l
Sleep
13(3):205-217, Raven Press, Ltd., New York
© 1990 Association of Professional Sleep Societies
Hypoxia-Induced Sleep Disturbance in Rats
Judit Laszy and Adam Sarkadi
Chemical Works of Gedeon Richter Ltd., Pharmacological Research Centre, Laboratory of
Neurophysiology, Budapest, Hungary
Summary: The effects of varying degrees of hypoxia on sleep-wake organization were studied in rats prepared for chronic electrophysiological recording.
The influence of Pirace tam (75, 50, and 500 mg/kg, Lp.) and Hydergine (0.5,1,
and 3 mg/kg, i.p.) on sleep-wake organization in 10.5% oxygen was also investigated. The sleep-wake organization of rats under the effect of 15.5%
oxygen content was unchanged, compared to that of normoxic control. More
extreme hypoxia (12.6 and 10.5% oxygen) produced dramatic changes in sleep
organization without influencing gross behavior. Paradoxical sleep (PS) stages
became less frequent and shortened and were totally absent in 10.5% oxygen.
Frequent wakings caused disturbed and superficial sleep. Central biochemical
mechanisms, peripheral chemoreceptor reflex pathways and, as a consequence, decrease of duration of deep sleep periods, may contribute to the
development of hypoxic sleep disturbances. Piracetam alleviated and Hydergine moderately reversed the hypoxic sleep disturbance. Key Words:
Sleep--Hypoxia-Piracetam-Hydergine-Rat.
Cerebral hypoxia due to organic and functional reasons modifies normal sleep organization. Hypoxic sleep disturbance in rats was first systematically and quantitatively
evaluated by Pappenheimer in 19770), and his findings were confirmed and extended
later (2-6). These studies showed that decrease in oxygen concentration of inspired air
counteracts the development of paradoxical sleep (PS) and increases wakefulness.
Reduction of slow wave sleep (SWS) was found in some studies, exposing rats to 10%
oxygen 0-3), whereas alterations of SWS under the effect of mild hypoxia were not
described in other investigations (5,6). In one study (6), more severe hypoxia 00% 02)
caused only a slight decrease in SWS in rats; indeed, similar oxygen concentration
resulted in an increase in SWS in kittens. In these sleep investigations, light sleep
(SWSl) and deep sleep (SWS2) stages were not distinguished within SWS. No data are
available with regard to different levels of hypoxia affecting fine sleep structure.
The second aim of this study was to show whether Piracetam and Hydergine can
alleviate hypoxic sleep disturbance. These two nootropic drugs-compounds of varyAccepted for publication December 1989.
Address correspondence and reprint requests to Judit Laszy, Chemical Works of Gedeon Richter Ltd.,
Pharmacological Research Centre, Laboratory of Neurophysiology, H-1475 Budapest 10, P.D.B. 27, Hungary.
205
206
1. LASZY AND A. SARKADI
ing chemical structure, which improve elderly mental functions and enhance learning
acquisition and resistance to impairing agents (7)-successfully protect against the
effect of hypoxia (8,9).
METHODS
Adult male outbred RG-Wistar rats of the Hannover strain were maintained at 26 ±
1°C, on a 12: 12-h light/dark cycle in a laboratory animal care unit. The animals were
prepared for chronic electrophysiological sleep investigations. Under pentobarbitone
anesthesia (40 mg/kg, i.p.), electrodes were implanted over the cortex of both hemispheres lstereotaxic coordinates according to atlas of Pellegrino et al. (10) (A:O.O;
L:3.0)] and in the dorsal neck muscles. The uninsulated spiral cortical disc electrodes
of 1.0 mm diameter were made of 0.25-mm-diameter nichrome wire. The outer ends of
the electrodes were crimped into the pins of a subminiature connector, and the connector was fixed to the skull with acrylate cement. The first technical control measurements were performed 2 weeks after surgery.
The experiments were carried out in transparent experimental chambers consisting of
two separate compartments, measuring 250 x 250 x 250 mm. Diffuse low-intensity
artificial light and masking noise (54 ± 10 dB) were applied when recording. Temperature was held at 27 ± 1°C. Measurements commenced after 2 weeks of habituation to
the sleep situation, following the stabilization of the sleep-wake organization that is
characteristic for each rat. Electrocorticograms and electromyograms of two animals
were recorded simultaneously (Medicor EEG-16) for 120 min beginning at 9:00 a.m.
