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Biomedical subjects

S Thanos

Publications and source records attributed to S Thanos.

At least 19 recordsLinked to original sources
PubMed2005

Unilateral optic nerve crush induces bilateral retinal glial cell proliferation.

The post-injury responses of retinal ganglion cells elicit a number of glial reactions which have not been completely understood. The bilateral pattern of non-neuronal retinal cell proliferation was examined in association with the differential fates of unilaterally injured adult retinal ganglion cells by means of bromodeoxyuridine (BrdU) immunocytochemistry. Lateralization of the glioproliferative events was studied by analysing both the experimental and the uninjured contralateral as well as matched retinas of sham-operated animals. Control adult rat retina included very few BrdU-positive cells within the nerve fibre and ganglion cell layers; however, experimental retinas of degenerating groups exhibited statistically significantly higher densities of newborn cells in most layers. Clusters of labelled cells were found in the inner plexiform layer related to OX-42 staining, indicating their microglial nature. Indeed, double-labelling experiments, after short-term unilateral optic nerve crushing, identified proliferating retinal glial cells in vivo. Both types of glia, astroglial and microglial cells, exhibited BrdU-positive labelling in injured as well as uninjured experimental rat retinas. Moreover, microglial proliferating cells were also identified in explanted retinal pieces after 2 days in culture. Affected and contralateral retinas responded similarly to the unilateral experimental manipulations applied with respect to BrdU labelling. The acute glial responses observed suggest that bilateral glial proliferation might represent a common response related to degeneration events in both retinas, i.e. ipsi- and contralateral to the experimental injury.

Animals↗
PubMed2004

Can lenticular factors improve the posttrauma fate of neurons?

The intraocular lens has recently been recognized as a potential source for neuroprotective and neurite-promoting activities. The lens is ontogenetically and functionally a peculiar intraocular tissue with the unique feature of performing incomplete cellular apoptosis throughout the lifetime. The ectodermally derived epithelial cells permanently divide to produce the nuclei- and organelle-free lens fibre cells that allow for the optical transparency. The underlying extremely specific physical, biochemical, metabolic and structural mechanism lead to efficient protection from photo-oxidative stress caused by exposure to short-wavelength light. The fact that fibre cells undergo incomplete apoptosis is also of crucial importance to other cellular systems. In particular, injured nerve cells such as axotomized retinal ganglion cells may profit from the apoptosis-blocking mechanisms operating within the lens fibres. In this review we first discuss some factors involved in the lens differentiation and partial apoptosis as a basic principle of long-term survival. We then present recent experimental evidence that lenticular factors also operate outside the lens, and in particular within the retina to contribute to axonal regeneration, e.g. after a trauma. In turn, factors such as GAP-43 that were thought to be exclusively expressed within nervous tissue have now also been discovered within the lenticular tissue. Experiments of the direct confrontation of lenticular epithelial and fibre cells with regenerating ganglion cell axons in vitro are presented. It is concluded that survival factors supplied by the lens might be used to facilitate survival within neuronal tissue.

Animals↗
PubMed2004

Role of a neuronal small non-messenger RNA: behavioural alterations in BC1 RNA-deleted mice.

BC1 RNA is a small non-messenger RNA common in dendritic microdomains of neurons in rodents. In order to investigate its possible role in learning and behaviour, we compared controls and knockout mice from three independent founder lines established from separate embryonic stem cells. Mutant mice were healthy with normal brain morphology and appeared to have no neurological deficits. A series of tests for exploration and spatial memory was carried out in three different laboratories. The tests were chosen as to ensure that different aspects of spatial memory and exploration could be separated and that possible effects of confounding variables could be minimised. Exploration was studied in a barrier test, in an open-field test, and in an elevated plus-maze test. Spatial memory was investigated in a Barnes maze and in a Morris water maze (memory for a single location), in a multiple T-maze and in a complex alley maze (route learning), and in a radial maze (working memory). In addition to these laboratory tasks, exploratory behaviour and spatial memory were assessed under semi-naturalistic conditions in a large outdoor pen. The combined results indicate that BC1 RNA-deficient animals show behavioural changes best interpreted in terms of reduced exploration and increased anxiety. In contrast, spatial memory was not affected. In the outdoor pen, the survival rates of BC1-depleted mice were lower than in controls. Thus, we conclude that the neuron-specific non-messenger BC1 RNA contributes to the aptive modulation of behaviour.

