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. 2002 Feb 1;538(Pt 3):947–955. doi: 10.1113/jphysiol.2001.012908

Life-long impairment of hypoxic phrenic responses in rats following 1 month of developmental hyperoxia

D D Fuller *, R W Bavis *, E H Vidruk *, Z-Y Wang *, E B Olson Jr *, G E Bisgard *, G S Mitchell *
PMCID: PMC2290109  PMID: 11826178

Abstract

Hypoxic ventilatory and phrenic responses are reduced in adult rats (3–5 months old) exposed to hyperoxia for the first month of life (hyperoxia treated). We previously reported that hypoxic phrenic responses were normal in a small sample of 14- to 15-month-old hyperoxia-treated rats, suggesting slow, spontaneous recovery. Subsequent attempts to identify the mechanism(s) underlying this spontaneous recovery of hypoxic phrenic responses led us to re-evaluate our earlier conclusion. Experiments were conducted in two groups of aged Sprague-Dawley rats (14–15 months old) which were anaesthetized, vagotomized, neuromuscularly blocked and ventilated: (1) a hyperoxia-treated group raised in 60 % O2 for the first 28 postnatal days; and (2) an age-matched control group raised in normoxia. Increases in minute phrenic activity and integrated phrenic nerve amplitude (∫Phr) during isocapnic hypoxia (arterial partial pressures of O2, 60, 50 and 40 ± 1 mmHg) were greater in aged control (n = 15) than hyperoxia-treated rats (n = 11; P≤ 0.01). Phrenic burst frequency during hypoxia was not different between groups. To examine the central integration of carotid chemoafferent inputs, steady-state relationships between carotid sinus nerve (electrical) stimulation frequency and phrenic nerve activity were compared in aged control (n = 7) and hyperoxia-treated rats (n = 7). Minute phrenic activity, ∫Phr and burst frequency were not different between groups at any stimulation frequency between 0.5 and 20 Hz. Carotid body chemoreceptor function was examined by recording whole carotid sinus nerve responses to cessation of ventilation or injection of cyanide in aged control and hyperoxia-treated rats. Electrical activity of the carotid sinus nerve did not change in five out of five hyperoxia-treated rats in response to stimuli that evoked robust increases in carotid sinus nerve activity in five out of five control rats. Estimates of carotid body volume were lower in aged hyperoxia-treated rats (4.4 (± 0.2) × 106μm3) compared to controls (17.4 (± 1.6) × 106μm3; P <0.01). We conclude that exposure to hyperoxia for the first month of life causes life-long impairment of carotid chemoreceptor function and, consequently, blunted phrenic responses to hypoxia.

Development of the hypoxic ventilatory response is impaired by prolonged increases or decreases in ambient oxygen during developmental periods (Eden & Hanson, 1986, 1987; Hansen et al. 1989; Okubo & Mortola, 1990; Sladek et al. 1993; Ling et al. 1997c; Peyronnet et al. 2000). For example, hyperoxia (fractional inspired oxygen, FI,O2 = 0.60) for the first month of life reduces both ventilatory and phrenic responses to acute hypoxia in young adult rats (age 3–4 months; Ling et al. 1996, 1997b). This functional impairment can be accounted for by impaired carotid body chemoreceptor structure and function (Ling et al. 1997c; Erickson et al. 1998). To investigate the persistence of this impairment, our laboratory examined hypoxic phrenic responses in a small sample of 15-month-old rats that had spent the first month of life in hyperoxia (Ling et al. 1998). The results of this study suggested slow, spontaneous functional recovery of hypoxic responses, reaching levels near normal in aged, hyperoxia-treated rats.

Based on the data of Ling et al. (1998), we hypothesized that functional recovery in aged hyperoxia-treated rats resulted from either augmented carotid chemosensitivity (peripheral mechanism) or an enhanced central integration of carotid chemoafferent inputs (central mechanism), or both. Experiments designed to test this hypothesis suggested that our earlier conclusion was incorrect and that hypoxic phrenic responses in hyperoxia-treated rats do not spontaneously recover with age. In this paper, we present the results of comprehensive experiments designed to re-evaluate the extent of spontaneous functional recovery in aged (14- to 15-month-old) hyperoxia-treated rats. The results uniformly refute our earlier report (Ling et al. 1998) and suggest that exposure to hyperoxia for the first 28 postnatal days causes life-long impairment of hypoxic phrenic responses.

