Biomedical subjects

P Mestres

Publications and source records attributed to P Mestres.

At least 37 records · Page 2Linked to original sources

PubMed1998

Protein extracts from the gut wall influence the postnatal development of rat myenteric neurons in vitro.

Neurons migrating from the neural crest into the presumptive gut wall need an appropriate microenvironment to survive and differentiate during ontogenesis. The rat enteric nervous system (ENS) keeps developing beyond birth. The aim of this study was to investigate the neurotrophic effects of the postnatal microenvironment. Myenteric plexus was isolated from the smooth muscle layer of newborn rats at different ages (postnatal day (p)1, p7, p14) and grown as dissociated cell cultures. The culture medium (hormone-supplemented, serum-free) was supplemented either with glial-cell-line-derived-neurotrophic factor (GDNF) or with protein extracts from homogenized smooth muscle layer of p7 rats. Cultures kept in defined medium alone were used as controls. After 18 h in vitro both GDNF and protein extract had a neuritogenic effect upon p1 and p7 neurons, while p14 neurons were only stimulated by the protein extracts. The GDNF effect upon these neurons did not differ significantly from the effects in defined medium alone. The average neurite outgrowth in extract-supplemented cultures was always longer than that seen in those treated with GDNF. Although GDNF influences the postnatal development of myentric neurons in vitro it could not be detected immunohistochemically either in Western blots of the protein extracts or in cryostat sections of the gut.

Abdominal Muscles↗

PubMed1997

GDNF is a trophic factor for adult rat corticospinal neurons and promotes their long-term survival after axotomy in vivo.

Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for several neuronal populations involved in motor control. The present study evaluates the trophic actions of GDNF on corticospinal neurons, an important central nervous system motor projection into the spinal cord. Death of spinal motoneurons and corticospinal neurons is observed in the neurodegenerative disease amyotrophic lateral sclerosis. Axotomy of adult rat corticospinal neurons at internal capsule levels induces half of them to die, and the surviving population displays severe atrophy. To examine the trophic effects of GDNF on corticospinal neurons, Fast Blue-labelled corticospinal neurons were stereotaxically axotomized at internal capsule levels and GDNF was infused intracortically to lesioned corticospinal neurons at total doses of 2, 4, 10, 20, 40, 100 and 300 microg for 7 days. GDNF prevented axotomy-induced death of corticospinal neurons at doses between 2 and 40 microg and abolished or attenuated their atrophy at all doses examined. In addition, treatment with 8 microg GDNF for the first 2 weeks after axotomy resulted in the long-term survival of corticospinal neurons for 42 days. With regard to the development of treatment strategies for upper motoneuron degeneration in amyotrophic lateral sclerosis, application of GDNF via the cerebrospinal fluid may be more relevant than intracortical delivery as its diffusion within the brain parenchyma is limited. Intraventricular as well as intracisternal infusion of GDNF (300 microg over 7 days) completely prevented corticospinal neuron death. These results show that GDNF promotes the long-term survival of corticospinal neurons and has a positive effect on their size in vivo. Furthermore, the survival-promoting effect of GDNF on corticospinal neurons after delivery via cerebrospinal fluid has important clinical implications for potential treatment of the upper motoneuron degeneration seen in amyotrophic lateral sclerosis.

Animals↗

PubMed1997

Surface-treated catheters with ion beam-based process evaluation in rats.

Ion beam-based processes such as ion implantation (silicone rubber) and ion beam-assisted deposition (silver-based coatings) affect the outer micron layers of catheter surfaces. These processes were used on the common catheter materials of silicone and polyurethane. In 56 rats, surface (Spi-Silicone and Spi-Argent I and II) catheter segments were implanted for 1, 3, and 6 weeks. After removal, these pieces were investigated for bacterial colonization and fixed for scanning electron microscopic evaluation. As controls, untreated catheter segments were implanted in 28 rats. Bacterial colonization was found in 2.4% in the surface treated catheter pieces versus 7.1% in the control group. The scanning electron microscope investigations showed low thrombogenicity in all of the treated catheters independent of the implantation times.

Animals↗

PubMed1997

Human newborn and adult myenteric plexus grows in different patterns.