Rats were placed into the experimental chambers 30 min prior to the beginning of
recording. The air/nitrogen mixtures were administered through flow meters at a rate of
100 Llh. The oxygen and carbon dioxide concentrations were measured by an analyzer
(Normocap). CO 2 content in the boxes was 0.4% during the recording sessions.
Sleep stages were scored visually. Evaluation was based on the electrocorticograms,
electromyograms, and the behavioral signs according to the following criteria: (a)
Awake (A W): irregular cortical activity of 4-8 cis when the animal is moving or lying
quietly with eyes open; (b) Arousal reaction (Ar): a short awake-like electrographic
pattern, lasting for 5-15 s, accompanied by minimal or no movement, inserted into
sleep phases or between two sleep stages; (c) SWSI: alternation of awake-like background activity with the appearance of cortical sleep spindles mixed with slower e
waves when the animal is lying quietly with eyes dropping or closed; (d) SWS2: high
amplitude, irregular cortical activity of 2--6 cis, when the animal is in the characteristic
sleep posture; (e) PS: regular cortical rhythm of 5-7 cis when the animal is lying relaxed
with slack head and limbs; occasionally, ear-twitches may occur.
The time-stage data collected by visual analysis of recordings were processed by
computer (TPA-1140, KFKI). Total time spent in sleep (TST), recurrence (N) and mean
duration (MT) of each sleep stage, proportion of each stage to TST, and hypnograms
reflecting the time course of a measured recording period were printed. The frequency
of the transitions from different sleep-wake levels to others was calculated for each
animal. Statistical comparisons between parameters of normoxic and hypoxic sessions
were made by analysis of variance (ANDY A) and unpaired t-tests.
Rats were divided into two groups. The sleep-wake organization of one group (eight
rats) was studied in 21.0, 15.5, 12.6, and 10.5% oxygen mixture in a randomized order.
Sleep. Vol. 13. No.3. 1990
207
HYPOXIC SLEEP DISTURBANCE IN RATS
!-:
The animals of the second group (eight rats) slept in only 21.0 and 10.5% oxygen and
were treated intraperitoneally with Piracetam or Hydergine, respectively, 30 min prior
to the beginning of recordings in hypoxia. Piracetam was administered in doses of 75,
150, and 500 mg/kg; Hydergine was administered in doses of 0.5, 1, and 3 mg/kg. The
effect of Hydergine on sleep-wake organization was also studied in normoxia in doses
of 1, 3, and 5 mg/kg. Both compounds were dissolved in 0.5 ml distilled water. Injections were arranged in a randomized order every other day. In normoxic and hypoxic
control sessions, 0.5 ml physiological saline was injected intraperitoneally.
Independently from the two experimental groups, the rectal temperature (Tr) of eight
animals was measured in the AW state (at 5 cm depth in the rectum) at 8:30 a.m. in
normoxia and at 11:00 a.m. after staying in hypoxia at rates of 15.5, 12.6, and 10.5% 02'
The Tr values of these rats were also measured at 10:30 a.m. and at 1:00 p.m. under the
above circumstances to determine whether changes in Tr during hypoxia are related to
normal diurnal changes in body temperature.
RESULTS
Effect of hypoxia on Tr
Tr values are summarized in Table 1. In experiments conducted between 8:30 and
11 :00 a.m., significant decreases in the Tr were observed under the effect of 12.6 and
10.5% oxygen. When the experiments took place from 10:30 a.m. to 1:00 p.m., only
slight (not significant) Tr decreases were found at similar hypoxic levels. On the other
hand, the difference was significant between Tr values measured in normoxia at 8:30
and those measured at 10:30 a.m. That is, the decrease in body temperature during
hypoxia did not significantly exceed the normal diurnal chance in body temperature
from 8:30 to 10:30 a.m.
Effect of varying degrees of hypoxia on sleep organization
In 15.5% oxygen environment, the sleep-wake organization of the rats remained
practically unchanged (Fig. 1, top right). No significant deviations from the normoxic
control could be detected either in the sleep-wake proportion (Fig. 21) or in the proportion of each sleep phase to TST (Fig. 211).
Further reduction of oxygen content of the inspired air to 12.6 and 10.5%, however,
caused an increasing change in sleep organization (Fig. 1, bottom). Figure 21 shows that
the proportion of wakefulness increased significantly with decreasing oxygen content.