Analysis of Variance↗
PubMed2004

[Neuroprotection and regeneration after traumatic lesion of the optic nerve].

BACKGROUND: After a traumatic lesion of the optic nerve, retinal ganglion cells (RGC) undergo massive degeneration by apoptosis, which leads to loss of vision in the affected eye. Like other neurones in the central nervous system, RGC are not able to regenerate their damaged axons spontaneously. We used special surgical methods and pharmacological measures to achieve enhanced survival and regeneration of damaged RGC. MATERIALS AND METHODS: Studies were performed using the model of RGC degeneration induced by severing the optic nerve of adult rats. RGC were loaded with a fluorescent dye, and several drugs were applied intravitreally. The effects were evaluated after two weeks by counting the surviving RGC. For regeneration studies, an autologous peripheral nerve graft was sutured to the stump of the cut optic nerve, or the ends of the cut optic nerve were re-sutured. Recovery of RGC function was assessed by VEP measurements. RESULTS: The number of RGC surviving an axotomy increased significantly after intravitreal injections of aurintricarboxylic acid, cortisol, a caspase inhibitor, brimonidine or microglia-targeted substances. Regeneration of cut axons was enhanced by aurintricarboxylic acid or cortisol. In addition, considerable neuroprotective and regenerative effects including partial restoration of VEP were induced by lens injury, which results in a gradual release of crystallins into the vitreous, or by intravitreal injection of purified crystallins. CONCLUSION: The loss of vision after an optic nerve trauma can be reduced in this animal model by suitable neuroprotective measures, which raises hope for the treatment of patients.

Animals↗
PubMed2003

AMOG/beta2 and glioma invasion: does loss of AMOG make tumour cells run amok?

The beta2 subunit of Na,K-ATPase, initially described as adhesion molecule on glia (AMOG), has been shown to mediate neurone-astrocyte adhesion as well as neural cell migration in vitro. We have investigated the expression of AMOG/beta2 in human gliomas and its effect on glioma cell adhesion and migration. Compared to normal astrocytes of human brain, AMOG/beta2 expression levels of neoplastic astrocytes were down-regulated in biopsy specimens and inversely related to the grade of malignancy. One rat and four human glioma cell lines showed complete loss of AMOG. To investigate the function of AMOG/beta2, its expression was re-established by transfecting an expression plasmid into AMOG/beta2-negative C6 rat glioma cells. In vitro assays revealed increased adhesion and decreased migration on matrigel of AMOG/beta2-positive cells as compared to their AMOG/beta2-negative counterparts. We conclude that increasing loss of AMOG/beta2 during malignant progression parallels and may underlie the extensive invasion pattern of malignant gliomas.

Adenosine Triphosphatases↗
PubMed2002

Isolation of total-RNA from formalin-fixed rat retina.

The aim of this project was to establish a method for the purification of total-RNA from fixed rat-retina. Two different established methods were used for RNA purification, and successful isolation was verified with RT-PCR for amplification of beta-actin (two different product-lengths) and subsequent gel-electrophoresis. Total-RNA was successfully isolated from fixed rat-retina. The house keeping gene, beta-actin could be detected after fixing the retina either with 1% formalin or with 4% paraformaldehyde (PFA). Hexamer-primer based RT-PCR gave better results than the oligo-d(T)-primer based RT-PCR method. Both the 698 and 225 bp beta-actin-fragments could be successfully amplified, where amplification of the latter was more efficient. This approach shows that tissue fixation prior to RNA-isolation facilitates the rapid isolation of undamaged RNAs in tissues such as the retina, which are known to yield low levels of RNA and are vulnerable to RNases.

