Effect of progesterone on respiratory response to moderate hypoxia and apnea frequency in developing rats

Respiratory Physiology & Neurobiology 185 (2013) 515–525

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Effect of progesterone on respiratory response to moderate hypoxia and apnea frequency in developing rats Aida Bairam ∗ , Delphine Lumbroso, Vincent Joseph Centre Hospitalier Universitaire de Québec, Hôpital Saint-Franc¸ois d’Assise, Département de Pédiatrie, Université Laval, Québec, Canada

a r t i c l e

i n f o

Article history: Accepted 1 November 2012 Keywords: Apnea Ventilation Newborn rats Progesterone

a b s t r a c t We used whole-body plethysmography and pulse oximetry to assess the effects of acute administration of progesterone (4 mg/kg, i.p.) on normoxic ventilation, hypoxic ventilatory response (HVR: FiO2 = 12% over 20 min), metabolism, and apnea frequency in rats on postnatal (P) days P1, P4, P7, and P12. Arterial oxygen saturation was continuously measured, and apneas were discriminated based on the degree of associated desaturation, at least 5 units less than the value before the desaturation. In normoxia, progesterone did not alter ventilation, metabolism or the coefficient of variation of minute ventilation at any age studied when compared with the control group (saline). However, it decreased apnea frequency and apnea associated with desaturation only in P1 rats. In hypoxia: progesterone increased the peak HVR in P4 and P7 rats, increased the steady-state HVR (mean at 15–20 min of exposure) in P1, P4 and P7 without affecting the rats’ metabolic rate, decreased the coefficient of variation of minute ventilation in P4 and P7 rats, and finally, decreased apnea frequency only in the P1 rats with no effect on apnea associated with desaturation at any age. We conclude that acute administration of progesterone has no effect on baseline ventilation, but it increases HVR in rats younger than 7days, and decreased the frequency of apnea only in P1 rats. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The neural circuits regulating breathing undergo considerable maturation both in utero and after birth in mammals. An important corollary of the immature respiratory control system of the human newborn is periodic breathing and apnea (Abu-Shaweesh, 2004; Darnall et al., 2006). These events are frequently associated with a transient decrease in arterial oxygen content (O2 desaturation) and heart rate (bradycardia) (Abu-Shaweesh, 2004; Darnall et al., 2006), which are, in turn, associated with increased morbidity (including long-term neurocognitive deficits) and length of hospitalization (Janvier et al., 2004; Eichenwald et al., 2011). Although periodic breathing and apneic episodes in the infant reflect a physiologic and not a pathologic immaturity of the respiratory system (Abu-Shaweesh, 2004; Darnall et al., 2006), it is standard practice to treat infants with these conditions. Among the available treatments, the respiratory stimulant caffeine is more widely used than theophylline and represents the best therapeutic option (Mathew, 2011; Zhao et al., 2011). Nevertheless, a substantial percentage of the abnormal breathing episodes persist after therapeutic plasma

∗ Corresponding author at: Centre de Recherche, D0-717, Hôpital Saint-Franc¸ois d’Assise, 10, rue de l’Espinay, Québec, Qc, Canada G1L 3L5. Tel.: +1 418 525 4444/4402; fax: +1 418 525 4195. E-mail address: [email protected] (A. Bairam). 1569-9048/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.resp.2012.11.001

levels are reached, requiring further supportive therapy, such as positive pressure ventilatory support, particularly in very preterm infants (Mathew, 2011; Zhao et al., 2011). For these special cases, progesterone was recently proposed as a potential candidate for the treatment of apneas that persist despite treatment with methylxanthine (Finer et al., 2006; Mathew, 2011). Although progesterone is a well-known respiratory stimulant in adult humans (Behan and Wenninger, 2008), research into its effects on the immature control of breathing is still scarce. In 10-day-old rats, the chronic administration of progesterone via the lactating mother increases the hypoxic ventilatory response (HVR) and reduces apnea frequency during hypoxia (Lefter et al., 2007). A recent study assessing the dose–response curve for progesterone across a dose range of 0–8 mg/kg showed that 4 mg/kg progesterone increases both the peak and the steady-state phase of HVR in 4- but not in 12-dayold rats. There was no observed effect at 2 mg/kg, and the 8 mg/kg dose had no further effects on respiration at either age (Hishri et al., 2012). While there may be a link between enhanced respiratory drive (Julien et al., 2011; Niane et al., 2012) or HVR (Lefter et al., 2007) and reduced frequency of apneas, high HVR may also enhance apnea frequency (Julien et al., 2008), underlying the independence of these effects. Based on these studies, we tested the hypothesis that progesterone (1) decreases ventilatory variability and apnea frequency, (2) enhances HVR, and (3) that these effects are agedependent. To do so, we used rats on postnatal days 1, 4, 7 and 12.

