Skip to main content
NCBI home page
The Journal of Physiology logoLink to The Journal of Physiology
. 2001 Nov 1;536(Pt 3):917–926. doi: 10.1111/j.1469-7793.2001.00917.x

Induced recovery of hypoxic phrenic responses in adult rats exposed to hyperoxia for the first month of life

D D Fuller *, Z-Y Wang *, L Ling †, E B Olson *, G E Bisgard *, G S Mitchell *
PMCID: PMC2278901  PMID: 11691883

Abstract

  1. Adult rats exposed to hyperoxia for the first month of life have permanently attenuated ventilatory and phrenic nerve responses to hypoxia. We tested the hypothesis that the blunted hypoxic phrenic response in hyperoxia-treated rats (inspired O2 fraction, FI,O2 = 0.6 for 28 post-natal days) could be actively restored to normal by intermittent (alternating 12 % O2/air at 5 min intervals; 12 h per night for 1 week) or sustained (12 % O2 for 1 week) hypoxia.

  2. Phrenic responses to isocapnic hypoxia(Pa,O2 = 60, 50 and 40 ± 2 mmHg) were assessed in the following groups of anaesthetized, vagotomized adult Sprague-Dawley rats (age 4 months), treated with a neuromuscular blocking agent and ventilated: control, hyperoxia-treated and hyperoxia-treated exposed to either intermittent or sustained hypoxia as adults. Experiments on intermittent and sustained hypoxia-treated rats were performed on the morning following hypoxic exposures.

  3. Both intermittent and sustained hypoxia enhanced hypoxic phrenic responses in hyperoxia-treated rats when expressed as minute phrenic activity (P < 0.05). Increases in phrenic burst amplitude during hypoxia were greater in hyperoxia-treated rats after intermittent hypoxia (P < 0.05), and a similar but non-significant trend was observed after sustained hypoxia. Hypoxia-induced changes in phrenic burst frequency were not significantly different among groups.

  4. The estimated carotid body volume in control rats (11.5 (± 0.7) × 106μm3) was greater than in the other treatment groups (P < 0.05). However, carotid body volume was significantly greater in hyperoxia-treated rats exposed to sustained hypoxia (6.3 (± 0.3) × 106μm3; P < 0.05) compared to hyperoxia-treated rats (3.3 (± 0.2) × 106μm3) or hyperoxia-treated rats exposed to intermittent hypoxia (3.8 (± 0.3) × 106μm3).

  5. Hypoxic phrenic responses in hyperoxia-treated rats 1 week after intermittent hypoxia were similar to responses measured immediately after intermittent hypoxia, indicating persistant functional recovery.

  6. The results indicate that diminished hypoxic phrenic responses in adult rats due to hyperoxia exposure for the first 28 post-natal days can be reversed by intermittent or sustained activation of the hypoxic ventilatory control system. Although the detailed mechanisms of functional recovery are unknown, we suggest that sustained hypoxia restores carotid chemoreceptor sensitivity, whereas intermittent hypoxia primarily augments central integration of synaptic inputs from chemoafferent neurons.

Ventilatory control in adult mammals is determined partly by environmental influences. Among the earliest evidence in support of this concept was the observation that adult humans raised at altitude (> 2300 m), but living at sea level, have blunted ventilatory responses to acute hypoxia (Sorensen & Severinghaus, 1968). It was suggested that the blunted hypoxic ventilatory response could be (at least partially) explained by developmental exposure to hypobaric hypoxia (Sorensen & Severinghaus, 1968). Subsequently, a considerable body of work has firmly established that experience (e.g. respiratory disease) and–or environmental conditions (e.g. hypoxia) restricted to developmental periods can have a lasting impact on ventilation and respiratory control in adults (Eden & Hanson, 1986, 1987; Okubo & Mortola 1988, 1990; Barker et al. 1991; Shaheen et al. 1994; Ling et al. 1996, 1997a). For example, exposure to hypoxia during development has both immediate and long-lasting inhibitory effects on hypoxic ventilatory responses (Eden & Hanson, 1986, 1987; Okubo & Mortola 1988, 1990). Similarly, when rats are reared in an oxygen-enriched environment (FI,O2 = 0.60) for the first month of life, they exhibit blunted ventilatory (and phrenic) responses to acute hypoxia, even several months after a return to normoxia (Ling et al. 1996, 1997a).

Plasticity in respiratory control is not limited to developmental periods, since short-term hypoxic response can be enhanced in adult mammals by exposure to either chronic intermittent (Serebrovskaya et al. 1999; Ling et al. 2001) or sustained (Bisgard, 2000) hypoxia. Thus, it might be possible to enhance or restore hypoxic responses in adult animals that were exposed to hyperoxia during development. Consequently, our primary aim was to test the hypothesis that blunted hypoxic phrenic responses in adult rats reared in hyperoxia for the first 28 post-natal days (hyperoxia-treated rats) would be augmented following 1 week of either intermittent or sustained hypoxia. In addition, we tested the hypothesis that sustained, but not intermittent, hypoxia would reverse the carotid body hypoplasia observed in hyperoxia-treated rats (Erickson et al. 1998). Finally, we determined whether intermittent hypoxia would cause persistent functional recovery in hyperoxia-treated rats.

METHODS

Experiments were performed on 43 4-month-old male Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Madison, WI, USA). All experimental procedures were approved by the Animal Care and Use Committee of the School of Veterinary Medicine at the University of Wisconsin.

Experimental treatments

Hyperoxia

Hyperoxia exposures were essentially the same as described previously (Ling et al. 1996). To summarize, 30 rats were exposed to hyperoxia(FI,O2 = 0.6) from 2–3 days prior to birth through the first 28 post-natal days. All animals were then reared in normoxia until adulthood (4 months of age). Eleven of these animals remained in normoxia until acute neurophysiological experiments were performed (body mass, 439 ± 9 g at the time of the experiment). All other hyperoxia-treated rats were exposed (as adults) to 1 week of chronic intermittent or chronic sustained hypoxia.

