Petar Charalambous

Engrafted Chicken Neural Tube–Derived Stem Cells Support the Innate Propensity for Axonal Regeneration within the Rat Optic Nerve

PURPOSE Injury to the adult optic nerve, caused mechanically or by diseases, is still not reparable because the retinal ganglion cells (RGCs) are not allowed to regrow their axons and die retrogradely, although they possess the intrinsic propensity to regenerate axons in experimental conditions. METHODS In vitro propagated embryonic stem cells derived from the early chicken neural tube (NTSCs) were used to examine whether transplanted NTSCs produce growth-promoting factors and pave the microenvironment, thus facilitating axonal regeneration within the rat optic nerve. RESULTS NTSCs survived within the site where the optic nerve had been cut and continued to be nestin-positive, thus preserving their undifferentiated cell phenotype. Transplanted NTSCs activated the matrix metalloproteases (MMP)-2 and -14 in glial fibrillary acidic protein (GFAP)-positive optic nerve astrocytes. MMP2 production correlated with immunohistochemically visible degradation of inhibitory chondroitin sulfate proteoglycans (CSPGs). In addition, NTSCs produced a panoply of neurite-promoting factors including oncomodulin, ciliary neurotrophic factor, brain-derived neurotrophic factor and crystallins beta and gamma. Cut axons intermingled with NTSCs and passed through the zone of injury to enter the distal optic nerve over long distances, arriving at the thalamus and midbrain. CONCLUSIONS This study showed evidence that paving of the distal optic nerve microenvironment with proteolytically active MMPs and providing stem-cell-derived growth factors is a suitable method for facilitating regenerative repair of the optic nerve. Understanding the molecular mechanisms of this repair has fundamental implications for development of NTSC-based subsidiary therapy after neural injuries.

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.

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.

Cholinergic inhibition by botulinum toxin in a rat model of congenital glaucoma raises intraocular pressure

Background: Cholinergic receptors are crucially involved in the regulation of intraocular pressure (IOP). Muscarinic agonists in the trabecular meshwork tissue increase aqueous humour outflow facility by a direct stimulation of ciliary muscle contraction. We investigated the contribution of cholinergic state to IOP regulation. Methods: Intracameral injections of botulinum toxin A (BTA) were applied in a group with four normotensive rats and a group with four glaucoma rats (genetic glaucoma model). BTA is a potent neurotoxin which inhibits presynaptic cholinergic transmission for 6–8 weeks. The same amount of saline was injected in a third group of four normotensive rats (sham condition). IOP measurements were performed preoperatively, as well as 1, 2 and 4 weeks postoperatively. Afterwards, the rat eyes were removed and subjected to immunhistochemistry and western blotting analysis using antibodies against choline acetyltransferase (CHAT). Results: Mean IOP in both normotensive groups was unaltered compared with the preoperative status. The glaucoma group showed a significant increase in the mean IOP (Student test, p<0.05) and a signal reduction for CHAT by immunolabelling in the trabecular meshwork compared with the other two groups. Western blotting confirmed the decreased expression of CHAT. Conclusion: Our results suggest that modification of the cholinergic status in the normotensive eye does not significantly affect the IOP; cholinergic regulation of the ciliary trabecular meshwork may have differential levels of control, apart from the ciliary muscle contraction. Moreover, it seems that differential expression of the muscarinic receptors may be responsible for the decreased trabecular cholinergic state occurring in this rat model of glaucoma.

GM-CSF protects rat photoreceptors from death by activating the SRC-dependent signalling and elevating anti-apoptotic factors and neurotrophins

