Open Journal of Molecular and Integrative Physiology, 2011, 1, 1-7
doi: 10.4236/ojmip.2011.11001 Published Online May 2011 (
Published Online May 2011 in SciRes.
Selecting representative ages for developmental changes of
respiratory irregularities and hypoxic ventilatory response in
Lalah M Niane*, Aida Bairam
Unité de Recherche en Périnatologie, Centre Hospitalier Universitaire de Québec, Hôpital Saint-François d’Assise, Département de
Pédiatrie, Université Laval, Québec.
Email: *
Received 9 April 2011; revised 26 April 2011; accepted 5 May 2011.
Apnea frequency and the weak ventilatory response
to hypoxia are a major clinical correlates of the im-
maturity of respiratory control system in preterm
neonates. Rats are frequently used as model to study
the respiratory control during development. However,
little is known about the postnatal ages that best rep-
resent these respiratory irregularities and the hy-
poxic ventilatory response. Using plethysmography,
we assessed baseline minute ventilation, ventilatory
response to moderate hypoxia (FiO2 = 12%, 20 min)
and apnea frequency in awake and non-anesthetized
rats at the postnatal ages of 1, 4, 7, 12, 21 and 90 days
old (P1, P4, P7, P12, P21, and P90, respectively).
Baseline minute ventilation slightly increased in P4 (~
25% vs P1) then gradually decreased with age (age
effect: p < 0.05). The lowest level of ventilation was
observed in P90 (p < 0.01 vs all ages). Minute ventila-
tion (% from baseline) in response to hypoxia showed
the well-known biphasic pattern in all rats at 12 days
old or less. Minute ventilation at the initial phase of
the hypoxic response was not significantly different
between P1, P4, between P7, P12 and between P21,
P90. The late phase of the hypoxic response was
similar between P1, P4, and between P21, P90, but
was significantly different between P7 and P12 (p <
0.05). Under baseline or hypoxic condition, the higher
number of apnea frequency (spontaneous and post-
sigh) was observed in P1, it then decreased progres-
sively with age (age effect: p < 0.01 for baseline; p <
0.001 for hypoxia). These results suggest that when
P4, P7 and P12 are selected to represent the age-de-
pendent changes of the hypoxic ventilatory response
in rats, the P1 rats should be included to better de-
scribe the age-dependence of apnea frequency.
Keywords: Apnea; Hypoxia; Newborn Rat
The respiratory control system evolves rapidly in new-
born mammals during the neonatal period. The major
clinical correlates of the immaturity of this system are
periodic breathing and apnea, particularly in preterm
neonates. Accordingly, apnea in preterm neonates is sys-
tematically treated. The hypoxic ventilatory response
(HVR) is regularly used to evaluate the immaturity of
the respiratory control system. This response is not fully
developed at birth and undergoes significant changes
during the postnatal period. In newborn mammals, the
HVR presents as a biphasic pattern consisting of an ini-
tial increase in ventilation, resulting from a stimulation
of peripheral chemoreceptors (primarily located in the
carotid bodies), followed by a decline, in some cases to a
level below pre-hypoxic levels. It is likely that the de-
cline in ventilation originates centrally and ultimately
overrides the excitatory input from peripheral chemore-
ceptors. The maturation of the HVR during the postnatal
period may take days to weeks depending on the mam-
malian species studied [1-3]. Although the developmen-
tal pattern of the respiratory control system has been
described in multiple newborn animal models, such as
mice, rat, cat, rabbit, piglet, and lambs, longitudinal
studies assessing ventilation in awake and non-anesthe-
tized rats are few [4,5]. Indeed, respiratory irregularities
have yet to be assessed in a population of rats from birth
to adult ages. Using rats as an animal model, our aim
was to determine the frequency of spontaneous apnea, as
an index of respiratory irregularities, and to concurrently
evaluate the HVR, as an index of maturation of the
chemoreceptor reflex to hypoxia in specific postnatal