Methods

Experiments were performed on fifty 14- to 15-month-old male and four 15-month-old female Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., colony 236b, 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 groups

Hyperoxia

The hyperoxia treatment has been described previously (Ling et al. 1996). Briefly, rats were exposed to hyperoxia (FI,O2 = 0.60) from 2–3 days before birth, through the first 28 postnatal days. All animals were then returned to normoxia until acute neurophysiological experiments were performed at 14–15 months of age (n = 23 males, weight 565 ± 11 g; n = 2 females, weight 390 and 361 g).

Control

Age-matched control rats were reared in normoxia (n = 27 males, weight 538 ± 7 g; n = 2 females, weight 296 and 321 g).

Experimental preparation and measurements

Preparation

In acute neurophysiological experiments to investigate phrenic responses to hypoxia or electrical stimulation of the carotid sinus nerve, rats were initially placed in a closed chamber and anaesthetized with isoflurane. Anaesthesia (2.5–3.5 % isoflurane; FI,O2 = 0.5) was then maintained via a nose cone and then a tracheal cannula. Rats were converted to urethane anaesthesia (2.0 m, 5 ml, i.v.) over a period of 30 min. For experiments investigating carotid sinus nerve responses to asphyxia or cyanide, anaesthesia was induced via i.p. injection of isotonic (0.329 m) urethane (4 ml (100 g)−1). In all animals, the adequacy of anaesthesia was periodically confirmed by a lack of blood pressure and phrenic response to toe pinch; supplemental urethane (0.3 mg kg−1i.v.) was given as needed. Rats were tracheotomized and mechanically ventilated during all experiments.

A femoral venous catheter was inserted to allow injection of anaesthetic and fluids. A femoral arterial catheter enabled measurement of blood pressure (Statham P23ID, Gould Instruments) and blood sampling. Arterial partial pressures of O2 (Pa,O2) and CO2 (Pa,CO2) and pH were measured at selected times throughout the protocol (see below) from 0.2 ml arterial samples with a blood gas analysis system (ABL-500 or ABL-300, Radiometer, Copenhagen, Denmark); unused blood was returned to the animal. Neuromuscular blockade was established with either pancuronium bromide (phrenic responses; 2.5 mg kg−1, i.v.) or gallamine triethiodide (carotid sinus nerve responses; 5 mg kg−1, i.v.). The vagus nerves were cut in the midcervical region in all rats except those used for carotid sinus nerve recordings. The end-tidal CO2 partial pressure (PET,CO2) was measured using a rapidly responding, flow-through CO2 analyser (Novametrix, model 1265, Wallingford, CT, USA) placed on the expiratory line of the ventilator circuit. Rectal temperature was maintained at 37–39 °C using a rectal thermistor and a heated table or heating pad. At the conclusion of each experiment, the rat was killed by systemic perfusion (see below) or urethane overdose.

Phrenic nerve recording

The phrenic nerve was isolated using a dorsal approach, cut distally and desheathed. The whole 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).

Carotid sinus nerve stimulation and recording

The left carotid sinus nerve was isolated via a dorsal approach, cut distally and mounted on a fine bipolar silver wire electrode connected to a stimulator (Grass S88 Stimulator; Grass Instrument Co., Quincy, MA, USA). The nerve was stimulated (pulse duration 0.2 ms) with constant currents ranging from 20 to 180 μA at varied stimulation frequencies (0.5–20 Hz; see Protocols, below).

