PEDIATRICS Vol. 106 No. 6 December 2000, pp. 1452-1459

Lung Elastic Tissue Maturation and Perturbations During the Evolution of Chronic Lung Disease

Donald W. Thibeault, MD^*, , Sherry M. Mabry, MS^*, Ikechukwu I. Ekekezie, MD^*, , and William E. Truog, MD^*,

From the ^* Department of Pediatrics, Children's Mercy Hospital; and the  University of Missouri-Kansas City School of Medicine, Kansas City, Missouri.

	ABSTRACT

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Background.  Infants <30 weeks' gestation have difficulty maintaining adequate functional residual capacity after the first week of life without positive end-expiratory pressure. We hypothesized that this is caused, in part, by increased lung elastic recoil. Our aims were to quantitate parenchymal elastic tissue during normal fetal development and in infants born at 23 to 30 weeks' gestation with prolonged survival at risk for chronic lung disease (CLD).

Methods.  The controls were 22 to 42 weeks' gestation (n = 71), received ventilator care, and died within 48 hours of birth, plus 7 term infants who died at 43 to 50 weeks' postconceptional age from nonpulmonary causes. Infants who were 23 to 30 weeks' gestation, at risk for CLD, and who lived 5 to 59 days (n = 44), were separated into groups based on respiratory score (SCORE; The integrated area under the curve of the average daily fraction of inspired oxygen × mean airway pressure (cm H₂O) over the number of days lived). The SCORE groups, <20, 21 to 69 and 70 to 200, related clinically to mild to severe lung disease. The lungs were tracheally perfused and formalin-fixed and total lung volume (TLV) was measured by water displacement. The paraffin-embedded lung blocks were stained with Miller's elastic stain. The parenchyma and parenchymal elastic tissue were point-counted. The absolute elastic tissue was calculated by multiplying TLV by the parenchymal and elastic fractions. Septal width, alveoli and alveolar duct diameters, and internal surface area (ISA) were also measured.

Results.  In the controls, the volume density of parenchymal elastic tissue and absolute quantity of elastic tissue increased progressively from 22 to 50 weeks. In infants with CLD and SCORE 20, the volume density and absolute quantity of elastic tissue increased significantly. Mean absolute elastic tissue in the 20 to 69 group was 0.76 ± 0.20 cm³ greater than in the <20 group (0.46 ± 0.10 cm³) who were similar to the controls, and the 70 to 200 group was 1.32 ± 0.56 cm³ greater than the 20 to 69 group. Elastic tissue for infants at risk for CLD, as a percent of predicted for same-age controls, rose linearly with increasing SCORE (r = 0.73; r² = 0.55). Control TLV and ISA were linearly related to age. Thirty-nine of the 44 CLD-risk infants had TLVs greater than controls. However, 77% with SCORE 20 to 200 had ISAs less than or equal to the control 95% confidence interval. Control septal width decreased sharply from 23 to 30 weeks, then gradually decreased to term. All infants with SCORE 70 to 200 and 80% of those with SCORE 20 to 69 had widths more than the control 95% confidence interval. Control alveolar and duct diameters doubled from 23 to 50 weeks and were significantly greater in infants with SCORES 20 to 200.

Discussion.  Lung elastic tissue maturation is tightly controlled during fetal development. With increasing SCORE, elastic tissue increased >200%, accounting, in part, for the positive end-expiratory pressure needed to maintain end-expiratory lung volume in infants at risk for CLD. Saccule and duct diameters more than doubled, and septa thickened significantly in CLD. We propose the following sequence to be operative in CLD: at birth, the preterm infant (30 weeks) has inadequate elastic tissue and elastic recoil, but high surface tension recoil. After surfactant treatment, surface tension recoil markedly decreases, permitting the saccules and ducts, with very low elastic recoil, to be overstretched by volutrauma. The damaged lung responds with elastosis, distorted acinar growth, cellular influx, and upregulation of inflammatory and reparative proteins. This hypothesis can be summarized by the following terms: lung immaturity, inflammation, volutrauma, and elastic tissue alterations.  Key words:  lung elastic tissue, chronic lung disease.

