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Regeneration of the optic nerve

Prof. Solon Thanos
Image Solon Thanos

The optic nerve is part of the CNS and represents a unidirectional neuronal path consisting of retinal ganglion cell axons that connect the eye with central visual areas. One of the marked features of the optic nerve, equally advantageous for clinicians and experimentalists is its extracranial location providing diagnostic and experimental accessibility. In addition to axons, oligodendrocytes myelinate the optic nerve, whereas astrocytes, microglial cells and a dense capillary network is pre sent throughout it length. Its microvasculature is assumed to be a major player in numerous optic nerve diseases such as glaucoma, microinfarctions, ischemias, embolisations, injuries, infections and hereditary diseases

Injury to the optic nerve results in irreversible destruction of the axons cumulating in bidirectional atrophy. Dissolution of the neuronal components results in gliotic scar formation and substantial remodeling of extracellular matrix that forms an environment which is forbidden for ingrowth and motility of new axons. Based on this inhibitory environment, retrograde signals to the ganglion cell bodies confer catabolic metabolism, thus initiating apoptotic cell death and gliotic responses within the retina, too. There is no method to replace death ganglion cells yet.

Regeneration of ganglion cell axons

There are, however, increasing experimental lines of evidence delivered during the last decades that some of the ganglion cells can survive axotomy. They present encouraging findings that optic nerve repair may be possible at one time. The principal finding is that apoptosis can be prevented to some extent, and the catabolic responses of cells can be reversed towards a growth of axons. Ultimately, axons can grow as shown in a number of models including regrowth of axons within the inhospitable environment of the optic nerve. The approaches used are based on both neutralization of inhibitory substances at the site of axonal injury and neurotrophic/neuroprotective support at the ganglion cell bodies. On major impediment is the understanding of how acute axonal stump, as a prospective growth cone can be forced to become a motile growth cone. For this, first profound understanding of the molecular mechanisms determining interactions between growth cones and their micro-surround is essential. Then, knowledge of the panoply of molecular tools that are used for navigation through astrocytes, microglia oligodendrocytes and ECM is mandatory.

Next, the search for substances which increase the quantity of regenerating axons is necessary. Then, the mechanisms of signaling life or death is a fundamental requirement to apply such substances. Last not least, the question of reconnection of ganglion cell axons within the brain. Besides of acute injuries, similar aspects are applicable after chronic injuries and compressions like that of abnormal increase of intraocular pressure.

We have established in vitro and in vivo animal models of optic nerve regeneration and glaucoma. Together with our partners in proteomics and genomics we have been able to demonstrate that under certain circumstances ganglion cell axons can grow. We now begin to understand some of the signaling pathways of neuroprotection and created conditions for functional repair of the optic nerve.

Following aspects have been addressed in detail:

