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To investigate the role of pancreatic (group I) secretory PLA2 (sPLA2-I) in the pathogenesis of meconium aspiration syndrome, human particulate meconium or its supernatant either before or after extraction of PLA2-I was insufflated into rat lungs. In addition, the pulmonary effects of intra-tracheal human and bovine PLA2-I were studied. Lungs with saline instillation served as controls. Intrapulmonary particulate meconium (both before and after PLA2-I extraction), unlike meconium supernatant, resulted in markedly elevated lung tissue PLA2 catalytic activity and human PLA2-I concentrations when compared with controls. On the other hand, tissue concentrations of the group II PLA2 remained unchanged in all meconium lungs. Pulmonary PLA2-I concentrations further correlated positively with lung injury scores. Instillation of meconium-derived human PLA2-I, at a concentration of one-third of that in particulate meconium, did not raise PLA2 activity or concentrations of PLA2-I or PLA2-II in the lung tissue from the control level, but still resulted in significantly elevated lung wet/dry ratio and injury score. In contrast, insufflation of bovine pancreatic PLA2 increased the lung tissue enzyme activity and wet/dry ratio from the control level, but had no effect on the type II PLA2 concentration or lung injury score. Our data thus indicate that human pancreatic PLA2, introduced in high amounts within aspirated meconium especially in particulate form, is a potent inducer of lung tissue inflammatory injury.

Pulmonary inflammation and parenchymal apoptosis are implicated in the pathogenesis of the acute lung injury, but the mechanisms of these reactions are still unclear. Because inhibition of the proinflammatory cyclo-oxygenase (COX)-2 enzyme action is proposed to be useful in various inflammatory lung injuries, we decided to investigate the expression of COX-2 and the possible beneficial effects of its inhibition on pulmonary inflammation and apoptosis in surfactant-depleted lungs. The injury was induced in 2-mo-old rats by repeated lung lavage to remove alveolar surfactant. Eight of these rats were pretreated with a specific COX-2 inhibitor, NS-398. All rats, including control rats without lung lavage, were ventilated with 60% oxygen for 5 h, and the lungs were then studied histologically for tissue injury and with DNA nick-end labeling, cleaved caspase-3 immunohistochemistry, and electron microscopy for apoptotic cell death. Lung tissue myeloperoxidase activity and the expression of COX-2 protein and concentration of prostaglandin E2 were additionally analyzed. Lung lavage increased pulmonary neutrophil migration, histologic injury, and the occurrence of epithelial apoptosis. In contrast, expression of COX-2 and amount of PGE2 were significantly lower in surfactant-depleted lungs than controls. Pretreatment with the COX-2 inhibitor further increased the migration of neutrophils and occurrence of epithelial apoptosis in the surfactant-depleted lungs, compared with nontreated insulted lungs. These results suggest that specific inhibitors of COX-2 should be used cautiously in association with surfactant-deficient lung injuries.

Lung tissue inflammation and apoptosis are implicated in the pathogenesis of meconium aspiration-induced lung injury in the newborn, but the mechanisms of these reactions are still poorly known. We investigated the time-dependent leukocyte influx and appearance of apoptosis, as well as the contribution of angiotensin (ANG) II receptor action on these processes in the meconium-induced lung injury. Experimental meconium aspiration was induced by intratracheal instillation of human meconium in 18 rats, and eight rats were further pretreated with an unspecific ANG II receptor inhibitor saralasin. Rats were ventilated with 60% oxygen for 1, 3, or 5 h, and the lungs were then studied histologically for tissue injury and with DNA nick-end labeling and electron microscopy for apoptotic cell death. Lung tissue myeloperoxidase activity and expression of angiotensinogen mRNA and endothelial monocyte-activating polypeptide (EMAP) II protein were also analyzed. The meconium-instilled lungs showed increasing neutrophil migration and histologic injury after the first hour, whereas the number of epithelial apoptotic cells was elevated from the control level throughout the study. Myeloperoxidase activity was high, and the angiotensinogen mRNA and EMAP II protein was up-regulated at 5 h after the meconium insult. Pretreatment with saralasin significantly prevented the increase in lung tissue myeloperoxidase activity, EMAP II, and lung epithelial apoptosis. The results suggest that pulmonary meconium insult rapidly results in epithelial apoptosis, before significant neutrophil sequestration into the lungs. Apoptotic cell death is further connected with ANG II receptor action in the meconium-contaminated lung tissue.

Neonatal meconium aspiration often produces severe respiratory distress due to an inflammatory pulmonary injury, but the extension of this damaging reaction to the noncontaminated lung regions is still uncertain. To investigate the presence of generalized pulmonary inflammatory response, 31 anesthetized and ventilated neonatal piglets (1-3 d) were studied. Meconium (n = 16) or saline (n = 15) was instilled unilaterally into the right lung, and analysis of the lung tissue or bronchoalveolar lavage (BAL) fluid from both lungs was performed after 12 h. Meconium increased the wet/dry weight ratio, histologic tissue injury score and tissue myeloperoxidase activity as well as BAL fluid total cell count in the contaminated lung. Tumor necrosis factor-alfa concentrations in BAL fluid did not however differ significantly. Furthermore, in the meconium-instilled lungs the tissue and lavage fluid catalytic activity of phospholipase A2 (PLA2) and tissue PLA2 group-I and group-II concentrations were significantly elevated. Although BAL fluid catalytic activity of PLA2 was moderately increased also in the meconium noninstilled lung, significant inflammatory injury in this lung was absent. The results thus indicate that meconium aspiration induces severe local inflammation and lung injury, but significant generalized pulmonary inflammatory damage in the pathogenesis of meconium aspiration syndrome is unlikely.

