GLP-2 receptor expression in excitatory and inhibitory enteric neurons and its role in mouse duodenum contractility.

Background. Glucagon-like peptide 2 (GLP-2), a nutrient-responsive hormone, exerts various actions in the gastrointestinal tract that are mediated by a G-protein coupled receptor called GLP-2R. A little information is available on GLP-2R expression in enteric neurons and nothing on the interstitial cells of Cajal (ICC). Methods. We investigated presence and distribution of the GLP-2R in the mouse duodenum by immunohistochemistry and the potential motor effects of GLP-2 on the spontaneous and neurally evoked mechanical activity. Key Results. The GLP-2R was expressed by the myenteric and submucosal neurons. Labelling was also present in nerve varicosities within the circular muscular layer and at the deep muscular plexus (DMP). No immunoreactive nerve fiber was seen within the longitudinal muscle layer. The GLP-2R-positive neurons were either excitatory (SP- and choline-acetyltransferase-positive) or inhibitory (vasoactive intestinal polypeptide and nNOS-positive). The ICC, both at the myenteric plexus and at theDMP,never expressed GLP-2R but, especially those at the DMP, were surrounded by GLP-2R-positive nerve varicosities co-expressing either excitatory or inhibitory neurotransmitters. Quantitative analysis demonstrated a consistent prevalence of GLP-2R on the excitatory pathways. In agreement, the functional results showed that the administration of GLP-2 in vitro caused decrease of the spontaneous contractions mediated by nitric oxide release and reduction of the evoked cholinergic contractions. Conclusions & Inferences. The present findings indicate that the GLP-2R is expressed by inhibitory and excitatory neurons, the GLP-2 inhibits the muscle contractility likely decreasing cholinergic neurotransmission and increasing nitric oxide production, and this effect is possibly mediated by the ICC-DMP recruitment

Telocytes

AbstractHere, we review the history, morphology, immunohistochemical phenotype, and presumptive roles of a new type of interstitial tissue cells, formerly called interstitial Cajal-like cells (ICLC) and by 2010 named 'telocytes' (TC). Many different techniques have been used to characterize TC and provide their unequivocal identification: (i) in vitro, cultures and isolated cells; (ii) in situ, fixed specimens examined by light and fluorescence microscopy, transmission (TEM) and scanning electron microscopy, and electron tomography. TEM allowed sure identification and characterization of the most peculiar feature of TC: the long, thin, and convoluted prolongations named 'telopodes'. An enormous variety of antibodies have been tested, but presently none are reliable to specifically label TC. TC have a mesenchymal origin and are resident connective tissue (stromal) cells. Possible identification with 'already identified' stromal cell types (fibroblasts, fibrocytes, fibroblast-like cells, and mesenchymal stromal cells) is discussed. We conclude that in adulthood, most of the TC have the morphology of fibrocytes. Apparently, immunocytochemistry suggests that a variety of TC populations showing different, likely organ-specific, immunophenotypes might exist. Several roles have been hypothesized for TC: mechanical roles, intercellular signaling, guiding and nursing of immature cells during organogenesis, and being themselves a pool of precursors for many of the mesenchyme-derived cells in adulthood; however, none of these roles have been proven yet. On the basis of the available data, we propose TC may be key players in organ regeneration and repair

