Main

Meconium aspiration syndrome is a severe neonatal respiratory disorder frequently associated with fatal pulmonary hypertension(1). The pathophysiology of this vascular complication is still unclear, although failure in perinatal pulmonary adaptation supposedly contributes to the increased lung vasoreactivity(2). Other pathogenetic mechanisms of meconium aspiration-associated pulmonary hypertension may be closely related to those initiating the rather stereotypic hypertensive response in various acute lung injuries after the perinatal adaptation period(3). In these injuries, inflammation with sequestration of activated neutrophils in lung microvasculature has been identified as a key event in the development of acute tissue damage(4). The release of bioactive mediators from activated leukocytes may then lead to imbalance in the endothelial vasoregulation and thereby increase the circulatory resistance and the extent of the initial injury in the lungs. Experimental data indeed show that lung injury owing to meconium aspiration also shares many of the features of acute inflammation(5–7).

Glucocorticoids are potent anti-inflammatory agents which have been shown to be of benefit in some clinical and experimentally induced hypertensive lung injuries, especially if given early(8). Steroids are able to modify granulocyte action and the release of various bioactive mediators, including vasoactive substances, and may thereby modulate vasomotor activity and limit the degree of injury in the diseased lungs(9). In fact, high dose methylprednisolone given before or shortly after endotoxin infusions improves the gas exchange and counteracts the pressure response in lungs of several species(10, 11). Although there are some uncontrolled reports of steroids being beneficial in the treatment of meconium aspiration, conclusive data of their efficacy are still lacking(1). In mild meconium aspiration corticosteroids may not be of therapeutic value(12, 13), but little is known about their pulmonary effects on the hypertensive lung injury owing to severe aspiration of meconium. Therefore, the purpose of this experimental study was to evaluate the influence of high dose methylprednisolone pretreatment on meconium aspiration-induced acute pulmonary vascular hypertension and longitudinal distribution of the vascular resistances after injury. We hypothesized that methylprednisolone would alleviate the hypertensive changes and thereby improve oxygenation and limit lung damage. To avoid the influence of perinatal pulmonary changes we studied juvenile pigs with their lung structure and function already adapted to extrauterine life.

METHODS

Animal preparation. Eleven 10-wk-old pigs weighing 24.0-26.5 kg were studied. The animals were anesthetized with ketamine hydrochloride (40 mg/kg) i.m. and diazepam (0.4 mg/kg) i.v., tracheotomized, and placed in a volume controlled ventilator (Harvard apparatus, model 607, Millis, MA). The initial ventilator settings were: Fio2 0.21, rate 18 breaths/min, end-expiratory pressure 4-5 cm H2O, and tidal volume 15-18 mL/kg. Anesthesia was maintained by continuous infusion of ketamine hydrochloride (12 mg/kg/h). Paralysis was induced with pancuronium bromide (0.3 mg/kg) i.v. and maintained with continuous infusion (0.6 mg/kg/h). All the experiments were approved by the Committee of Animal Care in Research of the University of Turku.

A 5 F Swan-Ganz thermodilution catheter (Baxter Corp., Irvine, CA) was inserted via the right external jugular vein and placed into the pulmonary artery under pressure monitoring for measurement of MPAP, PWP, and CVP at end-expiration. PAOP was extrapolated from a pressure decay curve after pulmonary artery occlusion(14). The right femoral artery was cannulated and a small polyethylene catheter was placed in the abdominal aorta to monitor MABP and to collect blood samples. Mean airway pressure was monitored from the proximal end of the endotracheal tube. The pressures were measured using Baxter Uniflow transducers (model 59-4H4, Edwards Critical Care Division, Uden, Holland) and a hemodynamic and respiratory computer system(Olli 530, Kone Oy, Helsinki, Finland) and recorded on an eight-channel recorder (Mingograf 82, Siemens-Elema, Solna, Sweden). Surface ECG was recorded for heart rate. CO was determined by the thermodilution technique(model 952A, American Edwards Laboratories, Santa Ana, CA) and calculated as the mean value of three measurements.

Pulmonary resistances were calculated from the following equations: PVR =(MPAP - PWP)/CO, Ra = (MPAP - PAOP)/CO, Rv = PVR - Ra, and systemic vascular resistance = (MABP - CVP)/CO. Arterial and mixed venous pH, Pco2, and Po2 were measured by an automatic blood gas analyzer (Radiometer ABL 50, Copenhagen, Denmark) immediately after drawing the samples and corrected for temperature. Venous admixture(˙Qva/˙Qt) was calculated using the standard shunt equation. The white cell concentration in aortic blood was counted by an electronic analyzer (Coulter Stkr, Luton, England).

