Main

Meconium (the first stool passed by the fetus or neonate) is a viscid material containing desquamated cells from the skin and alimentary canal, lanugo hairs, amniotic fluid, fatty material from the vernix caseosa, and bile(1). Evidence of meconium in amniotic fluid is seen in 7-22% of live births(2). Meconium is detected below the vocal cords in 7-56% of these neonates and may cause a wide spectrum of clinical disease referred to as MAS. MAS develops in 10-32% of neonates with meconium aspiration and ranges from mild transient hypoxia to severe pulmonary insufficiency(3). The respiratory distress includes atelectasis, airway obstruction, impaired gas exchange, pulmonary hypertension(2), decreased lung compliance, increased airway resistance(4), and severe acidosis(5). The initial site of meconium contact is the oropharynx and airway, and a portion of the MAS appears to involve airway smooth muscle either directly or indirectly(6).

Meconium may alter airway resistance by a number of mechanisms such as mechanical obstruction(7–9), reflex alteration in airway tone(6), or production of bronchoreactive agents(8, 10–12). Lipids constitute more than 12% of meconium solids(13) and may be one of the factors that cause the airway response to meconium. Because lipids have been shown to alter smooth muscle tone via synthesis of prostaglandins(14, 15), direct activation of diacylglycerol(16), or increased sarcoplasmic reticulum membrane permeability to calcium(17), there are several ways that they could contribute to the increased airway resistance seen in the MAS.

The present experiments were carried out to determine the direct effects of meconium on airway smooth muscle tension. We used an isolated tracheal ring preparation to test the hypothesis that the fatty acids in meconium can increase airway tone directly.

METHODS

Tissue preparation. Tracheas obtained from normal male Sprague-Dawley rats (mean body weight, 232 ± 7 g) reared on filtered room air, were removed (taking care not to stretch the tissue) and placed in cold (6 °C) PSS gassed with 95% oxygen and 5% carbon dioxide (composition in mM: 130 NaCl, 14.9 NaHCO3, 5.5 dextrose, 4.7 KCl, 1.19 KH2PO4, 0.7 MgSO4·7H2O, 1.6 CaCl2·2H2O, 0.3 EDTA). Tracheas not used immediately were stored in cold PSS for no more than 48 h. Studies done in our laboratory comparing fresh tracheal rings with those stored for 24 or 48 h showed that tension generated in response to acetylcholine (10-6 M) was the same. Each trachea was cleaned of fascia and cut into segments 4-5 mm long while in cold PSS. Two pieces of plastic-coated wire were inserted through the lumen of each segment, bent, and then twisted into a hanger shape on each side of the segment. One hanger was tied with silk suture to a fixed glass rod and suspended in a 20-mL organ bath containing PSS (at 37 °C gassed with 95% O2-5% CO2). The other hanger was attached to a force displacement transducer (Grass FTO3C, Quincy, MA) to measure transverse tension which was recorded on a polygraph (Grass model 7). Segments were stretched to 1.5 g according to the work of others(18) and after initial experiments in our laboratory confirmed that this was the optimal passive tension for our preparation.

Solutions. Meconium was obtained from healthy, unfed newborn human infants and transported to the laboratory on ice. A 20 g/100 mL stock suspension was prepared by mixing meconium with PSS and straining it through gauze to remove large particles. The meconium stock suspension was used immediately or frozen in aliquots at -18 °C. Over the course of the study, fresh meconium solution was usually prepared each week. On the day experiments were done, meconium suspension was thawed, shaken, and diluted with PSS to achieve bath concentrations of 5, 1, or 0.1 mg/mL when 1-mL aliquots were added. Indomethacin (Merck, West Point, PA) was dissolved in PSS with additional NaHCO3 (1.5 × 10-2 M) to form a 1.5 × 10-3 M stock solution. PSS with additional NaHCO3 (1.2 × 10-2 M) was the vehicle control for indomethacin. Acetylcholine (Sigma Chemical Co., St. Louis, MO) and atropine sulfate (Sigma Chemical Co.) were dissolved in PSS to form stock solutions (10-2 M). Oleic acid (Cayman Chemical, Ann Arbor, MI) was dissolved in ethanol (3.5 × 10-3 M) and diluted 10-100-fold in ethanol before adding it to the PSS in the organ bath.