Chronic intermittent hypoxia

At 4 months of age, 14 hyperoxia-treated rats were exposed to 1 week of nocturnal intermittent hypoxia. Rat cages were placed in a 112 l Plexiglas chamber, which was flushed with a mixture of air, O2 and N2 between 18.00 h and 06.00 h to achieve quasi-square-wave intermittent hypoxia (5 min hypoxia (FI,O2 = 0.12)/5 min normoxia). Once the change in chamber O2 was initiated, it took approximately 45 s for O2 to equilibrate at 21 or 12 %. The flow of gases through the chamber was maintained at 2 l min−1 per animal, which is sufficient to maintain chamber CO2 below 0.5 % (Olson, 1978). No attempt was made to maintain isocapnia during hypoxic episodes. The temperature of the chamber was held at 22–24 °C. This intermittent hypoxia protocol was substantially the same as that used in previous studies of normal adult rats (Ling et al. 2001), with the exception that a FI,O2 of 0.12, rather than 0.11, was used to compensate for impaired ventilatory responses and gas exchange in hyperoxia-treated rats (see Ling et al. 1996). Six of the hyperoxia-treated rats were studied on the morning on which intermittent hypoxia ended (427 ± 4 g). The remaining eight rats were returned to normoxia and experiments were conducted 1 week after the cessation of intermittent hypoxia (395 ± 6).

Chronic sustained hypoxia

At 4 months of age, hyperoxia-treated rats (n = 5; 454 ± 15 g) were exposed to sustained hypoxia for 1 week. Rat cages were placed in a 2000 l chamber that was flushed continuously with a mixture of N2 and O2 to achieve a constant FI,O2 of 0.12. Airflow was maintained at 2 l min−1 per animal (Olson, 1978) and temperature was held at 22–24 °C. All rats were studied on the morning on which sustained hypoxia ended.

Control

Rats (n = 13; 408 ± 10 g) were reared in normoxia from birth and were not exposed to hyperoxia or hypoxia at any time prior to the acute neurophysiological experiments.

Experimental preparation and measurements

Preparation

Rats were initially placed in a closed chamber and anaesthetized with isoflurane (2.5–3.5 %; FI,O2 = 0.5). Isoflurane anaesthesia was maintained via a nose cone while a tracheal cannula was inserted; the animals were then ventilated mechanically throughout the experiment. A catheter was inserted into the femoral vein to allow injection of anaesthetic and fluids. A femoral artery was catheterized to enable measurement of blood pressure (Statham P23ID, Gould Instruments) and withdrawal of blood samples. Isoflurane anaesthesia was changed to urethane anaesthesia (1.6 g kg−1) over a period of 30 min. The adequacy of anaesthesia was periodically confirmed by the lack of a blood pressure and phrenic response to a toe pinch. Animals were treated with the neuromuscular blocker pancuronium bromide (2.5 mg kg−1). Partial pressures of O2(Pa,O2) and CO2(Pa,CO2) and pH were determined periodically (see ‘Experimental protocol’) from 0.2 ml arterial samples (ABL-500, Radiometer, Copenhagen, Denmark); unused blood was returned to the animal. The vagus nerves were cut in the midcervical region. The end-tidal CO2 pressure(PET,CO2) was measured using a rapidly responding, flowthrough CO2 analyser (Novametrix, Model 1265) placed on the expired air line of the ventilator circuit. Rectal temperature was checked with a rectal thermistor and maintained at 37–38 °C using a heated table.

Nerve recordings

The phrenic nerve was isolated using a dorsal approach, cut distally and desheathed. The nerve was placed on bipolar silver wire electrodes and the signal was amplified (× 10 000) and filtered (300–10 000 Hz; A-M Systems, Model 1800). The amplified signal was full-wave rectified and integrated (time constant = 50 ms, CWE Inc., Model MA-821RSP) and recorded on a computer using commercially available software (WINDAQ, Akron, OH, USA).

Experimental protocol

Following conversion to urethane anaesthesia, a minimum of 30 min were allowed for the preparation to stabilize. The CO2 apnoeic threshold for inspiratory phrenic nerve activity was measured by mechanically hyperventilating the rats until phrenic nerve activity ceased and then slowly decreasing the ventilation rate until inspiratory phrenic nerve activity reappeared. The PET,CO2 was then set at 3 mmHg above this apnoeic threshold. Isocapnia was maintained throughout the experimental protocol by monitoring the PET,CO2 and adjusting the ventilation rate and–or inspired CO2 content as needed. Thus, baseline phrenic activity was standardized relative to the CO2 apnoeic threshold. Once baseline phrenic activity was established, 30 min was allowed to elapse to ensure an adequate representation of stable baseline neural activity. Isocapnic hypoxic phrenic responses were then tested by exposing the rats to 5 min bouts of hypoxia that were separated by 5 min of hyperoxia (FI,O2 = 0.50). Arterial blood samples were drawn in the final minute of each hypoxic episode. During hypoxia, the target Pa,O2 values were 60, 50 and 40 mmHg, respectively, and were obtained by altering the inspired O2 content. A hypoxic trial was accepted if the Pa,O2 was within 2 mmHg of the target Pa,O2, and if Pa,CO2 was within 2 mmHg of baseline. It was usually necessary to expose the animals to three to five hypoxic episodes to obtain successful trials at all hypoxic levels. At the conclusion of each experiment, a maximum phrenic response was established by gradually increasing inspired CO2 until PET,CO2 reached 80–90 mmHg.

Estimation of carotid body volume

At the conclusion of an acute neurophysiological experiment, rats were perfused transcardially with heparinized saline followed by 4 % formaldehyde in 0.1 m phosphate-buffered saline (pH 7.4). The bifurcation of the common carotid artery was then removed en bloc and the excised tissue was immersed in Bouin's fixative overnight. Following routine paraffin processing, the tissue was sectioned serially at 5 μm with a microtome (≈200 sections). Every 20th section was stained with haematoxyline and eosin to select sections that contained the carotid body, Subsequently, every 4th section that contained the carotid body was processed with haematoxyline and eosin staining and images were obtained with a Nikon E600 microscope (× 200) and imported into a computer using a Diagnostics Instruments Spot digital camera. The area of each carotid body was then determined using Image-Pro Plus computer software (Media Cybernetics, Silver Spring, MD, USA). Calibrations were done using a micrometer. Carotid body volume was estimated based on the carotid body area in each section, section thickness and the total number of carotid body sections.

