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Infantile hypertrophic pyloric stenosis (IHPS) is a common condition requiring surgery in the first few months of life(1). It is characterized by hypertrophy of the pyloric muscle, causing pyloric channel narrowing and elongation. The pathogenesis of IHPS is not fully understood. Heredity and family predisposition have been implicated as important factors in the pathogenesis of IHPS, but no consistent morphologic abnormality has been identified(1). Abnormalities of pyloric innervation, particularly nitrergic innervation, have been reported in IHPS(2–4). It has been demonstrated that the relaxation mechanism of the pyloric smooth muscle appears to be dependent on nonadrenergic noncholinergic inhibitory innervation, which is mediated by nitric oxide (NO) and some neuropeptides(5). Several investigators have reported absent or markedly reduced NO synthase as well as low levels of neuronal NO synthase (nNOS) mRNA in hypertrophic pyloric stenosis(3,4,6). Abnormalities of extracellular matrix proteins(7,8), smooth muscle cells(9), and growth factors(10,11) have also been reported in IHPS.

Studies using ultrasonography have shown markedly increased pyloric muscle mass in IHPS(12–14). The increase in smooth muscle mass may be due to hypertrophy (an increase in cell size), hyperplasia (an increase of cell number), or an increase in extracellular matrix. Hypertrophy of smooth muscle cells and increase in extracellular matrix are reportedly responsible for hypertrophic pyloric muscle in IHPS(7–9). However, little is known regarding the role of hyperplasia in increasing muscle mass in IHPS.

Cell proliferation (mitotic) activity is an important indicator of cell hyperplasia. The MAb Ki-67 reacts with human nuclear cell proliferation-associated antigen that is expressed in all active parts of the cell cycle and is well established as a marker of cell proliferation(15,16). The MAb MIB-1, prepared against recombinant parts of the Ki-67 antigen, reacts in routinely fixed, paraffin-embedded tissues(17). MIB-1 immunostaining has been reported suitable for assessing the proliferative activity, because it has less background staining and more uniform and stronger positive signals(16). In the present study, we investigated proliferative activity of smooth muscle cells, using MIB-1 immunohistochemistry, and we also measured smooth muscle cell number and size, using an image analyzer to enable us to understand the mechanism of pyloric muscle enlargement in IHPS.

MATERIALS AND METHODS

Tissue preparation. Full-thickness muscle biopsy specimens were obtained from 18 IHPS patients (age range 15-101 d, mean 45.3 d) at pyloromyotomy and from 11 age-matched controls (age range 14 d to 4 months, mean 50.2 d) without gastrointestinal disease at autopsy. Autopsy was performed within 12 h of death, and no autolysis was observed in the tissues. Controls were born at term, and the causes of death included sudden infant death syndrome, pneumonia, and encephalitis (Table 1). There was no evidence of malnutrition in controls. The specimens were immediately fixed in 10% formalin and embedded in paraffin. Three-µm-thick paraffin sections were cut and mounted on polylysine-coated glass slides. The study was approved by the Research Council of our institution.

Table 1 Data of Control Patients
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Immunohistochemistry. Sections were stained by the standard streptavidin-biotin immunoperoxidase method (Universal LSAB 2 kit, Dako, Denmark). A MAb MIB-1 (Immunotech, France) was used as first antibody at a dilution of 1:40, at 4°C, overnight. For good immunostaining, the de-waxed sections were placed in 10 mM citrate buffer (pH 6.0) and heated in a microwave oven twice for 5 min before staining.

Evaluation of proliferative activity. MIB-1 immunoreactivity was confined to the nucleus of proliferating cells. At least 10 fields were chosen at random, and more than 1000 muscle cells were counted at high magnification (×400) in each case. The PI was calculated as the percentage of positive cell nuclei.

Smooth muscle morphology. In addition to routine hematoxylin-and-eosin staining, sections were stained with Victorian blue Van Gieson stain to distinguish smooth muscle from connective tissue. The method for morphologic analysis of cell number and size was similar to that of Blennerhassett et al.(18) and Srinathan et al.(19). Microscopic images were scanned with a Zeiss Axioskop microscope with color video camera and were analyzed with an image analyzer, Interactive Image Processing System (IPS ver. 4.01, Alcatel TITN Answare, Cedex, France). The area of each muscle bundle was measured in five randomly chosen regions in the circular muscle layer. The number of nuclei in the muscle bundle was determined, and mean cell size was estimated by dividing the total area of each muscle bundle by the number of nuclei in that bundle, and was expressed in µm2/nuclei.

Statistics. Results were expressed as mean ± 1 SD. The statistical significance between groups was determined by unpaired t test. Linear regression analysis was used to test the significance of correlations. To detect a significant difference, p < 0.05 was chosen.

RESULTS

MIB-1 immunohistochemistry. MIB-1 immunoreactivity was confined to the nucleus of smooth muscle cells. Positive nuclei were easily detectable in each case (Fig. 1, A and B). The mean PI in IHPS (9.6 ± 5.7%) was significantly higher than that in controls (1.3 ± 1.2%) (p < 0.001) (Fig. 2). The correlation between proliferating index and age at operation in IHPS is shown in Fig. 3. There was a significant inverse correlation between PI and age at operation (r = -0.72, p < 0.002). Patients under the age of 40 d demonstrated significantly higher mean PI (13.8 ± 3.1%) than those older than 40 d (6.1 ± 5.6%) (p < 0.01).