For carotid sinus nerve recordings, the left carotid sinus nerve was exposed with a ventral approach, separated from the glossopharyngeal nerve and then cut. The nerve was placed on one lead of a pair of bipolar platinum electrodes; the other lead was earthed to the animal. The electrical signal was amplified with a Grass P15 AC preamplifier (low-pass filter 300 Hz; high-pass filter 10 000 Hz) and recorded on a polygraph (Grass, model 7D). To standardize the baseline activity in each preparation, the tidal volume and frequency of the ventilator were adjusted to maintain Pa,O2 and Pa,CO2 at approximately 90 and 40 mmHg, respectively (Tables 1C and 2C). A window discriminator and ratemeter (Frederick Haer & Co., Bowdoinham, ME, USA) were then set to detect spontaneous carotid sinus nerve discharge rates of 200 Hz. Therefore, all animals had the same (arbitrarily determined) baseline discharge frequency. Subsequent experiments (see Protocols, below) determined how carotid sinus nerve discharge frequency changed during carotid body stimulation, but our recording technique did not provide information regarding the baseline discharge frequency of the carotid sinus nerve.

Table 1.

Pa,CO2 (mmHg) during each experimental protocol in aged control and hyperoxia-treated rats

Control Hyperoxia treated
A.
Baseline 45 ± 1 47 ± 1
60 mmHg 45 ± 1 47 ± 1
50 mmHg 45 ± 1 47 ± 1
40 mmHg 45 ± 1 47 ± 1
B.
Before 46 ± 2 46 ± 1
After 46 ± 2 46 ± 1
C.
Before NaCN 40 ± 1 40 ± 1
After NaCN 39 ± 1 40 ± 1
Before asphyxia 39 ± 1 40 ± 1
After asphyxia 40 ± 1 40 ± 1
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Strict isocapnia (relative to baseline conditions) was maintained at all times. A, values during baseline and at each of 3 levels of hypoxia. B, values just before and immediately following carotid sinus nerve electrical stimulation. C, values just before and immediately following intravenous NaCN injection and asphyxia.

Table 2.

Pa,O2 (mmHg) during each experimental protocol in aged control and hyperoxia-treated rats

Control Hyperoxia treated
A.
Baseline 237 ± 8 237 ± 12
60 mmHg 61 ± 1 59 ± 1
50 mmHg 50 ± 1 51 ± 1
40 mmHg 40 ± 1 40 ± 1
B.
Before 227 ± 9 222 ± 5
After 217 ± 5 210 ± 10
C.
Before NaCN 91 ± 2 93 ± 2
After NaCN 89 ± 4 98 ± 2
Before asphyxia 98 ± 2 89 ± 4
After asphyxia 91 ± 2 87 ± 3
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A, values during baseline conditions and at each of 3 levels of hypoxia. B, values just before and immediately following carotid sinus nerve electrical stimulation. C, values just before and immediately following intravenous NaCN injection and asphyxia.

Protocols

Hypoxic phrenic responses

Control (n = 15) and hyperoxia-treated rats (n = 11) were studied. Sixty minutes after conversion to urethane anaesthesia, the CO2 apnoeic threshold for inspiratory phrenic nerve activity was established by increasing the ventilator rate until inspiratory phrenic nerve activity ceased, and then slowly decreasing the rate of the ventilator until activity resumed. The PET,CO2 was then maintained at 3 mmHg above this level by adjusting the ventilator rate and/or inspired CO2 content as needed. After stable baseline phrenic activity was established (∼20–30 min), rats were exposed to 5 min bouts of isocapnic hypoxia, separated by 5–10 min of hyperoxia (FI,O2 = 0.50). Arterial blood samples were drawn before the first hypoxic episode and in the final minute of each hypoxic episode. Target Pa,O2 values of 60, 50 and 40 mmHg were achieved by adjusting the inspired O2 concentration. Hypoxic trials were only accepted if Pa,O2 was within 3 mmHg of the target and Pa,CO2 was within 2 mmHg of baseline. At the conclusion of each experiment a ‘maximum’ phrenic response was established by increasing PET,CO2 to 80–90 mmHg.

