Progress in Retinal and Eye Research 43 (2014) 17e75
Contents lists available at ScienceDirect
Progress in Retinal and Eye Research
journal homepage: www.elsevier.com/locate/prer
Cellular responses following retinal injuries and therapeutic
approaches for neurodegenerative diseases
s Cuenca a, b, *, 2, Laura Ferna
ndez-Sa
nchez a, 1, 2, Laura Campello a, 1, 2,
Nicola
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, Spain
Multidisciplinary Institute for Environmental Studies “Ramon Margalef”, University of Alicante, Alicante, Spain
Department of Optics, Pharmacology and Anatomy, University of Alicante, Alicante, Spain
d
, Alcala
de Henares, Spain
Department of Systems Biology, University of Alcala
e
Department of Ophthalmology, Lozano Blesa University Hospital, Aragon Institute of Health Sciences, Zaragoza, Spain
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 April 2014
Received in revised form
3 July 2014
Accepted 7 July 2014
Available online 17 July 2014
Retinal neurodegenerative diseases like age-related macular degeneration, glaucoma, diabetic retinopathy and retinitis pigmentosa each have a different etiology and pathogenesis. However, at the cellular
and molecular level, the response to retinal injury is similar in all of them, and results in morphological
and 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 all
frequently induce a set of cell signals that lead to well-established and similar morphological and
functional changes, including controlled cell death and retinal remodeling. Interestingly, an inflammatory response, oxidative stress and activation of apoptotic pathways are common features in all these
diseases. Furthermore, it is important to note the relevant role of glial cells, including astrocytes, Müller
cells and microglia, because their response to injury is decisive for maintaining the health of the retina or
its degeneration. Several therapeutic approaches have been developed to preserve retinal function or
restore 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 degeneration
to obtain successful results. This review provides an overview of the common and distinctive features of
retinal neurodegenerative diseases, including the molecular, anatomical and functional changes caused
by the cellular response to damage, in order to establish appropriate treatments for these pathologies.
© 2014 Elsevier Ltd. All rights reserved.
Keywords:
Retinal remodeling
Neurodegeneration
Glial cells
Retinal therapy
Neuroprotection
Retinal diseases
List of abbreviations: AAV, Adeno-associated virus; AGEs, Advanced glycation end products; AMD, Age-related macular degeneration; Apaf-1, Apoptotic proteaseactivating factor-1; BDNF, Brain-derived neurotrophic factor; bFGF, Basic fibroblast growth factor; BRB, Blood retinal barrier; CNS, Central nervous system; CNTF, Ciliaryderived neurotrophic factor; CNV, Choroidal neovascularization; DR, Diabetic retinopathy; EGCG, Epigallocatechin gallate; ERG, Electroretinogram; ESC, Embryonic stem cells;
FGF, Fibroblast growth factor; GCL, Ganglion cell layer; GDNF, Glial-derived neurotrophic factor; GFAP, Glial fibrillary acidic protein; hESC, Human embryonic stem cells;
hiPSC, Human induced pluripotent stem cells; IL, Interleukin; INL, Inner nuclear layer; IPL, Inner plexiform layer; iPSC, Induced pluripotent stem cells; LIRD, Light-induced
retinal degeneration; mGluR6, Metabotropic glutamate receptor; MOMP, Mitochondrial outer membrane pores; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine; NAC,
N-acetylcysteine; NMDA, N-methyl-D-aspartate; NO, Nitric oxide; NF-, B; Nuclear factor, B; Nrf2, Nuclear factor erythroid 2-related factor 2; ONL, Outer nuclear Llayer; OPL,
Outer plexiform layer; PEDF, Pigment epithelium derived factor; PVR, Proliferative vitreoretinopathy; RCS, Royal College Surgeon rats; RGC, Retinal ganglion cells; ROS,
Reactive oxygen species; RP, Retinitis pigmentosa; RPE, Retinal pigment epithelium; TGF-b, Transforming growth factor-b; TLR, Toll-like receptor; TNF, Tumor necrosis factor;
TUDCA, Tauroursodeoxycholic acid; UPS, Ubiquitin-proteasome system; VEGF, Vascular endothelial growth factor; VEPs, Visual evoked potentials.
* Corresponding author. Department of Physiology, Genetics and Microbiology, University of Alicante, San Vicente del Raspeig, E-03080 Alicante, Spain. Tel.: þ34
965909916; fax: þ34 965903943.
E-mail address: cuenca@ua.es (N. Cuenca).
1
These authors contributed equally to this work.
2
s Cuenca: 15%; Laura Fern
nchez: 15%; Laura Campello:
Percentage of work contributed by each author in the production of the manuscript is as follows: Nicola
andez-Sa
15%; Victoria Maneu1: 15%; Pedro De la Villa: 10%; Pedro Lax: 15%; Isabel Pinilla 15%.
http://dx.doi.org/10.1016/j.preteyeres.2014.07.001
1350-9462/© 2014 Elsevier Ltd. All rights reserved.
18
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Cellular responses induced by retinal injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.1.
Retinal neurons and circuitries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.1.1.
Photoreceptor morphology changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.1.2.
Bipolar and horizontal cells sprouting and remodeling as a consequence of photoreceptor loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.1.3.
Does OPL connectivity plays a crucial role in vision loss? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.1.4.
Amacrine cell types and retinal degeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.1.5.
From photoreceptor loss to ganglion cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.2.
Alterations in retinal homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.2.1.
Oxidative stress and retinal degeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.2.2.
Activation of apoptotic pathways: role of the mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.2.3.
Retinal protein homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.3.
Glial responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.3.1.
Inflammatory response: microglial activation in retinal dystrophies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.3.2.
Macroglial cells: Müller and astrocytes cells in healthy and diseased retinas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.4.
Degenerative events in retinal vascularization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.4.1.
Retinal vascular networks and the blood retinal barrier in health and disease . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 41
2.4.2.
Retinal degenerative diseases with relevant vascular changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.5.
Retinal pigment epithelium (RPE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.5.1.
RPE physiology and functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.5.2.
RPE changes in aging and pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.6.
Functional changes following retinal injuries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
2.6.1.
Electroretinogram (ERG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
2.6.2.
Visual evoked potentials (VEPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.6.3.
Psychophysical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Remodeling of the retina in retinal degenerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.1.
Phase 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.2.
Phase 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.3.
Phase 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.4.
Phase 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Therapeutic approaches in neurodegenerative diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4.1.
Efficacy of anti-apoptotic therapies for retinal diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4.1.1.
Tauroursodeoxycholic acid (TUDCA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.1.2.
Rasagiline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4.1.3.
Norgestrel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4.1.4.
Proinsulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4.2.
Efficacy of antioxidant and anti-inflammatory agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4.2.1.
Curcumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.2.2.
Lutein and zeaxanthin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.2.3.
Saffron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.2.4.
Catechins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.2.5.
Ginkgo biloba extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4.2.6.
Resveratrol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4.2.7.
Quercetin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4.2.8.
N-acetylcysteine (NAC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4.2.9.
Antioxidant cocktails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
4.3.
Efficacy of neurotrophic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
4.4.
Gene therapy approaches and clinical trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
4.4.1.
Viral-mediated therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
4.4.2.
Optogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.5.
Cell-based therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.5.1.
Human embryonic stem cells (hESCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.5.2.
Human induced pluripotent stem cells (hiPSC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.5.3.
Human fetal embryonic stem cells; retinal progenitor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.5.4.
Human umbilical tissue-derived stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.5.5.
Human central nervous system stem cells (HuCNS-SC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.5.6.
Bone marrow-derived stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.6.
Effectiveness of retinal transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
4.7.
Clinical trials for retinal diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
4.8.
Suitable therapies in each phase of retinal degeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Conclusion remarks and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
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1. Introduction
The retina is a light-sensitive tissue lining the inner surface of
the eye. It is formed by multiple layers of interconnected neurons
and is in charge of the first steps of visual processing (Fig. 1A). The
photoreceptors (rods and cones) are, together with the melanopsin
ganglion cells, the photosensitive cells of the retina (Figs. 1B and 2).
Photoreceptors are one of the most specialized and complex cells in
the nervous system, and they are located in the outer nuclear layer
(ONL) of the vertebrate retina. Rods and cones initiate the conversion of light energy into electrical signals through a process called
phototransduction. Retinal interneurons further codify the electrical signals into optic nerve impulses, which are subsequently
interpreted by the brain as visual images. This process enables the
recognition of shapes, sizes, colors and movements. The organization of the retina and visual system has been described in detail by
Kolb (Kolb, 2003) and on Webvision (http://webvision.med.utah.
edu/). Briefly, after cones and rods absorb the incident light, phototransduction takes place in their outer segments, and the
resulting electrical impulse is relayed to the bipolar and horizontal
cells. At the outer plexiform layer (OPL), the dendrites of rod and
cone bipolar cells make synaptic contacts with the axonal terminations of the rods (spherules) and cones (pedicles), respectively
(Figs. 1B and 2). In the next stage, amacrine cells (located in the
inner nuclear layer) and bipolar cells establish a complex network
of synaptic interconnections at the inner plexiform layer (IPL) with
ganglion cells (Fig. 1A). Lastly, the electrical information is
conveyed to the ganglion cells, which send out impulses through
their axonal prolongations connecting the retina to the brain via the
optic nerve.
In the context of visual function, it is important to mention the
fovea, a central region of the human and primate retina containing
a very high concentration of cones responsible for a great visual
acuity and color appreciation (Fig. 3). In the center of the fovea is
located the foveola which is approximately 0.35 mm in diameter.
Interestingly, the structure of the foveola is different from that of
the rest of the laminated retina and consists of a unique layer
containing only cone cells. Surrounding the fovea, in the parafoveal
area between the photoreceptor and outer plexiform layers, the
axons of the foveal cones are arranged obliquely, constituting the
anatomical region called the Henle fiber layer (HFL), which is not
present in peripheral retina (Fig. 3).
The structural and functional complexity of the retina makes
this tissue vulnerable to alterations from any sort of pathological
injury. Glaucoma is a leading cause of blindness and is characterized by retinal ganglion cell (RGC) degeneration, leading to optic
nerve damage. Intraocular pressure is one of the most important
risk factors. Age-related macular degeneration (AMD) is the leading
cause of severe and irreversible loss of vision in the elderly in
developed countries. Age is the most significant risk factor, and the
initial symptoms of this disease include a loss of central visual
acuity, a subjective impression of the curvature of straight lines or
metamorphopsia, and a gradually enlarging central scotoma. In this
disease, impairment of retinal pigment epithelial (RPE) cells and
photoreceptors, as well as vascular angiogenesis are the main cause
of visual loss. Diabetic retinopathy (DR) refers to a group of eye
problems that people with diabetes may face. It is caused by
changes in the vascular cells of the retina. In some people, blood
vessels may swell and leak fluid, while in others abnormal new
blood vessels grow on the surface of the retina. Retinitis pigmentosa (RP) is considered to be a group of inherited diseases causing
photoreceptor degeneration. In most forms of RP, the rods are
affected first, prior to cone damage. Because rods are concentrated
in the peripheral retina, people suffering this disease show a progressive diminution of the peripheral visual field, ending in a
Fig. 1. Retinal cytoarchitecture. (A) Vertical section of a monkey retina showing the
main retinal layers. Antibodies against alpha-synuclein (red) stained outer segments of
cones and rods, axon terminals in the outer plexiform layer, and a specific population
of bipolar, amacrine and ganglion cells. GABA (blue) labeled amacrine cells and glycine
(green) stained bipolar and amacrine cells. Note the variety of bipolar and amacrine
cell types, and the complex neuronal circuits at the inner plexiform layer. (B) High
magnification of the outer retina triple-immnunolabeled with antibodies against
alpha-synuclein (red), arrestin and rhodopsin (Rho) (both in green), showing the entire
morphology of cones (green, elongated cells) from the outer segment to their axon
terminals (pedicles), as well as rod outer segments (top green lines) and rod axon
terminals (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 nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer.
tunnel vision. Additionally, visual dysfunctions have been
described in human neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Patients suffering these pathologies show a marked reduction in the retinal nerve fiber layer
thickness, alterations in the electroretinogram responses and
sensitivity to the visual contrast, as well as prolonged latency in
visual evoked potentials. Color perception abnormalities, especially
in the blue-yellow hue discrimination, have also been described
associated to these diseases, in addition to aberrations in ‘higher’
visual processing capabilities, such as read, object recognition and
spatial localization (Bodis-Wollner, 2009; Kirby et al., 2010).
Like in the brain, the loss of pre and/or postsynaptic inputs to
the retinal neurons causes changes in their morphology and
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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 photoreceptor
cells. 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 and
rods. (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. Arrows
indicate the axon running in the optic nerve fiber layer.
function and, as a consequence, these neurons try to establish new
synaptic contacts. Thus, the retinal changes underlying different
diseases may modify the transmission of the information between
cells and, as a consequence, the retina can undergo a marked
remodeling. Due to this non-specific disease-remodeling phenomenon, some neuroprotective therapies applied in CNS disorders
may be useful in retinal pathologies, even if they do not share the
same etiology. However, it is becoming increasingly clear that
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
Fig. 3. Morphology of the fovea. Vertical section of a monkey fovea stained with antibodies against calbindin (blue), alpha-synuclein (red) and PNA (peanut agglutinin,
green). The foveola consists of only a layer of photoreceptors with a high concentration
of cones. RPE: Retinal pigment epithelium; OS: outer segments; IS: inner segments;
ONL: outer nuclear layer; HFL: Henle fiber layer; OPL: outer plexiform layer; INL: inner
nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer.
treatments for retinal neurodegenerative diseases may require a
combination of several types of therapies. A large body of studies
indicates that not only apoptotic, but also autophagic and necrotic
cellular pathways are involved in photoreceptor cell death, and
thus the combined modification of these pathways may be an
effective neuroprotective strategy for retinal diseases associated
with photoreceptor cell loss. Another therapeutic option is the
replacement of lost cells with new ones that are able to connect to
the still-functional part of the host retina. This approach might be
capable of repairing a damaged retina and restoring eyesight. Genebased therapies may be the most suitable approaches for inherited
retinal diseases.
In this review, we will discuss important aspects regarding the
remodeling underlying the retinal degenerative diseases: the
alteration events in retinal vascularization, the functional changes
of the retina affecting vision, and the cellular responses induced by
retinal injuries. We will also focus on the most current preventive
and therapeutic strategies in the treatment of retinal neurodegenerative disorders. Ultimately, this large amount of information
will illustrate how a better understanding of the destructive
mechanisms occurring in retinal diseases could potentially enable
the identification and validation of new targets for the neuroprotection of this tissue against neurodegenerative processes, and
it will also allow the development of the next generation of
therapies.
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 and
functional responses in the different retinal cells (remodeling).
However, remodeling is not widely considered in the treatment of
retinal degeneration. The mammalian retina clearly has a vast
repertoire of cellular responses to injury, and understanding these
may help us improve current therapies or devise new ones for
conditions resulting in blindness. In this sense, many studies show
that animal models of retinal diseases exhibit features of human
retinal degeneration and remodeling that can be extremely useful
in the study of human neurodegenerative retinal diseases.
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2.1.1. Photoreceptor morphology changes
Photoreceptors are highly differentiated and specialized neuroepithelial cells sensitive to light. Their structure comprises
several main parts: the outer and the inner segment, a cell body, an
axon and the axon terminal (Fig. 2AeC). Visual transduction takes
place in the outer segment, which borders the RPE, has a cylindrical
shape and is connected to the inner segment by a thin cilium. The
outer segment consist of an ordered stack of membrane disks,
formed by infoldings of the surface membrane in the case of cone
cells, and by disks superimposed like a pile of coins covered by the
plasma membrane in rod cells (Fig. 2). The inner segment is divided
into two parts: the ellipsoid, containing a large cluster of mitochondria under the outer segment, and the myoid, which contains
typical subcellular organelles, including rough and smooth endoplasmic reticulum and Golgi apparatus (Fig. 2B). The cell body is
located at the innermost end of this segment. The axon, which does
not conduct action potentials, ends in a bulb-shaped structure
called spherule in rods, and a pedicle in cones, and contains a large
number of synaptic vesicles, loaded with neurotransmitter that are
continually released into the synaptic cleft in conditions of darkness. The most characteristic structure in the axon terminals is the
synaptic ribbon, a protein structure surrounded by synaptic vesicles. These specialized synapses are called triads, because they
consist of a presynaptic ribbon and three postsynaptic processes:
two horizontal cell dendrites on the sides and one or two bipolar
cell dendrites in the center. In addition to these invaginating synapses, bipolar cells make many flat contacts (basal junctions) with
the cone pedicle base (Dowling and Boycott, 1966; Kolb et al., 2001).
Proper development and functioning of the retina requires a
precise balance between the processes of proliferation, differentiation and programmed cell death. Certain genetic mutations, age
and environmental factors can trigger specific pathways to induce
apoptosis in photoreceptors, contributing as a component of many
diseases. The changes responsible for dystrophic and degenerative
photoreceptor diseases, which cause structural and functional
damage, may occur at any level of the signal transduction cascade
or in any of the morphological components of these differentiated
cells. On the other hand, due to their intense metabolic activity,
photoreceptors generate free radicals and other oxidative agents
whose removal is crucial for cellular health. Oxidative stress occurs
when the balance between oxidizing agents and antioxidants is
altered, resulting in dysfunction and cell death caused by the
oxidation of proteins, lipids and DNA. The phototransduction
cascade, the high level of membrane protein and neurotransmitter
synthesis, and all these complex structures are encoded by a large
number of genes, which explains the great variety of possible
mutations 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 to
involve apoptosis, as revealed by TUNEL (Li et al., 1995). During the
degeneration process, certain morphological changes can be
observed before photoreceptor death (Fig. 4). As degeneration
proceeds, there is a progressive reduction in the thickness of the
ONL, which indicates a loss of photoreceptors (Fig. 4CeF). In RP, the
cones experience a progressive size reduction as the result of rod
death, losing their normal morphology with a shortening of the
inner and outer segments and axon (Fig. 4CeD, F).
In normal, fully differentiated rods, rhodopsin is synthesized in
the rough endoplasmic reticulum, packaged into vesicles in the
Golgi apparatus, transported inside membrane vesicles through the
inner segment cytoplasm to the connecting cilium, and inserted
into newly forming membrane discs at the base of the outer
segment. To maintain the outer segment length constant, shedding
of the outer segment tips are phagocytized and degraded by the
RPE. Intense rhodopsin immunolocalization is seen in the outer
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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 models
of 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 and
inner 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 alterations 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 and
near the distal ends of the inner segments, while all other parts of
the cell appear negative for anti-rhodopsin staining. During retinal
degeneration, translocation of rhodopsin down to the cell bodies
and axon terminals is common to all retinal diseases (Fig. 4C).
Another sign of rod and cone degeneration at the earliest stages is
the shortening or disorganization of their outer segments, which
can be visualized by immunocytochemistry (using antibodies
against rhodopsin, transducin or cone opsins) (Figs. 4 and 8A). The
outer segments of the cones are greatly shortened and swollen in
the detached retina, and antibodies against cone opsins now label
the plasma membrane of cone cells extending to the ONL (Fisher
et al., 2005). Similar changes with swollen and truncated cone
outer segments have been found in organotypic cultures of human
neuroretina (Fernandez-Bueno et al., 2012) and in animal models of
RP (Figs. 4CeD, F and 8A) (Garcia-Ayuso et al., 2013; MartinezNavarrete et al., 2011; Pinilla et al., 2007).
Changes in photoreceptors and their synaptic connectivity are
evident in several human neurodegenerative diseases, as well as in
animal models of neurodegeneration. In RP human retinas, surviving rods were reported to have sprouted rhodopsin-positive
neurites that were closely associated with gliotic Müller cell processes and extended to the inner limiting membrane. However, the
rods and cones located in the macula did not form neurites, rather
the axons of peripheral cones were abnormally elongated and
branched (Vugler, 2010). It is interesting to note that rods in RP
human 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 inner
nuclear layer (INL) and ganglion cell layer (CGL) (Fariss et al., 2000;
Li et al., 1995; Milam et al., 1998; Sanyal, 1993). This sprouting of rod
axons into the INL has not been described in animal models. Similar
sprouting of rod axons into the INL has been found in human dry
AMD with geographic atrophy (Gupta et al., 2003). This sprouting of
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
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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) and
bassoon (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 photoreceptor synaptic ribbons (red) and their synaptic contacts with bipolar cell dendrites (green) during the degenerative process. Few bassoon-immunopositive spots are found at the
OPL 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 bipolar
cells are scarce or absent. Scale bar: 20 mm.
rod axon terminals into the INL in late degeneration needs to be
taken into account for retinal therapeutic approaches, because it
might disable the establishment of correct synaptic contacts.
In human AMD, many rod photoreceptors retract their synaptic
processes into the ONL and lose their synaptic connections with rod
bipolar cells (Sullivan et al., 2007). The retraction of rod photoreceptor synapses is also evident in retinal detachment (Fisher et al.,
2005) and RP (Fariss et al., 2000). In DBA/2J mice models of ocular
hypertension it has been documented an alteration of rod photoreceptor ribbon structure (Fernandez-Sanchez et al., 2013; Fuchs
et al., 2012). Similar results were found in transgenic mice overexpressing the guanylate cyclase activating protein 2 (GCAP2) in
rods leading to a shortening of synaptic ribbons, and to a higher
than normal percentage of club-shaped and spherical ribbon
morphologies (Lopez-del Hoyo et al., 2012). Mice with chronic
hypoglycemia by a null mutation in the glucagon receptor gene
Gcgr also showed a loss of synaptic ribbon in the OPL (Umino et al.,
2012). Besides, it has also been demonstrated that null mice in the
insulin-like growth factor-I (Igf1/) suffered important structural
modifications in retinal synapses (Rodriguez-de la Rosa et al., 2012).
In the case of detached retinas, synaptic invaginations of the rod
spherules are shallower, and the postsynaptic processes are more
loosely organized than in normal retinas. At the electron microscopy level, atypical synapses have been found in an animal model
of retinal detachment (Fisher et al., 2005) and in human retinal
organotypic cultures (Fernandez-Bueno et al., 2012). Furthermore,
groups of 3 synaptic ribbons without their corresponding postsynaptic elements were observed in both cases. Abnormal synaptic
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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 with
PKC-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 bipolar
cell 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), and
mGluR6, 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 P90
RCS 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 parallel membrane alignment under normal conditions, changes to an
irregular distribution of disc membranes, a common feature of RP
in animal models and human organotypic cultures (Cuenca et al.,
2013; Fernandez-Bueno et al., 2012).
Diabetic retinopathy affects retinal vascularization, as well as
the retinal cells themselves, including neural and glial components.
Microvascular lesions may occur in the early stages of DR, in both
animal models and humans (Abu-El-Asrar et al., 2004; Lieth et al.,
2000), but there is increasing evidence that retinal degeneration
occurs before any microvascular alteration (Antonetti et al., 2006;
Barber, 2003; Villarroel et al., 2010). In rodent models of DR, ganglion cells have been reported to die by apoptosis, and a decrease in
the thickness of both the INL and ONL has been observed 10 weeks
after the onset of the disease (Barber et al., 1998; Martin et al.,
2004). An elevated rate of apoptosis has also been observed in
the ONL and in the RPE (Aizu et al., 2002; Park et al., 2003). In 2003,
Park and collaborators described the apoptotic death of photoreceptors in a streptozotocin model of diabetic rat as early as one
month after the onset of the disease. They also showed that there
were modifications in postsynaptic cells (degeneration of horizontal cell processes) and necrotic features in some amacrine and
horizontal cells. Gastinger and coworkers also described in the
retina of streptozotocin diabetic rats the loss of dopaminergic and
cholinergic amacrine cells during the early stages of neurodegeneration (Gastinger et al., 2006). These cellular changes can
contribute to blood-retinal barrier alterations and the development
of retinal vascular changes, and they can be crucial for detecting
cellular neurodegenerative changes prior to the appearance of
functional deficits in these patients. The confirmation of these
events in humans with DR would allow initiating early treatment
with neuroprotective drugs prior to the occurrence of vascular
changes, as soon as the first signs are detected (Simon et al., 2012).
Swelling and loss of photoreceptors have been described in
chronic 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 glaucoma
using optical coherence tomography (OCT), as well as a loss in cone
density along with the expected inner retinal changes (Choi et al.,
2011; Fan et al., 2011). There is also evidence demonstrating that
non-glaucomatous and glaucomatous optic neuropathies are
associated with outer retinal changes following long-term inner
retinal pathology (Werner et al., 2011). As an example, the number
of photoreceptors was significantly reduced in a mouse model of
ocular hypertension (Cuenca et al., 2010) and in the DBA/2J mouse
model of glaucoma (Fig. 4E) (Fernandez-Sanchez et al., 2011b). All
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
25
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 cells
immunostained with antibodies against calbindin (green). Synaptophysin (SYP) were used to label axon terminals of photoreceptors (A, D, E, G), bassoon stained photoreceptor
synaptic 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-type
animals (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 with
the electroretinogram (ERG) changes found in patients with optic
nerve atrophy and glaucoma (Vaegan et al., 1995). These results
show that remodeling also occurs when cells of the inner layers of
the retina die.
2.1.2. Bipolar and horizontal cells sprouting and remodeling as a
consequence of photoreceptor loss
Bipolar and horizontal cells are the second-order neurons in the
retinal circuitry that connect with photoreceptor spherules and
pedicles. The death of photoreceptors determines the response of
bipolar (Figs. 5, 6 and 8) and horizontal cells (Figs. 7 and 8) in
different ways. Retinal bipolar cell remodeling is a universal feature
in retinal degenerative diseases in humans, rodents, rabbits and
cats. All evidence indicates that as the degeneration of rod bipolar
cells 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 photoreceptor death, bipolar cells initially retract their dendrites
(Fig. 5CeF and 8A), but after the loss of their normal input, secondorder bipolar cells seek out new functional photoreceptors with
which to make contact, thus extending their dendrites. This
sprouting of bipolar cell dendrites (Figs. 6C and 8A) into the ONL
has 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 bipolar dendrites and their further sprouting into the ONL took place
before photoreceptor death (Dorfman et al., 2011). In addition, rod
bipolar cells in the parafoveal region showed dendrite sprouting in
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Fig. 8. Cellular remodeling during retinal degeneration. Schematic representation of the main changes in morphology (A) and connectivity (B) that take place in retinal neurons
during 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 both
outer 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 are
frequent, 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 retinal
degenerative 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 new
contact with the remained cones.
humans affected by AMD, indicating a certain degree of dendritic
and synaptic plasticity in this disease (Sullivan et al., 2007). Rod
bipolar cell dendrite sprouting has also been demonstrated in an
experimental model of retinal detachment (Fisher et al., 2005).
However, not all animal models of outer retinal degeneration
exhibit the sprouting of rod bipolar cells. For example, bipolar cell
dendritic sprouting has not been detected in the P23H rat model of
RP (Fig. 5F) (Cuenca et al., 2004). The real reason for this different
behavior in specific animal models remains unknown. The differences may lie in the speed of degeneration at the ONL, or may be
determined by different gene mutations. In RCS rats (Fig. 6), the
presence of sprouted dendritic terminals in the ONL (Fig. 6BeC),
where some photoreceptor cells remain alive, suggests that these
cells may still be capable of sending inputs to postsynaptic cells.