~J
TABLE 1. Tr (0C) of rats at different hypoxic levels
Oxygen concentration (%)
t~
21.0
15.5
21.0
12.6
21.0
10.5
(8:30 a.m.)
37.36 ± 0.33
(10:30 a.m.)
36.86 ± 0.24c
(11:00 a.m.)
37.10 ± 0.56
(1:00 p.m.)
36.91 ± 0.28
(8:30 a.m.)
37.66 ± 0.34
(10:30 a.m.)
36.92 ± 0.21d
(11:00 a.m.)
36.86 ± 0.440
(1:00 p.m.)
36.77 ± 0.41
(8:30 a.m.)
37.55 ± 0.38
(10:30 a.m.)
36.73 ± 0.27e
(11:00 a.m.)
36.48 ± 0.48 b
(1:00 p.m.)
36.41 ± 0.49
Data are means ± SD of eight rats. Time of measurement ofTr value is in parenthesis.
p < 0.005 compared to normoxic value (paired t-tests).
b p < 0.01 compared to normoxic value (paired t-tests).
c p < 0.01 compared to 8:30 a.m. value (paired t-tests).
d p < 0.005 compared to 8:30 a.m. value (paired t-tests).
e p < 0.001 compared to 8:30 a.m. value (paired t-tests).
o
"
Sleep, Vol. 13, No.3, 1990
J. LASZY A~'D A. S/lRKAD/
1\' '"'"
0,
........ 111'-, , _ , ....... '''''' of ....
t
.......... .
1001" PS. !' - '- '
b
!
swS,.
du. _
,-..,io
.......5""", ...... _ ..
"'TIlT_ ....... .
· ..... TIlT;t ........ _ofSWS! .... , _
of '1,'" """"' , ""
!bI'.,~_
SWS, .... _~
0....".......,,,,,,
ctea>odbl'_"to.*'_........::
•
,. H"
_ .. * .... U'T ,.... ,.,_
,...... ",.",.*",
U, - . .. _ _
.,--'
_ ••'' ''lJ.ml
... " ••
s.,'"_..,..
,•• ' ..". '"
,,,
_ "'T'IT.
... ___
.
.IO" __
,,' .
--" <'.,'<" ,-....", <"".
,,,-...,., <._,
'" . ,~.
_.~.)V
... ... --;
.
HYPOXIC SLEEP DISTURBANCE IN RATS
209
decrease in PS. During inspiration of 10.5% oxygen, the SWS 1 proportion increased by
152% and the SWS2 and PS proportions decreased by 45 and 99%, respectively.
The number of AWand SWS 1 states increased significantly, whereas the number of
both SWS2 and PS stages decreased parallel to reduction of oxygen level (Fig. 3, N).
The MT of SWS2 epochs diminished significantly, whereas that of SWSI increased;
these changes occurred only under the effect of 10.5% Oz. The mean duration of PS
epochs decreased even under the effect of 12.6% O2 (Fig. 3, MT). As hypoxia increased
PS latency values rose.
Frequency of transitions from one sleep-wake level to another also changed in hypoxia. Table 2 summarizes the mean frequency of stage changes. The frequency of
changes in sleep-wake states was not altered significantly by 15.5% oxygen, although
there was a tendency to decrease in transitions of SWS2 to PS. The frequency of
transitions of SWSI to AW, AW to SWSl, SWSI to Ar, and SWS2 to AW increased
significantly in 12.6 and 10.5% oxygen, whereas the change from SWS2 to PS and the
changes from PS to Ar and AW decreased. The transitions from SWS 1 to SWS2 also
decreased in 10.5% oxygen.
Neither degree of hypoxia caused changes in the gross behavior of the animals.
Effect of Piracetam and Hydergine on hypoxic sleep disturbances
Piracetam in doses of 75 and 150 mg/kg had no effect on sleep-wake organization
disturbed by hypoxia. No changes were detected in any of the measured parameters,
compared to the hypoxic solvent control session. In a much higher dose (500 mg/kg),
however, the drug shifted the sleep-wake proportion toward the normoxic value; the
SWSlITST proportion decreased by 25%, and the SWS2rrST proportion increased by
38% compared to the hypoxic solvent control value (Fig. 4, %). The number of SWS2
periods also increased significantly (Fig. 4, N). The transitions from SWSI to SWS2
became more frequent [14.50 ± 2.63 versus 22.00 ± 2.58; F(3,28) = 27.00; p < 0.005].
