Age protects from harmful effects produced by chronic intermittent hypoxia

J Physiol 594.6 (2016) pp 1773–1790 1773 The Journal of Physiology Age protects from harmful effects produced by chronic intermittent hypoxia M. Quintero1,5,E.Olea 1,5,S.V.Conde 3,A.Obeso 1,5, T. Gallego-Martin1,5,C.Gonzalez 1,5, J. M. Monserrat4,5,A.G´ omez-Ni˜ no2,5,S.Yubero 1,5 and T. Agapito1,5 1Department of Biochemistry and Molecular Biology and Physiology, School of Medicine, University of Valladolid and IBGM/CSIC, Valladolid, Spain 2Department of Cell Biology, Histology and Pharmacology, School of Medicine, University of Valladolid and IBGM/CSIC, Valladolid, Spain 3Chronic Diseases Research Center (CEDOC), Nova Medical School, Faculdade de Ciˆ encias M´ edicas, University of Nova Lisboa, Lisbon, Portugal 4Laboratori de la Son, Pneumologia, Hospital Cl´ ınic-IDIBAPS, Barcelona, Spain 5CIBERES, Instituto de Salud Carlos III, Madrid, Spain Key points rEpisodes of intermittent hypoxia, as produced in obstructive sleep apnoea, cause a carotid-driven sympathetic hyperactivity and an oxidative status leading to cardiovascular and metabolic pathologies, which are less pronounced in patients >60–65 years old. rIn young rats (3–4 months) chronic intermittent hypoxia augments carotid body hypoxic responses, plasma catecholamine, renal catecholamine content and turnover, produces an oxidative status, and causes hypertension. rInagedrats(22–24 months)chronicintermittent hypoxiadoesnot alter carotidbodyresponses, catecholamine-related parameters or redox status, and does not cause hypertension. rIt is concluded that age affords protection to harmful effects produced by chronic intermittent hypoxia. rPossible mechanisms involved in age protection and the significance of our findings in the diagnosis and therapeutic approaches to obstructive sleep apneoa in the elderly are considered. Abstract Obstructive sleep apnoea (OSA) affects an estimated 3–7% of the adult population, the frequency doubling at ages >60–65 years. As it evolves, OSA becomes frequently associated with cardiovascular, metabolic and neuropsychiatric pathologies defining OSA syndrome (OSAS). Exposing experimental animals to chronic intermittent hypoxia (CIH) can be used as a model of the recurrent hypoxic and O2desaturation patterns observed in OSA patients. CIH is an important OSA event triggering associated pathologies; CIH induces carotid body (CB)-driven exaggerated sympathetic tone and overproduction of reactive oxygen species, related to the pathogenic mechanisms of associated pathologies observed in OSAS. Aiming to discover why OSAS is clinically less conspicuous in aged patients, the present study compares CIH effects in young (3–4months) andaged (22–24 months)rats. Todefine potentialdistinctive patternsofthese pathogenic mechanisms, mean arterial blood pressure as the final CIH outcome was measured. In young rats, CIH augmented CB sensory responses to hypoxia, decreased hypoxic ventilation and augmented sympathetic activity (plasma catecholamine levels and renal artery content and synthesis rate). An increased brainstem integration of CB sensory input as a trigger of sympathetic activity is suggested. CIH also caused an oxidative status decreasing aconitase/fumarase ratio and superoxide dismutase activity. In aged animals, CIH minimally affected CB responses, ventilation and sympathetic-related parameters leaving redox status unaltered. In young animals, CIH caused hypertension and in aged animals, whose baseline blood pressure was augmented, CIH did not augment it further. Plausible mechanisms of the differences and potential significance of these findings for the diagnosis and therapy of OSAS are discussed. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society DOI: 10.1113/JP270878 1774 M. Quintero and others J Physiol 594.6 (Received 6 May 2015; accepted after revision 6 January 2016; first published online 11 January 2016) Corresponding author S. Yubero: Department of Biochemistry and Molecular Biology and Physiology, School of Medicine, University of Valladolid c/ Ram´ on y Cajal no. 7 47005 Valladolid, Spain. E-mail: [email protected] Abbreviations 24M, rats aged 22–24 months; 24MCIH, rats aged 22–24 months exposed to chronic intermittent hypoxia; 3M, rats aged 3–4 months; 3MCIH, rats aged 3–4 months exposed to chronic intermittent hypoxia; A, adrenaline; AP, arterial pressure; CA, catecholamine; CB, carotid body; CI, confidence interval; CIH, chronic intermittent hypoxia; CRP, C-reactive protein; CuZnSOD, cytoplasmic superoxide dismutase; DA, dopamine; EGSH, glutathione redox potential; GPx, glutathione peroxidase; GSH, reduced glutathione; GSSG, oxidized glutathione; LPO, lipid peroxide; MnSOD, mitochondrial superoxide dismutase; MV, minute ventilation; NA, noradrenaline; NTS, nucleus of the tractus solitarius; OSA, obstructive sleep apnoea; OSAS, obstructive sleep apnoea syndrome; PaO2, arterial oxygen pressure; RA, renal artery; ROS, reactive oxygen species; RVLM, rostroventrolateral medulla; SaO2, arterial haemoglobin saturation; SOD, superoxide dismutase; SSA, 5-sulfosalicylic acid; TV, tidal volume; UA, upper airway. Introduction Obstructive sleep apnoea (OSA) consists of a repetitive obstruction of the upper airways (UAs) during sleep. Physiologically, in phases 3 and 4 of non-rapid eye movement sleep and mainly during the rapid eye movement periods of sleep, there is a decreased drive to inspiratory and UA opener muscles (Dempsey et al. 2010) resulting in a diminished air-pumping power and increased airways resistance. An abnormally high decrease in the drive to airways dilator