Remodeling of bipolar cells after retinal degeneration may also
affect their axon terminals (Fig. 6BeC and 8A). The axonal endings
of rod bipolar cells establish synaptic contacts with AII amacrine
cells at the ON strata of the IPL. In rd/rd mice, bipolar cell axonal
endings are small in size, have atrophic varicosities and also show
synaptic ribbons with an anomalous round shape that resembles
the morphology of immature synapses (Strettoi et al., 2002).
Similar alterations have been reported in RP rats and other mice
models of RP (Barhoum et al., 2008; Cuenca et al., 2004; MartinezNavarrete et al., 2011). Signs of synapse number reduction between
AII amacrine cells and rod bipolar cell axons were also found in
monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (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 immunostaining of both ON and OFF cone bipolar cells showed the dendrites of these cells forming a continuous thin layer at the ONL
(Strettoi et al., 2002). In the same way, two types of recoverin
immunoreactive cone bipolar cells have been reported to lose their
dendrites in the OPL and change their axon morphology in the IPL
during retinal degeneration in P23H and RCS rats (Cuenca et al.,
2004, 2005b).
Remodeling of retinal cells along the degenerative process may
also affect horizontal cells (Figs. 7 and 8). It is well known that the
dendrites of the single horizontal cell type in rats, the B-type cell,
contact cone terminals, whereas the axon terminal makes contact
with rod spherules (Kolb et al., 2001; Linberg et al., 2001). In RCS rat
retinas, beyond a certain stage of retinal degeneration, the horizontal cells retract their dendrites, but the somas are not grossly
swollen or shrunken and appear with a normal density (Figs. 7H
and 8A) (Chu et al., 1993; Cuenca et al., 2005b). Similarly, in
mutant mice (rd/rd and rd/bcl2) displaying severe retinal abnormalities, horizontal cell processes are impaired, but the mosaic
distribution resists photoreceptor degeneration (Rossi et al., 2003).
During the degenerative process (Fig. 7CeD, F), horizontal cells also
seek out new contacts at the ONL level, with the sprouting of
dendrites and axon terminals (Fig. 7H) (Cuenca et al., 2005b).
Outgrowing horizontal cells and the formation of ectopic synapses
in the ONL have also been described in other RP animal models,
such as the CNGA3/CNGB1 double-knockout mouse (Michalakis
et al., 2013). Horizontal cells also extend processes down into the
IPL (Cuenca et al., 2005b; Jones et al., 2011; Park et al., 2001; Strettoi
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
and Pignatelli, 2000; Strettoi et al., 2003, 2002). All these results
indicate that bipolar and horizontal cells postsynaptic to photoreceptors have the ability to seek out new contacts during degeneration, but they retract their dendrites if they fail to establish correct
synapses.
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 images. The first level where this process takes place is the OPL. Any
changes in the organization of synaptic contacts at this level may
lead to severe loss of vision. In this layer, photoreceptors make
synaptic contacts with bipolar and horizontal cells, releasing
glutamate as neurotransmitter. Metabotropic glutamate receptor 6
(mGluR6) is located in the dendritic tips of rod and cone ONbipolar cells (Fig. 6A, D). Labeling for mGluR6 in mouse and rat
models of RP shows a loss of normal localization of mGluR6 receptors at the OPL (Fig. 6B, E) and clusters of the receptor in the
apical parts of bipolar cell bodies, while intense immunoreactivity
was also observed at the INL and in axon terminals (Fig. 6B, arrowheads) (Cuenca et al., 2004; Strettoi and Pignatelli, 2000). Visual deafferentation in retinal detachment also leads to an
alteration of the glutamatergic pathway (de Souza et al., 2012). It
appears that bipolar cell loss of presynaptic inputs from photoreceptors induces mislocalization of mGluR6 receptors. Since
proper dendritic localization of mGluR6 is essential for synaptic
transmission, this issue must be addressed during cell transplantation to permit the recovery of bipolar cells.
Bassoon is a presynaptic protein located at synaptic ribbon in
cone and rod axon terminals. In the OPL, a continuous distribution
of punctate staining marks the synaptic ribbons of rod spherules,
and when double stained, they can be seen paired with mGluR6
granules (Fig. 6D). Bassoon and synaptophysin, two presynaptic
proteins, are diminished in many retinal diseases (Figs. 5e7), such
as in the model of retinal detachment described in 2005 by Fisher
and coworkers (Fisher et al., 2005).
The literature dealing with OPL remodeling in human retinal
degenerative diseases is scarce, although some laboratories have
demonstrated bipolar cell sprouting and synaptic abnormalities in
AMD (Sullivan et al., 2007). Similar results have been found in
animal models of AMD (Marc et al., 2008). The same behavior of
bipolar cell dendrites can be observed in older animals: in normal
C57BL/6 mice during their second of life, retinal rod bipolar and
horizontal cells undergo sprouting and form ectopic synapses at the
ONL (Terzibasi et al., 2009). These studies suggest that maintaining
viable photoreceptors is crucial to the health and maintenance of
normal second-order neurons. Indeed, direct experimental evidence supporting the hypothesis that ectopic bipolar cell synaptogenesis requires functional presynaptic photoreceptors is
provided by the work of Haverkamp's group on the CNGA3/
mouse, characterized by the deactivation and loss of cones with
intact rod function. In these mice, cone bipolar cells switch to
establish contacts with the remaining rods, a phenomenon that
does not occur in double mutant mice (CNGA3/CNGB1), where both
cone and rod function is lacking (Michalakis et al., 2013).
All these studies appear to confirm that, after losing their
normal input, bipolar cells will seek out new functional photoreceptors to make contact with them. When rods are absent and most
photoreceptors appear to be cones, cone photoreceptors look for
connections with the neurons of the rod pathway (Fig. 8B) (Cuenca
et al., 2004; Peng et al., 2000; Strettoi et al., 2004). To date, it has
been difficult to determine whether rod bipolar cell dendrites look
for new contacts with a retracted rod axon terminal in the ONL or if
the progressive retraction of the rod axon is accompanied by
sprouting of rod bipolar dendrites without any apparent purpose.
27
2.1.4. Amacrine cell types and retinal degeneration
Additional remodeling of amacrine cells has been reported in
several diseases (Fig. 8A). AII amacrine cells are postsynaptic to rod
bipolar cells, and are important neurons that drive rod information
to the cone bipolar pathways. AII amacrine cells receive excitatory
inputs from ON-rod bipolar cells in S5 strata of the IPL and transfer
the rod signal to the cone pathway by means of conventional
chemical synapses with OFF-cone bipolar cells and gap junctionmediated electrical synapses with ON type cone bipolar cells
(Kolb, 2003; Kolb et al., 2002; Linberg et al., 2001). Since they
receive a major synaptic input from rod bipolar cells, it can be expected that AII amacrine cells show morphological changes during
retinal degeneration. These cells conserve their typical morphological features and appear well preserved at all the ages tested in
rd/rd mice (Strettoi et al., 2002). However, in rd10 mice and P23H
rats, AII amacrine cells lose their lobular appendages in the OFF
strata of the IPL as degeneration progresses (Barhoum et al., 2008;
Cuenca et al., 2004). These differences could be attributed to the
diversity among animal models or to the later occurrence of AII cell
changes in rd/rd mice. In a rat model of oxygen-induced retinopathy, AII amacrine cells also lose their typical lobular appendages,
which reveals significant morphological changes and decreased
contact with rod ON-bipolar cells. Clear changes in the dendritic
morphology of AII amacrine cells (the main neuronal subtype
postsynaptic to dopaminergic cells in the retina) have also been
reported in a monkey model of Parkinson disease treated with
MPTP, where dopaminergic cells are impaired (Cuenca et al.,
2005a). In animal models of DR, both dopaminergic and cholinergic amacrine cells are lost at early stages of retinal degeneration,
and this loss has been associated with visual deficits (Gastinger
et al., 2006). Findings in both diseases have been linked to patient abnormalities, such as the thinning of the optic nerve fiber
layer, ERG changes and an increase in the latency of the pupillary
light reflex (Dutsch et al., 2004; Inzelberg et al., 2004; Shinoda
et al., 2007). Early aberrant neurite sprouting in the glycinergic
and GABAergic amacrine cell populations have been found in a
porcine animal model of RP in the early stages of degeneration.
Finally, remodeling events in both glycinergic and GABAergic
amacrine cells in human geographic atrophy, with aberrant
sprouting in both cell signals, have also been described (Jones et al.,
2012).
2.1.5. From photoreceptor loss to ganglion cell death
The survival and maintenance of the normal dendritic
morphology of ganglion cells is essential for transmitting the correct information to the CNS. The structural and functional integrity
of RGCs is a prerequisite for any therapeutic strategy for human
retinal diseases.
Significant preservation of RGC structure was found in rd10 mice
retinas, with projections to higher visual centers still present in
older animals even after the death of all photoreceptors. Unlike the
second-order neurons (i.e., bipolar and horizontal cells), RGCs
appear to be a considerably stable cell population (Mazzoni et al.,
2008). This preservation potentially constitutes a favorable substrate for restoring vision in RP patients by means of electronic
prostheses or direct expression of photosensitive proteins through
optogenetics.
Like RCS and P23H rats (Garcia-Ayuso et al., 2013), rd mice
experience a focal loss of RGCs with reduced ganglion cell size and
compromised axonal transport (Grafstein et al., 1972), which could
also occur in tandem with vascular abnormalities (Wang et al.,
2000). The same discrepancies between mouse and rat models
have been found for melanopsin-expressing intrinsically photosensitive RGC loss. Studies show that rd10 (Mazzoni et al., 2008)
and rd/rd cl (rodless/coneless) (Semo et al., 2003) mice fail to
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N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
exhibit significant abnormalities in these photosensitive RGCs,
whereas a significant loss of these cells have been reported to occur
in both RCS and P23H rat models (Esquiva et al., 2013; Vugler et al.,
2008b). The differences between animal models need to be
clarified.
It has been described the presence of neurites in both epiretinal
and subretinal membranes in animal models of retinal detachment
and reattachment and in human membranes removed during vitrectomy (Lewis et al., 2007). Horizontal and ganglion neurites cells
were observed in epiretinal and subretinal membranes from the
feline retinas. However, in human retinas the majority of the neurites correspond to RGCs observed with high frequency in epiretinal
membranes and less common in subretinal membranes (Lewis
et al., 2007).
2.2. Alterations in retinal homeostasis
In retinal tissues, both neuronal and glial cells respond to all
forms of injury and disease, independently of the etiology of the
damage. Cellular responses to injury represent a cellular attempt to
protect the tissue from damage and/or to preserve tissue function,
even though an excessive or inappropriate cell response may
contribute to neurodegeneration. Protective and regenerative responses of retinal cells involve, among others, the stimulation of
the antioxidant machinery, the activation of the mechanisms of
programmed cell death, and the promotion of the inflammatory
response.
2.2.1. Oxidative stress and retinal degeneration
The imbalance between the generation and elimination of
reactive oxygen species (ROS) is defined as oxidative stress. Besides
its role in aging, oxidative stress has been associated with various
pathological conditions. Brain tissue is the most sensitive tissue to
oxidative stress injuries. The high oxygen-consumption rates in the
nervous tissue (around 20% of the oxygen intake), and the fact that
the brain has a high percentage of polyunsaturated fatty acids make
this organ vulnerable to oxidative damage (Shukla et al., 2011).
Thereby, ROS have been postulated as an important contributor to
the damage associated to neurodegenerative diseases, such as
Parkinson's, Alzheimer's and Huntington's diseases, as well as
amyotrophic lateral sclerosis (Dias et al., 2013; Shukla et al., 2011).
The retina is a highly specialized neural tissue, and one of the
tissues most susceptible to ROS damage. Photoreceptor cells are
continuously exposed to varying degrees of light photons and are
one of the highest consumers of oxygen in the CNS, mainly due to a
large 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 and
lipid peroxidation due to the large surface area of membranes
enriched with polyunsaturated fats (Panfoli et al., 2012; Winkler
et al., 1999). The high metabolic rate of photoreceptors, together
with recent evidence for the presence of aerobic metabolism in the
membranous 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 in
retinal degeneration. For example, it has been shown that oxidative
stress in the RPE is the output that triggers the development of
AMD (Ardeljan and Chan, 2013). AMD is an age-related degenerative disease affecting choroid, RPE and photoreceptor cells (Kevany
and Palczewski, 2010). Environmental or genetic features that
might increase the oxidative stress in RPE cells can potentially
provoke AMD. Smoking, for example, is a habit known to cause
oxidative stress (Carnevali et al., 2003) and appears to be one of the
most 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 fractions
isolated from AMD patients, thus indicating their relevance in AMD
pathology (Kenney et al., 2013b; Udar et al., 2009). The capability of
these mitochondria to synthesize ATP, ROS and lactate may affect
the balance between aerobic and anaerobic mitochondrial metabolisms (Kenney et al., 2013a). In this context, we have observed a
reduction in the content of the 5A subunit of cytochrome c oxidase
and b subunit of ATP synthase in the retina of parkinsonian monkeys, two enzymes constituents of mitochondrial complexes IV and
V, respectively, that are correlated with a reduced respiratory capacity of mitochondria (Campello et al., 2013a). Although the increase in ROS has been shown to trigger AMD, a decrease in the
activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)
pathway has been identified as a factor increasing vulnerability to
oxidative damage in aging RPE cells (Sachdeva et al., 2014).
ROS may also be important in the pathogenesis of RP and
glaucoma. The photoreceptor cells in RP and the ganglion cells in
glaucoma are highly sensitive to oxidative stress during the early
stages of cell degeneration. In both cells types, the apoptotic
stimuli, which trigger their apoptotic death, are exacerbated by
oxidative 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 glucose
metabolism. The high concentration of circulating glucose drives
mitochondria to increase their activity (Du et al., 2003; Kowluru
et al., 2001), which results in an overproduction of superoxide
from mitochondrial complexes I and III (Muller et al., 2004).
Mitochondria are not the only source of ROS under hyperglycemic
conditions. The excess of glucose also activates the polyol
pathway, in which aldose reductase metabolizes glucose into
sorbitol, thus increasing oxidative stress by depletion of NADPH
(Ola et al., 2012). In addition, hyperglycemia increases the formation of advanced glycation end products (AGEs), which upon
binding to their receptor trigger ROS generation (Santos et al.,
2011). The oxidative stress in DR is not only due to the excessive
production of ROS; it is also mediated by impairment of Nrf2
signaling (Xu et al., 2014; Zhong et al., 2013). Diabetes increases
the binding of Nrf2 to the cytosolic Kelch-like ECH-associated
protein 1 (Keap 1), preventing Nrf2 translocation to the nucleus,
where it regulates the expression of antioxidant genes. This was
shown to occur in streptozotocin-treated rats, in isolated retinal
endothelial cells exposed to high levels of glucose, and in retinas
from human donors with DR (Zhong et al., 2013). Nrf2 knockout
mice have decreased expression of antioxidant enzymes and are
more susceptible to streptozotocin diabetic treatment (Xu et al.,
2014). Moreover, the expression of antioxidant enzymes such as
Mn-containing superoxide dismutase, glutathione peroxidase and
catalase are decreased in diabetic patients with retinopathy, as
compared to diabetic patients without retinopathy or non-diabetic
subjects (El-Bab et al., 2013).
2.2.2. Activation of apoptotic pathways: role of the mitochondria
Most defective, unwanted and potentially dangerous cells die by
apoptosis, an exquisitely controlled genetic program for removing
such cells without damaging the surrounding tissue (for a review,
see (Murakami et al., 2013)). The life-or-death decision seems to be
the result of a complex balance between pro- and anti-apoptotic
signals (Fig. 9) at several levels: extracellular, mitochondrial, nuclear and cytoplasmic (Kuan et al., 2000; Strasser et al., 2000). There
are two modes of apoptosis, which have been shown to be mediated by caspase-dependent and -independent pathways (Doonan
et al., 2005; Kroemer and Martin, 2005). Nonapoptotic forms of
programmed cell death (PCD) include those with features of
autophagy, and they can be activated simultaneously to apoptosis
during a neurodegenerative disease (Boya and Kroemer, 2008).
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
2.2.2.1. Caspase-dependent apoptosis. All pathways of apoptosis
converge upon the activation of cysteineeaspartic acid proteases
called caspases. These proteins have been functionally classified
into two groups, initiator caspases (caspases 2, 8, 9 and 10) and
executor caspases (caspases 3, 6 and 7) (Pop and Salvesen, 2009).
Caspases are present in the cell in their inactive form, and are
activated in the presence of apoptotic stimuli. Two main pathways
leading to caspase activation have been characterized: the extrinsic
route initiated by cell surface receptors, and the intrinsic path that
is regulated by mitochondria (Fig. 9).
In the extrinsic caspase-dependent pathway, activation of
membrane “death receptors” (usually by cytokines from the tumor
necrosis factor (TNF) family) drives the apoptotic cascade by activation of caspases 8 and/or 10, which then activates downstream
effector caspases, such as caspase 3, 6, and 7 (Tait and Green, 2010).
Additionally, external activation of caspase 8 leads to the generation of further internal signals that translocate to the mitochondrial
membrane (Fig. 9) to trigger the cell death process (Doonan et al.,
2007).
In contrast, an intrinsic pathway for apoptosis may be activated
by various cellular stress stimuli. Along this pathway, the mitochondria appear to be the primary target. Mitochondrial outer
29
membrane permeabilization (see below) leads to the release of
cytochrome c, which binds apoptotic protease-activating factor-1
(Apaf-1), thus inducing its conformational change and oligomerization (Fig. 9). This complex cytochrome c-Apaf-1, referred to as
“apoptosome”, recruits, dimerizes and activates the initiator caspase 9, which cleaves and activates caspases 3 and 7 (Tait and
Green, 2010).
Most of the apoptotic pathways converge at the permeabilization of the mitochondrial outer membrane, a step known as the
point of no return for cell death (Keeble and Gilmore, 2007).
Mitochondria play an important role in apoptosis, due to their
content rich in pro-apoptotic proteins (Fig. 9). These proteins serve
a dual function; on the one hand, they play a part on the electron
transport chain. This is the case of cytochrome c, which transports
electrons to complex IV (reviewed in Garrido et al. (2006)), or the
apoptosis-inducing factor (AIF), which stabilizes and eliminates
ROS production from complex I of the electron chain (reviewed in
Polster (2013)). However, their release into the cytoplasmic space
has fatal consequences, as they activate different proteases, which
eventually results in apoptosome formation, or translocate to the
nucleus, directly cleaving the DNA. In this sense, preserving the
integrity of the mitochondrial membrane by preventing the
Fig. 9. Apoptotic pathways in the retina. Schematic representation of the most important pathways involved in programmed cell death (PCD) in the retina. Most retinal cells die as
the result of extrinsic and/or intrinsic caspase-dependent pathways, although non-apoptotic forms of regulated cell death are also present. Caspase-independent apoptotic
pathways involve calpains and/or cathepsins. Among the major causes of stress and cell death in the retina are the accumulation of reactive oxygen species (ROS) associated with
pathological conditions and damage to both mitochondria and lysosomes. MOMP: Mitochondrial outer membrane permeabilization; ER: endoplasmic reticulum; DL: Death ligand;
Apaf-1: apoptotic protease-activating factor-1; EndoG: endonuclease G; AIF: apoptosis-inducing factor; ROS: reactive oxygen species; PCD: programmed cell death; Bcl-2: B-cell
lymphoma 2; Bcl-xL: B-cell lymphoma-extra large; BAX: Bcl-2-associated X protein; BAK: Bcl-2 antagonist or killer; Bid: BCL-2 interacting domain death agonist; JNK: c-Jun Nterminal kinases.
30
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
formation of mitochondrial outer membrane pores (MOMP) is one
of the anti-apoptotic mechanisms that protect cells from death
(Garrido et al., 2006).
The Bcl-2 (B-cell lymphoma 2) family is the best characterized
protein family involved in the regulation of MOMP (Fig. 9). The clan
includes four other anti-apoptotic proteins (Bcl-xL, Bcl-w, A1 and
Mcl1), and two groups of proteins that promote cell death: the
effector molecules BAX (Bcl-2-associated X protein) and BAK (Bcl-2
antagonist or killer), which permeabilize the outer mitochondrial
membrane; and the BH3-only family, which functions indistinctly
along the cellular stress pathways. BAX and BAK promote MOMP,
while Bcl-2 and Bcl-XL expression within the outer mitochondrial
membrane protects against MOMP formation (Keeble and Gilmore,
2007). BH3-only proteins, such as Bim (Bcl-2 interacting mediator
of cell death), Bid (BCL-2 interacting domain death agonist) and
Puma (p53-upregulated modulator of apoptosis), are unable to
trigger apoptosis by themselves, but it is thought that they act as
switch 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 other
types of programmed cell death PCD; for example, Bid has been
described as one of the main connections between intrinsic and
extrinsic apoptotic pathways (Kroemer and Martin, 2005) and can
increase BAX/BAK-induced MOMP formation.
Caspase-dependent pathways are the main mechanisms
involved in apoptosis in cases of retinal cell degeneration. In
glaucoma, it has been shown that ganglion cell death occurs primarily through the apoptotic intrinsic pathways, and is dependent
on the release of cytochrome c from mitochondria and the formation of the apoptosome complex (Nickells, 2012), although caspase
6 and 8 activity has also been described following the severing of
the optic nerve (Monnier et al., 2011). Degeneration of the ganglion
cell soma in DBA/2J and optic nerve crush models is mainly
mediated by BAX, whereas BAX does not seem to be involved in the
case of N-methyl-D-aspartate (NMDA)-induced toxicity (Libby et al.,
2005). In addition, it has been shown that glial cells become activated and release cytokines after an initial phase of ganglion cell
death. TNF-a activates secondary degenerative events mediated by
the extrinsic pathway in ganglion cells (Lebrun-Julien et al., 2009;
Nickells, 2012; Tezel et al., 2001). Both phases of ganglion cell
degeneration finally result in caspase 3 activation and show downregulation of the anti-apoptotic proteins Bcl-2 and Bcl-XL and upregulation of BAX and BAD (Levkovitch-Verbin et al., 2010).
The Bcl-2 family plays an important role in the progression of
apoptosis in photoreceptor cells. In experimental models of retinal
degeneration, it has been shown that photoreceptor death is mainly
mediated by changes in the balance between BAX and Bcl-XL (Jones
et al., 2003; Zheng et al., 2006). The Rpe65-deficient mouse, an
experimental model of Leber's congenital amaurosis, shows upregulated BAX and decreased Bcl-2 proteins, with decreased Bcl2/BAX ratio during the progression of the disease (Cottet and
Schorderet, 2008; Hamann et al., 2009). In RP, the high diversity
of gene mutations leads to the activation of a variety of apoptotic
pathways (Doonan et al., 2005; Sancho-Pelluz et al., 2008). In most
cases of RP, as well as in other retinal dystrophies, cell death occurs
after endoplasmic reticulum stress (Lin and Lavail, 2010). In this
sense, cells have evolved a set of intracellular signaling pathways,
cumulatively called unfolded protein response (UPR), that detect
protein misfolding within the endoplasmic reticulum (ER) and
direct protective and pro-apoptotic responses. It has been
demonstrated that Puma, a BH3-only member of the Bcl-2 family, is
transcriptionally activated and is essential for ER-stress-induced
neuronal death (Galehdar et al., 2010). In mouse models of RP, ER
stress triggers an increase in Ca2þ levels, and up-regulation of
caspase 12 (Yang et al., 2007), which in turn, activates caspase 3.
Furthermore, a decrease in the Bcl-xL/BAX ratio has been evidenced
in these animal models, thus indicating the implication of mitochondria in the process of apoptosis (Kunte et al., 2012; Sizova et al.,
2014). Caspase 12 has a leading role in ER-stress-induced neuronal
death, but accumulation of misfolded proteins and increased
cytosolic Ca2þ involves the activation of additional apoptotic factors
that reinforce each other during the apoptotic process, confirming
that mitochondria and ER can influence each other in the apoptotic
event (Sanges and Marigo, 2006).
Mutations or insults affecting the RPE or its phagocytic function
lead to photoreceptor cell death. In RCS rats, disabled photoreceptor outer segment phagocytosis drives the apoptotic photoreceptor cell death (Tso et al., 1994). Increased expression of c-Jun and
BAX proteins has been reported during the course of this process
(Katai et al., 2006), which points to mitochondrial involvement and
the activation of caspases (Perche et al., 2008), although it seems
that the apoptotic process is not mediated by Bcl-2 (Katai et al.,
2006; Sharma, 2001).
In the case of retinal detachment, photoreceptor degeneration
seems to be a process mediated by TNF-a (Nakazawa et al., 2011)
through the activation of apoptotic FAS (a death domain-containing
member of the TNF receptor) signaling and the downstream
cascade 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 during
this process, with the consequent involvement of the intrinsic
caspase-dependent apoptotic pathways (Zacks et al., 2004).
There is less evidence regarding death mechanisms in AMD, but
it has been suggested that photoreceptor death is also caused by
apoptosis (Osborne and Wood, 2006; Wang et al., 2011c). The same
death mechanisms have been proposed for DR (Cho et al., 2000;
Park et al., 2003).
2.2.2.2. Caspase-independent apoptosis. There is currently evidence
that caspase activation is not the only protease mechanism
involved 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 of
caspase-dependent processes does not prevent neuronal cell death,
other proteases must be involved in carrying out the apoptotic
program (Nguyen et al., 2012). Furthermore, in addition to
apoptosis, 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, ER
stress and the subsequent Ca2þ release can activate calciumdependent cysteine proteases known as calpains (Fig. 9) (Nguyen
et al., 2012; Suzuki et al., 2004). Calpains are present in the cytosolic portion of the cell, and caspase 12 (Tan et al., 2006) and other
pro-apoptotic proteins (Nguyen et al., 2012) may amplify the death
signal. Activation of calpains have been related to various retinal
diseases, and is considered one of the most important caspaseindependent apoptotic pathways in photoreceptor cell death
associated with RP (Doonan et al., 2005; Ozaki et al., 2012; PaquetDurand et al., 2006; Sanvicens et al., 2004), and diseases involving
ischemic conditions, such as DR (Nakajima et al., 2011) and glaucoma (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 proapoptotic proteins with a clear role in apoptotic events (Fig. 9)
(Boya and Kroemer, 2008; Garrido et al., 2006). MOMP can drive
apoptosis even when caspases are inhibited (Kroemer and Martin,
2005). The main factors involved in these processes are AIF and
endonuclease G (EndoG). Both are present within the mitochondrial intermembrane space under normal conditions, but with
apoptotic stimuli and MOMP, AIF and EndoG are able to translocate
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
into the cell nucleus and to fragment nuclear DNA (Li et al., 2001;
Polster, 2013). In this sense, nuclear translocation of AIF and
EndoG in various retinal degenerations has already been shown to
occur (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 are
receiving increasing attention among the mechanisms involved in
retinal degeneration (Chinskey et al., 2014; Kunchithapautham and
Rohrer, 2007; Murakami et al., 2013). Complete disruption of lysosomes provokes uncontrolled cell death by necrosis, but partial
and selective lysosomal membrane permeabilization induces the
controlled dismantling of the cell by apoptosis. The proteins mainly
responsible for autophagy are the cathepsins, which are also the
main proteases in lysosomes (Boya and Kroemer, 2008; Uchiyama,
2001). ROS accumulation may also induce lysosomal membrane
permeabilization and the release of cathepsins (Boya and Kroemer,
2008; Metrailler et al., 2012; Sanvicens and Cotter, 2006). In addition, ROS may induce the permeabilization of lysosomes only in
subcellular regions near mitochondria, the major ROS-generating
organelles. Cathepsin activation has been described in several
retinal diseases (Chahory et al., 2010; Sancho-Pelluz et al., 2008).
