ROLE OF OREXIN IN RESPIRATORY AND SLEEP DURING UPPER AIRWAY OBSTRUCTION
http://dx.doi.org/10.5665/sleep.3676
Role of Orexin in Respiratory and Sleep Homeostasis during Upper Airway
Obstruction in Rats
Ariel Tarasiuk, PhD1; Avishag Levi, MSc1,2; Nilly Berdugo-Boura, MSc1,2; Ari Yahalom, BSc1,2; Yael Segev, PhD2
Sleep-Wake Disorders Unit, Soroka University Medical Center and Department of Physiology, Faculty of Health Sciences, Ben-Gurion University of
the Negev, Beer-Sheva, Israel; 2Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University of the
Negev, Beer-Sheva, Israel
1
Study Objectives: Chronic upper airway obstruction (UAO) elicits a cascade of complex endocrine derangements that affect growth, sleep, and
energy metabolism. We hypothesized that elevated hypothalamic orexin has a role in maintaining ventilation during UAO, while at the same time
altering sleep-wake activity and energy metabolism. Here, we sought to explore the UAO-induced changes in hypothalamic orexin and their role in
sleep-wake balance, respiratory activity, and energy metabolism.
Interventions: The tracheae of 22-day-old Sprague-Dawley rats were surgically narrowed; UAO and sham-operated control animals were monitored
for 7 weeks. We measured food intake, body weight, temperature, locomotion, and sleep-wake activity. Magnetic resonance imaging was used
to quantify subcutaneous and visceral fat tissue volumes. In week 7, the rats were sacrificed and levels of hypothalamic orexin, serum leptin, and
corticosterone were determined. The effect of dual orexin receptor antagonist (almorexant 300 mg/kg) on sleep and respiration was also explored.
Measurements and Results: UAO increased hypothalamic orexin mRNA and protein content by 64% and 65%, respectively. UAO led to
30% chronic sleep loss, excessive active phase sleepiness, decreased body temperature, increased food intake, reduction of abdominal and
subcutaneous fat tissue volume, and growth retardation. Administration of almorexant normalized sleep but induced severe breathing difficulties in
UAO rats, while it had no effect on sleep or on breathing of control animals.
Conclusions: In upper airway obstruction animals, enhanced orexin secretion, while crucially important for respiratory homeostasis maintenance,
is also responsible for chronic partial sleep loss, as well as considerable impairment of energy metabolism and growth.
Keywords: upper airway loading, orexin, sleep, rat
Citation: Tarasiuk A, Levi A, Berdugo-Boura N, Yahalom A, Segev Y. Role of orexin in respiratory and sleep homeostasis during upper airway
obstruction in rats. SLEEP 2014;37(5):987-998.
INTRODUCTION
Chronic upper airway obstruction (UAO) during sleep in
children elicits a cascade of complex endocrine derangements
that affect growth, sleep, and energy metabolism.1,2 We have
previously shown that chronic UAO by tracheal narrowing in
rats leads to acute and chronic adaptive changes in the respiratory system, including large swings in pleural pressure and
respiratory muscle contractility.3-10 These adaptations are critical for proper ventilation maintenance, especially during sleep,
a condition where respiratory muscle force may not be sufficient
to support normal breathing. We have further shown that UAO
in rats causes growth retardation, sleep disorder, and hypothermia that were related to abnormalities in growth hormone
releasing hormone/growth hormone axis.7-10 The mechanisms
that link UAO and its neuroendocrine consequences are largely
unclear.
Looking for the central regulatory factor that may explain
sleep/wake abnormalities, hypothermia, and growth retardation in UAO,9,10 we focused our attention on the hypothalamic
neuropeptide orexin. Orexin emerged as a key orchestrator of
brain states and adaptive behaviors: both arousal and breathing
centers receive stimulatory inputs from orexin neurons.11 In
the respiratory system, orexin has a primary role in carbon
dioxide chemoreception.12-14 Orexin provides an important integrative link between peripheral metabolism and homeostatic
challenges of forced exercise15-18 that occurs in UAO rats.3,4
Orexin is also associated with multiple functions such as sleepwake balance, increased caloric intake, body temperature, and
locomotion activity,19-27 all of which are affected by UAO in
animals. Little is known, however, about the role of orexin in
respiratory adaptation to UAO, where the respiratory system is
chronically challenged to maintain homeostasis.
In the current study we investigated the effect of UAO in rats
on hypothalamic orexin level and its role in sleep-wake activity,
respiration, and energy metabolism. We tested the hypothesis
that hypothalamic orexin has a role in maintaining ventilation during UAO, while at the same time altering sleep-wake
balance and energy metabolism. Here, we report that orexin
mediates respiratory adaptations that are necessary to compensate for UAO, but in parallel triggers a cascade of neurohumoral
signaling events that cause partial sleep loss, derangements of
energy metabolism, and growth retardation.
METHODS
See the supplemental material for more information
regarding the methods of this study.
Submitted for publication August, 2013
Submitted in final revised form November, 2013
Accepted for publication December, 2013
Address correspondence to: Ariel Tarasiuk, PhD, Department of Physiology, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O.
Box 105, Beer-Sheva 84105, Israel; Tel: +972-8-640-3049; Fax: +972-8640-3886; E-mail: [email protected]
SLEEP, Vol. 37, No. 5, 2014
Animals
Male Sprague-Dawley rats, 22 days old (53-55 g), were
used. Animals were kept on 12-12 light-dark cycle with lights
on 13:00. Rats were housed individually in Plexiglas cages at
23 ± 1.0°C. Food and water were given ad libitum. The study
was approved by the Ben-Gurion University of the Negev
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Orexin in Upper Airway Obstruction—Tarasiuk et al.
