Abstract
Brief oxygen therapy is commonly used for resuscitation at birth or prevention of hypoxaemia before procedures during the neonatal period. However, O2 may severely depress breathing, especially when administered repeatedly. The aim of the present study was to test the effects of repeated hyperoxia on breathing control in newborn mice.
A total of 97 Swiss mouse pups were assigned to O2 or air on post-natal day 0, 1 or 2. Each pup in the O2 group was subjected to four hyperoxic tests (100% O2 for 3 min followed by 12 min normoxia), whereas pups in the air group were maintained in normoxia. Breathing variables were measured using flow-through barometric plethysmography.
O2 significantly decreased minute ventilation as seen in a decrease in respiratory rate. This decrease became significantly larger with repeated exposure and ranged -17– -26% for all ages combined. Furthermore, hyperoxia increased total apnoea duration, as compared with the baseline value.
In newborn mice, repeated hyperoxia increasingly depressed breathing. This finding further supports a need for stringent control of oxygen therapy, most notably repeated oxygen administration in the neonatal period for premature newborn infants and those carried to term.
Oxygen is the most commonly used treatment at birth and is an integral part of the respiratory support provided in neonatal units. Approximately 5–10% of neonates require resuscitation at birth 1, and most of these neonates are born prematurely 2. Furthermore, short periods of O2 therapy are used in critically ill neonates to prevent hypoxaemia when mechanical ventilation is not available, especially during transfer to the neonatal intensive care unit and before lumbar puncture 3, tracheal suction 4, 5, bronchoscopy 6, 7 and bottle feeding 5, 8.
The use of 100% O2 instead of room air has been challenged 1 based on studies showing cerebral blood flow reduction 9, generation of oxygen free radicals that cause or worsen brain injury 2, 10–12, increased rates of bronchopulmonary dysplasia 13 and retinopathy 14. In addition, the inhibitory effects of hyperoxia on breathing may compromise oxygenation after O2 administration, particularly in pre-term humans, who are more susceptible to apnoeas 15. Although this inhibitory effect of hyperoxia may be considered minor compared to other adverse effects of oxygen therapy, it may become worrisome when O2 is administered repeatedly, for instance to nonventilated pre-term infants with recurrent cyanosis after O2 withdrawal. The present authors reasoned that repeated hyperoxia, which alternately inhibits and stimulates chemoreceptors (upon return to normoxia), may induce potentiation, which is commonly observed with a variety of respiratory stimuli such as hypoxia 16, 17. For ethical reasons, these effects are difficult to investigate in human neonates.
The aim of the present study was to assess the inhibitory effects of repeated hyperoxia (administration of 100% O2) on ventilation in newborn mice. Breathing variables were measured noninvasively in freely moving pups to mimic human infants. The newborn mice were tested from birth (post-natal day 0 (P0)) to P2. The post-natal resetting of peripheral chemoreceptors occurs during this period in mice 18. P2 is a period of high vulnerability for white matter injury, which is a major consequence of hypoxia in the newborn brain, and corresponds to the high-risk period from 23–32 weeks post-conceptional age in humans 19.
METHODS
Animals
Mouse pups from Swiss female mice (IFFA-CREDO, L'Arbresle, France) were housed at 24°C with a 12-h/12-h light/dark cycle and fed ad libitum. The experimental protocols complied with the animal research guidelines established by the Institut National de la Santé et de la Recherche Médicale (French National Institute for Health and Medical Research). Pups were examined on the first day of life (P0), with a 12-h uncertainty regarding time from birth to testing, on P1 or on P2. At each age, the pups were randomly assigned to O2 or air with a 2:1 ratio. The weight differences between the O2 and air groups were not significant at any of the three ages. Two complementary experiments in 2-day-old pups were run.
Whole-body flow plethysmography
Respiratory variables in unrestrained newborn mice were measured noninvasively using whole-body flow barometric plethysmography as previously described 20. The mice were left unrestrained since restraint may affect baseline ventilation and ventilatory responses to chemical stimuli, as previously shown in adult mice 21.
