© 2003 American Thoracic Society
Airway Alveolar Attachment Points and Exposure to Cigarette Smoke In UteroDepartment of Pulmonary Physiology, Sir Charles Gairdner Hospital, Perth, Western Australia; and Department of Respiratory Medicine, Royal Children's Hospital, Melbourne, Victoria, Australia Correspondence and requests for reprints should be addressed to Dr. Philip Robinson, Department of Respiratory Medicine, Royal Children's Hospital, Melbourne, VIC, Australia 3052. E-mail: philrob{at}cryptic.rch.unimelb.edu.au
The harmful effects of in utero cigarette smoke exposure include increased asthma symptoms and reduced lung function during the neonatal period, increased airway responsiveness to inhaled stimuli, and an increased risk of sudden infant death syndrome. Altered lung function may result from altered airway/lung structure. Airway dimensions, alveolar attachment points, and parenchymal elastin content were measured in 32 infants who died from sudden infant death syndrome and were grouped according to their perinatal cigarette smoke exposure. Compared with those without any exposure to cigarette smoke, the distance between alveolar attachments on airways was greater (p < 0.001) in infants exposed to cigarette smoke only in utero or both in utero and during the postnatal period but not different in those with only postnatal exposure. The percentage of elastin within the alveolar walls was similar in all the exposure groups. These findings suggest that in utero cigarette smoke exposure may result in abnormal airway function due to a reduction of the forces opposing airway narrowing.
Key Words: alveolar attachments • in utero cigarette smoke exposure • sudden infant death syndrome
Maternal cigarette smoking during pregnancy is known to be a significant health risk to infants (1, 2). It is associated with restricted growth (3), reduced respiratory function at birth (4, 5) that persists into childhood (6), increased airway responsiveness (7), an increased likelihood of developing wheeze and asthma (8, 9), and an increased risk of sudden infant death syndrome (SIDS) (10–14). Epidemiologic studies that have adjusted for the effects of postnatal smoke exposure suggest that increased responsiveness in these infants is primarily associated with cigarette smoke exposure in utero (6, 15, 16). Gilliland and coworkers (8) have shown that although cigarette smoke exposure in utero increases the occurrence of doctor-diagnosed asthma and wheeze in school children, postnatal exposure operates as a cofactor that triggers wheezing attacks.
The mechanisms that result in abnormal postnatal lung function due to exposure to cigarette smoke in utero are unknown. The exposure is a unique form of passive smoke exposure in that there is no direct exposure to the fetal lung. However, components of cigarette smoke such as cotinine can cross the placental barrier (17). Prenatal subcutaneous administration of nicotine to pregnant monkeys results in decreased expression, but increased function, of We have recently shown an increase in airway responsiveness in guinea pigs after exposure to cigarette smoke in utero (21). Morphometric examination of the lungs of these animals showed differences in airway structure and a significant increase in the mean distance between alveolar attachments to the airway wall compared with control animals (21). Other animal studies have identified structural differences in the lungs of animals exposed to cigarette smoke in utero. Collins and coworkers (22) studied the offspring of pregnant rats exposed to cigarette smoke from Day 5 to Day 20 of gestation and found reduced birth weight, reduced lung elastic tissue, and a reduced number but increased size of alveolar saccules. Saetta and coworkers (23) have previously shown that the numbers of alveolar attachments to the surrounding airway adventitia are reduced in current smokers and that this reduction is associated with a reduction in lung elastic recoil. To our knowledge, morphometric analyses of alveolar attachments in infants exposed to cigarette smoke in utero have not been reported previously. Therefore, airway structure and alveolar attachments in infants who were exposed to cigarette smoke in utero were examined and compared with nonexposed infants. In a subgroup of infants exposed to cigarette smoke in utero, elastin content in the lung parenchyma was examined and compared with nonexposed infants.