Sleep-Wake Activity, MA, and Tb
Raw electroencephalogram (EEG) and electromyography
(EMG) outputs from the skull and skeletal muscle electrodes
were sampled at 256 Hz, filtered at 0.1-40 Hz and 10-300
Hz, respectively, using the DSI system (DSI, St. Paul, MN,
USA).9,10 The vigilance states were scored using DSI NeuroScore v. 2.1 software and were edited visually for 10-sec epochs
on the basis of the predominant state within the epoch.10,28,29
The duration of sleep-wake states was calculated in 1-h time
intervals. These were categorized as: (1) W (wake), (2) SWS
(slow wave sleep), and (3) PS (paradoxical sleep); light slow
wave sleep—high-voltage slow cortical waves (0.5-4 Hz) interrupted by low-voltage fast EEG activity (spindles, 6-15 Hz);
and deep slow wave sleep—continuous (> 70% epoch) highamplitude slow cortical waves (0.5-4 Hz) with reduced EMG
and motor activity. The power density values for 0.5-4.0 Hz
were integrated and used to calculate slow wave activity during
non-rapid-eye-movement sleep.
Measured Tb and MA were analyzed using previously
described methods.9,10 Tb (± 0.1°C) and MA were continuously
monitored using the Dataquest A.R.T. system (DSI, St. Paul,
MN, USA). The signal emitted by the transmitter is proportional to Tb. MA (counts) is obtained by counting the number
of impulses, detected by changes in signal strength, per unit
time. The signal is received by an antenna under each animal’s
cage and transferred to a peripheral processor connected to
a personal computer. All transmitters were calibrated before
surgery and at the completion of experimentation to ensure
validity of biotelemetry measurements. Tb and MA raw data
were collected at 1-sec intervals in unrestrained rats. Raw Tb
and MA data are graphically presented as 1-h averages for ease
of presentation.
Animal Use and Care Committee and complied with the American Physiological Society Guidelines.
Surgery
The technique used for sham surgery and to induce upper
airway obstruction (UAO) in 22-day-old male rats was as previously described.3,8-10 Animals were anesthetized with tribromoethanol (200 mg/kg) administered intraperitoneally. A midline
ventral cervical incision was made, and the trachea exposed and
dissected so as not to damage adjacent structures. A circumferential silicon band 0.5 cm long was placed around the trachea
to induce tracheal narrowing. Two sutures were looped around
the band and tightened, thus constricting the trachea so as to
increase inspiratory esophageal pressure swings 2- to 3-fold.
Controls underwent sham surgery with no tracheal narrowing.
On day 38 after UAO/sham surgery, a telemetric transmitter
(TL11M2-F20-EET Data Sciences International, DSI, St. Paul,
MN, USA) was implanted (under sterile conditions), enabling
recording of electroencephalography (EEG), dorsal neck electromyography (EMG), and body temperature. Leads from the
electrodes for EEG recording were placed over the frontal (1.1
mm anterior and 1.1 mm lateral to the bregma) and parietal (3
mm posterior and 1.5 mm lateral to the bregma) cortices. EEG
electrodes were anchored to the skull with dental cement.10 For
body temperature (Tb) and locomotion activity (MA) recording,
a free-floating transmitter (model TA10TA-F20, DSI, St. Paul,
MN) was inserted into the abdominal cavity one week after
UAO/sham surgery. The transmitter was able to freely move
among the peritoneal organs because it was not attached to the
peritoneum. The peritoneal muscle and skin layers were closed
with interrupted sutures.9 Following surgery, prophylactic
enrofloxacin 5 mg/mL (subcutaneously) and water containing
ibuprofen (0.1 mg/mL) were given for 3 days.3
Almorexant Study
Almorexant (2R)-2-{(1S)-6,7-dimethoxy-1-[2-(4-trifluoro­
methylphenyl)-ethyl]-3,4-dihydro-1Hisoquinolin-2-yl}-Nmethyl-2-phenyl-acetamide), a dual orexin receptor antagonist,
was supplied by Actelion Pharmaceuticals, Ltd. Almorexant
was dissolved in 0.25% methylcellulose solution in water.
Vehicle control was prepared with 0.25% methylcellulose solution in water alone. Almorexant (300 mg/kg) in was delivered
at 5 mL/kg final volume. UAO and control rats were treated on
day 49 with a dual orexin receptor antagonist, almorexant (300
mg/kg), or vehicle administered as oral gavage at lights on.14,25
Rodent Multiple Sleep Latency Test (rMSLT)
This test was performed according to previously described
methods (n = 6 in each group).30 It includes 6 separate sleep
latency tests/trials starting in the last 4 h of lights-off phase.
For each of the 6 tests, the rat was initially kept awake for 5
min by means of gentle handling (primarily auditory and light
tactile stimulation, without explicit handling). Rats were then
left alone for 25 min while data were collected. This test was
then repeated 5 more times at 30-min intervals. An elevation
of the homeostatic sleep drive to fall asleep was assessed by
comparing the average across rats of the 6 sleep latency trials
between control and UAO groups.
Respiratory Measurement
Inspiratory swings in pleural pressure and respiratory rate
were measured on day 49. Ten breaths were used to analyze
inspiratory swings for pleural pressure and respiratory rate.
Pleural pressure, as approximated by esophageal pressure,
was measured by means of a saline-filled catheter placed in
the lower one-third of the esophagus and connected to a pressure transducer.3,5-10Arterial blood gases (pH, PCO2, PO2, and
HCO3-) were determined 49 days after surgery. These rats were
killed after this procedure.9,10 A video recording was used to
semi-quantify the effects of dual orexin receptor antagonist
(almorexant) on breathing at day 49 after surgery (see the
supplemental material).
Food and Water Intake
Twenty-four hour food intake, expressed as grams of food/kg
of body weight, was measured (n = 25 UAO and n = 21 controls)
on days 14, 28, 35, 42, and 49 post-surgery.6,8,9Animals were
given 40 g/d (> 40% of maximal daily food intake) of standard rodent chow (Harlan, Jerusalem, Israel) containing protein
(21%), fat (4%), carbohydrate (53.5%), moisture (13%); energy
3.95 (Kcal/kg). Food was placed into the feeder at the beginning, and any remaining at the end of each 24-h period was
weighed. Any visible food in the cage was scavenged and
included in the measurements. Twenty-four-hour water intake,
expressed as mL water/kg of body weight, was measured on
days 28 and 49.
