Professor Damian Miles Bailey
email: damian.bailey@southwales.ac.uk

Julien V Brugniaux

Teresa Filipponi

Christopher Marley

Benjamin Stacey

Rodrigo Soria

Stefano F Rimoldi

David Cerny

Emrush Rexhaj

Lorenza Pratali

Carlos Salmon

Carla Murillo Jáuregui

Mercedes Villena

Jonathan D Smirl

Shigehiko Ogoh

Sylvia Pietri

Urs Scherrer

MD Claudio Sartori

Placide Nicod

Carlos Salinas Salmòn

Carla Murillo Jáuregui

Ogoh Shigehiko

Exaggerated systemic oxidative-inflammatory-nitrosative stress in chronic mountain sickness is associated with cognitive decline and depression

Keywords: chronic mountain sickness, free radicals, cerebrovascular function, cognition, dementia, depression Redox-regulation of cerebrovascular function in highlanders

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INTRODUCTION

The human brain has evolved a much higher rate of obligatory oxygen (O 2 ) consumption since, unlike most other organs, its evolutionary "drive for size" means that the brain is committed to a continually active state, demanding a disproportionate 20% of the body's basal O 2 budget in the resting state, more than 10 times that expected from its mass alone (Bailey et al., 2017b). The requirement to process large amounts of O 2 over a relatively small tissue mass supports the high rate of ATP formation to fuel the maintenance of ionic equilibria and uptake of neurotransmitters for synaptic transmission [START_REF] Alle | Energy-efficient action potentials in hippocampal mossy fibers[END_REF]. That cognitive function is impaired by acute hypoxia is thus not surprising and an extensive literature has since documented proportional deficits in executive function, attention, mental speed, language and memory [START_REF] Virues-Ortega | Neuropsychological functioning associated with high-altitude exposure[END_REF][START_REF] Rimoldi | Acute and Chronic Altitude-Induced Cognitive Dysfunction in Children and Adolescents[END_REF][START_REF] Mcmorris | Effect of acute hypoxia on cognition: A systematic review and meta-regression analysis[END_REF] including lasting damage to white and grey matter motor architecture [START_REF] Paola | Reduced oxygen due to high-altitude exposure relates to atrophy in motor-function brain areas[END_REF].

However, few studies have considered how lifelong exposure to hypoxia affects cognitive function and whether compensatory adaptations to sustain adequate tissue O 2 delivery prevent acute impairments from potentially progressing to irreversible dementia. The lack of information is surprising given an estimated 140 million high-altitude (HA) dwellers permanently living above 2,500 meters (m) through economic and social necessity [START_REF] Bailey | A Systematic Review and Meta-Analysis Reveals Altered Drug Pharmacokinetics in Humans During Acute Exposure to Terrestrial JP-R2 28 High Altitude-Clinical Justification for Dose Adjustment? High altitude medicine & biology[END_REF] and an evolving body of literature indicating that hypoxaemia is responsible for the higher prevalence of cognitive impairment and dementia observed in patients living at sea-level with cardiopulmonary disease [START_REF] Peers | Hypoxia and neurodegeneration[END_REF][START_REF] Bagge | Risk of Dementia in Adults With Congenital Heart Disease: Population-Based Cohort Study[END_REF].

In the few studies published to date, cognitive function has been shown to be slightly impaired in well-adapted elderly Andean HA dwellers relative to sea-level based lowlander controls [START_REF] Yan | Adaptive influence of long term high altitude residence on spatial working memory: an fMRI study[END_REF][START_REF] Hill | Cognitive performance in high-altitude Andean residents compared JP-R2 31 with low-altitude populations: from childhood to older age[END_REF][START_REF] Davis | Cognitive and psychomotor responses to high-altitude exposure in sea level and high-altitude residents of Ecuador[END_REF]. In contrast albeit in the only study conducted to date, the authors observed an inverse relationship between altitude of residence and (ageadjusted) dementia mortality rate. However, this study examined patients living at considerably lower altitudes (up to 1,800 m) in California with no control of potential confounders such as comorbidities and air pollution, highlighting the need for additional studies.

Furthermore, the mechanism underpinning altitude-induced cognitive impairment is unclear and to what extent further impairments occur in highlanders suffering from chronic mountain sickness (CMS+), a maladaptation syndrome characterised by exaggerated erythrocytosis and more pronounced hypoxaemia [START_REF] Villafuerte | Chronic Mountain Sickness: Clinical Aspects, Etiology, Management, and Treatment[END_REF] has not been examined. We have JP-R2 5 previously identified that compared to lowlander (normoxic) controls, systemic oxidativenitrosative-stress (OXNOS), defined by an increase in free radical formation and corresponding decrease in vascular nitric oxide (NO) bioavailability, was permanently elevated in healthy welladapted Andeans living at 3,600 m without CMS (CMS-). The observation that systemic vascular endothelial function remained intact suggested that physiological concentrations of ONS may prove hormetically beneficial for lifelong adaptation to the hypoxia of HA. In contrast, highlanders with CMS (CMS+) exhibited more exaggerated increases in ONS and impaired systemic vascular endothelial function, thus implying a potential metabolic basis to HA maladaptation [START_REF] Bailey | Oxidative-nitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia[END_REF].

In light of these findings, we sought to determine the potential relationships between metabolic (oxidative-inflammatory-nitrosative-stress herein referred to as OXINOS), haemodynamic (cerebrovascular function) and clinical (cognition and depression) correlates in CMS+ and CMSin order to provide more integrated mechanistic insight into the potential pathophysiology and consequences of neurological maladaptation to HA. We hypothesised that compared to lowlander controls, systemic OXINOS would be moderately elevated in CMS-in the absence of any impairments in cerebrovascular function, cognition or symptoms of depression. We further hypothesised that the systemic OXINOS response would be further exaggerated in CMS+ subsequent to more pronounced arterial hypoxaemia and associated with impairments in cerebrovascular function, cognition and symptoms of depression. Figure 1 provides a schematic summary of the proposed mechanisms.

MATERIALS AND METHODS

Ethical approval

The experimental protocol was approved by the Institutional Review Boards for Human Investigation at the University of San Andres, La Paz, Bolivia (CNB #52/04), University of Lausanne, Lasusanne, Switzerland (#89/06, #94/10), and University of Glamorgan, Pontypridd, UK (#4/07), and subsequently registered (clinicaltrials.gov; Identifier: NCT01182792). All participants were informed of the purpose/risks of the experiment and signed an informed consent form, with all procedures adhering to guidelines set forth in the Declaration of Helsinki.

Experimental design

The study was a cross-sectional population-based observational study in accordance with the STROBE statement [START_REF] Von Elm | The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement: guidelines for reporting observational studies[END_REF]. Figure 2

Participants

For all participants, inclusion criteria specified that they were born and had lived permanently at their resident altitude and sedentary defined as no formal recreational activity outside of everyday living (Bailey et al., 2013b). Exclusion criteria included those with significant developmental delay or learning difficulties, diagnosis of any central neurological disease such as aneurysm, stroke, transient ischemic attack, epilepsy, multiple sclerosis and psychiatric disorders including any history of traumatic brain injury and hypertension. None of the participants were taking nutritional supplements including over-the-counter antioxidant or antiinflammatory medications. We specifically chose to exclude females given our inability to control for differences in circulating estrogen known to affect cerebral blood flow (CBF) and cognition [START_REF] Yao | Frontal cerebral blood flow changes after hormone replacement therapy in depressed postmenopausal women[END_REF]. Prior to inclusion into the study, all participants were subject to an extensive clinical examination that consisted of a thorough medical history, chest auscultation and 12-lead ECG. Participants were subsequently familiarised with the equipment and procedures.

