INHIBITORY EFFECTS OF HYPOCAPNL\ ON RAPID

EYE MOVEMENT (REM) SLEEP

by

PRADEEP DINAKAR. M.B.B.S.

A THESIS

IN

PHYSIOLOGY

Submitted to the Graduate Faculty of Texas Tech University Health Sciences Center

in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE

Advisory Committee

Cynthia Jumper (Chairperson) Melvin Laski

Kathryn McMahon Jean Strahlendorf

Accepted

T3

ACKNOWLEDGEMENTS

^^^ I would like to gratefully acknowledge Texas Tech University Health Sciences

Co9'^ Center and the Department of Physiology for providing the facilities to carry out my

studies. I would like to thank all those who helped me accomplish this work. First, I

would like to express my deep gratitude to my mentor, Dr. Cynthia A. Jumper, for her

support and advice during my graduate studies and research. Her patience, friendship and

camaraderie have meant more to me than she shall ever know. If not for her guidance and

encouragement, I would not be here today. I would also like to acknowledge my

committee Drs. Jean C. Strahlendorf, Kathryn K. McMahon, and Melvin E. Laski for

their patience, time, and helpful suggestions. My deep appreciation goes to the faculty of

the Physiology Department for their commitment and involvement in my training. I

would also like to thank Dr. John M. Orem for allowing me use his lab for my graduate

studies. I also thank Andrew T. Lovering for helping me design and run the experiments.

I would also like to acknowledge Dr. Jerome Dempsey for his insights on the study

design. This study was supported in part by NIH HL-21257, HL-62589, and GAANN

P200A80102.

\\

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

ABSTRACT vi

LIST OF TABLES viii

LIST OF FIGURES ix

LIST OF ABBREVIATIONS x

CHAPTER

L INTRODUCTION 1

Overview 1

Sleep Disruption at High Altitude 2

Role of Hypoxia and Hypocapnia in High Altitude Sleep Disruption 5

n. METHODS AND MATERIALS 10

Experimental Model 10

Surgical Procedure 10

Preparation for Surgery 10

Fabrication of headcap 10

Animal preparation 11

Anesthesia 11

Surgery 11

Tracheostomy 11

Diaphragmatic EMG electrodes 12

111

EEG electrodes 12

PGO electrodes 12

Nuchal EMG electrodes 12

Post-Operative Period 13

Sleep and Ventilation Recording Procedures 13

Adaptation to the Apparatus 13

Preparation Before an Experiment 13

Experiment 14

Protocol 1 15

Protocol II 15

Protocol III 17

Protocol IV 18

Determinationof Sleep and Wakefulness 18

Data Analysis 19

m. RESULTS 20

Sleep in Ventilated Animals with Cycling CO2 (Protocol I) 20

REM Sleep Reduced by Normoxic Hypocapnia (Protocol II) 22

Sleep Study During Isocapnic and Hypocapnic Hypoxia

(Protocol III and IV) 23

Effect on REM sleep 25

Effect on NREM sleep 25

IV

Effect on Wakefulness 25

IV. DISCUSSION 31

Overview of the Results 31

Significance of this study with respect to current literature 33

Role of Hypoxia in Sleep Disruption 33

Role of Hypocapnia in Sleep Disruption 34

Reasons for Role of Hypocapnia in Sleep Disruption 36

BIBLIOGRAPHY 38

ABSTRACT

Sleep disturbances at altitude are accompanied by reductions in end tidal CO2 that

are caused by the ventilatory response to hypoxia. To determine whether hypocapnia

could contribute to the sleep disturbance, the effects of different levels of CO2 on REM

sleep both during apnea caused by mechanical hyperventilation and during hypoxia in

spontaneous breathing were studied. Adult cats were prepared for chronic recordings of

sleep and respiratory parameters (airflow, tidal CO2, intratracheal pressure and

diaphragmatic EMG). The animals were intubated through a tracheal fistula, allowing a

connection to a ventilator or allowing for the adjustment of the FIO2. Computerized

control of CO2 was achieved by injecting CO2 into the inspired air and thus maintaining

a selected end tidal or inspired CO2 level. Four different protocols in five intact,

unanaesthetized adult cats were used. In the first two protocols the animals were

mechanically hyperventilated to apnea and in the last two protocols they were allowed to

breathe spontaneously during three-hour recording periods. In the first protocol, CO2 was

cycled continuously from extreme hypocapnia (end tidal level ~ 3.5%) to just above the

eupneic level (~ 5%). In the second protocol, CO2 was held constant at 65%, 75%, 85%

and 95%) of eupneic levels (hypocapnic normoxia). In the third protocol, two levels of

hypoxia (FIO2 = 0.10 & 0.15) were studied both individually (hypocapnic hypoxia) and

with CO2 added back to maintain isocapnia (isocapnic hypoxia). The fourth protocol was

similar to the third except the inspired CO2 was maintained constant instead of endtidal

CO2 levels. Spontaneously breathing animals were used as the control. The results

VI

obtained from observations of more than 300 REM periods revealed that, irrespective of

the fraction of inspired oxygen, REM sleep was least at lower levels of CO2. In both

hypocapnic normoxia and hypocapnic hypoxia, REM sleep was reduced both in time and

episodes as the CO2 levels decreased. But in isocapnic hypoxia both REM time and

episodes were not significantly affected. NREM sleep was significantly affected at 10%

hypoxia but not at 15% hypoxia. Wakefiilness was not significantly affected by

hypocapnia in our studies. Hence the current results suggest that the sleep disruption at

high altitude may be fully or in part caused by hypocapnia associated with hypoxia.

Vll

LIST OF TABLES

2.1 An example of the counterbalance design used in protocols II, III and IV 16

2.2 Shows the comparison between EEG, PGO, EMG and breathing criteria in REM, NREM sleep and wakefulness 19

Vll l

LIST OF FIGURES

2.1 Illustrates the experimental setup of protocols I and II 16

2.2 Illustrates the experimental setup of protocols III and IV 17

3.1 Effect of cycling CO2 levels on time and episodes of REM sleep 24

3.2 Reduction of REM sleep in normoxic hypocapnia 26

3.3 Effect of hypocapnia on time spent and episodes of REM sleep 27

3.4 Comparison between 3-hour sleep recordings at control conditions

(top) and hypoxic hypocapnia (bottom) conditions 28

3.5 Isocapnic hypoxia prevented REM sleep disruption 29

3.6 Effect ofhypoxia and CO2 on NREM sleep 29

3.7 Effect of hypocapnic hypoxia and isocapnic hypoxic on wakefulness 30

IX

LIST OF ABBREVIATIONS

AMS Acute Mountain Sickness Syndrome

CO Carbon mono-oxide

CO2 Carbon di-oxide

CSF Cerebro Spinal Fluid

EEG Electroencephalography

EMG Electromyography

FIO2 Fraction of Inspired Oxygen

HVR Hypoxic Ventilatory Response

Kg Kilogram

Mg Milligram

Min Minutes

mm/Hg Millemeters of Mercury (Unit of Pressure)

NREM Sleep Non Rapid Eye Movement Sleep

O2 Oxygen

PCO2 Partial Pressure of Carbon di-oxide

PGO Ponto-geniculo-occipital

PO2 Partial Pressure of Oxygen

REM Sleep Rapid Eye Movement Sleep

Sa02 Arterial Saturation of Oxygen

SWS Slow Wave Sleep

CHAPTER I

INTRODUCTION

Overview

Disturbance in sleep architecture is an important feature of an acute high altitude

ascent. It is a part of a larger constellation of symptoms of the acute mountain sickness

syndrome (AMS), which includes nausea, vomiting, loss of appetite, headache, sleep

disturbances, decreased daytime performance, pulmonary edema, coma and death (Kryger,

2000). It is well known that the partial pressure of oxygen (POT) decreases with increasing

altitude (Hultgren, 1997). Previously the symptoms of AMS including sleep disturbances at

high altitude have predominantly been attributed to a lack of O2 (West, 1998). This concept

was reinforced by a normal sleep pattern in subjects who are administered oxygen in

simulated high altitude conditions (Luks et al., 1998). But hypoxia (deficiency of oxygen in

inspired air or in arterial blood and/or in the tissues) at altitude is associated with a

physiological hyperventilation that causes hypocapnia (lower than normal partial pressure

of carbon dioxide in the blood). This hypocapnia has also been attributed to many

symptoms of AMS including sleep disturbances. It has been shown that sleep disruption at

altitude can be relieved by administration of carbon dioxide (CO2) (West, 1998). But many

newer studies have refuted the claims of older studies which state that CO2 administration

does not have an effect in improving sleep architecture (Maher et al., 1975; Pappenheimer

et al., 1977). Thus it is uncertain from current literature whether sleep disruption at altitude

results from hypoxia alone, from hypocapnia caused by hypoxia or from a combination of

the two. This study was designed to investigate the effect ofhypoxia and hypocapnia in

sleep disturbances at simulated high altitude conditions in the intact unanaesthetized cat

model.