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Orexin in Upper Airway Obstruction—Tarasiuk et al.
Magnetic Resonance Imaging (MRI)
We used high performance 1T compact M2 MRI, 60 mm ID
solenoid coil (Aspect Imaging, Shoham, Israel). Images were
acquired with a gradient spin echo sequence, with TR/TE/
NEX = 13.4/3/2. Multi-slice axial scans were collected with a
5 cm field-of-view and data matrix of 256 × 256, resulting in
a 0.195 mm in-plane resolution, slice thickness of 1 mm. The
total scan time per animal was 3 min 13 sec. A global threshold
was applied to the volume of interest (VOI) in order to create a
binary adipose object. Connected objects (6-connected neighborhood) were then computed and labeled. This procedure was
used to identify relatively homogeneous groups. The relevant
visceral adipocyte tissue (VAT) and subcutaneous adipocyte
tissue (SAT) objects were manually selected. The selected VAT
and SAT objects were used as seeds for the following growing
procedure of close intensity neighbor search. The neighbor
voxels with intensities of 15% ± each seed’s mean intensity were
appended. The described procedure was run iteratively until no
more voxels could be added. Z-slice tracking of 2D images was
performed starting 5 mm rostral to the kidney bottom up to 25
mm caudal to the kidney. Adiposity volume was calculated using
MatLab software (The MathWorks, Inc., Natick, MA, USA).
Table 1—Respiratory parameters 7 weeks post-surgery
Variable
PO2 (mm Hg)
PCO2 (mm Hg)
pH (units)
HCO3- (mEq/L)
Hemoglobin (g/dL)
ΔPes (cm H2O)
Respiratory rate
(breaths/min)
Obstructive
(n = 9)
P value
90.5 (3.7)
0.34
50.5 (4.6)
0.05
7.33 (0.02)
0.2
24.1 (1.3)
0.3
15.4 (0.6)
0.09
-19.1 ± 9.4
0.002
77.4 ± 18.6 < 0.001
PO2, arterial O2 pressure; PCO2, arterial CO2 pressure; pH, arterial
pH; HCO3, calculated arterial bicarbonate; ΔPes, inspiratory swings in
esophageal pressure. Values are mean ± SD.
vehicle study was performed on day 48 and almorexant (300
mg/kg) study was performed on day 49. Respiratory testing
was performed on day 49 immediately before animal death;
tissues and serum were harvested between 1 and 2 h after light
onset and were frozen at −80°C until analysis. The effect of
almorexant on respiratory activity was measured at week 7 by
video observation. Animals (n = 6 in each group) underwent
standard magnetic resonance imaging (MRI) scanning under
1.5% isoflurane anesthesia to calculate adiposity volumes at 8
and 14 days post-surgery.
Tissues Harvested
At the conclusion of study, the abdominal wall was wide
open and the internal organs were photographed (iPhone 4S,
Apple, Israel). Serum was collected and liver, diaphragm, soleus
muscle, and hypothalamus were dissected and frozen in liquid
nitrogen and stored at −80°C. Organs were weighed immediately after removal. The small intestine length was measured
between the pylorus and the junction with the cecum.31
Data Analysis
Significance was analyzed by unpaired t-test. Two-way analysis of variance for repeated measures was used to determine
significance between time and group using post hoc comparisons by Student-Newman-Keuls test. Null hypotheses were
rejected at the 5% level.
Tissue and Biomarker Measurements
Frozen aliquots of liver, diaphragm, and soleus muscle were
prepared for determination of tissue composition of lipids,
protein, and moisture (see the supplemental material).31 Serum
and tissue samples were collected and frozen at −80°C until
analysis. Serum leptin, corticosterone, and orexin concentrations were measured using specific commercially available
ELISA kits according to the manufacturer’s instructions.7-10
Serum leptin and corticosterone concentrations were measured
using ELISA kits RLB00, R2000, and MOB00 (R&D systems
Minneapolis, MN, USA) and EC3001-1 (ASSAYPRO, Saint
Charles, MO, USA), respectively. Hypothalamic orexin level
was measured using ELISA kit (MBS727107; MyBioSource,
San Diego, CA, USA). Protein expressions of orexin 1 and
orexin 2 were determined by Western immunoblot,9,10 and
mRNA extraction was determined using quantitative real time
PCR assay (see the supplemental material).9,10
RESULTS
During the 7-week observation period, UAO group behavior
was similar to that of controls; they explored their cage and
engaged in social activity such as grooming. As expected,
immediately following UAO, inspiratory swings in esophageal
pressure increased by ~200% and respiratory rate decreased
by ~25% (Table 1). Thus, the measured changes in inspiratory
swings in esophageal pressure and respiratory rate indicate that
resistive loading had been produced. The UAO group demonstrated audible wheezing, especially after activity, but no signs
of respiratory distress, gasping, or stress were observed during
routine activity; see video capture at baseline conditions (Videos
2 and 3, supplemental material). Arterial PO2 and pH were
in normal range in both groups (Table 1). Arterial PCO2 was
significantly greater in UAO animals by 12.4 mm Hg (P = 0.05).
Hemoglobin and bicarbonate concentrations were unchanged
between groups (Table 1). No significant differences were found
in serum corticosterone levels between control and UAO groups
(2873 ± 528 vs. 3035 ± 535 pg/mL, respectively, P = 0.824).
Experimental Schedule
UAO or sham control surgery was performed on 22-day-old
rats (day 0), and animals were followed for 7 weeks, a period
comparable to > 20 years in humans. Tb, MA, and food intake
were measured on days 14, 28, 35, 42, and 49 following UAO
or sham surgery. Sleep was recorded for 24 h on day 48, and on
the following day rMSLT was performed starting at 09:00, 4 h
before lights on. The effect of almorexant (dual orexin receptor
antagonist) on sleep was studied on a separate group. Baseline
SLEEP, Vol. 37, No. 5, 2014
Control
(n = 9)
91.8 (4.8)
38.4 (1.4)
7.38 (0.01)
22.4 (0.5)
13.9 (0.2)
-9.7 ± 5
103.3 ± 12.7
Hypothalamic Orexin Level
Both hypothalamic orexin mRNA (Figure 1A) and protein
level (Figure 1B) increased by 66% and 64% in the UAO group,
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Orexin in Upper Airway Obstruction—Tarasiuk et al.
