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Published ahead of print on January 7, 2005, doi:10.1164/rccm.200408-1053OC
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American Journal of Respiratory and Critical Care Medicine Vol 171. pp. 743-752, (2005)
© 2005 American Thoracic Society
doi: 10.1164/rccm.200408-1053OC

Original Article

Lung Development and Susceptibility to Ventilator-induced Lung Injury

Alik Kornecki, Shinya Tsuchida, Hari Kumar Ondiveeran, Doreen Engelberts, Helena Frndova, A. Keith Tanswell, Martin Post, Colin McKerlie, Jaques Belik, Alison Fox-Robichaud and Brian P. Kavanagh

Lung Biology Program, Departments of Critical Care Medicine and Pediatrics, Hospital for Sick Children; Departments of Anesthesia, Pediatrics, Physiology, and the Interdepartmental Division of Critical Care Medicine, University of Toronto, Toronto; Intestinal Disease Research Program, Department of Medicine, McMaster University, Hamilton; Department of Pediatrics (Critical Care Unit, Children's Hospital of Western Ontario), University of Western Ontario, London, Ontario, Canada

Correspondence and requests for reprints should be addressed to Brian P. Kavanagh, M.D., Department of Critical Care Medicine, Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8. E-mail: brian.kavanagh{at}sickkids.ca


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Ventilator-induced lung injury has been predominantly studied in adults. Objectives: To explore the effects of age and lung development on susceptibility to such injury. Methods: Ex vivo isolated nonperfused rat lungs (infant, juvenile, and adult) were mechanically ventilated where VT was based on milliliters per kilogram of body weight or as a percentage of the measured total lung capacity (TLC). In vivo anesthetized rats (infant, adult) were mechanically ventilated with pressure-limited VTs. Allocation to ventilation strategy was randomized. Measurements: Ex vivo injury was assessed by pressure–volume analysis, reduction in TLC, and histology, and in vivo injury by lung compliance, cytokine production, and wet- to dry-weight ratio. Main Results: Ex vivo ventilation (VT 30 ml · kg–1) resulted in a significant reduction (36.0 ± 10.1%, p < 0.05) in TLC in adult but not in infant lungs. Ex vivo ventilation (VT 50% TLC) resulted in a significant reduction in TLC in both adult (27.8 ± 2.8%) and infant (10.6 ± 7.0%) lungs, but more so in the adult lungs (p < 0.05); these changes were paralleled by histology and pressure–volume characteristics. After high stretch in vivo ventilation, adult but not infant rats developed lung injury (total lung compliance, wet/dry ratio, tumor necrosis factor {alpha}). Surface video microscopy demonstrated greater heterogeneity of alveolar distension in ex vivo adult versus infant lungs. Conclusion: These data provide ex vivo and in vivo evidence that comparable ventilator settings are significantly more injurious in the adult than infant rat lung, probably reflecting differences in intrinsic susceptibility or inflation pattern.

Key Words: infant • lung injury • mechanical ventilation • pediatric • ventilator-induced lung injury

Lung injury, or worsened lung injury during mechanical ventilation, is clearly a matter of major concern. Clinicians caring for ventilated patients, as well as researchers investigating the responsible mechanisms, attempt to understand the optimal approach to ventilation to lessen lung injury. A current approach whereby excessive tidal volumes (VTs) are avoided (1, 2) has evolved over time on the basis of laboratory studies performed largely in adult animals (3). Furthermore, the demonstration that the manner of application of mechanical ventilation directly affects survival attests to the importance of this area of research and has been validated in a landmark randomized clinical trial in adults (4).

Compared with adults, pediatric patients demonstrate a spectrum of lung development spanning neonatal, infant, youth, and adult stages. Beyond neonatal age, there are important differences between infant and adult lungs (e.g., alveolar structure, matrix composition, angiogenesis) (5). In fact, maturation in the human lung continues well after the newborn period until between the ages of 2 and 8 years (5). Although lung injury in preterm neonates has been extensively investigated and characterized (6), there has been limited laboratory investigation (7–9) and no prospective clinical investigation of ventilator-associated lung injury affecting infancy or youth. Instead, clinical research concerning mechanical ventilation in infants and young children (nonneonatal, nonadult) has focused not on VT or airway pressure but on adjuvant therapies, including inhaled nitric oxide (10), surfactant (11), and the use of high-frequency oscillation (12). As a result, evidence-based age-specific guidelines for the use of conventional mechanical ventilation in pediatric patients have not been possible; furthermore, any recommendations that exist have been extrapolated from adult data (13, 14).

