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Perinatal aspiration of meconium often causes severe respiratory failure with high morbidity and mortality in mostly term infants. Pathophysiologically, meconium aspiration is characterized by initial obstruction of the airways resulting in ventilation-perfusion mismatching in the lungs, hypoxemia, and increase in pulmonary vascular resistance. Within a few hours of exposure to meconium, a severe inflammatory response in the lungs is initiated (1, 2), supposedly by meconium-induced activation of the pulmonary macrophages (3). This reaction is associated with increased pulmonary vascular permeability leading to proteinous exudation into the alveolar spaces, inactivation of the pulmonary surfactant, and decrease in lung compliance (1, 4). Direct toxic effects of meconium on the alveolocapillary membrane may further play an important contributing role to the pulmonary tissue damage (4). Despite the well described histologic alterations of the pulmonary tissue in experimental meconium aspiration and infants dying from this disease, the mechanisms of acute cellular injury in the meconium-exposed lungs are still unclear.

Phospholipase A2 (PLA2) is a ubiquitous enzyme that generates, by hydrolyzing phospholipids, biologically active FFA and lysophospholipids. In the lungs, activation of PLA2 by direct or indirect insults leads to formation of proinflammatory lipid-derived mediators (5, 6), and because PLA2 may also directly damage alveolar cells and inactivate surfactant (7, 8), its activity is proposed to be important in the pathogenesis of acute inflammatory lung injury (5–12). Pulmonary and circulating PLA2 activity is in fact increased in adults with acute respiratory distress syndrome and correlates positively with the severity of the lung disease (11, 12). In the meconium, the activity of this enzyme is supposed to be high (13), but PLA2 activity in the lungs of neonates with aspiration of meconium is unknown and has not, to our knowledge, been previously reported.

We hypothesized that induction of PLA2 activity could contribute to the pulmonary injury and inflammatory response to aspiration of meconium and therefore measured lung tissue and meconium PLA2 activity and searched for ultrastructural evidence of lung damage in neonatal piglets after intratracheal instillation of human meconium. Further, because activation of PLA2 has been implicated to mediate cellular apoptosis, programmed cell death of noninflammatory genesis (14), we also looked for signs of apoptotic cellular alterations in meconium-instilled piglet lungs. Newborn pig was chosen as the experimental animal because of its similar lung morphology and enzyme activity to that in human (15).

METHODS

Animals.

Twenty 10–12-d-old pigs (mean weight 4.6 kg, range 3.5–5.2 kg) were anesthetized and catheterized, as described earlier (16). Briefly, animals were premedicated with ketamine hydrochloride (40 mg/kg) and diazepam (2 mg/kg, intramuscularly) and placed on supine position. They were then intubated and connected to a volume-controlled ventilator. Mechanical ventilation was started with room air, ventilatory rate being 28 breaths/min, end expiratory pressure 2–3 cm H2O, and tidal volume 16–20 mL/kg. Anesthesia was maintained by continuous i.v. infusion of ketamine hydrochloride (10 mg/kg/h). Paralysis was induced with pancuronium bromide (0.1 mg/kg) and maintained with continuous infusion (0.25 mg/kg/h). A 5F Swan-Ganz thermodilution catheter was inserted via the right external jugular vein and placed under pressure monitoring into the pulmonary artery for measurement of pulmonary vascular pressures and cardiac output. Further, to monitor systemic hemodynamic values and to collect blood samples, a small polyethylene catheter was placed in the abdominal aorta via the right femoral artery. The experiments were approved by the Committee of Animal Care in Research of the University of Turku. Animals were cared for in accordance with procedures outlined in the “Guide for the Care and Use of Laboratory Animals” (National Institutes of Health Publication No. 85–23).

Experimental protocol.

