Biomedical subjects

S Thanos

Publications and source records attributed to S Thanos.

At least 19 recordsLinked to original sources

PubMed1992

Sick photoreceptors attract activated microglia from the ganglion cell layer: a model to study the inflammatory cascades in rats with inherited retinal dystrophy.

Understanding of neuron-glial interactions in neurodegenerative diseases remains limited, but is of crucial importance for unravelling the etiology of such disorders both in humans and in animals. The present work employed a new, function-dependent technique for examining the role of microglia in rats afflicted with inherited retinal photoreceptor degeneration (strain: royal college of surgeons, RCS). In this rat strain, which served as a surrogate for human inherited retinal photoreceptor dystrophy, the optic nerve was cut and the ganglion cells were retrogradely labelled with the fluorescent dye 4Di-10ASP. The experiment was performed under three different conditions: (1) at the 50th day of postnatal age (P50) when there is ongoing degeneration of photoreceptor cells, (2) at P110 when most photoreceptors were degenerated and (3) at P50 in non-dystrophic rats of the Sprague-Dawley strain. After axotomy-induced ganglion cell death and labelling of activated microglia by phagocytosis of the ganglion cell debris, this study monitored whether the labelled and therefore identifiable microglial cells within the severed ganglion cell layer (GCL) are prompted to migrate and to participate in phagocytosis of debris produced within the endogenously degenerating photoreceptor cell layer (PRL). Massive migration of microglial cells from the GCL to the PRL occurred in dystrophic animals with optic nerve transection at P50. Double-labelling of microglial cells with the fluorescent dye ingested within the GCL and with lipofuscin ingested within the PRL indicated the ability of these cells to perform double-phagocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Effect of bilateral tectum lesions on retinal ganglion cell morphology in rats.

We have examined morphological changes of retinal ganglion cells (RGCs) during postnatal development in albino rats. Somatic diameter, dendritic field diameter, and branching frequencies of RGCs of normal rats were compared with those of animals that had received bilateral lesions of the tectum immediately after birth. Bilateral lesions of the tectum at P1 (first postnatal day) induced a dramatic increase in RGC death during the time of naturally occurring cell death in the first postnatal week. RGC densities in adult experimental animals were found to be reduced to about 55% of normal. RGCs of normal and operated animals were retrogradely stained with crystals of the fluorescent dye DiI, which was applied to the optic disc of flat mounted and fixed retinae. In normal rats, the somatic and the dendritic field diameters of the RGCs increased and the branching frequency of type I and III RGCs decreased from P1 to P14. By P14, neither the somatic diameter nor the dendritic field size had yet reached adult values and the branching frequencies were still higher than those of adult rat RGCs. In animals with bilateral lesions of the tectum, all cell types showed an increase in somatic sizes, and in type I and II RGCs an expansion of dendritic territories could be observed. The branching frequencies, however, were significantly lower than those of normal rats of the same age. The dendritic morphology in type III RGCs in operated animals was not significantly different from controls. These findings demonstrate a potential plasticity of type I and II RGCs, which respond to a loss of neighbouring cells by expansion of their dendritic field during postnatal development.

Aging↗

PubMed1992

Ganglion cells in the juvenile chick retina and their ability to regenerate axons in vitro.

Ganglion cells in the chicken retina fail to regenerate their axons upon mechanical injury. In order to determine whether this failure to regenerate axons is intrinsic to the neurons or is mediated by the environment, we asked whether ganglion cells possess an ability to regrow their injured axons in the absence of their natural environment, namely in vitro. Since the retina contains morphologically different types of ganglion cells, it became desirable to investigate whether all types of ganglion cells contribute to regeneration of axons. Ganglion cells were labelled post-mortem with the fluorescent dye DiI and described morphologically. Morphometric parameters like the sizes of their perikarya, their dendrites, and the patterns of dendritic ramification and stratification were considered for grouping cells. Although a strong classification of the cells could not be achieved because of the high diversity among this population of neurons, the chick retinal ganglion cells could be separated into seven regular groups which have their somata located within the ganglion cell layer and into one group with the somata located within the inner nuclear layer (displaced ganglion cells). The experimental procedure for regeneration combines crush injury of the chick optic nerve in situ with explanation of retinal pieces 1 week later for organ cultures in a serum-free medium. Under these conditions, the ganglion cells extended axons 1 day after explanation on polylysine/laminin. The densities of ganglion cells contributing to the axonal regrowth reached up to 1447 cells mm-2 (mean 1028 cells mm-2; S.D. 237). This density corresponds to 13% of the ganglion cell density in the normal retina, averaged across the total retina area. Although the dendrites of some cells whose axons had regenerated were altered in comparison with the normal ganglion cells, all morphological types of ganglion cells including those with displaced cell bodies contributed proportionally to the regrowth of axons from the explants.