PS stages of several seconds appeared more often, but this change was not significant.
Hydergine decreased the elevated waking time characteristic of hypoxic sleep disturbance by only 16% in the highest dose (3 mg/kg), but the distribution of the different
sleep stages in the TST was already modified, even at a dose of 0.5 mg/kg. The SWSI
proportion decreased by 38, and the SWS2 proportion increased by 76% compared to
the hypoxic solvent control, i.e., the SWS2 proportion became even higher than that of
HT
min
6-
roFIG. 3. MT (SWSl, F = 5.45;
SWS2, F = 3.45; PS, F = 23.49) and
recurrence (N) of each sleep-wake
episode (AW, F = 28.42;SWSI, F =
3.43; SWS2, F = 4.83; PS, F =
24.19). (For abbreviations and conventions, see text and Fig. 2.)
ps
I ....
PS
....
59NS2
4-
9NS1
3-
Ar
502AW
1-
0-
PW
21.0
15,5 12,6 10,5 V% 02
0-
21,0
15,5 12,6 10,5 V% 02
Sleep, Vol. 13, No.3, 1990
210
J. LASZY AND A. SARKADI
TABLE 2. Frequency of transitions from various sleep-wake levels
Oxygen content (%)
Stage
transitions
21.0
AWto SWSI
Ar to SWSI
Ar to SWS2
SWSI to AW
SWSI to Ar
SWSI to SWS2
SWSI to PS
SWS2 to AW
SWS2 to Ar
SWS2 to SWSI
SWS2 to PS
PS to AW
PS to Ar
PS to SWSI
PS to SWS2
11.37
19.75
8.37
5.25
7.25
21.75
0.37
2.87
14.00
2.25
11.25
3.12
7.00
1.25
0.25
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
15.5
3.46
6.15
1.92
2.60
4.06
3.84
0.74
2.03
3.20
2.18
3.57
1.12
3.38
1.66
0.77
12.12
22.37
6.25
4.25
8.62
23.50
0.62
4.50
13.75
2.12
9.37
3.37
6.25
0.37
0.00
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
10.5
12.6
1.95
8.58
2.49
2.31
4.80
5.58
0.74
2.13
3.24
1.64
2.77
1.40
2.49
0.51
0.00
19.25
21.25
5.37
10.00
13.00
22.00
0.12
7.12
13.37
3.25
3.75
2.25
1.37
0.12
0.12
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
2.86a
6.58
3.11 b
3.33 d
2.77 d
7.54
0.35
2.58 c
6.56
1.83
2.86 a
1.66
1.50a
0.35
0.35
23.75
17.62
4.00
18.00
12.12
14.25
0.00
5.37
9.37
3.00
0.62
0.37
0.25
0.00
0.00
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
4.09a
6.69
3.02 c
6.14a
5.16b
4.83 c
0.00
3.81
3.96
1.30
0.91a
0.51 a
0.46a
0.00
0.00
F(3,28)
(ANOYA)
27.56
0.67
3.74
20.64
3.27
4.38
1.99
3.35
1.91
0.78
26.24
9.38
18.39
3.19
0.73
Data are means ± SD of 8 rats.
p < 0.001 compared to normoxia (unpaired t-test).
b p < 0.05 compared to normoxia (unpaired t-test).
c p < 0.005 compared to normoxia (unpaired t-test).
d p < 0.01 compared to normoxia (unpaired t-test).
a
the normoxic control (Fig. 5, %). The changes were similar at the two higher doses (1
and 3 mg/kg). The increase of the SWS2 proportion as an effect of Hydergine doses of
0.5 and 1 mg/kg may be due to the increase ofrecurrence rather than to that of duration
(Fig. 5, N, MT). At a dose of 3 mg/kg, Hydergine caused longer, uninterrupted SWS2
periods (Fig. 5, MT). This high dose significantly decreased the number of wakings
compared to hypoxic solvent control. Short paradoxical sleep stages appeared sporadically only at lower doses. Hydergine decreased the frequency of transitions from states
of SWSI to AW and increased the incidence of transitions into SWS2. The animals
went into SWS2 more frequently from short arousals (Table 3).
.... ....
%
-100-
100-
'PS
80TST
.