After 1 h of stabilization and subsequent baseline measurements, 12 piglets received via the endotracheal tube a bolus (3 mL/kg) of a 20-mg/mL (thin, n= 6) or 65-mg/mL (thick, n= 6) meconium mixture. Control piglets (n= 5) were given the same amount (3 mL/kg) of sterile saline intratracheally, and an additional three piglets were studied without instillation of any material via the airways. The meconium was obtained from the first stools of several healthy term human infants. It was then pooled, lyophilized, and irradiated for sterility before the experiment was diluted with sterile saline. We have shown previously that intratracheal insufflation of this amount of meconium induces a progressive and concentration-dependent pulmonary hypertension in neonatal piglets (16). The hemodynamic changes were registered, and blood gas samples were taken at baseline and serially for 6 h after meconium or saline administration. To avoid hypoxemia, the piglets received supplemental oxygen to maintain partial pressure of arterial oxygen above 8 kilopascals. The partial pressure of carbon dioxide in arterial gas was maintained below 5 kilopascals by adjusting the frequency of the ventilator. At the end of the experiment, the animals were killed with an overdose of potassium chloride, and pulmonary tissue samples were obtained.

Histologic examinations.

For histologic analysis of the lungs, 2 × 2-cm pieces of pulmonary tissue from the right lower lobe were fixed in buffered formalin. The tissue samples were dehydrated, cleared, and embedded to paraffin according to a routine process. Five-micromolar sections were stained with hematoxylin and eosin for light microscopic analysis. To determine the extension and severity of the lung tissue injury, the histologic samples were assessed by a pathologist blinded to the grouping of the piglets. A score from 0 to 4 was assigned for three different characteristics:1) extension of leukocyte infiltration, 2) amount of intra-alveolar leukocytes, and 3) amount of exudative debris, including fibrin, edema fluid, and meconium. The calculated total injury score means the sum of these scores.

Electron microscopy.

For ultrastructural analysis of the lungs, small pieces (1 mm3) of pulmonary tissue were taken from the lateral diaphragmatic edge of the right lower lobe. The samples were fixed in 3.0% phosphate-buffered glutaraldehyde, postfixed in 1% osmium tetroxide, and embedded in Epon 812. Sections 1 μm in thickness were cut and stained with toludine blue. The slides were then examined under the light microscope, and selected areas were processed for ultrathin sections, which were contrasted by uranyl acetate and lead citrate. The grids were studied using a Jeol JEM 1200 EX microscope.

Phospholipase A2 activity and concentrations.

The catalytic activity of phospholipase A2 in lung tissue homogenates was measured, as earlier described (17). Briefly, lung tissues were homogenized in 50 mM sodium acetate buffer, pH 5.0, with 1 M NaCl and 1 tablet of protease inhibitor cocktail (Complete by Boehringer, Mannheim, Germany), 1 g of lung tissue per 4 mL of buffer. Homogenized samples were centrifuged for 20 min with 1000 × g, and the supernatant was separated and used for PLA2 catalytic activity and concentration assays. In the activity measurement, the substrate was prepared by mixing unlabeled 1,2-dipalmitoylphosphatidylcholine (Sigma) with 1-palmitoyl-2-[14C]-arachidonoylphosphatidylethanolamine (DuPont) in a ratio of 6 mM/1.325 μM (250 nCi), dissolved in a mixture of chloroform and methanol (2:1), dried under a flow of nitrogen, and redissolved in 10 mL of 0.1 M glycine buffer (pH 8.1). 10-μl samples of lung tissue supernatant were incubated with 100 μl of substrate buffer for 3 h at 40°C. The reaction was stopped by adding 100 μl of Dole's reagent. Released [14C]arachidonic acid was segmented by SiO2/water/heptane phase extraction, as reported previously (18), and detected by a liquid scintillation spectrometer (Wallac, Turku, Finland) (17).

The concentrations of human PLA2 group I (PLA2-I, pancreatic type) and group II (PLA2-II, synovial type) were measured in the supernatant of the homogenized lung tissue by time-resolved fluoroimmunoassays, as described earlier (19, 20). PLA2-I immunoassay uses monoclonal antibody-coated microtitre plates and europium chelate-labeled polyclonal (rabbit) antibody as tracer. Both antibodies were raised against purified human pancreatic PLA2. PLA2-II immunoassay uses a polyclonal antibody, raised in rabbits against human recombinant group II enzyme, both immobilized on the microtitre plate and also detecting tracer antibody. Fluorescence was measured with an Arcus fluorometer (Wallac, Turku, Finland).