An empiric sense that infant lungs might be more susceptible to ventilator-induced lung injury (VILI) compared with adult lungs is supported by a single laboratory investigation that compared juvenile with adult rabbits (7). That report, which demonstrated greater injury in the lungs of younger animals, concluded that the findings may have reflected the larger VTs delivered because of greater respiratory system compliance (7). However, there is an important reason to believe that infant lungs may be less susceptible to VILI. VT is usually expressed in terms of milliliters per kilogram of body weight, but the ratio of lung volume compared with body weight varies with development. In rats, it increases (15), and in humans, it may decrease (16). Rather than attempt to define locally applied mechanical stress, the current study evaluated the pulmonary responses to comparable levels of VT on the basis of body weight (ml · kg–1) and lung volume (ml/ml, % total lung capacity [TLC]), as well as on inflation pressure.

Our main objective in this study was to explore whether lung maturation beyond the neonatal age range has an effect on susceptibility to VILI in the rat lung. The rat was chosen because of the well-characterized developmental morphology (15, 17, 18), which, after 4 days of age, is comparable to human lung development (19, 20). To remove any influence of age-related chest wall compliance or lung–heart interaction, an ex vivo, isolated, nonperfused rat lung model was studied (21, 22). We then reproduced our experiments in the in vivo anesthetized rat. Some of the results of these studies have been previously reported in the form of an abstract (23).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Male Sprague-Dawley rats of three maturity levels: adult (250–360 g, age {approx} 75 days), juvenile (72–100 g, age {approx} 35 days), and infant (26–45 g, age {approx} 17 days) were used. The study was conducted according to the guidelines of the Canadian Council for Animal Care and was approved by the Animal Care Committee of the Hospital for Sick Children. Complete details of the experimental protocol are provided on the online supplement. The animals were anesthetized and ventilated (24) with peak inspiratory pressure of 9 to 10 cm H2O, positive end-expiratory pressure of 1 cm H2O, inspired oxygen of 0.21, and respiratory rates of 45 (adult), 60 (young), and 80 (infant) breaths/minute. For ex vivo experiments, lungs and heart were removed en bloc and suspended in a warm, humidified chamber (21, 25, 26).

Experimental Outline
After determination of baseline TLC and pressure–volume (P-V) curves, lungs in Series 1 and 2 were randomly allocated to receive either no ventilation (control) or mechanical ventilation for 60 minutes. In Series 1, ex vivo lungs were ventilated with a VT of 30 ml · kg–1, a positive end-expiratory pressure of 0, a respiratory rate of 40 minutes–1, an FiO2 of 0.21, and an FiCO2 of 0.05 for 60 minutes. In Series 2, ex vivo lungs were ventilated with a VT set to 50% of baseline TLC. After the experiments, lung weight was recorded. In Series 3, anesthesia was maintained and the intact in vivo infant and adult animals ventilated with a peak inspiratory pressure of 20 or 30 cm H2O for 90 minutes, and compared with nonventilated control animals. In the in vivo experiments, respiratory rate was the same in all groups (34 minutes–1), and added dead space was used to obtain comparable PaCO2 levels (35–45 mm Hg) in each group. In Series 4, lungs were ventilated as in Series 2, and with a respiratory rate of 30 minutes–1. Alveolar inflation and deflation were measured using inverted microscopy and analyzed offline. Static P-V curves were constructed, before and after ventilation (21, 27). Lungs were excluded from the experiments in the event of air leaks or technical problems with preparation. After in vivo experiments (Series 3), the decrement in quasi-static respiratory compliance, bronchoalveolar lavage concentration of tumor necrosis factor {alpha} (TNF-{alpha}), and the lung wet/dry weight were determined.

Morphologic Analysis
The left lung was isolated and inflation-fixed in formalin (inflation pressure 20 cm H2O for 48 hours) (22). An assessment was performed independently by two observers, who were blinded as to group allocation and the mean values reported (22).

