Immunostaining of ganglioside GD1b, GD3 and GM1 in rat cerebellum: cellular layer and cell type specific associations.

We have studied the cellular distribution of gangliosides GD1b, GD3 and GM1 in rat cerebellum by immunostaining, using monoclonal antibodies and confocal microscopy. Antibodies against astroglial, neuronal and synaptic vesicle associated molecules were used for colocalization analyses. In the gray matter, the anti-GD1b antibody stained thin strands in the molecular layer (ML), interpreted as Bergman glia fibers based on colocalized staining with anti-glial fibrillary acidic protein (GFAP). The neuropil in the granule (GL) and Purkinje (PL) cell layers was also anti-GD1b positive. The anti-GD3 antibody stained the ML, the neuropil in the GL and PL and also the granule and Purkinje cell bodies, appearing intracytoplasmically and vesicle associated. Anti-GD1b and anti-GD3 staining in the GL glomeruli were colocalized with anti-synaptophysin staining. The anti-GM1 antibody stained cell bodies in the ML but they could not be characterized in colocalization experiments. The GL and PL were not stained with the anti-GM1 antibody. In the white matter, different staining patterns were seen for the gangliosides, the anti-GM1 staining being the most intense. This study shows cellular layer and cell type specific associations of the investigated gangliosides and localization of GD1b and GD3 at synaptic sites, warranting further studies on their role in synaptic mechanisms.

A heterozygous frameshift mutation in the endothelin-3 (EDN-3) gene in isolated Hirschsprung's disease.

Hirschsprung's disease, affecting one in 5000 live newborns, is the most common cause of neonatal intestinal obstruction. The obstruction or, later in life, constipation arises from the lack of enteric ganglia in the hindgut, thus resulting in poor coordination of peristalsis. Mutations in Hirschsprung patients have so far been reported in five genes associated in two different receptor-ligand systems, RET-GDNF/NTN and EDNRB-EDN-3, and an additional gene with yet unknown precise function, SOX10. We report the results of single-stranded conformation polymorphism screening of the endothelin-3 gene in a Swedish population-based material of 66 sporadic and nine familial Hirschsprung's disease cases. We have found a novel heterozygous mutation in exon 2, c.262insG, in a patient with sporadic short segment Hirschsprung's disease without any Waardenburg features. This frameshift results in a premature stop two codons further on. Because this stop is introduced 5' of the biologically active protein, this mutation can hence be predicted to result in haplo-insufficiency.

Phenotypic variation in a family with mutations in two Hirschsprung-related genes (RET and endothelin receptor B).

Hirschsprung disease is a congenital malformation affecting 1 in 5000 live births. The absence of parasympathetic neuronal ganglia (Meissner, Auerbach) in the hindgut results in poor coordination of peristaltic movement, and a varying degree of constipation. Four different genes have been implicated in the pathogenesis of Hirschsprung disease: the RET tyrosine kinase receptor gene; one of its ligands, the glial cell line-derived neurotrophic factor (GDNF) gene; the endothelin receptor B (EDNRB) gene; and its ligand, endothelin-3 (EDN3). Recently, combinations of mutations in two of these genes (RET and GDNF) have been reported in Hirschsprung patients. We report a family with missense mutations in both the RET gene (R982C) and the EDNRB gene (G57S). In this family, three out of five members have the two mutations, but only one, a boy, has the Hirschsprung disease phenotype. This illustrates the complexity of the molecular background of Hirschsprung disease.

Low frequency of RET mutations in Hirschsprung disease in Sweden.

Hirschsprung disease is a congenital malformation, where absence of intramural ganglia in the hindgut results in a defect in the coordination of peristaltic movement. This leads to ileus in the newborn or, more often, constipation in children and adults. The disease affects one in 5000 live births. Siblings of affected cases are at an increased risk (4%) of developing the disease. Among cases. males are affected more often than females. The first major susceptibility gene for Hirschsprung disease is the RET proto-oncogene on 10q11.2. Germline RET mutations in Hirschsprung disease are mainly inactivating, and have been reported to account for up to 20 and 50% of sporadic and familial cases, respectively. We have screened Swedish population-based samples from 62 sporadic cases and seven familial cases of Hirschsprung disease with single strand conformation polymorphism (SSCP), and found five mutations.

Monosialoganglioside (GM1) immunofluorescence in rat spinal roots studied with a monoclonal antibody.

Gangliosides are characteristic glycolipid components of plasma cell membranes, especially enriched in the CNS and PNS. In some diseases involving the PNS, in particular motor neuropathies associated with conduction block, IgM autoantibodies against ganglioside GM1 have been implicated as a pathogenic factor. In order to study the GM1 distribution in peripheral nerves we have investigated its in situ localization using a new anti-GM1 monoclonal antibody, GM1:1. Immunization and production of the monoclonal antibody was made by common protocols and binding specificity was investigated by using structurally related glycolipids and modified GM1-molecules. The result showed that an alpha 2-3 bound sialic acid together with a terminal galactose moiety were essential for GM1:1 binding. In situ localization of GM1 in rat dorsal and ventral spinal roots was investigated by conventional immunomicroscopy. GM1 immunoreactivity was the same in both roots and appeared like a finely granular, in places confluent, material confined to Schmidt-Lanterman's incisures, to myelin sheath paranodal end segments and to some extent to the abaxonal Schwann cell cytoplasm; all of these structures are likely to be the target for GM1 antibodies in peripheral neuropathies. Nodal gaps and fibre contours showed a weak non-specific fluorescence. The localization of GM1 to the incisures of Schmidt-Lanterman and the paranodal end segments of the myelin sheaths might indicate a role of gangliosides as adhesion molecules.