Figure 1— (A) Hypothalamic orexin relative mRNA level determined by real time PCR. (B) Hypothalamic orexin protein level determined by specific ELISA.
Orexin level was normalized to grams of tissue. * P < 0.05.
Figure 2— (A) Hypothalamic orexin 1 receptor relative mRNA level and protein level (B). (C) Hypothalamic orexin 2 receptor relative mRNA level and protein
level (D). Representative Western blot analysis of two controls and two obstructive animals are shown at the bottom of B and D. * P < 0.05, ** P < 0.001.
respectively (P < 0.05). Hypothalamic orexin 1 receptor mRNA
and protein (Figure 2A and B) decreased by 28% (P = 0.02)
and 43% (P = 0.03), respectively. Similarly, orexin 2 receptor
mRNA and protein decreased by 37% (P = 0.002) and 70%
(P = 0.003), respectively, in the UAO group (Figure 2C and D).
the UAO group exhibited a significant reduction of 51.7% in
deep SWS duration compared to control (P < 0.001, ANOVA2). During 12 h of lights-off phase, the UAO group was awake
8.9% less time (P = 0.023, ANOVA-2) and spent 46.6% more
time in SWS (P < 0.01, ANOVA-2). During the last 5 h of
lights-on, the UAO group exhibited 133% more SWS duration
(P < 0.001, ANOVA-2). The time course of slow wave activity
during non-rapid eye movement sleep showed a normal pattern
in the control group and was considerably lower and flat and in
UAO group (P < 0.01, ANOVA-2; Figure 4).
Sleep-Wake Activity
As expected for nocturnal animals, there was more sleep in
the light period than in the dark period in both groups (Figure 3
and Table 2). There were, however, several significant differences in sleep between groups. The UAO group was awake
30% more time during 12-h lights on (P < 0.001, ANOVA-2),
had 12.6% less SWS (P = 0.023, ANOVA-2), and 56.7% less
PS (P < 0.001, ANOVA-2). During the first 2 h of lights-on,
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rMSLT Study
Significantly lower sleep latency was found in all 6 trials
(P < 0.001, ANOVA-2) of the UAO group (Figure 5A). On
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Orexin in Upper Airway Obstruction—Tarasiuk et al.
Table 2—Spontaneous sleep 49 days post-surgery
12-h light phase (n = 14)
Wake (%)
SWS (%)
PS (%)
Control
42.1 ± 2.1
46.0 ± 2.2
11.5 ± 1.4
Obstructive
54.8 ± 2.0
40.2 ± 1.8
4.9 ± 0.7
12-h dark phase (n = 14)
% Change
30.0%***
-12.6%*
-56.7%***
Control
75.5 ± 1.8
18.2 ± 1.4
6.5 ± 0.8
Obstructive
68.9 ± 2.1
27.6 ± 2.2
4.6 ± 0.9
% Change
-8.9%*
46.6***
-29.3%
Average % of time spent in each sleep stage for light phase (09:00-21:00) and dark phase (21:00-09:00). SWS, slow wave sleep; PS, paradoxical sleep;
* P < 0.023; *** P < 0.001 comparing baseline (vehicle) control with baseline (vehicle). Significant differences were determined by two-way ANOVA. Values
are mean ± SEM.
average, rMSLT was shorter by 64% in the UAO group
(Figure 5B, P < 0.001, ANOVA-2).
Almorexant Study
The effect of dual orexin antagonist on sleep was explored
(Figure 6). Acute administration of almorexant significantly
decreased (P < 0.05, ANOVA-2) wake duration during lightson in UAO rats to levels statistically similar to those of baseline control values. Post hoc tests revealed almorexant reduced
wake duration for 4 and 8 h in the control and UAO rats, respectively (Figure 6). Almorexant significantly increased SWS
duration in both groups (P < 0.05, ANOVA-2). Post hoc tests
revealed almorexant increased SWS duration for 4 and 8 h in
the control and UAO rats, respectively (Figure 6). Almorexant
did not significantly affect PS duration in either group.
Video Observation
Administration of vehicle and/or inserting the feeding
needle without the drug did not affect respiration; both groups
had similar respiratory movement activity at baseline (Videos
1-3, supplemental material). Following dual orexin antagonist
(almorexant 300 mg/kg), the UAO group exhibited notable
changes in respiratory pattern indicating distressed breathing.
Substantial intercostal retractions and mouth opening during
inspiration were noted in video observation, while it had no
effect on the controls’ respiratory activity (Videos 1-3, supplemental material). Almorexant treatment decreased respiratory
rate in the UAO group from 144 ± 13 (breaths/min) at baseline
to 100 ± 12 (breaths/min) (P = 0.002). Almorexant treatment
did not affect (P = 0.06) respiratory rate of the control group,
146 ± 7 (breaths/min) and 134 ± 11 (breaths/min) on baseline
and almorexant, respectively.
Body Temperature (Tb) and Locomotion Activity (MA)
One-hour average of Tb and MA in the control (n = 9) and
UAO (n = 9) groups 2 and 7 weeks post-surgery are presented
in Figure 7. At 2 weeks, in both light and dark phases Tb was
significantly lower in the UAO group than control by 0.6°C
(P < 0.001, ANOVA-2; Figure 7A and C). Light phase MA was
similar in both groups (P = 0.6, ANOVA-2), and dark phase MA
was significantly lower by 27% in the UAO group (P < 0.001,
ANOVA-2). At 7 weeks (Figure 7B and C), both light and dark
phase Tb declined by 1.1°C in the UAO group (P < 0.0001,
ANOVA-2). The average 24-h MA increased significantly by
33% in the UAO compared to control group (4.0 ± 0.7 vs.
