Respiratory plasticity after perinatal hypercapnia in rats

Respiratory Physiology & Neurobiology 153 (2006) 78–91

Respiratory plasticity after perinatal hypercapnia in rats Ryan W. Bavis a,∗ , Rebecca A. Johnson b , Kari M. Ording a , Jessica P. Otis a , Gordon S. Mitchell b b

a Department of Biology, Bates College, 44 Campus Ave., Carnegie Science Hall, Lewiston, ME 04240, USA Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706, USA

Accepted 7 September 2005

Abstract Environmental conditions during early life may have profound effects on respiratory control development. We hypothesized that perinatal hypercapnia would exert lasting effects on the mammalian hypercapnic ventilatory response, but that these effects would differ between males and females. Rats were exposed to 5% CO2 from 1 to 3 days before birth through postnatal week 2 and ventilation was subsequently measured by whole-body plethysmography. In both male and female rats exposed to perinatal hypercapnia, a rapid, shallow breathing pattern was observed for the first 2 weeks after return to normocapnia, but ventilation was unchanged. Acute hypercapnic ventilatory responses (3% and 5% CO2 ) were reduced 27% immediately following perinatal hypercapnia, but these responses were normal after 2 weeks of recovery in both sexes and remained normal as adults. Collectively, these data suggest that perinatal hypercapnia elicits only transient respiratory plasticity in both male and female rats. This plasticity appears similar to that observed after chronic hypercapnia in adult animals and, therefore, is not unique to development. © 2005 Elsevier B.V. All rights reserved. Keywords: Control of breathing, development; Control of breathing, plasticity; Control of breathing, hypercapnia; Permissive hypercapnia

1. Introduction Early life experiences influence development of many neural systems, including the respiratory control system (Carroll, 2003; Bavis, 2005). For example, humans raised at high altitude acquire an attenuated hypoxic ventilatory response during childhood, and this effect may persist years after return to sea level ∗ Corresponding author. Tel.: +1 207 786 8269; fax: +1 207 786 8334. E-mail address: [email protected] (R.W. Bavis).

1569-9048/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2005.09.002

(Lahiri, 1981; Moore, 2000). Likewise, infants born with cyanotic heart disease exhibit prolonged blunting of hypoxic ventilatory responses (Sørensen and Severinghaus, 1968; Blesa et al., 1977), as do rats and sheep raised in normobaric hypoxia (Okubo and Mortola, 1990; Sladek et al., 1993; Bavis et al., 2004). Other experimental manipulations of perinatal oxygen, such as perinatal intermittent hypoxia (Reeves and Gozal, 2006) or sustained hyperoxia (Ling et al., 1996; Bavis, 2005), confirm that oxygen availability profoundly influences development of ventilatory control.

R.W. Bavis et al. / Respiratory Physiology & Neurobiology 153 (2006) 78–91

Hypercapnia often accompanies hypoxia clinically, and arterial carbon dioxide levels may be permitted to rise in mechanically ventilated infants in an effort to reduce chronic lung disease (i.e., permissive hypercapnia; Thome and Carlo, 2002; Varughese et al., 2002). However, relatively little is known about the effects of carbon dioxide on the developing respiratory control system. In adult humans (Schaefer et al., 1963; Dempsey and Forster, 1982), rats (Lai et al., 1981; Kondo et al., 2000) and ducks (Bebout and Hempleman, 1999), chronic hypercapnia elicits a moderate reduction in the hypercapnic ventilatory response which has been attributed to changes in blood buffering capacity. This plasticity, sometimes referred to as acclimation or respiratory adaptation, persists for only a short period after return to normocapnia (Dempsey and Forster, 1982). Rezzonico and Mortola (1989) observed that young rats also exhibit an attenuated hypercapnic ventilatory response 2 days after exposure to hypercapnia for the first postnatal week, but their study did not address the persistence of the plasticity. Birchard et al. (1984a) exposed male rats to chronic CO2 throughout perinatal development and found no lasting effects on resting ventilation or the hypercapnic ventilatory response measured at least 6 weeks later. Collectively, these data suggest that perinatal hypercapnia elicits only transient respiratory plasticity in mammals. However, more recent studies on developmental hypercapnia in birds indicate that this question should be revisited. Hypercapnia-induced developmental plasticity has been studied in two bird species. Both zebra finches and Japanese quail exhibit attenuated hypercapnic ventilatory responses as adults after mild to moderate hypercapnia during the embryonic and/or nestling periods (Williams and Kilgore, 1992; Bavis and Kilgore, 2001); this effect persists for months after returning to normocapnic conditions. However, only female birds express this plasticity; developmental hypercapnia did not cause similarly long-lasting changes in the hypercapnic ventilatory responses of male finches or quail (Bavis and Kilgore, 2001). Since Birchard et al. (1984a) studied only male rats, it remains possible that developmental hypercapnia will elicit plasticity in female rats. Thus, the purpose of the current study was to examine the effects of perinatal hypercapnia on respiratory control in both male and female rats. Resting ventilation and hypercapnic ventilatory

79

responses were measured immediately at the end of a 2-week, perinatal hypercapnic exposure. These measurements were repeated in young rats to determine the persistence of respiratory plasticity and in adult rats to determine whether sex differences would appear post-puberty.