Study protocol. After instrumentation and a stabilization period of 1 h, baseline measurements were done. All animals received a bolus of 3 mL/kg 20% meconium through the endotracheal tube. The meconium was obtained from the first stools of several healthy term human neonates of both genders. It was pooled, filtered, and lyophilized, and before the experiment diluted to a 20% mixture (equivalent to 65 mg/mL lyophilized meconium) with sterile saline. Six of the pigs received no medication, and five were pretreated with 30 mg/kg methylprednisolone (Solomet®, Lääkefarmos, Turku, Finland) 30 min before the meconium administration.

The hemodynamic changes were registered 15 and 30 min, and 1, 2, 4, and 6 h after the meconium administration. Samples for blood gas analysis and leukocyte counts were drawn at the same time points. To avoid hypoxemia Fio2 was increased to maintain arterial Po2 above 8 kPa. Arterial Pco2 was maintained below 5 kPa by adjusting the frequency of the ventilator.

Six hours after meconium insufflation the animals were killed by an overdose of potassium chloride. The diaphragmatic lobe of the right lung was removed for determination of lung wet/dry ratio. Tissue specimens from the same lung lobe were excised, fixed in 10% formalin, processed, and embedded in paraffin. Sections 5 μm in diameter were stained with hematoxylin and eosin for histologic examination. A pathologist blinded to the grouping evaluated the specimens for the presence of atelectasis, exudative debris, and alveolar inflammation.

Data analysis. Analysis of variance with repeated measures was used to compare the two groups at each time point. Comparisons between the groups were then made using the Mann-Whitney rank sum test, and comparisons within the group with Wilcoxon signed rank sum test. A p value < 0.05 was considered statistically significant. The results are expressed as mean ± SEM.

RESULTS

Meconium administration resulted initially in a simultaneous decrease in arterial Po2/Fio2 ratio and increase in venous admixture in both groups after aspiration (Fig. 1). In the untreated group, the arterial Po2/Fio2 ratio remained low, whereas˙Qva/˙Qt progressively increased from 2 h on. Methylprednisolone pretreatment, on the other hand, improved oxygenation after 2 h with a simultaneous fall in ˙Qva/˙Qt to the baseline level (Fig. 1). There were no significant changes in arterial Pco2 and pH (data not shown).

Figure 1
figure 1

Changes in arterial Po2(Pao2)/Fio2 and ˙Qva/˙Qt in six untreated(solid circles) and five methylprednisolone pretreated (open circles) pigs at baseline and for 6 h after meconium aspiration. Data are expressed as mean ± SEM. The asterisk (*) indicates a significant difference between the groups. BL = baseline.

Full size image

Insufflation of meconium induced a biphasic increase in MPAP and PAOP with a concomitant decrease in CO (Fig. 2 and Table 1). PWP did not change significantly. Pretreatment with methylprednisolone only slightly attenuated the first phase (0-1 h) increase in MPAP, prevented the increase in PAOP, and had no marked effect on CO. Instead, it significantly prevented the late phase (1-6 h) increase in both MPAP and PAOP (Fig. 2). No significant changes were detected in MABP and CVP (Table 1). Mean airway pressure increased rapidly after aspiration in both groups, but its late phase increase was significantly inhibited by methylprednisolone (Table 1).

Figure 2
figure 2

Changes in MPAP, PAOP, PWP, and pulmonary vascular resistance in six untreated (solid circles) and five methylprednisolone-pretreated (open circles) pigs at baseline and for 6 h after meconium aspiration. Data are expressed as mean ± SEM. The asterisk (*) indicates a significant difference between the groups. BL = baseline.

Full size image
Table 1 Hemodynamic changes (mean ± SEM) in six untreated and five methylprednisolone-pretreated pigs at baseline (BL) and for 6 h after aspiration of meconium
Full size table

Similar to MPAP, PVR showed a biphasic increase after meconium aspiration, whereas systemic vascular resistance did not change significantly (Fig. 2 and Table 1). The longitudinal distribution of PVR in the untreated group was characterized by an immediate increase in postarterial resistance after aspiration and a further progressive increase in both arterial and postarterial resistances during the later phase (Fig. 3). Methylprednisolone pretreatment attenuated significantly the increase in PVR in both phases, especially in the postarterial segment.

