| Objective:Hischsprung’s disease (HD) is commonly digestive malformation in pediatric surgery with pathological manifestations of the absence of ganglion cells in diseased colonic segments, which is also regarded as a typical neuronal intestinal malformation. Because of the absence of ganglion cells, intestines peristalsis is lost in the diseased segments and then the megacolon in the proximal end colon is caused by the excessive peristalsis. Although there are some theories on the pathogenesis of HD, such as the theory of genes abnormality (such as RET, SOX10, GDNE, EDNRB and ECE1), the theory of intestinal micro-environment abnormality and the theory of intestinal pacing cells abnormality, the actual etiology is still uncertain, which causes the difficulties of diagnosis, differential diagnosis and classification of HD.In this study, from the view of abnormality of nerve synapse, we firstly investigated the expression of Neuroligins protein in the inters titial cells of cajal within the myenefic region (ICC-MY) in the enteric nervous system (ENS) of HD, then further investigated the expression level of Neuroligin-1, a protein subunits and the related Glutamate (Glu) in the ENS and serum of HD, and discuss the pathogenesis and clinical significance of HD.Methods:Colonic samples and blood samples were collected from 90 HD children (short-type HD 50 cases and long-type HD 40 cases) treated and pathologically diagnosed in the Department of Pediatric Surgery of Qilu Hospital, Shandong University from 2010 to 2013.Another 50 blood samples were collected from 50 children with indirect inguinal hernia (IIH) as the Control group. In order to reduce error, nutritional status (serum total protein, serum albumin, hemoglobin, blood urea nitrogen, body length and weight), age and basal metabolic index (BMI) among groups were controlled similar and no statistical difference.For colonic samples, stenotic segments (aganglionic segments), transitional segments and proximal end of dilated segments (normal segments, ganglionic segments) were collected from the surgically excised colon of children with HD and parts fresh tissues were stored at -80℃ for Western-blot and qRT-PCR for the quantitative analysis and comparison of neuroligins, neuroligin-land Glu. Parts tissues were used to prepare Longitudinal muscle with adherent myenteric plexus (LMMP) which were used to detect the expression of neuroligins, neuroligin-1 and Glu in ENS by immunochemical staining and also for the isolation and culture of ICC-MY; Parts tissues were used to prepare paraffin-embedded sections which were used to detect the expression of neuroligin-1 among different segments by immunohistochernical staining; Magnetic-activated Cell Sorting (MACS) and flow cytometry (FCM) were used for the isolation and count of ICC-MY.Serum samples of HD and IIH were collected to detect the concentration of serum Glu by ELISA which may provide a valuable adjunct measure for diagnosing of HSCR or for determining the classification of HD (long type or short type HD)The averaged data were summarized as the mean±SD, and P values less than 0.05 were considered to be significant. For comparisons of two groups, unpaired T tests was performed. One-way ANOVA and the Tukey’s test were performed to compare three groups. All the Statistical analysis was performed by SPSS 11.5 and GraphPad Prism 5.0 software.Results:(1) ICC-MY were identified to be expressed as network structure on LMMP by immunohistochemical staining and no obvious disruptions of ICC-MY network were showed in aganglionic segments. (2) On LMMP neuroligins were expressed in ICC-MY also as network structure and neuroligins network was disrupted in aganglionic segments which was different from the ICC-MY network. (3) After the dissociation, culture, isolation and purification of ICC-MY from fresh LMMP, FCM showed that ICC-MY with high purity could be obtained and there was no statistical difference of the ICC-MY percentages among aganglionic, transitional and ganglionic segments.(4) ICC-MY with the shape of fusiform or triangular were identified by immunofluorescence labeling which showed a successful purification of ICC-MY.(5) By Western-blot with a inner reference of c-kit, the specific antibody of ICC-MY, neuroligins were expressed significantly in ICC-MY of ganglionic colonic segment, moderately in transitional segment, and obviously downed-regulated in aganglionic colonic segment. (6) On LMMP neuroligin-1 and Glu were co-expressed in the same site showed by double-labeled immunofluorescent staining of neuroligin-1 and Glu. (7) On paraffin-embedded sections, neuroligin-1 was expressed highest to lowest in the ganglionic, transitional and aganglionic segments showed by immunohistochemical staining. (8) Neuroligin-1 and Glu were co-expressed highest to lowest in the ganglionic, transitional and aganglionic segments based on Western-blot, which accorded with what immunohistochemical staining showed. (9) The serum Glu level was the highest to lowest in the non-HSCR, short-type HSCR and long-type HSCR samples based on ELISA.Conclutions:Neuroligins were expressed in ICC-MY of HD, and the different expression from different segments may play an important role in the pathogenesis of this disease through affecting the synaptic function of ICC-MY. And also the subunits protein neuroligin-1 and its related Glu may represent new markers of ganglion cells especially of the excitatory synapses, whose expression may correlate with the pathogenesis, diagnosis, differential diagnosis or classification of HSCR. As a laboratory detecting technique, the detection of Glu combining with the imaging examination could raise the diagnosis accuracy of HD especially the new-infant HD and supply a new reference basis for the classification of HD(long type, short type or common type). From a new view, we investigate a new pathogenesis and also a new lab testing method of HD, which supply a new way for the basic research and clinical practice of HD. |
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