3.0 ± 0.7 counts/min, respectively, P < 0.01). Light phase MA
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Figure 3—Spontaneous sleep in control and obstructive rats. Hourly values
of wake (W), slow wave sleep (SWS), and paradoxical sleep (PS) are shown.
Black horizontal bars represent the light-off (active) period on a 12:12-h
cycle. Obstructive group had significantly more wake and less SWS and PS
than controls during light period. During dark period obstructive group had
significantly more SWS and less PS than controls. Data are from 12 control
and 13 obstructive rats. Statistically significant (* P < 0.005; *** P < 0.001)
difference between the groups, ANOVA-2. Values are mean (SEM).
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Orexin in Upper Airway Obstruction—Tarasiuk et al.
increased significantly by 243% (P < 0.01, ANOVA-2) in the
UAO compared to control group (3.4 ± 0.70 vs. 1.40 ± 0.30
counts/min, respectively); no significant differences between
groups were seen in the dark phase (P = 0.55, ANOVA-2).
increased by 20% in the UAO group (P < 0.01, Figure 8B,
ANOVA-2). Water intake of the UAO group tended to increase
in week 4 by 15% (P = 0.082) compared to controls and increased
significantly by 34% (P = 0.008) in week 7 (106.8 ± 42.5 vs.
79.9 ± 11.6; mL/kg of body weight, in UAO and control groups
in week 7, respectively).
At week 7 body length was 25% smaller (P = 0.001) and
small intestine length to body length ratio of the UAO group was
24.5% longer than that of controls (P = 0.005, Table 3). Tissue
compositions for water, fat, and protein are summarized in
Table 4. No significant differences between groups were found
in liver and soleus muscle wet weights to body weight ratios.
However, diaphragm wet weight to body weight ratio increased
significantly by 19% (P < 0.0001) in the UAO group (Table 4).
Interestingly, MRI analysis revealed that both abdominal and
subcutaneous adiposity volume was strikingly low in UAO rats
on day 8 (Figure 9). Both subcutaneous and visceral adipocyte
volume were significantly reduced by 69% (P = 0.0003) and 72%
(P < 0.0001) in the UAO group compared to controls, respectively
(Figures 9B, C). Similar findings were observed on day 16 postsurgery (data are not presented). At 7 weeks abdominal adipocytes
were minimal or missing in UAO rats (Figure S1, supplemental
material) and serum leptin level was significantly lower by 67%
in the UAO group (2574 ± 279 pg/mL vs. 846 ± 135 pg/mL in
control and UAO groups, respectively, P < 0.0001).
Body Weight, Food and Water Intake, Tissue Composition, and MRI
Both groups had similar baseline body weight (Figure 8A).
Both groups exhibited significant body weight gain over time
(P < 0.001, ANOVA-2). However, the UAO group gained 40%
less body weight than the control group at all time intervals
(P < 0.001, ANOVA-2). Daily food intake (Figure 8B) was 22%
less in the UAO group than the controls at 2 weeks (P < 0.01,
ANOVA-2). However, food intake in weeks 5 through 7
Table 3—Small intestine and body lengths
Body weight (g)
Body length (cm)
Intestine length (cm)
Intestine length/
body length
Figure 4—Hourly average of electroencephalogram slow wave activity
(SWA, integrated power densities in delta range). SWA was significantly
lower and flat in obstructive rats compared with controls. Data are from
12 control and 13 obstructive rats. *** Statistically significant (P < 0.001)
difference between groups, ANOVA-2. Values are mean ± SEM.
Control
(n = 12)
307 ± 26.6
22.2 ± 0.9
118.8 ± 8.8
5.3 ± 0.1
Obstructive
(n = 14)
202 ± 28
17.7 ± 0.3
114.3 ± 9.7
6.5 ± 1.4
P value
0.001
0.001
0.22
0.005
Body length is measured from nose to anus. Small intestine length is
measured between the pylorus and the junction with the cecum.
Figure 5—Rodent multiple sleep latency test. (A) Left, rodent latency to sleep onset in six trials. (B) Right, rodent multiple sleep latency tests (rMSLT). Data
was collected from n = 6 in each group. *** Statistically significant (P < 0.001) difference between groups, ANOVA-2.
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Orexin in Upper Airway Obstruction—Tarasiuk et al.
Figure 6—Effect of dual orexin receptor antagonist (almorexant, ALM, 300 mg/kg) on spontaneous sleep. Hourly values of wake (W), slow wave sleep
(SWS), and paradoxical sleep (PS) are shown; left column control and right column obstructive (UAO). On day one animals were given vehicle (0.25%
methylcellulose solution in water), and on day two animals were treated with almorexant at lights on. Almorexant or vehicle was administered at light on
(arrow). Black horizontal bars represent the light-off (active) period on a 12:12-h cycle. Data are from n = 6 in both groups. * Statistically significant (P < 0.05).
Values are mean (SEM).
DISCUSSION
Here we present evidence indicating that enhanced orexin
secretion, while crucially important for respiratory homeostasis maintenance, is also responsible for chronic partial sleep
loss in UAO animals. The sleep abnormalities, in turn, cause
hypothermia, loss of adipocyte volume, and growth retardation despite significant increase in food intake. Administration
of almorexant normalized sleep but induced severe breathing
difficulties, while it affected neither sleep nor breathing in
control animals.
SLEEP, Vol. 37, No. 5, 2014
Model Strength and Limitation
To our knowledge this is the first study exploring the effects
of UAO on sleep-wake and energy metabolism from weaning
to adulthood. Upper airway obstruction was induced in 22-dayold rats, and animals were followed for 7 weeks, a period
that is comparable to age six months to about twenty years in
humans. The reduced respiratory rate and inspiratory swings
in esophageal pressure in the current study indicate that the
trachea was mildly to moderately obstructed, and the effects
were not exclusively sleep related.3-10 Similar to earlier studies,
993
Orexin in Upper Airway Obstruction—Tarasiuk et al.
