- Email: [email protected]
Postnatal ventilatory response to CO2 in awake piglets
Respiratory Physiology & Neurobiology 175 (2011) 49–54
Contents lists available at ScienceDirect
Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Postnatal ventilatory response to CO2 in awake piglets M.R. Dwinell ∗ , G.E. Hogan, E. Sirlin, D.L. Mayhew, H.V. Forster Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, United States
a r t i c l e
i n f o
Article history: Accepted 7 September 2010 Keywords: Hypercapnic ventilatory response Postnatal development Control of breathing Piglets
a b s t r a c t Abnormal ventilatory responses to increased levels of inspired CO2 during postnatal development may pose a risk for Sudden Infant Death Syndrome, primarily during periods of vulnerability. The purpose of this study was to test the hypothesis that in awake piglets the ventilatory response to hypercapnia would be attenuated between 10 and 15 days of age relative to younger and older ages. To test this hypothesis, we measured the ventilatory response to 5% inspired CO2 in piglets from postnatal (PN) days 1 through PN28. Piglets were divided into groups and exposed to 5% CO2 daily, every 3rd day or on and after PN20–21 only to avoid any plasticity that may result from repeated exposure to CO2 . Room air ventilation normalized to body weight (V˙ E , ml/min/kg) declined with postnatal age in piglets from all groups. The ventilatory response to 5% inspired CO2 (expressed as % change from control) was present at birth, and we did not find an age-dependent change from PN1 to PN28 (p > 0.1). In addition, we did not find that repeated exposure (daily or every 3rd day) to 5% inspired CO2 altered the ventilatory response during this period of development. We conclude that the previously documented apparent critical period of development in piglets between 10 and 15 days of age is not associated with attenuation of the ventilatory response to 5% inspired CO2 . © 2010 Elsevier B.V. All rights reserved.
1. Introduction The ventilatory responses to hypoxia and hypercapnia vary among different newborn mammals depending on the level of maturity at birth. Peripheral oxygen sensitivity is weak at birth and gradually increases to adult levels during the 1st weeks to months of life in many species (Bairam and Carroll, 2005). On the other hand, the ventilatory response to elevated levels of inspired CO2 had been thought to be relatively mature at birth and change little during development. However, recent studies have demonstrated that the level of maturity at birth may affect the CO2 response. For example, relatively immature species at birth (i.e. rats) appear to have an initial response to CO2 at birth that then declines at postnatal (PN) days 6–7 (Serra et al., 2001; Stunden et al., 2001; Wickstrom et al., 2002) or remains flat (Davis et al., 2006) until about PN14–15, when it begins to increase to reach adult levels by about PN21. Sudden Infant Death Syndrome (SIDS) is the leading cause of infant mortality between 1 month and 1 year of life. Although the exact pathophysiology of SIDS remains unknown, a triple-risk model has been proposed for the pathogenesis of SIDS (Filiano and Kinney, 1992, 1994; Filiano, 1994; Kinney et al., 1992). The three factors that must be present to result in death are (1) an underlying vulnerability, (2) a critical period of development, and (3) an exoge-
nous stressor. According to this model, SIDS may be caused in part by the inability of the respiratory control system to respond to an exogenous stressor during this critical window. Past studies have utilized peripheral chemoreceptor denervation to create an underlying vulnerability. In 1 week old rats, mortality following carotid body denervation (CBD) was increased suggesting a critical window of development in the respiratory control system which corresponds to the period of minimal sensitivity to CO2 in rats (Serra et al., 2001). In piglets, a more mature species at birth, CBD alone did not lead to significant mortality, but when combined with aortic denervation, mortality was increased between PN10 and PN15 (Serra et al., 2002). Therefore, the objective of this study was to test the hypothesis that CO2 sensitivity would be reduced (relative to younger and older ages) during postnatal days 10–15 in piglets. The piglet model was chosen as an alternate to the rodent models to extend our understanding of the maturation of the hypercapnic ventilatory response in a species whose maturation is more comparable to human than rodents. These findings will thereby provide insight into the importance of CO2 sensitivity to sustain breathing in vulnerable infants. 2. Methods 2.1. Experimental groups
∗ Corresponding author. Tel.: +1 414 456 4498; fax: +1 414 456 6546. E-mail address: [email protected] (M.R. Dwinell). 1569-9048/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2010.09.005
A total of 71 (n = 29 males, n = 42 females) piglets from 12 litters were randomly divided into five experimental groups. Outbred
50
M.R. Dwinell et al. / Respiratory Physiology & Neurobiology 175 (2011) 49–54
pregnant sows were allowed to deliver naturally in the Medical College of Wisconsin Biomedical Resource Center. The piglets were housed with the sow throughout the course of the studies and allowed to nurse freely. All protocols were reviewed and approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee. The piglets were divided into five experimental groups with each group containing 6–15 piglets. Group 1 piglets were exposed to CO2 daily. To explore the potential affect on the hypercapnic ventilatory response of daily CO2 exposure, three groups of piglets (Groups 2–4) were exposed to CO2 on every 3rd day beginning on either PN1 (Group 2), PN2 (Group 3) or PN3 (Group 4), respectively. Group 5 piglets were studied daily only under normoxic conditions through PN19. On PN20–21, these piglets were exposed to CO2 . A subset of piglets (n = 15) from Groups 1–5 (n = 15 total, 6 piglets from Group 1, 2 piglets from Group 2, 4 piglets from Group 3, 1 piglet from Group 4, and 1 piglet from Group 5) had a femoral catheter surgically implanted for arterial blood gas sampling. Due to a limited period for maintaining patent catheters, the number of piglets for blood sampling in specific postnatal age window varied between 3 and 8. Studies on another subset of piglets (n = 13 total; 2 piglets from Group 1, 4 piglets from Groups 2–4, and 7 piglets from Group 5) were extended into the 4th week of life (PN22–28). Nine of these piglets were exposed daily to 5% CO2 for 10 min, while the remaining four piglets were studied under room air conditions and exposed to 5% CO2 on days PN27–28 only. 2.2. Surgery Piglets (n = 13) from Groups 1–4 underwent surgery between PN5 and PN10. Piglets (n = 4) from Group 5 had surgery on PN17. Surgeries were performed after studies for that day were completed. Piglets were preanesthetized with Telazol (1–3 mg/kg IM) and then connected to a breathing mask and spontaneously ventilated with continuous 1–2% isoflurane in 2 l of 100% O2 throughout the procedure. Semi-rigid polyethylene catheters (PE50, Intramedic, Sparks, MD) were inserted into the femoral artery with additional length to loop under the skin and exteriorized through the back of the neck. The femoral catheter was used to measure arterial blood pressure and heart rate, and for arterial blood sampling for PaCO2 , PaO2 and pH measurement. After recovering from anesthesia, piglets were returned to the sow and allowed to nurse freely. The piglets received cefazolin (25 mg/kg IM) daily for antibiotic prophylaxis and buprenorphine (0.01–0.02 mg/kg bid IM) for analgesia. The piglets wore jackets made of elastic material to protect the catheter. 2.3. Physiological studies All piglets were studied in a 100-l whole body plethysmograph connected to a transducer signal conditioner (Quintron InstruTable 1 Eupneic PaO2 , PaCO2 and pH values in awake neonatal piglets from postnatal (PN) days 1 through 21. PN age (days)
PaCO2 (Torr)
3–4 5–6 7–8 9–10 11–12 13–14 15–16 17–18 19–21
26.5 31.5 31.6 31.5 29.3 31.4 34.9 31.7 34.4
Values are means ± SE.
