Biomedical subjects

R Saxod

Publications and source records attributed to R Saxod.

At least 19 recordsLinked to original sources

PubMed1998

Merkel complexes of human digital skin: three-dimensional imaging with confocal laser microscopy and double immunofluorescence.

Three-dimensional (3-D) reconstruction of images provided by confocal scanning laser microscopy (CSLM) is a powerful tool in a morpho-functional approach to cutaneous innervation studies. To investigate mechanoreceptors in the hand, a study of Merkel complexes was performed in human finger. A double fluorescent-conjugated immunolabeling with antibodies against neurofilament (NF 200) and cytokeratin (CK 20) on floating, thick cutaneous samples (80 to 100 microm), was used. After acquisition of serial optical planes by CSLM, reconstruction was performed with 3-D reconstruction software tools. Merkel cells were clearly labeled with CK 20, whereas nerve components were only NF 200 reactive. The cells, localized on the basal lamina of the epidermis, were usually arranged in clusters of five to eight cells. Each cell was connected to a nerve process ramification originating from a unique fiber. Quantitative data, compiled from a sample of 25 Merkel complexes, gave a mean cell diameter of 13 +/- 1 microm and a mean nerve fiber size of 3 +/- 1 microm. Surface measurements were done on a single reconstructed cluster with a mean and standard error which only refers to the optical 3-D resolution. It gives a surface of 12 +/- 1 microm2 for the contact zone between cell and nerve fiber and a cluster area of about 500 microm2. The great precision of reconstructed images provides a detailed analysis of spatial relationships between abutting nerve fibers and Merkel cells. Data interpretation is improved with complementary ultrastructural and physiological studies results, and this allows an accurate investigation of cutaneous sensory endings.

Antibodies↗

PubMed1998

Mosaic distribution of chondroitin and keratan sulphate in the developing rat striatum: possible involvement of proteoglycans in the organization of the nigrostriatal system.

The striatum of the mammalian basal ganglia is composed of two neurochemically distinct compartments termed patches and matrix that contribute overall to a mosaic organization. Glycosaminoglycans (GAGs), the sugar moieties of proteoglycans, provide specific spatio-temporal guidance cues during the development of several functional neural systems. However, their distribution within the nigrostriatal system has not been investigated yet. Here, the immunohistochemical distributions of unsulphated (C0S), 4-sulphated (C4S) and 6-sulphated chondroitin (C6S) and keratan sulphate (KS) were examined in the developing neostriatum of rat and compared with the distribution of dopaminergic terminals. All the chondroitin sulphate (CS) isomers are homogeneously expressed in the embryonic striatum. After birth, C0S and C6S reveal the striatal mosaic in being preferentially expressed within the matrix compartment and in boundaries around patches whereas the C4S epitope is present in both compartments, with a slight patchy distribution. KS expression is detected first in the patches during the early postnatal period and subsequently only in the matrix compartment. All these GAG expressions disappear as the brain matures except for C4S which remains high throughout adult life. Furthermore, studies within the developing medial forebrain bundle reveal that CS isomers, but not KS, are expressed in and around the dopamine axonal tract but show similar developmental patterns of distribution which do not appear to be specifically associated with the nigrostriatal pathway. These results suggest a possible implication of proteoglycans during the development of the striatum and may be useful for understanding the complex cellular and molecular interactions in degeneration and plasticity of the nigrostriatal circuit in Parkinson's disease.

Animals↗

PubMed1998

Transient compartmental expression of neurocan in the developing striatum of the rat.

The expression of the chondroitin sulfate proteoglycan neurocan was examined in the developing striatum of the rat and compared with the distribution of dopaminergic terminals. Neurocan immunoreactivity shows a homogeneous pattern in the embryonic striatum. In the postnatal striatum, neurocan was first expressed within the matrix but not the patch compartments, and subsequently within both. These results suggest that chondroitin sulfate proteoglycans are involved in formation of connections between the substantia nigra and striatum.

Age Factors↗

PubMed1998

Regulated expression of keratan sulphate and peanut agglutinin binding sites during organogenesis in the developing chick.

