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HIGHLIGHTED TOPICS
Oxygen Sensing in Health and Disease
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106
Submitted 18 August 2003 ; accepted in final form 12 July 2004
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ABSTRACT |
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100 s). In contrast, carotid body sensory response to hypoxia was greater and the time course of the response was faster (30 s) in IH compared with control pups. The magnitude of the hypoxic ventilatory response was greater in IH compared with control pups, whereas changes in O2 consumption and CO2 production during hypoxia were comparable between both groups. The magnitude of ventilatory stimulation by hyperoxic hypercapnia (7% CO2-balance O2), however, was the same between both groups of pups. These results demonstrate that neonatal IH facilitates carotid body sensory response to hypoxia and augments hypoxic ventilatory chemoreflex. neonates; apneas; hypoxic ventilatory response
Intermittent hypoxia (IH) is more often experienced in neonatal life than is continuous hypoxia. Prematurely born infants often experience IH as a consequence of recurrent apneas (17). Recently, Nock et al. (14) reported that preterm infants experiencing recurrent apneas exhibit increased hypoxic ventilatory response (HVR) and that the magnitude of the response was greater in infants having more number of apneic episodes. Because recurrent apneas are associated with periodic decreases in arterial O2 saturation, the enhanced hypoxic ventilatory response might in part be due to chronic IH. Indeed, 1 wk of IH (24 min of hypoxia/day) has been reported to increase HVR in experimental animals such as piglets (20). These studies suggest that neonatal IH leads to enhanced HVR. Whether the enhanced HVR caused by IH is due to augmented peripheral chemoreceptor sensitivity to hypoxia and/or due to enhanced processing of peripheral chemoreceptor inputs at the central nervous system, however, is not known. Our laboratory recently reported that IH (10 days; 8 h/day; 9 episodes/h) augments hypoxic sensitivity of the carotid bodies in adult rats (15, 16). Consequently, in the present study, we tested the hypothesis that IH enhances carotid body response to hypoxia and augments HVR in neonatal animals. We tested this possibility in 2-day-old rats exposed to IH. Our results showed that neonatal IH as short as 16 h results in marked enhancement of carotid body sensitivity to hypoxia and HVR.
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MATERIALS AND METHODS |
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Exposure to IH. Rat pups (2–6 h old) along with their mothers were exposed to IH by using the procedures described previously (15, 16). Briefly, animals housed in feeding cages were placed in a special chamber for exposure to IH, and the details of the chamber are described in detail elsewhere (18). The animals were unrestrained, freely mobile, and fed ad libitum. The chamber was flushed with alternating cycles of pure N2 and compressed air. Inspired O2 levels reached a nadir of 5% O2 during hypoxia within 68–75 s and 21% O2 during normoxia within 70–85 s. Animals were exposed to nine episodes of IH per hour for 16 h. Ambient O2 levels in the chamber were continuously monitored by an O2 analyzer (model OM-11, Beckman) by sampling the air in the chamber. Inspired CO2 levels were monitored continuously by an infrared analyzer (model LB-2, Beckman) and maintained between 0.2 and 0.5%. The duration of the gas flow during each hypoxic and normoxic episode was regulated by timer controlled solenoid valves. Control experiments were performed on rat pups (also 2–6 h after the birth) along with their mother exposed to alternating cycles of compressed room air instead of hypoxia in the same chamber. Acute experiments were performed 6–10 h after IH or normoxia.
Recording of ex vivo carotid body sensory activity.