In situ detection of apoptotic cells.

In situ detection of apoptotic cells in paraffin sections of the lung tissue (right lower lobe) was performed as reported earlier (17), with slight modifications. Briefly, deparaffinized tissue sections were treated with 10 μg/mL proteinase K (Boehringer) at 37°C in 2 mM CaCl2 and 20 mM Tris-HCl, pH 7.4, for 30 min. DNA 3′-end labeling was performed after 10 min of incubation with terminal transferase buffer (Promega). The labeling mixture contained fresh terminal transferase buffer, 5 μM nonradioactive digoxigenin-dideoxy-UTP (dig-ddUTP, Boehringer), 45 μM ddATP (Pharmacia, Uppsala, Sweden), and 0.34 U/μl terminal transferase (Promega). The reaction was allowed to continue for 1 h at 37°C in a humidified chamber. After washing, the slides were incubated with blocking buffer containing 2% (wt/vol) blocking reagent and 0.05% (wt/vol) sodium azide (Boehringer) for 30 min. Antidigoxigenin antibody, conjugated to alkaline phosphatase (1:4000, Boehringer), in 2% (wt/vol) blocking buffer was added and incubated for 2 h in a humidified chamber. The slides were treated with alkaline phosphatase buffer (0.1 M NaCl, 0.05 M MgCl, and 0.1 M Tris-HCl, pH 9.5) for 10 min. Thereafter, 337 μg/mL nitroblue tetrazolium salt (Boehringer) and 175 μg/mL 5-bromo-4-chloro-3-inodyl-phosphate (Boehringer) were added in fresh alkaline phosphatase buffer, and the reaction was terminated 16 h later by 1 mM EDTA and 10 mM Tris-HCl, pH 8.0. Finally, the slides were mounted with Gurr Aquamount (BDH Chemicals, Poole, England). For controls, terminal transferase, dig-ddUTP, or antidigoxigenin antibody was omitted from the reaction. Piglet lymphocytes undergoing apoptosis in the lymph nodes adjacent to lung tissue served as a positive control.

Apoptotic cells were counted in lung sections stained with the antidigoxigenin antibody. A distinct intensively dark color reaction within lung tissue cells was regarded to represent apoptotic DNA fragmentation. The results are expressed as the number of positive cells per mm2 of tissue section area in 20 fields of view of a ×10 objective lens at each time point. The method is well established in detection of apoptotic cellular changes and has been validated by simultaneous electrophoretic DNA analysis in pancreatic tissue (17).

Myeloperoxidase activity.

As a measure of pulmonary neutrophil influx, tissue specimens of the right lung lower lobe were initially frozen (−20°C) and later measured for myeloperoxidase (MPO) activity. After homogenization of the tissue, MPO activity was assayed spectrophotometrically using a method in which the enzyme catalyzes the oxidation of 3,3′,5,5′-tetramethylbenzidine by H2O2 to yield a blue chromogen that possesses a wavelength maximum at 655 nm (21).

Meconium localization.

For immunohistochemical localization of components of meconium within the lungs, the avidin-biotin peroxidase complex method (22) was applicated by using the Vectastain ABC peroxidase kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. The lung sections were incubated either with a polyclonal placental alkaline phosphatase (Dako, Glostrup, Denmark), dilution 1:20 with prokinase K pretreatment, or carcinoembryonal antigen (Dako, Glostrup, Denmark), dilution 1:10 000 with microwave and proteinase K pretreatment. Diaminobenzidine was used as chromogen. The sections were counterstained with Mayer's hematoxylin.

Data analysis.

Analysis of variance was used to compare the groups. If the overall analysis of variance was significant, post hoc comparisons between the groups were made using the Student-Newman-Keuls test. A level of p< 0.05 was considered statistically significant. The results are expressed as mean (SEM).

RESULTS

Physiologic changes.