Statistical Analysis
The data are expressed as mean ± SD. Statistical comparisons were performed with either analysis of variance followed by Student-Neumann-Keuls tests (Series 1), or with Student's t test (Series 2, 3). TNF-{alpha} (Series 3) was analyzed using the nonparametric Kruskal-Wallis test, and post hoc analysis was with Dunn's method. Histology scores between observers were compared using interclass correlation coefficient (0.88, 95% confidence intervals, 0.817–0.924). We considered differences significant where p was less than 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experimental Series 1: Ex Vivo VT Determined by Body Weight
Baseline characteristics.
A total of 54 animals were selected; 50 were randomized, and 48 completed the protocol. Baseline variables are reported in Table 1. The TLC corrected for body weight was ranked as follows: infant > juvenile > adult (Table 1; p < 0.05). This pattern was conserved for baseline chord compliance, which was also corrected for body weight (Table 1; p < 0.05). The baseline peak inspiratory pressure, recorded 10 minutes after commencement of ventilation, was significantly higher in the adult group than in the other groups, with a rank order as follows: adult > juvenile {approx} infant lungs (Table 1; p < 0.05).


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  		TABLE 1. Baseline characteristics of infant, juvenile, and adult lungs in series 1 (vT 30 ml/kg–1)

 
Impact of ventilation on lung mechanics.
After 60 minutes of ventilation with high VT, the TLC decreased significantly from the baseline values in the adult and juvenile groups, but not in the infant group (Figure 1). The TLC (expressed in ml · kg–1) decreased from 39.2 ± 3.0 to 25.0 ± 3.6 ml · kg–1, a reduction of 36.0 ± 10.1%, in the adult group (Figure 1; p < 0.05). In the juvenile group, the TLC decreased from 56.4 ± 4.7 to 47.5 ± 6.1 ml · kg–1, a reduction of 15.4 ± 10.1% (Figure 1; p < 0.05). In the infant group, the TLC was not significantly reduced after ventilation (Figure 1; 79.8 ± 9.2 vs. 75.9 ± 7.4 ml · kg–1, p = 0.32). The rank order of high VT–induced decrease in TLC was as follows: adult > juvenile {approx} infant (Figure 1; p < 0.05).



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  		Figure 1. Total lung capacity (TLC; ml · kg–1) before (open bars) and after (solid bars) 60 minutes of mechanical ventilation with VT of 30 ml · kg–1 in Series 1. The rank order of baseline TLC (ml · kg–1) was as follows: infant > juvenile > adult (*p < 0.05). TLC was significantly reduced after ventilation in adult and juvenile lungs ({dagger}p < 0.05), but not in infant lungs. The magnitude of the reduction in TLC was as follows: adult > juvenile > infant ({ddagger}p < 0.05). All data are mean ± SD. ns = not significant.

 
In addition to the TLC data, the complete P-V characteristics are presented for each group. Because the P-V characteristics change with age (28), the baseline and final P-V curves for each of the groups were expressed in the following three ways: pressure (cm H2O) versus uncorrected volume (ml; Figure 2A), pressure (cm H2O) versus volume per kilogram of animal body weight (ml · kg–1; Figure 2B) (7), and as pressure (cm H2O) versus volume expressed as a percentage of the preventilation TLC (Figure 2C) (29). Comparison of these P-V curves demonstrates the following features, which are independent of how the inflation and deflation volumes are expressed. First, the P-V curves displayed postventilation downward shift, reflecting a reduction in chord compliance as follows: adult > juvenile > infant (Figure 2). Second, there was no inflection point demonstrable in any of the preventilation curves, and after ventilation, a lower inflection point developed in the adult lungs, but not in the infant or juvenile lungs (Figure 2).



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  		Figure 2. Pressure–volume (P-V) curves before (solid circles) and after (open circles) mechanical ventilation for 60 minutes with VT of 30 ml · kg–1 in Series 1. The three columns represent the P-V curves from lungs of infant (left), juvenile (center), and adult (right) rats. The P-V curves are expressed in the three panels as follows: (A) absolute inflation volumes (ml; uncorrected), (B) inflation volumes corrected for body weight (ml · kg–1), (C) inflation volumes expressed as a percentage of the preventilation TLC). After ventilation, a lower inflection point is demonstrable in adult (but not the juvenile or infant) P-V curves; and the juvenile and adult (but not infant) P-V curves were shifted downward (*p < 0.05). All data are mean ± SD.

