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Developmental hyperoxia alters CNS mechanisms underlying hypoxic ventilatory depression in neonatal rats
Respiratory Physiology & Neurobiology 189 (2013) 498–505
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Developmental hyperoxia alters CNS mechanisms underlying hypoxic ventilatory depression in neonatal rats Corey B. Hill, Samuel H. Grandgeorge, Ryan W. Bavis ∗ Department of Biology, Bates College, Lewiston, ME 04240, USA
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Article history: Accepted 22 August 2013 Keywords: Biphasic hypoxic ventilatory response Hypoxic ventilatory depression Carotid body denervation Development Phenotypic plasticity Control of breathing
a b s t r a c t Newborn mammals exhibit a biphasic hypoxic ventilatory response (HVR), but the relative contributions of carotid body-initiated CNS mechanisms versus central hypoxia on ventilatory depression during the late phase of the HVR are not well understood. Neonatal rats (P4-5 or P13-15) were treated with a nonselective P2 purinergic receptor antagonist (pyridoxalphosphate-6-azophenyl-2 ,4 -disulfonic acid, or PPADS; 125 mg kg−1 , i.p.) to pharmacologically denervate the peripheral chemoreceptors. At P4-5, rats reared in normoxia showed a progressive decline in ventilation during a 10-min exposure to 12% O2 (21–28% decrease from baseline). No hypoxic ventilatory depression was observed in the older group of neonatal rats (i.e., P13-15), suggesting that the contribution of central hypoxia to hypoxic ventilatory depression diminishes with age. In contrast, rats reared in moderate hyperoxia (60% O2 ) from birth exhibited no hypoxic ventilatory depression at either age studied. Systemic PPADS had no effect on the ventilatory response to 7% CO2 , suggesting that the drug did not cross the blood–brain barrier. These findings indicate that (1) CNS hypoxia depresses ventilation in young, neonatal rats independent of carotid body activation and (2) hyperoxia alters the development of CNS pathways that modulate the late phase of the hypoxic ventilatory response. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Mammals generally respond to an acute hypoxic challenge by increasing ventilation. Newborn mammals exhibit a biphasic ventilatory response (HVR) in which an initial increase in ventilation (early phase) is followed by a subsequent decline in ventilation (late phase) toward or below baseline (Bissonnette, 2000; Teppema and Dahan, 2010). Adult mammals may also exhibit a biphasic HVR under isocapnic conditions, but the magnitude of the secondary ventilatory decline (or “roll-off”) is diminished such that ventilation remains elevated throughout the hypoxic exposure (Teppema and Dahan, 2010). Thus, postnatal maturation of the HVR is characterized by a shift from a strongly biphasic HVR to a more sustained HVR; this transition occurs within the second postnatal week in rats (Eden and Hanson, 1987), although the timing varies across species (Bissonnette, 2000). The early phase of the biphasic HVR reflects excitatory input from the peripheral chemoreceptors, primarily the carotid bodies. Indeed, the initial ventilatory increase is virtually abolished immediately after carotid body denervation (e.g., Martin-Body
∗ Corresponding author at: Department of Biology, Bates College, 44 Campus Avenue, Carnegie Science Hall, Lewiston, ME 04240, USA. Tel.: +1 207 786 8269; fax: +1 207 786 8334. E-mail address: [email protected] (R.W. Bavis). 1569-9048/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.resp.2013.08.017
et al., 1985; Serra et al., 2001), or after pharmacological blockade of glutamatergic signaling in the nucleus tractus solitarius (nTS) which serves as the first central synapse for carotid chemoafferent neurons (Vardhan et al., 1993). Although carotid chemoafferent input to the central nervous system (CNS) may decline somewhat during the late phase of the HVR, it is generally accepted that CNS mechanisms are critical to hypoxic ventilatory depression that begins to develop during the first few minutes of hypoxia (Bissonnette, 2000; Teppema and Dahan, 2010). These central effects could include metabolic depression of respiratory neurons (e.g., decreased ATP production; LaManna et al., 1996) and the release/accumulation of inhibitory neurochemicals (e.g., ␥-aminobutyric acid (GABA), adenosine, platelet-derived growth factor (PDGF)) (Simakajornboon and Kuptanon, 2005; Teppema and Dahan, 2010). Numerous CNS sites have been implicated in hypoxic ventilatory depression, including the medulla (particularly the nTS), pons, and midbrain structures (Bissonnette, 2000; Simakajornboon and Kuptanon, 2005; Teppema and Dahan, 2010). An unresolved question, however, is whether afferent input from the carotid bodies is necessary to initiate the central mechanisms of hypoxic ventilatory depression. On the one hand, hypoxia has been reported to depress ventilation in anesthetized animals following surgical denervation of the carotid bodies (reviewed in Teppema and Dahan, 2010); there appears to be a similar trend in conscious neonatal rats studied two days after carotid body denervation (Serra et al., 2001), but the authors did not
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report whether this was statistically significant. Similar respiratory depression has been noted in reduced preparations that lack peripheral chemoreceptors altogether (e.g., brainstem-spinal cord preparations) during in vitro hypoxic exposures (e.g., Okada et al., 1998; Khemiri et al., 2012). These observations suggest that hypoxia can act directly at the CNS to depress ventilation. However, other studies report that hypoxic ventilatory depression is abolished after carotid body denervation (Teppema and Dahan, 2010), including studies on lightly anesthetized neonatal rabbits (Schramm and Grunstein, 1987), conscious neonatal lambs (Bureau et al., 1985), and conscious adult rats (Maxová and Vízek, 2001). These observations suggest that carotid body input to the brainstem is necessary to reveal hypoxic ventilatory depression. Interestingly, rats reared in moderate hyperoxia (60% O2 ) from birth exhibit a sustained HVR at 4 and 6–7 days of age (Bavis et al., 2010; R.W. Bavis, K.J. DeAngelis, T.C. Horowitz, L.M. Reedich, and R.J. March, unpublished data), much younger than expected. Peripheral effects of developmental hyperoxia are well established and include decreased carotid body size, diminished carotid body O2 sensitivity, and degeneration of chemoafferent neurons (Bavis et al., 2013). Thus, it is unclear whether the lack of hypoxic ventilatory depression in hyperoxia-reared rats simply reflects decreased input from carotid body chemoreceptors (and consequently failure to initiate CNS mechanisms), or whether it actually reflects plasticity in the CNS mechanisms underlying the late phase of the HVR. In the present study, we tested the hypothesis that central hypoxia contributes to hypoxic ventilatory depression in neonatal rats independent of input from carotid chemoreceptors. We further hypothesized that hyperoxia-reared rats would exhibit less CNS-mediated hypoxic ventilatory depression than rats reared in room air. To isolate the respiratory effects of central hypoxia from peripheral chemoreceptor activation, we used a purinergic receptor antagonist (pyridoxalphosphate-6-azophenyl-2 ,4 -disulfonic acid; PPADS) to “pharmacologically denervate” the carotid bodies. This approach has several advantages over traditional surgical denervation. First, we were able to study conscious animals, thereby eliminating potentially confounding effects of anesthesia on the late phase of the HVR (Teppema and Dahan, 2010). Second, we were able to study rats immediately after disruption of carotid body input to the CNS; the recovery period required after surgical denervation may be sufficient for the initiation of compensatory plasticity in other peripheral chemoreceptors and/or the CNS (Martin-Body et al., 1986; Serra et al., 2001; Forster, 2003).
2. Methods 2.1. Experimental animals Experiments were conducted on neonatal Sprague-Dawley rats (SAS SD; Charles River Laboratories) of both sexes. In the initial experiments, neonatal rats were born and raised in room air until studied at 4–5 days of age (i.e., P4-5; day of birth = P0) or 14–15 days of age (P14-15). These rats were used to prepare a dose–response curve for the effects of PPADS on the hypoxic ventilatory response (HVR) or to assess the effects of systemic PPADS on the hypercapnic ventilatory response (HCVR); to ensure genetic diversity, individuals studied in these experiments were derived from 5 and 7 distinct litters, respectively. To assess the effects of developmental hyperoxia on the HVR, timed-pregnant rats (housed singly in standard cages) were placed into large (∼275 l) clear acrylic chambers 1–2 days prior to parturition. Chambers were flushed with gases at sufficient flow rates to maintain target gas levels of 21% O2 (“Control”; 12 litters) or 60% O2 (“Hyperoxia”; 6 litters) (CO2 < 0.4%). Offspring were reared with their mothers until studied at P4-5 or P13-15.