Preterm infants <30 weeks' gestation with chronic lung disease (CLD) have difficulty maintaining adequate lung volume without some mode of positive end-expiratory pressure, as demonstrated on chest radiographs. This low resting lung volume or functional residual capacity (FRC), which has its onset late in the first week of life, may persist for weeks and does not seem to be related to surfactant deficiency. With adequate surfactant, the FRC is mainly dependent on the elastic recoil pressures of the chest wall and lungs. Lung elastic tissue is a dominant force in determining the FRC in mechanically-ventilated infants. During fetal lung development, lung elastic growth is thought to provide a template for the acinar architecture.^1,2 CLD, also known as bronchopulmonary dysplasia, is common in infants <30 weeks' gestational age (GA), and abnormalities of alveolar septation and elastic tissue metabolism are important in its pathogenesis.^3-5 In chronically ventilated preterm lambs and baboons, there is decreased complexity of the acini and increased amounts and abnormal distribution of elastic tissue.^6-8

The preterm infant is subjected to a number of factors that are known to influence elastic tissue growth and development, with the most prominent being hyperoxia,^9,10 assisted ventilation,^5,11 and steroid therapy.¹² We hypothesized that net increases in lung elastic tissue are partially responsible for the difficulty in maintaining an adequate lung volume in infants with CLD.

The objectives of this study were:

1. to quantitate normal parenchymal elastic tissue during fetal development;
2. to quantitate parenchymal elastic tissue in infants at risk for CLD, born at 23 to 30 weeks' gestation, who lived 5 to 59 days;
3. to define the relationship, if any, between elastic tissue, quantified postmortem, and previous exposure to mean airway pressure (MAP) and oxygen concentration; and
4. to correlate lung elastic tissue with total lung volume (TLV) and surface area.

	MATERIALS AND METHODS

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The 71 control infants (GA range: 22-42 weeks) were appropriate weight for GA, received ventilator care, and died within 48 hours of birth (Table 1). Infants were excluded if they had prolonged rupture of fetal membranes (>48 hours), widespread bronchopneumonia, or if all lung lobes could not be inflated postmortem, leaked, or had extensive hemorrhage seen on lung histology. (Ten lungs were not used). Seven additional infants, 44.9 ± 3.4 weeks' postconceptional age (PCA) (range: 43-50 weeks), were born at term, lived 1 to 7 weeks, then died from nonpulmonary causes, receiving a maximum of 48 hours of ventilator care.

There were 44 infants, 25.8 ± 1.7 weeks' GA (range: 23-30 weeks) at risk to develop CLD. These infants died at PCA 28.6 ± 2.4 weeks, lived a mean of 19.8 days with a range of 5 to 59 days, and were ventilated for various durations (Table 1). These 44 infants were separated into 3 arbitrarily chosen groups based on a respiratory score (SCORE). The SCORE for each infant was determined by multiplying the average daily fraction of inspired oxygen (FIO₂) by the average daily MAP in cm H₂O and integrating the area under the curve, using the trapezoidal rule, for the total number of days lived. The 3 SCORE groups, <20, 21 to 69, and 70 to 200, generally related clinically to mild, moderate, and severe lung disease, respectively.

Lung Preparation

The lungs, collected over a 10-year period, were perfused and fixed the same way. The time from death to autopsy was 12 ± 12 hours. The lungs were weighed wet. The left lung was deflated by vacuum, warmed to 38°C, and tracheally inflated 72 hours at 24 cm H₂O pressure with 10% formalin. The left lung TLV was measured by water displacement. The TLV of both lungs was calculated by assuming a constant ratio between the wet weights and lung volumes. The following sections were cut: an entire cross section of the left upper lobe, the base of the lingula, two 1.5 × 1.0 cm pieces from the left lower lobe and one 1.5 × 1.0 cm longitudinal cut (including the central hilar structures surrounded by the parenchyma. After immersion in 10% buffered formalin, the area of the cut surface of each section was measured by computerized image analysis (Media Cybernetics, L.P., Silver Spring, MD). The area of the same section was measured again after processing, paraffin embedding, and staining to determine shrinkage factors, which were used to correct linear and areal measurements. Sections, 5-µm thick, were stained with hematoxylin and eosin, or Miller's elastic stain, counterstained lightly with eosin.

