Current Prospects in Optic Nerve Protection and Regeneration: Sixth ARVO/Pfizer Ophthalmics Research Institute Conference

The sixth annual conference of the ARVO/Pfizer Ophthalmic Research Institute was held in Fort Lauderdale, Florida, on April 30 and May 1, 2010. Funded by the ARVO Foundation for Eye Research through a grant from Pfizer Ophthalmics, the conference brought together several specialist scientists and clinicians in the fields of glaucoma, neural regeneration, gene therapy, and stem cell therapy to discuss current knowledge and formulate new strategies for the design and implementation of therapies to maintain, repair, and regenerate optic nerve function. The conference was divided into four main sessions, with themes introduced by a keynote presentation of 20 minutes, followed by a 10-minute discussion and then discussions on five to six topics. Each topic was introduced by a 5- to 7-minute overview by a specialist in the field who then led a 20-minute discussion. Each session was moderated by two experts in each area of discussion. These lively discussions were intended to identify the most important questions that are still unanswered or need further exploration. This report provides a synopsis of discussions and introduces guidelines for future research. 

The four themes of discussion were as follows:

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  Session I: Contribution of Retinal Ganglion Cell Damage and Increased Intraocular Pressure to Optic Nerve Degeneration
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  Session II: Neurotrophin Mediation of RGC Survival and Optic Nerve Function
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  Session III: Genetic Intervention for Optic Nerve Regeneration and Protection
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  Session IV: Prospects of Optic Nerve Regeneration and Protection by Stem Cells

Contribution of RGC Damage and Increased IOP to Optic Nerve Degeneration

Unraveling the Mechanisms of Optic Nerve and Retina Damage from Elevated IOP

The cellular mechanisms by which elevated intraocular pressure (IOP) results in visual loss in glaucoma remain poorly understood. Damage at the level of the optic nerve head (ONH) appears to be a key event, but it is likely that many other factors influence the response of retinal ganglion cells (RGCs) to injury and their chances of survival and continued function. However, analysis of the sequence of events involved in glaucomatous neurodegeneration is complicated, not least because glaucoma is a chronic, progressive disease, and therefore at any given time point individual RGCs are likely to be at different stages of the injury process. Animal models of glaucoma provide a useful way to explore RGC responses to pressure-induced injury, and the first part of this session involved a discussion of some of the models and approaches that can help to clarify the pathologic responses to elevated IOP. 

Elevated IOP develops spontaneously or can be induced in many different species, and animal models have been used extensively to study pathogenic mechanisms potentially relevant to human glaucoma. Primate models are perhaps the most directly relevant to human glaucoma and are particularly useful for studies of ONH biomechanics, ¹ and for the late preclinical testing of potential new treatments before use in humans. ² However, financial and ethical considerations mean that primate models are impractical for many other types of study in glaucoma. The tree shrew has been proposed as a potentially useful glaucoma model because, in contrast to most nonprimate species, these animals have a clearly defined lamina cribrosa with structural similarities to the human lamina. However, tree shrews are expensive, are difficult to handle, and require special housing, which has limited their use in glaucoma research. Although other models in as widely different species as zebrafish ³ and mutant quails ⁴ have been described, most current models involve rats and mice. Of the mouse models that develop spontaneously, the DBA/2J stain has been most widely used. This model has similarities to human pigment dispersion syndrome, with development of chronically elevated IOP in most animals over the first year of life. Pressure elevation is associated with progressive changes in electroretinography ⁵ and visual function. ⁶ A disadvantage of DBA/2J and similar strains is that the pressure elevation is variable, and it is not possible to determine accurately when the insult at the level of the RGC is initiated. Although these features arguably mimic aspects of human glaucoma, they do make it difficult to study the time course of RGC changes in response to pressure-induced injury. Nevertheless, spontaneous mouse models have been an extremely fertile source of new insights into pathogenic mechanisms potentially relevant to glaucoma, from detailed localization of the site of injury ⁷ to studying the effect of IOP on mitochondrial fission. ⁸ A key advantage of mouse models is the facility to exploit transgenic modifications in experimental design. Thus, the effect of knocking out individual genes can be studied. Such techniques have been widely used recently—for example, to dissociate RGC axonal and cell body degeneration in DBA/2J mice by knockout of Bax (reducing RGC body loss) ⁹ or overexpressing WldS (slowing axonal loss). ⁷  

Rodent models where IOP is experimentally elevated at a discrete point in time have also been widely used and comprehensively reviewed. ¹⁰ These models allow more control over the timing of the initiation of pressure-induced injury and thus can be useful in studies of the time course of degenerative changes at the level of the RGCs. The most widely used rat models at present involve obstruction of aqueous outflow by hypertonic saline injection into the episcleral vessels ¹¹ or laser treatment to the trabecular meshwork (TM), ¹² but models in which microbeads are used to obstruct outflow have shown recent promise. ¹³ Induced IOP elevation has also been achieved in mice by using laser ¹⁴ and microbead ¹⁵ techniques. 

One important use of animal models is to explore the genetic changes that occur in response to injury. As an example of this approach, the use of the rat hypertonic saline model to explore gene changes in response to IOP elevation was discussed. When combined with gene array analysis and laser capture microdissection of the RGC layer, this model can be used to identify the earliest axonal, glial, and other retinal responses to IOP elevation, as opposed to changes occurring secondary to RGC loss. ¹⁶ Such techniques show that far more genes are downregulated than upregulated in the early stages of glaucomatous injury. Mitochondrial and axonal transport dysfunction may be particularly important in early RGC injury. Genes that appear upregulated in early glaucomatous damage include immediate response genes such as Atf3, neurotrophic factor, and cytokine genes, and genes involved in the regulation of the cell cycle, extracellular matrix, and cytoskeleton. ¹⁷ It is likely that at least some of these early responses are protective and that some are injurious, and therefore an important goal of future research is to determine how the protective mechanisms can be therapeutically manipulated as an adjunct to IOP control. 

Changes in response to elevated IOP involve glial and neuronal components of the ONH. Studies suggest that ONH cell proliferation and increased interleukin-6 type cytokine expression are very early responses to chronically elevated IOP, whereas, simultaneously, astrocytic marker gene expression is unchanged or downregulated. ¹⁶ Together, these observations suggest that astrocytic proliferation, accompanied by the suppression of differentiated astrocytic functions, are very early responses to elevated IOP exposure and thus may be therapeutic targets. 

Further discussions highlighted some of the limitations of current animal models in the determination of the signals from the ONH that indicate stress to the RGC soma. An important issue remains the characterization of the IOP profile in individual animals. Most anesthetic agents reduce IOP significantly and thus awake IOP measurement may be more useful when practical. However, rodents also have significant circadian fluctuation in IOP, and high nighttime pressure spikes may be missed if IOP is measured only during the day. Exposure to constant low light levels can greatly reduce circadian IOP fluctuation and does not cause retinal degeneration in pigmented rodents. ¹⁸ Other problems identified include the age of the animals used in the experiments and gene changes in response to IOP. Gene changes can vary in different rat strains and thus findings in one strain cannot necessarily be extrapolated to other strains, let alone to different species. 

Other key issues highlighted by the group for future research included the mechanisms by which the extracellular matrix of the ONH is remodelled in glaucoma, ¹⁹ the consequences of this remodelling for axonal–glial–vascular functional relationships and the degree to which these changes are reversible by control of IOP. The importance of the biomechanical properties of the ONH ^20,21 and sclera ²² in determining the extent of pressure-induced optic nerve injury was emphasized, and it was agreed that the topic is an important one for future studies. In addition, it was proposed that therapeutic intervention targeting the earliest axonal injury responses could be most powerful, and thus mechanisms such as the modulation of stretch-sensitive ion channels responsive to RGC axonal deformation must be better understood. ²³  

 

Sixth ARVO/Pfizer Ophthalmics Research Institute Conference Working Group Participants

 

Organizers

 

G. Astrid Limb, Institute of Ophthalmology, University College London, London, UK

 

Keith Martin, Centre for Brain Repair, Cambridge University, Cambridge, UK

 

Participants from the Ophthalmology Field

 

Keynote Speakers

 

Adriana Di Polo, University of Montreal, Montreal, Canada

 

Alan R, Harvey, The University of Western Australia, Crawley, WA, Australia

 

John C. Morrison, Casey Eye Institute, Oregon Health and Science University, Portland, OR

 

Thomas Reh, University of Washington, Seattle, WA

 

Discussion Leaders

 

James Bainbridge, Moorfields Eye Hospital and Institute of Ophthalmology, University College London NIHR Biomedical Research Centre, London, UK

 