Data analyses

Except where noted, a blind experimental design was employed to conceal the treatment from the experimenter during data collection and analysis. Phrenic nerve activity was averaged over 30 s periods immediately prior to the first hypoxic episode (baseline), at the end of each hypoxic episode and at the end of the hypercapnic response. The following variables were determined: peak integrated phrenic amplitude (∫Phr), phrenic burst frequency (bursts min−1) and minute phrenic activity (∫ Phr × frequency). Changes in peak phrenic amplitude during hypoxia were expressed relative to phrenic nerve activity during both baseline and the maximum hypercapnic response. Expressing changes in phrenic nerve activity relative to both the baseline and maximal levels minimizes concerns regarding potential normalization artifacts that can occur when comparing neurograms within and between experimental preparations. However, in this paper, all data are presented as the percentage change relative to baseline, because all responses were qualitatively similar irrespective of whether they were expressed relative to the baseline or the maximum response.

Statistical comparisons of hypoxic responses within and between experimental groups were made using two-way ANOVA with a repeated measures design, followed by the Student-Neuman-Keuls post hoc test (SigmaStat, SPSS Scientific, Chicago, IL, USA). Statistical comparisons between experimental groups for variables with only one level (e.g. CO2 apnoeic threshold, carotid body volume) were made using one-way ANOVA. Effects were considered statistically significant when the P value was ≤ 0.05.

RESULTS

Blood gases

Pa,CO2 was maintained isocapnic relative to baseline conditions throughout all protocols (Table 1). Hypoxic Pa,O2 values were within 2 mmHg of the target values for each group, namely 40, 50 and 60 mmHg (Table 2).

Table 1.

Pa,CO2 during baseline and hypoxia

Baseline 60 mmHg 50 mmHg 40 mmHg
Control 46 ± 1 45 ± 2 46 ± 2 47 ± 2
Hyperoxia treated 44 ± 1 44 ± 1 45 ± 1 44 ± 1
Hyperoxia treated + intermittent hypoxia 44 ± 1 43 ± 1 44 ± 1 44 ± 1
Hyperoxia treated + intermittent hypoxia (1 week post) 48 ± 1 48 ± 1 47 ± 1 47 ± 2
Hyperoxia treated + sustained hypoxia 41 ± 2 41 ± 2 42 ± 2 41 ± 2
Open in a new tab

Table 2.

Pa,O2 during baseline and hypoxia

Baseline 60 mmHg 50 mmHg 40 mmHg
Control 238 ± 6 60 ± 1 49 ± 1 40 ± 1
Hyperoxia treated 246 ± 3 61 ± 1 49 ± 1 41 ± 1
Hyperoxia treated + intermittent hypoxia 246 ± 5 61 ± 1 50 ± 1 40 ± 1
Hyperoxia treated + intermittent hypoxia (1 week post) 251 ± 5 58 ± 1 50 ± 1 41 ± 1
Hyperoxia treated + sustained hypoxia 251 ± 7 61 ± 1 48 ± 1 40 ± 1
Open in a new tab

Apnoeic threshold

The end-tidal CO2 apnoeic threshold for inspiratory phrenic activity in hyperoxia-treated rats (40 ± 3 mmHg) was not significantly different from that of any other group. However, the CO2 apnoeic threshold in hyperoxia-treated rats exposed to sustained hypoxia (36 ± 5 mmHg) was significantly lower than that of the controls (43 ± 3 mmHg) and hyperoxia-treated rats immediately (42 ± 3 mmHg) and 1 week after intermittent hypoxia (43 ± 3 mmHg; P < 0.05 for all).

Hypoxic phrenic responses

Representative records depicting ∫ Phr and arterial blood pressure during baseline and hypoxia are presented in Fig. 1. These examples illustrate that increases in ∫Phr during hypoxia were greater in hyperoxia-treated rats that had received intermittent or sustained hypoxia than in hyperoxia-treated rats. Furthermore, the decrease in arterial pressure normally seen in control and hyperoxia-treated rats was blunted in rats exposed to intermittent or sustained hypoxia. The mean hypoxic phrenic response data (Fig. 2) show that intermittent and sustained hypoxia significantly enhanced minute phrenic activity during hypoxia (P < 0.05 vs. hyperoxia-treated rats without hypoxic exposures). These responses were not different from those of the controls. Intermittent hypoxia significantly enhanced the increase in ∫Phr during hypoxia in hyperoxia-treated rats (Fig. 2; P = 0.007). A similar, but non-significant trend was observed in hyperoxia-treated rats after sustained hypoxia (Fig. 2; P = 0.11). An apparent difference in the ∫Phr response to hypoxia between control and hyperoxia-treated rats exposed to intermittent and sustained hypoxia was not statistically significant (Fig. 2; control vs. intermittent hypoxia, P = 0.07; control vs. sustained hypoxia, P = 0.06). Sustained hypoxia increased the burst frequency response at 40 mmHg Pa,O2 in hyperoxia-treated rats (P < 0.05; Fig. 2). There were no other differences among treatment groups in the burst frequency response to hypoxia (P > 0.05; Fig. 2).

Figure 1. Representative recordings of integrated phrenic nerve activity (∫Phr) and arterial blood pressure.

Figure 1

Open in a new tab

Although peak ∫Phr increased during hypoxia in the hyperoxia-treated rat, the response was substantially lower than in the other three groups.

Figure 2. Hypoxic phrenic responses in untreated (control) rats (⋄), hyperoxia-treated rats (▪), and hyperoxia-treated rats exposed to 1 week of either chronic intermittent (▴) or sustained hypoxia (▵).