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Animals were maintained in a 12:12 light cycle with food and water ad libitum. All experimental procedures were approved by the Animal Care and Use Committee at Bates College. 2.2. Drug treatments The non-selective P2 purinergic receptor antagonist PPADS (pyridoxalphosphate-6-azophenyl-2 ,4 -disulfonic acid tetrasodium salt; Tocris Bioscience) was used to block synaptic transmission between the carotid body and its afferent neurons (Rong et al., 2003; Nurse and Piskuric, 2013). PPADS was prepared in saline (0.9% NaCl; VEDCO) and injected intraperitoneally (0.1 ml per 10 g body weight). In an initial dose–response study, rats received 0 (saline), 75, 100, 125, or 150 mg kg−1 PPADS prior to measuring the HVR to 12% O2 . Based on these data (see Section 3.1), P4-5 and P13-15 rats received 125 mg kg−1 PPADS in subsequent experiments. In the HCVR study, some rats received saline only (0.1 ml per 10 g body weight). Following injection, rat pups were placed in an incubator at 32–34 ◦ C (i.e., within the thermoneutral zone for neonatal rats; Malik and Fewell, 2003) for 10 min. Due to the time required to seal animals into the plethysmograph (approximately 5 min) and establish a calm baseline (12–16 min; see Section 2.3), the total time between injection and ventilation recordings was approximately 30 min. 2.3. Ventilation measurements Ventilation was measured using a head-body plethysmograph chamber, as previously described (Bavis et al., 2010). The chamber was separated into head and body compartments using a flexible collar; any gaps in the collar were sealed using petroleum jelly. Test gases were delivered to the head compartment at 1.5 L/min using a gas mixing mass flow controller (MFC-4; Sable Systems) and valves (series 840; Sierra Instruments). Respiratory-induced airflows into and out of the body compartment were detected using a pneumotach coupled to a differential pressure transducer (MLT1L and ML141; ADInstruments); the pneumotach was calibrated before each animal by injecting 0.5 ml of air. Respiratory airflows were recorded to a computer at a sampling rate of 1000 Hz, integrated, and digitally filtered (high-pass, 0.1 Hz) to obtain respiratory volumes (PowerLab 8SP and LabChart 7 software; ADInstruments). Air temperature in the body compartment was monitored continuously with a T-type thermocouple probe (Physitemp Instruments) and maintained at 32–34 ◦ C by placing the plethysmograph chamber inside an incubator (Precision 818; Thermo Scientific). Ten minutes after PPADS (or saline) injection, the neonatal rats were sealed into the plethysmograph chamber and allowed approximately 10 min to adjust. Once the animal appeared calm (based on stability of breathing pattern), baseline ventilation (21% O2 , 0% CO2 , balance N2 ) was recorded for 2–6 min. Rats were then exposed to either hypoxia (12% O2 , 0% CO2 , balance N2 ) or hypercapnia (21% O2 , 7% CO2 , balance N2 ) for 10 min (or 5 min for the dose–response experiment); a subset of Control rats was maintained under baseline conditions (21% O2 , 0% CO2 , balance N2 ) for this 10-min period to serve as a “time control” group. 2.4. Data analysis Ventilatory parameters were calculated for each breath and averaged over 45–60 s at the end of the baseline, excluding movement artifacts and sighs. To characterize both the early and late phases of the hypoxic and hypercapnic ventilatory responses, ventilation was calculated over 10–15 s of data 0.5, 1, and 10 min into the challenges.
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Fig. 1. Dose–response curve for the effects of intraperitoneal PPADS administration on the hypoxic ventilatory response (HVR) of neonatal rats (P4 and P14-15); all rats were reared in room air. Panels A and B show ventilation under baseline (BL) conditions and the time course for changes in ventilation during a 5-min exposure to 12% O2 ; no statistical comparisons were made with these data. In panels C and D, the change in ventilation during the early phase of the HVR (0.5 min) is expressed as a percentage of baseline to facilitate statistical comparisons across PPADS dosages. *P < 0.05 vs. saline-injected rats (i.e., 0 mg kg−1 ); † P < 0.05 vs. baseline. All values are mean ± SEM. Sample sizes are 8 at P4 and 6 at P14-15 for 0 mg kg−1 , and 3 per age for all other PPADS dosages.
Changes in ventilation (ml min−1 100 g−1 ) over time were compared between treatment groups by two-way repeated measures analysis of variance (ANOVA) [factor 1: drug (Fig. 2) or developmental O2 treatment (Figs. 3 and 4); factor 2: time] and Tukey’s post hoc tests. The HVR was also expressed as a percentage change from baseline. These values were compared between developmental O2 treatment groups or across PPADS dosages by independent samples t-tests or one-way ANOVA (followed by Dunnett’s multiple comparisons test), as appropriate, and compared to baseline (i.e., zero) by one-sample t-tests. Statistical tests were run using SigmaStat 3.11 (Systat Software) or Prism 6.00 (GraphPad Software), and P < 0.05 was considered significant. Values in the text are reported as mean ± SEM.
into the 12% O2 challenge was used to characterize the effects of PPADS on the early, carotid body-mediated phase of the HVR. There was a significant effect of PPADS in both age groups (both P < 0.001; Fig. 1C and D), and post hoc analysis revealed that the HVR was significantly reduced in rats receiving 100, 125, or 150 mg kg−1 PPADS compared to saline-injected rats (all P < 0.01). Partial inhibition of the early phase of the HVR was apparent at 75 mg kg−1 PPADS, and no significant increase in ventilation was detected at PPADS dosages ≥100 mg kg−1 relative to baseline (Fig. 1C and D). Therefore, 125 mg kg−1 PPADS was used to pharmacologically denervate the carotid body in subsequent experiments. 3.2. Effects of systemic PPADS on the hypercapnic ventilatory responses (HCVR)
3. Results 3.1. Effects of systemic PPADS on the hypoxic ventilatory response (HVR) A dose–response curve was constructed for the effects of systemic PPADS on the HVR of rats reared in room air (Fig. 1). PPADS tended to lower baseline ventilation, but this was only significant in P4 rats and only at doses >75 mg kg−1 (one-way ANOVA, P < 0.01). After saline injection (0 mg kg−1 PPADS), P4 rats exhibited the biphasic HVR typical of newborn mammals (Fig. 1A) whereas P14-15 rats exhibited a sustained increase in ventilation (Fig. 1B). The magnitude of the ventilatory increase 0.5 min
Administering PPADS directly to the brainstem greatly reduces central CO2 chemosensitivity (Thomas and Spyer, 2000) and the HCVR (da Silva et al., 2012). Therefore, the HCVR was assessed as an indirect test of whether PPADS penetrates the blood–brain barrier following systemic (intraperitoneal) injections. Ventilation was measured in P4-5 and P13-14 rats (all reared in room air) while they breathed 0 and 7% CO2 (Fig. 2). PPADS tended to decrease baseline respiratory frequency relative to that observed in salineinjected rats (main effect for treatment, P < 0.001 and 0.009 for P4-5 and P13-14, respectively), but the resulting decrease in minute ventilation was only significant at P13-14 (main effect for treatment, P = 0.03) (Fig. 2F). Despite this effect on baseline ventilation,
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Fig. 2. Hypercapnic ventilatory responses for neonatal rats (P4-5 and P13-14) after intraperitoneal administration of PPADS (125 mg kg−1 ) or saline; all rats were reared in room air. Respiratory frequency (A, B), tidal volume (C, D), and minute ventilation (E, F) are reported under baseline (“BL”) conditions and during a 10-min exposure to 7% CO2 . Sample sizes are 11 per group at P4-5 and 9 per group at P13-14. Values are mean ± SEM. *Main effect for drug treatment (P < 0.05).
systemic PPADS injection had no significant effect on the ventilatory response to 7% CO2 in either age group (drug × inspired CO2 level, P = 0.85 and 0.36, respectively) (Fig. 2E and F). Likewise, respiratory frequency and tidal volume responses to hypercapnia were similar between saline- and PPADS-injected rats (drug × inspired CO2 level, all P > 0.05) (Fig. 2A–D). 3.3. Effects of developmental hyperoxia on the late phase of the HVR Ventilatory responses to 12% O2 were measured for Control and Hyperoxia rats after systemic administration of PPADS. In the P4-5 age group, there was a significant interaction between developmental O2 treatment and time for ventilation during the hypoxic exposure (treatment × time, P < 0.001) (Fig. 3C). Hyperoxia rats exhibited lower baseline ventilation than Control rats due to lower respiratory frequencies (Fig. 3A) and tidal volumes (Fig. 3B), as previously reported (Bavis et al., 2010). Neither treatment group increased ventilation in the first minute of hypoxia, suggesting that PPADS abolished the early phase of the HVR. Instead, ventilatory
depression was evident within the first minute of hypoxia in Control rats and became more pronounced throughout the hypoxic exposure (Fig. 3C); ventilation was reduced by 28% in Control rats by the 10th minute of hypoxia (P < 0.001 vs. baseline) (Fig. 3D). In contrast, no ventilatory depression was observed in Hyperoxia rats at P4-5 (Fig. 3C and D). Consequently, ventilatory depression was significantly greater in Control rats than in Hyperoxia rats at P4-5 (P < 0.001) (Fig. 3D). Hypoxic ventilatory depression in the P4-5 Control rats reflected time-dependent decreases in respiratory frequency and tidal volume. For respiratory frequency, there was an interaction between developmental O2 treatment and time (treatment × time, P < 0.001), and post hoc analysis revealed a significant decrease in frequency by the 10th minute of hypoxia in the Control group only P < 0.001 vs. baseline (Fig. 3A). Although the interaction term for tidal volume was not quite significant (treatment × time, P = 0.08), visual inspection of the data suggests that the significant main effect for time (P = 0.003) is driven by diminished tidal volumes of Control rats by the 10th minute of hypoxia (Fig. 3B); indeed, the mean tidal volume was identical for Hyperoxia rats at baseline
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Fig. 3. Hypoxic ventilatory response (HVR) for P4-5 rats reared in 21% O2 (Control) or 60% O2 (Hyperoxia) from birth; all rats received 125 mg kg−1 PPADS (i.p.) prior to ventilation measurements. Respiratory frequency (A), tidal volume (B), and minute ventilation (C) are reported under baseline (“BL”) conditions and during a 10-min exposure to 12% O2 . In panel D, ventilation is expressed as the percentage change from baseline during the 10th minute of hypoxia. Values are mean ± SEM. Sample sizes are 18 Control and 18 Hyperoxia. *P < 0.05 vs. Control within the same time period; † P < 0.05 vs. baseline (BL or 0%) within the same treatment group. No treatment × time interaction was detected for tidal volume (panel B), so the symbols (with brackets) denote a significant difference between treatment groups when pooled across all time points (i.e., main effect for treatment) (*) or a significant difference from baseline when pooled across treatment groups (i.e., main effect for time, followed by post hoc tests) (†).