Using point-counting, the volume density of the parenchyma was determined.¹³ The parenchyma included saccular or alveolar and alveolar ductal air space and septal tissue. The volume density of the parenchymal elastic tissue (V_Vel) was then point-counted under oil (×100) by 2 separate observers and the average value was used. Each field was brought into focus, and without refocusing during the count, the hits on elastic tissue were counted. One hundred sixty sequential fields were assessed, but in lungs with cystic lesions, 400 fields were counted. Fields containing nonparenchyma or a blood vessel larger than a capillary were skipped. Because the sections have a finite thickness, elastic fibers within the sample, as well as the surface fibers, can be projected onto the observational plane, thereby overestimating the volume density of elastic fibers. Elastic fibers have a tubular shape, with the length many times longer than the diameter. A theoretical correction factor (k) has been derived to be k = 1/(1 + t/d), where t is the thickness of the observational depth and d is the average diameter of the elastic fibers.¹³ Focal depth, or thickness (t) was considered to be 0.2 µm.¹³ The average elastic fiber diameter was 0.5 µm. The calculated correction factor was 0.73, which reduced the observed volume density by 27%. The intraobserver error (coefficient of variation) was determined by 1 observer blindedly measuring the volume density of elastic tissue 10 times on a subject. The coefficient of variation was computed as the ratio of the standard deviation to the mean of the 10 samples and expressed as a percentage. The intraobserver error was 11%. The absolute amount of elastic tissue was calculated by multiplying the TLV by the parenchymal volume and the volume density of parenchymal elastic tissue. Interalveolar or intersaccular septal diameter was measured on hematoxylin and eosin stained sections with an eyepiece ruler on 20 consecutive fields of each of the 5 blocks and the average calculated to give the apparent septal width. The chord length of alveoli or saccules and alveolar ducts were measured similarly. The mean linear intercept was measured using crossed hairlines of known length. The internal surface area (ISA) of lungs was calculated using the mean linear intercept method (ISA = 4v/LM, where v is the displacement volume of the lungs and  = the fraction of lung containing parenchyma).¹⁴ Measurements were done with the observers blinded to the infant's grouping.

Statistical Analysis

Values are given as mean ± standard deviation (SD) in Table 1. Statistical analysis in Table 1 and text was performed with t tests or analysis of variance when there were >2 groups. A value of P < .05 was considered significant. Regression analysis and best-fit equations were determined by computer analysis (SigmaStat: SigmaPlot, SPSS, Inc, Chicago, IL). All confidence intervals are for the population.¹⁵

	RESULTS

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In the controls, the V_Vel increased progressively from 0.3% at 22 weeks' gestation to 1.5% at 50 weeks with the steepest slope between 22 and 30 weeks (Fig 1A). The V_Vel in infants at risk for developing CLD, increased with higher SCORES. The V_Vel were 0.99 ± 0.17; 1.35 ± 0.38, and 2.41 ± 0.54, with SCORE <20, 20 to 69, and 70 to 200, respectively. SCORES of 20 to 69 were significantly higher than those <20, and SCORES 70 to 200 were significantly higher than those 20 to 69. 

View larger version (29K): [in this window] [in a new window]   Fig. 1.   A, Volume density of elastic tissue (VVel), is plotted as a function of age for the control lungs. The best-fit equation is: VVel × 100 = ln(1.34 + 0.135 × age) with r = 0.97 and r2 = 0.94 (P < .0001). Each control lung is depicted by  and the lines define the 95% CI. The mean and SD are shown for the lungs of infants at risk for CLD, by SCORE. The VVel in infants with SCORES 20 to 69 was significantly greater than in infants with SCORE <20 (P < .05; *). VVel in infants with SCORE 70 to 200 was greater than in infants with SCORES 20 to 69 (P < .025;**). B, The quantity of parenchymal elastic tissue is plotted as a function of age for the control lungs. The best-fit equation is: elastic tissue = 0.52 + (0.005 age) + (0.0014 × age2), r = 0.98; r2 = 0.97 (P < .0001). Each control lung is depicted by  and the lines define the 95% CI. The mean and SD are shown for the lungs of infants at risk for CLD, by SCORE. Same symbols as Fig 1A, but + are the individual lungs of infants with SCORES 70 to 200.