As previously reported (16), pulmonary artery pressure and vascular resistance increased progressively after instillation of thin and thick meconium. Also, oxygenation of these piglets deteriorated acutely and remained low throughout the study.

Histologic findings.

The control lungs (both saline-instilled and uninsufflated) were normal and essentially well aerated (Fig. 1). In lungs instilled with thin or thick meconium, patchy areas of severe acute inflammation were found (Fig. 2). The alveoli were partly atelectatic, usually without plugging of the larger airways, and contained varying amounts of leukocytes (Fig. 3), fibrin, and also occasional epithelial cells of the instilled meconium, detected by immunohistochemistry (Fig. 4). Hyaline membranes were detected in some of the samples. Bronchi contained leukocytes, cell debris, and epithelial cells of meconium (Fig. 5). No pulmonary hemorrhages or necrosis of pulmonary parenchyma were observed. The total injury score was similar in lungs insufflated with thin or thick meconium (Table 1).

Figure 1
figure 1

Normal control lung with well-aerated alveoli. No changes are seen in a bronchus (b) or in a blood vessel (v) (H&E, ×70).

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Figure 2
figure 2

Pneumonitis of varying intensity after meconium instillation (H&E, ×140).

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Figure 3
figure 3

Alveolar spaces contain neutrophils and fibrin (H&E, ×280).

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Figure 4
figure 4

Alveoli after meconium instillation. Placental alkaline phosphatase immunohistochemistry is positive in alveoli containing meconium-derived material (arrow) (ABC technique, ×280).

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Figure 5
figure 5

Bronchus (b) after meconium instillation. Neutrophils and cell debris inside the bronchus. Respiratory epithelium is intact (H&E, ×140).

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Table 1 Lung tissue wet/dry ratio, injury score, and myeloperoxidase (MPO) activity (mU/mg protein) 6 h after saline (n = 5) or meconium (20 or 65 mg/mL, n= 6 each) instillation in neonatal piglets* * Three piglets were ventilated for 6 h and received no material intratracheally (controls). Mean, SEM.
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Electron microscopic findings.

In the control piglets, the structure of the alveolar epithelium, basement membrane, and vascular endothelium was intact (Fig. 6), whereas instillation of thick meconium resulted in reversible changes in the pulmonary tissue. Cellular debris and meconium were found in the alveolar spaces; macrophages, lymphocytes, and neutrophils were present in both alveolar spaces and in the pulmonary vessels. In many of these pulmonary tissue samples, alveolar epithelium was edematous with intact type II cells (Fig. 7). Some autophagocytotic bodies were found in the pulmonary endothelium, indicating sublethal cell injury (Fig. 8). Neutrophils were detected in the pulmonary vessel lumen, sometimes being in intimate contact with the pulmonary endothelium. Also, thrombocytes were seen in the pulmonary vessels, but no thrombus formation was found in the capillaries. These ultrastructural changes were essentially similar, although less severe, after thin meconium administration.

Figure 6
figure 6

Normal alveolar capillaries (c) containing red blood cells. Type I pneumocyte cytoplasm is normal (arrow) (×4000).

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Figure 7
figure 7

The cytoplasm of type I pneumocyte (arrow) is swollen, indicating edema. L, lymphocyte (×7000).

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Figure 8
figure 8

Autophagocytosis in endothelium (arrow). Normal lymphocyte in alveolar capillary (L). Cell debris in alveolar space (D) (×7000).

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PLA2 activity.

The catalytic PLA2 activity in lung tissue was significantly elevated after administration of thick meconium when compared with the other groups (Fig. 9). To evaluate the origin of this PLA2 activity, we measured the catalytic activity of the enzyme in the pool of lyophilized meconium and found a very high value (140.4 U/g meconium). Subsequent analysis of group I and group II PLA2 concentrations indicated that the main enzyme activity in meconium and meconium-instilled porcine lung tissue was because of human pancreatic (group I) PLA2 (>90%), whereas only very low amounts of group II enzyme were found (data not shown). In control and saline-instilled lungs, both PLA2 groups were undetectable. No cross-reaction was further found between the human and porcine or rat group I PLA2.