Affinity Labels↗

PubMed1992

Specific transcellular staining of microglia in the adult rat after traumatic degeneration of carbocyanine-filled retinal ganglion cells.

The present work was undertaken to assess the fate of ganglion cell debris in the axotomized retina of adult rats and employed a new technique to label phagocytosing microglia via the internalized material. In the main experiment, transection axotomy was performed on the intraorbital segment of the optic nerve, and a fast-transported, vital fluorescent styryl dye (4Di-10ASP) was deposited at the ocular stump of the nerve in order to pre-label retrogradely the ganglion cells destined to die because of the axotomy. Optic nerve transection resulted in progressive degradation of ganglion cell axons, perikarya, and dendrites within the retina and in release of fluorescent material, which was then incorporated into cells identified as microglia. No other retinal cells stained, although astrocytes and Müller's cells also responded to neuron degeneration by accumulating glial fibrillary acidic protein. Incorporation of labelled material into microglia topo-chronologically paralleled the ganglion cell degeneration starting within the optic fibre layer (OFL) and proceeding towards the ganglion cell layer (GCL) and the inner plexiform layer (IPL) of the affected retina. Long-term labelling of microglia monitored up to 3 months after optic nerve transection indicated that labelled microglial cells persisted within the retina. Microglia displayed a strong territorial arrangement within the GCL and IPL, and staggered, bilaminated distribution in both layers. These studies directly prove that microglia in the retina can be transcellularly labelled during traumatic degeneration of ganglion cells. The findings suggest that microglial cells play an important role in axotomy-induced wound healing and removal of cell debris.

Animals↗

PubMed1992

Development of the visual system of the chick--a review.

This article reviews recent and earlier findings that yield the present knowledge about the embryonic development of retina, tectum, and the retinotectal projection in the chick. Data and concepts dealing with cell proliferation, migration, and differentiation, the processes underlying the generation of cytoarchitecture in the nervous system are discussed for the avian visual system. Emphasis is also laid on the presentation of hypotheses and experiments about directed axonal growth along the visual pathway and concerning the mechanisms responsible for the establishment of specific connections between retinal ganglion cells and their central targets. Among the results, the following topics deserve special attention: 1) Investigations of morphogenetic factors in vitro, and the application of recombinant retroviruses in vivo to study cell lineages rendered new insights into the processes of cell determination and differentiation. The evolving picture in this progressing field is discussed. At present, however, the research of retinal and tectal histogenesis is still largely in the state of morphological description. 2) Both systems, retina and optic tectum, develop independently from each other but in corresponding spatio-temporal patterns, which provide that ingrowing retinal axons encounter receptive target tissue at appropriate locations at the time when connections are due to be formed. 3) Possible mechanisms of directed fibre growth are being elucidated by increasing efforts in research devoted to cell surface molecules, neurotrophic, and inhibitory substances, and their receptors. The axons of the primary visual pathway seem to be guided by local cues on glial endfeet and perhaps in the extracellular matrix, but so far, instructive molecules to which functional significance can be assigned have eluded discovery. 4) The question, how the retinotopic projection upon the tectum is created during development, remains still unsolved, although most results point to modified forms of the chemoaffinity hypothesis for its explanation. Sequential maturation and growth, selective fasciculation of orderly entering axons, recognition of positional tectal markers, and functionally controlled refinement may together contribute to the correct retinotectal projection.

Animals↗

PubMed1991

Specific transcellular carbocyanine-labelling of rat retinal microglia during injury-induced neuronal degeneration.