Animals↗
PubMed2001

Perforated microelectrode arrays implanted in the regenerating adult central nervous system.

Adult mammalian optic nerve axons are able to regenerate, when provided with the permissive environment of an autologous peripheral nerve graft, which is usually the sciatic nerve. This study demonstrates the ability of adult rat optic nerve axons to regenerate through the preformed perforations of a polyimide electrode carrier implanted at the interface between the proximal stump of the cut optic nerve and the stump of the peripheral nerve piece used for grafting. Evidence that retinal ganglion cells regenerated their axons through the perforated electrode carrier was obtained by retrograde labeling with a fluorescent dye deposited into the sciatic nerve graft beyond the nerve-carrier-nerve junction. The number of regenerating cells could be enhanced by injecting neuroprotective drugs like aurintricarboxylic acid and cortisol intravitreally. A second line of evidence was obtained by immunohistochemical staining with antibodies to neurofilament. Third, electrical activity of the regenerating nerves was recorded after stimulating the retina with a flash of light. The results suggest that a regenerating central nerve tract may serve as an experimental model to implant artificial microdevices to monitor the physiological and topographical properties of neurites passing through the device or to stimulate them, thus interfering with their potential to grow. This study reports for the first time that the optic nerve has unique properties, which aids in the realization of these goals.

Animals↗
PubMed2001

Lens-injury-stimulated axonal regeneration throughout the optic pathway of adult rats.

Axonal regrowth and restoration of visual function were studied in adult rats. The optic nerve was completely cut behind the eye. The proximal and distal nerve stumps were realigned and the meninges sutured back together. During the same surgical procedure, the lens was lesioned in order to induce secondary cellular cascades, which are known to strongly support the survival of retinal ganglion cells (RGCs) and to promote axonal regeneration. The anatomical and topographic restoration of the visual pathway was assessed neuroanatomically with the aid of anterograde and retrograde tracing using fluorescent dyes. It appeared that the axons formed growth cones at the junction of the suture soon after injury, before glial cells and extracellular matrix proteins were able to cause local scar formation. Growth cones first entered the distal optic nerve stump 3 days after injury, grew through it to reach the optic chiasm approximately 3 weeks after the lesion was made, and terminated within the retinoreceptive layers of the superior colliculus 5 weeks after lesioning. Quantification of the retrogradely labeled cell bodies within the regenerating retina revealed that up to 30% of the RGCs, which includes all major cell types, were capable of regenerating their axons along the entire visual pathway. To assess whether topography was restored, double-labeling experiments were performed, revealing only crude topographic restoration during the initial stages of regeneration. However, visual-evoked potentials could be recorded, indicating that synaptic transmission in higher visual areas was relatively intact. The data show, in principle, that cut axons can regenerate over long distances within the white matter of a central nerve like the adult optic nerve, spanning over 11 mm to the chiasm and between 12 and 15 mm to the thalamus and midbrain. The findings suggest, for the first time, that lentogenic stimulation of RGCs is sufficient to induce the formation of growth cones that can override inhibitors at the site of injury, grow through the white matter of the optic nerve, pass through the optic chiasm, and make synaptic connections within the brain.

Animals↗
PubMed2001

Exposure to a solar eclipse causes neuronal death in the retina.