2. Methods This study is composed of two series of experiments with somewhat different experimental protocols (see below): Series 1 includes measurements on rats at 2 and 4 weeks of age, and Series 2 includes measurements on rats at 5–6 and >13 weeks of age. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the institution where the experiments were completed, the School of Veterinary Medicine of the University of Wisconsin (Series 1) or Bates College (Series 2). 2.1. Experimental animals Pregnant female Sprague–Dawley rats were obtained from a commercial supplier (colony 217 and 236b in Series 1 and 2, respectively; Harlan Sprague–Dawley, Madison, WI). For perinatal hypercapnia exposures, acrylic environmental chambers were flushed with air, O2 and CO2 at sufficient flow rates to maintain 5% CO2 and 21% O2 (balance N2 ) and to maintain humidity and temperature near ambient. Pregnant rats were placed into the environmental chambers 1–3 days before delivery and resulting litters (Series 1, 4 litters; Series 2, 4 litters) were raised in the chambers with their mothers for the first 14 postnatal days. Rats were subsequently housed in room air in standard animal facilities (12:12 light cycle, food and water ad libitum) until studied. Age-matched control rats (Series 1, 4 litters; Series 2, 3 litters) were born and raised in the same room as hypercapnia rats but in room air (Series 1) or in an environmental chamber flushed with room air (Series 2). Barometric pressure averaged 735 and 759 mmHg in Series 1 and 2, respectively, during the developmental exposure periods. During the perinatal exposures, environmental chambers were opened briefly (<10 min) for routine animal care as needed. In Series 1 only, 16 hypercapnia-

80

R.W. Bavis et al. / Respiratory Physiology & Neurobiology 153 (2006) 78–91

reared and 16 control rats were removed from their cages for 35–45 min on postnatal days 5, 7 and 9 for ventilation measurements (data not presented). During this time, rats were exposed to 3–5% CO2 for 20 min and to 0% CO2 for the remainder of the time. 2.2. Series 1: ventilation in 2- and 4-week-old rats Ventilation measurements were made on 15 control rats (7 males, 8 females) and 16 hypercapnia-reared rats (8 males, 8 females) at 2 weeks of age; hypercapniareared rats were studied immediately upon removal from the chronic CO2 environmental chamber. These rats were re-studied at 4 weeks of age, as were an additional group of 8 control rats (5 males, 3 females) and 8 hypercapnia-reared rats (4 males, 4 females) that had not previously been studied. 2.2.1. Ventilation measurements Ventilation was measured in unrestrained rats using whole-body, flow-through plethysmographs (models PLY3211 and PLY3213 for 2- and 4-week-old rats, respectively; Buxco Electronics, Wilmington, NC). Air or hypercapnic gas mixtures were forced through the plethysmograph chamber at a known rate (0.6 and 3.3 l min−1 for 2- and 4-week-old rats, respectively) and exited through pneumotachographs in the chamber wall. Pressure differences between the animal chamber and a reference chamber were monitored and the resulting pressure signal was used to derive respiratory variables (see below); the system was calibrated by injecting known volumes of air with a syringe. Sensors in the plethysmograph continuously monitored chamber temperature and humidity. For 2-week-old rats, chamber temperature was regulated at 31 ◦ C (e.g., Stunden et al., 2001; Wickstr¨om et al., 2002) using a heat lamp (indirect light only); 4-week-old rats were studied at room temperature (23–25 ◦ C). Given the elevated chamber temperature, body temperature was assumed to be constant at 37 ◦ C at 2 weeks of age (Mortola and Lanthier, 1996; Serra et al., 2001). At 4 weeks of age, rectal temperature was measured at the beginning of the experiment and assumed not to change during hypercapnia (Mortola and Lanthier, 1996). Temperature, humidity and pressure signals were recorded to computer to calculate tidal volume (VT ) (Drorbaugh and Fenn, 1955), respiratory frequency (fR ) and ventilation (V˙ E ) using commercially available

software (BioSystem XA 2.5.0, Buxco Electronics); rejection criteria in the software were adjusted to exclude movement artifacts and sighs prior to analysis. For 2-week-old rats, known gas mixtures were delivered to the plethysmograph chamber from premixed gas cylinders. For 4-week-old rats, O2 , CO2 and N2 were mixed using a mass flow controller and valves (AFC2600D; Aalborg, Orangeburg, NY). In the larger chambers used at 4 weeks of age, composition of incurrent gas was regulated dynamically during the step change to hypercapnia to ensure rapid (∼1–2 min) equilibration at the intended CO2 level. To verify gas concentrations, gas was drawn continuously from the chamber, passed through O2 and CO2 gas analyzers (AUT4042, Buxco Electronics) and subsequently returned to the chamber. 2.2.2. Protocol Rats were weighed and placed unrestrained into the plethysmograph chamber; rectal temperature was measured prior to placing rats in the chamber in 4-week-old rats. Rats were initially exposed to 0% CO2 (21% O2 , balance N2 ). The duration of the 0% CO2 exposure was based on the time it took for rats to adjust to the chamber; data were only recorded if the rat was resting quietly. At 2 weeks of age, rats were exposed to 0% CO2 for a minimum of 15 min, with exposures typically lasting 20–45 min. At 4 weeks of age, rats were exposed to 0% CO2 for a minimum of 30 min, with exposures typically lasting 45–60 min. After recording this baseline ventilation, rats were exposed to 3% and 5% CO2 (21% O2 , balance N2 ), without returning to 0% CO2 between hypercapnic exposures; each hypercapnic exposure lasted 10 min. Respiratory variables were averaged over the final minute at each level of inspired CO2 . 2.3. Series 2: ventilation and metabolism in 5–6 and >13-week-old rats Ventilation and metabolism measurements were made on 14 control rats (7 males, 7 females) and 14 hypercapnia-reared rats (7 males, 7 females) at 5–6 weeks of age. Similar measurements were made on a separate group of 19 control rats (11 males, 8 females) and 17 hypercapnia-reared rats (8 males, 9 females) at >13 weeks of age; these rats were siblings to those studied at 5–6 weeks of age.