BackgroundThe term retinitis pigmentosa (RP) comprises a heterogeneous group of hereditary and sporadic human retinal degenerative diseases. The molecular and cellular events still remain obscure, thus hiding effective therapies. Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a hematopoietic factor which plays a crucial role in protecting neuronal cells. Binding of GM-CSF to its receptor induces several intracellular signaling pathways and kinases. Here we examined whether GM-CSF has a neuroprotective effect on photoreceptor degeneration in Royal College of Surgeons (RCS) rats.MethodsGM-CSF was injected into the vitreous body of RCS rats either once at the onset of photoreceptor degeneration at day 21, or twice at day 21 and day 42. At day 84, when photoreceptor degeneration is completed, the rats were sacrificed, their eyes enucleated and processed for histological staining and counting the surviving photoreceptor nuclei. The expression of apoptosis-related factors, such as BAD, APAF1 and BCL-2 was examined by Western blot analysis. The expression of neurotrophins such as ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and glia-derived neurotrophic actor (GDNF), as well as glial fibrillary acidic protein (GFAP) was analysed by Western blots and immunohistochemistry. The expression of JAK/STAT, ERK1/2 and SRC pathway proteins was assessed by Western blot analysis.ResultsGM-CSF protects significantly against photoreceptor degeneration in comparison to control group. After a single injection of GM-CSF at P21, a 4-fold increase of photoreceptors was observed, whereas eyes which received a repeated injection of GM-CSF at P42 showed a 10-fold increase of photoreceptors. Western blot analysis revealed a decreased BAD and an increased pBAD and BCL-2 expression, indicating changed expression profiles of apoptosis-related proteins. Neurotrophic factors examined are up-regulated, whereas GFAP was also modulated. At cell signalling levels, GM-CSF activates SRC-dependent STAT3 which is independent of JAK2, while proteins of the ERK1/2 pathway are not affected.ConclusionsThe data suggest that GM-CSF is a potent therapeutic agent in photoreceptor degeneration caused by mutation of the receptor tyrosine kinase gene (Mertk), and may be also effective in other photoreceptor degeneration.

Evidence of oxidative stress in Schnyder corneal dystrophy

Schnyder corneal dystrophy (SCD) is a rare, autosomal, dominantly inherited corneal disorder affecting the central and peripheral cornea and is characterised by cholesterol and phospholipid deposition in the corneal epithelium and stroma, leading to progressive corneal opacification, which may result in glare and disproportionate loss of photopic vision.1–3 Nitric oxide serves as a mediator in diverse and complex cellular processes throughout the eye.4 Increased production of nitric oxide, however, can lead to cytotoxic effects mediated by peroxynitrate, a potent oxidant that finally forms the stable product nitrotyrosine (NT). NT is considered a specific marker for nitrogen-related oxidants; therefore, its presence provides evidence of oxidative damage.5 Malondialdehyde (MDA) is the end product of free-radical–initiated lipid peroxidation, indicating oxygen-dependent degradation of cholesterol and phospholipids.6 7 In view of this evidence, the aim of our study was to examine the possible contribution of the nitric oxide pathway and lipid peroxidation to the pathogenesis of SCD. We examined three eyes from three patients with SCD who underwent a penetrating keratoplasty (PK) in our department (figure 1). The demographic and clinical data of the patients …

Erratum to: Growth/differentiation factor-15 and its role in peripheral nervous system lesion and regeneration

Growth/differentiation factor-15 (GDF-15) is a distant member of the transforming growth factor beta (TGF-β) superfamily. It is widely distributed in the nervous system, where it has been shown to play an important role in neuronal maintenance. The present study investigates the role of endogenous GDF-15 in sciatic nerve (SN) lesions using wild-type (WT) and GDF-15 knock-out (KO) mice. SN of 5–6-monthold mice were crushed or transected. Dorsal root ganglia (DRG) and nerve tissue were analyzed at different time points from 6 h to 9 weeks post-lesion. Both crush and transection induced GDF-15 mRNA and protein in the distal portion of the nerve, with a peak at day 7. DRG neuron death did not significantly differ between the genotypes; similarly, remyelination of regenerating axons was not affected by the genotype. Alternative macrophage activation and macrophage recruitment were more pronounced in the KO nerve. Protrusion speed of axons was similar in the two genotypes but WT axons showed better maturation, as indicated by larger caliber at 9 weeks. Furthermore, the regenerated WT nerve showed better performance in the electromyography test, indicating better functional recovery. We conclude that endogenous GDF-15 is beneficial for axon regeneration following SN crush.