Movements were detected based on changes in the baseline respiratory signal, using a previously validated criterion:
(Vi–Ve)–(Vi+Ve) (1)
where Vi and Ve, respectively, were the magnitudes of the inspiratory and expiratory limbs of the volume signal 20.
The plethysmograph was composed of two Plexiglas cylinders serving as the animal (40 mL) and reference (70 mL) chambers, immersed in a thermoregulated water-bath that maintained the temperature at 32.8°C. A 100-mL·min-1 flow of dry air (Hi-Tec airflow stabiliser; Bronkhorst, Uurlo, The Netherlands) was divided into two 50-mL·min-1 flows through the chambers, thus avoiding CO2 and water accumulation. The differential pressure between the two chambers (transducer; DRUCK-EFFA, Asnières, France; range±0.1 mb) was filtered (bandwidth 0.05–15 Hz at -3 dB), converted to a digital signal (Instrunet model 200 14-bits converter; GW-Instruments, Somerville, MA, USA) at a sample rate of 100 Hz, and processed by custom-written software (Software Superscope II; GW-Instruments). The time constant of the pressure decay within the system (0.35 s) was measured by injecting 2 µL into the measurement chamber. This allowed measurement of breathing frequencies within the 0.5 Hz–10 Hz range at −3 dB. Calibration was carried out before each session by injecting 2 µL of air into the animal chamber from a microsyringe (Ito Corporation, Tokyo, Japan). The pressure rise induced by this injection was of similar magnitude to that induced by a pup. Body temperature was not continuously recorded during ventilatory measurements but was measured immediately after the plethysmographic recordings. Considering the limitations of flow barometric plethysmography in newborn mice, the absolute values of tidal volume (VT) and minute ventilation (V’E) presented here should be considered with caution, whereas the absolute total respiratory time (ttot) values are valid.
Design
Each pup was tested once on P0, P1 or P2. At each age, the pups were randomly assigned to oxygen or air with ratios of 3:1 (P0 and P1) or 2:1 (P2). Mean±sd weights in the O2 group on days P0, P1 and P2 were: 1.49±0.26 g (n = 22); 1.72±0.11 g (n = 11); and 1.95±0.27 g (n = 26), respectively. In the air group, mean weights on days P0, P1 and P2 were: 1.62±0.11 g (n = 17); 1.74±0.17 g (n = 8); and 2.01±0.28 g (n = 13), respectively. The weight differences between the O2 and air groups were not significant at any of the three ages.
The experimental design is summarised in figure 1⇓. After ∼3-min waiting time to allow adaptation to the chamber, baseline V’E was recorded for 3 min. In the O2 group, the airflow through the plethysmograph was then switched to a 100% O2 flow for 3 min and back to air for the next 12 min. This sequence was repeated three times (total duration 63 min). The normoxic control pups were constantly maintained in normoxia in the plethysmograph to look for possible effects of isolation on breathing pattern, and for drifts in breathing variables. In preliminary experiments, it was ensured that breathing variables were identical when pups were exposed to continuous airflow from the hospital compressed air system or to alternation of compressed air from the hospital system and an air bottle (to mimic O2 administration). Simultaneous measurements of breathing variables and body temperature were carried out in 3 additional pups on P2 (mean weight 2.20±0.16 g).
Furthermore, two complementary experiments were run in 2-day-old pups. First, in a follow-up experiment, an independent sample of 10 pups (weight 1.84±0.09 g) were exposed to an additional O2 test 1 h after completing the 100% O2 protocol (fig. 1b⇓). Secondly, to examine the effects of repeated exposures to lower O2 concentrations, an independent sample of 16 pups (2.13±0.21 g) were exposed to 30% O2, instead of 100% O2 (fig. 1c⇓).
Ventilatory response to hyperoxia
Breathing variables and apnoeas were determined using a recently developed automatic classification method 20. Briefly, apnoeas were defined as ventilatory pauses longer than twice the duration of the preceding breath, whereas ttot in s, VT in µL·g−1, and V’E calculated as VT·ttot-1 and expressed in µL·s-1·g-1 were calculated on apnoea-free periods. Breathing variables were averaged over consecutive 30-s periods. For each test, baseline values for these variables were calculated as the mean value over the 3 min of air exposure preceding the test (or the corresponding period in the air group).
