J Physiol 2010 Slessarev 1591 606

J Physiol 588.9 (2010) pp 15911606 1591

Differences in the control of breathing between Himalayanand sea-level residents

M. Slessarev1, E. Prisman1, S. Ito1, R. R. Watson2, D. Jensen3, D. Preiss4, R. Greene5, T. Norboo6,T. Stobdan6, D. Diskit7, A. Norboo7, M. Kunzang8, O. Appenzeller9, J. Duffin1 and J. A. Fisher1

1Departments of Anesthesia, Medicine and Physiology, University Health Network, University of Toronto, Toronto, Canada2Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA3School of Kinesiology and Health Studies, Clinical Exercise Physiology Lab, Respiratory Investigation Unit, Queens University, Kingston, ON, Canada4Boston University School of Medicine, Boston, MA 02118, USA5New Mexico Highlands University, Las Vegas, NM 87701, USA6Ladakh Institute of Prevention and Ladakh Heart Foundation, 7Sonam Norboo Memorial Hospital Leh and 8Ladakh Autonomous Hill DevelopmentCouncil, Ladakh, India9NMHEMC Research Foundation, Albuquerque, NM 87122, USA

We compared the control of breathing of 12 male Himalayan highlanders with that of21 male sea-level Caucasian lowlanders using isoxic hyperoxic (PO2 = 150mmHg) andhypoxic (PO2 = 50mmHg) Duffins rebreathing tests. Highlanders had lower mean s.e.m.ventilatory sensitivities to CO2 than lowlanders at both isoxic tensions (hyperoxic: 2.3 0.3vs. 4.2 0.3 l min1 mmHg1, P = 0.021; hypoxic: 2.8 0.3 vs. 7.1 0.6 l min1 mmHg1,P < 0.001), and the usual increase in ventilatory sensitivity to CO2 induced by hypoxia inlowlanders was absent in highlanders (P = 0.361). Furthermore, the ventilatory recruitmentthreshold (VRT) CO2 tensions in highlanders were lower than in lowlanders (hyperoxic:33.8 0.9 vs.48.9 0.7 mmHg,P < 0.001;hypoxic: 31.2 1.1 vs.44.7 0.7 mmHg,P < 0.001).Both groups had reduced ventilatory recruitment thresholds with hypoxia (P < 0.001) andthere were no differences in the sub-threshold ventilations (non-chemoreflex drives to breathe)between lowlanders and highlanders at both isoxic tensions (P = 0.982), with a trend forhigher basal ventilation during hypoxia (P = 0.052). We conclude that control of breathingin Himalayan highlanders is distinctly different from that of sea-level lowlanders. Specifically,Himalayan highlanders have decreased central and absent peripheral sensitivities to CO2. Theirresponse to hypoxia was heterogeneous, with the majority decreasing their VRT indicatingeither a CO2-independent increase in activity of peripheral chemoreceptor or hypoxia-inducedincrease in [H+] at the central chemoreceptor. In some highlanders, the decrease in VRT wasaccompanied by an increase in sensitivity to CO2, while in others VRT remained unchanged andtheir sub-threshold ventilations increased, although these were not statistically significant.

(Received 1 December 2009; accepted after revision 26 February 2010; first published online 1 March 2010)Corresponding author J. A. Fisher: Toronto General Hospital, Department of Anesthesiology 3EN, 200 Elizabeth St,Toronto, Canada, M5G 2C4. Email: [email protected]

Abbreviations CBF, cerebral blood flow;HCVR, hypercapnic ventilatory response; HVR, hypoxic ventilatory response;P aO2 , arterial PO2 ; PET,CO2 , end-tidal PCO2 ; PET,O2 , end-tidal PO2 ; VEB, sub-ventilatory recruitment threshold basalventilation; VES, ventilatory sensitivity to CO2; VRT, ventilatory recruitment threshold.

Introduction

Andean andTibetan highlanders exhibit different patternsof adaptation to chronic hypoxia of their environment(Leon-Velarde & Richalet, 2006; Beall, 2007; Brutsaert,2007). Andean highlanders, compared to Tibetans, havelower resting minute ventilations (Beall et al. 1997),

higher end-tidalPCO2 (PET,CO2) (Moore, 2000) andbluntedhypoxic ventilatory responses (HVRs) (Chiodi, 1957;Severinghaus et al. 1966; Sorensen & Severinghaus,1968a,b; Velasquez et al. 1968; Lahiri et al. 1969;Cudkowicz et al. 1972; Leon-Velarde et al. 1996; Beallet al. 1997; Gamboa et al. 2003; Brutsaert et al. 2005).Blunting of HVRs in Andeans is thought to be acquired

C 2010 The Authors. Journal compilation C 2010 The Physiological Society DOI: 10.1113/jphysiol.2009.185504

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1592 M. Slessarev and others J Physiol 588.9

rather than genetic and develops in adolescence (Lahiriet al. 1976; Lahiri, 1980), although recent studies suggestthat the acquired blunting may depend on age and on thealtitude of exposure (Curran et al. 1995). Tibetans, on theother hand, have HVRs comparable to sea-level residentsat sea-level (Hackett et al. 1980; Zhuang et al. 1993; Geet al. 1994; Beall et al. 1997), so that Tibetan ancestrymay actually protect against blunting of the HVR (Curranet al. 1997). The reasons for differences in ventilatoryadaptation patterns between these two populations areunclear, but they probably involve differences in thecontrol of breathing (Moore, 2000; Brutsaert et al.2005).

Control of breathing in humans can be broadlydivided into chemoreflex and non-chemoreflex drivesto breathe (Fig. 1) (Lloyd & Cunningham, 1963).Non-chemoreflex breathing stimuli include a wakefulnessdrive (Longobardo et al. 2002), voluntary (cortical) drive(Shea, 1996) and hormonal factors (Jensen et al. 2008),as well as neural and humoral mediating factors that areespecially important in the control of breathing duringexercise (Bell, 2006; Dempsey, 2006; Haouzi, 2006). The

chemoreflex drive to breathe can be further divided intocentral and peripheral chemoreceptor drives. Both centraland peripheral chemoreceptors respond to changes in thehydrogen ion concentration ([H+]) in their immediateenvironments (Torrance, 1996; Nattie & Li, 2009). Incontrast to the central chemoreceptors, peripheral chemo-receptors are also sensitive to changes in arterial PO2(PaO2) via a hypoxia-mediated increase in their sensitivityto [H+] (Cunningham, 1987; Torrance, 1996; Kumar& Bin-Jaliah, 2007), and hyperoxia (PO2 150mmHg)effectively silences this response (Lloyd & Cunningham,1963; Mohan & Duffin, 1997). Central and peripheralchemoreceptor neural drives are integrated in themedullato provide the total chemoreflex neural drive (Fink, 1961;Shea, 1996;Mohan&Duffin, 1997; Orem et al. 2002) that,in combination with non-chemoreflex drives, providesventilatory drive to respiratory muscles. It is importantto point out that central and peripheral chemoreceptordrives only affect ventilation if their total sum exceeds adrive threshold so that a ventilatory recruitment threshold(VRT) to [H+] orPCO2 is established (Duffin, 2005). BelowVRT, changes in [H+] or PaO2 have no effect on ventilation

