Solon Thanos

Axonal regeneration and synapse formation in the superior colliculus by retinal ganglion cells in the adult rat

In adult rats, one optic nerve was transected and replaced by a 4 cm segment of autologous peripheral nerve (PN) that linked one eye and the superior colliculus (SC) along a predominantly extracranial course. Retrograde and orthograde studies with the tracers HRP or rhodamine-B- isothiocyanate (RITC), as well as immunocytochemical neuronal labels, indicated the following: (1) Regenerating axons from the axotomized retinal ganglion cells extended along the entire PN grafts, covering a distance nearly twice that of the normal retinotectal projection of intact rats. (2) Some of these axons penetrated the SC and formed terminal arborizations up to 500 microns from the end of the graft. (3) By electron microscopy, the arborizations of these regenerated axons in the SC were seen as small HRP-labeled axonal profiles that contacted neuronal processes in the SC; some of these contacts showed pre- and postsynaptic membrane specializations. These findings indicate that injured retinal ganglion cells in the adult rat are not only able to regrow lengthy axons, but may also form synapses in the SC.

Intravitreal injections of neurotrophic factors support the survival of axotomized retinal ganglion cells in adult rats in vivo.

After transection of the optic nerve (ON) in adult rats, retinal ganglion cells (RGC) progressively degenerate until, after two months, a residual population of only about 5% of these cells survives. In this study, we investigated the effect of regeneration-associated factors from sciatic nerve (ScN), BDNF, and CNTF on the survival of adult rat RGC after intraorbital ON transection. Neurotrophic factors were injected into the vitreous body. Rats were allowed to survive 3, 5, or 7 weeks, and the remaining viable RGC were then labelled by retrograde staining with the carbocyanine dye, 4Di-10Asp, which was applied onto the proximal nerve stump in vivo. The animals were sacrificed 3 days later and RGC counted in retinal whole mounts. Due to progressive degeneration following nerve transection the number of surviving RGC decreased to about 10% of the initially labelled population after 3 weeks, to about 8% after 5 weeks, and to about 5% after 7 weeks. Survival of axotomized cells could be prolonged using either of the neurotrophic factors: after 3 weeks a 2-3-fold increase in the number of viable RGC could be obtained compared to uninjected controls and to those which received injection of buffer. The prolonged survival effect vanished after 5 and 7 weeks, and no additive effect could be seen when combining brain-derived neurotrophic factor (BDNF) and ciliary neuronotrophic factor (CNTF) treatment. Morphometric analysis of labelled cells revealed that all neurotrophic factors supported predominantly large RGC with somal areas > 250 micron 2. In retinae from rats that survived the ON transection for several months, a characteristic population of axotomy-resistant RGC remained alive. Their few, very large, and often curled dendrites showed signs of placticity in the depleted inner nuclear layer of the adult rat retina. We conclude that the intraocular injection of CNTF, BDNF, and ScN-derived medium, which retard the process of lesion-induced RGC degeneration, may be successfully used as a subsidiary strategy in transplatation protocols. This would result in larger populations of RGC which can be recruited to regenerate their axons and provide a basis for functional recovery.

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.

Microglia-targeted pharmacotherapy in retinal neurodegenerative diseases.

Microglial cells, members of the monocytic lineage, represent the resident immunocompetent cells of the central nervous system including the retina with its peculiarities like a double blood retinal barrier. Microglial cells invade the retina in response to naturally occurring neuronal death during embryonic development and remodelling. Resident microglial cells are extremely sensitive to changes in their microenvironment arising from either traumatic or chronic neurodegeneration, inproper wiring, hereditary diseases or infection and become rapidly activated. In their activated state, the cells undergo drastic morphological changes, upregulate a variety of receptors and secrete soluble factors, which contribute to recognition and phagocytotic cleareance of dying or malfunctioning neurons. In this review, we aim to summarise the current knowledge of microglial involvement in experimentally induced or naturally occurring retinal neurodegenerations with emphasising on mechanisms of microglia activation. Expanding on the mechanisms, we shall discuss on approaches to pharmacologically interfere with the microglial activation and neurophagy. The protagonistic role of these cells in the outcome of certain diseases may help designing microglial targeted treatments with potential benefit for neuronal survival and regeneration in clinically relevant conditions.

The use of rhodamine-B-isothiocyanate (RITC) as an anterograde and retrograde tracer in the adult rat visual system

We have used the fluorescent dye Rhodamine-B-Isothiocyanate (RITC) as an anterograde and retrograde marker of retinal neurons in the adult rat. Introduced into the vitreous body, RITC is taken up by retinal ganglion cells and transported anterogradely along their axons. It labels the axons and their terminal arborizations. The dye stains axonal projections for at least 30 days without detectable extracellular leakage or transneuronal passage. Furthermore, the soma and dendrites of retinal ganglion cells can also be labeled retrogradely for at least 30 days when RITC is instilled into the superior colliculus. We conclude that RITC, used previously in studies of chick embryo visual pathways, is also suitable for similar neuroanatomical investigations in the adult rat.

Rocks are expressed in brain tumors and are required for glioma‐cell migration on myelinated axons

The interactions between migrating glioma cells and myelinated fiber tracts are poorly understood. We identified that C6 glioma cells can migrate along myelinated chicken retinal axons in a novel coculture, thereby expressing small GTPases of the Rho family and serine/threonine Rho‐associated kinases (ROCKs). We found that the ROCK1 isoform is also highly expressed in native human high‐grade gliomas. Glioma cells migrated faster in vitro along myelinated axons than on laminin‐1, with the former but not the latter being specifically and reversibly blocked by the ROCK inhibitor Y27632. These data suggest that the mechanisms underlying the migration of glioma cells on myelinated axons differ from those underlying the migration on extracellular matrix molecules such as laminin‐1.