The ventilatory response to hyperoxia was evaluated based on the maximal V’E decline, i.e. the minimal V’E value (min. V’E) over the 3 min of O2 exposure expressed as the percentage of the baseline level 22, according to the formula:
100 × (min. V’E-baseline V’E)/baseline V’E (2)
This formula accounts for possible interindividual differences in the time course of the V’E response to O2. The VT and the ttot responses to O2 were determined using the same formula with the values of VT and ttot corresponding to min. V’E. The same calculations were calculated in the air group. This yielded a negative value for the V’E response because min. V’E was smaller than the sample mean.
Total apnoea duration was calculated as the total apnoeic time during each 3-min period, i.e., before, during and after O2 exposure in the O2 group (or air in the air group). The apnoea response to O2 was calculated as the difference between air and O2; percentages could not be calculated, because some pups had no apnoeas during air breathing, i.e. had an apnoea duration of 0 s.
Statistics
Breathing variables and apnoeas were subject to ANOVAs with group (O2 versus air) and age (P0, P1 and P2) as the between-subject factors and test (from 1–4) as the within-subject factor. To take into account the heterogeneous correlations among the repeated time measurements, the degrees of freedom were adjusted using the Greenhouse and Geisser factor, which is a conservative downward correction to the degrees of freedom 23. Within-subject main effects and interactions are presented, together with p-values based on these adjusted degrees of freedom. When the interaction analysis was significant, contrast analyses were conducted to detect where the significant differences lay. Values are presented as mean±sd in the text and tables and mean±sem in the figures.
RESULTS
Baseline breathing variables
Baseline V’E levels were similar in the two groups (table 1⇓). These values did not show any significant group or age effects. However, in both groups, a small but significant V’E increase was found from the first to the fourth test (p<0.0016; the group differences were not significant). This drift was ascribable to a VT increase (main effect for test: p<0.0001, not shown), whereas the corresponding change in ttot was not significant (not shown).
Ventilatory response to hyperoxia
Large proportions of the 63-min recordings were free of movement artefacts in both the O2 (mean±sd P0: 90±4; P1: 86±7; and P2: 92±3%) and the air group (P0: 92±4; P1: 90±5; and P2: 92±3%).
At all ages, on average over the four tests, hyperoxia compared to air caused a significant decline in V’E (table 2⇓). The V’E decline in the O2 group was chiefly ascribable to a ttot increase, whereas changes in VT were small (table 2⇓). Because of the time needed to flush the chamber with O2, the min. V’E was achieved during the second 30-s period of O2 exposure. The V’E decline was reversed within a 3-min period upon return to normoxia: the analysis of post-hyperoxic values (calculated over successive 3-min periods from the return to normoxia) did not indicate significant differences between the O2 group and the air group.
The comparison between the O2 and air groups (∼10% difference between groups on P0 and P1 and 20% on P2) showed a significant increase in the O2-induced V’E decline on P1 and P2. This effect was supported by a significant group-by-age interaction and by the between-group and within-group pairwise comparisons described in detail in table 2⇓. The group differences were not accounted for by changes in baseline levels, which were small and similar in the two groups, as previously mentioned (table 1⇓). Thus, a significant response to hyperoxia, consisting mainly of ttot changes, was present on the day of birth and increased over the next 2 days. This effect was abolished by a return to normoxia at all ages and for all test numbers (data not shown). As for V’E, reversal of the apnoea effect occurred within 3 min after the return to normoxia. Post-hypoxic values of apnoeas calculated over successive 3-min periods did not show significant differences between the O2 group and the air group.
Finally, it was determined whether VT and V’E values were influenced by body temperature changes during hyperoxia. Body temperatures (measured in a separate sample of three pups subjected to the same protocol as the O2 group) showed remarkable stability throughout the experiment. Individual ranges of temperature changes were 0.7, 0.5 and 0.7°C, respectively, and variation coefficients were 0.5, 0.3 and 0.4%, respectively.
