Defining distal splenopancreatectomy by the mesopancreas.

BACKGROUND: The implementation of the pathologic CRM (circumferential resection margin) staging system for pancreatic head ductal adenocarcinomas (hPDAC) resulted in a dramatic increase of R1 resections at the dorsal resection margin, presumably because of the high rate of mesopancreatic fat (MP) infiltration. Therefore, mesopancreatic excision (MPE) during pancreatoduodenectomy has recently been promoted and has demonstrated better local disease control, fueling the discussion of neoadjuvant downsizing regimes in MP + patients. However, it is unknown to what extent the MP is infiltrated in patients with distal pancreatic (tail/body) carcinomas (dPDAC). It is also unknown if the MP infiltration status affects surgical margin control in distal pancreatectomy (DP). The aim of our study was to histopathologically analyze MP infiltration and elucidate the influence of resection margin clearance on recurrence and survival in patients with dPDAC. Furthermore, the results were compared to a collective receiving MPE for hPDAC. METHOD: Clinicopathological and survival parameters of 295 consecutive patients who underwent surgery for PDAC (n = 63 dPDAC and n = 232 hPDAC) were evaluated. The CRM evaluation was performed in a standardized fashion and the specimens were examined according to the Leeds pathology protocol (LEEPP). The MP area was histopathologically evaluated for cancerous infiltration. RESULTS: In 75.4% of dPDAC patients the MP fat was infiltrated by vital tumor cells. The rates of MP infiltration and R0CRM- resections were similar between dPDAC and hPDAC patients (p = 0.497 and 0.453 respectively). MP- infiltration status did not correlate with CRM implemented resection status in dPDAC patients (p = 0.348). In overall survival analysis, resection status and MP status remained prognostic factors for survival. In follow up analysis. surgical margin clearance in dPDAC patients was associated with a significant improvement in local recurrence rates (5.2% in R0CRM- resected vs. 33.3 in R1/R0CRM + resected, p = 0.002). CONCLUSION: While resection margin status was not affected by the MP status in dPDAC patients, the high MP infiltration rate, as well as improved survival in MP- dPDAC patients after R0CRM- resection, justify mesopancreatic excision during splenopancreatectomy. Larger scale studies are urgently needed to validate our results and to study the effect on neoadjuvant treatment in dPDAC patients.

Homer1 (VesL-1) in the rat esophagus: focus on myenteric plexus and neuromuscular junction.

Homer1, a scaffolding protein of the postsynaptic density (PSD), enriched at excitatory synapses is known to anchor and modulate group I metabotropic glutamate receptors (mGluRs) and different channel- and receptor-proteins. Homer proteins are expressed in neurons of different brain regions, but also in non-neuronal tissues like skeletal muscle. Occurrence and location of Homer1 and mGluR5 in myenteric plexus and neuromuscular junctions (NMJ) of rat esophagus have yet not been characterized. We located Homer1 and mGluR5 immunoreactivity (-iry) in rat esophagus and focused on myenteric neurons, intraganglionic laminar endings (IGLEs) and NMJs, using double- and triple-label immunohistochemistry and confocal laser scanning microscopy. Homer1-iry was found in a subpopulation of vesicular glutamate transporter 2 (VGLUT2) positive IGLEs and cholinergic varicosities within myenteric ganglia, but neither in nitrergic nor cholinergic myenteric neuronal cell bodies. Homer1-iry was detected in 63% of esophageal and, for comparison, in 35% of sternomastoid NMJs. Besides the location in the PSD, Homer1-iry colocalized with cholinergic markers, indicating a presynaptic location in coarse VAChT/CGRP/NF200- immunoreactive (-ir) terminals of nucleus ambiguus neurons supplying striated esophageal muscle. mGluR5-iry was found in subpopulations of myenteric neuronal cell bodies, VGLUT2-ir IGLEs and cholinergic varicosities within the myenteric neuropil and NMJs of esophagus and sternomastoid muscles. Thus, Homer1 may anchor mGluR5 at presynaptic sites of cholinergic boutons at esophageal motor endplates, in a small subpopulation of VGLUT2-ir IGLEs and cholinergic varicosities within myenteric ganglia possibly modulating Ca2+-currents and neurotransmitter release.

