Crucial Role of the Carotid Body Chemoreceptors on the Development of High Arterial Blood Pressure During Chronic Intermittent Hypoxia

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(1)Crucial Role of the Carotid Body Chemoreceptors on the Development of High Arterial Blood Pressure During Chronic Intermittent Hypoxia. 29. Rodrigo Iturriaga, David C. Andrade, and Rodrigo Del Rio. Abstract. Exposure to chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea, produces autonomic and cardiorespirartory alterations, and leads to systemic hypertension. These alterations are associated with enhanced carotid body (CB) chemosensory and ventilatory hypoxic reflexes and a decrease baroreflex (BRS) efficiency. The aim of this study was to determine the therapeutic effect of CB ablation on the elevated arterial blood pressure, the reduced BRS and the potentiated ventilatory response induced by CIH in conscious rats. Arterial blood pressure (BP) was continuous measured by telemetry in male Sprague-Dawley rats exposed to CIH (5 % O2, 12 times/h, and 8 h/day). After 21 days of CIH, the CBs were selectively cryodestroyed, and rats were kept one more week in CIH. Ventilatory responses to hypoxia were assessed by whole body plethysmography and spontaneous BRS measured by the sequence method. Exposure to CIH produces hypertension, increased the chemoreflex ventilatory hypoxic responses, and decreased BRS. The ablation of the CBs normalized the elevated BP, and the altered ventilatory response and BRS. Present results suggest that the CB play a crucial role in the development of high arterial pressure and autonomic alterations induced by CIH. Keywords. Obstructive sleep apnea • Carotid body • Intermittent hypoxia • Chemoreflex • Baroreflex. R. Iturriaga (*) • D.C. Andrade Laboratorio de Neurobiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile e-mail: riturriaga@bio.puc.cl. R. Del Rio Laboratorio de Control Cardiorrespiratorio. Centro de Investigación Biomédica, Universidad Autónoma de Chile, Santiago, Chile. © Springer International Publishing Switzerland 2015 C. Peers et al. (eds.), Arterial Chemoreceptors in Physiology and Pathophysiology, Advances in Experimental Medicine and Biology 860, DOI 10.1007/978-3-319-18440-1_29. 255.

(2) R. Iturriaga et al.. 256. 29.1. Introduction. The obstructive sleep apnea (OSA) syndrome, a worldwide sleep-breathing disorder, is recognized as an independent risk factor for the development of systemic hypertension (Dempsey et al. 2010; Garvey et al. 2009; Somers et al. 2008). Among the disturbances produced by OSA, the chronic intermittent hypoxia (CIH) is considered the main factor for the progression of the hypertension (Dempsey et al. 2010; Iturriaga et al. 2009; Somers et al. 2008). Although the link between OSA and hypertension is well established, the pathophysiological mechanisms responsible for the hypertension are not entirely understood. It has been proposed that CIH produces oxidative stress, inflammation, and sympathetic hyperactivity, which lead to endothelial dysfunction and hypertension (Dempsey et al. 2010; Garvey et al. 2009; Iturriaga et al. 2009). However, a growing body of evidences suggests that the carotid body (CB) plays a pivotal role in the development of the hypertension. Indeed, CIH selectively enhances the CB chemosensory responsiveness to hypoxia (Del Rio el at. 2010, 2011, 2012; Pawar et al. 2009; Peng et al. 2003; Rey et al. 2004), which in turn may increase the sympathetic outflow to the arterial blood vessels (Prabhakar et al. 2005; Iturriaga et al. 2009). It has been shown that OSA patients exhibit enhanced sympathetic drive (Dempsey et al. 2010; Somers et al. 2008). Similarly, animals exposed to CIH show enhanced sympathetic responses to hypoxia, and develop systemic hypertension (Del Rio et al. 2010; Greenberg et al. 1999; Prabhakar et al. 2005; Rey et al. 2008). The autonomic dysfunction characterized by enhanced sympathetic outflow, a reduction of the efficiency of the cardiac baroreflex sensitivity (BRS) and alterations of heart rate variability has been proposed to play a pivotal role in the development of high arterial blood pressure (Rey et al. 2008; Lai et al. 2006). We hypothesized that the CIH-induced hypertension is critically dependent on the enhanced. CB chemoreflex and the decrease in BRS. To determine the contribution of the CB to the hypertension induced by CIH in conscious rats, we performed selective ablation of the CB in hypertensive rats exposed to CIH. In addition, we measured the spontaneous baroreflex (sBRS) and ventilatory responses to hypoxia.. 29.2. Methods. 29.2.1 A nimals and Intermittent Hypoxic Exposure Experiments were performed on male Sprague- Dawley rats (250 g), fed with standard diet ad libitum and kept on a 12-h light/dark schedule (07:30–19:30). The protocol was approved by the Bioethical Committee of the Biological Science Faculty of Pontificia Universidad Católica de Chile, Santiago. Unrestrained, freely moving rats housed in individual chambers (12 cm × 35 cm, 2.2 L) were exposed to 5 % inspired O2 for 20 s followed by room air for 280 s; 12 episodes/h; 8 h/day for 21 days. The hypoxic pattern was applied from 08:30 to 16:30 h. A computerize system based on solenoid valves controls the alternating cycles of N2 and room air. In the Sham condition (7 days), rats were exposed to air by air flushing an equal flow of compressed air into chambers. Room temperature was kept at 22–23 °C.. 29.2.2 Evaluation of Ventilatory Chemoreflex Function Tidal volume (VT) was determined by unrestrained whole body plethysmography (Fine Pointe, Buxco Research Systems, USA). Resting breathing was recorded for 15 min while the rats breathe room air. Peripheral chemoreceptors were stimulated by allowing the rats to breathe hypoxic gas (10 % and 15 % O2 in balance N2) for 10 min. The frequency of sampling was 1,000 Hz..

