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(Chest. 2003;123:530-538.)
© 2003 American College of Chest Physicians

Neonatal Exposure to 65% Oxygen Durably Impairs Lung Architecture and Breathing Pattern in Adult Mice*

Stéphane Dauger, MD, PhD; Latifa Ferkdadji, MD; Georges Saumon, MD; Guy Vardon, Eng; Michel Peuchmaur, MD, PhD; Claude Gaultier, MD, PhD and Jorge Gallego, PhD

* From E9935 (Dr. Gallego) and IFR 02 (Dr. Saumon), the Institut National de la Santé et de la Recherche Médicale, Paris; Service de Pédiatrie-Réanimation (Dr. Dauger), Service d’Anatomie et Cytologie Pathologiques (Drs. Ferkdadji and Peuchmaur), and Service de Physiologie (Dr. Gaultier), Hôpital Robert-Debré, Paris; and Faculté de Médecine d’Amiens (Dr. Vardon), Unité de Recherches sur les Adaptations Physiologiques et Comportementales, Amiens, France.

Correspondence to: Jorge Gallego, PhD, Laboratoire de Neurologie et Physiologie du Développement, INSERM E9935, Hôpital Robert Debré, 75019 Paris, France; e-mail: gallego{at}idf.inserm.fr


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Study objective: To test the hypothesis that exposure to hyperoxia during the postnatal period of rapid alveolar multiplication by septation would cause permanent impairments, even with moderate levels of hyperoxia.

Design: We exposed mouse pups to 65% O2 (hyperoxic mice) or normoxia (normoxic mice) during their first postnatal month, and we analyzed lung histology, pulmonary mechanics, blood gas, and breathing pattern during normoxia or in response to chemical stimuli in adulthood, when they reached 7 to 8 months of postnatal age.

Results: Hyperoxic mice had fewer and larger alveoli than normoxic mice (number of alveoli per unit surface area of parenchyma, 266 ± 62/mm2 vs 578 ± 77/mm2, p < 0.0001) [mean ± SD], the cause being impaired alveolarization (radial alveolar count, 5.8 ± 0.2 in hyperoxic mice vs 10.5 ± 0.5 in normoxic mice, p < 0.0001). Respiratory system compliance was higher in hyperoxic mice (0.098 ± 0.006 mL/cm H2O) than in normoxic mice (0.064 ± 0.006 mL/cm H2O, p < 0.016). Baseline tidal volume (VT) and breath duration (TTOT]) measured noninvasively by whole-body plethysmography were larger in hyperoxic mice than in normoxic mice (VT, + 15%, p < 0.01; TTOT, + 12%, p < 0.01). Despite these impairments, blood gas, baseline minute ventilation E, and E responses to hypoxia and hypercapnia were normal in hyperoxic mice, compared with normoxic mice.

Conclusion: Hyperoxic exposure during lung septation in mice may cause irreversible lung injury and breathing pattern abnormalities in adulthood at O2 concentrations lower than previously thought. However, ventilatory function and body growth were preserved, and ventilatory function showed no major abnormalities, at least at rest, despite early oxygen-induced injuries.

Key Words: development • histology • lung • newborn • plethysmography • respiration


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Prolonged neonatal exposure to high O2 concentrations dramatically impairs lung development in newborn mammals.1 2 3 4 5 6 7 8 9 10 Mice exposed to 85% O2 during their first postnatal month had increases in terminal airspace size, lung inflammation, and fibrosis, and decreases in lung volumes immediately after exposure.2 These effects are not seen in adults.11 In rats, exposure to hyperoxia ranging from 40% to 95% O2 for the first 6 postnatal days elicited transient impairments in alveolarization, with a full recovery after 2 weeks of breathing air.7 Studies on neonatal hyperoxia are of major clinical relevance to the impairments in lung mechanics and breathing control seen in infants with chronic lung disease.12 13 14