Sleep Disruption at High Altitude

The effects of high altitude on the human body were recorded as early as 1783

when the first balloon ascensions began. In 1785, Jean Pierre Francois Blanchard flew

across the English Channel in a balloon and wrote about the deleterious effects of high

altitude. In 1787, physicist and geologist Horace-Benedict de Saussure climbed Mt.Blanc

and vividly described AMS. In 1890 Joseph Vallot constructed an observatory known as

Observatoire Vallot on Mt.Blanc for extensive field studies of life at high altitude (West,

1998).

The inability to obtain sound and restful sleep is one of the most common

discomforts experienced by the visitor to high altitude. The major sleep disruption

symptoms are frequent arousals, periodic breathing (abnormal respiration in which

periods of shallow and deep breathing alternate), shortness of breath, frequent dreams,

and headache (Hultgren, 1997). Objective observation indicates that sleep stages are

generally shifted from deeper to lighter stages while maintaining the total sleep time

constant (Kryger, 2000). Subsequent acclimatization results in sleep improvement and

better quality sleep over a period of days to weeks in most people.

In 1925, Joseph Barcroft conducted his glass chamber experiments to study sleep

quality at simulated high altitude. Even though from observations he seemed to

experience sound sleep, he felt that he had been awake half the night with incessant

dreams, and he felt unrefreshed in the morning. Joem et al. (1970) and Natini et al.

(1970) found serious sleep disruptions in men stationed in Antarctica. The disorder was

termed the polar red eye and presented a marked decrease in the stages 3 and 4 of NREM

sleep. This effect was predominantly attributed to the low partial pressures of oxygen

caused by low barometric pressures (485-525 mmHg) that are created due to geographic

elevation and terrestrial spin of the area. But the artifacts that plagued this study were the

Antarctic environment, stress of isolation, changes in the circadian zeitgebers

(environmental cues that usually help keep the circadian cycle) and possible influences

from the magnetic pole.

In spite of the subjective severity of the sleep symptomatology and the relative

abundance of studies in other aspects of high altitude physiology, there were no objective

studies on altitude induced sleep disturbances until 1974 when Reite et al. (1975)

performed a simultaneous monitoring of sleep state and respiratory pattern on Pike's

Peak. They studied normal healthy subjects both at sea level and at the summit of Pike's

Peak (4300m). On the night of arrival on the mountain it was observed that most subjects

exhibited a curious pattern of breathing consisting of three to four deep breaths followed

by a cessation of breathing for approximately 10 seconds (a form of periodic breathing),

often associated with sleep apnea syndromes. This occurred for at least half the time in

the night and the subject showed a reduction in the time spent in stages 3 and 4 of NREM

sleep and increased number of arousals. Even though sleep shifted towards lighter sleep

(stages 1 and 2), the total time spent in sleep (time spent in all four stages of sleep)

remained constant. Arousals occurred at the transition from apnea to hyperpnea, proving

that sleep disruption at altitude was similar in some ways to that of periodic breathing in

sleep apnea syndromes at lower altitudes. This indicated that periodic breathing, frequent

arousals, and poor quality of sleep are interrelated. O2 administration decreased periodic

breathing but did not decrease the arousals from sleep (Reite et al., 1975). Studies done

later to estimate the negative influences of periodic breathing on arterial O2 saturation

(Sa02) and sleep proved that periodic breathing at high altitude reduced both REM and

NREM sleep (Normand et al., 1990; Goldberg et al, 1992; Mizuno et al., 1993).

Even though periodic breathing is not fully understood, one of the plausible

explanations relates this to increased ventilation associated with the opposing effects of

hypoxia and inhibitory effects of hypocapnic alkalosis at high altitude (Harvey et al.,

1988). The outcome is respiratory oscillation. Hypocapnic alkalosis induces apnea, which

in turn lessens alkalotic inhibition and augments hypoxic stimulation. This stimulates

hyperpnea, which reduces respiratory stimulation by decreasing hypoxia and increasing

hypocapnia and inducing recurrent apnea. The fact that arousals commonly occur at the

transition from the end of apnea to the onset of hyperpnea makes periodic breathing at

altitude one of the most important factors responsible for sleep disruption (Kryger, 2000).

Ventilatory response to hypoxia, also known as hypoxic ventilatory response (HVR) is

directly related to the level of periodic breathing and hence lowlanders (westerners) who

have a greater HVR than highlanders (sherpas) have greater periodic breathing and hence

greater sleep disruption at high altitude (Lahiri et al., Jun 1983; Lahiri et al., 1984; Moore

etal., 1986).

Miller and Horvath in 1977 studied the effects of hypobaric hypoxia (493

Torr~3800m) upon the electrophysiologically assessed sleep of four male and four

female subjects 18-29 yrs old during 2 successive nights. They found the time spent in

synchronized sleep to be the same but REM sleep time was decreased and ascribed this

effect to hypobaric conditions and sleep hypoventilation (Miller et al., 1977).

In 1984, Pappenheimer compared the effects ofhypoxia (10.5%) O2) and carbon

monoxide (0.05%)) on sleep and found that both severely decreased NREM and REM

sleep. Hypoxia increased the respiratory rate and alveolar ventilation but these parameters

were unaffected by CO. Hence it was inferred that hypoxic sleep disruption was not

mediated by the peripheral chemoreceptors for breathing. Recovery of sleep was seen in

1-2 weeks but REM was affected even after a month (Pappenheimer, 1984).

In 1992, Anholm et al. in Operation Everest II studied five subjects exposed to a

progressive increase in simulated altitude for six weeks. Total time spent in sleep and

REM time decreased while the number of nighttime awakenings increased as altitude

increased showing the deleterious role of high altitude on sleep (Anholm et al., 1992).

Role of Hypoxia and Hvpocapnia in High Altitude Sleep Disruption

Hypoxia is an important consequence of high altitude. Thomas Beddoes in 1793

suggested that lack of oxygen was an important reason for symptoms of AMS including

sleep disturbances at high altitude. This was later confirmed by many studies that have

emphasized the role of only hypoxia in sleep disruption. In 1868, Eduard Friedrich

Wilhelm Pfluger (1829-1910) showed that ventilation is increased by hypoxia at high

altitude (West, 1998). This hyperventilation due to hypoxia blows off CO2 from the lungs

and results in hypocapnia or a reduction in pC02 in the blood. King and Robinson re-

emphasized this finding by proving an inverse relation between ventilatory response to

hypoxia and pC02 (King et al., 1972).

Even though carbon dioxide is thought to be less important than oxygen at high

altitude it is considered to have a role under some conditions (Hultgren, 1997). Harvey et

al. 1988 stated that CO2 (in appropriate concentration) is no less vital to life than oxygen.

Angelo Mosso in 1897 proposed that the hypocapnia of acute high altitude exposure

might cause disruption in the sleep pattern. He coined the term acapnia for this condition.

He administered CO2 gas mixtures to relieve the symptoms of AMS including sleep

disruptions in subjects exposed to pressures as low as 250 torr (~ 8800m) in a hypobaric

chamber. Douglas et al. (1909), Douglas et al. (1913) and Henderson et al. (1938) re-

emphasized the role of hypocapnia in AMS (West, 1998). Childs et al. in 1935 and

McFarland et al. in 1938 suggested that inhalation of 3%) CO2 may be beneficial in the

relief of symptoms of AMS. Harvey et al. showed that addition of levels of CO2 lower

than 3%) reduces symptoms of AMS (Harvey et al., 1988).

Opposition to the Mosso concept came from Maher in 1975 who maintained

hypocapnia in 4 subjects and normocapnia by addition of 3.8%) CO2 in 5 subjects at high

altitude for four days to examine the hypothesis that prevention of hypocapnia and

alkalosis would ameliorate the symptoms of AMS. AMS affected the subjects with

normocapnia to a greater extent than the hypocapnic subjects. The reason for this was

hypothesized to be an increased cerebral vasodilatation and spinal fluid pressure (Maher

etal., 1975)

Probably the most important study refuting the beneficial role of adding CO2 in

high altitude condition was that done by Pappenheimer in 1977. He observed the effects

ofhypoxia on slow wave sleep (SWS or NREM sleep) in rats and observed that SWS

under the simulated high altitude condition of 10%) O2 (5500m) was reduced by almost

half Addition of CO2 to the inspired gas failed to prevent the reduction of SWS during

hypoxia. Hence he hypothesized that the effect of altitude on sleep depended on changes

in O2 pressure rather than upon changes in CO2. He fiirther stated that stimulation for

breathing under hypoxia was greater during SWS than wakefulness. It was also his

impression that REM sleep was severely affected during prolonged hypoxia.

