FGF-2 Low Molecular Weight Selectively Promotes Neuritogenesis of Motor Neurons In Vitro

In this study, we screened in vitro the different capabilities of trophic factors with promising effect for enhancing selective regeneration and thus promoting specific reinnervation of target organs after peripheral nerve regeneration. We found that FGF-2 (18 kDa) was the trophic factor that exerted the most selective effect in promoting neurite outgrowth of spinal motoneurons both in terms of elongation and arborization. The mechanism underlying this effect on neuritogenesis seems related to FGF-2 enhancing the interaction between FGFR-1 and PSA-NCAM. The interaction of these two receptors is important during the early stages of neuritogenesis and pathfinding, while integrin alpha7B subunit seems to play a role during neurite stabilization.     

The Hemolymph of the ascidian Styela plicata (Chordata-Tunicata) contains heparin inside basophil-like cells and a unique sulfated galactoglucan in the plasma

The hemolymph of ascidians (Chordata-Tunicata) contains different types of hemocytes embedded in a liquid plasma. In the present study, heparin and a sulfated heteropolysaccharide were purified from the hemolymph of the ascidian Styela plicata. The heteropolysaccharide occurs free in the plasma, is composed of glucose ( approximately 60%) and galactose ( approximately 40%), and is highly sulfated. Heparin, on the other hand, occurs in the hemocytes, and high performance liquid chromatography of the products formed by degradation with specific lyases revealed that it is composed mainly by the disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4)) (39.7%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(6SO(4)) (38.2%). Small amounts of the 3-O-sulfated disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4)) (9.8%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4))(6SO(4)) (3.8%) were also detected. These 3-O-sulfated disaccharides were demonstrated to be essential for the binding of the hemocyte heparin to antithrombin III. Electron microscopy techniques were used to characterize the ultrastructure of the hemocytes and to localize heparin and histamine in these cells. At least five cell types were recognized and classified as univacuolated and multivacuolated cells, amebocytes, hemoblasts, and granulocytes. Immunocytochemistry showed that heparin and histamine co-localize in intracellular granules of only one type of hemocyte, the granulocyte. These results show for the first time that in ascidians, a sulfated galactoglucan circulates free in the plasma, and heparin occurs as an intracellular product of a circulating basophil-like cell.     

Identification and tissue-specific distribution of sulfated glycosaminoglycans in the blood-sucking bug Rhodnius prolixus (Linnaeus)

We have previously characterized heparan sulfate (HS) as the major ovarian sulfated glycosaminoglycan (GAG) in females of Rhodnius prolixus, while chondroitin sulfate (CS) was the minor component. Using histochemical procedures we found that GAGs were concentrated in the ovarian tissue but not found inside the oocytes. Here, we extend our initial observations of GAG expression in R. prolixus by characterizing these molecules in other organs: the fat body, intestinal tract, and the reproductive tracts. Only HS and CS were found in the three organs analyzed, however CS was the major GAG species in these tissues. We also determined the compartmental distribution of GAGs in these organs by histochemical analysis using 1,9-dimethylmethylene blue, and evaluated the specific distribution of CS within both male and female reproductive tracts by immunohistochemistry using an anti-CS antibody. We also determined the GAG composition in eggs at days 0 and 6 of embryonic development. Only HS and CS were found in eggs at day 6, while no sulfated GAGs were detected at day 0. Our results demonstrate that HS and CS are the only sulfated GAG species expressed in the fat body and in the intestinal and reproductive tracts of Rhodnius male and female adults. Both sulfated GAGs were also identified in Rhodnius embryos. Altogether, these results show no qualitative differences in the sulfated GAG composition regarding tissue-specific or development-specific distribution.     

Cytoarchitectural features of Ucides cordatus (Crustacea Decapoda) hepatopancreas: structure and elemental composition of electron-dense granules

Hepatopancreal tissue of the crab Ucides cordatus was investigated by light and electron microscopy. The observed epithelial cells were: E-cells (embryonic), located in the distal portion of the hepatopancreal tubules, R-cells (resorptive) F-cells (fibrillar) and B-cells (blister or secretory), found in its intermediate and proximal regions. Two types of electron-dense granules (EDGs) were found frequently in the cells of the proximal portion of the hepatopancreal tubule. Both types of EDGs presented alternating concentric electron-dense and electron-lucent layers. In order to better characterize these granules, energy dispersive X-ray analysis (EDXA) and glucose-6-phosphatase (G6Pase) cytochemistry were performed. One type of spherical granule was seen inside vacuoles surrounded by an association of myelin-like membranes as well as some small membrane-bound vesicles. This type of granule neither presented detectable Ca and P on EDXA spectra nor G6Pase cytochemical reaction products. The second type of granule had O, P and Ca characteristic peaks. G6Pase cytochemical products were observed inside these structures and showed that this mineralized type was surrounded by endoplasmic reticulum membranes. This result suggests that in U. cordatus the endoplasmic reticulum is associated with the genesis of mineralized EDGs. While amorphous mineral granules may be associated with a storage of Ca and P for the new carapace synthesis, EDGs covered by the non-mineralized spherical multi-layered membranes may be associated with late endosomes. No specific secretory pathway however was determined for the EDGs at the epithelial proximal portion.     

Colocalization of heparin and histamine in the intracellular granules of test cells from the invertebrate Styela plicata (Chordata-Tunicata)

In most ascidian species the oocytes are surrounded by two types of accessory cells called follicle cells and test cells. Test cells are located on the periphery of oocytes and remain in the perivitelline space during egg development until hatching. Heparin and histamine were previously described in the test cells of the ascidian Styela plicata. In the present study, electron microscopy techniques were used to characterize the ultrastructure of the S. plicata test cells and to localize heparin and histamine in these cells. Test cells contain several intracellular granules with unique ultrastructural features. They are formed by elongated filaments composed of serial globules with an electron-lucent circle, containing a central electron-dense spot. Immunocytochemistry showed that heparin and histamine colocalize at the border of granule filaments in the test cell. Compound 48/80, a potent secretagogue of heparin-containing mast cells, also induced degranulation of test cells. According to these results, we suggest that test cells represent ancient effector cells of the innate immunity in primitive chordates.     

The effects of UV radiation on the visual system of the crab Neohelice granulata: a protective role of melatonin

The first and main target-structure of ultraviolet (UV) radiation in animals is the body surface, including the skin and eyes. Here, we investigated cell damage in the visual system of the crab Neohelice granulata acclimated to constant light and exposed to UVA or UVB at 12:00 h for 30 min. The reactive oxygen species (ROS) production, antioxidant capacity against peroxyl radicals (ACAP), lipid peroxidation (LPO) damage, catalase (CAT) activity, and the melatonin immunohistochemical reactivity in the eyestalks were evaluated. The animals that received melatonin and were exposed to UVA and UVB radiation showed a decreased ROS concentration (p < 0.05).The ACAP test showed a decrease (p < 0.05) in their values when the animals received 2 pmol/crab of melatonin (physiological dose) before the exposure to UVA radiation. The animals exposed to UVB radiation after receiving the same dose of melatonin showed an increase (p < 0.05) in the ACAP test compared with the animals exposed to UVB radiation after receiving only crab physiological saline. The CAT activity increased (p < 0.05) in the animals that received melatonin and were exposed to UVA and UVB radiation. Animals exposed to UVA and UVB displayed an increase (p < 0.05) in the LPO levels, whereas animals treated with melatonin showed lower (p < 0.05) LPO levels when irradiated. The results indicate that the specific oxidative parameters altered by UV radiation can be modulated by a physiological dose of melatonin. Moreover, the melatonin regularly produced by virtually all eyestalk cells suggests that it may function to modulate the noxious effects of radiation, at least in the crab N. granulata.     

Crustacean visual system: an investigation on glial cells and their relation to extracellular matrix.

Glial cells in higher invertebrate groups are usually recognized on the basis of their location and general morphological or functional criteria. In this study of the crustacean visual system, we have approached the analysis of the relations between glial cells and the extracellular matrix by classical histochemical methods for carbohydrates at the light and electron microscopic levels, carbonic anhydrase histochemistry and by the biochemical characterization of sulphated polysaccharides. Periodic acid-Schiff-positive glial cells and processes were observed in the retina, basement membrane below the retina and in the optic ganglia. Carbonic anhydrase was not detected in the retina but it was demonstrated in all optic ganglia. The biochemical analysis of the extracellular matrix confirmed the alcian blue reaction and showed that sulphated polysaccharides are not abundant in the optic neuropils. This article describes into more details the crustacean visual system glial cells classification, and the relation between them and the extracellular matrix. In addition, they show that glial cells are the main components of the retinal basement membrane.     

Adult neurogenesis in the crayfish brain: Proliferation,migration, and possible origin of precursor cells

The birth of new neurons and their incorporation into functional circuits in the adult brain is a characteristic of many vertebrate and invertebrate organisms, including decapod crustaceans. Precursor cells maintaining life‐long proliferation in the brains of crayfish (Procambarus clarkii, Cherax destructor) and clawed lobsters (Homarus americanus) reside within a specialized niche on the ventral surface of the brain; their daughters migrate to two proliferation zones along a stream formed by processes of the niche precursors. Here they divide again, finally producing interneurons in the olfactory pathway. The present studies in P. clarkii explore (1) differential proliferative activity among the niche precursor cells with growth and aging, (2) morphological characteristics of cells in the niche and migratory streams, and (3) aspects of the cell cycle in this lineage. Morphologically symmetrical divisions of neuronal precursor cells were observed in the niche near where the migratory streams emerge, as well as in the streams and proliferation zones. The nuclei of migrating cells elongate and undergo shape changes consistent with nucleokinetic movement. LIS1, a highly conserved dynein‐binding protein, is expressed in cells in the migratory stream and neurogenic niche, implicating this protein in the translocation of crustacean brain neuronal precursor cells. Symmetrical divisions of the niche precursors and migration of both daughters raised the question of how the niche precursor pool is replenished. We present here preliminary evidence for an association between vascular cells and the niche precursors, which may relate to the life‐long growth and maintenance of the crustacean neurogenic niche. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Effects of seasonality and moult cycle on the proliferation of nerve cells and on the labelling of ecdysone receptors in an estuarine crab

Decapod crustaceans show proliferation of the nerve cells in the olfactory lobe throughout their lives. However, the regulation of this process is still poorly understood, since it may vary with endogenous and exogenous factors. The objective of the present investigation was to quantify the proliferation of nerve cells and number of nerve cells with ecdysone receptors in the clusters of the central olfactory system in Neohelice granulata, according to moult stages and in different seasons (summer and winter). Three injections of bromodeoxyuridine (BrdU) were administered to the crabs. Brains were sectioned by microtome and fixed on slides for immunohistochemistry with anti-BrdU and anti-EcR antibodies. The proliferation of nerve cells was higher in winter than in summer, probably because in winter the crabs do not breed and the premoult and postmoult periods are longer. Crabs in postmoult exhibited more BrdU-labelled cells than crabs in premoult or intermoult in winter, because of a greater number of mitoses related to an increase in body size and addition of olfactory receptor neurons. The number of EcR-labelled cells was higher in premoult than in postmoult or intermoult in winter. The proliferation of nerve cells is regulated seasonally and according to moult stages.     

The evaluation of chronic psychiatric care

During the past two decades the reduction in the size of the mental hospital population in this country has closely followed statistical predictions for bed requirements both in Canada and in the province of Ontario. The decrease has been most pronounced among chronic non-retarded patients. While these patients are now less evident in hospital statistics, it is suggested that improvements in their state may be more apparent than real, owing to differences in the terminology and numerators employed in presenting mental health statistics. Further, concern has been expressed about the quality of care received by former chronic patients now scattered through the community in a variety of after-care programs. Readmission rates have greatly increased in absolute terms and as a proportion of total admissions. The evaluation of the management of chronic psychiatric patients is discussed.