and in the 10th minute of hypoxia (0.48 ± 0.02 and 0.48 ± 0.01 ml 100 g−1 , respectively). An additional group of P4-5 Control rats (n = 14) was injected with PPADS but was maintained in 21% O2 throughout the ventilation protocol (i.e., no hypoxia). Ventilation did not change significantly over the course of the protocol in these “time control” rats (−3.1 ± 4.4% change from baseline; one-sample t-test, P = 0.49). Therefore, changes in ventilation in P4-5 Control rats (Fig. 1) represents hypoxic ventilatory depression, not time-dependent effects of PPADS or drift in ventilation measurements. The HVR was also assessed in Control and Hyperoxia rats at P1315; an additional group of P4 Control rats was studied alongside the P13-15 rats to enable quantitative comparisons between age groups. Again there was a significant interaction between treatment group and time for ventilation during the hypoxic exposure (treatment × time, P < 0.001), but this was driven by differences between P4 and P13-15 rats (Fig. 4C). Specifically, baseline ventilation was significantly lower in P13-15 Control rats than in P4 Control rats (P < 0.001), but no differences were detected between P13-15 Control and P13-15 Hyperoxia rats (P = 0.46). No increase in ventilation was evident in the first minute of hypoxia in any group. As in the earlier round of experiments, hypoxic ventilatory depression developed in P4 Control rats (Fig. 4C and D), with ventilation decreasing by 21% by the 10th minute of hypoxia (P = 0.01 vs. baseline) (Fig. 4D). However, hypoxia did not cause ventilatory depression in either the Control or Hyperoxia groups at P13-15 (Fig. 4C and D). Instead, ventilation tended to increase progressively during the hypoxic exposure in P13-15 rats, although this only reached statistical significance in the Hyperoxia group by
the 10th minute of hypoxia (Control, 13% increase from baseline, P = 0.07; Hyperoxia, 23% increase, P < 0.001) (Fig. 4D). Accordingly, the magnitude of the hypoxic ventilatory depression decreased significantly with age in Control rats (P < 0.01) (Fig. 4D). The progressive increase in hypoxic ventilation in P13-15 Hyperoxia rats, was associated with an increase in respiratory frequency (P = 0.009 for the 10th minute of hypoxia vs. baseline) (Fig. 4A); a similar trend was observed in P13-15 Control rats, but this was not significant (P = 0.11). Tidal volume did not change significantly over time in any group (main effect for time, P = 0.99) (Fig. 4B).
4. Discussion Hypoxic ventilatory depression was observed in young neonatal rats (P4-5) after pharmacological denervation of the carotid bodies with the P2 purinergic receptor antagonist PPADS. This finding suggests that the late phase of the biphasic HVR in conscious neonatal rats results, at least in part, from direct actions of hypoxia on respiratory neurons in the CNS. In contrast, rats reared in 60% O2 from birth lacked any measurable hypoxic ventilatory depression after systemic PPADS administration. This is consistent with the absence of a biphasic HVR in neonatal, hyperoxia-reared rats (Bavis et al., 2010; R.W. Bavis, K.J. DeAngelis, T.C. Horowitz, L.M. Reedich, and R.J. March, unpublished data). Thus, plasticity in the CNS regions governing hypoxic ventilatory depression likely explains the premature appearance of a sustained HVR after developmental hyperoxia.
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Fig. 4. Hypoxic ventilatory response (HVR) for P13-15 rats reared in 21% O2 (Control) or 60% O2 (Hyperoxia) from birth; all rats received 125 mg kg−1 PPADS (i.p.) prior to ventilation measurements. Sample sizes (n) are 16 Control and 16 Hyperoxia at P13-15; an additional group of P4 Control rats (n = 8) was studied alongside the P13-15 rats to facilitate quantitative comparisons between age groups. Respiratory frequency (A), tidal volume (B), and minute ventilation (C) are reported under baseline (“BL”) conditions and during a 10-min exposure to 12% O2 . In panel D, ventilation is expressed as the percentage change from baseline during the 10th minute of hypoxia. Values are mean ± SEM. † P < 0.05 vs. baseline (BL or 0%) within the same treatment group; # P < 0.05 vs. P13-15 (Control group only). No treatment × time interaction was detected for tidal volume (panel B), so # (with bracket) denotes a significant difference between P4 Control and P13-15 Control rats when pooled across all time points (i.e., main effect for treatment group, followed by post hoc tests).
4.1. Critique of methods Pharmacological blockade has many advantages to surgical denervation of the carotid bodies, most notably the ability to assess ventilation in conscious rats immediately after “denervation”. However, the validity of this approach depends on the capacity of the selected drug to block peripheral chemoreceptors without directly targeting respiratory neurons in the CNS. Several lines of evidence support these assumptions. First, the PPADS dosage in the present study effectively abolished the early, carotid bodymediated phase of the HVR (Figs. 1, 3 and 4), consistent with its blockade of synaptic signaling between carotid body glomus cells and their afferent neurons (Rong et al., 2003; Nurse and Piskuric, 2013). Although some increase in ventilation was detected during the late phase of the HVR in older neonates (i.e., P13-15), the slow onset of this response argues against a contribution from the carotid bodies; indeed, a small residual ventilatory increase is also common after surgical denervation of peripheral chemoreceptors and likely represents activation of central O2 sensors (e.g., MartinBody et al., 1985). Furthermore, PPADS also tended to decrease baseline ventilation (e.g., Figs. 1 and 2), consistent with the withdrawal of the peripheral contribution to eupneic ventilatory drive (e.g., Dejours, 1963; Teppema and Dahan, 2010). Finally, PPADS should not penetrate the blood–brain barrier given its large, polar structure (Pardridge, 2005). This appears to have been confirmed in the present study since systemic administration of PPADS had no effect on the hypercapnic ventilatory response (HCVR): if PPADS had crossed the blood–brain barrier, it would have attenuated the
HCVR (Thomas and Spyer, 2000; da Silva et al., 2012). In retrospect, it is somewhat surprising that systemic PPADS had no measurable effect on the HCVR since input from carotid chemoreceptors is proposed to modulate central CO2 chemosensitivity in adult mammals (Smith et al., 2010); the carotid bodies apparently contribute very little (if at all) to the HCVR of neonatal rats. In order to characterize the early phase of the HVR, high gas flow rates were necessary to ensure a rapid transition between normoxia and hypoxia. This approach precluded simultaneous measurement of metabolic O2 consumption and CO2 production which are important determinants of respiratory drive. Neonatal rats, like other animals, tend to decrease aerobic metabolism in response to hypoxia (e.g., Bavis et al., 2010; for review, see also Mortola, 2004; Teppema and Dahan, 2010), and it is possible that this hypoxic hypometabolism contributes to decreased ventilation during the late phase of the HVR of neonatal rats. However, there is strong evidence that neural mechanisms, rather than (or in addition to) changes in whole-body metabolism, are critical to the origin of hypoxic ventilatory depression. This evidence includes the rapid onset of hypoxic ventilatory depression in peripherally chemodenervated animals (e.g., ≤1 min in PPADS-injected P4-5 rats) and the repeated demonstration that pharmacological and/or physical disruption of specific groups of respiratory neurons can block hypoxic ventilatory depression in neonatal and adult mammals (reviewed in Bissonnette, 2000; Teppema and Dahan, 2010). Although the metabolic effects of systemic PPADS administration have not been assessed directly, experiments using other purinergic receptor antagonists indicate that it is unlikely that this drug
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would increase the metabolic reduction during hypoxia (and, thus, enhance hypoxic ventilatory depression). Indeed, systemic administration of other P2X receptor antagonists (suramin or A-317491) decreases resting metabolism in neonatal rats (Niane et al., 2011) but causes, if anything, a modest reduction in the magnitude of the hypoxic metabolic response (L.M. Niane and A. Bairam, personal communication). Moreover, developmental hyperoxia does not alter the magnitude of the metabolic response to hypoxia in neonatal rats at any of the ages studied (i.e., P4–P14) (Bavis et al., 2010). Therefore, it is unlikely that differences in whole-body metabolism explain the differences in hypoxic ventilatory depression observed between treatment groups in the present study. 