Chronic intermittent hypoxia increases sympathetic control of blood pressure: role of neuronal activity in the hypothalamic paraventricular nucleus.

Like humans with sleep apnea, rats exposed to chronic intermittent hypoxia (CIH) experience arterial hypoxemias and develop hypertension characterized by exaggerated sympathetic nerve activity (SNA). To gain insights into the poorly understood mechanisms that initiate sleep apnea/CIH-associated hypertension, experiments were performed in rats exposed to CIH for only 7 days. Compared with sham-treated normoxic control rats, CIH-exposed rats (n = 8 rats/group) had significantly increased hematocrit (P < 0.001) and mean arterial pressure (MAP; P < 0.05). Blockade of ganglionic transmission caused a significantly (P < 0.05) greater reduction of MAP in rats exposed to CIH than control rats (n = 8 rats/group), indicating a greater contribution of SNA in the support of MAP even at this early stage of CIH hypertension. Chemical inhibition of neuronal discharge in the hypothalamic paraventricular nucleus (PVN) (100 pmol muscimol) had no effect on renal SNA but reduced lumbar SNA (P < 0.005) and MAP (P < 0.05) more in CIH-exposed rats (n = 8) than control rats (n = 7), indicating that CIH increased the contribution of PVN neuronal activity in the support of lumbar SNA and MAP. Because CIH activates brain regions controlling body fluid homeostasis, the effects of internal carotid artery injection of hypertonic saline were tested and determined to increase lumbar SNA more (P < 0.05) in CIH-exposed rats than in control rats (n = 9 rats/group). We conclude that neurogenic mechanisms are activated early in the development of CIH hypertension such that elevated MAP relies on increased sympathetic tonus and ongoing PVN neuronal activity. The increased sensitivity of Na(+)/osmosensitive circuitry in CIH-exposed rats suggests that early neuroadaptive responses among body fluid regulatory neurons could contribute to the initiation of CIH hypertension.

Central losartan attenuates increases in arterial pressure and expression of FosB/ΔFosB along the autonomic axis associated with chronic intermittent hypoxia.

Chronic intermittent hypoxia (CIH) increases mean arterial pressure (MAP) and FosB/ΔFosB staining in central autonomic nuclei. To test the role of the brain renin-angiotensin system (RAS) in CIH hypertension, rats were implanted with intracerebroventricular (icv) cannulae delivering losartan (1 µg/h) or vehicle (VEH) via miniosmotic pumps and telemetry devices for arterial pressure recording. A third group was given the same dose of losartan subcutaneously (sc). Two groups of losartan-treated rats served as normoxic controls. Rats were exposed to CIH or normoxia for 7 days and then euthanized for immunohistochemistry. Intracerebroventricular losartan attenuated CIH-induced increases in arterial pressure during CIH exposure (0800-1600 during the light phase) on days 1, 6, and 7 and each day during the normoxic dark phase. FosB/ΔFosB staining in the organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus (MnPO), paraventricular nucleus of the hypothalamus (PVN), the rostral ventrolateral medulla (RVLM), and the nucleus of the solitary tract (NTS) was decreased in icv losartan-treated rats. Subcutaneous losartan also reduced CIH hypertension during the last 2 days of CIH and produced bradycardia prior to the effect on blood pressure. Following sc losartan, FosB/ΔFosB staining was reduced only in the OVLT, MnPO, PVN, and NTS. These data indicate that the central and peripheral RAS contribute to CIH-induced hypertension and transcriptional activation of autonomic nuclei and that the contribution of the central RAS is greater during the normoxic dark phase of CIH hypertension.

Chronic intermittent hypoxia increases blood pressure and expression of FosB/DeltaFosB in central autonomic regions.