To investigate the pulmonary effects of steroid treatment in neonates with meconium aspiration, 25 10- to 12-d-old piglets were studied for 6 h after an intratracheal bolus of human meconium. Dexamethasone (0.5 mg/kg) was given in two treatment schedules, either 1 h before (n = 6) or 1 h after meconium instillation (n = 8). Eight piglets served as controls. Three additional piglets were given dexamethasone without meconium instillation. Pulmonary hemodynamics and oxygenation were followed, and lung tissue samples investigated for signs of inflammation and ultrastructural injury, including apoptosis. Pulmonary artery pressure and vascular resistance increased after meconium instillation, but this rise was significantly prevented after prophylactic dexamethasone. This treatment also improved the acutely deteriorated oxygenation of the piglets after meconium insufflation. Prophylactic, but not early, dexamethasone treatment further protected the lungs from the ultrastructural changes caused by meconium instillation. Additionally, the increase of apoptotic epithelial cell deaths was significantly prevented by both dexamethasone treatments. These results show that prophylactic dexamethasone treatment significantly attenuates the early pulmonary hemodynamic deterioration and structural lung damage caused by meconium aspiration. Further studies on the apoptosis-inhibiting effect of dexamethasone administration in neonatal lungs exposed to heavy meconium are warranted.

Aspiration of meconium produces an inflammatory reaction resulting in necrotic changes in lung tissue. To further investigate the mechanisms of the meconium-induced early pulmonary injury, twenty 10-12-d-old piglets were studied for lung tissue ultrastructural and apoptotic changes and phospholipase A2 activity. Twelve piglets received an intratracheal bolus (3 mL/kg) of a 20-mg/mL (thin, n = 6) or 65-mg/mL (thick, n = 6) mixture of human meconium, and control piglets (n = 5) received the same amount of intratracheal saline. Three ventilated piglets with no aspiration were also studied. Pulmonary hemodynamics and systemic oxygenation were followed for 6 h after meconium or saline insufflation. In the control groups, the pulmonary tissue showed open alveolar spaces and intact vascular walls, whereas meconium administration resulted in severe pneumonitis, with alveolar spaces filled with inflammatory exudate. Meconium instillation additionally resulted in edematous changes in the vascular walls and alveolar epithelium, whereas type II pneumocytes were intact. The amount of apoptotic cells was increased, especially in the respiratory epithelium, and the catalytic activity of phospholipase A2 in lung tissue samples was significantly elevated after thick meconium instillation. This activity rise proved to be mainly because of human group I phospholipase A2, introduced by meconium. Our data thus show that aspiration of meconium leads to severe lung tissue inflammation with early ultrastructural changes in the pulmonary alveolar walls and is associated with apoptotic cell death in the epithelium, already during the first hours after the insult. These results further suggest that high phospholipase A2 activity, mainly introduced into the lungs within the meconium, may have an important role in the initiation of these alterations in neonatal lungs.

Severe neonatal aspiration of meconium is frequently complicated by fatal pulmonary hypertension. The protective effect of an i.v. bolus of methylprednisolone on meconium aspiration-induced hypertensive lung injury was studied in anesthetized pigs with adapted lung circulation. Eleven 10-wk-old pigs received 3 mL/kg 20% human meconium via the endotracheal tube. Five of them were pretreated with 30 mg/kg methylprednisolone 30 min before aspiration. Ventilator settings were adjusted to keep arterial PO2 above 8 kPa and arterial PCO2 below 5 kPa. Meconium insufflation induced a biphasic pulmonary pressor response during the 6 h follow-up. Methylprednisolone tended to prevent the early (0-1 h) increase in pulmonary artery pressure and inhibited significantly the second phase (1-6 h) progressive rise in pulmonary artery pressure and pulmonary vascular resistance. This inhibition of resistance increase was most profound in the postarterial segment of the lung circulation, as determined by pulmonary artery occlusion. Additionally, the methylprednisolone pretreated group demonstrated a significant decrease in venous admixture together with improved oxygenation during the late phase after the insult, and further showed evidence of diminished lung edema formation. Although meconium aspiration-induced fall in blood leukocyte concentration was inhibited by methylprednisolone pretreatment, no histologic difference was found in pulmonary leukocyte sequestration. Our results thus show that in adapted porcine lungs methylprednisolone pretreatment improves oxygenation and attenuates the meconium aspiration-induced pulmonary hypertensive response by preventing the increase in the postarterial resistance.