Carotid sinus nerve stimulation

Control (n = 7) and hyperoxia-treated rats (n = 7) were studied. Initially the CO2 apnoeic threshold was established as described above (see Hypoxic phrenic responses). The threshold stimulus current was then established by determining the minimum current necessary to elicit a detectable phrenic response to brief 20 Hz (0.2 ms pulse duration) stimulation. The stimulus current (3 times threshold) and pulse duration (0.2 ms) were then held constant, but stimulus frequency was changed in seven 45 s episodes of carotid sinus nerve stimulation: 0.5, 1, 2, 4, 8, 16 and 20 Hz, separated by a 4 min interval. The 45 s duration of stimulation was chosen as a compromise to minimize blood pressure and blood gas disturbances, while enabling phrenic amplitude to reach a plateau. Blood pressure always returned to prestimulation levels during the 4 min interval. After the final stimulus episode, the phrenic response to a 5 min episode of severe hypercapnia (PET,CO2 80–90 mmHg) was determined. To test whether phrenic responses to carotid sinus nerve stimulation were due to current spread, the proximal end of the carotid sinus nerve was crushed with forceps at the conclusion of each experiment; in all cases phrenic responses could no longer be elicited by carotid sinus nerve stimulation.

Carotid sinus nerve recordings

Control (n = 5) and hyperoxia-treated rats (n = 5) were studied. After stable baseline conditions had been established (see above), carotid sinus nerve chemoafferent responses to two different stimuli were assessed as previously reported (Ling et al. 1997c; see Fig. 4). The first stimulus was an i.v. injection of NaCN (20 μg in 0.2 ml saline); the injection was made three times with at least 2 min separating the trials. The second challenge was a 10 s bout of asphyxia produced by stopping the mechanical ventilator. This test was repeated three times at 3 min intervals.

Figure 4. Effects of NaCN (20 μg i.v.) and asphyxia (10 s) on the activity of the whole carotid sinus nerve.

Figure 4

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The upper records are from a control rat, the lower from a hyperoxia-treated rat. Injections of NaCN are indicated by the arrows beneath the ratemater records. Asphyxia is marked by the fall in tracheal pressure (PTr) to 0 cmH2O caused by stopping the ventilator for 10 s. At least 2 min elapsed between the NaCN injections and 3 min between the asphyxic trials. The 10 s time bar applies to all records. See text for description of carotid sinus nerve recording techniques.

Estimation of carotid body volume

The volume of the carotid body was estimated in five control and five hyperoxia-treated rats. Following acute neurophysiological experiments, rats were perfused through the heart 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 placed in Bouin's fixative overnight. Following routine paraffin embedding, the tissue was sectioned serially at 5 μm with a microtome (approximately 200 sections). Every fourth section containing the carotid body was processed with Haematoxylin and Eosin staining and images were obtained with a Nikon E600 microscope (×200) and imported to a computer using a Diagnostics Instruments Spot digital camera. The area of each carotid body section was determined using Image-Pro Plus computer software (Media Cybernetics, Silver Spring, MD, USA); calibrations were made via micrometer. Carotid body volume was estimated based on the area of each section, section thickness and the total number of carotid body sections.

Data analysis

In all experiments, a blinded design was employed to conceal the treatment (i.e. hyperoxia treated vs. control) from the experimenter during data collection and analysis. For the hypoxia experiments, phrenic nerve activity was averaged over 30 s periods immediately before the first hypoxic episode (baseline), during the fifth minute of hypoxia and at the end of the hypercapnic response. For the carotid sinus nerve stimulation experiments, phrenic nerve activity was averaged over 15 s periods immediately before the first stimulation episode and at the conclusion of each 45 s stimulation period. The following variables were determined from the phrenic recordings: peak integrated phrenic amplitude (∫Phr), phrenic burst frequency (bursts per minute) and minute phrenic activity (∫Phr × frequency). Changes in peak phrenic amplitude were expressed relative to phrenic nerve activity during both baseline conditions and hypercapnia (Bach & Mitchell, 1996). For the carotid sinus nerve recording experiments, the responses to three separate NaCN and asphyxia trials were averaged and expressed relative to baseline activity and the peak response. All data are presented as percentage change from baseline since all results were qualitatively similar when expressed in either manner (% baseline or % maximum).

Statistical comparisons of phrenic responses, blood gases and blood pressure during hypoxia and CSN stimulation within and between groups were made using two-way repeated measures analyses of variance followed by Student-Neuman-Keuls post hoc tests (Sigma Stat v. 2.03; Jandel Scientific, St Louis, MO, USA). Statistical differences between groups for CO2 apnoeic threshold, carotid body volume and carotid sinus nerve activity were determined using Student's unpaired t test. Differences were considered significant when P < 0.05. All data are presented as the means ± 1 s.e.m.