  1. Long term morphometric stabilization of regenerating retinal ganglion cells in the adult rat
    Adult retinal ganglion cells (RGCs) can regenerate their cut axons within peripheral nerve grafts used to replace the distal optic nerve stump. We examined the long-term stabilization of RGCs by guiding their regenerating axons into different termination areas. This reconnection stabilizes the cells at morphological and functional levels for a long period of time (Chiwitt et al, 2011, Rest Neurol Neurosci: 29(2): 127-139).
  2. Aquired glaucoma models
    We have generated and characterized a glaucoma model in rats by cauterizing two episcleral veins to chronically elevate the IOP. Staining of RGCs with retrograde markers and of microglial cells with IHC revealed that activation of retinal microglial cells coincides with degeneration of RGCs Therefore assigning them a role in glaucomatous neurodegeneartion (Naskar et al. 2002, Inv.Ophth.Vis.Sci.43 (9):2962-2968).
    At same time we examined RGCs in advanced human glaucomatous retinas and characterized IOP-resistant cells even in blind eyes to conclude that some cells express resistance mechanisms (Pavlidis et al. 2003, Invest Ophth.Vis.Sci 44:5916-5205). Monitoring of dying RGCS and phagocytotic microglial staining could be assessed both in vivo and in retinal whole mounts and sections ex vivo, respectively (Thanos et al. 2002, Trends.Neurosci.25:441-444).
  3. Inherited glaucoma model
    In order to confirm the data obtained from experimentally elevated IOP with other models, we have generated a second rat model. It consists of inherited glaucoma based on a spontaneous mutation that lasts over animal´s life and permits examination of RGCs at different stages of the disease. Besides of determining RGC-density with time, some genes and proteins were found to correlate with duration of the disease (Thanos, Naskar, 2004, Exp. Eye Res. 79:119-129).
  4. Gene profiling in glaucoma and optic nerve regeneration
    Retinal gene profiling in this hereditary model of elevated IOP showed a shifted retinal program of gene expression with 75 genes being up-regulated and 45 genes being down-regulated. Subsequent analysis with qRT-PCR and Western blotting confirmed the regulation of genes associated with glaucoma (Naskar, Thanos, 2006, Mol.Vis.18 (12)1199-1210). Using the MWG Rat 10k array which comprises 9715 rat genes spotted onto one array, we could identify that a number of specific genes are regulated within subsets of neurons and glial cells in the retina. These data suggest that retinal regenerative repair involves the orchestrated responses of all retinal neurons, glia, microglia and immigrating macrophages (Liedtke et al. 2007, Glia 55(2)189-201). Among the genes HMGB-1 and the toll-like receptor 4 was up-regulated. Some of the proteins can be detected with sensitive proteomic methods such as MALDI-MS including HMGB-1 which seems down-regulated in degenerating rat retina when compared with a regenerating one (Prokosch et al. 2010, Expert Rev. Proteomics 7(5):775-795; Rose et al. 2008, Rest. Neurol. 26(4):249-266; Rose, 2004, PhD thesis).
  5. α2-adrenergic receptors in the retina
    Besides of toll-like receptor expression in the retina α2-adrenergic receptors (α2-AR) are expressed both in normal and degenerated retinas. Their binding characteristics of α2-AR and their responsiveness to brimonidine point to a prominent role in degenerative and regenerative processes within the glaucomatous retinae most likely by interfering with metabolism of cells (Prokosch et al. 2010, Invest. Ophth. Sci. 51(12): 6688-6699). It is, however, not clear yet whether the α2-AR and TLRs interact with each other with respect to activation of intracellular signalling cascades.
  6. Membrane channels in retina
    Within a collaborative DFG grant with the University of Oldenburg we have been able to characterize voltage-gated sodium channels in regenerating axons in organotypic cultures. Using IHC and whole cell, patch lamp recordings sodium channels were localized in axonal growth cones and responded to depolarizing voltage steps with fast, transient, inward currents mediated by sodium ions. These experiments were the first to describe biophysical properties of Na(v) channels in regenerating RGCs and contrast their properties with Na(v) channels in adult RGCs (Feigenspan et al. 2010, Invest.Ophthalm.Vis.Sci.51(3):1789-1799). These results suggest that, in addition to metabolic proteome, changes at channel levels are to be expected in the diseased retina. Their potential association to HMGB-1 or other mediators of disease have to be elucidated.