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These ages were selected as representative of the postnatal development of HVR and apnea frequency in rats (Niane and Bairam, 2011, 2012). Furthermore, the effect of progesterone on oxygen desaturation and bradycardia during apnea was evaluated under baseline conditions and during the steady state of hypoxia using a pulse oximetry system for small rodents (MouseOx, STARR Life Sciences, Oakmont, PA, USA). Circulating progesterone levels were measured to identify any potential correlation with ventilatory response at each age. Our data show that progesterone increases the HVR in P1, P4, and P7 rats but decreases the frequency of apnea only in P1 rats during normoxia and hypoxia suggesting that different mechanisms are involved in mediating the effects of progesterone on HVR and apnea frequency. 2. Materials and methods 2.1. Ethics Our local animal care committee at Laval University approved the experimental protocols, in accordance with the guidelines of the Canadian Council on Animal Care. Moreover, a careful effort was made to reduce the number of animals used in the experiments. Twenty-four rats from our previous study describing the dose response curve of progesterone in P4 and P12 (Hishri et al., 2012) were included here (i.e. at P4: 6 pups for each saline and progesterone group at 4 mg/kg; and, at P12: 6 pups for each of saline and progesterone group at 4 mg/kg). All recordings were performed by the same trained experimenter (see Acknowledgment section), each dose of progesterone was tested on only one rat, and while analyzed and published separately, these recordings have been made as one single block of experiments (Hishri et al., 2012). 2.2. Animals A total of 144 newborn Sprague-Dawley rats born to 28 virgin females and fathered by 14 males that were purchased from Charles River (St. Constant, Québec, Canada) were used. All of the animals were supplied with food and water ad libitum and maintained under standard laboratory conditions (21 ◦ C, 12:12 h dark–light cycle: lights on at 07:00 and off at 19:00). On the day of mating, the males and females were placed together overnight. Mating was confirmed the next day by the presence of spermatozoid on vaginal smears, and pregnancy was confirmed by weighing the females weekly. Upon delivery, litters were culled to 12 pups, with a preference for males. The date of birth was considered P0. Recordings on P1 were taken between 20 and 28 h of life. We used both the males and females for studies performed with the P1 and P4 pups, but studies involving the P7 and P12 rats were performed using only males. This choice was made because subtle (but not significant) differences in HVR were observed between male and female P10 rats subjected to chronic neonatal intermittent hypoxia (Julien et al., 2008). To avoid pup- and litter-specific effects, we used the rats from one litter randomly in control or progesterone groups at P1, P4, P7 or P12. Each rat was used only once, receiving either progesterone or saline, and once the recording was achieved, the pup was re-placed with his mother to mitigate any changes in the mother and/or litter behavior. The mother and her pups were euthanized after postnatal day 12. The number of pups per age and experimental group is provided in the corresponding tables and figures. 2.3. Progesterone preparation The progesterone was purchased from Sigma Aldrich Canada (Oakville, ON, Canada), freshly dissolved in saline and conserved at 4 ◦ C. Progesterone solutions were used within 3–4 h. Based on the

dose–response curve from our earlier study (Hishri et al., 2012), progesterone was given as 4 mg/kg with a total injection volume of 2 ␮l/g. The dose was administered intraperitoneally approximately 30 min prior to ventilatory assessment. An equivalent volume of saline was administered to the control group. 2.4. Measurements of respiratory parameters and metabolic rate Respiratory and metabolic variables were assessed in awake and unrestrained pups using whole-body plethysmography (IOX, Emka Technologies, Paris, France), as previously described (Lefter et al., 2007; Julien et al., 2008, 2011; Niane and Bairam, 2011). Oral (P1 and P4 rats) or rectal (P7 and P12 rats) body temperature was measured before and at the end of each experiment using a thermocouple for small rodents (Harvard, Holiston, MA, USA). The gas flow through the chamber was set at 100 ml/min for P1 rats and 150 ml/min for P4, P7 and P12 rats. The flow was continuously measured with a TSI model 4140 mass flowmeter (TSI Inc., Shoreview, MN, USA). The temperature inside the plethysmograph was fixed at thermoneutrality for each age using a temperature control system (Physitemp, Clifton, NJ, USA) and was set at 34 ◦ C (P1), 32 ◦ C (P4), 31 ◦ C (P7), or 30 ◦ C (P12) (Mortola and Naso, 1998). The inflow and outflow of oxygen and carbon dioxide in the plethysmograph were continuously measured throughout the experiment using an oxygen (Model S-3A, AEI Technologies Naperville, IL, USA) and CO2 (Model CD-3A, AEI Technologies Naperville, IL, USA) analyzers. These measures were later used to calculate oxygen consumption and carbon dioxide production. The calibration of the analyzer was performed in accordance with the manufacturer’s specifications using a one- or two-point method with certified gases. Relative humidity was continuously monitored from the outflowing gas stream with a high-precision water-vapor pressure analyzer (RH-300 Sable Systems International, Las Vegas, NV, USA). Flow tracings from the plethysmograph were integrated using the IOX software (Emka Technologies, Paris, France) and used to calculate tidal volumes (BTPS) after calibration, using barometric pressure, plethysmograph and body temperature, and humidity using the Bartlett and Tenney equation (Bartlett and Tenney, 1970). Minute ventilation was then calculated as the product of respiratory frequency and tidal volume. Metabolic rates were calculated using the formulas provided by the manufacturers of the gas flow and analyzing system (Mortola and Frappell, 1998; Lighton, 2008). Oxygen consumption was calculated as follows: O2 consumption = flow ×

[(O2

in

− O2

out ) − O2 out

(1 − O2

× (CO2 out )

out

− CO2

in )]

Carbon dioxide production was calculated as follows: CO2 production = flow ×

[(CO2

out

− CO2

in ) − CO2 out

(1 − CO2

× (O2

in

− O2

out )]

out )