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Highlanders

We recruited 23 male patients with primary CMS (CMS+) and 14 healthy age, gender and education (consecutive years in secondary and University education)-matched controls without CMS (CMS-) native to La Paz, Bolivia (Table 1). We scored symptoms of CMS and confirmed clinical diagnosis by an excessive erythrocytosis [haemoglobin (Hb) > 20 g/dL] in the presence of normal pulmonary function and no history of working in the mining industry [START_REF] Leon-Velarde | Consensus statement on chronic and subacute high altitude diseases[END_REF]. All participants identified themselves as Aymaras and were from similar socioeconomic backgrounds having been born and bred in La Paz with Spanish spoken as their first language.

Lowlanders

We also recruited 17 age and education-matched healthy Caucasian males born and bred close to sea-level (~80 m) in the UK (Table 1) as a sea-level (normoxic) comparator.

Educational status

Data were collected on level of full time education attained.

Metabolic assessments

Participants were asked to refrain from physical activity, caffeine and alcohol and to follow a low nitrate/nitrite (NO  3 /NO  2 ) diet 24 h prior to formal experimentation [START_REF] Woodside | Systemic oxidative-nitrosative-inflammatory stress during acute exercise in hypoxia; implications for microvascular oxygenation and aerobic capacity[END_REF] and were 12 h overnight fasted when they attended the laboratory at 8 am. We obtained blood samples without stasis following 20 min of seated rest to control for plasma volume shifts.

Chemicals

All chemicals were of the highest available purity from Sigma-Aldrich ® (UK).

Blood sampling

We collected blood from an indwelling cannula located in a forearm antecubital vein into Vacutainers® (Becton, Dickinson and Company, Oxford, UK) before centrifugation at 600g (4°C) for 10 minutes. We decanted plasma and serum samples into cryogenic vials (Nalgene® JP-R2 8 Labware, Thermo Fisher Scientific Inc, Waltham, MA, USA) and immediately snap-frozen in liquid nitrogen (N 2 ) and shipped/stored under N 2 gas (Cryopak, Taylor-Wharton, Theodore, AL, USA) prior to analysis in the UK. We left samples to defrost at 37°C in the dark for 5 min before batch analysis.

Oxidative stress

Antioxidants: We assayed plasma concentrations of reduced and oxidised glutathione (GSH/GSSG) according to the methods established by N'Guessan et al. [START_REF] N'guessan | Absence of tumor suppressor tumor protein 53-induced nuclear protein 1 (TP53INP1) sensitizes mouse thymocytes and embryonic fibroblasts to redox-driven apoptosis[END_REF] with modifications [START_REF] Stocker | A fluorescent homogeneous assay for myeloperoxidase measurement in biological samples. A positive correlation between myeloperoxidase-generated HOCl level and oxidative status in STZdiabetic rats[END_REF]. The intra-and inter-assay coefficients of variation (CVs) were both <5%.

Free radicals: The ascorbate free radical (A •-) was employed as a direct measure of systemic free radical formation [START_REF] Buettner | Ascorbate free radical as a marker of oxidative stress: an EPR study[END_REF]. We injected 1 mL of plasma into a highsensitivity multiple-bore sample cell (AquaX, Bruker Daltonics Inc., Billerica, MA, USA) housed within a TM 110 cavity of an EPR spectrometer operating at X-band (9.87 GHz). We recorded samples by cumulative signal averaging of 10 scans using the following instrument parameters: resolution, 1024 points: microwave power, 20 mW; modulation amplitude, 0.65 G; receiver gain, 2  10 5 ; time constant, 40.96 ms; sweep rate, 0.14 G/s; sweep width, 6 G; centre field, 3486 G. We filtered spectra identically (moving average, 15 conversion points) using WINEPR software (Version 2.11, Bruker, Karlsruhe, Germany) and determined the double integral of each doublet using specialist software (OriginLab Corps, MA, USA). The intra-and inter-assay CVs were both <5 %.

Inflammatory stress

Myeloperoxidase (MPO) activity:

We employed a high-throughput, sensitive and homogeneous fluorescence-based method for detection of MPO chlorination activity using 7-hydroxy-2-oxo-2Hchromene-8-carbaldehyde oxime as a selective probe for hypochlorous acid as recently described [START_REF] Stocker | A fluorescent homogeneous assay for myeloperoxidase measurement in biological samples. A positive correlation between myeloperoxidase-generated HOCl level and oxidative status in STZdiabetic rats[END_REF]. The intra-and inter-assay CVs were both <5%.

Nitrosative stress

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We measured plasma NO metabolites using ozone-based chemiluminescence outlined below (Bailey et al., 2017a).

S-Nitrosothiols (RSNO):

Plasma (400 µL) was mixed with 5% acidified sulphanilamide and left to incubate in the dark at 21º C for 15 min to remove nitrite (NO  2 ) prior to injection into triiodide reagent for direct measurement of RSNO. calculated by subtracting the concentration of RSNO. We performed all calculations using Origin/Peak Analysis software.

Nitrite (NO

The intra-and inter-assay CVs were 7% and 10% respectively.

Total bioactive NO: Calculated as the sum of RSNO + NO  2 .

Haemodynamic assessments

We performed all resting measurements following 10 min of seated rest breathing room air at the prevailing barometric pressures in all groups (normoxic normocapnia for lowlanders, hypoxic hypocapnia for highlanders). Measurements were also repeated following the administration of hyperoxia (F I O 2 = 1.0, ~10 L/min for 10 min) to the inspired air in the highlanders only (hyperoxic normocapnia).

Cardiopulmonary function

We employed finger photoplethysmography (Finometer ® PRO, Finapres Medical Systems, Amsterdam, The Netherlands) to monitor beat-to-beat mean arterial pressure (MAP) using the Model Flow method that incorporates participant sex, age, stature and mass (BeatScope 1.0 software; TNO; TPD Biomedical Instruments) to calculate stroke volume (SV) and cardiac output (Q). We corrected for vertical displacement of the finger cuff relative to heart level using a reference probe placed on the chest at the fourth intercostal space in the mid-clavicular line.

We measured heart rate (HR) using a lead II electrocardiogram (Dual BioAmp; ADInstruments).

We sampled end-tidal partial pressures of oxygen and carbon dioxide (PET O2/CO2 ) from a leakfree mask and analysed via capnography (ML 206, ADInstruments Ltd, Oxford, UK). Pulse oximetry (Nonin 9550 Onyx II, Nonin Medical, Inc., Plymouth, MI, USA) monitored arterial oxyhaemoglobin saturation (SaO 2 ) on the third digit of the right hand.

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Cerebrovascular function

Cerebral blood flow (CBF):

We insonated the M1 segment of the right middle cerebral artery (MCA) at a depth ranging between 40-60 mm using a 2 MHz pulsed trans-cranial Doppler (TCD) ultrasound system (Multi-Dop X4, DWL Elektroniche Systeme GmbH, Sipplingen, Germany) to yield MCA velocity (MCAv). A headband device (Spencer Technologies, Nicolet Instruments, Madison, WI, USA) secured the Doppler probe over the trans-temporal window to achieve optimal insonation position and maintained in this position for the duration of the study to avoid movement artefact. We calculated cerebrovascular and total peripheral resistance (CVR and TPR) as MAP/MCAv or Q and cerebrovascular conductance index (CVCi) as MCAv/MAP. We calculated pulsatility index (PI) as systolic MCAv-diastolic MCAv/MCAv and further normalised the PI relative to the prevailing MAP. We calculated cerebral O 2 delivery (CDO 2 ) as the product of (arterial) O 2 content [c(a)O 2 ] (1.39 × Hb × SaO 2 /100) and MCAv. In order to normalise for the cerebral vasoconstrictor effects of polycythaemia and hypocapnia in the highlanders, we adjusted absolute CBF values for differences in (elevated) Hct and (lower) PET CO2 using the following equations:

Polycythaemia: CBF /Hct = 90 × CBF (measured)/(135 -Hct) [START_REF] Severinghaus | Cerebral circulation at altitude[END_REF] Hypocapnia: CBF /PETCO2 = CBF (measured)/[1 + (PET CO2HYPOXIA -PET CO2NORMOXIA* ) × 0.03] (Bailey et al., 2009b) where *refers to fixed PET CO2 of 40 mmHg.