BACKGROUND: A solar eclipse was observed in Europe on 11 August 1999. Several individuals suffered from transient or persisting retinal damage, caused by gazing at the eclipse without adequate eye protection. Retinal damage is the most serious hazard of exposure to light. but the mechanisms by which this type of exposure produces retinal damage and its cellular correlates are not yet established. We used an animal model to monitor the mechanisms of retinal damage following excessive light exposure, and in particular to study whether observation of the eclipse induces death of retinal cells. METHODS: In the geographic area where the experiment was conducted, a partial (90%) solar eclipse was observed. Experimental albino rats were exposed to these eclipse conditions, and control rats were exposed to normal sunlight. Another group of control animals was exposed to the same conditions, but was provided with protective light filters of the type recommended for human use. The DNA fragmentation in retinal sections of the various groups was analysed by terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labelling. This analysis revealed that exposure to both normal sunlight and to the eclipse resulted in neuronal apoptosis. Immunohistochemical techniques were used to evaluate possible glial-vascular alterations. RESULTS: Dying cells could first be detected 24 h after exposure, the largest number of which were found 6 days later in the photoreceptor layer. Control levels were attained 14 days after the exposure. Retinal ganglion cells underwent apoptosis in both groups (normal sunlight and eclipse exposure), whereas in the neuroglial cells there was an up-regulation of the intermediate filament content. The number of dying cells in both groups was greater in animals whose pupils had been dilated pharmacologically during exposure. On the other hand, the protective filters were effective in preserving the rat retinal cells from apoptosis. CONCLUSIONS: These results show, for the first time, that the cellular correlates of solar retinopathy are neuronal apoptosis accompanied by glio-vascular responses. Cellular apoptosis is an irreversible process, which could manifest itself as permanent visual impairment. The activation of non-neuronal cells, such as glial and endothelial cells, could be responsible for the more transient clinical symptoms.

Animals↗
PubMed2001

Development of the visual system of the chick. II. Mechanisms of axonal guidance.

The quest to understand axonal guidance mechanisms requires exact and multidisciplinary analyses of axon navigation. This review is the second part of an attempt to synthesise experimental data with theoretical models of the development of the topographic connection of the chick retina with the tectum. The first part included classic ideas from developmental biology and recent achievements on the molecular level in understanding cytodifferentiation and histogenesis [J. Mey, S. Thanos, Development of the visual system of the chick. (I) Cell differentiation and histogenesis, Brain Res. Rev. 32 (2000) 343-379]. The present part deals with the question of how millions of fibres exit from the eye, traverse over several millimetres and spread over the optic tectum to assemble a topographic map, whose precision accounts for the sensory performance of the visual system. The following topics gained special attention in this review. (i) A remarkable conceptual continuity between classic embryology and recent molecular biology has revealed that positional cellular specification precedes and determines the formation of the retinotectal map. (ii) Graded expression of asymmetric genes, transcriptional factors and receptors for signal transduction during early development seem to play a crucial role in determining the spatial identity of neurons within surface areas of retina and optic tectum. (iii) The chemoaffinity hypothesis constitutes the conceptual framework for development of the retinotopic organisation of the primary visual pathway. Studies of repulsive factors in vitro developed the original hypothesis from a theoretical postulate of chemoattraction to an empirically supported concept based on chemorepulsion. (iv) The independent but synchronous development of retina and optic tectum in topo-chronologically corresponding patterns ensures that ingrowing retinal axons encounter receptive target tissue at appropriate locations, and at the time when connections are due to be formed. (v) The growth cones of the retino-fugal axons seem to be guided both by local cues on glial endfeet and within the extracellular matrix. On the molecular level, the ephrins and their receptors have emerged as the most likely candidates for the material substrate of a topographic projection along the anterior-posterior axis of the optic tectum. Yet, since a number of alternative molecules have been proposed for the same function, it remains the challenge for the near future to define the proportional contribution of each one of the individual mechanisms proposed by matching theoretical predictions with the experimental evidence.

Animals↗
PubMed2000

Glioanatomy assessed by cell-cell interactions and phagocytotic labelling.