Pulmonary ventilation

Respiratory control centre

Central chemoreceptors

Peripheral chemoreceptors

CBF

Ventilatory drive

Chemoreflex drive

Non-chemoreflex drive

Arterial PCO2 / H

+

Arterial PO2

Brain tissue

PCO2 / H+

Central drive Peripheral drive

+ +

+/-

+

Figure 1. The control of breathing model (Lloyd & Cunningham, 1963)The total ventilatory drive is a sum of non-chemoreflex and chemoreflex drives to breathe that are integrated in therespiratory centre. The ventilatory drive exerts its action on the respiratory muscles that affect pulmonary ventilationand result in changes in arterial PCO2 and PO2 . Arterial PCO2 is sampled by the peripheral chemoreceptors locatedin the carotid bodies, where it determines the peripheral chemoreflex drive. Hypoxia exerts its effect on ventilationvia peripheral chemoreceptors where it acts indirectly via increasing the ventilatory sensitivity to CO2 in mostindividuals but may also act directly by increasing the overall activity of the receptor. Central chemoreceptorsrespond to changes in the local [H+] environment, which is mediated by brain tissue PCO2 . Brain tissue PCO2 isa function of both arterial PCO2 and cerebral blood flow (CBF), which acts to decrease the brain tissue PCO2 athigher CBF (Ainslie & Duffin, 2009). The central and peripheral chemoreflex drives add together to form a totalchemoreflex drive to breathe.

C 2010 The Authors. Journal compilation C 2010 The Physiological Society


J Physiol 588.9 Respiratory control at altitude 1593

Table 1. Summary of different methods used to measure hypoxic (HVR) and hypercapnic (HCVR) ventilatory responses in highlanders

Method Studies Advantages Disadvantages

HVR2 Non-isocapnic Acute hypoxia orhyperoxia

(Lahiri et al. 1967;Lefrancois et al. 1968;Velasquez et al. 1968;Zhuang et al. 1993; Curranet al. 1995, 1997)

Simple, fast, notaffected by HVD

Variable O2 stimulus1

Variable CO2 confoundsinterpretation of results

Effect of acutehypoxia/hyperoxiaon (non-isocapnic)ventilatoryresponse to CO2

(Severinghaus et al. 1966;Milledge & Lahiri, 1967;Sorensen & Severinghaus,1968a,b; Lahiri et al. 1969)

Simple, fast Variable CO2O2stimuli1

Confounding effects ofCO2 and O2 onventilation

Isocapnic Progressiveisocapnic hypoxia

(Forster et al. 1971; Weilet al. 1971; Byrne-Quinnet al. 1972; Hackett et al.1980; Huang et al. 1984;Zhuang et al. 1993; Curranet al. 1995, 1997; Beallet al. 1997; Gamboa et al.2003)

Isocapnia ensuresconstant CO2 stimulusMore than 1 point onVE vs. SaO2 graphAllows measurement ofHVDChoice of isocapnialevel may affect HVRcalculation (see Fig. 3)

Longer than acute HVRtestsPotentially confoundedby HVDAffected by choice ofhypercapnea (see Fig. 3)

HCVR Steady state2 CO2 inhalation (Forster et al. 1971) Simple, fast, notaffected by HVD

Variable CO2 stimulus1

Variable O2 confoundsinterpretation of results

Isocapnic changesin CO2

(Weil et al. 1971; Fatemianet al. 2003)

Constant CO2 stimulus Variable O2 confoundsresult interpretation

Rebreathing Reads rebreathing (Byrne-Quinn et al. 1972;Zhuang et al. 1993; Curranet al. 1995, 1997)

Ventilatory sensitivitynot affected by changesin CBF2

No separation betweencentral and peripheralchemoreceptorsresponsesNo measurement ofnon-chemoreflex drivesto breatheNo measurement ofVRT

Note that all of the described methods do not measure non-chemoreflex drives to breathe, fail to separate central and peripheralcomponents of the total chemoreflex drive to breathe (except Fatemian et al. 2003), and do not measure ventilatory recruitmentthreshold (VRT) at which the chemoreflex drive starts to affect ventilation. Detailed discussion of these topics can be found in Duffin(2007). HVD, hypoxic ventilatory decline. 1These methods rely on delivering fixed inspired concentration of O2 and CO2 in attempt tochange SaO2 and/or PET,CO2 , respectively. However, the actual change in any given subject will depend on their breathing pattern andventilatory sensitivity to O2 and CO2. 2Measurement of ventilatory response to CO2O2 using steady-state (non-rebreathing) methodsis confounded by changes in CBF (see Berkenbosch et al. 1989; Mohan et al. 1999).

which is solelydependentonnon-chemoreflexdrives, suchas the wakefulness drive (Duffin, 2005).

Control of breathing studies in high altitude nativeshave attempted to elucidate the observed differencesin ventilation between high altitude natives using twomethods: the hypoxic ventilatory response (HVR) andthe hypercapnic ventilatory response (HCVR) (Moore,2000; Brutsaert, 2007). As their names imply, theHVR method measures the ventilatory response toa hypoxic stimulus and thereby primarily assesses aperipheral-chemoreceptor-mediated response, while the

HCVR method measures ventilatory response to hyper-capnia and therefore assesses the combined central andperipheral responses (see Fig. 1 and Duffin, 2007). Severalmethods of measuring HVR and HCVR have been usedto study control of breathing in highlanders and theseare summarized in Table 1 and in the Methodologicalconsiderations section of the Discussion.

Despite numerous HVR studies, interpretation oftheir findings is problematic as HVR per se is not ameasure of individual components of the control ofbreathing model (Fig. 1), but is rather an indicator




of the integrated response from several components(Duffin, 2007). Furthermore, methodological differencesbetween HVR studies (Moore, 2000), as well as theirinability to separately study the individual componentsof the ventilatory control model (Moore, 2000; Duffin,2007), limit the usability of HVR in comparing controlof breathing between high altitude populations. Withreference to a control of breathing model (Lloyd &Cunningham, 1963) (Fig. 1), fundamental limitations ofthe HVR methods include their inability to: (1) separatethe contribution of chemoreflex from non-chemoreflexdrives to breathe, (2) delineate the contribution of centraland peripheral chemoreceptors, (3) identify VRT at whichchemoreceptor drives start to produce an increase inpulmonary ventilation, and (4) prevent attenuation ofmeasured ventilatory sensitivities due to cerebral bloodflow (CBF)-induced washout of the [H+] from thecentral chemoreceptor (for steady-state methods only)(Berkenbosch et al. 1989; Mohan et al. 1999).

In the present study, we used Duffins rebreathingmethod (Duffin et al. 2000) to fill in the missinginformation about control of breathing in highlanders bycomparing ventilatory control in Himalayan highlandersto that of lowlanders at sea-level.