± ± ± ± ± ± ± ± ±
0.5 1.4 1.9 2.0 2.9 0.9 0.4 0.6 1.2
PaO2 (Torr) 94.8 80.7 89.8 94.6 83.8 89.9 96.4 89.5 93.5
± ± ± ± ± ± ± ± ±
7.9 6.3 9.9 7.5 7.9 4.2 5.6 6.3 4.4
pH 7.507 7.473 7.490 7.454 7.461 7.464 7.483 7.481 7.467
n= ± ± ± ± ± ± ± ± ±
.018 .031 .01 .021 .007 .005 .005 .004 .011
3 4 8 6 3 6 4 7 8
ments, Milwaukee, WI). The pressure signal was recorded on a CODAS/Windaq (DATAQ Instruments, Akron, OH) system and used to measure frequency and tidal volume. Relative humidity and temperature in the plethysmograph were measured continuously using a calibrated Omega RX-93 temperature and relative humidity probe. The average temperature in the plethysmograph during the studies was 25.6 ± 1.0 ◦ C. The average relative humidity in the plethysmograph was 64.4 ± 6.8 (%). Temperature and relative humidity were recorded during normocapnia and hypercapnia and used for the tidal volume calculations. There were no significant differences in temperature or relative humidity between the normoxia and hypercapnia as the piglets size increased during development. Rectal temperature was recorded by a rectal thermometer (Omega Engineering, Stamford, CT). The piglets were placed in the plethysmograph for a 15 min adaptation period. Throughout this period and during data collection, we ensured that the piglets were in the awake state, thus avoiding complications in data interpretation by change in arousal state. The plethysmograph was flushed with room air for 2–5 min to eliminate expired CO2 . Control ventilation was measured for 10 min while the piglets breathed room air. After 5 l of CO2 was added to the plethysmograph to increase the inspired CO2 to 5% CO2 , ventilation was measured for 10 min. However, reported values for room air and CO2 inhalation were only for the last 2 min of each period. Following a 5 min flush to eliminate the elevated level of CO2 , recovery data was collected for 10 min. In piglets with an arterial catheter, arterial blood gas samples were drawn in the 7th min of the normoxic period and analyzed using a Chiron RapidLab Model 840 blood gas analyzer. The concentrations of O2 and CO2 in the plethysmograph were measured in initial studies only. In piglets with arterial catheters, blood was withdrawn during the 7th min of the control and hypercapnic periods. 2.4. Data analysis The ventilatory data (expressed as means ± SEM) were analyzed using a two-way analysis of variance (not repeated measures) for baseline and hypercapnic conditions. Within-group data were analyzed over time using a one-way analysis of variance (repeated measures) with Bonferroni post hoc test. Values are listed as mean values ± standard error of the mean. Results with p < 0.05 were considered significant. 3. Results 3.1. Growth and development The average weight at birth for the piglets (groups and genders combined) was 1.7 kg. There was a linear increase with age in body weight in all groups of piglets. The piglets grew an average of 280 g/day (Fig. 1) The mean body weight in Group 5 piglets (daily normoxia only until PN20) was greater than Group 1 (daily CO2 ) and Groups 2–4 (CO2 exposure every 3rd day), but Group 5 also had the smallest average litter size. Group 5 average litter size was 10.0 piglets/litter, while Groups 1 and 2–4 had average litter sizes of 12.9 and 11.4 piglets/litter, respectively. Rectal temperature increased slightly, but significantly (p < 0.01), in all groups with increasing postnatal age (Fig. 2). 3.2. Control, room air conditions When normalized for body weight, pulmonary ventilation (V˙ E , ml/min/kg) while breathing room air (control) decreased with age in all groups of piglets (Fig. 3). This decline with increasing postnatal age was due primarily to a decrease in tidal volume (VT ,
M.R. Dwinell et al. / Respiratory Physiology & Neurobiology 175 (2011) 49–54
51
Fig. 1. Weight gain in piglets between postnatal (PN) days 1 and 21. Group 1: piglets were exposed to 10 min of 5% CO2 daily from PN1 to PN21; Groups 2–4: piglets were exposed to 10 min of 5% CO2 every 3rd day beginning on PN1, 2 or 3; Group 5: piglets were exposed to normoxia (NX) daily and 10 min of 5% CO2 on PN20–21. Values are means ± SE.
ml/kg). Breathing frequency (f, breaths/min) did not change significantly during this window of postnatal development. Between PN3 and PN5, ventilation had significantly decreased under control conditions compared to PN1 (p < 0.01). There were no significant differences in control ventilation between piglets with arterial catheters (surgical) and non-instrumented piglets. There was an age-dependent 8 mm Hg increase (p < 0.01) in PaCO2 between PN3–4 and PN19–21 (Table 1). 3.3. Ventilatory response to hypercapnia There were no significant differences in the ventilatory response to CO2 between male and female piglets on any day between PN1 and PN21. Therefore, data from all piglets within a group were combined. V˙ E , f, VT eased from control levels during hypercapnia (FICO 0.05) 2
in all piglets from PN1 to PN21 (Fig. 4). When the hypercapnic ventilatory response is expressed as the percent increase in ventilation above the control ventilation, it is clear that there was no significant (p = 0.137) age-dependent change in the ventilatory response
Fig. 3. (A) Pulmonary ventilation (V˙ E , ml/min/kg) normalized for body weight during room air breathing from PN1 to PN21. (B) Tidal volume (VT , ml/kg) normalized for body weight during room air breathing from PN1 to PN21. (C) Breathing frequency (f, breaths/min) during room air breathing from PN1 to PN21. Values are means ± SE.
Fig. 2. Body temperature in piglets between PN1 and PN21. Group 1: piglets were exposed to 10 min of 5% CO2 daily from PN1 to PN21; Groups 2–4: piglets were exposed to 10 min of 5% CO2 every 3rd day beginning on PN1, 2 or 3; Group 5: piglets were exposed to normoxia (NX) daily and 10 min of 5% CO2 on PN20–21. Values are means ± SE.
to CO2 (Fig. 5A). In addition, exposure to 10 min of CO2 , either daily or every 3rd day, did not elicit any type of plasticity in the hypercapnic ventilatory response. The hypercapnic ventilatory response in piglets exposed to CO2 only on PN20–21 was not different than the response on those days in piglets exposed to CO2 throughout development. The increase in ventilation during 5% inspired CO2 was due to both an increase in tidal volume (VT , Fig. 5B) and breathing frequency (f, Fig. 5C) from birth to PN21. The PaCO2 while breathing 5% CO2 increased from room air randomly over all ages between 6.2 and 9.3 mm Hg. All piglets had an increased PaO2 during the increased inspired CO2 ; thus, the hyperpnea was not due to a change in the O2 stimulus.
