| Hirschsprung's disease (HD) is characterized by absence of ganglion cells in intramural plexus including myenteric plexus and submucosal plexus of the stricture segments of the distal colon, and hypertrophy of pre-ganglion parasym-pathetic nervous fibers arranged in haphazard ways, in short is the absence of intrinsic component and the hypertrophy of the extrinsic component of enteric nervous system (ENS) . Recently it is proved that the absence of interstitial cells of Cajal (ICCs) plays important role in the motility dysfunction of HD. ENS with complex 3-dimensional structure were studied by thin paraffin sectioning in the past which could only show the structure of ganglion cells, glial cells or related interstitial cells partly and locally. However whole-mount preparation (WMP) has provided whole view of the distribution of ganglion cells and nervous fibers. The aim of the study was to find a new way to study HD and its allied disorders through the investigation of intestinal innervation and ICCs using the technique of WMP and immunohistochemistry.Materials and MethodsThe specimens of 30 cases of rectosigmoid HD patients were obtained from definitive operations, in which 25 were boys and 5 were girls, and average age was 10 months old. The whole removed specimens were kept for the study. The normal, dilated, transitional, and stricture segments were differentiated for making internal controls according to the morphological and HE examinations. All the specimens were fixed in Zamboni solution (4% , paraformalin, 0.2% picric acid, 0.1M PBS pH7. 3, 900mOsm). The full-thick 2cm x2cm samples wereobtained at the same site of the bowels. WMP was performed under dissection microscope with microsurgical scissors and forceps. The prepared longitudinal muscular layers and submucosal layers, fixed on the self-make silica gel glass, were pre-incubated in 3% Triton X-100 (0.01M PBS) , then stained with S100 (1: 500 ) and c-kit ( 1 :500 ) immunohistochemistry by Strept-Avidin-Biotin-Complex (SABC) method. The stained samples were observed under light microscope and photographs were taken with Olympus camera.ResultsThis method of fixation in the experiment had lessened the contraction of specimens. The segments of HD were differentiate accurately with the combination of whole view and HE examination of ter specimens. And the incubation in Triton X-100 and digestive enzyme complex made the antigen exposed fully. In addition, some adjustments were made of the staining such as the high dosage, the high dilution of primary antibody and the lengthening of reaction time. The self-made silica gel glass was fit for the staining performance of intestinal WMP samples. The SlOO is expressed in the nervous fibers of all the four segments of HD colon. And the nerve fibers were stained rust-like color, c-kit staining was positive in the normal and dilated segments while that in the stricture parts were negative. This method of immunohistochemistry could provide the distribution and morphological features of ENS and ICCs in WMP intesinal samples. There was uniformly distributed network of intramural plexus extending between the longitudinal and circular muscular layers. Nerve fibers develop well with fine branches forming a network. Abundant ganglia were located along these fibers. And there were a uniform network around the crypts of the submucosal layers. In stricture segments there were thick nerve strands running along the long axis of the intestine with few branches. Tne interconnection between those branches was significantly less than that of the normal parts. These thick nerve strands were distributed in a haphazard way. And there was no regular network as in normal segments around the crypts. Tne myenteric plexus of the transitional segments were similar to that of the stricture segments, but the nervous strands gave morebranches and there were a few ganglion cells. The farther it is from the anus, the less hypertrophy the strands is. In the dilated segments the nervous fibers were almost the same as th...
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