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2Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas; and 1Department of Pediatrics, Yale University, New Haven, Connecticut
Submitted 14 September 2004 ; accepted in final form 16 February 2005
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ABSTRACT |
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normoxia; development; chemoreceptor; in vitro; inspired oxygen fraction
This ventilatory impairment can be largely attributed to chemoreceptor dysfunction in that whole carotid sinus nerve responses to asphyxia, cyanide, and moderate hypoxia are reduced (3). Part of this reduction appears to be due to axonal loss. Postnatal hyperoxia was associated with a 41% reduction in the number of sinus nerve C fibers, which, if confined to chemoreceptor fibers, may implicate a loss of chemoreceptor nerve fibers that is far in excess of 41% (8). This raises the question of whether those chemoreceptor fibers that survive postnatal hyperoxia have impaired responses or whether their response characteristics are normal. If the hyperoxia-induced ventilatory impairment were due solely to carotid sinus nerve axonal loss, then responses of the surviving chemoreceptor units may be relatively normal.
The present work was designed to begin to answer this question by examining the response of single-unit chemoreceptor activity of rat carotid bodies to graded hypoxia for rats treated with postnatal hyperoxia for 2 wk starting at postnatal day 0 or 14. Recordings were confined to the period immediately after hyperoxia exposure, and the issue of recovery of activity after resumption of normoxia was not addressed in this study. Experimental results show clear hyperoxia-induced impairments of single-unit chemoreceptor nerve activities at both ages, during normoxia and during graded hypoxia, and part of the impairment may extend to alterations in the excitability of the axonal fibers projecting to the carotid body. Some of these results were presented in abstract form (4).
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METHODS |
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Animal Model
Experiments were conducted on two experimental groups and two control groups. In the first group, pregnant Sprague-Dawley rats, at 20 days of gestation, were placed in an environment chamber in which the inspired O2 fraction (FIO2) was enriched to 60%, and they were allowed to give birth. Chamber O2 tension and CO2 were automatically controlled (Oxycycler model A84XOV, Reming Bioinstruments, Redfield, NY) and a record of O2 and CO2 tensions recorded to computer (AnaWin software). Rat pups were raised in this environment from postnatal days 0–14, and experiments undertaken in the immediate time period of postnatal days 14–17. Animals remained in the hyperoxia environment until use. In the second group, pregnant Sprague-Dawley rats were allowed to deliver in normoxia, and the pups were allowed to mature from postnatal days 0 to 14. At postnatal day 14, the environment was enriched to 60% FIO2 and maintained from postnatal days 14 to 28. Experiments were undertaken in the period of postnatal days 28 to 33, and animals remained in the hyperoxia environment until use. Control rats were similarly handled but were not exposed to the hyperoxia.
Tissue Harvest and Recording
Before tissue harvest, the rats were deeply anesthetized by placement in a closed chamber whose atmosphere was saturated with isoflurane. While anesthetized, the animals were removed and decapitated. After a midline neck incision, the trachea was retracted rostrally and the carotid arteries dissected free past the carotid bifurcation. After cutting of the internal and external carotid arteries and removal of the superior cervical ganglion, the vagus nerve was dissected free, centrally, past its junction with the glossopharyngeal nerve, and the combined nerve was cut near its entrance to the brain stem. The ganglion was reflected over the carotid bifurcation, and the tissue was removed and placed in oxygenated (21% O2, 5% CO2, balance NL) saline solution (in mM: 120 NaCl, 3 KCl, 2 CaCl2, 1 Na2HPO4, 1 MgSO4, 24 NaHCO3, and 10 glucose). In the bath, the vagus nerve and carotid arteries were dissected free from the glossopharyngeal nerve and carotid body. To aid in tissue cleaning, the complex (carotid body-sinus nerve-glossopharyngeal nerve and ganglia) was transferred to chamber filled with saline containing collagenase (0.01%, type P, Boeheringer) and trypsin (0.02% type IX, Sigma) at room temperature for 30 min with gentle agitation. The complex was further cleaned and transferred to a perfusion chamber (RC-22C, Warner Instruments; chamber volume 
140 µl) mounted on the stage of an inverted microscope. The complex was superfused with oxygenated saline at 37°C (TC344 temperature controller, Warner Instruments). Perfusion rate was 3 ml/min and was limited by the flow resistance of the perfusion tubing (stainless steel, <<1/16. ID x 5 ft., Upchurch Scientific). Greater details regarding dissection and cleaning were previously given (6).