Localization of receptors for calcitonin-gene-related peptide to intraganglionic laminar endings of the mouse esophagus: peripheral interaction between vagal and spinal afferents?

The calcitonin-gene-related peptide (CGRP) receptor is a heterodimer of calcitonin-receptor-like receptor (CLR) and receptor-activity-modifying protein 1 (RAMP1). Despite the importance of CGRP in regulating gastrointestinal functions, nothing is known about the distribution and function of CLR/RAMP1 in the esophagus, where up to 90 % of spinal afferent neurons contain CGRP. We detected CLR/RAMP1 in the mouse esophagus using immunofluorescence and confocal laser scanning microscopy and examined their relationship with neuronal elements of the myenteric plexus. Immunoreactivity for CLR and RAMP1 colocalized with VGLUT2-positive intraganglionic laminar endings (IGLEs), which were contacted by CGRP-positive varicose axons presumably of spinal afferent origin, typically at sites of CRL/RAMP1 immunoreactivity. This provides an anatomical basis for interaction between spinal afferent fibers and IGLEs. Immunoreactive CLR and RAMP1 also colocalized in myenteric neurons. Thus, CGRP-containing spinal afferents may interact with both vagal IGLEs and myenteric neurons in the mouse esophagus, possibly modulating motility reflexes and inflammatory hypersensitivity.

Interstitial cells of Cajal: crucial for the development of megacolon in human Chagas' disease?

AIM: Megacolon, chronic dilation of a colonic segment,is accompanied by extensive myenteric neuron loss. However, this fails to explain unequivocally the formation of megacolon. We aimed to study further enteric structures that are directly or indirectly involved in colonic motility. METHOD: From surgically removed megacolon segments of seven Chagasic patients, three sets of cryosections from oral, megacolonic and anal zones were immunohistochemically quadruple-stained for smooth-muscle actin (SMA), synaptophysin (SYN, for nerve fibres), S100 (glia) and c-Kit (interstitial cells of Cajal, ICCs). Values of area measurements were related to the appropriate muscle layer areas and these proportions were compared with those of seven non-Chagasic control patients. RESULTS: Whereas nerve and glia profile proportions did not mirror unequivocally the changes of Chagasic colon calibre (nondilation/dilation/nondilation), the proportions of SMA (i.e. muscle tissue density) and c-Kit (i.e. ICC density) did so: they decreased from the oral to the megacolonic segment but increased to the anal zones (muscle tissue density: control 68.3%, oral 54.3%, mega 42.1%, anal 47.6%; ICC-density: control 1.8%, oral 1.1%, mega 0.4, anal 0.8%). CONCLUSION: Of the parameters evaluated, muscle tissue and ICC densities may be involved in the formation of Chagasic megacolon, although the mechanism of destruction cannot be deduced.

Muscarinic acetylcholine receptors in the mouse esophagus: focus on intraganglionic laminar endings (IGLEs).