Carotid bodies along with the sinus nerves were harvested from rats pups anesthetized with urethane (1.5 g/kg ip). After the connective tissue was cleaned, the carotid body along with the sinus nerve was placed in a recording chamber (volume 250 µl) and superfused with warm physiological saline (36.5°C) at a rate of 2 ml/min. The lag time for the superfusing medium from the reservoir to the inlet port in the recording chamber was 25 s. The composition of the medium was as follows (in mM): 125 NaCl 125, 5 KCl, 1.8 CaCl2, 2 MgSO4, 1.2 NaH2PO4, 25 NaHCO3, 10 D-glucose, and 5 sucrose, and the solution was bubbled with 95% O2-5% CO2. To facilitate recording of "single" chemoreceptor fibers, sinus nerve was treated with 0.1% collagenase for 5 min. Action potentials (2–5 active units) were recorded from one of the nerve bundles with a suction electrode, amplified (alternating-current preamplifier; model P511K, Grass Instrument; bandwidth of 100–3,000 Hz), displayed on an oscilloscope (model 5B12N, Tektronix), and stored in a computer via an analog-to-digital translation board (PowerLab/8P, ADInstruments, New Castle, Australia). The following test was done to ensure that the action potentials were of carotid body origin. If the discharge frequency increased >20% above the baseline in response to stagnant hypoxia (i.e., interrupting the superfusion for 5 min) and return to baseline after the superfusion was resumed, then the action potentials were considered originating from the carotid body. Single units were selected on the basis of the height and duration of the individual action potentials by using a spike discrimination program (Spike Histogram Program, PowerLab). In each carotid body, at least two chemoreceptor units were analyzed.
The protocols for determining the carotid body sensory response to hypoxia were as follows. Baseline sensory activity was recorded for 5 min while the carotid body (control, n = 11 carotid bodies from 6 pups; IH, n = 10 carotid bodies from 6 pups) was superfused with medium equilibrated with 95% O2-5% CO2 (medium PO2 = 410 ± 13 Torr, and PCO2 = 36 ± 3 Torr). Then the medium was switched to that equilibrated with either 21% O2-5% CO2-balance N2 (PO2 =141 ± 2 Torr), 10% O2-5% CO2-balance N2 (PO2 = 67 ± 3 Torr), or 1% O2-5% CO2-balance N2 (PO2 = 35 ± 2 Torr). Each challenge was given for 3 min, followed by 5-min recovery period. The protocols were repeated twice in a given experiment. The PO2 and PCO2 of the superfusion medium were determined by a blood-gas analyzer (model ABL 5, Radiometer, Copenhagen, Denmark). The carotid body response to sodium cyanide (3 µg/ml) was also analyzed in the same experiments as above.
Ventilatory measurements.
Breathing was monitored in unanesthetized, unrestrained pups by whole body plethysmograph as described previously (13). Briefly, animals were placed in a 600-ml Lucite chamber connected to a high-gain differential pressure transducer (model MP45, Validyne, North Ridge, CA). As the animals breathed, changes in pressure within the chamber were converted to signals representing tidal volume (VT), which were amplified (model BMA 830, CWE, Ardmore, PA), recorded on a strip-chart recorder (Dash 10, Astro-Med, West Warwick, RI), and stored in a computer with respiratory acquisition software for further analysis. O2 consumption (
O2) and CO2 production (CO2) were determined by the open-circuit method by using O2 and CO2 analyzers (models CA-1 and FC-1, Sable Systems). All recordings were obtained at an ambient temperature of 26 ± 1°C. Animals were allowed to acclimate for 30 min in room air before the ventilatory response to hypoxia or hypercapnia was recorded.
The protocols for measurements of hypoxic and hypercapnic ventilatory response were as follows. Baseline ventilation was recorded for 5 min while the animals breathed 100% O2, and then the inspiring gas was switched to 21% followed by 12% O2-balance N2 (n = 7 in each group). Each gas challenge was maintained for 5 min. O2 and CO2 were measured at the end of each inspired O2. Hypercapnic ventilatory response was determined in the same animals. Baseline ventilation was recorded while breathing 100% O2 for 5 min, followed by 7% CO2-93% O2 for additional 5 min. The protocols for hypoxia and hypercapnia were repeated twice in a given experiment.
Data analysis.