Data sampling:

We sampled beat-by-beat data continuously at 1 kHz using an analog-to-digital converter (Powerlab/16SP ML795; ADInstruments, Colorado Springs, CO, USA) stored on a personal computer for off-line analysis (Chart version 7.2.2, ADInstruments, Colorado Springs, CO, USA). We gave chart files a coded number (not named) by an investigator blinded to the study. We "time-aligned" the MAP and TCD channels given the time-delay (1.07 s) associated with MAP signal processing when using the Finometer ® PRO.

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11 Cerebrovascular reactivity to CO 2 (CVR CO2 ): Following 10 min breathing room air, the inspirate was rapidly changed to 5% CO 2 with 21% O 2 and balanced nitrogen for 5 min at the prevailing barometric pressure. Following a 5 min recovery breathing room air, participants hyperventilated at 15 breaths/min for 5 min. From this, we calculated CVR CO2 as the % increase/decrease in MCAv from baseline per 1 mmHg increase/decrease in PET CO2 recorded during the final 30 s (average taken) of the hypercapneic/hypocapneic challenge having achieved steady-state: et al., 2013a) From these data, we derived the CVR CO2 range as a useful indication of the cerebral circulation's combined ability to respond to differential changes in CO 2 . We calculated the CVR CO2 range as the sum of the fractional vasodilation and vasoconstriction incurred during the respective hypercapnea and hypocapnia challenges as described: (Bailey et al., 2013a)

)] ( ) ( /[ ) ( ) ( ) ( 100 (%) 2 2 2 baseline PET final PET baseline MCA baseline MCA final MCA x CVR CO CO v v v CO    (Bailey
CVR CO2 range (%) = CVR CO2 [Hypercapnia (%)] + CVR CO2 [Hypocapnia (%) ]

Dynamic cerebral autoregulatory capacity (dCA):

We employed a combination of spontaneous (seated) and driven (repeated squat-stands) oscillations in blood pressure (BP) and MCAv in order to assess dCA via transfer function analysis (TFA). Following 10 min of rest in the seated position, we obtained a 5-min segment of BP and MCAv data for spectral analysis of spontaneous oscillations. In order to increase BP variability and improve the reliability and interpretation of the TFA metrics [START_REF] Katsogridakis | Detection of impaired cerebral autoregulation improves by increasing arterial blood pressure variability[END_REF], participants then performed 5-min periods of repeated squat-stand maneuvers at randomly assigned frequencies of 0.05 Hz [10-sec squat, 10-sec standing) and 0.10 Hz (5-sec squat, 5-sec standing) with 5 min of standing rest between frequencies [START_REF] Smirl | Methodological comparison of active-and passive-driven oscillations in blood pressure; implications for the assessment of cerebral pressure-flow relationships[END_REF]. During these maneuvers, we instructed participants to maintain normal breathing and to avoid Valsalva. Beat-to-beat MAP and MCAv signals were calculated across each cardiac cycle, linearly interpolated, and resampled at 2 Hz for TFA [START_REF] Zhang | Transfer function analysis of dynamic cerebral autoregulation in humans[END_REF] in accordance with the recommendations of the Cerebral Autoregulation Research Network [START_REF] Claassen | Transfer function analysis of dynamic cerebral autoregulation: A white paper from the International Cerebral Autoregulation Research Network[END_REF]. Spontaneous MAP and MCAv power spectrum JP-R2 12 density and the mean value of TFA coherence, gain, and phase of the spontaneous oscillations were band averaged across the very low frequency (VLF: 0.02-0.07 Hz, 50 to 14.3-second cycles) and low frequency (LF: 0.07-0.2 Hz, 14.3 to 5-second cycles) ranges where CA is most operant [START_REF] Zhang | Transfer function analysis of dynamic cerebral autoregulation in humans[END_REF]. The TFA coherence, gain, and phase of the driven MAP oscillations were sampled at the driven frequencies (0.05 or 0.10 Hz). To ensure we entered robust phase and gain estimates for analysis, we averaged only those gain and phase (positive to eliminate wraparound) values where the corresponding coherence was ≥ 0.5. Accordingly, we interpreted an increase in gain and decrease in phase to reflect impaired dCA indicative of a more pressurepassive relationship between MAP and MCAv.

Cognitive function

Psychometric tests

We conducted a battery of psychometric tests with 3 consecutive testing periods to ensure habituation and to avoid the confounding influence of learning effects as previously outlined [START_REF] Marley | Failure to account for practice effects leads to clinical misinterpretation of cognitive outcome following carotid endarterectomy[END_REF]:

a]. Learning & Memory
Rey Auditory Verbal Learning consists of 2 lists containing 15 unrelated words. We read List 1 aloud at a rate of 1 word per second. The participant recalled as many of the 15 words in any order. We repeated this 4 times (total of 5 recalls, A1-A5). We read List 2 aloud and the participant recalled as many of the 15 words as possible, again in any order (B1). Finally, we asked the participant to recall as many words as possible from List 1 (delayed recall, A6) [START_REF] Rey | L'examen clinique en psychologie[END_REF].

b]. Working Memory

Repetition of Digits Backwards (RDB) is based on the Repetition of Digits test [START_REF] Wechsler | Manual For The Wechsler Adult Intelligence Scale[END_REF] which comprises digits forwards (RDF) and RDB. Both tests consist of 7 pairs of random number sequences that the researcher reads aloud at the rate of one per second. RDF required the participant to recall the numbers in the correct sequence until they got the sequence wrong. The sequence began with 3 numbers and increased by 1 number every other pair. RDB required the participant to recall the numbers in the reverse order from the sequences they were presented, JP-R2 13 until they got the sequence wrong. The sequence began with 2 numbers and increased by 1 every other pair. We scored the tests by the total number of sequences re-called. Trail Making Test B (TMT-B) is based on the Army Individual Test Battery (1944) that consists of two parts, A (TMT-A) and TMT-B constructed with 25 circles distributed over a sheet of A4 paper. In part A, the circles are numbered 1-25; the participant connected the numbers in ascending order. In part B, the circles include both numbers (1-13) and letters (A-L); again participants connected the circles in the ascending pattern, but alternating between number and letter (i.e. 1-A-2-B-3-C, etc.). We iasked the participant to complete the task as quickly as possible without removing the pen from the paper until completion. We scored the tests as the time taken in seconds to complete each trail.

c]. Attention & Information Processing

We assessed RDF and TMT-A as outlined above. Digit Symbol Substitution Test (DSST) consists of matching 9 digits with their corresponding symbols. We gave the participant a piece of A4 paper with 9 digits and their corresponding symbol and a grid of 93 digits, under each digit the participant drew as many of the corresponding symbols as possible within 90 seconds. We scored the test by how many they drew correctly within the alloted time [START_REF] Wechsler | Manual For The Wechsler Adult Intelligence Scale[END_REF].

Stroop test (ST) consists of two parts A & B, with 24 words written in different colours on a 4 × 6 grid. Test A required the participants to read aloud the word written as quickly as possible.

Test B required the participant to read aloud the colour of the word written and not what the word said as quickly as possible [START_REF] Stroop | Studies of the interference in serial verbal reactions[END_REF]. This test was assessed in the highlander subgroups only and not the lowlanders due to logistical constraints.

d]. Visuo-Motor Coordination

The grooved pegboard dexterity test (GPDT) consists of two parts and required the participant to place 25 pegs in a pegboard containing equally size holes with randomly positioned slots.