The absolute quantity of parenchymal elastic tissue (cm³) increased progressively with age (0.03 cm³ at 22 weeks and 2.4 cm³ at 50 weeks). The steepest slope occurred between 30 to 50 weeks, in general, after the increase in lung volume and V_Vel (Fig 1B). With SCORES 20 to 200, the elastic tissue was significantly elevated above those infants with SCORES <20. With SCORES <20, mean elastic tissue was 0.46 ± 0.10 cm³, which overlapped controls. Mean elastic tissue in lungs with SCORES between 20 to 69 was 0.76 ± 0.20 cm³, significantly higher than those with SCORES <20, P < .05. Mean elastic tissue in lungs with SCORES 70 to 200 was 1.32 ± 0.56 cm³, significantly higher than the 20 to 69 group, P < .001. Two infants in the chronic lung group had histologic evidence of cystic lung disease interspersed with atelectasis and relatively normal lung tissue, a classical bronchopulmonary dysplasia presentation.¹⁶ Both were in the 70 to 200 SCORE group, and had low quantities of elastic tissue (Fig 1B).

To determine whether SCORE had a unique relationship to the V_Vel, the V_Vel for each infant at risk for CLD was plotted as a percentage of the V_Vel predicted for controls at the same age (Fig 2). The percent elastic tissue rose linearly with increasing SCORE with r = 0.73 and r² = 0.55 (P < .01). This regression analysis was done without using the 2 infants with cystic lung disease, who both had very low levels of elastic tissue with high SCORES. When the area under the curve was calculated using the MAP alone, without FIO₂, the correlation was r = 0.62; r² = 0.38; (P < .01); using only FIO₂, without MAP, the linear relationship was r = 0.55 and r² = 0.30 (P < .01).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   The VVel for each infant at risk for CLD is plotted as a percentage of that predicted for same age control elastic tissue. The independent variable is the integrated area under the curve for each infant, calculated from the average daily MAP × FIO2, and summed for the days lived (SCORE). The percent predicted for controls was calculated from the equation for Fig 1A. Infants with chronic lung disease that had cystic lung disease are depicted by the  and those without cystic disease by the .

The TLV per cm of crown-heel length for the controls is linearly related to age (Fig 3A). Thirty-nine of the 43 infants at risk for CLD had lung volumes more than or equal to the 95% confidence interval (CI) of the controls. The 2 largest volumes occurred in infants in the 70 to 200 score group, who had received low FIO₂ (<0.3) and low MAP (8 cm H₂O) for prolonged periods (40 and 50 days). Lung histology showed little interstitial thickening, with good septation, suggesting accelerated lung growth in these 2 infants. The parenchymal air space volume was also linearly related to age in the controls. All but 2 of the infants with SCORES 70 to 200 had an air space volume below the 95% CI of the controls (data not shown). The 2 infants described above, with largest TLVs, had air space volumes above the 95% CI of the controls, again suggesting that with low pressures and FIO₂, lungs had accelerated growth.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   A, The lung volume per crown-heel length (LV/C-H) is plotted as a function of age. The 95% CI contains the controls which fit the linear equation LV/C-H = 1.96 + 0.12 age; (r = 0.98; r2 = 0.96; P < .0001). B, The ISA/crown-heel length (ISA/C-H) is plotted as a function of age. The 95% CI contains the controls. The best-fit linear equation for the controls is ISA/C-H = 0.07 + 0.003 × age (r = 0.98; r2 = 0.97; P < .001).

ISA per crown-heel length was also linearly related to age in the controls (Fig 3B). Despite the fact that the lung volume in the infants at risk for CLD was large, the ISA was low or normal. Infants with scores <20 had normal ISAs, but 10 of the 15 infants with SCORES 20 to 69 and 13 of the 15 with SCORES 70 to 200 had ISAs less than or equal to the 95% CI for the controls. The 2 lowest ISAs were infants with cystic lung disease, and the 2 highest ISAs were those infants described above with very large lung volumes and relatively normal looking lung histology.