Chronic intermittent hypoxia (CIH) models repetitive bouts of arterial hypoxemia that occur in humans suffering from obstructive sleep apnea. CIH has been linked to persistent activation of arterial chemoreceptors and the renin-angiotensin system, which have been linked to chronic elevations of sympathetic nerve activity (SNA) and mean arterial pressure (MAP). Because Fos and FosB are transcription factors involved in activator protein (AP)-1 driven central nervous system neuronal adaptations, this study determined if CIH causes increased Fos or FosB staining in brain regions that regulate SNA and autonomic function. Male Sprague Dawley rats were instrumented with telemetry transmitters for continuous recording of MAP and heart rate (HR). Rats were exposed to continuous normoxia (CON) or to CIH for 8 h/day for 7 days. CIH increased MAP by 7-10 mmHg without persistently affecting HR. A separate group of rats was killed 1 day after 7 days of CIH for immunohistochemistry. CIH did not increase Fos staining in any brain region examined. Staining for FosB/ΔFosB was increased in the organum vasculosum of the lamina terminalis (CON: 9 ± 1; CIH: 34 ± 3 cells/section), subfornical organ (CON: 7 ± 2; CIH: 31 ± 3), median preoptic nucleus (CON 15 ± 1; CIH: 38 ± 3), nucleus of the solitary tract (CON: 9 ± 2; CIH: 28 ± 4), A5 (CON: 3 ± 1; CIH: 10 ± 1), and rostral ventrolateral medulla (CON: 5 ± 1; CIH: 17 ± 2). In the paraventricular nucleus, FosB/ΔFosB staining was located mainly in the dorsal and medial parvocellular subnuclei. CIH did not increase FosB/ΔFosB staining in caudal ventrolateral medulla or supraoptic nucleus. These data indicate that CIH induces an increase in FosB/ΔFosB in autonomic nuclei and suggest that AP-1 transcriptional regulation may contribute to stable adaptive changes that support chronically elevated SNA.

Induction of c-Fos and DeltaFosB immunoreactivity in rat brain by Vagal nerve stimulation.

Vagus nerve stimulation (VNS) is used as therapy for treatment-resistant depression or epilepsy. This study used immunohistochemistry for biomarkers of short-term (c-Fos) and long-term (DeltaFosB) neuronal activation to map regions in brain that are activated by acute (2 h) or chronic (3 weeks) VNS in conscious Sprague-Dawley rats. Electrodes (Cyberonics Inc.) were implanted on the left vagus nerve and 1 week after surgery, stimulation began using parameters employed clinically (one burst of 20 Hz, 250 micros pulse width, 0.25 mA stimulation for 30 s every 5 min). Radio telemetry transmitters were used for monitoring blood pressure, heart rate, activity, and respiratory rate during VNS; neither acute nor chronic VNS significantly affected these parameters. Acute VNS significantly increased c-Fos staining in the nucleus of the solitary tract, paraventricular nucleus of the hypothalamus, parabrachial nucleus, ventral bed nucleus of the stria terminalis, and locus coeruleus but not in the cingulate cortex or dorsal raphe nucleus (DRN). Acute VNS did not affect DeltaFosB staining in any region. Chronic VNS significantly increased DeltaFosB and c-Fos staining bilaterally in each region affected by acute VNS as well as in the cingulate cortex and DRN. Using these stimulation parameters, VNS was tested for antidepressant-like activity using the forced swim test (FST). Both VNS and desipramine significantly decreased immobility in the FST; whereas desipramine decreased immobility by increasing climbing behavior, VNS did so by increasing swimming behavior. This study, then, identified potential sites in brain where VNS may produce its clinical effects.

An Essential role for DeltaFosB in the median preoptic nucleus in the sustained hypertensive effects of chronic intermittent hypoxia.

One of the main clinical features of obstructive sleep apnea is sustained hypertension and elevated sympathetic activity during waking hours. Chronic intermittent hypoxia (CIH), animal model of the hypoxemia associated with obstructive sleep apnea, produces a similar sustained increase in blood pressure. This study determined the role of ΔFosB in the median preoptic nucleus (MnPO) in the sustained increase in mean arterial pressure associated with CIH. Rats were injected in the MnPO with viral vectors that expressed green fluorescent protein alone or green fluorescent protein plus a dominant-negative construct that inhibits the transcriptional effects of ΔFosB. In green fluorescent protein-injected rats and uninjected controls, 7-day exposure to CIH increased mean arterial pressure by 7 to 10 mm Hg during both intermittent hypoxia exposure and normoxia. Dominant-negative inhibition of MnPO ΔFosB did not affect changes in mean arterial pressure during intermittent hypoxia exposure but significantly reduced the sustained component of the blood pressure response to CIH during the normoxic dark phase. Inhibition of MnPO ΔFosB reduced the FosB/ΔFosB staining in the paraventricular nucleus and rostral ventrolateral medulla but not the nucleus of the solitary tract. PCR array analysis identified 6 activator protein 1-regulated genes expressed in the MnPO that were increased by CIH exposure, ace, ace2, nos1, nos3, prdx2, and map3k3. Dominant-negative inhibition of ΔFosB in the MnPO blocked increased expression of each of these genes in rats exposed to CIH except for Prdx2. ΔFosB may mediate transcriptional activity in MnPO necessary for sustained CIH hypertension, suggesting that neural adaptations may contribute to diurnal hypertension in obstructive sleep apnea.

Sex differences in blood pressure response to intermittent hypoxia in rats.