52
M.R. Dwinell et al. / Respiratory Physiology & Neurobiology 175 (2011) 49–54
Fig. 4. Pulmonary (V˙ E , ml/min/kg) normalized for body weight during minutes 7–9 of hypercapnia (inspired CO2 fraction = 0.05) PN1–21. Values are means ± SE.
The robust hypercapnic ventilatory response continued between PN22 and PN28 (Fig. 5) without any significant differences between days. A similar hypercapnic ventilatory response on PN27–28 was measured in piglets exposed to either room air or CO2 daily prior to PN27. 4. Discussion Postnatal changes in the hypercapnic ventilatory response have been reported in premature human infants as well as rodent models. However, the major finding of this study is that in piglets, the ventilatory response to hypercapnia did not change between PN1 and PN28. A second important finding is that repeated CO2 exposure, either daily or every 3rd day, did not alter the hypercapnic ventilatory response compared to normoxic control piglets. 4.1. Development of the respiratory control system Although the respiratory system must be fully functional at birth, it is not fully mature until well after birth. Postnatal maturation occurs in many aspects of respiratory control (Gaultier and Gallego, 2005) which are influenced by many factors. Developmental plasticity can result when environmental interactions alter the structure or function of the respiratory network. In the neonate, plasticity can occur as a result of environmental factors that have little or no effect in the adult. A specific period, referred to as the critical period, may exist when specific stressors may have a dramatic effect, whereas the same stressor prior to or following the critical period will have little or no effect. Many previous studies focusing on developmental plasticity of the respiratory control system have used the neonatal rat model which is relatively immature at birth. In the present study, neonatal piglets were studied since they have been used as a model for postnatal development of ventilation and sleep patterns in humans (Cote et al., 1996; Messier et al., 2004; Penatti et al., 2006; Scott et al., 1990; Waters and Tinworth, 2001) and are more mature at birth than rodents. Our findings are in agreement with findings of Messier et al. (2004) and Penatti et al. (2006) that CO2 sensitivity in piglets does not change during the neonatal period. This finding in piglets contrasts to neonatal rats in whom some studies have found an attenuated CO2 sensitivity at P6–P7 relative to older and younger rats (Serra et al., 2002; Stunden et al., 2001; Wickstrom et al., 2002). Moreover, the robust and stable CO2 sensitivity in piglets from P0 to P30 contrasts to rats who have a relatively low CO2 sensitivity until about P15 after which there is a dramatic increase that in many strains reaches adult levels by P21 (Davis et al., 2006). These
Fig. 5. Pulmonary ventilation (V˙ E , ml/min/kg) (A), tidal volume (VT , ml/kg) (B), and breathing frequency (f, breaths/min) (C) expressed as a percent of room air breathing during minutes 7–9 of hypercapnia (inspired CO2 fraction = 0.05) from PN1 to PN28. Values are means ± SE.
differences between rats and piglets may be due to the precocious level of maturity of piglets at birth, whose eyes are open, have fur and are able to walk almost immediately after birth. Thus, piglets are rather mature compared to human infants and rodents at birth. However, the hypercapnic ventilatory response in unanesthetized kittens which are less mature at birth than piglets apparently does not change during development (PN3 through 39 days) (Watanabe et al., 1996). Level of maturity at birth within a species has been shown to alter both hypoxic and hypercapnic sensitivity during the postnatal period (Davey et al., 1996). Preterm birth eliminated the normal postnatal maturation of the hypoxic ventilatory response
M.R. Dwinell et al. / Respiratory Physiology & Neurobiology 175 (2011) 49–54
and temporarily decreased hypercapnic sensitivity in developing lambs. The mechanisms involved in CO2 sensitivity and the development of the ventilatory responses to CO2 likely varies between full term newborns and late preterm infants (Darnall, 2010); thus, for understanding human development, careful consideration is required to identify the most appropriate animal model to study. 4.2. Influence of carotid body denervation on ventilation during development Previous studies investigating the effect of CBD on neonatal rats and piglets suggest different critical periods of development in these species. CBD in neonatal rats between PN6 and PN8 resulted in greater mortality than younger or older rats (Serra et al., 2001). During this window of development, several groups have shown that the ventilatory response to CO2 is attenuated (Serra et al., 2001; Stunden et al., 2001; Wickstrom et al., 2002). In contrast, at PN6–8, CBD alone was not enough to cause mortality in the piglet (Serra et al., 2002), perhaps due to excitatory input from other chemoreceptors able to compensate for the loss of the carotid body input. Indeed, we have documented significant aortic hypoxic chemoreception in neonatal piglets which is enhanced after CBD (Serra et al., 2002). When aortic denervation was combined with CBD, significant mortality occurred between PN10 and PN15 in the piglet (Serra et al., 2002). However, as presently shown a concomitant decrease in the ventilatory response to CO2 was not found during this window of development. Some factors other than low CO2 sensitivity must contribute to this apparent critical period in piglets. 4.3. Developmental changes in ventilation Control, room air ventilation did undergo developmental changes, especially during the 1st week of life. V˙ E and VT , normalized for body weight, declined significantly from PN1 to PN21 (p < 0.05). These data agree with the finding in this and a previous study (Lowry et al., 1999) that PaCO2 increases (4–7 mm Hg) significantly between PN1 and PN21. Clearly then the level around which PaCO2 is regulated is changed without an apparent change in the gain of the chemoreflex. This change seems to indicate that the balance between inhibitory and excitatory influences on respiratory neurons is shifting toward greater inhibition and/or less excitation. In piglets, it seems clear that this shift is not due to an overall change in CO2 sensitivity, but could be due to a combination of changes in a variety of brainstem respiratory controllers, a change in sensitivity and balance of the central and peripheral chemoreceptors, or changes in the effectors of the respiratory system (Darnall, 2010). However, it does not appear due to a change in peripheral chemoreceptor activity as we have shown in piglets that the ventilatory response to NaCN injected into the jugular vein is brisk by P2 and this response does not change between P2 and P30 (Serra et al., 2002). In addition to a combination of peripheral and central CO2 chemoreception, central CO2 sensitivity has been hypothesized to be redundant and plastic to provide stability during developmental stage, arousal state, level and duration of stimulus, pathophysiological changes, and gender differences (Nattie and Li, 2009). 4.4. Changes in ventilation and CO2 sensitivity during development It is also clear that there is, in piglets and rats, a major dissociation during the neonatal period between control ventilation and eucapnic PaCO2 vs CO2 sensitivity. In other words, as these species grow and mature, room air ventilation decreases relative to size and metabolic rate. On the other hand, as summarized by Davis et al. (2006), numerous studies have shown that body size