Septal width in the controls decreased sharply from 23 to 30 weeks and thereafter gradually decreased to ~4 µm at 50 weeks (Fig 4A). All the infants with SCORES 70 to 100 and 80% of those with SCORES 20 to 69 had widths more than the 95% CI of the controls. However, only 58% of the low SCORES (<20 µm) were more than the 95% CI of the controls.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   A, Apparent septal width plotted as a function of age. Controls are contained within the 95% confidence interval shown. Infants with SCORE <20 to 200 are at risk for CLD. B, Saccular or alveolar chord diameter plotted as a function of age. Controls are shown as open circles. Infants with SCORES <20 to 200 are at risk for CLD.

The alveolar or saccular diameter in the controls almost doubled throughout development from 50 µm at 23 weeks to ~90 µm at 50 weeks PCA (Fig 4B). The mean saccular diameter of infants with SCORES 20 to 69 (120 ± 47 µm), and 70 to 200 (119 ± 40 µm) was significantly greater than of those with SCORES <20 (67 ± 23µm), P < .01, which overlapped the controls.

Alveolar duct diameters increased from 60 µm at 22 weeks to ~150 µm at 50 weeks. Infants with SCORES 70 to 200 had significantly greater diameter (208 ± 75 µm) than the <20 SCORE group (P < .01). Fifteen of the 16 infants with SCORES 70 to 200 had diameters more than the 95% CI of the controls. Those with scores 20 to 69, because of the wide scatter of data (183 ± 112 µm), had diameters not significantly different from either the low or high scores.

Fifty-three percent (9/17) of the infants with severe CLD (SCORE: 70-200) received postnatal steroids for various durations. There were no significant differences in the absolute elastic tissue or V_Vel between those receiving or not receiving steroids.

	DISCUSSION

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The time of appearance and quantity of elastic tissue (elastin) is tightly regulated during fetal life. The volume density of elastic tissue in the parenchyma doubles from 22 to 30 weeks, but requires another 20 weeks to double again. Considering the low rate of CLD in preterm infants >30 weeks' gestation, it seems the 30-week lung has sufficient parenchymal elastic recoil and connective tissue support, in the presence of low surface tension forces after surfactant, to tolerate short-term ventilator exposure, without severe damage or CLD development. The absolute amount of parenchymal elastic tissue, in contrast to the volume density, increases slowly from 22 to 30 weeks, and rapidly thereafter. This latter increase in elastic tissue correlates with the sharp rise in parenchymal lung volume, secondary septation, surface area, and potential for gas exchange that occurs after 30 weeks. Elastic tissue develops in the distal acinus before the development of septation and alveoli formation, which begins around 30 weeks' gestation.¹⁷ This early appearance of elastic tissue is theorized to act both as a template and as the structural framework for the later development of alveoli and gas-exchanging surface area.² Control lungs have thickened intersaccular interstitium that decreases considerably from 22 to 30 weeks, and then progressively thins out as term is approached. This thickened interstitium in infants <30 weeks seems to be void of elastic fibers and bears no histologic resemblance to the mature alveolar septum.¹⁸ Among other functions, it provides a potential space for the formation of new distal lung units. The presence of this thick interstitium also implies that fetal and preterm lungs have decreased lung-interdependent forces. Forces in one part of the lung will not be transmitted to neighboring parts as they would be in juxtaposed alveoli connected by an elastic network.¹⁹ This deficiency is compounded by lack of collateral flow through the pores of Kohn, bronchiole-alveolar channels of Lambert, and interbronchiolar channels of Martin that help prevent atelectasis in the mature lung, but are absent in the immature lung.^20,21 In the fluid-filled fetal lung, with low interfacial saccular surface tension and controlled airway fluid pressure, decreased lung-interdependent forces are not a problem,²² but in the immature, air-ventilated lung with altered connective tissue and surface tension forces, mechanical ventilation poses major obstacles to homogeneous filling and emptying of acini. Events that alter elastic recoil pressure or distort the elastic template can therefore be expected to disturb lung development. The more immature the lung and the earlier the insult, the greater the perturbation.