Apnoeas
In normoxia, total apnoea duration was significantly longer on P1, compared to P0 and P2, with no significant difference between the O2 and air groups (table 3⇓). Hyperoxia significantly increased mean total apnoea duration at all ages (fig. 2⇓), as compared to the pre-oxygen period (group-by-period interaction, p<0.0001; fig. 3⇓). The increase in mean apnoea duration was not significantly affected by age (group-by-period interaction nonsignificant). When the combined mean values for all four tests were considered, the mean±sd increase in apnoea duration from air to O2 was 8.7±10.8 s , 8.4±16.4 s and 5.0±8.9 s on P0, P1 and P2, respectively (nonsignificant differences). In the air group, there were virtually no changes at any of the three ages. The apnoea increase was reversed by the return to normoxia, at all ages and for all test numbers (data not shown).
Finally, neither the linear correlation coefficient nor the Spearman rank correlation between the total apnoea duration and the mean V’E decline was significant overall or in any of the age groups.
Effect of repeated exposure to oxygen
The V’E decline caused by O2 (expressed as % of baseline) increased significantly with repeated exposure in the O2 group (group-by-test interaction, p<0.0005; fig. 3a⇓). In the O2 group, the V’E drop was about 70% larger during the fourth test than during the first test, with no significant effect of age. The small changes in baseline normoxic V’E did not account for the V’E decline (table 2⇓).
The increase in apnoea duration caused by O2 exposure was significantly greater during the second than the first test in the O2 group and remained elevated thereafter (group-by-test interaction, p<0.03, with no significant effect of age; fig. 3b⇓). This increase was not ascribable to differences in baseline levels, which were similar for the first and second tests (12.5±6.9 s and 11.7±16.9 s, respectively; fig. 3b⇓).
Follow-up
The protocol of the main experiment (i.e. four successive 100% O2 tests) was replicated in a smaller, independent sample of 10 P2 pups, which were re-exposed to a follow-up test 1 h after completing the last O2 test. This experiment confirmed the repetition effects on V’E (p<0.030, effects on apnoeas were not significant). After 1 h, the V’E decline was partially reversed, and its value was not significantly different from the baseline value on test 1 (fig. 4⇓).
30% O2 effects
To determine whether repeated exposure to lower concentrations of O2 also magnified the V’E decline, an independent sample of 16 P2 pups were exposed to the same protocol after replacing the 100% O2 stimulus by 30% O2. The V’E declines were -22.1±14.1, -23.9±13.2, -24.2±7.1, and -22.2±13.3% from test 1–4, respectively. In contrast to the 100% O2 tests, repeated exposure had no significant effect on the V’E decline, which remained the same over the four tests. As with the 100% O2 test, the V’E decline was chiefly ascribable to an increase in ttot (27.0±14.4% on average over the four tests), whereas changes in VT were small −2.1±4.5%. The V’E decline was slightly smaller than that caused by 100% O2, but the difference was not significant.
The 30% O2 stimulus slightly, but significantly, increased mean total apnoea duration at all ages, compared with the pre-oxygen period (p<0.008). The mean±sd increase in apnoea duration from air to O2 was 1.9±4.9 s, 2.6±4.9 s, 1.5±4.0 s and 2.5±5.8 s from test 1–4, respectively. The repeated-exposure effect was not significant.
DISCUSSION
Repeated exposure to 100% O2 was associated with increasing inhibitory effects of hyperoxia on V’E, as a result of increases in ttot and total apnoea duration, which is consistent with potentiating effects.
Methodological considerations
Whole-body plethysmography is the only method for measuring breathing variables in unrestrained newborn mice. This method has been validated against pneumotachography in larger animals but not in newborn mice, due to the lack of pneumotachographs designed for small animals. Furthermore, body temperature, which is inherent to equations for calculating VT and V’E, was not continuously measured during plethysmographic recordings, as this would have required restraining the pups. Thus, the absolute VT and V’E values in the present study should be considered with caution. However, these limitations do not invalidate the results, because the ventilatory depression caused by hyperoxia was explained by increases in ttot and apnoea duration, two variables known to be reliably measured by plethysmography 20. Furthermore, the body temperatures measured in three animals during plethysmography showed very small variations.