BACKGROUND: IGLEs represent the only low-threshold vagal mechanosensory terminals in the tunica muscularis of the esophagus. Previously, close relationships of vesicular glutamate transporter 2 (VGLUT2) immunopositive IGLEs and cholinergic varicosities suggestive for direct contacts were described in almost all mouse esophageal myenteric ganglia. Possible cholinergic influence on IGLEs requires specific acetylcholine receptors. In particular, the occurrence and location of neuronal muscarinic acetylcholine receptors (mAChR) in the esophagus were not yet characterized. METHODS: This study aimed at specifying relationships of VGLUT2 immunopositive IGLEs and vesicular acetylcholine transporter (VAChT)-immunopositive varicosities using pre-embedding electron microscopy and the location of mAChR1-3 (M1-3) within esophagus and nodose ganglia using multilabel immunofluorescence and retrograde tracing. KEY RESULTS: Electron microscopy confirmed synaptic contacts between cholinergic varicosities and IGLEs. M1- and M2-immunoreactivities (-iry; -iries) were colocalized with VGLUT2-iry in subpopulations of IGLEs. Retrograde Fast Blue tracing from the esophagus showed nodose ganglion neurons colocalizing tracer and M2-iry. M1-3-iries were detected in about 80% of myenteric ganglia and in about 67% of myenteric neurons. M1- and M2-iry were present in many fibers and varicosities within myenteric ganglia. Presynaptic M2-iry was detected in all, presynaptic M3-iry in one-fifth of motor endplates of striated esophageal muscles. M1-iry could not be detected in motor endplates of the esophagus, but in sternomastoid muscle. CONCLUSIONS & INFERENCES: Acetylcholine probably released from varicosities of both extrinsic and intrinsic origin may influence a subpopulation of esophageal IGLEs via M2 and M1-receptors.

Gastrin mediated down regulation of ghrelin and its pathophysiological role in atrophic gastritis.

The gastric hormone ghrelin is known as an important factor for energy homeostasis, appetite regulation and control of body weight. So far, ghrelin has mainly been examined as a serological marker for gastrointestinal diseases, and only a few publications have highlighted its role in local effects like mucus secretion. Ghrelin can be regarded as a gastroprotective factor, but little is known about the distribution and activity of ghrelin cells in pathologically modified tissues. We aimed to examine the morphological changes in ghrelin expression under several inflammatory, metaplastic and carcinogenic conditions of the upper gastrointestinal tract. In particular, autoimmune gastritis showed interesting remodeling effects in terms of ghrelin expression within neuroendocrine cell hyperplasia by immunohistochemistry. Using confocal laser microscopy, the gastrin/cholecystokinin receptor (CCKB) could be detected on normal ghrelin cells as well as in autoimmune gastritis. Functionally, we found evidence for a physiological interaction between gastrin and ghrelin in a primary rodent cell culture model. Additionally, we gathered serological data from patients with different basic gastrin levels due to long-term autoimmune gastritis or short-term proton pump inhibitor treatment with slightly reactive plasma gastrin elevations. Total ghrelin plasma levels showed a significantly inverse correlation with gastrin under long-term conditions. Autoimmune gastritis as a relevant condition within gastric carcinogenesis therefore has two effects on ghrelin-positive cells due to hypergastrinemia. On the one hand, gastrin stimulates the proliferation of ghrelinpositive cells as integral part of neuroendocrine cell hyperplasia, while on the other hand, plasma ghrelin is reduced by gastrin and lost in pseudopyloric and intestinal metaplastic areas. Ghrelin is necessary for the maintenance of the mucosal barrier and might play a role in gastric carcinogenesis, if altered under these pre neoplastic conditions.

Pitfalls using tyramide signal amplification (TSA) in the mouse gastrointestinal tract: endogenous streptavidin-binding sites lead to false positive staining.

Highly sensitive immunohistochemical detection systems such as tyramide signal amplification (TSA) are widely used, since they allow using two primary antibodies raised in the same species. Most of them are based on the streptavidin-biotin-peroxidase system and include streptavidin-coupled secondary antibodies. Using TSA in cryostat-sectioned tissues of mouse esophagus, we were puzzled by negative controls with unexpected staining mostly in the ganglionic areas. This prompted us to search for the causing agent and to include also other parts of the mouse gastrointestinal tract for comparison. Streptavidin-coupled antibodies bound to endogenous binding sites yet to be characterized, which are present throughout the mouse intestines. Staining was mainly localized around neuronal cell bodies of enteric ganglia. Thus, caution is warranted when applying streptavidin-coupled antibodies in the mouse gastrointestinal tract. The use of endogenous biotin-blocking kits combined with a prolonged post-fixation time could significantly reduce unintentional staining.