Methods

Subjects

The study was approved by the Ethics Review Boardof the University Health Network, Toronto, Canada;Queens University Ethics Review Board, Kingston,Canada; and Ladakh Institute of Prevention for theStudy of Environmental, Occupational, Life Style andHigh Altitude Related Diseases, Ladakh, India. All studieswere performed in accordance with the Declaration ofHelsinki of the World Medical Association (2004). Forlowlander studies, 21 healthy Caucasian male subjectswere recruited from the Queens University studentpopulation. Their mean S.D. age, height and body masswere 21.7 2.5 years, 180.2 5.4 cm and 80.4 10.4 kg,respectively. For highlander studies, 12 healthy malelong-term residents of Korzok village, Ladakh, India(altitude, 4550m) were recruited. Their mean S.D.age, height and body mass were 40.8 9.8 years,159.9 5.3 cm and 60.4 10.6 kg, respectively. Eachsubject gave informedwritten consentbefore commencingthe study.

General protocol

Each lowlander subject visited the laboratory atQueens University (altitude, 90m; barometric pressure,750mmHg; ambient air temperature, 2025C) for an

initial (1.5 h) familiarization test on all the rebreathingapparatus and upon return to the laboratory they eachperformed either two (10 subjects) or four (11 subjects)pairs of hypoxic andhyperoxic rebreathing tests in randomorder, with each test separated by a 20min rest period.At high altitude, the experiments were conducted in aheated research tent outside the Korzok village (altitude,4550m; barometric pressure, 442mmHg; air temperatureinside the tent, 1825C). On the first day of experiments,highlander subjects were familiarized with the equipmentand the test by first watching one of the research scientistsperform both hypoxic and hyperoxic rebreathing testson themselves, and then breathing on the rebreathingapparatus and performing several trial rebreathing runs.At the same time, the nature of the experiments andspecific instructions were provided in the highlandersnative language through an interpreter, and all questionswere answered. Highlanders returned to the tent on thefollowing days and completed at least two pairs of hypoxicand hyperoxic tests in random order separated by at least20min of rest time between the tests.

Chemoreflex assessment

Testing method. Duffins rebreathing tests (Duffin &McAvoy, 1988;Mohan &Duffin, 1997) were used to assessthe chemoreflexes. Their interpretation is given in Duffinet al. (2000), and they have been extensively discussedpreviously (Duffin et al. 2000; Mahamed & Duffin, 2001;Mateika et al. 2004). In thismodification of Reads originalmethods (Read, 1967), the rebreathing stage is precededby 5min of hyperventilation and isoxia is maintainedthroughout the test (Fig. 2).

The hyperventilation ensures that the rebreathing startsat a CO2 level below VRT, and enables both the VRTand the sub-threshold (basal) ventilation to be measured(Mohan et al. 1999). The latter measures the contributionof the non-chemoreflex drives to breathe (Shea, 1996),such as the wakefulness drive (Fink, 1961). Rebreathing isrepeated at twodifferent isoxicPO2 tensions: onehyperoxic(PO2 = 150mmHg) to silence peripheral chemoreceptorand allow isolated measurement of the central chemo-receptor response (Lloyd & Cunningham, 1963), andone hypoxic (PO2 = 50mmHg) to allow measurementof the combined central and peripheral chemoreceptorresponses (Fig. 3). The difference between hypoxic andhyperoxic responses represents the contribution of theperipheral chemoreflex and can be used to calculate HVRat any given isocapnic PET,CO2 (Fig. 3).

In contrast to previously used HVR and HCVRmethods, Duffins rebreathing technique allows directmeasurement of the individual components of the controlof breathing model (Fig. 1) (Lloyd & Cunningham, 1963)including: (1) measurement of non-chemoreflex drives to




breathe, (2) separate measurement of central and peri-pheral chemoreceptor drives, and (3) identification ofVRTs for both central and peripheral chemoreceptors.Furthermore, Duffins rebreathing ensures equilibrationbetween arterial, venous and tissue PCO2 (Read, 1967;Read & Leigh, 1967) and thereby avoids attenuation ofthe measured ventilatory response due to CBF-inducedwashout of the PCO2 and consequent lowering of [H

+] atthe central chemoreceptors that occurs with steady-stateHVR techniques (Berkenbosch et al. 1989; Mohan et al.1999; Ainslie & Duffin, 2009).

Testing protocol. The test order was randomly assigned.All subjects were seated comfortably upright during therebreathing tests and wore a finger pulse oximetry probe.They breathed via a mouthpiece, with nose clips, througha bacterial filter (Allegiance, Healthcare Corp., McGawPark, IL, USA) connected to one side of a Series 2870three-way sliding valve (Hans Rudolph Inc., Kansas City,MO, USA) that provided either ambient air or gas from

the rebreathing bag. The rebreathing bag had a volumeof 5 l and an inlet for oxygen. It was primed with aCO2O2mixture to ensure appropriate equilibrationat thebeginning of a rebreathing test (Mohan & Duffin, 1997).Inspired and expired partial pressures of CO2 andO2 weresampled at the mouth and monitored throughout thetest using a respiratory mass spectrometer (Perkin ElmerMGA 1100) at the Queens University laboratory andinfra-red gas analyser (RespirAct, Thornhill Research Inc.,Toronto, Canada) at Ladakh. Ventilation was measuredusing bi-directional volume turbines (VMM-2A; AlphaTechnologies, Laguna Niguel, CA, USA at QueensUniversity and Universal Ventilation Meter, VacuMed,Ventura, CA, USA at Ladakh). Continuous data wereinput to a computer via an analog-to-digital converter(DAQCard-6024E, National Instruments, Austin, TX,USA). A specially written program (LabVIEW, NationalInstruments, Austin, TX, USA; source code availableon request) analysed the data to provide a file ofbreath-by-breath PET,CO2 , end-tidal PO2 (PET,O2) and

Reads Duffins

rebreathingrebreathingrest hyper-ventilation

equilibrationequilibration

VE VE

PCO2 PCO2

VRT

VEB

VES=

030 40 50 60

End-tidal PCO2 (mmHg)30 40

VES=

50 60End-tidal PCO2 (mmHg)

25

50

75

V E (l/m

in)

0

25

50

75

V E (l/m

in)

Figure 2. Differences between Reads (Read,1967) and Duffins (Duffin et al. 2000)rebreathing methods (Mohan et al. 1999)The top two graphs for each method illustratethe change in ventilation and PCO2 with timeduring both tests, while the bottom graphssummarize the ventilatory response to CO2. InReads method, rebreathing is initiated above aeucapnic level. As a result, the ventilatoryresponse to CO2 is measured in the hypercapnicrange only. In Duffins method, rebreathing ispreceded by 5 min of hyperventilation, so therebreathing starts at a hypocapnic PCO2 belowthe ventilatory recruitment threshold (VRT). Asthe bottom right graph illustrates, following thestart of rebreathing, ventilation remainsunchanged below the VRT despite the risingPCO2 . Duffins method allows measurement ofsub-VRT ventilation, which corresponds to thenon-chemoreflex drive to breathe, and the VRT.Since the test is repeated at two isoxic PO2tensions, one hypoxic (PO2 of 50 mmHg) andone hyperoxic (PO2 of 150 mmHg), the testallows us to measure separately the contributionof central and peripheral chemoreceptors (sincehyperoxia effectively silences the peripheralchemoreflex).




ventilation. In addition, the program operated a solenoidvalve controlling the flow of oxygen to the rebreathingbag to maintain PO2 at 150mmHg (hyperoxic) or at50mmHg (hypoxic) during rebreathing. The PCO2 andPO2 analysers were calibrated using gas from cylinders ofanalysed medical grade compressed gases, and ventilationwas calibrated using a 3 l calibration syringe (ModelR5530B, Vacumed, Ventura, CA, USA). Portable pulseoximeters (OXI; Radiometer Copenhagen, Copenhagen,Denmark in lowlanders, and Autocorr Plus Vital SignsMonitor, BCI international, USA in highlanders) wereused to measure arterial oxygen saturation and heart rate.