Figure 2

Open in a new tab

Mean (±s.e.m.) changes from baseline in minute phrenic activity (∫Phr × frequency), phrenic burst frequency and peak amplitude of integrated phrenic activity (∫Phr) were assessed at three levels of isocapnic hypoxia (Pa,O2 = 60, 50 and 40 ± 2 mmHg). The symbols indicate significant differences at a particular level of hypoxia: * hyperoxia-treated group vs. other three groups; # hyperoxia-treated group vs. control and intermittent hypoxia-treated group; † hyperoxia-treated group vs. control group; ‡ hyperoxia-treated group vs. sustained hypoxia-treated group.

Data presented in Fig. 1 and Fig. 2 were collected using a blinded experimental design. To increase the sample size, these data were pooled with preliminary data collected from earlier experiments with a non-blinded design. During acute hypoxia (Pa,O2 = 40 ± 1 mmHg), minute phrenic activity was greater in both control (n = 27) and hyperoxia-treated rats exposed to intermittent hypoxia (n = 14), relative to hyperoxia-treated rats (n = 18, P < 0.05; Fig. 3). Intermittent hypoxia restored increases in ∫Phr during hypoxia to control (untreated) levels in hyperoxia-treated rats (Fig. 3). Indeed, both control and intermittent hypoxia-treated rats had ∫Phr responses to hypoxia that were greater than those of hyperoxia-treated rats (P < 0.05; Fig. 3). The pooled hypoxic frequency responses were greater after intermittent hypoxia (P < 0.05 vs. control and hyperoxia-treated rats; Fig. 3). These data suggest that full recovery of hypoxic phrenic responses is possible following intermittent hypoxia.

Figure 3. Pooled phrenic responses to hypoxia (Pa,O2 = 40 ± 1 mmHg) in hyperoxia-treated rats, hyperoxia-treated rats exposed to 1 week of intermittent hypoxia (IH) and untreated control rats.

Figure 3

Open in a new tab

Mean (±s.e.m.) changes from baseline in minute phrenic activity (∫Phr × frequency), phrenic burst frequency and peak amplitude of integrated phrenic activity (∫Phr) were quantified in a pooled data set in which a non-blind experimental design was used. * Significantly greater than hyperoxia-treated animals; † significantly greater than both hyperoxia-treated and control animals.

Persistence of intermittent hypoxia-induced recovery

Hypoxic phrenic responses were tested in hyperoxia-treated rats 1 week after the final day of intermittent hypoxia. Figure 4 shows that increases in ∫Phr during hypoxia in hyperoxia-treated rats persisted for at least 1 week after intermittent hypoxia. Increases in burst frequency and minute phrenic activity during hypoxia tended to be greater immediately vs. 1 week after intermittent hypoxia (both P > 0.25).

Figure 4. Hypoxic phrenic responses in hyperoxia-treated rats exposed as adults to 1 week of intermittent hypoxia and studied immediately (▴) or 1 week after treatment (▵).

Figure 4

Open in a new tab

Changes from baseline in minute phrenic activity (∫Phr × frequency), phrenic burst frequency and peak amplitude of integrated phrenic activity (∫Phr) were assessed at three levels of isocapnic hypoxia (Pa,O2 = 60, 50 and 40 ± 2 mmHg). Apparent differences in minute phrenic activity and burst frequency between groups were not significant.

Arterial blood pressure

Mean arterial pressure (MAP) was elevated during baseline and hypoxia in hyperoxia-treated rats that had received sustained hypoxia (P < 0.05 vs. all other groups; Table 3). In all groups, MAP decreased progressively as hypoxia became more severe, a response characteristic of anaesthetized rats (Bao et al. 1997; Greenberg et al. 1999). Hypoxia-induced hypotension was significantly reduced after intermittent or sustained hypoxia (P < 0.05 for both). However, hypoxia-induced hypotension was transient, because 1 week after intermittent hypoxia, hyperoxia-treated rats had decrements in blood pressure similar to those of the controls (Table 3).

Table 3.

MAP during baseline and hypoxic episodes

Baseline 60 mmHg 50 mmHg 40 mmHg
Control 129 ± 3 99 ± 6 90 ± 8 81 ± 8
Hyperoxia treated 123 ± 3 92 ± 5 82 ± 5 64 ± 4
Hyperoxia treated + intermittent hypoxia 124 ± 7 110 ± 8* 104 ± 8* 89 ± 8
Hyperoxia treated + intermittent hypoxia (1 week post) 123 ± 9 96 ± 10 83 ± 10 63 ± 17
Hyperoxia treated + sustained hypoxia 148 ± 7* 141 ± 7* 137 ± 6* 131 ± 9*
Open in a new tab
*

Significantly different from Control.

Carotid body volume

The representative carotid body sections presented in Fig. 5 demonstrate that hyperoxia induced carotid body hypoplasia. The volume occupied by type I cells and vascular structures appeared to decrease proportionally, similar to findings in a previous report (Erickson et al. 1998). Sustained hypoxia induced vasodilatation and an increase in total carotid body volume in hyperoxia-treated rats (P < 0.05 vs. hyperoxia-treated rats; Fig. 5 and Fig. 6), although carotid body volume remained below control values (P > 0.05; Fig. 6). Intermittent hypoxia did not influence carotid body volume in hyperoxia-treated rats (Fig. 6).

Figure 5. Representative images of the carotid body in a control and hyperoxia-treated rat, and hyperoxia-treated rats exposed to 1 week of either intermittent (IH) or sustained (SH) hypoxia.

Figure 5

Open in a new tab

In each panel, the boundaries of the carotid body are marked with a dashed line. The arrow denotes a blood vessel. The images show that hyperoxia exposure for the first month of life resulted in carotid body hypoplasia that was partially reversed following treatment with sustained hypoxia. See text for a description of tissue preparation and carotid body volume quantification.

Figure 6. Carotid body volume in hyperoxia-treated rats (n = 7), hyperoxia-treated rats exposed to 1 week of either intermittent (IH; n = 6) or sustained (SH; n = 5) hypoxia, and untreated (control) rats (n = 4).