Results

Apnoeic threshold

The end-tidal CO2 at which phrenic inspiratory activity began was not different between aged control (42 ± 1 mmHg) and hyperoxia-treated rats (44 ± 1 mmHg; P = 0.13).

Blood gases

The arterial partial pressure of CO2 was isocapnic relative to baseline conditions throughout all protocols (Table 1). During hypoxia, mean Pa,O2 was within 1 mmHg of the target values (i.e. 40, 50 and 60 mmHg) in all groups (Table 2A). Hyperoxia was maintained throughout carotid sinus nerve stimulation protocols (Pa,O2 > 200 mmHg; Table 2B). Carotid sinus nerve afferent recordings were made in normoxia (Pa,O2 87–100 mmHg; Table 2C).

Blood pressure

Baseline mean arterial pressure was not different between groups (control, 109 ± 5 mmHg; hyperoxia treated, 112 ± 7 mmHg). During hypoxia, mean arterial pressure declined to a similar extent in both groups (at 40 mmHg Pa,O2: control, 86 ± 6 mmHg; hyperoxia treated, 86 ± 7 mmHg). Changes in mean arterial pressure induced by carotid sinus nerve stimulation, NaCN injection or asphyxia were not significantly different between groups.

Hypoxic phrenic responses

Representative records depicting the ∫Phr response to hypoxia are presented in Fig. 1 and illustrate that increases in ∫Phr during hypoxia were smaller in aged hyperoxia-treated rats. Indeed, ∫Phr during hypoxia was significantly lower in aged hyperoxia-treated rats at all hypoxic levels (P = 0.008; Fig. 2). Although increases in phrenic burst frequency during hypoxia were not significantly different between groups (P = 0.18; Fig. 2), hypoxia-induced increases in minute phrenic activity were significantly greater in control than hyperoxia-treated rats (P = 0.011; Fig. 2). Hypoxic phrenic responses were also assessed in two control and two hyperoxia-treated 14-month-old female rats. These data were not pooled with data from the males because hypoxic phrenic responses are greater in aged female than male Sprague-Dawley rats (A.G. Zabka & G. S. Mitchell, unpublished observations). Nevertheless, the data were qualitatively similar to males: at 40 mmHg Pa,O2, increases in ∫Phr were 61 and 82 % baseline in the two hyperoxia-treated female rats compared to 210 and 129 % in controls.

Figure 1. Representative recordings of integrated phrenic nerve activity in two aged (14-month-old) rats, one that was reared in normoxia (control) and one that was exposed to hyperoxia for the first 28 postnatal days.

Figure 1

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In contrast to the control animals, the hyperoxia-treated rats showed little change (relative to baseline) in the amplitude of the integrated phrenic burst during hypoxia (Pa,O2, 40 mmHg). No consistent differences in phrenic burst frequency were observed between control and hyperoxia-treated rats.

Figure 2. Hypoxic phrenic responses in aged control (•) and hyperoxia-treated rats (▪).

Figure 2

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Mean changes from baseline in the peak amplitude of integrated phrenic activity (Δ∫Phr, % baseline), phrenic burst frequency (Δf, bursts min−1) and minute phrenic activity (Δ(∫Phr × frequency), % baseline) were assessed at 3 levels of isocapnic hypoxia (Pa,O2, 60, 50 and 40 ± 1 mmHg). Asterisk indicates that hyperoxia-treated is significantly less than control value.

Carotid sinus nerve electrical stimulation

The threshold current (20 Hz, 0.2 ms duration pulse) which evoked a phrenic response was not significantly different between hyperoxia-treated (54 ± 8 μA) and control rats (32 ± 11 μA; P = 0.13). At fixed stimulus current (3 times threshold) and pulse duration (0.2 ms), ∫Phr, burst frequency and minute phrenic activity all increased with stimulation frequency in both groups (all P < 0.05). Mean values of ∫Phr, burst frequency and minute phrenic activity were not significantly different between groups at any stimulation frequency (all P > 0.05; Fig. 3).