4.2. Potential mechanisms for age- and hyperoxia-induced changes in hypoxic ventilatory depression Hypoxic ventilatory depression after systemic PPADS is consistent with many previous studies that observed hypoxic ventilatory depression in anesthetized animals after surgical denervation of the carotid bodies (reviewed in Teppema and Dahan, 2010), but contradicts other studies of conscious or lightly anesthetized mammals (e.g., Bureau et al., 1985; Schramm and Grunstein, 1987; Maxová and Vízek, 2001). The HVR was assessed in conscious rats in the present study, so anesthesia cannot explain the discrepancy between this and the latter studies. Rather, it seems likely that these differences reflect the relative maturity of the study animals. Indeed, the magnitude of the hypoxic ventilatory depression decreased with age in rats (Fig. 4D; also compare the 0 mg kg−1 PPADS dosages in Fig. 1A and B). This may explain why hypoxic ventilatory depression was not observed in adult rats (Maxová and Vízek, 2001) or relatively precocial lambs (Bureau et al., 1985) after carotid body denervation. Likewise, Schramm and Grunstein (1987) observed that the magnitude of hypoxic ventilatory depression diminished greatly over the first two postnatal weeks in neonatal rabbits, but they only studied the effects of carotid body denervation on hypoxic ventilatory depression in older neonates (i.e., 18–29 days of age). Importantly, the finding that hypoxia directly contributes to ventilatory depression does not preclude additional mechanisms that depend on carotid body activation (Simakajornboon and Kuptanon, 2005; Teppema and Dahan, 2010). We speculate that carotid body-dependent mechanisms contribute some portion of the hypoxic ventilatory depression (or roll-off) observed in intact animals, and that the relative contribution of these mechanisms increases during postnatal maturation (as the contribution from CNS hypoxia diminishes). Although these findings indicate that hypoxia acts directly on the CNS to initiate ventilatory depression (at least in the youngest neonates), the specific mechanisms underlying this response are not fully understood. Many neurons in the CNS are inhibited by cellular hypoxia, including subsets of neurons in respiratory regions such as the rostral ventrolateral medulla, pre-Botzinger complex, and nTS (reviewed in Teppema and Dahan, 2010). Alternatively, hypoxia could cause O2 -sensitive cells to release inhibitory neuromodulators or neurotransmitters, or to recruit postsynaptic neurons that inhibit breathing. Numerous neurochemicals have been implicated in hypoxic ventilatory depression, including GABA, adenosine, PDGF, substance P, and various catecholamines (Bissonnette, 2000; Teppema and Dahan, 2010). Unfortunately, few experiments have considered whether these molecules are synthesized and/or released in hypoxia independent of carotid chemoafferent activation. The question has been asked for GABA, but this does not seem to be the case: carotid body denervation prevents the rise in extracellular GABA that normally occurs in the nTS of rats during hypoxia (Tabata et al., 2001). There is indirect evidence, however, that hypoxia induces PDGF signaling without input from peripheral chemoreceptors. Specifically, hypoxia influences
synaptic transmission between nTS neurons differently in brainstem slices from wild type and PDGF- receptor gene-knockout mice (Ohi et al., 2010); although PDGF release was not assessed in that study, the results suggest that hypoxia directly modulates PDGF signaling pathways in the brainstem. The expression of the PDGF-B chain, the PDGF subunit which binds with high affinity to the PDGF- receptor, does increase in nTS neurons during hypoxia, and the activation of the PDGF- receptors contributes to hypoxic ventilatory depression in intact rats (Gozal et al., 2000; Vlasic et al., 2001). Interestingly, recent work in our laboratory indicates that the late phase of the HVR is less sensitive to pharmacological blockade of PDGF- receptors in neonatal rats reared in hyperoxia compared to age-matched controls (R.W. Bavis, K.J. DeAngelis, T.C. Horowitz, L.M. Reedich, and R.J. March, unpublished data); thus, changes in PDGF signaling could contribute to the absence of hypoxic ventilatory depression in hyperoxia-reared rats after functional denervation of the carotid bodies in the present study. The actions of inhibitory neurochemicals in the brainstem during the late phase of the HVR are opposed by the release of excitatory neurochemicals, and the postnatal maturation of the biphasic HVR is thought to reflect the shifting balance between these inhibitory and excitatory influences (Bissonnette, 2000; Simakajornboon and Kuptanon, 2005; Teppema and Dahan, 2010). The release of glutamate in the nTS requires activation of the carotid body chemoreceptors (Mizusawa et al., 1994), but hypoxia initiates the release of ATP throughout the ventral medulla in anesthetized, peripherally chemodenervated adult rats (Gourine et al., 2005). Importantly, the endogenous release of ATP at or near respiratory neurons in the ventrolateral medulla (VLM) helps to maintain respiratory motor output during hypoxia (Gourine et al., 2005). Although it is possible that extracellular ATP is converted to adenosine in the nTS and VLM, the relatively slow accumulation of adenosine does not appear to correlate well with the rapid onset of hypoxic ventilatory depression in adult rats (Gourine et al., 2002, 2005). Thus, ATP release during CNS hypoxia is unlikely to explain the observed hypoxic ventilatory depression in room-air reared rats after pharmacological denervation of the carotid bodies. However, enhanced ATP release during CNS hypoxia could contribute to the age-dependent decrease in hypoxic ventilatory depression during normal postnatal maturation and/or to the sustained HVR observed in young, hyperoxia-reared rats. Additional studies are clearly needed to understand the potential roles for this and other CNS mechanisms in developmental plasticity of the HVR. Acknowledgments This study was supported in part by the Bates Student Research Fund. The authors would like to thank Sarah Logan and Halward J. Blegen for assisting with data analysis, and to thank Lalah M. Niane and Dr. Aida Bairam for sharing unpublished data. References Bavis, R.W., Young, K.M., Barry, K.J., Boller, M.R., Kim, E., Klein, P.M., Ovrutsky, A.R., Rampersad, D.A., 2010. Chronic hyperoxia alters the early and late phases of the hypoxic ventilatory response in neonatal rats. J. Appl. Physiol. 109, 796–803. Bavis, R.W., Fallon, S.C., Dmitrieff, E.F., 2013. Chronic hyperoxia and the development of the carotid body. Respir. Physiol. Neurobiol. 185, 94–104. Bissonnette, J.M., 2000. Mechanisms regulating hypoxic respiratory depression during fetal and postnatal life. Am. J. Physiol. Regul. Integr. Comp. Physiol. 278, R1391–R1400. Bureau, M.A., Lamarche, J., Foulon, P., Dalle, D., 1985. The ventilatory response to hypoxia in the newborn lamb after carotid body denervation. Respir. Physiol. 60, 109–119. da Silva, G.S., Moraes, D.J., Giusti, H., Dias, M.B., Glass, M.L., 2012. Purinergic transmission in the rostral but not caudal medullary raphe contributes to the hypercapnia-induced ventilatory response in unanesthetized rats. Respir. Physiol. Neurobiol. 184, 41–47. Dejours, P., 1963. Control of respiration by arterial chemoreceptors. Ann. N. Y. Acad. Sci. 109, 682–695.
C.B. Hill et al. / Respiratory Physiology & Neurobiology 189 (2013) 498–505 Eden, G.J., Hanson, M.A., 1987. Maturation of the respiratory response to acute hypoxia in the newborn rat. J. Physiol. 392, 1–9. Forster, H.V., 2003. Plasticity in the control of breathing following sensory denervation. J. Appl. Physiol. 94, 784–794. Gourine, A.V., Llaudet, E., Thomas, T., Dale, N., Spyer, K.M., 2002. Adenosine release in nucleus tractus solitarii does not appear to mediate hypoxia-induced respiratory depression in rats. J. Physiol. 544, 161-107. Gourine, A.V., Llaudet, E., Dale, N., Spyer, K.M., 2005. Release of ATP in the ventral medulla during hypoxia in rats: role in the hypoxic ventilatory response. J. Neurosci. 25, 1211–1218. Gozal, D., Simakajornboon, N., Czapla, M.A., Xue, Y.D., Gozal, E., Vlasic, V., Lasky, J.A., Liu, J.Y., 2000. Brainstem activation of platelet-derived growth factor- receptor modulates the late phase of the hypoxic ventilatory response. J. Neurochem. 74, 310–319. Khemiri, H., Seaborn, T., Gestreau, C., Soliz, J., 2012. Erythropoietin and its antagonist regulate hypoxic fictive breathing in newborn mice. Respir. Physiol. Neurobiol. 183, 115–121. LaManna, J.C., Haxhiu, M.A., Kutina-Nelson, K.L., Pundik, S., Erokwu, B., Yeh, E.R., Lust, W.D., Cherniack, N.S., 1996. Decreased energy metabolism in brain stem during central respiratory depression in response to hypoxia. J. Appl. Physiol. 81, 1772–1777. Malik, S.S., Fewell, J.E., 2003. Thermoregulation in rats during early postnatal maturation: importance of nitric oxide. Am. J. Physiol. Regul. Integr. Comp. Physiol. 285, R1366–R1372. Martin-Body, R.L., Robson, G.J., Sinclair, J.D., 1985. Respiratory effects of sectioning the carotid sinus glossopharyngeal and abdominal vagal nerves in the awake rat. J. Physiol. 361, 35–45. Martin-Body, R.L., Robson, G.J., Sinclair, J.D., 1986. Restoration of hypoxic respiratory responses in the awake rat after carotid body denervation by sinus nerve section. J. Physiol. 380, 61–73. Maxová, H., Vízek, M., 2001. Ventilatory response to sustained hypoxia in carotid body denervated rats. Physiol. Res. 50, 327–331. Mizusawa, A., Ogawa, H., Kikuchi, Y., Hida, W., Kurosawa, H., Okabe, S., Takishima, T., Shirato, K., 1994. In vivo release of glutamate in nucleus tractus solitarii of the rat during hypoxia. J. Physiol. 478, 55–66. Mortola, J.P., 2004. Implications of hypoxic hypometabolism during mammalian ontogenesis. Respir. Physiol. Neurobiol. 141, 345–356. Niane, L.M., Donnelly, D.F., Joseph, V., Bairam, A., 2011. Ventilatory and carotid body chemoreceptor responses to purinergic P2X receptor antagonists in newborn rats. J. Appl. Physiol. 110, 83–94.