age groups and as adult. The secondary aim was to select
ages that could longitudinally reflect the development of
the respiratory control system as well as reduce the
age-population of rat pups used for these types of invest-
L. M Niane et al. / Open Journal of Molecular and Integrative Physiology 1 (2011) 1-7
tigations. We choose rats for the following reasons: the
central nervous system of rat pups born at term is im-
mature in comparison with humans and has been roughly
compared to that of infants born at ~ 28 weeks of gesta-
tion [6]. Indeed, the HVR of rat pups at 3 - 5 days old is
similar to that observed in preterm humans below 30
weeks of gestational age; at about 2 weeks old, it is
similar to full term human neonates [1]. Finally, the
maturation of the central nervous system as a whole in
rats occurs during the first three weeks of age [6], thus
providing a highly reliable and practical model to deter-
mine the developmental pattern of the HVR at a very
specific postnatal age. Using plethysmography, we re-
corded baseline ventilation and the HVR by exposing
rats to moderate hypoxia (FiO2 = 12%, 20 min). We as-
sessed the frequency of apnea during the baseline and
the steady state of hypoxic response. Rats at ages 1, 4, 7,
12, and 21 days old and adults at 90 days old were stud-
2.1. Animals
The local Animal Care Committee at Laval University
approved the experimental protocol. The study was per-
formed on 94 Sprague-Dawley male rat pups born in our
animal care facility from 14 virgin females and males. At
birth (P1), litters were culled to 12 pups with a prefer-
ence for males. Because steroidal sex hormones may
affect the HVR [7], we preferred to use males to ensure
the homogeneity of our study at all ages. Those that
were used at adulthood (90 days old) were weaned from
their mother at 21 days old and were placed 2 per cage to
be studied at 90 days old.
2.2. Plethysmography Recording
Respiratory and metabolic indexes were recorded in P1
(12-24 h following birth; n = 9), P4 (n = 20), P7 (n = 13),
P12 (n = 17), P21 (n = 24) rats using a whole body
flow-through plethysmograph (IOX, Emka Technologies,
Paris, France) as we regularly used in newborn rats [8,9].
The gas flow through the plethysmograph was set at 100
ml/min for P1-P7 rats and 200 ml/min for P12 and P21
rats (flowmeter, model 4140; TSI, Shoreview, MN). The
temperature inside the plethysmograph was set at 34˚C
(P1), 32˚C (P4 and P7) and 30˚C (P12) using a tempera-
ture-control system (Physitemp, Clifton, NJ, USA), and
the relative humidity was continuously measured from
the out-flowing air stream. In P90 rats (n = 11), ventila-
tion was recorded using a double-chamber plethys-
mograph (model PLY 3023, Buxco Electronics, Sharon,
CT, USA) where the gas flows through the front (head)
and rear chambers were set at about 100 and 500 ml/min,
respectively[10]. The respiratory flow tracing, recorded
using the rear chamber of the plethysmography, was
calibrated by injecting a known volume of air into the
chamber [9,10]. The inflow and outflow of oxygen were
continuously recorded using a dedicated oxygen ana-
lyzer (AEI Technologies; Naperville, IL, USA). Body
temperature was measured via the mouth in P1, P4 and
P7 rats or via the rectum for the older rats before and at
the end of the experiment using a thermocouple for
small or adult rodents (Harvard, Holliston, MA, USA).
Respiratory frequency and tidal volume were recorded
from the plethysmograph signal. Tidal volume was first
corrected depending on barometric pressure, room and
body temperature, and humidity (BTPS) using the Bart-
lett and Tenney equation [11], and afterward, it was used
to calculate minute ventilation (respiratory frequency X
tidal volume). Because the aim of this study was to de-
scribe the developmental pattern of minute ventilation in
response to hypoxia, the changes in respiratory fre-
quency and tidal volume during development are not
presented for brevity. As an index of metabolism, oxy-
gen consumption was calculated as follows: flow ×
[(O2,in O2,out) – O2,out × (CO2,out CO2,in)]/(1 O2,out).