The present work employed a new technique for labelling phagocytizing microglia in the axotomized retinal of adult rats. Transection axotomy was performed within the intraorbital segment of the optic nerve, and the fast-transported, vital fluorescent carbocyanine dyes DiI and 4Di-10ASP were deposited at the ocular stump of the nerve in order to retrogradely prelabel the ganglion cells which were destined to die. Optic nerve transection resulted in progressive degradation of ganglion cell axons, perikarya and dendrites within the retina and in release of fluorescent material which was then incorporated into cells identified as microglia but not into other cells of the retina. Incorporation of labelled material into microglia occurred only when the ganglion cells degenerated and not when the non-lesioned ganglion cells were labelled from the superior colliculus. Double-staining of microglia with both dyes helped to compare the pattern of labelling for each dye. After progression of ganglion cell degeneration, microglia displayed a staggered, bilaminated distribution within the ganglion cell layer and within the inner plexiform layer. Fluorescent microglia were not found within the deeper layers of the retina indicating that transneuronal degeneration and subsequent labelling of microglial cells do not occur. The results show that one major function of microglia within the ganglion cell and inner plexiform layers of the lesioned retina is to remove debris produced after degradation of neurons.

Animals↗

PubMed1991

Ontogenetic changes in the regenerative ability of chick retinal ganglion cells as revealed by organ explants.

Whereas mature neurons in the central nervous system (CNS) of birds lack the capability of regenerating axons after injury, embryonic nerve cells are able to do so. The time course of this decline of regenerative ability was investigated in ganglion cells from embryonic chick retinae. Retinal strips from 7- to 19-day-old embryos (E7-E19) were explanted and cultured in vitro. The numbers of retinal ganglion cell (RGC) neurites that had extended during the first 22-23 h incubation, their elongation rates, and morphometric parameters of the growth cones were measured to characterize the regenerative behavior. We observed two periods of decline in neuritic growth: the first from E7 to E9, and another from E14 to E19. The first decrease may reflect a gradually disappearing portion of neurons which produced their axons de novo. The second decline coincides with the major period of synaptogenesis by ganglion cell axons in ovo. The time required for initiation of axonal outgrowth increased, accordingly, from less than 3 h in explants from younger retinae (E7-E16) to 10-12 h for E17 and E19 explants. Axonal elongation rates ranged between 36 micron/h and 56 micron/h (mean values) for E7-E13 explants, but were significantly lower for cells from E14-E19 retinae (13-21 microns/h). Morphologically, neurites and growth cones for RGC explanted before E17 were characterized by their high variability. They possessed more filopodia than mature neurons (E17, E19), fasciculated to a higher degree and branched more frequently. In addition, older neurites appeared "stiffer" and were morphologically simpler.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Regenerating axons from adult chick retinal ganglion cells recognize topographic cues from embryonic central targets.

We investigated whether regenerating mature axons recapitulate embryonic features essential to successful reconnectivity within the injured nervous system. Strips from embryonic and adult chick retinae were cultured, and outgrowing axons were examined morphometrically and immunohistochemically. In addition, the target-recognition properties of adult neurites were analyzed. Regenerating adult axons elongate on a poly-L-lysine/laminin substratum with a speed about one order of magnitude slower than that of embryonic axons. Morphologically, adult axonal tips differ dramatically from embryonic growth cones in that they possess only filopodial extensions whereas embryonic growth cones possess both lamellipodial and filopodial processes. Both embryonic and adult neurites express the growth-associated protein GAP-43. When cultured on alternating stripes of anterior and posterior embryonic tectal membranes, both adult and embryonic retinal axons distinguish between the two membrane preparations. Our results demonstrate that during axonal regeneration the mature neurons express embryonic properties that are involved in the recognition of tectal positional cues.

Animals↗

PubMed1991

[Monoclonal antibody HMB-45 in diagnosis of intra-ocular melanoma].