In 1991, the Victorian Sudden Infant Death Research Foundation commenced a study into the epidemiology of SIDS in Victoria, Australia. Parents of children who had died from SIDS were approached and invited to complete a questionnaire on demographic factors including infant gestation; birth weight; sex and age at death; and maternal smoking history before pregnancy, during the first, second, and third trimester of pregnancy and between the birth and death of their child. Daily cigarette consumption was scored: 0 = no smoking; 1 = fewer than 10 cigarettes/day; 2 = 10 to 20 cigarettes/day; 3 = 20 to 30 cigarettes/day and; 4 = more than 30 cigarettes/day. At postmortem examination, four to five blocks of lung parenchyma were taken at random from both lungs and fixed in 10% buffered formalin for more than 24 hours, dehydrated through a series of graded alcohols, and embedded in paraffin. Serial sections (5 µm) were cut and stained with hematoxylin and eosin, and Resorcin-Fuchsin. Transverse sections of airways were assessed using a Quantimet 500+ color image analyzer (24). The internal area (Ai) and perimeter defined by the luminal surface, the basement membrane perimeter (Pbm), the outer muscle area and perimeter defined by the outer edge of the smooth muscle, and the outer airway wall area and outer airway wall perimeter defined by the airway adventitia were measured (Figure 1) . The area of airway smooth muscle was measured by direct tracing. Inner wall area (outer muscle area - inner airway wall area) and outer wall area (outer airway wall area - outer muscle area) were calculated. Smooth muscle area was normalized for airway size by dividing it by Pbm, and the percent of smooth muscle shortening was calculated (25). At a magnification of x200, alveolar attachments to the airway wall were counted and divided by Po, minus the length of any section that was contiguous with an adjacent pulmonary vessel (Figure 1). If two septa joined before attaching to the airway wall at one point, this was counted as one attachment. In a subgroup of infants (n = 25), the amount of elastin in the alveolar walls was assessed using an 81-point grid at x400 magnification on 10 high-powered fields. All measurements were made by the one observer (J.E.) who was blinded to the case classification.
The mean distance between alveolar attachments and airway dimensions, for airways with a Pbm less than 5 mm (predominantly membranous bronchioles and small intraparenchymal cartilaginous bronchi) were analyzed. In addition, gestation, birth weight, and age were compared between smoke exposure groups using a one-way analysis of variance. The independent effects of smoke exposure, gestation, birth weight, sex, and age on attachments and dimensions were examined using multiple linear regression analysis. The proportion of positive to negative elastin staining of parenchymal tissue was compared in smoking groups using two tailed, unpaired t tests. Intraobserver error was calculated as the coefficient of variation (mean/SD x 100) for six repeated measurements on six separate airways. The study was reviewed and approved by the Royal Children's Hospital and the Victorian Institute of Forensic Medicine Ethics Committees.
Airways and parenchymal tissues were examined in 32 infants dying from SIDS. For descriptive purposes, infants were grouped according to the level of maternal cigarette smoke exposure: no smoke exposure (n = 8), in utero exposure only (n = 4), only postnatal exposure (n = 4), and in utero and postnatal exposure (n = 16). There were no differences between groups regarding gestational age at birth, sex, age at death, and birth weight (Table 1) .
For mothers who had smoked, the mean (± SD) daily score for cigarette smoking in the prenatal period was 1.25 ± 0.17 for infants exposed in utero only and 2.46 ± 1.26 for infants exposed both in utero and during the postnatal period (p = 0.08 for difference between groups). The cigarette smoking scores for the postnatal period were 1.75 ± 1.5 for infants with only postnatal exposure compared with 2.38 ± 1.15 for infants with both in utero and postnatal exposure (p = 0.3). Three hundred nineteen airways were examined with a mean (± SD) of 10 ± 6 airways per case. Pbm, outer perimeter (Po), outer perimeter minus any accompanying vessel length (Po - v), and the absolute number of alveolar attachment points were not significantly different between the four smoke exposure groups (Table 2) . However, the mean distance between alveolar attachment points was significantly increased in infants exposed to cigarette smoke either exclusively in utero or both in utero and postnatal exposure, compared with those with only postnatal or no exposure (Table 2). The mean distance between attachment points was not related to airway size (Pbm) but was consistently greater in the groups with any exposure in utero (Figure 2) .
