Objective
Epidemiologic studies have highlighted an association between maternal smoking and persistent pulmonary hypertension of the newborn infant. However, the impact of exposure to tobacco smoke on the perinatal pulmonary circulation is currently unknown. The purpose of this study was to assess the pulmonary vascular effects of environmental tobacco smoke in the fetus.
Study Design
We performed surgery on 16 fetal lambs and placed catheters in the main pulmonary artery, aorta, and left atrium to measure pressures. An ultrasonic blood flow transducer was placed around the left pulmonary artery to measure blood flow. The ewes were exposed to tobacco smoke for 2 hours. In another set of experiments, the pulmonary vascular response to increased fetal O 2 tension has been assessed after 2 hours of tobacco smoke inhalation or not (control group).
Results
Exposure to tobacco smoke decreased pulmonary blood flow by 30% and elevated pulmonary vascular resistance by 40%. The vasodilator response to increased O 2 tension was blunted after smoke inhalation compared with control animals. Smoke inhalation was associated with a decrease in fetal PaO 2 and SaO 2 .
Conclusion
Exposure to environmental tobacco smoke in pregnant ewes impairs both basal tone and vascular reactivity of the fetal lung.
Cigarette smoking during pregnancy is associated with multiple health problems that include spontaneous abortion, prematurity, growth restriction, and sudden infant death syndrome. It alters the development of the respiratory system and increases the risk of a wheeze and asthma in childhood. Studies of lungs that were exposed in utero to cigarette smoke showed marked structural and functional changes, such as decreased alveolar attachments to the airway wall and increased airways responsiveness. Moreover, growing evidence has emerged that in utero smoke exposure may also impair the perinatal pulmonary circulation. Cigarette smoking during pregnancy increases the risk of persistent pulmonary hypertension of the newborn infant (PPHN). PPHN results from the failure of the pulmonary circulation to dilate at birth and may cause severe hypoxemia. Little is known regarding the potential mechanisms to explain the link between cigarette smoking during pregnancy and PPHN.
In adults, pulmonary hypertension may worsen the course of tobacco smoke-induced chronic obstructive pulmonary disease. Endothelial dysfunction in intrapulmonary arteries was found during exposure to tobacco smoke that contributed, at least in part, to vasoconstriction and smooth muscle cells proliferation. Chronic hypoxia has also been involved in the mechanism of pulmonary vasoconstriction and vascular remodeling in heavy smokers. In vitro studies suggest that in utero tobacco smoke may alter pulmonary vasorelaxation. Indeed, prenatal exposure to cigarette smoke extract impairs endothelium-dependent relaxation of isolated pulmonary arteries of chicken embryo. However, the impact of maternal cigarette smoking on the perinatal pulmonary circulation currently is unknown.
Therefore, the aim of this study was to assess the effects of in utero cigarette smoke on the pulmonary vascular resistance (PVR) in the fetus. We performed serial studies on the acute hemodynamic effects of passive cigarette smoke in late-gestation fetal lambs.
Materials and Methods
All animal procedures and protocols were reviewed and approved by the French “Ministère de l’Alimentation, de l’agriculture et de la Pêche.” Surgery was performed on 16 mixed-breed pregnant ewes at 130-132 days’ gestation (term = 145 days). Ewes were sedated with intramuscular xylazine (Sedaxylan; 20 mg/mL, 40 mg), intravenous propofol (Diprivan; 2%, 4 mL), and nalbuphine (Nubain; 20 mg) before intubation and mechanical ventilation. The ewes were kept anesthetized by inhaled isoflurane (Aerrane; 1-5%). The left forelimb of the fetus was delivered through a uterine incision. A skin incision was made after subcutaneous injection of lidocaine (Xylocaine; 1%, 4 mL) and nalbuphine (Nubain; 5 mg). Polyvinyl catheters (20 gauge) were advanced into the fetal aorta and vena cava through the axillary vessels. A left thoracotomy exposed the heart and great vessels. A catheter (20 gauge) was inserted into the main pulmonary artery by direct puncture through purse string suture, as previously described. An ultrasonic flow transducer, size 6 (Transonic Systems; Transonic Systems Inc, Ithaca, NY), was placed around the left pulmonary artery (LPA). Another catheter was placed in the amniotic cavity. The flow transducer and catheters were exteriorized through a subcutaneous tunnel to an external flank pouch. The ewes received amoxicillin (Clamoxyl; 1 g) and nalbuphine (20 mg) daily until day 3 after surgery. Studies were performed after a recovery time of 72 hours (ie, at 133-135 days’ gestation. Previous lung morphometric studies suggest that this gestational age in the fetal lamb corresponds to full-term for lung maturation in human fetus.
