Selecting representative ages for developmental changes of respiratory irregularities and hypoxic ventilatory response in rats

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Open Journal of Molecular and Integrative Physiology, 2011, 1, 1-7

doi: 10.4236/ojmip.2011.11001 Published Online May 2011 (http://www.SciRP.org/journal/ojmip/

OJMIP

Published Online May 2011 in SciRes. http://www.scirp.org/journal/OJMIP

Selecting representative ages for developmental changes of

respiratory irregularities and hypoxic ventilatory response in

rats

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: *[email protected]

Received 9 April 2011; revised 26 April 2011; accepted 5 May 2011.

ABSTRACT

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

1. INTRODUCTION

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-

ied.

2. MATERIALS AND METHODS

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

[8-10,12].

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

L. M Niane et al. / Open Journal of Molecular and Integrative Physiology 1 (2011) 1-7

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.

100

150

200

250

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

Age/day

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.

(a)

Oxygen consumption

(VO

, ml/min/100g)

P1P4P7P12 P21P90

††††

† p < 0.05 vs P1, P4

$ p < 0.05 vs P7

Age/day

3. RESULTS

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)).

(b)

Figure 1. Baseline minute ventilation (a) and meta-

bolism (b) across ages studied in rats. Data are means

 SEM.

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

OJMIP

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

100

120

140

160

180

0510 15 20

100

120

140

160

180

05101520

100

120

140

160

180

P1 (n = 9)P4 (n = 20)P7 (n = 13)

Time (min)

0510 15 20

100

120

140

160

180 P1 2 (n = 17)

Time (min)

0510 15 20

100

120

140

160

180

Time (min)

0510 15 20

100

120

140

160

180

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).

4. DISCUSSION

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)

–100

–80

–60

–40

–20

P1P4P7P12 P21P90

†*

†† †

† p < 0.05 vs P1 and P90

* P < 0.05 vs P90

Age/day

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.

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)

–1

–3

–5

Pos t-s igh apn ea

–1

–3

–5

Flow (ml/s)

(a) (b)

Apnea frequency/10 min

during baseline

P1P4P7 P12P21P90

†

†$

†

Age/day

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

†

†$

†

Age/day

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

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.

5. ACKNOWLEDGEMENTS

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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