GM-CSF regulates the ERK1/2 pathways and protects injured retinal ganglion cells from induced death.

Granulocyte-macrophage-colony-stimulating-factor (GM-CSF) is a potent hematopoietic cytokine. In the present study, we examined whether GM-CSF is neuroprotective in retinal ganglion cells (RGCs). First, we studied the expression of GM-CSF and the GM-CSF-alpha-receptor in rat and human retina and in RGC-5 cells. Then, RGC-5 cells were incubated with apoptosis-inducing agents (e.g., staurosporine, glutamate and NOR3). The cell death was assessed by Live-Death-Assays and apoptosis-related-proteins were examined by immunoblotting. In addition, the expression of phosphorylated ERK1/2-pathway-proteins after incubation with GM-CSF and after inhibiting MEK1/2 with U0126 was analyzed. To assess the in vivo-effect, first staurosporine or GM-CSF plus staurosporine was injected into the vitreous body of Sprague-Dawley rats. In a second axotomy model the optic nerve was cut and GM-CSF was injected into the vitreous body. In both models, the RGCs were labeled retrogradely with either Fluoro-Gold or 4-Di-10-Asp and counted. As a first result, we identified GM-CSF and the GM-CSF-alpha-receptor in rat and human retina as well as in RGC-5 cells. Then, in the RGC-5 cells GM-CSF counteracts induced cell death in a dose-and time-dependent manner. With respect to apoptosis, Western blot analysis revealed a decreased Bad-expression and an increased Bcl-2-expression after co-incubation with GM-CSF. Concerning signaling pathways, incubation with GM-CSF activates the ERK1/2 pathway, whereas inhibition of MEK1/2 with U0126 strongly decreased the phosphorylation downstream in the ERK1/2 pathway, and the antiapoptotic activity of GM-CSF in vitro. Like in vitro, GM-CSF counteracts the staurosporine-induced cell death in vivo and protects RGCs from axotomy-induced degeneration. Our data suggest that GM-CSF might be a novel therapeutic agent in neuropathic disease of the eye.

Opposing Signaling of ROCK1 and ROCK2 Determines the Switching of Substrate Specificity and the Mode of Migration of Glioblastoma Cells

Despite current advances in therapy, the prognosis of patients with glioblastoma has not improved sufficiently in recent decades. This is due mainly to the highly invasive capacity of glioma cells. Little is known about the mechanisms underlying this particular characteristic. While the Rho-kinase (ROCK)-dependent signaling pathways involved in glioma migration have yet to be determined, they show promise as one of the candidates in targeted glioblastoma therapy. There are two ROCK isoforms: ROCK1, which is upregulated in glioblastoma tissue compared to normal brain tissue, and ROCK2, which is also expressed in normal brain tissue. Blockage of both of these ROCK isoforms with pharmacologic inhibitors regulates the migration process. We examined the activities of ROCK1 and ROCK2 using knockdown cell lines and the newly developed stripe assay. Selective knockdown of either ROCK1 or ROCK2 exerted antidromic effects on glioma migration: while ROCK1 deletion altered the substrate-dependent migration, deletion of ROCK2 did not. Furthermore, ROCK1 knockdown reduced cell proliferation, whereas ROCK2 knockdown enhanced it. Along the signaling pathways, key regulators of the ROCK pathway are differentially affected by ROCK1 and ROCK2. These data suggest that the balanced activation of ROCKs is responsible for the substrate-specific migration and the proliferation of glioblastoma cells.

Regulation of retinal proteome by topical antiglaucomatous eye drops in an inherited glaucoma rat model.

Examination of the response of the retinal proteome to elevated intraocular pressure (IOP) and to the pharmacological normalization of IOP is crucial, in order to develop drugs with neuroptorective potential. We used a hereditary rat model of ocular hypertension to lower IOP with travaprost and dorzolamide applied topically on the eye surface, and examine changes of the retinal proteome. Our data demonstrate that elevated IOP causes alterations in the retinal protein profile, in particular in high-mobility-group-protein B1 (HMGB1), calmodulin, heat-shock-protein (HSP) 70 and carbonic anhydrase II expression. The changes of the retinal proteome by dorzolamide or travoprost are different and independent of the IOP lowering effect. This fact suggests that the eye drops exert a direct IOP-independent effect on retinal metabolism. Further investigations are required to elucidate the potential neuroprotective mechanisms signaled through changes of HMGB1, calmodulin, HSP70 and carbonic anhydrase II expression in glaucoma. The data may facilitate development of eye drops that exert neuroprotection through direct pharmacological effect.

A coculture assay to visualize and monitor interactions between migrating glioma cells and nerve fibers

Glioma-cell migration is usually assessed in dissociated cell cultures, spheroid cultures, acute brain slices and intracranial implantation models. However, the interactions between migrating glioma cells and neuronal tracts remain poorly understood. We describe here a protocol for the coculture of glioma cells with myelinated axons in vitro. Unlike other methods, this protocol allows the creation of in vitro conditions that largely mimic the complex in vivo environment. First, long retinal axons from embryonic chicken are formed in an organotypic culture. Glioma cells are then positioned in the vicinity of the explants to allow them to contact the axons, interact with them and eventually migrate along them. High-resolution video microscopy and confocal microscopy can be used to monitor the migratory behavior. This protocol, which takes about 5 days to complete, could be applied to different types of tumor cells that interact with neurites, and is suitable for pharmacological and genetic approaches aimed at elucidating mechanisms underlying tumor migration.