Figure 3. Changes in phrenic nerve activity during electrical stimulation of the carotid sinus nerve in aged control (•) and hyperoxia-treated rats (▪).

Figure 3

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Mean changes from baseline in peak amplitude of integrated phrenic activity (Δ∫Phr, % baseline), phrenic burst frequency (Δf, bursts min−1) and minute phrenic activity (Δ(∫Phr × frequency), % baseline) were quantified at stimulation frequencies between 0.5 and 20 Hz. No significant differences were present between groups.

Carotid sinus nerve recordings

Examples of carotid sinus nerve responses to asphyxia and NaCN are presented in Fig. 4. Electrical activity of the carotid sinus nerve did not increase during asphyxia or intravenous NaCN in five out of five hyperoxia-treated rats (Fig. 5). In contrast, both of these stimuli caused a substantial increase in carotid sinus nerve electrical activity in five out of five control rats (Fig. 5). Differences in the carotid sinus nerve response to NaCN (P = 0.003) and asphyxia (P = 0.008) were highly significant between control and hyperoxia-treated rats (Fig. 5).

Figure 5. Increases in carotid sinus whole nerve activity (% baseline) during asphyxia and intravenous NaCN injection in aged control (▪) and hyperoxia-treated rats (□).

Figure 5

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Both of these stimuli evoked a robust response in control animals but did not alter carotid sinus nerve discharge in hyperoxia-treated rats. Asterisk indicates value significantly less than control.

Carotid body volume

Carotid body volume was significantly less in hyperoxia-treated (4.4 (± 0.2) × 106μm3) than in control rats (17.4 (± 1.6) × 106μm3; P = 0.003; see Fig. 6). The representative carotid body sections presented in Fig. 7 demonstrate that hyperoxia induced carotid body hypoplasia. Moreover, the volume occupied by type I cells and vascular structures was proportionally decreased, similar to young adult (age 3–5 months) hyperoxia-treated rats (Erickson et al. 1998; D. D. Fuller, Z.-Y. Wang, G. E. Bisgard & G. S. Mitchell, unpublished observations).

Figure 6. Calculated carotid body volume in aged control and hyperoxia-treated rats.

Figure 6

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Consistent with previous reports in young adult rats (Erickson et al. 1998), carotid body volume was significantly lower in aged hyperoxia-treated rats compared to age-matched controls. Asterisk indicates value significantly less than control.

Figure 7. Representative images of tissue sections taken from the carotid body of two 15-month-old rats: a control rat, and a rat which was exposed to hyperoxia (FI,O2 = 0.60) for the first 28 postnatal days (hyperoxia treated).

Figure 7

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In each panel the boundaries of the carotid body are marked with a dashed line. The images show that exposure to hyperoxia for the first month of life resulted in carotid body hypoplasia. See text for a description of tissue preparation and carotid body volume quantification.

DISCUSSION

Summary

In contrast to a previous report from our laboratory (Ling et al. 1998), four separate sets of experiments converge on the conclusion that functionally impaired hypoxic phrenic responses do not spontaneously recover with age in rats exposed hyperoxia for the first month of life. Hypoxic phrenic responses were lower in a large group of aged (14- to 15-month-old) hyperoxia-treated rats relative to control rats. Furthermore, aged hyperoxia-treated rats lacked carotid chemoafferent responses to NaCN and asphyxia, and their carotid body volumes were significantly below normal, just as in younger hyperoxia-treated rats (see Ling et al. 1997b; Erickson et al. 1998). Finally, ∫Phr responses to electrical stimulation of the carotid sinus nerve were similar between aged control and hyperoxia-treated rats, indicating that hyperoxia-treated rats did not have a compensatory increase in the central integration of carotid chemoafferent inputs. Collectively, these experiments provide strong evidence that impaired hypoxic responses in animals raised in hyperoxia for the first 28 postnatal days do not exhibit slow, spontaneous functional recovery, at least by 15 months of age. Potential reasons underlying the discrepancy between this and our earlier report are discussed below.