 
Impact of ventilation on lung histology.
There was no difference in alveolar injury, expressed as hyaline membrane score, among the nonventilated (control) adult, juvenile, or infant lungs (Figure 3). The hyaline membrane score was greater in ventilated than in nonventilated lungs in the adult group only, and not in the juvenile or infant groups. In ventilated lungs, the rank order of hyaline membrane score was as follows: adult > juvenile {approx} infant groups (Figure 3; p < 0.05). There were no differences detected in hyaline membrane score between proximal and distal airspaces in any of the groups (data not presented). Airway injury scores were not different among the nonventilated (control) lungs from adult juvenile or infant animals (Figure 4). The airway injury score was significantly greater in ventilated lungs compared with nonventilated lungs in adult, but not in juvenile or infant groups (Figure 4; p < 0.05). In ventilated lungs, the rank order of airway injury score was as follows: adult > juvenile {approx} infant groups (Figure 4; p < 0.05). There were no differences detected in airway injury score between proximal and distal airways (data not presented).



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  		Figure 3. Airspace injury, expressed as hyaline membrane score, in nonventilated lungs (open bars; control) and ventilated lungs (solid bars; VT 30 ml · kg–1, 60 minutes) in Series 1. The control values are comparable among all ages (adult, juvenile, infant). Hyaline membrane scores were greater in ventilated versus control in adult and juvenile (but not infant) lungs (*p < 0.05), and the rank order of score after ventilation was adult > juvenile > infant ({dagger}p < 0.05). All data are mean ± SD.

 


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  		Figure 4. Airway injury in nonventilated lungs (open bars; control) and ventilated lungs (solid bars; VT 30 ml · kg–1, 60 minutes) in Series 1. The control values are comparable among all ages (adult, juvenile, infant). Airway injury score was greater in ventilated versus control values in adult (but not juvenile or infant) lungs (*p < 0.05), and the rank order of score after ventilation was adult > juvenile {approx} infant ({dagger}p < 0.05). All data are mean ± SD.

 
Series 2: Ex Vivo VT Determined by TLC
In Series 2, VT was dictated not by body weight (i.e., ml · kg–1) but by the baseline lung volume (i.e., VT administered was 50% of baseline TLC). Pilot studies indicated that this approach was associated with lower peak airway pressures in the adult versus infant lungs. The greatest differences in Series 1 were between the adult and the infant lungs, and so these groups were the focus of Series 2.

Baseline characteristics.
In this series, seven adult and seven infant animals were selected; six from each group were studied, and all completed the experiments. The baseline variables are reported in Table 2.


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  		TABLE 2. Baseline characteristics of infant and adult lungs in series 2 (vT 50% total lung capacity)

 
Impact of ventilation on lung mechanics.
Despite the higher VT (ml · kg–1) and peak inspiratory pressure in the infant lungs, 60 minutes of mechanical ventilation with a VT at 50% of baseline TLC was associated with a significantly smaller reduction in TLC in the infant versus the adult lungs (Figure 5; p < 0.05). In the adult lungs, TLC corrected for body weight decreased from 39.8 ± 2.88 to 28.5 ± 2.82 ml · kg–1 (Figure 5; p < 0.05), whereas in the infant group, the TLC decreased from 68.3 ± 10.5 to 61.1 ± 10.9 ml · kg–1 (Figure 5; p < 0.05). The percentage of reduction in TLC (% baseline value) was greater in the adult versus the infant lungs (28.7 ± 2.84 vs. 10.6 ± 7.0%; Figure 5; p < 0.05). This pattern was maintained with expression of the P-V curves in terms of the following: pressure (cm H2O) versus uncorrected volume (ml), pressure (cm H2O) versus volume corrected for body weight (ml · kg–1), and as pressure (cm H2O) versus volume expressed as a percentage of the baseline volume at the same static pressure (Figure 6).