Ethanol absorption across human skin measured by in vivo microdialysis technique.

Microdialysis, a new bioanalytical sampling technique enables measurement of substances in the extracellular space. This initial study investigates the technique's usefulness in the field of percutaneous absorption of solvents, using ethanol as test substance. Microdialysis probes are equipped at the tip with a semi-permeable polycarbonate membrane which permits passive diffusion of substances. Ethanol does not damage the membrane. In vitro recovery for ethanol is good. Probes were inserted via a guide into the skin of the ventral forearm in 7 volunteers. 99.5% ethanol was applied to the skin in excess in a glass reservoir. The probe was perfused at a flow of 1 microliter/min. 50 microliters samples were analysed by gas chromatography. Absorption of ethanol was demonstrated in all subjects. Values from the 9 probes inserted ranged from 10 micrograms/ml to 800 micrograms/ml. The variation may be explained by inter-test or inter-individual variability in ethanol absorption. Individual metabolic capacity may be of importance. The method opens new possibilities in the investigation of skin barrier function in man.

Hirschsprung's disease: one year follow-up after modified Soave's operation.

At the Department of Paediatric surgery in Stockholm the choice of operative method for Hirschsprung's disease has since 1975 been endorectal pull-through. In 1985 some modifications were introduced, i.e. anal approach of the mucosal dissection, resection of the internal anal sphincter, total parenteral nutrition and fewer colostomies. Thirteen children treated accordingly have been compared to children treated before 1985. It was found that the children gained normal bowel habits earlier and had fewer postoperative problems than the children operated before introduction of the technical modifications.

Anal sphincter function after surgery for high imperforate anus--a long term follow-up investigation.

37 children were treated for high imperforate anus during the years 1944-1965. Eighteen of these 37 participated in a follow-up examination at the age of 18-35 years. The examination consisted of an anorectal manometry, EMG of the external sphincter, a digital anal examination, and an interview. Special attention was paid to anal continence. Fourteen patients did not admit any major anal problems, and eight of them had a good continence. Anorectal manometry showed an absence of internal sphincter function in all patients. During voluntary squeeze the continent patients increased anal pressure significantly more than the incontinent ones, and they were also aware of significantly smaller distensions of the rectal ampulla. The present investigation shows the necessity of well-functioning striated sphincter muscles to achieve good continence, and therefore also the importance of careful dissection and identification of the striated sphincter muscles during the operation.

A solitary ileal diverticulum in a child. A case report.

This is a report on a torquated solitary ileal diverticulum in a boy of 2 1/2 years of age who was operated on a suspicion of an appendiceal abscess.

Electrical activity of the external anal sphincter at different ages in childhood.

In EMG recordings of the external anal sphincter there is a brief contraction in response to rapid rectal distension, and a preserved or increased activity during a prolonged substantial rectal distension in healthy adults. In order to determine if this activity develops during childhood 30 healthy children, aged 2 months to 15 years, were examined with their parents' consent. EMG of the external anal sphincter during rest and during rectal distension was performed. All those children who had gained voluntary anal control showed an EMG recording similar to the adults. The 10 youngest children who had not gained voluntary control showed another EMG pattern. Instead of a brief contraction they had a brief loss of activity in response to rectal distension. During a prolonged rectal distension the external sphincter activity decreased and finally ceased. An intermediate state was found in the two youngest children who had gained voluntary control. The EMG pattern showed a good correlation with the maturation and gain of voluntary control of defaecation.

Fracture of the intercondylar eminence of the tibia: a review of 35 patients.

Thirty-five patients (28 children and seven adults) were reviewed six months to 22 years after sustaining the common childhood fracture of the intercondylar eminence of the tibia. The aim was to assess both short-term and long-term results and prognosis by clinical and radiological examination and to discover whether conservative treatment was adequate for those severe fractures where a fragment of the tibial crest had been totally displaced. All the less severe fractures and 14 of the 17 severe fractures were treated conservatively. Early improvement occurred in more patients when the knee was immobilised in extension rather than in flexion, but long-term results were similar. Seven patients had slight discomfort, two of whom had had operative treatment. Radiographs of all patients showed a projection at the fracture site but its size did not correlate with the extent of displacement of the fragment nor with the degree of discomfort suffered; in the severe group a smaller projection developed after open reduction and after immobilisation in extension than after immobilisation in flexion. There was no instability due to functional lengthening of the cruciate ligaments and no increased incidence of degenerative change.

Activity of the internal anal sphincter during the first days of life.

Manometric recordings of the internal anal sphincter activity are frequently used in the diagnosis of Hirschprung's disease. Normally, there is a relaxation of the internal sphincter in response to a rectal distension, but this is absent in Hirschprung's disease. In order to study the normal physiology during the first days of life, 17 healthy full-term infants were examined daily with ano-rectal manometry during the first four days of life. Resting anal pressure was lower than in older children. Internal sphincter relaxation was recorded in all infants but one on the first day, in all on the second and finally in all but one on the third and fourth days. The results indicate that anorectal manometry may be used in the diagnosis of Hirschprung's disease also in the newborn period. However, repeated examinations may be necessary and the diagnostic reliability may be somewhat lower than in older children.