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Nurse, C.A., Piskuric, N.A., 2013. Signal processing at mammalian carotid body chemoreceptors. Semin. Cell Dev. Biol. 24, 22–30. Ohi, Y., Ishii, Y., Sasahara, M., Haji, A., 2010. Involvement of platelet-derived growth factor-BB and its receptor-ˇ in hypoxia-induced depression of excitatory synaptic transmission in the nucleus tractus solitarius of mice. J. Pharmacol. Sci. 112, 477–481. Okada, Y., Kawai, A., Mückenhoff, K., Scheid, P., 1998. Role of the pons in hypoxic respiratory depression in the neonatal rat. Respir. Physiol. 111, 55–63. Pardridge, W.M., 2005. The blood–brain barrier: bottleneck in brain drug development. NeuroRx 2, 3–14. Rong, W., Gourine, A.V., Cockayne, D.A., Xiang, Z., Ford, A.P.D.W., Spyer, K.M., Burnstock, G., 2003. Pivotal role of nucleotide P2X2 receptor subunit of the ATPgated ion channel mediating ventilatory responses to hypoxia. J. Neurosci. 23, 11315–11321. 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. Schramm, C.M., Grunstein, M.M., 1987. Respiratory influence of peripheral chemoreceptor stimulation in maturing rabbits. J. Appl. Physiol. 63, 1671–1680. Simakajornboon, N., Kuptanon, T., 2005. Maturational changes in neuromodulation of central pathways underlying hypoxic ventilatory response. Respir. Physiol. Neurobiol. 149, 273–286. Smith, C.A., Forster, H.V., Blain, G.M., Dempsey, J.A., 2010. An interdependent model of central/peripheral chemoreception: evidence and implications for ventilatory control. Respir. Physiol. Neurobiol. 173, 288–297. Tabata, M., Kurosawa, H., Kikuchi, Y., Hida, W., Ogawa, H., Okabe, S., Tun, Y., Hattori, T., Shirato, K., 2001. Role of GABA within the nucleus tractus solitarii in the hypoxic ventilatory decline of awake rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, R1411–R1419. Teppema, L.J., Dahan, A., 2010. The ventilatory response to hypoxia in mammals: mechanisms, measurement, and analysis. Physiol. Rev. 90, 675–754. Thomas, T., Spyer, K.M., 2000. ATP as a mediator of mammalian central CO2 chemoreception. J. Physiol. 523, 441–447. Vardhan, A., Kachroo, A., Sapru, H.N., 1993. Excitatory amino acid receptors in commissural nucleus of the NTS mediate carotid chemoreceptor responses. Am. J. Physiol. 264, R41–R50. Vlasic, V., Simakajornboon, N., Gozal, E., Gozal, D., 2001. PDGF- receptor expression in the dorsocaudal brainstem parallels hypoxic ventilatory depression in the developing rat. Pediatr. Res. 50, 236–241.
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Developmental hyperoxia alters CNS mechanisms underlying hypoxic ventilatory depression in neonatal rats Corey B. Hill, Samuel H. Grandgeorge, Ryan W. Bavis ∗ Department of Biology, Bates College, Lewiston, ME 04240, USA
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Article history: Accepted 22 August 2013 Keywords: Biphasic hypoxic ventilatory response Hypoxic ventilatory depression Carotid body denervation Development Phenotypic plasticity Control of breathing
a b s t r a c t Newborn mammals exhibit a biphasic hypoxic ventilatory response (HVR), but the relative contributions of carotid body-initiated CNS mechanisms versus central hypoxia on ventilatory depression during the late phase of the HVR are not well understood. Neonatal rats (P4-5 or P13-15) were treated with a nonselective P2 purinergic receptor antagonist (pyridoxalphosphate-6-azophenyl-2 ,4 -disulfonic acid, or PPADS; 125 mg kg−1 , i.p.) to pharmacologically denervate the peripheral chemoreceptors. At P4-5, rats reared in normoxia showed a progressive decline in ventilation during a 10-min exposure to 12% O2 (21–28% decrease from baseline). No hypoxic ventilatory depression was observed in the older group of neonatal rats (i.e., P13-15), suggesting that the contribution of central hypoxia to hypoxic ventilatory depression diminishes with age. In contrast, rats reared in moderate hyperoxia (60% O2 ) from birth exhibited no hypoxic ventilatory depression at either age studied. Systemic PPADS had no effect on the ventilatory response to 7% CO2 , suggesting that the drug did not cross the blood–brain barrier. These findings indicate that (1) CNS hypoxia depresses ventilation in young, neonatal rats independent of carotid body activation and (2) hyperoxia alters the development of CNS pathways that modulate the late phase of the hypoxic ventilatory response. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Mammals generally respond to an acute hypoxic challenge by increasing ventilation. Newborn mammals exhibit a biphasic ventilatory response (HVR) in which an initial increase in ventilation (early phase) is followed by a subsequent decline in ventilation (late phase) toward or below baseline (Bissonnette, 2000; Teppema and Dahan, 2010). Adult mammals may also exhibit a biphasic HVR under isocapnic conditions, but the magnitude of the secondary ventilatory decline (or “roll-off”) is diminished such that ventilation remains elevated throughout the hypoxic exposure (Teppema and Dahan, 2010). Thus, postnatal maturation of the HVR is characterized by a shift from a strongly biphasic HVR to a more sustained HVR; this transition occurs within the second postnatal week in rats (Eden and Hanson, 1987), although the timing varies across species (Bissonnette, 2000). The early phase of the biphasic HVR reflects excitatory input from the peripheral chemoreceptors, primarily the carotid bodies. Indeed, the initial ventilatory increase is virtually abolished immediately after carotid body denervation (e.g., Martin-Body
∗ Corresponding author at: Department of Biology, Bates College, 44 Campus Avenue, Carnegie Science Hall, Lewiston, ME 04240, USA. Tel.: +1 207 786 8269; fax: +1 207 786 8334. E-mail address: [email protected] (R.W. Bavis). 1569-9048/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.resp.2013.08.017
et al., 1985; Serra et al., 2001), or after pharmacological blockade of glutamatergic signaling in the nucleus tractus solitarius (nTS) which serves as the first central synapse for carotid chemoafferent neurons (Vardhan et al., 1993). Although carotid chemoafferent input to the central nervous system (CNS) may decline somewhat during the late phase of the HVR, it is generally accepted that CNS mechanisms are critical to hypoxic ventilatory depression that begins to develop during the first few minutes of hypoxia (Bissonnette, 2000; Teppema and Dahan, 2010). These central effects could include metabolic depression of respiratory neurons (e.g., decreased ATP production; LaManna et al., 1996) and the release/accumulation of inhibitory neurochemicals (e.g., ␥-aminobutyric acid (GABA), adenosine, platelet-derived growth factor (PDGF)) (Simakajornboon and Kuptanon, 2005; Teppema and Dahan, 2010). Numerous CNS sites have been implicated in hypoxic ventilatory depression, including the medulla (particularly the nTS), pons, and midbrain structures (Bissonnette, 2000; Simakajornboon and Kuptanon, 2005; Teppema and Dahan, 2010). An unresolved question, however, is whether afferent input from the carotid bodies is necessary to initiate the central mechanisms of hypoxic ventilatory depression. On the one hand, hypoxia has been reported to depress ventilation in anesthetized animals following surgical denervation of the carotid bodies (reviewed in Teppema and Dahan, 2010); there appears to be a similar trend in conscious neonatal rats studied two days after carotid body denervation (Serra et al., 2001), but the authors did not
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report whether this was statistically significant. Similar respiratory depression has been noted in reduced preparations that lack peripheral chemoreceptors altogether (e.g., brainstem-spinal cord preparations) during in vitro hypoxic exposures (e.g., Okada et al., 1998; Khemiri et al., 2012). These observations suggest that hypoxia can act directly at the CNS to depress ventilation. However, other studies report that hypoxic ventilatory depression is abolished after carotid body denervation (Teppema and Dahan, 2010), including studies on lightly anesthetized neonatal rabbits (Schramm and Grunstein, 1987), conscious neonatal lambs (Bureau et al., 1985), and conscious adult rats (Maxová and Vízek, 2001). These observations suggest that carotid body input to the brainstem is necessary to reveal hypoxic ventilatory depression. Interestingly, rats reared in moderate hyperoxia (60% O2 ) from birth exhibit a sustained HVR at 4 and 6–7 days of age (Bavis et al., 2010; R.W. Bavis, K.J. DeAngelis, T.C. Horowitz, L.M. Reedich, and R.J. March, unpublished data), much younger than expected. Peripheral effects of developmental hyperoxia are well established and include decreased carotid body size, diminished carotid body O2 sensitivity, and degeneration of chemoafferent neurons (Bavis et al., 2013). Thus, it is unclear whether the lack of hypoxic ventilatory depression in hyperoxia-reared rats simply reflects decreased input from carotid body chemoreceptors (and consequently failure to initiate CNS mechanisms), or whether it actually reflects plasticity in the CNS mechanisms underlying the late phase of the HVR. In the present study, we tested the hypothesis that central hypoxia contributes to hypoxic ventilatory depression in neonatal rats independent of input from carotid chemoreceptors. We further hypothesized that hyperoxia-reared rats would exhibit less CNS-mediated hypoxic ventilatory depression than rats reared in room air. To isolate the respiratory effects of central hypoxia from peripheral chemoreceptor activation, we used a purinergic receptor antagonist (pyridoxalphosphate-6-azophenyl-2 ,4 -disulfonic acid; PPADS) to “pharmacologically denervate” the carotid bodies. This approach has several advantages over traditional surgical denervation. First, we were able to study conscious animals, thereby eliminating potentially confounding effects of anesthesia on the late phase of the HVR (Teppema and Dahan, 2010). Second, we were able to study rats immediately after disruption of carotid body input to the CNS; the recovery period required after surgical denervation may be sufficient for the initiation of compensatory plasticity in other peripheral chemoreceptors and/or the CNS (Martin-Body et al., 1986; Serra et al., 2001; Forster, 2003).