In 1994, Yang et al. designed a study to determine the role of added CO2 in

cerebral hemodynamic, metabolic, and fluid shift responses in a conscious sheep model

of acute mountain sickness (AMS). They maintained two groups of animals; a

hypocapnic group and a eucapnic group at the same level ofhypoxia. It was found that

CO2 supplementation at constant p02 did not reduce symptoms of AMS, intracranial

pressure or cerebral edema (Yang et al., 1994).

In 1998, Gundel et al. in their study for NASA studied sleep quality and sleep

architecture in 4 volunteers in a closed system under elevated ambient CO2 levels of 0.7%)

and 1.2% for 23 days each. This study was done to verify the hypothesis that elevated

CO2 levels reduced quality and quantity of sleep. They found that elevated ambient CO2

levels did not affect sleep quantity or latency of first REM sleep. The sleep architecture

was slightly abnormal and showed that the amount of SWS increased with increasing

duration of exposure to CO2 (Gundel et al, 1998).

Modem concepts of sleep disruption at altitude revolve around oxygen, which is

considered the 'vital spirit' and CO2 as a potentially dangerous component, which must

be removed (West, 1998). This concept is further strengthened by the study done by

Lukas et al in 1998, who reported that enrichment of room air with oxygen improves

sleep quality and daytime performance (Lukas et al., 1998). But Mosso's theory in 1897

stated that hypocapnia is the culprit in many AMS symptoms including sleep

disturbances. Ohi et al. (1994) suggested that the sleepiness at altitude is due to

hypocapnia and supports this hypothesis by pointing out that brief voluntary

hyperventilation at low altitude makes people markedly sleepy (Ohi et al., 1994). In 1985

Weil reported that sleep disruption at altitude is related to both hypoxia and hypocapnia

(Weil et al, 1985). According to Ohi et al. (1994), it was Weil's impression that

hypocapnia increases sleepiness by increasing the frequency of onset and time spent in

NREM sleep. Imray et al. in 2001 reported the beneficial effect of CO2 on increasing the

regional cerebral oxygenation and peripheral oxygen saturation. Studies of cerebral

regional oxygenation and peripheral oxygen saturation were done in a portable

hyperbaric chamber at altitude during pressurization. From ambient levels, pressurization

increased inspired CO2 levels, oxygen saturation and cerebral oxygenation. CO2 was

scavenged using soda lime from the inspired air and oxygen saturation and cerebral

oxygenation dropped down. Hence, they believed that CO2 is responsible for one third of

the beneficial effects of a hyperbaric chamber at high altitude for treatment of AMS

8

(Imray et al, 2001). Moreover with the studies showing elimination of the apneic pauses

through inhalation of CO2 during periodic breathing (Lahiri et al, 1983; Lahiri et al,

1984; West et al, 1986) in AMS at altitude, it can be hypothesized that there exists an

interaction between quality of sleep and pC02.

This hypothesis was further strengthened following a fortuitous observation in our

previous experiments of reduced incidence of REM sleep at low endtidal CO2 levels. This

along with the fact that the relation between CO2 levels and quality of sleep is not very

well established and because there is conflicting evidence, a study on the role of CO2 on

sleep, and in particular REM sleep, could help us understand sleep disruption at altitude.

Hence, I propose to study the effect of varying levels of CO2 and oxygen on REM sleep,

at simulated high altitude conditions using the intact unanaesthetized cat model.

9

CHAPTER II

METHODS AND MATERIALS

Experimental Model

The experimental model chosen for the current studies was the intact

unanaesthetized adult cat studied during normal sleep. These cats (n=5) were prepared

and adapted for chronic recordings of electroencephalographic (EEG), diaphragmatic and

nuchal electromyographic (EMG) and pontogeniculo-occipital (PGO) activity. A headcap

containing a connector for these electrodes was attached to their skulls. With the help of

standoffs on the headcap the animal's head was immobilized during sleep recordings. A

tracheal fistula was also created on the ventral aspect of the neck for measuring tracheal

pressure and inspired and end tidal O2 and CO2 levels. In addition, the tracheal fistula

allowed intubation of the animal for mechanical ventilation and for measurements of

airflow. In all experiments ventilation, whether spontaneous or as the result of

mechanical ventilation, occurred through a tracheal tube and not naturally through the

extra-thoracic airway. The Animal Care and Use Committee of Texas Tech University

Health Sciences Center approved the surgical and experimental procedures. The welfare

of the animal was monitored at all stages of the experiment.

Surgical Procedure

Preparation for Surgerv

Fabrication of headcap. A headcap was molded with dental cement. The headcap

10

contained two screws to attach a connector for electrodes (Cinch 19 pin) and three

standoffs for head restraint. The headcap was left to harden before use.

Animal preparation. The experimental animals were treated with 2.5 ml

Chloromycetin IM (Intra Muscular) and 0.3 ml dexamethasone IM, to reduce incidence of

infection and inflammation, for a week before the surgery. The night before the surgery

they were housed in separate cages with no overnight food or water. This helps prevent

operative and postoperative vomiting and aspiration of gastric contents. On the day of the

surgery the surgical fields were shaved and cleaned.

Anesthesia. The animals were anesthetized with acepromazine maleate (2.5 mg,

IM) and ketamine (17 mg kg\ IM). Halothane (1-2 %) in O2 was used as maintenance

anesthesia.

Surgerv

Surgery was performed in an operating room under aseptic/antiseptic conditions.

Depending on the site of electrode implantation, the animal was placed either in the

supine posture on heating pads and its four limbs fastened to the table with sterile cords

or in the prone position in a stereotaxic frame.

Tracheostomy. Under good illumination, with the cat in the supine position, the

neck was hyperextended with a support under it. The shaved area on the ventral side of

the neck was cleaned with betadine solution. The trachea was traced and an incision was

made from below the cricoid cartilage to just above the suprasternal notch. The

sternothyroid, sternohyoid and stemomastoid muscles were retracted to expose the

11

trachea. The trachea was opened longitudinally for a length of five cartilaginous rings.

After ensuring a bloodless field, the cut tracheal rings were stitched to the skin of the

corresponding side to create a fistula. The tracheal fistula was then cleaned and

suctioned, and a small silastic endotracheal tube was passed through it and left inside for

delivery of halothane (Orem et al, 1998).

Diaphragmatic EMG electrodes. The animal was placed in the supine position

with the head and body elevated to displace the abdominal contents caudally. An incision

was made caudal to the costal margin from the xiphoid process to the mid-axillary line.

The costal margin was elevated to provide access to the diaphragm. The right costal and

semitendinous regions were implanted with four electrodes to measure diaphragmatic

activity. These wires were then run under the skin to the skull (Orem et al, 1998).

EEG electrodes. The animal was placed in a stereotaxic frame, and the shaved

area on the dorsal aspect of the skull was cleaned with betadine. A midline incision was

made on the skin to expose the dorsal skull. The periosteum was removed and the skull

was cleaned with hydrogen peroxide. Six burr holes were made on the skull, three on

each side. EEG electrodes and 4-40 stainless steel screws were threaded into the skull in

contact with the cerebral cortex and secured with dental cement (Orem et al, 1998).

PGO electrodes. With the animal in the stereotaxic frame, tripolar stainless steel

electrodes were placed in the lateral geniculate bodies bilaterally to record PGO waves

(Orem etal , 1998).

Nuchal EMG electrodes. Teflon-coated multistranded stainless steel wires

(Cooner AS 632) were implanted in the nuchal muscles through an incision on the back

12

of the neck with the animal in the stereotaxic frame. The insulation was scraped in the

area where the wire was directly exposed to the muscle. EEG, EMG wires and the

prefabricated headcap were fixed to the skull with dental cement (Orem et al, 1998).

Post-Operative Period

The animals recovered from surgery for a minimum of 1 month before

experimentation. After recovery, they were adapted to the experimental apparatus.