R.W. Bavis et al. / Respiratory Physiology & Neurobiology 153 (2006) 78–91

2.3.1. Surgical preparation for >13-week-old rats Temperature transponders (E-mitter; Mini-Mitter, Bend, OR) were implanted into the abdominal cavity of all rats studied as adults. At least 1 week prior to ventilation measurements (i.e., when rats were at least 12 weeks of age), anesthesia was induced with isoflurane in a closed box and subsequently maintained via nose cone (∼2.5% isoflurane, balance O2 ); adequacy of anesthesia was assessed by lack of response to toe pinch, and the concentration of isoflurane was adjusted as necessary. Transponders were placed into the abdominal cavity through a ventral incision. During the same surgical session, femoral arterial catheters were placed in most rats (26 out of 36). Catheters were exteriorized through the back of the neck, filled with heparinized (10 units ml−1 ) saline and capped for later use. Body temperature was maintained throughout surgery with a heating pad. Carprofen (5 mg kg−1 , s.c.; Pfizer, New York, NY) was provided after surgery as an analgesic. To protect the catheter prior to use, exteriorized tubing was loosely taped to the back of the rat. 2.3.2. Ventilation and metabolism measurements Ventilation was measured in unrestrained rats by whole-body barometric plethysmography. Rats rested on a platform in a cylindrical plexiglass chamber (inner diameter: 14 cm and 19 cm at 5–6 and >13 weeks, respectively), the bottom of which was filled with water. Warm, humidified air was forced through the chamber at 1.5 and 2.5 l min−1 (STPD) at 5–6 and >13 weeks, respectively; flow rate and gas concentrations were regulated using a gas mixing mass flow controller (MFC-4; Sable Systems, Las Vegas, NV) and valves (Series 840; Sierra Instruments, Monterey, CA). To record ventilation, the plethysmogaph chamber was briefly sealed and pressure fluctuations within the chamber were measured with a differential pressure transducer (DP45; Validyne Engineering, Northridge, CA); the system was calibrated by injecting known volumes of air with a syringe. Respiratory-related pressure fluctuations, body temperature (obtained via telemetry; Series 4000, Mini-Mitter), chamber temperature (T-type thermocouple) and barometric pressure were recorded to computer (Chart 5, ADInstruments, Colorado Springs, CO; Vital View 4.1, Mini-Mitter) and used to calculate VT (Drorbaugh and Fenn, 1955), fR and V˙ E . For VT calculations, chamber air was

81

assumed to be saturated with water vapor; condensation occurred inside the plethysmograph chamber and in the excurrent tubing throughout each experiment. Fractional concentrations of O2 and CO2 in dried air entering and exiting the plethysmograph chamber were measured (S-3A and CD-3A analyzers, respectively; AEI Technologies, Pittsburgh, PA). Oxygen consumption (V˙ O2 ) was calculated using Eq. (1d) from Withers (1977), assuming a respiratory quotient (R) of 0.85; the error associated with this assumption should not exceed ±3% if R = 0.7–1.0. 2.3.3. Protocol Rats were weighed and placed unrestrained into the plethysmograph chamber which was initially flushed with air containing 0% CO2 (21% O2 , balance N2 ). After approximately 1 h, gas concentrations in the excurrent air were recorded and the chamber was sealed briefly to record ventilation; the chamber was sealed approximately 1 min to obtain a minimum of 30 s of data free from movement artifacts and sighs. After resuming gas flow through the chamber, rats were exposed to 5% CO2 (21% O2 , balance N2 ) for 15 min before repeating the measurements. Respiratory variables were averaged over a 30 s interval during the final minute at each level of inspired CO2 . The protocol was modified slightly for adult rats (>13-week-old) with patent arterial catheters. Before being placed into the plethysmograph chamber, rats were lightly anesthetized with isoflurane for just enough time to remove surgical tape and expose the catheter. Once rats recovered from anesthesia, they were placed into the plethysmograph chamber and exposed to air (0% CO2 ) for at least 1 h. Baseline ventilation and metabolism measurements were made as above. After resuming air flow for 5–10 min, a 0.2–0.3 ml blood sample was collected and immediately analyzed for PO2 , PCO2 and pH (i-STAT Portable Clinical Analyzer and CG4+ cartridges; Abbott Laboratories, Abbott Park, IL); unused blood was returned to the animal. Rats were then exposed to 5% CO2 and measurements were repeated. 2.4. Statistical analysis Body mass and baseline (0% CO2 ) body temperature (when measured), ventilation and metabolism measurements were compared between treatment

82

R.W. Bavis et al. / Respiratory Physiology & Neurobiology 153 (2006) 78–91

groups and sexes by two-way ANOVA (treatment, sex); body mass was transformed (logarithm) for 2and 4-week-old rats to meet the assumptions for parametric tests. Hypercapnic ventilatory and metabolic responses were calculated as a percentage of the baseline value and compared between treatment groups and sexes by two-way ANOVA (treatment, sex). Using ratios to represent ventilatory and metabolic responses can be misleading since differences between groups may be caused by different values in the denominator (Packard and Boardman, 1999), but baseline values for V˙ E , V˙ O2 , V˙ E /V˙ O2 did not differ between treatment groups in the present study (see Section 3.2, below) and preliminary analyses of the non-normalized data by two-way repeated measures ANOVA (treatment, CO2 level) produced qualitatively similar results. Sample sizes for arterial blood samples were insufficient for statistical comparison of males and females within treatment groups; preliminary analyses revealed no differences between males and females at either inspired CO2 level when treatment groups were pooled (Student’s t-test, P > 0.05). Therefore, arterial blood gases

and pH were compared by two-way repeated measures ANOVA (treatment, CO2 level) after pooling data for both sexes. In all analyses, differences between groups were confirmed using Student–Newman–Keuls multiple comparison procedures when statistically significant main effects were detected. All statistical tests were run using SigmaStat 3.0 (SPSS, Chicago, IL), and P < 0.05 was considered statistically significant.

3. Results 3.1. Body mass Rats exposed to perinatal hypercapnia (hypercapnia-reared) weighed slightly, but significantly, less than age-matched control rats at the end of the exposure (∼8% less at 2 weeks; P = 0.03) (Table 1), but differences were no longer detected by 4 weeks of age (Table 1) or in older rats (Table 2). Differences in body mass between males and females were evident by 4 weeks of age (Tables 1 and 2).