(3) 29 Crucial Role of the Carotid Body Chemoreceptors on the Development…. 29.2.3 A rterial Blood Pressure and Spontaneous Baroreflex Sensitivity. 257. 29.2.5 Statistics Analysis. We performed arterial blood pressure (BP) measurements in conscious freely moving rats using a radiotelemetry monitoring system (Data Science International, USA). Briefly, rats were anesthetized with 2 % isoflurane in O2, and a skin incision was made to expose the femoral artery. The tip of a pressure transmission catheter was guided into the femoral artery, and the transmitter body was placed into subcutaneous pocket. The sBRS was measured by sequence method (Hemolab Software). Slopes were calculated ratio to ΔR-R/ΔBP with a correlation coefficient r >0.8.. The data are expressed as means ± SEM. Differences between two groups were assessed by Student T-test comparisons. Differences between more groups were assessed with oneway ANOVA or Kruskal Wallis tests, followed by appropriate posthoc comparisons. The level was set at p < 0.05 for statistical significance. The tidal volume (VT) at different O2 levels was fitted to the following exponential curve. VT = bas + ( max − bas ) e − k / PO2. (29.1). where bas is the ventilatory response to 21 % O2 and max is the ventilatory response to 10 % O2. All statistical analysis was performed using GraphPad Prisma 6.0 (GraphPad Software, Inc., San Diego, CA, USA).. 29.2.4 Carotid Body Ablation After 21 days of CIH the rats were anesthetized with 2 % isoflurane in O2 and the CBs were exposed and cryogenically destroyed as previously described (Del Rio et al. 2013). The effectiveness of this maneuver was confirmed by the disappearance of the CB-mediated ventilatory reflex response to NaCN.. 29.3. Results. 29.3.1 E ffects of CB ablation on the CIH-Induced Hypertension The mean arterial pressure increases (~10 mmHg) after 3 days of CIH (Fig. 29.1). To determine the. 130 M A B P (m m H g ). CB ablation 120 110 100 CIH. 90 80 0. 4. 8. 12. 16. 20. 24. 28. 32. Time (days) Fig. 29.1 Effect of bilateral CB ablation on the elevated mean arterial blood pressure (MABP) in rats exposed to chronic intermittent hypoxia (CIH). n= 5.

(4) R. Iturriaga et al.. 258. contribution of the CB to the elevated BP, both CBs were destroyed. The maneuver abolished the hypertension even in the presence of the hypoxic stimulus, suggesting that the BP elevation is critically dependent on functional CBs.. (CIH), and following 7 days of the CB ablation in the presence of the hypoxic stimulus (CIH + CBA condition). The bilateral CB ablation reduced both the enhanced normoxic and hypoxic VT response induced by CIH.. 29.3.2 E ffects of CB Ablation on the Enhanced Hypoxic Ventilatory Chemoreflex Response. 29.3.3 E ffects of CB Ablation on Spontaneous Baroreflex Changes induced by CIH. Figure 29.2 shows the effects of the CB ablation on ventilatory responses (VT) measured in response to 10 % FiO2 in 4 rats before (Sham condition), after 20 days of intermittent hypoxia. We measured sBRS in rats exposed to Sham conditions, after 20 days of CIH, and after 7 days of the CB ablation. CIH reduced the sBRS sensitivity related to the Sham condition (Fig. 29.3). Notably, CB ablation increased the sBRS to. Fig. 29.2 Tidal volume (VT) measured at 10 % O2 and 21 % O2 in the same rats in Sham condition, following 20 days of intermittent hypoxia (CIH) and after 7 days after the CB ablation (CBA+ CIH). * p < 0.05 vs. Sham, CIH. n = 4. +. sBRS (% change). 120. * 80. 40. 0 Sham. CIH. CIH+CBA. Fig. 29.3 Spontaneous baroreflex sensitivity (sBRS) in Sham, (CIH) and CIH + CBA conditions. * p < 0.05 vs. Sham; + p < 0.05 vs. CIH, n = 4.