Both values were corrected for standard temperature and pressure for dry (STPD) conditions and were then converted to the ventilatory equivalent for oxygen and carbon dioxide exchange (Mortola and Frappell, 1998). The respiratory quotient (RQ) was calculated as CO2 production/O2 consumption. Tidal volume, minute ventilation, oxygen consumption and carbon dioxide production were expressed as units per 100 g body weight. 2.4.1. Ventilatory recording during normoxia (FiO2 21%, baseline) The rats received first the ip injection of either saline or progesterone and were habituated to the plethysmograph for approximately 10 min, after which the core body temperature was assessed. The rats were then habituated for additional 20 min. Once

A. Bairam et al. / Respiratory Physiology & Neurobiology 185 (2013) 515–525

their respiration was regular baseline recordings (normoxia; 21% O2 ) were initiated for 10 min. 2.5. Hypoxic ventilatory response (HVR) Once the 10-min recording of baseline were taken, the inlet flow was mixed with nitrogen to reach 12% oxygen inside the plethysmograph, and the desired O2 level was obtained within 2–3 min. Body temperature was assessed again at the end of the hypoxic exposure. This standard hypoxic test allows for the assessment of both phases of the HVR: the initial increase in ventilation that depends primarily on peripheral carotid body chemoreceptor activation, while the late phase reflects the balance between excitatory carotid body input and central inhibitory modulation (Powell et al., 1998).

517

occurred when the amplitude of the breath preceding the apnea was at least twice the resting tidal volume (sigh). In some pups in each age and treatment group, the respiratory recordings were combined with the measure of arterial oxygen saturation (SpO2 ) and heart rate (HR) using an oximeter for small rodents (MouseOx, STARR Life Sciences, Oakmont, PA, USA). Apnea associated with a drop of SpO2 equal or more than 5 units of the value before the apnea under both normoxic and hypoxic conditions was considered to be pathologic. This is an “arbitrary” definition based on our recent studies in P4 and P12 to define a threshold of “pathological” apnea in rats (Bairam et al., 2012). Apnea duration was expressed in seconds. The number of pups at each age and treatment group that were recorded with the oximeter is indicated under the histogram of the corresponding figures (Figs. 2C, 3D and E, 6D and E).

2.6. Plasma progesterone levels

2.8. Statistics

Following hypoxic exposure and an approximate 30-min normoxic recovery period, blood samples were collected by cardiac puncture under anesthesia (ketamine/xylasine, 100/15 ␮g/100 g) in 6–8 rats randomly selected from the control and progesterone groups at each age. In the P1 rats, blood was collected after decapitation to obtain a sufficient sample volume for plasma hormone assessment. The samples were kept at room temperature for 5 min to ensure blood coagulation and then centrifuged for 5 min at 13,000 rpm. The plasma was immediately separated and stored at −80 ◦ C for further analysis. Progesterone assays were performed in duplicate from each sample using an enzyme immunoassay kit in accordance with the manufacturer’s instructions (Progesterone EIA kit, ref: 582601, Caymen Chemical, Michigan USA; detection limit 10 pg/ml; 50% sensitivity at 70 pg/ml).

We used a two-way ANOVA to describe the effect of age, treatment, and their interactions (age × treatment). When a treatment effect or an age × treatment interaction was significant (p < 0.05), we used a Fisher test to determine the effect of progesterone into each age group (treatment effect). When an age effect appeared, we used a Tukey test to compare values between ages. When no treatment or treatment × age interaction appeared the Tukey test was conducted without separating the data by treatment. A spearman correlation test followed by linear regression analysis was used to describe correlation between apnea frequency and minute ventilation. p values < 0.05 were considered significant. All analyses were performed using Sigma plot (v11.0, Systat Software Inc., Chicago, IL, USA), and the data are presented as the mean ± SEM.

2.7. Data collection and analysis

3. Results

2.7.1. Respiratory variables Respiratory frequency, tidal volume, and minute ventilation were calculated on a minute-by-minute basis using the IOX software (V1.8.9 Emka Technologies, Paris, France). The variables were averaged over 5 min of data recorded at a stable baseline. HVR was expressed as both peak and steady-state values. The respiratory variables were averaged every 1 min for the first 10 min. The peak response represented the average of the two highest successive values between 3 and 5 min of hypoxic exposure (i.e. the mean of the 3rd and 4th min or the mean of the 4th and 5th min). While, the steady-state HVR represented the average of the last 5 min (15–20 min). Both the peak and steady-state responses were expressed as the percent change from the baseline. Metabolic variables were measured at baseline and at the end of HVR.

3.1. Progesterone plasma levels

2.7.2. Coefficient of variation The coefficient of variation of minute ventilation during the baseline and during the last 5 min of HVR (late phase) was calculated as CV = standard deviation/mean × 100, as used previously in the newborn rats (Julien et al., 2011; Niane and Bairam, 2012). This calculation was performed after exclusion of any movement artifact, sigh and apnea. 2.7.3. Apnea frequency and duration Spontaneous and post-sigh apnea frequencies were analyzed during the 10 min of baseline recording and the last 10 min of HVR, and the results are expressed as the number of apnea/10 min (Lefter et al., 2007; Julien et al., 2008, 2011; Niane and Bairam, 2011, 2012). We used the criteria proposed by Mendelson (Mendelson et al., 1988) to define spontaneous apnea as an interruption of flow for at least two normal respiratory cycles. While, post-sigh apnea

Plasma progesterone levels are presented in Table 1. There was an age × treatment interaction for progesterone level (p < 0.004). Plasma progesterone significantly increased after injection as compared with control rats at each age studied (treatment effect: p < 0.0002). In control rats and following progesterone injection, plasma level of progesterone declined with age reaching in P12 roughly half the values observed in P1 rats (age effect: p < 0.0001) suggesting differences in the pharmacokinetic properties (Table 1).

