Valdir A. Braga

Increased sympathetic outflow in juvenile rats submitted to chronic intermittent hypoxia correlates with enhanced expiratory activity

Chronic intermittent hypoxia (CIH) in rats produces changes in the central regulation of cardiovascular and respiratory systems by unknown mechanisms. We hypothesized that CIH (6% O2 for 40 s, every 9 min, 8 h day−1) for 10 days alters the central respiratory modulation of sympathetic activity. After CIH, awake rats (n= 14) exhibited higher levels of mean arterial pressure than controls (101 ± 3 versus 89 ± 3 mmHg, n= 15, P < 0.01). Recordings of phrenic, thoracic sympathetic, cervical vagus and abdominal nerves were performed in the in situ working heart–brainstem preparations of control and CIH juvenile rats. The data obtained in CIH rats revealed that: (i) abdominal (Abd) nerves exhibited an additional burst discharge in late expiration; (ii) thoracic sympathetic nerve activity (tSNA) was greater during late expiration than in controls (52 ± 5 versus 40 ± 3%; n= 11, P < 0.05; values expressed according to the maximal activity observed during inspiration and the noise level recorded at the end of each experiment), which was not dependent on peripheral chemoreceptors; (iii) the additional late expiratory activity in the Abd nerve correlated with the increased tSNA; (iv) the enhanced late expiratory activity in the Abd nerve unique to CIH rats was accompanied by reduced post‐inspiratory activity in cervical vagus nerve compared to controls. The data indicate that CIH rats present an altered pattern of central sympathetic–respiratory coupling, with increased tSNA that correlates with enhanced late expiratory discharge in the Abd nerve. Thus, CIH alters the coupling between the central respiratory generator and sympathetic networks that may contribute to the induced hypertension in this experimental model.

Involvement of l‐glutamate and ATP in the neurotransmission of the sympathoexcitatory component of the chemoreflex in the commissural nucleus tractus solitarii of awake rats and in the working heart–brainstem preparation

Peripheral chemoreflex activation with potassium cyanide (KCN) in awake rats or in the working heart–brainstem preparation (WHBP) produces: (a) a sympathoexcitatory/pressor response; (b) bradycardia; and (c) an increase in the frequency of breathing. Our main aim was to evaluate neurotransmitters involved in mediating the sympathoexcitatory component of the chemoreflex within the nucleus tractus solitarii (NTS). In previous studies in conscious rats, the reflex bradycardia, but not the pressor response, was reduced by antagonism of either ionotropic glutamate or purinergic P2 receptors within the NTS. In the present study we evaluated a possible dual role of both P2 and NMDA receptors in the NTS for processing the sympathoexcitatory component (pressor response) of the chemoreflex in awake rats as well as in the WHBP. Simultaneous blockade of ionotropic glutamate receptors and P2 receptors by sequential microinjections of kynurenic acid (KYN, 2 nmol (50 nl)−1) and pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonate (PPADS, 0.25 nmol (50 nl)−1) into the commissural NTS in awake rats produced a significant reduction in both the pressor (+38 ± 3 versus+8 ± 3 mmHg) and bradycardic responses (−172 ± 18 versus−16 ± 13 beats min−1; n= 13), but no significant changes in the tachypnoea measured using plethysmography (270 ± 30 versus 240 ± 21 cycles min−1, n= 7) following chemoreflex activation in awake rats. Control microinjections of saline produced no significant changes in these reflex responses. In WHBP, microinjection of KYN (2 nmol (20 nl)−1) and PPADS (1.6 nmol (20 nl)−1) into the commissural NTS attenuated significantly both the increase in thoracic sympathetic activity (+52 ± 2%versus+17 ± 1%) and the bradycardic response (−151 ± 17 versus−21 ± 3 beats min−1) but produced no significant changes in the increase of the frequency of phrenic nerve discharge (+0.24 ± 0.02 versus+0.20 ± 0.02 Hz). The data indicate that combined microinjections of PPADS and KYN into the commissural NTS in both awake rats and the WHBP are required to produce a significant reduction in the sympathoexcitatory response (pressor response) to peripheral chemoreflex activation. We conclude that glutamatergic and purinergic mechanisms are part of the complex neurotransmission system of the sympathoexcitatory component of the chemoreflex at the level of the commissural NTS.

Adipokines, diabetes and atherosclerosis: an inflammatory association.

Cardiovascular diseases can be considered the most important cause of death in diabetic population and diabetes can in turn increase the risk of cardiovascular events. Inflammation process is currently recognized as responsible for the development and maintenance of diverse chronic diseases, including diabetes and atherosclerosis. Considering that adipose tissue is an important source of adipokines, which may present anti and proinflammatory effects, the aim of this review is to explore the role of the main adipokines in the pathophysiology of diabetes and atherosclerosis, highlighting the therapeutic options that could arise from the manipulation of these signaling pathways both in humans and in translational models.