Each rebreathing test began with a 5min hyper-ventilation of ambient air, with subjects coached tomaintain their PET,CO2 between 19 and 25mmHg forlowlanders and between 10 and 15mmHg for highlanders.

V E (l/m

in)

PO2 = 50 mmHg

PCO2(mmHg)

PO2 = 150 mmHg

Rest+2

RestRest+4

HVR+2

HVR+4

VEB

VRT HVRVES =

Figure 3. Hypercapnic ventilatory responseThe graph displays two isoxic responses: hyperoxic (PO2 = 150 mmHg)representing central chemoreflex response, and hypoxic(PO2 = 50 mmHg) representing the addition of central and peripheralchemoreflexes responses. The slope of each isoxic response representssensitivity of the chemoreflex to CO2. The inflection point at whichventilation starts to increase in response to increasing PCO2 is theventilatory recruitment threshold (VRT), where the chemoreflex neuraldrive to breathe exceeds a drive threshold and starts to produce anincrease in pulmonary ventilation. Ventilation below VRT representsnon-chemoreflex drives to breathe and is known as the basalventilation. The differences in ventilation between isoxic rebreathinglines at any given isocapnic PCO2 can be used to calculate the hypoxicventilatory response (indicated by vertical arrows). Note that thechoice of isocapnic PCO2 affects the magnitude of the measured HVReven within the same subject (Duffin, 2007), with higher HVRsmeasured at higher isocapnic PCO2 values in the illustrated example.Note also that the magnitude of HVR provides little information aboutthe characteristics of the control of breathing model, as HVRmagnitude is dependent on the combination of central and peripheralchemoreflex responses.

The target PCO2 range was selected based on the restingmixed venous PCO2 in each population. Since restingPET,CO2 in highlanders was on average 10mmHg less thanthat in lowlanders and assuming similar mixed venousto end-tidal PCO2 gradients in both populations, thehyperventilation PET,CO2 target for highlanders was set at10mmHg lower than that for lowlanders.

Subjects then exhaled completely and were switched tothe rebreathing bag where they took three deep breathsto facilitate rapid equilibration of PCO2 in the bag, lungsand arterial blood to that of mixed venous blood. Thisequilibration was verified by observing a plateau in theend-tidal PCO2 , and was a prerequisite for continuingthe test. The rebreathing test ended when ventilationexceeded 100 l min1, or PET,CO2 exceeded 60mmHg, orif discomfort occurred. The testing environment wasquiet, with minimal distractions. Subjects were instructedto relax and close their eyes during the rebreathingexperiments.

Rebreathing test analysis. Rebreathing test data wereanalysed using a specially written program (LabVIEW).After eliminating the initial three equilibration breaths, aswell as sighs, swallows and breaths incorrectly detected bythe acquisition software, breath-by-breath PET,CO2 valueswere plotted against time and fitted with a least squaresregression line. This line provided a predicted valueof PET,CO2 as a function of time, thereby minimizinginter-breath variability, and tidal volume (ml BTPS),respiratory rate (breaths min1) and ventilation (l min1

BTPS)wereplotted against thepredictedPET,CO2 (mmHg).These plots (see Fig. 4 for an example) were fitted by

dividing theminto two segments separatedbyabreakpointcorresponding to the VRT, defined as the PET,CO2 whereventilation starts to increase in response to rising PET,CO2(Duffin et al. 2000). The first segment was fitted witheither an exponential decline to a final value, or a mean,and measured sub-VRT ventilation (VEB) that representsnon-chemoreflex ventilation drive. The second segmentwas fitted with a straight line whose slope measured thesensitivity (VES). Model fitting was based on minimizingthe sumof least squares for non-linear regression using theLabVIEW software (Levenberg-Marquardt algorithm).The furthest outlying points were automatically discardeduntil an r2 value of > 0.95 was achieved.

Statistical analyses

All results are reported as mean S.E.M. except wherenoted, with significance set at P < 0.05. The measuresobtained from the repeated tests on lowlanders wereaveraged. A two-way, repeated measures ANOVA withfactors population (highlanders vs. lowlanders) and isoxia(hypoxic vs. hyperoxic) and post hoc Tukeys tests were




used to detect differences in sub-threshold ventilations,ventilatory recruitment thresholds and chemoreflexsensitivities to CO2.

Results

Each subject completed the rebreathing protocol. Onehighlander subject (039) was unable to complete thehyperoxic protocol and his data were withdrawn fromanalysis. Table 2 shows values of VEB, VRT and VES for allsubjects.

There was no difference in basal ventilation betweenhighlanders and lowlanders during both isoxic tensions(hyperoxic: 12.9 0.9 vs. 12.4 1.6 l min1; hypoxic:13.5 1.2 vs. 14.9 1.9 l min1, P = 0.982), with a trendfor higher basal ventilation during hypoxia (P = 0.052).Highlanders had lower ventilatory sensitivities to CO2compared to lowlanders at both isoxic tensions (hyper-oxic: 2.3 0.3 vs. 4.2 0.3 l min1 mmHg1, P = 0.021;hypoxic: 2.8 0.3 vs. 7.1 0.6 l min1 mmHg1,P < 0.001). Interestingly, the hypoxia-induced increasein ventilatory sensitivity to CO2 observed in lowlanders(P < 0.001) was absent in highlanders (P = 0.361).Furthermore, ventilatory recruitment thresholds werelower in highlanders compared to lowlanders (hyperoxic:33.8 0.9 vs. 48.9 0.7 mmHg, P < 0.001; hypoxic:31.2 1.1 vs. 44.7 0.7 mmHg, P < 0.001), althoughthe ventilatory recruitment thresholds decreased withhypoxia in both groups (P < 0.001). Figure 5 summarizesthese findings for both populations.

Discussion

This study is the first to independently examine allthree components of the Oxford control of breathingmodel depicted in Fig. 1 (Lloyd & Cunningham, 1963) inhighlanders, namely the central and peripheral chemo-

reflex drives to breathe and the non-chemoreflex drive tobreathe, and compare them to those of lowlanders residingat sea-level. As Fig. 5 illustrates, the control of breathingin highlanders differs from that in lowlanders in severalaspects that are discussed below.