Participants placed identical pegs with a key on one side that had to be rotated to match the hole before they could be inserted, into the 25 holes on the pegboard working from left to right, front to back. The test consisted of two parts, the first test completed using the dominant hand (GPDT-D) and repeated using the non-dominant hand (GPDT-ND). The test was scored by the time taken to complete in seconds [START_REF] Klove | Clinical Neuropsychology[END_REF].

Questionnaires

The original English questionnaires outlined below were used for the lowlanders whereas the equivalent validated Spanish versions were used for the highlanders.

Montreal Cognitive Assessment (MoCA):

The MoCA measures visuospatial/executive, naming, memory, attention, language, abstraction, delayed recall and orientation with each section having a designated point value with a maximum score of 30 points [START_REF] Nasreddine | The Montreal Cognitive Assessment, MoCA: a brief screening tool for mild cognitive impairment[END_REF]. We scored the test as the number of questions answered correctly and participants with 12 years or less of education had a point added to their total score. We graded MoCA on 3 levels; normal: 25.2-29.6 points, mild cognitive impairment: 19-25.2 points and Alzheimer's disease: 11.4-21 points according to established guidelines [START_REF] Nasreddine | The Montreal Cognitive Assessment, MoCA: a brief screening tool for mild cognitive impairment[END_REF]. Since MoCA assesses executive function, it is particularly useful for patients with vascular impairment, including vascular dementia [START_REF] Sheehan | Assessment scales in dementia[END_REF].

Depression:

We assessed depressive symptoms using the Becks Depression Inventory (BDI)

shown to be a reliable and valid measure of self-reported depression in both normal and psychiatric populations [START_REF] Beck | An inventory for measuring depression[END_REF]. The BDI is a self-report inventory with 21 items assessing the behavioural and cognitive symptoms of depression. Each item consists of four statements numbered from 0-3 with higher numbers indicating more severe depressive symptoms. We scored the questionnaire by the sum of all the responses with a maximum score of 63 points. A score of 0-9 points indicated minimal depression whereas scores of 10-18 points, 19-29 points and 30-63 points indicated mild, moderate and severe depression respectively.

Statistical analysis

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Power calculation: We analysed these data using G* Power 3.1 software. Assuming comparable differences and corresponding effect sizes previously observed in plasma A •-(η² = 0.54) and NO  2 (η² = 0.67) [START_REF] Bailey | Oxidative-nitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia[END_REF] our primary end-outcome variables for OXNOX stress, the present study required a (minimum) sample size ranging between 24-36 participants (8-12 per group) in order to achieve a power of 0.80 at P < 0.05. We chose to further inflate this during recruitment given the potential for incomplete data collection.

Inferential statistics:

We analysed these data using using the Statistics Package for Social Scientists (IBM SPSS Statistics Version 24.0). Shapiro-Wilk W tests (P > 0.05) confirmed that all data sets were normally distributed. We analysed demographic (Table 1), metabolic (Table 2), haemodynamic (Table 3), cognition and depression (Table 5), CVR CO2 (Figure 2) and TFA (Figure 3) datasets using one-way analyses of variance (ANOVAs) and post-hoc Bonferonniadjusted independent samples t-tests if we observed a main effect. We analysed education data (Table 1) using Pearson Chi-square tests. We analysed responses to hyperoxia in the highlanders (Table 4) using two-way (subgroup × inspirate) ANOVAs and post-hoc Bonferonni-adjusted paired samples (within subgroups) or independent (between subgroups) samples t-tests if we observed an interaction effect. We determined relationships between metabolic, haemodynamic and clinical variables (Figure 4) using Pearson Product Moment Correlations. We established significance at P < 0.05 for all two-tailed tests (with individual P values for all comparisons shown) and data are expressed as mean ± standard deviation (SD).

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RESULTS

Anthropometric data

All groups were of a comparable age and body mass whereas the highlanders were generally shorter with a corresponding elevation in BMI in CMS+ (Table 1).

Metabolic data

Oxygenation and erythrocytosis

As anticipated, highlanders presented with arterial hypoxaemia with the most pronounced erythrocytosis observed in CMS+ (Table 1).

Oxidative stress

We observed lower GSH:GSSG in CMS+ due to the combination of lower GSH and higher GSSG (Table 2). We observed a reciprocal elevation in A •-that was generally more pronounced in highlanders and further exaggerated in CMS+ (Table 2). Figure 3, A-C provides typical examples of EPR doublets observed exhibiting hydrogen hyperfine coupling constants (a H ) of ~1.8 G (g = 2.0052).

Inflammatory stress

MPO activity was higher in CMS+ (Table 2).

Nitrosative stress

Plasma NO  2 and RSNO were consistently lower in highlanders (Table 2) but not different between CMS+ and CMS-. Figure 3, D-F provides typical examples of OBC traces observed.

Haemodynamic data

Cardiopulmonary

As anticipated, highlanders were more hypocapneic whereas we observed no between group/subgroup differences in MAP, SBP, DBP, Q, HR, SV or TPR (Table 3). Hyperoxia and the corresponding normalisation of SaO 2 decreased Q in the highlanders due to the combined decrease in both HR and SV and as a consequence was associated with elevated TPR (Table 4).

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Cerebrovascular

Cerebral perfusion was generally lower in highlanders and was associated with a corresponding elevation in CVR and PI and reciprocal decrease in CVCi and CDO 2 that was most marked in CMS+ (Table 3). While hyperoxia failed to normalise perfusion, it tended to restore CDO 2 subsequent to an elevation in caO 2 (Table 4). The lower cerebral perfusion still persisted in CMS+ when CBF was (retrospectively) adjusted for the independent vasoconstrictor effect of hypocapnea but not polycythaemia (Table 4). Highlanders exhibited a lower CVR CO2 range due primarily to a blunted response to hypercapnea (Figure 4) whereas we observed no differences between CMS+ and CMS-. Figure 5 illustrates typical waveform data for a representative patient with CMS+ and corresponding spectral analysis for both spontaneous (resting) and driven (squat-stand maneuvers) oscillations in MAP and MCAv. Spontaneous and driven TFA metrics are illustrated in Figure 6 (A-F). During spontaneous TFA with moderate coherence values (Figure 6A), LF phase was higher in CMS+ (Figure 6B) with no (between group) differences observed in gain at either frequency (Figure 6C). The driven protocols while not altering PET CO2 (P < 0.05 vs. spontaneous, data now shown) resulted in markedly amplified oscillations culminating in an augmented signal-to-noise ratio as confirmed by the 106-238 fold (0.05 Hz) and 665-1,320 fold (0.10 Hz) increase in BP spectral power and 133-374 fold (0.05 Hz) and 424-1,039 fold (0.10 Hz) increase in MCAv spectral power (compared to spontaneous VLF/LF measures at the respective frequency of interest) resulting in TFA coherence values exceeding 0.95 AU in all cases (Figure 6D). Despite no differences in phase (Figure 6E), we observed consistently lower (35-40 %) point-estimates of VLF and LF gain in the highlanders with no differences observed between CMS+ and CMS-(Figure 6F).

Clinical data

Cognitive function

We observed consistently lower performances on RAVLT, RDB, RDF, TMT-B and DSST in the highlanders with the lowest performance recorded for RAVLT-A6 and Stroop Task-A in CMS+ (Table 5). We observed comparable MoCA scores between CMS-and lowlanders and consistently lower scores in CMS+ with 87 % of the group (20/23 participants) scoring ≤ 25 points (Table 5).

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Depression

Likewise, we observed comparable BDI scores between CMS-and lowlanders and consistently lower scores in CMS+ (Table 5).

Correlations

We observed inverse relationships between A •-and the following variables: SaO 2 , total bioactive NO, MCAv and CVR CO2Hyper (Figure 7 A-D). We observed positive relationships between total bioactive NO and the following variables: CDO 2 and MoCA (Figure 7 E-F) and some aspects of cognitive function, namely performance on the RAVLT-A1-A5 (r = 0.438, P = 0.001), RAVLT-B1 (r = 0.358, P = 0.008), RAVLT-A6 (r = 0.270, P = 0.048), RDF (r = 0.536, P = <0.001), RDB (r = 0.565, P = <0.001) and DSST (r = 0.266, P = 0.052).

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19

DISCUSSION

More than 150 million HA dwellers are permanently exposed to hypoxaemia, a problem predisposing lowlanders suffering from cardiopulmonary disease to cognitive dysfunction and dementia. Here, we show for the first time that systemic OXINOS was permanently elevated in healthy well-adapted (CMS-) highlanders and accompanied by a proportional decrease in cerebral perfusion and blunted reactivity to hypercapnia. These alterations were associated with a mild decrease in cognitive performance with learning/memory and attention/information processing the domains most affected, but without any clinical evidence for depression. Second and in stark contrast, the sustained elevation in systemic OXINOS was further exaggerated in maladapted (CMS+) highlanders and accompanied by more pronounced impairments in cerebrovascular function, cognition and clinical symptoms of depression. Collectively, these findings are the first to suggest that a physiological continuum may exist for hypoxaemiainduced systemic OXINOS that when excessive, is associated with accelerated cognitive decline and depression, helping identify those in need of more specialist neurological follow-up and targeted support.

Metabolic function

Since the concentration of ascorbate in human plasma is orders of magnitude greater than any oxidising free radical combined with the low one-electron reduction potential for the A •- /ascorbate monanion (AH -) couple (E  ΄ = 282 mV) [START_REF] Williams | Outer-sphere electron-transfer reactions of ascorbic anions[END_REF], any oxidising species (R • ) generated within the systemic circulation will result in the one-electron oxidation of ascorbate to form the distinctive EPR-detectable A [START_REF] Buettner | The pecking order of free radicals and antioxidants: lipid peroxidation, αtocopherol, and ascorbate[END_REF]. Thus, the elevations observed in plasma A •- provide direct evidence that systemic free radical formation was permanently elevated in the highlanders, approximately double that observed in the lowlanders, with the highest concentrations recorded in the comparatively more hypoxaemic CMS+ patients.

•-doublet (AH -+ R •  A •-+ R-H, Supplemental Figure 2 A-C)
These observations combined with the inverse relationships consistently observed between A •- and SaO 2 suggest that hypoxaemia may have been the upstream stimulus for oxidative catalysis thus confirming our original hypothesis proposed in Supplemental Figure 1. Our findings agree with an evolving body of literature indicating that hypoxaemia catalyses not only systemic but also local cerebral free radical formation (Bailey et al., 2009c;Bailey et al., 2018a) classically JP-R2 20 attributed to mitochondrial superoxide (O 2 •-) release by complex III of the electron transport chain notwithstanding additional contributions from other (i.e. extra-mitochondrial) sources [START_REF] Waypa | Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells[END_REF]. Systemic free radical formation coincided with the selective oxidation of GSH, the most abundant intracellular thiol found in the brain that likely reflects an attempt to constrain OXINOS through the targeted scavenging of reactive oxygen, nitrogen and carboncentered species [START_REF] Rae | Glutathione in the human brain: Review of its roles and measurement by magnetic resonance spectroscopy[END_REF].

Oxidative stress coincided with both inflammatory and nitrosative stress confirmed by the combined, sustained elevation in MPO and lower NO  2 and RSNO respectively, culminating in OXINOS that was most pronounced in CMS+. These findings extend our previous observations of a permanent and graded elevation in systemic OXNOS stress ranging from moderate in CMSto severe in CMS+ [START_REF] Bailey | Oxidative-nitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia[END_REF]. Since plasma NO  2 is a source of NO with conversion catalyzed by deoxyhaemoglobin-mediated reduction and acidic disproportionation [START_REF] Gladwin | Role of circulating nitrite and Snitrosohemoglobin in the regulation of regional blood flow in humans[END_REF][START_REF] Cosby | Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation[END_REF] we interpreted the more exaggerated decrease in CMS+ to be one of the contributory factors responsible for the observed impairment in systemic vascular function in the form of blunted flow-mediated dilation and increased arterial stiffness [START_REF] Bailey | Oxidative-nitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia[END_REF]. To further extend these observations, we sought to determine if the same concept applied more locally to the hypoxic cerebrovasculature, including to what extent exaggerated OXINOS associates with the neurological deficits underpinning CMS.

Haemodynamic function

Consistent with previous studies [START_REF] Jansen | Brain blood flow in Andean and Himalayan high-altitude populations: evidence of different traits for the same environmental constraint[END_REF], highlanders, in particular CMS+, exhibited ~30 % lower cerebral perfusion notwithstanding the interpretive constraints associated with TCD ultrasound [START_REF] Liu | A longitudinal study of cerebral blood flow under hypoxia at high altitude using 3D pseudo-continuous arterial spin labeling[END_REF] and corresponding decrease in CDO 2 that persisted even following correction for the independent vasoconstrictor effects of polycythaemia-induced alteration in blood rheology and hypocapnia. The decrease in cerebral perfusion coincided with an impaired ability of the cerebrovasculature to respond to vasodilator stimuli, notably hyperoxia and hypercapnia, the latter consistently more pronounced in CMS+. The consistent relationships observed between total bioactive NO, MCAv and CVR CO2Hyper, though not disassociating cause from effect, support a potential contributory role for OXINOS and corresponding decrease in vascular NO availability, consistent with previous studies that have highlighted endothelialderived NO as an important, albeit not exclusive, mediator of cerebral (hyper)perfusion (Lavi et JP-R2 21 al., 2003). Interestingly, OXINOS appeared to exert more of an effect on the cerebral, than systemic circulation given the lack of difference in MAP and TPR in CMS+.

Furthermore, we have previously identified that NO plays an important role in the maintenance of cerebral vasomotor tone and blood-brain barrier integrity by dynamically buffering changes in CBF in response to spontaneous changes in MAP (Bailey et al., 2009a;Bailey et al., 2009c;[START_REF] Bailey | Exercise-induced oxidativenitrosative stress is associated with impaired dynamic cerebral autoregulation and bloodbrain barrier leakage[END_REF] justifying a complementary examination of the pressure-flow coupling dynamic. Contrary to our original expectations based on the published literature [START_REF] Jansen | Cerebral Autoregulation in Subjects Adapted and Not Adapted to High Altitude[END_REF], the elevated LF phase observed in CMS+ during spontaneous oscillations was indicative of improved pressure-flow coupling potentially related to the elevated CVR or hypocapnia as observed subsequent to changes in the respiratory chemoreflex [START_REF] Ogoh | The effect of oxygen on dynamic cerebral autoregulation: critical role of hypocapnia[END_REF] or equally, to increased sympathetic tone [START_REF] Lundby | Sustained sympathetic activity in altitude acclimatizing lowlanders and high-altitude natives[END_REF]. The phase and gain findings associated with the elevated coherence during the driven oscillations revealed a similar mechanism, namely, gain was found to be lower in the highlanders with the lowest values observed in CMS+. Lower gain is consistent with findings in Himalayan Sherpa [START_REF] Smirl | Cerebral pressure-flow relationship in lowlanders and natives at high altitude[END_REF] and may reflect an improved ability to buffer perfusion during rapid alterations in BP to protect against vasogenic oedema in the face of exaggerated OXINOS. However, it would seem unlikely that this constitutes a functionally neuroprotective adaptation given that our participants, in particular CMS+, exhibited clinical signs of neurodegeneration (see below).