Protocols. Common to all protocols in this study, tracheal segments were equilibrated for 2-5 h in the organ bath before adding acetylcholine (10-6 M) or KCl (15-30 mM) to cause a sustained, reproducible contraction. When tension plateaued, agonists were added. At the completion of each experiment, atropine sulfate (bath concentration 10-6 M) was added to confirm the amount of active tension gained in response to acetylcholine. Tracheal segments were discarded if they did not develop at least 125 mg of tension in response to 10-6 M acetylcholine. A comparison of the segments' response to acetylcholine (10-6 M) based on their position relative to the larynx showed no difference in the tension gained between any of the groups.

Protocol 1. Meconium suspension was added to the organ bath and left for 40 min before washing it from the bath with fresh PSS. After a recovery period of 1 h, the protocol was repeated. The epithelium was removed from some segments by inserting and rotating a wooden stick or plastic catheter inside the tracheal lumen before placing the segments in the organ bath. At the conclusion of four experiments, six tracheal segments with the epithelium removed and four with the epithelium undisturbed were fixed in 10% formalin for at least 24 h, then dehydrated in graded series ethanol, and embedded in paraffin. Sections 4.0 μm thick were cut from each end, and the center of each segment was cut with a Reichert-Jung microtome (Heidelburg, Germany) and stained with hematoxylin and eosin for examination by light microscopy to determine the integrity of the epithelium. Tracheal segments that were not rubbed showed only minimal damage (<5% denudation) in the regions where the coated wire had been, whereas the rubbed segments demonstrated ≈90% denudation of the epithelium.

Protocol 2. Meconium suspension was added as in protocol 1, but after segments were washed, indomethacin (final concentration 3 × 10-6 M) or vehicle was added and left in the bath for 1-2 h before adding acetylcholine and meconium for a second time.

Protocol 3. Instead of adding meconium as in protocol 1, oleic acid was added to the bath (3.5 × 10-6 M to 3.5 × 10-4 M) and left for 40 min before removal with fresh PSS. After 1 h, acetylcholine and oleic acid were added for a second time. In some segments, indomethacin (final concentration 3 × 10-6 M) or vehicle was added before the second addition of oleic acid.

Protocol 4. An acetylcholine dose response was obtained after the segments had stabilized in the tissue bath. After washing four times with fresh PSS, meconium (5 mg/mL) or PSS (vehicle control) was added to the tissue bath and left for either 1 h or 6 h or more. Baseline tension was adjusted back to 1.5 g, and the acetylcholine dose response was again obtained.

Protocol 5. To determine whether the effects of meconium on tracheal smooth muscle were due to a cholinesterase-like protein in meconium, in separate experiments tension was generated by increasing the KCl in the tissue bath to 15 or 30 mM, and meconium was added when tension plateaued. In further experiments, meconium was heated above 60 °C for 1 h before being adding to tracheal segments in which tone had been generated by acetylcholine(10-6 M).

Data analysis. Tension was recorded continuously, and peak changes were measured. Results are normalized as the percent change from the tension induced by acetylcholine and reported as the group mean plus or minus the SEM. Groups were compared using a two-tailed t test for two groups or a one-way analysis of variance for three or more groups. A Tukeypost hoc analysis was performed for any significant analysis of variance. A Fisher's exact test was used to compare the responses of tracheal segments to different doses of meconium or fatty acid. An α less than 0.05 was considered significant.

RESULTS

Meconium (0.1-5 mg/mL) usually caused tracheal smooth muscle relaxation when it was added to the muscle bath, although contraction sometimes occurred at the intermediate and low doses (Fig. 1). The incidence of relaxation and contraction at each concentration is summarized inTable 1. The incidence of segments which contracted to meconium (as well as those that did not respond) increased significantly at 0.1 mg/mL compared with 5 mg/mL meconium. The degree of relaxation increased significantly in a concentration-dependent manner when the net response at each concentration was compared (Fig. 2). Epithelial removal did not alter the response of tracheal smooth muscle to meconium at any dose tested (data not shown).

Figure 1
figure 1

Changes in tracheal smooth muscle tension caused by adding meconium to the tissue bath. At 5 mg/mL meconium, relaxation usually occurred; in some segments (A) relaxation was sustained during the exposure, but more frequently tension returned near the original baseline(B). At the lowest concentration of meconium (0.1 mg/mL) tested, tension usually decreased (C), but contraction occurred in a few segments (D). Baseline tension refers to the amount of tension generated in response to acetylcholine (ACH, 10-6 M) added as indicated by the mark on the time line. Arrows indicate where meconium was added.