Intermittent hypoxia is used to mimic the arterial hypoxemia that occurs during sleep apnea. The present study examined the blood pressure and heart rate responses to exposure to intermittent hypoxia in male rats and in female rats before and after ovariectomy. Rats were instrumented with telemetry transmitters and blood pressure, heart rate, and activity measured during 7 days of exposure to intermittent hypoxia (3 minutes of normoxia [21% oxygen] alternating with 3 minutes 10% oxygen between 8 am and 4 pm, remainder of day at normoxia). Blood pressure increased in males, females, and ovariectomized females in response to 7 days of intermittent hypoxia during the hours of exposure to hypoxia. Blood pressure increased less in intact females (average change in blood pressure 1.6+/-0.6 mm Hg, n=11) than in females studied after ovariectomy (5.1+/-1.1 mm Hg, n=6) or males (5.4+/-1.0 mm Hg, n=10). This elevated blood pressure persisted throughout the remainder of the day when the animals were not exposed to intermittent hypoxia and remained significantly attenuated in female rats. Ovariectomy abolished the protection against the elevated blood pressure response to intermittent hypoxia in females. Heart rate increased only in males, and only during the period of the day associated with intermittent hypoxia. Female rats were protected against this tachycardia independent of the ovarian hormones. These results indicate that females are protected from the hypertensive and tachycardia effects of intermittent hypoxia.

Nitric oxide modulation of glutamatergic, baroreflex, and cardiopulmonary transmission in the nucleus of the solitary tract.

The neuromodulatory effect of NO on glutamatergic transmission has been studied in several brain areas. Our previous single-cell studies suggested that NO facilitates glutamatergic transmission in the nucleus of the solitary tract (NTS). In this study, we examined the effect of the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) on glutamatergic and reflex transmission in the NTS. We measured mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) from Inactin-anesthetized Sprague-Dawley rats. Bilateral microinjections of L-NAME (10 nmol/100 nl) into the NTS did not cause significant changes in basal MAP, HR, or RSNA. Unilateral microinjection of (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA, 1 pmol/100 nl) into the NTS decreased MAP and RSNA. Fifteen minutes after L-NAME microinjections, AMPA-evoked cardiovascular changes were significantly reduced. N-methyl-D-aspartate (NMDA, 0.5 pmol/100 nl) microinjection into the NTS decreased MAP, HR, and RSNA. NMDA-evoked falls in MAP, HR, and RSNA were significantly reduced 30 min after L-NAME. To examine baroreceptor and cardiopulmonary reflex function, L-NAME was microinjected at multiple sites within the rostro-caudal extent of the NTS. Baroreflex function was tested with phenylephrine (PE, 25 microg iv) before and after L-NAME. Five minutes after L-NAME the decrease in RSNA caused by PE was significantly reduced. To examine cardiopulmonary reflex function, phenylbiguanide (PBG, 8 microg/kg) was injected into the right atrium. PBG-evoked hypotension, bradycardia, and RSNA reduction were significantly attenuated 5 min after L-NAME. Our results indicate that inhibition of NOS within the NTS attenuates baro- and cardiopulmonary reflexes, suggesting that NO plays a physiologically significant neuromodulatory role in cardiovascular regulation.

Effect of nitric oxide on excitatory amino acid-evoked discharge of neurons in NTS.

N-methyl-d-aspartate (NMDA) and non-NMDA excitatory amino acid (EAA) receptor subtypes are involved in the integration of visceral afferent inputs within the nucleus of the solitary tract (NTS). Microinjection studies indicate interactions between nitric oxide (NO) and EAA receptors within the NTS. To examine these interactions at the single cell level, this study characterized the effects of the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) and the NO donor 3-[2-hydroxy-2-nitroso-1-propylhydrazino]-1-propanamine (PAPA-NONOate) on the excitatory responses of vagus nerve (VN)-evoked NTS neurons to the activation of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA receptors in rats. Iontophoresis of l-NAME did not alter spontaneous or VN-evoked discharges, but significantly decreased the number of action potentials (APs) evoked by iontophoretic application of AMPA. The effects of l-NAME on NMDA-evoked discharge were variable; for the population, l-NAME did not change the number of APs evoked by NMDA. PAPA-NONOate enhanced the spontaneous discharge and the number of APs elicited by AMPA but not NMDA. Iontophoresis of the inactive enantiomers N(G)-nitro-d-arginine methyl ester and hydroxydiazenesulfonic acid 1-oxide disodium salt had no effect on AMPA-evoked discharge. Our data suggest that NO facilitates AMPA-mediated neuronal transmission within the NTS.