Larry Benowitz, Laboratories for Neuroscience Research in Neurosurgery, Children's Hospital, Boston, MA

 

Michael Coleman, Laboratory of Molecular Signaling, the Babraham Institute, Cambridge, UK

 

Annegret H. Dahlmann-Noor, Institute of Ophthalmology, London, UK

 

Andy Fischer, Neuroscience, College of Medicine, The Ohio State University, Columbus, OH

 

Dietmar Fischer, Experimentalle Neurologie, Ulm University, Ulm, Germany

 

Jeffrey L. Goldberg, Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, FL

 

Pedro Gonzales, Duke Eye Center, Duke University, Durham, NC

 

Franz Grus, Johannes Gutenberg University, Mainz, Germany

 

Stefanie M. Hauck, Department of Protein Science, Helmholtz Zentrum München, Neuherberg, Germany

 

Elaine C. Johnson, Casey Eye Institute, Oregon Health and Science University, Portland, OR

 

Paul Kaufman, Ophthalmology and Visual Science, University of Wisconsin School of Medicine and Public Health, Madison, WI

 

Paul Lingor, University Medicine Göttingen, Georg-August-University Göttingen, Göttingen, Germany

 

James E. Morgan, University Hospital of Wales, Cardiff, UK

 

Natik Piri, Jules Stein Eye Institute, University of California Los Angeles, Los Angeles, CA

 

Harry Quigley, Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD

 

Michael Sendtner, Institute of Clinical Neurobiology, Universität Würzburg, Würzburg, Germany

 

Gülgün Tezel, Department of Ophthalmology and Visual Sciences, University of Louisville, Louisville, KY

 

Arthur J. Weber, Michigan State University, East Lansing, MI

 

Participants from Nonophthalmology Fields

 

Moses V. Chao, Department of Physiology and Neuroscience, New York University School of Medicine, New York, NY

 

Francine F. Behar-Cohen, Department of Ophthalmology, Hôtel Dieu de Paris, Université Paris Descartes, Paris, France

 

Ashim Mitra, School of Pharmacy, University of Missouri, Kansas City, MO

 

Daniela Ferrari, Biotechnologie, University Milano Bicocca, Milan, Italy

The Role of Inflammation and Implications for the Preservation of RGC Function

Evidence from clinical and experimental studies suggests that failures in the regulation of innate and adaptive immune responses can be associated with neurodegeneration. However, the role of inflammatory and immune mechanisms in RGC degeneration remain incompletely understood. ^24,25 In glaucoma, the demonstration of glial activation and cytokine changes has been used to support a role for inflammation in the disease. ^26,27 However, different subtypes of glia exhibit different states of activation at different stages of degeneration and it is likely that some of these responses are beneficial whereas others may be harmful. Similarly, lymphocyte-mediated responses may be beneficial or harmful in the context of optic nerve disease. As an example, lymphocytes can produce brain-derived neurotrophic factor (BDNF) in optic nerve injury models, ²⁸ but it is unknown whether this occurs in chronic diseases such as glaucoma. In addition, T cells can enter the normal central nervous system (CNS) via the blood–brain barrier. It has been proposed this ability is part of a protective role for the immune system, but the role of protective immunity in glaucoma remains controversial. ²⁹  

Despite limited evidence to support a definite pathogenic role for autoimmunity in glaucoma, it has been proposed that immunologic responses can be detrimental to RGCs. ³⁰ It is suggested that autoreactive T-cell activation contributes to the neurodegenerative process in glaucoma, possibly by stimulation of regulatory T cells and protective immunity. However, there is a need to understand these factors, to design immunomodulatory treatment strategies to control the immune response and prevent RGC damage. The group questioned whether cytokine changes were always necessarily related to inflammation. Cytokine changes occur early in adaptive immune responses and are important in the recruitment and activation of T cells, and neurons may also use cytokines to signal to each other without other evidence of inflammation. ³¹ Key questions indentified for future research included the role of glial cells in detecting “danger signals” from stressed RGCs and regulating immune responses in glaucoma. Whether modulation of immune responses toward tissue healing and the preservation of neuronal function is a realistic treatment strategy was also discussed, given the potential of immune responses to contribute to neurodegeneration. It was agreed that a much better understanding of the immune mechanisms involved in optic nerve diseases such as glaucoma would be required before such treatments could be contemplated. 

Clinical Assessment of Ganglion Cell Loss in Human Glaucoma

Retinal imaging has been revolutionized by development of techniques such as high-resolution optical coherence tomography (OCT), confocal scanning laser ophthalmoscopy (SLO), scanning laser polarimetry (SLP), and methods for the neutralization of optical aberration in the human eye using adaptive optics. ^32,33 There is now good evidence that commercially available imaging devices using these technologies can reproducibly detect and quantify glaucoma progression based on changes in optic disc topography and/or retinal nerve fiber layer thickness. ³⁴ OCT technology is undergoing particularly rapid development at present, with recently introduced spectral domain devices capable of higher resolution, faster image acquisition, and the ability to measure other potentially useful markers of glaucoma damage such as the macular ganglion cell complex. Recent advances in combining OCT and adaptive optics technology have allowed imaging of individual retinal nerve fiber bundles in cross section in the human retina, with resolution of bundles of <15 μm in diameter. ³⁵ Such advances in resolution may suggest that routine imaging of individual RGCs in the human retina is only a matter of time. That high-resolution imaging of individual rods and cones in the human retina is already possible may add further weight to this idea. However, there are particular problems in imaging RGCs that make these cells more of a challenge to image individually than other cells within the retina. As an example, identification of individual cells by OCT requires a signal to be detected from the optical interface between the cell and neighboring structures. In the case of individual RGCs, this signal is extremely weak. Alternative technologies such as SLO have proved to be extremely useful for the identification of photoreceptor outer segments, which form a regular array occupying a significant proportion of the thickness of the outer retina. However, RGC bodies lie in a much thinner layer with irregular distribution, masked by the overlying nerve fiber layer. Resolution of individual cells is therefore considerably more challenging. 

 

Observers

 

Catherine Abbadie, Alcon Ltd., Fort Worth, TX

 

Ron Adelman, Yale University School of Medicine, New Haven, CT

 

Nicolas Bazan, LSU Health Sciences Center, New Orleans, LA

 

Katharina Bell, Universitats Augenklinik, Mainz, Germany

 

Roger Beuerman, Singapore Eye Research Institute, Singapore

 

Sanjoy Bhattacharya, University of Miami Miller School of Medicine, Miami, FL

 

Pierre Bitoun, Hôpital Jean Verdier CHU Paris-Nord, Bondy, France

 

Thomas Brunner, Glaucoma Research Foundation, San Francisco, CA

 

Natalie Bull, University of Cambridge, Cambridge, UK

 

Jingtai Cao, Regeneron Pharmaceuticals, Inc., Tarrytown, NY

 

Ian Catchpole, GSK Medicine Research Centre, Stevenage, UK

 

Sumit Dhingra, Institute of Ophthalmology, University College London, London, UK

 

David Eveleth, Pfizer Ophthalmics, San Diego, CA

 

Gerald Gough, GlaxoSmithKline, Stevenage, UK

 

Ruslan Grishanin, Rinat Laboratories, Pfizer, Inc., San Francisco, CA

 

John Grunden, Pfizer Ophthalmics, Pittstown, NJ

 

Donald Hood, Columbia University, New York, NY

 

Lauren James, Institute of Ophthalmology, University College London, London, UK

 

Hari Jayaram, Institute of Ophthalmology, University College London, London, UK

 

Thomas Johnson, National Eye Institute, Rockville, MD

 

Peng Khaw, Moorfields Eye Hospital and Institute of Ophthalmology, University College London, London, UK

 

Jeffrey Kiel, University of Texas Health Science Center, San Antonio, TX

 

Takuji Kurimoto, Osaka Medical College, Takatsuki, Japan

 

Hani Levkovitch-Verbin, Ophthal-Goldschleger Eye Institute, Tel-Aviv University, Tel-Hashomer, Israel

 

Barbara Lorber, Centre for Brain Repair, University of Cambridge, Cambridge, UK

 

Colm O'Brien, Mater Misericordiae University Hospital, Dublin, Ireland

 

Dario Paggiarino, Pfizer Ophthalmics, Escondido, CA

 

John Penn, Vanderbilt University School of Medicine, Nashville, TN

 

J. Mark Petrash, University of Colorado Denver, Aurora, CO

 

Renata Puertas, Moorfields Eye Hospital, Dubai Healthcare City, Dubai, United Arab Emirates

 

Robert Ritch, New York Eye and Ear Infirmary, New York, NY

 

Steve Roberds, Pfizer Ophthalmics, St. Louis, MO

 