Angiotensin-II-induced reactive oxygen species along the SFO-PVN-RVLM pathway: implications in neurogenic hypertension

Neurogenic hypertension has been the subject of extensive research worldwide. This review is based on the premise that some forms of neurogenic hypertension are caused in part by the formation of angiotensin-II (Ang-II)-induced reactive oxygen species along the subfornical organ-paraventricular nucleus of the hypothalamus-rostral ventrolateral medulla pathway (SFO-PVN-RVLM pathway). We will discuss the recent contribution of our laboratory and others regarding the mechanisms by which neurons in the SFO (an important circumventricular organ) are activated by Ang-II, how the SFO communicates with two other important areas involved in sympathetic activity regulation (PVN and RVLM) and how Ang-II-induced reactive oxygen species participate along the SFO-PVN-RVLM pathway in the pathogenesis of neurogenic hypertension.

Sympathoexcitatory response to peripheral chemoreflex activation is enhanced in juvenile rats exposed to chronic intermittent hypoxia

In the present study, we tested the hypothesis that chronic intermittent hypoxia (CIH) produces changes in the autonomic and respiratory responses to acute peripheral chemoreflex activation. To attain this goal, 3‐week‐old rats were exposed to 10 days of CIH (6% O2 for 40 s at 9 min intervals; 8 h day−1). They were then used to obtain a working heart–brainstem preparation and, using this unanaesthetized experimental preparation, the chemoreflex was activated with potassium cyanide (0.05%, injected via the perfusion system), and the thoracic sympathetic nerve activity (tSNA), heart rate and phrenic nerve discharge (PND) were recorded. Rats subjected to CIH (n= 12), when compared with control animals (n= 12), presented the following significant changes in response to chemoreflex activation: (a) an increase in tSNA (78 ± 4 versus 48 ± 3%); (b) a long‐lasting increase in the frequency of the PND at 20 (0.52 ± 0.03 versus 0.36 ± 0.03 Hz) and 30 s (0.40 ± 0.02 versus 0.31 ± 0.02 Hz) after the stimulus; and (c) a greater bradycardic response (−218 ± 20 versus−163 ± 16 beats min−1). These results indicate that the autonomic and respiratory responses to chemoreflex activation in juvenile rats previously submitted to CIH are greatly increased.

Dietary salt enhances angiotensin-II-induced superoxide formation in the rostral ventrolateral medulla

We investigated the association of dietary salt and angiotensin-II infusion on hypertension and superoxide formation in the RVLM. Male Wistar rats were subcutaneously infused with Ang-II (150ng/kg/min) or saline (0.9% NaCl) for 14days. In addition, rats had free access to drinking water containing 0.4% or 2.0% NaCl. On the 15th day, rats that received Ang-II+0.4% NaCl exhibited higher levels of baseline arterial blood pressure than rats that received saline+0.4% NaCl (118+/-5mm Hg vs 98+/-4mm Hg, n=9, P<0.05). Rats that received Ang-II+2% NaCl had a significantly greater hypertension compared to Ang-II+0.4% NaCl (165+/-7mm Hg vs 118+/-5mm Hg, n=10, P<0.05). On the other hand, rats treated with saline+2% NaCl or saline+0.4% NaCl did not become hypertensive (96+/-5mm Hg, n=8 and 98+/-4mm Hg, n=7, respectively). Furthermore, administration of hexamethonium (30mg/kg i.v.) evoked larger decreases in mean arterial pressure in rats treated with Ang-II+2% NaCl and rats treated with Ang-II+0.4% NaCl (-100+/-5 Deltamm Hg and -72+/-10 Deltamm Hg, P<0.05). The magnitude of superoxide formation measured by the dihydroethidium technique in the RVLM was greater in the RVLM of rats treated with Ang-II+2% NaCl (123+/-10 Delta%, P<0.05%), than with Ang-II+0.4% (67+/-9 Delta%) and saline+2% NaCl (5+/-3 Delta%,). The findings indicate that dietary salt potentiates Ang-II-derived superoxide formation in the RVLM, resulting in a more severe hypertension. We suggest that this effect could be mediated by an increase in inputs within the forebrain-PVN-RVLM axis.

Acute superoxide scavenging restores depressed baroreflex sensitivity in renovascular hypertensive rats.