Table 1 Baseline respiratory characteristics in control rats and in rats exposed to perinatal hypercapnia at 2 and 4 weeks of age Group

n

Age (days)

Mass (g)

Tb (◦ C)

VT (ml 100 g−1 )

fR (breaths min−1 )

V˙ E (ml min−1 100 g−1 )

2 Weeks Control Male Female

7 8

14 ± 0 14 ± 0

33.9 ± 1.9 33.3 ± 1.1

– –

0.98 ± 0.03 1.03 ± 0.05

121 ± 5 125 ± 5

116.3 ± 4.7 126.1 ± 5.0

All

15

14 ± 0

33.6 ± 1.0

–

1.01 ± 0.03

123 ± 4

121.5 ± 3.6

8 8

14 ± 0 14 ± 0

31.4 ± 0.9 30.4 ± 0.7

– –

0.91 ± 0.03 0.92 ± 0.04

128 ± 4 137 ± 8

116.1 ± 4.9 122.4 ± 6.0

All

16

14 ± 0

30.9 ± 0.6*

–

0.92 ± 0.03*

133 ± 4

119.3 ± 3.8

4 Weeks Control Male Female

12 11

29 ± 0 29 ± 0

94 ± 4 82 ± 2†

37.0 ± 0.1 37.2 ± 0.1

0.90 ± 0.03 1.00 ± 0.04†

103 ± 3 102 ± 3

90.7 ± 2.6 99.3 ± 3.7

All

23

29 ± 0

88 ± 3

37.1 ± 0.1

0.94 ± 0.02

102 ± 2

94.8 ± 2.3

Hypercapnia Male 12 Female 12

29 ± 0 29 ± 0

89 ± 3 81 ± 1†

37.2 ± 0.1 37.3 ± 0.1

0.89 ± 0.03 0.93 ± 0.03†

113 ± 2 111 ± 4

100.1 ± 4.3 102.6 ± 4.8

29 ± 0

85 ± 2

37.2 ± 0.1

0.91 ± 0.02

112 ± 2*

101.3 ± 3.2

Hypercapnia Male Female

All * †

24

P < 0.05 vs. control (i.e., main effect for treatment). P < 0.05 vs. male in same treatment group (i.e., main effect for sex).

Group

n

Age (days)

Mass (g)

Tb (◦ C)

VT (ml 100 g−1 )

fR (breaths min−1 )

V˙ E (ml min−1 100 g−1 )

V˙ O2 (ml O2 min−1 )

V˙ E /V˙ O2

5–6 Weeks Control Male Female

7 7

39 ± 1 38 ± 1

145 ± 6 119 ± 5†

37.5 ± 0.1 37.8 ± 0.1†

0.73 ± 0.04 0.78 ± 0.02

120 ± 6 101 ± 7

87.5 ± 6.6 84.7 ± 4.4

4.1 ± 0.4 3.7 ± 0.2

21.5 ± 1.2 23.6 ± 1.5

All

14

38 ± 1

132 ± 5

37.6 ± 0.1

0.75 ± 0.02

114 ± 4

86.1 ± 3.8

3.9 ± 0.2

22.6 ± 1.0

7 7

38 ± 1 39 ± 1

144 ± 10 123 ± 5†

37.3 ± 0.1 37.6 ± 0.1†

0.76 ± 0.05 0.74 ± 0.05

121 ± 6 124 ± 11

91.3 ± 5.8 88.6 ± 3.1

4.1 ± 0.4 4.4 ± 0.4

22.9 ± 2.0 21.2 ± 2.0

All

14

39 ± 1

133 ± 6

37.4 ± 0.1

0.75 ± 0.03

123 ± 6

89.9 ± 3.2

4.2 ± 0.3

22.1 ± 1.4

>13 Weeks Control Male Female

11 8

117 ± 7 106 ± 4

406 ± 14 251 ± 4†

37.7 ± 0.1 37.6 ± 0.3

0.53 ± 0.02 0.78 ± 0.07†

86 ± 5 69 ± 5†

45.3 ± 1.8 51.7 ± 2.5†

2.4 ± 0.1 3.0 ± 0.2†

18.8 ± 0.9 17.9 ± 1.8

All

19

112 ± 4

341 ± 20

37.7 ± 0.1

0.64 ± 0.04

79 ± 4

48.0 ± 1.6

2.7 ± 0.1

18.4 ± 0.9

8 9

108 ± 3 106 ± 3

388 ± 9 243 ± 6†

37.7 ± 0.1 37.5 ± 0.2

0.58 ± 0.03 0.73 ± 0.04†

84 ± 7 74 ± 7†

47.9 ± 2.2 52.8 ± 3.5†

2.6 ± 0.1 3.3 ± 0.2†

18.8 ± 0.9 16.3 ± 0.9

17

107 ± 2

311 ± 19

37.6 ± 0.1

0.66 ± 0.03

79 ± 5

50.5 ± 2.1

2.9 ± 0.1

17.5 ± 0.8

Hypercapnia Male Female

Hypercapnia Male Female All †

R.W. Bavis et al. / Respiratory Physiology & Neurobiology 153 (2006) 78–91

Table 2 Baseline respiratory characteristics in control rats and in rats exposed to perinatal hypercapnia at 5–6 and >13 weeks of age

P < 0.05 vs. male in same treatment group (i.e., main effect for sex).

83

84

R.W. Bavis et al. / Respiratory Physiology & Neurobiology 153 (2006) 78–91

3.2. Resting (normocapnic) ventilation and metabolism Resting ventilation (V˙ E ) was not affected by perinatal hypercapnia at any age (Tables 1 and 2), however these rats tended to have a more rapid, shallow breathing pattern for at least 2 weeks after the hypercapnic exposure. At 2 weeks of age, hypercapnia-reared rats exhibited significantly lower VT (P = 0.04) and tended to have higher fR (P = 0.10) relative to age-matched controls (Table 1). A similar pattern was also observed at 4 weeks of age, at which time hypercapnia-reared rats exhibited ∼10 breaths min−1 higher fR (P < 0.01) (Table 1). VT was no longer significantly lower at 4 weeks (Table 1), although female rats generally had higher VT relative to their body size at 4 weeks in both treatment groups (P = 0.04). Breathing pattern appeared normal by 5 weeks of age (Table 2). Although no differences were detected between sexes in any ventilatory variable measured at 5–6 weeks of age (main effects and interactions, all P > 0.05), females exhibited larger VT (P < 0.001) and V˙ E (P = 0.033) than males at >13 weeks reflecting a somewhat higher V˙ O2 (P < 0.001). Oxygen consumption, measured at 5–6 weeks and >13 weeks of age, and oxygen convection requirement (V˙ E /V˙ O2 ) did not differ between treatment groups (Table 2).