The present study is an attempt to provide an integrative analysis of the long-term effects of postnatal hyperoxia on lung architecture and ventilatory function in mice. Few previous studies have addressed these effects, especially at O2 concentrations close to those used for clinical purposes, and these studies were generally confined to lung histology and mechanics. Here, we hypothesized that exposure to hyperoxia during the postnatal period of rapid alveolar multiplication by septation would cause permanent impairments,15 even with moderate levels of hyperoxia. To test this hypothesis, we exposed mouse pups to 65% O2 during their first postnatal month and we analyzed lung histology, pulmonary mechanics, blood gas, and breathing pattern during normoxia or in response to chemical stimuli in adulthood.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Mice
Six Swiss female mice (IFFA-CREDO; L’Arbresle, France) were housed at 24°C with a 12 h/12h light/dark cycle and food and water ad libitum. These female mice were mated with Swiss male mice. Three pregnant females were randomly assigned to the hyperoxic group, and three pregnant females were assigned to the normoxic group. The hyperoxic female mice gave birth to 24 pups (10 males and 14 females), and the normoxia female mice gave birth to 19 pups (10 males and 9 females). Experimental protocols met the animal research guidelines established by the Institut National de la Santé et de la Recherche Médicale (a national institute for health and medical research).

Neonatal Exposure to Hyperoxia
Seventy-two hours before the normal day of delivery, ie, 17 days after mating, each group of three pregnant mice (hyperoxia and normoxia) was placed in a box covered by a Hood-like system (30 cm by 21 cm by 15 cm) and containing water, food, and sawdust. In the box containing the hyperoxic female mice, hyperoxia was created by introducing a continuous 100% O2 flow into the box, which maintained a constant uniform level of 65% O2 with < 1.5% CO2 (Arelco CO2/O2 analyzer PR O/A2008 DSH; Arelco; Fontenay-sous-Bois, France). Exposure to 65% O2 was initiated before delivery to familiarize the pregnant mice with the experimental setup and to avoid stress at the time of delivery. A similar flow of air was introduced continuously in the box containing the three normoxic females. Delivery occurred at full term in both groups, and the litters remained in their respective boxes until 28 days of postnatal age (PNA). Confinement was briefly interrupted (< 3 min) every week (three times during the 28-day period) for cleaning the boxes and providing food (water was provided without opening the box).

Design
All the animals underwent baseline recordings, with restraint at PNA of 30 days, PNA of 45 days (PNA45), and PNA of 100 days (PNA100), and without restraint at PNA of 130 days (4.5 months) and PNA of 210 days (7 months). All mice underwent one hypercapnic and one hypoxic test with restraint at PNA45 and PNA100. Data were incomplete in two hyperoxia pups: one pup injured itself in the restraint cage and was not tested further, and one pup died suddenly after the PNA45 hypoxic test, for unknown reasons. Separate groups of mice were used for lung histology, blood gas analyses, and compliance measurements. We performed lung histology at PNA of 220 days (7.5 months) in four normoxic and four hyperoxic mice (two males and two females in each group). We analyzed venous blood gas in 12 normoxic mice (6 males and 6 females) and 10 hyperoxic mice (5 males and 5 females) at PNA of 260 days (8.5 months) and arterial blood gas in 2 normoxic mice (1 male and 1 female) and 6 hyperoxic mice (4 males and 2 females) at PNA of 290 days (PNA290) [9.5 months]. We measured respiratory system compliance (Crs) in three male normoxic mice and three male hyperoxic mice at PNA290 (9.5 months).

Plethysmography
Ventilatory parameters were measured noninvasively using whole-body flow barometric plethysmography.16 The plethysmograph was composed of two superimposed, cylindrical Plexiglas chambers with capacities of 3.6 L and 3.0 L, respectively. The upper chamber served as a reference for pressure measurements, and the lower chamber contained the animal. A 1,000 mL/min flow of dry air (Bronkhorst Hi-Tec airflow stabilizer; Bronkhorst High-Tech B.V.; Ruurlo, the Netherlands) was divided into two 500 mL/min flows through the reference and measurement chambers, respectively. The pressure difference between reference and measurement chambers was measured (EFFA pressure transducer LPM9431; Druck; Asnières, France) [range ± 0.1 millibars], filtered (bandwidth, 0.4 to 15 Hz at - 3 decibels), and converted into a digital signal. The plethysmograph was calibrated before each experiment by injecting 100 µL of air into the measurement chamber. The pressure rise induced by this injection was of similar magnitude to that induced by the tidal volume (VT) of an adult mouse. Body temperature was not recorded inside the plethysmograph and was assumed stable at 37°C.17 Fractional CO2 and O2 concentrations in the measurement chamber were determined by sampling 100 mL/min of the plethysmograph outflow (Arelco CO2/O2 analyzer PR O/A20008 DSH; Arelco). We assumed that the dry gas flushing the plethysmograph maintained relative humidity near zero.