53
and metabolic rate do not affect CO2 sensitivity. In both species, an increasing PaCO2 during the neonatal period coexists with either a stable (piglet) or a low and then increasing (rat) CO2 sensitivity. This dissociation is not unique to the neonatal period. For example, after experimental lesions in the medulla of goats (Forster et al., 1998; Hodges et al., 2004) and in the clinical condition of congenital central alveolar hypoventilation (Shea et al., 1993), there is a normal eucapnic PaCO2 coexisting with an attenuated CO2 sensitivity. This dissociation indicates that the CO2 excitatory drive is not critical for eupneic breathing, and it seems to reflect a high degree of plasticity in the ventilatory control system that enables the system to function optimally for any condition irrespective of the degree of excitatory or inhibitory input from any single source. 4.5. Effect of repeated exposure to CO2 on ventilation Daily CO2 exposure does not affect the V˙ E response to hypercapnia. Because of the high degree of plasticity in neonates, we were concerned that repeated exposure to CO2 would alter the V˙ E response to hypercapnia (Putnam et al., 2005). However, there was no apparent effect on the V˙ E response of daily or every 3rd day exposure for to 5% CO2 . At PN20 and PN21, CO2 sensitivity did not differ between piglets not previously exposed to CO2 and those exposed daily or every 3rd day to a brief period of CO2 exposure. Of interest is that growth of the piglets was significantly greater in piglets not exposed to CO2 . Moreover, there was a tendency for greater growth in those exposed every 3rd day than in those exposed daily. All piglets were studied daily for the same time period to minimize the period without access to food and control for potential circadian variation. Litter size appears to be a greater determinant of weight gain as the piglets from the smallest litters had the greatest weight gain. The major point is that in spite of the high potential for plasticity in neonates, differences in CO2 exposure and major differences in surgical procedures in this and in a previous study did not seem to affect ventilatory CO2 sensitivity. In other words, CO2 sensitivity is highly robust throughout development in neonatal piglets. 4.6. Significance of results The importance of the findings reported herein is that they emphasize several factors critical to understanding the control of breathing. First, there are major species differences in development of the ventilatory response to CO2 . Second, a dissociation exists between eupneic breathing (i.e. PaCO2 ) and CO2 sensitivity. These results provide evidence that eupneic breathing is not critically dependent on CO2 drive. Finally, the robustness of CO2 sensitivity is not affected by growth, age, or repeated exposure to elevated inspired CO2 levels. Acknowledgements These studies were supported by the SIDS Foundation of Wisconsin, the Department of Veterans Affairs, the Parker B. Francis Foundation (MRD), and NIH grant HL 25739. References Bairam, A., Carroll, J.L., 2005. Neurotransmitters in carotid body development. Respir. Physiol. Neurobiol. 149, 217–232. Cote, A., Porras, H., Meehan, B., 1996. Age-dependent vulnerability to carotid chemodenervation in piglets. J. Appl. Physiol. 80, 323–331. Darnall, R.A., 2010. The role of CO2 and central chemoreception in the control of breathing in the fetus and the neonate. Respir. Physiol. Neurobiol., April 15 [Epub ahead of print]. Davey, M.G., Moss, T.J., McCrabb, G.J., Harding, R., 1996. Prematurity alters hypoxic and hypercapnic ventilatory responses in developing lambs. Respir. Physiol. 105, 57–67.
54
M.R. Dwinell et al. / Respiratory Physiology & Neurobiology 175 (2011) 49–54
Davis, S.E., Solhied, G., Castillo, M., Dwinell, M.R., Brozoski, D., Forster, H.V., 2006. Postnatal developmental changes in CO2 sensitivity in rats. J. Appl. Physiol. 101, 1097–1103. Filiano, J.J., Kinney, H.C., 1994. A perspective on neuropathologic findings in victims of the sudden infant death syndrome: the triple risk model. Biol. Neonate 65, 194–197. Filiano, J.J., Kinney, H.C., 1992. Arcuate nucleus hypoplasia in the sudden infant death syndrome. J. Neuropathol. Exp. Neurol. 51, 394–403. Filiano, J.J., 1994. Arcuate nucleus hypoplasia in sudden infant death syndrome: a review. Biol. Neonate 65, 156–159. Forster, H.V., Pan, L.G., Lowry, T.F., Feroah, T., Gershan, W.M., Whaley, A.A., Forster, M.M., Sprtel, B., 1998. Breathing of awake goats during prolonged dysfunction of caudal M ventrolateral medullary neurons. J. Appl. Physiol. 84, 129–140. Gaultier, C., Gallego, J., 2005. Development of respiratory control: evolving concepts and perspectives. Respir. Physiol. Neurobiol. 149, 3–15. Hodges, M.R., Opansky, C., Qian, B., Davis, S., Bonis, J., Bastasic, J., Leekley, J., Pan, L.G., Forster, H.V., 2004. Transient attenuation of CO2 sensitivity after neurotoxic lesions in the medullary raphe area of awake goats. J. Appl. Physiol. 97, 2236–2247. Kinney, H.C., Filiano, J.J., Harper, R.M., 1992. The neuropathology of the sudden infant death syndrome: a review. J. Neuropathol. Exp. Neurol. 51, 115–126. Lowry, T.F., Forster, H.V., Pan, L.G., Serra, A., Wenninger, J., Nash, R., Sheridan, D., Franciosi, R.A., 1999. Effect on breathing of carotid body denervation in neonatal piglets. J. Appl. Physiol. 87, 2128–2135. Messier, M.L., Li, A., Nattie, E.E., 2004. Inhibition of medullary raphe serotonergic neurons has are-dependent effects on the CO2 response in newborn piglets. J. Appl. Physiol. 96, 1909–1919. Nattie, E., Li, A., 2009. Central chemoreception is a complex system function that involves multiple brain stem sites. J. Appl. Physiol. 106, 1464–1466.
Penatti, E.M., Berniker, A.V., Kereshi, B., Cafaro, C., Kelly, M.L., Niblock, M.M., Gao, H.G., Kinney, H.C., Li, A., Nattie, E.E., 2006. Ventilatory response to hypercapnia and hypoxia after extensive lesion of medullary serotonergic neurons in newborn conscious piglets. J. Appl. Physiol. 101, 1177–1188. 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. Scott, S.C., Inman, J.D., Moss, I.R., 1990. Ontogeny of sleep/wake and cardiorespiratory behavior in unanesthetized piglets. Respir. Physiol. 80, 83–101. 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. Serra, A., Brozoski, D., Hodges, M., Roethle, S., Franciosi, R., Forster, H.V., 2002. Effects of carotid and aortic chemoreceptor denervation in newborn piglets. J. Appl. Physiol. 92, 893–900. Shea, S.A., Andrez, L.P., Shannon, D.C., Banzett, R.B., 1993. Ventilatory responses to exercises in humans lacking ventilatory chemosensitivity. J. Physiol. 468, 623–640. 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. Watanabe, T., Kumar, P., Hanson, M.A., 1996. Development of respiratory chemoreflexes to hypoxia and CO2 in unanaesthetized kittens. Respir. Physiol. 106, 247–254. Waters, K.A., Tinworth, K.D., 2001. Depression of ventilatory responses after daily, cyclic hypercapnic hypoxia in piglets. J. Appl. Physiol. 90, 1065–1073. Wickstrom, R., Hokfelt, T., Langercrantz, H., 2002. Development of CO2 -response in the early newborn period in rat. Respir. Physiol. Neurobiol. 132, 145–158.