Serotonin-immunoreactive neurons and mast cells in the mouse esophagus suggest involvement of serotonin in both motility control and neuroimmune interactions.

BACKGROUND: Serotonin is a major transmitter in the gastrointestinal tract, but little is known about the serotonergic system in the esophagus. METHODS: The aim of this study was to use multilabel immunofluorescence to characterize serotonin-positive nerve cell bodies and fibers and their relationship with other neuronal and non-neuronal elements in the mouse esophagus. Antibodies against serotonin, vesicular acetylcholine transporter (VAChT), choline acetyltransferase (ChAT), protein gene product 9.5 (PGP 9.5), and α-bungarotoxin (α-BT), were used. KEY RESULTS: Serotonin-containing perikarya represented ∼10% of all PGP 9.5-positive myenteric neurons. Serotonin-positive varicose nerve fibers were found in the lamina muscularis mucosae and present on ∼13% of α-BT-labeled motor endplates in addition to VAChT-immunoreactive motor terminals. As ChAT-positive neurons of the compact formation of the nucleus ambiguus were negative for serotonin, serotonin-positive varicosities on motor endplates are presumed to be of enteric origin. On the other hand, cholinergic ambiguus neurons were densely supplied with serotonin-positive varicosities. The tela submucosa and tunica adventitia contained large numbers of serotonin-positive mast cells, a few of which were in close association with serotonin-positive nerve fibers. CONCLUSIONS & INFERENCES: The mouse esophagus is endowed with a rich serotonin-positive intrinsic innervation, including enteric co-innervation of striated muscles. Serotonin may modulate vagal motor innervation of esophageal-striated muscles not only at the central level via projections of the raphe nuclei to the nucleus ambiguus but also at the peripheral level via enteric co-innervation. In addition, mast cells represent a non-neuronal source of serotonin, being involved in neuroimmune processes.

Proline-rich synapse-associated protein-1 and 2 (ProSAP1/Shank2 and ProSAP2/Shank3)-scaffolding proteins are also present in postsynaptic specializations of the peripheral nervous system.

Proline-rich synapse-associated protein-1 and 2 (ProSAP1/Shank2 and ProSAP2/Shank3) were originally found as synapse-associated protein 90/postsynaptic density protein-95-associated protein (SAPAP)/guanylate-kinase-associated protein (GKAP) interaction partners and also isolated from synaptic junctional protein preparations of rat brain. They are essential components of the postsynaptic density (PSD) and are specifically targeted to excitatory asymmetric type 1 synapses. Functionally, the members of the ProSAP/Shank family are one of the postsynaptic key elements since they link and attach the postsynaptic signaling apparatus, for example N-methyl-d-aspartic acid (NMDA)-receptors via direct and indirect protein interactions to the actin-based cytoskeleton. The functional significance of ProSAP1/2 for synaptic transmission and the paucity of data with respect to the molecular composition of PSDs of the peripheral nervous system (PNS) stimulated us to investigate neuromuscular junctions (NMJs), synapses of the superior cervical ganglion (SCG), and synapses in myenteric ganglia as representative synaptic junctions of the PNS. Confocal imaging revealed ProSAP1/2-immunoreactivity (-iry) in NMJs of rat and mouse sternomastoid and tibialis anterior muscles. In contrast, ProSAP1/2-iry was only negligibly found in motor endplates of striated esophageal muscle probably caused by antigen masking or a different postsynaptic molecular anatomy at these synapses. ProSAP1/2-iry was furthermore detected in cell bodies and dendrites of superior cervical ganglion neurons and myenteric neurons in esophagus and stomach. Ultrastructural analysis of ProSAP1/2 expression in myenteric ganglia demonstrated that ProSAP1 and ProSAP2 antibodies specifically labelled PSDs of myenteric neurons. Thus, scaffolding proteins ProSAP1/2 were found within the postsynaptic specializations of synapses within the PNS, indicating a similar molecular assembly of central and peripheral postsynapses.