Clinical function

Researchers have suggested that the Andean model of HA living, defined by cerebral hypoperfusion and polycythaemia, is phenotypically maladaptive given that it carries a higher risk of stroke and migraine and is associated with increased morbidity and earlier mortality [START_REF] Virues-Ortega | Survival and mortality in older adults living at high altitude in Bolivia: a preliminary report[END_REF][START_REF] Jansen | Brain blood flow in Andean and Himalayan high-altitude populations: evidence of different traits for the same environmental constraint[END_REF]. In support, Bolivians born and bred at the same altitude and location to that employed in the present study exhibited slower psychomotor speed in attention and digit symbol coding tasks that persisted across the lifespan reflecting a "speed-accuracy" trade-off such that slower may be surer [START_REF] Hill | Cognitive performance in high-altitude Andean residents compared JP-R2 31 with low-altitude populations: from childhood to older age[END_REF]. The present findings further extend the albeit limited literature identifying impairments in learning/memory and attention/information processing that were especially pronounced in CMS+.

Thus, it would appear that the highlander's brain, especially with CMS+, is unable to compensate for its hypoperfusion-induced CDO 2 constraint. Any global or local cerebral O 2 JP-R2 22 deficit has the potential to impact cognition potentially due to impaired neurotransmitter release that animal studies suggest is related to depressed monoamine synthesis [START_REF] Freeman | Dopamine, acetylcholine, and glutamate interactions in aging. Behavioral and neurochemical correlates[END_REF], elevated glutamate excitotoxicity [START_REF] Hota | NR1 and GluR2 expression mediates excitotoxicity in chronic hypobaric hypoxia[END_REF] and perturbations in choline acetyltranserase/acetyl cholinesterase expression [START_REF] Guerra-Narbona | Altitude acclimatization improves submaximal cognitive performance in mice and involves an imbalance of the cholinergic system[END_REF].

The observation that clinical symptoms of depression were absent in CMS-and only apparent in CMS+, in whom cognitive/haemodynamic impairments were most pronounced, suggests that a physiological continuum for hypoxaemia-induced systemic OXINOS may potentially exist that when surpassed may prove maladaptive and contribute to the neurological complications associated with CMS. This hypothesis is not unreasonable given that hypoxia and OXINOS contribute to the pathophysiology of dementia [START_REF] Sun | Hypoxia facilitates Alzheimer's disease pathogenesis by up-regulating BACE1 gene expression[END_REF][START_REF] Wojsiat | Oxidant/Antioxidant Imbalance in Alzheimer's Disease: Therapeutic and Diagnostic Prospects[END_REF] and depression [START_REF] Salim | Oxidative stress and psychological disorders[END_REF] in lowlander patient groups and extends our previous observations albeit confined to the systemic vascular circulation [START_REF] Bailey | Oxidative-nitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia[END_REF]. Alternatively, if systemic OXINOS/hypoxaemia are less pronounced it may simply take more time to develop clinical symptoms. Furthermore, our measurements obtained at rest may have underestimated the true magnitude of hypoxic stress encountered given that hypoxaemia in CMS+ is further compounded by physical activity [START_REF] Stuber | Exaggerated pulmonary hypertension during mild exercise in chronic mountain sickness[END_REF][START_REF] Pratali | Exercise induces rapid interstitial lung water accumulation in patients with chronic mountain sickness[END_REF] and sleep-disordered breathing [START_REF] Rexhaj | Sleep-Disordered Breathing and Vascular Function in Patients With Chronic Mountain Sickness and Healthy High-Altitude Dwellers[END_REF].

We observed a similar profile for performance on MoCA with consistently lower scores recorded in CMS+. Although originally developed as a measure of global cognitive function [START_REF] Nasreddine | The Montreal Cognitive Assessment, MoCA: a brief screening tool for mild cognitive impairment[END_REF], MoCA is frequently used as a clinical screening tool for the dementias [START_REF] Ballard | Helping you assess cognition: a practical toolkit for clinicians[END_REF] with a cut-off score of 25 points or lower widely used as the threshold for detecting mild cognitive impairment and possible dementia (Davis et al., 2015a). Thus, with 87 % of the CMS+ group fulfilling these clinical criteria, our findings may help identify those HA dwellers most "at risk" and in need of more specialist neurological assessment to diagnose an emerging dementia syndrome. Clinical diagnosis is inherently complex depending on the triad of cognitive function, patient report and informant history, notwithstanding complementary assessments of cerebrospinal fluid, blood-borne and structural biomarkers to exclude inflammatory, infective and malignancy related causes of dementia (Burns & Iliffe, 2009;[START_REF] Robinson | Dementia: timely diagnosis and early intervention[END_REF].

Experimental limitations

We need to consider several limitations when interpreting the present findings. First, the JP-R2 23 OXINOX assays employed, despite taking advantage of the most direct analytical techniques currently available, ultimately rely on ex-vivo detection of relatively stable reactants confined to circulating plasma/RBC formed downstream of the primary source/reaction pathway that we assume reflects dynamic events in-vivo (Bailey et al., 2009a). Given that these metabolites, especially GSH:GSSG, partition heterogenously across different tissues, our conclusions only apply to what we observed in circulating blood. In addition, there remains the inevitable translational challenge when attempting to determine the clinical outcomes associated with elevated OXINOS given our current inability to differentiate between physiologically adaptive and pathologically maladaptive concentration thresholds [START_REF] Bailey | Oxidative-nitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia[END_REF]. Furthermore, we encourage interventional studies incorporating targeted antioxidant prophylaxis to disassociate cause from effect and confirm the mechanisms proposed herein. Second, we need to be cautious when interpreting the perfusion data in HA dwellers given that we relied on differences in MCAv as an indirect surrogate of global CBF that fails to take into account the antagonistic dilatory/constricting effects caused by prevailing hypoxia/hypocapnea [START_REF] Wilson | Cerebral artery dilatation maintains cerebral oxygenation at extreme altitude and in acute hypoxia-an ultrasound and MRI study[END_REF].

Finally, future researchers need to consider more specialist follow-up neurological assessments to complement the current approaches taken to determine the prevalence of dementia in the most vulnerable CMS+ patient subgroup.

Conclusions

Notwithstanding the experimental limitations as outlined, these findings indicate that a chronic state of disequilibrium potentially exists between free radical formation and antioxidant defence in highlanders causing systemic OXINOS to be permanently elevated and especially exaggerated in more hypoxaemic CMS+ patients. OXINOS was associated with blunted perfusion and reactivity to hypercapnia, impaired cognition and in CMS+, symptoms of depression.

Collectively, these findings are the first to suggest that a physiological continuum may exist for hypoxaemia-induced systemic OXINOS that when excessive, is associated with accelerated cognitive decline and depression, helping identify those HA dwellers, especially CMS+, who may require more specialist neurological follow-up and targeted support. Future investigators need to consider the potential neuroprotective benefits of targeted antioxidant prophylaxis and cognitive training in this patient population. Finally, since arterial hypoxaemia is a hallmark feature of circulatory diseases including those that affect the brain, the current findings in

LEGENDS Table 1. Demographics

Values are mean ± SD; CMS-/CMS+, highlanders without/with chronic mountain sickness; Hb, haemoglobin; Hct, haematocrit; SaO 2 , arterial oxyhaemoglobin saturation; caO 2 , arterial oxygen content; CMS, chronic mountain sickness; BMI, body mass index. *different vs. Lowlanders (P < 0.05); †different vs. CMS-(P < 0.05). Clinical and anthropometric data analysed using one-way analyses of variance and post-hoc Bonferonni-adjusted independent samples t-tests. Education data analysed using Pearson Chi-square tests.