Breathing variables were measured with or without restraint. Restraint was achieved by placing the mouse in a cylindrical, wire-mesh cage (9 cm long by 3.5 cm in diameter) that prevented respiratory signal artifacts caused by animal movement. Restraint increases baseline breathing but does not affect ventilatory responses to hypercapnia or hypoxia calculated as percentage of changes in breathing variables from baseline levels.17 The ventilatory response to chemical stimuli was calculated under restrained conditions only. Unrestrained mice moved freely inside a box (8 cm by 11 cm by 4 cm) placed inside the plethysmograph.

Respiratory Stimuli
The respiratory stimuli were administered by replacing the gas flowing through the plethysmograph by an 8% CO2, 21% O2, and 71% N2 (hypercapnic mixture), or by a 10% O2, 3% CO2, and 87% N2 (hypoxic mixture) [Air Products; Paris, France]. The switch from air to the hypoxic or hypercapnic mixture was controlled by a computer via electrovalves. The hypoxic mixture contained 3% CO2 to maintain the mice near the isocapnic range during the hypoxic stimulus.18 After baseline recording, the mice were exposed to hypercapnia for 13 min (ventilation reached steady state 10 min after electrovalve opening) and to hypoxia for 55 min (steady state at 18 min). This prolonged exposure to hypoxia allowed us to look for a hypoxic ventilatory decline (which was not found in any mice). Breathing variables (minute ventilation [E], VT, and breath duration [TTOT]) were calculated during steady-state ventilation, ie, during the last 3 min of hypercapnia and the last 15 min of hypoxia. VT, VT/inspiratory duration [TI], and E were standardized for body weight (the mean of pretest and posttest weights).

Crs
Mice were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg of body weight), and the trachea was catheterized with a 20-gauge cannula. The mouse was then paralyzed with succinyl choline (Sigma; Saint-Quentin Fallavier, France), and the tracheal cannula was connected to a mouse ventilator (model 687; Harvard; Les Ulis, France). Airway pressure was measured via a sideport using a liquid pressure transducer (Viggo-Spectramed; Oxnard, CA) with no air dead space. The volume insufflated was obtained using a linear displacement transducer (model 0244; Trans-Tek; Ellington, CT) connected to the piston of the ventilator. Frequency was set at 15 breaths/min, and the volume insufflated was 1 mL for all mice. Pressure and volume signals were digitized at 100 Hz and entered into a personal computer. The five insufflations following connection of the animal to the respirator were discarded to normalize for volume history. Pressure and volume data from three successive insufflations were registered 1 min later. The values corresponding to the linear part of the inspiratory limb of the pressure-volume curve were used to calculate the quasistatic Crs.

Blood Gas Analysis
The blood samples were obtained by percutaneous left ventricle puncture with a heparin-coated 1-mL syringe19 after light anesthesia with ketamine, 50 mg/kg intraperitoneally. The animals were killed immediately after blood sampling, by neck elongation. Because of the technical difficulties raised by sample collection for arterial blood gas measurement, we also collected venous blood samples by orbital sinus puncture using a heparin-coated capillary tube in hyperoxic and normoxic awake mice.20 All the animals were apparently unaffected and showed normal behavior within a few minutes after this puncture. PaO2, PaCO2, pH, and HCO3 were measured using a clinical blood gas analyzer (Rapid Lab 844; Chiron Diagnostics; Paris, France).

Lung Histology
The mice were killed by intraperitoneal injection of sodium pentobarbital. The thorax was opened, and the heart and lungs were removed en bloc. We cannulated the trachea with a 24-gauge Silastic catheter (Dow Corning; Lyon, France) and instilled paraformaldehyde at 25 cm H2O of pressure. Then, the lungs were fixed overnight in 4% paraformaldehyde. The next day, the lungs were serially dehydrated in increasing concentrations of ethanol, and then embedded in paraffin. To standardize analysis, we obtained lung tissue blocks from apical, azygous (or cardiac), and diaphragmatic lobes of the right lung and from upper and lower regions of the left lung. Five-micrometer tissue sections from each of these blocks were stained with hematoxylin, eosin, and saffron, Sirius red for collagen, or Weigert stain for elastic fibers.21 This yielded five sections per mouse, all from different lung regions, for each stain. Each section was completely analyzed (> 20 fields per section).

