Morphogenetic events in the perinodal connective tissue in a metastatic cancer model.

BACKGROUND: The modifications of connective tissue surrounding metastatic lymph nodes in a murine model of rectal cancer are described. METHODS: Athymic nude mice (n=36) were inoculated with 10×10(5) ht-29 cancer cells into the submucosal layer of the rectum. Control mice (n=5) were treated with a sterile buffer. Tumor and the involved lymph nodes were visualized in vivo by magnetic resonance imaging at 1 to 4 weeks after cell injection. After the sacrifice, the excised samples were processed for histology. RESULTS: After one week from cell injection all treated animals developed rectal cancer. Since the first week, neoplastic cells were visible in the nodes. In the surrounding connective tissue, the diameter of the adipocytes was reduced and a mesenchymal-like pattern with stellate cells embedded in an oedematous environment was visible. Since the second week, in the perinodal connective an enlargement of the stroma was present. The tissue was organized in cords and areas with extracellular accumulation of lipids were found. At the fourth week, we observed an enlargement of multilocular areas and lobules of elongated elements almost devoid of lipid droplets. In control animals, in absence of neoplastic masses, pelvic nodes were surrounded by a typical connective tissue characterized by unilocular adipocytes with groups of multilocular adipocytes. CONCLUSIONS: We have developed a model of rectal cancer with nodal metastases. Using this model, the work demonstrates that around secondary lesions, the morphogenetic events follow a standard evolution characterized by an early phase with lipolysis and mesenchymalization and later phases with a brown-like phenotype acquisition.

The solitary chemosensory cells and the diffuse chemosensory system of the airway.

Solitary chemosensory cells (SCCs), which resemble taste bud cells, are present in the epidermis and oropharynx of most primary aquatic vertebrates. Recent studies have led to the description of SCCs also in mammals too. In the airway and digestive apparatus, these elements form a diffuse chemosensory system. SCCs do not aggregate into groups and in SCCs, as in taste bud cells, immunoreactivity forthe G-protein subunit alpha-gustducin and for other molecules of the chemoreceptive cascade was found. Questions remain about the role of the diffuse chemosensory system in control of complex functions (e.g. airway surface liquid secretion) and about the involvement of chemoreceptors in respiratory diseases. Therapeutic actions targeting chemoreceptors could be tested in the treatment of respiratory diseases.

Allelochemical communication in vertebrates: kairomones, allomones and synomones.

Communication between different species by means of chemicals (allelomones) is widespread among prokaryotes, plants and invertebrates. This study reviews data suggesting that allelochemically mediated communication also exists among vertebrates. The work aims to provide a concise, interdisciplinary review of communication mediated by infochemicals, with a focus on interspecies and interkingdom signaling. A definition of infochemicals is given, with a brief review of the general principles of chemical communication in different kingdoms in nature. Findings are reported which suggest that interspecies chemical signaling is important for vertebrates also. It is proposed that the general laws of chemical ecology are valid for mammals too, and that the terms indicating the different types of allelomones (i.e. kairomone, allomone and synomone) might also be used in medicine. In particular, the microchemical environment at the airway and digestive interfaces are discussed from an infochemical point of view.

The taste cell-related diffuse chemosensory system.

Elements expressing the molecular mechanisms of gustatory transduction have been described in several organs in the digestive and respiratory apparatuses. These taste cell-related elements are isolated cells, which are not grouped in buds, and they have been interpreted as chemoreceptors. Their presence in epithelia of endodermal origin suggests the existence of a diffuse chemosensory system (DCS) sharing common signaling mechanisms with the "classic" taste organs. The elements of this taste cell-related DCS display a site-related morphologic polymorphism, and in the past they have been indicated with various names (e.g., brush, tuft, caveolated, fibrillo-vesicular or solitary chemosensory cells). It may be that the taste cell-related DCS is like an iceberg: the taste buds are probably only the most visible portion, with most of the iceberg more caudally located in the form of solitary chemosensory cells or chemosensory clusters. Comparative anatomical studies in lower vertebrates suggest that this 'submerged' portion may represent the most phylogenetically ancient component of the system, which is probably involved in defensive or digestive mechanisms. In the taste buds, the presence of several cell subtypes and of a wide range of molecular mechanisms permits precise food analysis. The larger, 'submerged' portion of the iceberg is composed of a polymorphic population of isolated elements or cell clusters in which the molecular cascade of cell signaling needs to be explored in detail. The little data we have strongly suggests a close relationship with taste cells. Morphological and biochemical considerations suggest that the DCS is a potential new drug target. Modulation of the respiratory and digestive apparatuses through substances, which act on the molecular receptors of this chemoreceptive system, could be a new frontier in drug discovery.