muscles, or their abnormal response, leads to UA obstruction (Morrison et al. 1993). Typically, in OSA patients, a decrease in genioglossi activity causes the tongue to drop backwards and UAs close. Each obstruction causes an episode of hypoxia leading to a picture of recurrent or chronic intermittent hypoxia (CIH) causing a fall in arterial oxygen pressure (PaO2) and arterial haemoglobin saturation (SaO2). Hypoxic episodes stimulate the carotid body (CB) chemoreceptors, triggering an increased motor output towards the inspiratory and UA dilator muscles and an arousal reaction, which together solve the obstruction (Dempsey et al. 2010, 2014). Once blood gases are restored, another cycle of UA obstruction is generated. As OSA progresses, it frequently generates the obstructive sleep apnoea syndrome (OSAS), with associated pathologies: cardiovascular (hypertension and augmented acute vascular accidents; Dempsey et al. 2010; Gopalakrishnan & Tak, 2011), hepato-metabolic (insulin resistance, glucose intolerance, fatty liver disease; Morselli et al. 2012) and neuropsychiatric (anxiety, depression and cognitive-executive deficits; Sateia, 2003; Almendros et al. 2010; Gagnon et al. 2014). Epidemiological data show that OSAS-linked pathologies have a substantial worldwide economic impact on healthcare systems (Banno et al. 2009; Tarasiuk and Reuveni, 2013), and thus there is interest to understand their mechanisms with the aim to rationally address their therapies. Clinical (Adamson, 2009; Gami et al. 2013) and experimental studies (Fletcher et al. 1992) indicate that CIH is an important event in causing OSAS-associated pathologies. CIH causes CB sensitization (Peng et al. 2003; Del Rio et al. 2010), probably increasing CB chemoreceptor input to the brainstem (Zoccal et al. 2008; Kline, 2010; Gonzalez-Mart´ ın et al. 2011; Prabhakar et al. 2012; Ciriello and Moreau, 2013), leading to an exaggerated sympathetic tone, which generates hypertension and subsequent cardiovascular pathologies (Lesske et al. 1997; Kumar et al. 2006; Prabhakar et al. 2012; Olea et al. 2014). The swings in PaO2with each obstruction mimic ischaemia-reperfusion events, augmenting the production of reactive oxygen species (ROS). ROS generate a permanent status of vascular endothelium inflammation and dysfunction, lessening of its vasorelaxant and anti-adhesive phenotype, and facilitating cardiovascular pathologies (Lavie & Lavie, 2009, 2012; Jelic et al. 2010; Quintero et al. 2013). Age is another factor to consider in OSAS. Several studies indicate that in aged subjects (>65 years) OSAS is more prevalent than in middle ages (30–65 years) but the clinical picture is less sharp and somehow paradoxical (Lavie and Lavie, 2009; Kobayashi et al. 2010; Mart´ ınez-Garc´ ıa et al. 2010; Gagnon et al. 2014). Comparison of mortality in sleep laboratory cohorts of aged patients (>65 years) with matched general populations revealed that OSAS patients had a significantly lower mortality rate than expected (Lavie & Lavie, 2009), suggesting that oxidative stress, so closely associated with OSA, was implicated in the genesis of that protection by mechanisms similar to those responsible for heart and brain ischaemic preconditioning (Brzecka, 2005; Lavie and Lavie, 2009). Experimental studies of CIH effects in aged animals are scarce. The present study aims to define potential distinctive patterns or intensities in alterations of the two main pathogenic mechanisms (sympathetic tone and oxidative status) involved in OSAS-linked pathologies by comparing the effects of CIH in young adult and aged rats. The findings indicate that CIH causes less intense modifications in CB function–sympathetic tone and oxidative stress in aged than in young adult animals. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society J Physiol 594.6 Chronic intermittent hypoxia in aged rats 1775 Methods Animals and anaesthesia Young adult and old male Wistar rats (3–4 and 22–24 months old, respectively) were used, with free access to food and water and maintained under controlled conditions of temperature, humidity and a stationary light–dark cycle. Rats were distributed in four different groups: young 3–4 months old (3M), young chronic intermittent hypoxic (3MCIH), aged 22–24 months old (24M) and aged chronic intermittent hypoxic (24MCIH) rats. Except for CIH exposure and whole body plethysmography, experimental procedures were performed in animals anaesthetized with sodium pentobarbital (60 mg kg–1 body weight; I.P.) or ketamine plus diazepam (100 and 1.6 mg kg–1, respectively; I.P.). Experiments were performed in the morning (from 08.30 to 13.00 h) of the day after the last intermittent hypoxic exposure, with animals fasting overnight. In handling the animals, we followed the European Community Council directive of 24 November 1986 (86/609/EEC) for the Care and Use of Laboratory Animals. The Institutional Committee of the University of Valladolid for Animal Care and Use approved the protocols. Animals were killed by a cardiac overdose of sodium pentobarbital. Exposure to CIH The specific protocol of CIH used in the present study consisted of cycles of 40 s, 5% O2/80 s, air, equivalent to an apnoea–hypopnoea index of 30. The intermittent hypoxia was applied for 8 h day−1(from 08.00 to 16.00 h) for 14 days. General design and functioning of our equipment to expose rats to intermittent hypoxia have been described in detail in previous publications (Gonzalez-Martin et al. 2011; Quintero et al. 2013). In recent experiments carried out on young rats and using the same protocol (Quintero et al. 2013), we have found that the lowest value of PaO2 measured was 37.2 ±0.9 mmHg, yielding mean lowest haemoglobin O2saturation of 73.5 ±1.4%; haemoglobin O2saturation prior to hypoxic episodes was 96.5 ±0.75%. The duration that SaO2was below 90% represented 25% of the 8 h duration of the hypoxic exposure. Recording of carotid sinus nerve (CSN) activity To record CSN activity, the CB–CSN preparation was exposed under a dissecting microscope and a block of tissue, including the carotid bifurcation and the glossopharyngeal nerve, was removed and placed in a Lucite chamber with ice-cold 100% O2-equilibrated Tyrode solution (in mM: NaCl, 140; KCl, 5; CaCl2, 2; MgCl2, 1.1; Hepes, 10; glucose, 5; pH 7.40) for further dissection, freeing CB and CSN of surrounding connective tissue. The CB–CSN preparation was digested in collagenase type I (1 mg ml–1; Warthington) solution to facilitate later isolation of single sensory units or paucifibre filaments of the CSN for neural activity recording (Rigual et al. 2002; Conde et al. 2006a). The experiments were performed using the times of digestion needed to obtain a maximal activity on the recording and therefore different times of collagenase digestion were used: 3–7 min in 3-month-old animals (3M and 3MCIH) and 20–30 min in 24-month-old animals (24M and 24MCIH), as we have observed that different times of digestion affect electrical activity of the nerve, mainly due to the increase in the epineurium and perineurium thickness of the CSN with age. These intervals represent also small changes produced by the different batches of the type 1 collagenase used. The CB–CSN preparation was maintained in ice-cold 100% O2-equilibrated Tyrode solution until it was transferred to the recording chamber. Recording solution was Tyrode bicarbonate equilibrated with gas mixtures containing 5% CO2unless specified otherwise. The CB–CSN preparation was transferred to a recording chamber mounted on a dissection microscope (Nikon) and perfused with normoxic Tyrode solution (equilibrated with 20% O2–5% CO2–balanced N2at 37°C). Extracellular recordings from single or multiple fibre filaments of CSN were made using a suction electrode. The pipette potential was amplified (NeurologDigimiter, Welwyn Garden City, England), filtered (1 KHz), digitized at 6 kHz (Axonscope, Axon Instruments, Molecular Devices, Wokingham, UK) and stored on a computer. Chemoreceptor activity was identified (spontaneous generation of action potentials at irregular intervals) and confirmed by its increase in response to hypoxia (perfusion with solutions equilibrated with 2% or 7% O2+5% CO2+balanced N2). The acidic hypercapnic stimulation consisted of perfusion of the preparation with solutions equilibrated with 20% O2+20% CO2, balanced N2(pH 6.8). Chemoreceptor activity was discriminated off-line for height and timing (Clampex 9.0, Molecular Devices). Oxygen tension in the recording chamber was measured with a needle electrode (no. 760, Diamond Micro Sensors, Ann Arbor, MI, USA) polarized to −0.8 V against an Ag/AgCl reference electrode also placed in the recording chamber. Plethysmography Ventilation was measured in conscious freely moving rats by whole body plethysmography as described in detail by Olea et al. (2011). The system (Emka Technologies, Paris, France; BUXCO Research Systems, Wilmington, NC, USA) consists of 5-litre metacrylate chambers continuously fluxed (2 l min−1) with desired gas mixtures. Temperature was maintained in the chamber within the thermo-neutral range (22–24°C). Tidal volume (TV; ml kg–1), respiratory frequency (breaths min–1)and minute ventilation (MV; ml min–1 kg–1)weremeasured. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society 1776 M. Quintero and others J Physiol 594.6 Animals were placed in the plethysmographic chamber and breathed room air for at least 30 min until adapted to the ambient chamber conditions and they had acquired a standard resting behaviour. Thereafter recording of basal ventilatory parameters over 20 min started. Ventilatory parameters were measured in different atmospheres by fluxing the chamber during 10 min at 2 l min−1with gas mixtures containing 12, 10 or 7% O2(rest N2)and5%CO 2 in air. Each recording in a special atmosphere was followed by a 10–20 min recovery period in air. The pressure changes within the chamber reflecting tidal volume were measured with a high-gain differential pressure transducer. Ideally the frequency of pressure fluctuations is identical to breathing movements; spurious fluctuations of the pressure due to animal movements were electronically rejected. The amplitude of the pressure oscillations is proportionally related to TV; a calibration of the system by injections of 5 ml of air into the chamber allowed direct estimation of TV. Pressure signals were fed to a computer for visualization, storage and offline analysis with Buxco software. Measurement of endogenous catecholamine (CA) in plasma and renal artery (RA) tissue: measurement oftherateof3H-CA synthesis in renal arteries Special care was taken in handling the animals used to measure plasma CA due to the lability of plasma CA levels. After anaesthesia rats were tracheostomized and pump ventilated with air for around 30 min. The chest was opened and blood was slowly withdrawn by direct puncture in the left ventricle. Citrated blood was centrifuged at 1000 gfor 5 min at room temperature. Supernatant was transferred to tubes containing 60 mg ml−1sodium metabisulfite and frozen at −80°C until the HPLC analysis of CA. For analysis of endogenous CA in RA, extra-renal segments of the arteries were removedandglasstoglasshomogenizedin0.1Nperchloric acid (PCA) containing 0.1 mMEDTA and centrifuged. Supernatants were stored at −80°C until HPLC analysis. General procedures to study the rate of CA synthesis have been described in previous publications (Olea et al. 2014). In brief, RAs were incubated (37°C; 2 h) in Tyrode solution (in mM: NaCl, 140; KCl, 5; CaCl2,2; MgCl2, 1.1; Hepes, 10; glucose, 5; pH 7.40) containing 30 μMof 3,5-3H-tyrosine (the natural precursor of CA; 6 Ci mmol–1; Perkin Elmer, Boston, MA, USA) and the cofactors for tyrosine hydroxylase and dopamine beta hydroxylase, 100 μM6-methyl-tetrahydropterine and 1m Mascorbic acid, respectively. Tissues were washed in precursor-free Tyrode solution (4°C; 5 min), homogenized and processed for HPLC analysis. Plasma CAs were extracted from 500 μl of plasma using Waters Oasis-HLB cartridges after adequate conditioning following the instructions of the supplier (Waters Corporation, Milford, MA, USA). After convenient washings, CAs were eluted with 0.5 ml of citric acid (26.7 mMand 2.5% MeOH; pH 2.9) with a recovery of 98%.Eluateswereeither immediatelyinjectedin theHPLC system or refrozen at −80°C. The HPLC system consisted of a Waters 600 controller pump, automatic injector Waters 717 plus Autosampler and BAS LC-4C Amperometric Detector. The mobile phase was: 25 mMNa2HPO4,0.65mM1-octane sodium sulfonate acid, 0.1 mMEDTA, pH 3.46, and 6% MeOH. The sensitivity of the detector was modified as required. Amine identification was made against external standards and quantification was made with Peak Sample Data Chromatography System software (Buck Scientific, East Norwalk, CT, USA). Identification of 3H-CA was done against internal standards and quantification by collecting of the HPLC column effluents and scintillation counting. Plasma C-reactive protein (CRP) levels CRP levels were measured in plasma of EDTA anticoagulated blood plasma using a commercial rat-specific ELISA (Cusabio Biotech; bioNovaCientifica, Madrid, Spain) following the instructions of the supplier. Plasma adenosine levels Adenosine quantification has been described in detail by Conde & Monteiro (2004). In brief, blood samples were collected from rat heart puncture to EDTA-precoated tubes and centrifuged during 10 min at 3000 g.Plasmawas collected and adenosine was extracted and concentrated using Amicon Ultra-4 Centrifugal Filter Units (Millipore, Lisbon, Portugal). Adenosine was quantified by HPLC with UV detection. The HPLC system consisted of an LC 9-A pump, 7725i injector, SPD-6 AV UV-VIS wavelength detector and Class VP software to analyse the chromatograms (Shimadzu, Kyoto, Japan). The analytical column was a Lichrospher 100 RP-18 (125 4 mm, i.d., particle size 5 μm, Merck, Rahway, NJ, USA) protected by LichroCART 4–4 guard-columns (Merck). Columns and guard-columns were incorporated in the HPLC system through a ManuCART (Merck). The mobile phase was 100 mMKH2PO4with 15% methanol, pH 6.5, run at a flux of 1.75 ml min−1. External standards were prepared under the same conditions as the biological samples and adenosine identification and quantification was made against the standards. Measurement of redox status parameters a. Aconitase and fumarase activities. Activities of the two enzymes were determined as described by Quintero et al. (2013). Fresh tissues were minced in ice cold TES buffer (10 mMTris, pH 7.4, 250 mMsucrose and 1 mM C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society J Physiol 594.6 Chronic intermittent hypoxia in aged rats 1777 EDTA) and mitochondria were isolated as previously described (Quintero et al. 2013). The final mitochondrial pellet was re-suspended in the same TES buffer and mitochondria were lysed by the addition of triton X-100 at a final concentration of 0.5% (w/v). Twenty, 40 and 10 μl of mitochondrial lysates from liver, lung and brain, respectively, were used for enzymatic measurements. The enzymatic activities were assessed following the increase in optical density at 240 nm for 8 min in the appropriate media: 30 mMsodium isocitrate, 50 mMTris-HCl, pH 7.4, and 0.6 mMMnCl2for aconitase, and 50 mMsodium L-malate and 50 mMsodium phosphate buffer, pH 7.4, for fumarase. The linear rates during the last 3 min were used to evaluate enzymatic activities. The aconitase/fumarase ratiowasexpressedastheratioofrespectiveratesofoptical density increase. b. Reduced and oxidized glutathione (GSH and GSSG): glutathione redox potential. Tissues (liver, lung and brain) were quickly excised from the animals, washed in ice-cold saline, dry-blotted on filter paper, weighed and placed in Eppendorf tubes containing a solution of 5-sulfosalicylic acid (SSA) at 5% and 0.25 mMEDTA; SSA solution volume was adjusted to ×5tissueweight. Tissues were stored at −80°C until assay or immediately glass-to-glass homogenized at 0–4°C, centrifuged in a microfuge (4°C, 10 min) and the supernatant used to measure GSH and GSSG. Reduced and oxidized glutathione was determined by the Griffith method as described in detail by Gonzalez et al. (2004). From GSH and GSSG levels, the glutathione redox potential (EGSH) was calculated using the Nernst equation EGSH =EºGSH –RT/zF ×Ln [GSH]2/[GSSG] and a value for EºGSH of −24 mV. c. Measurement of lipid peroxides (LPOs). Tissues (brain, liver, lung) were homogenized in 20 mMphosphate buffer (pH 7.4; 0.2 w/v) and separated in three aliquots: one aliquot was used to determine LPO, the second to determine protein levels (Lowry) and the third to determine glutathione peroxidase (GPx) activity. The LPO aliquot was treated with 10 μlml –1 of 0.5 Mbutylated hydroxytoluene in acetonitrile to prevent oxidation of the sample. LPO levels were determined