Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Background: In Hirschsprung's disease, the trend has been for earlier performance of definitive surgery. In our institution, primary Swenson pull- through has become the preferred procedure. Methods: Retrospective review of the patients treated for Hirschsprung's disease from January 1988 through March 1998 was performed. Sixty-five patients were identified. Median values, analysis of variance and χ2 were used for comparisons. Results: The multiple-stage group (M, n = 47) was similar to the primary group (P, n = 18) for gestational age (40 v 39 weeks), time to meconium passage (37.9 v 35.5 hours), and age at diagnosis (median, M 27 v P 3.5 days). Age (median, M 268 v P 5 days) and weight (mean, M 9.4 v P 3.7 kg; P < .001) at pull-through were lower in the primary group. Length of stay (LOS) was lower in the primary group (mean, M 40,8 v P 20.3 days; P < .05). Operating time for pull- through was decreased in P (mean, M 305.2 v P 272.2 minutes; P = .02). Total complications were lower in the primary group (P = .03), with no differences in mortality or enterocolitis rates. Conclusions: At our institution there were no increases in total complications or enterocolitis in the group undergoing primary Swenson. Primary pull-through is a viable option for the treatment of Hirschsprung's disease.

published_or_final_version / abstract / Surgery / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Surgery / Doctoral / Doctor of Philosophy

Introduction: Hirschsprung’s Disease (HD) is the congenital absence of ganglion cells (GCs) within the distal intestine. Our objectives are to determine if apoptosis of myenteric GCs occurs during human development and to determine if myenteric GC apoptosis or injury contributes to HD. Materials and Methods: Apoptosis of myenteric GCs was assessed in archived fetal intestinal tissue (n = 4; 15-41 weeks gestational age) and in HD at the transition zone (TZ) (n = 6) using anti-cleaved caspase-3. Immunohistochemistry for GFAP, CD68, HLA-DR and APP was used to assess the presence of enteric reactive changes. Results: No activated caspase-3 expression was present in the myenteric GCs of the developing human intestine or the TZ of HD. No significant increase in GFAP, CD68, HLA-DR or APP expression was present. Conclusions: Apoptosis does not appear to occur during the development of the human myenteric plexus or, in conjunction with GC injury, in HD.

Introduction: Hirschsprung’s Disease (HD) is the congenital absence of ganglion cells (GCs) within the distal intestine. Our objectives are to determine if apoptosis of myenteric GCs occurs during human development and to determine if myenteric GC apoptosis or injury contributes to HD. Materials and Methods: Apoptosis of myenteric GCs was assessed in archived fetal intestinal tissue (n = 4; 15-41 weeks gestational age) and in HD at the transition zone (TZ) (n = 6) using anti-cleaved caspase-3. Immunohistochemistry for GFAP, CD68, HLA-DR and APP was used to assess the presence of enteric reactive changes. Results: No activated caspase-3 expression was present in the myenteric GCs of the developing human intestine or the TZ of HD. No significant increase in GFAP, CD68, HLA-DR or APP expression was present. Conclusions: Apoptosis does not appear to occur during the development of the human myenteric plexus or, in conjunction with GC injury, in HD.