69 eyes, which had to be enucleated because of an uveal melanoma, were investigated immunohistologically. HMB-45-antigen, which is supposed to be highly specific for melanocytic tumours, was found in 68 cases (sensitivity 99%) with a monoclonal antibody. S 100-antigen was positive in 63 cases (sensitivity 91%) using a polyclonal antibody. The addition of HMB-45-immunohistochemistry to routine histological diagnostic procedures is valuable, when a non-melanocytic uveal tumour cannot be ruled out or when the degree of melanoma-invasion has to be determined with greater accuracy. At the site of melanoma-invasion changes of cellular reactivity seem to occur, as 11 of the 69 studied tumours (16%) showed an increased HMB-45-expression in this area. HMB-45-antigen was detectable even in paraffin embedded material obtained from eyes enucleated 30 years ago.

Antibodies, Monoclonal↗

PubMed1991

Postmortem preservation of ganglion cells in the human retina. A morphometric investigation with the carbocyanine dye DiI.

Postmortem morphometric investigation of autopsy material is helpful for understanding the alterations cells undergo during life. The present work was undertaken to determine the morphologic features of human retinal ganglion cells during the first 2 days after death. Ganglion cells of 16 retinas were obtained from eyes, the corneas of which had been retrieved for keratoplasties. The ganglion cells were stained with the fluorescent carbocyanine dye Dil and investigated 12 to 36 hours postmortem. All labeled cells identified as ganglion cells had well preserved, type-specific dendritic profiles that made it possible to catalog them according to the established classes of ganglion cells. The various types of cells have in common some typical morphologic changes that occur during the postmortem period of observation: swelling and varicosity in the cell bodies and along the dendritic branches and the axons. These swellings were consistent throughout the postmortem period and did not hinder the identification of particular types of ganglion cells. The two major types (parasol and midget) and various subtypes of ganglion cells were identified morphologically. There are two distinct morphologic types of midget cells which differ from each other in the pattern of dendritic branching. Particular emphasis was given to analyze the dendritic geometry of the large parasol cells. A new finding in the human retina is the frequent presence of large parasol ganglion cells, the axons of which course along aberrant intraretinal paths, especially in the extreme periphery of the retina.

Axons↗

PubMed1991

Mechanisms governing neuronal degeneration and axonal regeneration in the mature retinofugal system.

The ability of mammalian CNS neurons to regrow their lesioned axons declines during late embryonic and postnatal development. Consequently, adult retinal ganglion cells of mammals respond to injuries with rapid anterograde and protracted retrograde (Wallerian) degeneration. To monitor the cascade of events initiated by neuronal injuries, and to explore whether the regressive events of this cascade can be blocked or reversed, axotomy-induced ganglion cell responses were investigated in adult rats. The aim of the experiments was to block degradation of axotomized ganglion cells with enzymes which inhibit proteolytic activities within the retina (protease inhibitors). To achieve this goal, a new fluorescence technique was employed to assess both the chronotopological pattern of degradation and the efficacy of the protease inhibitors and anti-inflammatory treatment in preventing cell death. Injection of protease inhibitors alone or combined with dexamethasone into the vitreous body of animals whose optic nerves were transected, protected ganglion cells from degradation and prevented endocytosis-dependent tracing of microglia. Two major functions of rescued ganglion cells proved their viability: (1) the numbers of ganglion cell axons extended from retinal stripes that were explanted 1 week after axotomy and cultured in vitro, were significantly higher when the retinal pieces originated from retinae pretreated with protease inhibitors and dexamethasone at the time of optic nerve transection than from untreated retinae; (2) the numbers of ganglion cells which regenerated axons into transplanted peripheral nerve pieces were more than doubled when the eyes were injected with protease inhibitors and dexamethasone during axotomy. The results show that blocking of the retinal proteases, which are presumably localized in microglial cells, and simultaneous treatment of the intraretinal inflammation, are key steps in understanding the intraretinal responses to axotomy and for beneficially manipulating the numbers of surviving neurons. In addition to the supporting influence of neurotrophic factors and to nonpermissive features of oligodendroglia, the microglia co-regulate whether neurons can regenerate their axons.

Animals↗

PubMed1991

[Glial and neuronal cellular changes in the glaucomatous human retina].