A new fate for old cells: brush cells and related elements.

Over the past 50 years, hundreds of studies have described those cells that are characterized by a brush of rigid apical microvilli with long rootlets, and which are found in the digestive and respiratory apparatuses. These cells have been given names such as brush cells, tuft cells, fibrillovesicular cells, multivesicular cells and caveolated cells. More recently, it has been realized that all these elements may represent a single cell type, probably with a chemosensory role, even if other functions (e.g. secretory or absorptive) seem to be possible. Very recent developments have permitted a partial definition of the chemical code characterizing these elements, revealing the presence of molecules involved in chemoreceptorial cell signalling. A molecular cascade, similar to those characterizing the gustatory epithelium, seems to be present in these elements. These new data suggest that these elements can be considered solitary chemosensory cells with the presence of the apical 'brush' as an inconsistent feature. They seem to comprise a diffuse chemosensory system that covers large areas (probably the whole digestive and respiratory apparatuses) with analogies to chemosensory systems described in aquatic vertebrates.

Neurochemistry of the gustatory subgemmal plexus.

Nerve fibers present in the basal plexus of the vallate papilla of the rat tongue were analyzed using cytochemical, immunocytochemical and ultrastructural methods to investigate whether the subgemmal plexus is subdivided into neurochemical compartments and to provide a clear definition of the reciprocal spatial relationships between nitrergic, peptidergic and acetylesterase positive structures. Several neuronal fibers were detected under the chemoreceptorial epithelium. Some of these fibers were in contact with the taste buds and in some cases neuronal projections were also present between the buds or inside them; some others fibers were present below this layer but in a more peripheral area. Antibodies against CGRP, SP and CCK stained fibers just below the chemoreceptorial epithelium, whereas fibers more distally located were immunolabeled by anti VIP, NOS-1 and NF-200 antibodies. Some double staining experiments were conducted using confocal microscopy. Other sections were processed cytochemically for AChE and subsequently for NADPH-d in colocalization experiments. All the data obtained using these techniques confirmed the results obtained with single immunostaining, as did the ultrastructural results. In conclusion, the present work demonstrates that the subgemmal plexus is a bilayered structure, suggesting that the complex relationship between the two layers plays a pivotal role in taste and in the control of processes ancillary to taste, such as control of vascular or secretory mechanisms.

Solitary chemosensory cells in mammals?

In fish, solitary chemosensory cells (SCCs) occur in the oropharynx, gills and skin and have often been found in association with taste buds. Among amphibia, a diffuse chemosensory system has been described on the ventral skin of toads, and a structural resemblance of SCCs to taste bud cells has been reported in frogs. Putative solitary chemoreceptors have been described in mammals too, at specific sites in the digestive or respiratory apparatus. In newborn rodents, a specific set of SCCs (composed of elements positive for alpha-gustducin, a marker of chemosensory cells) is associated with the gustatory epithelium. In conclusion, the available data suggest that a SCC system is not restricted to fish but is present in amphibia and mammals as well. At our present level of knowledge, establishing a precise homology between different species is difficult. However, the data from mammals and amphibia fully confirm previous findings in fish, and the use of chemical markers to study the diffuse chemosensory systems of vertebrates seems promising.

Correlation MRI/ultrastructure in cerebral ischemic lesions: application to the interpretation of cortical layered areas.