Sleep and Ventilation Recording Procedures

Adaptation to the Apparatus

Adaptation to the apparatus helps to reduce abnormal breathing patterns due to

fear and anxiety and enables the animal to have a normal sleep architecture. The animal

was placed in a veterinary cat bag, and the head was immobilized in the stereotaxic frame

with the help of standoffs. The animal could lie either in the sphinx position or in the

right or left lateral position. The EEG, EMG, PGO waves and diaphragmatic activity

were recorded to select the best montage. The recording chamber was closed and the

room was made dark and quiet. The animal was kept in the apparatus for 3 hours each

day during these adaptation sessions.

Preparation Before an Experiment

Once the animal was adequately adapted to the apparatus the actual experiments

were done. The night before each experiment the animal was housed in a cold chamber at

13

30-32°F to consolidate sleep during recording sessions on the following day (Orem et al.

1998). Each recording was 3 hours in duration. During recordings, the animal was

intubated through the tracheal fistula with a silastic 4.0 mm (inner diameter) endotracheal

tube, and the cuff was inflated. The endotracheal tube was attached to a Validyne

pneumotachograph. Tracheal pressures, CO2, and O2 concentrations, as well as airflow

were measured in the tracheal tube. Tracheal pressure was measured using a Volumetric

Pressure Transducer. An Infrared CO2 Analyzer was used to measure tidal CO2 and an

Oxygen Analyzer was used to measure the oxygen levels. In some experiments, the

animals were mechanically ventilated to apnea resulting from low CO2 levels. A two-

position valve switched the animal from spontaneous breathing to mechanical ventilation.

The ventilator delivered 40-50 ml tidal volumes at the rate of 50 per min. The setup also

had a computerized CO2 control program that used pulse-width modulation of a CO2

injector to control the level of CO2. The EEG, EMG, PGO, CO2, O2, airflow and tracheal

pressure signals were recorded on Astro-Med 9500 paper and also on magnetic tape.

Experiment

The literature suggests the effect of altitude is on the quality of sleep rather than

the duration. Thus this study was divided into four protocols to study sleep quality,

especially REM sleep, under various combinations of O2 and CO2. The different

combinations were normoxic hypocapnia, hypoxic hypocapnia and hypoxic normocapnia.

The first protocol was a preliminary study aimed at looking at the probability of REM

episodes at different levels of cycling CO2. The second protocol studied the effects of

14

constant CO2 levels on REM sleep episodes. The third protocol looked at the role of

hypoxia (inspired O2 level) and varying levels of CO2 on REM sleep. The fourth protocol

was same as the third protocol except that the end tidal O2 rather than inspired O2 was

controlled.

Protocol I. In the mechanically ventilated animal, CO2 was cycled from NREM

sleep eupneic level (control level) of CO2 to 65% of the eupneic level (a level below

which sleep quality and quantity are poor) once every twenty minutes. The computer

program cycled CO2 up and down in such a way as to allow the animal to spend a fixed

amount of time at each of the different desired CO2 levels (65%), 75%), 85%, 95% and

105%)) and then the program retraced the steps to 65%) again to complete the cycle in 20

minutes and thus spending equal time at the various levels). Figure 2.1 shows the

experimental setup for protocol I.

Protocol II. In this protocol, the animal was mechanically ventilated as shown in

Figure 2.1 and was exposed to constant preselected end tidal CO2 levels, which were

65%, 75%o, 85%), 95%) of the eupneic value for a 3-hour recording period. A

counterbalanced design was used in this protocol, which is illustrated in Table 2.1. This

required five weeks with five experimental days each week. Sleep during each of the four

different levels of CO2 was recorded on all five days of a week. For example, sleep at

65%, 75%), 85%), 95%) and control was recorded on consecutive Mondays to remove the

effect of the day on the results.

15

PROTOCOL I & II

pCO 2ET

Figure 2.1. Illustrates the experimental setup of protocols I and II. PCO2 ET-partial pressure of CO2 in the endotracheal tube.

Table 2.1. An example of the counterbalance design used in protocols II, III and IV. The percentage values indicate the end tidal CO2 percent of NREM eupneic threshold

WEEK 1 II III IV V

DAY MON

C 95% 85% 75% 65%

TUE 65%

C 95% 85% 75%

WED 75% 65%

C 95% 85%

THU 85% 75% 65%

C 95%

FRI 95% 85% 75% 65%

C

16

Protocol III. In protocol III, the effect of hypocapnia accompanying hypoxia on

sleep was studied. Spontaneously breathing animals were exposed to an inspired fraction

of 10%) and \5% O2. The level of hypocapnia associated with \0% and 15% hypoxia

were approximately 70-75 %> and 80-85 % of NREM eupneic CO2 value, respectively.

The next set of experiments consisted of adding back the CO2 during 10 and \5%

hypoxia to maintain eupneic levels of CO2 (cf Fig. 2.2).

PROTOCOL III & IV

Figure 2.2. Illustrates the experimental setup of protocols 111 and IV

17

Protocol IV. This study was developed to correct a possible artifact in protocol

III. The experimental setup was the same as protocol III except for one change. End tidal

O2 level was controlled instead of inspired O2 level. When CO2 was added back in

conditions of hypocapnia following hypoxia, p02 level increases by about lOmm/Hg due

to increased central stimulation of respiration by CO2. To compensate for the reduction in

hypoxia following CO2 addition, hypoxia had to be increased (more nitrogen had to be

bled into the ventilation). As both protocols III and IV represented similar conditions and

did not differ significantly in their outcomes they were assumed to be the same condition

(cf Fig. 2.2).

Determination of Sleep and Wakefulness

In the hypoxia and CO2 study in protocols III and IV, the 3-hour recordings were

scored. Each recording was scored based on EEG, PGO, EMG and CO2 levels, as REM,

NREM, wakefulness and off hypoxia conditions. Scoring each page is based on the stage

of sleep predominant on that page (in 1.57 minutes). Table 2.2 compares the criteria for

scoring each page as REM sleep, NREM sleep and wakefulness. Hypnograms were

created based on the scoring for the 3-hour recording. A hypnogram is a figure

constructed using a series of dots, each of which represent a page of the recording Astro-

Med paper (1.57 min) and is divided into three stages namely, REM sleep, NREM sleep

and wakefulness. The number of spikes indicates the number of REM sleep periods and

the duration of the spikes indicate the time spent in REM sleep (cf Fig. 3.2).

18

Table 2.2. Shows the comparison between EEG, PGO, EMG and breathing criteria in REM, NREM sleep and wakefulness.

EEG Waves

PGO Waves

EMG Waves Breathing

REM Sleep Desynchnanizatioi and low

inampUtude H^i-density and h i ^

amplitude

Absence of nuchal EMG Irregular breathing

NREM Sleep Synchnmized and h i ^

amplitude Low density and low

amplitude

Presence of nuchal EMG Slow regular breathing

Wakefulness Desynchixxiization and low

in amplitude Low daisity and low

amphtude Presence of nuchal EMG Variable taieathing

Data Analysis

Analysis of the number of episodes and the duration of REM sleep was done from

the data recorded on paper. For protocols I and II, total number of REM periods in every

three-hour recording period was counted and the time spent in REM sleep was

determined. States of sleep and wakefulness were scored using 1.57-minute epochs (1.57

minutes is the time it takes to record one sheet of the Astro-Med paper). The time and

episodes of REM sleep were averaged with each level of CO2 and across various sessions

and animals, and the relation between end tidal CO2 % and total time and periods of

REM sleep was plotted with a standard error of the mean. Statistical significance (p<

0.05) of the difference between the various levels ofhypoxia and hypoxia with CO2

added back was calculated using Psi Plot statistical tools.

19

CHAPTER III

RESULTS

Over 300 REM sleep periods during spontaneous breathing and mechanical

ventilation were obtained from 5 cats in four different protocols. The animals were

housed in a cold environment from 1800 hrs to 900 hrs in the morning. They were

removed at 900hrs and were ad lib till the recording time at 1400 hrs. They had access to

food and water continuously. A 3-hour recording time started as soon as the animals

adapted to the apparatus and the animals were ad lib for an hour before they were

exposed to the cold again.

Sleep in Ventilated Animals with Cycling CO? (Protocol I)

Protocol I was a pilot study done following a previous fortuitous observation of

reduced incidence of REM sleep at low end tidal CO2 levels. It was done in two cats over

a period of 3 months and data from one of the cats is shown here. Statistical analysis was

not done on the data shown. The study was based on the principle of cycling the end tidal

CO2 in a mechanically ventilated cat which was allowed to sleep for a three hour period.

