PVN ‐ projecting MnPO neurons are involved in blood pressure and sympathetic nerve activity regulation in CIH hypertension

Abstract

Sleep apnea (SA) is characterized by repeated interruptions in breathing resulting in hypoxia, hypercapnia, and increased thoracic pressure. Chronic intermittent hypoxia (CIH) in rats mimics the repetitive hypoxemias that occur with SA leading to a sustained increase in blood pressure associated with increased sympathetic activity and chemoreceptor sensitivity. CIH increases the peripheral renin-angiotensin system (RAS) activating the subfornical organ (SFO) which in turn projects to the hypothalamic paraventricular nucleus (PVN) and the median preoptic nucleus (MnPO). Angiotensin receptor knockdown in SFO or MnPO neurons and virally –mediated caspase lesions PVN-projecting MnPO neurons prevents CIH hypertension. These results suggest that PVN-projecting MnPO neurons play a significant role in blood pressure regulation in CIH hypertension. However, the contribution of MnPO to increased sympathetic nerve activity in CIH hypertension has not been directly tested. Our hypothesis is that the chronic activation of PVN by SFO via MnPO contributes to increased sympathetic outflow sustaining CIH hypertension through the diurnal cycle. In order to selectively inhibit PVN-projecting MnPO neurons, a retrograde Cre-dependent virus was microinjected bilaterally into PVN (AAV9.hSyn.HI.eGFP-Cre.WPRE.SV40) and a Cre-dependent inhibitory DREADD (PAAV.Syn.DIO.hM4D(Gi)-mCherry) was injected into MnPO. Two weeks later, rats were submitted to a 7-day CIH protocol (3 min of 10% oxygen followed by 3 min of 21% oxygen, 8 h/day) or normoxia. Rats were anesthetized (urethane 800 mg/kg and α-chloralose 80 mg/kg ip) and the trachea was cannulated for artificial ventilation. A femoral vein catheter (PE-50 tubing) was implanted for continuous infusion of gallamine triethiodide (20 mg/mL, 0.25 mL/h iv) for neuromuscular blockade. Blood pressure was measured by a Millar catheter system in the femoral artery. The rats were placed in a stereotaxic frame and a craniotomy was performed for intracerebroventricular (ICV) injections. Renal and splanchnic nerves were isolated and placed in a bipolar electrode. Noise levels were determined after a bolus injection of hexamethonium (30 mg/kg iv). Using a pneumatic picopump (WPI) connected to a single-barreled glass micropipette, DREADD agonist CNO (1µg/1µl) was injected ICV and responses were collected after 30 min of the injection. In rats exposed to normoxia, chemogenetic inhibition of PVN-projecting MnPO neurons slightly increased splanchnic SNA (4.9±21, n=3), renal SNA (17± 17, n=3) and increased blood pressure (Normoxia: 4.3±4, n=3; CIH; -0.3± 4, n=3). In rats exposed to CIH, CNO reduced splanchnic SNA (-42.9±37, n=4) and renal SNA (-88±49, n=3). These preliminary results suggest that CIH triggers a neuroadaptation that changes the relationship between the SFO-MnPO-PVN pathway and SNA.