However, the calculation was corrected to STPD condi-
tions as had been done previously [9,10]. All details for
the sources of apparatus were set as reported previously
2.3. Baseline Ventilation and Hypoxic Response
Each rat studies only once and was placed into the
plethysmograph at least 20 - 30 min before experiment
for adaptation. After the body temperature measurement,
baseline normoxic variables (FiO2 = 21%) were recorded
for 10 min. Then, the pup was exposed to moderate hy-
poxia (FiO2 = 12%, 20 min) by mixing pure nitrogen
with air flowing through the plethysmograph to achieve
12% O2 concentration in approximately 2 min. These
first 2 min were not considered for calculation. At the
end of the recording, the body temperature was immedi-
ately remeasured.
2.4. Apnea Frequency
The frequency of apnea was calculated during the last 10
min of baseline recording and the last 10 min of hypoxic
response (steady state) using the standardized criteria of
Mendelson [13], as we have used previously [8,12,14].
Two types of apnea were selected. Spontaneous apnea
was defined as the interruption of flow for at least two
normal respiratory cycles, and post-sigh apnea occurred
when the breath amplitude was at least twice the resting
tidal volume [13] (See examples, Figures 4(a) and (b)).
Because all newborn rats showed a very low frequency
of post- sigh apnea (about 10%) during baseline, in ac-
cordance with previous studies [14] but a very low
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L. M Niane et al. / Open Journal of Molecular and Integrative Physiology 1 (2011) 1-7
Copyright © 2011 SciRes.
spontaneous apneas (about 15%) during hypoxia, we
combined both types of apnea (total apnea) under each
conditions studied. Respiration is largely dependent on
the sleep stage [15]. Although we did not record the
sleep-wake state during recording, the rats were closely
observed, and if necessary we gently knocked on the
wall of the plethymograph to keep them awake.
P1P4P7 P12P21P90
V E (m l /m i n /100g)
† p < 0.05 vs P1, P7, P12, P21, P90
# p < 0.01 vs all ages
2.5. Data Collection and Statistical Analyses.
The ventilatory variables collected on a minute-by-
minute basis by IOX software (Version 1.8.9 EMKA
technology, Paris, France) were averaged over the last 5
min for the baseline values, every 2 min between 2 - 10
min for the initial phase of the hypoxic response and
every 5 min for the last 10 min, during the late phase of
the hypoxic response. Oxygen consumption was meas-
ured at the baseline and at the end of the hypoxic re-
sponse. A one-way ANOVA was used to compare dif-
ferent ages, and a p-value < 0.05 was considered sig-
nificant. Data are presented as mean ± SEM.
Oxygen consumption
, ml/min/100g)
P1P4P7P12 P21P90
† p < 0.05 vs P1, P4
$ p < 0.05 vs P7
3.1. Baseline Minute Ventilation and Metabolism
Minute ventilation increased slightly at P4 compared
with P1 as has been previously observed [5]. Minute
ventilation was then gradually decreased with age and
the lowest level was observed in P90 (adult) rats as
compared with all of the other ages studied (Figure
1(a)). Interestingly, the level of minute ventilation ob-
served in adult rats was comparable to a previous study
that used a similar double plethysmography chamber [10]
and with a study that used a whole body plethysmograph
room [12]. Oxygen consumption decreased with age
corresponding with a higher metabolic rate in newborns
compared with adults. Then, it decreased progressively
following the pattern of minute ventilation decreasing
with age. Oxygen consumption was not different be-
tween P1 and P4 or between P7, P12 and P21. The low-
est oxygen consumption was observed in P90 compared
with all other ages (Figure 1(b)).