immediately according to the assay kit supplier (Bioxytech LPO-586; OxisHealt Products, Portland, OR, USA). In the assay, malonaldehyde and 4-hydroxyalkenals present in the tissue sample react with a chromogen reagent at low temperature (45°C) and yield a stable chromophore with peak absorbance at 586 nm. This assay has been found to provide a convenient, sensitive and reliable index of lipid peroxidation. Low-temperature formation of the chromophore and prevention of tissue homogenate oxidation ensure the adequate stability of the analyses and the specificity of the assay. d. Measurement of GPx activity. GPx activity was assayed as described by Quintero et al. (2013). The assay measures the disappearance of NADPH in the coupled reaction: GPx oxidizes GSH to GSSG in the presence of peroxide and GSSG is back-reduced to GSH by glutathione reductase using NADPH as a donor of reduction equivalents. The activity of GPx in the sample (࢞absorbance min−1) was calculated on the basis of the molarextinctioncoefficientforNADPH(6220UA mol−1). One unit of activity is equal to one micromole NADPH oxidized min−1mg−1protein. e. Catalase activity in tissues. Catalase activity was assayedusing a modified version ofthemethodof Aebi(see Quintero et al. 2013). Tissues were homogenized in cold 50 mMphosphate buffer (pH 7; 0.05–0.10 w/v) containing 1m MEDTA and 0.1% Triton X-100, centrifuged (10,000 g; 15 min; 4°C) and the supernatant was immediately used to measurecatalase activity or stored at−80°C until theassay. An aliquot of the supernatant (10–20 μl) was incubated for 1 min at 25°C with an excess of H2O2(50 mM) generating water and oxygen. The reaction was stopped by addition of 900 μlof15m Msodium azide and 10 μl was assayed to evaluate remaining H2O2. Remaining H2O2 reacts with a colour reagent [150 mMpotassium phosphate buffer (pH 7) containing 0.25 mM4-aminoantipyrine, 2m M3,5-dichloro-2-hydroxybenzenesulfonic acid and 0.7Uml−1peroxidase], generatingaredquinoneimidedye that absorbs at 520 nm. Standard curves were constructed in parallel with concentrations of H2O2between 1.25 and 7.5 mM. Samples without tissue homogenate, but with H2O2(50 mM), were used to obtain the maximum rate of formation of red quinoneimide and by subtraction we calculated the H2O2that was destroyed by catalase present in the tissue homogenates and transformed to enzymatic activity. One unit of catalase activity equals 1 μmol H2O2 degraded per minute per milligram of protein at pH 7 at 25°C at a substrate concentration of 50 mMH2O2. f. Superoxide dismutase (SOD) activity in tissues. Tissues (liver, lung and brain) were homogenized in cold 0.25 Msucrose buffer (pH 7.4; 0.1 w/v) containing 10 mMTris and 1 mMEDTA. The homogenates were centrifuged and the supernatant was used to measure SOD activity immediately or stored at −80°C. SOD activity was determined with an indirect method (SOD determination kit; Fluka, Madrid, Spain). Potassium cyanide (3 mM), which inhibits cytosolic SOD, allowed us to differentially determine mitochondrial and cytosolic SOD activities. SOD is expressed as activity units, one unit being the amount of enzyme capable of inhibiting by 50% the reduction of cytochrome cin a coupled system with xanthine oxidase at pH 7.8 and 25°C in a 3 ml reaction mixture. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society 1778 M. Quintero and others J Physiol 594.6 Arterial pressure (AP) measurements AP was measured in ketamine +diazepam anaesthetized rats. Animals were tracheostomized and pump ventilated with room air (CL Palmer, London, UK) (60 cycles min–1 and a positive expiratory pressure of 2 cmH2O). The depressor effect of hypoxia was recorded by connecting the inlet of the respirator to a balloon filled with a 10% O2+90% N2mixture. AP was recorded from the right common carotid artery with a calibrated (with a mercury manometer) pressure transducer (Transpac IV; ICU Medical, Inc., San Clemente, CA, USA) connected to an acquisition card (Power Lab 16SP; ADI Instruments, Castle Hill, Australia). Pressure signals were constantly monitored and data were stored for offline analysis. Mean arterial blood pressures are plotted. Data presentation and statistical analysis Along with some sample recording, data are presented as mean ±SD with their 95% confidence intervals (CIs). Comparison of the data obtained in each experimental group was made by one-way ANOVA, two-way ANOVA and two-way ANOVA for repeated measures as required. The data were considered to be significantly different when the statistical test yielded a Pvalue <0.05. Results General characteristics of the animals Young control animals weighed 366.2 ±44.9 g and young animals exposed to CIH 369.8 ±42.0 g. Aged 22–24 control rats had a mean body weight of 542.5 ±73.0 g, and 24MCIH animals weighed 530.1 ±75.6 g. Haematocrit values were measured in groups of eight animals. Young 3Mand aged24M rats had haematocritvalues of 45.7±4.7 and 42.9 ±1.1%, respectively, and while in young rats CIH exposure did not alter this (46.1 ±3.0%), in aged animals it produced a significant increase to 47.4 ±3.2% (P<0.05). Glycaemia was not different among groups, but there was a tendency to increase in both CIH groups (75.9 and 76.3 mg dl−1in young and aged animals vs. 79.3 and 81.0 mg dl−1in young and aged animals exposed to CIH). Effects of CIH and age on CB chemoreceptor activity Figure 1 depicts the effect of different times of digestion of the CB–CSN preparation on the chemosensory activity of the CSN in response to hypoxia in 3M and 24M rats. The responses of the CSN to hypoxia were influenced by the times of digestion, with the young