Non-chemoreflex drives to breathe

There was no difference in the non-chemoreflex drivesto breathe between highlanders and lowlanders, asindicated by similar basal (below VRT) ventilations inthe two populations (Table 2). According to the control ofbreathing model (Lloyd & Cunningham, 1963) (Fig. 1),any differences in the control of breathing betweenHimalayanhighlanders and lowlanders are therefore solelydue to differences in their chemoreflexes. Note that thebasal ventilations in our subjects fall at the higher endof the range of those reported previously (2.522 l min1,see Table 3). Since basal ventilation is a measure of thenon-chemoreflexdrive tobreathe, this findingmay suggestthat the non-chemoreflex drive to breathe was increasedin our subjects, possibly due to increased experimentalanxiety secondary to unfamiliarity with the respiratoryexperiments. It therefore follows that familiarizing thesubjects with the experimental protocol by repeating theexperiments multiple times would reduce experimentalanxiety and basal ventilations. However, Jensen et al.(2010), whose study agrees well with our data, recentlyshowed that basal ventilation in a given subject wasnot affected by multiple repetitions of the rebreathingexperiments within and between days, suggesting that asignificant familiarization effect does not exist (Jensenet al. 2010). Furthermore, even if significant experimentalanxiety was present in the current study, our lowlanderand highlander subjects were equally nave to the natureof respiratory experiments and were therefore probablyaffected to the same degree, with the familiarizationprotocols during their first visit to the research tent

0

20

40

60

80

20 25 30 35 40 45 50Predicted PCO2 (mmHg)

Ventil

atio

n (l/m

in)

VRT

raw dataselected datafitted data

VEB

Slope = VES

Isoxia 50 mmHg

Figure 4Breath-by-breath ventilation vs. end-tidal PCO2during an isoxic hypoxic Duffins rebreathing testfor one highlander illustrating the measuresderived.




Table 2. Individual subject values for non-chemoreflex drives to breathe (VEB), ventilatory recruitmentthresholds (VRT) and sensitivities to CO2 (VES) for all rebreathing tests

Highlanders Isoxic hyperoxic tests (PO2 = 150 mmHg) Isoxic hypoxic tests (PO2 = 50 mmHg)

VEB VRT VES VEB VRT VES(l min1) (mmHg) (l min1 mmHg1) (l min1) (mmHg) (l min1 mmHg1)

001 11.5 30.5 3.5 23.5 27.0 2.8011 23.5 40.0 2.8 18.0 38.0 2.1014 7.2 34.0 5.3 24.7 33.0 5.4019 6.1 33.0 1.1 4.9 30.5 2.2020 14.0 35.0 2.1 13.2 33.0 3.8039 n/a n/a n/a 5.3 23.5 3.1051 3.5 37.0 1.9 9.8 37.0 1.7061 11.8 36.0 1.4 9.4 30.7 1.9062 19.4 33.0 2.0 21.9 34.0 2.2065 12.3 30.0 2.0 17.8 30.0 3.2066 12.7 31.0 2.1 10.6 28.7 2.9068 14.7 32.5 1.6 19.7 29.0 2.7

Mean (S.E.M.) 12.4(1.6) 33.8(0.9) 2.3(0.3) 14.9(1.9) 31.2(1.1) 2.8(0.3)

Lowlanders Isoxic hyperoxic tests (PO2 = 150 mmHg) Isoxic hypoxic tests (PO2 = 50 mmHg)

VEB VRT VES VEB VRT VES(l min1) (mmHg) (l min1 mmHg1) (l min1) (mmHg) (l min1 mmHg1)

BY 18.2 53.2 4.2 19.2 45.8 8.3EV 16.6 54.7 4.4 17.0 50.7 4.9GM 14.3 49.0 4.6 10.4 45.5 5.0ID 14.6 42.3 5.3 16.9 38.6 12.1KM 11.6 46.0 2.6 7.8 41.0 5.0KMM 21.4 52.3 2.9 10.2 43.5 7.3MD 15.9 47.4 4.2 20.2 44.4 9.5PR 12.1 53.0 8.2 13.8 47.0 13.5RG 18.8 52.3 4.0 14.3 47.9 4.7SC 9.6 52.6 4.3 11.1 49.8 9.1SM 18.8 47.0 3.9 28.6 48.7 10.4BS 10.3 47.0 4.4 10.1 43.7 7.6DG 11.2 47.1 6.1 15.4 43.1 7.1DJ 7.1 48.7 5.6 7.7 46.5 8.8GM 9.4 49.2 1.9 9.0 44.8 2.9JS 9.1 45.2 3.8 7.2 40.4 5.8KD 14.2 48.1 2.5 21.5 41.9 5.8MF 6.9 48.1 4.3 7.0 43.5 5.8MH 6.7 46.5 1.9 7.9 42.7 3.5MO 9.3 46.3 2.3 10.7 43.1 2.9PJ 14.2 51.4 6.3 17.9 47.0 8.8

Mean (S.E.M.) 12.9(0.9) 48.9(0.7) 4.2(0.3) 13.5(1.2) 44.7(0.7) 7.1(0.6)Mean S.E.M. values for each condition and subject populations are compared with () values indictingdifferences between hypoxic and hyperoxic tests within populations and () values indicating differencebetween populations determined by repeated measures ANOVA. Note that highlander subject 039 wasunable to complete hyperoxic rebreathing tests and his data were withdrawn from analysis.

aimed at reducing this anxiety. Lastly, other methodsof studying the control of breathing, including classicHVR tests, are affected by experimental anxiety tothe same extent as current experiments. As a result,

the reported basal ventilations are probably the bestrepresentation of the non-chemoreflex drives to breathein the studied populations during the described testingconditions.




Table 3. Summary of basal ventilation measurements with Duffins rebreathing method in lowlanders residing at sea-level

Study Subjects Basal ventilation (mean S.D., l min1)

Hyperoxic (150 mmHg) Hypoxic (50 mmHg)

Mohan & Duffin, 1997 7 males Basal ventilations were measured at different isoxic PO2 values(100, 80, 60 and 40 mmHg) and varied between subjects from 3.1to 14.7 l min1. Change in PO2 had no effect on basal ventilation(correlation coefficient of 0.0248)

Mahamed & Duffin, 2001 5 males and 2 females 6.1 0.8 8.5 1.0Mahamed et al. 2003 6 males and 3 females 3.9 0.6 6.0 0.8Somogyi et al. 2005 6 males 5.0 1.03 5.9 0.9Jensen et al. 2005 14 males and 14 females 16.2 2.1 in males 9.9 1.1

in females17.7 2.1 in males 10.5 1.3in females

Fan et al. 2010 11 males and 6 females 10.5 7.4 (sea level)11.1 6.3 (altitude)

Not available

Jensen et al. 2010 12 males and 8 females Mean, 9 (range, 2.5 to 19) Mean, 9 (range, 1.5 to 22)Present study 21 male lowlanders 12.9 0.9 (range, 3.5 to 23.5) 13.5 1.2 (range, 4.9 to 24.7)

12 male highlanders 12.4 1.6 (range, 6.7 to 21.4) 14.9 1.9 (range, 7.0 to 28.6)Note that more recent studies had higher averages and wider ranges of basal ventilations. Also note that the level of hypoxicrebreathing has no effect on basal ventilation (Mohan & Duffin, 1997).