Figure 9
figure 9

PLA2 activity (U/g tissue) in lung tissue of piglets without aspiration (control, n= 3) and 6 h after intratracheal instillation of saline (n= 5), or meconium of a concentration of 20 mg/mL (meconium 20, n= 6) or 65 mg/mL (meconium 65, n= 6). Mean, SEM. *, p< 0.05 vs control; §, p< 0.05 vs all of the other groups.

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Apoptosis.

Occasional apoptotic cells were seen in the control lungs (Fig. 10), whereas their number was significantly (p< 0.05) increased, mainly in the epithelium and in some places also in the endothelium, in the lungs instilled with thick meconium (Figs. 11 and 12).

Figure 10
figure 10

An occasional apoptotic cell (arrow) in an alveolar septum of control lung (TUNEL, ×140).

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Figure 11
figure 11

Several dark-staining apoptotic bodies with severe inflammatory reaction in the meconium-instilled lung (TUNEL, ×140).

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Figure 12
figure 12

The number of apoptotic cells (/mm2) in lung tissue of piglets without aspiration (control, n= 3) and 6 h after intratracheal instillation of saline (n= 5), or meconium of a concentration of 20 mg/mL (meconium 20, n= 6) or 65 mg/mL (meconium 65, n= 6). Mean, SEM. *, p< 0.05 vs saline.

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MPO activity.

The MPO activity in the saline-instilled lungs was on the control level. Administration of meconium (both thin and thick) tended to increase the MPO activity, but owing to high variation of the results, the rise did not reach statistical significance (Table 1).

DISCUSSION

The present data show that intrapulmonary meconium induces an early intense inflammatory reaction with simultaneous ultrastructural alterations in the alveolar epithelium and vascular endothelium in neonatal lungs. The mechanisms that initiate the pulmonary inflammation after aspiration of meconium are complex and still poorly identified. Although meconium itself does not possess any significant neutrophil chemotaxis (4), it may activate lung inflammatory cells to produce cytokines (3, 23), known to promote neutrophil accumulation and local inflammatory reaction (24). On the other hand, several lines of evidence implicate that activation of pulmonary PLA2, generating proinflammatory eicosanoids and lysophospholipids, may be important in the pathogenesis of various inflammatory diseases of the adult lungs (11, 12, 25). Our piglet data accordingly show that in meconium-exposed neonatal lungs, the catalytic activity of PLA2 is elevated, which, however, is almost exclusively due to human pancreatic (group I) enzyme activity. Thus, the present results suggest that intrapulmonary aspirated meconium challenge the lungs with high pancreatic PLA2 activity, demonstrated to be elevated also in a previous study (13), and may thereby contribute to the pulmonary inflammatory response. We cannot, however, exclude some contribution of endogenous PLA2 activation in cytokine-stimulated alveolar macrophages of meconium-exposed piglet lungs (10, 23, 25, 26). Furthermore, the severity of the pulmonary inflammatory reaction, in contrast to the catalytic PLA2 activity, was similar after both concentrations of human meconium, suggesting that additional mechanisms, like induction of leukocyte adhesion molecule expression and promotion of neutrophil adhesion to pulmonary vascular endothelial cells (3), may also have a significant role in the meconium aspiration-induced pulmonary inflammation.

The pathogenesis of the meconium instillation-induced early epithelial and endothelial ultrastructural injury in the piglet lungs remains unclear, but it may be related to the intense inflammatory reaction in the lung tissue and/or direct toxic injury to the airway epithelium, presumably through the action of bile salts in meconium (1, 27). These early structural alterations are considered to be still reversible but may eventually result in irreversible necrosis, observed in the alveolar epithelium not until after the first 24 h of meconium aspiration (1). Similar to our findings, injury to the type I alveolar epithelial cells with much less severe change in other lung cells is a common feature in acute damage of adult lungs from a variety of inhaled and blood-borne toxins (7, 9). Alveolar type II epithelial cells, although damaged by high concentrations of meconium (>1%) in vitro (26), were indeed intact in the lungs of our piglets. Still, meconium is documented to alter type II cell function and cause surfactant inactivation, not assessed in the present study, in a dose-dependent manner both in vitro (28, 29) and in vivo (29). Although the mechanisms of this selective type I pneumocyte injury are still poorly known, clinical and experimental data suggest that excess PLA2 activity, possibly through lysophosphatidyl formation, may contribute to the pulmonary cellular destruction (5–11). The fact that this damage is observed after intratracheal, in contrast to i.v., exposure of isolated adult lungs to PLA2 (7, 9) further suggests that the meconium-introduced PLA2 activity could play a role in the epithelial injury in neonatal lungs. The pathogenetic significance of this enzyme in meconium aspiration-induced pulmonary injury, however, needs to be established in further studies.