Single-Unit Recording
Single-unit activity was recorded by using a suction electrode advanced into the petrosal ganglion. Electrode tip size was 30 µm in diameter, which allowed individual ganglion cells to enter the tip. The pipette potential was voltage clamped at 0 mV (model 200B amplifier, Axon Instruments, Burlingame, CA), and the current signal was amplified, filtered (0–2 kHz), displayed on an oscilloscope, digitized (10-kHz sample rate), and stored on computer (Axoscope, Axon Instruments). Unit chemoreceptor activity was discriminated and timed, post hoc, by using an event detection program that identified the timing and magnitude of action potential events (FETCHAN, Axon Instruments). The number of individual spikes per second was calculated and graphed as a function of time (Origin 6.1, Microcal).
Nerve Conduction Velocity
For identification of axons that project to the carotid body and for measurement of nerve conduction time, a stimulus electrode (pipette filled with 1 M NaCl; 1-M
impedance) was advanced into the carotid body. A constant-current stimulus (100 µA, 0.1-ms duration; model BSI-2, BAK Instruments) was delivered under computer control (model pCLAMP6, Axon Instruments), and the success of the stimulus in initiating an orthodromic action potential was determined in the postspike period. If an action potential was successfully evoked, then the stimulus current was reduced to near threshold and 10 sweeps were stored on disk. Conduction time was measured as the time period from the start of the stimulus artifact to the time of arrival of the afferent spike at the soma. The conduction time was not translated to velocity owing to uncertainty of actual axonal length because it could not be immediately discerned where within the carotid body the action potential was initiated.
Experimental Protocol
After measurement of nerve conduction time, unit activity was recorded in 7-min epochs. The first minute of each epoch was baseline activity during normoxia perfusion; the perfusate was switched at the start of minute 2 to saline equilibrated with 12%, 5% or 0% O2-5% CO2-balance N2 and switched back to normoxia at the end of minute 5. Chamber O2 tension was not measured during the experiments, but measurements in the postexperimental period under identical perfusion conditions yielded an estimate of 25 Torr during perfusion from the 0% O2 reservoir. A rest period of 5 min was given between epochs, and the order of presentation of different hypoxia mixtures was varied between studies.
Data Analysis
Unit baseline activity was quantified as the average spiking rate measured over 60 s of normoxia perfusion. Peak discharge activity was quantified as the peak spiking rate, averaged over 5 s, during presentation of the hypoxic stimulus. Time to peak was quantified as the time period between the switch to hypoxia perfusion and the occurrence of the peak spiking rate. It was not corrected for the flow delay between the switch valve and chamber or for the dilution time in the chamber. However, chamber volume and perfusion rates were relatively constant across studies and would not be expected to induce any biasing effects. A "silent" chemoreceptor was defined as an afferent fiber that could be orthodromically evoked by a 100-µA stimulus within the carotid body but that had no spontaneous activity during normoxia (21% O2).
Comparison of baseline and peak discharge rates were between hyperoxia-treated and control rats and for each age group by using Student's t-test. Comparison of conduction time (velocity) was separately calculated for the 14-day age group and 28-day age group, because the conduction distance was larger in the 28-day group. However, within an age group, the conduction distance did not appear different between controls and hyperoxia-treated (within our visual resolution estimate). Comparison of proportions was made using the 
2 test with the null hypothesis that the group frequencies were the same. Significance level for all tests was set at P < 0.05.