animal preparations having maximal recording activity at 5–7 min and decreasing afterwards (Fig. 1Aand B,leftpanel)and the old animals having its maximum between 25 and 30 min of digestion (Fig. 1Aand B, right panel). Note also that young animals when submitted to lengthy digestion exhibit a decrease in the hypoxia-evoked chemosensory activity (Fig. 1Aand B, left panel) and that in response to greater periods of digestion, such as 30 min, it is impossible to record (Fig. 1A, left panel), probably due to a loss of axon integrity. The low activity described herein when the CB–CSN preparation is submitted to low digestion times in aged animals (Fig. 1Aand B, right panel) and its increase with the time of digestion correlates very well with the description that the epineurium and perineurium thickness increase with age as well as with perineurial thickness/fasciclesizeratio (Kundalic et al.2014; Campbell et al. 2013). We highlight that we have performed the experiments to compare the effect of CIH on the CSN activity in 3M and 24M animals, in conditions that allow us to obtain maximal recording activity, and therefore 3–7 min in 3M animals and 20–30 min in 24M animals. Figure2 showstheoutputoftheCB measuredaschemosensory activity (action potential frequency) in the CSN in the four groups of rats. Figure 2Aand Bshows the time course of the CSN activity during application of an intense hypoxic stimulus in 3M versus 24M rats (left) and 3MCIH versus 24MCIH rats (right). The most salient difference is the great reduction in the response to hypoxia in the aged animals. The lower panels show mean values of the activity recorded in the CSN in different situations. Figure 2Cshows that mean basal activity in 3M rats amounted to 3.7 ±2.8 spikes s–1 [95% confidence interval )CI), 2.4–5.0; n=20] being markedly diminished in 3MCIH rats, 1.4 ±0.5 spikes s−1(95% CI, 1.2–1.7; n=19; ∗∗∗P<0.001). Comparable findings were obtained in 24M versus 24MCIH rats (3.5 ±2.6 and 1.5 ±0.9 spikes s–1;∗∗∗P<0.001; n=21–23). Figure 2Dshows the CSN responses elicited by intense and moderate hypoxia expressed as the value times basal. In 3M rats intense hypoxic stimulus caused neural activity to increase by a factor of 13.8 ±6.6 (95% CI, 10.9–16.8) to a mean frequency of 51.5 Hz while in 3MCIH rats it increased by a factor of 44.1 ±12.2 (95% CI, 34.0–53.3; ∗∗∗P<0.001) to 64.1 Hz. In 24M and 24MCIH rats the increases in activity elicited by intense hypoxia were also different and amounted, respectively, to 6.2 ±5.5 (95% CI 3.5–8.9) and 22.3 ±15.3 times basal (95% CI, 10.5–24.0; ∗∗∗P<0.001) or to 21.8 and 33.4 Hz. These increases were statistically smaller than those seen in young animals (++P<0.001, 3M vs. 24M and +P<0.05, 3MCIH vs. 24MCIH). As expected, the responses to less intense hypoxia (5% O2) were smaller in all groups but the pattern of the response seen with intense hypoxia was maintained, i.e. aged rats respond to hypoxia less than young rats and at both ages the increment in discharges produced by hypoxia was higher in CIH rats. Figure 2Eshows the responses C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society J Physiol 594.6 Chronic intermittent hypoxia in aged rats 1779 elicited by an acidic-hypercapnic stimulus, revealing a multiplication of basal activity by a comparable factor in all groups, albeit higher in CIH. Yet, considering that basal activity in 3MCIH and 24MCIH rats was about half that seen in corresponding 3M and 24M controls, the actual total activity elicited by the hypercapnic stimulus also was smaller in CIH than in control animals. Effects of CIH and age on ventilation in different atmospheres Figure 3 shows MV in the four groups of animals while breathing in atmospheres containing 20 (room air), 12, 10 and 7% O2balance N2, and air with 5% CO2. In room air (basal conditions) 3M rats breathed 554.8 ±111.9 ml min–1 kg–1 (95% CI, 451.3–638.4; n=8), 3MCIH animals 447.9 ±121.8 ml min–1 kg–1 (95% CI, 296.7–599.2; n=8), 24M rats 331.7 ±53.5mlmin –1 kg–1 (95% CI, 282.2–381.3; n=8; P<0.05 vs. 3M) and 24MCIH 354.1 ±51.9mlmin –1 kg–1 (95% CI, 306.0–402.1; n=8). In low O2atmospheres MV increased in all groups and reached a maximum in 7% O2atmosphere in the 3M group of 1293.0 ±87.6 ml min–1 kg–1 (95% CI, 1212.0–1374.0). In air/5% CO2atmosphere MV was also higher than in air atmosphere in all groups and again reached a maximum in the 3M group of 1213.0 ±149.7 ml min–1 kg–1 (95% CI, 1088.1–1339.3). Exposure to CIH caused around a 20% decrease in the ventilation response at all atmospheres in young animals, making the comparison between 3M and 0 20 40 60 Frequency of discharges (Hz) 7 min (n=2) 15 min (n=3) 0 100 200 300 0 10 20 30 7 min (n=2) 15 min (n=3) 30 min (n=2) Time (min) 0 100 200 300 Time (min) Frequency of discharges (Hz) 0 25 50 75 Frequency of discharges (Hz) 30s 0 25 50 75 30s 30s 30s 30s 30s 30s 30s 30s 30s Frequency of discharges (Hz) 0 25 50 75 Frequency of discharges (Hz) 0 25 50 75 Frequency of discharges (Hz) 0 25 50 75 Frequency of discharges (Hz) 0 25 50 75 Frequency of discharges (Hz) 0 25 50 75 Frequency of discharges (Hz) 0 25 50 75 Frequency of discharges (Hz) 0 25 50 75 Frequency of discharges (Hz) 0 25 50 75 Frequency of discharges (Hz) 3 months 24 months 7' 15' impossible to record, preparation to much digested A B 30' Figure 1. Effect of different times of digestion of the CB–CSN preparation on the electrical activity of the CSN evoked by hypoxia in 3M and 24M rats Left and right panels represent, respectively, the recordings obtained in 3M and 24M animals. A, typical recordings of the chemosensory activity of the CSN evoked by hypoxia in CB–CSN preparation submitted to three different times of incubation: 5–7, 15 and 20–30 min in 3M and 24M animals. B, mean response of the CSN to hypoxia in CB–CSN preparation treated to different times of digestion, obtained in 2–3 different animals. Note that young animals submitted to lengthy digestion (15 min) exhibit a decrease in activity and in response to higher periods (30 min) of digestion it is impossible to obtain a good recording. The hypoxia used to increase the CSN response was 0% O2+5% CO2(N2) applied over 3 min. Data are means ±SD. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society 1780 M. Quintero and others J Physiol 594.6 3MCIH rats statistically different (∗P<0.5and ∗∗P<0.01) in every case except breathing air. The control aged rats (24M group) ventilated less than control 3M rats, the difference in ventilation being highly significant in all atmospheres (+++P<0.001) except in air (+P<0.05). CIH did not cause any further decrease in ventilation in aged rats such that MV was statistically identical in 24M and 24MCIH rats in all atmospheres. Table 1 shows breathing frequencies and tidal volumes (means ±SD) in the four groups of animals at different atmospheres (n=8 in all cases). The effect of age is shown in Table 1A, indicating that in every atmosphere 24M animals breathed at lower frequencies and with lower tidal volumes, reaching statistical differences as indicated in the table (+P<0.05 and ++P<0.01). To perceive the effect of intermittent hypoxia, the data in Table 1Aand B should be compared. Note that in young animals (3M vs. 3MIH) intermittent hypoxia diminished by around 10% both breathing frequencies and tidal volumes in all atmospheres, leading to the nearly 20% decrease in MV shown in Fig. 3 (see Gonzalez-Martin et al. 2011; Olea et al. 2014). In aged rats the effect of CIH (24M vs. 24MCIH) was less obvious, breathing frequencies and tidal volumes fluctuating without a definite pattern. Effects of CIH and age on plasma CA levels, RA CA content and rate of 3H-CA synthesis These three parameters were studied as indices of sympathetic activity. Figure 4 shows plasma noradrenaline (NA), adrenaline (A) and dopamine (DA) levels as well as NA/A ratios. Mean NA levels in 3M and 3MCIH rats were, respectively, 20.8 ±10.4 (95% CI, 14.5–27.0; n=13) and 39.9 ±28.0pmolml −1plasma (95% CI, 23.0–56.8;n=13; ∗P<0.05). In 24M and 24MCIH rats, NA levels were, respectively, 17.3 ±8.7 (95% CI, 9.3–25.3; n=8) and 0 2 4 6 8 10 0 20 40 60 +++ + * +++ Δ CNS activity (times basal) 0% O 2 0% O 2 0% O 2 5% O 2 20% CO 2 *** *** *** ++ 0 2 4 6 8 150 100 50 0 150 3MCIH 24MCIH 3M 24M 100 50 0 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 Time (s) Frequency (Hz) Time (s) *** CSN activity (Hz) Normoxic *** C Δ CNS activity (times basal) 24M 3MCIH 3M 24MCIH A B ED Figure 2. Effects of chronic intermittent hypoxia and age on carotid body chemoreceptor activity Aand B, time courses of the evolution of CSN activity in the four experimental groups on switching the superfusing solution from air to N2-equilibrated (intense hypoxia). The most salient difference is the diminution of the response in 24M animals whether control or exposed to CIH; CIH causes an increase in the response (peak response) and differences in the time course of the response. C, mean basal activity obtained in the four groups of animals: young adult control, 3-month-old (3M); young experimental, exposed to CIH for 15 days (3MCIH); aged control, 24 months old (24M); and aged CIH-exposed animals (24MCIH). D, mean evoked activity elicited by mild and intense hypoxic stimuli expressed as times basal in the four experimental groups. Eshows mean evoked activity elicited by a hypercapnic acidotic stimulus (superfusion with a solution equilibrated with 20% CO2/pH 6.8). Bar charts represent mean ±SD. ∗P<0.05 and ∗∗∗P<0.001 refer to significant differences due to intermittent hypoxia exposure (i.e. 3M vs. 3MCIH and 24M vs. 24MCIH). +P<0.05, ++P<0.01 and +++P<0.001 refer to age effects 3M vs. 24M and 3MCIH vs. 24MCIH. Statistical differences were assessed using a two-way ANOVA followed by Bonferroni’s multiple comparisons test. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society J Physiol 594.6 Chronic intermittent hypoxia in aged rats 1781 13.9 ±6.8 pmol ml−1plasma (95% CI, 8.3–19.6; n=8), neither values being statistically different. Levels of A were similarly increased in 3MCIH vs. 3M rats (∗∗P<0.01) and showed a tendency to increase, albeit non-significantly, in 24MCIH vs. 24M rats. Plasma NA/A ratios were highest in 24M animals with CIH causing a tendency to increase in young animals and to decrease in aged animals. Plasma DA levels were less than a 20th those of NA and A. In 3M rats DA levels amounted to 0.48 ±0.15 pmol ml−1plasma (95% CI, 0.40–0.57) and in 3MCIH rats were comparable and not statistically different. In 24M rats DA levels were higher, amounting to 0.75 ±0.29 pmol ml−1(95% CI, 0.44–1.05; +P<0.05) and CIH did not further increase them. Plasma CA levels represent parameters intimately related to the activity of the sympathetic system: sympathetic endings spillover 90% of NA encountered in plasma and the adrenal medulla secretes all A and the remaining NA; the origin of plasma DA is multiple and not well defined (Goldstein et al. 1983, 2003). Yet, their rate of catabolism might distort the significance of plasma NA and A levels independently of their rate of spillover or secretion. To more precisely evaluate sympathetic activity, we have measured NA content and rate of synthesis in the sympathetic fibres/endings in the renal arteries. Figure 5A shows the