Contents lists available at ScienceDirect
Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Postnatal ventilatory response to CO2 in awake piglets M.R. Dwinell ∗ , G.E. Hogan, E. Sirlin, D.L. Mayhew, H.V. Forster Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, United States
a r t i c l e
i n f o
Article history: Accepted 7 September 2010 Keywords: Hypercapnic ventilatory response Postnatal development Control of breathing Piglets
a b s t r a c t Abnormal ventilatory responses to increased levels of inspired CO2 during postnatal development may pose a risk for Sudden Infant Death Syndrome, primarily during periods of vulnerability. The purpose of this study was to test the hypothesis that in awake piglets the ventilatory response to hypercapnia would be attenuated between 10 and 15 days of age relative to younger and older ages. To test this hypothesis, we measured the ventilatory response to 5% inspired CO2 in piglets from postnatal (PN) days 1 through PN28. Piglets were divided into groups and exposed to 5% CO2 daily, every 3rd day or on and after PN20–21 only to avoid any plasticity that may result from repeated exposure to CO2 . Room air ventilation normalized to body weight (V˙ E , ml/min/kg) declined with postnatal age in piglets from all groups. The ventilatory response to 5% inspired CO2 (expressed as % change from control) was present at birth, and we did not find an age-dependent change from PN1 to PN28 (p > 0.1). In addition, we did not find that repeated exposure (daily or every 3rd day) to 5% inspired CO2 altered the ventilatory response during this period of development. We conclude that the previously documented apparent critical period of development in piglets between 10 and 15 days of age is not associated with attenuation of the ventilatory response to 5% inspired CO2 . © 2010 Elsevier B.V. All rights reserved.
1. Introduction The ventilatory responses to hypoxia and hypercapnia vary among different newborn mammals depending on the level of maturity at birth. Peripheral oxygen sensitivity is weak at birth and gradually increases to adult levels during the 1st weeks to months of life in many species (Bairam and Carroll, 2005). On the other hand, the ventilatory response to elevated levels of inspired CO2 had been thought to be relatively mature at birth and change little during development. However, recent studies have demonstrated that the level of maturity at birth may affect the CO2 response. For example, relatively immature species at birth (i.e. rats) appear to have an initial response to CO2 at birth that then declines at postnatal (PN) days 6–7 (Serra et al., 2001; Stunden et al., 2001; Wickstrom et al., 2002) or remains flat (Davis et al., 2006) until about PN14–15, when it begins to increase to reach adult levels by about PN21. Sudden Infant Death Syndrome (SIDS) is the leading cause of infant mortality between 1 month and 1 year of life. Although the exact pathophysiology of SIDS remains unknown, a triple-risk model has been proposed for the pathogenesis of SIDS (Filiano and Kinney, 1992, 1994; Filiano, 1994; Kinney et al., 1992). The three factors that must be present to result in death are (1) an underlying vulnerability, (2) a critical period of development, and (3) an exoge-
nous stressor. According to this model, SIDS may be caused in part by the inability of the respiratory control system to respond to an exogenous stressor during this critical window. Past studies have utilized peripheral chemoreceptor denervation to create an underlying vulnerability. In 1 week old rats, mortality following carotid body denervation (CBD) was increased suggesting a critical window of development in the respiratory control system which corresponds to the period of minimal sensitivity to CO2 in rats (Serra et al., 2001). In piglets, a more mature species at birth, CBD alone did not lead to significant mortality, but when combined with aortic denervation, mortality was increased between PN10 and PN15 (Serra et al., 2002). Therefore, the objective of this study was to test the hypothesis that CO2 sensitivity would be reduced (relative to younger and older ages) during postnatal days 10–15 in piglets. The piglet model was chosen as an alternate to the rodent models to extend our understanding of the maturation of the hypercapnic ventilatory response in a species whose maturation is more comparable to human than rodents. These findings will thereby provide insight into the importance of CO2 sensitivity to sustain breathing in vulnerable infants. 2. Methods 2.1. Experimental groups
∗ Corresponding author. Tel.: +1 414 456 4498; fax: +1 414 456 6546. E-mail address: [email protected] (M.R. Dwinell). 1569-9048/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2010.09.005
A total of 71 (n = 29 males, n = 42 females) piglets from 12 litters were randomly divided into five experimental groups. Outbred
50
M.R. Dwinell et al. / Respiratory Physiology & Neurobiology 175 (2011) 49–54
pregnant sows were allowed to deliver naturally in the Medical College of Wisconsin Biomedical Resource Center. The piglets were housed with the sow throughout the course of the studies and allowed to nurse freely. All protocols were reviewed and approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee. The piglets were divided into five experimental groups with each group containing 6–15 piglets. Group 1 piglets were exposed to CO2 daily. To explore the potential affect on the hypercapnic ventilatory response of daily CO2 exposure, three groups of piglets (Groups 2–4) were exposed to CO2 on every 3rd day beginning on either PN1 (Group 2), PN2 (Group 3) or PN3 (Group 4), respectively. Group 5 piglets were studied daily only under normoxic conditions through PN19. On PN20–21, these piglets were exposed to CO2 . A subset of piglets (n = 15) from Groups 1–5 (n = 15 total, 6 piglets from Group 1, 2 piglets from Group 2, 4 piglets from Group 3, 1 piglet from Group 4, and 1 piglet from Group 5) had a femoral catheter surgically implanted for arterial blood gas sampling. Due to a limited period for maintaining patent catheters, the number of piglets for blood sampling in specific postnatal age window varied between 3 and 8. Studies on another subset of piglets (n = 13 total; 2 piglets from Group 1, 4 piglets from Groups 2–4, and 7 piglets from Group 5) were extended into the 4th week of life (PN22–28). Nine of these piglets were exposed daily to 5% CO2 for 10 min, while the remaining four piglets were studied under room air conditions and exposed to 5% CO2 on days PN27–28 only. 2.2. Surgery Piglets (n = 13) from Groups 1–4 underwent surgery between PN5 and PN10. Piglets (n = 4) from Group 5 had surgery on PN17. Surgeries were performed after studies for that day were completed. Piglets were preanesthetized with Telazol (1–3 mg/kg IM) and then connected to a breathing mask and spontaneously ventilated with continuous 1–2% isoflurane in 2 l of 100% O2 throughout the procedure. Semi-rigid polyethylene catheters (PE50, Intramedic, Sparks, MD) were inserted into the femoral artery with additional length to loop under the skin and exteriorized through the back of the neck. The femoral catheter was used to measure arterial blood pressure and heart rate, and for arterial blood sampling for PaCO2 , PaO2 and pH measurement. After recovering from anesthesia, piglets were returned to the sow and allowed to nurse freely. The piglets received cefazolin (25 mg/kg IM) daily for antibiotic prophylaxis and buprenorphine (0.01–0.02 mg/kg bid IM) for analgesia. The piglets wore jackets made of elastic material to protect the catheter. 2.3. Physiological studies All piglets were studied in a 100-l whole body plethysmograph connected to a transducer signal conditioner (Quintron InstruTable 1 Eupneic PaO2 , PaCO2 and pH values in awake neonatal piglets from postnatal (PN) days 1 through 21. PN age (days)
PaCO2 (Torr)
3–4 5–6 7–8 9–10 11–12 13–14 15–16 17–18 19–21
26.5 31.5 31.6 31.5 29.3 31.4 34.9 31.7 34.4
Values are means ± SE.