In the last three decades of research in neuroscience, fluorescent probes have gone from technical tools in the studies of physicochemical reactions, to being versatile tools in developmental neurobiology, neuroanatomy, angiography, neuromorphology, connectivity, cell death and even photodynamic therapy. Fluorescent dyes belong to heterogeneous groups of substances, but the feature to emit light of a certain wavelength depends on the energy status of the corresponding chemical bond. Therefore, light emission can range from the blue to the infrared spectrum, thus allowing multiple stains of the same cell, or event. The heterogeneity in their structure allows application of some fluorescent dyes for anterograde long-tract labelling, whereas others can be used for retrograde tracing. Lipophilic dyes are suitable for intramembraneous diffusion along cell membranes post-mortem, whereas hydrophilic stains seem more suitable for genealogic cell studies over several cell divisions. In the same time, less attention has been paid by most researchers to the use of fluorescent dyes to monitor neuroglial interactions and glioanatomy in the healthy and diseased brain. Studies of cell-cell-interactions during apoptosis can now be carried out with sequestration and subsequent phagocytosis of intracellular dyes. The present review focuses on recent developments that include the use of fluorescent probes. These probes make it possible to transneuronally assess functions of glial cells during programmed cell death, or induced degeneration. The high variety of available dyes, and their particular accumulation within subcellular compartments, is promising to shed light on some glial cell geometry and functions. The lessons obtained from the vast number of studies in neurons are of increasing importance for cells too, as their functions are not directly accessible. In short, some glial-glial and neuroglial negotiations will be analysed in near future by developing new, or by modifying existing fluorescent probes.

Animals↗
PubMed2000

Aurintricarboxylic acid promotes survival and regeneration of axotomised retinal ganglion cells in vivo.

Aurintricarboxylic acid (ATA) has been used as an anti-apoptotic drug to counteract ischemic or cytotoxic injury to neurons. We investigated whether ATA has a neuroprotective effect on axotomized, adult retinal ganglion cells (RGC) as a model for traumatic neuronal cell death. A solution of ATA was injected into the vitreous body of rat eyes whose optic nerves had been cut. In controls, 14% of RGC survived 14 days after axotomy, whereas 44% of RGC survived after a single injection of ATA solution, and 59% survived when the injection was repeated after 7 days. A single injection of ATA 1 day after axotomy rescued 58% of RGC. However, injection of ATA 4 days after axotomy did not influence the survival of RGC, indicating that crucial, irreversible cascades of death are initiated prior to this point in time. The TUNEL technique was used to visualise apoptotic ganglion cells and revealed that 4 days after axotomy their number was significantly less in retinas whose optic nerves were axotomized and treated with ATA, than those of controls. As a consequence of neuroprotection, more RGC were recruited to regenerate into a peripheral nerve graft used to replace the cut optic nerve. In this paradigm, ATA-treated RGC extended significantly more axons within the graft than control RGC. This number could be increased by a second injection of ATA 7 days after axotomy. These data show that ATA is not only able to delay post-traumatic neuronal death but also enhances the extent of axonal regeneration in vivo.

Animals↗
PubMed2000

Migration of phagocytotic cells and development of the murine intraretinal microglial network: an in vivo study using fluorescent dyes.

This work was undertaken to study whether retinal ganglion cell (RGC) death, which occurs during postnatal development of the mouse retina could aid in assessing the topological and chronological pattern of microglial cell migration. The study was conducted from postnatal day 0 (P0) to adulthood. The fluorescent dyes Fluorogold (FG) or (4-[4-didecylaminostyryl]-N-methylpyridinium iodide (4Di-10ASP) used in this study, were transported retrogradely to the RGC soma when either dye was injected into the superior colliculus (SC) at P0. Some of these labeled RGCs die due to natural apoptosis during this stage of development and are phagocytosed by microglial cells, which move to the site of RGC death, to become labeled with the same dye. The retinas were examined to quantify the microglial cells from P5 to adulthood. In addition, the reaction of microglia to optic nerve crush was studied in adult animals. Both dyes labeled RGCs in the contralateral retina and a few RGCs in the retina ipsilateral to the injected SC. The density of labeled RGCs decreased by 22% between P5 and P7. During this phase, microglial cells become visible as they ingested the fluorescent detritus of the dying RGCs. Microglial cells were evenly distributed across the entire retinal surface and migrated to the outer plexiform layer. Migrating microglia consecutively altered their morphology from the amoeboid to the ramified form. In terms of intracellular storage of the dyes, resident microglial cells retained the fluorescent dye 4Di-10ASP over a period of 12 months. In contrast, FG was completely transferred from the RGCs and microglial cells to intramural cells (pericytes) of the retinal capillaries after 10 months. This resulted in delineation of the entire intraretinal vascular network. Finally, resident retinal microglial cells were also activated by injury to the adult optic nerve and phagocytosed degenerating neurons. Retinal microglial cells can be monitored with vital fluorescent dyes while they migrate across the retina and establish their intra-retinal network. It is possible to label microglia with lipophilic dyes and they remain labeled for a long time. In addition, intramural pericytes can be labeled by slow release of FG from RGCs and microglial cells. The findings suggest that ingested fluorescent dyes having different properties can be used to study different populations of retinal cells in vivo.

