Laser microdissection as a new tool to investigate site-specific gene expression in enteric ganglia of the human intestine.

BACKGROUND: Myenteric ganglia are key-structures for the control of intestinal motility and their mRNA expression profiles might be altered under pathological conditions. A drawback of conventional RT-PCR from full-thickness specimens is that gene expression analysis is based on heterogeneously composed tissues. To overcome this problem, laser microdissection combined with real-time RT-PCR can be used to detect and quantify low levels of gene expression in isolated enteric ganglia. METHODS: Fresh unfixed full-thickness specimens of sigmoid colon were obtained from patients (n = 8) with diseases unrelated to intestinal motility disorders. 10 microm cryo-sections were mounted on membrane-coated slides and ultra-rapidly stained with toluidine blue. Myenteric ganglia were isolated by laser microdissection and catapulting for mRNA isolation. Real-time RT-PCR was performed for selected growth factors, neurotransmitter receptors and specific cell type markers. KEY RESULTS: Collection of 0.5 mm(2) of ganglionic tissue was sufficient to obtain positive RT-PCR results. Collection of 4 mm(2) resulted in ct-values allowing a reliable quantitative comparison of gene expression levels. mRNA analysis revealed that neurotrophic growth factor, neurotrophin-3, serotonin receptor 3A, PGP 9.5 and S100 beta are specifically expressed in myenteric ganglia of the human colon. CONCLUSIONS & INFERENCES: Laser microdissection combined with real-time RT-PCR is a novel technique to reliably detect and quantify site-specific expression of low-abundance mRNAs (e.g. growth factors, neurotransmitter receptors) related to the human enteric nervous system. This technical approach expands the spectrum of available tools to characterize enteric neuropathologies underlying human gastrointestinal motility disorders at the molecular biological level.

Cell-free supernatants of Escherichia coli Nissle 1917 modulate human colonic motility: evidence from an in vitro organ bath study.

Abstract Clinical studies have shown that probiotics influence gastrointestinal motility, e.g. Escherichia coli Nissle 1917 (EcN) (Mutaflor) proved to be at least as efficacious as lactulose and more potent than placebo in constipated patients. As the underlying mechanisms are not clarified, the effects of EcN culture supernatants on human colonic motility were assessed in vitro. Human colonic circular smooth muscle strips (n = 94, 17 patients) were isometrically examined in an organ bath and exposed to different concentrations of EcN supernatants. Contractility responses were recorded under (i) native conditions, (ii) electrical field stimulation (EFS), (iii) non-adrenergic non-cholinergic conditions, and (iv) enteric nerve blockade by tetrodotoxin (TTX). As concentrations of acetic acid were increased in EcN supernatants, contractility responses to acetic acid were additionally tested. EcN supernatants significantly increased the maximal tension forces both at low and high concentrations. Neither blockade of both adrenergic and cholinergic nerves nor application of TTX abolished these effects. EFS-induced contractility responses were not altered after exposure to EcN supernatants. Acetic acid elicited effects comparable to EcN supernatants only under TTX conditions. EcN supernatants modulate in vitro contractility of the human colon. As neither partial nor TTX blockade of enteric nerves abolished these effects, EcN supernatants appear to enhance colonic contractility by direct stimulation of smooth muscle cells. Active metabolites may include other substances than acetic acid, as acetic acid only partially resembled the effects elicited by EcN supernatants. The data provide a rationale for therapeutical application of probiotics in gastrointestinal motility disorders.

[Quantitative volumetric determinations on MR tomograms in communicating hydrocephalus]. / Quantitative Volumenbestimmungen auf MR-Tomogrammen beim kommunizierenden Hydrozephalus.

For patients with communicating hydrocephalus the implantation of a cardioventricular shunt is mostly an approved therapy. However, indications are frequently problematic and therefore a shunt operation will not be always successful. The proof of periventricular oedemas in addition to clinical signs seems to be a criterion for an indication for implantation and for the selection of a special valve. With the aid of a newly developed computer programme volume estimation of the ventricular system and the periventricular oedemas has been effected for six patients on T2-weighted MR-tomograms (1.5 tesla). Quotients of the different volumes are correlated with cerebrospinal fluid (CSF) pressure obtained during shunt implantation or lumbar measurement. Distinct indications have been found for a correlation between the volume of the periventricular oedemas and the pressure measurements.

Vestibular adaptation to long-term stimuli.

Experimental procedures are described which respectively enlarge stimulus duration in vestibular peracceleratory tests, and allow to rule out direct thermal effects on the vestibular nerve during long term calorisations. First experimental results indicate that time course of nystagmus during prolonged stimulations differs markedly in rotational and caloric tests. Whereas there is a distinct decline of response during rotation (in accordance to the predictions of current mathematical models), in caloric tests nystagmus reaches a steady state level, maintained for at least 15 min.