The purpose of the present study was analysis of the cellular response to glaucoma in the human retina. Retinal strips obtained from two enucleated eyes with therapy-resistant, absolute glaucoma were explanted and cultured in vitro. The morphology of the remaining cell populations was assessed with the DiI method in non-cultured, formalin-fixed retinal tissue from both retinas. We found immunohistochemically identifiable glia cells migrating out from the explants and glial processes formed on the substrate. Labelling of representative retinal areas with the fluorescent dye DiI applied to the nerve fiber layer resulted in delineation of growth cone-bearing glial processes, of occasional non-atrophied ganglion cells, and of amacrines and horizontal cells in deeper layers of the retina. The results demonstrate that glaucoma leads both to selective damage of ganglion cells and to a glial proliferation characterized by the formation of processes both in the retina and following explantation.

Culture Techniques↗

PubMed1990

Simultaneous bilateral diffuse melanocytic uveal hyperplasia.

A 52-year-old woman noted loss of vision in August 1984. Clinical examination disclosed iris cysts and ciliary body cysts, macular edema, and uveal nevi. Cataract extraction and pressure-lowering operations were required in both eyes because of a tumor-induced angle-closure glaucoma. Vision, however, progressively decreased to light perception in each eye. Both eyes were finally enucleated because a malignant melanoma could not be ruled out, though iris tissue obtained in 1985 suggested a nevuslike process. Histologic study indicated a bilateral uveal hyperplasia. Results of light and electron microscopy, immunologic studies, and suspension cell culture suggested that the uveal hyperplasia was more likely a melanoma of low malignancy than a nevuslike process. We could not detect an extraocular primary tumor and assumed that this condition constituted an oncogenic syndrome.

Diagnosis, Differential↗

PubMed1990

The developing chick isthmo-optic nucleus forms a transient efferent projection to the optic tectum.

The present work describes the formation of a transient efferent axonal projection from the isthmo-optic nucleus (ION) to the ipsilateral optic tectum of the chick embryo. Local application of either the carbocyanine dye DiI or rhodamine-B-isothiocyanate (RITC) to the superficial layers of the optic tectum resulted in retrograde labeling of the corresponding retinal region, and in anterograde staining of tectal axons projecting to the ION. In addition to these known projections, retrogradely labeled ION neurons appeared to be filled from the tectum. This projection, called the isthmo-tectal projection, could be characterized by means of various staining techniques: (i) It first appears at embryonic day E9 and gradually disappears after day E16. It is absent in the hatched chick. (ii) Both the cells inside the ION and those situated outside the border of the ION, the so-called ectopic cells, contribute to the formation of the isthmo-tectal projection. (iii) Double labeling from the contralateral retina (Fast blue) and from the ipsilateral tectum (DiI or RITC) revealed that some of the ION fibers projecting to the tectum are collaterals of axons normally directed to the retina. (iv) Microsurgical removal of the eye anlage early in development resulted in a numerical increase of the ION-tectal fibers. The results are discussed in terms of the role of transient projections during development.

Animals↗

PubMed1990

Target dependence of chick retinal ganglion cells during embryogenesis: cell survival and dendritic development.

The survival of retinal ganglion cells and the dendritic development were investigated a) in normal chick embryos, b) in embryos whose primordial optic lobes and adjacent areas were removed (target reduced embryos), and c) in embryos whose optic nerves were transected (target deprived embryos) in order to study the influences of central targets on developing ganglion cells. The ganglion cells were stained postmortem with the carbocyanine dye DiI. Cell body and dendritic field diameters were measured in whole-mounted retinae before and after the period of cell death at embryonic day 10 (E10) and E16. The cell densities within the ganglion cell layer were counted in cresyl violet/thionine stained retinae. The central retinal projection in target reduced embryos was studied with the anterogradely transported fluorescent marker rhodamine-B-isothiocyanate (RITC). In normal embryos, the earliest dendritic processes were observed at E6 in the central retina, whereas at E10 elaborate dendritic branching was found across the retina. Different morphological types of ganglion cells could be identified at E16. In both, target reduced embryos and target deprived embryos, the initial dendritic growth and pattern of ramification were indistinguishable from those of normal embryos up to E10. Cell body diameters, dendritic tree diameters, and cell densities were not significantly different. At the end of the naturally occurring cell death period (E16), the ganglion cell density was strongly reduced in both experimental groups compared to controls. In particular, when the optic nerve was transected, it resulted in the almost complete degeneration of ganglion cells. In target reduced embryos, a small population (about 5% of the normal number) of ganglion cells survived. The proportion of large cells was increased within the total population compared to normal retinae. Displaced ganglion cells were not affected by partial target removal but strongly affected by transection of the optic nerve. Anterograde labelling from the retina revealed that in target reduced embryos the remaining ganglion cells innervated non-tectal primary visual nuclei. The present results suggest the following: a) Before the onset of the cell death period, the growth and ramification of ganglion cell dendrites occur independently of central visual targets. b) In target reduced embryos, a small population of ganglion cells survives, namely, those cells that project to remaining central areas. Complete disconnection from central targets by transecting the optic nerve leads to the degeneration of almost all ganglion cells. c) The surviving ganglion cell population consists mainly of large ganglion cells.