The results of the multiple linear regression analyses showed that both cigarette smoke exposure (p < 0.001) and decreased gestational age (p < 0.001) were associated with a greater distance between attachments. Examination of the data showed that the relationship between gestational ages was due mainly to a single case where the gestational age was only 28 weeks. This infant was also the oldest of the group at death (13 months). Therefore, the data were reanalyzed with this infant excluded. The reanalysis showed that postnatal exposure to cigarette smoke was also associated with an increased distance between alveolar attachments (p = 0.025); however, gestational age was no longer significant (p = 0.062). The area of the inner airway wall was increased in the group with only postnatal exposure compared with the group with no smoke exposure (p = 0.02) (Table 3) . The outer airway wall, the area of airway smooth muscle, and the amount of smooth muscle shortening were not significantly different between the smoke exposure groups. The proportion of positively stained elastin to the total lung parenchyma was not significantly different between the smoke exposure groups (Table 4) . The coefficient of variation for airway dimensions ranged from less than 1 to 3.2% with a mean value of 1.8 ± 0.5%, whereas the coefficient of variation for counts of alveolar attachments was 3.6 ± 3.6% with a range varying from 0 to 7%.
In this study there was a significantly greater mean distance between alveolar attachments in intraparenchymal airways (perimeter < 5 mm) in infants exposed to cigarette smoke in utero compared with infants who had no exposure to cigarette smoke in utero. This effect was not seen in infants exposed only during the postnatal period. The thickness of the inner airway wall in infants with postnatal exposure to cigarette smoke was greater compared with infants not exposed to cigarette smoke; however, there were no significant differences in other airway dimensions between the smoke exposure groups. Cigarette smoke exposure was not associated with changes in the parenchymal elastin content, as a proportion of total tissue. We found no effect of airway size on alveolar attachments in any of the exposure groups (Figure 2). Smoking histories were based on reported levels of smoking given by mothers at interviews shortly after the death of their infants. The number of cigarettes smoked might be under-reported, given public sentiment toward maternal smoking. If present, under-reporting was likely to be similar between smoke exposure groups and would tend to lead to an underestimate of the effect of cigarette smoke exposure. However, it would not materially affect the categories that we used or the observed differences between groups. We were unable to assess differences between cases of SIDS and control cases (infants dying from other causes and with a carefully documented smoking history). Nevertheless our results still show an effect of cigarette smoke within this SIDS group. All postmortem examinations and subsequent coronial findings were conducted at the Victorian Institute of Forensic Medicine under the same autopsy protocol and to our knowledge by the same pediatric pathologist. This has reduced the chance of misdiagnosis or crossover of control infants to SIDS. We have previously observed an increase in the thickness of the inner airway wall in airways between 2 and 4 mm Pbm in infants exposed to more than 20 cigarettes a day in utero and during the postnatal period (24). This was not observed in the present study where the mean level of smoking was less than 20 cigarettes per day. When the current data were analyzed in the same size groups as used previously (24) and confined to the same levels of exposure (> 20 cigarettes a day), similar results were observed (data not shown). This may indicate that the level of change in the thickness of the inner airway wall is dose-related. The current data suggest that the increase in the inner airway wall thickness may be associated with postnatal smoke exposure, whereas the increase in distance between attachment points was seen with exposure only in utero or both in utero and during the postnatal period. Young and coworkers (7) examined postnatal lung function in 63 infants at 4.5 weeks of age and found a strong association between increased airway reactivity and exposure to cigarette smoke in utero. Other investigators have also documented abnormalities in neonatal respiratory functions that are associated with maternal smoking during pregnancy (4, 16). In guinea pigs whose mothers had been exposed to cigarette smoke by inhalation only during pregnancy, an increase in airway responsiveness to acetylcholine at Day 27 of the postnatal period and an increase in the mean distance between alveolar attachments were observed (21). This suggests that the mechanisms that result in increased airway responsiveness occur in utero, in response to passive smoke exposure. Saetta and coworkers (23) have previously shown a reduction in the number of alveolar attachment points, an increase in the distance between attachments, and an increase in the percentage of abnormal attachments in cigarette smokers compared with nonsmokers. This reduction was associated with an increase in the score for airway inflammation and with reduced elastic recoil pressure in smokers. Airway responsiveness was not assessed in the study of Saetta and coworkers (23). Petty and