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RESULTS |
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Baseline Activities
Baseline (normoxia) spiking rates were calculated as the average spiking rate for the minute before the switching of perfusate to the test solution. To limit unequal bias between population samples, only runs employing the 5% O2 test solution were used. Although baseline activities were present in all recordings from C14 and C28 animals (Fig. 1), the majority of chemoreceptor fibers from hyperoxia-treated animals failed to generate spontaneous spikes until challenged with a hypoxic stimulus (Fig. 2). On average, the baseline spiking rates for normoxia-treated animals was 1 Hz [1.2 ± 0.6 Hz (C14), and 1.1 ± 0.5 Hz (C28); Fig. 3 ]. The averages for hyperoxia-treated rats were 0.12 ± 0.08 Hz (mean ± SE, H14) and 0.03 ± 0.02 Hz (H28). The proportion of afferents that were silent in normoxia was also significantly higher in hyperoxia-treated animals. As stated, all normoxia-treated animals showed baseline discharge activity (percent silent = 0% for C14 and C28), but more than half of hyperoxia-treated failed to generate spontaneous action potentials during superfusion with saline equilibrated with 21% O2 (8/12 = 67% for H14; 7/13 = 54% for H28). The proportion of silent units was significantly higher in the hyperoxia-treated group (P < 0.001 for each group, 2 test).
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The peak response was defined as the peak discharge frequency over 5 s during the period of hypoxia perfusion. As was the case for baseline spiking rates, peak firing rates for hyperoxia-treated animals were considerably below that for normoxia-treated ones. Some normoxia treated units reached peak discharge rates of 30 Hz followed by a reduction in spiking rate, which suggests a depolarization-block (Fig. 1). In response to 12% O2, peak spiking rates for normoxia-treated rats were 8.9 ± 3.2 (C14) and 9.3 ± 1.7 Hz (C28) compared with 2.9 ± 0.6 (H14) and 1.1 ± 0.3 (Hz) (H28). The reductions in peak rates were significant for both treatment groups (P < 0.001; Fig. 3). The 5% O2 stimulus produced elevated spiking rates in both groups. Peak rates during 5% O2 were 19.1 ± 4.2 (C14) and 9.3 ± 1.7 Hz (C28) for normoxia-treated and 3.2 ± 0.8 (H14) and 3.1 ± 0.6 Hz (H28) for hyperoxia-treated animals. The treatment effect of hyperoxia was significant for both groups (Fig. 3). The apparently larger response to 5% O2 in the C14 group was due to some fibers that produced peak rates far above the mean (e.g., Fig. 1). Peak spiking rates during superfusion with 0% O2 were no higher than that obtained during 5% O2, suggesting a maximum response had already been achieved. Peak rates during 0% O2 were 12.5 ± 2.2 (C14) and 8.3 ± 1.4 Hz (C28) for normoxia-treated and 2.9 ± 0.6 (H14) and 2.9 ± 0.4 Hz (H28) for hyperoxia-treated animals.
Time to Peak
The effect of hyperoxia treatment on the speed of response was less than the effect on the magnitude of the response. The time to peak was measured as the time period between the start of hypoxia perfusion and the peak nerve response. In response to 0% O2, the time to peak response was not significantly different between the C14 and H14 groups or between the C28 and H28 groups (Fig. 4). At lesser degrees of hypoxia, the response of the hyperoxia-treated group was significantly slower for the H28 group compared with the C28 group during perfusion with 5% and 12% O2 (Fig. 4).
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Nerve conduction time, that is the time between the electrical stimuli in the carotid body to the arrival of the action potentials at the soma, were significantly longer in both hyperoxia-treated groups. Conduction time for the H14 group was 4.49 ± 0.22 ms compared with 3.48 ± 0.23 ms for the C14 group (P < 0.05). Conduction time for the H28 group was 5.44 ± 0.16 ms compared with 4.24 ± 0.28 ms for the C28 group (P < 0.05; Fig. 5).