Animals↗

PubMed1990

Regenerative and proliferative capacity of adult human retinal cells in vitro.

The present work was undertaken (a) to determine whether adult human retinal neurons possess the ability to survive and regrow neurites in organ cultures and (b) to investigate the maintenance and proliferative activity of retinal glial cells in vitro. Using material from retinae obtained from human eyes postenucleation we established in vitro organ cultures of retinal pieces in a chemically defined, serum-free medium, previously developed for culturing adult rat and chick retinae. The time course of glial cell migration and of neurite extension was compared with that of adult rat and chick retinae which have been extensively investigated in our laboratory. It appeared from four explanted retinae that the human retinal cells survive for up to 4 weeks in culture and exhibit their typical morphologies. Immunohistochemical investigation of the migrating cells revealed that both astrocytes and Müller-like cells exhibit their typical morphologies in vitro. In explants obtained from a retina 30 years after very traumatic violence to the eye cup, the glial cells but not the neurons extended lengthy fibers. In the explants obtained from a retina about 2 months after traumatic optic nerve injury and subsequent ocular complications, several lengthy fibres were extended from the transplant's edge. They could be labeled with neurofilament antibodies but not with glial fibrillary acidic protein antibodies, indicating their neuronal origin. The results suggest that some injured human retinal neurons respond to exposure to growth-permissive substrates by regeneration of their neurites. Although some morphological features of the fibers suggest that they originate from ganglion cells in the retinal explants, definitive proof of this is not yet available.

Aged↗

PubMed1990

Bidirectional fluorescent labelling techniques for the developing and regenerating visual system.

Fluorescent dyes like RITC or Dil (282) are convenient and highly efficient labelling substances for investigating developing and regenerating neurons. Either dye can be taken up by cell bodies and transported in an anterograde direction, resulting in a complete outlining of the axons and their growth cones. Additionally the dyes can be transported in a retrograde direction, resulting in labelling on the cell bodies and their dendrites. These dyes have several features in common, for instance their poor solubility in aqueous buffers, the absence of neurotoxicity and the long time persistence in the filled neurons. Dil can in particular be used for the staining of neurons post-mortem. These dyes have also been used to address several questions concerning fiber growth and pathfinding during embryogenesis of the visual system, and during the period of axonal regeneration of adult retinofugal fibers.

Animals↗

PubMed1989

Ability of adult rat ganglion cells to regrow axons in vitro can be influenced by fibroblast growth factor and gangliosides.

The ability of lesioned adult retina ganglion cells (RGC) to survive and regrow axons in vitro was investigated in retina organ cultures under chemically defined conditions. Factors which are known to either affect the RGC survival like the basic fibroblast growth factor (FGF) or influence neurite outgrowth like gangliosides were assayed by recording the course of prelabeled RGC degeneration in vitro and the number and length of regrowing RGC axons from explanted retinal pieces. Administration of basic FGF significantly slowed down the decrease in the number of RITC-prelabeled RGC in the cultured retinae. In addition, in the presence of gangliosides (GM1, GD1a, GD1b GT1b), the numbers of regrown RGC axons (Thy 1-immunostained) increased dramatically as compared to controls. The data indicate that adult neurons with an intrinsic ability to regenerate axons can respond to substances with neurotrophic or neurite-promoting activities in tissue cultures.

Animals↗