We Siah, Mater Misericordiae University Hospital, Dublin, Ireland

 

Carla Starita, Pfizer Ophthalmics, Surrey, UK

 

Carol Toris, University of Nebraska Medical Center, Omaha, NE

 

Charles Tressler, Yorktown Heights, NY

 

Hannu Uusitalo, University of Tampere, Tampere, Finland

 

Tracy Valorie, Pfizer, Inc., New York, NY

 

Sauparnika Vijay, Institute of Ophthalmology University College London, London, UK

 

Deborah Wallace, Mater Misericordiae University Hospital, Dublin, Ireland

 

Larry Wheeler, Allergan, Inc., Irvine, CA

 

Barbara Wirostko, University of Utah, Park City, UT

 

Li Wu, Columbia University, New York, NY

 

Zhiyong Yang, Johns Hopkins School of Medicine, Baltimore, MD

 

Camila Zaverucha do Valle, University Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Although it is uncertain whether the imaging of individual RGCs is possible with OCT, important surrogate measures are possible. Recent studies suggest that the contrast sensitivity of ultrahigh resolution OCT may allow the detection of subcellular changes that correlate with neuronal health or possibly even with neuronal activity. ³⁶ The ability to detect sick RGCs at a stage when rescue is still possible would be extremely attractive, as all current imaging techniques in clinical use are sensitive only to changes in the structure of the ONH and retinal nerve fiber layer that occur mainly after RGCs have died. As a further application of OCT technology, long-wavelength (1050 nm) OCT may provide important measures of events that occur within the ONH and at the level of the lamina cribrosa. 

Discussion during this session focused on the relative merits of detection of RGC death versus monitoring and quantifying RGC survival. Suggestions of cellular structures and processes amenable to imaging that could give an indication of neuronal dysfunction before death included cytoskeletal components, axonal transport, early events in the apoptotic cascade, and possibly electrical function using voltage-sensitive dyes. However a more immediate issue is that, although existing imaging techniques have been shown to detect glaucoma progression, imaging endpoints are not recognized by the U.S. Food and Drug Administration when it comes to assessing the efficacy of new glaucoma treatments. A key issue is therefore to establish and quantify the ability of existing and future imaging technologies to predict loss of visual function in glaucoma. ³⁴  

Clinical Biomarkers of Optic Nerve Disease

The official National Institute of Health definition of a biomarker is “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.” ³⁷ The search for biomarkers predictive of glaucoma onset or progression has been undertaken by several groups, and some promising candidates have emerged. Multiple genetic loci associated with glaucoma have been found, but account for only a small proportion of the total number of glaucoma cases and therefore have low sensitivity and specificity when applied to populations. ³⁸ The complex profiles of natural occurring autoantibodies have been studied in glaucoma and control subjects, and in some studies, this approach has been shown to identify glaucoma cases with a sensitivity and specificity of up to 90%. ³⁹ Whether these changes occur as a cause or a consequence of glaucoma remains unknown. 

As an alternative to antibody profiling, very sensitive methods, such as two-dimensional (2D) gel electrophoresis and surface enhanced laser desorption/ionization–time of flight mass spectrometry (SELDI-TOF-MS), can be used for protein analysis in aqueous. ⁴⁰ Again, sensitivity and specificity of around 90% has been achieved. As an example, transthyretin concentrations were found to be selectively elevated in glaucoma samples. However, the clinical applicability of such techniques is currently limited by the requirement for complex handling procedures for aqueous samples and the lack of “local samples” from the environment of the retina and optic nerve. Discussions within the group highlighted the potential benefits of finding new markers of glaucoma onset and progression. However, it was emphasized that validation of putative biomarkers in multiple different large populations would be essential and that, to date, this remains to be achieved. Further work to establish and validate the most predictive biomarkers and to explore their role in the pathogenesis of glaucoma is needed. (Biomarkers in Glaucoma was the subject of the Seventh ARVO/Pfizer Ophthalmics Research Institute Conference, held April 29 and 30, 2011.) 

Neurotrophin Mediation of RGC Survival and Optic Nerve Function

Neuron–Glial Interactions in the Injured Retina

Neurotrophins modulate many key RGC processes from development through adulthood. RGC growth, synapse formation, survival, response to injury, and regenerative capacity may all be affected by neurotrophin signaling. ⁴¹ In many of these processes, the interaction between RGC and glia is critical. Müller cells, the most abundant glial cell type in the retina, play crucial roles in many aspects of neural function, including metabolic and neurotrophic support and neurotransmission, ion and water homeostasis, blood flow, and neurogenesis. ⁴² Müller cells become reactive in human glaucomatous eyes, but their response to ocular hypertension damage and whether they play a direct role in RGC death is unknown. Recent work has suggested novel pathways by which retinal glia can exacerbate neuronal death after injury. As an example, the pro form of nerve growth factor (pro-NGF) has been shown to induce RGC death via activation of p75NTR-signaling in Müller cells, leading to tumor necrosis factor (TNF)-α production. ⁴³ Müller cell–derived TNFα appears to cause neuronal loss by increasing RGC surface expression of calcium-permeable AMPA receptors, rather than activation of caspase-8. Similar mechanisms may also occur during RGC death in experimental glaucoma, and there is some evidence that inhibitors of TNFα protects RGCs from degeneration in animal models of the disease. ⁴⁴  

Additional studies related to the neuroprotective role of Müller glia in the retina based on proteomic analysis have revealed that Müller cells secrete at least 250 different proteins, including neurotrophins, such as glial-derived neurotrophic factor (GDNF), pigment epithelial–derived growth factor (PEDF), and ciliary neurotrophic factor (CNTF). ^45,46 A major therapeutic challenge is to identify and exploit those molecules that are beneficial for RGC survival and regeneration while avoiding glial signaling pathways that may be detrimental. An in-depth understanding of the functional consequences of glial cell responses in retinal and optic nerve neurodegeneration is paramount in developing successful strategies for neural protection and repair. 

Transcriptional Repression of Axon Growth in RGCs

RGCs and other neurons in the CNS exhibit a decrease in their intrinsic capacity for rapid axon growth during early development, and the decrease correlates with a loss of regenerative ability in vivo. As an illustration of this difference, embryonic RGCs in culture grow axons at least 10 times faster than do cultured adult RGCs. In vivo observations in rodents show that axonal growth and regrowth are markedly decreased after birth, ⁴⁷ suggesting that signals delivered by other cells in the retina inhibit axonal growth. Investigations into the type of cells that may promote this inhibition showed that amacrine cells, which are immediately presynaptic to RGCs, are the only neurons that inhibit RGC axonal growth. This finding coincided with RGCs' reaching their targets and acquiring axon growth ability. ⁴⁸  

Expression of genes that regulate axonal growth shows high variability during different stages of retinal development, which makes developmental analysis of gene expression in these cells very difficult. Furthermore, in rats at least, on any given day after conception, developing RGCs that are born on different embryonic days are also at different stages of maturation. ⁴⁹ Nonetheless, a role for the Kruppel-like factor (KLF) family of transcription factors that control the regulation of axon growth and regeneration in RGCs and cortical neurons has been described recently. ⁵⁰ Other transcription factors, including p53, NFκB, CREB, STAT3, cjun, Sox 11, and ATF3, have also been linked to the regenerative ability of RGC, usually as positive regulators of axon growth. Interestingly, many of the KLFs suppress axon growth, and knocking out at least one, KLF4, enhances regeneration in vivo. At present, it is not known how the KLF family of factors exert transcriptional control of axon growth. Whether they are regulated by neurotrophins or electrical activity or they have specific effects in different RGC subtypes is not yet clear and merits further investigation. In contrast to their effect on axonal regeneration during retinal development, KLF changes have not been found to affect axonal survival after optic nerve crush (ONC). In this context, it is important to consider evidence that the ability of the superior colliculus to receive synapses is developmentally regulated, suggesting that, for KLF factors to induce axonal growth in adulthood, it may be necessary to revert RGCs to early development. Further investigation in this field would help to clarify the role of KLF factors and to identify molecules in this family that could be targeted for the development of small-molecule therapies. 

