In the control samples, a homogenous immunoreactivity to the α-SMA antibody was observed in all of the muscle layers: the muscularis mucosae, circular muscle layer, and the longitudinal muscle layers (data not shown). In the proximal segment of the obstructed site in samples of patients with DA, a moderately to severely hypertrophic area was observed in the circular and longitudinal muscle layers, particularly in the circular muscle layers, compared with those of the distal segments or the controls (Fig. 2A, B, C). The innermost layer of the circular muscle layer in the proximal segments was also thinner and was nearly undetectable in some sections, compared with those in the distal segments or the controls. In addition, in all of the patients, unusual ectopic muscle bundles were found around the submucosal connective tissue near the innermost layer of the circular muscle layers (Fig. 2B). These smooth muscle bundles originated from the muscle bundle in the thin innermost circular muscle layer. In addition, the muscularis mucosae were also found to be hypertrophic (Fig. 2C). In contrast, in the distal segments of the obstructed site in DA, the staining pattern of α-SMA antibody was similar to that observed in the control samples (Fig. 2D, E, F). There was no significant difference in quantitative analyses of the thickness of the circular muscle layers, the longitudinal muscle layers, and the muscularis mucosae between the distal segments of obstructed sites and control tissues (P = 0.646, P = 598, and P = 0.395, respectively). In contrast, quantitative analyses revealed a significant difference in the thickness of the muscularis mucosae of the proximal segments compared with the distal segments of the obstructed site (proximal 36.46 ± 13.51 μm, distal 12.52 ± 6.06 μm, P < 0.001, Table 2). Quantitative analyses also revealed a significant difference between the circular muscle layers of the proximal and distal segments of the obstructed site (proximal 492.91 μm [range 263.19–733.16 μm], distal 164.94 μm [range 135.61–199.37 μm], P < 0.001, Table 2). There was also a significant difference between the proximal and distal segments of the longitudinal muscle layers (proximal 317.64 μm [range 110.73–369.18 μm], distal 96.28 μm [range 73.83–121.20 μm], P < 0.001, Table 2).
In the control samples, c-kit-positive cells were observed between the intermuscular space of the circular and longitudinal muscle layers, particularly around Auerbach plexus (data not shown). A small number of positive cells also were localized to the circular and longitudinal muscle layers and in the innermost layer of the circular muscle layers. In the proximal segment, the number of c-kit-positive cells was markedly decreased (Fig. 3A, B). Moreover, in some samples, the ICC were barely detectable, even around the myenteric plexus. The positive cells were bipolar in shape. Macrophage-like cells positive for c-kit staining were also observed within the muscularis propria and the submucosal area. Unlike the proximal segments, the distribution pattern and c-kit immunoreactivity in the ICC of the segment distal to the obstructed site were similar to those of the control samples (Fig. 3C, D; P = 0.133). These cells formed a network with cell-cell contacts and their shape was multipolar. The quantitative analyses revealed a significantly smaller c-kit-positive area in the proximal segments compared with the distal segments of the obstructed site (proximal 933.45 μm2/mm2 [range 297.87–3149.62 μm2/mm2], distal 12006.42 μm2/mm2 [range 2473.79–22458.1 μm2/mm2], P < 0.001, Table 2).
As a part of the normal growth process in the embryo from the fourth to the seventh week of gestation (embryo length 8.6–14.5 mm), the epithelial cells of the duodenum begin to proliferate and completely plug the lumen (solid phase). Therefore, between the 15- and 65-mm stages (from the end of the 7th week to the 12th week of gestation), a process of vacuolization, coalescence of vacuoles, and recanalization occurs. DAs, stenosis, and intraluminal webs are believed to be caused by insults resulting in recanalization failure during the lengthening and rotation of the primitive foregut. A resultant obstruction is then believed to cause the dilatation in the proximal duodenum (20–22).
Using histochemical techniques, we have herein provided the first study of the potential differences in abnormalities of the smooth muscle, nervous system, and ICC between the segments proximal and distal to the obstructed site in samples from patients with DA. These 3 enteric components, which are involved in peristaltic activity, were immunohistochemically analyzed.
Although the etiology of DA is not the same as that in other IAs, the morphologic changes of the intestine in DA are thought to be similar. The morphologic change of the proximal segment seems to depend on the postobstructive dilatation during the fetal period. Similarly, in other forms of IA, morphologic change in the proximal segments also depends on the change after the formation of the obstruction.
In an experimental model of IA, several studies showed that the dilatation of the proximal segment induces the involution and lysis of the ganglion cells after initial hyperplasia of the myenteric ganglia has occurred and irreversible distension continues to develop (23). Another possible cause of nerve alteration could be ischemic influence during fetal life through vascular disruption as shown in IA models (19,23). In the present study, we observed that in the area in which muscular layers are severely dilated, the distribution of ganglion and plexus is also less than that of the area with moderately dilated muscular layers. This finding supported a previous IA study (19), which observed marked abnormal neuronal changes in the distended proximal segments. In contrast, the neuronal distribution in the distal segment was close to normal in these studies, as in our study of human DA. The influence of muscular distention on the neuronal cell alteration is also shown in our previous case report of IA (24). In this case report, an improvement in numbers of neuronal cells and fiber distribution between primary operation of IA at 2 days after birth and second operation for the reconstruction of the dilated proximal segment at 6 months of age was observed (24). These abnormalities are probably a result of the developmental delay in the nervous system or of the dilatation of the proximal segment in DA, as mentioned in IA studies, which also found an alteration of enteric nerves in a severely dilated area of the proximal segment (11).
