INTRODUCTION

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The underlying pathophysiological cause of the sudden infant death syndrome (SIDS) remains uncertain. Several pathologic abnormalities have been identified in several organ systems including the nervous and respiratory systems, and the pulmonary circulation in some infants dying of SIDS; however, the direct relationship between these changes and the actual mechanisms of death remains unclear (1). Previous studies examining the airways of infants who died of SIDS have failed to identify a consistent pulmonary pathological feature. Baxendine and Moore (9) found an increase in eosinophil numbers in the lungs of SIDS infants while Howat and coworkers (10) found a T lymphocyte-mediated pulmonary inflammatory response present in the parenchyma and in the peribronchial area.

Small airway closure has been suggested as a pathophysiological abnormality in SIDS (11). We postulated that exaggerated small airway closure could result from increased airway smooth muscle mass. We therefore examined the structure of small airways in infants who died of SIDS with particular attention to the amount of airway smooth muscle and compared these findings with findings from age-matched control infants.

	METHODS

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In the state of Victoria, cases of sudden unexplained death are reviewed by the Victorian Institute of Forensic Pathology. A postmortem examination is performed in all cases by an experienced pediatric pathologist and diagnosis of SIDS is only made if the postmortem examination and circumstances surrounding death do not suggest an alternative cause. Routinely, sections of several organ systems are taken and stored in paraffin in the event that further review of the case is required.

The investigators of this present report obtained permission from the Victorian Institute of Forensic Pathology to examine stored lung blocks from infants who had died of SIDS. A total of 90 cases of SIDS were made available. From each lung block one 5-µm section was taken and stained with hematoxylin and eosin. Slides were examined using a video-linked microscope Leica Laborlux D (Leica, Wetzlar, Germany), with the image being projected onto the monitor screen of a 486 DX computer. Images were assessed using the color image analysis program Quantimet 500+ (Leica Cambridge Ltd, Cambridge, UK). Airways were subjected to standard airway morphometric analysis, as described subsequently.

Control Cases

Permission was obtained from the Victorian Institute of Forensic Pathology to access the records of infant deaths reviewed by the Institute from 1991. In age-matched cases where no previous pulmonary pathology was likely, sections were obtained from the stored lung blocks as described in the previous section. Cases in which significant pulmonary damage was likely to be a feature of the cause of death such as pneumonia, congenital heart disease, or history of premature birth, were excluded. Cases included were those in which previously healthy children had died suddenly, e.g., as a result of motor vehicle accidents or homicide. A further eight control cases with similar causes of death were included from a study in Western Australia by three of the investigators (A.J., N.C., J.E.). Sections of tissue had been obtained in a standardized way as part of a protocol for the pathological examination of all (including SIDS) deaths. Measurements of airway dimensions in these cases were performed by the same investigator (J.E.) using identical techniques.

Airway Morphometry

Morphometric analysis was performed on all airways that were cut in cross section, or near cross section. To avoid errors arising from tangential sectioning, airways with a short to long axis ratio of less than 0.6 were excluded from subsequent analysis (12). Four perimeters and areas were measured: (1) the internal perimeter (P_i) and area (A_i), defined by the luminal border of the epithelial; (2) the perimeter (P_bm) and area (A_bm), defined by the outer border of the basement membrane; (3) the perimeter (P_mo) and area (A_mo), defined by the outer border of the smooth muscle; and (4) the total perimeter (P_o), and area (A_o), defined by the outer edge of the adventitia surrounding the airway (Figure 1). In addition, the area of smooth muscle was traced.

View larger version (108K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   Figure 1.   (A) Photomicrograph of an airway from an infant who died of SIDS. The smooth muscle is clearly evident in the airway wall. (B) Schematic representation of an airway showing dimensions measured in this study. The airway wall is divided into inner and outer areas by the outer perimeter of the airway smooth muscle. Standard nomenclature as described by Bai and coworkers (12) has been employed.

Airways that showed > 50% epithelial detachment or branching were excluded; however, where smaller sections of epithelium were missing, the border was interpolated between two intact areas (12).

Data Analysis

The areas defined by the perimeters were used to calculate wall areas, i.e., the epithelial wall area (Wa_epi = A_bm  A_i), the inner wall area (Wa_i = A_mo  A_i) the outer wall area (Wa_o = A_o  A_mo), and the total wall area (Wa_t = A_o  A_i) as has been previously outlined by Bai and coworkers (12). Because the airway wall areas, and proportion of smooth muscle within the airway wall, are a function of airway size, the areas were divided by the basement membrane perimeter (12, 13).

Statistics. To assess the effects of SIDS, age, and sex on airway dimensions, a weighted least squares model was applied to the data. In brief, airway wall areas and smooth muscle area data were linearized by plotting the square root of area against the P_bm for each of the airway wall compartments. Using linear regression analysis, the data for each case were plotted and the slope and intercept, together with their standard errors (SE) calculated (14).