Another signaling system that is crucial in development of the optic pathway, but is also potentially important in RGC pathology, is the Eph/ephrin system. Eph receptors are a large family of tyrosine kinases for which ephrins are the ligands. This signaling pathway plays a key role in RGC axonal guidance during development, with EphB helping to direct axons to the ONH and onward toward the brain. However Eph/ephrin signaling occurs as an early response to RGC injury, and early changes in ephrin and the EphB receptor have been described in experimental models of glaucoma and in human glaucoma tissue. ⁵¹ In the ONH, both ephrinB1 and EphB1 were localized to astrocytes, and EphB1 was also localized to lamina cribrosa cells and perivascular cells, an example of how glial–axonal signaling may be mediated in response to IOP elevation. In the retina, ephrinB1 localized to Müller cells and astrocytes and was found in RGCs. It was therefore suggested that the Eph/ephrin pathway plays a protective role by limiting axonal damage and inflammatory cell invasion during glaucomatous injury, and that inhibition of axonal regeneration by ephrin signaling could be a less desirable strategy. More recent work in mouse models of ocular hypertension has provided further evidence that Eph/ephrin changes may occur early after IOP elevation, before morphologic signs of axonal damage. ⁵² Thus, it has been proposed that modulation of Eph/ephrin signaling is an important neuroprotective strategy that may also be relevant to strategies for stimulating RGC axonal regeneration. ⁵³  

Modulation of RGC Regeneration

RGCs do not normally regenerate axons after optic nerve injury, but instead they undergo apoptosis. Multiple mechanisms contribute to the failure of axonal regeneration including myelin-related factors, extracellular matrix proteoglycans, glial scarring, and the relatively poor regenerative capacity of mature RGCs. However, RGCs can be transformed into an active regenerative state, when exposed to certain conditions. For example, intraocular inflammation can dramatically increase RGC axonal regeneration, although there is some controversy regarding the relative importance of glial- and macrophage-derived factors in this situation. ^{54 –57} Macrophages are known to secrete oncomodulin (Ocm), a small calcium-binding protein that, in the presence of appropriate co-factors, shows high-affinity binding to RGCs and stimulates extensive axon regeneration in culture and in vivo. Conversely, agents that prevent Ocm from binding to its receptor suppress most inflammation-induced regeneration, but do not diminish the effects of intraocular inflammation on RGC survival. ⁵⁸ Additional manipulations of RGCs, such as ectopically induced expression of genes that make them “blind” to cell-extrinsic inhibitors of growth and/or enhancing complementary signaling pathways such as cAMP, can augment regeneration even further. When such combined approaches are experimentally used, some regenerating axons may extend for the whole length of the optic nerve, cross in the optic chiasm and enter the thalamus. 

CNTF and leukemia inhibitory factor (LIF), which are released from retinal cells, have also been proposed as important mediators of RGC regeneration. ^56,57,59 There is currently a debate in the literature regarding the role of Ocm and the extent to which CNTF alone at physiologically relevant concentrations can induce regeneration in mature RGCs and regarding possible differential effects of native and recombinant CNTF. ^58,59 Further work is ongoing to clarify these issues, but at the very least, CNTF seems to modulate responses to other growth factors, to mediate cell survival, and to have important additional effects that contribute to RGC regeneration. 

Functional optic nerve regeneration remains an important goal, and the group identified long-range axonal guidance as a key challenge. It also remains uncertain whether specific subsets of RGCs have increased regenerative potential. New types of cell-specific assays are needed to answer this question, although recent evidence suggests that the RGC's birth date may influence subsequent regenerative abilities after injury. ⁴⁹ Other experimental approaches suggested by the group included modulation of multiple transcription factors simultaneously, to determine whether the regenerative capacity can be enhanced further. However, it remains to be seen which, if any, transcription factors will be the most appropriate “drugable” targets. 

Neuroprotective Effects of BDNF

Previous work showing that intravitreal application of trophic factors after optic nerve injury is neuroprotective has generated considerable interest in their use as potential therapeutic agents. ⁶⁰ Of the different factors studied, BDNF is perhaps the best understood. BDNF and its receptors, TrkB and p75NTR, have been demonstrated at all levels of the central visual pathway, as has their transport within the optic nerve. However, because to date BDNF has been shown only to slow, and not to prevent, ganglion cell death after trauma to the optic nerve, its role as a potential retinal therapeutic remains equivocal. ^41,61 Since BDNF does not promote optic nerve regeneration after injury, ⁶² it has been suggested that it blocks the ability of other factors to promote regeneration. Alternatively, BDNF or its pro form pro-BDNF, which is also released from producing cells can exert proapoptotic functions via the p75NTR receptor, which is upregulated under pathophysiological conditions. BDNF is a highly basic protein that does not easily diffuse in tissues after injection or local production. An additional issue is that BDNF appears to have different effects, depending on how it is delivered to cells. It was suggested in discussion that BDNF delivered to cell-surface receptors on RGC bodies appears to potentially induce rapid signaling via Erk 1/2 pathways and transient neuroprotection, whereas BDNF transported retrogradely from the nerve terminals may result in signaling via Erk 5 that leads to longer term survival. Further work on this topic is therefore needed. The group agreed that BDNF delivery is a potential promising neuroprotective approach, but concerns remain about long-term effectiveness and the degree of protection that may be expected in chronic disease. 

Trk Signaling as an Integrator of Neuroprotective Mechanisms

Enthusiasm for the use of neurotrophic factors as therapeutic agents has been tempered to a degree by observations in some of the clinical trials conducted to date. Therapeutic use of CNTF in amyotrophic lateral sclerosis has been limited because the hepatic binding of CNTF causes fever and cachexia. It was also noted that the CNTF used was immunogenic, with many patients developing an antibody response. ⁶³ BDNF was also found to be immunogenic during clinical trials in neurodegenerative disease. ^64,65 Thus, there has been recent interest in the possibility of peptide use as an alternative to neurotrophic factors, such as BDNF and CNTF, that could be less immunogenic, could cross the blood–brain barrier more easily, and thus could constitute a more useful therapeutic agent. 

Neurotrophins mediate their effects on neuronal survival, maintenance, and regeneration via receptor complexes including Trk transmembrane proteins. TrkB, the mediator of BDNF effects, can also be activated by other receptors including G-protein-coupled proteins such as the adenosine type 2A receptor, or other transmembrane tyrosine kinase receptors for which small agonistic molecules have been identified. Use of ligands for the adenosine type 2A receptor can protect lesioned motor neurons via trkB signaling. ⁶⁶ These observations suggest that it may be worth exploring the ability of small molecules such as activators of G-protein-coupled receptors to protect RGCs in disease models via Trk transactivation. 

Genetic Intervention for Optic Nerve Regeneration and Protection

The Effects of Viral Vector–Mediated Delivery of Growth-Associated Factors on Survival, Morphology, and Regeneration of Adult Neurons

The impact of viral vector–mediated delivery of growth factors on the survival and differentiation of adult rat RGCs after ONC and peripheral nerve (PN) transplantation have provided some understanding of the benefits and limitations that gene therapy may offer. Since most neurons in the retina are terminally differentiated, appropriate vectors that do not actively replicate and are not infectious or highly immunogenic should be used. Of the seven different serotypes of adenoassociated viral (AAV) vectors studied, AAV-2 has been shown to predominantly transduce RGCs within the retina. ⁶⁷ Restoration of RGC function is undoubtedly a major therapeutic target, and recent studies have been conducted to investigate how gene therapy can improve the viability and regenerative capacity of these cells. Unlike the individual RPE65 mutations that cause various retinal dystrophies, ⁶⁸ single gene defects have not been associated with glaucoma, which makes the development of such therapies rather difficult. However, gene therapy can target major factors associated with the disease. 

The effects of AAV-2 encoding GFP alone, CNTF-GFP, BDNF-GFP, or GAP-43-GFP have been examined in ONC models and after grafting of autologous PN onto the cut optic nerve. ^69,70 AAV-2 vectors encoding BDNF temporarily increase cell survival of axotomized RGCs but do not promote regeneration, whereas vectors encoding CNTF promote longer term RGC survival and axonal regrowth, as demonstrated by the presence of fibers that cross the crush site. These observations support the idea that there is a dissociation between the mechanisms that mediate increased cell survival and subsequent axonal regeneration. 

Studies on Bcl-2 transgenic mice (resistant to axotomy) have shown that RGC viability and axonal regeneration are further potentiated by AAV-CNTF injections after crush injury. Combined therapy using AAV-BDNF with recombinant (r)CNTF and cAMP analogues increases survival of RGCs but reduces the axonal regeneration that is observed with rCNTF alone. ⁶² This contrasts with observations that co-injection of factors such as rCNTF and the cAMP analogue CPT-cAMP cause survival and extensive axonal regeneration of RGCs, although single injections of rCNTF at 3 days after PN autograft cause an increase in the expression of suppressor of cytokine signaling (SOCS) mRNA and proteins. ⁷¹ Unlike that seen with rCNTF vitreal injections, AAV-CNTF does not increase upregulation of SOCS3 mRNA, suggesting that vector-delivered CNTF causes less activation of SOCS signaling pathways, known to be repressors of RGC regeneration. ⁷²  

Investigation into the long-term effect of sustained overexpression of growth factors has shown that AAV2-BDNF, -CNTF, and -GAP-43 induce changes in the dendritic architecture of RGCs, as judged by changes in three types of RGCs examined 9 months after AAV injection and PN transplantation. BDNF and CNTF transduced and nontransduced (bystander) type I RGCs displayed bigger cell bodies compared with control GFP or non-GFP matches. However, CNTF bystander cells show a reduced complexity of dendritic architecture when compared with BDNF bystander and controls. In contrast, type II RGCs transduced with BDNF showed the greater dendritic changes, ⁷³ suggesting that although modulation of the dendritic architecture may be feasible, it is not known whether dendritic changes are beneficial or detrimental during disease development. It has been proposed that the development of gene delivery systems with promoters capable of exogenous regulation would help to clarify whether functional activity is altered as a consequence of morphologic changes in transduced and neighboring nontransduced RGCs. 

Intraocular Gene Transfer by AAV Vectors: Potential Application to Ganglion Cell Survival

The protection of RGCs by vector-mediated intraocular expression of neurotrophic factors in experimental models suggests that this approach protects vision in glaucoma. However, not all cells are transduced with the same efficiency, and the kinetics of transduction by different vectors depends on the route of delivery. AAV vector serotypes 2, 6, and 8 preferably transduce RGCs, whereas serotypes 7, 8, and 9 transduce peripheral retina, RPE, and RGCs. ⁷⁴ The role of the vitreous gel in limiting access of intravitreally injected vectors to retinal cells was also discussed, leading to the proposition that vitrectomy and peeling of the inner limiting membrane (ILM) for transduction of the inner retina would be preferable. It was concluded that it is important to identify vectors that specifically target RGCs in experimental models and that the underlying effect of age should be taken into consideration when designing such therapies. 

Because of the existence of multiple genes associated with glaucoma, together with the high variability in transduction efficiency and relatively small packaging size of existing AAA vectors, it may be appropriate to investigate the suitability of inducible systems controlling gene expression, such as the tetracycline-inducible recombination system in which gene expression can be manipulated at specific times with systemic antibiotics. Furthermore, development of vectors that can specifically target RGCs with larger genes may lead to their application in glaucoma therapy. 

Identification of Targets for Glaucoma Therapy

Taking into consideration that several pathophysiological factors determine the loss of neural function in glaucoma and that investigations are at present addressing different aspects of neuroprotection and regeneration, ⁷⁵ it was proposed that research in the field should target the most appropriate strategies that would be most beneficial and rapidly applicable to glaucoma therapies. Proposed targets included the protection of individual soma and axons and axonal connections to the brain, the prevention of RGC death, and the induction of regeneration of damaged RGCs. In addition, intravascular, intraocular, or local brain delivery of neuroprotective factors such as cytokines, neurotrophins or apoptotic inhibitors were considered as potential therapeutic strategies. Proposals for retrograde delivery of neurotrophins were made, based on observations that RGCs improve their function after experimental injection of BDNF directly into the lateral geniculate nucleus (LGN), especially in secondary degenerations. ⁷⁶ Since recent studies have shown that combined intravitreal injections of BDNF with 1 to 2 weeks of continuous delivery of this factor to the visual cortex enhances RGC survival after optic nerve injury, ⁷⁷ it was suggested that targeting both the eye and the visual cortex may constitute a realistic approach and should be considered in the development of future glaucoma therapies. 

The group agreed that efforts should be concentrated on preventing RGC death, promoting axonal regeneration of dying cells, reconnecting RGC axons to the optic nerve, and targeting neural connections to central visual structures. Examples were given of experimental data in which the RGC soma can be induced to survive without improving axonal function, and proposals for targets such as the BCL2 family of antiapoptotic molecules were formulated. ⁹ It was concluded that since single neuroprotective therapy in rodents is not sufficient to induce functional recovery of axons unless IOP is controlled, the establishment of multiple strategies for therapy in humans may be necessary. Because of the lack of evidence that terminal RGC damage can be reversed, significant interventions should be aimed at preventing death of the last surviving RGCs before blindness ensues. If successful, these approaches may constitute a real difference between blindness and vision in some individuals. More efficient clinical trial designs for the assessment of potential neuroprotective strategies in smaller and shorter studies remain a key unmet need. 

Gene Therapy to Suppress Apoptosis and Enhance Neural Survival in the Optic Nerve

Apoptotic cell death plays a major role in the pathogenesis of inflammatory and degenerative disorders of the retina and the optic nerve. To alter disease progression in experimental models, overexpression or downregulation of genes regulating apoptosis has been investigated. New imaging technologies have allowed the in vivo visualization of single RGC axons in real time using AAV-mediated expression of fluorescent dyes. By applying these techniques in the ONC model, early axonal degeneration can be observed before damage to the cell soma occurs. In addition, axonal vacuolation, corresponding to autophagosome formation, and intracellular influx of calcium are seen immediately after injury. ⁷⁸ Inhibition of autophagy attenuates acute axonal degeneration, whereas increased calcium influx promotes degeneration. ⁷⁹ Targeting of the antiapoptotic molecule BAG1 (Bcl-2-associated athanogene-1) using the AAV vector (AAV.BAG1) enhances RGC survival and increases the number and length of regenerating axons when compared with AAV.EGFP controls, ⁸⁰ suggesting that BAG1 has a dual role of inhibiting lesion-induced apoptosis and interacting with the inhibitory ROCK signaling cascade. ⁸¹  

The discussion addressed the possibility of using RNAi in preference to viral vectors to target genes that are difficult to modify by conventional methods. It is anticipated that although RNAi may have lower cellular toxicity than AAV vectors, the latter can be combined with promoters for specific cell types, such as synapsin, to prevent immunogenicity and widespread transfection. Trials using RNAi to suppress VEGF expression in wet AMD and caspase-2 in ischemic optic neuropathy are in progress, and methodology used to implement these therapies could be applied to silence the genes involved in RGC degeneration. Alternative therapies to promote RGC survival using calcium blockers were discussed, and the proposal that lithium could be used to block calcium influx in RGCs was received with caution, due to the side effects and narrow therapeutic range of this molecule, which also targets protein phosphorylation in RGCs. Silencing expression of kinases by RNAi was also suggested, based on recent reports that inhibition of the Mst3b kinase using shRNA prevents RGC from regenerating axons in response to growth factors in vitro. ⁵⁵ However, knockdown of kinases is not always appropriate, as downstream targets may remain active, and signaling pathways may be preserved. Duration of gene silencing after administration of the RNAi should also be evaluated. Compensatory changes in endogenous gene expression resulting in upregulation of other genes may counteract the desired effects, and this should be taken into consideration when attempting to develop long-term gene therapy. 

Potential Gene Therapy for Protection of RGC against Oxidative Stress

Oxidative stress has been implicated in RGC death after optic nerve transection, hypoxia, ischemia, axonal transport disruption, and glaucomatous neurodegeneration. Although the role of oxidative stress in glaucoma is not well understood, it is thought to contribute to TM degeneration and consequently to alterations in the aqueous outflow pathway, increased IOP, and axonal injury at the ONH. Molecules responsible for the protective mechanisms against cell oxidative damage include the superoxide dismutase, glutathione (GSH), and thioredoxin (TRX) systems. ⁸² The TRX protein system is a ubiquitous thiol-reducing complex that regulates the cellular redox state. Downregulation of this system has been demonstrated in RGC degeneration, whereas induced overexpression of TRX1 and -2 proteins in the retina in vivo has been shown to increase the survival of RGCs. ⁸³  

Mitochondrial dysfunction is associated with optic neuropathies such as Leber's hereditary optic neuropathy. In addition, autosomal dominant optic atrophy and increased mutations in mitochondrial DNA have been observed in patients with POAG. ⁸⁴ Strategies to reduce the damaging effects of oxidative stress in glaucoma that rely on the antioxidant activities of natural substances and vitamins have been proposed. However, it was acknowledged that at present there are challenges and limitations in the implementation of antioxidant gene therapy, partly due to the high metabolic activity of RGCs. These cells require high levels of ATP to power axonal transport and electrical activity as well as other cellular functions. Since an important source of ATP appears to be the mitochondria found in high numbers along the RGC axons ⁸⁵ and mitochondrial DNA mutations accompanied by reduced cellular respiratory activity are seen in a large number of glaucoma patients, ⁸⁶ further studies should be conducted to clarify the implications of mitochondrial mutations on ATP metabolism. 