Together with the pulmonary inflammatory reaction, development of pulmonary hypertension often complicates the symptoms of infants with meconium aspiration syndrome. This hypertensive reaction, thought to result from meconium-induced release of vasoactive mediators and altered pulmonary vasoreactivity (2, 30), is associated with microvascular endothelial alteration in the meconium-exposed lungs already during the first hours after the insult, also evident from our data (1, 16). Similar early pulmonary hypertension and ultrastructural evidence of vascular endothelial damage, connected to increased PLA2 activity in the lung tissue, have been found in endotoxin-induced lung injury (6). Further corroborating our previous (16) and present findings, intratracheally administered PLA2 may, through generation of vasoactive metabolites of arachidonic acid, produce pulmonary vasoconstriction (10, 31) and is also able to induce pronounced morphologic changes in the pulmonary endothelium, associated with sequestration of neutrophils and platelets within alveolar capillaries (9). Recent data from our laboratory have additionally shown that administration of glucocorticoids, known to induce the biosynthesis of inhibitors of PLA2 (10), is able to diminish the pulmonary hypertensive response and edema formation in porcine lungs following intrapulmonary instillation of human meconium (32). Thus, although the histology of the lung tissue is often discordant with the degree of pulmonary hypertension in meconium-exposed lungs (30), our results suggest that PLA2 activation may contribute to the development of the functional and structural components of the hypertensive injury in meconium-insufflated piglet lungs (16).

Apoptosis is a programmed cell death that, in contrast to necrosis, does not result in inflammatory tissue injury but rather is regarded as a cell clearance mechanism promoting resolution of inflammation (33). Nevertheless, there is indirect evidence to suggest that apoptosis may also play a role in acute lung injury. Oxidative stress during therapeutic use of high inspiratory concentrations of oxygen may in fact be associated with apoptotic cell death in the lungs (33, 34). Although apoptosis is unlikely a direct effect of oxygen and rather the result of increased release of apoptosis-mediating oxidants by inflammatory cells (34), we cannot exclude the possible effect of administered oxygen on the apoptotic cell deaths in the meconium lungs, especially in the epithelium, the main site of apoptosis in our study. Human meconium, despite having potent antioxidative capacity in vitro (35), is further able to activate alveolar macrophages to oxygen radical production (3). On the other hand, meconium aspiration-induced intrapulmonary production of cytokines, including tumor necrosis factor α (23), could be associated with pulmonary cellular apoptosis, as is demonstrated to occur in endotoxin-induced lung injury (36). This tumor necrosis factor α cytotoxicity may, possibly through increased intracellular levels of Ca2+, stimulate PLA2 activity (26) and thereby trigger apoptosis (14). Alike, bile salts extracted from meconium may directly induce Ca2+ accumulation in alveolar epithelial cells (27) and thus contribute to the apoptotic death of these cells.

In conclusion, intrapulmonary meconium causes within a few hours a severe lung inflammation, which is associated with ultrastructural changes in the pulmonary epithelium and endothelium, combined with apoptotic cell death in the respiratory epithelium. Because lung tissue PLA2 activity is simultaneously and concentration-dependently elevated, primarily due to a meconium-introduced high concentration of human pancreatic-type PLA2, this enzyme may have an important role in the pathogenesis of the observed functional and structural changes in neonatal lungs. Further, the role of the pulmonary apoptosis in neonatal lungs after meconium aspiration still needs to be established.