Table 1 Plasma progesterone concentration (ng/ml) in developing rats. Age

Saline

Progesterone (4 mg/kg, ip)

P1

37.2 ± 5.6 (11.3–50.8) n=6

223.7 ± 50.8† (106.8–506.1) n=7

P4

21.0 ± 6.3 (8.4–36.7) n=7

91.2 ± 17.5† (41.5–164.3) n=6

P7

24.6 ± 7.4 (2.7–75.0) n=8

129.4 ± 9.5† (84.7–165.1) n=7

P12

16.8 ± 3.6* (3.5–29.3) n=7

54.8 ± 6.3* , † (39.6–71.0) n=7

Data are mean ± SEM. * p < 0.01 vs. P1 of the corresponding group, saline or progesterone. † p < 0.001 vs. saline.

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Table 2 Baseline body weight, body temperature, ventilation and metabolism in developing rats. Age

Saline

Progesterone (4 mg/kg, ip) n (rats)

n (rats)

Body weight (g)

P1 P4 P7 P12

6.4 11.1 19.6 26.5

± ± ± ±

0.6 0.6 0.6 0.5

(20) (15) (15) (23)

6.8 11.1 20.2 27.3

± ± ± ±

0.6 0.6 0.6 0.6

(20) (15) (19) (17)

Body temperature (◦ C)

P1 P4 P7 P12

34.2 33.3 33.7 34.5

± ± ± ±

0.2 0.2 0.2 0.2

(20) (15) (15) (23)

33.9 33.7 34.0 35.3

± ± ± ±

0.2 0.2 0.2 0.2

(20) (15) (19) (17)

Breathing frequency (breath/min)

P1 P4 P7 P12

131 166 184 180

± ± ± ±

4 5* 4* , $ 4*

(20) (15) (15) (23)

120 161 180 182

± ± ± ±

4 4* 4* , $ 4* , $

(20) (15) (19) (17)

Tidal volume (ml/100 g)

P1 P4 P7 P12

1.35 1.11 0.89 0.91

± ± ± ±

0.05 0.07* 0.06* 0.04* , $

(20) (15) (15) (23)

1.34 1.16 0.96 0.81

± ± ± ±

0.05 0.05* 0.05* , $ 0.06* , $

(20) (15) (19) (17)

O2 consumption (ml/min/100 g)

P1 P4 P7 P12

4.7 5.0 4.2 4.0

± ± ± ±

0.2 0.2 0.3 0.2 $

(14) (10) (10) (12)

5.1 4.8 4.2 3.8

± ± ± ±

0.2 0.2 0.2* 0.2* , $

(11) (12) (14) (10)

CO2 production (ml/min/100 g)

P1 P4 P7 P12

2.1 3.5 2.8 2.6

± ± ± ±

0.2 0.2* 0.2 0.2$

(14) (10) (10) (12)

2.4 3.0 2.9 2.6

± ± ± ±

0.2 0.2 0.2 0.2

(11) (12) (14) (10)

Respiratory quotient (RQ: CO2 production/O2 consumption)

P1 P4 P7 P12

0.45 0.72 0.64 0.67

± ± ± ±

0.03 0.02* 0.04* 0.02*

(9) (10) (10) (9)

0.48 0.59 0.69 0.68

± ± ± ±

0.05 0.03 0.03* 0.04*

(11) (7) (14) (8)

No difference between progesterone and saline for each of the above variables were studied. Data are mean ± SEM. * p < 0.01 vs. P1 of the corresponding group. $ p < 0.01 vs. P4 of the corresponding group.

3.2. Progesterone effects at baseline Body temperature and body weight were similar between the control and progesterone groups at each age studied (Table 2). Baseline ventilation. Compared to control, progesterone did not affect the baseline ventilatory variables, metabolism or coefficient of variation of minute ventilation (Fig. 1A–D and Table 2). There was no interaction between age and treatment for ventilatory and metabolic variables under baseline conditions. In both groups, breathing frequency increased from P1 to P4 (p < 0.0001), while tidal volume relative to body weight (p < 0.0001) and oxygen consumption (p < 0.01) decreased with age. Minute ventilation was lower in P12 compared to P4 rats (Fig. 1). Baseline apnea. The effects of progesterone on apnea are shown in Figs. 2 and 3. Examples of respiratory traces, SpO2 and heart rate (HR) on a typical recording of apnea with (Fig. 2B) or without (Fig. 2A) desaturation are presented. In control and progesterone rats, the SpO2 and heart rate were higher in P4, P7 and P12 than P1 rats (age effect: p < 0.01) but there was no effect of treatment or interaction between age and treatment for the SpO2 or HR (Fig. 2C and D). In control rats the predominant type of apnea was spontaneous apnea (Fig. 3B) rather than post-sigh apnea (Fig. 3C), as observed previously (Lefter et al., 2007; Julien et al., 2011; Niane and Bairam, 2012). There was an age × treatment interaction for each apnea variables except for apnea duration (the p value for the interaction is noted on the top of each panel in Fig. 3A–F). Compared to the controls, progesterone significantly decreased the total apnea frequency (spontaneous and post-sigh) in P1 rats and had no effect in P4 or P7 rats. However, total apnea frequency was higher in P12 rats after progesterone injection (Fig. 3A and B), but this is attributable to an increase in “non-pathological” apnea (Fig. 3E).