The individual slopes and intercepts are weighted by calculating (1/SE²) (15). The slopes and intercepts were then regressed, adjusting for the effects of exposure (SIDS or no SIDS), sex (male or female), and age (mo). This is designed to adjust for the effects of airway growth when comparing airway dimensions. A probability of < 5% is considered significant.

Coefficient of variation. Intraobserver error was assessed by calculating the coefficient of variation, for measurements of airway dimensions made on 10 different measurements on separate occasions (15). All measurements were made by the one observer who was blinded to the case classification.

	RESULTS

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Results were obtained from 57 infants who had died of SIDS, 36 males and 21 females (age 4.8 ± 3.1 mo [mean ± SD]), and 21 control infants, 13 males and 8 females (age 7.2 ± 5 mo [mean ± SD]) (Figure 2). In a further 33 cases of SIDS no airways suitable for morphometric analysis were seen on the obtained section. All control infants had had a postmortem examination performed by a pediatric pathologist and in all cases the cause of the sudden death was not associated with evidence of significant lung pathology. The causes of death are outlined in Table 1. There was no significant difference in the age or sex distribution between the control groups from Victoria (age 7.6 ± 4.7) mo; 9 males, 4 females) and Western Australia (age 6.5 ± 5.8 mo; 4 males, 4 females), nor between the group of SIDS cases included in this study and those where morphometric assessment was not possible. There was no significant difference in any of the measured morphometric parameters between the two control groups and their results are thus pooled for comparison with the SIDS cases.

View larger version (14K): [in this window] [in a new window]   Figure 2.   Age distribution of patients studied. Age is expressed in months for both SIDS cases (solid bars) and control cases (clear bars). In the SIDS group one patient was older than 12 mo of age (17 mo); in the control group three patients were older than 12 mo of age (one 17-mo-old and two 18-mo-old infants).

Airway Morphometry

The coefficient of variation for airway measurements ranged from 0.44% to 3.7% with an overall mean value of 2% ± 0.7%.

A total of 587 airways from infants dying of SIDS were examined and compared with 227 airways from control infants. The intercepts of regression lines were similar between case groups. There was a significant effect of SIDS and sex on the slope of the airway smooth muscle area plot. Cases of SIDS had greater smooth muscle area for any given airway size and males had a steeper slope than females (Figure 3). There was no difference between SIDS and control cases for inner airway wall area, outer airway wall area, or epithelial thickness; however, there was an independent effect of sex on the slopes for the inner and outer wall area, with males having a steeper slope for inner wall area and females a steeper slope for outer wall area. This may be due to the effect of the larger airway smooth muscle area in the male infants.

View larger version (23K): [in this window] [in a new window]   Figure 3.   Plot of the square root of area of airway smooth muscle against basement membrane perimeter (Pbm) from 57 infants who died of SIDS (open squares) and 21 age-matched control infants who died suddenly from other causes (closed circles). There is a significantly increased amount of airway smooth muscle in the SIDS group compared with the control group (p < 0.01).

	DISCUSSION

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This study shows that airways obtained from infants who died of SIDS have a larger proportion of smooth muscle in their airways than age- and sex-matched infants dying from other causes. SIDS describes the unexpected and unexplained death of an apparently previously well infant (16). The diagnosis is one of exclusion and is made after postmortem examination and a detailed assessment of the environment in which death occurred. Although many postmortem examinations have been performed on infants whose death has subsequently been attributed to SIDS, there is no uniform pathological feature that aids in the diagnosis of this condition. The postmortem examination in cases of SIDS is, in many ways, to exclude other causes of death such as undiagnosed infections, e.g., meningitis or pneumonia. By examining tissue obtained at postmortem from infants dying of SIDS, many investigators have tried to identify the pathophysiological mechanism or mechanisms underlying the cause of death. Most interest has centered on two organ systems, the neurological (1) and respiratory systems (5, 6). Investigators have postulated abnormalities of the neural centers responsible for the control of breathing or have examined pulmonary tissue for evidence of altered structure or function. Haque and coworkers examined 25 cases of SIDS and 18 control cases in their study on airway morphometry in SIDS (17). They examined over 1,000 airways, predominantly membranous bronchioles from SIDS cases and found a significantly increased index of airway wall thickness compared with control cases. Smooth muscle content of the airway wall was not measured.