± ± ± ± ± ± ± ± ±
0.5 1.4 1.9 2.0 2.9 0.9 0.4 0.6 1.2
PaO2 (Torr) 94.8 80.7 89.8 94.6 83.8 89.9 96.4 89.5 93.5
± ± ± ± ± ± ± ± ±
7.9 6.3 9.9 7.5 7.9 4.2 5.6 6.3 4.4
pH 7.507 7.473 7.490 7.454 7.461 7.464 7.483 7.481 7.467
n= ± ± ± ± ± ± ± ± ±
.018 .031 .01 .021 .007 .005 .005 .004 .011
3 4 8 6 3 6 4 7 8
ments, Milwaukee, WI). The pressure signal was recorded on a CODAS/Windaq (DATAQ Instruments, Akron, OH) system and used to measure frequency and tidal volume. Relative humidity and temperature in the plethysmograph were measured continuously using a calibrated Omega RX-93 temperature and relative humidity probe. The average temperature in the plethysmograph during the studies was 25.6 ± 1.0 ◦ C. The average relative humidity in the plethysmograph was 64.4 ± 6.8 (%). Temperature and relative humidity were recorded during normocapnia and hypercapnia and used for the tidal volume calculations. There were no significant differences in temperature or relative humidity between the normoxia and hypercapnia as the piglets size increased during development. Rectal temperature was recorded by a rectal thermometer (Omega Engineering, Stamford, CT). The piglets were placed in the plethysmograph for a 15 min adaptation period. Throughout this period and during data collection, we ensured that the piglets were in the awake state, thus avoiding complications in data interpretation by change in arousal state. The plethysmograph was flushed with room air for 2–5 min to eliminate expired CO2 . Control ventilation was measured for 10 min while the piglets breathed room air. After 5 l of CO2 was added to the plethysmograph to increase the inspired CO2 to 5% CO2 , ventilation was measured for 10 min. However, reported values for room air and CO2 inhalation were only for the last 2 min of each period. Following a 5 min flush to eliminate the elevated level of CO2 , recovery data was collected for 10 min. In piglets with an arterial catheter, arterial blood gas samples were drawn in the 7th min of the normoxic period and analyzed using a Chiron RapidLab Model 840 blood gas analyzer. The concentrations of O2 and CO2 in the plethysmograph were measured in initial studies only. In piglets with arterial catheters, blood was withdrawn during the 7th min of the control and hypercapnic periods. 2.4. Data analysis The ventilatory data (expressed as means ± SEM) were analyzed using a two-way analysis of variance (not repeated measures) for baseline and hypercapnic conditions. Within-group data were analyzed over time using a one-way analysis of variance (repeated measures) with Bonferroni post hoc test. Values are listed as mean values ± standard error of the mean. Results with p < 0.05 were considered significant. 3. Results 3.1. Growth and development The average weight at birth for the piglets (groups and genders combined) was 1.7 kg. There was a linear increase with age in body weight in all groups of piglets. The piglets grew an average of 280 g/day (Fig. 1) The mean body weight in Group 5 piglets (daily normoxia only until PN20) was greater than Group 1 (daily CO2 ) and Groups 2–4 (CO2 exposure every 3rd day), but Group 5 also had the smallest average litter size. Group 5 average litter size was 10.0 piglets/litter, while Groups 1 and 2–4 had average litter sizes of 12.9 and 11.4 piglets/litter, respectively. Rectal temperature increased slightly, but significantly (p < 0.01), in all groups with increasing postnatal age (Fig. 2). 3.2. Control, room air conditions When normalized for body weight, pulmonary ventilation (V˙ E , ml/min/kg) while breathing room air (control) decreased with age in all groups of piglets (Fig. 3). This decline with increasing postnatal age was due primarily to a decrease in tidal volume (VT ,
M.R. Dwinell et al. / Respiratory Physiology & Neurobiology 175 (2011) 49–54
51
Fig. 1. Weight gain in piglets between postnatal (PN) days 1 and 21. Group 1: piglets were exposed to 10 min of 5% CO2 daily from PN1 to PN21; Groups 2–4: piglets were exposed to 10 min of 5% CO2 every 3rd day beginning on PN1, 2 or 3; Group 5: piglets were exposed to normoxia (NX) daily and 10 min of 5% CO2 on PN20–21. Values are means ± SE.
ml/kg). Breathing frequency (f, breaths/min) did not change significantly during this window of postnatal development. Between PN3 and PN5, ventilation had significantly decreased under control conditions compared to PN1 (p < 0.01). There were no significant differences in control ventilation between piglets with arterial catheters (surgical) and non-instrumented piglets. There was an age-dependent 8 mm Hg increase (p < 0.01) in PaCO2 between PN3–4 and PN19–21 (Table 1). 3.3. Ventilatory response to hypercapnia There were no significant differences in the ventilatory response to CO2 between male and female piglets on any day between PN1 and PN21. Therefore, data from all piglets within a group were combined. V˙ E , f, VT eased from control levels during hypercapnia (FICO 0.05) 2
in all piglets from PN1 to PN21 (Fig. 4). When the hypercapnic ventilatory response is expressed as the percent increase in ventilation above the control ventilation, it is clear that there was no significant (p = 0.137) age-dependent change in the ventilatory response
Fig. 3. (A) Pulmonary ventilation (V˙ E , ml/min/kg) normalized for body weight during room air breathing from PN1 to PN21. (B) Tidal volume (VT , ml/kg) normalized for body weight during room air breathing from PN1 to PN21. (C) Breathing frequency (f, breaths/min) during room air breathing from PN1 to PN21. Values are means ± SE.
Fig. 2. Body temperature in piglets between PN1 and PN21. Group 1: piglets were exposed to 10 min of 5% CO2 daily from PN1 to PN21; Groups 2–4: piglets were exposed to 10 min of 5% CO2 every 3rd day beginning on PN1, 2 or 3; Group 5: piglets were exposed to normoxia (NX) daily and 10 min of 5% CO2 on PN20–21. Values are means ± SE.
to CO2 (Fig. 5A). In addition, exposure to 10 min of CO2 , either daily or every 3rd day, did not elicit any type of plasticity in the hypercapnic ventilatory response. The hypercapnic ventilatory response in piglets exposed to CO2 only on PN20–21 was not different than the response on those days in piglets exposed to CO2 throughout development. The increase in ventilation during 5% inspired CO2 was due to both an increase in tidal volume (VT , Fig. 5B) and breathing frequency (f, Fig. 5C) from birth to PN21. The PaCO2 while breathing 5% CO2 increased from room air randomly over all ages between 6.2 and 9.3 mm Hg. All piglets had an increased PaO2 during the increased inspired CO2 ; thus, the hyperpnea was not due to a change in the O2 stimulus.