Carotid body sensory activity (impulses/s) was analyzed every 10 s during baseline as well as with each medium PO2. The time course of the response was analyzed in each experiment. The peak response was calculated from each carotid body and plotted against medium PO2 (stimulus response). The following respiratory variables were analyzed: 1) respiratory rate (RR; breaths/min), 2) VT (µl), and 3) minute ventilation [E (ml/min) = RR x VT]. Respiratory variables (RR and VT) were averaged over 5 min during baseline and with each inspired O2 or CO2. Changes in VT and E were normalized to grams of body weight. Changes in metabolic variables (O2, CO2, and O2/CO2) were expressed as a percentage of baseline values. All data are expressed as means ± SE. Two-way ANOVA with repeated measures followed by Tukey's test was used for assessing the statistical significance. Values of P < 0.05 were considered significant.
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RESULTS |
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100s after the onset of the stimulus (including the lag time 25 s). On the other hand, in IH pups (n = 6 pups; 22 fibers from 10 carotid bodies), sensory response was faster and started to increase within 30 s (including the lag time 25 s) after the onset of hypoxia (PO2 = 35 ± 2 Torr). Average data of the stimulus-response relation are summarized in Fig. 2B. The baseline activity during hyperoxia was significantly higher in IH-conditioned pups compared with control pups (IH 1.2 ± 0.1 vs. control 0.5 ± 0.07 impulses/s; P < 0.01). Furthermore, the magnitude of the sensory response was significantly higher at all three levels of medium PO2 tested in IH compared with control pups (Fig. 2B).
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E from both groups of pups are presented in Table 1. Baseline E (ml·min–1g·body wt–1) under hyperoxia was significantly higher in IH compared with control pups. The elevated resting E in IH pups was due to greater VT. In control pups, 21% O2 (normoxia) had no effect on E, whereas in IH pups there was a significant increase in E compared with baseline value. This increase in E during normoxia in IH pups was due to increased RR. Both groups of pups responded with significant increases in E in response to hypoxia (12% O2). The magnitude of increase in E during hypoxia was significantly greater in IH compared with control pups (expressed as percentage of baseline; Fig. 4A). The greater increase in E during hypoxia in IH pups was due to increases in RR. (Fig. 4A, Table 1). In both groups of pups, O2 fell progressively in response to the change of inspired O2 from 100 to 12% O2 (Fig. 4B). The magnitude of decrease in O2, however, was comparable between both groups of pups (P > 0.05). CO2 was unaffected by hypoxia in both groups of pups, and as a consequence, changes in O2/CO2 were nearly the same between both groups of pups (Fig. 4B).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Consistent with a previous report (12), we also found that carotid bodies from neonatal rat pups (2 days old) responded weakly to hypoxia (Figs. 1 and 2). On the other hand, carotid bodies from IH-exposed pups showed significantly enhanced hypoxic response, and the onset of excitation was much faster than in control pups. This enhanced hypoxic sensory response could be due to several factors, including cardiovascular alterations, which are known to influence the hypoxic sensory response (8). Because the present studies were performed on ex vivo carotid bodies, wherein influences from cardiovascular alterations are effectively absent, changes in cardiovascular variables are unlikely to account for the observed enhancement in the hypoxic sensory response. Prolonged hypoxia lasting several days to weeks results in hypertrophy of the carotid bodies (see Ref. 2 for references), which by increasing the diffusion gradient of O2 from the superfusate to the glomoid tissue, can lead to the enhanced hypoxic sensory response. Therefore changes in the morphology (in terms of either increased glomus cells or the volume of the organ) and/or ultrastructural changes in the carotid body (e.g., improved synaptic contact between glomus cells and the afferent nerve ending) might have contributed in part to the IH-induced enhancement of the hypoxic sensory response. Sensory response of the IH-conditioned carotid bodies was relatively faster than the control carotid bodies (Fig. 2). The differences in the speed of the responses could be attributed to the placement of the carotid bodies in a poorly perfused location in the recording chamber. However, care was taken to place the carotid bodies close to the inlet port of the recording chamber, and thus it is unlikely that the slower response of the control carotid bodies was due to the positioning in the recording chamber. Furthermore, the time course and the magnitude of cyanide-induced sensory response was nearly the same in both groups of pups, suggesting that IH selectively affected the speed and the magnitude of the hypoxic sensory response. Cellular mechanisms underlying IH-induced enhanced hypoxic sensory response are beyond the scope of the present study, and they may include alterations in the transduction process (e.g., changes in ionic conductances and/or Ca2+ homeostasis in the glomus cells) and/or the sensory transmission from glomus cell to the afferent nerve ending (e.g., alterations in the neurotransmitters and/or receptors). These possibilities, however, require further investigation. Nonetheless, the present observations demonstrate that exposure of rat pups to as short as 16 h of IH markedly facilitates the hypoxic sensory response of the neonatal carotid bodies, an effect that is opposite to that reported with continuous hypoxia (11).
Another major finding of the present study is that neonatal IH augmented the hypoxic ventilatory response. We monitored ventilation in unanesthetized pups by using the whole body plethysmograph. As pointed by Enhorning et al. (7), there are some limitations with the use of whole body plethysmography in small animals, and it may not provide accurate changes in VT. Despite this technical limitation, we believe that IH selectively enhanced hypoxic ventilatory response, because the magnitude of hypercapnic ventilatory response, which was mainly due to increased VT, was found to be the same between IH and control pups. We chose to determine baseline ventilation under hyperoxia because, if IH already increased breathing during normoxia, then any further increase during hypoxia might be small and thus might underestimate the magnitude of the HVR. Baseline ventilation during hyperoxia was significantly elevated in IH pups. The elevated baseline carotid body activity (Fig. 2) might contribute in part to the increased baseline ventilation. Other factors such as altered cerebral blood flow, lung pathology if any, and change in hemoglobin CO2-buffering capacity, which are known to occur during hyperoxia (5), might have equally contributed to the increased baseline ventilation. Hypoxia is known to affect O2 and CO2 production, especially in neonatal animals, which in turn can influence the HVR (9). However, the magnitude of changes in O2 during hypoxia was comparable between both groups of pups (i.e., IH and controls; Fig. 3), indicating that the augmented HVR is not secondary to metabolic alterations. Hypoxia can affect arterial blood pressure and body temperature, and alterations in these variables can also influence the HVR. We did not monitor these variables in our experiments because of technical limitations and thus cannot exclude their contributions, if any, to the augmented HVR. A recent study by Gozal and Gozal (10), however, reported that exposure of 2- to 3-day-old rat pups to IH affected mainly the "late" but not "early" HVR. However, these investigators exposed rat pups to 5 min of hypoxia followed by 10 min of normoxia (8 cycles) and then exposed the pups to acute hypoxia lasting 30 min, an experimental paradigm distinctly different from that employed in the present study. Thus the differences in experimental paradigms might explain the differences in the results of the present study from that of Gozal and Gozal.
In summary, the present study demonstrates that neonatal IH facilitates hypoxic sensing at the carotid body and augments ventilatory response to hypoxia. The effects of neonatal IH appear opposite of that elicited by neonatal sustained hypoxia, which has been shown to depress hypoxic sensing at the carotid body (11). Whether the effects of neonatal IH are reversible or persist even in adult life remains to be established. Although strict comparisons cannot be made between human infants and rat pups, the present observations might be of considerable clinical relevance to recurrent apneas in premature infants. Because carotid bodies respond weakly to hypoxia at birth, it is conceivable that hypoxia resulting from recurrent apneas might affect the central nervous system, leading to respiratory depression. In contrast neonatal IH by way of improving the hypoxic sensing ability of the carotid bodies leads to augmented HVR, which in turn improves oxygenation and thus protects the central nervous system from the deleterious effects of hypoxia.
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FOOTNOTES |
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