Figure 7—One-hour average of body temperature (top panel) and locomotion activity (bottom panel) in control and obstructive rats 2 weeks (A) and 7 weeks
(B) days post-surgery. Black horizontal bars represent the light-off period. (C) Mean body temperature during 12-h lights-on period (upper panel) and 12-hour
dark period (lower panel) during 7-week observation period. n = 9 in each group, ** P < 0.01, *** P < 0.0001, # P < 0.01. Values are mean (SEM).
UAO led to an increase in arterial PCO2 without change in arterial pH, suggesting renal compensation of acid balance.5,6,9 In
this model, both inspiratory and expiratory loading, which may
resemble subglottic stenosis in children and not be exclusively
sleep related, were introduced, while in clinical sleep disordered breathing, airway loading is mainly inspiratory and sleep
related. Obstructive sleep apnea is associated with intermittent
upper airway obstruction at night, primarily during inspiration.
It seems likely that our model also has implications for this
condition since, as in sleep apnea, obstructed animals exhibited sleep fragmentation, and as in children with sleep apnea,
growth retardation.1,2,9,10 We did not find evidence for stress in
this animal model; serum corticosterone level was similar in
both groups during early light onset. Measurements of serum
corticosterone were performed during the nadir of the circadian cycle 1-2 hours after lights onset.32 Under these conditions animals maintain PO2 in the normal range.5,6,8-10 Chronic
hypoxia can reduce food intake and induce erythropoiesis.33,34
In this study hemoglobin and lactate levels were similar to those
of controls, confirming previous findings,6,8-10 and dietary intake
was significantly higher.6 In children, however, oxygen saturation may decrease with airway loading during sleep.35
Respiratory Pattern during UAO
Orexin neurons provide an important integrative link
between peripheral metabolism and central regulation of behaviors required for adaptive response to homeostatic challenges of
exercise and breathing.11,15 In our study we found significant
elevation of hypothalamic orexin (Figure 1) and reduction of
both hypothalamic orexin receptors (Figure 2). This observation
suggests that orexin has a role in regulating its own synthesis
Figure 8—(A) Growth curves of control (n = 12) and obstructed (n = 13)
rats. The growth curve of obstructive rats was significantly less than that
of the controls over the duration of the observation period. (B) Daily food
intake expressed as grams of food per kilogram body weight. Values
are mean (SD). # Statistically significant (P < 0.001) difference between
groups, ANOVA-2.
SLEEP, Vol. 37, No. 5, 2014
994
Orexin in Upper Airway Obstruction—Tarasiuk et al.
Figure 9—(A) Example of 2D abdominal MRI and z-slices tracking of 2D images eight days after surgery; (B) VAT, visceral adipocyte tissue volume (red
color); (C) SAT, subcutaneous adipocyte tissue volume (blue color); n = 6 in each group; *** P < 0.001, values are mean (SD).
Table 4—Tissue composition of water, protein, and fat
Liver
Weight (g)
Weight/body Weight (× 10-3)
Water (%)
Protein (%)
Fat (%)
Fat/Water (%)
Control
(n = 9)
10.7 ± 1.3
45.5 ± 4.7
70.8 ± 1.2
16.4 ± 2.2
5.2 ± 1.7
7.4 ± 2.5
Soleus muscle
Obstructive
(n = 10)
5.9 ± 1.9***
42.5 ± 5.2
71 ± 1.6
17.4 ± 1.5
4.5 ± 1.7
6.3 ± 2.3
Control
(n = 7)
0.2 ± 0.01
0.9 ± 0.1
75.3 ± 0.8
13.4 ± 2.4
0.8 ± 0.3
1.1 ± 0.5
Diaphragm (n = 8)
Obstructive
(n = 7)
0.1 ± 0.01***
0.9 ± 0.3
75.4 ± 1.4
14.4 ± 3.7
1.1 ± 0.5
1.5 ± 0.3
Control
(n = 8)
0.6 ± 0.1
2.7 ± 0.2
74 ± 1.1
10.3 ± 2.1
2.2 ± 1.0
3.1 ± 1.8
Obstructive
(n = 8)
0.4 ± 0.1***
3.2 ± 0.5***
74.4 ± 1.3
8.7 ± 2.4
2.9 ± 1.3
4.3 ± 2.4
*** P < 0.0001. Values are mean ± SD.
by negative feedback at the level of the hypothalamus. Further
studies should explore this possibility. Orexin neurons can stimulate central control of respiration.12-14 In vitro, orexin neurons
increase their discharge when stimulated by CO2/H+.36 Also
prepro-orexin knockout mice, with a deficiency of both orexin
A and B, have a significantly attenuated hypercapnic ventilatory response in wakefulness but not in sleep, a defect that is
SLEEP, Vol. 37, No. 5, 2014
partially restored by injection of orexin A and B via the cerebral ventricles.13,37 In our study administration of almorexant
during lights on normalized sleep but induced in parallel severe
breathing difficulties and 30% reduction (P = 0.002) of respiratory rate in UAO rats, suggesting increased upper airway resistance,6,9,10 while it did not affect sleep or breathing in control
animals. Further studies should explore the role of orexin on
995
Orexin in Upper Airway Obstruction—Tarasiuk et al.
ventilation using whole body plethysmography and/or electrophysiological methods.6,14,38 Previous studies reported no significant differences between control and almorexant-treated rats in
CO2 response during lights on in any of the vigilance states.14
Thus our findings suggest that orexin has a role in the adaptive
response of the respiratory system to UAO to maintain ventilation and upper airway patency during the sleep phase. During
UAO the respiratory muscles are forced to contract harder in
order to preserve ventilation.3,4 A possible explanation of this
elevation of orexin may include respiratory muscle exercise,16-18
central load compensation,39 and/or role of the orexin system in
central chemoreception in a vigilance state and diurnal cycle
dependent manner.14 The dose of almorexant used in this study
(300 mg/kg) was similar to that used in prior studies exploring
the control of respiration14 and sleep,25 and was larger than that
used in the original studies of almorexant effects on sleep and
wakefulness (20-100 mg/kg).26
An impressive reduction of abdominal and subcutaneous
adipocyte tissue and serum leptin level was found in our UAO
animals (Figure 9 and Figure S1). Orexin and leptin systems
have non-overlapping expression in the brain.11 Leptin, a
satiety-promoting hormone secreted by adipocytes, has an
important role in central chemoreception. Adult C57BL/6JLepob has considerably reduced CO2 response.38,40 This suggests
that leptin has little effect on maintaining respiratory homeostasis in UAO rats.