levels of NA in renal arteries. In control 3M rats it amounted to 6.0 ±1.8 pmol mg−1tissue (95% CI, 5.2–6.8; n=21) and exposure to CIH increased it to 9.4 ±2.4 pmol mg−1tissue (95% CI, 8.1–10.7; n=16; ∗∗P<0.01). In aged rats, control and CIH animals have renal artery NA levels comparable to those seen in young rats. The rate of 3H-NA synthesis in renal artery (Fig. 5B) was 0.50 ±0.16 pmol mg−1tissue h–1 (95% CI, 0.43–0.57) MV (ml min–1kg–1) Air 12%O 2 10%O 2 7%O 2 5%CO 2 0 500 1000 1500 * +++ * +++ * +++ ** +++ + + ++ +++ + 24M3MCIH3M 24MCIH Figure 3. Effects of chronic intermittent hypoxia and age on ventilation at different atmospheres Each group of four columns corresponds to minute ventilation (MV) in the indicated atmosphere. Data are means ±SD. Comparisons for statistical significance of the differences were made with a two-way ANOVA for repeated measures followed by Bonferroni’s multiple comparisons test. ∗P<0.05 and ∗∗P<0.01 refer to significant differences due to CIH exposure (3M vs. 3MCIH). No statistical differences existed between 24M and 24MCIH. +P<0.05, ++P<0.01 and +++P<0.001 refer to age effects 3M vs. 24M and 3MCIH vs. 24MCIH. in 3M rats; in 3MCIH animals the rate of synthesis increased to 0.71 ±0.08 pmol mg−1tissue h–1 (95% CI, 0.64–0.78; ∗P<0.05). Control 24M rats exhibited a higher rate of 3H-NA synthesis, 0.73 ±0.21 pmol mg−1tissue h–1 (95% CI, 0.57–0.88; ++P<0.01 vs. 3M);contrarytothe situation with young animals, in aged animals exposure to CIH tended to decrease the rate of 3H-NA synthesis. Effects of age and CIH on CRP and adenosine CRP is an acute-phase reactant primarily synthesized in liver in response to inflammation mediators and released to plasma; in turn, CRP binding to endothelial cells may trigger generation of ROS, creating a vicious cycle that leads to the development of chronic inflammation and loss of the endothelial vase-relaxant and anti-adherent phenotype (Zhang et al. 2012). Figure 6Ashows that levels of CRP in plasma of 3M rats amounted to 68.9 ±20.2 μgml −1(95% CI, 56.1–81.7; n=12) and in 3MCIH rats to 121.5 ±12.5 μgml −1(95% CI, 111.0–131.9; n=8; ∗∗∗P<0.001). In control aged rats (24M) CRP plasma levels were 55.2 ±21.5 μgml −1(95% CI, 28.6–81.9) not different from those encountered in control young adult rats. CIH did not cause modifications in aged rats (57.7 ±28.3 μgml −1; 95% CI, 28.0–87.4; n=6). Adenosine is a product of ATP metabolism, which can be recycled to resynthesize ATP itself (Conde et al. 2009). In addition to being a neurotransmitter–neuromodulator substance, adenosine has been postulated to substantially contribute to the ROS generation and inflammation encountered in OSAS patients, it being reported that the purine nucleoside is augmented in the plasma of these patients (see Lavie & Lavie, 2009). Recently it has been proposed that adenosine may promote an anti-inflammatory immunological status (Pettengill et al. 2013). Therefore, we felt it was important to ascertain whether CIH and/or age altered plasma adenosine levels. As shown in Fig. 6B, 3M rats had plasma adenosine levels of 599.5 ±287.7 nmol l−1(95% CI, 483.3–715.7; n=26) and CIH exposure did not modify these levels. In 24M rats plasma adenosine levels amounted to 316.2 ±116.4 nmol l−1(95% CI, 261.7–370.7; n=20; +++P<0.001 vs. 3M) and were not modified by exposure to CIH. Effects of CIH and age on redox status parameters Table 2 shows values for several indicators of the redox status monitored in brain, liver and lung tissues. The initial rows in the table correspond to GSH, GSSG and EGSH in the tissues. Note that in young control 3M rats all tissues had GSH levels in the millimolar range with liver having the highest GSH levels approaching 10 mM. Note also that brain has the highest GSH/GSSG ratio so that in spite of C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society 1788 M. 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Erratum in: Proc Natl Acad Sci USA 1998; 95(4): 1968. Zhang Z, Yang Y, Hill MA & Wu J (2012). Does C-reactive protein contribute to atherothrombosis via oxidant-mediated release of pro-thrombotic factors and activation of platelets? Front Physiol 3, 433. Zoccal DB, Simms AE, Bonagamba LG, Braga VA, Pickering AE, Paton JF & Machado BH (2008). Increased sympathetic outflow in juvenile rats submitted to chronic intermittent hypoxia correlates with enhanced expiratory activity. JPhysiol586, 3253–3265. Additional information Competing interests None of the authors has any conflict of interest. Author contributions The experiments were performed in the Department of Biochemistry and Molecular Biology and Physiology of the University of Valladolid. M.Q. and T.A. managed all redox experiments; E.O., S.Y., A.G.-N. and T.G.-M. carried on the plethysmography, blood pressure measurements, HPLC analyses of catecholamines and performed the statistical tests; S.C. performed the electrophysiological recordings in the carotid sinus nerve and analyses of adenosine. A.O. carried out most of the surgical procedures and designed the figures and C.G. and J.M.M. designed the experiments. C.G. wrote the first draft of the manuscript; all authors read and made suggestions to the manuscript. A.G.-N. and S.Y. reviewed the manuscript and incorporated the referees’ suggested changes. Acknowledgements This work was supported by grants BFU2012-37459 from the Ministry of Economy and Competitiveness (Spain) and grant CIBER CB06/06/0050 from the Institute of Health Carlos III (Spain) and grant EXP/NEU-SCC/2813/2013 from Portuguese Foundation for Science and Technology. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society