[Disorders in urinary bladder micturition after rectal operations]. / Harnblasenentleerungsstörungen nach Rektumoperationen.

AIM: Functional disorders of micturition after rectal resection were investigated on an in-house patient collective. Because the neuro-anatomy of the small pelvis is very complex, the aim of this study was to evaluate the incidence and form of disorders in micturition. MATERIALS AND METHODS: A total of 536 patients were operated on for a rectal carcinoma during the period 2000 to 2004 at the Surgical University Clinic in Erlangen. Patients with a recurrent tumor and patients who died during the study were excluded (140 patients). All patients were retrospectively questioned on the pre-operative and postoperative bladder function using a standard questionnaire. RESULTS: The patient collective consisted of 167 males and 111 females with an average age of 63 years and a mean follow-up time of 2.6 years. Age (<65 years versus >65 years) was found not to play a significant role with respect to micturition (p>0.05). Of the patients 20 already had a pollakisuria before the operation and 63 patients after the operation. Also 9 patients already had a nycturia before the operation and 55 after the operation. An imperative urgency to urinate was experienced by 9 patients before and 47 after the operation. Stress incontinence grade I was present in 21 patients before the operation, grade II in 4 patients and grade III in 1 patient. Of the patients 43 complained of stress incontinence grade I after the operation, 20 patients of a grade II and 15 of a grade III. The postoperative quality of life was assessed as very limited by 57 patients and moderately limited by 85. CONCLUSIONS: Functional disorders of micturition after rectal resection in rectal cancer patients are commonly occurring complications which have received relatively little attention in the literature considering the clinical significance. Only few affected patients have received a urological treatment. Further studies are necessary, also experimental in nature, to understand the neuro-anatomy of the small pelvis in order to avoid intra-operative neurolesions as far as possible.

The influence of concentric and eccentric loading on the finger pulley system.

In this study we investigated the influence of the loading condition (concentric vs. eccentric loading) on the pulley system of the finger. For this purpose 39 cadaver finger (14 hands, 10 donors) were fixed into an isokinetic loading device. The forces in the flexor tendons and at the fingertip were recorded. In the concentric loading condition A2 and A4 ruptures as well as alternative events such as fracture of a phalanx or avulsion of the flexor tendons were almost equally distributed, whereas the A2 pulley rupture was the most common event (59%) in the eccentric loading condition and alternative events were rare (23.5%). The forces in the deep flexor tendon, the fingertip and in the pulleys were significantly lower in the eccentric loading condition. As the ruptures occurred at lower loads in the eccentric than in the concentric loading condition it can be concluded that friction may be an advantage for climbers, supporting the holding force of their flexor muscles but may also increase the susceptibility to injury.

The influence of the crimp and slope grip position on the finger pulley system.

In this study the influence of the grip position (crimp grip vs. slope grip position) on the pulley system of the finger was investigated. For this purpose 21 cadaver finger (11 hands, 10 donors) were fixed into an isokinetic loading device. Nine fingers were loaded in the slope grip position and 12 fingers in the crimp grip position. The forces in the flexor tendons and at the fingertip were recorded. A rupture of the A4 pulley occurred most often in the crimp grip position (50%) but did not occur in the slope grip position, in which alternative events were the most common (67%). The forces in the deep flexor tendon (FDP) (slope grip: 371 N, crimp grip: 348 N) and at the fingertip (slope grip: 105 N, crimp grip: 161 N) were not significantly different between the 2 finger positions, but the forces acting on the pulleys were higher in the crimp grip position (A2 pulley: 287 N, A4 pulley: 226 N) than in the slope grip position (A2 pulley: 121 N, A4 pulley: 103 N). The crimp grip position may be the main cause for A4 pulley ruptures but the slope grip position may be hazardous for other injuries as the forces recorded in the flexor tendons and at the fingertip were comparable at the occurrence of a terminal event.

