Cadiovascular Effect Of Intracerebroventricular Injection Of Orexin-1 Receptor Antagonist

Introduction: Orexins include orexinA and orexinB, which are also calledhypocretin 1 and hypocretin 2 respectively, are novel hypothalamic neuropeptides that were discovered in 1998.They are expressed in a population of orexinergic neurons in the lateral hypothalamus and the perifornical area (LH/PFA). Although the localization of orexin-containing cell bodies is restricted to the hypothalamus, orexin receptors and orexin immunoreactive neuronal fibres are widely distributed throughout the central nervous system, suggesting that the orexinergic neurons may play important roles in the coordination of integrated physiological systems. Two different orexin receptors have been described, the orexin-1 receptor (OX1R) and orexin-2 receptor (OX2R), both of which belong to the G-protein coupled receptor family. SB408124 is a selective OX1R antagonist, which can cross the blood-brain barrier. The biological actions of orexins are widely involved in physiological functional regulation, including feeding behavior,energy homeostasis, sleep-wake cycle, and the autonomic and neuroendocrine systems. Recently, more and more reports indicate that orexinergic system plays important role in modulation of cardiovascular activity. Orexins upon intracerebroventricular injection increased arterial blood pressure, heart rate, renal sympathetic nerve activity, and plasma catecholamines in conscious, unrestrained rats. Microinjection of orexin-A to the nucleus tractus solitarius (NTS) or the rostral ventrolateral medulla(RVLM) increased blood pressure and heart rate in anesthetized and awake rats. Orexinergic innervation was immunohistochemically detected in the NTS and RVLM. Moreover, basal blood pressure in prepro-orexin knockout mice was lower by about 20mmHg than wild-type controls. Taken together, the above observations suggest a possible contribution of orexin to the central cardiovascular regulation. Although a considerable amount of experimental work has been done in this respect, in addition to orexinA and orexinB ,their antibodies have always been selected as the research tools. so far, few experimental results have been reported in examining the effects of orexin receptor antagonists on regulating the cardiovascular activity. For this reason, the present study was done to investigate the cardiovascular effects of microinjection of selective OX1R antagonist SB408124 into the lateral intracerebroventricle in anesthetized rats and analyse the mechanism of activity underlying the cardiovascular respones in order to further clarify the complex relationship between the neuropeptides and the cardiovascular system.Objective: To study the cardiovascular effect of selective OX1R antagonist SB408124 in anesthetized rats and explore the underlying mechanism by using intracerebroventricular (I.C.V) microinjection combined with immunohistchemical assay.Methods: In the first series of experiments, in order to study the cardiovascular effect of microinjection of SB408124 into the lateral intracerebroventricle, male Sprague-Dawley rats weighing 250～300g were anesthetized with intraperitoneal injection of urethane (1.2 g/kg). A piece of metal cannular for microinjection was placed into left lateral intracerebroventricle through the skull and a catheter for measuring blood pressure was placed in left carotid artery. The changes in arterial blood pressure (ABP) and heart rate (HR) were measured and recording with RM6240CD phsiological data acquiring system for 60 minutes after I.C.V microinjection of SB408124. To determine the component of the autonomic nervous involved in cardiovascular reaction elicited by OX1R antagonist, the cardiovascular responses were re-tested after pretreatment with intravenous injection of atropine, a muscarinic receptor antagonist methyl nitrate, or hexamethonium bromide, a nicotinic receptor antagonist. Furthermore, in order to define whether the cardiovascular effects induced by SB408124 is mediated via alterations of activity of the sympathetic neurons in rat RVLM, tyrosine hydroxylase immunopositive neurons were detected with immunohistochemical assay.Results: 1.I.C.V administered SB408124 resulted in a significant decrease in MAPin anesthetized rats, which was companied with a mild decrease in HR. 2. The cardiovascular responses elicited by SB408124 were not abolished by prereatment of atropine methyl bromide whereas fully abolished by pretreatment of hexamethonium bromide. 3. The number of TH-immunopositive neurons in rat RVLM were significantly decreased following I.C.V administration of SB408124.Conclusions: I.C.V microinjection of selective OX1R antagonist SB408124 cancause decreases of MAP and HR mediated by inhibiting sympathetic activity in anesthetized rats.