Although necrosis was traditionally thought to be an uncontrolled process of cell death, it is now known to also have regulated
components in certain instances (Murakami et al., 2013). This
regulated type of necrosis, termed as “necroptosis” or “programmed necrosis”, has been demonstrated in several models of
retinal disease, including retinal detachment, retinal ischemiareperfusion injury and achromatopsia (Dong and Sun, 2011;
Rosenbaum et al., 2010; Viringipurampeer et al., 2014).
2.2.3. Retinal protein homeostasis
The retina is a highly specialized and well-structured neural
tissue. For this reason, maintaining homeostasis in all the different
retinal cell types is necessary for proper vision. Furthermore, the
retina has to withstand a variety of environmental insults, such as
light-induced injury, and the stress derived from oxidative damage
and inherited mutations. All of the previous factors can lead to
protein misfolding cytotoxicity, among other pathologies, and cells
have developed several mechanisms to cope with this. These
mechanisms are responsible for maintaining protein homeostasis,
and include the heat shock response (HSR), the ubiquitinproteasome system (UPS), the unfolded protein response (UPR)
and the ER-associated degradation (ERAD) (Fig. 10) (Athanasiou
et al., 2013). Cellular chaperones play an important role in detecting misfolded proteins for trying to refold them, but under certain
circumstances, correct refolding is not possible, and the cell must
remove the proteins to avoid protein aggregation or cellular
toxicity. The decreased levels of molecular chaperones are related
to several neurodegenerative diseases affecting the retina, as occurs
with the molecular chaperones HSC70 and GRP78 in the retina of
parkinsonian MPTP-treated monkeys (Campello et al., 2013a). In
other cases, misfolded proteins are removed from cells without
chaperone supervision. The degradation of proteins, which includes not only misfolded proteins, but also oxidized or denatured
proteins, can be carried out by several proteolytic systems,
including lysosomal degradation, chaperone-mediated autophagy
and substrate specific degradation by the UPS (Fig. 10). The latter is
the principal molecular machinery of the cell responsible for protein turnover, and it plays a pivotal role in cellular homeostasis. In
this regard, the UPS exerts a cellular protein quality control and, as a
consequence, it is involved in a wide variety of biological processes
where modulation of protein levels is crucial. Proteins are
commonly tagged with a tail of covalently-joined ubiquitin molecules in a reaction catalyzed by ubiquitin ligases, and are eventually
degraded by the proteasome. Moreover, the ubiquitination of a
31
protein, in addition to targeting it for proteasomal degradation, can
also affect its stability, activity, ability to interact with other molecules within the cell and/or its intracellular distribution. Examples
of all these effects are known to exist for retinal proteins. Accordingly, the UPS allows the cell to modulate its protein expression and
distribution pattern in response to different physiological conditions, and thus it plays a fundamental protective role in retinal
health and disease (Campello et al., 2013b; Shang and Taylor, 2012).
The functions of the UPS in the retina include roles in differentiation and retinal development, modulation of the visual cycle, responses 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 proteasome and the multitude of processes involved, dysfunction of
the UPS in the retina is involved in the pathogenesis of many
inherited and acquired visual pathologies. In this context, mutations in genes encoding retinal UPS components may directly
trigger a general pathological accumulation of a vast majority of
noxious proteins. On the other hand, in retinal diseases caused by
the expression of mutant proteins that are not directly related to
the UPS, intracellular accumulation of aberrant mutant protein
variants lacking an adequate structural conformation may overload the UPS, indirectly contributing to the disease process. In
autosomal dominant RP patients, for example, mutations have
been described in the genes coding for the TOPORS E3 and CUL3based (KLHL7) E3 ubiquitin ligases. It has also been demonstrated
that the P23H mutation of the RHO gene found in autosomal
dominant RP patients generates a misfolded variant of rhodopsin
that is not efficiently degraded by the UPS. As a consequence, the
UPS machinery becomes overwhelmed and aggresomes of
mutated and recruited normal protein are formed. The UPS also
modulates pathways associated with oxidative stress and inflammation, two pathogenic events closely related to AMD. Upon aging, oxidative stress leads to the malfunction of the proteasome
machinery, and the resulting accumulation of highly-ubiquitinated
proteins activates the heat shock factor 1 (HSF1), a transcription
factor associated with the heat shock protein response. The
oxidative inactivation of the proteasome also serves as a link between oxidative stress and the upregulation of inflammation in
RPE cells. This inflammation downregulates rhodopsin expression
levels via UPS-mediated degradation promoted by the STAT3dependent E3 ubiquitin ligase UBR1. In addition, in RPE cells, the
UPS plays an important role in modulating the activities of the
hypoxia inducible factor (HIF) and the nuclear factor kB (NFekB),
an important transcription factor that mediates hypoxic and inflammatory responses. The UPS is also involved in DR, a condition
in which angiotensin II decreases in an UPS-mediated fashion the
levels of synaptophysin, a protein essential for vision, as it is a
main constituent of synaptic vesicles in the two plexiform layers
of the retina. Furthermore, hyperglycemia-induced oxidative
stress decreases the glucose transport activity of retinal endothelial cells, due to increased internalization of the glucose
transporter 1 (GLUT1) in a proteasome-dependent mechanism.
The UPS also modulates the HIF-mediated signaling cascade that
regulates the expression of angiogenic growth factors, and is
involved in the breakdown of the blood-retinal barrier characteristic of DR through the turnover regulation of endothelial connexins. Finally, the proteasome machinery also contributes to the
progression of glaucoma, a condition in which prolonged ischemic
retinal injury promotes the ubiquitination of a set of antiapoptotic proteins, the degradation by the proteasome of which
triggers cell death.
The contents of this section have been analyzed in detail in the
following three reviews (Athanasiou et al., 2013; Campello et al.,
2013b; Shang and Taylor, 2012).
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Fig. 10. Retinal protein homeostasis networks. Schematic showing the proteostasis mechanisms in retinal cells related to misfolded proteins as the result of mutations, environmental insults and/or several types of stress. ERAD: Misfolded proteins are detected by the endoplasmic reticulum quality control machinery and shuttled by the retrotranslocon channel to the cytoplasm, where they are ubiquitinated before being degraded by the proteasome. UPR: Misfolded proteins in the endoplasmic reticulum are recognized by three sensors: IRE1a, PERK and ATF6, which inhibit protein synthesis and stimulate the production of chaperones and the ERAD machinery. HSR: Molecular chaperones
Hsp70, 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, which
can trimerize and become activated via phosphorylation. This results in traffic to the nucleus leading to increased chaperone expression. Autophagy: Misfolded proteins can be
degraded by three modes of autophagy: macroautophagy, microautophagy or chaperone-mediated autophagy (CMA).
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
2.3. Glial responses
2.3.1. Inflammatory response: microglial activation in retinal
dystrophies
As part of the CNS, the human retina has a population of resident
phagocytes called microglia (Fig. 11) that have an immunological
capacity comparable to that of the monocytes and macrophages in
other tissues (Graeber and Streit, 1990). The main function of
microglial cells in the retina is that of immune surveillance. They
take part in immune-mediated defense mechanisms (Fig. 14),
acting as phagocytes, clearing damaged cell debris from the inner
retinal layers and forming a network of potential immune effector
cells throughout the CNS, along with the perivascular cells (Hanisch
and Kettenmann, 2007; Kreutzberg, 1996; Raivich et al., 1999; Shin
et al., 2000). However, the role of microglia in the retinal pathophysiology goes far beyond their relevant function in infectious
injuries per se. Although many aspects have yet to be understood,
nowadays it is generally accepted that microglia are important for
maintaining photoreceptor survival in retinal dystrophies.
2.3.1.1. Microglia in healthy retinas. In a healthy CNS, microglial
cells are in an apparent state of rest, but they continuously scan
their environment, moving extensively and monitoring the surrounding area to clear away metabolic products and tissue debris.
This non-activated state is characterized by a highly arborescent
morphology, plasticity and a pluristratified distribution in the INL
and OPL (Fig. 11A). In this state, microglial cells express low levels of
co-stimulatory molecules and demonstrate relatively low levels of
phagocytic activity (Dick et al., 2003; Hume et al., 1983; Langmann,
2007; Streit et al., 1999). Microglial cells make specific and direct
contact with neuronal synapses and respond to their functional
status (Nimmerjahn et al., 2005; Wake et al., 2009). The apparent
quiescent state of the microglia in an uninjured retina is maintained by intercellular contacts and soluble factors secreted by
neurons, 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 cytokines, antioxidants and growth factors, including brain-derived
neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF),
glial cell line-derived neurotrophic factor (GDNF), nerve growth
factor (NGF), neurotrophin-3 (NT3) and basic fibroblast growth
factor (bFGF) (Langmann, 2007). Given the fact that photoreceptors
33
lack the receptors for some of these neurotrophic factors, the
beneficial action stems from the modulation of growth factors
produced and released by Müller cells, which contain receptors for
most of the molecules involved in photoreceptor rescue and which
are stimulated after retinal insults, such as mechanical injury and
light-induced degeneration. In this sense, NGF, BDNF and CNTF
modulate bFGF and GDNF production and release from Müller glia,
and also contribute to the protection of photoreceptors or increase
photoreceptor apoptosis (reviewed in Harada et al. (2002); Harry
(2013); Karlstetter et al. (2010a)).
Apart from the capacity to secrete anti-inflammatory molecules
when removing apoptotic cells or myelin debris, microglia can also
secrete inflammatory mediators in the event they are challenged by
a microorganism invasion (Hanisch and Kettenmann, 2007). The
effect of these inflammatory mediators in retinal pathophysiology
is related to retinal dystrophies and is discussed in the following
sections.
2.3.1.2. Regulation of microglial homeostasis. Microglia communicate with other glial cells and neurons, which regulate its activation
status and their capacity for clearing away cellular debris by
phagocytosis (Dick et al., 2003). In a healthy CNS, a bidirectional
microglia-neuron communication takes place: microglial activity is
modulated by neuronal signals and, reciprocally, microglia signals
are also sent to neurons. Microglia perceive their environment
through a great variety of surface receptors, as they express receptors for cytokines, chemokines, neurotransmitters, neurohormones and neuromodulators, as well as several ion channels
(Harry, 2013). Signaling mechanisms include direct physical contact
between microglial processes and neuronal elements, as well as
microglial regulation of neuronal synapses and circuits by several
soluble factors (Eyo and Wu, 2013; Garden and Moller, 2006;
Langmann, 2007; Polazzi and Monti, 2010; Ransohoff and
Cardona, 2010).
Under healthy conditions, retinal and brain microglia are
controlled by several inhibitory molecules, such as chemokine
CX3CL1, lectin CD22 and other membrane proteins, including
CD200, CD47 and neural cell adhesion molecules, that restrain
microglial 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 microglial
homeostasis is the chemokine fractalkine. It is secreted by healthy
Fig. 11. Microglial activation in retinal diseases. Vertical sections of a Sprague Dawley (SD) (A) and a P23H (B) rat retina labeled with antibodies against Iba-1 (green), a marker of
microglia, and MHC class-II RT1B (red), used to detect activated microglia. Nuclei stained with TO-PRO. Note the increase in the number of microglial cells and amoeboid shape
activated microglia in the P23H rat retina (B). RPE: Retinal pigment epithelium; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; GCL: ganglion cell
layer.Scale bar: 20 mm.
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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 P23H
rats (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 P23H
rat 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 the
presence of inflammation, it promotes the activation of both
microglia and astrocytes, thus making the microglia both neuroprotective 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 resting
state through the secretion of cytokines, such as transforming
growth factor-b (TGF-b), that predispose microglia to the preferential production of interleukin (IL)-10. This in turn down-regulates
the expression of molecules such as major histocompatibility
complex class II (MHCII), CD80 or CD86 and blocks inflammatory
gene expression, contributing to the normal retinal immune
regulation (Langmann, 2007; Paglinawan et al., 2003).
2.3.1.3. Retinal microglia after an injurious stimulus. Microglial cells
are rapidly alerted by a variety of injurious signal inputs, triggered
by either genetic or environmental factors, as the result of external
damage from ocular infections or due to cellular malfunction in the
neural retina or RPE. Furthermore, retinal microglia can be also
activated by systemic infections, either of viral (Zinkernagel et al.,
2013) or fungal origin (Maneu et al., 2014). The lack of cellecell
communication or other stimuli, such as the presence of high levels
of ATP, oxidized DNA, proteins, lipids, AGEs, damaged extracellular
matrix molecules, antibodies, complements, cytokines, nucleotides
or ions, may activate the microglia (Hanisch and Kettenmann,
2007; Xu et al., 2009).
The activation signal is mediated by Toll-like receptors (TLR). TLR
are 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 a
family of pattern recognition receptors that participate in the
recognition of microbial patterns and in innate and adaptive responses (Kawai and Akira, 2006). These pattern recognition receptors
are capable of promoting the production of pro-inflammatory cytokines, chemokines and molecules such as ROS, which are essential for
pathogen elimination in peripheral cells, and in astrocytes and
microglia. Apart from microorganism-associated molecular patterns,
many endogenous molecules from mammals are also ligands for TLR,
such as heat shock proteins or products derived from the hydrolysis
of the extracellular matrix (Langmann, 2007).
Upon activation, microglial cells undergo a morphological
change from a ramified to an amoeboid shape (Fig. 11B), with a
graded response according to the degree of activation (Hanisch and
Kettenmann, 2007; Raivich et al., 1999). In the activated state,
microglial cells proliferate, migrate to the site of the stimulus, and
display greater phagocytic capacity (Fig. 14). Blood precursors are
also 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 blood
macrophages can differentiate into a multitude of phenotypes,
depending on the surrounding micro-environmental signals, which
can change over time (Harry, 2013; Kigerl et al., 2009; Michelucci
et al., 2009; Perego et al., 2011).
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
35
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) stained
with antibodies against GFAP (red), an intermediate filament protein expressed in astrocytes. Blood vessels have been labeled with Griffonia simplicifolia lectin (green). (C, D) Highmagnification 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 hypertrophic 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 by
either soluble factors, which generate concentration gradients that
promote and direct microglia migration, or by changes in the
extracellular matrix of injured or diseased CNS tissues (Garden and
Moller, 2006). Many of the migratory factors are chemokines
(Cartier et al., 2005). Several growth factors also influence microglial migration. Some authors have shown that the angiogenic
peptide vascular endothelial growth factor (VEGF) acts as a trophic
factor, inducing the migration of microglial cells (Forstreuter et al.,
2002). Furthermore, NGF has been shown to induce microglial
migration, and this cell migration is modulated by TGF-b, which
also has a chemotactic activity (Ambrosini and Aloisi, 2004; De
Simone et al., 2007; Paglinawan et al., 2003). Migration can also
be stimulated by nucleotides, such as extracellular ATP and ADP,
which are released in response to ischemic and traumatic CNS injuries and interact with several purinoceptors (Honda et al., 2001;
Inoue et al., 2007; Luongo et al., 2014). Moreover, the microglial
response depends on initial cytokine stimulation (Carter and Dick,
2003).
Following activation, microglia are capable of entering the cell
cycle and proliferating. Among the cytokines that stimulate
microglia 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 such
as BDNF and NT-3 are released by activated microglia and act in a
paracrine 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 several
surface 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 peripheral macrophages and CNS microglia, retinal microglia also seem to
display the M1/M2 stage distinction, which could better explain
their heterogeneous functions. Hence, microglial cells can be
induced to express the pro-inflammatory M1 or the M2deactivated (also known as M2-alternatively activated or antiinflammatory) phenotypes (Ardeljan and Chan, 2013; Kigerl et al.,
2009; Lucin and Wyss-Coray, 2009; Michelucci et al., 2009; Perego et al., 2011; Polazzi and Monti, 2010; Xu et al., 2009).
Another consequence of microglial activation is the increased
phagocytic capacity of microglia (Fig. 14), which can now phagocytize not only microbes, but also pathological proteins, such as
beta-amyloid. Among the microglia receptors involved in engulfment during developmental apoptotic processes and under
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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 the
normal 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 by
secretion 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 DAP12associated receptors and CD36 (Garden and Moller, 2006; Harry,
2013; Langmann, 2007). After phagocytosis of microorganisms,
microglial cells have the capacity to present the engulfed proteins
as antigens to T cells, stimulating an adaptive immune response.
Thus, microglia appear to play an important role as antigenpresenting cells, even if they may require some additional signals
from circulating dendritic cells to generate a complete response. In
this sense, by both invading from peripheral blood and differentiating from the available pool of resting cells, microglia may act as
antigen-presenting cells to perform the neuroprotective function of
the 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 not
necessarily result in a shift to an amoeboid shape, and microglia can
return to an inactivated status following the disappearance of the
stimulus upon reception of a down-regulating signal (Harry, 2013).
The inflammatory response elicited after an injurious stimulus is
regulated by several diffusible factors originating from the microglia. These include cytokines, chemokines, trophic factors and small
molecule mediators of inflammation, such as prostaglandins
(Garden and Moller, 2006). If the harmful stimulus is prolonged or
severe, the secretion of pro-inflammatory cytokines, ROS, nitric
oxide (NO), TNF-a, glutamate or caspases is capable of inducing
photoreceptor death (Harada et al., 2002; Roque et al., 1999). Under
these conditions, microglial cells have been associated with
neurodegenerative diseases, either as an initiating factor or as an
aid in their development (Karlstetter et al., 2010a; Langmann,
2007).
2.3.1.4. Microglia in degenerating diseases. Although the exact
trigger stimulus involved in degenerative diseases remains unknown, it is recognized that microglial activation, as well as
expression of chemokines and microglia-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; Zheng
et al., 2005). Activated microglial cells have been shown to induce
photoreceptor death, at least in in vitro experiments (Harada et al.,
2002; Roque et al., 1999). In the first stages of retinal neurodegeneration, microglia trigger repair mechanisms, such as glial
scar formation (Muzio et al., 2007). But excessive or prolonged
microglial activation in the CNS and the retina may lead to chronic
inflammation, with severe pathological side effects that can result
in irreversible neuronal death (Fig. 14) (Hanisch and Kettenmann,
2007; Langmann, 2007; Schuetz and Thanos, 2004). There is also
the possibility that an alteration in neurons, glia or both may be
eventually amplified by a microglial response, ultimately affecting
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
the survival of neurons. On the other hand, it must be taken into
account that microglial dysfunction, with the loss of their protective functions (the secretion of trophic factors, antioxidants and
cytokines and the removal of cellular debris), could by itself lead to
neuronal cell death (reviewed in Polazzi and Monti (2010)).
Activation of the microglia has been demonstrated in association with several neurodegenerative diseases, such as Alzheimer's
and Parkinson's diseases, amyotrophic lateral sclerosis, and multiple sclerosis, although it remains unclear whether microglial activation is a cause or a consequence of neuronal damage (Muzio et al.,
2007; Polazzi and Monti, 2010). In Alzheimer's disease, activated
microglial cells have been associated with amyloid plaques in the
brain. These amyloid plaques have been found also in AMD drusen.
It is plausible that, in the early stages of the disease, microglial
activation could help remove amyloid plaques, while in later phases, pro-inflammatory cytokines induced by microglia could
contribute to the neurodegenerative process (Bornemann et al.,
2001; Griffin et al., 2006). In Parkinson's disease, an increased
number of activated microglia are found in the brain, accompanied
by increased expression of pro-inflammatory cytokines (Hirsch and
Hunot, 2009; Tansey et al., 2007).
In the same way, retinal neurodegenerative diseases are also
associated with chronic microglial activation and neuroinflammation. In the degenerating retina, endogenous signals
activate microglial cells, leading to their local proliferation, migration, enhanced phagocytosis and secretion of cytokines, chemokines, and neurotoxins. These immunological responses and the
loss of limiting control mechanisms may contribute significantly to
retinal tissue damage and pro-apoptotic events in retinal dystrophies (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 and
Horner, 2007; Johnson and Morrison, 2009; Luo et al., 2010).
Microglia in glaucomatous ocular tissues show an altered
morphology and upregulation of microglia-derived inflammatory
proteins, with increased secretion of inflammatory proteins, such
as TNF-a (Johnson and Morrison, 2009). Different studies have
suggested that inhibition of reactive microglia through physical
techniques, such as irradiation, or by anti-inflammatory drugs, such
as minocycline, may be a promising potential approach in glaucoma therapies, increasing the survival rate of functional RGC
(Bosco et al., 2012; Seitz et al., 2013). In this sense, the inhibition of
the signaling cascades initiated by reactive microglia (such as NO
synthase or TNF-a) is also considered as a potential therapy for the
treatment of glaucoma (Neufeld, 2004; Roh et al., 2012; Seitz et al.,
2013). Other researchers have shown that in human glaucoma, the
immunostimulatory signaling can also be initiated through glial
TLRs (Luo et al., 2010), what may involve another therapeutic
target.
In RP, Gupta and coworkers showed that human microglia are
activated in response to primary rod photoreceptor death, migrate
to the outer retina and phagocytize rod cell debris. The release of
cytotoxic factors such as NO can kill adjacent photoreceptors. Once
again, treatment targeting activated microglia could save cones in
human inherited diseases involving primary rod photoreceptor
degeneration (Gupta et al., 2003). As is the case in other retinopathies, microglial activation has been shown to be mediated by
TLR4 signaling in RP (Kohno et al., 2013).
Microglial activation also contributes to tissue degeneration in
AMD (Ardeljan and Chan, 2013). Immunologic responses in neural
retinal microglia and vascular elements appear to be related to
early changes in RPE pigmentation and drusen formation (Penfold
et al., 2001). In a model of AMD, the microglia was found to display
RPE cytotoxicity and increased production of inflammatory chemokines/cytokines after co-incubation with ligands that activate
37
innate immunity, and elevated mRNA levels of TLR2 and TLR4 were
also observed (Kohno et al., 2013). In AMD, as well as in other
degenerative diseases, an imbalance between the M1 and M2
macrophage populations, together with activation of retinal
microglia, have been observed and are thought to potentially
contribute to tissue degeneration (Ardeljan and Chan, 2013). In a
mouse model of AMD, naloxone was found to modulate the accumulation and activation of microglia at the site of retinal degeneration, which may be mediated by the inhibition of the proinflammatory molecules NO, TNF-a, and IL-b, and also ameliorated the clinical progression and severity of retinal lesions (Shen
et al., 2011). In this regard, naloxone was unable to prevent
apoptosis of photoreceptors in in vitro experiments, suggesting that
modulating the functions of microglia, rather than inhibiting their
activation, could be a good therapeutic approach for preventing
photoreceptor degeneration (Jiang et al., 2013).
Other non-inherited retinal diseases, such as degeneration
produced by trauma, axotomy or ischemia, involve microglial
activation, as in the case of light-induced photoreceptor degeneration. During light-induced retinal degeneration (LIRD), microglial
cells assume an activated state, migrate from the inner to the outer
retina and alter the production of trophic factors, which may also
affect photoreceptor cell survival (Harada et al., 2002; Zhang et al.,
2005). Moreover, microglia-derived factors influence the production of secondary trophic factors by Müller cells. Functional interactions between microglia and Müller glial cells may be
bidirectional and regulate photoreceptor cell survival during retinal
degeneration (Harada et al., 2002). In this sense, chemokine ligand
2 expression by Müller cells seems to play a role in promoting the
infiltration of monocytes/microglia and contribute to the neuroinflammatory response and photoreceptor death following retinal
injury (Rutar et al., 2012). Still other researchers have observed that
after photodegeneration, microglia fail to return to their original
state, rather they continued to show some degree of activation
(Santos et al., 2010). Suppression of the pro-inflammatory effect of
microglia, either by drugs such as naloxone or minocycline (Ni
et al., 2008; Zhang et al., 2004) or by physical methods, such as
electrical stimulation (Zhou et al., 2012), can contribute to reducing
photoreceptor degeneration in light-induced degeneration models.
Microglia have also been observed to become activated after
damage to ganglion cells. RGC neurodegeneration was found to be
associated with microgliosis, characterized by an increase in cell
density with concomitant morphologic changes from ramified to
amoeboid forms. The authors also demonstrated that microglia
density gradually declined to near-baseline level, and cell
morphology returned to ramified forms after approximately 4
weeks (Liu et al., 2012). Microglia activation has also been found in
a model of optic neuritis, where inflammation and cell death in the
optic nerve led to subsequent damage of RGC in the retina,
accompanied by gliosis, which could be prevented by protecting
myelin from degradation using a calpain inhibitor treatment (Das
et al., 2013).
Using primary retinal microglia from retinoschisin-deficient
(Rs1h-/Y) mice, a prototypic model for rapid retinal apoptosis and
degeneration, and the BV-2 cell line, Karlstetter and his group
identified a new protein called AMWAP (activated microglia/
macrophage WAP domain protein) that can act as a modulator of
microglial activation in neurodegenerative disorders. Moreover,
they demonstrated that AMWAP expression is rapidly induced by
ligands for TLR2, -4, and -9 and IFN-g, thereby reducing proinflammatory cytokine expression (Karlstetter et al., 2010b).
The fractalkine/CX3CR1 signaling pathway has also been
postulated as relevant in the control of retinal inflammation. In this
sense, under oxidative and ischemia/reperfusion conditions and in
the absence of CX3CR1, uncontrolled retinal inflammation results
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in extensive retinal degeneration (Chen et al., 2013), and it has been
suggested that CX3CL1 can exert a protective effect on the lightinjured retina (Huang et al., 2013a). On the other hand, the
expression of fractalkine was found to be significantly upregulated
after exposure to light, and was located mainly at the photoreceptors (Zhang et al., 2012a). This suggests that fractalkine/CX3CR1
signaling exerts multiple effects on the cross-talk between microglia and photoreceptors, and may play an important role in the
process of retinal microglia activation and migration in lightinduced photoreceptor degeneration. These authors also showed
that the soluble form of fractalkine released from photoreceptors
may function as a chemotactic factor to trigger the activation and
migration of retinal microglia, while low concentrations of fractalkine in the membrane-bound form would serve as a regulator of
the beneficial balance between microglia and photoreceptors
(Zhang et al., 2012a).
2.3.1.5. The controversial role of microglia in inflammation and
neurodegeneration. The true role of microglia in neurodegenerative
diseases as either a beneficial or harmful factor still remains
controversial, and a large body of results has been obtained to
support both hypotheses. Upon activation, different functions are
associated with microglia (Fig. 14), some clearly protective, some
harmful, and still others with dual effects, possibly due to specific
environmental conditions in each case, in such a way that the
singular circumstances that evoke microglia activation determine
the final net contribution to the disease (Streit et al., 1999). In this
sense, different stimuli can trigger different microglial responses.
For example, both IFN-g and IL-4 can make microglia neuroprotective, while aggregated beta-amyloid and lipopolysaccharide
provoke a cytotoxic response from the microglia (Butovsky et al.,
2005). Hence, different authors have suggested that the beneficial
or harmful expression of the local immune response in a damaged
CNS depends on the circumstances (Butovsky et al., 2005; Schwartz
et al., 2006). A plausible explanation for this dual role is the wide
phenotypic variation upon an injurious stimulus, which is likely to
lead to functional diversity (Hanisch and Kettenmann, 2007;
Schwartz et al., 2006).
Also relevant is the observation that microglial activation often
precedes astrogliosis (Carson et al., 2006) and the fact that
microglial deterioration is observed with age in retinal dystrophies
and in CX3CR1-deficient mice with dystrophic microglia and abnormalities in their cytoplasmic structure (Streit et al., 2004; Xu
et al., 2009). Among the aspects that determine whether full
microglial activation becomes a harmful stimulus is the release of
molecules such as pro-inflammatory cytokines, ROS, NO and TNFa, which have neurotoxic effects and are evoked in a chronically
activated state (Nimmerjahn et al., 2005; van Rossum and Hanisch,
2004).
In this sense, in some neurological diseases, such as multiple
sclerosis and Parkinson's disease, the attenuation of microglial
activation has a protective effect (Hanisch and Kettenmann, 2007;
Huitinga et al., 1990; Liu, 2006; Mount et al., 2007). In a similar
manner, in animal models of light-induced damage, different
groups have demonstrated that the inhibition of retinal microglia
activation by either minocycline (Zhang et al., 2004) or electrical
stimulation (Zhou et al., 2012) has a neuroprotective effect against
the loss of photoreceptors, through suppression of the secretion of
several pro-inflammatory cytokines and upregulation of neurotrophic factors (Zhou et al., 2012).
Hence, the selective inhibition of the overactive microglial activity and the preservation of their trophic and homeostatic functions appears to be a promising treatment for degenerative diseases
(Langmann, 2007). But it must also be noted that reactive microglial
cells can also have a protective effect in damaged retina and that
the inhibition of microglial activation can have harmful effects at
the same time.
Microglia participate in regenerative processes by removing
dendritic structures (Cullheim and Thams, 2007; Trapp et al., 2007).
In the early stages of the neurodegenerative process, microglial
activation can display a protective function (Fig. 14) through the
phagocytosis of cell debris and the release of protective molecules
(Kreutzberg, 1996; Neumann et al., 2009; Nimmerjahn et al., 2005;
Polazzi and Monti, 2010). In this sense, accumulation of microglia in
ischemic areas correlates with a reduction of neuronal damage and
confers neuroprotection (reviewed in Hanisch and Kettenmann
(2007)). Activated microglia secrete neurotrophic factors that protect and regulate the survival of photoreceptors (Arroba et al., 2011;
Carwile et al., 1998; Langmann, 2007). Microglia can also participate in proteolytic processes involved in tissue remodeling. As an
example, TGF-b1 produced by activated microglia can promote
tissue repair, either directly or indirectly, by reducing astrocytic
scar formation (Kreutzberg, 1996). Also, age-related microglia
activation is likely to represent a protective response against
harmful stimulus produced in the retinal microenvironment, and
thus may play an important role in retinal homeostasis (Xu et al.,
2009). Another beneficial function exerted by microglia is the
removal of glutamate. Under pathological conditions, astrocytic
glutamate uptake is impaired, and microglial cells are able to help
remove the excess of glutamate resulting from synaptic activity by
expressing the glutamate transporter protein GLT-1 (Hanisch and
Kettenmann, 2007; Persson et al., 2005).
To date, we have yet to completely understand the conditions
under which reactive glial cells mediate detrimental or protective
effects and the mechanisms that initiate the protective or damaging
effects by reactive glial cells. Moreover, in neurodegenerative diseases such as glaucoma, both effects seem to occur at the same time
(Seitz et al., 2013). Furthermore, in Alzheimer's disease, microglial
activation may have a dual effect. Microglial phagocytic activity is
beneficial, whereas the inflammation is detrimental, and it is the
inflammatory state of microglia that serves as an important condition for the disease (reviewed in (Hanisch and Kettenmann,
2007)). Concerning adult neurogenesis, inflammation-associated
microglia can attenuate neurogenesis, whereas microglia activated by certain T helper cell cytokines promote neurogenesis
(Hanisch and Kettenmann, 2007). It has even been observed that
microglia can amplify pro-inflammatory immune responses due to
their capacity to act as antigen-presenting cells, while their ability
to produce anti-inflammatory mediators could play the opposite
role (Aloisi et al., 2000).
In conclusion, to date, the real contribution of microglia to the
progression of neurodegenerative diseases and the optimal therapeutic intervention to halt or reduce their progression still remain
unclear.
2.3.2. Macroglial cells: Müller and astrocytes cells in healthy and
diseased retinas
Retinal macroglial cells, which include astrocytes and Müller
cells, are responsible for maintaining the homeostasis of the retinal
extracellular microenvironment, thus ensuring proper functioning
of the healthy retina. In the early stages of degeneration, glial cells
are activated as part of a process called gliosis. Reactive gliosis has a
direct neuroprotective effect on the retina, increasing the expression of cytoprotective factors or restoring neurotransmitter balance
and ion and water concentration, among other benefits. In contrast,
proliferative gliosis can accelerate the neurodegeneration during a
chronic disease, causing direct and indirect damage to neurons and
vasculature. Chronic gliosis exacerbates disease progression,
increasing vascular permeability, infiltration of toxic compounds
and even neovascularization (Penn et al., 2008).
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
2.3.2.1. The important role of Müller cells in retinal homeostasis and
degeneration
2.3.2.1.1. Müller cells in the healthy retina. Müller cells, the
specific and largest glial cell type in the vertebrate retina, span the
entire thickness of the retina and are in contact with all retinal
neuronal somata and processes, constituting an anatomical link
between the retinal neurons and the compartments with which
these need to exchange molecules. They are primarily responsible
for maintaining the homeostasis of the retina by regulating retinal
glucose metabolism, retinal blood flow, neuronal signaling processes, ion and water homeostasis and pH, among other aspects
(Fig. 14A). Moreover, Müller cells are involved in the regulation of
synaptic activity in the inner retina. More specifically, they are
responsible of the uptake and clearance of neurotransmitters,
mainly glutamate, but also GABA and glycine. In this sense, it has
been demonstrated that a failure to clear extracellular glutamate
reduces the scotopic b-wave (Barnett and Pow, 2000) and makes
glutamate neurotoxic (Izumi et al., 1999). Another function of
Müller cells is that of providing neurotransmitter precursors to
neurons, such as glutamine for glutamate synthesis. For more
detailed explanations see (Bringmann et al., 2006; Reichenbach and
Bringmann, 2013).
On the other hand, as previously stated, the retina has an
elevated need for antioxidant protection, due to its exposure to
light, high oxygen consumption and the presence of large amounts
of 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 or
hyperoxia during light exposure (Schutte and Werner, 1998).
Moreover, Müller cells protect photoreceptors and retinal neurons
from death (Fig. 14) through the secretion of neurotrophic factors,
growth factors and cytokines (Bringmann et al., 2009, 2006). The
ablation of Müller cells in the retina of adult mice causes vascular
abnormalities and photoreceptor cell death, which is reverted
following the administration of CNTF (Shen et al., 2012).
During the last decade, new roles for retinal Müller cells have
been described that are both relevant and important (Fig. 14A).
Briefly, it has been shown that Müller cells could be acting as living
optical fibers that guide light through the inner retinal layers toward the photoreceptors, minimizing the scattering of light (Franze
et al., 2007). Moreover, because Müller cells are softer than neurons, they may act as soft, pliant material for the embedding of
neurons, and as deformable substrates for neurite outgrowth and
branching (Lu et al., 2006). In addition, Müller cells may protect
neurons from mechanical stress caused, for example, by traumatic
retinal injuries (Lu et al., 2006). On the other hand, Müller cells can
regulate neuronal activity by releasing glutamate, adenosine or
ATP. Müller cells are connected to adjacent Müller cells through gap
junctions, forming a small syncytium of direct Müller-Müller cell
communication (Ball and McReynolds, 1998; Ceelen et al., 2001;
Zahs and Ceelen, 2006). Moreover, distant communications between neurons and Müller cells through the extracellular
messenger ATP (Newman, 2001, 2004) have been described, which
are associated with the capability of macroglial cells to regulate
local 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 cells
play a crucial role in the presence of injurious stimuli, providing a
rapid response to any alteration of the retinal microenvironment, as
they 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 hydrostatic
pressure. This resistance is conferred in part by their energy reserve
39
in the form of glycogen, their high antioxidant capacity and their
capacity to proliferate and regenerate, among other special properties (for a review, see (Bringmann et al., 2009)).
Almost all retinal diseases are associated with the gliosis of
Müller cells. Thus, in DR, for example, reactive changes in Müller
cells, such as up-regulation of glial fibrillary acidic protein (GFAP),
occur early in the course of the disease (Barber et al., 2000). Also, a
distinctive variation of intermediate filament expression in retinal
macroglia is associated with the pathogenesis of AMD, in which
discrete regions of GFAP upregulation in Müller cells can be associated with drusen formation (Wu et al., 2003). In the P23H rat
model of RP, Müller cells express the GFAP marker in response to
degeneration of the retina (Fernandez-Sanchez et al., 2010), and
glial cells show changes in number and morphology associated
with the progression of the pathology (Figs. 12 and 14B, D) (Cuenca
et al., 2011; Pinilla et al., 2010). Müller cell gliosis has also been
detected through the expression of GFAP in the DBA/2J mouse
model of glaucoma (Fernandez-Sanchez et al., 2013). For review the
cited information, see (Bringmann et al., 2006).
Reactive changes in Müller cells in response to damage may
have both cytoprotective and cytotoxic effects on retinal neurons.
Especially in the early stages after damage, Müller cell gliosis can be
neuroprotective (Fig. 14). In this case, retinal insults result in
functional and biochemical changes in Müller cells that have been
described as “conservative” or nonproliferative. But this usually
beneficial reaction can lead to a greater level of Müller cell response
described as “massive” or proliferative, in which case gliosis is
detrimental to the retinal tissue and exacerbates neuronal death. A
possible trigger for the transition from “conservative” to “massive”
gliosis is the breakdown of the blood-retinal barrier, resulting in an
increase in the retinal and vitreal contents of growth factors, cytokines and inflammatory factors, as well as an infiltration of bloodderived immune cells (Bringmann et al., 2009).
Müller cell gliosis involves a series of cellular and molecular
events that may or may not be dependent on the kind of stimulus.
The most sensitive non-specific response to retinal disease and
injury is the upregulation of the intermediate filaments nestin,
vimentin and GFAP (Figs. 12 and 14B, D). The upregulation of GFAP
is so sensitive that it can be used as an indicator of retinal stress,
retinal injury and Müller cell activation (Luna et al., 2010). There is
evidence that intermediate filaments may contribute to the
biomechanical properties of Müller cells (Lu et al., 2011), and thus
their upregulation could increase the stiffness of the tissue. This
upregulation seems to be crucial for many responses in Müller cell
gliosis (Fig. 14B), such as glial scar formation, monocyte infiltration,
neurite growth, neovascularization and cell integration in retinal
transplants, since all these traits were seen to be attenuated in mice
deficient in GFAP and vimentin, an experimental model of retinal
detachment (Bringmann et al., 2009; Nakazawa et al., 2007).
Other important non-specific Müller cell responses are cellular
hypertrophy, proliferation and migration of these cells to establish
a glial scar that fills retinal breaks, replacing degenerated neurons
and photoreceptors (Fig. 12AeC). Glial scars are involved in the
formation of epiretinal membranes (Bringmann and Wiedemann,
2009; Buchholz et al., 2013; Kase et al., 2006), frequently
described in retinal detachment, DR and AMD, and in the appearance of proliferative retinopathy. The proliferation of Müller cells is
needed for retinal regeneration and also serves as guide for neurite
growth. However, the development of a scar formed by these
Müller cells would appear to be the primary cause of failed retinal
detachment surgery, stem cell transplants or electronic device
implants in cases of advanced retinal degeneration (Bringmann
et al., 2009).
Another important feature in gliotic Müller cells is their intense
crosstalk with cells from the immune system. Müller cells have
40
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
been described to have the capacity to act as immunocompetent
cells 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 pathological
conditions, Müller cells can also act as antigen-presenting cells via
the processing of antigens into immunogenic forms that are then
presented in association with MHCII molecules (Bringmann et al.,
2009). Moreover, the increase in Müller cell-microglia adhesion
molecules also allows activated microglia to translocate intraretinally in a radial direction, using Müller cell processes as an
adhesive scaffold (Wang et al., 2011b). In addition, there are evidences that microglia-Müller cell interactions could serve as a
trophic factor-controlling system during retinal degeneration
(Harada et al., 2002), and may contribute to the protection of
photoreceptors or increased photoreceptor apoptosis.
Some evidence indicates that in proliferative gliosis, Müller cells
can promote neuronal cell death through the synthesis and secretion of TNF-a (Cotinet et al., 1997; Giaume et al., 2007; LebrunJulien et al., 2009; Tezel et al., 2001), monocyte chemoattractant
protein-1 and NO (Cotinet et al., 1997; Giaume et al., 2007). Excess
production of NO by Müller cells and the formation of free nitrogen
radicals result in protein nitrosylation, which has toxic effects on
surrounding neurons. Attenuated cell death after retinal detachment has been observed in GFAP- and vimentin-deficient mice, in
association with decreased gliosis and monocyte infiltration into
the retina (Nakazawa et al., 2007). The cytotoxic effects of Müller
cells contribute to retinal degeneration in various retinopathies,
such as DR (Bringmann et al., 2009, 2006).
On the other hand, proliferative gliosis of Müller cells might
impede tissue repair and regular neuroregeneration by forming
glial scars. But Müller cells are also capable of dedifferentiating to
cells with characteristics similar to pluripotent retinal cells
(Fig. 14B). After retinal detachment, Müller cells are known to
migrate to the outer retinal layer and undergo mitosis. Some of
these subpopulations of Müller cells appear to stop expressing
commonly accepted Müller cell marker proteins, which suggests a
potential dedifferentiation of some of these cells over time (Lewis
et al., 2010). The proliferation of Müller cells to form glial scars or
to transdifferentiate into cells with a neuronal phenotype may
depend on their expression profile of intermediated filament proteins. The upregulation of nestin has been described as being
indicative of the dedifferentiation of Müller cells into progenitor
cells (Luna et al., 2010). After retinal injury, when Müller cells
dedifferentiate and begin to act as progenitor cells, they also express specific neuronal stem cell markers, such as Chx10, Sox2, Ki67 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 a
neuronal phenotype, if it would be confirmed, will represent a
promising mechanism for therapeutic interventions since transdifferentiated Müller cells can be obtained from many sources, such
as immortalized human cell lines or epiretinal membranes surgically removed from the eyes of patients with proliferative retinopathies (Reichenbach and Bringmann, 2013). More knowledge is
necessary to elucidate the exact molecular mechanisms required to
obtain neural progenitors from Müller cells. So far, proliferation and
migration of transdifferentiated Müller cells after glutamateinduced 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 been
successfully achieved (Reyes-Aguirre et al., 2013; Tackenberg et al.,
2009).
2.3.2.2. Retinal astrocytes and astroglyosis. Astrocytes are the main
glial 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 almost
entirely restricted to the nerve fiber layer of the retina (Fig. 13A, C).
Although many of the functions of astrocytes in healthy retinas are
poorly understood, it is widely accepted that they play an essential
role 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 the
clearance of neurotransmitters, and they support synapse formation, function and elimination through the activation of microglial
cells (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 neurons, leading to either enhancement or depression of neuronal
spiking (Newman, 2004). However, astrocytes do not occur across
the retina in species with non vascularized retinas like rabbits
where the described astrocyte functions could be carried out by
other glial cells.
In the retina, astrocytes and Müller cells are associated with the
development of retinal blood vessels (Fig. 14A). During physiological hypoxia, astrocytes and Müller cells secrete VEGF, inducing the
formation of superficial vessels from astrocyte and vascular precursor cells (Chan-Ling et al., 2004; Kubota and Suda, 2009; Kur
et al., 2012). Müller cells then drive the formation of the deep
vascular plexus, an astrocyte-independent process, through
vascular 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 are
both involved in the development of the new neovascularization
associated with pathological processes such as AMD, DR or retinopathy of prematurity, releasing angiogenic factors in response to
pathogenic stimuli (Coorey et al., 2012).
But one of the most important functions of astrocyte cells is the
formation and support of the blood retinal barrier (BRB) (Fig. 14A).
Retinal capillaries that form the BRB consist of a single layer of
tightly adhered endothelial cells, a basal lamina and surrounding
pericytes, astrocytes and microglia, forming a functional complex
called the neurovascular unit (for a review, see (Klaassen et al.,
2013)). This structure selectively regulates the transport of molecules through the BRB. In the neurovascular unit, endothelial cells,
pericytes, astrocytes, microglia and neurons are actively connected
over a functional network. Thus, for example, neural activity can
regulate the blood flow by means of glial communication, through
vasoactive factors produced by astrocytes, which may control the
blood flow in local regions of the retina (Metea and Newman, 2006,
2007). Like Müller cells, astrocytes form a network where cells
communicate by means of calcium waves through gap junctions or
using extracellular messengers, such as ATP (Metea and Newman,
2006, 2007). Functional changes in pericytes and astrocytes
further facilitate BRB leakage (Fig. 14B, D). In fact, glial cell
dysfunction in retinal pathologies is associated with retinal
swelling and BRB breakdown (Bringmann et al., 2006; Klaassen
et al., 2013; Shen et al., 2010). Hyperglycemia, hypoxia, oxidative
stress and/or inflammation are the main underlying processes in
human ocular diseases in which BRB dysfunction is a major cause of
vision loss.
Reactive gliosis (Fig. 13B, D) in the retina is characterized by
changes in astrocyte morphology as production of GFAP and
vimentin increases (Anderson et al., 2008; Luna et al., 2010). A
growing body of evidence suggests that reactive astroglia can be
both beneficial and harmful to damaged neurons (Nakazawa et al.,
2007). Reactive astroglia either release neurotrophic factors to
support cell survival or produce molecules that inhibit axon
regeneration and repair, triggering neurocytotoxicity or secondary
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
damage in nearby neurons and glial cells (Fig. 14B). Thus, for
example, the attenuation of reactive responses in retinal glial cells
of GFAP- and vimentin-deficient mice improves the integration of
retinal grafts in the hippocampus (Widestrand et al., 2007). The
absence of GFAP and vimentin also protects against photoreceptor
cell degeneration in cases of retinal detachment (Nakazawa et al.,
2007). Moreover, the chemical inhibition of gliosis by glial toxins
can protect against ganglion cell death from excitotoxicity (Ganesh
and Chintala, 2011). On the other hand, reactive gliosis has been
associated with the up-regulation of enzymatic and non-enzymatic
antioxidant defenses that may increase the ability of the astrocytes
to protect neurons from free radicals (Wilson, 1997).
Virtually all forms of retinal injury or disease trigger reactive
gliosis. Thus, in AMD, for example, large numbers of hypertrophic
and reactive astrocytes have been observed to phagocytize the
residues of ganglion cells that have died through necrosis or
apoptosis (Ramirez et al., 2001). In end-stages of RP, when significant death of the RGCs has occurred, many of the nuclei in the inner
retina belong to astrocytes that have undergone reactive hyperplasia (Milam et al., 1998). In the DBA/2J mouse model of glaucoma,
retinal changes engender a reactive gliosis response (Inman and
Horner, 2007), and there is evidence that astrocytes are the cells
responsible for many of the pathological changes in the glaucomatous optic nerve head (Hernandez et al., 2008). In DR, GFAP induction has been reported in both the astrocytes and Müller cells of
streptozotocin diabetic rats (Lieth et al., 1998). Early astrocyte
changes, including decreased astrocyte gap junction protein and
gene expression, have also been correlated with inner retinal
hypoxia and ganglion cell functional deficits during the progression
of diabetes (Ly et al., 2011).
2.4. Degenerative events in retinal vascularization
The vascular supply to the retina depends on two distinct
vascular systems: the retinal vasculature, which supplies blood to
the inner two-thirds of the retina, and the choriocapillaris, which
supplies blood to the outer retina (Fig. 15A). Because of the high
metabolic activity of the retina, the tissue with the highest oxygen
demand in the body, the ability to regulate blood flow is an
essential feature of the mammalian retina (Kur et al., 2012). The
microenvironment of the retina is also regulated by the BRB system.
2.4.1. Retinal vascular networks and the blood retinal barrier in
health and disease
The retinal vasculature enters the retina through the central
retinal artery via the optic nerve, and after being distributed
throughout the retinal tissue, it leaves the tissue through the retinal
vein. Inside the retina, the blood is distributed by means of parallel
and interconnected vascular plexuses (Fig. 15A). The most superficial plexus is located in the ganglion cell layer, whereas the inner
plexuses are located in the OPL (Coorey et al., 2012). The retina
tends to maintain a constant blood flow. Autoregulation in the
retina is effective within a wide range of perfusion pressures, while
the influence of autonomic innervation with circulating hormones
and neurotransmitters on retinal vascular resistance is negligible
due to the BRB (Kur et al., 2012). The autoregulatory myogenic
response is intrinsic to smooth muscle cells, and pericytes are
capable of initiating vasomotor signals that can be propagated
along the length of capillaries.
The choroidal circulation arises from the ciliary arteries. In
contrast to the retina, choroidal microvessels are fenestrated, and
choroidal circulation is under neurogenic control (Coorey et al.,
2012). The basal lamina and the RPE tight junctions form the
outer BRB, and are the structures responsible for transport regulation at this level (Kaur et al., 2008).
41
BRB disruption is a frequent event in many retinal diseases, and
a major cause of loss of vision. Hyperglycemia, hypoxia, oxidative
stress and inflammation are known to increase BRB permeability
and can eventually lead to its breakdown by damaging some of the
elements of the neurovascular unit (Kaur et al., 2008; Klaassen
et al., 2013). Two basic vascular responses to retinal damage have
been described: (1) Neovascular formation, when new vessels are
formed in the retina in response to hypoxic conditions (Fig. 15CeD),
as in the case of DR, AMD or retinopathy of prematurity (Coorey
et al., 2012; Penn et al., 2008). The origin of the vessels can be
either the retina itself or the choroidal vascularization, depending
on the pathology. (2) Vascular degeneration, when retinal vessels
degenerate as a result of a reduction in oxygen consumption
(Fig. 15B), such as in RP (Fernandez-Sanchez et al., 2012a; Pennesi
et al., 2008).
2.4.1.1. Retinal neovascularization under hypoxic conditions.
Retinal diseases that impose local hypoxia or retinal ischemia
usually cause BRB disruption and result in the formation of new
vessels (Kaur et al., 2008). Retinal vascular disease represents a
leading cause of visual impairment and acquired blindness in infants (retinopathy of prematurity), working-age adults (DR) and the
elderly (AMD) in industrialized countries (Coorey et al., 2012).
Oxidative stress, impairment of the antioxidant machinery and
hyperglycemia have been proposed as the main underlying
mechanisms of endothelial cell damage and dysfunction of the BRB
in DR (El-Remessy et al., 2003, 2013; Klaassen et al., 2013). The
disruption of the BRB associated with DR promotes up-regulation
of VEGF (Fan et al., 2010), a prominent angiogenic factor also
referred to as vascular permeability factor. This mechanism seems
to have a positive feedback regulation, since VEGF is able to increase vascular permeability in hypoxic conditions. In this regard,
the permeability changes in BRB have been reported as a contributory factor in the development of macular edema (Kaur et al.,
2008). Furthermore, increased production of AGEs has been
shown to upregulate the expression of VEGF in glial cells (Hirata
et al., 1997), which promotes neovascularization in the advanced
stages of proliferative DR. The growth of new vessels in DR occurs at
the inner retinal layers, using the vitreous body structure for its
growth (Fig. 15D). The loss of the inner and outer BRB is also a
common finding in exudative AMD deriving in an increased fluid
leakage. New blood vessels grow from the choriocapillaris plexus
and enter the retina through Bruch's membrane (Fig. 15C), thus
increasing the risk of detachment of the RPE and/or neurosensory
retina (Hoffmann et al., 2002; Klaassen et al., 2013; Schlingemann,
2004).
Retinal vascular development is mediated by a gradient of VEGF
that is generated by the macroglial cells and, is mainly regulated by
retinal oxygen levels in the developing retina. In this context, the
secretion of VEGF by astrocytes contributes to develop the superficial vasculature of the retina while the VEGF gradient generated
by Müller cells helps to create the formation of the deep vascular
plexus by promoting the sprouting of superficial vessels to the
outer layers of the retina (Eichler et al., 2000; Stone et al., 1995). In
retinopathy of prematurity, treatment with a hyperoxic environment removes the physiological hypoxia of the retina leading to
insufficient vascularization, which, in turn, induces up-regulation
of angiogenic factors, particularly VEGF, and consequent neovascularization (Coorey et al., 2012). In oxygen-induced ischemic
retinopathy, it has also been demonstrated that neovascularization
is associated with increased expression of VEGF in glial cells (Akula
et al., 2008; Liang et al., 2012; Weidemann et al., 2010). The
development of neovascular glaucoma, one of the most important
complications that can appear during the course of retinal diseases
such as proliferative DR, ischemic retinal disease, vascular occlusive
42
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
Fig. 15. Vascular alterations associated with retinal disease. (A) Illustration of the vasculature of the retina and choroid. (B) Schematic representation of the progressive degeneration of retinal vasculature associated with retinitis pigmentosa. (C) Representation of the changes in choroidal vasculature observed in age-related macular degeneration. New
blood 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 to
be related with increased expression of VEGF (Hayreh, 2007).
2.4.1.2. Vascular
degeneration
under
hyperoxic conditions.
Vascular degeneration has been described in various retinal
degenerative diseases, such as RP, and the early loss of photoreceptors in some retinal degenerations affect the vascular development of the retina. Thus, a close relationship has been shown to
exist between the number of photoreceptors and vessel profiles in
the 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 photoreceptor cells are protected from oxidative-stress-induced death
(Fernandez-Sanchez et al., 2012a). But alterations in the deep
capillary plexus are not the only change evidenced in these pathologies; modification of the inner plexus has been shown to occur
in advanced stages of RP, once all photoreceptor cells have been lost
and a profound remodeling of the retina has occurred (GarciaAyuso et al., 2010, 2011; Wang et al., 2000). A sequence of the
retinal degenerative events in RP is shown in Fig. 15B. In a rat model
of oxygen-induced retinopathy, postnatal exposure to hyperoxia
destroys the plexiform layers, resulting in significant
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
electroretinographic anomalies, cell death and synaptic retraction
affecting principally the inner retina, pointing to the inner retina as
the primary target of hyperoxic injury (Dorfman et al., 2011).
43
ophthalmic flows have been associated with ophthalmic artery
stenosis.
2.5. Retinal pigment epithelium (RPE)
2.4.2. Retinal degenerative diseases with relevant vascular changes
2.4.2.1. Disorders of the retinal circulation. The retinal vasculature
lacks autonomic innervation, but responds to changes in the surrounding partial pressure of carbon dioxide and oxygen. Impairment of retinal vascular autoregulation is a common feature in a
number of ocular disorders, including DR and glaucoma.
Retinal circulatory changes precede overt clinical DR. Changes in
basal blood flow are characterized by retinal hypoperfusion in early
diabetes, with a shift to retinal hyperperfusion as DR progresses
(Kur et al., 2012). However, there are quantitative and qualitative
inconsistencies in the data. Measurements indicate a correlation
between the severity of DR and decreased flow velocity in the
central retinal artery (Goebel et al., 1995). Ocular perfusion pressure
is also reduced, which can exacerbate retinal hypoxia. Hemodynamic abnormalities may generate macular ischemia and/or neovascularization at different levels (optic disc, peripheral retinal, iris
or the anterior chamber angle).
Abnormal vascular regulation at the retinal level is also associated with pathogenesis of glaucoma. Elevated intraocular pressure
associated with glaucoma is expected to cause decreased ocular
blood flow, as has been proven in several studies showing
decreased blood flow in the optic nerve head, retina and choroid in
patients with glaucoma (Kur et al., 2012).
Late stages of retinal degenerative diseases can also show
vascular changes linked to anatomical modifications. Remodeling
in the late stages of RP after cell loss or in long-established retinal
detachment modifies the retinal anatomy. Retinal blood flow is
significantly decreased in patients with RP (Fig. 15B), probably as a
result of vascular remodeling in response to reduced metabolic
demand (Grunwald et al., 1996).
2.4.2.2. Disorders of the choroidal circulation. Central serous chorioretinopathy and AMD are disorders characterized by changes in
choroidal circulation. Patients with central serous chorioretinopathy show decreased choroidal blood flow and hyperpermeability
of the choroidal vessels, resulting in retinal edema (Kitaya et al.,
2003). Several studies have also demonstrated changes in
choroidal vascularization in AMD, including reduced blood flow
and abnormal choroidal perfusion patches (Friedman et al., 1995;
Kiel and Reiner, 1997). The subsequent hypoxia induces the secretion of growth factors, such as vascular permeability factor and
fibroblast growth factor (FGF) (Frank et al., 1996). These factors are
found in different retinal cells, including glial cells, RPE and
vascular endothelial cells. Secondary to the increase in growth
factors, in exudative AMD forms, there is neovascularization from
choroidal vessels that can cross Bruch's membrane, localizing
either under or over the RPE and generating a loss of fluid, hemorrhage and lipid exudation (Fig. 15C).
2.4.2.3. Disorders of the retrobulbar circulation. Vascular occlusions, such as occlusion of the central retinal artery or the central
retinal vein, result in circulation anomalies that are revealed by
Doppler imaging (Williamson and Baxter, 1994). Decreased blood
velocity and resistivity index in the retrobulbar arteries and
increased blood velocity and resistivity index in the central retinal
vein have been reported in patients with DR (Dimitrova et al.,
2003). Reduced blood velocities in the ophthalmic artery have
also been shown in ocular ischemic syndrome and carotid stenosis.
A reverse flow, indicating collateral perfusion, has been seen in
other cases of severe carotid stenosis, while increases in
2.5.1. RPE physiology and functions
The 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 the
outer segments of the photoreceptors on its apical portion. The cells
in this layer are connected by tight junctions, and constitute the
outer components of the blood-retinal barrier (Nag and Wadhwa,
2012; Spitznas, 1974). In the subretinal space, filled with the
interphotoreceptor matrix, microvilli from RPE cells appose to the
outer segments of the photoreceptors, forming the physical components that can contribute, with others, to the maintenance of
retinal adhesion (Nag and Wadhwa, 2012).
The RPE has several physiological functions that are indispensable for neural retina survival, including: (i) the delivery of
nutrients such as glucose, retinol and fatty acids to photoreceptors and (ii) the transfer of metabolic end products from the
subretinal space to the blood. Since photoreceptors do not have
their own blood supply, nutrients must travel to the photoreceptors from the choriocapillaris, through Bruch's membrane and
the RPE. The reverse path is followed for the elimination of
cellular debris (Taylor, 2012). The RPE also: (iii) regenerates
components of the visual cycle; (iv) buffers ion composition in
the subretinal space, which maintains photoreceptors excitability; (v) phagocytizes the shed photoreceptor outer segments
and recycles essential substances; (vi) absorbs the light focused
on the retina and protects against photo-oxidation; (vii) secretes
growth factors, such as fibroblast growth factors (FGF-1, FGF-2,
and FGF-5), TGF-b, insulin-like growth factor-I, ciliary neurotrophic factor, platelet-derived growth factor, VEGF and pigment
epithelium-derived factor; and (viii) regulates T cell activation in
the eye (Strauss, 2005; Sun et al., 2003). All these functions are
essential, and a failure of any of them can lead to photoreceptors
death, degeneration of the retina, loss of visual function and
blindness. Moreover, several studies have shown the RPE to be a
non-homogeneous population of cells that retains proliferative
potential, with subsets of cells that have an unusual capacity to
transdifferentiate into various cell types and to produce a new
retina, 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 in
the retina and its involvement in early AMD-associated damage, a
great amount of research has focused on this layer.
2.5.2. RPE changes in aging and pathology
With age, the eye undergoes several changes, some of which
affect the RPE. The accumulation of lipofuscin, a reduction in
melanin, diminished antioxidant capacity and the progressive
accumulation of deposits underlying Bruch's membrane have all
been described in aging eyes (Boulton and Dayhaw-Barker, 2001;
Nag and Wadhwa, 2012). The death of RPE cells is associated with
aging-pathophysiology; both acute and chronic progressive
dysfunction of RPE cells and the age-related deterioration of this
tissue have been shown to play a relevant role in retinal degenerative diseases, primarily AMD. Regardless of age, four main processes contribute to the pathogenesis of AMD: lipofuscin
accumulation and drusen formation, local inflammation and neovascularization (Nowak, 2006). The inflammatory responses
observed in AMD retinas are similar to, but more severe than those
observed in normal aging retinas. Different lifestyle factors, environmental conditions and gene alterations are likely to explain why
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N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
certain individuals develop AMD with age, while others do not (Xu
et al., 2009).
AMD is a multifactorial disease that is the leading cause of vision
loss in the industrialized world, with at least sixty million people
estimated to be affected (Taylor, 2012). The two most prevalent
AMD forms are atrophic and neovascular. Approximately 10e15% of
AMD cases correspond to the exudative, “wet” or neovascular form
of the disease. Nevertheless, neovascular AMD is responsible for the
majority of cases of severe vision loss. This severe form of AMD is
characterized by choroidal neovascularization (CNV). Abnormal
choroidal blood vessels start to grow through Bruch's membrane
underneath the macula, and this is accompanied by vascular
leakage or hemorrhage and scarring. In some cases, abnormal blood
vessels cause disciform scars, leading to the permanent loss of
central vision (Bhutto and Lutty, 2012). Most AMD patients suffer
the atrophic, non-exudative, “dry” form of the disease, which
progresses more slowly and is characterized by the formation of
yellow deposits (called drusen) in the macula and atrophic areas of
RPE, accompanied by varying degrees of choriocapillaris loss. As the
disease progress, a loss of RPE and photoreceptor cells can be
detected in the macula. It is not unusual to observe a progression
from atrophic to neovascular AMD (Bhutto and Lutty, 2012; Taylor,
2012). In both the atrophic and neovascular forms of AMD, the
mutualistic and symbiotic relationship between the photoreceptors, RPE, Bruch's membrane and choriocapillaris is lost, which
results in the death and dysfunction of all of the components in the
complex, through mechanisms involving apoptosis (Bhutto and
Lutty, 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 the
pathogenesis of AMD. Among them, there is an increase in the
formation of lipofuscin granules, an accumulation of AGEs, drusen
formation and changes in pigmentation, accompanied by a reduction in melanosomes, increased thickness of Bruch's membrane
and 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 from
the enzymatic oxidation of tyrosine and dihydroxyphenyl-alanine,
is contained in cytoplasmic granules called melanosomes. Lipofuscin, a heterogeneous group of complex fluorescent lipidprotein aggregates, represents the accumulation of nondegradable end products from the phagocytosis of the outer segments of photoreceptors (Ardeljan and Chan, 2013; Delori et al.,
2001; Sparrow and Boulton, 2005). Lipofuscin granules contain
lipids, proteins and photoreactive molecules, such as bisretinoid
fluorophore (A2E), which are potent photoinducible generators of
ROS with phototoxic effects (Sparrow and Boulton, 2005). In aging
RPE, there is a linear increase in lipofuscin formation in the cytoplasm, which can contribute to cell dysfunction. The exposure of
RPE cells with accumulated lipofuscin to light produces extragranular oxidation of lipids and inactivation of lysosomal and
antioxidant enzymes, which may result in lipid peroxidation, the
loss of lysosomal integrity, DNA damage and RPE cell death (Davies
et al., 2001; Godley et al., 2005; Shamsi and Boulton, 2001; Sparrow
and Boulton, 2005).
Besides lipofuscin accumulation, aging causes other alterations
in RPE pigmentation, such as a decrease in melanosomes and the
appearance of new pigmented organelles (melanolysosomes) as
the 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 and
reduce the levels of light entering the RPE. The decline in melanin
granules in the macular RPE may result in decreased light absorption and reduced antioxidant potential (Boulton and DayhawBarker, 2001). The fact that the blue light photoreactivity of melanosomes increases significantly with age can result in toxicity to
the RPE (Sparrow and Boulton, 2005).
2.5.2.2. Advanced glycation end products (AGEs) accumulation.
AGEs accumulation occurs with aging, and may have a significant
impact on age-related dysfunction of the RPE and also play a role in
AMD. AGEs accumulation in Bruch's membrane, drusen, RPE, and
choroidal extracellular matrix may be a consequence of incomplete
degradation of metabolic end products, such as lipoproteins from
both photoreceptors and RPE (Ardeljan and Chan, 2013; Bhutto and
Lutty, 2012; Crabb et al., 2002; McFarlane et al., 2005). AGEs
accumulation can also be related to a limited choroidal perfusion
and the inability of aged Bruch's membrane to transport material
(Harris et al., 1999; Stefansson et al., 2011). Moreover, Bruch's
membrane deposits and drusen may interfere with transport between the choriocapillaris and retina, and thus may be a contributing factor to retinal hypoxia (Stefansson et al., 2011).
It has been associated a decrease in the risk of AMD to patients
consuming low glycemic index foods. Likewise, a higher accumulation of AGEs and an acceleration of the appearance of age-related
retinal lesions have been demonstrated in the retinas of animal
models consuming high glycemic index foods, thus suggesting a
relationship with the disease etiology (Chiu et al., 2011; Handa
et al., 1999; Uchiki et al., 2012; Weikel et al., 2012). When RPE
cells are exposed to AGEs, a para-inflammation state may develop
in conjunction with an adaptive response from RPE cells, but when
the exposure becomes chronic, it is possible that the stresses
overpower the adaptive mechanisms of RPE cells, resulting in
dysfunction and death (Lin et al., 2013).
2.5.2.3. Drusen formation. The appearance of drusen is a widely
known characteristic of AMD. Drusen are extracellular deposits that
accumulate between the RPE basal lamina and the inner collagenous layer of Bruch's membrane in aging human eyes (Green and
Enger, 1993). They are likely composed of incompletely digested
material from the RPE, which cannot traverse Bruch's membrane
for removal. Drusogenesis is a complex, multifactorial process
affected by genetic, environmental, dietary and aging factors that
takes place slowly over manys. Several theories have attempted to
explain drusen formation, focusing on inflammatory, immune, and
cell-mediated events (Hageman et al., 2001; Nowak, 2006). Gene
expression analysis has revealed the existence of local synthesis
and differential expression of a number of drusen-associated
molecules (Hageman et al., 2001). Biochemically, deposits contain
cellular debris, lipids, proteins, lipoproteins, phospholipids, triglycerides, 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). Lowgrade monocyte infiltration within the choriocapillaris is often
present in the underlying areas of the deposits (Cherepanoff et al.,
2010), and macrophages can be also found along the outer side of
Bruch's membrane in areas of neovascularization and drusen
(Grossniklaus et al., 2002). Drusen contain inflammation-related
proteins, as well as complement components. The negative
impact of the formation of drusen on RPE cells and photoreceptors
is most likely due not only to the physical displacement of the RPE
monolayer and photoreceptors, but also to their indirect influence,
most likely via the activation of the immune system and local
inflammation (Anderson et al., 2002; Nowak, 2006).
Despite the numerous classification systems, drusen can be
broadly divided into hard and soft types. Hard drusen are small
punctate refractile lesions with sharp borders that appear in the
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
fundus as yellowish-white deposits. They are present in both the
macula and on the periphery of the retina in both normal and AMD
retinas, and are considered a consequence of normal aging. The
presence of hard drusen alone does not carry an adverse prognostic
significance, although in large numbers, hard drusen are an independent risk factor for vision loss in AMD (Bhutto and Lutty, 2012).
Soft drusen are larger deposits, with indistinct borders and a fluffy
appearance in the cross-section of the macula, and are considered
to be a precursor of advanced AMD, as much higher rates of AMD
progression are found in individuals with baseline soft distinct
drusen (Ardeljan and Chan, 2013; Buch et al., 2005). The histopathological examination of clinically identified “drusen” has
defined three main types of sub-RPE deposits, depending on their
location, thickness, and content: basal laminar deposits and basal
linear deposits (both diffuse deposits) and nodular drusen (discrete,
dome-shaped deposits within the inner collagenous zone of
Bruch's membrane) (Ardeljan and Chan, 2013; Bhutto and Lutty,
2012; Bressler et al., 1988; Mettu et al., 2012). The histological
correlation with clinically-observed drusen remains controversial
(Mettu et al., 2012). Depending on the retinal location, hard drusen
have a different composition, with those located on the periphery
being much more homogenous than those located in the macula.
On the other hand, soft drusen have a homogeneous composition
and are filled with a single amorphous material that resembles
membranous debris (Rudolf et al., 2008). The composition of drusen 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 membrane thickens with age. This thickening is greater at the posterior
pole than on the periphery, and is mainly produced by increased
deposition and cross-linking of less soluble collagen fibers and
increased deposition of biomolecules, mainly waste products from
RPE metabolism. This change eventually leads to several functional
disturbances, such as changes in elasticity and hydraulic conductivity (Bhutto and Lutty, 2012; Ramrattan et al., 1994). Bruch's
membrane also undergoes other biochemical and anatomical alterations with aging, such as calcification and lipid infiltration in
the absence of any apparent retinal dysfunction (Grossniklaus et al.,
2013; Mettu et al., 2012). AMD patients also experience alterations
in retinal layer thickness, which is of importance since the measurement of retinal structures is known to correlate with visual
acuity (Farsiu et al., 2014; Pappuru et al., 2011). In early AMD, when
visual defects are present, there is a significant thinning of the OS
layer and thickening and elevation of the RPE (Acton et al., 2012).
RPE-Bruch's membrane thickness is also negatively correlated with
visual acuity (Karampelas et al., 2013).
2.5.2.5. Increased susceptibility to cell damage. Aging RPE has a
limited capacity to respond to oxidative stress, which may
contribute to the development of AMD. Vulnerability to oxidative
damage in the RPE is due, at least in part, to impaired Nrf2 signaling
(Sachdeva et al., 2013). Nrf2 has been recognized as a key factor
regulating an array of genes that defend cells against the deleterious effects of environmental insults. Furthermore, it seems to play
the main regulatory role in the protective response to cellular
oxidative stress, coordinating the expression of antioxidant genes
and promoting cell survival, as Nrf2 deficiency has been associated
with increased susceptibility to oxidative stress (Sachdeva et al.,
2013; Zhang, 2006).
Many hypotheses have been proposed to explain the etiology
and mechanisms of AMD. Like many other complex multifactorial
diseases, several genetic, environmental and other factors influence
its development (Jarrett and Boulton, 2012; Mettu et al., 2012;
45
Taylor, 2012). As previously stated, the retina is one of the highest
oxygen-consuming tissues in the human body, although oxidative
damage is normally minimized by the presence of a range of antioxidant and efficient repair systems. A reduction in the protective
mechanisms of RPE or an increase in the number and concentration
of species involved in photooxidative reactions can increase the
oxidative stress and contribute to the pathogenesis of AMD (Bhutto
and Lutty, 2012).
2.6. Functional changes following retinal injuries
Early detection and reliable assessment of visual capacity are
key elements in preventing or slowing the progress of vision loss
associated with retinal diseases. Electrophysiological and psychophysical methods for testing retinal function provide valuable information in both experimental and clinical ophthalmological
procedures. Moreover, disease-associated morphological changes
in the retina, if independently measured, are not always directly
correlated with functional alterations, in terms of time course or
spatial location. Thus, in many retinal disorders (e.g., glaucoma, RP
and DR) there is a disease stage where functional disturbances may
precede 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 in
normal subjects and in patients with retinal disorders. The ERG
reflects contributions from many different retinal neurons and glial
cells, and provides a non-invasive technique to evaluate retinal
responses and visual signal processing. Considerable variability
exists in the onset and evolution of retinal pathologies. However,
there are a number of disorders in which the ERG can be used to
distinguish qualitative abnormalities.
Most retinal disorders are characterized by an attenuation of
both a- and b-wave amplitudes (Creel, 1995). Another value, implicit time, is also usually altered in a- and b-waves. Thus, for
example, in RP patients, ERGs characteristically show reduced aand b-wave amplitudes, as well as delayed rod and/or cone b-wave
implicit times (Berson, 1987; Pinilla et al., 2005). Based on these
parameters, ERGs have provided criteria for establishing the diagnosis of RP in early life, even when fundus abnormalities visible
with an ophthalmoscope are minimal or absent. Patients with
widespread progressive forms of RP have shown not only reduced
amplitudes, but also delayed cone and/or rod b-wave implicit
times, while patients with self-limited sector RP or stationary forms
of night blindness have evidenced reduced amplitudes with normal
b-wave implicit times (Berson, 1981). In contrast to RP, the ERGs of
patients with cone dystrophy characteristically exhibit good, albeit
slower rod b-waves, and reduced or absent cone ERG responses
(Kellner and Foerster, 1993).
In animal models of retinal degeneration, a- and b-wave amplitudes provide key information about disease-associated functional changes in the retina, but also about morphological
alterations occurring during degenerative processes, including the
progressive loss of photoreceptors and synaptic connectivity
impairment in both the OPL and IPL. In this sense, it has been
shown that the mean number of rows of photoreceptor cell bodies
positively correlates with the scotopic b-wave amplitude recorded
in P23H rats (Fernandez-Sanchez et al., 2012b; Lax et al., 2014), a
model of RP. Also, the thickness of the ONL was found to be proportional to the scotopic a- and b-wave amplitudes in a rat rotenone model of Parkinson's disease (Esteve-Rudd et al., 2011).
Moreover, ERG recordings provide key information to assess the
progress of retinal degeneration in animal models (Cuenca et al.,
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N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
2004, 2005b; Pinilla et al., 2007) and to evaluate on them the
neuroprotective 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 amplitudes also provides key information about the functional integrity
of the retina (Perlman, 1983). Any significant deviation from the
normal relationship can therefore be regarded as pathological, and
can be interpreted accordingly. For example, in most cases of RP, the
rods are affected more severely than the cones (Arden et al., 1983;
Pinilla et al., 2005), causing an increase in the b-/a-wave ratio. In
most cases of retinal ischemia, the ERG b-wave amplitude and
implicit time are selectively depressed, while the a-wave remains
normal or becomes larger than normal. The b-/a-wave ratio varies,
and the variation is believed to depend upon the degree of retinal
ischemia (Breton et al., 1989; Karpe and Germanis, 1962; Karpe and
Uchermann, 1955). Thus, the ERG b-/a-wave ratio can be considered
a 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 light
source is primarily dependent upon the speed with which photopigments can regenerate. Therefore, any disease compromising the
integrity of the photoreceptor/RPE complex, or limiting the supply
of metabolites to the outer retina, is likely to prolong the recovery
time of both rods and cones. This is exemplified in AMD, DR, cystoid
macular edema, RP and systemic diseases such as diabetes and
hypertension (Binns and Margrain, 2005).
Examination with flicker ERG provides valuable information
from a clinical point of view. Different rates of stimulus allow
separate rod and cone contributions to the ERG to be identified. The
primate ERG recorded on the cornea in response to fast flickering
light is thought to reflect primarily the cone photoreceptor potential (Bush and Sieving, 1996). Even under ideal conditions, rods
cannot follow a light that flashes up to 20 times per second,
whereas cones can easily follow a 30 Hz flicker (Creel, 1995). There
is evidence that the fast flicker (33-Hz) ERG is generated primarily
in the inner retina by the same neurons that are responsible
(directly or indirectly) for the b-wave and the d-waves of the
photopic flash ERG (Bush and Sieving, 1996).
The usefulness of oscillatory potentials in the analysis of retinal
disease has been largely demonstrated (Algvere, 1968;
Wachtmeister, 1998; Yonemura et al., 1962). Oscillatory potentials
are strongly dependent on normal retinal circulation and are
drastically affected by acute disturbances occurring in areas supplied by central retinal vessels (Speros and Price, 1981). A pathological oscillatory potential response indicates a neuronal
dysfunction affecting the inhibitory feedback pathways and/or reveals a pathological microcirculation in the inner retina that induces neuronal damage (Wachtmeister, 1998). In some cases of DR
with severe microangiopathy, the oscillatory potentials may be
selectively reduced or absent, while the amplitude of the a- and bwaves of the ERG remains normal (Speros and Price, 1981).
Ganglion cells have very little influence on the scotopic ERG
responses to bright stimulus flashes. However, both positive and
negative components of the scotopic threshold responses depend
directly upon intact ganglion cell function (Bui and Fortune, 2004).
Thus, the scotopic threshold response is reduced after substantial
RGC loss following the induction of experimental glaucoma (Bui
and Fortune, 2004; Frishman et al., 1996). On the other hand, the
photopic ERG also reflects ganglion cell signals and may serve as an
additional useful test of ganglion cell function (Bui and Fortune,
2004). Thereby, the photopic negative response has been found
to be sensitive to experimental glaucoma in monkeys (Viswanathan
et 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 electroencephalographic activity in the visual cortex, in response to
visual stimulation (Odom et al., 2010; Sokol, 1976). Since the visual
cortex is activated primarily by the central part of the visual field,
VEPs depend on the functional integrity of central vision at any
level of the visual pathway, including the eye, retina, the optic
nerve, optic radiations, and occipital cortex (Odom et al., 2010). In
addition to detecting anterior visual pathway dysfunctions, chiasmal and retro-chiasmal dysfunctions can be assessed by examining
the distribution of the VEP over the posterior regions of the scalp
(Holder, 2001). Thus, VEPs can be valuable in diagnosing optic
neuropathies, non-organic visual loss and assessing visual function
in infants or children (Young et al., 2012). Moreover, VEP results can
be predictive of visual recovery in traumatic optic neuropathy
(Young et al., 2012).
Multifocal VEPs allow identify spatially localized damage and
pathologies that may be missed with a traditional single VEP (Creel,
2012; Hood et al., 2003). The multifocal VEP is used to study visual
field defects caused by ganglion cell or optic nerve damage (Betsuin
et 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; Hood
and Greenstein, 2003; Klistorner et al., 2007).
2.6.3. Psychophysical methods
Visual acuity, the single most-widely used eye test, is the capacity for spatial resolution of the visual system (Kalloniatis and
Luu, 1995; Westheimer, 1965), namely, the ability of the visual
system to discriminate between two stimuli separated in space,
with a high contrast in relation to the background (Kniestedt and
Stamper, 2003). Visual acuity represent a practical tool for tracing
the course of ophthalmic dysfunction and therapy (Westheimer,
2009).
More recently, contrast sensitivity has been proposed as a
valuable addition to the psychophysical tests currently available.
Contrast sensitivity refers to a measure of how much contrast a
person requires to see a target (Owsley, 2003). Contrast sensitivity
plays a role in many aspects of vision, specifically motion detection,
visual field, pattern recognition, adaptation to darkness and visual
acuity (Richman et al., 2013). Contrast sensitivity loss is not specific
to any particular diagnosis, as many diseases have similar effects on
the contrast sensitivity function. However, contrast sensitivity
testing is a valuable tool for identifying ocular disease and guiding
treatment (Richman et al., 2013). Studies have been conducted on
the use of contrast sensitivity to evaluate intraocular lenses, and
pathologies such as cataracts, glaucoma, optic neuritis, DR and
AMD, 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 different
approaches. The optomotor test lets generate a psychophysical
threshold in a reduced amount of time, and does not involve the
failure of older animals to learn a task (Douglas et al., 2005; Prusky
et al., 2004). Pigmented animal responses are stronger and easier to
recognize than those of albino mice or rats, which do not show clear
responses to the optomotor test. The optomotor test has been used
as 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 and
metabolic pathways. These alterations occurring at molecular
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
levels produce changes in the function and morphology of the
retinal cells generating, as a consequence, retinal dysfunction
accompanied in most of cases with an evident abnormal structure
of the retina (Fig. 8). This is the general retinal remodeling phenomenon, however, depending on the nature and progression of
the disease, variations exist in the cell types that undergo this
remodeling, as well as in the rate at which these changes occur.
A better knowledge of the remodeling alterations underlying
the different types of retinal diseases and injuries could serve to
clarify how is retinal degeneration in each particular situation,
being useful for the proper diagnoses and prognoses of each pathology. In this context, it is important an adequate characterization of the evolution of the degenerative process (retinal
remodeling stage) occurring in each pathology with the aim to
apply the best therapeutic strategies to maintain or to rescue visual
function. Interestingly, the scientific community has coined the
‘retinal degeneration phase’ term in order to standardize appropriate links between morphological changes of the different cell
types of the retina and the degeneration stage in each type of
disease (Jones et al., 2012; Jones and Marc, 2005; Marc and Jones,
2003; Marc et al., 2007, 2003; Vugler, 2010). Thus, the establishment of the different degeneration phases in each disease using the
high resolution OCT technology could allow ophthalmologists to
determine the patient retinal degeneration stage for providing the
most effective treatment.
We propose the following 4 phases of retinal remodeling
(Fig. 16). The appropriate therapy for each degeneration phase will
be discussed later in section 4.8.
3.1. Phase 1
During this phase, retinal function and morphology seem
normal. No visual clinical symptoms can be recognized at this stage,
as occurs in early RP and the first stages of AMD or DR. Increased
intraocular pressure may induce cellular stress at the beginning of
glaucoma onset, but no signs of the disease are observed. Photoreceptor stress induces a cascade of events that culminates in
molecular changes and eventual cell death at the end of this phase.
Although photoreceptors are subject to a cell stress caused by genetic mutations (RP), disruption of the RPE (AMD) or changes in
glucose concentration (DR), these circumstances do not affect the
function and morphology of photoreceptor cells, and retinal neurons and layering appear normal (Fig. 16).
3.2. Phase 2
Cellular stress and the activation of apoptotic pathways lead to
progressive photoreceptor loss during this phase. At this stage
(Fig. 16), photoreceptor pyknotic nuclei may be present in different
numbers, depending on the disease or injury, and reactive changes
in glial cells (gliosis) can be detected.
Prior to photoreceptor cell death, one of the earliest histological
indicators of pathology in this phase is the delocalization of both
rhodopsin in rods and transducin in cones (Roof et al., 1994). Opsin
delocalization is a common feature in the majority of retinal
degenerative diseases in both animal models and humans, as has
been 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 outer
segments of rods. However, in animal models of RP (FernandezSanchez et al., 2011a; Martinez-Navarrete et al., 2011) and in human RP retinas (Fariss et al., 2000) rhodopsin extends from the
photoreceptor inner segments down to the cell bodies, axon processes and axon terminals (Fig. 4C). Rhodopsin redistribution to the
inner segments and cell bodies in the ONL, in addition to a decrease
47
in the length of rod outer segments, has been described in cases of
human retinal detachment with proliferative vitreoretinopathy
(PVR). Cone photoreceptors also displayed redistribution of opsins to
the 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 specimens
also showed outer retina degeneration. The outer and inner photoreceptor segments were markedly disrupted, with swollen inner
segments and pyknotic nuclei (Charteris et al., 2007). Besides, it
seems that in diseases where cones degenerate secondarily to rods,
the lack of a trophic factor secreted by rods called rod-derived cone
viability factor play an important role (Leveillard et al., 2004).
Stressed photoreceptors begin to deconstruct their synaptic
terminals, with a loss of bassoon and synaptophysin immunostaining (Figs. 5e7). The lack of synaptic signaling input also triggers a range of rewiring events, including retraction of bipolar and
horizontal cell dendrites, switching of synaptic targets by bipolar
cells, anomalous extension of horizontal cell processes into the IPL
and retraction of horizontal cell axon tip terminals. The retraction
of rod photoreceptor synaptic spherules and the subsequent
sprouting of rod bipolar dendrites that selectively reconnect to
appropriate target neurons suggest the existence of functional
plasticity, even in the aged human retina (Fig. 8). Neurons in retinal
tissues from AMD human eyes have the capacity to remodel by
sprouting processes and to re-form demonstrable synaptic complexes with appropriate targets (Sullivan et al., 2007). Retinectomy
specimens also demonstrated disruption of rod bipolar cell body
stratification 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 organotypic culture retinas and in different animal models of retinal
detachment, such as cat (Cook et al., 1995), rabbit (Lewis et al.,
2009) and primate models (Kroll and Machemer, 1968, 1969). The
degeneration process in primates is slower than in the cat model,
suggesting that human and other primate photoreceptors may be
more resistant than other species to the degenerative pathology
induced by detachment. In this phase, molecular changes in
glutamate receptor expression at bipolar cell dendrites begin to
modify the physiology of bipolar cells, shifting their functional
phenotypes from ON to OFF responses (Jones et al., 2012). The
degree of degeneration of the inner retina during this period depends on the characteristics of the degenerative pathology. In many
of the animal models examined, there is extensive rewiring or
reprogramming of inner retinal cells (Jones et al., 2003; Marc and
Jones, 2003; Marc et al., 2007, 2003).
The glial response in this phase is characterized by a marked
hypertrophy of the cell processes. In association with hypertrophy
and hyperplasia, glial cells enhance their protein production, as
shown by the increased expression of the intermediate filament
protein GFAP. Glial cell activation can be identified through GFAP
expression immunostaining (Fig. 12DeE and 13). Both animal
models and humans show a high level of GFAP in glial cells in all
retinal diseases, including RP, glaucoma, retinal detachment and
AMD (Madigan et al., 1994; Rodrigues et al., 1987; Wang et al., 2002;
Wu et al., 2003). Microglia activation is also noteworthy in animal
models of RP (Fig. 11), retinal detachment and glaucoma (Bosco
et al., 2011; Lewis et al., 2005), and in the retinas of patients with
RP (Gupta et al., 2003). In early anterior PVR, Müller cells express
upregulated 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 maintain
some function, but are in a process of degeneration. The other cells
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N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
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 changes
occurring in these cells during retinal degeneration. In the early stages (Phase 1) of retinal degenerative processes, changes in cellular and environmental homeostasis induce
molecular 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 are
neuroprotection 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 characterized 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 hypertrophy 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 retinal
degeneration (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 of
amacrine 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 remodeling
between all postsynaptic neurons occurs, forming microneuromas. In advanced stages of degeneration, cell death progresses, hypertrophy of Müller cells continues and microglial
activation 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, the
most suitable therapies are neuroprotection and electronic retinal implants. CR: Choroid, RPE: Retinal pigment epithelium; OS: outer segments; IS: inner segments; ONL: outer
nuclear 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 increases, as does the number of activated microglial cells with an
amoeboid shape. Apoptosis in second-order retinal neurons is
common during this period (Fig. 16).
Animal models of RP show a severe loss of photoreceptors, with
a 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 distal
scaffolding of Müller cells in the absence of photoreceptor and bipolar cells form a cell seal that isolates the neural retina from the
RPE and choroid. At this stage, blood vessels deteriorate and
respond to the lack of oxygen by retracting or producing new
vessels (see section 2.4 and Fig. 15). The loss of the photoreceptor
layer may result in the disappearance of the external limiting
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
membrane, formed by zonula adherens junctions between photoreceptors and Müller cell processes. Along with astrocytes and
migrated RPE cells, Müller glia proliferate and extend their processes to the INL, becoming part of the cellular components of
epiretinal membranes in eyes with RP (Bringmann and
Wiedemann, 2009; Szamier, 1981). Another common finding during this phase is the translocation of cell bodies to other retinal
layers (e.g., Müller cell bodies located outside the ONL) (Marc et al.,
2003). Finally, microglia can play an active role in phagocytosis of
degenerated retinal cells, including neurons. Bipolar cells are
completely deafferented in phase 3, not only physiologically,
through the redistribution of glutamate receptors in the bipolar cell
bodies and axons, but also anatomically, through the physical
retraction of all bipolar cell dendrites, resulting in severely altered
morphologies (Figs. 8 and 16) (Barhoum et al., 2008; Cuenca et al.,
2005b; Jones and Marc, 2005). These changes result in a complete
loss of glutamatergic input after degeneration of rod photoreceptors, as seen in different animal models, such as P23H or rd mice
(Cuenca et al., 2004; Strettoi et al., 2004), which undergo programmed cell death in various retinal diseases.
Photoreceptor death is evident in severe human PVR, and the
remaining patchy photoreceptors show abnormalities, such as the
loss of their outer segments. Synaptophysin staining is weak at the
IPL 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 cell
processes extending into the ONL (Sethi et al., 2005).
3.4. Phase 4
Clinical examination at this stage shows the absence of visual
function due to the loss of all retinal photoreceptors (Fig. 16). A
persistent and global remodeling and rewiring of retinal circuitries
characterize this phase. Amacrine and bipolar cells migrate into the
ganglion 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 between them. This new rewiring of retinal cells leads to unusual
visual circuitries (Jones and Marc, 2005). Conversely, in many retinas at this phase, RGCs can be observed migrating into the INL, and
some processes form fascicles that can run in bundles for great
distances within the neural retina (Jones et al., 2012; Jones and
Marc, 2005). The hypertrophy and migration of Müller cells
generate a distort lamination of the INL and OPL (Jones and Marc,
2005).
Retinas in patients with late-stage RP show dramatic changes in
their architecture, with a complete loss of rod and cone photoreceptors and bipolar cells, and subsequent topological restructuring
of the retina. New microneuromas with amacrine cells abutting the
choroid can be seen with no clear barrier in between, due to the
absence of RPE (Jones et al., 2012). During this phase, RPE cells
migrate into the retina, often with accompanying choroidal vessels,
passing through gaps in the glial seal, and displacing INL cells (Jones
rez et al., 1998).
and Marc, 2005; Villegas-Pe
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 epiretinal membranes is also evident (Sethi et al., 2005). Long-term
rhegmatogenous retinal detachment also leads to retinal remodeling, characterized by the loss of first- and second-order retinal
neurons, disruption of the entire retinal lamination and gliosis
(Ghosh and Johansson, 2012).
In the later stage of degeneration, breaks in Bruch's membrane
provide opportunities for some neurons to migrate out of the
neural retina into the choriocapillaris membrane complex (Fig. 16).
49
It is unclear whether similar migration events outside the neural
retina occur in human diseases, such as AMD. Breaches of Bruch's
membrane certainly occur in the late forms of AMD that have
vascular involvement, and choroidal vessels use these holes to
enter the retina (Fig. 15C). Calcification of Bruch's membrane has
also been shown following the breakdown of elastin and collagen in
non-vascular or dry AMD (Spraul et al., 1999).
Commonly, retinal remodeling is a secondary process that takes
place after cell death. However, it has been described remodeling of
retinal synaptic circuitries without neuronal death. Cell disappearance, cell migration, disruption of spatial cell patterning,
deregulation of structural stability, de novo synaptic repatterning
and glial activation are common features during remodeling (Marc
et al., 2003). Changes similar to those observed in the animal
models have also been found in human tissue samples, indicating
that retinal remodeling is a general principle in all retinal diseases
and species.
Knowledge of the exact phase of retinal degeneration in each
pathology is essential in order to choose the appropriate therapy
aimed at recovering vision (Fig. 16). The therapeutic approach has
to be selected based on the exact anatomical status. If there are still
functional cells and the circuitries remain in place, the probability
of success in the applied therapies is higher than in more advanced
retinal degenerative stages. After the loss of retinal cells and gliosis,
once synaptic markers have been lost and the connections among
neural retinal cells are impaired, the options are clearly reduced,
because no information will be transmitted through the remaining
altered circuitries. Regardless of the therapeutic approaches available, a careful study of the status of the retina should first be performed to ensure that the treatment will actually work. As an
example, a cell-based therapy using a selective cell type like photoreceptors would only work if the remaining cells are in good
shape. If the normal architecture of the retina has clearly disappeared, other options should be chosen, which do not require the
presence of a normal retinal circuitry; these might focus on
improving and preserving the remaining cells with neurotrophic
factors or using other approaches, such as retinal implants.
The key window for treatment will always be the type and
phase of the degeneration. For all therapeutic approaches, the
sooner they are begun, the better the results will be. The
mammalian retina clearly has a vast repertoire of cellular responses
to injury, the understanding of which may help us improve current
therapies or devise new ones to treat conditions resulting in
blindness.
4. Therapeutic approaches in neurodegenerative diseases
Neurodegeneration is a common process in several retinal diseases. In this context, neuroprotective treatments provide therapeutic strategies independent of the etiology of the degeneration.
The aim of neuroprotective mechanisms is to provide an adequate
environment in which to prolong the viability of retinal cells
through their effects on a number of biochemical pathways. This
can be achieved by either delivering neurotrophic growth factors to
retinal tissues, inhibiting pro-apoptotic pathways or implementing
viability factors, such as the rod-derived cone viability factor.
Therapeutic approaches in the fight against retinal diseases and
vision loss also include gene- and cell-based therapies, as well as
retinal transplants.
4.1. Efficacy of anti-apoptotic therapies for retinal diseases
The final common pathway of cell death in retinal diseases is
apoptosis, which initially affects only certain retinal cells, such as
photoreceptors, followed by the apoptosis of all remaining cells in
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the retina. In this context, the pharmacologic inhibition of cell
death through the use of anti-apoptotic agents may prevent
disease-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 for
thousands ofs. Synthetic TUDCA has been shown to exhibit antiapoptotic properties in neurodegenerative diseases, including
those affecting the retina. Systemic administration of TUDCA has
been demonstrated to slow retinal degeneration in both the rd10
autosomal recessive RP mouse model (Boatright et al., 2006; Drack
et al., 2012; Oveson et al., 2011; Phillips et al., 2008) and in a LIRD
mouse model (Boatright et al., 2006; Oveson et al., 2011). In these
two retinal degeneration models, TUDCA-treated animals were
shown to maintain better visual function, thicker ONL and better
preservation of outer segments than untreated animals. TUDCA
also prevented retinal degeneration in the P23H autosomal dominant RP rat model (Figs. 17G and 18C) (Fernandez-Sanchez et al.,
2011a). P23H treated rats showed higher a- and b-wave amplitudes under both photopic and scotopic conditions than untreated
rats. Furthermore, TUDCA decreased photoreceptor apoptosis
(Figs. 17G and 18C) and maintained synaptic connectivity (Fig. 18C,
F, J) among retinal cells (Fernandez-Sanchez et al., 2011a). In
addition to its anti-apoptotic properties, TUDCA has also been
shown to exert anti-inflammatory, antioxidant and chaperone activities. In this context, TUDCA suppressed the formation of laserinduced CNV in rats by decreasing the number and size of CNV
lesions, probably due to its anti-inflammatory properties, which
diminished VEGF levels in the retina after the laser treatment (Woo
et al., 2010). Additionally, systemic administration of TUDCA preserved photoreceptors after retinal detachment in rats, preventing
the reduction in ONL thickness, and this was accompanied by
decreased oxidative stress and inhibition of the increase in caspase
3 and 9 activity (Mantopoulos et al., 2011). TUDCA also protected
retinal neural cell cultures from high glucose-induced death by
decreasing mitochondrial-nuclear translocation of the apoptosis
inducing factor (AIF). This inhibition of the release of AIF from the
mitochondria was probably due to the antioxidant properties of
TUDCA, as corroborated by the marked decrease in oxidative stress
biomarkers with TUDCA treatment (Gaspar et al., 2013). These
findings may have relevance in the treatment of DR. Furthermore,
systemic injection of TUDCA diminished endoplasmic reticulum
stress, prevented apoptosis and reduced cone degeneration in the
Fig. 17. Neuroprotective treatments prevent photoreceptor cell degeneration during retinal diseases. Vertical sections of Sprague Dawley (SD) and P23H rat retinas (P120) labeled
with antibodies against g-transducin (cone cells; green) and recoverin (cones, rods and some bipolar cells; red). Few rows of photorecpetors remain at 4 month of age in P23H rats
(B). Note that retinas from P23H rats treated with the neuroprotective compounds tauroursodeoxycholic acid (TUDCA) (G) or safranal (C, F) show more rows of photoreceptors than
those from untreated P23H rats (B, E). The morphology of cones is preserved in P23H rats treated with safranal (F) and TUDCA (G). OS: outer segments; IS: inner segments; ONL:
outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar: 20 mm.
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
retina of the Lrat/ mouse model of Leber congenital amaurosis
(Zhang et al., 2012b).
4.1.2. Rasagiline
Rasagiline (N-propargyl-1-(R)-aminoindan) is a selective
monoamine oxidase inhibitor with proven neuroprotective effects
in the retina of prph2/rds mice, an animal model of RP, through a
delay in the activation of caspase 3 dependent apoptotic pathways
and the induction of the anti-apoptotic protein Bcl-XL (EigeldingerBerthou et al., 2012). This study also showed that rasagiline affects
the induction of autophagy and reduces inflammatory activity in
the retina. Rasagiline has also provided for significantly enhanced
survival of RGCs in the translimbal photocoagulation model of
experimental glaucoma in Wistar rats (Levkovitch-Verbin et al.,
2011).
4.1.3. Norgestrel
Norgestrel, a synthetic progestin used in oral contraceptives
with effects similar to progesterone, exhibited neuroprotective
properties in two distinct animal models of RP: the acute lightinduced degeneration model and the more chronic rd10 mouse
model. In both models, norgestrel preserved both photoreceptor
cell number and morphology to a significant degree and, as a
51
consequence, improved ERG responses (Doonan and Cotter, 2012;
Doonan et al., 2011). The neuroprotective mechanism of action is
likely to involve the increased expression and activation of bFGF
and the extracellular signal-regulated kinases 1/2 (Erk1/2).
4.1.4. Proinsulin
Transgenic expression of human proinsulin in the rd10 mouse
model of RP attenuated retinal degeneration, as determined by the
histological preservation of photoreceptors and ERG responses.
Systemic proinsulin was able to reach the retinal tissue, delay the
apoptotic death of photoreceptors and decrease oxidative damage
(Corrochano et al., 2008). Furthermore, intramuscular injection of
an adeno-associated viral vector serotype 1 expressing human
proinsulin in the P23H rat model of RP attenuated retinal degeneration by preserving cone and rod structure and function, together
with their contacts with postsynaptic neurons (Fernandez-Sanchez
et al., 2012b).
4.2. Efficacy of antioxidant and anti-inflammatory agents
There are numerous compounds found in nature that contain
active ingredients with beneficial properties for the improvement
and/or prevention of various eye diseases affecting vision. In this
Fig. 18. Synaptic connectivity is preserved by neuroprotective treatments. Vertical sections of Sprague Dawley (SD) and P23H rat retinas (P120) labeled with antibodies against PKCa (ON-rod bipolar cells), calbindin (horizontal cells) and bassoon (synaptic ribbons). Nuclei stained with TO-PRO. Treatment of P23H rats with the neuroprotective compounds
tauroursodeoxycholic acid (TUDCA) (C, F, J) or safranal (G, K) reduces the photoreceptor cell death (compare photoreceptor rows in C and B) and the loss of synaptic contacts
between synaptic ribbons of photoreceptors (red) and dendrites of bipolar cells (compare F and G with E), and between synaptic ribbons of photoreceptors (red) and terminal tips of
horizontal cells (compare J and K with I). ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar:
20 mm.
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N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
context, antioxidant and anti-inflammatory attributes make them
useful compounds for the pharmacological treatment of retinal
diseases.
4.2.1. Curcumin
Curcumin is a natural polyphenolic yellow pigment isolated
from the rhizomes of the plant Curcuma longa with well-known
anti-inflammatory and antioxidant properties. In rat models of
LIRD, dietary addition of 0.2% curcumin during a period of two
weeks evidenced retinal neuroprotection through the inhibition of
NFekB activation and down regulation of inflammatory genes
(Mandal et al., 2009). Further studies in retina-derived cell lines
661W and ARPE-19 showed that treatment with curcumin protected cells from hydrogen peroxide (H2O2) oxidative stressinduced cell death through the reduction of ROS levels, mediated
by an increase in the expression of the oxidative stress defense
enzymes 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 H2O2
oxidative stress showed that curcumin alters the expression of
H2O2-modulated miRNAs and, as a consequence, regulates the
expression of their target genes, resulting in an increased expression of antioxidant genes and a reduction of angiotensin II type 1
receptor, NFekB and VEGF expression at the mRNA and protein
levels (Howell et al., 2013). DR studies on streptozotocin-induced
diabetic rats have evidenced the protective effects of curcumin in
the retina, where it exerts a positive modulation of the antioxidant
system through the regulation of glutathione, superoxide dismutase and catalase levels (Gupta et al., 2011). On the other hand,
curcumin also prevents the increase in pro-inflammatory cytokines
IL-1b, NFekB, TNF-a and VEGF, as well as the structural degeneration of the diabetic rat retina (Gupta et al., 2011; Kowluru and
Kanwar, 2007; Mrudula et al., 2007). Furthermore, recent studies
have shown that curcumin protects retinal Müller cells in rats
suffering from the early stages of diabetes (Zuo et al., 2013). Curcumin has also demonstrated its neuroprotective activity against
NMDA toxicity in primary retinal cell cultures, possibly related to an
increase in the NMDA receptor type 2A subunit (Matteucci et al.,
2011). Curcumin also exhibits beneficial effects on neuronal and
vascular degeneration in the retina after ischemia and reperfusion
injury. In this sense, in the presence of ischemia and reperfusion
stimuli, curcumin was able to inhibit ganglion cell loss, and to
prevent the degeneration of retinal capillaries, probably through its
inhibitory effects on the injury-induced activation of NFekB and
STAT3, and on the overexpression of monocyte chemoattractant
protein-1 (Wang et al., 2011a). Curcumin also suppressed Nmethyl-N-nitrosourea-induced photoreceptor cell apoptosis in
SpragueeDawley rats through inhibition of DNA oxidative stress
(Emoto et al., 2013). Besides its anti-inflammatory and antioxidant
properties, curcumin also exhibits anti-protein aggregating activity.
In this context, an improvement in retinal morphology, physiology
and gene expression and localization of rhodopsin has been
observed in the P23H transgenic rat model of autosomal dominant
RP treated with curcumin (Vasireddy et al., 2011).
4.2.2. Lutein and zeaxanthin
Lutein and zeaxanthin are carotenoids that are referred to as
macular pigments, due to their presence in the human macula. It is
suggested that these carotenoids may protect the macula and outer
retinal photoreceptor segments from oxidative stress by triggering
the antioxidant cascade that disables ROS (Krinsky et al., 2003;
Ozawa et al., 2012). Studies on cultured ARPE-19 cells showed evidence that supplementation with lutein and zeaxanthin reduced
photo-oxidative damage and inhibited the expression of
inflammation-related genes in RPE cells (Bian et al., 2012a, 2012b).
Similarly, both carotenoids protected photoreceptors from oxidative stress-induced apoptosis in rat retinal neurons in culture
(Chucair et al., 2007). Additionally, lutein and zeaxanthin both act
as light filters in the eye, absorbing the blue-light that enters the
retina, hence effectively protecting the retina from LIRD during
acute light exposure and in the presence of bright light (Barker
et al., 2011; Ozawa et al., 2012). Furthermore, results from various
epidemiological studies have shown inverse associations between
the amount of macular pigment and the incidence of AMD. Along
the same lines, clinical studies have shown that supplementation
with lutein and zeaxanthin improves visual function and prevents
the progression of the pathology in patients with early AMD (Chew
et al., 2014; Ma et al., 2012a, 2012b; Sabour-Pickett et al., 2012).
Similarly, clinical studies evidenced that zeaxanthin improves visual function in older male patients with AMD (Richer et al., 2011).
Likewise, the neuroprotective effect of lutein on outer retinal cells
in AMD may also play a relevant role in the protection of the inner
retina from acute ischemic damage, as demonstrated by the
reduction in oxidative stress and apoptotic death in a rodent model
of ischemia/reperfusion (Dilsiz et al., 2006; Li et al., 2009).
4.2.3. Saffron
The active ingredients of the spice saffron (safranal, crocin and
crocetin) are known antioxidant carotenoids. Crocetin prevented
retinal degeneration induced by oxidative and endoplasmic reticulum stresses via the inhibition of caspase 3 and 9 activity in the
RGC-5 retinal ganglion cell line in vitro and in a LIRD mice model
in vivo (Yamauchi et al., 2011). Crocin protected retinal photoreceptors against light-induced cell death in primary cell cultures
from primate and bovine retinas (Laabich et al., 2006). In albino rats
fed saffron supplements, the effects of continuous bright light
exposure were significantly diminished, and the morphology and
function of the retina were maintained (Maccarone et al., 2008;
Marco et al., 2013; Natoli et al., 2010). Crocetin inhibited retinal
ischemic damage in mice, preventing the apoptotic death of ganglion cells and the reduction of the INL by inhibiting the activation
of p38, JNK, NFekB and c-Jun, while maintaining at the same time
the functional activity of the retina (Ishizuka et al., 2013). In a
similar manner, crocin prevented retinal ischemia/reperfusion
injury-induced apoptosis of RGCs in rats by activating the PI3K/AKT
signaling pathway (Qi et al., 2013). Dietary supplementation with
safranal in the P23H rat model of RP slowed photoreceptor cell
degeneration (Fig. 17C, F and 18G, K) and ameliorated the loss of
retinal function and vascular network disruption (FernandezSanchez et al., 2012a). Crocetin also prevented NMDA-induced
murine retinal damage by inhibiting both caspases 3/7 activation
and the increased expression of cleaved caspase 3 in the GCL and
INL (Ohno et al., 2012). Additionally, in clinical trials involving
human patients with early AMD, 20 mg per day of saffron supplementation for 90 days significantly improved some parameters of
the macular photopic flash electroretinogram, such as amplitude
and modulation threshold (Falsini et al., 2010).
4.2.4. Catechins
Catechins are a group of polyphenolic antioxidants commonly
found in green tea. The most abundant catechin in green tea is
epigallocatechin gallate (EGCG), which has extremely strong antioxidant properties. Previous studies involving intraocular injection
of EGCG with sodium nitroprusside showed a protective effect on
rat retinal photoreceptors, indicating that EGCG may benefit patients suffering from ocular diseases involving oxidative stress
(Zhang and Osborne, 2006). Oral administration of EGCG to
ischemia/reperfusion rat models reduced many of the induced
damaging effects, including the activation of caspases, the reduction in the ERG a- and b- wave amplitudes, the decrease in RGC and
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
photoreceptor specific proteins, the increase in GFAP protein and
the decrease in optic nerve proteins associated with ganglion cell
axons (Zhang et al., 2008, 2007). Moreover, dietary administration
of EGCG reduced light-induced retinal neuronal death in albino rat
models (Costa et al., 2008). EGCG also reduced apoptotic death in
the 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 has
the ability to inhibit RPE cell migration and adhesion, thereby
providing potential preventive actions against AMD (Alex et al.,
2010; Chan et al., 2010). Furthermore, administration of EGCG in
rats 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 to Wistar
rats resulted in a significantly lower NMDA-related loss of RGCs
(Chen et al., 2012). Interestingly, studies in patients with openangle glaucoma treated with oral EGCG suggest a beneficial influence of this compound on inner retinal function (Falsini et al.,
2009).
4.2.5. Ginkgo biloba extract
Ginkgo leaves contain two main active ingredients: flavonoids
and terpenoids. The main properties of G. biloba extract are protection against free radical damage and lipid peroxidation. In
Sprague Dawley rats that were administered G. biloba extract prior
to and/or on a daily basis following experimental optic nerve crush,
the survival rate of RGCs was significantly higher than in control
animals (Ma et al., 2009, 2010). Studies on rodents showed that the
standardized extract of G. biloba EGb761 partially inhibited
apoptosis of photoreceptor cells, increasing the cell survival rate
after light-induced retinal damage of photoreceptors (Qiu et al.,
2012; Xie et al., 2007). Similarly, EGb761 showed a protective effect on human RPE cells (ARPE-19) under light-induced damage
stimuli by acting on HSP70, cathepsin B and cytochrome c reductase modulation (Zhou et al., 2014). EGb761 also protected the
retina against ischemia-reperfusion damage via its free radicalscavenging and anti-lipoperoxidative properties, as well as its
regulation of mitochondrial respiratory function (Clostre, 2001). In
addition, EGb761 was found to provide a neuroprotective benefit
for RGCs in a rat model of chronic glaucoma (Hirooka et al., 2004).
G. biloba is also believed to have good therapeutic potential in cases
of normal tension glaucoma, where the disease continues to
progress despite surgically normalized intraocular pressure
(Cybulska-Heinrich et al., 2012). In retinal explants, ginkgolide B, a
component of EGb761, has also been demonstrated to promote RGC
axon growth and to decrease cellular RGC apoptosis through the
inhibition of caspase 3 activity (Wang et al., 2012). In addition, a
fortified extract containing G. biloba extract (among other components) attenuated retinal inflammation in early streptozotocininduced diabetic rats by decreasing TNF-a and VEGF cytokine
levels (Bucolo et al., 2013).
4.2.6. Resveratrol
Resveratrol is a polyphenol contained in red wine with strong
antioxidant properties. Many studies have evidenced that resveratrol reduces diabetes-induced early vascular lesion, VEGF, and
oxidative stress in rat and mice models (Kim et al., 2012; Yar et al.,
2012). Resveratrol administered to streptozotocin-induced diabetic
rats significantly reduced the enhancement of oxidative markers
and superoxide dismutase activity in the retina. Moreover, resveratrol improved the elevated levels of NFekB activity and the
apoptosis rate, and prevented the reduction in thickness of the
retina (Soufi et al., 2012). Resveratrol has also been shown to be
effective in decreasing vascular lesions and VEGF induction in
mouse retinas during the early stages of diabetes (Kim et al., 2012).
This polyphenol also prevented diabetes-induced RGC death via
53
CaMKII down-regulation in streptozotocin-diabetic mice (Kim
et al., 2010). Trans-resveratrol inhibited hyperglycemia-induced
low-grade inflammation and connexin down-regulation in RPE
cultured cells through inhibition of VEGF, TGF-b1, COX-2, IL-6 and
IL-8 accumulation, PKCb activation, Cx43 degradation and
enhanced intercellular gap junction communication (Losso et al.,
2010). Resvega, a nutritional complex containing resveratrol, has
also been demonstrated to provide beneficial effects for the prevention of CNV in a murine model of laser-induced CNV (Fernandez
et al., 2013). It has also been demonstrated that resveratrol
administration to rat models of retinal detachment prevents
photoreceptor cell death via the upregulation of FoxO family protein levels (FoxO1a, FoxO3a, FoxO4) and by blocking caspase 3, 8
and 9 activation (Huang et al., 2013b). In an ongoing clinical study,
preliminary observations on the human retina in octogenarian
AMD patients taking a daily resveratrol-based nutritional supplement showed an anatomic restoration of retinal structure, which
suggested an improvement in choroidal blood flow, and as a
consequence, better visual function with the treatment (Richer
et al., 2013). RPE cell death occurs early in the pathogenesis of
AMD, and for this reason, protecting these cells is essential in
treating the disease. In this context, in RPE cell lines, resveratrol has
been shown to defend against oxidative stress (King et al., 2005;
Pintea et al., 2011; Sheu et al., 2008), oxysterol-induced cell death
and VEGF secretion (Dugas et al., 2010), and to inhibit RPE cell
migration in a dose-dependent manner (Chan et al., 2013). On the
other hand, resveratrol prevented LIRD in mouse models by suppressing the activation of retinal activator protein-1 and promoting
retinal sirtuin activity (Kubota et al., 2010). Furthermore, resveratrol prophylactic treatment reduced ischemia-mediated thinning of
the entire retina and in particular the inner retinal layers, attenuating ischemic-induced loss of retinal function in rats (Vin et al.,
2013). Since retinal ischemia is a major factor in close-angle glaucoma and DR, resveratrol could be a potentially useful drug for
vascular dysfunction in the retina (Li et al., 2012a). The aforementioned properties of resveratrol could prove essential in establishing innovative treatments and preventive interventions for major
ocular diseases, such as AMD, glaucoma and DR, since oxidative
stress is an integral part of the pathophysiology of those diseases.
4.2.7. Quercetin
Quercetin is a flavonoid with anti-inflammatory and antioxidative properties found in a variety of plant foods, including
black and green teas, Brassica vegetables and many types of berries.
Recent studies on the human RPE cell line ARPE-19 have demonstrated the protective effects of quercetin against oxidative stress
through the inhibition of pro-inflammatory molecules, as well as
direct inhibition of the intrinsic apoptosis pathway (Cao et al.,
2010). In vitro studies using the RF/6A rhesus choroids-retina
endothelial cell line treated with quercetin showed a dosedependent inhibition of cellular migration and tube formation,
which are important steps in retinal angiogenesis, characteristic of
AMD (Chen et al., 2008). Further studies on human cultured RPE
cells showed similar results. In this context, quercetin treatment
followed by oxidative damage reduced cellular deterioration and
senescence occurring in AMD in a dose-dependent manner, probably by inhibiting the up-regulation of caveolin-1 (Kook et al.,
2008). Similarly, quercetin significantly reduced ROS production
by 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 reducing
oxidative damage (Lee et al., 2011). In addition, NAC administered
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N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
to a rat model of DR diminished the plasma markers of oxidative
stress and inflammation (15-F(2t)-isoprostane and TNF-a, respectively), helping to minimize early events in DR (Tsai et al., 2009).
Moreover, NAC supplementation to rats with induced ocular hypertension ameliorated the retinal oxidative damage through the
maintenance of glutathione peroxidase and catalase levels, and the
inhibition of retinal peroxidation (Ozdemir et al., 2009). NAC also
prevented the increased expression of p53 and caspase 8, induced
by long-term maintained hypoxia in bovine RPE primary cell cultures (Gerona et al., 2010), which makes it of great interest in oxygen stress-related diseases, such as AMD and other senescenceassociated pathologies. In this context, hypoxia-induced cell
death in the RGC-5 cell line was significantly counteracted by pretreating cells with NAC, which targets the hypoxia-inducible factor1a pathway via the BNIP3 and PI3K/Akt/mTOR pathways (Yang
et al., 2012).
4.2.9. Antioxidant cocktails
The administration of a cocktail of antioxidants (including atocopherol, ascorbic acid, Mn(III)tetrakis (4-benzoic acid)
porphyrin, and a-lipoic acid) to three mouse models of RP (rd1, rd10
and Q344ter) reduced the levels of oxidative damage markers in
cones and, as a consequence, preserved cone density (Komeima
et al., 2007, 2006). Additionally, this mixture of antioxidants
slowed rod cell death, thus maintaining photoreceptor function, as
demonstrated by the larger a- and b-wave amplitudes as compared
to untreated animals (Komeima et al., 2007, 2006). The use of antioxidants in a combination consisting of lutein, zeaxanthin, alipoic acid and reduced L-glutathione (GSH) in rd1 mice drastically
reduced the number of rod photoreceptors displaying oxidatively
damaged DNA, and significantly delayed the degeneration process
(Miranda et al., 2010; Sanz et al., 2007). Thiol contents and thioldependent peroxide metabolism seem to be directly related to
the 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 to
promote cell proliferation, maturation, survival and/or regeneration, thereby maintaining overall cell homeostasis (Snider and
Johnson, 1989; von Bartheld, 1998). In the eye, the major sources
of these molecules are retinal RPE and Müller cells. Exogenous
administration of these pro-survival factors, either singly or in
combination, has been used in attempt to ameliorate retinal
degeneration. It is important to note that the short half-life of
neurotrophic factors makes iterative intravitreal injections necessary. To solve this problem, researchers have developed new strategies for the long-term delivery of trophic factor in the eye. The use
of nanoparticles, viral-mediated transference and implants of
encapsulated cells producing neurotrophic factors inserted in the
vitreous cavity are examples of improved delivery methods.
Several neurotrophic factors have demonstrated success in
preventing or delaying retinal degeneration in different LIRD animal models; these include BDNF (LaVail et al., 1992), GDNF (Read
et al., 2010), acidic FGF (LaVail et al., 1992), bFGF (Lau and
Flannery, 2003; LaVail et al., 1992; Li et al., 2003), CNTF (LaVail
et al., 1992) and pigment epithelium derived factor (PEDF) (Cao
et al., 2001; Imai et al., 2005). A wide variety of neurotrophic factors have also been shown to have neuroprotective effects on
degenerating photoreceptors through the morphological and
functional protection of rods in models of RP. Examples of this are
GDNF (Andrieu-Soler et al., 2005; Frasson et al., 1999; McGee
Sanftner 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 factors
provides synergistic neuroprotection in photoreceptor rescue
(Miyazaki et al., 2008). Furthermore, they also have demonstrated
beneficial properties in the treatment of animal models of ischemia,
intraocular pressure and retinal detachment, among other pathologies. An extended review of the in vivo effects of the exogenous
administration of trophic factors in animal models of retinal
degeneration has been recently published (Kolomeyer and Zarbin,
2014). Rod-derived cone viability factor is a diffusible molecule
secreted by rod cells that promotes cone survival. In this context,
rod-derived cone viability factor injected into the eye of the P23H
transgenic rat model of RP preserved the vision by increasing cone
survival and function (Yang et al., 2009). Clinical trials with
encapsulated ARPE-19 cells secreting CNTF into the vitreous of
patients with RP and AMD showed evidence of photoreceptor
protection 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 therapies
The most widely used vectors for ocular gene delivery are based
on adeno-associated virus (AAV). AAV vectors do not integrate into
the host genome, rather they exist as extragenomic circular episomes, which significantly decreases the risk of insertional oncogenesis. They typically elicit minimal immune responses and allow
for stable, long-term transgene expression in a variety of retinal
cells, such as photoreceptors, RPE, Müller and ganglion cells.
Adenoviral vectors are also non-integrative vectors; however, they
elicit robust cytotoxic T lymphocyte-mediated immune responses
that limit the duration of transgene expression. Lentiviral vectors
can induce stable, long-term transgene expression in anterior
ocular structures, including the corneal endothelium and the
trabecular meshwork, in addition to retinal tissues. Because they
are integrating vectors, justifiable concerns have been raised over
the risk of insertional oncogenesis. A variety of non-viral ocular
gene transfer methods have also been studied, including the use of
DNA nanoparticles (Conley and Naash, 2010; Farjo et al., 2006), the
fC31 integrase system (Chalberg et al., 2005), and electroporation
and lipofection (Kachi et al., 2005). Although they have seen some
tangible success in ocular applications, they will not be discussed
further in this review.
4.4.1.1. Diabetic retinopathy. Neovascularization associated with
DR and AMD is a leading cause of visual impairment and adultonset blindness. Gene transfer of anti-angiogenic proteins is an
approach that has the potential to provide long-term suppression
of neovascularization and/or excessive vascular leakage in the eye.
Gene transfer of anti-angiogenic PEDF via AAV2 injected in the
vitreous has been reported to have positive effects on a transgenic
mouse model that mimics the chronic progression of human DR
(Haurigot et al., 2012). Long-term production of PEDF produced a
striking inhibition of intravitreal neovascularization, normalization
of retinal capillary density, prevention of retinal detachment, and
reduction in the intraocular levels of VEGF. Furthermore, as a
consequence of the latter, there was a down-regulation of downstream effectors of angiogenesis, such as the activity of matrix
metalloproteinases 2 and 9 and the content of connective tissue
growth factor (Haurigot et al., 2012). Other researchers constructed an AAVrh.10 coding for bevacizumab (an anti-VEGF
monoclonal antibody), which was injected in the vitreous of
transgenic mice overexpressing human VEGF165 in photoreceptors. Directed long-term bevacizumab expression in the RPE
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
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) (angiotensin
1e7) genes by intravitreal injection mediated by AAV in the retina
of streptozotocin-induced diabetic eNOS/- mice and SpragueeDawley rats has also been confirmed (Verma et al., 2012). The
increased levels of ACE2/Ang-(1e7) resulted in a significant
reduction of diabetes-induced retinal vascular leakage, acellular
capillaries, infiltrating inflammatory cells and oxidative damage.
On the other hand, AAV2-mediated intravitreal gene delivery of
the high-affinity soluble VEGF receptor hybrid called sFLT01 efficiently inhibited angiogenesis in the mouse oxygen-induced retinopathy model (Pechan et al., 2009). AAV-mediated gene
expression of sFLT1 injected into the subretinal space also proved
to be efficient in the spontaneously diabetic non-obese Torii rat
model of human DR (Ideno et al., 2007). Auricchio and coworkers
used AAV vectors for the gene transfer to the eye of three antiangiogenic factors: PEDF, tissue inhibitor of metalloproteinase-3
and endostatin. They observed that, in all cases, the treatment
inhibited retinal neovascularization in a mouse model of retinopathy of prematurity (Auricchio et al., 2002).
4.4.1.2. Age-related macular degeneration. The use of antiangiogenic therapy is also effective in the treatment of AMD. In
fact, the most effective treatment for AMD-associated CNV is
currently the administration of anti-VEGF compounds (monoclonal antibodies, siRNA, RNA oligonucleotides, among others).
There are treatments already approved for wet AMD. Of the new
antivascular inhibitors undergoing testing, three anti-VEGF therapies are approved by the US Food and Drug Administration (FDA)
at this time for the treatment of AMD-associated CNVs: pegaptanib (Gragoudas et al., 2004), ranibizumab (Brown et al., 2009;
Rosenfeld et al., 2006), and aflibercept (Heier et al., 2012). Bevacizumab is also widely used, although it is not approved
(Chakravarthy et al., 2012; Martin et al., 2012). Meanwhile, the
search for other therapy strategies continues in various animal
models. In this regard, subretinal AAV delivery vehicles with short
hairpin RNAs have been employed, targeting the pro-angiogenic
growth factor VEGF mRNAs. This approach has successfully
inhibited endogenous mouse VEGF protein expression in the laserinduced murine model of CNV, and as a consequence, reduced the
formation of CNV (Askou et al., 2012). Moreover, AAV-mediated
gene expression of sFLT1 injected into the subretinal space has
been demonstrated to be successful in CNV-induced mice (Igarashi
et al., 2010). Subretinal or intravitreal delivery of an AAV vector
expressing a transgene for a soluble non-membrane binding form
of human CD59, a naturally occurring membrane bound inhibitor
of membrane attack complex, attenuated the formation of laserinduced CNV and murine membrane attack complex formation
(Cashman et al., 2011). Oxidative stress in RPE cells is another key
to fighting the AMD pathology. In this context, overexpression of
the human X-linked inhibitor of apoptosis using recombinant AAV
in ARPE-19 cells exposed to H2O2-induced oxidative death protected the cells from death by acting on the apoptotic pathway
(Shan et al., 2011). Moreover, the use of PEDF in therapies
designed to prevent or reduce the neovascularization associated
with AMD and DR is very common. Viral-mediated PEDF intraocular injection has demonstrated beneficial properties in animal
models 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 1
clinical trial, a single intravitreal injection of AdPEDF.11 in 28 patients with advanced neovascular AMD maintained or reduced the
CNV size after up to 12 months of follow up (Campochiaro et al.,
2006).
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4.4.1.3. Retinitis pigmentosa. Different strategies are used to treat
RP disease, according to its etiology. A suitable approach for cases of
autosomal dominant RP is ‘gene silencing and replacement’. First,
the levels of both mutant and wild type mRNA are knocked down
using allele non-specific ribozymes or siRNAs (mutation-independent suppression). Second, it is essential to deliver an allele cDNA
resistant to ribozyme or siRNA mediated degradation. In cases of
autosomal recessive RP, the common strategy is to add a wild-type
allele to increase the level of functional protein.
Greenwald and collaborators developed a mouse model of
autosomal dominant RP expressing a pathogenic mutant human
rhodopsin gene on a rhodopsin knockout background. These authors observed higher ERG a-wave responses in the eyes where the
photoreceptors were transduced with AAV containing a microRNA
sequence targeting the human mutant rhodopsin gene, which
silenced 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 and
normal retinal dimensions and morphology, including the preservation of photoreceptor cells, have been observed in the P23H
transgenic mouse model of autosomal dominant RP injected with a
single dose of an AAV expressing both a small interfering RNA
(siRNA301), which cleaves rhodopsin mRNA at nucleotide 301, and
a modified rhodopsin cDNA with five silent base changes surrounding position 301, which is resistant to siRNA digestion (Mao
et al., 2012). A delay in retinal degeneration has also been reported in the P347S rhodopsin transgenic mouse model of autosomal dominant RP using two AAV subretinally co-injected and
expressing an interference RNA to suppress rhodopsin and a codonmodified rhodopsin gene resistant to suppression due to nucleotide
alterations at degenerate positions over the interference RNA target
site (Millington-Ward et al., 2011). Another method used to develop
a mutation-independent treatment for autosomal dominant RP is
the use of ribozymes. It has been demonstrated that AAV delivery of
rhodopsin-specific ribozyme (Rz525) rescues vision in P23H line 3
rats by diminishing the expression of the P23H transgene
(Gorbatyuk et al., 2007). Consequently, Rz525 is a candidate ribozyme for RNA replacement gene therapy when combined with a
ribozyme-resistant rhodopsin gene.
Conlon and his group demonstrated the potency and safety of
ocular injection of AAV vectors expressing human MERKT cDNA in
the RCS rat model of autosomal recessive RP. Vector-injected eyes
showed improved ERG responses as compared to untreated eyes.
Furthermore, funduscopic analysis and postmortem retinal
morphology of vector-injected eyes were normal as compared to
the controls (Conlon et al., 2013). Subretinal administration of AAV
expressing the wild-type mouse Mfrp (membrane-type frizzledrelated protein) gene prevented retinal degeneration in the Rd6
Mfrp mutant mouse model of autosomal recessive RP (Dinculescu
et al., 2012). CNGB1a (subunit of rod cyclic nucleotide-gated
(CNG) channel) gene replacement via subretinal space AAV delivery restored the rod CNG channel expression and localization,
improving retinal function and vision-guided behavior, and delaying retinal degeneration in autosomal recessive RP mouse model
CNGB1/ (Koch et al., 2012).
Beltran and collaborators evaluated the retina of two blinding
canine photoreceptor diseases that model the common X-linked
form 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 injected
with an AAV2/5 vector carrying a full-length normal human RPGR
gene. Gene augmentation rescued photoreceptors from death and
reversed mislocalization of rod and cone opsins in both XLPRA
models, thus alleviating the characteristic features of photoreceptor degeneration, such as the progressive changes that take
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N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
place in the OPL, bipolar cells and inner retinal layers (Beltran
et al., 2012).
As mentioned earlier, due to the broad genetic heterogeneity of
RP disease, gene-specific therapies are impractical, and thus the
development of mutation-independent treatments to slow
photoreceptor cell death is required. One promising strategy for
photoreceptor neuroprotection is neurotrophin secretion from
Müller cells, the primary retinal glia. Müller glia are excellent
targets for secreting neurotrophins, as they span the entire tissue,
connect with all neuronal populations, are numerous and persist
throughout retinal degeneration. An AAV variant (ShH10) has been
engineered which efficiently and selectively transduces glial cells
through intravitreal injection (Dalkara et al., 2011). ShH10mediated-GDNF secretion from the glia generated high GDNF
levels in treated retinas, leading to sustained functional rescue
over 5 months in the TgS334-4ter rat model of RP. Other researchers have argued that manipulating cell death and prosurvival pathways and shifting the balance in photoreceptor cells
toward cell survival could be a reliable therapeutic approach for
preserving vision in RP patients, and may represent a more
promising approach than gene replacement therapy. Along these
lines, researchers have assayed subretinal injections of P23H rats
with AAV expressing human GRP78 (Bip) or functional rescue of
photoreceptors with HSF-1 (Gorbatyuk et al., 2012). Both GRP78
and HSF-1 overexpression increased the a- and b-wave response
amplitudes and the integrity of the retina as compared to untreated eyes.
AAV-mediated gene replacement has also been used to restore
visual function in a dog model of Leber congenital amaurosis, a
retinal degeneration that can cause severe childhood visual loss
(Acland et al., 2001; Annear et al., 2013). The gene defect in the
naturally occurring dog model, a mutation in the RPE65 gene that
codes for an RPE cell membrane-associated protein involved in
retinoid metabolism, also occurs in human Leber congenital
amaurosis. An AAV carrying wild-type RPE65 was able to restore
vision as assessed by electroretinography, pupillometry, and psychophysical and behavioral tests. Human clinical trials using a
similar vector are currently ongoing. Trials in human patients with
RPE65-associated Leber congenital amaurosis have shown that
gene therapy leads to substantial visual improvement (Cideciyan
et al., 2008, 2013; Jacobson et al., 2012).
4.4.2. Optogenetics
The severe loss of photoreceptor cells caused by retinal
degenerative diseases such as RP can result in partial or complete
blindness. As a new strategy to treat blindness caused by retinal
degeneration, researchers have developed optogenetic tools in an
attempt to restore retinal photosensitivity by creating new photosensors and coupling them to the remaining retinal circuitry.
The two best-known optogenetic tools are channelrhodopsin-2
(ChR2), from the algae Chlamydomonas reinhardtii, and halorhodopsin (NpHR), from the archaebacterium Natronomonas
pharaonis. These proteins are photosensitive and can be activated
at specific light wavelengths. For this reason, it was suggested
that introduction of these molecules through gene transfer can
render the cells of the inner retina photosensitive, thus imparting
light 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 retinal
degeneration (Bi et al., 2006; Doroudchi et al., 2011; Lagali et al.,
2008). Using the chloride pump halorhodopsin, visual function
was restored in animal models of RP at the level of the retina and
cortex, as well as behaviorally (Busskamp et al., 2010). The
translational potential of this optogenetic approach has been
supported by the efficacy of the transduced halorhodopsin
expression in human photoreceptors in tissue explants from
postmortem human retinas, while clinical examinations in blind
patients confirmed the presence of dormant cone photoreceptors
that could be reactivated through this approach (Fig. 19B)
(Busskamp et al., 2010). Moreover, the co-expression of Channelrhodopsin2/HaloR in RGCs restored both ON and OFF light
responses in the retina after the death of rod and cone photoreceptors (Zhang et al., 2009).
Researchers have also used melanopsin (OPN4) as an intrinsic
light-sensitive protein. In this context, AAV have been successfully
used to ectopically express mouse melanopsin in RGCs of a mouse
model of photoreceptor degeneration (Lin et al., 2008). OPN4transfected ganglion cells provided an enhancement of visual
function in the mice, such that the pupillary light reflex returned to
a nearly normal condition, the mice showed behavioral avoidance
of light in an open-field test and they were able to discriminate a
light from a dark stimulus in a two-choice visual discrimination
behavioral test.
4.4.1.4. Glaucoma. Neuroprotection of RGCs is an important goal
in glaucoma therapy. PEDF is a potent anti-angiogenic, neuroprotective and anti-inflammatory factor for neurons. Intravitreal
injection of AAV expressing PEDF in the DBA/2J mouse model of
inherited glaucoma reduced the loss of RGC and nerve fiber layer,
delayed vision loss and reduced TNF, IL-18 and GFAP expression
in the retina and optic nerve (Zhou et al., 2009b). In a rat model
of experimental glaucoma, the use of recombinant AAV to
transduce to RGCs genes encoding constitutively active or wildtype MEK1, the upstream activator of Erk1/2, markedly
increased neuronal survival (Zhou et al., 2005). Thus, selective
activation of the Erk1/2 pro-survival pathway protected RGCs.
Martin and collaborators used an AAV incorporating cDNA for
BDNF to transfect RGCs in a rat model of glaucoma, and observed
that intravitreal AAV-BDNF rescued RGCs (Martin et al., 2003).
Interestingly, the final common pathway of RGC apoptosis involves the activation of caspase enzymes. In this context, an AAV
vector coding for human baculoviral IAP repeat-containing protein-4 (BIRC4), a potent caspase inhibitor, injected into one eye of
a rat model of experimental glaucoma, allowed the researchers to
conclude that BIRC4 delivery significantly promoted optic nerve
axon survival in a rat chronic ocular hypertensive model of
glaucoma (McKinnon et al., 2002).
4.5. Cell-based therapies
Advanced therapies are different from conventional chemicalor protein-based therapies. The European Medicines Agency classifies advanced therapies in three main groups, depending on the
origin of their products: genes (gene therapy), cells (cell therapy) or
tissues (tissue engineering) (European Commission News; http://
ec.europa.eu/health/human-use/advanced-therapies/index_en.
htm). One such cell therapy, the Regenerative Medicine, attempts to
find ways to replace cells in the body that have degenerated.
The central focus of regenerative medicine is human cells. The
cells used for cell therapy may be somatic, adult stem or embryoderived cells. Currently, there are cells that have been reprogrammed from adult cells so that they can be conveniently driven to
become ‘pluripotent cells’ (Mason and Dunnill, 2008).
The field of stem cell based-therapy holds great potential for the
treatment of retinal degenerative diseases. The retina is one of the
best places to treat, not only with gene therapy, but also with cellbased approaches, due to easy access, immune privilege and relative isolation from other body systems, as well as the different
options available to check therapeutic benefits and possible secondary effects, both anatomical and functional. This is real when
the eye is healthy, but becomes a utopia in the eyes affected by a
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
57
Fig. 19. Optogenetic therapy for retinal degeneration. (A) Schematic representation of a healthy retina with sensitive photoreceptors and functional bipolar and ganglion cells. Two
subpopulations of bipolar cells respond differently to the synaptic inputs from photoreceptors (ON and OFF visual pathways). (B) In pathological conditions, the remnant unhealthy
rods 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 the
disease, 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 because
the BRB is compromised.
Among the patients who may benefit directly from cellular
replacement strategies are those suffering from what are currently
incurable eye diseases or other conditions with progressive visual
impairment due to the loss of specific cells, such as RP, AMD, DR,
ischemic retinopathy and glaucoma. RPE cells and photoreceptors
seem to be good candidates for replacement, and are preferable to
the integration of RGCs, which need to redirect and extend their
processes towards the central nervous system after forming the
optic nerve. This redirection is clearly difficult to obtain, although
there is a great deal of interest because of its potential for the
treatment of optic neuropathies, such as glaucoma.
In order to be a potential source for the treatment of retinal
diseases, the cells must meet a number of conditions. Cell based-
therapies for photoreceptor or RPE regeneration should have a
high level of efficacy and reproducibility, a low failure rate and,
ideally, they should not require immunosuppression. The tissue
needs to be easy to propagate, with a low harvest rate and few
ethical issues. Additionally, the cost should be low and the transplantation technique easy to perform (Ramsden et al., 2013). The
subretinal space and vitreous cavity are known as immunoprivileged sites for transplantation, due to the blood-retina barrier
(Jiang et al., 1993). The last, but not the least important, condition is
that it should be possible to obtain cells through a proper
manufacturing process, and in sufficient quantities to allow for
patient transplantation. Although there are still some issues to be
solved with regard to human pluripotent stem cells, both embryonic and pluripotent stem cells seem to be the best bet for cell
replacement. They fulfill almost all the requirements and, in the
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future, induced pluripotent stem cells will allow autologous cell
therapies, if needed. This approach will solve the problem of immune rejection, once all other pending issues have been addressed.
Stem cell transplantation for retinal diseases is currently transitioning from over a decade of preclinical research to phase 1-2
clinical trials. The main focus of these trials is safety, with efficacy
being a secondary concern, and they are design to determine the
required levels of immunosuppression, the best delivery method,
etc. Potential sources of these cells include pluripotent and multipotent 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 to
almost unlimited numbers and their pluripotency can be guided to
differentiate into any cell of the body. The potential of hESCs for
treating different diseases is unlimited. The progress made in recents in terms of the safety and efficacy of transplanted hESCs in
animal models of both neural and retinal degeneration has brought
the field to the brink of clinical trials.
ESCs represent one of the most promising sources of cells for
transplantation, and considerable progress has been made in their
differentiation in the laboratory towards photoreceptor and RPE
lineages. There are different protocols that drive the cells in the
same direction they follow during embryonic development, going
through an anterior neuroectoderm fate to an eye field stage,
forming optic vesicles and, at the end, differentiating into neuroretina and RPE. Initial attempts to generate retinal cells tried to
force embryonic stem cells to chose their fate by default, using
either bFGF, retinoid acid or ITSFn (insulin transferrin selenium and
fibronectin) (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 were
adopted, such as adding N2 supplement and manually selecting the
spheres with optic vesicle structure. The increase in MITF expression through the addition of FGF-inhibitors (e.g., activin A) directs
the cells to an RPE fate (Meyer et al., 2009). Other methods use the
same factors that promote retinal induction (Nodal antagonist or
inhibition of Notch pathway) and increase RPE fate by adding
activin A (Davis et al., 2000; del Barco Barrantes et al., 2003; Ikeda
et al., 2005; Osakada et al., 2008, 2009) or morphogens to induce
neural differentiation (Lamba et al., 2006).
4.5.1.1. Deriving photoreceptors from ESCs. Considering the
different ways in which retinal cells can be produced, ESCs could be
an easy way to obtain photoreceptor and/or RPE cells to replace the
lost ones in the retina. After being generated, it was necessary to
prove that the cells could integrate into the retina and be functional. One of the first demonstrations of their function was done by
Kwan and collaborators in 1999, after transplanting normal mice
photoreceptors in rd1 mice and showing synaptic formation and
functional rescue (Kwan et al., 1999). MacLaren and coworkers
showed that it was possible to transplant photoreceptor cells into
an 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 generate
cells suitable for transplantation. They successfully restored vision
in a mouse model of stationary night blindness, thus demonstrating
that the cells could be functional (Pearson et al., 2012). Some
concerns 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 internal
limiting membrane and the glial changes that appear with degeneration, which may interfere with the cell integration. Another
issue to be addressed is that of the optimal stage of differentiation
of the transplanted cells (Gamm and Wright, 2013); donor photoreceptors from more mature mice are able to integrate into the host
retina, but their efficacy decreases with maturity (Gust and Reh,
2011). It would be ideal to have a cell maintain part of its pluripotency, but avoid teratoma formation and other problems associated
with its differentiation. All the above mentioned studies demonstrated the feasibility of transplanting photoreceptors derived from
mouse ESCs. Ongoing efforts towards clinical translation are
needed to develop human ESC lines in order to provide a potentially unlimited source of transplantation-competent photoreceptor precursors with good integration, despite the use of an
injection method (Banin et al., 2006; Lamba et al., 2009).
4.5.1.2. Deriving RPE from ESCs. hESCs derived into RPE, either in a
spontaneous way, directed by blocking Wnt and Nodal signaling
pathways or through incubation with Activin A, replicate the
morphology and function of RPE cells in the retina, maintaining
their appearance, polarization and protein expression in vitro
(Klimanskaya et al., 2004; Vugler et al., 2008a). These RPE cells
derived from hESCs have been successfully transplanted in animal
models of retinal degeneration, especially the RCS rat (Idelson et al.,
2009; Lu et al., 2009; Lund et al., 2006; Vugler et al., 2008a). An US
Food and Drug Administration Phase 1/2 clinical trial has been
approved in patients with Stargardt Disease and AMD, using injections of hESCs-derived RPE cells. The preliminary results published indicated the safety and tolerability of their stem cell
implantation, 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 RPE
are a promising advance in retinal replacement therapies. Their
safety and efficacy have been shown in a large number of preclinical studies in animal models. However, there are ethical and
rejection issues, as well as the possibility of teratoma formation,
which should be addressed by keeping under control their pluripotency 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 induced
Pluripotent Stem Cells (iPSC). In 2006, it was first reported how to
reprogram adult somatic mouse cells (Takahashi and Yamanaka,
2006); one later, Takahashi and Yu groups were able to drive human fibroblasts to a pluripotent state with the ability to generate
the three embryonic layers (Takahashi et al., 2007; Yu et al., 2007).
The process of generating pluripotent cells from somatic cells was
termed “reprogramming” and the resultant cells were called
induced pluripotent stem cells. The iPSCs shared properties with
hESCs, including the ability to self-renew and to be differentiated
into the germ layers. Cell therapy using iPSC-derived cells still has
many hurdles to overcome before they can be used in clinical applications. However, since they are able to recapitulate the phenotypes of a wide variety of diseases, patient-specific iPSCs might
become useful in the future for disease analysis, allowing to shed
light on their pathogenesis and discover effective new drugs
(Egashira et al., 2013). The ultimate goal would be the generation of
individual pluripotent lines to correct any individual genetic defects ex vivo and to transplant the required cell type back (Wright
et al., 2014).
The eye is an ideal target to explore the potential of hiPSC
technology, not only to understand the disease pathways, but also
to explore novel therapeutic strategies (Borooah et al., 2013). There
are still some concerns about iPSCs. Transcribed genes, the epigenetic landscape, differentiation potential and mutational load show
small, yet distinctive dissimilarities between iPSCs and ESCs, which
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
are considered the gold standard for in vitro pluripotency (Bilic and
Izpisua Belmonte, 2012). However, murine iPSCs have been
expanded stably and homogeneously for over 30 passages changing
the reprogramming strategy (Zhou et al., 2009a).
4.5.2.1. RPE production from hiPSCs. RPE cells can be generated
either by following the same steps as with ESCs or through the
formation of embryoid bodies, followed by plating the suspended
embryoid bodies onto a coated surface, dissecting them and letting
them grow into pigmented spheroids (Buchholz et al., 2009, 2013;
Carr et al., 2009; Gamm et al., 2008; Hirami et al., 2009; Meyer
et al., 2009; Phillips et al., 2012). RPE cells from both hESCs and
iPSCs are similar to RPE not only in their morphology, but also in
their function, polarity and gene and protein expression.
The replacement of RPE using iPSC should be easier than the
replacement of the inner retinal cells because of the less complex
connection established by the cells. RPE can be generated from
iPSCs in large amounts, with relative simplicity. The cells have been
transplanted into RCS rats and Rpe65 (rd12)/Rpe65 (rd12) mice
with cell integration, functional response and the absence of tumor
formation (Carr et al., 2009; Li et al., 2012b). Efficacy has been such
that research has progressed to the clinical trial stage in the case of
ESCs 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 hiPSCderived neuroretinal progenitor cells differentiate in a similar
manner, mimicking the order and time course of normal retinogenesis (Meyer et al., 2011, 2009; Phillips et al., 2012). Pluripotent
cells pass through an anterior neuroectoderm-like stage when
recapitulating retinal differentiation in vitro, but the culture needs
further processing than that required to obtain RPE (Meyer et al.,
2009). Retinal differentiation has been obtained using both
adherent and 3-D aggregate differentiation methods.
The future of hiPSC-derived neuroretinal differentiation lies
perhaps in the generation of a three-dimensional whole neuroretina which recapitulates retinal development in vitro. This was
first tried using murine ESCs (Eiraku et al., 2011) and confirmed
using hESCs (Nakano et al., 2012). It was demonstrated that hiPSCs
and ESCs could generate neuroepithelial-like clusters similar to
developing optic vesicles (Meyer et al., 2011).
4.5.3. Human fetal embryonic stem cells; retinal progenitor cells
Fetal stem cells are derived from embryonic and extraembryonic tissue. Retinal progenitor cells can be derived from
either fetal or neonatal retinas and comprise an immature cell
population that is responsible for the generation of all retinal cells
during development (Reh, 2006). Human prenatal retinal tissue
was one of the first donor sources used in patients. Humayun and
collaborators used a subretinal injection of a suspension of prenatal neuroretinal cells in RP patients, with transient functional
improvement (Humayun et al., 2000). Radtke and coworkers used
neuroretina sheets with attached RPE in AMD and RP patients,
transplanted into the submacular area (Radtke et al., 2008). Human fetal neural stem cells isolated from donated aborted fetuses
aged 16e20 weeks have been used to prepare a suspension of
neutralized stem cells that were then injected into the subretinal
space of RCS rats, with good anatomical and functional results
(McGill et al., 2012). The cells were unable to integrate into the
retina, so the exact working mechanism is unknown. Based on this
study, it has initiated a clinical trial for treating dry AMD using
subretinal injection of these cells (Clinicaltrial.gov identifier
NCT01632527).
59
4.5.4. Human umbilical tissue-derived stem cells
Human umbilical tissue has multipotent stem cells that are
considered to be adult stem cells. Suspensions of these cells have
been able to improve anatomical degeneration and function in RCS
rats due to a paracrine effect, with no cell integration (Lund et al.,
2007). The repairing mechanism cannot be justified by the integration of the transferred cells because no morphological changes
were observed in the injected cells; the reason for improving vision
seems to be paracrine, being the cells able to secrete BDNF. Based
on these findings, another clinical trial for dry AMD treatment was
initiated but at this time is not longer ongoing.
Stem cells from the blood of human umbilical cords have been
extensively used in studies on neurological pathologies, including
traumatic optic nerve neuropathy. Some reports have shown that
the cells can integrate into the retina (Koike-Kiriyama et al., 2007),
while others have evidenced just the opposite, again suggesting a
neuroprotective 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) transplanted into the subretinal space in RCS rat maintain an immature
phenotype throughout 7 months and undergo very limited proliferation with no evidence of uncontrolled growth or tumor-like
formation (McGill et al., 2012). Another study reveals that transplant of these stem cells also preserves the synaptic contacts between photoreceptors and second order neurons bipolar and
horizontal cells, as well as phagocytosis of photoreceptor outer
segments (Cuenca et al., 2013). This study indicated that the neuroprotective transplantation of HuCNS-SC cells results in the stabilization of photoreceptor degeneration and slowing of
progressive visual loss. The Food and Drug Administration (FDA)authorized phase 1/2 clinical trial in AMD with geographic atrophy
using these stem cells and is underway.
4.5.6. Bone marrow-derived stem cells
Bone marrow-derived stem cells may be divided into hematopoietic and mesenchymal types. Bone marrow-derived hematopoietic stem cells are able to migrate after retinal damage, not only
from endogenous locations, but also post-injection, reaching the
damaged retina and expressing the same RPE markers as RPE65
(Atmaca-Sonmez et al., 2006; Li et al., 2007, 2006). The cells also
have a demonstrated ability to stabilize and rescue retinal blood
vessels in rd mice and to induce neurotrophic rescue, preserving
retinal layers (Otani et al., 2004). These aspects, and the fact that no
safety issues have been identified after injecting them into the
vitreous humor of RP patients (although with no functional effect),
have moved research forward to various clinical trials in order to
evaluate the effects of these cells on RP, dry AMD, retina vein occlusion, DR, retinal and optic nerve diseases, glaucoma and
ischemic retinopathy (www.Clinicaltrials.gov).
Mesenchymal stem cells are also a good source for cellular
therapy. While bone marrow remains the primary source of
mesenchymal stem cells for most preclinical and clinical studies, fat
sources are gaining increasing importance because cells can be
easily isolated in large numbers. Bone-marrow mesenchymal stem
cells injected into the subretinal space of the rhodopsin knockout
mice and RCS rats are able to forestall functional decline with no
immune rejection problems, showing a lower immunogenic status
than other stem cells (Arnhold et al., 2007, 2006; Lu et al., 2010).
They are able to express RPE65 after systemic injection, with no
other RPE features (Atmaca-Sonmez et al., 2006). Intravitreal injection of these cells was experimented with in three patients with
RP and two patients with cone-rod dystrophies, with no side effects
but limited functional improvement (Siqueira et al., 2011). This
group is experimenting with the use of these cells in AMD, DR and
60
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
retinal vein occlusion, and is currently moving into the clinical trial
stage. Additional clinical trials have been registered for these pathologies and others, such as RP, AMD and ischemia (www.
Clinicaltrials.gov).
Stem cell transplantation for retinal diseases is currently transitioning to clinical trials. The primary outcome of the first clinical
trials is the safety of the procedure. Secondary outcomes will check
the efficacy, the number of transplanted cells and the procedure.
Early results are encouraging, and a real clinical application seems
to be closer every day. ESC-derived RPE is being used in ongoing
clinical trials to repair the damage of outer retina diseases.
Although multiple animal studies have shown that the transplantation of photoreceptors is able to prevent degeneration, their
integration and function need to be clear for initiating human
treatments. There are points that need to be elucidated, such as the
best cell type and the best moment during the course of the disease.
Immunosuppression is still an issue for long-term survival and
human treatment. Another challenge is the survival of the cell in a
retina with extensive remodeling and changes in circuitries. iPSCs
would constitute a revolutionary, personalized treatment if all the
issues regarding their production, expensive cost and correction of
genetic defects in patient-specific cells were addressed. In the
meantime, they might be best used to analyze the physiopathology
and potential pharmacological treatment of each disease. As
reprogramming, differentiation and cell characterization protocols
continue to improve, it is likely that stem cell technology will
become easier to use and more widely accessible and available.
Advanced therapies, through both gene therapy and stem cellbased therapy, provide hope for retinal degenerative diseases that
currently have no cure. However, it remains clear that a customized
approach will be needed to adequately treat any retinal disease.
4.6. Effectiveness of retinal transplantation
Retinal transplantation is another potential therapeutic
approach to restore vision in patients with advanced degenerative
disease. In this process, sheets of developing retina and RPE cells
are inserted into the subretinal space. Acceptable efficacy and
safety levels have been reported for human fetal retina implants
with 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 in
animal models of retinal degeneration (Radtke et al., 2008).
Photoreceptor sheets or whole retinal sheets (both outer and inner
layers) from postnatal P8 healthy rat retinas transplanted into 3month-old P23H rats improved the amplitude of the ERG b-wave
and exerted a positive paracrine effect which enabled the rescue of
host cones (Yang et al., 2010). Surprisingly, in AMD patients, it has
been demonstrated that RPE-choroid graft transplantation may
maintain macular function for up to 7s after surgery, with relatively
low complication and recurrence rates (van Zeeburg et al., 2012).
However, this technique must be used with caution. Transplantation of adult and fetal retinal photoreceptors, in addition to
RPE cells, has been achieved safely in patients with retinal degenerative diseases, but unfortunately, in the vast majority of preclinical studies and human trials, the cells transplanted failed to
establish functional connectivity with the host tissue, resulting in
moderate or null restoration of visual function. Additional studies
are needed to determine new cell sources, in order to avoid problems associated with transplant rejection.
4.7. Clinical trials for retinal diseases
A large number of clinical trials are currently under way to find a
treatment or cure for various retinal diseases. Due to the large
number of trials being carried out, only the most relevant are cited
below. Extensive information on clinical trials is available from the
website clinicaltrials.gov.
In DR, the aim of clinical trials is primarily to diminish or block
neovascularization or to diminish the increase permeability of the
capillary network. Accordingly, several drugs have been employed
with different administration routes: intravitreal injection (bevacizumab, ranibizumab, pegaptanib, dexametason, triamcinolone,
fluometolone), topical application (somatostatin) and different dietary supplements. Vitrectomy surgery is also a common procedure
in the treatment of DR depending on the retinal status. The presence of epiretinal membranes, vitreous hemorrhage or fibrovascular proliferations threating the macula are common indications
for the surgery with good anatomical and functional results and
with the possibility of using adjunctive antinflamatory or antivascular intravitreal drugs at the end of the surgery.
As in DR, the primary objective of the wet AMD trials is to find a
successful anti-angiogenic therapy. The anti-VEGFs bevacizumab,
ranibizumab, pegaptanib and aflibercept are being used, as well as
the AAV-mediated VEGF receptor FLT01. Also under testing for the
treatment of AMD are adeno-associated viral mediated PEDF delivery or endostatin and angiostatin, topical drugs for inhibiting
tirosinkinase receptors, encapsulated human cells secreting CNTF
neurotrophic factor, among others. For dry forms, antibodies
against amyloid substance and serotonin agonists are also tested.
For both wet and dry forms, human stem cells sub-retinal transplantation is another therapeutic option, as well as the dietary
supplementation with antioxidants such as lutein zeaxanthin,
omega-3 fatty acids and vitamin D. Rheopheresis procedures are
also being tested with different results.
Clinical trials performed with RP patients include pharmacologic treatment with vitamin A and E, valproic acid, lutein or docosahexaenoic acid. Stem cell-based therapies are also being
analyzed, as well as encapsulated cell technology to deliver CNTF
and gene therapy via AAV-mediated MERTK replacement. Gene
therapies are also applied to other retinal diseases. In that sense
modification in the following genes are being tested: MY07A in type
1B Usher, RPE65 in Leber Congenital amaurosis, ABCR in Stargardt
disease and XLRS1 in X linked retinosquisis. Retinal transplantation
with fetal tissue, transcorneal electrostimulation and electrical
implants are other therapeutic options in assay.
4.8. Suitable therapies in each phase of retinal degeneration
Degenerative retinal diseases involve structural and functional
changes, many of which result from tissue remodeling and the
functional reprogramming of the neural retina (Fig. 16). Trying to
generalize to all the retinal diseases, we are proposing a fourphased process of neural remodeling is common to all retinal
degenerative diseases, the progress of the events is different in each
type of degeneration and necessarily influences the optimal treatment, which depends primarily on the stage of cell degeneration.
In phase 1, while there are still no evident structural signs of the
disease, neuroprotection of the retina is essential. Patients who will
suffer future eye disease signs could be identified before the
appearance of retinal anatomic or functional changes by means of
medical tests or genetic analysis. The detection of an intraocular
pressure increase in glaucoma, the high glucose levels in DR or the
existence of DNA mutations in RP are crucial in earlier stages of the
diseases. The aim in this stage is to provide a protective environment, independently of the etiology of the disease, to preserve the
structure and recover the normal function of retinal cells. This may
be achieved by delivering neurotrophic factors, vitamins, antioxidants, anti-apoptotic and anti-inflammatory compounds (Fig. 16).
These pharmacological agents help counteract the emerging
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
apoptotic death cascade and the inflammatory responses generated
by the biochemical defects caused by the diseases. The use of gene
therapy is also highly recommended at this stage of degeneration to
correct known defective genes (Fig. 16). Gene silencing and
replacement is the proper approach for diseases caused by dominant mutations, while wild-type allele addition is the appropriate
strategy when recessive mutations are the origin of the pathology.
At this point, the use of filters to block short-wavelength light and
intense visible light can be of benefit in protecting the retina from
photopic damage, thus delaying retinal degeneration.
In phase 2, morphological and functional alterations of the
retina become evident and the first clinical signs can be observed.
The recent use of high resolution optical coherence tomography
provides a unique tool to detect the early retinal structural changes
related to different pathologies, such as AMD, RP, DR and glaucoma
(Acton et al., 2012; Aizawa et al., 2009; Giani et al., 2010; Hagiwara
et al., 2011; Hoerster et al., 2011; Kotowski et al., 2013; Lupo et al.,
2011; Oishi et al., 2009; Taliantzis et al., 2009). Using this technology, ophthalmologists can measure the thickness of each patient's retinal cell layer, thus being able to detect outer segment
damage to rods and cones, as well as RPE alterations. Furthermore,
studies performed in animal models with different types of retinal
degeneration corroborate the rod and cone morphology modifications, as well as the rod bipolar and horizontal cell dendrite
retraction. Hypertrophy of Müller cells and activation of microglia
are other noticeable signs in this phase. At this stage, gene therapy
is advisable to silence mutated genes and to replace them with the
correct ones, as well as the use of optogenetic tools to make
remnant unhealthy photoreceptors responsive to light (Fig. 16). The
neuroprotective agents cited in phase 1 must be also administered
in order to keep retinal cells alive and prolong the visual function.
Another method that can be successful at this point is the application of cell-based therapies (Fig. 16). For example, retinal repair
via the transplantation of photoreceptors or stem cells may be a
good option, because the remaining bipolar and horizontal cells,
which are losing their normal photoreceptor input without major
changes in their morphology, will seek out new functional photoreceptors with which to establish contact. Additionally, proper
maintenance of the RPE function is a requirement to preserve
photoreceptors in good health. Within this framework, stem cell
therapy applied to reestablish RPE function may offer a very good
chance of success, in some cases even better than the photoreceptor replacement therapy. The reason for this difference is that
the transplanted photoreceptors need to establish highly specialized synaptic connections with bipolar and horizontal cells, as well
as proper interactions with RPE cells. Another option would be
transplanting both cell types, already developed on a membranelike surface.
Phase 3 is characterized by a profound loss of vision, evidenced
by abnormal results in morphological and functional tests. At this
stage, there are few or no functional photoreceptors and the inner
retina has been irreversibly damaged. Dramatic alterations in the
vascularization of the retina occur in AMD and DR. The use of gene
therapy no longer makes sense at this phase, when almost no
photoreceptors remain alive. However, the optogenetic technique
applied to bipolar and RGCs to compensate for the loss of photosensitive photoreceptor cells may be a good approach (Fig. 16). The
stem cell transplantation to replace the lost photoreceptors is also a
suitable therapeutic option at this stage. The application of subretinal, epiretinal or suprachoroidal electronic retinal implants is
another possibility, but the rate of success will depend on the stage
of retinal degeneration of the patient (Fig. 16). The employment of
these visual prosthetic designs in a highly remodeled retina will be
more difficult than the use of the same approach in retinas with
most of their circuitry still intact (the use of these devices will not
61
be discussed in this review). Therefore, the efficacy of either
treatment increases to the extent that the integrity of the retina has
been preserved by the previous administration of neuroprotective
compounds.
In the most advanced stage of disease (phase 4), the retina is in a
marked state of remodeling, due to the loss of many of its neurons.
As a consequence, there is no visible structure and the visual
function is almost absent. Cell transplantation must not be
considered at this stage, at least with the actual knowledge, due to
the development of a new non-sense rewiring of the retinal circuitry that does not allow for correct processing of visual information, even if the new cells make contact with the remaining
functional retinal cells. Furthermore, the existence of a thick glial
scar formed by the Müller cells in the outer retina (Fig. 12AeC)
constitutes a mechanical barrier to synapse formation between the
transplanted photoreceptors and the remaining bipolar and horizontal cells, and also between the photoreceptor outer segments
and the RPE. Optogenetic tools applied to the remaining ganglion
cells are a therapeutic option, but the low number of these cells
could compromise the success of this approach. In these cases,
artificial vision, combined with the administration of neurotrophic
factors that contribute to prolonging the life of the surviving RGCs,
may be the best strategy (Fig. 16). It is clear that new scientific
developments could change these options in the future.
Interestingly, the administration of neuroprotective factors is
crucial in all degeneration phases, even when vision has been
completely lost. RGCs are not only important because they are
responsible for transmitting through their axons electrical signals
from the retina to the brain to form visual images, a subset of retinal
photosensitive ganglion cells expressing the photopigment melanopsin 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 is
not only essential for proper visual function, but also to avoid disturbances in non-visual functions, such as sleepewake cycles, daily
activities and the mood of people who have retinal disorders.
Hence, with our present knowledge of the use of different approaches in the treatment of retinal pathologies, the combination
of several types of therapies is considered essential to achieve
success in delaying the progression of retinal degeneration. Moreover, it is important to emphasize that all retinal disorders,
regardless of their etiology, have in common the activation of
oxidative stress, inflammation and apoptosis pathways. For this
reason, all the neuroprotective compounds described (neurotrophic factors, vitamins, antioxidants, anti-apoptotic and antiinflammatory molecules) must be administered in the treatment
of any retinal disease (Fig. 16). As stated earlier, the administration
of the neuroprotective compounds mentioned in all degeneration
phases of retinal diseases is crucial for maintaining retinal health
and, as a consequence, for strengthening and prolonging the
beneficial effects of the applied therapy(s) (Cideciyan et al., 2013).
On the other hand, we must not forget that the success of the
treatments in improving visual function depends on proper patient
selection, the etiology and stage of the disease, the right choice of
treatment(s) and the age of the patient, among other factors.
In conclusion, it is critical to remember that despite all the scientific advances made in an attempt to cure or slow down retinal
degenerative diseases, there are still several barriers to overcome.
The successful translation of new therapies requires the development of appropriate animal models of the diseases, as certain
mutations are not similar between humans and the existing animal
models. There is vast genetic heterogeneity associated with some
disease phenotypes, and thus an accurate genetic characterization
is necessary for specific gene therapies. Furthermore, it is important
to maintain proper RPE function regardless of the disease;
62
N. Cuenca et al. / Progress in Retinal and Eye Research 43 (2014) 17e75
otherwise, the therapeutic approaches applied to photoreceptor
cells will be useless. Finally, the blood-retinal barrier prevents most
molecules administered systemically from reaching an effective
dose in the retina, making the development of effective delivery
methods necessary; perhaps smaller or polarized molecules can
pass through this barrier with the improvement of the actual
knowledge.
5. Conclusion remarks and future directions
Human retinal degenerative diseases are currently incurable
and retinal degeneration, once initiated, is irreversible. The therapies applied at present in the treatment of retinal dystrophies delay
the onset or progression of degeneration, but no therapies are
available to replace lost retinal cells or restore accurate vision. The
search for effective treatments has stimulated the development of a
large number of animal models that mimic the different human
retinal diseases, as well as the isolation of an increasing number of
retinal cell lines that are of great interest for the study of the
cellular pathways involved in the progression of these diseases.
Currently, the potential therapeutic approaches aimed at finding a
cure for blinding diseases focus on three main lines of action. First is
the use of preventive strategies that attempt to counteract the
underlying disease mechanisms, either by manipulating cellular
pathways through the use of pharmacological compounds or genetic modification by gene silencing and/or gene replacement. The
second approach is not concerned as much with the causes of the
diseases as it is with ways to prevent cell death, such as are the
administration of anti-apoptotic, anti-inflammatory and neurotrophic factors compounds. The third approach focuses on retinal
cell replacement through the transplantation of stem cell or
differentiated retinal cell types and artificial vision. However,
despite the efforts of researchers to identify a therapy capable of
preventing retinal degeneration or restoring vision, current therapies entail difficulties that need to be addressed to achieve safe,
effective treatments. In this context, the administration of antioxidants (alone or in cocktails), anti-apoptotics, anti-inflammatories,
neurotrophic factors or viability factors unfortunately only slows
the neurodegeneration of the retina by delaying retinal cell death,
but fail to prevent the progression of the disease. On the other
hand, to design a proper gene-based therapy, it is necessary to
identify all the genes and loci that cause inherited retinal diseases,
but the enormous mutational heterogeneity makes this task very
difficult. Thus, despite the growing body of knowledge about these
diseases, a thorough understanding of the molecular mechanisms
underlying retinal degeneration and the identification of all the
genetic causes of these disorders is still needed to improve the
prospects of therapies. Another hindrance to be overcome is finding
a suitable way to get the genetic material to specific cell types in the
retina. In this sense, the development of viral vectors with modified
tropisms has facilitated this access, but further progress is still
needed.
Certain aspects of cell replacement also need to be considered to
succeed in improving vision. These include the choice of the cell
type to be transplanted, the proper degeneration phase in which to
perform the transplant, the selection of the site of the cell transplant (vitreous humor, subretinal space, periphery of the retina,
fovea) and the number of cells needed. It is also important to note
that the success of cell replacement depends on the survival of the
transplanted cells in the retina, the prevention of tissue rejection,
migration and integration of cells in the remaining retinal circuitry
in a mosaic arrangement and the establishment of adequate synaptic connections capable of restoring visual function. Questions
like how long the transplanted cells will live and whether they are
safe for the healthy cells are yet to be answered, and it has not yet
been clarified whether it is better to transplant differentiated or
undifferentiated cells. Interestingly, although cell migration and
integration in the retina, together with expression of cell-type
specific proteins have been observed in several published studies,
the reports of synapse formation and cell function improvement
are still exceptionally rare. For this reason, the ever-increasing hypothesis is that the main mechanism for the beneficial effect of cell
transplantation appears to be the secretion of neurotrophic factors
that prolong retinal cell survival. Furthermore, photoreceptor and
RPE cell transplantation is relatively easy as compared to ganglion
cell transplantation, which presents additional difficulties because
of their need to extend long processes to form the optic nerve so
that it can make proper contact with the geniculate nucleus in the
brain to achieve image composition. Another obstacle to cell
transplantation is crossing the barrier formed by extracellular
matrix molecules, such as chondroitin sulfate proteoglycans produced by activated microglial cells and astrocytes.
In summary, retinal remodeling in response to alterations in
molecular pathways and the activation of cellular responses underlying retinal disease entail the impairment of visual function. It
seems clear that the treatment of the different pathologies affecting
the retina must involve a combination of several therapeutic approaches, and that the administration of neuroprotective compounds is essential from the time the disease is detected and
throughout the course of treatment. Furthermore, the success of
current or new therapies for blinding conditions will depend on the
detailed knowledge of the genetic causes behind each retinal disorder, the mechanisms underlying cellular homeostasis and
controlled cell death, the morphological and functional changes of
the different retinal cells in response to injury, and the stage of
degeneration of each structure in the retina at the moment of the
therapeutic intervention. Further studies are needed to more
exactly unravel the mechanisms involved in retinal neurodegeneration. This information could eventually be useful in
developing pharmacotherapies targeting fundamental biochemical
defects aimed at retarding disease progression, and/or alleviating
neurodegenerative symptoms in the retina, as well as designing
new and effective gene-based therapies to prevent the diseases.
To conclude, it is important to remark that the combined use of
the current ophthalmologic surgery techniques with therapies
derivative of a deep knowledge of the factors involved in cellular
responses could be the key for increase the success in visual
restoration in different retinal diseases.
Acknowledgments
This work was supported by project grants from the Spanish
Ministry of Economy and Competitiveness-FEDER (BFU201236845), Plan Nacional de IþDþI 2008-2011, Instituto de Salud
n General de Redes y Centros de Investigacio
n
Carlos III, Subdireccio
Cooperativa (RETICS RD07/0062/0008-0012, RETICS RD12/0034/
0006-0010, PS0901854, PI13/01124), ONCE and FUNDALUCE.
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