When examining smooth muscle morphology in the present study, we observed that the muscle layers in the proximal segments of the obstructed site are moderately to severely hypertrophic, unlike in the distal segments, which are indistinguishable from those of the control samples. This muscular hypertrophy has been well documented and results from a compensatory process following obstruction during the prenatal period, and it is localized exclusively to the circular musculature of the distended proximal intestinal segment (23,25,26). As mentioned in the literature, dysmotility of the intestine is often encountered in a severely dilated muscle of the segment proximal to the obstructed site. To prevent recurrence of the dilated segment, the markedly dilated duodenum must be completely reconstructed surgically. Even after reconstruction surgery, however, a proximal segment consisting of a hypertrophic area remains because of the difficulty in visualizing the healthy area and minimizing the resected segment. Previous investigators have shown that the contractile pressure in the dilated proximal intestine of an IA model is lower than that in normal intestines. Moreover, physiologic studies of humans and animal models of IA have also shown a decrease in the motor activity in both the proximal and distal segments. These studies suggest that low contractile pressure was involved in the postoperative dysmotility (17). In addition, the existence of unusual muscle bundles, which have an oblique configuration, likely contributes to the disturbed bowel rhythms. Similar findings have been described in our previous study of cases with IA (17). Therefore, the existence of both hypertrophy of the circular muscle layers and unusual muscle bundles in the submucosal layers likely contributes to the development of motility disorders later in life, even after a successful initial operation. Additional procedures, such as intestinal plication or tapering the dilated intestine, are sometimes needed to produce efficient peristalsis of the proximal intestines.
Of particular interest with regard to the occurrence of unusual muscle bundles, our previous study of human IA showed that the smooth muscle bundles which emerged from the innermost layer of the circular musculature could be of either an oblique or vertical orientation to the long axis of the intestines, stretching toward mucosae, forming a coarse, irregular meshwork in the submucosa (17). Based on the chronologic view of our previous study of myogenesis in chick embryos, it is supposed that these muscle bundles may be a remnant of early developmental stages during the formation of the muscularis mucosae (27). Another study also proposed that a possible explanation for this phenomenon is that the ectopic muscle bundles are a secondary reaction of muscle cells to the chronic and progressive dilatation of the proximal segments. Moreover, these bundles also could indicate a secondary regressive reaction, which the proliferating reaction of regressive smooth muscle cells undergo when they first emerge in the inner layer of the circular muscle layer; thereafter, these smooth muscle cells protrude from the inner layer of the circular muscle layer to the layer of muscularis mucosae, according to the rules of normal development. The real causality regarding the development of ectopic muscle bundles in the proximal segment of the DA remains unclear, and the proposed reasons are based mostly on experimental IA, which is probably not appropriate for DA because of differences in the underlying etiology (27,28).
The small intestine exhibits rhythmic and phasic contractions that form the basis for propagating and segmental contraction. These rhythmic and phasic contractions are generated by the ICC surrounding the myenteric plexus (ICC-MY) between the longitudinal and circular muscular layers and the ICC lining the septa separating the CM bundles (ICC-SEP). Each ICC-MY and ICC-SEP generate a spontaneous electrical slow-wave pacemaker activity that is actively propagated through the ICC network, in addition to regulating smooth muscle membrane potential and mediating enteric neurotransmission. The loss or abnormalities of ICC have been described in a variety of human motility disorders, including hypoganglionosis, Hirschsprung disease, and jejunal and ileal atresia but not in DA (29–31). A reduction in the distribution of pacemaking cells has also been reported in a dilated colon of 2 neonates with atresia of the colon (32). In the present study, we observed that the proximal segments of the obstructed site showed not only a decreased immunoreactivity to c-kit protein but also a markedly reduction in the number of ICC. The distribution of ICC also showed a discrete distribution without connection of ICC cells and a bipolar shape. This finding may be associated with the reduction of pacemaker activity and enteric neurotransmission, thus resulting in hypoperistalsis of the proximal segments. Several physiologic studies also showed that peristalsis and spontaneous contraction were disturbed in the proximal segments. Therefore, the abnormality of ICC in the proximal segments may lead to postoperative dysmotility in DA.
In the present study, abnormalities of the enteric nerves, smooth muscle cells, and ICC were predominantly observed in the proximal segments. It has been pointed out that a tight connection existed between the ICC, enteric nervous system, and the smooth muscle to produce a synchronized and sustainable contraction of the duodenum (30). Therefore, the observed abnormalities in these 3 enteric components of the proximal duodenum suggest that duodenal motility disorders may occur later in postnatal life.
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Keywords:Copyright 2012 by ESPGHAN and NASPGHAN
α-smooth muscle actin; duodenal atresia; interstitial cells of Cajal; neural cells