OSA.Materials and MethodsHealthy adult male SD rats were used in all experiments. In the present study, we sought to clarify the effect of orexin on genioglossus muscle by microinjection different doses of exogenous orexin A and different specific OXR antagonists on the level of hypoglossal nucleus, to observe role of endogenous orexins in genioglossus EMG and hypoglossal nerve discharge after lesions of orexin neurons in bilateral lateral hypothalamus (LH) or hypoglossal motoneurons in medulla, to investigate role of endogenous orexin on genioglossus muscle in sleep/wake states after lesions of bilateral lateral hypothalamic orexin neurons, and to observe change of genioglossus EMG and hypoglossal nerve discharge as well as expression of orexin neurons in LH and OXRs in medullary hypoglossal nucleus in a rat model of CIH treatment. Data are presented as the means±S.E.M.. Statistical analyses were performed by one-way ANOVA, paired sample t-test and independent sample t-test. A P value less than 0.05 was assumed to denote a significant difference.ResultsOur results revealed microinjection with different concentrations of orexin A (50 μmol/l,100 μmol/l,200 μmol/l,0.1 μl/each) into hypoglossal nucleus of anesthetized rat increased the integral respiratory-related GG activity (n=5, P<0.05) without affecting respiratory rate. Orexin A at 200 μmol/1 enhanced respiratory-related GG-EMG more than orexin A at 50 μmol/1 (P<0.05).Bilateral LH lesions with orexin-saporin (0.43 mg/ml,400 nl/side) significantly impaired respiratory-related GG activity (n=6,.P<0.001) and respiratory-related discharge of hypoglossal nerve (n=5, P<0.001). Bilateral hypoglossal nucleus lesions with orexin-saporin (0.43 mg/ml,200 nl/side) markedly impaired respiratory-related GG activity (n=4, P<0.001) and respiratory-related discharge of hypoglossal nerve (n=3, P<0.001).And GG EMG significantly decreased during slow wave sleep (n=4, P<0.05) either at daytime or night after bilateral LH lesions. But GG EMG during wake and REM sleep at either daytime or night had a decrease tendency without a significant difference (n=4, P>0.05)Microinjection with SB 334867 (10 mmol/l,0.1 μl), OX1R antagonist, into hypoglossal nucleus decreased respiratory-related GG activity (n=4, P<0.01). Both tonic (n=4, P<0.05) and respiratory-related GG activity (n=5, P<0.01) were markedly attenuated by microinjection with TCS PX2 29 (1 mmol/l,0.1 μl), OX2R antagonist.Both tonic (n=6, P<0.001) and respiratory-related (n=6, P<0.05) GG activity attenuated in a rat model of CIH. And respiratory-related hypoglossal nerve discharge also decreased (n=7, P<0.05). And relative optical density (ROD) of orexin neurons in LH decreased (P<0.05) in CIH model rats. Howerver, ROD of OX2R in medullary hypoglossal nucleus increased (P>0.05) and ROD of OX1R decreased. But the two results did not denote significant differences.ConclusionThe results suggested that both endogenous and exogenous orexin A enhanced GG muscle and hypoglossal nerve activity, which may be mediated by both OX1R and OX2R at the level of hypoglossal motoneurons in medulla. And endogenous orexin enhanced GG activity during slow wave sleep. CIH attenuated genioglossus muscle and hypoglossal nerve activity, which may be caused by decrease expression of orexin neurons in LH. |
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