Developmental plasticity of the hypoxic ventilatory response

It is now well established that the hypoxic ventilatory response is susceptible to plasticity elicited by increases or decreases in ambient oxygen during development (Eden & Hanson, 1986, 1987; Hansen et al. 1989; Okubo & Mortola, 1990; Sladek et al. 1993; Ling et al. 1997c; Peyronnet et al. 2000). Of particular relevance to the present results, our laboratory previously demonstrated that hypoxic ventilatory (Ling et al. 1996) and phrenic (Ling et al. 1997b) responses are suppressed in adult rats exposed to hyperoxia (FI,O2 = 0.60) for the first 28 postnatal days, an effect that is unique to development (Ling et al. 1996). 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. 1997c). In contrast, phrenic responses to electrical stimulation of the carotid sinus nerve are similar between hyperoxia-treated and control rats, suggesting that the central neural integration of chemoafferent inputs is not impaired in hyperoxia-treated animals (Ling et al. 1997a). Thus, exposure to hyperoxia for the first month of life results in a blunted hypoxic ventilatory response that persists beyond development, and which arises from inadequate development of peripheral oxygen-sensing mechanisms.

Permanent impairment of hypoxic responses

Our laboratory previously reported that hypoxic phrenic responses in a smaller sample (n = 5) of 14- to 15-month-old hyperoxia-treated rats were similar to age-matched controls, a result that suggested slow, spontaneous functional recovery (Ling et al. 1998). Subsequently, experiments were conducted to identify the mechanism(s) underlying the spontaneous functional recovery of the hypoxic phrenic response. We initially tested the hypothesis that the smaller carotid chemoafferent response to chemical stimulation returned to normal as the animals aged. However, increases in carotid sinus nerve electrical activity during NaCN or asphyxia in aged hyperoxia-treated rats remained well below those in age-matched controls (Fig. 4 and Fig. 5). Moreover, carotid body volumes in treated rats were significantly less than in age-matched controls (Fig. 6 and Fig. 7), a hallmark of functional impairment in young hyperoxia-treated rats (Erickson et al. 1998). Thus, carotid chemoafferent responses to physiological stimulation do not improve with age in hyperoxia-treated rats. Accordingly, we hypothesized that functional recovery in geriatric hyperoxia-treated rats resulted from an enhanced central neural integration of carotid chemoafferent inputs (i.e. an increase in the central reflex ‘gain’). Contrary to expectations, no such enhancement was observed (Fig. 3). Moreover, the relationship between carotid sinus nerve stimulation frequency and phrenic motor output was nearly identical to values previously reported for young adult (age 4–6 months) hyperoxia-treated rats (Ling et al. 1997a).

Our inability to find a mechanism that could explain the functional recovery of hypoxic phrenic responses (Ling et al. 1998) led us to re-evaluate the earlier study with a more comprehensive experimental design and a larger sample size. The results of this larger study do not support our earlier conclusion that spontaneous functional recovery of hypoxic responses occurs in aged hyperoxia-treated rats. Indeed, the functional deficit in the hypoxic phrenic response in these aged rats was actually greater than in an earlier study on young adult (age 3–5 months) hyperoxia-treated rats (Ling et al. 1997b). At this time, the reasons underlying the discrepancy between the present study and our previous report (Ling et al. 1998) are not completely clear. However, the most plausible explanation is that biological variability, combined with the (relatively) small sample size in the earlier study resulted in an incorrect acceptance of the null hypothesis (i.e. a type II statistical error; Sokal & Rohlf, 1995). In support of this suggestion, Fig. 8 presents the range of phrenic responses in aged control and hyperoxia-treated rats when Pa,O2 was decreased to 40 mmHg. These data reveal a degree of overlap in the hypoxic phrenic response of these two groups. It is possible that the sample in the earlier study (Ling et al. 1998) was drawn from the region of greatest overlap between the control and hyperoxia-treated rats.

Figure 8. Scatter plot demonstrating the range of hypoxic phrenic responses in control and hyperoxia-treated rats.

Figure 8

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Values are expressed as percentage change in ∫Phr amplitude from baseline (Pa,O2 = 40 mmHg). Although the mean data are significantly different (e.g. Fig. 2), the magnitude of the hypoxic phrenic response is somewhat variable within treatment groups.