2. Methods 2.1. Experimental animals Experiments were conducted on neonatal Sprague-Dawley rats (SAS SD; Charles River Laboratories) of both sexes. In the initial experiments, neonatal rats were born and raised in room air until studied at 4–5 days of age (i.e., P4-5; day of birth = P0) or 14–15 days of age (P14-15). These rats were used to prepare a dose–response curve for the effects of PPADS on the hypoxic ventilatory response (HVR) or to assess the effects of systemic PPADS on the hypercapnic ventilatory response (HCVR); to ensure genetic diversity, individuals studied in these experiments were derived from 5 and 7 distinct litters, respectively. To assess the effects of developmental hyperoxia on the HVR, timed-pregnant rats (housed singly in standard cages) were placed into large (∼275 l) clear acrylic chambers 1–2 days prior to parturition. Chambers were flushed with gases at sufficient flow rates to maintain target gas levels of 21% O2 (“Control”; 12 litters) or 60% O2 (“Hyperoxia”; 6 litters) (CO2 < 0.4%). Offspring were reared with their mothers until studied at P4-5 or P13-15.
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Animals were maintained in a 12:12 light cycle with food and water ad libitum. All experimental procedures were approved by the Animal Care and Use Committee at Bates College. 2.2. Drug treatments The non-selective P2 purinergic receptor antagonist PPADS (pyridoxalphosphate-6-azophenyl-2 ,4 -disulfonic acid tetrasodium salt; Tocris Bioscience) was used to block synaptic transmission between the carotid body and its afferent neurons (Rong et al., 2003; Nurse and Piskuric, 2013). PPADS was prepared in saline (0.9% NaCl; VEDCO) and injected intraperitoneally (0.1 ml per 10 g body weight). In an initial dose–response study, rats received 0 (saline), 75, 100, 125, or 150 mg kg−1 PPADS prior to measuring the HVR to 12% O2 . Based on these data (see Section 3.1), P4-5 and P13-15 rats received 125 mg kg−1 PPADS in subsequent experiments. In the HCVR study, some rats received saline only (0.1 ml per 10 g body weight). Following injection, rat pups were placed in an incubator at 32–34 ◦ C (i.e., within the thermoneutral zone for neonatal rats; Malik and Fewell, 2003) for 10 min. Due to the time required to seal animals into the plethysmograph (approximately 5 min) and establish a calm baseline (12–16 min; see Section 2.3), the total time between injection and ventilation recordings was approximately 30 min. 2.3. Ventilation measurements Ventilation was measured using a head-body plethysmograph chamber, as previously described (Bavis et al., 2010). The chamber was separated into head and body compartments using a flexible collar; any gaps in the collar were sealed using petroleum jelly. Test gases were delivered to the head compartment at 1.5 L/min using a gas mixing mass flow controller (MFC-4; Sable Systems) and valves (series 840; Sierra Instruments). Respiratory-induced airflows into and out of the body compartment were detected using a pneumotach coupled to a differential pressure transducer (MLT1L and ML141; ADInstruments); the pneumotach was calibrated before each animal by injecting 0.5 ml of air. Respiratory airflows were recorded to a computer at a sampling rate of 1000 Hz, integrated, and digitally filtered (high-pass, 0.1 Hz) to obtain respiratory volumes (PowerLab 8SP and LabChart 7 software; ADInstruments). Air temperature in the body compartment was monitored continuously with a T-type thermocouple probe (Physitemp Instruments) and maintained at 32–34 ◦ C by placing the plethysmograph chamber inside an incubator (Precision 818; Thermo Scientific). Ten minutes after PPADS (or saline) injection, the neonatal rats were sealed into the plethysmograph chamber and allowed approximately 10 min to adjust. Once the animal appeared calm (based on stability of breathing pattern), baseline ventilation (21% O2 , 0% CO2 , balance N2 ) was recorded for 2–6 min. Rats were then exposed to either hypoxia (12% O2 , 0% CO2 , balance N2 ) or hypercapnia (21% O2 , 7% CO2 , balance N2 ) for 10 min (or 5 min for the dose–response experiment); a subset of Control rats was maintained under baseline conditions (21% O2 , 0% CO2 , balance N2 ) for this 10-min period to serve as a “time control” group. 2.4. Data analysis Ventilatory parameters were calculated for each breath and averaged over 45–60 s at the end of the baseline, excluding movement artifacts and sighs. To characterize both the early and late phases of the hypoxic and hypercapnic ventilatory responses, ventilation was calculated over 10–15 s of data 0.5, 1, and 10 min into the challenges.
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Fig. 1. Dose–response curve for the effects of intraperitoneal PPADS administration on the hypoxic ventilatory response (HVR) of neonatal rats (P4 and P14-15); all rats were reared in room air. Panels A and B show ventilation under baseline (BL) conditions and the time course for changes in ventilation during a 5-min exposure to 12% O2 ; no statistical comparisons were made with these data. In panels C and D, the change in ventilation during the early phase of the HVR (0.5 min) is expressed as a percentage of baseline to facilitate statistical comparisons across PPADS dosages. *P < 0.05 vs. saline-injected rats (i.e., 0 mg kg−1 ); † P < 0.05 vs. baseline. All values are mean ± SEM. Sample sizes are 8 at P4 and 6 at P14-15 for 0 mg kg−1 , and 3 per age for all other PPADS dosages.
Changes in ventilation (ml min−1 100 g−1 ) over time were compared between treatment groups by two-way repeated measures analysis of variance (ANOVA) [factor 1: drug (Fig. 2) or developmental O2 treatment (Figs. 3 and 4); factor 2: time] and Tukey’s post hoc tests. The HVR was also expressed as a percentage change from baseline. These values were compared between developmental O2 treatment groups or across PPADS dosages by independent samples t-tests or one-way ANOVA (followed by Dunnett’s multiple comparisons test), as appropriate, and compared to baseline (i.e., zero) by one-sample t-tests. Statistical tests were run using SigmaStat 3.11 (Systat Software) or Prism 6.00 (GraphPad Software), and P < 0.05 was considered significant. Values in the text are reported as mean ± SEM.
into the 12% O2 challenge was used to characterize the effects of PPADS on the early, carotid body-mediated phase of the HVR. There was a significant effect of PPADS in both age groups (both P < 0.001; Fig. 1C and D), and post hoc analysis revealed that the HVR was significantly reduced in rats receiving 100, 125, or 150 mg kg−1 PPADS compared to saline-injected rats (all P < 0.01). Partial inhibition of the early phase of the HVR was apparent at 75 mg kg−1 PPADS, and no significant increase in ventilation was detected at PPADS dosages ≥100 mg kg−1 relative to baseline (Fig. 1C and D). Therefore, 125 mg kg−1 PPADS was used to pharmacologically denervate the carotid body in subsequent experiments. 3.2. Effects of systemic PPADS on the hypercapnic ventilatory responses (HCVR)
3. Results 3.1. Effects of systemic PPADS on the hypoxic ventilatory response (HVR) A dose–response curve was constructed for the effects of systemic PPADS on the HVR of rats reared in room air (Fig. 1). PPADS tended to lower baseline ventilation, but this was only significant in P4 rats and only at doses >75 mg kg−1 (one-way ANOVA, P < 0.01). After saline injection (0 mg kg−1 PPADS), P4 rats exhibited the biphasic HVR typical of newborn mammals (Fig. 1A) whereas P14-15 rats exhibited a sustained increase in ventilation (Fig. 1B). The magnitude of the ventilatory increase 0.5 min
Administering PPADS directly to the brainstem greatly reduces central CO2 chemosensitivity (Thomas and Spyer, 2000) and the HCVR (da Silva et al., 2012). Therefore, the HCVR was assessed as an indirect test of whether PPADS penetrates the blood–brain barrier following systemic (intraperitoneal) injections. Ventilation was measured in P4-5 and P13-14 rats (all reared in room air) while they breathed 0 and 7% CO2 (Fig. 2). PPADS tended to decrease baseline respiratory frequency relative to that observed in salineinjected rats (main effect for treatment, P < 0.001 and 0.009 for P4-5 and P13-14, respectively), but the resulting decrease in minute ventilation was only significant at P13-14 (main effect for treatment, P = 0.03) (Fig. 2F). Despite this effect on baseline ventilation,
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Fig. 2. Hypercapnic ventilatory responses for neonatal rats (P4-5 and P13-14) after intraperitoneal administration of PPADS (125 mg kg−1 ) or saline; all rats were reared in room air. Respiratory frequency (A, B), tidal volume (C, D), and minute ventilation (E, F) are reported under baseline (“BL”) conditions and during a 10-min exposure to 7% CO2 . Sample sizes are 11 per group at P4-5 and 9 per group at P13-14. Values are mean ± SEM. *Main effect for drug treatment (P < 0.05).