In some pathological conditions such as hypertension, there is an impairment in the autonomic control of blood pressure resulting in changes in baroreflex sensitivity. In the present study we tested the hypothesis that acute superoxide scavenging would restore the reduced baroreflex sensitivity in renovascular hypertension. Male Wistar rats underwent 2-Kidney-1-Clip (2K1C) or sham surgery and were maintained untouched for six weeks to develop hypertension. After six weeks, animals from the 2K1C group were hypertensive when compared to the sham group (165±9 vs. 108±7mm Hg, P<0.05). As a proof of principle for the hypertension model adopted, animals from the 2K1C group presented increased non-clipped kidney and cardiac mass index and reduced clipped kidney mass index. Regarding baroreflex, 2K1C rats presented diminished baroreflex sensitivity when compared to the sham group (2K1C+saline: -1.61±0.15 vs. sham+saline: -2.79±0.24bpm mm Hg(-1), p<0.05). Moreover, acute administration of Vitamin C (150mg/Kg, i.v.) restored baroreflex sensitivity in 2K1C rats (2K1C+Vit C: -3.08±0.37 vs. 2K1C+saline: -1.61±0.15bpm mm Hg(-1), p<0.05). Furthermore, administration of apocynin (30μg/Kg, i.v.), a NADPH oxidase inhibitor, also improved baroreflex sensitivity in the 2K1C group (2K1C+apocynin: -2.81±0.24 vs. 2K1C+saline: -1.61±0.15bpm mm Hg(-1), p<0.05). In addition, autonomic blockade with either methylatropine or propranolol reduced the changes in heart rate to the same extent in all groups suggesting that improved baroreflex sensitivity by antioxidants were mediated by improvement in autonomic function. Taken together, these data suggest that NADPH oxidase-derived reactive oxygen species are involved in the blunted baroreflex sensitivity in renovascular hypertension and that acute scavenging of superoxide restores baroreflex sensitivity.

In Vivo Bioluminescence Imaging Reveals Redox-Regulated Activator Protein-1 Activation in Paraventricular Nucleus of Mice With Renovascular Hypertension

Renovascular hypertension in mice is characterized by an elevation in hypothalamic angiotensin II levels. The paraventricular nucleus (PVN) is a major cardioregulatory site implicated in the neurogenic component of renovascular hypertension. Increased superoxide (O2−·) production in the PVN is involved in angiotensin II–dependent neurocardiovascular diseases such as hypertension and heart failure. Here, we tested the hypothesis that excessive O2−· production and activation of the redox-regulated transcription factor activator protein-1 (AP-1) in PVN contributes to the development and maintenance of renovascular hypertension. Male C57BL/6 mice underwent implantation of radiotelemeters, bilateral PVN injections of an adenovirus (Ad) encoding superoxide dismutase (AdCuZnSOD) or a control gene (LacZ), and unilateral renal artery clipping (2-kidney, one-clip [2K1C]) or sham surgery. AP-1 activity was longitudinally monitored in vivo by bioluminescence imaging in 2K1C or sham mice that had undergone PVN-targeted microinjections of an Ad encoding the firefly luciferase (Luc) gene downstream of AP-1 response elements (AdAP-1Luc). 2K1C evoked chronic hypertension and an increase in O2−· production in the PVN. Viral delivery of CuZnSOD to the PVN not only prevented the elevation in O2−· but also abolished renovascular hypertension. 2K1C also caused a surge in AP-1 activity in the PVN, which paralleled the rise in O2−· production in this brain region, and this was prevented by treatment with AdCuZnSOD. Finally, Ad-mediated expression of a dominant-negative inhibitor of AP-1 activity in the PVN prevented 2K1C-evoked hypertension. These results implicate oxidant signaling and AP-1 transcriptional activity in the PVN as key mediators in the pathogenesis of renovascular hypertension.

Cardiovascular Responses to Peripheral Chemoreflex Activation and Comparison of Different Methods to Evaluate Baroreflex Gain in Conscious Mice Using Telemetry

Peripheral chemoreceptors located in the carotid bodies are the primary sensors of systemic hypoxia. Although the pattern of responses elicited by peripheral chemoreceptor activation is well established in rats, lambs, and rabbits, the cardiovascular responses to peripheral chemoreflex activation in conscious mice have not been delineated. Here we report that stimulation of peripheral chemoreceptors by potassium cyanide (KCN) in conscious mice elicits a unique biphasic response in blood pressure that is characterized by an initial and robust rise followed by a decrease in blood pressure, which is accompanied by a marked reduction in heart rate. The depressor and bradycardic responses to KCN were abolished by muscarinic receptor blockade with atropine, and the pressor response was abolished by alpha-adrenergic receptor blockade with prazosin, suggesting that vagal and sympathetic drive to the heart and sympathetic drive to the vasculature mediate these cardiovascular responses. These studies characterized the chemoreflex in conscious mice and established the reliability of using them for studying hypoxia-related diseases such as obstructive sleep apnea. In another series of experiments, two methods for analyzing baroreflex sensitivity were compared: the classical pharmacological approach using phenylephrine and sodium nitroprusside (i.e., the Oxford technique) or the sequence method for analyzing spontaneous baroreflex activity. Our findings indicate that both methods are reliable, and the sequence method certainly has its benefits as a predictive tool in the context of long-term noninvasive studies using telemetry. However, for absolute determination of baroreflex function, analysis of spontaneous baroreflex activity should be complemented by the classical pharmacological method.

Autonomic and respiratory responses to microinjection of ATP into the intermediate or caudal nucleus tractus solitarius in the working heart-brainstem preparation of the rat.

1. Activation of peripheral chemoreceptors with KCN in the working heart–brainstem preparation from young male Wistar rats (70–90 g) increases phrenic (PNA; +105 ± 18%) and thoracic (tSNA; +44 ± 6%) sympathetic nerve activity compared with baseline and reduces heart rate (HR; from 377 ± 27 to 83 ± 6 b.p.m.).