Table 2. Metabolic data

Values are mean ± SD; CMS-/CMS+, highlanders without/with chronic mountain sickness; GSH/GSSH, reduced/oxidised glutathione; A •-, ascorbate radical; NO, nitric oxide; NO  2 , nitrite; RSNO, S-Nitrosothiols; Total bioactive NO (NO  2 + RSNO); MPO, myeloperoxidase; N/A, not assessed. *different vs. Lowlanders (P < 0.05); †different vs. CMS-(P < 0.05). Data analysed using independent samples t-tests (where lowlander data were unavailable) and one-way analyses of variance with post-hoc Bonferonni-adjusted independent samples t-tests.

Table 3. Haemodynamic data

Values are mean ± SD; CMS-/CMS+, highlanders without/with chronic mountain sickness; HR, heart rate; MAP, mean arterial pressure; SBP, systolic blood pressure; DBP, diastolic blood pressure; Q, cardiac output; SV, stroke volume; TPR, total peripheral resistance; SaO 2 , arterial oxyhemoglobin saturation; PET O2/CO2 , end-tidal partial pressure of oxygen/carbon dioxide; MCAv, middle cerebral artery velocity; SMCAv/DMCAv, systolic/diastolic MCAv; MCAv /Hct , MCAv (retrospectively) adjusted for differences in haematocrit (Hct) and MCAv /PETCO2 , MCAv (retrospectively) adjusted for differences in end-tidal partial pressure of carbon dioxide, see Methods; AU, arbitrary units; CVR, cerebrovascular resistance; CVCi, cerebrovascular conductance index; CDO 2 , cerebral oxygen delivery. *different vs. Lowlanders (P < 0.05); **different vs. hypoxia for given Highlanders subgroup (P < 0.05); †different vs. CMS-for given inspirate (P < 0.05). Data analysed using one-way analyses of variance and post-hoc Bonferonniadjusted independent samples t-tests.

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Table 4. Responses to hyperoxia in highlanders

Values are mean ± SD; CMS-/CMS+, highlanders without/with chronic mountain sickness; HR, heart rate; MAP, mean arterial pressure; SBP, systolic blood pressure; DBP, diastolic blood pressure; Q, cardiac output; SV, stroke volume; TPR, total peripheral resistance; SaO 2 , arterial oxyhemoglobin saturation; PET O2/CO2 , end-tidal partial pressure of oxygen/carbon dioxide; MCAv, middle cerebral artery velocity; SMCAv/DMCAv, systolic/diastolic MCAv; MCAv /Hct , MCAv (retrospectively) adjusted for differences in haematocrit (Hct) and MCAv /PETCO2 , MCAv (retrospectively) adjusted for differences in end-tidal partial pressure of carbon dioxide, see Methods; AU, arbitrary units; CVR, cerebrovascular resistance; CVCi, cerebrovascular conductance index; CDO 2 , cerebral oxygen delivery. **different vs. hypoxia for given subgroup (P < 0.05); †different vs. CMS-for given inspirate (P < 0.05). Data analysed using two-way (subgroup × inspirate) analyses of variance and post-hoc Bonferonni-adjusted paired samples (within subgroups) or independent (between subgroups) samples t-tests.

Table 5. Cognition and depression

Values are mean ± SD; CMS-/CMS+, highlanders without/with chronic mountain sickness; n, number correct; s, seconds. *different vs. Lowlanders (P < 0.05); †different vs. CMS-(P < 0.05).

Data analysed using one-way analyses of variance and post-hoc Bonferonni-adjusted independent samples t-tests with the exception of MoCA cut-point (≤ 25 points) analysed using Pearson Chisquare tests with standardised residuals.

S-nitrosothiols) nitric oxide metabolites (D-F) at rest in the systemic circulation of a lowlander and highlanders without (CMS-) and with chronic mountain sickness (CMS).

A-C: Oxidation of the ascorbate monoanion (AH -) by any free radical (R • ) with a one electron reduction potential that exceeds +282 mV will yield A •-(schematic illustrated above). The unpaired electron is delocalised over a highly conjugated tri-carbonyl π-system rendering it resonance stabilized thereby facilitating direct detection by EPR spectroscopy. At the current settings, A •-appears as a (filtered) doublet with a hydrogen hyperfine coupling constant (a H Values are mean ± SD; CMS-/CMS+, highlanders without/with chronic mountain sickness; *different vs. Lowlanders for respective challenge (P < 0.05). Individual hypocapnia and hypercapnia datasets analysed using one-way analyses of variance and post-hoc Bonferonniadjusted independent samples t-tests. Values are mean ± SD; CMS-/CMS+, highlanders without/with chronic mountain sickness; *different vs. Lowlanders for given frequency (P < 0.05). Individual frequency data sets analysed using using one-way analyses of variance and post-hoc Bonferonni-adjusted independent samples t-tests.

  provides a schematic of the experimental design. The Neurovascular Research Laboratory in the UK (~80 m) was the site of investigation for the lowlanders and the Instituto Boliviano de Biologia de Altura in La Paz, Bolivia (~3,600 m) for the highlanders.

 2 )

 2 : A separate sample (200 µL) was also injected into tri-iodide reagent for the combined measurement of NO

  the RAVLT-A/B, RDB/RDF tests and lower scores in the TMT-A/B, GPDT-D/GPDT-ND and Stroop A/B tests indicated superior performance.

Figure 1 .

 1 Figure 1. Functionally-integrated translational hypothesisCMS-/CMS+, highlanders without/with chronic mountain sickness; +++comparatively more hypoxaemic; OXINOS, oxidative-inflammatory-nitrosative stress.

Figure 2 .

 2 Figure 2. Experimental design CMS-/CMS+, highlanders without/with chronic mountain sickness; NO, nitric oxide; O 2 , oxygen; CVR, cerebrovascular reactivity; CO 2 , carbon dioxide; dCA, dynamic cerebral autoregulation.

Figure 3 .

 3 Figure 3. Typical electron paramagnetic resonance (EPR) spectra of the plasma ascorbate radical (A-C) and ozone-based chemiluminescence-based detection of bioactive (nitrite +

  Gauss (see insert of A for simulated spectrum). D-F: Filtered traces of bioactive nitric oxide (NO) metabolites (nitrite + S-nitrosothiols) generated via ozone-based chemiluminescence involving the reaction of NO with ozone (O 3 ) that yields a photon (hv) and subsequent conversion to a potential difference. Insert top right highlights the composite signals (before and after sulphanilamide incubation) for separate measurement of nitrite (NO  2 ) and S-nitrosothiols (RSNO). Note general elevations in the signal intensity (AU, arbitrary units) of A •-and reciprocal decrease in the circulating concentration of bioactive NO metabolites in the highlanders especially in the patient with CMS. Spectra chosen to best reflect the average signal intensities observed in each of the respective groups.

Figure 4 .

 4 Figure 4. Cerebrovascular reactivity to carbon dioxide (CVR CO2 )

Figure 5 .Figure 6 .

 56 Figure 5. Typical waveforms and spectral analysis of haemodynamic responses observed spontaneously during spontaneous (A) and repeated squat-stand maneuvers (B-C) in a representative patient with chronic mountain sickness. BP, blood pressure; MCAv, middle cerebral artery velocity; PSD, power spectral density; V/LF, very/low frequency. Note the markedly amplified and coherent oscillations in MAP and MCAv during the repeated squat-stand maneuvers compared to resting (spontaneous) measures leading to improved estimation of transfer function of dynamic cerebral autoregulation at the frequency of interest.

Figure 7 .

 7 Figure 7. Relationships between metabolic, hemodynamic and clinical correlates in lowlanders and highlanders with and without chronic mountain sickness (CMS). A •-, ascorbate radical; SaO 2 , arterial oxyhemoglobin saturation; NO, nitric oxide; MCAv, middle cerebral artery blood flow velocity; CDO 2 , cerebral oxygen delivery; CVR CO2Hyper , cerebrovascular reactivity to carbon dioxide in the hypercapnic range; MoCA, Montreal Cognitive Assessment. Data analysed using a Pearson Product Moment Correlation.