Age Factors↗
PubMed2000

Development of the visual system of the chick. I. Cell differentiation and histogenesis.

This review summarizes present knowledge on the embryonic development of the avian visual projections, based on the domestic chick as a model system. The reductionist goal to understand formation and function of complex neuroanatomical systems on a causal level requires a synthesis of classic developmental biology with recent advances on the molecular mechanisms of cell differentiation and histogenesis. It is the purpose of this article. We are discussing the processes underlying patterning of the anterior neural tube, when the retina and optic tectum are specified and their axial polarity is determined. Then the development of these structures is described from the molecular to the anatomical level. Following sections deal with the establishment of secondary visual connections, and the developmental interactions between compartments of the retinotectal system. Using this latter pathway, from the retina to the optic tectum, many investigations aimed at mechanisms of axonal pathfinding and connectivity have accumulated a vast body of research, which will be covered by a following review.

Animals↗
PubMed2000

Current concepts in neuroanatomical tracing.

The development of new axonal tract tracing and cell labelling methods has revolutionised neurobiology in the last 30 years. The aim of this review is to consider some of the key methods of neuroanatomical tracing that are currently in use and have proved invaluable in charting the complex interconnections of the central nervous system. The review begins with a short overview of the most frequently used tracers, including enzymes, peptides, biocytin, latex beads, plant lectins and the ever-increasing number of fluorescent dyes. This is followed by a more detailed consideration of both well established and more recently introduced neuroanatomical tracing methods. Technical aspects of the application, uptake mechanisms, intracellular transport of tracers, and the problems of subsequent signal detection, are also discussed. The methods that are presented and discussed in detail include: (1) anterograde and retrograde neuroanatomical labelling with fluorescent dyes in vivo, (2) labelling of post mortem tissue, (3) developmental studies, (4) transcellular tracing (phagocytosis-dependent staining of glial cells), (5) electrophysiological mapping combined with neuronal tract tracing, and (6) simultaneous detection of more than one axonal tracer. (7) Versatile protocols for three-colour labelling have been developed to study complex patterns of connections. It is envisaged that this review will be used to guide the readers in their selection of the most appropriate techniques to apply to their own particular area of interest.

Animals↗
PubMed2000

Restoration of the retinofugal pathway.

In a relatively short period of time covering the last 2 decades, regeneration of retinofugal axons has become one of most prominent experimental models in restorative neurobiology. There is now a significant knowledge both on the mechanisms governing retinal ganglion cell responses to transection of the optic nerve, and the subsequent cell-cell interactions accumulating in death of the neurons. In addition, retinofugal axons served as an excellent model to examine whether, and to conclude that these axons have remarkable abilities for re-growth. This last issue was of invaluable importance, because axons could regenerate in vivo, into peripheral nerve grafts, and last but not least within the white matter of the cut optic nerve. As it stands to date, the extremely complex aspects of axonal regeneration will probably be understood within the retinofugal pathway. Final elucidation of this delicate system will essentially lead to some revision of our knowledge concerning neurotraumatology and CNS-repair.

Animals↗