The origin and fate of cortical ischemic lesions, showing a stratified appearance at in vivo MRI-examination, was studied on rats in which a focal brain ischemia was induced by occlusion of the middle cerebral artery. One week after ischemia induction, six rats were selected in which three layers of different intensity were visible in the lesioned cortex. Two animals were sacrificed and studied by histology and electron microscopy. The external hyperintense layer was composed of pial and lesioned nervous tissue, the intermediate of degenerating nervous tissue in which an accumulation of macrophages was found, the deepest of edematous nerve tissue without a marked accumulation of macrophages. The remaining rats underwent further MRI examinations showing that, in the lesioned areas, cerebral blood volume was 14-69% lower than the contralateral healthy cortex. At histological and ultrastructural examination, a large part of the lesion was occupied by enlarged pial tissue and marginal glia. A dilatation of the ventricular cavity and cystic structures were also visible. In three animals an increase of the transverse diameter of the caudo-putamen ipsilateral to the lesion was found. The study suggests that the layered appearance is mainly due to an accumulation of macrophages in the intermediate layer and that several processes contribute to the occlusion of the space created by the removal of the necrotic tissue in stratified ischemic lesions (i.e. expansion of the pial tissue, thickening of the marginal glia; expansion of the caudo-putamen, enlargement of the ventricular cavity and development of cystic structures).

Ultrastructural phenotype of "intestinal-type" cells in columnar-lined esophagus.

An intestinal-type epithelium is often present at columnar-lined esophagus, gastroesophageal junction or within the so-called short segment Barrett's esophagus, but ultrastructural study failed to detect enterocytes in columnar-lined esophagus. The authors have analyzed the intestinal aspects present in areas of columnar-lined esophagus in a population of patients with reflux esophagitis to better understand the morphology and histogenesis of the proliferating elements. Columnar-lined mucosa was studied in 35 patients. Columnarsurface cells displayed a wide spectrum of ultrastructural features. Well-differentiated columnar secretory cells, secretory-absorptive cells, poorly differentiated columnar cells, atypical columnar cells, and goblet cells were detected. Well-differentiated absorptive cells were never found, These results demonstrate that the areas of intestinal metaplasia show a wide spectrum of ultrastructural phenotypes, ranging from poorly to well-differentiated cells. However, true enterocytes were not found and the most represented phenotype is that of secretory-absorptive cells, whose principal characteristic is the presence of secretory and absorptive aspects together. They can be described as secretory enterocytes or cells with double specialization. To the authors' knowledge, similar cells were not previously described in normal intestinal mucosa, and ultrastructural studies are consistent in describing a broad spectrum of ultrastructural features, suggesting that Barrett's specialized metaplasia is derived from cells with the capacity for a wide range of differentiation. Therefore, despite the wide use of term intestinal metaplasia in the medical literature, experimental data clearly failed to detect enterocytes in the columnar-lined esophagus, and ultrastructural data do not support the concept of intestinal metaplasia. The cellular heterogeneity seems to be the result of a "phenotypic shift" of undifferentiated elements, which show a different pattern of evolution. The result of this process is the formation of new cell types dissimilar from those normally present in esophageal, gastric, or duodenal mucosa.

Polyunsaturated fatty acids mapping by (1)H MR-chemical shift imaging.

Parametric mapping of polyunsaturated fatty acids (PUFA) distribution in adipose tissues was obtained by (1)H chemical shift imaging (CSI). A matrix of spectra, acquired with a CSI sequence having two spatial and one spectroscopic dimension, was processed with ad hoc algorithms. The protocol was applied to phantoms containing different lipids in which the degree of polyunsaturation was determined by high-resolution nuclear magnetic resonance (NMR). High correlation (R(2) = 0.998) between degrees of polyunsaturation given by our protocol and that measured by high-resolution NMR was found. The thoracic region of rats was also examined. Parametric maps of the polyunsaturation degree were obtained for the brown adipose tissue and the white axillary fat: the first deposit was found more polyunsaturated than the second. Finally, in vivo mapping of the inguinal region of the rat was produced that allowed us to individuate PUFA-rich areas in adipose tissue. This work demonstrates the feasibility of PUFA imaging in vivo.

Delayed muscle injuries in arterial insufficiency: contrast-enhanced MR imaging and 31P spectroscopy in rats.