This adjustable hypocapnia, produced by the ventilator, was controlled by an automated

system that made use of pulse width modulation by a CO2 injector, which injected CO2

cyclically. This caused CO2 to cycle in steps from just above NREM sleep eupneic

threshold to 65% of it (65%, 75%, 85%, 95% and 105%). The number of REM sleep

episodes and their duration were recorded at each of the different CO2 levels during

20

cycling. The data shown in Figure 3.1 was from a ventilated animal. The mechanically

ventilated animals developed spontaneous apnea immediately after the start of ventilation

(identified by the absence of diaphragmatic activity). Hypocapnia at 65% of the NREM

sleep CO2 eupneic threshold was achieved within a minute. The cats were normoxic.

Figure 3.1 represents the average total REM time and probability of occurrence of

REM episodes per hour on the ventilator at the different levels of end tidal CO2 in 1

ventilated cat. The x-axis has the various levels of end tidal CO2 values (65%, 75%, 85%,

95%), 105%)) and the y-axis shows probability of occurrence of REM episodes (top) and

percentage of total sleep time spent in REM sleep originating at each level per hour on

the ventilator (bottom). As the experimental setup involved cycling of end tidal CO2

levels, the rate of occurrence of REM episodes were more important than their

maintenance. Hence the REM episodes were calculated as a probability of occurrence at

that particular end tidal CO2 level.

It was observed that average time and episodes of REM sleep was greatest at 85%

end tidal CO2 level. The values at 65% and 95%) end tidal CO2 were much lower than the

85%). The control values (100%) end tidal CO2) were less than that at 85% end tidal CO2,

but much greater than 65% and 95% values. At 105% end tidal CO2 the animal failed to

go into REM sleep. As the end tidal CO2 levels became hypocapnic (lesser than NREM

eupneic CO2 threshold) or hypercapnic (greater than NREM eupneic CO2 threshold), the

time and probability of episodes of REM sleep decreased by up to 50%) of that in eupneic

NREM threshold. Hence, protocol I suggested a possible predilection of lesser REM

sleep at lower and higher levels of end tidal CO2.

21

Cats typically have a REM period once every 15-20 minutes (Coren et al. 1996).

The major artifact that might have affected this study was that the pump cycling time was

also twenty minutes. This may have caused more than normal amounts of REM to occur

in any one stage of CO2 cycling. The effect of the ventilator on this kind of sleep pattern

was also a point under consideration as the levels were given in order and not randomly.

In a three-hour period the ventilator cycled 9 times at 65% and 105% but cycled 18 times

at the other levels. Hence 75%, 85%, and 95% occurred with twice the probability as

65%) and 105%). This was corrected for by taking into consideration the length of time at

the different levels and expressing the data as a probability of REM per hour.

REM Sleep Reduced by Normoxic Hypocapnia (Protocol ID

Protocol II was designed to avoid the artifact that affected the first protocol. This

protocol made use of 2 mechanically ventilated cats over a period of 6 months, in which

the end tidal CO2 levels were held constant in each of the different levels of CO2 (65%,

75%), 85%), 95%)) for a three-hour period. Control conditions were defined as an animal

breathing ambient air spontaneously at Lubbock, Texas (altitude 1000m), under a quiet

and comfortable environment. Figure 3.2 shows the comparison between the hypnograms

of 3-hour recordings in control conditions and in conditions of sustained normoxic

hypocapnia. The control recording had eight REM sleep periods and about 35 minutes of

REM sleep time. When the same animal was exposed to normoxic hypocapnia (65% end

tidal CO2), severe sleep disruption was noticed. The cat had only two REM sleep

22

episodes lasting for 5 minutes over a 3-hour recording period with all other conditions

being the same.

Figure 3.3 shows the effect of constant CO2 levels on REM sleep. REM sleep

episodes and time in REM sleep were highest at the control, where the cats breathe

ambient air spontaneously. Among the various levels of end tidal CO2, 85% end tidal

CO2 had the maximum REM time and episodes compared to the other levels. This was

still less than the control, showing the negative effect of the ventilator on REM sleep.

These values decreased in levels lower and higher than 85%) eupneic CO2. The decrease

in REM sleep at 65%) of eupneic CO2 was statistically significant when compared to the

control values.

Sleep Study During Isocapnic and Hypocapnic Hypoxia

(Protocol III and IV)

Hypoxia associated with high altitude causes a state of hypoxic hypocapnia secondary to

physiological hyperventilation (West, 1998). The significant sleep disruption associated

with high altitude is often ascribed to hypoxia (Reite et al, 1975; Barcroft, 1925;

Pappenheimer, 1977). Figure 3.4 represents a comparison between hypnograms in control

and hypoxic hypocapnic (high altitude conditions) in which significant REM sleep

disruption was seen (bottom figure). The results of protocol II showed that normoxic

hypocapnia caused a significant reduction in REM sleep. The extent of effect ofhypoxia

in high altitude REM sleep disruption was studied in protocol III and IV using two cats

over a 9 month period, under various conditions ofhypoxia (10% and 15%) O2) and

hypoxia with CO2 added back (10%) O2+CO2 and 15% 02+C02)to produce normocapnia.

23

(0 UJ Q O (0 Q. lU

s _

U-T-

LLj a.

U Q .

>-iu t o .

CQ < ffi O Q.

0.400 - |

0.300 -

0.200

0.100 -

0.000

(6) n = 16

(4)

55 I r

75 85

ENDTIDAL C02 %

105

X

rrr io.o n LU

» -

o

z UJ >

3

ai a m

LU

ENDTIDAL C02 %

Figure 3.1. Effect of cycling CO2 levels on time and episodes of REM sleep. The top figure shows the effect of end tidal CO2 on the probability of occurrence of REM sleep episodes and the figure in the bottom shows the effect of end tidal CO2 levels on REM time. The numbers in parentheses represent average REM episodes for that level. Average control REM time 23.5 min, average control REM episodes is 5. Total REM episodes are 16 (n).

24

Bleeding nitrogen into the ventilation system induced hypoxic hypocapnia and adding

CO2 back created normocapnic hypoxia. A spontaneously room air breathing animal

recorded for a three-hour period served as the control.

Effect on REM sleep. Figure 3.5 compares isocapnic hypoxia and hypocapnic

hypoxia at both 10%) and 15% O2 on REM sleep time. In both 10%) and 15% hypoxia,

isocapnic hypoxia (top curve) had greater REM episodes and greater time spent in REM

sleep than hypocapnic hypoxia (bottom curve). Even though the 15% hypoxia values

were not statistically significant, REM sleep values at 10%) hypocapnic hypoxia was

significant statistically from that at 10% isocapnic hypoxia and also from control values.

Effect on NREM sleep. Figure 3.6 shows the effect of hypocapnic hypoxia and

isocapnic hypoxia on NREM sleep. Two cats were studied in this experiment. Both these

conditions did not differ much in time spent in NREM at the level of the control or 15%

O2, but were significantly different at 10% O2.

Effect on Wakefulness. Figure 3.7 represents the effect ofhypoxia and hypoxia

with added back CO2 on wakefulness. Two cats were studied under this experiment. The

hypocapnic hypoxia and isocapnic hypoxia conditions did not differ significantly at any

of the levels ofhypoxia.

25

Control REM

NREM

Wakefulness

n

60 120

Time In minutes

180

Normoxic Hypocapnia

REM

NREM

^

Wakefulness I 0 60 120

Time in minutes on the ventilator

180

Figure 3.2. Reduction of REM sleep in normoxic hypocapnia. The figure on the top represents a control hypnogram with the animal in spontaneous breathing and the figure on the bottom represents that of normoxic hypocapnia at 65% endtidal CO2.

26

J3

o

o

10.0 -n

-

5.0 -

0.0 -

(C)

1(33)

T

(20)

(29)

^ \

'

(19) *

' 1 105

(15)

95 85 75 65

End Tidal CO2 (% Normocapnia)

u JS f^ X 30.0 c

'i g - 20.0 -

10.0 -

H

(C)

(33)

(15)

' \ ' 1 ' \ ' I 105 95 85 75 65

End Tidal CO2 (% Normocapnia)

Figure 3.3. Effect of hypocapnia on time spent and episodes of REM sleep. The left figure shows the comparison between end tidal CO2 levels and REM sleep episodes per three hours and the right figure shows the comparison between end tidal CO2 levels and REM sleep time per three hours. Both the 65% values were significantly reduced compared to the control (C) and it corresponds to a FIO2 of 0.10. Values are means +/-SEM.*p<0.05, Student's t-test. The numbers in parentheses represent number of REM period for that level.