Figure 6

Open in a new tab

*Significantly greater than hyperoxia-treated and hyperoxia-treated + intermittent hypoxia; † significantly greater than the other three groups.

DISCUSSION

The results of the present study indicate that impairment of the hypoxic phrenic response due to hyperoxia can be at least partially reversed by either intermittent or sustained hypoxia. The effects of intermittent hypoxia were persistent and lasted for at least 1 week following the final hypoxic exposure. Since sustained (but not intermittent) hypoxia increased carotid body volume, there is at least some indication that intermittent and sustained hypoxia elicit functional recovery via distinct mechanisms. Although this suggestion is consistent with the literature (cf. Mitchell et al. 2001), it remains to be determined whether intermittent and–or sustained hypoxia induce functional recovery of hypoxic phrenic responses via central or peripheral mechanisms (or both).

Developmental plasticity of the hypoxic ventilatory response

The hypoxic ventilatory response is susceptible to developmental plasticity elicited by post-natal increases (hyperoxia) or decreases (hypoxia) in ambient oxygen (Okuba & Mortola, 1988, 1990; Ling et al. 1996). Okuba & Mortola (1988, 1990) demonstrated that neonatal hypoxia blunts the hypoxic ventilatory response in rats and that this effect lasted for at least 50 days post-hypoxia. Similarly, neonatal hypoxia attenuates the hypoxic ventilatory response in sheep for up to 5 weeks after a return to normoxia (Sladek et al. 1993). It has been suggested that blunted hypoxic responses following neonatal hypoxia result from improper carotid body development (Hanson et al. 1989a). Indeed, carotid chemoreceptor responses to hypoxia are blunted in kittens reared in hypoxic conditions (Hanson et al. 1989b) and chronic hypoxia from birth eliminates the normal developmental increase in carotid body type I cell Ca2+ increases during hypoxia (Sterni et al. 1999).

Both hypoxic ventilatory and carotid chemoafferent responses are suppressed in neonatal rats immediately after prolonged hyperoxia (Eden & Hanson, 1986). Moreover, hypoxic phrenic (Ling et al. 1997a) and ventilatory (Ling et al. 1996) responses are impaired in adult rats raised in hyperoxia. Similar deficits are not observed in rats exposed to the same level and duration of hyperoxia during adulthood, suggesting that the effect is unique to development. Adult hyperoxia-treated rats exhibit carotid body hypoplasia, chemoafferent degeneration (Erickson et al. 1998) and reduced carotid chemoafferent responses to intravenous NaCN and acute asphyxia (Ling et al. 1997b). By contrast, phrenic responses to electrical stimulation of carotid sinus nerves are similar in hyperoxia-treated and control rats, suggesting that central neural integration of chemoafferent inputs is not impaired by hyperoxia (Ling et al. 1997c). Our laboratory reported previously that hypoxic phrenic responses in a small sample (n = 5) of hyperoxia-treated rats recovered slowly and spontaneously over the course of normal ageing (Ling et al. 1998). However, subsequent experiments on a much larger sample of animals (n > 30) have called this finding into question (D. D. Fuller, R. W. Bavis, E. H. Vidruk & G. S. Mitchell, unpublished observations). At this time, we believe that the conclusion of Ling et al. (1998) was incorrect and, instead, that hyperoxia during development results in a life-long impairment of the hypoxic ventilatory response. Therefore, the fact that functional impairment can be actively reversed, as demonstrated in the present study, is highly significant from both a biological and, potentially, an applied perspective.

The hypoxic phrenic responses of hyperoxia-treated rats reported here are qualitatively consistent with earlier reports (see above), but suggest that functional impairment might be even greater than previously reported. In an earlier study, hyperoxia-treated rats exhibited significantly blunted hypoxic phrenic responses at a Pa,O2 of 50 mmHg, but not 40 mmHg (Ling et al. 1997a). The present study indicates that hypoxic responses are impaired at both 50 and 40 mmHg Pa,O2. The reasons for this discrepancy are unclear, but could include the smaller sample size in the earlier study, different techniques for establishing baseline (Ling et al. 1997a) and biological variability.

Sustained and intermittent hypoxia augment hypoxic ventilatory responses

Seven weeks of sustained hypoxia (partial pressure of inspired O2, PI,O2 = 80 mmHg) augments the short-term hypoxic ventilatory response in rats by approximately 50 % (Aaron & Powell, 1993). A shorter duration of sustained hypoxia also augments hypoxic ventilatory responses in goats (Engwall & Bisgard, 1990) and humans (Sato et al. 1992; Howard & Robbins, 1995). Most studies indicate that sustained hypoxia augments the carotid chemoafferent response to hypoxia and that this augmentation is sufficient to explain the increased hypoxic ventilatory response (Nielsen et al. 1988; Bisgard, 2000). Indeed, changes in the carotid body of normal animals after sustained hypoxia include increased tyrosine hydroxylase expression, enhanced release and turnover of dopamine, type I cell hypertrophy, vascular growth and an increase in vascular endothelial growth factor (reviewed in Bisgard, 2000). Recent carotid sinus nerve stimulation experiments suggest that the central neural integration of carotid chemoafferent inputs might also be enhanced following sustained hypoxia (Dwinell & Powell, 1999). Therefore, sustained hypoxia appears to evoke peripheral chemoreceptor plasticity with at least some central neural enhancement.

Chronic exposure to intermittent hypoxia augments ventilatory responses to hypoxia in humans (Serebrovskaya et al. 1999) and phrenic responses in rats (Ling et al. 1998b, 2000). The effects of short-term (that is, minutes to hours) intermittent hypoxia have been the subject of several recent reviews (Powell et al. 1998; Fuller et al. 2000; Mitchell et al. 2001). A chronic intermittent hypoxia treatment protocol, similar to that used in the present study, increases the short-term hypoxic phrenic response in rats by more than 100 % when Pa,O2 < 40 mmHg (Ling et al. 1998). We found recently that the central neural integration of carotid chemoafferent inputs is enhanced following intermittent hypoxia to an extent sufficient to account for all augmentation of the hypoxic phrenic response in normal rats (Ling et al. 2000). Therefore, a central mechanism appears to be the primary contributor to augmented hypoxic phrenic responses following intermittent hypoxia, although an additional effect at the carotid body chemoreceptors cannot be ruled out.