Physiologic measurements
The blood flow transducer cable was connected to an internally calibrated flow meter (T201; Transonic Systems Inc) for continuous measurements of LPA blood flow. The zero blood flow was defined as the measured blood flow value immediately before the beginning of systole. The catheters were connected to a pressure transducer (Philips HP Merlin; Soma Technology Inc, Bloomfield, CT). Pressures were referenced to the amniotic cavity pressure. PVR in the left lung was calculated as the difference between mean pulmonary artery pressure (PAP) and left atrial pressures divided by mean LPA blood flow. Based on our past studies in which left atrial pressure was found to be consistently close to 2-cm H 2 O, a value of 2-cm H 2 O was used as an estimate of left atrial pressure. Blood samples from the main pulmonary artery catheter were used for blood gas analysis, carboxyhemoglobin, lactate, and oxygen saturation (SaO 2 ) measurements (OSM3 Hemoximeter and ABL 800 FLEX; Radiometer, Copenhagen, Denmark; i-Stat, Abbott Laboratories, Chicago, IL).
Experimental design
The animals were assigned randomly either in an experimental group (tobacco smoke exposure) or in a control group. Two different experimental protocols were included in this study: (1) the effects of maternal exposure to environmental cigarette smoke on the basal pulmonary vascular tone (protocol 1) and (2) the effects of maternal exposure to environmental cigarette smoke on the pulmonary vascular response to an increase in O 2 tension (protocol 2). Experimental protocols were performed successively (protocol 1 then protocol 2). A minimum recovery period of 48 hours was required between each protocol. To ensure that complete recovery was achieved before another protocol began, we checked that the measured parameters, arterial blood gases, and carboxyhemoglobin levels had returned to the baseline values. Mean PAP, aortic pressure (AoP), amniotic pressure, LPA blood flow, and heart rate were recorded at 10-minute intervals. Blood gas analysis, carboxyhemoglobin, and SaO 2 measurements in both the ewe and fetus were performed before and at the end of each protocol.
Protocol 1: effects of maternal exposure to environmental cigarette smoke on the basal pulmonary vascular tone. After 30 minutes of baseline hemodynamic measurements, a large helmet (self-made light and transparent plastic, volume approximately 125 L) was placed around the ewe’s head and held around the neck. The helmet was ventilated by an air/O 2 blender and flow meter (Sechrist Industries Inc, Anaheim, CA) that was set at a rate of 18 L/min and fraction of inspired oxygen (FiO 2 ) of 21%. FiO 2 and carbon monoxide (CO) concentration inside the helmet was monitored continuously (Puritan-Bennett 7820 oxygen monitor; Puritan-Bennett Inc, Overland Park, KS; TIF8500A carbon monoxide analyzer; TIF Instruments, Owatonna, MN). After 30 minutes of quiet breathing in the helmet, tobacco smoke was delivered into it. The tobacco smoke generator system consisted of a device which lit and presented a cigarette (Gauloise disque bleu, no filter: tar, 10.7mg/cigarette; nicotine, 0.59 mg/cigarette; CO, 16.5 mg/cigarette) to a tube that was connected to a precision air flow meter (0-100 mL/min; Sechrist Industries Inc). The entire air flow passed over the cigarette. Smoke was delivered into a mixing chamber where it was diluted with the fresh gas that was driven by the air/O 2 blender and flow meter (rate, 18 L/min). The blender was then adjusted to maintain the FiO 2 at 21% in the helmet. The rate of airflow that generated the cigarette smoke was adjusted to obtain a target CO concentration inside the helmet of 40 parts per million (ppm; 80 mL/min [range, 75–85 mL/min]). This CO concentration and the 2-hour exposure duration were chosen to mimic actual environmental cigarette smoke in which pregnant women potentially can be exposed in various private or public facilities. Each cigarette was puffed for 10 minutes before replacement. Duration of the smoking period was 120 minutes. The helmet was then removed, and the smoke generator was stopped. The ewe breathed room air for an additional 30 minutes. Special care was taken to ventilate the room for the duration of the experiment (CO, <2 ppm).
Protocol 2: effects of maternal exposure to environmental cigarette smoke on the pulmonary vascular response to increased O 2 tension. The purpose of this protocol was to determine whether in utero cigarette smoke would alter the pulmonary vasodilator response to increase in fetal PaO 2 . The ewes were exposed to 120 minutes of tobacco smoke, as described earlier. Then, the smoke generator was discontinued, and the air/O 2 blender and flow meter was set at FiO 2 of 100% (18 L/min) for 30 minutes. Hemodynamic measurements (mean PAP, AoP, pulmonary blood flow, PVR) were recorded at 10-minute intervals that started 30 minutes before O 2 inhalation, during O 2 inhalation, and for 30 minutes after O 2 discontinuation. Control animals were subjected to a similar protocol, excluding tobacco smoke inhalation.
Statistical analysis
The results are presented as means ± SEM. The data were analyzed with repeated-measures and factorial analysis of variance. Intergroup differences were analyzed with the Fisher’s, Scheffe’s, and Bonferroni/Dunn’s least significant tests (Stat View for PC; Abacus Concepts, Berkeley, CA). The Mann-Whitney test (independent values) and paired Wilcoxon rank test (paired values) were also performed on the quantitative data to test for statistical differences between the groups. A probability value of < .05 was considered to be statistically significant.
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