  7. Retina degeneration and angiogenesis
    We have examined retinal angiogenesis and receptor expression for VEGF, Arg-2 and SRIF in the rat strain Royal-College-of Surgeons (RCS) which is characterized by a genetic defect in the Mertk gene and a progressive dystrophy of photoreceptors due to failure of RPE-cells to phagocytose. We found that during the period of P14 and P45 malperfused retinas expressed VEGF, SRIF and Arg-2 as well as their receptors. At later stages of disease networks of neovascularising capillaries were observed throughout the retina (Prokosch et al. 2011, Exp.Eye Res. 92(2):128-137). These data indicate that angiogenesis-related factors and their receptors may be targets for treatment if ischemic proliferative retinal diseases with complex etiologies. These data point to a close relationship between retinal cell survival and VEGF-dependent neovascularization.
  8. Neuroprotective strategies
    Rescue of RGCs in glaucoma and after acute optic nerve injuries has been the purpose of work throughout the last 2 decades in our laboratory. While focussing on regeneration of damaged ganglion cell axons we discovered that lens injury supports growth of cut axons at unpresented quantities (Fischer et al. 2000, 2001). Expanding on these data we searched for lens-derived and regeneration and identified that crystallins of the beta/ gamma-superfamily mimic the effects of lens injury and enhance the regeneration ability of axons both in vivo and in culture (Fischer et al. 2008, Mol. Cell. Neuroscience, 37: 371-379). Moreover, proteomic analysis of the regenerating retina and the culture medium revealed secretion of crybb2 that has been closer analysed to find that this protein supports elongation of retinal axons (Liedtke et al. 2007, Mol. Cell. Proteomics, 6(5):895-907). These data were the first to examine molecules with unexpected involvement in retinal repair. They deserve further attention because they represent innate molecules of the retina with high potential of supporting cell survival and axonal growth.
    During the same time the laboratory of Schneider and Schäbitz exploited endogeneous death-preventing mechanisms and discovered that the hemopoietic factor GM-CSF protects cortical neurons from stroke-injury (Krüger et al. 2006; Schäbitz and Schneider, 2997; 2008). We identified GM-CSF-receptors within the retina and in particular within RGCs and performed experiments in vitro and in vivo. We found that GM-CSF counteracts both staurosporin-induced and axotomy-induced cell death. GM-CSF binds on its α-receptor both in rat and human retina and regulates the ERK1/ 2 pathways as shown with different methods (Schallenberg et al. 2009, Exp. Eye Res. 89(5):665-677). These data support our hypothesis that the search for innate, receptor-binding molecules are involved in neuroprotection.
    In a collaborative work with the groups of Lagreze and DiGiovanni we further explored whether intracellular inhibition of histone-diacetylases (HDAC) delays apoptosis of RGCs. We found that intravitreal valproic acid (VPA) enhances the numbers of RGCs that survive optic nerve crush. In addition, VPA enhanced the numbers of regenerating axons in organotypic retinal cultures interfering with different intracellular pathways such as caspase-3, CREB induction and PERK (Biermann et al. 2010, Inv. Ophthalmol. Vis. Sci. 51(1):526-534). Also these data support the view that a multifactorial analysis of the signalling pathways resulting in cell death will help to rescue damaged ganglion cells.
  9. GM-CSF protects rat photoreceptors from death by activating the SRC-dependent signalling and elevating anti-apoptotic factors and neurotrophins (2012, in press)
    GM-CSF was injected into the vitreous body of RCS rats either once at the onset of photoreceptor degeneration at day 21, or twice a 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-factor (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.

Selected Bibliography

  1. Naskar R, Thanos S (2006) Retinal gene profiling in a hereditary rodent model of elevated intraocular pressure. Mol Vis. 12: 1199-1210.
  2. Liedtke T, Naskar R, Eisenacher M, Thanos S (2007) Transformation of adult retina from the regenerative to the axonogenesis state activates specific genes in various subsets of neurons and glial cells. Glia. 55(2): 189-201.
  3. Pavlidis M, Stupp T, Hummeke M, Thanos S (2006) Morphometric examination of human and monkey retinal ganglion cells within the papillomacular area. Retina. 26(4): 445-453.
  4. Stupp T, Pavlidis M, Busse H, Thanos S (2005) Lens epithelium supports axonal regeneration of retinal ganglion cells in a coculture model in vitro. Exp Eye Res. 81(5): 530-538.
  5. Panagis L, Thanos S, Fischer D, Dermon CR (2005) Unilateral optic nerve crush induces bilateral retinal glial cell proliferation. Eur J Neurosci. 21(8): 2305-2309.
  6. Fischer D, Petkova V, Thanos S, Benowitz LI (2004) Switching mature retinal ganglion cells to a robust growth state in vivo: gene expression and synergy with RhoA inactivation. J Neurosci. 24(40): 8726-8740.
  7. Cristofanilli M, Thanos S, Brosius J, Kindler S, Tiedge H (2004) Neuronal MAP2 mRNA: species-dependent differential dendritic targeting competence. J Mol Biol. 341(4): 927-934.
  8. Thanos S, Naskar R (2004) Correlation between retinal ganglion cell death and chronically developing inherited glaucoma in a new rat mutant. Exp Eye Res. 79(1): 119-129.
  9. Thanos S, Püttmann S, Naskar R, Rose K, Langkamp-Flock M, Paulus W (2004) Potential role of Pax-2 in retinal axon navigation through the chick optic nerve stalk and optic chiasm. J Neurobiol 59(1): 8-23.
  10. Pavlidis M, Stupp T, Naskar R, Cengiz C, Thanos S (2003) Retinal ganglion cells resistant to advanced glaucoma: a postmortem study of human retinas with the carbocyanine dye DiI. Invest Ophthalmol Vis Sci. 44(12): 5196-205.
  11. Thanos S, Indorf L, Naskar R (2002) In vivo FM: using conventional fluorescence microscopy to monitor retinal neuronal death in vivo. Trends Neurosci. 25(9): 441-444.
  12. Hafezi W, Eing BR, Lorentzen EU, Thanos S, Kühn JE (2002) Reciprocal transmission of herpes simplex virus type 1 (HSV-1) between corneal epithelium and trigeminal neurites in an embryonic chick organ culture. FASEB J. 16(8): 878-880.
  13. Fischer D, Heiduschka P, Thanos S (2001) Lens-injury-stimulated axonal regeneration throughout the optic pathway of adult rats. Exp Neurol. 172(2): 257-72.
  14. Heiduschka P, Romann I, Stieglitz T, Thanos S (2001) Perforated microelectrode arrays implanted in the regenerating adult central nervous system. Exp Neurol. 171(1): 1-10.
  15. Bodeutsch N, Thanos S (2000) Migration of phagocytotic cells and development of the murine intraretinal microglial network: an in vivo study using fluorescent dyes. Glia. 32(1): 91-101.
  16. Heiduschka P, Thanos S (2000) Restoration of the retinofugal pathway. Prog Retin Eye Res. 19(5): 577-606.
  17. Köbbert C, Apps R, Bechmann I, Lanciego JL, Mey J, Thanos S (2000) Current concepts in neuroanatomical tracing. Prog Neurobiol. 62(4): 327-351.
  18. Thanos S (1999) Genesis, neurotrophin responsiveness, and apoptosis of a pronounced direct connection between the two eyes of the chick embryo: a natural error or a meaningful developmental event? J Neurosci. 19(10): 3900-3917.
  19. Bodeutsch N, Siebert H, Dermon C, Thanos S (1999) Unilateral injury to the adult rat optic nerve causes multiple cellular responses in the contralateral site. J Neurobiol. 38(1): 116-128.
  20. Moore S, Thanos S (1996) The concept of microglia in relation to central nervous system disease and regeneration. Prog Neurobiol. 48(4-5): 441-460.

Professor Solon Thanos

Neuroregeneration

[vision-research.eu-Profile]

Westfalian Wilhelms-University Münster

Institute of Experimental Ophthalmology

School of Medicine
Albert-Schweitzer-Campus-1, D15
48149 Münster, Germany

Phone: 0049 251 8356915 or 6033
Fax: 0049 251 8356916

E-mail:
solon[at]uni-muenster.de

Website:
campus.uni-muenster.de/expoph_start.html