PURPOSE: To evaluate whether the vascular system resulting from an arterial lesion shows differences in permeability to a tracer with respect to the normal vascular system and whether eventual differences are maintained for long periods. MATERIALS AND METHODS: Permanent ischemia was induced in rats with femoral arterial removal, and magnetic resonance (MR) imaging was performed after 1, 7, 14, and 90 days. Gadopentetate dimeglumine was injected, and the kinetics of its penetration in the leg were studied. Phosphorus 31 spectroscopy was performed to determine the bioenergetic characteristics of the gastrocnemius muscle at rest and stimulation. Ischemic muscles were then processed for electron microscopy. RESULTS: After ischemia induction, a hyperintense area that progressively decreased was present on T2-weighted images. Gadopentetate dimeglumine improved the signal intensity of the area. Three months after arterial occlusion, the contrast-enhanced images still showed microvessels highly permeable to the tracers. Spectroscopic data revealed that 3 months after arterial removal, the bioenergetic reserve of the gastrocnemius muscle was reduced, suggesting that the contrast-enhanced MR imaging-visible area is functionally relevant. Ultrastructural examination revealed persistent muscle damage and signs of chronic microangiopathy. CONCLUSION: After ischemia induction, the restitutio ad integrum is not complete, and delayed muscle injuries can result from arterial insufficiency.

A brief survey of the modifications in sensory-secretory organs of the neonatal rat tongue.

Recent data obtained on rats suggest that in the days immediately following birth several events take place in the circumvallate papillae of the oral cavity. A phylogenetically primitive system of solitary chemosensory cells develops and is rapidly replaced by taste buds. The lipase-secreting von Ebner gland, which is associated with taste organs, begins to develop by forming short tubules. The intrinsic nervous system of the gustatory organs rapidly completes its maturation showing fast proliferation of fibers and immunocytochemical maturation. Intraepithelial lipid accumulation is visible in the non-receptorial mucosa of the tongue, showing aspects which suggest an active lipid secretion. These data demonstrate that in the rat the structure of the sensory-secretory organs of the newborn's tongue shows a typical conformation with respect to the adult and rapidly changes its organization in the first week after the birth. At the present level of knowledge, it is difficult to link the anatomical structures to peculiar functional roles but the rather simple organization of the neonatal gustatory epithelium could be in relation to the dietary regimen. The data obtained in laboratory animals underline the necessity of studies on human newborns to update the anatomical knowledge of the oral chemoceptive system.

The neuroprotective activity of the glycine receptor antagonist GV150526: an in vivo study by magnetic resonance imaging.

The neuroprotective activity of GV150526 (3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxylic acid sodium salt), a selective glycine receptor antagonist of the NMDA receptor, has been evaluated by magnetic resonance imaging (MRI) in a rat model of middle cerebral artery occlusion. The aim of the work was to evaluate, using an in vivo method, whether GV150526 was able to reduce the extent of ischemic brain damage when administered both before and after (6 h) middle cerebral artery occlusion. GV150526 was administered at a dose of 3 mg/kg i.v. T2-weighted (T2W) and diffusion weighted (DW) images were acquired at 6, 24 and 144 h after the establishment of the cerebral ischemia. Substantial neuroprotection was demonstrated at all investigated time points when GV150526 was administered before the ischemic insult. The ischemic volume was reduced by 84% and 72%, compared to control values, when measured from T2W and DW images, acquired 24 h after middle cerebral artery occlusion. Administration of the same dose of GV150526, 6 h post-ischemia, also resulted in a significant (p < 0.05) neuroprotection. The ischemic volume was reduced by 48% from control values when measured from T2W images and by 45% when measured from DW images. No significant difference was found between volumes of brain ischemia obtained by either MRI or triphenyltetrazolium chloride staining. These data confirm the potential neuroprotective activity of the glycine receptor antagonist GV150526 when administered either before or up to 6 h after ischemia.

Postnatal development of the intrinsic nervous system in the circumvallate papilla-vonEbner gland complex.