Table 3 Arterial blood gases in control and perinatal hypercapnia-reared rats while breathing 0 and 5% CO2 0% CO2

5% CO2

Pa O2 (mmHg) Control Perinatal hypercapnia

85 ± 4 86 ± 1

113 ± 4* 119 ± 4*

Pa CO2 (mmHg) Control Perinatal hypercapnia

38.9 ± 1.0 39.8 ± 1.7

46.9 ± 1.0* 47.2 ± 0.6*

pHa Control Perinatal hypercapnia

7.46 ± 0.01 7.45 ± 0.01

7.41 ± 0.01* 7.41 ± 0.00*

Sample sizes are 7 control (4 males, 3 females), 6 perinatal hypercapnia (3 males, 3 females). * P < 0.05 vs. baseline (0% CO ) within the same treatment group. 2

To determine whether changes in metabolism obscured ventilatory effects of perinatal hypercapnia, or whether effects are revealed only after sexual maturity, hypercapnic ventilatory responses were measured in older rats as well. V˙ E , V˙ O2 and V˙ E /V˙ O2 responses to 5% CO2 were similar to those of control rats at 5–6 weeks of age (Fig. 3) and at >13 weeks of age (Fig. 4). Hypercapnic ventilatory responses did not differ between sexes (main effects and interactions, all P > 0.05).

3.3. Hypercapnic ventilatory response

3.4. Blood gases

At 2 weeks of age, ventilatory responses to 3% CO2 and 5% CO2 were 27% smaller in hypercapnia-reared rats when compared to age-matched controls (Fig. 1). The smaller increase in V˙ E to 3% CO2 (P = 0.05) resulted from a reduced fR response (P = 0.03), whereas the smaller increase in V˙ E to 5% CO2 (P = 0.01) resulted from non-significant reductions in both VT and fR responses compared to controls (Fig. 1). Hypercapnic ventilatory responses did not differ between sexes (main effects and interactions, all P > 0.05). Hypercapnic ventilatory responses no longer differed between hypercapnia-reared and control rats by 4 weeks of age (i.e., 2 weeks after return to normocapnia) (Fig. 2). Aside from a smaller ventilatory response to 3% CO2 in female rats versus males (24% lower, P = 0.03), independent of treatment group, these results did not differ between sexes (main effects and interactions, all P > 0.05).

Arterial blood gases and pH were similar between adult hypercapnia-reared and control rats while breathing both 0% and 5% CO2 (all P > 0.05; Table 3). Arterial PO2 increased during hypercapnia, consistent with hyperventilation, and this increase was similar between treatment groups (P > 0.05). We were unable to obtain blood samples for all rats with indwelling catheters, so there were insufficient sample sizes to statistically compare males and females within each treatment group. However, there were no obvious differences between sexes (data not shown).

4. Discussion Perinatal hypercapnia elicits plasticity in resting breathing and the acute hypercapnic ventilatory response of rats, but these effects are relatively

R.W. Bavis et al. / Respiratory Physiology & Neurobiology 153 (2006) 78–91

85

Fig. 1. Ventilatory responses to 3% CO2 (left panels) and 5% CO2 (right panels) at 2 weeks of age. Changes in ventilation (means ± S.E.M.) are presented for control rats (filled bars) and for rats exposed to perinatal hypercapnia (open bars). To facilitate comparisons between sexes and between treatment groups, responses are presented separately for males and females as well as combined (all). Sample sizes are given in Table 1. * P < 0.05 vs. the control group (i.e., main effect for treatment) by two-way ANOVA; no significant differences were detected between sexes.

86

R.W. Bavis et al. / Respiratory Physiology & Neurobiology 153 (2006) 78–91

Fig. 2. Ventilatory responses to 3% CO2 (left panels) and 5% CO2 (right panels) at 4 weeks of age. Changes in ventilation (means ± S.E.M.) are presented for control rats (filled bars) and for rats exposed to perinatal hypercapnia (open bars). Sample sizes are given in Table 1. † P < 0.05 vs. the male rats (i.e., main effect for sex) by two-way ANOVA; no significant differences were detected between control and hypercapnia treatment groups.

R.W. Bavis et al. / Respiratory Physiology & Neurobiology 153 (2006) 78–91

87

Fig. 3. Ventilatory responses to 5% CO2 at 5–6 weeks of age. Changes in ventilation (means ± S.E.M.) are presented for control rats (filled bars) and for rats exposed to perinatal hypercapnia (open bars). Sample sizes are given in Table 2. No significant differences were detected between control and hypercapnia treatment groups or between sexes by two-way ANOVA.