Intermittent hypoxia might have a different effect on hypoxic respiratory responses if administered during development (Moss, 2000). For example, 21- to 32-day-old piglets exposed to 30 min of hypoxia daily for 5 consecutive days had a blunted hypoxic ventilatory response (Waters et al. 1997). However, this protocol is considerably different from that used in the present study and, therefore, might have invoked mechanisms that are more consistent with the effects of sustained developmental hypoxic exposures (Okuba & Mortola, 1988; Hanson et al. 1989a,b). Other reports suggest possible beneficial effects of intermittent hypoxia during development. Indeed, an intermittent hypoxia protocol more similar to that used in the present study (although of shorter duration) diminished ventilatory roll-off during hypoxia in neonatal rats (Gozal & Gozal, 1999, 2001).

Functional recovery of hypoxic phrenic responses

The results of the present study suggest that short-term hypoxic phrenic responses in adult rats raised in hyperoxia are augmented immediately after intermittent and sustained hypoxia and that these treatments are equally effective. The data are equivocal as to whether 1 week of intermittent hypoxia is sufficient to return hypoxic phrenic responses of hyperoxia-treated rats to normal (control) levels, because the results from the blind experimental protocol suggest that there was partial recovery (e.g. Fig. 2), whereas an increased sample size (in the non-blinded protocol) indicated complete functional recovery following intermittent hypoxia (Fig. 3).

Critique of methods

Acute hypoxic phrenic responses were assessed in anaesthetized, vagotomized rats, treated with a neuromuscular blocking agent and mechanically ventilated. Major advantages of this preparation include the precise regulation of blood gasses and the ability to assess integrated neural responses to hypoxia, which bypasses potentially confounding influences from changes in pulmonary mechanics (Ling et al. 1997a). However, we are unable to state with certainty that our findings concerning phrenic motor output in anaesthetized rats are applicable to ventilatory responses in awake, spontaneously breathing animals. Nevertheless, the decrements in hypoxic responses in hyperoxia-treated rats are qualitatively and quantitatively similar to those in awake, spontaneously breathing rats assessed via plethysmography (Ling et al. 1996) and anaesthetized rats assessed via phrenic recordings (the present study). Therefore, there is no compelling reason to suspect that the functional recovery reported here would be different in awake, spontaneously breathing animals.

Carotid body morphology

Experiments on normal, untreated animals (see above) suggest that the primary mechanism of enhanced hypoxic responses following sustained hypoxia is carotid body chemoafferent sensitization/recovery. The carotid body volume data (Fig. 6) indicate that such a mechanism is also operative in hyperoxia-treated rats, although these data must be interpreted with caution, because carotid body volume does not necessarily provide a reliable indicator of carotid body function. Nevertheless, because sustained hypoxia increases carotid body volume and hypoxic sensitivity in normal animals (Bisgard & Neubauer, 1995), and carotid body volume is diminished in hyperoxia-treated rats (Fig. 5; Erickson et al. 1998), it is tempting to speculate that increased carotid body volume in hyperoxia-treated rats after sustained hypoxia reflects carotid body plasticity that might invoke functional recovery.

In contrast to sustained hypoxia, chronic intermittent hypoxia did not influence carotid body volume in the present study (Fig. 6), yet evoked a significant increase in hypoxic phrenic responsiveness (Fig. 3). Although this observation does not rule out an effect on carotid body function, it is consistent with the interpretation that intermittent hypoxia enhances the central neural integration of carotid chemoafferent inputs in hyperoxia-treated rats. This interpretation is consistent with the effects of intermittent hypoxia in control rats (Ling et al. 2000) and could occur in the brainstem or spinal cord.

Possible significance

In recent years, an appreciation of the plastic nature of the respiratory neural control system has emerged (Eden & Hanson, 1986, 1987; Eldridge & Milhorn, 1986; Okubo & Mortolo, 1988, 1990; Mitchell et al. 1990; McCrimmon et al. 1995; Poon, 1996; Ling et al. 1997b). This study indicates that respiratory plasticity in adult rats can be used to offset functional deficits caused by abnormal experiences during development, such as hyperoxia. Katz-Salomon and colleagues examined peripheral chemoreflexes in healthy infants and infants with bronchopulmonary displasia following long-term oxygen therapy (Katz-Salomon & Lagercrantz, 1994; Katz-Salomon et al. 1995, 1996). Their findings suggest that experiences in early life, as well as treatment with supplemental oxygen, can have deleterious effects, because these infants displayed significantly blunted peripheral chemoreflexes. It is not known whether these deficits in peripheral chemosensitivity have a significant impact on ventilation during normal breathing or whether they persist throughout adulthood. However, if deficits in peripheral chemosensitivity do persist throughout adulthood, our data suggest that the inherent plasticity of the respiratory neural control system might allow (at least partial) restoration of chemosensory function. Moreover, because the effects of intermittent hypoxia appear to persist for at least 1 week following treatment, this might represent a viable therapeutic strategy. On the other hand, because some intermittent hypoxia protocols are known to elicit significant pathology, such as systemic hypertension (Greenberg et al. 1999) or hippocampal apoptosis (Gozal et al. 2001), its direct use as a therapeutic tool might be limited. Regardless, an understanding of spontaneous and evoked functional recovery following early life exposure to hyperoxia will advance our understanding of plasticity in respiratory motor control in general.

Acknowledgments

This work was supported by NIH grant HL53319 and Training Grant HL 07654.