Ventilatory recruitment thresholds

The highlanders had decreased VRTs comparedto lowlanders during both hypoxic and hyperoxicrebreathing tests. The leftward shift of the VRTs inhighlanders suggests that a lower PCO2 was required toexceed the VRT in highlanders compared to lowlanders.Since both central and peripheral chemoreceptors areactually [H+] sensors, interpretation of this result shouldconsider the acidbase status in both populations.According to theHendersonHasselbach equation (Nunn,1993), the relationship between [H+] and PCO2 can bedescribed as follows:

[H+] = 24(PCO2)/[HCO3]

where [HCO3] is the bicarbonate ion concentration.In a hypothetical sea-level resident at sea-level, [H+]is approximately 40 nM l1, PCO2 is 40 mmHg and[HCO3] is 24mM l1. At altitude, hypoxia-inducedhyperventilation results in a reduction of PCO2 thatleads to a reduction in [H+] and respiratory alkalosisaccording to the above equation. Highlanders compensatefor respiratory alkalosis by presumably reducing their[HCO3] through increased renal excretion, therebyrestoring the PCO2/[HCO3

] ratio to sea-level values andnormalizing [H+]. Although the PCO2/[HCO3

] ratio in

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Figure 5. Mean S.E.M. breath-by-breathventilation vs. PCO2 during isoxic hyperoxic andhypoxic Duffins rebreathing tests for allhighlander and lowlander subjectsIn both populations, the continuous lines representhyperoxic (PO2 = 150 mmHg) and dashed linesrepresent hypoxic (PO2 = 50 mmHg) rebreathing tests.Dotted lines for each test represent S.E.M. responses.Note that highlanders had lower ventilatoryrecruitment thresholds (VRT) than lowlanders at bothisoxic tensions probably due to differences inacidbase status between the two populations.Additionally, highlanders had lower centralchemoreflex sensitivities to CO2 compared tolowlanders and responded to acute hypoxia bydecreasing their VRT rather than increasing theventilatory CO2 sensitivity like lowlanders.




adapted highlanders is comparable to that of lowlandersat sea-level, the lower [HCO3] in highlanders alters therelationship between [H+] and PCO2 in this populationsuch that a lower PCO2 is required to attain the [H

+] of40 nM l1 in highlanders compared to lowlanders (Duffin,2005). For example, if hypoxia-induced hyperventilationreduced CO2 from 40 to 30mmHg, and HCO3 fell from24 to 18mM l1, then the overall ratio of CO2/HCO3

would be maintained at 5/3 as in sea-level lowlanders,but normal [H+] of 40 nM l1 would be achieved at alower PCO2 of 30mmHg rather than 40mmHg, as atsea-level. Considering that the highlanders in our studyhave an adapted acidbase status (Santolaya 1989), theobserveddifference inVRTs canbe explainedby the altered[H+]PCO2 relationship in highlanders compared tolowlanders, with the assumption that the chemoreceptorthresholds [H+] are similar (Duffin, 2005).However, sincewedidnotperformacidbasemeasurements in thepresentstudy, the above explanation requires further experimentalvalidation.

Central chemoreflex sensitivity to CO2

The sensitivity of the central chemoreceptor to CO2,as indicated by the ventilatory sensitivity during hyper-oxic rebreathing, was lower in highlanders comparedto lowlanders (2.5 0.4 vs. 4.2 0.3 l min1 mmHg1,P = 0.011). Hyperoxia effectively silences the peripheralchemoreceptor (Lloyd & Cunningham, 1963; Mohan &Duffin, 1997), and therefore the ventilatory sensitivitymeasured during hyperoxic rebreathing can be taken asa measure of central chemoreceptor sensitivity (Duffin,2007). The difference in central chemoreceptor sensitivitytoCO2 betweenhighlanders and lowlandersmay representan intrinsic difference between central chemoreceptors intwo populations, or as shown previously,may be related tothedifference in anthropomorphic characteristics betweenhighlanders and lowlanders. Highlanders were older(40.8 2.6 vs. 21.7 0.5 years), lighter (60.4 2.9 vs.80.4 2.2 kg) and shorter (159.9 1.5 vs.180.2 1.2 cm)than lowlanders (P < 0.001 for all comparisons). Since theventilatory response to hypercapnia decreases with age(Nishimura et al. 1991; Jones et al. 1993; Poulin et al.1993; McGurk et al. 1995) and increases with weight(Marcus et al. 1994), the observed lower central CO2chemosensivity in our highlander subjects may be theresult of their older age and smaller body size. Repeatingthe experiments with age-matched controls may clarifythe mechanism responsible for this observation.

Previous studies of the ventilatory response to CO2reported similar ventilatory sensitivities to CO2 betweenhighlanders and lowlanders (Weil et al. 1971; Zhuanget al. 1993; Curran et al. 1995), but these studiesmeasured the total ventilatory sensitivity to CO2 andfailed to separate contributions from the central and peri-

pheral chemoreceptors. Fatemian et al. (2003) employed amultifrequency binary sequence in PET,CO2 (Pedersen et al.1999) using an end-tidal forcing technique in an attemptto study the central and peripheral components of thechemoreflex response to CO2 separately. They found thatduring euoxia (PO2 of 100mmHg), the total and peri-pheral chemoreceptor sensitivities to CO2 in highlanderswere double those of the sea-level residents at sea-level,with hypoxia (PO2 of 50mmHg) abolishing this differencethrough proportionally greater hypoxia-induced increasesin total and peripheral chemoreceptor sensitivities inhighlanders. Our results contradict the findings ofFatemian et al. (2003) in that we found the centralchemoreceptor drive to breathe to be depressed inhighlanders compared to lowlanders. These differencescould potentially be attributed to the different highaltitude populations studied (Himalayans in our studyvs. Andeans in the study of Fatemian et al. 2003), tothe different levels of hyperoxia used (PO2 150mmHg inours vs. 100mmHg in their study) and to the potentialconfounding effect of CBF on the measured ventilatorysensitivity of the central chemoreceptor (Berkenboschet al. 1989; Mohan et al. 1999). Fatemian et al. (2003) didnotmeasureCBF in their study, but if theirhighlandershadan impaired cerebrovascular response, as shown in otherstudies (Norcliffe et al. 2005), then the washout of PCO2and subsequent decrease in [H+] at the central chemo-receptors would be decreased, leading to higher measuredcentral chemoreflex sensitivity.