demonstrating increased intestine surface area to absorb water
and nutrients during chronic partial sleep loss.31 In contrast,
chronic partial sleep loss in humans has been associated with
an increased risk for obesity, type 2 diabetes mellitus, and
cardiovascular events.43,46 The apparent differential metabolic response to chronic partial sleep loss between humans
and rats may therefore simply reflect a species difference,44
although several additional and possibly contributing factors
differ between our UAO rats and humans with obstructive
sleep apnea.2,47 Moreover, it has been suggested that humans
may be less active, while our UAO animals showed 33%
increase in their 24-hour MA. The difference in response of
feeding and energy expenditure may represent a fundamental
species difference between rats and humans, or sleep loss in
humans may cause adverse metabolic consequences due to
other factors such as circadian disruption, increased feeding,
and decreased activity level.44 The slow body weight gain in
UAO was mostly related to a striking reduction of adiposity
tissue. Previous studies employing sleep deprivation in rodents
have also reported slower weight gain (or even weight loss),
but the reports of food intake were inconsistent. While most
studies claimed an increase in food intake,45,48 others reported
no change in food intake after sleep loss.44 The increased
multi-locular adipocytes, known to be rich with mitochondria, supports the possibility of high-energy production during
chronic partial sleep loss in rats.31 In rats orexin-A has a role
in regulating lipolytic processes through facilitation of sympathetic nervous system activity.49 Further studies are needed to
explore the role of sympathetic nervous system activity in regulating lipolytic processes in UAO. We found a gradual decline
in body temperature of 1.1°C in the UAO animals during the
7-week observation period, and at 7 weeks the decline in body
temperature was accompanied by elevation of MA. Sleep is
essential for regulation of body temperature. Partial sleep deprivation leads to hypothermia and difficulty retaining body heat
in spite of increased food intake.50-53 Our findings strengthen the
hypothesis that sleep has an energy-conserving role, regardless
of the methods used.31,48-55
Sleep-Wake Activity during UAO
Orexin neurons are typically active during wakefulness
(lights off) but show little or no activity when animals are asleep
(lights on).17-23 The discharge of orexin neurons is synchronized
with arousal states, being greater with increased arousal. We
found that UAO increased arousal and led to fragmented sleep
during sleep-time phase. In our study, the UAO group slept
30% less during lights on and had increased whole-day MA
due to enhanced hypothalamic orexin secretion; administration
of almorexant restored sleep and reduced locomotion activity.
This observation confirms our earlier report of 36% sleep loss
two weeks following UAO surgery.10 Intracerebroventricular
administration of orexin A to mice significantly increased MA
and almorexant attenuated this finding.25 Sleep loss in our UAO
animals was associated with significant reduction of awake
time and increased SWS during the second half of lights-off
phase. The 64% reduction of rMSLT sleep latency strongly
indicates that UAO animals have excessive sleepiness during
active phase.30 Our findings are in accordance with prior studies
demonstrating that obstructive apnea can lead to excessive
sleepiness in adult patients41 and in severe cases of pediatric
obstructive sleep apnea.42
CONCLUSION
Our study provides evidence that in upper airway obstruction
(UAO) the respiratory system is challenged to maintain homeostasis, consequently resulting in sleep loss, and that hypothalamic orexin has a role in this process. Partial sleep loss in UAO
is associated with energy metabolism abnormalities and growth
retardation. These metabolic changes appear to be an adaptive
response to high-energy production, but ultimately are insufficient to compensate for inadequate sleep and maintain health
during UAO.
Metabolic Effects
UAO induces orexin-mediated chronic partial sleep loss
from early life to adulthood. Disruption of sleep, either by sleep
deprivation or partial sleep loss, will lead to adverse health
outcomes in rodents, i.e., increased MA, loss of body temperature, increased food intake, loss of adipocyte tissue.31,43-45 Our
UAO animals had low weight gain and growth despite 20%
increased food intake and lengthening of the intestine. The
lengthening of the intestine is consistent with earlier reports
SLEEP, Vol. 37, No. 5, 2014
ACKNOWLEDGMENTS
The authors thank Ms Svetlana Lublinsky for conducting the
MRI study. The authors thank Actelion Pharmaceuticals Ltd.
for their generous donation of almorexant.
DISCLOSURE STATEMENT
This was not an industry supported study. This research was
supported by Israel Science Foundation Grant No. 160/10.
The authors have indicated no financial conflicts of interest.
996
Orexin in Upper Airway Obstruction—Tarasiuk et al.
Authors’ contributions to the study: A. Tarasiuk, PhD, principal
investigator: recruitment of funds, acquisition of sleep and
metabolic study, writing the manuscript. A. Levi, MSc student:
biochemical and molecular analysis; N. Berdugo-Boura, PhD
student: sleep and body temperature analysis, almorexant study;
A. Yahalom, MSc student: MRI and almorexant study, writing
part of the manuscript; Y. Segev, PhD, principal investigator:
recruitment of funds, acquisition of molecular endocrinology
data & analysis, writing the manuscript.
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24. Kotz CM. Integration of feeding and spontaneous physical activity: role
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SUPPLEMENTAL MATERIAL
METHODS
The study was approved by the Ben-Gurion University of the
Negev Animal Use and Care Committee and complied with the
American Physiological Society Guidelines.