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  		Figure 5. TLC (ml · kg–1) before (open bars) and after (solid bars) 60 minutes of mechanical ventilation with VT of 50% TLC in Series 2. The baseline TLC (ml · kg–1) was greater in the infant versus adult lungs (*p < 0.05). TLC was significantly reduced after ventilation in both infant and adult lungs ({dagger}p < 0.05), and the magnitude of that reduction was significantly greater in the adult versus the infant lungs ({ddagger}p < 0.05). All data are mean ± SD.

 


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  		Figure 6. P-V curves before (solid circles) and after (open circles) mechanical ventilation for 60 minutes with VT, 50% of baseline TLC, in Series 2. The two columns represent the P-V curves from lungs of infant (left) and adult (right) rats. The P-V curves are expressed in the three panels as follows: (A) absolute inflation volumes (ml; uncorrected), (B) inflation volumes corrected for the body weight (ml · kg–1), (C) inflation volumes expressed as a percentage of the preventilation TLC. After ventilation, a lower inflection is demonstrable in adult (but not infant) P-V curves, and whereas both adult and infant P-V curves are shifted downward, the change in chord compliance is greater in the adult than in the infant curves (*p < 0.05). All data are mean ± SD.

 
Impact of ventilation on lung histology.
Hyaline membrane scores and airway injury scores were both significantly greater after ventilation in the adult versus the infant lungs (Figure 7; see Figure E1 in the online supplement). No significant differences in hyaline membrane scores or airway injury score were observed between proximal and distal areas (data not presented).



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  		Figure 7. Histology data after mechanical ventilation for 60 minutes with VT, 50% of baseline TLC, in Series 2. (A) Airspace injury, expressed as hyaline membrane score, was greater in adult than infant lungs (*p < 0.05). (B) Airway injury score was greater in adult than in infant lungs (*p < 0.05). All data are mean ± SD.

 
Series 3: In Vivo Ventilation
Baseline characteristics.
Thirty-two animals were selected, and all completed the experiment. The baseline variables are reported in Table 3. When ventilated with 20 cm H2O, the resultant VTs in the infants versus adults were 37.5 ± 1.2 versus 18.4 ± 1.3 ml · kg–1, respectively (Table 3; p < 0.05). Similarly, when ventilated with 30 cm H2O, the resultant VTs in the infants versus adults were 42.4 ± 4.4 compared with 29.7 ± 3.8 ml · kg–1 (Table 3; p < 0.05).


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  		TABLE 3. Baseline characteristics of infant and adult in vivo animals in series 3

 
Impact on lung mechanics and edema.
After 90 minutes of mechanical ventilation, no change in total lung compliance was detected in the adult animals ventilated with a pressure of 20 cm H2O, but a significant reduction in lung compliance occurred in those ventilated with a pressure of 30 cm H2O (Figure 8). Despite being ventilated with comparatively higher VTs (in terms of ml · kg–1), no reductions in lung compliance occurred in any of the infant animals, whether ventilated with 20 or 30 cm H2O (Figure 8). The magnitude of the effect of ventilation on compliance in the adult and infant animals paralleled the changes in lung wet/dry ratio (Figure 9).



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  		Figure 8. Decrement in compliance after in vivo mechanical ventilation for 90 minutes with peak inspiratory pressures of either 20 or 30 cm H2O in adult and infant animals in Series 3. Compared with baseline compliance, there was no decrement in either of the infant groups (Infant-20, Infant-30) or in the adult group (Adult-20); in contrast, there was a significant decrease in compliance in the Adult-30 group (*p < 0.05). All data are mean ± SD.

 


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  		Figure 9. Lung wet/dry weight ratio in infants and adults after in vivo mechanical ventilation for 90 minutes with peak inspiratory pressures of either 20 or 30 cm H2O, compared with nonventilated controls (C), in Series 3. In infants, the wet/dry ratio was not different among either of the ventilated groups (Infant-20, Infant-30) or compared with nonventilated infant controls. In adults, wet/dry ratio was significantly greater in Adult-30 compared with Adult-20 and nonventilated adult controls (*p < 0.05). All data are mean ± SD.

 
Impact on pulmonary TNF-{alpha}.
The volume of bronchoalveolar lavage fluid retrieved was similar within each age group. An illustrative proinflammatory cytokine, TNF-{alpha} (30), was not detected in nonventilated control animals and was significantly greater in ventilated adults ventilated with 30 cm H2O versus all other groups, with no other among-group differences (Figure 10; p < 0.05).