Next, the influences on the migration of neural crest cell from the microenvironment of the hindgut through which the neural crest cells migrate were studied. An organ culture system was established to recombine different gut segments together at E11.5 for gut culture in order to trace the migration of neural crest cells from the midgut of the +/+ or Dom/+ embryo to the hindgut of the same or different genotypes. At E11.5, the midgut of both +/+ and Dom/+ embryos had already been fully colonized by neural crest cells, thus an explanted midgut segment (donor midgut) could serve as the source of the neural crest cells, while the caudal half of the hindgut (recipient hindgut) acted as the recipient of the neural crest cells from the donor midgut segment because at this stage, the caudal half of the hindgut was completely devoid of neural crest cells. After three days of culture, when a segment of midgut from the +/+ embryo was used as the donor of migratory vagal neural crest-derived cells and combined with an aneural segment of the hindgut (segment without neural crest-derived cells) from Dom/+ or Dom/Dom embryos, neural crest-derived cells from the midgut segment successfully crossed the combination junction and migrated normally along the hindgut segment to reach its caudal end within a normal developmental time frame. However, the migration of neural crest-derived donor cells from the Dom/+ midgut segment was abnormal in the recipient hindgut with a genotype of +/+, Dom/+ or Dom/Dom as evidenced by the retarded rostrocaudal progression of the vagal neural crest-derived cells and the reduced number of migratory cells in the recipient hindgut segment. These results thus indicate that the migration of the vagal neural crest-derived cells is minimally influenced by the migratory environment of the hindgut of the Dom embryo, and that the neural crest cells themselves may be defective in migration leading to the retarded migration in the hindgut of Dom mouse embryos. / The vagal neural crest cells originating from the region of the neural tube adjacent to somites 1 to 7 migrate along defined pathways to the gastrointestinal tract and then colonize the gut to give rise to the majority of neurons and glia of the enteric nervous system. Mutation of Sox10 in the Dominant megacolon (Dom) mouse, which is an animal model of Hirschsprung's disease, leads to aganglionosis (absence of ganglia) in varying lengths of the hindgut. To investigate the underlying cellular mechanism of aganglionosis, the migration of vagal neural crest cells from the neural tube to the gut (pre-enteric migration) in Dom mouse embryos at E8.5 was firstly traced with extrinsic cell markers, such as wheat germ agglutinin gold conjugates (WGA-Au) or fluorescent dye DiI. After the vagal neural crest cells entered the gut at E9.5, their migration was then followed by the examination of the expression of specific markers for undifferentiated neural crest cells with immunohistochemical staining. It was found that, although vagal neural crest cells in embryos of the three genotypes examined migrated along similar pre-enteric pathways at a similar migratory rate, the numbers of neural crest cells in embryos heterozygous (Dom/+) and homozygous (Dom/Dom) for the Sox10 mutation were significantly reduced when compared with the number of neural crest cells in wild-type (+/+) embryos. After vagal neural crest had entered the gut and from E10.5 onwards, no neural crest-derived cells were found in the gut of Dom/Dom embryos, and the migration of neural crest cells along the Dom/+ gut was significantly retarded from E12.5 onwards as compared with the migration in stage-matched +/+ embryos. / To further trace the cause of defective migration of neural crest cells in the Dom embryo, the proliferation and survival of neural crest cells were investigated with BrdU labeling and TUNEL assay. It was found that, although there was no obvious difference in the proliferating ability of vagal neural crest cells in embryos of all the three Dom genotypes studied during the pre-enteric migration and the migration in the gut, more apoptotic neural crest cells were found along the pre-enteric migratory pathway of Dom/Dom embryos than Dom/+ and +/+ embryos. Therefore, the decreased surviving ability, but possibly not the reduced proliferating ability, of neural crest cells during their pre-enteric migration may be partly responsible for aganglionosis in the hindgut of the Dom mouse. / Wang Liang. / "June 2006." / Adviser: W. Y. Chan. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1380. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 287-307). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Hirschsprung’s disease (HSCR) is the major enteric nervous system anomaly affecting newborns with high incidence in Asians. HSCR is a congenital complex genetic disorder characterized by a lack of enteric ganglia along a variable length of the intestine. The receptor tyrosine kinase gene (RET) is the major HSCR gene and cis-elements in the promoter and intron of RET gene are crucial for RET expression. Abnormal RET expression leading to insufficient RET activity causes defective development of the enteric nervous system and is implicated in the pathogenesis of the Hirschsprung’s disease. The human homeobox B5, HOXB5, has an important role in the development of enteric neural crest cells, and perturbation of HOXB5 signaling causes reduced RET expression and HSCR phenotypes in mice. To investigate the roles of HOXB5 in the regulation of RET expression and in the aetiology of HSCR, I sought to(i) elucidate the underlying mechanisms that HOXB5 mediates RET expression, and (ii) to examine the interactions between HOXB5 and other transcription factors including SOX10 and NKX2-1 that have been implicated in RET expression and HSCR. In this study, I demonstrated that HOXB5 binds to the RET promoter and regulates RET expression. HOXB5 and NKX2-1 forma protein complex and mediate RET expression in a synergistic manner. In contrast, HOXB5 cooperates in an additive manner with SOX10in trans-activation from RET promoter. ChIP assay further revealed that HOXB5 and NKX2-1 interact with the same chromatin region proximate to the transcription start site of RET, suggesting that these two factors may interact with each other and regulate the transcription of RET. In silico analysis, EMSA and ChIP analysis showed that HOXB5 also binds to an enhancer element (MCS+9.7)in the intron 1 of RET gene, and HSCR-associated SNPs have been identified in this enhancer element. To further access the HOXB5 trans-activity onMCS+9.7, RET mini-gene was constructed by ligating the RET promoter to the 5’and MCS+9.7 to the 3’of a luciferase gene. Luciferase assay indicated that MCS+9.7 enhances the HOXB5 trans-activation from the RET promoter. In addition, previously identified HSCR-associated SNPs inintron 1 markedly reduce the HOXB5 trans-activation from the RET mini-gene. Moreover, the result of IP-LC-MS/MS indicated that HOXB5 could form protein-protein complexes with nuclear proteins involved in the transcription initiation of genes with TATA-less promoter. This evidence suggested that HOXB5 may cooperate with other activators or co-factors in the remodeling of chromatin conformation, local histone modification and recruitment of essential transcription factors for RNA Polymerase II based transcription from TATA-less promoter, such as RET. My data indicated that HOXB5 in coordination with other transcription factors mediates RET expression. Therefore, defects in cis-or trans-regulation of RET by HOXB5 could lead to a reduction of RET expression and contribute to the manifestation of the HSCR phenotype. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy