Progress in Retinal and Eye Research - ua · Cellular responses following retinal injuries and therapeutic approaches for neurodegenerative diseases Nicolas Cuenca a, b, *, 2, Laura

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Progress in Retinal and Eye Research 43 (2014) 17e75

Contents lists avai

Progress in Retinal and Eye Research

journal homepage: www.elsevier .com/locate/prer

Cellular responses following retinal injuries and therapeuticapproaches for neurodegenerative diseases

Nicol�as Cuenca a, b, *, 2, Laura Fern�andez-S�anchez a, 1, 2, Laura Campello a, 1, 2,Victoria Maneu c, 2, Pedro De la Villa d, 2, Pedro Lax a, 2, Isabel Pinilla e, 2

a Department of Physiology, Genetics and Microbiology, University of Alicante, Alicante, Spainb Multidisciplinary Institute for Environmental Studies “Ramon Margalef”, University of Alicante, Alicante, Spainc Department of Optics, Pharmacology and Anatomy, University of Alicante, Alicante, Spaind Department of Systems Biology, University of Alcal�a, Alcal�a de Henares, Spaine Department of Ophthalmology, Lozano Blesa University Hospital, Aragon Institute of Health Sciences, Zaragoza, Spain

a r t i c l e i n f o

Article history:Received 23 April 2014Received in revised form3 July 2014Accepted 7 July 2014Available online 17 July 2014

Keywords:Retinal remodelingNeurodegenerationGlial cellsRetinal therapyNeuroprotectionRetinal diseases

List of abbreviations: AAV, Adeno-associated viruactivating factor-1; BDNF, Brain-derived neurotrophicderived neurotrophic factor; CNV, Choroidal neovasculFGF, Fibroblast growth factor; GCL, Ganglion cell layehiPSC, Human induced pluripotent stem cells; IL, Interetinal degeneration; mGluR6, Metabotropic glutamatN-acetylcysteine; NMDA, N-methyl-D-aspartate; NO, NOuter plexiform layer; PEDF, Pigment epithelium deReactive oxygen species; RP, Retinitis pigmentosa; RPETUDCA, Tauroursodeoxycholic acid; UPS, Ubiquitin-pr* Corresponding author. Department of Physiolog

965909916; fax: þ34 965903943.E-mail address: [email protected] (N. Cuenca).

1 These authors contributed equally to this work.2 Percentage of work contributed by each author in

15%; Victoria Maneu1: 15%; Pedro De la Villa: 10%; Pe

http://dx.doi.org/10.1016/j.preteyeres.2014.07.0011350-9462/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Retinal neurodegenerative diseases like age-related macular degeneration, glaucoma, diabetic retinop-athy and retinitis pigmentosa each have a different etiology and pathogenesis. However, at the cellularand molecular level, the response to retinal injury is similar in all of them, and results in morphologicaland functional impairment of retinal cells. This retinal degeneration may be triggered by gene defects,increased intraocular pressure, high levels of blood glucose, other types of stress or aging, but they allfrequently induce a set of cell signals that lead to well-established and similar morphological andfunctional changes, including controlled cell death and retinal remodeling. Interestingly, an inflamma-tory response, oxidative stress and activation of apoptotic pathways are common features in all thesediseases. Furthermore, it is important to note the relevant role of glial cells, including astrocytes, Müllercells and microglia, because their response to injury is decisive for maintaining the health of the retina orits degeneration. Several therapeutic approaches have been developed to preserve retinal function orrestore eyesight in pathological conditions. In this context, neuroprotective compounds, gene therapy,cell transplantation or artificial devices should be applied at the appropriate stage of retinal degenerationto obtain successful results. This review provides an overview of the common and distinctive features ofretinal neurodegenerative diseases, including the molecular, anatomical and functional changes causedby the cellular response to damage, in order to establish appropriate treatments for these pathologies.

© 2014 Elsevier Ltd. All rights reserved.

s; AGEs, Advanced glycation end products; AMD, Age-related macular degeneration; Apaf-1, Apoptotic protease-factor; bFGF, Basic fibroblast growth factor; BRB, Blood retinal barrier; CNS, Central nervous system; CNTF, Ciliary-arization; DR, Diabetic retinopathy; EGCG, Epigallocatechin gallate; ERG, Electroretinogram; ESC, Embryonic stem cells;r; GDNF, Glial-derived neurotrophic factor; GFAP, Glial fibrillary acidic protein; hESC, Human embryonic stem cells;rleukin; INL, Inner nuclear layer; IPL, Inner plexiform layer; iPSC, Induced pluripotent stem cells; LIRD, Light-inducede receptor; MOMP, Mitochondrial outer membrane pores; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine; NAC,itric oxide; NF-, B; Nuclear factor, B; Nrf2, Nuclear factor erythroid 2-related factor 2; ONL, Outer nuclear Llayer; OPL,rived factor; PVR, Proliferative vitreoretinopathy; RCS, Royal College Surgeon rats; RGC, Retinal ganglion cells; ROS,, Retinal pigment epithelium; TGF-b, Transforming growth factor-b; TLR, Toll-like receptor; TNF, Tumor necrosis factor;oteasome system; VEGF, Vascular endothelial growth factor; VEPs, Visual evoked potentials.y, Genetics and Microbiology, University of Alicante, San Vicente del Raspeig, E-03080 Alicante, Spain. Tel.: þ34

the production of the manuscript is as follows: Nicol�as Cuenca: 15%; Laura Fern�andez-S�anchez: 15%; Laura Campello:dro Lax: 15%; Isabel Pinilla 15%.

N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e7518

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192. Cellular responses induced by retinal injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2.1. Retinal neurons and circuitries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212.1.1. Photoreceptor morphology changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212.1.2. Bipolar and horizontal cells sprouting and remodeling as a consequence of photoreceptor loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252.1.3. Does OPL connectivity plays a crucial role in vision loss? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272.1.4. Amacrine cell types and retinal degeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272.1.5. From photoreceptor loss to ganglion cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

2.2. Alterations in retinal homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282.2.1. Oxidative stress and retinal degeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282.2.2. Activation of apoptotic pathways: role of the mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282.2.3. Retinal protein homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

2.3. Glial responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332.3.1. Inflammatory response: microglial activation in retinal dystrophies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332.3.2. Macroglial cells: Müller and astrocytes cells in healthy and diseased retinas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

2.4. Degenerative events in retinal vascularization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412.4.1. Retinal vascular networks and the blood retinal barrier in health and disease . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 412.4.2. Retinal degenerative diseases with relevant vascular changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

2.5. Retinal pigment epithelium (RPE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432.5.1. RPE physiology and functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432.5.2. RPE changes in aging and pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

2.6. Functional changes following retinal injuries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452.6.1. Electroretinogram (ERG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452.6.2. Visual evoked potentials (VEPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462.6.3. Psychophysical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

3. Remodeling of the retina in retinal degenerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463.1. Phase 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473.2. Phase 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473.3. Phase 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473.4. Phase 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

4. Therapeutic approaches in neurodegenerative diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494.1. Efficacy of anti-apoptotic therapies for retinal diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

4.1.1. Tauroursodeoxycholic acid (TUDCA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504.1.2. Rasagiline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514.1.3. Norgestrel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514.1.4. Proinsulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

4.2. Efficacy of antioxidant and anti-inflammatory agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514.2.1. Curcumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 524.2.2. Lutein and zeaxanthin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 524.2.3. Saffron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 524.2.4. Catechins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 524.2.5. Ginkgo biloba extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534.2.6. Resveratrol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534.2.7. Quercetin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534.2.8. N-acetylcysteine (NAC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534.2.9. Antioxidant cocktails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

4.3. Efficacy of neurotrophic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544.4. Gene therapy approaches and clinical trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

4.4.1. Viral-mediated therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544.4.2. Optogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

4.5. Cell-based therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564.5.1. Human embryonic stem cells (hESCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584.5.2. Human induced pluripotent stem cells (hiPSC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584.5.3. Human fetal embryonic stem cells; retinal progenitor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594.5.4. Human umbilical tissue-derived stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594.5.5. Human central nervous system stem cells (HuCNS-SC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594.5.6. Bone marrow-derived stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

4.6. Effectiveness of retinal transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604.7. Clinical trials for retinal diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604.8. Suitable therapies in each phase of retinal degeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

5. Conclusion remarks and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Fig. 1. Retinal cytoarchitecture. (A) Vertical section of a monkey retina showing themain retinal layers. Antibodies against alpha-synuclein (red) stained outer segments ofcones and rods, axon terminals in the outer plexiform layer, and a specific populationof bipolar, amacrine and ganglion cells. GABA (blue) labeled amacrine cells and glycine(green) stained bipolar and amacrine cells. Note the variety of bipolar and amacrinecell types, and the complex neuronal circuits at the inner plexiform layer. (B) Highmagnification of the outer retina triple-immnunolabeled with antibodies againstalpha-synuclein (red), arrestin and rhodopsin (Rho) (both in green), showing the entiremorphology of cones (green, elongated cells) from the outer segment to their axonterminals (pedicles), as well as rod outer segments (top green lines) and rod axonterminals (spherules, red dots). These images were awarded for Vision Research (www.vision-research.eu) in 2009. RPE: Retinal pigment epithelium; OS: outer segments; IS:inner segments; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nu-clear layer; IPL: inner plexiform layer; GCL: ganglion cell layer.

N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75 19

1. Introduction

The retina is a light-sensitive tissue lining the inner surface ofthe eye. It is formed by multiple layers of interconnected neuronsand is in charge of the first steps of visual processing (Fig. 1A). Thephotoreceptors (rods and cones) are, together with the melanopsinganglion cells, the photosensitive cells of the retina (Figs. 1B and 2).Photoreceptors are one of the most specialized and complex cells inthe nervous system, and they are located in the outer nuclear layer(ONL) of the vertebrate retina. Rods and cones initiate the conver-sion of light energy into electrical signals through a process calledphototransduction. Retinal interneurons further codify the elec-trical signals into optic nerve impulses, which are subsequentlyinterpreted by the brain as visual images. This process enables therecognition of shapes, sizes, colors and movements. The organiza-tion of the retina and visual system has been described in detail byKolb (Kolb, 2003) and on Webvision (http://webvision.med.utah.edu/). Briefly, after cones and rods absorb the incident light, pho-totransduction takes place in their outer segments, and theresulting electrical impulse is relayed to the bipolar and horizontalcells. At the outer plexiform layer (OPL), the dendrites of rod andcone bipolar cells make synaptic contacts with the axonal termi-nations of the rods (spherules) and cones (pedicles), respectively(Figs. 1B and 2). In the next stage, amacrine cells (located in theinner nuclear layer) and bipolar cells establish a complex networkof synaptic interconnections at the inner plexiform layer (IPL) withganglion cells (Fig. 1A). Lastly, the electrical information isconveyed to the ganglion cells, which send out impulses throughtheir axonal prolongations connecting the retina to the brain via theoptic nerve.

In the context of visual function, it is important to mention thefovea, a central region of the human and primate retina containinga very high concentration of cones responsible for a great visualacuity and color appreciation (Fig. 3). In the center of the fovea islocated the foveola which is approximately 0.35 mm in diameter.Interestingly, the structure of the foveola is different from that ofthe rest of the laminated retina and consists of a unique layercontaining only cone cells. Surrounding the fovea, in the parafovealarea between the photoreceptor and outer plexiform layers, theaxons of the foveal cones are arranged obliquely, constituting theanatomical region called the Henle fiber layer (HFL), which is notpresent in peripheral retina (Fig. 3).

The structural and functional complexity of the retina makesthis tissue vulnerable to alterations from any sort of pathologicalinjury. Glaucoma is a leading cause of blindness and is character-ized by retinal ganglion cell (RGC) degeneration, leading to opticnerve damage. Intraocular pressure is one of the most importantrisk factors. Age-relatedmacular degeneration (AMD) is the leadingcause of severe and irreversible loss of vision in the elderly indeveloped countries. Age is the most significant risk factor, and theinitial symptoms of this disease include a loss of central visualacuity, a subjective impression of the curvature of straight lines ormetamorphopsia, and a gradually enlarging central scotoma. In thisdisease, impairment of retinal pigment epithelial (RPE) cells andphotoreceptors, as well as vascular angiogenesis are the main causeof visual loss. Diabetic retinopathy (DR) refers to a group of eyeproblems that people with diabetes may face. It is caused bychanges in the vascular cells of the retina. In some people, bloodvessels may swell and leak fluid, while in others abnormal newblood vessels grow on the surface of the retina. Retinitis pigmen-tosa (RP) is considered to be a group of inherited diseases causingphotoreceptor degeneration. In most forms of RP, the rods areaffected first, prior to cone damage. Because rods are concentratedin the peripheral retina, people suffering this disease show a pro-gressive diminution of the peripheral visual field, ending in a

tunnel vision. Additionally, visual dysfunctions have beendescribed in human neurodegenerative disorders such as Alz-heimer's and Parkinson's diseases. Patients suffering these pa-thologies show a marked reduction in the retinal nerve fiber layerthickness, alterations in the electroretinogram responses andsensitivity to the visual contrast, as well as prolonged latency invisual evoked potentials. Color perception abnormalities, especiallyin the blue-yellow hue discrimination, have also been describedassociated to these diseases, in addition to aberrations in ‘higher’visual processing capabilities, such as read, object recognition andspatial localization (Bodis-Wollner, 2009; Kirby et al., 2010).

Like in the brain, the loss of pre and/or postsynaptic inputs tothe retinal neurons causes changes in their morphology and

Fig. 2. Photosensitive cells in the retina. Confocal images (A, C) and schematic drawings (B) of a monkey rod (left) and cone cell (right) showing the main parts of photoreceptorcells. Antibodies against recoverin (green) were used to stain both rod and cone cells. Anti-alpha-synuclein antibodies (red) stained outer segments and axon terminals of cones androds. (D) Human melanopsin-positive intrinsically photosensitive retinal ganglion cell with cell body located in the amacrine cell layer and dendrites in strata S1 of the IPL. Arrowsindicate the axon running in the optic nerve fiber layer.


function and, as a consequence, these neurons try to establish newsynaptic contacts. Thus, the retinal changes underlying differentdiseases may modify the transmission of the information betweencells and, as a consequence, the retina can undergo a marked

remodeling. Due to this non-specific disease-remodeling phe-nomenon, some neuroprotective therapies applied in CNS disordersmay be useful in retinal pathologies, even if they do not share thesame etiology. However, it is becoming increasingly clear that

Fig. 3. Morphology of the fovea. Vertical section of a monkey fovea stained with an-tibodies against calbindin (blue), alpha-synuclein (red) and PNA (peanut agglutinin,green). The foveola consists of only a layer of photoreceptors with a high concentrationof cones. RPE: Retinal pigment epithelium; OS: outer segments; IS: inner segments;ONL: outer nuclear layer; HFL: Henle fiber layer; OPL: outer plexiform layer; INL: innernuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer.


treatments for retinal neurodegenerative diseases may require acombination of several types of therapies. A large body of studiesindicates that not only apoptotic, but also autophagic and necroticcellular pathways are involved in photoreceptor cell death, andthus the combined modification of these pathways may be aneffective neuroprotective strategy for retinal diseases associatedwith photoreceptor cell loss. Another therapeutic option is thereplacement of lost cells with new ones that are able to connect tothe still-functional part of the host retina. This approach might becapable of repairing a damaged retina and restoring eyesight. Gene-based therapies may be the most suitable approaches for inheritedretinal diseases.

In this review, we will discuss important aspects regarding theremodeling underlying the retinal degenerative diseases: thealteration events in retinal vascularization, the functional changesof the retina affecting vision, and the cellular responses induced byretinal injuries. We will also focus on the most current preventiveand therapeutic strategies in the treatment of retinal neurode-generative disorders. Ultimately, this large amount of informationwill illustrate how a better understanding of the destructivemechanisms occurring in retinal diseases could potentially enablethe identification and validation of new targets for the neuro-protection of this tissue against neurodegenerative processes, andit will also allow the development of the next generation oftherapies.

2. Cellular responses induced by retinal injury

2.1. Retinal neurons and circuitries

In ocular diseases, retinal tissues may be the target of physical,chemical or biological insults that induce morphological andfunctional responses in the different retinal cells (remodeling).However, remodeling is not widely considered in the treatment ofretinal degeneration. The mammalian retina clearly has a vastrepertoire of cellular responses to injury, and understanding thesemay help us improve current therapies or devise new ones forconditions resulting in blindness. In this sense, many studies showthat animal models of retinal diseases exhibit features of humanretinal degeneration and remodeling that can be extremely usefulin the study of human neurodegenerative retinal diseases.

2.1.1. Photoreceptor morphology changesPhotoreceptors are highly differentiated and specialized neu-

roepithelial cells sensitive to light. Their structure comprisesseveral main parts: the outer and the inner segment, a cell body, anaxon and the axon terminal (Fig. 2AeC). Visual transduction takesplace in the outer segment, which borders the RPE, has a cylindricalshape and is connected to the inner segment by a thin cilium. Theouter segment consist of an ordered stack of membrane disks,formed by infoldings of the surface membrane in the case of conecells, and by disks superimposed like a pile of coins covered by theplasmamembrane in rod cells (Fig. 2). The inner segment is dividedinto two parts: the ellipsoid, containing a large cluster of mito-chondria under the outer segment, and the myoid, which containstypical subcellular organelles, including rough and smooth endo-plasmic reticulum and Golgi apparatus (Fig. 2B). The cell body islocated at the innermost end of this segment. The axon, which doesnot conduct action potentials, ends in a bulb-shaped structurecalled spherule in rods, and a pedicle in cones, and contains a largenumber of synaptic vesicles, loaded with neurotransmitter that arecontinually released into the synaptic cleft in conditions of dark-ness. The most characteristic structure in the axon terminals is thesynaptic ribbon, a protein structure surrounded by synaptic vesi-cles. These specialized synapses are called triads, because theyconsist of a presynaptic ribbon and three postsynaptic processes:two horizontal cell dendrites on the sides and one or two bipolarcell dendrites in the center. In addition to these invaginating syn-apses, bipolar cells make many flat contacts (basal junctions) withthe cone pedicle base (Dowling and Boycott,1966; Kolb et al., 2001).

Proper development and functioning of the retina requires aprecise balance between the processes of proliferation, differenti-ation and programmed cell death. Certain genetic mutations, ageand environmental factors can trigger specific pathways to induceapoptosis in photoreceptors, contributing as a component of manydiseases. The changes responsible for dystrophic and degenerativephotoreceptor diseases, which cause structural and functionaldamage, may occur at any level of the signal transduction cascadeor in any of the morphological components of these differentiatedcells. On the other hand, due to their intense metabolic activity,photoreceptors generate free radicals and other oxidative agentswhose removal is crucial for cellular health. Oxidative stress occurswhen the balance between oxidizing agents and antioxidants isaltered, resulting in dysfunction and cell death caused by theoxidation of proteins, lipids and DNA. The phototransductioncascade, the high level of membrane protein and neurotransmittersynthesis, and all these complex structures are encoded by a largenumber of genes, which explains the great variety of possiblemutations that lead to retinal degeneration.

As has been found in several animal models of retinal diseases,the mechanism of photoreceptor death in human RP appears toinvolve apoptosis, as revealed by TUNEL (Li et al., 1995). During thedegeneration process, certain morphological changes can beobserved before photoreceptor death (Fig. 4). As degenerationproceeds, there is a progressive reduction in the thickness of theONL, which indicates a loss of photoreceptors (Fig. 4CeF). In RP, thecones experience a progressive size reduction as the result of roddeath, losing their normal morphology with a shortening of theinner and outer segments and axon (Fig. 4CeD, F).

In normal, fully differentiated rods, rhodopsin is synthesized inthe rough endoplasmic reticulum, packaged into vesicles in theGolgi apparatus, transported insidemembrane vesicles through theinner segment cytoplasm to the connecting cilium, and insertedinto newly forming membrane discs at the base of the outersegment. To maintain the outer segment length constant, sheddingof the outer segment tips are phagocytized and degraded by theRPE. Intense rhodopsin immunolocalization is seen in the outer

Fig. 4. Photoreceptor cell changes during retinal degeneration. Vertical sections of mouse (A, C, E) and rat (B, D, F) retinas labeled for g-transducin (cone cells; green) recoverin(cones, rods and some bipolar cells; red) and rhodopsin (Rho; rod outer segments, red), showing the structure of photoreceptors in wild-type animals (A, B) and in different modelsof retinal degeneration (CeF). Retinitis pigmentosa models (C, D, F) show drastic changes in morphology of rod and cone photoreceptors, including the shortening of both outer andinner segments. Note mislocalization of rhodopsin in the photoreceptor cell bodies of rd10 mice (C). The DBA/2J mouse (E), a model of intraocular hypertension, also shows al-terations at the ONL and OPL level. SD: Sprague Dawley; OS: outer segments; IS: inner segments; ONL: outer nuclear layer; OPL: outer plexiform layer. Scale bar: 20 mm.


segment discs of rods, and to a certain extent, in the Golgi area andnear the distal ends of the inner segments, while all other parts ofthe cell appear negative for anti-rhodopsin staining. During retinaldegeneration, translocation of rhodopsin down to the cell bodiesand axon terminals is common to all retinal diseases (Fig. 4C).Another sign of rod and cone degeneration at the earliest stages isthe shortening or disorganization of their outer segments, whichcan be visualized by immunocytochemistry (using antibodiesagainst rhodopsin, transducin or cone opsins) (Figs. 4 and 8A). Theouter segments of the cones are greatly shortened and swollen inthe detached retina, and antibodies against cone opsins now labelthe plasma membrane of cone cells extending to the ONL (Fisheret al., 2005). Similar changes with swollen and truncated coneouter segments have been found in organotypic cultures of humanneuroretina (Fernandez-Bueno et al., 2012) and in animal models ofRP (Figs. 4CeD, F and 8A) (Garcia-Ayuso et al., 2013; Martinez-Navarrete et al., 2011; Pinilla et al., 2007).

Changes in photoreceptors and their synaptic connectivity areevident in several human neurodegenerative diseases, as well as inanimal models of neurodegeneration. In RP human retinas, sur-viving rods were reported to have sprouted rhodopsin-positiveneurites that were closely associated with gliotic Müller cell pro-cesses and extended to the inner limiting membrane. However, therods and cones located in the macula did not form neurites, ratherthe axons of peripheral cones were abnormally elongated andbranched (Vugler, 2010). It is interesting to note that rods in RPhuman retinas behave differently than those in RP animal models,as they experience a characteristic growth at their axon terminals.Rod axonal sprouting extends from the OPL down into the innernuclear layer (INL) and ganglion cell layer (CGL) (Fariss et al., 2000;Li et al., 1995; Milam et al., 1998; Sanyal, 1993). This sprouting of rodaxons into the INL has not been described in animal models. Similarsprouting of rod axons into the INL has been found in human dryAMDwith geographic atrophy (Gupta et al., 2003). This sprouting of

Fig. 5. Morphological changes in bipolar cells during retinal degeneration in several animal models of retinal disease. Immunostaining against PKC-a (ON-rod bipolar cells) andbassoon (synaptic ribbons) in retinas from C57BL/6 mice (A), Long Evans (LE) rats (B) and both rat and mouse models of retinal degeneration (CeF) evidence the loss of photo-receptor synaptic ribbons (red) and their synaptic contacts with bipolar cell dendrites (green) during the degenerative process. Few bassoon-immunopositive spots are found at theOPL level in degenerative retinas, as compared to the number of immunoreactive puncta present in the retina of wild-type animals. In diseased retinas, dendritic branches in bipolarcells are scarce or absent. Scale bar: 20 mm.


rod axon terminals into the INL in late degeneration needs to betaken into account for retinal therapeutic approaches, because itmight disable the establishment of correct synaptic contacts.

In human AMD, many rod photoreceptors retract their synapticprocesses into the ONL and lose their synaptic connections with rodbipolar cells (Sullivan et al., 2007). The retraction of rod photore-ceptor synapses is also evident in retinal detachment (Fisher et al.,2005) and RP (Fariss et al., 2000). In DBA/2J mice models of ocularhypertension it has been documented an alteration of rod photo-receptor ribbon structure (Fernandez-Sanchez et al., 2013; Fuchset al., 2012). Similar results were found in transgenic mice over-expressing the guanylate cyclase activating protein 2 (GCAP2) inrods leading to a shortening of synaptic ribbons, and to a higherthan normal percentage of club-shaped and spherical ribbon

morphologies (Lopez-del Hoyo et al., 2012). Mice with chronichypoglycemia by a null mutation in the glucagon receptor geneGcgr also showed a loss of synaptic ribbon in the OPL (Umino et al.,2012). Besides, it has also been demonstrated that null mice in theinsulin-like growth factor-I (Igf1�/�) suffered important structuralmodifications in retinal synapses (Rodriguez-de la Rosa et al., 2012).

In the case of detached retinas, synaptic invaginations of the rodspherules are shallower, and the postsynaptic processes are moreloosely organized than in normal retinas. At the electron micro-scopy level, atypical synapses have been found in an animal modelof retinal detachment (Fisher et al., 2005) and in human retinalorganotypic cultures (Fernandez-Bueno et al., 2012). Furthermore,groups of 3 synaptic ribbons without their corresponding post-synaptic elements were observed in both cases. Abnormal synaptic

Fig. 6. Glutamate receptors changes in retinal degeneration. Retinal degeneration is associated with the loss of connectivity between photoreceptors and bipolar cells in the OPL. (A)Vertical section of a rat retina stained with antibodies against the metabotropic glutamate receptor 6 (mGluR6; green) located on the dendritic tips of rod bipolar cells (stained withPKC-a antibodies, red). (B) Confocal image showing mislocalization of mGluR6 from the dendritic tips of bipolar cells to the cell bodies and axon terminal (arrowheads) and bipolarcell sprouting (C) in a model of retinal degeneration, RCS. (D, E) Double immunostaining with bassoon (red), to stain synaptic ribbons in spherules and pedicles (arrows), andmGluR6, to stain dendritic tips of bipolar cells. The paired bassoon/mGluR6 profiles in the OPL disappear and mGluR6 immunoreactivity is located around bipolar cell bodies in P90RCS rats (E). OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer. Scale bar: A-C, 20 mm; D-E, 10 mm.


ribbons have also been found in several animal models of RP,including Royal College Surgeon (RCS) rats (Cuenca et al., 2013).Moreover, the organization of the outer segment discs, with par-allel membrane alignment under normal conditions, changes to anirregular distribution of disc membranes, a common feature of RPin animal models and human organotypic cultures (Cuenca et al.,2013; Fernandez-Bueno et al., 2012).

Diabetic retinopathy affects retinal vascularization, as well asthe retinal cells themselves, including neural and glial components.Microvascular lesions may occur in the early stages of DR, in bothanimal models and humans (Abu-El-Asrar et al., 2004; Lieth et al.,2000), but there is increasing evidence that retinal degenerationoccurs before any microvascular alteration (Antonetti et al., 2006;Barber, 2003; Villarroel et al., 2010). In rodent models of DR, gan-glion cells have been reported to die by apoptosis, and a decrease inthe thickness of both the INL and ONL has been observed 10 weeksafter the onset of the disease (Barber et al., 1998; Martin et al.,2004). An elevated rate of apoptosis has also been observed inthe ONL and in the RPE (Aizu et al., 2002; Park et al., 2003). In 2003,Park and collaborators described the apoptotic death of photore-ceptors in a streptozotocin model of diabetic rat as early as onemonth after the onset of the disease. They also showed that therewere modifications in postsynaptic cells (degeneration of hori-zontal cell processes) and necrotic features in some amacrine and

horizontal cells. Gastinger and coworkers also described in theretina of streptozotocin diabetic rats the loss of dopaminergic andcholinergic amacrine cells during the early stages of neuro-degeneration (Gastinger et al., 2006). These cellular changes cancontribute to blood-retinal barrier alterations and the developmentof retinal vascular changes, and they can be crucial for detectingcellular neurodegenerative changes prior to the appearance offunctional deficits in these patients. The confirmation of theseevents in humans with DR would allow initiating early treatmentwith neuroprotective drugs prior to the occurrence of vascularchanges, as soon as the first signs are detected (Simon et al., 2012).

Swelling and loss of photoreceptors have been described inchronic human and monkey experimental models of glaucoma,with patchy loss of red/green cones and rods (Nork et al., 2000).Changes in the outer retina were found in patients with glaucomausing optical coherence tomography (OCT), as well as a loss in conedensity along with the expected inner retinal changes (Choi et al.,2011; Fan et al., 2011). There is also evidence demonstrating thatnon-glaucomatous and glaucomatous optic neuropathies areassociated with outer retinal changes following long-term innerretinal pathology (Werner et al., 2011). As an example, the numberof photoreceptors was significantly reduced in a mouse model ofocular hypertension (Cuenca et al., 2010) and in the DBA/2J mousemodel of glaucoma (Fig. 4E) (Fernandez-Sanchez et al., 2011b). All

Fig. 7. Morphological changes in horizontal cells during retinal degeneration in mouse (A, C, E, G) and rat (B, D, F, H) models. Confocal images of retinas showing horizontal cellsimmunostained with antibodies against calbindin (green). Synaptophysin (SYP) were used to label axon terminals of photoreceptors (A, D, E, G), bassoon stained photoreceptorsynaptic ribbons (B, F), and C-terminal binding protein 2 (CtBP2) labeled synaptic ribbons within the OPL (C). In the different animal models of retinal degeneration (CeH),horizontal cells showed retraction of the dendrite tips, a decreased number of terminal tips, and a loss of contact with the photoreceptor axon terminals with respect to wild-typeanimals (A, B). RCS model presents horizontal sprouting into debris zone (H). LE: Long Evans. Scale bar: 10 mm.


these morphological alterations in the outer retina correlate withthe electroretinogram (ERG) changes found in patients with opticnerve atrophy and glaucoma (Vaegan et al., 1995). These resultsshow that remodeling also occurs when cells of the inner layers ofthe retina die.

2.1.2. Bipolar and horizontal cells sprouting and remodeling as aconsequence of photoreceptor loss

Bipolar and horizontal cells are the second-order neurons in theretinal circuitry that connect with photoreceptor spherules andpedicles. The death of photoreceptors determines the response ofbipolar (Figs. 5, 6 and 8) and horizontal cells (Figs. 7 and 8) indifferent ways. Retinal bipolar cell remodeling is a universal featurein retinal degenerative diseases in humans, rodents, rabbits and

cats. All evidence indicates that as the degeneration of rod bipolarcells progresses, they display early retraction and loss of dendrites(Fig. 5CeF) (Barhoum et al., 2008; Cuenca et al., 2004, 2005b;Martinez-Navarrete et al., 2011; Strettoi et al., 2004). After photo-receptor death, bipolar cells initially retract their dendrites(Fig. 5CeF and 8A), but after the loss of their normal input, second-order bipolar cells seek out new functional photoreceptors withwhich to make contact, thus extending their dendrites. Thissprouting of bipolar cell dendrites (Figs. 6C and 8A) into the ONLhas been described in animal models of RP, such as the RCS rat(Cuenca et al., 2005b). In a rat model of hyperoxia, the loss of bi-polar dendrites and their further sprouting into the ONL took placebefore photoreceptor death (Dorfman et al., 2011). In addition, rodbipolar cells in the parafoveal region showed dendrite sprouting in

Fig. 8. Cellular remodeling during retinal degeneration. Schematic representation of the main changes in morphology (A) and connectivity (B) that take place in retinal neuronsduring degenerative processes, regardless of the origin of the damage. (A) Signs of rod and cone degeneration are the reduction in the size of photoreceptors, the shortening of bothouter and inner segments, and the loss of synaptic connections with second-order neurons. Bipolar cells display early retraction and loss of dendrites during retinal degeneration,with further sprouting of ON rod bipolar cell dendrites into the ONL in some degenerative diseases. In advanced degenerative processes, the retraction of dendrites may also occur.The axonal endings of bipolar cells are shortened. Horizontal cells retract their dendrites during retinal degeneration, although the sprouting of dendrites and axon terminals arefrequent, with the formation of ectopic synapses in the ONL. Remodeling of AII amacrine cells involves the loss of lobular appendages in the OFF strata of the IPL in several retinaldegenerative diseases. (B) Summary of the connectivity changes occurring in retinal neurons during the course of the degenerative process at the OPL. Rod bipolar cells make newcontact with the remained cones.


humans affected by AMD, indicating a certain degree of dendriticand synaptic plasticity in this disease (Sullivan et al., 2007). Rodbipolar cell dendrite sprouting has also been demonstrated in anexperimental model of retinal detachment (Fisher et al., 2005).

However, not all animal models of outer retinal degenerationexhibit the sprouting of rod bipolar cells. For example, bipolar celldendritic sprouting has not been detected in the P23H rat model ofRP (Fig. 5F) (Cuenca et al., 2004). The real reason for this differentbehavior in specific animal models remains unknown. The differ-ences may lie in the speed of degeneration at the ONL, or may bedetermined by different gene mutations. In RCS rats (Fig. 6), thepresence of sprouted dendritic terminals in the ONL (Fig. 6BeC),where some photoreceptor cells remain alive, suggests that thesecells may still be capable of sending inputs to postsynaptic cells.

Remodeling of bipolar cells after retinal degeneration may alsoaffect their axon terminals (Fig. 6BeC and 8A). The axonal endingsof rod bipolar cells establish synaptic contacts with AII amacrinecells at the ON strata of the IPL. In rd/rd mice, bipolar cell axonalendings are small in size, have atrophic varicosities and also showsynaptic ribbons with an anomalous round shape that resemblesthe morphology of immature synapses (Strettoi et al., 2002).Similar alterations have been reported in RP rats and other micemodels of RP (Barhoum et al., 2008; Cuenca et al., 2004; Martinez-Navarrete et al., 2011). Signs of synapse number reduction betweenAII amacrine cells and rod bipolar cell axons were also found inmonkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetra-hydro-pyridine (MPTP), a model of Parkinson disease (Cuenca et al.,2005a). Cone bipolar cells also lose their dendrites during the

degeneration process in the rd/rd mouse. Caldendrin immuno-staining of both ON and OFF cone bipolar cells showed the den-drites of these cells forming a continuous thin layer at the ONL(Strettoi et al., 2002). In the same way, two types of recoverinimmunoreactive cone bipolar cells have been reported to lose theirdendrites in the OPL and change their axon morphology in the IPLduring retinal degeneration in P23H and RCS rats (Cuenca et al.,2004, 2005b).

Remodeling of retinal cells along the degenerative process mayalso affect horizontal cells (Figs. 7 and 8). It is well known that thedendrites of the single horizontal cell type in rats, the B-type cell,contact cone terminals, whereas the axon terminal makes contactwith rod spherules (Kolb et al., 2001; Linberg et al., 2001). In RCS ratretinas, beyond a certain stage of retinal degeneration, the hori-zontal cells retract their dendrites, but the somas are not grosslyswollen or shrunken and appear with a normal density (Figs. 7Hand 8A) (Chu et al., 1993; Cuenca et al., 2005b). Similarly, inmutant mice (rd/rd and rd/bcl2) displaying severe retinal abnor-malities, horizontal cell processes are impaired, but the mosaicdistribution resists photoreceptor degeneration (Rossi et al., 2003).During the degenerative process (Fig. 7CeD, F), horizontal cells alsoseek out new contacts at the ONL level, with the sprouting ofdendrites and axon terminals (Fig. 7H) (Cuenca et al., 2005b).Outgrowing horizontal cells and the formation of ectopic synapsesin the ONL have also been described in other RP animal models,such as the CNGA3/CNGB1 double-knockout mouse (Michalakiset al., 2013). Horizontal cells also extend processes down into theIPL (Cuenca et al., 2005b; Jones et al., 2011; Park et al., 2001; Strettoi


and Pignatelli, 2000; Strettoi et al., 2003, 2002). All these resultsindicate that bipolar and horizontal cells postsynaptic to photore-ceptors have the ability to seek out new contacts during degener-ation, but they retract their dendrites if they fail to establish correctsynapses.

2.1.3. Does OPL connectivity plays a crucial role in vision loss?The processing of visual information in the retina is essential

for the central nervous system (CNS) to be able to interpret im-ages. The first level where this process takes place is the OPL. Anychanges in the organization of synaptic contacts at this level maylead to severe loss of vision. In this layer, photoreceptors makesynaptic contacts with bipolar and horizontal cells, releasingglutamate as neurotransmitter. Metabotropic glutamate receptor 6(mGluR6) is located in the dendritic tips of rod and cone ON-bipolar cells (Fig. 6A, D). Labeling for mGluR6 in mouse and ratmodels of RP shows a loss of normal localization of mGluR6 re-ceptors at the OPL (Fig. 6B, E) and clusters of the receptor in theapical parts of bipolar cell bodies, while intense immunoreactivitywas also observed at the INL and in axon terminals (Fig. 6B, ar-rowheads) (Cuenca et al., 2004; Strettoi and Pignatelli, 2000). Vi-sual deafferentation in retinal detachment also leads to analteration of the glutamatergic pathway (de Souza et al., 2012). Itappears that bipolar cell loss of presynaptic inputs from photo-receptors induces mislocalization of mGluR6 receptors. Sinceproper dendritic localization of mGluR6 is essential for synaptictransmission, this issue must be addressed during cell trans-plantation to permit the recovery of bipolar cells.

Bassoon is a presynaptic protein located at synaptic ribbon incone and rod axon terminals. In the OPL, a continuous distributionof punctate staining marks the synaptic ribbons of rod spherules,and when double stained, they can be seen paired with mGluR6granules (Fig. 6D). Bassoon and synaptophysin, two presynapticproteins, are diminished in many retinal diseases (Figs. 5e7), suchas in the model of retinal detachment described in 2005 by Fisherand coworkers (Fisher et al., 2005).

The literature dealing with OPL remodeling in human retinaldegenerative diseases is scarce, although some laboratories havedemonstrated bipolar cell sprouting and synaptic abnormalities inAMD (Sullivan et al., 2007). Similar results have been found inanimal models of AMD (Marc et al., 2008). The same behavior ofbipolar cell dendrites can be observed in older animals: in normalC57BL/6 mice during their second of life, retinal rod bipolar andhorizontal cells undergo sprouting and form ectopic synapses at theONL (Terzibasi et al., 2009). These studies suggest that maintainingviable photoreceptors is crucial to the health and maintenance ofnormal second-order neurons. Indeed, direct experimental evi-dence supporting the hypothesis that ectopic bipolar cell syn-aptogenesis requires functional presynaptic photoreceptors isprovided by the work of Haverkamp's group on the CNGA3�/�

mouse, characterized by the deactivation and loss of cones withintact rod function. In these mice, cone bipolar cells switch toestablish contacts with the remaining rods, a phenomenon thatdoes not occur in double mutant mice (CNGA3/CNGB1), where bothcone and rod function is lacking (Michalakis et al., 2013).

All these studies appear to confirm that, after losing theirnormal input, bipolar cells will seek out new functional photore-ceptors tomake contact with them.When rods are absent andmostphotoreceptors appear to be cones, cone photoreceptors look forconnections with the neurons of the rod pathway (Fig. 8B) (Cuencaet al., 2004; Peng et al., 2000; Strettoi et al., 2004). To date, it hasbeen difficult to determine whether rod bipolar cell dendrites lookfor new contacts with a retracted rod axon terminal in the ONL or ifthe progressive retraction of the rod axon is accompanied bysprouting of rod bipolar dendrites without any apparent purpose.

2.1.4. Amacrine cell types and retinal degenerationAdditional remodeling of amacrine cells has been reported in

several diseases (Fig. 8A). AII amacrine cells are postsynaptic to rodbipolar cells, and are important neurons that drive rod informationto the cone bipolar pathways. AII amacrine cells receive excitatoryinputs from ON-rod bipolar cells in S5 strata of the IPL and transferthe rod signal to the cone pathway by means of conventionalchemical synapses with OFF-cone bipolar cells and gap junction-mediated electrical synapses with ON type cone bipolar cells(Kolb, 2003; Kolb et al., 2002; Linberg et al., 2001). Since theyreceive a major synaptic input from rod bipolar cells, it can be ex-pected that AII amacrine cells show morphological changes duringretinal degeneration. These cells conserve their typical morpho-logical features and appear well preserved at all the ages tested inrd/rd mice (Strettoi et al., 2002). However, in rd10 mice and P23Hrats, AII amacrine cells lose their lobular appendages in the OFFstrata of the IPL as degeneration progresses (Barhoum et al., 2008;Cuenca et al., 2004). These differences could be attributed to thediversity among animal models or to the later occurrence of AII cellchanges in rd/rd mice. In a rat model of oxygen-induced retinop-athy, AII amacrine cells also lose their typical lobular appendages,which reveals significant morphological changes and decreasedcontact with rod ON-bipolar cells. Clear changes in the dendriticmorphology of AII amacrine cells (the main neuronal subtypepostsynaptic to dopaminergic cells in the retina) have also beenreported in a monkey model of Parkinson disease treated withMPTP, where dopaminergic cells are impaired (Cuenca et al.,2005a). In animal models of DR, both dopaminergic and cholin-ergic amacrine cells are lost at early stages of retinal degeneration,and this loss has been associated with visual deficits (Gastingeret al., 2006). Findings in both diseases have been linked to pa-tient abnormalities, such as the thinning of the optic nerve fiberlayer, ERG changes and an increase in the latency of the pupillarylight reflex (Dutsch et al., 2004; Inzelberg et al., 2004; Shinodaet al., 2007). Early aberrant neurite sprouting in the glycinergicand GABAergic amacrine cell populations have been found in aporcine animal model of RP in the early stages of degeneration.Finally, remodeling events in both glycinergic and GABAergicamacrine cells in human geographic atrophy, with aberrantsprouting in both cell signals, have also been described (Jones et al.,2012).

2.1.5. From photoreceptor loss to ganglion cell deathThe survival and maintenance of the normal dendritic

morphology of ganglion cells is essential for transmitting the cor-rect information to the CNS. The structural and functional integrityof RGCs is a prerequisite for any therapeutic strategy for humanretinal diseases.

Significant preservation of RGC structurewas found in rd10miceretinas, with projections to higher visual centers still present inolder animals even after the death of all photoreceptors. Unlike thesecond-order neurons (i.e., bipolar and horizontal cells), RGCsappear to be a considerably stable cell population (Mazzoni et al.,2008). This preservation potentially constitutes a favorable sub-strate for restoring vision in RP patients by means of electronicprostheses or direct expression of photosensitive proteins throughoptogenetics.

Like RCS and P23H rats (Garcia-Ayuso et al., 2013), rd miceexperience a focal loss of RGCs with reduced ganglion cell size andcompromised axonal transport (Grafstein et al., 1972), which couldalso occur in tandem with vascular abnormalities (Wang et al.,2000). The same discrepancies between mouse and rat modelshave been found for melanopsin-expressing intrinsically photo-sensitive RGC loss. Studies show that rd10 (Mazzoni et al., 2008)and rd/rd cl (rodless/coneless) (Semo et al., 2003) mice fail to


exhibit significant abnormalities in these photosensitive RGCs,whereas a significant loss of these cells have been reported to occurin both RCS and P23H rat models (Esquiva et al., 2013; Vugler et al.,2008b). The differences between animal models need to beclarified.

It has been described the presence of neurites in both epiretinaland subretinal membranes in animal models of retinal detachmentand reattachment and in human membranes removed during vit-rectomy (Lewis et al., 2007). Horizontal and ganglion neurites cellswere observed in epiretinal and subretinal membranes from thefeline retinas. However, in human retinas the majority of the neu-rites correspond to RGCs observedwith high frequency in epiretinalmembranes and less common in subretinal membranes (Lewiset al., 2007).

2.2. Alterations in retinal homeostasis

In retinal tissues, both neuronal and glial cells respond to allforms of injury and disease, independently of the etiology of thedamage. Cellular responses to injury represent a cellular attempt toprotect the tissue from damage and/or to preserve tissue function,even though an excessive or inappropriate cell response maycontribute to neurodegeneration. Protective and regenerative re-sponses of retinal cells involve, among others, the stimulation ofthe antioxidant machinery, the activation of the mechanisms ofprogrammed cell death, and the promotion of the inflammatoryresponse.

2.2.1. Oxidative stress and retinal degenerationThe imbalance between the generation and elimination of

reactive oxygen species (ROS) is defined as oxidative stress. Besidesits role in aging, oxidative stress has been associated with variouspathological conditions. Brain tissue is the most sensitive tissue tooxidative stress injuries. The high oxygen-consumption rates in thenervous tissue (around 20% of the oxygen intake), and the fact thatthe brain has a high percentage of polyunsaturated fatty acids makethis organ vulnerable to oxidative damage (Shukla et al., 2011).Thereby, ROS have been postulated as an important contributor tothe damage associated to neurodegenerative diseases, such asParkinson's, Alzheimer's and Huntington's diseases, as well asamyotrophic lateral sclerosis (Dias et al., 2013; Shukla et al., 2011).

The retina is a highly specialized neural tissue, and one of thetissues most susceptible to ROS damage. Photoreceptor cells arecontinuously exposed to varying degrees of light photons and areone of the highest consumers of oxygen in the CNS, mainly due to alarge accumulation of mitochondria in the ellipsoid (Fig. 2AeC)(Fernandez-Sanchez et al., 2011a; Stone et al., 2008). Moreover,photoreceptors are particularly sensitive to high ROS levels andlipid peroxidation due to the large surface area of membranesenriched with polyunsaturated fats (Panfoli et al., 2012; Winkleret al., 1999). The high metabolic rate of photoreceptors, togetherwith recent evidence for the presence of aerobic metabolism in themembranous disks of photoreceptor outer segments (Panfoli et al.,2012), make the retina a perfect target for ROS.

It is widely accepted that oxidative stress plays a central role inretinal degeneration. For example, it has been shown that oxidativestress in the RPE is the output that triggers the development ofAMD (Ardeljan and Chan, 2013). AMD is an age-related degenera-tive disease affecting choroid, RPE and photoreceptor cells (Kevanyand Palczewski, 2010). Environmental or genetic features thatmight increase the oxidative stress in RPE cells can potentiallyprovoke AMD. Smoking, for example, is a habit known to causeoxidative stress (Carnevali et al., 2003) and appears to be one of themost important risk factors in the development of AMD (Khan et al.,2006; Tomany et al., 2004). Some mitochondrial polymorphisms

have also been found to be increased in mitochondrial fractionsisolated from AMD patients, thus indicating their relevance in AMDpathology (Kenney et al., 2013b; Udar et al., 2009). The capability ofthese mitochondria to synthesize ATP, ROS and lactate may affectthe balance between aerobic and anaerobic mitochondrial metab-olisms (Kenney et al., 2013a). In this context, we have observed areduction in the content of the 5A subunit of cytochrome c oxidaseand b subunit of ATP synthase in the retina of parkinsonian mon-keys, two enzymes constituents of mitochondrial complexes IV andV, respectively, that are correlated with a reduced respiratory ca-pacity of mitochondria (Campello et al., 2013a). Although the in-crease in ROS has been shown to trigger AMD, a decrease in theactivation of the nuclear factor erythroid 2-related factor 2 (Nrf2)pathway has been identified as a factor increasing vulnerability tooxidative damage in aging RPE cells (Sachdeva et al., 2014).

ROS may also be important in the pathogenesis of RP andglaucoma. The photoreceptor cells in RP and the ganglion cells inglaucoma are highly sensitive to oxidative stress during the earlystages of cell degeneration. In both cells types, the apoptoticstimuli, which trigger their apoptotic death, are exacerbated byoxidative stress (Chrysostomou et al., 2013; Himori et al., 2013;Oveson et al., 2011; Sanvicens et al., 2004).

In DR, there is an increase in ROS production linked to glucosemetabolism. The high concentration of circulating glucose drivesmitochondria to increase their activity (Du et al., 2003; Kowluruet al., 2001), which results in an overproduction of superoxidefrom mitochondrial complexes I and III (Muller et al., 2004).Mitochondria are not the only source of ROS under hyperglycemicconditions. The excess of glucose also activates the polyolpathway, in which aldose reductase metabolizes glucose intosorbitol, thus increasing oxidative stress by depletion of NADPH(Ola et al., 2012). In addition, hyperglycemia increases the for-mation of advanced glycation end products (AGEs), which uponbinding to their receptor trigger ROS generation (Santos et al.,2011). The oxidative stress in DR is not only due to the excessiveproduction of ROS; it is also mediated by impairment of Nrf2signaling (Xu et al., 2014; Zhong et al., 2013). Diabetes increasesthe binding of Nrf2 to the cytosolic Kelch-like ECH-associatedprotein 1 (Keap 1), preventing Nrf2 translocation to the nucleus,where it regulates the expression of antioxidant genes. This wasshown to occur in streptozotocin-treated rats, in isolated retinalendothelial cells exposed to high levels of glucose, and in retinasfrom human donors with DR (Zhong et al., 2013). Nrf2 knockoutmice have decreased expression of antioxidant enzymes and aremore susceptible to streptozotocin diabetic treatment (Xu et al.,2014). Moreover, the expression of antioxidant enzymes such asMn-containing superoxide dismutase, glutathione peroxidase andcatalase are decreased in diabetic patients with retinopathy, ascompared to diabetic patients without retinopathy or non-diabeticsubjects (El-Bab et al., 2013).

2.2.2. Activation of apoptotic pathways: role of the mitochondriaMost defective, unwanted and potentially dangerous cells die by

apoptosis, an exquisitely controlled genetic program for removingsuch cells without damaging the surrounding tissue (for a review,see (Murakami et al., 2013)). The life-or-death decision seems to bethe result of a complex balance between pro- and anti-apoptoticsignals (Fig. 9) at several levels: extracellular, mitochondrial, nu-clear and cytoplasmic (Kuan et al., 2000; Strasser et al., 2000). Thereare two modes of apoptosis, which have been shown to be medi-ated by caspase-dependent and -independent pathways (Doonanet al., 2005; Kroemer and Martin, 2005). Nonapoptotic forms ofprogrammed cell death (PCD) include those with features ofautophagy, and they can be activated simultaneously to apoptosisduring a neurodegenerative disease (Boya and Kroemer, 2008).


2.2.2.1. Caspase-dependent apoptosis. All pathways of apoptosisconverge upon the activation of cysteineeaspartic acid proteasescalled caspases. These proteins have been functionally classifiedinto two groups, initiator caspases (caspases 2, 8, 9 and 10) andexecutor caspases (caspases 3, 6 and 7) (Pop and Salvesen, 2009).Caspases are present in the cell in their inactive form, and areactivated in the presence of apoptotic stimuli. Two main pathwaysleading to caspase activation have been characterized: the extrinsicroute initiated by cell surface receptors, and the intrinsic path thatis regulated by mitochondria (Fig. 9).

In the extrinsic caspase-dependent pathway, activation ofmembrane “death receptors” (usually by cytokines from the tumornecrosis factor (TNF) family) drives the apoptotic cascade by acti-vation of caspases 8 and/or 10, which then activates downstreameffector caspases, such as caspase 3, 6, and 7 (Tait and Green, 2010).Additionally, external activation of caspase 8 leads to the genera-tion of further internal signals that translocate to themitochondrialmembrane (Fig. 9) to trigger the cell death process (Doonan et al.,2007).

In contrast, an intrinsic pathway for apoptosis may be activatedby various cellular stress stimuli. Along this pathway, the mito-chondria appear to be the primary target. Mitochondrial outer

Fig. 9. Apoptotic pathways in the retina. Schematic representation of the most important pathe result of extrinsic and/or intrinsic caspase-dependent pathways, although non-apoppathways involve calpains and/or cathepsins. Among the major causes of stress and cell deapathological conditions and damage to both mitochondria and lysosomes. MOMP: MitochonApaf-1: apoptotic protease-activating factor-1; EndoG: endonuclease G; AIF: apoptosis-indulymphoma 2; Bcl-xL: B-cell lymphoma-extra large; BAX: Bcl-2-associated X protein; BAK:terminal kinases.

membrane permeabilization (see below) leads to the release ofcytochrome c, which binds apoptotic protease-activating factor-1(Apaf-1), thus inducing its conformational change and oligomeri-zation (Fig. 9). This complex cytochrome c-Apaf-1, referred to as“apoptosome”, recruits, dimerizes and activates the initiator cas-pase 9, which cleaves and activates caspases 3 and 7 (Tait andGreen, 2010).

Most of the apoptotic pathways converge at the permeabiliza-tion of the mitochondrial outer membrane, a step known as thepoint of no return for cell death (Keeble and Gilmore, 2007).Mitochondria play an important role in apoptosis, due to theircontent rich in pro-apoptotic proteins (Fig. 9). These proteins servea dual function; on the one hand, they play a part on the electrontransport chain. This is the case of cytochrome c, which transportselectrons to complex IV (reviewed in Garrido et al. (2006)), or theapoptosis-inducing factor (AIF), which stabilizes and eliminatesROS production from complex I of the electron chain (reviewed inPolster (2013)). However, their release into the cytoplasmic spacehas fatal consequences, as they activate different proteases, whicheventually results in apoptosome formation, or translocate to thenucleus, directly cleaving the DNA. In this sense, preserving theintegrity of the mitochondrial membrane by preventing the

thways involved in programmed cell death (PCD) in the retina. Most retinal cells die astotic forms of regulated cell death are also present. Caspase-independent apoptoticth in the retina are the accumulation of reactive oxygen species (ROS) associated withdrial outer membrane permeabilization; ER: endoplasmic reticulum; DL: Death ligand;cing factor; ROS: reactive oxygen species; PCD: programmed cell death; Bcl-2: B-cellBcl-2 antagonist or killer; Bid: BCL-2 interacting domain death agonist; JNK: c-Jun N-


formation of mitochondrial outer membrane pores (MOMP) is oneof the anti-apoptotic mechanisms that protect cells from death(Garrido et al., 2006).

The Bcl-2 (B-cell lymphoma 2) family is the best characterizedprotein family involved in the regulation of MOMP (Fig. 9). The clanincludes four other anti-apoptotic proteins (Bcl-xL, Bcl-w, A1 andMcl1), and two groups of proteins that promote cell death: theeffector molecules BAX (Bcl-2-associated X protein) and BAK (Bcl-2antagonist or killer), which permeabilize the outer mitochondrialmembrane; and the BH3-only family, which functions indistinctlyalong the cellular stress pathways. BAX and BAK promote MOMP,while Bcl-2 and Bcl-XL expression within the outer mitochondrialmembrane protects against MOMP formation (Keeble and Gilmore,2007). BH3-only proteins, such as Bim (Bcl-2 interacting mediatorof cell death), Bid (BCL-2 interacting domain death agonist) andPuma (p53-upregulated modulator of apoptosis), are unable totrigger apoptosis by themselves, but it is thought that they act asswitch regulators of the pro- and anti-apoptotic Bcl-2 members,tilting the balance towards life or death (Keeble and Gilmore, 2007).In addition, the BH3-only members can act as a link between othertypes of programmed cell death PCD; for example, Bid has beendescribed as one of the main connections between intrinsic andextrinsic apoptotic pathways (Kroemer and Martin, 2005) and canincrease BAX/BAK-induced MOMP formation.

Caspase-dependent pathways are the main mechanismsinvolved in apoptosis in cases of retinal cell degeneration. Inglaucoma, it has been shown that ganglion cell death occurs pri-marily through the apoptotic intrinsic pathways, and is dependenton the release of cytochrome c from mitochondria and the forma-tion of the apoptosome complex (Nickells, 2012), although caspase6 and 8 activity has also been described following the severing ofthe optic nerve (Monnier et al., 2011). Degeneration of the ganglioncell soma in DBA/2J and optic nerve crush models is mainlymediated by BAX, whereas BAX does not seem to be involved in thecase of N-methyl-D-aspartate (NMDA)-induced toxicity (Libby et al.,2005). In addition, it has been shown that glial cells become acti-vated and release cytokines after an initial phase of ganglion celldeath. TNF-a activates secondary degenerative events mediated bythe extrinsic pathway in ganglion cells (Lebrun-Julien et al., 2009;Nickells, 2012; Tezel et al., 2001). Both phases of ganglion celldegeneration finally result in caspase 3 activation and show down-regulation of the anti-apoptotic proteins Bcl-2 and Bcl-XL and up-regulation of BAX and BAD (Levkovitch-Verbin et al., 2010).

The Bcl-2 family plays an important role in the progression ofapoptosis in photoreceptor cells. In experimental models of retinaldegeneration, it has been shown that photoreceptor death is mainlymediated by changes in the balance between BAX and Bcl-XL (Joneset al., 2003; Zheng et al., 2006). The Rpe65-deficient mouse, anexperimental model of Leber's congenital amaurosis, shows up-regulated BAX and decreased Bcl-2 proteins, with decreased Bcl-2/BAX ratio during the progression of the disease (Cottet andSchorderet, 2008; Hamann et al., 2009). In RP, the high diversityof gene mutations leads to the activation of a variety of apoptoticpathways (Doonan et al., 2005; Sancho-Pelluz et al., 2008). In mostcases of RP, as well as in other retinal dystrophies, cell death occursafter endoplasmic reticulum stress (Lin and Lavail, 2010). In thissense, cells have evolved a set of intracellular signaling pathways,cumulatively called unfolded protein response (UPR), that detectprotein misfolding within the endoplasmic reticulum (ER) anddirect protective and pro-apoptotic responses. It has beendemonstrated that Puma, a BH3-only member of the Bcl-2 family, istranscriptionally activated and is essential for ER-stress-inducedneuronal death (Galehdar et al., 2010). In mouse models of RP, ERstress triggers an increase in Ca2þ levels, and up-regulation ofcaspase 12 (Yang et al., 2007), which in turn, activates caspase 3.

Furthermore, a decrease in the Bcl-xL/BAX ratio has been evidencedin these animal models, thus indicating the implication of mito-chondria in the process of apoptosis (Kunte et al., 2012; Sizova et al.,2014). Caspase 12 has a leading role in ER-stress-induced neuronaldeath, but accumulation of misfolded proteins and increasedcytosolic Ca2þ involves the activation of additional apoptotic factorsthat reinforce each other during the apoptotic process, confirmingthat mitochondria and ER can influence each other in the apoptoticevent (Sanges and Marigo, 2006).

Mutations or insults affecting the RPE or its phagocytic functionlead to photoreceptor cell death. In RCS rats, disabled photore-ceptor outer segment phagocytosis drives the apoptotic photore-ceptor cell death (Tso et al., 1994). Increased expression of c-Jun andBAX proteins has been reported during the course of this process(Katai et al., 2006), which points to mitochondrial involvement andthe activation of caspases (Perche et al., 2008), although it seemsthat the apoptotic process is not mediated by Bcl-2 (Katai et al.,2006; Sharma, 2001).

In the case of retinal detachment, photoreceptor degenerationseems to be a process mediated by TNF-a (Nakazawa et al., 2011)through the activation of apoptotic FAS (a death domain-containingmember of the TNF receptor) signaling and the downstreamcascade of caspases 3, 7, 8 and 9 (Besirli et al., 2012; Lo et al., 2011;Zacks et al., 2004). Activation of Bid has also been described duringthis process, with the consequent involvement of the intrinsiccaspase-dependent apoptotic pathways (Zacks et al., 2004).

There is less evidence regarding death mechanisms in AMD, butit has been suggested that photoreceptor death is also caused byapoptosis (Osborne andWood, 2006; Wang et al., 2011c). The samedeath mechanisms have been proposed for DR (Cho et al., 2000;Park et al., 2003).

2.2.2.2. Caspase-independent apoptosis. There is currently evidencethat caspase activation is not the only protease mechanisminvolved in retinal cell apoptosis (Fig. 9) (Chahory et al., 2010;Doonan et al., 2005; Lo et al., 2011; McKernan et al., 2007;Mizukoshi et al., 2010; Nickells, 2012). Since specific inhibition ofcaspase-dependent processes does not prevent neuronal cell death,other proteases must be involved in carrying out the apoptoticprogram (Nguyen et al., 2012). Furthermore, in addition toapoptosis, programmed cell death can be activated by autophagy(Boya and Kroemer, 2008; Kunchithapautham and Rohrer, 2007).

It has been shown that, besides the activation of caspase 12, ERstress and the subsequent Ca2þ release can activate calcium-dependent cysteine proteases known as calpains (Fig. 9) (Nguyenet al., 2012; Suzuki et al., 2004). Calpains are present in the cyto-solic portion of the cell, and caspase 12 (Tan et al., 2006) and otherpro-apoptotic proteins (Nguyen et al., 2012) may amplify the deathsignal. Activation of calpains have been related to various retinaldiseases, and is considered one of the most important caspase-independent apoptotic pathways in photoreceptor cell deathassociated with RP (Doonan et al., 2005; Ozaki et al., 2012; Paquet-Durand et al., 2006; Sanvicens et al., 2004), and diseases involvingischemic conditions, such as DR (Nakajima et al., 2011) and glau-coma (McKernan et al., 2007).

On the other hand, ROS accumulation can induce both MOMP(Garrido et al., 2006) and lysosomal membrane permeabilization(Boya and Kroemer, 2008), releasing cytochrome c and other pro-apoptotic proteins with a clear role in apoptotic events (Fig. 9)(Boya and Kroemer, 2008; Garrido et al., 2006). MOMP can driveapoptosis even when caspases are inhibited (Kroemer and Martin,2005). The main factors involved in these processes are AIF andendonuclease G (EndoG). Both are present within the mitochon-drial intermembrane space under normal conditions, but withapoptotic stimuli and MOMP, AIF and EndoG are able to translocate


into the cell nucleus and to fragment nuclear DNA (Li et al., 2001;Polster, 2013). In this sense, nuclear translocation of AIF andEndoG in various retinal degenerations has already been shown tooccur (Hisatomi et al., 2001; Leal et al., 2009; Mizukoshi et al., 2010;Munemasa et al., 2010; Sizova et al., 2014; Zanna et al., 2005).Alternative mechanisms of cell death such as autophagy arereceiving increasing attention among the mechanisms involved inretinal degeneration (Chinskey et al., 2014; Kunchithapautham andRohrer, 2007; Murakami et al., 2013). Complete disruption of ly-sosomes provokes uncontrolled cell death by necrosis, but partialand selective lysosomal membrane permeabilization induces thecontrolled dismantling of the cell by apoptosis. The proteins mainlyresponsible for autophagy are the cathepsins, which are also themain proteases in lysosomes (Boya and Kroemer, 2008; Uchiyama,2001). ROS accumulation may also induce lysosomal membranepermeabilization and the release of cathepsins (Boya and Kroemer,2008; Metrailler et al., 2012; Sanvicens and Cotter, 2006). In addi-tion, ROS may induce the permeabilization of lysosomes only insubcellular regions near mitochondria, the major ROS-generatingorganelles. Cathepsin activation has been described in severalretinal diseases (Chahory et al., 2010; Sancho-Pelluz et al., 2008).

Although necrosis was traditionally thought to be an uncon-trolled process of cell death, it is now known to also have regulatedcomponents in certain instances (Murakami et al., 2013). Thisregulated type of necrosis, termed as “necroptosis” or “pro-grammed necrosis”, has been demonstrated in several models ofretinal disease, including retinal detachment, retinal ischemia-reperfusion injury and achromatopsia (Dong and Sun, 2011;Rosenbaum et al., 2010; Viringipurampeer et al., 2014).

2.2.3. Retinal protein homeostasisThe retina is a highly specialized and well-structured neural

tissue. For this reason, maintaining homeostasis in all the differentretinal cell types is necessary for proper vision. Furthermore, theretina has to withstand a variety of environmental insults, such aslight-induced injury, and the stress derived from oxidative damageand inherited mutations. All of the previous factors can lead toprotein misfolding cytotoxicity, among other pathologies, and cellshave developed several mechanisms to cope with this. Thesemechanisms are responsible for maintaining protein homeostasis,and include the heat shock response (HSR), the ubiquitin-proteasome system (UPS), the unfolded protein response (UPR)and the ER-associated degradation (ERAD) (Fig. 10) (Athanasiouet al., 2013). Cellular chaperones play an important role in detect-ing misfolded proteins for trying to refold them, but under certaincircumstances, correct refolding is not possible, and the cell mustremove the proteins to avoid protein aggregation or cellulartoxicity. The decreased levels of molecular chaperones are relatedto several neurodegenerative diseases affecting the retina, as occurswith the molecular chaperones HSC70 and GRP78 in the retina ofparkinsonian MPTP-treated monkeys (Campello et al., 2013a). Inother cases, misfolded proteins are removed from cells withoutchaperone supervision. The degradation of proteins, which in-cludes not only misfolded proteins, but also oxidized or denaturedproteins, can be carried out by several proteolytic systems,including lysosomal degradation, chaperone-mediated autophagyand substrate specific degradation by the UPS (Fig. 10). The latter isthe principal molecular machinery of the cell responsible for pro-tein turnover, and it plays a pivotal role in cellular homeostasis. Inthis regard, the UPS exerts a cellular protein quality control and, as aconsequence, it is involved in a wide variety of biological processeswhere modulation of protein levels is crucial. Proteins arecommonly tagged with a tail of covalently-joined ubiquitin mole-cules in a reaction catalyzed by ubiquitin ligases, and are eventuallydegraded by the proteasome. Moreover, the ubiquitination of a

protein, in addition to targeting it for proteasomal degradation, canalso affect its stability, activity, ability to interact with other mol-ecules within the cell and/or its intracellular distribution. Examplesof all these effects are known to exist for retinal proteins. Accord-ingly, the UPS allows the cell tomodulate its protein expression anddistribution pattern in response to different physiological condi-tions, and thus it plays a fundamental protective role in retinalhealth and disease (Campello et al., 2013b; Shang and Taylor, 2012).The functions of the UPS in the retina include roles in differentia-tion and retinal development, modulation of the visual cycle, re-sponses to a variety of stresses (oxidative and nitrosative stress),protection from injury and/or damage repair, among others.

Given the numerous substrate proteins targeted to the pro-teasome and the multitude of processes involved, dysfunction ofthe UPS in the retina is involved in the pathogenesis of manyinherited and acquired visual pathologies. In this context, muta-tions in genes encoding retinal UPS components may directlytrigger a general pathological accumulation of a vast majority ofnoxious proteins. On the other hand, in retinal diseases caused bythe expression of mutant proteins that are not directly related tothe UPS, intracellular accumulation of aberrant mutant proteinvariants lacking an adequate structural conformation may over-load the UPS, indirectly contributing to the disease process. Inautosomal dominant RP patients, for example, mutations havebeen described in the genes coding for the TOPORS E3 and CUL3-based (KLHL7) E3 ubiquitin ligases. It has also been demonstratedthat the P23H mutation of the RHO gene found in autosomaldominant RP patients generates a misfolded variant of rhodopsinthat is not efficiently degraded by the UPS. As a consequence, theUPS machinery becomes overwhelmed and aggresomes ofmutated and recruited normal protein are formed. The UPS alsomodulates pathways associated with oxidative stress and inflam-mation, two pathogenic events closely related to AMD. Upon ag-ing, oxidative stress leads to the malfunction of the proteasomemachinery, and the resulting accumulation of highly-ubiquitinatedproteins activates the heat shock factor 1 (HSF1), a transcriptionfactor associated with the heat shock protein response. Theoxidative inactivation of the proteasome also serves as a link be-tween oxidative stress and the upregulation of inflammation inRPE cells. This inflammation downregulates rhodopsin expressionlevels via UPS-mediated degradation promoted by the STAT3-dependent E3 ubiquitin ligase UBR1. In addition, in RPE cells, theUPS plays an important role in modulating the activities of thehypoxia inducible factor (HIF) and the nuclear factor kB (NFekB),an important transcription factor that mediates hypoxic and in-flammatory responses. The UPS is also involved in DR, a conditionin which angiotensin II decreases in an UPS-mediated fashion thelevels of synaptophysin, a protein essential for vision, as it is amain constituent of synaptic vesicles in the two plexiform layersof the retina. Furthermore, hyperglycemia-induced oxidativestress decreases the glucose transport activity of retinal endo-thelial cells, due to increased internalization of the glucosetransporter 1 (GLUT1) in a proteasome-dependent mechanism.The UPS also modulates the HIF-mediated signaling cascade thatregulates the expression of angiogenic growth factors, and isinvolved in the breakdown of the blood-retinal barrier character-istic of DR through the turnover regulation of endothelial con-nexins. Finally, the proteasome machinery also contributes to theprogression of glaucoma, a condition in which prolonged ischemicretinal injury promotes the ubiquitination of a set of anti-apoptotic proteins, the degradation by the proteasome of whichtriggers cell death.

The contents of this section have been analyzed in detail in thefollowing three reviews (Athanasiou et al., 2013; Campello et al.,2013b; Shang and Taylor, 2012).

Fig. 10. Retinal protein homeostasis networks. Schematic showing the proteostasis mechanisms in retinal cells related to misfolded proteins as the result of mutations, envi-ronmental insults and/or several types of stress. ERAD: Misfolded proteins are detected by the endoplasmic reticulum quality control machinery and shuttled by the retro-translocon channel to the cytoplasm, where they are ubiquitinated before being degraded by the proteasome. UPR: Misfolded proteins in the endoplasmic reticulum are recog-nized by three sensors: IRE1a, PERK and ATF6, which inhibit protein synthesis and stimulate the production of chaperones and the ERAD machinery. HSR: Molecular chaperonesHsp70, Hsp40 and Hsp90 form a complex in the cytosol with the transcription factor HSF1. Upon binding misfolded proteins, Hsp70, Hsp40 and Hsp90 dissociate from HSF1, whichcan trimerize and become activated via phosphorylation. This results in traffic to the nucleus leading to increased chaperone expression. Autophagy: Misfolded proteins can bedegraded by three modes of autophagy: macroautophagy, microautophagy or chaperone-mediated autophagy (CMA).



2.3. Glial responses

2.3.1. Inflammatory response: microglial activation in retinaldystrophies

As part of the CNS, the human retina has a population of residentphagocytes called microglia (Fig. 11) that have an immunologicalcapacity comparable to that of the monocytes and macrophages inother tissues (Graeber and Streit, 1990). The main function ofmicroglial cells in the retina is that of immune surveillance. Theytake part in immune-mediated defense mechanisms (Fig. 14),acting as phagocytes, clearing damaged cell debris from the innerretinal layers and forming a network of potential immune effectorcells throughout the CNS, alongwith the perivascular cells (Hanischand Kettenmann, 2007; Kreutzberg, 1996; Raivich et al., 1999; Shinet al., 2000). However, the role of microglia in the retinal patho-physiology goes far beyond their relevant function in infectiousinjuries per se. Although many aspects have yet to be understood,nowadays it is generally accepted that microglia are important formaintaining photoreceptor survival in retinal dystrophies.

2.3.1.1. Microglia in healthy retinas. In a healthy CNS, microglialcells are in an apparent state of rest, but they continuously scantheir environment, moving extensively and monitoring the sur-rounding area to clear away metabolic products and tissue debris.This non-activated state is characterized by a highly arborescentmorphology, plasticity and a pluristratified distribution in the INLand OPL (Fig.11A). In this state, microglial cells express low levels ofco-stimulatory molecules and demonstrate relatively low levels ofphagocytic activity (Dick et al., 2003; Hume et al., 1983; Langmann,2007; Streit et al., 1999). Microglial cells make specific and directcontact with neuronal synapses and respond to their functionalstatus (Nimmerjahn et al., 2005; Wake et al., 2009). The apparentquiescent state of the microglia in an uninjured retina is main-tained by intercellular contacts and soluble factors secreted byneurons, the RPE and astrocytes (Langmann, 2007).

Microglial cells play a key role in the survival of neurons (Fig.14).They secrete protective factors, such as anti-inflammatory cyto-kines, antioxidants and growth factors, including brain-derivedneurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF),glial cell line-derived neurotrophic factor (GDNF), nerve growthfactor (NGF), neurotrophin-3 (NT3) and basic fibroblast growthfactor (bFGF) (Langmann, 2007). Given the fact that photoreceptors

Fig. 11. Microglial activation in retinal diseases. Vertical sections of a Sprague Dawley (SD) (microglia, and MHC class-II RT1B (red), used to detect activated microglia. Nuclei stained wactivated microglia in the P23H rat retina (B). RPE: Retinal pigment epithelium; ONL: outerlayer.Scale bar: 20 mm.

lack the receptors for some of these neurotrophic factors, thebeneficial action stems from the modulation of growth factorsproduced and released by Müller cells, which contain receptors formost of the molecules involved in photoreceptor rescue and whichare stimulated after retinal insults, such as mechanical injury andlight-induced degeneration. In this sense, NGF, BDNF and CNTFmodulate bFGF and GDNF production and release from Müller glia,and also contribute to the protection of photoreceptors or increasephotoreceptor apoptosis (reviewed in Harada et al. (2002); Harry(2013); Karlstetter et al. (2010a)).

Apart from the capacity to secrete anti-inflammatory moleculeswhen removing apoptotic cells or myelin debris, microglia can alsosecrete inflammatory mediators in the event they are challenged bya microorganism invasion (Hanisch and Kettenmann, 2007). Theeffect of these inflammatory mediators in retinal pathophysiologyis related to retinal dystrophies and is discussed in the followingsections.

2.3.1.2. Regulation of microglial homeostasis. Microglia communi-cate with other glial cells and neurons, which regulate its activationstatus and their capacity for clearing away cellular debris byphagocytosis (Dick et al., 2003). In a healthy CNS, a bidirectionalmicroglia-neuron communication takes place: microglial activity ismodulated by neuronal signals and, reciprocally, microglia signalsare also sent to neurons. Microglia perceive their environmentthrough a great variety of surface receptors, as they express re-ceptors for cytokines, chemokines, neurotransmitters, neurohor-mones and neuromodulators, as well as several ion channels(Harry, 2013). Signalingmechanisms include direct physical contactbetween microglial processes and neuronal elements, as well asmicroglial regulation of neuronal synapses and circuits by severalsoluble factors (Eyo and Wu, 2013; Garden and Moller, 2006;Langmann, 2007; Polazzi and Monti, 2010; Ransohoff andCardona, 2010).

Under healthy conditions, retinal and brain microglia arecontrolled by several inhibitory molecules, such as chemokineCX3CL1, lectin CD22 and other membrane proteins, includingCD200, CD47 and neural cell adhesion molecules, that restrainmicroglial activation (reviewed in Chavarria and Cardenas (2013);Perry and Teeling (2013); Ransohoff and Cardona (2010); Xu et al.(2009)). Among these, one of the main regulators of microglialhomeostasis is the chemokine fractalkine. It is secreted by healthy

A) and a P23H (B) rat retina labeled with antibodies against Iba-1 (green), a marker ofith TO-PRO. Note the increase in the number of microglial cells and amoeboid shapenuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; GCL: ganglion cell

Fig. 12. Müller cells in healthy and diseased retina. Confocal images of whole-mount (A, B, C) and vertical sections (D, E) of retinas from normal Sprague Dawley (SD) (D) and P23Hrats (A, B, C, E) stained with antibodies against GFAP (glial fibrillary acidic protein). High level of GFAP expression were found in Müller cells in response to retinal damage in P23Hrat retinas (E), a protein expressed abundantly in normal astrocytes (D). In ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer;GCL: ganglion cell layer. Scale bar: (A) 1 mm; (B) 200 mm; (C) 40 mm; (D, E) 20 mm.


neurons and binds to the CX3CR1 receptor. In the absence of injury,fractalkine prevents excessive activation of the microglia, but in thepresence of inflammation, it promotes the activation of bothmicroglia and astrocytes, thus making the microglia both neuro-protective and neurotoxic at the same time (Sheridan and Murphy,2013). The CX3CR1 deficiency dysregulates microglial responses,which results in neurotoxicity and degeneration (Cardona et al.,2006; Langmann, 2007). RPE also influences the microglia restingstate through the secretion of cytokines, such as transforminggrowth factor-b (TGF-b), that predispose microglia to the prefer-ential production of interleukin (IL)-10. This in turn down-regulatesthe expression of molecules such as major histocompatibilitycomplex class II (MHCII), CD80 or CD86 and blocks inflammatorygene expression, contributing to the normal retinal immuneregulation (Langmann, 2007; Paglinawan et al., 2003).

2.3.1.3. Retinal microglia after an injurious stimulus. Microglial cellsare rapidly alerted by a variety of injurious signal inputs, triggeredby either genetic or environmental factors, as the result of externaldamage from ocular infections or due to cellular malfunction in theneural retina or RPE. Furthermore, retinal microglia can be alsoactivated by systemic infections, either of viral (Zinkernagel et al.,2013) or fungal origin (Maneu et al., 2014). The lack of cellecellcommunication or other stimuli, such as the presence of high levelsof ATP, oxidized DNA, proteins, lipids, AGEs, damaged extracellularmatrix molecules, antibodies, complements, cytokines, nucleotidesor ions, may activate the microglia (Hanisch and Kettenmann,2007; Xu et al., 2009).

The activation signal is mediated by Toll-like receptors (TLR). TLRare broadly expressed in microglial cells in the brain (Bsibsi et al.,2002; Kielian et al., 2002) and the retina (Halder et al., 2013;Kohno et al., 2013; Maneu et al., 2011; Xu et al., 2009). TLR are afamily of pattern recognition receptors that participate in therecognition of microbial patterns and in innate and adaptive re-sponses (Kawai andAkira, 2006). These pattern recognition receptorsare capable of promoting the production of pro-inflammatory cyto-kines, chemokines andmolecules such asROS,which are essential forpathogen elimination in peripheral cells, and in astrocytes andmicroglia. Apart frommicroorganism-associatedmolecular patterns,many endogenousmolecules frommammals are also ligands for TLR,such as heat shock proteins or products derived from the hydrolysisof the extracellular matrix (Langmann, 2007).

Upon activation, microglial cells undergo a morphologicalchange from a ramified to an amoeboid shape (Fig. 11B), with agraded response according to the degree of activation (Hanisch andKettenmann, 2007; Raivich et al., 1999). In the activated state,microglial cells proliferate, migrate to the site of the stimulus, anddisplay greater phagocytic capacity (Fig. 14). Blood precursors arealso enrolled to assist in the damaged zones. In the effector phase,microglia accumulate in the nuclear layers and the subretinal space,where they act as phagocytes, clearing away the dying cells(Langmann, 2007). Both activated microglia and recruited bloodmacrophages can differentiate into a multitude of phenotypes,depending on the surroundingmicro-environmental signals, whichcan change over time (Harry, 2013; Kigerl et al., 2009; Michelucciet al., 2009; Perego et al., 2011).

Fig. 13. Morphological changes in astrocytes during retinal degeneration. (A, B) Confocal images of whole-mount retinas from a Sprague Dawley (SD) (A) and a P23H rat (B) stainedwith antibodies against GFAP (red), an intermediate filament protein expressed in astrocytes. Blood vessels have been labeled with Griffonia simplicifolia lectin (green). (C, D) High-magnification images of the top panels (A) and (B), respectively. Nuclei stained with TO-PRO. Note that in P23H rats (B, D) activated astrocytes become less ramified and hyper-trophic than in SD rats (A, C). Scale bar: (A,B) 40 mm; (C,D) 10 mm.


Microglia migration to the site of neural injury is regulated byeither soluble factors, which generate concentration gradients thatpromote and direct microglia migration, or by changes in theextracellular matrix of injured or diseased CNS tissues (Garden andMoller, 2006). Many of the migratory factors are chemokines(Cartier et al., 2005). Several growth factors also influence micro-glial migration. Some authors have shown that the angiogenicpeptide vascular endothelial growth factor (VEGF) acts as a trophicfactor, inducing the migration of microglial cells (Forstreuter et al.,2002). Furthermore, NGF has been shown to induce microglialmigration, and this cell migration is modulated by TGF-b, whichalso has a chemotactic activity (Ambrosini and Aloisi, 2004; DeSimone et al., 2007; Paglinawan et al., 2003). Migration can alsobe stimulated by nucleotides, such as extracellular ATP and ADP,which are released in response to ischemic and traumatic CNS in-juries and interact with several purinoceptors (Honda et al., 2001;Inoue et al., 2007; Luongo et al., 2014). Moreover, the microglialresponse depends on initial cytokine stimulation (Carter and Dick,2003).

Following activation, microglia are capable of entering the cellcycle and proliferating. Among the cytokines that stimulatemicroglia division, we find IL-1b, IL-4, interferon gamma (IFN-g),macrophage colony-stimulating factors (M-CSF) and granulocyte

macrophage CSF (GM-CSF). In addition, neurotrophic factors suchas BDNF and NT-3 are released by activated microglia and act in aparacrine fashion, as microglial mitogens (Garden and Moller,2006).

Activated microglial cells can display a variety of distinct,functional phenotypes with a large spectrum of potential markers.When activated, microglial cells increase the expression of severalsurface markers, such as antigen F4/80, complement receptor 3(CD11b/CD18), MHC-II and CD68 (Guillemin and Brew, 2004;Kreutzberg, 1996; Langmann, 2007). In a way similar to periph-eral macrophages and CNS microglia, retinal microglia also seem todisplay the M1/M2 stage distinction, which could better explaintheir heterogeneous functions. Hence, microglial cells can beinduced to express the pro-inflammatory M1 or the M2-deactivated (also known as M2-alternatively activated or anti-inflammatory) phenotypes (Ardeljan and Chan, 2013; Kigerl et al.,2009; Lucin and Wyss-Coray, 2009; Michelucci et al., 2009; Per-ego et al., 2011; Polazzi and Monti, 2010; Xu et al., 2009).

Another consequence of microglial activation is the increasedphagocytic capacity of microglia (Fig. 14), which can now phago-cytize not only microbes, but also pathological proteins, such asbeta-amyloid. Among the microglia receptors involved in engulf-ment during developmental apoptotic processes and under

Fig. 14. The role of glial cells in the retina. Schematic representation of the main morphological and functional features of glial cells in normal (A, C) and injured retinas (B, D). In thenormal retina, glial cells play a key role in maintaining homeostasis and preserving the survival of neurons (A). Retinal injury triggers the activation of glial cells, characterized bysecretion of pro-inflammatory factors and phagocytosis (B), and by the decrease or absence of their normal functions in healthy retina.


pathological conditions, we find transmembrane adapter DAP12-associated receptors and CD36 (Garden and Moller, 2006; Harry,2013; Langmann, 2007). After phagocytosis of microorganisms,microglial cells have the capacity to present the engulfed proteinsas antigens to T cells, stimulating an adaptive immune response.Thus, microglia appear to play an important role as antigen-presenting cells, even if they may require some additional signalsfrom circulating dendritic cells to generate a complete response. Inthis sense, by both invading from peripheral blood and differenti-ating from the available pool of resting cells, microglia may act asantigen-presenting cells to perform the neuroprotective function ofthe activated T-helper cells (Aloisi et al., 2000; Byram et al., 2004;Garden and Moller, 2006).

An activated state induced by a harmful stimulus may notnecessarily result in a shift to an amoeboid shape, andmicroglia canreturn to an inactivated status following the disappearance of thestimulus upon reception of a down-regulating signal (Harry, 2013).

The inflammatory response elicited after an injurious stimulus isregulated by several diffusible factors originating from the micro-glia. These include cytokines, chemokines, trophic factors and smallmolecule mediators of inflammation, such as prostaglandins(Garden and Moller, 2006). If the harmful stimulus is prolonged orsevere, the secretion of pro-inflammatory cytokines, ROS, nitricoxide (NO), TNF-a, glutamate or caspases is capable of inducing

photoreceptor death (Harada et al., 2002; Roque et al., 1999). Underthese conditions, microglial cells have been associated withneurodegenerative diseases, either as an initiating factor or as anaid in their development (Karlstetter et al., 2010a; Langmann,2007).

2.3.1.4. Microglia in degenerating diseases. Although the exacttrigger stimulus involved in degenerative diseases remains un-known, it is recognized that microglial activation, as well asexpression of chemokines andmicroglia-derived toxic factor TNF-a,often precedes overt astrogliosis, changes in neuronal physiology,photoreceptor apoptosis and retinal degeneration (Gehrig et al.,2007; Karlstetter et al., 2010a; Zeiss and Johnson, 2004; Zhenget al., 2005). Activated microglial cells have been shown to inducephotoreceptor death, at least in in vitro experiments (Harada et al.,2002; Roque et al., 1999). In the first stages of retinal neuro-degeneration, microglia trigger repair mechanisms, such as glialscar formation (Muzio et al., 2007). But excessive or prolongedmicroglial activation in the CNS and the retina may lead to chronicinflammation, with severe pathological side effects that can resultin irreversible neuronal death (Fig. 14) (Hanisch and Kettenmann,2007; Langmann, 2007; Schuetz and Thanos, 2004). There is alsothe possibility that an alteration in neurons, glia or both may beeventually amplified by a microglial response, ultimately affecting


the survival of neurons. On the other hand, it must be taken intoaccount that microglial dysfunction, with the loss of their protec-tive functions (the secretion of trophic factors, antioxidants andcytokines and the removal of cellular debris), could by itself lead toneuronal cell death (reviewed in Polazzi and Monti (2010)).

Activation of the microglia has been demonstrated in associa-tion with several neurodegenerative diseases, such as Alzheimer'sand Parkinson's diseases, amyotrophic lateral sclerosis, and multi-ple sclerosis, although it remains unclear whether microglial acti-vation is a cause or a consequence of neuronal damage (Muzio et al.,2007; Polazzi and Monti, 2010). In Alzheimer's disease, activatedmicroglial cells have been associated with amyloid plaques in thebrain. These amyloid plaques have been found also in AMD drusen.It is plausible that, in the early stages of the disease, microglialactivation could help remove amyloid plaques, while in later pha-ses, pro-inflammatory cytokines induced by microglia couldcontribute to the neurodegenerative process (Bornemann et al.,2001; Griffin et al., 2006). In Parkinson's disease, an increasednumber of activated microglia are found in the brain, accompaniedby increased expression of pro-inflammatory cytokines (Hirsch andHunot, 2009; Tansey et al., 2007).

In the same way, retinal neurodegenerative diseases are alsoassociated with chronic microglial activation and neuro-inflammation. In the degenerating retina, endogenous signalsactivate microglial cells, leading to their local proliferation, migra-tion, enhanced phagocytosis and secretion of cytokines, chemo-kines, and neurotoxins. These immunological responses and theloss of limiting control mechanisms may contribute significantly toretinal tissue damage and pro-apoptotic events in retinal dystro-phies (Gupta et al., 2003; Karlstetter et al., 2010a; Langmann, 2007).

Several studies have linked microglial activation with glaucoma(Bosco et al., 2012; Fan et al., 2010; Gallego et al., 2012; Inman andHorner, 2007; Johnson and Morrison, 2009; Luo et al., 2010).Microglia in glaucomatous ocular tissues show an alteredmorphology and upregulation of microglia-derived inflammatoryproteins, with increased secretion of inflammatory proteins, suchas TNF-a (Johnson and Morrison, 2009). Different studies havesuggested that inhibition of reactive microglia through physicaltechniques, such as irradiation, or by anti-inflammatory drugs, suchas minocycline, may be a promising potential approach in glau-coma therapies, increasing the survival rate of functional RGC(Bosco et al., 2012; Seitz et al., 2013). In this sense, the inhibition ofthe signaling cascades initiated by reactive microglia (such as NOsynthase or TNF-a) is also considered as a potential therapy for thetreatment of glaucoma (Neufeld, 2004; Roh et al., 2012; Seitz et al.,2013). Other researchers have shown that in human glaucoma, theimmunostimulatory signaling can also be initiated through glialTLRs (Luo et al., 2010), what may involve another therapeutictarget.

In RP, Gupta and coworkers showed that human microglia areactivated in response to primary rod photoreceptor death, migrateto the outer retina and phagocytize rod cell debris. The release ofcytotoxic factors such as NO can kill adjacent photoreceptors. Onceagain, treatment targeting activated microglia could save cones inhuman inherited diseases involving primary rod photoreceptordegeneration (Gupta et al., 2003). As is the case in other retinop-athies, microglial activation has been shown to be mediated byTLR4 signaling in RP (Kohno et al., 2013).

Microglial activation also contributes to tissue degeneration inAMD (Ardeljan and Chan, 2013). Immunologic responses in neuralretinal microglia and vascular elements appear to be related toearly changes in RPE pigmentation and drusen formation (Penfoldet al., 2001). In a model of AMD, the microglia was found to displayRPE cytotoxicity and increased production of inflammatory che-mokines/cytokines after co-incubation with ligands that activate

innate immunity, and elevated mRNA levels of TLR2 and TLR4 werealso observed (Kohno et al., 2013). In AMD, as well as in otherdegenerative diseases, an imbalance between the M1 and M2macrophage populations, together with activation of retinalmicroglia, have been observed and are thought to potentiallycontribute to tissue degeneration (Ardeljan and Chan, 2013). In amouse model of AMD, naloxone was found to modulate the accu-mulation and activation of microglia at the site of retinal degen-eration, which may be mediated by the inhibition of the pro-inflammatory molecules NO, TNF-a, and IL-b, and also amelio-rated the clinical progression and severity of retinal lesions (Shenet al., 2011). In this regard, naloxone was unable to preventapoptosis of photoreceptors in in vitro experiments, suggesting thatmodulating the functions of microglia, rather than inhibiting theiractivation, could be a good therapeutic approach for preventingphotoreceptor degeneration (Jiang et al., 2013).

Other non-inherited retinal diseases, such as degenerationproduced by trauma, axotomy or ischemia, involve microglialactivation, as in the case of light-induced photoreceptor degener-ation. During light-induced retinal degeneration (LIRD), microglialcells assume an activated state, migrate from the inner to the outerretina and alter the production of trophic factors, which may alsoaffect photoreceptor cell survival (Harada et al., 2002; Zhang et al.,2005). Moreover, microglia-derived factors influence the produc-tion of secondary trophic factors by Müller cells. Functional in-teractions between microglia and Müller glial cells may bebidirectional and regulate photoreceptor cell survival during retinaldegeneration (Harada et al., 2002). In this sense, chemokine ligand2 expression by Müller cells seems to play a role in promoting theinfiltration of monocytes/microglia and contribute to the neuro-inflammatory response and photoreceptor death following retinalinjury (Rutar et al., 2012). Still other researchers have observed thatafter photodegeneration, microglia fail to return to their originalstate, rather they continued to show some degree of activation(Santos et al., 2010). Suppression of the pro-inflammatory effect ofmicroglia, either by drugs such as naloxone or minocycline (Niet al., 2008; Zhang et al., 2004) or by physical methods, such aselectrical stimulation (Zhou et al., 2012), can contribute to reducingphotoreceptor degeneration in light-induced degeneration models.

Microglia have also been observed to become activated afterdamage to ganglion cells. RGC neurodegeneration was found to beassociated with microgliosis, characterized by an increase in celldensity with concomitant morphologic changes from ramified toamoeboid forms. The authors also demonstrated that microgliadensity gradually declined to near-baseline level, and cellmorphology returned to ramified forms after approximately 4weeks (Liu et al., 2012). Microglia activation has also been found ina model of optic neuritis, where inflammation and cell death in theoptic nerve led to subsequent damage of RGC in the retina,accompanied by gliosis, which could be prevented by protectingmyelin from degradation using a calpain inhibitor treatment (Daset al., 2013).

Using primary retinal microglia from retinoschisin-deficient(Rs1h-/Y) mice, a prototypic model for rapid retinal apoptosis anddegeneration, and the BV-2 cell line, Karlstetter and his groupidentified a new protein called AMWAP (activated microglia/macrophage WAP domain protein) that can act as a modulator ofmicroglial activation in neurodegenerative disorders. Moreover,they demonstrated that AMWAP expression is rapidly induced byligands for TLR2, -4, and -9 and IFN-g, thereby reducing pro-inflammatory cytokine expression (Karlstetter et al., 2010b).

The fractalkine/CX3CR1 signaling pathway has also beenpostulated as relevant in the control of retinal inflammation. In thissense, under oxidative and ischemia/reperfusion conditions and inthe absence of CX3CR1, uncontrolled retinal inflammation results


in extensive retinal degeneration (Chen et al., 2013), and it has beensuggested that CX3CL1 can exert a protective effect on the light-injured retina (Huang et al., 2013a). On the other hand, theexpression of fractalkine was found to be significantly upregulatedafter exposure to light, and was located mainly at the photore-ceptors (Zhang et al., 2012a). This suggests that fractalkine/CX3CR1signaling exerts multiple effects on the cross-talk between micro-glia and photoreceptors, and may play an important role in theprocess of retinal microglia activation and migration in light-induced photoreceptor degeneration. These authors also showedthat the soluble form of fractalkine released from photoreceptorsmay function as a chemotactic factor to trigger the activation andmigration of retinal microglia, while low concentrations of frac-talkine in the membrane-bound formwould serve as a regulator ofthe beneficial balance between microglia and photoreceptors(Zhang et al., 2012a).

2.3.1.5. The controversial role of microglia in inflammation andneurodegeneration. The true role of microglia in neurodegenerativediseases as either a beneficial or harmful factor still remainscontroversial, and a large body of results has been obtained tosupport both hypotheses. Upon activation, different functions areassociated with microglia (Fig. 14), some clearly protective, someharmful, and still others with dual effects, possibly due to specificenvironmental conditions in each case, in such a way that thesingular circumstances that evoke microglia activation determinethe final net contribution to the disease (Streit et al., 1999). In thissense, different stimuli can trigger different microglial responses.For example, both IFN-g and IL-4 can make microglia neuro-protective, while aggregated beta-amyloid and lipopolysaccharideprovoke a cytotoxic response from the microglia (Butovsky et al.,2005). Hence, different authors have suggested that the beneficialor harmful expression of the local immune response in a damagedCNS depends on the circumstances (Butovsky et al., 2005; Schwartzet al., 2006). A plausible explanation for this dual role is the widephenotypic variation upon an injurious stimulus, which is likely tolead to functional diversity (Hanisch and Kettenmann, 2007;Schwartz et al., 2006).

Also relevant is the observation that microglial activation oftenprecedes astrogliosis (Carson et al., 2006) and the fact thatmicroglial deterioration is observed with age in retinal dystrophiesand in CX3CR1-deficient mice with dystrophic microglia and ab-normalities in their cytoplasmic structure (Streit et al., 2004; Xuet al., 2009). Among the aspects that determine whether fullmicroglial activation becomes a harmful stimulus is the release ofmolecules such as pro-inflammatory cytokines, ROS, NO and TNF-a, which have neurotoxic effects and are evoked in a chronicallyactivated state (Nimmerjahn et al., 2005; van Rossum and Hanisch,2004).

In this sense, in some neurological diseases, such as multiplesclerosis and Parkinson's disease, the attenuation of microglialactivation has a protective effect (Hanisch and Kettenmann, 2007;Huitinga et al., 1990; Liu, 2006; Mount et al., 2007). In a similarmanner, in animal models of light-induced damage, differentgroups have demonstrated that the inhibition of retinal microgliaactivation by either minocycline (Zhang et al., 2004) or electricalstimulation (Zhou et al., 2012) has a neuroprotective effect againstthe loss of photoreceptors, through suppression of the secretion ofseveral pro-inflammatory cytokines and upregulation of neuro-trophic factors (Zhou et al., 2012).

Hence, the selective inhibition of the overactive microglial ac-tivity and the preservation of their trophic and homeostatic func-tions appears to be a promising treatment for degenerative diseases(Langmann, 2007). But it must also be noted that reactivemicroglialcells can also have a protective effect in damaged retina and that

the inhibition of microglial activation can have harmful effects atthe same time.

Microglia participate in regenerative processes by removingdendritic structures (Cullheim and Thams, 2007; Trapp et al., 2007).In the early stages of the neurodegenerative process, microglialactivation can display a protective function (Fig. 14) through thephagocytosis of cell debris and the release of protective molecules(Kreutzberg, 1996; Neumann et al., 2009; Nimmerjahn et al., 2005;Polazzi andMonti, 2010). In this sense, accumulation of microglia inischemic areas correlates with a reduction of neuronal damage andconfers neuroprotection (reviewed in Hanisch and Kettenmann(2007)). Activated microglia secrete neurotrophic factors that pro-tect and regulate the survival of photoreceptors (Arroba et al., 2011;Carwile et al., 1998; Langmann, 2007). Microglia can also partici-pate in proteolytic processes involved in tissue remodeling. As anexample, TGF-b1 produced by activated microglia can promotetissue repair, either directly or indirectly, by reducing astrocyticscar formation (Kreutzberg, 1996). Also, age-related microgliaactivation is likely to represent a protective response againstharmful stimulus produced in the retinal microenvironment, andthus may play an important role in retinal homeostasis (Xu et al.,2009). Another beneficial function exerted by microglia is theremoval of glutamate. Under pathological conditions, astrocyticglutamate uptake is impaired, and microglial cells are able to helpremove the excess of glutamate resulting from synaptic activity byexpressing the glutamate transporter protein GLT-1 (Hanisch andKettenmann, 2007; Persson et al., 2005).

To date, we have yet to completely understand the conditionsunder which reactive glial cells mediate detrimental or protectiveeffects and themechanisms that initiate the protective or damagingeffects by reactive glial cells. Moreover, in neurodegenerative dis-eases such as glaucoma, both effects seem to occur at the same time(Seitz et al., 2013). Furthermore, in Alzheimer's disease, microglialactivation may have a dual effect. Microglial phagocytic activity isbeneficial, whereas the inflammation is detrimental, and it is theinflammatory state of microglia that serves as an important con-dition for the disease (reviewed in (Hanisch and Kettenmann,2007)). Concerning adult neurogenesis, inflammation-associatedmicroglia can attenuate neurogenesis, whereas microglia acti-vated by certain T helper cell cytokines promote neurogenesis(Hanisch and Kettenmann, 2007). It has even been observed thatmicroglia can amplify pro-inflammatory immune responses due totheir capacity to act as antigen-presenting cells, while their abilityto produce anti-inflammatory mediators could play the oppositerole (Aloisi et al., 2000).

In conclusion, to date, the real contribution of microglia to theprogression of neurodegenerative diseases and the optimal thera-peutic intervention to halt or reduce their progression still remainunclear.

2.3.2. Macroglial cells: Müller and astrocytes cells in healthy anddiseased retinas

Retinal macroglial cells, which include astrocytes and Müllercells, are responsible for maintaining the homeostasis of the retinalextracellular microenvironment, thus ensuring proper functioningof the healthy retina. In the early stages of degeneration, glial cellsare activated as part of a process called gliosis. Reactive gliosis has adirect neuroprotective effect on the retina, increasing the expres-sion of cytoprotective factors or restoring neurotransmitter balanceand ion and water concentration, among other benefits. In contrast,proliferative gliosis can accelerate the neurodegeneration during achronic disease, causing direct and indirect damage to neurons andvasculature. Chronic gliosis exacerbates disease progression,increasing vascular permeability, infiltration of toxic compoundsand even neovascularization (Penn et al., 2008).


2.3.2.1. The important role of Müller cells in retinal homeostasis anddegeneration

2.3.2.1.1. Müller cells in the healthy retina. Müller cells, thespecific and largest glial cell type in the vertebrate retina, span theentire thickness of the retina and are in contact with all retinalneuronal somata and processes, constituting an anatomical linkbetween the retinal neurons and the compartments with whichthese need to exchange molecules. They are primarily responsiblefor maintaining the homeostasis of the retina by regulating retinalglucose metabolism, retinal blood flow, neuronal signaling pro-cesses, ion and water homeostasis and pH, among other aspects(Fig. 14A). Moreover, Müller cells are involved in the regulation ofsynaptic activity in the inner retina. More specifically, they areresponsible of the uptake and clearance of neurotransmitters,mainly glutamate, but also GABA and glycine. In this sense, it hasbeen demonstrated that a failure to clear extracellular glutamatereduces the scotopic b-wave (Barnett and Pow, 2000) and makesglutamate neurotoxic (Izumi et al., 1999). Another function ofMüller cells is that of providing neurotransmitter precursors toneurons, such as glutamine for glutamate synthesis. For moredetailed explanations see (Bringmann et al., 2006; Reichenbach andBringmann, 2013).

On the other hand, as previously stated, the retina has anelevated need for antioxidant protection, due to its exposure tolight, high oxygen consumption and the presence of large amountsof polyunsaturated fatty acids in the photoreceptors. In this sense,Müller cells produce antioxidant molecules, such as glutathione(Pow and Crook, 1995), which are released under oxidative stress,for example, that resulting from hypoxia during darkness orhyperoxia during light exposure (Schutte and Werner, 1998).Moreover, Müller cells protect photoreceptors and retinal neuronsfrom death (Fig. 14) through the secretion of neurotrophic factors,growth factors and cytokines (Bringmann et al., 2009, 2006). Theablation of Müller cells in the retina of adult mice causes vascularabnormalities and photoreceptor cell death, which is revertedfollowing the administration of CNTF (Shen et al., 2012).

During the last decade, new roles for retinal Müller cells havebeen described that are both relevant and important (Fig. 14A).Briefly, it has been shown that Müller cells could be acting as livingoptical fibers that guide light through the inner retinal layers to-ward the photoreceptors, minimizing the scattering of light (Franzeet al., 2007). Moreover, because Müller cells are softer than neu-rons, they may act as soft, pliant material for the embedding ofneurons, and as deformable substrates for neurite outgrowth andbranching (Lu et al., 2006). In addition, Müller cells may protectneurons from mechanical stress caused, for example, by traumaticretinal injuries (Lu et al., 2006). On the other hand, Müller cells canregulate neuronal activity by releasing glutamate, adenosine orATP. Müller cells are connected to adjacent Müller cells through gapjunctions, forming a small syncytium of direct Müller-Müller cellcommunication (Ball and McReynolds, 1998; Ceelen et al., 2001;Zahs and Ceelen, 2006). Moreover, distant communications be-tween neurons and Müller cells through the extracellularmessenger ATP (Newman, 2001, 2004) have been described, whichare associated with the capability of macroglial cells to regulatelocal blood flow. For more detailed review of these concepts see(Reichenbach and Bringmann, 2013).

2.3.2.1.2. Müller cell activation in retinal disease. Müller cellsplay a crucial role in the presence of injurious stimuli, providing arapid response to any alteration of the retinal microenvironment, asthey are usually one of the first glial cells to detect retinal damage(Fig. 12DeE and 14C-D), because of their radial distribution.Moreover, Müller cells are highly resistant to pathogenic stimuli,such as ischemia, anoxia, hypoglycemia and elevated hydrostaticpressure. This resistance is conferred in part by their energy reserve

in the form of glycogen, their high antioxidant capacity and theircapacity to proliferate and regenerate, among other special prop-erties (for a review, see (Bringmann et al., 2009)).

Almost all retinal diseases are associated with the gliosis ofMüller cells. Thus, in DR, for example, reactive changes in Müllercells, such as up-regulation of glial fibrillary acidic protein (GFAP),occur early in the course of the disease (Barber et al., 2000). Also, adistinctive variation of intermediate filament expression in retinalmacroglia is associated with the pathogenesis of AMD, in whichdiscrete regions of GFAP upregulation in Müller cells can be asso-ciated with drusen formation (Wu et al., 2003). In the P23H ratmodel of RP, Müller cells express the GFAP marker in response todegeneration of the retina (Fernandez-Sanchez et al., 2010), andglial cells show changes in number and morphology associatedwith the progression of the pathology (Figs. 12 and 14B, D) (Cuencaet al., 2011; Pinilla et al., 2010). Müller cell gliosis has also beendetected through the expression of GFAP in the DBA/2J mousemodel of glaucoma (Fernandez-Sanchez et al., 2013). For review thecited information, see (Bringmann et al., 2006).

Reactive changes in Müller cells in response to damage mayhave both cytoprotective and cytotoxic effects on retinal neurons.Especially in the early stages after damage, Müller cell gliosis can beneuroprotective (Fig. 14). In this case, retinal insults result infunctional and biochemical changes in Müller cells that have beendescribed as “conservative” or nonproliferative. But this usuallybeneficial reaction can lead to a greater level of Müller cell responsedescribed as “massive” or proliferative, in which case gliosis isdetrimental to the retinal tissue and exacerbates neuronal death. Apossible trigger for the transition from “conservative” to “massive”gliosis is the breakdown of the blood-retinal barrier, resulting in anincrease in the retinal and vitreal contents of growth factors, cy-tokines and inflammatory factors, as well as an infiltration of blood-derived immune cells (Bringmann et al., 2009).

Müller cell gliosis involves a series of cellular and molecularevents that may or may not be dependent on the kind of stimulus.The most sensitive non-specific response to retinal disease andinjury is the upregulation of the intermediate filaments nestin,vimentin and GFAP (Figs. 12 and 14B, D). The upregulation of GFAPis so sensitive that it can be used as an indicator of retinal stress,retinal injury and Müller cell activation (Luna et al., 2010). There isevidence that intermediate filaments may contribute to thebiomechanical properties of Müller cells (Lu et al., 2011), and thustheir upregulation could increase the stiffness of the tissue. Thisupregulation seems to be crucial for many responses in Müller cellgliosis (Fig. 14B), such as glial scar formation, monocyte infiltration,neurite growth, neovascularization and cell integration in retinaltransplants, since all these traits were seen to be attenuated inmicedeficient in GFAP and vimentin, an experimental model of retinaldetachment (Bringmann et al., 2009; Nakazawa et al., 2007).

Other important non-specific Müller cell responses are cellularhypertrophy, proliferation and migration of these cells to establisha glial scar that fills retinal breaks, replacing degenerated neuronsand photoreceptors (Fig. 12AeC). Glial scars are involved in theformation of epiretinal membranes (Bringmann and Wiedemann,2009; Buchholz et al., 2013; Kase et al., 2006), frequentlydescribed in retinal detachment, DR and AMD, and in the appear-ance of proliferative retinopathy. The proliferation of Müller cells isneeded for retinal regeneration and also serves as guide for neuritegrowth. However, the development of a scar formed by theseMüller cells would appear to be the primary cause of failed retinaldetachment surgery, stem cell transplants or electronic deviceimplants in cases of advanced retinal degeneration (Bringmannet al., 2009).

Another important feature in gliotic Müller cells is their intensecrosstalk with cells from the immune system. Müller cells have


been described to have the capacity to act as immunocompetentcells and are a known source of inflammatory molecules (Fig. 14B),such as TNF-a, IL, interferon and intercellular adhesion molecule-1(Bringmann et al., 2009; Wang et al., 2011b). Under pathologicalconditions, Müller cells can also act as antigen-presenting cells viathe processing of antigens into immunogenic forms that are thenpresented in association with MHCII molecules (Bringmann et al.,2009). Moreover, the increase in Müller cell-microglia adhesionmolecules also allows activated microglia to translocate intra-retinally in a radial direction, using Müller cell processes as anadhesive scaffold (Wang et al., 2011b). In addition, there are evi-dences that microglia-Müller cell interactions could serve as atrophic factor-controlling system during retinal degeneration(Harada et al., 2002), and may contribute to the protection ofphotoreceptors or increased photoreceptor apoptosis.

Some evidence indicates that in proliferative gliosis, Müller cellscan promote neuronal cell death through the synthesis and secre-tion of TNF-a (Cotinet et al., 1997; Giaume et al., 2007; Lebrun-Julien et al., 2009; Tezel et al., 2001), monocyte chemoattractantprotein-1 and NO (Cotinet et al., 1997; Giaume et al., 2007). Excessproduction of NO byMüller cells and the formation of free nitrogenradicals result in protein nitrosylation, which has toxic effects onsurrounding neurons. Attenuated cell death after retinal detach-ment has been observed in GFAP- and vimentin-deficient mice, inassociation with decreased gliosis and monocyte infiltration intothe retina (Nakazawa et al., 2007). The cytotoxic effects of Müllercells contribute to retinal degeneration in various retinopathies,such as DR (Bringmann et al., 2009, 2006).

On the other hand, proliferative gliosis of Müller cells mightimpede tissue repair and regular neuroregeneration by formingglial scars. But Müller cells are also capable of dedifferentiating tocells with characteristics similar to pluripotent retinal cells(Fig. 14B). After retinal detachment, Müller cells are known tomigrate to the outer retinal layer and undergo mitosis. Some ofthese subpopulations of Müller cells appear to stop expressingcommonly accepted Müller cell marker proteins, which suggests apotential dedifferentiation of some of these cells over time (Lewiset al., 2010). The proliferation of Müller cells to form glial scars orto transdifferentiate into cells with a neuronal phenotype maydepend on their expression profile of intermediated filament pro-teins. The upregulation of nestin has been described as beingindicative of the dedifferentiation of Müller cells into progenitorcells (Luna et al., 2010). After retinal injury, when Müller cellsdedifferentiate and begin to act as progenitor cells, they also ex-press specific neuronal stem cell markers, such as Chx10, Sox2, Ki-67 or cyclin D1 (Bringmann et al., 2009; Fischer and Bongini, 2010;Fischer and Reh, 2001; Kohno et al., 2006; Wohl et al., 2009).

The capacity of Müller cells to dedifferentiate to cells with aneuronal phenotype, if it would be confirmed, will represent apromising mechanism for therapeutic interventions since trans-differentiatedMüller cells can be obtained frommany sources, suchas immortalized human cell lines or epiretinal membranes surgi-cally removed from the eyes of patients with proliferative reti-nopathies (Reichenbach and Bringmann, 2013). More knowledge isnecessary to elucidate the exact molecular mechanisms required toobtain neural progenitors fromMüller cells. So far, proliferation andmigration of transdifferentiated Müller cells after glutamate-induced toxicity or laser injury have been described (Kohno et al.,2006; Reyes-Aguirre et al., 2013; Tackenberg et al., 2009),although differentiation to new retinal neurons has not yet beensuccessfully achieved (Reyes-Aguirre et al., 2013; Tackenberg et al.,2009).

2.3.2.2. Retinal astrocytes and astroglyosis. Astrocytes are the mainglial cells in the brain, where they accomplish many of the

functions that Müller cells perform in the retina (Figs. 13 and 14)(Coorey et al., 2012). Astrocyte cell bodies and processes are almostentirely restricted to the nerve fiber layer of the retina (Fig. 13A, C).Although many of the functions of astrocytes in healthy retinas arepoorly understood, it is widely accepted that they play an essentialrole in the development and function of the retinal vasculature,blood flow and blood-retinal barrier (Kur et al., 2012). In addition,astrocytes help Müller cells to maintain ionic homeostasis and theclearance of neurotransmitters, and they support synapse forma-tion, function and elimination through the activation of microglialcells (Kimelberg, 2010; Stasi et al., 2006; Stevens et al., 2007).Moreover, they help neurons to modulate synaptic transmission,since neurotransmitters can evoke calcium transients in astrocytes,which in turn can modulate the electrical activity of retinal neu-rons, leading to either enhancement or depression of neuronalspiking (Newman, 2004). However, astrocytes do not occur acrossthe retina in species with non vascularized retinas like rabbitswhere the described astrocyte functions could be carried out byother glial cells.

In the retina, astrocytes and Müller cells are associated with thedevelopment of retinal blood vessels (Fig. 14A). During physiolog-ical hypoxia, astrocytes and Müller cells secrete VEGF, inducing theformation of superficial vessels from astrocyte and vascular pre-cursor cells (Chan-Ling et al., 2004; Kubota and Suda, 2009; Kuret al., 2012). Müller cells then drive the formation of the deepvascular plexus, an astrocyte-independent process, throughvascular sprouting transversely into the retina (Kur et al., 2012).Only species with astrocytes in the retina have retinal vasculature(Kur et al., 2012). In mature retinas, astrocytes and Müller cells areboth involved in the development of the new neovascularizationassociated with pathological processes such as AMD, DR or reti-nopathy of prematurity, releasing angiogenic factors in response topathogenic stimuli (Coorey et al., 2012).

But one of the most important functions of astrocyte cells is theformation and support of the blood retinal barrier (BRB) (Fig. 14A).Retinal capillaries that form the BRB consist of a single layer oftightly adhered endothelial cells, a basal lamina and surroundingpericytes, astrocytes and microglia, forming a functional complexcalled the neurovascular unit (for a review, see (Klaassen et al.,2013)). This structure selectively regulates the transport of mole-cules through the BRB. In the neurovascular unit, endothelial cells,pericytes, astrocytes, microglia and neurons are actively connectedover a functional network. Thus, for example, neural activity canregulate the blood flow by means of glial communication, throughvasoactive factors produced by astrocytes, which may control theblood flow in local regions of the retina (Metea and Newman, 2006,2007). Like Müller cells, astrocytes form a network where cellscommunicate by means of calciumwaves through gap junctions orusing extracellular messengers, such as ATP (Metea and Newman,2006, 2007). Functional changes in pericytes and astrocytesfurther facilitate BRB leakage (Fig. 14B, D). In fact, glial celldysfunction in retinal pathologies is associated with retinalswelling and BRB breakdown (Bringmann et al., 2006; Klaassenet al., 2013; Shen et al., 2010). Hyperglycemia, hypoxia, oxidativestress and/or inflammation are the main underlying processes inhuman ocular diseases inwhich BRB dysfunction is a major cause ofvision loss.

Reactive gliosis (Fig. 13B, D) in the retina is characterized bychanges in astrocyte morphology as production of GFAP andvimentin increases (Anderson et al., 2008; Luna et al., 2010). Agrowing body of evidence suggests that reactive astroglia can beboth beneficial and harmful to damaged neurons (Nakazawa et al.,2007). Reactive astroglia either release neurotrophic factors tosupport cell survival or produce molecules that inhibit axonregeneration and repair, triggering neurocytotoxicity or secondary


damage in nearby neurons and glial cells (Fig. 14B). Thus, forexample, the attenuation of reactive responses in retinal glial cellsof GFAP- and vimentin-deficient mice improves the integration ofretinal grafts in the hippocampus (Widestrand et al., 2007). Theabsence of GFAP and vimentin also protects against photoreceptorcell degeneration in cases of retinal detachment (Nakazawa et al.,2007). Moreover, the chemical inhibition of gliosis by glial toxinscan protect against ganglion cell death from excitotoxicity (Ganeshand Chintala, 2011). On the other hand, reactive gliosis has beenassociated with the up-regulation of enzymatic and non-enzymaticantioxidant defenses that may increase the ability of the astrocytesto protect neurons from free radicals (Wilson, 1997).

Virtually all forms of retinal injury or disease trigger reactivegliosis. Thus, in AMD, for example, large numbers of hypertrophicand reactive astrocytes have been observed to phagocytize theresidues of ganglion cells that have died through necrosis orapoptosis (Ramirez et al., 2001). In end-stages of RP, when signifi-cant death of the RGCs has occurred, many of the nuclei in the innerretina belong to astrocytes that have undergone reactive hyper-plasia (Milam et al., 1998). In the DBA/2J mouse model of glaucoma,retinal changes engender a reactive gliosis response (Inman andHorner, 2007), and there is evidence that astrocytes are the cellsresponsible for many of the pathological changes in the glaucom-atous optic nerve head (Hernandez et al., 2008). In DR, GFAP in-duction has been reported in both the astrocytes andMüller cells ofstreptozotocin diabetic rats (Lieth et al., 1998). Early astrocytechanges, including decreased astrocyte gap junction protein andgene expression, have also been correlated with inner retinalhypoxia and ganglion cell functional deficits during the progressionof diabetes (Ly et al., 2011).

2.4. Degenerative events in retinal vascularization

The vascular supply to the retina depends on two distinctvascular systems: the retinal vasculature, which supplies blood tothe inner two-thirds of the retina, and the choriocapillaris, whichsupplies blood to the outer retina (Fig. 15A). Because of the highmetabolic activity of the retina, the tissue with the highest oxygendemand in the body, the ability to regulate blood flow is anessential feature of the mammalian retina (Kur et al., 2012). Themicroenvironment of the retina is also regulated by the BRB system.

2.4.1. Retinal vascular networks and the blood retinal barrier inhealth and disease

The retinal vasculature enters the retina through the centralretinal artery via the optic nerve, and after being distributedthroughout the retinal tissue, it leaves the tissue through the retinalvein. Inside the retina, the blood is distributed by means of paralleland interconnected vascular plexuses (Fig. 15A). The most super-ficial plexus is located in the ganglion cell layer, whereas the innerplexuses are located in the OPL (Coorey et al., 2012). The retinatends to maintain a constant blood flow. Autoregulation in theretina is effective within a wide range of perfusion pressures, whilethe influence of autonomic innervation with circulating hormonesand neurotransmitters on retinal vascular resistance is negligibledue to the BRB (Kur et al., 2012). The autoregulatory myogenicresponse is intrinsic to smooth muscle cells, and pericytes arecapable of initiating vasomotor signals that can be propagatedalong the length of capillaries.

The choroidal circulation arises from the ciliary arteries. Incontrast to the retina, choroidal microvessels are fenestrated, andchoroidal circulation is under neurogenic control (Coorey et al.,2012). The basal lamina and the RPE tight junctions form theouter BRB, and are the structures responsible for transport regu-lation at this level (Kaur et al., 2008).

BRB disruption is a frequent event in many retinal diseases, anda major cause of loss of vision. Hyperglycemia, hypoxia, oxidativestress and inflammation are known to increase BRB permeabilityand can eventually lead to its breakdown by damaging some of theelements of the neurovascular unit (Kaur et al., 2008; Klaassenet al., 2013). Two basic vascular responses to retinal damage havebeen described: (1) Neovascular formation, when new vessels areformed in the retina in response to hypoxic conditions (Fig. 15CeD),as in the case of DR, AMD or retinopathy of prematurity (Cooreyet al., 2012; Penn et al., 2008). The origin of the vessels can beeither the retina itself or the choroidal vascularization, dependingon the pathology. (2) Vascular degeneration, when retinal vesselsdegenerate as a result of a reduction in oxygen consumption(Fig. 15B), such as in RP (Fernandez-Sanchez et al., 2012a; Pennesiet al., 2008).

2.4.1.1. Retinal neovascularization under hypoxic conditions.Retinal diseases that impose local hypoxia or retinal ischemiausually cause BRB disruption and result in the formation of newvessels (Kaur et al., 2008). Retinal vascular disease represents aleading cause of visual impairment and acquired blindness in in-fants (retinopathy of prematurity), working-age adults (DR) and theelderly (AMD) in industrialized countries (Coorey et al., 2012).

Oxidative stress, impairment of the antioxidant machinery andhyperglycemia have been proposed as the main underlyingmechanisms of endothelial cell damage and dysfunction of the BRBin DR (El-Remessy et al., 2003, 2013; Klaassen et al., 2013). Thedisruption of the BRB associated with DR promotes up-regulationof VEGF (Fan et al., 2010), a prominent angiogenic factor alsoreferred to as vascular permeability factor. This mechanism seemsto have a positive feedback regulation, since VEGF is able to in-crease vascular permeability in hypoxic conditions. In this regard,the permeability changes in BRB have been reported as a contrib-utory factor in the development of macular edema (Kaur et al.,2008). Furthermore, increased production of AGEs has beenshown to upregulate the expression of VEGF in glial cells (Hirataet al., 1997), which promotes neovascularization in the advancedstages of proliferative DR. The growth of new vessels in DR occurs atthe inner retinal layers, using the vitreous body structure for itsgrowth (Fig. 15D). The loss of the inner and outer BRB is also acommon finding in exudative AMD deriving in an increased fluidleakage. New blood vessels grow from the choriocapillaris plexusand enter the retina through Bruch's membrane (Fig. 15C), thusincreasing the risk of detachment of the RPE and/or neurosensoryretina (Hoffmann et al., 2002; Klaassen et al., 2013; Schlingemann,2004).

Retinal vascular development is mediated by a gradient of VEGFthat is generated by the macroglial cells and, is mainly regulated byretinal oxygen levels in the developing retina. In this context, thesecretion of VEGF by astrocytes contributes to develop the super-ficial vasculature of the retina while the VEGF gradient generatedby Müller cells helps to create the formation of the deep vascularplexus by promoting the sprouting of superficial vessels to theouter layers of the retina (Eichler et al., 2000; Stone et al., 1995). Inretinopathy of prematurity, treatment with a hyperoxic environ-ment removes the physiological hypoxia of the retina leading toinsufficient vascularization, which, in turn, induces up-regulationof angiogenic factors, particularly VEGF, and consequent neo-vascularization (Coorey et al., 2012). In oxygen-induced ischemicretinopathy, it has also been demonstrated that neovascularizationis associated with increased expression of VEGF in glial cells (Akulaet al., 2008; Liang et al., 2012; Weidemann et al., 2010). Thedevelopment of neovascular glaucoma, one of the most importantcomplications that can appear during the course of retinal diseasessuch as proliferative DR, ischemic retinal disease, vascular occlusive

Fig. 15. Vascular alterations associated with retinal disease. (A) Illustration of the vasculature of the retina and choroid. (B) Schematic representation of the progressive degen-eration of retinal vasculature associated with retinitis pigmentosa. (C) Representation of the changes in choroidal vasculature observed in age-related macular degeneration. Newblood vessels grow from the choriocapillaris plexus and enter the retina through Bruch's membrane. (D) Neovascularization of the inner retinal layers during diabetic retinopathy.RPE: Retinal pigment epithelium; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer.


disease or, less frequently, ocular ischemic syndrome, seems also tobe related with increased expression of VEGF (Hayreh, 2007).

2.4.1.2. Vascular degeneration under hyperoxic conditions.Vascular degeneration has been described in various retinaldegenerative diseases, such as RP, and the early loss of photore-ceptors in some retinal degenerations affect the vascular develop-ment of the retina. Thus, a close relationship has been shown toexist between the number of photoreceptors and vessel profiles inthe deep capillary plexus of the retina in animal models of RP(Pennesi et al., 2008). Furthermore, the loss of capillary loops

observed in these animal models can be ameliorated if photore-ceptor cells are protected from oxidative-stress-induced death(Fernandez-Sanchez et al., 2012a). But alterations in the deepcapillary plexus are not the only change evidenced in these pa-thologies; modification of the inner plexus has been shown to occurin advanced stages of RP, once all photoreceptor cells have been lostand a profound remodeling of the retina has occurred (Garcia-Ayuso et al., 2010, 2011; Wang et al., 2000). A sequence of theretinal degenerative events in RP is shown in Fig.15B. In a rat modelof oxygen-induced retinopathy, postnatal exposure to hyperoxiadestroys the plexiform layers, resulting in significant


electroretinographic anomalies, cell death and synaptic retractionaffecting principally the inner retina, pointing to the inner retina asthe primary target of hyperoxic injury (Dorfman et al., 2011).

2.4.2. Retinal degenerative diseases with relevant vascular changes

2.4.2.1. Disorders of the retinal circulation. The retinal vasculaturelacks autonomic innervation, but responds to changes in the sur-rounding partial pressure of carbon dioxide and oxygen. Impair-ment of retinal vascular autoregulation is a common feature in anumber of ocular disorders, including DR and glaucoma.

Retinal circulatory changes precede overt clinical DR. Changes inbasal blood flow are characterized by retinal hypoperfusion in earlydiabetes, with a shift to retinal hyperperfusion as DR progresses(Kur et al., 2012). However, there are quantitative and qualitativeinconsistencies in the data. Measurements indicate a correlationbetween the severity of DR and decreased flow velocity in thecentral retinal artery (Goebel et al., 1995). Ocular perfusion pressureis also reduced, which can exacerbate retinal hypoxia. Hemody-namic abnormalities may generate macular ischemia and/or neo-vascularization at different levels (optic disc, peripheral retinal, irisor the anterior chamber angle).

Abnormal vascular regulation at the retinal level is also associ-ated with pathogenesis of glaucoma. Elevated intraocular pressureassociated with glaucoma is expected to cause decreased ocularblood flow, as has been proven in several studies showingdecreased blood flow in the optic nerve head, retina and choroid inpatients with glaucoma (Kur et al., 2012).

Late stages of retinal degenerative diseases can also showvascular changes linked to anatomical modifications. Remodelingin the late stages of RP after cell loss or in long-established retinaldetachment modifies the retinal anatomy. Retinal blood flow issignificantly decreased in patients with RP (Fig. 15B), probably as aresult of vascular remodeling in response to reduced metabolicdemand (Grunwald et al., 1996).

2.4.2.2. Disorders of the choroidal circulation. Central serous cho-rioretinopathy and AMD are disorders characterized by changes inchoroidal circulation. Patients with central serous chorioretinop-athy show decreased choroidal blood flow and hyperpermeabilityof the choroidal vessels, resulting in retinal edema (Kitaya et al.,2003). Several studies have also demonstrated changes inchoroidal vascularization in AMD, including reduced blood flowand abnormal choroidal perfusion patches (Friedman et al., 1995;Kiel and Reiner, 1997). The subsequent hypoxia induces the secre-tion of growth factors, such as vascular permeability factor andfibroblast growth factor (FGF) (Frank et al., 1996). These factors arefound in different retinal cells, including glial cells, RPE andvascular endothelial cells. Secondary to the increase in growthfactors, in exudative AMD forms, there is neovascularization fromchoroidal vessels that can cross Bruch's membrane, localizingeither under or over the RPE and generating a loss of fluid, hem-orrhage and lipid exudation (Fig. 15C).

2.4.2.3. Disorders of the retrobulbar circulation. Vascular occlu-sions, such as occlusion of the central retinal artery or the centralretinal vein, result in circulation anomalies that are revealed byDoppler imaging (Williamson and Baxter, 1994). Decreased bloodvelocity and resistivity index in the retrobulbar arteries andincreased blood velocity and resistivity index in the central retinalvein have been reported in patients with DR (Dimitrova et al.,2003). Reduced blood velocities in the ophthalmic artery havealso been shown in ocular ischemic syndrome and carotid stenosis.A reverse flow, indicating collateral perfusion, has been seen inother cases of severe carotid stenosis, while increases in

ophthalmic flows have been associated with ophthalmic arterystenosis.

2.5. Retinal pigment epithelium (RPE)

2.5.1. RPE physiology and functionsThe RPE is a hexagonal monolayer of columnar pigmented

epithelial cells that lies between the choroid and the neural retina.It is flanked by Bruch's membrane on its basal surface, and by theouter segments of the photoreceptors on its apical portion. The cellsin this layer are connected by tight junctions, and constitute theouter components of the blood-retinal barrier (Nag and Wadhwa,2012; Spitznas, 1974). In the subretinal space, filled with theinterphotoreceptor matrix, microvilli from RPE cells appose to theouter segments of the photoreceptors, forming the physical com-ponents that can contribute, with others, to the maintenance ofretinal adhesion (Nag and Wadhwa, 2012).

The RPE has several physiological functions that are indis-pensable for neural retina survival, including: (i) the delivery ofnutrients such as glucose, retinol and fatty acids to photorecep-tors and (ii) the transfer of metabolic end products from thesubretinal space to the blood. Since photoreceptors do not havetheir own blood supply, nutrients must travel to the photore-ceptors from the choriocapillaris, through Bruch's membrane andthe RPE. The reverse path is followed for the elimination ofcellular debris (Taylor, 2012). The RPE also: (iii) regeneratescomponents of the visual cycle; (iv) buffers ion composition inthe subretinal space, which maintains photoreceptors excit-ability; (v) phagocytizes the shed photoreceptor outer segmentsand recycles essential substances; (vi) absorbs the light focusedon the retina and protects against photo-oxidation; (vii) secretesgrowth factors, such as fibroblast growth factors (FGF-1, FGF-2,and FGF-5), TGF-b, insulin-like growth factor-I, ciliary neuro-trophic factor, platelet-derived growth factor, VEGF and pigmentepithelium-derived factor; and (viii) regulates T cell activation inthe eye (Strauss, 2005; Sun et al., 2003). All these functions areessential, and a failure of any of them can lead to photoreceptorsdeath, degeneration of the retina, loss of visual function andblindness. Moreover, several studies have shown the RPE to be anon-homogeneous population of cells that retains proliferativepotential, with subsets of cells that have an unusual capacity totransdifferentiate into various cell types and to produce a newretina, at least in amphibians and chicks (Fuhrmann et al., 2013;Machalinska et al., 2013).

Due to the fact that the RPE performs such relevant functions inthe retina and its involvement in early AMD-associated damage, agreat amount of research has focused on this layer.

2.5.2. RPE changes in aging and pathologyWith age, the eye undergoes several changes, some of which

affect the RPE. The accumulation of lipofuscin, a reduction inmelanin, diminished antioxidant capacity and the progressiveaccumulation of deposits underlying Bruch's membrane have allbeen described in aging eyes (Boulton and Dayhaw-Barker, 2001;Nag and Wadhwa, 2012). The death of RPE cells is associated withaging-pathophysiology; both acute and chronic progressivedysfunction of RPE cells and the age-related deterioration of thistissue have been shown to play a relevant role in retinal degener-ative diseases, primarily AMD. Regardless of age, four main pro-cesses contribute to the pathogenesis of AMD: lipofuscinaccumulation and drusen formation, local inflammation and neo-vascularization (Nowak, 2006). The inflammatory responsesobserved in AMD retinas are similar to, but more severe than thoseobserved in normal aging retinas. Different lifestyle factors, envi-ronmental conditions and gene alterations are likely to explainwhy


certain individuals develop AMD with age, while others do not (Xuet al., 2009).

AMD is amultifactorial disease that is the leading cause of visionloss in the industrialized world, with at least sixty million peopleestimated to be affected (Taylor, 2012). The two most prevalentAMD forms are atrophic and neovascular. Approximately 10e15% ofAMD cases correspond to the exudative, “wet” or neovascular formof the disease. Nevertheless, neovascular AMD is responsible for themajority of cases of severe vision loss. This severe form of AMD ischaracterized by choroidal neovascularization (CNV). Abnormalchoroidal blood vessels start to grow through Bruch's membraneunderneath the macula, and this is accompanied by vascularleakage or hemorrhage and scarring. In some cases, abnormal bloodvessels cause disciform scars, leading to the permanent loss ofcentral vision (Bhutto and Lutty, 2012). Most AMD patients sufferthe atrophic, non-exudative, “dry” form of the disease, whichprogresses more slowly and is characterized by the formation ofyellow deposits (called drusen) in the macula and atrophic areas ofRPE, accompanied by varying degrees of choriocapillaris loss. As thedisease progress, a loss of RPE and photoreceptor cells can bedetected in the macula. It is not unusual to observe a progressionfrom atrophic to neovascular AMD (Bhutto and Lutty, 2012; Taylor,2012). In both the atrophic and neovascular forms of AMD, themutualistic and symbiotic relationship between the photorecep-tors, RPE, Bruch's membrane and choriocapillaris is lost, whichresults in the death and dysfunction of all of the components in thecomplex, through mechanisms involving apoptosis (Bhutto andLutty, 2012; Dunaief et al., 2002).

The most important retinal changes associated with aging and/or AMD are summarized below. These mainly affect the RPE,leading to photoreceptor death, and thus contributing to thepathogenesis of AMD. Among them, there is an increase in theformation of lipofuscin granules, an accumulation of AGEs, drusenformation and changes in pigmentation, accompanied by a reduc-tion in melanosomes, increased thickness of Bruch's membraneand mitochondrial DNA deletions.

2.5.2.1. Lipofuscin accumulation and other changes in pigmentation.The RPE contains two kinds of pigment: melanin and lipofuscin.Melanin, an insoluble high-molecular weight polymer derived fromthe enzymatic oxidation of tyrosine and dihydroxyphenyl-alanine,is contained in cytoplasmic granules called melanosomes. Lip-ofuscin, a heterogeneous group of complex fluorescent lipid-protein aggregates, represents the accumulation of non-degradable end products from the phagocytosis of the outer seg-ments of photoreceptors (Ardeljan and Chan, 2013; Delori et al.,2001; Sparrow and Boulton, 2005). Lipofuscin granules containlipids, proteins and photoreactive molecules, such as bisretinoidfluorophore (A2E), which are potent photoinducible generators ofROS with phototoxic effects (Sparrow and Boulton, 2005). In agingRPE, there is a linear increase in lipofuscin formation in the cyto-plasm, which can contribute to cell dysfunction. The exposure ofRPE cells with accumulated lipofuscin to light produces extra-granular oxidation of lipids and inactivation of lysosomal andantioxidant enzymes, which may result in lipid peroxidation, theloss of lysosomal integrity, DNA damage and RPE cell death (Davieset al., 2001; Godley et al., 2005; Shamsi and Boulton, 2001; Sparrowand Boulton, 2005).

Besides lipofuscin accumulation, aging causes other alterationsin RPE pigmentation, such as a decrease in melanosomes and theappearance of new pigmented organelles (melanolysosomes) asthe result of melanin degradation, and melanolipofuscin granules,due to the accumulation of lipofuscin in the melanosomes (Feeney,1978; Strauss, 2005). Melanin granules act as a density filter andreduce the levels of light entering the RPE. The decline in melanin

granules in the macular RPE may result in decreased light absorp-tion and reduced antioxidant potential (Boulton and Dayhaw-Barker, 2001). The fact that the blue light photoreactivity of mela-nosomes increases significantly with age can result in toxicity tothe RPE (Sparrow and Boulton, 2005).

2.5.2.2. Advanced glycation end products (AGEs) accumulation.AGEs accumulation occurs with aging, and may have a significantimpact on age-related dysfunction of the RPE and also play a role inAMD. AGEs accumulation in Bruch's membrane, drusen, RPE, andchoroidal extracellular matrix may be a consequence of incompletedegradation of metabolic end products, such as lipoproteins fromboth photoreceptors and RPE (Ardeljan and Chan, 2013; Bhutto andLutty, 2012; Crabb et al., 2002; McFarlane et al., 2005). AGEsaccumulation can also be related to a limited choroidal perfusionand the inability of aged Bruch's membrane to transport material(Harris et al., 1999; Stefansson et al., 2011). Moreover, Bruch'smembrane deposits and drusen may interfere with transport be-tween the choriocapillaris and retina, and thus may be a contrib-uting factor to retinal hypoxia (Stefansson et al., 2011).

It has been associated a decrease in the risk of AMD to patientsconsuming low glycemic index foods. Likewise, a higher accumu-lation of AGEs and an acceleration of the appearance of age-relatedretinal lesions have been demonstrated in the retinas of animalmodels consuming high glycemic index foods, thus suggesting arelationship with the disease etiology (Chiu et al., 2011; Handaet al., 1999; Uchiki et al., 2012; Weikel et al., 2012). When RPEcells are exposed to AGEs, a para-inflammation state may developin conjunction with an adaptive response from RPE cells, but whenthe exposure becomes chronic, it is possible that the stressesoverpower the adaptive mechanisms of RPE cells, resulting indysfunction and death (Lin et al., 2013).

2.5.2.3. Drusen formation. The appearance of drusen is a widelyknown characteristic of AMD. Drusen are extracellular deposits thataccumulate between the RPE basal lamina and the inner collage-nous layer of Bruch's membrane in aging human eyes (Green andEnger, 1993). They are likely composed of incompletely digestedmaterial from the RPE, which cannot traverse Bruch's membranefor removal. Drusogenesis is a complex, multifactorial processaffected by genetic, environmental, dietary and aging factors thattakes place slowly over manys. Several theories have attempted toexplain drusen formation, focusing on inflammatory, immune, andcell-mediated events (Hageman et al., 2001; Nowak, 2006). Geneexpression analysis has revealed the existence of local synthesisand differential expression of a number of drusen-associatedmolecules (Hageman et al., 2001). Biochemically, deposits containcellular debris, lipids, proteins, lipoproteins, phospholipids, tri-glycerides, cholesterol, unsaturated fatty acids and carbohydrates,among other components (Crabb et al., 2002; D'Souza et al., 2008;Hageman et al., 2001; Mettu et al., 2012; Wang et al., 2010). Low-grade monocyte infiltration within the choriocapillaris is oftenpresent in the underlying areas of the deposits (Cherepanoff et al.,2010), and macrophages can be also found along the outer side ofBruch's membrane in areas of neovascularization and drusen(Grossniklaus et al., 2002). Drusen contain inflammation-relatedproteins, as well as complement components. The negativeimpact of the formation of drusen on RPE cells and photoreceptorsis most likely due not only to the physical displacement of the RPEmonolayer and photoreceptors, but also to their indirect influence,most likely via the activation of the immune system and localinflammation (Anderson et al., 2002; Nowak, 2006).

Despite the numerous classification systems, drusen can bebroadly divided into hard and soft types. Hard drusen are smallpunctate refractile lesions with sharp borders that appear in the


fundus as yellowish-white deposits. They are present in both themacula and on the periphery of the retina in both normal and AMDretinas, and are considered a consequence of normal aging. Thepresence of hard drusen alone does not carry an adverse prognosticsignificance, although in large numbers, hard drusen are an inde-pendent risk factor for vision loss in AMD (Bhutto and Lutty, 2012).Soft drusen are larger deposits, with indistinct borders and a fluffyappearance in the cross-section of the macula, and are consideredto be a precursor of advanced AMD, as much higher rates of AMDprogression are found in individuals with baseline soft distinctdrusen (Ardeljan and Chan, 2013; Buch et al., 2005). The histo-pathological examination of clinically identified “drusen” hasdefined three main types of sub-RPE deposits, depending on theirlocation, thickness, and content: basal laminar deposits and basallinear deposits (both diffuse deposits) and nodular drusen (discrete,dome-shaped deposits within the inner collagenous zone ofBruch's membrane) (Ardeljan and Chan, 2013; Bhutto and Lutty,2012; Bressler et al., 1988; Mettu et al., 2012). The histologicalcorrelation with clinically-observed drusen remains controversial(Mettu et al., 2012). Depending on the retinal location, hard drusenhave a different composition, with those located on the peripherybeing much more homogenous than those located in the macula.On the other hand, soft drusen have a homogeneous compositionand are filled with a single amorphous material that resemblesmembranous debris (Rudolf et al., 2008). The composition of dru-sen remains similar among different pathologies, such as AMD,glaucoma, chorioretinitis and malignant melanoma (D'Souza et al.,2008).

2.5.2.4. Thickening of Bruch's membrane. Generally, Bruch's mem-brane thickens with age. This thickening is greater at the posteriorpole than on the periphery, and is mainly produced by increaseddeposition and cross-linking of less soluble collagen fibers andincreased deposition of biomolecules, mainly waste products fromRPE metabolism. This change eventually leads to several functionaldisturbances, such as changes in elasticity and hydraulic conduc-tivity (Bhutto and Lutty, 2012; Ramrattan et al., 1994). Bruch'smembrane also undergoes other biochemical and anatomical al-terations with aging, such as calcification and lipid infiltration inthe absence of any apparent retinal dysfunction (Grossniklaus et al.,2013; Mettu et al., 2012). AMD patients also experience alterationsin retinal layer thickness, which is of importance since the mea-surement of retinal structures is known to correlate with visualacuity (Farsiu et al., 2014; Pappuru et al., 2011). In early AMD, whenvisual defects are present, there is a significant thinning of the OSlayer and thickening and elevation of the RPE (Acton et al., 2012).RPE-Bruch's membrane thickness is also negatively correlated withvisual acuity (Karampelas et al., 2013).

2.5.2.5. Increased susceptibility to cell damage. Aging RPE has alimited capacity to respond to oxidative stress, which maycontribute to the development of AMD. Vulnerability to oxidativedamage in the RPE is due, at least in part, to impaired Nrf2 signaling(Sachdeva et al., 2013). Nrf2 has been recognized as a key factorregulating an array of genes that defend cells against the delete-rious effects of environmental insults. Furthermore, it seems to playthe main regulatory role in the protective response to cellularoxidative stress, coordinating the expression of antioxidant genesand promoting cell survival, as Nrf2 deficiency has been associatedwith increased susceptibility to oxidative stress (Sachdeva et al.,2013; Zhang, 2006).

Many hypotheses have been proposed to explain the etiologyand mechanisms of AMD. Like many other complex multifactorialdiseases, several genetic, environmental and other factors influenceits development (Jarrett and Boulton, 2012; Mettu et al., 2012;

Taylor, 2012). As previously stated, the retina is one of the highestoxygen-consuming tissues in the human body, although oxidativedamage is normally minimized by the presence of a range of anti-oxidant and efficient repair systems. A reduction in the protectivemechanisms of RPE or an increase in the number and concentrationof species involved in photooxidative reactions can increase theoxidative stress and contribute to the pathogenesis of AMD (Bhuttoand Lutty, 2012).

2.6. Functional changes following retinal injuries

Early detection and reliable assessment of visual capacity arekey elements in preventing or slowing the progress of vision lossassociated with retinal diseases. Electrophysiological and psycho-physical methods for testing retinal function provide valuable in-formation in both experimental and clinical ophthalmologicalprocedures. Moreover, disease-associated morphological changesin the retina, if independently measured, are not always directlycorrelated with functional alterations, in terms of time course orspatial location. Thus, in many retinal disorders (e.g., glaucoma, RPand DR) there is a disease stage where functional disturbances mayprecede visible morphological changes (Berson, 1981; Falsini et al.,2008; Scholl and Zrenner, 2000).

2.6.1. Electroretinogram (ERG)The electrical response of the eye to a light stimulus has been

established for the objective examination of retinal function innormal subjects and in patients with retinal disorders. The ERGreflects contributions frommany different retinal neurons and glialcells, and provides a non-invasive technique to evaluate retinalresponses and visual signal processing. Considerable variabilityexists in the onset and evolution of retinal pathologies. However,there are a number of disorders in which the ERG can be used todistinguish qualitative abnormalities.

Most retinal disorders are characterized by an attenuation ofboth a- and b-wave amplitudes (Creel, 1995). Another value, im-plicit time, is also usually altered in a- and b-waves. Thus, forexample, in RP patients, ERGs characteristically show reduced a-and b-wave amplitudes, as well as delayed rod and/or cone b-waveimplicit times (Berson, 1987; Pinilla et al., 2005). Based on theseparameters, ERGs have provided criteria for establishing the diag-nosis of RP in early life, even when fundus abnormalities visiblewith an ophthalmoscope are minimal or absent. Patients withwidespread progressive forms of RP have shown not only reducedamplitudes, but also delayed cone and/or rod b-wave implicittimes, while patients with self-limited sector RP or stationary formsof night blindness have evidenced reduced amplitudes with normalb-wave implicit times (Berson, 1981). In contrast to RP, the ERGs ofpatients with cone dystrophy characteristically exhibit good, albeitslower rod b-waves, and reduced or absent cone ERG responses(Kellner and Foerster, 1993).

In animal models of retinal degeneration, a- and b-wave am-plitudes provide key information about disease-associated func-tional changes in the retina, but also about morphologicalalterations occurring during degenerative processes, including theprogressive loss of photoreceptors and synaptic connectivityimpairment in both the OPL and IPL. In this sense, it has beenshown that the mean number of rows of photoreceptor cell bodiespositively correlates with the scotopic b-wave amplitude recordedin P23H rats (Fernandez-Sanchez et al., 2012b; Lax et al., 2014), amodel of RP. Also, the thickness of the ONL was found to be pro-portional to the scotopic a- and b-wave amplitudes in a rat rote-none model of Parkinson's disease (Esteve-Rudd et al., 2011).Moreover, ERG recordings provide key information to assess theprogress of retinal degeneration in animal models (Cuenca et al.,


2004, 2005b; Pinilla et al., 2007) and to evaluate on them theneuroprotective effects of antioxidant and anti-apoptotic agents(Fernandez-Sanchez et al., 2012a, 2012b, 2011a; Lax et al., 2014,2011).

The relationship between the b-wave and the a-wave ampli-tudes also provides key information about the functional integrityof the retina (Perlman, 1983). Any significant deviation from thenormal relationship can therefore be regarded as pathological, andcan be interpreted accordingly. For example, inmost cases of RP, therods are affected more severely than the cones (Arden et al., 1983;Pinilla et al., 2005), causing an increase in the b-/a-wave ratio. Inmost cases of retinal ischemia, the ERG b-wave amplitude andimplicit time are selectively depressed, while the a-wave remainsnormal or becomes larger than normal. The b-/a-wave ratio varies,and the variation is believed to depend upon the degree of retinalischemia (Breton et al., 1989; Karpe and Germanis, 1962; Karpe andUchermann,1955). Thus, the ERG b-/a-wave ratio can be considereda good indicator of retinal ischemia in central retinal vein occlusion(Matsui et al., 1994; Sabates et al., 1983).

Recovery of visual function after adaptation to a bright lightsource is primarily dependent upon the speed with which photo-pigments can regenerate. Therefore, any disease compromising theintegrity of the photoreceptor/RPE complex, or limiting the supplyof metabolites to the outer retina, is likely to prolong the recoverytime of both rods and cones. This is exemplified in AMD, DR, cystoidmacular edema, RP and systemic diseases such as diabetes andhypertension (Binns and Margrain, 2005).

Examination with flicker ERG provides valuable informationfrom a clinical point of view. Different rates of stimulus allowseparate rod and cone contributions to the ERG to be identified. Theprimate ERG recorded on the cornea in response to fast flickeringlight is thought to reflect primarily the cone photoreceptor po-tential (Bush and Sieving, 1996). Even under ideal conditions, rodscannot follow a light that flashes up to 20 times per second,whereas cones can easily follow a 30 Hz flicker (Creel, 1995). Thereis evidence that the fast flicker (33-Hz) ERG is generated primarilyin the inner retina by the same neurons that are responsible(directly or indirectly) for the b-wave and the d-waves of thephotopic flash ERG (Bush and Sieving, 1996).

The usefulness of oscillatory potentials in the analysis of retinaldisease has been largely demonstrated (Algvere, 1968;Wachtmeister, 1998; Yonemura et al., 1962). Oscillatory potentialsare strongly dependent on normal retinal circulation and aredrastically affected by acute disturbances occurring in areas sup-plied by central retinal vessels (Speros and Price, 1981). A patho-logical oscillatory potential response indicates a neuronaldysfunction affecting the inhibitory feedback pathways and/or re-veals a pathological microcirculation in the inner retina that in-duces neuronal damage (Wachtmeister, 1998). In some cases of DRwith severe microangiopathy, the oscillatory potentials may beselectively reduced or absent, while the amplitude of the a- and b-waves of the ERG remains normal (Speros and Price, 1981).

Ganglion cells have very little influence on the scotopic ERGresponses to bright stimulus flashes. However, both positive andnegative components of the scotopic threshold responses dependdirectly upon intact ganglion cell function (Bui and Fortune, 2004).Thus, the scotopic threshold response is reduced after substantialRGC loss following the induction of experimental glaucoma (Buiand Fortune, 2004; Frishman et al., 1996). On the other hand, thephotopic ERG also reflects ganglion cell signals and may serve as anadditional useful test of ganglion cell function (Bui and Fortune,2004). Thereby, the photopic negative response has been foundto be sensitive to experimental glaucoma inmonkeys (Viswanathanet al., 1999) and humans (Colotto et al., 2000; Huang et al., 2012;Viswanathan et al., 2001).

2.6.2. Visual evoked potentials (VEPs)VEPs are electrophysiological signals extracted from the elec-

troencephalographic activity in the visual cortex, in response tovisual stimulation (Odom et al., 2010; Sokol, 1976). Since the visualcortex is activated primarily by the central part of the visual field,VEPs depend on the functional integrity of central vision at anylevel of the visual pathway, including the eye, retina, the opticnerve, optic radiations, and occipital cortex (Odom et al., 2010). Inaddition to detecting anterior visual pathway dysfunctions, chias-mal and retro-chiasmal dysfunctions can be assessed by examiningthe distribution of the VEP over the posterior regions of the scalp(Holder, 2001). Thus, VEPs can be valuable in diagnosing opticneuropathies, non-organic visual loss and assessing visual functionin infants or children (Young et al., 2012). Moreover, VEP results canbe predictive of visual recovery in traumatic optic neuropathy(Young et al., 2012).

Multifocal VEPs allow identify spatially localized damage andpathologies that may bemissed with a traditional single VEP (Creel,2012; Hood et al., 2003). The multifocal VEP is used to study visualfield defects caused by ganglion cell or optic nerve damage (Betsuinet al., 2001; Holder et al., 2009; Hood et al., 2000; Klistorner et al.,1998), and has been considered a powerful tool for the detection,management and study of glaucoma (Fortune et al., 2007; Hoodand Greenstein, 2003; Klistorner et al., 2007).

2.6.3. Psychophysical methodsVisual acuity, the single most-widely used eye test, is the ca-

pacity for spatial resolution of the visual system (Kalloniatis andLuu, 1995; Westheimer, 1965), namely, the ability of the visualsystem to discriminate between two stimuli separated in space,with a high contrast in relation to the background (Kniestedt andStamper, 2003). Visual acuity represent a practical tool for tracingthe course of ophthalmic dysfunction and therapy (Westheimer,2009).

More recently, contrast sensitivity has been proposed as avaluable addition to the psychophysical tests currently available.Contrast sensitivity refers to a measure of how much contrast aperson requires to see a target (Owsley, 2003). Contrast sensitivityplays a role in many aspects of vision, specifically motion detection,visual field, pattern recognition, adaptation to darkness and visualacuity (Richman et al., 2013). Contrast sensitivity loss is not specificto any particular diagnosis, as many diseases have similar effects onthe contrast sensitivity function. However, contrast sensitivitytesting is a valuable tool for identifying ocular disease and guidingtreatment (Richman et al., 2013). Studies have been conducted onthe use of contrast sensitivity to evaluate intraocular lenses, andpathologies such as cataracts, glaucoma, optic neuritis, DR andAMD, among others (Bailey, 1993; Ginsburg, 2006; Howes et al.,1982; Richman et al., 2013; Ross et al., 1984; Umino and Solessio,2013; Woo, 1985).

Visual discrimination in animals has been tested using differentapproaches. The optomotor test lets generate a psychophysicalthreshold in a reduced amount of time, and does not involve thefailure of older animals to learn a task (Douglas et al., 2005; Pruskyet al., 2004). Pigmented animal responses are stronger and easier torecognize than those of albinomice or rats, which do not showclearresponses to the optomotor test. The optomotor test has been usedas a visual test in different animal models of retinal degeneration(e.g., (Barabas et al., 2011; Umino and Solessio, 2013)).

3. Remodeling of the retina in retinal degenerations

The retinal cells respond against different types of damages,regardless of their origin, by modifying various cell signaling andmetabolic pathways. These alterations occurring at molecular


levels produce changes in the function and morphology of theretinal cells generating, as a consequence, retinal dysfunctionaccompanied in most of cases with an evident abnormal structureof the retina (Fig. 8). This is the general retinal remodeling phe-nomenon, however, depending on the nature and progression ofthe disease, variations exist in the cell types that undergo thisremodeling, as well as in the rate at which these changes occur.

A better knowledge of the remodeling alterations underlyingthe different types of retinal diseases and injuries could serve toclarify how is retinal degeneration in each particular situation,being useful for the proper diagnoses and prognoses of each pa-thology. In this context, it is important an adequate characteriza-tion of the evolution of the degenerative process (retinalremodeling stage) occurring in each pathology with the aim toapply the best therapeutic strategies to maintain or to rescue visualfunction. Interestingly, the scientific community has coined the‘retinal degeneration phase’ term in order to standardize appro-priate links between morphological changes of the different celltypes of the retina and the degeneration stage in each type ofdisease (Jones et al., 2012; Jones and Marc, 2005; Marc and Jones,2003; Marc et al., 2007, 2003; Vugler, 2010). Thus, the establish-ment of the different degeneration phases in each disease using thehigh resolution OCT technology could allow ophthalmologists todetermine the patient retinal degeneration stage for providing themost effective treatment.

We propose the following 4 phases of retinal remodeling(Fig. 16). The appropriate therapy for each degeneration phase willbe discussed later in section 4.8.

3.1. Phase 1

During this phase, retinal function and morphology seemnormal. No visual clinical symptoms can be recognized at this stage,as occurs in early RP and the first stages of AMD or DR. Increasedintraocular pressure may induce cellular stress at the beginning ofglaucoma onset, but no signs of the disease are observed. Photo-receptor stress induces a cascade of events that culminates inmolecular changes and eventual cell death at the end of this phase.Although photoreceptors are subject to a cell stress caused by ge-netic mutations (RP), disruption of the RPE (AMD) or changes inglucose concentration (DR), these circumstances do not affect thefunction and morphology of photoreceptor cells, and retinal neu-rons and layering appear normal (Fig. 16).

3.2. Phase 2

Cellular stress and the activation of apoptotic pathways lead toprogressive photoreceptor loss during this phase. At this stage(Fig. 16), photoreceptor pyknotic nuclei may be present in differentnumbers, depending on the disease or injury, and reactive changesin glial cells (gliosis) can be detected.

Prior to photoreceptor cell death, one of the earliest histologicalindicators of pathology in this phase is the delocalization of bothrhodopsin in rods and transducin in cones (Roof et al., 1994). Opsindelocalization is a common feature in the majority of retinaldegenerative diseases in both animal models and humans, as hasbeen observed in RCS rats for both opsins (Barhoum et al., 2008;Fernandez-Sanchez et al., 2011a; Martinez-Navarrete et al., 2011).Rhodopsin is normally located at high concentrations in the outersegments of rods. However, in animal models of RP (Fernandez-Sanchez et al., 2011a; Martinez-Navarrete et al., 2011) and in hu-man RP retinas (Fariss et al., 2000) rhodopsin extends from thephotoreceptor inner segments down to the cell bodies, axon pro-cesses and axon terminals (Fig. 4C). Rhodopsin redistribution to theinner segments and cell bodies in the ONL, in addition to a decrease

in the length of rod outer segments, has been described in cases ofhuman retinal detachment with proliferative vitreoretinopathy(PVR). Cone photoreceptors also displayed redistribution of opsins tothe inner segments and decreased outer segment length (Fig. 4CeF).Some synaptophysin redistribution in the ONL was also found(Fig. 7D) (Sethi et al., 2005). Early anterior PVR human specimensalso showed outer retina degeneration. The outer and inner photo-receptor segments were markedly disrupted, with swollen innersegments and pyknotic nuclei (Charteris et al., 2007). Besides, itseems that in diseases where cones degenerate secondarily to rods,the lack of a trophic factor secreted by rods called rod-derived coneviability factor play an important role (Leveillard et al., 2004).

Stressed photoreceptors begin to deconstruct their synapticterminals, with a loss of bassoon and synaptophysin immuno-staining (Figs. 5e7). The lack of synaptic signaling input also trig-gers a range of rewiring events, including retraction of bipolar andhorizontal cell dendrites, switching of synaptic targets by bipolarcells, anomalous extension of horizontal cell processes into the IPLand retraction of horizontal cell axon tip terminals. The retractionof rod photoreceptor synaptic spherules and the subsequentsprouting of rod bipolar dendrites that selectively reconnect toappropriate target neurons suggest the existence of functionalplasticity, even in the aged human retina (Fig. 8). Neurons in retinaltissues from AMD human eyes have the capacity to remodel bysprouting processes and to re-form demonstrable synaptic com-plexes with appropriate targets (Sullivan et al., 2007). Retinectomyspecimens also demonstrated disruption of rod bipolar cell bodystratification and numerous dendrite extensions into the ONL.However, these extensions were absent in advanced degeneration(Sethi et al., 2005). Similar results were found in human organo-typic culture retinas and in different animal models of retinaldetachment, such as cat (Cook et al., 1995), rabbit (Lewis et al.,2009) and primate models (Kroll and Machemer, 1968, 1969). Thedegeneration process in primates is slower than in the cat model,suggesting that human and other primate photoreceptors may bemore resistant than other species to the degenerative pathologyinduced by detachment. In this phase, molecular changes inglutamate receptor expression at bipolar cell dendrites begin tomodify the physiology of bipolar cells, shifting their functionalphenotypes from ON to OFF responses (Jones et al., 2012). Thedegree of degeneration of the inner retina during this period de-pends on the characteristics of the degenerative pathology. In manyof the animal models examined, there is extensive rewiring orreprogramming of inner retinal cells (Jones et al., 2003; Marc andJones, 2003; Marc et al., 2007, 2003).

The glial response in this phase is characterized by a markedhypertrophy of the cell processes. In association with hypertrophyand hyperplasia, glial cells enhance their protein production, asshown by the increased expression of the intermediate filamentprotein GFAP. Glial cell activation can be identified through GFAPexpression immunostaining (Fig. 12DeE and 13). Both animalmodels and humans show a high level of GFAP in glial cells in allretinal diseases, including RP, glaucoma, retinal detachment andAMD (Madigan et al.,1994; Rodrigues et al., 1987;Wang et al., 2002;Wu et al., 2003). Microglia activation is also noteworthy in animalmodels of RP (Fig. 11), retinal detachment and glaucoma (Boscoet al., 2011; Lewis et al., 2005), and in the retinas of patients withRP (Gupta et al., 2003). In early anterior PVR, Müller cells expressupregulated levels of GFAP and form epiretinal membranes,together with other cell types (Charteris et al., 2007).

3.3. Phase 3

During phase 3, the few remaining photoreceptors still maintainsome function, but are in a process of degeneration. The other cells

Fig. 16. Phases of retinal remodeling and suitable theraphies in each phase. Illustration of the most relevant cell types in the retina, and representation of the major changesoccurring in these cells during retinal degeneration. In the early stages (Phase 1) of retinal degenerative processes, changes in cellular and environmental homeostasis inducemolecular and cellular responses that do not significantly affect the function and morphology of the retina. At this stage of the degenerative process, the most suitable therapies areneuroprotection and gene therapy. In the second stage of the remodeling process (Phase 2), cellular changes involve truncation of the outer segments of rods and cones, rod death,retraction and loss of dendrites of bipolar and horizontal cells, with a reduction of cell connectivity in the OPL. Glial cells appear activated, including Müller cells and microglia.Therapies that may offer a good chance of success at this stage are neuroprotection, gene therapy and cell transplantation. Advanced stages of remodeling (Phase 3) are charac-terized by cone degeneration and death, reduction in cell numbers within the INL and neurite remodeling in both ONL and IPL. Gliosis is more evident at this stage, with hy-pertrophy of Müller cells. In this phase, the use of neuroprotectors, optogenetics, cell transplants and electronic retinal implants may be good approaches. The later stages of retinaldegeneration (Phase 4) are associated with the absence of visual capacity due to the loss of all photoreceptor cells. Neuronal cells migrate within the retina, with translocation ofamacrine and bipolar cells into the inner plexiform and ganglion cell layers, resulting in a topological restructuring of the retina. During this phase, a deep synaptic remodelingbetween all postsynaptic neurons occurs, forming microneuromas. In advanced stages of degeneration, cell death progresses, hypertrophy of Müller cells continues and microglialactivation increases. The retinal blood barrier deteriorates, RPE and Brunch's membrane break, and choroidal vessels enter the retina. At this stage of the degenerative process, themost suitable therapies are neuroprotection and electronic retinal implants. CR: Choroid, RPE: Retinal pigment epithelium; OS: outer segments; IS: inner segments; ONL: outernuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer.


become vulnerable to cell death. The gliosis of Müller cells in-creases, as does the number of activated microglial cells with anamoeboid shape. Apoptosis in second-order retinal neurons iscommon during this period (Fig. 16).

Animal models of RP show a severe loss of photoreceptors, witha concomitant loss of visual response, as measured by ERG(Fernandez-Sanchez et al., 2012a, 2012b; Strettoi et al., 2002).

Additionally, Müller cell hypertrophy and the collapse of the distalscaffolding of Müller cells in the absence of photoreceptor and bi-polar cells form a cell seal that isolates the neural retina from theRPE and choroid. At this stage, blood vessels deteriorate andrespond to the lack of oxygen by retracting or producing newvessels (see section 2.4 and Fig. 15). The loss of the photoreceptorlayer may result in the disappearance of the external limiting


membrane, formed by zonula adherens junctions between photo-receptors and Müller cell processes. Along with astrocytes andmigrated RPE cells, Müller glia proliferate and extend their pro-cesses to the INL, becoming part of the cellular components ofepiretinal membranes in eyes with RP (Bringmann andWiedemann, 2009; Szamier, 1981). Another common finding dur-ing this phase is the translocation of cell bodies to other retinallayers (e.g., Müller cell bodies located outside the ONL) (Marc et al.,2003). Finally, microglia can play an active role in phagocytosis ofdegenerated retinal cells, including neurons. Bipolar cells arecompletely deafferented in phase 3, not only physiologically,through the redistribution of glutamate receptors in the bipolar cellbodies and axons, but also anatomically, through the physicalretraction of all bipolar cell dendrites, resulting in severely alteredmorphologies (Figs. 8 and 16) (Barhoum et al., 2008; Cuenca et al.,2005b; Jones and Marc, 2005). These changes result in a completeloss of glutamatergic input after degeneration of rod photorecep-tors, as seen in different animal models, such as P23H or rd mice(Cuenca et al., 2004; Strettoi et al., 2004), which undergo pro-grammed cell death in various retinal diseases.

Photoreceptor death is evident in severe human PVR, and theremaining patchy photoreceptors show abnormalities, such as theloss of their outer segments. Synaptophysin staining is weak at theIPL and thin at the OPL, with a dropout of rod terminals (Sethi et al.,2005). Remodeling of inner cell types takes place during this phase,with a reduction in cell numbers at the INL and some horizontal cellprocesses extending into the ONL (Sethi et al., 2005).

3.4. Phase 4

Clinical examination at this stage shows the absence of visualfunction due to the loss of all retinal photoreceptors (Fig. 16). Apersistent and global remodeling and rewiring of retinal circuitriescharacterize this phase. Amacrine and bipolar cells migrate into theganglion cell layer and induce the formation of microneuromas(Jones and Marc, 2005). These structures are tangles of amacrine,bipolar and ganglion cells processes that form new synapses be-tween them. This new rewiring of retinal cells leads to unusualvisual circuitries (Jones and Marc, 2005). Conversely, in many ret-inas at this phase, RGCs can be observed migrating into the INL, andsome processes form fascicles that can run in bundles for greatdistances within the neural retina (Jones et al., 2012; Jones andMarc, 2005). The hypertrophy and migration of Müller cellsgenerate a distort lamination of the INL and OPL (Jones and Marc,2005).

Retinas in patients with late-stage RP show dramatic changes intheir architecture, with a complete loss of rod and cone photore-ceptors and bipolar cells, and subsequent topological restructuringof the retina. Newmicroneuromas with amacrine cells abutting thechoroid can be seen with no clear barrier in between, due to theabsence of RPE (Jones et al., 2012). During this phase, RPE cellsmigrate into the retina, oftenwith accompanying choroidal vessels,passing through gaps in the glial seal, and displacing INL cells (Jonesand Marc, 2005; Villegas-P�erez et al., 1998).

The Müller component of the retina is significantly expanded,filling areas previously occupied by degenerated retinal neurons.Wrinkling of the inner limiting membrane and formation of epi-retinal membranes is also evident (Sethi et al., 2005). Long-termrhegmatogenous retinal detachment also leads to retinal remod-eling, characterized by the loss of first- and second-order retinalneurons, disruption of the entire retinal lamination and gliosis(Ghosh and Johansson, 2012).

In the later stage of degeneration, breaks in Bruch's membraneprovide opportunities for some neurons to migrate out of theneural retina into the choriocapillaris membrane complex (Fig. 16).

It is unclear whether similar migration events outside the neuralretina occur in human diseases, such as AMD. Breaches of Bruch'smembrane certainly occur in the late forms of AMD that havevascular involvement, and choroidal vessels use these holes toenter the retina (Fig. 15C). Calcification of Bruch's membrane hasalso been shown following the breakdown of elastin and collagen innon-vascular or dry AMD (Spraul et al., 1999).

Commonly, retinal remodeling is a secondary process that takesplace after cell death. However, it has been described remodeling ofretinal synaptic circuitries without neuronal death. Cell disap-pearance, cell migration, disruption of spatial cell patterning,deregulation of structural stability, de novo synaptic repatterningand glial activation are common features during remodeling (Marcet al., 2003). Changes similar to those observed in the animalmodels have also been found in human tissue samples, indicatingthat retinal remodeling is a general principle in all retinal diseasesand species.

Knowledge of the exact phase of retinal degeneration in eachpathology is essential in order to choose the appropriate therapyaimed at recovering vision (Fig. 16). The therapeutic approach hasto be selected based on the exact anatomical status. If there are stillfunctional cells and the circuitries remain in place, the probabilityof success in the applied therapies is higher than in more advancedretinal degenerative stages. After the loss of retinal cells and gliosis,once synaptic markers have been lost and the connections amongneural retinal cells are impaired, the options are clearly reduced,because no information will be transmitted through the remainingaltered circuitries. Regardless of the therapeutic approaches avail-able, a careful study of the status of the retina should first be per-formed to ensure that the treatment will actually work. As anexample, a cell-based therapy using a selective cell type like pho-toreceptors would only work if the remaining cells are in goodshape. If the normal architecture of the retina has clearly dis-appeared, other options should be chosen, which do not require thepresence of a normal retinal circuitry; these might focus onimproving and preserving the remaining cells with neurotrophicfactors or using other approaches, such as retinal implants.

The key window for treatment will always be the type andphase of the degeneration. For all therapeutic approaches, thesooner they are begun, the better the results will be. Themammalian retina clearly has a vast repertoire of cellular responsesto injury, the understanding of which may help us improve currenttherapies or devise new ones to treat conditions resulting inblindness.

4. Therapeutic approaches in neurodegenerative diseases

Neurodegeneration is a common process in several retinal dis-eases. In this context, neuroprotective treatments provide thera-peutic strategies independent of the etiology of the degeneration.The aim of neuroprotective mechanisms is to provide an adequateenvironment in which to prolong the viability of retinal cellsthrough their effects on a number of biochemical pathways. Thiscan be achieved by either delivering neurotrophic growth factors toretinal tissues, inhibiting pro-apoptotic pathways or implementingviability factors, such as the rod-derived cone viability factor.Therapeutic approaches in the fight against retinal diseases andvision loss also include gene- and cell-based therapies, as well asretinal transplants.

4.1. Efficacy of anti-apoptotic therapies for retinal diseases

The final common pathway of cell death in retinal diseases isapoptosis, which initially affects only certain retinal cells, such asphotoreceptors, followed by the apoptosis of all remaining cells in


the retina. In this context, the pharmacologic inhibition of celldeath through the use of anti-apoptotic agents may preventdisease-associated retinal degeneration.

4.1.1. Tauroursodeoxycholic acid (TUDCA)Bear bile, the major component of which is TUDCA, has been

used in traditional Chinese medicine to treat visual disorders forthousands ofs. Synthetic TUDCA has been shown to exhibit anti-apoptotic properties in neurodegenerative diseases, includingthose affecting the retina. Systemic administration of TUDCA hasbeen demonstrated to slow retinal degeneration in both the rd10autosomal recessive RP mouse model (Boatright et al., 2006; Dracket al., 2012; Oveson et al., 2011; Phillips et al., 2008) and in a LIRDmouse model (Boatright et al., 2006; Oveson et al., 2011). In thesetwo retinal degeneration models, TUDCA-treated animals wereshown to maintain better visual function, thicker ONL and betterpreservation of outer segments than untreated animals. TUDCAalso prevented retinal degeneration in the P23H autosomal domi-nant RP rat model (Figs. 17G and 18C) (Fernandez-Sanchez et al.,2011a). P23H treated rats showed higher a- and b-wave ampli-tudes under both photopic and scotopic conditions than untreatedrats. Furthermore, TUDCA decreased photoreceptor apoptosis

Fig. 17. Neuroprotective treatments prevent photoreceptor cell degeneration during retinalwith antibodies against g-transducin (cone cells; green) and recoverin (cones, rods and som(B). Note that retinas from P23H rats treated with the neuroprotective compounds tauroursothose from untreated P23H rats (B, E). The morphology of cones is preserved in P23H rats touter nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexifo

(Figs. 17G and 18C) and maintained synaptic connectivity (Fig. 18C,F, J) among retinal cells (Fernandez-Sanchez et al., 2011a). Inaddition to its anti-apoptotic properties, TUDCA has also beenshown to exert anti-inflammatory, antioxidant and chaperone ac-tivities. In this context, TUDCA suppressed the formation of laser-induced CNV in rats by decreasing the number and size of CNVlesions, probably due to its anti-inflammatory properties, whichdiminished VEGF levels in the retina after the laser treatment (Wooet al., 2010). Additionally, systemic administration of TUDCA pre-served photoreceptors after retinal detachment in rats, preventingthe reduction in ONL thickness, and this was accompanied bydecreased oxidative stress and inhibition of the increase in caspase3 and 9 activity (Mantopoulos et al., 2011). TUDCA also protectedretinal neural cell cultures from high glucose-induced death bydecreasing mitochondrial-nuclear translocation of the apoptosisinducing factor (AIF). This inhibition of the release of AIF from themitochondria was probably due to the antioxidant properties ofTUDCA, as corroborated by the marked decrease in oxidative stressbiomarkers with TUDCA treatment (Gaspar et al., 2013). Thesefindings may have relevance in the treatment of DR. Furthermore,systemic injection of TUDCA diminished endoplasmic reticulumstress, prevented apoptosis and reduced cone degeneration in the

diseases. Vertical sections of Sprague Dawley (SD) and P23H rat retinas (P120) labelede bipolar cells; red). Few rows of photorecpetors remain at 4 month of age in P23H ratsdeoxycholic acid (TUDCA) (G) or safranal (C, F) show more rows of photoreceptors thanreated with safranal (F) and TUDCA (G). OS: outer segments; IS: inner segments; ONL:rm layer; GCL: ganglion cell layer. Scale bar: 20 mm.


retina of the Lrat�/� mouse model of Leber congenital amaurosis(Zhang et al., 2012b).

4.1.2. RasagilineRasagiline (N-propargyl-1-(R)-aminoindan) is a selective

monoamine oxidase inhibitor with proven neuroprotective effectsin the retina of prph2/rds mice, an animal model of RP, through adelay in the activation of caspase 3 dependent apoptotic pathwaysand the induction of the anti-apoptotic protein Bcl-XL (Eigeldinger-Berthou et al., 2012). This study also showed that rasagiline affectsthe induction of autophagy and reduces inflammatory activity inthe retina. Rasagiline has also provided for significantly enhancedsurvival of RGCs in the translimbal photocoagulation model ofexperimental glaucoma in Wistar rats (Levkovitch-Verbin et al.,2011).

4.1.3. NorgestrelNorgestrel, a synthetic progestin used in oral contraceptives

with effects similar to progesterone, exhibited neuroprotectiveproperties in two distinct animal models of RP: the acute light-induced degeneration model and the more chronic rd10 mousemodel. In both models, norgestrel preserved both photoreceptorcell number and morphology to a significant degree and, as a

Fig. 18. Synaptic connectivity is preserved by neuroprotective treatments. Vertical sections oa (ON-rod bipolar cells), calbindin (horizontal cells) and bassoon (synaptic ribbons). Nucletauroursodeoxycholic acid (TUDCA) (C, F, J) or safranal (G, K) reduces the photoreceptor cbetween synaptic ribbons of photoreceptors (red) and dendrites of bipolar cells (compare F ahorizontal cells (compare J and K with I). ONL: outer nuclear layer; OPL: outer plexiform laye20 mm.

consequence, improved ERG responses (Doonan and Cotter, 2012;Doonan et al., 2011). The neuroprotective mechanism of action islikely to involve the increased expression and activation of bFGFand the extracellular signal-regulated kinases 1/2 (Erk1/2).

4.1.4. ProinsulinTransgenic expression of human proinsulin in the rd10 mouse

model of RP attenuated retinal degeneration, as determined by thehistological preservation of photoreceptors and ERG responses.Systemic proinsulin was able to reach the retinal tissue, delay theapoptotic death of photoreceptors and decrease oxidative damage(Corrochano et al., 2008). Furthermore, intramuscular injection ofan adeno-associated viral vector serotype 1 expressing humanproinsulin in the P23H rat model of RP attenuated retinal degen-eration by preserving cone and rod structure and function, togetherwith their contacts with postsynaptic neurons (Fernandez-Sanchezet al., 2012b).

4.2. Efficacy of antioxidant and anti-inflammatory agents

There are numerous compounds found in nature that containactive ingredients with beneficial properties for the improvementand/or prevention of various eye diseases affecting vision. In this

f Sprague Dawley (SD) and P23H rat retinas (P120) labeled with antibodies against PKC-i stained with TO-PRO. Treatment of P23H rats with the neuroprotective compoundsell death (compare photoreceptor rows in C and B) and the loss of synaptic contactsnd G with E), and between synaptic ribbons of photoreceptors (red) and terminal tips ofr; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar:


context, antioxidant and anti-inflammatory attributes make themuseful compounds for the pharmacological treatment of retinaldiseases.

4.2.1. CurcuminCurcumin is a natural polyphenolic yellow pigment isolated

from the rhizomes of the plant Curcuma longa with well-knownanti-inflammatory and antioxidant properties. In rat models ofLIRD, dietary addition of 0.2% curcumin during a period of twoweeks evidenced retinal neuroprotection through the inhibition ofNFekB activation and down regulation of inflammatory genes(Mandal et al., 2009). Further studies in retina-derived cell lines661W and ARPE-19 showed that treatment with curcumin pro-tected cells from hydrogen peroxide (H2O2) oxidative stress-induced cell death through the reduction of ROS levels, mediatedby an increase in the expression of the oxidative stress defenseenzymes heme oxygenase-1 (Mandal et al., 2009; Woo et al., 2012)and thioredoxin (Mandal et al., 2009). The analysis of microRNAs(miRNAs) on curcumin pre-treated ARPE-19 cells exposed to H2O2oxidative stress showed that curcumin alters the expression ofH2O2-modulated miRNAs and, as a consequence, regulates theexpression of their target genes, resulting in an increased expres-sion of antioxidant genes and a reduction of angiotensin II type 1receptor, NFekB and VEGF expression at the mRNA and proteinlevels (Howell et al., 2013). DR studies on streptozotocin-induceddiabetic rats have evidenced the protective effects of curcumin inthe retina, where it exerts a positive modulation of the antioxidantsystem through the regulation of glutathione, superoxide dismut-ase and catalase levels (Gupta et al., 2011). On the other hand,curcumin also prevents the increase in pro-inflammatory cytokinesIL-1b, NFekB, TNF-a and VEGF, as well as the structural degenera-tion of the diabetic rat retina (Gupta et al., 2011; Kowluru andKanwar, 2007; Mrudula et al., 2007). Furthermore, recent studieshave shown that curcumin protects retinal Müller cells in ratssuffering from the early stages of diabetes (Zuo et al., 2013). Cur-cumin has also demonstrated its neuroprotective activity againstNMDA toxicity in primary retinal cell cultures, possibly related to anincrease in the NMDA receptor type 2A subunit (Matteucci et al.,2011). Curcumin also exhibits beneficial effects on neuronal andvascular degeneration in the retina after ischemia and reperfusioninjury. In this sense, in the presence of ischemia and reperfusionstimuli, curcumin was able to inhibit ganglion cell loss, and toprevent the degeneration of retinal capillaries, probably through itsinhibitory effects on the injury-induced activation of NFekB andSTAT3, and on the overexpression of monocyte chemoattractantprotein-1 (Wang et al., 2011a). Curcumin also suppressed N-methyl-N-nitrosourea-induced photoreceptor cell apoptosis inSpragueeDawley rats through inhibition of DNA oxidative stress(Emoto et al., 2013). Besides its anti-inflammatory and antioxidantproperties, curcumin also exhibits anti-protein aggregating activity.In this context, an improvement in retinal morphology, physiologyand gene expression and localization of rhodopsin has beenobserved in the P23H transgenic rat model of autosomal dominantRP treated with curcumin (Vasireddy et al., 2011).

4.2.2. Lutein and zeaxanthinLutein and zeaxanthin are carotenoids that are referred to as

macular pigments, due to their presence in the human macula. It issuggested that these carotenoids may protect the macula and outerretinal photoreceptor segments from oxidative stress by triggeringthe antioxidant cascade that disables ROS (Krinsky et al., 2003;Ozawa et al., 2012). Studies on cultured ARPE-19 cells showed ev-idence that supplementation with lutein and zeaxanthin reducedphoto-oxidative damage and inhibited the expression ofinflammation-related genes in RPE cells (Bian et al., 2012a, 2012b).

Similarly, both carotenoids protected photoreceptors from oxida-tive stress-induced apoptosis in rat retinal neurons in culture(Chucair et al., 2007). Additionally, lutein and zeaxanthin both actas light filters in the eye, absorbing the blue-light that enters theretina, hence effectively protecting the retina from LIRD duringacute light exposure and in the presence of bright light (Barkeret al., 2011; Ozawa et al., 2012). Furthermore, results from variousepidemiological studies have shown inverse associations betweenthe amount of macular pigment and the incidence of AMD. Alongthe same lines, clinical studies have shown that supplementationwith lutein and zeaxanthin improves visual function and preventsthe progression of the pathology in patients with early AMD (Chewet al., 2014; Ma et al., 2012a, 2012b; Sabour-Pickett et al., 2012).Similarly, clinical studies evidenced that zeaxanthin improves vi-sual function in older male patients with AMD (Richer et al., 2011).Likewise, the neuroprotective effect of lutein on outer retinal cellsin AMD may also play a relevant role in the protection of the innerretina from acute ischemic damage, as demonstrated by thereduction in oxidative stress and apoptotic death in a rodent modelof ischemia/reperfusion (Dilsiz et al., 2006; Li et al., 2009).

4.2.3. SaffronThe active ingredients of the spice saffron (safranal, crocin and

crocetin) are known antioxidant carotenoids. Crocetin preventedretinal degeneration induced by oxidative and endoplasmic retic-ulum stresses via the inhibition of caspase 3 and 9 activity in theRGC-5 retinal ganglion cell line in vitro and in a LIRD mice modelin vivo (Yamauchi et al., 2011). Crocin protected retinal photore-ceptors against light-induced cell death in primary cell culturesfrom primate and bovine retinas (Laabich et al., 2006). In albino ratsfed saffron supplements, the effects of continuous bright lightexposure were significantly diminished, and the morphology andfunction of the retina were maintained (Maccarone et al., 2008;Marco et al., 2013; Natoli et al., 2010). Crocetin inhibited retinalischemic damage in mice, preventing the apoptotic death of gan-glion cells and the reduction of the INL by inhibiting the activationof p38, JNK, NFekB and c-Jun, while maintaining at the same timethe functional activity of the retina (Ishizuka et al., 2013). In asimilar manner, crocin prevented retinal ischemia/reperfusioninjury-induced apoptosis of RGCs in rats by activating the PI3K/AKTsignaling pathway (Qi et al., 2013). Dietary supplementation withsafranal in the P23H rat model of RP slowed photoreceptor celldegeneration (Fig. 17C, F and 18G, K) and ameliorated the loss ofretinal function and vascular network disruption (Fernandez-Sanchez et al., 2012a). Crocetin also prevented NMDA-inducedmurine retinal damage by inhibiting both caspases 3/7 activationand the increased expression of cleaved caspase 3 in the GCL andINL (Ohno et al., 2012). Additionally, in clinical trials involvinghuman patients with early AMD, 20 mg per day of saffron supple-mentation for 90 days significantly improved some parameters ofthe macular photopic flash electroretinogram, such as amplitudeand modulation threshold (Falsini et al., 2010).

4.2.4. CatechinsCatechins are a group of polyphenolic antioxidants commonly

found in green tea. The most abundant catechin in green tea isepigallocatechin gallate (EGCG), which has extremely strong anti-oxidant properties. Previous studies involving intraocular injectionof EGCG with sodium nitroprusside showed a protective effect onrat retinal photoreceptors, indicating that EGCG may benefit pa-tients suffering from ocular diseases involving oxidative stress(Zhang and Osborne, 2006). Oral administration of EGCG toischemia/reperfusion rat models reduced many of the induceddamaging effects, including the activation of caspases, the reduc-tion in the ERG a- and b- wave amplitudes, the decrease in RGC and


photoreceptor specific proteins, the increase in GFAP protein andthe decrease in optic nerve proteins associated with ganglion cellaxons (Zhang et al., 2008, 2007). Moreover, dietary administrationof EGCG reduced light-induced retinal neuronal death in albino ratmodels (Costa et al., 2008). EGCG also reduced apoptotic death inthe RGC-5 cell line generated by light damage (Zhang et al., 2008)or H2O2 (Fan et al., 2008; Zhang et al., 2007). In addition, EGCG hasthe ability to inhibit RPE cell migration and adhesion, therebyproviding potential preventive actions against AMD (Alex et al.,2010; Chan et al., 2010). Furthermore, administration of EGCG inrats prior to axotomy promoted RGC survival through nitric oxide,anti-apoptotic and cell survival signaling pathways modulation(Peng et al., 2010). On the other hand, EGCG administered toWistarrats resulted in a significantly lower NMDA-related loss of RGCs(Chen et al., 2012). Interestingly, studies in patients with open-angle glaucoma treated with oral EGCG suggest a beneficial influ-ence of this compound on inner retinal function (Falsini et al.,2009).

4.2.5. Ginkgo biloba extractGinkgo leaves contain two main active ingredients: flavonoids

and terpenoids. The main properties of G. biloba extract are pro-tection against free radical damage and lipid peroxidation. InSprague Dawley rats that were administered G. biloba extract priorto and/or on a daily basis following experimental optic nerve crush,the survival rate of RGCs was significantly higher than in controlanimals (Ma et al., 2009, 2010). Studies on rodents showed that thestandardized extract of G. biloba EGb761 partially inhibitedapoptosis of photoreceptor cells, increasing the cell survival rateafter light-induced retinal damage of photoreceptors (Qiu et al.,2012; Xie et al., 2007). Similarly, EGb761 showed a protective ef-fect on human RPE cells (ARPE-19) under light-induced damagestimuli by acting on HSP70, cathepsin B and cytochrome c reduc-tase modulation (Zhou et al., 2014). EGb761 also protected theretina against ischemia-reperfusion damage via its free radical-scavenging and anti-lipoperoxidative properties, as well as itsregulation of mitochondrial respiratory function (Clostre, 2001). Inaddition, EGb761 was found to provide a neuroprotective benefitfor RGCs in a rat model of chronic glaucoma (Hirooka et al., 2004).G. biloba is also believed to have good therapeutic potential in casesof normal tension glaucoma, where the disease continues toprogress despite surgically normalized intraocular pressure(Cybulska-Heinrich et al., 2012). In retinal explants, ginkgolide B, acomponent of EGb761, has also been demonstrated to promote RGCaxon growth and to decrease cellular RGC apoptosis through theinhibition of caspase 3 activity (Wang et al., 2012). In addition, afortified extract containing G. biloba extract (among other compo-nents) attenuated retinal inflammation in early streptozotocin-induced diabetic rats by decreasing TNF-a and VEGF cytokinelevels (Bucolo et al., 2013).

4.2.6. ResveratrolResveratrol is a polyphenol contained in red wine with strong

antioxidant properties. Many studies have evidenced that resver-atrol reduces diabetes-induced early vascular lesion, VEGF, andoxidative stress in rat and mice models (Kim et al., 2012; Yar et al.,2012). Resveratrol administered to streptozotocin-induced diabeticrats significantly reduced the enhancement of oxidative markersand superoxide dismutase activity in the retina. Moreover, resver-atrol improved the elevated levels of NFekB activity and theapoptosis rate, and prevented the reduction in thickness of theretina (Soufi et al., 2012). Resveratrol has also been shown to beeffective in decreasing vascular lesions and VEGF induction inmouse retinas during the early stages of diabetes (Kim et al., 2012).This polyphenol also prevented diabetes-induced RGC death via

CaMKII down-regulation in streptozotocin-diabetic mice (Kimet al., 2010). Trans-resveratrol inhibited hyperglycemia-inducedlow-grade inflammation and connexin down-regulation in RPEcultured cells through inhibition of VEGF, TGF-b1, COX-2, IL-6 andIL-8 accumulation, PKCb activation, Cx43 degradation andenhanced intercellular gap junction communication (Losso et al.,2010). Resvega, a nutritional complex containing resveratrol, hasalso been demonstrated to provide beneficial effects for the pre-vention of CNV in amurine model of laser-induced CNV (Fernandezet al., 2013). It has also been demonstrated that resveratroladministration to rat models of retinal detachment preventsphotoreceptor cell death via the upregulation of FoxO family pro-tein levels (FoxO1a, FoxO3a, FoxO4) and by blocking caspase 3, 8and 9 activation (Huang et al., 2013b). In an ongoing clinical study,preliminary observations on the human retina in octogenarianAMD patients taking a daily resveratrol-based nutritional supple-ment showed an anatomic restoration of retinal structure, whichsuggested an improvement in choroidal blood flow, and as aconsequence, better visual function with the treatment (Richeret al., 2013). RPE cell death occurs early in the pathogenesis ofAMD, and for this reason, protecting these cells is essential intreating the disease. In this context, in RPE cell lines, resveratrol hasbeen shown to defend against oxidative stress (King et al., 2005;Pintea et al., 2011; Sheu et al., 2008), oxysterol-induced cell deathand VEGF secretion (Dugas et al., 2010), and to inhibit RPE cellmigration in a dose-dependent manner (Chan et al., 2013). On theother hand, resveratrol prevented LIRD in mouse models by sup-pressing the activation of retinal activator protein-1 and promotingretinal sirtuin activity (Kubota et al., 2010). Furthermore, resvera-trol prophylactic treatment reduced ischemia-mediated thinning ofthe entire retina and in particular the inner retinal layers, attenu-ating ischemic-induced loss of retinal function in rats (Vin et al.,2013). Since retinal ischemia is a major factor in close-angle glau-coma and DR, resveratrol could be a potentially useful drug forvascular dysfunction in the retina (Li et al., 2012a). The aforemen-tioned properties of resveratrol could prove essential in establish-ing innovative treatments and preventive interventions for majorocular diseases, such as AMD, glaucoma and DR, since oxidativestress is an integral part of the pathophysiology of those diseases.

4.2.7. QuercetinQuercetin is a flavonoid with anti-inflammatory and anti-

oxidative properties found in a variety of plant foods, includingblack and green teas, Brassica vegetables and many types of berries.Recent studies on the human RPE cell line ARPE-19 have demon-strated the protective effects of quercetin against oxidative stressthrough the inhibition of pro-inflammatory molecules, as well asdirect inhibition of the intrinsic apoptosis pathway (Cao et al.,2010). In vitro studies using the RF/6A rhesus choroids-retinaendothelial cell line treated with quercetin showed a dose-dependent inhibition of cellular migration and tube formation,which are important steps in retinal angiogenesis, characteristic ofAMD (Chen et al., 2008). Further studies on human cultured RPEcells showed similar results. In this context, quercetin treatmentfollowed by oxidative damage reduced cellular deterioration andsenescence occurring in AMD in a dose-dependent manner, prob-ably by inhibiting the up-regulation of caveolin-1 (Kook et al.,2008). Similarly, quercetin significantly reduced ROS productionby ascorbate/Feþ2-induced oxidative stress in retinal cell cultures(Areias et al., 2001).

4.2.8. N-acetylcysteine (NAC)Oral administration of NAC to rd1 and rd10 mouse models of RP

decreased cone cell death and preserved cone function by reducingoxidative damage (Lee et al., 2011). In addition, NAC administered


to a rat model of DR diminished the plasma markers of oxidativestress and inflammation (15-F(2t)-isoprostane and TNF-a, respec-tively), helping to minimize early events in DR (Tsai et al., 2009).Moreover, NAC supplementation to rats with induced ocular hy-pertension ameliorated the retinal oxidative damage through themaintenance of glutathione peroxidase and catalase levels, and theinhibition of retinal peroxidation (Ozdemir et al., 2009). NAC alsoprevented the increased expression of p53 and caspase 8, inducedby long-term maintained hypoxia in bovine RPE primary cell cul-tures (Gerona et al., 2010), which makes it of great interest in ox-ygen stress-related diseases, such as AMD and other senescence-associated pathologies. In this context, hypoxia-induced celldeath in the RGC-5 cell line was significantly counteracted by pre-treating cells with NAC, which targets the hypoxia-inducible factor-1a pathway via the BNIP3 and PI3K/Akt/mTOR pathways (Yanget al., 2012).

4.2.9. Antioxidant cocktailsThe administration of a cocktail of antioxidants (including a-

tocopherol, ascorbic acid, Mn(III)tetrakis (4-benzoic acid)porphyrin, and a-lipoic acid) to threemousemodels of RP (rd1, rd10and Q344ter) reduced the levels of oxidative damage markers incones and, as a consequence, preserved cone density (Komeimaet al., 2007, 2006). Additionally, this mixture of antioxidantsslowed rod cell death, thus maintaining photoreceptor function, asdemonstrated by the larger a- and b-wave amplitudes as comparedto untreated animals (Komeima et al., 2007, 2006). The use of an-tioxidants in a combination consisting of lutein, zeaxanthin, a-lipoic acid and reduced L-glutathione (GSH) in rd1 mice drasticallyreduced the number of rod photoreceptors displaying oxidativelydamaged DNA, and significantly delayed the degeneration process(Miranda et al., 2010; Sanz et al., 2007). Thiol contents and thiol-dependent peroxide metabolism seem to be directly related tothe survival of photoreceptors in rd1 mouse retinas (Miranda et al.,2010).

4.3. Efficacy of neurotrophic factors

Trophic or growth factors are endogenously secreted substances(either proteins or steroid hormones) that generally function topromote cell proliferation, maturation, survival and/or regenera-tion, thereby maintaining overall cell homeostasis (Snider andJohnson, 1989; von Bartheld, 1998). In the eye, the major sourcesof these molecules are retinal RPE and Müller cells. Exogenousadministration of these pro-survival factors, either singly or incombination, has been used in attempt to ameliorate retinaldegeneration. It is important to note that the short half-life ofneurotrophic factors makes iterative intravitreal injections neces-sary. To solve this problem, researchers have developed new stra-tegies for the long-term delivery of trophic factor in the eye. The useof nanoparticles, viral-mediated transference and implants ofencapsulated cells producing neurotrophic factors inserted in thevitreous cavity are examples of improved delivery methods.

Several neurotrophic factors have demonstrated success inpreventing or delaying retinal degeneration in different LIRD ani-mal models; these include BDNF (LaVail et al., 1992), GDNF (Readet al., 2010), acidic FGF (LaVail et al., 1992), bFGF (Lau andFlannery, 2003; LaVail et al., 1992; Li et al., 2003), CNTF (LaVailet al., 1992) and pigment epithelium derived factor (PEDF) (Caoet al., 2001; Imai et al., 2005). A wide variety of neurotrophic fac-tors have also been shown to have neuroprotective effects ondegenerating photoreceptors through the morphological andfunctional protection of rods in models of RP. Examples of this areGDNF (Andrieu-Soler et al., 2005; Frasson et al., 1999; McGeeSanftner et al., 2001), bFGF (Lau et al., 2000), CNTF (Cayouette

et al., 1998; LaVail et al., 1998) and PEDF (Cayouette et al., 1999).Interestingly, the combination of some of these trophic factorsprovides synergistic neuroprotection in photoreceptor rescue(Miyazaki et al., 2008). Furthermore, they also have demonstratedbeneficial properties in the treatment of animalmodels of ischemia,intraocular pressure and retinal detachment, among other pathol-ogies. An extended review of the in vivo effects of the exogenousadministration of trophic factors in animal models of retinaldegeneration has been recently published (Kolomeyer and Zarbin,2014). Rod-derived cone viability factor is a diffusible moleculesecreted by rod cells that promotes cone survival. In this context,rod-derived cone viability factor injected into the eye of the P23Htransgenic rat model of RP preserved the vision by increasing conesurvival and function (Yang et al., 2009). Clinical trials withencapsulated ARPE-19 cells secreting CNTF into the vitreous ofpatients with RP and AMD showed evidence of photoreceptorprotection and/or improved visual acuity (Birch et al., 2013;Emerich and Thanos, 2008; Sieving et al., 2006; Talcott et al.,2011; Zhang et al., 2011).

4.4. Gene therapy approaches and clinical trials

4.4.1. Viral-mediated therapiesThe most widely used vectors for ocular gene delivery are based

on adeno-associated virus (AAV). AAV vectors do not integrate intothe host genome, rather they exist as extragenomic circular epi-somes, which significantly decreases the risk of insertional onco-genesis. They typically elicit minimal immune responses and allowfor stable, long-term transgene expression in a variety of retinalcells, such as photoreceptors, RPE, Müller and ganglion cells.Adenoviral vectors are also non-integrative vectors; however, theyelicit robust cytotoxic T lymphocyte-mediated immune responsesthat limit the duration of transgene expression. Lentiviral vectorscan induce stable, long-term transgene expression in anteriorocular structures, including the corneal endothelium and thetrabecular meshwork, in addition to retinal tissues. Because theyare integrating vectors, justifiable concerns have been raised overthe risk of insertional oncogenesis. A variety of non-viral oculargene transfer methods have also been studied, including the use ofDNA nanoparticles (Conley and Naash, 2010; Farjo et al., 2006), thefC31 integrase system (Chalberg et al., 2005), and electroporationand lipofection (Kachi et al., 2005). Although they have seen sometangible success in ocular applications, they will not be discussedfurther in this review.

4.4.1.1. Diabetic retinopathy. Neovascularization associated withDR and AMD is a leading cause of visual impairment and adult-onset blindness. Gene transfer of anti-angiogenic proteins is anapproach that has the potential to provide long-term suppressionof neovascularization and/or excessive vascular leakage in the eye.Gene transfer of anti-angiogenic PEDF via AAV2 injected in thevitreous has been reported to have positive effects on a transgenicmouse model that mimics the chronic progression of human DR(Haurigot et al., 2012). Long-term production of PEDF produced astriking inhibition of intravitreal neovascularization, normalizationof retinal capillary density, prevention of retinal detachment, andreduction in the intraocular levels of VEGF. Furthermore, as aconsequence of the latter, there was a down-regulation of down-stream effectors of angiogenesis, such as the activity of matrixmetalloproteinases 2 and 9 and the content of connective tissuegrowth factor (Haurigot et al., 2012). Other researchers con-structed an AAVrh.10 coding for bevacizumab (an anti-VEGFmonoclonal antibody), which was injected in the vitreous oftransgenic mice overexpressing human VEGF165 in photorecep-tors. Directed long-term bevacizumab expression in the RPE


efficiently suppressed VEGF-induced retinal neovascularization(Mao et al., 2011). The protective effect of overexpressing ACE2(angiotensin I converting enzyme 2) and Ang-(1-7) (angiotensin1e7) genes by intravitreal injection mediated by AAV in the retinaof streptozotocin-induced diabetic eNOS�/- mice and Spra-gueeDawley rats has also been confirmed (Verma et al., 2012). Theincreased levels of ACE2/Ang-(1e7) resulted in a significantreduction of diabetes-induced retinal vascular leakage, acellularcapillaries, infiltrating inflammatory cells and oxidative damage.On the other hand, AAV2-mediated intravitreal gene delivery ofthe high-affinity soluble VEGF receptor hybrid called sFLT01 effi-ciently inhibited angiogenesis in the mouse oxygen-induced reti-nopathy model (Pechan et al., 2009). AAV-mediated geneexpression of sFLT1 injected into the subretinal space also provedto be efficient in the spontaneously diabetic non-obese Torii ratmodel of human DR (Ideno et al., 2007). Auricchio and coworkersused AAV vectors for the gene transfer to the eye of three anti-angiogenic factors: PEDF, tissue inhibitor of metalloproteinase-3and endostatin. They observed that, in all cases, the treatmentinhibited retinal neovascularization in a mouse model of retinop-athy of prematurity (Auricchio et al., 2002).

4.4.1.2. Age-related macular degeneration. The use of anti-angiogenic therapy is also effective in the treatment of AMD. Infact, the most effective treatment for AMD-associated CNV iscurrently the administration of anti-VEGF compounds (mono-clonal antibodies, siRNA, RNA oligonucleotides, among others).There are treatments already approved for wet AMD. Of the newantivascular inhibitors undergoing testing, three anti-VEGF ther-apies are approved by the US Food and Drug Administration (FDA)at this time for the treatment of AMD-associated CNVs: pegapta-nib (Gragoudas et al., 2004), ranibizumab (Brown et al., 2009;Rosenfeld et al., 2006), and aflibercept (Heier et al., 2012). Bev-acizumab is also widely used, although it is not approved(Chakravarthy et al., 2012; Martin et al., 2012). Meanwhile, thesearch for other therapy strategies continues in various animalmodels. In this regard, subretinal AAV delivery vehicles with shorthairpin RNAs have been employed, targeting the pro-angiogenicgrowth factor VEGF mRNAs. This approach has successfullyinhibited endogenous mouse VEGF protein expression in the laser-induced murine model of CNV, and as a consequence, reduced theformation of CNV (Askou et al., 2012). Moreover, AAV-mediatedgene expression of sFLT1 injected into the subretinal space hasbeen demonstrated to be successful in CNV-induced mice (Igarashiet al., 2010). Subretinal or intravitreal delivery of an AAV vectorexpressing a transgene for a soluble non-membrane binding formof human CD59, a naturally occurring membrane bound inhibitorof membrane attack complex, attenuated the formation of laser-induced CNV and murine membrane attack complex formation(Cashman et al., 2011). Oxidative stress in RPE cells is another keyto fighting the AMD pathology. In this context, overexpression ofthe human X-linked inhibitor of apoptosis using recombinant AAVin ARPE-19 cells exposed to H2O2-induced oxidative death pro-tected the cells from death by acting on the apoptotic pathway(Shan et al., 2011). Moreover, the use of PEDF in therapiesdesigned to prevent or reduce the neovascularization associatedwith AMD and DR is very common. Viral-mediated PEDF intra-ocular injection has demonstrated beneficial properties in animalmodels of laser-induced CNV and transgenic VEGF (Mori et al.,2002; Murakami et al., 2010), retinal ischemia/reperfusion(Takita et al., 2003) and LIRD (Imai et al., 2005). In a phase 1clinical trial, a single intravitreal injection of AdPEDF.11 in 28 pa-tients with advanced neovascular AMD maintained or reduced theCNV size after up to 12 months of follow up (Campochiaro et al.,2006).

4.4.1.3. Retinitis pigmentosa. Different strategies are used to treatRP disease, according to its etiology. A suitable approach for cases ofautosomal dominant RP is ‘gene silencing and replacement’. First,the levels of both mutant and wild type mRNA are knocked downusing allele non-specific ribozymes or siRNAs (mutation-indepen-dent suppression). Second, it is essential to deliver an allele cDNAresistant to ribozyme or siRNA mediated degradation. In cases ofautosomal recessive RP, the common strategy is to add a wild-typeallele to increase the level of functional protein.

Greenwald and collaborators developed a mouse model ofautosomal dominant RP expressing a pathogenic mutant humanrhodopsin gene on a rhodopsin knockout background. These au-thors observed higher ERG a-wave responses in the eyes where thephotoreceptors were transduced with AAV containing a microRNAsequence targeting the human mutant rhodopsin gene, whichsilenced its expression, and a ‘codon-modified’ rhodopsin (RhoR2)resistant to degradation by the microRNA (Greenwald et al., 2013).Similarly, long-term preservation of normal retinal function andnormal retinal dimensions and morphology, including the preser-vation of photoreceptor cells, have been observed in the P23Htransgenic mouse model of autosomal dominant RP injected with asingle dose of an AAV expressing both a small interfering RNA(siRNA301), which cleaves rhodopsin mRNA at nucleotide 301, anda modified rhodopsin cDNA with five silent base changes sur-rounding position 301, which is resistant to siRNA digestion (Maoet al., 2012). A delay in retinal degeneration has also been re-ported in the P347S rhodopsin transgenic mouse model of auto-somal dominant RP using two AAV subretinally co-injected andexpressing an interference RNA to suppress rhodopsin and a codon-modified rhodopsin gene resistant to suppression due to nucleotidealterations at degenerate positions over the interference RNA targetsite (Millington-Ward et al., 2011). Another method used to developa mutation-independent treatment for autosomal dominant RP isthe use of ribozymes. It has been demonstrated that AAV delivery ofrhodopsin-specific ribozyme (Rz525) rescues vision in P23H line 3rats by diminishing the expression of the P23H transgene(Gorbatyuk et al., 2007). Consequently, Rz525 is a candidate ribo-zyme for RNA replacement gene therapy when combined with aribozyme-resistant rhodopsin gene.

Conlon and his group demonstrated the potency and safety ofocular injection of AAV vectors expressing human MERKT cDNA inthe RCS rat model of autosomal recessive RP. Vector-injected eyesshowed improved ERG responses as compared to untreated eyes.Furthermore, funduscopic analysis and postmortem retinalmorphology of vector-injected eyes were normal as compared tothe controls (Conlon et al., 2013). Subretinal administration of AAVexpressing the wild-type mouse Mfrp (membrane-type frizzled-related protein) gene prevented retinal degeneration in the Rd6Mfrp mutant mouse model of autosomal recessive RP (Dinculescuet al., 2012). CNGB1a (subunit of rod cyclic nucleotide-gated(CNG) channel) gene replacement via subretinal space AAV de-livery restored the rod CNG channel expression and localization,improving retinal function and vision-guided behavior, and delay-ing retinal degeneration in autosomal recessive RP mouse modelCNGB1�/� (Koch et al., 2012).

Beltran and collaborators evaluated the retina of two blindingcanine photoreceptor diseases that model the common X-linkedform of RP caused by mutations in the RP GTPase regulator(RPGR) gene, which encodes a photoreceptor ciliary protein.XLPRA1 and XLPRA2 canine models were subretinally injectedwith an AAV2/5 vector carrying a full-length normal human RPGRgene. Gene augmentation rescued photoreceptors from death andreversed mislocalization of rod and cone opsins in both XLPRAmodels, thus alleviating the characteristic features of photore-ceptor degeneration, such as the progressive changes that take


place in the OPL, bipolar cells and inner retinal layers (Beltranet al., 2012).

As mentioned earlier, due to the broad genetic heterogeneity ofRP disease, gene-specific therapies are impractical, and thus thedevelopment of mutation-independent treatments to slowphotoreceptor cell death is required. One promising strategy forphotoreceptor neuroprotection is neurotrophin secretion fromMüller cells, the primary retinal glia. Müller glia are excellenttargets for secreting neurotrophins, as they span the entire tissue,connect with all neuronal populations, are numerous and persistthroughout retinal degeneration. An AAV variant (ShH10) has beenengineered which efficiently and selectively transduces glial cellsthrough intravitreal injection (Dalkara et al., 2011). ShH10-mediated-GDNF secretion from the glia generated high GDNFlevels in treated retinas, leading to sustained functional rescueover 5 months in the TgS334-4ter rat model of RP. Other re-searchers have argued that manipulating cell death and pro-survival pathways and shifting the balance in photoreceptor cellstoward cell survival could be a reliable therapeutic approach forpreserving vision in RP patients, and may represent a morepromising approach than gene replacement therapy. Along theselines, researchers have assayed subretinal injections of P23H ratswith AAV expressing human GRP78 (Bip) or functional rescue ofphotoreceptors with HSF-1 (Gorbatyuk et al., 2012). Both GRP78and HSF-1 overexpression increased the a- and b-wave responseamplitudes and the integrity of the retina as compared to un-treated eyes.

AAV-mediated gene replacement has also been used to restorevisual function in a dog model of Leber congenital amaurosis, aretinal degeneration that can cause severe childhood visual loss(Acland et al., 2001; Annear et al., 2013). The gene defect in thenaturally occurring dog model, a mutation in the RPE65 gene thatcodes for an RPE cell membrane-associated protein involved inretinoid metabolism, also occurs in human Leber congenitalamaurosis. An AAV carrying wild-type RPE65 was able to restorevision as assessed by electroretinography, pupillometry, and psy-chophysical and behavioral tests. Human clinical trials using asimilar vector are currently ongoing. Trials in human patients withRPE65-associated Leber congenital amaurosis have shown thatgene therapy leads to substantial visual improvement (Cideciyanet al., 2008, 2013; Jacobson et al., 2012).

4.4.1.4. Glaucoma. Neuroprotection of RGCs is an important goalin glaucoma therapy. PEDF is a potent anti-angiogenic, neuro-protective and anti-inflammatory factor for neurons. Intravitrealinjection of AAV expressing PEDF in the DBA/2J mouse model ofinherited glaucoma reduced the loss of RGC and nerve fiber layer,delayed vision loss and reduced TNF, IL-18 and GFAP expressionin the retina and optic nerve (Zhou et al., 2009b). In a rat modelof experimental glaucoma, the use of recombinant AAV totransduce to RGCs genes encoding constitutively active or wild-type MEK1, the upstream activator of Erk1/2, markedlyincreased neuronal survival (Zhou et al., 2005). Thus, selectiveactivation of the Erk1/2 pro-survival pathway protected RGCs.Martin and collaborators used an AAV incorporating cDNA forBDNF to transfect RGCs in a rat model of glaucoma, and observedthat intravitreal AAV-BDNF rescued RGCs (Martin et al., 2003).Interestingly, the final common pathway of RGC apoptosis in-volves the activation of caspase enzymes. In this context, an AAVvector coding for human baculoviral IAP repeat-containing pro-tein-4 (BIRC4), a potent caspase inhibitor, injected into one eye ofa rat model of experimental glaucoma, allowed the researchers toconclude that BIRC4 delivery significantly promoted optic nerveaxon survival in a rat chronic ocular hypertensive model ofglaucoma (McKinnon et al., 2002).

4.4.2. OptogeneticsThe severe loss of photoreceptor cells caused by retinal

degenerative diseases such as RP can result in partial or completeblindness. As a new strategy to treat blindness caused by retinaldegeneration, researchers have developed optogenetic tools in anattempt to restore retinal photosensitivity by creating new pho-tosensors and coupling them to the remaining retinal circuitry.The two best-known optogenetic tools are channelrhodopsin-2(ChR2), from the algae Chlamydomonas reinhardtii, and hal-orhodopsin (NpHR), from the archaebacterium Natronomonaspharaonis. These proteins are photosensitive and can be activatedat specific light wavelengths. For this reason, it was suggestedthat introduction of these molecules through gene transfer canrender the cells of the inner retina photosensitive, thus impartinglight sensitivity to retinas lacking rods and cones (Fig. 19).Channelrhodopsin2 was thus used to sensitize either RGCs(Fig. 19D) or ON-bipolar cells (Fig. 19C) in mice with retinaldegeneration (Bi et al., 2006; Doroudchi et al., 2011; Lagali et al.,2008). Using the chloride pump halorhodopsin, visual functionwas restored in animal models of RP at the level of the retina andcortex, as well as behaviorally (Busskamp et al., 2010). Thetranslational potential of this optogenetic approach has beensupported by the efficacy of the transduced halorhodopsinexpression in human photoreceptors in tissue explants frompostmortem human retinas, while clinical examinations in blindpatients confirmed the presence of dormant cone photoreceptorsthat could be reactivated through this approach (Fig. 19B)(Busskamp et al., 2010). Moreover, the co-expression of Chan-nelrhodopsin2/HaloR in RGCs restored both ON and OFF lightresponses in the retina after the death of rod and cone photo-receptors (Zhang et al., 2009).

Researchers have also used melanopsin (OPN4) as an intrinsiclight-sensitive protein. In this context, AAV have been successfullyused to ectopically express mouse melanopsin in RGCs of a mousemodel of photoreceptor degeneration (Lin et al., 2008). OPN4-transfected ganglion cells provided an enhancement of visualfunction in the mice, such that the pupillary light reflex returned toa nearly normal condition, the mice showed behavioral avoidanceof light in an open-field test and they were able to discriminate alight from a dark stimulus in a two-choice visual discriminationbehavioral test.

4.5. Cell-based therapies

Advanced therapies are different from conventional chemical-or protein-based therapies. The European Medicines Agency clas-sifies advanced therapies in three main groups, depending on theorigin of their products: genes (gene therapy), cells (cell therapy) ortissues (tissue engineering) (European Commission News; http://ec.europa.eu/health/human-use/advanced-therapies/index_en.htm). One such cell therapy, the RegenerativeMedicine, attempts tofind ways to replace cells in the body that have degenerated.

The central focus of regenerative medicine is human cells. Thecells used for cell therapy may be somatic, adult stem or embryo-derived cells. Currently, there are cells that have been reprog-rammed from adult cells so that they can be conveniently driven tobecome ‘pluripotent cells’ (Mason and Dunnill, 2008).

The field of stem cell based-therapy holds great potential for thetreatment of retinal degenerative diseases. The retina is one of thebest places to treat, not only with gene therapy, but also with cell-based approaches, due to easy access, immune privilege and rela-tive isolation from other body systems, as well as the differentoptions available to check therapeutic benefits and possible sec-ondary effects, both anatomical and functional. This is real whenthe eye is healthy, but becomes a utopia in the eyes affected by a

Fig. 19. Optogenetic therapy for retinal degeneration. (A) Schematic representation of a healthy retina with sensitive photoreceptors and functional bipolar and ganglion cells. Twosubpopulations of bipolar cells respond differently to the synaptic inputs from photoreceptors (ON and OFF visual pathways). (B) In pathological conditions, the remnant unhealthyrods and cones can be transduced with the rhodopsin-2 (ChR2) channel and halorhodopsin (NpHR) genes, respectively, to recover light sensitivity. (C, D) In advanced stages of thedisease, when the absence of photoreceptors is evident, expression of ChR2 and NpHR by ON and OFF bipolar (C) and ganglion (D) cells can restore the visual function.


disease or as soon as virus or cells are injected into the eye becausethe BRB is compromised.

Among the patients who may benefit directly from cellularreplacement strategies are those suffering from what are currentlyincurable eye diseases or other conditions with progressive visualimpairment due to the loss of specific cells, such as RP, AMD, DR,ischemic retinopathy and glaucoma. RPE cells and photoreceptorsseem to be good candidates for replacement, and are preferable tothe integration of RGCs, which need to redirect and extend theirprocesses towards the central nervous system after forming theoptic nerve. This redirection is clearly difficult to obtain, althoughthere is a great deal of interest because of its potential for thetreatment of optic neuropathies, such as glaucoma.

In order to be a potential source for the treatment of retinaldiseases, the cells must meet a number of conditions. Cell based-

therapies for photoreceptor or RPE regeneration should have ahigh level of efficacy and reproducibility, a low failure rate and,ideally, they should not require immunosuppression. The tissueneeds to be easy to propagate, with a low harvest rate and fewethical issues. Additionally, the cost should be low and the trans-plantation technique easy to perform (Ramsden et al., 2013). Thesubretinal space and vitreous cavity are known as immunoprivi-leged sites for transplantation, due to the blood-retina barrier(Jiang et al., 1993). The last, but not the least important, condition isthat it should be possible to obtain cells through a propermanufacturing process, and in sufficient quantities to allow forpatient transplantation. Although there are still some issues to besolved with regard to human pluripotent stem cells, both embry-onic and pluripotent stem cells seem to be the best bet for cellreplacement. They fulfill almost all the requirements and, in the


future, induced pluripotent stem cells will allow autologous celltherapies, if needed. This approach will solve the problem of im-mune rejection, once all other pending issues have been addressed.

Stem cell transplantation for retinal diseases is currently tran-sitioning from over a decade of preclinical research to phase 1-2clinical trials. The main focus of these trials is safety, with efficacybeing a secondary concern, and they are design to determine therequired levels of immunosuppression, the best delivery method,etc. Potential sources of these cells include pluripotent and multi-potent stem cells from both fetal and adult tissues.

4.5.1. Human embryonic stem cells (hESCs)Embryonic stem cells (ESCs) are derived from the inner cell mass

of blastocyst embryos. The cells can be multiplied in culture toalmost unlimited numbers and their pluripotency can be guided todifferentiate into any cell of the body. The potential of hESCs fortreating different diseases is unlimited. The progress made in re-cents in terms of the safety and efficacy of transplanted hESCs inanimal models of both neural and retinal degeneration has broughtthe field to the brink of clinical trials.

ESCs represent one of the most promising sources of cells fortransplantation, and considerable progress has been made in theirdifferentiation in the laboratory towards photoreceptor and RPElineages. There are different protocols that drive the cells in thesame direction they follow during embryonic development, goingthrough an anterior neuroectoderm fate to an eye field stage,forming optic vesicles and, at the end, differentiating into neuro-retina and RPE. Initial attempts to generate retinal cells tried toforce embryonic stem cells to chose their fate by default, usingeither bFGF, retinoid acid or ITSFn (insulin transferrin selenium andfibronectin) (Aoki et al., 2006; Hirano et al., 2003; Meyer et al.,2006; Sugie et al., 2005; Tabata et al., 2004; Zhao et al., 2006).Results improved after a number of different methods wereadopted, such as adding N2 supplement andmanually selecting thespheres with optic vesicle structure. The increase in MITF expres-sion through the addition of FGF-inhibitors (e.g., activin A) directsthe cells to an RPE fate (Meyer et al., 2009). Other methods use thesame factors that promote retinal induction (Nodal antagonist orinhibition of Notch pathway) and increase RPE fate by addingactivin A (Davis et al., 2000; del Barco Barrantes et al., 2003; Ikedaet al., 2005; Osakada et al., 2008, 2009) or morphogens to induceneural differentiation (Lamba et al., 2006).

4.5.1.1. Deriving photoreceptors from ESCs. Considering thedifferent ways inwhich retinal cells can be produced, ESCs could bean easy way to obtain photoreceptor and/or RPE cells to replace thelost ones in the retina. After being generated, it was necessary toprove that the cells could integrate into the retina and be func-tional. One of the first demonstrations of their functionwas done byKwan and collaborators in 1999, after transplanting normal micephotoreceptors in rd1 mice and showing synaptic formation andfunctional rescue (Kwan et al., 1999). MacLaren and coworkersshowed that it was possible to transplant photoreceptor cells intoan adult mouse retina, depending on the stage of development,using a post-mitotic photoreceptor precursor (Maclaren et al.,2006). They achieved poor integration, but were able to generatecells suitable for transplantation. They successfully restored visionin amousemodel of stationary night blindness, thus demonstratingthat the cells could be functional (Pearson et al., 2012). Someconcerns arose about the experiments where cells were injected,one of them being the decreased viability of the cells in suspension.Other problems were the lack of ability to pass through the internallimiting membrane and the glial changes that appear with degen-eration, which may interfere with the cell integration. Anotherissue to be addressed is that of the optimal stage of differentiation

of the transplanted cells (Gamm and Wright, 2013); donor photo-receptors frommore mature mice are able to integrate into the hostretina, but their efficacy decreases with maturity (Gust and Reh,2011). It would be ideal to have a cell maintain part of its pluripo-tency, but avoid teratoma formation and other problems associatedwith its differentiation. All the above mentioned studies demon-strated the feasibility of transplanting photoreceptors derived frommouse ESCs. Ongoing efforts towards clinical translation areneeded to develop human ESC lines in order to provide a poten-tially unlimited source of transplantation-competent photore-ceptor precursors with good integration, despite the use of aninjection method (Banin et al., 2006; Lamba et al., 2009).

4.5.1.2. Deriving RPE from ESCs. hESCs derived into RPE, either in aspontaneous way, directed by blocking Wnt and Nodal signalingpathways or through incubation with Activin A, replicate themorphology and function of RPE cells in the retina, maintainingtheir appearance, polarization and protein expression in vitro(Klimanskaya et al., 2004; Vugler et al., 2008a). These RPE cellsderived from hESCs have been successfully transplanted in animalmodels of retinal degeneration, especially the RCS rat (Idelson et al.,2009; Lu et al., 2009; Lund et al., 2006; Vugler et al., 2008a). An USFood and Drug Administration Phase 1/2 clinical trial has beenapproved in patients with Stargardt Disease and AMD, using in-jections of hESCs-derived RPE cells. The preliminary results pub-lished indicated the safety and tolerability of their stem cellimplantation, with no signs of hyperproliferation, tumorigenicity,ectopic tissue formation or apparent rejection after 4 months(Schwartz et al., 2012).

In summary, ESCs driven to become photoreceptors and/or RPEare a promising advance in retinal replacement therapies. Theirsafety and efficacy have been shown in a large number of preclin-ical studies in animal models. However, there are ethical andrejection issues, as well as the possibility of teratoma formation,which should be addressed by keeping under control their plu-ripotency and lineage-specific differentiation.

4.5.2. Human induced pluripotent stem cells (hiPSC)As soon as ESCs became familiar to the scientific world, a new

source of pluripotent cells appeared on scene: the inducedPluripotent Stem Cells (iPSC). In 2006, it was first reported how toreprogram adult somatic mouse cells (Takahashi and Yamanaka,2006); one later, Takahashi and Yu groups were able to drive hu-man fibroblasts to a pluripotent state with the ability to generatethe three embryonic layers (Takahashi et al., 2007; Yu et al., 2007).The process of generating pluripotent cells from somatic cells wastermed “reprogramming” and the resultant cells were calledinduced pluripotent stem cells. The iPSCs shared properties withhESCs, including the ability to self-renew and to be differentiatedinto the germ layers. Cell therapy using iPSC-derived cells still hasmany hurdles to overcome before they can be used in clinical ap-plications. However, since they are able to recapitulate the phe-notypes of a wide variety of diseases, patient-specific iPSCs mightbecome useful in the future for disease analysis, allowing to shedlight on their pathogenesis and discover effective new drugs(Egashira et al., 2013). The ultimate goal would be the generation ofindividual pluripotent lines to correct any individual genetic de-fects ex vivo and to transplant the required cell type back (Wrightet al., 2014).

The eye is an ideal target to explore the potential of hiPSCtechnology, not only to understand the disease pathways, but alsoto explore novel therapeutic strategies (Borooah et al., 2013). Thereare still some concerns about iPSCs. Transcribed genes, the epige-netic landscape, differentiation potential and mutational load showsmall, yet distinctive dissimilarities between iPSCs and ESCs, which


are considered the gold standard for in vitro pluripotency (Bilic andIzpisua Belmonte, 2012). However, murine iPSCs have beenexpanded stably and homogeneously for over 30 passages changingthe reprogramming strategy (Zhou et al., 2009a).

4.5.2.1. RPE production from hiPSCs. RPE cells can be generatedeither by following the same steps as with ESCs or through theformation of embryoid bodies, followed by plating the suspendedembryoid bodies onto a coated surface, dissecting them and lettingthem grow into pigmented spheroids (Buchholz et al., 2009, 2013;Carr et al., 2009; Gamm et al., 2008; Hirami et al., 2009; Meyeret al., 2009; Phillips et al., 2012). RPE cells from both hESCs andiPSCs are similar to RPE not only in their morphology, but also intheir function, polarity and gene and protein expression.

The replacement of RPE using iPSC should be easier than thereplacement of the inner retinal cells because of the less complexconnection established by the cells. RPE can be generated fromiPSCs in large amounts, with relative simplicity. The cells have beentransplanted into RCS rats and Rpe65 (rd12)/Rpe65 (rd12) micewith cell integration, functional response and the absence of tumorformation (Carr et al., 2009; Li et al., 2012b). Efficacy has been suchthat research has progressed to the clinical trial stage in the case ofESCs and, in spite of reservations expressed by some researchers,the use of RPE derived from iPSC has been already announced(Cyranoski, 2013).

4.5.2.2. Neuroretina production from hiPSCs. Both hESC- and hiPSC-derived neuroretinal progenitor cells differentiate in a similarmanner, mimicking the order and time course of normal retino-genesis (Meyer et al., 2011, 2009; Phillips et al., 2012). Pluripotentcells pass through an anterior neuroectoderm-like stage whenrecapitulating retinal differentiation in vitro, but the culture needsfurther processing than that required to obtain RPE (Meyer et al.,2009). Retinal differentiation has been obtained using bothadherent and 3-D aggregate differentiation methods.

The future of hiPSC-derived neuroretinal differentiation liesperhaps in the generation of a three-dimensional whole neuro-retina which recapitulates retinal development in vitro. This wasfirst tried using murine ESCs (Eiraku et al., 2011) and confirmedusing hESCs (Nakano et al., 2012). It was demonstrated that hiPSCsand ESCs could generate neuroepithelial-like clusters similar todeveloping optic vesicles (Meyer et al., 2011).

4.5.3. Human fetal embryonic stem cells; retinal progenitor cellsFetal stem cells are derived from embryonic and extra-

embryonic tissue. Retinal progenitor cells can be derived fromeither fetal or neonatal retinas and comprise an immature cellpopulation that is responsible for the generation of all retinal cellsduring development (Reh, 2006). Human prenatal retinal tissuewas one of the first donor sources used in patients. Humayun andcollaborators used a subretinal injection of a suspension of pre-natal neuroretinal cells in RP patients, with transient functionalimprovement (Humayun et al., 2000). Radtke and coworkers usedneuroretina sheets with attached RPE in AMD and RP patients,transplanted into the submacular area (Radtke et al., 2008). Hu-man fetal neural stem cells isolated from donated aborted fetusesaged 16e20 weeks have been used to prepare a suspension ofneutralized stem cells that were then injected into the subretinalspace of RCS rats, with good anatomical and functional results(McGill et al., 2012). The cells were unable to integrate into theretina, so the exact working mechanism is unknown. Based on thisstudy, it has initiated a clinical trial for treating dry AMD usingsubretinal injection of these cells (Clinicaltrial.gov identifierNCT01632527).

4.5.4. Human umbilical tissue-derived stem cellsHuman umbilical tissue has multipotent stem cells that are

considered to be adult stem cells. Suspensions of these cells havebeen able to improve anatomical degeneration and function in RCSrats due to a paracrine effect, with no cell integration (Lund et al.,2007). The repairing mechanism cannot be justified by the inte-gration of the transferred cells because no morphological changeswere observed in the injected cells; the reason for improving visionseems to be paracrine, being the cells able to secrete BDNF. Basedon these findings, another clinical trial for dry AMD treatment wasinitiated but at this time is not longer ongoing.

Stem cells from the blood of human umbilical cords have beenextensively used in studies on neurological pathologies, includingtraumatic optic nerve neuropathy. Some reports have shown thatthe cells can integrate into the retina (Koike-Kiriyama et al., 2007),while others have evidenced just the opposite, again suggesting aneuroprotective action through GDNF (Zwart et al., 2009).

4.5.5. Human central nervous system stem cells (HuCNS-SC)Human central nervous system stem cells (HuCNS-SC) trans-

planted into the subretinal space in RCS rat maintain an immaturephenotype throughout 7 months and undergo very limited prolif-eration with no evidence of uncontrolled growth or tumor-likeformation (McGill et al., 2012). Another study reveals that trans-plant of these stem cells also preserves the synaptic contacts be-tween photoreceptors and second order neurons bipolar andhorizontal cells, as well as phagocytosis of photoreceptor outersegments (Cuenca et al., 2013). This study indicated that the neu-roprotective transplantation of HuCNS-SC cells results in the sta-bilization of photoreceptor degeneration and slowing ofprogressive visual loss. The Food and Drug Administration (FDA)-authorized phase 1/2 clinical trial in AMD with geographic atrophyusing these stem cells and is underway.

4.5.6. Bone marrow-derived stem cellsBone marrow-derived stem cells may be divided into hemato-

poietic and mesenchymal types. Bone marrow-derived hemato-poietic stem cells are able to migrate after retinal damage, not onlyfrom endogenous locations, but also post-injection, reaching thedamaged retina and expressing the same RPE markers as RPE65(Atmaca-Sonmez et al., 2006; Li et al., 2007, 2006). The cells alsohave a demonstrated ability to stabilize and rescue retinal bloodvessels in rd mice and to induce neurotrophic rescue, preservingretinal layers (Otani et al., 2004). These aspects, and the fact that nosafety issues have been identified after injecting them into thevitreous humor of RP patients (although with no functional effect),have moved research forward to various clinical trials in order toevaluate the effects of these cells on RP, dry AMD, retina vein oc-clusion, DR, retinal and optic nerve diseases, glaucoma andischemic retinopathy (www.Clinicaltrials.gov).

Mesenchymal stem cells are also a good source for cellulartherapy. While bone marrow remains the primary source ofmesenchymal stem cells for most preclinical and clinical studies, fatsources are gaining increasing importance because cells can beeasily isolated in large numbers. Bone-marrow mesenchymal stemcells injected into the subretinal space of the rhodopsin knockoutmice and RCS rats are able to forestall functional decline with noimmune rejection problems, showing a lower immunogenic statusthan other stem cells (Arnhold et al., 2007, 2006; Lu et al., 2010).They are able to express RPE65 after systemic injection, with noother RPE features (Atmaca-Sonmez et al., 2006). Intravitreal in-jection of these cells was experimented with in three patients withRP and two patients with cone-rod dystrophies, with no side effectsbut limited functional improvement (Siqueira et al., 2011). Thisgroup is experimenting with the use of these cells in AMD, DR and


retinal vein occlusion, and is currently moving into the clinical trialstage. Additional clinical trials have been registered for these pa-thologies and others, such as RP, AMD and ischemia (www.Clinicaltrials.gov).

Stem cell transplantation for retinal diseases is currently tran-sitioning to clinical trials. The primary outcome of the first clinicaltrials is the safety of the procedure. Secondary outcomes will checkthe efficacy, the number of transplanted cells and the procedure.Early results are encouraging, and a real clinical application seemsto be closer every day. ESC-derived RPE is being used in ongoingclinical trials to repair the damage of outer retina diseases.Although multiple animal studies have shown that the trans-plantation of photoreceptors is able to prevent degeneration, theirintegration and function need to be clear for initiating humantreatments. There are points that need to be elucidated, such as thebest cell type and the bestmoment during the course of the disease.Immunosuppression is still an issue for long-term survival andhuman treatment. Another challenge is the survival of the cell in aretina with extensive remodeling and changes in circuitries. iPSCswould constitute a revolutionary, personalized treatment if all theissues regarding their production, expensive cost and correction ofgenetic defects in patient-specific cells were addressed. In themeantime, they might be best used to analyze the physiopathologyand potential pharmacological treatment of each disease. Asreprogramming, differentiation and cell characterization protocolscontinue to improve, it is likely that stem cell technology willbecome easier to use and more widely accessible and available.Advanced therapies, through both gene therapy and stem cell-based therapy, provide hope for retinal degenerative diseases thatcurrently have no cure. However, it remains clear that a customizedapproach will be needed to adequately treat any retinal disease.

4.6. Effectiveness of retinal transplantation

Retinal transplantation is another potential therapeuticapproach to restore vision in patients with advanced degenerativedisease. In this process, sheets of developing retina and RPE cellsare inserted into the subretinal space. Acceptable efficacy andsafety levels have been reported for human fetal retina implantswith accompanying RPE in AMD and RP patients with vision of 20/200 or worse. Using this approach, seven of ten patients (three RP,four AMD) showed improved visual acuity, corroborating results inanimal models of retinal degeneration (Radtke et al., 2008).Photoreceptor sheets or whole retinal sheets (both outer and innerlayers) from postnatal P8 healthy rat retinas transplanted into 3-month-old P23H rats improved the amplitude of the ERG b-waveand exerted a positive paracrine effect which enabled the rescue ofhost cones (Yang et al., 2010). Surprisingly, in AMD patients, it hasbeen demonstrated that RPE-choroid graft transplantation maymaintain macular function for up to 7s after surgery, with relativelylow complication and recurrence rates (van Zeeburg et al., 2012).However, this technique must be used with caution. Trans-plantation of adult and fetal retinal photoreceptors, in addition toRPE cells, has been achieved safely in patients with retinal degen-erative diseases, but unfortunately, in the vast majority of preclin-ical studies and human trials, the cells transplanted failed toestablish functional connectivity with the host tissue, resulting inmoderate or null restoration of visual function. Additional studiesare needed to determine new cell sources, in order to avoid prob-lems associated with transplant rejection.

4.7. Clinical trials for retinal diseases

A large number of clinical trials are currently under way to find atreatment or cure for various retinal diseases. Due to the large

number of trials being carried out, only the most relevant are citedbelow. Extensive information on clinical trials is available from thewebsite clinicaltrials.gov.

In DR, the aim of clinical trials is primarily to diminish or blockneovascularization or to diminish the increase permeability of thecapillary network. Accordingly, several drugs have been employedwith different administration routes: intravitreal injection (bev-acizumab, ranibizumab, pegaptanib, dexametason, triamcinolone,fluometolone), topical application (somatostatin) and different di-etary supplements. Vitrectomy surgery is also a common procedurein the treatment of DR depending on the retinal status. The pres-ence of epiretinal membranes, vitreous hemorrhage or fibrovas-cular proliferations threating the macula are common indicationsfor the surgery with good anatomical and functional results andwith the possibility of using adjunctive antinflamatory or anti-vascular intravitreal drugs at the end of the surgery.

As in DR, the primary objective of the wet AMD trials is to find asuccessful anti-angiogenic therapy. The anti-VEGFs bevacizumab,ranibizumab, pegaptanib and aflibercept are being used, as well asthe AAV-mediated VEGF receptor FLT01. Also under testing for thetreatment of AMD are adeno-associated viral mediated PEDF de-livery or endostatin and angiostatin, topical drugs for inhibitingtirosinkinase receptors, encapsulated human cells secreting CNTFneurotrophic factor, among others. For dry forms, antibodiesagainst amyloid substance and serotonin agonists are also tested.For both wet and dry forms, human stem cells sub-retinal trans-plantation is another therapeutic option, as well as the dietarysupplementation with antioxidants such as lutein zeaxanthin,omega-3 fatty acids and vitamin D. Rheopheresis procedures arealso being tested with different results.

Clinical trials performed with RP patients include pharmaco-logic treatment with vitamin A and E, valproic acid, lutein or do-cosahexaenoic acid. Stem cell-based therapies are also beinganalyzed, as well as encapsulated cell technology to deliver CNTFand gene therapy via AAV-mediated MERTK replacement. Genetherapies are also applied to other retinal diseases. In that sensemodification in the following genes are being tested:MY07A in type1B Usher, RPE65 in Leber Congenital amaurosis, ABCR in Stargardtdisease and XLRS1 in X linked retinosquisis. Retinal transplantationwith fetal tissue, transcorneal electrostimulation and electricalimplants are other therapeutic options in assay.

4.8. Suitable therapies in each phase of retinal degeneration

Degenerative retinal diseases involve structural and functionalchanges, many of which result from tissue remodeling and thefunctional reprogramming of the neural retina (Fig. 16). Trying togeneralize to all the retinal diseases, we are proposing a four-phased process of neural remodeling is common to all retinaldegenerative diseases, the progress of the events is different in eachtype of degeneration and necessarily influences the optimal treat-ment, which depends primarily on the stage of cell degeneration.

In phase 1, while there are still no evident structural signs of thedisease, neuroprotection of the retina is essential. Patients whowillsuffer future eye disease signs could be identified before theappearance of retinal anatomic or functional changes by means ofmedical tests or genetic analysis. The detection of an intraocularpressure increase in glaucoma, the high glucose levels in DR or theexistence of DNA mutations in RP are crucial in earlier stages of thediseases. The aim in this stage is to provide a protective environ-ment, independently of the etiology of the disease, to preserve thestructure and recover the normal function of retinal cells. This maybe achieved by delivering neurotrophic factors, vitamins, antioxi-dants, anti-apoptotic and anti-inflammatory compounds (Fig. 16).These pharmacological agents help counteract the emerging


apoptotic death cascade and the inflammatory responses generatedby the biochemical defects caused by the diseases. The use of genetherapy is also highly recommended at this stage of degeneration tocorrect known defective genes (Fig. 16). Gene silencing andreplacement is the proper approach for diseases caused by domi-nant mutations, while wild-type allele addition is the appropriatestrategy when recessive mutations are the origin of the pathology.At this point, the use of filters to block short-wavelength light andintense visible light can be of benefit in protecting the retina fromphotopic damage, thus delaying retinal degeneration.

In phase 2, morphological and functional alterations of theretina become evident and the first clinical signs can be observed.The recent use of high resolution optical coherence tomographyprovides a unique tool to detect the early retinal structural changesrelated to different pathologies, such as AMD, RP, DR and glaucoma(Acton et al., 2012; Aizawa et al., 2009; Giani et al., 2010; Hagiwaraet al., 2011; Hoerster et al., 2011; Kotowski et al., 2013; Lupo et al.,2011; Oishi et al., 2009; Taliantzis et al., 2009). Using this tech-nology, ophthalmologists can measure the thickness of each pa-tient's retinal cell layer, thus being able to detect outer segmentdamage to rods and cones, as well as RPE alterations. Furthermore,studies performed in animal models with different types of retinaldegeneration corroborate the rod and cone morphology modifica-tions, as well as the rod bipolar and horizontal cell dendriteretraction. Hypertrophy of Müller cells and activation of microgliaare other noticeable signs in this phase. At this stage, gene therapyis advisable to silence mutated genes and to replace them with thecorrect ones, as well as the use of optogenetic tools to makeremnant unhealthy photoreceptors responsive to light (Fig. 16). Theneuroprotective agents cited in phase 1 must be also administeredin order to keep retinal cells alive and prolong the visual function.Another method that can be successful at this point is the appli-cation of cell-based therapies (Fig. 16). For example, retinal repairvia the transplantation of photoreceptors or stem cells may be agood option, because the remaining bipolar and horizontal cells,which are losing their normal photoreceptor input without majorchanges in their morphology, will seek out new functional photo-receptors with which to establish contact. Additionally, propermaintenance of the RPE function is a requirement to preservephotoreceptors in good health. Within this framework, stem celltherapy applied to reestablish RPE function may offer a very goodchance of success, in some cases even better than the photore-ceptor replacement therapy. The reason for this difference is thatthe transplanted photoreceptors need to establish highly special-ized synaptic connections with bipolar and horizontal cells, as wellas proper interactions with RPE cells. Another option would betransplanting both cell types, already developed on a membrane-like surface.

Phase 3 is characterized by a profound loss of vision, evidencedby abnormal results in morphological and functional tests. At thisstage, there are few or no functional photoreceptors and the innerretina has been irreversibly damaged. Dramatic alterations in thevascularization of the retina occur in AMD and DR. The use of genetherapy no longer makes sense at this phase, when almost nophotoreceptors remain alive. However, the optogenetic techniqueapplied to bipolar and RGCs to compensate for the loss of photo-sensitive photoreceptor cells may be a good approach (Fig. 16). Thestem cell transplantation to replace the lost photoreceptors is also asuitable therapeutic option at this stage. The application of sub-retinal, epiretinal or suprachoroidal electronic retinal implants isanother possibility, but the rate of success will depend on the stageof retinal degeneration of the patient (Fig. 16). The employment ofthese visual prosthetic designs in a highly remodeled retina will bemore difficult than the use of the same approach in retinas withmost of their circuitry still intact (the use of these devices will not

be discussed in this review). Therefore, the efficacy of eithertreatment increases to the extent that the integrity of the retina hasbeen preserved by the previous administration of neuroprotectivecompounds.

In the most advanced stage of disease (phase 4), the retina is in amarked state of remodeling, due to the loss of many of its neurons.As a consequence, there is no visible structure and the visualfunction is almost absent. Cell transplantation must not beconsidered at this stage, at least with the actual knowledge, due tothe development of a new non-sense rewiring of the retinal cir-cuitry that does not allow for correct processing of visual infor-mation, even if the new cells make contact with the remainingfunctional retinal cells. Furthermore, the existence of a thick glialscar formed by the Müller cells in the outer retina (Fig. 12AeC)constitutes a mechanical barrier to synapse formation between thetransplanted photoreceptors and the remaining bipolar and hori-zontal cells, and also between the photoreceptor outer segmentsand the RPE. Optogenetic tools applied to the remaining ganglioncells are a therapeutic option, but the low number of these cellscould compromise the success of this approach. In these cases,artificial vision, combined with the administration of neurotrophicfactors that contribute to prolonging the life of the surviving RGCs,may be the best strategy (Fig. 16). It is clear that new scientificdevelopments could change these options in the future.

Interestingly, the administration of neuroprotective factors iscrucial in all degeneration phases, even when vision has beencompletely lost. RGCs are not only important because they areresponsible for transmitting through their axons electrical signalsfrom the retina to the brain to formvisual images, a subset of retinalphotosensitive ganglion cells expressing the photopigment mela-nopsin is also involved in processes that control circadian rhythms,pupil contraction, memory and depression (Fig. 2D) (Esquiva et al.,2013; Roecklein et al., 2013). Thus, the neuroprotection of RGCs isnot only essential for proper visual function, but also to avoid dis-turbances in non-visual functions, such as sleepewake cycles, dailyactivities and the mood of people who have retinal disorders.

Hence, with our present knowledge of the use of different ap-proaches in the treatment of retinal pathologies, the combinationof several types of therapies is considered essential to achievesuccess in delaying the progression of retinal degeneration. More-over, it is important to emphasize that all retinal disorders,regardless of their etiology, have in common the activation ofoxidative stress, inflammation and apoptosis pathways. For thisreason, all the neuroprotective compounds described (neuro-trophic factors, vitamins, antioxidants, anti-apoptotic and anti-inflammatory molecules) must be administered in the treatmentof any retinal disease (Fig. 16). As stated earlier, the administrationof the neuroprotective compounds mentioned in all degenerationphases of retinal diseases is crucial for maintaining retinal healthand, as a consequence, for strengthening and prolonging thebeneficial effects of the applied therapy(s) (Cideciyan et al., 2013).On the other hand, we must not forget that the success of thetreatments in improving visual function depends on proper patientselection, the etiology and stage of the disease, the right choice oftreatment(s) and the age of the patient, among other factors.

In conclusion, it is critical to remember that despite all the sci-entific advances made in an attempt to cure or slow down retinaldegenerative diseases, there are still several barriers to overcome.The successful translation of new therapies requires the develop-ment of appropriate animal models of the diseases, as certainmutations are not similar between humans and the existing animalmodels. There is vast genetic heterogeneity associated with somedisease phenotypes, and thus an accurate genetic characterizationis necessary for specific gene therapies. Furthermore, it is importantto maintain proper RPE function regardless of the disease;


otherwise, the therapeutic approaches applied to photoreceptorcells will be useless. Finally, the blood-retinal barrier prevents mostmolecules administered systemically from reaching an effectivedose in the retina, making the development of effective deliverymethods necessary; perhaps smaller or polarized molecules canpass through this barrier with the improvement of the actualknowledge.

5. Conclusion remarks and future directions

Human retinal degenerative diseases are currently incurableand retinal degeneration, once initiated, is irreversible. The thera-pies applied at present in the treatment of retinal dystrophies delaythe onset or progression of degeneration, but no therapies areavailable to replace lost retinal cells or restore accurate vision. Thesearch for effective treatments has stimulated the development of alarge number of animal models that mimic the different humanretinal diseases, as well as the isolation of an increasing number ofretinal cell lines that are of great interest for the study of thecellular pathways involved in the progression of these diseases.Currently, the potential therapeutic approaches aimed at finding acure for blinding diseases focus on threemain lines of action. First isthe use of preventive strategies that attempt to counteract theunderlying disease mechanisms, either by manipulating cellularpathways through the use of pharmacological compounds or ge-netic modification by gene silencing and/or gene replacement. Thesecond approach is not concerned as much with the causes of thediseases as it is with ways to prevent cell death, such as are theadministration of anti-apoptotic, anti-inflammatory and neuro-trophic factors compounds. The third approach focuses on retinalcell replacement through the transplantation of stem cell ordifferentiated retinal cell types and artificial vision. However,despite the efforts of researchers to identify a therapy capable ofpreventing retinal degeneration or restoring vision, current thera-pies entail difficulties that need to be addressed to achieve safe,effective treatments. In this context, the administration of antiox-idants (alone or in cocktails), anti-apoptotics, anti-inflammatories,neurotrophic factors or viability factors unfortunately only slowsthe neurodegeneration of the retina by delaying retinal cell death,but fail to prevent the progression of the disease. On the otherhand, to design a proper gene-based therapy, it is necessary toidentify all the genes and loci that cause inherited retinal diseases,but the enormous mutational heterogeneity makes this task verydifficult. Thus, despite the growing body of knowledge about thesediseases, a thorough understanding of the molecular mechanismsunderlying retinal degeneration and the identification of all thegenetic causes of these disorders is still needed to improve theprospects of therapies. Another hindrance to be overcome is findinga suitableway to get the genetic material to specific cell types in theretina. In this sense, the development of viral vectors with modifiedtropisms has facilitated this access, but further progress is stillneeded.

Certain aspects of cell replacement also need to be considered tosucceed in improving vision. These include the choice of the celltype to be transplanted, the proper degeneration phase in which toperform the transplant, the selection of the site of the cell trans-plant (vitreous humor, subretinal space, periphery of the retina,fovea) and the number of cells needed. It is also important to notethat the success of cell replacement depends on the survival of thetransplanted cells in the retina, the prevention of tissue rejection,migration and integration of cells in the remaining retinal circuitryin a mosaic arrangement and the establishment of adequate syn-aptic connections capable of restoring visual function. Questionslike how long the transplanted cells will live and whether they aresafe for the healthy cells are yet to be answered, and it has not yet

been clarified whether it is better to transplant differentiated orundifferentiated cells. Interestingly, although cell migration andintegration in the retina, together with expression of cell-typespecific proteins have been observed in several published studies,the reports of synapse formation and cell function improvementare still exceptionally rare. For this reason, the ever-increasing hy-pothesis is that the main mechanism for the beneficial effect of celltransplantation appears to be the secretion of neurotrophic factorsthat prolong retinal cell survival. Furthermore, photoreceptor andRPE cell transplantation is relatively easy as compared to ganglioncell transplantation, which presents additional difficulties becauseof their need to extend long processes to form the optic nerve sothat it can make proper contact with the geniculate nucleus in thebrain to achieve image composition. Another obstacle to celltransplantation is crossing the barrier formed by extracellularmatrix molecules, such as chondroitin sulfate proteoglycans pro-duced by activated microglial cells and astrocytes.

In summary, retinal remodeling in response to alterations inmolecular pathways and the activation of cellular responses un-derlying retinal disease entail the impairment of visual function. Itseems clear that the treatment of the different pathologies affectingthe retina must involve a combination of several therapeutic ap-proaches, and that the administration of neuroprotective com-pounds is essential from the time the disease is detected andthroughout the course of treatment. Furthermore, the success ofcurrent or new therapies for blinding conditions will depend on thedetailed knowledge of the genetic causes behind each retinal dis-order, the mechanisms underlying cellular homeostasis andcontrolled cell death, the morphological and functional changes ofthe different retinal cells in response to injury, and the stage ofdegeneration of each structure in the retina at the moment of thetherapeutic intervention. Further studies are needed to moreexactly unravel the mechanisms involved in retinal neuro-degeneration. This information could eventually be useful indeveloping pharmacotherapies targeting fundamental biochemicaldefects aimed at retarding disease progression, and/or alleviatingneurodegenerative symptoms in the retina, as well as designingnew and effective gene-based therapies to prevent the diseases.

To conclude, it is important to remark that the combined use ofthe current ophthalmologic surgery techniques with therapiesderivative of a deep knowledge of the factors involved in cellularresponses could be the key for increase the success in visualrestoration in different retinal diseases.

Acknowledgments

This work was supported by project grants from the SpanishMinistry of Economy and Competitiveness-FEDER (BFU2012-36845), Plan Nacional de IþDþI 2008-2011, Instituto de SaludCarlos III, Subdirecci�on General de Redes y Centros de Investigaci�onCooperativa (RETICS RD07/0062/0008-0012, RETICS RD12/0034/0006-0010, PS0901854, PI13/01124), ONCE and FUNDALUCE.

References

Abu-El-Asrar, A.M., Dralands, L., Missotten, L., Al-Jadaan, I.A., Geboes, K., 2004.Expression of apoptosis markers in the retinas of human subjects with diabetes.Invest Ophthalmol. Vis. Sci. 45, 2760e2766.

Acland, G.M., Aguirre, G.D., Ray, J., Zhang, Q., Aleman, T.S., Cideciyan, A.V., Pearce-Kelling, S.E., Anand, V., Zeng, Y., Maguire, A.M., Jacobson, S.G., Hauswirth, W.W.,Bennett, J., 2001. Gene therapy restores vision in a canine model of childhoodblindness. Nat. Genet. 28, 92e95.

Acton, J.H., Smith, R.T., Hood, D.C., Greenstein, V.C., 2012. Relationship betweenretinal layer thickness and the visual field in early age-related maculardegeneration. Invest Ophthalmol. Vis. Sci. 53, 7618e7624.

Aizawa, S., Mitamura, Y., Baba, T., Hagiwara, A., Ogata, K., Yamamoto, S., 2009.Correlation between visual function and photoreceptor inner/outer segmentjunction in patients with retinitis pigmentosa. Eye (Lond.) 23, 304e308.


Aizu, Y., Oyanagi, K., Hu, J., Nakagawa, H., 2002. Degeneration of retinal neuronalprocesses and pigment epithelium in the early stage of the streptozotocin-diabetic rats. Neuropathology 22, 161e170.

Akula, J.D., Mocko, J.A., Benador, I.Y., Hansen, R.M., Favazza, T.L., Vyhovsky, T.C.,Fulton, A.B., 2008. The neurovascular relation in oxygen-induced retinopathy.Mol. Vis. 14, 2499e2508.

Alex, A.F., Spitznas, M., Tittel, A.P., Kurts, C., Eter, N., 2010. Inhibitory effect of epi-gallocatechin gallate (EGCG), resveratrol, and curcumin on proliferation ofhuman retinal pigment epithelial cells in vitro. Curr. Eye Res. 35, 1021e1033.

Algvere, P., 1968. Clinical studies on the oscillatory potentials of the human elec-troretinogram with special reference to the scotopic b-wave. Acta Ophthalmol.(Copenh.) 46, 993e1024.

Aloisi, F., Serafini, B., Adorini, L., 2000. Glia-T cell dialogue. J. Neuroimmunol. 107,111e117.

Ambrosini, E., Aloisi, F., 2004. Chemokines and glial cells: a complex network in thecentral nervous system. Neurochem. Res. 29, 1017e1038.

Anderson, D.H., Mullins, R.F., Hageman, G.S., Johnson, L.V., 2002. A role for localinflammation in the formation of drusen in the aging eye. Am. J. Ophthalmol.134, 411e431.

Anderson, P.J., Watts, H., Hille, C., Philpott, K., Clark, P., Gentleman, M.C., Jen, L.S.,2008. Glial and endothelial blood-retinal barrier responses to amyloid-beta inthe neural retina of the rat. Clin. Ophthalmol. 2, 801e816.

Andrieu-Soler, C., Aubert-Pouessel, A., Doat, M., Picaud, S., Halhal, M., Simonutti, M.,Venier-Julienne, M.C., Benoit, J.P., Behar-Cohen, F., 2005. Intravitreous injectionof PLGA microspheres encapsulating GDNF promotes the survival of photore-ceptors in the rd1/rd1 mouse. Mol. Vis. 11, 1002e1011.

Annear, M.J., Mowat, F.M., Bartoe, J.T., Querubin, J., Azam, S.A., Basche, M.,Curran, P.G., Smith, A.J., Bainbridge, J.W., Ali, R.R., Petersen-Jones, S.M., 2013.Successful gene therapy in older Rpe65-deficient dogs following subretinalinjection of an adeno-associated vector expressing RPE65. Hum. Gene Ther. 24,883e893.

Antonetti, D.A., Barber, A.J., Bronson, S.K., Freeman, W.M., Gardner, T.W.,Jefferson, L.S., Kester, M., Kimball, S.R., Krady, J.K., LaNoue, K.F., Norbury, C.C.,Quinn, P.G., Sandirasegarane, L., Simpson, I.A., 2006. Diabetic retinopathy:seeing beyond glucose-induced microvascular disease. Diabetes 55, 2401e2411.

Aoki, H., Hara, A., Nakagawa, S., Motohashi, T., Hirano, M., Takahashi, Y., Kunisada, T.,2006. Embryonic stem cells that differentiate into RPE cell precursors in vitrodevelop into RPE cell monolayers in vivo. Exp. Eye Res. 82, 265e274.

Ardeljan, D., Chan, C.C., 2013. Aging is not a disease: Distinguishing age-relatedmacular degeneration from aging. Prog. Retin Eye Res. 37, 68e89.

Arden, G.B., Carter, R.M., Hogg, C.R., Powell, D.J., Ernst, W.J., Clover, G.M., Lyness, A.L.,Quinlan, M.P., 1983. Rod and cone activity in patients with dominantly inheritedretinitis pigmentosa: comparisons between psychophysical and electroretino-graphic measurements. Br. J. Ophthalmol. 67, 405e418.

Areias, F.M., Rego, A.C., Oliveira, C.R., Seabra, R.M., 2001. Antioxidant effect of fla-vonoids after ascorbate/Fe(2þ)-induced oxidative stress in cultured retinalcells. Biochem Pharmacol. 62, 111e118.

Arnhold, S., Absenger, Y., Klein, H., Addicks, K., Schraermeyer, U., 2007. Trans-plantation of bone marrow-derived mesenchymal stem cells rescue photore-ceptor cells in the dystrophic retina of the rhodopsin knockout mouse. GraefesArch. Clin. Exp. Ophthalmol. 245, 414e422.

Arnhold, S., Heiduschka, P., Klein, H., Absenger, Y., Basnaoglu, S., Kreppel, F., Henke-Fahle, S., Kochanek, S., Bartz-Schmidt, K.U., Addicks, K., Schraermeyer, U., 2006.Adenovirally transduced bone marrow stromal cells differentiate into pigmentepithelial cells and induce rescue effects in RCS rats. Invest Ophthalmol. Vis. Sci.47, 4121e4129.

Arroba, A.I., Alvarez-Lindo, N., van Rooijen, N., de la Rosa, E.J., 2011. Microglia-mediated IGF-I neuroprotection in the rd10 mouse model of retinitis pigmen-tosa. Invest Ophthalmol. Vis. Sci. 52, 9124e9130.

Askou, A.L., Pournaras, J.A., Pihlmann, M., Svalgaard, J.D., Arsenijevic, Y., Kostic, C.,Bek, T., Dagnaes-Hansen, F., Mikkelsen, J.G., Jensen, T.G., Corydon, T.J., 2012.Reduction of choroidal neovascularization in mice by adeno-associated virus-delivered anti-vascular endothelial growth factor short hairpin RNA. J. GeneMed. 14, 632e641.

Athanasiou, D., Aguila, M., Bevilacqua, D., Novoselov, S.S., Parfitt, D.A.,Cheetham, M.E., 2013. The cell stress machinery and retinal degeneration. FEBSLett. 587, 2008e2017.

Atmaca-Sonmez, P., Li, Y., Yamauchi, Y., Schanie, C.L., Ildstad, S.T., Kaplan, H.J.,Enzmann, V., 2006. Systemically transferred hematopoietic stem cells home tothe subretinal space and express RPE-65 in a mouse model of retinal pigmentepithelium damage. Exp. Eye Res. 83, 1295e1302.

Auricchio, A., Behling, K.C., Maguire, A.M., O'Connor, E.M., Bennett, J., Wilson, J.M.,Tolentino, M.J., 2002. Inhibition of retinal neovascularization by intraocularviral-mediated delivery of anti-angiogenic agents. Mol. Ther. 6, 490e494.

Bailey, I.L., 1993. New procedures for detecting early vision losses in the elderly.Optom. Vis. Sci. 70, 299e305.

Ball, A.K., McReynolds, J.S., 1998. Localization of gap junctions and tracer coupling inretinal Muller cells. J. Comp. Neurol. 393, 48e57.

Banin, E., Obolensky, A., Idelson, M., Hemo, I., Reinhardtz, E., Pikarsky, E., Ben-Hur, T., Reubinoff, B., 2006. Retinal incorporation and differentiation of neuralprecursors derived from human embryonic stem cells. Stem Cells 24,246e257.

Barabas, P., Huang, W., Chen, H., Koehler, C.L., Howell, G., John, S.W., Tian, N.,Renteria, R.C., Krizaj, D., 2011. Missing optomotor head-turning reflex in theDBA/2J mouse. Invest Ophthalmol. Vis. Sci. 52, 6766e6773.

Barber, A.J., 2003. A new view of diabetic retinopathy: a neurodegenerative diseaseof the eye. Prog. Neuropsychopharmacol. Biol. Psychiatry 27, 283e290.

Barber, A.J., Antonetti, D.A., Gardner, T.W., 2000. Altered expression of retinaloccludin and glial fibrillary acidic protein in experimental diabetes. The PennState Retina Research Group. Invest Ophthalmol. Vis. Sci. 41, 3561e3568.

Barber, A.J., Lieth, E., Khin, S.A., Antonetti, D.A., Buchanan, A.G., Gardner, T.W., 1998.Neural apoptosis in the retina during experimental and human diabetes. Earlyonset and effect of insulin. J. Clin. Invest 102, 783e791.

Barhoum, R., Martinez-Navarrete, G., Corrochano, S., Germain, F., Fernandez-Sanchez, L., de la Rosa, E.J., de la Villa, P., Cuenca, N., 2008. Functional andstructural modifications during retinal degeneration in the rd10 mouse.Neuroscience 155, 698e713.

Barker 2nd, F.M., Snodderly, D.M., Johnson, E.J., Schalch, W., Koepcke, W., Gerss, J.,Neuringer, M., 2011. Nutritional manipulation of primate retinas, V: effects oflutein, zeaxanthin, and n-3 fatty acids on retinal sensitivity to blue-light-induced damage. Invest Ophthalmol. Vis. Sci. 52, 3934e3942.

Barnett, N.L., Pow, D.V., 2000. Antisense knockdown of GLAST, a glial glutamatetransporter, compromises retinal function. Invest Ophthalmol. Vis. Sci. 41,585e591.

Beltran, W.A., Cideciyan, A.V., Lewin, A.S., Iwabe, S., Khanna, H., Sumaroka, A.,Chiodo, V.A., Fajardo, D.S., Roman, A.J., Deng, W.T., Swider, M., Aleman, T.S.,Boye, S.L., Genini, S., Swaroop, A., Hauswirth, W.W., Jacobson, S.G., Aguirre, G.D.,2012. Gene therapy rescues photoreceptor blindness in dogs and paves the wayfor treating human X-linked retinitis pigmentosa. Proc. Natl. Acad. Sci. U. S. A.109, 2132e2137.

Berson, E.L., 1981. Retinitis pigmentosa and allied diseases: applications of elec-troretinographic testing. Int. Ophthalmol. 4, 7e22.

Berson, E.L., 1987. Electroretinographic findings in retinitis pigmentosa. Jpn. J.Ophthalmol. 31, 327e348.

Besirli, C.G., Zheng, Q.D., Reed, D.M., Zacks, D.N., 2012. ERK-mediated activation ofFas apoptotic inhibitory molecule 2 (Faim2) prevents apoptosis of 661W cells ina model of detachment-induced photoreceptor cell death. PLoS One 7, e46664.

Betsuin, Y., Mashima, Y., Ohde, H., Inoue, R., Oguchi, Y., 2001. Clinical application ofthe multifocal VEPs. Curr. Eye Res. 22, 54e63.

Bhutto, I., Lutty, G., 2012. Understanding age-related macular degeneration (AMD):relationships between the photoreceptor/retinal pigment epithelium/Bruch'smembrane/choriocapillaris complex. Mol. Asp. Med. 33, 295e317.

Bi, A., Cui, J., Ma, Y.P., Olshevskaya, E., Pu, M., Dizhoor, A.M., Pan, Z.H., 2006. Ectopicexpression of a microbial-type rhodopsin restores visual responses in mice withphotoreceptor degeneration. Neuron 50, 23e33.

Bian, Q., Gao, S., Zhou, J., Qin, J., Taylor, A., Johnson, E.J., Tang, G., Sparrow, J.R.,Gierhart, D., Shang, F., 2012a. Lutein and zeaxanthin supplementation reducesphotooxidative damage and modulates the expression of inflammation-relatedgenes in retinal pigment epithelial cells. Free Radic. Biol. Med. 53, 1298e1307.

Bian, Q., Qin, T., Ren, Z., Wu, D., Shang, F., 2012b. Lutein or zeaxanthin supple-mentation suppresses inflammatory responses in retinal pigment epithelialcells and macrophages. Adv. Exp. Med. Biol. 723, 43e50.

Bilic, J., Izpisua Belmonte, J.C., 2012. Concise review: Induced pluripotent stem cellsversus embryonic stem cells: close enough or yet too far apart? Stem Cells 30,33e41.

Binns, A., Margrain, T.H., 2005. Evaluation of retinal function using the DynamicFocal Cone ERG. Ophthalmic Physiol. Opt. 25, 492e500.

Birch, D.G., Weleber, R.G., Duncan, J.L., Jaffe, G.J., Tao, W., 2013. Randomized trial ofciliary neurotrophic factor delivered by encapsulated cell intraocular implantsfor retinitis pigmentosa. Am. J. Ophthalmol. 156, 283e292 e281.

Boatright, J.H., Moring, A.G., McElroy, C., Phillips, M.J., Do, V.T., Chang, B.,Hawes, N.L., Boyd, A.P., Sidney, S.S., Stewart, R.E., Minear, S.C., Chaudhury, R.,Ciavatta, V.T., Rodrigues, C.M., Steer, C.J., Nickerson, J.M., Pardue, M.T., 2006. Toolfrom ancient pharmacopoeia prevents vision loss. Mol. Vis. 12, 1706e1714.

Bodis-Wollner, I., 2009. Retinopathy in parkinson disease. J. Neural Transm. 116,1493e1501.

Bornemann, K.D., Wiederhold, K.H., Pauli, C., Ermini, F., Stalder, M., Schnell, L.,Sommer, B., Jucker, M., Staufenbiel, M., 2001. Abeta-induced inflammatoryprocesses in microglia cells of APP23 transgenic mice. Am. J. Pathol. 158, 63e73.

Borooah, S., Phillips, M.J., Bilican, B., Wright, A.F., Wilmut, I., Chandran, S., Gamm, D.,Dhillon, B., 2013. Using human induced pluripotent stem cells to treat retinaldisease. Prog. Retin Eye Res. 37, 163e181.

Bosco, A., Crish, S.D., Steele, M.R., Romero, C.O., Inman, D.M., Horner, P.J.,Calkins, D.J., Vetter, M.L., 2012. Early reduction of microglia activation by irra-diation in a model of chronic glaucoma. PLoS One 7, e43602.

Bosco, A., Steele, M.R., Vetter, M.L., 2011. Early microglia activation in a mousemodel of chronic glaucoma. J. Comp. Neurol. 519, 599e620.

Boulton, M., Dayhaw-Barker, P., 2001. The role of the retinal pigment epithelium:topographical variation and ageing changes. Eye (Lond) 15, 384e389.

Boya, P., Kroemer, G., 2008. Lysosomal membrane permeabilization in cell death.Oncogene 27, 6434e6451.

Bressler, N.M., Bressler, S.B., Fine, S.L., 1988. Age-related macular degeneration. Surv.Ophthalmol. 32, 375e413.

Breton, M.E., Quinn, G.E., Keene, S.S., Dahmen, J.C., Brucker, A.J., 1989. Electroreti-nogram parameters at presentation as predictors of rubeosis in central retinalvein occlusion patients. Ophthalmology 96, 1343e1352.

Bringmann, A., Iandiev, I., Pannicke, T., Wurm, A., Hollborn, M., Wiedemann, P.,Osborne, N.N., Reichenbach, A., 2009. Cellular signaling and factors involved inMuller cell gliosis: neuroprotective and detrimental effects. Prog. Retin Eye Res.28, 423e451.


Bringmann, A., Pannicke, T., Grosche, J., Francke, M., Wiedemann, P., Skatchkov, S.N.,Osborne, N.N., Reichenbach, A., 2006. Muller cells in the healthy and diseasedretina. Prog. Retin Eye Res. 25, 397e424.

Bringmann, A., Wiedemann, P., 2009. Involvement of Muller glial cells in epiretinalmembrane formation. Graefes Arch. Clin. Exp. Ophthalmol. 247, 865e883.

Brown, D.M., Michels, M., Kaiser, P.K., Heier, J.S., Sy, J.P., Ianchulev, T., 2009. Rani-bizumab versus verteporfin photodynamic therapy for neovascular age-relatedmacular degeneration: two-year results of the ANCHOR study. Ophthalmology116, 57e65 e55.

Bsibsi, M., Ravid, R., Gveric, D., van Noort, J.M., 2002. Broad expression of Toll-likereceptors in the human central nervous system. J. Neuropathol. Exp. Neurol.61, 1013e1021.

Buch, H., Nielsen, N.V., Vinding, T., Jensen, G.B., Prause, J.U., la Cour, M., 2005. 14-year incidence, progression, and visual morbidity of age-related maculopathy:the Copenhagen City Eye Study. Ophthalmology 112, 787e798.

Buchholz, D.E., Hikita, S.T., Rowland, T.J., Friedrich, A.M., Hinman, C.R., Johnson, L.V.,Clegg, D.O., 2009. Derivation of functional retinal pigmented epithelium frominduced pluripotent stem cells. Stem Cells 27, 2427e2434.

Buchholz, D.E., Pennington, B.O., Croze, R.H., Hinman, C.R., Coffey, P.J., Clegg, D.O.,2013. Rapid and efficient directed differentiation of human pluripotent stemcells into retinal pigmented epithelium. Stem Cells Transl. Med. 2, 384e393.

Bucolo, C., Marrazzo, G., Platania, C.B., Drago, F., Leggio, G.M., Salomone, S., 2013.Fortified extract of red berry, Ginkgo biloba, and white willow bark in experi-mental early diabetic retinopathy. J. Diabetes Res. 2013, 432695.

Bui, B.V., Fortune, B., 2004. Ganglion cell contributions to the rat full-field elec-troretinogram. J. Physiol. 555, 153e173.

Bush, R.A., Sieving, P.A., 1996. Inner retinal contributions to the primate photopicfast flicker electroretinogram. J. Opt. Soc. Am. a Opt. Image Sci. Vis. 13, 557e565.

Busskamp, V., Duebel, J., Balya, D., Fradot, M., Viney, T.J., Siegert, S., Groner, A.C.,Cabuy, E., Forster, V., Seeliger, M., Biel, M., Humphries, P., Paques, M., Mohand-Said, S., Trono, D., Deisseroth, K., Sahel, J.A., Picaud, S., Roska, B., 2010. Geneticreactivation of cone photoreceptors restores visual responses in retinitis pig-mentosa. Science 329, 413e417.

Butovsky, O., Talpalar, A.E., Ben-Yaakov, K., Schwartz, M., 2005. Activation ofmicroglia by aggregated beta-amyloid or lipopolysaccharide impairs MHC-IIexpression and renders them cytotoxic whereas IFN-gamma and IL-4 renderthem protective. Mol. Cell. Neurosci. 29, 381e393.

Byram, S.C., Carson, M.J., DeBoy, C.A., Serpe, C.J., Sanders, V.M., Jones, K.J., 2004.CD4-positive T cell-mediated neuroprotection requires dual compartment an-tigen presentation. J. Neurosci. 24, 4333e4339.

Campello, L., Esteve-Rudd, J., Bru-Martinez, R., Herrero, M.T., Fernandez-Villalba, E.,Cuenca, N., Martin-Nieto, J., 2013a. Alterations in energy metabolism, neuro-protection and visual signal transduction in the retina of Parkinsonian, MPTP-treated monkeys. PLoS One 8, e74439.

Campello, L., Esteve-Rudd, J., Cuenca, N., Martin-Nieto, J., 2013b. The ubiquitin-proteasome system in retinal health and disease. Mol. Neurobiol. 47, 790e810.

Campochiaro, P.A., Nguyen, Q.D., Shah, S.M., Klein, M.L., Holz, E., Frank, R.N.,Saperstein, D.A., Gupta, A., Stout, J.T., Macko, J., DiBartolomeo, R., Wei, L.L., 2006.Adenoviral vector-delivered pigment epithelium-derived factor for neovascularage-related macular degeneration: results of a phase I clinical trial. Hum. GeneTher. 17, 167e176.

Cao, W., Tombran-Tink, J., Elias, R., Sezate, S., Mrazek, D., McGinnis, J.F., 2001. In vivoprotection of photoreceptors from light damage by pigment epithelium-derivedfactor. Invest Ophthalmol. Vis. Sci. 42, 1646e1652.

Cao, X., Liu, M., Tuo, J., Shen, D., Chan, C.C., 2010. The effects of quercetin in culturedhuman RPE cells under oxidative stress and in Ccl2/Cx3cr1 double deficientmice. Exp. Eye Res. 91, 15e25.

Cardona, A.E., Pioro, E.P., Sasse, M.E., Kostenko, V., Cardona, S.M., Dijkstra, I.M.,Huang, D., Kidd, G., Dombrowski, S., Dutta, R., Lee, J.C., Cook, D.N., Jung, S.,Lira, S.A., Littman, D.R., Ransohoff, R.M., 2006. Control of microglial neurotox-icity by the fractalkine receptor. Nat. Neurosci. 9, 917e924.

Carnevali, S., Petruzzelli, S., Longoni, B., Vanacore, R., Barale, R., Cipollini, M.,Scatena, F., Paggiaro, P., Celi, A., Giuntini, C., 2003. Cigarette smoke extract in-duces oxidative stress and apoptosis in human lung fibroblasts. Am. J. Physiol.Lung Cell. Mol. Physiol. 284, L955eL963.

Carr, A.J., Vugler, A.A., Hikita, S.T., Lawrence, J.M., Gias, C., Chen, L.L., Buchholz, D.E.,Ahmado, A., Semo, M., Smart, M.J., Hasan, S., da Cruz, L., Johnson, L.V.,Clegg, D.O., Coffey, P.J., 2009. Protective effects of human iPS-derived retinalpigment epithelium cell transplantation in the retinal dystrophic rat. PLoS One4, e8152.

Carson, M.J., Thrash, J.C., Walter, B., 2006. The cellular response in neuro-inflammation: the role of leukocytes, microglia and astrocytes in neuronaldeath and survival. Clin. Neurosci. Res. 6, 237e245.

Carter, D.A., Dick, A.D., 2003. Lipopolysaccharide/interferon-gamma and nottransforming growth factor beta inhibits retinal microglial migration fromretinal explant. Br. J. Ophthalmol. 87, 481e487.

Cartier, L., Hartley, O., Dubois-Dauphin, M., Krause, K.H., 2005. Chemokine receptorsin the central nervous system: role in brain inflammation and neurodegener-ative diseases. Brain Res. Brain Res. Rev. 48, 16e42.

Carwile, M.E., Culbert, R.B., Sturdivant, R.L., Kraft, T.W., 1998. Rod outer segmentmaintenance is enhanced in the presence of bFGF, CNTF and GDNF. Exp. Eye Res.66, 791e805.

Cashman, S.M., Ramo, K., Kumar-Singh, R., 2011. A non membrane-targeted humansoluble CD59 attenuates choroidal neovascularization in a model of age relatedmacular degeneration. PLoS One 6, e19078.

Cayouette, M., Behn, D., Sendtner, M., Lachapelle, P., Gravel, C., 1998. Intraoculargene transfer of ciliary neurotrophic factor prevents death and increasesresponsiveness of rod photoreceptors in the retinal degeneration slow mouse.J. Neurosci. 18, 9282e9293.

Cayouette, M., Smith, S.B., Becerra, S.P., Gravel, C., 1999. Pigment epithelium-derivedfactor delays the death of photoreceptors in mouse models of inherited retinaldegenerations. Neurobiol. Dis. 6, 523e532.

Ceelen, P.W., Lockridge, A., Newman, E.A., 2001. Electrical coupling between glialcells in the rat retina. Glia 35, 1e13.

Chahory, S., Keller, N., Martin, E., Omri, B., Crisanti, P., Torriglia, A., 2010. Lightinduced retinal degeneration activates a caspase-independent pathwayinvolving cathepsin D. Neurochem Int. 57, 278e287.

Chakravarthy, U., Harding, S.P., Rogers, C.A., Downes, S.M., Lotery, A.J.,Wordsworth, S., Reeves, B.C., 2012. Ranibizumab versus bevacizumab to treatneovascular age-related macular degeneration: one-year findings from theIVAN randomized trial. Ophthalmology 119, 1399e1411.

Chalberg, T.W., Genise, H.L., Vollrath, D., Calos, M.P., 2005. phiC31 integrase confersgenomic integration and long-term transgene expression in rat retina. InvestOphthalmol. Vis. Sci. 46, 2140e2146.

Chan, C.M., Chang, H.H., Wang, V.C., Huang, C.L., Hung, C.F., 2013. Inhibitory effectsof resveratrol on PDGF-BB-induced retinal pigment epithelial cell migration viaPDGFRbeta, PI3K/Akt and MAPK pathways. PLoS One 8, e56819.

Chan, C.M., Huang, J.H., Chiang, H.S., Wu, W.B., Lin, H.H., Hong, J.Y., Hung, C.F., 2010.Effects of (-)-epigallocatechin gallate on RPE cell migration and adhesion. Mol.Vis. 16, 586e595.

Chan-Ling, T., McLeod, D.S., Hughes, S., Baxter, L., Chu, Y., Hasegawa, T., Lutty, G.A.,2004. Astrocyte-endothelial cell relationships during human retinal vasculardevelopment. Invest Ophthalmol. Vis. Sci. 45, 2020e2032.

Charteris, D.G., Downie, J., Aylward, G.W., Sethi, C., Luthert, P., 2007. Intraretinal andperiretinal pathology in anterior proliferative vitreoretinopathy. Graefes Arch.Clin. Exp. Ophthalmol. 245, 93e100.

Chavarria, A., Cardenas, G., 2013. Neuronal influence behind the central nervoussystem regulation of the immune cells. Front. Integr. Neurosci. 7, 64.

Chen, F., Jiang, L., Shen, C., Wan, H., Xu, L., Wang, N., Jonas, J.B., 2012. Neuro-protective effect of epigallocatechin-3-gallate against N-methyl-D-aspartate-induced excitotoxicity in the adult rat retina. Acta Ophthalmol. 90,e609e615.

Chen, M., Luo, C., Penalva, R., Xu, H., 2013. Paraquat-induced retinal degeneration isexaggerated in CX3CR1-deficient mice and is associated with increased retinalinflammation. Invest Ophthalmol. Vis. Sci. 54, 682e690.

Chen, Y., Li, X.X., Xing, N.Z., Cao, X.G., 2008. Quercetin inhibits choroidal and retinalangiogenesis in vitro. Graefes Arch. Clin. Exp. Ophthalmol. 246, 373e378.

Cherepanoff, S., McMenamin, P., Gillies, M.C., Kettle, E., Sarks, S.H., 2010. Bruch'smembrane and choroidal macrophages in early and advanced age-relatedmacular degeneration. Br. J. Ophthalmol. 94, 918e925.

Chew, E.Y., Clemons, T.E., Sangiovanni, J.P., Danis, R.P., Ferris 3rd, F.L., Elman, M.J.,Antoszyk, A.N., Ruby, A.J., Orth, D., Bressler, S.B., Fish, G.E., Hubbard, G.B.,Klein, M.L., Chandra, S.R., Blodi, B.A., Domalpally, A., Friberg, T., Wong, W.T.,Rosenfeld, P.J., Agron, E., Toth, C.A., Bernstein, P.S., Sperduto, R.D., 2014. Sec-ondary analyses of the effects of Lutein/Zeaxanthin on age-related maculardegeneration Progression: AREDS2 report No. 3. JAMA Ophthalmol. 132,142e149.

Chinskey, N.D., Zheng, Q.D., Zacks, D.N., 2014. Control of photoreceptor autophagyafter retinal detachment: the switch from survival to death. Invest Ophthalmol.Vis. Sci. 55, 688e695.

Chiu, C.J., Liu, S., Willett, W.C., Wolever, T.M., Brand-Miller, J.C., Barclay, A.W.,Taylor, A., 2011. Informing food choices and health outcomes by use of the di-etary glycemic index. Nutr. Rev. 69, 231e242.

Cho, N.C., Poulsen, G.L., Ver Hoeve, J.N., Nork, T.M., 2000. Selective loss of S-cones indiabetic retinopathy. Arch. Ophthalmol. 118, 1393e1400.

Choi, S.S., Zawadzki, R.J., Lim, M.C., Brandt, J.D., Keltner, J.L., Doble, N., Werner, J.S.,2011. Evidence of outer retinal changes in glaucoma patients as revealed byultrahigh-resolution in vivo retinal imaging. Br. J. Ophthalmol. 95, 131e141.

Chrysostomou, V., Rezania, F., Trounce, I.A., Crowston, J.G., 2013. Oxidative stressand mitochondrial dysfunction in glaucoma. Curr. Opin. Pharmacol. 13, 12e15.

Chu, Y., Humphrey, M.F., Constable, I.J., 1993. Horizontal cells of the normal anddystrophic rat retina: a wholemount study using immunolabelling for the 28-kDa calcium-binding protein. Exp. Eye Res. 57, 141e148.

Chucair, A.J., Rotstein, N.P., Sangiovanni, J.P., During, A., Chew, E.Y., Politi, L.E., 2007.Lutein and zeaxanthin protect photoreceptors from apoptosis induced byoxidative stress: relation with docosahexaenoic acid. Invest Ophthalmol. Vis.Sci. 48, 5168e5177.

Cideciyan, A.V., Aleman, T.S., Boye, S.L., Schwartz, S.B., Kaushal, S., Roman, A.J.,Pang, J.J., Sumaroka, A., Windsor, E.A., Wilson, J.M., Flotte, T.R., Fishman, G.A.,Heon, E., Stone, E.M., Byrne, B.J., Jacobson, S.G., Hauswirth, W.W., 2008.Human gene therapy for RPE65 isomerase deficiency activates the retinoidcycle of vision but with slow rod kinetics. Proc. Natl. Acad. Sci. U. S. A. 105,15112e15117.

Cideciyan, A.V., Jacobson, S.G., Beltran, W.A., Sumaroka, A., Swider, M., Iwabe, S.,Roman, A.J., Olivares, M.B., Schwartz, S.B., Komaromy, A.M., Hauswirth, W.W.,Aguirre, G.D., 2013. Human retinal gene therapy for Leber congenital amaurosisshows advancing retinal degeneration despite enduring visual improvement.Proc. Natl. Acad. Sci. U. S. A. 110, E517eE525.

Clostre, F., 2001. Protective effects of a Ginkgo biloba extract (EGb 761) on ischemia-reperfusion injury. Therapie 56, 595e600.


Colotto, A., Falsini, B., Salgarello, T., Iarossi, G., Galan, M.E., Scullica, L., 2000. Phot-opic negative response of the human ERG: losses associated with glaucomatousdamage. Invest Ophthalmol. Vis. Sci. 41, 2205e2211.

Conley, S.M., Naash, M.I., 2010. Nanoparticles for retinal gene therapy. Prog. RetinEye Res. 29, 376e397.

Conlon, T.J., Deng, W.T., Erger, K., Cossette, T., Pang, J.J., Ryals, R., Clement, N.,Cleaver, B., McDoom, I., Boye, S.E., Peden, M.C., Sherwood, M.B., Abernathy, C.R.,Alkuraya, F.S., Boye, S.L., Hauswirth, W.W., 2013. Preclinical potency and safetystudies of an AAV2-mediated gene therapy vector for the treatment of MERTKassociated retinitis pigmentosa. Hum. Gene Ther. Clin. Dev. 24, 23e28.

Cook, B., Lewis, G.P., Fisher, S.K., Adler, R., 1995. Apoptotic photoreceptor degenerationin experimental retinal detachment. Invest Ophthalmol. Vis. Sci. 36, 990e996.

Coorey, N.J., Shen, W., Chung, S.H., Zhu, L., Gillies, M.C., 2012. The role of glia inretinal vascular disease. Clin. Exp. Optom. 95, 266e281.

Corrochano, S., Barhoum, R., Boya, P., Arroba, A.I., Rodriguez-Muela, N., Gomez-Vicente, V., Bosch, F., de Pablo, F., de la Villa, P., de la Rosa, E.J., 2008. Attenuationof vision loss and delay in apoptosis of photoreceptors induced by proinsulin ina mouse model of retinitis pigmentosa. Invest Ophthalmol. Vis. Sci. 49,4188e4194.

Costa, B.L., Fawcett, R., Li, G.Y., Safa, R., Osborne, N.N., 2008. Orally administeredepigallocatechin gallate attenuates light-induced photoreceptor damage. BrainRes. Bull. 76, 412e423.

Cotinet, A., Goureau, O., Hicks, D., Thillaye-Goldenberg, B., de Kozak, Y., 1997. Tumornecrosis factor and nitric oxide production by retinal Muller glial cells from ratsexhibiting inherited retinal dystrophy. Glia 20, 59e69.

Cottet, S., Schorderet, D.F., 2008. Triggering of Bcl-2-related pathway is associatedwith apoptosis of photoreceptors in Rpe65-/- mouse model of Leber'scongenital amaurosis. Apoptosis 13, 329e342.

Crabb, J.W., Miyagi, M., Gu, X., Shadrach, K., West, K.A., Sakaguchi, H., Kamei, M.,Hasan, A., Yan, L., Rayborn, M.E., Salomon, R.G., Hollyfield, J.G., 2002. Drusenproteome analysis: an approach to the etiology of age-related macular degen-eration. Proc. Natl. Acad. Sci. U. S. A. 99, 14682e14687.

Creel, D.J., 1995. Clinical electrophysiology. In: Kolb, H., Fernandez, E., Nelson, R.(Eds.), Webvision: the Organization of the Retina and Visual System, 2011/03/18ed, Salt Lake City.

Creel, D.J., 2012. Visually Evoked Potentials, Webvision: the Organization of theRetina and Visual System, 2012/10/05 ed.

Cuenca, N., Fernandez-Sanchez, L., Angulo, A., Pinilla, I., Martin-Nieto, J., Lax, P.,2011. Müller and astrocyte cell changes with aging in the P23H rat retina andTUDCA neuroprotective effects. Invest Ophthalmol. Vis. Sci. 52. E-Abstract 5467.

Cuenca, N., Fernandez-Sanchez, L., McGill, T.J., Lu, B., Wang, S., Lund, R., Huhn, S.,Capela, A., 2013. Phagocytosis of photoreceptor outer segments by transplantedhuman neural stem cells as a neuroprotective mechanism in retinal degener-ation. Invest Ophthalmol. Vis. Sci. 54, 6745e6756.

Cuenca, N., Herrero, M.T., Angulo, A., de Juan, E., Martinez-Navarrete, G.C., Lopez, S.,Barcia, C., Martin-Nieto, J., 2005a. Morphological impairments in retinal neu-rons of the scotopic visual pathway in a monkey model of Parkinson's disease.J. Comp. Neurol. 493, 261e273.

Cuenca, N., Pinilla, I., Fernandez-Sanchez, L., Salinas-Navarro, M., Alarcon-Martinez, L., Aviles-Trigueros, M., de la Villa, P., Miralles de Imperial, J., Villegas-Perez, M.P., Vidal-Sanz, M., 2010. Changes in the inner and outer retinal layersafter acute increase of the intraocular pressure in adult albino Swiss mice. Exp.Eye Res. 91, 273e285.

Cuenca, N., Pinilla, I., Sauve, Y., Lu, B., Wang, S., Lund, R.D., 2004. Regressive andreactive changes in the connectivity patterns of rod and cone pathways of P23Htransgenic rat retina. Neuroscience 127, 301e317.

Cuenca, N., Pinilla, I., Sauve, Y., Lund, R., 2005b. Early changes in synaptic connec-tivity following progressive photoreceptor degeneration in RCS rats. Eur. J.Neurosci. 22, 1057e1072.

Cullheim, S., Thams, S., 2007. The microglial networks of the brain and their role inneuronal network plasticity after lesion. Brain Res. Rev. 55, 89e96.

Cybulska-Heinrich, A.K., Mozaffarieh, M., Flammer, J., 2012. Ginkgo biloba: anadjuvant therapy for progressive normal and high tension glaucoma. Mol. Vis.18, 390e402.

Cyranoski, D., 2013. Stem cells cruise to clinic. Nature 494, 413.D'Souza, Y., Jones, C.J., Bonshek, R., 2008. Glycoproteins of drusen and drusen-like

lesions. J. Mol. Histol. 39, 77e86.Dalkara, D., Kolstad, K.D., Guerin, K.I., Hoffmann, N.V., Visel, M., Klimczak, R.R.,

Schaffer, D.V., Flannery, J.G., 2011. AAV mediated GDNF secretion from retinalglia slows down retinal degeneration in a rat model of retinitis pigmentosa.Mol. Ther. 19, 1602e1608.

Das, A., Guyton, M.K., Smith, A., Wallace, G.t., McDowell, M.L., Matzelle, D.D.,Ray, S.K., Banik, N.L., 2013. Calpain inhibitor attenuated optic nerve damage inacute optic neuritis in rats. J. Neurochem. 124, 133e146.

Davies, S., Elliott, M.H., Floor, E., Truscott, T.G., Zareba, M., Sarna, T., Shamsi, F.A.,Boulton, M.E., 2001. Photocytotoxicity of lipofuscin in human retinal pigmentepithelial cells. Free Radic. Biol. Med. 31, 256e265.

Davis, A.A., Matzuk, M.M., Reh, T.A., 2000. Activin A promotes progenitor differ-entiation into photoreceptors in rodent retina. Mol. Cell. Neurosci. 15, 11e21.

De Simone, R., Ambrosini, E., Carnevale, D., Ajmone-Cat, M.A., Minghetti, L., 2007.NGF promotes microglial migration through the activation of its high affinityreceptor: modulation by TGF-beta. J. Neuroimmunol. 190, 53e60.

de Souza, C.F., Kalloniatis, M., Polkinghorne, P.J., McGhee, C.N., Acosta, M.L., 2012.Functional and anatomical remodeling in human retinal detachment. Exp. EyeRes. 97, 73e89.

del Barco Barrantes, I., Davidson, G., Grone, H.J., Westphal, H., Niehrs, C., 2003. Dkk1and noggin cooperate in mammalian head induction. Genes. Dev. 17,2239e2244.

Delori, F.C., Goger, D.G., Dorey, C.K., 2001. Age-related accumulation and spatialdistribution of lipofuscin in RPE of normal subjects. Invest Ophthalmol. Vis. Sci.42, 1855e1866.

Dias, V., Junn, E., Mouradian, M.M., 2013. The role of oxidative stress in Parkinson'sdisease. J. Park. Dis. 3, 461e491.

Dick, A.D., Carter, D., Robertson, M., Broderick, C., Hughes, E., Forrester, J.V.,Liversidge, J., 2003. Control of myeloid activity during retinal inflammation.J. Leukoc. Biol. 74, 161e166.

Dilsiz, N., Sahaboglu, A., Yildiz, M.Z., Reichenbach, A., 2006. Protective effects ofvarious antioxidants during ischemia-reperfusion in the rat retina. GraefesArch. Clin. Exp. Ophthalmol. 244, 627e633.

Dimitrova, G., Kato, S., Yamashita, H., Tamaki, Y., Nagahara, M., Fukushima, H.,Kitano, S., 2003. Relation between retrobulbar circulation and progression ofdiabetic retinopathy. Br. J. Ophthalmol. 87, 622e625.

Dinculescu, A., Estreicher, J., Zenteno, J.C., Aleman, T.S., Schwartz, S.B., Huang, W.C.,Roman, A.J., Sumaroka, A., Li, Q., Deng, W.T., Min, S.H., Chiodo, V.A., Neeley, A.,Liu, X., Shu, X., Matias-Florentino, M., Buentello-Volante, B., Boye, S.L.,Cideciyan, A.V., Hauswirth, W.W., Jacobson, S.G., 2012. Gene therapy for retinitispigmentosa caused by MFRP mutations: human phenotype and preliminaryproof of concept. Hum. Gene Ther. 23, 367e376.

Dong, K., Sun, X., 2011. Targeting death receptor induced apoptosis and necroptosis:a novel therapeutic strategy to prevent neuronal damage in retinal detachment.Med. Hypotheses 77, 144e146.

Doonan, F., Cotter, T.G., 2012. Norgestrel may be a potential therapy for retinaldegenerations. Expert Opin. Investig. Drugs 21, 579e581.

Doonan, F., Donovan, M., Cotter, T.G., 2005. Activation of multiple pathways duringphotoreceptor apoptosis in the rd mouse. Invest Ophthalmol. Vis. Sci. 46,3530e3538.

Doonan, F., Donovan, M., Gomez-Vicente, V., Bouillet, P., Cotter, T.G., 2007. Bimexpression indicates the pathway to retinal cell death in development anddegeneration. J. Neurosci. 27, 10887e10894.

Doonan, F., O'Driscoll, C., Kenna, P., Cotter, T.G., 2011. Enhancing survival ofphotoreceptor cells in vivo using the synthetic progestin Norgestrel.J. Neurochem 118, 915e927.

Dorfman, A.L., Cuenca, N., Pinilla, I., Chemtob, S., Lachapelle, P., 2011. Immunohis-tochemical evidence of synaptic retraction, cytoarchitectural remodeling, andcell death in the inner retina of the rat model of oygen-induced retinopathy(OIR). Invest Ophthalmol. Vis. Sci. 52, 1693e1708.

Doroudchi, M.M., Greenberg, K.P., Liu, J., Silka, K.A., Boyden, E.S., Lockridge, J.A.,Arman, A.C., Janani, R., Boye, S.E., Boye, S.L., Gordon, G.M., Matteo, B.C.,Sampath, A.P., Hauswirth, W.W., Horsager, A., 2011. Virally deliveredchannelrhodopsin-2 safely and effectively restores visual function in multiplemouse models of blindness. Mol. Ther. 19, 1220e1229.

Douglas, R.M., Alam, N.M., Silver, B.D., McGill, T.J., Tschetter, W.W., Prusky, G.T.,2005. Independent visual threshold measurements in the two eyes of freelymoving rats and mice using a virtual-reality optokinetic system. Vis. Neurosci.22, 677e684.

Dowling, J.E., Boycott, B.B., 1966. Organization of the primate retina: electron mi-croscopy. Proc. R. Soc. Lond B Biol. Sci. 166, 80e111.

Drack, A.V., Dumitrescu, A.V., Bhattarai, S., Gratie, D., Stone, E.M., Mullins, R.,Sheffield, V.C., 2012. TUDCA slows retinal degeneration in two different mousemodels of retinitis pigmentosa and prevents obesity in Bardet-Biedl syndrometype 1 mice. Invest Ophthalmol. Vis. Sci. 53, 100e106.

Du, Y., Miller, C.M., Kern, T.S., 2003. Hyperglycemia increases mitochondrial su-peroxide in retina and retinal cells. Free Radic. Biol. Med. 35, 1491e1499.

Dugas, B., Charbonnier, S., Baarine, M., Ragot, K., Delmas, D., Menetrier, F.,Lherminier, J., Malvitte, L., Khalfaoui, T., Bron, A., Creuzot-Garcher, C.,Latruffe, N., Lizard, G., 2010. Effects of oxysterols on cell viability, inflammatorycytokines, VEGF, and reactive oxygen species production on human retinalcells: cytoprotective effects and prevention of VEGF secretion by resveratrol.Eur. J. Nutr. 49, 435e446.

Dunaief, J.L., Dentchev, T., Ying, G.S., Milam, A.H., 2002. The role of apoptosis in age-related macular degeneration. Arch. Ophthalmol. 120, 1435e1442.

Dutsch, M., Marthol, H., Michelson, G., Neundorfer, B., Hilz, M.J., 2004. Pupillog-raphy refines the diagnosis of diabetic autonomic neuropathy. J. Neurol. Sci.222, 75e81.

Egashira, T., Yuasa, S., Fukuda, K., 2013. Novel insights into disease modeling usinginduced pluripotent stem cells. Biol. Pharm. Bull. 36, 182e188.

Eichler, W., Kuhrt, H., Hoffmann, S., Wiedemann, P., Reichenbach, A., 2000. VEGFrelease by retinal glia depends on both oxygen and glucose supply. Neuroreport11, 3533e3537.

Eigeldinger-Berthou, S., Meier, C., Zulliger, R., Lecaude, S., Enzmann, V., Sarra, G.M.,2012. Rasagiline interferes with neurodegeneration in the Prph2/rds mouse.Retina 32, 617e628.

Eiraku, M., Takata, N., Ishibashi, H., Kawada, M., Sakakura, E., Okuda, S., Sekiguchi, K.,Adachi, T., Sasai, Y., 2011. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51e56.

El-Bab, M.F., Zaki, N.S., Mojaddidi, M.A., Al-Barry, M., El-Beshbishy, H.A., 2013.Diabetic retinopathy is associated with oxidative stress and mitigation of geneexpression of antioxidant enzymes. Int. J. Gen. Med. 6, 799e806.

El-Remessy, A.B., Behzadian, M.A., Abou-Mohamed, G., Franklin, T., Caldwell, R.W.,Caldwell, R.B., 2003. Experimental diabetes causes breakdown of the blood-


retina barrier by a mechanism involving tyrosine nitration and increases inexpression of vascular endothelial growth factor and urokinase plasminogenactivator receptor. Am. J. Pathol. 162, 1995e2004.

El-Remessy, A.B., Franklin, T., Ghaley, N., Yang, J., Brands, M.W., Caldwell, R.B.,Behzadian, M.A., 2013. Diabetes-induced superoxide anion and breakdown ofthe blood-retinal barrier: role of the VEGF/uPAR pathway. PLoS One 8, e71868.

Emerich, D.F., Thanos, C.G., 2008. NT-501: an ophthalmic implant of polymer-encapsulated ciliary neurotrophic factor-producing cells. Curr. Opin. Mol.Ther. 10, 506e515.

Emoto, Y., Yoshizawa, K., Uehara, N., Kinoshita, Y., Yuri, T., Shikata, N., Tsubura, A.,2013. Curcumin suppresses N-methyl-N-nitrosourea-induced photoreceptorapoptosis in Sprague-Dawley rats. In Vivo 27, 583e590.

Esquiva, G., Lax, P., Cuenca, N., 2013. Impairment of intrinsically photosensitiveretinal ganglion cells associated with late stages of retinal degeneration. InvestOphthalmol. Vis. Sci. 54, 4605e4618.

Esteve-Rudd, J., Fernandez-Sanchez, L., Lax, P., De Juan, E., Martin-Nieto, J.,Cuenca, N., 2011. Rotenone induces degeneration of photoreceptors and impairsthe dopaminergic system in the rat retina. Neurobiol. Dis. 44, 102e115.

Eyo, U.B., Wu, L.J., 2013. Bidirectional microglia-neuron communication in thehealthy brain. Neural Plast. 2013, 456857.

Falsini, B., Marangoni, D., Salgarello, T., Stifano, G., Montrone, L., Campagna, F.,Aliberti, S., Balestrazzi, E., Colotto, A., 2008. Structure-function relationship inocular hypertension and glaucoma: interindividual and interocular analysis byOCT and pattern ERG. Graefes Arch. Clin. Exp. Ophthalmol. 246, 1153e1162.

Falsini, B., Marangoni, D., Salgarello, T., Stifano, G., Montrone, L., Di Landro, S.,Guccione, L., Balestrazzi, E., Colotto, A., 2009. Effect of epigallocatechin-gallateon inner retinal function in ocular hypertension and glaucoma: a short-termstudy by pattern electroretinogram. Graefes Arch. Clin. Exp. Ophthalmol. 247,1223e1233.

Falsini, B., Piccardi, M., Minnella, A., Savastano, C., Capoluongo, E., Fadda, A.,Balestrazzi, E., Maccarone, R., Bisti, S., 2010. Influence of saffron supplementa-tion on retinal flicker sensitivity in early age-related macular degeneration.Invest Ophthalmol. Vis. Sci. 51, 6118e6124.

Fan, B., Li, G.Y., Li, Y.P., Cui, J.Z., 2008. Neuroprotective effect of epigallocatechingallate on oxidative-stress-injured retinal cells. Zhonghua Yi Xue Za Zhi 88,1711e1714.

Fan, N., Huang, N., Lam, D.S., Leung, C.K., 2011. Measurement of photoreceptor layerin glaucoma: a spectral-domain optical coherence tomography study.J. Ophthalmol. 2011, 264803.

Fan, W., Li, X., Wang, W., Mo, J.S., Kaplan, H., Cooper, N.G., 2010. Early involvement ofimmune/inflammatory response genes in retinal degeneration in DBA/2J mice.Ophthalmol. Eye Dis. 1, 23e41.

Fariss, R.N., Li, Z.Y., Milam, A.H., 2000. Abnormalities in rod photoreceptors, ama-crine cells, and horizontal cells in human retinas with retinitis pigmentosa. Am.J. Ophthalmol. 129, 215e223.

Farjo, R., Skaggs, J., Quiambao, A.B., Cooper, M.J., Naash, M.I., 2006. Efficient non-viral ocular gene transfer with compacted DNA nanoparticles. PLoS One 1, e38.

Farsiu, S., Chiu, S.J., O'Connell, R.V., Folgar, F.A., Yuan, E., Izatt, J.A., Toth, C.A., 2014.Quantitative classification of eyes with and without intermediate age-relatedmacular degeneration using optical coherence tomography. Ophthalmology121, 162e172.

Feeney, L., 1978. Lipofuscin and melanin of human retinal pigment epithelium.Fluorescence, enzyme cytochemical, and ultrastructural studies. Invest Oph-thalmol. Vis. Sci. 17, 583e600.

Fernandez, P., Recalde, S., Ivanescu, A., Garcia-Garcia, L., Redondo-Exposito, A.,Moreno-Ordu~na, M., Fernandez-Garcia, V., Garcia-Layana, A., 2013. Preventiveeffect of a nutritional supplement with resveratrol and DHA in CNV laser-induced model. Invest Ophthalmol. Vis. Sci. 54. E-Abstract 1195.

Fernandez-Bueno, I., Fernandez-Sanchez, L., Gayoso, M.J., Garcia-Gutierrez, M.T.,Pastor, J.C., Cuenca, N., 2012. Time course modifications in organotypic cultureof human neuroretina. Exp. Eye Res. 104, 26e38.

Fernandez-Sanchez, L., Esquiva, G., Pinilla, I., Martin-Nieto, J., Cuenca, N., 2010. Theantiapoptotic TUDCA protects against mitochondrial dysfunction, glial cellchanges and loss of the capillary network in the transgenic rat model of retinitispigmentosa P23H. Invest Ophthalmol. Vis. Sci. 51. E-Abstract 3721.

Fernandez-Sanchez, L., Lax, P., Esquiva, G., Martin-Nieto, J., Pinilla, I., Cuenca, N.,2012a. Safranal, a saffron constituent, attenuates retinal degeneration in P23Hrats. PLoS One 7, e43074.

Fernandez-Sanchez, L., Lax, P., Isiegas, C., Ayuso, E., Ruiz, J.M., de la Villa, P., Bosch, F.,de la Rosa, E.J., Cuenca, N., 2012b. Proinsulin slows retinal degeneration andvision loss in the P23H rat model of retinitis pigmentosa. Hum. Gene Ther. 23,1290e1300.

Fernandez-Sanchez, L., Lax, P., Pinilla, I., Martin-Nieto, J., Cuenca, N., 2011a. Taur-oursodeoxycholic acid prevents retinal degeneration in transgenic P23H rats.Invest Ophthalmol. Vis. Sci. 52, 4998e5008.

Fernandez-Sanchez, L., Perez de Sevilla Müller, L., Brecha, N., Cuenca, N., 2013.Morphological and functional study of retinal astrocytes in DBA/2J mice. InvestOphthalmol. Vis. Sci. 54. E-Abstract 5096.

Fernandez-Sanchez, L., Raymond, I., Brecha, N., Cuenca, N., 2011b. Cellular changesin the outer retina of DBA/2J mice with aging. Invest Ophthalmol. Vis. Sci. 52. E-Abstract 706.

Fischer, A.J., Bongini, R., 2010. Turning Muller glia into neural progenitors in theretina. Mol. Neurobiol. 42, 199e209.

Fischer, A.J., Reh, T.A., 2001. Muller glia are a potential source of neural regenerationin the postnatal chicken retina. Nat. Neurosci. 4, 247e252.

Fisher, S.K., Lewis, G.P., Linberg, K.A., Verardo, M.R., 2005. Cellular remodeling inmammalian retina: results from studies of experimental retinal detachment.Prog. Retin Eye Res. 24, 395e431.

Forstreuter, F., Lucius, R., Mentlein, R., 2002. Vascular endothelial growth factorinduces chemotaxis and proliferation of microglial cells. J. Neuroimmunol. 132,93e98.

Fortune, B., Demirel, S., Zhang, X., Hood, D.C., Patterson, E., Jamil, A.,Mansberger, S.L., Cioffi, G.A., Johnson, C.A., 2007. Comparing multifocal VEP andstandard automated perimetry in high-risk ocular hypertension and earlyglaucoma. Invest Ophthalmol. Vis. Sci. 48, 1173e1180.

Frank, R.N., Amin, R.H., Eliott, D., Puklin, J.E., Abrams, G.W., 1996. Basic fibroblastgrowth factor and vascular endothelial growth factor are present in epiretinaland choroidal neovascular membranes. Am. J. Ophthalmol. 122, 393e403.

Franze, K., Grosche, J., Skatchkov, S.N., Schinkinger, S., Foja, C., Schild, D.,Uckermann, O., Travis, K., Reichenbach, A., Guck, J., 2007. Muller cells are livingoptical fibers in the vertebrate retina. Proc. Natl. Acad. Sci. U. S. A. 104,8287e8292.

Frasson, M., Picaud, S., Leveillard, T., Simonutti, M., Mohand-Said, S., Dreyfus, H.,Hicks, D., Sabel, J., 1999. Glial cell line-derived neurotrophic factor induceshistologic and functional protection of rod photoreceptors in the rd/rd mouse.Invest Ophthalmol. Vis. Sci. 40, 2724e2734.

Friedman, E., Krupsky, S., Lane, A.M., Oak, S.S., Friedman, E.S., Egan, K.,Gragoudas, E.S., 1995. Ocular blood flow velocity in age-related maculardegeneration. Ophthalmology 102, 640e646.

Frishman, L.J., Shen, F.F., Du, L., Robson, J.G., Harwerth, R.S., Smith 3rd, E.L., Carter-Dawson, L., Crawford, M.L., 1996. The scotopic electroretinogram of macaqueafter retinal ganglion cell loss from experimental glaucoma. Invest Ophthalmol.Vis. Sci. 37, 125e141.

Fuchs, M., Scholz, M., Sendelbeck, A., Atorf, J., Schlegel, C., Enz, R., Brandstatter, J.H.,2012. Rod photoreceptor ribbon synapses in DBA/2J mice show progressive age-related structural changes. PLoS One 7, e44645.

Fuhrmann, S., Zou, C., Levine, E.M., 2013. Retinal pigment epithelium development,plasticity, and tissue homeostasis. Exp. Eye Res..

Galehdar, Z., Swan, P., Fuerth, B., Callaghan, S.M., Park, D.S., Cregan, S.P., 2010.Neuronal apoptosis induced by endoplasmic reticulum stress is regulated byATF4-CHOP-mediated induction of the Bcl-2 homology 3-only member PUMA.J. Neurosci. 30, 16938e16948.

Gallego, B.I., Salazar, J.J., de Hoz, R., Rojas, B., Ramirez, A.I., Salinas-Navarro, M.,Ortin-Martinez, A., Valiente-Soriano, F.J., Aviles-Trigueros, M., Villegas-Perez, M.P., Vidal-Sanz, M., Trivino, A., Ramirez, J.M., 2012. IOP induces upre-gulation of GFAP and MHC-II and microglia reactivity in mice retina contra-lateral to experimental glaucoma. J. Neuroinflam. 9, 92.

Gamm, D.M., Melvan, J.N., Shearer, R.L., Pinilla, I., Sabat, G., Svendsen, C.N.,Wright, L.S., 2008. A novel serum-free method for culturing human prenatalretinal pigment epithelial cells. Invest Ophthalmol. Vis. Sci. 49, 788e799.

Gamm, D.M., Wright, L.S., 2013. From embryonic stem cells to mature photore-ceptors. Nat. Biotechnol. 31, 712e713.

Ganesh, B.S., Chintala, S.K., 2011. Inhibition of reactive gliosis attenuatesexcitotoxicity-mediated death of retinal ganglion cells. PLoS One 6, e18305.

Garcia-Ayuso, D., Ortin-Martinez, A., Jimenez-Lopez, M., Galindo-Romero, C.,Cuenca, N., Pinilla, I., Vidal-Sanz, M., Agudo-Barriuso, M., Villegas-Perez, M.P.,2013. Changes in the photoreceptor mosaic of P23H-1 rats during retinaldegeneration: implications for rod-cone dependent survival. Invest Oph-thalmol. Vis. Sci. 54, 5888e5900.

Garcia-Ayuso, D., Salinas-Navarro, M., Agudo, M., Cuenca, N., Pinilla, I., Vidal-Sanz, M., Villegas-Perez, M.P., 2010. Retinal ganglion cell numbers anddelayed retinal ganglion cell death in the P23H rat retina. Exp. Eye Res. 91,800e810.

Garcia-Ayuso, D., Salinas-Navarro, M., Agudo-Barriuso, M., Alarcon-Martinez, L.,Vidal-Sanz, M., Villegas-Perez, M.P., 2011. Retinal ganglion cell axonalcompression by retinal vessels in light-induced retinal degeneration. Mol. Vis.17, 1716e1733.

Garden, G.A., Moller, T., 2006. Microglia biology in health and disease.J. Neuroimmune Pharmacol. 1, 127e137.

Garrido, C., Galluzzi, L., Brunet, M., Puig, P.E., Didelot, C., Kroemer, G., 2006.Mechanisms of cytochrome c release from mitochondria. Cell. Death Differ. 13,1423e1433.

Gaspar, J.M., Martins, A., Cruz, R., Rodrigues, C.M., Ambrosio, A.F., Santiago, A.R.,2013. Tauroursodeoxycholic acid protects retinal neural cells from cell deathinduced by prolonged exposure to elevated glucose. Neuroscience 253,380e388.

Gastinger, M.J., Singh, R.S., Barber, A.J., 2006. Loss of cholinergic and dopaminergicamacrine cells in streptozotocin-diabetic rat and Ins2Akita-diabetic mouseretinas. Invest Ophthalmol. Vis. Sci. 47, 3143e3150.

Gehrig, A., Langmann, T., Horling, F., Janssen, A., Bonin, M., Walter, M., Poths, S.,Weber, B.H., 2007. Genome-wide expression profiling of the retinoschisin-deficient retina in early postnatal mouse development. Invest Ophthalmol.Vis. Sci. 48, 891e900.

Gerona, G., Lopez, D., Palmero, M., Maneu, V., 2010. Antioxidant N-acetyl-cysteineprotects retinal pigmented epithelial cells from long-term hypoxia changes ingene expression. J. Ocul. Pharmacol. Ther. 26, 309e314.

Ghosh, F., Johansson, K., 2012. Neuronal and glial alterations in complex long-termrhegmatogenous retinal detachment. Curr. Eye Res. 37, 704e711.

Giani, A., Cigada, M., Choudhry, N., Deiro, A.P., Oldani, M., Pellegrini, M.,Invernizzi, A., Duca, P., Miller, J.W., Staurenghi, G., 2010. Reproducibility of


retinal thickness measurements on normal and pathologic eyes by differentoptical coherence tomography instruments. Am. J. Ophthalmol. 150, 815e824.

Giaume, C., Kirchhoff, F., Matute, C., Reichenbach, A., Verkhratsky, A., 2007. Glia: thefulcrum of brain diseases. Cell. Death Differ. 14, 1324e1335.

Ginsburg, A.P., 2006. Contrast sensitivity: determining the visual quality andfunction of cataract, intraocular lenses and refractive surgery. Curr. Opin.Ophthalmol. 17, 19e26.

Godley, B.F., Shamsi, F.A., Liang, F.Q., Jarrett, S.G., Davies, S., Boulton, M., 2005. Bluelight induces mitochondrial DNA damage and free radical production inepithelial cells. J. Biol. Chem. 280, 21061e21066.

Goebel, W., Lieb, W.E., Ho, A., Sergott, R.C., Farhoumand, R., Grehn, F., 1995. ColorDoppler imaging: a new technique to assess orbital blood flow in patients withdiabetic retinopathy. Invest Ophthalmol. Vis. Sci. 36, 864e870.

Gorbatyuk, M., Justilien, V., Liu, J., Hauswirth, W.W., Lewin, A.S., 2007. Preservationof photoreceptor morphology and function in P23H rats using an allele inde-pendent ribozyme. Exp. Eye Res. 84, 44e52.

Gorbatyuk, M.S., Gorbatyuk, O.S., LaVail, M.M., Lin, J.H., Hauswirth, W.W.,Lewin, A.S., 2012. Functional rescue of P23H rhodopsin photoreceptors by genedelivery. Adv. Exp. Med. Biol. 723, 191e197.

Graeber, M.B., Streit, W.J., 1990. Microglia: immune network in the CNS. BrainPathol. 1, 2e5.

Grafstein, B., Murray, M., Ingoglia, N.A., 1972. Protein synthesis and axonal transportin retinal ganglion cells of mice lacking visual receptors. Brain Res. 44, 37e48.

Gragoudas, E.S., Adamis, A.P., Cunningham Jr., E.T., Feinsod, M., Guyer, D.R., 2004.Pegaptanib for neovascular age-related macular degeneration. N Engl. J. Med.351, 2805e2816.

Green, W.R., Enger, C., 1993. Age-related macular degeneration histopathologicstudies. The 1992 Lorenz E. Zimmerman Lecture. Ophthalmology 100,1519e1535.

Greenwald, D.L., Cashman, S.M., Kumar-Singh, R., 2013. Mutation-independentrescue of a novel mouse model of Retinitis Pigmentosa. Gene Ther. 20,425e434.

Griffin, W.S., Liu, L., Li, Y., Mrak, R.E., Barger, S.W., 2006. Interleukin-1 mediatesAlzheimer and Lewy body pathologies. J. Neuroinflammation 3, 5.

Grossniklaus, H.E., Ling, J.X., Wallace, T.M., Dithmar, S., Lawson, D.H., Cohen, C.,Elner, V.M., Elner, S.G., Sternberg Jr., P., 2002. Macrophage and retinal pigmentepithelium expression of angiogenic cytokines in choroidal neovascularization.Mol. Vis. 8, 119e126.

Grossniklaus, H.E., Nickerson, J.M., Edelhauser, H.F., Bergman, L.A., Berglin, L., 2013.Anatomic alterations in aging and age-related diseases of the eye. InvestOphthalmol. Vis. Sci. 54, ORSF23e27.

Grunwald, J.E., Maguire, A.M., Dupont, J., 1996. Retinal hemodynamics in retinitispigmentosa. Am. J. Ophthalmol. 122, 502e508.

Guillemin, G.J., Brew, B.J., 2004. Microglia, macrophages, perivascular macrophages,and pericytes: a review of function and identification. J. Leukoc. Biol. 75,388e397.

Gupta, N., Brown, K.E., Milam, A.H., 2003. Activated microglia in human retinitispigmentosa, late-onset retinal degeneration, and age-related macular degen-eration. Exp. Eye Res. 76, 463e471.

Gupta, S.K., Kumar, B., Nag, T.C., Agrawal, S.S., Agrawal, R., Agrawal, P., Saxena, R.,Srivastava, S., 2011. Curcumin prevents experimental diabetic retinopathy inrats through its hypoglycemic, antioxidant, and anti-inflammatory mecha-nisms. J. Ocul. Pharmacol. Ther. 27, 123e130.

Gust, J., Reh, T.A., 2011. Adult donor rod photoreceptors integrate into the maturemouse retina. Invest Ophthalmol. Vis. Sci. 52, 5266e5272.

Hageman, G.S., Luthert, P.J., Victor Chong, N.H., Johnson, L.V., Anderson, D.H.,Mullins, R.F., 2001. An integrated hypothesis that considers drusen as bio-markers of immune-mediated processes at the RPE-Bruch's membrane inter-face in aging and age-related macular degeneration. Prog. Retin Eye Res. 20,705e732.

Hagiwara, A., Yamamoto, S., Ogata, K., Sugawara, T., Hiramatsu, A., Shibata, M.,Mitamura, Y., 2011. Macular abnormalities in patients with retinitis pigmentosa:prevalence on OCT examination and outcomes of vitreoretinal surgery. ActaOphthalmol. 89, e122e125.

Halder, S.K., Matsunaga, H., Ishii, K.J., Akira, S., Miyake, K., Ueda, H., 2013. Retinal celltype-specific prevention of ischemia-induced damages by LPS-TLR4 signalingthrough microglia. J. Neurochem. 126, 243e260.

Hamann, S., Schorderet, D.F., Cottet, S., 2009. Bax-induced apoptosis in Leber'scongenital amaurosis: a dual role in rod and cone degeneration. PLoS One 4,e6616.

Handa, J.T., Verzijl, N., Matsunaga, H., Aotaki-Keen, A., Lutty, G.A., te Koppele, J.M.,Miyata, T., Hjelmeland, L.M., 1999. Increase in the advanced glycation endproduct pentosidine in Bruch's membrane with age. Invest Ophthalmol. Vis. Sci.40, 775e779.

Hanisch, U.K., Kettenmann, H., 2007. Microglia: active sensor and versatile effectorcells in the normal and pathologic brain. Nat. Neurosci. 10, 1387e1394.

Harada, T., Harada, C., Kohsaka, S., Wada, E., Yoshida, K., Ohno, S., Mamada, H.,Tanaka, K., Parada, L.F., Wada, K., 2002. Microglia-Muller glia cell interactionscontrol neurotrophic factor production during light-induced retinal degenera-tion. J. Neurosci. 22, 9228e9236.

Harris, A., Chung, H.S., Ciulla, T.A., Kagemann, L., 1999. Progress in measurement ofocular blood flow and relevance to our understanding of glaucoma and age-related macular degeneration. Prog. Retin Eye Res. 18, 669e687.

Harry, G.J., 2013. Microglia during development and aging. Pharmacol. Ther. 139,313e326.

Haurigot, V., Villacampa, P., Ribera, A., Bosch, A., Ramos, D., Ruberte, J., Bosch, F.,2012. Long-term retinal PEDF overexpression prevents neovascularization in amurine adult model of retinopathy. PLoS One 7, e41511.

Hayreh, S.S., 2007. Neovascular glaucoma. Prog. Retin Eye Res. 26, 470e485.Heier, J.S., Brown, D.M., Chong, V., Korobelnik, J.F., Kaiser, P.K., Nguyen, Q.D.,

Kirchhof, B., Ho, A., Ogura, Y., Yancopoulos, G.D., Stahl, N., Vitti, R., Berliner, A.J.,Soo, Y., Anderesi, M., Groetzbach, G., Sommerauer, B., Sandbrink, R., Simader, C.,Schmidt-Erfurth, U., 2012. Intravitreal aflibercept (VEGF trap-eye) in wet age-related macular degeneration. Ophthalmology 119, 2537e2548.

Hernandez, M.R., Miao, H., Lukas, T., 2008. Astrocytes in glaucomatous optic neu-ropathy. Prog. Brain Res. 173, 353e373.

Himori, N., Yamamoto, K., Maruyama, K., Ryu, M., Taguchi, K., Yamamoto, M.,Nakazawa, T., 2013. Critical role of Nrf2 in oxidative stress-induced retinalganglion cell death. J. Neurochem. 127, 669e680.

Hirami, Y., Osakada, F., Takahashi, K., Okita, K., Yamanaka, S., Ikeda, H.,Yoshimura, N., Takahashi, M., 2009. Generation of retinal cells from mouse andhuman induced pluripotent stem cells. Neurosci. Lett. 458, 126e131.

Hirano, M., Yamamoto, A., Yoshimura, N., Tokunaga, T., Motohashi, T., Ishizaki, K.,Yoshida, H., Okazaki, K., Yamazaki, H., Hayashi, S., Kunisada, T., 2003. Generationof structures formed by lens and retinal cells differentiating from embryonicstem cells. Dev. Dyn. 228, 664e671.

Hirata, C., Nakano, K., Nakamura, N., Kitagawa, Y., Shigeta, H., Hasegawa, G.,Ogata, M., Ikeda, T., Sawa, H., Nakamura, K., Ienaga, K., Obayashi, H., Kondo, M.,1997. Advanced glycation end products induce expression of vascular endo-thelial growth factor by retinal Muller cells. Biochem Biophys. Res. Commun.236, 712e715.

Hirooka, K., Tokuda, M., Miyamoto, O., Itano, T., Baba, T., Shiraga, F., 2004. TheGinkgo biloba extract (EGb 761) provides a neuroprotective effect on retinalganglion cells in a rat model of chronic glaucoma. Curr. Eye Res. 28, 153e157.

Hirsch, E.C., Hunot, S., 2009. Neuroinflammation in Parkinson's disease: a target forneuroprotection? Lancet Neurol. 8, 382e397.

Hisatomi, T., Sakamoto, T., Murata, T., Yamanaka, I., Oshima, Y., Hata, Y., Ishibashi, T.,Inomata, H., Susin, S.A., Kroemer, G., 2001. Relocalization of apoptosis-inducingfactor in photoreceptor apoptosis induced by retinal detachment in vivo. Am. J.Pathol. 158, 1271e1278.

Hoerster, R., Muether, P.S., Hermann, M.M., Koch, K., Kirchhof, B., Fauser, S., 2011.Subjective and functional deterioration in recurrences of neovascular AMD areoften preceded by morphologic changes in optic coherence tomography. Br. J.Ophthalmol. 95, 1424e1426.

Hoffmann, S., Friedrichs, U., Eichler, W., Rosenthal, A., Wiedemann, P., 2002.Advanced glycation end products induce choroidal endothelial cell prolifera-tion, matrix metalloproteinase-2 and VEGF upregulation in vitro. Graefes Arch.Clin. Exp. Ophthalmol. 240, 996e1002.

Holder, G.E., 2001. Pattern electroretinography (PERG) and an integrated approachto visual pathway diagnosis. Prog. Retin Eye Res. 20, 531e561.

Holder, G.E., Gale, R.P., Acheson, J.F., Robson, A.G., 2009. Electrodiagnostic assess-ment in optic nerve disease. Curr. Opin. Neurol. 22, 3e10.

Honda, S., Sasaki, Y., Ohsawa, K., Imai, Y., Nakamura, Y., Inoue, K., Kohsaka, S., 2001.Extracellular ATP or ADP induce chemotaxis of cultured microglia through Gi/o-coupled P2Y receptors. J. Neurosci. 21, 1975e1982.

Hood, D.C., Greenstein, V.C., 2003. Multifocal VEP and ganglion cell damage: ap-plications and limitations for the study of glaucoma. Prog. Retin Eye Res. 22,201e251.

Hood, D.C., Odel, J.G., Chen, C.S., Winn, B.J., 2003. The multifocal electroretinogram.J. Neuroophthalmol. 23, 225e235.

Hood, D.C., Zhang, X., Greenstein, V.C., Kangovi, S., Odel, J.G., Liebmann, J.M.,Ritch, R., 2000. An interocular comparison of the multifocal VEP: a possibletechnique for detecting local damage to the optic nerve. Invest Ophthalmol. Vis.Sci. 41, 1580e1587.

Howell, J.C., Chun, E., Farrell, A.N., Hur, E.Y., Caroti, C.M., Iuvone, P.M., Haque, R.,2013. Global microRNA expression profiling: curcumin (diferuloylmethane)alters oxidative stress-responsive microRNAs in human ARPE-19 cells. Mol. Vis.19, 544e560.

Howes, S.C., Caelli, T., Mitchell, P., 1982. Contrast sensitivity in diabetics with reti-nopathy and cataract. Aust. J. Ophthalmol. 10, 173e178.

Huang, L., Shen, X., Fan, N., He, J., 2012. Clinical application of photopic negativeresponse of the flash electroretinogram in primary open-angle Glaucoma. EyeSci. 27, 113e118.

Huang, L., Xu, W., Xu, G., 2013a. Transplantation of CX3CL1-expressing mesen-chymal stem cells provides neuroprotective and immunomodulatory effects ina rat model of retinal degeneration. Ocul. Immunol. Inflamm. 21, 276e285.

Huang, W., Li, G., Qiu, J., Gonzalez, P., Challa, P., 2013b. Protective effects ofresveratrol in experimental retinal detachment. PLoS One 8, e75735.

Huitinga, I., van Rooijen, N., de Groot, C.J., Uitdehaag, B.M., Dijkstra, C.D., 1990.Suppression of experimental allergic encephalomyelitis in Lewis rats afterelimination of macrophages. J. Exp. Med. 172, 1025e1033.

Humayun, M.S., de Juan Jr., E., del Cerro, M., Dagnelie, G., Radner, W., Sadda, S.R., delCerro, C., 2000. Human neural retinal transplantation. Invest Ophthalmol. Vis.Sci. 41, 3100e3106.

Hume, D.A., Perry, V.H., Gordon, S., 1983. Immunohistochemical localization of amacrophage-specific antigen in developing mouse retina: phagocytosis of dyingneurons and differentiation of microglial cells to form a regular array in theplexiform layers. J. Cell. Biol. 97, 253e257.

Idelson, M., Alper, R., Obolensky, A., Ben-Shushan, E., Hemo, I., Yachimovich-Cohen, N., Khaner, H., Smith, Y., Wiser, O., Gropp, M., Cohen, M.A., Even-Ram, S.,


Berman-Zaken, Y., Matzrafi, L., Rechavi, G., Banin, E., Reubinoff, B., 2009.Directed differentiation of human embryonic stem cells into functional retinalpigment epithelium cells. Cell. Stem Cell. 5, 396e408.

Ideno, J., Mizukami, H., Kakehashi, A., Saito, Y., Okada, T., Urabe, M., Kume, A.,Kuroki, M., Kawakami, M., Ishibashi, S., Ozawa, K., 2007. Prevention of diabeticretinopathy by intraocular soluble flt-1 gene transfer in a spontaneously dia-betic rat model. Int. J. Mol. Med. 19, 75e79.

Igarashi, T., Miyake, K., Masuda, I., Takahashi, H., Shimada, T., 2010. Adeno-associ-ated vector (type 8)-mediated expression of soluble Flt-1 efficiently inhibitsneovascularization in a murine choroidal neovascularization model. Hum. GeneTher. 21, 631e637.

Ikeda, H., Osakada, F., Watanabe, K., Mizuseki, K., Haraguchi, T., Miyoshi, H.,Kamiya, D., Honda, Y., Sasai, N., Yoshimura, N., Takahashi, M., Sasai, Y., 2005.Generation of Rxþ/Pax6þ neural retinal precursors from embryonic stem cells.Proc. Natl. Acad. Sci. U. S. A. 102, 11331e11336.

Imai, D., Yoneya, S., Gehlbach, P.L., Wei, L.L., Mori, K., 2005. Intraocular gene transferof pigment epithelium-derived factor rescues photoreceptors from light-induced cell death. J. Cell. Physiol. 202, 570e578.

Inman, D.M., Horner, P.J., 2007. Reactive nonproliferative gliosis predominates in achronic mouse model of glaucoma. Glia 55, 942e953.

Inoue, K., Koizumi, S., Tsuda, M., 2007. The role of nucleotides in the neuronegliacommunication responsible for the brain functions. J. Neurochem 102,1447e1458.

Inzelberg, R., Ramirez, J.A., Nisipeanu, P., Ophir, A., 2004. Retinal nerve fiber layerthinning in Parkinson disease. Vis. Res. 44, 2793e2797.

Ishizuka, F., Shimazawa, M., Umigai, N., Ogishima, H., Nakamura, S., Tsuruma, K.,Hara, H., 2013. Crocetin, a carotenoid derivative, inhibits retinal ischemicdamage in mice. Eur. J. Pharmacol. 703, 1e10.

Izumi, Y., Kirby, C.O., Benz, A.M., Olney, J.W., Zorumski, C.F., 1999. Muller cellswelling, glutamate uptake, and excitotoxic neurodegeneration in the isolatedrat retina. Glia 25, 379e389.

Jacobson, S.G., Cideciyan, A.V., Ratnakaram, R., Heon, E., Schwartz, S.B., Roman, A.J.,Peden, M.C., Aleman, T.S., Boye, S.L., Sumaroka, A., Conlon, T.J., Calcedo, R.,Pang, J.J., Erger, K.E., Olivares, M.B., Mullins, C.L., Swider, M., Kaushal, S.,Feuer, W.J., Iannaccone, A., Fishman, G.A., Stone, E.M., Byrne, B.J.,Hauswirth, W.W., 2012. Gene therapy for leber congenital amaurosis caused byRPE65 mutations: safety and efficacy in 15 children and adults followed up to3s. Arch. Ophthalmol. 130, 9e24.

Jarrett, S.G., Boulton, M.E., 2012. Consequences of oxidative stress in age-relatedmacular degeneration. Mol. Asp. Med. 33, 399e417.

Jiang, L.Q., Jorquera, M., Streilein, J.W., 1993. Subretinal space and vitreous cavity asimmunologically privileged sites for retinal allografts. Invest Ophthalmol. Vis.Sci. 34, 3347e3354.

Jiang, X., Ni, Y., Liu, T., Zhang, M., Ren, H., Xu, G., 2013. Inhibition of LPS-inducedretinal microglia activation by naloxone does not prevent photoreceptordeath. Inflammation 36, 42e52.

Johnson, E.C., Morrison, J.C., 2009. Friend or foe? Resolving the impact of glial re-sponses in glaucoma. J. Glaucoma 18, 341e353.

Jones, B.W., Kondo, M., Terasaki, H., Lin, Y., McCall, M., Marc, R.E., 2012. Retinalremodeling. Jpn. J. Ophthalmol. 56, 289e306.

Jones, B.W., Kondo, M., Terasaki, H., Watt, C.B., Rapp, K., Anderson, J., Lin, Y.,Shaw, M.V., Yang, J.H., Marc, R.E., 2011. Retinal remodeling in the Tg P347Lrabbit, a large-eye model of retinal degeneration. J. Comp. Neurol. 519,2713e2733.

Jones, B.W., Marc, R.E., 2005. Retinal remodeling during retinal degeneration. Exp.Eye Res. 81, 123e137.

Jones, B.W., Watt, C.B., Frederick, J.M., Baehr, W., Chen, C.K., Levine, E.M.,Milam, A.H., Lavail, M.M., Marc, R.E., 2003. Retinal remodeling triggered byphotoreceptor degenerations. J. Comp. Neurol. 464, 1e16.

Kachi, S., Oshima, Y., Esumi, N., Kachi, M., Rogers, B., Zack, D.J., Campochiaro, P.A.,2005. Nonviral ocular gene transfer. Gene Ther. 12, 843e851.

Kalloniatis, M., Luu, C., 1995. Visual Acuity, 2011/03/18 ed.Karampelas, M., Sim, D.A., Keane, P.A., Papastefanou, V.P., Sadda, S.R., Tufail, A.,

Dowler, J., 2013. Evaluation of retinal pigment epithelium-Bruch's membranecomplex thickness in dry age-related macular degeneration using opticalcoherence tomography. Br. J. Ophthalmol. 97, 1256e1261.

Karlstetter, M., Ebert, S., Langmann, T., 2010a. Microglia in the healthy and degen-erating retina: insights from novel mouse models. Immunobiology 215,685e691.

Karlstetter, M., Walczak, Y., Weigelt, K., Ebert, S., Van den Brulle, J., Schwer, H.,Fuchshofer, R., Langmann, T., 2010b. The novel activated microglia/macrophageWAP domain protein, AMWAP, acts as a counter-regulator of proinflammatoryresponse. J. Immunol. 185, 3379e3390.

Karpe, G., Germanis, M., 1962. The prognostic value of the electroretinogram inthrombosis of the retinal veins. Acta Ophthalmol. Suppl. Suppl. 70, 202e229.

Karpe, G., Uchermann, A., 1955. The clinical electroretinogram. VII. The electroret-inogram in circulatory disturbances of the retina. Acta Ophthalmol. (Copenh.)33, 493e516.

Kase, S., Saito, W., Yokoi, M., Yoshida, K., Furudate, N., Muramatsu, M., Saito, A.,Kase, M., Ohno, S., 2006. Expression of glutamine synthetase and cell pro-liferation in human idiopathic epiretinal membrane. Br. J. Ophthalmol. 90,96e98.

Katai, N., Yanagidaira, T., Senda, N., Murata, T., Yoshimura, N., 2006. Expression of c-Jun and Bcl-2 family proteins in apoptotic photoreceptors of RCS rats. Jpn. J.Ophthalmol. 50, 121e127.

Kaur, C., Foulds, W.S., Ling, E.A., 2008. Blood-retinal barrier in hypoxic ischaemicconditions: basic concepts, clinical features and management. Prog. Retin EyeRes. 27, 622e647.

Kawai, T., Akira, S., 2006. TLR signaling. Cell. Death Differ. 13, 816e825.Keeble, J.A., Gilmore, A.P., 2007. Apoptosis commitmentetranslating survival signals

into decisions on mitochondria. Cell. Res. 17, 976e984.Kellner, U., Foerster, M.H., 1993. Pattern of dysfunction in progressive cone dys-

trophiesean extended classification. Ger. J. Ophthalmol. 2, 170e177.Kenney,M.C., Chwa,M.,Atilano,S.R., Pavlis, J.M., Falatoonzadeh,P.,Ramirez,C.,Malik,D.,

Hsu, T., Woo, G., Soe, K., Nesburn, A.B., Boyer, D.S., Kuppermann, B.D.,Jazwinski, S.M., Miceli, M.V., Wallace, D.C., Udar, N., 2013a. Mitochondrial DNAvariantsmediate energy production and expression levels for CFH, C3 and EFEMP1genes: implications for age-related macular degeneration. PLoS One 8, e54339.

Kenney, M.C., Hertzog, D., Chak, G., Atilano, S.R., Khatibi, N., Soe, K., Nobe, A.,Yang, E., Chwa, M., Zhu, F., Memarzadeh, M., King, J., Langberg, J., Small, K.,Nesburn, A.B., Boyer, D.S., Udar, N., 2013b. Mitochondrial DNA haplogroupsconfer differences in risk for age-related macular degeneration: a case controlstudy. BMC Med. Genet. 14, 4.

Kevany, B.M., Palczewski, K., 2010. Phagocytosis of retinal rod and cone photore-ceptors. Physiol. (Bethesda) 25, 8e15.

Khan, J.C., Thurlby, D.A., Shahid, H., Clayton, D.G., Yates, J.R., Bradley, M., Moore, A.T.,Bird, A.C., 2006. Smoking and age related macular degeneration: the number ofpacks of cigarette smoking is a major determinant of risk for both geographicatrophy and choroidal neovascularisation. Br. J. Ophthalmol. 90, 75e80.

Kiel, J.W., Reiner, A.J., 1997. Morphometric analysis of the choroid, Bruch's mem-brane, and retinal pigment epithelium in eyes with age-related maculardegeneration. Invest Ophthalmol. Vis. Sci. 38, 1290e1292.

Kielian, T., Mayes, P., Kielian, M., 2002. Characterization of microglial responses toStaphylococcus aureus: effects on cytokine, costimulatory molecule, and Toll-like receptor expression. J. Neuroimmunol. 130, 86e99.

Kigerl, K.A., Gensel, J.C., Ankeny, D.P., Alexander, J.K., Donnelly, D.J., Popovich, P.G.,2009. Identification of two distinct macrophage subsets with divergent effectscausing either neurotoxicity or regeneration in the injured mouse spinal cord.J. Neurosci. 29, 13435e13444.

Kim, Y.H., Kim, Y.S., Kang, S.S., Cho, G.J., Choi, W.S., 2010. Resveratrol inhibitsneuronal apoptosis and elevated Ca2þ/calmodulin-dependent protein kinase IIactivity in diabetic mouse retina. Diabetes 59, 1825e1835.

Kim, Y.H., Kim, Y.S., Roh, G.S., Choi, W.S., Cho, G.J., 2012. Resveratrol blocks diabetes-induced early vascular lesions and vascular endothelial growth factor inductionin mouse retinas. Acta Ophthalmol. 90, e31e37.

Kimelberg, H.K., 2010. Functions of mature mammalian astrocytes: a current view.Neuroscientist 16, 79e106.

King, R.E., Kent, K.D., Bomser, J.A., 2005. Resveratrol reduces oxidation and prolif-eration of human retinal pigment epithelial cells via extracellular signal-regulated kinase inhibition. Chem. Biol. Interact. 151, 143e149.

Kirby, E., Bandelow, S., Hogervorst, E., 2010. Visual impairment in Alzheimer'sdisease: a critical review. J. Alzheimers Dis. 21, 15e34.

Kitaya, N., Nagaoka, T., Hikichi, T., Sugawara, R., Fukui, K., Ishiko, S., Yoshida, A.,2003. Features of abnormal choroidal circulation in central serous chorior-etinopathy. Br. J. Ophthalmol. 87, 709e712.

Klaassen, I., Van Noorden, C.J., Schlingemann, R.O., 2013. Molecular basis of theinner blood-retinal barrier and its breakdown in diabetic macular edema andother pathological conditions. Prog. Retin Eye Res. 34, 19e48.

Klimanskaya, I., Hipp, J., Rezai, K.A., West, M., Atala, A., Lanza, R., 2004. Derivationand comparative assessment of retinal pigment epithelium from human em-bryonic stem cells using transcriptomics. Cloning Stem Cells 6, 217e245.

Klistorner, A., Graham, S.L., Martins, A., Grigg, J.R., Arvind, H., Kumar, R.S.,James, A.C., Billson, F.A., 2007. Multifocal blue-on-yellow visual evoked poten-tials in early glaucoma. Ophthalmology 114, 1613e1621.

Klistorner, A.I., Graham, S.L., Grigg, J.R., Billson, F.A., 1998. Multifocal topographicvisual evoked potential: improving objective detection of local visual field de-fects. Invest Ophthalmol. Vis. Sci. 39, 937e950.

Kniestedt, C., Stamper, R.L., 2003. Visual acuity and its measurement. Ophthalmol.Clin. North Am. 16, 155e170 v.

Koch, S., Sothilingam, V., Garcia Garrido, M., Tanimoto, N., Becirovic, E., Koch, F.,Seide, C., Beck, S.C., Seeliger, M.W., Biel, M., Muhlfriedel, R., Michalakis, S., 2012.Gene therapy restores vision and delays degeneration in the CNGB1(-/-) mousemodel of retinitis pigmentosa. Hum. Mol. Genet. 21, 4486e4496.

Kohno, H., Chen, Y., Kevany, B.M., Pearlman, E., Miyagi, M., Maeda, T., Palczewski, K.,Maeda, A., 2013. Photoreceptor proteins initiate microglial activation via Toll-like receptor 4 in retinal degeneration mediated by all-trans-retinal. J. Biol.Chem. 288, 15326e15341.

Kohno, H., Sakai, T., Kitahara, K., 2006. Induction of nestin, Ki-67, and cyclin D1expression in Muller cells after laser injury in adult rat retina. Graefes Arch.Clin. Exp. Ophthalmol. 244, 90e95.

Koike-Kiriyama, N., Adachi, Y., Minamino, K., Iwasaki, M., Nakano, K., Koike, Y.,Yamada, H., Mukaide, H., Shigematsu, A., Mizokami, T., Matsumura, M.,Ikehara, S., 2007. Human cord blood cells can differentiate into retinal nervecells. Acta Neurobiol. Exp. (Wars) 67, 359e365.

Kolb, H., 2003. How the retina works. Am. Sci. 91, 28e35.Kolb, H., Nelson, R., Ahnelt, P., Cuenca, N., 2001. Cellular organization of the

vertebrate retina. Prog. Brain Res. 131, 3e26.Kolb, H., Zhang, L., Dekorver, L., Cuenca, N., 2002. A new look at calretinin-

immunoreactive amacrine cell types in the monkey retina. J. Comp. Neurol.453, 168e184.


Kolomeyer, A.M., Zarbin, M.A., 2014. Trophic factors in the pathogenesis and ther-apy for retinal degenerative diseases. Surv. Ophthalmol..

Komeima, K., Rogers, B.S., Campochiaro, P.A., 2007. Antioxidants slow photoreceptorcell death in mouse models of retinitis pigmentosa. J. Cell. Physiol. 213,809e815.

Komeima, K., Rogers, B.S., Lu, L., Campochiaro, P.A., 2006. Antioxidants reduce conecell death in a model of retinitis pigmentosa. Proc. Natl. Acad. Sci. U. S. A. 103,11300e11305.

Kook, D., Wolf, A.H., Yu, A.L., Neubauer, A.S., Priglinger, S.G., Kampik, A., Welge-Lussen, U.C., 2008. The protective effect of quercetin against oxidative stress inthe human RPE in vitro. Invest Ophthalmol. Vis. Sci. 49, 1712e1720.

Kotowski, J., Wollstein, G., Ishikawa, H., Schuman, J.S., 2013. Imaging of the opticnerve and retinal nerve fiber layer: an essential part of glaucoma diagnosis andmonitoring. Surv. Ophthalmol..

Kowluru, R.A., Kanwar, M., 2007. Effects of curcumin on retinal oxidative stress andinflammation in diabetes. Nutr. Metab. (Lond.) 4, 8.

Kowluru, R.A., Tang, J., Kern, T.S., 2001. Abnormalities of retinal metabolism indiabetes and experimental galactosemia. VII. Effect of long-term administrationof antioxidants on the development of retinopathy. Diabetes 50, 1938e1942.

Kreutzberg, G.W., 1996. Microglia: a sensor for pathological events in the CNS.Trends Neurosci. 19, 312e318.

Krinsky, N.I., Landrum, J.T., Bone, R.A., 2003. Biologic mechanisms of the protectiverole of lutein and zeaxanthin in the eye. Annu. Rev. Nutr. 23, 171e201.

Kroemer, G., Martin, S.J., 2005. Caspase-independent cell death. Nat. Med. 11,725e730.

Kroll, A.J., Machemer, R., 1968. Experimental retinal detachment in the owl monkey.3. Electron microscopy of retina and pigment epithelium. Am. J. Ophthalmol. 66,410e427.

Kroll, A.J., Machemer, R., 1969. Experimental retinal detachment and reattachmentin the rhesus monkey. Electron microscopic comparison of rods and cones. Am.J. Ophthalmol. 68, 58e77.

Kuan, C.Y., Roth, K.A., Flavell, R.A., Rakic, P., 2000. Mechanisms of programmed celldeath in the developing brain. Trends Neurosci. 23, 291e297.

Kubota, S., Kurihara, T., Ebinuma, M., Kubota, M., Yuki, K., Sasaki, M., Noda, K.,Ozawa, Y., Oike, Y., Ishida, S., Tsubota, K., 2010. Resveratrol prevents light-induced retinal degeneration via suppressing activator protein-1 activation.Am. J. Pathol. 177, 1725e1731.

Kubota, Y., Suda, T., 2009. Feedback mechanism between blood vessels and astro-cytes in retinal vascular development. Trends Cardiovasc. Med. 19, 38e43.

Kunchithapautham, K., Rohrer, B., 2007. Autophagy is one of the multiple mecha-nisms active in photoreceptor degeneration. Autophagy 3, 65e66.

Kunte, M.M., Choudhury, S., Manheim, J.F., Shinde, V.M., Miura, M., Chiodo, V.A.,Hauswirth, W.W., Gorbatyuk, O.S., Gorbatyuk, M.S., 2012. ER stress is involvedin T17M rhodopsin-induced retinal degeneration. Invest Ophthalmol. Vis. Sci.53, 3792e3800.

Kur, J., Newman, E.A., Chan-Ling, T., 2012. Cellular and physiological mechanismsunderlying blood flow regulation in the retina and choroid in health and dis-ease. Prog. Retin Eye Res. 31, 377e406.

Kwan, A.S., Wang, S., Lund, R.D., 1999. Photoreceptor layer reconstruction in a ro-dent model of retinal degeneration. Exp. Neurol. 159, 21e33.

Laabich, A., Vissvesvaran, G.P., Lieu, K.L., Murata, K., McGinn, T.E., Manmoto, C.C.,Sinclair, J.R., Karliga, I., Leung, D.W., Fawzi, A., Kubota, R., 2006. Protective effectof crocin against blue light- and white light-mediated photoreceptor cell deathin bovine and primate retinal primary cell culture. Invest Ophthalmol. Vis. Sci.47, 3156e3163.

Lagali, P.S., Balya, D., Awatramani, G.B., Munch, T.A., Kim, D.S., Busskamp, V.,Cepko, C.L., Roska, B., 2008. Light-activated channels targeted to ON bipolarcells restore visual function in retinal degeneration. Nat. Neurosci. 11, 667e675.

Lamba, D.A., Gust, J., Reh, T.A., 2009. Transplantation of human embryonic stemcell-derived photoreceptors restores some visual function in Crx-deficient mice.Cell. Stem Cell. 4, 73e79.

Lamba, D.A., Karl, M.O., Ware, C.B., Reh, T.A., 2006. Efficient generation of retinalprogenitor cells from human embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A.103, 12769e12774.

Langmann, T., 2007. Microglia activation in retinal degeneration. J. Leukoc. Biol. 81,1345e1351.

Lau, D., Flannery, J., 2003. Viral-mediated FGF-2 treatment of the constant lightdamage model of photoreceptor degeneration. Doc. Ophthalmol. 106, 89e98.

Lau, D., McGee, L.H., Zhou, S., Rendahl, K.G., Manning, W.C., Escobedo, J.A.,Flannery, J.G., 2000. Retinal degeneration is slowed in transgenic rats by AAV-mediated delivery of FGF-2. Invest Ophthalmol. Vis. Sci. 41, 3622e3633.

LaVail, M.M., Unoki, K., Yasumura, D., Matthes, M.T., Yancopoulos, G.D.,Steinberg, R.H., 1992. Multiple growth factors, cytokines, and neurotrophinsrescue photoreceptors from the damaging effects of constant light. Proc. Natl.Acad. Sci. U. S. A. 89, 11249e11253.

LaVail, M.M., Yasumura, D., Matthes, M.T., Lau-Villacorta, C., Unoki, K., Sung, C.H.,Steinberg, R.H., 1998. Protection of mouse photoreceptors by survival factors inretinal degenerations. Invest Ophthalmol. Vis. Sci. 39, 592e602.

Lax, P., Esquiva, G., Altavilla, C., Cuenca, N., 2014. Neuroprotective effects of thecannabinoid agonist HU210 on retinal degeneration. Exp. Eye Res. 120, 175e185.

Lax, P., Otalora, B.B., Esquiva, G., Rol Mde, L., Madrid, J.A., Cuenca, N., 2011. Circadiandysfunction in P23H rhodopsin transgenic rats: effects of exogenous melatonin.J. Pineal Res. 50, 183e191.

Leal, E.C., Aveleira, C.A., Castilho, A.F., Serra, A.M., Baptista, F.I., Hosoya, K.,Forrester, J.V., Ambrosio, A.F., 2009. High glucose and oxidative/nitrosative

stress conditions induce apoptosis in retinal endothelial cells by a caspase-independent pathway. Exp. Eye Res. 88, 983e991.

Lebrun-Julien, F., Duplan, L., Pernet, V., Osswald, I., Sapieha, P., Bourgeois, P., Dickson, K.,Bowie, D., Barker, P.A., Di Polo, A., 2009. Excitotoxic death of retinal neurons in vivooccurs via a non-cell-autonomous mechanism. J. Neurosci. 29, 5536e5545.

Lee, S.Y., Usui, S., Zafar, A.B., Oveson, B.C., Jo, Y.J., Lu, L., Masoudi, S.,Campochiaro, P.A., 2011. N-Acetylcysteine promotes long-term survival of conesin a model of retinitis pigmentosa. J. Cell. Physiol. 226, 1843e1849.

Leveillard, T., Mohand-Said, S., Lorentz, O., Hicks, D., Fintz, A.C., Clerin, E.,Simonutti, M., Forster, V., Cavusoglu, N., Chalmel, F., Dolle, P., Poch, O.,Lambrou, G., Sahel, J.A., 2004. Identification and characterization of rod-derivedcone viability factor. Nat. Genet. 36, 755e759.

Levkovitch-Verbin, H., Dardik, R., Vander, S., Melamed, S., 2010. Mechanism ofretinal ganglion cells death in secondary degeneration of the optic nerve. Exp.Eye Res. 91, 127e134.

Levkovitch-Verbin, H., Vander, S., Melamed, S., 2011. Rasagiline-induced delay ofretinal ganglion cell death in experimental glaucoma in rats. J. Glaucoma 20,273e277.

Lewis, G.P., Betts, K.E., Sethi, C.S., Charteris, D.G., Lesnik-Oberstein, S.Y., Avery, R.L.,Fisher, S.K., 2007. Identification of ganglion cell neurites in human subretinaland epiretinal membranes. Br. J. Ophthalmol. 91, 1234e1238.

Lewis, G.P., Chapin, E.A., Byun, J., Luna, G., Sherris, D., Fisher, S.K., 2009. Muller cellreactivity and photoreceptor cell death are reduced after experimental retinaldetachment using an inhibitor of the Akt/mTOR pathway. Invest Ophthalmol.Vis. Sci. 50, 4429e4435.

Lewis, G.P., Chapin, E.A., Luna, G., Linberg, K.A., Fisher, S.K., 2010. The fate of Muller'sglia following experimental retinal detachment: nuclear migration, cell divi-sion, and subretinal glial scar formation. Mol. Vis. 16, 1361e1372.

Lewis, G.P., Sethi, C.S., Carter, K.M., Charteris, D.G., Fisher, S.K., 2005. Microglial cellactivation following retinal detachment: a comparison between species. Mol.Vis. 11, 491e500.

Li, C., Wang, L., Huang, K., Zheng, L., 2012a. Endoplasmic reticulum stress in retinalvascular degeneration: protective role of resveratrol. Invest Ophthalmol. Vis.Sci. 53, 3241e3249.

Li, F., Cao, W., Anderson, R.E., 2003. Alleviation of constant-light-induced photo-receptor degeneration by adaptation of adult albino rat to bright cyclic light.Invest Ophthalmol. Vis. Sci. 44, 4968e4975.

Li, L.Y., Luo, X., Wang, X., 2001. Endonuclease G is an apoptotic DNase when releasedfrom mitochondria. Nature 412, 95e99.

Li, S.Y., Fu, Z.J., Ma, H., Jang, W.C., So, K.F., Wong, D., Lo, A.C., 2009. Effect of lutein onretinal neurons and oxidative stress in a model of acute retinal ischemia/reperfusion. Invest Ophthalmol. Vis. Sci. 50, 836e843.

Li, Y., Atmaca-Sonmez, P., Schanie, C.L., Ildstad, S.T., Kaplan, H.J., Enzmann, V., 2007.Endogenous bone marrow derived cells express retinal pigment epithelium cellmarkers and migrate to focal areas of RPE damage. Invest Ophthalmol. Vis. Sci.48, 4321e4327.

Li, Y., Reca, R.G., Atmaca-Sonmez, P., Ratajczak, M.Z., Ildstad, S.T., Kaplan, H.J.,Enzmann, V., 2006. Retinal pigment epithelium damage enhances expression ofchemoattractants and migration of bone marrow-derived stem cells. InvestOphthalmol. Vis. Sci. 47, 1646e1652.

Li, Y., Tsai, Y.T., Hsu, C.W., Erol, D., Yang, J., Wu, W.H., Davis, R.J., Egli, D., Tsang, S.H.,2012b. Long-term safety and efficacy of human-induced pluripotent stem cell(iPS) grafts in a preclinical model of retinitis pigmentosa. Mol. Med. 18,1312e1319.

Li, Z.Y., Kljavin, I.J., Milam, A.H., 1995. Rod photoreceptor neurite sprouting inretinitis pigmentosa. J. Neurosci. 15, 5429e5438.

Liang, X.J., Yuan, L., Hu, J., Yu, H.H., Li, T., Lin, S.F., Tang, S.B., 2012. Phospho-mannopentaose sulfate (PI-88) suppresses angiogenesis by downregulatingheparanase and vascular endothelial growth factor in an oxygen-inducedretinal neovascularization animal model. Mol. Vis. 18, 1649e1657.

Libby, R.T., Li, Y., Savinova, O.V., Barter, J., Smith, R.S., Nickells, R.W., John, S.W., 2005.Susceptibility to neurodegeneration in a glaucoma is modified by Bax genedosage. PLoS Genet. 1, 17e26.

Lieth, E., Barber, A.J., Xu, B., Dice, C., Ratz, M.J., Tanase, D., Strother, J.M., 1998. Glialreactivity and impaired glutamate metabolism in short-term experimentaldiabetic retinopathy. Penn State Retina Research Group. Diabetes 47, 815e820.

Lieth, E., Gardner, T.W., Barber, A.J., Antonetti, D.A., 2000. Retinal neuro-degeneration: early pathology in diabetes. Clin. Exp. Ophthalmol. 28, 3e8.

Lin, B., Koizumi, A., Tanaka, N., Panda, S., Masland, R.H., 2008. Restoration of visualfunction in retinal degeneration mice by ectopic expression of melanopsin.Proc. Natl. Acad. Sci. U. S. A. 105, 16009e16014.

Lin, J.H., Lavail, M.M., 2010. Misfolded proteins and retinal dystrophies. Adv. Exp.Med. Biol. 664, 115e121.

Lin, T., Walker, G.B., Kurji, K., Fang, E., Law, G., Prasad, S.S., Kojic, L., Cao, S., White, V.,Cui, J.Z., Matsubara, J.A., 2013. Parainflammation associated with advancedglycation endproduct stimulation of RPE in vitro: implications for age-relateddegenerative diseases of the eye. Cytokine 62, 369e381.

Linberg, K., Cuenca, N., Ahnelt, P., Fisher, S., Kolb, H., 2001. Comparative anatomy ofmajor retinal pathways in the eyes of nocturnal and diurnal mammals. Prog.Brain Res. 131, 27e52.

Liu, B., 2006. Modulation of microglial pro-inflammatory and neurotoxic activity forthe treatment of Parkinson's disease. AAPS J. 8, E606eE621.

Liu, S., Li, Z.W., Weinreb, R.N., Xu, G., Lindsey, J.D., Ye, C., Yung, W.H., Pang, C.P.,Lam, D.S., Leung, C.K., 2012. Tracking retinal microgliosis in models of retinalganglion cell damage. Invest Ophthalmol. Vis. Sci. 53, 6254e6262.


Lo, A.C., Woo, T.T., Wong, R.L., Wong, D., 2011. Apoptosis and other cell deathmechanisms after retinal detachment: implications for photoreceptor rescue.Ophthalmologica 226 (Suppl. 1), 10e17.

Lopez-del Hoyo, N., Fazioli, L., Lopez-Begines, S., Fernandez-Sanchez, L., Cuenca, N.,Llorens, J., de la Villa, P., Mendez, A., 2012. Overexpression of guanylate cyclaseactivating protein 2 in rod photoreceptors in vivo leads to morphologicalchanges at the synaptic ribbon. PLoS One 7, e42994.

Losso, J.N., Truax, R.E., Richard, G., 2010. trans-resveratrol inhibits hyperglycemia-induced inflammation and connexin downregulation in retinal pigmentepithelial cells. J. Agric. Food Chem. 58, 8246e8252.

Lu, B., Malcuit, C., Wang, S., Girman, S., Francis, P., Lemieux, L., Lanza, R., Lund, R.,2009. Long-term safety and function of RPE from human embryonic stem cellsin preclinical models of macular degeneration. Stem Cells 27, 2126e2135.

Lu, B., Wang, S., Girman, S., McGill, T., Ragaglia, V., Lund, R., 2010. Human adult bonemarrow-derived somatic cells rescue vision in a rodent model of retinaldegeneration. Exp. Eye Res. 91, 449e455.

Lu, Y.B., Franze, K., Seifert, G., Steinhauser, C., Kirchhoff, F., Wolburg, H., Guck, J.,Janmey, P., Wei, E.Q., Kas, J., Reichenbach, A., 2006. Viscoelastic properties ofindividual glial cells and neurons in the CNS. Proc. Natl. Acad. Sci. U. S. A. 103,17759e17764.

Lu, Y.B., Iandiev, I., Hollborn, M., Korber, N., Ulbricht, E., Hirrlinger, P.G., Pannicke, T.,Wei, E.Q., Bringmann, A., Wolburg, H., Wilhelmsson, U., Pekny, M.,Wiedemann, P., Reichenbach, A., Kas, J.A., 2011. Reactive glial cells: increasedstiffness correlates with increased intermediate filament expression. FASEB J.25, 624e631.

Lucin, K.M., Wyss-Coray, T., 2009. Immune activation in brain aging and neuro-degeneration: too much or too little? Neuron 64, 110e122.

Luna, G., Lewis, G.P., Banna, C.D., Skalli, O., Fisher, S.K., 2010. Expression profiles ofnestin and synemin in reactive astrocytes and Muller cells following retinalinjury: a comparisonwith glial fibrillar acidic protein and vimentin. Mol. Vis. 16,2511e2523.

Lund, R.D., Wang, S., Klimanskaya, I., Holmes, T., Ramos-Kelsey, R., Lu, B.,Girman, S., Bischoff, N., Sauve, Y., Lanza, R., 2006. Human embryonic stemcell-derived cells rescue visual function in dystrophic RCS rats. Cloning StemCells 8, 189e199.

Lund, R.D., Wang, S., Lu, B., Girman, S., Holmes, T., Sauve, Y., Messina, D.J., Harris, I.R.,Kihm, A.J., Harmon, A.M., Chin, F.Y., Gosiewska, A., Mistry, S.K., 2007. Cellsisolated from umbilical cord tissue rescue photoreceptors and visual functionsin a rodent model of retinal disease. Stem Cells 25, 602e611.

Luo, C., Yang, X., Kain, A.D., Powell, D.W., Kuehn, M.H., Tezel, G., 2010. Glaucomatoustissue stress and the regulation of immune response through glial Toll-likereceptor signaling. Invest Ophthalmol. Vis. Sci. 51, 5697e5707.

Luongo, L., Guida, F., Imperatore, R., Napolitano, F., Gatta, L., Cristino, L., Giordano, C.,Siniscalco, D., Di Marzo, V., Bellini, G., Petrelli, R., Cappellacci, L., Usiello, A., deNovellis, V., Rossi, F., Maione, S., 2014. The A1 adenosine receptor as a newplayer in microglia physiology. Glia 62, 122e132.

Lupo, S., Grenga, P.L., Vingolo, E.M., 2011. Fourier-domain optical coherence to-mography and microperimetry findings in retinitis pigmentosa. Am. J. Oph-thalmol. 151, 106e111.

Ly, A., Yee, P., Vessey, K.A., Phipps, J.A., Jobling, A.I., Fletcher, E.L., 2011. Early innerretinal astrocyte dysfunction during diabetes and development of hypoxia,retinal stress, and neuronal functional loss. Invest Ophthalmol. Vis. Sci. 52,9316e9326.

Ma, K., Xu, L., Zhan, H., Zhang, S., Pu, M., Jonas, J.B., 2009. Dosage dependence of theeffect of Ginkgo biloba on the rat retinal ganglion cell survival after optic nervecrush. Eye (Lond) 23, 1598e1604.

Ma, K., Xu, L., Zhang, H., Zhang, S., Pu, M., Jonas, J.B., 2010. The effect of ginkgo bilobaon the rat retinal ganglion cell survival in the optic nerve crush model. ActaOphthalmol. 88, 553e557.

Ma, L., Dou, H.L., Huang, Y.M., Lu, X.R., Xu, X.R., Qian, F., Zou, Z.Y., Pang, H.L.,Dong, P.C., Xiao, X., Wang, X., Sun, T.T., Lin, X.M., 2012a. Improvement of retinalfunction in early age-related macular degeneration after lutein and zeaxanthinsupplementation: a randomized, double-masked, placebo-controlled trial. Am.J. Ophthalmol. 154, 625e634 e621.

Ma, L., Yan, S.F., Huang, Y.M., Lu, X.R., Qian, F., Pang, H.L., Xu, X.R., Zou, Z.Y.,Dong, P.C., Xiao, X., Wang, X., Sun, T.T., Dou, H.L., Lin, X.M., 2012b. Effect of luteinand zeaxanthin on macular pigment and visual function in patients with earlyage-related macular degeneration. Ophthalmology 119, 2290e2297.

Maccarone, R., Di Marco, S., Bisti, S., 2008. Saffron supplement maintainsmorphology and function after exposure to damaging light in mammalianretina. Invest Ophthalmol. Vis. Sci. 49, 1254e1261.

Machalinska, A., Kawa, M.P., Pius-Sadowska, E., Roginska, D., Klos, P., Baumert, B.,Wiszniewska, B., Machalinski, B., 2013. Endogenous regeneration of damagedretinal pigment epithelium following low dose sodium iodate administration:an insight into the role of glial cells in retinal repair. Exp. Eye Res. 112, 68e78.

Maclaren, R.E., Pearson, R.A., Macneil, A., Douglas, R.H., Salt, T.E., Akimoto, M.,Swaroop, A., 2006. Retinal repair by transplantation of photoreceptor pre-cursors, 444, 203e207.

Madigan, M.C., Penfold, P.L., Provis, J.M., Balind, T.K., Billson, F.A., 1994. Intermediatefilament expression in human retinal macroglia. Histopathologic changesassociated with age-related macular degeneration. Retina 14, 65e74.

Mandal, M.N., Patlolla, J.M., Zheng, L., Agbaga, M.P., Tran, J.T., Wicker, L., Kasus-Jacobi, A., Elliott, M.H., Rao, C.V., Anderson, R.E., 2009. Curcumin protects retinalcells from light-and oxidant stress-induced cell death. Free Radic. Biol. Med. 46,672e679.

Maneu, V., Noailles, A., Megias, J., Gomez-Vicente, V., Carpena, N., Gil, M.L.,Gozalbo, D., Cuenca, N., 2014. Retinal microglia are activated by systemic fungalinfection. Invest Ophthalmol. Vis. Sci. 55, 3578e3585.

Maneu, V., Yanez, A., Murciano, C., Molina, A., Gil, M.L., Gozalbo, D., 2011. Dectin-1mediates in vitro phagocytosis of Candida albicans yeast cells by retinalmicroglia. FEMS Immunol. Med. Microbiol. 63, 148e150.

Mantopoulos, D., Murakami, Y., Comander, J., Thanos, A., Roh, M., Miller, J.W.,Vavvas, D.G., 2011. Tauroursodeoxycholic acid (TUDCA) protects photoreceptorsfrom cell death after experimental retinal detachment. PLoS One 6, e24245.

Mao, H., Gorbatyuk, M.S., Rossmiller, B., Hauswirth, W.W., Lewin, A.S., 2012. Long-term rescue of retinal structure and function by rhodopsin RNA replacementwith a single adeno-associated viral vector in P23H RHO transgenic mice. Hum.Gene Ther. 23, 356e366.

Mao, Y., Kiss, S., Boyer, J.L., Hackett, N.R., Qiu, J., Carbone, A., Mezey, J.G.,Kaminsky, S.M., D'Amico, D.J., Crystal, R.G., 2011. Persistent suppression ofocular neovascularization with intravitreal administration of AAVrh.10 codingfor bevacizumab. Hum. Gene Ther. 22, 1525e1535.

Marc, R.E., Jones, B.W., 2003. Retinal remodeling in inherited photoreceptor de-generations. Mol. Neurobiol. 28, 139e147.

Marc, R.E., Jones, B.W., Anderson, J.R., Kinard, K., Marshak, D.W., Wilson, J.H.,Wensel, T., Lucas, R.J., 2007. Neural reprogramming in retinal degeneration.Invest Ophthalmol. Vis. Sci. 48, 3364e3371.

Marc, R.E., Jones, B.W., Watt, C.B., Strettoi, E., 2003. Neural remodeling in retinaldegeneration. Prog. Retin Eye Res. 22, 607e655.

Marc, R.E., Jones, B.W., Watt, C.B., Vazquez-Chona, F., Vaughan, D.K.,Organisciak, D.T., 2008. Extreme retinal remodeling triggered by light damage:implications for age related macular degeneration. Mol. Vis. 14, 782e806.

Marco, F.D., Romeo, S., Nandasena, C., Purushothuman, S., Adams, C., Bisti, S.,Stone, J., 2013. The time course of action of two neuroprotectants, dietarysaffron and photobiomodulation, assessed in the rat retina. Am. J. Neuro-degener. Dis. 2, 208e220.

Martin, D.F., Maguire, M.G., Fine, S.L., Ying, G.S., Jaffe, G.J., Grunwald, J.E., Toth, C.,Redford, M., Ferris 3rd, F.L., 2012. Ranibizumab and bevacizumab for treatmentof neovascular age-related macular degeneration: two-year results. Ophthal-mology 119, 1388e1398.

Martin, K.R., Quigley, H.A., Zack, D.J., Levkovitch-Verbin, H., Kielczewski, J.,Valenta, D., Baumrind, L., Pease, M.E., Klein, R.L., Hauswirth, W.W., 2003. Genetherapy with brain-derived neurotrophic factor as a protection: retinal ganglioncells in a rat glaucoma model. Invest Ophthalmol. Vis. Sci. 44, 4357e4365.

Martin, P.M., Roon, P., Van Ells, T.K., Ganapathy, V., Smith, S.B., 2004. Death of retinalneurons in streptozotocin-induced diabetic mice. Invest Ophthalmol. Vis. Sci.45, 3330e3336.

Martinez-Navarrete, G., Seiler, M.J., Aramant, R.B., Fernandez-Sanchez, L., Pinilla, I.,Cuenca, N., 2011. Retinal degeneration in two lines of transgenic S334ter rats.Exp. Eye Res. 92, 227e237.

Mason, C., Dunnill, P., 2008. A brief definition of regenerative medicine. Regen. Med.3, 1e5.

Matsui, Y., Katsumi, O., Sakaue, H., Hirose, T., 1994. Electroretinogram b/a wave ratioimprovement in central retinal vein obstruction. Br. J. Ophthalmol. 78, 191e198.

Matteucci, A., Cammarota, R., Paradisi, S., Varano, M., Balduzzi, M., Leo, L.,Bellenchi, G.C., De Nuccio, C., Carnovale-Scalzo, G., Scorcia, G., Frank, C.,Mallozzi, C., Di Stasi, A.M., Visentin, S., Malchiodi-Albedi, F., 2011. Curcuminprotects against NMDA-induced toxicity: a possible role for NR2A subunit.Invest Ophthalmol. Vis. Sci. 52, 1070e1077.

Mazzoni, F., Novelli, E., Strettoi, E., 2008. Retinal ganglion cells survive and maintainnormal dendritic morphology in a mouse model of inherited photoreceptordegeneration. J. Neurosci. 28, 14282e14292.

McFarlane, S., Glenn, J.V., Lichanska, A.M., Simpson, D.A., Stitt, A.W., 2005. Charac-terisation of the advanced glycation endproduct receptor complex in the retinalpigment epithelium. Br. J. Ophthalmol. 89, 107e112.

McGee Sanftner, L.H., Abel, H., Hauswirth, W.W., Flannery, J.G., 2001. Glial cell linederived neurotrophic factor delays photoreceptor degeneration in a transgenicrat model of retinitis pigmentosa. Mol. Ther. 4, 622e629.

McGill, T.J., Cottam, B., Lu, B., Wang, S., Girman, S., Tian, C., Huhn, S.L., Lund, R.D.,Capela, A., 2012. Transplantation of human central nervous system stem cells eneuroprotection in retinal degeneration. Eur. J. Neurosci. 35, 468e477.

McKernan, D.P., Guerin, M.B., O'Brien, C.J., Cotter, T.G., 2007. A key role for calpainsin retinal ganglion cell death. Invest Ophthalmol. Vis. Sci. 48, 5420e5430.

McKinnon, S.J., Lehman, D.M., Tahzib, N.G., Ransom, N.L., Reitsamer, H.A., Liston, P.,LaCasse, E., Li, Q., Korneluk, R.G., Hauswirth, W.W., 2002. Baculoviral IAP repeat-containing-4 protects optic nerve axons in a rat glaucoma model. Mol. Ther. 5,780e787.

Metea, M.R., Newman, E.A., 2006. Glial cells dilate and constrict blood vessels: amechanism of neurovascular coupling. J. Neurosci. 26, 2862e2870.

Metea, M.R., Newman, E.A., 2007. Signalling within the neurovascular unit in themammalian retina. Exp. Physiol. 92, 635e640.

Metrailler, S., Schorderet, D.F., Cottet, S., 2012. Early apoptosis of rod photoreceptorsin Rpe65(-/-) mice is associated with the upregulated expression of lysosomal-mediated autophagic genes. Exp. Eye Res. 96, 70e81.

Mettu, P.S., Wielgus, A.R., Ong, S.S., Cousins, S.W., 2012. Retinal pigment epitheliumresponse to oxidant injury in the pathogenesis of early age-related maculardegeneration. Mol. Asp. Med. 33, 376e398.

Meyer, J.S., Howden, S.E., Wallace, K.A., Verhoeven, A.D., Wright, L.S., Capowski, E.E.,Pinilla, I., Martin, J.M., Tian, S., Stewart, R., Pattnaik, B., Thomson, J.A.,Gamm, D.M., 2011. Optic vesicle-like structures derived from human


pluripotent stem cells facilitate a customized approach to retinal diseasetreatment. Stem Cells 29, 1206e1218.

Meyer, J.S., Katz, M.L., Maruniak, J.A., Kirk, M.D., 2006. Embryonic stem cell-derivedneural progenitors incorporate into degenerating retina and enhance survival ofhost photoreceptors. Stem Cells 24, 274e283.

Meyer, J.S., Shearer, R.L., Capowski, E.E., Wright, L.S., Wallace, K.A., McMillan, E.L.,Zhang, S.C., Gamm, D.M., 2009. Modeling early retinal development with hu-man embryonic and induced pluripotent stem cells. Proc. Natl. Acad. Sci. U. S. A.106, 16698e16703.

Michalakis, S., Schaferhoff, K., Spiwoks-Becker, I., Zabouri, N., Koch, S., Koch, F.,Bonin, M., Biel, M., Haverkamp, S., 2013. Characterization of neurite outgrowthand ectopic synaptogenesis in response to photoreceptor dysfunction. Cell. Mol.Life Sci. 70, 1831e1847.

Michelucci, A., Heurtaux, T., Grandbarbe, L., Morga, E., Heuschling, P., 2009. Char-acterization of the microglial phenotype under specific pro-inflammatory andanti-inflammatory conditions: effects of oligomeric and fibrillar amyloid-beta.J. Neuroimmunol. 210, 3e12.

Milam, A.H., Li, Z.Y., Fariss, R.N., 1998. Histopathology of the human retina in reti-nitis pigmentosa. Prog. Retin Eye Res. 17, 175e205.

Millington-Ward, S., Chadderton, N., O'Reilly, M., Palfi, A., Goldmann, T., Kilty, C.,Humphries, M., Wolfrum, U., Bennett, J., Humphries, P., Kenna, P.F., Farrar, G.J.,2011. Suppression and replacement gene therapy for autosomal dominantdisease in a murine model of dominant retinitis pigmentosa. Mol. Ther. 19,642e649.

Miranda, M., Arnal, E., Ahuja, S., Alvarez-Nolting, R., Lopez-Pedrajas, R., Ekstrom, P.,Bosch-Morell, F., van Veen, T., Romero, F.J., 2010. Antioxidants rescue photore-ceptors in rd1 mice: relationship with thiol metabolism. Free Radic. Biol. Med.48, 216e222.

Miyazaki, M., Ikeda, Y., Yonemitsu, Y., Goto, Y., Kohno, R., Murakami, Y., Inoue, M.,Ueda, Y., Hasegawa, M., Tobimatsu, S., Sueishi, K., Ishibashi, T., 2008. Synergisticneuroprotective effect via simian lentiviral vector-mediated simultaneous genetransfer of human pigment epithelium-derived factor and human fibroblastgrowth factor-2 in rodent models of retinitis pigmentosa. J. Gene Med. 10,1273e1281.

Mizukoshi, S., Nakazawa, M., Sato, K., Ozaki, T., Metoki, T., Ishiguro, S., 2010. Acti-vation of mitochondrial calpain and release of apoptosis-inducing factor frommitochondria in RCS rat retinal degeneration. Exp. Eye Res. 91, 353e361.

Monnier, P.P., D'Onofrio, P.M., Magharious, M., Hollander, A.C., Tassew, N.,Szydlowska, K., Tymianski, M., Koeberle, P.D., 2011. Involvement of caspase-6and caspase-8 in neuronal apoptosis and the regenerative failure of injuredretinal ganglion cells. J. Neurosci. 31, 10494e10505.

Mori, K., Gehlbach, P., Ando, A., McVey, D., Wei, L., Campochiaro, P.A., 2002.Regression of ocular neovascularization in response to increased expression ofpigment epithelium-derived factor. Invest Ophthalmol. Vis. Sci. 43, 2428e2434.

Mount, M.P., Lira, A., Grimes, D., Smith, P.D., Faucher, S., Slack, R., Anisman, H.,Hayley, S., Park, D.S., 2007. Involvement of interferon-gamma in microglial-mediated loss of dopaminergic neurons. J. Neurosci. 27, 3328e3337.

Mrudula, T., Suryanarayana, P., Srinivas, P.N., Reddy, G.B., 2007. Effect of curcuminon hyperglycemia-induced vascular endothelial growth factor expression instreptozotocin-induced diabetic rat retina. Biochem Biophys. Res. Commun.361, 528e532.

Muller, F.L., Liu, Y., Van Remmen, H., 2004. Complex III releases superoxide to bothsides of the inner mitochondrial membrane. J. Biol. Chem. 279, 49064e49073.

Munemasa, Y., Kitaoka, Y., Kuribayashi, J., Ueno, S., 2010. Modulation of mito-chondria in the axon and soma of retinal ganglion cells in a rat glaucoma model.J. Neurochem. 115, 1508e1519.

Murakami, Y., Ikeda, Y., Yonemitsu, Y., Miyazaki, M., Inoue, M., Hasegawa, M.,Sueishi, K., Ishibashi, T., 2010. Inhibition of choroidal neovascularization viabrief subretinal exposure to a newly developed lentiviral vector pseudotypedwith Sendai viral envelope proteins. Hum. Gene Ther. 21, 199e209.

Murakami, Y., Notomi, S., Hisatomi, T., Nakazawa, T., Ishibashi, T., Miller, J.W.,Vavvas, D.G., 2013. Photoreceptor cell death and rescue in retinal detachmentand degenerations. Prog. Retin Eye Res. 37, 114e140.

Muzio, L., Martino, G., Furlan, R., 2007. Multifaceted aspects of inflammation inmultiple sclerosis: the role of microglia. J. Neuroimmunol. 191, 39e44.

Nag, T.C., Wadhwa, S., 2012. Ultrastructure of the human retina in aging and variouspathological states. Micron 43, 759e781.

Nakajima, E., Hammond, K.B., Rosales, J.L., Shearer, T.R., Azuma, M., 2011. Calpain,not caspase, is the causative protease for hypoxic damage in cultured monkeyretinal cells. Invest Ophthalmol. Vis. Sci. 52, 7059e7067.

Nakano, T., Ando, S., Takata, N., Kawada, M., Muguruma, K., Sekiguchi, K.,Saito, K., Yonemura, S., Eiraku, M., Sasai, Y., 2012. Self-formation of optic cupsand storable stratified neural retina from human ESCs. Cell. Stem Cell. 10,771e785.

Nakazawa, T., Kayama, M., Ryu, M., Kunikata, H., Watanabe, R., Yasuda, M.,Kinugawa, J., Vavvas, D., Miller, J.W., 2011. Tumor necrosis factor-alpha mediatesphotoreceptor death in a rodent model of retinal detachment. Invest Oph-thalmol. Vis. Sci. 52, 1384e1391.

Nakazawa, T., Takeda, M., Lewis, G.P., Cho, K.S., Jiao, J., Wilhelmsson, U., Fisher, S.K.,Pekny, M., Chen, D.F., Miller, J.W., 2007. Attenuated glial reactions and photo-receptor degeneration after retinal detachment in mice deficient in glialfibrillary acidic protein and vimentin. Invest Ophthalmol. Vis. Sci. 48,2760e2768.

Natoli, R., Zhu, Y., Valter, K., Bisti, S., Eells, J., Stone, J., 2010. Gene and noncoding RNAregulation underlying photoreceptor protection: microarray study of dietary

antioxidant saffron and photobiomodulation in rat retina. Mol. Vis. 16,1801e1822.

Neufeld, A.H., 2004. Pharmacologic neuroprotection with an inhibitor of nitric oxidesynthase for the treatment of glaucoma. Brain Res. Bull. 62, 455e459.

Neumann, H., Kotter, M.R., Franklin, R.J., 2009. Debris clearance by microglia: anessential link between degeneration and regeneration. Brain 132, 288e295.

Newman, E.A., 2001. Propagation of intercellular calciumwaves in retinal astrocytesand Muller cells. J. Neurosci. 21, 2215e2223.

Newman, E.A., 2004. Glial modulation of synaptic transmission in the retina. Glia47, 268e274.

Nguyen, A.T., Campbell, M., Kenna, P.F., Kiang, A.S., Tam, L., Humphries, M.M.,Humphries, P., 2012. Calpain and photoreceptor apoptosis. Adv. Exp. Med. Biol.723, 547e552.

Ni, Y.Q., Xu, G.Z., Hu, W.Z., Shi, L., Qin, Y.W., Da, C.D., 2008. Neuroprotective effects ofnaloxone against light-induced photoreceptor degeneration through inhibitingretinal microglial activation. Invest Ophthalmol. Vis. Sci. 49, 2589e2598.

Nickells, R.W., 2012. The cell and molecular biology of glaucoma: mechanisms ofretinal ganglion cell death. Invest Ophthalmol. Vis. Sci. 53, 2476e2481.

Nimmerjahn, A., Kirchhoff, F., Helmchen, F., 2005. Resting microglial cells are highlydynamic surveillants of brain parenchyma in vivo. Science 308, 1314e1318.

Nork, T.M., Ver Hoeve, J.N., Poulsen, G.L., Nickells, R.W., Davis, M.D., Weber, A.J.,Vaegan, Sarks, S.H., Lemley, H.L., Millecchia, L.L., 2000. Swelling and loss ofphotoreceptors in chronic human and experimental glaucomas. Arch. Oph-thalmol. 118, 235e245.

Nowak, J.Z., 2006. Age-related macular degeneration (AMD): pathogenesis andtherapy. Pharmacol. Rep. 58, 353e363.

Odom, J.V., Bach, M., Brigell, M., Holder, G.E., McCulloch, D.L., Tormene, A.P., Vaegan,2010. ISCEV standard for clinical visual evoked potentials (2009 update). Doc.Ophthalmol. 120, 111e119.

Ohno, Y., Nakanishi, T., Umigai, N., Tsuruma, K., Shimazawa, M., Hara, H., 2012. Oraladministration of crocetin prevents inner retinal damage induced by N-methyl-D-aspartate in mice. Eur. J. Pharmacol. 690, 84e89.

Oishi, A., Otani, A., Sasahara, M., Kojima, H., Nakamura, H., Kurimoto, M.,Yoshimura, N., 2009. Photoreceptor integrity and visual acuity in cystoidmacular oedema associated with retinitis pigmentosa. Eye (Lond.) 23,1411e1416.

Ola, M.S., Nawaz, M.I., Siddiquei, M.M., Al-Amro, S., Abu El-Asrar, A.M., 2012. Recentadvances in understanding the biochemical and molecular mechanism of dia-betic retinopathy. J. Diabetes Complicat. 26, 56e64.

Osakada, F., Ikeda, H., Mandai, M., Wataya, T., Watanabe, K., Yoshimura, N.,Akaike, A., Sasai, Y., Takahashi, M., 2008. Toward the generation of rod and conephotoreceptors from mouse, monkey and human embryonic stem cells. Nat.Biotechnol. 26, 215e224.

Osakada, F., Jin, Z.B., Hirami, Y., Ikeda, H., Danjyo, T., Watanabe, K., Sasai, Y.,Takahashi, M., 2009. In vitro differentiation of retinal cells from humanpluripotent stem cells by small-molecule induction. J. Cell. Sci. 122, 3169e3179.

Osborne, N.N., Wood, J.P., 2006. The beta-adrenergic receptor antagonist metipra-nolol blunts zinc-induced photoreceptor and RPE apoptosis. Invest Ophthalmol.Vis. Sci. 47, 3178e3186.

Otani, A., Dorrell, M.I., Kinder, K., Moreno, S.K., Nusinowitz, S., Banin, E.,Heckenlively, J., Friedlander, M., 2004. Rescue of retinal degeneration byintravitreally injected adult bone marrow-derived lineage-negative hemato-poietic stem cells. J. Clin. Invest. 114, 765e774.

Oveson, B.C., Iwase, T., Hackett, S.F., Lee, S.Y., Usui, S., Sedlak, T.W., Snyder, S.H.,Campochiaro, P.A., Sung, J.U., 2011. Constituents of bile, bilirubin and TUDCA,protect against oxidative stress-induced retinal degeneration. J. Neurochem.116, 144e153.

Owsley, C., 2003. Contrast sensitivity. Ophthalmol. Clin. North Am. 16, 171e177.Ozaki, T., Nakazawa, M., Yamashita, T., Sorimachi, H., Hata, S., Tomita, H., Isago, H.,

Baba, A., Ishiguro, S., 2012. Intravitreal injection or topical eye-drop applicationof a mu-calpain C2L domain peptide protects against photoreceptor cell deathin Royal College of Surgeons' rats, a model of retinitis pigmentosa. Biochim.Biophys. Acta 1822, 1783e1795.

Ozawa, Y., Sasaki, M., Takahashi, N., Kamoshita, M., Miyake, S., Tsubota, K., 2012.Neuroprotective effects of lutein in the retina. Curr. Pharm. Des. 18, 51e56.

Ozdemir, G., Tolun, F.I., Gul, M., Imrek, S., 2009. Retinal oxidative stress induced byintraocular hypertension in rats may be ameliorated by brimonidine treatmentand N-acetyl cysteine supplementation. J. Glaucoma 18, 662e665.

Paglinawan, R., Malipiero, U., Schlapbach, R., Frei, K., Reith, W., Fontana, A., 2003.TGFbeta directs gene expression of activated microglia to an anti-inflammatoryphenotype strongly focusing on chemokine genes and cell migratory genes. Glia44, 219e231.

Panfoli, I., Calzia, D., Ravera, S., Morelli, A.M., Traverso, C.E., 2012. Extra-mito-chondrial aerobic metabolism in retinal rod outer segments: new perspectivesin retinopathies. Med. Hypotheses 78, 423e427.

Pappuru, R.R., Ouyang, Y., Nittala, M.G., Hemmati, H.D., Keane, P.A., Walsh, A.C.,Sadda, S.R., 2011. Relationship between outer retinal thickness substructuresand visual acuity in eyes with dry age-related macular degeneration. InvestOphthalmol. Vis. Sci. 52, 6743e6748.

Paquet-Durand, F., Azadi, S., Hauck, S.M., Ueffing, M., van Veen, T., Ekstrom, P., 2006.Calpain is activated in degenerating photoreceptors in the rd1 mouse.J. Neurochem 96, 802e814.

Park, S.H., Park, J.W., Park, S.J., Kim, K.Y., Chung, J.W., Chun, M.H., Oh, S.J., 2003.Apoptotic death of photoreceptors in the streptozotocin-induced diabetic ratretina. Diabetologia 46, 1260e1268.


Park, S.J., Kim, I.B., Choi, K.R., Moon, J.I., Oh, S.J., Chung, J.W., Chun, M.H., 2001.Reorganization of horizontal cell processes in the developing FVB/N mouseretina. Cell. Tissue Res. 306, 341e346.

Pearson, R.A., Barber, A.C., Rizzi, M., Hippert, C., Xue, T., West, E.L., Duran, Y.,Smith, A.J., Chuang, J.Z., Azam, S.A., Luhmann, U.F., Benucci, A., Sung, C.H.,Bainbridge, J.W., Carandini, M., Yau, K.W., Sowden, J.C., Ali, R.R., 2012. Restora-tion of vision after transplantation of photoreceptors. Nature 485, 99e103.

Pechan, P., Rubin, H., Lukason, M., Ardinger, J., DuFresne, E., Hauswirth, W.W.,Wadsworth, S.C., Scaria, A., 2009. Novel anti-VEGF chimeric molecules deliveredby AAV vectors for inhibition of retinal neovascularization. Gene Ther. 16, 10e16.

Penfold, P.L., Madigan, M.C., Gillies, M.C., Provis, J.M., 2001. Immunological andaetiological aspects of macular degeneration. Prog. Retin Eye Res. 20, 385e414.

Peng, P.H., Chiou, L.F., Chao, H.M., Lin, S., Chen, C.F., Liu, J.H., Ko, M.L., 2010. Effects ofepigallocatechin-3-gallate on rat retinal ganglion cells after optic nerve axot-omy. Exp. Eye Res. 90, 528e534.

Peng, Y.W., Hao, Y., Petters, R.M., Wong, F., 2000. Ectopic synaptogenesis in themammalian retina caused by rod photoreceptor-specific mutations. Nat. Neu-rosci. 3, 1121e1127.

Penn, J.S., Madan, A., Caldwell, R.B., Bartoli, M., Caldwell, R.W., Hartnett, M.E., 2008.Vascular endothelial growth factor in eye disease. Prog. Retin Eye Res. 27,331e371.

Pennesi, M.E., Nishikawa, S., Matthes, M.T., Yasumura, D., LaVail, M.M., 2008. Therelationship of photoreceptor degeneration to retinal vascular development andloss in mutant rhodopsin transgenic and RCS rats. Exp. Eye Res. 87, 561e570.

Perche, O., Doly, M., Ranchon-Cole, I., 2008. Transient protective effect of caspaseinhibitors in RCS rat. Exp. Eye Res. 86, 519e527.

Perego, C., Fumagalli, S., De Simoni, M.G., 2011. Temporal pattern of expression andcolocalization of microglia/macrophage phenotype markers following brainischemic injury in mice. J. Neuroinflam. 8, 174.

Perlman, I., 1983. Relationship between the amplitudes of the b wave and the awave as a useful index for evaluating the electroretinogram. Br. J. Ophthalmol.67, 443e448.

Perry, V.H., Teeling, J., 2013. Microglia and macrophages of the central nervoussystem: the contribution of microglia priming and systemic inflammation tochronic neurodegeneration. Semin. Immunopathol. 35, 601e612.

Persson, M., Brantefjord, M., Hansson, E., Ronnback, L., 2005. Lipopolysaccharideincreases microglial GLT-1 expression and glutamate uptake capacity in vitro bya mechanism dependent on TNF-alpha. Glia 51, 111e120.

Phillips, M.J., Walker, T.A., Choi, H.Y., Faulkner, A.E., Kim, M.K., Sidney, S.S., Boyd, A.P.,Nickerson, J.M., Boatright, J.H., Pardue, M.T., 2008. Tauroursodeoxycholic acidpreservation of photoreceptor structure and function in the rd10 mousethrough postnatal day 30. Invest Ophthalmol. Vis. Sci. 49, 2148e2155.

Phillips, M.J., Wallace, K.A., Dickerson, S.J., Miller, M.J., Verhoeven, A.D., Martin, J.M.,Wright, L.S., Shen, W., Capowski, E.E., Percin, E.F., Perez, E.T., Zhong, X., Canto-Soler, M.V., Gamm, D.M., 2012. Blood-derived human iPS cells generate opticvesicle-like structures with the capacity to form retinal laminae and developsynapses. Invest Ophthalmol. Vis. Sci. 53, 2007e2019.

Pinilla, I., Cuenca, N., Sauve, Y., Wang, S., Lund, R.D., 2007. Preservation of outerretina and its synaptic connectivity following subretinal injections of humanRPE cells in the Royal College of Surgeons rat. Exp. Eye Res. 85, 381e392.

Pinilla, I., Fernandez-Sanchez, L., Esquiva, G., Cuenca, N., 2010. Retinal vasculardegeneration and macroglia changes in the transgenic P23H rat model ofretinitis pigmentosa. Invest Ophthalmol. Vis. Sci. 51. E-Abstract 4069.

Pinilla, I., Lund, R.D., Sauve, Y., 2005. Enhanced cone dysfunction in rats homozy-gous for the P23H rhodopsin mutation. Neurosci. Lett. 382, 16e21.

Pintea, A., Rugina, D., Pop, R., Bunea, A., Socaciu, C., Diehl, H.A., 2011. Antioxidanteffect of trans-resveratrol in cultured human retinal pigment epithelial cells.J. Ocul. Pharmacol. Ther. 27, 315e321.

Polazzi, E., Monti, B., 2010. Microglia and neuroprotection: from in vitro studies totherapeutic applications. Prog. Neurobiol. 92, 293e315.

Polster, B.M., 2013. AIF, reactive oxygen species, and neurodegeneration: a “com-plex” problem. Neurochem Int. 62, 695e702.

Pop, C., Salvesen, G.S., 2009. Human caspases: activation, specificity, and regulation.J. Biol. Chem. 284, 21777e21781.

Pow, D.V., Crook, D.K., 1995. Immunocytochemical evidence for the presence of highlevels of reduced glutathione in radial glial cells and horizontal cells in therabbit retina. Neurosci. Lett. 193, 25e28.

Prusky, G.T., Alam, N.M., Beekman, S., Douglas, R.M., 2004. Rapid quantification ofadult and developing mouse spatial vision using a virtual optomotor system.Invest Ophthalmol. Vis. Sci. 45, 4611e4616.

Qi, Y., Chen, L., Zhang, L., Liu, W.B., Chen, X.Y., Yang, X.G., 2013. Crocin preventsretinal ischaemia/reperfusion injury-induced apoptosis in retinal ganglion cellsthrough the PI3K/AKT signalling pathway. Exp. Eye Res. 107, 44e51.

Qiu, Q.H., Xie, Z.G., Xu, X., Liang, S.X., Gao, Y., 2012. EGB761 on retinal light injury inrats. Chin. Med. J. Engl. 125, 2306e2309.

Radtke, N.D., Aramant, R.B., Petry, H.M., Green, P.T., Pidwell, D.J., Seiler, M.J., 2008.Vision improvement in retinal degeneration patients by implantation of retinatogether with retinal pigment epithelium. Am. J. Ophthalmol. 146, 172e182.

Raivich, G., Bohatschek, M., Kloss, C.U., Werner, A., Jones, L.L., Kreutzberg, G.W.,1999. Neuroglial activation repertoire in the injured brain: graded response,molecular mechanisms and cues to physiological function. Brain Res. Brain Res.Rev. 30, 77e105.

Ramirez, J.M., Ramirez, A.I., Salazar, J.J., de Hoz, R., Trivino, A., 2001. Changes ofastrocytes in retinal ageing and age-related macular degeneration. Exp. Eye Res.73, 601e615.

Ramrattan, R.S., van der Schaft, T.L., Mooy, C.M., de Bruijn, W.C., Mulder, P.G., deJong, P.T., 1994. Morphometric analysis of Bruch's membrane, the chorioca-pillaris, and the choroid in aging. Invest Ophthalmol. Vis. Sci. 35, 2857e2864.

Ramsden, C.M., Powner, M.B., Carr, A.J., Smart, M.J., da Cruz, L., Coffey, P.J., 2013.Stem cells in retinal regeneration: past, present and future. Development 140,2576e2585.

Ransohoff, R.M., Cardona, A.E., 2010. The myeloid cells of the central nervous sys-tem parenchyma. Nature 468, 253e262.

Read, S.P., Cashman, S.M., Kumar-Singh, R., 2010. POD nanoparticles expressingGDNF provide structural and functional rescue of light-induced retinal degen-eration in an adult mouse. Mol. Ther. 18, 1917e1926.

Reh, T.A., 2006. Neurobiology: right timing for retina repair. Nature 444, 156e157.Reichenbach, A., Bringmann, A., 2013. New functions of Muller cells. Glia 61,

651e678.Reyes-Aguirre, L.I., Ferraro, S., Quintero, H., Sanchez-Serrano, S.L., Gomez-

Montalvo, A., Lamas, M., 2013. Glutamate-induced epigenetic and morpholog-ical changes allow rat Muller cell dedifferentiation but not further acquisition ofa photoreceptor phenotype. Neuroscience 254, 347e360.

Richer, S., Stiles, W., Ulanski, L., Carroll, D., Podella, C., 2013. Observation of humanretinal remodeling in octogenarians with a resveratrol based nutritional sup-plement. Nutrients 5, 1989e2005.

Richer, S.P., Stiles, W., Graham-Hoffman, K., Levin, M., Ruskin, D., Wrobel, J.,Park, D.W., Thomas, C., 2011. Randomized, double-blind, placebo-controlledstudy of zeaxanthin and visual function in patients with atrophic age-relatedmacular degeneration: the Zeaxanthin and Visual Function Study (ZVF) FDAIND #78, 973. Optometry 82, 667e680 e666.

Richman, J., Spaeth, G.L., Wirostko, B., 2013. Contrast sensitivity basics and a critiqueof currently available tests. J. Cataract. Refract Surg. 39, 1100e1106.

Rodrigues, M.M., Bardenstein, D., Wiggert, B., Lee, L., Fletcher, R.T., Chader, G., 1987.Retinitis pigmentosa with segmental massive retinal gliosis. An immunohisto-chemical, biochemical, and ultrastructural study. Ophthalmology 94, 180e186.

Rodriguez-de la Rosa, L., Fernandez-Sanchez, L., Germain, F., Murillo-Cuesta, S.,Varela-Nieto, I., de la Villa, P., Cuenca, N., 2012. Age-related functional andstructural retinal modifications in the Igf1-/- null mouse. Neurobiol. Dis. 46,476e485.

Roecklein, K.A., Wong, P.M., Miller, M.A., Donofry, S.D., Kamarck, M.L., Brainard, G.C.,2013. Melanopsin, photosensitive ganglion cells, and seasonal affective disor-der. Neurosci. Biobehav Rev. 37, 229e239.

Roh, M., Zhang, Y., Murakami, Y., Thanos, A., Lee, S.C., Vavvas, D.G., Benowitz, L.I.,Miller, J.W., 2012. Etanercept, a widely used inhibitor of tumor necrosis factor-alpha (TNF-alpha), prevents retinal ganglion cell loss in a rat model of glau-coma. PLoS One 7, e40065.

Roof, D.J., Adamian, M., Hayes, A., 1994. Rhodopsin accumulation at abnormal sitesin retinas of mice with a human P23H rhodopsin transgene. Invest Ophthalmol.Vis. Sci. 35, 4049e4062.

Roque, R.S., Rosales, A.A., Jingjing, L., Agarwal, N., Al-Ubaidi, M.R., 1999. Retina-derived microglial cells induce photoreceptor cell death in vitro. Brain Res. 836,110e119.

Rosenbaum, D.M., Degterev, A., David, J., Rosenbaum, P.S., Roth, S., Grotta, J.C.,Cuny, G.D., Yuan, J., Savitz, S.I., 2010. Necroptosis, a novel form of caspase-independent cell death, contributes to neuronal damage in a retinalischemia-reperfusion injury model. J. Neurosci. Res. 88, 1569e1576.

Rosenfeld, P.J., Rich, R.M., Lalwani, G.A., 2006. Ranibizumab: phase III clinical trialresults. Ophthalmol. Clin. North Am. 19, 361e372.

Ross, J.E., Bron, A.J., Clarke, D.D., 1984. Contrast sensitivity and visual disability inchronic simple glaucoma. Br. J. Ophthalmol. 68, 821e827.

Rossi, C., Strettoi, E., Galli-Resta, L., 2003. The spatial order of horizontal cells is notaffected by massive alterations in the organization of other retinal cells.J. Neurosci. 23, 9924e9928.

Rudolf, M., Clark, M.E., Chimento, M.F., Li, C.M., Medeiros, N.E., Curcio, C.A., 2008.Prevalence and morphology of druse types in the macula and periphery of eyeswith age-related maculopathy. Invest Ophthalmol. Vis. Sci. 49, 1200e1209.

Rutar, M., Natoli, R., Provis, J.M., 2012. Small interfering RNA-mediated suppressionof Ccl2 in Muller cells attenuates microglial recruitment and photoreceptordeath following retinal degeneration. J. Neuroinflam. 9, 221.

Sabates, R., Hirose, T., McMeel, J.W., 1983. Electroretinography in the prognosisand classification of central retinal vein occlusion. Arch. Ophthalmol. 101,232e235.

Sabour-Pickett, S., Nolan, J.M., Loughman, J., Beatty, S., 2012. A review of the evi-dence germane to the putative protective role of the macular carotenoids forage-related macular degeneration. Mol. Nutr. Food Res. 56, 270e286.

Sachdeva, M.M., Cano, M., Handa, J.T., 2013. Nrf2 signaling is impaired in the agingRPE given an oxidative insult. Exp. Eye Res..

Sachdeva, M.M., Cano, M., Handa, J.T., 2014. Nrf2 signaling is impaired in the agingRPE given an oxidative insult. Exp. Eye Res. 119, 111e114.

Sancho-Pelluz, J., Arango-Gonzalez, B., Kustermann, S., Romero, F.J., van Veen, T.,Zrenner, E., Ekstrom, P., Paquet-Durand, F., 2008. Photoreceptor cell deathmechanisms in inherited retinal degeneration. Mol. Neurobiol. 38, 253e269.

Sanges, D., Marigo, V., 2006. Cross-talk between two apoptotic pathways activatedby endoplasmic reticulum stress: differential contribution of caspase-12 andAIF. Apoptosis 11, 1629e1641.

Santos, A.M., Martin-Oliva, D., Ferrer-Martin, R.M., Tassi, M., Calvente, R., Sierra, A.,Carrasco, M.C., Marin-Teva, J.L., Navascues, J., Cuadros, M.A., 2010. Microglialresponse to light-induced photoreceptor degeneration in the mouse retina.J. Comp. Neurol. 518, 477e492.


Santos, J.M., Mohammad, G., Zhong, Q., Kowluru, R.A., 2011. Diabetic retinopathy,superoxide damage and antioxidants. Curr. Pharm. Biotechnol. 12, 352e361.

Sanvicens, N., Cotter, T.G., 2006. Ceramide is the key mediator of oxidative stress-induced apoptosis in retinal photoreceptor cells. J. Neurochem. 98, 1432e1444.

Sanvicens, N., Gomez-Vicente, V., Masip, I., Messeguer, A., Cotter, T.G., 2004.Oxidative stress-induced apoptosis in retinal photoreceptor cells is mediated bycalpains and caspases and blocked by the oxygen radical scavenger CR-6. J. Biol.Chem. 279, 39268e39278.

Sanyal, S., 1993. Synaptic growth in the rod terminals after partial photoreceptorcell loss. Prog. Retin Res. 12, 247e270.

Sanz, M.M., Johnson, L.E., Ahuja, S., Ekstrom, P.A., Romero, J., van Veen, T., 2007.Significant photoreceptor rescue by treatment with a combination of antioxi-dants in an animal model for retinal degeneration. Neuroscience 145,1120e1129.

Schlingemann, R.O., 2004. Role of growth factors and the wound healing responsein age-related macular degeneration. Graefes Arch. Clin. Exp. Ophthalmol. 242,91e101.

Scholl, H.P., Zrenner, E., 2000. Electrophysiology in the investigation of acquiredretinal disorders. Surv. Ophthalmol. 45, 29e47.

Schuetz, E., Thanos, S., 2004. Microglia-targeted pharmacotherapy in retinalneurodegenerative diseases. Curr. Drug. Targets 5, 619e627.

Schutte, M., Werner, P., 1998. Redistribution of glutathione in the ischemic ratretina. Neurosci. Lett. 246, 53e56.

Schwartz, M., Butovsky, O., Bruck, W., Hanisch, U.K., 2006. Microglial phenotype: isthe commitment reversible? Trends Neurosci. 29, 68e74.

Schwartz, S.D., Hubschman, J.P., Heilwell, G., Franco-Cardenas, V., Pan, C.K.,Ostrick, R.M.,Mickunas, E., Gay, R., Klimanskaya, I., Lanza, R., 2012. Embryonic stemcell trials for macular degeneration: a preliminary report. Lancet 379, 713e720.

Seitz, R., Ohlmann, A., Tamm, E.R., 2013. The role of Muller glia and microglia inglaucoma. Cell. Tissue Res. 353, 339e345.

Semo, M., Lupi, D., Peirson, S.N., Butler, J.N., Foster, R.G., 2003. Light-induced c-fos inmelanopsin retinal ganglion cells of young and aged rodless/coneless (rd/rd cl)mice. Eur. J. Neurosci. 18, 3007e3017.

Sethi, C.S., Lewis, G.P., Fisher, S.K., Leitner, W.P., Mann, D.L., Luthert, P.J.,Charteris, D.G., 2005. Glial remodeling and neural plasticity in human retinaldetachment with proliferative vitreoretinopathy. Invest Ophthalmol. Vis. Sci.46, 329e342.

Shamsi, F.A., Boulton, M., 2001. Inhibition of RPE lysosomal and antioxidant activityby the age pigment lipofuscin. Invest Ophthalmol. Vis. Sci. 42, 3041e3046.

Shan, H., Ji, D., Barnard, A.R., Lipinski, D.M., You, Q., Lee, E.J., Kamalden, T.A., Sun, X.,MacLaren, R.E., 2011. AAV-mediated gene transfer of human X-linked inhibitorof apoptosis protects against oxidative cell death in human RPE cells. InvestOphthalmol. Vis. Sci. 52, 9591e9597.

Shang, F., Taylor, A., 2012. Roles for the ubiquitin-proteasome pathway in proteinquality control and signaling in the retina: implications in the pathogenesis ofage-related macular degeneration. Mol. Asp. Med. 33, 446e466.

Sharma, R.K., 2001. Bcl-2 expression during the development and degeneration ofRCS rat retinae. Brain Res. Dev. Brain Res. 132, 81e86.

Shen, D., Cao, X., Zhao, L., Tuo, J., Wong, W.T., Chan, C.C., 2011. Naloxone amelioratesretinal lesions in Ccl2/Cx3cr1 double-deficient mice via modulation of micro-glia. Invest Ophthalmol. Vis. Sci. 52, 2897e2904.

Shen, W., Fruttiger, M., Zhu, L., Chung, S.H., Barnett, N.L., Kirk, J.K., Lee, S.,Coorey, N.J., Killingsworth, M., Sherman, L.S., Gillies, M.C., 2012. ConditionalMullercell ablation causes independent neuronal and vascular pathologies in anovel transgenic model. J. Neurosci. 32, 15715e15727.

Shen, W., Li, S., Chung, S.H., Gillies, M.C., 2010. Retinal vascular changes after glialdisruption in rats. J. Neurosci. Res. 88, 1485e1499.

Sheridan, G.K., Murphy, K.J., 2013. Neuron-glia crosstalk in health and disease:fractalkine and CX3CR1 take centre stage. Open. Biol. 3, 130181.

Sheu, S.J., Bee, Y.S., Chen, C.H., 2008. Resveratrol and large-conductance calcium-activated potassium channels in the protection of human retinal pigmentepithelial cells. J. Ocul. Pharmacol. Ther. 24, 551e555.

Shin, D.H., Lee, H.Y., Lee, H.W., Lee, K.H., Lim, H.S., Jeon, G.S., Cho, S.S., Hwang, D.H.,2000. Activation of microglia in kainic acid induced rat retinal apoptosis.Neurosci. Lett. 292, 159e162.

Shinoda, K., Rejdak, R., Schuettauf, F., Blatsios, G., Volker, M., Tanimoto, N., Olcay, T.,Gekeler, F., Lehaci, C., Naskar, R., Zagorski, Z., Zrenner, E., 2007. Early electro-retinographic features of streptozotocin-induced diabetic retinopathy. Clin. Exp.Ophthalmol. 35, 847e854.

Shukla, V., Mishra, S.K., Pant, H.C., 2011. Oxidative stress in neurodegeneration. Adv.Pharmacol. Sci. 2011, 572634.

Sieving, P.A., Caruso, R.C., Tao, W., Coleman, H.R., Thompson, D.J., Fullmer, K.R.,Bush, R.A., 2006. Ciliary neurotrophic factor (CNTF) for human retinal degen-eration: phase I trial of CNTF delivered by encapsulated cell intraocular im-plants. Proc. Natl. Acad. Sci. U. S. A. 103, 3896e3901.

Simon, D.J., Weimer, R.M., McLaughlin, T., Kallop, D., Stanger, K., Yang, J.,O'Leary, D.D.M., Hannoush, R.N., Tessier-Lavigne, M., 2012. A caspase cascaderegulating developmental axon degeneration. J. Neurosci. : Off. J. Soc. Neurosci.32, 17540e17553.

Siqueira, R.C., Messias, A., Voltarelli, J.C., Scott, I.U., Jorge, R., 2011. Intravitreal in-jection of autologous bone marrow-derived mononuclear cells for hereditaryretinal dystrophy: a phase I trial. Retina 31, 1207e1214.

Sizova, O.S., Shinde, V.M., Lenox, A.R., Gorbatyuk, M.S., 2014. Modulation of cellularsignaling pathways in P23H rhodopsin photoreceptors. Cell. Signal 26,665e672.

Snider, W.D., Johnson Jr., E.M., 1989. Neurotrophic molecules. Ann. Neurol. 26,489e506.

Sokol, S., 1976. Visually evoked potentials: theory, techniques and clinical applica-tions. Surv. Ophthalmol. 21, 18e44.

Soufi, F.G., Mohammad-Nejad, D., Ahmadieh, H., 2012. Resveratrol improves dia-betic retinopathy possibly through oxidative stress e nuclear factor kappaB e

apoptosis pathway. Pharmacol. Rep. 64, 1505e1514.Sparrow, J.R., Boulton, M., 2005. RPE lipofuscin and its role in retinal pathobiology.

Exp. Eye Res. 80, 595e606.Speros, P., Price, J., 1981. Oscillatory potentials. History, techniques and potential use

in the evaluation of disturbances of retinal circulation. Surv. Ophthalmol. 25,237e252.

Spitznas, M., 1974. The fine structure of the chorioretinal border tissues of the adulthuman eye. Adv. Ophthalmol. 28, 78e174.

Spraul, C.W., Lang, G.E., Grossniklaus, H.E., Lang, G.K., 1999. Histologic andmorphometric analysis of the choroid, Bruch's membrane, and retinal pigmentepithelium in postmortem eyes with age-related macular degeneration andhistologic examination of surgically excised choroidal neovascular membranes.Surv. Ophthalmol. 44 (Suppl. 1), S10eS32.

Stasi, K., Nagel, D., Yang, X., Wang, R.F., Ren, L., Podos, S.M., Mittag, T., Danias, J.,2006. Complement component 1Q (C1Q) upregulation in retina of murine,primate, and human glaucomatous eyes. Invest Ophthalmol. Vis. Sci. 47,1024e1029.

Stefansson, E., Geirsdottir, A., Sigurdsson, H., 2011. Metabolic physiology in agerelated macular degeneration. Prog. Retin Eye Res. 30, 72e80.

Stevens, B., Allen, N.J., Vazquez, L.E., Howell, G.R., Christopherson, K.S., Nouri, N.,Micheva, K.D., Mehalow, A.K., Huberman, A.D., Stafford, B., Sher, A., Litke, A.M.,Lambris, J.D., Smith, S.J., John, S.W., Barres, B.A., 2007. The classical complementcascade mediates CNS synapse elimination. Cell 131, 1164e1178.

Stone, J., Itin, A., Alon, T., Pe'er, J., Gnessin, H., Chan-Ling, T., Keshet, E., 1995.Development of retinal vasculature is mediated by hypoxia-induced vascularendothelial growth factor (VEGF) expression by neuroglia. J. Neurosci. 15,4738e4747.

Stone, J., van Driel, D., Valter, K., Rees, S., Provis, J., 2008. The locations of mito-chondria in mammalian photoreceptors: relation to retinal vasculature. BrainRes. 1189, 58e69.

Strasser, A., O'Connor, L., Dixit, V.M., 2000. Apoptosis signaling. Annu Rev. Biochem69, 217e245.

Strauss, O., 2005. The retinal pigment epithelium in visual function. Physiol. Rev. 85,845e881.

Streit, W.J., Sammons, N.W., Kuhns, A.J., Sparks, D.L., 2004. Dystrophic microglia inthe aging human brain. Glia 45, 208e212.

Streit, W.J., Walter, S.A., Pennell, N.A., 1999. Reactive microgliosis. Prog. Neurobiol.57, 563e581.

Strettoi, E., Mears, A.J., Swaroop, A., 2004. Recruitment of the rod pathway by conesin the absence of rods. J. Neurosci. 24, 7576e7582.

Strettoi, E., Pignatelli, V., 2000. Modifications of retinal neurons in a mouse model ofretinitis pigmentosa. Proc. Natl. Acad. Sci. U. S. A. 97, 11020e11025.

Strettoi, E., Pignatelli, V., Rossi, C., Porciatti, V., Falsini, B., 2003. Remodeling ofsecond-order neurons in the retina of rd/rd mutant mice. Vis. Res. 43, 867e877.

Strettoi, E., Porciatti, V., Falsini, B., Pignatelli, V., Rossi, C., 2002. Morphological andfunctional abnormalities in the inner retina of the rd/rd mouse. J. Neurosci. 22,5492e5504.

Sugie, Y., Yoshikawa, M., Ouji, Y., Saito, K., Moriya, K., Ishizaka, S., Matsuura, T.,Maruoka, S., Nawa, Y., Hara, Y., 2005. Photoreceptor cells from mouse ES cells byco-culture with chick embryonic retina. Biochem Biophys. Res. Commun. 332,241e247.

Sullivan, R.K., Woldemussie, E., Pow, D.V., 2007. Dendritic and synaptic plasticity ofneurons in the human age-related macular degeneration retina. Invest Oph-thalmol. Vis. Sci. 48, 2782e2791.

Sun, D., Enzmann, V., Lei, S., Sun, S.L., Kaplan, H.J., Shao, H., 2003. Retinal pigmentepithelial cells activate uveitogenic T cells when they express high levels ofMHC class II molecules, but inhibit T cell activation when they expressrestricted levels. J. Neuroimmunol. 144, 1e8.

Suzuki, K., Hata, S., Kawabata, Y., Sorimachi, H., 2004. Structure, activation, andbiology of calpain. Diabetes 53 (Suppl. 1), S12eS18.

Szamier, R.B., 1981. Ultrastructure of the preretinal membrane in retinitis pig-mentosa. Invest Ophthalmol. Vis. Sci. 21, 227e236.

Tabata, Y., Ouchi, Y., Kamiya, H., Manabe, T., Arai, K., Watanabe, S., 2004. Specifi-cation of the retinal fate of mouse embryonic stem cells by ectopic expression ofRx/rax, a homeobox gene. Mol. Cell. Biol. 24, 4513e4521.

Tackenberg, M.A., Tucker, B.A., Swift, J.S., Jiang, C., Redenti, S., Greenberg, K.P.,Flannery, J.G., Reichenbach, A., Young, M.J., 2009. Muller cell activation, prolif-eration and migration following laser injury. Mol. Vis. 15, 1886e1896.

Tait, S.W., Green, D.R., 2010. Mitochondria and cell death: outer membrane per-meabilization and beyond. Nat. Rev. Mol. Cell. Biol. 11, 621e632.

Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K.,Yamanaka, S., 2007. Induction of pluripotent stem cells from adult human fi-broblasts by defined factors. Cell 131, 861e872.

Takahashi, K., Yamanaka, S., 2006. Induction of pluripotent stem cells from mouseembryonic and adult fibroblast cultures by defined factors. Cell 126, 663e676.

Takita, H., Yoneya, S., Gehlbach, P.L., Duh, E.J., Wei, L.L., Mori, K., 2003. Retinalneuroprotection against ischemic injury mediated by intraocular gene transferof pigment epithelium-derived factor. Invest Ophthalmol. Vis. Sci. 44,4497e4504.


Talcott, K.E., Ratnam, K., Sundquist, S.M., Lucero, A.S., Lujan, B.J., Tao, W., Porco, T.C.,Roorda, A., Duncan, J.L., 2011. Longitudinal study of cone photoreceptors duringretinal degeneration and in response to ciliary neurotrophic factor treatment.Invest Ophthalmol. Vis. Sci. 52, 2219e2226.

Taliantzis, S., Papaconstantinou, D., Koutsandrea, C., Moschos, M.,Apostolopoulos, M., Georgopoulos, G., 2009. Comparative studies of RNFLthickness measured by OCT with global index of visual fields in patients withocular hypertension and early open angle glaucoma. Clin. Ophthalmol. 3,373e379.

Tan, Y., Dourdin, N., Wu, C., De Veyra, T., Elce, J.S., Greer, P.A., 2006. Ubiquitouscalpains promote caspase-12 and JNK activation during endoplasmic reticulumstress-induced apoptosis. J. Biol. Chem. 281, 16016e16024.

Tansey, M.G., McCoy, M.K., Frank-Cannon, T.C., 2007. Neuroinflammatory mecha-nisms in Parkinson's disease: potential environmental triggers, pathways, andtargets for early therapeutic intervention. Exp. Neurol. 208, 1e25.

Taylor, A., 2012. Introduction to the issue regarding research regarding age relatedmacular degeneration. Mol. Asp. Med. 33, 291e294.

Terzibasi, E., Calamusa, M., Novelli, E., Domenici, L., Strettoi, E., Cellerino, A.,2009. Age-dependent remodelling of retinal circuitry. Neurobiol. Aging 30,819e828.

Tezel, G., Li, L.Y., Patil, R.V., Wax, M.B., 2001. TNF-alpha and TNF-alpha receptor-1 inthe retina of normal and glaucomatous eyes. Invest Ophthalmol. Vis. Sci. 42,1787e1794.

Tomany, S.C., Wang, J.J., Van Leeuwen, R., Klein, R., Mitchell, P., Vingerling, J.R.,Klein, B.E., Smith, W., De Jong, P.T., 2004. Risk factors for incident age-relatedmacular degeneration: pooled findings from 3 continents. Ophthalmology111, 1280e1287.

Trapp, B.D., Wujek, J.R., Criste, G.A., Jalabi, W., Yin, X., Kidd, G.J., Stohlman, S.,Ransohoff, R., 2007. Evidence for synaptic stripping by cortical microglia. Glia55, 360e368.

Tsai, G.Y., Cui, J.Z., Syed, H., Xia, Z., Ozerdem, U., McNeill, J.H., Matsubara, J.A., 2009.Effect of N-acetylcysteine on the early expression of inflammatory markers inthe retina and plasma of diabetic rats. Clin. Exp. Ophthalmol. 37, 223e231.

Tso, M.O., Zhang, C., Abler, A.S., Chang, C.J., Wong, F., Chang, G.Q., Lam, T.T., 1994.Apoptosis leads to photoreceptor degeneration in inherited retinal dystrophy ofRCS rats. Invest Ophthalmol. Vis. Sci. 35, 2693e2699.

Uchiki, T., Weikel, K.A., Jiao, W., Shang, F., Caceres, A., Pawlak, D., Handa, J.T.,Brownlee, M., Nagaraj, R., Taylor, A., 2012. Glycation-altered proteolysis as apathobiologic mechanism that links dietary glycemic index, aging, and age-related disease (in nondiabetics). Aging Cell. 11, 1e13.

Uchiyama, Y., 2001. Autophagic cell death and its execution by lysosomal cathep-sins. Arch. Histol. Cytol. 64, 233e246.

Udar, N., Atilano, S.R., Memarzadeh, M., Boyer, D.S., Chwa, M., Lu, S., Maguen, B.,Langberg, J., Coskun, P., Wallace, D.C., Nesburn, A.B., Khatibi, N., Hertzog, D.,Le, K., Hwang, D., Kenney, M.C., 2009. Mitochondrial DNA haplogroups associ-ated with age-related macular degeneration. Invest Ophthalmol. Vis. Sci. 50,2966e2974.

Umino, Y., Cuenca, N., Everhart, D., Fernandez-Sanchez, L., Barlow, R.B., Solessio, E.,2012. Partial rescue of retinal function in chronically hypoglycemic mice. InvestOphthalmol. Vis. Sci. 53, 915e923.

Umino, Y., Solessio, E., 2013. Loss of scotopic contrast sensitivity in the optomotorresponse of diabetic mice. Invest Ophthalmol. Vis. Sci. 54, 1536e1543.

Vaegan, Graham, S.L., Goldberg, I., Buckland, L., Hollows, F.C., 1995. Flash andpattern electroretinogram changes with optic atrophy and glaucoma. Exp. EyeRes. 60, 697e706.

van Rossum, D., Hanisch, U.K., 2004. Microglia. Metab. Brain Dis. 19, 393e411.van Zeeburg, E.J., Maaijwee, K.J., Missotten, T.O., Heimann, H., van Meurs, J.C., 2012.

A free retinal pigment epithelium-choroid graft in patients with exudative age-related macular degeneration: results up to 7s. Am. J. Ophthalmol. 153, 120e127e122.

Vasireddy, V., Chavali, V.R., Joseph, V.T., Kadam, R., Lin, J.H., Jamison, J.A.,Kompella, U.B., Reddy, G.B., Ayyagari, R., 2011. Rescue of photoreceptor degen-eration by curcumin in transgenic rats with P23H rhodopsin mutation. PLoSOne 6, e21193.

Verma, A., Shan, Z., Lei, B., Yuan, L., Liu, X., Nakagawa, T., Grant, M.B., Lewin, A.S.,Hauswirth, W.W., Raizada, M.K., Li, Q., 2012. ACE2 and Ang-(1-7) confer pro-tection against development of diabetic retinopathy. Mol. Ther. 20, 28e36.

Villarroel, M., Ciudin, A., Hernandez, C., Simo, R., 2010. Neurodegeneration: an earlyevent of diabetic retinopathy. World J. Diabetes 1, 57e64.

Villegas-P�erez, M.P., Lawrence, J.M., Vidal-Sanz, M., Lavail, M.M., Lund, R.D., 1998.Ganglion cell loss in RCS rat retina: a result of compression of axons by con-tracting intraretinal vessels linked to the pigment epithelium. J. Comp. Neurol.392, 58e77.

Vin, A.P., Hu, H., Zhai, Y., Von Zee, C.L., Logeman, A., Stubbs Jr., E.B., Perlman, J.I.,Bu, P., 2013. Neuroprotective effect of resveratrol prophylaxis on experimentalretinal ischemic injury. Exp. Eye Res. 108, 72e75.

Viringipurampeer, I.A., Shan, X., Gregory-Evans, K., Zhang, J.P., Mohammadi, Z.,Gregory-Evans, C.Y., 2014. Rip3 knockdown rescues photoreceptor cell death inblind pde6c zebrafish. Cell. Death Differ..

Viswanathan, S., Frishman, L.J., Robson, J.G., Harwerth, R.S., Smith 3rd, E.L., 1999.The photopic negative response of the macaque electroretinogram: reductionby experimental glaucoma. Invest Ophthalmol. Vis. Sci. 40, 1124e1136.

Viswanathan, S., Frishman, L.J., Robson, J.G., Walters, J.W., 2001. The photopicnegative response of the flash electroretinogram in primary open angle glau-coma. Invest Ophthalmol. Vis. Sci. 42, 514e522.

von Bartheld, C.S., 1998. Neurotrophins in the developing and regenerating visualsystem. Histol. Histopathol. 13, 437e459.

Vugler, A., Carr, A.J., Lawrence, J., Chen, L.L., Burrell, K., Wright, A., Lundh, P.,Semo, M., Ahmado, A., Gias, C., da Cruz, L., Moore, H., Andrews, P., Walsh, J.,Coffey, P., 2008a. Elucidating the phenomenon of HESC-derived RPE: anatomyof cell genesis, expansion and retinal transplantation. Exp. Neurol. 214,347e361.

Vugler, A.A., 2010. Progress toward the maintenance and repair of degeneratingretinal circuitry. Retina 30, 983e1001.

Vugler, A.A., Semo, M., Joseph, A., Jeffery, G., 2008b. Survival and remodeling ofmelanopsin cells during retinal dystrophy. Vis. Neurosci. 25, 125e138.

Wachtmeister, L., 1998. Oscillatory potentials in the retina: what do they reveal.Prog. Retin Eye Res. 17, 485e521.

Wake, H., Moorhouse, A.J., Jinno, S., Kohsaka, S., Nabekura, J., 2009. Restingmicroglia directly monitor the functional state of synapses in vivo and deter-mine the fate of ischemic terminals. J. Neurosci. 29, 3974e3980.

Wang, L., Cioffi, G.A., Cull, G., Dong, J., Fortune, B., 2002. Immunohistologic evidencefor retinal glial cell changes in human glaucoma. Invest Ophthalmol. Vis. Sci. 43,1088e1094.

Wang, L., Clark, M.E., Crossman, D.K., Kojima, K., Messinger, J.D., Mobley, J.A.,Curcio, C.A., 2010. Abundant lipid and protein components of drusen. PLoS One5, e10329.

Wang, L., Li, C., Guo, H., Kern, T.S., Huang, K., Zheng, L., 2011a. Curcumin inhibitsneuronal and vascular degeneration in retina after ischemia and reperfusioninjury. PLoS One 6, e23194.

Wang, M., Ma, W., Zhao, L., Fariss, R.N., Wong, W.T., 2011b. Adaptive Muller cellresponses to microglial activation mediate neuroprotection and coordinateinflammation in the retina. J. Neuroinflam. 8, 173.

Wang, S., Villegas-Perez, M.P., Vidal-Sanz, M., Lund, R.D., 2000. Progressive opticaxon dystrophy and vacuslar changes in rd mice. Invest Ophthalmol. Vis. Sci. 41,537e545.

Wang, W., Noel, J., Kaplan, H.J., Dean, D.C., 2011c. Circulating reactive oxidant causesapoptosis of retinal pigment epithelium and cone photoreceptors in the mousecentral retina. Ophthalmol. Eye Dis. 3, 45e54.

Wang, Z.Y., Mo, X.F., Jiang, X.H., Rong, X.F., Miao, H.M., 2012. Ginkgolide B promotesaxonal growth of retina ganglion cells by anti-apoptosis in vitro. Sheng Li XueBao 64, 417e424.

Weidemann, A., Krohne, T.U., Aguilar, E., Kurihara, T., Takeda, N., Dorrell, M.I.,Simon, M.C., Haase, V.H., Friedlander, M., Johnson, R.S., 2010. Astrocyte hypoxicresponse is essential for pathological but not developmental angiogenesis ofthe retina. Glia 58, 1177e1185.

Weikel, K.A., Fitzgerald, P., Shang, F., Caceres, M.A., Bian, Q., Handa, J.T., Stitt, A.W.,Taylor, A., 2012. Natural history of age-related retinal lesions that precede AMD inmice fedhighor lowglycemic indexdiets. InvestOphthalmol. Vis. Sci. 53, 622e632.

Werner, J.S., Keltner, J.L., Zawadzki, R.J., Choi, S.S., 2011. Outer retinal abnormalitiesassociated with inner retinal pathology in nonglaucomatous and glaucomatousoptic neuropathies. Eye (Lond.) 25, 279e289.

Westheimer, G., 1965. Visual acuity. Annu Rev. Psychol. 16, 359e380.Westheimer, G., 2009. Visual acuity: information theory, retinal image structure

and resolution thresholds. Prog. Retin Eye Res. 28, 178e186.Widestrand, A., Faijerson, J., Wilhelmsson, U., Smith, P.L., Li, L., Sihlbom, C.,

Eriksson, P.S., Pekny, M., 2007. Increased neurogenesis and astrogenesis fromneural progenitor cells grafted in the hippocampus of GFAP-/- Vim-/- mice.Stem Cells 25, 2619e2627.

Williamson, T.H., Baxter, G.M., 1994. Central retinal vein occlusion, an investigationby color Doppler imaging. Blood velocity characteristics and prediction of irisneovascularization. Ophthalmology 101, 1362e1372.

Wilson, J.X., 1997. Antioxidant defense of the brain: a role for astrocytes. Can. J.Physiol. Pharmacol. 75, 1149e1163.

Winkler, B.S., Boulton, M.E., Gottsch, J.D., Sternberg, P., 1999. Oxidative damage andage-related macular degeneration. Mol. Vis. 5, 32.

Wohl, S.G., Schmeer, C.W., Kretz, A., Witte, O.W., Isenmann, S., 2009. Optic nervelesion increases cell proliferation and nestin expression in the adult mouse eyein vivo. Exp. Neurol. 219, 175e186.

Woo, G.C., 1985. Contrast sensitivity function as a diagnostic tool in low vision. Am.J. Optom. Physiol. Opt. 62, 648e651.

Woo, J.M., Shin, D.Y., Lee, S.J., Joe, Y., Zheng, M., Yim, J.H., Callaway, Z., Chung, H.T.,2012. Curcumin protects retinal pigment epithelial cells against oxidative stressvia induction of heme oxygenase-1 expression and reduction of reactive oxy-gen. Mol. Vis. 18, 901e908.

Woo, S.J., Kim, J.H., Yu, H.G., 2010. Ursodeoxycholic acid and tauroursodeoxycholicacid suppress choroidal neovascularization in a laser-treated rat model. J. Ocul.Pharmacol. Ther. 26, 223e229.

Wright, L.S., Phillips, M.J., Pinilla, I., Hei, D., Gamm, D.M., 2014. Induced pluripotentstem cells as custom therapeutics for retinal repair: progress and rationale. Exp.Eye Res..

Wu, K.H., Madigan, M.C., Billson, F.A., Penfold, P.L., 2003. Differential expression ofGFAP in early v late AMD: a quantitative analysis. Br. J. Ophthalmol. 87,1159e1166.

Xie, Z., Wu, X., Gong, Y., Song, Y., Qiu, Q., Li, C., 2007. Intraperitoneal injection ofGinkgo biloba extract enhances antioxidation ability of retina and protectsphotoreceptors after light-induced retinal damage in rats. Curr. Eye Res. 32,471e479.

Xu, H., Chen, M., Forrester, J.V., 2009. Para-inflammation in the aging retina. Prog.Retin Eye Res. 28, 348e368.


Xu, Z., Wei, Y., Gong, J., Cho, H., Park, J.K., Sung, E.R., Huang, H., Wu, L., Eberhart, C.,Handa, J.T., Du, Y., Kern, T.S., Thimmulappa, R., Barber, A.J., Biswal, S., Duh, E.J.,2014. NRF2 plays a protective role in diabetic retinopathy in mice. Diabetologia57, 204e213.

Yamauchi, M., Tsuruma, K., Imai, S., Nakanishi, T., Umigai, N., Shimazawa, M.,Hara, H., 2011. Crocetin prevents retinal degeneration induced by oxidative andendoplasmic reticulum stresses via inhibition of caspase activity. Eur. J. Phar-macol. 650, 110e119.

Yang, L., Tan, P., Zhou, W., Zhu, X., Cui, Y., Zhu, L., Feng, X., Qi, H., Zheng, J., Gu, P.,Fan, X., Chen, H., 2012. N-acetylcysteine protects against hypoxia mimetic-induced autophagy by targeting the HIF-1alpha pathway in retinal ganglioncells. Cell. Mol. Neurobiol. 32, 1275e1285.

Yang, L.P., Wu, L.M., Guo, X.J., Tso, M.O., 2007. Activation of endoplasmic reticulumstress in degenerating photoreceptors of the rd1 mouse. Invest Ophthalmol. Vis.Sci. 48, 5191e5198.

Yang, Y., Mohand-Said, S., Danan, A., Simonutti, M., Fontaine, V., Clerin, E., Picaud, S.,Leveillard, T., Sahel, J.A., 2009. Functional cone rescue by RdCVF protein in adominant model of retinitis pigmentosa. Mol. Ther. 17, 787e795.

Yang, Y., Mohand-Said, S., Leveillard, T., Fontaine, V., Simonutti, M., Sahel, J.A., 2010.Transplantation of photoreceptor and total neural retina preserves cone func-tion in P23H rhodopsin transgenic rat. PLoS One 5, e13469.

Yar, A.S., Menevse, S., Dogan, I., Alp, E., Ergin, V., Cumaoglu, A., Aricioglu, A.,Ekmekci, A., Menevse, A., 2012. Investigation of ocular neovascularization-related genes and oxidative stress in diabetic rat eye tissues after resveratroltreatment. J. Med. Food 15, 391e398.

Yonemura, D., Tsuzuki, K., Aoki, T., 1962. Clinical importance of the oscillatory po-tential in the human ERG. Acta Ophthalmol. Suppl. Suppl. 70, 115e123.

Young, B., Eggenberger, E., Kaufman, D., 2012. Current electrophysiology inophthalmology: a review. Curr. Opin. Ophthalmol. 23, 497e505.

Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S.,Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin II, , Thomson, J.A., 2007.Induced pluripotent stem cell lines derived from human somatic cells. Science318, 1917e1920.

Zacks, D.N., Zheng, Q.D., Han, Y., Bakhru, R., Miller, J.W., 2004. FAS-mediatedapoptosis and its relation to intrinsic pathway activation in an experimentalmodel of retinal detachment. Invest Ophthalmol. Vis. Sci. 45, 4563e4569.

Zahs, K.R., Ceelen, P.W., 2006. Gap junctional coupling and connexin immunore-activity in rabbit retinal glia. Vis. Neurosci. 23, 1e10.

Zanna, C., Ghelli, A., Porcelli, A.M., Martinuzzi, A., Carelli, V., Rugolo, M., 2005.Caspase-independent death of Leber's hereditary optic neuropathy cybrids isdriven by energetic failure and mediated by AIF and Endonuclease G. Apoptosis10, 997e1007.

Zeiss, C.J., Johnson, E.A., 2004. Proliferation of microglia, but not photoreceptors, inthe outer nuclear layer of the rd-1 mouse. Invest Ophthalmol. Vis. Sci. 45,971e976.

Zhang, B., Osborne, N.N., 2006. Oxidative-induced retinal degeneration is attenu-ated by epigallocatechin gallate. Brain Res. 1124, 176e187.

Zhang, B., Rusciano, D., Osborne, N.N., 2008. Orally administered epigallocatechingallate attenuates retinal neuronal death in vivo and light-induced apoptosisin vitro. Brain Res. 1198, 141e152.

Zhang, B., Safa, R., Rusciano, D., Osborne, N.N., 2007. Epigallocatechin gallate, anactive ingredient from green tea, attenuates damaging influences to the retinacaused by ischemia/reperfusion. Brain Res. 1159, 40e53.

Zhang, C., Lei, B., Lam, T.T., Yang, F., Sinha, D., Tso, M.O., 2004. Neuroprotection ofphotoreceptors by minocycline in light-induced retinal degeneration. InvestOphthalmol. Vis. Sci. 45, 2753e2759.

Zhang, C., Shen, J.K., Lam, T.T., Zeng, H.Y., Chiang, S.K., Yang, F., Tso, M.O., 2005.Activation of microglia and chemokines in light-induced retinal degeneration.Mol. Vis. 11, 887e895.

Zhang, D.D., 2006. Mechanistic studies of the Nrf2-Keap1 signaling pathway. Drug.Metab. Rev. 38, 769e789.

Zhang, K., Hopkins, J.J., Heier, J.S., Birch, D.G., Halperin, L.S., Albini, T.A., Brown, D.M.,Jaffe, G.J., Tao, W., Williams, G.A., 2011. Ciliary neurotrophic factor delivered byencapsulated cell intraocular implants for treatment of geographic atrophy inage-related macular degeneration. Proc. Natl. Acad. Sci. U. S. A. 108, 6241e6245.

Zhang, M., Xu, G., Liu, W., Ni, Y., Zhou, W., 2012a. Role of fractalkine/CX3CR1interaction in light-induced photoreceptor degeneration through regulatingretinal microglial activation and migration. PLoS One 7, e35446.

Zhang, T., Baehr, W., Fu, Y., 2012b. Chemical chaperone TUDCA preserves conephotoreceptors in a mouse model of Leber congenital amaurosis. Invest Oph-thalmol. Vis. Sci. 53, 3349e3356.

Zhang, Y., Ivanova, E., Bi, A., Pan, Z.H., 2009. Ectopic expression of multiple microbialrhodopsins restores ON and OFF light responses in retinas with photoreceptordegeneration. J. Neurosci. 29, 9186e9196.

Zhao, X., Liu, J., Ahmad, I., 2006. Differentiation of embryonic stem cells to retinalcells in vitro. Methods Mol. Biol. 330, 401e416.

Zheng, H., Schuetz, E., Zeevi, A., Zhang, J., McCurry, K., Webber, S., Iacono, A.,Lamba, J., Burckart, G.J., 2005. Sequential analysis of tacrolimus dosing in adultlung transplant patients with ABCB1 haplotypes. J. Clin. Pharmacol. 45,404e410.

Zheng, L., Anderson, R.E., Agbaga, M.P., Rucker 3rd, E.B., Le, Y.Z., 2006. Loss of BCL-XLin rod photoreceptors: increased susceptibility to bright light stress. InvestOphthalmol. Vis. Sci. 47, 5583e5589.

Zhong, Q., Mishra, M., Kowluru, R.A., 2013. Transcription factor Nrf2-mediatedantioxidant defense system in the development of diabetic retinopathy.Invest Ophthalmol. Vis. Sci. 54, 3941e3948.

Zhou, H., Wu, S., Joo, J.Y., Zhu, S., Han, D.W., Lin, T., Trauger, S., Bien, G., Yao, S.,Zhu, Y., Siuzdak, G., Scholer, H.R., Duan, L., Ding, S., 2009a. Generation ofinduced pluripotent stem cells using recombinant proteins. Cell. Stem Cell. 4,381e384.

Zhou, W.T., Ni, Y.Q., Jin, Z.B., Zhang, M., Wu, J.H., Zhu, Y., Xu, G.Z., Gan, D.K., 2012.Electrical stimulation ameliorates light-induced photoreceptor degenerationin vitro via suppressing the proinflammatory effect of microglia and enhancingthe neurotrophic potential of Muller cells. Exp. Neurol. 238, 192e208.

Zhou, X., Li, F., Kong, L., Chodosh, J., Cao, W., 2009b. Anti-inflammatory effect ofpigment epithelium-derived factor in DBA/2J mice. Mol. Vis. 15, 438e450.

Zhou, Y., Pernet, V., Hauswirth, W.W., Di Polo, A., 2005. Activation of the extracel-lular signal-regulated kinase 1/2 pathway by AAV gene transfer protects retinalganglion cells in glaucoma. Mol. Ther. 12, 402e412.

Zhou, Y.Y., Chen, C.Z., Su, Y., Li, L., Yi, Z.H., Qi, H., Weng, M., Xing, Y.Q., 2014. Effect ofEGb761 on light-damaged retinal pigment epithelial cells. Int. J. Ophthalmol. 7,8e13.

Zinkernagel, M.S., Chinnery, H.R., Ong, M.L., Petitjean, C., Voigt, V., McLenachan, S.,McMenamin, P.G., Hill, G.R., Forrester, J.V., Wikstrom, M.E., Degli-Esposti, M.A.,2013. Interferon gamma-dependent migration of microglial cells in the retinaafter systemic cytomegalovirus infection. Am. J. Pathol. 182, 875e885.

Zuo, Z.F., Zhang, Q., Liu, X.Z., 2013. Protective effects of curcumin on retinal Mullercell in early diabetic rats. Int. J. Ophthalmol. 6, 422e424.

Zwart, I., Hill, A.J., Al-Allaf, F., Shah, M., Girdlestone, J., Sanusi, A.B., Mehmet, H.,Navarrete, R., Navarrete, C., Jen, L.S., 2009. Umbilical cord blood mesenchymalstromal cells are neuroprotective and promote regeneration in a rat optic tractmodel. Exp. Neurol. 216, 439e448.

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Progress in Retinal and Eye Research - ua · Cellular responses following retinal injuries and therapeutic approaches for neurodegenerative diseases Nicolas Cuenca a, b, *, 2, Laura