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  		Figure 10. Bronchoalveolar lavage fluid tumor necrosis factor {alpha} (TNF-{alpha}) concentration in infants and adults after in vivo mechanical ventilation for 90 minutes with peak inspiratory pressures of either 20 or 30 cm H2O, compared with nonventilated controls, in Series 3. TNF-{alpha} was not detected in nonventilated controls and was significantly greater in ventilated adults ventilated with 30 cm H2O versus all other groups, with no other among-group differences (*p < 0.05).

 
Series 4: Ex Vivo Ventilation—Lung Imaging
Three animals from each group (infant 43 ± 1.8 g, adult 331 ± 33 g) completed the experiment. The mean TLC (infant 38.2 ± 3.2 ml · kg–1, adult 69 ± 7.2 ml · kg–1) was similar to the respective values in Series 1 and 2. Mean alveolar diameter at the end-expiration was 39.7 ± 8.4 µm in adults compared with a mean of 35.3 ± 4.5 µm in infants (p = not significant). Mean alveolar diameter at end-inspiration was significantly greater in the adult versus infant lungs (77.3 ± 6.7 vs. 57.3 ± 2.0 µm, p < 0.05). The mean variance of the alveolar diameter at the end of inspiration was significantly greater in adult versus infant lungs (824 ± 240 vs. 318 ± 40, p < 0.05; Figure 11).



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  		Figure 11. Ex vivo photomicrographs of a field (350 x 260 µm) in adult lung at end-expiration (A), adult lung at end-inspiration (B), infant lung at end-expiration (C), and infant lung at end-inspiration (D). The end-inspiration alveoli size appears more uniform in infant compared with adult lung. The variability of the alveolar diameter at the end of inspiration is significantly greater in adult versus infant lungs (see text).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The principal finding of this study is that, whether VT is based on body weight (ml · kg–1) or baseline TLC (%), comparable ventilator settings resulted in greater injury in adult versus infant (nonneonatal, nonadult) lungs. The lessened lung injury in infants was confirmed in vivo, despite exposure to larger VT (relative to body weight) and higher peak inspiratory pressure. In addition, adult lungs appeared to have a more heterogeneous distribution of alveolar ventilation. Although the current study did not directly address the issues of local lung stress or strain (31), the overall data may suggest that intrinsic properties of the lung are responsible for the lesser susceptibility in infants.

Development and Susceptibility to Lung Injury
Laboratory or clinical studies of VILI have been performed mostly in surfactant-deficient preterm newborns (32, 33) or in adults (3, 4, 22, 34). Two articles have recently reported important findings that may relate to mechanisms of VILI in nonpremature newborn models (35, 36). Newborn rats exposed to high VT demonstrate less cytokine mRNA production compared with adult animals (36). In addition, Rau and coworkers (35) have demonstrated that, in a porcine model, the newborn has different surfactant composition—and superior dynamic function—than the adolescent.

Such comparative studies are important because they recognize the potential impact of development on susceptibility to VILI between newborn versus adult lungs. However, beyond the newborn or neonatal period, only three laboratory studies have addressed VILI in young animals (7–9). The only study (7) that directly compared juveniles with adults demonstrated that, with comparably elevated peak inspiratory pressures, younger animals developed more lung injury. The authors speculated that, because of greater respiratory system compliance, the younger animals might have been exposed to disproportionately larger VT (7).

The current study focuses on the infant versus adult susceptibility, where infancy in this model is characterized by almost complete alveolarization (i.e., having attained the adult number of alveoli) (15, 18), and with the principal differences between infant (nonneonate) and adult lungs being the degree of matrix development (28, 37).

Choice of VT
Where VT is considered in the clinical context, it is usually specified as milliliters per kilogram of body weight. Although the relationship between VT, body weight, and ventilator-associated lung injury is complex in adults (38), there is an additional layer of complexity in children. This is because even where weights can be calculated with high degrees of accuracy, the ratio of lung volume (TLC) to body weight changes with age and development. The ratio of TLC to body weight in the present study decreased over maturation from 74.0 ± 8.8 ml · kg–1 in infant, to 53.4 ± 5.6 ml · kg–1 in juvenile, and to 39.7 ± 4.0 ml · kg–1 in adult groups (data averaged from Tables 1 and 2). These findings are comparable to previously reported data for the developing rat (15, 28, 39, 40). Although the optimal measurement of lung volume in young children is not established, the available data suggest that TLC/body weight ratio increases with age from 52 ± 13 ml · kg–1 in infants to 87 ± 11 ml · kg–1 in older children (16, 29).