Figure 1. Baseline minute ventilation (a) and meta-
bolism (b) across ages studied in rats. Data are means
3.2. HVR Across Ages
of hypoxia), minute ventilation was significantly lower
than the baseline in P1 and P4 (p < 0.05), not different
from the baseline in P7 and significantly higher than the
baseline in P12 (p < 0.05), P21 and P90 rats (p < 0.0001,
for clarity the labels are not included in the figures).
Again, its level was not different between P1 and P4 but
was lower than P7 (Figure 2). In P12, minute ventilation
at 20 min of HVR was higher than P7 but lower than P21
and P90 rats. Finally, neither the initial nor the late phase
of HVR was different between P21 and P90 rats (Figure
2). At the end of HVR, the lowest oxygen consumption
Because of the difference in baseline ventilation across
ages studied (Figure 1(a)), the HVR was expressed as
the percentage of increase from the baseline. The HVR
was biphasic in all pups less or equal to 12 days old
(Figure 2). At 4 min of HVR, minute ventilation was
significantly higher than baseline in all ages studied (P
varied from 0.01 to 0.001; for clarity, the labels are not
included on the figures). Its level was similar between
P1 and P4, between P7 and P12, and between P21 and
P90 (Figure 2). During the late phase of HVR (at 20 min
L. M Niane et al. / Open Journal of Molecular and Integrative Physiology 1 (2011) 1-7
Minute ventilation
(% from bas el in e )
0510 15 20
0510 15 20
P1 (n = 9)P4 (n = 20)P7 (n = 13)
Time (min)
0510 15 20
180 P1 2 (n = 17)
Time (min)
0510 15 20
Time (min)
0510 15 20
P21 (n = 24)Ad u l t (n = 11)
Minute ventilation
(% from baseline)
* P < 0.05 vs P1, P4
** p < 0.05 vs P7, P12
$ P < 0.05 vs P1, P4 P12
$$ p < 0.05 vs P7, P12
Figure 2. Minute ventilation in response to moderate hypoxia (FiO2 = 12%, 20 min) across ages studied in rats. Minute ventilation
is expressed as percentage change from the baseline. Data are means SEM.
was observed in P12 as compared with all other ages
(Figure 3).
3.3. Apnea Frequency across Ages.
Figure 4 showed examples of spontaneous (A) and post-
sigh (B) apnea recorded in P12 rats. The occurrence of
apnea was significantly higher during hypoxia than that
observed during baseline (Figures 4(c) and (d)) in each
of age studied (p values were: 0.001, 0.005, 0.008, 0.009,
0.02, 0.01 for P1, P4, P7, P12, P21 and P90, respectively).
Apnea frequency decreased gradually with age (Figures
4(c) and (d)) showing a highly significant age-dependent
correlation, whether the P90 rats were included or not
during baseline (R2 = –0.599; Correlation p < 0.0001) or
hypoxia (R2 = –0.605; Correlation p < 0.0001).
The respiratory control system undergoes intense devel-
opmental changes during postnatal life [1,2,16]. One
major question that is debated regularly is what are the
most appropriate ages of animal models that best reflect
the developmental pattern of respiratory control. On the
basis of our current results, we suggest that P4, P7 and
(% from bas el in e)
P1P4P7P12 P21P90
† p < 0.05 vs P1 and P90
* P < 0.05 vs P90
Figure 3. Oxygen consumption at the end of hy-
poxic exposure across ages studied in rats. Oxygen
consumption is expressed as percentage change
from the baseline. Data are means SEM.
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L. M Niane et al. / Open Journal of Molecular and Integrative Physiology 1 (2011) 1-7 5
S p on tan e ou s ap n ea
Flow (ml/s)
Pos t-s igh apn ea
Flow (ml/s)
(a) (b)
Apnea frequency/10 min
during baseline
P1P4P7 P12P21P90
Apnea frequency/10 min
during hypoxia
P1P4P7 P12P21P90
12 † p < 0.05 vs P1
$ p < 0.05 vs P4, P7, P12
# p < 0.05 vs P21
(c) (d)
Figure 4. Examples of spontaneous (a) and post-sigh (b) apnea recorded during last 10 min of baseline and hypoxia in P12 rats.