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Table 1 Incidence of responses to increased concentrations of meconium
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Figure 2
figure 2

Net change in tracheal smooth muscle tension to different concentrations of meconium. Percent change refers to the change from baseline tension obtained by adding acetylcholine (10-6 M) to the bath. Each strip was exposed to one concentration, and the response shown is the mean ± SEM, n = the number of segments exposed to each concentration of meconium. Asterisks denote a significant relaxation.

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To determine whether responses to meconium were mediated by products of the cyclooxygenase pathway, we examined effects of indomethacin on the changes in tracheal smooth muscle tension caused by 0.1 and 5 mg/mL meconium (protocol 2). The net responses to these doses of meconium were not different after indomethacin treatment (Fig. 3).

Figure 3
figure 3

Tracheal responses to meconium before and after exposure to indomethacin. Black bars represent responses to the first administration of meconium, and striped bars represent repeated administration of meconium after 1-h exposure to indomethacin (3× 10-6 M). Percent change refers to the change from baseline tension obtained by adding acetylcholine (10-6 M) to the bath.n = the number of tracheal segments before and after indomethacin.

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To determine whether a fatty acid can cause similar responses to meconium on tracheal smooth muscle in vitro, we examined the effects of oleic acid (3.5 × 10-6 M to 3.5 × 10-4 M) in a separate group of tracheal segments (protocol 3). The net response was contraction at the lowest dose (3.5 × 10-6 M) and relaxation at the highest dose(3.5 × 10-4 M) (Fig. 4). Repeated exposure of tracheal segments to oleic acid had no significant effect on the incidence of relaxation and contraction or on the magnitudes of the changes in tension at each dose tested (data not shown).

Figure 4
figure 4

Changes in tracheal smooth muscle tension caused by different concentrations of oleic acid. Note that contraction predominated at 3.5 × 10-6 M and relaxation predominated at 3.5 × 10-4 M, n = the number of segments tested at each dose. Theasterisk denotes a significant difference from control.

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To investigate the effect that prolonged exposure to meconium might have on airway tension and responsiveness to acetylcholine, tracheal segments were left in meconium (5 mg/mL) after an initial dose response to acetylcholine was obtained (protocol 4). Incubation of tracheal segments for 1 h with meconium did not significantly change tension from baseline (tension increased 100± 166 mg, n = 6) and was not different from from the change in tension from baseline of other segments incubated with PSS for 1 h (117± 191 mg). Prolonging the incubation of the tracheal segments with meconium for 6 h or more also did not cause a significant difference in baseline tension (tension increased 8 ± 86 mg, n = 13) and again was not different from the change in tension of other segments after incubation in PSS for 6 h or more (tension increased 104 ± 126 mg,n = 12). In addition, the response to acetylcholine before and after meconium for 1 h (Table 2) or 6 h(Table 3) was not significantly different.

Table 2 Change in tracheal smooth muscle tension in response to increasing concentrations of acetylcholine before and after incubating with meconium for 1 h
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Table 3 Change in tracheal smooth muscle tension in response to increasing concentrations of acetylcholine before and after incubating with meconium for 6
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To determine whether the relaxation of tracheal segments to meconium was due to a cholinesterase-like material in meconium, we examined the effect of meconium (5 mg/mL) on tracheal segments in which tone was generated by increasing the bath concentration of KCl to 15-30 mM KCl. KCl (n = 6) increased tracheal smooth muscle tone by 800 ± 245 mg. Meconium (5 mg/mL) when added to the tissue bath decreased tension by 65 ± 13%. This was not significantly different from the results in protocol 1. In other experiments in which tracheal smooth muscle tone was increased with a different concentration of acetylcholine (10-5 M, n = 4, tension increased 660 ± 220 mg), addition of meconium (5 mg/mL), which had previously been heated above 60 °C for 1 h and then cooled, also caused relaxation (tension decreased 70 ± 18%) that was similar to what was observed in protocol 1.