The integrated area under the curve of MAP × FIO₂, over time, gives a general quantitation of stress to the immature lung during mechanical ventilation. However, a high SCORE can be achieved with low MAP and FIO_2, if the course is prolonged, or with high pressures and oxygen over a short period. The data show that high SCORE, achieved with either scenario, is associated with increased V_Vel and absolute quantity of lung elastic tissue, increased TLV, and decreased ISA. Two infants had high SCORES, with low FIO₂ (<0.3) and MAP (<8) for 40 to 50 days. Their lung geometry and septations were relatively preserved, and their lung volume and ISA were increased. These findings resembled those in weanling ferrets, where room air continuous positive airway pressure for 2 weeks increased total lung air capacity by 40%.²³ The elastic recoil pressure remained normal in these animals, whereas the preterm infants significantly increased their elastic tissue volume density. This may have been related to the mechanical tidal ventilation and oxygen administered to the infants, but not to the ferrets. In the majority of the CLD infants with SCORES 70 to 200, ISAs decreased because of decreased septation and increased saccule diameter.^5,24,25 High scores were associated with severe distortion of the distal acinar structures, that is, the alveolar ducts and saccules and interstitial septa increased in diameter. These changes were associated with elastic tissue increases, mainly at the intersections of gas exchanging units and alveolar ducts. In large diameter saccules, in infants with CLD, there were evenly placed elastic tissue deposits around the wall, presumably where septation should have occurred. Although septa, especially the portions connected to alveolar ducts, are normally the sites of the largest amount of elastic tissue, immature lungs with high scores without septation still had increased elastic tissue. Therefore, although failure to septate occurred in lungs with high scores, elastic tissue was still increased over controls, indicating excessive increases of elastic tissue in aberrant sites. These data suggest that the saccular-alveolar duct junction receives the highest stress, the site of large accumulations of elastic tissue in mechanically-ventilated infants.

In adult animals at low lung volumes, the lung surface area is highly dependent on surface tension forces and less dependent on tissue elastic forces.^26,27 The delicate alveolar septa and their capillaries, at normal lung volume, are protected by the surface tension forces on both sides of septa with little stress to the septal tissue. At lung volumes >80% of total lung capacity, the stress is transmitted to the elastic fiber system, which is mainly at the mouth of air sacs and at the alveolar duct level.²⁷ However, this also increases the stress on the sparse elastic and collagen fibers of alveolar septa.²⁶ Larger animals have larger alveolar diameters than smaller animals, which decreases surface tension forces, according to Laplace's Law.²⁸ Large animals compensate for this decrease in surface tension recoil pressure by thickening the interseptal interstitium, and by increasing the quantity of elastic and collagen tissue in the septa.²⁸

We propose the following hypothesis to account for these distortions in lung development in infants 30 weeks with CLD. The immature, surfactant-deficient lung at birth has large saccular surface tension recoil forces with collapsed saccules and alveolar ducts. If large repetitive inflation pressures are applied to these lungs, acinar airways proximal to the saccules and ducts distend and are damaged by the shear forces described by Nilsson et al.²⁹ However, in recent years, surfactant is administered to virtually all immature infants shortly after birth. Surfactant reduces the surface tension forces in the distal acinus and permits inflation of the ducts and saccules. The benefits of surfactant are obvious, permitting lower inflation pressures and lower oxygen requirements. However, the reduced saccular and alveolar duct surface tension recoil pressures after surfactant therapy expose the immature lung with inadequate elastic and connective tissues to the full brunt of the mechanical inflating pressures. If the inflating pressures or volumes are not reduced in conjunction with surfactant treatment, the saccules and ducts will be at risk for volutrauma.³⁰ Over time, the peripheral acinar airspace diameters are increased by overstretching, possibly exceeding tensile limits of collagen tissue and the elastic limit of elastic tissue, which further decreases the surface tension forces, according to Laplace.²⁸ If saccules were spherical, a doubling of the diameter, as occurred in infants with the highest SCORE, would markedly increase the volume eightfold. The interstitium remains thick, and in most infants, thickens further to give support to the large air spaces. The elastic template is deformed by the volutrauma. Septation, which is anatomically related to elastic tissue, is compromised by the connective tissue disruptions. Capillary development occurs in tandem with septation, and therefore, as expected, is seriously impaired by these tissue deformations.^8,31 A major, chronic stress site in the system seems to be at the saccular-duct junction where elastic tissue is overproduced. The thickened interstitium and elastic tissue increases can be considered as lung defensive responses to mechanical ventilation, which in most instances succeed because the majority of these infants survive. However, without positive end-expiratory pressure, these lungs tend to collapse at end expiration and have a small FRC. Any form of mechanical ventilation that exposes the distal acinar units to overstretching would be expected to produce these changes.