Whilst embedded within the gut wall the inaccessibility of the enteric nervous system (ENS) is a major drawback for the establishment of an in vitro model for the human ENS. Using a method which combines collagenase digestion, mechanical agitation and manual dissection it was possible to dissect myenteric plexus from human colon of patients at all ages, from newborn to old-age. While complex networks of ganglia and their interconnecting strands could be isolated from newborn gut, the adult tissue allowed only single or small groups of ganglia to be dissected coherently. Pieces of plexus were cultivated on glass coverslips or on plastic sheets respectively. Explants from newborn or older patients displayed different growth patterns. The cytological behaviour of the newborn explants is characterized by an intensive neurite outgrowth. After 1 to 2 days in culture, glial cells start to leave the explant while proliferating and forming a dense cellular carpet. The axons appear partly arranged in bundles, expanding above the glial carpet. Single neurites can leave the carpet and attach directly on the substrate. Semithin sections and EM studies reveal the existence of numerous neurons within the ganglia. The characteristic dense arrangement of neurons, glial cells and neuropil of the myenteric ganglia in vivo was only partly conserved in newborns while in cultured adult ganglia the cells were sparsely scattered throughout the explant with large clefts between the single cells. The ganglia of the adults do not show any considerable neurite outgrowth, which correlates with the low amount of neurons within the ganglia. The migratory behaviour of the adult glial cells is rather moderate, also the proliferation rate compared to the newborn cultures. In general single cells leave the explant without forming an glial carpet as seen in the newborn cultures. The described method delivers an in vitro paradigm for the study of human myenteric plexus. It underlines the differences in growth pattern, neurite outgrowth and glial proliferation from newborn and older children, as well as from adult patients, thus establishing a base line for future studies.

Adolescent↗

PubMed1996

Scanning electron microscopic investigation of catheters for blood access.

Using blood-contacting catheters, infections, thromboses, and stenoses are among the most frequent complications. They are caused by surface properties of the basic material. Ion beam based processes such as ion implantation (silicone rubber) and ion beam assisted deposition (silver-based coatings) affect only the outer micron of the treated material surface; there is little effect on bulk properties. These processes were employed also in the production of large-bore catheters used for extra-corporeal detoxification. In a prospective study in 56 patients, 72 large-bore catheters were inserted into the internal jugular and subclavian veins and investigated after removal for bacterial colonization using a scanning electron microscope. In 24 patients 30 surface-treated catheters (Spi-Argent I n = 14; Spi-Silicone n = 16) were used. 42 untreated catheters used in 32 patients served as controls. Bacterial colonization was observed in 8.9% in contrast to 38.1% in untreated catheters. The scanning electron microscopic investigations showed in all catheters a low thrombogenicity. The ion beam based processes make the surface-treated catheters thrombus and infection resistant.

Catheterization, Central Venous↗

PubMed1995

Somatostatin-mRNA expression in brainstem projections into the medial preoptic nucleus.

The medial preoptic nucleus plays an important role in the regulation of neuroendocrine processes, vegetative functions, sexual behaviour and the modulation of the somatomotoric system. The connections of the medial preoptic nucleus to other areas of the central nervous system are very complex, and the area receives afferents using numerous transmitters and neuropeptides. Previous investigations have shown that this nucleus receives afferents from various brainstem nuclei that also contain somatostatinergic neurons. This study was carried out to investigate if somatostatin-projecting neurons of the brainstem are afferents to the medial preoptic nucleus. This was approached by combining somatostatin-mRNA in situ hybridisation with True Blue retrograde tracing. Our results demonstrate somatostinergic brainstem projections into the medial preoptic nucleus mainly in the pedunculopontine nucleus and in the nucleus of the solitary tract (50% together). Other important somatostatinergic afferents into the medial preoptic nucleus originate in the cuneiform area, the dorsal parabrachial nucleus and in the lateral reticular nucleus (37% together). Less important are the somatostatinergic projections coming from the central grey, the laterodorsal tegmental nucleus, the locus coeruleus and the nucleus raphe magnus. Considering that these areas are involved in diverse functions such as cardiovascular regulation (nucleus of the solitary tract), transmission of visceral sensibility (dorsal parabrachial nucleus), modulation of the somatomotoric system (pedunculopontine nucleus) and in the regulation of neuroendocrine mechanisms (locus coeruleus), it seems tenable that the somatostatin projections demonstrated here also have a diverse functional quality within the medical preoptic nucleus where they terminate.

Animals↗

PubMed1995

Scanning electron microscopic investigations of surface treated large-bore catheters used for extracorporeal detoxification methods.