Figure 1
figure 1

Immunostaining of Ki-67 antigen using MAb MIB-1 (× 200). MIB-1 immunohistochemistry was confined to the nucleus, and positive nuclei were easily detectable in each case. (A) In normal pyloric muscle, a few MIB-1-positive cells were observed. (B) There were many MIB-1-positive smooth muscle cells in pyloric muscle in IHPS patients.

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Figure 2
figure 2

Mean PI in IHPS and normal controls. The mean PI in IHPS was significantly higher than that in controls (p < 0.01).

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Figure 3
figure 3

The relation between proliferating index and age at operation. There was a significantly inverse correlation between PI and age at operation in IHPS patients (r = -0.72, p < 0.01).

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Smooth muscle morphology. In the circular layer of pyloric muscle, there were significantly more cells per muscular bundle (hyperplasia) in IHPS (240.6 ± 129.4) than there were in controls (134.1 ± 49.8) (p < 0.05) (Fig. 4). Estimated smooth muscle cell size in IHPS was significantly larger (hypertrophy) in IHPS (298.5 ± 59.0 µm2) than in controls (154.3 ± 21.5 µm2) (p < 0.01) (Fig. 5).

Figure 4
figure 4

Smooth muscle cell number per bundle in IHPS and normal controls. In the circular layer of pyloric muscle, there were significantly more cells per muscular bundle in IHPS, compared with controls (p < 0.05).

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Figure 5
figure 5

Smooth muscle cell size in IHPS and normal controls. Estimated smooth muscle cell sizes in IHPS were significantly larger in IHPS than in controls (p < 0.01).

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DISCUSSION

Morphologic studies and measurement by ultrasonography have shown a marked increase of pyloric smooth muscle mass in IHPS(12–14). Pyloric muscle in IHPS is reported to be more than twice as thick as that of normal controls. An increase of smooth muscle volume is known to occur in various pathologic states in various organs, such as blood vessels during hypertension(20), bladder muscle in outlet obstruction(21), and airway smooth muscle in asthma(22). In these conditions, both hypertrophy and hyperplasia are reported responsible for the smooth muscle growth. Similar smooth muscle changes reportedly occur in the gastrointestinal tract. Extensive hypertrophy and hyperplasia associated with an increased amount of mitosis was observed in the intestinal smooth muscle after partial intestinal obstruction(23). However, very little is known regarding the possible role of hyperplasia in causing an increase in pyloric muscle mass in IHPS. In the present study, smooth muscle morphometry with an image analyzer demonstrated that both the number of cells per muscle bundle and smooth muscle cell size were about twice that of normal controls.

Recently, as the result of a morphologic study using electron microscopy, it was reported that pyloric smooth muscle cells from patients with IHPS are often in a proliferative phase, with large amounts of dilated rough endoplasmic reticulum and a lower proportion of contractile filaments(9). This finding suggests that increased proliferative activity may contribute to an increase in muscle mass in IHPS. To evaluate the role of hyperplasia in the pathogenesis of IHPS, we performed the first quantitative evaluation of proliferative activity in pyloric muscle in IHPS. Our results showed that the percentage of MIB-1-positive pyloric muscle cells is significantly higher in IHPS than in normal controls, suggesting that increased proliferative activity of the smooth muscle plays an important role in the increasing pyloric muscle mass in IHPS. Our findings, therefore, indicate that both hypertrophy and hyperplasia contribute to the pyloric muscle thickness in IHPS.

A striking finding in the present study was the significantly inverse correlation between PI and age at operation in IHPS patients. Patients less than 40 d old demonstrated a significantly higher mean PI than did patients older than 40 d. This may indicate that proliferative activity becomes normalized within a few months after birth. Studies in which ultrasonography was used have revealed that pyloric muscle regresses after medical treatment or surgery. Nagita et al.(24) reported the regression of pyloric muscle after medical treatment. All of the infants who recovered after receiving i.v. atropine experienced normalization of pyloric muscle caliber 4-12 months after treatment. Sauerbrei et al.(12) and Okorie et al.(13) measured pyloric muscle size after pyloromyotomy, using ultrasonography, and showed that hypertrophy regresses at variable rates, but a normal size is consistently reached by 12 wk postoperatively. Our finding of reduced proliferative activity in older infants with IHPS may explain the regression of hypertrophic pyloric muscle reported in these studies, as well as the spontaneous regression of IHPS observed in some infants(25).

Proliferation and growth of cultured smooth muscle cells can be stimulated by a number of peptide growth factors, such as insulin-like growth factor-I (IGF-I), platelet-derived growth factor (PDGF), epidermal growth factor, and transforming growth factor-β1 (TGF-β1)(26–28). It is thus possible that the local production of these peptide growth factors is involved in the development of pyloric muscle hypertrophy and hyperplasia in IHPS. However, it is not clear which growth factors account for pyloric muscle enlargement in IHPS. Recent studies from our laboratory have shown that IGF-I, PDGF-BB, and their receptors, as well as TGF-β1, were markedly increased in hypertrophic pyloric muscle in IHPS(2,10,11). It is interesting to speculate that the upregulated local IGF-I, PDGF, and TGF-β1 systems may induce altered autocrine growth regulation in pyloric smooth muscle cells, contributing to the development of pyloric muscle hypertrophy and hyperplasia in IHPS. Although many factors have been identified that influence the growth of cultured smooth muscle cells, the precise signals that mediate hypertrophic versus hyperplastic growth of smooth muscle cells in vivo have not been defined; neither is it clear how the control of these two processes is related.