Progesterone decreased the frequency of pathological apnea (spontaneous and post-sigh) (treatment effect: p = 0.007) (Fig. 3D) only in the P1 rats with slight but not significant effects in P4 and P12 (not P7) rats. Progesterone did not affect apnea duration (Fig. 3F). Finally, there was no correlation between total or pathological apnea frequency and baseline minute ventilation (data not shown). 3.3. Progesterone effects during hypoxia Arterial SpO2 during hypoxia. Minute-by-minute changes in arterial SpO2 and HR during the 20-min exposure to hypoxia for each age and group are shown in Fig. 4. SpO2 decreased in response to the decreased partial pressure of inhaled oxygen. Heart rates increased initially but decreased to a stable rate during the steady state of HVR. There was no difference of SpO2 or HR between progesterone and control at any age studied (Fig. 4). In P4 SpO2 decreased substantially less than in other ages, this result has been consistently repeated throughout the study (see Section 4). Peak HVR. There was significant, age (p < 0.0001) and treatment (p = 0.0001) effect for peak HVR expressed as % changes of minute ventilation (Fig. 5A), but no age × treatment interaction. Progesterone enhanced minute ventilation in P4 and P7 rats as compared to the controls. Progesterone had no effect on the peak respiratory frequency of HVR (Table 3); however it increased tidal volume in P7 rats (treatment effect: p < 0.01 – age × treatment: p < 0.005 – Table 3). There was no age and treatment interaction for the other ventilatory variables at peak HVR. Steady state HVR. There was significant, age (p < 0.0001) and treatment (p < 0.0001) effect for steady state HVR expressed as % changes of minute ventilation (Fig. 5B), but no age × treatment interaction. Progesterone enhanced minute ventilation in P1, P4

A. Bairam et al. / Respiratory Physiology & Neurobiology 185 (2013) 515–525

baseline minute ventilation (ml/min/100g)

A

Table 3 Breathing frequency and tidal volume at peak HVR in developing rats.

200

$

160

Age

Saline

Breathing frequency (% from baseline)

P1 P4 P7 P12

122 132 131 123

± ± ± ±

3 4 4 3

124 133 133 130

± ± ± ±

3 4 3 4

Tidal volume (% from baseline)

P1 P4 P7 P12

111 109 117 108

± ± ± ±

4 5 5 4

109 120 135 112

± ± ± ±

4 5 4* , $ , # 4

Saline Progesterone

120

*: p<0.01 vs P1

80

$: p<0.01 vs. P4

40 0 P1

P4

P7

P12

100

B

519

Progesterone (4 mg/kg, ip)

Data are mean ± SEM. * p < 0.01 vs. P1 of the corresponding progesterone group. $ p < 0.01 vs. P12 of the corresponding progesterone group. # p < 0.05 vs. saline of the corresponding saline group.

. .

baseline VE /VO

2

80 60 40 20 0 P1

C 2

.

baseline VE /VCO

.

P4

P7

P12

100

*

80

*

60

There was a significant interaction between age and treatment for the coefficient of variation of minute ventilation during the steady-state phase of HVR (p = 0.005). It decreased with age (p < 0.0001), and progesterone injection at P4 and P7 reduced the coefficient of variation (treatment effect: p = 0.008, Fig. 5E). Progesterone injection did not affect oxygen consumption and carbon dioxide production (Table 4). Finally, there were tendencies to higher V˙ E /V˙ O2 and V˙ E /V˙ CO2 at the end of HVR after progesterone injection but there was no significant treatment effects or age × treatment interactions (Fig. 5C and D). Apnea frequency during steady-state hypoxia. In contrast to normoxic ventilation, during steady-state hypoxia, the predominant type of apnea was post-sigh apneas (Fig. 6A–C) as observed previously (Lefter et al., 2007; Niane and Bairam, 2012). Progesterone

40 Table 4 Ventilatory and metabolism at steady state of the HVR.

20

Age

Saline

Progesterone (4 mg/kg, ip)

0 P1

P4

P7

P12

Baseline coefficient variation of minute ventilation (%)

D

Body temperature ( from baseline, ◦ C)

P1 P4 P7 P12

−0.2 −0.7 −0.4 −0.5

± ± ± ±

0.2 0.2 0.2 0.2

−0.1 −1.0 −0.4 −0.8

± ± ± ±

0.2 0.2* 0.2 0.2*

Breathing frequency (% from baseline)

P1 P4 P7 P12

94 108 117 125

± ± ± ±

3 3* 3* 3* , $

102 114 126 138

± ± ± ±

3 4* 3* , $ 3* , $ , #

Tidal volume (% from baseline)