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Six units that were tentatively identified as chemoreceptor afferent fibers on the basis of the ability to orthodromically activate the fibers using an electrical stimulus were not included in the above averages because no spontaneous activity was generated during the hypoxia perfusion periods. Thus it was unclear whether they qualified as chemoreceptor afferent fibers. This included three units from the H14 group and two units from the H28 group but only one unit in the C14 group and none in the C28. The frequency of encountering these silent units was significantly higher in the H14 group compared with the C14 group and higher in the H28 group compared with the C28 group (P < 0.05, 2).
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DISCUSSION |
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Previous studies have indicated that hyperoxia produces blunted chemoreceptor responses in older animals. In adult cats, exposure to normobaric O2 (100% O2 x 60–67 h) (11, 12) or high-pressure O2 (100% O2 x 5 atm x 2 h) (15) resulted in a depressed acute chemoreceptor response to hypoxia. However, baseline activity was either enhanced (12) or unchanged (15) by hyperoxia treatment, a result different from that found in the present study in which chemoreceptor activity was depressed at all levels of O2 tension. The differences in results may be related to species, age of the animal, or relative toxicity of the hyperoxia. In the case of rats, exposure to 60% FIO2 can be tolerated for long periods of time without the animals suffering deleterious consequences (13).
Critique of Methodology
The present experiments were undertaken using a superfused carotid body, in vitro, which allows for an independence from vascular effects but presents abnormal tissue O2 gradients because O2 is only delivered from around the organ. To some extent, these gradients are minimized in carotid bodies from small animals because the distance to a free surface is also small (100 µM). The spontaneous discharge frequencies observed in our experiments are also relatively similar reported for rat carotid bodies in situ. In adult male rats recorded in situ, unit discharge activity increased from 0.7 to 10 Hz as arterial PO2 decreased from 115 to 35 Torr (16). This is in general agreement with the discharge rates observed in the present study during superfusion with saline equilibrated with 21% O2 (normoxia) and 5% O2 (Fig. 3). It is also likely that 5% O2 evoked a maximal increase in activity because no further increase was apparent during superfusion with saline equilibrated with 0% O2 (Fig. 3).
An electrical stimulus within the carotid body to identify axons projecting to the carotid body, including those that lack spontaneous activity, also has the potential to evoke spikes in fibers of passage (e.g., baroreceptors). Several factors argue that the contamination of the sample with nonchemoreceptor axons is relatively minor. First, only a small proportion of axons tentatively identified as chemoreceptor axons by the method of orthodromic stimulation failed to generate spontaneous action potentials during hypoxia stimulation. Second, all fibers recorded in this study were C fibers with conduction velocities <1 m/s. Because initiation of an action potential by electrical stimulation is dependent on axial current flow through the axon, C fibers are relatively resistant to electrical stimulation owing to their high axial electrical resistance. Thus spike initiation probably occurred at nerve terminals that evidence higher excitability than the conducting portion of the axon.
Immediate Effects of Postnatal Hyperoxia
The present results are generally consistent with those of Hanson and colleagues (7), who recorded whole sinus nerve activity in control rats and rats exposed to 30% FIO2 for 5–10 wk (7). Whereas control sinus nerve recordings evidenced an 500% increase in overall activity, the nerve activity of treated rats failed to change during acute hypoxia. Similar results were obtained from multiunit recordings obtained on control kittens and kittens exposed to 30% FIO2 for 12–23 days (9). However, neither previous study examined a developmental window or single-unit resolution of chemoreceptor activity levels. Furthermore, the chemoreceptor activity in control and hyperoxic kittens was not significantly different at high arterial PO2 levels, a result in contrast to the present results that showed a significant decrease in baseline levels of chemoreceptor activity.
Long-term consequences of postnatal hyperoxia.
Much of the rationale for performance of this work arose from the work of Mitchell and colleagues (1, 2, 13), who demonstrated that exposure to 60% FIO2 at birth for a period of 2–4 wk followed by a return to normoxia for a period of months resulted in a marked reduction in the ventilatory response to acute hypoxia but not to acute hypercapnia. However, this is not a universal observation. Prieto-Lloret and colleagues (14) found the respiratory frequency responses to 10% FIO2 in 5-mo-old rats were identical between control rats and those exposed to hyperoxia for the first month of life. In an attempt to reconcile the observations, they suggested that the respiratory response to hypoxia may be reduced at moderate levels but eventually reaches the same "maximal" level during severe hypoxia; however, this conjecture was not experimentally tested.