coworkers (26) showed that in patients with mild emphysema (i.e., destruction and loss of the alveolar walls), elastic recoil pressure is reduced but this reduction is not associated with airflow limitation. The destruction of alveolar walls observed in patients with moderate to severe emphysema (associated with cigarette smoking) is associated with airflow obstruction and increased airway hyperresponsiveness, in conjunction with elastin degradation (27). These effects are thought to result from the actions of elastases released by activated neutrophils (28). These studies have generally been performed in adults; however, our study examined the effects of in utero cigarette smoke exposure on lung structure in a developing lung system. Possible mechanisms for the decreased number of alveolar attachment points in smoke-exposed infants include destruction of existing alveoli or abnormal growth of alveoli. To assess the effects of cigarette smoke exposure on parenchymal structure the elastin content in the parenchyma was measured. No differences between the groups were seen. Unlike the study by Saetta and coworkers (23), the degree of damage to existing alveolar attachments was not assessed. It was not possible to assess alveolar dimensions or number because the lungs were not fixed in inflation. However, there are a number of studies that show a relationship between airway alveolar attachments and alveolar number. Lamb and coworkers (29) examined 42 patients who underwent surgery for solitary tumors and showed a negative relationship between the mean distance between alveolar attachments and the airspace wall surface area per unit volume and FEV1, as a percentage of the predicted value. Petty and coworkers (30) reported a positive relationship between the mean number of alveolar attachments per bronchiole and FEV1% and a negative relationship between emphysema score and the mean number of alveolar attachments per bronchiole. Nagai and coworkers (31) also showed a negative relationship between the number of attachments per airway circumference and the severity of emphysema. In the present study a maximum difference of 25% in the distance between alveolar attachments was observed between exposure groups, and this is likely to be clinically significant. Linhartová and coworkers (32), in a study of cases of moderate and severe emphysema, showed a 24% difference in the number of alveolar attachments to nonrespiratory bronchioles in emphysematous lungs compared with nondiseased lungs.
Other studies have reported emphysema-like changes and a reduction in the elastic tissue in animal models of nicotine exposure during pregnancy and lactation, compared with control animals (33). Sekhon and coworkers (18) reported a significant increase in alveolar airspace in rhesus monkey fetuses that had been exposed to nicotine in utero. Their study reported an upregulation of An increased distance between alveolar attachments may result from reduced development of alveoli (alveolerization) in utero and during the postnatal period or the loss of alveoli as part of a general process of alveolar destruction that has been described previously by Saetta and coworkers (23). It is believed that the latter is less likely because the present study found that in utero, rather than postnatal, exposure to cigarette smoke was more closely related to distance between alveolar attachments. We have previously reported similar findings in guinea pigs (21). Reduced alveolerization may occur as part of a general reduction in somatic growth (35, 36). In a rat model of cigarette smoke exposure in utero, Collins and coworkers (22) observed a significant growth restriction shown by reduced birth weight and lung weight. They also showed alveolar saccules were fewer in number (p = < 0.005) and larger in size (p = < 0.025), which results in a reduction in the surface area available for gas exchange. We did not observe differences in birth weight between our exposure groups. In utero cigarette smoke exposure could be considered a distinct form of "passive" cigarette smoking in that the fetus is not directly exposed to cigarette smoke. A number of mechanisms may result in the altered airway structure that we have observed in infants and that have been observed in animal studies. These include alterations in placental blood flow, altered cortisol levels, and changes in fetal breathing patterns, all of which may influence fetal lung development (37, 38). Whatever the mechanism, these studies show that maternal smoking during pregnancy does alter airway structure. The alterations in airway structure are those likely to result in excessive airway narrowing in response to irritants encountered during the postnatal period, which may account for symptoms and abnormal lung function.
The authors wish to thank Prof. Nick de Klerk for helpful statistical advice, Ms. Peta Maxwell for preparation of the manuscript, SIDS and Kids Victoria (formally known as Victorian SIDS Foundation) for assistance in accessing the epidemiologic data, and The Victorian Institute of Forensic Medicine for assistance in histologic preparations.
Supported by the National Health and Medical Research Council of Australia. Received in original form October 1, 2001; accepted in final form July 31, 2002
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ABSTRACT
INTRODUCTION
-7 nicotinic cholinergic receptors and increased airway collagen content in the lungs of the newborn (