Figure 3
figure 3

PVR and its distribution to arterial (Ra) and postarterial (Rv) components in six untreated and five methylprednisolone-pretreated pigs at baseline and for 6 h after meconium aspiration. The percentage distributions of these resistances(%Ra/%Rv) are shown above the bars. The asterisk (*) indicates a significant difference vs baseline. BL = baseline.

Full size image

The second phase after meconium aspiration was associated with a significant fall in circulating leukocyte concentration, which was prevented by methylprednisolone pretreatment (Fig. 4). In fact, methylprednisolone tended to increase the peripheral leukocyte concentration initially. On histologic examination the severity of lung damage, assessed by histologic scoring of the tissue specimens, was equal in both groups (data not shown). Nevertheless, the mean lung wet/dry weight ratio was significantly lower in the methylprednisolone-pretreated group (7.27) when compared with the untreated group (8.06, p < 0.03).

Figure 4
figure 4

Effect of methylprednisolone on meconium aspiration-induced changes in blood leukocytes in six untreated (solid circles) and five methylprednisolone-pretreated (open circles) pigs at baseline and for 6 h after meconium aspiration. Data are expressed as mean ± SEM. The asterisk (*) indicates a significant difference between the groups. BL = baseline.

Full size image

DISCUSSION

The data of this study indicate that in adapted porcine lungs high dose methylprednisolone pretreatment attenuates the meconium aspiration-induced pulmonary hypertensive response by preventing the early and late phase increase in postarterial resistance. This is associated with improved oxygenation and diminished pulmonary edema formation, which may be secondary to the suppression of the leukocyte activity in the lungs.

Aspiration of meconium is a neonatal disorder that occurs during adaptation of the lung to extrauterine life. In this work, however, we studied 10-wk-old juvenile pigs with adapted lungs to determine the effects of aspirated meconium on hemodynamics in lungs without perinatal structural or functional alterations. Consequently, this model limits our ability to apply the results to the neonatal meconium aspiration syndrome, but enables us to compare our findings with those in other experimental lung injuries, mostly accomplished after the perinatal adaptation period(5, 8). Typical of these acute injury models, usually associated with an intense inflammatory reaction in the lungs, is a biphasic hypertensive response in pulmonary hemodynamics, regardless of the inducing insult(8). The initial pulmonary pressor response is followed by a pathogenetically separable phase of often progressive hypertension with sustained increase in lung vascular permeability(15, 16). These pulmonary alterations are indeed closely mimicked by the hemodynamic responses to intratracheal administration of human meconium in the lungs of our juvenile pigs and may superimpose on the transitional vascular changes in the clinical neonatal disease.

Experimental evidence indicates that corticosteroids may prevent or diminish the rise in vascular resistance and microvascular permeability after a wide variety of injuries in adult lungs(9) and some damages to neonatal lungs(17). Steroids can inhibit the activation of phospholipase A2, thus preventing the release of cell membrane phospholipids to form arachidonic acid with its vasoactive cyclooxygenase and lipooxygenase products(16, 18, 19), and may thereby modulate also the pulmonary vasomotor activity(20). In fact, high dose methylprednisolone pretreatment can blunt the initial rise in the canine and porcine pulmonary vascular resistance in response to endotoxin, thought to be related to vasoconstrictor thromboxane A2 release(15, 21). These results corroborate our findings obtained after endotracheal meconium insufflation in juvenile pigs. This early circulatory effect of methylprednisolone, however, may not only be mediated via suppressed formation of vasoconstrictor eicosanoids, but also through increased production of a predominantly vasodilator prostanoid, such as prostacyclin, by still unknown mechanisms(21).

The beneficial effects of corticosteroids on the second hypertensive phase after the insult, characterized also by sustained increase in microvascular permeability, may not be due to inhibition of prostanoid production, but seem to be related to the ability of steroids to suppress increased release of lipooxygenase products of arachidonate and to block granulocyte aggregation in the lungs(10, 15, 19). To preclude this progressive impairment in pulmonary vascular and ventilatory function, early or prophylactic administration of steroids is emphasized. In fact, pretreatment with methylprednisolone prevents or reduces the late phase pulmonary hypertension and hypoxemia in porcine(15) and ovine(16) septic lung injury, thus corroborating our findings. This is also partially true for oleic acid-induced lung injury in dogs(22). The beneficial impact of methylprednisolone on pulmonary gas exchange is likely to be mediated, at least in part, by improvement in ventilation and perfusion matching, as also indicated in our study. Glucocorticoids may indeed have a permissive effect on the local vascular tone(23), and by enhancement of the vascular responsiveness to hypoxic vasoconstriction cause redistribution of the pulmonary blood flow to the better ventilated areas(20, 24). Further, prevention of lung edema formation through diminished vascular permeability may also play a role(15). In addition to pretreatment, delayed therapy with methylprednisolone in porcine acute lung injuries may also prevent further deterioration in pulmonary gas exchange and even restore it toward baseline(11, 19). However, once a maximal tissue response to the insult has occurred, steroid therapy is not beneficial(10).

It is known from previous works that glucocorticoids may affect pulmonary arterial and venous vasomotion in the injured lungs(11, 25, 26). Methylprednisolone reduces pulmonary venous resistance in various experimental models of lung injury, resulting in a fall in pulmonary microvascular pressures, and, together with a decrease in microvascular permeability, diminished edema formation(9, 11, 25). In our model, methylprednisolone pretreatment also protected, similar to the effects in endotoxemic sheep and pig lungs(10, 15) and oleic acid injury in isolated dog lungs(25), against a rise in microvascular pressures and postarterial pulmonary vascular resistance. This may have contributed to the decreased edema production in our steroid-treated pig lungs. In contrast, lung edema per se does not affect pressure changes in the pulmonary fluid-exchange vessels and may therefore not explain the microvascular depressor effect of methylprednisolone in the injured lung(25).

Considerable evidence has now accumulated to suggest that sequestration of granulocytes in lung microcirculation with subsequent mediator release is an important precipitating event in the development of a variety of acute lung injuries, including endotoxemia and fat embolism in adult lungs(4, 8, 15) and hyperoxia and meconium aspiration in neonatal lungs(6, 17). In fact, the degree of granulocytopenia associated with pulmonary trapping of leukocytes has been shown to correlate with the degree of lung microvascular injury(15, 27). Early steroid treatment, on the other hand, attenuates significantly the late phase leukopenia in experimentally induced lung injuries, as also shown in our study, and partially prevents the accumulation of granulocytes in peripheral lung tissue(11, 15, 16). The protective effects of steroids on vascular integrity may be therefore largely mediated by inhibiting granulocyte trapping in the lungs, possibly through reduced pulmonary synthesis of chemotactic lipooxygenase products(15, 16). Inasmuch as an accumulation of leukocytes in lung tissue in our study was not substantially inhibited by methylprednisolone, as also found in endotoxemic pigs(11), its beneficial pulmonary action may also be mediated by blocking effects on leukocyte function. Glucocorticoids may indeed bind directly to the nuclear receptors and thereby suppress neutrophil activation and subsequent mediator release, known to be involved in the pathophysiology of acute tissue injury in adult and neonatal lungs(1, 4). Consequently, inhibition of neutrophil activation, by administration of glucocorticoids or more specific blockers, may play an important role in the therapeutic approaches of acute lung injuries in the future(4).

Despite favorable effects of corticosteroids in some experimental acute lung injury models, the results in clinical studies have been mostly disappointing. In fact, early or prophylactic steroid treatment is ineffective in adults with acute lung injury owing to severe sepsis or aspiration(8, 28), but may be of help in fat embolism(29). Although a diffuse inflammatory reaction in the pulmonary microvasculature is a common feature to these injuries, pulmonary hemodynamics is not consistently benefited by administration of steroids(8, 28). In newborn infants, controlled trials of prophylactic (prenatal) or early dexamethasone administration have demonstrated significant improvements in the course of the respiratory distress syndrome(30, 31), but early hydrocortisone therapy seems to be ineffective in the mild form of meconium aspiration syndrome(12). In contrast, our data, although obtained from a postneonatal animal model, suggest that early, possibly even antenatal, administration of high doses of methylprednisolone could help to interrupt the injury cycle in the lungs, specifically through action on the local vasculature. Future trials on this subject are therefore clearly indicated.

In conclusion, we have shown that methylprednisolone pretreatment attenuates the hypertensive pulmonary response induced by meconium aspiration in pigs. Interestingly, this same protective effect has been described also in other experimental acute injury models in adapted lungs, suggesting similar pathophysiologic mechanisms behind the pulmonary dysfunction. Presently, steroid treatment is not recommended in meconium aspiration syndrome owing to previous inconclusive data. Our results, however, suggest that further evaluation of early high dose steroid treatment in severe meconium aspiration is warranted, also in neonatal animal models.