References

  1. Aaron EA, Powell FL. Effect of chronic hypoxia on hypoxic ventilatory response in awake rats. Journal of Applied Physiology. 1993;74:1635–1640. doi: 10.1152/jappl.1993.74.4.1635. [DOI] [PubMed] [Google Scholar]
  2. Bao G, Randhawa PM, Fletcher EC. Acute blood pressure elevation during repetitive hypocapnic and eucapnic hypoxia in rats. Journal of Applied Physiology. 1997;82:1071–1078. doi: 10.1152/jappl.1997.82.4.1071. [DOI] [PubMed] [Google Scholar]
  3. Barker DJ, Godfrey KM, Fall C, Osmond C, Winter PD, Shaheen SO. Relation of birth weight and childhood respiratory infection to adult lung function and death from chronic obstructive airways disease. British Medical Journal. 1991;303:671–675. doi: 10.1136/bmj.303.6804.671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bisgard GE. Carotid body mechanisms in acclimatization to hypoxia. Respiration Physiology. 2000;121:237–246. doi: 10.1016/s0034-5687(00)00131-6. [DOI] [PubMed] [Google Scholar]
  5. Bisgard GE, Neubauer JA. Peripheral and central effects of hypoxia. In: Dempsey JA, Pack AI, editors. Regulation of Breathing. New York: Marcel Dekker; 1995. pp. 617–618. [Google Scholar]
  6. Dwinell MR, Powell FL. Chronic hypoxia enhances the phrenic nerve response to arterial chemoreceptor stimulation in anaesthetised rats. Journal of Applied Physiology. 1999;87:817–823. doi: 10.1152/jappl.1999.87.2.817. [DOI] [PubMed] [Google Scholar]
  7. Eden GJ, Hanson MA. Effect of hyperoxia from birth on the carotid chemoreceptor and ventilatory responses of rats to acute hypoxia. Journal of Physiology. 1986;374:24. P. [Google Scholar]
  8. Eden GJ, Hanson MA. Effects of chronic hypoxia from birth on the ventilatory response to acute hypoxia in the newborn rat. Journal of Physiology. 1987;392:11–19. doi: 10.1113/jphysiol.1987.sp016766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Engwall MJA, Bisgard GE. Ventilatory responses to chemoreceptor stimulation after hypoxic acclimatization in awake goats. Journal of Applied Physiology. 1990;69:1236–1243. doi: 10.1152/jappl.1990.69.4.1236. [DOI] [PubMed] [Google Scholar]
  10. Erickson JT, Mayer C, Jawa A, Ling L, Olson EB, Jr, Vidruk EH, Mitchell GS, Katz DM. Chemoafferent degeneration and carotid body hypoplasia following chronic hyperoxia in newborn rats. Journal of Physiology. 1998;509:519–526. doi: 10.1111/j.1469-7793.1998.519bn.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gozal D, Daniel JM, Dohanich GP. Behavioral and anatomical correlates of chronic episodic hypoxia during sleep in the rat. Journal of Neuroscience. 2001;21:2442–2450. doi: 10.1523/JNEUROSCI.21-07-02442.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gozal D, Gozal E. Episodic hypoxia enhances late hypoxic ventilation in the developing rat: putative role of neuronal NO synthase. American Journal of Physiology. 1999;276:R17–22. doi: 10.1152/ajpregu.1999.276.1.R17. [DOI] [PubMed] [Google Scholar]
  13. Gozal E, Gozal D. Intermittent hypoxia during development: critical windows of vulnerability and plasticity. Journal of Applied Physiology. 2001;90:1995–1999. doi: 10.1152/jappl.2001.90.5.1995. [DOI] [PubMed] [Google Scholar]
  14. Greenberg HE, Sica A, Batson D, Scharf SM. Chronic intermittent hypoxia increases sympathetic responsiveness to hypoxia and hypercapnia. Journal of Applied Physiology. 1999;86:298–305. doi: 10.1152/jappl.1999.86.1.298. [DOI] [PubMed] [Google Scholar]
  15. Hanson MA, Eden GJ, Nijhuis JG, Moore PJ. Peripheral chemoreceptors and other oxygen sensors in the fetus and newborn. In: Lahiri S, Forster RE, Davies RO, Pack AI, editors. Chemoreceptors and Reflexes in Breathing: Cellular and Molecular Aspects. New York: Oxford University Press; 1989a. pp. 113–120. [Google Scholar]
  16. Hanson MA, Kumar P, Williams BA. The effects of chronic hypoxia upon the development of respiratory chemoreflexes in the newborn kitten. Journal of Physiology. 1989b;411:563–574. doi: 10.1113/jphysiol.1989.sp017590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Howard LSGE, Robbins PA. Alterations in respiratory control during 8 h of isocapnic and poikilocapnic hypoxia in humans. Journal of Applied Physiology. 1995;78:1098–1107. doi: 10.1152/jappl.1995.78.3.1098. [DOI] [PubMed] [Google Scholar]
  18. Katz-Salamon M, Eriksson M, Jonsson B. Development of peripheral chemoreceptor function in infants with chronic lung disease and initially lacking hyperoxia response. Archives of Disease in Childhood. 1996;75:F4–9. doi: 10.1136/fn.75.1.f4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Katz-Salamon M, Jonsson B, Lagercrantz H. Blunted peripheral chemoreceptor response to hyperoxia in a group of infants with bronchopulmonary dysplasia. Pediatric Pulmonology. 1995;20:101–106. doi: 10.1002/ppul.1950200209. [DOI] [PubMed] [Google Scholar]