Typical complications caused by surface properties of synthetic catheter implants are infection, thrombosis, and stenosis. New methods for surface modification with the aim of reducing such complications are ion beam-based technologies. In our study 109 large-bore catheters without (n = 42) and with treated surfaces with silver (n = 39) or silicone (n = 28) were inserted into the interna jugular and the subclavian veins and were used for extracorporeal detoxification methods. After removal, the catheters were investigated with scanning electron microscopy (SEM) and for bacterial colonization. In 42 large-bore catheters without surface treatment deposits of fibrin, protein and blood cells were seen on the inner and outer surface. Bacterial colonization was observed in 38.1%. In contrast, the catheters with treated outer surfaces showed a very low thrombogenicity and a low contamination rate of 8.9%. The ion beam-based technologies reduce the thrombogenicity and infection rates of the catheter surfaces. In comparison to catheters without treated surfaces, catheters with surface treatment are good alternatives in blood contacting applications ranging from hemodialysis to oncology.

Adsorption↗

PubMed1994

Scanning electron microscopic investigation of catheters for blood access.

Large-bore catheters for extracorporeal detoxification methods without and with treated surface with silver or silicone were investigated after removal with a scanning electron microscope and for bacterial colonization. In 42 large-bore catheters of three different materials, small deposits of fibrin and protein on the inner and outer surface were seen. This second layer covered the entire surface after 3 days and increased to a thickness of 3 to 60 microns during the following days. Bacterial colonization was observed in 38.1%. In contrast to these results, the catheters with the treated outer surface showed a very low thrombogenicity and a low contamination rate of 6.7%.

Bacteria↗

PubMed1994

Expression of casein kinase 2 during mouse embryogenesis.

This paper deals with the expression and distribution of casein kinase 2 (CK-2) subunits in mouse embryos at different developmental stages. Expression was investigated at the mRNA level of CK-2 alpha- and beta-subunits by in situ hybridization and distribution at the protein level by immunohistochemistry using CK-2-alpha- and CK-2-beta-specific antibodies, respectively. In general both methods gave similar results. In earlier stages of mouse embryonic development (day 10.5 after coitus) CK-2 was more expressed in neuroepithelia than in all other tissues. From day 11.5 after coitus on, high expression of CK-2 was detected in all epithelia. From day 16.5 on, all tissues and anlagen of the fetus involved in organogenesis revealed a higher CK-2 expression as compared with secondary mesenchyma. The only difference between in situ hybridization and immunocytochemistry was observed in the skin. Transcripts of CK-2 were found mostly in the basal layer of the epidermis and in the nuclei of keratinocytes, whereas CK-2 protein was almost exclusively found in the cytoplasm of epidermal cells.

Animals↗

PubMed1994

The ventricular system of the pigeon brain: a scanning electron microscope study.

The fine structural features and regional differences of the ependyma in adult pigeons have been investigated by scanning electron microscopy. Pigeons of either sex were fixed with buffered glutaraldehyde (3%) and formaldehyde (0.5%) by intravascular perfusion. The brain was dissected using section planes adequate to expose each part of the ventricular system. The specimens were then dehydrated, critical point dried and sputtered with gold. Depending upon the distribution of cilia, microvilli and single cilia, different areas were recognised in the 4 ventricles. The topographic locations of these areas were determined using the atlas of Karten & Hodos (1967). The medial surfaces of the 1st and 2nd lateral ventricles are more densely ciliated than the lateral surfaces. In the floor of the 4th ventricle the medial part is less ciliated than the lateral parts. The circumventricular organs (subseptal organ, organum vasculosum of the lamina terminalis, infundibulum, choroid plexus, subcommissural organ, area postrema) show very characteristic surfaces and are surrounded by a transitional zone with the nonspecialized ependyma. In contrast, in the paraventricular organ the transition to the nonspecialized ependyma is rather abrupt. The ependyma covering the trochlear nucleus appears densely ciliated, differing from that of the classic circumventricular organs. Finally, the existence of openings in the caudal medullary velum, which represent direct communications between the ventricles and the subarachnoid space, was demonstrated.

Animals↗

PubMed1992

Direct and indirect actions of HgCl2 and methyl mercury chloride on permeability and chloride secretion across the rat colonic mucosa.