Table 3 . Haemodynamic data

 3

	Group:	Lowlanders	Highlanders			P values
	Subgroup:	Controls	CMS-	CMS+	CMS-vs.	CMS+ vs.	CMS+ vs.
		(n = 17)	(n = 14)	(n = 23)	Controls	Controls	CMS-
	MAP (mmHg)	79 ± 21	80 ± 10	87 ± 14	0.238 (between groups)
	SBP (mmHg)	118 ± 30	118 ± 18	128 ± 22	0.309 (between groups)
	DBP (mmHg)	61 ± 18	61 ± 8	67 ± 11	0.228 (between groups)
	Q (L/min)	7.42 ± 1.33	6.99 ± 0.80	7.24 ± 1.38	0.627 (between groups)
	HR (b/min)	71 ± 14	71 ± 12	72 ± 14	0.990 (between groups)
	SV (mL)	105 ± 13	101 ± 22	103 ± 22	0.853 (between groups)
	TPR (mmHg/L/min)	10.97 ± 2.93	11.60 ± 1.73	12.41 ± 2.80	0.231 (between groups)
	SaO 2 (%)	97 ± 0	91 ± 5*	88 ± 4* †	<0.001	<0.001	0.044
	PET O2 (mmHg)	107 ± 5	54 ± 3*	51 ± 5*	<0.001	<0.001	0.236
	PET CO2 (mmHg)	39 ± 3	34 ± 5*	33 ± 4*	0.019	0.022	1.000
	Cerebrovascular
	MCAv (cm/s)	49 ± 12	39 ± 12*	35 ± 12* †	0.044	0.001	0.048
	MCAv /Hct (cm/s)	50 ± 11	42 ± 13*	43 ± 14*	0.039	0.037	1.000
	MCAv /PETCO2 (cm/s)	51 ± 11	48 ± 16	44 ± 15*	1.000	0.040	1.000
	PI (AU)	1.04 ± 0.18	1.15 ± 0.41	1.45 ± 0.39* †	1.000	0.002	0.048
	Normalised PI (AU)	0.014 ± 0.006	0.014 ± 0.005	0.017 ± 0.004	0.216 (between groups)
	CVR (mmHg/cm/s)	1.74 ± 0.69	2.27 ± 0.80	2.89 ± 0.57* †	0.667	0.010	0.037
	CVCi (cm/s/mmHg)	0.70 ± 0.39	0.49 ± 0.16	0.42 ± 0.18* †	0.088	0.003	0.010
	CDO 2 (mL/cm/s)	983 ± 209	850 ± 305*	889 ± 303*	0.046	0.047	1.000

Table 4 . Responses to hyperoxia in highlanders

 4

	Subgroup:	CMS-(n = 14)	CMS+ (n = 23)		P values
	Inspirate:	Hypoxia	Hyperoxia	Hypoxia	Hyperoxia	Subgroup	Inspirate Interaction
	Cardiopulmonary
	MAP (mmHg)	80 ± 10	83 ± 8	87 ± 14	87 ± 16	0.188	0.500	0.405
	SBP (mmHg)	118 ± 18	121 ± 13	128 ± 22	125 ± 24	0.284	0.885	0.267
	DBP (mmHg)	61 ± 8	64 ± 7	67 ± 11	68 ± 13	0.167	0.241	0.426
	Q (L/min)	6.99 ± 0.80	6.11 ± 0.77	7.24 ± 1.38	6.17 ± 1.22	0.663	<0.001	0.540
	HR (b/min)	71 ± 12	68 ± 11	72 ± 14	67 ± 14	0.993	<0.001	0.456
	SV (mL)	101 ± 22	92 ± 21	103 ± 22	95 ± 28	0.746	0.001	0.863
	TPR (mmHg/L/min)	11.60 ± 1.73	13.81 ± 2.00	12.41 ± 2.80	14.55 ± 3.99	0.385	<0.001	0.948
	SaO 2 (%)	91 ± 5	97 ± 1**	88 ± 4 †	97 ± 1**	0.043	<0.001	0.043
	PET O2 (mmHg)	54 ± 3	374 ± 81	51 ± 5	380 ± 51	0.887	<0.001	0.699
	PET CO2 (mmHg)	34 ± 5	35 ± 6	33 ± 4	39 ± 4	0.007	0.385	0.847
	Cerebrovascular
	MCAv (cm/s)	39 ± 12	39 ± 9	35 ± 12 †	34 ± 7 †	0.052	0.756	0.029
	MCAv /Hct (cm/s)	42 ± 13	43 ± 11	43 ± 14	42 ± 8	0.939	0.759	0.626
	MCAv /PETCO2 (cm/s)	48 ± 16	48 ± 14	44 ± 15	36 ± 11** †	0.045	0.009	0.011
	PI (AU)	1.15 ± 0.41	0.99 ± 0.18	1.45 ± 0.39 †	1.21 ± 0.27**	0.013	0.001	0.041
	Normalised PI (AU)	0.014 ± 0.005	0.012 ± 0.003	0.017 ± 0.004	0.015 ± 0.006	0.068	0.010	0.803
	CVR (mmHg/cm/s)	2.27 ± 0.80	2.23 ± 0.53	2.89 ± 0.57	2.67 ± 0.74	0.054	0.518	0.640
	CVCi (cm/s/mmHg)	0.49 ± 0.16	0.47 ± 0.11	0.42 ± 0.18	0.40 ± 0.12	0.119	0.504	0.861
	CDO 2 (mL/cm/s)	850 ± 305	909 ± 243	889 ± 303	951 ± 193	0.626	0.083	0.981

Aymaras may provide complementary insight into the pathophysiology and treatment of patients suffering from early-onset neurodegeneration at sea-level.

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Acknowledgements

We thank Mrs Catherine Romero and staff of the Instituto Boliviano de Biologia de Altura (La Paz, Bolivia) for technical support. ADInstruments provided exceptional technical input/support during the period of data logging. Finally, we appreciate the participants and patients' enthusiasm and commitment to this study. Our study is dedicated to the memory of the late Dr Christopher K Willie (University of British Columbia Okanagan, Kelowna, Canada) whose memory remains a constant source of inspiration.

JP-R2

for the work; Drafting the work or revising it critically for important intellectual data for the work; Drafting the work or revising it critically for important intellectual content; Final approval of the version to be published; data for the work; Drafting the work or revising it critically for important intellectual content; Final approval of the version data for the work; Drafting the work or revising it critically for important intellectual interpretation of data for the work; Drafting the work or revising it critically for important intellectual content; Final approval of the version to be published; interpretation of data for the work; Drafting the work or revising it critically for important intellectual content; Final approval of the version to be published; data for the work; Drafting the work or revising it critically for important intellectual content; Final approval of the version for the work; Drafting the work or revising it critically for important intellectual for the work; Drafting the work or revising it critically for important intellectual for the work; Drafting the work or revising it critically for important intellectual interpretation of data for the work; Drafting the work or revising it critically for important intellectual content; Final approval of the version to be published; for the work; Drafting the work or revising it critically for important intellectual

Funding

Supported by a Royal Society Wolfson Research Fellowship (#WM170007) to DMB and grants from the Swiss National Science Foundation, Cloëtta Foundation, Eagle Foundation, Leenaards Foundation and Placide Nicod Foundation to US and CS.

Competing interests

The authors declare that they have no competing interests.

Author contributions

DMB, US and CS conceived and designed the research. DMB, US and CS obtained funding. All authors contributed to data collection and analysis. All authors interpreted the results of the experiments. DMB drafted the manuscript and revisions thereof. All authors edited and revised the manuscript(s) and approved the final version submitted for publication. All authors agree to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.