systemic PPADS injection had no significant effect on the ventilatory response to 7% CO2 in either age group (drug × inspired CO2 level, P = 0.85 and 0.36, respectively) (Fig. 2E and F). Likewise, respiratory frequency and tidal volume responses to hypercapnia were similar between saline- and PPADS-injected rats (drug × inspired CO2 level, all P > 0.05) (Fig. 2A–D). 3.3. Effects of developmental hyperoxia on the late phase of the HVR Ventilatory responses to 12% O2 were measured for Control and Hyperoxia rats after systemic administration of PPADS. In the P4-5 age group, there was a significant interaction between developmental O2 treatment and time for ventilation during the hypoxic exposure (treatment × time, P < 0.001) (Fig. 3C). Hyperoxia rats exhibited lower baseline ventilation than Control rats due to lower respiratory frequencies (Fig. 3A) and tidal volumes (Fig. 3B), as previously reported (Bavis et al., 2010). Neither treatment group increased ventilation in the first minute of hypoxia, suggesting that PPADS abolished the early phase of the HVR. Instead, ventilatory
depression was evident within the first minute of hypoxia in Control rats and became more pronounced throughout the hypoxic exposure (Fig. 3C); ventilation was reduced by 28% in Control rats by the 10th minute of hypoxia (P < 0.001 vs. baseline) (Fig. 3D). In contrast, no ventilatory depression was observed in Hyperoxia rats at P4-5 (Fig. 3C and D). Consequently, ventilatory depression was significantly greater in Control rats than in Hyperoxia rats at P4-5 (P < 0.001) (Fig. 3D). Hypoxic ventilatory depression in the P4-5 Control rats reflected time-dependent decreases in respiratory frequency and tidal volume. For respiratory frequency, there was an interaction between developmental O2 treatment and time (treatment × time, P < 0.001), and post hoc analysis revealed a significant decrease in frequency by the 10th minute of hypoxia in the Control group only P < 0.001 vs. baseline (Fig. 3A). Although the interaction term for tidal volume was not quite significant (treatment × time, P = 0.08), visual inspection of the data suggests that the significant main effect for time (P = 0.003) is driven by diminished tidal volumes of Control rats by the 10th minute of hypoxia (Fig. 3B); indeed, the mean tidal volume was identical for Hyperoxia rats at baseline
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Fig. 3. Hypoxic ventilatory response (HVR) for P4-5 rats reared in 21% O2 (Control) or 60% O2 (Hyperoxia) from birth; all rats received 125 mg kg−1 PPADS (i.p.) prior to ventilation measurements. Respiratory frequency (A), tidal volume (B), and minute ventilation (C) are reported under baseline (“BL”) conditions and during a 10-min exposure to 12% O2 . In panel D, ventilation is expressed as the percentage change from baseline during the 10th minute of hypoxia. Values are mean ± SEM. Sample sizes are 18 Control and 18 Hyperoxia. *P < 0.05 vs. Control within the same time period; † P < 0.05 vs. baseline (BL or 0%) within the same treatment group. No treatment × time interaction was detected for tidal volume (panel B), so the symbols (with brackets) denote a significant difference between treatment groups when pooled across all time points (i.e., main effect for treatment) (*) or a significant difference from baseline when pooled across treatment groups (i.e., main effect for time, followed by post hoc tests) (†).
and in the 10th minute of hypoxia (0.48 ± 0.02 and 0.48 ± 0.01 ml 100 g−1 , respectively). An additional group of P4-5 Control rats (n = 14) was injected with PPADS but was maintained in 21% O2 throughout the ventilation protocol (i.e., no hypoxia). Ventilation did not change significantly over the course of the protocol in these “time control” rats (−3.1 ± 4.4% change from baseline; one-sample t-test, P = 0.49). Therefore, changes in ventilation in P4-5 Control rats (Fig. 1) represents hypoxic ventilatory depression, not time-dependent effects of PPADS or drift in ventilation measurements. The HVR was also assessed in Control and Hyperoxia rats at P1315; an additional group of P4 Control rats was studied alongside the P13-15 rats to enable quantitative comparisons between age groups. Again there was a significant interaction between treatment group and time for ventilation during the hypoxic exposure (treatment × time, P < 0.001), but this was driven by differences between P4 and P13-15 rats (Fig. 4C). Specifically, baseline ventilation was significantly lower in P13-15 Control rats than in P4 Control rats (P < 0.001), but no differences were detected between P13-15 Control and P13-15 Hyperoxia rats (P = 0.46). No increase in ventilation was evident in the first minute of hypoxia in any group. As in the earlier round of experiments, hypoxic ventilatory depression developed in P4 Control rats (Fig. 4C and D), with ventilation decreasing by 21% by the 10th minute of hypoxia (P = 0.01 vs. baseline) (Fig. 4D). However, hypoxia did not cause ventilatory depression in either the Control or Hyperoxia groups at P13-15 (Fig. 4C and D). Instead, ventilation tended to increase progressively during the hypoxic exposure in P13-15 rats, although this only reached statistical significance in the Hyperoxia group by
the 10th minute of hypoxia (Control, 13% increase from baseline, P = 0.07; Hyperoxia, 23% increase, P < 0.001) (Fig. 4D). Accordingly, the magnitude of the hypoxic ventilatory depression decreased significantly with age in Control rats (P < 0.01) (Fig. 4D). The progressive increase in hypoxic ventilation in P13-15 Hyperoxia rats, was associated with an increase in respiratory frequency (P = 0.009 for the 10th minute of hypoxia vs. baseline) (Fig. 4A); a similar trend was observed in P13-15 Control rats, but this was not significant (P = 0.11). Tidal volume did not change significantly over time in any group (main effect for time, P = 0.99) (Fig. 4B).
4. Discussion Hypoxic ventilatory depression was observed in young neonatal rats (P4-5) after pharmacological denervation of the carotid bodies with the P2 purinergic receptor antagonist PPADS. This finding suggests that the late phase of the biphasic HVR in conscious neonatal rats results, at least in part, from direct actions of hypoxia on respiratory neurons in the CNS. In contrast, rats reared in 60% O2 from birth lacked any measurable hypoxic ventilatory depression after systemic PPADS administration. This is consistent with the absence of a biphasic HVR in neonatal, hyperoxia-reared rats (Bavis et al., 2010; R.W. Bavis, K.J. DeAngelis, T.C. Horowitz, L.M. Reedich, and R.J. March, unpublished data). Thus, plasticity in the CNS regions governing hypoxic ventilatory depression likely explains the premature appearance of a sustained HVR after developmental hyperoxia.
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Fig. 4. Hypoxic ventilatory response (HVR) for P13-15 rats reared in 21% O2 (Control) or 60% O2 (Hyperoxia) from birth; all rats received 125 mg kg−1 PPADS (i.p.) prior to ventilation measurements. Sample sizes (n) are 16 Control and 16 Hyperoxia at P13-15; an additional group of P4 Control rats (n = 8) was studied alongside the P13-15 rats to facilitate quantitative comparisons between age groups. Respiratory frequency (A), tidal volume (B), and minute ventilation (C) are reported under baseline (“BL”) conditions and during a 10-min exposure to 12% O2 . In panel D, ventilation is expressed as the percentage change from baseline during the 10th minute of hypoxia. Values are mean ± SEM. † P < 0.05 vs. baseline (BL or 0%) within the same treatment group; # P < 0.05 vs. P13-15 (Control group only). No treatment × time interaction was detected for tidal volume (panel B), so # (with bracket) denotes a significant difference between P4 Control and P13-15 Control rats when pooled across all time points (i.e., main effect for treatment group, followed by post hoc tests).
4.1. Critique of methods Pharmacological blockade has many advantages to surgical denervation of the carotid bodies, most notably the ability to assess ventilation in conscious rats immediately after “denervation”. However, the validity of this approach depends on the capacity of the selected drug to block peripheral chemoreceptors without directly targeting respiratory neurons in the CNS. Several lines of evidence support these assumptions. First, the PPADS dosage in the present study effectively abolished the early, carotid bodymediated phase of the HVR (Figs. 1, 3 and 4), consistent with its blockade of synaptic signaling between carotid body glomus cells and their afferent neurons (Rong et al., 2003; Nurse and Piskuric, 2013). Although some increase in ventilation was detected during the late phase of the HVR in older neonates (i.e., P13-15), the slow onset of this response argues against a contribution from the carotid bodies; indeed, a small residual ventilatory increase is also common after surgical denervation of peripheral chemoreceptors and likely represents activation of central O2 sensors (e.g., MartinBody et al., 1985). Furthermore, PPADS also tended to decrease baseline ventilation (e.g., Figs. 1 and 2), consistent with the withdrawal of the peripheral contribution to eupneic ventilatory drive (e.g., Dejours, 1963; Teppema and Dahan, 2010). Finally, PPADS should not penetrate the blood–brain barrier given its large, polar structure (Pardridge, 2005). This appears to have been confirmed in the present study since systemic administration of PPADS had no effect on the hypercapnic ventilatory response (HCVR): if PPADS had crossed the blood–brain barrier, it would have attenuated the
HCVR (Thomas and Spyer, 2000; da Silva et al., 2012). In retrospect, it is somewhat surprising that systemic PPADS had no measurable effect on the HCVR since input from carotid chemoreceptors is proposed to modulate central CO2 chemosensitivity in adult mammals (Smith et al., 2010); the carotid bodies apparently contribute very little (if at all) to the HCVR of neonatal rats. In order to characterize the early phase of the HVR, high gas flow rates were necessary to ensure a rapid transition between normoxia and hypoxia. This approach precluded simultaneous measurement of metabolic O2 consumption and CO2 production which are important determinants of respiratory drive. Neonatal rats, like other animals, tend to decrease aerobic metabolism in response to hypoxia (e.g., Bavis et al., 2010; for review, see also Mortola, 2004; Teppema and Dahan, 2010), and it is possible that this hypoxic hypometabolism contributes to decreased ventilation during the late phase of the HVR of neonatal rats. However, there is strong evidence that neural mechanisms, rather than (or in addition to) changes in whole-body metabolism, are critical to the origin of hypoxic ventilatory depression. This evidence includes the rapid onset of hypoxic ventilatory depression in peripherally chemodenervated animals (e.g., ≤1 min in PPADS-injected P4-5 rats) and the repeated demonstration that pharmacological and/or physical disruption of specific groups of respiratory neurons can block hypoxic ventilatory depression in neonatal and adult mammals (reviewed in Bissonnette, 2000; Teppema and Dahan, 2010). Although the metabolic effects of systemic PPADS administration have not been assessed directly, experiments using other purinergic receptor antagonists indicate that it is unlikely that this drug
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would increase the metabolic reduction during hypoxia (and, thus, enhance hypoxic ventilatory depression). Indeed, systemic administration of other P2X receptor antagonists (suramin or A-317491) decreases resting metabolism in neonatal rats (Niane et al., 2011) but causes, if anything, a modest reduction in the magnitude of the hypoxic metabolic response (L.M. Niane and A. Bairam, personal communication). Moreover, developmental hyperoxia does not alter the magnitude of the metabolic response to hypoxia in neonatal rats at any of the ages studied (i.e., P4–P14) (Bavis et al., 2010). Therefore, it is unlikely that differences in whole-body metabolism explain the differences in hypoxic ventilatory depression observed between treatment groups in the present study. 4.2. Potential mechanisms for age- and hyperoxia-induced changes in hypoxic ventilatory depression Hypoxic ventilatory depression after systemic PPADS is consistent with many previous studies that observed hypoxic ventilatory depression in anesthetized animals after surgical denervation of the carotid bodies (reviewed in Teppema and Dahan, 2010), but contradicts other studies of conscious or lightly anesthetized mammals (e.g., Bureau et al., 1985; Schramm and Grunstein, 1987; Maxová and Vízek, 2001). The HVR was assessed in conscious rats in the present study, so anesthesia cannot explain the discrepancy between this and the latter studies. Rather, it seems likely that these differences reflect the relative maturity of the study animals. Indeed, the magnitude of the hypoxic ventilatory depression decreased with age in rats (Fig. 4D; also compare the 0 mg kg−1 PPADS dosages in Fig. 1A and B). This may explain why hypoxic ventilatory depression was not observed in adult rats (Maxová and Vízek, 2001) or relatively precocial lambs (Bureau et al., 1985) after carotid body denervation. Likewise, Schramm and Grunstein (1987) observed that the magnitude of hypoxic ventilatory depression diminished greatly over the first two postnatal weeks in neonatal rabbits, but they only studied the effects of carotid body denervation on hypoxic ventilatory depression in older neonates (i.e., 18–29 days of age). Importantly, the finding that hypoxia directly contributes to ventilatory depression does not preclude additional mechanisms that depend on carotid body activation (Simakajornboon and Kuptanon, 2005; Teppema and Dahan, 2010). We speculate that carotid body-dependent mechanisms contribute some portion of the hypoxic ventilatory depression (or roll-off) observed in intact animals, and that the relative contribution of these mechanisms increases during postnatal maturation (as the contribution from CNS hypoxia diminishes). Although these findings indicate that hypoxia acts directly on the CNS to initiate ventilatory depression (at least in the youngest neonates), the specific mechanisms underlying this response are not fully understood. Many neurons in the CNS are inhibited by cellular hypoxia, including subsets of neurons in respiratory regions such as the rostral ventrolateral medulla, pre-Botzinger complex, and nTS (reviewed in Teppema and Dahan, 2010). Alternatively, hypoxia could cause O2 -sensitive cells to release inhibitory neuromodulators or neurotransmitters, or to recruit postsynaptic neurons that inhibit breathing. Numerous neurochemicals have been implicated in hypoxic ventilatory depression, including GABA, adenosine, PDGF, substance P, and various catecholamines (Bissonnette, 2000; Teppema and Dahan, 2010). Unfortunately, few experiments have considered whether these molecules are synthesized and/or released in hypoxia independent of carotid chemoafferent activation. The question has been asked for GABA, but this does not seem to be the case: carotid body denervation prevents the rise in extracellular GABA that normally occurs in the nTS of rats during hypoxia (Tabata et al., 2001). There is indirect evidence, however, that hypoxia induces PDGF signaling without input from peripheral chemoreceptors. Specifically, hypoxia influences
synaptic transmission between nTS neurons differently in brainstem slices from wild type and PDGF- receptor gene-knockout mice (Ohi et al., 2010); although PDGF release was not assessed in that study, the results suggest that hypoxia directly modulates PDGF signaling pathways in the brainstem. The expression of the PDGF-B chain, the PDGF subunit which binds with high affinity to the PDGF- receptor, does increase in nTS neurons during hypoxia, and the activation of the PDGF- receptors contributes to hypoxic ventilatory depression in intact rats (Gozal et al., 2000; Vlasic et al., 2001). Interestingly, recent work in our laboratory indicates that the late phase of the HVR is less sensitive to pharmacological blockade of PDGF- receptors in neonatal rats reared in hyperoxia compared to age-matched controls (R.W. Bavis, K.J. DeAngelis, T.C. Horowitz, L.M. Reedich, and R.J. March, unpublished data); thus, changes in PDGF signaling could contribute to the absence of hypoxic ventilatory depression in hyperoxia-reared rats after functional denervation of the carotid bodies in the present study. The actions of inhibitory neurochemicals in the brainstem during the late phase of the HVR are opposed by the release of excitatory neurochemicals, and the postnatal maturation of the biphasic HVR is thought to reflect the shifting balance between these inhibitory and excitatory influences (Bissonnette, 2000; Simakajornboon and Kuptanon, 2005; Teppema and Dahan, 2010). The release of glutamate in the nTS requires activation of the carotid body chemoreceptors (Mizusawa et al., 1994), but hypoxia initiates the release of ATP throughout the ventral medulla in anesthetized, peripherally chemodenervated adult rats (Gourine et al., 2005). Importantly, the endogenous release of ATP at or near respiratory neurons in the ventrolateral medulla (VLM) helps to maintain respiratory motor output during hypoxia (Gourine et al., 2005). Although it is possible that extracellular ATP is converted to adenosine in the nTS and VLM, the relatively slow accumulation of adenosine does not appear to correlate well with the rapid onset of hypoxic ventilatory depression in adult rats (Gourine et al., 2002, 2005). Thus, ATP release during CNS hypoxia is unlikely to explain the observed hypoxic ventilatory depression in room-air reared rats after pharmacological denervation of the carotid bodies. However, enhanced ATP release during CNS hypoxia could contribute to the age-dependent decrease in hypoxic ventilatory depression during normal postnatal maturation and/or to the sustained HVR observed in young, hyperoxia-reared rats. Additional studies are clearly needed to understand the potential roles for this and other CNS mechanisms in developmental plasticity of the HVR. Acknowledgments This study was supported in part by the Bates Student Research Fund. The authors would like to thank Sarah Logan and Halward J. Blegen for assisting with data analysis, and to thank Lalah M. Niane and Dr. Aida Bairam for sharing unpublished data. References Bavis, R.W., Young, K.M., Barry, K.J., Boller, M.R., Kim, E., Klein, P.M., Ovrutsky, A.R., Rampersad, D.A., 2010. Chronic hyperoxia alters the early and late phases of the hypoxic ventilatory response in neonatal rats. J. Appl. Physiol. 109, 796–803. Bavis, R.W., Fallon, S.C., Dmitrieff, E.F., 2013. Chronic hyperoxia and the development of the carotid body. Respir. Physiol. Neurobiol. 185, 94–104. Bissonnette, J.M., 2000. Mechanisms regulating hypoxic respiratory depression during fetal and postnatal life. Am. J. Physiol. Regul. Integr. Comp. Physiol. 278, R1391–R1400. Bureau, M.A., Lamarche, J., Foulon, P., Dalle, D., 1985. The ventilatory response to hypoxia in the newborn lamb after carotid body denervation. Respir. Physiol. 60, 109–119. da Silva, G.S., Moraes, D.J., Giusti, H., Dias, M.B., Glass, M.L., 2012. Purinergic transmission in the rostral but not caudal medullary raphe contributes to the hypercapnia-induced ventilatory response in unanesthetized rats. Respir. Physiol. Neurobiol. 184, 41–47. Dejours, P., 1963. Control of respiration by arterial chemoreceptors. Ann. N. Y. Acad. Sci. 109, 682–695.
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