The actions of two mercury compounds, the inorganic HgCl2 and the organic methyl mercury chloride (MeHg), and of CdCl2 on ion transport across the rat colon were studied with the Ussing chamber and the everted sac method. The mercury compounds (5-50 microM), but not CdCl2, administered to the luminal side, induced a large, concentration-dependent increase of tissue conductance (Gt). The transepithelial movement of the extracellular marker, mannitol, was enhanced in the presence of the mercury compounds, indicating that they cause an increase in the permeability of the epithelium. Morphological studies revealed that at least for HgCl2 this increase of the permeability was associated with a loosening of the tight junctions, severe alterations of the enterocytes, and a loss of the continuity of the epithelium. After washing out HgCl2, cells neighboring the altered enterocytes developed lateral processes and in this way restored the continuity of the epithelial layer. In parallel, the mercury compounds induced an increase of short-circuit current (Isc), which is indicative of an induction of Cl- secretion. The increase of Isc, but not that of Gt, was suppressed by indomethacin and, in the case of MeHg, also by tetrodotoxin. These results suggest that MeHg and HgCl2 induce Cl- secretion by an indirect effect on the epithelium, which is mediated by prostaglandins.

Animals↗

PubMed1992

Histo- and immunocytochemical characterization of the neurons of the mucosal plexus in the rat colon.

The mucosal plexus of the rat colon descendens is constituted of a network of nerves that, in contrast to most other segments of the digestive tract, contains also ganglia. The ganglia, consisting of neurons and glial cells, are located in the basal part of the lamina propria at distances between 100 and 1,200 microns. They are not vascularized. The neurons in these ganglia were characterized by means of: (1) the histochemical demonstration of acetylcholinesterase (AChE) activity, (2) the immunocytochemical identification of neurofilament proteins (NFP; 200 kD) and (3) their ultrastructure. The glial cells, which were AChE negative, could be distinguished from the neurons by differences in size and chromatin pattern. All neurons of the mucosal plexus reveal AChE activity in the perikaryon, but only parts of the axons are AChE positive. NFP-like immunoreactivity was detected in the perikarya but only in a minor part of the axons. These findings confirm previous light-microscopical observations and add new evidence for the existence of neurons (ganglia) in the mucosal plexus of the rat colon.

Acetylcholinesterase↗

PubMed1992

Electron microscopy of the mucosal plexus of the rat colon.

The ultrastructure of neurons, glial cells and axons of the mucosal plexus of the rat colon descendens was studied. Serial semithin sections and a re-embedding technique were used in order to localize the ganglia. The ganglia are free of blood vessels and connective tissue. The ratio of neurons to glial cells is approximately 1. Ganglia and nerve strands are enclosed by a basement membrane, without a well-defined perineural connective tissue. The neurons show a structure similar to other enteric plexus. Synaptic contacts were observed frequently in the neuropil, where nerve endings and varicosities show a diverse outfit in vesicles. The glial cells, which contain immunocytochemically detectable glial fibrillary protein, possess the same ultrastructural attributes in the intra- and extraganglionic localizations. In the nerves, axonic profiles and varicosities appear in close relation with glial cells or their processes. The distance between the nerves and their target cells, i.e. the enterocytes, is 0.5 microns or more with interposed basement membranes and fibroblasts.

Animals↗

PubMed1991

Segmental heterogeneity of the rat colon in the response to activators of secretion on the cAMP-, the cGMP- and the Ca(2+)-pathway.

The electrolyte transport was compared in proximal and distal segments of the rat colon under control conditions and after induction of secretion on the cAMP-, the cGMP- and the Ca(2+)-pathway. Baseline short-circuit current was decreased by indomethacin and tetrodotoxin in the distal colon, indicating a spontaneous production of neuronally acting prostaglandins. In contrast, baseline short-circuit current in the proximal colon was decreased only by indomethacin, but not by tetrodotoxin. Unidirectional flux measurements revealed that in the distal colon sodium and chloride were absorbed, while the proximal colon secreted chloride. A morphological comparison between the distal and proximal epithelium revealed that the zonulae occludentes and the microvilli were longer in the distal colon. The size of the Golgi apparatus was several times larger in the crypt than in the surface region without differences between proximal and distal colon. Distal segments were more sensitive to an activator of the Ca(2+)-pathway, carbachol, or activators of the cAMP-pathway such as forskolin and a cAMP-analogue. In contrast, the activation of the cGMP-pathway by a cGMP-analogue or by the heat-stable enterotoxin of E. coli (STa) was more effective in the proximal colon. The results give evidence for a segmental specificity with regard to the intracellular pathways responsible for the activation of secretion.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

The epithelial basal lamina of the isolated colonic mucosa: scanning and transmission electron microscopy.