Ventilatory response to hypoxia

Ventilatory response to hypoxia in highlanders wasmarkedly different from that in lowlanders. Unlikelowlanders, who responded to hypoxia by increasing thesensitivity of their ventilatory response to CO2 (Mohan& Duffin, 1997), the highlanders seemed to decreasetheir VRT in response to hypoxia with no change inthe sensitivity to CO2. It should be noted, however,that highlanders displayed marked heterogeneity in theirventilatory response to hypoxia, from which we identifiedthree distinct patterns (Fig. 6). While most highlandersseemed to decrease their VRT in response to hypoxia(Pattern B the only change that was statisticallysignificant), some also increased their basal ventilationwithout a change in sensitivity or VRT (Pattern A), whileothers increased their sensitivity in addition to a change inVRT (PatternC).Our limited sample size prevents us fromcommenting on whether these observations representcertain subsets within the highlander population, but itdemonstrates that the ventilatory response to hypoxia inhighlanders may be heterogeneous, which could furtherconfound interpretation of ventilatory control studies.More studies with larger sample sizes are required tofurther elucidate the nature of this heterogeneity.




The ventilatory response to hypoxia in sea-levelresidents is mediated by peripheral chemoreceptorsvia an increase in the peripheral chemoreceptorsensitivity to [H+] (Cunningham, 1987; Torrance, 1996;Kumar & Bin-Jaliah, 2007). A lack of increase inventilatory sensitivity to CO2 with induction of hypoxiain highlanders suggests that their peripheral chemo-receptors are relatively insensitive to CO2. However, thehypoxia-induced decrease in VRT in highlanders suggeststhat their ventilation is sensitive to hypoxia, albeit by adifferent mechanism than in lowlanders. One possiblemechanism is a complete lackof peripheral chemoreceptorresponsiveness in highlanders due to dysfunctional carotidbodies. In this case, hypoxia might induce a central lacticacidosis that would alter the relationship between [H+]and PCO2 such that a given [H

+] concentration wouldnow be achieved at a lower PCO2 , shifting the VRT to alower value (Duffin, 2005).On theotherhand, consideringthat the peripheral chemoreceptors and carotid bodies areintact in highlanders, the peripheral chemoreceptors mayrespond to hypoxia via CO2-independent mechanisms byincreasing their overall activity and shifting the VRT tolower values (Duffin, 2005). Other possible mechanismsof CO2-independent peripheral responses to hypoxia may

include a hypoxia-induced increase in the carotid bodytonic drive to breathe, changes in systemic hormonalmediators, an altered cerebral spinal fluid-bufferingcapacity at the central chemoreceptor or an alteration incerebral vascular reactivity leading to a higher [H+] atcentral chemoreceptor. What specific changes in carotidbody function have occurred in highlanders to producethe observed differences in the chemoreflex response tohypoxia is beyond the scope of this investigation; however,other studies have shown that cellular and molecularmechanisms play a major role in adaptation to chronichypoxia in many species (Powell, 2007), and that thecarotid bodies of highlanders have altered gene expressionand hypertrophy of carotid body glomus cells (Arias-Stella& Valcarcel, 1976; Kay & Laidler, 1977; Heath et al. 1985;Khan et al. 1988; Lahiri et al. 2000).

Decreased central and absent peripheral chemoreflexsensitivities toCO2 inhighlanders compared to lowlanderscould account for the previously observed blunting ofthe HVR in highlanders (Moore, 2000; Leon-Velarde& Richalet, 2006). As seen from Fig. 5, the differencebetween hypoxic and hyperoxic rebreathing lines is less inhighlanders than in lowlanders, which could account fora lower HVR in highlanders measured during previous

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Figure 6. Heterogeneity of the observed responses to hypoxia in highlandersThree patterns of ventilatory response to hypoxia were observed in the highlander population and these arerepresented by the top panel of graphs. The bottom panel represents individual (dotted) and mean (continuous)responses to hypoxia in basal ventilation (VEB), ventilatory recruitment threshold (VRT) and ventilatory sensitivityto CO2 (VES) among all highlanders, with () indicating statistically significant differences (P < 0.05). Pattern Awas characterized by an increase in basal ventilation during hypoxic rebreathing, with no change in ventilatoryrecruitment threshold (VRT) or sensitivity to CO2 (VES). Pattern B was characterized by a decrease in VRT withhypoxia, and no change in VEB or VES. Pattern C was characterized by increase in VES with hypoxia, and no changein VEB or VRT. Note that only the decrease in VRT with hypoxia (pattern B) was statistically significant across allsubjects.




studies. However, this is only an extrapolation of ourresults, as we did not measure HVR directly during thepresent study.

Genetic versus environmental considerations

An important question that arises from the currentstudy is whether the observed differences in thecontrol of breathing between Himalayan highlanders andCaucasian lowlanders residing at sea-level are due togenetic mechanisms or environmental factors. There isa mounting body of evidence suggesting that geneticmechanismshave an effect on the phenotype of ventilatoryresponses to hypoxia. For example, it has been shown thatblunting of the HVR in Andean highlanders is linked tothe degree of native Indian (Quechua) ancestry (Brutsaertet al. 2005), that certain HVR traits are heritable (Beallet al. 1997), and that the offspring of Tibetan and Chineseparents resemble the Tibetan population in terms of theirresting ventilation and the Chinese population in terms oftheir HVR (Curran et al. 1997).

However, there is also evidence that environmentalfactors including altitude (Curran et al. 1995) and lengthof residence at high altitude (Lahiri, 1980) determinethe magnitude of the HVR in Tibetan and Andeanhighlanders, although a more recent study (Beall et al.1997) failed to identify an association between the lengthof life at high altitude and the HVR. Furthermore,although older studies reported sustained blunting ofHVR in Andean highlanders when theymoved to sea-level(Milledge&Lahiri, 1967; Sorensen&Severinghaus, 1968b;Lahiri et al. 1969), more recent studies suggest thatblunting of the HVR in highlanders is at least partiallyreversed after at least 5 years residence at sea-level (Vargaset al. 1998), indicating plasticity of the mechanismsresponsible for the HVR blunting.

There is therefore evidence for both genetic andenvironmental factors determining the control ofbreathing in highlanders. Although specific genesresponsible for adaptation to high altitude have yet tobe identified, there is evidence that the expression ofhypoxic genes is probably related to the duration ofhigh altitude residence in a given population, such thatTibetan highlanders, who have resided at high altitudefor at least 25,000 years compared to 11,000 years fortheirAndeancounterparts (Aldenderfer, 2003), hadhigherhypoxic gene expression than Andeans residing at similaraltitude (Xing et al. 2008). Our study was not intendedto discern the relative contribution of environmental andgenetic factors on the control of breathing in highlanders,but rather to observe any phenotypic differences in thecontrol of breathing between Himalayan highlanders andCaucasian lowlanders residing at sea-level. Whether theidentified differences are due to environmental or genetic

factors requires further evaluation with dedicated studiesthat examine both control of breathing parameters usingDuffins rebreathing method and genetic factors.

Methodological considerations

Previous control of breathing studies in highlanders usedHVR and HCVR methods, and are briefly summarizedbelow. More detailed discussion about these methods canbe found elsewhere (Duffin, 2007).