At present, it is not clear whether primary mitochondrial dysfunctions occur in the axons or the soma of RGCs. Drosophila studies have shown that reduction of mitochondria in axons and dendrites do not induce severe neurodegeneration. ⁸⁷ It is thought that differences between species may occur, and therefore functional investigation to elucidate the involvement of mitochondria in human glaucoma is needed. Application of these studies to therapy in humans is still unclear, and it may be possible to target proteins involved in the regulatory cascade of TRX such as TRXNIP to prevent oxidative stress damage. However, because of the high levels of mitochondria mutations observed in glaucoma patients, the targeting of specific molecules may be a difficult task. In the context of oxidative stress, cigarette smoking was discussed, and it was concluded that currently there is no clear evidence that smoking predisposes to glaucoma. Known trials for antioxidant therapy in glaucoma patients have been scarce, and no evidence of protection by antioxidant consumption has been found in primary open-angle glaucoma. ⁸⁸  

Gene Therapy for Overexpression of CNTF and BDNF to Induce RGC Survival

Several potential areas for neuroprotective treatment using gene therapy have been identified. RGC survival has been induced by intravitreal injection of AAV-BDNF vectors into the vitreous of rats subjected to laser-induced glaucoma. ⁶⁰ Similar effects have been observed with AAV-induced overexpression of CNTF, albeit with short-term effects. Since these neuroprotective effects cannot be sustained, the use of this approach in glaucoma treatment may be limited. It was proposed that encapsulated cells overexpressing CNTF, which are at present used in clinical trials for retinitis pigmentosa, may be tried in glaucoma therapy. ⁸⁹  

Since early RGC activation may reflect a nonspecific RGC response to injury and not a feature of glaucoma itself, effective neuroprotective agents may have to be identified in early stages of experimental glaucoma models. There was a general consensus that interventions to prevent RGC death as early as possible were most likely to be effective. Recent work in a model of raised IOP using microbead injections into the anterior chamber has identified several molecules that are rapidly activated after injection. These molecules include caspase-9 and -3 and TNFα, which could be targeted by existing inhibitors (Quigley H, personal communication, May 2010). Other molecules that may justify further investigation include β-amyloids, which induce neural apoptosis in Alzheimer's disease and which have been strongly associated with glaucoma, ⁹⁰ and calcium channel blockers, which have been shown to be highly neuroprotective when RGC health is compromised. ⁹¹ However, the exact mechanisms by which calcium channel blockers may act in neuroprotection are not yet known, and their effectiveness remains controversial. 

Analysis of the variability of results obtained by different groups and the age of the animals used in glaucoma models would be important for translational studies. Differences in IOP levels and axonal damage between young and old rats after glaucoma induction have been observed, suggesting that the use of older animals would be more relevant to human glaucoma. Furthermore, species differences should also be considered in the interpretation of experimental data, as supported by in vitro observations that mouse and human embryonic stem cells differ in their ability to differentiate into motor neurons. ⁹² Although differentiated neurons obtained from both cell types have similar capacities for axonal growth, the axon growth capacity of mouse cells is much higher than that of human cells, which suggests that regeneration may be more difficult to achieve in the human than in the mouse. 

Strategies for Axonal Protection

Retrograde axonal transport of key neuroprotective molecules is vital for neuronal survival, and axons can survive only if anterograde axonal transport delivers essential cargoes from the soma. Impairment of axonal transport has been shown to constitute an early feature of a wide range of neurodegenerative conditions including glaucoma, retinal ischemia, and optic atrophy. ⁹³ Transgenic rats where Wallerian degeneration is significantly delayed by expression of a fusion protein (Wld^S) have shown delayed axonal degeneration of RGCs when subjected to experimental glaucoma, suggesting that axonal degeneration observed in glaucoma involves a Wallerian-like mechanism. ⁹⁴  

A major determinant of axon survival is Nmnat2, a labile, rapidly transported axonal enzyme and key axonal survival factor whose depletion is sufficient to trigger Wallerian-like degeneration. ⁹⁵ Nmnat2 has a short half life in vitro, and its levels fall relatively rapidly after axonal transport interruption, triggering Wallerian degeneration. Wld^S appears able to functionally replace Nmnat2, so that axonal degeneration after transport blockade is delayed. Strategies to enhance the protective effects of Wld^S merit further exploration as part of a combined therapeutic approach. 

When axonal transport is disrupted, the distal end of the axon is particularly vulnerable to deprivation of essential factors. On this basis, strategies to improve the transport and longevity of survival factors would be of prime importance. It was suggested that pharmacologic therapy rather than gene therapy would be more apt to attain this effect, as the latter may need a longer time for protein transcription. Stabilization of proteins by degradation of proteasomes slightly prolongs axonal survival. It was therefore proposed that to exploit this mechanism therapeutically, it would be necessary to identify the specific ubiquitin ligases that target neuroprotective proteins. Since TGFβ inhibits proteasome function without causing detrimental effects, ⁹⁶ this factor could be potentially used to stabilize proteins. Alternative approaches to promote axonal transport could include stabilization of proteins by blocking autophagy, a regulatory process of protein turnover, or phosphorylation of specific protein targets, as inferred by the observations in which BDNF transport is enhanced by phosphorylation of Huntingdon protein. ⁹⁷  

The half-life of neuroprotective factors should be considered if they are to be used in glaucoma therapy. Further investigation of the extent of axonal damage in glaucoma models in young and old animals, as well as in chronic and acute models of glaucoma, also should be pursued. At present, it is not known how to measure axonal transport in humans, and studies in this field may have to be undertaken. It was speculated that short-term elevation of IOP in humans without the risk of retinal damage may be feasible and would allow development of such methods. 

Prospects of Optic Nerve Regeneration and Protection by Stem Cells

Stem Cell Therapy for Retinal Degeneration

Loss of retinal neurons is the ultimate cause of visual impairment in retinal disease. Once a significant number of neurons are lost, blindness ensues and becomes irreversible. In fish and amphibians, endogenous repair mechanisms can restore normal retinal structure and function, but these mechanisms are largely absent in mammals. At present, retinal neurons, including RGCs and photoreceptors, can be efficiently derived from human embryonic stem cells (ESs), inducible pluripotent stem (iPS) cells, ⁹⁸ and Müller stem cells (Limb GA, personal communication, May 2010). Transplanted ES- or iPS-derived retinal cells have been shown to integrate into the retina and restore light responses in mouse models of retinal disease. ⁹⁹ However, very little is still known about the potential of these cells to restore RGC function by transplantation. It was suggested that in addition to their potential for cell replacement, iPS-derived retinal cells could be used to generate patient-specific ganglion cells for use in high-throughput screening for survival factors, as well as for drug screening by creating disease model–derived cells. 

Although studies investigating differentiation of ES cells into RGCs are limited, protocols for generation of photoreceptor cells from ES in the presence of growth factors and feeder cells ¹⁰⁰ have been extensively investigated. ES cells can be differentiated into retinal neurons by the addition of factors such as insulin growth factor (IGF), DKK1 (an inhibitor of Wnt), and Noggin. They typically form rosettes in culture and express markers of photoreceptor cells such as Chx10, Pax6, Six3, Crx, Nrl, and recoverin, as well as markers of ganglion and amacrine cells, including β-tubulin, Brn3b, and HuD/C. ^98,100 Application of ES cells to RGC replacement therapy requires differentiation methods that generate RGCs capable of achieving adequate synapses between the retina and the visual targets in the brain. It is recognized that RGC degeneration in glaucoma occurs over a long period, for which there is the need to better understand the early mechanisms that induce long-term RGC damage. At present, it is accepted that transplantation of photoreceptor precursors constitutes an easier alternative to treat photoreceptor dystrophies than transplantation aimed at replacing RGCs. 

The extent of retinal regeneration mediated by Müller stem cells in fish and amphibians does not parallel that seen in birds or mice after injury. NMDA-induced damage in birds only gives rise to regeneration of amacrine cells, and this process can be controlled by growth factors. A study ¹⁰¹ in the GAD67-GFP mouse, in which Müller glia are permanently labeled with GFAP-Cre-EYFP to trace their progeny, showed that NMDA injury alone does not induce regeneration unless the eyes are also injected with EGF and FGF1. In the presence of these factors, only amacrine and RGCs are regenerated. 

The discussion addressed practical issues related to the potential for iPS cell transplantation. It is recognized that generation of iPS cells for autologous transplantation is a long-term process, because of the need to undertake appropriate controls to validate their biological properties. Moreover, it is possible that, if RGCs are derived from glaucoma patients, they can be more susceptible to degeneration, as suggested by in vitro studies. Because of these restrictions, it may be more appropriate to create iPS cell banks that could be sourced for HLA compatibility. The possibility that the retina could be denuded of cells and repopulated by stem cell–derived neurons was dismissed because of the small amount of extracellular matrix present in this tissue. The design of cellular scaffolds for transplantation of RGCs is potentially useful, provided that cells can be assembled with the right orientation and lamination and that electrical connectivity of retinal neurons can be achieved. 