short-lived (<2 weeks) and do not differ between males and females. Therefore, our data generally support the conclusion of Birchard et al. (1984a) that perinatal hypercapnia has no permanent effect on CO2 sensitivity in rats. 4.1. Changes in normocapnic ventilation Changes in normocapnic ventilation have previously been reported after developmental hypercapnia. Rezzonico and Mortola (1989) found that rats exposed to 7% CO2 for postnatal days 1–7 hyperventilated 2 days later due to small increases in both tidal volume and respiratory frequency. This differs from the present study, however, since rats exposed to 5% CO2 through postnatal week 2 tended to have lower tidal volumes and higher respiratory frequencies without a change in overall ventilation, and presumably no

increase in alveolar ventilation, under normocapnic conditions. Similarly, we recently observed that rats exposed to combined hyperoxia and hypercapnia (60% O2 , 5% CO2 ) for the first 2 postnatal weeks also exhibit increased respiratory frequency, and tend to have lower tidal volumes, while breathing room air immediately after the exposure (Bavis, R.W., Otis, J.P., unpublished observations). It is unclear why our results differ from the earlier study by Rezzonico and Mortola (1989), but this discrepancy could reflect the level of CO2 or duration of the CO2 exposure, the duration of recovery before ventilation was measured or genetic differences in the populations of Sprague–Dawley rats used in these experiments (e.g., Fuller et al., 2001). Changes in normocapnic ventilation after perinatal hypercapnia have also been reported in adult humans after chronic hypercapnia. Schaefer et al. (1963) observed that humans exposed to 1.5% CO2 for

88

R.W. Bavis et al. / Respiratory Physiology & Neurobiology 153 (2006) 78–91

Fig. 4. Ventilatory responses to 5% CO2 at >13 weeks of age. Changes in ventilation (means ± S.E.M.) are presented for control rats (filled bars) and for rats exposed to perinatal hypercapnia (open bars). Sample sizes are given in Table 2. No significant differences were detected between control and hypercapnia treatment groups or between sexes by two-way ANOVA.

42 days had increased tidal volumes and reduced respiratory frequencies for the first 1–2 weeks after returning to room air. As a result, pulmonary and alveolar ventilation initially returned to normal, but a secondary increase in tidal volume and gradual recovery in respiratory frequency resulted in hyperventilation by day 9 after return to normocapnia; ventilation and respiratory pattern had normalized when restudied after 4 weeks of recovery. Thus, normocapnic ventilation may be normal or slightly elevated for up to 2 weeks after chronic hypercapnia in adult humans (Dempsey and Forster, 1982). Less is known about the time course of changes in normocapnic ventilation after chronic hypercapnia in adult rats. After a 3-week exposure to 5% CO2 , ventilation returned to normal within 30 min in adult rats, although respiratory frequency was significantly reduced (Lai et al., 1981); measurements were not made at subsequent time points. Taken together,

these data suggest that chronic hypercapnia leads to a short-term decrease in respiratory frequency in adult mammals, opposite to what occurs after developmental hypercapnia; changes in tidal volume and overall ventilation are somewhat more variable and may change over time. Thus, in both adults and developing mammals, chronic hypercapnia causes transient (∼2 weeks) changes in normocapnic ventilation, although the qualitative expression of this plasticity may differ among developmental stages or genetic backgrounds. The mechanism and functional significance of altered normocapnic ventilation after perinatal hypercapnia are not obvious. Rezzonico and Mortola (1989) suggested that greater normoxic ventilation after developmental hypercapnia could be a response to increased respiratory dead space. Although they did not measure respiratory dead space in their study, chronic hypercapnia does increase dead space in adult rats, possibly by

R.W. Bavis et al. / Respiratory Physiology & Neurobiology 153 (2006) 78–91

inducing bronchodilation (Lai et al., 1981). However, the altered breathing pattern in the present study was not associated with an increase in overall ventilation, and rapid, shallow breathing would not be an effective compensation for increased dead space. Humans may exhibit elevated arterial PCO2 more than a week after chronic hypercapnic exposures (Schaefer et al., 1963), but the lack of change in overall ventilation in the present study makes persistent hypercapnia or acidosis an unlikely explanation for altered breathing patterns in young rats. Alternatively, changes in breathing pattern may reflect developmental plasticity in respiratory mechanics. Rats exposed to chronic neonatal hypercapnia develop higher compliance and lower resistance of the respiratory system (Rezzonico et al., 1990). While respiratory mechanics can influence breathing pattern, changes in respiratory mechanics persist at least 6 weeks after return to normocapnia (Rezzonico et al., 1990), whereas changes in breathing pattern normalized within 3 weeks in the present study. 4.2. Changes in acute hypercapnic ventilatory response Immediately after perinatal hypercapnia, ventilatory responses to acute changes in the inspired CO2 were attenuated in 2-week-old rats, but the ventilatory response returned to normal within 2 weeks. These data are generally consistent with other studies on the effects of developmental hypercapnia in rats. Rezzonico and Mortola (1989) reported that the acute ventilatory response to 10% CO2 was reduced 2 days after being exposed to a week of neonatal hypercapnia, although their data must be interpreted cautiously since their rats had significantly higher baseline ventilation (i.e., although the change in ventilation was smaller, absolute ventilation at 10% CO2 was similar to the untreated control group). In a recent review on the development of CO2 sensitivity in mammals, Putnam et al. (2005) refer to unpublished data from their laboratory on 1–3-week-old rats (P7–20) previously exposed to 7.5% CO2 for the first postnatal week. These authors report that the acute hypercapnic ventilatory response was abolished after this hypercapnic exposure, but they did not study rats >P20; no details are provided on normocapnic ventilation. Finally, Birchard et al. (1984a) observed that hypercapnic ventilatory responses were normal in rats after 6 weeks of recovery from perina-