To evaluate the ventilatory response to hyperoxia, a min. V’E over the 3-min O2 exposure period was used instead of the mean decrease over the total O2 exposure period 22. Min. V’E during hyperoxia is thought to depend only on suppression of the peripheral chemoreceptor drive. However, the increase in metabolism caused by hyperoxia 24 may elicit excitatory inputs to breathing that may partially counteract the ventilatory fall caused by peripheral chemoreceptor silencing. Thus, the mean V’E value calculated over the entire hyperoxic period may confound excitatory and inhibitory inputs to breathing. Furthermore, the current method for assessing the ventilatory response to hyperoxia accounted for possible interindividual differences in the timecourse of the V’E response to O2.
The study period (P1–P3) encompassed major post-natal changes in respiratory sensitivity to hypoxia. Previous studies showed that significant changes in ventilatory responses to chemical stimuli take place around 12 h after birth in mice 18. In human infants, post-natal resetting of peripheral chemoreceptors occurs within days to weeks after birth 25. The effects of hyperoxia beyond this critical period were not examined in the present study. Oxygen causes ventilatory depression in mature mammals 26, and may yield similar potentiation effects to those found here. However, the present study focused on the early post-natal period during which ventilatory depression may be aggravated by other sources of respiratory instability, in particular immaturity of the central chemoreceptors 27.
Development of the hyperoxic response
Hyperoxia induced depression of ventilation in the present study, as previously reported in mouse pups 1–2 days after birth 28, as well as in other newborn mammals including humans 29. The present results establish that the V’E decline is present on P0 and is accompanied by apnoeas. A previous study has suggested that the post-natal resetting of peripheral chemoreceptors occurs within 6–12 h after birth in mice 18. This maturation effect may account for the increase in the V’E decline after P0 in the present study.
The V’E decline in the O2 group was chiefly ascribable to a ttot increase, whereas VT changes were small. In a study of human pre-term infants, the V’E decline after 3 min of O2 exposure was due to a significant decrease in respiratory rate with little or no change in VT on P2, whereas the opposite breathing pattern changes were found on P6 15 and in term infants during P2–P6 29. Taken together, these results suggest that the breathing strategy during hyperoxia undergoes rapid developmental changes and that, in this respect, newborn mice resemble pre-term infants shortly after birth. This similarity further supports the validity of newborn mice as a model for studying early breathing disorders in pre-term human infants and at a lesser stage in neonatal period in term.
Effects of repeated O2 exposure
The V’E decline and total apnoea duration tended to increase with repeated 100% O2 exposure, whereas both V’E and apnoea duration returned to baseline levels between 100% O2 exposures. These effects were transient; after 1 h, re-exposure to 100% O2 did not induce a significantly larger decline in V’E compared to the first test.
Finally, the increase in ventilatory depression observed with repeated 100% O2 was not found with a lower O2 concentration (30%). Exposure to 30% O2 significantly inhibited ventilation, but this effect did not become stronger with repeated exposure.
Repeated hyperoxia was associated with alternating inhibition (during hyperoxia) and stimulation of peripheral chemoreceptors (upon return to normoxia). However, the effects shown in the current study departed from potentiation effects induced by repeated hypoxia in that the V’E changes induced by repeated hyperoxia did not outlast the exposure to the stimulus 16. Sustained changes in metabolic rate from one test to the other may also contribute to the increase in ventilatory depression with repeated hyperoxia. Hyperoxia has previously been found to increase O2 consumption and CO2 production in newborn mammals 24, 30. However, the rapid return of V’E and apnoea duration to pre-O2 baseline levels upon return to normoxia after each O2 exposure in the current experiments militates against a sustained increase in metabolic rate.
Conclusion
Repeated O2 administration exacerbated O2-induced ventilatory depression by increasing the minute ventilation decline and apnoea duration in newborn mice. These findings support that systematic preventive oxygenotherapy should be avoided. They may indicate a need to revise current resuscitation strategies for premature infants. Other studies are warranted to determine the age at which this detrimental effect disappears.
Footnotes
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For editorial comments see page 4.
- Received September 26, 2005.
- Accepted August 29, 2006.
- © ERS Journals Ltd