Keratan sulphate proteoglycans are potentially important during development and are possible binding molecules for the lectin, peanut agglutinin, a marker for areas that are inhibitory for axonal growth in early embryos. The present study describes the spatiotemporal distributions of keratan sulphate epitopes and peanut agglutinin binding sites during organogenesis in the developing chick from E5 to hatching. The widespread distributions of these molecules did not often overlap but clearly delimited different carbohydrate compartments demonstrating that peanut agglutinin does not necessarily bind to keratan sulphate proteoglycans. These markers were mostly extracellular but keratan sulphate, in particular, was found within certain specific cells in cartilage, gonad, heart and pancreas, at certain ages. The presence of keratan sulphate in putative germ cells during their migrations and in the gonads may be of particular importance. Their distributions generally evoke modulation of adhesion allowing cell migrations or morphogenetic movements related to epitheliomesenchymal interactions, but may also suggest an involvement in axonal guidance in skin, cartilage, gut and possibly heart. Furthermore, in the kidney, peanut agglutinin binding sites seem to be related to the functional differentiation of the nephrons.

Animals↗

PubMed1997

Keratan sulphate is present in developing chick skin in vivo where it could constitute a barrier to advancing neurites as observed in vitro.

Proteoglycans play an important role in axonal guidance. The glycosaminoglycan chondroitin sulphate is known to be inhibitory. Keratan sulphate is structurally similar and may play a similar role. The lectin peanut agglutinin potentially labels chondroitin or keratan sulphate rich areas. In order to elucidate the mechanisms by which the regular nerve pattern develops in chick skin, keratan sulphate epitopes and peanut agglutinin binding sites were localized in chick embryos and compared to previous studies of chondroitin sulphate. The markers all display specific, developmentally regulated staining patterns, thus reflecting their individuality. Their maximal expression coincides with the formation of feathers and their nerve pattern in both time and space. Furthermore, culture experiments using dorsal root ganglia demonstrate the avoidance of keratan sulphate by the growing sensory neurites, although outgrowth and elongation occur if they grow directly on the keratan sulphate substrate. Thus, keratan sulphate proteoglycans in developing chick skin are potentially involved in guidance of sensory neurites or maintenance of the new nerve pattern, but are clearly different to the chondroitin sulphate proteoglycans which have a complementary distribution. Furthermore, peanut agglutinin binding sites are independent of both proteoglycans.

Agglutinins↗

PubMed1997

Close link between cutaneous nerve pattern development and feather morphogenesis demonstrated by experimental production of neo-apteria and ectopic feathers: implication of chondroitin sulphate proteoglycans and other matrix molecules.

In chick skin, nerve arcades develop around the base of feathers. In order to understand the mechanisms of their formation, we have tried to dissociate arcade formation from feather morphogenesis in various ways. Nerve patterns were analysed (1) in hydrocortisone-treated embryos that are partially devoid of feathers, (2) after retinoic acid treatment that produces ectopic feathers, (3) in dorsal root ganglia-skin co-cultures. Whenever tested, immunochemistry revealed that nerve arcades form around chondroitin sulphate proteoglycan-rich areas. Hydrocortisone treatment modifies the distribution of two out of three chondroitin sulphate proteoglycan epitopes tested, as well as the shapes of the feathers and nerve arcades, but not fibronectin, tenascin or laminin localizations. Chondroitinase digestion in co-cultures eliminated the nerve arcade formation and produced abnormally thin feathers, but nevertheless with a normal spatial distribution. Thus, chondroitin sulphate proteoglycans are probably not involved in the overall arrangement of feathers, but appear to play a fundamental role in both the formation of nerve arcades and the morphogenesis of the feather.

Animals↗

PubMed1997

Free radical production after exposure of astrocytes and astrocytic C6 glioma cells to ethanol. Preliminary results.

Formation of the alpha-hydroxyethyl radical (CH3 degree CHOH) has already been extensively demonstrated after ethanol metabolism in the liver. Despite favourable conditions, this formation in the brain has remained speculative since there is no direct experimental evidence in intact brain cells. In this preliminary study, the formation of such a radical was observed after exposure of astrocytes and astrocytic C6 glioma cells to ethanol. These cells were studied because astrocyte integrity is essential for normal growth and functioning of neurons. The free radicals were detected by EPR spectroscopy using the spin trapping technique. Astrocytes appeared to be more sensitive than the C6 cells to free radical formation as the intensity of the signal was higher after exposure of the astrocytes and increased with time, a fact not observed after exposure of the C6 cells.