89

tal hypercapnia. Collectively, these studies suggest that perinatal hypercapnia causes an immediate blunting of the acute hypercapnic ventilatory response and that this blunting persists for at most 2 weeks after return to normocapnia. Transient blunting of the hypercapnic ventilatory response after perinatal hypercapnia is similar to the effects of chronic hypercapnia in adult animals (Schaefer et al., 1963; Lai et al., 1981; Dempsey and Forster, 1982; Bebout and Hempleman, 1999; Kondo et al., 2000), and it is likely that similar mechanisms underlie this plasticity. Blunting in adult animals is usually attributed to changes in H+ buffering capacity via increased bicarbonate concentrations (i.e., partial metabolic compensation) (Dempsey and Forster, 1982; Bebout and Hempleman, 1999). Thus, changes in PCO2 cause smaller changes in pH and, therefore, are less effective at stimulating chemoreceptors and eliciting the ventilatory response. Neonatal rats already have a well developed ability to compensate for metabolic acidosis by altering plasma and cerebrospinal fluid bicarbonate concentrations (Johanson et al., 1976), so changes in buffering capacity likely contribute to blunted hypercapnic ventilatory responses during and immediately following perinatal hypercapnia. In both bird species studied to date, zebra finch and Japanese quail, chronic exposure to 2–5% CO2 throughout embryonic and/or nestling (postnatal) development attenuates the hypercapnic ventilatory response of adult females (Williams and Kilgore, 1992; Bavis and Kilgore, 2001). Although Birchard et al. (1984a) found no persistent effects of perinatal hypercapnia on respiratory control in rats, this group only studied male rats. In the present study, hypercapnic ventilatory responses were normal in both male and female rats within 2 weeks following perinatal hypercapnia. Metabolic rates and arterial blood gases and pH (adults only) were similar to untreated control rats while breathing air or 5% CO2 , confirming that ventilatory responses were measured under comparable conditions. Moreover, similar results were obtained for both young and adult rats, indicating that latent, sexspecific effects of perinatal hypercapnia do not emerge with sexual maturity. Therefore, these data demonstrate that perinatal hypercapnia does not have permanent effects on the hypercapnic ventilatory response in rats. Thus, in contrast to birds, perinatal hypercapnia does not elicit developmental plasticity in the hypercapnic

90

R.W. Bavis et al. / Respiratory Physiology & Neurobiology 153 (2006) 78–91

ventilatory response in rats, where developmental plasticity is defined as a form of (long-lasting) plasticity unique to development (cf. Carroll, 2003). 4.3. Implications for human infants and burrowing mammals The influence of perinatal hypercapnia on respiratory control development is relevant to human infants that are mechanically ventilated or experience chronic hypercapnia as the result of cardiorespiratory disease. Trauma caused by mechanical ventilation contributes to chronic lung disease. “Permissive hypercapnia” is a strategy in which ventilation is reduced to minimize lung damage at the cost of rising arterial PCO2 (e.g., 45–55 mmHg) (Thome and Carlo, 2002; Varughese et al., 2002). Although these mild to moderate levels of hypercapnia are considered safe or even beneficial (Thome and Carlo, 2002; Varughese et al., 2002), effects of permissive hypercapnia on cardiorespiratory control have not been studied to date. In the present study, rats were exposed to 5% CO2 which, based on arterial blood gases in adult rats (Table 3), is likely to produce an arterial PCO2 similar to those observed clinically in permissive hypercapnia or cardiorespiratory disease. Although perinatal hypercapnia may have no permanent consequences for respiratory control in mammals, hypercapnic ventilatory responses are blunted during hypercapnic exposures and remain blunted for a period of days to weeks (Birchard et al., 1984a; Rezzonico and Mortola, 1989; Putnam et al., 2005; present study). Even transient changes in CO2 sensitivity may be clinically important, such as in some cases of sudden infant death syndrome (Hunt and Brouillette, 1987; Richerson, 2004). Indeed, the time course of changes in respiratory control during and following the hypercapnic exposure may determine a period of heightened vulnerability. Another group of animals naturally exposed to hypercapnia during development are burrowing animals (Birchard et al., 1984b; Tenney and Boggs, 1986). Burrowing mammals and birds consistently exhibit low hypercapnic ventilatory responses relative to nonburrowing species (Boggs et al., 1984). It has long been questioned whether the ventilatory responses of burrowing species are inherited or acquired during development in hypercarbic burrows. Since hypercap-

nic ventilatory responses are qualitatively similar in burrowing birds and in non-burrowing birds raised in high CO2 , a role for developmental plasticity remains possible for burrowing birds. In contrast, the results of the present study suggest a predominant role for genetic factors in the blunted hypercapnic ventilatory responses of burrowing mammals. Additional studies with burrowing birds and mammals are required to test these hypotheses directly.

Acknowledgments This research was supported in part by National Institutes of Health grants HL65383 and HL53319 to G.S.M., HL03874 to R.A.J. and HL70506 to R.W.B., and by a Bates Student Research Grant to K.M.O. We thank Brad A. Hodgeman for technical assistance.

References Bavis, R.W., 2005. Developmental plasticity of the hypoxic ventilatory response after perinatal hyperoxia and hypoxia. Respir. Physiol. Neurobiol. 149, 287–299. Bavis, R.W., Kilgore Jr., D.L., 2001. Effects of embryonic CO2 exposure on the adult ventilatory response in quail: does gender matter? Respir. Physiol. 126, 183–199. Bavis, R.W., Olson Jr., E.B., Vidruk, E.H., Fuller, D.D., Mitchell, G.S., 2004. Developmental plasticity of the hypoxic ventilatory response in rats induced by neonatal hypoxia. J. Physiol. 557, 645–660. Bebout, D.E., Hempleman, S.C., 1999. Chronic hypercapnia resets CO2 sensitivity of avian intrapulmonary chemoreceptors. Am. J. Physiol. 276, R317–R322. Birchard, G.F., Boggs, D.F., Tenney, S.M., 1984a. Effect of perinatal hypercapnia on the adult ventilatory response to carbon dioxide. Respir. Physiol. 57, 341–347. Birchard, G.F., Kilgore Jr., D.L., Boggs, D.F., 1984b. Respiratory gas concentrations and temperatures within the burrows of three species of burrow-nesting birds. Wilson Bull. 96, 451–456. Blesa, M.I., Lahiri, S., Rashkind, W.J., Fishman, A.P., 1977. Normalization of the blunted ventilatory response to acute hypoxia in congenital cyanotic heart disease. N. Engl. J. Med. 296, 237–241. Boggs, D.F., Kilgore Jr., D.L., Birchard, G.F., 1984. Respiratory physiology of burrowing mammals and birds. Comp. Biochem. Physiol. 77A, 1–7. Carroll, J.L., 2003. Developmental plasticity in respiratory control. J. Appl. Physiol. 94, 375–389. Dempsey, J.A., Forster, H.V., 1982. Mediation of ventilatory adaptations. Physiol. Rev. 62, 262–346. Drorbaugh, J.E., Fenn, W.O., 1955. A barometric method for measuring ventilation in newborn infants. Pediatrics 16, 81–87.

