Preoperative enzymo-histochemical diagnosis of dysganglionoses associated with anorectal malformations (ARM) with recto-vestibular and recto-perineal fistula.

The posterior sagittal anorectoplasty (PSARP) is widely recognized as the best technique available today for the surgical treatment of anorectal malformations (ARM). However, different retrospective studies on the functional results of PSARP in the treatment of ARM have shown different postoperative degrees of constipation. In particular, even in patients with normal sacrum, about 70% of operated ARM with vestibular fistula and about 50% with perineal fistula can be complicated by fecal constipation and pseudoincontinence. In order to identify preoperatively whether ARM patients present abnormal innervation patterns of rectal pouch and fistula (as reported by Holschneider et al [7]), we decided to study suction rectal biopsies performed by introducing SBT-100 rectal suction biopsy tool into the fistula at 6, 4, 3 and 2 cm from the meatus. To date, this approach has been adopted in 22 ARM cases (15 females and 7 males, age range 7 days-4 years), 6 of them with recto-vestibular fistula and 13 with recto-perineal fistula. Biopsies were frozen in isopentane at liquid nitrogen temperature and cryostatic sections were studied by acetylcholinesterase (AChE), succinic-dehydrogenase (SDH) and alpha-naphthyl-esterase (ANE) enzymo-histochemical techniques. The results concerning the innervation-type of fistula and proximal rectal pouch were confirmed by the biopsies obtained during PSARP. Our overall incidence of rectal innervation intrinsic disorders was 81.82%. In particular, all our cases of vestibular fistula presented associated dysganglionoses. The incidence of associated Hirschsprung's disease was high, corresponding to 18% of cases. Our results suggest that the high frequency of constipation in low forms of ARMS depends on primary intestinal neuronal malformations and it cannot be ascribed to a denervation secondary to rectal dissection and to PSARP procedure. We propose the introduction of this type of preoperative enzymo-histochemical diagnosis in ARM cases because it can select those patients with severe associated dysganglionoses. In our opinion, if this diagnosis is available preoperatively, PSARP can be performed without using abnormally innervated structures and reducing postoperative functional complications.

GDNF deficit in Hirschsprung's disease.

BACKGROUND/PURPOSE: In 1996, the glial cell line-derived neurotrophic factor (GDNF) was identified as one of the ligands of the RET transmembrane receptor. In the same year, GDNF mutations were found in association with RET protooncogene mutations in Hirschsprung patients. Mutations in GDNF per se are thought neither necessary nor sufficient to cause Hirschsprung's disease (HD). To date, our study group has identified GDNF mutations only in 2 of 98 cases of intestinal dysganglionosis. The aim of our study was to investigate a possible expression deficit of GDNF in the enteric nervous system of Hirschsprung patients not mutated for the GDNF gene. METHODS: We used rabbit polyclonal antibodies raised against a peptide corresponding to amino acids 186-205 mapping within the carboxy-terminal domain of human GDNF. GDNF expression was studied immunohistochemically in surgical specimens from 30 HD cases (27 classic forms and 3 ultralong forms) and from 10 age-matched controls. Serial sections from the same full-thickness specimens were investigated with the following histochemical and immunohistochemical techniques: acetylcholinesterase, lactate dehydrogenase, succinic dehydrogenase, alpha-naphthyl-esterase, glial fibrillary acid protein, S-100 protein, and neuron-specific enolase. RESULTS: A high level of GDNF expression was found in normal intestine and in Hirschsprung ganglionic segment. Satellite elements of myenteric ganglia presented a strong immunoreactivity to GDNF. Conversely, the aganglionic segment showed cholinergic hyperinnervation and hypertrophic trunks of nerve fibers in the muscular interstitium with complete absence of GDNF expression. The small ganglia of the hypoganglionic segment showed a reduced GDNF immunoreactivity. CONCLUSIONS: GDNF, a distantly related member of the transforming growth factor-beta superfamily, is a potent neurotrophic and survival factor for neurons and enteric ganglion cells. Mutations of the GDNF gene or GDNF expression deficit interrupt the faithful GDNF signaling via Ret, contributing to HD pathogenesis.

[Constipation and chronic intestinal pseudoobstruction as a clinical expression of intestinal neuronal dysplasia (IND)]. / Stipsi e pseudo-ostruzione intestinale cronica come espressione clinica di displasia neuronale intestinale (IND).

BACKGROUND: Intestinal neuronal dysplasia (IND) belongs to the group of dysganglionosis. It may occur as part of a syndrome of early chronic constipation, neonatal intestinal occlusion, chronic intestinal pseudo obstruction. The aim of this study was to report the cases examined by the Istituto G. Gaslini in Genoa and to discuss the numerous aspects of this disease which are still unclear. METHODS: 787 children were included in the study and underwent biopsy between 1984 and 1997. Rectal biopsies were obtained by suction or in some cases during surgery and were treated using enzymohistochemical techniques, such as acetylcholinesterase, rapid acetylcholinesterase and alpha-naphthylesterase. RESULTS: 574 children were found to be suffering from innervative alterations: 348 (60.6%) presented isolated Hirschsprung's disease, IND was found in 83 (14.5%), in 8 of the latter in association with other dysganglionosis. IND was accompanied by other diseases in 40 cases (48.2%). Over the past three years (since October 1994) a total of 164 dysganglionosis have been diagnosed, including 55 cases of aganglia. During this period IND was the most frequently observed alteration and affected 61 children. CONCLUSIONS: Rectal biopsy is the essential diagnostic test for the diagnosis of intestinal dysganglionosis. Biopsies are performed in outpatient clinics without sedation, and do not represent an invasive procedure for the young patients. Radiological and manometric examinations cannot provide reliable data for the diagnosis of IND. In our experience, the incidence of IND over the past few years has increased and its diagnosis is essential for correct treatment which is not surgical in the majority of cases. The real incidence of IND and its pathogenesis still need to be clarified.