27

REM

NREM

Wakefulness

Control

120 180

Time in minutes

REM

NREM

Wakefulness

Hypocapnic Hypoxia

U 60 120

Time in minutes

180

Figure 3.4. Comparison between 3-hour sleep recordings at control conditions (top) and hypoxic hypocapnia (bottom) conditions. The area from the last spike to the 180-minute mark in both these hypnograms represents wakefulness.

28

120 120

O

o

m

I r

I a. o £ w Z 111

80

40 -

0 25.0

' p<0.05 (ten) ' p<0.05 (cont)

20.0 15.0

FI02

IH

HH

10.0 5.0

Figure 3.5. Isocapnic hypoxia prevented REM sleep disruption. Comparison between the time spent in REM (left) and REM episodes (right) with the various inspired oxygen levels (10%), 15% and control) in both conditions of isocapnia and hypocapnia. The values in parentheses are number of REM episodes at each level. Values are means +/- SEM.* p<0.05, IH - Isocapnic Hypoxia, HH -Hypocapnic Hypoxia

80 - ,

£ 70

s LU

•^ 60

C « a w 0)

E

50 -

40 -

30

IH

HH

20.0 15.0 10.0 so

FI02

Figure 3.6. Effect ofhypoxia and CO2 on NREM sleep. Comparison between the time spent in NREM at various inspired oxygen levels (10%, 15% and 21%) in both conditions of isocapnia and hypocapnia. Values are means +/- SEM.* p<0.05. IH - Isocapnic Hypoxia, HH - Hypocapnic Hypoxia

29

(A in a> c

0)

re

c 0) Q. M

E

120 -

110 -

~ 100 -

>0 -

80

HH

IH

20.0 16,0 10.0 M

FI02

Figure 3.7. Effect of hypocapnic hypoxia and isocapnic hypoxic on wakefulness. IH - Isocapnic Hypoxia, HH -Hypocapnic Hypoxia

30

CHAPTER IV

DISCUSSION

Overview of the Resuhs

Hypoxia associated with high altitude causes hyperventilation and hypocapnia.

Various combinations of O2 and CO2 were used to look at the role of CO2 in high akitude

sleep problems. It is also known that hypoxia at altitude is associated with a sleep

disruption, daytime sleepiness and reduced performance (West. 1998). Four protocols

were developed to study this effect. Protocol I and II dealt with normoxic hypocapnia and

protocol III and IV studied the effects of hypocapnic and isocapnic hypoxia.

In protocol I, CO2 was cycled from hypercapnia to hypocapnia in a ventilated

animal. There appeared to be a predilection for reduced probability of occurrence of

REM sleep episodes at low end tidal CO2 levels. It was also seen that at these low end

tidal CO2 levels, REM episodes were invariably shorter when compared to those, which

developed at higher end tidal CO2 levels. The maximum probability of occurrence of

REM sleep was at 85%) of NREM eupneic threshold and decreased on either side (Figure

3.1).

To rule out the most significant possible artifact in protocol I, protocol II was

developed to study the effect of constant CO2 levels on sleep disruption (65%, 75%, 85%,

95%), 100%) or control). In general, protocol II produced the same results observed using

protocol I and indicated that low CO2 causes sleep disruption.

To rule out the possible negative effect of the ventilator on sleep in the first two

experiments, a third protocol was developed which involved hypoxia under spontaneous

31

breathing with and without maintaining a constant end tidal CO2. Protocol IV differed

from the third protocol in maintaining a constant inspired CO2 instead of end tidal CO2.

As the results of Protocol III and IV did not differ significantly they were considered as

one protocol (Figure 3.5). The results from protocol III and IV suggest that the amount of

REM sleep and the number of episodes were significantly lower at 10% O2 compared to

the 10%o O2 with CO2 added back and the control values. The amount of NREM sleep

also was significantly greater at 10% O2 than when CO2 was added back. Wakefulness

was not different in the two conditions. In other words breathing 10%) O2 prevented

NREM sleep from converting into REM sleep but did not significantly affect

wakefiilness. In the case of 15%) O2, it was seen that the total number of REM episodes

and the total time spent in REM was not significantly greater at 15% O2 with CO2 added

back. NREM sleep at \5% O2 was not significantly different than with CO2 added back.

The change in wakefulness at 15% O2 was also not significant.

Summarizing the results of the current study, we found that REM sleep was

significantly lower at 10%) O2 levels compared to the control values and when isocapnic

conditions were maintained the REM sleep levels return to control values. 15% O2 level

values were similar to that of the 10%o O2 values, but were not statistically significant.

The lost REM sleep was re-distributed as an increase in NREM sleep. Thus the present

study suggested that hypocapnia associated with high altitude played an important role

independent of oxygen in causing the deleterious effects of high altitude on the induction

of REM sleep.

32

Significance of This Study With Respect to Current Literature

Hypoxia at high altitudes causes hypocapnia and leads to the condition of hypoxic

hypocapnia. Sleep disruption at altitude may therefore be the result of either hypoxia or

hypocapnia individually or a combination of both. Protocols III and IV without a constant

end tidal CO2 were equivalent to a high altitude condition, in which both hypoxia and

hypocapnia coexist. Severe reduction in REM sleep and increase in NREM sleep were

seen under these conditions. The first two protocols created a condition of isolated

hypocapnia, which also caused a severe sleep disruption at low end tidal CO2. The last

condition, hypoxia alone, was achieved by maintaining a constant end tidal CO2 levels.

This condition was associated with near normal sleep amounts and architecture.

Role of Hypoxia in Sleep Disruption

After Torricelli noted a decrease in the barometric pressure of O2 with increasing

altitude in the early seventeenth century, Thomas Beddoes in 1793 suggested that

mountain sickness is caused by lack of oxygen (Hultgren, 1997). This was thought to be

confirmed through the realization of Paul Bert's theories in the beginning of the twentieth

century (West, 1998). These theories show that the effect of O2 inhalation relieves the

deleterious effects of high altitude. The study by Miller and Horvath (1977) showed a

role for hypobaric hypoxia in sleep disruption. Pappenhiemer (1977) proved that hypoxia

causes a 50%) reduction SWS (comparable to NREM sleep) in rats and showed that CO2

addition does not reduce the SWS found in hypoxia. Sutton et al. in 1979 re-emphasized

the role ofhypoxia in sleep disruption. West et al. in 1998 proved through his room

33

oxygen enrichment experiments that removal ofhypoxia at altitude improves sleep and

improves AMS score in the morning. However, Reite et al. (1975) and many others

(Normand et al. 1990; Goldberg et all992; Mizuno et al. 1993) have proved that

administration of O2 at altitude decreases periodic breathing but not arousals from sleep.

Generally these studies support a role for hypoxia as the causative factor in sleep

disruption observed in high altitude conditions. However, it is imperative to determine

the contribution of hypocapnia in high altitude sleep disruption. The observed

improvement in nocturnal sleep architecture following oxygen administration might be

the result of a decrease in hypocapnia caused by a reduction in hypoxia.

It is also interesting to note that the conditions of hypoxic normocapnia, which

caused REM sleep values to be equal to the control values, is also found in nature in

utero. The fetus in utero has an arterial poj of 28 mmHg, PCO2 ^^ ̂ ^ ™^ ^8 ^"^ P^ of

7.32 compared to the mother who has an arterial p02 of 100mm Hg, pC02 of 32 mm Hg

and pH 7.42 (Bonica, 1980). This acidotic fetal condition is similar to spending 10

months on the top of Mt.Everest in normocapnia. It is also worth pointing out that the last

trimester fetus spends a majority of its time in REM sleep (Schmidt et al, 2000). Thus

this observation in nature supports the results of the current study.

Role of Hypocapnia in Sleep Disruption

According to Mosso's theory in 1897, the hypocapnia associated with altitude

hypoxia is considered a culprit in causing symptoms of AMS including sleep

disturbances. He relieved symptoms of AMS by CO2 administration. Douglas, Haldane

34

and Henderson et al re-emphasized the role of hypocapnia in AMS (West 1998). In the

1930s several investigators proved that 3% CO2 administration relieved the severity of

AMS (Childs et al, 1935; McFarland et al, 1938; Ohi et al, 1994). In 1985 Weil

reported that sleep disruption at altitude is related to both hypoxia and hypocapnia.

According to Ohi et al. (1994), it was Weil's impression that hypocapnia increases

sleepiness by increasing the frequency of onset and time spent in NREM sleep. Harvey et

al. in 1988 also re-emphasized the improvement in symptoms of AMS through addition

of 3%) CO2 and thus maintaining eucapnia. Ohi et al. (1994) suggested that the sleepiness

at altitude is due to hypocapnia and supported this hypothesis by pointing out that brief

voluntary hyperventilation at low altitude makes people markedly sleepy. In 2001 Imray

et al confirmed the beneficial role of CO2 in the alleviation of sleep disruption and

showed that increased CO2 levels improved cerebral oxygenation and peripheral oxygen

saturation.

However other studies argue against the positive effect of addition of CO2 in sleep

improvement (Forwand et al, 1968; King et al, 1972). Maher et al. in 1975 showed that

administration of 3.8%) of CO2 in hypoxia (to make subjects eucapnic) worsens symptoms

of AMS. Gundel et al in 1998 showed that prolonged exposure to high levels of CO2

leads to sleep disruptions. In 1994 Yang et al. designed a study to determine the role of

CO2 in symptoms of AMS and sleep disturbance and showed CO2 supplementation at

constant p02 did not reduce AMS or cerebral edema. But the most important study

showing a negative effect of CO2 was done by Pappenhiemer in 1977, in which he

showed the addition of CO2 during hypoxia in rats does not reduce SWS during hypoxia.

35

The current study found that NREM sleep (SWS) significantly decreased on

administration of CO2 at low p02 (simulation of high altitudes) but did not decrease

significantly at more moderate p02 level (a simulation of lower altitude). It is noteworthy

that the main difference between the Pappenhiemer study and the current study was the

subject animal. Pappenhiemer used rats, which have a sleep pattern different from cats

(and humans). Moreover, unlike the current study, hypoxia was induced using CO in his

studies and thus a resulting reflex increase in ventilatory response causing hypocapnia

was absent.

Reasons for the Role of Hypocapnia in Sleep Disruption

From the present study and sleep literature it can be inferred that hypocapnia with

or with out hypoxia disrupts sleep. It can be extrapolated further that addition of CO2 is

beneficial to sleep at altitude. It can be hypothesized that the effectiveness of CO2 relates

to increased cerebral blood flow. Isolated hypocapnia is a powerfiil constrictor of

mammalian cerebral blood vessels (Grubb et al, 1974). As the brain in REM sleep is

highly active, comparable to the brain in wakefulness, an increase in cerebral blood flow

is observed (Lenzi et al, 2000). Hypocapnia may reduce this flow resulting in a loss of

REM sleep. This would explain the findings of the first two protocols that normoxic

hypocapnia disrupts REM sleep at altitude. But this fails to explain the sleep disruption in

hypoxic hypocapnia where cerebral blood flow would be expected to be increased due to

hypoxia (Kannurpatti et al, 2002). Another suggestion is the respiratory stimulant action

36

of C02. Imray et al in 2001 shows that the administration of CO2 improves cerebral

oxygenation and peripheral oxygen saturation through increased breathing and proves

beneficial in sleep disruption. Another possible explanation for this effect of hypocapnia

is the acid trigger hypothesis. It is known that hypoxic hypocapnia causes a state of

alkalosis, and this alkalosis by unknown mechanisms, inhibits REM sleep. This

hypothesis would predict that addition of CO2 would provide an acid trigger, and thereby

increase REM sleep to control values.

Further studies are required to determine if one or more possible explanations

account for the REM sleep changes observed. Studies measuring the pH of CSF and also

intra cerebral pH during hypoxia and addition of CO2 would be a next confirmatory study

to test the acid trigger hypothesis. Doppler flow studies of the cerebral vessels during

hypoxia and REM could be used to define the role of carbon dioxide in REM sleep.

Further the different mechanisms affecting sleep may not be mutually exclusive and may

operate to different degrees depending on the presence and absence ofhypoxia.

37

BIBLIOGRAPHY

Articles

Anholm J.D, Powles A.C.P, Downey III R, Houston C.S, Sutton J.R, Bonnet M.H, Cymerman A. Operation everest II: Arterial oxygen saturation and sleep extreme simulated altitude. Am rev respir dis 1992; 145: 817-26

Aserinsky E, Kleitman N. Regulariy occurring periods of eye motility and concomitant Phenomena, during sleep. Science 1953; 118: 273 - 274

Aserinsky E, Kleitman N. Two types of ocular motility occurring in sleep. J Appl Physiol 1955; 8: 11-18

Barcroft J. The Respiratory function of the Blood, part I: Lessons from High Altitudes. Cambridge : Cambridge University Press, 1925.

Childs, Hamlin H, Henderson Y. Possible value of inhalation of CO2 in climbing great altitudes. Nature 1935; 135:457-458

Chin K, Hirai M, Kuriyama T, Fukui M, Kuno K, Sagawa Y, Ohi M. Changes in the arterial PC02 during a single night's sleep in patients with obstructive sleep apnea. Intern med 1997 Jul;36(7):454-60

Cohen G, Henderson-Smart DJ. The characteristics and frequency of augmented breaths during C02-induced hyperpnoea of newbom infants. J Physiol 1996 Jan 15;490 (Pt2):551-7

Corfield DR, Roberts CA, Griffiths MJ, Adams L. Sleep-related changes in the human 'neuromuscular' ventilatory reponse to hypoxia. Respir physiol 1999 Sep 15;117(2-3):109-20

Dement W, Kleitman N. Cyclic variations in EEG during sleep and their relation to eye movements, body motility, and dreaming. Electroencephalogr Clin Neurophysiol 1957; 9:673-90

Douglas C.G, Haldane J.S. The regulation of normal breathing. J. Physiol 1909; 38: 420-40

Evarts E. Effects of sleep and waking on spontaneous and evoked discharge of single units in visual cortex. Fed proc. 1960; 4 (suppl): 828-37

38

Fitzgerald D, Van Asperen P, Leslie G, Arnold J, Sullivan C. Higher Sa02 in chronic neonatal lung disease: does it improve sleep? Pediatr Pulmonol 1998 Oct;26(4):235-40

Forwand S, Landsdwne M, Folansbee J, et al Effect of acetazolamide on acute mountain sickness. N. Engl. J. Med. 1968; 279: 839-45

Goldberg F, Richalet J.P, Onnen I. Sleep apneas and high altitude newcomers. Int Sports Med. 1992;13:S34-S36

Grubb R.L, Raichle M.E, Eichling J.O, Ter-Pergossian M.M. The effects of changes in PaC02 on cerebral blood volume, blood flow and vascular mean transit time. Stroke 1974; 5 : 630-39

Gundel A, Parisi RA, Strobel R, Weihrauch MR. Joint NASA-ESA-DARA Study. Part three: characterization of sleep under ambient C02-levels of 0.7% and 1.2%. Aviat Space Environ Med 1998 May;69(5):491-5

Harvey T, Winterbom M, Lassen N, et al. Effect of C02 in acute mountain sickness: A rediscovery. Lancet 1988; 17:639-41

Hajack G, Klingelhofer J, Schulz-Varszegi M, Sander D, Staedt J, Conrad B, Ruther E. Cerebral perfusion during sleep-disordered breathing. J Sleep Res 1995 Jun;4(Sl):135-144

Holditch - Davis D, Edwards L.J. Modeling development of sleep-wake behaviors. II. Results of two cohorts of preterms. Physiol Behav. 1998 Feb 1;63(3):319-28.

Imray C.H.E, Clarke T, Forster P.J.G, Harvey T.C, Hoar H, Walsh S, Wright A.D. Carbon dioxide contributes to the beneficial effect of pressurization in a portable hyperbaric chamber at high altitude. Clin Sci (Lond). 2001 Feb; 100(2): 151-7

Joren A.T, Shurley J.T, Brooks R.E,Guenter C.A,Pierce CM. Short term changes in sleep patterns on arrival at the south polar plateau. Arch. Intern. Med. 1970, 125:649-54

Jouvet M. Recherches sur les structures nerveuses et les mecanismes responsales des differentes phases du sommeil physiologique. Arch Ital Biol. 1962; 100: 125-206

Kannurpatti SS, Biswal BB, Hudetz AG. Differential fMRI-BOLD signal response to apnea in humans and anesthetized rats. : Magn Reson Med 2002 May;47(5):864-70

39

Kellog R. The role of C02 in altitude acclimatization. In D Cunnigham and B. Lloyd, eds.. The regulation of human respiration: The proceedings of the J.S Haldane centenary Symposium. Oxford: Blackwell Scientific Publications, 1963: 379-95

Kim WS, Champagne VL, Gottfried SB, Kimoff RJ. Effect of inspiratory pressure support on the arousal response to C02 in sleeping dogs. J Appl physiology 1995 Nov;79(5): 1419-25

King A, Robinson S. Ventilation Response To Hypoxia and Acute Mountain Sickness. Aero Med 1972;43:419-24

Lahiri S, Barnard P. Role of arterial chemoreflex in breathing during sleep at high altitude. Prog Clin Biol Res. 1983;136:75-85.

Lahiri S, Maret K, Sherpa M.G. Dependence of high altitude sleep apnea on ventilatory sensitivity to hypoxia. Respir Physiol 1983 Jun;52(3):281-301

Lenzi P, Zoccoli G, Walker A.M, Franzini C. Cerebral circulation in rem sleep: is oxygen a main regulating factor? Sleep Res Online. 2000;3(2):77-85

Luks A.M, Van Melick H, Batarse R.R, Powell F.L, Grant I, West JB. Room oxygen enrichment improves sleep and subsequent day-time performance at high altitude. Respir Physiol. 1998 Sep;113(3):247-58

Lutz W, Wendt H.J, Werz R Von, Zimgibl M. Uber die werkung von Kohlensaiire auf die erholung aus sauerstoffmangel Luftfahrt med 1943; 14 : 250-61

Maher J, Cymerman A, Reeves J, et al. Acute Mountain Sickness: Increased severity in eucapneic hypoxia. Aviat Space Environ med. 1975; 46:826-29

McFariand and Dill D. Comparative study of the effects of 02 pressure on man during acclimatization. J Aviat Med 1938; 9:18-44

McNicholas W. Impact of Sleep in COPD. Chest. 2000;117: 48S-53S

Meurice JC, Marcl, Series F. Influence of sleep on ventilatory and upper airway response to C02 in normal subjects and patients with COPD. Am J Respir Crit Care Med 1995Nov;152(5Ptl):1620-6

Milledge J, and Lahiri S. Respiratory control in lowlanders and Sherpa highlanders at altitude. Respir. Physiol. 1967; 2:310-22

Miller J.C, Horvath S.M. Sleep at Altitude. Aviation, Space, and Environmental Medicine. July 1977

40

Moore L.G, Harrison G.L, McCullough R.E. Low acute hypoxic ventilatory response and hypoxic depression in acute altitude sickness. J Appl Physiol 1986; 60: 1407-12

Mizuno K, Asano K, Okudaira N. Sleep and Respiration under Acute Hypobaric Hypoxia. Jap J Physiol, 43, 161-75, 1993

Natani K, Shurley J.T, Pierce CM, Brooks R.E. Long-term changes in sleep patterns in man on the south polar pleateau. Arch. Intern. Med. 1970, 125:655-59

Nattie EE, Li A. C02 dialysis in the medullary raphe of the rat increases ventilation in sleep. J Appl Physiol 2001 Apr;90(4): 1247-57

Nicholson A.N, Smith P.A, Stone B.M, Bradwell A.R, Coote J.H. Ahitude insomnia: Studies during an Expedition to the Himalayas. Sleep 11(4):354-361, Raven press,Ltd., New York

Nielsen A.M, Bisgard G.E, Vidruk E.H.Carotid chemoreceptor activity during acute and sustained hypoxia in goats. J Appl Physiol. 1988; 65:1796-1802

Nims L.F. Anoxia in Aviation. Annu Rev Physiol. 1948; 10: 305-14

Normand H, Barragan M,Benoit O, Bailliart O, Raynaud J. Periodic breathing and 02 saturation in relation to sleep stages at high altitude. Aviat Space Environ Med. 1990Mar;61(3):229-35

Ohi M, Chin K, Hirai M, Kuriyama T, Fuhui M, Sagawa Y, Kuno K. Oxygen desaturation following voluntary hyperventilation in normal subjects. Am J Respir Crit Care Med. 1994 Mar;149(3 Pt l):73l-8

Orem J, Vidruk EH. Activity of medullary respiratory neurons during ventilator-induced apnea in sleep and wakefulness. J Appl Physiol 1998 Mar;84(3):922-32

Pappenheimer J.R. Sleep and respiration of rats during hypoxia. J Physiol 1977 Mar;266(l): 191-207

Pappenheimer J.R. Hypoxic insomnia: effects of carbon monoxide and acclimatization. J Appl Physiol. 1984 Dec;57(6): 1696-703

Phillipson E.A, Kozar L.F, Rebuck A.S, Murphy E. Ventilatory and waking responses to C02 in sleeping dogs. Am Rev Respir Dis. 1977 Feb;l 15(2):251-9.

Reite M, Jackson D, Gaboon R.L, Weil J. Sleep physiology at high altitude. Electroencephalography and Clinical Neurophysiology, 1975, 38: 463-71

41

Reivich M, Kety S. Blood flow metabolism couple in brain. In: Plum F, ed. Brain Dysfunction in Metabolic Disorders. New York, NY : Raven Press; 1968 : 125-40

Schafer T. Variability of vigilance and ventilation: studies on the control of respiration during sleep. Respir Physiol 1998 Oct;114(l):37-48

Schafer T, Schlafke M. Respiratory changes associated with rapid eye movements in normo- and hypercapnia during sleep. J Appl Physiol 1998 Dec;85(6):2213-2219

Schmidt K, Kott M, Muller T, Schubert H, Schwab M. Developmental changes in the complexity of the electrocortical activity in foetal sheep. J Physiol Paris. 2000 Sep-Dec;94(5-6):435-43

Smith CA, Saupe K.W, Henderson K.S, Dempsey J.A. Ventilatory effects of specific carotid body hypocapnia in dogs during wakefulness and sleep. J Appl Physiol. 1995 Sep;79(3):689-99

Stein MB, Millar TW, Larsen DK, Kryger MH. Irregular breathing during sleep in patients with panic disorder. Am J Psychiatry 1995 Aug; 152(8): 1168-73

Straub H. Alveolargasanalysen. I. Uber schwankungen in der tatigkeit des atemzentrums, speziell im schlaf Dsch. Arch. Klin. Med. 117: 397-417, 1915

Sutton J.R, Houston C.S, Mansell A.L, McFadden M.D, Hackett P.M, Rigg J.R, Powles A.C Effect of acetazolamide on hypoxemia during sleep at high altitude. N Engl J Med. 1979 Dec 13;301(24): 1329-31

Tenny S. Physiological adaptations to life at high altitude. Modem Concepts CV Dis. 1962;31:713-18

Weil J, Kryger H, and Scoggin C Sleep and breathing at high altitude. In C Guilleminauh and W dement, eds.. Sleep apnea syndromes. New York: A.R. Liss, 1978: 119-23

Weil J.V. Sleep at Altitude. Clin Chest Med 1985 Dec;6(4):615-21

Weil J. Ventilatory Control At High Altitude.In: Chemiack N.S, Widdicombe J.G, eds.Handbook of Physiology: The Respiratory System II. Bethesda, Md: American physiological Society; 1986

West J.B, Peters R.M, Aksnes G, Maret KH, Milledge J.S, Schoene R.B. Nocturnal periodic breathing at altitudes of 6,300 and 8,050 m. J Appl Physiol. 1986 Jul;61(l):280-7

42

Yang SP, Bergo GW, Krasney E, Krasney JA. Cerebral pressure-flow and metabolic responses to sustained hypoxia: effect of C02. J Appl Physiol 1994 Jan;76(l):303-13

Zielenski J, Koziej M, Mankowski M, Sarybaev A, Tursalieva J, Sabirov I, Karamuratov A, Mirrakhimov M. The quality of sleep and periodic breathing in healthy subjects at an altitude of 3200 m. High Altitude Medicine and Biology; Vol 1, Number 4, 2000,331-36

Books

Bonica JJ: Obstetric Analgesia and Anesthesia. 2"'' ed. Amsterdam: Worid Federation of Societies of Anesthesiologists, 1980, p29

Coren, Stanley. Sleep Thieves: an Eye-Opening Exploration into the Science & Mysteries of Sleep. New York: The Free Press, 1996. p. 43

Hultgren H. High Altitude Medicine, 1997. Hultgren Publications

Kryger, Roth, Dement. Principles and Practice of Sleep Medicine, 3'̂ '̂ ed, 2000. W.B. Saunders Company

Lahiri S, Maret K, Sherpa M.G, Peters R.M.Jr. Sleep and periodic breathing at high altitude: Sherpa natives versus sojourners. In: High Altitude and Man, edited by J.B.West and S. Lahiri. Bethesda, M.D: American physiological society, 1984, pp. 73-90

West J.B. High Life. A History Of High Altitude Physiology And Medicine. New York. Oxford University Press. 1998

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