We have studied the postnatal development of the intrinsic nervous system in the circumvallate papilla-vonEbner gland complex using NADPH-diaphorase cytochemistry, immunocytochemistry (for nitric oxide synthase-1 and alpha-internexin) and electron microscopy. In rats sacrificed in their first day post partum (1 p.p.), only isolated NADPH-diaphorase positive neurons were visible in the organ. At 2 p.p., a small group of neurons was visible at the base of the papillae and positive neurons formed short chains close to the developing glandular tubules. In the following days, the NADPH-diapharase positive cells increased in number and nerve fibres were associated to small ganglia located at the base of the papilla or in the gland. After the first week of extrauterine life, the intrinsic nervous system was similar to the intrinsic system of adult animals. An immunocytochemical positivity for nitric oxide synthase-1 appeared at 4 p.p. in neurons located in the gland and at 7 p.p. in cells located at the base of the papilla. Immunocytochemical staining for alpha-internexin showed that at 1 p.p. developing nerve fibres were present in the connective tissue of the tongue's muscle layer. At 2-3 p.p., developing nerve fibres were also present at the bases and in the core of the papilla. In the following days, the positivity for alpha-internexin was reduced and one week after birth was virtually absent. Ultrastructural examination revealed that since 1 p.p. isolated neurons can be found at the base of the papilla. In conclusion, the intrinsic nervous system originates from neurons present in the organ at the birth which, in the first days, undergo a biochemical and morphological maturation while the nerve fibres rapidly grow. These findings support the hypothesis that the intrinsic nervous system of the circumvallate papilla has a role in the maturation of the vonEbner gland.

The glio-pial system in cortical ischemic lesions.

The ultrastructural features of the glio-pial system have been analyzed in an animal model of brain focal ischemic lesion. Aim of the work was to describe the relationship between glial and pial cells in a condition of ischemic neurodegeneration and the eventual involvement of these cells in removal of debris and reconstruction of the glial-limiting lamina. The study was performed in rats in which the right middle cerebral artery was occluded. The development of a lesion and its extension was controlled in vivo 24 h after the middle cerebral artery occlusion by magnetic resonance imaging. The rats were sacrificed 10 or 15 days after the occlusion and their brain was processed for transmission and scanning electron microscopy. The ultrastructural examination of the lesion revealed that the nervous parenchyma was replaced by cicatricial tissue composed by glial and pial areas. The pial areas were composed mainly by leaf-like cells forming a network. Their main cytoplasmic feature was the presence of large lysosomes in which ferritine-like particles were often enclosed. The glial areas were composed by strictly packed cells which displayed at scanning electron microscopy a spider-like shape with a central body and elongated cell processes. These results suggest a role for the glio-pial system in acute ischemia. In particular, the pial cells seem to be directly involved in removal of cell debris while glial cells seem mainly devoted to the reconstruction of the glial limiting lamina.

Regional cerebral blood volume mapping after ischemic lesions.

The possible persistence of a microvascular deficit at long time intervals after cerebral ischemia induction is not well established. In rats, we have generated in vivo maps of the regional cerebral blood volume (rCBV) at different time intervals after middle cerebral artery occlusion (MCAo) with the aim to evaluate the persistence of a rCBV deficit in the damaged area or in the surrounding regions. The rats were examined by magnetic resonance imaging (MRI) at different time intervals, starting from the first day until three months after ischemia and postmortem histological and ultrastructural correlation was obtained. All MRI experiments were carried out using an imager-spectrometer equipped with a 4.7 Tesla magnet. To produce the susceptibility-weighted rCBV images, a suspension of superparamagnetic iron oxide nanoparticles (AMI-25) was injected to the rat. In a control group (nonoperated or sham-operated rats), a symmetrical distribution of rCBV values was found between the two hemispheres (differences between left and right cortex below 8%). In the rats with MCAo an evident vascular asymmetry was found 24 h after ischemia (differences between left and right ranging from 22 and 77%) and reduced rCBV values were evident in the ischemic areas. In a time range following the 15th day most of the rats showed a complete recovery of the lesion while only four animals still had a small residual lesion, as probed by T2-weighted (T2W) images. In three of these four cases, the reduction of rCBV in the ipsilateral cortex with respect to the contralateral was greater than 20%. Correlation was found (Y > 0.8) between late rCBV measurement and the initial volume of the lesion (hyperintense region in T2W images). The postmortem measurements correlate much better with the rCBV data than with the T2W ones. In conclusion, the present work demonstrates that cortical lesions may result in a deficit of rCBV for long periods and that a mismatch between T2w and rCBV data can be present during the repair process.

