| BackgroundHirschsprung's disease is a conmon congenital condition in children,which means it is present from birth.It is characterized by an absence of enteric ganglia in the myenteric and the submucosal plexus of the distal colon and a failure of innervation in the gastrointestinal tract,and the unmyelinated parasympathetic fibers increase and become thicker.Because of the lack of ganglion cells in the myenteric and submucosal plexus,the segment lacking neurons can't have a bowel movement and becomes constricted,result in blockages in the bowel because the feces does not move through the bowel normally,causing the normal,proximal section of bowel to become distended with feces.Clinical manifestations,barium enema and anorectal manometry can diagnose typical cases,but it is difficult for basic hospital to do it early and accurately · Definitive diagnosis is made by suction biopsy of the distally narrowed segment.A small sample of tissue from the rectum can be taken easily during surgery in basic hospital,which will have a histologic examination.But improper placement where the sample of tissue were taken may cause missed diagnosis or misdiagnosis.Surgery is the only way to cure Hirschsprung's disease.However,no matter what kind of operation they have,postoperative gastrointestinal dysfunction,enterocolitis,and constipation recurrence are common postoperative complications,which may be related to the extent of bowel resection.ObjectiveIn this study,the distribution and mor:phological characteristics of ganglion cells and nerve fibers in different segments of distal intestinal above dentate line and the staining of Calretinin were observed to explore the accurate biopsy range of congenital megacolon in children of different ages.The morphological changes and distribution of nerves and smooth muscles in the proximal intestinal wall of children with HD were observed in order to provide reference for defining the extent of resection of the diseased intestine.Materials and methods1 Case selection and SubgroupResearch One:The patients clinically diagnosed with HD in Zhongshan Boai Hospital of Southern Medical University between January,2014 and December,2017was selected as the observation group,and they was performed transanal rectal myometrium biopsy.The HD patients who was diagnosed by pathological examination were a study group,and they were indived into three groups,group A(?1 month),group B(>1month,<1year),group C(>1year).Research Two:All the children with hirschsprung's disease were selected as the study group,and 25 cases died of non-gastrointestinal and nervous system diseases were selected as the control group.The changes of nervous system and smooth muscle in different proximal segments of the two groups were observed and analyzed.2 Biopsy methodFinger anal dilatation preoperative.0.5 cm in diameter of intestinal tissues were taken jfrom deep rectal muscular layer,neonates in the anal dentate line 2.0 cm,infants in the anal dentate line 3.0 cm,infants in the anal dentate line 4.0 cm.The samples were performed pathological examination with HE staining,observing ganglion cells.ganglion-free cells for positive results,they diagnosed HD.3 Surgical methodsThe HD patients were performed Transanal radic.al megacolon surgery(Soave)or transabdominal Transanal Radical megacolon surgery or laparoscopic assisted Transanal Radical megacolon surgery.4 Sample Treatment4.1 Biopsy specimens were embedded and sectioned and stained with HE.4.2 Operative specimen(the distal bowel)was fixed along the longitudinal axis of the bowel.About 20 consecutive sections were taken from the dentate line to the dilated bowel every 0.5 cm.They were performed HE staining and immunohistochemical Calretinin staining(SP method).4.3 About 20 consecutive sections were taken from the proximal intestine every 2 cm,and they were performed HE staining and immunohistochemical Calretinin staining(SP method)labeling Syn?NCAM.5 Observed Parameters5.1 The distal bowel:At the low power microscope(100 times),10 visual fields of the sections stained with HE were randomly selected to observe the myenteric and the submucosal plexus.,and the data were recorded by individual/visual fields,Myenteric and the submucosal plexus were randomly selected under high power microscope(400 times).The number of ganglion cells in the plexus was observed.The data were recorded by individual/nerve plexus.The hypertrophic plexus was recorded as'+',and the non-hypertrophic plexus was recorded as'-'.5.2 Immunohistochemical staining of distal bowel:Calretinin staining,brown-yellow,brown-brown was positive,light yellow was weak positive,non-staining was negative.5.3 The proximal intestine:HE staining(1)the number of ganglion cells in the myenteric plexus and plexus of two groups was recorded by the same method.(2)Myometrial thickness and thickness ratio:After HE staining,10 visual fields were randomly selected,and the thickness of longitudinal and circular muscles were measured by micrometer,and the thickness ratio was calculated.(3)myometrial lesions:the degree of vacuolar degeneration of the muscular layer is divided into four grades.No(-);a small quantity(+);a moderate quantity(++);a large quantity(+ ++).5.4 Immunohistochemical staining of proximal bowel(Syn,NCAM staining):the distribution density was divided into four grades,negative(-):no staining,the number of positive cells does not exceed 10%;weak positive(+):yellow,the number of positive cells does not exceed 25%;moderate positive(++):brown,the nunber of positive cells does not exceed 50%;strong positive(+++):brownish brown,the number of positive cells was more than 50%.6 Statistics AnalysisData were analyzed by SPSS20.0 statistical software,the measurement data were expressed by x s,and the comparison between groups was performed by independent sample t test;the counting data was expressed by percentage,and the comparison between groups was performed by x2 test or Fisher exact probability method;P<0.05 was statistically significant.ResultsResearch One:No ganglion cells in the myenteric plexus jfrom dentate line to spasmodic segments in three groups were observated,and that significantly differenced with transitional segments(P<0.01).few myenteric plexus and nerve fiber above the dentate line 1 cm in group A,2 cm in groupB,2.5 cm in group C were observed,and thickening and denaturating nerve fiber and hyperplastic nerve plexus over that segments were observed,and there were significant differences intwo segments of each group(P<0.01).Calretinin dyeing played an important role in observing ganglion cells.Research Two:the enteric ganglion cells observed in different intestinal segments,ganglion cell adhesion molecule(NCAM),nerve fiber count,synaptophysin(Syn),in the expansion of distal 8cm of every index has not yet reached it Often,but compared with the control group no significant difference(P>0.05);compared with the control group,the observation group of different segments of the ring,different degrees of longitudinal muscle layer thickening were in 8cm far from the thickness of the dilated segment did not recover normal,significant difference(P<0.01);two group ring the longitudinal muscle layer,vacuolar degeneration,no statistically significant difference(P>0.05).Conclusion1 There was a "physiological non-ganglion cells" area above the dentate line of Hirschsprung's disease.The distance of this area was different in different ages,and avoided this areawhen specimens were taken.By analyzing the morphology of nerve plexus and nerve fiber,with the results of Calretinin dyeing,"physiological non-ganglion cells" area and intestine of Hirschsprung disease were effectively distinguished,avoiding misdiagnosis.2 The intestinal nerve of HD in resected segment,smooth muscle lesions in varying degrees,but in the position of enteric nerves from the dilated segment of distal 8cm,smooth muscle lesions alleviated.Surgical resection can be combined with their own children the situation allows up to,or Over the area. |
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