DISCUSSION

At the doses tested, meconium predominantly caused tracheal smooth muscle relaxation in a dose-dependent fashion in vitro. As the concentration of meconium decreased, the magnitude of the relaxation decreased, and the number of segments which constricted or failed to respond increased. Tracheal segments which were not constricted with acetylcholine were incubated with meconium for either 1 or 6 h. Incubation with meconium did not affect either resting tracheal smooth muscle tension or the amount of tension generated when acetylcholine was added. The MAS is characterized by airway obstruction(2), decreased lung compliance, and increased airway resistance(4); therefore in view of our results, these clinical observations do not appear to be caused by a direct acute affect of meconium on tracheal smooth muscle. It must be appreciated, however, that MAS is a neonatal disease, whereas these experiments were conducted in vitro on adult rat tracheas. Furthermore, there may be interspecies differences in the response to human meconium and nonhuman airways as well as differences in distal airway smooth muscle responses compared with the more proximal airways tested in these experiments.

Removal of the epithelium did not alter the response of the segments to meconium, suggesting that meconium acts directly on the tracheal smooth muscle. In addition, our observations are probably not due to products of the cyclooxygenase pathway, because blockade of cyclooxygenase with indomethacin and heating meconium did not significantly alter the response of tracheal smooth muscle to meconium.

Interestingly, at the low dose of oleic acid (a fatty acid which is not a prostaglandin precursor), tracheal smooth muscle contraction predominated, whereas relaxation predominated at the highest concentration. The response to the highest concentration of oleic acid was similar to the response to the highest concentration of meconium; however, at the lower concentration it caused much more contraction than meconium. Oleic acid has been previously identified in relatively high concentrations in meconium(19) and may activate relaxation pathways within smooth muscle by several different mechanisms. Oleic acid directly activates adenyl cyclase in A431 cells and rat glioma C6 cells(20). Activation of adenyl cyclase in smooth muscle cells leads to decreased smooth muscle tone and could account for part of the tracheal relaxation that we observed in response to meconium. Casals et al.(21) reported decreased de novo synthesis of diacylglycerol in lung tissue membranes from rabbits with oleic acid-induced adult respiratory distress syndrome. A role for diacylglycerol activation of myosin light chain kinase via protein kinase C, independent of the calcium-calmodulin complex leading to smooth muscle contraction, has been proposed(22). Therefore, decreased production of diacylglycerol might reduce the level of smooth muscle tone. Oleic acid has also been reported to enhance sarcoplasmic reticulum permeability to calcium, which would increase smooth muscle tone(17).

Acetylcholine was used to generate tone in this study because it is similar to the primary source of airway tone in vivo(23). The mechanism by which airway smooth muscle tone is increased may affect the airway's response to agonists. For example, the inhibitory effects of prostaglandin E2 on airway smooth muscle contraction are reduced when canine tracheal smooth muscle is contracted with acetylcholine compared with histamine(24). Inhibitory responses to meconium do not appear to be specific to cholinergic agonists or to the presence of residual acetylcholinesterase or pseudocholinesterase enzymes, because meconium had the same effect on segments that were constricted by KCl. In addition, heated meconium caused the same degree of relaxation of segments constricted by acetylcholine, suggesting that enzyme activity was not an important factor. A state of enhanced tracheal smooth muscle tone may more closely resemble the airway milieu at the time shortly before or during parturition when MAS occurs due to the complex interaction ofin utero fetal hypoxia/asphyxia(25), chemical pneumonitis(2), chorioamnionitis(26), and or atelectasis from mechanical obstruction or surfactant displacement(27) proposed to occur in MAS.

Some of the variability observed in the response of tracheal smooth muscle to meconium could also be due to the heterogenous nature of meconium. The composition of meconium varies considerably between healthy infants(13). Although meconium was pooled in our study to decrease this variability, several fresh batches were used over the course of this study, and tracheal smooth muscle responses may still have been affected.

In summary, we found that meconium had a dose-dependent effect on tracheal smooth muscle, with relaxation predominating at high concentrations and both relaxation and contraction occurring at lower concentrations. Neither airway epithelium nor prostaglandin production appeared to contribute to these observations. Like meconium, oleic acid caused a dose-dependent response, but there was a higher incidence of contraction with the low dose of oleic acid than with meconium. Although meconium does directly affect tracheal smooth muscle in vitro, the clinical characteristics of the MAS suggest other factors, such as airway reflexes, hypoxia, or nonprostaglandin inflammatory mediators probably cause the increased airway resistance associated the MAS. Further studies are needed to examine mechanisms by which meconium may directly alter airway smooth muscle tension and to determine the factors which determine relaxation or contraction.