Animals↗

PubMed1997

Regulation of the chick cutaneous innervation pattern in retinoic acid-induced ectopic feathers and in the naked neck mutant.

In chick skin, nerve fibers develop in a typical network formed by arcades around the base of feathers. In this study, we tried to dissociate the morphogenesis of nerve arcades and feathers, and to clarify the implication of several matricial molecules in these two developmental events. For this purpose, cutaneous nerve pattern and distribution of fibronectin, tenascin, and three epitopes of chondroitin sulfate proteoglycans (CSPGs) have been immunohistologically studied in the skin of the specific apteria of naked neck chick mutants, which lack feathers in the neck area, and in the tarso-metatarsal zone of retinoic acid-treated embryos where ectopic feathers grow. The presence of feathers was always associated with nerve arcades; no arcades were present in featherless areas. Specific immunofluorescence for tenascin and two epitopes of CSPGs revealed different distributions in the naked-neck neo-apteria as compared to control apteria. Moreover, the only difference in matricial composition in ectopic feathers concerned a CSPG isoform, bringing additional evidence that extracellular matrix molecules, and especially some (but not all) CSPGs, are involved both directly and indirectly in the cutaneous nerve pattern development.

Animals↗

PubMed1997

Characterization of the production of acetaldehyde by astrocytes in culture after ethanol exposure.

The nervous system is one of the main targets of ethanol toxicity. Astrocytes might play an important role in ethanol-induced brain toxicity, because their integrity is essential for the normal growth and functioning of neurons. On the other hand, acetaldehyde has been implicated as a mediator in some of the biochemical, pharmacological, and behavioral effects of ethanol. The present study aimed at demonstrating the ability of astrocytes in culture to produce acetaldehyde from ethanol. Significant metabolization of ethanol with production of acetaldehyde was demonstrated in the primary culture of astrocytes. This production was quite low, compared with that usually observed in hepatocytes, but was in the same range as that measured in whole brain homogenates and corresponded to biologically active levels. Such a demonstration could bring new elements for understanding of ethanol neurotoxicity.

Acetaldehyde↗

PubMed1996

Ontogeny of the cutaneous sensory organs.

The ontogeny of cutaneous sensory nerve organs is described in higher vertebrates, and includes the lamellated corpuscles of Meissner, Pacini and Herbst, and the Merkel cell-neurite complex with bird Merkel and Grandry corpuscles, and mammalian Merkel cells. The main common feature is that for most corpuscles there is an inside-out order of assembly around the nerve ending which is present from the beginning of end-organ ontogeny. The exception is the mammalian Merkel cell which is present in the epidermis before the entrance of nerve fibers, and could play a promotional role in the development of skin innervation. The developmental origin of Herbst and Merkel corpuscles in birds is reported as demonstrated using embryological experiments with cell markers. Conclusions are that inner bulb cells of Herbst corpuscles and bird Merkel cells are of neural crest origin, whereas other cells (inner space and capsular cells for Herbst corpuscle and capsular cells for Merkel corpuscles) are provided by the local mesenchyme. The question of the ontogeny of mammalian Merkel cells is discussed in relation to the two debated hypothesis of epidermal and neural crest origins. Morphogenetic interactions during the development of cutaneous sensory end organs are also discussed.

Animals↗

PubMed1996

Characterization of the morphological variations of astrocytes in culture following ethanol exposure.

The nervous system is one of the main targets of ethanol toxicity and it has been suggested that astrocytes might play an important role as their integrity is essential for the normal growth and functioning of neurons. Morphological variations of astrocyte cultures were therefore examined after exposure to various doses of ethanol (0.5, 1 and 2%) for different durations (24, 48, 72 and 96 h). The percentage of cell viability and the cell density were calculated and the changes in astrocyte morphology were assessed by an image analysis system (Samba 2005) allowing the characterization of 5 parameters (perimeter, surface, elongation factor, convexity factor and the form factor) of a great number of cells (over 6500). This was necessary because of the high variability in normal cultured astrocyte morphology. A two-way statistical approach (2-factors ANOVA completed by stepwise discriminant analysis) was adopted to emphasize the differences between control and exposed cells. In such conditions, ethanol treated cells became more elongated, less circular and more concave and did not grow like non-exposed cells. The mean pooled values of these parameters tended to be modified as a function of the dose of ethanol. The relationships between parameters clearly separated the groups as a function of the different doses. Finally no significant difference was observed in cell viability and cell density despite lower scores in the groups exposed to the highest dose of ethanol for the longest time. Our results suggest that ethanol might affect astrocytes in two different but probably complementary ways by modifying the cell shape and by altering normal cell development.

Animals↗

PubMed1996

[Development of the cutaneous nervous system].

Skin of vertebrates is richly innervated, mainly by sensory nerve fibres which form a well organized pattern, particularly around phaners. This innervation develops segmentally (dermatomes) from cutaneous branches provided by spinal nerves. The innervation begins at 13 days (E 13) in the mouse embryo and, although hair buds form at E 16, follicles are only innervated from 5 days postnatally being complete at about 20 days. In the chick skin, innervation forms a regular and characteristic pattern around feathers, and can be visualized on whole mounts. Its development can be traced from 6 days of development in relation to feather morphogenesis. Experiments producing non formation of spinal ganglia (X-ray irradiation or neural tube ablation) or production of neoapteria (hydrocortisone treatment) or ectopic feathers on scales (retinoic acid treatment) show there is a close link between feather development and nerve pattern formation. In vitro co-cultures of dorsal root ganglia and epidermis combined with the use of synthesis inhibitors and antibodies, showed that epidermis has a repulsive effect on nerve fibres mediated, at least in part, by chondroitin sulphate proteoglycans. These compounds have been localized, using antibodies mainly at the base of the feather buds and seem to play a key role in the construction of the fine nerve pattern around feather follicles. In conclusion, the specific nerve patterns are the final result of selective responses of growing nerve endings to unique combinations of local cues and conflicting interactions which are developmentally regulated in parallel with the morphogenesis of phaners.

Aggrecans↗

PubMed1994

Development of sensory innervation in chick skin: comparison of nerve fibre and chondroitin sulphate distributions in vivo and in vitro.

In bird skin, nerve fibres develop in the dermis but do not enter the epidermis. In co-cultures of 7-day-old chick embryo dorsal root ganglia and epidermis, the neurites also avoid the epidermis. Previous studies have shown that chondroitin sulphate proteoglycans may be involved. Chondroitin sulphate has therefore been visualized by immunocytochemistry, using the monoclonal antibody CS-56, both in vivo and in vitro using light and electron microscopy. Its distribution was compared to those of 2 other chondroitin sulphate epitopes and to that of the growing nerve fibres. In cultures of epidermis from 7-day-old embryonic chicks, immunoreactivity is found uniformly around the epidermal cells while at 7.5 days the distribution in dermis is heterogeneous, and particularly marked in feather buds. In vivo, chondroitin sulphate immunoreactivity is detected in the epidermis, on the basal lamina, on the surfaces of fibroblasts and along collagen fibrils. This localization is complementary to the distribution of cutaneous nerves. Chondroitin sulphate in the basal lamina could prevent innervation of the epidermis and the dermal heterogeneities could partly explain the nerve fibres surrounding the base of the feathers. Chondroitin sulphate could therefore be important for neural guidance in developing chick skin.

Animals↗

PubMed1992

Morphometric and biochemical studies in trigeminal nerve of rat after trichloroethylene or dichloroacetylene oral administration.

Trigeminal nerve impairment is one of the main features of the clinical spectrum observed after trichloroethylene (Tce) exposure. A morphometric study of teased fibres, an analysis of the fatty acid composition of total lipids of the trigeminal trunk, measurements of myelin basic protein (MBP) and of 2'-3'-cyclic nucleotide 3' phosphohydrolase (CNP) activity were used to characterize this lesion. Furthermore, the effects of Tce and of dichloroacetylene (Dca), a breakdown product supposed to be the main cause of the trigeminal neuropathy, were compared under the same experimental conditions. Results showed a significant decrease of the internode length and of the mean fibre diameter in the Dca group whereas these values, although also diminished, were not significantly affected in the Tce group. In the trigeminal nerve, this was associated with a significant (46%) decrease in sigma. (n-6) fatty acid and a 22% decrease in sigma. (n-3) fatty acid contents in the Tce group whereas these decreases were respectively 32% in sigma. (n-6) fatty acid and 27% (significant) in sigma. (n-3) fatty acid contents in the Dca group. In this group, a 64% increase in sigma. Fatty aldehydes was also observed. Finally, Tce or Dca had only a slight effect on the overall profile of fatty acid content in the brain and no significant variation was observed in MBP level and CNP activity. Both compounds can thus induce trigeminal nerve alterations, but with a higher intensity for Dca.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1991

A morphometric evaluation of the effects of trichloroethylene and dichloroacetylene on the rat mental nerve. Preliminary results.

Morphometric analysis was used to compare the effects of trichloroethylene (Tri) and dichloroacetylene (Dca) on the fibre parameters of the trigeminal nerve. Treated animals were clearly separated from controls according to a discriminant analysis. Furthermore, in the class of nerve fibres defined by a clustering analysis and corresponding to the largest fibres, myelin thickness was significantly decreased in the Dca group, but less so in the Tri group. In the group of the smallest fibres however, the myelin thickness was significantly increased by the treatments, but especially by Tri. Such a variability in the effects of Tri has already been demonstrated. Mechanisms for this are quite unclear although demyelination could be involved as already suggested. Our results thus show the ability of Tri and Dca to alter nerve parameters but probably with different modes of action depending on the size of the fibre.

Acetylene↗

PubMed1991

Involvement of a chondroitin sulfate proteoglycan in the avoidance of chick epidermis by dorsal root ganglia fibers: a study using beta-D-xyloside.

In 7-day chick embryo dorsal root ganglia and epidermis cocultures, nerve fibers avoid the epidermis. Previous studies have indicated that glycoproteic factors, secreted by epidermis, could be involved in this phenomenon. Treatment of epidermis by beta-D-xyloside, a specific proteoglycan synthesis inhibitor, abolishes the avoidance reaction. The same result is obtained when anti-chondroitin sulfate antibodies are added to the culture medium. Using HPLC and 35SO4 labeling combined with chondroitinase and hyaluronidase treatment, it has been demonstrated that chondroitin sulfate is present in the epidermal conditioned medium. This suggests that a chondroitin sulfate proteoglycan secreted by the epidermis is implicated in the neurite avoidance reaction and that epidermis could therefore control its own "noninnervation". In vivo, inhibitory influences by local extracellular components may control the guidance of growth cones during nerve pattern formation.

Animals↗

PubMed1991

Morphometry of human nerve biopsies by means of automated cytometry: assessment with reference to ultrastructural analysis.

The morphometric analysis of myelinated fibres is a tool for diagnosis of neuropathies and for assessing nerve regeneration after reconstructive surgery. In order to obtain reliable information, sampling techniques should be avoided and the measurement of all the fibres within a nerve fascicle requiring the use of automated cytometry is necessary. We have developed a programme on the SAMBA cytological image analyser for the automated measurement of myelinated fibres. Different techniques of segmentation were tested and adaptive grey level thresholding gave the more reliable result. Accuracy and reproducibility were tested using a set of five human superficial peroneal nerve biopsies that were previously analysed by means of semi-automatic ultrastructural morphometry. A maximum difference of 9% in the number of fibres counted was obtained when comparing the automatic and the semi-automatic methods. In all cases the histograms of the morphometrical variables (fibre and axon diameters, myelin sheath thickness) were found to be identical to the reference histograms obtained by the semi-automatic method.

Evaluation Studies as Topic↗

PubMed1990

Effects of tunicamycin on the avoidance reaction of epidermis by sensory neurites in co-cultures.

In 7-day chick embryo dorsal root ganglia and epidermis or dermis co-cultures, nerve fibres establish contacts with dermis while avoiding epidermis. Previous results have indicated that factor(s) secreted by epidermis could be involved in this avoidance reaction. The present study demonstrates that the avoidance reaction is abolished when epidermal cells are treated by the N-linked glycoproteins synthesis inhibitor, tunicamycin. The same result is obtained after monensin treatment. The epidermal cell viability, development and total protein secretion are not significantly affected by tunicamycin, as demonstrated by trypan blue exclusion, electron microscopy and SDS-PAGE electrophoresis after 35S-methionine labelling. It has thus been concluded that the avoidance factor is glycoproteic in nature. It is also suggested that this factor possibly contains chondroitin-6-sulphate moieties.

Animals↗