Light microscopy was performed by an investigator unaware of the treatment of each mouse. For each field, the number of alveoli was counted once visually and once using Histolab software (Microvision Instruments; Evry, France). The difference between the two countings was < 1% for any given field. Histolab software was used to measure the surface area of each alveolar parenchyma field and of regions with fibrotic lesions. Alveolarization was estimated by the radial alveolar count (RAC) method.22 23 Briefly, this method consists in counting the number of distal air sacs that are transected by a line drawn from a terminal respiratory bronchiole to the nearest pleura. All the terminal bronchioles of each of the five sections were used for RAC. At least 2 RACs were performed on each lung section for each mouse, yielding at least 10 RACs per mouse.

Statistical Analysis
Morphometric and breathing variables were submitted to analyses of variance (Superanova Software; Abacus Concepts; Berkeley, CA) with group (two levels, hyperoxic vs normoxic) as the between-subject factor and gas (two levels, air vs hypercapnia or air vs hypoxia) as a within-subject factor. The main effect for gas reflected the ventilatory response to a given stimulus. Consequently, the difference between hyperoxic and normoxic mice with respect to these responses were evaluated on the basis of the group-by-gas interaction. PNA was used as a within-subject factor for breathing variables. Data are summarized as the group means ± SD.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Weight
The hyperoxic mice showed normal aspect and behavior. Neither weight, which increased normally with PNA (Fig 1 ), nor rectal temperature, which remained steady throughout the study period, was significantly different in hyperoxic and normoxic mice (37.5 ± 0.6°C vs 37.4 ± 0.3°C).



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  		Figure 1.. Body weight vs postnatal age in mice exposed to hyperoxia (HX; filled squares, n = 24) or normoxia (NX; empty circles, n = 19) for the first 28 postnatal days. Values are means ± SD.

 
Breathing Pattern
Baseline VT and TTOT were slightly but significantly larger in hyperoxic mice than in normoxic mice at all PNAs (Fig 2 ). The longer TTOT values in hyperoxic mice were caused by significantly longer TI and expiratory duration (TE) at all PNAs (Table 1 ). In contrast, E, VT/TI, and TI/TTOT were not significantly different between hyperoxic and normoxic mice. Thus, hyperoxic and normoxic mice showed similar ventilation levels with different breathing patterns.



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  		Figure 2.. Ventilatory traces in a 7-month-old mouse exposed during the first 28 postnatal days to 65% O2 (hyperoxia, bottom trace) compared to a 7-month-old mouse exposed to room air (normoxia, top trace). The hyperoxic mouse show longer TTOTs and larger VTs than the normoxic mouse. These differences were confirmed by statistical tests on group comparisons. See Figure 1 legend for expansion of abbreviations.

 

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  		Table 1.. Baseline Breathing Variables in Mice Exposed to 65% O2 or Normoxia During the First 28 Postnatal Days*

 
Neither the E response, the VT response, nor the TTOT response to hypoxia and hypercapnia (Tables 2 , 3 ), nor the time course of these responses (data not shown), were significantly different between hyperoxic and normoxic mice at any PNA. Both responses increased between PNA45 and PNA100.


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  		Table 2.. Ventilatory Responses to Hypercapnia in Mice Exposed to 65% O2 or Normoxia During the First 28 Postnatal Days*

 

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  		Table 3.. Ventilatory Responses to Hypoxia in Mice Exposed to 65% O2 or Normoxia During the First 28 Postnatal Days*

 
Blood Gas
PO2, PCO2, pH, and HCO3 were not significantly different in hyperoxic and normoxic mice in arterial or venous blood samples (although this may be due to the small sample sizes for this analysis; Table 4 ). We noted, however, that arterial PO2 was somewhat low in hyperoxic mice (86.2 ± 11.6 mm Hg).


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  		Table 4.. Blood Gas in Mice Exposed to 65% O2 or Normoxia During the First 28 Postnatal Days*

 
Lung Histology
Hyperoxic mice had fewer and larger alveoli than normoxic mice (Fig 3 ). Morphometric analysis showed that the number of alveoli per unit surface area of parenchyma (266 ± 62/mm2 vs 578 ± 77/mm2, p < 0.0001) and the RAC (5.8 ± 0.2 vs 10.5 ± 0.5, p < 0.0001) were significantly smaller in hyperoxic than in normoxic mice (Fig 3) .