Comparison of results of scanning electron microscopy and magnetic resonance imaging before and after administration of a radiographic contrast agent in the tendon of the deep digital flexor muscle obtained from horse cadavers.

OBJECTIVE: To analyze the tendon of the deep digital flexor (TDDF) muscle of the forelimb in horses by use of a contrast radiographic agent (gadopentate dimeglumine [Gd-DTPA/Dimeg]) and magnetic resonance imaging (MRI) and to determine the concentration of water protons in the tendons by use of MRI. SAMPLE POPULATION: 8 TDDF harvested from the forelimbs of 6 horse cadavers. PROCEDURE: Examinations were performed on the same portion of each tendon. Tendons were examined by use of two techniques: MRI before and after treatment with Gd-DTPA/Dimeg as well as scanning electron microscopy. RESULTS: Tendons did not have detectable signal intensity on MRI before treatment with Gd-DTPA/Dimeg; however, intravascular injection of Gd-DTPA/Dimeg allowed evaluation of the internal structure of the tendons Scanning electron microscopy images correlated well with images obtained by use of MRI before and after administration of Gd-DTPA/Dimeg. Localized spectra revealed the concentration of water protons in the TDDF. CONCLUSIONS AND CLINICAL RELEVANCE: The techniques used in this study provided information about internal organization of the TDDF in horses. Analysis of results revealed that the best technique involved vascular injection of contrast medium. Results of MRI correlated well with results for scanning electron microscopy. After administration of Gd-DTPA/Dimeg, MRI provided additional information about tendon morphologic characteristics. This technique may be of value for examination of tendons in lame horses.

alpha-Gustducin expression in the vomeronasal organ of the mouse.

The expression of alpha-gustducin, a G protein alpha subunit involved in bitter and sweet taste transduction, was investigated in chemosensory tissues of adult mice. By immunohistochemistry, alpha gustducin was absent in the olfactory neuroepithelium. Instead, alpha gustducin was expressed in a subset of bipolar cells in the proliferative zone of the vomeronasal neuroepithelium as well as in taste buds. Northern blot analysis confirmed the presence of alpha gustducin in isolated vomeronasal organs. Moreover, immunohisto- chemistry revealed the expression of alpha gustducin in scattered cells of the nasal respiratory epithelium. These results show for the first time that alpha gustducin is expressed in chemosensory tissue outside the alimentary tract, suggesting that common transduction mechanisms could be shared by apparently unrelated chemosensory tissues.

Alpha-gustducin-immunoreactive solitary chemosensory cells in the developing chemoreceptorial epithelium of the rat vallate papilla.

The presence of solitary chemosensory cells was studied in rat vallate papillae during the first week of post-natal life by alpha-gustducin immunocytochemistry. In 1- to 3-day-old rats, isolated alpha-gustducin-immunoreactive cells were found within the epithelium of the vallate papilla. These cells, mainly located in the basal layer, were scattered among keratocytes and wrapped in alpha-gustducin-negative epithelial cells in a glia-like fashion. The alpha-gustducin-immunoreactive cells were usually round and some of them gave rise to short, large processes directed towards the lumen of the oral cavity or the basal lamina. Rarely, some cells showed an evident bipolar shape. Small taste buds containing either alpha-gustducin-immunoreactive or alpha-gustducin-negative cells appeared in the vallate papillae of 4-day-old rats in which isolated, bipolar-shaped alpha-gustducin-immunoreactive cells were also found. After the first week of post-natal life, the taste buds appeared basically similar to those of adult animals. In conclusion, the present study demonstrates that the presence of epithelial cells with characteristics of solitary chemosensory cells precedes the development of the taste buds.

Use of magnetic resonance imaging for diagnosis of a spinal tumor in a cat.

Magnetic resonance imaging was used to identify a spinal mass at the level of the 6th-7th cervical vertebral body in a cat. The MRI images were most consistent with the presence of an intradural, extramedullary meningioma that was confirmed by subsequent histological examination.