Potential Mechanisms
There are several possible explanations for the current findings. The absolute VT was different among the groups. However, in Series 1, the VT was identical in terms of milliliters per kilogram of body weight and in Series 2 was dictated by TLC (50% TLC). In addition, airway pressure, although slighter higher in the adult group in Series 1, was significantly lower (~ 2 cm H2O) in the adult group in Series 2. Thus, neither differences in VT nor in airway pressure explain the among-group differences in injury; in fact, the study was biased against finding worse injury in the adult lungs in Series 2, because both the VT (in ml · kg–1) and airway pressures were lower in the adult lungs.

The inclusion of in vivo experiments provides additional important information, because the presence of the intact chest wall, pulmonary perfusion with intact blood, as well as the potential for cardiopulmonary interactions corroborate the ex vivo findings. There are significant differences in baseline TLC, which, when corrected for body weight, are far larger in infant versus adult lungs (Tables 1 and 2). This result could certainly explain the findings in Series 1 where VT based on body weight would occupy a smaller fraction of TLC in infants versus adults and therefore cause less stretch-induced injury, but this could not explain the differences observed in Series 2. Lung compliance represents the interaction of pressure and volume and determines how a given VT alters pressure and vice versa. However, comparing baseline compliance among different age groups is complex, and although correction for either body weight or lung weight has been reported (41), there is no universally agreed-on approach. Both approaches have been used in the current experiments, and when corrected for body weight, the compliance is greater in infants, but when corrected for lung weight, is greater in adults. Overall, the complexity of correcting or normalizing lung parameters for either body weight or lung weight is apparent from the current study, as well as from previous publications (15, 28, 39–41). It seems logical therefore to expect a different impact of "body weight–directed" versus "lung volume–directed" prescription of VT.

The pattern of alveolar distension might provide additional explanation. Real-time direct visualization of the lung has been recently described (42), and has provided important insights into alveolar micromechanics. Notwithstanding the sampling limitations of such surface visualization of subpleural alveoli (43), the current study describes a greater degree of heterogeneous subpleural alveolar inflation in adult versus infant lungs. This finding raises, but does not prove, the possibility that either underdistension (22) and/or overdistension (44) may account for the differences in injury between the two groups. Furthermore, the downward and rightward shift of the adult P-V curves after ventilation, as well as the appearance of a lower inflection point, may support the previously described findings and suggest an atelectasis-associated mechanism of injury in the adult lung (Figures 2 and 6) (45).

It is possible that the infant lung, which at 17 days has almost full alveolarization but with smaller alveoli, has an increased quantity of surfactant relative to the alveolar size. If true, this could play a role in the increased uniformity of inflation and lesser susceptibility to VILI observed in the infant lungs. Unfortunately, surfactant analysis was not performed in the current study.

An integrated explanation of these phenomena may involve appreciation of the differences in the matrix of infant versus adult lungs. The levels of collagen and elastin, the major matrix components, undergo significant changes between 4 and 40 days (28). Collagen concentration increases linearly over the interval from infancy to adulthood, whereas elastin concentration increases 10-fold over the first 20 days and rises less rapidly thereafter (46). In addition, the cross-linking of collagen changes with age. Furthermore, data from lung slice experiments indicate that mechanical stretch changes lung matrix properties (47). Thus, it is possible that the age-related change in ratio of elastin to collagen could contribute to altered susceptibility to stretch-induced lung injury. Finally, the recent studies cited previously (35, 36) may provide additional explanation. If the relatively blunted cytokine response (36) or enhanced surfactant function (35) that were reported in the newborn are also present in older infants, then the lowered susceptibility to ventilator-associated lung injury of infants may be, in part, attributable to these phenomena.

Pulmonary Cytokine Release
The role of cytokines in general and TNF-{alpha} in particular in the genesis of VILI is controversial (48, 49). The observation that the concentration of TNF-{alpha} increases significantly after high VT ventilation in vivo in the adult but not the infant (Figure 10) mirrors a previous report (36) where mechanical ventilation promoted less production of cytokine in newborn compared with adult rats. That finding may be explained by a difference in immunologic maturation between adult versus infant rats, or by the known differences in the matrix composition that may affect the way that global lung stretch is transmitted to individual cells.

Choice of Experimental Models and Study Limitations
The use of an ex vivo preparation alone would limit the applicability of the data. The current data, however, include in vivo confirmation, and the ex vivo data control for the impact of pulmonary vascular pressure or flow on injury (50, 51). The TLC in this study was measured in nondegassed lung. In fact, we measured the volume above the FRC volume. Furthermore, the value of FRC may differ across progressive stages of lung development, and an increased ratio of FRC to TLC has been described in newborn versus adult rats (41). We know of no data regarding true FRC values in the isolated nonperfused rat lung at infancy, but (at any age) they are almost certainly far smaller than in vivo values. Because each lung served as its own control, baseline differences in FRC among the groups should not alter the conclusions.

All animal models of human development are necessarily limited. Although there are important differences in development between the human and rat lung (52), the rat lung provides a good model for human lung development after the age of 4 days (19). At 1 month, the human lung is morphologically comparable to a rat lung aged 1 week, and at 2 to 5 years, the human lung is comparable to a rat lung aged 17 to 21 days (20). It is possible that development confers differences in viability on ex vivo lung preparations. Furthermore, data indicate that infants may be less susceptible than adults to organ ischemia or reperfusion (53). However, the current ex vivo experiments were performed within the known limits of lung viability (54), and such issues would not affect the in vivo data.

We have tested three different sets of comparisons (i.e., VT according to body weight, TLC, or by pressure); however, the current study did not directly address the concepts of stress or strain at the local tissue level. The central nature of these concepts has been commented on previously (31). Lung weight was measured in Series 2, and the higher ratio of VT (ml) to lung weight (g) in the adult versus the infant lungs raises the possibility that stress per unit of lung tissue mass might contribute to the increased injury in the adult lungs (Table 2).

Because neither airway resistance nor plateau pressure were recorded, it is possible that the lesser injury observed in the infant may be attributed to higher airway resistance or to auto-positive end-exipirator pressure. However, as previously suggested by Gomes and coworkers (40), airway resistance in rats, when corrected for weight, is not significantly different between the infant and adult. In addition, the length of the endotracheal tube was designed to abolish any differences in endotracheal tube resistance (see online supplement for details). Furthermore, from the clinical perspective, lung mechanical stretch is extrapolated from the magnitude of VT and/or plateau pressure. Thus, inclusion of plateau pressure, especially in the ex vivo preparations, should be considered in future work.

Potential Significance
This study, the first study to make age-specific comparisons of VILI between infants and adults, suggests four potential lessons. First, infant lungs may be less susceptible to VILI when exposed to comparable ventilation settings of VT or pressure. Second, development or propagation of stretch may be different in infant than adult lungs. Furthermore, if the differences observed in inflation pattern (Figure 11) are confirmed in other studies, then the utility of positive end-expiratory pressure or recruitment maneuvers may be different in infants versus adults. Third, we suggest caution in extrapolating recommendations about ventilator settings from adult studies to children, especially where the VT is prescribed on the basis of milliliters per kilogram of body weight. Finally, because understanding preclinical phenomena is an important factor in the design of successful clinical trials (55), studies such as the current report might eventually influence clinical evaluation of pediatric ventilatory strategies that are distinct from adult studies.


   Acknowledgments
 
The authors thank Dr. A.C. Bryan for his insightful comments and Derek Stephens (Program in Population Health Sciences, Hospital for Sick Children) for statistical expertise.


   FOOTNOTES
 
Supported by the Canadian Institutes of Health Research (CHIR). B.P.K. is the recipient of a New Investigator Award (CHIR) and a PREA award (Ontario Ministry of Science and Technology).

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Conflict of Interest Statement: A.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; H.K.O. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; D.E. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; H.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.K.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; C.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.F.-R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; B.P.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form August 13, 2004; accepted in final form January 1, 2005


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