Ultrastructural differences between diabetic and idiopathic gastroparesis

The ultrastructural changes in diabetic and idiopathic gastroparesis are not well studied and it is not known whether there are different defects in the two disorders. As part of the Gastroparesis Clinical Research Consortium, full thickness gastric body biopsies from 20 diabetic and 20 idiopathic gastroparetics were studied by light microscopy. Abnormalities were found in many (83%) but not all patients. Among the common defects were loss of interstitial cells of Cajal (ICC) and neural abnormalities. No distinguishing features were seen between diabetic and idiopathic gastroparesis. Our aim was to provide a detailed description of the ultrastructural abnormalities, compare findings between diabetic and idiopathic gastroparesis and determine if patients with apparently normal immunohistological features have ultrastructural abnormalities. Tissues from 40 gastroparetic patients and 24 age‐ and sex‐matched controls were examined by transmission electron microscopy (TEM). Interstitial cells of Cajal showing changes suggestive of injury, large and empty nerve endings, presence of lipofuscin and lamellar bodies in the smooth muscle cells were found in all patients. However, the ultrastructural changes in ICC and nerves differed between diabetic and idiopathic gastroparesis and were more severe in idiopathic gastroparesis. A thickened basal lamina around smooth muscle cells and nerves was characteristic of diabetic gastroparesis whereas idiopathic gastroparetics had fibrosis, especially around the nerves. In conclusion, in all the patients TEM showed abnormalities in ICC, nerves and smooth muscle consistent with the delay in gastric emptying. The significant differences found between diabetic and idiopathic gastroparesis offers insight into pathophysiology as well as into potential targeted therapies.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92113/1/jcmm1451.pd

From conception to birth: ancient library sources of embryology and women anatomy kept in the Biblioteca Biomedica of the Università degli Studi di Firenze (Biomedical Library of Florence University)

The Biomedical Library of the University of Florence boasts a prestigious group of books collected at first in 1679 at the hospital “Santa Maria Nuova” and then continuously enriched in the course of time up today. The “Antique Collection” consists of 13 incunabola, hundreds of 16th-century books, more than one thousand books on medical subject from the 1600’s, about six thousand 18th-century volumes and several large, valuable anatomical atlases. In this paper the most important, curious and fascinating books dealing with human ontogeny (from embryo generation to birth) and with female anatomy (mostly concerning pregnancy and childbirth) are reported in chronological order starting from the work of Hippocrates. Among the ancient sources useful for the reconstruction of the opinions about obstetrics there are also outstanding handbooks specifically edited for midwives. Many of these antique books are especially precious because they embed a great number of didactic pictures, some of which may compete against any modern book of anatomy, embryology and obstetric. Selected images from these books are shown

NK-receptors, Substance P, Ano1 expression and the ultrastructural features of the muscle coat are modified in the Cav-1-/- mouse ileum

Caveolin (Cav)-1 is an integral membrane protein of caveolae playing a crucial role in various signal transduction pathways. Caveolae represent the sites for calcium entry and storage especially in smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Cav-1-/- mice lack caveolae and show abnormalities in pacing and contractile activity of the small intestine. In particular, the absence of caveolae in ICC compromised their ability to maintain frequencies of contraction. Presently, we investigated, by transmission electron microscopy (TEM) and immunohistochemistry, whether the absence of Cav-1 in Cav-1-/- mouse small intestine affects ICC, SMC and neuronal morphology, the expression of NK1 and NK2 receptors, and of Ano1 (also called Dog1 or TMEM16A) an essential molecule for slow wave activity in gastrointestinal muscles. ICC were also labeled with c-Kit and tachykinergic neurons with Substance P (SP). Immunohistochemical results showed that in Cav-1-/- mice: i) ICC were Ano1-negative but maintained c-Kit expression, ii) NK1 and NK2 receptor immunoreactivity was increased and, in the SMC, mainly intracytoplasmatic, iii) SP-immunoreactivity was significantly reduced. Under TEM: i) ICC and SMC lacked typical caveolae but had few and large flask-shaped vesicles we called large-sized caveolae; ii) SMC and ICC contained an extraordinary high number of mitochondria; iii) neurons were unchanged. In conclusion, the present study shows important changes in SMC, ICC and neurons of the Cav-1-/- mice. Loss of Ano1 expression in the ICC and rearrangement of NK receptors in the SMC are interpretable as consequence of Cav-1/caveolae loss and possibly responsible for the impaired contractile activity. However, the impressive richness in mitochondria and the decrease in SP content might represent the ways to compensate the reduced calcium availability and the increased expression of NKr, allowing the maintenance of a certain cell function