In the present study, the thickness of the airway wall and the area occupied by airway smooth muscle were measured directly. We did not find any significant difference in airway wall thickness between SIDS cases and control cases, contrasting our results with those of Haque and coworkers. We have previously examined the airways from 38 infants who died of SIDS and shown that in the 19 infants who were exposed to high levels of cigarette smoke (maternal smoking > 20 cigarettes a day) both in utero and postnatally there was a significantly increased inner airway wall thickness when compared with the 19 infants who had died of SIDS whose mothers did not smoke either during pregnancy or postnatally (18). In this study we also examined the amount of smooth muscle in the airways and found that there was no difference between smoke-exposed and nonsmoke-exposed groups of infants who died of SIDS. In the study by Haque and coworkers no allowance was made for the number of smoke-exposed infants in the SIDS group, and it is likely given our recent findings that the finding of increased inner airway wall thickness in the SIDS group as a whole in Haque's study reflects the number of smoke-exposed infants in their study group. Further, given that we previously have not shown any difference in airway smooth muscle mass within a group of SIDS infants when infants exposed to high levels of smoke are compared with those infants not exposed to any maternal smoke, the findings of this present study of increased airway smooth muscle in lungs from infants who died of SIDS compared with control infants would suggest that this difference is not smoke-related effect and may indeed reflect a SIDS effect (18).

In the present study, we were given access to stored lung blocks from the coroner's court. We therefore had no control over the site of sampling of the lung, and further, no control over the degree of lung inflation that occurred before tissue sampling. These limitations prevent us from using the alternative form of tissue morphometry, stereology, as pioneered by Cruz-Orive, Gundersen, and Weibel and more recently reviewed by Bolender (19). This technique permits assessment of epithelial and smooth muscle volume providing that full lung inflation to TLC is achieved before lung sectioning commences. From this starting point, isotropic uniform random sections can be obtained by vertical sectioning of the lung, thereby ensuring that all orientations and positions of airways are sampled. Point counting using a cycloid grid placed over the sections permits a very detailed and accurate assessment of epithelial and smooth muscle volume. This detailed and precise method of measurement may well provide a more accurate way of measuring airway smooth muscle mass than that employed by us in this present study, and ideally our results will need to be confirmed by future studies using this technique. In reality, however, the ability of researchers to ensure full lung inflation at a coronial postmortem is not only difficult to achieve as a matter of pure practicality, but in this era is further limited by both ethical and legal constraints. The ability of researchers to obtain any tissue from a postmortem on an infant dying of SIDS is in many areas, including our own state of Victoria, now very tightly restricted by legal directives. In this climate the use of the more detailed methods of stereology, while desirable, is for most cases unachievable.

The present study has shown that airway smooth muscle is increased in infants who die of SIDS. What effect could this increase in airway smooth muscle produce? For a given airway size, an increase in the degree of airway smooth muscle may predispose to excessive airway narrowing owing to increased force development during stimulation (22). Increased activation of smooth muscle, independent of any actual increase in airway smooth muscle content, has been shown by Moreno and colleagues to predispose to small airway closure (22). Martinez (11) has postulated that small airway closure could be associated with SIDS, and our present findings suggest that in some cases this mechanism may be an important factor in the cause of death. Although this study has shown an increase in airway smooth muscle mass within the airway wall of infants who die of SIDS, it is not possible to draw conclusions from these data regarding the level of maximal smooth muscle contraction. To answer these questions it would be necessary to obtain fresh tissue for in vitro studies as well as submitting resected tissue to a maximal contractile stimulus before sectioning to allow for analysis of maximal contractile ability (13).

What is the underlying mechanism for this observed increase in smooth muscle? The present study is unable to answer whether the observed increase in airway smooth muscle is a primary effect that directly produces the pathophysiological abnormalities resulting in sudden death which leads to SIDS, or whether the alterations in airway smooth muscle are secondary to other factors that may cause death in SIDS through other pathophysiological mechanisms. While unrecognized genetic factors may be responsible for producing this increased amount of airway smooth muscle, environmental factors may also be responsible. It is further possible that the observed increase in airway smooth muscle in the SIDS lungs in this group is reflecting pathological mechanisms occurring elsewhere. Chronic upper airway obstruction has frequently been postulated to be an important mechanisms in the pathogenesis of SIDS (23). Although Naeye has previously described an increase in smooth muscle content in pulmonary arteries in infants dying of SIDSa finding also seen in other forms of chronic upper airway obstruction such as obstructive sleep apnea (OSA), there has never been a description from any other cause of chronic upper airway obstruction (including OSA) of increased airway smooth muscle. In the absence of any precedence from other conditions, as well as the difficulty in explaining such a relationship on physiological principles, it is hard to postulate that the observed increase in airway smooth muscle seen in this study could be secondary to chronic upper airway obstruction.

In summary, this study has shown that infants who die of SIDS have a higher proportion of airway smooth muscle in their small airways than age-matched infants who die suddenly from causes no associated with underlying cardiorespiratory pathology. The increase in smooth muscle may contribute to excessive airway narrowing which, along with other factors such as immature ventilatory control mechanisms, may result in sudden death, but the precise significance of this finding remains unknown.