52
M.R. Dwinell et al. / Respiratory Physiology & Neurobiology 175 (2011) 49–54
Fig. 4. Pulmonary (V˙ E , ml/min/kg) normalized for body weight during minutes 7–9 of hypercapnia (inspired CO2 fraction = 0.05) PN1–21. Values are means ± SE.
The robust hypercapnic ventilatory response continued between PN22 and PN28 (Fig. 5) without any significant differences between days. A similar hypercapnic ventilatory response on PN27–28 was measured in piglets exposed to either room air or CO2 daily prior to PN27. 4. Discussion Postnatal changes in the hypercapnic ventilatory response have been reported in premature human infants as well as rodent models. However, the major finding of this study is that in piglets, the ventilatory response to hypercapnia did not change between PN1 and PN28. A second important finding is that repeated CO2 exposure, either daily or every 3rd day, did not alter the hypercapnic ventilatory response compared to normoxic control piglets. 4.1. Development of the respiratory control system Although the respiratory system must be fully functional at birth, it is not fully mature until well after birth. Postnatal maturation occurs in many aspects of respiratory control (Gaultier and Gallego, 2005) which are influenced by many factors. Developmental plasticity can result when environmental interactions alter the structure or function of the respiratory network. In the neonate, plasticity can occur as a result of environmental factors that have little or no effect in the adult. A specific period, referred to as the critical period, may exist when specific stressors may have a dramatic effect, whereas the same stressor prior to or following the critical period will have little or no effect. Many previous studies focusing on developmental plasticity of the respiratory control system have used the neonatal rat model which is relatively immature at birth. In the present study, neonatal piglets were studied since they have been used as a model for postnatal development of ventilation and sleep patterns in humans (Cote et al., 1996; Messier et al., 2004; Penatti et al., 2006; Scott et al., 1990; Waters and Tinworth, 2001) and are more mature at birth than rodents. Our findings are in agreement with findings of Messier et al. (2004) and Penatti et al. (2006) that CO2 sensitivity in piglets does not change during the neonatal period. This finding in piglets contrasts to neonatal rats in whom some studies have found an attenuated CO2 sensitivity at P6–P7 relative to older and younger rats (Serra et al., 2002; Stunden et al., 2001; Wickstrom et al., 2002). Moreover, the robust and stable CO2 sensitivity in piglets from P0 to P30 contrasts to rats who have a relatively low CO2 sensitivity until about P15 after which there is a dramatic increase that in many strains reaches adult levels by P21 (Davis et al., 2006). These
Fig. 5. Pulmonary ventilation (V˙ E , ml/min/kg) (A), tidal volume (VT , ml/kg) (B), and breathing frequency (f, breaths/min) (C) expressed as a percent of room air breathing during minutes 7–9 of hypercapnia (inspired CO2 fraction = 0.05) from PN1 to PN28. Values are means ± SE.
differences between rats and piglets may be due to the precocious level of maturity of piglets at birth, whose eyes are open, have fur and are able to walk almost immediately after birth. Thus, piglets are rather mature compared to human infants and rodents at birth. However, the hypercapnic ventilatory response in unanesthetized kittens which are less mature at birth than piglets apparently does not change during development (PN3 through 39 days) (Watanabe et al., 1996). Level of maturity at birth within a species has been shown to alter both hypoxic and hypercapnic sensitivity during the postnatal period (Davey et al., 1996). Preterm birth eliminated the normal postnatal maturation of the hypoxic ventilatory response
M.R. Dwinell et al. / Respiratory Physiology & Neurobiology 175 (2011) 49–54
and temporarily decreased hypercapnic sensitivity in developing lambs. The mechanisms involved in CO2 sensitivity and the development of the ventilatory responses to CO2 likely varies between full term newborns and late preterm infants (Darnall, 2010); thus, for understanding human development, careful consideration is required to identify the most appropriate animal model to study. 4.2. Influence of carotid body denervation on ventilation during development Previous studies investigating the effect of CBD on neonatal rats and piglets suggest different critical periods of development in these species. CBD in neonatal rats between PN6 and PN8 resulted in greater mortality than younger or older rats (Serra et al., 2001). During this window of development, several groups have shown that the ventilatory response to CO2 is attenuated (Serra et al., 2001; Stunden et al., 2001; Wickstrom et al., 2002). In contrast, at PN6–8, CBD alone was not enough to cause mortality in the piglet (Serra et al., 2002), perhaps due to excitatory input from other chemoreceptors able to compensate for the loss of the carotid body input. Indeed, we have documented significant aortic hypoxic chemoreception in neonatal piglets which is enhanced after CBD (Serra et al., 2002). When aortic denervation was combined with CBD, significant mortality occurred between PN10 and PN15 in the piglet (Serra et al., 2002). However, as presently shown a concomitant decrease in the ventilatory response to CO2 was not found during this window of development. Some factors other than low CO2 sensitivity must contribute to this apparent critical period in piglets. 4.3. Developmental changes in ventilation Control, room air ventilation did undergo developmental changes, especially during the 1st week of life. V˙ E and VT , normalized for body weight, declined significantly from PN1 to PN21 (p < 0.05). These data agree with the finding in this and a previous study (Lowry et al., 1999) that PaCO2 increases (4–7 mm Hg) significantly between PN1 and PN21. Clearly then the level around which PaCO2 is regulated is changed without an apparent change in the gain of the chemoreflex. This change seems to indicate that the balance between inhibitory and excitatory influences on respiratory neurons is shifting toward greater inhibition and/or less excitation. In piglets, it seems clear that this shift is not due to an overall change in CO2 sensitivity, but could be due to a combination of changes in a variety of brainstem respiratory controllers, a change in sensitivity and balance of the central and peripheral chemoreceptors, or changes in the effectors of the respiratory system (Darnall, 2010). However, it does not appear due to a change in peripheral chemoreceptor activity as we have shown in piglets that the ventilatory response to NaCN injected into the jugular vein is brisk by P2 and this response does not change between P2 and P30 (Serra et al., 2002). In addition to a combination of peripheral and central CO2 chemoreception, central CO2 sensitivity has been hypothesized to be redundant and plastic to provide stability during developmental stage, arousal state, level and duration of stimulus, pathophysiological changes, and gender differences (Nattie and Li, 2009). 4.4. Changes in ventilation and CO2 sensitivity during development It is also clear that there is, in piglets and rats, a major dissociation during the neonatal period between control ventilation and eucapnic PaCO2 vs CO2 sensitivity. In other words, as these species grow and mature, room air ventilation decreases relative to size and metabolic rate. On the other hand, as summarized by Davis et al. (2006), numerous studies have shown that body size