In any case, there appears to be a compromise of chemoreceptor responsiveness downfield of the hyperoxia exposure. Whole nerve recordings showed little response to asphyxia, cyanide, or moderate hypoxia when examined at 1–2 mo of age after a period of postnatal hyperoxia (3). At least part of this reduction is due to axonal degeneration associated with perinatal hyperoxia (8), and it is currently uncertain to what extent the remaining chemoreceptors are compromised. The present work demonstrates that perinatal hyperoxia exposure greatly reduces chemoreceptor response to acute hypoxia in the immediate posthyperoxia period; however, recovery of function is possible or even expected if a period of normoxia was allowed to follow the period of hyperoxia. This is supported by the recent work of Prieto-Lloret et al. (14), who had difficulty in finding chemoreceptor activity in vitro in rats treated with hyperoxia followed by recovery in normoxia; however, when activity was found, it was deemed to respond normally to acute hypoxia. However, methodological limitations restrict comparison between our results and those of Prieto-Lloret et al. Their results were an amalgamation of single-unit and multiunit recordings, and results are expressed only as a change in discharge frequency during superfusion with 0 or 5% O2. Thus no information is presently available on absolute spiking levels for chemoreceptors subjected to postnatal hyperoxia followed by a period of recovery.
Potential Implications of Perinatal Hyperoxia for Developmental Plasticity of Respiratory Control
Developmental window for hyperoxia effect. The ability of hyperoxia to cause long-lasting impairment of the ventilatory response to acute hypoxia appears to diminish with age. The initial observation involved an exposure lasting 1 mo (13), and subsequent work narrowed the developmental window to the first 2 wk of life with little effect of hyperoxia beyond the first 2 wk (1, 2). However, the present data show relatively the same impairment of chemoreceptor function after a 2-wk exposure to hyperoxia regardless of whether the exposure started around postnatal day 0 or 14. Because our data were obtained in the immediate posthyperoxia period, we have no information regarding activity levels 3–4 mo later. However, it would suggest that chemoreceptor activity can recover in the older age group but not in the younger age group. This speculation is currently being addressed in our laboratory.
Mechanism of hyperoxic suppression of chemoreceptor activity. The present study offers limited insight to the mechanism altered by hyperoxia exposure. One parameter that did change is an increase in conduction time between the carotid body and ganglion soma. This may reflect a general decrease in axonal excitability. Previously, our laboratory has shown that a decrease in excitability induced by an isotonic reduction in extracellular sodium caused a profound reduction in chemoreceptor discharge frequency (5). Alternatively, presynaptic mechanisms involved in hypoxia transduction and nerve excitation, presumably undertaken by glomus cells, may be impaired in the posthyperoxic period, a speculation that is supported by preliminary data are that currently being developed in our laboratory (10).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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This article has been cited by other articles:
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  D. F. Donnelly Assisting Mother Nature in postnatal chemoreceptor maturation J Appl Physiol, May 1, 2008; 104(5): 1260 - 1261. [Full Text] [PDF]
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R. W. Bavis and G. S. Mitchell Long-term effects of the perinatal environment on respiratory control J Appl Physiol, April 1, 2008; 104(4): 1220 - 1229. [Abstract] [Full Text] [PDF]
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 R. W. Bavis, F. L. Powell, A. Bradford, C. C.W. Hsia, J. E. Peltonen, J. Soliz, B. Zeis, E. K. Fergusson, Z. Fu, M. Gassmann, et al. Respiratory plasticity in response to changes in oxygen supply and demand Integr. Comp. Biol., October 1, 2007; 47(4): 532 - 551. [Abstract] [Full Text] [PDF]
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