Functionalized silicon quantum dots tailored for targeted siRNA delivery.

For RNA interference (RNAi) mediated silencing of the ABCB1 gene in Caco-2 cells biocompatible luminescent silicon quantum dots (SiQDs) were developed to serve as self-tracking transfection tool for ABCB1 siRNA. While the 2-3nm sized SiQD core exhibits green luminescence, the QD surfaces are completely saturated with covalently linked 2-vinylpyridine that may electrostatically bind siRNA. For down-regulating P-glycoprotein (Pgp) expression of the ABCB1 gene the SiQDs were complexed with siRNA. The cellular uptake and allocation of SiQD-siRNA complexes in Caco-2 cells were monitored using confocal laser scanning microscopy and transmission electron microscopy. The release of siRNA to the cytoplasm was verified through real-time PCR quantification of the reduced ABCB1 mRNA level. Additional evidence was obtained from time-resolved in situ fluorescence spectroscopic monitoring of the Pgp efflux dynamics in transfected Caco-2 cells which yielded significantly reduced transporter efficiencies for the Pgp substrate Rhodamine 123.

Olfactory bulb ventricles as a frequent finding in magnetic resonance imaging studies of the olfactory system.

UNLABELLED: Background. In some species an embryologic cavity inside the olfactory bulb (OB) persists and is called olfactory bulb ventricle (OBV). It is generally assumed that OBVs in humans are solitary findings representing remnants of embryologic structures that were not fully regressed, although the incidence of OBVs was never examined. Using magnetic resonance imaging (MRI), the present study aimed to study the incidence of OBVs in healthy human subjects. Material and methods. A total of 122 individuals participated. Volumes of the right and left OB were determined using MRI scans and a standardized protocol for OB analysis. For comparison, OBs of 42 cadavers were collected and sectioned. Results. The main finding of this study was the high incidence of OBV-like structures in our study group. Seventy-two out of 122 (59%) participants yielded signs for an OBV whereas three out of 42 postmortem OBs contained histologically detectable OBV. DISCUSSION: This stands in disagreement with the previous assumption of complete obliteration at the time of birth. This discrepancy may be explained by the fact that our present findings are based on modern MRI techniques with much higher resolution than 10 or 20 years ago. Another possible explanation for the discrepancy between studies based on MRI and histopathology might relate to postmortem resorption of cerebrospinal fluid from OBVs. Especially with a long postmortem interval OBVs may collapse and may no longer appear as an open cavity.

Increase in NADPH-diaphorase-positive and neuronal NO synthase immunoreactive neurons in the rat spinal trigeminal nucleus following infusion of a NO donor--evidence for a feed-forward process in NO production involved in trigeminal nociception.

Nitric oxide (NO) donors, which cause delayed headaches in migraineurs, have been shown to activate central trigeminal neurons with meningeal afferent input in animal experiments. Previous reports indicate that this response may be due to up-regulation of NO-producing cells in the trigeminal brainstem. To investigate this phenomenon further, we determined nitric oxide synthase (NOS)-containing neurons in the rat spinal trigeminal nucleus (STN), the projection site of nociceptive trigeminal afferents, following infusion of the NO donor sodium nitroprusside (SNP). Barbiturate anaesthetized rats were infused intravenously with SNP (50 microg/kg) or vehicle for 20 min or 2 h, and after periods of 3-8 h fixed by perfusion. Cryostat sections of the medulla oblongata containing the caudal STN were histochemically processed for detection of nicotineamide adenine dinucleotide phosphate (NADPH)-diaphorase or immunohistochemically stained for NOS isoforms and examined by light and fluorescence microscopy. The number of neurons positive for these markers was determined. Various forms of neurons positive for NADPH-diaphorase or immunoreactive to neuronal NOS (nNOS) were found in superficial and deep laminae of the STN caudalis and around the central canal. Neurons were not immunopositive for endothelial (eNOS) or inducible (iNOS) NOS isoforms. The number of NADPH-diaphorase-positive neurons increased time dependently after SNP infusion by a factor of more than two. Likewise, the number of nNOS-immunopositive neurons was increased after SNP compared with vehicle infusion. Around the central canal the number of NADPH-diaphorase-positive neurons was slightly increased and the number of nNOS+ neurons not changed after SNP treatment. NO donors increase the number of neurons that produce NO in the STN, possibly by induction of nNOS expression. Increased NO production may facilitate neurotransmitter release and promote nociceptive transmission in the STN. This mechanism may explain the delayed increase in neuronal activity and headache after infusion of NO donors.