Total apnea frequency during baseline (c) and hypoxia (d) across ages studied in rats. Data are means SEM.
clinical correlation of these observations, we further
suggest that P4 rats may be representative of very im-
mature babies of less than 28 weeks of gestation, P7 rats
of immature babies less than 36 weeks of gestation and
P12 rats to term babies [1,3,17]. However, the P1 rats
may need to be considered when studying respiratory
irregularities such as apnea.
Two main studies have described the effects of age on
the HVR to hypoxia in newborn rats using whole body
plethysmography in awake and non-anesthetized new-
born rats; however, in either study adult rats are not in-
cluded. Liu et al. [5] focused on the changes in the res-
piratory pattern cycle in rats from birth to 21 days old
while Eden et al. [4] mainly studied the HVR to differ-
ent levels of inspired oxygen. For example, in Liu et al.,
the same rat was tested at different postnatal ages to as-
sess the HVR during 5 min exposures to 10% oxygen. In
Eden et al., the same rat was also used at different post-
natal ages and was exposed in each experiment to multi-
ple levels of inspired oxygen, 8, 12, and 15%. In our
experimental design we recorded each rat just once, be-
cause repeated handling [18] and repeated exposure to
stimuli [19] may disrupt the developmental process of
the respiratory control system in a newborn, and such
disruption may persist well into adult life [2,20,21]. In-
deed, the level of hypoxia was moderate but sustained
for 20 min, allowing us better evaluate the developmen-
tal pattern of the biphasic HVR and metabolism. It was
clear that while the late phase progressively increased
with age, the initial phase showed different relationships
with age. P1 and P4 rats significantly increased their
ventilation in response to hypoxia, although this increase
was for a very short duration. Later, at P7 and P12, the
level of the initial increase of ventilation was lower than
that observed in younger rats, but it was more sustained.
Hence, we proposed that ages of P4, P7 and P12 could
be representative of the developmental aspect of both the
peripheral and central components of the HVR. Finally,
as indices of respiratory irregularities during the neona-
tal period we counted apnea events during the baseline
and the steady state of hypoxic response. Under each
condition, the frequency of apnea was highly dependent
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L. M Niane et al. / Open Journal of Molecular and Integrative Physiology 1 (2011) 1-7
on age providing a useful index of immaturity of the
respiratory control system. It is suggested that apnea
frequency can be used as an efficient parameter to study
respiratory irregularities in rats and an additional vari-
able to study the development of respiratory control.
One pitfall related to the use of whole body plethys-
mography is the accuracy of tidal volume measurements.
Despite this limitation, this method remains the best
available for measuring ventilatory variables (including
tidal volume) in awake, unrestrained small animals
[11,22,23]. As discussed previously [8,9,14,12], all ex-
periments were conducted under similar conditions, and
the tidal volume was corrected by considering baro-
metric pressure, humidity in the plethysmograph, body
temperature and body weight. Conversely, ventilation is
affected by sleep state [15], and rats during the first 15
days of life spend about 70% of their time asleep [24,25].
However, rats were carefully observed during recording,
and if they showed signs of falling asleep, we gently
knocked on the wall of the recording chamber to keep
the rat awake [12,14].
In conclusion, respiratory irregularities such as apnea
and periodic breathing are frequently observed in pre-
term infants. These irregularities are related to an imma-
turity of the breathing control system [17], and rats are
regularly used as a model. We propose that rats at 4, 7
and 12 days old could be used to study the developmen-
tal pattern of mechanisms, factors or drugs that affect the
HVR. However, rats at P1 old would be included to bet-
ter describe the age-dependence of apnea frequency.
This study was supported, in part, by the CIHR operating grant MOP-
81101 to A. Bairam. We thank Mrs. Melanie Pelletier and Sylvie Viger
for animal care.
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