Another possibility is that the two samples (i.e. Ling et al. 1998; present study) were drawn from genetically distinct rat populations. Considerable evidence indicates that hypoxic ventilatory responses are influenced by genetic factors (Han & Strohl, 2000; Tankersley et al. 2000; Fuller et al. 2001a), and developmental plasticity of the hypoxic response may also be subject to genetic influences. Sprague-Dawley rats in both studies were obtained from the same vendor (Harlan); however, the possibility of genetic drift within or between rat colonies must be acknowledged.

It is worth commenting that hyperoxia-treated rats retained a (smaller) hypoxic phrenic response, even though carotid sinus nerve responses to i.v. NaCN and brief asphyxia were absent (Ling et al. 1997c). There are several possible explanations for this observation. First, the relationship between carotid sinus nerve discharge and phrenic motor output may be sufficiently non-linear that (relatively) small changes in sinus nerve discharge significantly affect phrenic motor output. Indeed, within a physiological range, small changes in carotid sinus nerve stimulation frequency (e.g. 1–5 Hz; Vidruk et al. 2001) significantly increase phrenic motor output in a non-linear way (Fig. 3; see also Ling et al. 1997a). Thus, (relatively) small increases in carotid sinus nerve discharge during hypoxia (vs. NaCN or asphyxia) could substantially impact phrenic motor output. A second possibility is that stimulation of glossopharyngeal or central neurons by hypoxia (Martin-Body et al. 1985; Smith et al. 1993) contributed to phrenic responses in hyperoxia-treated rats. However, in 3- to 5-month-old hyperoxia-treated rats, carotid sinus nerve section completely abolishes hypoxic phrenic responses (Ling et al. 1997b). Thus non-carotid body-mediated mechanisms do not appear to contribute substantially to the hypoxic response in hyperoxia-treated rats. A resolution of this issue awaits further investigation.

Recovery of functional deficits

Permanent functional impairment following sensory deprivation occurs in other neural systems. The most prominent example comes from the classic studies of Hubel & Wiesel, who demonstrated that 7–12 weeks of monocular deprivation in kittens results in permanent blindness in the affected eye (Hubel & Wiesel, 1970). Other studies have shown that neonatal anoxia results in a permanent deficit in spatial memory in rats (Dell'Anna et al. 1991). Interestingly, the (permanent) functional deficits in both of the aforementioned examples can be at least partially reversed by a variety of treatments in the adult animal (Kupperman & Kasamatsu, 1984; Shirokawa & Kasamatsu, 1987; Fox & Zahs, 1994; Iuvone et al. 1996). The same is true of the reduced hypoxic responses in adult rats exposed to hyperoxia for the first 28 postnatal days. We recently demonstrated that chronic activation of the hypoxic ventilatory control system by 1 week of chronic intermittent or sustained hypoxia partially restores lost chemoresponsiveness in adult (3- to 5-month-old) hyperoxia-treated rats (Fuller et al. 2001b). The mechanism of such ‘active’ functional recovery remains to be explored.

Significance

Improper functional development of the respiratory neural control network has been implicated in a range of pathological conditions, such as sudden infant death syndrome and central alveolar hypoventilation syndrome (Mellins et al. 1990; Weese-Mayer et al. 1992; Richerson, 1997). Accordingly, there is much interest in determining how developmental experiences lead to functional impairment of respiratory control mechanisms. Katz-Salamon & Lagercrantz (1994) have examined peripheral chemoreflexes in human infants that had received long-term supplemental oxygen therapy. Their findings suggest that treatment with clinical levels of supplemental oxygen can have deleterious effects, inasmuch as these infants displayed significantly reduced peripheral chemoreflexes. However, it is not yet known if these deficits in peripheral chemosensitivity have a significant impact on ventilation during normal breathing, or if they persist through adulthood and beyond. Our data suggest that at least some deficits in respiratory control that arise from hyperoxia experiences may be permanent. Their impact on morbidity and mortality remains to be determined.

Acknowledgments

These experiments were supported by NIH grant HL 53319. D.D.F. and R. W. B. were supported by training grant HL 07654.

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