P1 P4 P7 P12

89 90 79 95

± ± ± ±

3 5 4 4

92 98 105 93

± ± ± ±

4 5 4* , # 4

O2 consumption ( from baseline, ml/min/100 g)

P1 P4 P7 P12

−1.3 −1.7 −1.1 −1.2

± ± ± ±

0.4 0.2 0.4 0.2

−1.7 −1.1 −1.5 −1.1

± ± ± ±

0.4 0.2 0.2 0.3

CO2 production ( from baseline, ml/min/100 g)

P1 P4 P7 P12

−1.3 −1.6 −0.9 −0.9

± ± ± ±

0.3 0.1 0.2 0.2

−1.3 −1.3 −1.5 −1.0

± ± ± ±

0.2 0.2 0.1 0.2

SpO2 (%)

P1 P4 P7 P12

64.9 80.0 72.7 73.5

± ± ± ±

2.7 2.7* 2.5$ 2.7$

63.5 82.9 71.3 71.7

± ± ± ±

2.8 2.4* 2.5$ 3.0$

Heart rate (beat/min)

P1 P4 P7 P12

292 322 349 397

± ± ± ±

12 12 11* 12* , $

280 329 364 414

± ± ± ±

12 11* 11* 13* , $

40 35 30

*

25 20

*

*

15 10 5 0

P1

P4

P7

P12

Fig. 1. The baseline minute ventilation (A), ventilatory to oxygen consumption (B) and carbon dioxide (C) ratio and coefficient of variations of minute ventilation after saline or progesterone-injections in rats on postnatal days 1, 4, 7 and 12. Progesterone produced no changes in minute ventilation or metabolism independent of age. $ p < 0.01 vs. P4: *p < 0.01 vs. P1. Symbols above one bar indicate effect for the corresponding group (tested if significant group effect or age × group interaction appeared); symbols for two bars indicate effect for both group combined (no significant group effect or age × group interaction). The number of pups in each group is shown in Table 2. The data are reported as the mean ± SEM.

and P7 rats (not P12) as compared to the controls. Progesterone injection increased the tidal volume response to hypoxia exposure in P7 rats (treatment effect: p < 0.002 – treatment × age: p < 0.004) (Table 4), and increased the breathing frequency response to hypoxia in the P12 rats (treatment effect: p < 0.01) (Table 4)

Data are mean ± SEM. * p < 0.01 vs. P1 of the corresponding progesterone group. $ p < 0.01 vs. P4 of the corresponding progesterone group. # p < 0.05 vs. saline of the corresponding saline group.

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Fig. 2. Typical baseline recording of respiratory flow, arterial oxygen saturation (SpO2 ) and heart rate (HR) obtained from a P4 rat demonstrating non-pathological apnea (A) and pathological apnea (B). Baseline arterial SpO2 (C) was less in the control P1 rats than in the older rats. Heart rate (D) did not differ after saline or progesterone injection in rats for the P1, P4, P7 and P12 groups. *p < 0.01 vs. P1. Symbols for two bars indicate effect for both group combined (no significant group effect or age × group interaction). The number of pups for which pulse oximetry values are reported is indicated in the histograms. The data are reported as the mean ± SEM.

significantly decreased the frequency of spontaneous and post-sigh apnea (Fig. 6B,C and E) in P1 rats only (age × treatment: p < 0.01). Progesterone had no effect on “pathological” apnea frequency during hypoxia at any age (Fig. 6D). There was age × treatment interaction for total (Fig. 6A) and “non-pathological” (Fig. 6E) apnea frequency (p < 0.01) during the steady state HVR. Finally, there was no correlation between total or “pathological” apnea frequency and minute ventilation at steady-state HVR (data not shown). 4. Discussion This study shows that between postnatal days 1 and 12 in rats, an acute injection of progesterone (4 mg/kg i.p.) had no effect on baseline ventilation, but decreased the frequency of “pathological” apnea in 1-day-old rats. Under hypoxic conditions, progesterone enhanced the peak HVR in P4 and P7 rats and the steady-state HVR in P1, P4, and P7 rats. Progesterone decreased the overall apnea frequency during hypoxia in 1-day-old rats, but this was mostly due to the effect on “non-pathologic”, rather than on “pathologic” apneas”. These results suggest that progesterone exerts age-specific effects on HVR and apnea frequency, and that these effects are independent, and appear at different time-window during development. Methodological considerations. The relative imprecision of tidal volume measurements using whole-body plethysmography in

non-anesthetized newborn rats is still the major shortcoming of this approach. However, whole-body plethysmography is the only non-invasive method for unrestrained small animals (Gallego and Matrot, 2010) and remains valid for identifying respiratory frequency and apneic episodes (Gallego and Matrot, 2010). We have already discussed this limitation in many of our previous studies (Lefter et al., 2007; Julien et al., 2008, 2011; Niane and Bairam, 2011). RQ values are low compared to the expected values, and it is commonly agreed that indirect calorimetry studies in newborn rodents might give varying results between independent studies (Dominguez et al., 2009). The fact that RQ is lower in P1 than to P4 suggests (a) preferred utilization of fat vs. carbohydrate as primary source of metabolites for oxidation and (b) hypoventilation relatively to the amount of CO2 produced by the metabolism. The lower baseline SpO2 in P1 compared to other ages is consistent with hypoventilation. However, previous studies in rat pups did not report similar changes of RQ between P1 and P4 (Liu et al., 2009). It is striking to observe that in P4 rats SpO2 decreased substantially less than in other ages. However, we observed a similar result in another series of experiments in which we recorded SpO2 in P4 (n = 12) and P14 (n = 16) male rats at various FiO2 (21–9% O2 ): P4 rats maintained a higher SpO2 at 12% compared to P14 rats (not shown). So far we can only speculate that hemoglobin oxygen affinity is

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Fig. 3. Baseline total apnea frequency (A), spontaneous (B), post-sigh (C), pathological-apnea (D), non-pathological apnea (E) and apnea duration (F) after saline or progesterone injection in rats on postnatal days 1, 4, 7 and 12. Progesterone decreased total apnea frequency and pathological apnea only in P1 rats. Progesterone injection was associated with the decreased frequency of pathological apnea in P4 and P12 rats. # p < 0.01 vs. saline. Symbols above one bar indicate effect for the corresponding group (tested if significant group effect or age × group interaction appeared). The number of pups for which pulse oximetry values are reported is indicated in the histograms. The data are reported as the mean ± SEM.

higher around P4, probably due to varying 2,3-diphosphoglycerate levels (Petschow et al., 1978), while in P1 this higher affinity would be offset by hypoventilation (basal SpO2 being lower in P1). The other limitation is the use of the arbitrary definition of “pathological” apnea applied under normoxic and hypoxic conditions, and the application of this definition for each of age studied. The criterion of a “pathological” threshold was established during recent studies performed under normoxia in P4 and P12 rats (Bairam et al., 2012). These experiments helped setting a threshold after which apnea is considered “pathologic”: a drop in arterial oxygen saturation by at least 5 units and/or a decrease in heart rate for at least 5% of the values before apnea. Surprisingly there is no clear relationship between the duration of apnea and the degree of desaturation (Bairam et al., 2012). Nevertheless, it is worth noting that no normative standards exist for defining pathological apnea in the human newborn. In both premature and full-term infants, apnea is generally defined as the cessation of breathing for at least 20 s or less if it is accompanied with a heart rate below 100 min–1 or SpO2 below 85% (Darnall et al., 2006; Finer et al., 2006; Mathew, 2011). Finally, the plasma levels of progesterone in the P1 control rats are in a range similar to those found in fetal rats (Ward and Weisz, 1984). The decrease in plasma progesterone levels after birth is consistent with the trend observed in human neonates (Hughes

et al., 1979). However, it should be acknowledged that blood samples for progesterone assays were taken 30 min after the end of the exposure to hypoxia, and may represent, at least in part, a nonspecific stress response, which includes progesterone release from the adrenals in prepubertal rats (Romeo et al., 2005). Progesterone and normoxic ventilation. The lack of effects of progesterone on baseline ventilation or metabolism are consistent with those from another study, in which progesterone was administered for 10 consecutive days to rat pups via the lactating mother (Lefter et al., 2007). Although progesterone did not change the baseline coefficient variation of minute ventilation, it decreased the frequency of apnea (pathological and non-pathological) only in P1 rats under normoxia with no effect on apnea duration at any age studied. The fact that progesterone had no effects on apnea duration corroborates with data obtained in adult aged rats (Yamazaki et al., 2005). Apnea is a common manifestation of an immature respiratory control system in newborn human and rats, and its incidence declines with age in both species (Darnall et al., 2006; Finer et al., 2006; Mathew, 2011; Niane and Bairam, 2011, 2012). Preterm birth deprives the newborn of steroid hormones synthesized by the placenta, including estradiol and progesterone (Trotter et al., 1999b). In human neonates, plasma progesterone levels are very high in the umbilical cord and serum at birth but then decrease abruptly, dropping to values that are 5 times less by the seventh day of life

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Fig. 4. Minute-by-minute changes in arterial oxygen saturation (left panels) and heart rate (right panels) under hypoxic conditions (FiO2 12%, 20 min) after saline or progesterone injection in rats on postnatal days 1, 4, 7 and 12. Oxygen saturation and heart rate during the 20 min of exposure to hypoxia were similar between groups. The data are reported as the mean ± SEM.

(Hughes et al., 1979). Raising steroid hormone levels to the fetal level through exogenous administration (Trotter et al., 1999b) has been proposed as a method of preventing osteoporosis in preterm babies (Trotter et al., 1999a). Progesterone has also been proposed as a treatment option for apnea of prematurity that remains resistant to methylxanthines (Finer et al., 2006), but its adoption for this use has been limited by a lack of conclusive evidence. The present findings indicate that progesterone injection reduced the frequency of apneas associated with desaturation in the very early stages of life. It is worth noting that at birth, full-term newborn rats exhibit significant neurological immaturity that roughly corresponds to that of a human fetus between 24 and 28 weeks of gestation, while the P12 rats’ levels of neurological development correspond to that of a full-term human newborn up to 2 months old (Clancy et al., 2001, 2007). Our results (limited to the first 12 days of life) suggest that acute progesterone administration for apnea in the human newborn might be efficacious during the first days (to a few weeks) after birth.