HVR methods can be divided into isocapnic andnon-isocapnic methods. Isocapnic methods clamp PCO2and allow the isolated effect of hypoxia on ventilationto be studied. However, these methods have severaldisadvantages. First, they take more time to complete,and since the ventilatory response to hypoxia varies withthe length of hypoxic exposure (Powell et al. 1998), themeasured ventilatory responses may be confounded bythe ventilatory decline that occurs minutes after hypoxicexposure (Moore, 2000). However, recent studies suggestthat the decline does not occur in the 57min required tocomplete an isocapnic test (Huang et al. 1984). Second, thechosen level of isocapnia may affect the magnitude of theHVR(see Fig. 3) (Duffin, 2007). Third, the increase inCBFsecondary tohypoxiaorhypercapniawillwashout thePCO2and lower [H+] at the central chemoreceptor, attenuatingthe measured ventilatory response (Berkenbosch et al.1989;Mohan et al. 1999). Furthermore, the CBF reactivityaffects the magnitude of this attenuation, and CBFreactivity to vasodilating stimuli has been shown tobe different between different highlander populations(Norcliffe et al. 2005; Appenzeller et al. 2006; Claydonet al. 2008).

HCVR methods can be separated into steady-state andrebreathing methods, with the former measuring theventilatory response to progressively increasing isocapnicCO2 levels, and the latter measuring the ventilatoryresponse to continuously rising PET,CO2 . Rebreathing testshave an advantage over steady-state methods, because thedifference between arterial and tissue PCO2 is probablyreduced during rebreathing (Read, 1967; Read & Leigh,1967), so that the effect of changes in CBF on [H+] atthe central chemoreceptor are reduced (Berkenbosch et al.1989; Mohan et al. 1999).

In the present study, we used the Duffins rebreathingtechnique to compare individual components of controlof the breathing model (Fig. 1) between Himalayanhighlanders and lowlanders at sea-level. Unlike previousHVR and HCVR methods, Duffins technique allowedfor independent measurement of the central and peri-pheral chemoreflexdrives, and thenon-chemoreflexdrivesto breathe and comparison of these between Himalayanhighlanders and sea-level lowlanders two populationsliving in their native environments. Measurement of the




change in control of breathing with sustained (hours todays) hypoxic exposure in these populations was beyondthe scope of this study.

Asmentioned earlier in theMethods section, inDuffinstechnique, rebreathing is carried out against a back-ground of both hyperoxia (PO2 of 150mmHg) andhypoxia (PO2 of 50mmHg). Hyperoxia blunts peripheralchemoreflexes and therefore emphasises measurementof the central chemoreflex response, while hypo-xic rebreathing measures the combined central andperipheral chemoreflex responses. In sea-level lowlanders,hypoxic rebreathing both increases the slope of theventilationCO2 relationship and shifts the VRT to theleft compared to hyperoxic rebreathing (Mohan &Duffin,1997). The change in VRT with hypoxia is thought tobe brought about by the increase in slope rather than anincreased tonic drive from the peripheral chemoreceptorsaccording to the current model of the chemoreflexes inlowlanders (Duffin, 2005; Duffin, 2007). As the thresholdof the peripheral chemoreceptors is below the chemo-reflex drive threshold in lowlanders the increased slopeof the peripheral response to CO2 in hypoxia resultsin a decreased VRT. Further detailed discussion of thisphenomenon can be found elsewhere (Duffin et al. 2000).

Future directions

In future studies, it will be important to compare the pre-sent results to those from other high altitude populations,including Andean and Ethiopian highlanders, as wellas acclimatizing lowlanders at different stages ofacclimatization (Moore, 2000). Control of breathingparameters should also be evaluated in highlanders witha varying degree of ancestry to look for correlations(Brutsaert et al. 2005), as well as compared betweengenerations to determine heritability (Curran et al. 1997).Lastly, the interaction between ventilation and otherphysiological parameters, such as cerebral blood flow,should also be considered (Ainslie & Duffin, 2009).

Relevance of our findings

This is the first study to examine the individualcomponents of the Oxford control of breathing model(Fig. 1) in highlanders. We showed that the chemo-reflex drives to breathe of Himalayan highlanders differfrom those of lowlanders, while their non-chemoreflexdrives to breathe are similar. Specifically, we showedthat Himalayan highlanders have decreased central andabsent peripheral chemoreceptor sensitivity to CO2, andthat they are sensitive to hypoxia, albeit via a differentmechanism than that observed in lowlanders at sea-level.A blunted central and an absent peripheral ventilatorysensitivity to CO2 in Himalayan highlanders may stabilize

their ventilatory controller by reducing the overall gainin the feedback part of the controller circuit, therebyreducing altitude-related breathing instability (Ainslie& Duffin, 2009) Furthermore, reduced CO2 sensitivitymay reduce their work of breathing and sensation ofdyspnoea, providing an energy-conserving adaptationin highlanders (Moore, 2000). However, this adaptationshould be compensated for by other adaptations in the O2delivery chain to ensure that O2 delivery to the tissues ismaintained, such as changes in O2 carrying capacity of theblood, blood flow to the organs, oxygen extraction, andenergy production and utilization at the tissues (Moore,2000; Beall, 2007).

Conclusion

Himalayan highlanders have an altered control ofbreathing relative to lowlanders residing at sea-level.They have lower ventilatory recruitment thresholds,decreased central chemoreflex sensitivity to CO2, andlittle or no peripheral chemoreflex sensitivity to CO2.The latter finding may account for the previous findingsof decreased HVR among highlanders. Hypoxia causesa decrease in the ventilatory recruitment threshold inHimalayan highlanders without a concomitant increase inthe sensitivity to CO2 that is seen in lowlanders, suggestinga CO2-independent O2 sensing mechanism by the peri-pheral chemoreceptor, or an increased central chemo-receptor drive secondary to hypoxia-induced central lacticacidosis. The non-chemoreflex drives to breathe weresimilar between Himalayan highlanders and sea-levellowlanders.

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Author contribution

All the authors were involved in all aspects of this work and allauthors approved the final version of the manuscript.

Acknowledgements

We thank Thornhill Research Inc., Toronto, Canada and NewMexico Health Enhancement and Marathon Clinics Research

Foundation, Albuquerque, USA for funding the expedition toLadakh, India, where a substantial part of the data presentedin this paper was acquired. We thank the doctors fromLadakh Institute of Prevention for the Study of Environmental,Occupational, Life Style and High Altitude Related Diseases,Ladakh, India for volunteering their time to assist with datacollection during the expedition. We also thank volunteersubjects from Korzok village, Ladakh, India and QueensUniversity, Canada for their time in the name of science. Fourof the study authors (M.S., E.P., D.P. and J.A.F.) contributedto the development of the RespirAct, a device used for dataacquisition during the Ladakh expedition. These authors standto gain financially if the device is successfully commercializedby Thornhill Research Inc., a University of Toronto/UniversityHealth Network-related company.



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