CLD has been repeatedly shown to be associated with proinflammatory proteins and influx of neutrophils into the tracheal fluids.^32-34 Furthermore, these proinflammatory proteins are decreased by steroid treatment, which simultaneously improves lung function. These associations suggest that inflammation is a primal causal factor in the cause of CLD. Lung inflammation, infection, and hyperoxia clearly are important factors in the degree and chronicity of CLD.³⁵ However, mechanical ventilation and volutrauma also have been shown to be associated with increased permeability and capillary damage with an influx of neutrophils and macrophages into the airways. These cells, when activated, have led to increases of tumor necrosis factor-, an early proinflammatory cytokine, in tracheal fluids.³⁶ Other animal studies have shown that, at high states of lung inflation, over just a few hours, there is increased lung gene expression for procollagen, fibronectin, basic fibroblast growth factors, and transforming growth factor-₁.³⁷ These findings support the view that volutrauma, lung deformation, and tissue disruption in the structurally immature lung, along with persistent mechanical ventilation, are early events that evoke inflammation and increase the need for hyperoxia, thus setting up a vicious cycle of recurrent injury and repair. This hypothesis can be summarized by the terms lung immaturity, inflammation, volutrauma, and elastic tissue alterations. The multifactorial cause of CLD is obvious, and the components of the lung immaturity, inflammation, volutrauma, and elastic tissue alterations hypothesis are only part of the cause. However, these anatomic alterations are fundamental in determining future lung development and function.

The results of this study suggest that maintaining a low to normal FRC and reducing large tidal volume swings should help mitigate these structural changes. Theoretically, liquid fluorocarbon ventilation would be useful, because there is a modest interfacial tension between the fluorocarbon and lung saccule liquid.³⁸ This would support the fragile connective tissue by increasing the tissue recoil.

Study Limitations

Because the lungs were selectively and not uniformly sampled, a biased sampling error of the estimated tissue volumes was introduced. Because it is doubtful that the peripheral structures of the growing or chronically damaged lung are isotropically oriented, surface area estimations are also biased.

Steroids, hyperoxia, and mechanical ventilation all retard alveolar septation and reduce acinar complexity in the growing lung.^5,12 Hyperoxia, in contrast to mechanical ventilation, does not seem to increase peripheral elastic tissue.^6,7,39 Indeed, animal studies suggest that hyperoxia may alter or inhibit it.⁴⁰ Steroids administered to rats during lung development have shown increased peripheral air spaces and static compliance, suggesting a decrease or alteration of lung elastic tissue.¹² Nonetheless, this study is not able to dissect out the separate effects of steroids, hyperoxia, or mechanical ventilation.

	ACKNOWLEDGMENTS

This work was supported, in part, by the Katharine B. Richardson Foundation (D.W.T.); by a Physician Scientist Award from Children's Mercy Hospital (W.E.T.); by National Institutes of Health Grants R-01 HL 58125 (W.E.T.) and K-23HL04264-01 (I.I.E.).

	FOOTNOTES

Received for publication Dec 8, 1999; accepted Apr 14, 2000.

This work was presented at the Annual Meeting of the Society of Pediatric Research, Pediatric Academic Society; May 1-4, 1999; San Francisco, CA.

Reprint requests to (D.W.T.) Children's Mercy Hospital, 2401 Gillham Rd, Kansas City, MO 64108. E-mail: dthibeault{at}cmh.edu

	ABBREVIATIONS

CLD, chronic lung disease; FRC, functional residual capacity; GA, gestational age; MAP, mean airway pressure; TLV, total lung volume; PCA, postconceptional age; SCORE, respiratory score; FIO₂, fraction of inspired oxygen; V_Vel, volume density of parenchymal elastic tissue; ISA, internal surface area; SD, standard deviation; CI, 95% confidence interval.

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