Problems associated with the potential implementation of iPS cell therapy include the reported teratogenicity of these cells and the high variability in the iPS cell response to protocols used for photoreceptor differentiation. However, recent studies have found that if differentiated photoreceptor cell populations are highly selected, they do not induce tumor formation. In addition, extensive studies are presently aimed at improving protocols for differentiation of these cells into retinal neurons. Ultimately, the ideal therapy to replace RGCs would be to stimulate Müller glial stem cells to endogenously differentiate into the missing neurons. However, we first need to understand the factors that control the proliferation and differentiation of these cells in high mammalian species, including humans. 

Potential of Müller Stem Cells for RGC Replacement

The potent retinal regenerative ability observed in fish and amphibians has been ascribed to the presence of Müller glia with stem cell characteristics in the retina of these species. ¹⁰² Of interest, the existence of Müller glial stem cells has also been identified in the adult human retina, despite the lack of evidence of retinal regeneration occurring in the human eye. ^103,104 These cells can be easily isolated from cadaveric donor eyes and can be induced to form neurospheres and to differentiate into cells expressing markers of most retinal neurons. They express markers of neural progenitors in vitro, including Sox2, Notch1, Pax 6, Shh, and Chx10. In the presence of FGF2 they acquire neural morphology, and a small proportion of cells express selective markers of all retinal neurons, depending on the culture conditions. Downregulation of Notch using the γ-secretase inhibitor DAPT induces differentiation of Müller glia into Brn3b⁺ cells, a marker of RGCs. Functional studies in these cells in vitro have shown that DAPT treatment induces loss of retinal stem cell markers, accompanied by acquisition of RGC markers and characteristic responsiveness to neurotransmitters. On transplantation of Müller-derived RGC precursors into NMDA-depleted rat retina, a partial restoration of RGC function in rats depleted of RGCs by NMDA has been observed (Limb GA, personal communication, May 2010). This effect could only be induced if cells were transplanted in the presence of microglia inhibitors and matrix-degrading enzymes. ¹⁰⁵  

The consequences of induction of proliferation and differentiation in situ were discussed, and it was suggested that in vivo induction of Müller glial differentiation would cause their depletion with consequent loss of structural support of the retina. However, if the population of Müller glia that differentiate into neurons is a real stem cell population, it should be expected that these cells would not only replace retinal neurons but would also maintain the Müller glia population. It was proposed that experimental models used to test stem cells for RGC replacement should resemble the features of glaucoma or optic nerve transection, since models of RGC depletion also affect other retinal cells and induce inflammation. The need to control microglia to promote transplant cell survival was also discussed, as microglia reactivity is a feature of glaucoma and other retinal degenerations. ¹⁰⁶  

A previous study attempting to replace cells in the cortex with stem cells showed that survival of transplanted cells partially depends on local microenvironment control signals. In this study, targeted neuronal apoptosis rather than necrosis resulted in a better environment for the successful engraftment and differentiation of neural precursors. ¹⁰⁷ It was noted that if there is a need to establish a topographic map of the neural visual network, current cell transplantation approaches are very limited, as the adult visual system in humans does not have the plasticity observed in the fish. It was also suggested that transplantation of RGC progenitors may be more efficient if a bridge such as a PN graft is implanted near the eye to provide a more conducive environment for the development and extension of axonal connections into the brain. 

Optic Nerve Protection by Transplantation of Oligodendrocyte Precursors

The discussion was focused on suggestions that new neuroprotective strategies could be developed as an alternative to stem cell transplantation. It was noted that, to date, published studies aimed at treating glaucomatous neurodegeneration by stem cell grafting have not shown evidence of migration into the RGC layer. Prolonged RGC survival, but not integration of grafted cells into the RGC layer, has been observed after intravitreal transplantation of oligodendrocyte precursors into the vitreous of rats with experimental glaucoma. Although transplanted oligodendrocytes extend long processes on the RGC surface, they induce neuroprotection only when injected in the presence of zymosan. ¹⁰⁸ Similar studies have shown that mesenchymal stem cells (MSCs), which do not undergo neural differentiation in the retina, are also neuroprotective of RGCs, ¹⁰⁹ while systemically injected MSCs do not migrate into the RGC layer. It was proposed that stem cell transplantation to induce neuroprotection could be justified if cells were shown to provide additional benefits, such as modulation of the immune system, induction of neural differentiation, or delivery of additional factors that cannot be delivered efficiently by other routes. 

The discussion addressed the consequences of RGC axon myelination on oligodendrocyte transplantation in the retina. It was proposed that the use of experimental animals that harbor oligodendrocytes in their retinas, such as the rabbit, would provide some information on the effect of myelination in RGC axonal function. However, it was noted that it is very difficult to induce glaucomatous optic nerve damage by experimental IOP elevation in this species. It was speculated that such resistance to glaucoma damage in the rabbit could also be related to the connective tissue properties of the sclera and optic nerve. Because of evidence that severe field defects can occur in some patients with a rare developmental condition characterized by intraocular myelination of RGC axons, it was suggested that these defects may be caused by myelin blocking both light transmission and electrical conduction in RGCs. On this basis, it was suggested that it would be safer not to use oligodendrocytes for direct transplantation into the retina or the optic nerve for glaucoma neuroprotection. However, on the basis of the evidence that oligodendrocytes are neuroprotective, further studies are worthwhile to identify the neurotrophic factors released by these cells, which may aid in the design of neuroprotective therapy for RGCs. It was also suggested that transplantation of oligodendrocytes directly into the brain regions that control vision may potentially constitute a source of neurotrophic factors that could help to re-establish synaptic functions. 

Survival of RGCs Induced by Embryonic Retinal Cells

The discussion addressed the potential of embryonic retinal progenitors to promote RGC survival on transplantation into the vitreous. In a study ¹¹⁰ in a posthatching chick model of RGC depletion by colchicine, transplantation of embryonic retinal cells from embryonic day (E)10 and E11 promoted the survival of RGCs, whereas cells from earlier or later embryonic stages did not have such an effect. Furthermore, the mature chick retina did not support the proliferation of embryonic retinal cells, unlike the environment provided by experimental conditions that promote their long-term proliferation. The longer embryonic retinal progenitors were maintained in culture, the better they survived when transplanted into the chick eye, whereas inflammation appeared to facilitate their integration into the retina. Transplanted cells that migrated into the retina did not express neuronal or glial markers, but cells that remained in the vitreous reaggregated and acquired laminar structure. This study, however, suggests that embryonic retinal progenitors produce factors that support the survival of damaged ganglion cells, and therefore embryonic retinal cells may be used in cell-based survival therapy to treat glaucomatous disease. 

During normal retinal development there is an overproduction of neurons that is corrected by programmed cell death. A balance is established between cues that regulate survival and death so that the appropriate number of neurons remain. In the case of developing ganglion cells, prosurvival factors are derived from target cells in higher visual centers and from within the retina. It was suggested that factors that promote the survival and differentiation of embryonic cells within neurospheres should be identified and their applicability to RGC survival therapies investigated. However, the idea that spheres themselves could be transplanted into the vitreous to release RGC survival factors was dismissed, as it is not known how long these cells may remain viable and actively secrete these factors. Promotion of RGC survival is thought not to be ascribable to a single molecule, such as CNTF, but to a combination of factors not yet identified in embryonic cells. 