  20. Katz-Salamon M, Lagercrantz H. Hypoxic ventilatory defense in very preterm infants: attenuation after long term oxygen treatment. Archives of Disease in Childhood. 1994;70:F90–95. doi: 10.1136/fn.70.2.f90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ling L, Fuller DD, Bach KB, Kinkead R, Olson EB, Jr, Mitchell GS. Chronic intermittent hypoxia elicits serotonin-dependent plasticity in the central neural control of breathing. Journal of Neuroscience. 2001;21:5381–5388. doi: 10.1523/JNEUROSCI.21-14-05381.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ling L, Olson EB, Jr, Vidruk EH, Mitchell GS. Attenuation of the hypoxic ventilatory response in adult rats following one month of hyperoxia. Journal of Physiology. 1996;495:561–571. doi: 10.1113/jphysiol.1996.sp021616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ling L, Olson EB, Jr, Vidruk EH, Mitchell GS. Phrenic responses to isocapnic hypoxia in adult rats following hyperoxia. Respiration Physiology. 1997a;109:107–116. doi: 10.1016/s0034-5687(97)00045-5. [DOI] [PubMed] [Google Scholar]
  24. Ling L, Olson EB, Jr, Vidruk EH, Mitchell GS. Developmental plasticity of the hypoxic ventilatory response. Respiration Physiology. 1997b;110:261–268. doi: 10.1016/s0034-5687(97)00091-1. [DOI] [PubMed] [Google Scholar]
  25. Ling L, Olson EB, Jr, Vidruk EH, Mitchell GS. Integrated phrenic responses to carotid afferent stimulation in adult rats following hyperoxia. Journal of Physiology. 1997c;500:787–796. doi: 10.1113/jphysiol.1997.sp022058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ling L, Olson EB, Jr, Vidruk EH, Mitchell GS. Slow recovery of impaired responses to hypoxia following hyperoxia in rats. Journal of Physiology. 1998;511:599–603. doi: 10.1111/j.1469-7793.1998.599bh.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mitchell GS, Baker TL, Nanda SA, Fuller DD, Zabka AG, Hodgeman BA, Bavis RW, Mack KI, Olson EB., Jr Intermittent hypoxia and respiratory plasticity. Journal of Applied Physiology. 2001;90:2466–2475. doi: 10.1152/jappl.2001.90.6.2466. [DOI] [PubMed] [Google Scholar]
  28. Mitchell GS, Douse MA, Foley KT. Receptor interactions in modulating ventilatory activity. American Journal of Physiology. 1990;259:R911–920. doi: 10.1152/ajpregu.1990.259.5.R911. [DOI] [PubMed] [Google Scholar]
  29. Moss IR. Respiratory responses to single and episodic hypoxia during development: mechanisms of adaptation. Respiration Physiology. 2000;121:185–197. doi: 10.1016/s0034-5687(00)00127-4. [DOI] [PubMed] [Google Scholar]
  30. Nielsen AM, Bisgard GE, Vidruk EH. Carotid chemoreceptor activity during acute and sustained hypoxia in goats. Journal of Applied Physiology. 1988;65:1796–1802. doi: 10.1152/jappl.1988.65.4.1796. [DOI] [PubMed] [Google Scholar]
  31. Okubo S, Mortola JP. Long-term respiratory effects of neonatal hypoxia in the rat. Journal of Applied Physiology. 1988;64:952–958. doi: 10.1152/jappl.1988.64.3.952. [DOI] [PubMed] [Google Scholar]
  32. Okubo S, Mortola JP. Control of ventilation in adult rats hypoxic in the neonatal period. American Journal of Physiology. 1990;259:R836–841. doi: 10.1152/ajpregu.1990.259.4.R836. [DOI] [PubMed] [Google Scholar]
  33. Olson EB, Jr, Dempsey JA. The rat as a model for human-like ventilatory adaptation to chronic hypoxia. Journal of Applied Physiology. 1978;44:763–769. doi: 10.1152/jappl.1978.44.5.763. [DOI] [PubMed] [Google Scholar]
  34. Poon CS. Synaptic plasticity and respiratory control. In: Khoo MCK, editor. Bioengineering Approaches to Pulmonary Physiology and Medicine. New York: Plenum Press; 1996. pp. 93–113. [Google Scholar]
  35. Sato M, Severinghaus JW, Powell FL, Xu FD, Spellman MJ., Jr Augmented hypoxic ventilatory response in men at altitude. Journal of Applied Physiology. 1992;77:313–316. doi: 10.1152/jappl.1992.73.1.101. [DOI] [PubMed] [Google Scholar]
  36. Serebrovskaya TV, Karaban IN, Kolesnikova EE, Mishunia TM, Kusiminskaya LA, Serbrovsky AN, Swanson RJ. Human hypoxic ventilatory response with blood dopamine content under intermittent hypoxic training. Canadian Journal of Physiology and Pharmacology. 1999;77:967–973. [PubMed] [Google Scholar]
  37. Shaheen SO, Barker DJ, Shiell AW, Crocker FJ, Wield GA, Holgate ST. The relationship between pneumonia in early childhood and impaired lung function in late adult life. American Journal of Respiratory and Critical Care Medicine. 1994;149:616–619. doi: 10.1164/ajrccm.149.3.8118627. [DOI] [PubMed] [Google Scholar]
  38. Sladek M, Parker RA, Grogaard JB, Sundell HW. Long-lasting effect of prolonged hypoxemia after birth on the immediate ventilatory response to changes in arterial partial pressure of oxygen in young lambs. Pediatric Research. 1993;34:821–828. doi: 10.1203/00006450-199312000-00025. [DOI] [PubMed] [Google Scholar]
  39. Sorensen SC, Severinghaus JW. Irreversible respiratory insensitivity to acute hypoxia in man born at high altitude. Journal of Applied Physiology. 1968;25:217–220. doi: 10.1152/jappl.1968.25.3.217. [DOI] [PubMed] [Google Scholar]
  40. Sterni LM, Bamford OS, Wasicko MJ, Carroll JL. Chronic hypoxia abolished the postnatal increase in carotid body type I cell sensitivity to hypoxia. American Journal of Physiology. 1999;277:L645. doi: 10.1152/ajplung.1999.277.3.L645. [DOI] [PubMed] [Google Scholar]
  41. Waters KA, Laferriere A, Paquette J, Goodyear C, Moss IR. Curtailed respiration by repeated vs isolated hypoxia in maturing piglets is unrelated to NTS ME or SP levels. Journal of Applied Physiology. 1997;83:522–529. doi: 10.1152/jappl.1997.83.2.522. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES