Extraction, Purification and Electrical Characterization of Gross Galactomannan and Purified Galactomannan Obtained from Adenanthera pavonina L. Seeds.

Aiming of self-sustainable production, the search for biodegradable and biocompatible materials has brought with it the need to know the physicochemical and dielectric characteristics of polysaccharide-based composite structures, which can be used as important and promising raw materials for biotechnology and electronic industries. Galactomannans are polysaccharides, extracted from seeds and microbiological sources, consisting of mannose and galactose. In this context, this work aimed to extract, purify and characterize by XRD, FTIR and impedance spectroscopy galactomannan obtained from seeds of Adenanthera pavonina L. The purification process was made with ethyl alcohol at concentrations of 70, 80 and 90 %. Polymeric films were prepared by solvent slow evaporation at low temperatures. XRD measurements revealed that Galactomannan from Adenanthera pavonina L., after purification, has a semi-crystalline structure due to the identification of two peaks the first between 5.849° and 6.118° and the second between 20.011° and 20.247°. FTIR spectra showed the functional groups associated with monosaccharides of the galactomannan from Adenanthera pavonina L. seeds, as well as the typical polysaccharide bands and peaks, confirmed by literature data. The impedance results give an increment on the state-of-the-art of this biomaterial by showing the existence of dielectric relaxations, independent of the degree of purification, using the dielectric modulus formalism. The permittivity analysis reveals the presence of water in the structure of the film, whose dipoles contribute to the relatively high value of the dielectric constant. From the results obtained, it can be concluded that purified galactomannan has the potential for possible applications in the electronics industry as a green and eco-friendly dielectric material.

Transient populations of presumptive macrophages in the brain of the developing hamster, as indicated by endocytosis of blood-borne horseradish peroxidase.

During postnatal development, clusters of cells associated with the mononuclear phagocytic system appear within the white matter of rodents and cats. We studied the distribution and morphology of these cells in the hamster's brain during the first 2 weeks after birth. In animals of different ages, horseradish peroxidase was injected into the heart. After 3-6 h survival, the animals were perfused with aldehydes and had their brains removed, cut and reacted. In another series, fixed brain sections from horseradish peroxidase-injected and non-injected animals were reacted for a non-specific esterase expressed by monocytes and macrophages. The horseradish peroxidase reaction-product was seen throughout the nervous tissue at the first postnatal day, appearing more concentrated in certain brain sectors from postnatal day 3 through 10, to finally become restricted to a few regions at postnatal day 16. Horseradish peroxidase-labeled cells appeared in increasing numbers from postnatal day 1 to 8, decreasing thereafter to disappear completely at postnatal day 16. Some labeled cells were roundish or elliptical with few, if any, processes; others had several clearly detectable processes. Horseradish peroxidase-labelled cells formed clusters within the dorsal subventricular zone, dorsal cortical white matter, corpus callosum and several other prosencephalic fiber tracts. The morphology of esterase-reactive cells was less clearly outlined but their distribution and relative density correlated with those of horseradish peroxidase-labeled cells. Also, many horseradish peroxidase-labeled cells were esterase-positive in most clusters. We conclude that (1) some cells in the developing brain selectively endocytose and accumulate blood-borne horseradish peroxidase in their cytoplasm, (2) these cells do not appear to be neurons but a particular cell type associated to the mononuclear phagocytic system and (3) they cluster transiently in particular sectors of the cortical and subcortical white matter during the first 2 weeks after birth.

CNPase expression in the developing opossum brain stem and cerebellum.

Immunoreactivity to 2',3' cyclic nucleotide 3' phosphohydrolase (CNPase; EC 3.1.4.37) was studied in the developing opossum brain stem and cerebellum. Regional differences were found in oligodendrocytes concerning the time of appearance (early: medical longitudinal fascicle [mlf]; intermediate: inferior colliculus [IC], deep layers of the superior colliculus [SC] and white matter of cerebellar folia; late: optic layer of SC) and duration of immunoreactivity (short: optic layer of SC; intermediate: mlf; long: cerebellar folia, etc). The results suggest that regional heterogeneities in CNPase expression are linked to intrinsic properties of local and afferent axons.

Differential patterns of laminin expression in lateral and medial midbrain glia.

An analysis of the extra cellular matrix (ECM) in regionally heterogeneous midbrain glia has been started. Immunoreactivity to laminin has been tested in confluent glial cultures from lateral (L) and medial (M) sectors of 14 days mouse embryos (E14) and in neurone-glia cocultures kept for 48 hours after plating of E14 midbrain freshly-dissociated neurones. Laminin is present in both types of glial cultures, but its distribution assumes a punctate pattern in glia that is not permissive for neurite growth (M-glia) and a fibrillar configuration in a favourable glial substrate (L-glia). Moreover, laminin expression is dramatically upregulated in co-cultures although fibrillar and punctate patterns are maintained.

Expression of the non-compact myelin protein 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in olfactory bulb ensheathing glia from explant cultures.

The non-compact myelin protein 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) may have a unique role in signaling pathways mediated by lipid-protein domains (Kim and Pfeiffer, 1999). We have tested for CNPase in olfactory bulb ensheathing glia (OBEG) in explant cultures. Migrating bipolar and multipolar cells with OBEG typical morphologies were inimunoreactive for both vimentin- and S100-like proteins. Although apparently devoid of myelin basic protein (MBP)-like immunoreactivity, these cells displayed weak but unambiguous CNPase-like immunoreactivity. Our results suggest a further resemblance to myelinating Schwann cells.

Glial cells with differential neurite growth-modulating properties probed by atomic force microscopy.

Lateral (L) and medial (M) midbrain astrocytes differ in their ability to support neuritic growth (L, permissive; M, non-permissive) with properties of M glia depending on heparan sulfate (HS). Here we show by atomic force microscopy that the surfaces of formaldehyde-fixed astrocytes differ by conspicuous 250 nm protrusions in L and by a HS-dependent fibrillar network in M glia, thus, demonstrating correlations between cell surface morphology and functional properties.

Subcellular localization of neuronal nitric oxide synthase in the superficial gray layer of the rat superior colliculus.

The superficial layers of the rat superior colliculus (sSC) receive innervation from retina and include nitric oxide synthase (NOS)-immunoreactive neurons. We used electron microscopic immunocytochemistry to assess the subcellular localization of neuronal NOS (nNOS) in the sSC. nNOS immunoreactivity was detected on the external membrane of mitochondria, endoplasmic reticulum, in pre- and postsynaptic profiles and also diffusely distributed in the cytosol. Postsynaptic labeled regions were often associated with presumptive retinal unlabeled terminals. Microtubules also appeared intensely labeled. These results show that NOS immunoreactive neurons may be innervated by retinal terminals and suggest an association of nNOS with cytoskeletal elements.

Postnatal development of retinogeniculate, retinopretectal and retinotectal projections in the opossum.

The postnatal development of retinogeniculate, retinopretectal and retinotectal projections has been studied by the anterograde transport of proline-labeled proteins in 20 pouch young opossums aged from 10 to 60 days. Radioautographical findings suggest delayed development of uncrossed as compared to crossed projections. There is a phase of overlapping projections from both eyes in thalamic and tectal target sites. Partial segregation of projections to the dorsal lateral geniculate nucleus (GLD) is preceded by differential distribution of crossed and uncrossed terminal fields along its dorsoventral axis (at age 23 days). The quasilaminar pattern of projections in the dorsocaudal region of GLD pars alpha is incipient by 42 days and is fully established at 60 days of age, as eye opening starts. The mature pattern of projections to the ventral lateral geniculate nucleus (GLV) is established much earlier, at 23 days of age. The development of retinopretectal projections is assessed mostly from the analysis of the olivary pretectal nucleus (PO). Distribution of silver grains into discrete areas coextensive with PO is relatively delayed (by 23 days of age) as compared to the nuclei of the lateral geniculate body. Soon after, however, the mature pattern of projections to PO is established (at 33 days of age). The early development of retinotectal projections (from 10 to 23 days) is compatible with an initial tangential course of crossed optic fibers in the superior colliculus (CS) but other alternatives remain open, such as a sequential outside-in arrangement of terminal fields of deeply coursing fibers. Arborization of uncrossed fibers is delayed at extreme rostromedial and caudolateral portion of the territory of the main uncrossed retinotectal projection. Segregation of uncrossed projections at different depths of CS is nearly complete by 42 days. Differences in the development of terminal fields in different target nuclei or in regions of a given target site are discussed in relation to retinal and local factors.

Retinofugal projections in the opossum, An anterograde degeneration and radioautographic study.

A study of anterograde degeneration and anterograde transport was undertaken in the opossum primary optic system in order to clarify several points regarding fiber organization and patterns of terminal fields. Through the radioautographic technique of axon tracing, it was demonstrated that the accessory optic system follows the generalized scheme of Hayhow, consisting of two fascicles the three terminal nuclei.

Observations on postnatal neurogenesis in the superior colliculus and the pretectum in the opossum.

Postnatal neurogenesis has been detected in the superior colliculus (CS) and caudal pretectum of the opossum in the period ranging from 2 to 13 days ( PND2 to PND13 ) of life in the pouch. Examination of the pattern of labeling in specimens exposed to a pulse of tritiated thymidine ( [3H]T) in PND4 or PND7 and allowed 1.5 h survival reveals that postnatal cell proliferation for the CS is virtually confined to the ventricular zone with no evidence for in situ[3H]T uptake in the collicular plate. Semi-quantitative analysis in long survival animals shows that postnatal neurogenesis peaks later in the CS ( PND7 ) than in the caudal pretectum ( PND4 ) and also persists longer in the former than in the latter. Comparisons of the numerical density of heavily labeled neurons suggest the occurrence of ventro-dorsal and rostro-caudal gradients of neurogenesis in the CS. Separate analysis of superficial, intermediate and deep layers shows, in addition, a combined rostrolateral-to-caudomedial gradient of neurogenesis in the superficial layers. Comparisons of the time schedules of neurogenesis for the superficial layers and of the deployment of optic fibers suggest that migration of neurons to their eventual destination is completed at or after the arrival of afferents.

Growth and restriction of the ipsilateral retinocollicular projection in the opossum.

The distribution of optic nerve fibers and terminals in the superior colliculus (SC) was followed throughout its development in pouch young opossums in order to establish the normal sequence of events leading to the formation of mature patterns. Up to 7 days of life in the pouch, labeled fibers can be followed only as far as the rostral aspect of the optic tract. The earliest evidence for crossed retinal projections in the SC is found at 10 days of age. In parasagittal sections, the label extends along the rostrocaudal tectal axis from the rostral border to the presumptive caudal pole of the SC. Unequivocal evidence for ipsilateral retinocollicular projection is found at 15 days extending to all but the caudal 5th of the rostrocaudal extent of the SC. The projections from both eyes overlap extensively in the SC at 22 days and after this age significant changes occur, mostly at the ipsilateral side: a sub-pial tier of fine label develops excluding both rostral and caudal collicular poles; a deeper tier of coarse label extends from the rostral to the caudal pole and a third, patchy tier of label is found at the prospective strata griseum superficiale and griseum intermediate. By 47 and 60 days the tangential distribution of the projections is virtually indistinguishable from the adult pattern although laminar segregation does not seem as sharp as in the adult. Comparisons of the changeable patterns of ipsilateral retinocollicular projections from 22 to 34 days with the invariant, aberrant pattern in adult animals submitted to uniocular enucleation at either age suggests that the preservation of a juvenile pattern does not provide a comprehensive explanation for the formation of aberrant projections.

Asynchronous changes in size, number, and shape of synapses in the developing superior colliculus.

The ultra-structural development of synapses in retino-receptive layers of the opossum superior colliculus was studied by the ethanolic phosphotungstic acid (E-PTA) method. There was a tendency for a slight reduction in the diameter of synaptic disks, a rise and fall of numerical densities and, except for an ephemeral period, a general increase in the proportion of "frown" among curve synapses. The lack of strict synchrony and the occurrence of different patterns of changes suggest that multiple factors contribute to synaptic maturation.

Expression of 9-O-acetylated gangliosides is correlated with tangential cell migration in the rat brain.

9-O-acetylated gangliosides (9-O-aGs) are expressed in regions of the developing brain during radial neuronal migration and an anti-9-O-aGs monoclonal antibody (JONES mAb) interferes with axonal growth. To determine the generality of 9-O-aGs expression in directional movements, we have examined their immunoreactivity with mAb JONES in the cell stream from the lateral ventricle rostral subventricular zone (LVSVZ) to the olfactory bulb (OB) in postnatal and adult rats. We show expression of 9-O-aGs both in LVSVZ, along the rostral migratory stream and in the OB in developing animals and, at lower levels, in adulthood. That suggests 9-O-aGs' involvement in tangential cell migration as well in other neural directional movements.

Fiber order in the opossum's optic tract.

The distribution of axons by size in the optic tract of the South American opossum, Didelphis marsupialis was studied. Thin and semi-thin sections were examined, and measurements of axonal diameter were made on electron micrographs taken from various locations across the optic tract of normal opossums. In order to determine the contributions of the different axon diameter classes to the crossed and uncrossed retinofugal pathways, measurements were also made from the tracts of opossums in which one eye had been enucleated 5 weeks previously. Within the opossum's optic tract, the axons are partially segregated by their size: the deepest parts of the tract contain only fine and medium-sized axons, whereas coarse axons are also present superficially. In the middle of the tract, all three size classes are present. At increasingly superficial positions, there is a steady reduction in the proportion of medium-diameter axons, and an increase in the number of the finest axons. Medium and coarse axons contribute to both the crossed and uncrossed pathways, and the uncrossed component is displaced superficially relative to the crossed component. The fine axons in the deeper parts of the tract arise from both retinae, while those in the superficial parts of the tract, near the pial surface, are virtually all crossed. The opossum's optic tract thus displays the segregation of axons by size found in placental mammals, and follows a pattern reminiscent of that found in carnivores. Such a common organizational plan, particularly the similarities between the didelphids and carnivores, is suggestive of an early acquisition of parallel visual pathways in mammalian phylogeny. Since the fiber order in the optic tract of eutherians is a chronological map of axonal arrival during development, these results suggest that a conserved developmental mechanism has led to a common organizational plan.

Patterns of myelination in the opossum superior colliculus with additional reference to the optic tract.

Patterns of myelination have been studied in the optic tract and the superior colliculus (SC), with special reference to the optic layer, in the opossum Didelphis marsupialis. Myelination in the optic tract starts far in precedence of eye opening and follows a rostrocaudal gradient. Myelination in the SC presents the following features: it proceeds according to a general inside-out pattern and follows both rostro-caudal and latero-medial gradients in the optic layer, and it accelerates in the SC optic layer soon after systematic exposure to visual input. The data presented here, together with other available information, suggest that myelination in the opossum optic tract starts in parallel with the stabilization in the number of optic fibers, and advances in the rostro-caudal mode common to most eutherian mammals, and also that myelogenesis in the SC neither correlates necessarily with, nor recapitulates, the sequence of acquisition of GFAP-positive astrocytes in a given set of layers. Changes in the rate of myelination in the optic layer after exposure to visual input are regionally-selective, and seem compatible with the recruitment of thin axons into the myelogenetic cycle rather than with the thickening of pre-existing myelin sheaths. It is concluded that the SC is a favorable structure for the study of the differentiation of glial cells, particularly in species with an extended time course of maturation such as the opossum.

Genesis of neurons of the retinal ganglion cell layer in the opossum.

In this study, we have examined the genesis of neurons of the retinal ganglion cell layer of the opossum Didelphis marsupialis by [3H]-thymidine autoradiography. Our results suggest that most neurons surviving to adulthood are generated in postnatal life from day 1 to day 23. Cells are generated according to a coarse gradient from the retinal geometric center to the periphery. Regional analysis of soma size distributions in different cohorts suggest that this gradient is actually formed by two partially-overlapping, concentric waves of cell proliferation. Most medium and large ganglion cells are formed during the early wave, whereas most displaced amacrine cells and small ganglion cells are formed during the late wave. Our results confirm the appropriateness of the opossum as a model for studies of development of the mammalian visual system.

Proliferation of differentiated glial cells in the brain stem.

Classical studies of macroglial proliferation in muride rodents have provided conflicting evidence concerning the proliferating capabilities of oligodendrocytes and microglia. Furthermore, little information has been obtained in other mammalian orders and very little is known about glial cell proliferation and differentiation in the subclass Metatheria although valuable knowledge may be obtained from the protracted period of central nervous system maturation in these forms. Thus, we have studied the proliferative capacity of phenotypically identified brain stem oligodendrocytes by tritiated thymidine radioautography and have compared it with known features of oligodendroglial differentiation as well as with proliferation of microglia in the opossum Didelphis marsupialis. We have detected a previously undescribed ephemeral, regionally heterogeneous proliferation of oligodendrocytes expressing the actin-binding, ensheathment-related protein 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), that is not necessarily related to the known regional and temporal heterogeneity of expression of CNPase in cell bodies. On the other hand, proliferation of microglia tagged by the binding of Griffonia simplicifolia B4 isolectin, which recognizes an alpha-D-galactosyl-bearing glycoprotein of the plasma membrane of macrophages/microglia, is known to be long lasting, showing no regional heterogeneity and being found amongst both ameboid and differentiated ramified cells, although at different rates. The functional significance of the proliferative behavior of these differentiated cells is unknown but may provide a low-grade cell renewal in the normal brain and may be augmented under pathological conditions.

Developmental changes in synapses of the retino-recipient layers of the opossum superior colliculus.

Changes in synaptic structure were examined in junctions of the retino-recipient layers of the opossum superior colliculus (SC) at critical developmental stages (30, 40 and 61 day-old pouch young and adult controls). Criteria of classification were the presence and direction of curvature (smile, flat, frown and irregular) and the degree of aggregation of paramembranous components (immature, intermediate and mature). The results favor the aggregation of dense components vis-a-vis curvature in the assessment of maturational changes.

Observations on the arrangement of axons according to diameter in the optic tract of the opossum Didelphis marsupialis.

The spectra of fiber sizes at different depths of the optic tract of the opossum Didelphis marsupialis were examined by electron microscopy in order to test for correlations between the eventual location of axons and relevant developmental events. Frequency histograms showed 1) a predominant representation of medium-sized axons and the virtual exclusion of coarse fibers from the deepest portion of that pathway, and 2) a progressive increase in the proportion of thin axons from deep to superficial sites of the tract. These findings are discussed in terms of the view of the optic tract as a chronological map of axon arrival.

The extracellular matrix of the midline and non-midline midbrain glia: correlations with neurite growth-supporting abilities.

The central nervous system (CNS) midline plays an important role in growth and guidance of axons. At the midline, a multiplicity of cell types establish boundaries that control the navigation of crossed and uncrossed axonal fibers. The extracellular matrix (ECM) molecules of the resident neuroepithelial or committed neuronal or glial cells could be involved in the control of axon growth and axon guidance. This review reports the recent advances in the study of the structure and functional role of the ECM at the midline locus of the CNS. In vivo and in vitro approaches are considered to provide new clues in the understanding of processes involved in the cellular decisions of the CNS midline.