R.W. Bavis et al. / Respiratory Physiology & Neurobiology 153 (2006) 78–91 Fuller, D.D., Baker, T.L., Behan, M., Mitchell, G.S., 2001. Expression of hypoglossal long-term facilitation differs between substrains of Sprague–Dawley rat. Physiol. Genomics 4, 175–181. Hunt, C.E., Brouillette, R.T., 1987. Sudden infant death syndrome: 1987 perspective. J. Pediatr. 110, 669–678. Johanson, C.E., Woodbury, D.M., Withrow, C.D., 1976. Distribution of bicarbonate between blood and cerebrospinal fluid in the neonatal rat in metabolic acidosis and alkalosis. Life Sci. 19, 691–700. Kondo, T., Kumagai, M., Ohta, Y., Bishop, B., 2000. Ventilatory responses to hypercapnia and hypoxia following chronic hypercapnia in the rat. Respir. Physiol. 122, 35–43. Lahiri, S., 1981. Adaptive respiratory regulation-lessons from high altitudes. In: Hovarth, S.M., Yousef, M.K. (Eds.), Environmental Physiology: Aging, Heat and Altitude. Elsevier North Holland, New York, pp. 341–350. Lai, Y.-L., Lamm, W.J.E., Hildebrandt, J., 1981. Ventilation during prolonged hypercapnia in the rat. J. Appl. Physiol. 51, 78–83. Ling, L., Olson Jr., E.B., Vidruk, E.H., Mitchell, G.S., 1996. Attenuation of the hypoxic ventilatory response in adult rats following 1 month of perinatal hyperoxia. J. Physiol. 495, 561–571. Moore, L.G., 2000. Comparative human ventilatory adaptation to high altitude. Respir. Physiol. 121, 257–276. Mortola, J.P., Lanthier, C., 1996. The ventilatory and metabolic response to hypercapnia in newborn mammalian species. Respir. Physiol. 103, 263–270. Okubo, S., Mortola, J.P., 1990. Control of ventilation in adult rats hypoxic in the neonatal period. Am. J. Physiol. 259, R836–R841. Packard, G.C., Boardman, T.J., 1999. The use of percentages and size-specific indices to normalize physiological data for variation in body size: wasted time, wasted effort? Comp. Biochem. Physiol. A 122, 37–44. Putnam, R.W., Conrad, S.C., Gdovin, M.J., Erlichman, J.S., Leiter, J.C., 2005. Neonatal maturation of the hypercapnic ventilatory response and central neural CO2 chemosensitivity. Respir. Physiol. Neurobiol. 149, 165–179. Reeves, S.R., Gozal, D., 2006. Changes in ventilatory adaptations associated with long-term intermittent hypoxia across the age spectrum in the rat. Respir. Physiol. Neurobiol. 150, 135–143. Rezzonico, R., Mortola, J.P., 1989. Respiratory adaptation to chronic hypercapnia in newborn rats. J. Appl. Physiol. 67, 311–315.

91

Rezzonico, R., Gleed, R.D., Mortola, J.P., 1990. Respiratory mechanics in adult rats hypercapnic in the neonatal period. J. Appl. Physiol. 68, 2274–2279. Richerson, G.B., 2004. Serotonergic neurons as carbon dioxide sensors that maintain pH homeostasis. Nat. Rev. Neurosci. 5, 449–461. Schaefer, K.E., Hastings, B.J., Carey, C.R., Nichols Jr., G., 1963. Respiratory acclimatization to carbon dioxide. J. Appl. Physiol. 18, 1071–1078. Serra, A., Brozoski, D., Hedin, N., Franciosi, R., Forster, H.V., 2001. Mortality after carotid body denervation in rats. J. Appl. Physiol. 91, 1298–1306. Sladek, M., Parker, R.A., Grogaard, J.B., Sundell, H.W., 1993. Longlasting effect of prolonged hypoxemia after birth on the immediate ventilatory response to changes in arterial partial pressure of oxygen in young lambs. Pediatr. Res. 34, 821–828. Sørensen, S.C., Severinghaus, J.W., 1968. Respiratory insensitivity to acute hypoxia persisting after correction of tetralogy of Fallot. J. Appl. Physiol. 25, 221–223. Stunden, C.E., Filosa, J.A., Garcia, A.J., Dean, J.B., Putnam, R.W., 2001. Development of in vivo ventilatory and single chemosensitive neuron responses to hypercapnia in rats. Respir. Physiol. 127, 135–155. Tenney, S.M., Boggs, D.F., 1986. Comparative mammalian respiratory control. In: Cherniack, N.S., Widdicombe, J.G. (Eds.), Handbook of Physiology, vol. II. Section 3: The Respiratory System, Part 2. Control of Breathing. American Physiological Society, Bethesda, MD, pp. 833–855. Thome, U.H., Carlo, W.A., 2002. Permissive hypercapnia. Semin. Neonatol. 7, 409–419. Varughese, M., Patole, S., Shama, A., Whitehall, J., 2002. Permissive hypercapnia in neonates: the case of the good, the bad, and the ugly. Pediatr. Pulmonol. 33, 56–64. Wickstr¨om, R., H¨okfelt, T., Lagercrantz, H., 2002. Development of CO2 -response in the early newborn period in rat. Respir. Physiol. Neurobiol. 132, 145–158. Williams Jr., B.R., Kilgore Jr., D.L., 1992. Ontogenetic modification of the hypercapnic ventilatory response in the zebra finch. Respir. Physiol. 90, 125–134. Withers, P.C., 1977. Measurements of V˙ O2 , V˙ CO2 , and water evaporative loss with a flow through mask. J. Appl. Physiol. 42, 120– 123.