The morphology of the rat colonic basement membrane was studied under control conditions and after induction of secretion by distension in the Ussing chamber. The basement membrane was visualized by removing the epithelium with the aid of Ca2+ chelation combined with vibration. Specimens with and without metallic coating were studied with SEM and TEM, respectively. The surface of the basement membrane in the surface region of the epithelium, i.e. between the mouths of the crypts, is rough. In contrast, the deeper parts of the crypts have a rather smooth basal lamina, which is finely pleated. Fenestrations of the basal lamina with a diameter of 0.5-1 microns were found frequently at the surface region, but less frequently in the crypts. Fenestrations with diameters larger than 2 microns were never observed. After the distension experiments and long-time incubation in vitro (5 h), the size of the fenestrations remained unchanged, however, their number increased compared to the controls.

Animals↗

PubMed1990

Storage of glycogen in rat colonic epithelium during induction of secretion and absorption in vitro.

Changes induced in the ultrastructure of the epithelium of the rat colon descendens by long-term electric field stimulation (EFS) in an Ussing chamber were investigated. The anion secretion, which was induced by EFS and was measured by the short-circuit current, fell continuously during a 5 h stimulation. At the end of the stimulation period, small particles were observed in the epithelium; these did not appear in unstimulated control tissue. They were localized predominantly in the apical part of the cell. By staining with periodic acid-thiosemicarbazide-silver proteinate and because of their sensitivity to alpha-amylase, they were identified as glycogen deposits. This storage of glycogen was time-dependent and was first visible after an EFS of 2 h. It did not appear if glucose was substituted in the bathing solution by sodium butyrate. Glycogen particles were also observed after addition of forskolin, which in contrast to EFS causes a high secretory activity that is stable over several hours. The surface cells contained significantly more glycogen than the crypt cells when secretion was stimulated by EFS or forskolin. The formation of glycogen during EFS was not prevented by tetrodotoxin (TTX). In contrast, TTX itself, which causes maximal absorptive activity by blocking secretomotor neurons, induced the appearance of glycogen in the enterocytes without EFS. However, in the presence of TTX, the amount of glycogen was the same in surface and crypt cells. The results demonstrate that the capacity to synthesize and store glycogen, which has up to now only been observed in embryonic or tumor epithelial cells, is still present in adult colonic mucosa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Raster-electron-microscopic investigations in large-bore catheters for extracorporeal detoxification.

Large bore catheters (n = 42, 31 patients) which remained in position for a period of between 2 to 73 days (mean = 26.8 days/patients) were investigated with raster electron-microscopy (scanning electron micrographic: SEM) and for bacterial colonisation. Microbiological findings indicated colonisation of the intravascular large bore catheters in 8 out of 42 catheters (19%). SEM morphological investigation showed bacterial colonisation in only 3 catheters (7%). The 3 different catheter materials had small deposits of fibrin and protein on the inner and outer surfaces after 2 days. This second layer covered the entire surface after 3 days and increased to a thickness of 3-60 microns in the following days. In this matrix erythrocytes, thrombocytes and granulocytes could also been seen.

Bacterial Adhesion↗

PubMed1989

Single chloride channels in colon mucosa and isolated colonic enterocytes of the rat.

Chloride channels from rat colonic enterocytes were studied using the patch-clamp technique. After removal of mucus, inside-out patches were excised from the apical membrane of intact epithelium located at the luminal surface. They contained spontaneously switching Cl- channels with a conductance of 35-40 pS. The channels were blocked reversibly by anthracene-9-carboxylic acid (1mM). In excised patches from single enterocytes, isolated by calcium removal, the Cl- channels were studied in more detail. The I-V relation was linear between +/- 80 mV. The selectivity was I- greater than Br- greater than Cl- = NO3- greater than F- = HCO3-. Thirty pS Cl- channels were also found on the basolateral membrane of crypts isolated by brief calcium removal. The I-V curve of these Cl- channels was also linear. The results provide direct evidence for the existence of Cl- channels in the apical membrane of surface cells in colonic mucosa. The properties of these channels are similar to those previously observed when incorporating membrane vesicles into planar lipid bilayers. Both results support the validity of the theoretical models describing intestinal secretion.

Animals↗