Ensheathing of RGC Axons by Grafted Olfactory Ensheathing Cells

The possibility that olfactory ensheathing cells (OECs) obtained from olfactory tissue may be transplanted into the vicinity of the ONH to repopulate the optic pathway and/or enhance RGC function was discussed. Olfactory tissue is unique in the CNS, in that there is continuous removal and regrowth of OECs and their central projections, possibly due to their constant exposure to the environment and perhaps to neurotoxic agents. Axons of olfactory sensory neurons project from the periphery into the CNS and are closely associated with the specialized ensheathing glia, which, during development, guide neural progenitor cells toward the olfactory bulb. After birth, OECs wrap around the axonal processes projecting into the olfactory bulb. Turnover of neurons is high, and the glia maintains permissive channels for newly growing axons to extend into the CNS to find their appropriate targets. ¹¹¹  

Various types of olfactory precursor and stem cells have been identified. Two types of cells, known as horizontal basal and global basal cells, have been well characterized in the rat, and a third type has been recently identified. The latter appears to be an MSC that can differentiate into bone and adipose tissue. It is thought that global basal cells maintain the continual renewal of the OECs, whereas horizontal basal cells constitute the real stem cell population, responsible for the generation of global basal cells. ¹¹¹  

Initial transplantation studies of these cells in experimental models of spinal cord injury promotes regeneration of dorsal root axons, which has led to further exploration of the regenerative ability of these cells in various neurodegenerative conditions. ¹¹² Transplantation studies using the ONC model have produced variable results, ^113,114 and evidence that these cells produce multiple neurotrophic factors, such as BDNF and CNTF, has been presented. ¹¹³  

Transplantation of OECs into the rat retina shows that, although these cells migrate into the nerve fiber layer and the different retinal cell layers, they do not express markers of retinal neurons. When grafted into the subretinal space, OECs remain at the site of the injection and do not migrate into the retina. In vitro studies, however, have shown that OECs cause ensheathment of RGCs without myelination. ¹¹⁴ It was acknowledged that the use of OECs is an innovative approach for development of cell-based therapy to treat glaucoma. It was also suggested that there is the need to identify whether fetal and adult OECs exhibit different characteristics and ability to ensheathe RGCs and induce myelination, as well as to examine whether OEC migration into the retina has any functional effects. 

Potential Cell Therapy Approaches to Target the TM in Glaucoma

An additional limiting factor in developing more effective treatment for glaucoma is the partial understanding of the cellular and molecular mechanisms involved in pathogenic events in the outflow pathway. At present, there is only circumstantial evidence of the presence of stem cells in the adult TM. Recent investigations have identified a cell population, isolated from the TM region of the anterior segment, that grows under nonadherent conditions as free-floating spheres. Addition of serum to the culture medium induced the attachment of spheres to the substrate, followed by migration of cells from the spheres. Cells that migrated from the spheres exhibited the phenotypic features of normal TM cells. Free-floating spheres cultured under nonadherent conditions could be grown for more than 3 months, expressed Nestin, a marker of undifferentiated progenitor cells, and appeared to contain undifferentiated progenitors. ¹¹⁵ The existence of stem cells in the TM has been further suggested by in vivo proliferation studies in the cat eye and ex vivo in explants of human eyes. After laser application to the TM, cells divide and locate along the Schwalbe's line before moving in the direction of the TM. ¹¹⁶ These observations suggested that the TM may contain progenitor cells that could be further investigated for their potential use in cell therapy to target the TM in glaucoma. 

Suggested options for the potential application of these cells included TM cell replacement, induction of proliferation of these cells in situ after identification of factors that promote their growth, and retinal transplantation. However, it was thought that the properties of these cells and the identification of mechanisms that induce their differentiation should be better understood and that in the event of therapeutic application, these should be used for TM therapy, but not for retinal therapy. 

It was suggested that injection of encapsulated TM cells into the anterior chamber or vitreous has some possible applications and that these methods should be explored for therapies that work well in the TM and the retina. However, the proposal to find a single therapy to treat both the TM and the neural retina was considered unrealistic, as these cells have different embryonic origin. Furthermore, the idea of cell-replacement to correct damaged TM was thought to be interesting and feasible due to the easy access to the TM. This could also be applied to the application of small molecules and gene therapy. Two different indications for cell replacement were also considered: to manipulate TM to induce lowering of the IOP and to replace aging damaged cells. To replace damaged cells, it is important to identify how much of the cell function is reduced before the global tissue function is lost. Identification of molecular mechanisms that govern TM functions would therefore have essential implications for glaucoma therapy. 

Concluding Remarks and Proposals for Further Research

Recent scientific developments have identified elements not previously implicated in the pathogenesis of ONH and RGC damage, and they should be considered in formulating new therapies. They include mitochondria, new transcription factors involved in the regulation of axonal function, and the participation of immune mechanisms involving microglia, cytokines, and complement. Further studies on the identification of biomarkers to detect disease progression, the identification of early events at the cellular level, and improvements in imaging techniques should be taken into consideration for future research. It is possible that in the pathogenesis of glaucoma, there is a window of opportunity in which new therapies may be applied to prevent disease progression. To this effect, investigation into gene expression changes in RGCs, other retinal neurons, Müller glia, microglia, and astrocytes may shed light on the early pathologic events and may aid in developing therapeutic targets. New research on the pathologic changes leading to RGC damage has also highlighted the importance of Müller glia as a source of TNFα-mediated events that cause microglia activation and RGC damage. On this basis, the use of genomic and proteomic methods to identify neuroprotective factors released by Müller glia may be important in ensuring RGC survival. 

Intraocular inflammation has been recognized as having a neuroprotective effect on RGCs and axonal regeneration, possibly through activation of macrophages, astrocytes, and Müller glia and their release of neurotrophic factors. Regulation of CNTF and LIF production by astrocytes and Ocm production by macrophages merits further investigation. Elucidation of the complexity of signaling induced by CNTF and BDNF may clarify whether these factors act at cell body or axonal levels and would provide an understanding of the reason that despite upregulation of these factors after optic nerve injury, they fail to protect most of the RGCs. It is recognized that BDNF prolongs survival of RGCs, but does not induce axonal regeneration. Therefore, it would be important to determine whether this response can be modulated. 

Further investigation into the most appropriate routes for application of neurotrophic factors should be considered in light of the present findings that retrograde transport of BDNF from the LGN to the RGCs induces their survival. Promotion of RGC survival was considered to be a more realistic approach to treatment than the long-term goal of RGC regeneration. Since the existing experimental models do not mimic the pathogenesis of human glaucoma accurately, comparison between existing models and live imaging of axonal transport may help us to better understand the pathologic features of human disease and the identification of targets for effective treatments. 

There is also the need to define the principal goals expected from potential gene therapies. Protection of RGC survival and preservation and restoration of function or induction of axonal regeneration may require different approaches. In a similar manner, the elements to be targeted—that is, oxidative stress, antiapoptosis, neurotrophins, or Wallerian degeneration— should be examined for their suitability. Compensatory pathways, development of tachyphylaxis, and the long-term consequences of sustained gene expression should be also investigated. Depending on the pathway targeted for gene therapy, it would be appropriate to examine cellular reactivity elicited by viral vectors, as well as to control gene expression and establish adequate doses for transduction. In this context, it would be essential to assess the risks or benefits of such therapies, for which robust experimental data in a relevant model would be most appropriate. Based on present evidence, it would also be important to consider the effect of bystander cells that are not transduced by the administered vectors. Intraretinal changes of the dendritic trees and in vivo retinal function studies at sites where RGCs are still present should provide more information on the efficacy of gene therapies. It was suggested that the ideal vector for use in neuroprotection should keep cells functionally and anatomically intact, exhibit target specificity, protect both soma and axons, have the ability to be packed in vectors at high titer, and show high efficiency of transduction and stability over time. Ideal vectors should have significant effects on neuromodulation without side effects that could outweigh the benefits. Moreover, they should have a permanent effect on the damaged optic nerve and RGCs, which could be assessed by in vivo imaging of the human eye. 

Development of stem cell therapies to treat glaucoma and regenerate damaged retina and optic nerve at present requires extensive investigation. Progress has been made on the establishment of protocols for differentiating embryonic stem cells and iPS cells into photoreceptors and RGCs, but very little has been achieved so far by transplantation of these cells into animal models of retinal disease. Although it has been thought that iPS cells can provide more efficient autologous and risk-free therapies, in practice they are highly teratogenic and need a very long time to undergo the process of differentiation and validation. Intravitreal transplantations of oligodendrocytes and MSCs have been shown to induce RGC neuroprotection but not regeneration, suggesting that some of these cells may instead constitute a source of neurotrophic factors. In contrast, transplantation of RGC precursors derived from Müller stem cells into an RGC-depletion model has resulted in migration of these cells into the RGC layer, accompanied by a partial recovery of the retinal electrophysiological response. However, it was suggested that validation of the effect of these cells should be performed using experimental models resembling the human disease. Innovative approaches using transplantation of OECs have also been investigated and suggest that these cells may be neuroprotective. Finally, the recent identification of TM stem cells provides an additional tool for potential use in cell-based therapies for the renewal and regeneration of the TM and merits further study. 

In conclusion, better understanding of the cellular and molecular processes that occur in the ONH and retina during the early stages of glaucoma is still required. Recent advances in live imaging, gene therapy, neuroprotection, and stem cell biology have opened other avenues in the investigation of advanced therapies to treat and prevent glaucoma, and they need further exploration. 

Footnotes

 Supported by the ARVO/Pfizer Institute.

Footnotes

 Disclosure: G.A. Limb, None; K.R. Martin, None