Galanin modulates vagally induced contractions in the mouse oesophagus.

Nitrergic myenteric neurons co-innervating motor endplates were previously shown to inhibit vagally induced contractions of striated muscle in the rodent oesophagus. Immunohistochemical demonstration of putative co-transmitters, e.g. galanin, in enteric neurons prompted us to study a possible role of galanin in modulating vagally mediated contractions in an in vitro vagus nerve-oesophagus preparation of the mouse. Galanin (1-16) (1-100 nmol L(-1)), in the presence of the peptidase inhibitor, phenanthroline monohydrate, inhibited vagally induced contractions in a concentration-dependent manner (control: 100%; galanin 1 nmol L(-1): 95.6 +/- 1.6%; galanin 10 nmol L(-1): 57.3 +/- 6.5%; galanin 100 nmol L(-1): 31.2 +/- 8.1%, n = 5). The non-selective galanin receptor antagonist, galantide (100 nmol L(-1)), blocked the inhibitory effect of galanin (10 nmol L(-1)) while the selective non-galanin receptor 1 and galanin receptor 3 antagonists, M871 (1 micromol L(-1)) and SNAP37889 (100 nmol L(-1)), respectively, and the nitric oxide synthase inhibitor, NG-nitro-l-arginine methyl ester (L-NAME) (200 micromol L(-1)), failed to affect this galanin-induced response. Simultaneous application of galantide (100 nmol L(-1)) and L-NAME (200 micromol L(-1)) significantly reduced the inhibitory effect of capsaicin (30 mumol L(-1)) on vagally induced contractions when compared with its effect in the presence of L-NAME alone or in combination with the selective galanin receptor 2 or 3 antagonists. An inhibitory effect of piperine on vagally induced contractions was reduced neither by galantide nor by L-NAME. Immunohistochemistry revealed galanin immunoreactive myenteric neurons and nerve fibres intermingling with cholinergic vagal terminals at motor endplates. These data suggest that galanin from co-innervating enteric neurons co-operates with nitric oxide in modulating vagally induced contractions in the mouse oesophagus.

Enteric co-innervation of striated muscle fibres in human oesophagus.

Oesophageal striated muscle of several mammalian species receives dual innervation from both vagal motor fibres originating in the brain stem and enteric nerve fibres originating in myenteric ganglia. The aim of this study was to investigate this so-called enteric co-innervation in the human oesophagus. Histochemical and immunohistochemical methods combined with confocal laser scanning microscopy were utilized to study innervation of 14 oesophagi obtained from body donors (age range 47-95 years). In addition, the distribution of striated and smooth muscle in longitudinal and circular layers of the tunica muscularis was studied semiquantitatively. The upper half of the oesophagus was built up of both muscle types with a predominance (>50-60%) of striated muscle, whereas the lower half consisted of smooth muscle only. The majority of motor endplates was compact and ovoid. Enteric nerve fibres on approximately 17% of motor endplates stained for neuronal nitric oxide synthase, vasoactive intestinal polypeptide, galanin and neuropeptide Y and were completely separated from vagal cholinergic nerve terminals. There was remarkable variability of co-innervation rates between striated muscle bundles with some reaching almost 50%. Myenteric neurons representing the putative source of enteric co-innervating nerve fibres, stained for all these markers, which were almost completely colocalized with NADPH-diaphorase. Our study provides evidence for enteric co-innervation of striated muscle in human oesophagus. From these and recent functional results in various rodent species, we suggest that this innervation component represents an integral part of an intramural reflex mechanism for local most likely inhibitory modulation of oesophageal motility.

Morphological characterization of rat Mas-related G-protein-coupled receptor C and functional analysis of agonists.

A recently described family of "orphan" receptors, called Mas-related G-protein-coupled receptors (Mrg), is preferentially expressed in small nociceptive neurons of the rodent and human dorsal root ganglia (DRG). Mrg are activated by high affinity peptide fragments derived from the proenkephalin A gene, e.g. BAM22 (bovine adrenal medullary). To study the histological distribution and functional properties of these receptors, we combined confocal immunohistochemistry in rat DRG and dermis whole mounts, using new antibodies against the rat Mas-related G-protein-coupled receptor C (MrgC), with single-fiber recordings and neurochemical experiments using isolated hind-paw skin and sciatic nerve. In lumbar DRG we found cytoplasmic MrgC labeling mainly in small- and medium-sized neurons; coexpression with isolectin B4 (46%) and transient receptor potential vanilloid receptor 1 channel protein (TRPV1) (52%) occurred frequently, whereas calcitonin gene-related peptide (CGRP) was rarely colocalized with MrgC in DRG (11%) and dermal nerve fibers (6%). One of the MrgC agonists, BAM22, more than doubled the heat-induced cutaneous CGRP release from rat and mouse skin. The effect of BAM22, also known to activate opioid receptors, was further enhanced by combination with naloxone that had no effect on its own. This sensitizing effect proved to be independent of secondary prostaglandin formation, mast cell degranulation, protein kinase C (PKC) activation and independent of TRPV1. Nonetheless, the capsaicin-induced CGRP release was also sensitized. Receptive fields of 26 mechano-heat sensitive C-fibers were treated with MrgC agonists. Only one unit was strongly and repeatedly excited and showed a profound sensitization to heat upon BAM22+naloxone. Two other established MrgC agonists (gamma2-melanocyte stimulating hormone and BAM8-22) were ineffective. Thus, BAM22 sensitizes the capsaicin- and heat-induced CGRP release in an apparently MrgC-unrelated way. The sensitization to heat appears unusually resistant against pharmacological interventions and does not involve TRPV1.

Distribution of vesicular glutamate transporter 1 (VGLUT1) in the mouse esophagus.

In rat and mouse esophagus, vesicular glutamate transporter 2 (VGLUT2) has been demonstrated to identify vagal intraganglionic laminar endings (IGLEs); this has recently also been shown for VGLUT1 in rat esophagus. In this study, we have investigated the distribution of VGLUT1 in the mouse esophagus and compared these results with the recently published data from the rat esophagus. Unexpectedly, we have discovered that VGLUT1 mostly fails to identify IGLEs in the mouse esophagus. This is surprising, since the distribution of VGLUT2 shows comparable results in both species. Confocal imaging has revealed substantial colocalization of VGLUT1 immunoreactivity (-ir) with cholinergic and nitrergic/peptidergic markers within the myenteric neuropil and in both cholinergic and nitrergic myenteric neuronal cell bodies. VGLUT1 and cholinergic markers have also been colocalized in fibers of the muscularis mucosae, whereas VGLUT1 and nitrergic markers have never been colocalized in fibers of the muscularis mucosae, although this does occur in fibers of the muscularis running to motor endplates. Thus, VGLUT1 is contained in the nitrergic innervation of mouse esophageal motor endplates, another difference from the rat esophagus. VGLUT1-ir is therefore present in extrinsic and intrinsic innervation of the mouse esophagus, but the significant differences from the rat indicate species variations concerning the distribution of VGLUTs in the peripheral nervous system.