Inner and Outer Portions of Colonic Circular Muscle: Ultrastructural and Immunohistochemical Changes in Rat Chronically Treated with Otilonium Bromide

Rat colonic circular muscle, main target of otilonium bromide (OB) spasmolytic activity, is subdivided in an inner and outer portion. Since the inner one is particularly rich in organelles involved in calcium availability (caveolae, smooth endoplasmic reticulum, mitochondria), the expression of specific markers (Caveolin-1, eNOS, calreticulin, calsequestrin) in comparison with the outer portion was investigated. The possible changes of these organelles and related markers, and of muscarinic receptors (Mr2) were then studied after OB chronic exposition. Rats were treated with 2-20 mg/kg/OB for 10 or 30 days. Proximal colon was processed by electron microscopy, immunohistochemistry, and western blot. In colon strips the stimulated contractility response to muscarinic agonist was investigated. The inner portion showed a higher expression of Caveolin-1 and Mr2, but not of eNOS, calreticulin and calsequestrin, compared to the outer portion. Chronic OB treatment caused similar ultrastructural and immunohistochemical changes in both portions. Organelles and some related markers were increased at 10 days; Mr2 expression and muscle contractility induced by methacholine was increased at 30 days. The present findings: 1) provide new information on the immunohistochemical properties of the inner portion of the circular layer that are in favour of a role it might play in colonic motility distinct from that of the outer portion; 2) demonstrate that chronically administered OB interferes with cell structures and molecules responsible for calcium handling and storage, and modifies cholinergic transmission. In conclusion, chronic OB administration in the colonic circular muscle layer directly interacts with the organelles and molecules calcium-related and with the Mr2

The wrap partial restrain stress, an animal model of the irritable bowel syndrome: immunohistochemical and functional characterization

Several animal models have been proposed to mimic the human irritable bowel syndrome (IBS) all based on two etio-pathogenic hypotheses: infection and stress, both responsible for the development of a local inflammation. We investigated the wrap partial restrain stress (WRS) animal model with the aim to evaluate its validity in understanding the human IBS. Male Wistar rats were used and WRS was maintained for 2h. Abdominal contractions (AC) were recorded by a distension balloon in the colon-rectum. The number of faecal pellets and their total weight were determined. Colonic specimens from both groups were examined by routine histology, immunohistochemistry and western blot (WB). WRS animals were characterized by: 1) a statistically significant increase in the number of AC and in the mean number and mean weight of faecal pellets; 2) the presence of large clusters of mononucleated cells and a significant increase in eosinophilic granulocytes and mast cells in the mucosa; 3) reduction of GLP1r-immunoreactivity (IR) located at the basolat- eral periphery and the Golgi level of the cells of the glandular funds; 4) an increase in CGRP-IR in the lamina propria; 5) no significant difference in the muscle wall for Cav1, L- type Ca+2-channels, Mr2, NK1r and NK2r; 6) a significant decrease in the myenteric and a significant increase in the submucous NK1-IR neuron number; 7) a significant decrease in Substance P-IR in the myenteric plexus and muscle coat; 8) a significant decrease in myenteric and submucous nNOS-IR neuron number; 9) no difference in ChAT-IR neurons of both enteric plexuses; 10) a reduction in S-100-IR in the entire colonic wall; 11) no difference in the total number of neurons evaluated by the pan-neuronal marker PGP 9.5; 12) no change of all the ICC populations. The functional data are in favor of a lowering in the colonic wall distention threshold; the morphological results obtained in the lamina propria demonstrate the presence of a local inflammation, particularly intense at the level of the mucosa. Both of these findings agree with the hypothesis that inflammation might have a main role in the insurgence and maintenance of the typical IBS symptoms and support the validity of our WRS model. Moreover, while the smooth muscle cells do not show any significant variation, numerous and consistent changes in the excitatory, inhibitory and NK1r-IR neurons are detected