(5) 29 Crucial Role of the Carotid Body Chemoreceptors on the Development…. v alues comparable to the ones obtained in sham conditions.. 29.4. Discussion. Chronic intermittent hypoxia enhances carotid body (CB) chemosensory and ventilatory responses to hypoxia, and leads to systemic hypertension. These alterations are attributed to oxidative stress since antioxidant treatment prevented the enhanced hypoxic chemosensory and ventilatory responses and the hypertension. (Del Rio et al. 2012, 2011, 2013; Peng et al. 2003; Pawar et al. 2009). We propose that the enhanced CB chemosensory tone plays a crucial role in the maintenance of the hypertension during CIH. The role played by the CB in the development and progression of the hypertension induced by OSA is an unanswered question. The CB is involved in several sympathetic-mediated diseases and the carotid sinus denervation or the selective CB ablation has been proposed as a feasible clinical treatment for severe and resistant hypertension in humans (Mc Bryde et al. 2013; Paton et al. 2013), and the cardiorespiratory alterations in chronic heart failure (Del Rio et al. 2013). Thus, it is plausible that CB ablation will improve the cardiovascular alterations in OSA. However, there is no available information showing the effects of CB denervation in OSA patients or animals exposed to CIH. To study the origin of the CIH-induced hypertension and the contribution of the enhanced CB chemosensory response to hypoxia, we performed selective ablation of both CBs in hypertensive rats exposed to CIH. The removal of the CB inputs markedly decreases BP, suggesting that the maintenance of the hypertension following CIH is critically dependent on the intermittent hypoxic stimulation of the CB chemoreceptors, and the resulting sympathetic activation. The effectiveness of this maneuver was confirmed by the absence of a ventilatory reflex response to NaCN. Remarkably, the selective ablation of the CBs restored normal BP values comparable to the ones observed in sham rats. However, we cannot preclude that the rela-. 259. tive contribution of the CB to the hypertension will diminish if the CIH stimulus is maintained for 2–3 more weeks, whereas the contribution of the vascular compartment may increase. In summary, our data using telemetry recordings show that BP increases 3 days after the beginning of CIH, which is consistent with the time required to establish enhanced CB chemosensory responses to hypoxia and oxidative stress in the CB, both of which remained elevated for 21 days of CIH (Del Rio et al. 2010, 2012). The CIH-induced hypertension, which occurs after 3–5 days of exposure, is not related to endothelial dysfunction and/or vascular remodeling since CB ablation totally normalized BP in animals exposed to CIH. It is likely that this fast hypertensive response is caused by a higher sympathetic outflow due to the cyclic hypoxic excitation of the CB. Thus, our results indicate that the CB plays a key role in the progression and maintenance of the CIH-induced hypertension. Acknowledgements This work was supported by grant 1100405 from the National Fund for Scientific and Technological Development of Chile (FONDECYT) and the project Puente 28/2014 of the VRI-PUC.. References Del Rio R, Moya EA, Iturriaga R (2010) Carotid body and cardiorespiratory alterations in intermittent hypoxia: the oxidative link. Eur Respir J 36:143–150 Del Rio R, Moya EA, Iturriaga R (2011) Differential expression of pro-inflammatory cytokines, endothelin1 and nitric oxide synthases in the rat carotid body exposed to intermittent hypoxia. Brain Res 1395:74–85 Del Rio R, Moya EA, Parga MJ, Madrid C, Iturriaga R (2012) Carotid body inflammation and cardiorespiratory alterations in intermittent hypoxia. Eur Respir J 39:1492–1500 Del Rio R, Marcus NJ, Schultz HD (2013) Carotid chemoreceptor ablation improves survival in heart failure: rescuing autonomic control of cardiorespiratory function. J Am Coll Cardiol 62:2422–2430 Dempsey JA, Veasey SC, Morgan BJ, O’Donnell CP (2010) Pathophysiology of sleep apnea. Physiol Rev 90:47–112 Garvey JF, Taylor CT, McNicholas WT (2009) Cardiovascular disease in obstructive sleep apnoea syndrome: the role of intermittent hypoxia and inflammation. Eur Respir J 33:1195–1205.

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