Video recording
Video camera was positioned downward from the cage and
additional video capture (720 × 576 pixels, 25 frames/sec) was
taken via the cage boundary to record breathing activity. Videos
were taken 15 minutes prior and up to 2 hours following administration of almorexant. Respiratory rate was calculated after
slowing the video time frames. Two independent reviewers who
were blinded to the study protocol reviewed the video captures.
They were instructed to observe the respiratory movements
and rate the retractions and gasping as normal activity, mild,
moderate, or severe respiratory difficulties. In case of disagreement (if any) they were instructed to meet and to agree on the
results.
Tissue composition
Frozen aliquots of liver, diaphragm, and soleus muscle were
prepared for determining tissue composition of fat, protein,
and moisture.1 Lipids were extracted by the 2:1 chloroform
methanol method.2 Chloroform extracts were evaporated using
the thermo centrifugal vacuum concentrator system (Savant
SpeedVac SC 110A, Hyannis, MA, USA). Protein content was
determined using protein assay (Bio-Rad Laboratories GmbH,
Munich Germany). Tissue water content was determined by
baking at 110°C in a dry-heat oven for 5 hours.
Western immunoblot analysis
Protein analysis of orexin 1 and orexin 2 receptors was
conducted Western immunoblot as previously described.3,4
Hypothalamic tissue was homogenized on ice with a polytron
(Kinetica, Littau, Switzerland) in lysis buffer (50mM Tris, pH
7.4, 0.2% Triton X-100) containing 20mM sodium pyrophosphate, 100mM NaF, 4mM EGTA, 4mM Na3VO4, 2mM PMSF,
0.25% aprotinin, and 0.02 mg/mL leupeptin. Extracts were
centrifuged for 20 minutes at 17,000 g at 4°C and the supernatants collected and frozen. Antibodies were purchased for
the detection of Hypothalamic Orexin R-1 (C-19), Orexin R-2
(C-20) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and
β-actin (MP Biomedical, Solon, OH, USA). Homogenates were
mixed with 5× sample buffer and boiled for 5 minutes. Then,
75 μg portions of sample protein were loaded in each gel lane,
subjected to 10% SDS polyacrylamide gel, and electroblotted
into nitrocellulose membranes. Blots were blocked for 1 hour
in TBST (0.05% Twin-20) buffer (10mM tris, pH 7.4, 138mM
NaCl) containing 5% non-fat dehydrated milk, followed by
overnight incubation with polyclonal antibody diluted in TBST
(0.05% Twin-20) containing 5% dry milk. After washing 3
times for 15 minutes in TBST (0.05% Twin-20), the blots were
incubated with a secondary anti-goat antibody conjugated to
horseradish peroxidase for 1 hour at room temperature and then
washed again 3 times. The band antibody was visualized by
enhanced chemiluminescence (ECL; Amersham, Life Sciences
SLEEP, Vol. 37, No. 5, 2014
Figure S1—Images show examples of post-mortum abdominal adiposity
tissue 7 weeks after UAO (right) or sham (left). Note the normal adiposity
tissues in control that is minimal in the UAO animal. Bar indicates 1
centimeter.
Inc.). Protein expression was quantitated densitometrically
using Image J software.
mRNA extraction and real-time PCR3,4
Animals were sacrificed by guillotine 1-2 hours after lights on;
the hypothalamus was quickly removed (< 1 minute) and frozen
at -80°C until analysis. Hypothalamic RNA was extracted, and
quantitative real time PCR assays were performed. Total RNA
was extracted using the PerfectPure RNA Tissue kit (Gentra
Systems, Minneapolis, MN) and used for cDNA synthesis by
High-Capacity cDNA reverse transcription (Applied Biosystems, Foster City, CA). The cDNA samples were then subjected
to PCR analysis. Quantitative real time PCR (qPCR) assays
were carried out for orexin, orexin 1 receptor, orexin 2 receptor,
and β-actin with the following primers:
• Orexin sense: 5′-TAGAGCCATATCCCTGCCC.
• Orexin anti-sense: 5′-GAGGAGAGGGGAAAGTTAGG.
• Orexin 1 receptor sense:
GCGCGATTATCTCTATCCGAA.
• Orexin 1 receptor anti-sense:
AAGGCTATGAGAAACACGGCC.
• Orexin 2 receptor sense: GAGTGCCATCTTCACTCCTG.
• Orexin 2 receptor anti-sense:
GATTCCATAAGGATGCTCGGG.
• β-actin sense: GGTCTCAAACATGATCTGGG.
• β-actin anti-sense: GGGTCAGAAGAATTCCTATG.
Primer optimized concentrations were chosen according to
primer optimized protocol (Applied Biosystems, Foster City,
CA, USA). Real time PCR reactions were performed with
power SYBR green PCR master mix (Applied Biosystems)
using the ABI Prism 7300 Sequence detection System (Applied
Biosystems). Each sample was analyzed in duplicate (final reaction volume 20 μL) in 96-well Micro Optical plates (Applied
Biosystems), each sample representing an individual assay. For
each sample, 200 ng of cDNA was added to power SYBR green
998A
Orexin in Upper Airway Obstruction—Tarasiuk et al.
PCR master mix containing Rox (Applied Biosystems, Foster
City, CA, USA) and 500nM primers. The PCR protocol was:
50°C for 2 min; 95°C for 10 min; and 40 cycles of 95°C for 15
s followed by 60°C for 1 min. The specificity of the reaction is
given by the detection of the melting temperatures (Tms) of the
amplification products immediately after the last reaction cycle.
The target gene expression value was calculated by the ΔΔct
method after normalization with a housekeeping gene (β-actin).
Video 2 shows obstructed animal – obstructive 1.
Video 3 shows obstructed animal – obstructive 2.
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Videos
Video 1 shows six seconds of activity of control animal at
baseline and the same animal 60 minutes following administration of dual orexin antagonist (oral gavage). Upper panel shows
the animal at downward and sideways angles from the cage;
lower panel shows via the cage boundary. Videos capture 720 ×
576 pixels and 25 frames/sec.
SLEEP, Vol. 37, No. 5, 2014
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Orexin in Upper Airway Obstruction—Tarasiuk et al.