53
and metabolic rate do not affect CO2 sensitivity. In both species, an increasing PaCO2 during the neonatal period coexists with either a stable (piglet) or a low and then increasing (rat) CO2 sensitivity. This dissociation is not unique to the neonatal period. For example, after experimental lesions in the medulla of goats (Forster et al., 1998; Hodges et al., 2004) and in the clinical condition of congenital central alveolar hypoventilation (Shea et al., 1993), there is a normal eucapnic PaCO2 coexisting with an attenuated CO2 sensitivity. This dissociation indicates that the CO2 excitatory drive is not critical for eupneic breathing, and it seems to reflect a high degree of plasticity in the ventilatory control system that enables the system to function optimally for any condition irrespective of the degree of excitatory or inhibitory input from any single source. 4.5. Effect of repeated exposure to CO2 on ventilation Daily CO2 exposure does not affect the V˙ E response to hypercapnia. Because of the high degree of plasticity in neonates, we were concerned that repeated exposure to CO2 would alter the V˙ E response to hypercapnia (Putnam et al., 2005). However, there was no apparent effect on the V˙ E response of daily or every 3rd day exposure for to 5% CO2 . At PN20 and PN21, CO2 sensitivity did not differ between piglets not previously exposed to CO2 and those exposed daily or every 3rd day to a brief period of CO2 exposure. Of interest is that growth of the piglets was significantly greater in piglets not exposed to CO2 . Moreover, there was a tendency for greater growth in those exposed every 3rd day than in those exposed daily. All piglets were studied daily for the same time period to minimize the period without access to food and control for potential circadian variation. Litter size appears to be a greater determinant of weight gain as the piglets from the smallest litters had the greatest weight gain. The major point is that in spite of the high potential for plasticity in neonates, differences in CO2 exposure and major differences in surgical procedures in this and in a previous study did not seem to affect ventilatory CO2 sensitivity. In other words, CO2 sensitivity is highly robust throughout development in neonatal piglets. 4.6. Significance of results The importance of the findings reported herein is that they emphasize several factors critical to understanding the control of breathing. First, there are major species differences in development of the ventilatory response to CO2 . Second, a dissociation exists between eupneic breathing (i.e. PaCO2 ) and CO2 sensitivity. These results provide evidence that eupneic breathing is not critically dependent on CO2 drive. Finally, the robustness of CO2 sensitivity is not affected by growth, age, or repeated exposure to elevated inspired CO2 levels. Acknowledgements These studies were supported by the SIDS Foundation of Wisconsin, the Department of Veterans Affairs, the Parker B. Francis Foundation (MRD), and NIH grant HL 25739. References Bairam, A., Carroll, J.L., 2005. Neurotransmitters in carotid body development. Respir. Physiol. Neurobiol. 149, 217–232. Cote, A., Porras, H., Meehan, B., 1996. Age-dependent vulnerability to carotid chemodenervation in piglets. J. Appl. Physiol. 80, 323–331. Darnall, R.A., 2010. The role of CO2 and central chemoreception in the control of breathing in the fetus and the neonate. Respir. Physiol. Neurobiol., April 15 [Epub ahead of print]. Davey, M.G., Moss, T.J., McCrabb, G.J., Harding, R., 1996. Prematurity alters hypoxic and hypercapnic ventilatory responses in developing lambs. Respir. Physiol. 105, 57–67.
54
M.R. Dwinell et al. / Respiratory Physiology & Neurobiology 175 (2011) 49–54
Davis, S.E., Solhied, G., Castillo, M., Dwinell, M.R., Brozoski, D., Forster, H.V., 2006. Postnatal developmental changes in CO2 sensitivity in rats. J. Appl. Physiol. 101, 1097–1103. Filiano, J.J., Kinney, H.C., 1994. A perspective on neuropathologic findings in victims of the sudden infant death syndrome: the triple risk model. Biol. Neonate 65, 194–197. Filiano, J.J., Kinney, H.C., 1992. Arcuate nucleus hypoplasia in the sudden infant death syndrome. J. Neuropathol. Exp. Neurol. 51, 394–403. Filiano, J.J., 1994. Arcuate nucleus hypoplasia in sudden infant death syndrome: a review. Biol. Neonate 65, 156–159. Forster, H.V., Pan, L.G., Lowry, T.F., Feroah, T., Gershan, W.M., Whaley, A.A., Forster, M.M., Sprtel, B., 1998. Breathing of awake goats during prolonged dysfunction of caudal M ventrolateral medullary neurons. J. Appl. Physiol. 84, 129–140. Gaultier, C., Gallego, J., 2005. Development of respiratory control: evolving concepts and perspectives. Respir. Physiol. Neurobiol. 149, 3–15. Hodges, M.R., Opansky, C., Qian, B., Davis, S., Bonis, J., Bastasic, J., Leekley, J., Pan, L.G., Forster, H.V., 2004. Transient attenuation of CO2 sensitivity after neurotoxic lesions in the medullary raphe area of awake goats. J. Appl. Physiol. 97, 2236–2247. Kinney, H.C., Filiano, J.J., Harper, R.M., 1992. The neuropathology of the sudden infant death syndrome: a review. J. Neuropathol. Exp. Neurol. 51, 115–126. Lowry, T.F., Forster, H.V., Pan, L.G., Serra, A., Wenninger, J., Nash, R., Sheridan, D., Franciosi, R.A., 1999. Effect on breathing of carotid body denervation in neonatal piglets. J. Appl. Physiol. 87, 2128–2135. Messier, M.L., Li, A., Nattie, E.E., 2004. Inhibition of medullary raphe serotonergic neurons has are-dependent effects on the CO2 response in newborn piglets. J. Appl. Physiol. 96, 1909–1919. Nattie, E., Li, A., 2009. Central chemoreception is a complex system function that involves multiple brain stem sites. J. Appl. Physiol. 106, 1464–1466.
Penatti, E.M., Berniker, A.V., Kereshi, B., Cafaro, C., Kelly, M.L., Niblock, M.M., Gao, H.G., Kinney, H.C., Li, A., Nattie, E.E., 2006. Ventilatory response to hypercapnia and hypoxia after extensive lesion of medullary serotonergic neurons in newborn conscious piglets. J. Appl. Physiol. 101, 1177–1188. 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. Scott, S.C., Inman, J.D., Moss, I.R., 1990. Ontogeny of sleep/wake and cardiorespiratory behavior in unanesthetized piglets. Respir. Physiol. 80, 83–101. 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. Serra, A., Brozoski, D., Hodges, M., Roethle, S., Franciosi, R., Forster, H.V., 2002. Effects of carotid and aortic chemoreceptor denervation in newborn piglets. J. Appl. Physiol. 92, 893–900. Shea, S.A., Andrez, L.P., Shannon, D.C., Banzett, R.B., 1993. Ventilatory responses to exercises in humans lacking ventilatory chemosensitivity. J. Physiol. 468, 623–640. 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. Watanabe, T., Kumar, P., Hanson, M.A., 1996. Development of respiratory chemoreflexes to hypoxia and CO2 in unanaesthetized kittens. Respir. Physiol. 106, 247–254. Waters, K.A., Tinworth, K.D., 2001. Depression of ventilatory responses after daily, cyclic hypercapnic hypoxia in piglets. J. Appl. Physiol. 90, 1065–1073. Wickstrom, R., Hokfelt, T., Langercrantz, H., 2002. Development of CO2 -response in the early newborn period in rat. Respir. Physiol. Neurobiol. 132, 145–158.