AW
~ II
sol¥ 500
'---"
21.0
Piracetam
10.5
-~-
-0-
~-
III
solv. 500
'---',
21.0
Piracetam
10,5
0-
solv 500
mg/~
Piracetam
21.0~'----::10:-:-.5~ V% Oz
FIG. 4. Effect of Piracetam in hypoxia on sleep-wake proportion, on share of different sleep stages from TST
(%) (SWSl, F = 9.53; SWS2, F = 9.64), and on recurrence of each sleep-wake episode (N) (SWS2, F =
12.42). 0, p < 0.05 compared to hypoxic solvent control (unpaired t-test). (For other abbreviations and
conventions see text and Fig. 2.)
Sleep. Vol. 13. No.3, 1990
HYPOXIC SLEEP DISTURBANCE IN RATS
f'1T
-,
PS
min
6%
10'
'00-
.
PS
•
PS
,
O.
,
,
,
0
so- SWS,
Ar
2-
,-
Ar
AW
,
(IS
3 III1/kg 0H)der9ine
1>.5 V%
°
°
0
~2
~'
&
SWS,
so-
21.0
lOO-
4-
3-
soIv.
.... ....
.fi0
0
SWS2
S
0-
,
,,
5-
0
0
0
0
211
soIv.
D2
21.0
0,5 1 3 III1/kg
H)dergi1e ,
0-
I
AW
SO(V,
3 III1/kg
Hydergine
10,5 yo;. 02
,
10.5 yo;. 02
21.0
FIG. 5. Effect of Hydergine in hypoxia on share of various sleep stages from TST (%) (SWSI, F = 7.75;
SWS2, F = 13.68), on MT of sleep-wake episodes (SWS2, F = 2.i4), and on N (SWS2, F = 6.43). (For
abbreviations and conventions, see text and Figs. 2 and 4.)
~:
Effect of Hydergine on normoxic sleep-wake organization
The sleep-wake proportion was unchanged under the effect of Hydergine doses of I,
3, and 5 mg/kg. The MT of SWS2, however, increased significantly, parallel to a slight
reduction in the frequency of SWS2 stages (Fig. 6, MT, N). Thus, the proportion of
SWS2 elevated considerably (Fig. 6, %). Hydergine had a dose-dependent suppressing
effect on the development of PS stages. The frequency of PS and the PSITST proportion decreased significantly (Fig. 6, N, %).
DISCUSSION
l"
Effect of different levels of hypoxia on sleep-wake organization
The sleep-wake organization of rats remained practically unaltered by their breathing
15.5% oxygen. Further reduction of the oxygen content of the environment to 12.6%
caused detectable disturbance of sleep-wake organization in rats. The most evident
change seemed to be a decrease in frequency and MT of PS and an increase of PS
latency, whereas the number of wakings and the time spent in AW were increased.
TABLE 3. Effect of Hydergine on stage transitions affected by hypoxia
t:J
Oxygen concentration (%)
10.5%
Stage
transitions
~
AWtoSWI
SWSI to AW
Ar to SWS2
SWSI to SWS2
SWS2 to PS
Hydergine doses (mg/kg)
21.0
12.37
4.75
5.12
18.50
7.87
±
±
±
±
±
10.5
4.24
2.86
4.18
6.00
2.90
19.50
12.62
2.75
14.62
0.00
±
±
±
±
±
0.5
5.50
3.73
2.76
4.86
0.00
17.75
6.75
6.25
19.37
0.50
1.0
5.39
5.00"
2.12"
4.40Q
1.41
17.37
6.37
6.00
19.50
0.62
F(3,28)
(ANOVA)
3.0
6.45
4.59"
3.42Q
4.24Q
1.18
14.00
5.37
5.37
15.62
0.00
±
±
±
±
±
2.82
I~
2.26c
5.25
2.54
2.76
1.01
2.92
3.54
0.00
Data are means ± SD of eight rats.
p < 0.05 compared to hypoxic control (unpaired t-test).
b p < 0.01 compared to hypoxic control (unpaired t-test).
c p < 0.001 compared to hypoxic control (unpaired t-test).
Q
"
Sleep. Vol. 13. No.3, 1990
212
1. LASZY AND A. SARKADI
MT
min
6-
%
100 -
PS
5-
100-
4-
350 -
1-
0-
SDlv
1
5 mg/kg
Hyderglne
21,0 V% 02
0-
AW
solv
01
5 mg/kg
H yderglne
21,0 V% 02
solv.