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  		Figure 3.. Lung architecture changes in a 7-month-old mouse exposed during the first 28 postnatal days to 65% O2 (hyperoxia, top right, b, and bottom right, d) compared to a 7-month-old mouse exposed to room air (normoxia, top left, a, and bottom left, c). Tissue sections were stained with hematoxylin, eosin, and saffron. Note that the alveoli are larger and less numerous in the hyperoxic mouse (top right, b) than in the normoxia mouse (top left, a). The RAC was defined as the number of air sacs transected by a line drawn from the center of a terminal bronchiole to the nearest pleura. Note the lower RAC in the hyperoxic mouse (bottom right, d) compared to the normoxic mice (bottom left, c) indicating abnormal alveolar development (original print magnification for all panels x 160). See Figure 1 legend for expansion of abbreviations.

 
The lung architecture of most hyperoxic mice was heterogeneous, with areas with airspace enlargement and foci of fibrosis (Fig 4 , top left, a, and top right, b). Fibrotic lesions were found in three of the four hyperoxic mice vs none of the four normoxic mice. In these three hyperoxic mice, the surface areas of these fibrotic lesions were 3%, 9%, and 16% of the total parenchyma surface area, indicating noticeable interindividual variability. The amount of elastic fibers within alveolar walls was decreased in hyperoxic mice (Fig 4 , bottom left, c, and bottom right, d).



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  		Figure 4.. Staining of collagen (Sirius red, top left, a, and top right, b) and elastic fibers (Weigert, bottom left, c, and bottom right, d) in a 7-month-old mouse exposed during the first 28 postnatal days to 65% O2 (hyperoxia, top right, b, and bottom right, d) compared to a 7-month-old mouse exposed to room air (normoxia, top left, a, and bottom left, c). Note the focal area of fibrosis (top right, b) in the hyperoxic mouse. Dark elastic fibers are seen within alveolar walls in the normoxic mouse (bottom left, c). Note the smaller amount of elastic fibers in the hyperoxia mouse (bottom right, d) [original print magnification for all panels x 400]. See Figure 1 legend for expansion of abbreviations.

 
Crs
Mean Crs was significantly higher in hyperoxic than in normoxic mice (0.098 ± 0.006 mL/cm H2O vs 0.064 ± 0.006 mL/cm H2O, p < 0.016; Fig 5 )



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  		Figure 5.. Illustrative examples of pressure-volume curves in a 9.5-month-old mouse exposed during the first 28 postnatal days to 65% O2 (hyperoxia) compared to a 9.5-month-old mouse exposed to room air (normoxia). Mice were anesthetized and paralyzed. Note the higher compliance in the hyperoxia mouse. See Figure 1 legend for expansion of abbreviations.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
This study shows that 28-day postnatal exposure to 65% O2 in mice causes long-term changes in pulmonary structure and breathing pattern. Seven months after 65% O2 hyperoxic exposure, the mice had reduced alveolarization, enlarged alveolar spaces, fibrotic lesions, increased Crs, lower baseline breathing frequency, and larger VT than normoxic control mice. In contrast, hyperoxic exposure did not cause major impairments of respiratory homeostasis, as suggested by blood gas values, baseline ventilation, and ventilatory responses to chemical stimuli.

Long-Lasting Changes in Lung Histology
In hyperoxic mice, histology showed regions of enlarged, overexpanded alveoli, alternating with a few regions of fibrotic, compressed alveoli. The reduced RAC suggested impairment of alveolar development. Because the alveoli are formed postnatally in the mouse, a role for the 3-day prenatal exposure to hyperoxia in these abnormalities is improbable. Our results show that septation may be durably impaired by weaker hyperoxic stimuli than used in most previous studies.2 3 6 7 24 25 26 In particular, we extended the observations obtained using 85% O2 for a similar period in mice2 or > 95% O2 for 8 days in rats.27 We cannot discard the possibility that postnatal hyperoxia increased the occurrence of lung diseases, which may partly account for some of the histologic changes observed in hyperoxia mice. However, to our knowledge, there are no published data to support this possibility. Importantly, the mice exposed to 65% hyperoxia in the present study showed normal growth (as attested by their normal weight-vs-age curve), whereas weights of mice exposed to 85% O2 were 26% smaller than in control mice at 4 weeks.2 Thus, 65% O2 hyperoxia is a valuable stimulus for evaluating the effects of neonatal hyperoxia on respiratory function while avoiding confounding related to major growth alterations.

Previous studies in 60- and 95-day-old mice showed gender-related differences in the size of alveoli.28 29 In the present study, we did not observe gender-related differences in the number of alveoli per surface unit and RAC in 7.5-month-old mice. However, alveolar size changes with aging,30 31 and this effect possibly leveled off gender-related differences in lung architecture observed in younger animals.

The arterial blood gas values in hyperoxic and normoxic mice were within the range previously reported in normal mice,19 32 33 34 although PaO2 values in hyperoxic mice were toward the bottom of this range. The small number of arterial blood data precludes definitive conclusions. The similar values in our hyperoxic and normoxic mice provide further support that postnatal hyperoxia had no major effects on gas exchange. We are not aware of previous reports of venous blood gas values in adult mice; the similar values in our hyperoxic and normoxic mice provide further support that ventilation was not affected by neonatal hyperoxia. The normal E and blood gas levels establish that the altered respiratory mechanics in hyperoxic mice did not impair respiratory homeostasis. These results are in line with previous studies providing evidence in favor of the lack of correlation between lung architecture damage and blood gas values. This evidence stems primarily from studies on aging, which showed drastic enlargement of alveoli in aging mice,35 rats,36 37 dogs,38 and humans,39 40 whereas blood gas values were generally unaltered in the absence of lung disease.30 41 In particular, lung damage with normal or nearly normal blood gas levels has been reported in 5- and 13-month-old hamsters with elastase-induced emphysema.31 The PaCO2 and pH were within the normal range, as observed in the present hyperoxic mice.

Increased Crs
Crs of normoxic mice was within the range of previously reported values in adult mice.42 Hyperoxic mice had higher compliances than normoxic mice 7 months after neonatal hyperoxic exposure, in keeping with previous results in rats exposed to a far higher level of hyperoxia (8-day neonatal exposure to 100% O2).27 The long-term increase in compliance contrasted with a short-term decrease in compliance observed consistently at discontinuation of hyperoxic exposure.2 43 44 This short-term decrease was ascribed to pulmonary edema, impaired synthesis of surfactant, and the pro-inflammatory effect of free radicals, which resolved over time. However, the decreased amount of elastic fibers observed in our hyperoxic mice, in line with previous quantitative studies,44 may increase compliance. The higher compliance in hyperoxic mice may also be ascribable to alterations in lung parenchyma resulting in the emphysema-like architecture seen in the hyperoxic mice. Compliance measured in gas-filled lungs depends on tissue elements (elastin and collagen fibers) and on surface forces. The alteration in the density of elastic fibers influences the distensibility of gas-filled lungs to the extent that there is an accompanying change in the size of air spaces.45

Abnormal Baseline Breathing Pattern
Ventilatory parameters were measured using whole-body flow barometric plethysmography, which is the only method currently available for measuring breathing variables in small mammals without restraining the animals.46 The underlying principle and the accuracy of the method are debated.47 However, the comparison of whole-body plethysmography with direct pneumotachography in adult mice showed a 5% difference in calculated VT values.48 The possible inaccuracy of absolute values of breathing variables in the present experiment does not detract from our results, which are all based on group comparisons. Furthermore, breathing frequency, which is accurately calculated by whole-body plethysmography, was also different between hyperoxic and normoxic mice.

Alterations in baseline breathing pattern—longer TTOT and larger VT with normal E—have been observed immediately after exposure to 85% O2 during the first postnatal month in mice.2 Interestingly, the pattern of breathing of hamsters with elastase-induced emphysema was also characterized by larger VTs and lower breathing frequencies.31 However, such changes in breathing pattern have not been reported previously among the long-term effects of hyperoxia. The mechanisms of this effect are unclear. The lower breathing frequency was probably not related to increased respiratory resistance in hyperoxic mice (which was not measured), since this would probably have affected TI/TTOT ratios, which were similar in hyperoxic and normoxic mice. In contrast, this effect may be related to the principle that mammals adjust frequency (and accordingly, VT) to minimize the mechanical work rate of breathing.49 For a given alveolar ventilation, the equation relating breathing variables and mechanical work predicts that the optimal frequency will be decreased if compliance increases.49 Therefore, the present observation that hyperoxia-exposed mice had lower breathing frequencies could theoretically be accounted for by the principle of minimal work of ventilation.

The normal E responses to hypoxia in hyperoxic mice are apparently at variance with previous studies in rats showing that neonatal exposure to 60% O2 for 1 month affected the response to hypoxia 2 to 4 months later.50 The stronger hypoxic stimulus used in the present study (10% O2 vs 13% O2) and the presence of 3% CO2 to maintain the mice near the isocapnic range during the hypoxic stimulus may account for this discrepancy. The responses to hypercapnia were similar in hyperoxic and normoxic mice, in line with previous studies in rats exposed to similar treatment.50 These normal hypoxic and hypercapnic responses in hyperoxic mice suggested that the baseline breathing pattern in hyperoxic mice was not caused by impaired chemical drive. However, we cannot discard the possibility that postnatal hyperoxia may affect central rhythmogenesis, given the importance of plasticity in the development of ventilatory control,51 although this hypothesis has not received support from this study or previous studies.

Implications for Animal Models of Early Lung Injury
The anatomic lesions caused by neonatal exposure to hyperoxia (enlarged airspace alternating with fibrotic lesions) are reminiscent of those observed in infants with chronic lung disease.52 53 Decreased alveolarization occurs not only in infants treated with supplemental oxygen and mechanical ventilation but also in extremely immature infants, as a consequence of the initiation of pulmonary gas exchange.54 Such lung injuries are also related with corticosteroid administration55 or malnutrition.56 O2 exposure of newborn animals has been validly used to study some of these pathophysiologic processes.2 Data from baboons, whose lung development is very similar to that of humans, are especially valuable in this respect.57 However, mice are particularly well suited to investigations into the contribution of genetic factors, most notably genetic susceptibility to hyperoxic injury.58 The long-term effects of neonatal hyperoxia documented here suggest new criteria (compliance, breathing pattern, and lung structure) for comparing mouse models to human diseases and for further assessment of the validity of these models.


   Conclusion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
Hyperoxic exposure during lung septation in mice may cause irreversible lung injury and breathing pattern abnormalities in adulthood at O2 concentrations lower than previously thought. However, ventilatory function and body growth were preserved, and ventilatory function showed no major abnormalities, at least at rest, despite early oxygen-induced injuries.


   Acknowledgements
 
We thank Dr. Caroline Rambaud (Service d’Anatomo-Pathologie, Hôpital Antoine-Béclère, Université Paris XI) for her careful reading of a previous version of the article.


   Footnotes
 
Abbreviations: Crs = respiratory system compliance; PNA = postnatal age; PNA45 = postnatal age of 45 days; PNA100 = postnatal age of 100 days; PNA290 = postnatal age of 290 days; RAC = radial alveolar count; TE = expiratory duration; TI = inspiratory duration; TTOT = breath duration; E = minute ventilation; VT = tidal volume

This study was supported by the Institut National de la Santé et de la Recherche Médicale (grant awarded to Dr. Dauger), and the Université Paris VII (Legs Poix).

Received for publication February 26, 2002. Accepted for publication July 23, 2002.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

  1. Gries, DM, Tam, EK, Blaisdell, JM, et al (2000) Differential effects of inhaled nitric oxide and hyperoxia on pulmonary dysfunction in newborn guinea pigs. Am J Physiol Regul Integr Comp Physiol 279,R1525-R1530[Abstract/Free Full Text]
  2. Warner, BB, Stuart, LA, Papes, RA, et al Functional and pathological effects of prolonged hyperoxia in neonatal mice. Am J Physiol 1998;275,L110-L117[Abstract/Free Full Text]
  3. Blanco, LN, Frank, L The formation of alveoli in rat lung during the third and fourth postnatal weeks: effect of hyperoxia, dexamethasone, and deferoxamine. Pediatr Res 1993;34,334-340[ISI][Medline]
  4. Blanco, LN, Frank, L Development of gas-exchange surface area in rat lung: the effect of alveolar shape. Am J Respir Crit Care Med 1994;149,759-766[Abstract]
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