Progesterone and hypoxic ventilation. In adult cats whose carotid body chemoreceptors and pulmonary mechanoreceptors are denervated, progesterone acts through hypothalamic and brainstem (nucleus tractus solitarius) areas to enhance phrenic nerve activity (Bayliss et al., 1987). In adult cats, progesterone also enhances the carotid body response to hypoxia (Hannhart et al., 1990). In adult rats, progesterone and estradiol combined reduce dopamine synthesis in the carotid bodies, suppressing its inhibitory effect on breathing (Joseph et al., 2002). In line with these observations, acute progesterone administration enhances both the central and peripheral components of HVR in rats 7 days old or younger. Immunohistochemical staining has revealed that the classical form of nuclear progesterone receptors are found in the carotid bodies of late-stage fetal, newborn, and adult male rats (Joseph et al., 2006); in the mid- and hindbrain tissues of newborn rats and mice (Quadros et al., 2008), and in the NTS of adult rats (Haywood et al., 1999; Francis et al., 2002). In addition, the localization and mechanisms of developmental regulation of the more

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Fig. 5. The changes in minute ventilation at peak HVR (A) and steady-state HVR (B); steady-state ventilation rate to oxygen consumption (C) and carbon dioxide production ratios (D); and the coefficient of variation of minute ventilation (E) after saline and progesterone injection in rats on postnatal days 1, 4, 7 and 12. Peak and steady-state minute ventilations are expressed as the percent change from the baseline in the control and progesterone-treated rats. *p < 0.01 vs. P1. # p < 0.05 vs. saline. Symbols above one bar indicate effect for the corresponding group (tested if significant group effect or age × group interaction appeared); symbols for two bars indicate effect for both group combined (no significant group effect or age × group interaction). The number of pups in each group is shown in Table 2. The data are reported as the mean ± SEM.

recently characterized membrane progesterone receptors, which are present in neural tissues (Labombarda et al., 2010), are also unclear. So far the developmental regulation of nuclear and membrane progesterone receptors in the central and peripheral arms of the respiratory control system, remains to be elucidated. It is tempting to speculate that the development-specific regulation of progesterone receptor expression explains why progesterone has no effect on HVR in P12 rats. However, the lack of a progesteronemediated effect on the first phase of HVR in P1 rats may be related to specific developmental aspects of peripheral chemoreceptors, including postnatal resetting (i.e., adaptation to the extra-uterine oxygen level), and the powerful effect of dopamine-mediated respiratory depression at this stage (Hertzberg et al., 1992; Holgert et al., 1993). Progesterone enhances the steady-state HVR in P1 rats, which lends support to the relevance of its centrally mediated effects.

It is puzzling that the respiratory effects of progesterone do not translate into higher arterial oxygen saturation during the hypoxic exposure (Fig. 4), but this is consistent with the lack of clear effect for V˙ E/V˙ O2 and V˙ E/V˙ CO2 . One possible (but speculative) explanation might be that progesterone (a pulmonary vasodilator in adults – (Aller et al., 2002; Smith et al., 2006)) acts on pulmonary vasculature in a way that counterbalances the benefits of the higher ventilation. Progesterone also decreases respiratory variability during steadystate hypoxia in P4 and P7 rats but failed to decrease “pathological” apneas during hypoxia at any age (Fig. 6D). However, the finding that progesterone decreased total apnea frequency in P1 rats is consistent with previous findings in 10-day-old rats that received progesterone from birth via the milk of their mothers (Lefter et al., 2007). In the previous studies by Lefter et al. there was no discrimination between “non-pathological” and “pathological” apneas, and the effects below 10 days of age were not reported. Because the

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Fig. 6. Steady-state total apnea frequency (A), spontaneous (B), post-sigh (C), pathological (D), non-pathological apnea (E) and apnea duration (F) after saline and progesterone injection in rats on postnatal days 1, 4, 7 and 12. In P1 rats, progesterone decreased total apnea frequency and apnea duration but had no effect on pathological apnea at any age. The number of pups is indicated in the histograms. The data are reported as the mean ± SEM.

gene expression of progesterone receptors is enhanced by estrogen (Bayliss et al., 1990, 1991) interactions between estradiol and progesterone might occur in newborn (Lefter et al., 2008). However independent and powerful effects of estradiol on the respiratory pattern have also been reported in newborn rats (Lefter et al., 2008) and adult mice (Gassmann et al., 2010). Accordingly, further experiments describing the acute effects of both estradiol and progesterone are clearly needed. Additionally, further investigation of the age-dependent effects of chronic progesterone administration on ventilation and respiratory stability is necessary. In conclusion, our results demonstrated that acute administration of progesterone (4 mg/kg i.p.) had no effect on baseline ventilation in P1–P12 rats but decreased apnea frequency in 1day-old rats. In hypoxia, progesterone enhanced the peak HVR in P4 and P7 rats and the steady-state HVR in P1, P4, and P7 rats. Progesterone also decreased the apnea frequency in 1-day-old rats under hypoxia. Collectively, the present and past (Lefter et al., 2007) results highlight the relevance to perform further studies in rat pups with different regimen of progesterone administration (acute vs. chronic) under conditions mimicking pathophysiological events unique to the preterm neonates population. Such studies will help designing clinical assay in human newborns. Acknowledgements This study was supported in part by an operating grant MOP119272 to A.B. from the Canadian Institutes of Health Research. We particularly thank Ms Cécile Julien for her meticulous technical assistance. We also thank Mss. Mélanie Pelletier and Sylvie Viger for their assistance with animal care.

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