1
5 mg/kg
Hydergine
21,0 V% 02
FIG. 6. Effect of Hydergine in normoxia on share of various sleep stages from TST (%) (SWS2, F = 12.01;
PS, F = 9.48), on MT of sleep-wake episodes (SWS2, F = 7.42), andonN (PS, F = 3.18). F(3;28),ANOVA.
(For abbreviations and conventions, see text and Fig. 2.)
More severe hypoxia 00.5% oxygen) caused drastic changes in sleep organization.
Sleep was interrupted frequently; paradoxical sleep phases and, as a consequence,
sleep cycles, were practically absent. The number, MT, and total proportion of SWS2
decreased substantially. The elevation of SWS 1 proportion and the increase in frequency of the transitions SWS 1 to AW, SWS2 to AW, and SWS 1 to Ar caused sleep to
be superficial.
Hale et al. (5) and Pollard et al. (6) found significantly altered sleep-wake pattern in
rats in 15% oxygen. They attributed this alteration largely to the influence of hypoxia
on the frequency of changes in sleep-wake states. The number of transitions from SWS
to AW increased, and the number of transitions from SWS to PS diminished. In accordance with this, the proportion of A W increased and that of PS decreased, compared to normoxic control. In this study, the change in similar parameters and the
change in the proportion of SWS1/SWS2 not mentioned by other researchers have not
reached the level of significance in 15.5% oxygen. The variation in the results might be
explained by different experimental circumstances. In the studies of Hale et al. (5) and
Pollard et al. (6), the rats slept in sound-attenuated chambers, whereas in our investigations, the rats were kept in well-habituated, normal laboratory circumstances with
masking background noise. In a stimulus-free environment, mild hypoxia, itself, may
represent a disturbing stimulus that is likely to be the cause for a change in a very
sensitive sleep organization.
In more severe hypoxia-12.6 and 10.5% 02-changes in sleep parameters can well
be compared to the results of Pollard et al. (6). Although, in our investigation, PS seems
to have been more affected by hypoxia, the decrease in its frequency and MT was more
drastic. Our study supports the results of Megirian et al. (2) and Ryan and Megirian (3),
who reported practical cessation ofPS and more pronounced decrease of SWS in 10.5%
O2 relative to the results found by Pollard et al. (6).
By distinguishing SWSI and SWS2 stages, we could analyze the changes within
SWS. Our results show that with decreasing O2 levels, the proportion of SWSI increased and that of SWS2 decreased, due to the increase in the frequency of SWS 1 to
Sleep, Vol. 13, No.3, 1990
HYPOXIC SLEEP DISTURBANCE IN RATS
f'
213
AW and AW to SWSI phase changes and the decrease in SWSI to SWS2 transitions.
The mean epoch duration of SWSI and SWS2 phases changed significantly only in the
most severe hypoxia (10.5% O2),
Summarizing the results of the different hypoxic sleep studies, mild hypoxia seems to
have influence primarily on the frequency of changes of sleep-wake stages. More
pronounced hypoxia influences the MT of sleep phases, as well.
Szymusiak and Satinoff (11) determined the thermoneutral zone (minimal metabolic
rate) as that lying between 25 and 31°C in rats; but measuring the maximal PS time, they
defined the neutral temperature more exactly, at 29°C. Data show that both Tr (12) and
core temperature (5) decrease at an ambient temperature of 25-30 and 29°C, respectively, in mild hypoxia. Our investigation confirmed the data (6) in which mild and
moderate hypoxia do not influence significantly the body temperature at the neutral
temperature of rats.
Sleep stages, especially the PS stage, are sensitive to decreases in body temperature
(13,14). The insignificant decrease in body temperature (0.2--O.3°C) is not comparable to
the rough disturbance in sleep organization in our experiments.
Our experiments prove that the decrease in the Tr observed in hypoxia at 27 ± 1°C
ambient temperature is connected with the diurnal changes in the body temperature of
rats.
The maintenance of normal brain function in hypoxia results in development of
different homeostatic mechanisms. The partial arterial oxygen tension (PaOz) decreases
to 58.9 mmHg at 15.3% oxygen in the inspired air in a conscious, spontaneously breathing rat (15). Simultaneously, the PaC02 falls significantly from 40.9 mmHg to a value of
36.8 mmHg, due to increased ventilation even in this very mild hypoxia. The vasodilatory autoregulation is functioning even at mildly reduced arterial oxygen tension (70-80
mmHg) in artificially ventilated, normocapnic rats (16), but marked brain blood flow
enhancement was observed at values under 50 mmHg (16,17). In spontaneous hyperventilation, hypocapnic alkalosis producing cerebral vasoconstriction attenuates the
hypoxia-induced increase in the cerebral blood flow. There is, however, no significant
difference in the increase of cerebral blood flow between normocapnic and hypocapnic
rats in more severe hypoxia (Pa0 2 = 25 mmHg) (16).
We did not find any change in sleep-wake organization in the mildest hypoxia investigated. It is probable that the oxygen supply of the brain remains relatively unchanged due to the circulatory/respiratory compensatory mechanisms. Thus, oxygen
tension in the extracellular space of the brain at 15.5% oxygen could not change to such
an extent to substantially modify brain function. In more severe hypoxia, however, the
compensation is insufficient and the brain function is damaged.
During 45 mmHg Pa02 (which corresponds to -10% oxygen in the inspired air), the
oxygen and glucose consumption of the whole brain and the tissue concentration of
energy-rich phosphates were found to be unchanged, whereas the cerebral metabolic
rate of lactate was raised (18). This indicates the increased conversion of pyruvate to
lactate during moderate hypoxia (18). The increase in glycolytic rate probably plays
only a minor role in preventing disturbances in the energy metabolism of the brain; the
dominating factor seems to be the increase in cerebral blood flow (19). Hypoxemia,
produced by 7% oxygen in the inspired air in spontaneously breathing conscious rats,
however, caused a complete redistribution of the local rates of glucose utilization,
whereas overall average glucose utilization remained unchanged (20). Thus, the sleepwake disturbance due to 10% oxygen content of inspired air may be explained by
Sleep, Vol. 13, No.3, 1990
214
1. LASZY AND A. SARKADI
metabolic and functional changes in specific regions of the brain, rather than by a
significant change in total brain energy supply.
Hypoxia also influences biochemical mechanisms fundamental in sleep-wake regulation. In moderate hypoxia (9% oxygen in the inspired air), disturbances of synthesis
and metabolism of the monoamines were already observed: Both tryptophan and tyrosine hydroxylase activity decreased, and there was a significant decrease in accumulation of 5-HTP and DOPA (21,22). In another experiment (23), rats were exposed
to 5.6% oxygen in air for up to 2 h. There was a decrease in tyrosine hydroxylase
activity in the whole brain, as well as in the hemispheres, striatum, and brain stem.
Tryptophan hydroxylase activity declined during the first hour. The levels of noradrenaline, dopamine, and serotonin were not changed significantly in the whole brain nor in
the brain regions. Gibson and Blass (24) observed damaged acetylcholine production
parallel to undisturbed brain energy metabolism in histotoxic hypoxic mice. The synthesis of acetylcholine in free-breathing rats declined substantially, even at 15% oxygen
(25). Although the most protected area against hypoxia is the brain stem-where the
ascending noradrenergic, serotonergic, and cholinergic pathways run-this region may
suffer a relative oxygen insufficiency at 10% or lower oxygen content in the air. The
hypoxia-induced imbalance of the metabolism of these neurotransmitters may explain
the disturbances in sleep-wake organization. We studied the hypoxic range, where the
first signs of sleep disturbace may be detected in connection with first appearance of an
imbalance in the neurotransmitter metabolism.
Peripheral chemoreceptor reflex pathways may also contribute to disruption of the
sleep-wake pattern in hypoxia. Ryan and Megirian (3) and Ryan et al. (26) reported that
the cutting of the carotid sinus nerve alleviates sleep disturbances by increasing the MT
of the various sleep stages in hypocapnic hypoxic rats. Bowes et al. (27) demonstrated
that carotid body denervation markedly impairs the hypoxic arousal response in sleeping dogs. The afferent impulses coming from the carotid chemoreceptors in hypoxia
may produce an excitation of the reticular activating system, which leads to frequent
wakings and interrupted sleep.
Our results, in accordance with earlier findings (2-4), prove the special sensitivity to
hypoxia of PS compared to other sleep stages. This special sensitivity may be related
to increased oxygen consumption of the brain during PS (28,29). In human and animal
experiments, increase in cerebral blood flow has been measured during PS (30). It is
also known that PS is always preceded by a SWS2 phase of certain duration-several
minutes, in rats. In hypoxia-as proved in our experiment (Table 2)-the transitions
from SWS2 to AW become more frequent, so the duration of the uninterrupted SWS2
phases is shortened substantia!ly. Thus, the shortness of SWS2 periods can decrease
the probability of development of PS stages.
Obviously, reducing the oxygen content of the inhaled air below a certain limit
impairs sleep-wake organization. The first disturbances in adult, healthy rats kept
under laboratory conditions but not in full isolation can be detected at oxygen levels
below 15%. An oxygen level of 10.5% leads to total disintegration of normal sleep-wake
organization; however, no behavioral changes can be detected. In mild hypoxia, circulatory/respiratory mechanisms can compensate for the deficiency in the oxygen supply to the brain. With oxygen concentration under 15%, this compensation is insufficient. Changes in the cerebral metabolic processes alter the biochemical background of
the sleep-wake regulation. Peripheral chemoreceptor pathways may also contribute to
hypoxic changes in sleep-wake organization.
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HYPOXIC SLEEP DISTURBANCE IN RATS
215
Effect of Piracetam and Hydergine on hypoxic sleep disturbance
Hypoxic sleep disturbance can be considered as an appropriate model of changes in
functions evoked by hypoxia.
Piracetam beneficially influenced the decline of memory and performance after brain
hypoxia in some human and animal studies (7,31-33) and speeded convalescence from
hypoxic damage. This drug contributed significantly to the recovery of normal EEG
pattern after "nitrogen hypoxia" in rabbits (8). Piracetam was administered in rather
high doses in these experiments. The mechanism of action of this drug is thought to be
a combination of metabolic and hemodynamic effects (34). The protective effect of
Piracetam against cerebral hypoxia is attributed to its adenylate kinase-stimulating
action (35) and to its beneficial effect on cerebral circulation (36). In our experiment,
the Piracetam doses of 75 and 150 mg/kg i.p. had no significant effect on hypoxic sleep
disturbance. Very short PS stages, however, developed in some animals already under
the effect of a Piracetam dose of 150 mg/kg. A Piracetam dose of 500 mg/kg had a more
pronounced effect: The sleep-wake pattern shifted toward normoxic values in the
proportion of AWand SWS 1 and SWS2 states. Considering the effect of Piracetam on
normal sleep-wake organization, it seems to have selective action on PS (37). Piracetam slightly increased the percentage and MT of PS.
Hydergine had a reactivating effect on brain metabolism after transient ischemic
periods and decreased the energy loss in every frequency range in isolated perfused cat
brain (38). In acute experiments, it accelerated the normalization of evoked cortical
potentials damaged by ischemia (39). Hydergine improved the active avoidance learning in rats in 9% oxygen (38). The beneficial effect of Hydergine on impaired brain
function does not appear to be related to total or regional cerebral blood flow enhancement. It may result from an enhanced effect of the compound on brain metabolism (38).
According to our results, Hydergine very characteristically altered the hypoxic sleepwake organization. It increased the ratio of SWS2 even at a dose of 0.5 mg/kg. The
uninterrupted long SWS2 episodes became more pronounced with high doses (3 mg/kg)
of Hydergine. It counteracted the effect of hypoxia in this respect. With Hydergine as
well, PS stages did not appear at the highest dose.
Our findings in connection with the effect of Hydergine on the normoxic sleep-wake
organization correspond to the results of Susic and Masirevic (40) obtained in experiments with cats. In their investigations, Hydergine caused significant increase in the
MT of SWS epochs. During these SWS periods, the animals were awakened by external
and internal stimuli with more difficulty. Hydergine decreased the time spent awake, as
well as the proportion of PS. These results suggest that Hydergine alleviates hypoxic
sleep disturbance, primarily by its marked contribution to the formation of SWS2
phases. In this way, Hydergine moderates one of the characteristic symptoms of hypoxic sleep disturbance, namely superficial sleep interrupted by frequent wakings. However, it cannot restore the normal cyclicity of sleep. In low doses, Hydergine evokes
only short and sporadic PS phases, by suppressing their formation.
In the hypoxic range, where the air contains 15.5-10.5% oxygen, significant changes
in sleep-wake organization occurred in rats. These changes offer an appropriate model
for testing drugs to improve the brain's resistance to hypoxia.
Acknowledgment: The authors thank Mrs. M. Szoke and Miss K. Gerber for careful technical
assistance, and Miss Elizabeth Cunningham for editing the manuscript.
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1. LASZY AND A. SARKADI
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